Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

Oligonucleotides composed of 2′-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5′-disulphide-linked conjugates of 3′-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48–58), Penetratin and R(9)F(2) was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48–58), Penetratin and R(9) conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R(9)F(2) and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide

Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells

The trans-activation response (TAR) RNA stem–loop that occurs at the 5′ end of HIV RNA transcripts is an important antiviral target and is the site of interaction of the HIV-1 Tat protein together with host cellular factors. Oligonucleotides and their analogues targeted to TAR are potential antiviral candidates. We have investigated a range of cell penetrating peptide (CPP) conjugates of a 16mer peptide nucleic acid (PNA) analogue targeted to the apical stem–loop of TAR and show that disulfide-linked PNA conjugates of two types of CPP (Transportan or a novel chimeric peptide R(6)-Penetratin) exhibit dose-dependent inhibition of Tat-dependent trans-activation in a HeLa cell assay when incubated for 24 h. Activity is reached within 6 h if the lysosomotropic reagent chloroquine is co-administered. Fluorescein-labelled stably-linked conjugates of Tat, Transportan or Transportan TP10 with PNA were inactive when delivered alone, but attained trans-activation inhibition in the presence of chloroquine. Confocal microscopy showed that such fluorescently labelled CPP–PNA conjugates were sequestered in endosomal or membrane-bound compartments of HeLa cells, which varied in appearance depending on the CPP type. Co-administration of chloroquine was seen in some cases to release fluorescence from such compartments into the nucleus, but with different patterns depending on the CPP. The results show that CPP–PNA conjugates of different types can inhibit Tat-dependent trans-activation in HeLa cells and have potential for development as antiviral agents. Endosomal or membrane release is a major factor limiting nuclear delivery and trans-activation inhibition

A bi-functional siRNA construct induces RNA interference and also primes PCR amplification for its own quantification

RNA interference (RNAi) is a process of post-transcriptional gene silencing initiated by double-stranded RNAs, including short interfering RNA (siRNA). Silencing is sequence-specific and RNAi has rapidly become central to the study of gene function. RNAi also carries promise for selective silencing of viral and endogenous genes causal for disease. To detect the very low levels of siRNA effective for RNAi we modified the 3′ end of the sense strand of siRNA with a nuclease-resistant DNA hairpin. We show that the modified siRNA-DNA construct (termed ‘crook’ siRNA) functions as a primer for the PCR and describe a novel, yet simple PCR protocol for its quantification (amolar levels/cell). When transfected into mammalian cells, crook siRNA induces selective mRNA knock-down equivalent to its unmodified siRNA counterpart. This new bifunctional siRNA construct will enable future in vivo studies on the uptake, distribution and pharmacokinetics of siRNA, and is particularly important for the development of siRNA-based therapeutics. More generally, PCR-based detection of siRNA carries wide-ranging applications for RNAi reverse genetics

Measurement of the neutron lifetime using a gravitational trap and a low-temperature Fomblin coating

We present a new value for the neutron lifetime of 878.5 +- 0.7 stat. +- 0.3 syst. This result differs from the world average value (885.7 +- 0.8 s) by 6.5 standard deviations and by 5.6 standard deviations from the previous most precise result. However, this new value for the neutron lifetime together with a beta-asymmetry in neutron decay, Ao, of -0.1189(7) is in a good agreement with the Standard Model.Comment: 11 pages, 9 figures; extended content with some correction

On the Protection of Intangible Heritage of Russian Folk Oral Music in the Context of Intercultural Communication (Hulunbuir, Inner Mongolia)

The article discusses the issues of preserving the genre of Russian folk oral musical creativity in the village of the Argun and the city of Hulunbuir, Inner Mongolia, located in the northern part of China. The issue is viewed in the context of intercultural communication between descendants of Russian immigrants and Chinese locals. The article considers the factors in the formation of the ethnic group of Chinese Russians in Hulunbuir, an area of Russian immigrants’ compact settlement, and the markers of their distinct ethnic identity. Chinese Russians are a specific ethnic group since over several generations they fused with the local ethnicities yet preserved their unique cultural background. One of the peculiar aspects of Russian culture observed within the community of Chinese Russians is chastushka, or ditty, a short witty song expressing an individual’s attitude to any happening. The authors give records of the texts of ditties on various subjects, such as love, daily life, politics, etc. The folk genre of chastushka indicates intercultural communication between China and Russia and the integration of ethnic Russians into Chinese society. The article reveals the problems of protection of the Russian chastushka in the region and possible measures of state provision of its protection as intangible cultural heritage. Both national and local authorities take steps to ensure the continuity of various identities within the national identity of China. Several proposals have been put forward for the protection of the local heritage of this genre of the city of Hulunbuir. Such measures may include further research of cultural materials, enhancing tourism in the region, and incorporating ditties into local festivities Based on the historical and cultural significance, the research points to the real impact of Russian folk oral musical creativity in the processes of Russian-Chinese intercultural communication. The authors underline the significance of the ditty as intangible cultural heritage and the need to include the ditty in the list of the cultural heritage of Chinese Russians. The need to create conditions and state-organizational support for various forms of popularization of this genre, especially for those studying the Russian language, to preserve the oral folk musical creativity of Chinese ethnic Russians is substantiated

Systematic screening of LNA/2′-O-methyl chimeric derivatives of a TAR RNA aptamer

AbstractWe synthesized and evaluated by surface plasmon resonance 64 LNA/2′-O-methyl sequences corresponding to all possible combinations of such residues in a kissing aptamer loop complementary to the 6-nt loop of the TAR element of HIV-1. Three combinations of LNA/2′-O-methyl nucleoside analogues where one or two LNA units are located on the 3′ side of the aptamer loop display an affinity for TAR below 1nM, i.e. one order of magnitude higher than the parent RNA aptamer. One of these combinations inhibits the TAR-dependent luciferase expression in a cell assay

A measurement of the neutron lifetime using the method of storage of ultracold neutrons and detection of inelastically up-scattered neutrons

AbstractWe present estimations of systematic corrections and results of their experimental studies for our neutron lifetime experiment carried out in 2008–2010 at ILL. Taking into account these systematic corrections, we reduce the data of three independent sets of measurements (obtained during period 2008–2010) performed with different energy spectra of ultracold neutrons (UCNs) at different trap temperatures to the mean neutron lifetime value equal to 880.2(1.2) s

UCN anomalous losses and the UCN capture cross-section on material defects

Experimental data shows anomalously large Ultra Cold Neutrons (UCN) reflection losses and that the process of UCN reflection is not completely coherent. UCN anomalous losses under reflection cannot be explained in the context of neutron optics calculations. UCN losses by means of incoherent scattering on material defects are considered and cross-section values calculated. The UCN capture cross-section on material defects is enhanced by a factor of 10^4 due to localization of UCN around defects. This phenomenon can explain anomalous losses of UCN.Comment: 13 pages, 4 figure

The Evaluation of V_{ud}, Experiment and Theory

The value of the V_{ud} matrix element of the Cabibbo-Kobayashi-Maskawa (CKM) matrix can be derived from nuclear superallowed beta decays, neutron decay, and pion beta decay. We survey current world data for all three. Today, the most precise value of V_{ud} comes from the nuclear decays; however, the precision is limited not by experimental error but by the estimated uncertainty in theoretical corrections. Experimental uncertainty does limit the neutron-decay result, which, though statistically consistent with the nuclear result, is approximately a factor of three poorer in precision. The value obtained for $V_{ud}$ leads to a result that differs at the 98% confidence level from the unitarity condition for the CKM matrix. We examine the reliability of the small calculated corrections that have been applied to the data, and assess the likelihood of even higher quality nuclear data becoming available to confirm or deny the discrepancy. Some of the required experiments depend upon the availability of intense radioactive beams. Others are possible today.Comment: 21 pages, 1 figure, LaTe

Simultaneous pancreas‐kidney transplantation in type 1 diabetes mellitus. Clinical options

Simultaneous pancreas-kidney transplantation (SPKT) is the most promising treatment option for patients with type 1 diabetes mellitus (T1DM) and end-stage renal disease (ESRD) due to diabetic nephropathy (DN). Successful SPKT eliminates uremic intoxication and hyperglycemia – the leading trigger of vascular diabetic complications. Therefore, euglycemia is an important metabolic change in patients after surgery and remains only one of the factors for the saved renal allograft functioning. In the case of resuming renal replacement therapy by dialysis after SPKT, the management and monitoring of the pancreatic graft remains open. Special attention to the pancreatic graft’s function is due to both the potential risk of surgical complications, and some probability of T1DM relapse with the need to resume insulin therapy. In patients with saved function of both transplants, the assessment of the dynamics of diabetic complications in general becomes more important. The results of few studies in this regard remain contradictory. Thus, clinical options can be unpredictably diverse and require not only search for the root cause, but also optimization of rehabilitation tactics, even if the expected results are achieved