24-h Incubation Research Articles

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.

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.

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.

Synthetic free fatty acid receptor (FFAR) 2 agonist 4-CMTB and FFAR4 agonist GSK13764 inhibit colon cancer cell growth and migration and regulate FFARs expression in in vitro and in vivo models of colorectal cancer.

Free fatty acid receptors (FFARs) are G protein-coupled receptors that divide into 4 subtypes; FFAR2 and FFAR3 are activated by short-chain fatty acids, while FFAR1 and FFAR4 - by long-chain fatty acids. Recent studies show the potential involvement of FFARs in the pathophysiology of colorectal cancer (CRC). A decrease in FFAR2 and FFAR4 gene expression is observed in patients with CRC. The aim of our study was to evaluate the anti-cancer effect of FFAR2 and FFAR4 stimulation by selective synthetic agonists in in vitro and in vivo models of CRC. FFAR2 agonist, 4-CMTB, and FFAR4 agonist, GSK137647 were used. Cell viability (CCD 841 CoN and SW-480) was determined after 48h incubation with tested compounds using MTT assay. Real-time qPCR and Western Blot were used to identify changes in FFARs expression. Migration and invasion were characterized by commercially available tests. Colitis-associated CRC (CACRC) mouse model was induced by azoxymethane and dextran sodium sulfate. 4-CMTB and GSK137647 significantly reduced cancer cell growth as well as migration and invasion capacities. Both synthetic compounds increased FFAR2 and FFAR4 expression in SW-480 cells. Neither 4-CMTB nor GSK137647 influenced the course of AOM/DSS-induced CACRC in mice, however, 4-CMTB elevated FFAR2 protein expression in mouse tissues. We presented that stimulation of FFAR2 and FFAR4 may inhibit CRC cell viability and migration and that the FFAR2 and FFAR4 expression decreased in CRC can be restored by treatment with respective agonists, indicating new promising pharmacological targets in CRC treatment.

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 < 0.05), protozoa count (p < 0.05), and ammonia nitrogen concentrations in rumen fluid (p < 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 < 0.05). Specifically, the addition of green tea waste extract has led to a highly significant reduction in acetic acid, (p < 0.01) and the addition of both extracts has resulted in a significant increase in propionic acid (p < 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

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.

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

Background and Aim: 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). Materials and Methods: 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. Results: 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. Conclusion: 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. Keywords: Enterobacteriaceae, histamine, Histaminogenic bacteria, polymerase chain reaction, sardine, seafood.

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 < 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 < 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.

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.

Functional characterization and biotechnological applications of exopolysaccharides produced by newly isolated Enterococcus hirae MLG3-25-1.

This study investigated the potential applications of Enterococcus hirae MLG3-25-1 exopolysaccharides (EPS), with a focus on their isolation, identification, production, and functional characteristics. After the bacterial strain was cultured in De Man-Rogosa-Sharpe (MRS) medium containing 1% glucose at 37°C, the EPS was refined, and the highest yield of 0.85mg/mL was achieved at the 24-h incubation period. Enterococcus hirae MLG3-25-1 was found to be able to produce EPS. The study explored the microstructure of the EPS, which resembles polysaccharide sheets with smooth surfaces, through scanning electron microscope (SEM) analysis. Through Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance(NMR) analysis, the chemical composition, aligning with glycosidic bond characteristics, has been deciphered. Furthermore, the antimicrobial and antibiofilm activities against pathogenic bacteria, particularly Bacillus sp., demonstrated potential applications in combating antibiotic resistance. The EPS exhibited notable antioxidant activity (89.36% DPPH scavenging), along with high water-holding capacity (575%), emulsifying activity, and flocculation activity, suggesting its potential as a stabilizing agent in the food industry. Overall, this study provides a comprehensive characterization of Enterococcus hirae MLG3-25-1 EPS, emphasizing its diverse applications in antimicrobial, antioxidant, and food-related industries. These findings lay the groundwork for further exploration and utilization of this EPS in various sectors.

Dopamine-modified hyaluronic acid (DA-HA) as a novel dopamine-mimetics with minimal autoxidation and cytotoxicity

Dopamine-modified hyaluronic acid (DA-HA) has been initially developed as an efficient coating and adhesion material for industrial uses. However, the biological activity and safety of DA-HA in the brain have not been explored yet. Here, we report a series of evidence that DA-HA exhibits similar functionality as dopamine (DA), but with much lower toxicity arising from autoxidation. DA-HA shows very little autoxidation even after 48-h incubation. This is profoundly different from DA and its derivatives including l-DOPA, which all induce severe neuronal death after pre-autoxidation, indicating that autoxidation is the cause of neuronal death. Furthermore, in vivo injection of DA-HA induces significantly lower toxicity compared to 6-OHDA, a well-known oxidized and toxic form of DA, and alleviates the apomorphine-induced rotational behavior in the 6-OHDA animal model of Parkinson's disease. Our study proposes that DA-HA with DA-like functionalities and minimal toxicity has a great potential to treat DA-related disease.

Performance of common primary and chromogenic culture media for MALDI-TOF MS identification of clinically relevant yeasts.

Timely and accurate identification of yeasts is essential for adequate treatment, considering the increase in antifungal resistance of some species, particularly for C. auris. Current matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) manufacturer's protocol for identification of yeasts requires 24- to 72-h cultivation on Sabouraud dextrose media (SAB), but not some of the mainstay primary culture media used in mycology such as inhibitory mold agar (IMA), Mycosel, CHROMagar Candida Plus, and CHROMagar Candida. As culture media can influence MALDI-TOF MS identification results, this study evaluated the accuracy and performance of identification of clinically relevant yeasts on these first-line media using the VITEK-MS MALDI-TOF MS system.IMPORTANCEIn this study, a panel of 140 strains (21 species) was used to assess the performance of the selected media. Although not in the manufacturer's list of accepted media, IMA and chromogenic media are suitable for the identification of yeasts on the VITEK-MS systems. CHROMagar Candida Plus allowed the identification of 135/140 isolates tested after 24-h incubation similar to SAB reference media (137/140). Yeast isolates that grew on Mycosel selective media were also reliably identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. VITEK-MS system with IVD database V3.2 correctly identified C. auris strains to the species level on CHROMagar Candida Plus alleviating the need for subcultivation and reduced turnaround time (24-72 h) to identification for patient screening.

Hg2+ removal characteristics of a strain of mercury-tolerant bacteria screened from heavy metal-contaminated soil in a molybdenum-lead mining area.

In this study, the mercury-tolerant strain LTC105 was isolated from a contaminated soil sample collected from a molybdenum-lead mine in Luanchuan County, Henan Province, China. The strain was shown to be highly resistant to mercury, with a minimum inhibitory concentration (MIC) of 32 mg·L-1. After a 24-h incubation in LB medium with 10 mg·L-1 Hg2+, the removal, adsorption, and volatilization rates of Hg2+ were 97.37%, 7.3%, and 90.07%, respectively, indicating that the strain had significant influence on mercury removal. Based on the results of Fourier infrared spectroscopy (FTIR) and scanning electron microscopy (SEM), the investigation revealed that the primary function of LTC105 was to encourage the volatilization of mercury. The LTC105 strain also showed strong tolerance to heavy metals such as Mn2+, Zn2+, and Pb2+. According to the results of the soil incubation test, the total mercury removal rate of the LTC105 inoculation increased by 16.34% when the initial mercury concentration of the soil was 100 mg·L-1 and by 62.28% when the initial mercury concentration of the soil was 50 mg·kg-1. These findings indicate that LTC105 has certain bioremediation ability for Hg-contaminated soil and is a suitable candidate strain for microbial remediation of heavy metal-contaminated soil in mining areas.

Assessment of the effects of citric acid and EDTA on cell viability of cultured human periodontal ligament cells attached to simulated avulsed permanent tooth using a spectrofluorometer-An invitro study.

The delayed re-implantation of avulsed teeth results in ankylosis, followed by replacement resorption and eventual loss of the tooth within 2-4 years. To prevent tooth loss, the root surface can be etched with acid to expose the collagen fibers present in the cementum layer. This process facilitates normal reattachment and regeneration of the periodontal ligament. This in-vitro study aimed to assess the viability and number of attached cultured Human Periodontal Ligament Cells (HPLC) on the dehydrated root surface of simulated avulsed teeth treated with citric acid and EDTA solutions. Sound human permanent teeth were included in the study. The root portions of the teeth were sectioned into slices, air-dried for 1 h, and divided into the following three groups: Group A-control; Group B-Citric acid treated for 30 min; Group C-EDTA treated for 5 min. The slices were then placed in cultured HPLC. After a 24-h incubation period, the slices were visualized under the microscope and prepared for reading the viable and dead HPLC using a spectrofluorometer, as well as for counting HPLC in a Neubauer Chamber. The spectrofluorometer intensity for viable and dead HPLC showed a statistically significant difference (p = .003 and p = .002), with the mean intensity for viable HPLC greater in citric acid group (69.52 ± 74.51), followed by EDTA group (31.39 ± 9.12), and control group (-130.93 ± 30.99). The dead HPLC intensity was greater in the EDTA group (19.43 ± 47.31), followed by the citric acid group (1.28 ± 1.85), and the control group (-2.77 ± 0.76). The total number of cells in the Neubauer chamber showed a statistically significant difference (p < 0.001), with a higher count in the citric acid group (10.83 ± 4.08) followed by EDTA group (2.92 ± 2.92). The application of citric acid for 30 min on the dehydrated root surface of avulsed teeth demonstrated superior outcomes compared to both EDTA treatment for 5 min and the control group.