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Exposure to microgravity conditions causes cardiovascular deconditioning in astronauts during spaceflight. Until now, no specific drugs are available for countermeasure, since the underlying mechanism is largely unknown. Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in various vascular functions, many of which are regulated by purinergic 2 (P2) receptors. However, their function in ECs and SMCs under microgravity conditions is still unclear. In this study, primary ECs and SMCs were isolated from bovine aorta and verified with specific markers. We show for the first time that the P2 receptor expression pattern is altered in ECs and SMCs after 24 h exposure to simulated microgravity using a clinostat. However, conditioned medium compensates this change in specific P2 receptors, for example, P2X7. Notably, P2 receptors such as P2X7 might be the important players during the paracrine interaction. Additionally, ECs and SMCs secreted different cytokines under simulated microgravity, leading into a pathogenic proliferation and migration. In conclusion, our data indicate P2 receptors might be important players responding to gravity changes in ECs and SMCs. Since some artificial P2 receptor ligands are applied as drugs, it is reasonable to assume that they might be promising candidates against cardiovascular deconditioning in the future.
Human mesenchymal stem cells (hMSCs) are considered a promising cell source for regenerative medicine, because they have the potential to differentiate into a variety of lineages among which the mesoderm-derived lineages such adipo- or osteogenesis are investigated best. Human MSCs can be harvested in reasonable to large amounts from several parts of the patient’s body and due to this possible autologous origin, allorecognition can be avoided. In addition, even in allogenic origin-derived donor cells, hMSCs generate a local immunosuppressive microenvironment, causing only a weak immune reaction. There is an increasing need for bone replacement in patients from all ages, due to a variety of reasons such as a new recreational behavior in young adults or age-related diseases. Adipogenic differentiation is another interesting lineage, because fat tissue is considered to be a major factor triggering atherosclerosis that ultimately leads to cardiovascular diseases, the main cause of death in industrialized countries. However, understanding the differentiation process in detail is obligatory to achieve a tight control of the process for future clinical applications to avoid undesired side effects. In this review, the current findings for adipo- and osteo-differentiation are summarized together with a brief statement on first clinical trials.
Background: Human mesenchymal stem cells (hMSCs) have shown their multipotential including differentiating towards endothelial and smooth muscle cell lineages, which triggers a new interest for using hMSCs as a putative source for cardiovascular regenerative medicine. Our recent publication has shown for the first time that purinergic 2 receptors are key players during hMSC differentiation towards adipocytes and osteoblasts. Purinergic 2 receptors play an important role in cardiovascular function when they bind to extracellular nucleotides. In this study, the possible functional role of purinergic 2 receptors during MSC endothelial and smooth muscle differentiation was investigated. Methods and Results: Human MSCs were isolated from liposuction materials. Then, endothelial and smooth muscle-like cells were differentiated and characterized by specific markers via Reverse Transcriptase-PCR (RT-PCR), Western blot and immunochemical stainings. Interestingly, some purinergic 2 receptor subtypes were found to be differently regulated during these specific lineage commitments: P2Y4 and P2Y14 were involved in the early stage commitment while P2Y1 was the key player in controlling MSC differentiation towards either endothelial or smooth muscle cells. The administration of natural and artificial purinergic 2 receptor agonists and antagonists had a direct influence on these differentiations. Moreover, a feedback loop via exogenous extracellular nucleotides on these particular differentiations was shown by apyrase digest. Conclusions: Purinergic 2 receptors play a crucial role during the differentiation towards endothelial and smooth muscle cell lineages. Some highly selective and potent artificial purinergic 2 ligands can control hMSC differentiation, which might improve the use of adult stem cells in cardiovascular tissue engineering in the future.
Cytokine-induced killer cells (CIK) in combination with dendritic cells (DCs) have shown favorable outcomes in renal cell carcinoma (RCC), yet some patients exhibit recurrence or no response to this therapy. In a broader perspective, enhancing the antitumor response of DC-CIK cells may help to address this issue. Considering this, herein, we investigated the effect of anti-CD40 and anti-CTLA-4 antibodies on the antitumor response of DC-CIK cells against RCC cell lines. Our analysis showed that, a) anti-CD40 antibody (G28.5) increased the CD3+CD56+ effector cells of CIK cells by promoting the maturation and activation of DCs, b) G28.5 also increased CTLA-4 expression in CIK cells via DCs, but the increase could be hindered by the CTLA-4 inhibitor (ipilimumab), c) adding ipilimumab was also able to significantly increase the proportion of CD3+CD56+ cells in DC-CIK cells, d) anti-CD40 antibodies predominated over anti-CTLA-4 antibodies for cytotoxicity, apoptotic effect and IFN-g secretion of DC-CIK cells against RCC cells, e) after ipilimumab treatment, the population of Tregs in CIK cells remained unaffected, but ipilimumab combined with G28.5 significantly reduced the expression of CD28 in CIK cells. Taken together, we suggest that the agonistic anti-CD40 antibody rather than CTLA-4 inhibitor may improve the antitumor response of DC-CIK cells, particularly in RCC. In addition, we pointed towards the yet to be known contribution of CD28 in the crosstalk between anti-CTLA-4 and CIK cells.
When the Artemis missions launch, NASA's Orion spacecraft (and crew as of the Artemis II mission) will be exposed to the deep space radiation environment beyond the protection of Earth's magnetosphere. Hence, it is essential to characterize the effects of space radiation, microgravity, and the combination thereof on cells and organisms, i.e., to quantify any correlations between the deep space radiation environment, genetic variation, and induced genetic changes in cells. To address this, the Artemis I mission will include the Peristaltic Laboratory for Automated Science with Multigenerations (PLASM) hardware containing the Deep Space Radiation Genomics (DSRG) experiment. The scientific aims of DSRG are (i) to identify the metabolic and genomic pathways in yeast affected by microgravity, space radiation, and their combination, and (ii) to differentiate between gravity and radiation exposure on single-gene deletion/overexpressing strains' ability to thrive in the spaceflight environment. Yeast is used as a model system because 70% of its essential genes have a human homolog, and over half of these homologs can functionally replace their human counterpart. As part of the experiment preparation towards spaceflight, an Experiment Verification Test (EVT) was performed at the Kennedy Space Center to verify that the experiment design, hardware, and approach to automated operations will enable achieving the scientific aims. For the EVT, fluidic systems were assembled, sterilized, loaded, and acceptance-tested, and subsequently integrated with the engineering parts to produce a flight-like PLASM unit. Each fluidic system consisted of (i) a Media Bag, (ii) four Culture Bags loaded with Saccharomyces cerevisiae (two with deletion series and the remaining two with overexpression series), and (iii) tubing and check valves. The EVT PLASM unit was put under a temperature profile replicating the anticipated different phases of flight, including handover to launch, spaceflight, and splashdown to handover back to the science team, for a 58-day period. At EVT completion, the rate of activation, cellular growth, RNA integrity, and sample contamination were interrogated. All of the experiment's success criteria were satisfied, encouraging our efforts to perform this investigation on Artemis I. This manuscript thus describes the process of spaceflight experiment design maturation with a focus on the EVT, its results, DSRG's preparation for its planned launch on Artemis I in 2022, and how the PLASM hardware can enable other scientific goals on future Artemis missions and/or the Lunar Orbital Platform – Gateway.
Extremophiles are optimal models in experimentally addressing questions about the effects of cosmic radiation on biological systems. The resistance to high charge energy (HZE) particles, and helium (He) ions and iron (Fe) ions (LET at 2.2 and 200 keV/µm, respectively, until 1000 Gy), of spores from two thermophiles, Bacillushorneckiae SBP3 and Bacilluslicheniformis T14, and two psychrotolerants, Bacillus sp. A34 and A43, was investigated. Spores survived He irradiation better, whereas they were more sensitive to Fe irradiation (until 500 Gy), with spores from thermophiles being more resistant to irradiations than psychrotolerants. The survived spores showed different germination kinetics, depending on the type/dose of irradiation and the germinant used. After exposure to He 1000 Gy, D-glucose increased the lag time of thermophilic spores and induced germination of psychrotolerants, whereas L-alanine and L-valine increased the germination efficiency, except alanine for A43. FTIR spectra showed important modifications to the structural components of spores after Fe irradiation at 250 Gy, which could explain the block in spore germination, whereas minor changes were observed after He radiation that could be related to the increased permeability of the inner membranes and alterations of receptor complex structures. Our results give new insights on HZE resistance of extremophiles that are useful in different contexts, including astrobiology.
Cytokine-induced killer (CIK) cells are an ex vivo expanded heterogeneous cell population with an enriched NK-T phenotype (CD3+CD56+). Due to the convenient and relatively inexpensive expansion capability, together with low incidence of graft versus host disease (GVHD) in allogeneic cancer patients, CIK cells are a promising candidate for immunotherapy. It is well known that natural killer group 2D (NKG2D) plays an important role in CIK cell-mediated antitumor activity; however, it remains unclear whether its engagement alone is sufficient or if it requires additional co-stimulatory signals to activate the CIK cells. Likewise, the role of 2B4 has not yet been identified in CIK cells. Herein, we investigated the individual and cumulative contribution of NKG2D and 2B4 in the activation of CIK cells. Our analysis suggests that (a) NKG2D (not 2B4) is implicated in CIK cell (especially CD3+CD56+ subset)-mediated cytotoxicity, IFN-γ secretion, E/T conjugate formation, and degranulation; (b) NKG2D alone is adequate enough to induce degranulation, IFN-γ secretion, and LFA-1 activation in CIK cells, while 2B4 only provides limited synergy with NKG2D (e.g., in LFA-1 activation); and (c) NKG2D was unable to costimulate CD3. Collectively, we conclude that NKG2D engagement alone suffices to activate CIK cells, thereby strengthening the idea that targeting the NKG2D axis is a promising approach to improve CIK cell therapy for cancer patients. Furthermore, CIK cells exhibit similarities to classical invariant natural killer (iNKT) cells with deficiencies in 2B4 stimulation and in the costimulation of CD3 with NKG2D. In addition, based on the current data, the divergence in receptor function between CIK cells and NK (or T) cells can be assumed, pointing to the possibility that molecular modifications (e.g., using chimeric antigen receptor technology) on CIK cells may need to be customized and optimized to maximize their functional potential.
Healing of large bone defects requires implants or scaffolds that provide structural guidance for cell growth, differentiation, and vascularization. In the present work, an agarose-hydroxyapatite composite scaffold was developed that acts not only as a 3D matrix, but also as a release system. Hydroxyapatite (HA) was incorporated into the agarose gels in situ in various ratios by a simple procedure consisting of precipitation, cooling, washing, and drying. The resulting gels were characterized regarding composition, porosity, mechanical properties, and biocompatibility. A pure phase of carbonated HA was identified in the scaffolds, which had pore sizes of up to several hundred micrometers. Mechanical testing revealed elastic moduli of up to 2.8 MPa for lyophilized composites. MTT testing on Lw35human mesenchymal stem cells (hMSCs) and osteosarcoma MG-63 cells proved the biocompatibility of the scaffolds. Furthermore, scaffolds were loaded with model drug compounds for guided hMSC differentiation. Different release kinetic models were evaluated for adenosine 5′-triphosphate (ATP) and suramin, and data showed a sustained release behavior over four days.
Bone tissue engineering is an ever-changing, rapidly evolving, and highly interdisciplinary field of study, where scientists try to mimic natural bone structure as closely as possible in order to facilitate bone healing. New insights from cell biology, specifically from mesenchymal stem cell differentiation and signaling, lead to new approaches in bone regeneration. Novel scaffold and drug release materials based on polysaccharides gain increasing attention due to their wide availability and good biocompatibility to be used as hydrogels and/or hybrid components for drug release and tissue engineering. This article reviews the current state of the art, recent developments, and future perspectives in polysaccharide-based systems used for bone regeneration.
Renewable resources are gaining increasing interest as a source for environmentally benign biomaterials, such as drug encapsulation/release compounds, and scaffolds for tissue engineering in regenerative medicine. Being the second largest naturally abundant polymer, the interest in lignin valorization for biomedical utilization is rapidly growing. Depending on its resource and isolation procedure, lignin shows specific antioxidant and antimicrobial activity. Today, efforts in research and industry are directed toward lignin utilization as a renewable macromolecular building block for the preparation of polymeric drug encapsulation and scaffold materials. Within the last five years, remarkable progress has been made in isolation, functionalization and modification of lignin and lignin-derived compounds. However, the literature so far mainly focuses lignin-derived fuels, lubricants and resins. The purpose of this review is to summarize the current state of the art and to highlight the most important results in the field of lignin-based materials for potential use in biomedicine (reported in 2014⁻2018). Special focus is placed on lignin-derived nanomaterials for drug encapsulation and release as well as lignin hybrid materials used as scaffolds for guided bone regeneration in stem cell-based therapies.
Polyether and polyether/ester based TPU (thermoplastic polyurethanes) were investigated with wide-angle XRD (X-ray diffraction) and SAXS (small angle X-ray scattering). Furthermore, SAXS measurements were performed in the temperature range of 30 °C to 130 °C. Polyether based polymers exhibit only one broad diffraction signal in a region of 2 θ 15° to 25°. In case of polyurethanes with ether/ester modification, the broad diffraction signal arises with small sharp diffraction signals. SAXS measurements of polymers reveal the size and shape of the crystalline zones of the polymer. Between 30 °C and 130 °C the size of the crystalline zone changes significantly. The size decreases in most of investigated TPU. In the case of Desmopan 9365D an increase of the particle size was observed.
Approximately 45% of global greenhouse gas emissions are caused by the construction and use of buildings. Thermal insulation of buildings in the current context of climate change is a well-known strategy to improve the energy efficiency of buildings. The development of renewable insulation material can overcome the drawbacks of widely used insulation systems based on polystyrene or mineral wool. This study analyzes the sustainability and thermal conductivity of new insulation materials made of Miscanthus x giganteus fibers, foaming agents, and alkali-activated fly ash binder. Life cycle assessments (LCA) are necessary to perform benchmarking of environmental impacts of new formulations of geopolymer-based insulation materials. The global warming potential (GWP) of the product is primarily determined by the main binder component sodium silicate. Sodium silicate's CO2 emissions depend on local production, transportation, and energy consumption. The results, which have been published during recent years, vary in a wide range from 0.3 kg to 3.3 kg CO2-eq. kg-1. The overall GWP of the insulation system based on Miscanthus fibers, with properties according to current thermal insulation regulations, reaches up to 95% savings of CO2 emissions compared to conventional systems. Carbon neutrality can be achieved through formulations containing raw materials with carbon dioxide emissions and renewable materials with negative GWP, thus balancing CO2 emissions.
Background: the potency of drugs that interfere with glucose metabolism, i.e., glucose transporters (GLUT) and nicotinamide phosphoribosyltransferase (NAMPT) was analyzed in neuroendocrine tumor (NET, BON-1, and QPG-1 cells) and small cell lung cancer (SCLC, GLC-2, and GLC-36 cells) tumor cell lines. (2) Methods: the proliferation and survival rate of tumor cells was significantly affected by the GLUT-inhibitors fasentin and WZB1127, as well as by the NAMPT inhibitors GMX1778 and STF-31. (3) Results: none of the NET cell lines that were treated with NAMPT inhibitors could be rescued with nicotinic acid (usage of the Preiss–Handler salvage pathway), although NAPRT expression could be detected in two NET cell lines. We finally analyzed the specificity of GMX1778 and STF-31 in NET cells in glucose uptake experiments. As previously shown for STF-31 in a panel NET-excluding tumor cell lines, both drugs specifically inhibited glucose uptake at higher (50 μM), but not at lower (5 μM) concentrations. (4) Conclusions: our data suggest that GLUT and especially NAMPT inhibitors are potential candidates for the treatment of NET tumors.
In thyroid carcinoma cells, the soluble βgalactosidespecific lectin, galectin3, is extra and intracellularly expressed and plays a significant role in thyroid cancer diagnosis. The functional relevance of this molecule, particularly in its extracellular environment however, warrants further elucidation. To gain insight into this topic, the present study characterized principal functional properties of galectin3 in 3 commonly used thyroid carcinoma cell lines (BCPAP, Cal62 and FTC133) that express the molecule intra and extracellulary. Cellintrinsic galectin3 harbors a functional carbohydrate recognition domain as determined by affinity purification. Moreover, cell surface expressed galectin3 can be partially removed by treatment with lactose or asialofetuin, but not with sucrose. Thyroid carcinoma cells adhere to substratebound galectin3 in a βgalactosidespecific manner, whereby only cell adhesion, but not cell migration is promoted. Thus, thyroid tumor cells harbor functional active galectin3 that, inter alia, specifically interacts with cell surfaceexpressed molecular ligands in a βgalactosidedependent manner, whereby the molecule can at least interfere with cell adhesion. The modulation of galectin3 expression level or its ligands in such tumor cells could be of therapeutic interest and needs further experimental clarification.
Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion.
The synthesis and characterization of a new class of 1,2,4-oxadiazolylpyridinium as a cationic scaffold for fluorinated ionic liquid crystals is herein described. A series of 12 fluorinated heterocyclic salts based on a 1,2,4-oxadiazole moiety, connected through its C(5) or C(3) to an N-alkylpyridinium unit and a perfluoroheptyl chain, differing in the length of the alkyl chain and counterions, has been synthesized. As counterions iodide, bromide and bis(trifluoromethane)sulfonimide have been considered. The synthesis, structure, and liquid crystalline properties of these compounds are discussed on the basis of the tuned structural variables. The thermotropic properties of this series of salts have been investigated by differential scanning calorimetry and polarized optical microscopy. The results showed the existence of an enantiotropic mesomorphic smectic liquid crystalline phase for six bis(trifluoromethane)sulfonimide salts.
In this work, the surface reactions of the homemade explosive triacetone triperoxide on tungsten oxide (WO3) sensor surfaces are studied to obtain detailed information about the chemical reactions taking place. Semiconductor gas sensors based on WO3 nanopowders are therefore produced and characterized by scanning electron microscopy, X-ray diffraction, and Raman spectroscopy. To analyze the reaction mechanisms at the sensor surface, the sensor is monitored online under operation conditions using Raman spectroscopy, which allows to identify the temperature-dependent sensor reactions. By combining information from the Raman spectra with data on the changing resistivity of the underlying semiconductor, it is possible to establish a correlation between the adsorbed gas species and the physical properties of the WO3 layer. In the results, it is indicated that a Lewis acid–base reaction is the most likely mechanism for the increase in resistance observed at temperatures below 150 °C. In the results, at higher temperatures, the assumption of a radical mechanism that causes a decrease in resistance is supported.
The development of sustainable, environmentally friendly insulation materials with a reduced carbon footprint is attracting increased interest. One alternative to conventional insulation materials are foamed geopolymers. Similar to foamed concrete, the mechanical properties of geopolymer foams can also be improved by using fibers for reinforcement. This paper presents an overview of the latest research findings in the field of fiber-reinforced geopolymer foam concrete with special focus on natural fibers reinforcement. Furthermore, some basic and background information of natural fibers and geopolymer foams are reported. In most of the research, foams are produced either through chemical foaming with hydrogen peroxide or aluminum powder, or through mechanical foaming which includes a foaming agent. However, previous reviews have not sufficiently addresses the fabrication of geopolymer foams by syntactic foams. Finally, recent efforts to reduce the fiber degradation in geopolymer concrete are discussed along with challenges for natural fiber reinforced-geopolymer foam concrete.
Due to increased emissions of palladium nanoparticles in recent years, it is important to develop analytical techniques to characterize these particles. The synthesis of defined and stable particles plays a key role in this process, as there are not many materials commercially available yet which could act as reference materials. Polyvinylpyrrolidone- (PVP-) stabilized palladium nanoparticles were synthesized through the reduction of palladium chloride by tetraethylene glycol (TEG) in the presence of KOH. Four different methods were used for particle size analysis of the palladium nanoparticles. Palladium suspensions were analyzed by scanning electron microscopy (SEM), small angle X-ray scattering (SAXS), single-particle ICP-MS (SP-ICP-MS), and X-ray diffraction (XRD). Secondary particles between 30 nm and 130 nm were detected in great compliance with SAXS and SP-ICP-MS. SEM analysis showed that the small particulates tend to form agglomerates.
New sustainable, environmentally friendly materials for thermal insulation of buildings are necessary to reduce their carbon footprints. In this study, Miscanthus fiber-reinforced geopolymer composites, foamed with sodium dodecyl sulfate (SDS), were developed using fly ash as a geopolymer precursor. The effects of fiber content, fiber size, curing temperature, foaming agent content, fumed silica specific surface area and fumed silica content on thermal conductivity and compressive strength were evaluated using a Plackett-Burman design of experiment. Furthermore, the microstructure of geopolymer composites was investigated using X-ray diffraction (XRD), X-ray micro-computed tomography (μCT) and scanning electron microscopy (SEM). The measured characteristic values were in the following ranges: Thermal conductivity 0.057 W (m K)−1 to 0.127 W (m K)−1, compressive strength 0.007 MPa–0.719 MPa and porosity 49 vol% to 76 vol%. The results reveal an enhancement of thermal conductivity by elevated fiber size and foaming agent content. In contrast, the compressive strength is enhanced by high fiber content. Additionally, SEM images indicate a good interaction between the fibers and the geopolymer matrix, because nearly the whole fiber surface is covered by the geopolymer.
Aim: To understand how transcriptional factors Pdr1 and Pdr3, belonging to the pleiotropic drug resistance system, are activated, and regulated after introducing chemical toxins to the cell in the model organism Saccharomyces cerevisiae.
Methods: Series of molecular methods were applied using different strains of S. cerevisiae over-expressing proteins of interest as a eukaryotic cell model. The chemical stress introduced to the cell is represented by menadione. Results were obtained performing protein detection and analysis. Additionally, the regulation of the DNA binding of the transcriptional activators after stimulation is quantified using chromatin immunoprecipitation, employing epitope-tagged factors and real-time qPCR.
Results: Our results indicated higher expression levels of the Pdr1 transcriptional factor, compared to its homologous Pdr3 after treatment with menadione. The yeast-cell defence system was tested against various organic solvents to exclude the possibility of their presence potentially affecting the results. The results indicate that Pdr1 is most abundant after 30 minutes from the beginning of the treatment, compared with 240 minutes after the treatment when the function of the transcription factor is faded. It appears that Pdr1 binding to the PDR5 and SNQ2 promoters, which are both activated by Pdr1, peaks around the same time, or more precisely after 40 minutes from the start of the treatment.
Conclusion: The tendency of Pdr1 reduction after its activation by menadione is detected. One possibility is that Pdr1, after recognizing the xenobiotic menadione, is removed by a degradation mechanism. Given the fact that Pdr1 directly binds the xenobiotic molecule, its destruction might help the cells to remove toxic levels of menadione. It is possible that overexpressing the part of Pdr1 which recognizes menadione alone was sufficient to detoxify and hence produce a tolerance towards menadione.
Therapeutic Treatments for Osteoporosis-Which Combination of Pills Is the Best among the Bad?
(2022)
Osteoporosis is a chronical, systemic skeletal disorder characterized by an increase in bone resorption, which leads to reduced bone density. The reduction in bone mineral density and therefore low bone mass results in an increased risk of fractures. Osteoporosis is caused by an imbalance in the normally strictly regulated bone homeostasis. This imbalance is caused by overactive bone-resorbing osteoclasts, while bone-synthesizing osteoblasts do not compensate for this. In this review, the mechanism is presented, underlined by in vitro and animal models to investigate this imbalance as well as the current status of clinical trials. Furthermore, new therapeutic strategies for osteoporosis are presented, such as anabolic treatments and catabolic treatments and treatments using biomaterials and biomolecules. Another focus is on new combination therapies with multiple drugs which are currently considered more beneficial for the treatment of osteoporosis than monotherapies. Taken together, this review starts with an overview and ends with the newest approaches for osteoporosis therapies and a future perspective not presented so far.
Intimate swabs taken for examination in sexual assault cases typically yield mixtures of sperm and epithelial cell types. While powerful, differential extraction protocols to overcome such cell type mixtures by separate lysis of epithelial cells and spermatozoa can still prove ineffective, in particular if only few sperm cells are present or if swabs contain sperm from more than one individual leading to complex low level DNA mixtures. A means to avoid such mixtures consists in the analysis of single micromanipulated sperm cells. However, the quantity of DNA from single sperm cells is not sufficient for conventional STR analysis. Here, we describe a simple method for micromanipulating individual sperm cells from intimate swabs and show that whole genome amplification can generate sufficient amounts of DNA from single cells for subsequent DNA profiling. We recovered over 80% of alleles of haploid autosomal STR profiles from the majority of individual sperm cells. Furthermore, we demonstrate that in mixtures of sperm from two contributors, Y-STR and X-STR profiles of individual sperm cells can be used to sort the haploid autosomal profiles to develop the diploid consensus STR profiles of the individual donors. Finally, by analysing single sperm cells from mock sexual assault swabs with one or two sperm donors, we showed that our protocols enabled the identification of the unknown male contributors.
Tamoxifen therapy of invasive breast cancer has been associated with increased levels of endothelin-1 (ET-1) so that an endothelin-1 receptor (ETR) blockade has been suggested as a new therapeutic approach. This study analyzed the relationship between Tamoxifen and ET-1 signalling in invasive breast cancer. Using paraffinized tissue from 50 randomly chosen cases of invasive and in-situ ductal carcinoma from our archive, the expression of ETRs was analyzed by immune histology. ETRs were regularly detectable in normal breast tissue, but rarely in adjacent tumor areas (3/50 cases). By immunoprecipitation, a complex was found consisting of ET-1, estrogen receptors and Tamoxifen. Consequently, transcription of several target genes of ET-1 and estrogen receptors was detectable (interleukin-6, wnt-11 and a vimentin spliceform). In particular, the combination of Tamoxifen, ET-1, and estrogen receptors induced further increasing levels of these target genes. Some of these genes have been found upregulated in metastatically spreading breast cancer cells. We conclude: i) ETRs do not play a role in invasive or in-situ ductal breast cancer; ii) estrogen receptors and Tamoxifen build a complex with ET-1; and iii) upregulated transcription of target genes by ET-1–estrogen receptor–Tamoxifen complex may negatively influence breast cancer prognosis. These results indicate a role for ET-1 in Tamoxifen treated breast cancer patients leading to a potentially worsening prognosis.
The French–Italian Concordia Research Station, situated on the Antarctic Polar Plateau at an elevation of 3233 m above sea level, offers a unique opportunity to study the presence and variation of microbes introduced by abiotic or biotic vectors and, consequently, appraise the amplitude of human impact in such a pristine environment. This research built upon a previous work, which explored microbial diversity in the surface snow surrounding the Concordia Research Station. While that study successfully characterized the bacterial assemblage, detecting fungal diversity was hampered by the low DNA content. To address this knowledge gap, in the present study, we optimized the sampling by increasing ice/snow collected to leverage the final DNA yield. The V4 variable region of the 16S rDNA and Internal Transcribed Spacer (ITS1) rDNA was used to evaluate bacterial and fungal diversity. From the sequencing, we obtained 3,352,661 and 4,433,595 reads clustered in 930 and 3182 amplicon sequence variants (ASVs) for fungi and bacteria, respectively. Amplicon sequencing revealed a predominance of Basidiomycota (49%) and Ascomycota (42%) in the fungal component; Bacteroidota (65.8%) is the main representative among the bacterial phyla. Basidiomycetes are almost exclusively represented by yeast-like fungi. Our findings provide the first comprehensive overview of both fungal and bacterial diversity in the Antarctic Polar Plateau’s surface snow/ice near Concordia Station and to identify seasonality as the main driver of microbial diversity; we also detected the most sensitive microorganisms to these factors, which could serve as indicators of human impact in this pristine environment and aid in planetary protection for future exploration missions.
The biological effects of bilirubin, still poorly understood, are concentration-dependent ranging from cell protection to toxicity. Here we present data that at high nontoxic physiological concentrations, bilirubin inhibits growth of proliferating human coronary artery smooth muscle cells by three events. It impairs the activation of Raf/ERK/MAPK pathway and the cellular Raf and cyclin D1 content that results in retinoblastoma protein hypophosphorylation on amino acids S608 and S780. These events impede the release of YY1 to the nuclei and its availability to regulate the expression of genes and to support cellular proliferation. Moreover, altered calcium influx and calpain II protease activation leads to proteolytical degradation of transcription factor YY1. We conclude that in the serum-stimulated human vascular smooth muscle primary cell cultures, bilirubin favors growth arrest, and we propose that this activity is regulated by its interaction with the Raf/ERK/MAPK pathway, effect on cyclin D1 and Raf content, altered retinoblastoma protein profile of hypophosphorylation, calcium influx, and YY1 proteolysis. We propose that these activities together culminate in diminished 5 S and 45 S ribosomal RNA synthesis and cell growth arrest. The observations provide important mechanistic insight into the molecular mechanisms underlying the transition of human vascular smooth muscle cells from proliferative to contractile phenotype and the role of bilirubin in this transition.
Multiple myeloma is the second most common hematological malignancy. Despite all the progress made in treating multiple myeloma, it still remains an incurable disease. Patients are left with a median survival of 4-5 years. The combined treatment of multiple myeloma with histone deacetylase inhibitors and cytokine-induced killer cells provides a promising targeted treatment option for patients. This study investigated the impact of a combined treatment compared to treatment with histone deacetylase inhibitors. The experiments revealed that a treatment with histone deacetylase (HDAC) inhibitors could reduce cell viability to 59% for KMS 18 cell line and 46% for the U-266 cell line. The combined treatment led to a decrease of cell viability to 33% for KMS 18 and 27% for the U-266 cell line, thus showing a significantly better efficacy than the single treatment.
A series of reactive binaphthyl‐diimine‐based dopants is prepared and investigated with respect to their potential for the chiral induction of structural coloration in nematic liquid crystal mixture E7 and the selective photonic sensing of nitrogen dioxide (NO2). Studies of the helical twisting power (HTP) in 4‐cyano‐4′‐pentylbiphenyl (5CB) reveal HTP values as high as 375 µm‐1 and the tremendous impact of structural compatibility and changes of the dihedral binaphthyl angle on the efficiency of the chiral transfer. Detailed investigation of the sensing capabilities of the systems reveals an extraordinarily high selectivity for NO2 and a response to concentrations as low as 100 ppm. The systems show a direct response to the analyte gas leading to a concentration‐dependent shift of the reflectance wavelength of up to several hundred nanometers. Incorporation of copper ions remarkably improves the sensor's properties in terms of sensitivity and selectivity, enabling the tailored tweaking of the system's properties.
Background: Bile acids, end products of the pathway for cholesterol elimination, are required for dietary lipid and fatsoluble vitamin absorption and maintain the balance between cholesterol synthesis in the liver and cholesterol excretion. They are composed of a steroid structure and are primarily made in the liver by the oxidation of cholesterol. Cholesterol is also highly abundant in the human ovarian follicle, where it is used in the formation of the sex steroids.
Methodology/Principal Findings: Here we describe for the first time evidence that all aspects of the bile acid synthesis pathway are present in the human ovarian follicle, including the enzymes in both the classical and alternative pathways, the nuclear receptors known to regulate the pathway, and the end product bile acids. Furthermore, we provide functional evidence that bile acids are produced by the human follicular granulosa cells in response to cholesterol presence in the culture media.
Conclusions/Significance: These findings establish a novel pathway present in the human ovarian follicle that has the capacity to compete directly with sex steroid synthesis.
Introduction: Matrix metalloproteinases (MMPs) are important in tissue remodelling. Here we investigate the role of collagenase-3 (MMP-13) in antibody-induced arthritis.
Methods: For this study we employed the K/BxN serum-induced arthritis model. Arthritis was induced in C57BL/6 wild type (WT) and MMP-13-deficient (MMP-13–/–) mice by intraperitoneal injection of 200 μl of K/BxN serum. Arthritis was assessed by measuring the ankle swelling. During the course of the experiments, mice were sacrificed every second day for histological examination of the ankle joints. Ankle sections were evaluated histologically for infiltration of inflammatory cells, pannus tissue formation and bone/cartilage destruction. Semi-quantitative PCR was used to determine MMP-13 expression levels in ankle joints of untreated and K/BxN serum-injected mice.
Results: This study shows that MMP-13 is a regulator of inflammation. We observed increased expression of MMP-13 in ankle joints of WT mice during K/BxN serum-induced arthritis and both K/BxN serum-treated WT and MMP-13–/– mice developed progressive arthritis with a similar onset. However, MMP-13–/– mice showed significantly reduced disease over the whole arthritic period. Ankle joints of WT mice showed severe joint destruction with extensive inflammation and erosion of cartilage and bone. In contrast, MMP-13–/– mice displayed significantly decreased severity of arthritis (50% to 60%) as analyzed by clinical and histological scoring methods.
Conclusions: MMP-13 deficiency acts to suppress the local inflammatory responses. Therefore, MMP-13 has a role in the pathogenesis of arthritis, suggesting MMP-13 is a potential therapeutic target.
Dental stem cells have been isolated from the medical waste of various dental tissues. They have been characterized by numerous markers, which are evaluated herein and differentiated into multiple cell types. They can also be used to generate cell lines and iPSCs for long-term in vitro research. Methods for utilizing these stem cells including cellular systems such as organoids or cell sheets, cell-free systems such as exosomes, and scaffold-based approaches with and without drug release concepts are reported in this review and presented with new pictures for clarification. These in vitro applications can be deployed in disease modeling and subsequent pharmaceutical research and also pave the way for tissue regeneration. The main focus herein is on the potential of dental stem cells for hard tissue regeneration, especially bone, by evaluating their potential for osteogenesis and angiogenesis, and the regulation of these two processes by growth factors and environmental stimulators. Current in vitro and in vivo publications show numerous benefits of using dental stem cells for research purposes and hard tissue regeneration. However, only a few clinical trials currently exist. The goal of this review is to pinpoint this imbalance and encourage scientists to pick up this research and proceed one step further to translation.
The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.
Hox genes are an evolutionary highly conserved gene family. They determine the anterior-posterior body axis in bilateral organisms and influence the developmental fate of cells. Embryonic stem cells are usually devoid of any Hox gene expression, but these transcription factors are activated in varying spatial and temporal patterns defining the development of various body regions. In the adult body, Hox genes are among others responsible for driving the differentiation of tissue stem cells towards their respective lineages in order to repair and maintain the correct function of tissues and organs. Due to their involvement in the embryonic and adult body, they have been suggested to be useable for improving stem cell differentiations in vitro and in vivo. In many studies Hox genes have been found as driving factors in stem cell differentiation towards adipogenesis, in lineages involved in bone and joint formation, mainly chondrogenesis and osteogenesis, in cardiovascular lineages including endothelial and smooth muscle cell differentiations, and in neurogenesis. As life expectancy is rising, the demand for tissue reconstruction continues to increase. Stem cells have become an increasingly popular choice for creating therapies in regenerative medicine due to their self-renewal and differentiation potential. Especially mesenchymal stem cells are used more and more frequently due to their easy handling and accessibility, combined with a low tumorgenicity and little ethical concerns. This review therefore intends to summarize to date known correlations between natural Hox gene expression patterns in body tissues and during the differentiation of various stem cells towards their respective lineages with a major focus on mesenchymal stem cell differentiations. This overview shall help to understand the complex interactions of Hox genes and differentiation processes all over the body as well as in vitro for further improvement of stem cell treatments in future regenerative medicine approaches.
Although p27 plays a central role in cell cycle regulation, its role in breast cancer prognosis is controversial. Furthermore, the p27 gene CDKN1B carries a polymorphism with unknown functional relevance. This study was designed to evaluate p27 expression and p27 genotyping with respect to early breast cancer prognosis. 279 patients with infiltrating metastasis-free breast cancer were included in this study. p27 expression was determined in tumor tissue specimens from 261 patients by immunohistochemistry. From 108 patients, the CDKN1B genotype was examined by PCR and subsequent direct sequencing. 55.2% of the tumors were considered p27 positive. p27 expression did not correlate with any of the established parameters except for nodal involvement but significantly correlated to prolonged disease-free survival. In 35% of the tumors analyzed, the CDKN1B gene showed a polymorphism at codon 109 (V109G). The V109G polymorphism correlated with greater nodal involvement. In the node-negative subgroup, V109G correlated significantly with a shortened disease-free survival. In conclusion, the determination of the CDKN1B genotype might be a powerful tool for the prognosis of patients with early breast cancer.
With the increasing demand for ultrapure water in the pharmaceutical and semiconductor industry, the need for precise measuring instruments for those applications is also growing. One critical parameter of water quality is the amount of total organic carbon (TOC). This work presents a system that uses the advantage of the increased oxidation power achieved with UV/O3 advanced oxidation process (AOP) for TOC measurement in combination with a significant miniaturization compared to the state of the art. The miniaturization is achieved by using polymer-electrolyte membrane (PEM) electrolysis cells for ozone generation in combination with UV-LEDs for irradiation of the measuring solution, as both components are significantly smaller than standard equipment. Conductivity measurement after oxidation is the measuring principle and measurements were carried out in the TOC range between 10 and 1000 ppb TOC. The suitability of the system for TOC measurement is demonstrated using the oxidation by ozonation combined with UV irradiation of defined concentrations of isopropyl alcohol (IPA).
The solvent exchange as one of the most important steps during the manufacturing process of organic aerogels was investigated. This step is crucial as a preparatory step for the supercritical drying, since the pore solvent must be soluble in supercritical carbon dioxide to enable solvent extraction. The development and subsequent optimization of a suitable system with a peristaltic pump for automatic solvent exchange proved to be a suitable approach. In addition, the influence of zeolites on the acceleration of the process was found to be beneficial. To investigate the process, the water content in acetone was determined at different times using Karl Fischer titration. The shrinkage, densities, as well as the surface areas of the aerogels were analyzed. Based on these, the influence of various process parameters on the final structure of the obtained aerogels was investigated and evaluated. Modeling on diffusion in porous materials completes this study.
Fabry disease (FD) is an X‐linked lysosomal storage disorder. Deficiency of the lysosomal enzyme alpha‐galactosidase (GLA) leads to accumulation of potentially toxic globotriaosylceramide (Gb3) on a multisystem level. Cardiac and cerebrovascular abnormalities as well as progressive renal failure are severe, life‐threatening long‐term complications. The complete pathophysiology of chronic kidney disease (CKD) in FD and the role of tubular involvement for its progression are unclear.
We established human renal tubular epithelial cell lines from the urine of male FD patients and male controls. The renal tubular system is rich in mitochondria and involved in transport processes at high energy costs. Our studies revealed fragmented mitochondria with disrupted cristae structure in FD patient cells. Oxidative stress levels were elevated and oxidative phosphorylation was up‐regulated in FD pointing at enhanced energetic needs. Mitochondrial homeostasis and energy metabolism revealed major changes as evidenced by differences in mitochondrial number, energy production and fuel consumption. The changes were accompanied by activation of the autophagy machinery in FD. Sirtuin1, an important sensor of (renal) metabolic stress and modifier of different defense pathways, was highly expressed in FD.
Our data show that lysosomal FD impairs mitochondrial function and results in severe disturbance of mitochondrial energy metabolism in renal cells. This insight on a tissue‐specific level points to new therapeutic targets which might enhance treatment efficacy.
Nitrile-type inhibitors are known to interact with cysteine proteases in a covalent-reversible manner. The chemotype of 3-cyano-3-aza-β-amino acid derivatives was designed in which the N-cyano group is centrally arranged in the molecule to allow for interactions with the nonprimed and primed binding regions of the target enzymes. These compounds were evaluated as inhibitors of the human cysteine cathepsins K, S, B, and L. They exhibited slow-binding behavior and were found to be exceptionally potent, in particular toward cathepsin K, with second-order rate constants up to 52 900 × 103 M–1 s–1.
Polymer fibers with liquid crystals (LCs) in the core have potential as autonomous sensors of airborne volatile organic compounds (VOCs), with a high surface-to-volume ratio enabling fast and sensitive response and an attractive non-woven textile form factor. We demonstrate their ability to continuously and quantitatively measure the concentration of toluene, cyclohexane, and isopropanol as representative VOCs, via the impact of each VOC on the LC birefringence. The response is fully reversible and repeatable over several cycles, the response time can be as low as seconds, and high sensitivity is achieved when the operating temperature is near the LC-isotropic transition temperature. We propose that a broad operating temperature range can be realized by combining fibers with different LC mixtures, yielding autonomous VOC sensors suitable for integration in apparel or in furniture that can compete with existing consumer-grade electronic VOC sensors in terms of sensitivity and response speed.
Major progress occurred in understanding inborn errors of ketone body transport and metabolism between the International Congresses on Inborn Errors of Metabolism in Barcelona (2013) and Rio de Janeiro (2017). These conditions impair either ketogenesis (presenting as episodes of hypoketotic hypoglycemia) or ketolysis (presenting as ketoacidotic episodes); for both groups, immediate intravenous glucose administration is the most critical and (mHGGCS, HMGCS2) effective treatment measure.
3-Hydroxy-3-methylglutaryl-coenzyme A lyase (HMGCL, HMGCL) deficiency is a rare inborn error of ketogenesis. Even if the ketogenic enzyme is fully disrupted, an elevated signal for the ketone body acetoacetic acid is a frequent observation in the analysis of urinary organic acids, at least if derivatization is performed by methylation. We provide an explanation for this phenomenon and trace it back to degradation of the derivatized 3-hydroxy-3-methylglutaric acid and high temperature of the injector of the gas chromatograph.
Pozzolanic properties of Pennisetum purpureum grass ash were tested on Portland cement. Results show that the ash can be blended with cements without compromising binding strength of the cement. It was found that Portland cement could be blended with Pennisetum purpureum up to a ratio of 3:2 compromising compressive strength of mortar.Mortar with lower cement replacement took longer to set as evidenced by lower compressive strength within the 28-day aging time. Mortar with higher cement replacement had lower water absorption capacity, an indication that the test pozzolan was of smaller particulate size. XRF analysis and the FTIR spectrum showed that the ash has a higher content of silica. The XRD pattern of the ash showed that the ash was predominantly amorphous. SEM images showed that the ash produced at 600 o C had residual carbon material.
This research studies in detail four different assays, namely DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), FRAP (ferric ion reducing antioxidant potential) and FC (Folin-Ciocalteu), to determine the antioxidant capacity of standard substances as well as 50 organosolv lignins, and two kraft lignins. The coefficient of variation was determined for each method and was lowest for ABTS and highest for DPPH. The best correlation was found for FRAP and FC, which both rely on a single electron transfer mechanism. A good correlation between ABTS, FRAP and FC, respectively, could be observed, even though ABTS relies on a more complex reaction mechanism. The DPPH assay merely correlates with the others, implying that it reflects different antioxidative attributes due to a different reaction mechanism. Lignins obtained from paulownia and silphium have been investigated for the first time regarding their antioxidant capacity. Paulownia lignin is in the same range as beech wood lignin, while silphium lignin resembles wheat straw lignin. Miscanthus lignin is an exception from the grass lignins and possesses a significantly higher antioxidant capacity. All lignins possess a good antioxidant capacity and thus are promising candidates for various applications, e. g. as additives in food packaging or for biomedical purposes.
We examined the cytotoxic effects of chaetocin on clear cell renal cell carcinoma (ccRCC) cells and the possibility to combine the effects of chaetocin with the effects of cytokine-induced killer cells (CIK) assayed by MTT assay and FACS analysis. Chaetocin is a thiodioxopiperazine produced by fungi belonging to the chaetomiaceae family. In 2007, it was first reported that chaetocin shows potent and selective ex vivo anti-cancer activity by inducing reactive oxygen species. CIK cells are generated from CD3+/CD56- T lymphocytes with double negative CD4-/CD8- phenotype that are isolated from human blood. The addition of distinct interleukins and antibodies results in the generation of CIK cells that are able to specifically target and destroy renal carcinoma cells. The results of this research state that the anti-ccRCC activity of chaetocin is weak and does not show a high grade of selectivity on clear cell renal cell carcinoma cells. Although the CIK cells show a high grade of selective anti-ccRCC activity, this effect could not be improved by the addition of chaetocin. So chaetocin seems to be no suitable agent for specific targeting ccRCC cells or for the combination therapy with CIK cells in renal cancer.
A main factor hampering life in space is represented by high atomic number nuclei and energy (HZE) ions that constitute about 1% of the galactic cosmic rays. In the frame of the “STARLIFE” project, we accessed the Heavy Ion Medical Accelerator (HIMAC) facility of the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. By means of this facility, the extremophilic species Haloterrigena hispanica and Parageobacillus thermantarcticus were irradiated with high LET ions (i.e., Fe, Ar, and He ions) at doses corresponding to long permanence in the space environment. The survivability of HZE-treated cells depended upon either the storage time and the hydration state during irradiation; indeed, dry samples were shown to be more resistant than hydrated ones. With particular regard to spores of the species P. thermantarcticus, they were the most resistant to irradiation in a water medium: an analysis of the changes in their biochemical fingerprinting during irradiation showed that, below the survivability threshold, the spores undergo to a germination-like process, while for higher doses, inactivation takes place as a consequence of the concomitant release of the core’s content and a loss of integrity of the main cellular components. Overall, the results reported here suggest that the selected extremophilic microorganisms could serve as biological model for space simulation and/or real space condition exposure, since they showed good resistance to ionizing radiation exposure and were able to resume cellular growth after long-term storage.
Apple replant disease (ARD) is a soil-borne disease, which is of particular importance for fruit tree nurseries and fruit growers. The disease manifests by a poor vegetative development, stunted growth, and reduced yield in terms of quantity and quality, if apple plants (usually rootstocks) are replanted several times at the same site. Genotype-specific differences in the reaction of apple plants to ARD are documented, but less is known about the genetic mechanisms behind this symptomatology. Recent transcriptome analyses resulted in a number of candidate genes possibly involved in the plant response. In the present study, the expression of 108 selected candidate genes was investigated in root and leaf tissue of four different apple genotypes grown in untreated ARD soil and ARD soil disinfected by γ-irradiation originating from two different sites in Germany. Thirty-nine out of the 108 candidate genes were differentially expressed in roots by taking a p-value of < 0.05 and a fold change of > 1.5 as cutoff. Sixteen genes were more than 4.5-fold upregulated in roots of plants grown in ARD soil. The four genes MNL2 (putative mannosidase); ALF5 (multi antimicrobial extrusion protein); UGT73B4 (uridine diphosphate (UDP)-glycosyltransferase 73B4), and ECHI (chitin-binding) were significantly upregulated in roots. These genes seem to be related to the host plant response to ARD, although they have never been described in this context before. Six of the highly upregulated genes belong to the phytoalexin biosynthesis pathway. Their genotype-specific gene expression pattern was consistent with the phytoalexin content measured in roots. The biphenyl synthase (BIS) genes were found to be useful as early biomarkers for ARD, because their expression pattern correlated well with the phenotypic reaction of the Malus genotypes investigated.
Modeling of Creep Behavior of Particulate Composites with Focus on Interfacial Adhesion Effect
(2022)
Evaluation of creep compliance of particulate composites using empirical models always provides parameters depending on initial stress and material composition. The effort spent to connect model parameters with physical properties has not resulted in success yet. Further, during the creep, delamination between matrix and filler may occur depending on time and initial stress, reducing an interface adhesion and load transfer to filler particles. In this paper, the creep compliance curves of glass beads reinforced poly(butylene terephthalate) composites were fitted with Burgers and Findley models providing different sets of time-dependent model parameters for each initial stress. Despite the finding that the Findley model performs well in a primary creep, the Burgers model is more suitable if secondary creep comes into play; they allow only for a qualitative prediction of creep behavior because the interface adhesion and its time dependency is an implicit, hidden parameter. As Young’s modulus is a parameter of these models (and the majority of other creep models), it was selected to be introduced as a filler content-dependent parameter with the help of the cube in cube elementary volume approach of Paul. The analysis led to the time-dependent creep compliance that depends only on the time-dependent creep of the matrix and the normalized particle distance (or the filler volume content), and it allowed accounting for the adhesion effect. Comparison with the experimental data confirmed that the elementary volume-based creep compliance function can be used to predict the realistic creep behavior of particulate composites.
This study presents a microindentation system which allows spatially resolved local as well as bulk viscoelastic material information to be obtained within one instrument. The microindentation method was merged with dynamic mechanical analysis (DMA) for a tungsten cone indenter. Three tungsten cone indenters were investigated: tungsten electrode, tungsten electrode + 2% lanthanum, and tungsten electrode + rare earth elements. Only the tungsten electrode + 2% lanthanum indenter showed the sinusoidal response, and its geometry remained unaffected by the repeated indentations. Complex moduli obtained from dynamic microindentation for high-density polyethylene, polybutylene terephthalate, polycarbonate, and thermoplastic polyurethane are in agreement with the literature. Additionally, by implementing a specially developed x-y-stage, this study showed that dynamic microindentation with a tungsten cone indenter was an adequate method to determine spatially resolved local viscoelastic surface properties.
Background: Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues.
Methodology/Principal Findings: Here, we present evidence that raft-associated endocytic proteins (flotillins) are preassembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions.
Conclusions: Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization.
Matrix metalloproteinases (MMPs) are matrix-degrading enzymes that are over-expressed in joints of rheumatoid arthritis (RA) patients. However, the contribution of specific MMPs for the development of arthritic joints is unknown. This study is aimed at studying the role of matrix metalloproteinase-9 (MMP-9) in mice, using the K/BxN serum-transfer model of RA. Arthritis was induced in Balb/c mice by injecting K/BxN serum. Development of arthritis was followed in these mice by measuring ankle thickness and clinical index score. MMP-9 expression in the joints of mice killed at various time points during the disease progression was determined by gelatin zymography using ankle lysates. We found that MMP-9 expression increased with the severity of arthritis. Importantly MMP-9 deficient mice injected with K/BxN serum showed a milder form of arthritis in comparison to the control C57BL/6 mice injected with K/BxN serum. We therefore conclude that MMP-9 promotes arthritis in mice.
DT-13 attenuates inflammation by inhibiting NLRP3-inflammasome related genes in RAW264.7 macrophages
(2024)
Plant derived saponins or other glycosides are widely used for their anti-inflammatory, antioxidant, and anti-viral properties in therapeutic medicine. In this study, we focus on understanding the function of the less known steroidal saponin from the roots of Liriope muscari L. H. Bailey – saponin C (also known as DT-13) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages in comparison to the well-known saponin ginsenoside Rk1 and anti-inflammatory drug dexamethasone. We proved that DT-13 reduces LPS-induced inflammation by inhibiting nitric oxide (NO) production, interleukin-6 (IL-6) release, cycloxygenase-2 (COX-2), tumour necrosis factor-alpha (TNF-α) gene expression, and nuclear factor kappa-B (NFκB) translocation into the nucleus. It also inhibits the inflammasome component NOD-like receptor family pyrin domain containing protein 3 (NLRP3) regulating the inflammasome activation. This was supported by the significant inhibition of caspase-1 and interleukin-1 beta (IL-1β) expression and release. This study demonstrates the anti-inflammatory effect of saponins on LPS-stimulated macrophages. For the first time, an in vitro study shows the attenuating effect of DT-13 on NLRP3-inflammasome activation. In comparison to the existing anti-inflammatory drug, dexamethasone, and triterpenoid saponin Rk1, DT-13 more efficiently inhibits inflammation in the applied cell culture model. Therefore, DT-13 may serve as a lead compound for the development of new more effective anti-inflammatory drugs with minimised side effects.
The analysis of Δ9-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) from blood serum is a routine task in forensic toxicology laboratories. For examination of consumption habits, the concentration of the phase I metabolite THC-COOH is used. Recommendations for interpretation of analysis values in medical-psychological assessments (regranting of driver’s licenses, Germany) include threshold values for the free, unconjugated THC-COOH. Using a fully automated two-step liquid-liquid extraction, THC, 11-OH-THC, and free, unconjugated THC-COOH were extracted from blood serum, silylated with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), and analyzed by GC/MS. The automation was carried out by an x-y-z sample robot equipped with modules for shaking, centrifugation, and solvent evaporation. This method was based on a previously developed manual sample preparation method. Validation guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) were fulfilled for both methods, at which the focus of this article is the automated one. Limits of detection and quantification for THC were 0.3 and 0.6 μg/L, for 11-OH-THC were 0.1 and 0.8 μg/L, and for THC-COOH were 0.3 and 1.1 μg/L, when extracting only 0.5 mL of blood serum. Therefore, the required limit of quantification for THC of 1 μg/L in driving under the influence of cannabis cases in Germany (and other countries) can be reached and the method can be employed in that context. Real and external control samples were analyzed, and a round robin test was passed successfully. To date, the method is employed in the Institute of Legal Medicine in Giessen, Germany, in daily routine. Automation helps in avoiding errors during sample preparation and reduces the workload of the laboratory personnel. Due to its flexibility, the analysis system can be employed for other liquid-liquid extractions as well. To the best of our knowledge, this is the first publication on a comprehensively automated classical liquid-liquid extraction workflow in the field of forensic toxicological analysis.
Optical gas sensors based on chiral-nematic liquid crystals (N* LCs) forming one-dimensional photonic crystals do not require electrical energy and have a considerable potential to supplement established types of sensors. A chiral-nematic phase with tunable selective reflection is induced in a nematic host LC by adding reactive chiral dopants. The selective chemical reaction between dopant and analyte is capable to vary the pitch length (the lattice constant) of the soft, self-assembled, one-dimensional photonic crystal. The progress of the ongoing chemical reaction can be observed even by naked eye because the color of the samples varies. In this work, we encapsulate the responsive N* LC in microscale polyvinylpyrrolidone (PVP) fibers via coaxial electrospinning. The sensor is, thus, given a solid form and has an improved stability against nonavoidable environmental influences. The reaction behavior of encapsulated and nonencapsulated N* LC toward a gaseous analyte is compared, systematically. Making use of the encapsulation is an important step to improve the applicability.
The application of Raman and infrared (IR) microspectroscopy is leading to hyperspectral data containing complementary information concerning the molecular composition of a sample. The classification of hyperspectral data from the individual spectroscopic approaches is already state-of-the-art in several fields of research. However, more complex structured samples and difficult measuring conditions might affect the accuracy of classification results negatively and could make a successful classification of the sample components challenging. This contribution presents a comprehensive comparison in supervised pixel classification of hyperspectral microscopic images, proving that a combined approach of Raman and IR microspectroscopy has a high potential to improve classification rates by a meaningful extension of the feature space. It shows that the complementary information in spatially co-registered hyperspectral images of polymer samples can be accessed using different feature extraction methods and, once fused on the feature-level, is in general more accurately classifiable in a pattern recognition task than the corresponding classification results for data derived from the individual spectroscopic approaches.
Forensic DNA profiles are established by multiplex PCR amplification of a set of highly variable short tandem repeat (STR) loci followed by capillary electrophoresis (CE) as a means to assign alleles to PCR products of differential length. Recently, CE analysis of STR amplicons has been supplemented by high-throughput next generation sequencing (NGS) techniques that are able to detect isoalleles bearing sequence polymorphisms and allow for an improved analysis of degraded DNA. Several such assays have been commercialised and validated for forensic applications. However, these systems are cost-effective only when applied to high numbers of samples. We report here an alternative, cost-efficient shallow-sequence output NGS assay called maSTR assay that, in conjunction with a dedicated bioinformatics pipeline called SNiPSTR, can be implemented with standard NGS instrumentation. In a back-to-back comparison with a CE-based, commercial forensic STR kit, we find that for samples with low DNA content, with mixed DNA from different individuals, or containing PCR inhibitors, the maSTR assay performs equally well, and with degraded DNA is superior to CE-based analysis. Thus, the maSTR assay is a simple, robust and cost-efficient NGS-based STR typing method applicable for human identification in forensic and biomedical contexts.
Background: Type 2 diabetes mellitus is associated with increased cardiovascular risk. One laboratory marker for cardiovascular risk assessment is high-sensitivity C-reactive protein (hsCRP).
Methods: This cross-sectional study attempted to analyze the association of hsCRP levels with insulin resistance, β-cell dysfunction and macrovascular disease in 4270 non-insulin-treated patients with type 2 diabetes [2146 male, 2124 female; mean age ±SD, 63.9±11.1years; body mass index (BMI) 30.1±5.5kg/m2; disease duration 5.4±5.6years; hemoglobin A1c (HbA1c) 6.8±1.3%]. It consisted of a single morning visit with collection of a fasting blood sample. Observational parameters included several clinical scores and laboratory biomarkers.
Results: Stratification into cardiovascular risk groups according to hsCRP levels revealed that 934 patients had low risk (hsCRP <1mg/L), 1369 patients had intermediate risk (hsCRP 1–3mg/L), 1352 patients had high risk (hsCRP >3–10mg/L), and 610 patients had unspecific hsCRP elevation (>10mg/L). Increased hsCRP levels were associated with other indicators of diabetes-related cardiovascular risk (homeostatic model assessment, intact proinsulin, insulin, BMI, β-cell dysfunction, all p<0.001), but showed no correlation with disease duration or glucose control. The majority of the patients were treated with diet (34.1%; hsCRP levels 2.85±2.39mg/L) or metformin monotherapy (21.1%; 2.95±2.50mg/L hsCRP). The highest hsCRP levels were observed in patients treated with sulfonylurea (17.0%; 3.00±2.43mg/L).
Conclusions: Our results indicate that hsCRP may be used as a cardiovascular risk marker in patients with type 2 diabetes mellitus and should be evaluated in further prospective studies.
Background and Objectives: In advanced β-cell dysfunction, proinsulin is increasingly replacing insulin as major component of the secretion product. It has been speculated that proinsulin has at least the same adipogenic potency than insulin, leading to an increased tendency of lipid tissue formation in patients with late stage β-cell dysfunction. Methods and Results: Mesenchymal stem cells obtained from liposuction material were grown in differentiation media containing insulin (0.01 μmol), proinsulin (0.01 μmol) or insulin+proinsulin (each 0.005 μmol). Cell culture supernatants were taken from these experiments and an untreated control at weeks 1, 2, and 3, and were stored at -80°C until analysis. Cell differentiation was microscopically supervised and adiponectin concentrations were measured as marker for differentiation into mature lipid cells. This experiment was repeated three times. No growth of lipid cells and no change in adiponectin values was observed in the negative control group (after 7/14/12 days: 3.2±0.5/3.3±0.1/4.4±0.5 ng/ml/12 h). A continuous differentiation into mature adipocytes (also confirmed by Red-Oil-staining) and a corresponding increase in adiponectin values was observed in the experiments with insulin (3.6±1.9/5.1±1.4/13.3±1.5 ng/ml/12 h; p<0.05 week 1 vs. week 3) and proinsulin (3.3±1.2/3.5±0.3/12.2±1.2 ng/ml/12 h; p<0.05). Comparable effects were seen with the insulin/proinsulin combination. Conclusions: Proinsulin has the same adipogenic potential than insulin in vitro. Proinsulin has only 10∼20% of the glucose-lowering effect of insulin. It can be speculated that the adipogenic potential of proinsulin may be a large contributor to the increased body weight problems in patients with type 2 diabetes and advanced β-cell dysfunction.
Host-derived succinate accumulates in the airways during bacterial infection. Here, we show that luminal succinate activates murine tracheal brush (tuft) cells through a signaling cascade involving the succinate receptor 1 (SUCNR1), phospholipase Cβ2, and the cation channel transient receptor potential channel subfamily M member 5 (TRPM5). Stimulated brush cells then trigger a long-range Ca2+ wave spreading radially over the tracheal epithelium through a sequential signaling process. First, brush cells release acetylcholine, which excites nearby cells via muscarinic acetylcholine receptors. From there, the Ca2+ wave propagates through gap junction signaling, reaching also distant ciliated and secretory cells. These effector cells translate activation into enhanced ciliary activity and Cl- secretion, which are synergistic in boosting mucociliary clearance, the major innate defense mechanism of the airways. Our data establish tracheal brush cells as a central hub in triggering a global epithelial defense program in response to a danger-associated metabolite.
The modern concept of the evolution of Mars assumes that life could potentially have originated on the planet Mars, possibly during the end of the late heavy bombardment, and could then be transferred to other planets. Since then, physical and chemical conditions on Mars changed and now strongly limit the presence of terrestrial-like life forms. These adverse conditions include scarcity of liquid water (although brine solutions may exist), low temperature and atmospheric pressure, and cosmic radiation. Ionizing radiation is very important among these life-constraining factors because it damages DNA and other cellular components, particularly in liquid conditions where radiation-induced reactive oxidants diffuse freely. Here, we investigated the impact of high doses (up to 2 kGy) of densely-ionizing (197.6 keV/µm), space-relevant iron ions (corresponding on the irradiation that reach the uppermost layer of the Mars subsurface) on the survival of an extremophilic terrestrial organism-Cryomyces antarcticus-in liquid medium and under atmospheric conditions, through different techniques. Results showed that it survived in a metabolically active state when subjected to high doses of Fe ions and was able to repair eventual DNA damages. It implies that some terrestrial life forms can withstand prolonged exposure to space-relevant ion radiation.
One of the primary current astrobiological goals is to understand the limits of microbial resistance to extraterrestrial conditions. Much attention is paid to ionizing radiation, since it can prevent the preservation and spread of life outside the Earth. The aim of this research was to study the impact of accelerated He ions (150 MeV/n, up to 1 kGy) as a component of the galactic cosmic rays on the black fungus C. antarcticus when mixed with Antarctic sandstones—the substratum of its natural habitat—and two Martian regolith simulants, which mimics two different evolutionary stages of Mars. The high dose of 1 kGy was used to assess the effect of dose accumulation in dormant cells within minerals, under long-term irradiation estimated on a geological time scale. The data obtained suggests that viable Earth-like microorganisms can be preserved in the dormant state in the near-surface scenario for approximately 322,000 and 110,000 Earth years within Martian regolith that mimic early and present Mars environmental conditions, respectively. In addition, the results of the study indicate the possibility of maintaining traces within regolith, as demonstrated by the identification of melanin pigments through UltraViolet-visible (UV-vis) spectrophotometric approach.
Non-Destructive Sensor-Based Prediction of Maturity and Optimum Harvest Date of Sweet Cherry Fruit
(2017)
(1) Background: The aim of the study was to use innovative sensor technology for non-destructive determination and prediction of optimum harvest date (OHD), using sweet cherry as a model fruit, based on different ripening parameters. (2) Methods: Two cherry varieties in two growing systems viz. field and polytunnel in two years were employed. The fruit quality parameters such as fruit weight and size proved unsuitable to detect OHD alone due to their dependence on crop load, climatic conditions, cultural practices, and season. Coloration during cherry ripening was characterized by a complete decline of green chlorophyll and saturation of the red anthocyanins, and was measured with a portable sensor viz. spectrometer 3-4 weeks before expected harvest until 2 weeks after harvest. (3) Results: Expressed as green NDVI (normalized differential vegetation index) and red NAI (normalized anthocyanin index) values, NAI increased from -0.5 (unripe) to +0.7 to +0.8 in mature fruit and remained at this saturation level with overripe fruits, irrespective of variety, treatment, and year. A model was developed to predict the OHD, which coincided with when NDVI reached and exceeded zero and the first derivative of NAI asymptotically approached zero. (4) Conclusion: The use of this sensor technology appears suitable for several cherry varieties and growing systems to predict the optimum harvest date.
Small Molecules Enhance Scaffold-Based Bone Grafts via Purinergic Receptor Signaling in Stem Cells
(2018)
The need for bone grafts is high, due to age-related diseases, such as tumor resections, but also accidents, risky sports, and military conflicts. The gold standard for bone grafting is the use of autografts from the iliac crest, but the limited amount of accessible material demands new sources of bone replacement. The use of mesenchymal stem cells or their descendant cells, namely osteoblast, the bone-building cells and endothelial cells for angiogenesis, combined with artificial scaffolds, is a new approach. Mesenchymal stem cells (MSCs) can be obtained from the patient themselves, or from donors, as they barely cause an immune response in the recipient. However, MSCs never fully differentiate in vitro which might lead to unwanted effects in vivo. Interestingly, purinergic receptors can positively influence the differentiation of both osteoblasts and endothelial cells, using specific artificial ligands. An overview is given on purinergic receptor signaling in the most-needed cell types involved in bone metabolism-namely osteoblasts, osteoclasts, and endothelial cells. Furthermore, different types of scaffolds and their production methods will be elucidated. Finally, recent patents on scaffold materials, as wells as purinergic receptor-influencing molecules which might impact bone grafting, are discussed.
In silico Epitope Mapping of Glucose-6-Phosphate Isomerase: A Rheumatoid Arthritis Autoantigen
(2017)
Rheumatoid arthritis-like symptoms can be initiated experimentally in naive K/BxN mice by simultaneously administering the two monoclonal antibodies 11H3 and 46H9. Both antibodies specifically recognize Glucose-6-Phosphate Isomerase (GPI), a known auto antigen in RA patients. Amino acid sequences of the Fv parts of the antibodies were determined by translating the respective hybridoma DNA sequences and served for threedimensional structure modeling of the paratope regions. In silico docking of both Fv antibody structure models to the X-ray structures of the homodimeric murine GPI as well as to the homodimeric human GPI predicted the murine epitope of the 11H3 antibodies to comprise partial amino acid sequences QRVRSGDWKGYTGKS (aa134-148) and AAKDPSAVAK (aa232-241), generating an assembled (conformational) epitope. The 11H3 epitope on human GPI encompasses the matching partial amino acid sequences QRVRSGDWKGYTGKT (aa134-148) and AAKDPSAVAK (aa232-241). The epitope of the 46H9 antibody was determined to consist of the partial murine GPI amino acid sequence RKELQAAGKSPEDLEK (aa446-461) and the human GPI amino acid sequence RKELQAAGKSPEDLER (aa446-461), respectively, resembling consecutive (linear) epitopes. The predicted epitopes were verified by mass spectrometric epitope mapping using synthetic epitope peptides. Peptide QRVRSGDWKGYTGKS[GSMSGS] AAKDPSAAK included a small spacer sequence in between the epitope sequences, mimicking the assembled epitope for the 11H3 antibody. The peptide RKELQAAGKSPEDLEK represented the consecutive epitope for the 46H9 antibody. The determined B-cell epitopes of GPI and their interactions with the monoclonal antibodies provide a detailed structural understanding of immunological disease onset mechanisms in a mouse model of rheumatoid arthritis.
Indoor spaces exhibit microbial compositions that are distinctly dissimilar from one another and from outdoor spaces. Unique in this regard, and a topic that has only recently come into focus, is the microbiome of hospitals. While the benefits of knowing exactly which microorganisms propagate how and where in hospitals are undoubtedly beneficial for preventing hospital-acquired infections, there are, to date, no standardized procedures on how to best study the hospital microbiome. Our study aimed to investigate the microbiome of hospital sanitary facilities, outlining the extent to which hospital microbiome analyses differ according to sample-preparation protocol. For this purpose, fifty samples were collected from two separate hospitals—from three wards and one hospital laboratory—using two different storage media from which DNA was extracted using two different extraction kits and sequenced with two different primer pairs (V1–V2 and V3–V4). There were no observable differences between the sample-preservation media, small differences in detected taxa between the DNA extraction kits (mainly concerning Propionibacteriaceae), and large differences in detected taxa between the two primer pairs V1–V2 and V3–V4. This analysis also showed that microbial occurrences and compositions can vary greatly from toilets to sinks to showers and across wards and hospitals. In surgical wards, patient toilets appeared to be characterized by lower species richness and diversity than staff toilets. Which sampling sites are the best for which assessments should be analyzed in more depth. The fact that the sample processing methods we investigated (apart from the choice of primers) seem to have changed the results only slightly suggests that comparing hospital microbiome studies is a realistic option. The observed differences in species richness and diversity between patient and staff toilets should be further investigated, as these, if confirmed, could be a result of excreted antimicrobials.
ESKAPEE Pathogen Biofilm Control on Surfaces with Probiotic Lactobacillaceae and Bacillus species
(2023)
Combatting the rapidly growing threat of antimicrobial resistance and reducing prevalence and transmission of ESKAPEE pathogens in healthcare settings requires innovative strategies, one of which is displacing these pathogens using beneficial microorganisms. Our review comprehensively examines the evidence of probiotic bacteria displacing ESKAPEE pathogens, with a focus on inanimate surfaces. A systematic search was conducted using the PubMed and Web of Science databases on 21 December 2021, and 143 studies were identified examining the effects of Lactobacillaceae and Bacillus spp. cells and products on the growth, colonization, and survival of ESKAPEE pathogens. While the diversity of study methods limits evidence analysis, results presented by narrative synthesis demonstrate that several species have the potential as cells or their products or supernatants to displace nosocomial infection-causing organisms in a variety of in vitro and in vivo settings. Our review aims to aid the development of new promising approaches to control pathogen biofilms in medical settings by informing researchers and policymakers about the potential of probiotics to combat nosocomial infections. More targeted studies are needed to assess safety and efficacy of different probiotic formulations, followed by large-scale studies to assess utility in infection control and medical practice.
The non-filarial and non-communicable disease podoconiosis affects around 4 million people and is characterized by severe leg lymphedema accompanied with painful intermittent acute inflammatory episodes, called acute dermatolymphangioadenitis (ADLA) attacks. Risk factors have been associated with the disease but the mechanisms of pathophysiology remain uncertain. Lymphedema can lead to skin lesions, which can serve as entry points for bacteria that may cause ADLA attacks leading to progression of the lymphedema. However, the microbiome of the skin of affected legs from podoconiosis individuals remains unclear. Thus, we analysed the skin microbiome of podoconiosis legs using next generation sequencing. We revealed a positive correlation between increasing lymphedema severity and non-commensal anaerobic bacteria, especially Anaerococcus provencensis, as well as a negative correlation with the presence of Corynebacterium, a constituent of normal skin flora. Disease symptoms were generally linked to higher microbial diversity and richness, which deviated from the normal composition of the skin. These findings show an association of distinct bacterial taxa with lymphedema stages, highlighting the important role of bacteria for the pathogenesis of podoconiosis and might enable a selection of better treatment regimens to manage ADLA attacks and disease progression.
Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as Blautia, Faecalibacterium, and Parabacteroides. However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance.
The Concordia Research Station provides a unique location for preparatory activities for future human journey to Mars, to explore microbial diversity at subzero temperatures, and monitor the dissemination of human-associated microorganisms within the pristine surrounding environment. Amplicon sequencing was leveraged to investigate the microbial diversity of surface snow samples collected monthly over a two-year period, at three distances from the Station (10, 500, and 1000 m). Even when the extracted total DNA was below the detection limit, 16S rRNA gene sequencing was successfully performed on all samples, while 18S rRNA was amplified on 19 samples out of 51. No significant relationships were observed between microbial diversity and seasonality (summer or winter) or distance from the Concordia base. This suggested that if present, the anthropogenic impact should have been below the detectable limit. While harboring low microbial diversity, the surface snow samples were characterized by heterogeneous microbiomes. Ultimately, our study corroborated the use of DNA sequencing-based techniques for revealing microbial presence in remote and hostile environments, with implications for Planetary Protection during space missions and for life-detection in astrobiology relevant targets.
Cytokine-induced killer (CIK) cells are heterogeneous, major histocompatibility complex (MHC)-unrestricted T lymphocytes that have acquired the expression of several natural killer (NK) cell surface markers following the addition of interferon gamma (IFN-γ), OKT3 and interleukin-2 (IL-2). Treatment with CIK cells demonstrates a practical approach in cancer immunotherapy with limited, if any, graft versus host disease (GvHD) toxicity. CIK cells have been proposed and tested in many clinical trials in cancer patients by autologous, allogeneic or haploidentical administration. The possibility of combining them with specific monoclonal antibodies nivolumab and ipilimumab will further expand the possibility of their clinical utilization. Initially, phenotypic analysis was performed to explore CD3, CD4, CD56, PD-1 and CTLA-4 expression on CIK cells and PD-L1/PD-L2 expression on tumor cells. We further treated CIK cells with nivolumab and ipilimumab and measured the cytotoxicity of CIK cells cocultured to renal carcinoma cell lines, A-498 and Caki-2. We observed a significant decrease in viability of renal cell lines after treating with CIK cells (p < 0.0001) in comparison to untreated renal cell lines and anti-PD-1 or anti-CTLA-4 treatment had no remarkable effect on the viability of tumor cells. Using CCK-8, Precision Count Beads™ and Cell Trace™ violet proliferation assays, we proved significant increased proliferation of CIK cells in the presence of a combination of anti-PD-1 and anti-CTLA-4 antibodies compared to untreated CIK cells. The IFN-γ secretion increased significantly in the presence of A-498 and combinatorial blockade of PD-1 and CTLA-4 compared to nivolumab or ipilimumab monotreatment (p < 0.001). In conclusion, a combination of immune checkpoint inhibition with CIK cells augments cytotoxicity of CIK cells against renal cancer cells.
Sweet sorghum (Sorghum bicolor (L.) moench), a crop that is grown by subsistence farmers in Zimbabwe was used to extract silica gel in order to assess its possible use as a raw material for the production of silica-based products. The gel was prepared from sodium silicate extracted from sweet sorghum bagasse ash by sodium hydroxide leaching. Results show that maximum yield can be obtained at pH 5 and with 3 M sodium concentration. The silica gel prepared at optimum pH 5 had a bulk density of 0.5626 g/cm3 and anestimated porosity of 71.87%. Silica gel aged over 10 h had improved moisture adsorption properties. X-ray fluorescence (XRF) determinations show that the silica content in the ash is 40.1%. Characterization of sweet sorghum ash and silica gels produced at pH 5, 7 and 8.5 by Fourier Transform Infrared spectroscopy gave absorption bands similar to those reported by other researchers.Transmission electron micrographs show that silica prepared under optimum conditions is amorphous and consisted of irregular particles. Sweet sorghum proved to be a potential low cost raw material for the production of silica gel.
A precise characterization of substances is essential for the safe handling of explosives. One parameter regularly characterized is the impact sensitivity. This is typically determined using a drop hammer. However, the results can vary depending on the test method and even the operator, and it is not possible to distinguish the type of decomposition such as detonation and deflagration. This study monitors the reaction progress by constructing a drop hammer to measure the decomposition reaction of four different primary explosives (tetrazene, silver azide, lead azide, lead styphnate) in order to determine the reproducibility of this method. Additionally, further possible evaluation methods are explored to improve on the current binary statistical analysis. To determine whether classification was possible based on extracted features, the responses of equipped sensor arrays, which measure and monitor the reactions, were studied and evaluated. Features were extracted from this data and were evaluated using multivariate methods such as principal component analysis (PCA) and linear discriminant analysis (LDA). The results indicate that although the measurements show substance specific trends, they also show a large scatter for each substance. By reducing the dimensions of the extracted features, different sample clusters can be represented and the calculated loadings allow significant parameters to be determined for classification. The results also suggest that differentiation of different reaction mechanisms is feasible. Testing of the regressor function shows reliable results considering the comparatively small amount of data.
Trade of wild-caught animals is illegal for many taxa and in many countries. Common regulatory procedures involve documentation and marking techniques. However, these procedures are subject to fraud and thus should be complemented by routine genetic testing in order to authenticate the captive-bred origin of animals intended for trade. A suitable class of genetic markers are SNPSTRs that combine a short tandem repeat (STR) and single nucleotide polymorphisms (SNPs) within one amplicon. This combined marker type can be used for genetic identification and for parentage analyses and in addition, provides insight into haplotype history. As a proof of principle, this study establishes a set of 20 SNPSTR markers for Athene noctua, one of the most trafficked owls in CITES Appendix II. These markers can be coamplified in a single multiplex reaction. Based on population data, the percentage of observed and expected heterozygosities of the markers ranged from 0.400 to 1.000 and 0.545 to 0.850, respectively. A combined probability of identity of 5.3*10-23 was achieved with the whole set, and combined parentage exclusion probabilities reached over 99.99%, even if the genotype of one parent was missing. A direct comparison of an owl family and an unrelated owl demonstrated the applicability of the SNPSTR set in parentage testing. The established SNPSTR set thus proved to be highly useful for identifying individuals and analysing parentage to determine wild or captive origin. We propose to implement SNPSTR-based routine certification in wildlife trade as a way to reveal animal laundering and misdeclaration of wild-caught animals.
Electrical signal transmission in power electronic devices takes place through high-purity aluminum bonding wires. Cyclic mechanical and thermal stresses during operation lead to fatigue loads, resulting in premature failure of the wires, which cannot be reliably predicted. The following work presents two fatigue lifetime models calibrated and validated based on experimental fatigue results of an aluminum bonding wire and subsequently transferred and applied to other wire types. The lifetime modeling of Wöhler curves for different load ratios shows good but limited applicability for the linear model. The model can only be applied above 10,000 cycles and within the investigated load range of R = 0.1 to R = 0.7. The nonlinear model shows very good agreement between model prediction and experimental results over the entire investigated cycle range. Furthermore, the predicted Smith diagram is not only consistent in the investigated load range but also in the extrapolated load range from R = −1.0 to R = 0.8. A transfer of both model approaches to other wire types by using their tensile strengths can be implemented as well, although the nonlinear model is more suitable since it covers the entire load and cycle range.
Bonding wires made of aluminum are the most used materials for the transmission of electrical signals in power electronic devices. During operation, different cyclic mechanical and thermal stresses can lead to fatigue loads and a failure of the bonding wires. A prediction or prevention of the wire failure is not yet possible by design for all cases. The following work presents meaningful fatigue tests in small wire dimensions and investigates the influence of the R-ratio on the lifetime of two different aluminum wires with a diameter of 300 μm each. The experiments show very reproducible fatigue results with ductile failure behavior. The endurable stress amplitude decreases linearly with an increasing stress ratio, which can be displayed by a Smith diagram, even though the applied maximum stresses exceed the initial yield stresses determined by tensile tests. A scaling of the fatigue results by the tensile strength indicates that the fatigue level is significantly influenced by the strength of the material. Due to the very consistent findings, the development of a generalized fatigue model for predicting the lifetime of bonding wires with an arbitrary loading situation seems to be possible and will be further investigated.
The utilization of simulation procedures is gaining increasing attention in the product development of extrusion blow molded parts. However, some simulation steps, like the simulation of shrinkage and warpage, are still associated with uncertainties. The reason for this is on the one hand a lack of standardized interfaces for the transfer of simulation data between different simulation tools, and on the other hand the complex time-, temperature- and process-dependent material behavior of the used semi crystalline polymers. Using a new vendor neutral interface standard for the data transfer, the shrinkage analysis of a simple blow molded part is investigated and compared to experimental data. A linear viscoelastic material model in combination with an orthotropic process- and temperature-dependent thermal expansion coefficient is used for the shrinkage prediction. A good agreement is observed. Finally, critical parameters in the simulation models that strongly influence the shrinkage analysis are identified by a sensitivity study.
3-Hydroxyisobutyrate Dehydrogenase (HIBADH) deficiency - a novel disorder of valine metabolism
(2021)
3-Hydroxyisobutyric acid (3HiB) is an intermediate in the degradation of the branched-chain amino acid valine. Disorders in valine degradation can lead to 3HiB accumulation and its excretion in the urine. This article describes the first two patients with a new metabolic disorder, 3-hydroxyisobutyrate dehydrogenase (HIBADH) deficiency, its phenotype and its treatment with a low-valine diet. The detected mutation in the HIBADH gene leads to nonsense-mediated mRNA decay of the mutant allele and to a complete loss-of-function of the enzyme. Under strict adherence to a low-valine diet a rapid decrease of 3HiB excretion in the urine was observed. Due to limited patient numbers and intrafamilial differences in phenotype with one affected and one unaffected individual, the clinical phenotype of HIBADH deficiency needs further evaluation.
Background: Staurosporine-dependent single and collective cell migration patterns of breast carcinoma cells MDA-MB-231, MCF-7, and SK-BR-3 were analysed to characterise the presence of drug-dependent migration promoting and inhibiting yin-yang effects. Methods: Migration patterns of various breast cancer cells after staurosporine treatment were investigated using Western blot, cell toxicity assays, single and collective cell migration assays, and video time-lapse. Statistical analyses were performed with Kruskal–Wallis and Fligner–Killeen tests. Results: Application of staurosporine induced the migration of single MCF-7 cells but inhibited collective cell migration. With the exception of low-density SK-BR-3 cells, staurosporine induced the generation of immobile flattened giant cells. Video time-lapse analysis revealed that within the borderline of cell collectives, staurosporine reduced the velocity of individual MDA-MB-231 and SK-BR-3, but not of MCF-7 cells. In individual MCF-7 cells, mainly the directionality of migration became disturbed, which led to an increased migration rate parallel to the borderline, and hereby to an inhibition of the migration of the cell collective as a total. Moreover, the application of staurosporine led to a transient activation of ERK1/2 in all cell lines. Conclusion: Dependent on the context (single versus collective cells), a drug may induce opposite effects in the same cell line.
Characterization methods of pressure sensitive adhesives (PSA) originate from technical bonding and do not cover relevant data for the development and quality assurance of medical applications, where PSA with flexible backing layers are adopted to human skin. In this study, a new method called RheoTack is developed to determine (mechanically and optically) an adhesion and detaching behavior of flexible and transparent PSA based patches. Transdermal therapeutic systems (TTS) consisting of silicone-based PSAs on a flexible and transparent backing layer were tested on a rotational rheometer with an 8 mm plate as a probe rod at retraction speeds of 0.01, 0.1, and 1 mm/s with respect to their adhesion and detaching behavior in terms of force-retraction displacement curves. The curves consist of a compression phase to affirm wetting; a tensile deformation phase intercepting stretching, cavity, and fibril formation; and a failure phase with detaching. Their analysis provides values for stiffness, force, and displacement of the beginning of fibril formation, force and displacement of the beginning of a failure due to fibril breakage and detaching, as well as corresponding activation energies. All these parameters exhibit the pronounced dependency on the retraction speed. The force-retraction displacement curves together with the simultaneous video recordings of the TTS deformation from three different angles (three cameras) provide deeper insight into the deformation processes and allow for interpreting the properties’ characteristics for PSA applications.
In 2018, in the US alone, it is estimated that 268,670 people will be diagnosed with breast cancer, and that 41,400 will die from it. Since breast cancers often become resistant to therapies, and certain breast cancers lack therapeutic targets, new approaches are urgently required. A cell-stress response pathway, the unfolded protein response (UPR), has emerged as a promising target for the development of novel breast cancer treatments. This pathway is activated in response to a disturbance in endoplasmic reticulum (ER) homeostasis but has diverse physiological and disease-specific functions. In breast cancer, UPR signalling promotes a malignant phenotype and can confer tumours with resistance to widely used therapies. Here, we review several roles for UPR signalling in breast cancer, highlighting UPR-mediated therapy resistance and the potential for targeting the UPR alone or in combination with existing therapies.
Pitfalls of using sequence databases for heterologous expression studies - a technical review
(2023)
Synthesis of DNA fragments based on gene sequences available in public resources has become an efficient and affordable method that gradually replaced traditional cloning efforts such as PCR cloning from cDNA. However, database entries based on genome sequencing results are prone to errors which can lead to false sequence information and, ultimately, errors in functional characterization of proteins such as ion channels and transporters in heterologous expression systems. We have identified five common problems that repeatedly appear in public resources: 1) Not every gene has yet been annotated; 2) Not all gene annotations are necessarily correct; 3) Transcripts may contain automated corrections; 4) There are mismatches between gene, mRNA, and protein sequences; and 5) Splicing patterns often lack experimental validation. This technical review highlights and provides a strategy to bypass these issues in order to avoid critical mistakes that could impact future studies of any gene/protein of interest in heterologous expression systems. Abstract figure legend Projects involving heterologous gene expression are often characterised by similar steps. Initially, database research (A) is necessary to retrieve information of full of partial sequences of a gene of interest. A multitude of genome assemblies are annotated and deposited in public databases or that are available for refined search options using individual sequence information. The search results need to be scrutinised and compared to already available information (B). Once the sequence has been determined, DNA synthesis (C) by PCR or commercial synthesis are necessary for further cloning procedures (D). Eventually, the DNA needs to be transfected (E) and expressed in, e.g., eukaryotic cells (F). Finally, the expression of the gene of interest needs to be documented and its function analysed (G). This article is protected by copyright. All rights reserved.
Background: Atypical myopathy (AM), an acquired multiple acyl-CoA dehydrogenase deficiency (MADD) in horses, induce changes in mitochondrial metabolism. Only few veterinary laboratories offer diagnostic testing for this disease. Inborn and acquired MADD exist in humans, therefore determination of organic acids (OA) in urine and acylcarnitines (AC) in blood by assays available in medical laboratories can serve as AM diagnostics. The evolution of OA and AC profiles in surviving horses is unreported.
Methods: AC profiles using electrospray ionization tandem mass spectrometry (ESI-MS/MS) and OA in urine using gas chromatography mass spectrometry (GC–MS) were determined in dried blot spots (DBS, n = 7) and urine samples (n = 5) of horses with AM (n = 7) at disease presentation and in longitudinal samples from 3/4 survivors and compared to DBS (n = 16) and urine samples (n = 7) from control horses using the Wilcoxon test.
Results: All short- (C2-C5) and medium-chain (C6-C12) AC in blood differed significantly (p < 0.008) between horses with AM and controls, except for C5:1 (p = 0.45) and C5OH + C4DC (p = 0.06). In AM survivors the AC concentrations decreased over time but were still partially elevated after 7 days. 14/62 (23%) of OA differed significantly between horses with AM and control horses. Concentrations of ethylmalonic acid, 2-hydroxyglutaric acid and the acylglycines (butyryl-, valeryl-, and hexanoylglycine) were highly elevated in the urine of all horses with AM at the day of disease presentation. In AM survivors, concentrations of those metabolites were initially lower and decreased during remission to approach normalization after 7 days.
Conclusion: OA and AC profiling by specialized human medical laboratories was used to diagnose AM in horses. Elevation of specific metabolites were still evident several days after disease presentation, allowing diagnosis via analysis of samples from convalescent animals.
Hydrogen‐Bonded Cholesteric Liquid Crystals—A Modular Approach Toward Responsive Photonic Materials
(2022)
A supramolecular approach for photonic materials based on hydrogen-bonded cholesteric liquid crystals is presented. The modular toolbox of low-molecular-weight hydrogen-bond donors and acceptors provides a simple route toward liquid crystalline materials with tailor-made thermal and photonic properties. Initial studies reveal broad application potential of the liquid crystalline thin films for chemo- and thermosensing. The chemosensing performance is based on the interruption of the intermolecular forces between the donor and acceptor moieties by interference with halogen-bond donors. Future studies will expand the scope of analytes and sensing in aqueous media. In addition, the implementation of the reported materials in additive manufacturing and printed photonic devices is planned.
Explorative experiments were done to figure out differences in the emission of volatile organic compounds (VOCs) of not infested trees and trees infested by Anoplophora glabripennis (Asian longhorn beetle, ALB), a quarantine pest. Therefore, VOCs from some native insect species, Anoplophora glabripennis infested Acer, stressed Acer, healthy Acer, Populus and Salix were obtained by enrichment on adsorbents. Qualitative analysis was done by thermal desorption gas chromatography coupled with a mass selective detector (TD-GC/MS). Altogether 169 substances were identified. 11 substances occur from ALB infested or mechanically damaged trees i.e. stressed trees, but not from healthy trees. (+)-Cyclosativene, (+)-α-longipinene, copaene and caryophyllene are detectable only from ALB-infested Acer not from mechanically damaged or healthy Acer. However, these substances are also emitted by healthy Salix. 2,4-Dimethyl-1-heptene is among all tree samples exclusively present in the ambience of ALB-infested trees. It´s rarely detectable from native insect species’ samples.
There is an unmet need for the development and validation of biomarkers and surrogate endpoints for clinical trials in propionic acidemia (PA) and methylmalonic acidemia (MMA). This review examines the pathophysiology and clinical consequences of PA and MMA that could form the basis for potential biomarkers and surrogate endpoints. Changes in primary metabolites such as methylcitric acid (MCA), MCA:citric acid ratio, oxidation of 13C-propionate (exhaled 13CO2), and propionylcarnitine (C3) have demonstrated clinical relevance in patients with PA or MMA. Methylmalonic acid, another primary metabolite, is a potential biomarker, but only in patients with MMA. Other potential biomarkers in patients with either PA and MMA include secondary metabolites, such as ammonium, or the mitochondrial disease marker, fibroblast growth factor 21. Additional research is needed to validate these biomarkers as surrogate endpoints, and to determine whether other metabolites or markers of organ damage could also be useful biomarkers for clinical trials of investigational drug treatments in patients with PA or MMA. This review examines the evidence supporting a variety of possible biomarkers for drug development in propionic and methylmalonic acidemias.
Triple-negative breast cancer (TNBC) lacks targeted therapies and has a worse prognosis than other breast cancer subtypes, underscoring an urgent need for new therapeutic targets and strategies. IRE1 is an endoplasmic reticulum (ER) stress sensor, whose activation is predominantly linked to the resolution of ER stress and, in the case of severe stress, to cell death. Here we demonstrate that constitutive IRE1 RNase activity contributes to basal production of pro-tumorigenic factors IL-6, IL-8, CXCL1, GM-CSF, and TGFβ2 in TNBC cells. We further show that the chemotherapeutic drug, paclitaxel, enhances IRE1 RNase activity and this contributes to paclitaxel-mediated expansion of tumor-initiating cells. In a xenograft mouse model of TNBC, inhibition of IRE1 RNase activity increases paclitaxel-mediated tumor suppression and delays tumor relapse post therapy. We therefore conclude that inclusion of IRE1 RNase inhibition in therapeutic strategies can enhance the effectiveness of current chemotherapeutics.
The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.
We investigated graphene structures grafted with fullerenes. The size of the graphene sheets ranges from 6400 to 640,000 atoms. The fullerenes (C60 and C240) are placed on top of the graphene sheets, using different impact velocities we could distinguish three types of impact. Furthermore, we investigated the changes of the vibrational properties. The modified graphene planes show additional features in the vibronic density of states.
It is know that mesenchymal stem cells (MSCs) actively secretemultiple biologically-active factors during their process of differentiation which gives rise to a variey of cytotypes including bone and fatcells. It is also acknowledged that the chemokines secreted throughoutMSC differentiation may play an important role in the development and growth of tumor cells, although literature data appear somewhat indeterminate due to the contradictory evidence often found.
Background: Cancer heterogeneity poses a serious challenge concerning the toxicity and adverse effects of therapeutic inhibitors, especially when it comes to combinatorial therapies that involve multiple targeted inhibitors. In particular, in non-small cell lung cancer (NSCLC), a number of studies have reported synergistic effects of drug combinations in the preclinical models, while they were only partially successful in the clinical setup, suggesting those alternative clinical strategies (with genetic background and immune response) should be considered. Herein, we investigated the antitumor effect
of cytokine-induced killer (CIK) cells in combination with ALK and PD-1 inhibitors in vitro on genetically variable NSCLC cell lines.
Methods: We co-cultured the three genetically different NSCLC cell lines NCI-H2228 (EML4-ALK), A549 (KRAS mutation), and HCC-78 (ROS1 rearrangement) with and without nivolumab (PD-1 inhibitor) and crizotinib (ALK inhibitor). Additionally, we profiled the variability of surface expression multiple immune checkpoints, the concentration of absolute dead cells, intracellular granzyme B on CIK cells using flow cytometry as well as RT-qPCR. ELISA and Western blot were performed to verify the activation of CIK cells.
Results: Our analysis showed that (a) nivolumab significantly weakened PD-1 surface expression on CIK cells without impacting other immune checkpoints or PD-1 mRNA expression, (b) this combination strategy showed an effective response on cell viability, IFN-g production, and intracellular release of granzyme B in CD3+ CD56+ CIK cells, but solely in NCI-H2228, (c) the intrinsic expression of Fas ligand (FasL) as a T-cell activation marker in CIK cells was upregulated by this additive effect, and (d) nivolumab induced Foxp3 expression in CD4+CD25+ subpopulation of CIK cells significantly increased. Taken together, we could show that CIK cells in combination with crizotinib and nivolumab can enhance the anti-tumor immune response through FasL activation, leading to increased IFN-g and granzyme B, but only in NCI-H2228 cells with EML4-ALK rearrangement. Therefore, we hypothesize that CIK therapy may be a potential alternative in NSCLC patients harboring EML4-ALK rearrangement, in addition, we support the idea that combination therapies offer significant potential when they are optimized on a patient-by-patient basis.
Introduction: A multitude of findings from cell cultures and animal studies are available to support the anti-cancer properties of cannabidiol (CBD). Since CBD acts on multiple molecular targets, its clinical adaptation, especially in combination with cancer immunotherapy regimen remains a serious concern.
Methods: Considering this, we extensively studied the effect of CBD on the cytokine-induced killer (CIK) cell immunotherapy approach using multiple non-small cell lung cancer (NSCLC) cells harboring diverse genotypes.
Results: Our analysis showed that, a) The Transient Receptor Potential Cation Channel Subfamily V Member 2 (TRPV2) channel was intracellularly expressed both in NSCLC cells and CIK cells. b) A synergistic effect of CIK combined with CBD, resulted in a significant increase in tumor lysis and Interferon gamma (IFN-g) production. c) CBD had a preference to elevate the CD25+CD69+ population and the CD62L_CD45RA+terminal effector memory (EMRA) population in NKT-CIK cells, suggesting early-stage activation and effector memory differentiation in CD3+CD56+ CIK cells. Of interest, we observed that CBD enhanced the calcium influx, which was mediated by the TRPV2 channel and elevated phosphor-Extracellular signal-Regulated Kinase (p-ERK) expression directly in CIK cells, whereas ERK selective inhibitor FR180204 inhibited the increasing cytotoxic CIK ability induced by CBD. Further examinations revealed that CBD induced DNA double-strand breaks via upregulation of histone H2AX phosphorylation in NSCLC cells and the migration and invasion ability of NSCLC cells suppressed by CBD were rescued using the TRPV2 antagonist (Tranilast) in the absence of CIK cells. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation.
Conclusions: Taken together, CBD holds a great potential for treating NSCLC with CIK cell immunotherapy. In addition, we utilized NSCLC with different driver mutations to investigate the efficacy of CBD. Our findings might provide evidence for CBD-personized treatment with NSCLC patients.
Silicon carbide and graphene possess extraordinary chemical and physical properties. Here, these different systems are linked and the changes in structural and dynamic properties are investigated. For the simulations performed a classical molecular dynamic (MD) approach was used. In this approach, a graphene layer (N = 240 atoms) was grafted at different distances on top of a 6H-SiC structure (N = 2400 atoms) and onto a 3C-SiC structure (N = 1728 atoms). The distances between the graphene and the 6H are 1.0, 1.3 and 1.5 Å and the distances between the graphene layer and the 3C-SiC are 2.0, 2.3, and 2.5 Å. Each system has been equilibrated at room temperature until no further relaxation was observed. The 6H-SiC structure in combination with graphene proves to be more stable compared to the combination with 3C-SiC. This can be seen well in the determined energies. Pair distribution functions were influenced slightly by the graphene layer due to steric and energetic changes. This becomes clear from the small shifts of the C-C distances. Interactions as well as bonds between graphene and SiC lead to the fact that small shoulders of the high-frequency SiC-peaks are visible in the spectra and at the same time the high-frequency peaks of graphene are completely absent.
The complex nature of multifactorial diseases, such as Morbus Alzheimer, has produced a strong need to design multitarget-directed ligands to address the involved complementary pathways. We performed a purposive structural modification of a tetratarget small-molecule, that is contilisant, and generated a combinatorial library of 28 substituted chromen-4-ones. The compounds comprise a basic moiety which is linker-connected to the 6-position of the heterocyclic chromenone core. The syntheses were accomplished by Mitsunobu- or Williamson-type ether formations. The resulting library members were evaluated at a panel of seven human enzymes, all of which being involved in the pathophysiology of neurodegeneration. A concomitant inhibition of human acetylcholinesterase and human monoamine oxidase B, with IC50 values of 5.58 and 7.20 μM, respectively, was achieved with the dual-target 6-(4-(piperidin-1-yl)butoxy)-4H-chromen-4-one (7).
The epithelial sodium channel (ENaC) is a key regulator of sodium homeostasis that contributes to blood pressure control. ENaC open probability is adjusted by extracellular sodium ions, a mechanism referred to as sodium self-inhibition (SSI). With a growing number of identified ENaC gene variants associated with hypertension, there is an increasing demand for medium- to high-throughput assays allowing the detection of alterations in ENaC activity and SSI. We evaluated a commercially available automated two-electrode voltage-clamp (TEVC) system that records transmembrane currents of ENaC-expressing Xenopus oocytes in 96-well microtiter plates. We employed guinea pig, human and Xenopus laevis ENaC orthologs that display specific magnitudes of SSI. While demonstrating some limitations over traditional TEVC systems with customized perfusion chambers, the automated TEVC system was able to detect the established SSI characteristics of the employed ENaC orthologs. We were able to confirm a reduced SSI in a gene variant, leading to C479R substitution in the human α-ENaC subunit that has been reported in Liddle syndrome. In conclusion, automated TEVC in Xenopus oocytes can detect SSI of ENaC orthologs and variants associated with hypertension. For precise mechanistic and kinetic analyses of SSI, optimization for faster solution exchange rates is recommended.
Development of colored surfaces by formation of nano-structured aggregates is a widely used strategy in nature to color lightweight structures (e.g. butterflies) without the use of dye pigments. The deposition of nanoscale particles mimics nature in it’s approach coloring surfaces. This work presents sol-gel modification of cellulose surfaces used to form a template for growth of Cu/Cu2O core-shell particles with defined size-distributions. Besides improving the adhesion of the deposited particulate material, the sol-gel matrix serves as a template for the control of particle sizes of the Cu/Cu2O structures, and as a consequence of particle size variation the surface color is tunable. As an example, red color was achieved with an average particle size of 35 nm, and shifts gradually to blue appearance when particles have grown to 80 nm on the sol-gel modified fabric. The copper concentration on representative fabrics is kept low to avoid modifying the textile characteristics and were all in the range of 150–170 mg per g of cellulose material. As a result of copper deposition on the surface of the material, the cellulose fabric also became electrically conductive. Remarkably, the electrical conductivity was found to be dependent on the average particle sizes of the deposits and thus related to the change in observed color. The generation of color by growth of nano-sized particles on sol-gel templates provides a highly promising approach to stain surfaces by physical effects without use of synthetic colorants, which opens a new strategy to improve environmental profile of coloration.
Isovaleric acidemia (IVA), due to isovaleryl-CoA dehydrogenase (IVD) deficiency, results in the accumulation of isovaleryl-CoA, isovaleric acid and secondary metabolites. The increase in these metabolites decreases mitochondrial energy production and increases oxidative stress. This contributes to the neuropathological features of IVA. A general assumption in the literature exists that glycine N-acyltransferase (GLYAT) plays a role in alleviating the symptoms experienced by IVA patients through the formation of N-isovalerylglycine. GLYAT forms part of the phase II glycine conjugation pathway in the liver and detoxifies excess acyl-CoA’s namely benzoyl-CoA. However, very few studies support GLYAT as the enzyme that conjugates isovaleryl-CoA to glycine. Furthermore, GLYATL1, a paralogue of GLYAT, conjugates phenylacetyl-CoA to glutamine. Therefore, GLYATL1 might also be a candidate for the formation of N-isovalerylglycine. Based on the findings from the literature review, we proposed that GLYAT or GLYATL1 can form N-isovalerylglycine in IVA patients. To test this hypothesis, we performed an in-silico analysis to determine which enzyme is more likely to conjugate isovaleryl-CoA with glycine using AutoDock Vina. Thereafter, we performed in vitro validation using purified enzyme preparations. The in-silico and in vitro findings suggested that both enzymes could form N-isovaleryglycine albeit at lower affinities than their preferred substrates. Furthermore, an increase in glycine concentration does not result in an increase in N-isovalerylglycine formation. The results from the critical literature appraisal, in-silico, and in vitro validation, suggest the importance of further investigating the reaction kinetics and binding behaviors between these substrates and enzymes in understanding the pathophysiology of IVA.
Gas chromatography with flame-ionization detection (FID) and gas chromatography-mass spectrometry (GC/MS) with electron impact ionization (EI) and chemical ionization (PCI and NCI) were successfully used for separation and identification of commercially available longchain primary alkyl amines. The investigated compounds were used as corrosion inhibiting and antifouling agents in a water-steam circuit of energy systems in the power industry. Solidphase extraction (SPE) with octadecyl bonded silica (C18) sorbents followed by gas chromatography were used for quantification of the investigated Primene JM-T™ alkyl amines in boiler water, condensate and superheated steam samples from the power plant. Amine formulations from Kotamina group favor formation of protective layers on internal surfaces and keep them free from corrosion and scale. Alkyl amines contained in those formulations both render the environment alkaline and limit the corrosion impact of ionic and gaseous impurities by formation of protective layers. Moreover, alkyl amines limit scaling on heating surfaces of boilers and in turbine, ensuring failure-free operation. Application of alkyl amine formulation enhances heat exchange during boiling and condensation processes. Alkyl amines with branched structure are more thermally stable than linear alkyl amines, exhibit better adsorption and effectiveness of surface shielding. As a result, application of thermostable long-chain branched alkyl amines increases the efficiency of anti-corrosive protection. Moreover, the concentration of ammonia content in water and in steam was also considerably decreased.
Analytical pyrolysis technique hyphenated to gas chromatography/mass spectrometry (Py-GC/MS) has extended the range of possible tools for characterization of synthetic polymers/copolymers. Pyrolysis involves thermal fragmentation of the analytical sample at elevated temperature between 500 and 1400 °C. In the presence of an inert gas, reproducible decomposition products characteristic for the original polymer/copolymer sample are formed. The pyrolysis products are chromatographically separated by using a fused silica capillary column and subsequently identified by interpretation of the obtained mass spectra or by using mass spectra libraries. The analytical technique eliminate the need for pre-treatment by performing analyses directly on the solid or liquid polymer sample.
In this paper, application examples of the analytical pyrolysis hyphenated to gas chromatography/mass spectrometry for the identification of different polymeric materials in the plastic and automotive industry, dentistry and occupational safety are demonstrated. For the first time results of identification of commercially light-curing dental filling material and a car wrapping foil by pyrolysis-GC/MS are presented.