Comparison of different methods of molecular-genetic analysis of somatic mutations in K-ras gene in patients with colorectal cancer

Two approaches to somatic point mutations in 12 and 13 codones of K-ras gene were analyzed: PCR/SSCP/ACRS/sequencing and allele-specific PCR in the real-life regimen (Russian set "KRAS-7M"). The comparison was carried out on 62 examples of genomic DNA extracted from frozen colon carcinomas, which underwent manual dissection. The results obtained in two attempts were consistent in 95,2% (N=59). Specificity and sensitivity of K-ras mutations detection using "KRAS-7M" set were 100 and 96,4% respectively, and 94,1 and 100% respectievly using PCR/SSCP/ACRS/automatic sequencing. False positive results were absent when detecting with "KRAS-7M" and accounted for 2 cases (5,9%) when using PCR/SSCP/ ACRS/automatic sequencing. The only false negative response (3,6%) was obtained analyzing mutations using "KRAS-7M".     

Incorporation of scaffolding protein gpO in bacteriophages P2 and P4

Scaffolding proteins act as chaperones for the assembly of numerous viruses, including most double-stranded DNA bacteriophages. In bacteriophage P2, an internal scaffolding protein, gpO, is required for the assembly of correctly formed viral capsids. Bacteriophage P4 is a satellite phage that has acquired the ability to take control of the P2 genome and use the P2 capsid protein gpN to assemble a capsid that is smaller than the normal P2 capsid. This size determination is dependent on the P4 external scaffolding protein Sid. Although Sid is sufficient to form morphologically correct P4-size capsids, the P2 internal scaffolding protein gpO is required for the formation of viable capsids of both P2 and P4. In most bacteriophages, the scaffolding protein is either proteolytically degraded or exits intact from the capsid after assembly. In the P2/P4 system, however, gpO is cleaved to an N-terminal fragment, O(*), that remains inside the mature capsid after DNA packaging. We previously showed that gpO exhibits autoproteolytic activity, which is abolished by removal of the first 25 amino acids. Co-expression of gpN with this N-terminally truncated version of gpO leads to the production of immature P2 procapsid shells. Here, we use protein analysis and mass spectroscopy to show that P2 and P4 virions as well as procapsids isolated from viral infections contain O(*) and that cleavage occurs between residues 141 and 142 of gpO. By co-expression of gpN with truncated gpO proteins, we show that O(*) binds to gpN and retains the proteolytic activity of gpO and that the C-terminal 90 residues of gpO (residues 195-284) are sufficient to promote the formation of P2-size procapsids. Using mass spectrometry, we have also identified the head completion protein gpL in the virions.     

Robust T-tubulation and maturation of cardiomyocytes using tissue-engineered epicardial mimetics

Complex three-dimensional (3-D) heart structure is an important determinant of cardiac electrical and mechanical function. In this study, we set to develop a versatile tissue-engineered system that can promote important aspects of cardiac functional maturation and reproduce variations in myofiber directions present in native ventricular epicardium. We cultured neonatal rat cardiomyocytes within a 3-D hydrogel environment using microfabricated elastomeric molds with hexagonal posts. By varying individual post orientations along the directions derived from diffusion tensor magnetic resonance imaging (DTMRI) maps of human ventricle, we created large (2.5 × 2.5 cm²) 3-D cardiac tissue patches with cardiomyocyte alignment that replicated human epicardial fiber orientations. After 3 weeks of culture, the advanced structural and functional maturation of the engineered 3-D cardiac tissues compared to age-matched 2-D monolayers was evident from: 1) the presence of dense, aligned and electromechanically-coupled cardiomyocytes, quiescent fibroblasts, and interspersed capillary-like structures, 2) action potential propagation with near-adult conduction velocity and directional dependence on local cardiomyocyte orientation, and 3) robust formation of T-tubules aligned with Z-disks, co-localization of L-type Ca²⁺ channels and ryanodine receptors, and accelerated Ca²⁺ transient kinetics. This biomimetic tissue-engineered platform can enable systematic in vitro studies of cardiac structure–function relationships and promote the development of advanced tissue engineering strategies for cardiac repair and regeneration.     

MHV68 Latency Modulates the Host Immune Response to Influenza A Virus

Murine gammaherpesvirus 68 (MHV68) is a natural rodent pathogen that has been used as a model to study the pathogenesis of human gammaherpesviruses. Like other herpesviruses, MHV68 causes acute infection and establishes life-long latency in the host. Recently, it has been shown that mice latently infected with MHV68 have resistance to unrelated pathogens in secondary infection models. We therefore hypothesized that latent MHV68 infection could modulate the host response to influenza A virus. To test this hypothesis, mice were infected intranasally with influenza virus following the establishment of MHV68 latency. Mice latently infected with MHV68 showed significantly higher survival to influenza A virus infection than did PBS mock-infected mice. Latent MHV68 infection led to lower influenza viral loads and decreased inflammatory pathology in the lungs. Alveolar macrophages of mice latently infected with MHV68 showed activated status, and adoptive transfer of those activated macrophages into mice followed the infection with influenza A virus had significantly greater survival rates than control mice, suggesting that activated alveolar macrophages are a key mechanistic component in protection from secondary infections.     

Videothoracosopy in diagnosis and surgical treatment of tuberculosis

From 1993 to May 2001, 795 psychiatric patients were treated including 563 with pleural effusion, 98 with multiple or solitary tuberculoma, 69 with tuberculous empyema, 14 with fibrotic-cavitary tuberculosis, 51 with disseminated tuberculosis. Mean age of the patients was 32 years. Survey of pleural cavity with pleural or lung biopsy was performed in 691 patients. In tuberculoma 14 lobectomies were performed, 23 patients underwent atypical lung resection without stapler (including with precise technique). Bilateral on-stage interventions were made in 32 patients. In empyema necrectomy and lung decortication were performed. 14 patients underwent videocavernoscopy with sequestrectomy and cavity drainage. In disseminated tuberculosis lung biopsy without staplers was usually performed. Aerohemostasis was achieved with plasma stream. One patient with empyema and one patient with pleural effusion died (lethality was 0.25%). Rate of postoperative complications was 7.5% in tuberculoma and 1.5% in disseminated tuberculosis. Conversion to thoracotomy was necessary in 3 (3%) patients with tuberculoma and 12 (17%) patients with empyema. Mean hospital stay was 4 days after diagnostic surgery and 7 days after lung resection. In pleural effusion diagnosis was verified in 98% cases, in disseminated tuberculosis--in 100%. Videothoracoscopy is the best diagnostic method for pleural effusion and disseminated forms of lung tuberculosis and operation of choice in tuberculoma and empyema. Videothoracoscopy in tuberculosis is highly effective and associates with low rate of postoperative complications and lethality.     

Space-time water monitoring system at the Iriklinsk hydroelectric power station

The Microbiosensor B 17677 F test system was applied to make a space-time monitoring of the biotoxicity of water used for production and everyday purposes at the Iriklinsk hydroelectric power station (IHEPS) and to identify the leading causes determining the biotoxicity of tested samples. There were seasonal variations in the biotoxicity with the maximum in spring and with minimum in winter and spring and a relationship of the spring rise in the biotoxicity to water pH changes. There was also an association of the certain values of the biotoxicity of industrial water with the concentration of petroleum products that are major pollutants at the IHEPS. The datum points that characterize the maximum level of technogenic exposure were identified.     

Rarity of CDK4 germline mutations in familial melanoma

To date, two genes have been implicated in melanoma pathogenesis. The first, CDKN2A, is a tumour suppressor gene with germline mutations detected in 20% of melanoma-prone families. The second, CDK4, is an oncogene with co-segregating germline mutations detected in only three kindreds worldwide. We examined 16 American melanoma-prone families for mutations in all coding exons of CDK4 and screened additional members of two previously reported families with the Arg24Cys germline CDK4 mutation to evaluate the penetrance of the mutation. No new CDK4 mutations were identified. In the two Arg24Cys families, the penetrance was estimated to be 63%. Overall, 12 out of 12 invasive melanoma patients, none out of one in situ melanoma patient, five out of 13 dysplastic naevi patients, two out of 15 unaffected family members, and none out of 10 spouses carried the Arg24Cys mutation. Dysplastic naevi did not strongly co-segregate with the Arg24Cys mutation. Thus the phenotype observed in melanoma-prone CDK4 families appears to be more complex than just the CDK4 mutation. Both genetic and environmental factors are likely to contribute to the occurrence of melanoma and dysplastic naevi in these families. In summary, although CDK4 is a melanoma susceptibility gene, it plays a minor role in hereditary melanoma.     

Extravesicle ureterocystoanastomosis

Extravesical ureterocystoanastomosis without wide opening of the urinary bladder was practised since 1986. A total of 79 patients were operated: 71 children at the age from 1 to 14 years and 8 adults aged 14 to 58 years. Surgical intervention was performed on 112 ureters for vesicoureteral reflux and supravesical ureteral obstruction. 6 of 79(7.6%) patients on postoperative day 11-15 after removal of intubating drains had an attack of acute pyelonephritis. 3 of these 6 patients were reoperated with a good effect, in 2 patients urine passage recovered 2-3 months after removal of transcutaneous puncture nephrostoma. In one child this technique was used for the first time at the third operation on vesicoureteral anastomosis. This prevented subsequent removal of the nephrostomic drain because of vesicoureteral reflux in the presence of severe irreversible retention changes of both ureters. The advantages of the above technique are reduced operative injury, no contraindications in primary operations and reoperations, feasibility in various forms of vesicoureteral anastomosis failure. This makes the technique of extravesical ureterocystoanastomosis rather promising.