The diversity of a collection of 49 Lactococcus garvieae strains, including isolates of dairy, fish, meat, vegetable and cereal origin, was explored using a molecular polyphasic approach comprising PCR-ribotyping, REP and RAPD-PCR analyses and a multilocus restriction typing (MLRT) carried out on six partial genes (atpA, tuf, dltA, als, gapC, and galP). This approach allowed high-resolution cluster analysis in which two major groups were distinguishable: one group included dairy isolates, the other group meat isolates. Unexpectedly, of the 12 strains coming from fish, four grouped with dairy isolates, whereas the others with meat isolates. Likewise, strains isolated from vegetables allocated between the two main groups. These findings revealed high variability within the species at both gene and genome levels. The observed genetic heterogeneity among L. garvieae strains was not entirely coherent with the ecological niche of origin of the strains, but rather supports the idea of an early separation of L. garvieae population into two independent genomic lineages.
Lactococcus garvieae is a fish pathogen and an emerging zoonotic opportunistic pathogen as well as a component of natural microbiota in dairy products. Here, we present the first report of a genome sequence of L. garvieae TB25, isolated from a dairy source, and that of L. garvieae LG9, isolated from rainbow trout.
Previous studies have reported that compounds bearing an arylamide linked to a heterocyclic planar ring have successfully inhibited the hemopexin-like domain (PEX9) of matrix metalloproteinase 9 (MMP9). PEX9 has been suggested to be more selectively targeted than MMP9’s catalytic domain in a degrading extracellular matrix under some pathologic conditions, especially in cancer. In this study, we aim to synthesize and evaluate 10 arylamide compounds as MMP9 inhibitors through an enzymatic assay as well as a cellular assay. The mechanism of inhibition for the most active compounds was investigated via molecular dynamics simulation (MD). Molecular docking was performed using AutoDock4.0 with PEX9 as the protein model to predict the binding of the designed compounds. The synthesis was carried out by reacting aniline derivatives with 3-bromopropanoyl chloride using pyridine as the catalyst at room temperature. The MMP9 assay was conducted using the FRET-based MMP9 kits protocol and gelatin zymography assay. The cytotoxicity assay was done using the MTT method, and the MD simulation was performed using AMBER16. Assay on MMP9 demonstrated activities of three compounds (2, 7, and 9) with more than 50% inhibition. Further inhibition on MMP9 expressed by 4T1 showed that two compounds (7 and 9) inhibited its gelatinolytic activity more than 50%. The cytotoxicity assay against 4T1 cells results in the inhibition of the cell growth with an EC50 of 125 μM and 132 μM for 7 and 9, respectively. The MD simulation explained a stable interaction of 7 and 9 in PEX9 at 100 ns with a free energy of binding of −8.03 kcal/mol and −6.41 kcal/mol, respectively. Arylamides have potential effects as selective MMP9 inhibitors in inhibiting breast cancer cell progression.
Matrix metalloproteinase9 (MMP9) is known to be highly expressed during metastatic cancer where most known potential inhibitors failed in the clinical trials. This study aims to select local plants in our state, as anti-breast cancer agent with hemopexin-like domain of MMP9 (PEX9) as the selective protein target. In silico screening for PEX9 inhibitors was performed from our in house-natural compound database to identify the plants. The selected plants were extracted using methanol and then a step-by-step in vitro screening against MMP9 was performed from its crude extract, partitions until fractions using FRET-based assay. The partitions were obtained by performing liquid–liquid extraction on the methanol extract using n-hexane, ethylacetate, n-butanol, and water representing nonpolar to polar solvents. The fractions were made from the selected partition, which demonstrated the best inhibition percentage toward MMP9, using column chromatography. Of the 200 compounds screened, 20 compounds that scored the binding affinity −11.2 to −8.1 kcal/mol toward PEX9 were selected as top hits. The binding of these hits were thoroughly investigated and linked to the plants which they were reported to be isolated from. Six of the eight crude extracts demonstrated inhibition toward MMP9 with the IC50 24 to 823 µg/mL. The partitions (1 mg/mL) of Ageratum conyzoides aerial parts and Ixora coccinea leaves showed inhibition 94% and 96%, whereas their fractions showed IC50 43 and 116 µg/mL, respectively toward MMP9. Using MTT assay, the crude extract of Ageratum exhibited IC50 22 and 229 µg/mL against 4T1 and T47D cell proliferations, respectively with a high safety index concluding its potential anti-breast cancer from herbal.
Objective: Ursolic acid was a compound found in Hedyotis corymbosa L., (HCoL) while berberine found in Tinospora crispa (TCa) which are proven to have cytotoxic effect to cancer cells. This research aims to review the effect of cisplatin, ethanolic extract of HCoL and TCa to the sensitivity increase on breast cancer cells, which will be confirmed through apoptosis induction and cell cycle modulation.Methods: The cytotoxic effect was tested using 3-(4,5-dimethylthiazol-2-il)-2,5-diphenyltetrazolium bromide assay on T47D cell using the IC50 parameter. The combination was tested by determining their combination index (CI) and cell viability. The combination effect of apoptosis induction and cell cycle modulation was observed using flow cytometry method.Results: The cytotoxic test result of the combination shows CI value of below 1 at the concentration of HCoL ethanolic extract as much as 1 μg/mL, TCa ethanolic extract as much as 6 μg/mL, and cisplatin as much as 2,5 μM. The combination of HCoL ethanolic extract, TCa ethanolic extract, and cisplatin results in phase S cell accumulation (29.98%) on breast cancer cell T47D and was able to induce apoptosis.Conclusion: The result proves that ethanolic extract of HCoL and TCa can be developed as a cochemotherapeutic agent with cisplatin to increase the effectivity of breast cancer treatment.
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