[Transoral endoscopic thyroid surgery]. / Transoral'nyi éndoskopicheskii dostup k shchitovidnoi zheleze.

OBJECTIVE: To analyze own initial experience of transoral thyroid surgery. MATERIAL AND METHODS: There were 7 patients thyroid nodules who underwent surgery for the period from March 2018 to May 2019. All patients signed an informed consent to be included in the study. Surgical approach was performed through three incisions in the lower arch of the vestibule of the mouth with deployment of 10 mm endoscope and two 5 mm tools. Gas insufflation was used. All patients were females aged 43.3±11.8 years. Thyroidectomy was performed in 2 cases, hemithyroidectomy - in 5 patients. Dimensions of nodules varied from 10 to 42 mm. RESULTS: Mean time of hemithyroidectomy and thyroidectomy was 206.4±63.8 and 232±37.5 min, respectively. Papillary carcinoma was histologically verified in 1 case. Injuries of recurrent laryngeal nerve, postoperative hypocalcemia and local complications were absent. Drainage was not applied. Postoperative hospital-stay was 3.7±1.1 days. CONCLUSION: Transoral approach to the thyroid gland is technically feasible with standard endoscopic instruments, safe for important anatomical structures and more precise due to the optical capabilities of endoscopic equipment. Any types of procedures are possible. Undoubtedly, aesthetic outcome is also favorable.

[Evolution of complex treatment of patients with non-specific pleural empyema]. / Évoliutsiia metodov kompleksnogo lecheniia bol'nykh s nespetsificheskoi émpiemoi plevry.

AIM: To analyze long-term own experience of NPE treatment in view of evolution of surgical sanitation of pleural cavity. MATERIAL AND METHODS: The analysis included 5115 patients with NPE for the last 39 years (1977-2015). Morbidity, features of microflora of purulent exudate, changes in the structure of surgical methods were assessed. The role of computed tomography in the diagnostic algorithm and treatment of NPE was studied. RESULTS: The evolution of NPE surgical management includes introduction of video technologies, thermal surgical instruments and widespread use of computed tomography in the diagnosis of pleural empyema. So, significant reduction of patients who were discharged with residual cavities was observed. Postoperative mortality was 19.5% for the period 1977-1996 when traumatic open surgery was used. At the same time there were no deaths within 1997-2015 due to introduction of VATS pleural drainage. Overall mortality decreased from 4.9% to 3.2% for the same period due to reduced postoperative complications. CONCLUSION: The introduction of minimally invasive technologies, new thermal surgical instruments changed management of NPE patients, reduced the number of traumatic open procedures. So, improved outcomes were achieved.

[Transoral approach to thyroid gland in the experiment]. / Transoral'nyi dostup k shchitovidnoi zheleze v eksperimente (s kommentariem P.S. Vetsheva).

AIM: To develop minimally invasive and safe endoscopic access to thyroid gland. MATERIAL AND METHODS: Transoral pre-mandibular video-assisted gas-free access to thyroid gland was developed in experimental study that included 19 human cadavers. Stereometric modeling defined the evaluation criteria including the form of basal arch of lower jaw and its height. There was no conflict of instruments in working chamber under platysma. Additional trocar was deployed to resolve the conflict between working parts of instruments during thyroid gland mobilization. The angle of operative action between the instruments is close to 90°. Trocar hole is used for drainage. RESULTS: The access provides good visualization of recurrent laryngeal nerve, upper and lower thyroid arteries and parathyroid gland. It is less traumatic compared with other extra-cervical accesses to thyroid glands.

[Transhiatal esophagectomy for cardia and esophagus cancer]. / Transkhiatal'naya ezofagektomiya pri rake kardii i pishchevoda.

AIM: To present the result of transhiatal esophagectomies with simultaneous repair. MATERIAL AND METHODS: The study included 67 procedures. In 35 cases surgery was carried out for adenocarcinoma of distal esophagus or cardia with high transition to esophagus, in 32 cases - for epidermal carcinoma of the esophagus. Gastric graft and left half of the colon were used in 60 and 7 cases respectively for simultaneous repair. 29 patients underwent transhiatal instrumental esophagectomy using author's original technique.

[Risk of hypocalcemia after thyroid surgery].

AIM: To reveal calcium metabolism disorders that frequently occur after thyroid surgery. MATERIAL AND METHODS: The study included 202 patients who underwent thyroid surgery for different diseases and had normal calcium level in peripheral blood at baseline. RESULTS: Based on laboratory data postoperative hypocalcemia was diagnosed in 57 (28.8%) patients. It was not always accompanied by clinical symptoms. Clinical picture depended on degree of hypocalcemia. Symptoms was diagnosed more frequently if calcium concentration was less than 2.1 mmol/l. Clinical manifestations were absent in 64.9% of cases on background of hypocalcemia. Incidence of hypocalcemia was higher after thyroidectomy compared to organ-preserving surgery. Symptoms of hypocalcemia occurred after thyroidectomy only. Casual parathyroidectomy does not always cause hypocalcemia. Only in 14% of patients with hypocalcemia excised parathyroid was identified in specimen. At the same time 7.6% of patients with postoperative normocalcaemia also had excised parathyroids in specimens. Symptoms of hypocalcemia does not always occur at 1 day after surgery. They can appear later, for example at 5 days postoperatively and depend on severity of hypocalcemia. Thyroidectomy has high risk of postoperative hypocalcemia with clinical symptoms (19.6%) that is transient in 15.5% of cases and permanent in 4.1% of patients.

Targeting homologous recombination and telomerase in Barrett's adenocarcinoma: impact on telomere maintenance, genomic instability and tumor growth.

Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in Barrett's esophageal adenocarcinoma (BAC). The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for ß-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller (P=0.001) than those that resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contributes to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase renders telomeres more vulnerable to degradation and significantly increases/expedites their attrition, leading to apoptosis. We therefore demonstrate that a therapy targeting HR and telomerase has the potential to prevent both tumor growth and genomic evolution in BAC.

Telomerase inhibitor GRN163L inhibits myeloma cell growth in vitro and in vivo.

Human telomerase, the reverse transcriptase which extends the life span of a cell by adding telomeric repeats to chromosome ends, is expressed in most cancer cells but not in the majority of normal somatic cells. Inhibition of telomerase therefore holds great promise as anticancer therapy. We have synthesized a novel telomerase inhibitor GRN163L, a lipid-attached phosphoramidate oligonucleotide complementary to template region of the RNA subunit of telomerase. Here, we report that GRN163L is efficiently taken up by human myeloma cells without any need of transfection and is resistant to nucleolytic degradation. The exposure of myeloma cells to GRN163L led to an effective inhibition of telomerase activity, reduction of telomere length and apoptotic cell death after a lag period of 2-3 weeks. Mismatch control oligonucleotides had no effect on growth of myeloma cells. The in vivo efficacy of GRN163L was confirmed in two murine models of human multiple myeloma. In three independent experiments, significant reduction in tumor cell growth and better survival than control mice was observed. Furthermore, GRN163L-induced myeloma cell death could be significantly enhanced by Hsp90 inhibitor 17AAG. These data provide the preclinical rationale for clinical evaluation of GRN163L in myeloma and in combination with 17AAG.

Telomerase inhibitors--oligonucleotide phosphoramidates as potential therapeutic agents.

We have designed, synthesized, and evaluated using physical, chemical and biochemical assays various oligonucleotide N3'-->P5' phosphoramidates, as potential telomerase inhibitors. Among the prepared compounds were 2'-deoxy, 2'-hydroxy, 2'-methoxy, 2'-ribo-fluoro, and 2'-arabino-fluoro oligonucleotide phosphoramidates, as well as novel N3'-->P5' thio-phosphoramidates. The compounds demonstrated sequence specific and dose dependent activity with IC50 values in the sub-nM to pM concentration range.

RNA and N3'-->P5' kissing aptamers targeted to the trans-activation responsive (TAR) RNA of the human immunodeficiency virus-1.

We used in vitro selection to identify RNA aptamers able to selectively bind to the TAR RNA motif of HIV-1, an unperfect RNA hairpin involved in the transcription of the retroviral genome. We selected aptameric RNA hairpins giving rise to kissing complexes with TAR. The N3'-->P5' phosphoramidate variant of the aptamer bind to TAR with a Kd in the low nanomolar range. However, only the RNA-RNA loop-loop complex is recognized by the Rop protein of E. coli which is specific for kissing complexes.

Telomerase can inhibit the recombination-based pathway of telomere maintenance in human cells.

Telomere length can be maintained by telomerase or by a recombination-based pathway. Because individual telomeres in cells using the recombination-based pathway of telomere maintenance appear to periodically become extremely short, cells using this pathway to maintain telomeres may be faced with a continuous state of crisis. We expressed telomerase in a human cell line that uses the recombination-based pathway of telomere maintenance to test whether telomerase would prevent telomeres from becoming critically short and examine the effects that this might have on the recombination-based pathway of telomere maintenance. In these cells, telomerase maintains the length of the shortest telomeres. In some cases, the long heterogeneous telomeres are completely lost, and the cells now permanently contain short telomeres after only 40 population doublings. This corresponds to a telomere reduction rate of 500 base pairs/population doubling, a rate that is much faster than expected for normal telomere shortening but is consistent with the rapid telomere deletion events observed in cells using the recombination-based pathway of telomere maintenance (Murnane, J. P., Sabatier, L., Marder, B. A., and Morgan, W. F. (1994) EMBO J. 13, 4953-4962). We also observed reductions in the fraction of cells containing alternative lengthening of telomere-associated promyelocytic leukemia bodies and extrachromosomal telomere repeats; however, no alterations in the rate of sister chromatid exchange were observed. Our results demonstrate that human cells using the recombination-based pathway of telomere maintenance retain factors required for telomerase to maintain telomeres and that once the telomerase-based pathway of telomere length regulation is engaged, recombination-based elongation of telomeres can be functionally inhibited.

Binding enhancement by tertiary interactions and suicide inhibition of a Candida albicans group I intron by phosphoramidate and 2'-O-methyl hexanucleotides.

Candida albicans is one of many infectious pathogens that are evolving resistance to current treatments. RNAs provide a large class of targets for new therapeutics for fighting these organisms. One strategy for targeting RNAs uses short oligonucleotides that exhibit binding enhancement by tertiary interactions in addition to Watson-Crick pairing. A potential RNA target in C. albicans is the self-splicing group I intron in the LSU rRNA precursor. The recognition elements that align the 5' exon splice site for a ribozyme derived from this precursor are complex [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. These recognition elements have been used to guide design of hexanucleotide mimics of the 5' exon that have backbones modified for nuclease stability. These hexanucleotides bind as much as 100000-fold more tightly to a ribozyme derived from the intron than to a hexanucleotide mimic of the intron's internal guide sequence, r(GGAGGC). Several of these oligonucleotides inhibit precursor self-splicing via a suicide inhibition mechanism. The most promising suicide inhibitor is the ribophosphoramidate rn(GCCUC)rU, which forms more trans-spliced than cis-spliced product at oligonucleotide concentrations of >100 nM at 1 mM Mg(2+). The results indicate that short oligonucleotides modified for nuclease stability can target catalytic RNAs when the elements of tertiary interactions are complex.

A phosphoramidate substrate analog is a competitive inhibitor of the Tetrahymena group I ribozyme.

BACKGROUND: Phosphoramidate oligonucleotide analogs containing N3'-P5' linkages share many structural properties with natural nucleic acids and can be recognized by some RNA-binding proteins. Therefore, if the N-P bond is resistant to nucleolytic cleavage, these analogs may be effective substrate analog inhibitors of certain enzymes that hydrolyze RNA. We have explored the ability of the Tetrahymena group I intron ribozyme to bind and cleave DNA and RNA phosphoramidate analogs. RESULTS: The Tetrahymena group I ribozyme efficiently binds to phosphoramidate oligonucleotides but is unable to cleave the N3'-P5' bond. Although it adopts an A-form helical structure, the deoxyribo-phosphoramidate analog, like DNA, does not dock efficiently into the ribozyme catalytic core. In contrast, the ribo-phosphoramidate analog docks similarly to the native RNA substrate, and behaves as a competitive inhibitor of the group I intron 5' splicing reaction. CONCLUSIONS: Ribo-N3'-P5' phosphoramidate oligonucleotides are useful tools for structural and functional studies of ribozymes as well as protein-RNA interactions.

Synthetic oligonucleotides as RNA mimetics: 2'-modified Rnas and N3'-->P5' phosphoramidates.

Significant interest in synthetic DNA and RNA oligonucleotides and their analogues has marked the past two decades of research in chemistry and biochemistry. This attention was largely determined by the great potential of these compounds for various therapeutic applications such as antisense, antigene and ribozyme-based agents. Modified oligonucleotides have also become powerful molecular biological and biochemical research tools that allow fast and efficient regulation of gene expression and gene functions in vitro and in vivo. These applications in turn are based on the ability of the oligonucleotides to form highly sequence-specific complexes with nucleic acid targets of interest. This review summarizes recent advances in the design, synthesis, biochemical and structural properties of various RNA analogues. These comprise 3'-modified oligonucleotide N3'-->P5' phosphoramidates, analogues with modifications at the 2'-position of nucleoside sugar rings, or combinations of the two. Among the properties of the RNA minetics reviewed here are the thermal stability of their duplexes and triplexes, hydrolytic resistance to cellular nucleases and biological activity in in vitro and in vivo systems. In addition, key structural aspects of the complexes formed by the RNA analogues, including interaction with water molecules and ions, are analyzed and presented.

Contributions of individual nucleotides to tertiary binding of substrate by a Pneumocystis carinii group I intron.

Pneumocystis carinii is a mammalian pathogen that infects and kills immunocompromised hosts such as cancer and AIDS patients. The LSU rRNA precursor of P. carinii contains a conserved group I intron that is an attractive drug target because humans do not contain group I introns. The oligonucleotide r(AUGACU), whose sequence mimics the 3'-end of the 5'-exon, binds to a ribozyme derived from the intron with a K(d) of 5.2 nM, which is 61000-fold tighter than expected from base-pairing alone [Testa, S. M., Haidaris, G. C., Gigliotti, F., and Turner, D. H. (1997) Biochemistry 36, 9379-9385]. Thus, oligonucleotide binding is enhanced by tertiary interactions. To localize interactions that give rise to this tertiary stability, binding to the ribozyme has been measured as a function of oligonucleotide length and sequence. The results indicate that 4.3 kcal/mol of tertiary stability is due to a G.U pair that forms at the intron's splice junction. Eliminating nucleotides at the 5'-end of r(AUGACU) does not affect intron binding more than expected from differences in base-pairing until r((___)ACU), which binds much more tightly than expected. Adding a C at the 5'- or 3'-end that can potentially form a C-G pair with the target has little effect on binding affinity. Truncated oligonucleotides were tested for their ability to inhibit intron self-splicing via a suicide inhibition mechanism. The tetramer, r((__)GACU), retains similar binding affinity and reactivity as the hexamer, r(AUGACU). Thus oligonucleotides as short as tetramers might serve as therapeutics that can use a suicide inhibition mechanism to inhibit self-splicing. Results with a phosphoramidate tetramer and thiophosphoramidate hexamer indicate that oligonucleotides with backbones stable to nuclease digestion retain favorable binding and reactivity properties.

Study of HIV-2 primer-template initiation complex using antisense oligonucleotides.

HIV-2 reverse transcription is initiated by the retroviral DNA polymerase (reverse transcriptase) from a cellular tRNALys3 partially annealed to the primer binding site in the 5'-region of viral RNA. The HIV-2 genome has two A-rich regions upstream of the primer binding site. In contrast to HIV-1 RNA, no direct evidence of interactions with the U-rich anticodon loop of tRNALys3 has been described to date. Here we address the question of the potential role of the interactions between these highly structured regions in the initiation of viral DNA synthesis. To evaluate this we used an antisense approach, first validated in our in vitro HIV-1 reverse transcription system. Annealing of the antisense oligonucleotides to the pre-primer binding site (the upstream region contiguous to the HIV-2 primer binding site) was determined in the presence of native tRNALys3 or synthetic primers. Using natural and chemically modified antisense oligonucleotides we found that interactions between the anticodon of tRNALys3 and an A-rich loop of viral RNA led to an important destabilization of the pre-primer binding site; this region became accessible to anti-pre-primer binding site oligonucleotides in a cooperative manner. These studies allowed to identify an A-rich region in HIV-2ROD RNA capable of interacting with tRNALys3. Better knowledge of these interactions is very important for understanding the primer/template positioning in the early steps of HIV-2 reverse transcription.

A remarkable stabilization of complexes formed by 2,6-diaminopurine oligonucleotide N3'-->P5' phophoramidates.

2'-Deoxyribo- and ribo-oligonucleotide N3'-->P5'phosphoramidates containing 2,6-diaminopurine nucleosides were synthesized. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed by these compounds with DNA and RNA complementary strands, relative to adenosine-containing phosphoramidate counterparts. The increase in melting temperature of the complexes reached up to 6.9 degrees C per substitution. The observed stabilization was attributed to the apparent synergistic effects of N-type sugar puckering of the oligonucleotide N3'-->P5' phosphoramidate backbone, and the ability of 2,6-diaminopurine bases to form three hydrogen bonds.

Antisense PNA tridecamers targeted to the coding region of Ha-ras mRNA arrest polypeptide chain elongation.

We have previously described the rational design of mutation-selective antisense oligonucleotides targeted to codon 12 of oncogenic Ha-ras mRNA. In order to further improve the biological efficacy of these unmodified oligonucleotides, we have studied three different classes of modifications: peptide nucleic acid backbone (PNA), sugar modification (2'-O-methyl) and phosphoramidate linkage (PN). We show that PNA is unique among the investigated steric blocking agents in its ability to specifically inhibit the translation of Ha-ras mRNA in vitro. The PNA-RNA hybrid (Tm=86 degrees C), which is not dissociated by cellular proteins and resists phenol extraction and urea denaturing conditions, specifically blocks the translation of mutated Ha-ras mRNA. A PNA tridecamer which forms with wild-type Ha-ras mRNA a duplex with a central mismatch had little effect on mRNA translation. Codon 12 is located close to the translation initiation site and hybridization of the PNA at this position may interfere with the assembly of the translation initiation complex. To test whether polypeptide chain elongation can also be blocked, we have targeted PNA tridecamers to codons in the 74, 128 and 149 regions. These PNAs form equally stable duplexes as that formed by the PNA targeted to the codon 12 region (ten G.C base-pairs out of 13). We show that PNA-RNA duplexes block the progression of the 80 S ribosome. Therefore, it is possible to arrest translation with concomitant production of a truncated protein by using duplex-forming PNA oligonucleotides targeted to a G+C-rich sequences. Our data demonstrate for the first time that a non-covalent duplex can arrest the translation machinery and polypeptide chain elongation.

Inhibition of IL-6 in mice by anti-NF-kappaB oligodeoxyribonucleotide N3'-->oligodeoxyribonnucleotide N3' --> P5' phosphoramidates.

Oligonucleotide N3'->P5' Phosphoramidates (PN) may confer advantages over unmodified phosphodiester compounds for therapeutic applications (1). Previous in vitro data demonstrated that PN Oligodeoxynucleotides (ODNs) possess several advantageous features, including RNase H-independence, an improved resistance to nuclease degradation, decreased protein binding, and high affinity sequence-specific binding to complementary RNAs (1, 2). Consequently, we undertook a study to investigate the effects of PN antisense (AS) oligos targeted against the p65 subunit of the Nuclear Factor Kappa beta (NF-kappaB) transcription factor in vivo, in mice. The ability of the antisense molecules to inhibit IL-6 elevation induced by lipopolysaccharide (LPS) in mice, was studied. A 16 mer uniformly modified PN and a chimeric phosphoramidate-phosphodiester oligodeoxynucleotide complementary to the region surrounding the starting codon, (PN-PO-PN) of the NK-kappaB p65 subunit mRNA, both caused a sequence specific reduction of the serum IL-6 level in mice. A scrambled oligodeoxynucleotide showed much lower IL-6 inhibition in mice. These results show that the p65 PN-AS can modulate expression of IL-6 in mice without uptake enhancers and therefore may be a useful prototype for RNAse-H independent therapeutic agents.

Synthesis and properties of RNA analogs-oligoribonucleotide N3'-->P5' phosphoramidates.

The synthesis and characterization of RNA mimetics, uniformly modified oligoribonucleotide N3'-->P5' phosphoramidates containing all four natural bases (uracil, cytosine, adenine and guanine) as well as thymidine and 2,6-diaminopurine, are described. These RNA analogs contain N3'-->P5' phosphoramidate internucleotide linkages which replaced natural RNA O3'-->P5' phosphodiester groups. These oligonucleo-tides were constructed from novel monomeric units (2'- t -butyldimethylsilyl)-3'-(monomethoxyltrityl)-amino-nucleoside-5'- phos phoramidites, the preparation of which is also presented. Several mixed base 9-13mer oligoribonucleotide phosphoramidates were synthesized with step-wise coupling yields of 96-98%. Thermal denaturation experiments demonstrated that ribo-N3'-->P5' phosphoramidates form stable duplexes with a complementary RNA strand. Thus, the melting temperature ( T (m)) of a duplex formed by a 13mer ribo-N3'-->P5' phosphoramidate (84 degrees C) was higher than that observed for the isosequential natural RNA oligomer (64.0 degrees C), or for the 2'-deoxy-N3'-->P5' phosphoramidate counterpart (71.7 degrees C). Moreover, substitution of adenine by 2, 6-diaminopurine in an oligoribophosphoramidate pentamer resulted in a very significant increase in the duplex melting temperature ( approximately 7 degrees C per base substitution). The RNA phosphoramidates also showed similar rates of hydrolysis by both RNase A and RNase T(1)as compared to natural RNA oligomers. The data presented indicate that this class of RNA analogs may be used as structural and functional RNA mimetics.

In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N3' --> P5' phosphoramidate hexanucleotide.

Binding enhancement by tertiary interactions is a strategy that takes advantage of the higher order folding of functionally important RNAs to bind short nucleic acid-based compounds tightly and more specifically than possible by simple base pairing. For example, tertiary interactions enhance binding of specific hexamers to a group I intron ribozyme from the opportunistic pathogen Pneumocystis carinii by 1,000- to 100,000-fold relative to binding by only base pairing. One such hexamer, d(AnTnGnAnCn)rU, contains an N3' --> P5' phosphoramidate deoxysugar-phosphate backbone (n) that is resistant to chemical and enzymatic decay. Here, it is shown that this hexamer is also a suicide inhibitor of the intron's self-splicing reaction in vitro. The hexamer is ligated in trans to the 3' exon of the precursor, producing dead-end products. At 4 mM Mg2+, the fraction of trans-spliced product is greater than normally spliced product at hexamer concentrations as low as 200 nM. This provides an additional level of specificity for compounds that can exploit the catalytic potential of complexes with RNA targets.