Biology subjects

Fan, L.

Publications and source records attributed to Fan, L..

136 records · Page 8Linked to original sources

bioRxiv2018

Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

The gene mutated in Huntingtons disease (HD) patients encodes the 348 kDa huntingtin (HTT) protein. The pathogenic HD CAG-expansion mutation causes a polyglutamine (polyQ) tract at the N-terminus of the HTT protein to expand above a critical threshold of ~35 glutamine residues. The effect of HD mutations on HTT is not well understood, in part due to difficulties in carrying out biochemical, biophysical and structural studies of this large protein. To facilitate such studies, we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprises both N and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, juvenile HD patients as well as the more extreme polyQ expansions used in some HD tissue and animal models. These reagents yield milligram quantities of pure recombinant HTT protein, including many of the previously mapped posttranslational modifications. We have characterised both apo and HTT-HAP40 complex samples produced using this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful complexes of HTT. We demonstrate how these resources can produce sufficient material for protein-intensive experiments such as small angle X-ray scattering (SAXS), providing biochemical insight into HTT protein structure. The work outlined in this manuscript and the tools generated, lay a foundation for further biochemical and structural work on the HTT protein and its functional interactions with other biomolecules.

biochemistry↗

bioRxiv2018

Characterization of functional diversity of the human brain based on intrinsic connectivity networks

Spontaneous fluctuations underlying the brain activity can reflect the intrinsic organization of the system, such as the functional brain networks. In large scale, a network perspective has emerged as a new avenue to explore the functional properties of human brain. Here, we studied functional diversity in healthy subjects based on the network perspective. We hypothesized that the patterns of participation of different functional networks were related with the functional diversity of particular brain regions. Independent component analysis (ICA) was adopted to detect the intrinsic connectivity networks (ICNs) based on the data of resting-state functional MRI. An index of functional diversity (FD index) was proposed to quantitatively describe the degree of anisotropic distribution related with participation of various ICNs. We found that FD index continuously varied across the brain, for example, the primary motor cortex with low FD value and the precuneus with significantly high FD value. The FD values indicated the different functional roles of the corresponding brain regions, which were reflected by the various patterns of participation of ICNs. The FD index can be used as a new approach to quantitatively characterize the functional diversity of human brain, even for the changed functional properties caused by the psychiatric disorders.

neuroscience↗

bioRxiv2018

Genome-wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed

Brassica napus (AACC, 2n=38), is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic clusters, such as European and Asian rapeseed, and impacts of historical large-sale introgression events in this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7 and its four progenitors and carried out de novo assembly-based comparison, pedigree and population analysis with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed, but it had subsequently significantly diverged, with rapid genome differentiation after intensive local breeding selection. The first historical introgression of B. rapa dramatically broadened the allelic pool of Asian B. napus, but decreased their deleterious variations. The secondary historical introgression of European rapeseed (canola-quality) has reshaped Asian rapeseed into two groups, accompanied by an increase in genetic load. This study demonstrates distinctive genomic footprints by recent intra- and inter-species introgression events for local adaptation, and provide novel insights for understanding the rapid genome evolution of a young allopolyploid crop.

genomics↗

bioRxiv2018

Structural differences between pri-miRNA paralogs promotes alternative Drosha cleavage and expands target repertoires

MicroRNA (miRNA) processing begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. To understand how pri-miRNA sequence and structure influence Drosha cleavage, we studied the maturation of three pri-miR-9 paralogs, which encode the same mature miRNA but differ in the surrounding scaffold. We show that pri-miR-9-1 has a unique Drosha cleavage profile due to its distorted and flexible stem structure. Cleavage of pri-miR-9-1, but not pri-miR-9-2 or pri-miR-9-3, generates an alternative-miR-9 with a shifted seed sequence that expands the scope of its target RNAs. Analyses of low grade glioma patient samples indicate that the alternative-miR-9 plays a distinct role in preventing tumor progression. To generalize our model, we provide evidence that distortion of pri-miRNA stems correlates with Drosha cleavage at non-canonical sites. Our studies reveal that pri-miRNA paralogs can have distinct functions via differential Drosha processing.

molecular biology↗

bioRxiv2018

A Novel AuNPs-based Glucose Oxidase Mimic with Enhanced Activity and Selectivity Constructed by Molecular Imprinting and O2-Containing Nanoemulsion Embedding

In spite of the competitive advantages, inorganic nanoparticle mimic enzymes exhibit inherent disadvantages of limited catalytic efficiency and lacking selectivity. Here a AuNPs based mimic enzyme with significantly enhanced glucose selectivity and catalytic activity was constructed and demonstrated for the first time. Aminophenylboronic acid was employed to increase the affinity to glucose, as well as build molecular imprinted polymer shells to realize the selectivity for template molecules of glucose. Besides that, heptadecafluoro-n-octyl bromide nanoemulsion with the function of providing oxygen was introduced to gain a further improvement in catalytic activity, which successfully enhanced the catalytic efficiency (kcat/Km) up to about 270-fold. Based on the demonstrated catalytic properties, AuNPs based glucose oxidase mimics have been successfully applied in practical glucose detection of drinks and blood glucose.

biochemistry↗

bioRxiv2018

MLL1 minimal catalytic complex is a dynamic conformational ensemble susceptible to pharmacological allosteric disruption

Histone H3K4 methylation is an epigenetic mark associated with actively transcribed genes. This modification is catalyzed by the mixed lineage leukaemia (MLL) family of histone methyltransferases including MLL1, MLL2, MLL3, MLL4, SET1A and SET1B. Catalytic activity of MLL proteins is dependent on interactions with additional conserved proteins but the structural basis for subunit assembly and the mechanism of regulation is not well understood. We used a hybrid methods approach to study the assembly and biochemical function of the minimally active MLL1 complex (MLL1, WDR5 and RbBP5). A combination of small angle X-ray scattering (SAXS), cross-linking mass spectrometry (XL-MS), NMR spectroscopy, and computational modeling were used to generate a dynamic ensemble model in which subunits are assembled via multiple weak interaction sites. We identified a new interaction site between the MLL1 SET domain and the WD40 repeat domain of RbBP5, and demonstrate the susceptibility of the catalytic function of the complex to disruption of individual interaction sites.

biophysics↗

bioRxiv2018

A multicellular rosette-mediated collective dendrite extension

Coordination of neurite morphogenesis with surrounding tissues is crucial to the establishment of neural circuits, but the underlying cellular and molecular mechanisms remain poorly understood. We show that neurons in a C. elegans sensory organ, called the amphid, undergo a collective dendrite extension to initiate formation of the sensory nerve. The amphid neurons first assemble into a multicellular rosette. The vertex of the rosette, which becomes the dendrite tips, is attached to the anteriorly migrating epidermis and carried to the sensory depression, extruding the dendrites away from the neuronal cell bodies. Multiple adhesion molecules including DYF-7, SAX-7, HMR-1 and DLG-1 function redundantly in rosette-to-epidermis attachment. PAR-6 is localized to the rosette vertex and dendrite tips, and promotes DYF-7 localization and dendrite extension. Our results suggest a collective mechanism of neurite extension that is distinct from the classical pioneer-follower model and highlight the role of mechanical cues from surrounding tissues in shaping neurites.

developmental biology↗

bioRxiv2018

Xist Intron 1 Repression by TALE Transcriptional Factor Improves Somatic Cell Reprogamming in Mice

Xist is the master regulator of X chromosome inactivation (XCI). In order to further understand the Xist locus in reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) and in somatic cell nuclear transfer (SCNT), we tested transcription-factor-like effectors (TALE)-based designer transcriptional factors (dTFs), which were specific to numerous regions at the Xist locus. We report that the selected dTF repressor 6 (R6) binding the intron 1 of Xist, which did not affect Xist expression in mouse embryonic fibroblasts (MEFs), substantially improved the iPSC generation and the SCNT preimplantation embryo development. Conversely, the dTF activator targeting the same genomic region of R6 decreased iPSC formation, and blocked SCNT-embryo development. These results thus uncover the critical requirement for the Xist locus in epigenetic resetting, which is not directly related to Xist transcription. This may provide a unique route to improving the reprogramming.

developmental biology↗

bioRxiv2018

Consistency in predicting functions from anatomical and functional connectivity profiles across the cortical cortex

More and more studies had used connectivity profiles to predict functions of the brain. However, whether anatomical connectivity can predict functions consistently with functional connectivity in various functional domains and whether the connectivity-function relationship is universal across the whole cortex are unknown. Using a linear model, we discovered that anatomical connectivity was comparative to functional connectivity in explaining the variance of functions in most cortical regions, with the exception that anatomical connectivity had poor explaining abilities in brain areas which had high individual task variations. In addition, anatomical connectivity were not that good at capturing individual functional differences and had less inter-subject variation than functional connectivity, however anatomical connectivity could be regarded as more stable in the perspective of parcellation. The current results provided the first comprehensive picture of the relationships between functions and connectivity in the whole human cortex at a fine-grained brain atlas.

neuroscience↗

bioRxiv2018

E-cadherin bridges cell polarity and spindle orientation to ensure prostate epithelial integrity and prevent carcinogenesis in vivo

Cell polarity and correct mitotic spindle positioning are essential for the maintenance of a proper prostate epithelial architecture, and disruption of the two biological features occurs at early stages in prostate tumorigenesis. However, whether and how these two epithelial attributes are connected in vivo is largely unknown. We herein report that conditional genetic deletion of E-cadherin, a key component of adherens junctions, in a mouse model results in loss of prostate luminal cell polarity and randomization of spindle orientations. Critically, E-cadherin ablation causes prostatic hyperplasia which progresses to invasive adenocarcinoma. Mechanistically, E-cadherin and the spindle positioning determinant LGN interacts with the PDZ domain of cell polarity protein SCRIB and form a ternary protein complex to bridge cell polarity and cell division orientation. These findings provide a novel mechanism by which E-cadherin acts an anchor to maintain prostate epithelial integrity and to prevent carcinogenesis in vivo.

developmental biology↗