Bee venom genotoxicity on Saccharomyces cerevisiae cells - The role of mitochondria and YAP1 transcription factor.

The present work aims to clarify the genotype differences of a model organism Saccharomyces cerevisiae in response to bee venom. The study evaluated various endpoints including cell survival, induction of physiologically active superoxide anions, mitotic gene conversion, mitotic crossing-over, reverse mutations, DNA double-strand breaks, and Ty1 retrotransposition. The role of the intact mitochondria and the YAP1 transcription factor was also evaluated. Our results indicate a genotype-specific response. The first experimental evidence has been provided that bee venom induces physiologically active superoxide anions and DNA double-strand breaks in S. cerevisiae. The lack of oxidative phosphorylation due to disrupted or missing mitochondrial DNA reduces but not diminishes the cytotoxicity of bee venom. The possible modes of action could be considered direct damage to membranes (cytotoxic effect) and indirect damage to DNA through oxidative stress (genotoxic effect). YAP1 transcription factor was not found to be directly involved in cell defense against bee venom treatment.

Analysis of Results After Surgical Application of Bone Marrow Aspirate Stem Cell Concentrate in the Treatment of Avascular Necrosis of the Femoral Head.

Introduction: Avascular necrosis or osteonecrosis of the femoral head is defined as a pathological process that results in a critical reduction in the blood supply to the hip head with increased intraosseous pressure. Avascular necrosis is a multifactorial disease. The treatment used so far consists of transosseous decompression of the femoral head and is the most commonly used method in the early stages of osteonecrosis. Aim: The aim of this study was to evaluate the results after application of bone marrow stem cells obtained from the pelvic ridge, which was applied to the necrotic zone after previous decompression. Material and methods. The study is of prospective character and included 30 patients with first, second, and third degrees of AVN according to the Ficat classification, determined with X-ray. The range of motion in the hip was examined preoperatively by using a goniometer, a radiological evaluation of the degree of AVN was performed, according to the Ficat classification, and the VAS scale and the Harris Hip Score were examined preoperatively and postoperatively. Results: The average value for the Harris Hip Score (HHS) preoperatively was: for Ficat I -66.32±5.1, for Ficat II - 49.19±3.4 and for Ficat III - 33.71±2.1. At the 3-month postoperative follow-up, the average HHS values were: 87.92±4.3 for Ficat I, 78.64±6.6 for Ficat II and 76.48±2.6 for Ficat III. The same values for HHS were obtained in the control examinations at the 6th and 12th month postoperatively, indicating the fact that good bone regeneration was achieved and the progression of the condition was prevented. A decrease in the HHS value was observed at the control examination at 1 year after the surgical treatment, in 3 patients according to the Ficat classification of grade III, in whom a total hip replacement surgery was performed. Conclusion: The use of stem cells in the treatment of avascular necrosis of the femoral head has achieved good functional results and reduced pain in operated patients. Radiographically, good bone regeneration was achieved and the progression of necrosis to a higher degree was prevented.

Apricot kernels' extract and amygdalin alter bleomycin-induced Ty1 retrotransposition, mitotic gene conversion in the trp-5 locus and reverse point mutations in ilv1-92 allele in Saccharomyces cerevisiae.

The present study aims to analyze the effect of apricot kernels' extract (AKE) and amygdalin (AMY) on bleomycin-induced genetic alternations. Five endpoints were analyzed: cell survival, Ty1 retrotransposition, mitotic gene conversion in the trp-5 locus, reverse point mutations in ilv1-92 allele, and mitotic crossing-over in the ade2 locus. The present work provides the first experimental evidence that bleomycin induces Ty1 retrotransposition in Saccharomyces cerevisiae. New data is obtained that the degree of DNA protection of AMY and AKE depends on the studied genetic event. AKE has been found to provide significant protection against bleomycin-induced Ty1 retrotransposition due to better-expressed antioxidant potential. On the other side, AMY better-expressed protection against bleomycin-induced mitotic gene conversion and reverse mutations may be attributed to the activation of the repair enzymes.

Phytochemical characterization and biological activity of apricot kernels' extract in yeast-cell based tests and hepatocellular and colorectal carcinoma cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Bitter apricot kernels' extract contains a broad spectrum of biologically active substances with a lot of attention to amygdalin - cyanogenic glycoside. The extract has been used in the pharmaceutical industry for years as an ingredient of different pharmaceuticals with anti-inflammatory, antimicrobial, or regenerative properties. In traditional medicine, the bitter apricot kernels are known as a remedy for respiratory disorders and skin diseases. The apricot kernels and amygdalin are often prescribed by practitioners for the prevention and treatment of various medical conditions, including colorectal cancer. THE PRESENT STUDY AIMS: to evaluate the phytochemical composition and the potential antimutagenic, antirecombinogenic, and antitumor effect of apricot kernels' extract at very low concentrations in yeast cell-based tests and mammalian hepatocellular and colon carcinoma cell lines. MATERIALS AND METHODS: Phytochemical analysis was performed by LC-MS profiling. Reverse-phase HPLC and UV detection were applied for the determination of amygdalin quantity in the extract. Biological activity was evaluated by Zimmermann's mutagenicity and Ty1 retrotransposition test. Cytotoxic/antiproliferative activity of apricot kernels' extract was performed on four types of cell lines - HepG2, HT-29, BALB/3T3, clone A31, and BJ using the standard MTT-dye reduction assay. RESULTS: Data revealed the presence of more than 1000 compounds and 4 cyanogenic glycosides among them - Amygdalin, Deidaclin, Linamarin and Prulaurasin. The Amygdalin concentration was measured to be 57.8 µg/ml. All extract concentrations demonstrated a strong antigenotoxic, antirecombinogenic, antimutagenic, and anticarcinogenic effect in the yeast cell-based tests. High selectivity of the extract action is established among different mammalian cell lines. Normal cell line BJ is found to be resistant to the extract action. HepG2 was found to be the most sensitive to apricot kernels' action. CONCLUSION: The present study provides the first phytochemical analysis of Bulgarian bitter apricot kernels. Three new cyanogenic glycosides were reported. Evidence is obtained that the apricot kernels' extract at low concentrations is not able to induce some of the events related to the initial steps of tumorigenesis. Additionally, a high selectivity of the extract action is established among different cell lines. The most sensitive cell line was found to be HepG2.

Sacrectomy with Ilio-Lumbar Stabilization Due to Low-Grade Chondrosarcoma of Sacrum and Iliac Wing. Case Report of the First Surgery Performed in the Republic of North Macedonia.

Low - grade chondrosarcomas are primary malignant bone tumors that are resistant to chemo- and radiotherapy and are treated surgically. Sacral localization makes surgical resection technically difficult due to position, anatomic structures involved, and large tumor size at detection. The risk of complications is high. We present the introduction of a novel surgical technique in our country, sacrectomy with ilio-lumbar stabilization. This was performed on a 67-year-old man with low-grade chondrosarcoma of the sacrum with sacroiliac joint involvement. The procedure was performed via an antero-posterior approach in two stages. Ilio-lumbar fixation with a mesh cage bridge was used to obtain spinopelvic continuity and stability. Sacrectomy is a technically demanding procedure that requires careful preoperative planning and a multidisciplinary approach, as well as high level of surgical experience.

DNA susceptibility of Saccharomyces cerevisiae to Zeocin depends on the growth phase.

The aim of this study was to evaluate the level of Zeocin-induced double-strand breaks (DSBs) in Saccharomyces cerevisiae cells in a different growth phase, using constant-field gel electrophoresis (CFGE). Saccharomyces cerevisiae diploid strain D7ts1 with enhanced cellular permeability was used. The effects of growth phase and treatment time were evaluated based on Zeocin-induced DSBs, measured by CFGE. Survival assay was also applied. No protoplast isolation was necessary for the detection of DSBs in strain D7ts1. Differences in the response of cells depending on the growth phase were obtained. Cells in exponential growth phase had increased DSB levels only after Zeocin treatment with concentrations equal or higher than 200 µgml-1. Increasing treatment time did not result in higher DSB levels. Oppositely, treatment of cells at the beginning of stationary phase with Zeocin concentrations resulted in more than 1.5-fold increase in DSB levels in comparison with those in untreated cells. Increased DSB levels were measured for all the treatment times. A dose-dependent decrease in cell survival was observed after Zeocin treatment with concentrations in the range of lethality LD20-LD50. A strong negative correlation was calculated between the levels of DSBs and cell survival. New information is provided concerning DNA susceptibility depending on the growth phase. DNA susceptibility is higher in cells at the beginning of stationary phase than those in exponential phase. Data presented here illustrate that the optimized by us CFGE protocol is sensitive and could be used successfully for DSB measurement in Saccharomyces cerevisiae strains with enhanced cellular permeability.

Antimutagenic, Antirecombinogenic, and Antitumor Effect of Amygdalin in a Yeast Cell-Based Test and Mammalian Cell Lines.

Amygdalin is a major component of the seeds of Rosaceae family of plants such as apricots, peaches, cherry, nectarines, apples, plums, and so on, as well as almonds. It is used in alternative medicine for cancer prevention, alleviation of fever, cough suppression, and quenching thirst. The aim of the present study is to determine the mutagenic and recombinogenic effects of amygdalin in a test system Saccharomyces cerevisiae and to evaluate its potential antitumor effect in a yeast cell-based test and colon cancer cell lines. Results obtained show that concentrations 25, 50, and 100 µg/mL did not have any cytotoxic, mutagenic, and carcinogenic effect in yeast cell-based tests. Pretreatment with amygdalin at concentration 100 µg/mL leads to around twofold of the cell survival and decrease of reverse mutation frequency, induced by the alkylating agent methyl methanesulfonate. The frequency of gene conversion and mitotic crossing-over is around threefold lower. The anticarcinogenic potential of amygdalin at the same concentration is presented as around fourfold reduction of Ty1 retrotransposition induced by hexavalent chromium. In summary, data presented in this study provide evidence concerning the inability of amygdalin itself to provoke events related to the initial steps of tumorigenesis. In addition, the observed antimutagenic/antirecombinogenic effect could be activation of error-free and error-prone recombination events. Based on the high selectivity toward normal or tumor cell lines, it could be speculated that amygdalin has higher cytotoxic effect in cell lines with higher proliferative and metabolic activity, which are the majority of fast developing tumors.

Mutational Status of CDKN2A and TP53 Genes in Laryngeal Squamous Cell Carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is the second most common tumour of the head and neck. It is characterized by frequent aberrations in two cell-cycle regulators--CDKN2A and TP53. However, LSCC has been often studied as a part of the group of head and neck cancers and not as an individual entity. In the current study we aimed to examine mutation status of CDKN2A and TP53 genes in 108 LSCC patients. DNA was extracted from fresh-frozen tumour tissues; exons 1-3 of CDKN2A and exons 5-8 of TP53 were screened for mutations by direct sequencing. Genetic aberrations in CDKN2A were found in 16 (14.2%) and those in TP53--in 56/108 (51.9%) tumours. Seven mutations (two insertions, three deletions, one missense and one silent) detected in CDKN2A were not described previously. Also, we found seven novel deletions and a novel indel in TP53. No significant associations with clinical features were found. However, TP53 mutations were predominantly observed in smokers with advanced stage tumours. Screening for genetic aberrations in a defined group of LSCC contributes to the knowledge about laryngeal carcinogenesis. Further investigations are required to confirm the observed trends in associations with clinical features.

Antioxidant, antimutagenic, and anticarcinogenic effects of Papaver rhoeas L. extract on Saccharomyces cerevisiae.

The aim of this work was to analyze the antioxidant and antimutagenic/anticarcinogenic capacity of Papaver rhoeas L. water extract against standard mutagen/carcinogen methyl methanesulfonate (MMS) and radiomimetic zeocin (Zeo) on a test system Saccharomyces cerevisiae. The following assays were used: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, quantitative determination of superoxide anion (antireactive oxygen species [antiROS test]), DNA topology assay, D7ts1 test--for antimutagenic--and Ty1 transposition test--for anticarcinogenic effects. Strong pro-oxidative capacity of Zeo was shown to correlate with its well-expressed mutagenic and carcinogenic properties. The mutagenic and carcinogenic effects of MMS were also confirmed. Our data concerning the antioxidant activity of P. rhoeas L. extract revealed that concentration corresponding to IC(50) in the DPPH assay possessed the highest antioxidant activity in the antiROS biological assay. It was also observed that a concentration with 50% scavenging activity expressed the most pronounced antimutagenic properties decreasing Zeo-induced gene conversion twofold, reverse mutation fivefold, and total aberrations fourfold. The same concentration possessed well-expressed anticarcinogenic properties measured as reduction of MMS-induced Ty1 transposition rate fivefold and fourfold when Zeo was used as an inductor. Based on the well-expressed antioxidant, antimutagenic, and anticarcinogenic properties obtained in this work, the P. rhoeas L. extract could be recommended for further investigations and possible use as a food additive.

Reversible photoswitching between nitrito-N and nitrito-O isomers in trans-[Ru(py)(4)(NO(2))(2)].

Nitro-nitrito photoisomerisation is investigated in solid samples and solutions of trans-[Ru(py)(4)(NO(2))(2)]. Using light of wavelength 325 nm 50% of the N-bound Ru-NO(2) ligands can be switched to the O-bound Ru-ONO configuration (nitrito-N to nitrito-O isomerisation) at temperatures below T = 250 K in solids. The population of the isomeric configurations is determined with infrared spectroscopy from the decrease of the area of the nu(NO) stretching and delta(NO) deformation modes. In a frozen methanol-ethanol solution nearly 100% can be converted to the nitrito-O configuration. Upon heating above T = 250 K the Ru-NO(2) configuration is restored. The nitrito-O Ru-ONO configuration can be partially transferred back to the nitrito-N configuration by irradiation with light in the spectral range 405-442 nm. Using absorption spectroscopy on a frozen methanol-ethanol solution, two new bands at 447 and 380 nm are observed in the nitrito-O configuration compared to one at 334 nm of the nitrito-N ground state configuration. The photoconversion is initiated by the metal-to ligand charge transfer transition Ru(d) -->pi*(NO(2),py) as shown by the calculated partial density of states using Density Functional Theory. The calculations yield also the structure of the nitrito-N and nitrito-O isomer as well as the corresponding vibrational densities. The experimental structure of the ground state is determined using powder diffraction.

Photogeneration of nitrosyl linkage isomers in octahedrally coordinated platinum complexes in the red spectral range.

Octahedrally coordinated platinum nitrosyl complexes [Pt(NH(3))(4)(NO(3))(NO)](NO(3))(2) (1) and [Pt(NH(3))(4)(SO(4))(NO)](HSO(4))(CH(3)CN) (2) undergo linkage isomerization at temperatures below 130 K when excited with red light. Irradiation in the spectral range of 570-800 nm results in an inversion of the NO ligand from a Pt-NO to a Pt-ON configuration. The metastable state Pt-ON can be reverted back to the ground state (GS) Pt-NO by irradiation with blue-green or infrared light or by heating above 130 K. The characteristic shift of the nu(NO) stretching vibration from 1744 to 1815 cm(-1) in 1 and from 1714 to 1814 cm(-1) in 2 allowed the unambiguous identification of the respective nitrosyl isomers. Up to 26% of the complexes of 1 and 20% of 2 may be photochemically excited toward the metastable state (MS). Using X-ray crystallography and DFT calculations, it is shown that the Pt-NO in these {MNO}(8) complexes exhibits a bent arrangement with a Pt-N-O angle in the range of 117-120 degrees. As a consequence and in contrast to the known {MNO}(6) complexes only one metastable linkage isomer Pt-ON with a correspondingly bent Pt-O-N arrangement is formed, as evidenced by spectroscopy and DFT calculations. The calculated partial density of states shows that the charge transfer transition Pt(5d) --> pi(star)(NO) is responsible for the formation of the metastable state.

[Ru(py)4Cl(NO)](PF6)2.0.5H2O: a model system for structural determination and ab initio calculations of photo-induced linkage NO isomers.

Structure analysis of ground state (GS) and two light-induced (SI and SII) metastable linkage NO isomers of [Ru(py)4Cl(NO)](PF6)2.0.5H2O is presented. Illumination of the crystal by a laser with lambda = 473 nm at T = 80 K transfers around 92% of the NO ligands from Ru-N-O into the isomeric configuration Ru-O-N (SI). A subsequent irradiation with lambda = 980 nm generates about 48% of the side-on configuration Ru<(N)(O) (SII). Heating to temperatures above 200 K or irradiation with light in the red spectral range transfers both metastable isomers reversibly back to the GS. Photodifference maps clearly show the N-O configurations for both isomers and they could be used to find a proper starting model for subsequent refinements. Both metastable isomers have slightly but significantly different cell parameters with respect to GS. The main structural changes besides the Ru-O-N and RU<(N)(O) linkage are shortenings of the trans Ru-Cl bonds and the equatorial Ru-N bonds. The experimental results are compared with solid-state calculations based on density functional theory (DFT), which reproduce the observed structures with high accuracy concerning bond lengths and angles. The problem of how the different occupancies of SI and GS could affect refinement results was solved by a simulation procedure using the DFT data as starting values.

The Creutz-Taube complex revisited: DFT study of the infrared frequencies.

The structural parameters, electronic properties, and infrared frequencies of three binuclear ruthenium complexes, [(NH(3))(5)Ru(pyrazine)Ru(NH(3))(5)](n+), n = 4-6, have been investigated with density functional theory. Structural analysis demonstrates that the structure of the mixed-valence 5+, or [II,III], is not an intermediate of the reduced 4+, or [II,II], and the oxidized 6+, or [III,III], compounds. Electronic structure comparison shows that the Ru d(yz) antibonding orbital is empty when n = 5 and 6 and occupied, when n = 4. The infrared frequencies have been calculated for a sequence of models with increasingly detailed accounts of counterions, ranging from the free Creutz-Taube 5+ ion, over jellium embedded, COnductor-like Screening MOdel (COSMO), to the experimental structure of the triclinic [II,III](tos)(5).4H(2)O (tos = p-toluenesulfonate) crystal. Analysis of the Ru vibrations shows that the spectra for the two symmetry-inequivalent Ru atoms are essentially the same. We find that Ru-Ru modes exist near three well-defined frequencies in the solid: at 145, 285, and 345 cm(-1). Similar results are also obtained for the simplified jellium and COSMO models. The spectral properties of these vibrational correlations testify to the existence of two coupled Ru atoms in the same charge state.

Car-Parrinello Molecular Dynamics Simulations of CaCl2 Aqueous Solutions.

Car-Parrinello molecular dynamics (CPMD) simulations are used to investigate the structural properties of 1 and 2 molal (m) CaCl2 aqueous solutions and, in particular, the radial distribution functions, coordination numbers, and dipole moments of water molecules in the first solvation shell. According to these simulations, the first solvation shell of the Ca(2+) ion consists of six water molecules, that are characterized by an increased averaged dipole moment compared to that of bulk water, and a first-shell Ca-O radial distribution function peak at 2.39 Å. The results are compared to those of CPMD simulations of Ca(2+) (no counterions), and no significant differences are found. This indicates that the homogeneous neutralizing background charge density implicitly included in simulations of non-neutral systems appropriately mimics the presence of the counterions (at least in terms of reproducing the solvation structure properties and for the box sizes considered). Classical molecular dynamics (MD) simulations of aqueous Ca(2+) using varying box sizes confirm this suggestion. The CPMD simulations at 2 m concentration also reveal additional possibilities for the structural arrangement of water molecules and chloride ions around Ca(2+). In particular, they support the stability of Ca(2+)-Cl(-) (contact) and Ca(2+)-H2O-Cl(-) (solvent-separated) ion pairs. In addition, the solvent-separated cation pair is found to occur in a deprotonated Ca(2+)-OH(-)-Ca(2+) form. The existence of such a species has, to our knowledge, never been invoked previously to account for experimental data on CaCl2 solutions.

Delay to surgery and mortality after hip fracture.

BACKGROUND: Hip fractures in elderly patients are associated with increased mortality. These patients frequently experience a delay to surgery for both medical and non-medical reasons. The effect of this delay on patient morbidity and mortality is controversial. METHODS: We conducted an observational study of 222 consecutive patients greater than 50 years of age who underwent surgical management of a hip fracture at one tertiary hospital. Baseline measures recorded were age, sex, time to theatre, American Society of Anesthesiology (ASA) scores, type of surgery, number of theatre cancellations and the reason for any cancellation. Our primary outcome was 30-day mortality after surgery. RESULTS: The 30-day mortality after hip fracture was 7.2%. Patients who were operated on within 2 days of admission had a 30-day postoperative mortality rate of 5.8% versus 9.4% in those patients who experience a delay of more than 2 calendar days. This was not a statistically significant difference, and the effect of surgical delay was less on multivariate analysis. In multivariate analysis, the only statistically significant predictors of 30-day mortality were an increasing ASA score and having a fracture treated with an arthroplasty procedure. CONCLUSIONS: Our study did not show a significant increase in mortality in patients whose surgery was delayed by more than 2 days. However, in the context of other published work, we would continue to recommend early surgery to minimize length of stay and complications.

Molecular dynamics simulation of liquid water: hybrid density functionals.

The structure, dynamical, and electronic properties of liquid water utilizing different hybrid density functionals were tested within the plane wave framework of first-principles molecular dynamics simulations. The computational approach, which employs modified functionals with short-ranged Hartree-Fock exchange, was first tested in calculations of the structural and bonding properties of the water dimer and cyclic water trimer. Liquid water simulations were performed at the state point of 350 K at the experimental density. Simulations included three different hybrid functionals, a meta-functional, four gradient-corrected functionals, and the local density and Hartree-Fock approximations. It is found that hybrid functionals are superior in reproducing the experimental structure and dynamical properties as measured by the radial distribution function and self-diffusion constant when compared to the pure density functionals. The local density and Hartree-Fock approximations show strongly over- and understructured liquids, respectively. Hydrogen bond analysis shows that the hybrid functionals give slightly smaller average numbers of hydrogen bonds than pure density functionals but similar hydrogen bond populations. The average molecular dipole moments in the liquid from the three hybrid functionals are lower than those of the corresponding pure density functionals.