Novel Role of Mammalian Cell Senescence-Sustenance of Muscle Larvae of Trichinella spp.

Muscle larva of the parasitic nematode Trichinella spp. lives in a portion of muscle fibre transformed to a nurse cell (NC). Based on our previous transcriptomic studies, NC growth arrest was inferred to be accompanied by cellular senescence. In the current study, NC was proven to display the following markers of senescence: high senescence-associated ß-galactosidase activity, lipid deposition, DNA damage, and cell cycle inhibition. Moreover, the nuclear localization of Activator Protein 1 (c-Fos, c-Jun, and FosB), as well as the upregulation of numerous AP-1 target genes in the NC, remained in accord with AP-1 recently identified as a master transcription factor in senescence. An increase in reactive oxygen species generation and the upregulation of antioxidant defence enzymes, including glutathione peroxidases 1 and 3, catalase, superoxide dismutases 1 and 3, and heme oxygenase 1, indicated an ongoing oxidative stress to proceed in the NC. Interestingly, antioxidant defence enzymes localized not only to the NC but also to the larva. These results allowed us to hypothesize that oxidative stress accompanying muscle regeneration and larval antigenic properties lead to the transformation of a regenerating myofibre into a senescent cell. Cellular senescence apparently represents a state of metabolism that sustains the long-term existence of muscle larva and ultimately provides it with the antioxidant capacity needed during the next host colonization. Senotherapy, a therapeutic approach aimed at selective elimination of senescent cells, can thus be viewed as potentially effective in the treatment of trichinosis.

Development of a Sensitive Screening Method for Simultaneous Determination of Nine Genotoxic Nitrosamines in Active Pharmaceutical Ingredients by GC-MS.

A worldwide crisis with nitrosamine contamination in medical products began in 2018. Therefore, trace-level analysis of nitrosamines is becoming an emerging topic of interest in the field of quality control. A novel GC-MS method with electron ionization and microextraction was developed and validated for simultaneous determination of nine carcinogenic nitrosamines (NDMA, NMEA, NDEA, NDBA, NMOR, NPYR, NPIP, NDPA, and N-methyl-npz) in active pharmaceutical ingredients (APIs): cilostazol, sunitinib malate, and olmesartan medoxomil. The method was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines, demonstrating good linearity in the range of LOQ up to 21.6 ng/mL (120% of specification limit). The limits of detection for the nine nitrosamines were determined to be in the range 0.15-1.00 ng/mL. The developed trace level GC-MS method turned out to be specific, accurate, and precise. The accuracy of all the tested APIs ranged from 94.09% to 111.22% and the precision evaluated by repeatability, intermediate precision, and system precision was RSD ≤ 7.65%. Nitrosamines were not detected in cilostazol and sunitinib, whereas in olmesartan medoxomil NDEA was detected at the level of LOQ. The novel protocol was successfully applied for nitrosamines determination in selected APIs and can be used for the routine quality control of APIs under Good Manufacturing Practices rules, ensuring the safety and effectiveness of pharmaceutical products.

Kinetics and Dynamics of the Stiff and Flexible Tines with the Duckfoot and the Coulter after Impact with Stones Embedded in Compacted Soil. Part II.

The kinetics and dynamics of the stiff and flexible tines with the duckfoot and the coulter after impact with stones embedded in compacted soil were examined. The beak of the duckfoot was positioned in the axis of the row of stones embedded in the soil at the depth of stones thickness. The coulter covered the stone or impact the edge of the stone halfway along its length. The tools worked at a speed of 0.83-2.22 m·s-1 and a working depth of 0.05-0.10 m. The results of specific parameters were compared to the response of the tools to loads in soil without stones. For both soil conditions, the kinetics of the flexible tine was 24 times more reactive, and the dynamic loads were two times lower than for the stiff tine. The responses of both tines were suppressed along with the working depth because of the more favorable place of impact of the duckfoot beak with the stone. Along with the working speed, for a stiff tine, the specific accelerations decreased significantly, by ten times, and the specific forces increased slightly, by 1.6 times. Among the two systems of setting the coulter, the impact of the cutting edge of the coulter with the stone in the middle of its length was more unfavorable than the work of the coulter covering the stone.

Effect of Stone Impacts on Various Ground Engaging Tools (Flexible/Stiff Tines and Coulter): Part I.

Analysis of the state of knowledge showed a gap in the description of tool-stone feedback. Therefore, the aim of this study was to investigate tool-stone interactions. Spherical-like silicate stones were hit by stiff and flexible tines with a duckfoot or a coulter. The tools worked with various parameters in the depth range of 0.05-0.10 m and a speed of 0.83-2.22 m·s-1. The characteristics of stone movement were specific to the type of tool and were described by the Numerical Stone Movement Scale developed for the purpose of the research. After the impact with the stiff tine, the stones were thrown the greatest distance of 0.26-1.08 m, and these distances were strongly dependent on the working speed and slightly dependent on the working depth. Large vibrations of the flexible tine and the location of the contact point of the tine in relation to the centre of the stone thickness contributed to the random behaviour of stones that were slightly moved, rotated or displaced. The specific work required to remove the stone reflected the distance travelled by the stone as well as the specific force which largely contributed to increasing the differences in this work between both tines.

Inhibition of Arabidopsis stomatal development by plastoquinone oxidation.

Stomata are the pores in the epidermal surface of plant leaves that regulate the exchange of water and CO2 with the environment thus controlling leaf gas exchange.1 In the model dicot plant Arabidopsis thaliana, the transcription factors SPEECHLESS (SPCH) and MUTE sequentially control formative divisions in the stomatal lineage by forming heterodimers with ICE1.2 SPCH regulates entry into the stomatal lineage and its stability or activity is regulated by a mitogen-activated protein kinase (MAPK) signaling cascade, mediated by its interaction with ICE1.3-6 This MAPK pathway is regulated by extracellular epidermal patterning factor (EPFs) peptides, which bind a transmembrane receptor complex to inhibit (EPF1 and EPF2) or promote (STOMAGEN/EPFL9) stomatal development.7-9 MUTE controls the transition to guard mother cell identity and is regulated by the HD-ZIP transcription factor HDG2, which is expressed exclusively in stomatal lineage cells.10,11 Light signals acting through phytochrome and cryptochrome photoreceptors positively regulate stomatal development in response to increased irradiance.12,13 Here we report that stomatal development is also regulated by the redox state of the photosynthetic electron transport chain (PETC). Oxidation of the plastoquinone (PQ) pool inhibits stomatal development by negatively regulating SPCH and MUTE expression. This mechanism is dependent on MPK6 and forms part of the response to lowering irradiance, which is distinct to the photoreceptor dependent response to increasing irradiance. Our results show that environmental signals can act through the PETC, demonstrating that photosynthetic signals regulate the development of the pores through which CO2 enters the leaf.

Influence of the Die Height on the Density of the Briquette Produced from Shredded Logging Residues.

An alternative to plant biomass of various origins are forest logging residues. They differ significantly from other, previously used plant materials. This difference is due to the heterogeneous composition and relatively large size of individual particles. This research on the compaction of this type of shredded material was aimed at determining the influence of the die height on the density and relaxation of briquettes. This parameter is crucial for the proper construction of compaction devices. The measurements were carried out for the same fractional composition of the shredded logging residues, with variable input parameters of the material and process. It was found that the briquette density and relaxation are influenced by the die height, as well as the material moisture content and process temperature. The highest density at maximum compaction pressure (1.40 g·cm-3) was obtained at a moisture content of 16%, temperature of 80 °C, and the lowest die height (195 mm). In the case of the briquette density after ejection from the die, the best results were obtained at the same temperature and die height but at a moisture content of 9%. The tests confirmed that, regardless of the process temperature and material moisture, the briquette density increases as the die height is reduced. The relaxation coefficient of compacted logging residues ranges from 21.7% to 50.1% and depends mainly on the material moisture content and the temperature of the process. The lowest value of the relaxation coefficient (21.7 ± 1.61) was obtained at 9% moisture content, 60 °C temperature, and 220 mm die height.

Antigen presentation capability and AP-1 activation accompany methotrexate-induced colon cancer cell senescence in the context of aberrant ß-catenin signaling.

Reversible cellular senescence was demonstrated previously to constitute colon cancer cell response to methotrexate. The current study presents a comparison of two senescent states of colon cancer cells, arrested and reversing, resulting from respectively, 120 h exposure to the drug, and 48 h exposure followed by 96 h regrowth in drug-free media. The upregulation of immunoproteasome subunit-coding genes and the increase in human leukocyte antigen HLA-A/B/C membrane level indicated MHC-I-restricted antigen presentation as common to both senescent states. Nuclear factor NF-κB p65 level decreased and activating protein AP-1: c-Jun, Fra2 and JunB nuclear levels increased in both senescent cell populations. Notably, the increase in AP-1- dependent transcription occurred after 48 h exposure to methotrexate. ß-catenin nuclear level increased after 48 h exposure to the drug and remained as such only in senescence-arrested cells. ß-catenin level was found uncoupled from the protein phosphorylation status indicating the deregulation of ß-catenin signaling in colon cancer cells employed in the study. These findings carry implications for both, a general mechanism of senescence establishment and putative advantages for colon cancer treatment.

Paving the way towards precise and safe CRISPR genome editing.

As the possibilities of CRISPR-Cas9 technology have been revealed, we have entered a new era of research aimed at increasing its specificity and safety. This stage of technology development is necessary not only for its wider application in the clinic but also in basic research to better control the process of genome editing. Research during the past eight years has identified some factors influencing editing outcomes and led to the development of highly specific endonucleases, modified guide RNAs and computational tools supporting experiments. More recently, large-scale experiments revealed a previously overlooked feature: Cas9 can generate reproducible mutation patterns. As a result, it has become apparent that Cas9-induced double-strand break (DSB) repair is nonrandom and can be predicted to some extent. Here, we review the present state of knowledge regarding the specificity and safety of CRISPR-Cas9 technology to define gRNA, protein and target-related problems and solutions. These issues include sequence-specific off-target effects, immune responses, genetic variation and chromatin accessibility. We present new insights into the role of DNA repair in genome editing and define factors influencing editing outcomes. In addition, we propose practical guidelines for increasing the specificity of editing and discuss novel perspectives in improvement of this technology.

Surface-Related Kinetic Models for Anaerobic Digestion of Mi-crocrystalline Cellulose: The Role of Particle Size.

In this work, for modelling the anaerobic digestion of microcrystalline cellulose, two surface-related models based on cylindrical and spherical particles were developed and compared with the first-order kinetics model. A unique dataset consisting of particles with different sizes, the same crystallinity and polymerisation degree was used to validate the models. Both newly developed models outperformed the first-order kinetics model. Analysis of the kinetic constant data revealed that particle size is a key factor determining the anaerobic digestion kinetics of crystalline cellulose. Hence, crystalline cellulose particle size should be considered in the development and optimization of lignocellulose pre-treatment methods. Further research is necessary for the assessment of impact of the crystalline cellulose particle size and surface properties on the microbial cellulose hydrolysis rate.

Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and translation inhibition of mutant transcripts.

Polyglutamine (polyQ) diseases are incurable neurological disorders caused by CAG repeat expansion in the open reading frames (ORFs) of specific genes. This type of mutation in the HTT gene is responsible for Huntington's disease (HD). CAG repeat-targeting artificial miRNAs (art-miRNAs) were shown as attractive therapeutic approach for polyQ disorders as they caused allele-selective decrease in the level of mutant proteins. Here, using polyQ disease models, we aimed to demonstrate how miRNA-based gene expression regulation is dependent on target sequence features. We show that the silencing efficiency and selectivity of art-miRNAs is influenced by the localization of the CAG repeat tract within transcript and the specific sequence context. Furthermore, we aimed to reveal the events leading to downregulation of mutant polyQ proteins and found very rapid activation of translational repression and HTT transcript deadenylation. Slicer-activity of AGO2 was dispensable in this process, as determined in AGO2 knockout cells generated with CRISPR-Cas9 technology. We also showed highly allele-selective downregulation of huntingtin in human HD neural progenitors (NPs). Taken together, art-miRNA activity may serve as a model of the cooperative activity and targeting of ORF regions by endogenous miRNAs.

The effects of solid lignin on the anaerobic digestion of microcrystalline cellulose and application of smoothing splines for extended data analysis of its inhibitory effects.

Lignocellulose is an abundant substrate for biogas production; however, for efficient utilization, proper pre-treatment is required to enhance the biomethane yield and hydrolysis rate significantly. Phenolic compounds from dissolved lignin, produced during alkali pre-treatment, have inhibitory effects on the anaerobic digestion; however, the possible inhibitory effects of solid lignin have not gathered enough interest. Especially, the effect of solid lignin on methanogenesis remains a knowledge gap. In this study, kraft lignin was used as a model solid lignin substrate for its co-digestion with microcrystalline cellulose. A new approach of modelling biomethane production curves using smoothing splines was developed to describe the long-term inhibitory effects of solid lignin on hydrolysis and methanogenesis. The method gives possibility to describe long-term inhibitory effects by using batch instead of continuous test data. Results revealed that kraft lignin showed mild inhibitory effects on methanogens. However lignin impact combined with volatile fatty accumulation can prolong hydrolysis and reactor recovery start-up by 47.3% and 75.3%, respectively. For small dosages of solid lignin adaptation of methanogens is possible.

Influence of Fraction Particle Size of Pure Straw and Blends of Straw with Calcium Carbonate or Cassava Starch on Pelletising Process and Pellet.

The aim of this study was to investigate the pressure agglomeration process of wheat straw (WS) and the blends of WS with calcium carbonate (CC) or cassava straw (CS) with a ratio of 6% wt./wt. from seven separate fractions with sizes in the range of 0.21-2.81 mm. The agglomeration was performed at a moisture of 30% wb and a material temperature of 78 °C, with a dose of 0.1 g, in a die of diameter 8 mm and height 80 mm. The effects of the process were evaluated based on the compaction parameters and the pellets' density, tensile strength, and water absorption. The incorporation of additives into the WS improved the pellet process and quality. Refined results were achieved after adding CC, as compared to those achieved after adding CS, and the preferred particle size was in the range of 1.00-1.94 mm. This was because, under the given conditions, the back pressure in the die chamber significantly increased, allowing the achievement of a single pellet density of 800 kg·m-3. The pellets were resistant to compressive loads and cracked only at tensile strength of 6 MPa and a specific compression work of 6.5 mJ·mm-2. The addition of CC to the WS improved the strength of the adhesive and the cohesive bonds between the particles. The water absorption for the uncrushed pellets was considerably less than that for crushed pellets, which results in the safer storage of uncrushed pellets and excellent moisture absorption of crushed pellets. The addition of CC to the WS offers benefits in the form of pellet strength with a high water absorption capability. Notably, a study of crushed pellet litter under broiler rearing conditions and an analysis of the operational costs of using WS additives are required for implementing this study.

The Correlation between Physical Crosslinking and Water-Soluble Drug Release from Chitosan-Based Microparticles.

Microparticles containing water-soluble zidovudine were prepared by spray-drying using chitosan glutamate and beta-glycerophosphate as an ion crosslinker (CF). The Box-Behnken design was applied to optimize the microparticles in terms of their drug loading and release behavior. Physicochemical studies were undertaken to support the results from dissolution tests and to evaluate the impact of the crosslinking ratio on the microparticles' characteristics. The zidovudine dissolution behavior had a complex nature which comprised two phases: an initial burst effect followed with a prolonged release stage. The initial drug release, which can be modulated by the crosslinking degree, was primarily governed by the dissolution of the drug crystals located on the microparticles' surfaces. In turn, the further dissolution stage was related to the drug diffusion from the swollen polymer matrix and was found to correlate with the drug loading. Differential Scanning Calorimetry (DSC) studies revealed the partial incorporation of a non-crystallized drug within the polymer matrix, which correlated with the amount of CF. Although CF influenced the swelling capacity of chitosan glutamate microparticles, surprisingly a higher amount of CF did not impact the time required for 80% of the drug to be released markedly. The formulation with the lowest polymer:CF ratio, 3:1, was selected as optimal, providing satisfactory drug loading and displaying a moderate burst effect within the first 30 min of the study, followed with a prolonged drug release of up to 210 min.

Generation of New Isogenic Models of Huntington's Disease Using CRISPR-Cas9 Technology.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the expansion of CAG repeats in exon 1 of the huntingtin gene (HTT). Despite its monogenic nature, HD pathogenesis is still not fully understood, and no effective therapy is available to patients. The development of new techniques such as genome engineering has generated new opportunities in the field of disease modeling and enabled the generation of isogenic models with the same genetic background. These models are very valuable for studying the pathogenesis of a disease and for drug screening. Here, we report the generation of a series of homozygous HEK 293T cell lines with different numbers of CAG repeats at the HTT locus and demonstrate their usefulness for testing therapeutic reagents. In addition, using the CRISPR-Cas9 system, we corrected the mutation in HD human induced pluripotent stem cells and generated a knock-out of the HTT gene, thus providing a comprehensive set of isogenic cell lines for HD investigation.

The influence of scar on the spatio-temporal relationship between electrical and mechanical activation in heart failure patients.

AIMS: The aim of this study was to determine the relationship between electrical and mechanical activation in heart failure (HF) patients and whether electromechanical coupling is affected by scar. METHODS AND RESULTS: Seventy HF patients referred for cardiac resynchronization therapy or biological therapy underwent endocardial anatomo-electromechanical mapping (AEMM) and delayed-enhancement magnetic resonance (CMR) scans. Area strain and activation times were derived from AEMM data, allowing to correlate mechanical and electrical activation in time and space with unprecedented accuracy. Special attention was paid to the effect of presence of CMR-evidenced scar. Patients were divided into a scar (n = 43) and a non-scar group (n-27). Correlation between time of electrical and mechanical activation was stronger in the non-scar compared to the scar group [R = 0.84 (0.72-0.89) vs. 0.74 (0.52-0.88), respectively; P = 0.01]. The overlap between latest electrical and mechanical activation areas was larger in the absence than in presence of scar [72% (54-81) vs. 56% (36-73), respectively; P = 0.02], with smaller distance between the centroids of the two regions [10.7 (4.9-17.4) vs. 20.3 (6.9-29.4) % of left ventricular radius, P = 0.02]. CONCLUSION: Scar decreases the association between electrical and mechanical activation, even when scar is remote from late activated regions.

Gene Therapy for Huntington's Disease Using Targeted Endonucleases.

Huntington's disease (HD) is a hereditary neurological disorder caused by expansion of the CAG repeat tract in the huntingtin gene (HTT). The mutant protein with a long polyglutamine tract is toxic to cells, especially neurons, leading to their progressive degeneration. Similar to many other monogenic diseases, HD is a good target for gene therapy approaches, including the use of programmable endonucleases. Here, we describe a protocol for HTT gene knock out using a modified Cas9 protein (nickase, Cas9n) and a pair of sgRNAs flanking the repeats. Recently, we showed that excision of the CAG repeat tract resulted in a frameshift mutation and premature translation termination. As a model, we used HD patient-derived fibroblasts electroporated with a pair of plasmid vectors expressing CRISPR-Cas9n tools. Efficient HTT inactivation independent of the CAG tract length was confirmed by Western blotting. A modified version of this protocol involving precise excision of the CAG repeats and insertion of a new DNA sequence by homology directed repair may also be used for the generation of new isogenic cellular models of HD.

Methotrexate-induced senescence of human colon cancer cells depends on p53 acetylation, but not genomic aberrations.

Human colon cancer C85 cell response to methotrexate has been documented previously to take on a form of reversible premature senescence. Seeking genomic aberrations encompassing candidate genes whose functional impairment could determine such a response to the drug, an array Comparative Genomic Hybridization method was applied, complemented by expression microarray data set searching. In the C85 cell genome, only short aberrations were identified, classified as focal chromosomal aberrations. 62% of the aberrant regions, selected by referral to normal human colon epithelium, were not carrying any gene. Out of the genes, subject to aberrations, 50% were protein-coding ones. Expression of those that could serve a signaling or a growth-regulatory function was found to be either downregulated or unchanged during C85 cell progression into methotrexate-induced senescence. Lack of extensive chromosomal instability in C85 cells is hypothesized to be attributed to the presence of the wild-type tumor suppressor p53 protein. Although two p53 protein isoforms were detected in C85 cells, stabilization and acetylation of the full-length p53 isoform were shown to underpin progression of the cells into premature senescence upon methotrexate treatment.

qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites.

Genome editing technology based on engineered nucleases has been increasingly applied for targeted modification of genes in a variety of cell types and organisms. However, the methods currently used for evaluating the editing efficiency still suffer from many limitations, including preferential detection of some mutation types, sensitivity to polymorphisms that hamper mismatch detection, lack of multiplex capability, or sensitivity to assay conditions. Here, we describe qEva-CRISPR, a new quantitative method that overcomes these limitations and allows simultaneous (multiplex) analysis of CRISPR/Cas9-induced modifications in a target and the corresponding off-targets or in several different targets. We demonstrate all of the advantages of the qEva-CRISPR method using a number of sgRNAs targeting the TP53, VEGFA, CCR5, EMX1 and HTT genes in different cell lines and under different experimental conditions. Unlike other methods, qEva-CRISPR detects all types of mutations, including point mutations and large deletions, and its sensitivity does not depend on the mutation type. Moreover, this approach allows for successful analysis of targets located in 'difficult' genomic regions. In conclusion, qEva-CRISPR may become a method of choice for unbiased sgRNA screening to evaluate experimental conditions that affect genome editing or to distinguish homology-directed repair from non-homologous end joining.

Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases.

Huntington's disease (HD) is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of CAG repeats in the first exon of the huntingtin gene (HTT). The accumulation of polyglutamine-rich huntingtin proteins affects various cellular functions and causes selective degeneration of neurons in the striatum. Therapeutic strategies used to date to silence the expression of mutant HTT include antisense oligonucleotides, RNA interference-based approaches and, recently, genome editing with the CRISPR/Cas9 system. Here, we demonstrate that the CAG repeat tract can be precisely excised from the HTT gene with the use of the paired Cas9 nickase strategy. As a model, we used HD patient-derived fibroblasts with varied numbers of CAG repeats. The repeat excision inactivated the HTT gene and abrogated huntingtin synthesis in a CAG repeat length-independent manner. Because Cas9 nickases are known to be safe and specific, our approach provides an attractive treatment tool for HD that can be extended to other polyQ disorders.

Oxidative stress and inhibition of nitric oxide generation underlie methotrexate-induced senescence in human colon cancer cells.

The response of human colon cancer C85 cells to methotrexate takes the form of reversible growth arrest of the type of stress-induced senescence. In the present study it is shown that during C85 cell progression into methotrexate-induced senescence, dihydrofolate reductase, the primary intracellular target for the drug, is stabilized at the protein level and its enzymatic activity, assayed in crude cellular extracts, decreases by 2-fold. Dihydrofolate reductase inhibition results in an increase in dihydrobiopterin level and an ultimate decrease in the tetrahydrobiopterin: dihydrobiopterin ratio in senescent cells. Endothelial nitric oxide synthase expression declines. Despite concomitant upregulation of inducible nitric oxide synthase expression, no nitric oxide generation in senescent cells is detected. Progressing oxidative stress accompanies establishment of the state of senescence. DNA damage, in the form of double strand-breaks, occurs at the highest level at the senescence initiation phase and decreases as cells progress into the senescence maintenance phase.