Capture of a Hyena-Specific Retroviral Envelope Gene with Placental Expression Associated in Evolution with the Unique Emergence among Carnivorans of Hemochorial Placentation in Hyaenidae

ABSTRACT Capture of retroviral envelope genes from endogenous retroviruses has played a role in the evolution of mammals, with evidence for the involvement of these genes in the formation of the maternofetal interface of the placenta. It has been shown that the diversity of captured genes is likely to be responsible for the diversity of placental structures, ranging from poorly invasive (epitheliochorial) to highly invasive (hemochorial), with an intermediate state (endotheliochorial) as found in carnivorans. The latter recapitulate part of this evolution, with the hyena being the sole carnivoran with a hemochorial placenta. In this study, we performed RNA sequencing on hyena placental transcripts and searched for endogenous retroviral envelope genes that have been captured specifically in the Hyaenidae clade and are not found in any other carnivoran. We identified an envelope gene that is expressed in the placenta at the level of the maternofetal interface, as evidenced by in situ hybridization/immunohistochemistry. The gene entry is coincidental with the emergence of the Hyaenidae clade 30 million years ago (Mya), being found at the same genomic locus in all 4 extant hyena species. Its coding sequence has further been maintained during all of Hyaenidae evolution. It is not found in any of the 30 other carnivorans—both Felidae and Canidae—that we screened. This envelope protein does not disclose any fusogenic activity in ex vivo assays, at variance with the syncytin-Car1 gene, which is found in all carnivorans, including the hyena, in which it is still present, transcriptionally active in the placenta, and fusogenic. Together, the present results illustrate the permanent renewal of placenta-specific genes by retroviral capture and de facto provide a candidate gene for the endotheliochorial to hemochorial transition of Hyaenidae among carnivorans. IMPORTANCE The placenta is the most diverse organ among mammals, due in part to stochastic capture of retroviral envelope genes. In carnivorans, capture of syncytin-Car1 took place 80 Mya. It is fusogenic, expressed at the syncytialized placental maternofetal interface, and conserved among all carnivorans, consistent with their shared endotheliochorial placenta. Hyenas are a remarkable exception, with a highly invasive hemochorial placenta, as found in humans, where disruption of maternal blood vessels results in maternal blood bathing the syncytial maternofetal interface. In this study, we identified a retroviral envelope gene capture and exaptation that took place about 30 Mya and is coincident with the emergence of the Hyaenidae, being conserved in all extant hyena species. It is expressed at the maternofetal interface in addition to the shared syncytin-Car1 gene. This new env gene, not present in any other carnivoran, is a likely candidate to be responsible for the specific structure of the hyena placenta.

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A host gene regulates the structure of the transmembrane envelope protein of murine leukemia viruses.

Journal of Experimental Medicine ◽

10.1084/jem.171.5.1739 ◽

1990 ◽

Vol 171 (5) ◽

pp. 1739-1752 ◽

Cited By ~ 3

Author(s):

M A Coppola ◽

C Y Thomas

Keyword(s):

Gene Structure ◽

Envelope Protein ◽

Leukemia Virus ◽

Biological Significance ◽

Endogenous Retroviruses ◽

Host Gene ◽

Class I ◽

Chromosome 17 ◽

Envelope Gene ◽

Murine Leukemia

Heterogeneity in the structure of the envelope proteins has been observed in many human and animal retroviruses and may influence pathogenicity. However, the biological significance of this heterogeneity and the mechanisms by which it is generated are poorly understood. We have studied a mouse model in which the envelope gene structure of lymphoma-associated viruses appears to be controlled by a single host gene. The inoculation of HRS and CWD mice with a leukemogenic murine leukemia virus (MuLV) results in recombination between the injected virus and envelope gene sequences of endogenous retroviruses. The genomes of HRS (class I) env recombinants and CWD (class II) env recombinants differ in the sequences encoding the NH2-terminal portion of the transmembrane envelope protein (TM). We have shown that an HRS gene linked to the MHC on chromosome 17 mediates a dominant selection for recombinant retroviruses with the class I envelope gene structure. CBA mice, which share the H-2k haplotype with HRS, also carry the dominant allele at this locus. This system provides a useful model for studies of host factors involved in the selection of specific variants of pathogenic retroviruses.

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Differential Expression Pattern of Retroviral Envelope Gene in the Equine Placenta

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.693416 ◽

2021 ◽

Vol 8 ◽

Author(s):

Valentina Stefanetti ◽

Luisa Pascucci ◽

Sandra Wilsher ◽

Katia Cappelli ◽

Stefano Capomaccio ◽

...

Keyword(s):

Expression Pattern ◽

Placental Tissue ◽

Endogenous Retroviruses ◽

Envelope Gene ◽

Chorionic Villi ◽

Beneficial Effects ◽

Uterine Endometrium ◽

Differential Expression Pattern ◽

Reverse Transcription Quantitative Pcr

Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses, which have coevolved with vertebrate genomes for millions of years. The conservation of ERV genes throughout evolution suggests their beneficial effects on their hosts' survival. An example of such positive selection is demonstrated by the syncytin gene, which encodes a protein with affinity for various mammalian placentas that is involved in the formation of syncytiotrophoblasts. Although the horse has an epitheliochorial placenta, in which the fetal trophoblasts are simply apposed to the intact uterine epithelium, we have previously demonstrated that the equine ERV (EqERV) env RNA is unexpectedly expressed in placental tissue. In the present study, we investigated the mRNA expression pattern of the EqERV env gene in different parts of the equine placenta, to gain more insight into its putative role in the fetal–maternal relationship. To this end, we used reverse transcription–quantitative PCR (RT–qPCR) and in situ hybridization assays to analyze different target areas of the equine placenta. The retroviral env gene is expressed in the equine placenta, even though there is no syncytium or erosion of the uterine endometrium. The gene is also expressed in all the sampled areas, although with some quantitative differences. We suggest that these differences are attributable to variations in the density, height, and degree of morphological complexity of the chorionic villi forming the microcotyledons. The involvement of the EqERV env gene in different functional pathways affecting the fetus–mother relationship can be hypothesized.

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An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714590114 ◽

2017 ◽

Vol 114 (51) ◽

pp. E10991-E11000 ◽

Cited By ~ 28

Author(s):

Guillaume Cornelis ◽

Mathis Funk ◽

Cécile Vernochet ◽

Francisca Leal ◽

Oscar Alejandro Tarazona ◽

...

Keyword(s):

Ex Vivo ◽

Transmembrane Protein ◽

Chinese Hamster ◽

Endogenous Retroviruses ◽

Molecular Tools ◽

Screening Assay ◽

Cognate Receptor ◽

Fusogenic Activity ◽

Membrane Bound

Syncytins are envelope genes from endogenous retroviruses that have been captured during evolution for a function in placentation. They have been found in all placental mammals in which they have been searched, including marsupials. Placental structures are not restricted to mammals but also emerged in some other vertebrates, most frequently in lizards, such as the viviparous Mabuya Scincidae. Here, we performed high-throughput RNA sequencing of a Mabuya placenta transcriptome and screened for the presence of retroviral env genes with a full-length ORF. We identified one such gene, which we named “syncytin-Mab1,” that has all the characteristics expected for a syncytin gene. It encodes a membrane-bound envelope protein with fusogenic activity ex vivo, is expressed at the placental level as revealed by in situ hybridization and immunohistochemistry, and is conserved in all Mabuya species tested, spanning over 25 My of evolution. Its cognate receptor, required for its fusogenic activity, was searched for by a screening assay using the GeneBridge4 human/Chinese hamster radiation hybrid panel and found to be the MPZL1 gene, previously identified in mammals as a signal-transducing transmembrane protein involved in cell migration. Together, these results show that syncytin capture is not restricted to placental mammals, but can also take place in the rare nonmammalian vertebrates in which a viviparous placentotrophic mode of reproduction emerged. It suggests that similar molecular tools have been used for the convergent evolution of placentation in independently evolved and highly distant vertebrates.

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Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽

10.3390/pathogens10010054 ◽

2021 ◽

Vol 10 (1) ◽

pp. 54

Author(s):

Christine Landlinger ◽

Lenka Tisakova ◽

Vera Oberbauer ◽

Timo Schwebs ◽

Abbas Muhammad ◽

...

Keyword(s):

Bacterial Vaginosis ◽

Bactericidal Activity ◽

High Selectivity ◽

Ex Vivo ◽

Vaginal Microbiome ◽

Wild Type ◽

Wild Type Enzyme ◽

Promising Alternative ◽

First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

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A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals

Function ◽

10.1093/function/zqab012 ◽

2021 ◽

Vol 2 (3) ◽

Cited By ~ 1

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+]).

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Capsazepine decreases corneal pain syndrome in severe dry eye disease

Journal of Neuroinflammation ◽

10.1186/s12974-021-02162-7 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Darine Fakih ◽

Adrian Guerrero-Moreno ◽

Christophe Baudouin ◽

Annabelle Réaux-Le Goazigo ◽

Stéphane Mélik Parsadaniantz

Keyword(s):

In Situ Hybridization ◽

Inflammatory Pain ◽

Transient Receptor Potential ◽

Ex Vivo ◽

Receptor Potential ◽

Eye Disease ◽

Ocular Pain ◽

Trpv1 Antagonist ◽

Transient Receptor

Abstract Background Dry eye disease (DED) is a multifactorial disease of the ocular surface accompanied by neurosensory abnormalities. Here, we evaluated the effectiveness of transient receptor potential vanilloid-1 (TRPV1) blockade to alleviate ocular pain, neuroinflammation, and anxiety-like behavior associated with severe DED. Methods Chronic DED was induced by unilateral excision of the Harderian and extraorbital lacrimal glands of adult male mice. Investigations were conducted at 21 days after surgery. The mRNA levels of TRPV1, transient receptor potential ankyrin-1 (TRPA1), and acid-sensing ion channels 1 and 3 (ASIC1 and ASIC3) in the trigeminal ganglion (TG) were evaluated by RNAscope in situ hybridization. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous and stimulated (cold, heat, and acid) corneal nerve responsiveness in ex vivo eye preparations. DED mice received topical instillations of the TRPV1 antagonist (capsazepine) twice a day for 2 weeks from d7 to d21 after surgery. The expression of genes involved in neuropathic and inflammatory pain was evaluated in the TG using a global genomic approach. Chemical and mechanical corneal nociception and spontaneous ocular pain were monitored. Finally, anxiety-like behaviors were assessed by elevated plus maze and black and white box tests. Results First, in situ hybridization showed DED to trigger upregulation of TRPV1, TRPA1, ASIC1, and ASIC3 mRNA in the ophthalmic branch of the TG. DED also induced overexpression of genes involved in neuropathic and inflammatory pain in the TG. Repeated instillations of capsazepine reduced corneal polymodal responsiveness to heat, cold, and acidic stimulation in ex vivo eye preparations. Consistent with these findings, chronic capsazepine instillation inhibited the upregulation of genes involved in neuropathic and inflammatory pain in the TG of DED animals and reduced the sensation of ocular pain, as well as anxiety-like behaviors associated with severe DED. Conclusion These data provide novel insights on the effectiveness of TRPV1 antagonist instillation in alleviating abnormal corneal neurosensory symptoms induced by severe DED, opening an avenue for the repositioning of this molecule as a potential analgesic treatment for patients suffering from chronic DED.

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In Situ Saturating Mutagenesis Screening Identifies a Functional Genomic Locus that Regulates Ucp1 Expression

Phenomics ◽

10.1007/s43657-020-00006-7 ◽

2021 ◽

Vol 1 (1) ◽

pp. 15-21

Author(s):

Yan Qiu ◽

Xiaojian Liu ◽

Yingmin Sun ◽

Shuang Li ◽

Yuda Wei ◽

...

Keyword(s):

Functional Genomic ◽

Genomic Locus ◽

Ucp1 Expression

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Generation of vascular chimerism within donor organs

Scientific Reports ◽

10.1038/s41598-021-92823-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shahar Cohen ◽

Shirly Partouche ◽

Michael Gurevich ◽

Vladimir Tennak ◽

Vadym Mezhybovsky ◽

...

Keyword(s):

Ex Vivo ◽

Patient Specific ◽

Machine Perfusion ◽

Organ Perfusion ◽

Perfusion Process ◽

Hind Limbs ◽

Porcine Organs ◽

Immunological Barrier

AbstractWhole organ perfusion decellularization has been proposed as a promising method to generate non-immunogenic organs from allogeneic and xenogeneic donors. However, the ability to recellularize organ scaffolds with multiple patient-specific cells in a spatially controlled manner remains challenging. Here, we propose that replacing donor endothelial cells alone, while keeping the rest of the organ viable and functional, is more technically feasible, and may offer a significant shortcut in the efforts to engineer transplantable organs. Vascular decellularization was achieved ex vivo, under controlled machine perfusion conditions, in various rat and porcine organs, including the kidneys, liver, lungs, heart, aorta, hind limbs, and pancreas. In addition, vascular decellularization of selected organs was performed in situ, within the donor body, achieving better control over the perfusion process. Human placenta-derived endothelial progenitor cells (EPCs) were used as immunologically-acceptable human cells to repopulate the luminal surface of de-endothelialized aorta (in vitro), kidneys, lungs and hind limbs (ex vivo). This study provides evidence that artificially generating vascular chimerism is feasible and could potentially pave the way for crossing the immunological barrier to xenotransplantation, as well as reducing the immunological burden of allogeneic grafts.

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