Synovial Sarcoma Microvesicles Harbor the SYT-SSX Fusion Gene Transcript: Comparison of Different Methods of Detection and Implications in Biomarker Research.

Background. Synovial sarcoma is an aggressive soft-tissue malignancy. This study examines the presence of the SYT-SSX fusion transcript in synovial sarcoma microvesicles as well as its potential role as a biomarker for synovial sarcoma. Patients and Methods. Microvesicle release of synovial sarcoma cells was examined by transmission electron microscopy. RNA-content was analyzed by qPCR, nested PCR, nested qPCR, and droplet digital PCR to compare their sensitivity for detection of the SYT-SSX fusion gene transcript. Whole blood RNA, RNA of mononuclear cells, and microvesicle RNA of synovial sarcoma patients were analyzed for the presence of the fusion gene transcripts. Results. Electron microscopic analysis revealed synovial sarcoma cells releasing membrane-enclosed microvesicles. In vitro, the SYT-SSX fusion gene transcript was detected in both synovial sarcoma cells and microvesicles. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA. In contrast, the fusion gene transcript was not detected in peripheral blood cells and microvesicles of synovial sarcoma patients. Conclusion. Synovial sarcoma cells release microvesicles harboring the SYT-SSX fusion transcript. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA; however, more sensitive assays are needed to detect cancer-specific microvesicles in the peripheral blood of cancer patients.

A novel method of HPV genotyping using Hybrid Capture sample preparation method combined with GP5+/6+ PCR and multiplex detection on Luminex XMAP.

A novel DNA detection assay comprising Hybrid Capture sample preparation, GP5+/6+ PCR with modifications and Luminex 100 detection was developed and applied to genotyping of human papillomavirus (HPV) in cervical samples. Target-specific sample preparation was performed using magnetic beads conjugated with Hybrid Capture (HC) antibody. DNA-RNA hybrids were formed between DNA target and RNA probes and captured on HC-beads. DNA on magnetic beads was amplified without elution using consensus GP5+/6+ PCR and then genotyped on Luminex beads using hybridization probes for the 17 high-risk HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, 82 and an internal control. This new sequence-specific Hybrid Capture sample preparation is fast, efficient and allows direct HPV genotyping by PCR. Compared to traditional non-sequence-specific sample preparation methods, HC sample preparation demonstrated slightly better detection of multiple HPV infections. The clinical utility of this method was demonstrated on cervical samples positive for HR HPV by the Hybrid Capture 2 (hc2) screening assay.

[Development of postresuscitation morphological changes of hippocampal and cerebellar neurons: common regularities and specific features]. / Razvitie postreanimatsionnykh morfologicheskikh izmenenii neironov gippokampa i mozzhechka: obshchie zakonomernosti i osobennosti.

A comparative morphometric study of postresuscitation changes in the neuronal populations of the pyramidal cells from hyppocampal sector CA1 and Purkinje cells of the lateral cerebellar region in the course of postresuscitation period after 12-minute cardiac arrest in rats has shown that the changes differ in severity and pattern. In the pyramidal cells there were reversible dystrophic alterations of the neurons. Purkinje cells showed death of some neurons, this process progressed in the course of postresuscitation period. A positive effect of the peptide kyotorphin on the brain condition after resuscitation was found but its efficacy in different neuronal populations varied.

[Effect sandostatin on functional and structural recovery of the central nervous system after clinical death]. / Vliianie sandostatina na funktsional'no-strukturnoe vosstanovlenie tsentral'noi nervnoi sistemy posle klinicheskoi smerti.

A behavior examination set was used to reveal a decreased anxiety as well as increased locomotor and exploratory activities and changes in resuscitated animals undergoing different learning tests. A single injection of sandostatin--an analogue of regulatory peptide somatostatin--was found to improve the structural and functional recovery of the central nervous system after a 12-minute cardiac arrest.

Postresuscitation recovery of functional activity of central nervous system in rats during combination treatment with mexidol and neuropeptides delta sleep-inducing peptide and oxytocin.

In experiments on rats we studied the effects of antioxidant and membrane-protecting agent mexidol and neuropeptides delta sleep-inducing peptide and oxytocin administered during resuscitation after 12-min clinical death. Individual and combination treatment with these substances accelerated recovery of the neurological status and partially or completely corrected behavioral disorders associated with changes in the emotional and motivational status. Combined administration of mexidol and oxytocin most significantly promoted postresuscitation recovery of functional activity in the central nervous system.

[Therapy of postresuscitation behavioral disorders with mexidol and kyotorphin]. / Korrektsiia postreanimatsionnykh narushenii povedencheskikh reaktsii meksidolom i kiotorfinom.

A complex of behavioral tests revealed diminished anxiety, increased locomotor and exploratory activities, and changes in different learning tests in resuscitated animals. Mexidol alone and in combination with kyotorphin exhibited antistressogenic and nootropic activities, and led to a compensation of ischemic lesions in rats. It can be stated that a the injection of mexidol in combination with kyotorphin yielded better results due to its neuroprotective effect in the CA1 and CA4 fields of the hippocampus.

[Effect of perftoran on post-resuscitation recovery of the central nervous system]. / Vliianie perftorana na postreanimatsionnoe vosstanovlenie tsentral'noi nervnoi sistemy.

The effect of perfluorane on survival and restorative process in the brain were studied in rats subjected to 12-min arrest of systemic circulation. Perfluorane in a single dose of 5-10 ml/kg was injected intraperitoneally 30 min after the beginning of reanimation. The drug did not affect the postreanimation death of animals and time course of neurologic deficiency disappearance. Perfluorane activated behavioral reactions and prevented development of dystrophic changes in the brain structures of rats highly sensitive to hypoxia.

[Functional-morphologic evaluation of the effect of the regulatory peptide kyotorphin on the status of the CNS in the post-resuscitation period]. / Funktsional'no-morfologicheskaia otsenka vliianiia reguliatornogo peptida kiotorfina na sostoianie TsNS v postreanimatsionnom periode.

Functional and morphological analysis of changes in white rat CNS was made throughout 4 months after 12 minute interruption of circulation. Within 3 months after clinical death and resuscitation the rats' neurological status was normal except some abnormalities in emotions and motives especially noticeable under stress. Moreover, neurons in the hippocampus and cerebellum were destroyed. A single intranasal 0.05 mg/kg dose of kyotorphin--a peptide isolated from the brain of winter-sleeping animals--given 30 min after beginning of resuscitation increased survival, accelerated restoration of neurological status, normalized emotional reactivity, orientation and search behavior, prevented death of neurons in the hippocamp and cerebellum.

Detection of telomerase activity utilizing energy transfer primers: comparison with gel- and ELISA-based detection.

We have developed a closed-tube format telomeric repeat amplification protocol (TRAP) assay for direct quantification of telomerase activity within the PCR vessel. The assay utilizes energy transfer (ET) primers, which emit fluorescence only upon incorporation into PCR products. This novel ET primer system (Amplifluor primers) has major advantages over existing detection methods because it eliminates the need for post-PCR processing and thus reduces greatly the risk of carryover contamination and the time required for the sample analysis. The assay is as sensitive, specific and quantitative as the polyacrylamide gel-based or ELISA-based TRAP assay.

In situ amplification using universal energy transfer-labeled primers.

We developed an amplification detection system in which a universal energy transfer-labeled primer (UniPrimer) is used in combination with any target-specific primer pair. The target specific primers each have a 5' tail sequence, which is homologous to the 3' end of the UniPrimer which, in turn, has a hairpin structure on the 5' end. The hairpin structure brings the fluorophore and quencher into close proximity when the primer is free in solution, providing efficient quenching. When the primer is incorporated into the PCR product, the hairpin structure is unfolded and a fluorescent signal can be detected. Using hepatitis C and human papillomavirus as model systems, this study demonstrates several advantages in the hot-start in situ PCR technique with the UniPrimer system, including target specific detection of one DNA copy per cell without a separate in situ hybridization step and detection of an RNA target by RT in situ PCR without overnight DNase digestion. The UniPrimer-based in situ PCR allows rapid and simple detection of any DNA or RNA target without concern for the background from DNA repair invariably evident in paraffin-embedded tissue when a labeled nucleotide is used.

[The apoB genotype and the efficacy of hypolipidemic therapy]. / Genotip apoB i éffektivnost' gipolipidemicheskoi terapii.

Several men were examined for association between restriction fragment length polymorphism (RELP) Xba I (exon 26) number of tandem repeats in 3'-hypervariable region of the apolipoprotein-B gene and serum levels of cholesterol and triglyceride. These two types of polymorphism were studied. An association of the Xba I site and alleles containing more repeats in the 3'-hypervariable region with higher cholesterol and triglyceride was observed. 32 patients with CHD aged 24-56 years were examined. All the patients are males with clinical picture of CHD (stable angina pectoris of II-III functional classes) and dyslipoproteinemia of II a, II b and IV types by D. S. Fredrickson. Xba-I polymorphism of apo-B gene was detected by DNA polymerase reaction method. The following Xba-I genotypes were distinguished: X1X1 (absence of Xba I site); X1X2 (heterozygosity on Xba I site) and X2X2 (homozygosity on Xba-I site). Lipantil (fenofibratte) was prescribed in a dose of 300 mg daily after meals (100 mg three times a day). Data obtained show that DNA polymorphism of apo-B gene not only influences plasma lipids concentration but also determines effectiveness of hypolipidemic therapy. Hypolipidemic effect of lipantil depends on Xba-I site presents in apo-B gene and is significantly expressed in homozygous patients with X1X1 genotype.

A closed tube format for amplification and detection of DNA based on energy transfer.

A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.

[The effect of beta-casomorphin-7 on the level of food and defense motivations in different types of learning]. / Vliianie beta-kazomorfina-7 na uroven' pishchevoi i oboronitel'noi motivatsii pri razlichnykh vidakh obucheniia.

The effects of intraperitoneal administration of the heptapeptide beta-casomorphin-7 (beta-C-7) on learning were investigated in white rats using T-maze, active and passive avoidance tests. The substance injected in the dose of 5 mg/kg 5 min prior to training accelerated the acquisition of food-reinforced task increasing the number of correct trials in T-maze. The same injection in the dose of 1 mg/kg impaired the acquisition of avoidance tasks. The heptapeptide didn't influence food motivation and consolidation of memory trace. The data obtained support the idea that beta-C-7 attenuated manifestations of defensive motivation.

Defining a smaller RNA substrate for elongation factor Tu.

A nuclease protection assay was used to obtain equilibrium dissociation constants of Thermus thermophilus EF-Tu with two well-characterized internal deletions of Escherichia coli Ala-tRNA(Ala) and yeast Phe-tRNA(Phe). Aminoacylated tRNAs with the anticodon hairpin substituted by a tetranucleotide bind to EF-Tu as well as the corresponding full-sized tRNAs. However, the Ala minihelix, where residue A7 is joined directly to A49, binds to EF-Tu less well than the full-sized Ala-tRNA(Ala). Similar data were obtained for Escherichia coli EF-Tu. An in vitro selection strategy was used to isolate a substrate for EF-Tu from an RNA library where nine random nucleotides inserted between A7 and A49 in the Ala minihelix. After six rounds of enrichment, two groups of RNA were obtained that bound T. thermophilus EF-Tu as well as Ala-tRNA(Ala). Group I molecules have the consensus sequence UNDUGACUY (N = U, C, A, G; D = U, G; Y = U, C) in the randomized region, and Group II molecules generally have 5'-terminal GUG, but are more variable in the remaining six nucleotides. The selected RNAs bind EF-Tu better than the minihelix either because they provide additional function groups for protein binding or because they have a structure more similar to the aminoacyl acceptor branch of tRNA.

Many of the conserved nucleotides of tRNA(Phe) are not essential for ternary complex formation and peptide elongation.

An RNase protection assay was used to show that the dissociation rate constants and equilibrium constants of unmodified yeast and Escherichia coli phenylalanyl-tRNA(Phes) to elongation factor Tu from E.coli were very similar to each other and to their fully modified counterparts. The affinity of aminoacylated tRNA to elongation factor Tu was substantially lower when GTP analogues were used in place of GTP, emphasizing the importance of the beta-gamma phosphate linkage in the function of G-proteins. Fourteen different mutations in conserved and semi-conserved nucleotides of yeast phenylalanyl-tRNA(Phe) were tested for binding to elongation factor Tu.GTP and assayed for activity in the ribosomal A- and P-sites. Most of the mutations did not severely impair the function of these tRNAs in any of the assays. This suggests that the translational machinery does not form sequence-specific interactions with the conserved nucleotides of tRNA.