scholarly journals Toll signaling enhances mosquito antiplasmodial immunity by promoting differentiation of hemocytes to the Megacyte lineage

2021 ◽  
Author(s):  
Carolina Barillas Mury ◽  
Ana Beatriz Ferreira Barletta ◽  
Banhisikha Saha ◽  
Nathanie Trisnadi ◽  
Gianmarco Raddi
Keyword(s):  
In Situ Hybridization ◽  
Anopheles Gambiae ◽  
Morphological Analysis ◽  
Plasmodium Infection ◽  
System A ◽  
Toll Signaling ◽  
Local Release

Activation of Toll signaling in Anopheles gambiae, by silencing Cactus, eliminates Plasmodium ookinetes by enhancing local release of hemocytes-derived microvesicles that promote activation of the mosquito complement-like system. A new effector hemocyte subpopulation of large granulocytes, the megacytes, was recently identified. We report that Cactus silencing dramatically increases the proportion of megacytes, from 5 to 79% of circulating granulocytes. Transcriptomic and morphological analysis, as well as in situ hybridization and expression of cell-specific markers, indicate that Cactus silencing triggers granulocyte differentiation into megacytes. Megacytes are very plastic cells that can extend long filopodia and tend to form clusters in vivo. Moreover, megacytes are massively recruited to the basal midgut surface in response to bacterial feeding and Plasmodium infection. We propose that Toll signaling promotes differentiation of granulocytes to the megacyte lineage, a major cellular effector of antibacterial and antiplasmodial immunity.

scholarly journals Can Anopheles gambiae Be Infected with Wolbachia pipientis? Insights from an In Vitro System

2006 ◽  
Vol 72 (12) ◽  
pp. 7718-7722 ◽  
Author(s):  
Jason L. Rasgon ◽  
Xiaoxia Ren ◽  
Michael Petridis
Keyword(s):  
In Situ Hybridization ◽  
Anopheles Gambiae ◽  
Cytoplasmic Incompatibility ◽  
Potential Mechanism ◽  
In Vitro System ◽  
Wolbachia Pipientis ◽  
Genetic Block

ABSTRACT Wolbachia pipientis are maternally inherited endosymbionts associated with cytoplasmic incompatibility, a potential mechanism to drive transgenic traits into Anopheles populations for malaria control. W. pipientis infections are common in many mosquito genera but have never been observed in any Anopheles species, leading to the hypothesis that Anopheles mosquitoes are incapable of harboring infection. We used an in vitro system to evaluate the ability of Anopheles gambiae cells to harbor diverse W. pipientis infections. We successfully established W. pipientis infections (strains wRi and wAlbB) in the immunocompetent Anopheles gambiae cell line Sua5B. Infection was confirmed by PCR, antibiotic curing, DNA sequencing, and direct observation using fluorescence in situ hybridization. The infections were maintained at high passage rates for >30 passages. Our results indicate that there is no intrinsic genetic block to W. pipientis infection in A. gambiae cells, suggesting that establishment of in vivo W. pipientis infections in Anopheles mosquitoes may be feasible.

scholarly journals circHMGCS1–016 reshapes immune environment by sponging miR-1236-3p to regulate CD73 and GAL-8 expression in intrahepatic cholangiocarcinoma

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Ya-Ping Xu ◽  
Ze-Ning Dong ◽  
Si-Wei Wang ◽  
Yi-Min Zheng ◽  
Chi Zhang ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Intrahepatic Cholangiocarcinoma ◽  
Molecular Mechanisms ◽  
Prognostic Significance ◽  
Luciferase Reporter ◽  
Mouse Tumor ◽  
Rna Seq ◽  
Potential Biomarker

Abstract Background Accumulating evidence indicates that circRNAs may serve as essential regulators in the progression of several human cancers, but the function and mechanism of circRNAs in intrahepatic cholangiocarcinoma (ICC) are largely unknown. Methods RNA-seq was used to assess differentially expressed circRNAs between 4 ICC and peritumor tissues. Quantitative RT-PCR and in situ hybridization were used to determine the circHMGCS1–016 expression in ICC tissues. The function and mechanism of circHMGCS1–016 were further identified via in vivo experiments. The clinical characteristics and prognostic significance of circHMGCS1–016 were analyzed by a retrospective study. The functions of circHMGCS1–016 were assessed via modifying circRNA expression in ICC cells. Moreover, the molecular mechanisms of circHMGCS1–016 in ICC cells were explored by circRNA precipitation, miRNA immunoprecipitation, SILAC and luciferase reporter assays. Results We identified that compared with peritumor tissues, ICC tissues expressed hsa_circ_0008621 (circHMGCS1–016) high by RNA-seq, which was further identified by qRT-PCR and in situ hybridization. Moreover, the expression of circHMGCS1–016 was revealed to be associated with survival and recurrence of ICC patients. By regulating circHMGCS1–016 expression, we found that elevated circHMGCS1–016 promoted ICC development both in vitro and in vivo. By SILAC and circRNA-pull down, we demonstrated that circHMGCS1–016 induced ICC cell invasion and reshaped the tumor immune microenvironment via the miR-1236-3p/CD73 and GAL-8 axis. In ICC tissues, we uncovered that a high level of circHMGCS1–016 was positively associated with CD73 and GAL-8 expression and negatively related to the CD8+ T cells infiltration, which was further validated by establishing a humanized mouse tumor model. Importantly, we displayed that ICC patients with high levels of circHMGCS1–016 in tumor tissues benefited less from anti-PD1 treatment compared to those with low levels of circHMGCS1–016. Conclusions CircHMGCS1–016 is a forceful contributor in ICC development and immune tolerance via miR-1236-3p/CD73 and GAL-8 axis. CircHMGCS1–016 can be explored as a new potential biomarker and therapeutic target for PD1-resistant ICC.

Use of cross-species in-situ hybridization (ZOO-FISH) to assess chromosome abnormalities in day-6 in-vivo- or in-vitro-produced sheep embryos

2007 ◽  
Vol 15 (3) ◽  
pp. 399-408 ◽  
Author(s):  
Gianfranco Coppola ◽  
Basil Alexander ◽  
Dino Di Berardino ◽  
Elizabeth St John ◽  
Parvathi K. Basrur ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Chromosome Abnormalities

Interactions of human cytomegalovirus with leukocytes in vivo: analysis by in situ hybridization

1987 ◽  
Vol 3 (4) ◽  
pp. 287-297 ◽  
Author(s):  
Lloyd W. Turtinen ◽  
Robin Saltzman ◽  
M.Colin Jordan ◽  
Ashley T. Haase
Keyword(s):  
In Situ Hybridization ◽  
Human Cytomegalovirus ◽  
In Vivo Analysis

Analysis of HIV-1 expression in vivo with in situ hybridization and the polymerase chain reaction

1992 ◽  
Vol 6 (3) ◽  
pp. 215-221 ◽  
Author(s):  
B. Delord ◽  
M. Ottmann ◽  
M.-H. Schrive ◽  
J.-M. Ragnaud ◽  
J.-M. Seigneurin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Hiv 1

scholarly journals Nonrandom Transduction of Recombinant Adeno-Associated Virus Vectors in Mouse Hepatocytes In Vivo: Cell Cycling Does Not Influence Hepatocyte Transduction

2000 ◽  
Vol 74 (8) ◽  
pp. 3793-3803 ◽  
Author(s):  
Carol H. Miao ◽  
Hiroyuki Nakai ◽  
Arthur R. Thompson ◽  
Theresa A. Storm ◽  
Winnie Chiu ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Gene Transfer ◽  
Genetic Diseases ◽  
Critical Event ◽  
Dimer Formation ◽  
Adeno Associated Virus ◽  
Virus Vectors ◽  
Preclinical Trials

ABSTRACT Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about ∼5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only ∼5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5′-bromo-2′-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic β-galactosidase. Colabeling for β-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.

scholarly journals Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization

1988 ◽  
Vol 106 (2) ◽  
pp. 441-450 ◽  
Author(s):  
S Nomura ◽  
AJ Wills ◽  
DR Edwards ◽  
JK Heath ◽  
BL Hogan
Keyword(s):  
In Situ Hybridization ◽  
Bone Development ◽  
Mouse Cell ◽  
Sensory Epithelium ◽  
Developmental Expression ◽  
Granulated Metrial Gland Cells ◽  
Metrial Gland ◽  
Temporal And Spatial

2ar has been identified as a gene inducible by tumor promoters and growth factors in a variety of cultured mouse cell lines (Smith, J. H., and D. T. Denhardt. 1987. J. Cell. Biochem. 34:13-22). Sequence analysis shows that it codes for mouse osteopontin, an RGDS-containing, phosphorylated, sialic acid-rich Ca++-binding protein originally isolated from bone (Oldberg, A., A. Franzen, and D. Heinegard. 1986. Proc. Natl. Acad. Sci. USA. 83:8819-8823; Prince, C. W., T. Oosawa, W. T. Butler, M. Tomana, A. S. Brown, and R. E. Schrohenloer. 1987. J. Biol. Chem. 262:2900-3907.). In this paper we use Northern blot analysis and in situ hybridization to localize expression of 2ar during mouse embryogenesis. 2ar RNA is first detected in developing limb bones and calvaria at 14.5 d p.c., in a population of cells distinct from those expressing SPARC (osteonectin). High levels of 2ar expression are also seen in the bone marrow-derived granulated metrial gland cells of the deciduum and placenta, and in a number of epithelial tissues, including embryonic and postnatal kidney tubules, uterine epithelium and sensory epithelium of the embryonic ear. The temporal and spatial pattern of 2ar expression seen in vivo suggests that the protein plays a wider role than previously realized, in processes which are not confined to bone development.

Expression of endo-oligopeptidase A in the rat central nervous system: a non-radioactive in situ hybridization study

2001 ◽  
Vol 89 (1-2) ◽  
pp. 86-93 ◽  
Author(s):  
Mirian A.F. Hayashi ◽  
Raquel S. Pires ◽  
Nancy A. Rebouças ◽  
Luiz R.G. Britto ◽  
Antonio C.M. Camargo
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
In Situ Hybridization ◽  
Hybridization Study ◽  
System A
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