An Efficient DNA Extraction Method for Lactobacillus casei, a Difficult-to-Lyse Bacterium

Alimolaei, Mojtaba; Golchin, Mehdi

doi:10.17795/ijep32472

Cited by 12 publications

(9 citation statements)

References 18 publications

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“…The strains of molds, yeasts and LAB were identified by sequencing of the internal transcribed spacer (ITS) region using the ITS1 and ITS4 primers for molds and yeasts, and the 16S rRNA gene fragment using the 27F and 1492R primers for LAB [23]. Genomic DNA from each strain was isolated as previously described [22,24] and used as the template for ITS or 16S rRNA amplification. DNA amplification was performed in a thermocycler with an initial denaturation step for 5 min at 95 o C, followed by 35 cycles of denaturation for 30 sec at 95 o C, annealing for 30 sec at 55 o C, and extension for 1 min at 72 o C and then a final extension for 5 min at 72 o C. PCR products were sequenced by Macrogen, South Korea.…”

Section: Isolation and Identification Of Microorganisms In The Loog-pmentioning

confidence: 99%

Fermented Unpolished Black Rice (Oryza sativa L.) Inhibits Melanogenesis via ERK, p38, and AKT Phosphorylation in B16F10 Melanoma Cells

Sangkaew

Yompakdee

2020

J. Microbiol. Biotechnol.

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Melanin is a skin pigment that plays an important role in protection from the harmful effects caused by ultraviolet (UV) light. Overproduction and accumulation of melanin can result in pigmented patches and skin discolorations, such as chloasma, solar lentigo and freckles, that lead to perceived esthetic problems [1, 2]. The production of melanin pigment, also called "melanogenesis, " is a complicated process regulated by various melanogenesis enzymes, such as tyrosinase, and tyrosinase-related protein 1 (TYRP1) and TYRP2. Among these enzymes, tyrosinase is the essential enzyme in melanogenesis. It catalyzes two initial steps in this process; namely hydroxylation of L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and the oxidation of L-DOPA into dopaquinone. These melanogenesis enzymes are regulated by microphthalmia-associated transcription factor (MITF) [3-5]. In addition, melanogenesis is modulated by mitogen-activated protein kinase (MAPK) signaling via extracellular signal-regulated kinases (ERK) and p38 MAPK. The phosphorylation of ERK (Thr202/Tyr204) and p38 (Thr180/Tyr182) leads to MITF degradation, which plays a pivotal role in suppressing melanin production [6-8]. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is also known to be involved in the regulation of melanogenesis, where the phosphorylation of Akt leads to negative regulation of melanogenesis [9, 10]. Additionally, UV light radiation causes the synthesis of reactive oxygen species (ROS) that are also involved in the regulation of melanin synthesis. Hence, ROS scavengers and inhibitors of ROS generation may down-regulate melanogenesis [11]. Currently, known depigmenting agents, such as kojic acid, arbutin and linoleic acid, are used as cosmetic agents Melanin is a major factor that darkens skin color as one of the defense systems to prevent the harmful effects of UV light. However, darkened skin from the localized or systemic accumulation of melanin is viewed in many cultures as an esthetic problem. Consequentially, searching for antimelanogenic agents from natural sources is very popular worldwide. Previous screening of fermented rice products, obtained from various rice cultivars fermented with different sources of loog-pang (Thai traditional fermentation starter), revealed that the highest ability to reduce the melanin content in B16F10 melanoma cells was from unpolished black rice fermented with a defined starter mixture of microbes isolated from loog-pang E11. The aim of this study was to investigate the mechanism of the fermented unpolished black rice (FUBR) on the inhibition of melanogenesis in B16F10 melanoma cells. The strongest reduction of cellular melanin content was found in the FUBR sap (FUBRS). The melanin reduction activity was consistent with the significant decrease in the intracellular tyrosinase activity. The FUBRS showed no cytotoxic effect to B16F10 melanoma or Hs68 human fibroblast cell lines. It also significantly reduced the transcript and protein expression levels of tyrosinase, tyrosinase-related pr...

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Section: Isolation and Identification Of Microorganisms In The Loog-pmentioning

confidence: 99%

Fermented Unpolished Black Rice (Oryza sativa L.) Inhibits Melanogenesis via ERK, p38, and AKT Phosphorylation in B16F10 Melanoma Cells

Sangkaew

Yompakdee

2020

J. Microbiol. Biotechnol.

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“…After centrifugation (12000 rpm, 10 min) the bacterial pellets were used for total DNA extraction. 56 These pellets were washed with NaCl-EDTA (30 mM NaCl, 2 mM EDTA, pH = 8.0) and resuspended in lysis buffer (Tris-HCl 20 mM, EDTA 2 mM, pH = 8.0), lysozyme (20 mg/mL) and triton X-100 (1% v/v). After incubation for 2h at 37°C, proteinase K (20 mg/ mL) and RNase A (0.2 mg/mL) were added and incubated for 1h at 55°C.…”

Section: Lgg Conditioned Media Preparationmentioning

confidence: 99%

Lactobacillus rhamnosus GG prevents epithelial barrier dysfunction induced by interferon-gamma and fecal supernatants from irritable bowel syndrome patients in human intestinal enteroids and colonoids

et al. 2018

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Disruption of intestinal barrier homeostasis is an important pathogenic factor in conditions such as irritable bowel syndrome (IBS). Lactobacillus rhamnosus GG (LGG) improves IBS symptoms through unclear mechanisms. Previous studies utilizing colorectal adenocarcinoma cell lines showed that LGG metabolites prevented interferon gamma (IFN-gamma) induced barrier damage but the model employed limited these findings. We aimed to interrogate the protective effects of LGG on epithelial barrier function using human intestinal epithelial cultures (enteroids and colonoids) as a more physiologic model. To investigate how LGG affects epithelial barrier function, we measured FITC-Dextran (FD4) flux across the epithelium as well as tight junction zonula occludens 1 (ZO-1) and occludin (OCLN) expression. Colonoids were incubated with fecal supernatants from IBS patients (IBS-FSN) and healthy controls in the presence or absence of LGG to examine changes in gut permeability. Enteroids incubated with IFN-gamma demonstrated a downregulation of OCLN and ZO-1 expression by 67% and 50%, respectively (p<0.05). This was accompanied by increased paracellular permeability as shown by leakage of FD4. Pretreatment of enteroids with LGG prevented these changes and normalized OCLN and ZO-1 to control levels. These actions were independent of its action against apoptosis. However, these protective effects were not seen with LGG cell wall extracts, LGG DNA, or denatured (boiled) LGG. Intriguingly, IBS-FSN injected into colonoids increased paracellular permeability, which was prevented by LGG. LGG, likely due to secreted proteins, protects against epithelial barrier dysfunction. Bacterial-derived factors to modulate gut barrier function may be a treatment option in disorders such as IBS.

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“…Lactobacillus casei (Nagaoka et al . 1990 ; Alimolaei and Golchin 2016 ). In general, mechanical lysis and bead beating increase the extraction efficiency of Gram positive bacteria (Costea et al .…”

Section: Discussionmentioning

confidence: 99%

Evaluation of commercially available DNA extraction kits for the analysis of the broiler chicken cecal microbiota

Pankoke

Maus

Loh

et al. 2019

FEMS Microbiology Letters

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16S rRNA gene amplicon sequencing is a state of the art technology to analyze bacterial communities via microbiome profiling. Choosing an appropriate DNA extraction protocol is crucial for characterizing the microbial community and can be challenging, especially when preliminary knowledge about the sample matrix is scarce. The aim of the present study was to evaluate seven commercial DNA extraction kits suitable for 16S rRNA gene amplicon sequencing of the bacterial community of the chicken cecum, taking into account different criteria such as high technical reproducibility, high bacterial diversity and easy handling. The DNA extraction kits differed strongly with respect to extractable DNA quantity, DNA quality, technical reproducibility and bacterial diversity determined after 16S rRNA gene amplicon sequencing and subsequent bioinformatic and biostatistical data processing. While some of the DNA extraction protocols under-represented specific bacterial community members, the removal of PCR inhibitors supported technical reproducibility and subsequently enhanced the recovered bacterial diversity from the chicken cecum community. In conclusion, the removal of PCR inhibitors from the sample matrix seemed to be one of the main drivers for a consistent representation of the bacterial community even of low abundant taxa in chicken cecum samples.

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An Efficient DNA Extraction Method for Lactobacillus casei, a Difficult-to-Lyse Bacterium

Cited by 12 publications

References 18 publications

Fermented Unpolished Black Rice (Oryza sativa L.) Inhibits Melanogenesis via ERK, p38, and AKT Phosphorylation in B16F10 Melanoma Cells

Fermented Unpolished Black Rice (Oryza sativa L.) Inhibits Melanogenesis via ERK, p38, and AKT Phosphorylation in B16F10 Melanoma Cells

Lactobacillus rhamnosus GG prevents epithelial barrier dysfunction induced by interferon-gamma and fecal supernatants from irritable bowel syndrome patients in human intestinal enteroids and colonoids

Evaluation of commercially available DNA extraction kits for the analysis of the broiler chicken cecal microbiota

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