Impact of COVID-19 Pandemic on Young Children With Feeding and Eating Problems and Disorders and Their Families.

OBJECTIVES: The incidence of feeding and eating problems and disorders (FEPD) in children increased during the coronavirus disease 2019 (COVID-19) pandemic. The aim of this study was to assess the impact of the COVID-19 pandemic on young children with FEPD and their parents. METHODS: Cross-sectional survey: parents of children with FEPD (0-11 years) in the Netherlands completed an online questionnaire (January-April 2021). This questionnaire included 4 demographic questions (including criteria of pediatric feeding disorder [PFD] and/or avoidant/restrictive food intake disorder [ARFID]) and 11 questions related to experienced impact of the COVID-19 pandemic. Parental responses regarding children with FEPD (including PFD and ARFID) were compared with those of healthy controls (HCs). RESULTS: In total, 240 children (median age, 5.5 years; interquartile range [IQR], 3.5-7.9 years; 53.3% female) were included; 129 children with FEPD and 111 HC. Most children with FEPD fulfilled criteria for PFD (n = 119; 92.2%) and/or ARFID (n = 117; 90.7%). Parents of children with FEPD reported more stress (of their child [ P = 0.014] and parental stress [ P = 0.014]), worse eating by the child ( P < 0.001), more negative relations within the family ( P = 0.006), and less support from the environment ( P = 0.001) compared with parents of HC during the COVID-19 pandemic than before. CONCLUSIONS: It seems that the COVID-19 pandemic had great impact on young children with FEPD and their parents because parents of children with FEPD reported significantly more perceived stress within both the child and parents, more difficult eating behavior of the child, more negative behavior between family members, and less support from the environment as compared with HC.

A polarized view on DNA under tension.

In the past decades, sensitive fluorescence microscopy techniques have contributed significantly to our understanding of the dynamics of DNA. The specific labeling of DNA using intercalating dyes has allowed for quantitative measurement of the thermal fluctuations the polymers undergo. On the other hand, recent advances in single-molecule manipulation techniques have unraveled the mechanical and elastic properties of this intricate polymer. Here, we have combined these two approaches to study the conformational dynamics of DNA under a wide range of tensions. Using polarized fluorescence microscopy in conjunction with optical-tweezers-based manipulation of YOYO-intercalated DNA, we controllably align the YOYO dyes using DNA tension, enabling us to disentangle the rapid dynamics of the dyes from that of the DNA itself. With unprecedented control of the DNA alignment, we resolve an inconsistency in reports about the tilted orientation of intercalated dyes. We find that intercalated dyes are on average oriented perpendicular to the long axis of the DNA, yet undergo fast dynamics on the time scale of absorption and fluorescence emission. In the overstretching transition of double-stranded DNA, we do not observe changes in orientation or orientational dynamics of the dyes. Only beyond the overstretching transition, a considerable depolarization is observed, presumably caused by an average tilting of the DNA base pairs. Our combined approach thus contributes to the elucidation of unique features of the molecular dynamics of DNA.

Introduction to Optical Tweezers: Background, System Designs, and Commercial Solutions.

Optical tweezers are a means to manipulate objects with light. With the technique, microscopically small objects can be held and steered, while forces on the trapped objects can be accurately measured and exerted. Optical tweezers can typically obtain a nanometer spatial resolution, a picoNewton force resolution, and a millisecond time resolution, which makes them excellently suited to study biological processes from the single-cell down to the single-molecule level. In this chapter, we will provide an introduction on the use of optical tweezers in single-molecule approaches. We will introduce the basic principles and methodology involved in optical trapping, force calibration, and force measurements. Next we describe the components of an optical tweezers setup and their experimental relevance in single-molecule approaches. Finally, we provide a concise overview of commercial optical tweezers systems. Commercial systems are becoming increasingly available and provide access to single-molecule optical tweezers experiments without the need for a thorough background in physics.

Introduction to optical tweezers: background, system designs, and commercial solutions.

Optical tweezers are a means to manipulate objects with light. With the technique, microscopically small objects can be held and steered while forces on the trapped objects can be accurately measured and exerted. Optical tweezers can typically obtain a nanometer spatial resolution, a piconewton force resolution, and a millisecond time resolution, which make them excellently suited to study biological processes from the single-cell down to the single-molecule level. In this chapter, we provide an introduction on the use of optical tweezers in single-molecule approaches. We introduce the basic principles and methodology involved in optical trapping, force calibration, and force measurements. Next, we describe the components of an optical tweezers setup and their experimental relevance in single-molecule approaches. Finally, we provide a concise overview of commercial optical tweezers systems. Commercial systems are becoming increasingly available and provide access to single-molecule optical tweezers experiments without the need for a thorough background in physics.

Visualizing the formation and collapse of DNA toroids.

In living organisms, DNA is generally confined into very small volumes. In most viruses, positively charged multivalent ions assist the condensation of DNA into tightly packed toroidal structures. Interestingly, such cations can also induce the spontaneous formation of DNA toroids in vitro. To resolve the condensation dynamics and stability of DNA toroids, we use a combination of optical tweezers and fluorescence imaging to visualize in real-time spermine-induced (de)condensation in single DNA molecules. By actively controlling the DNA extension, we are able to follow (de)condensation under tension with high temporal and spatial resolution. We show that both processes occur in a quantized manner, caused by individual DNA loops added onto or removed from a toroidal condensate that is much smaller than previously observed in similar experiments. Finally, we present an analytical model that qualitatively captures the experimentally observed features, including an apparent force plateau.

Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging.

Single-molecule manipulation studies have revealed that double-stranded DNA undergoes a structural transition when subjected to tension. At forces that depend on the attachment geometry of the DNA (65 pN or 110 pN), it elongates approximately 1.7-fold and its elastic properties change dramatically. The nature of this overstretched DNA has been under debate. In one model, the DNA cooperatively unwinds, while base pairing remains intact. In a competing model, the hydrogen bonds between base pairs break and two single DNA strands are formed, comparable to thermal DNA melting. Here, we resolve the structural basis of DNA overstretching using a combination of fluorescence microscopy, optical tweezers, and microfluidics. In DNA molecules undergoing the transition, we visualize double- and single-stranded segments using specific fluorescent labels. Our data directly demonstrate that overstretching comprises a gradual conversion from double-stranded to single-stranded DNA, irrespective of the attachment geometry. We found that these conversions favorably initiate from nicks or free DNA ends. These discontinuities in the phosphodiester backbone serve as energetically favorable nucleation points for melting. When both DNA strands are intact and no nicks or free ends are present, the overstretching force increases from 65 to 110 pN and melting initiates throughout the molecule, comparable to thermal melting. These results provide unique insights in the thermodynamics of DNA and DNA-protein interactions.

Leveraging single protein polymers to measure flexural rigidity.

The micrometer-scale length of some protein polymers allows them to be mechanically manipulated in single-molecule experiments. This provides a direct way to measure persistence length. We have used a double optical trap to elastically deform single microtubules and actin filaments. Axial extensional force was exerted on beads attached laterally to the filaments. Because the attachments are off the line of force, pulling the beads apart couples to local bending of the filament. We present a simple mechanical model for the resulting highly nonlinear elastic response of the dumbbell construct. The flexural rigidities of the microfilaments that were found by fitting the model to the experimentally observed force-distance curves are (7.1 +/- 0.8) x 10(4) pN nm2 (persistence length L(p) = 17.2 microm) for F-actin and (6.1 +/- 1.3) x 10(6) pN nm2 (L(p) = 1.4 mm) for microtubules.

Counting RAD51 proteins disassembling from nucleoprotein filaments under tension.

The central catalyst in eukaryotic ATP-dependent homologous recombination consists of RAD51 proteins, polymerized around single-stranded DNA. This nucleoprotein filament recognizes and invades a homologous duplex DNA segment. After strand exchange, the nucleoprotein filament should disassemble so that the recombination process can be completed. The molecular mechanism of RAD51 filament disassembly is poorly understood. Here we show, by combining optical tweezers with single-molecule fluorescence microscopy and microfluidics, that disassembly of human RAD51 nucleoprotein filaments results from the interplay between ATP hydrolysis and the release of the tension stored in the filament. By applying external tension to the DNA, we found that disassembly slows down and can even be stalled. We quantified the fluorescence of RAD51 patches and found that disassembly occurs in bursts interspersed by long pauses. After relaxation of a stalled complex, pauses were suppressed resulting in a large burst. These results indicate that tension-dependent disassembly takes place only from filament ends, after tension-independent ATP hydrolysis. This integrative single-molecule approach allowed us to dissect the mechanism of this principal homologous recombination reaction step, which in turn clarifies how disassembly can be influenced by accessory proteins.

See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins.

Direct visualization of DNA and proteins allows researchers to investigate DNA-protein interactions with great detail. Much progress has been made in this area as a result of increasingly sensitive single-molecule fluorescence techniques. At the same time, methods that control the conformation of DNA molecules have been improving constantly. The combination of both techniques has appealed to researchers ever since single-molecule measurements have become possible and indeed first implementations of such combined approaches have proven useful in the study of several DNA-binding proteins in real time. Here, we describe the technical state-of-the-art of various integrated manipulation-and-visualization methods. We first discuss methods that allow only little control over the DNA conformation, such as DNA combing. We then describe DNA flow-stretching approaches that allow more control, and end with the full control on position and extension obtained by manipulating DNA with optical tweezers. The advantages and limitations of the various techniques are discussed, as well as several examples of applications to biophysical or biochemical questions. We conclude with an outlook describing potential future technical developments in combining fluorescence microscopy with DNA micromanipulation technology.

Visualizing single DNA-bound proteins using DNA as a scanning probe.

Many biological processes involve enzymes moving along DNA. Such motion might be impeded by DNA-bound proteins or DNA supercoils. Current techniques are incapable of directly measuring forces that such 'roadblocks' might impose. We constructed a setup with four independently moveable optical traps, allowing us to manipulate two DNA molecules held between beads. By tightly wrapping one DNA around the other, we created a probe that can be scanned along the contour of the second DNA. We found that friction between the two polymers remains below 1 pN. Upon encountering DNA-bound proteins substantial friction forces are measured, allowing accurate localization of protein positions. Furthermore, these proteins remained associated at low probe tensions but could be driven off using forces greater than 20 pN. Finally, the full control of the orientation of two DNA molecules opens a wide range of experiments on proteins interacting with multiple DNA regions.

Fluorescent human RAD51 reveals multiple nucleation sites and filament segments tightly associated along a single DNA molecule.

The DNA strand-exchange reactions defining homologous recombination involve transient, nonuniform allosteric interactions between recombinase proteins and their DNA substrates. To study these mechanistic aspects of homologous recombination, we produced functional fluorescent human RAD51 recombinase and visualized recombinase interactions with single DNA molecules in both static and dynamic conditions. We observe that RAD51 nucleates filament formation at multiple sites on double-stranded DNA. This avid nucleation results in multiple RAD51 filament segments along a DNA molecule. Analysis of fluorescent filament patch size and filament kinks from scanning force microscopy (SFM) images indicate nucleation occurs minimally once every 500 bp. Filament segments did not rearrange along DNA, indicating tight association of the ATP-bound protein. The kinetics of filament disassembly was defined by activating ATP hydrolysis and following individual filaments in real time.

Analysis of scanning force microscopy images of protein-induced DNA bending using simulations.

Bending of DNA is a feature essential to the function of many DNA-binding proteins. Bending angles can be estimated with a variety of techniques, but most directly from images obtained using scanning force microscopy (SFM). Direct measurement of the bending angle using a tangent method often produces angles that deviate significantly from values obtained using other techniques. Here, we describe the application of SFM in combination with simulations of DNA as a means to estimate protein-induced bending angles in a reliable and unbiased fashion. In this manner, we were able to obtain accurate estimates for the bending angles induced by nuclear factor I, octamer-binding transcription factor 1, the human XPC-Rad23B complex and integration host factor [correction]