A modified Sindbis vector for prolonged gene expression in neurons

Sindbis viruses have been widely used in neurobiology to express a variety of genes in cultured neurons, in cultured slices, and in vivo. They provide fast onset and high levels of expression of foreign genes, but the expression is limited to a short time window due to a shut-off of host protein synthesis. We have used a mutation in an essential gene (nsP2) of the life cycle of Sindbis, which allows the functional analysis of changes in protein expression for >/=6 days after infection. This Sindbis mutant (nsP2) was used to express enhanced green fluorescent protein (EGFP) in hippocampal neurons in culture and in vivo without any sign of toxicity, based on two-photon imaging and electrophysiology. In addition, the EGFP mutant virus can be injected in vivo to visualize spines and other details of neuronal structure. The Sindbis mutant described here provides an improved tool in neurobiology with reduced cytotoxicity and a prolonged time window of expression for novel applications in imaging and behavior. In addition, the use of this vector for the functional expression of mammalian voltage-gated ion channels in organotypic slices is demonstrated.     

Rapid Myopic Progression in Childhood Is Associated With Teenage High Myopia

PurposeThe purpose of this study was to evaluate the association of childhood progression of spherical equivalent (SE) with high myopia (HM) in teenagers in the Singapore Cohort of Risk factors for Myopia (SCORM).MethodsWe included 928 SCORM children followed over a mean follow-up of 6.9 ± 1.0 years from baseline (6–11 years old) until their teenage years (12–19 years old). Cycloplegic autorefraction and axial length (AL) measurements were performed yearly. The outcomes in teenagers were HM (SE ≤ −5 diopter [D)], AL ≥ 25 mm, SE and AL. Three-year SE and AL progression in childhood and baseline SE and AL with outcomes were evaluated using multivariable logistic or linear regression models, with predictive performance of risk factors assessed using the area under the curve (AUC).ResultsAt the last visit, 9.8% of teenagers developed HM and 22.7% developed AL ≥ 25 mm. In multivariate regression analyses, every −0.3 D/year increase in 3-year SE progression and every 0.2 mm/year increase in 3-year AL progression were associated with a −1.14 D greater teenage SE and 0.52 mm greater teenage AL (P values < 0.001). The AUC (95% confidence interval [CI]) of a combination of 3-year SE progression and baseline SE for teenage HM was 0.97 (95% CI = 0.95 – 0.98). The AUC of 3-year AL progression and baseline AL for teenage AL ≥ 25 mm was 0.91 (95% CI = 0.89 – 0.94).ConclusionsThree-year myopia progression in childhood combined with baseline SE or AL were good predictors of teenage HM. Clinicians may use this combination of factors to guide timing of interventions, potentially reducing the risk of HM later in life.     

Counterpoint: the myeloperoxidase -463G-->a polymorphism does not decrease lung cancer susceptibility in Caucasians.

The myeloperoxidase (MPO) G-to-A substitution polymorphism in the promoter region of the MPO gene has been associated with a 40-70% reduction in lung cancer risk in several studies, although a recent nested case-control study disputes these findings. MPO is involved in the activation of a number of procarcinogens, including benzo(a)pyrene. The variant A allele has been shown to reduce MPO mRNA expression, thus potentially decreasing carcinogen activation. To confirm results from smaller studies, we evaluated this MPO polymorphism in 988 incident Caucasian lung cancer cases and 1128 controls. Logistic regression evaluated the association between MPO genotype and lung cancer risk, adjusting for age, gender, smoking status, time since quitting smoking, and pack-years of smoking. In the controls, the A allele frequency was 21%, and genotype distribution was in the Hardy-Weinberg equilibrium. Compared with the wild-type G/G genotype, the adjusted odds ratios for the A/A and A/G genotypes were 1.15 (95% confidence interval 0.7-1.9, P > 0.2) and 1.03 (95% confidence interval 0.8-1.3, P > 0.20), respectively. A similar lack of association was seen in analyses stratified by smoking status, median age, a number of smoking variables, disease stage, tumor grade, and histological subtype. These findings are in contrast with earlier studies suggesting a protective effect of carrying the variant A allele.     

Calcium signaling at single mossy fiber presynaptic terminals in the rat hippocampus

We investigated internal Ca(2+) release at mossy fiber synapses on CA3 pyramidal neurons (mossy fiber terminals, MFTs) in the hippocampus. Presynaptic Ca(2+) influx was induced by giving a brief train of 20 stimuli at 100 Hz to the mossy fiber pathway. Using Ca(2+) imaging techniques, we recorded the Ca(2+) response as DeltaF/F, which increased rapidly with stimulation, but was often accompanied by a delayed peak that occurred after the train. The rise in presynaptic [Ca(2+)] could be completely blocked by application of 400 microM Cd(2+). Furthermore, the evoked Ca(2+) signals were reduced by group II mGluR agonists. Under the same experimental conditions, we investigated the effects of several agents on MFTs that disrupt regulation of intracellular Ca(2+) stores resulting in depletion of internal Ca(2+). We found that ryanodine, cyclopiazonic acid, thapsigargin, and ruthenium red all decreased both the early and the delayed increase in the Ca(2+) signals. We applied D,L-2-amino-5-phosphonovaleric acid (D,L-APV; 50 microM) and 6,7-Dinitroquinoxaline-2,3-dione (DNQX; 20 microM) to exclude the action of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Experiments with alternative lower affinity indicators for Ca(2+) (fura-2FF and calcium green-2) and the transient K(+) channel blocker, 4-aminopyridine were performed to control for the possible saturation of fura-2. Taken together, these results strongly support the hypothesis that the recorded terminals were from the mossy fibers of the dentate gyrus and suggest that a portion of the presynaptic Ca(2+) signal in response to brief trains of stimuli is due to release of Ca(2+) from internal stores.     

Activation of M2 muscarinic receptors leads to sustained suppression of hippocampal transmission in the medial prefrontal cortex

Cholinergic innervation of the prefrontal cortex is critically involved in arousal, learning and memory. Dysfunction of muscarinic acetylcholine receptors and their downstream signalling pathways has been identified in mental retardation. To assess the role played by the muscarinic receptors at the hippocampal–frontal cortex synapses, an important relay in information storage, we used a newly developed frontal slice preparation in which hippocampal afferent fibres are preserved. Transient activation of muscarinic receptors by carbachol results in a long-lasting depression of synaptic efficacy at the hippocampal but not cortical pathways or local circuitry. On the basis of a combination of electrophysiological, pharmacological and anatomical results, this input-specific muscarinic modulation can be partially attributed to the M2 subtype of muscarinic receptors, possibly through a combination of pre- and postsynaptic mechanisms.     

An OspA-based genospecies identification of Lyme disease spirochetes (Borrelia burgdorferi) isolated in Taiwan

To further identify the genospecies of Lyme disease spirochetes (Borrelia burgdorferi) isolated in Taiwan, we analyzed the genomic identities of these Taiwan isolates (TWKM1-7) by genospecies-specific polymerase chain reaction (PCR) assay, restriction fragment length polymorphism (RFLP) analysis, and gene sequencing based on the OspA gene sequences of B. burgdorferi sensu lato. PCR analysis indicates that all of these Taiwan isolates were genetically related to the genospecies of B. burgdorferi sensu stricto by their differential reactivities with genospecies-specific PCR primers. After cleavage by DraI, three different RFLP patterns in relation to three different genospecies of Lyme disease spirochetes were observed, and all of these Taiwan isolates were affiliated with the genospecies of B. burgdorferi sensu stricto. The phylogenetic analysis also reveals that the sequence similarity of PCR-amplified OspA gene of these Taiwan isolates is highly homogeneous, with a homogeneity of more than 99.8% within the genospecies of B. burgdorferi sensu stricto. These results confirm that the genomic identities of these Taiwan isolates belong to the genospecies of B. burgdorferi sensu stricto.     

早期PRP与复方樟柳碱颞浅动脉旁穴位注射治疗RR的疗效

目的：观察早期视网膜激光光凝（PRP）联合复方樟柳碱颞浅动脉旁皮下注射治疗放射性视网膜病变（RR）的临床疗效。方法：在我院确诊为因鼻咽癌外照射后发生RR的患者21例41眼,早期行双眼视网膜激光光凝联合复方樟柳碱颞浅动脉旁穴位注射治疗,观察治疗后3mo最佳矫正视力（BCVA）、毛细血管无灌注区变化、视网膜新生血管及并发症情况。结果：治疗后观察3mo,视力提高6眼（14.6%）,视力不变31眼（75.7%）,下降4眼（9.7%）。2眼虹膜新生血管消退,1眼行睫状体光凝术;9眼（75%）视网膜新生血管消退;22眼（68.7%）原视网膜无灌注区消失,7眼（21.8%）原视网膜无灌注区（NPA）缩小〉5个DA,总有效率90.5%。1眼发生玻璃体出血,未见视盘及虹膜新生血管和新生血管性青光眼等并发症以及与治疗相关的并发症。结论：早期PRP与复方樟柳碱颞浅动脉旁穴位注射治疗是有效的、合理的中西医结合治疗方法。     

糖尿病视网膜病变合并视网膜静脉阻塞的临床分析

目的: 分析糖尿病视网膜病变(DR)合并视网膜静脉阻塞(RVO)的眼底特征及荧光素眼底血管造影(FFA)的图像特征。方法: 回顾性分析118例129眼DR合并RVO患者的视力、眼底、荧光素眼底血管造影(FFA)的图像特征及相关临床资料。结果: DR合并RVO的患者118例中,双眼同时发病有11例22眼,其余皆为单眼,其中66眼表现为视网膜中央静脉阻塞(CRVO),占51.2%,58眼表现为颞上分支静脉阻塞,占45.0%,其它分支静脉阻塞有5眼,占3.9%。FFA表现为:静脉阻塞区视网膜有大量神经纤维层出血,相应黄斑区荧光渗漏,掩盖了此眼DR的改变,对侧眼均可见DR不同级别的改变。结论: 糖尿病视网膜病变合并视网膜静脉阻塞眼底表现复杂,应与单一的糖尿病视网膜病变和视网膜静脉阻塞甄别。     

不同胎龄早产儿生后早期对脂肪乳的耐受性研究

目的分析不同出生胎龄早产儿生后早期对脂肪乳的耐受性。方法 98例早产儿分为超早产儿组(n=17)、早期早产儿组(n=48)和中晚期早产儿组(n=33),再根据脂肪乳剂量分为低剂量脂肪乳与高剂量两个亚组,留取脐血及生后前3 d的血干滤纸片,用串联质谱法检测短、中、长链酰基肉碱含量。结果超早产儿组与早期早产儿组脐血及生后前3 d长链酰基肉碱浓度均低于中晚期早产儿组(P0.05),且长链酰基肉碱浓度与胎龄呈正相关(P0.01)。超早产儿低剂量脂肪乳组生后第2天的短、中、长链酰基肉碱浓度均高于高剂量组(P0.05),而早期早产儿与中晚期早产儿不同剂量脂肪乳亚组的生后3 d短、中、长链酰基肉碱浓度差异均无统计学意义。结论超早产儿和早期早产儿生后前3 d对长链脂肪酸的代谢能力均低于中晚期早产儿;早期早产儿与中晚期早产儿生后早期可以耐受高剂量脂肪乳,但超早产儿生后早期对高剂量脂肪乳代谢能力可能不足。     

Cytotoxic C21 and C22 terpenoid-derived metabolites from the sponge Ircinia sp

One novel C21 terpenoidal natural product, ircinolin A (2), two new C22 furanoterpene metabolites, 15-acetylirciformonin B (3) and 10-acetylirciformonin B (4), and two known compounds, irciformonin B (1) and irciformonin F (5), were isolated from the sponge Ircinia sp. The structures of these compounds were elucidated on the basis of their spectroscopic data. Moreover, the absolute configuration of 1 was determined by Mosher's method. Among these metabolites, 2 is the first C21 terpenoid-derived metabolite to be reported from this genus. Compounds 1 and 3-5 exhibited significant cytotoxic activity against K562, DLD-1, HepG2, and Hep3B cancer cell lines.