The G285S mutation in nsP1 is sufficient to render Sindbis virus as a stable vector for gene delivery

Neuroscience, gene therapy, and vaccine have all benefited from the increased use of viral vectors. Sindbis virus (SINV) is a notable candidate among these vectors. However, viral vectors commonly suffer from a loss of expression of the transgene, especially RNA viral vectors. In this study, we used a directed evolution approach by continuous passage of selection to identify adaptive mutations that help SINV to stably express exogenous genes. As a result, we found two adaptive mutations that are located at aa 285 (G to S) of nsP1 and aa 422 (D to G) of nsP2, respectively. Further study showed that G285S was sufficient for SINV to stabilize the expression of the inserted gene, while D422G was not. Combined with AlphaFold2 and sequence alignment with the genus Alphavirus, we found that G285S is conserved. Based on this mutation, we constructed a new vector for the applications in neural circuits mapping. Our results indicated that the mutant SINV maintained its anterograde transsynaptic transmission property. In addition, when the transgene was replaced by another gene, granulocyte-macrophage colony-stimulating factor (GM-CSF), the vector still showed stable expression of the inserted gene. Hence, using SINV as an example, we have demonstrated an efficient approach to greatly augment the gene delivery capacity of viral vectors, which will be useful to neuroscience and oncolytic therapy

Elevated glutamate, glutamine and GABA levels and reduced taurine level in a schizophrenia model using an in vitro proton nuclear magnetic resonance method

Accumulating evidence suggests that brain metabolic changes may be associated with the pathophysiology of schizophrenia. Both and studies have found glutamatergic and GABAergic abnormalities in different brain regions of individuals with schizophrenia. We report a longitudinal behavioral study in a methylazoxymethanol acetate (MAM) rat model of schizophrenia at three different age periods: prepuberty, late-puberty and early-adulthood. MAM-treated rats showed stable hypolocomotive activity, anxiety and cognitive deficits from late-puberty to early-adulthood. Therefore we detected the metabolites changes of adult MAM-treated rats using an proton nuclear magnetic resonance ( H-NMR) method. In the MAM-treated rats, glutamate was increased in the thalamus and hypothalamus, glutamine was increased in the hippocampus and GABA was increased in the hippocampus and prefrontal cortex, while taurine showed a decrease in the striatum, temporal cortex and parietal cortex. These abnormalities may be helped further understanding the pathophysiology of schizophrenia. [Abstract copyright: AJTR Copyright © 2019.

Diagenetic–Porosity Evolution and Reservoir Evaluation in Multiprovenance Tight Sandstones: Insight from the Lower Shihezi Formation in Hangjinqi Area, Northern Ordos Basin

AbstractThe reservoir property of tight sandstones is closely related to the provenance and diagenesis, and multiprovenance system and complex diagenesis are developed in Hangjinqi area. However, the relationship between provenance, diagenesis, and physical characteristics of tight reservoirs in Hangjinqi area has not yet been reported. The Middle Permian Lower Shihezi Formation is one of the most important tight gas sandstone reservoirs in the Hangjinqi area of Ordos Basin. This research compared the diagenesis-porosity quantitative evolution mechanisms of Lower Shihezi Formation sandstones from various provenances in the Hangjinqi area using thin-section descriptions, cathodoluminescence imaging, X-ray diffraction (XRD), scanning electron microscopy (SEM), and homogenization temperature of fluid inclusions, along with general physical data and high-pressure mercury intrusion (HPMI) data. The sandstones mainly comprise quartzarenite, sublitharenite, and litharenite with low porosity and low permeability and display obvious zonation in the content of detrital components as a result of multiprovenance. Pore space of those sandstone mainly consists of primary pores, secondary pores, and microfractures, but their proportion varies in different provenances. According to HPMI, the order of the pore-throat radius from largest to smallest is central provenance, eastern provenance, and western provenance, which is consistent with the change tend of porosity (middle part>northern part>western part) in Hangjinqi region. The diagenetic evolution path of those sandstones is comparable, with compaction, cementation, dissolution, and fracture development. The central provenance has the best reservoir quality, followed by the eastern provenance and the western provenance, and this variation due to the diverse diagenesis (diagenetic stage and intensity) of different provenances. These findings reveal that the variations in detrital composition and structure caused by different provenances are the material basis of reservoir differentiation, and the main rationale for reservoir differentiation is varying degrees of diagenesis during burial process

Rabies virus pseudotyped with CVS-N2C glycoprotein as a powerful tool for retrograde neuronal network tracing

Abstract Background: Efficient viral vectors for mapping and manipulating long projection neuronal circuits are crucial in brain structural and functional studies. The glycoprotein gene-deleted SAD strain rabies virus pseudotyped with the N2C glycoprotein (SAD-RV(ΔG)-N2C(G)) shows high neuro-tropism in cell culture, but its in vivo retrograde infection efficiency and neuro-tropism have not been systematically characterized. Methods: SAD-RV(ΔG)-N2C(G) and two other broadly used retrograde tracers, SAD-RV(ΔG)-B19(G) and rAAV2-retro were respectively injected into the VTA or DG in C57BL/6 mice. The neuron numbers labeled across the whole brain regions were counted and analyzed by measuring the retrograde infection efficiencies and tropisms of these viral tools. The labeled neural types were analyzed using fluorescence immunohistochemistry or GAD67-GFP mice. Result: We found that SAD-RV (ΔG)-N2C (G) enhanced the infection efficiency of long-projecting neurons by ~ 10 times but with very similar neuro-tropism, compared with SAD-RV (ΔG)-B19(G). On the other hand, SAD-RV(ΔG)-N2C(G) showed comparable infection efficiency with rAAV2-retro, but had a more restricted diffusion range, and broader tropism to different types and regions of long-projecting neuronal populations. Conclusions: These results demonstrate that SAD-RV(ΔG)-N2C(G) can serve as an effective retrograde vector for studying neuronal circuits. Key words：Viral vector, N2C Glycoprotein, Neuronal circuits, Retrograde tracin