Elucidation of the full genetic information of Japanese rubella vaccines and the genetic changes associated with in vitro and in vivo vaccine virus phenotypes

Introducing small genetic changes to study specific mutations or reverting clinical mutations to wild type has been an area of interest in precision genomics for several years. In fact, it has been found that nearly 90% of all human pathogenic mutations are caused by small genetic variations, and the methods to efficiently and precisely correct these errors are critically important. One common way to make these small DNA changes is to provide a single stranded DNA (ssDNA) donor containing the desired alteration together with a targeted double-strand break (DSB) at the genomic target. The donor is typically flanked by regions of homology that are often ~30-100bp in length to leverage the homology directed repair (HDR) pathway. Coupling a ssDNA donor with a CRISPR-Cas9 to produce a targeted DSB is one of the most streamlined approaches to introduce small changes. However, in many cell types this approach results in a low rate of incorporation of the desired alteration and has undesired imprecise repair at the 5' or 3' junction due to artifacts of the DNA repair process. We herein report a technology that couples the spatial temporal localization of an ssDNA repair template and leverages the nucleic acid components of the CRISPR-Cas9 system. We show that by direct fusion of an ssDNA template to the trans activating RNA (tracrRNA) to generate an RNA-DNA chimera, termed Donorguide, we recover precise integration of genetic alterations, with both increased integration rates and decreased imprecision at the 5' or 3' junctions relative to an ssODN donor in vitro in HEK293T cells as well as in vivo in zebrafish. Further, we show that this technology can be used to enhance gene conversion with other gene editing tools such as TALENs.

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Live-Attenuated Measles Virus Vaccine Targets Dendritic Cells and Macrophages in Muscle of Nonhuman Primates

Journal of Virology ◽

10.1128/jvi.02924-14 ◽

2014 ◽

Vol 89 (4) ◽

pp. 2192-2200 ◽

Cited By ~ 35

Author(s):

Linda J. Rennick ◽

Rory D. de Vries ◽

Thomas J. Carsillo ◽

Ken Lemon ◽

Geert van Amerongen ◽

...

Keyword(s):

Measles Virus ◽

Nonhuman Primates ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Vaccine Virus ◽

Target Cells ◽

Recombinant Viruses ◽

Green Fluorescent

ABSTRACTAlthough live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replicationin vivoare still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MVEZ. Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMVEZEGFP(1), rMVEZEGFP(3), and rMVEZEGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were comparedin vitroby growth curves, which indicated that rMVEZEGFP(1) was overattenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMVEZEGFP(1) and rMVEZEGFP(6) did not induce satisfactory serum antibody responses, whereas bothin vitroandin vivorMVEZEGFP(3) was functionally equivalent to the commercial MVEZ-containing vaccine. Intramuscular vaccination of macaques with rMVEZEGFP(3) resulted in the identification of EGFP+cells in the muscle at days 3, 5, and 7 postvaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV.IMPORTANCEEven though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicatesin vivo. This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious.

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Vitamin D and prostate cancer

Medicinski pregled ◽

10.2298/mpns1306259m ◽

2013 ◽

Vol 66 (5-6) ◽

pp. 259-262

Author(s):

Goran Marusic ◽

Dimitrije Jeremic ◽

Sasa Vojinov ◽

Natasa Filipovic ◽

Milan Popov

Keyword(s):

Prostate Cancer ◽

Vitamin D ◽

Physiological Role ◽

In Vivo Studies ◽

Vitamin D Metabolism ◽

Genetic Changes ◽

Metabolic Role

In addition to the metabolic role of vitamin D, which is well known and clearly defined, there have been many hypotheses regarding its anti-proliferative and pro-apoptotic role. Epidemiology and Significance of Prostate Cancer. Prostate cancer is the second most common malignancy in men. Long period of cancerogenesis, available tumor markers and high incidence make this cancer ideal for preventive measures. Physiological Role of Vitamin D and its Effect on Prostate Cancer Cells. In vitro and in vivo studies have shown the anti-proliferative and pro-apoptopic role of vitamin D. Disorders of vitamin D metabolism are noted in vitamin D gene level, vitamin D receptor, vitamin D responsive elements and androgen receptors. We present the most important effect of those changes on vitamin D metabolism. Conclusion. Available studies on vitamin D level in serum, prostate tissue, observed activity of vitamin D enzymes and genetic changes give us only a slight insight into the basic mechanisms of vitamin D action in the development of prostate cancer; therefore, further investigations are needed.

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In Vitro Matured Oocytes Are More Susceptible than In Vivo Matured Oocytes to Mock ICSI Induced Functional and Genetic Changes

PLoS ONE ◽

10.1371/journal.pone.0119735 ◽

2015 ◽

Vol 10 (3) ◽

pp. e0119735 ◽

Cited By ~ 2

Author(s):

Shubhashree Uppangala ◽

Shilly Dhiman ◽

Sujit Raj Salian ◽

Vikram Jeet Singh ◽

Guruprasad Kalthur ◽

...

Keyword(s):

Genetic Changes

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Levamisole and bovine immunity: in vitro and in vivo effects on immune responses to herpesvirus immunization

Canadian Journal of Microbiology ◽

10.1139/m81-201 ◽

1981 ◽

Vol 27 (12) ◽

pp. 1312-1319 ◽

Cited By ~ 11

Author(s):

L. A. Babiuk ◽

V. Misra

Keyword(s):

Vaccine Virus ◽

Pokeweed Mitogen ◽

Infectious Bovine Rhinotracheitis ◽

Purified Protein Derivative ◽

Macrophage Activity ◽

Bovine Lymphocytes ◽

Humoral Responses ◽

In Vivo Effects

Levamisole was shown to enhance in vitro blastogenic responses of bovine lymphocytes to nonspecific mitogens (phytohemagglutinin and pokeweed mitogen) as well as to infectious bovine rhinotracheitis virus and purified protein derivative. Greatest enhancement was observed at suboptimal concentrations of viral antigen. In addition to enhancing lymphocyte reactivity levamisole also affected macrophage activity as determined by increased Fc receptor activity and [3H]glucosamine incorporation. Levamisole (5–50 μg/mL) enhanced type II immune (or γ) interferon production by macrophage–lymphocyte cultures. Administration of levamisole and attenuated infectious bovine rhinotracheitis vaccine virus in vivo did not elevate cellular or humoral responses.

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Trehalose improves PPR vaccine virus stability in diluent

10.33109/bjvmjd19rm1 ◽

2020 ◽

Vol 17 (2) ◽

Author(s):

N. Mohanto ◽

A. Khatun ◽

J. A. Begum ◽

M. M. Parvin ◽

M. S. I. Siddiqui ◽

...

Keyword(s):

Ambient Temperature ◽

Vero Cells ◽

Vaccine Virus ◽

Virus Infectivity ◽

Different Temperatures ◽

Ppr Virus ◽

Pcr Targeting ◽

Infectivity Titer

Background: Specialized freeze-drying process is being used in the field for different thermostable vaccine preparation worldwide. The thermostability remains only in undiluted conditions. If dilution is made at the morning and used for the whole day, the vaccine efficacy is compromised at high ambient temperature. In this study, trehalose based specialized vaccine diluent was used to improve the stability of Peste des Petits Ruminants (PPR) vaccine in diluted condition. Methods: The available PPR vaccine was reconstituted with conventional diluent and with trehalose based test diluent. The diluted vaccine was kept at ambient temperature without maintaining any cool chain. Stability of diluted vaccine virus was further assessed in vivo and in vitro at different temperatures. Goats were vaccinated and Vero cells were infected with reconstituted vaccines and were assessed at 0, 3, 6, 9 and 24 hours post dilution. Antibody titer was measured and virus infectivity titer was determined in both cultured cell lysate and supernatant. The presence of the virus particles in Vero cell was confirmed by standard RT-PCR targeting Fusion (F) gene of PPR virus. Results: In vivo results revealed that the number of goats possessed antibodies to PPR virus was higher in trehalose based vaccine formulation than the conventional PBS based diluent. Reconstituted vaccine virus (using PBS and trehalose diluent) infected Vero cells produced 70-80% cytopathic effect (CPE) in 5th days of post infection. Both diluents produced and maintained infectivity titer from log10 TCID50 5.5 to log10 TCID50 3.6, until the use of vaccines incubated for 9 hours after dilution. On the other hand, at 24 hours of post dilution only trehalose formulated vaccine produced log10 TCID502.5 whereas no infectivity titer was observed at the same time using conventional one. Conclusion: The present study suggests that trehalose preserves the quality of reconstituted vaccine in terms of infectivity titers. Trehalose can be a diluent of choice for reconstitution of PPR vaccine in field.

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LncRNA IDH1-AS1 suppresses cell proliferation and tumor growth in glioma

Biochemistry and Cell Biology ◽

10.1139/bcb-2019-0465 ◽

2020 ◽

Vol 98 (5) ◽

pp. 556-564

Author(s):

Jubo Wang ◽

Yu Quan ◽

Jian Lv ◽

Quan Dong ◽

Shouping Gong

Keyword(s):

Cell Proliferation ◽

Cell Growth ◽

Protein Expression ◽

Caspase 9 ◽

Isocitrate Dehydrogenase 1 ◽

Genetic Changes ◽

Primary Glioblastoma ◽

U251 Cells

Glioma is a type of brain tumor that is common globally, and is associated with a variety of genetic changes. It has been reported that isocitrate dehydrogenase 1 (IDH1) is overexpressed in glioma and in HeLa cells. The lncRNA IDH1-AS1 is believed to interact with IDH1, and when IDH1-AS1 is overexpressed, HeLa cell proliferation is inhibited. However, the effects of IDH1-AS1 on glioma were relatively unknown. The results from this work show that IDH1-AS1 is downregulated in the glioma tissues. We used primary glioblastoma cell lines U251 and U87-MG to study the effects of IDH1-AS1 on glioma cell growth, in vitro and in vivo. We found that when IDH1-AS1 is overexpressed cell proliferation is inhibited, cell cycle is arrested at the G1 phase, and the protein expression levels of cyclinD1, cyclinA, cyclinE, CDK2, and CDK4 are decreased. We found that cell apoptosis was increased when IDH1-AS1 was overexpressed, as evidenced by increases in the levels of cleaved caspase-9 and -3. Conversely, knockdown of IDH1-AS1 promoted cell proliferation. Moreover, we proved that overexpression of IDH1-AS1 inhibits the tumorigenesis of U251 cells, in vivo. Furthermore, IDH1-AS1 did not affect IDH1 protein expression, but altered its enzymatic activities in glioma cells. Silencing of IDH1 reversed the effects of IDH1-AS1 upregulation on cell viability. Hence, our study provides first-hand evidence for the effects of lncRNA IDH1-AS1 on gliomas. Because overexpressing IDH1-AS1 inhibited cell growth, IDH1-AS1 could also be considered as a potential target for glioma treatment.

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Monkeypox Transmission, Need and Treatment of Humans with an Antiviral Drug

International Journal of Social Sciences and Management ◽

10.3126/ijssm.v4i2.16684 ◽

2017 ◽

Vol 4 (2) ◽

pp. 77-79

Author(s):

Chanda Jabeen ◽

Gulshan Umbreen

Keyword(s):

Direct Contact ◽

Infected Animal ◽

Antiviral Drug ◽

Zoonotic Disease ◽

Genetic Changes ◽

The Family

Monkeypox is a viral zoonotic disease belongs to the family Poxviridae and Orthopoxvirus genus. Transmission of monkey pox is through direct contact with infected animal and blood. Human to human transmission occur through respiratory route but previously so many studies are conducted to prove that monkey pox viruse was not transmitted through the respiratory route both in animals and humans. But now monkey pox is able to survive in humans due to genetic changes and human to human transmission is possible. Because it can be used as bioweapon, So there is a great need of having an antiviral drug which is effective against monkey pox virus.ST 246 proved effective in vivo and in vitro in infected animals and trials done safely on non-infected humans but no data is available about the effectiveness of ST 246 on monkey pox or Orthopox infected human treated with ST 246.Int. J. Soc. Sc. Manage. Vol. 4, Issue-2: 77-79

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Differential Gene Expression Patterns ofYersinia pestisandYersinia pseudotuberculosisduring Infection and Biofilm Formation in the Flea Digestive Tract

mSystems ◽

10.1128/msystems.00217-18 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 8

Author(s):

Iman Chouikha ◽

Daniel E. Sturdevant ◽

Clayton Jarrett ◽

Yi-Cheng Sun ◽

B. Joseph Hinnebusch

Keyword(s):

Digestive Tract ◽

Biofilm Formation ◽

Yersinia Pseudotuberculosis ◽

Expression Patterns ◽

Type Vi Secretion System ◽

Genetic Changes ◽

Content Type ◽

Biofilm Development

ABSTRACTYersinia pestis, the etiologic agent of plague, emerged as a fleaborne pathogen only within the last 6,000 years. Just five simple genetic changes in theYersinia pseudotuberculosisprogenitor, which served to eliminate toxicity to fleas and to enhance survival and biofilm formation in the flea digestive tract, were key to the transition to the arthropodborne transmission route. To gain a deeper understanding of the genetic basis for the development of a transmissible biofilm infection in the flea foregut, we evaluated additional gene differences and performedin vivotranscriptional profiling ofY. pestis, aY. pseudotuberculosiswild-type strain (unable to form biofilm in the flea foregut), and aY. pseudotuberculosismutant strain (able to produce foregut-blocking biofilm in fleas) recovered from fleas 1 day and 14 days after an infectious blood meal. Surprisingly, theY. pseudotuberculosismutations that increased c-di-GMP levels and enabled biofilm development in the flea did not change the expression levels of thehmsgenes responsible for the synthesis and export of the extracellular polysaccharide matrix required for mature biofilm formation. TheY. pseudotuberculosismutant uniquely expressed much higher levels ofYersiniatype VI secretion system 4 (T6SS-4) in the flea, and this locus was required for flea blockage byY. pseudotuberculosisbut not for blockage byY. pestis. Significant differences between the two species in expression of several metabolism genes, the Psa fimbrial genes, quorum sensing-related genes, transcription regulation genes, and stress response genes were evident during flea infection.IMPORTANCEY. pestisemerged as a highly virulent, arthropod-transmitted pathogen on the basis of relatively few and discrete genetic changes fromY. pseudotuberculosis. Parallel comparisons of thein vitroandin vivotranscriptomes ofY. pestisand twoY. pseudotuberculosisvariants that produce a nontransmissible infection and a transmissible infection of the flea vector, respectively, provided insights into howY. pestishas adapted to life in its flea vector and point to evolutionary changes in the regulation of metabolic and biofilm development pathways in these two closely related species.

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Synthetic Life with Alternative Nucleic Acids as Genetic Materials

Molecules ◽

10.3390/molecules25153483 ◽

2020 ◽

Vol 25 (15) ◽

pp. 3483

Author(s):

Peng Nie ◽

Yanfen Bai ◽

Hui Mei

Keyword(s):

Nucleic Acids ◽

Genetic Information ◽

Structural Parameters ◽

Living Cells ◽

Information Storage ◽

Chemically Modified ◽

Synthetic Life ◽

Living Organisms

DNA, the fundamental genetic polymer of all living organisms on Earth, can be chemically modified to embrace novel functions that do not exist in nature. The key chemical and structural parameters for genetic information storage, heredity, and evolution have been elucidated, and many xenobiotic nucleic acids (XNAs) with non-canonical structures are developed as alternative genetic materials in vitro. However, it is still particularly challenging to replace DNAs with XNAs in living cells. This review outlines some recent studies in which the storage and propagation of genetic information are achieved in vivo by expanding genetic systems with XNAs.

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