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1.
Foods ; 10(11)2021 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-34829100

RESUMO

Clanis bilineata tsingtauica Mell, 1922 (Lepidoptera, Sphingidae), also known as "Doudan" in China, is an important pest in legume crops. As an edible insect, it is most commonly consumed in Jiangsu, Shandong, and Henan Provinces. Mass rearing requires access to large numbers of eggs. This stage, however, is of short duration and supplies are frequently not sufficient for insect production. Therefore, we identified the cold storage conditions for C. bilineata tsingtauica that can effectively prolong the storage time of the eggs, to make supplies more readily available. We found that when stored at 4 °C, only 7.5% of the eggs hatched after 7 days, while at 10 °C the hatch rate was 78.3%. At 15 °C, the egg hatch rate remained at this same level (77.8% even after 14-20 days). Considering various combinations, we found that optimal egg hatch occurred if eggs were stored at 15 °C for 11 days, and then held at 15-20 °C under dark conditions. Stored as described above, the egg hatch rate was not significantly different from the control group (at 28 °C). These conditions allow for easier mass rearing of C. bilineata tsingtauica by providing a stable supply of eggs.

2.
J Am Chem Soc ; 143(46): 19330-19340, 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34780151

RESUMO

The efficient isolation of immune cells with high purity and low cell damage is important for immunotherapy and remains highly challenging. We herein report a cell capture DNA network containing polyvalent multimodules for the specific isolation and in situ incubation of T lymphocytes (T-cells). Two ultralong DNA chains synthesized by an enzymatic amplification process were rationally designed to include functional multimodules as cell anchors and immune adjuvants. Mutually complementary sequences facilitated the formation of a DNA network and encapsulation of T-cells, as well as offering cutting sites of a restriction enzyme for the responsive release of T-cells and immune adjuvants. The purity of captured tumor-infiltrating T-cells reached 98%, and the viability of T-cells maintained ∼90%. The T-cells-containing DNA network was further administrated to a tumor lesion for localized immunotherapy. Our work provides a robust nanobiotechnology for efficient isolation of immune cells and other biological particles.

3.
Insect Biochem Mol Biol ; 139: 103665, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34624466

RESUMO

The black cutworm (BCW), Agrotis ipsilon, is a worldwide polyphagous and underground pest that causes a high level of economic loss to a wide range of crops through the damage of roots. This species performs non-directed migration throughout East and Southeast Asia seasonally. Lack of a genome information has limited further studies on its unique biology and the development of novel management approaches. In this study, we present a 476 Mb de novo assembly of BCW, along with a consensus gene set of 14,801 protein-coding gene models. Quality controls show that both genome assembly and annotations are high-quality and mostly complete. We focus manual annotation and comparative genomics on gene families that related to the unique attributes of this species, such as nocturnality, long-distance migration, and host adaptation. We find that the BCW genome encodes a similar gene repertoire in various migration-related gene families to the diural migratory butterfly Danaus plexiipus, with additional copies of long wavelength opsin and two eye development-related genes. On the other hand, we find that the genomes of BCW and many other polyphagous lepidopterans encode many more gustatory receptor genes, particularly the lineage-specific expanded bitter receptor genes, than the mono- or oligo-phagous species, suggesting a common role of gustatory receptors (GRs) expansion in host range expansion. The availability of a BCW genome provides valuable resources to study the molecular mechanisms of non-directed migration in lepidopteran pests and to develop novel strategies to control migratory nocturnal pests.

4.
Insect Biochem Mol Biol ; 138: 103638, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34428581

RESUMO

The Asian corn borer (ACB) is the most devastating pest on maize in the western Pacific region of Asia. Despite broad interests in insecticide resistance, seasonal adaptation, and larval color mimicry regarding the ACB system, lacking of reference genomic information and a powerful gene editing approach have hindered the in-depth studies of these aspects. Here we present a 455.7 Mb draft genome of ACB with 98.4% completeness. Comparative genomics analysis showed an evident expansion in gene families of gustatory receptors (105), which is related to polyphagous characteristics. Based on the comparative transcriptome analysis of resistant and susceptible ACB against Bt Cry1Ab toxin, we identified 26 genes related to Cry1Ab resistance. Additionally, transcriptomics of insects exposed to conditions of low temperature and diapause (LT) vs. room temperature and diapause (RT) provided insights into the genetic mechanisms of cold adaptation. We also successfully developed an efficient CRISPR/Cas9-based genome editing system and applied it to explore the role of color pattern genes in the ecological adaptation of ACB. Taken together, our study provides a fully annotated high-quality reference genome and efficient gene editing system to realize the potential of ACB as a study system to address important biological questions such as insecticide resistance, seasonal adaptation, and coloration. These valuable genomic resources will also benefit the development of novel strategies for maize pest management.


Assuntos
Adaptação Biológica , Genoma de Inseto , Herbivoria/genética , Mariposas/genética , Animais , Zea mays
5.
Nat Commun ; 12(1): 4108, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226550

RESUMO

DNA glycosylases must distinguish the sparse damaged sites from the vast expanse of normal DNA bases. However, our understanding of the nature of nucleobase interrogation is still limited. Here, we show that hNEIL1 (human endonuclease VIII-like 1) captures base lesions via two competing states of interaction: an activated state that commits catalysis and base excision repair, and a quarantine state that temporarily separates and protects the flipped base via auto-inhibition. The relative dominance of the two states depends on key residues of hNEIL1 and chemical properties (e.g. aromaticity and hydrophilicity) of flipped bases. Such a DNA repair mechanism allows hNEIL1 to recognize a broad spectrum of DNA damage while keeps potential gratuitous repair in check. We further reveal the molecular basis of hNEIL1 activity regulation mediated by post-transcriptional modifications and provide an example of how exquisite structural dynamics serves for orchestrated enzyme functions.


Assuntos
DNA Glicosilases/química , DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , Triagem , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , DNA/química , Dano ao DNA , DNA Glicosilases/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Especificidade por Substrato
6.
Pest Manag Sci ; 77(7): 3588-3596, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33843144

RESUMO

BACKGROUND: Genetic manipulation of sex determination pathways in insects provides the basis for a broad range of strategies to benefit agricultural security and human health. The P-element somatic inhibitor (PSI) protein, an exon splicing silencer that promotes male-specific splicing of dsx, plays a critical role in male sexual differentiation and development. The functions of PSI have been characterized in the lepidopteran model species Bombyx mori. However, the molecular mechanism and functions of PSI in Plutella xylostella, a worldwide agricultural pest and taxonomically basal species, are still unknown. RESULTS: Here we identified PxPSI transcripts and analyzed their spatiotemporal expression pattern in P. xylostella. Multiple sequence alignment revealed that PxPSI contains four KH domains and is highly conserved in lepidopterans. We used the CRISPR-Cas9 system to generate mutations of the PxPSI genomic locus. Disruptions of PxPSI caused male-specific defects in internal and external genitals. In addition, we detected female-specific Pxdsx transcripts in PxPSI male mutants. Mutations also caused changes in expression of several sex-biased genes and induced male sterility. CONCLUSION: Our study demonstrates that PxPSI plays a key role in male sex determination in P. xylostella and suggests a potential molecular target for genetic-based pest management in lepidopteran pests. © 2021 Society of Chemical Industry.


Assuntos
Infertilidade Masculina , Mariposas , Animais , Feminino , Proteínas de Insetos/genética , Masculino , Mariposas/genética , Mutação
7.
Nat Methods ; 18(3): 283-292, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33589836

RESUMO

Genome-wide profiling of histone modifications can reveal not only the location and activity state of regulatory elements, but also the regulatory mechanisms involved in cell-type-specific gene expression during development and disease pathology. Conventional assays to profile histone modifications in bulk tissues lack single-cell resolution. Here we describe an ultra-high-throughput method, Paired-Tag, for joint profiling of histone modifications and transcriptome in single cells to produce cell-type-resolved maps of chromatin state and transcriptome in complex tissues. We used this method to profile five histone modifications jointly with transcriptome in the adult mouse frontal cortex and hippocampus. Integrative analysis of the resulting maps identified distinct groups of genes subject to divergent epigenetic regulatory mechanisms. Our single-cell multiomics approach enables comprehensive analysis of chromatin state and gene regulation in complex tissues and characterization of gene regulatory programs in the constituent cell types.


Assuntos
Lobo Frontal/metabolismo , Regulação da Expressão Gênica/genética , Hipocampo/metabolismo , Código das Histonas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Linhagem Celular Tumoral , Cromatina/metabolismo , Epigênese Genética/genética , Lobo Frontal/citologia , Perfilação da Expressão Gênica , Células HeLa , Hipocampo/citologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional , Análise de Célula Única , Transcriptoma/genética
8.
Int J Clin Exp Pathol ; 13(11): 2720-2726, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33284867

RESUMO

Ulinastatin (UTI) is a trypsin inhibitor observed in urine. UTI can treat some diseases by inhibiting the broad-spectrum hydrolysis activity of various enzymes and other pharmacological effects. UTI can widely treat pancreatitis, systemic multiple organ dysfunction syndrome, circulatory failure, and toxic shock clinically. The liver is a major metabolic organ of the human body. Various biological metabolic reactions require the liver's participation. When various physical and chemical factors drive the body, it will damage the liver to varying degrees. As a clinically effective drug, UTI is also known to treat some liver diseases. This article mainly describes UTI's research progress in treating septic liver injury, hepatitis, liver fibrosis, autoimmune liver disease with liver failure, and liver ischemia-reperfusion injury.

9.
Elife ; 92020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32701057

RESUMO

Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase-assisted RNA/DNA hybrids Co-tagmEntation (termed 'TRACE-seq') are compared to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis. At the meantime, TRACE-seq enables a cost-effective one-tube library construction protocol and hence is more rapid (within 6 hr) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.


Assuntos
DNA/genética , RNA/genética , Análise de Sequência/métodos , Transposases/genética , Células HEK293 , Humanos , Transposases/metabolismo
11.
Nat Chem Biol ; 16(2): 160-169, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31819270

RESUMO

Pseudouridine synthases (PUSs) are responsible for installation of pseudouridine (Ψ) modification in RNA. However, the activity and function of the PUS enzymes remain largely unexplored. Here we focus on human PUS10 and find that it co-expresses with the microprocessor (DROSHA-DGCR8 complex). Depletion of PUS10 results in a marked reduction of the expression level of a large number of mature miRNAs and concomitant accumulation of unprocessed primary microRNAs (pri-miRNAs) in multiple human cells. Mechanistically, PUS10 directly binds to pri-miRNAs and interacts with the microprocessor to promote miRNA biogenesis. Unexpectedly, this process is independent of the catalytic activity of PUS10. Additionally, we develop a sequencing method to profile Ψ in the tRNAome and report PUS10-dependent Ψ sites in tRNA. Collectively, our findings reveal differential functions of PUS10 in nuclear miRNA processing and in cytoplasmic tRNA pseudouridylation.


Assuntos
Hidroliases/metabolismo , MicroRNAs/metabolismo , RNA de Transferência/metabolismo , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células/fisiologia , Citoplasma/genética , Citoplasma/metabolismo , Regulação da Expressão Gênica , Humanos , Hidroliases/genética , Processamento Pós-Transcricional do RNA
12.
Nat Struct Mol Biol ; 26(11): 1063-1070, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31695190

RESUMO

Simultaneous profiling of transcriptome and chromatin accessibility within single cells is a powerful approach to dissect gene regulatory programs in complex tissues. However, current tools are limited by modest throughput. We now describe an ultra high-throughput method, Paired-seq, for parallel analysis of transcriptome and accessible chromatin in millions of single cells. We demonstrate the utility of Paired-seq for analyzing the dynamic and cell-type-specific gene regulatory programs in complex tissues by applying it to mouse adult cerebral cortex and fetal forebrain. The joint profiles of a large number of single cells allowed us to deconvolute the transcriptome and open chromatin landscapes in the major cell types within these brain tissues, infer putative target genes of candidate enhancers, and reconstruct the trajectory of cellular lineages within the developing forebrain.


Assuntos
Encéfalo/citologia , Cromatina/genética , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Transcriptoma , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Perfilação da Expressão Gênica/economia , Células HEK293 , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Análise de Célula Única/economia
13.
Angew Chem Int Ed Engl ; 58(52): 18987-18993, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31617293

RESUMO

Together with the more intuitive and commonly recognized conductance mechanisms of charge-hopping and tunneling, quantum-interference (QI) phenomena have been identified as important factors affecting charge transport through molecules. Consequently, establishing simple and flexible molecular-design strategies to understand, control, and exploit QI in molecular junctions poses an exciting challenge. Here we demonstrate that destructive quantum interference (DQI) in meta-substituted phenylene ethylene-type oligomers (m-OPE) can be tuned by changing the position and conformation of methoxy (OMe) substituents at the central phenylene ring. These substituents play the role of molecular-scale taps, which can be switched on or off to control the current flow through a molecule. Our experimental results conclusively verify recently postulated magic-ratio and orbital-product rules, and highlight a novel chemical design strategy for tuning and gating DQI features to create single-molecule devices with desirable electronic functions.

14.
Methods Mol Biol ; 1979: 251-267, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028643

RESUMO

Active DNA demethylation plays important roles in the epigenetic reprogramming of developmental processes. 5-formylcytosine (5fC) is produced during active demethylation of 5-methylcytosine (5mC). Here, we describe a technique called CLEVER-seq (Chemical-labeling-enabled C-to-T conversion sequencing), which detects the whole genome 5fC distribution at single-base and single-cell resolution. CLEVER-seq is suitable for the analysis of precious samples such as early embryos and laser microdissection captured samples.


Assuntos
Citosina/análogos & derivados , Metilação de DNA , DNA/química , DNA/genética , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Animais , Sequência de Bases , Citosina/análise , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos
15.
Angew Chem Int Ed Engl ; 58(1): 130-133, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30407705

RESUMO

The emergence of unnatural DNA bases provides opportunities to demystify the mechanisms by which DNA polymerases faithfully decode chemical information on the template. It was previously shown that two unnatural cytosine bases (termed "M-fC" and "I-fC"), which are chemical labeling adducts of the epigenetic base 5-formylcytosine, can induce C-to-T transition during DNA amplification. However, how DNA polymerases recognize such unnatural cytosine bases remains enigmatic. Herein, crystal structures of unnatural cytosine bases pairing to dA/dG in the KlenTaq polymerase-host-guest complex system and pairing to dATP in the KlenTaq polymerase active site were determined. Both M-fC and I-fC base pair with dA/dATP, but not with dG, in a Watson-Crick geometry. This study reveals that the formation of the Watson-Crick geometry, which may be enabled by the A-rule, is important for the recognition of unnatural cytosines.


Assuntos
Citosina/química , DNA Polimerase Dirigida por DNA/química , DNA/química , Timina/química , Humanos , Estrutura Molecular
16.
J Am Chem Soc ; 140(41): 13190-13194, 2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30278133

RESUMO

High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples remains challenging. Here, we present hmC-CATCH, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. hmC-CATCH is based on selective 5hmC oxidation, chemical labeling and subsequent C-to-T transition during PCR. Requiring only nanoscale input genomic DNA samples, hmC-CATCH enabled us to detect genome-wide hydroxymethylome of human embryonic stem cells in a cost-effective manner. Further application of hmC-CATCH to cell-free DNA (cfDNA) of healthy donors and cancer patients revealed base-resolution hydroxymethylome in the human cfDNA for the first time. We anticipate that our chemical biology approach will find broad applications in hydroxymethylome analysis of limited biological and clinical samples.


Assuntos
5-Metilcitosina/análogos & derivados , Ácidos Nucleicos Livres/química , Genômica/métodos , 5-Metilcitosina/análise , 5-Metilcitosina/química , Ácidos Nucleicos Livres/genética , Células-Tronco Embrionárias/química , Genoma , Humanos , Técnicas de Amplificação de Ácido Nucleico , Oxirredução , Análise de Sequência de DNA
17.
Nat Chem Biol ; 14(7): 680-687, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29785056

RESUMO

Uracil in DNA can be generated by cytosine deamination or dUMP misincorporation; however, its distribution in the human genome is poorly understood. Here we present a selective labeling and pull-down technology for genome-wide uracil profiling and identify thousands of uracil peaks in three different human cell lines. Surprisingly, uracil is highly enriched at the centromere of the human genome. Using mass spectrometry, we demonstrate that human centromeric DNA contains a higher level of uracil. We also directly verify the presence of uracil within two centromeric uracil peaks on chromosomes 6 and 11. Moreover, centromeric uracil is preferentially localized within the binding regions of the centromere-specific histone CENP-A and can be excised by human uracil-DNA glycosylase UNG. Collectively, our approaches allow comprehensive analysis of uracil in the human genome and provide robust tools for mapping and future functional studies of uracil in DNA.


Assuntos
Centrômero/metabolismo , Mapeamento Cromossômico , DNA/metabolismo , Desoxiuridina/metabolismo , Uracila/metabolismo , Linhagem Celular , Centrômero/genética , DNA/genética , Humanos , Espectrometria de Massas
18.
Nat Commun ; 8(1): 901, 2017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-29026078

RESUMO

DNA recognition by transcription activator-like effector (TALE) proteins is mediated by tandem repeats that specify nucleotides through repeat-variable diresidues. These repeat-variable diresidues form direct and sequence-specific contacts to DNA bases; hence, TALE-DNA interaction is sensitive to DNA chemical modifications. Here we conduct a thorough investigation, covering all theoretical repeat-variable diresidue combinations, for their recognition capabilities for 5-methylcytosine and 5-hydroxymethylcytosine, two important epigenetic markers in higher eukaryotes. We identify both specific and degenerate repeat-variable diresidues for 5-methylcytosine and 5-hydroxymethylcytosine. Utilizing these novel repeat-variable diresidues, we achieve methylation-dependent gene activation and genome editing in vivo; we also report base-resolution detection of 5hmC in an in vitro assay. Our work deciphers repeat-variable diresidues for 5-methylcytosine and 5-hydroxymethylcytosine, and provides tools for TALE-dependent epigenome recognition.Transcription activator-like effector proteins recognise specific DNA sequences via tandem repeats. Here the authors demonstrate TALEs can recognise the methylated bases 5mC and 5hmC, enabling them to detect epigenetic modifications.


Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , DNA/metabolismo , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , 5-Metilcitosina/química , Sequência de Bases , DNA/genética , Epigênese Genética , Células HEK293 , Células HeLa , Humanos , Estrutura Molecular , Ligação Proteica , Sequências Repetitivas de Aminoácidos , Efetores Semelhantes a Ativadores de Transcrição/genética
19.
Cell Stem Cell ; 20(5): 720-731.e5, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28343982

RESUMO

Active DNA demethylation in mammals involves ten-eleven translocation (TET) family protein-mediated oxidation of 5-methylcytosine (5mC). However, base-resolution landscapes of 5-formylcytosine (5fC) (an oxidized derivative of 5mC) at the single-cell level remain unexplored. Here, we present "CLEVER-seq" (chemical-labeling-enabled C-to-T conversion sequencing), which is a single-cell, single-base resolution 5fC-sequencing technology, based on biocompatible, selective chemical labeling of 5fC and subsequent C-to-T conversion during amplification and sequencing. CLEVER-seq shows intrinsic 5fC heterogeneity in mouse early embryos, Epi stem cells (EpiSCs), and embryonic stem cells (ESCs). CLEVER-seq of mouse early embryos also reveals the highly patterned genomic distribution and parental-specific dynamics of 5fC during mouse early pre-implantation development. Integrated analysis demonstrates that promoter 5fC production precedes the expression upregulation of a clear set of developmentally and metabolically critical genes. Collectively, our work reveals the dynamics of active DNA demethylation during mouse pre-implantation development and provides an important resource for further functional studies of epigenetic reprogramming in single cells.


Assuntos
Citosina/análogos & derivados , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Animais , Citosina/metabolismo , Metilação de DNA/genética , Regulação da Expressão Gênica , Camundongos , Regiões Promotoras Genéticas/genética
20.
Langmuir ; 32(49): 13159-13166, 2016 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-27951712

RESUMO

A novel approach for the preparation of interconnected macroporous polymers with a controllable pore structure was reported. The method was based on the polymerization of water-in-oil Pickering high internal phase emulsion (HIPE) stabilized by polystyrene (PS)/silica composite particles. The composite Pickering stabilizers were facilely obtained by mixing positively charged PS microspheres and negatively charged silica nanoparticles, and their amphiphilicity could be delicately tailored by varying the ratio of PS and silica. The droplet size of Pickering HIPEs was characterized using an optical microscope. The pore structure of polymer foams was observed using a scanning electron microscope. The interconnectivity of macroporous polymers was evaluated upon their gas permeability, which was greatly improved after etching PS microspheres included in the Pickering stabilizers with tetrahydrofuran. As a result, fine tailoring of the pore structure of polymer foams could be realized by simply tuning the ratio of PS to silica particles in the composite stabilizer.

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