Hours Of Culture Research Articles

Antibacterial effect of semiconductor laser radiation against the strains of S. aureus and P. aeruginosa, leading pathogens in osteomyelitis

Introduction The study of the antibacterial effect of photodynamic therapy against the leading pathogens of chronic osteomyelitis is one of the promising directions today.Purpose of the work was to evaluate the antibacterial effect against the strains of Staphylococcus aureus and Pseudomonas aeruginosa with the ALOD-01 laser system in the presence of photodithazine.Materials and methods The object of the study was 24-hour archival cultures of gram-positive and gram-negative microorganisms belonging to two taxa: Staphylococcus aureus (25923), Pseudomonas aeruginosa (27853). The antibacterial effect after the exposure to laser radiation in the presence of photodithazine on the microbial cells of the studied cultures was assessed by the absence of microorganism growth in the area of the light beam.Results Laser exposure in combination with photodithazine (concentration 0.5–1.0 mg/ml) on S. aureus for two minutes at 200–300 J achieved a bactericidal effect in the beam area. A bactericidal effect on the entire surface of the Petri dish was achieved with light exposure of 400 J for 5 minutes and a photodithazine concentration of 1.0 mg/ml. Laser exposure for 2 minutes in the presence of photodithazine at a concentration of 0.5 mg/ml and 1 mg/ml did not have an antibacterial effect on P. aeruginosa strains. Continuous growth of microorganisms was observed on the dish. Increasing the light dose and exposure time contributed to a decrease in the growth of microbial cells. A bactericidal effect was obtained only in the center of the dish in treating the bacterial suspension with photodithazine at a concentration of 5 mg/ml.Discussion The effectiveness of PDT depends on the type of microorganisms, the anatomical location of the infection site, as well as the properties of the photosensitizer and the laser used. Depending on the structure of the cell wall, different susceptibility of bacteria to photodynamic effects is observed.Conclusion S. aureus strains can be successfully photoinactivated using photodithazine. For P. aeruginosa strains, it was not possible to find a regime in which microbial cell growth was absent throughout the dish. The photodynamic reaction occurs only when adequate doses of light energy act on the photosentisizer in the presence of oxygen in the medium, while the photodynamic damage is local and the bactericidal effect is limited by the zone of light exposure.

Bone Marrow and Peripheral Blood Mononuclear Cell Phenotype Changes after Cultivation and Autologous Infusion in Patients with Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is a condition affecting the liver and immune system. In this study, the impact of autologous bone marrow-derived mononuclear cell (BM-MNC) transplantation on PBC patients was investigated. Sixteen eligible PBC patients participated at the National Scientific Medical Center in Astana, Kazakhstan, between 2017 and 2022, and BM-MNCs were harvested from their anterior iliac crest. After isolating and cultivating the BM-MNCs, they were infused back into the patient's peripheral veins. Changes in BM-MNC and peripheral blood mononuclear cell (PB-MNC) phenotypes were assessed before and after a 24-hour cultivation period and 72 hours post-transplantation. We monitored liver function parameters over 6-month intervals and conducted flow cytometry analysis to assess CD markers on BM-MNCs before and after cultivation and PB-MNCs before and after transplantation. Statistical analysis included the Friedman test for liver parameters and the Wilcoxon signed-rank test for BM-MNC and PB-MNC comparisons. Our findings revealed significant reductions in liver function tests after multiple transplantations. Flow cytometry analysis before and after a 24-hour culture and autologous BM-MNC infusion revealed the expansion of specific cell populations, with significant increases in CD3+, CD4+, CD16+, CD20+, CD25+, CD34+, CD105+, CD73+, СD117+, and CD34+populations, while CD4+25+, CD34+105+, and CD4+FOXP3+ populations decreased. Interestingly, a contradictory finding was observed with a decrease in bone marrow CD34+105+ cell lines (P=0.03) alongside an increase in peripheral CD34+105+ population (P=0.03). In summary, our study shows that BM-MNC transplantation in PBC patients leads to changes in immune cell populations and liver function. These findings suggest potential therapeutic applications of BM-MNC transplantation in managing PBC and offer insights into the dynamics of immune cells associated with this treatment approach.

Oxygenator assisted dynamic microphysiological culture elucidates the impact of hypoxia on valvular interstitial cell calcification

IntroductionMicrophysiological systems (MPS) offer simulation of (patho)physiological parameters. Investigation includes items which lead to fibrosis and calcification in development and progress of calcific aortic valve disease, based e.g. on culturing of isolated valvular interstitial cells (VICs). Hypoxia regulated by hypoxia inducible factors impacts pathological differentiation in aortic valve (AV) disease. This is mimicked via an MPS implemented oxygenator in combination with calcification inducing medium supplementation.MethodsHuman valvular interstitial cells were isolated and dynamically cultured in MPS at hypoxic, normoxic, arterial blood oxygen concentration and cell incubator condition. Expression profile of fibrosis and calcification markers was monitored and calcification was quantified in induction and control media with and without hypoxia and in comparison to statically cultured counterparts.ResultsHypoxic 24-hour culture of human VICs leads to HIF1α nuclear localization and induction of EGLN1, EGLN3 and LDHA mRNA expression but does not directly impact expression of fibrosis and calcification markers. Dependent on medium formulation, induction medium induces monolayer calcification and elevates RUNX2, ACTA2 and FN1 but reduces SOX9 mRNA expression in dynamic and static MPS culture. But combining hypoxic oxygen concentration leads to higher calcification potential of human VICs in calcification and standard medium formulation dynamically cultured for 96 h.ConclusionIn hypoxic oxygen concentration an increased human VIC calcification in 2D VIC culture in an oxygenator assisted MPS was detected. Oxygen regulation therefore can be combined with calcification induction media to monitor additional effects of pathological marker expression. Validation of oxygenator dependent VIC behavior envisions future advancement and transfer to long term aortic valve tissue culture MPS.

Heterotrophic activity and hydrocarbon degradation in crude oil-contaminated coastal soil augmented with indigenous biosurfactant producing Pseudomonas sp.

The enhanced bioremediation of the petroleum-contaminated soil from the Ibeno coastal area was investigated using standard microbiological and biotechnological methods. Soil samples were simulated with 200ml, 400ml, and 800ml of Bonny Light crude oil representing 5%, 10%, and 20 % contamination levels respectively, and allowed to mimic natural crude oil degradation for 48 hours. The contaminated soil was bioaugmented with 15 ml of 24-hour culture of potent biosurfactant-producing bacteria – Pseudomonas aeruginosa with a viable cell count of 2.6 x 103 CFU/ml and monitored for 12 weeks. Analysis of the fate of the hydrocarbon contamination in soil augmented with the biosurfactant-producing pseudomonad revealed that the degradation of total petroleum hydrocarbon (TPH) and polycyclic aromatic hydrocarbons (PAHs) was faster in augmented soils. At the end of the degradation, the augmentation process induced a reduction in the TPH content and PAH levels of soil exposed to 20% contamination from 32.85 mg/kg to 15.14 mg/kg and from 16.34 mg/kg to 5.35 mg/kg respectively. These represent 45.9% and 32.7% remediation rates of TPH and PAH, respectively. The findings of this study also indicate that bioaugmentation of crude oil-contaminated soil with a culture of P. aeruginosa, does not only influence the density of heterotrophic and hydrocarbon-degrading bacteria in the soil but increases the natural attenuation potentials of the contaminated soil.

Antibacterial Effect of Nitric Oxide on the Causative Agents of Hospital-Acquired Pneumonia (Experimental Study)

The aim of the study was to evaluate the antimicrobial effect of single and repeated nitric oxide (NO) exposure on the major pathogens of nosocomial pneumonia isolated from the sputum of cardiac surgery patients.Materials and Methods. A 24-hour culture of microorganisms from pan-resistant isolates of Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae from the sputum of inpatient cardiac surgery patients with nosocomial pneumonia, as well as strains of P. aeruginosa and E. coli from the American Type Culture Collection (ATCC), were exposed to 200 ppm NO (experimental sample) or medical air (control sample) in a sealed chamber for 30 minutes. After a single or 4 repeated gas exposure at 4 h intervals, Petri dishes were placed in a thermostat at 37°C and the results were evaluated at 24 and 48 h or at 12, 24, 36 and 48 h, respectively. Grown colonies were counted using an automated colony counter and recorded as CFU/mL.Results. No growth of clinical isolates of P. aeruginosa and E. coli was observed 24 and 48 h after a single exposure to NO. Growth of A. baumannii was lower compared to controls at 24 h but continued at 48 h. No effect of a single exposure to 200 ppm NO on other microorganisms was observed. After 4 exposures to NO, the growth of ATCC E. coli was not detected, the growth of other experimental strains was significantly lower compared to the control (P<0.05).Conclusion. Our results provide a rationale for the use of multiple intermittent inhalation of 220 ppm NO for the treatment of patients with hospital-acquired bacterial pneumonia.

Extending culture time to improve Mitotic Index for cytogenetic dosimetry

Purpose To analyze the effects of extending lymphocyte cultivation time on the Mitotic Index, frequency of first-division cells, and dose estimation after irradiating blood samples with different doses of radiation. Materials and methods Blood samples from two healthy male volunteers were separately irradiated with three doses (3, 5, and 6 Gy) using a 60Co gamma source (average dose rate: 1.48 kGy.h−1) and cultivated in vitro for conventional (48 h) and extended (56, 68, and 72 h) amounts of time. Colcemid (0.01 µg.mL−1) was added at the beginning of the culture period. Cells were fixed, stained with fluorescence plus Giemsa (FPG), and analyzed under a light microscope. The effects of prolonged culture duration on the Mitotic Index (MI), frequency of first-division cells (M1 cells), and the First-Division Mitotic Index (FDMI) were investigated. The estimation of delivered doses was conducted using a conventional 48h-culture calibration curve. Results Overall, cells presented higher MI (up to 12-fold) with the extension of culture, while higher radiation doses led to lower MI values (up to 80% reduction at 48 h). Cells irradiated with higher doses (5 and 6 Gy) had the most significant increase (5- to 12-fold) of MI as the cultivation was prolonged. The frequency of M1 cells decreased with the prolongation of culture for all doses (up to 75% reduction), while irradiated cells presented higher frequencies of M1 cells than non-irradiated ones. FDMI increased for all irradiated cultures but most markedly in those irradiated with higher doses (up to 10-fold). The conventional 48h-culture calibration curve proved adequate for assessing the delivered dose based on dicentric frequency following a 72-hour culture. Conclusion Compared to the conventional 48-hour protocol, extending the culture length to 72 hours significantly increased the Mitotic Index and the number of first-division metaphases of irradiated lymphocytes, providing slides with a better scorable metaphase density. Extending the culture time to 72 hours, combined with FPG staining to score exclusively first-division metaphases, improved the counting of dicentric chromosomes. The methodology presented and discussed in this study can be a powerful tool for dicentric-based biodosimetry, especially when exposure to high radiation doses is involved.

Anti-blastocystis activity of hop extracts in vitro

Blastocystis sp. are the most common, unicellular, anaerobic parasites of the intestinal tract of many animal and human species that can cause various digestive diseases. Metronidazole has long been used as a first-line treatment for blastocystosis, but recent clinical and in vitro studies have demonstrated its low efficacy against Blastocystis sp. The aim of this study was to determine the in vitro sensitivity of Blastocystis sp. clinical isolates to carbonic acid hop extracts and alcohol hop extracts in comparison with metronidazole. Five cultures of Blastocystis sp. were isolated from faecal samples from patients with irritable bowel syndrome with predominant diarrhoea (IBS-D, Rome IV). The parasites were identified by microscopy of faecal smears permanently stained with trichrome, Wheatley’s modification and Heidenhain’s iron-haematoxylin. Blastocystis sp. was cultivated at 37°C under anaerobic conditions on RPMI-1640 with antibiotics and horse serum, taking into account their growth characteristics. To detect the anti­blastocystic activity hop extracts and metronidazole were tested in the range from 1000 μg/ml to 1 μg/ml.The presence and number of viable Blastocystis sp. cells were determined after 24, 48, 72 and 96 hours. Blastocystis sp. cells were counted in a hemocytometer using the trypan blue dye exclusion test. All experiments were performed in triplicate. According to the results of in vitro sensitivity of 5 clinical isolates of Blastocystis sp. to the action of carbonic acid hop extract, alcohoicl hop extract and metronidazole, a direct positive pattern in the dose-response and contact time-response effects was established. Alcoholic hop extract showed the highest level of antiblastocystic activity with indicators (for 72-hour parasite cultures) of the minimum inhibitory concentration (which inhibits the parasite cultures growth by 50%) (2.8±0,8) μg/ml and the minimum lethal concentration (which destroys parasite cells by 100%) – 8 μg/ml, being 2.4 and 4.5 times lower than the minimum inhibitory concentration and 8 and 62.5 times lower, than the minimum lethal concentration for hop extract and metronidazole, respectively (p<0.05). It has been shown that alcoholic hop extract (≥16 μg/ml) causes gradual morphological changes in Blastocystis sp. cells, leading to their complete destruction.In contrast to metronidazole, subinhibitory concentrations of hop extract (<2 μg/ml) do not stimulate the proliferation of Blastocystis sp. cells in vitro.

Viability and Proliferation Assessment of Gingival Fibroblasts Cultured on Silver Nanoparticle-Doped Ti-6Al-4V Surfaces.

To investigate the biocompatibility of silver nanoparticle (AgNP)-doped Ti-6Al-4V surfaces by evaluating the viability and proliferation rate of human gingival fibroblasts (HGFs)-as the dominant cells of peri-implant soft tissues-seeded on the modified surfaces. AgNPs (sizes 8 nm and 30 nm) were incorporated onto Ti-6Al-4V specimen surfaces via electrochemical deposition, using colloid silver dispersions with increasing AgNP concentrations of 100 ppm, 200 ppm, and 300 ppm. One control and six experimental groups were included in the study: (1) control (Ti-6Al-4V), (2) 8 nm/100 ppm, (3) 8 nm/200 ppm, (4) 8 nm/300 ppm, (5) 30 nm/100 ppm, (6) 30 nm/200 ppm, and (7) 30 nm/300 ppm. HGF cell primary cultures were isolated from periodontally healthy donor patients and cultured in direct contact with the group specimens for 24 and 72 hours. The cytotoxicity of AgNP-doped Ti-6Al-4V specimens toward HGF was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and BrdU (5-bromo-2'-deoxyuridine) assay tests. Calcein AM and ethidium homodimer (EthD-1) fluorescent stains were used to determine the live and dead cells. The morphology and attachment properties of the HGFs were determined via scanning electron microscopy (SEM). Energy dispersive x-ray (EDX) analysis confirmed the presence of AgNPs on the specimens. The MTT test revealed that AgNPs of both sizes and all concentrations presented a decreased cellular metabolic activity compared to the control discs. All concentrations of both sizes of AgNPs affected the cell proliferation rate compared to the control group, as revealed by the BrdU assay. Overall, cytotoxicity of the modified Ti-6Al-4V surfaces depended on cell exposure time. Observation via confocal microscopy confirmed the results of the MTT and BrdU assay tests. Specifically, most cells remained alive throughout the 72-hour culture period. SEM images revealed that adjacent cells form bonds with each other, creating confluent layers of conjugated cells. The findings of the present study indicate that Ti-6Al-4V surfaces modified with 8 nm and 30 nm AgNPs at concentrations of 100 ppm, 200 ppm, and 300 ppm do not produce any serious cytotoxicity toward HGFs. The initial arrest of the HGF proliferation rate recovered at 72 hours. These results on the antibacterial activity against common periodontal pathogens, in combination with the results found in a previous study by the same research group, suggest that AgNP-doped Ti-6Al-4V surfaces are potential candidates for use in implant abutments for preventing peri-implant diseases.

Evaluation of Mex A' Mex B Efflux Pump Gene in Pseudomonas aeruginosa Isolated from Milk in Qom Province, Iran

Background:: Pseudomonas aeruginosa is an opportunistic pathogen protected from antibiotics' harmful effects by various mechanisms. One of the most important mechanisms is the activation of exudation systems. Therefore, identifying such targets in food contaminating bacteria can help us target them and eliminate bacteria resistant to several drugs, making food safe. Therefore, the present study aimed to trace Mex A and Mex B efflux pump genes in Pseudomonas aeruginosa isolated from dairy products in Qom City, Iran. Methods:: 300 samples of raw milk, 30 samples of pasteurized milk, and 30 samples of local cream were collected. After a 24-hour culture on nutrient agar and blood agar and performing warm staining, confirmatory biochemical tests were performed to identify bacteria. The disk method was used according to CLSI guidelines to determine the antibiotic sensitivity pattern. Then Mex A and Mex B efflux pump genes were detected using specific primers with PCR technique. Results:: 117 strains of Pseudomonas aeruginosa were isolated from 300 raw milk samples, and 7 strains were resistant to ciprofloxacin. Out of 117 Pseudomonas aeruginosa isolates, 51 isolates (43.5%) had Mex A gene and 10 isolates (8.5%) had Mex B gene. Three isolates had both Mex A and Mex B genes. Among the strains with resistance to ciprofloxacin (7 isolates), 4 isolates did not have any of the genes. One isolate had Mex A and Mex B genes; 2 isolates were Mex A positive and Mex B negative, and Mex B positive and Mex A negative isolates were not observed. Conclusions:: Based on the results, among the studied isolates, the Mex A gene was more abundant. In addition, no significant relationship was found between the existence of Mex A and Mex B genes and ciprofloxacin resistance.

Getting the drop on Staphylococcus aureus: Semiquantitative Staphylococcus aureus nasal colony reduction in orthopedic surgery reduces surgical site infection

Surgical site infection (SSI) is a frequent health care-associated infection. We aimed to reduce SSI risk after joint arthroplasty and spine surgery by reducing Staphylococcus aureus colonization burden with presurgery intranasal povidone-iodine (PVP-I) application in conjunction with skin antisepsis ("the intervention"). Retrospective case-control study; postintervention cohort versus a historical cohort. Adults who underwent joint arthroplasty or spine surgery during February 2018 through October 2021 ("post-intervention cohort") included. In the analysis cases any patient who underwent surgery and developed SSI within 90 days postsurgery, controls had no SSI. Postintervention cohort data were compared with a similar retrospective 2016 to 2017 patient cohort that did not use intranasal PVP-I. The postintervention cohort comprised 688 consecutive patients aged 65y/o, 48.8% male, 28 cases, and 660 controls. Relatively more cases than controls had diabetes mellitus (P=.019). There was a 39.6% eradication rate of S aureus nasal colonization post intranasal PVP-I (P<.0001). SSI rate was higher in patients positive versus those negative for S aureus on a 24-hour postsurgery nasal culture (P<.0001). The deep SSI rate per 100 operations postintervention versus the historical cohort decreased for all surgical procedures. Semiquantitative S aureus nasal colony reduction using intranasal PVP-I is effective for decreasing SSI rate in joint arthroplasty and spine surgery. In patients with presurgery S aureus nasal colonization additional intranasal PVP-I postsurgery application should be considered.

Abstract 45: Driving AMPK signaling in CD4+ CART cells enhances metabolic capacity, memory formation, and anti-leukemia activity

Abstract Background: While Chimeric Antigen Receptor (CAR)T cell therapy has seen great success in pediatric and young adult acute lymphoblastic leukemia, up to 50% of patients will relapse following treatment. Increased memory formation and mitochondrial capacity, particularly in the CD4+ subset, have been linked to improved CART cell function and enhanced in vivo persistence, leading to long-term relapse-free survival. AMP-activated protein kinase (AMPK) is a cellular energy sensor responsible for promoting metabolic efficiency and T cell memory formation. We have previously demonstrated AMPK activity can be increased in human T cells via overexpression of the AMPKγ2 domain. We hypothesized that overexpression of AMPKγ2 in CART cells would enhance their metabolic capacity and improve their anti-leukemia function. Methods: Human CART cells bearing a CD19-CAR utilizing a 41BB costimulatory domain were generated from healthy human donors via lentiviral transduction and co-transduced with AMPKγ2 (AMPK) or an empty vector (EV) control. Flow sorting for the co-transduced populations yielded &amp;gt;95% CD4+ CART cells. CARTs were expanded in IL-2 for approximately 2 weeks, followed by overnight stimulation and subsequent metabolic analysis using the Seahorse Metabolic Analyzer. Memory status was assessed by CD62L and CCR7 co-expression. In vitro CART function was assessed using Zs-green+ NALM6 targets, with loss of green fluorescence over time measured on the Incucyte analyzer. Where indicated, tumor conditioned media was harvested from 72-hour NALM6 cultures. For in vivo experiments, immunodeficient NOD-scid IL2Rγnull (NSG) mice were injected with luciferase+ NALM6 targets, followed one week later by AMPK- or EV-CARTs. Leukemia burden, via IVIS imaging, and survival were followed over time. Results: CD4+ CARTs co-transduced with AMPKγ2 (AMPK-CARTs) increased oxygen consumption, maximal respiration, and spare respiratory capacity following overnight stimulation by ~20% compared to CARTs co-transduced with the EV (EV-CARTs) (p&amp;lt;0.05). Following expansion, CD62L+CCR7+ central memory cells were 4.5-fold more abundant in the AMPK-CART group (2.4-6.2-fold, p&amp;lt;0.01). In vitro, while AMPK-CARTs demonstrated similar cytotoxicity against NALM6 targets as EV-CARTs in standard media, they mediated faster killing of NALM6 targets when challenged in tumor conditioned media (p&amp;lt;0.001). In vivo, recipients of AMPK-CARTs showed a consistent decrease in tumor burden as measured by median radiance values, translating to a significant survival advantage (p&amp;lt;0.05). Conclusions: Increasing AMPK signaling in CD4+ CARTs via overexpression of AMPKγ2 improved metabolic profile, increased memory generation, and enhanced toxicity against leukemia targets both in vitro and in vivo. Together, these features make elevating AMPK signaling an attractive method to increase the efficacy of CART therapy. Citation Format: Erica Braverman, Herbert Schuler, Mengtao Qin, Craig Byersdorfer. Driving AMPK signaling in CD4+ CART cells enhances metabolic capacity, memory formation, and anti-leukemia activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 45.

Preliminary evaluation of in-Vivo and in-Vitro antifungal activity of Piper longum, Origanum majorana, Embelia ribes and Butea monosperma with Gas chromatography-mass spectrometry analysis of phytochemical property of plant extracts against Candida species causing dermatological

: Globally, fungi infections cause more than 1.5 million fatalities annually, mostly in those with weakened immune systems with Candida albicans found to be the chief pathogen. Approximately one to two billion people are affected by dermatological fungal infections involving skin and skin appendages, perhaps very difficult to estimate its exact prevalence due to under-reporting, this augments the requirements for more attention towards new molecules to combat resistance.: Evaluation of antifungal activity of Piper longum, Origanum majorana, Embelia ribes, Butea monosperma both in-vivo and in-vitro analysis against Candida species. This experimental study conducted for evaluating both in-vitro as well as in-vivo anti-fungal activity of Piper longum, Butea monosperma, Embelia ribes and Origanum majorana, and evaluation of phytoconstituent through Gas Chromatography and Mass Spectrometry analysis by GC-MS-QP-2010 plus system to identify the active bio-component. Fluconazole used as the standard control in-vivo and Itraconazole used as a standard drug for in-vitro infection and analysis. Ethical Committee approval was taken (IMS, BHU Dean/2018/CAEC/818). The antifungal activity of the extracts in-vitro was evaluated by Resazurin microtiter assay against Candida sp. Micro broth dilution methods. Rats were given an injection of 0.2 mL (intravenously/i.v) of a 10° UFC/mL inoculum made from a fresh 48-hour Candida albicans culture in sterile saline to cause infection. Induction of candidemia in rats was confirmed using qualitative Gram-stained smear. : The extracts of Butea monosperma (Alc.), Piper longum (Alc.), Origanum majorana (Aq.), and Piper longum (Aq.) showed effective antifungal activity against Candida sp. in-vitro research with a minimum inhibitory concentration (MIC) of less than 0.25 mg/mL. : This study is contributing towards the search for a natural drug candidate to combat resistance caused by the Candida genus, as it is one of the highest contributors to fungal dermatological/skin infections.

Determination of Raman spectrum under different culture conditions: preliminary research on bacterial fish pathogens

The intensive labour and time required for conventional methods to identify bacterial fish pathogens have revealed the need to develop alternative methods. Raman spectroscopy has been used in the rapid optical identification of bacterial pathogens in recent years as an alternative method in microbiology. Strains of bacterial fish pathogens (Vibrio anguillarum, Lactococcus garvieae and Yersinia ruckeri) that often cause infectious diseases in fish were here identified and analyzed in terms of their biochemical structures in different media and at different incubation times, and the data were specified by using Raman spectroscopy. The results demonstrated that Raman spectroscopy presents species-specific Raman spectra of each disease-causing bacteria and that it would be more appropriate to choose general microbiological media over selective media for routine studies. Additionally, it was found that species-specific band regions did not differ in 24- and 48-hour cultures, but there could be a difference in peak intensity which may lead to difficult characterization of spectrum. The current study, conducted for the first time with bacterial fish pathogens under different incubation conditions, is believed to provide a basis for the routine use of Raman spectroscopy for quick pathogen identification and the precise determination of the methodology for further research.

The effect of BMSCs on implantation mechanisms and pregnancy process in the experimental Asherman model.

In this study, we aimed to investigate the role of bone marrow-derived mesenchymal stem cells (BMSC) on the molecular mechanisms involved in the removal of endometrial adhesions and the implantation process in the experimentally-created Asherman syndrome model in rats. An experimental model chemical agent was used. Culture medium (CM), only BMSC, 48-hour culture medium (Niche), and BMSC+ niche were used as therapy. Each group was divided into two subgroups, and changes in the endometrial tissue were investigated by histochemical and immunohistochemical staining. The results of the study show that BMSCs exhibit therapeutic properties in endometrial damage, increase endometrial thickness and vascularization, and contribute to the implantation of embryos by reducing fibrous areas. These results are promising for the prevention of Asherman-induced infertility. It has been shown that BMSC and Niche applications can be helpful in preventing adhesion molecules in the mechanisms that cause infertility.

A Randomized Double-blind Placebo-controlled Study on the Efficacy of Oregano (Origanum Vulgare) Leaf Ointment on the Reduction of 24-hour Colony Forming Units of Staphylococcus Aureus in Infected Open Wounds of Children Aged 8-12 Years Old

The practice of traditional medicine has been a vital part of Philippine medical practice, and, as a consequence, has led to innumerable researches conducted by the Department of Health (DOH) as to their role in the Filipino well-being. Out of the 10 named herbal plants studied by the department, oregano (Origanum vulgare) was chosen for its uses on the disorders of the skin, gastrointestinal and respiratory tract in the communities; and was therefore used in this study for the evaluation of its antimicrobial property, formulated as a leaf ointment against Staphylococcus aureus. OBJECTIVES: Specifically, this research aimed to determine a significant difference between the colony forming units (CFUs) on infected wounds treated with the placebo and oregano (Origanum vulgare) ointments. METHODOLOGY: A randomized double-blind placebo-controlled research study was conducted to the 60 infected wounds of the students of Dona Juana Actub Lluch Memorial Central School and Ubaldo Laya Memorial Central School with respondents aging 8-12 years old. Pre-treatment wound swabbing was done for 24-hour microbial culture, from which CFUs were counted. Post-treatment swabbing was performed after 5 days of treatment and subsequent CFUs were obtained. Using 0.05 level of significance, Mann-Whitney Pairwise Comparison Test was used to determine the significant difference in CFU count reduction between the two treatments. Findings showed reduction of CFUs in both ointments with greater reduction of CFUs in wounds treated Oregano leaf ointment as compared to those with the placebo. Statistical analysis of the study revealed that there was a significant difference between Placebo and Oregano (Origanum vulgare) on the reduction of CFUs in a 24-hour bacterial culture of infected open wounds.

116 Evaluation of frozen–thawed bovine embryos before and after 24-hour culture

116 Evaluation of frozen–thawed bovine embryos before and after 24-hour culture

B16F10.OVA cell에서 Atorvastatin에 의한 Ferroptosis 유도 규명

In this study, we identified whether atorvastatin could induce ferroptosis or not in B16F10.OVA cells, which is being used to treat hypercholesterolemia. To demonstrate this, we treated the mouse melanoma cell line B16F10.OVA with atorvastatin at varying concentrations, spanning from low to high, followed by a 24-hour culture period. In the group treated with atorvastatin in our experiment, we observed a decrease in cell viability that was dependent on the concentration of the treatment, when compared to the control group. The IC50 value of atorvastatin in B16F10.OVA cells was determined to be 49.89 μM. Compared to the control group, treatment with atorvastatin resulted in a concentrationdependent increase in the generation of reactive oxygen species and the accumulation of iron ions. Moreover, atorvastatin treatment exhibited a concentration-dependent increase in the population of apoptotic cells and the expression of calreticulin, significantly surpassing that of the control group. The analysis of ferroptosis-related protein expression levels revealed a notable decrease in the expression of GPX4 in the group treated with atorvastatin, as compared to the control group. These findings strongly suggest that atorvastatin holds promise as a potent anti-cancer agent capable of inducing ferroptosis in melanoma cells.

Systemic production of cytokines and growth factors in rats with metabolic syndrome

ABSTRACT. An important characteristic of the state of the immune system is the production of cytokines, which determine the development of metabolic processes and are aimed at the immune response. Changes in the immune response occur with the development of homeostasis system disorders that change the rheological characteristics of the blood and contribute to the development of the metabolic syndrome (MS). The aim of the study was to study changes in the functional activity of cytokines and angiogenic growth factors (GF) during experimental MS in rats depending on age. Мaterials. MS modeling was carried out on 21 rats, which were divided into 3 groups, 7 animals each, of the same age (1a subgroup - 6 months of age, 2a subgroup - 9 months of age, 3a subgroup - 12 months). In the pathogenesis of the development of MS The role of MS in the development of systemic production of cytokines and FR has been studied, and data on the content of pro-inflammatory cytokines in the peripheral blood depending on the age of rats have been presented. The results of the study of the content of growth factor (GF) and endothelin in peripheral blood in MS tended to decrease with increasing age of rats providing morpho- and angiogenesis (p&lt;0.01). An increase in the concentration of endothelin in the blood serum of rats with MS may contribute to the activation of vascular endothelial cells and an increase in the level of fibrinoid in the vascular system with increasing age. The level of cytokines circulating in the peripheral blood in rats with MS significantly differed from that, which was manifested by an increase in the level of IL-4, IL-8 and IL-10. Spontaneous production of cytokines by peripheral blood lymphocytes in MS increased with the age of rats, which indicated an increase in the secretion of IL-6 and IL-10, and a decrease in the production of IFN-1 and TNF-α. The decrease in spontaneous production of IL-1β and IL-10 in 24-hour cultures of peripheral blood monocytes in rats was significantly higher compared to IL-6 (p&lt;0.001) compared to the 2nd and 3rd - observation groups. The content of IL-8 and TNF-α did not differ significantly (p&gt;0.05), and IL-12 was below the sensitivity limit of the test system (less than 3 pg/ml). Conclusion. The data obtained indicate that in the peripheral blood of rats with MS, the secretion of pro-inflammatory and regulatory cytokines, as well as FR, decreases with age. Hormones also have an inhibitory effect on the production of cytokines by macrophages, the level of which changes significantly during MS. The results of the study indicate various pathogenetic mechanisms of the formation of MS in rats, accompanied by a violation of the immune response of mononuclear cells and angiogenic RF at the system level.

P-706 Non-invasive analysis of mitochondrial DNA mutation for embryo evaluation.

Abstract Study question Is it possible to amplify whole mitochondrial DNA (mtDNA) and detect all mtDNA mutation in human embryo from spent media after embryo culture? Summary answer We were able to amplify whole mtDNA from 43% of spent medium after 24-hour culture. The most of mtDNA mutation in embryo could be detected. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) with embryo biopsy is now commonly used to improve pregnancy rate. However, invasive embryo biopsy requires highly trained embryologist and induced higher risk of pre-eclampsia. Therefore, non-invasive methods with cell-free DNA in spent medium have been studied. Our group previously showed euploid or viable embryos at post-implantation stage have lower number of non-synonymous mtDNA mutations and this may help to select the best blastocyst to transfer. Although cell-free DNA is reported to contain mtDNA, it remains unclear whether mtDNA mutations number can be assessed from cell-free DNA. Study design, size, duration Donated low grade blastocysts were cultured for additional 24 hours with new media, cell-free mtDNA were obtained from the spent culture medium (non-invasive samples). For comparison, trophectoderm cells were biopsied from each embryo (invasive sample). The mtDNA selective amplifications were done to both samples. We performed mtDNA sequencing by next generation sequencing (NGS) and compared mtDNA mutations between non-invasive and invasive samples to clarify the capability of non-invasive assessment for mtDNA mutation. Participants/materials, setting, methods Under the ethical review of Yokohama City University and informed consent with patients, we collected human blastocysts which were discarded because of slow development or morphologically low-grade. We extracted whole DNA from biopsy and medium samples and selectively amplified mtDNA by long-range PCR for two overlapping amplicons. The mtDNA sequencing with NGS was performed to successfully amplified mtDNA. The 90% or more heteroplasmy level non-synonymous mutations were investigated. Main results and the role of chance We collected 34 discarded embryos from 13 patients. All embryos were cultured individually for additional 24 hours with 20 μl drop of newly prepared media. Amplified mtDNA were visualized by the presence of 8 kb band following agarose gel-apheresis for two fragments PCR. Both fragments were detected in 41% (14/34) of non-invasive samples and all invasive samples. Only one fragment was detected in 14% (5/34) of non-invasive samples. The completely amplified non-invasive samples and corresponding invasive samples were performed mtDNA sequencing with NGS. In results, 13 non-synonymous mutations were identified in invasive samples. All of these mutations were also detected in non-invasive samples. Although the number and location of mutations completely matched between the invasive and non-invasive samples in 78.5% (11/14) embryos, in other 3 embryos, mtDNA mutation numbers were higher in non-invasive samples even though no mutation existed in invasive samples. This means, if selective mtDNA amplification is completed, mtDNA mutations can be evaluated from the non-invasive samples with a probability of about 80% but include 20% false positive mtDNA mutations. To realize this, we need to improve the mtDNA amplification method. Limitations, reasons for caution We can used only clinically discarded embryos because ethical restriction. And it was necessary to additional 24-hours culture to obtain individually cultured spent media because embryos are co-cultured until they are donated. Because of small number of samples available, we keep collecting more samples for more accurate evaluation. Wider implications of the findings If the mtDNA mutations can be analyzed from spent medium, high-quality embryos can be selected without invasive embryo biopsy and complicated procedure. If this non-invasive mtDNA analysis can be performed to clinical embryo for embryo transfer, it may contribute to further improvement of pregnancy rate without any invasion to embryo. Trial registration number the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research C (Grant Number JP21K09474) and Early-Career Scientists (Grant Number JP20K18169)

10-year stability of magnetite nanopowder prepared by the exploding wire method: is it a useful feature for environment safety and biomedical applications?

Aim. To analyze the structural, magnetic, and cytotoxic features of magnetite nanoparticles (MNPs) prepared by the exploding wire method and stored in a dark place at ambient temperature (65 ± 15% humidity, air pressure 760± 20 mm Hg., temperature 22 ± 4 °C) for 10 years.Materials and methods. The properties of MNPs were analyzed by X-ray diffraction (XRD), transmission electron microscopy (TEM) and selected area electron diffraction (SAED), and vibrating-sample magnetometer (VSM). Viability of human blood mononuclear leukocytes was determined using 0.4% trypan blue staining after 24-hour culture with the nanopowder.Results. The calculated size of the particles remained almost unchanged after 10 years of storage. The XRD and SAED patterns showed that crystallinity was preserved for 10 years. The diameter of the crystalline component of MNPs (DXRD) was close to the particle size determined by TEM. It confirms high crystallinity of the tested nanoparticles. Saturation magnetization (MS) of the MNP powder after 10 years of storage was unexpectedly higher than that of the as-prepared MNP powder. Reduced remanent magnetization (MR / MS) was equal for both samples within the margin of error. No cytotoxic effect of MNPs in vitro was detected in the long-term study.Conclusion. No dramatic changes in the structural, magnetic, and cytotoxic features of MNPs were noted after 10 years of storage. It indicated 10-year stability of MNP powder that may be a useful feature for environment safety and biomedical applications.