Diaphragmatic hernia in a term newborn with congenital myotonic dystrophy: case report.

Background and aim Myotonic dystrophy (DM) is a genetic disorder determined by an amplified trinucleotide CTG repeat in the untranslated region of the DMPK gene on chromosome 19q13.3. The incidence of the congenital form is 1 in 47619 live births and the mortality in the neonatal period is up to 40%.

Safety use of high frequency oscillatory ventilation in transport of newborn infants affected by severe respiratory failure: preliminary data in central Tuscany.

Background: Neonatal Emergency Transport Services play a fundamental role in neonatal care. Stabilization before transport of newborns suffering from severe respiratory failure is often a challenging problem and some critically ill infants may benefit from High Frequency Oscillatory Ventilation (HFOV) as rescue treatment. In these cases, transition to conventional ventilation for transport may cause a deterioration in clinical conditions.

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics.

Self-reported prior night's sleep quantity on baseline symptom factors and computerized neurocognitive testing in high school athletes.

Little is known regarding sleep and baseline concussion performance and symptoms in athletes. We explored the effects of self-reported prior night's sleep quantity on baseline symptoms and computerized neurocognitive testing in high school athletes. A retrospective analysis of 958 high school athletes between the ages of 13-19 years was completed on Immediate Post-Concussion Assessment and Cognitive Testing (ImPACT) performance.

High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow.

Optical crosstalk in SPAD arrays for high-throughput single-molecule fluorescence spectroscopy.

Single-molecule fluorescence spectroscopy (SMFS), based on the detection of individual molecules freely diffusing through the excitation spot of a confocal microscope, has allowed unprecedented insights into biological processes at the molecular level, but suffers from limited throughput. We have recently introduced a multispot version of SMFS, which allows achieving high-throughput SMFS by virtue of parallelization, and relies on custom silicon single-photon avalanche diode (SPAD) detector arrays. Here, we examine the premise of this parallelization approach, which is that data acquired from different spots is uncorrelated.

Feeding Practices in Very Preterm and Very Low Birth Weight Infants in an Area Where a Network of Human Milk Banks Is in Place.

Great variability in enteral feeding practices for very preterm (<32 weeks gestational age-GA) and very low birth weight infants (VLBW; ≤1,500 g) have been reported. We aimed to describe data on enteral feeding in Tuscany (Italy), where a network of 6 donor milk banks is in place. A 4-years (2012-2015) observational study was performed analyzing the database "TIN Toscane online" on very preterm and VLBW infants.

Monte Carlo Diffusion-Enhanced Photon Inference: Distance Distributions and Conformational Dynamics in Single-Molecule FRET.

Single-molecule Förster resonance energy transfer (smFRET) is utilized to study the structure and dynamics of many biomolecules, such as proteins, DNA, and their various complexes. The structural assessment is based on the well-known Förster relationship between the measured efficiency of energy transfer between a donor (D) and an acceptor (A) dye and the distance between them. Classical smFRET analysis methods called photon distribution analysis (PDA) take into account photon shot-noise, D-A distance distribution, and, more recently, interconversion between states in order to extract accurate distance information.

Characterizing highly dynamic conformational states: The transcription bubble in RNAP-promoter open complex as an example.

Bio-macromolecules carry out complicated functions through structural changes. To understand their mechanism of action, the structure of each step has to be characterized. While classical structural biology techniques allow the characterization of a few "structural snapshots" along the enzymatic cycle (usually of stable conformations), they do not cover all (and often fast interconverting) structures in the ensemble, where each may play an important functional role.

48-spot single-molecule FRET setup with periodic acceptor excitation.

Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples.

Membrane insertion of-and membrane potential sensing by-semiconductor voltage nanosensors: Feasibility demonstration.

We developed membrane voltage nanosensors that are based on inorganic semiconductor nanoparticles. We provide here a feasibility study for their utilization. We use a rationally designed peptide to functionalize the nanosensors, imparting them with the ability to self-insert into a lipid membrane with a desired orientation.

16-Ch Time-resolved Single-Molecule Spectroscopy Using Line Excitation.

Single-molecule spectroscopy on freely-diffusing molecules allows detecting conformational changes of biomolecules without perturbation from surface immobilization. Resolving fluorescence lifetimes increases the sensitivity in detecting conformational changes and overcomes artifacts common in intensity-based measurements. Common to all freely-diffusing techniques, however, are the long acquisition times.

Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules.

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription.

Different types of pausing modes during transcription initiation.

In many cases, initiation is rate limiting to transcription. This due in part to the multiple cycles of abortive transcription that delay promoter escape and the transition from initiation to elongation. Pausing of transcription in initiation can further delay promoter escape.

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase.

Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.

FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET.

Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution.

Photon-HDF5: Open Data Format and Computational Tools for Timestamp-based Single-Molecule Experiments.

Archival of experimental data in public databases has increasingly become a requirement for most funding agencies and journals. These data-sharing policies have the potential to maximize data reuse, and to enable confirmatory as well as novel studies. However, the lack of standard data formats can severely hinder data reuse.

Photon-HDF5: An Open File Format for Timestamp-Based Single-Molecule Fluorescence Experiments.

We introduce Photon-HDF5, an open and efficient file format to simplify exchange and long-term accessibility of data from single-molecule fluorescence experiments based on photon-counting detectors such as single-photon avalanche diode, photomultiplier tube, or arrays of such detectors. The format is based on HDF5, a widely used platform- and language-independent hierarchical file format for which user-friendly viewers are available. Photon-HDF5 can store raw photon data (timestamp, channel number, etc.

Silicon photon-counting avalanche diodes for single-molecule fluorescence spectroscopy.

Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with applications in cell biology, biochemistry and biophysics. The basic feature of this technique is to excite and collect light from a very small volume and work in a low concentration regime resulting in rare burst-like events corresponding to the transit of a single molecule. Detecting photon bursts is a challenging task: the small number of emitted photons in each burst calls for high detector sensitivity.

8-spot smFRET analysis using two 8-pixel SPAD arrays.

Single-molecule Förster resonance energy transfer (smFRET) techniques are now widely used to address outstanding problems in biology and biophysics. In order to study freely diffusing molecules, current approaches consist in exciting a low concentration (<100 pM) sample with a single confocal spot using one or more lasers and detecting the induced single-molecule fluorescence in one or more spectrally- and/or polarization-distinct channels using single-pixel Single-Photon Avalanche Diodes (SPADs). A large enough number of single-molecule bursts must be accumulated in order to compute FRET efficiencies with sufficient statistics.

Single-molecule FRET experiments with a red-enhanced custom technology SPAD.

Single-molecule fluorescence spectroscopy of freely diffusing molecules in solution is a powerful tool used to investigate the properties of individual molecules. Single-Photon Avalanche Diodes (SPADs) are the detectors of choice for these applications. Recently a new type of SPAD detector was introduced, dubbed red-enhanced SPAD (RE-SPAD), with good sensitivity throughout the visible spectrum and with excellent timing performance.

Development of new photon-counting detectors for single-molecule fluorescence microscopy.

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences.