Directed gene modification via triple helix formation

The ability to selectively target mammalian genes and disrupt or restore their function would represent an important advance in gene therapy. Mutation of a single nucleotide can often result in a non-functional gene product. Reversion of defective genes to their correct sequences could lead to permanent cures for patients with many genetic diseases. Molecules such as triplex forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs) are currently being employed to bind to double-stranded DNA. Efficient targeting of genomic DNA with these molecules will be the initial step in gene modification.     

Angiopoietin-1 protects the adult vasculature against plasma leakage

Pathological increases in vascular leakage lead to edema and swelling, causing serious problems in brain tumors, in diabetic retinopathy, after strokes, during sepsis and also in inflammatory conditions such as rheumatoid arthritis and asthma. Although many agents and disease processes increase vascular leakage, no known agent specifically makes vessels resistant to leaking. Vascular endothelial growth factor (VEGF) and the angiopoietins function together during vascular development, with VEGF acting early during vessel formation, and angiopoietin-1 acting later during vessel remodeling, maturation and stabilization. Although VEGF was initially called vascular permeability factor, there has been less focus on its permeability actions and more effort devoted to its involvement in vessel growth and applications in ischemia and cancer. Recent transgenic approaches have confirmed the profound permeability effects of VEGF (refs. 12-14), and have shown that transgenic angiopoietin-1 acts reciprocally as an anti-permeability factor when provided chronically during vessel formation, although it also profoundly affects vascular morphology when thus delivered. To be useful clinically, angiopoietin-1 would have to inhibit leakage when acutely administered to adult vessels, and this action would have to be uncoupled from its profound angiogenic capabilities. Here we show that acute administration of angiopoietin-1 does indeed protect adult vasculature from leaking, countering the potentially lethal actions of VEGF and inflammatory agents.     

A Mechanism of Calmodulin Modulation of the Human Cardiac Sodium Channel

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Interplay between DNA repair and inflammation,and the link to cancer

Abstract

DNA damage and repair are linked to cancer. DNA damage that is induced endogenously or from exogenous sources has the potential to result in mutations and genomic instability if not properly repaired, eventually leading to cancer. Inflammation is also linked to cancer. Reactive oxygen and nitrogen species (RONs) produced by inflammatory cells at sites of infection can induce DNA damage. RONs can also amplify inflammatory responses, leading to increased DNA damage. Here, we focus on the links between DNA damage, repair, and inflammation, as they relate to cancer. We examine the interplay between chronic inflammation, DNA damage and repair and review recent findings in this rapidly emerging field, including the links between DNA damage and the innate immune system, and the roles of inflammation in altering the microbiome, which subsequently leads to the induction of DNA damage in the colon. Mouse models of defective DNA repair and inflammatory control are extensively reviewed, including treatment of mouse models with pathogens, which leads to DNA damage. The roles of microRNAs in regulating inflammation and DNA repair are discussed. Importantly, DNA repair and inflammation are linked in many important ways, and in some cases balance each other to maintain homeostasis. The failure to repair DNA damage or to control inflammatory responses has the potential to lead to cancer.     

Four novel Loci (19q13, 6q24, 12q24, and 5q14) influence the microcirculation in vivo

There is increasing evidence that the microcirculation plays an important role in the pathogenesis of cardiovascular diseases. Changes in retinal vascular caliber reflect early microvascular disease and predict incident cardiovascular events. We performed a genome-wide association study to identify genetic variants associated with retinal vascular caliber. We analyzed data from four population-based discovery cohorts with 15,358 unrelated Caucasian individuals, who are members of the Cohort for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, and replicated findings in four independent Caucasian cohorts (n = 6,652). All participants had retinal photography and retinal arteriolar and venular caliber measured from computer software. In the discovery cohorts, 179 single nucleotide polymorphisms (SNP) spread across five loci were significantly associated (p<5.0×10(-8)) with retinal venular caliber, but none showed association with arteriolar caliber. Collectively, these five loci explain 1.0%-3.2% of the variation in retinal venular caliber. Four out of these five loci were confirmed in independent replication samples. In the combined analyses, the top SNPs at each locus were: rs2287921 (19q13; p = 1.61×10(-25), within the RASIP1 locus), rs225717 (6q24; p?=?1.25×10(-16), adjacent to the VTA1 and NMBR loci), rs10774625 (12q24; p = 2.15×10(-13), in the region of ATXN2,SH2B3 and PTPN11 loci), and rs17421627 (5q14; p?=?7.32×10(-16), adjacent to the MEF2C locus). In two independent samples, locus 12q24 was also associated with coronary heart disease and hypertension. Our population-based genome-wide association study demonstrates four novel loci associated with retinal venular caliber, an endophenotype of the microcirculation associated with clinical cardiovascular disease. These data provide further insights into the contribution and biological mechanisms of microcirculatory changes that underlie cardiovascular disease.     

Effect of Soil Texture and Moisture on the Activity of Entomopathogenic Nematodes Against Female Boophilus annulatus Ticks

Soil texture, chemistry and moisture have a profound effect upon the activity and persistence of entomopathogenic nematodes (EPNs). Whereas nematodes’ natural habitat is within the soil, ticks and other arthropod pests prefer to stay on the soil surface and under stones or leaf litter; they spend much of their life cycle in the humid environment of the soil upper layer, therefore consideration of the effect of the soil environment on nematode activity is a pre-requisite for the sucessful use of EPNs against arthropod pests. In the present study we investigated the effects of soil type, and humidity on various nematode strains and on their effectiveness against ticks. Many infective juveniles (IJs) of Steinernema carpocapsae and S. riobrave were found in the uppermost soil layer whereas the heterorhabditid strains were almost absent from the upper 6 cm of the soil profile. The IJs of S. feltiae, and the S. carpocapsae strain S-20, exhibited an intermediate behavior. It was found that the activity of IJs of S. carpocapsae in the soil upper layer (1 cm depth) was strongly affected by soil type: the greatest number of IJs were recorded from sandy loam soil; less were found in the lighter soils – ‘Marine sand’ and ‘Calcareous sandstone’ – and only very few were recovered from heavy soils. Strikingly, even when the soil moisture was low and the number of nematodes found in the upper layer correspondingly low, tick mortality remained high. The results demonstrate: (a) the possible use of the nematodes as an anti-tick agent; (b) the importance of knowing the exact interaction of nematodes with the immediate environment of the pest, in order to optimize the pest-control activity of the nematode.     

Targeted cross-linking of the human beta-globin gene in living cells mediated by a triple helix forming oligonucleotide

Triple helix forming oligonucleotides (TFOs) may have utility as gene targeting reagents for "in situ" gene therapy of genetic disorders. Triplex formation is challenged by negative charge repulsion between third strand and duplex phosphates, and destabilizing positive charge repulsion between adjacent protonated cytosines within pyrimidine motif third strands. Here we describe the synthesis of TFOs designed to target a site in the human beta-globin gene, which is the locus for mutations that underlie the beta-globinopathies, including sickle cell anemia. The target is an uninterrupted polypurine:polypyrimidine sequence, containing four adjacent cytosines, next to a psoralen cross-link site. Pyrimidine motif TFOs that contained four adjacent cytosines or 5-methylcytosines did not form stable triplexes at physiological pH, despite the introduction of otherwise stabilizing base and sugar analogues. We synthesized a series of pso-TFOs containing 2'-O-methyl (OMe) and 2'-O-aminoethoxy substitutions (AE), as well as 8-oxo-adenine (A8) and 2'-O-methylpseudoisocytidine (P) as neutral cytosine replacements. Thermal stability measurements indicated that TFOs with A8 did not meet criteria established in previous work. However, TFOs with P did form triplexes with appropriate T(m) and k(ON) values. A pso-TFO with AE and P residues was sufficiently active to permit the determination of targeting in living cells by direct measurement of cross-link formation at the target site. Our results validate the modification format described in our previous studies and indicate that P substitutions are an effective solution to the problem of targeting genomic sequences containing adjacent cytosines.     

长春市儿童医院1998～2001年婴幼儿杯状病毒腹泻流行病学研究

人类杯状病毒(human calicivirus,HuCV)是引起儿童和成人非菌性胃肠炎的主要病原之一.为了掌握HuCV在我国的流行情况,1998年7月至2001年6月,从长春市儿童医院2343例5岁以下腹泻患儿中共收集粪便标本1264份,其中1056份来自2135例住院患儿.对轮状病毒检测为阴性的588份标本,经多价酶免疫试验(EIA)和两组引物反转录-聚合酶链反应(RT-PCR)检测HuCV,202份为阳性,其中住院患儿标本178份,HuCV检出率为16.9%.HuCV腹泻以2岁以下儿童为主(占96%),流行高峰季节为11月至次年3月.选择17株HuCV进行分子鉴定,15株属GⅡ-4群,1株属GⅡ-3群,另1株属GⅠ-2群,表明GⅡ-4群HuCV是我国流行的优势株.根据HuCV住院患儿的监测资料初步估计,HuCV腹泻住院率约为0.5‰～2.4‰.讨论了长春地区HuCV的流行趋势和疾病负担.以上结果为我国HuCV腹泻的预防和控制提供了科学依据.     

Evidence that high mobility group protein 17 is not phosphorylated in human colon carcinoma cells

The high mobility group proteins 14 and 17 were reported previously to be phosphorylated in murine and human tumor cell lines. Recently, it was suggested that subgroups of HMG-14, HMG-14a and 14b, but not HMG-17, were phosphorylated in situ in HeLa cells. In order to definitively determine whether HMG-17 is indeed phosphorylated or whether the protein previously identified as [³²P]HMG-17 was a subgroup of HMG-14, we have used the technique of electroblotting in conjunction with an immunochemical procedure utilizing anti-HMG-17 IgG. Our results indicate that HMG-17 was not phosphorylated in human colon carcinoma cell line HT-29 incubated for 18 h with ³²P_i, but that HMG-14a and HMG-14b were phosphorylated. In contrast, HMG-14a, -14b and -17 were phosphorylated in vitro in isolated nuclei incubated with [γ-³²P]ATP.     

超声治疗动脉粥样硬化性心血管疾病研究进展

超声干预治疗动脉粥样硬化性心血管疾病（atherosclerotic cardiovascular disease,ASCVD）是一种非侵入性治疗方法，其应用于临床治疗的前景广阔。超声波在身体组织中产生的机械、空化及生化等一系列作用可以有效清除血管中的斑块或血栓。但是，安全性是超声疗法应用于临床中亟需解决的首要问题。超声波在身体组织中的传播会引起组织损伤。另外，安全处理因超声刺激而产生的斑块或血栓碎片也是超声疗法应用中面临的挑战。除了确保安全性，合理制定治疗方案及治疗参数从而提高超声疗法疗效是超声疗法应用于临床中有待解决的重要问题。本文结合近年来超声干预治疗动脉粥样硬化性心血管疾病方面的各种临床、动物及体外模型实验研究结果，综述了超声干预治疗的机制、不同治疗方法和治疗参数的效果、如何确保安全性，以及提高超声疗法疗效需解决的一系列问题，进而提出可能的解决方案。