The lysosomal cell death (LCD) pathway is a caspase 3-independent cell death pathway that has been suggested as a possible target for cancer therapy, making the development of sensitive and specific high-throughput (HT) assays to identify LCD inducers highly desirable. In this study, we report a two-step HT screening platform to reliably identify such molecules. First, using a robust HT primary screen based on propidium iodide uptake, we identified compounds that kill through nonapoptotic pathways. A phenotypic image-based assay using a galectin-3 (Gal-3) reporter was then used to further classify hits based on lysosomal permeabilization, a hallmark of LCD. The identification of permeabilized lysosomes in our image-based assay is not affected by changes in the lysosomal pH, thus resolving an important limitation in currently used methods. We have validated our platform in a screen by identifying 24 LCD inducers, some previously known to induce LCD. Although most LCD inducers were cationic amphiphilic drugs (CADs), we have also identified a non-CAD LCD inducer, which is of great interest in the field. Our data also gave new insights into the biology of LCD, suggesting that lysosomal accumulation and acid sphingomyelinase inhibition are not sufficient or necessary for the induction of LCD. Overall, our results demonstrate a robust HT platform to identify novel LCD inducers that will also be very useful for gaining deeper insights into the molecular mechanism of LCD induction.
Probiotic has been widely used in functional food because of numerous advantages for health. MRS broth is commonly used as standard medium in studying lactobacilli. However, in some communities - like muslim and vegetarian society, components in MRS broth/medium become an issue. Beef extract and peptone – animal derived substances as nitrogen sources in the MRS medium should be avoided for the vegetarian. Meanwhile, for the muslim society, all components must be halal-certified including those animal derived ingredients. Therefore, several alternative sources for beef extract and peptone substitution were studied. Combination of alternative nitrogen sources was applied. In order to increase the effect of the alternative nitrogen sources, alternative carbon sources were also included. This is the first report about effects of L. brevis media components on cells growth to expression level of surface layer protein (Slp). Whey, lactose, sucrose, and galactose showed high contribution to L. brevis growth. However, the tested concentration of those substances were not sufficient to obtain equal bacterial growth and Slp expression than that of MRS broth. In addition, yeast extract appeared necessary to maintain cell wall and Slp expression.
Recombinant human epidermal growth factor (rh-EGF) has high value in therapies for h-EGF deficiency-related diseases. The expression of the h-EGF gene was designed by using the pET21b(+) vector and Escherichia coli BL21(DE3) as the expression host. In a previous study, the sequence of a 6xHis tag without any restriction sites was fused to the h-EGF gene, yet it was not possible to obtain a purified and single rh-EGF by this approach. In this study, we modified the rh-EGF expression vector using site-directed mutagenesis (SDM) to remove the sequence of the 6xHis tag. The vector modification was carried out by inserting a stop codon and the EcoRI restriction site, along with deleting the 6xHis tag sequence. The results of PCR showed non-specific bands, while 2-step cycles PCR produced one non-specific band, and 3-step cycles PCR produced two non-specific bands. After purification of the PCR products, the SDM-recombinant plasmids treated for template plasmid-free product were transformed into E. coli DH5a. Even though the transformation efficiency was low, the planned gene mutations including the deletion of the 6xHis tag and insertion of the stop codon and EcoRI restriction site in plasmid pET21b(+) were successfully carried out. When using this modified vector in expression studies, rh-EGF of a similar size to that of the rh-EGF standard and approximately 1 kDa smaller than the rh-EGF-6xHis of the previous study was obtained.
Human Epidermal Growth Factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices, especially in therapeutic uses of wound healing. Since it has a lot of benefits, production of recombinant hEGF (rhEGF) in a large scale is needed. Some methods have been used in this protein production, one of them was the production of rhEGF using extracellular secretion in Escherichia coli. Previous research have been done using co-expression method with phospholipase C from Bacillus cereus to increase the amount of rhEGF. Phospholipase C B. cereus have been used in several protein expression and was proved that it could increase the secretion of recombinant protein through hydrolytic mechanism of cell membrane. In addition, growth condition is one of some major factors which can affect the yields of produced protein. Different compositions of bacterial growth medium often lead to different result. This paper studies how rich-nutrient Terrifict Broth (TB) medium and Luria Bertani (LB) medium produced different rhEGF results when it was co-expressed with phospholipase C B. cereus. rhEGF was characterized using SDS-PAGE and confirmed by western blot using anti-mouse EGF, and its concentration was measured using ELISA. rhEGF was successfully characterized after co-expression in TB medium and the concentration was 503.48 μg/mL. rhEGF was better produced in TB medium rather than in LB medium since TB medium has richer composition.
The aim of this research is to know activity of ethanol extract from marine spons Aaptos suberitoides in total number and differentiation leukocyte cells which induce by carcinogenic agent Benzo(a)pyrene. METHODS. Mice (Mus musculus) were grouped in 6 group, group I (healthy mice), group II (induce by CMC Na), group III (treatment by cyvlophospamide), group IV (administered by spons extract 500 mg/kg BW), group V (administered by spons extract 1000 mg/kg BW) and group VI (administered by spons extract 1500 mg/kg BW). Benzo(a)pyrene induced for 10 days (5 times). The doses that given is 0,3 gram / 0,2 ml CMC Na. Mice were killed and the blood were taken. The sacrificed and blood sampling were taken. The total number and differentiation of leukocyte cells is made by smear method. RESULT AND DISCUSSION. The result of this examination is there were average total number of leukocyte cells in group I was 10,800 cells/μl, II and VI group were 3,700-5,500 cells/μl, the groups III, IV, V of 2,100-5,500 cells μl. The number of neutrophils and lymphocytes showed a significant difference (P <0.05) while the monocyte was no significant difference (P> 0.05).
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