Pressure-induced changes on the morphology and gene expression in mammalian cells

We evaluated the effect of high hydrostatic pressure on mouse embryonic fibroblasts (MEFs) and mouse embryonic stem (ES) cells. Hydrostatic pressures of 15, 30, 60, and 90 MPa were applied for 10 minutes, and changes in gene expression were evaluated. Among genes related to mechanical stimuli, death-associated protein 3 was upregulated in MEF subjected to 90 MPa pressure, however, other genes known to be upregulated by mechanical stimuli did not change significantly. Genes related to cell differentiation did not show a large change in expression. On the other hand, genes related to pluripotency, such as Oct4 and Sox2, showed a two-fold increase in expression upon application of 60 MPa hydrostatic pressure for 10 minutes. Although these changes did not persist after overnight culture, cells that were pressurized to 15 MPa showed an increase in pluripotency genes after overnight culture. When mouse ES cells were pressurized, they also showed an increase in the expression of pluripotency genes. These results show that hydrostatic pressure activates pluripotency genes in mammalian cells.

Growing overnight bacterial culture in 96WP (Cabreiro Lab) v1

Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. LB agar plates are frequently used to isolate individual (clonal) colonies of bacteria carrying a specific plasmid. However, a liquid culture is capable of supporting a higher density of bacteria and is used to grow up sufficient numbers of bacteria necessary to isolate enough plasmid DNA for experimental use. The following protocol is for inoculating an overnight culture of liquid LB with bacteria. This is a general protocol for making a liquid bacterial culture in 96-well plate format from frozen stock plates. The following parameters need to be mentioned for the specific type of bacterial strains inoculated: Parameters Example 1. Name/s of the bacterial strain OP50 2. Growth temperature 37C 3. Incubation time 16-18hrs 4. Antibiotic resistance (if any) None 5. Rpm of the shaking incubator (if needed) 200-220 rpm if required

In-Vitro Model of Scardovia wiggsiae Biofilm Formation and Effect of Nicotine

Recently, Scardovia wiggsiae has been reported to be strongly associated with caries formation. This study aimed to establish an in vitro model of S. wiggsiae biofilm and to investigate the effect of nicotine on S. wiggsiae colony-forming units (CFUs) growth. S. wiggsiae biofilm was grown overnight using brain-heart infusion (BHI) broth supplemented with 5 g of yeast extract/L (BHI-YE). The overnight culture was used as an inoculum to grow S. wiggsiae biofilm on standardized enamel and dentin samples. Samples were incubated with different nicotine concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 mg/mL) for 3 days. The dissociated biofilms were diluted, spiral plated on blood agar plates, and incubated for 24 h. CFUs/mL were quantified using an automated colony counter. A two-way ANOVA was used to compare the effect of different nicotine concentrations on S. wiggsiae CFUs. This study demonstrated that S. wiggsiae biofilm could be initiated and formed in vitro. Increased CFUs was observed through 0.5-4 mg/mL and 0.5-8 mg/mL of nicotine using enamel and dentin substrates, respectively. 16 and 32 mg/mL of nicotine were determined as the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), respectively. S. wiggsiae formed greater biofilm on enamel than dentin specimens in response to the nicotine stimulus. This study demonstrated the negative effect of smoking on increasing S. wiggsiae biofilm. Establishing S. wiggsiae biofilm in vitro may allow researchers in the future to have a better understanding of caries pathogenesis and bacterial interaction.

Comparative study of transferring frozen embryo with short culture vresus overnight culture and also assess prevalence rate of psychological distress amongs infertility patient

Frozen embryo transfer is a procedure which is widely preferable technique to transfer of embryos obtained in assisted reproductive technology. The transfer of cryopreserved embryos transfer which is less expensive than the second fresh cycle, fertility treatment costs, can get an increased implantation rate, and an increased rate of pregnancy. The primary negative emotional reaction to both infertility and assisted reproductive therapy is typically either anxiety or depression. To compare serum β-hCG value in fresh frozen-thawed embryo transfer with overnight cultured frozen-thawed embryo transfer. And prevalence rate of psychological distress. 52 FET Cycles performed in between June to December 2018 among these embryos transfer 36 were performed within 1 hour after thawing and 16 were performed after overnight incubation. We observed that patient in 1 hour incubation has higher β-hCG level than overnight culture patients and there is worse psychological status in infertile women and nearly half of our infertile sample (51.9%) displayed mild to extreme depressive symptoms. Obstetricians should consider that reducing the mental and social issues will lead to greater happiness and productivity. If psychiatry disorders are chronic, it can affect adversely.

696. Mechanism of Cefiderocol high MIC mutants obtained in non-clinical FoR studies

Background Cefiderocol (S-649266, CFDC) is a novel siderophore cephalosporin with activity against a wide variety of Gram-negative bacteria including carbapenem-resistant strains. We previously reported that CFDC is efficiently transported into Pseudomonas aeruginosa via iron transporter PiuA. In this study, we examined frequency of resistance of P. aeruginosa to CFDC, and investigated the resistance mechanisms of appeared colonies. Methods Frequency of resistance (FoR) was determined by plating an overnight culture of P. aeruginosa PAO1 on Mueller–Hinton Agar containing 4× or 10×MIC of CFDC or ceftazidime (CAZ). Appeared colonies were analyzed by whole-genome sequencing (WGS) to identify genomic mutations. The mRNA expression was determined by real-time RT-PCR, and pyoverdine production was determined by MALDI-TOF/MS and expression of outer membrane protein was analyzed by SDS–PAGE and proteomic analysis. Results The FoR to CFDC was 2.9 × 10–8 and <7.1 × 10–8, which were lower than those to CAZ (3.1 × 10–7 and 3.4 × 10–8) in the conditions of 4× and 10×MIC, respectively. MIC of CFDC against CFDC-derived mutant increased from 0.5 μg/mL (MIC against PAO1) to 2 μg/mL, and MICs of CAZ did not increase. In the case of CAZ-derived mutant, MICs of CAZ increased from 1 μg/mL (MIC against PAO1) to 16 μg/mL or higher, though MIC of CFDC did not increase, suggesting no cross-resistance between CFDC and CAZ. WGS identified mutations in upstream regions of pvdS (pvdS mutant), which regulates pyoverdine synthesis, or fecI (fecI mutant), which regulates the synthesis of iron transporter FecA contributing to the transport of iron citrate. The pvdS expression and pyoverdine production in the pvdS mutant were more than 4- and 6-fold higher than those in PAO1, respectively. The expression of fecA in the fecI mutant was more than ninefold higher than that in PAO1. Conclusion The MIC increase of CFDC against P. aeruginosa occurred due to the mutation of iron transporter-related genes. The resistance acquisition risks should be low as the frequency of resistance to CFDC was lower and the MIC increase of CFDC against the mutants was smaller than that of CAZ. In addition, no cross-resistance between CFDC and CAZ was observed. Disclosures A. Ito, Shionogi & Co., Ltd.: Employee, Salary. T. Nishikawa, Shionogi & Co., Ltd.: Employee, Salary. R. Ishii, Shionogi & Co., Ltd.: Employee, Salary. M. Kuroiwa, Shionogi & Co., Ltd.: Employee, Salary. Y. Ishioka, Shionogi & Co., Ltd.: Employee, Salary. N. Kurihara, Shionogi & Co., Ltd.: Employee, Salary. I. Sakikawa, Shionogi & Co., Ltd.: Employee, Salary. T. Ota, Shionogi & Co., Ltd.: Employee, Salary. M. Rokushima, Shionogi & Co., Ltd.: Employee, Salary. M. Tsuji, SHIONOGI & CO., LTD.: Employee, Salary. T. Sato, SHIONOGI & CO., LTD.: Employee, Salary. Y. Yamano, SHIONOGI & CO., LTD.: Employee, Salary.

Structural and proteomic changes in viable but non-culturableVibrio cholerae

Aquatic environments are reservoirs of the human pathogenVibrio choleraeO1, which causes the acute diarrheal disease cholera. Upon low temperature or limited nutrient availability, the cells enter a viable but non-culturable (VBNC) state. Characteristic of this state are an altered morphology, low metabolic activity and lack of growth under standard laboratory conditions. Here, for the first time, the cellular ultrastructure ofV. choleraeVBNC cells raised in natural waters was investigated using electron cryo-tomography complemented by comparison of the proteomes and the peptidoglycan composition of LB overnight culture and VBNC cells. The extensive remodeling of the VBNC cells was most obvious in the passive dehiscence of the cell envelope, resulting in improper embedment of flagella and pili. Only minor changes of the peptidoglycan and osmoregulated periplasmic glucans were observed. Active changes in VBNC cells included the production of cluster I chemosensory arrays and change of abundance of cluster II array proteins. Components involved in iron acquisition and storage, peptide import and arginine biosynthesis were overrepresented in VBNC cells, while enzymes of the central carbon metabolism were found at lower levels. Finally, several pathogenicity factors ofV. choleraewere less abundant in the VBNC state, potentially limiting their infectious potential.

Whole genome amplification and sequencing of low cell numbers directly from a bacteria spiked blood model

Whilst next generation sequencing is frequently used to whole genome sequence bacteria from cultures, it’s rarely applied directly to clinical samples. Therefore, this study addresses the issue of applying NGS microbial diagnostics directly to blood samples. To demonstrate the potential of direct from blood sequencing a bacteria spiked blood model was developed. Horse blood was spiked with clinical samples of E. coli and S. aureus, and a process developed to isolate bacterial cells whilst removing the majority of host DNA. One sample of each isolate was then amplified using ϕ29 multiple displacement amplification (MDA) and sequenced. The total processing time, from sample to amplified DNA ready for sequencing was 3.5 hours, significantly faster than the 18-hour overnight culture step which is typically required. Both bacteria showed 100% survival through the processing. The direct from sample sequencing resulted in greater than 92% genome coverage of the pathogens whilst limiting the sequencing of host genome (less than 7% of all reads). Analysis of de novo assembled reads allowed accurate genotypic antibiotic resistance prediction. The sample processing is easily applicable to multiple sequencing platforms. Overall this model demonstrates potential to rapidly generate whole genome bacterial data directly from blood.