scholarly journals Influence of Nutrient Media Compared to Human Synovial Fluid on the Antibiotic Susceptibility and Biofilm Gene Expression of Coagulase-Negative Staphylococci In Vitro

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 790
Author(s):  
Stephan Josef Maria Steixner ◽  
Christopher Spiegel ◽  
Dietmar Dammerer ◽  
Alexander Wurm ◽  
Michael Nogler ◽  
...  

Bacterial antibiotic resistance and biofilm formation are mechanisms usually involved in the pathogeny of implant-related infections. Worldwide, antibiotic susceptibility tests are usually carried out using nutrient-rich media. Clinical routine laboratories and even research centers use for example EUCAST or CLSI for guidelines. In this study, we investigated the effect of different nutrient media on the antibiotic susceptibility and icaADBC gene expression of bacteria in biofilm. As media, Müller-Hinton Bouillon (MHB), Tryptic Soy Broth (TSB) and human synovial fluid (SF) diluted 1:4 in phosphate buffered saline (PBS), each also supplemented with 1% glucose, were used. The influence of different nutrient media on the antibiotic susceptibility of coagulase-negative staphylococci (CoNS) was evaluated by counting of colony-forming units (CFU) and by checking the metabolic activity of the bacteria. We used reverse transcriptase and real-time qPCR to investigate the influence of nutrient media on the biofilm gene expression. We used two-way analysis of variance (ANOVA). p < 0.05 was considered to be statistically significant. Significant differences in growth and antibiotic susceptibility were detected in all strains tested among the different media used. The nutrient media showed influence on the cell viability of all bacteria after antibiotic treatment. IcaADBC gene expression was significantly influenced by glucose and all nutrient media. The results highlight the influence of glucose on the antibiotic susceptibility, growth and gene expression of all strains tested. For all strains, a significant difference in bacterial recovery, viability and gene expression were found when compared to biofilm grown in SF.

Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 385
Author(s):  
Alaa Baazeem ◽  
Alicia Rodriguez ◽  
Angel Medina ◽  
Naresh Magan

Pistachio nuts are an important economic tree nut crop which is used directly or processed for many food-related activities. They can become colonized by mycotoxigenic spoilage fungi, especially Aspergillus flavus, mainly resulting in contamination with aflatoxins (AFs), especially aflatoxin B1 (AFB1). The prevailing climate in which these crops are grown changes as temperature and atmospheric CO2 levels increase, and episodes of extreme wet/dry cycles occur due to human industrial activity. The objectives of this study were to evaluate the effect of interacting Climate Change (CC)-related abiotic factors of temperature (35 vs. 37 °C), CO2 (400 vs. 1000 ppm), and water stress (0.98–0.93 water activity, aw) on (a) growth (b) aflD and aflR biosynthetic gene expression and (c) AFB1 production by two strains A. flavus (AB3, AB10) in vitro on milled pistachio-based media and when colonizing layers of shelled raw pistachio nuts. The A. flavus strains were resilient in terms of growth on pistachio-based media and the colonisation of pistachio nuts with no significant difference when exposed to the interacting three-way climate-related abiotic factors. However, in vitro studies showed that AFB1 production was significantly stimulated (p < 0.05), especially when exposed to 1000 ppm CO2 at 0.98–0.95 aw and 35 °C, and sometimes in the 37 °C treatment group at 0.98 aw. The relative expression of the structural aflD gene involved in AFB1 biosynthesis was decreased or only slightly increased, relative to the control conditions at elevated CO, regardless of the aw level examined. For the regulatory aflR gene expression, there was a significant (p < 0.05) increase in 1000 ppm CO2 and 37 °C for both strains, especially at 0.95 aw. The in situ colonization of pistachio nuts resulted in a significant (p < 0.05) stimulation of AFB1 production at 35 °C and 1000 ppm CO2 for both strains, especially at 0.98 aw. At 37 °C, AFB1 production was either decreased, in strain AB3, or remained similar, as in strain AB10, when exposed to 1000 ppm CO2. This suggests that CC factors may have a differential effect, depending on the interacting conditions of temperature, exposure to CO2 and the level of water stress on AFB1 production.


2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.


2016 ◽  
Vol 24 ◽  
pp. S166
Author(s):  
C. Galeano-Garces ◽  
S.M. Riester ◽  
E.T. Camilleri ◽  
H.S. Ryan ◽  
J. Smith ◽  
...  

2010 ◽  
Vol 22 (1) ◽  
pp. 272
Author(s):  
E. S. Caixeta ◽  
P. Ripamonte ◽  
M. F. Machado ◽  
R. B. da Silva ◽  
C. Price ◽  
...  

Mammalian oocytes require pyruvate as an energy source for growth and resumption of meiosis. Because oocytes are not competent to carry out glycolysis, cumulus cells (CC) are responsible for metabolizing glucose into pyruvate and providing it to the oocyte through gap junctions. The understanding of the energetic metabolism of CC in culture conditions might provide basis for the improvement of COC in vitro maturation. The aim of this study was to determine the temporal patterns of mRNA expression of glycolytic enzymes [phosphofructokinase (PFKP), aldolase (ALDOA), triosephosphate isomerase (TPI), enolase (ENO1), pyruvate kinase (PKM2), and lactate dehydrogenase (LDHA)] in bovine CC during COC in vitro maturation with or without FSH. Immature COC (grades 1 and 2) were obtained from 2- to 8-mm follicles from abattoir ovaries (predominantly Bos indicus). Cumulus cells were separated from COC and frozen before (immature group) or after COC culture for 4, 8, 12, 16, and 20 hours with (10 ng/mL) or without FSH. Total RNA was extracted using RNeasy® (Qiagen, Valencia, CA, USA), and 100 ng of RNA was reverse transcribed using oligo dT primers and Omniscript® (Qiagen). Relative expression of target genes was assessed by real-time PCR using bovine-specific primers and Power SYBR green master mix in an ABI Prism® 7300. To select the most stable housekeeping gene for expression normalization, cyclophilin-A (CYC-A), GAPDH, and histone H2AFZ amplification profiles were compared using the geNorm applet for Microsoft Excel (Vandesompele J et al. 2002 Genome Biol. 3, 1-11); the most stable housekeeping gene was CYC-A. Relative expression values were calculated using the AACt method with efficiency correction (Pfaffl MW 2001 Nucleic Acids Res. 29, 2002-2007). Effects of time in culture and of FSH treatment were tested by ANOVA, and groups were compared by Tukey-Kramer Honestly Significant Difference test. Nonparametric analysis was used when data were not normally distributed. Abundance of mRNA of all glycolytic enzymes decreased during in vitro maturation with or without FSH. Expression of PFKP, ALDOA, TPI1, ENO1, and LDHA genes was decreased to around half of the initial value (time 0) by 4 to 8 h of culture (P < 0.05) and did not increase thereafter. A similar expression pattern was observed for PKM2, although mRNA abundance was reduced later in comparison with other enzymes; levels were decreased by 16 (without FSH) to 20 h (with FSH) of culture. The presence of FSH did not alter the overall temporal pattern of gene expression but decreased mRNA abundance for PFKP, ALDOA, and TPI1 at 20, 16 and 16 h of culture, respectively. In conclusion, gene expression of glycolytic enzymes decreased with time during COC in vitro maturation in cattle, and FSH did not have a major influence on this expression pattern. This study was supported by CAPES and FAPESP.


2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Arman Rahimmi ◽  
Ilaria Peluso ◽  
Aref Rajabi ◽  
Kambiz Hassanzadeh

There are still unknown mechanisms involved in the development of Parkinson’s disease (PD), which elucidating them can assist in developing efficient therapies. Recently, studies showed that genes located on the human chromosomal location 22q11.2 might be involved in the development of PD. Therefore, the present study was designed to evaluate the role of two genes located on the chromosomal location (miR-185 and SEPT5), which were the most probable candidates based on our bibliography. In vivo and in vitro models of PD were developed using male Wistar rats and SHSY-5Y cell line, respectively. The expression levels of miR-185, SEPT5, LRRK2, and PARK2 genes were measured at a mRNA level in dopaminergic areas of rats’ brains and SHSY-5Y cells using the SYBR Green Real-Time PCR Method. Additionally, the effect of inhibition on the genes or their products on cell viability and gene expression pattern in SHSY-5Y cells was investigated. The level of miR-185 gene expression was significantly decreased in the substantia nigra (SN) and striatum (ST) of the rotenone-treated group (control group) compared to the healthy normal group (P<0.05). In addition, there was a significant difference in the expression of SEPT5 gene (P<0.05) in the substantia nigra between two studied groups. The results of an in vitro study showed no significant change in the expression of the genes; however, the inhibition on miR-185 gene expression led to the increase in LRRK2 gene expression in SHSY-5Y cells. The inhibition on LRRK2 protein also decreased the cellular toxicity effect of rotenone on SHSY-5Y cells. The results suggested the protective role of miR-185 gene in preventing the development of PD.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4897-4897
Author(s):  
Nancy Day ◽  
Evan Shereck ◽  
Janet Ayello ◽  
Catherine McGuinn ◽  
Prakash Satwani ◽  
...  

Abstract Background: NK cells are characterized by absent CD3 but expression of CD56dim (90%, cytotoxic) and CD56bright (10%, mediator). NK cells may contribute to the immaturity in cord blood innate and adaptive immunity, and play an important role in the GVL effect post CBT. However, little is known regarding the NKR signaling pathways in CB vs PB CD56dim NK cells and its relationship to the cytotoxic activity. We previously demonstrated the ability to ex-vivo expand CB into NK subsets with profound NK in-vitro and in-vivo cytotoxic activity (Ayello/Cairo BBMT 2006). We further observed that there were 33 and 37 proteins over and under expressed by proteomic expression profiling studies of CB vs PB CD56dim (Shereck/Cairo, ASH 2007; ASPHO 2007; AACR 2007). The differential protein expressions included NKG2A, IP3R type 3, NCR3, MAPKAPK5, Notch 2, PLEK, and NF-X1 using both immunophenotype and proteomic profiling studies. Objectives: To understand the importance of NKR signaling pathways in mediating the differential protein expression and thus in regulating the NK cytotoxic activities in CB vs PB CD56dim, we compared the genomic expression pattern in CB vs PB CD56dim. Methods: For CD56dim isolation, first, NK cells were isolated indirectly by magnetic separation from non-NK cells. Second, the pre-enriched NK cells (CD56+/CD3−) from CB and PB were directly labeled with CD16 (FCGR3) MicroBeads, and the CD56+ CD16+ NK cells (CD56dim) were eluted after removing the column from the magnetic field (Miltenyi). Purity of CD56dim NK cells were then examined by flow cytometry (BD FACScan). For genomic studies, total RNA was isolated and reverse transcribed to cDNA using T7-Oligo (dT) primer. cRNA was Biotin-labeled by in vitro transcription. Fragmented biotin-labeled cRNA was hybridized to GeneChip U133A_2 in GCOS-operated Fluidics Station 450, and then scanned by GeneChip Scanner 3000 (Affymetrix). Data were analyzed using Agilent GeneSpring. Signal intensities were compared using one way ANOVA and Welch Test for statistical analysis. Results: There were 193 and 222 genes over and under expressed at the genomic level between CB vs PB CD56dim NK cells, respectively. CB vs PB CD56dim significantly overexpressed NKG2A (2.14F), CD16b (2.46F), KIR2D (2.13F), NKp44 (NCR2; 2.62F), PBX1 (4.29F), ENPEP (3.93F). There was no significant difference in NKR gene expression of CD16a, CD161, NKG2C, and NKp46 in CB vs PB CD56dim. CB vs PB CD56dim underexpressed the following NK genes: IP3R (1.32F), MAPKAPK5 (1.77F), NCR3 (1.24F), ACACB (3.23F), BBS1 (2.00F). Conclusion: CB vs PB CD56dim overexpressed NKG2A, CD16b, KIR2D, and NKp44 genes compared to only NKG2A was overexpressed at the protein level. These results suggest that NKR protein product levels in CB CD56dim may be directly regulated at the translation level, but not the transcription level. The discrepancy of IP3R, ENPEP, PBX1, and MAPKAPK5 gene expression suggest the involvements of IP3 and calcium ions in NKR signaling pathways. Since the Notch2, PLEK, and NF-X1 gene expression patterns were not increased, the augmented protein levels may result from the regulation of protein translation. The potential regulators of this process may include PBX1, ENPEP, ACACB, and BBS1 though the roles of these regulators need to be defined. We conclude that genomic differences between CB vs PB CD56dim may play an important role in regulating NKR signaling pathway, and thus contribute to disparate cytotoxic activity between CB vs PB and suggest a possible explanation for immaturity of cord blood innate and adaptive immunity.


2020 ◽  
Vol 21 (17) ◽  
pp. 6417 ◽  
Author(s):  
Alejandro Cuevas ◽  
Nicolás Saavedra ◽  
Luis A. Salazar ◽  
Marcela F. Cavalcante ◽  
Jacqueline C. Silva ◽  
...  

Atherosclerosis is a chronic inflammatory disease, whose progression and stability are modulated, among other factors, by an innate and adaptive immune response. Prodiginines are bacterial secondary metabolites with antiproliferative and immunomodulatory activities; however, their effect on the progression or vulnerability of atheromatous plaque has not been evaluated. This study assessed the therapeutic potential of prodigiosin and undecylprodigiosin on inflammatory marker expression and atherosclerosis. An in vitro and in vivo study was carried out. Migration, low-density lipoprotein (LDL) uptake and angiogenesis assays were performed on cell types involved in the pathophysiology of atherosclerosis. In addition, male LDL receptor null (Ldlr-/-) C57BL/6J mice were treated with prodigiosin or undecylprodigiosin for 28 days. Morphometric analysis of atherosclerotic plaques, gene expression of atherogenic factors in the aortic sinus and serum cytokine quantification were performed. The treatments applied had slight effects on the in vitro tests performed, highlighting the inhibitory effect on the migration of SMCs (smooth muscle cells). On the other hand, although no significant difference in atherosclerotic plaque progression was observed, gene expression of IL-4 and chemokine (C-C motif) ligand 2 (Ccl2) was downregulated. In addition, 50 µg/Kg/day of both treatments was sufficient to inhibit circulating tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in serum. These results suggested that prodigiosin and undecylprodigiosin modulated inflammatory markers and could have an impact in reducing atherosclerotic plaque vulnerability.


2008 ◽  
Vol 20 (1) ◽  
pp. 179
Author(s):  
M. Clemente ◽  
P. Lonergan ◽  
C. Borque ◽  
J. de La Fuente ◽  
D. Rizos

Preimplatation embryos grown in vitro are sensitive to their environment, and the conditions of culture can affect developmental potential. Progesterone (P4) is the key hormone responsible for maintenance of pregnancy in mammals, and circulating levels in the early postconception period have been associated with pregnancy success. It is not clear whether P4 acts directly or indirectly on the embryo to alter gene expression and development. The aim of this study was to assess the effect of varying levels of exogenous P4 on the development of bovine zygotes to the blastocyst stage in vitro. A preliminary study was conducted to analyze the media used for culture (stock of P4, SOF, SOF + 1 × 10–7 M P4) on Days 1 (day of culture), 4, and 7 for P4 concentration in 25-μL droplets overlain with mineral oil or 500 μL in wells with or without mineral oil. P4 was measured using an ELISA kit, prepared for human serum or plasma (DE1561 Dimeditec Diagnostics GmbH, Kiel, Germany). Inter- and intra-assay coefficients of variation were 6.63 and 6.42%, respectively, and recovery was 95%. P4 concentration on Day 1 in all media was the expected (40 ng mL–1). However, on Days 4 and 7 in media under mineral oil, the level of P4 was nearly zero (0.1 to 1.6 ng mL–1) compared with the media without mineral oil, which remained unchanged (39 to 40 ng mL–1) through the 7 days of culture. Zygotes (n = 1467) were produced in 8 replicates by in vitro oocyte maturation and fertilization, and were cultured in groups of 40 to 50 in wells of 500 μL without mineral oil in (1) SOF supplemented with 5% fetal calf serum (control–), (2) SOF with ethanol (control+), (3) SOF with P4 0.1 × 10–7 M, (4) SOF with P4 1 × 10–7 M, and (5) SOF with P4 10 × 10–7 M at 39°C, 5% CO2 and 5% O2, with maximum humidity. No significant difference was found between groups in cleavage rate or blastocyst yield on Days 6, 7, and 8 (Table 1). These results indicate that the addition of P4 to the in vitro culture medium (SOF) did not enhance the development of bovine embryos to the blastocyst stage. However, further studies on the quality of these embryos in terms of gene expression are in preparation. Table 1. Effect of P4 on bovine in vitro early embryo development


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Kocaman ◽  
B Ayas

Abstract Study question Does kisspeptin administration affect the motility parameters in sperm samples of subfertile cases? Summary answer Kisspeptin administration significantly increased gene expression levels related with sperm motility as well as intracellular calcium concentrations. What is known already Sperm motility problems are among the most important causes of male infertility. In recent years, a peptide named kisspeptin has been discovered that may have effects on sperm motility. Kisspeptin is known to trigger calcium release in hypothalamic neurons. In addition, kisspeptin administration increased sperm progressive motility in studies conducted on normozoospermic individuals. Furthermore, it is suggested that kisspeptin protein in seminal plasma is positively associated with semen quality. However, there is no evidence that how kisspeptin can affect sperm in men with infertility problems. Study design, size, duration This basic research study was an in vitro experimental approach involving the use of semen samples from an infertil cases between September to December in 2020. 40 men were included in both control and experimental groups. Participants/materials, setting, methods All analyses were performed on semen samples from 10 normozoospermic (NZ), 10 asthenozoospermic (AZ), 10 oligoasthenozoospermic (OAZ) and 10 oligoastenoteratozoospermic (OATZ) men, aging between (21-40) years. Basal serum and seminal kisspeptin levels were analyzed by ELISA. Sperm were divided into two groups. Kisspeptin-13 administered in vitro. KISS1, KISS1R, CATSPER1, AKAP4 gene expressions analyzed by qRT-PCR using 2−ΔΔCt algorithm. Intracellular calcium concentration was determined with floresence spectroflurometer and laser scanning confocal microscope. Main results and the role of chance The serum kisspeptin level of NZ was significantly higher than other groups (p &lt; 0.05). The semen kisspeptin level was significantly higher than OAZ and OATZ (p &lt; 0.05), but not in NZ (p &gt; 0.05). Also, KISS1 gene expression was higher in AZ compared to other groups (p &lt; 0.05). Biochemical and gene expression analysis of kisspeptin were consistent with each other. There was a significant increase in the expression of CATSPER1 gene in AZ compared to other groups (p &lt; 0.05). Also, AKAP4 gene expression was significantly higher in OATZ compared to other groups (p &lt; 0.05). No significant difference was documented for the expression of KISS1R (p &gt; 0.05). Intracellular calcium was significantly increased in AZ and NZ after kisspeptin administration. The intracellular calcium increase is consistent with increased CATSPER1 gene expression levels in AZ. Kisspeptin administration may have a significant effect on sperm motility parameters. Limitations, reasons for caution The biochemical and gene expression levels of KISS1 were consistent. However, gene expression was explored at the mRNA level for CATSPER1 and AKAP4. The protein expression analyses of these genes may confirm the results. Also, using kisspeptin antagonists may strength the results of intracellular calcium analysis. Wider implications of the findings Kisspeptin treatment for individuals diagnosed with asthenozoospermia may have therapeutic results. KISS1 quantitation may be a determining factor for the subfertility in routine semen analysis. Trial registration number OMU KAEK 2019/462


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