Targeting the Actions of Muscarinic Receptors on Dopamine Systems: New Strategies for Treating Neuropsychiatric Disorders.

Cholinergic regulation of dopamine (DA) signaling has significant implications for numerous disorders, including schizophrenia, substance use disorders, and mood-related disorders. The activity of midbrain DA neurons and DA release patterns in terminal regions are tightly regulated by cholinergic neurons found in both the striatum and the hindbrain. These cholinergic neurons can modulate DA circuitry by activating numerous receptors, including muscarinic acetylcholine receptor (mAChR) subtypes. This review specifically focuses on the complex role of M2, M4, and M5 mAChR subtypes in regulating DA neuron activity and DA release and the potential clinical implications of targeting these mAChR subtypes.

Forest restoration and fuels reduction work: Different pathways for achieving success in the Sierra Nevada.

Fire suppression and past selective logging of large trees have fundamentally changed frequent-fire-adapted forests in California. The culmination of these changes produced forests that are vulnerable to catastrophic change by wildfire, drought, and bark beetles, with climate change exacerbating this vulnerability. Management options available to address this problem include mechanical treatments (Mech), prescribed fire (Fire), or combinations of these treatments (Mech + Fire). We quantify changes in forest structure and composition, fuel accumulation, modeled fire behavior, intertree competition, and economics from a 20-year forest restoration study in the northern Sierra Nevada. All three active treatments (Fire, Mech, Mech + Fire) produced forest conditions that were much more resistant to wildfire than the untreated control. The treatments that included prescribed fire (Fire, Mech + Fire) produced the lowest surface and duff fuel loads and the lowest modeled wildfire hazards. Mech produced low fire hazards beginning 7 years after the initial treatment and Mech + Fire had lower tree growth than controls. The only treatment that produced intertree competition somewhat similar to historical California mixed-conifer forests was Mech + Fire, indicating that stands under this treatment would likely be more resilient to enhanced forest stressors. While Fire reduced modeled wildfire hazard and reintroduced a fundamental ecosystem process, it was done at a net cost to the landowner. Using Mech that included mastication and restoration thinning resulted in positive revenues and was also relatively strong as an investment in reducing modeled wildfire hazard. The Mech + Fire treatment represents a compromise between the desire to sustain financial feasibility and the desire to reintroduce fire. One key component to long-term forest conservation will be continued treatments to maintain or improve the conditions from forest restoration. Many Indigenous people speak of "active stewardship" as one of the key principles in land management and this aligns well with the need for increased restoration in western US forests. If we do not use the knowledge from 20+ years of forest research and the much longer tradition of Indigenous cultural practices and knowledge, frequent-fire forests will continue to be degraded and lost.

Ventral Tegmental Area M5 Muscarinic Receptors Mediate Effort-Choice Responding and Nucleus Accumbens Dopamine in a Sex-Specific Manner .

Optimization of effort-related choices is impaired in depressive disorders. Acetylcholine (ACh) and dopamine (DA) are linked to depressive disorders, and modulation of ACh tone in the ventral tegmental area (VTA) affects mood-related behavioral responses in rats. However, it is unknown if VTA ACh mediates effort-choice behaviors. Using a task of effort-choice, rats can choose to lever press on a fixed-ratio 5 (FR5) schedule for a more-preferred food or consume freely available, less-preferred food. VTA administration of physostigmine (1 µg and 2 µg/side), a cholinesterase inhibitor, reduced FR5 responding for the more-preferred food while leaving consumption of the less-preferred food intact. VTA infusion of the M5 muscarinic receptor negative allosteric modulator VU6000181 (3 µM, 10 µM, 30 µM/side) did not affect lever pressing or chow consumption. However, VU6000181 (30 µM/side) coadministration with physostigmine (2 µg/side) attenuated physostigmine-induced decrease in lever pressing in female and male rats and significantly elevated lever pressing above vehicle baseline levels in male rats. In in vivo voltammetry experiments, VTA infusion of combined physostigmine and VU6000181 did not significantly alter evoked phasic DA release in the nucleus accumbens core (NAc) in female rats. In male rats, combined VTA infusion of physostigmine and VU6000181 increased phasic evoked DA release in the NAc compared with vehicle, physostigmine, or VU6000181 infusion alone. These data indicate a critical role and potential sex differences of VTA M5 receptors in mediating VTA cholinergic effects on effort choice behavior and regulation of DA release. SIGNIFICANCE STATEMENT: Effort-choice impairments are observed in depressive disorders, which are often treatment resistant to currently available thymoleptics. The role of ventral tegmental area (VTA) acetylcholine muscarinic M5 receptors, in a preclinical model of effort-choice behavior, is examined. Using the selective negative allosteric modulator of the M5 receptor VU6000181, we show the role of VTA M5 receptors on effort-choice and regulation of dopamine release in the nucleus accumbens core. This study supports M5 receptors as therapeutic targets for depression.

Imidazole-based sphingosine-1-phosphate transporter Spns2 inhibitors.

Sphingosine-1-phosphate (S1P) is a chemotactic lipid that influences immune cell positioning. S1P concentration gradients are necessary for proper egress of lymphocytes from the thymus and secondary lymphoid tissues. This trafficking is interdicted by S1P receptor modulators, and it is expected that S1P transporter (Spns2) inhibitors, by reshaping S1P concentration gradients, will do the same. We previously reported SLF1081851 as a prototype Spns2 inhibitor, which provided a scaffold to investigate the importance of the oxadiazole core and the terminal amine. In this report, we disclose a structure-activity relationship study by incorporating imidazole as both a linker and surrogate for a positive charge in SLF1081851. In vitro inhibition of Spns2-dependent S1P transport in HeLa cells identified 7b as an inhibitor with an IC50 of 1.4 ± 0.3 µM. The SAR studies reported herein indicate that imidazolium can be a substitute for the terminal amine in SLF1081851 and that Spns2 inhibition is highly dependent on the lipid alkyl tail length.

Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in Preclinical Models of Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two U.S. Food and Drug Administration-approved antifibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Objectives: Using an in silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. Methods: We investigated the antifibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: 1) in vitro in normal human lung fibroblasts; 2) in vivo in bleomycin and recombinant Ad-TGF-ß (adenovirus transforming growth factor-ß) murine models of pulmonary fibrosis; and 3) ex vivo in mice and human precision-cut lung slices from these two murine models as well as patients with IPF and healthy donors. Measurements and Main Results: In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone. Transcriptomic analyses of TGF-ß-stimulated normal human lung fibroblasts identified specific gene sets associated with fibrosis, including epithelial-mesenchymal transition, TGF-ß, and WNT signaling that was uniquely altered by saracatinib. Transcriptomic analysis of whole-lung extracts from the two animal models of pulmonary fibrosis revealed that saracatinib reverted many fibrogenic pathways, including epithelial-mesenchymal transition, immune responses, and extracellular matrix organization. Amelioration of fibrosis and inflammatory cascades in human precision-cut lung slices confirmed the potential therapeutic efficacy of saracatinib in human lung fibrosis. Conclusions: These studies identify novel Src-dependent fibrogenic pathways and support the study of the therapeutic effectiveness of saracatinib in IPF treatment.

PTPα promotes fibroproliferative responses after acute lung injury.

The acute respiratory distress syndrome (ARDS) is a major healthcare problem, accounting for significant mortality and long-term disability. Approximately 25% of patients with ARDS will develop an overexuberant fibrotic response, termed fibroproliferative ARDS (FP-ARDS) that portends a poor prognosis and increased mortality. The cellular pathological processes that drive FP-ARDS remain incompletely understood. We have previously shown that the transmembrane receptor-type tyrosine phosphatase protein tyrosine phosphatase-α (PTPα) promotes pulmonary fibrosis in preclinical murine models through regulation of transforming growth factor-ß (TGF-ß) signaling. In this study, we examine the role of PTPα in the pathogenesis of FP-ARDS in a preclinical murine model of acid (HCl)-induced acute lung injury. We demonstrate that although mice genetically deficient in PTPα (Ptpra-/-) are susceptible to early HCl-induced lung injury, they exhibit markedly attenuated fibroproliferative responses. In addition, early profibrotic gene expression is reduced in lung tissue after acute lung injury in Ptpra-/- mice, and stimulation of naïve lung fibroblasts with the BAL fluid from these mice results in attenuated fibrotic outcomes compared with wild-type littermate controls. Transcriptomic analyses demonstrate reduced extracellular matrix (ECM) deposition and remodeling in mice genetically deficient in PTPα. Importantly, human lung fibroblasts modified with a CRISPR-targeted deletion of PTPRA exhibit reduced expression of profibrotic genes in response to TGF-ß stimulation, demonstrating the importance of PTPα in human lung fibroblasts. Together, these findings demonstrate that PTPα is a key regulator of fibroproliferative processes following acute lung injury and could serve as a therapeutic target for patients at risk for poor long-term outcomes in ARDS.

Influenza virus infection increases ACE2 expression and shedding in human small airway epithelial cells.

BACKGROUND: Patients with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrate high rates of co-infection with respiratory viruses, including influenza A (IAV), suggesting pathogenic interactions. METHODS: We investigated how IAV may increase the risk of COVID-19 lung disease, focusing on the receptor angiotensin-converting enzyme (ACE)2 and the protease TMPRSS2, which cooperate in the intracellular uptake of SARS-CoV-2. RESULTS: We found, using single-cell RNA sequencing of distal human nondiseased lung homogenates, that at baseline, ACE2 is minimally expressed in basal, goblet, ciliated and secretory epithelial cells populating small airways. We focused on human small airway epithelial cells (SAECs), central to the pathogenesis of lung injury following viral infections. Primary SAECs from nondiseased donor lungs apically infected (at the air-liquid interface) with IAV (up to 3×105 pfu; ∼1 multiplicity of infection) markedly (eight-fold) boosted the expression of ACE2, paralleling that of STAT1, a transcription factor activated by viruses. IAV increased the apparent electrophoretic mobility of intracellular ACE2 and generated an ACE2 fragment (90 kDa) in apical secretions, suggesting cleavage of this receptor. In addition, IAV increased the expression of two proteases known to cleave ACE2, sheddase ADAM17 (TACE) and TMPRSS2 and increased the TMPRSS2 zymogen and its mature fragments, implicating proteolytic autoactivation. CONCLUSION: These results indicate that IAV amplifies the expression of molecules necessary for SARS-CoV-2 infection of the distal lung. Furthermore, post-translational changes in ACE2 by IAV may increase vulnerability to lung injury such as acute respiratory distress syndrome during viral co-infections. These findings support efforts in the prevention and treatment of influenza infections during the COVID-19 pandemic.

Activation of the mGlu1 metabotropic glutamate receptor has antipsychotic-like effects and is required for efficacy of M4 muscarinic receptor allosteric modulators.

Recent clinical and preclinical studies suggest that selective activators of the M4 muscarinic acetylcholine receptor have potential as a novel treatment for schizophrenia. M4 activation inhibits striatal dopamine release by mobilizing endocannabinoids, providing a mechanism for local effects on dopamine signaling in the striatum but not in extrastriatal areas. G protein-coupled receptors (GPCRs) typically induce endocannabinoid release through activation of Gαq/11-type G proteins whereas M4 transduction occurs through Gαi/o-type G proteins. We now report that the ability of M4 to inhibit dopamine release and induce antipsychotic-like effects in animal models is dependent on co-activation of the Gαq/11-coupled mGlu1 subtype of metabotropic glutamate (mGlu) receptor. This is especially interesting in light of recent findings that multiple loss of function single nucleotide polymorphisms (SNPs) in the human gene encoding mGlu1 (GRM1) are associated with schizophrenia, and points to GRM1/mGlu1 as a gene within the "druggable genome" that could be targeted for the treatment of schizophrenia. Herein, we report that potentiation of mGlu1 signaling following thalamo-striatal stimulation is sufficient to inhibit striatal dopamine release, and that a novel mGlu1 positive allosteric modulator (PAM) exerts robust antipsychotic-like effects through an endocannabinoid-dependent mechanism. However, unlike M4, mGlu1 does not directly inhibit dopamine D1 receptor signaling and does not reduce motivational responding. Taken together, these findings highlight a novel mechanism of cross talk between mGlu1 and M4 and demonstrate that highly selective mGlu1 PAMs may provide a novel strategy for the treatment of positive symptoms associated with schizophrenia.

Bile diversion, a bariatric surgery, and bile acid signaling reduce central cocaine reward.

The gut-to-brain axis exhibits significant control over motivated behavior. However, mechanisms supporting this communication are poorly understood. We reveal that a gut-based bariatric surgery chronically elevates systemic bile acids and attenuates cocaine-induced elevations in accumbal dopamine. Notably, this surgery reduces reward-related behavior and psychomotor sensitization to cocaine. Utilizing a knockout mouse model, we have determined that a main mediator of these post-operative effects is the Takeda G protein-coupled bile acid receptor (TGR5). Viral restoration of TGR5 in the nucleus accumbens of TGR5 knockout animals is sufficient to restore cocaine reward, centrally localizing this TGR5-mediated modulation. These findings define TGR5 and bile acid signaling as pharmacological targets for the treatment of cocaine abuse and reveal a novel mechanism of gut-to-brain communication.

Disc degeneration spreads: long-term behavioural, histologic and radiologic consequences of a single-level disc injury in active and sedentary mice.

STUDY DESIGN: A multi-cohort, case-control rodent study. PURPOSE: Investigate the long-term behavioural, histologic and radiologic consequences on the complete lumbar spine of L4/5 intervertebral disc (IVD) injury in mice and determine if increased physical activity mitigates the observed changes. METHODS: Cohorts of 2-month-old CD1 female mice underwent a single ventral puncture of the L4/5 IVD. 0.5-, 3- or 12-months after injury, general health (body weight and locomotor capacity), behavioural signs of axial discomfort (tail suspension, grip strength and FlexMaze assays) and radiating pain (von Frey and acetone tests) were assessed. Experimental groups with free access to an activity wheel in their home cages were including in the 12-month cohort. Lumbar disc status was determined using colorimetric staining and radiologic (X-ray and T2-MRI) analysis. Innervation was measured by immunoreactivity for PGP9.5 and calcitonin gene-related peptide. RESULTS: No changes in general health or persistent signs of axial discomfort were observed up to one year post-injury. In contrast, signs of radiating pain developed in injured mice at 3 months post-injury, persisted up to 12 months and were reversed by long-term physical activity. At 12-months post-injury, degeneration was observed in non-injured lumbar discs. Secondary degenerating IVDs were similar to the injured discs by X-ray (narrowing) and T2-MRI (internal disc disruption) but did not show abnormal innervation. Increased physical activity had no impact on mechanically injured IVDs, but attenuated disc narrowing at other lumbar levels. CONCLUSIONS: Mechanical injury of L4/5-IVDs induces delayed radiating pain and degeneration of adjacent discs; increased physical activity positively mitigated both.

Protein tyrosine phosphatase-α amplifies transforming growth factor-ß-dependent profibrotic signaling in lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal, fibrosing lung disease for which treatment remains suboptimal. Fibrogenic cytokines, including transforming growth factor-ß (TGF-ß), are central to its pathogenesis. Protein tyrosine phosphatase-α (PTPα) has emerged as a key regulator of fibrogenic signaling in fibroblasts. We have reported that mice globally deficient in PTPα (Ptpra-/-) were protected from experimental pulmonary fibrosis, in part via alterations in TGF-ß signaling. The goal of this study was to determine the lung cell types and mechanisms by which PTPα controls fibrogenic pathways and whether these pathways are relevant to human disease. Immunohistochemical analysis of lungs from patients with IPF revealed that PTPα was highly expressed by mesenchymal cells in fibroblastic foci and by airway and alveolar epithelial cells. To determine whether PTPα promotes profibrotic signaling pathways in lung fibroblasts and/or epithelial cells, we generated mice with conditional (floxed) Ptpra alleles (Ptpraf/f). These mice were crossed with Dermo1-Cre or with Sftpc-CreERT2 mice to delete Ptpra in mesenchymal cells and alveolar type II cells, respectively. Dermo1-Cre/Ptpraf/f mice were protected from bleomycin-induced pulmonary fibrosis, whereas Sftpc-CreERT2/Ptpraf/f mice developed pulmonary fibrosis equivalent to controls. Both canonical and noncanonical TGF-ß signaling and downstream TGF-ß-induced fibrogenic responses were attenuated in isolated Ptpra-/- compared with wild-type fibroblasts. Furthermore, TGF-ß-induced tyrosine phosphorylation of TGF-ß type II receptor and of PTPα were attenuated in Ptpra-/- compared with wild-type fibroblasts. The phenotype of cells genetically deficient in PTPα was recapitulated with the use of a Src inhibitor. These findings suggest that PTPα amplifies profibrotic TGF-ß-dependent pathway signaling in lung fibroblasts.

High density is a property of slow-cycling and treatment-resistant human glioblastoma cells.

Slow-cycling and treatment-resistant cancer cells escape therapy, providing a rationale for regrowth and recurrence in patients. Much interest has focused on identifying the properties of slow-cycling tumor cells in glioblastoma (GBM), the most common and lethal primary brain tumor. Despite aggressive ionizing radiation (IR) and treatment with the alkylating agent temozolomide (TMZ), GBM patients invariably relapse and ultimately succumb to the disease. In patient biopsies, we demonstrated that GBM cells expressing the proliferation markers Ki67 and MCM2 displayed a larger cell volume compared to rare slow-cycling tumor cells. In optimized density gradients, we isolated a minor fraction of slow-cycling GBM cells in patient biopsies and tumorsphere cultures. Transcriptional profiling, self-renewal, and tumorigenicity assays reflected the slow-cycling state of high-density GBM cells (HDGCs) compared to the tumor bulk of low-density GBM cells (LDGCs). Slow-cycling HDGCs enriched for stem cell antigens proliferated a few days after isolation to generate LDGCs. Both in vitro and in vivo, we demonstrated that HDGCs show increased treatment-resistance to IR and TMZ treatment compared to LDGCs. In conclusion, density gradients represent a non-marker based approach to isolate slow-cycling and treatment-resistant GBM cells across GBM subgroups.

Evaluation of a Culturally Tailored Educational Video Intervention to Promote Bike Helmet Safety for Urban Children: A Pilot Study.

Bicycle-related falls are a significant cause of mortality and morbidity. Use of bicycle helmets substantially reduces risk of severe traumatic brain injury but compliance with this safety practice is particularly low in urban children. Given the lack of educational interventions for urban youth, our research team created a youth-informed, culturally relevant educational video on bike helmet safety, which was informed by focus groups with Baltimore City youth. This video, You Make the Call, linked the concept of use of cases to protect phones to use of helmets to protect heads and can be viewed at http://bit.ly/2Kr7UCN. The impact of the video as part of an intervention (coupled with a free helmet, fit instructions, and a parent guidance document) was tested with 20 parent-child dyads. The majority (80%) of youth (mean age 9.9 ± 1.8 years) reported not owning or wearing a helmet. At 1-month follow-up (n = 12, 60% response rate), helmet use was higher in the five youth reporting bike-riding after the intervention; 100% "always" used helmets compared to 0% preintervention. There were increases in youth reporting that parents required helmet use (35% pre vs. 67% post) and that is was possible to fall when bike-riding (60% pre vs. 92% post). These pilot results support the use of this video and educational intervention along with further evaluation in a larger sample size. This youth-informed and culturally tailored approach could be explored as a strategy to address other pediatric injury topics.

The Kinome of Human Alveolar Type II and Basal Cells, and Its Reprogramming in Lung Cancer.

The discovery of mutant tyrosine kinases as oncogenic drivers of lung adenocarcinomas has changed the basic understanding of lung cancer development and therapy. Yet, expressed kinases (kinome) in lung cancer progenitor cells, as well as whether kinase expression and the overall kinome changes or is reprogrammed upon transformation, is incompletely understood. We hypothesized that the kinome differs between lung cancer progenitor cells, alveolar type II cells (ATII), and basal cells (BC) and that their respective kinomes undergo distinct lineage-specific reprogramming to adenocarcinomas and squamous cell carcinomas upon transformation. We performed RNA sequencing on freshly isolated human ATII, BC, and lung cancer cell lines to define the kinome in nontransformed cells and transformed cells. Our studies identified a unique kinome for ATII and BC and changes in their kinome upon transformation to their respective carcinomas.

Interleukin-6 is required for Neuregulin-1 induced HER2 signaling in lung epithelium.

A clear understanding of the mechanisms that regulate the alveolar epithelium's barrier is critical to develop new therapeutic strategies to mitigate lung injury. The HER2/HER3 receptor tyrosine kinase complex plays a central role in maintaining the alveolar-capillary barrier. This receptor complex is activated by its ligand, neuregulin-1 (NRG-1). Interleukin-6 (IL-6) is also known to induce HER2 signaling through HER2 transphosphorylation by the IL-6 receptor (IL-6R) complex (1). Due to this interaction, we hypothesized that NRG-1 and IL-6 cooperatively interacted to activate the HER2/HER3 complex. Studies were performed in cultured pulmonary epithelial cells measuring the HER2/IL-6/IL-6R/GP130 interaction and receptor activation by western blotting and confocal microscopy, IL-6 production by ELISA, and IL-6 inhibition using specific antibodies, small molecule inhibitors and shRNA. We found that IL-6 was required for NRG-1 induced activation of HER2 in pulmonary epithelial cells. IL-6 inhibition led to a decrease in NRG-1 induced HER2 activation. The IL-6R and GP130, a subunit of the IL-6R complex, were physically associated with HER2 and were required for NRG-1 induced HER2 activation. Inhibition of GP130, the ß-subunit of the IL-6 receptor decreased NRG-1 induced HER2 activation lower than control by 38% Finally, HER2 activation increased IL-6 secretion more than two-fold over resting cells (526 ± 131 vs 231 ± 39.7 pg/ml), and inhibition of HER2 gene expression decreased basal IL-6 secretion over 80% (89 + 4.6 vs 1.3 + 0.8 pg/ml). These findings identify a requirement for IL-6 and the IL-6R complex to allow NRG-1 mediated HER2 activation, and a HER2 driven IL-6 production feedback loop.

Highly Speciated Measurements of Terpenoids Emitted from Laboratory and Mixed-Conifer Forest Prescribed Fires.

Wildland fires in the western United States are projected to increase in frequency, duration, and size. Characterized by widespread and diverse conifer forests, burning within this region may lead to significant terpenoid emissions. Terpenoids constitute a major class of highly reactive secondary organic aerosol (SOA) precursors, with significant structure-dependent variability in reactivity and SOA-formation potential. In this study, highly speciated measurements of terpenoids emitted from laboratory and prescribed fires were achieved using two-dimensional gas chromatography. Nearly 100 terpenoids were measured in smoke samples from 71 fires, with high variability in the dominant compounds. Terpenoid emissions were dependent on plant species and tissues. Canopy/needle-derived emissions dominated in the laboratory fires, whereas woody-tissue-derived emissions dominated in the prescribed fires. Such differences likely have implications for terpenoid emissions from high vs low intensity fires and suggest that canopy-dominant laboratory fires may not accurately represent terpenoid emissions from prescribed fires or wildland fires that burn with low intensity. Predicted SOA formation was sensitive to the diversity of emitted terpenoids when compared to assuming a single terpene surrogate. Given the demonstrated linkages between fuel type, fire terpenoid emissions, and the subsequent implications for plume chemistry, speciated measurements of terpenoids in smoke derived from diverse ecosystems and fire regimes may improve air quality predictions downwind of wildland fires.

Activity in LIP, But not V4, Matches Performance When Attention is Spread.

The enhancement of neuronal responses in many visual areas while animals perform spatial attention tasks has widely been thought to be the neural correlate of visual attention, but it is unclear whether the presence or absence of this modulation contributes to our striking inability to notice changes in change blindness examples. We asked whether neuronal responses in visual area V4 and the lateral intraparietal area (LIP) in posterior parietal cortex could explain the limited ability of subjects to attend multiple items in a display. We trained animals to perform a change detection task in which they had to compare 2 arrays of stimuli separated briefly in time and found that each animal's performance decreased as function of set-size. Neuronal discriminability in V4 was consistent across set-sizes, but decreased for higher set-sizes in LIP. The introduction of a reward bias produced attentional enhancement in V4, but this could not explain the vast improvement in performance, whereas the enhancement in LIP responses could. We suggest that behavioral set-size effects and the marked improvement in performance with focused attention may not be related to response enhancement in V4 but, instead, may occur in or on the way to LIP.

Promoting Bike Helmet Safety for Urban Children Through a Culturally Tailored Educational Video Intervention.

Bicycle-related falls are a significant cause of mortality and morbidity. Use of bicycle helmets substantially reduces risk of severe traumatic brain injury but compliance with this safety practice is particularly low in urban children. We recruited eleven 8- to 15-year-old youth to participate in focus groups to inform the creation of a video promoting helmet use. Key emerging themes included that youth were responsible for keeping themselves safe and that most youth had cell phones with cases to protect them. A video was created that linked the concept of use of cases to protect phones to use of helmets to protect heads. Soliciting information from urban youth was helpful for developing this educational video.

Probing Position-Dependent Diffusion in Folding Reactions Using Single-Molecule Force Spectroscopy.

Folding of proteins and nucleic acids involves a diffusive search over a multidimensional conformational energy landscape for the minimal-energy structure. When examining the projection of conformational motions onto a one-dimensional reaction coordinate, as done in most experiments, the diffusion coefficient D is generally position dependent. However, it has proven challenging to measure such position-dependence experimentally. We investigated the position-dependence of D in the folding of DNA hairpins as a simple model system in two ways: first, by analyzing the round-trip time to return to a given extension in constant-force extension trajectories measured by force spectroscopy, and second, by analyzing the fall time required to reach a given extension in force jump measurements. These methods yielded conflicting results: the fall time implied a fairly constant D, but the round-trip time implied variations of over an order of magnitude. Comparison of experiments with computational simulations revealed that both methods were strongly affected by experimental artifacts inherent to force spectroscopy measurements, which obscured the intrinsic position-dependence of D. Lastly, we applied Kramers's theory to the kinetics of hairpins with energy barriers located at different positions along the hairpin stem, as a crude probe of D at different stem positions, and we found that D did not vary much as the barrier was moved along the reaction coordinate. This work underlines the difficulties faced when trying to deduce position-dependent diffusion coefficients from experimental folding trajectories.

Monoubiquitination of survival motor neuron regulates its cellular localization and Cajal body integrity.

Low levels of the survival motor neuron (SMN) protein cause spinal muscular atrophy, the leading genetic disorder for infant mortality. SMN is ubiquitously expressed in various cell types and localizes in both the cytoplasm and the nucleus, where it concentrates in two subnuclear structures termed Cajal body (CB) and gems. In addition, SMN can also be detected in the nucleolus of neurons. Mechanisms that control SMN sorting in the cell remain largely unknown. Here, we report that the ubiquitin (Ub) ligase Itch directly interacts with and monoubiquitinates SMN. Monoubiquitination of SMN has a mild effect on promoting proteasomal degradation of SMN. We generated two SMN mutants, SMN(K0), in which all lysines are mutated to arginines and thereby abolishing SMN ubiquitination, and Ub-SMN(K0), in which a single Ub moiety is fused at the N-terminus of SMN(K0) and thereby mimicking SMN monoubiquitination. Immunostaining assays showed that SMN(K0) mainly localizes in the nucleus, whereas Ub-SMN(K0) localizes in both the cytoplasm and the nucleolus in neuronal SH-SY5Y cells. Interestingly, canonical CB foci and coilin/small nuclear ribonucleoprotein (snRNP) co-localization are significantly impaired in SH-SY5Y cells stably expressing SMN(K0) or Ub-SMN(K0). Thus, our studies discover that Itch monoubiquitinates SMN and monoubiquitination of SMN plays an important role in regulating its cellular localization. Moreover, mislocalization of SMN disrupts CB integrity and likely impairs snRNP maturation.