24-h Incubation Research Articles

Efect of N-acetylcysteine on HepG2 cells which were induced into fatty liver cells.

Non-alcoholic fatty liver disease is a prevalent liver condition that can progress to fibrosis and cirrhosis. It also poses a risk for hepatocellular carcinoma, underscoring the importance of identifying effective treatments. N-acetylcysteine, an inhibitor of glutathione depletion, shows promise in modulating intracellular glutathione biosynthesis and combating oxidative stress, making it a potentially beneficial therapy for liver fibrosis in non-alcoholic fatty liver disease. This study assesses the impact of N-acetylcysteine on HepG2 cells which were induced into fatty liver cells was evaluated. HepG2 cells were cultured in DMEM and seeded onto six-well plates at a density of 5 × 105 cells. Following a 24-h incubation period, the cells were exposed to a medium inducing fat accumulation. Subsequently, the cells were treated with varying concentrations of N-acetylcysteine for 48 h. Some plates were utilized for Real-Time-PCR tests, while others underwent Oil Red staining. The findings indicated a significant increase in the expression of fatty acid β-oxidation genes in the group treated with 10mM N-acetylcysteine (p < 0.05), along with reduced expression of lipogenesis-related genes (p < 0.05) in N-acetylcysteine-treated groups. Analysis of apoptotic gene expression revealed decreased BAX expression but increased BCL2 expression in the N-acetylcysteine-treated groups. Oil Red staining demonstrated a dose-dependent reduction in lipid droplets compared to the control group. This study's results suggest that N-acetylcysteine has the potential to decrease lipid droplets and modulate lipid metabolism effectively.

Plant-derived extracellular vesicles release combined with systemic DOX exhibits synergistic effects in 3D bioprinted triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer, lacking targeted therapeutic options. Hydrogels, particularly gelatin methacrylate (GelMA), have emerged as promising materials for localized drug delivery due to their biocompatibility and tunable properties. This study investigates a dual-delivery system for enhancing the treatment efficacy of triple-negative breast cancer (TNBC) using a combination of extracellular vesicles (EVs) derived from Citrus limon L. and the chemotherapeutic drug doxorubicin (DOX). We fabricated 3D bioprinted GelMA scaffolds to achieve localized and controlled release of EVs and evaluated their synergistic effects with systemic DOX delivery on both primary and metastatic 3D TNBC models.The GelMA scaffolds, especially those with 95 % methacrylation, exhibited higher stiffness, which enhanced their sustained release. Following 48-h incubation, the combination of EVs and DOX significantly increased cytotoxicity in the primary 3D TNBC model, reducing cell viability to approximately 30 % compared to controls. This was notably more effective than treatments with DOX or EVs alone. During the extended 7-day incubation period, the combination treatment continued to show superior efficacy, with persistently high levels of ROS generation and further reduction in cell viability. In a metastatic 3D TNBC model, a significant sensitivity to the combined treatment was observed, which notably inhibited aggregate formation and migration. Importantly, EVs-embedded scaffolds promoted the proliferation of human fibroblasts, highlighting their non-toxic nature, while concurrently inhibiting TNBC cell growth. This approach provides a promising strategy to improve the treatment outcomes of TNBC by exploiting the synergistic effects of local EVs release and systemic chemotherapy.

Antibiofilm activity of exopolysaccharide-mediated ZnO nanoparticle against Pseudomonas aeruginosa biofilm.

Exopolysaccharides (EPSs) are the group of biological macromolecules those play a potent role in protecting the bacteria from any sorts of stress. They exhibit multifunctional roles in natural and bioactive product science hence exhibits various types of medical and biochemical applications. EPS ensures the storage of nutrients, produce antigens to create defense mechanism during infection, and is also responsible for the formation of biofilm and cell adhesion. Green synthesis of ZnO nanoparticle mediated by EPS from Lactobacillus sp. which is a type of lactic acid bacteria (LAB) is a novel approach for its application in the food industry as it exhibits antimicrobial and antibiofilm potential. In this study, Lactobacillus sp. was cultivated in Lactobacillus broth media (LBM) and glucose mineral salt media (GMSM) to identify the best suitable media that would provide maximum amount of EPS, and it was observed that the two media exhibited maximum yield of 0.8g/L and 0.6g/L respectively after 48-h incubation. SEM, EDS, and XRD were used for characterizing the green synthesized ZnONPs from the EPS and was observed that the NPs were synthesized. 62.6% and 67.6% ZnONPs were observed in LBM-ZnONP and GMSM-ZnONP respectively from XRD analysis. UV spectroscopic detection showed corresponding peak of the nanoparticle formed at 349nm which confirmed the production of ZnO NPs. Scanning electron microscopic (SEM) images and Fourier transform infrared spectroscopy (FT-IR) established the average size, shape, and composition of the nanoparticles. The peaks of the FT-IR also revealed the presence of the C = H and N-H stretching (1 H). It was also observed that the average size of LBM ZnONPs were 60.578nm whereas GMSM ZnONPs were 53.09nm. Viability studies exhibited that the NPs brought considerable reduction of the sessile cells of P. aeuginosa. It was further observed that the cells treated with NPs did not show revival. The NPs were able to inhibit the quorum sensing (QS) mechanism of Pseudomonas aeruginosa thereby preventing the development of virulence. Out of the two NPs, it was observed that GMSM ZnONPs showed better efficacy in comparison to LBM ZnONPs. Thus, the study concludes that EPS-mediated NPs can be used effectively in the process of treating the biofilm.

Toxicological evaluation of Salmonella phage NINP13076 in BALB/c mice: Histopathological studies

Salmonella spp. represent a significant etiological agent of foodborne illnesses and constitute a notable public health challenge on a global scale. Effectively managing Salmonella contamination within the food supply chain is imperative, as it represents a significant threat to public health, potentially leading to severe illnesses with fatal consequences. A novel bio-preservative agent is required to combat Salmonella food contamination during different stages of the food supply chain. Bacteriophages are acknowledged for their safety and recognized for their efficacy in bio-food preservation. Given their consumption together alongside food, it becomes pivotal to assess their potential toxicity concerning the probiotic gut microbiota. In this research, BALB/c mice were employed to evaluate the sub-chronic oral toxicity of bacteriophages targeting Salmonella. Following 28 consecutive days of sub-chronic administration of the Salmonella phage through oral means, histopathological examinations of vital organs, including the lung, kidneys, heart, liver, and intestine, revealed normal organ structures and no significant pathological abnormalities, in both the control and experimental groups. No physical or behavioural change was noticed in the experimental mice. Furthermore, the proliferation of indicative probiotic bacteria persisted unchanged even following a 24-h incubation with the Salmonella phage. The body weight of both the control and experimental subjects, along with a comparative analysis of urine test results, exhibited no variations. Notably, the phages demonstrated no discernible impact on the probiotic microbiota, specifically the Lactobacillus and Bifidobacterium species isolated from the caecum of both mice that received treatment and those that did not. In untreated animals, the probiotic population (mean log CFU/ml) ranged from 1.9 to 2.4 log and 2.4 to 2.5 log, while in treated mice, it ranged from 2.0 to 2.3 log and 2.3 to 2.4 log for the Lactobacillus and Bifidobacterium species, respectively. The results of the investigation indicate that the oral administration of the lytic Salmonella phage showed no observable adverse effects on the animals, indicating an absence of harm.

Biochemical and proteomic approaches to investigating effects of IAA-aspartate in pea (Pisum sativum L.) seedlings during osmotic shock

Osmotic shock is the first step of high salt or drought action that involves biochemical and molecular changes during plant response to these unfavorable conditions. Indole-3-acetyl-aspartate (IAA-aspartate, IAA-Asp) is the main amide conjugate of auxin in pea (Pisum sativum L.) tissues. Although the exact molecular mechanism of the IAA-Asp action is unknown, this conjugate's indole-3-acetic acid (IAA)-independent biological activity has been observed during physiological and stress conditions. In this work, we investigated the effect of IAA-Asp alone, as well as in combination with NaCl or polyethylene glycol (PEG) (osmotic shock) on reduced/oxidized glutathione (GSH/GSSG) ratio, activities of enzymes modulating glutathione concentration, protein S-glutathionylation, and IAA homeostasis. We did not observe the hydrolysis of IAA-Asp to IAA in pea seedlings, which, together with other results, suggests that IAA-Asp modulates plant response to abiotic stimuli independently of IAA. Moreover, despite the effect of IAA-Asp on the enzymes responsible for IAA conjugation, no changes in this phytohormone level were visible. Furthermore, 3h plant treatment with IAA-Asp increased the activity of glutathione reductase (GR), which correlates with an elevated GSH/GSSG ratio. On the contrary, more extended (48h) incubation with IAA-Asp diminished the GSH/GSSG ratio and increased the activity of glutathione peroxidase (GPX). IAA-Asp reduced GR activity during salt treatment but did not affect the GSH/GSSG ratio. Similarly, under plant incubation with PEG, IAA-Asp did not change the GSH/GSSG ratio but increased glutathione S-transferase (GST) activity. We also analyzed the effect of IAA-Asp on pea protein S-glutathionylation. Increased S-glutathionylation of heat shock 70 kDa protein (HSP70) was observed after plant treatment with IAA-Asp, PEG, or IAA-Asp combined with PEG. The proteomic analysis also revealed that IAA-Asp diminished S-glutathionylation of lipoxygenase during plant incubation with PEG. Thus, we suggest that IAA-Asp modulates redox status in pea during oxidative stress and under normal physiological conditions.

Harnessing CRISPR/Cas12a Activity and DNA-Based Ultrabright FluoroCube for In Situ Imaging of Metabolically Labeled Cell Membrane Glycoproteins.

Fluorescence imaging of cell membrane glycoproteins based on metabolic labeling faces challenges including the sensitivity and spatial specificity and the use of a high concentration of unnatural sugars. To overcome these limitations, we developed a method for in situ imaging of cell membrane glycoproteins by operating Cas12a activity, and employing the ultrabright DNA nanostructure, FluoroCube (FC), as a signal reporter. Following Cas12a activation, we observed stable and intense fluorescence signals within 15 min. The combination of bright FC and Cas12a's amplification capability allows for effective imaging with only 5 μM of unnatural sugars and a brief 24-h incubation. Computational modeling demonstrates that Cas12a specifically cleaves FC in the 11-17 nm range of the glycosylation site, enabling spatially precise imaging. This approach successfully enabled fluorescence imaging of glycoproteins across various cell lines and the detection of changes in glycoprotein levels induced by drugs.

You can bring plankton to fecal indicator organisms, but you cannot make the plankton graze: particle contribution to E. coli and MS2 inactivation in surface waters.

Organisms that are associated with feces ("fecal indicator organisms") are monitored to assess the potential for fecal contamination of surface water bodies in the United States. However, the effect of the complex mixtures of chemicals and the natural microbial community within surface water ("particles") on fecal indicator organism persistence is not well characterized. We aimed to better understand how particles, including biological (e.g., potential grazers) and inert (e.g., minerals) types, affect the fecal indicator organisms Escherichia coli K-12 ("E. coli") and bacteriophage MS2 in surface waters. A gradient of particles captured by a 0.2-µm-pore-size filter ("large particles") was generated, and the additional particles and dissolved constituents that passed through the filter were deemed "small particles." We measured the ratio of MS2 and E. coli that survived over a 24-h incubation period for each condition (0%-1,000% large-particle concentration in raw water) and completed a linear regression that included large- and small-particle coefficients. Particles were characterized by quantifying plankton, total bacterial cells, and total solids. E. coli and MS2 persistence was not significantly affected by large particles, but small particles had an effect in most waters. Small particles in higher-salinity waters had the largest, negative effect on E. coli and MS2 survival ratios: Significant small-particle coefficients ranged from -1.7 to -5.5 day-1 in the marine waters and -0.89 to -3.2 day-1 in the fresh and estuarine waters. This work will inform remediation efforts for impaired surface water bodies.IMPORTANCEMany surface water bodies in the United States have organisms associated with fecal contamination that exceed regulatory standards and prevent safe recreation. The process to remediate impaired water bodies is complicated because these fecal indicator organisms are affected by the local environmental conditions. For example, the effect of particles in surface water on fecal indicator concentrations are difficult to quantify in a way that is comparable between studies and water bodies. We applied a method that overcomes this limitation to assess the effects of large particles, including natural plankton that could consume the seeded fecal indicator organisms. Even in environmental water samples with diverse communities of plankton present, no effect of large particles on fecal indicator concentrations was observed. These findings have implications for the interpretation and design of future studies, including that particle characterization of surface water may be necessary to assess the fate of fecal indicators.

Study on the anti-atherosclerosis mechanisms of Tanyu Tongzhi formula based on network pharmacology, Mendelian randomization, and experimental verification

Context Tanyu Tongzhi Formula (TTF) exhibits potential against atherosclerosis; however, its mechanisms remain unclear. Objective This study explores the pharmacological mechanisms of TTF in treating atherosclerosis. Materials and methods Network pharmacology, molecular docking, mendelian randomization (MR), and liquid chromatography-mass spectrometry (LC-MS) analyses were utilized to reveal potential targets and compounds of TTF against atherosclerosis. After exploring the appropriate concentration of TTF to treat HCAECs using Cell Counting Kit-8 (CCK-8), the HCAECs were divided into three groups: control, oxidized low-density lipoprotein (ox-LDL, 50 μg/mL), and ox-LDL (50 μg/mL) + TTF (1 mg/mL). After 24-h incubation, the efficacy of TTF was verified by CCK-8, Oil red O staining, and ELISA. The expression of key targets was detected by real-time polymerase chain reaction (qPCR) and western blotting. Results A total of 137 active compounds and 127 potential TTF targets against atherosclerosis were identified. MR identified ALB, TNF, PPARα, and PPARγ as key targets. Molecular docking indicated that baicalin, naringenin, and curcumin exhibited suitable binding activities to these targets, further confirming by LC-MS analysis. The IC50 of TTF in HCAECs was 18.25 mg/mL. TTF treatment significantly improved atherosclerosis by enhancing cell viability, reducing lipid accumulation, and inhibiting inflammation factors (IL6, IL1B and TNF-α) in ox-LDL-treated HCAECs. Moreover, qPCR or western blotting indicated that TTF could up-regulate PPARα and PPARγ while down-regulate TNF expression. Discussion and conclusions Our results revealed active compounds, key pathways, and core targets of TTF against atherosclerosis, providing experimental support for its application in treating of atherosclerosis.

IAA-producing bacteria from the rhizosphere of chickpea (Cicer arietinum L.): Isolation, characterization, and their effects on plant growth performance

Indole-3-acetic acid (IAA), a crucial plant hormone, regulates diverse physiological processes. This study aimed to isolate and characterize IAA-producing bacteria from the chickpea (Cicer arietinum L.) rhizosphere and evaluate their effects on plant growth. From 54 rhizosphere samples, 118 bacteria (designated as GAC) were isolated and screened for IAA production using a Salkowski colorimetric assay, and Bergey's manual was used for biochemical identification. Isolates were grown under various conditions and in vitro screened for their growth promotion traits. A PCR investigation was performed for IAA and nitrogen-fixing genes, and evaluated for greenhouse conditions. Among them, 27 isolates produced IAA, with eight high producers selected. Morphological and biochemical identification classified the six isolates as Pseudomonas and the other two as Bacillus. Optimal conditions for IAA production were observed at 500 μg/ml tryptophan, 35 °C, and pH 7.0. A 48-h incubation was ideal for IAA production, except for GAC-34 and GAC-73, which required 72 h. All the isolates achieved optimal IAA levels with tryptone and sucrose as nitrogen and carbon sources, respectively. Moreover, all isolates showed nitrogen fixation ability, and the six isolates exhibited phosphate solubilization. PCR confirmed the amplification of nifH (300 bp), nifK (360 bp), and ipdC (1170 bp) genes. Greenhouse experiments demonstrated that eight selected isolates significantly enhanced chickpea growth parameters (p < 0.001). These findings suggest that these IAA-producing bacteria have the potential to be used as biofertilizers to improve crop productivity, although further molecular identification and field studies are required.

The Effect of Adding Green and Black Tea Waste Extracts on Rumen Fermentation Parameters by In Vitro Techniques

The increase in global temperatures over the past few decades due to greenhouse gas emissions has raised concerns and necessitated further research in climate change mitigation and adaptation. Methane is a prominent greenhouse gas that significantly contributes to climate change, with a substantial amount generated through fermentation processes occurring in the rumen of ruminant animals. The potential of plant secondary metabolites, especially those derived from tannin-rich plants, warrants investigation to modify rumen fermentation and mitigate methane emissions in livestock diets. The objective of this study was to assess the impact of extracts obtained from green and black tea waste on rumen fermentation dynamics and gas (methane) production, utilizing in vitro methods. For this purpose, rumen fluid was collected from two fistulated sheep and subjected to three treatments: (1) a basal diet (control), (2) a basal diet + green tea waste extract (5% of dry matter), (3) a basal diet + black tea waste extract (5% of dry matter). The study assessed the effects of incorporating extracts from green and black tea waste on various parameters, including digestibility, protozoa population, ammonia nitrogen levels, volatile fatty acids, and methane gas production following a 24-h incubation period. Statistical analysis of the data was conducted using SAS software within a completely randomized design framework. The findings indicated that the addition of green and black tea waste extracts significantly decreased methane gas production (p &lt; 0.05), protozoa count (p &lt; 0.05), and ammonia nitrogen concentrations in rumen fluid (p &lt; 0.05) when compared to the control group. The addition of green and black tea waste extracts has significantly altered the concentration of VFAs in rumen fluid (p &lt; 0.05). Specifically, the addition of green tea waste extract has led to a highly significant reduction in acetic acid, (p &lt; 0.01) and the addition of both extracts has resulted in a significant increase in propionic acid (p &lt; 0.05). Consequently, the results suggest that the inclusion of green and black tea waste extracts in livestock diets may effectively mitigate methane emissions in the rumen, thereby reducing feed costs and reducing environmental pollution.

8660 Bodipy Labeled Vitamin D3 Associates With Lipid Droplets In Adipocytes

Abstract Disclosure: N. Ucar: None. J.T. Deeney: None. R. Pickering, PhD: None. M.T. Kirber: None. T. Fan: None. R. Loo, PhD: Research Investigator; Self; Carbogen Amcis BV. M.F. Holick: Grant Recipient; Self; Carbogen Amcis BV. Research Investigator; Self; Carbogen Amcis BV. Obese patients are often found to be deficient in vitamin D, requiring higher levels of supplementation than normal weight subjects. While this has been attributed to vitamin D3‘s lipid solubility and accumulation in fat tissue, little is known about how vitamin D3 enters adipocytes and associates with the intracellular lipid droplet. The goal of this study was to investigate this association using a novel fluorescently labelled vitamin D3. Bodipy, 4,4-difluoro-1,3,5, 7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503), was covalently linked to vitamin D3 and its structure was confirmed by NMR and mass spectroscopy. Murine 3T3L1 and primary adipocytes were used for our study. Mouse 3T3L1 preadipocytes were cultured in multiwell plates or glass bottom dishes in high glucose DMEM media and differentiated for 7-14 days. Primary adipocytes were obtained by collagenase digestion after surgical extraction of adipose tissue from male/female C57BL/6J mice and attached to glass bottom dishes. The cultured adipocytes were exposed to 10 microM BODIPY labelled Vitamin D3 (vitD-B). BODIPY was added to cultured cells and served as the control. Cells were imaged at various times on a wide-field epifluorescence microscope and analysed with NIH ImageJ software. VitD-B adipocyte uptake and accumulation in lipid droplets was observed over 24 hrs. Isolated lipid droplets were also incubated with vitD-B and BODIPY. VitD-B in mature 3T3L1 cells was observed in lipid droplets after 1h incubation and increased fluorescent intensity was observed over the next 24 hrs. BODIPY was observed in the lipid droplets after 15-20 mins. As a negative control, non-differentiated 3T3L1 cells did not exhibit staining by BODIPY even after 24-hrs. When vitD-B was added to primary cultured adipocytes, it appeared in the adipocytes within 1-hr. Fluorescence intensity of primary adipocytes was increased 1.2-fold at 8 hrs and 5.7-fold at 24 hrs compared to 1 hr. For comparison the positive control treated with BODIPY exhibited rapid fluorescence uptake into primary adipocytes that was increased 29-fold higher than that of vitD-B at 1 hr. VitD-B and BODIPY both demonstrated rapid uptake into isolated lipid droplets (less than 2 min) based on fluorescence microscopy.These results hold promising implications for understanding of the interaction between vitamin D and intracellular lipid droplets. Keywords: Adipocyte, Vitamin D3, BODIPY, adipocytes, lipid droplet, fluorescent labelled vitamin D3 Presentation: 6/2/2024

Microbiological profile and prevalence of histamine-producing bacteria in fresh sardines stored at different temperatures.

The European pilchard (Sardina pilchardus) is an important fish species for the Moroccan economy in terms of production and export. Biogenic amine histamine is a metabolite produced in the flesh of some fish species after death due to the decarboxylation of free histidine by histaminogenic bacteria. Failure to control the histamine risk in European pilchard may lead to public health and socioeconomic issues. This study aimed to estimate the prevalence of histaminogenic bacteria in association with histamine levels and the growth of microflora in Moroccan sardines (European pilchard). We conducted the study by monitoring Moroccan sardines of histamine content and microbiological profile (aerobic plate count [APC], coliforms, and thermo-tolerant coliforms [TTC]) during 6 days of storage at three different temperatures (0°C, 10°C, and ambient temperature [22°C]). The histamine assay was performed using a spectrofluorometric method, and the microbiological identification of histamine-producing bacteria was performed using a combination of biochemical and molecular tests. The histamine content in European pilchard stored at 0°C was negligible. However, high concentrations were observed at 10°C and 22°C. The microbiological profile showed a positive association between microflora counts and histamine content according to storage time. At 0°C, a moderate increase in the APC, a decrease in coliforms, and an absence of TTC were observed. The rapid proliferation of all microflora was observed at 10°C, whereas at 22°C, the proliferation was almost exponential. Bacterial identification revealed the exclusive presence of species belonging to the Enterobacteriaceae family at varying frequencies depending on storage temperature. Morganella morganii and Proteus mirabilis had the highest histamine induction rates in L-histidine-supplemented broth, with 1600 and 255 parts per million (ppm), respectively, after 48-h incubation at 35°C. Klebsiella ozaenae could produce 136 ppm and Serratia plymuthica 115 ppm. Reverse transcription polymerase chain reaction showed positive results for the presence of genes associated with histidine decarboxylase. The hdc genes of M. morganii, P. mirabilis, and K. ozaenae were successfully amplified and exhibited strong similarity with the reference gene of M. morganii. This study describes for the first time the hdc gene in bacteria that form histamine in Moroccan sardines. The results also confirm that respect for the cold chain integrity is a crucial factor in histamine management. This information should help stakeholders in the implementation of sound strategies for managing the hazards associated with seafood and their products.

A robust high-throughput screening system to assess bacterial tyrosine ammonia lyase activity in the context of tyrosine inherited metabolic disorders.

Inborn errors of tyrosine metabolism result in patient's inability to degrade tyrosine. Current treatment consists of a phenylalanine and tyrosine restricted diet and nitisinone, causing a block in the tyrosine degradation pathway. However, tyrosine levels will increase, leading to acquired hypertyrosinemia, implying the need for an add-on treatment. Tyrosine ammonia lyases (TAL) can provide such an add-on treatment as they catalyze the deamination of tyrosine into p-coumaric acid and ammonia. In this study, we developed a robust high-throughput screening (HTS) assay to assess the capacity of bacterial TAL enzymes to decrease excessive tyrosine. The assay is based on the spectrophotometric quantification of p-coumaric acid after conversion of tyrosine by bacterial TAL. As a benchmark, TAL from Flavobacterium johnsoniae (FjTAL) was used to optimize the assay. Optimal growth conditions for high-level protein expression were determined by incubating transformed Escherichia coli BL21 (DE3) cells at different temperatures during various incubation times. Subsequently, assay temperature and pH were optimized followed by testing different ratios of tyrosine assay mixes to bacterial lysate. Finally, assay robustness and functionality were evaluated. Optimal FjTAL expression was obtained after incubation for 24h at 22°C. Ideal assay conditions consist of a 80/20 ratio of 1mM tyrosine assay mix to FjTAL lysate performed at pH 9.2 and 37°C. The robustness test showed Z' values > 0.4 and signal window values > 2 without edge or drift effects. As proof-of-principle, we successfully determined the catalytic activity of two other bacterial TAL enzymes RsTAL (5.718.10-3 ± 0.21.10-3) and SeSAM8 (4.658.10-3 ± 0.37.10-3). A robust, simple and reliable HTS assay was thus developed to evaluate the tyrosine degradation capacity of bacterial TAL enzymes.

Molecular Mechanics Simulation and Ftir Spectrocopy Optimization of Secondary Structure of Aβ (25-35) Peptide

Abstract In this study, we probed the secondary structure of a soluble Aβ (25-35) peptide in water solution by applying a molecular mechanics method as well as infrared spectroscopy. We have used Fourier Transformed Infrared Spectroscopy (FTIR) to examine the secondary structure of this peptide. The amide I bands of Ab (25-35) obtained from transmission-FTIR spectra consists of one main band at 1658 cm-1, at both concentrations used (200 mM and 1 mM). This band shows us that Ab (25-35) in aqueous solution was mostly organized into a a-helical structure (48 %). The contribution of the unordered structure was found to be about 12 %. The proportion of b-sheet and b-turn structures are slightly lower, 12-15 % and 13-14 %, respectively for both concentrations. FTIR spectra of the peptide in water solution (100 µM) after incubation for 12h showed Aβ peptide conformation is critically dependent on the environmental conditions used. The simulation approach reveals that the C-terminal octapeptide of this molecule preferably adopts an α-helical structure, while the N-terminal tripeptide may exist as a β-turn and unordered structures.

Biocompatibility and antimicrobial effect of demineralised dentin matrix hydrogel for dental pulp preservation.

Regeneration of dentin and preserving pulp vitality are essential targets for vital pulp therapy. Our study aimed to evaluate a novel biomimetic pulp capping agent with increased dentin regenerative activities. To produce demineralised dentin matrix (DDM) particles, human extracted teeth were ground and treated with ethylene diamine tetra-acetic acid solution. DDM particles were added to sodium alginate and this combination was dripped into a 5% calcium chloride to obtain DDM hydrogel (DDMH). The eluants of both DDMH and mineral trioxide aggregate (MTA) were tested using an MTT assay to detect their cytotoxic effect on dental pulp stem cells (DPSC). Collagen-I (COL-I) gene expression was analysed on DPSC exposed to different dilutions of pulp capping material eluants by real-time quantitative polymerase chain reaction. Acridine orange staining was used to monitor the cell growth over the tested materials. Agar diffusion assay was utilised to test the antibacterial effect of DDMH and MTA compared to controls. MTT assay revealed that neat eluates of DDMH promoted DPSC viability. However, neat eluates of MTA were cytotoxic on DPSC after 72h of culture. Moreover, DPSC were capable of growth and attached to the surface of DDMH, while they showed a marked reduction in their number when cultured on the MTA surface for one week, as shown by the acridine orange stain. In DPSC cultured with DDMH eluates, the COL-I gene was overexpressed compared to those cultured with MTA eluants. DDMH had significant antimicrobial activity in comparison to MTA after 24h incubation. This in vitro study showed that DDMH could be an alternative pulp capping agent for regenerative endodontics.

PSVIII-6 Effect of adding plant extracted saponin (PES) on accumulative gas production kinetics and methane emission of the prairie blend pelleted feed products in ruminant system

Abstract The objectives of this study were to study the effect of adding plant extracted saponin (PES) as phytochemical feed additive on in vitro accumulative gas production kinetics and methane (CH4) emission of blend pelleted feed products in ruminant system. Four levels of PES (0, 1, 2, 3%) were added to canola meal and pea mixtures with two different ratios (CMP1: 50:50; CMP2: 70:30) which were used to make Prairie blend pellet feed products. The accumulative gas production kinetics and CH4 were determined using in vitro systems. Ruminal fluid for in vitro studies was obtained from rumen fistulated lactation dairy cows. The experimental design was a randomized complete block design with the PES level as a fixed effect and in vitro run with fistulated animals as a random effect. The data were analyzed using Mixed Model Procedure of SAS. Polynomial contract was used to determine linear and quadratic relationship. The results showed that adding PES did not significantly affect the asymptotic production (a) with an average of 104.06 ± 12.38 (SD) mL/g DM, the gas production fractional rate (c) with an average of 1.00 ± 0.90 %/h, the average production at one-half of asymptotic production (AP) with an average 11.26 ± 0.87 mL and lag time with an average of 2.08 ± 0.81 h. As to CH4 emission at different incubation times, the result showed that at short incubation times (2 h, 4 h, 12 h), adding PES showed numerically reduced CH4 from 277 to 1.08 mL/g (at 2 h) and 4.39 to 1.59 mL/g (at 4 h). With increasing PES level, CH4 was significantly linearly reduced (P = 0.019) from 8.31 to 6.30 mL/g at 12h incubation time. At long incubation (24 h), adding PES did not have significant impact on CH4. In conclusion, adding PES at the studied levels to Prairie blend pellet feed products did not significantly affect accumulative gas production kinetics but it had a high potential to decrease CH4 emission at short incubation times.

400 Effect of pH on in vitro ruminal metabolism of trans-10 18:1 and trans-11 18:1

Abstract Dietary C18 unsaturated fatty acids are extensively biohydrogenated by rumen microbiota into 18:0 and trans-18:1 isomers. Usually, the biohydrogenation (BH) pathways produce mostly 18:0 and trans-11 18:1 as the main intermediate. However, when fed low-forage diets that decrease the rumen pH, the main BH intermediate often is the trans-10 18:1 isomer. This change in BH, called the trans-10 shift, is associated with milk fat depression, but despite that, little is known about the triggers of the trans-10 shift and its ecological role in rumen microbiota. Microbiota stressed by low rumen pH may benefit from increased trans-18:1 availability favoring the production of the trans-18:1 isomer with a slower rate of BH. Thus, we hypothesize that at low rumen pH conditions, the BH of trans-10 18:1 will be slower than that of trans-11 18:1. We tested that with an in vitro experiment using batch incubation in Hungate tubes, replicated in 5 wk. In each run, 24 tubes were allocated at 6 treatments and 4 incubation times (0, 6, 24 and 48 h). The treatments resulted from the combination of 2 buffering solution pH (6.74 vs. 5.85) and 3 substrates: 1) Control (60 mg feed DM) 2) T10 (Control plus 1.2 mg of trans-10 18:1) and 3) T11 (Control plus 1.2 mg of trans-11 18:1). Volatile fatty acids (VFA) and long-chain fatty acids were analyzed by gas chromatography. Data were analyzed using the 2 × 3 factorial arrangement for each incubation time. Low pH resulted in less 30 to 37% DM disappearance than those with high pH at all incubation times (P &amp;lt; 0.05). The decrease in VFA with low pH was less expressive (≈17%) and only evident at 24 and 48 h incubation times. The low pH also decreased (P &amp;lt; 0.05) the BH of 18:1n-9 (less ≈47 to 53%), 18:2n-6 (less 30 to 34%) and 18:3n-3 (less 20 to 22%) at 24h and 48h incubation times, respectively. The disappearance of trans-11 18:1 was 14.6% at 6 h, 29.0% at 24 h and 32.8% at 48 h and was not affected by pH. The disappearance of trans-10 18:1 was dependent on pH. At 6 h and high pH the disappearance of both was similar, but at low pH the disappearance of trans-10 18:1 was much less (9.8%) than that of trans-11 18:1. However, at 48 h and low pH, the disappearance was similar between isomers and the disappearance of trans-10 18:1 was much greater (46.6%) than trans-11 18:1. In summary, the BH of trans-10 18:1 is less at low pH than at high pH, and this could in part explain its abundance in some practical ruminant feeding conditions. Acknowledgements: Fundação para a Ciência e a Tecnologia (FCT) funding through PTDC/CAL-ZOO/29654/2017(RumOmics), PTDC/CAL-ZOO/4515/2021(Gene2Rumen), UIDB/00276/2020 (CIISA), and LA/P/0059/2020 (AL4Animals) projects.

A New Method for Obtaining Monospecies and Binary Cultures of Staphylococcus spp. in Alginate Gel and the Study of the Action of Active Compounds on These Cultures on the Example of Catecholamines.

A simple and efficient method for obtaining monospecies and binary Staphylococcus aureus and Staphylococcus epidermidis cultures in sodium alginate gel matrix mimicking the natural microenvironment of the nasal cavity was proposed. The cultures were used for studying the effect of norepinephrine on monospecies and binary communities of two types of bacteria, S. aureus (invasive strain) and S. epidermis (commensal strain). After 24-h incubation, S. aureus predominated in the binary community, but later it was replaced by S. epidermis. Norepinephrine at higher concentrations accelerated this process without principally changing it. The model can be used to develop more effective complex antimicrobial drugs.

FERMENTATIVE CHARACTERISTICS OF MILLET-BASED DIETS FOR WEST AFRICAN DWARF GOATS IN THE SOUTHERN GUINEA SAVANNAH ZONE OF NIGERIA

The study is to investigate the in vitro fermentation pattern of the diets at inclusion levels of millet husk as feed for ruminants. Five experimental diets were formulated using millet husk as a replacement for wheat bran at graded levels of D1(0%), D2(25%), D3(50%) D4(75%) and D5(100%). The rumen fluid collected from five WAD goats through the suction tube before the morning feed was sieved with four layered cheesecloth and subjected to an in vitro fermentation study. Incubation was carried out in 120 ml calibrated syringes in three batches at 39 ± 1ºC. The gas production was measured at 3, 6, 9, 12, 15, 18, 21 and 24h. The metabolizable energy, organic matter digestibility and short-chain fatty acids were estimated at 24h incubation. At the end of 24 hours of incubation, 4 mL of NaOH (10 M) was introduced using a 5 mL capacity syringe in a completely randomized design. All the parameters measured and calculated in this study were significantly influenced by the experimental diets. The gas volume produced decreased as the millet husk inclusion level increased. The OMD values recorded indicate good digestibility of the diets. It is observed that the digestibility and methane production decrease as the inclusion level of millet husk increases. Hence, millet husk could be incorporated in the ruminant diet to mitigate methane emissions.

Combined antimicrobial activity of short peptide and phage-derived endolysin against antibiotic-resistant Salmonella Typhimurium

This study was designed to evaluate the combination effects of antimicrobial peptides (FK13 and FK16) and phage-encoded endolysin (LysPB32) on the inhibition of growth of polymyxin B-resistant Salmonella Typhimurium ATCC 19585 (STPMB). The inhibitory effects of FK13, FK16, and LysPB32 against STPMB were evaluated by using antimicrobial susceptibility, membrane permeability, biofilm reduction, cross-resistance, and mutant frequency assay. The minimum inhibitory concentrations (MICs) of FK13 and FK16 treated with LysPB32 (FK13+LysPB32 and FK16+LysPB32) against STPMB were decreased from more than 512 to 128 μg/ml and from 64 to 32 μg/ml, respectively. Compared to the control, the number of STPMB in the growing culture was reduced by 4.2 and 5.2 log CFU/ml, respectively, for FK13+LysPB32 and FK16+LysPB32 after 12-h incubation at 37 °C. All treatments (FK13, FK16, FK13+LysPB32, FK16+LysPB32) significantly increased the permeability of the outer membrane of STPMB. Biofilms were significantly decreased from OD600 of 0.6 to 0.16 for FK13+LysPB32 and from 0.6 to 0.13 for FK16+LysPB32. The ratios of MICs of erythromycin, ceftriaxone, polymyxin B, and ciprofloxacin to MIC of the control against STPMB were decreased to 0.50 for FK13+LysPB32 and FK16+LysPB32. The bactericidal activities of amikacin and gentamicin were enhanced for FK13+LysPB32 and FK16+LysPB32 (2-fold < MBC/MIC ratio). The mutant frequencies of STPMB to antibiotics were decreased when treated with FK13+LysPB32 and FK16+LysPB32. The results suggest that the combination of antimicrobial peptides and endolysins can be a promising strategy to control polymyxin B-resistant S. Typhimurium.