Formation of nickel-carbon heterofullerenes under electron irradiation.

A way to produce new metal-carbon nanoobjects by transformation of a graphene flake with an attached transition metal cluster under electron irradiation is proposed. The transformation process is investigated by molecular dynamics simulations by the example of a graphene flake with a nickel cluster. The parameters of the nickel-carbon potential (I. V. Lebedeva et al., J. Phys. Chem. C, 2012, 116, 6572) are modified to improve the description of the balance between the fullerene elastic energy and graphene edge energies in this process. The metal-carbon nanoobjects formed are found to range from heterofullerenes with a metal patch to particles consisting of closed fullerene and metal clusters linked by chemical bonds. The atomic-scale transformation mechanism is revealed by the local structure analysis. The average time of formation of nanoobjects and their lifetime under electron irradiation are estimated for the experimental conditions of high-resolution transmission electron microscopy (HRTEM). The sequence of images of nanostructure evolution with time during its observation by HRTEM is also modelled. Furthermore, the possibility of batch production of studied metal-carbon nanoobjects and solids based on these nanoobjects is discussed.

Dissipation and fluctuations in nanoelectromechanical systems based on carbon nanotubes.

The tribological characteristics of nanotube-based nanoelectromechanical systems (NEMS) exemplified by a gigahertz oscillator are studied. Various factors that influence the tribological properties of nanotube-based NEMS are quantitatively analyzed with the use of molecular dynamics calculations of the quality factor (Q-factor) of the gigahertz oscillator. We demonstrate that commensurability of the nanotube walls can increase the dissipation rate, while the structure of the wall ends and the nanotube length do not influence the Q-factor. It is shown that the dissipation rate depends on the interwall distance and the way of fixation of the outer wall, and is significant in the case of a poor fixation for nanotubes with a large interwall distance. Defects are found to strongly decrease the Q-factor due to the excitation of low-frequency vibrational modes. No universal correlation between the static friction forces and the energy dissipation rate is established. We propose an explanation of the obtained results on the basis of the classical theory of vibrational-translational relaxation. Significant thermodynamics fluctuations are revealed in the gigahertz oscillator by molecular dynamics simulations and they are analyzed in the framework of the fluctuation-dissipation theorem. The possibility of designing NEMS with a desirable Q-factor and their applications are discussed on the basis of the above results.

Targeting inhibition of K-ras enhances Ad.mda-7-induced growth suppression and apoptosis in mutant K-ras colorectal cancer cells.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, growth-suppressing and apoptosis-inducing gene with broad-spectrum antitumor activity. However, when administered by means of a replication-incompetent adenovirus, Ad.mda-7, several colorectal carcinoma cell lines are resistant to its antiproliferative and antisurvival effects. We have presently endeavored to determine if K-ras mutations, present in approximately 40-50% of colorectal cancers and which may mediate resistance to chemotherapy and radiotherapy, represent a predisposing genetic factor mitigating reduced sensitivity to Ad.mda-7. To suppress ras expression, three structurally different replication-incompetent adenoviral vectors were engineered that express (1) an intracellular, neutralizing single-chain antibody (scAb) to p21 ras (Ad.K-ras scAb), (2) an antisense (AS) K-ras gene (Ad.K-ras AS) or (3) both mda-7/IL-24 and a K-ras AS gene in a single bipartite virus (Ad.m7.KAS). Simultaneous inhibition of K-ras and expression of mda-7/IL-24 enhanced killing of colorectal carcinoma cells with mutated K-ras, but not with wild-type K-ras. The extent of killing depended on the degree of K-ras downregulation, with Ad.K-ras AS being generally more efficient than Ad.K-ras scAb in combination with Ad.mda-7. These findings support an effective dual-combinatorial approach for the therapy of colorectal cancers that employs a unique cancer-specific suppressor gene (mda-7/IL-24) with targeted inhibition of oncogene (ras) expression.

Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis.

Melanoma differentiation-associated gene-5 (mda-5) was the first molecule identified in nature whose encoded protein embodied the unique structural combination of an N-terminal caspase recruitment domain and a C-terminal DExD/H RNA helicase domain. As suggested by its structure, cumulative evidences documented that ectopic expression of mda-5 leads to growth inhibition and/or apoptosis in various cell lines. However, the signaling pathways involved in mda-5-mediated killing have not been elucidated. In this study, we utilized either genetically modified cloned rat embryo fibroblast cells overexpressing different functionally and structurally distinct oncogenes or human pancreatic and colorectal carcinoma cells containing mutant active ras to resolve the role of the Ras/Raf signaling pathway in mda-5-mediated growth inhibition/apoptosis induction. Rodent and human tumor cells containing constitutively activated Raf/Raf/MEK/ERK pathways were resistant to mda-5-induced killing and this protection was antagonized by intervening in this signal transduction cascade either by directly inhibiting ras activity using an antisense strategy or by targeting ras-downstream factors, such as MEK1/2, with the pharmacological inhibitor PD98059. The present findings provide a further example of potential cross-talk between growth-inhibitory and growth-promoting pathways in which the ultimate balance of these factors defines cellular homeostasis, leading to survival or induction of programmed cell death.

Ionizing radiation enhances therapeutic activity of mda-7/IL-24: overcoming radiation- and mda-7/IL-24-resistance in prostate cancer cells overexpressing the antiapoptotic proteins bcl-xL or bcl-2.

Subtraction hybridization applied to terminally differentiating human melanoma cells identified mda-7/IL-24, a cytokine belonging to the IL-10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 (Ad.mda-7) provokes apoptosis selectively in a wide spectrum of cancers in vitro in cell culture, in vivo in human tumor xenograft animal models and in patients with advanced carcinomas and melanomas. In human prostate cancer cells, a role for mitochondrial dysfunction and induction of reactive oxygen species in the apoptotic process has been established. Ectopic overexpression of bcl-xL and bcl-2 prevents these changes including apoptosis induction in prostate tumor cells by Ad.mda-7. We now document that this resistance to apoptosis can be reversed by treating bcl-2 family overexpressing prostate tumor cells with ionizing radiation in combination with Ad.mda-7 or purified GST-MDA-7 protein. Additionally, radiation augments apoptosis induction by mda-7/IL-24 in parental and neomycin-resistant prostate tumor cells. Radiosensitization to mda-7/IL-24 is dependent on JNK signaling, as treatment with the JNK 1/2/3 inhibitor SP600125 abolishes this effect. Considering that elevated expression of bcl-xL and bcl-2 are frequent events in prostate cancer development and progression, the present studies support the use of ionizing radiation in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in prostate cancer, particularly in the context of tumors displaying resistance to radiation therapy owing to bcl-2 family member overexpression.

[Hospitalization for morbidity with respiratory diseases in residents of an industrial city]. / Gospitalizirovannaia zabolevaemost' po bolezniam organov dykhaniia naseleniia promyshlennogo goroda.

In view of the growth of adverse epidemiological indices for respiratory diseases (RD) in Russia, studies are warranted to get insights into regularities of their dynamics in a large industrial urban centre. A paradoxical decline is shown in the level of RD morbidity, caused by economical problems of health care with the growing need for hospitalization still in place, as exemplified by the situation in Orenburg. Of a serious concern to us is also an increasing latent RD morbidity resulting in disability of patients.

Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties.

Abnormalities in cellular differentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell differentiation. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful effects occur when mda-7 is expressed in normal epithelial or fibroblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Hu-mda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-beta plus MEZ. Mda-7 expression is also induced during megakaryocyte differentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-beta+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-beta+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in significant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a differentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers.

A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells.

Pancreatic cancer is an extremely aggressive neoplasm whose incidence equals its death rate. Despite intensive analysis, the genetic changes that mediate pancreatic cancer development and effective therapies for diminishing the morbidity associated with this disease remain unresolved. Through subtraction hybridization, we have identified a gene associated with induction of irreversible growth arrest, cancer reversion, and terminal differentiation in human melanoma cells, melanoma differentiation associated gene-7 (mda-7). Ectopic expression of mda-7 when using a recombinant adenovirus, Ad.mda-7, results in growth suppression and apoptosis in a broad spectrum of human cancers with diverse genetic defects, without exerting deleterious effects in normal human epithelial or fibroblast cells. Despite the apparently ubiquitous antitumor effects of mda-7, pancreatic carcinoma cells are remarkably refractory to Ad.mda-7 induced growth suppression and apoptosis. In contrast, the combination of Ad.mda-7 with antisense phosphorothioate oligonucleotides, which target the K-ras oncogene (a gene that is mutated in 85 to 95% of pancreatic carcinomas), induces a dramatic suppression in growth and a decrease in cell viability by induction of apoptosis. In mutant K-ras pancreatic carcinoma cells, programmed cell death correlates with expression and an increase, respectively, in MDA-7 and BAX proteins and increases in the ratio of BAX to BCL-2 proteins. Moreover, transfection of mutant K-ras pancreatic carcinoma cells with an antisense K-ras expression vector and infection with Ad.mda-7 inhibits colony formation in vitro and tumorigenesis in vivo in nude mice. These intriguing observations demonstrate that a combinatorial approach, consisting of a cancer-specific apoptosis-inducing gene and an oncogene inactivation strategy, may provide the foundation for developing an effective therapy for pancreatic cancer.

Antisense oligonucleotides in cancer: recent advances.

The activation of dominant oncogenes and inactivation of tumour suppressor genes may result in cancer. These genetic events may represent novel targets for cancer therapy. Antisense nucleic acids can be used to modulate the expression of selected genes, and to suppress malignant behaviour in cancer cells. Nevertheless, in practice, the selection of suitable antisense targets still remains a trial-and-error procedure. Promising targets for antisense cancer therapy that have been extensively studied include proteases and protease receptors, telomerase, fusion genes, the Bcl family of proteins and various protein kinases. Combinations of antisense oligonucleotides with cytotoxic agents offer important advantages in cancer therapy. However, control oligonucleotides must be carefully chosen to separate the antisense effect from the many potential nonspecific effects. Several antisense drugs have been very effective in in vitro experiments, and have entered clinical trials. Successive generations of antisense drugs, including molecules with novel backbones or other structural modifications, chimeric oligonucleotides and peptide nucleic acids, are currently in development.

[Prevalence of ear diseases in children under 7 years of age in Orenburg]. / Rasprostranennost' patologii ukha u detei pervykh semi let zhizni v Orenburge.

Incidence rate of otitis media in children under 7 years of age by the number of outpatient consultations in Orenburg, implications of some social factors in this rate and in growing number of cases of chronic otitis media are analysed.

[Comprehensive assessment of the work of physicians at clinico-diagnostic laboratories of therapeutic-prophylactic institutions]. / Kompleksnaia otsenka truda vrachei kliniko-diagnosticheskikh laboratorii lechebno-profilakticheskikh uchrezhdenii.

Presents a system of quantitative criteria for comprehensive assessment of the labor of laboratory physicians based on the results of a comparative quantitative analysis of the actual labor, standards, and mean values. The labor of a laboratory physician is assessed by its quantity (intensity), difficulty, and quality, and an integral value is derived. A comprehensive approach, relative simplicity, and use of modern reference values make this assessment objective and help improve the labor organization for certain workplaces and for the whole laboratory.

[A new method of covalent immobilization of oligodeoxyribonucleotides on nylon membranes for hybridization with nucleic acids]. / Novyi metod kovalentnoi immobilizatsii oligodezoksiribonukleotidov na neilonovykh membranakh dlia gibridizatsii s nukleinovymi kislotami.

A new method of covalent immobilization of oligodeoxyribonucleotides on nylon membranes which contain surface amino groups was developed. The method consists in condensation between the amino group of the membrane and the carboxyl group of modified oligonucleotide by means of 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide. The carboxyl group was introduced into the oligonucleotide by means of postsynthetic attachment of peptide (reduced glutathione) at the terminal phosphate group of the oligonucleotide, using the N-hydroxybenzotriazole method of phosphate activation. Membranes containing a covalently immobilized 23-membered oligonucleotide were tested in hybridization with complementary oligonucleotide, and with single-stranded DNA of bacteriophage M13 which has a complementary sequence. The method of covalent immobilization is very simple and convenient. The membranes with covalently immobilized oligonucleotides may be used not only in hybridization analysis, but also for purification of nucleic acids and proteins which recognize nucleotide sequences and in sense biotechnology.

[A new method of nonradioactive labelling of oligonucleotides and their use as allele-specific probes for detecting mutations causing beta-thalassemia]. / Novyi metod neradioaktivnogo mecheniia oligonukleotidov i ispol'zovaniie ikh v kachestve allel'-spetsificheskikh zondov dlia viavleniia mutatsii vyzyvaiushchikh beta-talassemiiu.

We have developed simple and efficient methods for synthesis of biotin and horseradish peroxidase (HRP)-labeled oligonucleotides. Biotinylated oligonucleotides were obtained in quantitative yields, and oligonucleotide conjugates with HRP in 60-80% yields. Allele-specific oligonucleotide probes for the diagnostics of IVS 1-110 mutation in the beta-globin gene causing beta-thalassemia were thus obtained. Temperature conditions for the non-radioactive ASO hybridization with the amplified segment of the human beta-globin gene and wash conditions were selected. HRP-labelled probes were used in hybridization without preliminary separation after synthesis. To decrease nonspecific enzyme binding we have elaborated special conditions for membrane blocking. Detection of the biotinylated probe was carried out with the help of a streptavidin--HRP conjugate. O-Dianisidine was used as a chromogenic substrate. We have demonstrated the usefulness of this method in the analysis of amplified samples of DNA obtained from blood of patients homozygous in the mutant gene, and heterozygous carriers.

[Design of a universal biotin-containing oligonucleotide diagnostic kit for detecting viroid plant diseases]. / Konstruirovanie universal'nogo biotinsoderzhashchego oligonukleotidnogo diagnostikuma dlia vyiavleniia viroidnykh zabolevanii rastenii.

A non-radioactive diagnosticum for plant viroid diseases has been designed, based on hybridization of a biotin-labeled 26-member oligonucleotide probe with the viroid RNA site identical for potato spindle tuber viroid and chrysanthemum stunt viroid. The biotin label has been introduced into the synthetic oligonucleotide probe by the high-yield acylation of the oligonucleotide aminoalkylamide with the biotin imidazolide or N-hydroxysuccinimide ester. Hybridization techniques have been elaborated for nucleic acids isolated from plant sap. The hybrids obtained have been detected with streptavidin and biotinylated alkaline phosphatase or with the covalent conjugate of streptavidin and alkaline phosphatase, the sensitivity being as low as 1 ng. The methodology used can be applied for revealing viroids and for large scale and quick investigation of plant cell cultures.

[Determination of potato spindle tumor viroid and chrysanthenum stund viroid using biotinylated olideoxyribonucleotides]. / Opredelenie viroidov veretenovidnosti klubnei kartofelia i karlikovosti krizantem s ispol'zovaniem biotinilirovannogo oligodezoksiribonukleotida.

A 26 base long oligodeoxyribonucleotide complementary to a common RNA sequence of potato spindle tuber viroid (PSTV) and chrysantemum stunt viroid (CSV) was synthesized. The 3'-end biotinylated one was used for the detection of PSTV and CSV RNA immobilized on nitrocellulose filters by nucleic acid hybridization. Visualization of hybridization results was performed by two ways, either by streptavidin-alkaline phosphatase conjugate or streptavidine and biotinylated alkaline phosphatase. It was possible to detect 0.65 ng of purified CSV and PSTV RNA. The suggested system of viroid diseases detection can be used by agricultural and horticultural enterprises.

[Testing of G----A mutation in position 110 of a minor intron of beta-globin genes in patients with thalassemia in Azerbaijan]. / Testirovanie mutatsii G----A v 110-m polozhenii malogo introna beta-globinovogo gena u bol'nykh talassemiei iz Azerbaidzhana.

A point mutation G-A in the 110 position of the beta-globin gene small intron has been revealed by cloning and sequencing from the material of a homozygote beta-thalassemia patient in Azerbaijan. In the present study two allele-specific oligonucleotide probes for testing the mutation have been synthesized. Assessment frequency of the mutation among the beta-thalassemia patients in Azerbaijan has been performed with the use of the amplified beta-globin gene fragments obtained by using the thermostable DNA-polymerase from T. thermophilus with the subsequent dot-hybridization in gel of the amplified material with the oligonucleotide probes. The possibility to test the mutation by hybridization of the oligonucleotide probes with the donors and beta-thalassemia patients restricted genomic DNA has been analyzed. Only one of 50 thalassemia alleles of beta-globin genes under study has been shown to possess the mutation mentioned.