Lauryl Gallate Activity and Streptococcus mutans: Its Effects on Biofilm Formation, Acidogenicity and Gene Expression.

Streptococcus mutans bacterium is implicated in the pathogenesis of dental caries due to the production of biofilm and organic acids from dietary sucrose. Despite the availability of various means of prophylaxis, caries still has a high worldwide prevalence. Therefore, it is important to find new pharmaceuticals to inhibit S. mutans biofilm formation and acidogenicity. The aim of the current study was to evaluate the activity of lauryl gallate (dodecyl gallate) against S. mutans acidogenicity, the expression of biofilm-associated genes, and biofilm development on solid surfaces (polystyrene, glass). The biofilm quantities produced by S. mutans bacteria were assessed using colorimetric and optical profilometry techniques. Acidogenicity was evaluated by measuring the pH of the biofilm growth medium with microelectrode. Assessment of the expression of gene coding for glucan-binding protein B (gbpB), glucosyltranferases B, -C, -D (gtfB, -C, -D), and the F-ATPase ß subunit of F1 protein (atpD) was carried out using a quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The results demonstrate the capacity of lauryl gallate to significantly inhibit S. mutans acidogenicity and biofilm development on solid surfaces, in a dose-dependent manner, compared to untreated bacteria (p < 0.05). The highest activity of lauryl gallate occurred at a concentration of 98.98 µM, at which it suppressed biofilm formation by 100% and lowered pH levels by 98%. The effect of lauryl gallate treatment on gene expression changes, as demonstrated by our RT-qPCR data, was limited to the gtfD gene only, was a significant (48%) decrease in gene expression, obtained for the biofilm-producing bacteria, while a 300% increase in fold change for the same gene occurred in the planktonic cells. It is important to note that in previous studies we showed a broader effect of related derivatives. However, a similar magnitude of difference in effects between biofilm and planktonic cells for the atpD gene was obtained after treatment with octyl gallate and reverse magnitude for the same gene after treatment with ethyl gallate. Therefore, to ascertain the possible direct or indirect effects of lauryl gallate, as well as octyl gallate and ethyl gallate, more research is needed to examine the effects on the amount of enzymes and on the enzymatic activity of the products of the affected genes that are involved in the production and maintenance of biofilm by S. mutans.

Suppressive Effects of Octyl Gallate on Streptococcus mutans Biofilm Formation, Acidogenicity, and Gene Expression.

The accumulation of biofilm by Streptococcus mutans bacteria on hard tooth tissues leads to dental caries, which remains one of the most prevalent oral diseases. Hence, the development of new antibiofilm agents is of critical importance. The current study reports the results from testing the effectiveness of octyl gallate (C8-OG) against: (1) S. mutans biofilm formation on solid surfaces (polystyrene, glass), (2) acidogenicity, (3) and the expression of biofilm-related genes. The amount of biofilm formed by S. mutans bacteria was evaluated using the colorimetric method and optical profilometry. The pH of the biofilm growth medium was measured with microelectrode. A quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to assess the expression of genes encoding glucan binding protein B (gbpB), glucosyltransferases B, -C, -D (gtfB, -C, -D), and the F-ATPase ß subunit of the F1 protein (atpD). The results show that C8-OG significantly diminished biofilm formation by exposed S. mutans on solid surfaces and suppressed acidogenicity in a dose-dependent manner, compared to unexposed bacteria (p < 0.05). The C8-OG concentration of 100.24 µM inhibited S. mutans biofilm development on solid surfaces by 100% and prevented a decrease in pH levels by 99%. In addition, the RT-qPCR data demonstrate that the biofilm-producing bacteria treated with C8-OG underwent a significant reduction in gene expression in the case of the four genes under study (gbpB, gtfC, gtfD, and atpD), and there was a slight decrease in expression of the gtfB gene. However, C8-OG treatments did not produce significant expression change compared to the control for the planktonic cells, although there was a significant increase for the atpD gene. Therefore, C8-OG might be a potent antibiofilm and/or anticaries agent for oral formulations that aim to reduce the prevalence of dental caries.

Inhibitory Effects of Ethyl Gallate on Streptococcus mutans Biofilm Formation by Optical Profilometry and Gene Expression Analysis.

This study aimed to test the effectiveness of ethyl gallate (EG) against S. mutans biofilm formation on solid surfaces (polystyrene, glass) and acidogenicity, and to examine the effect on expression of related genes. The biofilm that is formed by S. mutans bacteria was evaluated using colorimetric assay and optical profilometry, while the pH of the biofilm growth medium was measured with microelectrode. The expression of genes encoding glucan binding protein B (gbpB), glucosyltranferases B, -C, -D (gtfB, -C, -D) and F-ATPase (atpD, atpF) was assessed using a quantitative reverse transcription-polymerase chain reaction (RT-qPCR). It was revealed that all of the EG concentrations significantly suppressed S. mutans biofilm build-up on polystyrene and glass surfaces, and inhibited acidogenicity, in a dose-dependent manner, compared to the activity of untreated bacteria (p < 0.05). The highest concentration of EG (3.53 mM) reduced biofilm formation on polystyrene and glass surfaces by 68% and more than 91%, respectively, and prevented a decrease in pH levels by 95%. The RT-qPCR data demonstrate that the biofilm-producing bacteria treated with EG underwent significant gene expression changes involving the gtfC (a 98.6 increase in fold change), gtfB gene (a 47.5 increase in fold change) and the gbpB gene (a 13.8 increase in fold change). However, for the other genes tested (gtfD, atpD and atpF), the EG treatments did not produce significant expression change compared to the control. EG produced significant gene expression change in three genes-gtfC, gtfB, and gbpB; it has the capacity to inhibit S. mutans biofilm formation on solid surfaces (polystyrene, glass), as well as acidogenicity. Therefore, EG might be used as an antibiofilm and/or anticaries agent for oral formulations in order to reduce the prevalence of dental caries.

Indexing Natural Products for Their Potential Anti-Diabetic Activity: Filtering and Mapping Discriminative Physicochemical Properties.

Diabetes mellitus (DM) poses a major health problem, for which there is an unmet need to develop novel drugs. The application of in silico techniques and optimization algorithms is instrumental to achieving this goal. A set of 97 approved anti-diabetic drugs, representing the active domain, and a set of 2892 natural products, representing the inactive domain, were used to construct predictive models and to index anti-diabetic bioactivity. Our recently-developed approach of 'iterative stochastic elimination' was utilized. This article describes a highly discriminative and robust model, with an area under the curve above 0.96. Using the indexing model and a mix ratio of 1:1000 (active/inactive), 65% of the anti-diabetic drugs in the sample were captured in the top 1% of the screened compounds, compared to 1% in the random model. Some of the natural products that scored highly as potential anti-diabetic drug candidates are disclosed. One of those natural products is caffeine, which is noted in the scientific literature as having the capability to decrease blood glucose levels. The other nine phytochemicals await evaluation in a wet lab for their anti-diabetic activity. The indexing model proposed herein is useful for the virtual screening of large chemical databases and for the construction of anti-diabetes focused libraries.

Inhibiting mutant KRAS G12D gene expression using novel peptide nucleic acid-based antisense: A potential new drug candidate for pancreatic cancer.

KRAS mutations, which are the main cause of the pathogenesis of lethal pancreatic adenocarcinomas, impair the functioning of the GTPase subunit, thus rendering it constitutively active and signaling intracellular pathways that end with cell transformation. In the present study, the AsPC-1 cell line, which has a G12D-mutated KRAS gene sequence, was utilized as a cellular model to test peptide nucleic acid-based antisense technology. The use of peptide nucleic acids (PNAs) that are built to exhibit improved hybridization specificity and have an affinity for complementary RNA and DNA sequences, as well as a simple chemical structure and high biological stability that affords resistance to nucleases and proteases, enabled targeting of the KRAS-mutated gene to inhibit its expression at the translation level. Because PNA-based antisense molecules should be capable of binding to KRAS mRNA sequences, PNAs were utilized to target the mRNA of the mutated KRAS gene, a strategy that could lead to the development of a novel drug for pancreatic cancer. Moreover, it was demonstrated that introducing new PNA to cells inhibited the growth of cancer cells and induced apoptotic death and, notably, that it can inhibit G12D-mutated KRAS gene expression, as demonstrated by RT-PCR and western blotting. Altogether, these data strongly suggest that the use of PNA-based antisense agents is an attractive therapeutic approach to treating KRAS-driven cancers and may lead to the development of novel drugs that target the expression of other mutated genes.

A New Approach for Indexing Honey for Its Heath/Medicinal Benefits: Visualization of the Concept by Indexing Based on Antioxidant and Antibacterial Activities.

Background: The goals of the current study were to address a new concept termed a health benefits' index (HBI) and to verify the type of correlation between the pricing of honey and its HBI/medicinal properties. Diverse types of honey from different origins and places were investigated for their antioxidant and antimicrobial activity. Methods: We have utilized a modified protocol of the DPPH assay for measuring free radical scavenging and the microdilution test for the determination of antibacterial/antifungal minimum inhibitory concentrations (MICs). MICs were determined against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Candida albicans microorganisms. Employing a "combined benefits approach" enabled us to attach to each honey type a unique number of HBI that correlate with honey health and medicinal values. Results: The various types of honey demonstrated significant but variable antioxidant, antibacterial, and antifungal activities. Types of wildflower-labeled honey were found to have a wide range of HBI values and medicinal properties, probably due to their containing different nectar contents/phytochemicals. Moreover, an inconsiderable correlation was detected between the market prices of different types of honey and their HBIs. Conclusions: The proposed index of health benefits could be recalculated/updated following measurement of more and more medicinal properties, such as anti-inflammatory, antidiabetic, and anticancer activities. This index could be used as an effective tool for consumers of honey to evaluate the real value of the purchased product.

Inhibitory capacity of Rhus coriaria L. extract and its major component methyl gallate on Streptococcus mutans biofilm formation by optical profilometry: Potential applications for oral health.

Streptococcus mutans (S. mutans) bacterium is the most well recognized pathogen involved in pathogenesis of dental caries. Its virulence arises from its ability to produce a biofilm and acidogenicity, causing tooth decay. Discovery of natural products capable to inhibit biofilm formation is of high importance for developing health care products. To the best of our knowledge, in all previous scientific reports, a colorimetric assay was applied to test the effect of sumac and methyl gallate (MG) on S. mutans adherence. Quantitative assessment of the developed biofilm should be further performed by applying an optical profilometry assay, and by testing the effect on both surface roughness and thickness parameters of the biofilm. To the best of our knowledge, this is the first study to report the effect of sumac extract and its constituent MG on biofilm formation using an optical profilometry assay. Testing antibacterial activity of the sumac extract and its fractions revealed that MG is the most bioactive component against S. mutans bacteria. It reduced S. mutans biofilm biomass on the polystyrene surface by 68­93%, whereas 1 mg/ml MG was able to decrease the biofilm roughness and thickness on the glass surface by 99%. MG also prevented a decrease in pH level by 97%. These bioactivities of MG occurred in a dose­dependent manner and were significant vs. untreated bacteria. The findings are important for the development of novel pharmaceuticals and formulations of natural products and extracts that possess anti­biofilm activities with primary applications for oral health, and in a broader context, for the treatment of various bacterial infections.

Tomato yellow leaf curl virus infection mitigates the heat stress response of plants grown at high temperatures.

Cultured tomatoes are often exposed to a combination of extreme heat and infection with Tomato yellow leaf curl virus (TYLCV). This stress combination leads to intense disease symptoms and yield losses. The response of TYLCV-susceptible and resistant tomatoes to heat stress together with viral infection was compared. The plant heat-stress response was undermined in TYLCV infected plants. The decline correlated with the down-regulation of heat shock transcription factors (HSFs) HSFA2 and HSFB1, and consequently, of HSF-regulated genes Hsp17, Apx1, Apx2 and Hsp90. We proposed that the weakened heat stress response was due to the decreased capacity of HSFA2 to translocate into the nuclei of infected cells. All the six TYLCV proteins were able to interact with tomato HSFA2 in vitro, moreover, coat protein developed complexes with HSFA2 in nuclei. Capturing of HSFA2 by viral proteins could suppress the transcriptional activation of heat stress response genes. Application of both heat and TYLCV stresses was accompanied by the development of intracellular large protein aggregates containing TYLCV proteins and DNA. The maintenance of cellular chaperones in the aggregated state, even after recovery from heat stress, prevents the circulation of free soluble chaperones, causing an additional decrease in stress response efficiency.