in vivo assays
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2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Milena do Amaral ◽  
Ana Camila Oliveira Freitas ◽  
Ariana Silva Santos ◽  
Everton Cruz dos Santos ◽  
Monaliza Macêdo Ferreira ◽  
...  

AbstractProtease inhibitors (PIs) are important biotechnological tools of interest in agriculture. Usually they are the first proteins to be activated in plant-induced resistance against pathogens. Therefore, the aim of this study was to characterize a Theobroma cacao trypsin inhibitor called TcTI. The ORF has 740 bp encoding a protein with 219 amino acids, molecular weight of approximately 23 kDa. rTcTI was expressed in the soluble fraction of Escherichia coli strain Rosetta [DE3]. The purified His-Tag rTcTI showed inhibitory activity against commercial porcine trypsin. The kinetic model demonstrated that rTcTI is a competitive inhibitor, with a Ki value of 4.08 × 10–7 mol L−1. The thermostability analysis of rTcTI showed that 100% inhibitory activity was retained up to 60 °C and that at 70–80 °C, inhibitory activity remained above 50%. Circular dichroism analysis indicated that the protein is rich in loop structures and β-conformations. Furthermore, in vivo assays against Helicoverpa armigera larvae were also performed with rTcTI in 0.1 mg mL−1 spray solutions on leaf surfaces, which reduced larval growth by 70% compared to the control treatment. Trials with cocoa plants infected with Mp showed a greater accumulation of TcTI in resistant varieties of T. cacao, so this regulation may be associated with different isoforms of TcTI. This inhibitor has biochemical characteristics suitable for biotechnological applications as well as in resistance studies of T. cacao and other crops.


2021 ◽  
Vol 12 (12) ◽  
Author(s):  
Ping Liu ◽  
Ruiting Fu ◽  
Kai Chen ◽  
Lu Zhang ◽  
Shasha Wang ◽  
...  

AbstractHigh-grade serous ovarian cancer (HGSOC) is a common and lethal cancer of the female reproductive system. Long non-coding RNAs (lncRNAs) are aberrantly expressed in various cancers and play crucial roles in tumour progression. However, their function and molecular mechanism in HGSOC remain largely unknown. Based on public databases and bioinformatics analyses, the overexpression of lncRNA CTBP1-DT in HGSOC tissues was detected and validated in a cohort of HGSOC tissues. High expression of lncRNA CTBP1-DT was associated with poor prognosis and was an independent risk factor for survival. Overexpression of lncRNA CTBP1-DT promoted malignant biological behaviour of HGSOC cells, whereas its depletion induced growth arrest of HGSOC cells by vitro and in vivo assays. Mechanistically, lncRNA CTBP1-DT could competitively bind to miR-188-5p to protect MAP3K3 from degradation. Moreover, our results revealed that ETV5 could specifically interact with the promoter of lncRNA CTBP1-DT and activate its transcription. Collectively, these results reveal a novel ETV5/lncRNA CTBP1-DT/miR-188-5p/MAP3K3 pathway for HGSOC progression and suggest that lncRNA CTBP1-DT might be a potential biomarker and therapeutic target for HGSOC.


2021 ◽  
Vol 14 (3) ◽  
pp. 165-172
Author(s):  
Juliane Felicissimo ◽  
◽  
Danielle Marconato ◽  
Nayara Emídio ◽  
Lara Carvalho ◽  
...  

In this study, Licochalcone A (LicoA) was investigated in in vitro and in vivo assays. The survival of worms in culture, the pattern of oviposition, the count of intact tubers and the integrity of the coat were adopted in the in vitro tests. After the animals were perfused, the number of worms recovered, their location and the oogram study were the parameters analyzed to signal the existence of potential schistosomicidal activity in vivo. We observed reduction on the survival, integument integrity and reproduction of adult worms in vitro. Murine models did not show a significant difference in the parasitological parameters analyzed that indicate activity against the worms with an oral single dose of 25 mg/ kg of LicoA or two intraperitoneal injection of 50 mg/ kg LicoA. Nevertheless, it is too early to completely exclude the schistosomicide activity of LicoA, considering that the used dosage form could not provide a regular absorption of the drug.


2021 ◽  
Author(s):  
Harshad R. Thacore ◽  
Abdul Gaffar ◽  
Seiyoung Yun ◽  
Agnes L. Chenine ◽  
Maria G. Ferrari ◽  
...  

Abstract SARS-CoV-2 and coronaviruses, enveloped RNA viruses, are major causes of acute human respiratory diseases. The aim of the study was to investigate the broad-spectrum antiviral effects of ethyl lauroyl arginine hydrochloride (ELAH) in in vitro and in vivo assays. Cell-based assays found that the pseudovirus VSV-SARS-CoV-2 was inhibited with an EC50 of 15 micrograms/ml, with complete inhibition achieved at 110 micrograms/ml. The effects were comparable to those observed with anti-SARS-CoV-2 antibody neutralization assays against VSV-SARS-CoV-2. Intranasal administration of the Wuhan strain of SARS-CoV-2 treated in vitro with ELAH inhibited the disease symptoms caused by the virus in a Syrian hamster model compared to that caused by the same dose of virus treated in vitro with medium alone. Subgenomic RNA and total RNA viral load were concomitantly reduced in the treated animals compared with the control group. In cell-based studies, pretreatment of susceptible cells with 1-10 micrograms/ml ELAH inhibited the attachment of the virus to the cells, as measured by cytopathic and high-resolution scanning electron microscopy (SEM) effects, suggesting that the primary mode of ELAH action was due to preventing the attachment of the virus to the cells. Collectively, the data suggest that ELAH could be a promising agent for the prevention of SARS infection through nasopharyngeal surfaces.


2021 ◽  
Vol 10 (3) ◽  
Author(s):  
Haasini Nandyala ◽  
Ariel Pham ◽  
Anushka Wagle ◽  
Hansika Daggolu ◽  
Amrita Guha ◽  
...  

Abstract: Trigonella foenum-graecum has been shown to have anti-diabetic potential through a wide variety of in-vivo assays as well as by inhibiting enzymes such as alpha-glucosidase and alpha-amylase. Studies have indicated the therapeutic potential of different phytoconstituents found in Trigonella foenum-graceum including diosgenin trigonelline, 4-hydroxyisoleucine, leucine, and L-lysine. This study aims to find novel protein targets that these specific phytoconstituents from fenugreek can bind to, thereby helping to treat diabetes mellitus. Through multiple stages of molecular docking and analyzing the binding sites in comparison to previously reported inhibitors, a suitable and novel target protein for four of the compounds was found and the relevance to diabetes was discussed, setting up these compounds as novel inhibitors for the target proteins.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Robin Gradin ◽  
Andy Forreryd ◽  
Ulrika Mattson ◽  
Anders Jerre ◽  
Henrik Johansson

AbstractHundreds of chemicals have been identified as skin sensitizers. These are chemicals that possess the ability to induce hypersensitivity reactions in humans, giving rise to a condition termed allergic contact dermatitis. The capacity to limit hazardous exposure to such chemicals depends upon the ability to accurately identify and characterize their skin sensitizing potency. This has traditionally been accomplished using animal models, but their widespread use offers challenges from both an ethical and a scientific perspective. Comprehensive efforts have been made by the scientific community to develop new approach methodologies (NAMs) capable of replacing in vivo assays, which have successfully yielded several methods that can identify skin sensitizers. However, there is still a lack of new approaches that can effectively measure skin sensitizing potency. We present a novel methodology for quantitative assessment of skin sensitizing potency, which is founded on the already established protocols of the GARDskin assay. This approach analyses dose–response relationships in the GARDskin assay to identify chemical-specific concentrations that are sufficient to induce a positive response in the assay. We here compare results for 22 skin sensitizers analyzed using this method with both human and LLNA potency reference data and show that the results correlate strongly and significantly with both metrics (rLLNA = 0.81, p = 9.1 × 10–5; rHuman = 0.74, p = 1.5 × 10–3). In conclusion, the results suggest that the proposed GARDskin dose–response methodology provides a novel non-animal approach for quantitative potency assessment, which could represent an important step towards reducing the need for in vivo experiments.


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