Co-expression of Interleukin-15 Enhances the Protective Immune Responses Induced by Immunization with a Murine Malaria MVA-Based Vaccine Encoding the Circumsporozoite Protein

Malaria remains a major global public health problem with an estimated 200 million cases detected in 2012. Although the most advanced candidate malaria vaccine (RTS,S) has shown promise in clinical trials, its modest efficacy and durability have created uncertainty about the impact of RTS,S immunization (when used alone) on global malaria transmission. Here we describe the development and characterization of a novel modified vaccinia virus Ankara (MVA)–based malaria vaccine which co-expresses the Plasmodium yoelii circumsporozoite protein (CSP) and IL-15. Vaccination/challenge studies showed that C57BL/6 mice immunized with the MVA-CSP/IL15 vaccine were protected significantly better against a P. yoelii 17XNL sporozoite challenge than either mice immunized with an MVA vaccine expressing only CSP or naïve controls. Importantly, the levels of total anti-CSP IgG were elevated about 100-fold for the MVA-CSP/IL15 immunized group compared to mice immunized with the MVA-CSP construct that does not express IL-15. Among the IgG subtypes, the IL-15 expressing MVA-CSP vaccine induced levels of IgG1 (8 fold) and IgG2b (80 fold) higher than the MVA-CSP construct. The significantly enhanced humoral responses and protection detected after immunization with the MVA-CSP/IL15 vaccine suggest that this IL-15 expressing MVA construct could be considered in the development of future malaria immunization strategies.     

Oryza sativa Chloroplast Signal Recognition Particle 43 (OscpSRP43) Is Required for Chloroplast Development and Photosynthesis

A rice chlorophyll-deficient mutant w67 was isolated from an ethyl methane sulfonate (EMS)–induced IR64 (Oryza sativa L. ssp. indica) mutant bank. The mutant exhibited a distinct yellow-green leaf phenotype in the whole plant growth duration with significantly reduced levels of chlorophyll and carotenoid, impaired chloroplast development and lowered capacity of photosynthesis compared with the wild-type IR64. Expression of a number of genes associated with chlorophyll metabolism, chloroplast biogenesis and photosynthesis was significantly altered in the mutant. Genetic analysis indicated that the yellow-green phenotype was controlled by a single recessive nuclear gene located on the short arm of chromosome 3. Using map-based strategy, the mutation was isolated and predicted to encode a chloroplast signal recognition particle 43 KD protein (cpSRP43) with 388 amino acid residuals. A single base substitution from A to T at position 160 resulted in a premature stop codon. OscpSRP43 was constitutively expressed in various organs with the highest level in the leaf. Functional complementation could rescue the mutant phenotype and subcellular localization showed that the cpSRP43:GFP fusion protein was targeted to the chloroplast. The data suggested that Oryza sativa cpSRP43 (OscpSRP43) was required for the normal development of chloroplasts and photosynthesis in rice.     

Peripheral Nerve Diffusion Tensor Imaging: Assessment of Axon and Myelin Sheath Integrity

Purpose

To investigate the potential of diffusion tensor imaging (DTI) parameters as in-vivo biomarkers of axon and myelin sheath integrity of the median nerve in the carpal tunnel as validated by correlation with electrophysiology.

Methods

MRI examinations at 3T including DTI were conducted on wrists in 30 healthy subjects. After manual segmentation of the median nerve quantitative analysis of fractional anisotropy (FA) as well as axial, radial and mean diffusivity (AD, RD, and MD) was carried out. Pairwise Pearson correlations with electrophysiological parameters comprising sensory nerve action potential (SNAP) and compound muscle action potential (CMAP) as markers of axon integrity, and distal motor latency (dml) and sensory nerve conduction velocity (sNCV) as markers of myelin sheath integrity were computed. The significance criterion was set at P=0.05, Bonferroni corrected for multiple comparisons.

Results

DTI parameters showed a distinct proximal-to-distal profile with FA, MD, and RD extrema coinciding in the center of the carpal tunnel. AD correlated with CMAP (r=0.50, p=0.04, Bonf. corr.) but not with markers of myelin sheath integrity. RD correlated with sNCV (r=-0.53, p=0.02, Bonf. corr.) but not with markers of axon integrity. FA correlated with dml (r=-0.63, p=0.002, Bonf. corr.) and sNCV (r=0.68, p=0.001, Bonf. corr.) but not with markers of axon integrity.

Conclusion

AD reflects axon integrity, while RD (and FA) reflect myelin sheath integrity as validated by correlation with electrophysiology. DTI parameters consistently indicate a slight decrease of structural integrity in the carpal tunnel as a physiological site of median nerve entrapment. DTI is particularly sensitive, since these findings are observed in healthy participants. Our results encourage future studies to evaluate the potential of DTI in differentiating axon from myelin sheath injury in patients with manifest peripheral neuropathies.     

Trans-Corneal Subretinal Injection in Mice and Its Effect on the Function and Morphology of the Retina

Purpose

To introduce a practical method of subretinal injection in mice and evaluate injection-induced retinal detachment (RD) and damage using a dynamic imaging system, electrophysiology, and histology.

Methods

After full dilation of a 2-month-old C57BL/6J mouse pupil, the cornea near the limbus was punctured with a 30 ½-gague disposable beveled needle. A 33 ½-gauge blunt needle was inserted through the corneal perforation into the anterior chamber, avoiding the lens before going deeper into the vitreous cavity, and penetrating the inner retina to reach the subretinal space. The mice were divided into four groups: in group 1, about 80–100% of the retina was filled with subretinally injected solution; in group 2, approximately 50–70% of the retina was filled with injected solution; in group 3, the procedures were stopped before solution injection; and non-injected eyes were used as the negative control in group 4. An optical coherence tomography (OCT) imaging system was used to monitor retinal reattachment during the first three days following the injections. Histological and functional changes were examined by light microscopy and electroretinography (ERG) at five weeks post-injection.

Results

After a short-term training, a 70% success rate with 50% or more coverage (i.e., retinal blebs occupied 50% or more retinal area and filled with the injected solution) with minimal injection-related damages can be achieved. Bleb formation was associated with retinal detachment (RD) between the neuroretina and the retinal pigment epithelium (RPE) layer. Partial RD could be observed at post-injection day 1, and by day 2 most of the retina had reattached. At 5 weeks post-injection, compared to uninjected control group 4, the b-wave amplitudes of ERG decreased 22% in group 1, 16% in group 2, and 7% in group 3; the b-wave amplitudes were statistically different between the uninjected group and the groups with either 50–70% or 80–100% coverage. The subretinal injection-induced RD reattached and became stable at five weeks post-injection, although some photoreceptor damage could still be observed in and around the injection sites, especially in 80–100% coverage group.

Conclusions

Trans-corneal subretinal injection is effective and practical, although subretinal injection-related damages can cause some morphological and functional loss.     

Cadmium-Induced Hydrogen Accumulation Is Involved in Cadmium Tolerance in Brassica campestris by Reestablishment of Reduced Glutathione Homeostasis

Hydrogen gas (H₂) was recently proposed as a therapeutic antioxidant and signaling molecule in clinical trials. However, the underlying physiological roles of H₂ in plants remain unclear. In the present study, hydrogen-rich water (HRW) was used to characterize the physiological roles of H₂ in enhancing the tolerance of Brassica campestris against cadmium (Cd). The results showed that both 50 μM CdCl₂ and 50%-saturated HRW induced an increase of endogenous H₂ in Brassica campestris seedlings, and HRW alleviated Cd toxicity related to growth inhibition and oxidative damage. Seedlings supplied with HRW exhibited increased root length and reduced lipid peroxidation, similar to plants receiving GSH post-treatment. Additionally, seedlings post-treated with HRW accumulated higher levels of reduced glutathione (GSH) and ascorbic acid (AsA) and showed increased GST and GPX activities in roots. Molecular evidence illustrated that the expression of genes such as GS, GR1 and GR2, which were down-regulated following the addition of Cd, GSH or BSO, could be reversed to varying degrees by the addition of HRW. Based on these results, it could be proposed that H₂ might be an important regulator for enhancing the tolerance of Brassica campestris seedlings against Cd, mainly by governing reduced glutathione homeostasis.     

Modeling changes in probabilistic reinforcement learning during adolescence

In the real world, many relationships between events are uncertain and probabilistic. Uncertainty is also likely to be a more common feature of daily experience for youth because they have less experience to draw from than adults. Some studies suggest probabilistic learning may be inefficient in youths compared to adults, while others suggest it may be more efficient in youths in mid adolescence. Here we used a probabilistic reinforcement learning task to test how youth age 8-17 (N = 187) and adults age 18-30 (N = 110) learn about stable probabilistic contingencies. Performance increased with age through early-twenties, then stabilized. Using hierarchical Bayesian methods to fit computational reinforcement learning models, we show that all participants’ performance was better explained by models in which negative outcomes had minimal to no impact on learning. The performance increase over age was driven by 1) an increase in learning rate (i.e. decrease in integration time scale); 2) a decrease in noisy/exploratory choices. In mid-adolescence age 13-15, salivary testosterone and learning rate were positively related. We discuss our findings in the context of other studies and hypotheses about adolescent brain development.     

猪链球菌1、2、14、1/2型荚膜多糖的单糖组成比较

【目的】猪链球菌1、2、14和1/2型间存在单向或双向的交叉抗原性,这种交叉抗原性的产生原因至今未被揭示。【方法】采用Sephacryl S-300凝胶层析柱对猪链球菌14和1/2型荚膜多糖进行分离纯化,经苯酚-硫酸检测和dot-ELISA辅助鉴定,确定荚膜多糖成分。采用高效凝胶渗透色谱法测定14和1/2型猪链球菌荚膜多糖分子量分别为487.38 kDa和512.72 kDa。【结果】经柱前衍生高效液相色谱法、荧光标记液相色谱法和核磁共振测定14和1/2型猪链球菌荚膜多糖单糖组成分别为:Glc/Gal/GlcNAc/Rha/Neu5Ac(1∶2.94∶1.35∶0.24∶0.37)和Glc/Gal/GlcNAc/GalNAc/Rha/Neu5Ac(1∶1.67∶1.05∶0.93∶0.72∶0.7)。并与已知的猪链球菌1、2型荚膜多糖的单糖组成进行比较分析,发现4种血清型荚膜多糖都具有Glc、GlcNAc、Gal和Neu5Ac,但单糖组成和比列并无明显相似性,这种交叉抗原性可能是由于荚膜多糖的空间结构相似性和(或)细胞表面的其他成分引起的。     

革兰氏阴性细菌β-桶状结构外膜蛋白体外折叠的研究进展

除了细胞质膜,革兰氏阴性细菌细胞还有一层组成细胞壁的外膜(Outer Membrane)。膜蛋白是外膜的主要组成成分之一,绝大多数外膜蛋白是由反向平行的β-折叠(β-Strands)通过相邻的氢键结合形成的β-桶状结构蛋白(β-Barrel Proteins)。这些蛋白既可作为通道蛋白、转运蛋白、酶、受体、毒力因子,也可作为结构蛋白发挥稳定外膜的重要作用,它们是否正确折叠并整合到外膜对革兰氏阴性细菌的生存至关重要。大多数外膜蛋白易于重组表达和体外重折叠(in vitro refolding),并且折叠状态可通过多种方法测定,因此β-桶状结构外膜蛋白被当着模式蛋白来研究各类生物和非生物因子对膜蛋白折叠的影响,是膜蛋白研究的一大热点。本文将从β-桶状结构外膜蛋白体外折叠的研究方法和影响折叠的因素角度对近年相关研究进展进行综合述评,最后总结了外膜蛋白体外折叠模式,并结合作者的相关研究结果和观点对该领域的研究前景进行了展望。     

Acidolysis-based component mapping of glycosaminoglycans by reversed-phase high-performance liquid chromatography with off-line electrospray ionization–tandem mass spectrometry: Evidence and tags to distinguish different glycosaminoglycans

Diverse monosaccharide analysis methods have been established for a long time, but few methods are available for a complete monosaccharide analysis of glycosaminoglycans (GAGs) and certain acidolysis-resistant components derived from GAGs. In this report, a reversed-phase high-performance liquid chromatography (RP–HPLC) method with pre-column 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization was established for a complete monosaccharide analysis of GAGs. Good separation of glucosamine/mannosamine (GlcN/ManN) and glucuronic acid/iduronic acid (GlcA/IdoA) was achieved. This method can also be applied to analyze the acidolysis-resistant disaccharides derived from GAGs, and the sequences of these disaccharides were confirmed by electrospray ionization–collision-induced dissociation–tandem mass spectrometry (ESI–CID–MS/MS). These unique disaccharides could be used as markers to distinguish heparin/heparan sulfate (HP/HS), chondroitin sulfate/dermatan sulfate (CS/DS), and hyaluronic acid (HA).     

Involvement of androgen receptor and glucose-regulated protein 78 kDa in human hepatocarcinogenesis

Previous studies demonstrated that androgen receptor (AR) is expressed in human hepatocellular carcinoma (HCC), one of the male-dominant diseases. Glucose-regulated protein 78 kDa (GRP78/Bip), which has a role in cancer development, is one of the androgen response genes in prostate cell lines. The aim of this study was to investigate the impact of AR on endoplasmic reticulum (ER)-stress signaling in human hepatoma. AR and GRP78 expressions were examined in human liver tissue panels. Human hepatoma cells stably expressing short hairpin RNA targeting AR and cells over-expressing AR were generated. The expressions of ER-stress molecules and AR were measured by real-time RT-PCR and Western blotting. The effect of AR on ER-stress responsive gene expression was examined by reporter assay. Strong positive correlation between AR mRNA and GRP78 mRNA was observed in stage I/II-HCCs. AR enhanced ER-stress responsive element activities and GRP78 expression, and regulated ER-stress response in hepatocytes. Sorafenib strongly induced significant apoptosis in HepG2 cells by the inhibition of AR and inhibition of the downstream GRP78. AR seems a co-regulator of GRP78 especially in earlier-stage HCC. AR plays a critical role in controlling ER-stress, providing new therapeutic options against HCC.