scholarly journals Programmed knockout mutation of liver fluke granulin, Ov-grn-1, attenuates virulence and impedes malignant transformation during chronic opisthorchiasis

2021 ◽  
Author(s):  
Sujittra Chaiyadet ◽  
Sirikachorn Tangkawattana ◽  
Michael J Smout ◽  
Wannaporn Ittiprasert ◽  
Victoria H. Mann ◽  
...  
Keyword(s):  
Growth Factor ◽  
Liver Fluke ◽  
Gene Knockout ◽  
Lao Pdr ◽  
Immunohistochemical Analysis ◽  
Hamster Model ◽  
Thin Sections ◽  
Tract Disease ◽  
Liver Flukes ◽  
Hepatobiliary Tract

AbstractInfection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA; bile duct cancer) in the Lower Mekong River Basin countries including Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using gene edited liver flukes in a hamster model of CCA involving concurrent infection and administration of sub-carcinogenic levels of nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We produced programmed gene knockout flukes (ΔOv-grn-1) by delivery of a CRISPR/Cas9/gRNA system by square wave electroporation. Targeted genome sequencing confirmed Cas9-catalyzed mutations in target parasite genes, and the rapid depletion of transcripts and the targeted proteins. Hamsters were infected with gene edited larval parasites and exposed to sub-carcinogenic levels of dimethyl nitrosamine in drinking water. Whereas Ov-grn-1 gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control parasites. Specifically, immunohistochemical analysis of thin sections of hamster livers revealed markedly less fibrosis surrounding flukes and less global liver fibrosis as a result of infection with ΔOv-grn-1 worms, minimal biliary epithelial cell proliferation, and markedly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high grade CCA when infected with ΔOv-grn-1 flukes compared to controls. The clinically-relevant pathophysiological phenotype of the livers of the hamsters confirmed a role for this parasite secreted growth factor in morbidity and malignancy during opisthorchiasis.

scholarly journals Discovery of Carcinogenic Liver Fluke Metacercariae in Second Intermediate Hosts and Surveillance on Fish-Borne Trematode Metacercariae Infections in Mekong Region of Myanmar

2020 ◽  
Vol 17 (11) ◽  
pp. 4108
Author(s):  
Ei Ei Phyo Myint ◽  
Amornpun Sereemaspun ◽  
Joacim Rocklöv ◽  
Choosak Nithikathkul
Keyword(s):  
Liver Fluke ◽  
Lao Pdr ◽  
Intermediate Hosts ◽  
Mekong Region ◽  
Zoonotic Infections ◽  
Lack Of Information ◽  
The Status ◽  
Control And Prevention ◽  
Liver Flukes ◽  
Trematode Infections

Countries of lower Mekong regions are highly alarmed by the spread of fish-borne trematode infections, i.e., small liver flukes and minute intestinal flukes especially in Thailand, Lao People’s Democratic Republic (Lao PDR), Vietnam, Cambodia and Myanmar. Moreover; the incidence of cholangiocarcinoma has also been increasing in the endemic area of liver fluke infections. Only a few reports have been published concerning the fish-borne trematodes infections in the central region of Myanmar. However; there is still a lack of information regarding the status of trematodes infections in second intermediate hosts in the Mekong region of Myanmar. Therefore, we conducted surveillance on the distribution of trematode metacercariae in small cyprinoid fishes collected from the Mekong region of Myanmar. A total of 689 fishes (12 different species of cyprinoid fishes) have been collected and examined by pepsin digestion methods. We discovered four species of fish-borne trematode metacercariae infections, i.e., carcinogenic liver fluke, Opisthorchis viverrini; minute intestinal flukes, Haplorchis taichui; Haplorchis pumilio and Haplorchoides sp. in Tachileik, the Mekong Region of Myanmar. The outcome of this study could be a useful index for the fish-borne zoonotic trematode epidemiology in the Mekong area. Besides, the results of our study contribute to filling the gap of information necessary for the control and prevention of fish-borne trematode zoonotic infections in the Mekong region.

scholarly journals Utilizing the ABC Transporter for Growth Factor Production by fleQ Deletion Mutant of Pseudomonas fluorescens

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 679
Author(s):  
Benedict-Uy Fabia ◽  
Joshua Bingwa ◽  
Jiyeon Park ◽  
Nguyen-Mihn Hieu ◽  
Jung-Hoon Ahn
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Pseudomonas Fluorescens ◽  
Abc Transporter ◽  
Transforming Growth Factor Beta ◽  
Deletion Mutant ◽  
Transforming Growth Factor ◽  
Gene Knockout ◽  
Type I ◽  
Double Deletion

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also secretes unnecessary background proteins that complicate protein purification and other downstream processes. One of the background proteins produced in large amounts is FliC, a flagellin protein. In this study, the master regulator of flagella gene expression, fleQ, was deleted from P. fluorescens Δtp, a lipase and protease double-deletion mutant, via targeted gene knockout. FleQ directs flagella synthesis, so the new strain, P. fluorescens ΔfleQ, does not produce flagella-related proteins. This not only simplifies purification but also makes P. fluorescens ΔfleQ an eco-friendly expression host because it will not survive outside a controlled environment. Six recombinant growth factors, namely, insulin-like growth factors I and II, beta-nerve growth factor, fibroblast growth factor 1, transforming growth factor beta, and tumor necrosis factor beta, prepared using our supercharging method, were successfully secreted by P. fluorescens ΔfleQ. Our findings demonstrate the potential of P. fluorescens ΔfleQ, combined with our supercharging process, as a PMF.

Pattern of the insulin-like growth factor II gene expression during early mouse embryogenesis

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 151-159 ◽  
Author(s):  
J.E. Lee ◽  
J. Pintar ◽  
A. Efstratiadis
Keyword(s):  
Growth Factor ◽  
Immunohistochemical Analysis ◽  
Primitive Streak ◽  
Insulin Like Growth Factor ◽  
Surface Ectoderm ◽  
Factor Ii ◽  
Extraembryonic Mesoderm ◽  
Early Mouse ◽  
Lateral Mesoderm

The mouse insulin-like growth factor II (IGF-II) gene encodes a polypeptide that plays a role in embryonic growth. We have examined the temporal and spatial pattern of expression of this gene in sections of the mouse conceptus between embryonic days 4.0 and 8.5 by in situ hybridization. Abundant IGF-II transcripts were detected in all the trophectodermal derivatives, after implantation. Labeling was then observed in primitive endoderm, but was transient and disappeared after formation of the yolk sac. Expression was next detected in extraembryonic mesoderm at the early primitive streak stage. Labeling in the embryo proper appeared first at the late primitive streak/neural plate stage in lateral mesoderm and in anterior-proximal cells located between the visceral endoderm and the most cranial region of the embryonic ectoderm. The position of the latter cells suggests that their descendants are likely to participate in the formation of the heart and the epithelium of the ventral and lateral walls of the foregut, where intense labeling was observed at the neural fold stage. Hybridization was also detected in cranial mesenchyme, including neural crest cells. The intensity of hybridization signal increased progressively in paraxial (presomitic and somitic) mesoderm, while declining in the ectoplacental cone. The neuroectoderm and surface ectoderm did not exhibit hybridization at any stage. Immunohistochemical analysis indicated co-localization of IGF-II transcripts, translated pre-pro-IGF-II, and the cognate IGF-II/mannose-6-phosphate receptor. These correlations are consistent with the hypothesis that IGF-II has an autocrine function.

Pirfenidone inhibits PDGF isoforms in bleomycin hamster model of lung fibrosis at the translational level

1999 ◽  
Vol 276 (2) ◽  
pp. L311-L318 ◽  
Author(s):  
G. Gurujeyalakshmi ◽  
M. A. Hollinger ◽  
S. N. Giri
Keyword(s):  
Growth Factor ◽  
Lung Fibrosis ◽  
Platelet Derived Growth Factor ◽  
Pcr Analysis ◽  
Saline Control ◽  
Protective Effects ◽  
Rt Pcr ◽  
Hamster Model ◽  
Pdgf Isoforms ◽  
Translational Level

Pirfenidone (PD) is known for its antifibrotic effects in the bleomycin (BL) hamster model of lung fibrosis. We evaluated whether pretreatment of hamsters with PD could influence the effects of BL-induced overexpression of platelet-derived growth factor (PDGF)-A and PDGF-B genes and proteins in the same model of lung fibrosis. We demonstrate elevated levels of PDGF-A and PDGF-B mRNAs in bronchoalveolar lavage (BAL) cells from lungs of BL-treated compared with saline control hamsters by RT-PCR analysis. However, these levels were not altered in BAL cells obtained from BL-treated hamsters on diets containing 0.5% PD. Western blot analysis of BAL fluid for PDGF isoforms demonstrated that PD treatment inhibited the synthesis of both PDGF-A and PDGF-B isoforms. PD treatment also decreased the mitogenic activity in the BAL fluid from BL-treated hamster lungs. Taken together, these data provide evidence that the protective effects of PD against BL-induced lung fibrosis may be mediated by a reduction in PDGF isoforms produced by lung macrophages.

Characterisation of two calmodulin-like proteins from the liver fluke, Fasciola hepatica

2007 ◽  
Vol 388 (6) ◽  
pp. 593-599 ◽  
Author(s):  
Sean L. Russell ◽  
Neil V. McFerran ◽  
Elizabeth M. Hoey ◽  
Alan Trudgett ◽  
David J. Timson
Keyword(s):  
Metal Binding ◽  
Metal Ion ◽  
Liver Fluke ◽  
Fasciola Hepatica ◽  
Expression Vectors ◽  
Metal Ion Binding ◽  
Ion Sensing ◽  
Calcium Magnesium ◽  
Ion Binding Sites ◽  
Liver Flukes

Abstract Calmodulin is a calcium ion-sensing signalling protein found in eukaryotics. Two calmodulin-like gene sequences were identified in an EST library from adult liver flukes. One codes for a protein (FhCaM1) homologous to mammalian calmodulins (98% identity), whereas the other protein (FhCaM2) has only 41% identity. These genes were cloned into expression vectors and the recombinant proteins were expressed in Escherichia coli. Gel shift assays showed that both proteins bind to calcium, magnesium and zinc ions. Homology models have been built for both proteins. As expected, FhCaM1 has a highly similar structure to other calmodulins. Although FhCaM2 has a similar fold, its surface charge is higher than FhCaM1. One of the potential metal ion-binding sites has lower metal-ion co-ordination capability, while another has an adjacent lysine residue, both of which may decrease the metal-binding affinity. These differences may reflect a specialised role for FhCaM2 in the liver fluke.

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