UFGI publication round-up week 5/21
Crystal Structure of Cleaved Serp-1, a Myxomavirus-Derived Immune Modulating Serpin: Structural Design of Serpin Reactive Center Loop Peptides with Improved Therapeutic Function.
Author information
- 1
- Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892, United States.
- 2
- Department of Biochemistry and Molecular Biology, University of Florida College of Medicine , Gainesville, Florida 32610-0277, United States.
- 3
- Department of Medicine, Divisions of Cardiovascular Medicine and Rheumatology, University of Florida , Gainesville, Florida 32610-0277, United States.
- 4
- Cloud Pharmaceuticals , 6 Davis Drive, Research Triangle Park, North Carolina 27709, United States.
- 5
- CGMBio Consulting , London, Ontario, Canada.
- 6
- Saint Joseph’s Hospital, Dignity Health , Phoenix, Arizona 85013, United States.
Abstract
The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a “first-in-class” therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII), and α-1 antitrypsin (A1AT), and target proteases. Using in silico modeling of the Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins because of the larger number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic-treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest interaction of the peptides with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step toward the development of a promising new class of anti-inflammatory serpin-based therapeutics.
The Arabidopsis ELP3/ELO3 and ELP4/ELO1 genes enhance disease resistance in Fragaria vesca L.
Author information
- 1
- Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611, USA.
- 2
- Department of Horticultural Sciences, University of Florida, Gainesville, FL, 32611, USA.
- 3
- Department of Plant Pathology, University of Florida, Gainesville, FL, 32611, USA.
- 4
- Department of Plant Pathology, Gulf Coast Research and Education Center, University of Florida, Wimauma, FL, 33598, USA.
- 5
- Department of Horticultural Sciences, University of Florida, Gainesville, FL, 32611, USA. kfolta@ufl.edu.
- 6
- Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611, USA. zhlmou@ufl.edu.
Abstract
BACKGROUND:
Plant immune response is associated with a large-scale transcriptional reprogramming, which is regulated by numerous transcription regulators such as the Elongator complex. Elongator is a multitasking protein complex involved in diverse cellular processes, including histone modification, DNA methylation, and tRNA modification. In recent years, Elongator is emerging as a key regulator of plant immune responses. However, characterization of Elongator’s function in plant immunity has been conducted only in the model plant Arabidopsis thaliana. It is thus unclear whether Elongator’s role in plant immunity is conserved in higher plants. The objective of this study is to characterize transgenic woodland strawberry (Fragaria vesca L.) overexpressing the Arabidopsis Elongator (AtELP) genes, AtELP3 and AtELP4, and to determine whether F. vesca carries a functional Elongator complex.
METHODS:
Transgenic F. vesca and Arabidopsis plants were produced via Agrobacterium-mediated genetic transformation and characterized by morphology, PCR, real-time quantitative PCR, and disease resistance test. The Student’s t test was used to analyze the data.
RESULTS:
Overexpression of AtELP3 and AtELP4 in F. vesca impacts plant growth and development and confers enhanced resistance to anthracnose crown rot, powdery mildew, and angular leaf spot, which are caused by the hemibiotrophic fungal pathogen Colletotrichum gloeosporioides, the obligate biotrophic fungal pathogen Podosphaera aphanis, and the hemibiotrophic bacterial pathogen Xanthomonas fragariae, respectively. Moreover, the F. vesca genome encodes all six Elongator subunits by single-copy genes with the exception of FvELP4, which is encoded by two homologous genes, FvELP4-1 and FvELP4-2. We show that FvELP4-1 complemented the Arabidopsis Atelp4/elo1-1 mutant, indicating that FvELP4 is biologically functional.
CONCLUSIONS:
This is the first report on overexpression of Elongator genes in plants. Our results indicate that the function of Elongator in plant immunity is most likely conserved in F. vesca and suggest that Elongator genes may hold potential for helping mitigate disease severity and reduce the use of fungicides in strawberry industry.
Genomic Differentiation during Speciation-with-Gene-Flow: Comparing Geographic and Host-Related Variation in Divergent Life History Adaptation in Rhagoletis pomonella.
Author information
- 1
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. mdoellma@nd.edu.
- 2
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. gregory.ragland@ucdenver.edu.
- 3
- Environmental Change Initiative, University of Notre Dame, Notre Dame, IN 46556, USA. gregory.ragland@ucdenver.edu.
- 4
- Department of Integrative Biology, University of Colorado, Denver, CO 80217, USA. gregory.ragland@ucdenver.edu.
- 5
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. glenrayhood@rice.edu.
- 6
- Department of Biosciences, Rice University, Houston, TX 77005, USA. glenrayhood@rice.edu.
- 7
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. pmeyers2@nd.edu.
- 8
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. scott.p.egan@rice.edu.
- 9
- Department of Biosciences, Rice University, Houston, TX 77005, USA. scott.p.egan@rice.edu.
- 10
- Advanced Diagnostics and Therapeutics Initiative, University of Notre Dame, Notre Dame, IN 46556, USA. scott.p.egan@rice.edu.
- 11
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. powellt@binghamton.edu.
- 12
- Department of Biological Sciences, State University of New York, Binghamton, NY 13902, USA. powellt@binghamton.edu.
- 13
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. lazorchakp@jhu.edu.
- 14
- Department of Computer Science, Johns Hopkins University, Baltimore, MD 21218, USA. lazorchakp@jhu.edu.
- 15
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. mglover@nd.edu.
- 16
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. ctait@nd.edu.
- 17
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. hannes.schuler@boku.ac.at.
- 18
- Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, USA. dahahn@ufl.edu.
- 19
- Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. stewartb@life.illinois.edu.
- 20
- Lyman Briggs College and Department of Entomology, Michigan State University, East Lansing, MI 48824, USA. jimsmith@msu.edu.
- 21
- Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK. p.nosil@sheffield.ac.uk.
- 22
- Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. Jeffrey.L.Feder.2@nd.edu.
- 23
- Environmental Change Initiative, University of Notre Dame, Notre Dame, IN 46556, USA. Jeffrey.L.Feder.2@nd.edu.
- 24
- Advanced Diagnostics and Therapeutics Initiative, University of Notre Dame, Notre Dame, IN 46556, USA. Jeffrey.L.Feder.2@nd.edu.
Abstract
A major goal of evolutionary biology is to understand how variation within populations gets partitioned into differences between reproductively isolated species. Here, we examine the degree to which diapause life history timing, a critical adaptation promoting population divergence, explains geographic and host-related genetic variation in ancestral hawthorn and recently derived apple-infesting races of Rhagoletis pomonella. Our strategy involved combining experiments on two different aspects of diapause (initial diapause intensity and adult eclosion time) with a geographic survey of genomic variation across four sites where apple and hawthorn flies co-occur from north to south in the Midwestern USA. The results demonstrated that the majority of the genome showing significant geographic and host-related variation can be accounted for by initial diapause intensity and eclosion time. Local genomic differences between sympatric apple and hawthorn flies were subsumed within broader geographic clines; allele frequency differences within the races across the Midwest were two to three-fold greater than those between the races in sympatry. As a result, sympatric apple and hawthorn populations displayed more limited genomic clustering compared to geographic populations within the races. The findings suggest that with reduced gene flow and increased selection on diapause equivalent to that seen between geographic sites, the host races may be recognized as different genotypic entities in sympatry, and perhaps species, a hypothesis requiring future genomic analysis of related sibling species to R. pomonella to test. Our findings concerning the way selection and geography interplay could be of broad significance for many cases of earlier stages of divergence-with-gene flow, including (1) where only modest increases in geographic isolation and the strength of selection may greatly impact genetic coupling and (2) the dynamics of how spatial and temporal standing variation is extracted by selection to generate differences between new and discrete units of biodiversity.
Hepatic ZIP14-mediated zinc transport is required for adaptation to endoplasmic reticulum stress.
Author information
- 1
- Food Science and Human Nutrition Department, Center for Nutritional Sciences, College of Agricultural and Life Sciences, University of Florida, Gainesville, FL 32611.
- 2
- Food Science and Human Nutrition Department, Center for Nutritional Sciences, College of Agricultural and Life Sciences, University of Florida, Gainesville, FL 32611; cousins@ufl.edu.
- 3
- Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32611.
Abstract
Extensive endoplasmic reticulum (ER) stress damages the liver, causing apoptosis and steatosis despite the activation of the unfolded protein response (UPR). Restriction of zinc from cells can induce ER stress, indicating that zinc is essential to maintain normal ER function. However, a role for zinc during hepatic ER stress is largely unknown despite important roles in metabolic disorders, including obesity and nonalcoholic liver disease. We have explored a role for the metal transporter ZIP14 during pharmacologically and high-fat diet-induced ER stress using Zip14-/- (KO) mice, which exhibit impaired hepatic zinc uptake. Here, we report that ZIP14-mediated hepatic zinc uptake is critical for adaptation to ER stress, preventing sustained apoptosis and steatosis. Impaired hepatic zinc uptake in Zip14 KO mice during ER stress coincides with greater expression of proapoptotic proteins. ER stress-induced Zip14 KO mice show greater levels of hepatic steatosis due to higher expression of genes involved in de novo fatty acid synthesis, which are suppressed in ER stress-induced WT mice. During ER stress, the UPR-activated transcription factors ATF4 and ATF6α transcriptionally up-regulate Zip14 expression. We propose ZIP14 mediates zinc transport into hepatocytes to inhibit protein-tyrosine phosphatase 1B (PTP1B) activity, which acts to suppress apoptosis and steatosis associated with hepatic ER stress. Zip14 KO mice showed greater hepatic PTP1B activity during ER stress. These results show the importance of zinc trafficking and functional ZIP14 transporter activity for adaptation to ER stress associated with chronic metabolic disorders.
Oncogenic PKC-ι activates Vimentin during epithelial-mesenchymal transition in melanoma; a study based on PKC-ι and PKC-ζ specific inhibitors.
Author information
- 1
- a Department of Chemistry , University of South Florida , Tampa , FL , USA.
- 2
- b Department of Pathology , Immunology and Laboratory Medicine, University of Florida, College of Medicine , Gainesville , FL , USA.
Abstract
Melanoma is one of the fastest growing cancers in the United States and is accompanied with a poor prognosis owing to tumors being resistant to most therapies. Atypical protein kinase Cs (aPKC) are involved in malignancy in many cancers. We previously reported that aPKCs play a key role in melanoma’s cell motility by regulating cell signaling pathways which induce epithelial-mesenchymal Transition (EMT). We tested three novel inhibitors; [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) which are specific to protein kinase C-iota (PKC-ι) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) which is specific to PKC-zeta (PKC-ζ) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular modeling was used to identify potential binding sites for the inhibitors and to predict selectivity. Kinase assay showed >50% inhibition for specified targets beyond 5 μM for all inhibitors. Both ICA-1 and ζ-Stat significantly reduced cell proliferation and induced apoptosis, while ICA-1 also significantly reduced migration and melanoma cell invasion. PKC-ι stimulated EMT via TGFβ/Par6/RhoA pathway and activated Vimentin by phosphorylation at S39. Both ICA-1 and ζ-Stat downregulate TNF-α induced NF-κB translocation to the nucleus there by inducing apoptosis. Results suggest that PKC-ι is involved in melanoma malignancy than PKC-ζ. Inhibitors proved to be effective under in-vitro conditions and need to be tested in-vivo for the validity as effective therapeutics. Overall, results show that aPKCs are essential for melanoma progression and metastasis and that they could be used as effective therapeutic targets for malignant melanoma.
Sensory and Flavor Characteristics of Tomato Juice from Garden Gem and Roma Tomatoes with Comparison to Commercial Tomato Juice.
Author information
- 1
- Food Science and Human Nutrition Dept., Univ. of Florida, Gainesville, Fla., U.S.A.
- 2
- Horticulture Dept., Univ. of Florida, Gainesville, Fla. 32611, U.S.A.
Abstract
The objective of this study was to characterize the flavor of a premium Florida tomato variety that has significant potential for producing a high quality processed juice product. A high-quality Florida plum tomato variety (Garden Gem), and a typical grocery-store plum tomato variety (Roma) were thermally processed into tomato juices without any additives. The 2 pilot products and a popular commercially available tomato juice (low sodium with sugar and flavor added) were compared using sensory evaluation and instrumental analysis. Flavor compounds in these products were identified using dynamic headspace purge and trap-gas chromatography-mass spectrometry (PT-GC-MS) by MS library match and retention index and were semi-quantitated using internal standards. Color, uniformity, overall liking, tomato flavor, sweetness and texture were rated using a hedonic scale. Analysis of variance, correlation and principal component analysis were used to analyze both sensory and flavor data. Among the 3 products, Garden Gem juice was rated significantly (P < 0.05) higher for overall liking, tomato flavor, and sweetness by the 119 consumer panelists in both seasons. Garden Gem juice was found to contain higher levels of 3 sweet/fruity related aroma compounds: 6-methyl-5-hepten-2-one, linalool, and β-ionone. The commercial tomato juice contained a high level of the Maillard reaction-related notes furfural, dimethyl sulfide, and the least amount of green-related notes (hexanal, E-2-hexenal and Z-2-heptenal). The flavor profile of the Roma tomato juice was similar to Garden Gem juice except it contained substantially lower amounts of hexanal and 2-isobutylthiazole. The compound β-ionone (fruity note) was not detected in either the commercial or Roma juice.
PRACTICAL APPLICATION:
This proof of concept study demonstrates that high flavor quality tomatoes can be used to create better tasting processed tomato products. The study also demonstrates how sensory preference can confer a potential market advantage over existing commercial products. The Garden Gem variety has potential to add desirable flavor attributes to processed tomato products. This research may also provide insights for product developers to which flavor volatiles best reflect sensory observations for different aspects of tomato flavor.
Childhood Trauma, DNA Methylation of Stress-related Genes, and Depression: Findings from Two Monozygotic Twin Studies.
Author information
- 1
- Department of Epidemiology, College of Public Health and Health Professions, College of Medicine, University of Florida, Gainesville, FL.
- 2
- Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, WA.
- 3
- Department of Pediatrics, University of Washington, Seattle, WA.
- 4
- Department of Epidemiology, School of Public health, University of Washington, Seattle, WA.
- 5
- Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, GA.
- 6
- Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA.
Abstract
OBJECTIVE:
DNA methylation has been associated with both early life stress and depression. This study examined the combined association of DNA methylation at multiple CpG probes in five stress-related genes with depressive symptoms, and tested whether these genes methylation mediated the association between childhood trauma and depression in two monozygotic (MZ) twin studies.
METHODS:
The current analysis comprised 119 MZ twin pairs (84 male pairs [mean 55 years], and 35 female pairs [mean 36 years]). Peripheral blood DNA methylation of five stress-related genes (BDNF, NR3C1, SLC6A4, MAOA, and MAOB) was quantified by bisulfite pyrosequencing or 450K BeadChip. We applied generalized Poisson linear mixed models to examine the association between each single CpG methylation and depressive symptoms. The joint associations of multiple CpGs in a single gene or all five stress-related genes as a pathway were tested by weighted truncated product method. Mediation analysis was conducted to test the potential mediating effect of stress gene methylation on the relationship between childhood trauma and depressive symptoms.
RESULTS:
Multiple CpG probes showed nominal individual associations, but very few survived multiple testing. Gene-based or gene-set approach, however, revealed significant joint associations of DNA methylation in all five stress-related genes with depressive symptoms in both studies. Moreover, two CpG probes in the BDNF and NR3C1 mediated ~20% of the association between childhood trauma and depressive symptoms.
CONCLUSION:
DNA methylation at multiple CpG sites are jointly associated with depressive symptoms, and partly mediates the association between childhood trauma and depression. Our results highlight the importance of testing the combined effects of multiple CpG loci on complex traits, and may unravel a molecular mechanism through which adverse early life experiences are biologically embedded.
Porphyromonas gingivalis strain dependent inhibition of uterine spiral artery remodeling in the pregnant rat.
Author information
- 1
- Microbiology & Immunology, A.T. Still University of Health Sciences, Kirksville College of Osteopathic Medicine, Kirksville, MO. USA.
- 2
- Infectious Disease and Immunology, College of Veterinary Medicine and D. H. Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL, USA.
- 3
- Center for Molecular Microbiology and Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL. USA.
- 4
- Department of Pathobiological Sciences, University of Wisconsin – Madison, School of Veterinary Medicine, Madison, WI. USA.
Abstract
Porphyromonas gingivalis (Pg), is an important periodontal pathogen that is also implicated in pregnancy complications involving defective deep placentation (DDP). We hypothesized that Pg invasion of the placental bed promotes DDP. Pregnant rats were intravenously inoculated with sterile vehicle, Pg strain W83, or A7436 at gestation day (GD) 14 (acute cohort). Non-pregnant rats received repeated oral inoculations for 3 months before breeding (chronic cohort). Tissues and/or sera were collected at GD18 for analysis. Pg infection status was determined by seroconversion (chronic cohort) and by presence of Pg antigen in utero-placental tissues processed for histology and morphometric assessment of spiral artery remodeling. Mesometrial tissues from seropositive dams were analyzed for expression of interleukin 1β, 6, and 10, TNF, TGF-β, follistatin-related protein 3, and inhibin beta A chain since these genes regulate extravillous trophoblast invasion. The in situ distribution of W83 and A7436 antigen in utero-placental tissues was similar in both cohorts. In the acute cohort, mesometrial stromal necrosis was more common with W83, but arteritis was more common with A7436 infection (P < 0.05). Increased vascular necrosis was seen in mesometrium of chronically infected groups (P < 0.05). Only A7436 infected animals had increased fetal deaths, reduced spiral artery remodeling, reduced inhibin beta A expression, and an increased proportion of FSLT3 positive extravillous trophoblasts within spiral arteries. While infection with both Pg strains produced varying pathology of the deep placental bed, only infection with strain A7436 resulted in impaired spiral artery remodeling.
Supramolecularly Engineered Circular Bivalent Aptamer for Enhanced Functional Protein Delivery.
Author information
- 1
- Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Life Sciences, Aptamer Engineering Center of Hunan Province , Hunan University , Changsha , Hunan 410082 , People’s Republic of China.
- 2
- Center for Research at Bio/Nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics, Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute , University of Florida , Gainesville , Florida 32611-7200 , United States.
- 3
- Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry , Chinese Academy of Sciences , Beijing 100190 , People’s Republic of China.
- 4
- University of Chinese Academy of Sciences , Beijing 100049 , People’s Republic of China.
- 5
- Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, and College of Chemistry and Chemical Engineering , Shanghai Jiao Tong University , Shanghai 200240 , People’s Republic of China.
Abstract
Circular bivalent aptamers (cb-apt) comprise an emerging class of chemically engineered aptamers with substantially improved stability and molecular recognition ability. Its therapeutic application, however, is challenged by the lack of functional modules to control the interactions of cb-apt with therapeutics. We present the design of a β-cyclodextrin-modified cb-apt (cb-apt-βCD) and its supramolecular interaction with molecular therapeutics via host-guest chemistry for targeted intracellular delivery. The supramolecular ensemble exhibits high serum stability and enhanced intracellular delivery efficiency compared to a monomeric aptamer. The cb-apt-βCD ensemble delivers green fluorescent protein into targeted cells with efficiency as high as 80%, or cytotoxic saporin to efficiently inhibit tumor cell growth. The strategy of conjugating βCD to cb-apt, and subsequently modulating the supramolecular chemistry of cb-apt-βCD, provides a general platform to expand and diversify the function of aptamers, enabling new biological and therapeutic applications.
Fluorinated DNA Micelles: Synthesis and Properties.
Author information
- 1
- Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Life Sciences , Aptamer Engineering Center of Hunan Province, Hunan University , Changsha , Hunan 410082 , China.
- 2
- Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, College of Chemistry and Chemical Engineering , Shanghai Jiao Tong University , Shanghai , 200240 , People’s Republic of China.
- 3
- MOE Key Laboratory for Analytical Science of Food Safety and Biology, College of Chemistry , Fuzhou University , Fuzhou 350116 , People’s Republic of China.
- 4
- Department of Chemistry and Department of Physiology and Functional Genomics , Center for Research at the Bio/Nano Interface, UF Health Cancer Center, UF Genetics Institute, McKnight Brain Institute, University of Florida , Gainesville , Florida 32611-7200 , United States.
Abstract
Creating new functional building blocks that expand the versatility of nanostructures depends on bottom-up self-assembly of amphiphilic biomolecules. Inspired by the unique physicochemical properties of hydrophobic perfluorocarbons, coupled with the powerful functions of nucleic acids, we herein report the synthesis of a series of diperfluorodecyl-DNA conjugates (PF-DNA) which can efficiently self-assemble into micelles in aqueous solution. On the basis of the micelle structure, both target binding affinity and enzymatic resistance of the DNA probe can be enhanced. In addition, based on the hydrophobic effect, the PF-DNA micelles (PFDM) can actively anchor onto the cell membrane, offering a promising tool for cell-surface engineering. Finally, the PFDM can enter cells, which is significant for designing carriers for intracellular delivery. The combined advantages of the DNA micelle structure and the unique physicochemical properties of perfluorocarbons make these PFDM promising for applications in bioimaging and biomedicine.
A Cyanine Dye to Probe Mitophagy: Simultaneous Detection of Mitochondria and Autolysosomes in Live Cells.
Author information
- 1
- Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences , Beijing, 100190, China.
- 2
- University of the Chinese Academy of Sciences , Beijing 100049, China.
- 3
- CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology , Beijing, 100190, China.
- 4
- Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Biology and College of Chemistry and Chemical Engineering, Hunan University , Changsha, 410082, China.
- 5
- Department of Chemistry, 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
Mitophagy is a process in which cells remove dysfunctional mitochondria and recycle their constituents in a lysosome-dependent manner. To probe this process, two different fluorescent dyes specific for mitochondria and lysosomes, respectively, are often used in combination. However, current fluorescent dyes for lysosomes cannot distinguish mitochondria-containing autolysosomes from other lysosomes. Therefore, we herein report a cyanine dye, HQO, which can simultaneously probe mitochondria and autolysosomes in live cells by exhibiting different fluorescence properties. HQO selectively accumulates in mitochondria but then transforms to the protonated HQOH(+) form with the decrease of pH when dysfunctional mitochondria evolve into autolysosomes. Since HQO and HQOH(+) exhibit different absorption and emission with Ex/Em at 530/650 and 710/750 nm, respectively, in a low polarity environment, such as that found in micelles, they are uniquely suited to monitor mitophagy with the ability to distinguish autolysosomes from other lysosomes.
A novel adeno-associated virus capsid with enhanced neurotropism corrects a lysosomal transmembrane enzyme deficiency.
Author information
- 1
- Department of Infectious Diseases, School of Immunology and Microbial Sciences, King’s College London, London, UK.
- 2
- Stem Cell and Neurotherapies, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester, UK.
- 3
- Molecular Neuromodulation, Wallenberg Neuroscience Center, Lund University, Lund, Sweden.
- 4
- Department of Genetics, UCL Institute of Ophthalmology, London, UK.
- 5
- Department of Biochemistry and Molecular Biology, Center for Structural Biology, McKnight Brain Institute, College of Medicine, University of Florida, Gainesville, FL, USA.
- 6
- CHU Ste-Justine, University of Montreal, Montreal, Canada.
- 7
- Centre Hospitalo-Universitaire de Toulouse, Institut Fédératif de Biologie, Laboratoire de Biochimie Métabolique, and Unité Mixte de Recherche (UMR) 1037 Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche en Cancérologie de Toulouse, Toulouse, France.
- 8
- Manchester Collaborative Centre for Inflammation Research, Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester, UK.
- 9
- Department of Pediatrics, Powell Gene Therapy Center, University of Florida, Gainesville, FL, USA.
- 10
- Department of Pharmacology, UCL School of Pharmacy, University College London, London, UK.
- 11
- Gene Transfer Technology Group, Institute for Women’s Health, University College London, London, UK.
- 12
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
Abstract
Recombinant adeno-associated viruses (AAVs) are popular in vivo gene transfer vehicles. However, vector doses needed to achieve therapeutic effect are high and some target tissues in the central nervous system remain difficult to transduce. Gene therapy trials using AAV for the treatment of neurological disorders have seldom led to demonstrated clinical efficacy. Important contributing factors are low transduction rates and inefficient distribution of the vector. To overcome these hurdles, a variety of capsid engineering methods have been utilized to generate capsids with improved transduction properties. Here we describe an alternative approach to capsid engineering, which draws on the natural evolution of the virus and aims to yield capsids that are better suited to infect human tissues. We generated an AAV capsid to include amino acids that are conserved among natural AAV2 isolates and tested its biodistribution properties in mice and rats. Intriguingly, this novel variant, AAV-TT, demonstrates strong neurotropism in rodents and displays significantly improved distribution throughout the central nervous system as compared to AAV2. Additionally, sub-retinal injections in mice revealed markedly enhanced transduction of photoreceptor cells when compared to AAV2. Importantly, AAV-TT exceeds the distribution abilities of benchmark neurotropic serotypes AAV9 and AAVrh10 in the central nervous system of mice, and is the only virus, when administered at low dose, that is able to correct the neurological phenotype in a mouse model of mucopolysaccharidosis IIIC, a transmembrane enzyme lysosomal storage disease, which requires delivery to every cell for biochemical correction. These data represent unprecedented correction of a lysosomal transmembrane enzyme deficiency in mice and suggest that AAV-TT-based gene therapies may be suitable for treatment of human neurological diseases such as mucopolysaccharidosis IIIC, which is characterized by global neuropathology.
Germline viral “fossils” guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.
Author information
- 1
- Laboratory of Molecular Virology and Gene Therapy, National Heart, Lung and Blood Institute, Bethesda, Maryland, United States of America.
- 2
- Gene Therapy Research Unit, Children’s Medical Research Institute and The Children’s Hospital at Westmead, Westmead, New South Wales, Australia.
- 3
- Department of Biochemistry and Genetics, La Trobe University, Bundoora, Victoria, Australia.
- 4
- The McKnight Brain Institute, University of Florida, Gainesville, Florida, United States of America.
- 5
- Evolutionary Genomics Research Group, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, United States of America.
- 6
- The University of Sydney, Discipline of Paediatrics and Child Health, Westmead, New South Wales, Australia.
Abstract
Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV “fossils” provide novel capsid sequences for use in translational research and clinical applications.
Warfarin Pharmacogenomics in Diverse Populations.
Author information
- 1
- Department of Pharmacy Practice and Science, University of Arizona College of Pharmacy, Tucson, Arizona.
- 2
- Department of Pharmacology and Toxicology, University of Arizona College of Pharmacy, Tucson, Arizona.
- 3
- Department of Public Health, University of Arizona College of Medicine, Tucson, Arizona.
- 4
- Department of Surgery, University of Arizona College of Medicine, Tucson, Arizona.
- 5
- Center for Applied Genetics and Genomic Medicine, University of Arizona College of Medicine, Tucson, Arizona.
- 6
- Department of Pharmacotherapy and Translational Research and Center for Pharmacogenomics, University of Florida, Gainesville, Florida.
- 7
- Sarver Heart Center, University of Arizona College of Medicine, Tucson, Arizona.
Abstract
Genotype-guided warfarin dosing algorithms are a rational approach to optimize warfarin dosing and potentially reduce adverse drug events. Diverse populations, such as African Americans and Latinos, have greater variability in warfarin dose requirements and are at greater risk for experiencing warfarin-related adverse events compared with individuals of European ancestry. Although these data suggest that patients of diverse populations may benefit from improved warfarin dose estimation, the vast majority of literature on genotype-guided warfarin dosing, including data from prospective randomized trials, is in populations of European ancestry. Despite differing frequencies of variants by race/ethnicity, most evidence in diverse populations evaluates variants that are most common in populations of European ancestry. Algorithms that do not include variants important across race/ethnic groups are unlikely to benefit diverse populations. In some race/ethnic groups, development of race-specific or admixture-based algorithms may facilitate improved genotype-guided warfarin dosing algorithms above and beyond that seen in individuals of European ancestry. These observations should be considered in the interpretation of literature evaluating the clinical utility of genotype-guided warfarin dosing. Careful consideration of race/ethnicity and additional evidence focused on improving warfarin dosing algorithms across race/ethnic groups will be necessary for successful clinical implementation of warfarin pharmacogenomics. The evidence for warfarin pharmacogenomics has a broad significance for pharmacogenomic testing, emphasizing the consideration of race/ethnicity in discovery of gene-drug pairs and development of clinical recommendations for pharmacogenetic testing.