Mesyl phosphoramidate antisense oligonucleotides as an alternative to phosphorothioates with improved biochemical and biological properties.

Here we describe a DNA analog in which the mesyl (methanesulfonyl) phosphoramidate group is substituted for the natural phosphodiester group at each internucleotidic position. The oligomers show significant advantages over the often-used DNA phosphorothioates in RNA-binding affinity, nuclease stability, and specificity of their antisense action, which involves activation of cellular RNase H enzyme for hybridization-directed RNA cleavage. Biological activity of the oligonucleotide analog was demonstrated with respect to pro-oncogenic miR-21. A 22-nt anti-miR-21 mesyl phosphoramidate oligodeoxynucleotide specifically decreased the miR-21 level in melanoma B16 cells, induced apoptosis, reduced proliferation, and impeded migration of tumor cells, showing superiority over isosequential phosphorothioate oligodeoxynucleotide in the specificity of its biological effect. Lower overall toxicity compared with phosphorothioate and more efficient activation of RNase H are the key advantages of mesyl phosphoramidate oligonucleotides, which may represent a promising group of antisense therapeutic agents.

[Modified Oligonucleotides for Guiding RNA Cleavage Using Bacterial RNase P].

The ability of a series of novel modified external guide sequences (EGS oligonucleotides) to induce the hydrolysis of target RNA with bacterial ribonuclease P has been studied; the most efficient modification variants have been selected. We have found patterns of the oligonucleotide sugar-phosphate backbone modi-fications that enhance oligonucleotide stability in the biological environment and do not violate the ability to interact with the enzyme and induce the RNA hydrolysis. It has been shown that analogues of EGS oligonucleotides selectively modified at 2'-position (2'-O-methyl and 2'-fluoro) or at internucleotide phosphates (phosphoryl guanidines) can be used for the addressed cleavage of a model RNA target by bacterial RNase P. The ability of new phosphoryl guanidine analogues of oligodeoxyribonucleotides that are stable in biological media to induce the hydrolysis of target RNA with bacterial ribonuclease P has been shown for the first time. The modified EGS oligonucleotides with an optimal balance between functional activity and stability in biological media can be considered as potential antibacterial agents.

The economic impact of anastomotic leakage after anterior resections in English NHS hospitals: are we adequately remunerating them?

AIM: Our aim was to determine the frequency and economic impact of anastomotic leakage (AL) at local and national levels in England. METHOD: All patients who underwent AR in Oxford between 2007 and 2009 were evaluated for AL. Hospital Episode Statistics (HES) data were used to determine reoperation rates after elective AR (n = 23 388) in England between 2000 and 2008. Hospital episode remuneration costs were calculated by the local commissioning department and compared with Department of Health (DH) reference index costs. RESULTS: The frequency of AL following anterior resection was 10.9% (31 out of 285) in Oxford. Laparotomy for leakage was performed in 5.6% of cases. The 30-day hospital mortality rate for all ARs was 2.1%, compared with 3.2% after AL. The national relaparotomy rate (within 28 days) and 30-day hospital mortality in English National Health Service (NHS) trusts following AR were 5.9% and 2.9%, respectively. Institutional remunerated tariffs (£6233 (SD ± 965)) were similar to DH reference costs (£6319 (SD ± 1830)) after uncomplicated AR. However, there was a significant (P = 0.008) discrepancy between the remunerated tariff for AL (£9605 (SD ± 6908)) and the actual cost (£17 220 (SD ± 9642)). AL resulted in an additional annual cost of approximately £1.1 million to £3.5 million when extrapolated nationally. CONCLUSION: The estimated economic burden of anastomotic leakage following AR is approximately double that of the remunerated tariff.

"Virtual patch clamp analysis" for predicting the functional significance of pathogenic variants in sodium channels.

OBJECTIVE: Opening of voltage-gated sodium channels is crucial for neuronal depolarization. Proper channel opening and influx of Na+ through the ion pore, is dependent upon binding of Na+ ion to a specific amino-acid motif (DEKA) within the pore. In this study we used molecular dynamic simulations, an advanced bioinformatic tool, to research the dysfunction caused by pathogenic variants in SCN1a, SCN2a and SCN8a genes. METHOD: Molecular dynamic simulations were performed in six patients: three patients with Dravet syndrome (p.Gly177Ala,p.Ser259Arg and p.Met1267Ile, SCN1a), two patients with early onset drug resistant epilepsy(p.Ala263Val, SCN2a and p.Ile251Arg, SCN8a), and a patient with autism (p.Thr155Ala, SCN2a). After predicting the 3D-structure of mutated proteins by homology modeling, time dependent molecular dynamic simulations were performed, using the Schrödinger algorithm. The opening of the sodium channel, including the detachment of the sodium ion to the DEKA motif and pore diameter were assessed. Results were compared to the existent patch clamp analysis in four patients, and consistency with clinical phenotype was noted. RESULTS: The Na+ ion remained attached to DEKA filter longer when compared to wild type in the p.Gly177Ala, p.Ser259Arg,SCN1a, and p.Thr155Ala, SCN2a variants, consistent with loss-of-function. In contrast, it detached quicker from DEKA than wild type in the p.Ala263Val,SCN2a variant, consistent with gain-of-function. In the p.Met1267Ile,SCN1a variant, detachment from DEKA was quicker, but pore diameter decreased, suggesting partial loss-of-function. In the p.Leu251Arg,SCN8a variant, the pore remained opened longer when compared to wild type, consistent with a gain-of-function. The molecular dynamic simulation results were consistent with the existing patch-clamp analysis studies, as well as the clinical phenotype. SIGNIFICANCE: Molecular dynamic simulation can be useful in predicting pathogenicity of variants and the disease phenotype, and selecting targeted treatment based on channel dysfunction. Further development of these bioinformatic tools may lead to "virtual patch-clamp analysis".

Localization in the nucleolus and coiled bodies of protein subunits of the ribonucleoprotein ribonuclease P.

The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors.

Selection of guide sequences that direct efficient cleavage of mRNA by human ribonuclease P.

Any RNA, when in a complex with another oligoribonucleotide known as an external guide sequence (EGS), can become a substrate for ribonuclease P. Simulation of evolution in vitro was used to select EGSs that bind tightly to a target substrate messenger RNA and that increase the efficiency of cleavage of the target by human ribonuclease P to a level equal to that achieved with natural substrates. The most efficient EGSs form transfer RNA precursor-like structures with the target RNA, in which the analog of the anticodon stem has been disrupted, an indication that selection for the optimal substrate for ribonuclease P yields an RNA structure different from that of present-day transfer RNA precursors.

Catalytic activity of an RNA molecule prepared by transcription in vitro.

Ribonuclease P is a ribonucleoprotein that cleaves precursors to transfer RNA (tRNA) molecules to yield the correct 5' terminal sequences of the mature tRNA's. The RNA moiety M1 RNA of ribonuclease P from Escherichia coli and the unprocessed transcript prepared in vitro of the gene for M1 RNA can both perform the cleavage reactions of the canonical enzyme in the absence of the protein moiety. When the transcript of the M1 RNA gene is combined with the protein moiety not only is a tRNA precursor cleaved but also the precursor to 4.5S RNA from Escherichia coli.

External guide sequences for an RNA enzyme.

Ribonuclease P (RNase P) from Escherichia coli or its catalytic RNA subunit can efficiently cleave small RNA substrates that lack the conserved features of natural substrates of RNase P if an additional small RNA is also present. This additional RNA must contain a sequence complementary to the substrate [external guide sequence (EGS)] and a 3'-proximal CCA sequence to ensure cleavage. The aminoacyl acceptor stem and some additional 5'- and 3'-terminal sequences of a precursor transfer RNA are sufficient to allow efficient cleavage by RNAase P, and the 2'-hydroxyl group at the cleavage site is not absolutely necessary for cleavage. In principle, any RNA could be targeted by a custom-designed EGS RNA for specific cleavage by RNase P in vitro or in vivo.

A catalytic RNA and its gene from Salmonella typhimurium.

The gene for the RNA subunit (M1 RNA) of ribonuclease P from Salmonella typhimurium directs the synthesis of an RNA that can cleave transfer RNA precursor molecules. The mature M1 RNA coded for by Salmonella typhimurium is 375 nucleotides long and has six nucleotide changes in comparison to M1 RNA from Escherichia coli. The regions for promotion and termination of transcription are closely conserved, but adjacent regions of nucleotide sequences show considerable drift.

Specific interactions in RNA enzyme-substrate complexes.

Analysis of crosslinked complexes of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli, and transfer RNA precursor substrates has led to the identification of regions in the enzyme and in the substrate that are in close physical proximity to each other. The nucleotide in M1 RNA, residue C92, which participates in a crosslink with the substrate was deleted and the resulting mutant M1 RNA was shown to cleave substrates lacking the 3' terminal CCAUCA sequence at sites several nucleotides away from the normal site of cleavage. The presence or absence of the 3' terminal CCAUCA sequence in transfer RNA precursor substrates markedly affects the way in which these substrates interact with the catalytic RNA in the enzyme-substrate complex. The contacts between wild-type M1 RNA and its substrate are in a region that resembles part of the transfer RNA "E" (exit) site in 23S ribosomal RNA. These data demonstrate that in RNA's with very different cellular functions, there are domains with similar structural and functional properties and that there is a nucleotide in M1 RNA that affects the site of cleavage by the enzyme.

Model substrates for an RNA enzyme.

M1 RNA, the catalytic RNA subunit of Escherichia coli ribonuclease P, can cleave novel transfer RNA (tRNA) precursors that lack specific domains of the normal tRNA sequence. The smallest tRNA precursor that was cleaved efficiently retained only the domain of the amino acid acceptor stem and the T stem and loop. The importance of the 3' terminal CCA nucleotide residues in the processing of both novel and normal tRNA precursors implies that the same enzymatic function of M1 RNA is involved.

Integration and decontamination of Bacillus cereus in Pseudomonas fluorescens biofilms.

AIMS: The hypothesis that surrogate planktonic pathogens (Bacillus cereus and polystyrene microspheres) could be integrated in biofilms and protected from decontamination was tested. METHODS AND RESULTS: Pseudomonas fluorescens biofilms were grown on polyvinyl chloride coupons in annular reactors under low nutrient conditions. After biofilm growth, B. cereus spores and polystyrene microspheres (an abiotic control) were introduced separately. Shear stress at the biofilm surface was varied between 0.15 and 1.5 N m(-2). The amount of surrogate pathogens introduced ranged from approximately 10(5) CFU ml(-1) to 10(10 )spheres ml(-1). The quantity of surrogate pathogens integrated in the biofilm was proportional to the amount introduced. In 14 of the 16 cases, 0.4-3.0% of the spores or spheres introduced were measured in the biofilms. The other two cases had 10% and 21% of the spores detected. Data suggested that the spores germinated in the system. The amount of surrogate pathogens detected in the biofilms was higher in the mid-shear range. Chlorine treatment reduced the quantity of both surrogate pathogens and biofilm organisms. In one experiment, the biofilms and B. cereus recovered when the chlorine treatment was terminated. CONCLUSIONS: Planktonic surrogate pathogens can be integrated in biofilms and protected from chlorination decontamination. SIGNIFICANCE AND IMPACT OF THE STUDY: This knowledge assists in understanding the impact of biofilms on harbouring potential pathogens in drinking-water systems and protecting the pathogens from decontamination.

Reducing catheter infections through use of the CD-1000: a retrospective review of a unique catheter specific composite dressing.

PURPOSE: Catheter-related blood stream infections pose a significant risk for patients living with vascular catheters. The cost to manage these infections is substantial. Although the etiology of these infections is multifactorial, tap water has been implicated as a significant causative factor. This retrospective review evaluates the effectiveness of a surgical dressing, the CD-1000, at protecting catheters and exit site wounds from fluid and debris when patients engage in high risk activities like showering. METHODS: All patients who received the CD-1000 from a single national medical supplier from September 2006 through to March 2007 were contacted to participate in this retrospective review; 209 patients, representing 34 states and 175 unique physicians, participated in this study. Effectiveness of the dressing along with prior and current history of catheter events was queried. RESULTS: The CD-1000 was 95% effective at keeping the catheter and exit site dry while patients engaged in high risk activities like showering. Prior to using the CD-1000, the 209 patients reported a historical catheter infection rate of 1.83 per 1000 catheter days. While using the CD-1000 the 209 patients reported a catheter infection rate of 0.47 per 1000 catheter days. CONCLUSION: The CD-1000 catheter specific composite dressing adequately protects vascular catheters and exit sites when patients engage in high risk activities like showering. In this geographically diverse retrospective review, use of the CD-1000 was associated with a 75% reduction in catheter associated infections.

Characterization of RNase P from Thermotoga maritima.

The protein subunit of RNase P from a thermophilic bacterium, Thermotoga maritima, was overexpressed in and purified from Escherichia coli. The cloned protein was reconstituted with the RNA subunit transcribed in vitro. The temperature optimum of the holoenzyme is near 50 degrees C, with no enzymatic activity at 65 degrees C or above. This finding is in sharp contrast to the optimal growth temperature of T.maritima, which is near 80 degrees C. However, in heterologous reconstitution experiments in vitro with RNase P subunits from other species, we found that the protein subunit from T.maritima was responsible for the comparative thermal stability of such complexes.

Tamoxifen and oophorectomy in the treatment of recurrent breast cancer: a Southwest Oncology Group Study.

The relatively short survival following chemotherapy in patients with metastatic carcinoma of the breast and the introduction of antiestrogens has led to renewed interest in the hormonal therapy of breast cancer. Pritchard et al. (Cancer Treat. Rep., 64: 787-796, 1980) have recently stated that response to the antiestrogen tamoxifen (TAM) strongly predicts a subsequent response to oophorectomy in premenopausal patients. The Southwest Oncology Group administered TAM to pre- and postmenopausal women with first recurrence of breast cancer. Following response and subsequent relapse, or after no response, patients underwent an oophorectomy while continuing on TAM. None of 14 premenopausal patients who responded to TAM had a response to oophorectomy plus TAM, while 5 of 22 had a remission with oophorectomy plus TAM after initially failing with TAM alone. The reverse was seen in postmenopausal women; 4 of 18 responders to TAM subsequently responded to oophorectomy plus TAM, but none of 18 TAM failures responded to oophorectomy plus TAM. These results suggest that in the premenopausal women the TAM dose may be insufficient to block all estrogen action and that oophorectomy, by removing the major source of estrogen, can result in a more effective antiestrogen action of TAM leading to a response. No explanation is readily available for the results in postmenopausal patients.

Identification of ribonuclease P activity from chick embryos.

RNAase P (EC 3.1.26.5) activity has been identified in chick embryo thigh tissue on the basis of specific cleavage of Escherichia coli 129 nucleotide tRNATyr precursor and has been partially purified by the procedure used for human tissue culture KB cell RNAase P. RNAase P from chick resembles the KB cell RNAase P in substrate specificity, requirement for a divalent cation (Mg2+) and a monovalent cation (K+, Na+ or NH4+) for activity, inhibition by bulk tRNA, ready inactivation by proteases, and increasing instability; with purification. RNAase P activity is also present in whole chick embryos, as well as in liver and heart tissues. Furthermore, crude preparations of RNAase P from chick embryo heart tissue are relatively free of contaminating nucleases.

One versus 2 years of CMFVP adjuvant chemotherapy in axillary node-positive and estrogen receptor-negative patients: a Southwest Oncology Group study.

PURPOSE: To determine if prolonged adjuvant treatment (2 years v 1 year) with combination chemotherapy (cyclophosphamide, methotrexate, fluorouracil [5-FU], vincristine, and prednisone [CMFVP]) in poor-prognosis breast cancer patients (estrogen receptor [ER]-negative, stage II to IIIA) would result in improved disease-free and overall survival rates. PATIENTS AND METHODS: Four hundred forty-five women with ER-negative node-positive breast cancer were enrolled by the Southwest Oncology Group (SWOG) over a period of 5 years (1979 to 1984). Randomized assignments were made to either 1 or 2 years of adjuvant CMFVP. Doses were daily oral cyclophosphamide 60 mg/m2, intravenous (i.v.) weekly methotrexate 15 mg/m2, i.v. weekly 5-FU 400 mg/m2, i.v. weekly vincristine .625 mg/m2 for the first 10 weeks, and prednisone weeks 1 through 6 with doses decreasing from 30 mg/m2 to 10 mg/m2. RESULTS: The median follow-up duration is 8.6 years, with a maximum of 11.3 years. Treatment arms were not significantly different as regards either survival or disease-free survival rates (P = .33 and P = .24, respectively). The five-year survival rate is 57% on the 1-year arm and 62% on the 2-year arm. Patients with three or fewer nodes and premenopausal status were associated with improved survival. Compliance on the 2-year arm was poor, with only 37% completing the full 2 years of treatment. SWOG grade 3 to 4 toxicity was experienced by 47% of patients on the 1-year arm and by 52% on the 2-year arm. There were no treatment-related deaths. CONCLUSION: We conclude that 2-year adjuvant treatment with CMFVP is not an improvement over 1-year treatment. Moreover, 2 years of CMFVP is difficult to complete. However, the results are not definitely negative. A moderate improvement attributed to prolonged chemotherapy, especially among patients with four or more positive nodes, cannot be ruled out.

A randomized trial of cisplatin, vinblastine, and bleomycin versus vinblastine, cisplatin, and etoposide in the treatment of advanced germ cell tumors of the testis: a Southwest Oncology Group study.

This is a Southwest Oncology Group (SWOG) prospective randomized trial of cisplatin, vinblastine, and bleomycin (PVB) versus vinblastine, cisplatin, and etoposide (VP-16) (VPV) in the treatment of advanced germ cell tumors of the testis. The study objective was to determine what effect the replacement of bleomycin with VP-16 has on complete response (CR), survival, and drug toxicity. One hundred sixty-nine patients were registered and randomized. Of these patients, 160 were assessable for response. All had histologically confirmed disseminated germ cell neoplasms of testicular origin. Forty-six had minimal metastatic disease, and 114 had maximal disease. Seventy-seven were randomized to PVB and 83 to VPV chemotherapy. There was no significant difference in pretreatment characteristics between the two arms with regard to tumor burden, histologic type, and overall performance status. Patients received four courses of induction chemotherapy, either PVB (cisplatin 120 mg/m2 day 3, vinblastine 12 mg/m2 day 1, bleomycin 15 U/m2 twice per week) or VPV (vinblastine 8 mg/m2 day 1, cisplatin 120 mg/m2 day 3, VP-16 50 mg/m2 days 2 to 5). Chemotherapy was given every 3 weeks. Cytoreductive surgery was done postinduction if a chemotherapy CR was not achieved. There was no difference in the percentage of patients achieving a disease-free status between PVB (77%) and VPV (73%). The mean leukocyte nadir was similar for both treatments, but the mean platelet nadir was significantly lower (P = .003) in the VPV arm. All bleomycin-related toxicities (pulmonary, mucositis, skin) were avoided in the VPV arm. We conclude that bleomycin can be replaced in first-line therapy for advanced germ cell tumors without sacrificing efficacy and with the advantage of avoiding unnecessary drug toxicity.

Selection and characterization of randomly produced mutants in the gene coding for M1 RNA.

The gene for M1 RNA, the catalytic subunit of RNase P of Escherichia coli, was subjected to random chemical mutagenesis in vitro. Mutations were selected by electrophoresis in denaturing gradient gels. Twenty-seven different mutants of the gene for M1 RNA were selected, and in 24 cases the mutations were identified as single base substitutions. The mutant forms of M1 RNA were analyzed in vitro for catalytic activity in the absence and in the presence of the protein subunit of RNase P (C5 protein). The structure of mutant RNAs was probed by limited digestion with ribonuclease T1; a correlation between reduced catalytic activity of mutant M1 RNAs and perturbations in secondary and tertiary structure was noted in many cases. The results indicate the involvement of specific regions of the M1 RNA molecule in the catalytic function of RNase P, in the binding of the C5 protein, and in substrate binding.

Kinetics of the processing of the precursor to 4.5 S RNA, a naturally occurring substrate for RNase P from Escherichia coli.

A study was made of the cleavage by M1 RNA and RNase P of a non-tRNA precursor that can serve as a substrate for RNase P from Escherichia coli, namely, the precursor to 4.5 S RNA (p4.5S). The overall efficiency of cleavage of p4.5S by RNase P is similar to that of wild-type tRNA precursors. However, unlike the reaction with wild-type tRNA precursors, the reaction catalyzed by the holoenzyme with p4.5S as substrate has a much lower Km value than that catalyzed by M1 RNA with the same substrate, indicating that the protein subunit plays a crucial role in the recognition of p4.5S. A model hairpin substrate, based on the sequence of p4.5S, is cleaved with greater efficiency than the parent molecule. The 3'-terminal CCC sequence of p4.5 S may be as important for cleavage of this substrate as the 3'-terminal CCA sequence is for cleavage of tRNA precursors.