Comparative analysis of miRNA expressions in different developmental stages of Echinococcus granulosus in mono-phasic and di-phasic culture systems

2020 ◽  
Vol 20 ◽  
Author(s):  
Seifollah Mortezaei ◽  
Ali Afgar ◽  
Balal Sadeghi ◽  
Mohammad Ali Mohammadi ◽  
Seyed Mohammad Mousavi ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
Drug Targets ◽  
Developmental Stages ◽  
Ex Vivo ◽  
Basic Research ◽  
Experimental Investigations ◽  
Intermediate Hosts ◽  
Germinal Layer ◽  
Probable Role

Background:: The dog tapeworm, Echinococcus granulosus, is a zoonotic parasite affecting human and livestock across the globe. Basic research on the molecular biology and genetics of E. granulosus improves our understanding of the biology and potential drug targets in various developmental stages of E. granulosus in both definitive and intermediate hosts. There has been increasing interest in identification of microRNAs in parasitic organisms. The purpose of the current study was to compare the activity of a selected profile of miRNAs in different developmental stages of E. granulosus. Methods:: Different developmental stages of the parasite were obtained from ex vivo as well as in vitro cultured E. granu-losus. MicroRNAs were extracted from the ex vivo germinal layer and invaginated protoscoleces as well as the in vitro gen-erated microcysts, evaginated protoscoleces and strobilated worms. Expression of the selected miRNAs was evaluated by RT-qPCR for each stage. Results:: Four out of five miRNAs were present and active in different developmental stages of E. granulosus. A significant over-expression of miR-61 was observed in germinal layer and during the protoscolex transformation into the microcysts, however miR-10 was more expressed in the mature strobilated forms than the other stages. Let-7 and miR-3489 showed a high expression in germinal layer. Conclusion:: Differential expression of four miRNAs among different in vitro and ex vivo developmental stages of E. granu-losus was documented in the present study. Further experimental investigations are required to elucidate the probable role of the miRNAs in bi-directional differentiation of protoscoleces either into the strobilated worm or to a secondary hydatid cyst and the potential of these miRNAs as drug targets.

Echinococcus granulosus: the effects of praziquantel, in vivo and in vitro, on the ultrastructure of equine strain murine cysts

Parasitology ◽  
1988 ◽  
Vol 96 (2) ◽  
pp. 323-336 ◽  
Author(s):  
K. Sylvia Richards ◽  
D. L. Morris ◽  
D. Daniels ◽  
E. M. Riley
Keyword(s):  
Echinococcus Granulosus ◽  
Structural Organization ◽  
Drug Withdrawal ◽  
Serum Levels ◽  
Germinal Layer ◽  
Ultrastructural Examination ◽  
Hepatic Cysts ◽  
Drug Treatments

SummaryPraziquantel (500 mg/kg) administered orally to BALB/c mice with secondary equine E. granulosus daily for 21, 30 or 30 + 30 days without the drug resulted in the majority of cysts, using bench criteria of turgidity and eosin exclusion, being assessed as ‘alive’. Ultrastructural examination of 54 of these ‘alive’ cysts did not support this conclusion. They all showed increased vesiculation of the germinal layer leading, in many, to the loss of its integrity. Increased mitochondrial numbers occurred frequently. The longer drug treatments appeared to have greater effects on the germinal layer of ‘alive’ cysts and there was no detectable re-establishment of structural organization within 30 days after drug withdrawal. Subjectively, there was no substantial difference between cysts from 4-month and 9-month infections or between affected peritoneal and hepatic cysts. Tissue from collapsed cysts was necrotic. Peak serum levels of praziquantel (6430–6136 μg/l) occurred 5–10 min after drug administration (500 mg/kg) and dropped rapidly to less than 10 μg/l at 3 h. In an in vitro study at praziquantel concentrations of 1000 and 5000 μg/l over a 10-day period, most cysts were judged ‘alive’ by bench criteria but showed ultrastructurally a time- and concentration-dependent loss of integrity identical to that seen in vivo. Turgidity and eosin exclusion therefore underestimate the effect of praziquantel and the results indicate that in vitro experiments can fulfil a legitimate preliminary role in a hydatid chemotherapy programme.

scholarly journals Development of A Novel Highly Selective TLR8 Agonist for Cancer Immunotherapy

2020 ◽  
Author(s):  
Yuxun Wang ◽  
Heping Yang ◽  
Huanping Li ◽  
Shuda Zhao ◽  
Yikun Zeng ◽  
...  
Keyword(s):  
Immune Responses ◽  
Drug Targets ◽  
Ex Vivo ◽  
Single Agent ◽  
Cancer Drug ◽  
Phase I Clinical Trials ◽  
Tlr Agonists ◽  
Favorable Safety

ABSTRACTToll-like receptors (TLRs) are a family of proteins that recognize pathogen associated molecular patterns (PAMPs). Their primary function is to activate innate immune responses while also involved in facilitating adaptive immune responses. Different TLRs exert distinct functions by activating varied immune cascades. Several TLRs are being pursued as cancer drug targets. We discovered a novel, highly potent and selective small molecule TLR8 agonist DN052. DN052 exhibited strong in vitro cellular activity with EC50 at 6.7 nM and was highly selective for TLR8 over other TLRs including TLR4, 7 and 9. The selectivity profile distinguished DN052 from all other TLR agonists currently in clinical development. DN052 displayed excellent in vitro ADMET and in vivo PK profiles. DN052 potently inhibited tumor growth as a single agent. Moreover, combination of DN052 with the immune checkpoint inhibitor, selected targeted therapeutics or chemotherapeutic drugs further enhanced efficacy of single agents. Mechanistically, treatment with DN052 resulted in strong induction of pro-inflammatory cytokines in ex vivo human PBMC assay and in vivo monkey study. GLP toxicity studies in rats and monkeys demonstrated favorable safety profile. This led to the advancement of DN052 into phase I clinical trials.

scholarly journals Development of a novel TLR8 agonist for cancer immunotherapy

2020 ◽  
Vol 1 (1) ◽  
Author(s):  
Yuxun Wang ◽  
Heping Yang ◽  
Huanping Li ◽  
Shuda Zhao ◽  
Yikun Zeng ◽  
...  
Keyword(s):  
Immune Responses ◽  
Drug Targets ◽  
Ex Vivo ◽  
Phase 1 ◽  
Single Agent ◽  
Cancer Drug ◽  
Favorable Safety ◽  
Molecular Patterns

Abstract Toll-like receptors (TLRs) are a family of proteins that recognize pathogen associated molecular patterns (PAMPs). Their primary function is to activate innate immune responses while also involved in facilitating adaptive immune responses. Different TLRs exert distinct functions by activating varied immune cascades. Several TLRs are being pursued as cancer drug targets. We discovered a novel, highly potent and selective small molecule TLR8 agonist DN052. DN052 exhibited strong in vitro cellular activity with EC50 at 6.7 nM and was highly selective for TLR8 over other TLRs including TLR4, 7 and 9. DN052 displayed excellent in vitro ADMET and in vivo PK profiles. DN052 potently inhibited tumor growth as a single agent. Moreover, combination of DN052 with the immune checkpoint inhibitor, selected targeted therapeutics or chemotherapeutic drugs further enhanced efficacy of single agents. Mechanistically, treatment with DN052 resulted in strong induction of pro-inflammatory cytokines in ex vivo human PBMC assay and in vivo monkey study. GLP toxicity studies in rats and monkeys demonstrated favorable safety profile. This led to the advancement of DN052 into phase 1 clinical trials.

The effect of albendazole sulfoxide on the expression of miR-61 and let-7 in different in vitro developmental stages of Echinococcus granulosus

Acta Tropica ◽  
2019 ◽  
Vol 195 ◽  
pp. 97-102 ◽  
Author(s):  
Seifollah Mortezaei ◽  
Ali Afgar ◽  
Mohammad Ali Mohammadi ◽  
Seyed Mohammad Mousavi ◽  
Balal Sadeghi ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
Developmental Stages ◽  
Albendazole Sulfoxide

scholarly journals Reduced calcium storage blunts calcium signaling in Toxoplasma bradyzoites and impedes motility and egress

2021 ◽  
Author(s):  
Yong Fu ◽  
Kevin M Brown ◽  
Nathaniel G Jones ◽  
Silvia N J Moreno ◽  
L. David Sibley
Keyword(s):  
Developmental Stages ◽  
Ex Vivo ◽  
Acute Infection ◽  
Gliding Motility ◽  
Lytic Cycle ◽  
Calcium Storage ◽  
Calcium Atpases ◽  
Dual Reporter

Toxoplasma gondii has evolved different developmental stages of tachyzoites for disseminating during acute infection and bradyzoites for establishing chronic infection. Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unknown. Here we show that Ca2+ signals and egress by bradyzoites in response to agonists are highly restricted. Development of dual-reporter parasites revealed dampened calcium responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of calcium ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisome. Once liberated from cysts by trypsin digestion, bradyzoites displayed weaker gliding motility associated with Ca2+ oscillations compared with tachyzoites, although gliding motility of bradyzoites was enhanced by uptake of exogenous Ca2+. Collectively, our findings indicate that bradyzoites exhibit dampened Ca2+ signaling due to a decreased amount of stored Ca2+, limiting microneme secretion and egress, likely constituting an adaptation to their long-term intracellular niche.

scholarly journals Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Yong Fu ◽  
Kevin M Brown ◽  
Nathaniel G Jones ◽  
Silvia NJ Moreno ◽  
L David Sibley
Keyword(s):  
Developmental Stages ◽  
Ex Vivo ◽  
Energy Levels ◽  
Acute Infection ◽  
Lytic Cycle ◽  
Physiological Plasticity ◽  
Dual Reporter ◽  
Energy Stores ◽  
Intracellular Ca

Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e. tachyzoites) and for establishing chronic infection (i.e. bradyzoites). Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca2+ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of Ca2+-ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca2+ rapidly restored their intracellular Ca2+ and ATP stores leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.

scholarly journals In Vitro and Ex Vivo Models for Functional Testing of Therapeutic Anti-scarring Drug Targets in Keloids

2019 ◽  
Vol 8 (12) ◽  
pp. 655-670
Author(s):  
Jyoti R. Sharma ◽  
Maribanyana Lebeko ◽  
Elvis B. Kidzeru ◽  
Nonhlanhla P. Khumalo ◽  
Ardeshir Bayat
Keyword(s):  
Drug Targets ◽  
Ex Vivo ◽  
Functional Testing

scholarly journals Amphiphilic Stilbene Derivatives Attenuate the Neurotoxicity of Soluble Aβ42 Oligomers by Controlling Their Interactions with Cell Membranes

2021 ◽  
Author(s):  
Zhengxin Yu ◽  
Weijie Guo ◽  
Hong-Jun Cho ◽  
Shrey Patel ◽  
Liviu M. Mirica
Keyword(s):  
Binding Affinity ◽  
Drug Targets ◽  
Ex Vivo ◽  
Amyloid Β ◽  
Docking Studies ◽  
Aβ Oligomers ◽  
Human Neuroblastoma ◽  
Amphiphilic Compounds ◽  
Stilbene Derivatives

<p>Misfolded proteins or polypeptides commonly observed in neurodegenerative diseases, including Alzheimer’s disease (AD), are promising drug targets for developing therapeutic agents. To target the amyloid-β (Aβ) plaques and oligomers, the hallmarks of AD, we have developed twelve amphiphilic small molecules with different hydrophobic and hydrophilic fragments. <i>In vitro</i> binding experiments (i.e., fluorescence saturation assays) demonstrated that these amphiphilic compounds show high binding affinity to both Aβ plaques and oligomers, and six of them exhibit even higher binding affinity toward Aβ oligomers. These amphiphilic compounds can also <a>label <i>ex vivo </i>Aβ species in the brain sections of transgenic AD mice, as shown by immunostaining with an Aβ antibody. </a>Molecular docking studies were performed to help understand the structure-affinity relationships. To our delight, four amphiphilic compounds can alleviate Cu<sup>2+</sup>-Aβ induced toxicity in mouse neuroblastoma N2a via cell toxicity assays. In addition, <a>confocal</a> fluorescence imaging studies provided evidence that compounds ZY-15-MT and ZY-15-OMe can disrupt <a>the interactions between Aβ oligomers and human neuroblastoma SH-SY5Y cell</a> membranes. Overall, these studies suggest that developing compounds with amphiphilic properties that target Aβ oligomers can be an effective strategy for small molecule AD therapeutics.</p>

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