scholarly journals The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

2021 ◽  
Author(s):  
Pavithra Aravamudhan ◽  
Camila Guzman-Cardozo ◽  
Kelly Urbanek ◽  
Olivia Welsh ◽  
Jennifer Konopka-Anstadt ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Target Cells ◽  
Viral Attachment ◽  
Genetic Ablation ◽  
Murine Homolog ◽  
Intramuscular Inoculation ◽  
Neural Tissues ◽  
Mammalian Orthoreovirus ◽  
The Brain

Engagement of host receptors is essential for viruses to enter target cells and initiate infection. Expression patterns of receptors in turn dictate host and tissue tropism and disease pathogenesis during infection. Mammalian orthoreovirus (reovirus) displays serotype-dependent patterns of tropism in the murine central nervous system (CNS) that are dictated by viral attachment protein σ1. However, the receptor that mediates reovirus CNS tropism is unknown. Two proteinaceous receptors have been identified for reovirus, junctional adhesion molecule-A (JAM-A) and Nogo 66 receptor 1 (NgR1). Engagement of JAM-A is required for reovirus hematogenous dissemination but is dispensable for neural spread. To determine whether NgR1 functions in reovirus neuropathogenesis, we compared virus replication and disease following inoculation of wild-type (WT) and NgR1-/- mice. Genetic ablation of NgR1 did not alter replication of neurotropic reovirus strain T3SA- in the intestine and transmission to the brain following peroral inoculation. Viral titers in neural tissues following intramuscular inoculation, which provides access to neural dissemination routes, also were comparable in WT and NgR1-/- mice, suggesting that NgR1 is dispensable for reovirus neural spread to the CNS. The absence of both NgR1 and JAM-A also did not alter replication, neural tropism, and virulence of T3SA- following direct intracranial inoculation. In agreement with these findings, we found that the human but not the murine homolog of NgR1 functions as a receptor and confers efficient reovirus binding and infection of nonsusceptible cells in vitro. These results eliminate functions for JAM-A and NgR1 in shaping CNS tropism in mice and suggest that other receptors, yet to be identified, support this function.

scholarly journals Effects of Melatonin on Peripheral Reproductive Function: Regulation of Testicular GnIH and Testosterone

Endocrinology ◽  
2011 ◽  
Vol 152 (9) ◽  
pp. 3461-3470 ◽  
Author(s):  
Nicolette L. McGuire ◽  
Kristina Kangas ◽  
George E. Bentley
Keyword(s):  
Expression Patterns ◽  
Reproductive Function ◽  
Melatonin Receptors ◽  
Sex Steroid ◽  
European Starlings ◽  
Steroid Secretion ◽  
Local Inhibition ◽  
Gonadotropin Inhibitory Hormone ◽  
The Brain

Study of seasonal reproduction has focused on the brain. Here, we show that the inhibition of sex steroid secretion can be seasonally mediated at the level of the gonad. We investigate the direct effects of melatonin on sex steroid secretion and gonadal neuropeptide expression in European starlings (Sturnus vulgaris). PCR reveals starling gonads express mRNA for gonadotropin inhibitory hormone (GnIH) and its receptor (GnIHR) and melatonin receptors 1B (Mel 1B) and 1C (Mel 1C). We demonstrate that the gonadal GnIH system is regulated seasonally, possibly via a mechanism involving melatonin. GnIH/ GnIHR expression in the testes is relatively low during breeding compared with outside the breeding season. The expression patterns of Mel 1B and Mel 1C are correlated with this expression, and melatonin up-regulates the expression of GnIH mRNA in starling gonads before breeding. In vitro, GnIH and melatonin significantly decrease testosterone secretion from LH/FSH-stimulated testes before, but not during, breeding. Thus local inhibition of sex steroid secretion appears to be regulated seasonally at the level of the gonad, by a mechanism involving melatonin and the gonadal GnIH system.

Neurovirulence of the UC-2 and UC-8 Strains of Bluetongue Virus Serotype 11 in Newborn Mice

1987 ◽  
Vol 24 (5) ◽  
pp. 404-410 ◽  
Author(s):  
A. S. Waldvogel ◽  
C. A. Anderson ◽  
R. J. Higgins ◽  
B. I. Osburn
Keyword(s):  
Bluetongue Virus ◽  
Immunofluorescent Staining ◽  
Target Cells ◽  
Neural Cells ◽  
Inoculation Site ◽  
Virus Serotype ◽  
The Difference ◽  
The Brain ◽  
Subcutaneous Inoculation

In vivo and in vitro experiments were done to investigate whether the difference in neurovirulence between the two strains of bluetongue virus 11, UC-2 and UC-8, is based on a different capability to gain access to the brain from the subcutaneous inoculation site or on a different tropism for neural cells. In newborn Balb/c mice subcutaneous inoculation of UC-8 at doses between 10−0.2 plaque forming units (PFU) and 104.8 PFU caused a severe necrotizing encephalitis whereas UC-2 at doses of up to 104.4 PFU did not affect newborn Balb/c mice. However, intracranial inoculation of 102.4 PFU of either virus strain produced severe necrotizing encephalitis. In vitro both virus strains infected dissociated brain cell cultures similarly. Double labelling immunofluorescent staining with markers specific for neural cells did not reveal differences in the target cells for the two viruses. The difference in neurovirulence between UC-2 and UC-8, therefore, appears to be determined by the ability of UC-8 to infect the brain from a subcutaneous inoculation site.

scholarly journals Peripheral clocks and their role in circadian timing: insights from insects

2001 ◽  
Vol 356 (1415) ◽  
pp. 1791-1799 ◽  
Author(s):  
Jadwiga M. Giebultowicz
Keyword(s):  
Feedback Loop ◽  
Clock Genes ◽  
Expression Patterns ◽  
Isolated Organs ◽  
Peripheral Organs ◽  
Peripheral Clocks ◽  
Molecular Components ◽  
The Brain

Impressive advances have been made recently in our understanding of the molecular basis of the cell–autonomous circadian feedback loop; however, much less is known about the overall organization of the circadian systems. How many clocks tick in a multicellular animal, such as an insect, and what are their roles and the relationships between them? Most attempts to locate clock–containing tissues were based on the analysis of behavioural rhythms and identified brain–located timing centres in a variety of animals. Characterization of several essential clock genes and analysis of their expression patterns revealed that molecular components of the clock are active not only in the brain, but also in many peripheral organs of Drosophila and other insects as well as in vertebrates. Subsequent experiments have shown that isolated peripheral organs can maintain self–sustained and light sensitive cycling of clock genes in vitro . This, together with earlier demonstrations that physiological output rhythms persist in isolated organs and tissues, provide strong evidence for the existence of functionally autonomous local circadian clocks in insects and other animals. Circadian systems in complex animals may include many peripheral clocks with tissue–specific functions and a varying degree of autonomy, which seems to be correlated with their sensitivity to external entraining signals.

scholarly journals GAPDH controls extracellular vesicle biogenesis and enhances therapeutic potential of EVs in silencing the Huntingtin gene in mice via siRNA delivery

2020 ◽  
Author(s):  
Ghulam Hassan Dar ◽  
Cláudia C. Mendes ◽  
Wei-Li Kuan ◽  
Mariana Conceição ◽  
Samir El-Andaloussi ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Therapeutic Potential ◽  
Sirna Delivery ◽  
Extracellular Vesicle ◽  
Glycolytic Enzyme ◽  
Target Cells ◽  
Binding Motif ◽  
Systemic Injection ◽  
The Brain

AbstractExtracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication and pathophysiology. Their capacity to transfer biomolecules between cells has sparked efforts to bioengineer EVs as drug delivery vehicles. However, a better understanding of EV biogenesis mechanisms and function is required to unleash their considerable therapeutic potential. Here we demonstrate a novel role for GAPDH, a glycolytic enzyme, in EV assembly and secretion, and we exploit these findings to develop a GAPDH-based methodology to load therapeutic siRNAs onto EVs for targeted drug delivery to the brain. In a series of experiments, we observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif, designated as G58, and discover that the tetrameric nature of GAPDH promotes extensive EV aggregation. Studies in a Drosophila EV biogenesis model demonstrate that GAPDH is absolutely required for normal generation of intraluminal vesicles in endosomal compartments and promotes vesicle clustering both inside and outside the cell. Fusing a GAPDH-derived G58 peptide to dsRNA-binding motifs permits highly efficient loading of RNA-based drugs such as siRNA onto the surface of EVs. Such vesicles efficiently deliver siRNA to target cells in vitro and into the brain of a Huntington’s disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in multiple anatomical regions of the brain and modulation of phenotypic features of disease. Taken together, our study demonstrates a novel role for GAPDH in EV biogenesis, and that the presence of free GAPDH binding sites on EVs can be effectively exploited to substantially enhance the therapeutic potential of EV-mediated drug delivery to the brain.

Phenomic Screen in Vivo and in Vitro to Explore Novel Pathogenesis of AML1-ETO-Positive Leukemia Using PSC-Derived Hematopoietic Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2370-2370
Author(s):  
Akira Niwa ◽  
Akitsu Hotta ◽  
Megumu K Saito ◽  
Tatsutoshi Nakahata
Keyword(s):  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Hematopoietic Cells ◽  
Genetic Alterations ◽  
Positive Control ◽  
Target Cells ◽  
Myeloid Lineage ◽  
Hematopoietic Stem

Abstract Onset of acute myeloid leukemia (AML) has been accounted for by cooperation between multiple genetic alterations. Among the previously listed leukemogenic lesions, AML1-ETO fusion (AE) generated by translocation (8;21) (q22;q22) is one of the common mutations observed in 20-40% of patients. AE affects transcriptional regulation associated with hematopoietic differentiation, while 60% of AE-positive AML cases are shown to have together other types of mutation of genes involved in cell proliferation, such as receptor tyrosine kinase (RTK) c-kit and FLT3. Those data are compatible with so-called “multi-step leukemogenesis” model. At the same time, variety in clinical phenotypes even among patients harboring same sets of mutation strongly indicates the yet unknown “cooperative ” cues in genetics or epigenetics. From this viewpoint, finely elucidating “What kinds of such mechanisms” give “What kinds of impact associating with AE fusion” on “What kinds of target cells” during leukemogenesis is important. In order to address those problems in reproducible manner, we developed the novel phenomic screen system by combining reverse and forward genetic modifications with pluripotent stem cell (PSC)-derived hematopoietic culture. We induced the PiggyBac-based random knockdown motifs into PSC-derived hematopoietic cells that harbour artificial expression cassettes for AE fusion gene, then applied them for in vitro and in vivo assays. We performed these platform-based trials in parallel to the work with positive control cells carrying AE in addition to c-kit or Flt3-ITD mutations, recently identified poor prognosis conveyers among AE-positive AML. First, serial replanting assays in methylcellulose-containing semisolid media as well as liquid culture revealed that a few numbers of clones acquired significantly reinforced potential in cell growth and colony forming efficacy. As for positive control cells, they showed reproducibly strong tendency toward both increased colony forming efficacy and suppressed differentiation. Those results suggested the successful recapitulation of pathogenic cooperation between AE and RTK mutations in our PSC-derived hematopoietic cells, and confirmed the feasibility of our screening system. Interestingly, AE-positive myeloid lineage-committed progenitors as well as immature multipotent hematopoietic stem and progenitor cells (HSPCs) showed higher replating colony forming efficacy, which may indicate the representation of higher incidence of myeloblastic leukemia in AE-positive AML. We are identifying the knocked down genes and evaluating the perturbation of expression patterns of genes involved in differentiation, proliferation and survival as well as epigenetics associating genes within affected cells. In addition to in vitro assays, we next evaluated the feasibility of in vivo phenomic screening by performing intra bone marrow transplantation of gene modified PSC-derived progenitors into immunodeficient NOD/Shi-scid,IL-2RγKO Jic(NOG) mice. Also in these experiments, we successfully observed the cooperation between AE and RTK mutations for prolonged engraftment in case HSPCs and myeloid lineage-committed progenitors were transplanted. After that, we are now performing these screenings to select the phenotype acquiring clones. In conclusion, we successfully established a novel phenomic screen in vivo and in vitro to explore novel pathogenesis of AE-positive leukemia using PSC-derived hematopoietic cells. After confirming the recapitulation of already known cooperation between AE and RTK mutation, we are currently extending the screening using this platform. We believe that our model must allow us to better match treatment to prognosis across the disease spectrum via comprehensive understanding of pathogenesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Combinatorial effects of odorants on mouse behavior

2016 ◽  
Vol 113 (23) ◽  
pp. E3300-E3306 ◽  
Author(s):  
Luis R. Saraiva ◽  
Kunio Kondoh ◽  
Xiaolan Ye ◽  
Kyoung-hye Yoon ◽  
Marcus Hernandez ◽  
...  
Keyword(s):  
Behavioral Effect ◽  
Predator Odor ◽  
Odorant Receptors ◽  
Odor Aversion ◽  
Genetic Ablation ◽  
Single Receptor ◽  
Trace Amine ◽  
Specific Receptors ◽  
The Brain

The mechanisms by which odors induce instinctive behaviors are largely unknown. Odor detection in the mouse nose is mediated by >1, 000 different odorant receptors (ORs) and trace amine-associated receptors (TAARs). Odor perceptions are encoded combinatorially by ORs and can be altered by slight changes in the combination of activated receptors. However, the stereotyped nature of instinctive odor responses suggests the involvement of specific receptors and genetically programmed neural circuits relatively immune to extraneous odor stimuli and receptor inputs. Here, we report that, contrary to expectation, innate odor-induced behaviors can be context-dependent. First, different ligands for a given TAAR can vary in behavioral effect. Second, when combined, some attractive and aversive odorants neutralize one another’s behavioral effects. Both a TAAR ligand and a common odorant block aversion to a predator odor, indicating that this ability is not unique to TAARs and can extend to an aversive response of potential importance to survival. In vitro testing of single receptors with binary odorant mixtures indicates that behavioral blocking can occur without receptor antagonism in the nose. Moreover, genetic ablation of a single receptor prevents its cognate ligand from blocking predator odor aversion, indicating that the blocking requires sensory input from the receptor. Together, these findings indicate that innate odor-induced behaviors can depend on context, that signals from a single receptor can block innate odor aversion, and that instinctive behavioral responses to odors can be modulated by interactions in the brain among signals derived from different receptors.

scholarly journals Hypersilencing of SRRM4 suppresses basal microexon inclusion and promotes tumor growth across cancers

2020 ◽  
Author(s):  
Sarah A. Head ◽  
Xavier Hernandez-Alias ◽  
Jae-Seong Yang ◽  
Violeta Beltran-Sastre ◽  
Antonio Torres-Méndez ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Neuronal Differentiation ◽  
Cancer Cell ◽  
Splicing Factor ◽  
Altered Expression ◽  
Intervention Point ◽  
Neural Tissues ◽  
The Brain ◽  
Low Expression

AbstractRNA splicing is widely dysregulated in cancer, frequently due to altered expression or activity of splicing factors. Microexons are extremely small exons (3-27 nucleotides long) that are highly evolutionarily conserved and play critical roles in promoting neuronal differentiation and development. Inclusion of microexons in mRNA transcripts is mediated by the splicing factor SRRM4, whose expression is largely restricted to neural tissues. However, microexons have been largely overlooked in prior analyses of splicing in cancer, as their small size necessitates specialized computational approaches for their detection. Here we demonstrate that despite having low expression in normal non-neural tissues, SRRM4 is hypersilenced in tumors, resulting in the suppression of basal microexon inclusion. Remarkably, SRRM4 is the most consistently silenced splicing factor across all tumor types analyzed, implying a general advantage of microexon downregulation in cancer independent of its tissue of origin. We show that this silencing is favorable for tumor growth, as decreased SRRM4 expression in tumors is correlated with an increase in mitotic gene expression, and upregulation of SRRM4 in cancer cell lines dose-dependently inhibits proliferation in vitro and in a mouse xenograft model. Further, this proliferation inhibition is accompanied by induction of neural-like expression and splicing patterns in cancer cells, suggesting that SRRM4 expression shifts the cell state away from proliferation and towards differentiation. We therefore conclude that SRRM4 acts as a proliferation brake, and tumors gain a selective advantage by cutting off this brake.SignificanceMicroexons are extremely small exons enriched in the brain that play important roles in neural development. Their inclusion is mediated by the splicing factor SRRM4, also predominantly expressed in the brain. Surprisingly, we find that low expression of SRRM4 outside of the brain is further decreased in tumors, and in fact SRRM4 is the most consistently silenced splicing factor in tumors across tissue types. We demonstrate that SRRM4 inhibits cancer cell proliferation in vitro and in vivo by inducing a neuronal differentiation program. Our findings add a new element to the overall picture of splicing dysregulation in cancer, reveal an antiproliferative function for SRRM4 and microexons outside of the brain, and may present a common therapeutic intervention point across cancer types.

An Abnormal Methylation Ratio Induces Hypomethylation In Vitro in the Brain of Pig and Man, But Not in Rat

1995 ◽  
Vol 88 (1) ◽  
pp. 73-79 ◽  
Author(s):  
M. McKeever ◽  
A. Molloy ◽  
D. G. Weir ◽  
P. B. Young ◽  
D. G. Kennedy ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
In Vitro Assay ◽  
Methyltransferase Activity ◽  
Vitro Assay ◽  
Neural Tissues ◽  
Protein Methyltransferases ◽  
The Brain ◽  
Methylation Ratio

1. The ratio of the methyl donor, S-adenosylmethionine, to the co-product, S-adenosylhomocysteine (the methylation ratio) is known to control the activity of methyltransferases in tissues. Inactivation of the vitamin B12-dependent enzyme, methionine synthase, reduces the methylation ratio in rats and pigs in vivo. 2. We have determined the effect that such alterations have on neural protein ‘O’ and ‘N’ methyltransferases using an in vitro assay in rats, pigs and humans in the presence of the normal methylation ratio and the abnormal methylation ratios found experimentally in vivo in rats and pigs. 3. The methylation ratio found in the neural tissues of vitamin B12-inactivated pigs significantly inhibits the protein methyltransferases of pigs and humans. 4. By contrast, the altered methylation ratio found in vitamin B12-inactivated rats only marginally inhibits the equivalent rat methyltransferases. 5. This is consistent with the induction of a myelopathy by such treatment in pigs and humans, but not in the rat. 6. Dietary supplements of methionine given to vitamin B12-inactivated pigs have been shown to prevent the myelopathy in vivo by both elevating the neural S-adenosylmethionine level and resetting the methylation ratio. We find in our in vitro assay that these events reinstate the methyltransferase activity to near normal levels, thus explaining its protective effect in vivo.

scholarly journals A Peptide-Based Nanocarrier for an Enhanced Delivery and Targeting of Flurbiprofen into the Brain for the Treatment of Alzheimer’s Disease: An In Vitro Study

Nanomaterials ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1590
Author(s):  
Shafq Al-azzawi ◽  
Dhafir Masheta ◽  
Anna Guildford ◽  
Gary Phillips ◽  
Matteo Santin
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
High Efficiency ◽  
Solid Phase ◽  
Amyloid Β ◽  
In Vitro Study ◽  
Target Cells ◽  
Age Related ◽  
The Brain

Alzheimer’s disease (AD) is an age-related disease caused by abnormal accumulation of amyloid-β in the brain leading to progressive tissue degeneration. Flurbiprofen (FP), a drug used to mitigate the disease progression, has low efficacy due to its limited permeability across the blood–brain barrier (BBB). In a previous work, FP was coupled at the uppermost branching of an ε-lysine-based branched carrier, its root presenting a phenylalanine moiety able to increase the hydrophobicity of the complex and enhance the transport across the BBB by adsorptive-mediated transcytosis (AMT). The present study explores a different molecular design of the FP-peptide delivery system, whereby its root presents an ApoE-mimicking peptide, a targeting ligand that could enhance transport across the BBB by receptor-mediated transcytosis (RMT). The functionalised complex was synthesised using a solid-phase peptide synthesis and characterised by mass spectrometry and FTIR. Cytotoxicity and permeability of this complex across an in vitro BBB model were analysed. Moreover, its activity and degradation to release the drug were investigated. The results revealed successful synthesis and grafting of FP molecules at the uppermost molecular branches of the lysine terminal without observed cytotoxicity. When covalently linked to the nanocarrier, FP was still active on target cells, albeit with a reduced activity, and was released as a free drug upon hydrolysis in a lysosome-mimicking medium. Noticeably, this work shows the high efficiency of RMT-driven FP delivery over delivery systems relying on AMT.

scholarly journals Identification, Expression, and Roles of the Cystine/Glutamate Antiporter in Ocular Tissues

2020 ◽  
Vol 2020 ◽  
pp. 1-10 ◽  
Author(s):  
Renita M. Martis ◽  
Luis J. Knight ◽  
Paul J. Donaldson ◽  
Julie C. Lim
Keyword(s):  
Knockout Mouse ◽  
Redox Balance ◽  
Ocular Tissues ◽  
Age Related ◽  
Neural Tissues ◽  
The Brain ◽  
Oxidative State ◽  
Shed Light

The cystine/glutamate antiporter (system xc-) is composed of a heavy chain subunit 4F2hc linked by a disulphide bond to a light chain xCT, which exchanges extracellular cystine, the disulphide form of the amino acid cysteine, for intracellular glutamate. In vitro research in the brain, kidney, and liver have shown this antiporter to play a role in minimising oxidative stress by providing a source of intracellular cysteine for the synthesis of the antioxidant glutathione. In vivo studies using the xCT knockout mouse revealed that the plasma cystine/cysteine redox couple was tilted to a more oxidative state demonstrating system xc- to also play a role in maintaining extracellular redox balance by driving a cystine/cysteine redox cycle. In addition, through import of cystine, system xc- also serves to export glutamate into the extracellular space which may influence neurotransmission and glutamate signalling in neural tissues. While changes to system xc- function has been linked to cancer and neurodegenerative disease, there is limited research on the roles of system xc- in the different tissues of the eye, and links between the antiporter, aging, and ocular disease. Hence, this review seeks to consolidate research on system xc- in the cornea, lens, retina, and ocular humours conducted across several species to shed light on the in vitro and in vivo roles of xCT in the eye and highlight the utility of the xCT knockout mouse as a tool to investigate the contribution of xCT to age-related ocular diseases.

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