Differences in degeneration of the apical scolopidial organ in the labial palp of Lepidoptera during pupal development

Summary The apical scolopidial organ (ASO) in the labial palp of six species from four families of Lepidoptera was studied in pupal and imaginal stages using electron microscopy. The organ houses three sensory units, each of which consists of one sensory cell and two enveloping cells at early pupal stage in all the species studied. The distal part of the ASO is connected with the epidermis of the tip of the labial palp. Proximally it is attached to the primordium of the palpal nerve. The axons of the sensory cells run within this nerve to the central nervous system. There are two main differences in the differentiation of the ASO in the species examined during postembryonic development: (1) the sensory cells of the ASO degenerate at different rates; and (2) the ASO may or may not change its position within the palp. In Pieris brassicae and Pieris napi (Pieridae), all three sensory cells undergo stepwise degeneration. Consequently, no sensory cells are left in the imago in these species. However, in the Rhodogastria sp. (Arctiidae), only one sensory cell of the ASO degenerates during pupal life. Two remain, therefore, in the imaginal stage. Their dendritic outer segments and axons are normal, and their appearance does not differ from that in early pupal life. The same process was also observed in Rhodogastria bubo (Arctiidae), Autographa gamma (Noctuidae) and Aglais urticae (Nymphalidae). In addition to the degeneration of the sensory cells the ASO turns through about 180° in P. brassicae and P. napi so that its tip points to the base of the palp in the imagines of these species.     

Regulation of inflammatory arthritis by the upstream kinase mitogen activated protein kinase kinase 7 in the c-Jun N-Terminal kinase pathway

Introduction

The c-Jun N-terminal kinase (JNK) is a key regulator of matrix metalloproteinase (MMP) and cytokine production in rheumatoid arthritis (RA) and JNK deficiency markedly protects mice in animal models of arthritis. Cytokine-induced JNK activation is strictly dependent on the mitogen-activated protein kinase kinase 7 (MKK7) in fibroblast-like synoviocytes (FLS). Therefore, we evaluated whether targeting MKK7 using anti-sense oligonucleotides (ASO) would decrease JNK activation and severity in K/BxN serum transfer arthritis.

Methods

Three 2''-O-methoxyethyl chimeric ASOs for MKK7 and control ASO were injected intravenously in normal C57BL/6 mice. PBS, control ASO or MKK7 ASO was injected from Day -8 to Day 10 in the passive K/BxN model. Ankle histology was evaluated using a semi-quantitative scoring system. Expression of MKK7 and JNK pathways was evaluated by quantitative PCR and Western blot analysis.

Results

MKK7 ASO decreased MKK7 mRNA and protein levels in ankles by about 40% in normal mice within three days. There was no effect of control ASO on MKK7 expression and MKK7 ASO did not affect MKK3, MKK4 or MKK6. Mice injected with MKK7 ASO had significantly less severe arthritis compared with control ASO (P < 0.01). Histologic evidence of synovial inflammation, bone erosion and cartilage damage was reduced in MKK7 ASO-treated mice (P < 0.01). MKK7 deficiency decreased phospho-JNK and phospho-c-Jun in ankle extracts (P < 0.05), but not phospho-MKK4. Interleukin-1beta (IL-1β), MMP3 and MMP13 gene expression in ankle joints were decreased by MKK7 ASO (P < 0.01).

Conclusions

MKK7 plays a critical regulatory role in the JNK pathway in a murine model of arthritis. Targeting MKK7 rather than JNK could provide site and event specificity when treating synovitis.     

Potent inhibition of microRNA in vivo without degradation

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.     

Metabolic Consequences of High-Fat Diet Are Attenuated by Suppression of HIF-1��

Obesity is associated with tissue hypoxia and the up-regulation of hypoxia inducible factor 1 alpha (HIF-1α). Prior studies in transgenic mice have shown that HIF-1α plays a role in the metabolic dysfunction associated with obesity. Therefore, we hypothesized that, after the development of diet-induced obesity (DIO), metabolic function could be improved by administration of HIF-1α antisense oligonucleotides (ASO). DIO mice were treated with HIF-1α ASO or with control ASO for 8 weeks and compared with an untreated group. We found that HIF-1α ASO markedly suppressed Hif-1α gene expression in adipose tissue and the liver. HIF-1α ASO administration induced weight loss. Final body weight was 41.6±1.4 g in the HIF-1α ASO group vs 46.7±0.9 g in the control ASO group and 47.9±0.8 g in untreated mice (p<0.001). HIF-1α ASO increased energy expenditure (13.3±0.6 vs 12±0.1 and 11.9±0.4 kcal/kg/hr, respectively, p<0.001) and decreased the respiratory exchange ratio (0.71±0.01 vs 0.75±0.01 and 0.76±0.01, respectively, p<0.001), which suggested switching metabolism to fat oxidation. In contrast, HIF-1a ASO had no effect on food intake or activity. HIF-1α ASO treatment decreased fasting blood glucose (195.5±8.4 mg/dl vs 239±7.8 mg/dl in the control ASO group and 222±8.2 mg/dl in untreated mice, p<0.01), plasma insulin, hepatic glucose output, and liver fat content. These findings demonstrate that the metabolic consequences of DIO are attenuated by HIF-1α ASO treatment.     

Understanding Opioid Actions,Pain and Analgesia: A Tribute to Dr. Gavril Pasternak

This special issue is a tribute to our mentor, colleague and friend, Gavril W. Pasternak, MD, PhD. Homage to the breadth and depth of his work (~?450 publications) over a 40 career in pharmacology and medicine cannot be captured fully in one special issue, but the 22 papers collected herein represent seven of the topics near and dear to Gav’s heart, and the colleagues, friends and mentees who held him near to theirs. The seven themes include: (1) sites and mechanisms of opioid actions in vivo; (2) development of novel analgesic agents; (3) opioid tolerance, withdrawal and addiction: mechanisms and treatment; (4) opioid receptor splice variants; (5) novel research tools and approaches; (6) receptor signaling and crosstalk in vitro; and (7) mentorship. This introduction to the issue summarizes contributions and includes formal and personal remembrances of Gav that illustrate his personality, warmth, and dedication to making a difference in patient care and people’s lives.

     

Predicting antisense oligonucleotide inhibitory efficacy: a computational approach using histograms and thermodynamic indices.

Antisense oligonucleotides (ASOs) are designed to bind to a specific mRNA and selectively suppress its translation. To facilitate selection of optimal ASO targets, we have developed three thermodynamic indices to evaluate putative structural complexes important in ASO action. These indices are: a secondary structure score (Sscore), which estimates the strength of local mRNA secondary structures at the ASO target site; a duplex score (Dscore), which estimates the delta Gformation for the ASO:mRNA target sequence duplex; and a competition score (Cscore), which is the difference between the Dscore and the Sscore. We also present two histograms to graphically display these indices from different regions of the mRNA. The indices are compared to the inhibition reported in five studies of ASO-mediated suppression of gene expression. The Dscore is the most consistent predictor of ASO efficacy in four of the five studies (r2 from 0.44 to 0.99), while the results of the fifth study could not be predicted by any thermodynamic or physical index. Thus the Dscores and their histogram may prove useful in selection of ASO targets.     

Population policies in developing countries and related international attitudes

Abstract

Huntington's disease (HD) is a chronic degenerative neurologic disorder inherited as an autosomal dominant trait. Symptom onset typically occurs at about age 40, and considerable effort has been expended in attempts to diagnose the disorder prior to the childbearing years. We review attempts to use psychological tests in early diagnosis. Specifically, we identify variables that show promise as early markers of the disorder, review evidence that a prodromal phase may exist, analyze the few prospective studies of persons at risk for HD, and consider the logical and methodological problems involved in early detection research.     

Antioxidant activity of 5-alkoxymethyl-6-chromanols

Abstract

The 5-alkoxymethyl-2,2,7,8-tetramethyl-6-chromanols (II) are excellent antioxidants against autoxidising safflower oil (ASO), although not as good as 2,2,5,7,8-pentamethyl-6-chromanol (I), the model compound of -tocopherol. The aim of this work was to determine whether the rate of reaction of (II) with the radicals diphenylpicrylhydrazyl (DPP^·) and galvinoxyl (ArO^·) was directly proportional to their antioxidant activity against ASO. Compounds (II) reacted faster with DPP^·. than with ArO^·. but, in each case, slower than compound (I). The rates of reaction of I and II with both radicals followed the order I > II (R = H) > II (R = CH₃) > II (R = other alkyls) and were directly proportional to their antioxidant activity against ASO.     

Comparison of Antistreptolysin O Latex Screening Test with the Antistreptolysin O Hemolytic Test

This report describes a comparison of the Behringwerke antistreptolysin O (ASO) latex screening test with the ASO hemolytic test. Agreement between the two tests was poor when Difco streptolysin O (SLO) reagent was employed in the hemolytic test; approximately 34% of the sera with ASO titers in the normal range of the hemolytic test gave false-positive latex test reactions. However, the percentage of false-positive latex test reactions was only 5% when Behringwerke SLO reagent was used in the hemolytic test. An assay of the Difco and Behringwerke SLO reagents against an ASO standard indicated that the Difco SLO reagent was more potent than the Behringwerke SLO reagent. The lack of agreement between the Behringwerke latex test and the hemolytic test using Difco SLO reagent is attributed to the potency of the SLO reagents.     

Effect of pH on anoxic sulfide oxidizing reactor performance

The effects of pH on the performance of anoxic sulfide oxidizing (ASO) reactor were evaluated. Performance was investigated under various operational conditions at influent pH range of 4-11. At the influent pH of 7-7.5 during loading tests and HRT tests, the sulfide oxidation was partial. In general, the amount of sulfate formed decreased with the increasing sulfide and nitrite loadings. The bacterial communities in ASO reactors were more sensitive to acidic pH compared with alkaline pH, as nitrite and sulfide removal rates dropped significantly when exposed to acidic pH 3. High dissolved bisulfide ions, nitrite and excess of sulfate (>300 mg/L) might have inhibited the sulfide oxidation under highly acidic and alkaline conditions in the ASO reactor. Based on sulfide and nitrite removal efficiencies, the ASO reactor can be operated in a wide range of pH, i.e. 5-11.     

Usefulness of F2-isoprostanes in early prognostication after cardiac arrest: a topical review of the literature and meta-analysis of preclinical data

Abstract

Prognostication after cardiac arrest (CA) represents a challenging issue, and several biomarkers have been proposed in the attempt to predict outcome. Among these, F2-isoprostanes stand out as potential biomarkers for early prognostication, providing information on the magnitude of global oxidative injury after return of spontaneous circulation (ROSC). We performed a topical review searching PubMed and Scopus databases to identify studies evaluating the modifications of F2-isoprostanes in the early period after CA, and a meta-analysis of studies providing curves of F2-isoprostanes plasma levels seeking to describe the biomarker’s kinetics after CA. Evidence suggests that plasma levels of F2-isoprostanes increase in the early post-resuscitation period and seem well correlated with the burden of ischaemia-reperfusion injury. Our meta-analysis shows a possible increase as early as 5?minutes after ROSC, which persists at 2?hours and is attenuated at 4?hours. Clinical studies are warranted to evaluate the utility of this biomarker for prognostication purposes in CA survivors.     

类风湿关节炎患者抗CCP，RF，AKA，ASO，RA33的临床价值分析

目的:分析抗抗环瓜氨酸肽(CCP)、类风湿因子(RF)、抗角蛋白抗体(AKA)、抗链球菌溶血素"O"(ASO、)抗RA33抗体对类风湿关节炎诊断的临床价值。方法:选取2015年3月至2016年2月本院收治的79例类风湿关节炎患者视为观察组,另选取同期本院收治的85例非类风湿关节炎自身免疫疾病者视为对照组。比较类风湿关节炎和非类风湿关节炎患者抗CCP、RF、AKA、ASO、RA33阳性情况,对抗CCP、RF、AKA、ASO、RA33的特异度和敏感度予以分析。结果:两组患者的ASO阳性率比较无显著性差异(P0.05),观察组的抗CCP、RF、AKA、RA33阳性率显著高于对照组(P0.05)。抗CCP抗体诊断类风湿关节炎患者的敏感度为64.56%、特异度为92.94%;RF敏感度为60.46%、特异度为80.00%;AKA敏感度为51.90%、特异度为96.47%;ASO敏感度为10.13%、特异度为89.41%;抗RA33抗体敏感度为30.38%、特异度为95.29%。结论:抗CCP、RF、AKA、RA33对类风湿关节炎患者均具有较高的诊断价值,而ASO在类风湿关节炎患者中的诊断价值不明显。     

Synthesis and biological evaluation of sialyl-oligonucleotide conjugates targeting leukocyte B trans-membranal receptor CD22 as delivery agents for nucleic acid drugs

Antisense oligonucleotides (ASOs) modified with ligands which target cell surface receptors have the potential to significantly improve potency in the target tissue. This has recently been demonstrated using triantennary N-acetyl d-galactosamine conjugated ASOs. CD22 is a cell surface receptor expressed exclusively on B cells thus presenting an attractive target for B cell specific delivery of drugs. Herein, we reported the synthesis of monovalent and trivalent ASO conjugates with biphenylcarbonyl (BPC) modified sialic acids and their study as ASO delivery agents into B cells. CD22 positive cells exhibited reduced potency when treated with ligand modified ASOs and mechanistic examination suggested reduced uptake into cells potentially as a result of sequestration of ASO by other cell-surface proteins.     

Topical delivery of interleukin‐13 antisense oligonucleotides with cationic elastic liposome for the treatment of atopic dermatitis

Background

Interleukin (IL)‐13, overproduced in the skin of atopic dermatitis (AD), has been shown to play an essential role in the pathogenesis of the disease. Thus, inhibition of IL‐13 production should provide a key step to alleviate disease conditions of the atopic skin. In the present study, IL‐13 antisense oligonucleotide (ASO) was designed and formulated with cationic elastic liposome (cEL) to improve transdermal delivery.

Methods

ASOs were generated against murine IL‐13 mRNA (+4 to + 23) and complexed with cEL. Physicochemical properties of IL‐13 ASO/cEL complex were examined by DNA retardation and DNase I protection assay. An in vitro inhibition study was performed in T‐helper 2 (Th2) cells and cytotoxicity was tested by the XTT assay. The in vivo effect of IL‐13 ASO/cEL complex was tested in a murine model of AD.

Results

In vitro, the IL‐13 ASO/cEL complex showed dose‐ and ratio‐dependent inhibition of IL‐13 secretion in Th2 cells. At the IL‐13 ASO/cEL ratio of 6, maximum inhibition of IL‐13 secretion was observed. When applied to the ovalbumin‐sensitized murine model of AD, topically administered IL‐13 ASO/cEL complex dramatically suppressed IL‐13 production (by up to 70% of the control) in the affected skin region. In addition, the levels of IL‐4 and IL‐5 were also significantly reduced. Moreover, IL‐13 ASO/cEL‐treated AD mice showed reduced infiltration of inflammatory cells into the epidermal and dermal areas, with concomitant reduction of skin thickness.

Conclusions

These data suggests that IL‐13 ASO/cEL complex can provide a potential therapeutic tool for the treatment of AD and also be applied to other immune diseases associated with the production of Il‐13. Copyright © 2008 John Wiley & Sons, Ltd.     

Hepatic ACAT2 Knock Down Increases ABCA1 and Modifies HDL Metabolism in Mice

Objectives

ACAT2 is the exclusive cholesterol-esterifying enzyme in hepatocytes and enterocytes. Hepatic ABCA1 transfers unesterified cholesterol (UC) to apoAI, thus generating HDL. By changing the hepatic UC pool available for ABCA1, ACAT2 may affect HDL metabolism. The aim of this study was to reveal whether hepatic ACAT2 influences HDL metabolism.

Design

WT and LXRα/β double knockout (DOKO) mice were fed a western-type diet for 8 weeks. Animals were i.p. injected with an antisense oligonucleotide targeted to hepatic ACAT2 (ASO6), or with an ASO control. Injections started 4 weeks after, or concomitantly with, the beginning of the diet.

Results

ASO6 reduced liver cholesteryl esters, while not inducing UC accumulation. ASO6 increased hepatic ABCA1 protein independently of the diet conditions. ASO6 affected HDL lipids (increased UC) only in DOKO, while it increased apoE-containing HDL in both genotypes. In WT mice ASO6 led to the appearance of large HDL enriched in apoAI and apoE.

Conclusions

The use of ASO6 revealed a new pathway by which the liver may contribute to HDL metabolism in mice. ACAT2 seems to be a hepatic player affecting the cholesterol fluxes fated to VLDL or to HDL, the latter via up-regulation of ABCA1.     

Reduction of liver tumor necrosis factor‐α expression by targeting delivery of antisense oligonucleotides into Kupffer cells protects rats from fulminant hepatitis

Background

Fulminant liver failure can cause extreme mortality due to the lack of effective and targeting therapeutics for the disease. Novel therapeutics using antisense technology require an efficient and safe delivery system with Kupffer cell targeting ability.

Methods

We explored the capacity of galactosylated low molecular weight chitosan (GLC) to efficiently mediate the antisense oligonucleotide (ASO) TJU‐2755 into Kupffer cells, enhance the effect of the oligonucleotides on the suppression of tumor necrosis factor (TNF)‐α and prolong the active time of the antisense drug in vivo. The protective and therapeutic effect of ASO/GLC in the animal model of D ‐galactosamine/lipopolysaccharide‐induced fulminant hepatitis was tested.

Results

ASOs delivered by GLC were concentrated in Kupffer cells and more potent in reducing the expression of TNF‐α mRNA, as well as reducing serum TNF‐α levels. Furthermore, the ASO/GLC complex successfully rescued animals from fulminant hepatitis and mortality. Compared to naked ASO, the complex notably reduced the dose administrated in animals and prolonged its effectiveness. A single dose of 5 mg ASO per kg body weight achieved a satisfactory effect after 5 days, and 20 mg ASO per kg body weight preserved 70% of the effect after more than 2 weeks. Its efficacy was affirmed through both pretreatment and therapeutic use after liver damage had begun.

Conclusions

Inhibiting TNF‐α expression in the liver by this strategy represents a novel therapeutic approach that may be valuable for the treatment of some inflammation‐related liver diseases. Copyright © 2009 John Wiley & Sons, Ltd.     

腔内治疗下肢动脉硬化闭塞症的临床研究

目的：下肢动脉硬化闭塞症（arteriosclerosis obliterans,ASO）是血管外科常见疾病,我们拟讨论腔内成形术在ASO治疗中的应用。方法：回顾2008年5月至2010年4月我科收治的76例ASO病例,对手术成功率、手术效果和糖尿病等合并症对疗效的影响进行分析。结果：总体技术成功61例,技术成功率80.3%,Fontaine II期组技术成功36例（成功率92.3%）,明显高于III期（82.1%）和IV期组（22.2%）;合并糖尿病组22例（28.9%）,技术成功15例（成功率68.2%）,低于无糖尿病组的85.2%;结论：腔内成形术是治疗ASO的有效微创手术方法,积极腔内手术干预有助提高早期患者成功率,同时对晚期患者可有效改善症状。     

Photoperiodic activation of brains in castrates and the role of the gonad in reproductive maturation ofLimax maximus

Summary Results obtained with castration and gonad implantation verify that a factor(s) from the gonad is required for development of the female accessory sex organs (ASO) inLimax maximus. In addition, they show that a gonadal factor promotes rapid gonadal growth and sperm production, and gradual or incomplete development of the penis. Although an immature slug that is castrated and then switched to inductive photoperiods fails to exhibit reproductive tract maturation, in two differently designed experimental tests, the brains of such animals were able to promote sexual maturation when subsequently paired with an immature gonad. We interpret the results of these experiments as evidence for photoperiodic induction of secretion of maturation-inducing factor (MH) by the brain in the absence of the gonad. One or more gonadal hormones appear to be both necessary and sufficient for production of sperm and for development of ASO, but they are not required for the photoperiodic stimulation of MH release.Abbreviations ASO accessory sex organs - MH maturation hormone     

Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides

The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5′-terminus of well-characterised 2′,4′-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.