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Minimally invasive determination of mRNA concentration in single living bacteria.

ABSTRACT: Fluorescence correlation spectroscopy (FCS) has permitted the characterization of high concentrations of noncoding RNAs in a single living bacterium. Here, we extend the use of FCS to low concentrations of coding RNAs in single living cells. We genetically fuse a red fluorescent protein (RFP) gene and two binding sites for an RNA-binding protein, whose translated product is the RFP protein alone. Using this construct, we determine in single cells both the absolute [mRNA] concentration and the associated [RFP] expressed from an inducible plasmid. We find that the FCS method allows us to reliably monitor in real-time [mRNA] down to approximately 40 nM (i.e. approximately two transcripts per volume of detection). To validate these measurements, we show that [mRNA] is proportional to the associated expression of the RFP protein. This FCS-based technique establishes a framework for minimally invasive measurements of mRNA concentration in individual living bacteria.

SUBMITTER: Guet CC

PROVIDER: S-EPMC2475643 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Minimally invasive determination of mRNA concentration in single living bacteria.

Guet Călin C CC Bruneaux Luke L Min Taejin L TL Siegal-Gaskins Dan D Figueroa Israel I Emonet Thierry T Cluzel Philippe P

Nucleic acids research 20080530 12

Fluorescence correlation spectroscopy (FCS) has permitted the characterization of high concentrations of noncoding RNAs in a single living bacterium. Here, we extend the use of FCS to low concentrations of coding RNAs in single living cells. We genetically fuse a red fluorescent protein (RFP) gene and two binding sites for an RNA-binding protein, whose translated product is the RFP protein alone. Using this construct, we determine in single cells both the absolute [mRNA] concentration and the as ...[more]

PMID: 18515347

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Project description:Background:To analyze the protective effect of single-dose del Nido cardioplegia (DNC) in adult minimally invasive valve surgery. Methods:From January to December 2017, 165 consecutive adult patients who underwent minimally invasive valve surgery by the same team of surgeons were divided into two cohorts based on the type of cardioplegia administered during surgery: (I) single-dose DNC (DNC group (n=76, male 41, female 35) used in patients from May to December, 2017 and (II) intermittent standard 4:1 blood cardioplegia based on St.Thomas solution (SBC group, n=89, male 45, female 44) used in patients from January to April, 2017. Preoperative baseline demographics, preoperative comorbidities, operative variables, postoperative complications, and patient outcomes were collected and compared between the two groups. Results:Preoperative characteristics were shown to be similar between the two groups before and after propensity matching. Patients in the DNC group required a significantly lower volume of cardioplegia. The volume of ultrafiltration in the DNC group was substantially higher than that in the SBC group. The spontaneous return of heartbeat rate in the DNC group was considerably higher than that in the SBC group (97.0% vs. 78.8%, P=0.006). The Euroscore II in the DNC group was markedly lower than that in the SBC group (2.00 vs. 3.00, P<0.05). The level of blood urea nitrogen (BUN) in the DNC group was significantly lower than that in the SBC group (6.20 vs. 6.95, P<0.05). There were no differences in surgery procedure, cross-clamp time, bypass time, Apache score, troponin T (cTnT), brain natriuretic peptide (BNP), liver and renal function, postoperative complications or patient outcomes between two groups. Regression analysis showed that cTnT increased with the prolongation of myocardial ischemia time, and was closely related to the type of operation, but had no significant correlation with the type of cardioplegia. Conclusions:In our initial experience, single-dose DNC in adult minimally invasive valve surgery in which the cross-clamp time was mostly less than 90 min, achieved equivalent myocardial protection and clinical outcomes when compared with standard whole blood cardioplegia. In addition, single-dose DNC made the minimally invasive valve surgery procedure progress in a smoother and more convenient fashion.

Project description:Minimally invasive esophagectomy is increasingly performed for the treatment of esophageal cancer, but it is unclear whether hybrid minimally invasive esophagectomy (HMIE) or totally minimally invasive esophagectomy (TMIE) should be preferred. The objective of this study was to perform a meta-analysis of studies comparing HMIE with TMIE. A systematic literature search was performed in MEDLINE, Embase, and the Cochrane Library. Articles comparing HMIE and TMIE were included. The Newcastle-Ottawa scale was used for critical appraisal of methodological quality. The primary outcome was pneumonia. Sensitivity analysis was performed by analyzing outcome for open chest hybrid MIE versus total TMIE and open abdomen MIE versus TMIE separately. Therefore, subgroup analysis was performed for laparoscopy-assisted HMIE versus TMIE, thoracoscopy-assisted HMIE versus TMIE, Ivor Lewis HMIE versus Ivor Lewis TMIE, and McKeown HMIE versus McKeown TMIE. There were no randomized controlled trials. Twenty-nine studies with a total of 3732 patients were included. Studies had a low to moderate risk of bias. In the main analysis, the pooled incidence of pneumonia was 19.0% after HMIE and 9.8% after TMIE which was not significantly different between the groups (RR: 1.46, 95% CI: 0.97-2.20). TMIE was associated with a lower incidence of wound infections (RR: 1.81, 95% CI: 1.13-2.90) and less blood loss (SMD: 0.78, 95% CI: 0.34-1.22) but with longer operative time (SMD:-0.33, 95% CI: -0.59--0.08). In subgroup analysis, laparoscopy-assisted HMIE was associated with a higher lymph node count than TMIE, and Ivor Lewis HMIE was associated with a lower anastomotic leakage rate than Ivor Lewis TMIE. In general, TMIE was associated with moderately lower morbidity compared to HMIE, but randomized controlled evidence is lacking. The higher leakage rate and lower lymph node count that was found after TMIE in sensitivity analysis indicate that TMIE can also have disadvantages. The findings of this meta-analysis should be considered carefully by surgeons when moving from HMIE to TMIE.

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Minimally invasive determination of mRNA concentration in single living bacteria.

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Minimally invasive determination of mRNA concentration in single living bacteria.

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