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Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

The single-stranded DNA binding protein (SSB) is essential to all aspects of DNA metabolism in bacteria. This protein performs two distinct, but closely intertwined and indispensable functions in the cell. SSB binds to single-stranded DNA (ssDNA) and at least 20 partner proteins resulting in their regulation. These partners comprise a family of genome guardians known as the SSB interactome. Essential to interactome regulation is the linker/OB-fold network of interactions. This network of interactions forms when one or more PXXP motifs in the linker of SSB bind to an OB-fold in a partner, with interactome members involved in competitive binding between the linker and ssDNA to their OB-fold. Consequently, when linker-binding occurs to an OB-fold in an interactome partner, proteins are loaded onto the DNA. When linker/OB-fold interactions occur between SSB tetramers, cooperative ssDNA-binding results, producing a multi-tetrameric complex that rapidly protects the ssDNA. Within this SSB-ssDNA complex, there is an extensive and dynamic network of linker/OB-fold interactions that involves multiple tetramers bound contiguously along the ssDNA lattice. The dynamic behavior of these tetramers which includes binding mode changes, sliding as well as DNA wrapping/unwrapping events, are likely coupled to the formation and disruption of linker/OB-fold interactions. This behavior is essential to facilitating downstream DNA processing events. As OB-folds are critical to the essence of the linker/OB-fold network of interactions, and they are found in multiple interactome partners, the SSB interactome is classified as the first family of prokaryotic, oligosaccharide/oligonucleotide binding fold (OB-fold) genome guardians.

Holographic optical tweezers (HOTs) use spatial light modulators (SLM) to modulate light beams, thereby enabling the dynamic control of optical trap arrays with complex intensity and phase distributions. This has provided exciting new opportunities for cell sorting, microstructure machining, and studying single molecules. However, the pixelated structure of the SLM will inevitably bring up the unmodulated zero-order diffraction possessing an unacceptably large fraction of the incident light beam power. This is harmful to optical trapping because of the bright, highly localized nature of the errant beam. In this paper and to address this issue, we construct a cost-effective, zero-order free HOTs apparatus, thanks to a homemade asymmetric triangle reflector and a digital lens. As there is no zero-order diffraction, the instrument performs excellently in generating complex light fields and manipulating particles.

DNA replication forks stall at least once per cell cycle in Escherichia coli. DNA replication must be restarted if the cell is to survive. Restart is a multi-step process requiring the sequential action of several proteins whose actions are dictated by the nature of the impediment to fork progression. When fork progress is impeded, the sequential actions of SSB, RecG and the RuvABC complex are required for rescue. In contrast, when a template discontinuity results in the forked DNA breaking apart, the actions of the RecBCD pathway enzymes are required to resurrect the fork so that replication can resume. In this review, we focus primarily on the significant insight gained from single-molecule studies of individual proteins, protein complexes, and also, partially reconstituted regression and RecBCD pathways. This insight is related to the bulk-phase biochemical data to provide a comprehensive review of each protein or protein complex as it relates to stalled DNA replication fork rescue.

Nonunion of fractures about the proximal humerus represents a rare and complex problem. The purpose of this study was to evaluate the clinical and functional outcomes following proximal humerus fracture (PHF) nonunion repair using a plate and screw construct with a direct comparison to those experienced following operative fixation of acute PHF using a plate and screw construct. Two separate patient cohorts were included in this study. The first consisted of 16 patients diagnosed with a non-united PHF who underwent operative nonunion repair treated with a standard algorithmic approach. The comparison group consisted of 173 patients who achieved union following initial open reduction and internal fixation of an acute PHF treated with a proximal humerus locking plate construct. All patients had at least 12 months of postoperative follow-up. Outcomes were assessed for both groups using American Shoulder and Elbow Surgeons (ASES) scores, visual analog scale (VAS) pain scores, and postoperative shoulder range of motion (ROM). Statistical analyses were used to compare these outcome measures between the two cohorts. The nonunion repair cohort consisted of eleven surgical neck nonunions and five nonunions of both the surgical neck and greater tuberosity. Ten patients had undergone surgical treatment for their original fracture, while six were initially treated non-operatively. All patients had Boileau type 3 sequelae of their proximal humerus fracture. Union was achieved in all patients at a mean of 5.4 months following nonunion repair. Complications included hardware failure requiring revision in two patients (12.5%) and avascular necrosis requiring conversion to anatomic total shoulder arthroplasty following union in one patient (6.3%). The nonunion repair and acute fracture cohorts did not differ with respect to mean ASES scores, VAS pain scores, or active shoulder ROM at any postoperative time point. Surgical repair of PHF nonunion is a viable treatment strategy that can lead to consistent bony healing with outcomes comparable to those of patients who achieve fracture union following initial surgical repair of an acute proximal humerus fracture. Surgeons should be cognizant of mechanical considerations that may lead to early failure.

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3′-to-5′ direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. Single-stranded DNA–binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5′- and 3′-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB.

The Escherichia coli single‐strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single‐stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide‐oligonucleotide binding folds (OB‐fold) present in SSB and its interactome partners. Not surprisingly, partner OB‐fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB‐fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C‐terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles. First, and consistent with the proposal of Dixon, to regulate the structure of the C‐terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB‐folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB‐folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB‐fold genome guardians identified in prokaryotes.

Purpose The 6-min walk test (6MWT) is broadly used to evaluate the functional ability of patients with heart failure (HF). The purpose of this study was to evaluate the accuracy and reliability of the wearable cardioverter defibrillator (WCD)-guided 6MWT performed at home by patients with HF versus in-clinic testing. Methods Patients (n = 197) with HF and a low ejection fraction prescribed a WCD were randomized to 2 groups. Group 1 completed an in-clinic clinician-guided 6MWT while wearing the WCD; results were recorded by the clinician. Group 2 completed a WCD-guided 6MWT, also performed in the clinic; results were recorded by the WCD accelerometer. Both groups performed weekly unsupervised WCD-guided 6MWTs at home, with results recorded by the WCD. Results The initial in-clinic 6MWT showed no significant group difference in distance walked (group 1 = 306 m; group 2 = 297 m). For patients in group 2 who completed at least one 6MWT at home, there was a 15-step decrease between the in-clinic WCD-guided 6MWT and the first at-home 6MWT, 558 and 543 median steps (P = .001), respectively. Among patients with at least 8 weekly home WCD-guided 6MWTs (n = 70), there was no significant difference in the number of steps walked during the 6MWT from week to week. Conclusions Results of the in-clinic 6MWT are similar between clinician-guided and WCD-guided patients across objective distances. Distances walked with a WCD-guided walk test were consistent whether conducted in the clinic or at home and were reliable over time.

The RecG DNA helicase is a guardian of the bacterial genome where it dominates stalled DNA replication fork rescue. The single-stranded DNA binding protein (SSB) is involved in this process and promotes the binding of RecG to stalled replication forks. Atomic force microscopy (AFM) was used to investigate the interaction of RecG and SSB on a mobile fork substrate capable of being regressed. In the absence of proteins, the fork undergoes spontaneous dynamics between two states defined by the length of the DNA complementarity at the fork. Binding of SSB does not affect these dynamics as it binds to single-stranded regions as expected. In contrast, RecG interacts with the two states quite differently. We demonstrate that RecG has two modes of interaction with fork DNA in the presence of SSB and ATP. In the first mode, RecG translocates over the duplex region and this activity is defined by SSB-mediated remodeling of the helicase. In the second mode, RecG utilizes its helicase activity to regress the fork, in an ATP-dependent manner, displacing SSB on the ssDNA. Overall, our results highlight two functions of RecG that can be employed in the regulation of stalled DNA replication fork rescue.

There are multiple assays available that can provide insight into the biochemical mechanism of DNA helicases. For the first 22 years since their discovery, bulk-phase assays were used. These include gel-based, spectrophotometric, and spectrofluorometric assays that revealed many facets of these enzymes. From 2001, single-molecule studies have contributed additional insight into these DNA nanomachines to reveal details on energy coupling, step size, processivity as well as unique aspects of individual enzyme behavior that were masked in the averaging inherent in ensemble studies. In this review, important aspects of the study of helicases are discussed including beginning with active, nuclease-free enzyme, followed by several bulk-phase approaches that have been developed and still find widespread use today. Finally, two single-molecule approaches are discussed, and the resulting findings are related to the results obtained in bulk-phase studies.

The DNA helicase PriA is a key protein for restarting stalled DNA replication forks in bacteria. With 3′ to 5′ helicase activity, PriA is important in primosome assembly. We used atomic force microscopy (AFM) and specifically employed time-lapse AFM to visualize the interaction of PriA with two DNA substrates. The results show that most of the PriA molecules are observed bound at the fork. However, PriA is capable of translocating over distances of about 400 bp. There is a preference for the long-range translocation of PriA depending on the fork type. For a fork with the nascent leading strand as single-stranded DNA (ssDNA; F4 substrate), PriA translocates preferentially on the parental arm of the fork. For the substrate F14, which contains an additional ssDNA segment between the parental and lagging arms (5 nt gap), PriA translocates on both the parental and lagging strand arms. These data suggest that transient formation of the single-stranded regions during the DNA replication can change the selection of the DNA duplex by PriA. Translocation of the helicase was directly visualized by time-lapse AFM imaging, which revealed that PriA can switch strands during translocation. These novel features of PriA shed new light on the mechanisms of PriA interaction with stalled replication forks. [Image: see text]

The RecG DNA helicase plays a crucial role in stalled replication fork rescue as the guardian of the bacterial genome. We have recently demonstrated that single-strand DNA binding protein (SSB) promotes binding of RecG to the stalled replication fork by remodeling RecG, enabling the helicase to translocate ahead of the fork. We also hypothesized that mispairing of DNA could limit such translocation of RecG, which plays the role of roadblocks for the fork movement. Here, we used atomic force microscopy (AFM) to directly test this hypothesis and investigate how sensitive RecG translocation is to different types of mispairing. We found that a C-C mismatch at a distance of 30 bp away from the fork position prevents translocation of RecG over this mispairing. A G-bulge placed at the same distance also has a similar roadblock efficiency. However, a C-C mismatch 10 bp away from the fork does not prevent RecG translocation, as 10 bp from fork is within the distance of footprint of RecG on fork DNA. Our findings suggest that retardation of RecG translocation ahead of the replication fork can be a mechanism for the base pairing control for DNA replication machinery.

Understanding protein-protein interactions is key to unraveling protein function in vivo. Here we describe a dual/triple-plasmid system that enables co-expression of two, or three, recombinant proteins harboring different affinity tags in the same Escherichia coli cell. This novel protein expression system provides a platform to understand protein-protein interactions and enables researchers to study protein complex formation and in vivo localization.

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks, protecting the ssDNA from nucleases. Research that is based on the assays for junction dissociation, surface plasmon resonance, single-molecule FRET, and x-ray crystal structure has revealed the helicase activity of PriA, the SSB-PriA interaction, and structural information of PriA helicase. Here, we used Atomic Force Microscopy (AFM) to visualize the interaction between PriA and DNA substrates with or without SSB in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. The protocol describes the steps to obtain high-resolution AFM images and the details of data analysis and presentation.

PriA is a member of the SuperFamily 2 helicase family. Its rolein vivois to reload the primosome onto stalled replication forks resulting in the restart of the previously stalled DNA replication process. SSB is known to play key roles in mediating activities at replication forks and it is known to bind to PriA. To gain mechanistic insight into the PriA-SSB interaction, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of PriAin vitroin the presence of fork substrates. The results demonstrate that SSB enhances the ability of PriA to discriminate between fork substrates 140-fold. This is due to a significant increase in the catalytic efficiency of the helicase induced by DNA-bound SSB. This interaction is species-specific as bacteriophage gene 32 protein cannot substitute for theE.coliprotein. SSB, while enhancing the activity of PriA on its preferred fork, both decreases the affinity of the helicase for other forks and decreases catalytic efficiency. Central to the stimulation afforded by SSB is the unique ability of PriA to bind with high affinity to the 3’-OH placed at the end of the nascent leading strand at the fork. When both the 3’-OH and SSB are present, the maximum effect is observed. This ensures that PriA will only load onto the correct fork, in the right orientation, thereby ensuring that replication restart is directed to only the template lagging strand.

Single-molecule studies involving DNA or RNA, require homogeneous preparations of nucleic acid substrates of exceptional quality. Over the past several years, a variety of methods have been published describing different purification methods but these are frustratingly inconsistent with variable yields even in the hands of experienced bench scientists. To address these issues, we present an optimized and straightforward, column-based approach that is reproducible and produces high yields of substrates or substrate components of exceptional quality. Central to the success of the method presented is the use of a non-porous anion exchange resin. In addition to the use of this resin, we encourage the optimization of each step in the construction of substrates. The fully optimized method produces high yields of a hairpin DNA substrate of exceptional quality. While this substrate is suitable for single-molecule, magnetic tweezer experiments, the described method is readily adaptable to the production of DNA substrates for the majority of single-molecule studies involving nucleic acids ranging in size from 70–15000 bp.

Nonunion of fractures about the proximal humerus represents a rare and complex problem. The purpose of this study was to evaluate the clinical and functional outcomes following proximal humerus fracture (PHF) nonunion repair using a plate and screw construct with a direct comparison to those experienced following operative fixation of acute PHF using a plate and screw construct.Two separate patient cohorts were included in this study. The first consisted of 16 patients diagnosed with a non-united PHF who underwent operative nonunion repair treated with a standard algorithmic approach. The comparison group consisted of 173 patients who achieved union following initial open reduction and internal fixation of an acute PHF treated with a proximal humerus locking plate construct. All patients had at least 12 months of postoperative follow-up. Outcomes were assessed for both groups using American Shoulder and Elbow Surgeons (ASES) scores, visual analog scale (VAS) pain scores, and postoperative shoulder range of motion (ROM). Statistical analyses were used to compare these outcome measures between the two cohorts.The nonunion repair cohort consisted of eleven surgical neck nonunions and five nonunions of both the surgical neck and greater tuberosity. Ten patients had undergone surgical treatment for their original fracture, while six were initially treated non-operatively. All patients had Boileau type 3 sequelae of their proximal humerus fracture. Union was achieved in all patients at a mean of 5.4 months following nonunion repair. Complications included hardware failure requiring revision in two patients (12.5%) and avascular necrosis requiring conversion to anatomic total shoulder arthroplasty following union in one patient (6.3%). The nonunion repair and acute fracture cohorts did not differ with respect to mean ASES scores, VAS pain scores, or active shoulder ROM at any postoperative time point.Surgical repair of PHF nonunion is a viable treatment strategy that can lead to consistent bony healing with outcomes comparable to those of patients who achieve fracture union following initial surgical repair of an acute proximal humerus fracture. Surgeons should be cognizant of mechanical considerations that may lead to early failure.

In Escherichia coli, DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so that repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls helicase-fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.

In Escherichia coli, DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so that repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls helicase-fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart.

In bacteria, the restart of stalled DNA replication forks requires the PriA DNA helicase. PriA recognizes and remodels abandoned DNA replication forks performing the DNA unwinding in 3’ to 5’-direction and facilitates loading of the DnaB helicase onto the DNA to restart replication. The single stranded DNA binding protein (SSB) is typically present at the abandoned forks, but there is gap in the knowledge on the interaction between SSB and PriA protein. Here, we used atomic force microscopy (AFM) to visualize the interaction of PriA with DNA substrates in the absence or presence of SSB. Results show that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Preferential binding occurs only on forked DNA structures as 5’- and 3’-tailed duplexes were bound equally well. Furthermore, in the absence of SSB, PriA bound exclusively to the fork regions of substrates. In contrast, fork bound SSB loads PriA onto the duplex DNA arms of forks. When the fork has a gap in the leading strand, PriA localizes to both the parental and lagging strand arms. When the gap is present in the lagging strand, PriA is loaded preferentially onto the leading strand arm of the fork. Remodeling of PriA requires a functional C-terminal domain of SSB.

Limitations of conventional treatment and dosage forms prompted the investigation of novel approaches that combine efficacy, selectivity and fewer adverse effects. These are the main promises of nanomedicine, generally defined as the application of nanotechnology to the biomedical field. Despite the considerable advances over the years and the large number of publications resulting from the growth of nanomedicine, there are still many hurdles and unknown factors limiting its successful translation from promise to reality. These factors range from the lack of standardization in the terminology and regulations, to the lack of understanding of interactions between nanocarriers and the biological system, and their influence on drug pharmacodynamics. In this manuscript, we aim at presenting and discussing concepts and terminologies used in the nanomedicine field and examining the main types of nanocarriers and their effects to modulate biological barriers and aid drug transport, bioavailability and targeting.

AIM To examine whether wearable cardioverter defibrillator (WCD) alarms for asystole improve patient outcomes and survival. METHODS All asystole episodes recorded by the WCD in 2013 were retrospectively analyzed from a database of device and medical record documentation and customer call reports. Events were classified as asystole episodes if initial presenting arrhythmia was asystole (< 10 beats/minor ≥ 5 s pause). Survival was defined as recovery at the scene or arrival to a medical facility alive, or not requiring immediate medical attention. Episodes occurring in hospitals, nursing homes, or ambulances were considered to be under medical care. Serious asystole episodes were defined as resulting in unconsciousness, hospital transfer, or death. RESULTS Of the total 51933 patients having worn the WCD in 2013, there were 257 patients (0.5%) who had asystole episodes and comprised the study cohort. Among the 257 patients (74% male, median age 69 years), there were 264 asystole episodes. Overall patient survival was 42%. Most asystoles were considered “serious” (n = 201 in 201 patients, 76%), with a 26% survival rate. All 56 patients with “non-serious” asystole episodes survived. Being under medical care was associated with worse survival of serious asystoles. Among acute survivors, 20% later died during WCD use (a median 4 days post asystole episode). Of the 86 living patients at the end of WCD use period, 48 (56%) received ICD/pacemaker and 17 (20%) improved their condition. CONCLUSION Survival rates after asystole in patients with WCD are higher than historically reported survival rates. Those under medical care at time of asystole exhibited lower survival.

Cancer is the disease with highest public health impact in developed countries. Particularly, breast cancer has the highest incidence in women worldwide and the fifth highest mortality in the globe, imposing a significant social and economic burden to society. The disease has a complex heterogeneous etiology, being associated with several risk factors that range from lifestyle to age and family history. Breast cancer is usually classified according to the site of tumor occurrence and gene expression profiling. Although mutations in a few key genes, such as BRCA1 and BRCA2, are associated with high breast cancer risk, the large majority of breast cancer cases are related to mutated genes of low penetrance, which are frequently altered in the whole population. Therefore, understanding the molecular basis of breast cancer, including the several deregulated genes and related pathways linked to this pathology, is essential to ensure advances in early tumor detection and prevention. In this review, we outline key cellular pathways whose deregulation has been associated with breast cancer, leading to alterations in cell proliferation, apoptosis, and the delicate hormonal balance of breast tissue cells. Therefore, here we describe some potential breast cancer-related nodes and signaling concepts linked to the disease, which can be positively translated into novel therapeutic approaches and predictive biomarkers.

The E. coli single stranded DNA binding protein (SSB) is essential to all aspects of DNA metabolism. Here, it has two seemingly disparate but equally important roles: it binds rapidly and cooperatively to single stranded DNA (ssDNA) and it binds to partner proteins that constitute the SSB interactome. These two roles are not disparate but are instead, intimately linked. A model is presented wherein the intrinsically disordered linker (IDL) is directly responsible for mediating protein-protein interactions. It does this by binding, via PXXP motifs, to the OB-fold (aka SH3 domain) of a nearby protein. When the nearby protein is another SSB tetramer, this leads to a highly efficient ssDNA binding reaction that rapidly and cooperatively covers and protects the exposed nucleic acid from degradation. Alternatively, when the nearby protein is a member of the SSB interactome, loading of the enzyme onto the DNA takes places.

In E. coli, the regression of stalled DNA replication forks is catalyzed by the DNA helicase RecG. One means of gaining access to the fork is by binding to the single strand binding protein or SSB. This interaction occurs via the wedge domain of RecG and the intrinsically disordered linker (IDL) of SSB, in a manner similar to that of SH3 domains binding to PXXP motif-containing ligands in eukaryotic cells. During loading, SSB remodels the wedge domain so that the helicase domains bind to the parental, duplex DNA, permitting the helicase to translocate using thermal energy. This translocation may be used to clear the fork of obstacles, prior to the initiation of fork regression.

The E. coli single strand DNA binding protein (SSB) is essential to viability where it functions in two seemingly disparate roles: it binds to single stranded DNA (ssDNA) and to target proteins that comprise the SSB interactome. The link between these roles resides in a previously under-appreciated region of the protein known as the intrinsically disordered linker (IDL). We present a model wherein the IDL is responsible for mediating protein-protein interactions critical to each role. When interactions occur between SSB tetramers, cooperative binding to ssDNA results. When binding occurs between SSB and an interactome partner, storage or loading of that protein onto the DNA takes place. The properties of the IDL that facilitate these interactions include the presence of repeats, a putative polyproline type II helix and, PXXP motifs that may facilitate direct binding to the OB-fold in a manner similar to that observed for SH3 domain binding of PXXP ligands in eukaryotic systems.

Various optical instruments have been developed for three-dimensional (3D) surface topography, including the white light interference, reflectance confocal microscopes, and digital holographic microscopes, etc. However, the steep local slope of objects may cause the light to be reflected in a way that it will not be captured by the objective lens because of the finite collection angle of the objective. To solve this "shadow problem," we report a method to enlarge the collection angle range of optical sectioning structured illumination microscopy by capturing sectioned images of the objects from multiple angle of views. We develop a multi-view image fusion algorithm to reconstruct a single 3D image. Using this method, we detect previously invisible details whose slopes are beyond the collection angle of the objective. The proposed approach is useful for height map measurement and quantitative analyses in a variety of fields, such as biology, materials science, microelectronics, etc.

TheE. colisingle stranded DNA binding protein (SSB) is essential to viability. It plays key roles in DNA metabolism where it binds to nascent single strands of DNA and to target proteins known as the SSB interactome. There are >2,000 tetramers of SSB per cell with perhaps 100-150 associated with genome at any one time, either at DNA replication forks or at sites of DNA repair. The remaining 1,900 tetramers could constantly diffuse throughout the cytosol or be associated with the inner membrane as observed for other DNA metabolic enzymes such as DnaA and RecA. To visualize SSB directly and to ascertain spatiotemporal changes in tetramer localization in response to DNA damage, SSB-GFP chimeras were visualized using a novel, super-resolution microscope optimized for visualization of prokaryotic cells. Results show that in the absence of DNA damage, SSB localizes to a small number of foci and the excess protein is observed associated with the inner membrane where it binds to the major phospholipids. Within five minutes following DNA damage, the vast majority of SSB disengages from the membrane and is found almost exclusively in the cell interior. Here, it is observed in a large number of foci, in discreet structures or, in diffuse form spread over the genome, thereby enabling repair events. In the process, it may also deliver interactome partners such as RecG or PriA to sites where their repair functions are required.

The E. coli single-stranded DNA-binding protein (SSB) binds to the fork DNA helicases RecG and PriA in vitro. Typically for binding to occur, 1.3 M ammonium sulfate must be present, bringing into question the validity of these data as these are non-physiological conditions. To determine whether SSB can bind to these helicases, we examined binding in vivo. First, using fluorescence microscopy, we show that SSB localizes PriA and RecG to the vicinity of the inner membrane in the absence of DNA damage. Localization requires that SSB be in excess over the DNA helicases and the SSB C-terminus and both PriA and RecG be present. Second, using purification of tagged complexes, our results demonstrate that SSB binds to PriA and RecG in vivo, in the absence of DNA. We propose that this may be the “storage form” of RecG and PriA. We further propose that when forks stall, RecG and PriA are targeted to the fork by SSB which, by virtue of its high affinity for single stranded DNA, allows these helicases to out compete other proteins. This ensures their actions in the early stages of the rescue of stalled replication forks.

PURPOSE The ability to predict contracture development after elbow fracture would benefit patients and physicians. This study aimed to identify factors associated with the development of posttraumatic elbow joint contracture. METHODS A retrospective review of elbow fractures (AO/Orthopaedic Trauma Association [OTA] type 13 and 21) treated at one institution between 2011 and 2015 was performed. Data collected included demographics, injury information, treatment, and postoperative elbow range of motion (ROM). Multivariate logistic regression analyses were performed to identify factors associated with contracture development. Notable contracture was defined as an arc of motion less than 100° flexion/extension, which has been associated with reduced ability to perform activities of daily living. RESULTS A total of 278 patients at least 1 8 years of age underwent surgical repair of an elbow fracture or fracture-dislocation during the study period and had at least 6 months of postoperative follow-up. Forty-two (15.1%) developed a clinically notable elbow contracture, of whom 29 (69.0%) developed heterotopic ossification (HO). Multivariate analysis of preoperative variables demonstrated that AO/OTA 13-C fracture classification (odds ratio [OR], 13.7, P = 0.025), multiple noncontiguous fractures (OR, 3.7, P = 0.010), and ulnohumeral dislocation at the time of injury (OR, 4.9, P = 0.005) were independently associated with contracture development. At 6 weeks postoperatively, an arc of elbow ROM less than 50° flexion/extension (OR, 23.0, P < 0.0005) and the presence of HO on radiographs (OR, 6.7, P < 0.0005) were found to be independent risk factors for significant elbow stiffness. DISCUSSION Ulnohumeral dislocation, multiple noncontiguous fractures, AO/OTA 13-C fracture classification, limited elbow ROM at 6 weeks postoperatively, and the presence of radiographic HO at 6 weeks postoperatively are associated with contracture development after surgical elbow fracture repair. Patients with these risk factors should receive aggressive physical therapy and be counseled as to the possible development of a contracture requiring surgical intervention.

The Rep and UvrD DNA helicases are proposed to act at stalled DNA replication forks to facilitate replication restart when RNA polymerase stalls forks. To clarify the role of these DNA helicases in fork rescue, we used a coupled spectrophotometric ATPase assay to determine how they act on model fork substrates. For both enzymes, activity is low on regressed fork structures, suggesting that they act prior to the regression step that generates a Holliday junction. In fact, the preferred cofactors for both enzymes are forks with a gap in the nascent leading strand, consistent with the 3′–5′ direction of translocation. Surprisingly, for Rep, this specificity is altered in the presence of stoichiometric amounts of a single-strand DNA-binding protein (SSB) relative to a fork with a gap in the nascent lagging strand. Even though Rep and UvrD are similar in structure, elevated concentrations of SSB inhibit Rep, but they have little to no effect on UvrD. Furthermore, Rep and UvrD antagonize one another at a fork. This is surprising given that these helicases have been shown to form a heterodimer and are proposed to act together to rescue an RNA polymerase-stalled fork. Consequently, the results herein indicate that although Rep and UvrD can act on similar fork substrates, they cannot function on the same fork simultaneously.

Background: Certain pediatric patients are at risk for sudden cardiac death. The wearable cardioverter-defibrillator (WCD) can be used in clinical situations in which implantable cardioverter-defibrillator placement is not ideal. The objectives of the study are to examine the effectiveness, safety, and compliance of the WCD in the identification and treatment of life-threatening ventricular arrhythmias in pediatric patients. Methods: All United States pediatric patients Results: In total, 455 patients were identified. The median age was 15 (3–17) years, median duration of WCD use was 33 (1–999) days and median patient wear time was 20.6 (0.3–23.8) hours per day. The population was divided into 2 groups: (1) patients with implantable cardioverter-defibrillator problem, n=63 and (2) patients with nonimplantable cardioverter-defibrillator problem, n=392. Wear time per day was >20 hours in both groups. Wear duration was shorter in the implantable cardioverter-defibrillator problem group, 26 days versus 35 days, P Conclusions: The WCD has overall adequate compliance with appropriate wear times and wear durations in pediatric patients. The WCD is safe and effective in treating ventricular arrhythmias that can lead to sudden cardiac death in pediatric patients.