scholarly journals The Novel Zoonotic Pathogen, Anaplasma capra, Infects Human Erythrocytes, HL-60, and TF-1 Cells In Vitro

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 600
Author(s):  
Yongshuai Peng ◽  
Chenyang Lu ◽  
Yaqun Yan ◽  
Jinxing Song ◽  
Zhiyang Pei ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Cell Lines ◽  
Human Infection ◽  
Human Erythrocytes ◽  
Giemsa Staining ◽  
In Vitro Cultivation ◽  
Chromogenic In Situ Hybridization ◽  
Anaplasma Capra

Anaplasma capra, a species of the family Anaplasmataceae, is zoonotic tick-borne obligate intracellular bacteria. There have been no reports of human infection with this pathogen since 2015. Therefore, the zoonotic characteristics of A. capra need to be further studied. To verify the ability of A. capra to infect human cells, A. capra were inoculated in human erythrocytes, HL-60, and TF-1 cell lines in vitro. Cell smears were taken after inoculation, using Giemsa staining, transmission electron microscope (TEM), chromogenic in situ hybridization and immunocytochemistry for detection. In the Giemsa staining, many dark colored corpuscles or purple granules were seen in the inoculated erythrocytes, HL-60, and TF-1 cells. The results of chromogenic in situ hybridization show that there were brown precipitates on the surface of most erythrocytes. Immunocytochemistry results show many dark brown vacuolar structures or corpuscles in the cytoplasm of erythrocytes, HL-60, and TF-1 cell lines. The A. capra morulae were seen in the cytoplasm of both HL-60 and TF-1 in TEM, and their diameter was about 295–518 nm. Both dense-cored (DC) and reticulate cell (RC) form morulae could be seen. This study confirmed the ability of A. capra to infect human erythrocytes, HL-60, and TF-1. This study is of profound significance in further verifying the zoonotic characteristics of the pathogen and for establishing an in vitro cultivation model.

scholarly journals Visualization of Alternative Epstein-Barr Virus Expression Programs by Fluorescent In Situ Hybridization at the Cell Level

1999 ◽  
Vol 73 (6) ◽  
pp. 5064-5069 ◽  
Author(s):  
Anna Szeles ◽  
Kerstin I. Falk ◽  
Stephan Imreh ◽  
George Klein
Keyword(s):  
In Situ Hybridization ◽  
Cell Lines ◽  
Fluorescent In Situ Hybridization ◽  
Epstein Barr Virus ◽  
Type I ◽  
Type Iii ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) transforms human B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). They regularly express six virally encoded nuclear proteins (EBNA1 to EBNA6) and three membrane proteins (LMP1, LMP2A, and LMP2B). In contrast, EBV-carrying Burkitt lymphoma (BL) cells in vivo and derived type I cell lines that maintain the BL phenotype express only EBNA1. During prolonged in vitro culturing, most EBV-carrying BL lines drift toward a more immunoblastic (type II or III) phenotype. Their viral antigen expression is upregulated in parallel. We have used fluorescent in situ hybridization to visualize viral transcripts in type I and III BL lines and LCLs. In type I cells, EBNA1 is encoded by a monocistronic message that originates from the Qp promoter. In type III cells, the EBNA1 transcript is spliced from a giant polycistronic message that originates from one of several alternative Wp or Cp promoters and encodes all six EBNAs. We have obtained a “track” signal with aBamHI W DNA probe that could hybridize with the polycistronic but not with the monocistronic message in two type III BL lines (Namalwa-Cl8 and MUTU III) and three LCLs (LCL IB4-D, LCL-970402, and IARC-171). A BamHI K probe that can hybridize to both the monocistronic and the polycistronic message visualized the same pattern in the type III BLs and the LCLs as the BamHI W probe. A positive signal was obtained with the BamHI K but not the BamHI W probe in the type I BL lines MUTU I and Rael. The RNA track method can thus distinguish between cells that use a type III and those that use a type I program. The former cells hybridize with both the W and the K probes, but the latter cells hybridize with only the K probe. Our findings may open the way for studies of the important but still unanswered question of whether cells with type I latency arise from immunoblasts with a full type III program or are generated by a separate pathway during primary infection.

Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica

2020 ◽  
Vol 27 (5) ◽  
pp. 432-446
Author(s):  
Akiko Yamamoto ◽  
Ken-ichiro Matsunaga ◽  
Toyoaki Anai ◽  
Hitoshi Kawano ◽  
Toshihisa Ueda ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Immunohistochemical Analysis ◽  
Protein Characterization ◽  
Intermediate Filament Protein ◽  
Tail Domain ◽  
Animal Cells ◽  
Dugesia Japonica ◽  
Function Relationship

Background: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. Objective: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. Method: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. Results: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. Conclusions: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

scholarly journals Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1518
Author(s):  
Maria Qatato ◽  
Vaishnavi Venugopalan ◽  
Alaa Al-Hashimi ◽  
Maren Rehders ◽  
Aaron D. Valentine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Human Thyroid ◽  
Apical Plasma Membrane ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Trace Amine ◽  
Rat Thyroid

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

scholarly journals Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

Foods ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1502
Author(s):  
Jorge García-Hernández ◽  
Manuel Hernández ◽  
Yolanda Moreno
Keyword(s):  
In Situ Hybridization ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent In Situ Hybridization ◽  
Potential Method ◽  
Viable Count ◽  
Hybridization Technique ◽  
Fish Technique ◽  
Viable Cells

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

scholarly journals Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

BMC Cancer ◽  
2009 ◽  
Vol 9 (1) ◽  
Author(s):  
Fabíola E Rosa ◽  
Sara M Silveira ◽  
Cássia GT Silveira ◽  
Nádia A Bérgamo ◽  
Francisco A Moraes Neto ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Breast Carcinoma ◽  
Real Time ◽  
Rt Pcr ◽  
Her 2 ◽  
Chromogenic In Situ Hybridization

Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes

1993 ◽  
Vol 104 (4) ◽  
pp. 1187-1197 ◽  
Author(s):  
R.W. Dirks ◽  
F.M. van de Rijke ◽  
S. Fujishita ◽  
M. van der Ploeg ◽  
A.K. Raap
Keyword(s):  
In Situ Hybridization ◽  
Cell Lines ◽  
Cultured Cells ◽  
Direct Detection ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Immediate Early ◽  
High Signal ◽  
Pretreatment Conditions

We have determined optimal conditions for the detection of mRNA sequences in cultured cells by nonradioactive in situ hybridization. For this purpose a number of different cell lines have been used: rat 9G cells for the detection of human cytomegalovirus immediate early mRNA, and HeLa as well as 5637 carcinoma cells for the detection of housekeeping gene mRNAs. Extensive optimization of fixation and pretreatment conditions revealed that most intense hybridization signals are obtained when cells are grown on glass microscope slides, fixed with a mixture of formaldehyde and acetic acid, pretreated with pepsin and denatured prior to hybridization. In addition, we also studied the potential of fluorochromized probes for the direct detection of multiple RNA sequences. The optimized in situ hybridization procedure revealed that immediate early mRNA transcripts are, in addition to a cytoplasmic localization, localized within nuclei of rat 9G cells. Double hybridization experiments showed that intron and exon sequences colocalize within the main nuclear signal. In addition, the presence of small, intron-specific, fluorescent spots scattered around the main nuclear signals indicates that intron sequences which are spliced out can be visualized. Additional information about the functioning of cells could be obtained by the detection of mRNA simultaneously with bromodeoxyuridine, incorporated during S-phase, or its cognate protein. The sensitivity of these methods is such that mRNAs of abundantly expressed housekeeping genes can be detected in a variety of cell lines with high signal to noise ratios.

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