Identification and Quantification of Nonviable Lactobacillus pentosus Cells in a Health Food Product

Background: The lack of formal protocol to verify the nonviable cell probiotic product authenticity blocks its registration and supervision process. Objective: To develop a protocol for identification, enumeration, and purity determination of a Lactobacillus pentosus nonviable cell product. Methods: The 16S ribosomal RNA (rRNA) sequencing, 16S rRNA metagenomic analysis, whole-genome sequencing, matrix-assisted laser desorption ionization–time of flight MS (MALDI-TOF-MS), and FACSMicroCount™ system were applied to establish a protocol of identification, enumeration and purity determination of a Lactobacillus pentosus nonviable cell product. Results: The 1530 bp of 16S rRNA sequence could only identify the bacteria at genus level, but the MALDI-TOF-MS could identify both the nonviable cell and fresh culture to species level with high confidence. Metagenomic analysis of the 16S rRNA amplicon could recognize Lactobacillus as the dominant genus in the nonviable cell product. The total number of matching k-mers between the nonviable cell product and the L. pentosus BGM48 in the GenBank was the highest. The 95% confidence interval of the nonviable cell concentration in the product was determined as 4.31–4.68 × 1010 cells/g through the BD FACSMicroCount system. Conclusions: This validation protocol offers an executable approach that can verify microbial contents in nonviable cell products and ensure the compliance with label claims. Highlights: The established validation protocol could determine the nonviable cell species through MALDI-TOF-MS, the concentration through FACSMicroCount system, and the purity and strain level identification through metagenomic analysis of 16S rRNA and the genomic deoxyribonucleic acid.

Extracts fromVatica diospyroidesType SS Fruit Show Low Dose Activity against MDA-MB-468 Breast Cancer Cell-Line via Apoptotic Action

Very strong antiproliferative action ofV. diospyroidestype SS fruit extracts (IC50range of 1.60-17.45 µg/mL) in MDA-MB-468 cell-line was observed in an MTT assay. After dosing of an extract concentration at half IC50to cell line for 24 to 72 hours, treated cells were subjected to Annexin V-FITC/PI binding assay, followed by FACS and western blot analyses. Significant apoptotic death was observed with all extract treatments and both exposure times. Dosing with acetone extract of pericarp and cotyledon induced the highest apoptotic populations (33 and 32%, resp.), with the lowest populations of viable cells (65 and 67%, resp.). During 24 to 72 hours of dosing with methanolic extract of pericarp, the populations of viable and early apoptotic cells decreased significantly from 72.40 to 71.32% and from 12.00 to 6.36%, respectively, while the late apoptotic and nonviable cell populations continuously increased from 15.30 to 19.18% and from 0.30 to 3.14%, respectively. The expression of Bax increased within 12–48 hours of dosing, confirming apoptosis induced by time-dependent responses. The mutant p53 of MDA-MB-468 cells was expressed. Our results indicate that apoptosis and time-dependent therapeutic actions contribute to the cytotoxic effects ofV. diospyroidestype SS fruit on MDA-MB-468 cell.

L929 cell response to root perforation repair cements: an in vitro cytotoxicity assay

This study compared the cytotoxicity of an experimental epoxy-resin and calcium hydroxide-based cement (MBPc), gray mineral trioxide aggregate (MTA) and white mineral trioxide aggregate (WMTA) using the agar overlay method with neutral red dye. L929 cells were seeded into 6-well culture plates where 48-h set test materials were placed on the agar overlay, in triplicate. Teflon and natural rubber served as negative and positive controls. After an incubation period of 24 h at 37ºC in a humidified atmosphere of 5% CO2 in air, a discolored area around the samples and the positive controls could be observed and measured per quadrant. The mean values were compared and converted into grades to classify the results according to the table of cytotoxicity grades according to the Standard Operating Procedures (SOP) of the Oswaldo Cruz Foundation, Brazil. The nonviable cell areas and the morphological changes in the cells were observed with an inverted microscope. The results showed grade 1 (slight) for the two types of MTA (p>0.05) and grade 2 (mild) for the MBPc (p<0.001). All samples met the requirements of the test as none of the cultures showed reactivity higher than grade 2.

Nonviable Burkholderia mallei Induces a Mixed Th1- and Th2-Like Cytokine Response in BALB/c Mice

ABSTRACT Nonviable cell preparations of Burkholderia mallei, the causative agent of glanders, were evaluated as potential vaccine candidates in a BALB/c murine model. Three different B. mallei cell preparations plus Alhydrogel were evaluated: a heat-killed preparation, an irradiation-inactivated preparation, and a preparation of a capsule-negative mutant strain which had been irradiation inactivated. BALB/c mice were vaccinated twice with the different B. mallei preparations, and spleens and sera were collected to determine their cellular and humoral immune responses. All three bacterial cell preparations had essentially the same results in two cellular immune response assays. In a splenocyte proliferation assay, the amount of cell proliferation in response to the homologous immunogen, concanavalin A, or lipopolysaccharide was similar for all the cell preparations. Also, splenocytes from the inoculated mice expressed interleukin 2 (IL-2), gamma interferon, and small amounts of IL-4 and IL-5, and more IL-10 cytokine in the presence of the homologous antigen. When the immunoglobulin subclasses from these mice were examined, they all produced higher levels of IgG1 than IgG2a subclasses. The higher ratio of IgG1 to IgG2a was not due to the amount of the immunogen or the adjuvant (Alhydrogel) used in the BALB/c mice. The cell preparations did not protect the vaccinated mice from a live challenge (>300 50% lethal doses). Our results suggest that in BALB/c mice, a mixed T-helper-cell-like response to nonviable B. mallei is obtained, as demonstrated by a Th1- and Th2-like cytokine response and a Th2-like subclass immunoglobulin response. This may be the reason for the inability of the B. mallei cells that were examined as candidate vaccines to protect the mice from a live challenge.