Biology subjects

Ji, G.

Publications and source records attributed to Ji, G..

22 records · Page 2Linked to original sources

bioRxiv2020

Amorphous nickel titanium alloy film: a new choice for cryo electron microscopy sample preparation

Cryo-electron microscopy (cryoEM) has become one of the most important approach for structural biology. However, barriers are still there for an increase successful rate, a better resolution and improved efficiency from sample preparation, data collection to image processing. CryoEM sample preparation is one of the bottlenecks with many efforts made recently, including the optimization of supporting substrate (e.g. ultra-thin carbon, graphene, pure gold, 2d crystal of streptavidin, and affinity modification), which was aimed to solve air-water interface problem, or reduce beam induced motion (BIM), or change particle distribution in the grid hole. Here, we report another effort of developing a new supporting substrate, the amorphous nickel-titanium alloy (ANTA) film, for cryoEM sample preparation. Our investigations showed advantages of ANTA film in comparison with conventional carbon film, including superior electron conductivity and trace non-specific interaction with protein. These advantages yield less BIM and significantly improved particle distribution during cryoEM experiment of human apo-ferritn, thus resulting an improved reconstruction resolution from a reduced number of micrographs and particles. Unlike the pure gold film, the usage of the ANTA film is just same with the carbon film, compatible to conventional automatic cryoEM data collection procedure.

biophysics↗

bioRxiv2019

VHUT-cryo-FIB, a method to fabricate frozen-hydrated lamella of tissue specimen for in situ cryo-electron tomography

ABSTACTCryo-electron tomography (cryo-ET) provides a promising technique to study high resolution structures of macromolecules in situ, opening a new era of structural biology. One major bottleneck of this technique is to prepare suitable cryo-lamellas of cell/tissue samples. The emergence of cryo-focused ion beam (cryo-FIB) milling technique provides a good solution of this bottleneck. However, there are still large limitations of using cryo-FIB to prepare cryo-lamella of tissue specimen because the thickness of tissue increases the difficulty of specimen freezing and cryo-FIB milling. Here we report a new workflow, VHUT-cryo-FIB (Vibratome - High pressure freezing - Ultramicrotome Trimming - cryo-FIB), aiming for efficient preparation of frozen hydrated tissue lamella for subsequent cryo-ET data collection. This workflow includes tissue slicing using vibratome, high pressure freezing, ultramicrotome cryo-trimming, cryo-FIB milling and the subsequent cryo-electron microscopy (cryo-EM). The modification of equipment in this workflow is highly eliminated. We developed two strategies with a special cryo-holder tip or carrier for loading cryo-lamella into side entry cryo-holder or Autoloader catridge. We tested this workflow using the tissue sample of rat skeleton muscle and spinach leaf and collected high quality cryo-ET tilt series, which enabled us to obtain an in situ structure of spinach ribosome by sub-tomogram averaging.

biophysics↗

bioRxiv2019

The full-length transcriptome of Spartina alterniflora reveals the complexity of high salt tolerance in monocotyledonous halophyte

Spartina alterniflora (Spartina) is the only halophyte in the salt marsh. However, the molecular basis of its high salt tolerance remains elusive. In this study, we used PacBio full-length single molecule long-read sequencing and RNA-seq to elucidate the transcriptome dynamics of high salt tolerance in Spartina by salt-gradient experiments (0, 350, 500 and 800 mM NaCl). We systematically analyzed the gene expression diversity and deciphered possible roles of ion transporters, protein kinases and photosynthesis in salt tolerance. Moreover, the co-expression network analysis revealed several hub genes in salt stress regulatory networks, including protein kinases such as SaOST1, SaCIPK10 and three SaLRRs. Furthermore, high salt stress affected the gene expression of photosynthesis through down-regulation at the transcription level and alternative splicing at the post-transcriptional level. In addition, overexpression of two Spartina salt-tolerant genes SaHSP70-I and SaAF2 in Arabidopsis significantly promoted the salt tolerance of transgenic lines. Finally, we built the SAPacBio website for visualizing the full-length transcriptome sequences, transcription factors, ncRNAs, salt-tolerant genes, and alternative splicing events in Spartina. Overall, this study sheds light on the high salt tolerance mechanisms of monocotyledonous-halophyte and demonstrates the potential of Spartina genes for engineering salt-tolerant plants.

plant biology↗

bioRxiv2018

Overdispersed gene expression characterizes schizophrenic brains

Schizophrenia (SCZ) is a severe, highly heterogeneous psychiatric disorder with varied clinical presentations. The polygenic genetic architecture of SCZ makes identification of causal variants daunting. Gene expression analyses have shown that SCZ may result in part from transcriptional dysregulation of a number of genes. However, most of these studies took the commonly used approach--differential gene expression analysis, assuming people with SCZ are a homogenous group, all with similar expression levels for any given gene. Here we show that the overall gene expression variability in SCZ is higher than that in an unaffected control (CTL) group. Specifically, we applied the test for equality of variances to the normalized expression data generated by the CommonMind Consortium (CMC) and identified 87 genes with significantly higher expression variances in the SCZ group than the CTL group. One of the genes with differential variability, VEGFA, encodes a vascular endothelial growth factor, supporting a vascular-ischemic etiology of SCZ. We also applied a Mahalanobis distance-based test for multivariate homogeneity of group dispersions to gene sets and identified 19 functional gene sets with higher expression variability in the SCZ group than the CTL group. Several of these gene sets are involved in brain development (e.g., development of cerebellar cortex, cerebellar Purkinje cell layer and neuromuscular junction), supporting that structural and functional changes in the cortex cause SCZ. Finally, using expression variability QTL (evQTL) analysis, we show that common genetic variants contribute to the increased expression variability in SCZ. Our results reveal that SCZ brains are characterized by overdispersed gene expression, resulting from dysregulated expression of functional gene sets pertaining to brain development, necrotic cell death, folic acid metabolism, and several other biological processes. Using SCZ as a model of complex genetic disorders with a heterogeneous etiology, our study provides a new conceptual framework for variability-centric analyses. Such a framework is likely to be important in the era of personalized medicine. (313 words)

genetics↗