An in vitro assay of the effect of lysine oxidation end-product, α-aminoadipic acid, on the redox status and gene expression in probiotic Lactobacillus reuteri PL503

AbstractThis study was designed to gain information about the underlying mechanisms of the effects of a food-occurring free oxidized amino acid, α-aminoadipic acid (AAA), on the probiotic Lactobacillus reuteri PL503. This bacterium was incubated in colonic-simulated conditions (37 °C for 24 h in microaerophilic conditions) and exposed to three food-compatible AAA concentrations, namely, 1 mM, 5 mM, and 10 mM. A control group with no AAA exposure was also considered. Each of the four experimental conditions was replicated three times and samplings were collected at 12, 16, 20, and 24 h. The downregulation of the uspA gene by AAA (0.5-fold decrease as compared to control) suggests that AAA is identified as a potential chemical threat. The dhaT gene, implicated in the antioxidant defense, was found to be upregulated in bacteria treated with 1 and 5 mM AAA (up to twofold increase, as compared to control), which suggest the ability of the oxidized amino acid to impair the redox status of the bacterium. In fact, AAA caused an increased production of reactive oxygen species (ROS) and the accretion of post-translational changes (protein carbonylation) in L. reuteri (up to 13 nmol allysine/mg protein vs 1.8 nmol allysine/mg protein in control). These results suggest that probiotic bacteria identify oxidized amino acids as harmful species and activate mechanisms that may protect themselves and the host against their noxious effects.

Download Full-text

Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

Viruses ◽

10.3390/v13061092 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1092

Author(s):

János András Mótyán ◽

Márió Miczi ◽

Stephen Oroszlan ◽

József Tőzsér

Keyword(s):

Amino Acid ◽

Zinc Finger ◽

Cleavage Site ◽

Potential Role ◽

Binding Mode ◽

Interaction Energies ◽

Vitro Assay ◽

Hiv 1

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

Download Full-text

In vitro activity of zinc oxide-eugenol and glass ionomer cements on Candida albicans

Brazilian Oral Research ◽

10.1590/s1806-83242005000200011 ◽

2005 ◽

Vol 19 (2) ◽

pp. 134-138 ◽

Cited By ~ 6

Author(s):

Anna Carolina Aguiar Cassanho ◽

Aletéia Massula Fernandes ◽

Luciane Dias de Oliveira ◽

Claudio Antonio Talge Carvalho ◽

Antonio Olavo Cardoso Jorge ◽

...

Keyword(s):

Zinc Oxide ◽

Candida Albicans ◽

Control Group ◽

Glass Ionomer ◽

Experimental Conditions ◽

Mean Values ◽

Zinc Oxide Eugenol ◽

Colony Forming Units ◽

And Control

The aim of this study was to evaluate in vitro the antimicrobial activity of glass ionomer (GIC) and zinc oxide-eugenol (ZOE) cements against Candida albicans. Standardized GIC and ZOE specimens were maintained in contact with C. albicans suspension (1 <FONT FACE=Symbol>´</FONT> 10(6) cells/ml) at 37°C for 24 h, 48 h or 7 days. A control group without any testing cement was included. After the incubation period, aliquots of 0.1 ml were plated on Sabouraud's agar, and then the number of colonies was counted. The results were expressed as values of logarithms of colony-forming units per milliliter (log CFU/mL) and were analyzed statistically by Kruskal-Wallis ANOVA. After 48 h of incubation, the ZOE group presented no growth of C. albicans. GIC and control groups presented similar mean values at all tested periods. According to the results obtained, it could be concluded that, under the experimental conditions, ZOE cement was more effective in vitro against C. albicans than GIC.

Download Full-text

A Novel Type V CRISPR System with Potential for Genome Editing in the Liver

Blood ◽

10.1182/blood-2021-154505 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1862-1862

Author(s):

Gregory J. Cost ◽

Morayma Temoche-Diaz ◽

Janet Mei ◽

Cristina N. Butterfield ◽

Christopher T. Brown ◽

...

Keyword(s):

Amino Acid ◽

Genome Editing ◽

Mammalian Cells ◽

Conflicts Of Interest ◽

Attractive Alternative ◽

Vitro Assay ◽

Crispr System ◽

Type V

Abstract RNA guided CRISPR genome editing systems can make specific changes to the genomes of mammalian cells and have the potential to treat a range of diseases including those that can be addressed by editing hepatocytes. Attempts to edit the liver in vivo have relied almost exclusively on the Cas9 nucleases derived from the bacteria S treptococcus pyogenes or Staphylococcus aureus to which humans are commonly exposed. Pre-existing immunity to both these proteins has been reported in humans which raises concerns about their in vivo application. In silico analysis of a large metagenomics database followed by testing in mammalian cells in culture identified MG29-1, a novel CRISPR system which is a member of the Type V family but exhibits only 41 % amino acid identity to Francisella tularensis Cas12a/cpf1. MG29-1 is a 1280 amino acid RNA programmable nuclease that utilizes a single guide RNA comprised of a 22 nucleotide (nt) constant region and a 20 to 25 nt spacer, recognizes the PAM KTTN (predicted frequency 1 in 16 bp) and generates staggered cuts. MG29-1 was derived from a sample taken from a hydrothermal vent and it is therefore unlikely that humans will have developed pre-existing immunity to this protein. A screen for sgRNA targeting serum albumin in the mouse liver cell line Hepa1-6 identified 6 guides that generated more than 80% INDELS. The MG29-1 system was optimized for in vivo delivery by screening chemical modifications to the guide that improve stability in mammalian cell lysates while retaining or improving editing activity. Two lead guide chemistries were evaluated in mice using MG29-1 mRNA and sgRNA packaged in lipid nanoparticles (LNP). Three days after a single IV administration on-target editing was evaluated in the liver by Sanger sequencing. The sgRNA that was the most stable in the in vitro assay generated INDELS that ranged from 20 to 25% while a sgRNA with lower in vitro stability failed to generate detectable INDELs. The short sgRNA and small protein size compared to spCas9 makes MG29-1 an attractive alternative to spCas9 for in vivo editing applications. Evaluation of the potential of MG29-1 to perform gene knockouts and gene additions via non-homologous end joining is ongoing. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Silica nanoparticles inducing the apoptosis of spermatocyte cell through microRNA-450b-3p targeting MTCH2-mediating mitochondrial signaling pathway

10.21203/rs.3.rs-24673/v1 ◽

2020 ◽

Author(s):

Guiqing Zhou ◽

Jianhui Liu ◽

Xiangyang Li ◽

Yujian Sang ◽

Yue Zhang ◽

...

Keyword(s):

Cytochrome C ◽

Silica Nanoparticles ◽

Caspase 3 ◽

Reproductive Toxicity ◽

Control Group ◽

Caspase 9 ◽

Protein Expressions ◽

Underlying Mechanisms

Abstract Background: Silica nanoparticles (SiNPs) are found in environmental particulate matter and are proven to have adverse effects on fertility. The relationship and underlying mechanisms between miRNAs and apoptosis induced by SiNPs during spermatogenesis is currently ambiguous. Experimental design: The present study was designed to investigate the role of miRNA-450b-3p in the reproductive toxicity caused by SiNPs. In vivo, 40 male mice were randomly divided into control and SiNPs groups, 20 per group. The mice in the SiNPs group were administrated 20 mg/kg SiNPs by tracheal perfusion once every 5 days, for 35 days, and the control group were given the equivalent of a normal luminal saline. In vitro, spermatocyte cells were divided into 0 and 5 μg/mL SiNPs groups, after passaged for 30 generations, the GC-2spd cells in 5 μg/mL SiNPs groups were transfected with miRNA-450b-3p and its mimic and inhibitor. Results: In vivo, the results showed that SiNPs damaged tissue structures of testis, decreased the quantity and quality of the sperm, reduced the expression of miR-450b-3p, and increased the protein expressions of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 in the testis. In vitro, SiNPs obviously repressed the viability and increased the LDH level and apoptosis rate, decreased the levels of the miR-450b-3p, significantly enhanced the protein expressions of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, Caspase-3; while the mimic of miR-450b-3p reversed the changes induced by SiNPs, but inhibitor further promoted the effects induced by SiNPs.Conclusion: The result suggested that SiNPs could induce the spermatocyte apoptosis by inhibiting the miR-450b-3p expression to target promoting the MTCH2 resulting in activating mitochondrial apoptotic signaling pathways in the spermatocyte cells.

Download Full-text

KIF23 Promotes Gastric Cancer by Stimulating Cell Proliferation

Disease Markers ◽

10.1155/2019/9751923 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 8

Author(s):

Xiao-Long Li ◽

Ya-Ming Ji ◽

Rui Song ◽

Xiao-Ning Li ◽

Lan-Shuan Guo

Keyword(s):

Gastric Cancer ◽

Cell Proliferation ◽

Malignant Tumors ◽

Vital Role ◽

Gastric Cancer Cells ◽

Vitro Assay ◽

Multiple Tumor ◽

Axon Elongation ◽

Underlying Mechanisms

Gastric cancer (GC) is one of the most aggressive malignant tumors with low early diagnosis and high metastasis. Despite progress in treatment, to combat this disease, a better understanding of the underlying mechanisms and novel therapeutic targets is needed. KIF23, which belongs to the KIF family, plays a vital role in various cell processes, such as cytoplasm separation and axon elongation. Nowadays, KIF23 has been found to be highly expressed in multiple tumor tissues and cells, suggesting a potential link between KIF23 and tumorigenesis. Herein, we reported that KIF23 expression was correlated with poor prognosis of gastric cancer and found an association between KIF23 and pTNM stage. An in vitro assay proved that the proliferation of gastric cancer cells was significantly inhibited, which is caused by KIF23 depletion. Additionally, knockdown of KIF23 resulted in a marked inhibition of cell proliferation of gastric cancer in mice, with significant downregulation of Ki67 and PCNA expression. In conclusion, these data indicate that KIF23 is a potential therapeutic target for gastric cancer treatment.

Download Full-text

Radiometric studies of mycobacterium tuberculosis

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46651987000100003 ◽

1987 ◽

Vol 29 (1) ◽

pp. 18-25

Author(s):

Edwaldo E. Camargo ◽

Judith A. Kertcher ◽

Marianne F. Chen ◽

Patricia Charache ◽

Henry N. Wagner Jr

Keyword(s):

Palmitic Acid ◽

Lauric Acid ◽

Bacterial Suspension ◽

Dilution Method ◽

Agar Dilution Method ◽

Experimental Conditions ◽

Vitro Assay ◽

Minimal Inhibitory Concentrations ◽

Different Strains

An in vitro assay system that included automated radiometric quantification of 14CO2 released as a result of oxidation of 14C- substrates was applied for studying the metabolic activity of M. tuberculosis under various experimental conditions. These experiments included the study of a) mtabolic pathways, b) detection times for various inoculum sizes, c) effect of filtration on reproducibility of results, d) influence of stress environment e) minimal inhibitory concentrations for isoniazid, streptomycin, ethambutol and rifampin, and f) generation times of M. tuberculosis and M. bovis. These organisms were found to metabolize 14C-for-mate, (U-14C) acetate, (U-14C) glycerol, (1-14C) palmitic acid, 1-14C) lauric acid, (U-14C) L-malic acid, (U-14C) D-glucose, and (U-14C) D-glucose, but not (1-14C) L-glucose, (U-14C) glycine, or (U-14C) pyruvate to 14CO2. By using either 14C-for-mate, (1-14C) palmitic acid, or (1-14C) lauric acid, 10(7) organisms/vial could be detected within 24 48 hours and as few as 10 organisms/vial within 16-20 days. Reproducible results could be obtained without filtering the bacterial suspension, provided that the organisms were grown in liquid 7H9 medium with 0.05% polysorbate 80 and homogenized prior to the study. Drugs that block protein synthesis were found to have lower minimal inhibitory concentrations with the radiometric method when compared to the conventional agar dilution method. The mean generation time obtained for M. bovis and different strains of M. tuberculosis with various substrates was 9 ± 1 hours.

Download Full-text

3-Pentylcatechol, a Non-Allergenic Urushiol Derivative, Displays Anti-Helicobacter pylori Activity In Vivo

Pharmaceuticals ◽

10.3390/ph13110384 ◽

2020 ◽

Vol 13 (11) ◽

pp. 384

Author(s):

Hang Yeon Jeong ◽

Tae Ho Lee ◽

Ju Gyeong Kim ◽

Sueun Lee ◽

Changjong Moon ◽

...

Keyword(s):

Helicobacter Pylori ◽

Triple Therapy ◽

Inflammatory Cells ◽

Control Group ◽

Vitro Assay ◽

Low Concentrations ◽

H Pylori ◽

Stomach Mucous Membrane

We previously reported that 3-pentylcatechol (PC), a synthetic non-allergenic urushiol derivative, inhibited the growth of Helicobacter pylori in an in vitro assay using nutrient agar and broth. In this study, we aimed to investigate the in vivo antimicrobial activity of PC against H. pylori growing in the stomach mucous membrane. Four-week-old male C57BL/6 mice (n = 4) were orally inoculated with H. pylori Sydney Strain-1 (SS-1) for 8 weeks. Thereafter, the mice received PC (1, 5, and 15 mg/kg) and triple therapy (omeprazole, 0.7 mg/kg; metronidazole, 16.7 mg/kg; clarithromycin, 16.7 mg/kg, reference groups) once daily for 10 days. Infiltration of inflammatory cells in gastric tissue was greater in the H. pylori-infected group compared with the control group and lower in both the triple therapy- and PC-treated groups. In addition, upregulation of cytokine mRNA was reversed after infection, upon administration of triple therapy and PC. Interestingly, PC was more effective than triple therapy at all doses, even at 1/15th the dose of triple therapy. In addition, PC demonstrated synergism with triple therapy, even at low concentrations. The results suggest that PC may be more effective against H. pylori than established antibiotics.

Download Full-text

In Vitro Assay for Predicting Protein Efficiency Ratio as Measured by Rat Bioassay: Collaborative Study

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/65.4.798 ◽

1982 ◽

Vol 65 (4) ◽

pp. 798-809 ◽

Cited By ~ 2

Author(s):

Lowell D Satterlee ◽

James G Kendrick ◽

Henry F Marshall ◽

Duane K Jewell ◽

Rida A Ali ◽

...

Keyword(s):

Amino Acid ◽

Protein Efficiency Ratio ◽

Muscle Protein ◽

Collaborative Study ◽

Protein Digestibility ◽

Efficiency Ratio ◽

Breakfast Cereal ◽

Vitro Assay ◽

Food Ingredients

Abstract Seven laboratories collaborated in testing the calculated protein efficiency ratio (C-PER and DC-PER). The collaborative study required each laboratory to analyze 6 foods and a control protein (ANRC casein) for in vitro apparent protein digestibility, amino acid composition, and PER via rat bioassay. The 6 foods or food ingredients tested were nonfat dry milk, cooked chicken muscle, protein-fortified dry breakfast cereal, textured soy protein, oat-based dry breakfast cereal, and durum wheat flour. Data obtained from the study were analyzed statistically for the intralaboratory variation for each method of analysis (i.e., amino acid analysis, PER, etc.). The ability of the C-PER to rapidly predict rat PER was also measured. The C-PER and DC-PER methods were adopted official first action.

Download Full-text

Probiotic properties of native Lactobacillus spp. strains for dairy calves

Beneficial Microbes ◽

10.3920/bm2017.0131 ◽

2018 ◽

Vol 9 (4) ◽

pp. 613-624 ◽

Cited By ~ 4

Author(s):

S. Fernández ◽

M. Fraga ◽

E. Silveyra ◽

A.N. Trombert ◽

A. Rabaza ◽

...

Keyword(s):

Lactobacillus Reuteri ◽

Bacterial Species ◽

Control Group ◽

Probiotic Properties ◽

Real Time Quantitative Pcr ◽

Treatment And Prevention ◽

Probiotic Strains ◽

Lactobacillus Spp

The use of native microorganisms with probiotic capacity is an alternative tool for the treatment and prevention of several diseases that affect animals, such as neonatal calf diarrhoea. The selection of probiotic strains within a collection is based on different in vitro and in vivo assays, which predict their potential. The aim of this study was to characterise a group of native Lactobacillus spp. strains isolated from faeces of healthy calves using an in vitro approach and to assess their ability to colonise the gastrointestinal tract (GIT) of calves. Native Lactobacillus spp. strains were evaluated on their capacity to survive low pH conditions and bile salts presence, biofilm formation and adhesion to both mucus and Caco-2 cells. Based on the in vitro characterisation, four strains (Lactobacillus johnsonii TP1.1, Lactobacillus reuteri TP1.3B, L. johnsonii TP1.6 and Lactobacillus amylovorus TP8.7) were selected to evaluate their capacity to colonise and persist in the GIT of calves. The assessment of enteric persistence involved an in vivo assay with oral administration of probiotics and quantification in faeces of the administered bacterial species with real-time quantitative PCR (qPCR). The study was conducted using 15 calves (1-month-old) which were divided into five groups of three animals, four of which were treated with four different selected strains and one was the control group. Strains TP1.3B and TP1.6 managed to persist in treated animals until ten days after the end of the administration period, indicating that they could be promising candidates for the design of probiotics for calves.

Download Full-text

Nutritional availability of methionine, lysine and tryptophan in fish meals, as assessed with biological, microbiological and dye-binding assay procedures

British Journal Of Nutrition ◽

10.1079/bjn19850067 ◽

1985 ◽

Vol 53 (3) ◽

pp. 575-586 ◽

Cited By ~ 2

Author(s):

D. Hewitt ◽

J. E. Ford

Keyword(s):

Amino Acid ◽

Tetrahymena Pyriformis ◽

Poor Quality ◽

Analysis Procedure ◽

Chick Growth ◽

Vitro Assay ◽

Similar Treatment ◽

Dye Binding ◽

Available Lysine

1. In vitro assay procedures were applied in the measurement of available amino acids in a selection of fish meals representing good- and poor-quality product. Results were assessed by comparing them with results from chick-growth assays.2. Available methionine and tryptophan were assayed microbiologically with Streptococcus zymogenes, after predigestion of the test samples with papain or pronase. Results for methionine were correlated with chick-growth assays (r 0.86 for papain, 0.91 for pronase; P < 0.01). Compared with the chick assays, papain tended to give lower, and pronase higher, results. Finer milling of the test samples did not influence the pronase values.3. Results for available tryptophan were also correlated with chick-growth assays (r 0.95 for papain, 0.96 for pronase; P < 0.001). Compared with the chick values, papain gave markedly lower results and pronasem marginally higher ones. Finer milling of the test samples increased the papain values by about 50% but had no effect with pronase.4. Available lysine was assayed microbiologically with Tetrahymena pyriformis and with a dye-binding procedure (DBL). The results correlated with the chick-growth assays (r 0.99 for DBL, P < 0.001; 0.85 for Tetrahymena, P < 0.01) but both methods overrated the poorer-quality samples.5. True nitrogen digestibilities and amino acid digestibilities were determined with chickens by the 'ileal analysis' procedure: the amino acid digestibilities were significantly higher and similar to the corresponding availabilities as measured in chick-growth assays.6. Ball milling a poor-quality fish meal caused a marked fall in its N digestibility, whereas similar treatment of a good-quality meal caused a slight increase. An explanation for this finding is proposed.7. Strep. zymogenes assays following pronase digestion of the test samples gave precise and acceptably accurate measures of the biologically available methionine and tryptophan in the test samples. For available lysine, Tetrahymena and DBL values for the poorer-quality samples were notably higher than the chick-growth assay; possible reasons for this are discussed. The ileal analysis procedure underestimated true N digestibility.

Download Full-text