[Investigation on status of dyslipidemia in Chinese females aged 35 years or above].

Objective: To investigate the prevalence, awareness, treatment and control status of dyslipidemia among females aged ≥35 years old across China. Methods: Participants were selected by stratified multistage random sampling method in the "Twelfth Five-Year Plan" National Science and Technology Support Project "Survey on the Prevalence of Important Cardiovascular Diseases and Key Technology Research in China" project. This study is a retrospective, cross-sectional study. A total of 17 418 females aged 35 years and over were included in the current study. The basic information such as age, medical history and menopause was collected by questionnaire. The blood lipid parameters were derived from clinical laboratory examinations. The prevalence of dyslipidemia and the rate of awareness, treatment, and control of dyslipidemia were analyzed in females aged 35 years and over. Results: The age of participants was (56.2±13.0) years old, and the prevalence of dyslipidemia was 33.1% (5 765/17 418). The prevalence rates of high total cholesterol, hypertriglyceridemia, low HDL-C and high LDL-C were 9.7% (1 695/17 418), 11.1% (1 925/17 418), 10.9% (1 889/17 418) and 7.3% (1 262/17 418), respectively. The prevalence of dyslipidemia increased with age and the prevalence of dyslipidemia in women who were not married, Han, menarche age>16 years, obesity, central obesity, alcohol consumption, diabetes, hypertension and family history of cardiovascular disease were higher than those without such characteristics (P<0.05). There were 10 432 (59.9%) menopausal females in this cohort and prevalence of dyslipidemia of these participants was 38.8% (4 048/10 432), which was higher than that of non-postmenopausal females (24.6%, 1 717/6 986) (P<0.05). The awareness rates, treatment rates and control rates of dyslipidemia were 33.9% (1 953/5 765), 15.1% (870/5 765) and 2.5% (143/5 765) respectively among females aged 35 years and over in China. Conclusion: The prevalence of dyslipidemia in Chinese females aged 35 years and over is high, and its awareness, treatment, and control rates need to be optimized.

[Effect of exogenous gonadotropin dosage on embryo aneuploidy rate and pregnancy outcome in patients of preimplantation genetic test].

Objective: To investigate the effect of gonadotropin (Gn) on embryo aneuploidy rate and pregnancy outcome during preimplanptation genetic testing for aneuploidy (PGT-A) cycles. Methods: The clinical data of patients undergoing PGT-A cycle at the First Medical Center of the PLA General Hospital from January 1, 2013 to May 31, 2019 were retrospectively analyzed. Patients were divided into younger patient group (<35 years old) and elder patient group (≥35 years old) by maternal age, then divided into two groups in line with Gn dosage (≤2 250 U, >2 250 U), and into four groups by number of oocytes retrieved (1-5, 6-10, 11-15 and ≥16 oocytes). The embryo aneuploidy rate and pregnancy outcome between the groups were compared. Logistic regression was used to analyze the relationship between the cumulative amount of Gn, embryo aneuploidy rate and live-birth rate. Results: A total of 402 cycles (338 patients) and 1 883 embryos were included in the study. (1) In the younger patients, the aneuploidy rate was 52.5% (304/579) in the group of Gn≤2 250 U and 48.6% (188/387) in the group of Gn>2 250 U, with no significant difference between them (P=0.232). In the elderly patients, the difference in embryo aneuploidy rate between the two Gn group [57.9% (208/359) versus 60.6% (319/526)] was not statistically significant (P=0.420). (2) The embryonic aneuploidy rate in different protocol of ovary stimulation was analyzed,in the younger group, the embryonic aneuploidy rate in patients using antagonist long protocol was 50.3% (158/314), it was 50.0% (121/242) in agonist long protocol, 52.1% (207/397) in agonist short protocol and 6/13 in luteal phase protocol, no statistical difference was found in above groups (P=0.923); in the elder group, embryonic aneuploidy rate was 60.8% (191/314) in antagonist protocol, 58.4% (132/226) in agonist long protocol, 59.2%(199/336) in agonist short protocol, 5/9 in luteal phase protocol, respectively,no significant difference was found (P=0.938). (3) In the younger patients, the aneuploidy rate in 1-5 oocytes group, 6-10 oocytes group, 11-15 oocytes group and ≥16 oocytes group was 37.9% (11/29), 54.0% (94/174), 52.5% (104/198) and 50.1% (283/565) respectively, no significant difference was found between the groups (P=0.652); while in the elder patients, the difference between aneuploidy rate in each retrieved oocytes group [73.6% (89/121), 57.5% (119/207), 56.3% (108/192), 57.8% (211/365)] was statistically significant (P=0.046). (4) Logistic regression analysis of age, cumulative dosage of Gn, number of oocytes obtained, and embryo aneuploidy rate showed that there was no association between the amount of Gn and embryo aneuploidy rate (P>0.05); the increase in maternal age would increase the risk of aneuploidy rate of embryos, which was statistically significant (OR=1.031, 95%CI: 1.010-1.054, P=0.004); the increase in oocytes retrived would significantly decrease the risk of aneuploidy (OR=0.981, 95%CI: 0.971-0.991, P<0.01). (5) There was no significant difference in biochemical pregnancy rate [55.6% (80/144) versus 52.1% (63/121)], clinical pregnancy rate [50.0% (72/144) versus 47.9% (58/121)] and live-birth rate [46.5% (67/144) versus 40.5% (49/121)] between different Gn dosage groups (P=0.613, P=0.738, P=0.324). The logistic regression analysis showed that the maternal age, the cumulative dosage of Gn, the number of oocytes obtained, and the ovarian stimulation protocol had no effect on the live-birth rate (all P>0.05). Conclusions: In PGT-A cycle, the dosage of Gn has no association with the embryo aneuploidy rate and pregnancy outcome. In the patients ≥35 years old, the increase in number of oocytes obtained may decrease the risk of aneuploidy. Age is an important factor affecting the embryo aneuploidy in PGT-A cycle.

[A real-world study of paritaprevir/ritonavir-ombitasvir combined with dasabuvir in the treatment of genotype 1b chronic hepatitis C].

Objective: To recognize the efficacy and safety of paritaprevir/ritonavir-ombitasvir combined with dasabuvir (OBV/PTV/RTV+DSV) in the treatment of genotype 1b chronic hepatitis C. Methods: Patients with genotype 1b chronic hepatitis C who were admitted to the People's Hospital of Henan Province, Huashan Hospital of Shanghai and the Fifth Medical Center of the General Hospital of the People's Liberation Army of China between November 2017 to August 2018 were enlisted. All patients received OBV/PTV/RTV+DSV antiviral therapy. HCV RNA levels were measured at baseline, weeks 1, 2, 3, 4, 8, 12, and 24, then 12 weeks, and 24 weeks after completion of treatment; patients' comorbidity, concomitant medications, and clinical adverse events were recorded. Results: 108 patients were enrolled in the study, with an average age of 49.1 years, 44 patients were male (40.8%), 96.3% (104/108) were newly diagnosed, and four patients had previous treatment history, of whom three were treated with IFN and one with IFN + DAA. Ninety-eight cases completed 12 weeks treatment and 89 cases were in follow up for 12 weeks, after discontinuation of the drug. Overall, 89 cases (100%) achieved SVR12.One patient treated with PR and DAA had HCV RNA level of 869175 IU/mL at 4 weeks of treatment, which was significantly higher than the baseline HCV RNA level (301776IU/ML), and was judged as failure of treatment; and follow-up was discontinued. Of all enrolled patients, 19 (17.6%) had underlying diseases and 15 (13.9%) had combined medications. During treatment, adverse events (AE) occurred in 11 patients (10.1%). The main adverse events were pruritus and elevated bilirubin. Conclusion: Combined antiviral therapy (OBV/PTV/RTV+DSV) of 12 weeks are highly effective with good safety profile in the treatment of Chinese patients with genotype 1b chronic hepatitis C.

[Analysis on the gender-specific risk factors of new-onset cerebral hemorrhage].

Objective: To explore the gender-specific risk factors of new-onset cerebral hemorrhage. Methods: In this prospective cohort study,a total of 98 961 participants((51.1±12.6)years old), who underwent the 2006 to 2007 physical examination and met the inclusion criteria, were enrolled from the Kailuanstudy cohort. There were 78 908 (79.7%) male,and 20 053 (20.3%) female.The incidence of cerebral hemorrhage was observed once per year until December 31, 2016.The difference on the incidence of cerebral hemorrhage between male and female was compared. Multivariate Cox regression analysis was applied to analyze therisk factors of cerebral hemorrhage events among different genders. Results: The participants were followed up for(10.00±0.73) years,and 860 cerebral hemorrhage events were recorded during follow up. The incidence of cerebral hemorrhage in the population was 86.90/10 million person years (standardized incidence rate of 47.85/10 million person years). The incidence of cerebral hemorrhage was significantly higher in male (49.61/10 million person years) than in female (34.07/10 million person years, P<0.05). Multivariate Cox regression analysis showed that 45-59 years old, ≥ 60 years old, diabetes,and waist-hip ratio were more strongly related to new-onset of cerebral hemorrhage events in female than in male, and the hazard ratios(95%CI) were 2.33 (1.23-4.43) ,2.71 (1.30-5.66) ,2.16 (1.24-3.74) and 8.79 (1.42-54.32) in female versus 1.55 (1.21-1.97) ,2.16 (1.68-2.78) ,1.19 (0.93-1.53) and 3.21 (1.09-9.41) in male, respectively. The risk of male cerebral hemorrhage increased by 29% (HR=1.29, 95%CI 1.19-1.40) in male and 24% (HR=1.24, 95%CI 1.20-1.28) in female,when the systolic blood pressure increased 10 mmHg (1 mmHg=0.133 kPa). Conclusions: The incidence of cerebral hemorrhage is higher in male than in female in this cohort.The association between systolic blood pressure and cerebral hemorrhage is stronger in male than that in female.The associations between age, waist-hip ratio, diabetes and cerebral hemorrhage are stronger in female than in male. Trial Registration: Chinese Clinical Trail Registry, ChiCTR-TNC-11001489.

Cloning and characterization of the dehydration-responsive element-binding protein 2A gene in Eruca vesicaria subsp sativa.

Eruca vesicaria subsp sativa is one of the most tolerant Cruciferae species to drought, and dehydration-responsive element-binding protein 2A (DREB2A) is involved in responses to salinity, heat, and particularly drought. In this study, a gene encoding EvDREB2A was cloned and characterized in E. vesicaria subsp sativa. The full-length EvDREB2A cDNA sequence contained a 388-bp 5'-untranslated region (UTR), a 348-bp 3'-UTR, and a 1002-bp open reading frame that encoded 334 amino acid residues. The theoretical isoelectric point of the EvDREB2A protein was 4.80 and the molecular weight was 37.64 kDa. The genomic sequence of EvDREB2A contained no introns. Analysis using SMART indicated that EvDREB2A contains a conserved AP2 domain, similar to other plant DREBs. Phylogenetic analysis revealed that EvDREB2A and DREB2As from Brassica rapa, Eutrema salsugineum, Arabidopsis thaliana, Arabidopsis lyrata, and Arachis hypogaea formed a small subgroup, which clustered with DREB2Bs from A. lyrata, A. thaliana, Camelina sativa, and B. rapa to form a larger subgroup. EvDREB2A is most closely related to B. rapa DREB2A, followed by DREB2As from E. salsugineum, A. thaliana, A. hypogaea, and A. lyrata. A quantitative real-time polymerase chain reaction indicated that EvDREB2A expression was highest in the leaves, followed by the roots and hypocotyls, and was lowest in the flower buds. EvDREB2A could be used to improve drought tolerance in crops.

Detection of Metabolism Function of Microbial Community of Corpses by Biolog-Eco Method.

OBJECTIVES: To detect the changes of microbial community functional diversity of corpses with different postmortem interval （PMI） and to evaluate forensic application value for estimating PMI. METHODS: The cultivation of microbial community from the anal swabs of a Sus scrofa and a human corpse placed in field environment from 0 to 240 h after death was performed using the Biolog-Eco Microplate and the variations of the absorbance values were also monitored. Combined with the technology of forensic pathology and flies succession, the metabolic characteristics and changes of microbial community on the decomposed corpse under natural environment were also observed. RESULTS: The diversity of microbial metabolism function was found to be negatively correlated with the number of maggots in the corpses. The freezing processing had the greatest impact on average well color development value at 0 h and the impact almost disappeared after 48 h. The diversity of microbial metabolism of the samples became relatively unstable after 192 h. The principal component analysis showed that 31 carbon sources could be consolidated for 5 principal components （accumulative contribution ratio >90%）.The carbon source tsquare-analysis showed that N-acetyl-D-glucosamine and L-serine were the dominant carbon sources for estimating the PMI （0=240 h） of the Sus scrofa and human corpse. CONCLUSIONS: The Biolog-Eco method can be used to reveal the metabolic differences of the carbon resources utilization of the microbial community on the corpses during 0-240 h after death, which could provide a new basis for estimating the PMI.

Methotrexate/mifepristone-combined with embryo removal in the treatment of caesarean scar pregnancy.

OBJECTIVE: The aim of this study was to compare the effect of different administration modalities of methotrexate (MTX)/mifepristone in the initial medication stage, followed by embryo transfer in the treatment of caesarean scar pregnancy (CSP). PATIENTS AND METHODS: A retrospective analysis of 66 CSP patients who received treatment in our hospital from January 2015 to July 2021 was performed, and participants were divided into three groups: Group one (n=14) received mifepristone followed by embryo removal treatment, Group two (n=29) received MTX followed by embryo removal, and Group three (n=23) received a methotrexate/mifepristone combined treatment followed by embryo removal. The basic findings were analysed, along with the curative effects between the three groups. Risk factors predicting additional treatment after initial intervention failure were analysed. RESULTS: There were statistically significant differences in gestational age, hospitalization days, costs, myometrial thickness, cardiac activity, and mean sac diameter between groups (p<0.05) after grouping by eight weeks. The initial intervention success rates were 92.86%, 89.66%, and 65.22% in Group one, two, and three, respectively (p<0.05), while the complication rates were 14.29%, 6.90%, and 26.87%, respectively (p>0.05). After grouping according to eight weeks of gestational age, the difference in initial serum ß-hCG between Group two and three was statistically significant (p<0.05). Mean sac diameter was a risk factor for additional treatment after initial intervention failure, with an odds ratio of 1.113 (p<0.05). A cut-off of 22.75 mm was a preferable indicator. CONCLUSIONS: MTX/mifepristone followed by embryo removal is a reliable way to treat CSP. Mean sac diameter was a risk factor for additional treatment after initial intervention failure.

A novel missense mutation in GTP cyclohydrolase I (GCH1) gene causes Dopa-responsive dystonia in Chinese Han population.

BACKGROUND: Dopa-responsive dystonia has been shown to be caused by a number of different mutations in the GCH1 gene. Up to now, only several genetic studies of Chinese patients with Dopa-responsive dystonia (DRD) have been reported. METHODS: We performed a genetic analysis by amplifying the entire coding region of GCH1 gene and direct sequencing in four DRD families from mainland China. RESULTS: A novel missense mutation, Gly155Ser, has been identified in a sporadic case from a consanguineous marriage family. Furthermore, two known mutations, Met137Arg and Gly203Arg, have also been detected in the other families. CONCLUSIONS: A novel missense mutation in the GCH1 gene can be associated with DRD. Our findings further expanded the mutational spectrum of GCH1 gene associated with DRD.

Let-7 derived from endometrial extracellular vesicles is an important inducer of embryonic diapause in mice.

Embryonic diapause is a maternally controlled phenomenon. The molecule controlling the onset of the phenomenon is unknown. We demonstrated that overexpression of microRNA let-7a or incubation with let-7g-enriched extracellular vesicles from endometrial epithelial cells prolonged the in vitro survival of mouse blastocysts, which developed into live pups after having been transferred to foster mothers. Similar to in vivo dormant blastocysts, let-7-induced dormant blastocysts exhibited low level of proliferation, apoptosis, and nutrient metabolism. Let-7 suppressed c-myc/mTORC1 and mTORC2 signaling to induce embryonic diapause. It also inhibited ODC1 expression reducing biosynthesis of polyamines, which are known to reactivate dormant embryos. Furthermore, the overexpression of let-7 blocked trophoblast differentiation and implantation potential of human embryo surrogates, and prolonged survival of human blastocysts in vitro, supporting the idea that embryonic diapause was an evolutionary conserved phenomenon. In conclusion, let-7 is the main factor inducing embryonic diapause.

Massive deletion in AZFb/b+c and azoospermia with Sertoli cell only and/or maturation arrest.

Recent studies have revealed that AZF deletion in Y chromosome is the most common known molecular genetic cause of spermatogenetic failure leading to male infertility. Characteristics of AZFa, AZFb and AZFc deletions and their association with spermatogenic impairment have been reported in a large number of populations. However, the distributions of those larger deletions resulted from P5/proximal-P1, P5/distal-P1 and P4/distal-P1 recombinations are still unclear as the literature on their frequencies is limited, and the contribution of these deletions to spermatogenetic failure remain to be confirmed by population studies. In this study, we investigated such massive deletions in 387 idiopathic azoospermic, 269 oligozoospermic patients and 315 men with normal spermatogenesis using 21 AZFb/c specific sequence-tagged sites. As a result, nine uninterrupted massive deletions were observed exclusively in men with azoospermia caused by either Sertoli cell only (SCO) or maturation arrest (MA). Prevalence of the deletion was 2.33%, in which five deletions arose from non-allelic homologous recombination between the palindromes P5 and P1 and two between P4 and P1. In other two deletions, novel proximal breakpoints in the interval region between P4 and P3 were observed. Our findings strongly support that the massive deletions in the AZFb or AZFb+c regions are important genetic causes of SCO and/or MA resulting in azoospermia and, besides the P5/proximal-P1, P5/distal-P1 and P4/distal-P1 deletions there may be other massive deletions in these regions resulting in severe spermatogenic impairment.

Effect of different doses of dexmedetomidine on median effective concentration of propofol for anesthesia induction: a randomized controlled trial.

OBJECTIVE: Dexmedetomidine, a highly selective α2-adrenergic receptor agonist with sedative and analgesic properties, is used as an anesthetic adjunct. We determined the effects of different dexmedetomidine doses on the median effective concentration (EC50) of propofol and bispectral index (BIS) values during anesthesia induction. PATIENTS AND METHODS: This randomized, prospective, case-control clinical trial involved 120 patients (56 women; physical status, American Society of Anesthesiologists grades I or II) scheduled to undergo surgery requiring general anesthesia from July 15th, 2014 to June 15th, 2015. The patients were divided into groups of 30 and received dexmedetomidine (0.5 µg/kg, group L; 0.75 µg/kg, group M; 1 µg/kg, group H) with propofol for loss of consciousness or propofol only (control group, group C). EC50, BIS, hemodynamics, and side effects were assessed. RESULTS: The EC50 of propofol was significantly lower in the dexmedetomidine groups than in group C, and decreased with increasing dexmedetomidine dose (p < 0.05). BIS values significantly decreased after 2 min of dexmedetomidine infusion in all dexmedetomidine groups; the values at 8 and 10 min were lower in the dexmedetomidine groups than in group C. The heart rate was lower in the dexmedetomidine groups than in group C. The incidence of bradycardia at loss of consciousness increased with increasing dexmedetomidine dose. CONCLUSIONS: Dexmedetomidine significantly and dose-dependently reduced the EC50 of propofol and BIS values during anesthesia induction. A loading dexmedetomidine dose of 0.5 µg/kg significantly reduced the EC50 of propofol and BIS value, and was associated with a lower incidence of bradycardia than higher doses.

Considerations in antimicrobial prescribing.

Soon after antimicrobial drugs became available, it was recognized that they were being overused and misused. Reasons for concern about the inappropriate use of antimicrobials include the emergence of resistant nosocomial bacteria, which have been identified in every hospital nationwide. The presence of resistant nosocomial bacteria presents the physician with a clinical problem and increases the cost of therapy. It is clear that methods need to be implemented to help physicians improve prescribing of antimicrobial agents. As health care practitioners in all fields strive to make patient care more cost-effective, one observation has become evident: The successfully treated patient consumes fewer resources and subsequently costs the hospital less than an otherwise similar, unsuccessfully treated patient. The goal in the 1990s is to provide optimal, cost-effective care for patients without compromising quality. This goal can be achieved by collaborative efforts of physicians, pharmacists, and microbiologists working together as a team to promote quality patient care.

The effect of control on the pattern of utilization of cefotaxime, moxalactam and cefoperazone in a teaching hospital.

In a 710-bed teaching hospital, the infectious disease section and pharmacy service initiated a joint programme to control and monitor the use of third-generation cephalosporins (TGC): cefotaxime, moxalactam and cefoperazone. The policy requires that an infectious disease physician approve the use of a TGC prior to its administration. This retrospective study sought to assess the pattern of utilization of these agents. From January to December 1983, 109 patient courses of cefotaxime (CT), moxalactam (MX), and cefoperazone (CP) were tabulated. Records from 92 patient courses were available for review (84.4%). The preliminary data show that 46.7% of TGC were used empirically to treat suspected infections without culture and sensitivity data, while 50.0% were used to treat various infections (central nervous system, pulmonary, wound, abdominal, biliary, urinary and blood) after culture and sensitivities were known. Only 3.3% of the TGC were used for surgical prophylaxis. In the empirical treatment group, the leading indications for use of TGC were central nervous system (CT = 40.0%, MX = 48.0%) and biliary (CP = 23.1%) infections. In the treatment group with culture and sensitivity data, the primary uses of TGC were for the treatment of Gram-negative pulmonary (MX = 28.0%, CP = 19.2%) and wound (CP = 15.4%) infections. This study shows that physician and pharmacy control does not eliminate empirical treatment with TGC; but it does narrow their use to situations in which their properties are best exploited.

Premature gestures: a speculative dialogue on Asian Pacific Islander lesbian and gay writing.

A collaborative exploration of the political realities and implications faced by self-identified Asian Pacific Islander lesbian and gay writers. Mixed-genre piece combining the essay and dialogue form, it contains sections co-written as well as individual pieces by the authors. The issues touched upon through this discussion are: available community-based and mainstream publishing venues, development of community-based writing, relation between grassroots political organizing and writing, API and lesbian/gay identity issues, internalized racism and homophobia, and other barriers for API lesbian and gay writers.

[Immunohistochemical study on myocardial acute ischemia reperfusion injury in rats with anti-FOS protein antibody].

To investigate law of FOS protein induced by myocardial ischemia reperfusion (MI/R) in acute period, a model in 20 anaethetized SD rats was established. Rats with normal and ischemia were used as control groups. Specimens were studied immunohistochemically with c-fos antibody. After ischemia 20 minutes, followed by 30 minutes reperfusion, the area of MI/R showed nuclei of myocytes positive staining in cryosection slides. In C2 group, the area showed nuclei of myocytes (37.76% +/- 9.66%) positive staining weakly. In C3 group, nuclei of cardiac myocytes(40.34% +/- 3.32%) was significantly positive. In C4 group it began to attenuate(35.36% +/- 4.81%). The myocardium in normal and ischemic control groups showed negative staining. No changes were seen with HE staining. Our data indicated that immunohistochemical method may reveal acute MI/R injury of ischemia 20 min and reperfusion 30 min with anti-FOS protein staining and there is problely a peak between 60-120 min after reperfusion. It is possible that the method be used to diagnose sudden cardiac death in forensic medicine.

Resistance of gram-negative bacilli as related to hospital use of antimicrobial agents.

The development of resistance of gram-negative bacilli, which are common nosocomial pathogens, is an increasing problem. It is generally accepted that this resistance may directly reflect the frequency of use of various antimicrobial agents. Because our institution experienced in 1976 a dramatic change in the pattern of antimicrobial use, primarily a marked decrease in prescribing cephalosporins, we attempted to evaluate retrospectively the effects of this change upon the resistance of gram-negative bacilli that are common nosocomial pathogens. Susceptibilities of Klebsiella and Providencia spp., Pseudomonas aeruginosa, and Serratia marcescens were determined for the years 1975 to 1979. Not unexpectedly, we observed a substantial decrease in cephalosporin resistance. An unexpected finding was a decrease in aminoglycoside resistance, despite increased use of these agents. The possibility that decreased cephalosporin use may lead to decreased aminoglycoside resistance is an intriguing and provocative thesis which can only be speculative at this time but which would seem worthy of additional formal investigation.

Nuclease-resistant external guide sequence-induced cleavage of target RNA by human ribonuclease P.

External guide sequences (EGSs) are short oligoribonucleotides, which are designed to bind to a given RNA target and form a precursor tRNA-like complex. This complex can be recognized by ribonuclease P (RNase P), resulting in specific cleavage of the RNA target. To explore the potential of this class of compounds as therapeutic agents and valuable tools for gene function analysis, various chemical modifications were introduced into an all-RNA EGS molecule to confer nuclease resistance. In particular, 2'-O-methyl substitutions were incorporated into the entire sequence (i.e., A-stem, D-stem, and T-stem) except the T-loop region without loss of cleavage-inducing activity. Replacement of rU (position 54) and rC (position 56) in the T-loop with their 2'-O-methyl counterparts caused pronounced decrease in activity. Moreover, phosphorothioate backbone modification of the T-loop did not provide sufficient protection against endonucleolytic attack at the ribopyrimidine residues. Systematic modification of the T-loop with a variety of modified nucleosides and the addition of a 3'-3' inverted T at the 3'-end have generated several lead EGS prototypes, which not only exhibit wild-type activity in inducing RNase P-mediated target cleavage as compared with the all-RNA control but also remain intact in human serum for more than 24 hours. These results should provide useful insights into the design and development of oligonucleotide-based EGSs as potential regulators of gene expression.

Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure.

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.