Biology subjects

Pei, D.

Publications and source records attributed to Pei, D..

26 records · Page 2Linked to original sources

bioRxiv2022

Three-dimensional molecular architecture of mouse organogenesis

Mammalian embryos have sophisticated cell organizations that are orchestrated by molecular regulation at cellular and tissue level. It has recently been appreciated that the cells that make up the animal body themselves harbor significant heterogeneity in the context of both cellular and particularly spatial dimension. However, current spatial transcriptomics profiling of embryonic tissues either lack three-dimensional representation or are restricted to limited depth and organs. Here, we reported a holistic spatial transcriptome atlas of all major organs at embryonic day 13.5 of mouse embryo and delineated a 3D rendering of the molecular regulation of embryonic patterning. By integrating with corresponding single-cell transcriptome data, the spatial organogenesis atlas provides rich molecular annotation of the dynamic organ nature, spatial cellular interaction, embryonic axes and divergence of cell fates underlying mammalian development, which would pave the way for precise organ-engineering and stem-cell based regenerative medicine.

developmental biology↗

bioRxiv2022

Cryo-EM structures of kalium channelrhodopsins KCRs

H. catenoides kalium channelrhodopsins (HcKCR1 and HcKCR2) are potassium-selective light-gated ion channels with unique properties for optogenetics. Here we report cryo-electron microscopy (cryo-EM) structures of HcKCR1 and HcKCR2 with endogenous retinal. Strikingly, one structure of HcKCR1 represents the conducting state with continuous potassium ion flow in the lumen, while another in non-conducting state with discontinuous potassium ion flow. Further electrophysiological studies identified extracellular side of ion permeation region as the major element for channel potassium selectivity and retinal binding region for channel gating. Our results uncover the unprecedented potassium selectivity and light-gating mechanism, and may facilitate the rational design of new optogenetic tools.

biophysics↗

bioRxiv2020

Placental microRNA Expression Associates with Birthweight through Control of Adipokines: Results from Two Independent Cohorts

MicroRNAs are non-coding RNAs that regulate gene expression post-transcriptionally. In the placenta, the master regulator of fetal growth and development, microRNAs shape the basic processes of trophoblast biology and specific microRNA have been associated with fetal growth. To comprehensively assess the role of microRNAs in placental function and fetal development, we have performed small RNA sequencing to profile placental microRNAs from two independent mother-infant cohorts: the Rhode Island Child Health Study (n=225) and the New Hampshire Birth Cohort Study (n=317). We modeled microRNA counts on infant birthweight percentile (BWP) in each cohort, while accounting for race, sex, parity and technical factors, using negative binomial generalized linear models. We identified microRNAs that were differentially expressed (DEmiRs) with BWP at false discovery rate (FDR) less than 0.05 in both cohorts. hsa-miR-532-5p (miR-532) was positively associated with BWP in both cohorts. By integrating parallel whole transcriptome and small RNA sequencing in the RICHS cohort, we identified putative targets of miR-532. These targets are enriched for pathways involved in adipogenesis, adipocytokine signaling, energy metabolism and hypoxia response, and included Leptin, which we further demonstrated to have decreasing expression with increasing BWP, particularly in male infants. Overall, we have shown a robust and reproducible association of miR-532 with BWP, which could influence BWP through regulation of adipocytokines Leptin and Adiponectin.

genetics↗

bioRxiv2020

Engineering Cell-Permeable Proteins through Insertion of Cell-Penetrating Motifs into Surface Loops

Effective delivery of proteins into the cytosol and nucleus of mammalian cells would open the door to a wide range of applications including treatment of many currently intractable diseases. However, despite great efforts from numerous investigators and the development of a variety of innovative methods, effective protein delivery in a clinical setting is yet to be accomplished. Herein we report a potentially general approach to engineering cell-permeable proteins by genetically grafting a short cell-penetrating peptide to an exposed loop region of a protein of interest. The grafted peptide is conformationally constrained by the protein structure, sharing the structural features of cyclic cell-penetrating peptides and exhibiting enhanced proteolytic stability and cellular entry efficiency. Insertion of an amphipathic motif, Arg-Arg-Arg-Arg-Trp-Trp-Trp, into the loop regions of enhanced green fluorescent protein (EGFP), protein-tyrosine phosphatase 1B (PTP1B), and purine nucleoside phosphorylase (PNP) rendered all three proteins cell-permeable and biologically active in cellular assays. When added into growth medium, the engineered PTP1B dose-dependently reduced the phosphotyrosine levels of intracellular proteins, while the modified PNP protected PNP-deficient mouse T lymphocytes (NSU-1) against toxic levels of deoxyguanosine, providing a potential enzyme replacement therapy for a rare genetic disease.

biochemistry↗

bioRxiv2019

Asymmetrical cleavages of Sleeping Beauty transposons generate multiple excised transposon fragments during transposition

Although the Sleeping Beauty (SB) transposon is the most validated DNA transposon used as a gene delivery vehicle in vertebrates, many details of the excision and integration steps in the transposition process are unclear. We have probed in detail the products of the excision step and apparent selective integration of a subset of those products during transposition. The standard model of SB transposase-mediated transposition includes symmetrical cleavages at both ends of the transposon for excision and re-integration in another DNA sequence. In our analysis of excised transposon fragments (ETFs), we found evidence for the requirement of certain flanking sequences for efficient cleavage and a significant rate of asymmetrical cleavage during the excision process that generates multiple ETFs. Our results suggest that the cleavage step by SB transposase is not as precise as indicated in most models. Repair of the donor ends can produce eight footprint sequences (TACTGTA, TACAGTA, TACATA, TACGTA, TATGTA, TACTA, TAGTA and TATA). Our data also suggest that mismatch repair (MMR) is not an essential requirement for footprint formation. Among the twenty liberated ETFs, only eight appear to effectively re-integrate into TA sites distributed across the genome, supporting earlier findings of unequal rates of excision and reintegration during SB transposition. These findings may be important in considerations of efficiency of SB transposon remobilization, selection of TA integration sites and detection of SB excision and integration loci, all of which may be important in human gene therapy.

molecular biology↗

bioRxiv2018

In Silico Identification of Three Types of Integrative and Conjugative Elements (ICEs) in Elizabethkingia anophelis Strains Isolated from Around the World

Elizabethkingia anophelis is an emerging global multidrug-resistant opportunistic pathogen. We assessed the diversity among 13 complete genomes and 23 draft genomes of E. anophelis derived from various environmental settings and human infections from different geographic regions around the world over past decades from 1950s. Thirty-one of these 36 strains harbor integrative and conjugative elements (ICEs). A total of 52 ICEs were identified, and categorized into three ICE types based on the architecture of signature genes in the conjugation module. The type II and III ICEs were found to integrate into regions adjacent to tRNA genes, while type I ICEs used a variety of integration sites, inserting into intergenic regions or even directly into a gene, sometimes disrupting gene function. Integrases such as tyrosine recombinases, serine recombinases and DDE transposases were found in most ICEs. The ICEs carry various cargo genes including transcription regulators and those involved in antibiotic resistance. The CRISPR-Cas system was found in nine strains, including four strains in which CRISPR-Cas machinery and ICEs co-exist. ICE distribution in the strains showed no geographic or temporal patterns. The ICEs in E. anophelis differ in gene structure and sequence from CTnDOT, a well-studied ICE prevalent in Bacteroides spp. This is the first set of ICEs identified in the family Flavobacteriaceae. As a prevalent type of mobile genetic elements in various strains of E. anophelis around the world, the categorization of ICEs will facilitate further investigations such as virulence, genome epidemiology and adaptation genomics of E. anophelis.\n\nImportanceElizabethkingia anophelis is an opportunistic human pathogen, and the genetic diversity between strains from around the world becomes apparent as more genomes are sequenced. The Integrative Conjugative Element (ICE), found in many bacterial species, contains genes for transfer via conjugation and integration into the chromosome, along with various cargo genes. ICEs are identified in 31 of 36 strains and categorized into three types based on architecture of modular genes, integrases, and integration sites. ICE distribution in different strains displays no spatial and temporal patterns. Several ICE-containing strains also possessed CRISPR-Cas units, considered to be the bacterial adaptive immune system providing protection against phage and predatory mobile genetic elements. This co-existence suggests that ICEs are beneficial or at least not harmful to the bacterial cells they inhabit. ICEs as a component of the mobile genetic repertoire enable recipients to resist antibiotics, survive disinfecting agents, and adapt to various ecological niches.

genomics↗

bioRxiv2018

Verified the effectiveness of AsCpf1 system in a variety of vertebrate species

CRISPR/Cpf1 system is a novel genomic editing tool. Because of its more sophisticated components, and lower off-target rate, it has the potential to become a better gene-editing tool. Previous reports showed that CRISPR/Cpf1 could work effectively in multiple species. But our data show that AsCpf1 activity has a big difference in different vertebrates. Using in vitro experiments, we finally learned that the difference between species is due to temperature.

developmental biology↗

bioRxiv2017

Resolution of Reprogramming Transition States by Single Cell RNA-Sequencing

The Yamanaka factors convert mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) through a highly heterogeneous process. Here we profile single cells undergoing an optimized 7-day reprogramming process and show that cells start reprogramming relatively in sync, but diverge into two branches around day 2. The first branch of cells expressing Cd34/Fxyd5/Psca become nonpluripotent. The second one contains cells that are first Oct4+, then Dppa5a+ and pluripotent. We show that IFN-{gamma} blocks this late transition. Our results reveal the heterogeneous nature of somatic cell reprogramming, identify Dppa5a as a marker for pluripotent and innate immunity as a potential barrier for reprogramming.\n\nOne Sentence SummarySingle cell RNA sequencing reveals a continuum of cell fates from somatic to pluripotent and Dppa5a as a marker for chimera-competent iPSCs.

cell biology↗