多重聚合酶链反应与荚膜肿胀试验对肺炎链球菌血清分型的比较研究

为评估多重聚合酶链反应（PCR ）对肺炎链球菌血清分型的可行性,分别采用多重PCR和荚膜肿胀试验对568株肺炎链球菌进行血清分型,并对分型结果进行比较分析。结果显示,568株肺炎链球菌中,213株通过荚膜肿胀试验分出16个血清群,主要有血清群19（23．1％,131／568）、6（5．3％,30／568）、23（1．6％,9／568）、14（1．4％,8／568）、9（1．1％,6／568）、15（1．1％,6／568）等,分型率为37．5％（213／568）;356株通过多重PCR分出21个血清群,主要有血清群19（27．8％,158／568）、23（8．5％,48／568）、6（7．4％,42／568）、14（4．4％,25／568）、3（4．2％,24／568）、15（3．5％,20／568）等,分型率为62．7％（356／568）。荚膜肿胀试验鉴定出血清群4和18,但多重PCR未能鉴定;多重PCR鉴定出血清群5、12、35、16、17和22,但荚膜肿胀试验未能鉴定。多重PCR与荚膜肿胀试验对19F、19A血清型的鉴定无显著差异。结果提示,这2种方法对肺炎链球菌血清分型结果有差别,多重PCR的分型率高于荚膜肿胀试验。对来源复杂的标本进行肺炎链球菌血清分型,2种方法可相互补充,以提高分型率。     

Alphavirus Replicon DNA Expressing HIV Antigens Is an Excellent Prime for Boosting with Recombinant Modified Vaccinia Ankara (MVA) or with HIV gp140 Protein Antigen

Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.     

红芽芋球茎片两步法离体快繁及其再生苗生理和光合特性研究

以江西铅山红芽芋(Colocasia esculenta L.Schott var.cormosus‘Hongyayu’)试管苗为材料,建立了芋球茎片两步法离体快繁体系,并对其再生苗的形态指标、染色体数目、生理和光合特性以及叶绿素荧光特性进行了检测。结果表明:(1)红芽芋球茎片单芽诱导的最佳培养基为MS+KT 2 mg/L+6-BA 1 mg/L+NAA0.1mg/L,诱导培养30d后将单芽从球茎片上分离,再接种到生根培养基(MS+KT 2mg/L+NAA 0.1mg/L)上培养30d即可形成完整植株,移栽成活率高达98%;(2)由球茎片单芽、丛生芽、不定芽离体快繁获得的红芽芋再生苗在形态指标、叶下表皮气孔参数、染色体数目、生理生化指标以及叶片光合特性参数和叶绿素荧光特性方面均无显著差异。说明红芽芋球茎片两步法离体培养的再生苗繁殖系数高、染色体数目稳定,该离体快繁体系可应用于江西铅山红芽芋的工厂化生产。     

Retinal Nerve Fiber and Optic Disc Morphology in Patients with Human Immunodeficiency Virus Using the Heidelberg Retina Tomography 3

Purpose

To use novel confocal scanning ophthalmoscopy technology to test hypothesis that HIV-seropositive patients without history of retinitis with a history of a low CD4 count are more likely to have damage to their retinal nerve fiber layer (RNFL) when compared to patients with high CD4 count. In addition, we compared optic disc morphologic changes with glaucoma.

Design

Cross-sectional study.

Participants and Controls

171 patients were divided into four groups. The control group consisted of 40 eyes of 20 HIV-seronegative patients. The second group consisted of 80 eyes of 41 HIV-positive patients whose CD4 cell count never dropped below 100 (1.0 x 10⁹/L). The third group consisted of 44 eyes of 26 HIV-positive patients with a history of low CD4 counts <100. Fourth group consisted of 79 eyes of 79 patients with confirmed glaucoma who served as positive controls.

Testing

Confocal scanning laser ophthalmoscopy was performed with the Heidelberg Retina Tomograph (HRT3) and data were analyzed with HRT3, software (Heyex version 1.5.10.0).

Main Outcome Measures

Disc area, cup area, cup volume, rim volume, mean cup depth, maximum cup depth, cup-to-disc ration, mean RNFL thickness, and RNFL cross-sectional area.

Results

Analysis of the global optic nerve and cup parameters showed no difference in disk area among the four groups. There was also no difference in cup, rim volume, mean cup depth, or maximum cup depth among the first three groups but they were all different from glaucoma group. The RNFL was thinner in glaucoma and both HIV-positive groups compared to HIV-seronegative subjects. The cross sectional RNFL area was thinner in both high and low CD4 HIV-positive groups compared to HIV-seronegative group in the nasal and temporal/inferior sectors, respectively. Glaucoma group showed thinning in all sectors.

Conclusions

HIV retinopathy results in retinal nerve fiber layer loss without structural optic nerve supportive tissue change. RNFL damage may occur early in HIV disease by mechanism different than in glaucoma.     

Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads

A high-sensitivity assay has been developed for the detection of human topoisomerase I with single molecule resolution. The method uses magnetic sepharose beads to concentrate rolling circle products, produced by the amplification of DNA molecules circularized by topoisomerase I and detectable with a confocal microscope as single and discrete dots, once reacted with fluorescent probes. Each dot, corresponding to a single cleavage–religation event mediated by the enzyme, can be counted due to its high signal/noise ratio, allowing detection of 0.3 pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples.     

Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: A new paradigm for understanding tumor metabolism,the field effect and genomic instability in cancer cells

Loss of stromal fibroblast caveolin-1 (Cav-1) is a powerful single independent predictor of poor prognosis in human breast cancer patients, and is associated with early tumor recurrence, lymph node metastasis and tamoxifen-resistance. We developed a novel co-culture system to understand the mechanism(s) by which a loss of stromal fibroblast Cav-1 induces a “lethal tumor microenvironment.” Here, we propose a new paradigm to explain the powerful prognostic value of stromal Cav-1. In this model, cancer cells induce oxidative stress in cancer-associated fibroblasts, which then acts as a “metabolic” and “mutagenic” motor to drive tumor-stroma co-evolution, DNA damage and aneuploidy in cancer cells. More specifically, we show that an acute loss of Cav-1 expression leads to mitochondrial dysfunction, oxidative stress and aerobic glycolysis in cancer associated fibroblasts. Also, we propose that defective mitochondria are removed from cancer-associated fibroblasts by autophagy/mitophagy that is induced by oxidative stress. As a consequence, cancer associated fibroblasts provide nutrients (such as lactate) to stimulate mitochondrial biogenesis and oxidative metabolism in adjacent cancer cells (the “Reverse Warburg effect”). We provide evidence that oxidative stress in cancer-associated fibroblasts is sufficient to induce genomic instability in adjacent cancer cells, via a bystander effect, potentially increasing their aggressive behavior. Finally, we directly demonstrate that nitric oxide (NO) over-production, secondary to Cav-1 loss, is the root cause for mitochondrial dysfunction in cancer associated fibroblasts. In support of this notion, treatment with anti-oxidants (such as N-acetyl-cysteine, metformin and quercetin) or NO inhibitors (L-NAME) was sufficient to reverse many of the cancer-associated fibroblast phenotypes that we describe. Thus, cancer cells use “oxidative stress” in adjacent fibroblasts (1) as an “engine” to fuel their own survival via the stromal production of nutrients and (ii) to drive their own mutagenic evolution towards a more aggressive phenotype, by promoting genomic instability. We also present evidence that the “field effect” in cancer biology could also be related to the stromal production of ROS and NO species. eNOS-expressing fibroblasts have the ability to downregulate Cav-1 and induce mitochondrial dysfunction in adjacent fibroblasts that do not express eNOS. As such, the effects of stromal oxidative stress can be laterally propagated, amplified and are effectively “contagious”—spread from cell-to-cell like a virus—creating an “oncogenic/mutagenic” field promoting widespread DNA damage.Key words: caveolin-1, cancer associated fibroblasts, oxidative stress, reactive oxygen species (ROS), mitochondrial dysfunction, autophagy, nitric oxide (NO), DNA damage, aneuploidy, genomic instability, anti-oxidant cancer therapy, the “field effect” in cancer biology     

不明原因男性不育人群中睾丸特异性乳酸脱氢酶基因突变的发现及其意义

为了研究睾丸特异性乳酸脱氢酶,即乳酸脱氢酶C4(LDH-C4)基因突变在男性不育发病中的作用,利用LDH-C4特异性底物对100名不明原因男性不育症患者的精子LDH-C4进行活性显色,用变性高效液相色谱(DHPLC)技术对LDH-C4活性低下的患者进行LDHC基因PCR产物的突变筛查,对DHPLC峰形异常的PCR产物进行序列测定.筛选到一组精子LDH-C4活性明显下降的患者,其中1名患者的LDHC基因PCR产物在DHPLC中呈异常洗脱峰.对这一PCR产物进行序列测定,发现患者LDHC基因第5外显子的115位碱基发生了T→A的杂合改变(GenBank登录号GU479375),该突变使LDHC基因的178位密码子由原来的TTG(编码亮氨酸)变为TAG(终止密码子),形成截短的C亚基.T克隆-测序进一步证实了该无义突变的杂合状态.这是在人类LDHC基因上发现的第一个突变,提示LDHC基因突变可能是男性不育发病的原因之一.     

青海湟水流域蝶类

1995～2003年调查并整理分析青海湟水流域的蝶类,有9科52属80种,其中凤蝶科1属2种,绢蝶科1属6种,粉蝶科8属22种,斑蝶科1属1种,眼蝶科12属13种,蛱蝶科15属18种,蚬蝶科1属3种,灰蝶科9属10种,弄蝶科4属5种.以古北界为主,青藏区最为丰富.     

Effects of experimental seismic shock on vasoactivity of arteries, integrity of the vascular endothelium and on primary stress hormones of the Atlantic salmon

The postulated harmful effects of underwater detonations of explosives on the vascular endothelium in farmed Atlantic salmon were assessed under controlled conditions. Acclimated salmon were exposed to a series of 10 underwater explosions over 70 min, each of ≃2 MPa in pressure amplitude, in a laboratory tank. No mortality occurred immediately or during the subsequent 7 days of observation. The response to each of the 10 detonations was cessation of swimming for a few minutes and failure to express a flight reaction.
Structurally, the vascular endothelium of the ventral aorta (VA) and the coeliaco mesenteric artery (CMA) revealed signs of injury within the first 30 min after the experimental shock. In contrast to vessels from the controls, the injury was further aggravated by the mounting procedure for tension recording. The endothelial impairment was temporary, persisting throughout the first days while being restored after 1 week.
Functionally, the cholinergic and adrenergic vasoconstrictor responses in the CMA were markedly reduced during the first day after the shock. These responses were similar to those observed in the endothelium probed CMA of controls. The loss of structural integrity and the reduced functional responses indicated a temporary impairment of the vascular endothelium in response to this experimental simulation of a seismic shock.
The primary stress hormones, adrenaline and cortisol, were not immediately elevated in plasma, but revealed different patterns of delayed increases. The head kidney content of catecholamines was not altered by the acoustic shock, while the atrial uptake of both calecholamines declined progressively during the 48 h of observation. Plasma chloride was not affected.     

Resolution of Holliday junction substrates by human topoisomerase I

Prompted by the close relationship between tyrosine recombinases and type IB topoisomerases we have investigated the ability of human topoisomerase I to resolve the typical intermediate of recombinase catalysis, the Holliday junction. We demonstrate that human topoisomerase I catalyzes unidirectional resolution of a synthetic Holliday junction substrate containing two preferred cleavage sites surrounded by DNA sequences supporting branch migration. Deleting part of the N-terminal domain (amino acid residues 1-202) did not affect topoisomerase I resolution activity, whereas a topoisomerase I variant lacking both the N-terminal domain and amino acid residues 660-688 of the linker domain was unable to resolve the Holliday junction substrate. The inability of the double deleted variant to mediate resolution correlated with the inability of this enzyme to introduce concomitant cleavage at the two preferred cleavage sites in a single Holliday junction substrate, which is a prerequisite for resolution. As determined by the gel electrophoretic mobility of native enzyme or enzyme crosslinked by disulfide bridging, the double deleted mutant existed almost entirely in a dimeric form. The impairment of this enzyme in performing double cleavages on the Holliday junction substrate may be explained by only one cleavage competent active site being formed at a time within the dimer. The assembly of only one active site within dimers is a well-known characteristic of the tyrosine recombinases. Hence, the obtained results may suggest a recombinase-like active site assembly of the double deleted topoisomerase I variant. Taken together the presented results consolidate the relationship between type IB topoisomerases and tyrosine recombinases.