Browsing by Author "Li, Ping"

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    Characterization of a broad-based mosquito yeast interfering RNA larvicide with a conserved target site in mosquito semaphorin-1a genes
    (Springer Nature, 2019-05-22) Mysore, Keshava; Li, Ping; Wang, Chien-Wei; Hapairai, Limb K.; Scheel, Nicholas D.; Realey, Jacob S.; Sun, Longhua; Severson, David W.; Wei, Na; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    BACKGROUND: RNA interference (RNAi), which has facilitated functional characterization of mosquito neural development genes such as the axon guidance regulator semaphorin-1a (sema1a), could one day be applied as a new means of vector control. Saccharomyces cerevisiae (baker's yeast) may represent an effective interfering RNA expression system that could be used directly for delivery of RNA pesticides to mosquito larvae. Here we describe characterization of a yeast larvicide developed through bioengineering of S. cerevisiae to express a short hairpin RNA (shRNA) targeting a conserved site in mosquito sema1a genes. RESULTS: Experiments conducted on Aedes aegypti larvae demonstrated that the yeast larvicide effectively silences sema1a expression, generates severe neural defects, and induces high levels of larval mortality in laboratory, simulated-field, and semi-field experiments. The larvicide was also found to induce high levels of Aedes albopictus, Anopheles gambiae and Culex quinquefasciatus mortality. CONCLUSIONS: The results of these studies indicate that use of yeast interfering RNA larvicides targeting mosquito sema1a genes may represent a new biorational tool for mosquito control.
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    Comparison of porcine corneal decellularization methods and importance of preserving corneal limbus through decellularization
    (PLOS, 2021-03-05) Isidan, Abdulkadir; Liu, Shaohui; Chen, Angela M.; Zhang, Wenjun; Li, Ping; Smith, Lester J.; Hara, Hidetaka; Cooper, David K. C.; Ekser, Burcin; Surgery, School of Medicine
    Background: The aim of this study is to compare the three previously applied, conventional porcine corneal decellularization methods and to demonstrate the importance of preserving the corneal limbus through decellularization. Methods: Fresh, wild-type (with or without) limbus porcine corneas were decellularized using three different methods, including (i) sodium dodecyl sulfate (SDS), (ii) hypertonic saline (HS), and (iii) N2 gas (NG). Post-treatment evaluation was carried out using histological, residual nuclear material, and ultrastructural analyses. Glycerol was used to help reduce the adverse effects of decellularization. The corneas were preserved for two weeks in cornea storage medium. Results: All three decellularization methods reduced the number of keratocytes at different rates in the stromal tissue. However, all methods, except SDS, resulted in the retention of large numbers of cells and cell fragments. The SDS method (0.1% SDS, 48h) resulted in almost 100% decellularization in corneas without limbus. Low decellularization capacity of the NG method (<50%) could make it unfavorable. Although HS method had a more balanced damage-decellularization ratio, its decellularization capacity was lower than SDS method. Preservation of the corneoscleral limbus could partially prevent structural damage and edema, but it would reduce the decellularization capacity. Conclusion: Our results suggest that SDS is a very powerful decellularization method, but it damages the cornea irreversibly. Preserving the corneoscleral limbus reduces the efficiency of decellularization, but also reduces the damage.
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    Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models
    (Wiley, 2020-03) Sego, T. J.; Prideaux, Matthew; Sterner, Jane; McCarthy, Brian Paul; Li, Ping; Bonewald, Lynda F.; Ekser, Burcin; Tovar, Andres; Smith, Lester Jeshua; Anatomy and Cell Biology, School of Medicine
    Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient‐rich oxygenated blood through the vasculature to support cell metabolism within most cell‐dense tissues. Since scaffold‐free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue‐like structures, we generated a generalizable biofabrication method resulting in self‐supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO‐A5 osteoblast‐based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico.
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    Corrigendum to "The Role of Costimulation Blockade in Solid Organ and Islet Xenotransplantation"
    (Hindawi, 2018-03-22) Samy, Kannan P.; Butler, James R.; Li, Ping; Cooper, David K. C.; Ekser, Burcin; Surgery, School of Medicine
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    Decellularization methods for developing porcine corneal xenografts and future perspectives
    (Wiley, 2019-11) Isidan, Abdulkadir; Liu, Shaohui; Li, Ping; Lashmet, Matthew; Smith, Lester J.; Hara, Hidetaka; Cooper, David K. C.; Ekser, Burcin; Ophthalmology, School of Medicine
    Corneal transplantation is the only option to cure corneal opacities. However, there is an imbalance between supply and demand of corneal tissues in the world. To solve the problem of corneal shortage, corneal xenotransplantation studies have been implemented in the past years using porcine corneas. The corneal xenografts could come from (a) wild-type pigs, (b) genetically engineered pigs, (c) decellularized porcine corneas, and (d) decellularized porcine corneas that are recellularized with human corneal cells, eventually with patients' own cells, as in all type of xenografts. All approaches except, the former would reduce or mitigate recipient immune responses. Although several techniques in decellularization have been reported, there is still no standardized protocol for the complete decellularization of corneal tissue. Herein, we reviewed different decellularization methods for porcine corneas based on the mechanism of action, decellularization efficacy, biocompatibility, and the undesirable effects on corneal ultrastructure. We compared 9 decellularization methods including: (a) sodium dodecyl sulfate, (b) triton x-100, (c) hypertonic saline, (d) human serum with electrophoresis, (e) high hydrostatic pressure, (f) freeze-thaw, (h) nitrogen gas, (h) phospholipase A2 , and (i) glycerol with chemical crosslinking methods. It appears that combined methods could be more useful to perform efficient corneal decellularization.
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    Erythrocytes from GGTA1/CMAH knockout pigs: implications for xenotransfusion and testing in non-human primates
    (Wiley, 2014-07) Wang, Zheng-Yu; Burlak, Christopher; Estrada, Jose L.; Li, Ping; Tector, Matthew F.; Tector, A. Joseph; Department of Surgery, IU School of Medicine
    Background Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement mediated lysis. Methods Distribution of anti Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. Results The pooled human AB serum contained 0.38 μg/ml anti Neu5Gc IgG and 0.085 μg/ml anti Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutinaion of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared to GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared to GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared to GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared to GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. Conclusions Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantion researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplation to the clinic.
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    Examination of the genetic basis for sexual dimorphism in the Aedes aegypti (dengue vector mosquito) pupal brain
    (BioMed Central, 2014-10-21) Tomchaney, Michael; Mysore, Keshava; Sun, Longhua; Li, Ping; Emrich, Scott J.; Severson, David W.; Duman-Scheel, Molly; Department of Medical and Molecular Genetics, IU School of Medicine
    BACKGROUND: Most animal species exhibit sexually dimorphic behaviors, many of which are linked to reproduction. A number of these behaviors, including blood feeding in female mosquitoes, contribute to the global spread of vector-borne illnesses. However, knowledge concerning the genetic basis of sexually dimorphic traits is limited in any organism, including mosquitoes, especially with respect to differences in the developing nervous system. METHODS: Custom microarrays were used to examine global differences in female vs. male gene expression in the developing pupal head of the dengue vector mosquito, Aedes aegypti. The spatial expression patterns of a subset of differentially expressed transcripts were examined in the developing female vs. male pupal brain through in situ hybridization experiments. Small interfering RNA (siRNA)-mediated knockdown studies were used to assess the putative role of Doublesex, a terminal component of the sex determination pathway, in the regulation of sex-specific gene expression observed in the developing pupal brain. RESULTS: Transcripts (2,527), many of which were linked to proteolysis, the proteasome, metabolism, catabolic, and biosynthetic processes, ion transport, cell growth, and proliferation, were found to be differentially expressed in A. aegypti female vs. male pupal heads. Analysis of the spatial expression patterns for a subset of dimorphically expressed genes in the pupal brain validated the data set and also facilitated the identification of brain regions with dimorphic gene expression. In many cases, dimorphic gene expression localized to the optic lobe. Sex-specific differences in gene expression were also detected in the antennal lobe and mushroom body. siRNA-mediated gene targeting experiments demonstrated that Doublesex, a transcription factor with consensus binding sites located adjacent to many dimorphically expressed transcripts that function in neural development, is required for regulation of sex-specific gene expression in the developing A. aegypti brain. CONCLUSIONS: These studies revealed sex-specific gene expression profiles in the developing A. aegypti pupal head and identified Doublesex as a key regulator of sexually dimorphic gene expression during mosquito neural development.
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    FABRICA: A Bioreactor Platform for Printing, Perfusing, Observing, & Stimulating 3D Tissues
    (Nature Publishing Group, 2018-05-15) Smith, Lester J.; Li, Ping; Holland, Mark R.; Ekser, Burcin; Radiology and Imaging Sciences, School of Medicine
    We are introducing the FABRICA, a bioprinter-agnostic 3D-printed bioreactor platform designed for 3D-bioprinted tissue construct culture, perfusion, observation, and analysis. The computer-designed FABRICA was 3D-printed with biocompatible material and used for two studies: (1) Flow Profile Study: perfused 5 different media through a synthetic 3D-bioprinted construct and ultrasonically analyzed the flow profile at increasing volumetric flow rates (VFR); (2) Construct Perfusion Study: perfused a 3D-bioprinted tissue construct for a week and compared histologically with a non-perfused control. For the flow profile study, construct VFR increased with increasing pump VFR. Water and other media increased VFR significantly while human and pig blood showed shallow increases. For the construct perfusion study, we confirmed more viable cells in perfused 3D-bioprinted tissue compared to control. The FABRICA can be used to visualize constructs during 3D-bioprinting, incubation, and to control and ultrasonically analyze perfusion, aseptically in real-time, making the FABRICA tunable for different tissues.
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    A functional requirement for sex-determination M/m locus region lncRNA genes in Aedes aegypti female larvae
    (Springer Nature, 2021-05-20) Mysore, Keshava; Hapairai, Limb K.; Li, Ping; Roethele, Joseph B.; Sun, Longhua; Igiede, Jessica; Misenti, Joi K.; Duman‑Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    Although many putative long non-coding RNA (lncRNA) genes have been identified in insect genomes, few of these genes have been functionally validated. A screen for female-specific larvicides that facilitate Aedes aegypti male sex separation uncovered multiple interfering RNAs with target sites in lncRNA genes located in the M/m locus region, including loci within or tightly linked to the sex determination locus. Larval consumption of a Saccharomyces cerevisiae (yeast) strain engineered to express interfering RNA corresponding to lncRNA transcripts resulted in significant female death, yet had no impact on male survival or fitness. Incorporation of the yeast larvicides into mass culturing protocols facilitated scaled production and separation of fit adult males, indicating that yeast larvicides could benefit mosquito population control strategies that rely on mass releases of male mosquitoes. These studies functionally verified a female-specific developmental requirement for M/m locus region lncRNA genes, suggesting that sexually antagonistic lncRNA genes found within this highly repetitive pericentromeric DNA sequence may be contributing to the evolution of A. aegypti sex chromosomes.
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    High-throughput cis-regulatory element discovery in the vector mosquito Aedes aegypti
    (BioMed Central, 2016-05-10) Behura, Susanta K.; Sarro, Joseph; Li, Ping; Mysore, Keshava; Severson, David W.; Emrich, Scott J.; Duman-Scheel, Molly; Department of Medical & Molecular Genetics, IU School of Medicine
    BACKGROUND: Despite substantial progress in mosquito genomic and genetic research, few cis-regulatory elements (CREs), DNA sequences that control gene expression, have been identified in mosquitoes or other non-model insects. Formaldehyde-assisted isolation of regulatory elements paired with DNA sequencing, FAIRE-seq, is emerging as a powerful new high-throughput tool for global CRE discovery. FAIRE results in the preferential recovery of open chromatin DNA fragments that are not bound by nucleosomes, an evolutionarily conserved indicator of regulatory activity, which are then sequenced. Despite the power of the approach, FAIRE-seq has not yet been applied to the study of non-model insects. In this investigation, we utilized FAIRE-seq to profile open chromatin and identify likely regulatory elements throughout the genome of the human disease vector mosquito Aedes aegypti. We then assessed genetic variation in the regulatory elements of dengue virus susceptible (Moyo-S) and refractory (Moyo-R) mosquito strains. RESULTS: Analysis of sequence data obtained through next generation sequencing of FAIRE DNA isolated from A. aegypti embryos revealed >121,000 FAIRE peaks (FPs), many of which clustered in the 1 kb 5' upstream flanking regions of genes known to be expressed at this stage. As expected, known transcription factor consensus binding sites were enriched in the FPs, and of these FoxA1, Hunchback, Gfi, Klf4, MYB/ph3 and Sox9 are most predominant. All of the elements tested in vivo were confirmed to drive gene expression in transgenic Drosophila reporter assays. Of the >13,000 single nucleotide polymorphisms (SNPs) recently identified in dengue virus-susceptible and refractory mosquito strains, 3365 were found to map to FPs. CONCLUSION: FAIRE-seq analysis of open chromatin in A. aegypti permitted genome-wide discovery of CREs. The results of this investigation indicate that FAIRE-seq is a powerful tool for identification of regulatory DNA in the genomes of non-model organisms, including human disease vector mosquitoes.