Obg and Membrane Depolarization Are Part of a Microbial Bet-Hedging Strategy that Leads to Antibiotic Tolerance.

Within bacterial populations, a small fraction of persister cells is transiently capable of surviving exposure to lethal doses of antibiotics. As a bet-hedging strategy, persistence levels are determined both by stochastic induction and by environmental stimuli called responsive diversification. Little is known about the mechanisms that link the low frequency of persisters to environmental signals. Our results support a central role for the conserved GTPase Obg in determining persistence in Escherichia coli in response to nutrient starvation. Obg-mediated persistence requires the stringent response alarmone (p)ppGpp and proceeds through transcriptional control of the hokB-sokB type I toxin-antitoxin module. In individual cells, increased Obg levels induce HokB expression, which in turn results in a collapse of the membrane potential, leading to dormancy. Obg also controls persistence in Pseudomonas aeruginosa and thus constitutes a conserved regulator of antibiotic tolerance. Combined, our findings signify an important step toward unraveling shared genetic mechanisms underlying persistence.

Imaging muscle as a potential biomarker of denervation in motor neuron disease.

OBJECTIVE: To assess clinical, electrophysiological and whole-body muscle MRI measurements of progression in patients with motor neuron disease (MND), as tools for future clinical trials, and to probe pathophysiological mechanisms in vivo. METHODS: A prospective, longitudinal, observational, clinicoelectrophysiological and radiological cohort study was performed. Twenty-nine patients with MND and 22 age-matched and gender-matched healthy controls were assessed with clinical measures, electrophysiological motor unit number index (MUNIX) and T2-weighted whole-body muscle MRI, at first clinical presentation and 4 months later. Between-group differences and associations were assessed using age-adjusted and gender-adjusted multivariable regression models. Within-subject longitudinal changes were assessed using paired t-tests. Patterns of disease spread were modelled using mixed-effects multivariable regression, assessing associations between muscle relative T2 signal and anatomical adjacency to site of clinical onset. RESULTS: Patients with MND had 30% higher relative T2 muscle signal than controls at baseline (all regions mean, 95% CI 15% to 45%, p<0.001). Higher T2 signal was associated with greater overall disability (coefficient -0.009, 95% CI -0.017 to -0.001, p=0.023) and with clinical weakness and lower MUNIX in multiple individual muscles. Relative T2 signal in bilateral tibialis anterior increased over 4 months in patients with MND (right: 10.2%, 95% CI 2.0% to 18.4%, p=0.017; left: 14.1%, 95% CI 3.4% to 24.9%, p=0.013). Anatomically, contiguous disease spread on MRI was not apparent in this model. CONCLUSIONS: Whole-body muscle MRI offers a new approach to objective assessment of denervation over short timescales in MND and enables investigation of patterns of disease spread in vivo. Muscles inaccessible to conventional clinical and electrophysiological assessment may be investigated using this methodology.

Effect of microstructure on population growth parameters of Escherichia coli in gelatin-dextran systems.

Current literature acknowledges the effect of food structure on bacterial dynamics. Most studies introduce this "structure" factor using a single gelling agent, resulting in a homogeneous environment, whereas in practice most food products are heterogeneous. Therefore, this study focuses on heterogeneous protein-polysaccharide mixtures, based on gelatin and dextran. These mixtures show phase separation, leading to a range of heterogeneous microstructures by adjusting relative concentrations of both gelling agents. Based on confocal microscope observations, the growth of Escherichia coli in gelatin-dextran systems was observed to occur in the dextran phase. To find a relation between microscopic and population behavior, growth experiments were performed in binary and singular gelatin-dextran systems and culture broth at 23.5°C, with or without adding 2.9% (wt/vol) NaCl. The Baranyi and Roberts growth model was fitted to the experimental data and parameter estimates were statistically compared. For salted binary mixtures, a decrease in the population maximum cell density was observed with increasing gelatin concentration. In this series, for one type of microstructure, i.e., a gelatin matrix phase with a disperse dextran phase, the maximum cell density decreased with decreasing percentage of dextran phase. However, this relation no longer held when other types of microstructure were observed. Compared to singular systems, adding a second gelling agent in the presence of NaCl had an effect on population lag phases and maximum cell densities. For unsalted media, the growth parameters of singular and binary mixtures were comparable. Introducing this information into mathematical models leads to more reliable growth predictions and enhanced food safety.

Synthesis and in vitro evaluation of a PDT active BODIPY-NLS conjugate.

Two new photosensitizers based on the BODIPY scaffold have been synthesized, of which one bears an NLS peptide, which is linked to the BODIPY's core using the copper catalysed azide-alkyne click reaction. The phototoxicities of these BODIPY based photosensitizers have been determined, as well as their dark toxicities. Although the conjugation of a single NLS peptide to the BODIPY did not lead to any observable nuclear localization, the photosensitizer did exhibit a superior photoxicity. Cellular co-localization experiments revealed a localization of both dyes in the lysosomes, as well as a partial localization within the ER (for the peptide-bearing BODIPY).

Mesoscale DNA structural changes on binding and photoreaction with Ru[(TAP)2PHEHAT]2+.

We used scanning force microscopy (SFM) to study the binding and excited state reactions of the intercalating photoreagent Ru[(TAP)(2)PHEHAT](2+) (TAP = 1,4,5,8-tetraazaphenanthrene; PHEHAT = 1,10-phenanthrolino[5,6-b]1,4,5,8,9,12-hexaazatriphenylene) with DNA. In the ground state, this ruthenium complex combines a strong intercalative binding mode via the PHEHAT ligand, with TAP-mediated hydrogen bonding capabilities. After visible irradiation, SFM imaging of the photoproducts revealed both the structural implications of photocleavages and photoadduct formation. It is found that the rate of photocleaving is strongly increased when the complex can interact with DNA via hydrogen bonding. We demonstrated that the photoadduct increases DNA rigidity, and that the photo-biadduct can crosslink two separate DNA segments in supercoiled DNA. These mechanical and topological effects might have important implications in future therapeutic applications of this type of compounds.

Spectroscopic characterization of Venus at the single molecule level.

Venus is a recently developed, fast maturating, yellow fluorescent protein that has been used as a probe for in vivo applications. In the present work the photophysical characteristics of Venus were analyzed spectroscopically at the bulk and single molecule level. Through time-resolved single molecule measurements we found that single molecules of Venus display pronounced fluctuations in fluorescence emission, with clear fluorescence on- and off-times. These fluorescence intermittencies were found to occupy a broad range of time scales, ranging from milliseconds to several seconds. Such long off-times can complicate the analysis of single molecule counting experiments or single-molecule FRET experiments.

Toward early screening for early management of postnatal depression? Relationships between clinical signs present in the infant and underlying maternal postnatal depression.

Objective: The objective was to screen for maternal postnatal depression (MPD) by administering the Edinburgh Postnatal Depression Scale (EPDS) during the first "peak" of incidence of MPD (i. e., between the 6th and the 10th week of the infant's life) and to therefore explore the relationship between mothers' EPDS scores and early clinical signs in the infant. We wanted to evaluate the relevance of a diagnostic tool that combines the EPDS with questions focused on clinical signs displayed by the infant. Participants: Seven hundred and sixty seven mothers aged 18-46 (M = 30.5, SD = 4.9) participated in the study, representing 49.2% of all women who delivered in the study area during the research inclusion period. Main outcome measures: Sociodemographic data were collected. MPD was measured by EPDS (score ≥ 12). The presence of clinical signs in the infant was investigated by closed (i.e., yes or no) questions inquiring into whether the infant has or has had difficulty sleeping, feeding difficulties, crying difficult to calm, or other difficulties. Results: The prevalence of MPD in our sample was 22.16%. The relationships between MPD and early clinical signs present in the infant, i.e., sleep difficulties, feeding problems, crying difficult to calm (p < 0.001), and other problems (p = 0.004), were very significant, as confirmed by a chi-square test of independence. In particular, sleep difficulties (OR = 2.05, CI 1.41-2.99) and feeding difficulties (OR = 1.59, CI 1.10-2.30) seemed to predict MPD. Conclusions: Early clinical signs in the infant can alert the medical team to potential psychological suffering on the part of the mother, at which time the EPDS can be proposed. The use of this method has the potential to improve screening for, and therefore early management of, MPD.

Prognosis of Type 2 Myocardial Infarction Patients Implanted With a Prophylactic Defibrillator (from the Very-High-Rate Registry).

An implantable cardioverter defibrillator (ICD) is recommended in primary prevention patients with a coronary artery disease (CAD) and reduced left ventricular ejection fraction. Benefits of ICD in CAD unrelated to coronary thrombosis are unknown. We sought to compare the prognosis of patients with CAD implanted with a prophylactic ICD according to the type of myocardial infarction (MI). Patients from the Very-High-Rate registry implanted with a prophylactic ICD for CAD between 2006 and 2016 were retrospectively included. Cardiac resynchronization therapy patients were excluded. Patients with type 2 MI were matched (1:4) with patients with type 1 MI using propensity score. The following events were collected: death, hospitalization for heart failure, cardiac transplantation, and appropriated therapies on ventricular arrhythmia (≥220 beats/min). Among 571 consecutive ischemic patients, 65 type 2 MI patients were matched to 260 type 1. After a mean follow up of 55 ± 36 months, 63 patients (24%) died in type 1 group, 18 (28%) in type 2 group (p = 0.19). Survival rate from appropriate therapies on high-rate ventricular arrhythmias was significantly lower in type 2 group (p = 0.04). In conclusion, patients implanted with a prophylactic ICD for severe CAD, whether type 1 or type 2 MI, have similar outcomes.

The effect of medium structure complexity on the growth of Saccharomyces cerevisiae in gelatin-dextran systems.

As most food systems are (semi-)solid, the effect of food structure on bacterial growth has been widely acknowledged. However, studies on the growth dynamics of yeasts have neglected the effect of food structure. In this paper, the growth dynamics of the spoilage yeast Saccharomyces cerevisiae was investigated at 23.5 °C in broth, singular, homogeneous biopolymer systems and binary biopolymer systems with a heterogeneous microstructure. The biopolymers gelatin and dextran were used to introduce the different levels of structure. The metabolizing ability of gelatin and dextran by S. cerevisiae was examined. To study microbial behavior in the binary systems at the micro level, mixtures were imaged with confocal laser scanning microscopy (CLSM). Growth dynamics and microscopic images of S. cerevisiae were compared with those obtained for Escherichia coli in the same model system (Boons et al., 2014). Different phase-separated, heterogeneous microstructures were obtained by changing the amount of added gelatin and dextran. Regardless of the microstructure, S. cerevisiae was preferentially located in the dextran phase. Metabolizing ability-tests indicated that gelatin could be consumed by S. cerevisiae but in the presence of glucose, no change in gelatin concentration was observed. No indication of dextran metabolizing ability was observed. When supplementing broth with gelatin or dextran alone, an enhanced growth rate and maximum cell density were observed. This enhancement was further increased by adding a second biopolymer, introducing a heterogeneous microstructure and hence increasing the medium structure complexity. The results obtained indicate that food structure complexity plays a significant role in the growth dynamics of S. cerevisiae, an important food spoiler.

Rational design of photoconvertible and biphotochromic fluorescent proteins for advanced microscopy applications.

Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. IrisFP was recently reported as the first fluorescent protein combining these two types of phototransformations. The introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging. However, the spectroscopic properties of IrisFP are far from being optimal and its tetrameric organization complicates its use as a fusion tag. Here, we demonstrate how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins and develop and characterize a new set of such enhanced optical highlighters derived from mEosFP and Dendra2. We present in particular NijiFP, a promising new fluorescent protein with photoconvertible and biphotochromic properties that make it ideal for advanced fluorescence-based imaging applications.