The effect of less invasive surfactant administration on cerebral oxygenation in preterm infants.

AIM: To determine the regional cerebral tissue oxygenation saturation (rcSO2 ) in a group of infants requiring less invasive surfactant administration (LISA) as compared to infants with continuous positive airway pressure (CPAP) only. METHODS: In preterm infants with a gestational age 26 0/7-31 6/7 weeks, we conducted an observational study using near-infrared spectroscopy (NIRS) in the first 120 hours of life. RESULTS: We analysed the data of 22 infants who never received surfactant (CPAP), 22 infants had LISA and CPAP (LISA) and 6 infants received surfactant via endotracheal tube (ETT). Four infants had both surfactant application modes including six LISA applications. In total, there were 32 successful LISA applications but 44 attempts; 13/44 (30%) of LISA attempts resulted in a 20% decrease of rcSO2 . During the first 120 hours of life, rcSO2 values of CPAP were similar to those of infants in the LISA group, that is median rcSO2 values 90% vs 85%, respectively (P = .126). Episodes with rcSO2 values <65% were 0.4% in the CPAP group as compared to 4.8% in the LISA group (P < .001). CONCLUSION: Our observational data indicate that rcSO2 values of infants in the LISA group were similar to the CPAP group.

Live cell imaging of SOS and prophage dynamics in isogenic bacterial populations.

Almost all bacterial genomes contain DNA of viral origin, including functional prophages or degenerated phage elements. A frequent but often unnoted phenomenon is the spontaneous induction of prophage elements (SPI) even in the absence of an external stimulus. In this study, we have analyzed SPI of the large, degenerated prophage CGP3 (187 kbp), which is integrated into the genome of the Gram-positive Corynebacterium glutamicum ATCC 13032. Time-lapse fluorescence microscopy of fluorescent reporter strains grown in microfluidic chips revealed the sporadic induction of the SOS response as a prominent trigger of CGP3 SPI but also displayed a considerable fraction (∼30%) of RecA-independent SPI. Whereas approx. 20% of SOS-induced cells recovered from this stress and resumed growth, the spontaneous induction of CGP3 always led to a stop of growth and likely cell death. A carbon source starvation experiment clearly emphasized that SPI only occurs in actively proliferating cells, whereas sporadic SOS induction was still observed in resting cells. These data highlight the impact of sporadic DNA damage on the activity of prophage elements and provide a time-resolved, quantitative description of SPI as general phenomenon of bacterial populations.

Development of a fully integrated falling film microreactor for gas-liquid-solid biotransformation with surface immobilized O2 -dependent enzyme.

Microstructured flow reactors are powerful tools for the development of multiphase biocatalytic transformations. To expand their current application also to O2 -dependent enzymatic conversions, we have implemented a fully integrated falling film microreactor that provides controllable countercurrent gas-liquid phase contacting in a multi-channel microstructured reaction plate. Advanced non-invasive optical sensing is applied to measure liquid-phase oxygen concentrations in both in- and out-flow as well as directly in the microchannels (width: 600 µm; depth: 200 µm). Protein-surface interactions are designed for direct immobilization of catalyst on microchannel walls. Target enzyme (here: d-amino acid oxidase) is fused to the positively charged mini-protein Zbasic2 and the channel surface contains a negatively charged γ-Al2 O3 wash-coat layer. Non-covalent wall attachment of the chimeric Zbasic2 _oxidase resulted in fully reversible enzyme immobilization with fairly uniform surface coverage and near complete retention of biological activity. The falling film at different gas and liquid flow rates as well as reactor inclination angles was shown to be mostly wavy laminar. The calculated film thickness was in the range 0.5-1.3 × 10(-4) m. Direct O2 concentration measurements at the channel surface demonstrated that the liquid side mass transfer coefficient (KL ) for O2 governed the overall gas/liquid/solid mass transfer and that the O2 transfer rate (≥0.75 mM · s(-1) ) vastly exceeded the maximum enzymatic reaction rate in a wide range of conditions. A value of 7.5 (±0.5) s(-1) was determined for the overall mass transfer coefficient KL a, comprising a KL of about 7 × 10(-5) m · s(-1) and a specific surface area of up to 10(5) m(-1) . Biotechnol. Bioeng. 2016;113: 1862-1872. © 2016 Wiley Periodicals, Inc.

Analysis of SOS-induced spontaneous prophage induction in Corynebacterium glutamicum at the single-cell level.

The genome of the Gram-positive soil bacterium Corynebacterium glutamicum ATCC 13032 contains three integrated prophage elements (CGP1 to -3). Recently, it was shown that the large lysogenic prophage CGP3 (∼187 kbp) is excised spontaneously in a small number of cells. In this study, we provide evidence that a spontaneously induced SOS response is partly responsible for the observed spontaneous CGP3 induction. Whereas previous studies focused mainly on the induction of prophages at the population level, we analyzed the spontaneous CGP3 induction at the single-cell level using promoters of phage genes (Pint2 and Plysin) fused to reporter genes encoding fluorescent proteins. Flow-cytometric analysis revealed a spontaneous CGP3 activity in about 0.01 to 0.08% of the cells grown in standard minimal medium, which displayed a significantly reduced viability. A PrecA-eyfp promoter fusion revealed that a small fraction of C. glutamicum cells (∼0.2%) exhibited a spontaneous induction of the SOS response. Correlation of PrecA to the activity of downstream SOS genes (PdivS and PrecN) confirmed a bona fide induction of this stress response rather than stochastic gene expression. Interestingly, the reporter output of PrecA and CGP3 promoter fusions displayed a positive correlation at the single-cell level (ρ = 0.44 to 0.77). Furthermore, analysis of the PrecA-eyfp/Pint2-e2-crimson strain during growth revealed the highest percentage of spontaneous PrecA and Pint2 activity in the early exponential phase, when fast replication occurs. Based on these studies, we postulate that spontaneously occurring DNA damage induces the SOS response, which in turn triggers the induction of lysogenic prophages.

Trendelenburg position in gynecologic robotic-assisted surgery.

OBJECTIVE: To estimate the necessity of routine patient positioning in steep Trendelenburg in robotic-assisted gynecologic surgery performed for benign indications. DESIGN: Descriptive study (Canadian Task Force classification II-2). SETTING: University-affiliated community hospital. PATIENTS: Twenty women undergoing robotic-assisted gynecologic surgery for benign indications. INTERVENTION: Robotic-assisted total hysterectomy, supracervical hysterectomy, myomectomy, and sacrocolpopexy. MEASUREMENTS AND MAIN RESULTS: Demographic data and perioperative variables were recorded including age, body mass index, procedure type, console time, perioperative complications, estimated blood loss, hospital length of stay, and degree of Trendelenburg position. The degree of Trendelenburg position was measured at the end of each procedure using an electronic level. The surgeons were blinded to the degree of Trendelenburg used. All procedures were performed successfully without conversion to laparotomy. All patients were discharged to home within 24 hours. No perioperative complications were noted. The mean (SD; 95% CI) Trendelenburg position used in this cohort was 16.4 (4.1; 14.4-18.3) degrees. Patient body mass index was 28.5 (5.3; 26.1-31.1). Median console time was 87.5 (27-112) minutes. CONCLUSION: Robotic-assisted benign gynecologic surgery can be effectively performed without use of the steep Trendelenburg position. The practice of routine adherence to steep Trendelenburg positioning in benign gynecologic robotic surgery should be questioned.

Epidemiological trends in mortality, event rates and case fatality of acute myocardial infarction from 2004 to 2015: results from the KORA MI registry.

AIM: This study examines epidemiological trends of acute myocardial infarction (AMI) in Germany from 2004-2015 across different age groups, using data of the population-based KORA myocardial infarction registry. METHODS: Annual age-standardised, age-group- and sex-specific mortality and event rates (incident and recurrent) per 100,000 population as well as 28-day case fatality were calculated from all registered cases of AMI and coronary heart disease deaths in 25-74-year-olds from 2004-2015 and 75-84-year-olds from 2009-2015. Average annual percentage changes (AAPC) were calculated by joinpoint regression. RESULTS: Mortality rates declined considerably among the elderly (75-84 years), in men by -6.0% annually, due to declines of case fatality by -3.0% and incidence rate by 3.4% and in women by -10.0%, driven by declines in incidence (-9.1%) and recurrence rate (-4.9%). Significant mortality declines also occurred in males, 65-74 years of age (AAPC -3.8%). Among the age groups 25-54 years and 55-64 years, there was no substantial decline in mortality, event rates or case fatality except for a decline of incidence rate in 55-64-year-old men (AAPC -1.8%). CONCLUSION: Inhomogeneous AMI trends across age-groups indicate progress in prevention and treatment for the population >64 years, while among <55-year-olds, we found no significant trend in AMI morbidity and mortality.KEY MESSAGESAge standardised AMI mortality continued to decline from 2009 to 2015 in the study region.Declines in AMI mortality were driven by declines in event rates (both incidence and recurrence rates) and case fatality.AMI trends were inconsistent across different age groups with the strongest declines in mortality and event rates among the elderly population (75-84 years of age).

Characterization of the vitamin D endocrine system in human sebocytes in vitro.

Sebocytes are sebum-producing cells that form the sebaceous glands. We investigated the role of sebocytes as target cells for vitamin D metabolites and the existence of an enzymatic machinery for the local synthesis and metabolism of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3), calcitriol], the biologically active vitamin D metabolite, in these cell types. Expression of vitamin D receptor (VDR), vitamin D-25-hydroxylase (25 OHase), 25-hydroxyvitamin D-1alpha-hydroxylase (1 alphaOHase), and 1,25-dihydroxyvitamin D-24-hydroxylase (24 OHase) was detected in SZ95 sebocytes in vitro using real time quantitative polymerase chain reaction. Splice variants of 1alphaOHase were identified by nested touchdown polymerase chain reaction. We demonstrated that incubation of SZ95 sebocytes with 1,25(OH)(2)D(3) resulted in a cell culture condition-, time-, and dose-dependent modulation of cell proliferation, cell cycle regulation, lipid content and interleukin-6/interleukin-8 secretion in vitro. RNA expression of VDR and 24 OHase was upregulated along with vitamin D analogue treatment. Although several other splice variants of 1alphaOHase were detected, our findings indicate that the full length product represents the major 1 alphaOHase gene product in SZ95 cells. In conclusion, SZ95 sebocytes express VDR and the enzymatic machinery to synthesize and metabolize biologically active vitamin D analogues. Sebocytes represent target cells for biologically active metabolites. Our findings indicate that the vitamin D endocrine system is of high importance for sebocyte function and physiology. We conclude that sebaceous glands represent potential targets for therapy with vitamin D analogues or for pharmacological modulation of 1,25(OH)(2)D(3) synthesis/metabolism.

Viable adhered Staphylococcus aureus highly reduced on novel antimicrobial sutures using chlorhexidine and octenidine to avoid surgical site infection (SSI).

BACKGROUND: Surgical sutures can promote migration of bacteria and thus start infections. Antiseptic coating of sutures may inhibit proliferation of adhered bacteria and avoid such complications. OBJECTIVES: This study investigated the inhibition of viable adhering bacteria on novel antimicrobially coated surgical sutures using chlorhexidine or octenidine, a critical factor for proliferation at the onset of local infections. The medical need, a rapid eradication of bacteria in wounds, can be fulfilled by a high antimicrobial efficacy during the first days after wound closure. METHODS: As a pretesting on antibacterial efficacy against relevant bacterial pathogens a zone of inhibition assay was conducted with middle ranged concentrated suture coatings (22 µg/cm). For further investigation of adhering bacteria in detail the most clinically relevant Staphylococcus aureus (ATCC®49230™) was used. Absorbable braided sutures were coated with chlorhexidine-laurate, chlorhexidine-palmitate, octenidine-laurate, and octenidine-palmitate. Each coating type resulted in 11, 22, or 33 µg/cm drug content on sutures. Scanning electron microscopy (SEM) was performed once to inspect the coating quality and twice to investigate if bacteria have colonized on sutures. Adhesion experiments were assessed by exposing coated sutures to S. aureus suspensions for 3 h at 37°C. Subsequently, sutures were sonicated and the number of viable bacteria released from the suture surface was determined. Furthermore, the number of viable planktonic bacteria was measured in suspensions containing antimicrobial sutures. Commercially available sutures without drugs (Vicryl®, PGA Resorba®, and Gunze PGA), as well as triclosan-containing Vicryl® Plus were used as control groups. RESULTS: Zone of inhibition assay documented a multispecies efficacy of novel coated sutures against tested bacterial strains, comparable to most relevant S. aureus over 48 hours. SEM pictures demonstrated uniform layers on coated sutures with higher roughness for palmitate coatings and sustaining integrity of coated sutures. Adherent S. aureus were found via SEM on all types of investigated sutures. The novel antimicrobial sutures showed significantly less viable adhered S. aureus bacteria (up to 6.1 log) compared to Vicryl® Plus (0.5 log). Within 11 µg/cm drug-containing sutures, octenidine-palmitate (OL11) showed the highest number of viable adhered S. aureus (0.5 log), similar to Vicryl® Plus. Chlorhexidine-laurate (CL11) showed the lowest number of S. aureus on sutures (1.7 log), a 1.2 log greater reduction. In addition, planktonic S. aureus in suspensions were highly inhibited by CL11 (0.9 log) represents a 0.6 log greater reduction compared to Vicryl® Plus (0.3 log). CONCLUSIONS: Novel antimicrobial sutures can potentially limit surgical site infections caused by multiple pathogenic bacterial species. Therefore, a potential inhibition of multispecies biofilm formation is assumed. In detail tested with S. aureus, the chlorhexidine-laurate coating (CL11) best meets the medical requirements for a fast bacterial eradication. This suture coating shows the lowest survival rate of adhering as well as planktonic bacteria, a high drug release during the first-clinically most relevant- 48 hours, as well as biocompatibility. Thus, CL11 coatings should be recommended for prophylactic antimicrobial sutures as an optimal surgical supplement to reduce wound infections. However, animal and clinical investigations are important to prove safety and efficacy for future applications.

Correction: Viable adhered Staphylococcus aureus highly reduced on novel antimicrobial sutures using chlorhexidine and octenidine to avoid surgical site infection (SSI).

[This corrects the article DOI: 10.1371/journal.pone.0190912.].

Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion.

Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 µM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour.

Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester.

Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single cell cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity. Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.