An Escherichia coli System Expressing Human Deoxyribonucleoside Salvage Enzymes for Evaluation of Potential Antiproliferative Nucleoside Analogs

Deoxyribonucleoside salvage in animal cells is mainly dependent on two cytosolic enzymes, thymidine kinase (TK1) and deoxycytidine kinase (dCK), while Escherichia coli expresses only one type of deoxynucleoside kinase, i.e., TK. A bacterial whole-cell system based on genetically modified E. coli was developed in which the relevant bacterial deoxypyrimidine metabolic enzymes were mutated, and the cDNA for human dCK or TK1 under the control of the lac promoter was introduced. The TK level in extract from induced bacteria with cDNA for human TK1 was found to be 20,000-fold higher than that in the parental strain, and for the strain with human dCK, the enzyme activity was 160-fold higher. The in vivo incorporation of deoxythymidine (Thd) and deoxycytidine (dCyd) into bacterial DNA by the two recombinant strains was 20 and 40 times higher, respectively, than that of the parental cells. A number of nucleoside analogs, including cytosine arabinoside, 5-fluoro-dCyd, difluoro-dCyd, and several 5-halogenated deoxyuridine analogs, were tested with the bacterial system, as well as with human T-lymphoblast CEM cells. The results showed a close correlation between the inhibitory effects of several important cytostatic and antiviral analogs on the recombinant bacteria and the cellular system. Thus, E. coli expressing human salvage kinases is a rapid and convenient model system which may complement other screening methods in drug discovery projects.     

Nucleoside and Nucleotide Analogs Select in Culture for Different Patterns of Drug Resistance in Human Immunodeficiency Virus Types 1 and 2

Recent findings suggest bidirectional antagonisms between the K65R mutation and thymidine analogue mutations in human immunodeficiency virus type 1 (HIV-1)-infected, treatment-experienced patients, yet little is known about HIV-2 in this regard. This study addressed the effects of innate polymorphisms in HIV-2 on emergent resistance to nucleoside/nucleotide analogues. Emergent drug resistance profiles in HIV-2 subtypes A (n = 3) and B (n = 1) were compared to those of HIV-1 subtypes B and C. Drug resistance was evaluated with cord blood mononuclear cells (CBMCs) and MT2 cells, using selective pressure with tenofovir (TFV), zidovudine (ZDV), stavudine (d4T), didanosine (ddI), abacavir (ABC), lamivudine (3TC), emtricitabine (FTC), or various dual-drug combinations. Resistance was evaluated using conventional and ultrasensitive sequencing approaches. In agreement with our previous findings, dual-drug combinations of TFV, ddI, ABC, d4T, ZDV, and 3TC preferentially selected for K65R in HIV-1 subtype C isolates. In HIV-1 subtype B, TFV-3TC and ZDV-3TC selected for M184I and D67N, respectively. In contrast, selections with all four HIV-2 cultures favored the development of M184I in dual-drug combinations that included either 3TC or FTC. Since HIV-2 cultures did not develop K65R, an ultrasensitive allele-specific real-time PCR assay was developed to distinguish the presence of 65R from wild-type K65 after 16 cycles with a discriminatory ability of 0.1% against a population of wild-type virus. These results underscore potential differences in emergent drug resistance pathways in HIV-1 and HIV-2 and show that polymorphisms may influence the development of the resistance pathways that are likely to emerge.     

Exacerbation of Influenza Virus Infections in Mice by Intranasal Treatments and Implications for Evaluation of Antiviral Drugs

Compounds lacking oral activity may be delivered intranasally to treat influenza virus infections in mice. However, intranasal treatments greatly enhance the virulence of such virus infections. This can be partially compensated for by giving reduced virus challenge doses. These can be 100- to 1,000-fold lower than infections without such treatment and still cause equivalent mortality. We found that intranasal liquid treatments facilitate virus production (probably through enhanced virus spread) and that lung pneumonia was delayed by only 2 days relative to a 1,000-fold higher virus challenge dose not accompanied by intranasal treatments. In one study, zanamivir was 90 to 100% effective at 10 mg/kg/day by oral, intraperitoneal, and intramuscular routes against influenza A/California/04/2009 (H1N1) virus in mice. However, the same compound administered intranasally at 20 mg/kg/day for 5 days gave no protection from death although the time to death was significantly delayed. A related compound, Neu5Ac2en (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid), was ineffective at 100 mg/kg/day. Intranasal zanamivir and Neu5Ac2en were 70 to 100% protective against influenza A/NWS/33 (H1N1) virus infections at 0.1 to 10 and 30 to 100 mg/kg/day, respectively. Somewhat more difficult to treat was A/Victoria/3/75 virus that required 10 mg/kg/day of zanamivir to achieve full protection. These results illustrate that treatment of influenza virus infections by the intranasal route requires consideration of both virus challenge dose and virus strain in order to avoid compromising the effectiveness of a potentially useful antiviral agent. In addition, the intranasal treatments were shown to facilitate virus replication and promote lung pathology.     

RNA Interference Inhibits DUX4-induced Muscle Toxicity In Vivo: Implications for a Targeted FSHD Therapy

No treatment exists for facioscapulohumeral muscular dystrophy (FSHD), one of the most common inherited muscle diseases. Although FSHD can be debilitating, little effort has been made to develop targeted therapies. This lack of focus on targeted FSHD therapy perpetuated because the genes and pathways involved in the disorder were not understood. Now, more than 2 decades after efforts to decipher the root cause of FSHD began, this barrier to translation is finally lowering. Specifically, several recent studies support an FSHD pathogenesis model involving overexpression of the myopathic DUX4 gene. DUX4 inhibition has therefore emerged as a promising therapeutic strategy for FSHD. In this study, we tested a preclinical RNA interference (RNAi)-based DUX4 gene silencing approach as a prospective treatment for FSHD. We found that adeno-associated viral (AAV) vector-delivered therapeutic microRNAs corrected DUX4-associated myopathy in mouse muscle. These results provide proof-of-principle for RNAi therapy of FSHD through DUX4 inhibition.     

生长抑素类似物治疗Gravesl眼病有效性的Meta分析

目的系统评价生长抑素类似物治疗Graves眼病（GO）的有效性。方法计算机检索PubMed、EMbase、The Cochrane Library、WanFang Data、CNKI、VIP和CBM中关于生长抑素类似物治疗Graves眼病的随机对照试验（RCT）,检索时间截至2012年3月,并追溯纳入文献的参考文献。由2位研究者按照纳入标准筛选文献、提取资料和评价纳入研究的方法学质量后,采用RevMan5．0软件进行Meta分析。结果最终纳入5个RCT,共210例患者。Meta分析结果显示：生长抑素类似物治疗GO可降低临床活动性评分[MD=0．58,95％CI（0．02,1．13）,P=0．04],但尚不能证实可降低突眼度（mm）[MD=0．21,95％CI（-0．14,0．56）,P=0．24];对复视程度、球后体积、眼压、视力及眼球活动受限未显示出明显疗效;目前证据尚不能说明生长抑素类似物治疗GO有效[OR=I．32,95％CI（0．45,3．9）,P=0．61].结论生长抑素类似物治疗GO可降低患者的临床活动性评分,但未能明显降低突眼度,目前证据尚不能证实生长抑素类似物治疗GO有效。受纳入研究的质量和数量限制,上述结论尚需开展更多高质量RCT加以验证。     

齐多夫定单用预防HIV母婴传播的有效性与安全性的系统评价

目的系统评价单独应用齐多夫定(zidovudine,ZDV)阻断HIV母婴传播的有效性和安全性。方法采用Cochrane系统评价方法,计算机检索Cochrane图书馆(2007第1期)、PubMed、EMbase、CINAHL、AIDSearch、AIDSLINE、AIDSTRIALS、AIDSDRUGS、AIDSinfo、CRD(center of review and dissemination)、CBMdisc,VIP和CNKI等数据库,以及全球或地区性AIDS相关的会议论文集、政府或非政府组织的相关文件等,检索日期截至2007年4月30日,全面收集全球抗艾滋病病毒药物预防HIV母婴传播的随机对照试验。由两名评价员独立筛查文献、评价质量和提取资料,然后交叉核对,若遇分歧则征求第三方意见讨论解决。使用RevMan软件进行Meta分析。结果共纳入8个RCT,包括24篇全文和13篇摘要,其方法学质量的Jadad评分≥3分。Meta分析显示:①ZDV与安慰剂比较共纳入4个RCTs(2385例),无论长短疗程、母乳或非母乳喂养人群,ZDV预防HIV母婴传播的效果均优于安慰剂组,降低HIV母婴传播风险43%～50%,且两组死产率、婴儿死亡率、母亲死亡率、早产、低体重儿、出生缺陷、母婴不良反应发生率和母亲产前、产时和产后并发症发生率差异均无统计学意义(P>0.05)。②1个大样本RCT(1437例)比较了ZDV不同疗程的效果,结果显示ZDV“长–长疗程”(从孕28周开始到产后6周)比“短–短疗程”(从孕35周开始到分娩后3天)降低HIV母婴传播风险61%[RR=0.39,95%CI(0.19,0.82)]。长–长疗程与长–短疗程(从孕28周开始到产后3天)及短-长疗程(从孕35周开始到产后6周)比较,其预防HIV母婴传播的效果差异均无统计学意义(P>0.05)。各组死产、新生儿死亡、1年内婴儿死亡、母亲死亡、早产、低体重儿、出生缺陷、母婴不良反应发生率相似(P>0.05)。③1个大样本RCT(1200例)显示:人工喂养 短程ZDV预防HIV母婴传播的效果优于母乳喂养 长程ZDV,可降低婴儿HIV感染风险的35%～39%,但提高了婴儿7个月时的死亡率(9.3%vs4.9%;P=0.003);两组婴儿早产率、低体重儿出生率、出生缺陷率、不良反应发生率相似(P>0.05)。④2个直接比较短程或超短程ZDV与单剂量奈韦拉平(Nevirapine,NVP)预防HIV母婴传播效果的RCT(702例)显示,NVP可降低HIV母婴传播风险的44%～48%,两组死产、6月内婴儿死亡、母亲死亡、低体重儿、母婴不良反应发生率相似(P>0.05)。结论无论长短疗程、母乳或非母乳喂养人群,ZDV预防HIV母婴传播的效果均优于安慰剂,且其妊娠结局和不良反应发生情况相似。ZDV“长–长疗程”比“短–短疗程”预防HIV母婴传播效果更好,但长–长疗程与长–短疗程、短–长疗程预防HIV母婴传播的效果相似;各组安全性相似。人工喂养 短程ZDV预防HIV母婴传播的效果优于母乳喂养 长程ZDV,但提高了婴儿7个月时的死亡率。单剂量NVP预防HIV母婴传播效果优于短程和超短程ZDV,且安全性相似。     

HIV Coreceptor Downregulation as Antiviral Principle: SDF-1α–dependent Internalization of the Chemokine Receptor CXCR4 Contributes to Inhibition of HIV Replication

Ligation of CCR5 by the CC chemokines RANTES, MIP-1α or MIP-1β, and of CXCR4 by the CXC chemokine SDF-1α, profoundly inhibits the replication of HIV strains that use these coreceptors for entry into CD4⁺ T lymphocytes. The mechanism of entry inhibition is not known. We found a rapid and extensive downregulation of CXCR4 by SDF-1α and of CCR5 by RANTES or the antagonist RANTES(9-68). Confocal laser scanning microscopy showed that CCR5 and CXCR4, after binding to their ligands, are internalized into vesicles that qualify as early endosomes as indicated by colocalization with transferrin receptors. Internalization was not affected by treatment with Bordetella pertussis toxin, showing that it is independent of signaling via G_i-proteins. Removal of SDF-1α led to rapid, but incomplete surface reexpression of CXCR4, a process that was not inhibited by cycloheximide, suggesting that the coreceptor is recycling from the internalization pool. Deletion of the COOH-terminal, cytoplasmic domain of CXCR4 did not affect HIV entry, but prevented SDF-1α–induced receptor downregulation and decreased the potency of SDF-1α as inhibitor of HIV replication. Our results indicate that the ability of the coreceptor to internalize is not required for HIV entry, but contributes to the HIV suppressive effect of CXC and CC chemokines.     

Self-Management of Recovery: Implications for Nursing Practice

This article describes and discusses differences in responses to a 60-item survey instrument, the Carolina Self-Regulation Inventory (CSRI) from a selected sample of university faculty and staff. The CSRI was designed to measure the types of self-regulation strategies (SRS) individuals use to facilitate their recovery from illness episodes. When the CSRI was developed, 1,306 university employees participated in the survey. Four hundred eighty (36%) employees classified themselves as faculty; 346 (26%), staff; 418 (32%), midlevel administrators; and 62 (6%), blue-collar workers. This report focuses on the differences between the faculty and staff groups in the survey. Analysis revealed that the staff used a variety of strategies and had higher statistically significant means on the following CSRI subscales: visualization (t = 2.53, P less than .01), self-talk (t = 6.801, P less than .001), and external attention deployment strategies (t = 4.08, P less than .001). No significant differences were found between faculty and staff in the use of exercise and physical activity or interactive self-regulation strategies. Higher-educated faculty women tended to use fewer SRS when compared with female staff (F = 4.70; df = 8.819; P = .0009). Faculty women were similar to faculty men in their use of SRS. Female staff used significantly more SRS when compared with faculty of both genders and male staff (t = 2.81, P = .05). Nursing implications of self-regulation nursing assessments are discussed in terms of nursing interventions and need for more research.     

Antiviral Action and Cellular Toxicity of Four Thymidine Analogues: 5-Ethyl-, 5-Vinyl-, 5-Propyl-, and 5-Allyl-2′-Deoxyuridine

5-Ethyl-, 5-vinyl-, 5-propyl-, and 5-allyl-2'-deoxyuridine (dUrd) had antiviral activity against herpes simplex type 1 and type 2 grown in HeLa TK(-) cells, in the order 5-vinyl-dUrd, 5-ethyl-dUrd, 5-propyl-dUrd, 5-allyl-dUrd, but they were inactive against a TK(-) mutant of herpes simplex type 1. The antiviral activity of these compounds could be partially reversed by thymidine. Except for 5-vinyl-dUrd, they were not toxic to WI-38 and HeLa TK(-) cells at a concentration of 25 muM. All four analogues inhibited the growth of herpes simplex type 1-transformed HeLa TK(-) cells at a concentration of 1 muM.     

Gut hormones: Implications for the treatment of obesity

Bariatric surgery is the only effective treatment for patients with morbid obesity. This is no solution to the present obesity pandemic however. Currently licensed non-surgical pharmaceuticals are of limited efficacy and alternatives are needed.Harnessing the body's own appetite-regulating signals is a desirable pharmacological strategy. The gastrointestinal tract has a prime role in sensing and signalling food intake to the brain. Gut hormones are key mediators of this information, including: peptide YY (PYY), pancreatic polypeptide (PP), glucagon-like peptide 1 (GLP-1), oxyntomodulin (OXM), ghrelin, amylin and cholecystokinin (CCK).This review summarises the latest knowledge regarding the physiological and pathophysiological role of gut hormones in regulating our food intake and how this knowledge could guide, or has guided, the development of weight-loss drugs. Up-to-date outcomes of clinical trials are evaluated and directions for the future suggested.     

Implications of cyberspace communication: a role for physicians

Through the presentation of three clinical case reports and subsequent discussion, it is demonstrated that physicians must begin to familiarize themselves with the health-related implications of online communication, and must proactively address Internet use as it relates to health and well-being. Included case presentations highlight the following: the established association between those seeking sexual partners through the Internet and an increased risk for sexually transmitted disease; the implications of cyber-communication for young people and concerns related to unsafe online behaviors including sharing identifying information with strangers; the potential use of strategically constructed virtual identities to facilitate sexual exploitation; the impact of accelerated intimacy and disinhibition evident in online communication; and the invasive nature of Internet sexual harassment or bullying. Although it is recognized that most online activities do not negatively affect health, doctors must be prepared to ask patients about Internet use and become involved in educating children, teenagers, and parents about safe online relationships to promote optimal physical, mental, and social health.