UFGI publication round-up week 6/11

18 Jun 2018

2018 Jun 15. doi: 10.1021/acs.est.8b02805. [Epub ahead of print]

In Silico Computational Transcriptomics Reveals Novel Endocrine Disruptors in Largemouth Bass ( Micropterus salmoides).

Basili D¹, Zhang JL^2,³, Herbert J¹, Kroll K³, Denslow ND³, Martyniuk CJ³, Falciani F¹, Antczak P¹.

Author information

1: Institute for Integrative Biology, University of Liverpool , L69 7ZB , Liverpool , United Kingdom.
2: Henan Open Laboratory of Key Subjects of Environmental and Animal Products Safety, College of Animal Science and Technology , Henan University of Science and Technology , Henan 471003 , China.
3: Center for Environmental and Human Toxicology, Department of Physiological Sciences, College of Veterinary Medicine and UF Genetics Institute , University of Florida , Gainesville , Florida 32611 , United States.

Abstract

In recent years, decreases in fish populations have been attributed, in part, to the effect of environmental chemicals on ovarian development. To understand the underlying molecular events we developed a dynamic model of ovary development linking gene transcription to key physiological end points, such as gonadosomatic index (GSI), plasma levels of estradiol (E2) and vitellogenin (VTG), in largemouth bass ( Micropterus salmoides). We were able to identify specific clusters of genes, which are affected at different stages of ovarian development. A subnetwork was identified that closely linked gene expression and physiological end points and by interrogating the Comparative Toxicogenomic Database (CTD), quercetin and tretinoin (ATRA) were identified as two potential candidates that may perturb this system. Predictions were validated by investigation of reproductive associated transcripts using qPCR in ovary and in the liver of both male and female largemouth bass treated after a single injection of quercetin and tretinoin (10 and 100 μg/kg). Both compounds were found to significantly alter the expression of some of these genes. Our findings support the use of omics and online repositories for identification of novel, yet untested, compounds. This is the first study of a dynamic model that links gene expression patterns across stages of ovarian development.

2018 Jun 13:1-11. doi: 10.1080/09593330.2018.1485749. [Epub ahead of print]

Hydrotalcite-based CeNiAl mixed oxides for SO₂ adsorption and oxidation.

Zhao L^1,², Kang Q¹, Guan X¹, Martyniuk CJ².

Author information

1: a School of Ecology and Environment , Inner Mongolia University , Hohhot , People’s Republic of China.
2: b Center for Environmental and Human Toxicology, Department of Physiological Sciences, College of Veterinary Medicine , University of Florida , Gainesville , FL , USA.

Abstract

The impact of Ce on SO₂ adsoption and oxidation was studied over a series of flower-like hydrotalcite-based CeNiAl mixed oxides. Combined with XRD, BET, pyridine chemisorption, CO₂-TPD, XPS and H₂-TPR results, it revealed that introduction of Ce into NiAlO generates new centres for oxygen storage and release, promotes the enhancement of Lewis acid strength, increases weakly and strongly alkaline sites, and increases ability for SO₂adsorption and oxidation. Furthermore, in situ Fourier transform infrared spectroscopy revealed that adsorbed SO₂molecules formed surface bidentate binuclear sulfate. Taken together, we propose that the addition of Ce⁴⁺ to NiAlO acts to improve this compound as major adsorbent for SO₂.

2016 Sep 6;6:32717. doi: 10.1038/srep32717.

Na2CO3-responsive mechanisms in halophyte Puccinellia tenuiflora roots revealed by physiological and proteomic analyses.

Zhao Q^1,², Suo J², Chen S³, Jin Y², Ma X², Yin Z², Zhang Y¹, Wang T⁴, Luo J⁵, Jin W⁵, Zhang X⁴, Zhou Z¹, Dai S^1,².

Author information

1: Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China.
2: Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin 150040, China.
3: Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610, USA.
4: Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.
5: AB Sciex Asia Pacific Application Support Center, Shanghai 200233, China.

Abstract

Soil alkalization severely affects crop growth and agricultural productivity. Alkali salts impose ionic, osmotic, and high pH stresses on plants. The alkali tolerance molecular mechanism in roots from halophyte Puccinellia tenuiflora is still unclear. Here, the changes associated with Na2CO3 tolerance in P. tenuiflora roots were assessed using physiological and iTRAQ-based quantitative proteomic analyses. We set up the first protein dataset in P. tenuiflora roots containing 2,671 non-redundant proteins. Our results showed that Na2CO3 slightly inhibited root growth, caused ROS accumulation, cell membrane damage, and ion imbalance, as well as reduction of transport and protein synthesis/turnover. The Na2CO3-responsive patterns of 72 proteins highlighted specific signaling and metabolic pathways in roots. Ca(2+) signaling was activated to transmit alkali stress signals as inferred by the accumulation of calcium-binding proteins. Additionally, the activities of peroxidase and glutathione peroxidase, and the peroxiredoxin abundance were increased for ROS scavenging. Furthermore, ion toxicity was relieved through Na(+) influx restriction and compartmentalization, and osmotic homeostasis reestablishment due to glycine betaine accumulation. Importantly, two transcription factors were increased for regulating specific alkali-responsive gene expression. Carbohydrate metabolism-related enzymes were increased for providing energy and carbon skeletons for cellular metabolism. All these provide new insights into alkali-tolerant mechanisms in roots.

2017 Nov;154(5):627-644. doi: 10.1530/REP-17-0345. Epub 2017 Aug 16.

Single-cell gene expression of the bovine blastocyst.

Negrón-Pérez VM¹, Zhang Y², Hansen PJ³.

Author information

1: Department of Animal SciencesD. H. Barron Reproductive and Perinatal Biology Research Program and Genetics Institute, University of Florida, Gainesville, Florida, USA.
2: Gene Expression and Genotyping CoreInterdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida, USA.
3: Department of Animal SciencesD. H. Barron Reproductive and Perinatal Biology Research Program and Genetics Institute, University of Florida, Gainesville, Florida, USA pjhansen@ufl.edu.

Abstract

The first two differentiation events in the embryo result in three cell types – epiblast, trophectoderm (TE) and hypoblast. The purpose here was to identify molecular markers for each cell type in the bovine and evaluate the differences in gene expression among individual cells of each lineage. The cDNA from 67 individual cells of dissociated blastocysts was used to determine transcript abundance for 93 genes implicated as cell lineage markers in other species or potentially involved in developmental processes. Clustering analysis indicated that the cells belonged to two major populations (clades A and B) with two subpopulations of clade A and four of clade B. Use of lineage-specific markers from other species indicated that the two subpopulations of clade A represented epiblast and hypoblast respectively while the four subpopulations of clade B were TE. Among the genes upregulated in epiblast were AJAP1, DNMT3A, FGF4, H2AFZ, KDM2B, NANOG, POU5F1, SAV1 and SLIT2Genes overexpressed in hypoblast included ALPL, FGFR2, FN1, GATA6, GJA1, HDAC1, MBNL3, PDGFRA and SOX17, while genes overexpressed in all four TE populations were ACTA2, CDX2, CYP11A1, GATA2, GATA3, IFNT, KRT8, RAC1 and SFN The subpopulations of TE varied among each other for multiple genes including the prototypical TE marker IFNT. New markers for each cell type in the bovine blastocyst were identified. Results also indicate heterogeneity in gene expression among TE cells. Further studies are needed to confirm whether subpopulations of TE cells represent different stages in the development of a committed TE phenotype.

2017 May 1;96(5):948-959. doi: 10.1093/biolre/iox037.

Role of chemokine (C-C motif) ligand 24 in spatial arrangement of the inner cell mass of the bovine embryo.

Negrón-Pérez VM¹, Vargas-Franco D², Hansen PJ¹.

Author information

1: Department of Animal Sciences, D. H. Barron Reproductive and Perinatal Biology Research Program and Genetics Institute, University of Florida, Gainesville, Florida, USA.
2: Department of Molecular Genetics and Microbiology, Center for Epigenetics and Genetics Institute, University of Florida, Gainesville, Florida, USA.

Abstract

The process of spatial rearrangement of cells of the inner cell mass (ICM) that are destined to become hypoblast is not well understood. The observation that the chemokine (C-C motif) ligand 24 (CCL24) and several other genes involved in chemokine signaling are expressed more in the ICM than in the trophectoderm of the bovine embryo resulted in the hypothesis that CCL24 participates in spatial organization of the ICM. Temporally, expression of CCL24 in the bovine embryo occurs coincidently with blastocyst formation: transcript abundance was low until the late morula stage, peaked in the blastocyst at Day 7 of development and declined by Day 9. Treatment of embryos with two separate antagonists of C-C motif chemokine receptor 3 (the prototypical receptor for CCL24) decreased the percent of GATA6+ cells (hypoblast precursors) that were located in the outside of the ICM. Similarly, injection of zygotes with a CCL24-specific morpholino decreased the percent of GATA6+ cells in the outside of the ICM. In conclusion, CCL24 assists in spatial arrangement of the ICM in the bovine embryo. This experiment points to new functions of chemokine signaling in the bovine embryo and is consistent with the idea that cell migration is involved in the spatial organization of hypoblast cells in the blastocyst.

2017 Oct 30;9(11). pii: E321. doi: 10.3390/v9110321.

Atomic Resolution Structure of the Oncolytic Parvovirus LuIII by Electron Microscopy and 3D Image Reconstruction.

Pittman N^1,², Misseldine A^3,⁴, Geilen L^5,⁶, Halder S^7,⁸, Smith JK^9,¹⁰, Kurian J^11,¹², Chipman P^13,¹⁴, Janssen M¹⁵, Mckenna R^16,¹⁷, Baker TS¹⁸, D’Abramo A Jr¹⁹, Cotmore S²⁰, Tattersall P^21,²², Agbandje-McKenna M^23,²⁴.

Author information

1: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA. nikea@ufl.edu.
2: Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA. nikea@ufl.edu.
3: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA. adammisseldine@ufl.edu.
4: Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA. adammisseldine@ufl.edu.
5: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA. lorenageilen@ufl.edu.
6: Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA. lorenageilen@ufl.edu.
7: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA. halder.sujata@gmail.com.
8: Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA. halder.sujata@gmail.com.
9: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA. jkennonsmith@ufl.edu.
10: Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA. jkennonsmith@ufl.edu.
11: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA. justinkurian@ufl.edu.
12: Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA. justinkurian@ufl.edu.
13: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA. pchipman@ufl.edu.
14: Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA. pchipman@ufl.edu.
15: Department of Chemistry and Biochemistry and Division of Biological Sciences, University of California-San Diego, San Diego, CA 92093, USA. mjanssen@nanoimagingservices.com.
16: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA. rmckenna@ufl.edu.
17: Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA. rmckenna@ufl.edu.
18: Department of Chemistry and Biochemistry and Division of Biological Sciences, University of California-San Diego, San Diego, CA 92093, USA. tsb@ucsd.edu.
19: Department of Laboratory Medicine, Yale University Medical School, New Haven, CT 06520, USA. anthony.dabramo@yale.edu.
20: Department of Laboratory Medicine, Yale University Medical School, New Haven, CT 06520, USA. susan.cotmore@yale.edu.
21: Department of Laboratory Medicine, Yale University Medical School, New Haven, CT 06520, USA. peter.tattersall@yale.edu.
22: Department of Genetics, Yale University Medical School, New Haven, CT 06510, USA. peter.tattersall@yale.edu.
23: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA. mckenna@ufl.edu.
24: Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA. mckenna@ufl.edu.

Abstract

LuIII, a protoparvovirus pathogenic to rodents, replicates in human mitotic cells, making it applicable for use to kill cancer cells. This virus group includes H-1 parvovirus (H-1PV) and minute virus of mice (MVM). However, LuIII displays enhanced oncolysis compared to H-1PV and MVM, a phenotype mapped to the major capsid viral protein 2 (VP2). This suggests that within LuIII VP2 are determinants for improved tumor lysis. To investigate this, the structure of the LuIII virus-like-particle was determined using single particle cryo-electron microscopy and image reconstruction to 3.17 Å resolution, and compared to the H-1PV and MVM structures. The LuIII VP2 structure, ordered from residue 37 to 587 (C-terminal), had the conserved VP topology and capsid morphology previously reported for other protoparvoviruses. This includes a core β-barrel and α-helix A, a depression at the icosahedral 2-fold and surrounding the 5-fold axes, and a single protrusion at the 3-fold axes. Comparative analysis identified surface loop differences among LuIII, H-1PV, and MVM at or close to the capsid 2- and 5-fold symmetry axes, and the shoulder of the 3-fold protrusions. The 2-fold differences cluster near the previously identified MVM sialic acid receptor binding pocket, and revealed potential determinants of protoparvovirus tumor tropism.

2018 Jun 11. pii: a033019. doi: 10.1101/cshperspect.a033019. [Epub ahead of print]

Repeat-Associated Non-ATG Translation in Neurological Diseases.

Zu T^1,², Pattamatta A^1,², Ranum LPW^1,^2,^3,⁴.

Author information

1: Center for Neuro-Genetics, University of Florida, Gainesville, Florida 32610.
2: Departments of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610.
3: Departments of Neurology, College of Medicine, University of Florida, Gainesville, Florida 32610.
4: Genetics Institute, University of Florida, Gainesville, Florida 32610.

Abstract

More than 40 different neurological diseases are caused by microsatellite repeat expansions that locate within translated or untranslated gene regions, including 5′ and 3′ untranslated regions (UTRs), introns, and protein-coding regions. Expansion mutations are transcribed bidirectionally and have been shown to give rise to proteins, which are synthesized from three reading frames in the absence of an AUG initiation codon through a novel process called repeat-associated non-ATG (RAN) translation. RAN proteins, which were first described in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1), have now been reported in a growing list of microsatellite expansion diseases. This article reviews what is currently known about RAN proteins in microsatellite expansion diseases and experiments that provide clues on how RAN translation is regulated.

2018 May 20;8(10). pii: e2845. doi: 10.21769/BioProtoc.2845.

In vitro Analysis of Ubiquitin-like Protein Modification in Archaea.

Fu X¹, Adams Z¹, Maupin-Furlow JA^1,².

Author information

1: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA.
2: Genetics Institute, University of Florida, Gainesville, Florida, USA.

Abstract

The ubiquitin-like (Ubl) protein is widely distributed in Archaea and involved in many cellular pathways. A well-established method to reconstitute archaeal Ubl protein conjugation in vitro is important to better understand the process of archaeal Ubl protein modification. This protocol describes the in vitro reconstitution of Ubl protein modification and following analysis of this modification in Haloferax volcanii, a halophilic archaeon serving as the model organism.

2018 Mar 26;18:913-919. doi: 10.1016/j.dib.2018.03.107. eCollection 2018 Jun.

Phenotypic characterization of an Arabidopsis T-DNA insertion line SALK_063500.

Sng NJ¹, Paul AL^1,², Ferl RJ^1,^2,³.

Author information

1: Plant Molecular and Cellular Biology, University of Florida, Fifield Hall, 2550 Hull Road, Gainesville, FL 32611, USA.
2: Horticultural Science Department, University of Florida, Fifield Hall, 2550 Hull Road, Gainesville, FL 32611, USA.
3: Interdisciplinary Center for Biotechnology Research (ICBR), University of Florida, 2033 Mowry Road, Gainesville, FL 32601, USA.

Abstract

In this article we report the identification of a homozygous lethal T-DNA (transfer DNA) line within the coding region of the At1G05290 gene in the genome of Arabidopsis thaliana (Arabidopsis) line, SALK_063500. The T-DNA insertion is found within exon one of the AT1G05290 gene, however a homozygous T-DNA allele is unattainable. In the heterozygous T-DNA allele the expression levels of AT1G05290 were compared to wild type Arabidopsis (Col-0, Columbia). Further analyses revealed an aberrant silique phenotype found in the heterozygous SALK_063500 plants that is attributed to the reduced rate of pollen tube germination. These data are original and have not been published elsewhere.

2017 May 1;96(5):1071-1084. doi: 10.1093/biolre/iox025.

Impact of gestational nicotine exposure on intrauterine and fetal infection in a rodent model.

von Chamier M¹, Reyes L^1,², Hayward LF³, Brown MB¹.

Author information

1: Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.
2: Department of Pathobiological Sciences, College of Veterinary Medicine, University of Wisconsin.
3: Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.

Abstract

We investigated the interaction between prenatal nicotine exposure and intrauterine infection using established rat models. Beginning at gestation day (GD) 6, dams were continuously infused with either saline or 6 mg/kg/day nicotine (Nic). At GD 14, dams received either sterile broth or 105 colony-forming units Mycoplasma pulmonis (MP), resulting in four treatment groups: control (4 dams, 33 fetal units); MP only (5 dams, 55 fetal units); Nic only (5 dams, 61 fetal units), and Nic + MP (7 dams, 82 fetal units). At GD 18, nicotine exposure significantly increased (P ≤ 0.02) the percentage of amniotic fluids and fetuses infected by MP but did not impact colonization rates of maternal sites. Nicotine exposure significantly reduced the numbers of MP in the placenta required for high microbial loads (≥104 color-changing units) in the amniotic fluid (P < 0.01). Fetal inflammatory response lesions were most extensive in the Nic only and Nic + MP groups (P < 0.0001). Control and MP only placentas were interleukin (IL)10-dominant, consistent with an M2/Th2 environment. Placentas exposed to nicotine shifted to a neutral environment, with equivalent levels of interferon gamma (IFNG) and IL10. Both IL6 and tumor necrosis factor (TNF) levels in amniotic fluid were highly elevated when both nicotine and infection were present. Our study suggests that prenatal exposure to nicotine increases the risk for intrauterine infection, lowers the infectious dose required to breach the placental barrier and infect the amniotic fluid and fetus, and alters the pathology and inflammatory profile associated with maternal and fetal sites.

2018 Jun 14. doi: 10.1111/tpj.13996. [Epub ahead of print]

Autonomous and nonautonomous functions of the maize Shohai1 gene, encoding a RWP-RK putative transcription factor, in regulation of embryo and endosperm development.

Mimura M¹, Kudo T¹, Wu S¹, McCarty DR¹, Suzuki M¹.

Author information

1: Horticultural Sciences Department, University of Florida, Gainesville, Florida, 32611, USA.

Abstract

In plants, establishment of the basic body plan during embryogenesis involves complex processes of axis formation, cell fate specification and organ differentiation. While molecular mechanisms of embryogenesis have been well studied in the eudicot Arabidopsis, only a small number of genes regulating embryogenesis has been identified in grass species. Here, we show that a RKD-type RWP-RK transcription factor encoded by Shohai1 (Shai1) is indispensable for embryo and endosperm development in maize. Loss of Shai1 function causes variable morphological defects in the embryo including small scutellum, shoot axis bifurcation and arrest during early organogenesis. Analysis of molecular markers in mutant embryos reveal disturbed patterning of gene expression and altered polar auxin transport. In contrast to typical embryo-defective (emb) mutants that expose a vacant embryo pocket in the endosperm, the endosperm of shai1 kernels conforms to the varied size and shape of the embryo. Indeed, genetic analysis confirms that Shai1 is required for autonomous formation of the embryo pocket in endosperm of emb mutants. Analyses of genetic mosaic kernels generated by B-A translocation revealed that expression of Shai1 in the endosperm could partially rescue a shai1 mutant embryo suggesting that Shai1 is involved in non-cell autonomous signaling from endosperm that supports normal embryo growth. Taken together, we propose that Shai1 gene functions in regulating embryonic patterning during grass embryogenesis partly by endosperm-to-embryo interaction. This article is protected by copyright. All rights reserved.

2017 Nov;27(11):1807-1815. doi: 10.1101/gr.217984.116. Epub 2017 Oct 12.

Disease-specific biases in alternative splicing and tissue-specific dysregulation revealed by multitissue profiling of lymphocyte gene expression in type 1 diabetes.

Newman JRB¹, Conesa A^2,³, Mika M⁴, New FN¹, Onengut-Gumuscu S⁴, Atkinson MA^5,⁶, Rich SS⁴, McIntyre LM^1,³, Concannon P^3,⁶.

Author information

1: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610, USA.
2: Department of Microbiology and Cell Science, Institute for Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32610, USA.
3: Genetics Institute, University of Florida, Gainesville, Florida 32610, USA.
4: Center for Public Health Genomics and Department of Public Health Sciences, University of Virginia, Charlottesville, Virginia 22908, USA.
5: Diabetes Institute, University of Florida, Gainesville, Florida 32610, USA.
6: Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, Florida 32610, USA.

Abstract

Genome-wide association studies (GWAS) have identified multiple, shared allelic associations with many autoimmune diseases. However, the pathogenic contributions of variants residing in risk loci remain unresolved. The location of the majority of shared disease-associated variants in noncoding regions suggests they contribute to risk of autoimmunity through effects on gene expression in the immune system. In the current study, we test this hypothesis by applying RNA sequencing to CD4⁺, CD8⁺, and CD19⁺ lymphocyte populations isolated from 81 subjects with type 1 diabetes (T1D). We characterize and compare the expression patterns across these cell types for three gene sets: all genes, the set of genes implicated in autoimmune disease risk by GWAS, and the subset of these genes specifically implicated in T1D. We performed RNA sequencing and aligned the reads to both the human reference genome and a catalog of all possible splicing events developed from the genome, thereby providing a comprehensive evaluation of the roles of gene expression and alternative splicing (AS) in autoimmunity. Autoimmune candidate genes displayed greater expression specificity in the three lymphocyte populations relative to other genes, with significantly increased levels of splicing events, particularly those predicted to have substantial effects on protein isoform structure and function (e.g., intron retention, exon skipping). The majority of single-nucleotide polymorphisms within T1D-associated loci were also associated with one or more cis-expression quantitative trait loci (cis-eQTLs) and/or splicing eQTLs. Our findings highlight a substantial, and previously underrecognized, role for AS in the pathogenesis of autoimmune disorders and particularly for T1D.

2017 Oct;175(2):619-627. doi: 10.1104/pp.17.00577. Epub 2017 Aug 14.

Identification of Novel Growth Regulators in Plant Populations Expressing Random Peptides.

Bao Z¹, Clancy MA¹, Carvalho RF¹, Elliott K¹, Folta KM^2,³.

Author information

1: Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611.
2: Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611 kfolta@ufl.edu.
3: Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, Florida 32611.

Abstract

The use of chemical genomics approaches allows the identification of small molecules that integrate into biological systems, thereby changing discrete processes that influence growth, development, or metabolism. Libraries of chemicals are applied to living systems, and changes in phenotype are observed, potentially leading to the identification of new growth regulators. This work describes an approach that is the nexus of chemical genomics and synthetic biology. Here, each plant in an extensive population synthesizes a unique small peptide arising from a transgene composed of a randomized nucleic acid sequence core flanked by translational start, stop, and cysteine-encoding (for disulfide cyclization) sequences. Ten and 16 amino acid sequences, bearing a core of six and 12 random amino acids, have been synthesized in Arabidopsis (Arabidopsis thaliana) plants. Populations were screened for phenotypes from the seedling stage through senescence. Dozens of phenotypes were observed in over 2,000 plants analyzed. Ten conspicuous phenotypes were verified through separate transformation and analysis of multiple independent lines. The results indicate that these populations contain sequences that often influence discrete aspects of plant biology. Novel peptides that affect photosynthesis, flowering, and red light response are described. The challenge now is to identify the mechanistic integrations of these peptides into biochemical processes. These populations serve as a new tool to identify small molecules that modulate discrete plant functions that could be produced later in transgenic plants or potentially applied exogenously to impart their effects. These findings could usher in a new generation of agricultural growth regulators, herbicides, or defense compounds.

2018 Jun 12;5:180106. doi: 10.1038/sdata.2018.106.

Pharmacological and genomic profiling of neurofibromatosis type 1 plexiform neurofibroma-derived schwann cells.

Ferrer M¹, Gosline SJC², Stathis M³, Zhang X¹, Guo X², Guha R¹, Ryman DA³, Wallace MR⁴, Kasch-Semenza L⁵, Hao H⁶, Ingersoll R⁵, Mohr D⁷, Thomas C¹, Verma S³, Guinney J², Blakeley JO³.

Author information

1: National Center for Advancing Translational Sciences (NCATS), Division of Pre-clinical Innovation, National Institutes of Health, Bethesda, MD, USA.
2: Sage Bionetworks, Seattle, WA, USA.
3: Neurofibromatosis Therapeutic Acceleration Program (NTAP), Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
4: Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, USA.
5: Genetic Resources Core Facility (GRCF), Johns Hopkins School of Medicine, Baltimore, MD, USA.
6: Deep Sequencing & Microarray Core, Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, USA.
7: Center for Inherited Disease Research (CIDR), Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Abstract

Neurofibromatosis type I (NF1) is an autosomal dominant genetic condition characterized by peripheral nervous system tumors (PNSTs), including plexiform neurofibromas (pNFs) that cause nerve dysfunction, deformity, pain damage to adjacent structures, and can undergo malignant transformation. There are no effective therapies to prevent or treat pNFs. Drug discovery efforts are slowed by the ‘benign’ nature of the Schwann cells that are the progenitor cells of pNF. In this work we characterize a set of pNF-derived cell lines at the genomic level (via SNP Arrays, RNAseq, and Whole Exome- Sequencing), and carry out dose response-based quantitative high-throughput screening (qHTS) with a collection of 1,912 oncology-focused compounds in a 1536-well microplate cell proliferation assays. Through the characterization and screening of NF1^-/-, NF1^+/+ and NF1^+/- Schwann cell lines, this resource introduces novel therapeutic avenues for the development for NF1 associated pNF as well as all solid tumors with NF1 somatic mutations. The integrated data sets are openly available for further analysis at http://www.synapse.org/pnfCellCulture.

2017;1654:361-375. doi: 10.1007/978-1-4939-7231-9_27.

Engineered Zinc Finger DNA-Binding Domains: Synthesis, Assessment of DNA-Binding Affinity, and Direct Protein Delivery to Mammalian Cells.

Hossain MA¹, Knudson IJ¹, Thakur S¹, Shen Y¹, Stees JR¹, Barrow JJ^2,³, Bungert J⁴.

Author information

1: Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, Genetics Institute, University of Florida, 1600 SW Archer Road, PO Box 100245, Gainesville, FL, 32610, USA.
2: Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
3: Department of Cell Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
4: Department of Biochemistry and Molecular Biology, College of Medicine, UF Health Cancer Center, Genetics Institute, University of Florida, 1600 SW Archer Road, PO Box 100245, Gainesville, FL, 32610, USA. jbungert@ufl.edu.

Abstract

Zinc finger proteins are the most common among families of DNA-binding transcription factors. Designer transcription factors generated by the fusion of engineered zinc finger DNA-binding domains (ZF-DBDs) to effector domains have been valuable tools for the modulation of gene expression and for targeted genome editing. However, ZF-DBDs without effector domains have also been shown to effectively modulate gene expression by competing with sequence-specific DNA-binding transcription factors. Here, we describe the methodology and provide a detailed workflow for the cloning, expression, purification, and direct cell delivery of engineered ZF-DBDs. Using this protocol, ZF-DBDs can be generated with high efficiency in less than 2 weeks. We also describe a nonradioactive method for measuring DNA binding affinity of the purified ZF-DBD proteins as well as a method for direct delivery of the purified ZF-DBDs to mammalian cells.

2018 Jun 14. doi: 10.1111/1462-2920.14288. [Epub ahead of print]

Novel bacterial lineages associated with boreal moss species.

Holland-Moritz H^1,², Stuart J³, Lewis LR⁴, Miller S³, Mack MC³, McDaniel SF⁴, Fierer N^1,².

Author information

1: Cooperative Institute for Research in Environmental Sciences, University of Colorado at Boulder, Boulder, CO, USA.
2: Department of Ecology and Evolutionary Biology, University of Colorado at Boulder, Boulder, CO, USA.
3: Center for Ecosystem Science and Society, Northern Arizona University, Flagstaff, AZ, USA.
4: Department of Biology, University of Florida, Gainesville, FL 32611-8525, USA.

Abstract

Mosses are critical components of boreal ecosystems where they typically account for a large proportion of net primary productivity and harbor diverse bacterial communities that can be the major source of biologically-fixed nitrogen in these ecosystems. Despite their ecological importance, we have limited understanding of how microbial communities vary across boreal moss species and the extent to which local site conditions may influence the composition of these bacterial communities. We used marker gene sequencing to analyze bacterial communities associated with seven boreal moss species collected near Fairbanks, AK USA. We found that host identity was more important than site in determining bacterial community composition and that mosses harbor diverse lineages of potential N₂ -fixers as well as an abundance of novel taxa assigned to understudied bacterial phyla (including candidate phylum WPS-2). We performed shotgun metagenomic sequencing to assemble genomes from the WPS-2 candidate phylum and found that these moss-associated bacteria are likely anoxygenic phototrophs capable of carbon fixation via RuBisCo with an ability to utilize byproducts of photorespiration from hosts via a glyoxylate shunt. These results give new insights into the metabolic capabilities of understudied bacterial lineages that associate with mosses and the importance of plant hosts in shaping their microbiomes. This article is protected by copyright. All rights reserved.

2017 Nov;92(5):1194-1205. doi: 10.1016/j.kint.2017.03.038. Epub 2017 Jul 12.

DNA methylation protects against cisplatin-induced kidney injury by regulating specific genes, including interferon regulatory factor 8.

Guo C¹, Pei L², Xiao X¹, Wei Q¹, Chen JK¹, Ding HF², Huang S³, Fan G⁴, Shi H², Dong Z⁵.

Author information

1: Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University and Charlie Norwood VA Medical Center, Augusta, Georgia 30912, USA.
2: Georgia Cancer Center, Augusta University, Augusta, Georgia 30912, USA.
3: Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32611, USA.
4: Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, California 90095.
5: Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China; Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University and Charlie Norwood VA Medical Center, Augusta, Georgia 30912, USA. Electronic address: zdong@augusta.edu.

Abstract

DNA methylation is an epigenetic mechanism that regulates gene transcription without changing primary nucleotide sequences. In mammals, DNA methylation involves the covalent addition of a methyl group to the 5-carbon position of cytosine by DNA methyltransferases (DNMTs). The change of DNA methylation and its pathological role in acute kidney injury (AKI) remain largely unknown. Here, we analyzed genome-wide DNA methylation during cisplatin-induced AKI by reduced representation bisulfite sequencing. This technique identified 215 differentially methylated regions between the kidneys of control and cisplatin-treated animals. While most of the differentially methylated regions were in the intergenic, intronic, and coding DNA sequences, some were located in the promoter or promoter-regulatory regions of 15 protein-coding genes. To determine the pathological role of DNA methylation, we initially examined the effects of the DNA methylation inhibitor 5-aza-2′-deoxycytidine and showed it increased cisplatin-induced apoptosis in a rat kidney proximal tubular cell line. We further established a kidney proximal tubule-specific DNMT1 (PT-DNMT1) knockout mouse model, which showed more severe AKI during cisplatin treatment than wild-type mice. Finally, interferon regulatory factor 8 (Irf8), a pro-apoptotic factor, was identified as a hypomethylated gene in cisplatin-induced AKI, and this hypomethylation was associated with a marked induction of Irf8. In the rat kidney proximal tubular cells, the knockdown of Irf8 suppressed cisplatin-induced apoptosis, supporting a pro-death role of Irf8 in renal tubular cells. Thus, DNA methylation plays a protective role in cisplatin-induced AKI by regulating specific genes, such as Irf8.

2017 Oct 31;21(5):1240-1252. doi: 10.1016/j.celrep.2017.10.018.

Aberrant Myokine Signaling in Congenital Myotonic Dystrophy.

Nakamori M¹, Hamanaka K², Thomas JD³, Wang ET³, Hayashi YK⁴, Takahashi MP⁵, Swanson MS³, Nishino I², Mochizuki H⁶.

Author information

1: Department of Neurology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan. Electronic address: mnakamor@neurol.med.osaka-u.ac.jp.
2: Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan.
3: Department of Molecular Genetics and Microbiology, Center for NeuroGenetics and the Genetics Institute, University of Florida, College of Medicine, Gainesville, FL 32610, USA.
4: Department of Pathophysiology, Tokyo Medical University, Shinjuku, Tokyo 160-0022, Japan.
5: Department of Neurology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan; Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
6: Department of Neurology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.

Abstract

Myotonic dystrophy types 1 (DM1) and 2 (DM2) are dominantly inherited neuromuscular disorders caused by a toxic gain of function of expanded CUG and CCUG repeats, respectively. Although both disorders are clinically similar, congenital myotonic dystrophy (CDM), a severe DM form, is found only in DM1. CDM is also characterized by muscle fiber immaturity not observed in adult DM, suggesting specific pathological mechanisms. Here, we revealed upregulation of the interleukin-6 (IL-6) myokine signaling pathway in CDM muscles. We also found a correlation between muscle immaturity and not only IL-6 expression but also expanded CTG repeat length and CpG methylation status upstream of the repeats. Aberrant CpG methylation was associated with transcriptional dysregulation at the repeat locus, increasing the toxic RNA burden that upregulates IL-6. Because the IL-6 pathway is involved in myocyte maturation and muscle atrophy, our results indicate that enhanced RNA toxicity contributes to severe CDM phenotypes through aberrant IL-6 signaling.

2018 Jun 11. doi: 10.2174/1389203719666180612082432. [Epub ahead of print]

Chinese medicine protein and peptide in gene and cell therapy.

Feng Y¹, Yin Z², Zhang D², Srivastava A², Ling C³.

Author information

1: Department of Traditional Chinese Medicine, 401 Hospital of the Chinese People’s Liberation Army, Qingdao, Shandong 266071. China.
2: Division of Cellular and Molecular Therapy, Department of Pediatrics, College of Medicine, University of Florida, Gainesville 32611. United States.
3: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida. United States.

Abstract

The success of gene and cell therapy in clinic during the past two decades as well as our expanding ability to manipulate these biomaterials are leading to new therapeutic options for a wide range of inherited and acquired diseases. Combining conventional therapies with this emerging field is a promising strategy to treat those previously-thought untreatable diseases. Traditional Chinese medicine (TCM) has evolved for thousands of years in China and still plays an important role in human health. As part of the active ingredients of TCM, proteins and peptides have attracted long-term enthusiasm of researchers. More recently, they have been utilized in gene and cell therapy, resulting in promising novel strategies to treat both cancer and non-cancer diseases. This manuscript presents a critical review on this field, accompanied with perspectives on the challenges and new directions for future research in this emerging frontier.

2017 Oct 17;21(3):628-640. doi: 10.1016/j.celrep.2017.09.078.

UTX/KDM6A Loss Enhances the Malignant Phenotype of Multiple Myeloma and Sensitizes Cells to EZH2 inhibition.

Ezponda T¹, Dupéré-Richer D², Will CM¹, Small EC¹, Varghese N¹, Patel T¹, Nabet B¹, Popovic R¹, Oyer J¹, Bulic M¹, Zheng Y³, Huang X⁴, Shah MY¹, Maji S², Riva A⁵, Occhionorelli M⁶, Tonon G⁶, Kelleher N⁴, Keats J⁷, Licht JD⁸.

Author information

1: Division of Hematology/Oncology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
2: Division of Hematology/Oncology, University of Florida Health Cancer Center, Gainesville, FL 2033, USA.
3: Department of Chemistry, Department of Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, Evanston, IL 60208, USA.
4: Division of Hematology/Oncology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Department of Chemistry, Department of Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, Evanston, IL 60208, USA.
5: Bioinformatics Core, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 2033, USA.
6: Functional Genomics of Cancer Unit, Division of Molecular Oncology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Raffaele Scientific Institute, Milan 70126, Italy.
7: Translational Genomics Research Institute (TGen), Phoenix, AZ 85004, USA.
8: Division of Hematology/Oncology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Division of Hematology/Oncology, University of Florida Health Cancer Center, Gainesville, FL 2033, USA. Electronic address: jdlicht@ufl.edu.

Abstract

Loss or inactivation of the histone H3K27 demethylase UTX occurs in several malignancies, including multiple myeloma (MM). Using an isogenic cell system, we found that loss of UTX leads to deactivation of gene expression ultimately promoting the proliferation, clonogenicity, adhesion, and tumorigenicity of MM cells. Moreover, UTX mutant cells showed increased in vitro and in vivo sensitivity to inhibition of EZH2, a histone methyltransferase that generates H3K27me3. Such sensitivity was related to a decrease in the levels of IRF4 and c-MYC and an activation of repressors of IRF4 characteristic of germinal center B cells such as BCL6 and IRF1. Rebalance of H3K27me3 levels at specific genes through EZH2 inhibitors may be a therapeutic strategy in MM cases harboring UTX mutations.

2018 May;154(6):1751-1763.e2. doi: 10.1053/j.gastro.2018.01.042. Epub 2018 Feb 1.

Microbiota-Derived Metabolic Factors Reduce Campylobacteriosis in Mice.

Sun X¹, Winglee K², Gharaibeh RZ³, Gauthier J³, He Z³, Tripathi P⁴, Avram D³, Bruner S⁴, Fodor A², Jobin C⁵.

Author information

1: Department of Medicine, University of Florida, Gainesville, Florida; Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas. Electronic address: xiaoluns@uark.edu.
2: Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, Charlotte, North Carolina.
3: Department of Medicine, University of Florida, Gainesville, Florida.
4: Department of Chemistry, University of Florida, Gainesville, Florida.
5: Department of Medicine, University of Florida, Gainesville, Florida; Department of Infectious Diseases and Pathology, University of Florida, Gainesville, Florida; Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida. Electronic address: Christian.Jobin@medicine.ufl.edu.

Abstract

BACKGROUND & AIMS:

Campylobacter jejuni, a prevalent foodborne bacterial pathogen, exploits the host innate response to induce colitis. Little is known about the roles of microbiota in C jejuni-induced intestinal inflammation. We investigated interactions between microbiota and intestinal cells during C jejuni infection of mice.

METHODS:

Germ-free C57BL/6 Il10^-/- mice were colonized with conventional microbiota and infected with a single dose of C jejuni (10⁹ colony-forming units/mouse) via gavage. Conventional microbiota were cultured under aerobic, microaerobic, or anaerobic conditions and orally transplanted into germ-free Il10^-/- mice. Colon tissues were collected from mice and analyzed by histology, real-time polymerase chain reaction, and immunoblotting. Fecal microbiota and bile acids were analyzed with 16S sequencing and high-performance liquid chromatography with mass spectrometry, respectively.

RESULTS:

Introduction of conventional microbiota reduced C jejuni-induced colitis in previously germ-free Il10^-/-mice, independent of fecal load of C jejuni, accompanied by reduced activation of mammalian target of rapamycin. Microbiota transplantation and 16S ribosomal DNA sequencing experiments showed that Clostridium XI, Bifidobacterium, and Lactobacillus were enriched in fecal samples from mice colonized with microbiota cultured in anaerobic conditions (which reduce colitis) compared with mice fed microbiota cultured under aerobic conditions (susceptible to colitis). Oral administration to mice of microbiota-derived secondary bile acid sodium deoxycholate, but not ursodeoxycholic acid or lithocholic acid, reduced C jejuni-induced colitis. Depletion of secondary bile acid-producing bacteria with antibiotics that kill anaerobic bacteria (clindamycin) promoted C jejuni-induced colitis in specific pathogen-free Il10^-/- mice compared with the nonspecific antibiotic nalidixic acid; colitis induction by antibiotics was associated with reduced level of luminal deoxycholate.

CONCLUSIONS:

We identified a mechanism by which the microbiota controls susceptibility to C jejuni infection in mice, via bacteria-derived secondary bile acids.

2018 Jun 14. doi: 10.1089/hum.2018.016. [Epub ahead of print]

Systemic delivery of AAVB1-GAA clears glycogen and prolongs survival in a mouse model of Pompe disease.

Keeler AM¹, Zieger M², Todeasa S³, Mccall A⁴, Gifford J⁵, Bircsak S⁶, Choudhury SR⁷, Byrne BJ^8,⁹, Sena-Esteves M¹⁰, ElMallah MK¹¹.

Author information

1: University of Massachusetts Medical School, Gene Therapy , 368 Plantation Street , Worcester, Massachusetts, United States , 01604 ; allison.keeler@umassmed.edu.
2: University of Massachusetts Medical School, Gene Therapy, Worcester, Massachusetts, United States ; marina.Zieger@umassmed.edu.
3: University of Massachusetts Medical School, Gene Therapy, Worcester, Massachusetts, United States ; sophia.todeasa@umassmed.edu.
4: Duke University, Pediatrics, Durham, North Carolina, United States ; angela.mccall440@duke.edu.
5: University of Massachusetts Medical School, Gene Therapy , 368 Plantation Street , Worcester, Massachusetts, United States , 01605 ; Jennifer.gifford@umassmed.edu.
6: Worcester Polytechnic Institute, 8718, Worcester, Massachusetts, United States ; sambircsak@gmail.com.
7: University of Massachusetts Medical School, 12262, Neurology , 368 Plantation St. , AS6-2001 , Worcester, Massachusetts, United States , 01605 ; sourav.rc@gmail.com.
8: University of Florida, School of Medicine, Pediatrics , 1600 SW Archer Road , RG-183 , Gainesville, Florida, United States , 32610.
9: United States ; bbyrne@ufl.edu.
10: University of Massachusetts Medical School, Neurology and Gene Therapy Center , 381 Plantation Street, Suite 250 , Worcester, Massachusetts, United States , 01605 ; Miguel.Esteves@umassmed.edu.
11: University of Massachusetts Medical School, Pediatrics and Gene Therapy Center , 55 Lake Avenue North , Worcester, Massachusetts, United States , 01655 ; mai.elmallah@duke.edu.

Abstract

Pompe disease is an autosomal recessive glycogen storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption in muscle and central nervous system (CNS). Adeno-associated virus (AAV) gene therapy is ideal for Pompe disease since a single systemic injection may correct both muscle and CNS pathologies. Using the Pompe mouse, we sought to explore if AAVB1, a newly engineered vector with a high affinity for muscle and CNS, reduces systemic weakness and improves survival in adult mice. 3-month-old Gaa-/- animals were injected with either AAVB1 or AAV9 vectors expressing GAA and tissues were harvested 6-months later. Both AAV vectors prolonged survival. AAVB1 treated animals had a robust weight gain compared to AAV9 treated group. Vector genome levels, GAA enzyme activity, and histological analysis indicated both vectors transduced the heart efficiently leading to glycogen clearance, and transduced the diaphragm and CNS at comparable levels. AAVB1 treated mice had higher GAA activity and greater glycogen clearance in the tongue. Finally, AAVB1 treated animals showed improved respiratory function comparable to WT animals. In conclusion, AAVB1-GAA offers a promising therapeutic option for the treatment of muscle and CNS in Pompe disease.

2018 Jun 15. doi: 10.1094/PHYTO-02-18-0052-R. [Epub ahead of print]

The type III effector AvrBsT enhances Xanthomonas perforans fitness in field-grown tomato.

Abrahamian P^1,², Timilsina S³, Minsavage GV⁴, Kc S⁵, Goss EM^6,⁷, Jones JB⁸, Vallad GE⁹.

Author information

1: University of Florida, Gulf Coast and Research Education Center, Wimauma, Florida, United States.
2: University of Florida, Plant Pathology, Gainesville, Florida, United States ; peterabrahamian@gmail.com.
3: University of Florida, Plant Pathology, Gainesville, Florida, United States ; sujan.timilsina@ufl.edu.
4: University of Florida, Plant Pathology, Gainesville, Florida, United States ; gvmins@ufl.edu.
5: University of Florida, Gulf Coast Research and Education Center, Wimauma, Florida, United States ; sushmitakc@ufl.edu.
6: University of Florida, Plant Pathology, Gainesville, Florida, United States.
7: University of Florida, Emerging Pathogens Institute, Gainesville, Florida, United States ; emgoss@ufl.edu.
8: University of Florida, Plant Pathology, Gainesville, Florida, United States ; jbjones@ufl.edu.
9: University of Florida, Gulf Coast and Research Education Center, Wimauma, Florida, United States ; gvallad@ufl.edu.

Abstract

Type III secretion system effectors contribute to pathogenicity through various mechanisms. Recent surveys showed an increasing prevalence of the type III secretion effector, avrBsT, among Xanthomonas perforans (Xp). We hypothesized that the acquisition of avrBsT has a fitness advantage for the pathogen. The contribution of avrBsT to fitness on tomato was evaluated based on disease severity, in planta growth, competition and recovery rates of wild-type (WT) and avrBsT mutant strains in greenhouse and field plants. GEV872 and GEV1001, representative strains of two phylogenomic groups of Xp, were selected for generating avrBsT mutants. Disease severity was higher for WT strains compared to the avrBsT mutant strains. Xp WT and avrBsT mutant strains did not differ following leaf infiltration of greenhouse plants in direct competition and in planta growth assays. The effect of avrBsT on pathogen fitness was noticeable under field conditions. Differences in strain recovery were significant, with WT being recovered two to eight times more than avrBsT mutant strains in the case of both strains GEV872 and GEV1001. WT strains were capable of spreading longer distance across field plots compared to avrBsT mutant strains. Findings suggest that the functional AvrBsT affects the fitness of Xp under field conditions, making it an ideal candidate for bacterial spot resistance breeding efforts in tomato.

2018 Jun 12;521:118-128. doi: 10.1016/j.virol.2018.05.026. [Epub ahead of print]

Degradation of DAXX by adenovirus type 12 E1B-55K circumvents chemoresistance of ovarian cancer to cisplatin.

Li Q¹, Wang J², Liao D³, Ai J¹, Jin L⁴, Gao Q⁵.

Author information

1: Department of Gynecology and Obstetrics, Tongji hospital, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
2: Department of Gynecology and Obstetrics, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou 450015, Henan Province, China.
3: Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610-0235, USA.
4: Department of Gynecology and Obstetrics, Tongji hospital, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China. Electronic address: ljin@tjh.tjmu.edu.cn.
5: Department of Gynecology and Obstetrics, Tongji hospital, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China. Electronic address: qlgao@tjh.tjmu.edu.cn.

Abstract

Adenovirus E1B 55-kilodalton (E1B-55K) mediated DAXX degradation represents a potential mechanism by which E1B-55K sensitizes cancer cells to chemotherapy. Here we report the effects of E1B-55K-mediated DAXX degradation in chemoresistant ovarian cancer cells on response to chemotherapy. Cells with E1B-55K expression were more sensitive to cisplatin than cells without E1B-55K expression. In vivo C13* xenograft studies showed that the combination of cisplatin and E1B-55K was markedly more effective to slow tumor growth and to confer prolonged survival of tumor-bearing mice than either cisplatin or E1B-55K alone. Our studies show that DAXX plays an important role in cisplatin resistance in ovarian cancer, and strategies that promote DAXX degradation such as E1B-55K expression in combination with cisplatin can overcome drug resistance and improve responses to standard chemotherapy. These results also indicate that E1B-55K might be a novel agent for enhancing treatment responses for cisplatin-resistant ovarian cancer.

2018 Jun 11. doi: 10.1093/gbe/evy116. [Epub ahead of print]

Evidence of the Red-Queen hypothesis from accelerated rates of evolution of genes involved in biotic interactions in Pneumocystis.

Delaye L¹, Ruiz-Ruiz S², Calderon E^3,⁴, Tarazona S^5,⁶, Conesa A^5,⁷, Moya A^2,⁸.

Author information

1: Departamento de Ingeniería Genética, CINVESTAV Irapuato, Km. 9.6 Libramiento Norte Carretera Irapuato-León 36821, Irapuato, Guanajuato, México.
2: Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (FISABIO)-Salud Pública, Avenida de Catalunya 21 46020, València, Spain.
3: Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/Consejo Superior de Investigaciones Científicas/Universidad de Sevilla.
4: Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP), Madrid, Spain.
5: Centro de Investigacion Principe Felipe, 46012 València, Spain.
6: Departamento de Estadística e Investigación Operativa Aplicadas y Calidad, Universitat Politècnica de València, València, Spain.
7: Microbiology and Cell Science, University of Florida, Gainesville, FL 32603, US.
8: Institute for Integrative Systems Biology, Universitat de València, Calle Catedrático José Beltrán 2, 46980 Paterna, València, Spain.

Abstract

Pneumocystis species are ascomycete fungi adapted to live inside the lungs of mammals. These ascomycetes show extensive stenoxenism, meaning that each species of Pneumocystis infects a single species of host. Here we study the effect exerted by natural selection on gene evolution in the genomes of three Pneumocystis species.We show that genes involved in host interaction evolve under positive selection. In the first place, we found strong evidence of episodic diversifying selection in Major surface glycoproteins (Msg). These proteins are located on the surface of Pneumocystis and are used for host attachment and probably for immune system evasion. Consistent with their function as antigens, most sites under diversifying selection in Msg code for residues with large relative surface accessibility areas. We also found evidence of positive selection in part of the cell machinery used to export Msg to the cell surface. Specifically, we found that genes participating in glycosylphosphatidylinositol (GPI) biosynthesis show an increased rate of non-synonymous substitutions (dN) versus synonymous substitutions (dS). GPI is a molecule synthesized in the endoplasmic reticulum that is used to anchor proteins to membranes.We interpret the aforementioned findings as evidence of selective pressure exerted by the host immune system on Pneumocystis species, shaping the evolution of Msg and several proteins involved in GPI biosynthesis. We suggest that genome evolution in Pneumocystis is well described by the Red-Queen hypothesis whereby genes relevant for biotic interactions show accelerated rates of evolution.

2018 Jun 11. doi: 10.1002/ps.5118. [Epub ahead of print]

Methionine as a safe and effective novel biorational mosquito larvicide.

Weeks ENI¹, Baniszewski J¹, Gezan SA², Allan SA³, Cuda JP¹, Stevens BR⁴.

Author information

1: Entomology and Nematology Department, University of Florida, Gainesville, Florida.
2: School of Forest Resources and Conservation, University of Florida, Gainesville, Florida.
3: USDA-ARS, Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida.
4: Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida.

Abstract

BACKGROUND:

Mosquito larvicides provide a source-reduction strategy to diminish adult females that bite and potentially spread pathogens. Demands are mounting for new and innovative effective biorational larvicides, due to the development of resistance to some currently utilized mosquito larvicides, undesirable non-target effects, and U.S. Environmental Protection Agency (EPA) restrictions. Methionine is a human nutrient essential amino acid that unexpectedly has been shown to be a valuable safe pest management tool against select insect pests that possess alkaline gut physiology. The present study evaluated larvicidal toxicity of methionine in several pestiferous mosquito (Diptera: Culicidae) genera.

RESULTS:

Concentration-dependent DL-methionine kinetics assays of survival and pupation were conducted in larvae of Aedes albopictus Skuse, Anopheles quadrimaculatus Say, and Culex tarsalis Coquillett in glass jars. Higher concentrations of DL-methionine yielded 100% mortality for all test species and prevented pupation at a rate equivalent to Bacillus thuringiensis israelensis (Bti) treatments. Concentration kinetics indicated that An. quadrimaculatus was 10-fold more sensitive to DL-methionine than Ae. albopictus and Cx. tarsalis.

CONCLUSIONS:

EPA regulations currently exempt methionine in pesticide formulations applied to agricultural crops. This study demonstrates that methionine is a highly effective mosquito larvicide that can provide a beneficial new biorational, environmentally sustainable tool to control pestiferous mosquitoes. This article is protected by copyright. All rights reserved.

2017 Sep 13;139(36):12410-12413. doi: 10.1021/jacs.7b07485. Epub 2017 Aug 30.

Facile Assembly/Disassembly of DNA Nanostructures Anchored on Cell-Mimicking Giant Vesicles.

Peng R¹, Wang H¹, Lyu Y^1,², Xu L¹, Liu H¹, Kuai H¹, Liu Q¹, Tan W^1,².

Author information

1: Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Life Sciences, and Aptamer Engineering Center of Hunan Province, Hunan University , Changsha, Hunan 410082, China.
2: Department of Chemistry and Department of Physiology and Functional Genomics, Center for Research at the Bio/Nano Interface, Health Cancer Center, UF Genetics Institute, McKnight Brain Institute, University of Florida , Gainesville, Florida 32611-7200, United States.

Abstract

DNA nanostructures assembled on living cell membranes have become powerful research tools. Synthetic lipid membranes have been used as a membrane model to study the dynamic behavior of DNA nanostructures on fluid soft lipid bilayers, but without the inherent complexity of natural membranes. Herein, we report the assembly and disassembly of DNA nanoprisms on cell-mimicking micrometer-scale giant membrane vesicles derived from living mammalian cells. Three-dimensional DNA nanoprisms with a DNA arm and a cholesterol anchor were efficiently localized on the membrane surface. The assembly and disassembly of DNA nanoprisms were dynamically manipulated by DNA strand hybridization and toehold-mediated strand displacement. Furthermore, the heterogeneity of reversible assembly/disassembly of DNA nanoprisms was monitored by Förster resonance energy transfer. This study suggests the feasibility of DNA-mediated functional biomolecular assembly on cell membranes for biomimetics studies and delivery systems.

2018 Jun 11. pii: S1055-7903(18)30198-2. doi: 10.1016/j.ympev.2018.06.002. [Epub ahead of print]

Phylogenetics of moth-like butterflies (Papilionoidea: Hedylidae) based on a new 13-locus target capture probe set.

Kawahara AY¹, Breinholt JW², Espeland M³, Storer C⁴, Plotkin D⁵, Dexter KM⁴, Toussaint EFA⁴, St Laurent RA⁶, Brehm G⁷, Vargas S⁸, Forero D⁸, Pierce NE⁹, Lohman DJ¹⁰.

Author information

1: Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA; Entomology and Nematology Department, University of Florida, Gainesville FL 32611, USA; Department of Biology, University of Florida, Gainesville FL 32611, USA.
2: Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA; RAPiD Genomics 747 SW 2nd Avenue IMB#14, Gainesville FL 32601, USA.
3: Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA; Arthropoda Department, Zoological Research Museum Alexander Koenig, Adenauer Allee 160, Bonn 53113, Germany.
4: Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA.
5: Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA; Entomology and Nematology Department, University of Florida, Gainesville FL 32611, USA.
6: Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA; Department of Biology, University of Florida, Gainesville FL 32611, USA.
7: Institute of Zoology and Evolutionary Biology with Phyletic Museum, Friedrich-Schiller-University Jena, Jena 07743, Germany.
8: Laboratorio de Entomología, Departamento de Biología, Pontificia Universidad Javeriana, Bogotá, Colombia.
9: Department of Organismic and Evolutionary Biology and Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138, USA.
10: Biology Department, City College of New York, New York, NY 10031, USA; Ph.D. Program in Biology, Graduate Center, City University of New York, New York, NY 10016, USA; Entomology Section, National Museum of the Philippines, Manila 1000, Philippines.

Abstract

The Neotropical moth-like butterflies (Hedylidae) are perhaps the most unusual butterfly family. In addition to being species-poor, this family is predominantly nocturnal and has anti-bat ultrasound hearing organs. Evolutionary relationships among the 36 described species are largely unexplored. A 13-gene anchored hybrid enrichment probe set (‘BUTTERFLY2.0’), that includes standard markers used in butterfly phylogenetics, captured sequences from decades-old museum specimens, and appears to be a cost-effective technique to infer phylogenetic relationships of the butterfly tree of life. Our dataset comprises up to 10,898 aligned base pairs from each of the 22 species of Hedylidae and 19 outgroups. Eleven of the thirteen loci were captured from 100% of the taxa, and the remaining loci were captured from ≥ 94% of taxa. The inferred phylogeny had robust support at 80% of nodes. Our results are consistent with morphological work, with Macrosoma tipulata sister to all remaining hedylids, followed by M. semiermis sister to the remaining species in the genus. We tested the hypothesis that nocturnality evolved only once from diurnality in Hedylidae, and showed that the ancestral condition was likely diurnal, with a shift to nocturnality early in the diversification of this family.

2018 Jun 13. pii: JVI.00616-18. doi: 10.1128/JVI.00616-18. [Epub ahead of print]

Immunization by replication-competent controlled herpesvirus vectors.

Bloom DC¹, Tran RK¹, Feller J¹, Voellmy R^2,³.

Author information

1: Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA.
2: HSF Pharmaceuticals SA, La Tour-de-Peilz, Switzerland and rvoellmy@hsfpharma.com.
3: Department of Physiological Sciences, University of Florida College of Veterinary Sciences, Gainesville, Florida, USA.

Abstract

Replication-competent controlled virus vectors were derived from virulent HSV-1 wildtype strain 17syn+ by placing one or two replication-essential genes under the stringent control of a gene switch that is co-activated by heat and an antiprogestin. Upon activation of the gene switch, the vectors replicate in infected cells with an efficacy that approaches that of the wildtype virus from which they were derived. Essentially no replication occurs in the absence of activation. When administered to mice, localized application of a transient heat treatment in the presence of systemic antiprogestin results in efficient but limited virus replication at the site of administration. The immunogenicity of these viral vectors was tested in a mouse footpad lethal challenge model. Unactivated viral vectors – which may be regarded as equivalents of inactivated vaccines – induced detectable protection against lethality caused by wildtype virus challenge. Single activation of the viral vectors at the site of administration (rear footpads) greatly enhanced protective immune responses, and second immunization resulted in complete protection. Once activated vectors also induced far better neutralizing antibody and HSV-1-specific cellular immune responses than unactivated vectors. To find out whether the immunogenicity of a heterologous antigen was also enhanced in the context of efficient transient vector replication, a virus vector constitutively expressing an equine influenza virus hemagglutinin was constructed. Immunization of mice with this recombinant induced detectable antibody-mediated neutralization of equine influenza virus as well as a hemagglutinin-specific cellular immune response. Single activation of viral replication resulted in a several-fold enhancement of these immune responses.IMPORTANCE We hypothesized that vigorous replication of a pathogen may be critical for eliciting the most potent and balanced immune response against it. Hence, attenuation/inactivation (as in conventional vaccines) should be avoided. Instead, necessary safety should be provided by placing replication of the pathogen under stringent control and of activating time-limited replication of the pathogen strictly in an administration region in which pathology cannot develop. Immunization will then occur in the context of highly efficient pathogen replication and uncompromised safety. We found that localized activation in mice of efficient but limited replication of a replication-competent controlled herpesvirus vector resulted in a greatly enhanced immune response to the virus or an expressed heterologous antigen. This finding supports the above hypothesis as well as suggests that the vectors may be promising novel agents worth exploring for the prevention/mitigation of infectious diseases for which efficient vaccination is lacking, in particular in immunocompromised patients.

2018 Jun 11. pii: S0305-4179(18)30425-X. doi: 10.1016/j.burns.2018.05.018. [Epub ahead of print]

Optimal skin regeneration after full thickness thermal burn injury in the spiny mouse, Acomys cahirinus.

Maden M¹.

Author information

1: Department of Biology & UF Genetics Institute, University of Florida, rm 326 Bartram Hall, P.O. Box 118525, Gainesville, FL 32611, USA. Electronic address: malcmaden@ufl.edu.

Abstract

The spiny mouse, Acomys cahirinus, shows remarkable regenerative abilities after excisional skin wounding by regrowing hair, sebaceous glands, smooth muscle, skeletal muscle and dermis without scarring. We have asked here whether this same regeneration can be seen after full thickness thermal burn injuries. Using a brass rod thermal injury model we show that in contrast to the lab mouse, Mus musculus, which forms a thick scar covered by a hairless epidermis, the spiny mouse regenerates all the tissues injured – skeletal muscle, dermis, hairs, sebaceous glands such that the skin is externally indistinguishable from its original appearance. Re-epithelialization is faster in Acomys than in Mus but ingression and proliferation of dermal fibroblasts is the same in both species. After 3 weeks the wound epithelium of Acomys has developed a covering of new hair follicles in contrast to Mus. The skeletal muscle of the panniculus carnosus in Mus shows some regeneration but it is incomplete and fibrotic whereas the Acomys muscle is replaced perfectly. There are differences in the macrophage profiles which invade the damaged tissues such as the absence of F4/80 or MOMA-2 +ve cells in Acomys which likely reflect different cytokine profiles resulting from the same injury in these two species.

2018 Apr-Jun;7(2):156-161. doi: 10.4103/ijmy.ijmy_33_18.

Mycobacterium tuberculosis Strains H37ra and H37rv have equivalent minimum inhibitory concentrations to most antituberculosis drugs.

Heinrichs MT¹, May RJ², Heider F³, Reimers T⁴, B Sy SK³, Peloquin CA⁵, Derendorf H³.

Author information

1: Department of Pharmaceutics, University of Florida; Emerging Pathogens Institute; Infectious Disease Pharmacokinetics Laboratory, Gainesville, Florida, USA.
2: Emerging Pathogens Institute, University of Florida, Florida, USA.
3: Department of Pharmaceutics, University of Florida, Gainesville, Florida, USA.
4: Department of Pharmaceutics, University of Florida; Emerging Pathogens Institute, Gainesville, Florida, USA.
5: Emerging Pathogens Institute, University of Florida; Infectious Disease Pharmacokinetics Laboratory; Department of Pharmacotherapy and Translational Research, Gainesville, Florida, USA.

Abstract

BACKGROUND:

Mycobacterium tuberculosis (Mtb) strains H37Ra and H37Rv are commonly used to study new and re-evaluate old antituberculous agents with respect to their pharmacodynamic effects in vitro. The differences in membrane proteins and, in particular, differences in carrier proteins between Mtb H37Ra and Mtb H37Rv may have an impact on antibiotic potency. The question of whether H37Ra can be used as a reliable surrogate for H37Rv and clinical strains has not been addressed sufficiently. The purpose of this study is to provide a full comparison of susceptibility data of the most common antituberculosis (TB) agents against both Mtb strains.

METHODS:

In addition to a literature review, in vitro checkerboard susceptibility study was conducted comparing the in vitro minimum inhibitory concentration (MIC) of 16 common antituberculous drugs against H37Ra and H37Rv. Heifets-Sanchez TB agar drug susceptibility plates were utilized.

RESULTS:

Half of the antibiotics demonstrated similar growth inhibition against both strains, while slightly differing MIC values were found for 7 of 16 drugs. With the exception of rifampicin, no marked difference in MIC against H37Ra and H37Rv was observed.

CONCLUSION:

While neither the attenuated (H37Ra) nor the virulent strain (H37Rv) is a clinical strain, both strains predicted MICs of clinical isolates equally well, when comparing the current in vitro results to clinical susceptibility data in the literature. H37Ra comes with the benefits of lower experimental costs and less administrative barriers including the requirement of a biosafety Level III environment.