肠毒素性大肠杆菌CS3纤毛抗原在痢疾菌中的表达及其免疫效果研究

首先通过体内外重组的方法,构建了福氏2a痢疾菌T32asd基因缺陷的突变体FaD,作为抗原载体菌;同时,构建包含asd基因的表达质粒pYX102,与FaD一起,构成宿主-载体平衡致死系统,用于在没有抗生素条件选择的情况下,稳定表达克隆在表达质粒上的外源抗原基因.将肠毒素性大肠杆菌的CS3菌毛抗原基因克隆至pYX102,构建成重组表达质粒pYX103,ELISA检测结果证实CS3在痢疾菌中可以很好地表达.免疫小鼠后可诱生相应的抗体,虽然口服免疫和注射免疫产生的CS3抗体效价有一定差别,但对痢疾菌的毒株攻击均可提供较好保护.该结果为细菌性腹泻疫苗的研制提供了候选株.     

Spina bifida cystica and severe congenital bilateral talipes equinovarus in one twin of a monoamniotic pair: a case report

Background

Spina bifida and congenital talipes equinovarus (CTEV) are common congenital malformations which may occur together and increase morbidity. Monozygous twins are particularly at risk of these malformations and discordance in one type of malformation is typical. The occurrence of both spina bifida and CTEV in one twin of a monozygotic pair is rare.

Case presentation

A 22 year-old Cameroonian primigravida at 36 weeks of a twin gestation was received in our district hospital at the expulsive phase of labour on a background of sub-optimal antenatal care. A caesarean section indicated for cephalo-pelvic disproportion was performed and life monoamniotic male twins were extracted. The first twin was normal. The second twin had spina bifida cystica and severe bilateral CTEV. Routine postnatal care was ensured and at day 2 of life, the affected twin was evacuated to a tertiary hospital for proper management. He was later on reported dead from complications of hydrocephalus.

Conclusions

Spina bifida cystica with severe bilateral CTEV in one twin of a monoamniotic pair illustrates the complexity in the interplay of causal factors of these malformations even among monozygotic twins who are assumed to share similar genetic and environmental features. The occurrence and poor outcome of the malformations was probably potentiated by poor antenatal care. With postnatal diagnoses, a better outcome was difficult to secure even with prompt referral. Early prenatal diagnoses and appropriate counseling of parents are cardinal.

     

Time and dose dependence of pluronic bioactivity in hyperthermia-induced tumor cell death

Pluronic block copolymers have been shown to sensitize cancer cells resulting in an increased activity of antineoplastic agents. In the current study we examined a new application of Pluronic bioactivity in potentiating hyperthermia-induced cancer cell injury. DHD/K12/TRb rat adenocarcinoma cells were exposed to low-grade hyperthermia at 43 degrees C with or without Pluronic P85 or Pluronic L61. A range of Pluronic doses, pre-exposure and heat exposure durations were investigated, and the test conditions were optimized. Treatment efficacy was assessed by measurement of intracellular ATP and mitochondrial dehydrogenase activity. Both P85 and L61 in synergy with heat reduced cell viability appreciably compared to either heat or Pluronic alone. Under optimal conditions, P85 (10 mg/ml, 240 mins) combined with 15 mins heat reduced intracellular ATP to 60.1 +/- 3.5% of control, while heat alone and P85 without heat caused a negligible decrease in ATP of 1.2% and 3.8%, respectively. Similarly, cells receiving 120 mins pre-exposure of L61 (0.3 mg/ml) showed reduction in intracellular ATP to 14.1 +/- 2.1% of control. Again, heat or L61 pre-exposure alone caused a minor decrease in levels of intracellular ATP (1.5% and 4.4%, respectively). Comparable results were observed when viability was assessed by mitochondrial enzyme activity. Survival studies confirmed that the loss of viability translates to a long-term reduction in proliferative activity, particularly for L61 treated cells. Based on these results, we conclude that Pluronic is effective in improving hyperthermic cancer treatment in vitro by potentiating heat-induced cytotoxicity in a concentration and time dependent manner.     

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

Background

Oxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not received sufficient attention in related studies. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H₂O₂).

Results

Using geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H₂O₂. Genes with the most stable expression according to geNorm were U6, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB.

Conclusion

Taken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression performed by qRT-PCR assays in HUVECs under oxidative stress study.

     

Intraclass diversification of immunoglobulin heavy chain genes in the African lungfish

Lungfish (Dipnoi) are the closest living relatives to tetrapods, and they represent the transition from water to land during vertebrate evolution. Lungfish are armed with immunoglobulins (Igs), one of the hallmarks of the adaptive immune system of jawed vertebrates, but only three Ig forms have been characterized in Dipnoi to date. We report here a new diversity of Ig molecules in two African lungfish species (Protopterus dolloi and Protopterus annectens). The African lungfish Igs consist of three IgMs, two IgWs, three IgNs, and an IgQ, where both IgN and IgQ originated evidently from the IgW lineage. Our data also suggest that the IgH genes in the lungfish are organized in a transiting form from clusters (IgH loci in cartilaginous fish) to a translocon configuration (IgH locus in tetrapods). We propose that the intraclass diversification of the two primordial gnathostome Ig classes (IgM and IgW) as well as acquisition of new isotypes (IgN and IgQ) has allowed lungfish to acquire a complex and functionally diverse Ig repertoire to fight a variety of microorganisms. Furthermore, our results support the idea that “tetrapod-specific” Ig classes did not evolve until the vertebrate adaptation to land was completed ~360 million years ago.     

Rupture of plasma membrane under tension

We present a study on the rupture behavior of single NIH 3T3 mouse fibroblasts under tension using micropipette aspiration. Membrane rupture was characterized by breaking and formation of an enclosed membrane linked to a tether at the cell apex. Three different rupture modes, namely: single break, initial multiple breaks, and continuous multiple breaks, were observed under similar loading condition. The measured mean tensile strengths of plasma membrane were 3.83 ± 1.94 and 3.98 ± 1.54mN/m for control cells and cells labeled with TubulinTracker, respectively. The tensile strength data was described by Weibull distribution. For the control cells, the Weibull modulus and characteristic strength were 1.86 and 4.40 mN/m, respectively; for cells labeled with TubulinTracker, the Weibull modulus and characteristic strength were 2.68 and 4.48 mN/m, respectively. Based on the experimental data, the estimated average transmembrane proteins-lipid cleavage strength was 2.64 ± 0.64 mN/m. From the random sampling of volume ratio of transmembrane proteins in cell membrane, we concluded that the Weibull characteristic of plasma membrane strength was likely to be originated from the variation in transmembrane proteins-lipid interactions.     

Genetic variations in Tibetan populations and high-altitude adaptation at the Himalayas

Modern humans have occupied almost all possible environments globally since exiting Africa about 100,000 years ago. Both behavioral and biological adaptations have contributed to their success in surviving the rigors of climatic extremes, including cold, strong ultraviolet radiation, and high altitude. Among these environmental stresses, high-altitude hypoxia is the only condition in which traditional technology is incapable of mediating its effects. Inhabiting at >3,000-m high plateau, the Tibetan population provides a widely studied example of high-altitude adaptation. Yet, the genetic mechanisms underpinning long-term survival in this environmental extreme remain unknown. We performed an analysis of genome-wide sequence variations in Tibetans. In combination with the reported data, we identified strong signals of selective sweep in two hypoxia-related genes, EPAS1 and EGLN1. For these two genes, Tibetans show unusually high divergence from the non-Tibetan lowlanders (Han Chinese and Japanese) and possess high frequencies of many linked sequence variations as reflected by the Tibetan-specific haplotypes. Further analysis in seven Tibetan populations (1,334 individuals) indicates the prevalence of selective sweep across the Himalayan region. The observed indicators of natural selection on EPAS1 and EGLN1 suggest that during the long-term occupation of high-altitude areas, the functional sequence variations for acquiring biological adaptation to high-altitude hypoxia have been enriched in Tibetan populations.