Precise druggability of the PTH type 1 receptor.

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates.

Children with dyslexia show no deficit in exogenous spatial attention but show differences in visual encoding.

In the search for mechanisms that contribute to dyslexia, the term "attention" has been invoked to explain performance in a variety of tasks, creating confusion since all tasks do, indeed, demand "attention." Many studies lack an experimental manipulation of attention that would be necessary to determine its influence on task performance. Nonetheless, an emerging view is that children with dyslexia have an impairment in the exogenous (automatic/reflexive) orienting of spatial attention. Here we investigated the link between exogenous attention and reading ability by presenting exogenous spatial cues in the multi-letter processing task-a task relevant for reading. The task was gamified and administered online to a large sample of children (N = 187) between 6 and 17 years. Children with dyslexia performed worse overall at rapidly recognizing and reporting strings of letters. However, we found no evidence for a difference in the utilization of exogenous spatial cues, resolving two decades of ambiguity in the field. Previous studies that claimed otherwise may have failed to distinguish attention effects from overall task performance or found spurious group differences in small samples. RESEARCH HIGHLIGHTS: We manipulated exogenous visual spatial attention using pre-cues in a task that is relevant for reading and we see robust task effects of exogenous attention. We found no evidence for a deficit in utilizing exogenous spatial pre-cues in children with dyslexia. However, children with dyslexia showed reduced recognition ability for all letter positions. Children with dyslexia were just as likely to make letter transposition errors as typical readers.

Shoulder Arthroplasty in the Upper Extremity Weight-bearing Patient: A Systematic Review of Clinical Outcomes and Complications.

BACKGROUND: Patients who rely on their upper extremities for ambulation, or upper extremity ambulators (UEAs), place considerable stress on their shoulders through the use of assistive devices like walkers, crutches, canes, and wheelchairs. It has been postulated that UEAs may be at increased risk for complications following shoulder arthroplasty. This study aimed to systematically review the literature related to (1) patient-reported outcomes measures (PROMs), (2) functional outcomes, and (3) complications in UEAs who undergo shoulder arthroplasty. METHODS: A systematic review of the PubMed/MEDLINE, Embase, and Cochrane databases was performed to identify studies reporting clinical outcomes of shoulder arthroplasty in UEAs. Patient demographics, clinical characteristics, PROMs, radiographic outcomes, and postoperative range of motion were collected and compared to control patients (i.e. bipedal ambulators) from the constituent studies. RESULTS: A total of eight studies evaluating 248 UEA cases and 206 control cases were included for review. Ambulatory assistive devices utilized by UEAs included walkers (39%), wheelchairs (38%), canes (22%), and a crutch (<1%). Among UEA cases, 197 (79%) reverse total shoulder arthroplasty, 37 (15%) anatomic total shoulder arthroplasty, and 14 (6%) hemiarthroplasty were performed. Overall, patients exhibited significant improvements in mean American Shoulder and Elbow Surgeons (ASES) scores, Constant-Murley scores, Simple Shoulder Test (SST) scores, and Visual Analog Scale (VAS) scores postoperatively. Among 3 studies that included comparison with control groups of bipedal ambulators, no significant differences in outcomes were identified. The overall clinical complication rate was 17% for UEAs compared to 9.1% for controls. The rate of revision surgery was 7.7% for UEAs and 4.9% for bipedal ambulators. CONCLUSIONS: UEAs experience satisfactory pain relief, functional improvements, and good subjective outcomes following shoulder arthroplasty. However, complication and revision rates are higher compared to those for bipedal ambulators, and the majority of UEAs undergo reverse shoulder arthroplasty (RSA) compared to anatomic total shoulder arthroplasty (aTSA).

Biased GPCR signaling by the native parathyroid hormone-related protein 1 to 141 relative to its N-terminal fragment 1 to 36.

The parathyroid hormone (PTH)-related protein (PTHrP) is indispensable for the development of mammary glands, placental calcium ion transport, tooth eruption, bone formation and bone remodeling, and causes hypercalcemia in patients with malignancy. Although mature forms of PTHrP in the body consist of splice variants of 139, 141, and 173 amino acids, our current understanding on how endogenous PTHrP transduces signals through its cognate G-protein coupled receptor (GPCR), the PTH type 1 receptor (PTHR), is largely derived from studies done with its N-terminal fragment, PTHrP1-36. Here, we demonstrate using various fluorescence imaging approaches at the single cell level to measure kinetics of (i) receptor activation, (ii) receptor signaling via Gs and Gq, and (iii) receptor internalization and recycling that the native PTHrP1-141 displays biased agonist signaling properties that are not mimicked by PTHrP1-36. Although PTHrP1-36 induces transient cAMP production, acute intracellular Ca2+ (iCa2+) release and ß-arrestin recruitment mediated by ligand-PTHR interactions at the plasma membrane, PTHrP1-141 triggers sustained cAMP signaling from the plasma membrane and fails to stimulate iCa2+ release and recruit ß-arrestin. Furthermore, we show that the molecular basis for biased signaling differences between PTHrP1-36 and properties of native PTHrP1-141 are caused by the stabilization of a singular PTHR conformation and PTHrP1-141 sensitivity to heparin, a sulfated glycosaminoglycan. Taken together, our results contribute to a better understanding of the biased signaling process of a native protein hormone acting in conjunction with a GPCR.

Gq/11-dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR.

cAMP production upon activation of Gs by G protein-coupled receptors has classically been considered to be plasma membrane-delimited, but a shift in this paradigm has occurred in recent years with the identification of several receptors that continue to signal from early endosomes after internalization. The molecular mechanisms regulating this aspect of signaling remain incompletely understood. Here, we investigated the role of Gq/11 activation by the parathyroid hormone (PTH) type 1 receptor (PTHR) in mediating endosomal cAMP responses. Inhibition of Gq/11 signaling by FR900359 markedly reduced the duration of PTH-induced cAMP production, and this effect was mimicked in cells lacking endogenous Gαq/11 We determined that modulation of cAMP generation by Gq/11 occurs at the level of the heterotrimeric G protein via liberation of cell surface Gßγ subunits, which, in turn, act in a phosphoinositide-3 kinase-dependent manner to promote the assembly of PTHR-ßarrestin-Gßγ signaling complexes that mediate endosomal cAMP responses. These results unveil insights into the spatiotemporal regulation of Gs-dependent cAMP signaling.

The Outcome of Hip Arthroscopy in the Setting of Lumbar Spine Disease Is Beneficial, Yet Limited: A Systematic Review of Existing Evidence.

PURPOSE: To compare hip arthroscopy outcomes in femoroacetabular impingement (FAI) patients with concurrent symptomatic lumbar spine disease to the outcomes of arthroscopic FAI patients without spine disease. METHODS: A systematic review was performed according to PRISMA guidelines via PubMed, Cochrane, Embase, and Google Scholar databases. Studies were valid for inclusion if they had an average follow-up ≥12 months and compared patient-reported outcome measures (PROMs) in hip arthroscopy patients with and without concurrent spinal disease. Data collected included study characteristics, patient demographics, follow-up intervals, surgical indications, spinal pathology, PROMs, and reoperation rates. RESULTS: Twelve studies were included in this systematic review. 3,107 patients who underwent hip arthroscopy were evaluated: 1,056 with coexisting lumbar spine disease (spine cohort) and 2,051 control subjects without spine disease (control cohort). The average follow-up period was 24 months. Across included studies, there were 35 instances wherein postoperative PROM scores reported by each cohort were compared. In all 35 instances, the spine cohort reported inferior postoperative PROM scores with the difference being significant (P < .05) on 23 PROMs. Collectively, 23 cases were available contrasting the proportion of each cohort to achieve the minimal clinically important difference (MCID). In 22 (95.65%) of these cases, the spine cohort achieved the MCID at a lower rate than the control cohort. There were 14 PROMs, wherein intragroup analyses were reported that compared the preoperative and postoperative score reported by the spine cohort. On all 14 PROMs, the spine cohort reported significant (P < .05) improvement after arthroscopic intervention. CONCLUSION: FAI patients with coexisting lumbar spine pathology experience significant improvement from baseline state after arthroscopic intervention. However, the postoperative outcomes reported are inferior, and the improvement from arthroscopy was limited when compared to surgical control subjects with FAI and normal spinal anatomy. LEVEL OF EVIDENCE: Level IV: systematic review of Level II, III, and IV studies.

Oculomotor freezing indicates conscious detection free of decision bias.

The appearance of a salient stimulus rapidly and automatically inhibits saccadic eye movements. Curiously, this "oculomotor freezing" response is triggered only by stimuli that the observer reports seeing. It remains unknown, however, whether oculomotor freezing is linked to the observer's sensory experience or their decision that a stimulus was present. To dissociate between these possibilities, we manipulated decision criterion via monetary payoffs and stimulus probability in a detection task. These manipulations greatly shifted observers' decision criteria but did not affect the degree to which microsaccades were inhibited by stimulus presence. Moreover, the link between oculomotor freezing and explicit reports of stimulus presence was stronger when the criterion was conservative rather than liberal. We conclude that the sensory threshold for oculomotor freezing is independent of decision bias. Provided that conscious experience is also unaffected by such bias, oculomotor freezing is an implicit indicator of sensory awareness.NEW & NOTEWORTHY Sometimes a visual stimulus reaches awareness, and sometimes it does not. To understand why, we need objective, bias-free measures of awareness. We discovered that a reflexive freezing of small eye movements indicates when an observer detects a stimulus. Furthermore, when we biased observers' decisions to report seeing the stimulus, the oculomotor response was unaltered. This suggests that the threshold for conscious perception is independent of the decision criterion and is revealed by oculomotor freezing.

Allosteric interactions in the parathyroid hormone GPCR-arrestin complex formation.

Peptide ligands of class B G-protein-coupled receptors act via a two-step binding process, but the essential mechanisms that link their extracellular binding to intracellular receptor-arrestin interactions are not fully understood. Using NMR, crosslinking coupled to mass spectrometry, signaling experiments and computational approaches on the parathyroid hormone (PTH) type 1 receptor (PTHR), we show that initial binding of the PTH C-terminal part constrains the conformation of the flexible PTH N-terminal signaling epitope before a second binding event occurs. A 'hot-spot' PTH residue, His9, that inserts into the PTHR transmembrane domain at this second step allosterically engages receptor-arrestin coupling. A conformational change in PTHR intracellular loop 3 permits favorable interactions with ß-arrestin's finger loop. These results unveil structural determinants for PTHR-arrestin complex formation and reveal that the two-step binding mechanism proceeds via cooperative fluctuations between ligand and receptor, which extend to other class B G-protein-coupled receptors.

Parallel spatial channels converge at a bottleneck in anterior word-selective cortex.

In most environments, the visual system is confronted with many relevant objects simultaneously. That is especially true during reading. However, behavioral data demonstrate that a serial bottleneck prevents recognition of more than one word at a time. We used fMRI to investigate how parallel spatial channels of visual processing converge into a serial bottleneck for word recognition. Participants viewed pairs of words presented simultaneously. We found that retinotopic cortex processed the two words in parallel spatial channels, one in each contralateral hemisphere. Responses were higher for attended than for ignored words but were not reduced when attention was divided. We then analyzed two word-selective regions along the occipitotemporal sulcus (OTS) of both hemispheres (subregions of the visual word form area, VWFA). Unlike retinotopic regions, each word-selective region responded to words on both sides of fixation. Nonetheless, a single region in the left hemisphere (posterior OTS) contained spatial channels for both hemifields that were independently modulated by selective attention. Thus, the left posterior VWFA supports parallel processing of multiple words. In contrast, activity in a more anterior word-selective region in the left hemisphere (mid OTS) was consistent with a single channel, showing (i) limited spatial selectivity, (ii) no effect of spatial attention on mean response amplitudes, and (iii) sensitivity to lexical properties of only one attended word. Therefore, the visual system can process two words in parallel up to a late stage in the ventral stream. The transition to a single channel is consistent with the observed bottleneck in behavior.

Ca2+ allostery in PTH-receptor signaling.

The parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only the PTH sustains GS-mediated adenosine 3',5'-cyclic monophosphate (cAMP) production after PTHR internalization into early endosomes. The mechanism of this unexpected behavior for a G-protein-coupled receptor is not fully understood. Here, we show that extracellular Ca2+ acts as a positive allosteric modulator of PTHR signaling that regulates sustained cAMP production. Equilibrium and kinetic studies of ligand-binding and receptor activation reveal that Ca2+ prolongs the residence time of ligands on the receptor, thus, increasing both the duration of the receptor activation and the cAMP signaling. We further find that Ca2+ allostery in the PTHR is strongly affected by the point mutation recently identified in the PTH (PTHR25C) as a new cause of hypocalcemia in humans. Using high-resolution and mass accuracy mass spectrometry approaches, we identified acidic clusters in the receptor's first extracellular loop as key determinants for Ca2+ allosterism and endosomal cAMP signaling. These findings coupled to defective Ca2+ allostery and cAMP signaling in the PTHR by hypocalcemia-causing PTHR25C suggest that Ca2+ allostery in PTHR signaling may be involved in primary signaling processes regulating calcium homeostasis.

Luminescence-activated nucleotide cyclase regulates spatial and temporal cAMP synthesis.

cAMP is a ubiquitous second messenger that regulates cellular proliferation, differentiation, attachment, migration, and several other processes. It has become increasingly evident that tight regulation of cAMP accumulation and localization confers divergent yet specific signaling to downstream pathways. Currently, few tools are available that have sufficient spatial and temporal resolution to study location-biased cAMP signaling. Here, we introduce a new fusion protein consisting of a light-activated adenylyl cyclase (bPAC) and luciferase (nLuc). This construct allows dual activation of cAMP production through temporally precise photostimulation or chronic chemical stimulation that can be fine-tuned to mimic physiological levels and duration of cAMP synthesis to trigger downstream events. By targeting this construct to different compartments, we show that cAMP produced in the cytosol and nucleus stimulates proliferation in thyroid cells. The bPAC-nLuc fusion construct adds a new reagent to the available toolkit to study cAMP-regulated processes in living cells.

The Effect of Patient Characteristics and Comorbidities on the Rate of Revision Rotator Cuff Repair.

PURPOSE: To describe the national rates of failed primary rotator cuff repair (RCR) requiring revision repair, using numerous patient characteristics previously defined in orthopaedic literature, including smoking history, diabetes mellitus (DM), hyperlipidemia (HLD), vitamin D deficiency, and osteoporosis to determine which factors independently affect the success of primary RCR. METHODS: A combined public and private national insurance database was searched from 2007 to 2016 for all patients who underwent RCR. Current Procedural Terminology codes were used to identify RCRs. Laterality modifiers for the primary surgery were used to identify subsequent revision RCRs. All patients who did not have a linked laterality modifier for the RCR Current Procedural Terminology code were excluded from the study. Basic demographics were recorded. International Classification of Diseases Ninth Revision codes were used to identify patient characteristics including Charlson Comorbidity Index, smoking status, DM, obesity, HLD, vitamin D deficiency, and osteoporosis. Patient age categorized as <60, 60-69, 70-74, or 75+ years old. Dichotomous data were analyzed with χ2 testing. Multivariable logistic regression was used to characterize independent associations with revision RCR. RESULTS: Included in the study were 41,467 patients (41,844 shoulders, 52.7% male patients) who underwent primary arthroscopic RCR. Of all arthroscopic RCRs, 3072 patients (3463 shoulders, 53.5% male patients) underwent revision RCR (8.38%). In both primary and revision RCR, patients age 60 to 69 years were most prevalent, accounting for 38.4% and 37.6% of the cohorts, respectively. The average time from primary RCR to revision was 414.9 days (median 214.0 days). Increasing age and male sex (odds ratio [OR] 1.10, P = .019, 95% confidence interval [CI] 1.02-1.19) were significantly predictive of revision RCR. Of the remaining patient characteristics, smoking most strongly predicted revision RCR (OR 1.36, P < .001, CI 1.23-1.49). Obesity (OR 1.32, P < .001, CI 1.21-1.43), hyperlipidemia (OR 1.09, P = .032, CI 1.01-1.18), and vitamin D deficiency (OR 1.18, P < .001, CI 1.08-1.28) also increased risk of revision RCR significantly. DM was found to be protective against revision surgery (OR 0.84, P < .001, CI 0.76-0.92). Overall comorbidity burden as measured by the Charlson Comorbidity Index was not predictive of revision RCR. CONCLUSIONS: Smoking, obesity, vitamin D deficiency, and HLD are shown to be independent risk factors for failure of primary RCR requiring revision RCR. However, despite the suggestions of previous studies, DM, osteoporosis, and overall comorbidity burden did not demonstrate independent associations in this study. LEVEL OF EVIDENCE: IV, Case Series.

Development of a return to play checklist following patellar instability surgery: a Delphi-based consensus.

PURPOSE: To date, there is no consensus for the appropriate timing or functional evaluation for safe return to play following patellar instability surgery. The purpose of this study is to develop a consensus-based return to play checklist following patellar stabilization surgery using the Delphi method. METHODS: A 3-part survey series was conducted following the systematic guidelines of the Delphi technique for gathering consensus from experts in the management of patellofemoral instability. All surveys were completed between July and November of 2017. A literature search was performed in SCOPUS and PubMed to identify existing sources on return to play following patellar instability surgery and determining patellofemoral joint strength in athletes, which served as the basis for the surveys. RESULTS: 12 of the 19 selected participants (63%) completed the first-round survey, 11 of those 12 participants (92%) completed the second-round survey, and 10 of these 11 participants (91%) completed the final survey. Of the final ten participants, there was representation from seven different states in the USA. Nine of the ten (90%) respondents endorsed the final checklist. The final checklist included eight overarching domains with defined and reproducible objective criteria. CONCLUSION: The standardized list of objective and reproducible criteria for rehabilitation outlined below should help practitioners focus more on patient-centred factors and less on arbitrary timelines. No prior study has gathered consensus from experts on this topic; therefore, this study should serve as a benchmark to help guide patients back to sport safely. LEVEL OF EVIDENCE: V.

Fluorogenic structure activity library pinpoints molecular variations in substrate specificity of structurally homologous esterases.

Cellular esterases catalyze many essential biological functions by performing hydrolysis reactions on diverse substrates. The promiscuity of esterases complicates assignment of their substrate preferences and biological functions. To identify universal factors controlling esterase substrate recognition, we designed a 32-member structure-activity relationship (SAR) library of fluorogenic ester substrates and used this library to systematically interrogate esterase preference for chain length, branching patterns, and polarity to differentiate common classes of esterase substrates. Two structurally homologous bacterial esterases were screened against this library, refining their previously broad overlapping substrate specificity. Vibrio cholerae esterase ybfF displayed a preference for γ-position thioethers and ethers, whereas Rv0045c from Mycobacterium tuberculosis displayed a preference for branched substrates with and without thioethers. We determined that this substrate differentiation was partially controlled by individual substrate selectivity residues Tyr-119 in ybfF and His-187 in Rv0045c; reciprocal substitution of these residues shifted each esterase's substrate preference. This work demonstrates that the selectivity of esterases is tuned based on transition state stabilization, identifies thioethers as an underutilized functional group for esterase substrates, and provides a rapid method for differentiating structural isozymes. This SAR library could have multifaceted future applications, including in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design.

Use of Backbone Modification To Enlarge the Spatiotemporal Diversity of Parathyroid Hormone Receptor-1 Signaling via Biased Agonism.

The type-1 parathyroid hormone receptor (PTHR1), which regulates calcium homeostasis and tissue development, has two native agonists, parathyroid hormone (PTH) and PTH-related protein (PTHrP). PTH forms a complex with the PTHR1 that is rapidly internalized and induces prolonged cAMP production from endosomes. In contrast, PTHrP induces only transient cAMP production, which primarily arises from receptors on the cell surface. We show that backbone modification of PTH(1-34)-NH2 and abaloparatide (a PTHrP derivative) with a single homologous ß-amino acid residue can generate biased agonists that induce prolonged cAMP production from receptors at the cell surface. This unique spatiotemporal profile could be useful for distinguishing effects associated with the duration of cAMP production from effects associated with the site of cAMP production.

Characterisation and screening of antimicrobial essential oil components against clinically important antibiotic-resistant bacteria using thin layer chromatography-direct bioautography hyphenated with GC-MS, LC-MS and NMR.

INTRODUCTION: The antimicrobial activity of many essential oils (EOs) is well established, indicating that EOs may be a source of compounds for antimicrobial drug development. Thin layer chromatography-direct bioautography (TLC-DB) can quickly identify antimicrobial components in complex mixtures and can be applied to the screening of EOs for lead compounds. OBJECTIVES: This study aimed to identify antimicrobial components of oregano, rosewood and cumin EOs against antibiotic-sensitive and -resistant bacteria using TLC-DB and a multi-faceted approach of GC-MS, LC-MS and NMR techniques to characterise bioactive compounds. The study also aimed to quantify the antimicrobial activity of bioactive compounds in order to evaluate their potential for the development of therapies against antibiotic-resistant bacteria. MATERIALS AND METHODS: EOs were eluted on TLC plates and sprayed with a suspension of Staphylococcus aureus, Enterococcus faecium, Escherichia coli or Pseudomonas aeruginosa (antibiotic-sensitive and -resistant isolates). Zones of inhibition, visualised with iodonitrotetrazolium chloride, were subject to GC-MS, LC-MS and NMR to characterise the bioactive compounds. RESULTS: Seven compounds were identified from the three EOs using GC-MS, while LC-MS and NMR failed to detect the presence of any further non-volatile or heat labile compounds. Carvacrol was most antimicrobial compound identified, with minimum inhibitory concentrations ranging 0.99-31.62 mM. CONCLUSION: The identified antimicrobial compounds present in oregano, rosewood and cumin EOs including carvacrol may be candidates for the development of novel antimicrobial therapies against antibiotic-resistant bacteria.

Evidence of Serial Processing in Visual Word Recognition.

To test the limits of parallel processing in vision, we investigated whether people can recognize two words at once. Participants viewed brief, masked pairs of words and were instructed in advance to judge both of the words (dual-task condition) or just one of the words (single-task condition). For judgments of semantic category, the dual-task deficit was so large that it supported all-or-none serial processing: Participants could recognize only one word and had to guess about the other. Moreover, participants were more likely to be correct about one word if they were incorrect about the other, which also supports a serial-processing model. In contrast, judgments of text color with identical stimuli were consistent with unlimited-capacity parallel processing. Thus, under these conditions, serial processing is necessary to judge the meaning of words but not their physical features. Understanding the implications of this result for natural reading will require further investigation.

Missed Thrower's Fracture of the Humerus in a Pediatric Athlete: A Case Report.

BACKGROUND: Humerus fractures caused by the throwing motion are extremely rare. They have been reported mostly in recreational adult athletes in their third or later decades of life. A pediatric thrower's fracture is even less common, with few reported cases. The pediatric version of this fracture is located in the proximal to midshaft humerus, distinguishing it from the adult type, which occurs in the middle to distal shaft. CASE REPORT: A 12-year-old male pitcher experienced a "snap" in his right arm while throwing a pitch in a baseball game. He presented to the Emergency Department with right arm pain and deformity. He was misdiagnosed with a right glenohumeral dislocation and a reduction maneuver was attempted prior to any radiographic imaging. Upon further review of the imaging and outpatient follow-up, he was found to have a humeral spiral fracture consistent with a "ball-thrower's fracture." The fracture healed with conservative treatment and he returned to unrestricted sports participation. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Recognition of this fracture is important to avoid unnecessary and potentially harmful treatment of the pediatric patient. A thrower's fracture of the pediatric humerus is rare, but glenohumeral dislocation without direct trauma is even less common and has never been reported as a result of the throwing motion in a pediatric patient. Radiographic imaging is important, and consideration of the thrower's fracture should be in the differential for any patient presenting with acute pain and deformity of the arm resulting from throwing any object.

In Vivo Delivery and Activation of Masked Fluorogenic Hydrolase Substrates by Endogenous Hydrolases in C.†elegans.

Protein expression and localization are often studied in vivo by tagging molecules with green fluorescent protein (GFP), yet subtle changes in protein levels are not easily detected. To develop a sensitive in vivo method to amplify fluorescence signals and allow cell-specific quantification of protein abundance changes, we sought to apply an enzyme-activated cellular fluorescence system in vivo by delivering ester-masked fluorophores to Caenorhabditis elegans neurons expressing porcine liver esterase (PLE). To aid uptake into sensory neuron membranes, we synthesized two novel fluorogenic hydrolase substrates with long hydrocarbon tails. Recombinant PLE activated these fluorophores in vitro. In vivo activation occurred in sensory neurons, along with potent activation in intestinal lysosomes quantifiable by imaging and microplate and partially attributable to gut esterase 1 (GES-1) activity. These data demonstrate the promise of biorthogonal hydrolases and their fluorogenic substrates as in vivo neuronal imaging tools and for characterizing endogenous C. elegans hydrolase substrate specificities.

Feature singletons attract spatial attention independently of feature priming.

People perform better in visual search when the target feature repeats across trials (intertrial feature priming [IFP]). Here, we investigated whether repetition of a feature singleton's color modulates stimulus-driven shifts of spatial attention by presenting a probe stimulus immediately after each singleton display. The task alternated every two trials between a probe discrimination task and a singleton search task. We measured both stimulus-driven spatial attention (via the distance between the probe and singleton) and IFP (via repetition of the singleton's color). Color repetition facilitated search performance (IFP effect) when the set size was small. When the probe appeared at the singleton's location, performance was better than at the opposite location (stimulus-driven attention effect). The magnitude of this attention effect increased with the singleton's set size (which increases its saliency) but did not depend on whether the singleton's color repeated across trials, even when the previous singleton had been attended as a search target. Thus, our findings show that repetition of a salient singleton's color affects performance when the singleton is task relevant and voluntarily attended (as in search trials). However, color repetition does not affect performance when the singleton becomes irrelevant to the current task, even though the singleton does capture attention (as in probe trials). Therefore, color repetition per se does not make a singleton more salient for stimulus-driven attention. Rather, we suggest that IFP requires voluntary selection of color singletons in each consecutive trial.