Publications by authors named "Yong-Ping Huang"

36 Publications

Disruption of egg-specific protein causes female sterility in Bombyx mori.

Insect Sci 2021 Feb 24. Epub 2021 Feb 24.

Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China.

Yolk proteins are the main source of nutrients during embryonic and early larval development in oviparous animals. Therefore, vitellogenesis is crucial for reproduction. The silkworm, Bombyx mori, is a model lepidopteran insect in which there are three yolk proteins: vitellin, 30-kDa protein, and egg-specific protein (Esp). In this study, we explored the gene function of Esp through transgenic clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 technology-mediated mutations in the silkworm. We found that Esp mutation resulted in female sterility but had no effect on male fertility. Female mutants could lay eggs after mating, but the eggs were smaller and lighter colored than those laid by wild-type females. The most important finding is that the eggs laid by female mutants did not hatch. Furthermore, we observed stable inheritance of female sterility caused by Esp mutation through successive generations. Thus, Esp encodes a yolk protein that is crucial for female reproductive success and is a potential target for pest control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12904DOI Listing
February 2021

MicroRNA-2738 regulates gene expression in the sex determination pathway in Bombyx mori.

Insect Sci 2020 Aug 17;27(4):646-654. Epub 2019 Jun 17.

CAS Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, CAS, Shanghai, China.

MicroRNAs (miRNAs) are a class of short, non-coding transcripts that bind to 3'-untranslated regions to trigger messenger RNA degradation or translational inhibition. Here we explored how miRNAs regulate sex determination in Bombyx mori, a lepidopteran model insect. Genes known to be involved in sex determination, BmPSI, Bmdsx, and BmMasc, are predicted targets of the species-specific miR-2738. Using a dual luciferase reporter assay in HEK293T cells, we confirmed that miR-2738 suppressed transcription of BmPSI, Bmdsx, and BmMasc. The levels of BmPSI and BmMasc were significantly down-regulated in B. mori miR-2738 overexpression. In contrast, the genetic disruption of miR-2738 using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 transgenic system increased the levels of BmPSI and BmMasc transcripts, whereas splicing of Bmdsx was unaltered by miR-2738 depletion or overexpression. Taken together, this study implicates miR-2738 as a minor regulator of sex determination genes in the silkworm.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12694DOI Listing
August 2020

Integrated Omics Reveals Tollip as an Regulator and Therapeutic Target for Hepatic Ischemia-Reperfusion Injury in Mice.

Hepatology 2019 11 22;70(5):1750-1769. Epub 2019 Jun 22.

College of Life Sciences, Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, China.

Hepatic ischemia-reperfusion (IR) injury is the leading cause of liver dysfunction and failure after liver resection or transplantation and lacks effective therapeutic strategies. Here, we applied a systematic proteomic analysis to identify the prominent contributors to IR-induced liver damage and promising therapeutic targets for this condition. Based on an unbiased proteomic analysis, we found that toll-interacting protein (Tollip) expression was closely correlated with the hepatic IR process. RNA sequencing analysis and phenotypic examination showed a dramatically alleviated hepatic IR injury by Tollip deficiency both in vivo and in hepatocytes. Mechanistically, Tollip interacts with apoptosis signal-regulating kinase 1 (ASK1) and facilitates the recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF6) to ASK1, leading to enhanced ASK1 N-terminal dimerization and the subsequent activation of downstream mitogen-activated protein kinase (MAPK) signaling. Furthermore, the Tollip methionine and phenylalanine motif and TRAF6 ubiquitinating activity are required for Tollip-regulated ASK1-MAPK axis activation. Conclusion: Tollip is a regulator of hepatic IR injury by facilitating ASK1 N-terminal dimerization and the resultant c-Jun N-terminal kinase/p38 signaling activation. Inhibiting Tollip or its interaction with ASK1 might be promising therapeutic strategies for hepatic IR injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.30705DOI Listing
November 2019

CRISPR/Cas9-mediated ebony knockout results in puparium melanism in Spodoptera litura.

Insect Sci 2019 Dec 5;26(6):1011-1019. Epub 2019 Mar 5.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12663DOI Listing
December 2019

Identification of a germline-expression promoter for genome editing in Bombyx mori.

Insect Sci 2019 Dec 27;26(6):991-999. Epub 2019 Feb 27.

CAS Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China.

Identification of stage- and tissue-specific cis-regulatory elements will enable more precise genomic editing. In previous studies of the silkworm Bombyx mori, we identified and characterized several tissue- and sex-specific cis-regulatory elements using transgenic technology, including a female- and fat body-specific promoter, vitellogenin, testis-specific promoters, Radial spoke head 1 (BmR1) and beta-tubulin 4 (Bmβ4). Here we report a cis-regulatory element specific for a somatic and germ cell-expressed promoter, nanos (Bmnos). We investigated activities of three truncated promoter sequences upstream of the transcriptional initiation site sequences of Bmnos in vitro (nos-0.6kb, nos-1kb and nos-2kb) and in vivo (nos-2kb). In BmN cultured cells, all three lengths drove expression of the gene encoding enhanced green fluorescence protein (EGFP), although nos-2kb had the highest fluorescence activity. In transgenic silkworms, nos-2kb drove EGFP expression at the early embryonic stage, and fluorescence was concentrated in the gonads at later embryonic stages. In addition, this cis-regulatory element was not sex differentiated. The fluorescence intensity gradually weakened following the larval developmental stage in the gonads and were broadly expressed in the whole body. The nos-2kb promoter drove the Cas9 system with efficiency comparable to that of the broad-spectrum strong IE1 promoter. These results indicate that Bmnos is an effective endogenous cis-regulatory element in the early embryo and in the gonad that can be used in applications involving the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12657DOI Listing
December 2019

CRISPR/Cas9-mediated Tyrosine hydroxylase knockout resulting in larval lethality in Agrotis ipsilon.

Insect Sci 2018 Dec;25(6):1017-1024

School of Life Science, East China Normal University, Shanghai, China.

Tyrosine hydroxylase (TH) is involved in insect melanin and the catecholamine biosynthesis pathway. TH as an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine is the first step reaction in the pathway. Although TH has been proven to affect the pigmentation of the epidermis and development in many insects, there is no report about physiological function of the TH gene in Agrotis ipsilon. Here we cloned the TH gene from A. ipsilon. Semi-quantitative real-time polymerase chain reaction (PCR) analysis showed that AiTH was expressed at all development stages. Moreover, its high expression levels in the head and epidermis suggest that it is mainly related to pigment deposition and insect development. Then, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system to target the AiTH gene: deletion events were detected at the target sites. Compared with the control group, a few mutants with the phenomenon of narrowing in the egg shell and embryos can develop but cannot hatch; the other hatched embryos were seriously dehydrated after hatching and died within the first day. Quantitative real-time PCR analysis revealed that TH was down-regulated in AiTH mutants. Here, our work demonstrated that AiTH plays an important role in growth and development of newly hatched larvae; meanwhile, it would be a promising target to explore a control strategy for A. ipsilon.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12647DOI Listing
December 2018

The Masc gene product controls masculinization in the black cutworm, Agrotis ipsilon.

Insect Sci 2019 Dec 17;26(6):1037-1044. Epub 2018 Sep 17.

CAS Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai, China.

Sex determination has been studied in the model lepidopteran species Bombyx mori, but it remains poorly understood in lepidopteran pests. In the present study, we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species, Agrotis ipsilon. Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280). We assessed the masculinizing activity of AiMasc in BmN cells and found that AiMasc induced expression of the male-specific doublesex isoform. Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A. ipsilon caused abnormalities in abdominal segments and external genitalia, resulting in male-specific sterility. These results suggest that Masc participates in the process of sex determination in A. ipsilon. Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12635DOI Listing
December 2019

BmHpo mutation induces smaller body size and late stage larval lethality in the silkworm, Bombyx mori.

Insect Sci 2018 Dec 24;25(6):1006-1016. Epub 2018 Jul 24.

School of Life Science, East China Normal University, Shanghai, China.

As a core member of the Hippo signaling pathway, Hpo plays a critical role in regulating growth and development. Previous studies reported that loss of function of Hpo results in increased proliferation, reduced apoptosis and induction of tissue overgrowth in Drosophila. In this study, we used CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) to study Hpo gene (BmHpo) function in the lepidopteran insect Bombyx mori, known commonly as the silkworm. Sequence analysis of BmHpo revealed an array of deletions in mutants. We found that BmHpo knockout resulted in defects in body size regulation, in developmental defects and pigment accumulation and early death. Our data show that BmHpo is essential for regulation of insect growth and development and that CRISPR/Cas9 technology can serve as a basis for functional analysis of target genes in lepidopteran insects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12607DOI Listing
December 2018

Identification and functional characterization of doublesex gene in the testis of Spodoptera litura.

Insect Sci 2019 Dec 19;26(6):1000-1010. Epub 2018 Jul 19.

Guangzhou Key Laboratory of Insect Development Regulation and Applied Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China.

Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific Sldsx and female-specific Sldsx isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12608DOI Listing
December 2019

CRISPR disruption of TCTP gene impaired normal development in the silkworm Bombyx mori.

Insect Sci 2019 Dec 19;26(6):973-982. Epub 2018 Feb 19.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

The translationally controlled tumor protein (TCTP) is a highly conserved and multifunctional protein with activities ranging from cytoskeletal regulation to transcription regulation in numerous organisms. In insects, TCTP is essential for cell growth and proliferation. Recently, TCTP has been reported to affect the innate intestinal immune pathway in the Bombyx mori silkworm, a lepidopteran model insect. However, the comprehensive physiological roles of TCTP in the silkworm remain poorly understood. Here, we performed functional analysis of BmTCTP by using a binary transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/RNA-guided CRISPER-associated protein 9 nucleases) system. Disruption of BmTCTP led to developmental arrestment and subsequent lethality in third instar larvae. Histological analysis revealed that growth impairment originated from decreased cell size, and the proliferation and differentiation of intestinal epithelial cells were also affected. RNA-seq analysis revealed that genes involved in carbohydrate metabolism, lipid metabolism and digestive system pathways were significantly affected by BmTCTP depletion. Together, the results demonstrated that BmTCTP plays a key role in controlling larval growth and development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380024PMC
December 2019

Metatranscriptome of the protistan community in Reticulitermes flaviceps.

Insect Sci 2016 Aug 29;23(4):543-7. Epub 2016 Jun 29.

Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

The hindgut of lower termites harbors various symbiotic protists, which perform varied functions in lignocellulose decomposition. As termites are social insects, the species and numbers of these flagellated protists in the termite gut vary among the different castes. Juvenile hormones (JHs) can regulate caste differentiation in termites. In this study, we used the juvenile hormone analog fenoxycarb to induce termite workers (Reticulitermes flaviceps) to differentiate into pre-soldiers. A metatranscriptomic investigation of the protistan community was then performed by 454 pyrosequencing. From a thorough analysis based on 597 312 generated reads, we found that the starch and sucrose metabolism pathway was the most abundant pathway across the metatranscriptome. The current study demonstrates that the metatranscriptome of the protistan community in termites contains an abundance of lignocellulase, which plays a vital role in termite nutrition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12363DOI Listing
August 2016

Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing.

Insect Biochem Mol Biol 2016 08 15;75:1-9. Epub 2016 May 15.

Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address:

Functional gene analysis by using genome editing techniques is limited only in few model insects. Here, we reported an efficient and heritable gene mutagenesis analysis in an important lepidopteran pest, Spodoptera litura, using the CRISPR/Cas9 system. By using this system, we successfully obtained the homozygous S. litura strain by targeting the pheromone binding protein 3 gene (SlitPBP3), which allowed us to elucidate the role of this gene in the olfaction of the female sex pheromones. By co-injection of Cas9 mRNA and sgRNA into S. litura eggs, highly efficient chimera mutation in SlitPBP3 loci was detected both in injected eggs (39.1%) and in the resulting individual moths (87.5%). We used the mutant moths as parents to obtain the G1 offspring and the homozygous mutant strain in G2. The function of SlitPBP3 was explored by Electroantennogram (EAG) recordings with a homozygous mutant strain. The result showed that the EAG responses were significantly decreased in mutant males than in control males when treated with the major sex pheromone component (Z9,E11-14:Ac) and a minor component (Z9-14:Ac) at higher dosages. The results demonstrate that s SlitPBP3 gene plays a minor role in the perception of the female sex pheromones. Furthermore, our study provides a useful methodology with the CRISPR/Cas9 system for gene in vivo functional study, particular for lepidopteran species in which the RNAi approach is not efficient.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ibmb.2016.05.006DOI Listing
August 2016

Leap forward with insect genomics.

Insect Sci 2016 Jun;23(3):332-4

Key Laboratory of Insect Development and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12355DOI Listing
June 2016

CRISPR/Cas9-mediated targeted gene mutagenesis in Spodoptera litura.

Insect Sci 2016 Jun 13;23(3):469-77. Epub 2016 May 13.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

Custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis mediated by the CRISPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricultural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A (Slabd-A) gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of insects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA (sgRNA) into S. litura embryos successfully induced the typical abd-A deficient phenotype, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1744-7917.12341DOI Listing
June 2016

Gossypol-enhanced P450 gene pool contributes to cotton bollworm tolerance to a pyrethroid insecticide.

Mol Ecol 2012 Sep 20;21(17):4371-85. Epub 2012 Apr 20.

National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

Cotton plants accumulate phytotoxins, including gossypol and related sesquiterpene aldehydes, to resist insect herbivores and pathogens. To counteract these defensive plant secondary metabolites, cotton bollworms (Helicoverpa armigera) elevate their production of detoxification enzymes, including cytochrome P450 monooxygenases (P450s). Besides their tolerance to phytotoxin, cotton bollworms have quickly developed resistance to deltamethrin, a widely used pyrethroid insecticide in cotton field. However, the relationship between host plant secondary metabolites and bollworm insecticide resistance is poorly understood. Here, we show that exogenously expressed CYP6AE14, a gossypol-inducible P450 of cotton bollworm, has epoxidation activity towards aldrin, an organochlorine insecticide, indicating that gossypol-induced P450s participate in insecticide metabolism. Gossypol-ingested cotton bollworm larvae showed higher midgut P450 enzyme activities and exhibited enhanced tolerance to deltamethrin. The midgut transcripts of bollworm larvae administrated with different phytochemicals and deltamethrin were then compared by microarray analysis, which showed that gossypol and deltamethrin induced the most similar P450 expression profiles. Gossypol-induced P450s exhibited high divergence and at least five of them (CYP321A1, CYP9A12, CYP9A14, CYP6AE11 and CYP6B7) contributed to cotton bollworm tolerance to deltamethrin. Knocking down one of them, CYP9A14, by plant-mediated RNA interference (RNAi) rendered the larvae more sensitive to the insecticide. These data demonstrate that generalist insects can take advantage of secondary metabolites from their major host plants to elaborate defence systems against other toxic chemicals, and impairing this defence pathway by RNAi holds a potential for reducing the required dosages of agrochemicals in pest control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-294X.2012.05548.xDOI Listing
September 2012

[Variations of the amount of sialic acids on hepatocellular carcinoma cell membrane].

Nan Fang Yi Ke Da Xue Xue Bao 2010 Oct;30(10):2323-6

Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.

Objective: To observe the change in the amount of sialic acids on hepatocellular carcinoma (HCC) cell membrane.

Methods: Surgical specimens of HCC and liver cirrhosis tissues were obtained from 28 patients to prepare carcinoma cell and hepatocyte suspensions by collagenase digestion. For assay of α2, 3 and α2, 6-sialic acids, the cells were suspended in the staining buffer containing either fluorescein isothiocyanate-Maackia amurensis lectin (FITC-MAL) or fluorescein isothiocyanate-Sambucus nigra bark lectin (FITC-SNA) and incubated for 1 h, respectively. Flow cytometric analysis was carried out to measure the mean fluorescence intensity (MFI) on the cell surface.

Results: In both FITC-MAL- and FITC-SNA-incubated HCC cells, the MFI on the cell surface was greater than that of the hepatocytes.

Conclusion: Both of α2, 3 and α2, 6- sialic acids increases significantly on the hepatocyte membrane after the carcinomatous change.
View Article and Find Full Text PDF

Download full-text PDF

Source
October 2010

[Intrahepatic transplantation of in vitro induced autologous bone marrow-derived liver stem cells in patients with posthepatitic cirrhosis].

Nan Fang Yi Ke Da Xue Xue Bao 2010 Mar;30(3):529-1

Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.

Objective: To evaluate the therapeutic effect of in vitro induced autologous bone marrow-derived liver stem cell transplantation for posthepatitic cirrhosis.

Methods: Between Jun 2008 and Mar 2009, 12 patients with posthepatitic cirrhosis and portal hypertensive underwent azygousportal disconnection and splenectomy in our department. The patients were then divided into two groups to receive autologous bone marrow-deprived liver stem cell infusion via the hepatic artery after in vitro induction for 7 days (n=6) or saline (n=6). The therapeutic effects of the operations on the liver functions and liver fibrosis index were evaluated.

Results: All the patients recovered uneventfully and no side effect of the operation was found. After the operation, the patients receiving bone marrow-deprived liver stem cell infusion showed better hepatic function improvement than those receiving saline infusion (P<0.05).

Conclusion: Transplantation of in vitro induced autologous bone marrow-derived liver stem cell via the hepatic artery is safe and effective for treatment of posthepatitic cirrhosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
March 2010

[Protocols for cloning human bone marrow-derived hepatic stem cells in vitro].

Nan Fang Yi Ke Da Xue Xue Bao 2010 Feb;30(2):318-20

Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.

Objective: To explore practical protocols for cloning bone marrow-derived hepatic stem cells in vitro.

Methods: The cell fraction rich in CD117(+) cells and CD184(+) cells was separated from fresh bone marrow by density gradient centrifugation and cultured for 0, 7 and 14 days in high-glucose DMEM supplemented with or without 10% autologous serum or in serum-free high-glucose DMEM. All the media were supplemented with different concentrations of hepatocyte growth promoting factors (HGPF), thrombopoietin (TPO) and interleukin-3 (IL-3). The quantitative changes of CD117(+) cells and CD184(+) cells were measured by flow cytometry.

Results: The optimal effect for cell cloning was achieved with high-glucose DMEM with 10% autologous serum group supplemented with 40 microg/ml HGPF, 50 ng/ml TPO, and 10 ng/ml IL-3. At day 7 of cell culture in this media, the quantity of CD117(+) cells and CD184(+) cells increased by 6.55 and 6.20 folds, and by 11.62 and 20.57 folds at day 14, respectively.

Conclusion: It is practical for cloning bone marrow-derived hepatic stem cells in high-glucose DMEM with 10% autologous serum supplemented with 40 microg/ml HGPF, 50 ng/ml TPO, and 10 ng/ml IL-3.
View Article and Find Full Text PDF

Download full-text PDF

Source
February 2010

[Mapping of non-lepis wing gene nlw in silkworm (Bombyx mori) using SSR and STS markers].

Yi Chuan 2010 Jan;32(1):54-8

Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China.

The non-lepis wing of silkworm (Bombyx mori) is controlled by the recessive gene, nlw. Owning to lack of crossing over in females, the reciprocal backcrossed F(1) (BC(1)) progenies were used for linkage analysis and mapping of nlw based on the SSR linkage map and STS markers using the wild type (+(nlw)/+(nlw)) silkworm strain P50 and U06 with scaleless wing (nlw/nlw). The nlw gene was linked to eight SSR markers and one STS marker. All the individuals with the wild type in the BC1F (Using F(1) as female to backcross to the recessive parent, that is (U06xP50)xU06) showed heterozygous profile of (U06xP50) F(1), and the ones with non-lepis wing in BC1F exhibited the homozygous profile of the strain U06. Using a reciprocal BC1M (Using F1 as male to backcross to the recessive parent, that is U06x(U06xP50))cross, we constructed a linkage map of 125.6 cM, and the distance between nlw and the nearest marker cash2p was 11.4 cM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3724/sp.j.1005.2010.00054DOI Listing
January 2010

[Mapping of the yellow inhibitor gene I in silkworm Bombyx mori using SSR markers].

Yi Chuan 2008 Aug;30(8):1039-42

College of Biotechnology and Environmental Engineering, Jiangsu University of Science and Technology, Zhenjiang 212018, China.

The yellow color of silkworm (Bombyx mori) cocoon is mainly controlled by three genes, Y (yellow blood), I (yellow inhibitor) and C (out-layer yellow cocoon) genes. I gene locates on the 9th chromosome of silkworm and prevents the transport of carotenoid from epithelia of midgut into hemolymph. Owning to a lack of crossing over in females, reciprocal backcrossed F1(BC1) progenies were used for linkage analysis and mapping of the I gene based on the SSR linkage map using silkworm strains Baghdad (Ba), which express white hemolymph (II+Y+Y), and KY, which express yellow hemolymph (+I+IYY). The gene of interest was linked to three (S0904, S0905, and S0906) SSR markers. All the individuals with white hemolymph in the BC1F (BC1 was generated using F1 as female) showed heterozygous profile of (BaxKY) F1, and the yellow ones in BC1F showed the homozygous profile of the strain KY. Using a reciprocal BC1M cross, we con-structed a linkage map of 38.4 cM, and the distance between I gene and the nearest marker S0904 is 7.4 cM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3724/sp.j.1005.2008.01039DOI Listing
August 2008

Differentially expressed genes in resistant and susceptible Bombyx mori strains infected with a densonucleosis virus.

Insect Biochem Mol Biol 2008 Sep 16;38(9):853-61. Epub 2008 Jul 16.

Institute of Insect Sciences, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, China.

We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ibmb.2008.06.004DOI Listing
September 2008

[Identification of necrophagous fly species from 12 different cities and regions in China using inter-simple sequence repeat melocular markers].

Nan Fang Yi Ke Da Xue Xue Bao 2008 Apr;28(4):524-8

Department of Pathogenic Biology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China.

Objective: To identify necrophagous fly species from different regions in China using inter-simple sequence repeat (ISSR) melocular markers and analyze their genetic difference and relationship.

Methods: Five carrion fly species were collected from 12 cities and regions in China, including M.domestica, Lucilia sericata, Chrysomyia megacephala, Helicophagella melanura, and Boetthcherisca peregrina. Twenty-two ISSR primers were designed and synthesized, from which 8 were selected to identify the necrophagous fly species. Cluster analysis was conducted based on distance matrices using unweighted pair group method.

Results: Totally 121 amplification samples were obtained using the 8 primers, and 679 clear and stable bands were visualized including 516 bands with polymorphisms. M.domestica, Lucilia sericata, Chrysomyia megacephala, Helicophagella melanura, and Boethcherisca peregrina from different regions in China produced their specific PCR band spectra. M. domestica from 10 different regions in China showed different inheritance patterns of the markers. Species-specific ISSR fragment was found among the necrophagous flys pecies. Cluster analysis among the most abundant carrion fly species demonstrated that M.domestica from 10 different regions could be divided into 4 groups at different levels. Most of the Chrysomyia megacephala and Lucilia sericata could be clustered in one tree.

Conclusion: This study represents the first identification of the common necrophagous fly species in China. ISSR-PCR-based identification of the species reveals the genetic diversity and genotypic difference among M.domestica from 10 cities and regions in China.
View Article and Find Full Text PDF

Download full-text PDF

Source
April 2008

Inheritance and linkage analysis of co-dominant SSR markers on the Z chromosome of the silkworm (Bombyx mori L.).

Genet Res (Camb) 2008 Apr;90(2):151-6

Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, People's Republic of China.

Microsatellites or simple sequence repeats (SSRs) are co-dominant molecular markers. When we used fluorescent SSR markers to construct a linkage map for the female heterogametic silkworm (Bombyx mori, ZW), we found that some loci did not segregate in a Mendelian ratio of 1:1 in a backcross population. These loci segregated in a 3:1 ratio of single bands compared with double bands. Further examination of band patterns indicated that three types of SSR bands were present: two homozygotes and one heterozygote. In the beginning, we considered to discard these markers. By scoring male and female F1 individuals, we confirmed that these loci were located on the Z chromosome. Using the sex-linked visible mutation sch (K05) and its wild-type (C108), we constructed an F1 male backcross (BC1M) mapping population. The combination of sch backcross and SSR data enabled us to map the SSR markers to the Z chromosome. By adjusting input parameters based on these data, we were able to use Mapmaker software to construct a linkage map. This strategy takes advantage of co-dominant markers for positional cloning of genes on the Z chromosome. We localized sch to the Z chromosome relative to six SSR markers and one PCR marker, covering a total of 76.1 cM. The sch mutation is an important sex-linked visible mutation widely used in breeding of commercial silkworms (e.g. male silkworm selection rearing). Localization of the sch gene may prove helpful in cloning the gene and developing strains for marker-assisted selection in silkworm breeding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1017/S0016672308009221DOI Listing
April 2008

Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol.

Nat Biotechnol 2007 Nov 4;25(11):1307-13. Epub 2007 Nov 4.

National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, P.R. China.

We identify a cytochrome P450 gene (CYP6AE14) from cotton bollworm (Helicoverpa armigera), which permits this herbivore to tolerate otherwise inhibitory concentrations of the cotton metabolite, gossypol. CYP6AE14 is highly expressed in the midgut and its expression correlates with larval growth when gossypol is included in the diet. When larvae are fed plant material expressing double-stranded RNA (dsRNA) specific to CYP6AE14, levels of this transcript in the midgut decrease and larval growth is retarded. Both effects are more dramatic in the presence of gossypol. As a glutathione-S-transferase gene (GST1) is silenced in GST1 dsRNA-expressing plants, feeding insects plant material expressing dsRNA may be a general strategy to trigger RNA interference and could find applications in entomological research and field control of insect pests.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nbt1352DOI Listing
November 2007

Proteomic analysis of silk gland programmed cell death during metamorphosis of the silkworm Bombyx mori.

J Proteome Res 2007 Aug 4;6(8):3003-10. Epub 2007 Jul 4.

Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, PR China.

The silk gland of the silkworm Bombyx mori undergoes programmed cell death (PCD) during pupal metamorphosis. On the basis of their morphological changes and the occurrence of a DNA ladder, the tissue cells were categorized into three groups: intact, committed, and dying. To identify the proteins involved in this process, we conducted a comparative proteomic analysis. Protein expression changes among the three different cell types were examined by two-dimensional gel electrophoresis. Among approximately 1000 reproducibly detected protein spots on each gel, 43 were down-regulated and 34 were up-regulated in PCD process. Mass spectrometry identified 17 differentially expressed proteins, including some well-studied proteins as well as some novel PCD related proteins, such as caspases, proteasome subunit, elongation factor, heat shock protein, and hypothetical proteins. Our results suggest that these proteins may participate in the silk gland PCD process of B. mori and, thus, provide new insights for this mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr070043fDOI Listing
August 2007

A flavonoid glycoside isolated from Smilax china L. rhizome in vitro anticancer effects on human cancer cell lines.

J Ethnopharmacol 2007 Aug 18;113(1):115-24. Epub 2007 May 18.

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China.

The anticancer activity of eight crude extracts of Smilax china L. rhizome (SCR) against HeLa cells was assessed by MTT assay and clonogenic assay, the fraction rich in flavonoids had show good activity against HeLa cells. A bioassay-guided separation on this extract lead to the detection of kaempferol-7-O-beta-D-glucoside (KG), which belongs to flavonoid glycoside, displayed marked anticancer activity. We evaluated its in vitro cytotoxicity and antiproliferative effect in a panel of established cancer cell lines by MTT assay and clonogenic assay. KG induces A375 and HL60 cells apoptosis, which was demonstrated by morphological changes, DNA fragmentation and flow cytometric analysis. Fluorescent staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the A375 and HL60 cells. Flow cytometric analysis shown that A375 and HL60 cells treated with KG resulted in the appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. Quantitation of the hypodiploid cells shows a dose-dependent response to KG, and this result is in good accordance with that of the DNA fragmentation assay by agarose gel electrophoresis. Our results suggested that cell cycle arrest at G(1) phase and induce apoptosis as a mechanism by which KG exerts an antiproliferative effect.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jep.2007.05.016DOI Listing
August 2007

Microbial communities in the larval midgut of laboratory and field populations of cotton bollworm (Helicoverpa armigera).

Can J Microbiol 2006 Nov;52(11):1085-92

Shanghai Institute of Plant Physiology and Ecology, Shanghai Institute Biological Sciences, The Chinese Academy of Sciences, Shanghai, China.

We compared the bacterial communities in the larval midgut of field and laboratory populations of a polyphagous pest, the cotton bollworm (Helicoverpa armigera), using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences and 16S library sequence analysis. DGGE profiles and 16S rDNA library sequence analysis indicated similar patterns of midgut microbial community structure and diversity: specific bacterial types existed in both populations, and a more diverse microbial community was observed in caterpillars obtained from the field. The laboratory population harbored a rather simple gut microflora consisting mostly of phylotypes belonging to Enterococcus (84%). For the field population, phylotypes belonging to Enterococcus (28%) and Lactococcus (11%), as well as Flavobacterium (10%), Acinetobacter (19%), and Stenotrophomonas (10%) were dominant members. These results provided the first comprehensive description of the microbial diversity of the midgut of the important pest cotton bollworm and suggested that the environment and food supply might influence the diversity of the gut bacterial community.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1139/w06-064DOI Listing
November 2006

Construction of fingerprinting and genetic diversity of mulberry cultivars in China by ISSR markers.

Yi Chuan Xue Bao 2006 Sep;33(9):851-60

Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China.

The ISSR fingerprintings of 24 mulberry cultivars were constructed. Totally 80 bands were produced using 17 primers selected from 20 primers. Of them, 40 bands showed polymorphism. From the bands amplified, there were three independent ways to identify the mulberry varieties, such as unique ISSR markers, unique band patterns and a combination of the band patterns provided by different primers. ISSRs were very effective in differentiating the mulberry varieties. The mean genetic similarity coefficient, the mean Nei's gene diversity (h), and the mean Shannon's Information index (I) of mulberry cultivars were 0.8731, 0.1210, and 0.1942, respectively. This suggests that the genetic diversity of mulberry cultivars was low and the genetic base was narrow. Both UPGMA cluster and PCA (Principal Coordinates Analysis) analysis showed clear genetic relationships among the 24 mulberry cultivars. The major clusters were related to known pedigree relationships.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S0379-4172(06)60119-4DOI Listing
September 2006

[Genetic diversity of Beauveria bassiana (Bals.) Vuill. in forest ecosystem assessed by inter-simple sequence repeat (ISSR) markers].

Yi Chuan 2006 Aug;28(8):977-83

Anhui Key Laboratory of Microbial Control, Anhui Agricultural University, Hefei 230036, China.

In the present paper, the genetic diversity of 48 Beauveria bassiana strains from different altitudes and at different seasons in Dabie Mountains of western Anhui was estimated using inter-simple sequence repeat (ISSR) markers. Twelve among 33 ISSR primers were chosen for their reproducibility and high polymorphism. Seven (2 - 11) markers per primer were scored, and a total of 84 fragments were amplified, in which 73 (81%) were polymorphic. Genetic diversity analysis revealed a relatively high level of intraspecific genetic diversity of B. bassiana in Dabie Mountains of western Anhui: the percentage of polymorphic loci (PPL) was 81%, Nei's genetic diversity (He) was 0.3187 and Shannon's genetic diversity index (I) was 0.4782. The genetic differentiation, Gst was 0.1028, indicating that a low degree of genetic differentiation occurred in the B. Bassiana among populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
August 2006

Simple sequence repeat-based consensus linkage map of Bombyx mori.

Proc Natl Acad Sci U S A 2005 Nov 1;102(45):16303-8. Epub 2005 Nov 1.

Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.

We established a genetic linkage map employing 518 simple sequence repeat (SSR, or microsatellite) markers for Bombyx mori (silkworm), the economically and culturally important lepidopteran insect, as part of an international genomics program. A survey of six representative silkworm strains using 2,500 (CA)n- and (CT)n-based SSR markers revealed 17-24% polymorphism, indicating a high degree of homozygosity resulting from a long history of inbreeding. Twenty-nine SSR linkage groups were established in well characterized Dazao and C108 strains based on genotyping of 189 backcross progeny derived from an F(1) male mated with a C108 female. The clustering was further focused to 28 groups by genotyping 22 backcross progeny derived from an F(1) female mated with a C108 male. This set of SSR linkage groups was further assigned to the 28 chromosomes (established linkage groups) of silkworm aided by visible mutations and cleaved amplified polymorphic sequence markers developed from previously mapped genes, cDNA sequences, and cloned random amplified polymorphic DNAs. By integrating a visible mutation p (plain, larval marking) and 29 well conserved genes of insects onto this SSR-based linkage map, a second generation consensus silkworm genetic map with a range of 7-40 markers per linkage group and a total map length of approximately 3431.9 cM was constructed and its high efficiency for genotyping and potential application for synteny studies of Lepidoptera and other insects was demonstrated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0507794102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1283447PMC
November 2005