scholarly journals Extracellular Vesicles promote host immunity during an M. tuberculosis infection through RNA Sensing

2018 ◽  
Author(s):  
Yong Cheng ◽  
Jeffery S. Schorey
Keyword(s):  
Extracellular Vesicles ◽  
Type I ◽  
Dependent Manner ◽  
Drug Resistant ◽  
Interferon Production ◽  
Bacterial Killing ◽  
Novel Approach ◽  
And Function ◽  
Dependent Pathway

AbstractExtracellular vesicles (EVs) have been shown to carry microbial components and function in the host defense against infections. In this study, we demonstrate that Mycobacterium tuberculosis (M.tb) RNA is delivered into macrophage-derived EVs through an M.tb SecA2-dependent pathway, and that EVs released from M.tb-infected macrophages stimulate a host RIG-I/MAVS/TBK1/IRF3 RNA sensing pathway, leading to type I interferon production in recipient cells. These EVs also promote, in a RIG-I/MAVS-dependent manner, the maturation of M.tb-containing phagosomes through a noncanonical LC3 modification, leading to increased bacterial killing. Moreover, treatment of M.tb-infected macrophages or mice with a combination of moxifloxacin and EVs, isolated from M.tb-infected macrophages, significantly lowered bacterial burden relative to either treatment alone. We propose that EVs, which are preferentially removed by macrophages in vivo, may be developed in combination with effective antibiotics as a novel approach to treat drug-resistant TB.

scholarly journals Detection of Spacer Precursors Formed In Vivo During Primed CRISPR Adaptation

2019 ◽  
Author(s):  
Anna A. Shiriaeva ◽  
Ekaterina Savitskaya ◽  
Kirill A. Datsenko ◽  
Irina O. Vvedenskaya ◽  
Iana Fedorova ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Type I ◽  
Dependent Manner ◽  
Dna Fragments ◽  
Crispr Array ◽  
Protospacer Adjacent Motif ◽  
Sequencing Method ◽  
Foreign Dna ◽  
And Function

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA1. Immunity involves “adaptation,” the integration of ~30-bp DNA fragments into the CRISPR array as “spacer” sequences, and “interference,” the targeted degradation of DNA containing a “protospacer” sequence mediated by a complex containing a spacer-derived CRISPR RNA (crRNA)1–4. Specificity for targeting interference to protospacers, but not spacers, occurs through recognition of a 3-bp protospacer adjacent motif (PAM)5 by the crRNA-containing complex6. Interference-driven DNA degradation of protospacer-containing DNA can be coupled with “primed adaptation,” ill which spacers are acquired from DNA surrounding the targeted protospacer in a bidirectional, orientation-dependent manner2,3,7. Here we construct a robust in vivo model for primed adaptation consisting of an Escherichia coli type I-E CRISPR-Cas “self-targeting” locus encoding a crRNA that targets a chromosomal protospacer. We develop a strand-specific, high-throughput-sequencing method for analysis of DNA fragments, “FragSeq,” and use this method to detect short fragments derived from DNA surrounding the targeted protospacer. The detected fragments have sequences matching spacers acquired during primed adaptation, contain ~3- to 4-nt overhangs derived from excision of genomic DNA within a PAM, are generated in a bidirectional, orientation-dependent manner relative to the targeted protospacer, require the functional integrity of machinery for interference and adaptation to accumulate, and function as spacer precursors when exogenously introduced into cells by transformation. DNA fragments with a similar structure accumulate in cells undergoing primed adaptation in a type I-F CRISPR-Cas self-targeting system. We propose the DNA fragments detected in this work are products of universal steps of spacer precursor processing in type I CRISPR-Cas systems.

scholarly journals Extracellular vesicles as mediators and markers of acute organ injury: current concepts

2021 ◽  
Author(s):  
Birte Weber ◽  
Niklas Franz ◽  
Ingo Marzi ◽  
Dirk Henrich ◽  
Liudmila Leppik
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Diseases ◽  
Organ Injury ◽  
Isolation And Characterization ◽  
Communication Processes ◽  
Incidence And Mortality ◽  
New Biomarkers ◽  
And Function

AbstractDue to the continued high incidence and mortality rate worldwide, there is a need to develop new strategies for the quick, precise, and valuable recognition of presenting injury pattern in traumatized and poly-traumatized patients. Extracellular vesicles (EVs) have been shown to facilitate intercellular communication processes between cells in close proximity as well as distant cells in healthy and disease organisms. miRNAs and proteins transferred by EVs play biological roles in maintaining normal organ structure and function under physiological conditions. In pathological conditions, EVs change the miRNAs and protein cargo composition, mediating or suppressing the injury consequences. Therefore, incorporating EVs with their unique protein and miRNAs signature into the list of promising new biomarkers is a logical next step. In this review, we discuss the general characteristics and technical aspects of EVs isolation and characterization. We discuss results of recent in vitro, in vivo, and patients study describing the role of EVs in different inflammatory diseases and traumatic organ injuries. miRNAs and protein signature of EVs found in patients with acute organ injury are also debated.

scholarly journals Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 730
Author(s):  
Biji Mathew ◽  
Leianne A. Torres ◽  
Lorea Gamboa Gamboa Acha ◽  
Sophie Tran ◽  
Alice Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Extracellular Vesicles ◽  
Time Course ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Paracrine Effects

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

scholarly journals Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 29
Author(s):  
Raghubendra Singh Dagur ◽  
Moses New-Aaron ◽  
Murali Ganesan ◽  
Weimin Wang ◽  
Svetlana Romanova ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Extracellular Vesicles ◽  
Treated Group ◽  
Human Hepatocytes ◽  
In Vivo Studies ◽  
People Living With Hiv ◽  
Humanized Mouse Model ◽  
And Function

Background: Alcohol abuse is common in people living with HIV-1 and dramaticallyenhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomaldysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles(EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. Methods:The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanizedfumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chainknockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus. Results: We observedan increase in the secretion of EVs associated with a decrease in lysosomal activity and expressionof lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary humanhepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulatedgenes in the alcohol–HIV-treated group. Upstream regulator analysis of differentially expressedgenes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstreamgenes associated with increased oxidative stress, lysosomal associated disease, and function andEVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplantedhumanized mice, indicating that intensive EVs’ generation by human hepatocytes andtheir secretion to serum was associated with increased oxidative stress and reduction in lysosomalactivities triggered by HIV infection and ethanol diet. Conclusion: HIV-and-ethanol-metabolisminducedEVs release is tightly controlled by lysosome status in hepatocytes and participates in thedevelopment of double-insult-induced liver injury.

scholarly journals Profound functional and molecular diversity of mitochondria revealed by cell type-specific profiling in vivo

2018 ◽  
Author(s):  
Caroline Fecher ◽  
Laura Trovò ◽  
Stephan A. Müller ◽  
Nicolas Snaidero ◽  
Jennifer Wettmarshausen ◽  
...  
Keyword(s):  
Molecular Diversity ◽  
Molecular Level ◽  
Cell Types ◽  
Long Chain Fatty Acids ◽  
Cell Type ◽  
Mitochondrial Proteome ◽  
Novel Approach ◽  
Cell Type Specific ◽  
And Function

AbstractMitochondria vary in morphology and function in different tissues, however little is known about their molecular diversity among cell types. To investigate mitochondrial diversity in vivo, we developed an efficient protocol to isolate cell type-specific mitochondria based on a new MitoTag mouse. We profiled the mitochondrial proteome of three major neural cell types in cerebellum and identified a substantial number of differential mitochondrial markers for these cell types in mice and humans. Based on predictions from these proteomes, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neurons. Moreover, we identified Rmdn3 as a major determinant of ER-mitochondria proximity in Purkinje cells. Our novel approach enables exploring mitochondrial diversity on the functional and molecular level in many in vivo contexts.

Isoform-specific expression and function of neuregulin

Development ◽  
1997 ◽  
Vol 124 (18) ◽  
pp. 3575-3586 ◽  
Author(s):  
D. Meyer ◽  
T. Yamaai ◽  
A. Garratt ◽  
E. Riethmacher-Sonnenberg ◽  
D. Kane ◽  
...  
Keyword(s):  
Biological Effects ◽  
Type I ◽  
Mouse Development ◽  
Specific Expression ◽  
Independent Functions ◽  
Cell Growth And Differentiation ◽  
Cranial Ganglia ◽  
And Function

Neuregulin (also known as NDF, heregulin, ARIA, GGF or SMDF), induces cell growth and differentiation. Biological effects of neuregulin are mediated by members of the erbB family of tyrosine kinase receptors. Three major neuregulin isoforms are produced from the gene, which differ substantially in sequence and in overall structure. Here we use in situ hybridization with isoform-specific probes to illustrate the spatially distinct patterns of expression of the isoforms during mouse development. Ablation of the neuregulin gene in the mouse has demonstrated multiple and independent functions of this factor in development of both the nervous system and the heart. We show here that targeted mutations that affect different isoforms result in distinct phenotypes, demonstrating that isoforms can take over specific functions in vivo. Type I neuregulin is required for generation of neural crest-derived neurons in cranial ganglia and for trabeculation of the heart ventricle, whereas type III neuregulin plays an important role in the early development of Schwann cells. The complexity of neuregulin functions in development is therefore due to independent roles played by distinct isoforms.

Orchestrated biomechanical, structural, and biochemical stimuli for engineering anisotropic meniscus

2019 ◽  
Vol 11 (487) ◽  
pp. eaao0750 ◽  
Author(s):  
Zheng-Zheng Zhang ◽  
You-Rong Chen ◽  
Shao-Jie Wang ◽  
Feng Zhao ◽  
Xiao-Gang Wang ◽  
...  
Keyword(s):  
Rabbit Model ◽  
Proper Function ◽  
Type I ◽  
Specific Expression ◽  
Tissue Organization ◽  
Meniscus Tissue ◽  
Biomimetic Scaffold ◽  
And Function

Reconstruction of the anisotropic structure and proper function of the knee meniscus remains an important challenge to overcome, because the complexity of the zonal tissue organization in the meniscus has important roles in load bearing and shock absorption. Current tissue engineering solutions for meniscus reconstruction have failed to achieve and maintain the proper function in vivo because they have generated homogeneous tissues, leading to long-term joint degeneration. To address this challenge, we applied biomechanical and biochemical stimuli to mesenchymal stem cells seeded into a biomimetic scaffold to induce spatial regulation of fibrochondrocyte differentiation, resulting in physiological anisotropy in the engineered meniscus. Using a customized dynamic tension-compression loading system in conjunction with two growth factors, we induced zonal, layer-specific expression of type I and type II collagens with similar structure and function to those present in the native meniscus tissue. Engineered meniscus demonstrated long-term chondroprotection of the knee joint in a rabbit model. This study simultaneously applied biomechanical, biochemical, and structural cues to achieve anisotropic reconstruction of the meniscus, demonstrating the utility of anisotropic engineered meniscus for long-term knee chondroprotection in vivo.

scholarly journals Isopsoralen Enhanced Osteogenesis by Targeting AhR/ERα

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2600 ◽  
Author(s):  
Luna Ge ◽  
Yazhou Cui ◽  
Kai Cheng ◽  
Jinxiang Han
Keyword(s):  
Osteoblast Differentiation ◽  
Estrogen Receptor Alpha ◽  
Biological Effects ◽  
Hormone Deficiency ◽  
In Vivo Studies ◽  
Osteogenic Potential ◽  
Type I ◽  
Dependent Manner

Isopsoralen (IPRN), one of the main effective ingredients in Psoralea corylifolia Linn, has a variety of biological effects, including antiosteoporotic effects. In vivo studies show that IPRN can increase bone strength and trabecular bone microstructure in a sex hormone deficiency-induced osteoporosis model. However, the mechanism underlying this osteogenic potential has not been investigated in detail. In the present study, we investigated the molecular mechanism of IPRN-induced osteogenesis in MC3T3-E1 cells. Isopsoralen promoted osteoblast differentiation and mineralization, increased calcium nodule levels and alkaline phosphatase (ALP) activity and upregulated osteoblast markers, including ALP, runt-related transcription factor 2 (RUNX2), and collagen type I alpha 1 chain (COL1A1). Furthermore, IPRN limited the nucleocytoplasmic shuttling of aryl hydrocarbon receptor (AhR) by directly binding to AhR. The AhR target gene cytochrome P450 family 1 subfamily A member 1 (CYP1A1) was also inhibited in vitro and in vivo. This effect was inhibited by the AhR agonists indole-3-carbinol (I3C) and 3-methylcholanthrene (3MC). Moreover, IPRN also increased estrogen receptor alpha (ERα) expression in an AhR-dependent manner. Taken together, these results suggest that IPRN acts as an AhR antagonist and promotes osteoblast differentiation via the AhR/ERα axis.

scholarly journals Detection of spacer precursors formed in vivo during primed CRISPR adaptation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Anna A. Shiriaeva ◽  
Ekaterina Savitskaya ◽  
Kirill A. Datsenko ◽  
Irina O. Vvedenskaya ◽  
Iana Fedorova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
High Throughput Sequencing ◽  
Dna Degradation ◽  
Type I ◽  
Dna Fragments ◽  
Crispr Array ◽  
Foreign Dna ◽  
And Function

Abstract Type I CRISPR-Cas loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA. Immunity involves adaptation, the integration of ~30-bp DNA fragments, termed prespacers, into the CRISPR array as spacers, and interference, the targeted degradation of DNA containing a protospacer. Interference-driven DNA degradation can be coupled with primed adaptation, in which spacers are acquired from DNA surrounding the targeted protospacer. Here we develop a method for strand-specific, high-throughput sequencing of DNA fragments, FragSeq, and apply this method to identify DNA fragments accumulated in Escherichia coli cells undergoing robust primed adaptation by a type I-E or type I-F CRISPR-Cas system. The detected fragments have sequences matching spacers acquired during primed adaptation and function as spacer precursors when introduced exogenously into cells by transformation. The identified prespacers contain a characteristic asymmetrical structure that we propose is a key determinant of integration into the CRISPR array in an orientation that confers immunity.

scholarly journals Rabies Virus Infection Induces Type I Interferon Production in an IPS-1 Dependent Manner While Dendritic Cell Activation Relies on IFNAR Signaling

2010 ◽  
Vol 6 (7) ◽  
pp. e1001016 ◽  
Author(s):  
Elizabeth J. Faul ◽  
Celestine N. Wanjalla ◽  
Mehul S. Suthar ◽  
Michael Gale ◽  
Christoph Wirblich ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Virus Infection ◽  
Rabies Virus ◽  
Cell Activation ◽  
Type I Interferon ◽  
Type I ◽  
Dependent Manner ◽  
Interferon Production ◽  
Dendritic Cell Activation ◽  
Rabies Virus Infection
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