Exploring the Biosafety Potential of Haberlea rhodopensis Friv. In Vitro Culture Total Ethanol Extract: A Comprehensive Assessment of Genotoxicity, Mitotoxicity, and Cytotoxicity for Therapeutic Applications.

The escalating elderly population worldwide has prompted a surge of interest in longevity medicine. Its goal is to interfere with the speed of ageing by slowing it down or even reversing its accompanying effects. As a field, it is rapidly growing and spreading into different branches. One of these is the use of nutraceuticals as anti-ageing drugs. This field is gaining massive popularity nowadays, as people are shifting towards a more natural approach to life and seeking to use natural products as a source of medicine. The present article focuses on the cellular effect of Haberlea rhodopensis Friv. in vitro culture total ethanol extract (HRT), produced by a sustainable biotechnological approach. The extract showed a similar phytochemical profile to plant leaf extract and was rich in primary bioactive ingredients-caffeoyl phenylethanoid glycosides, myconoside, and paucifloside. This study examined the biosafety potential, cytotoxicity, genotoxicity, and mitochondrial activity of the extract using in vitro cultures. The results showed high cell survival rates and minimal cytotoxic effects on Lep3 cells, with no induction of reactive oxygen species nor genotoxicity. Additionally, the extract positively influenced mitochondrial activity, indicating potential benefits for cellular health. The results are promising and show the beneficial effect of HRT without the observation of any adverse effects, which sets the foundation for its further testing and potential therapeutic applications.

Insight into the influence of natural deep eutectic solvents on the extraction of phenolic compounds from poplar type propolis: Composition and in vitro biological activity.

Natural deep eutectic solvents (NADESs) have been considered promising to replace traditional volatile and toxic organic solvents for the extraction of biologically active substances from natural sources. This work applied an efficient and ethanol-exclusion strategy for extraction of phenolic compounds from poplar type propolis using five known NADESs (lactic acid:1,2-propanediol 1:1, lactic acid:fructose 5:1, choline chloride:1,2-propanediol 1:3, choline chloride:1,2-propanediol:water 1:1:1 and betaine:malic acid:water 1:1:6). The selected NADESs' extractability was evaluated by measuring the concentrations of total phenolics and total flavones and flavonols in the propolis extracts obtained, which qualitative chemical composition was further determined in detail by gas chromatography-mass spectrometry (GC-MS) analysis. It demonstrated that the chemical profiles of NADES and 70% ethanolic propolis extracts are similar. To expand the knowledge about the role of the applied solvents in the poplar propolis extraction process, the in vitro antimicrobial, cytotoxic and genotoxic activity of both NADESs and liquid NADES extracts were evaluated. The results revealed that the use of the selected NADESs as an extraction media for phenolic compounds from poplar propolis not only delivered a good extraction yield in some cases, but generally led to the preservation of propolis extracts' biological activity and even to the enhancement of their antimicrobial effect in comparison with the hydroethanolic one. Besides, the tested NADESs except for lactic acid:fructose and betaine:malic acid:water exerted low to negligible toxicity against normal cells treated and apart from lactic acid:fructose the remaining solvents demonstrated concentration-dependent moderate to subtle genotoxicity. There is a probability that not the supramolecular structure of the NADESs, but their components, played a key role for the observed biological effects. The present study has demonstrated an alternative approach for extracting the biologically active complex from poplar type propolis using NADESs, which could be useful for further pharmaceutical and cosmeceutical applications.

Haberlea rhodopensis Extract Tunes the Cellular Response to Stress by Modulating DNA Damage, Redox Components, and Gene Expression.

Ionizing radiation (IR) and reactive oxygen species (ROS)-induced oxidative stress can cause damage to cellular biomolecules, including DNA, proteins, and lipids. These harmful effects can compromise essential cellular functions and significantly raise the risk of metabolic dysfunction, accumulation of harmful mutations, genome instability, cancer, accelerated cellular senescence, and even death. Here, we present an investigation of HeLa cancer cells' early response to gamma IR (γ-IR) and oxidative stress after preincubation of the cells with natural extracts of the resurrection plant Haberlea rhodopensis. In light of the superior protection offered by plant extracts against radiation and oxidative stress, we investigated the cellular defence mechanisms involved in such protection. Specifically, we sought to evaluate the molecular effects of H. rhodopensis extract (HRE) on cells subjected to genotoxic stress by examining the components of the redox pathway and quantifying the transcription levels of several critical genes associated with DNA repair, cell cycle regulation, and apoptosis. The influence of HRE on genome integrity and the cell cycle was also studied via comet assay and flow cytometry. Our findings demonstrate that HREs can effectively modulate the cellular response to genotoxic and oxidative stress within the first two hours following exposure, thereby reducing the severity of such stress. Furthermore, we observed the specificity of genoprotective HRE doses depending on the source of the applied genotoxic stress.

Homo- and heterometallic complexes of Zn(II), {Zn(II)Au(I)}, and {Zn(II)Ag(I)} with pentadentate Schiff base ligands as promising anticancer agents.

Two families of homo- and heterometallic complexes, [Zn2L1(µ-OH)(H2O)2](ClO4)2, [Zn2L2(µ-OH)(H2O)2](ClO4)2, [Zn2L3(µ-OH)(H2O)2](ClO4)2, 1∞[{L1Zn2(µ-OH)}{µ-[Ag(CN)2]}](ClO4), [{L1Zn2(µ-OH)}2{µ-[Au(CN)2]}{[Au(CN)2]2}](ClO4)·H2O, 1∞[{L2Zn2(µ3-OH)}2(H2O){µ-[Ag(CN)2]}](ClO4)3·THF·0.5MeOH, 1∞[{L2Zn2(µ3-OH)}2(H2O){µ-[Au(CN)2]}](ClO4)3·THF·H2O, and 1∞[{L3Zn2(µ-OH)}{µ-[Ag(CN)2]}][Ag(CN)2]·H2O, respectively, have been synthesized and characterized. The Schiff bases used as ligands were obtained by condensation reactions of 2,6-diformyl-p-cresol with N,N-dimethyl-ethylenediamine (HL1), 2-aminomethyl-pyridine (HL2), and 2-aminoethyl-pyridine (HL3), respectively. The cytotoxic/cytostatic and genotoxic effects in cultured human MCF-7 (luminal type A breast cancer), MDA-MB-231 (triple negative breast cancer), HeLa (cervical carcinoma), and Lep-3 (non-tumor embryonal fibroblastoid cells) were studied. The investigations were performed by thiazolyl blue tetrazolium bromide test (MTT test), neutral red uptake cytotoxicity assay, crystal violet staining, hematoxylin and eosin staining, double staining with acridine orange and propidium iodide, AnnexinV/FITC, and Comet assay in short-term experiments (24-72 h, with monolayer cell cultures) as well as by 3D colony-forming method in long-term experiments (28 days, with 3D cancer cell colonies). The results obtained revealed that: (i) applied at a concentration range of 0.1-100 µg mL-1, the compounds investigated decrease in a time- and concentration-dependent manner the viability and/or proliferation of the treated cells; (ii) complexes of {Zn(II)Au(I)} show relatively higher cytotoxic/genotoxic activity and antitumor potential as compared to {Zn(II)Ag(I)}; (iii) some of the complexes demonstrate more pronounced cytotoxic potential than commercially available antitumor agents cisplatin, oxaliplatin, and epirubicin.

Yeast Chromatin Mutants Reveal Altered mtDNA Copy Number and Impaired Mitochondrial Membrane Potential.

Mitochondria are multifunctional, dynamic organelles important for stress response, cell longevity, ageing and death. Although the mitochondrion has its genome, nuclear-encoded proteins are essential in regulating mitochondria biogenesis, morphology, dynamics and function. Moreover, chromatin structure and epigenetic mechanisms govern the accessibility to DNA and control gene transcription, indirectly influencing nucleo-mitochondrial communications. Thus, they exert crucial functions in maintaining proper chromatin structure, cell morphology, gene expression, stress resistance and ageing. Here, we present our studies on the mtDNA copy number in Saccharomyces cerevisiae chromatin mutants and investigate the mitochondrial membrane potential throughout their lifespan. The mutants are arp4 (with a point mutation in the ARP4 gene, coding for actin-related protein 4-Arp4p), hho1Δ (lacking the HHO1 gene, coding for the linker histone H1), and the double mutant arp4 hho1Δ cells with the two mutations. Our findings showed that the three chromatin mutants acquired strain-specific changes in the mtDNA copy number. Furthermore, we detected the disrupted mitochondrial membrane potential in their chronological lifespan. In addition, the expression of nuclear genes responsible for regulating mitochondria biogenesis and turnover was changed. The most pronounced were the alterations found in the double mutant arp4 hho1Δ strain, which appeared as the only petite colony-forming mutant, unable to grow on respiratory substrates and with partial depletion of the mitochondrial genome. The results suggest that in the studied chromatin mutants, hho1Δ, arp4 and arp4 hho1Δ, the nucleus-mitochondria communication was disrupted, leading to impaired mitochondrial function and premature ageing phenotype in these mutants, especially in the double mutant.

Small Heterocyclic Ligands as Anticancer Agents: QSAR with a Model G-Quadruplex.

G-quadruplexes (GQs) have become valid targets for anticancer studies in recent decades due to their multifaceted biological function. Herewith, we aim to quantify interactions of potential heterocyclic ligands (Ls) with model GQs. For seven 4-aminoquinazolines and three 2-heteroaryl perimidines, seven of this ten-membered group so far unknown, we use routine quantum chemical modeling. As shown in the literature, a preferred mode of interaction of heterocycles with cellular structures is stacking to exposable faces of G-quadruplexes. To exploit the energy of this interaction as a molecular descriptor and achieve the necessary chemical precision, we use state of the art large-scale density functional theory (DFT) calculations of stacked heterocycles to a GQ. Actually, the GQ has been simplified for the computation by stripping it off all pentose phosphate residues into a naked model of stacked guanine quartets. The described model thus becomes computable. The obtained heterocyclic ligand GQ.L stacking energies, that is, their GQ affinities, are the necessary ligand descriptors. Using the ligand biological inhibitory activities (IC50) on a human malignant melanoma A375 cell line, we obtain a good linear relationship between computed ligand stacking affinities to GQ, and experimental log (IC50) values. Based on the latter relationship, we discuss a putative mechanism of anticancer activity of heterocyclic ligands via stacking interactions with GQs and thereby controlling cell regulatory activity. This mechanism may tentatively be applied to other condensed five- and six-membered small heterocycles as well.

Natural Deep Eutectic Extracts of Propolis, Sideritis scardica, and Plantago major Reveal Potential Antiageing Activity during Yeast Chronological Lifespan.

Nowadays, the environmentally friendly approach to everyday life routines including body supplementation with pharma-, nutraceuticals and dietary supplements gains popularity. This trend is implemented in pharmaceutical as well as cosmetic and antiageing industries by adopting a newly developed green chemistry approach. Following this trend, a new type of solvents has been created, called Natural Deep Eutectic Solvents (NADES), which are produced by plant primary metabolites. These solvents are becoming a much better alternative to the already established organic solvents like ethanol and ionic liquids by being nontoxic, biodegradable, and easy to make. An interesting fact about NADES is that they enhance the biological activities of the extracted biological compounds. Here, we present our results that investigate the potential antiageing effect of CiAPD14 as a NADES solvent and three plant extracts with it. The tested NADES extracts are from propolis and two well-known medicinal plants-Sideritis scardica and Plantago major. Together with the solvent, their antiageing properties have been tested during the chronological lifespan of four Saccharomyces cerevisiae yeast strains-a wild type and three chromatin mutants. The chromatin mutants have been previously proven to exhibit characteristics of premature ageing. Our results demonstrate the potential antiageing activity of these NADES extracts, which was exhibited through their ability to confer the premature ageing phenotypes in the mutant cells by ameliorating their cellular growth and cell cycle, as well as by influencing the activity of some stress-responsive genes. Moreover, we have classified their antiageing activity concerning the strength of the observed bioactivities.

Actin-Related Protein 4 and Linker Histone Sustain Yeast Replicative Ageing.

Ageing is accompanied by dramatic changes in chromatin structure organization and genome function. Two essential components of chromatin, the linker histone Hho1p and actin-related protein 4 (Arp4p), have been shown to physically interact in Saccharomyces cerevisiae cells, thus maintaining chromatin dynamics and function, as well as genome stability and cellular morphology. Disrupting this interaction has been proven to influence the stability of the yeast genome and the way cells respond to stress during chronological ageing. It has also been proven that the abrogated interaction between these two chromatin proteins elicited premature ageing phenotypes. Alterations in chromatin compaction have also been associated with replicative ageing, though the main players are not well recognized. Based on this knowledge, here, we examine how the interaction between Hho1p and Arp4p impacts the ageing of mitotically active yeast cells. For this purpose, two sets of strains were used-haploids (WT(n), arp4, hho1Δ and arp4 hho1Δ) and their heterozygous diploid counterparts (WT(2n), ARP4/arp4, HHO1/hho1Δ and ARP4 HHO1/arp4 hho1Δ)-for the performance of extensive morphological and physiological analyses during replicative ageing. These analyses included a comparative examination of the yeast cells' chromatin structure, proliferative and reproductive potential, and resilience to stress, as well as polysome profiles and chemical composition. The results demonstrated that the haploid chromatin mutants arp4 and arp4 hho1Δ demonstrated a significant reduction in replicative and total lifespan. These findings lead to the conclusion that the importance of a healthy interaction between Arp4p and Hho1p in replicative ageing is significant. This is proof of the concomitant importance of Hho1p and Arp4p in chronological and replicative ageing.

The Photobiomodulation of MAO-A Affects the Contractile Activity of Smooth Muscle Gastric Tissues.

Nowadays, the utilized electromagnetic radiation (ER) in modalities such as photobiomodulation (PBM) finds broader applications in medical practice due to the promising results suggested by numerous reports. To date, the published data do not allow for the in-depth elucidation of the molecular mechanisms through which ER impacts the human organism. Furthermore, there is a total lack of evidence justifying the relation between the enzymatic activity of monoamine oxidase A (MAO-A) and the effect of 5-hydroxytryptamine (5-HT) on the spontaneous contractile activity of smooth muscle gastric tissues exposed to various light sources. We found that exposure of these tissues to lamps, emitting light with wavelengths of 254 nm and 350 nm, lasers, emitting light with 532 nm and 808 nm, and light-emitting diodes (LEDs) with ER at a wavelength of 660 nm, increased the 5-HT effect on the contractility. On the other hand, LEDs at 365 nm and 470 nm reduced it. The analysis of MAO-A enzymatic activity after exposure to the employed light emitters endorsed these findings. Furthermore, MAOA gene expression studies confirmed the possibility of its optogenetic regulation. Therefore, we concluded that the utilized emitters could alternate the functions of significant neuromediators by modulating the activity and gene transcription levels of enzymes that degrade them. Our investigations will help to disclose the selective conditions upon which PBM can effectively treat gastrointestinal and neurological disorders.

Bioactivity of PEGylated Graphene Oxide Nanoparticles Combined with Near-Infrared Laser Irradiation Studied in Colorectal Carcinoma Cells.

Central focus in modern anticancer nanosystems is given to certain types of nanomaterials such as graphene oxide (GO). Its functionalization with polyethylene glycol (PEG) demonstrates high delivery efficiency and controllable release of proteins, bioimaging agents, chemotherapeutics and anticancer drugs. GO-PEG has a good biological safety profile, exhibits high NIR absorbance and capacity in photothermal treatment. To investigate the bioactivity of PEGylated GO NPs in combination with NIR irradiation on colorectal cancer cells we conducted experiments that aim to reveal the molecular mechanisms of action of this nanocarrier, combined with near-infrared light (NIR) on the high invasive Colon26 and the low invasive HT29 colon cancer cell lines. During reaching cancer cells the phototoxicity of GO-PEG is modulated by NIR laser irradiation. We observed that PEGylation of GO nanoparticles has well-pronounced biocompatibility toward colorectal carcinoma cells, besides their different malignant potential and treatment times. This biocompatibility is potentiated when GO-PEG treatment is combined with NIR irradiation, especially for cells cultured and treated for 24 h. The tested bioactivity of GO-PEG in combination with NIR irradiation induced little to no damages in DNA and did not influence the mitochondrial activity. Our findings demonstrate the potential of GO-PEG-based photoactivity as a nanosystem for colorectal cancer treatment.

Changes in Chromatin Organization Eradicate Cellular Stress Resilience to UVA/B Light and Induce Premature Aging.

Complex interactions among DNA and nuclear proteins maintain genome organization and stability. The nuclear proteins, particularly the histones, organize, compact, and preserve the stability of DNA, but also allow its dynamic reorganization whenever the nuclear processes require access to it. Five histone classes exist and they are evolutionarily conserved among eukaryotes. The linker histones are the fifth class and over time, their role in chromatin has been neglected. Linker histones interact with DNA and the other histones and thus sustain genome stability and nuclear organization. Saccharomyces cerevisiae is a brilliant model for studying linker histones as the gene for it is a single-copy and is non-essential. We, therefore, created a linker histone-free yeast strain using a knockout of the relevant gene and traced the way cells age chronologically. Here we present our results demonstrating that the altered chromatin dynamics during the chronological lifespan of the yeast cells with a mutation in ARP4 (the actin-related protein 4) and without the gene HHO1 for the linker histone leads to strong alterations in the gene expression profiles of a subset of genes involved in DNA repair and autophagy. The obtained results further prove that the yeast mutants have reduced survival upon UVA/B irradiation possibly due to the accelerated decompaction of chromatin and impaired proliferation. Our hypothesis posits that the higher-order chromatin structure and the interactions among chromatin proteins are crucial for the maintenance of chromatin organization during chronological aging under optimal and UVA-B stress conditions.

Biological activity of quinazoline analogues and molecular modeling of their interactions with G-quadruplexes.

BACKGROUND: Quinazolines 1 to 6, with an aromatic or aryl-vinyl substituent in position 2 are selected with the aim to compare their structures and biological activity. The selection includes a natural alkaloid, schizocommunin, and the synthetic 2-(2'-quinolyl)-3H-quinazolin-4-one, known to interact with guanine-quadruplex dependent enzymes, respectively telomerase and topoisomerase. METHODS: Breast cancer cells of the MDA cell line have been used to study the bioactivity of the tested compounds by the method of Comet Assay and FACS analyses. We model observed effects assuming stacking interactions of studied heterocycles with a naked skeleton of G-quadruplex, consisting of guanine quartet layers and potassium ions. Interaction energies are computed using a dispersion corrected density functional theory method, and an electron-correlated molecular orbital theory method. RESULTS: Selected compounds do not remarkably delay nor change the dynamics of cellular progression through the cell cycle phases, while changing significantly cell morphology. Our computational models quantify structural effects on heterocyclic G4-complex stabilization energies, which directly correlate with observed biological activity. CONCLUSION: Our computational model of G-quadruplexes is an acceptable tool for the study of interaction energies of G-quadruplexes and heterocyclic ligands, predicting, and allowing design of novel structures. GENERAL SIGNIFICANCE: Genotoxicity of quinazolin-4-one analogues on human breast cancer cells is not related to molecular metabolism but rather to their interference with G-quadruplex regulatory mechanisms. Computed stabilization energies of heterocyclic ligand complexes of G-quadruplexes might be useful in the prediction of novel telomerase / helicase, topoisomerase and NA polymerase dependent drugs.

Extracts of medicinal plants with natural deep eutectic solvents: enhanced antimicrobial activity and low genotoxicity.

Natural deep eutectic solvents (NADES) are a new alternative to toxic organic solvents. Their constituents are primary metabolites, non-toxic, biocompatible and sustainable. In this study four selected NADES were applied for the extraction of two medicinal plants: Sideritis scardica, and Plantago major as an alternative to water-alcohol mixtures, and the antimicrobial and genotoxic potential of the extracts were studied. The extraction efficiency was evaluated by measuring the extracted total phenolics, and total flavonoids. Best extraction results for total phenolics for the studied plants were obtained with choline chloride-glucose 5:2 plus 30% water; but surprisingly these extracts were inactive against all tested microorganisms. Extracts with citric acid-1,2-propanediol 1:4 and choline chloride-glycerol 1:2 showed good activity against S. pyogenes, E. coli, S. aureus, and C. albicans. Low genotoxicity and cytotoxicity were observed for all four NADES and the extracts with antimicrobial activity. Our results confirm the potential of NADESs for extraction of bioactive constituents of medicinal plants and further suggest that NADES can improve the effects of bioactive extracts. Further studies are needed to clarify the influence of the studied NADES on the bioactivity of dissolved substances, and the possibility to use such extracts in the pharmaceutical and food industry.

Amination of Graphene Oxide Leads to Increased Cytotoxicity in Hepatocellular Carcinoma Cells.

Clinically, there is an urgent need to identify new therapeutic strategies for selectively treating cancer cells. One of the directions in this research is the development of biocompatible therapeutics that selectively target cancer cells. Here, we show that novel aminated graphene oxide (haGO-NH2) nanoparticles demonstrate increased toxicity towards human hepatocellular cancer cells compared to pristine graphene oxide(GO). The applied novel strategy for amination leads to a decrease in the size of haGO-NH2 and their zeta potential, thus, assuring easier penetration through the cell membrane. After characterization of the biological activities of pristine and aminated GO, we have demonstrated strong cytotoxicity of haGO-NH2 toward hepatic cancer cells - HepG2 cell line, in a dose-dependent manner. We have presented evidence that the cytotoxic effects of haGO-NH2 on hepatic cancer cells were due to cell membrane damage, mitochondrial dysfunction and increased reactive oxygen species (ROS) production. Intrinsically, our current study provides new rationale for exploiting aminated graphene oxide as an anticancer therapeutic.

Aminated Graphene Oxide as a Potential New Therapy for Colorectal Cancer.

Nanotechnology-based drug delivery systems for cancer therapy are the topic of interest for many researchers and scientists. Graphene oxide (GO) and its derivates are among the most extensively studied delivery systems of this type. The increased surface area, elevated loading capacity, and aptitude for surface functionalization together with the ability to induce reactive oxygen species make GO a promising tool for the development of novel anticancer therapies. Moreover, GO nanoparticles not only function as effective drug carriers but also have the potential to exert their own inhibitory effects on tumour cells. Recent results show that the functionalization of GO with different functional groups, namely, with amine groups, leads to increased reactivity of the nanoparticles. The last steers different hypotheses for the mechanisms through which this functionalization of GO could potentially lead to improved anticancer capacity. In this research, we have evaluated the potential of amine-functionalized graphene oxide nanoparticles (GO-NH2) as new molecules for colorectal cancer therapy. For the purpose, we have assessed the impact of aminated graphene oxide (GO) sheets on the viability of colon cancer cells, their potential to generate ROS, and their potential to influence cellular proliferation and survival. In order to elucidate their mechanism of action on the cellular systems, we have probed their genotoxic and cytostatic properties and compared them to pristine GO. Our results revealed that both GO samples (pristine and aminated) were composed of few-layer sheets with different particle sizes, zeta potential, and surface characteristics. Furthermore, we have detected increased cyto- and genotoxicity of the aminated GO nanoparticles following 24-hour exposure on Colon 26 cells. The last leads us to conclude that exposure of cancer cells to GO, namely, aminated GO, can significantly contribute to cancer cell killing by enhancing the cytotoxicity effect exerted through the induction of ROS, subsequent DNA damage, and apoptosis.

Linker histones and chromatin remodelling complexes maintain genome stability and control cellular ageing.

Linker histones are major players in chromatin organization and per se are essential players in genome homeostasis. As the fifth class of histone proteins the linker histones not only interact with DNA and core histones but also with other chromatin proteins. These interactions prove to be essential for the higher levels of chromatin organization like chromatin loops, transcription factories and chromosome territories. Our recent results have proved that Saccharomyces cerevisiae linker histone - Hho1p, physically interacts with the actin-related protein 4 (Arp4) and that the abrogation of this interaction through the deletion of the gene for the linker histone in arp4 mutant cells leads to global changes in chromatin compaction. Here, we show that the healthy interaction between the yeast linker histone and Arp4p is critical for maintaining genome stability and for controlling cellular sensitivity to different types of stress. The abolished interaction between the linker histone and Arp4p leads the mutant yeast cells to premature ageing phenotypes. Cells die young and are more sensitive to stress. These results unambiguously prove the role of linker histones and chromatin remodelling in ageing by their cooperation in pertaining higher-order chromatin compaction and thus maintaining genome stability.

Biocompatibility and Biological Efficiency of Inorganic Calcium Filled Bacterial Cellulose Based Hydrogel Scaffolds for Bone Bioengineering.

The principal focus of this work is the in-depth analysis of the biological efficiency of inorganic calcium-filled bacterial cellulose (BC) based hydrogel scaffolds for their future use in bone tissue engineering/bioengineering. Inorganic calcium was filled in the form of calcium phosphate (ß-tri calcium phosphate (ß-TCP) and hydroxyapatite (HA)) and calcium carbonate (CaCO3). The additional calcium, CaCO3 was incorporated following in vitro bio-mineralization. Cell viability study was performed with the extracts of BC based hydrogel scaffolds: BC-PVP, BC-CMC; BC-PVP-ß-TCP/HA, BC-CMC-ß-TCP/HA and BC-PVP-ß-TCP/HA-CaCO3, BC-CMC-ß-TCP/HA-CaCO3; respectively. The biocompatibility study was performed with two different cell lines, i.e., human fibroblasts, Lep-3 and mouse bone explant cells. Each hydrogel scaffold has facilitated notable growth and proliferation in presence of these two cell types. Nevertheless, the percentage of DNA strand breaks was higher when cells were treated with BC-CMC based scaffolds i.e., BC-CMC-ß-TCP/HA and BC-CMC-ß-TCP/HA-CaCO3. On the other hand, the apoptosis of human fibroblasts, Lep-3 was insignificant in BC-PVP-ß-TCP/HA. The scanning electron microscopy confirmed the efficient adhesion and growth of Lep-3 cells throughout the surface of BC-PVP and BC-PVP-ß-TCP/HA. Hence, among all inorganic calcium filled hydrogel scaffolds, 'BC-PVP-ß-TCP/HA' was recommended as an efficient tissue engineering scaffold which could facilitate the musculoskeletal (i.e., bone tissue) engineering/bioengineering.

Kluyveromyces lactis genome harbours a functional linker histone encoding gene.

Linker histones are essential components of chromatin in eukaryotes. Through interactions with linker DNA and nucleosomes they facilitate folding and maintenance of higher-order chromatin structures and thus delicately modulate gene activity. The necessity of linker histones in lower eukaryotes appears controversial and dubious. Genomic data have shown that Schizosaccharomyces pombe does not possess genes encoding linker histones while Kluyveromyces lactis has been reported to have a pseudogene. Regarding this controversy, we have provided the first direct experimental evidence for the existence of a functional linker histone gene, KlLH1, in K. lactis genome. Sequencing of KlLH1 from both genomic DNA and copy DNA confirmed the presence of an intact open reading frame. Transcription and splicing of the KlLH1 sequence as well as translation of its mRNA have been studied. In silico analysis revealed homology of KlLH1p to the histone H1/H5 protein family with predicted three domain structure characteristic for the linker histones of higher eukaryotes. This strongly proves that the yeast K. lactis does indeed possess a functional linker histone gene thus entailing the evolutionary preservation and significance of linker histones. The nucleotide sequences of KlLH1 are deposited in the GenBank under accession numbers KT826576, KT826577 and KT826578.

Random, double- and single-strand DNA breaks can be differentiated in the method of Comet assay by the shape of the comet image.

Comet assay is an invaluable tool in DNA research. It is widely used to detect DNA damage as an indicator of exposure to genotoxic stress. A canonical set of parameters and specialized software programs exist for Comet assay data quantification and analysis. None of them so far has proven its potential to employ a computer-based algorithm for assessment of the shape of the comet as an indicator of the exact mechanism by which the studied genotoxins cut in the molecule of DNA. Here, we present 14 unique measurements of the comet image based on the comet morphology. Their mathematical derivation and statistical analysis allowed precise description of the shape of the comet image which in turn discriminated the cause of genotoxic stress. This algorithm led to the development of the "CometShape" software which allowed easy discrimination among different genotoxins depending on the type of DNA damage they induce.

Methanol extracts from the resurrection plant Haberlea rhodopensis ameliorate cellular vitality in chronologically ageing Saccharomyces cerevisiae cells.

Bioactive substances that are found in many natural plant extracts are very important for the cosmetics, pharmaceutical industry and biotechnology. Especially interesting for these industries are the substances that possess cell revitalizing and anti-ageing properties. The endemic plant Haberlea rhodopensis is known for its ability to withstand drought and to revitalize when returned to optimal conditions after a long time in desiccation. It is a mere fact that this plant not only can completely resurrect from a dried state but is also able to bring back the natural biochemical compositions of its cells. As a result H. rhodopensis offers a wide field for investigation of the exact mechanisms of the revitalization process as well as broadens the search for unique bioactive chemical substances in its cells. Here, by using the yeast Saccharomyces cerevisiae as a model we have demonstrated that methanol extracts from the plant H. rhodopensis hold specific properties to revitalize and ameliorate cellular growth as well as to balance intracellular metabolic states. Our results add valuable knowledge on the effects of natural compounds on ageing and reinforce the idea of using yeast as a model organism in the development of rapid tests for studying the efficacy of different bioactive substances.