Subunit Communication within Dimeric SF1 DNA Helicases.

Monomers of the Superfamily (SF) 1 helicases, E. coli Rep and UvrD, can translocate directionally along single stranded (ss) DNA, but must be activated to function as helicases. In the absence of accessory factors, helicase activity requires Rep and UvrD homo-dimerization. The ssDNA binding sites of SF1 helicases contain a conserved aromatic amino acid (Trp250 in Rep and Trp256 in UvrD) that stacks with the DNA bases. Here we show that mutation of this Trp to Ala eliminates helicase activity in both Rep and UvrD. Rep(W250A) and UvrD(W256A) can still dimerize, bind DNA, and monomers still retain ATP-dependent ssDNA translocase activity, although with ∼10-fold lower rates and lower processivities than wild type monomers. Although neither wtRep monomers nor Rep(W250A) monomers possess helicase activity by themselves, using both ensemble and single molecule methods, we show that helicase activity is achieved upon formation of a Rep(W250A)/wtRep hetero-dimer. An ATPase deficient Rep monomer is unable to activate a wtRep monomer indicating that ATPase activity is needed in both subunits of the Rep hetero-dimer. We find the same results with E. coli UvrD and its equivalent mutant (UvrD(W256A)). Importantly, Rep(W250A) is unable to activate a wtUvrD monomer and UvrD(W256A) is unable to activate a wtRep monomer indicating that specific dimer interactions are required for helicase activity. We also demonstrate subunit communication within the dimer by virtue of Trp fluorescence signals that only are present within the Rep dimer, but not the monomers. These results bear on proposed subunit switching mechanisms for dimeric helicase activity.

A Model for Competency-Based Grading and Its Effect on Student Outcomes in a Biomechanics Course.

Competency-based grading (CBG) can take different forms in different subject areas. We present a method for implementing CBG in a biomechanics course with nine primary learning objectives. Competency in each learning objective is measured by the student's ability to correctly answer knowledge questions and solve analytical problems in the field of biomechanics. The primary goal of implementing CBG was to provide more opportunities for lower-performing students to learn the material and to demonstrate that learning. To determine the efficacy of CBG to improve student learning, the primary measure was course grade distribution before and after implementation of CBG. The course grade distribution data indicated that CBG has primarily helped midperforming students to improve their grades. Because of the limitations of course grades as a measure of learning, we also performed analysis of student performance on successive attempts which indicated initial and secondary attempts are best, with student success declining on subsequent attempts. Anecdotally, many students improved performance, and thus their grade, on the (optional) final exam attempts. Limitations of the study include the limited course offerings with CBG (three), and that effects of COVID-19 may be confounding CBG data. Also, the approach places nearly all the grade on quizzes or exams. However, the approach could be modified to include homework grades, projects, and the like. Overall, the student learning in this course and implementation appears to be only positively affected, so this approach appears to have benefits in a biomechanics course.

Safety Factors and Patterns of Bicycle Trauma in a Tertiary Level 1 Trauma Center in Boston, Massachusetts: A Retrospective Review and Survey Study.

Objectives: Increasing bicycle ridership is accompanied by ongoing bicycle-related accidents in many urban cities. There is a need for improved understanding of patterns and risks of urban bicycle usage. We describe the injuries and outcomes of bicycle-related trauma in Boston, Massachusetts, and determine accident-related factors and behaviors associated with injury severity. Methods: We conducted a retrospective review via chart review of 313 bicycle-related injuries presenting to a Level 1 trauma center in Boston, Massachusetts. These patients were also surveyed regarding accident-related factors, personal safety practices, and road and environmental conditions during the accident. Results: Over half of all cyclists biked for commuting and recreational purposes (54%), used a road without a bike lane (58%), and a majority wore a helmet (91%). The most common injury pattern involved the extremities (42%) followed by head injuries (13%). Bicycling for commuting rather than recreation, cycling on a road with a dedicated bicycle lane, the absence of gravel or sand, and use of bicycle lights were all factors associated with decreased injury severity (p<0.05). After any bicycle injury, the number of miles cycled decreased significantly regardless of cycling purpose. Conclusion: Our results suggest that physical separation of cyclists from motor vehicles via bicycle lanes, regular cleaning of these lanes, and usage of bicycle lights are modifiable factors protective against injury and injury severity. Safe bicycling practices and understanding of factors involved in bicycle-related trauma can reduce injury severity and guide effective public health initiatives and urban planning.

Kinetics of ATP/ADP binding to the gp16 ATPase.

The gp16 ATPase is the constituent subunit of the pentameric dsDNA (double-stranded deoxyribonucleic acid) translocation motor of the Bacillus subtilis Φ29 bacteriophage. Although recent single-molecule studies have provided tantalizing clues about the activity of this motor, the mechanism by which the gp16 subunits couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of dsDNA translocation remains unknown. To address this need, we have characterized the binding of fluorophore-labeled ATP and ADP to monomeric gp16 using a stopped-flow fluorescence assay. These experiments show that the binding of ATP/ADP occurs through a single-step mechanism with corresponding affinities of 523.8 ± 247.3 nM for ATP and a lower limit of 30 µM for ADP. When analyzed through the lens of changes in free energy of the system, this difference in binding affinities is reasonable for a cyclical process of binding, hydrolysis, and product release. In addition to answering questions about the activity of monomeric gp16, these results are also a necessary step in constructing a model for intersubunit communication within the pentameric gp16 motor.

Analysis of the Fire Behavior of Polymers (PP, PA 6 and PE-LD) and Their Improvement Using Various Flame Retardants.

The fire behavior of polymers is examined primarily with the time-dependent heat release rate (HRR) measured with a cone calorimeter. The HRR is used to examine the fire behavior of materials with and without flame retardants, especially Polypropylene (PP-Copo) and Polyethylene (PE-LD). Polypropylene is stored for up to 99 days under normal conditions and the heat release rate shows especially changes about 100 s after irradiation with cone calorimeter, which may be caused by aging effects. The effect of crosslinking to the burning behavior of PP was examined too. Polyamides (PA 6) are irradiated with a radiation intensity of 25 kW/m2 to 95 kW/m2 and fire-related principles between radiation intensity and time to ignition can be derived from the measurement results. In order to comprehensively investigate the fire behavior of PP (also with flame retardant additives), the samples were also exposed to a flame, according to UL 94 with small power (50 W) and is inflamed with the power of a few 100 W. The irradiation causes different trigger mechanisms for the flame retardant additives in a plastic than the flame exposure. It is shown that the compound, which is favorable for irradiation, is not necessarily good for flame exposure. It can be seen that expandable graphite alone or with the addition of other additives is a very effective flame retardant for PP.

Remodeler Catalyzed Nucleosome Repositioning: Influence of Structure and Stability.

The packaging of the eukaryotic genome into chromatin regulates the storage of genetic information, including the access of the cell's DNA metabolism machinery. Indeed, since the processes of DNA replication, translation, and repair require access to the underlying DNA, several mechanisms, both active and passive, have evolved by which chromatin structure can be regulated and modified. One mechanism relies upon the function of chromatin remodeling enzymes which couple the free energy obtained from the binding and hydrolysis of ATP to the mechanical work of repositioning and rearranging nucleosomes. Here, we review recent work on the nucleosome mobilization activity of this essential family of molecular machines.

Movement patterns and habitat use of tiger sharks (Galeocerdo cuvier) across ontogeny in the Gulf of Mexico.

The tiger shark (Galeocerdo cuvier) is globally distributed with established coastal and open-ocean movement patterns in many portions of its range. While all life stages of tiger sharks are known to occur in the Gulf of Mexico (GoM), variability in habitat use and movement patterns over ontogeny have never been quantified in this large marine ecosystem. To address this data gap we fitted 56 tiger sharks with Smart Position and Temperature transmitting tags between 2010 and 2018 and examined seasonal and spatial distribution patterns across the GoM. Additionally, we analyzed overlap of core habitats (i.e., 50% kernel density estimates) among individuals relative to large benthic features (oil and gas platforms, natural banks, bathymetric breaks). Our analyses revealed significant ontogenetic and seasonal differences in distribution patterns as well as across-shelf (i.e., regional) and sex-linked variability in movement rates. Presumably sub-adult and adult sharks achieved significantly higher movement rates and used off-shelf deeper habitats at greater proportions than juvenile sharks, particularly during the fall and winter seasons. Further, female maximum rate of movement was higher than males when accounting for size. Additionally, we found evidence of core regions encompassing the National Oceanographic and Atmospheric Administration designated Habitat Areas of Particular Concern (i.e., shelf-edge banks) during cooler months, particularly by females, as well as 2,504 oil and gas platforms. These data provide a baseline for future assessments of environmental impacts, such as climate variability or oil spills, on tiger shark movements and distribution in the region. Future research may benefit from combining alternative tracking tools, such as acoustic telemetry and genetic approaches, which can facilitate long-term assessment of the species' movement dynamics and better elucidate the ecological significance of the core habitats identified here.

Safety and Efficacy of Pembrolizumab With Chemoradiotherapy in Locally Advanced Head and Neck Squamous Cell Carcinoma: A Phase IB Study.

PURPOSE: Pembrolizumab is a humanized monoclonal antibody that blocks interaction between programmed death receptor-1 (PD-1) and its ligands (PD-L1, PD-L2). Although pembrolizumab is approved for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), its role in the management of locally advanced (LA) disease is not defined. We report a phase IB study evaluating the safety and efficacy of adding pembrolizumab to cisplatin-based chemoradiotherapy in patients with LA HNSCC. PATIENTS AND METHODS: Eligible patients included those with oral cavity (excluding lip), oropharyngeal, hypopharyngeal, or laryngeal stage III to IVB HNSCC (according to American Joint Committee on Cancer, 7th edition, staging system) eligible for cisplatin-based, standard-dose (70 Gy) chemoradiotherapy. Pembrolizumab was administered concurrently with and after chemoradiotherapy with weekly cisplatin. Safety was the primary end point and was determined by incidence of chemoradiotherapy adverse events (AEs) and immune-related AEs (irAEs). Efficacy was defined as complete response (CR) rate on end-of-treatment (EOT) imaging or with pathologic confirmation at 100 days postradiotherapy completion. Key secondary end points included overall (OS) and progression-free survival (PFS). RESULTS: The study accrued 59 patients (human papillomavirus [HPV] positive, n = 34; HPV negative, n = 25) from November 2015 to October 2018. Five patients (8.8%) required discontinuation of pembrolizumab because of irAEs, all of which occurred during concurrent chemoradiotherapy; 98.3% of patients completed the full planned treatment dose (70 Gy) of radiotherapy without any delays ≥ 5 days; 88.1% of patients completed the goal cisplatin dose of ≥ 200 mg/m2. EOT CR rates were 85.3% and 78.3% for those with HPV-positive and -negative HNSCC, respectively. CONCLUSION: Pembrolizumab in combination with weekly cisplatin-based chemoradiotherapy is safe and does not impair delivery of curative radiotherapy or chemotherapy in HNSCC. Early efficacy data support further investigation of this approach.

Anisotropy-Based Nucleosome Repositioning Assay.

Most eukaryotic DNA is tightly packaged into nucleosomes that render these sequences largely inaccessible for transcription or repair. Molecular motors called chromatin remodelers use an ATP-dependent mechanism to relieve the inhibition of these processes by sliding or disassembling the nucleosomes. This allows them to serve an essential role in the regulation of gene expression and genomic integrity. The sliding of nucleosomes along DNA can be studied directly by monitoring the associated changes in the fluorescence anisotropy of fluorophores attached to the ends of the DNA. Nucleosome repositioning can also be monitored indirectly through the ATP hydrolysis of the chromatin remodeler during the sliding reaction. Here we discuss how the kinetic data collected in these experiments can be analyzed by simultaneous global nonlinear least squares (NLLS) analysis using simple sequential "n-step" mechanisms to obtain estimates of the macroscopic rate of nucleosome repositioning and of the stoichiometry of coupling ATP binding and hydrolysis to this reaction.

First insights into the movements of young-of-the-year white sharks (Carcharodon carcharias) in the western North Atlantic Ocean.

In recent years, white sharks (Carcharodon carcharias) have become more accessible to researchers off the northeastern U.S. as feeding aggregation sites have emerged and the population has increased. However, there has been limited research on young-of-the-year (YOY) sharks relative to older age classes in this region. Previous research indicated that YOY white sharks were most frequently observed in the New York Bight, suggesting the region serves a nursery role. To further examine the species' use of this area, we deployed satellite and acoustic tags on ten YOY white sharks (138-166 cm total length) off Long Island, New York. The sharks remained resident in New York Bight waters through summer (August through October), further supporting the notion that the region is a nursery area. Southward movements were observed during fall, with overwintering habitat identified off North and South Carolina shelf waters. Return migrations toward the New York Bight were observed in some individuals the following spring. YOY white sharks in this heavily-populated region are exposed to anthropogenic impacts such as fisheries bycatch and coastal habitat degradation. As juvenile survival rates are important for long-term population sustainability, further research is necessary to assess the potential impacts of these activities on the western North Atlantic white shark population.

Effects of nucleosome stability on remodeler-catalyzed repositioning.

Chromatin remodelers are molecular motors that play essential roles in the regulation of nucleosome positioning and chromatin accessibility. These machines couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of manipulating chromatin structure through processes that are not completely understood. Here we present a quantitative analysis of nucleosome repositioning by the imitation switch (ISWI) chromatin remodeler and demonstrate that nucleosome stability significantly impacts the observed activity. We show how DNA damage induced changes in the affinity of DNA wrapping within the nucleosome can affect ISWI repositioning activity and demonstrate how assay-dependent limitations can bias studies of nucleosome repositioning. Together, these results also suggest that some of the diversity seen in chromatin remodeler activity can be attributed to the variations in the thermodynamics of interactions between the remodeler, the histones, and the DNA, rather than reflect inherent properties of the remodeler itself.

Molecular motor translocation kinetics: Application of Monte Carlo computer simulations to determine microscopic kinetic parameters.

Methods for studying the translocation of motor proteins along a filament (e.g., nucleic acid and polypeptide) typically monitor the total production of ADP, the arrival/departure of the motor protein at/from a particular location (often one end of the filament), or the dissociation of the motor protein from the filament. The associated kinetic time courses are often analyzed using a simple sequential uniform n-step mechanism to estimate the macroscopic kinetic parameters (e.g., translocation rate and processivity) and the microscopic kinetic parameters (e.g., kinetic step-size and the rate constant for the rate-limiting step). These sequential uniform n-step mechanisms assume repetition of uniform and irreversible rate-limiting steps of forward motion along the filament. In order to determine how the presence of non-uniform motion (e.g., backward motion, random pauses, or jumping) affects the estimates of parameters obtained from such analyses, we evaluated computer simulated translocation time courses containing non-uniform motion using a simple sequential uniform n-step model. By comparing the kinetic parameters estimated from the analysis of the data generated by these simulations with the input parameters of the simulations, we were able to determine which of the kinetic parameters were likely to be over/under estimated due to non-uniform motion of the motor protein.

Factitious Disorder in a Patient Claiming to be a Sexually Sadistic Serial Killer.

Factitious disorder involves the conscious simulation of psychological or physiological symptoms of illness, for the purpose of fulfilling the unconscious desire to be taken care of or to assume the "sick role." Typically patients with factitious disorder simulate conditions that are designed to arouse feelings of empathy in care providers with the intention to engage them in caretaking. However, patients might also simulate conditions that arouse revulsion or rejection and still meet full diagnostic criteria for factitious disorder. In this case report, we present a patient who fabricated an elaborate history of being a sexually sadistic serial killer with homicidal ideation with the intention of obtaining personal attention, nurturance, and empathy from his psychotherapist. However, given the nature of his feigned condition, the patient frightened the very person whom he sought to engage in caretaking.

Mitotic-dependent phosphorylation of leukemia-associated RhoGEF (LARG) by Cdk1.

Rho GTPases are integral to the regulation of actin cytoskeleton-dependent processes, including mitosis. Rho and leukemia-associated Rho guanine-nucleotide exchange factor (LARG), also known as ARHGEF12, are involved in mitosis as well as diseases such as cancer and heart disease. Since LARG has a role in mitosis and diverse signaling functions beyond mitosis, it is important to understand the regulation of the protein through modifications such as phosphorylation. Here we report that LARG undergoes a mitotic-dependent and cyclin-dependent kinase 1 (Cdk1) inhibitor-sensitive phosphorylation. Additionally, LARG is phosphorylated at the onset of mitosis and dephosphorylated as cells exit mitosis, concomitant with Cdk1 activity. Furthermore, using an in vitro kinase assay, we show that LARG can be directly phosphorylated by Cdk1. Through expression of phosphonull mutants that contain non-phosphorylatable alanine mutations at potential Cdk1 S/TP sites, we demonstrate that LARG phosphorylation occurs in both termini. Using phosphospecific antibodies, we confirm that two sites, serine 190 and serine 1176, are phosphorylated during mitosis in a Cdk1-dependent manner. In addition, these phosphospecific antibodies show phosphorylated LARG at specific mitotic locations, namely the mitotic organizing centers and flanking the midbody. Lastly, RhoA activity assays reveal that phosphonull LARG is more active in cells than phosphomimetic LARG. Our data thus identifies LARG as a phosphoregulated RhoGEF during mitosis.

A 3D paper-based enzymatic fuel cell for self-powered, low-cost glucose monitoring.

In this work, we demonstrate a novel low-cost, self-powered paper-based biosensor for glucose monitoring. The device operating mechanism is based on a glucose/oxygen enzymatic fuel cell using an electrochemical energy conversion as a transducing element for glucose monitoring. The self-powered glucose biosensor features (i) a 3D origami paper-based structure for easy system integration onto paper, (ii) an air-cathode on paper for low-cost production and easy operation, and (iii) a screen printed chitosan/glucose oxidase anode for stable current generation as an analytical signal for glucose monitoring. The sensor showed a linear range of output current at 1-5mM glucose (R(2)=0.996) with a sensitivity of 0.02 µA mM(-1). The advantages offered by such a device, including a low cost, lack of external power sources/sophisticated external transducers, and the capacity to rapidly generate reliable results, are well suited for the clinical and social settings of the developing world.

Genome-wide association study of colorectal cancer identifies six new susceptibility loci.

Genetic susceptibility to colorectal cancer is caused by rare pathogenic mutations and common genetic variants that contribute to familial risk. Here we report the results of a two-stage association study with 18,299 cases of colorectal cancer and 19,656 controls, with follow-up of the most statistically significant genetic loci in 4,725 cases and 9,969 controls from two Asian consortia. We describe six new susceptibility loci reaching a genome-wide threshold of P<5.0E-08. These findings provide additional insight into the underlying biological mechanisms of colorectal cancer and demonstrate the scientific value of large consortia-based genetic epidemiology studies.

Low processivity for DNA translocation by the ISWI molecular motor.

The motor protein ISWI (Imitation SWItch) is the conserved catalytic ATPase domain of the ISWI family of chromatin remodelers. Members of the ISWI family are involved in regulating the structure of cellular chromatin during times of transcription, translation, and repair. Current models for the nucleosome repositioning activity of ISWI and other chromatin remodelers require the translocation of the remodeling protein along double-stranded DNA through an ATP-dependent mechanism. Here we report results from spectrofluorometric stopped-flow experiments which demonstrate that ISWI displays very low processivity for free DNA translocation. By combining these results with those from experiments monitoring the DNA stimulated ATPase activity of ISWI we further demonstrate that the DNA translocation by ISWI is tightly coupled to ATP hydrolysis. The calculated coupling efficiency of 0.067±0.018 ATP/ISWI/bp is seemingly quite low in comparison to similar DNA translocases and we present potential models to account for this. Nevertheless, the tight coupling of ATP hydrolysis to DNA translocation suggests that DNA translocation is not energetically rate limiting for nucleosome repositioning by ISWI.

Trapping of intermediates with substrate analog HBOCoA in the polymerizations catalyzed by class III polyhydroxybutyrate (PHB) synthase from Allochromatium vinosum.

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the formation of biodegradable PHB polymers that are considered as an ideal alternative to petroleum-based plastics. To provide strong evidence for the preferred mechanistic model involving covalent and noncovalent intermediates, a substrate analog HBOCoA was synthesized chemoenzymatically. Substitution of sulfur in the native substrate HBCoA with an oxygen in HBOCoA enabled detection of (HB)nOCoA (n = 2-6) intermediates when the polymerization was catalyzed by wild-type (wt-)PhaECAv at 5.84 h(-1). This extremely slow rate is due to thermodynamically unfavorable steps that involve the formation of enzyme-bound PHB species (thioesters) from corresponding CoA oxoesters. Synthesized standards (HB)nOCoA (n = 2-3) were found to undergo both reacylation and hydrolysis catalyzed by the synthase. Distribution of the hydrolysis products highlights the importance of the penultimate ester group as previously suggested. Importantly, the reaction between primed synthase [(3)H]-sT-PhaECAv and HBOCoA yielded [(3)H]-sTet-O-CoA at a rate constant faster than 17.4 s(-1), which represents the first example that a substrate analog undergoes PHB chain elongation at a rate close to that of the native substrate (65.0 s(-1)). Therefore, for the first time with a wt-synthase, strong evidence was obtained to support our favored PHB chain elongation model.