Structural basis for allosteric regulation of human phosphofructokinase-1.

Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.

Joint contributions of preview and task instructions on visual search strategy selection.

People tend to employ suboptimal attention control strategies during visual search. Here we question why people are suboptimal, specifically investigating how knowledge of the optimal strategies and the time available to apply such strategies affect strategy use. We used the Adaptive Choice Visual Search (ACVS), a task designed to assess attentional control optimality. We used explicit strategy instructions to manipulate explicit strategy knowledge, and we used display previews to manipulate time to apply the strategies. In the first two experiments, the strategy instructions increased optimality. However, the preview manipulation did not significantly boost optimality for participants who did not receive strategy instruction. Finally, in Experiments 3A and 3B, we jointly manipulated preview and instruction with a larger sample size. Preview and instruction both produced significant main effects; furthermore, they interacted significantly, such that the beneficial effect of instructions emerged with greater preview time. Taken together, these results have important implications for understanding the strategic use of attentional control. Individuals with explicit knowledge of the optimal strategy are more likely to exploit relevant information in their visual environment, but only to the extent that they have the time to do so.

Cancer-associated somatic mutations in human phosphofructokinase-1 reveal a critical electrostatic interaction for allosteric regulation of enzyme activity.

Metabolic reprogramming, including increased glucose uptake and lactic acid excretion, is a hallmark of cancer. The glycolytic 'gatekeeper' enzyme phosphofructokinase-1 (PFK1), which catalyzes the step committing glucose to breakdown, is dysregulated in cancers. While altered PFK1 activity and expression in tumors have been demonstrated, little is known about the effects of cancer-associated somatic mutations. Somatic mutations in PFK1 inform our understanding of allosteric regulation by identifying key amino acid residues involved in the regulation of enzyme activity. Here, we characterized mutations disrupting an evolutionarily conserved salt bridge between aspartic acid and arginine in human platelet (PFKP) and liver (PFKL) isoforms. Using purified recombinant proteins, we showed that disruption of the Asp-Arg pair in two PFK1 isoforms decreased enzyme activity and altered allosteric regulation. We determined the crystal structure of PFK1 to 3.6 Šresolution and used molecular dynamic simulations to understand molecular mechanisms of altered allosteric regulation. We showed that PFKP-D564N had a decreased total system energy and changes in the electrostatic surface potential of the effector site. Cells expressing PFKP-D564N demonstrated a decreased rate of glycolysis, while their ability to induce glycolytic flux under conditions of low cellular energy was enhanced compared with cells expressing wild-type PFKP. Taken together, these results suggest that mutations in Arg-Asp pair at the interface of the catalytic-regulatory domains stabilizes the t-state and presents novel mechanistic insight for therapeutic development in cancer.

Definitions of Solitude in Everyday Life.

What does it mean to be in solitude? Researchers building this nascent field are learning much about the potential affordances of solitude, but lack an agreed-upon definition or set of definitions. Arriving at that meaning is crucial to forming a solid foundation for studies that use both naturalistic and laboratory designs to explore outcomes of solitude. This study identified themes from semi-structured interviews with adults aged 19 to 80 from diverse backgrounds. We concluded that solitude is a state in which the dominant relationship is with the self. If not physically alone, people in solitude are mentally distanced from others and away from active technology-mediated interactions. Complete solitude involves both physical separation and inner focus, but solitude is best defined through a taxonomy that recognizes physical separation and internal focus as independent, sufficient characteristics. An internal focus benefits from (but is not defined by) balancing solitude with social time, quiet, and choice.

Visual Joint Angle Assessment: Does Accuracy Improve with a Higher Level of Orthopaedic Surgery Training?

INTRODUCTION: The purpose of this study was to evaluate the accuracy of visual joint angle assessments by orthopaedic surgery trainees amongst various levels of training. METHODS: Sagittal plane photographs of several joints at various angles were distributed to trainees within an orthopaedic residency program. Joint angles were estimated and compared to those obtained with a goniometer. Inter-and intra-rater reliability and ANOVA were conducted to assess differences between groups. RESULTS: Twenty trainees were studied. The percent error for knee measurements differed at 23.1%, 26.2% and 11.1% for the PGY 2-3, PGY 4-5 and PGY 6 groups, respectively (P=0.024). Percentage error for ankles showed the greatest variability at 69.7-96.3%. Intra-rater reliabilities for all visual joint angle assessment were similar amongst groups. CONCLUSIONS: Visual joint angle assessments vary amongst trainees, with PGY 6s most accurately identifying knee joint angles. Visual assessment is inaccurate compared to goniometric measurements, thus limiting visual measurements during patient encounters.

Neural evidence for non-orofacial triggers in mild misophonia.

Misophonia, an extreme aversion to certain environmental sounds, is a highly prevalent yet understudied condition plaguing roughly 20% of the general population. Although neuroimaging research on misophonia is scant, recent work showing higher resting-state functional connectivity (rs-fMRI) between auditory cortex and orofacial motor cortex in misophonia vs. controls has led researchers to speculate that misophonia is caused by orofacial mirror neurons. Since orofacial motor cortex was defined using rs-fMRI, we attempted to theoretically replicate these findings using orofacial cortex defined by task-based fMRI instead. Further, given our recent work showing that a wide variety of sounds can be triggering (i.e., not just oral/nasal sounds), we investigated whether there is any neural evidence for misophonic aversion to non-orofacial stimuli. Sampling 19 adults with varying misophonia from the community, we collected resting state data and an fMRI task involving phoneme articulation and finger-tapping. We first defined "orofacial" cortex in each participant using rs-fMRI as done previously, producing what we call resting-state regions of interest (rsROIs). Additionally, we functionally defined regions (fROIs) representing "orofacial" or "finger" cortex using phoneme or finger-tapping activation from the fMRI task, respectively. To investigate the motor specificity of connectivity differences, we subdivided the rsROIs and fROIs into separate sensorimotor areas based on their overlap with two common atlases. We then calculated rs-fMRI between each rsROI/fROI and a priori non-sensorimotor ROIs. We found increased connectivity in mild misophonia between rsROIs and both auditory cortex and insula, theoretically replicating previous results, with differences extending across multiple sensorimotor regions. However, the orofacial task-based fROIs did not show this pattern, suggesting the "orofacial" cortex described previously was not capturing true orofacial cortex; in fact, using task-based fMRI evidence, we find no selectivity to orofacial action in these previously described "orofacial" regions. Instead, we observed higher connectivity between finger fROIs and insula in mild misophonia, demonstrating neural evidence for non-orofacial triggers. These results provide support for a neural representation of misophonia beyond merely an orofacial/motor origin, leading to important implications for the conceptualization and treatment of misophonia.

What Time Alone Offers: Narratives of Solitude From Adolescence to Older Adulthood.

Solitude - the state of being alone and not physically with another - can be rewarding. The present research explored the potential benefits of solitude from a pragmatist approach: a ground-up, top-down perspective that is receptive to new knowledge but informed by theory. Participant recruitment was stratified by age and gender, and the sample involved 2,035 individuals including adolescents (13-16 years), adults (35-55 years), or older adults (65+ years). Data were analyzed with a mixed-methods approach. Coded themes from brief narratives about solitude were extracted, and their frequencies (i.e., their salience to participants) were compared across the lifespan. Themes were then correlated with two indicators of well-being in solitude: self-determined motivation for solitude and peaceful mood. Several prominent themes emerged when talking about time spent in solitude. With the exception of feeling competent in solitude, which was described frequently but consistently unrelated to self-reported well-being regardless of age, benefits of solitude tended to shift over the lifespan. Some qualities, such as a sense of autonomy (self-connection and reliance; absence of pressure), were salient and consequential for everyone, but increasingly so from adolescence to older adulthood. Older adults also reported feeling most peaceful in solitude and described their social connection and alienation less frequently, suggesting they see solitude and social time as more distinct states. Findings are discussed in light of existing work on solitude across the lifespan, and theoretical frameworks that spoke well to the data (e.g., self-determination theory).

What sound sources trigger misophonia? Not just chewing and breathing.

OBJECTIVES: Misophonia is a highly prevalent yet understudied condition characterized by aversion toward particular environmental sounds. Oral/nasal sounds (e.g., chewing, breathing) have been the focus of research, but variable experiences warrant an objective investigation. Experiment 1 asked whether human-produced oral/nasal sounds were more aversive than human-produced nonoral/nasal sounds and non-human/nature sounds. Experiment 2 additionally asked whether machine-learning algorithms could predict the presence and severity of misophonia. METHOD: Sounds were presented to individuals with misophonia (Exp.1: N = 48, Exp.2: N = 45) and members of the general population (Exp.1: N = 39, Exp.2: N = 61). Aversiveness ratings to each sound were self-reported. RESULTS: Sounds from all three source categories-not just oral/nasal sounds-were rated as significantly more aversive to individuals with misophonia than controls. Further, modeling all sources classified misophonia with 89% accuracy and significantly predicted misophonia severity (r = 0.75). CONCLUSIONS: Misophonia should be conceptualized as more than an aversion to oral/nasal sounds, which has implications for future diagnostics and experimental consistency moving forward.

Adults vs. neonates: Differentiation of functional connectivity between the basolateral amygdala and occipitotemporal cortex.

The amygdala, a subcortical structure known for social and emotional processing, consists of multiple subnuclei with unique functions and connectivity patterns. Tracer studies in adult macaques have shown that the basolateral subnuclei differentially connect to parts of visual cortex, with stronger connections to anterior regions and weaker connections to posterior regions; infant macaques show robust connectivity even with posterior visual regions. Do these developmental differences also exist in the human amygdala, and are there specific functional regions that undergo the most pronounced developmental changes in their connections with the amygdala? To address these questions, we explored the functional connectivity (from resting-state fMRI data) of the basolateral amygdala to occipitotemporal cortex in human neonates scanned within one week of life and compared the connectivity patterns to those observed in young adults. Specifically, we calculated amygdala connectivity to anterior-posterior gradients of the anatomically-defined occipitotemporal cortex, and also to putative occipitotemporal functional parcels, including primary and high-level visual and auditory cortices (V1, A1, face, scene, object, body, high-level auditory regions). Results showed a decreasing gradient of functional connectivity to the occipitotemporal cortex in adults-similar to the gradient seen in macaque tracer studies-but no such gradient was observed in neonates. Further, adults had stronger connections to high-level functional regions associated with face, body, and object processing, and weaker connections to primary sensory regions (i.e., A1, V1), whereas neonates showed the same amount of connectivity to primary and high-level sensory regions. Overall, these results show that functional connectivity between the amygdala and occipitotemporal cortex is not yet differentiated in neonates, suggesting a role of maturation and experience in shaping these connections later in life.

Innate connectivity patterns drive the development of the visual word form area.

What determines the functional organization of cortex? One hypothesis is that innate connectivity patterns, either structural or functional connectivity, set up a scaffold upon which functional specialization can later take place. We tested this hypothesis by asking whether the visual word form area (VWFA), an experience-driven region, was already functionally connected to proto language networks in neonates scanned within one week of birth. Using the data from the Human Connectone Project (HCP) and the Developing Human Connectome Project (dHCP), we calculated intrinsic functional connectivity during resting-state functional magnetic resonance imaging (fMRI), and found that neonates showed similar functional connectivity patterns to adults. We observed that (1) language regions connected more strongly with the putative VWFA than other adjacent ventral visual regions that also show foveal bias, and (2) the VWFA connected more strongly with frontotemporal language regions than with regions adjacent to these language regions. These data suggest that the location of the VWFA is earmarked at birth due to its connectivity with the language network, providing evidence that innate connectivity instructs the later refinement of cortex.

Strain-Dependent Kinetics in the Cis-to-Trans Isomerization of Azobenzene in Bulk Elastomers.

The cis-to-trans isomerization of azobenzene is accelerated in a bulk PDMS elastomer under uniaxial tension. The kinetics are cleanly described by a single-exponential first-order process (k = 2.7 × 10-5 s-1) in the absence of tension but become multiexponential under constant strains of 40-90%. The complex kinetics can be reasonably modeled as a two-component process. The majority (∼92%) process is slower and occurs with a rate constant that is similar to that of the unstrained system (k = 2.3-2.7 × 10-5 s-1), whereas the rate constant of the minority (∼8%) process increases from k = 10.1 × 10-5 s-1 at 40% strain to k = 21.3 × 10-5 s-1 at 90% strain. Simple models of expected force-rate relationships suggest that the average force of tension per strand in the minority component ranges from 28 to 44 pN across strains of 40-90%.

Taking stock: The role of environmental appraisal in the strategic use of attentional control.

There are many strategies we can use to control attention when approaching a visual search task, but some are more effective than others. How do we choose the most optimal strategy? We have proposed that individuals must appraise the stimulus environment, taking in relevant statistical information about task-relevant features. In the present experiments, we examined whether interfering with the appraisal process via a secondary task decreases participants' use of the optimal strategy. We used a modified version of the Adaptive Choice Visual Search paradigm whereby individuals can freely search for either of two targets on every trial. Each search display was preceded by a colored environmental preview, offering participants time to appraise the display and determine which target would be more optimal to search for. On some blocks, participants also completed a secondary task - a central line-length judgment - either before or during this colored preview. We found that participants were significantly less likely to search optimally when the line task occurred during the colored preview than when it occurred beforehand or was absent. Insofar as the secondary task disrupts an individual's ability to engage in appraisal, these results support the need for such an appraisal mechanism in the optimal choice of attentional control settings.

Patient and Parent Satisfaction With Sling Use After Pediatric Upper Extremity Fractures: A Randomized Controlled Trial of a Customized Cast-Sling Versus Standard Cast and Sling.

BACKGROUND: Long arm cast immobilization after operative and nonoperative treatment of pediatric upper extremity fractures is common. The use of a sling to aid in carrying the casted extremity as well as provide further immobilization is also common practice. Off-the-shelf slings vary in quality and fit, can be confusing for parents/patients to apply, and lead to frustration and dissatisfaction with its use. The purpose of this investigation was to compare patient/parent centered outcomes after the use of a customized sling compared with a standard sling by utilizing a prospective, randomized-controlled trial. We hypothesized that patients and their parents would find the "Providence" Pedi Cast-Sling (PPCS) to be more convenient and be more satisfied with its use compare with a standard sling. METHODS: Eligible subjects included patients 0 to 18 years old, evaluated at an urban, tertiary care pediatric emergency department (ED), who sustained an upper extremity fracture that required placement of a long-arm cast. Exclusion criteria were: open fractures; fractures at multiple levels; fractures requiring urgent/emergent surgery; admitted patients; bivalved casts. Patients were randomized to receive a standard sling or a PPCS. Questionnaires assessing patient/parent satisfaction, preferences, sling-use, and pain level were collected at patients' first follow-up visit. RESULTS: A total of 100 patients were randomized with 71 questionnaires available for analysis (39 standard sling vs. 32 PPCS). There were statistically significant differences for satisfaction scores for all related questions between patients who received a PPCS compared with a standard sling. Patients and their parents were more likely to choose the PPCS (P=0.001), were more satisfied with the PPCS (P<0.006), and reported the PPCS to be more convenient than a standard sling (P<0.001). Patients who received a PPCS wore the sling for a greater number of hours during the day (10.3±7.1 h) compared with those who received a standard sling (5.9±5.0 h) (P=0.004). CONCLUSIONS: The "Providence" Pedi Cast-Sling is a superior alternative to an off-the-shelf sling when used after the application of a long-arm cast providing greater convenience, compliance, and satisfaction to both patients and families. LEVEL OF EVIDENCE: Level I-Prospective randomized-controlled trial.

Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates.

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

Infection Following Total Joint Arthroplasty Is the Main Cause of Litigation: Data From One Metropolitan Area.

BACKGROUND: A prior survey of members of the American Association of Hip and Knee Surgeons revealed that 78% of responding surgeons were named as a defendant in at least 1 lawsuit, and 69% of these lawsuits were dismissed or settled out of court. The most common sources of litigation were nerve injury, limb-length discrepancy, and infection. This study examined common reasons for lawsuits after total joint arthroplasty (TJA) in a single metropolitan area. METHODS: A retrospective review of lawsuits filed between 2009 and 2015 in a 5-county metropolitan area was performed, including 30 hospitals and 113 TJA surgeons. Complaints underwent a manual review to determine the number of lawsuits and the specific allegations filed against each surgeon. RESULTS: Thirty-one (27.4%) surgeons were named as a defendant in at least 1 lawsuit. Eighty-three total lawsuits were filed during the period, 50 of which were dismissed or settled outside of court. Top reasons for lawsuits were, in descending order, infection, nerve injury, chronic pain, vascular injury, periprosthetic fracture, retention of foreign body, dislocation, limb-length discrepancy, venous thromboembolism, loosening, compartment syndrome, and other medical complaints. CONCLUSION: Infection appears to be the basis of most lawsuits after TJA. Surgeons should be aware of the potential for a lawsuit for complications and should strive to better communicate with patients regarding preoperative informed consent and disclosure after adverse events. Surgeons should minimize performing surgery in patients at high risk of complications, such as patients with a higher likelihood of developing postoperative infection or patients on chronic pain medications.

Radiographic Evaluation of Common Pediatric Elbow Injuries.

Normal variations in anatomy in the skeletally immature patient may be mistaken for fracture or injury due to the presence of secondary centers of ossification. Variations in imaging exist from patient to patient based on sex, age, and may even vary from one extremity to the other on the same patient. Despite differences in the appearance of the bony anatomy of the elbow there are certain landmarks and relationships, which can help, distinguish normal from abnormal. We review common radiographic parameters and pitfalls associated in the evaluation of pediatric elbow imaging. We also review common clinical diagnoses in this population.

Does Digital Video Advertising Increase Population-Level Reach of Multimedia Campaigns? Evidence From the 2013 Tips From Former Smokers Campaign.

BACKGROUND: Federal and state public health agencies in the United States are increasingly using digital advertising and social media to promote messages from broader multimedia campaigns. However, little evidence exists on population-level campaign awareness and relative cost efficiencies of digital advertising in the context of a comprehensive public health education campaign. OBJECTIVE: Our objective was to compare the impact of increased doses of digital video and television advertising from the 2013 Tips From Former Smokers (Tips) campaign on overall campaign awareness at the population level. We also compared the relative cost efficiencies across these media platforms. METHODS: We used data from a large national online survey of approximately 15,000 US smokers conducted in 2013 immediately after the conclusion of the 2013 Tips campaign. These data were used to compare the effects of variation in media dose of digital video and television advertising on population-level awareness of the Tips campaign. We implemented higher doses of digital video among selected media markets and randomly selected other markets to receive similar higher doses of television ads. Multivariate logistic regressions estimated the odds of overall campaign awareness via digital or television format as a function of higher-dose media in each market area. All statistical tests used the .05 threshold for statistical significance and the .10 level for marginal nonsignificance. We used adjusted advertising costs for the additional doses of digital and television advertising to compare the cost efficiencies of digital and television advertising on the basis of costs per percentage point of population awareness generated. RESULTS: Higher-dose digital video advertising was associated with 94% increased odds of awareness of any ad online relative to standard-dose markets (P<.001). Higher-dose digital advertising was associated with a marginally nonsignificant increase (46%) in overall campaign awareness regardless of media format (P=.09). Higher-dose television advertising was associated with 81% increased odds of overall ad awareness regardless of media format (P<.001). Increased doses of television advertising were also associated with significantly higher odds of awareness of any ad on television (P<.001) and online (P=.04). The adjusted cost of each additional percentage point of population-level reach generated by higher doses of advertising was approximately US $440,000 for digital advertising and US $1 million for television advertising. CONCLUSIONS: Television advertising generated relatively higher levels of overall campaign awareness. However, digital video was relatively more cost efficient for generating awareness. These results suggest that digital video may be used as a cost-efficient complement to traditional advertising modes (eg, television), but digital video should not replace television given the relatively smaller audience size of digital video viewers.

Does Digital Ad Exposure Influence Information-Seeking Behavior Online? Evidence From the 2012 Tips From Former Smokers National Tobacco Prevention Campaign.

BACKGROUND: Measuring the impact of online health campaigns is challenging. Ad click-through rates are traditionally used to measure campaign reach, but few Internet users ever click on ads. Alternatively, self-reported exposure to digital ads would be prone to recall bias. Furthermore, there may be latency effects whereby people do not click on ads when exposed but visit the promoted website or conduct campaign-related searches later. Online panels that unobtrusively collect panelists' Web behavior data and link ad exposure to website visits and searches can more reliably assess the impact of digital ad exposure. From March to June 2012, the Centers for Disease Control and Prevention aired the national Tips From Former Smokers (Tips 2012) media campaign designed to encourage current smokers to quit. Advertisements ran across media channels, and the digital ads directed users to the Tips 2012 campaign website. OBJECTIVE: Our aim was to examine whether exposure to Tips 2012 digital ads influenced information-seeking behaviors online. METHODS: ComScore mined its panelists' Web behavior data for unique codes that would indicate exposure to Tips 2012 ads, regardless of whether panelists clicked the ad or not. A total of 15,319 US adults were identified as having been exposed to a Tips 2012 campaign ad. An equal number of unexposed adults (N=15,319) were identified and matched on demographics and Internet use behavior to the exposed group. Panelists' Web behavior data were mined for up to 4 weeks after initial Tips 2012 ad exposure to determine whether they visited the Tips 2012 campaign website or other cessation-related websites (eg, nicotine replacement therapy site) or conducted searches for campaign-related topics (eg, quit smoking). RESULTS: The proportion of exposed adults visiting the Tips 2012 sites increased from 0.4% in Week 1 to 0.9% 4 weeks after ad exposure, and these rates were significantly higher than in the unexposed group (0.1% in Week 1 to 0.4% in Week 4, P<.001) across all weeks examined. The proportion of exposed panelists visiting other cessation websites increased from 0.2% in Week 1 to 0.3% 4 weeks after initial ad exposure, and these rates were significantly higher than in the unexposed group (0.0% in Week 1 to 0.2% in Week 4, P=.001 to P=.019) across all weeks examined. There were no significant differences in searches for campaign-related topics between the exposed and unexposed group during most of the weeks examined. CONCLUSIONS: These results suggest that online ad exposure is associated with confirmed visits to the Tips 2012 campaign sites and visits to other cessation websites and that these information-seeking behaviors occur up to several weeks after ad exposure. Web behavior data from online panels are useful for examining exposure and behavioral responses to digital campaign ads.