scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

240 QUALITY AND VIABILITY OF IVF BOVINE EMBRYOS AFTER INTRACYTOPLASMIC INJECTION OF DNA–LIPOSOME COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 232
Author(s):  
L. N. Moro ◽  
G. Vichera ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Dna Fragmentation ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Egfp Expression ◽  
Bovine Embryos ◽  
Exogenous Dna ◽  
Intracytoplasmic Injection

Transgenic animals have important applications in agriculture and human medicine; nevertheless the available techniques still remain inefficient and technically difficult. We have recently developed a novel method to transfect bovine embryos that consists of intracytoplasmic injection of exogenous DNA–liposome complexes (eDNA-LC) in IVF zygotes. This study was designed to evaluate the quality and viability of IVF bovine embryos, after intracytoplasmic injection of pCX-EGFP–liposome complexes (EGFP-LC) or pBCKIP2.8-liposome complexes (plasmid that codifies the human insulin gene, HI-LC). First, we evaluated embryo development and enhanced green fluorescent protein (EGFP) expression of IVF embryos injected with both plasmids separately. This treatment was analysed by Fisher's Exact test (P ≤ 0.05). Cleavage rates for EGFP-LC, HI-LC and IVF embryos injected with liposomes alone (IVF-L) and IVF control (IVF-C) were 62% (63/102), 67% (67/100), 66% (67/101) and 79% (98/124); blastocysts rates were 17% (17/102), 21% (21/100), 21% (21/101) and 23% (28/124), respectively. No statistical differences were seen among groups. The percentage of EGFP-positive embryos (EGFP+) after EGFP-LC injection was 42.9% after 3 days of culture and 41.8% at the blastocyst stage. In the second experiment, the blastocysts obtained, EGFP+ or EGFP-negative (EGFP–), were analysed by TUNEL assay at Day 6 (Bd6), 7 (Bd7) and 8 (Bd8) of in vitro culture, in order to evaluate the effect of the transgene and culture length, on DNA fragmentation. This treatment was analysed by the difference of proportions test (P ≤ 0.05) using statistical INFOSTAT software. All EGFP+ blastocysts showed TUNEL positive cells (T+). The percentage of T+ in Bd6, Bd7 and Bd8 were 91, 73.7 and 99.5%, respectively (P ≤ 0.05). EGFP– blastocysts showed lower fragmented nuclei (0, 44.6 and 85%, respectively; P ≤ 0.05). Groups IVF-L and IVF-C were also evaluated. In both groups, there was no evidence of DNA fragmentation in Bd6 and Bd7, but T+ were detected in Bd8 (66.4 and 85.8%, respectively; P ≤ 0.05). In the third experiment, bovine blastocysts obtained from the HI-LC group were individually transferred to recipient cows after 6 (n = 11), 7 (n = 5) and 8 (n = 5) days of culture post-IVF and HI-LC injection. The pregnancies obtained were from Bd6 [18.2% (2/11)] and Bd7 [40% (2/5)], although none of the recipients receiving Bd8 were diagnosed pregnant. Two pregnancies developed to term, one derived from Bd6 and the other from Bd7. Analysis by PCR determined that none of the born cows were transgenic. In summary, IVF bovine embryos could be easily transfected after the injection of eDNA-LC and the technique did not affect offspring viability. The results indicate that extended time in in vitro culture increases the percentage of fragmented nuclei in blastocysts. Moreover, this parameter increases in blastocysts with transgene expression compared with those without expression. Finally, more transfers are required in order to obtain the real efficiency of this new technique and to overcome the drawbacks generated by in vitro culture length and transgene expression.

113. DEVELOPMENT OF A METHOD OF SEMINIFEROUS TUBULE TRANSFECTION IN VITRO TO DEFINE POSTMEIOTIC GENE REGULATION

2009 ◽  
Vol 21 (9) ◽  
pp. 32
Author(s):  
S. Danner ◽  
C. Kirchhoff ◽  
R. Ivell
Keyword(s):  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Cell Types ◽  
Seminiferous Tubules ◽  
Gene Promoters ◽  
Cell Type ◽  
Cell Type Specificity ◽  
High Throughput Analysis ◽  
Apparent Lack

Postmeiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. Here we report the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation. Culture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment. Successful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals. We have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.

Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 324-326 ◽  
Author(s):  
Xiaohong Mao ◽  
Yuko Fujiwara ◽  
Aimée Chapdelaine ◽  
Haidi Yang ◽  
Stuart H. Orkin
Keyword(s):  
Fluorescent Protein ◽  
Threshold Level ◽  
Egfp Expression ◽  
Mouse Strains ◽  
Reporter Strain ◽  
Egfp Gene ◽  
Reporter Mouse ◽  
Functional Studies

Abstract Reporter mouse strains are important tools for monitoring Cre recombinase-mediated excision in vivo. In practice, excision may be incomplete in a given population due to threshold level or variegated expression of Cre. Hence, it is desirable in many experimental contexts to isolate cells that have undergone excision to assess the consequences of gene ablation. To generate alternative reporter mice, an enhanced green fluorescent protein (EGFP) gene was targeted to the retroviral-trapped ROSA26 locus. Upon Cre-mediated excision of “Stop” sequences, EGFP was expressed ubiquitously during embryogenesis and in adult tissues (including T cells, B cells, and myeloid cells). Using this new reporter strain, separation of excised from nonexcised cells in vitro was achieved in thymocytes in a noninvasive manner based on activated EGFP expression. This new EGFP reporter strain should facilitate a variety of conditional gene-targeting experiments, including the functional studies of hematopoietic cells in lineage-specific knockout mice.

84 PAIRS OF BLASTOMERES FROM BOVINE DAY 5 MORULAE ARE ABLE TO CONTRIBUTE TO INNER CELL MASS AND TROPHECTODERM IN CHIMERIC EMBRYOS GENERATED BY AGGREGATION WITH TWO DAY 4 MORULAE

2015 ◽  
Vol 27 (1) ◽  
pp. 135
Author(s):  
K. Simmet ◽  
M. Reichenbach ◽  
S. Jung ◽  
R. Fries ◽  
T. Grupp ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Inner Cell Mass ◽  
Ex Vivo ◽  
Standard Procedure ◽  
Cell Mass ◽  
Breeding Value ◽  
Egfp Expression ◽  
Promising Alternative ◽  
Donor Embryo

The multiplication of high-value embryos by chimera formation using asynchronic aggregation is a promising alternative to embryonic cell nuclear transfer. Single blastomeres from a donor embryo are aggregated with 2 host embryos, thus several chimeras can be constructed per donor embryo. Due to the advanced developmental stage, the donor blastomeres are likely to contribute to the inner cell mass (ICM) and later give rise to the embryo proper, whereas the host embryos form extra-embryonic tissues. To test if pairs of blastomeres from Day 5 morulae are able to form the ICM when aggregated with 2 Day 4 host embryos, we produced transgenic donor embryos carrying a fluorescent reporter gene (enhanced green fluorescent protein, eGFP) by using semen from an eGFP transgenic bull (Reichenbach et al. 2010 Transgenic Res. 19, 549–556) for in vitro fertilization and in vitro host embryos produced by a standard procedure. The zona pellucida of all embryos was removed by treatment with 1 mg mL–1 pronase. Donor embryos were assessed for eGFP expression by fluorescence microscopy and disaggregated by gentle pipetting after incubation in Mg2+- and Ca2+-free medium. Pairs of blastomeres were then placed between 2 host embryos and cultured individually in a well-of-the-well culture dish. On Day 6 after aggregation, fully developed blastocysts were assessed for eGFP fluorescence. In 3 replicates, n = 30 chimeras were produced by aggregation; 13 (43%) developed to blastocysts, of which 2 (15%) showed local eGFP expression in the ICM and 7 (54%) showed a generalized expression. From the results of this study we conclude that Day 5 morulae may be multiplied in an efficient manner by using the chimera formation technique, which makes this approach applicable to ex vivo-derived embryos. In future investigations we will study the effect of using donor blastomeres from either the inside or outside of the donor morula and test the use of tetraploid host embryos to increase the rate of blastocysts with the desired genotype in the ICM. Finally, we aim to introduce this multiplication approach to the production of genotyped embryos with a genomic estimated breeding value (gEBV) and intend to produce calves with identical gEBV.Funded by the Bavarian Research Foundation (AZ-1031–1).

scholarly journals OP0001 BARICITINIB, TOFACITINIB, UPADACITINIB, FILGOTINIB, AND CYTOKINE SIGNALING IN HUMAN LEUKOCYTE SUBPOPULATIONS: AN UPDATED EX-VIVO COMPARISON

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1-2
Author(s):  
I. Mcinnes ◽  
G. Rocha ◽  
R. E. Higgs ◽  
D. Dairaghi ◽  
T. Wehrman ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Human Leukocyte ◽  
Parent Drug ◽  
Cell Types ◽  
Vertical Line ◽  
Cytokine Signaling ◽  
Cell Type ◽  
Research Support

Background:everal JAKi are now used for the treatment of RA; approved doses include baricitinib (bari) 2- and/or 4-mg QD, tofacitinib (tofa) 5-mg BID, upadacitinib (upa) 15-mg QD. The JAK selectivity these agents is proposed to vary across the class.Objectives:In vitro cellular pharmacology of bari to tofa, upa, and filgotinib (filgo) were compared.Methods:PBMCs from 6 healthy donors were incubated with the JAKis over a 7- to 8-point concentration range. Following cytokine stimulation, levels of pSTAT were measured and IC50 calculated in gated leukocyte subpopulations. Therapeutic dose relevance was assessed using calculated mean concentration-time (CT) profiles over 24 hours for bari 2- and 4-mg QD; tofa 5- and 10-mg BID; upa 15- and 30-mg QD; filgo 100- and 200-mg QD. Average daily % inhibition of pSTAT (%SI) was calculated for each JAKi, cytokine, and cell type; filgo %SI integrated parent drug + metabolite.Results:The cytokines did not signal in all cell types (Figure 1). When signaling was detected, IC50 and %SI for a particular JAKi were generally similar across cell types, with dose-dependent inhibition (Figures 1 & 2). Based on IC50s, upa was most and filgo/metabolite least potent across JAK2/2 or JAK2/TYK2-dependent (IL-3, GM-CSF, G-CSF), JAK1/3-dependent (IL-2, 4, 15, 21), and JAK1/2/TYK2 dependent (IL-6 & 10, IFN-α & γ) signaling pathways.Figure 1.IC50 values (nM) for baricitinib, tofacitinib, upadacitinib, filgotinib (parent and metabolite) in cytokine-stimulated human PBMC preparations. *p<0.01, **p<0.001, ***p<0.0001 vs. bari.Incorporating CT profiles, no agent potently or continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. Comparing bari 4-mg to tofa 5-mg BID, upa 15-mg QD, and filgo 100-mg QD, %SI of JAK2/2 or JAK2/TYK2-dependent cytokines was highest with bari 4-mg and upa. Inhibition of JAK1/2/TYK2 cytokines was highest with bari 4-mg. Inhibition of JAK2/2 or JAK2/TYK2, and of JAK1/3-dependent cytokines was lowest for filgo 100-mg QD. For bari 2-mg QD vs. these other JAKi doses, %SI of JAK2/2 or JAK2/TYK2 was highest with upa followed by bari 2-mg. The highest inhibitors of the JAK1/2/TYK2-dependent cytokines varied by cytokine / cell type though consistently included upa. Inhibition of JAK1/3 was highest for upa and tofa. Across comparisons, filgo 100-mg QD showed the least %SI of JAK2/2 or JAK2/TYK2-dependent, and of JAK1/3-dependent cytokines. Filgo reached the highest levels of %SI among agents only for 200-mg QD vs. lower doses of the other JAKi (for selected JAK1/2/TYK2-dependent cytokines).Conclusion:JAKis display different in vitro pharmacologic profiles which, coupled to their in vivo pharmacokinetics, suggest they modulate distinct cytokine pathways to differing degrees and durations over 24 hours. Ex vivo whole cell assays seem distinct from cell free kinase inhibition assays in determining the overall cytokine modulatory potential of members of the JAKi class.References:[1]McInnes IB, et al. Arthritis Res Ther. 2019 Aug 2;21(1):183Figure 2.Baricitinib, tofacitinib, upadacitinib, filgotinib: calculated average percent daily STAT inhibition for selected cytokines. -p<0.01, --p<0.001, ---p<0.0001 significantly lower compared to bari (vs. 2-mg if left of vertical line “|”, vs. 4-mg if right of vertical line “|”). +p<0.01, ++p<0.001, +++p<0.0001 significantly higher compared to bari (vs. 2-mg if left of vertical line “|”, vs. 4-mg if right of vertical line “|”).Disclosure of Interests:Iain McInnes Grant/research support from: Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Janssen, and UCB, Consultant of: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Gilead, Janssen, Novartis, Pfizer, and UCB, Guilherme Rocha Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Richard E Higgs Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Daniel Dairaghi Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Thomas Wehrman Shareholder of: An insignificant amount in AbbVie as part of a larger portfolio, Consultant of: Primity Bio Inc. works with many pharmaceutical and biotech companies and provides CRO services., Evan Wang Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Zhang Xin Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Jorge Ross Terres Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Terence Rooney Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Peter C. Taylor Grant/research support from: Celgene, Eli Lilly and Company, Galapagos, and Gilead, Consultant of: AbbVie, Biogen, Eli Lilly and Company, Fresenius, Galapagos, Gilead, GlaxoSmithKline, Janssen, Nordic Pharma, Pfizer Roche, and UCB

scholarly journals Effect of mRNA Delivery Modality and Formulation on Cutaneous mRNA Distribution and Downstream eGFP Expression

Pharmaceutics ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 151
Author(s):  
Aditya R. Darade ◽  
Maria Lapteva ◽  
Thomas Hoffmann ◽  
Markus Mandler ◽  
Achim Schneeberger ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Intradermal Injection ◽  
Laser Scanning Microscopy ◽  
Egfp Expression ◽  
Confocal Laser ◽  
Skin Sample ◽  
Messenger Ribonucleic Acid

In vitro transcribed messenger ribonucleic acid (mRNA) constitutes an emerging therapeutic class with several clinical applications. This study presents a systematic comparison of different technologies—intradermal injection, microneedle injection, jet injection, and fractional laser ablation—for the topical cutaneous delivery of mRNA. Delivery of Cy5 labeled mRNA and non-labeled enhanced green fluorescent protein (eGFP) expressing mRNA was investigated in a viable ex vivo porcine skin model and monitored for 48 h. Forty 10 µm-thick horizontal sections were prepared from each skin sample and Cy5 labeled mRNA or eGFP expression visualized as a function of depth by confocal laser scanning microscopy and immunohistochemistry. A pixel-based method was used to create a semi-quantitative biodistribution profile. Different spatial distributions of Cy5 labeled mRNA and eGFP expression were observed, depending on the delivery modality; localization of eGFP expression pointed to the cells responsible. Delivery efficiencies and knowledge of delivery sites can facilitate development of efficient, targeted mRNA-based therapeutics.

scholarly journals Astrocytes Exhibit a Protective Role in Neuronal Firing Patterns under Chemically Induced Seizures in Neuron–Astrocyte Co-Cultures

2021 ◽  
Vol 22 (23) ◽  
pp. 12770
Author(s):  
Annika Ahtiainen ◽  
Barbara Genocchi ◽  
Jarno M. A. Tanskanen ◽  
Michael T. Barros ◽  
Jari A. K. Hyttinen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Microelectrode Arrays ◽  
Cell Types ◽  
Protective Role ◽  
Neuronal Firing ◽  
Neuronal Homeostasis ◽  
Chemically Induced ◽  
Rat Cortical Neurons

Astrocytes and neurons respond to each other by releasing transmitters, such as γ-aminobutyric acid (GABA) and glutamate, that modulate the synaptic transmission and electrochemical behavior of both cell types. Astrocytes also maintain neuronal homeostasis by clearing neurotransmitters from the extracellular space. These astrocytic actions are altered in diseases involving malfunction of neurons, e.g., in epilepsy, Alzheimer’s disease, and Parkinson’s disease. Convulsant drugs such as 4-aminopyridine (4-AP) and gabazine are commonly used to study epilepsy in vitro. In this study, we aim to assess the modulatory roles of astrocytes during epileptic-like conditions and in compensating drug-elicited hyperactivity. We plated rat cortical neurons and astrocytes with different ratios on microelectrode arrays, induced seizures with 4-AP and gabazine, and recorded the evoked neuronal activity. Our results indicated that astrocytes effectively counteracted the effect of 4-AP during stimulation. Gabazine, instead, induced neuronal hyperactivity and synchronicity in all cultures. Furthermore, our results showed that the response time to the drugs increased with an increasing number of astrocytes in the co-cultures. To the best of our knowledge, our study is the first that shows the critical modulatory role of astrocytes in 4-AP and gabazine-induced discharges and highlights the importance of considering different proportions of cells in the cultures.

scholarly journals A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals

Function ◽  
2021 ◽  
Vol 2 (3) ◽  
Author(s):  
Nelly Redolfi ◽  
Elisa Greotti ◽  
Giulia Zanetti ◽  
Tino Hochepied ◽  
Cristina Fasolato ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Brain Slices ◽  
Resonance Energy ◽  
Mouse Line ◽  
Isolated Cells ◽  
Genomic Locus ◽  
Transgenic Mouse Line

AbstractMitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

scholarly journals In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1483
Author(s):  
Emily A. Bates ◽  
John R. Counsell ◽  
Sophie Alizert ◽  
Alexander T. Baker ◽  
Natalie Suff ◽  
...  
Keyword(s):  
Viral Vector ◽  
Ex Vivo ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Cell Types ◽  
In Vivo Evaluation ◽  
In Vivo Biodistribution ◽  
Adenovirus Type 5

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

scholarly journals The Carbamate, Physostigmine does not Impair Axonal Transport in Rat Cortical Neurons

2021 ◽  
Vol 16 ◽  
pp. 263310552110202
Author(s):  
Sean X Naughton ◽  
Wayne D Beck ◽  
Zhe Wei ◽  
Guangyu Wu ◽  
Peter W Baas ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Cortical Neurons ◽  
Acetylcholinesterase Inhibitors ◽  
Neurological Deficits ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rat Cortical Neurons ◽  
Toxicological Mechanism

Among the various chemicals that are commonly used as pesticides, organophosphates (OPs), and to a lesser extent, carbamates, are most frequently associated with adverse long-term neurological consequences. OPs and the carbamate, pyridostigmine, used as a prophylactic drug against potential nerve agent attacks, have also been implicated in Gulf War Illness (GWI), which is often characterized by chronic neurological symptoms. While most OP- and carbamate-based pesticides, and pyridostigmine are relatively potent acetylcholinesterase inhibitors (AChEIs), this toxicological mechanism is inadequate to explain their long-term health effects, especially when no signs of acute cholinergic toxicity are exhibited. Our previous work suggests that a potential mechanism of the long-term neurological deficits associated with OPs is impairment of axonal transport (AXT); however, we had not previously evaluated carbamates for this effect. Here we thus evaluated the carbamate, physostigmine (PHY), a highly potent AChEI, on AXT using an in vitro neuronal live imaging assay that we have previously found to be very sensitive to OP-related deficits in AXT. We first evaluated the OP, diisopropylfluorophosphate (DFP) (concentration range 0.001-10.0 µM) as a reference compound that we found previously to impair AXT and subsequently evaluated PHY (concentration range 0.01-100 nM). As expected, DFP impaired AXT in a concentration-dependent manner, replicating our previously published results. In contrast, none of the concentrations of PHY (including concentrations well above the threshold for impairing AChE) impaired AXT. These data suggest that the long-term neurological deficits associated with some carbamates are not likely due to acute impairments of AXT.

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