Mesenchymal stem cells conditioned media-chitosan nanoparticles against clinical carbapenem-resistant Acinetobacter baumannii: In-vitro study.

Background and Objectives: Stenotrophomonas maltophilia is an opportunistic pathogen causing nosocomial infections. Diclofenac is an anti-inflammatory drug that is considered a non-antibiotic drug. This study assessed the antibacterial and antibiofilm effects of diclofenac and levofloxacin/diclofenac combination against levofloxacin resistant isolates. Materials and Methods: Minimum inhibitory concentration was determined using broth microdilution method for levo- floxacin, diclofenac, and levofloxacin/diclofenac combination. Biofilm forming capacity and biofilm inhibition assay were determined. Relative gene expression was measured for efflux pump genes; smeB, and smeF genes and biofilm related genes rmlA, spgM, and rpfF without and with diclofenac and the combination. Results: Diclofenac demonstrated MIC of 1 mg/ml. The combination-with ½ MIC diclofenac- showed synergism where levofloxacin MIC undergone 16-32 fold decrease. All the isolates that overexpressed smeB and smeF showed a significant decrease in gene expression in presence of diclofenac or the combination. The mean percentage inhibition of biofilm for- mation with diclofenac and the combination was 40.59% and 46.49%, respectively. This agreed with biofilm related genes expression investigations. Conclusion: Diclofenac showed an antibacterial effect against Stenotrophomonas maltophilia. The combination showed in-vitro synergism, significant reduction in biofilm formation and in the relative level of gene expression. Furthermore, it can potentiate the levofloxacin activity or revert its resistance.

PCBs are persistent environmental contaminants that cause a variety of adverse health effects in wildlife and humans. This article describes the use of signature gene expression patterns that link increased PCB exposure with progressive, adverse biological effects. Developing Xenopus laevis tadpoles of two age classes were exposed to the PCB mixture Aroclor 1254 for 2 days. Real-time PCR was used to quantitate mRNA expression for 11 physiologically relevant, potential bioindicator genes. Younger tadpoles (5 days postfertilization) were resistant to Aroclor 1254 and showed few changes in gross morphology, swimming behavior, survival, or gene expression. Older tadpoles (11 days postfertilization) were more susceptible to Aroclor 1254. Exposure to 25 and 50 ppm Aroclor 1254 caused alterations in gross morphology and swimming behavior and statistically significant decreases in survival. These tadpoles showed statistically significant decreases in gene expression for 9 out of the 11 genes measured. Tadpoles exposed to 10 ppm showed incipient health changes but had gene expression profiles similar to the tadpoles treated with higher doses of Aroclor 1254. Tadpoles exposed to 1 ppm did not exhibit any observable adverse health effects, yet statistically significant decreases in gene expression occurred in these tadpoles (4 out of 11 genes). After prolonged exposure, tadpoles exposed to 1 and 10 ppm Aroclor 1254 exhibited health effects similar to those exposed to higher concentrations. Therefore, changes in expression of specific genes may serve not only as molecular bioindicators of Aroclor 1254 exposure but also as predictors of impending adverse health effects.

Background and Aim Apoptosis or programmed death is active biological process and reversible that the balance between growth and cell death in various tissues, particularly myocardial tissue is essential role. The aim of this study was the effect of 8 weeks High Intensity Interval Training (HIIT) with and without food restriction on gene expression of Caspase 3 and 9 in rats. Methods & Materials: This study was experimental multi group design with control group which conducted on 30 rats with two months old male. Subjects were divided in to five homogenous groups including base control, control, caloric restriction, interval exercise training and caloric restriction+interval exercise training. Training groups five sessions in weeks were participated in interval exercise training for 8 weeks. The level of gene expression of the proteins Caspase 3 and 9 was evaluated by Real Time-PCR. Dates were analyzed using the 1-way ANOVA at the level of (P<0.05). Ethical Considerations: The protocol of this study was approved by the Medical Committee of Sanandaj University of Medical Sciences (Code: IR.MYK.REC.1397.5022). Results: The results showed that the training group had a significant decrease in gene expression of Caspase 3 in comparison with caloric restriction+exercise training (P<0.05). Also, exercise training and exercise training+caloric restriction significantly decrease in the gene expression of Caspase 9 compared to caloric restriction, base control and control (P<0.05) and significantly decrease in the gene expression of Caspase 3 compared to base control and control (P<0.05). Conclusion: According to the results, it seems that high intensity interval training with and without calorie restriction provides the necessary adaptations to inhibit or stop apoptosis induced by aerobic exercise.

PS204. Mechanical (or “Gravitational”) Unloading Reduces Inflammatory and Cell Adhesion Molecule Gene Expression in Human Endothelial Cells

Objectives: Mechanical forces including gravity affect mechanotransduction and subsequent cell function. The goal of this study was to investigate the impact of mechanical unloading (MU) and loading (ML) of endothelial cells (ECs) with microgravity and hypergravity respectively, with the hypothesis that MU alters expression of inflammatory and adhesion molecule gene expression and these changes are reversed by ML. Methods: Human umbilical vascular endothelial cells (HUVECs) grown to confluency were studied. A desktop random positioning machine and a gravitational cell-loading apparatus provided MU and ML conditions, respectively. The experimental conditions included: 1) controls exposed to 1-gravity environment for 24 h (CL), 2) MU for 24 hours, 3) MU for 24 hours with three 30-minutes periods of ML of 12-gravity (MU/ML). Gene expression was studied with reverse transcription followed by real-time quantitative polymerase chain reaction (qRTPCR). Results: MU led to a significant decrease in gene expression of the adhesion molecules ICAM-1, VCAM-1, E-Selectin, as well as TNF-α, IL-6 and VEGF. In contrast, NOS-3, Caveolin-1 and -2 were significantly increased with MU. The changes observed in gene expression with MU were reversed by gravitational mechanical loading (MU/ML). Conclusions: Gravitational MU decreases inflammatory and adhesion molecule gene expression and these changes are reversed by short periods of ML. This points towards the importance of gravitational loading in ECs function and cellular interactions.

fliA, flrB, and fliR regulate adhesion by controlling the expression of critical virulence genes in Vibrio harveyi

Simple SummaryThe purpose of this study was to initially determine the role of methylation of the promoter region of Forkhead box O 1 (FOXO1) in regulating its transcriptional level and to further investigate the effect of FOXO1 on the proliferation and differentiation of bovine myogenic cells. In this study, we used bisulfite sequencing polymerase chain reaction, real-time quantitative PCR, western blot, cell counting kit-8 (CCK-8), and flow cytometry and found that the mRNA expression of the FOXO1 was low when the methylation of FOXO1 promoter region was high, and silencing the expression of the FOXO1 gene could promote the proliferation and differentiation of myoblasts.This study aimed to explore the regulatory role of FOXO1 promoter methylation on its transcriptional level and unravel the effect of FOXO1 on the proliferation and differentiation of bovine myoblasts. Bisulfite sequencing polymerase chain reaction (BSP) and real-time quantitative PCR were performed to determine the methylation status and transcript levels of the FOXO1 promoter region at different growth stages. BSP results showed that the methylation level in the calf bovine (CB) group was significantly higher than that in the adult bovine (AB) group (p < 0.05). On the other hand, qRT-PCR results indicated that the mRNA expression level in the AB group was significantly higher than that in the CB group (p < 0.05), suggesting a significant decrease in gene expression at high levels of DNA methylation. CCK-8 and flow cytometry were applied to determine the effect of silencing the FOXO1 gene on the proliferation of bovine myoblasts. Furthermore, qRT-PCR and Western blot were conducted to analyze the expression of genes associated with the proliferation and differentiation of bovine myoblasts. Results from CCK-8 revealed that the short hairpin FOXO1 (shFOXO1) group significantly promoted the proliferation of myoblasts compared to the short-hairpin negative control (shNC) group (p < 0.05). Flow cytometry results showed a significant decrease in the number of the G1 phase cells (p < 0.05) and a significant increase in the number of the S phase cells (p < 0.05) in the shFOXO1 group compared to the shNC group. In addition, the expression of key genes for myoblast proliferation (CDK2, PCNA, and CCND1) and differentiation (MYOG, MYOD, and MYHC) was significantly increased at both mRNA and protein levels (p < 0.05). In summary, this study has demonstrated that FOXO1 transcription is regulated by methylation in the promoter region and that silencing FOXO1 promotes the proliferation and differentiation of bovine myoblasts. Overall, our findings lay the foundation for further studies on the regulatory role of epigenetics in the development of bovine myoblasts.

Delivery LL37 by chitosan nanoparticles for enhanced antibacterial and antibiofilm efficacy

Objective: Bicuspid aortic valve (BAV) disease predisposes to early and more frequent ascending thoracic aortic aneurysm formation, but the mechanisms underlying this aortopathy remain incompletely characterized. The objective of this study was to identify genes predisposing to ascending aneurysm formation in patients with BAV aortopathy using a multipronged molecular approach. Methods: Ascending aortic aneurysm tissue samples were collected at the time of ascending aortic replacement in subjects with bicuspid (N=16) and trileaflet (N=16) aortic valves. All patients in the trileaflet valve group had degenerative aneurysms and those with connective tissue disorders were excluded. Using the aortic tissue samples, genome-wide DNA methylation status was determined using Illumina 450K methylation chips. Gene expression was determined using Illumina Whole-Genome DASL arrays. Gene methylation and expression were compared between bicuspid and trileaflet groups using Wilcoxon Rank Sum tests. Results: Twenty-six genes were differentially methylated between the bicuspid and trileaflet groups, and six of these genes also had significantly different gene expression between groups. Protein tyrosine phosphatase, non-receptor type 22 ( PTPN22 ) was strongly hypermethylated (beta: bicuspid 0.68 vs. trileaflet 0.55, P=4x10 -5 ) and had a resultant highly significant decrease in gene expression (log 2 gene expression intensity: bicuspid 5.1 vs. trileaflet 7.9, P=2x10 -5 ). Conclusions: In patients with ascending thoracic aortic aneurysms, there exist differential DNA methylation and gene expression signatures between those with BAV aortopathy versus acquired degenerative aneurysms and trileaflet aortic valves. The strongest finding was for PTPN22 , a gene involved in T-cell receptor signaling and associated with various autoimmune disorders. These differences may highlight novel mechanisms of aneurysm development in the BAV population, potentially serving as biomarkers and/or pharmacologic targets.

Iron Salts Perturb Biofilm Formation and Disrupt Existing Biofilms of Pseudomonas aeruginosa

In most ecosystems, bacteria exist primarily as structured surface-associated biofilms that can be highly tolerant to antibiotics and thus represent an important health issue. Here, we explored drug repurposing as a strategy to identify new antibiofilm compounds, screening over 1,000 compounds from the Prestwick Chemical Library of approved drugs for specific activities that prevent biofilm formation by Escherichia coli Most growth-inhibiting compounds, which include known antibacterial but also antiviral and other drugs, also reduced biofilm formation. However, we also identified several drugs that were biofilm inhibitory at doses where only a weak effect or no effect on planktonic growth could be observed. The activities of the most specific antibiofilm compounds were further characterized using gene expression analysis, proteomics, and microscopy. We observed that most of these drugs acted by repressing genes responsible for the production of curli, a major component of the E. coli biofilm matrix. This repression apparently occurred through the induction of several different stress responses, including DNA and cell wall damage, and homeostasis of divalent cations, demonstrating that biofilm formation can be inhibited through a variety of molecular mechanisms. One tested drug, tyloxapol, did not affect curli expression or cell growth but instead inhibited biofilm formation by suppressing bacterial attachment to the surface.IMPORTANCE The prevention of bacterial biofilm formation is one of the major current challenges in microbiology. Here, by systematically screening a large number of approved drugs for their ability to suppress biofilm formation by Escherichia coli, we identified a number of prospective antibiofilm compounds. We further demonstrated different mechanisms of action for individual compounds, from induction of replicative stress to disbalance of cation homeostasis to inhibition of bacterial attachment to the surface. Our work demonstrates the potential of drug repurposing for the prevention of bacterial biofilm formation and suggests that also for other bacteria, the activity spectrum of antibiofilm compounds is likely to be broad.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesCandida auris has emerged as a major multidrug-resistant nosocomial pathogen worldwide. The organism exhibits a persistent, colonizing phenotype, usually associated with biofilm formation on hospital surfaces, medical equipment, and indwelling medical devices. Biofilm formation by C. auris can further aggravate the infection acquisition and outcome, owing to the intrinsic resistance of biofilms to disinfectants, antiseptics, and antifungal drugs. The present study aimed to evaluate the preventive and therapeutic efficacy of select peptide derivative(s) from staphylococci against C. auris biofilms in vitro and in a mouse model of C. auris catheter-associated infection.MethodsThree potentially antimicrobial, staphylococcal alpha-helical amphipathic peptides (19-23 amino acids) were evaluated for antifungal and antibiofilm activity against clinical isolates of C. auris. The antifungal activity against C. auris planktonic cells was tested by broth microdilution assay according to the method of the Clinical and Laboratory Standards Institute. Biofilm assays were performed in 96 well, flat-bottomed microtiter plates in RPMI-1640, and the effect of the test agents on biofilm formation (MBPC, minimum biofilm preventive concentration) as well as pre-formed biofilms (MBIC, minimum biofilm inhibitory concentration) was determined by subjecting to a two-fold dilution range of the test agents (0.03-64 μm) for 24 h at 37°C, followed by XTT dye reduction test. Field-Emission Scanning Electron Microscopy was performed to observe the effect of peptides on biofilm morphology. The cytotoxicity of these compounds was elucidated on HeLa, HEK-293, and Raw 264.7 murine macrophages by MTT reduction test. The synergistic effect of the selected peptide with representative antifungal drugs belonging to three different classes (amphotericin B, caspofungin, and voriconazole) was tested by fractional inhibitory concentration assays. In vivo activity of the selected peptide was determined in a murine model of subcutaneous C. auris catheter-associated infection, alone and in combination with amphotericin B and caspofungin.ResultsBased on the antimicrobial activity, antibiofilm activity, and cytotoxicity data, the 19 amino acid, alpha helical staphylococcal peptide derivative (charge, +3; hydrophobicity, 0.634, and hydrophobic moment, 0.623) exhibited promising activity against C. auris biofilms. The peptide was particularly effective in preventing C. auris biofilm formation, with a median MBPC50 of 1 μm. It demonstrated synergistic activity with amphotericin B (FIC index, 0.3) as well as caspofungin (FIC index, 0.18), and an additive effect with voriconazole (FIC index, 0.71). When combined with 0.125 μm of the peptide derivative, nearly 4-to-8-fold lower amount of the drug was required to achieve results comparable to the drug-only controls. Furthermore, nearly 99% reduction in biofilm formation was noted at clinically-achievable trough levels of amphotericin B or caspofungin in combination with this peptide derivative. Similar results were noted in vivo, with nearly 99% reduction in biofilm formation of C. auris in catheter lumen using combination therapy with 0.125 μm of the peptide.ConclusionThe present study demonstrates that a 19 amino acid, alpha helical staphylococcal peptide derivative exhibits promising antibiofilm activity against C. auris, particularly in preventing biofilm formation, in vitro and in a murine model of subcutaneous catheter-associated infection.

This study was designed to investigate the precancerous lesions of gastric carcinoma (PLGC)-reversing mechanisms of astragaloside IV (ASIV) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PLGC rats. All rats were sacrificed after 10-week treatment. Gastric tissue was analyzed by using histopathology and electron microscope. To be fully evidenced, LDHA, p53, TIGAR, MCT1, MCT4, HIF-1α, CD147, and miRNA-34a were detected by Western blotting and Real-time Quantitative polymerase chain reaction (RT-qPCR). As histopathology and electron microscope showed, it can be clearly observed that the area of dysplasia was reduced in ASIV groups, indicating that MNNG-induced PLGC was markedly reversed by ASIV. Moreover, compared with model group, a significant decrease in gene expressions of LDHA, MCT1, MCT4, HIF-1α, CD147, and TIGAR was observed whereas miRNA-34a level was increased in ASIV groups. A significant up-regulation induced by MNNG in protein levels of LDHA, MCT1, MCT4, HIF-1α, and CD147 was attenuated in rats treated with ASIV. In contrast, the decreased expression of TIGAR was restored by ASIV. Interestingly, up-regulation of p53 expression induced by MNNG was further increased in ASIV groups. In brief, these results implied that abnormal glycolysis was relieved by ASIV via regulation of the expressions of LDHA, p53, TIGAR, MCT1, MCT4, HIF-1α, CD147, and miRNA-34a.

Recent evidence suggests that leukocytes infiltrate uterine tissues at or around the time of parturition, implicating inflammation as a key mechanism of human labor. MCP-1 (also known as C-C chemokine motif ligand 2, CCL-2) is a proinflammatory cytokine that is up-regulated in human myometrium during labor. Myometrium was collected from pregnant rats across gestation and at labor. Total RNA and proteins were subjected to real-time PCR and ELISA, respectively. Ccl-2 gene and protein expression was significantly up-regulated in the gravid rat myometrium before and during labor, which might suggest that it is regulated positively by mechanical stretch of the uterus imposed by the growing fetus and negatively by physiological withdrawal of progesterone (P4). We confirmed in vivo that: 1) administration of P4 receptor antagonist RU486 induced an increase in Ccl-2 mRNA and preterm labor, whereas 2) artificial maintenance of elevated P4 levels at late gestation caused a significant decrease in gene expression and blocked labor; 3) Ccl-2 was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium; 4) in vitro static mechanical stretch of primary rat myometrial smooth muscle cells (25% elongation) induced a release of Ccl-2 protein, which was repressed by pretreatment with P4 (1 microM); and 5) stretch enhanced their monocyte chemoattractant activity. These data indicate that Ccl-2 protein serves to integrate mechanical and endocrine signals contributing to uterine inflammation and the induction of labor and thus may represent a novel target for therapeutic prevention of preterm labor in humans.

BackgroundSkin aging is a complex biological phenomenon influenced by intrinsic and extrinsic factors. Photoaging can be prevented by applying phytochemicals that have sun-protective properties. This study aimed to evaluate the effect of saffron serum to restore collagen density and autophagy processes and reduce inflammatory gene expression in UVB-exposed Wistar rats. MethodsAn experimental laboratory study was conducted involving 20 male Wistar rats that were divided into 4 groups: control, UVB, UVB + base serum, UVB + saffron serum exposed to UVB radiation for 5 weeks with a total dose of 3100 mJ/cm2. The skin was extracted then underwent Masson Trichrome staining and real-time PCR to obtain collagen density and gene expression. ResultsThe gene expression of MMP1, IL6, TNFá, LC3, and p62 was significantly increased in the UVB group compared to the control group. Topical administration of saffron serum significantly increased collagen density (p=0.001). Induction by UVB significantly increased LC3 (p=0.020) and p62 (p=0.030) gene expression, indicating an inhibition of autophagy. The saffron serum might modulate autophagy by increasing LC3, but not significant (p=0.495) and significantly decreasing p62 gene expression (p=0.001). As for MMP1, IL6, and TNFá, no significant decrease in gene expression was found in the UVB + saffron serum group compared to the UVB group. ConclusionSaffron serum increases collagen density and modulates autophagy in the skin of UVB-exposed Wistar rats. Inflammatory markers were increased after UVB induction, but no differences were found after saffron serum topical administration.