Biology subjects

Gao, L.

Publications and source records attributed to Gao, L..

9 recordsLinked to original sources

bioRxiv2018

Tiling light sheet selective plane illumination microscopy using discontinuous light sheets

Tiling light sheet selective plane illumination microscopy (TLS-SPIM) improves 3D imaging ability of SPIM by using a real-time optimized tiling light sheet. However, the imaging speed decreases, and size of the raw image data increases proportionally to the number of tiling positions in TLS-SPIM. The decreased imaging speed and the increased raw data size could cause significant problems when TLS-SPIM is used to image large specimens at high spatial resolution. Here, we present a novel method to solve the problem. Discontinuous light sheets created by scanning coaxial beam arrays synchronized with camera exposures are used for 3D imaging to decrease the number of tiling positions required at each image plane without sacrificing the spatial resolution. We investigate the performance of the method via numerical simulation and discuss the technical details of the method.

biophysics↗

bioRxiv2018

MSTD: an efficient method for detecting multi-scale topological domains from symmetric and asymmetric 3D genomic maps

The chromosome conformation capture (3C) technique and its variants have been employed to reveal the existence of a hierarchy of structures in three-dimensional (3D) chromosomal architecture, including compartments, topologically associating domains (TADs), sub-TADs and chromatin loops. However, existing methods for domain detection were only designed based on symmetric Hi-C maps, ignoring long-range interaction structures between domains. To this end, we proposed a generic and efficient method to identify multi-scale topological domains (MSTD), including cis- and trans-interacting regions, from a variety of 3D genomic datasets. We first applied MSTD to detect promoter-anchored interaction domains (PADs) from promoter capture Hi-C datasets across 17 primary blood cell types. The boundaries of PADs are significantly enriched with one or the combination of multiple epigenetic factors. Moreover, PADs between functionally similar cell types are significantly conserved in terms of domain regions and expression states. Cell type-specific PADs involve in distinct cell type-specific activities and regulatory events by dynamic interactions within them. We also employed MSTD to define multi-scale domains from typical symmetric Hi-C datasets and illustrated its distinct superiority to the-state-of-art methods in terms of accuracy, flexibility and efficiency.

bioinformatics↗

bioRxiv2018

Distinct impacts of air and root-zone temperatures on leaf and root features of cucumber seedlings: resource acquisition capacity, organ size and carbon-nitrogen balance

Both low air (Tair) and root-zone (Troot) temperatures can inhibit resource (e.g. carbon and nutrients) acquisition by leaves and roots through various aspects, such as morphology, biomass allocation and assimilation/absorption capacity. However, it is still ambiguous whether Tair and Troot influence carbon (C) and nutrient acquisition via the same approach. To this end, in this study, cucumber (Cucumis sativus L.) seedlings were hydroponically grown under treatments arranged in complete factorial combination of two levels of Tair (26/18{degrees}C and 20/12{degrees}C, day/night) and two levels of Troot (19{degrees}C and 13{degrees}C, constant). In general, both Tair and Troot affected leaf and root sizes mainly by regulating their morphology rather than biomass investment. Under low Tair conditions (20/18{degrees}C), elevated Troot (compare 19{degrees}C versus 13{degrees}C) did not influence C acquisition, but increased nitrogen (N) acquisition mainly due to an increase in relative root length, resulting in decreased C : N acquisition ratio. However, under low Troot conditions (13{degrees}C), elevated Tair (compare 26/18{degrees}C versus 20/12{degrees}C) enhanced both C and N acquisition mainly because of an increase of both C assimilation in leaves and N absorption by roots, resulting in relatively constant C : N acquisition ratio. In addition, the Tair and Troot interaction was mainly observed in relative growth rate and root growth-related variables. Our results infer that Tair and Troot have distinct impacts on resource acquisition and carbon-nitrogen balance in plants.

plant biology↗

bioRxiv2018

Chromosome Removal Via Cellular Fragmentation and Aneuploid Blastomere Exclusion in Primate Embryos

Aneuploidy that arises during meiosis and/or mitosis is a major contributor to early embryo loss. We previously demonstrated that human preimplantation embryos encapsulate mis-segregated chromosomes into micronuclei while undergoing cellular fragmentation and that fragments can contain chromosomal material, but the source of this DNA was unknown. Here, we leveraged the use of a non-human primate model and single-cell DNA-sequencing (scDNA-seq) to examine the chromosomal content of 471 individual samples comprising 254 blastomeres, 42 polar bodies, and 175 cellular fragments from a large number (N=50) of disassembled rhesus cleavage-stage embryos. Our analysis revealed that the frequency of aneuploidy and micronucleation is conserved between humans and macaques and that cellular fragments encapsulate whole and/or partial chromosomes lost from blastomeres. Single-cell/fragment genotyping demonstrated that these chromosome-containing cellular fragments (CCFs) can be either maternal or paternal in origin and display DNA damage via double-stranded breaks. Chromosome breakage and abnormal cytokinesis resulted in reciprocal losses/gains at the terminal ends of chromosome arms, uniparental genome segregation, and mixoploidy between blastomeres. Combining time-lapse imaging with scDNA-seq, we also determined that multipolar divisions at the zygote or 2-cell stage generated chaotic aneuploidy encompassing a complex mixture of maternal and paternal chromosomes. Despite frequent chromosomal mis-segregation at the cleavage-stage, we show that CCFs and non-dividing aneuploid blastomeres exhibiting extensive DNA damage are prevented from incorporation at the blastocyst stage. These findings suggest that embryos respond to chromosomal errors by encapsulation into micronuclei, elimination by cellular fragmentation, and selection against highly aneuploid blastomeres to overcome chromosome instability during preimplantation development.

developmental biology↗

bioRxiv2017

A method to keep the excitation light sheet in focus in selective plane illumination microscopy

Keeping the excitation light sheet in focus is critical in selective plane illumination microscopy (SPIM) to ensure its 3D imaging ability. Unfortunately, an effective method that can be used in SPIM on general biological specimens to find the axial position of the excitation light sheet and keep it in focus is barely available. Here, we present a method to solve the problem. We investigate its mechanism and demonstrate its performance on a lattice light sheet microscope.

bioengineering↗

bioRxiv2017

Isolation And Characterization Of Key Genes That Promote Flavonoid Accumulation In Purple-Leaf Tea (Camellia sinensis L.)

There were several high concentrations of flavonoid components in tea leaves that present health benefits. A novel purple-leaf tea variety, Mooma1, was obtained from the natural hybrid population of Longjing 43 variety. The buds and young leaves of Mooma1 were displayed in bright red. HPLC and LC-MS analysis showed that anthocyanins and O-Glycosylated flavonols were remarkably accumulated in the leaves of Mooma1, while the total amount of catechins in purple-leaf leaves was slightly decreased compared with the control. A R2R3-MYB transcription factor (CsMYB6A) and a novel UGT gene (CsUGT72AM1), that were highly expressed in purple leaf were isolated and identified by transcriptome sequencing. The over-expression of transgenic tobacco confirmed that CsMYB6A can activate the expression of flavonoid-related structural genes, especially CHS and 3GT, controlling the accumulation of anthocyanins in the leaf of transgenic tobacco. Enzymatic assays in vitro confirmed that CsUGT72AM1 has catalytic activity as a flavonol 3-O-glucosyltransferase, and displayed broad substrate specificity. The results were useful for further elucidating the molecular mechanisms of the flavonoid metabolic fluxes in the tea plant.

plant biology↗

bioRxiv2017

NLR1-V, a CC-NBS-LRR encoding gene, is a potential candidate gene of the wheat powdery mildew resistance gene Pm21

Wheat powdery mildew caused by Blumeria graminisb f. sp. tritici is one of the most destructive diseases all over the world. Pm21, transferred from the wild Haynaldia villosa to wheat, confers broad spectrum resistance throughout the whole stage, and this gene has been widely used in wheat production for more than 20 years. Cloning the candidate gene of Pm21 is the prerequisite for elucidating the resistance mechanism, and is a valuable attempt to clone the target genes from the evolutionarily distant wild species. In this study, an innovative approach, which combined cytogenetic stocks development, mutagenesis, RenSeq and PacBio, was tried successfully to clone an NBS-LRR type gene NLR1-V from the Pm21 locus. Firstly, a powdery mildew resistant cryptic alien introgression line HP33 involved very small 6VS segment was developed, and 6 independent susceptible mutants of T6VS {middle dot} 6AL was identified. Then, the transcriptome of H. villosa was obtained by NGS and the full-length NBS-LRR gene database was constructed by RenSeq-PacBio. In the following study, two expressed NLR genes were located to the Pm21 locus using the HP33 as the mapping material, and only NLR1-V showed polymorphism between the wild T6VS {middle dot} 6AL and its six mutants. The functional analysis indicated that silencing of NLR1-V could compromise the resistance of T6VS {middle dot} 6AL completely, and could also decrease the resistance of T6VS {middle dot} 6DL dramatically. Moreover, NLR1-V could recover the resistance of the susceptible mutant and increase the resistance in the susceptible wheat. The study implied that NLR1-V, a CC-NBS-LRR encoding gene, is a potential candidate gene of the powdery mildew resistance gene Pm21.

plant biology↗

bioRxiv2016

Engineered Cpf1 Enzymes with Altered PAM Specificities

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1-5. Compared to other genome editing platforms, Cpf1 offers distinct advantages, such as the ability to easily target multiple genes simultaneously3, as well as low rates of off-target activity4, 5. However, the Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), which has been successfully harnessed for genome editing, can only robustly cleave target sites preceded by a TTTV protospacer adjacent motif (PAM), which may limit its practical utility. To address this limitation, we used a structure- guided saturation mutagenesis screen to increase the targeting range of Cpf1. We engineered two variants of AsCpf1 with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM-interacting domains. Together, these variants increase the targeting range of AsCpf1 to one cleavage site for every ~8.7 bp in non-repetitive regions of the human genome, providing a useful addition to the CRISPR/Cas genome engineering toolbox.

synthetic biology↗

bioRxiv2016

BLISS: quantitative and versatile genome-wide profiling of DNA breaks in situ

We present a method for genome-wide DNA double-strand Breaks (DSBs) Labeling In Situ and Sequencing (BLISS) which, compared to existing methods, introduces several key features: 1) high efficiency and low input requirement by in situ DSB labeling in cells or tissue sections directly on a solid surface; 2) easy scalability by performing in situ reactions in multi-well plates; 3) high sensitivity by linearly amplifying tagged DSBs using in vitro transcription; and 4) accurate DSB quantification and control of PCR biases by using unique molecular identifiers. We demonstrate the ability to use BLISS to quantify natural and drug-induced DSBs in low-input samples of cancer cells, primary mouse embryonic stem cells, and mouse liver tissue sections. Finally, we applied BLISS to compare the specificity of CRISPR-associated RNA-guided endonucleases Cas9 and Cpf1, and found that Cpf1 has higher specificity than Cas9. These results establish BLISS as a versatile, sensitive, and efficient method for genome-wide DSB mapping in many applications.

molecular biology↗