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Many essential proteins require pyridoxal 5'-phosphate, the active form of vitamin B6, as a cofactor for their activity. These include enzymes important for amino acid metabolism, one-carbon metabolism, polyamine synthesis, erythropoiesis, and neurotransmitter metabolism. A third of all mammalian pyridoxal 5'-phosphate-dependent enzymes are localized in the mitochondria; however, the molecular machinery involved in the regulation of mitochondrial pyridoxal 5'-phosphate levels in mammals remains unknown. In this study, we used a genome-wide CRISPR interference screen in erythroleukemia cells and organellar metabolomics to identify the mitochondrial inner membrane protein SLC25A38 as a regulator of mitochondrial pyridoxal 5'-phosphate. Loss of SLC25A38 causes depletion of mitochondrial, but not cellular, pyridoxal 5'-phosphate, and impairs cellular proliferation under both physiological and low vitamin B6 conditions. Metabolic changes associated with SLC25A38 loss suggest impaired mitochondrial pyridoxal 5'-phosphate-dependent enzymatic reactions, including serine to glycine conversion catalyzed by serine hydroxymethyltransferase-2 as well as ornithine aminotransferase. The proliferation defect of SLC25A38-null K562 cells in physiological and low vitamin B6 media can be explained by the loss of serine hydroxymethyltransferase-2-dependent production of one-carbon units and downstream de novo nucleotide synthesis. Our work points to a role for SLC25A38 in mitochondrial pyridoxal 5'-phosphate accumulation and provides insights into the pathology of congenital sideroblastic anemia. Pyridoxal 5'-phosphate (PLP, vitamin B6) is crucial for various metabolic processes. Here, the authors identified SLC25A38 as a key regulator of mitochondrial PLP levels using a genome-wide CRISPRi screen and organellar metabolomics, with insights into congenital sideroblastic anemia. [ABSTRACT FROM AUTHOR]

Application of stable isotopically labelled (SIL) molecules in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) over a series of time points allows the temporal and spatial dynamics of biochemical reactions to be tracked in a biological system. However, these large kinetic MSI datasets and the inherent variability of biological replicates presents significant challenges to the rapid analysis of the data. In addition, manual annotation of downstream SIL metabolites involves human input to carefully analyse the data based on prior knowledge and personal expertise. To overcome these challenges to the analysis of spatiotemporal MALDI-MSI data and improve the efficiency of SIL metabolite identification, a bioinformatics pipeline has been developed and demonstrated by analysing normal bovine lens glucose metabolism as a model system. The pipeline consists of spatial alignment to mitigate the impact of sample variability and ensure spatial comparability of the temporal data, dimensionality reduction to rapidly map regional metabolic distinctions within the tissue, and metabolite annotation coupled with pathway enrichment modules to summarise and display the metabolic pathways induced by the treatment. This pipeline will be valuable for the spatial metabolomics community to analyse kinetic MALDI-MSI datasets, enabling rapid characterisation of spatio-temporal metabolic patterns from tissues of interest. [ABSTRACT FROM AUTHOR]

A finite element model was developed for assessing the efficacy of rugby body padding in reducing the risk of sustaining cuts and abrasions. The model was developed to predict the onset of damage to a soft tissue simulant from concentrated impact loading (i.e., stud impact) and compared against a corresponding experiment. The damage modelling techniques involved defining an element deletion criterion, whereby those on the surface of the surrogate were deleted if their maximum principal stress reached a predefined value. Candidate maximum principal stress values for element deletion criteria were identified independently from puncture test simulations on the soft tissue simulant. Experimental impacts with a stud were carried out at three energies (2, 4 and 6 J), at three angular orientations (0°, 15° and 30°) and compared to corresponding simulations. Suitable maximum principal stress values for element deletion criteria settings were first identified for the 4 J impact, selecting the candidates that best matched the experimental results. The same element deletion settings were then applied in simulations at 2 and 6 J and the validity of the model was further assessed (difference < 15% for the force at tear and < 30% for time to tear). The damage modelling techniques presented here could be applied to other skin simulants to assess the onset of skin injuries and the ability of padding to prevent them. [ABSTRACT FROM AUTHOR]

Liquid Chromatography Mass Spectrometry (LC-MS) is a powerful method for profiling complex biological samples. However, batch effects typically arise from differences in sample processing protocols, experimental conditions, and data acquisition techniques, significantly impacting the interpretability of results. Correcting batch effects is crucial for the reproducibility of omics research, but current methods are not optimal for the removal of batch effects without compressing the genuine biological variation under study. We propose a suite of Batch Effect Removal Neural Networks (BERNN) to remove batch effects in large LC-MS experiments, with the goal of maximizing sample classification performance between conditions. More importantly, these models must efficiently generalize in batches not seen during training. A comparison of batch effect correction methods across five diverse datasets demonstrated that BERNN models consistently showed the strongest sample classification performance. However, the model producing the greatest classification improvements did not always perform best in terms of batch effect removal. Finally, we show that the overcorrection of batch effects resulted in the loss of some essential biological variability. These findings highlight the importance of balancing batch effect removal while preserving valuable biological diversity in large-scale LC-MS experiments. Liquid Chromatography Mass Spectrometry (LC-MS) is a powerful method for profiling biological samples. Here, the authors have developed a suit of Batch Effect Removal Neural Networks (BERNN) to remove batch effects in large LC-MS experiments to maximize sample classification between conditions. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a complex and heterogeneous neurodegenerative disorder with contributions from multiple pathophysiological pathways. One of the long-recognized and important features of AD is disrupted cerebral glucose metabolism, but the underlying molecular basis remains unclear. In this study, unbiased mass spectrometry was used to survey CSF from a large clinical cohort, comparing patients who are either cognitively unimpaired (CU; n = 68), suffering from mild-cognitive impairment or dementia from AD (MCI-AD, n = 95; DEM-AD, n = 72), or other causes (MCI-other, n = 77; DEM-other, n = 23), or Normal Pressure Hydrocephalus (NPH, n = 57). The results revealed changes related to altered glucose metabolism. In particular, two glycolytic enzymes, pyruvate kinase (PKM) and aldolase A (ALDOA), were found to be upregulated in CSF from patients with AD compared to those with other neurological conditions. Increases in full-length PKM and ALDOA levels in CSF were confirmed with immunoblotting. Levels of these enzymes furthermore correlated negatively with CSF glucose in matching CSF samples. PKM levels were also found to be increased in AD in publicly available brain-tissue data. These results indicate that ALDOA and PKM may act as technically-robust potential biomarkers of glucose metabolism dysregulation in AD. [ABSTRACT FROM AUTHOR]

Why are Homo sapiens the only human species living on this planet? Homo sapiens have lived on this planet for about 300,000 years. During most of their existence, early modern humans shared this planet with other archaic humans, such as Neanderthals and Denisovans. Why did the other archaic humans become extinct? To explore this question, this study develops a Malthusian model with natural selection of human species to explore how population dynamics of one group of humans may cause the extinction of another group. In my model, different groups of humans engage in hunting-gathering. The larger group of humans can occupy more land. Therefore, the expansion of one population causes the other population to shrink in a Malthusian economy. Which human population shrinks or even becomes extinct depends on structural parameters in the Malthusian model. [ABSTRACT FROM AUTHOR]

In this paper, we present a numerical model of a vertical-axis turbine (VAT) with active-pitch torque control. The model is based upon the Wind and Tidal Turbine Embedded Simulator (WATTES) and WATTES-V turbine realisations in conjunction with the actuator line method (ALM), and uses OpenFOAM to solve the unsteady Reynolds-averaged Navier-Stokes (URANS) equations with two-equation k - ε turbulence closure. Our novel pitch-controlled system is based on an even pressure drop across the entire rotor to mitigate against dynamic stall at low tip speed ratio. The numerical model is validated against experimental measurements and alternative numerical predictions of the hydrodynamic performance of a 1:6 scale UNH-RM2 hydrokinetic turbine. Simulations deploying the variable pitch mechanism exhibit improved turbine performance compared to measured data and fixed zero-pitch model predictions. Near-wake characteristics are investigated by examining the vorticity distribution near the turbine. The pitch-controlled system is demonstrated to theoretically decrease turbulence generated by turbine rotations, mitigate the intensity of vortex shedding and size of detached vortices, and significantly enhance the performance of a vertical-axis hydrokinetic turbine for rated tip-speed ratios. [ABSTRACT FROM AUTHOR]

Recent technological advances have opened the door to single-cell proteomics that can answer key biological questions regarding how protein expression, post-translational modifications, and protein interactions dictate cell state in health and disease. [ABSTRACT FROM AUTHOR]

A finite element model of an impact test on a soft tissue simulant, used as part of a shoulder surrogate, was developed in Ansys© LS-DYNA®. The surrogate consisted of a metal hemicylindrical core, with a diameter of 75 mm, covered with a 15 mm thick relaxed muscle simulant. The muscle simulant consisted of a 14 mm thick layer of silicone covered with 1 mm thick chamois leather to represent skin. The material properties of the silicone were obtained via quasi-static compression testing (curve fit with hyperelastic models) and compressive stress relaxation testing (curve fit with a Prony series). Outputs of the finite element models were compared against experimental data from impact tests on the shoulder surrogate at energies of 4.9, 9.8 and 14.7 J. The accuracy of the finite element models was assessed using four parameters: peak impact force, maximum deformation, impact duration and impulse. A 5-parameter Mooney-Rivlin material model combined with a 2-term Prony series was found to be suitable for modelling the soft tissue simulant of the shoulder surrogate. This model had under 10% overall mean deviation from the experimental values for the four assessment parameters across the three impact energies. Overall, the model provided a repeatable test method that can be adapted to help predict injuries to skin tissue and the performance/efficacy of personal protective equipment. [ABSTRACT FROM AUTHOR]

Padded clothing (shoulder padding) is worn in Rugby Union to give players an opportunity to protect themselves. A performance specification for padded clothing has been set out by World Rugby™, with the intention that padded clothing only protects against lacerations and abrasions. Test protocols in this specification provide an assessment of the impact force attenuative properties of the material, this itself will not indicate what injuries they may have the potential to prevent or lessen the severity of. The current study has used previously established biomechanical parameters to develop a mechanical test procedure to assess the ability of padded clothing to prevent or lessen the severity of stud-induced laceration and abrasion injuries. A synthetic skin and soft tissue surrogate was developed and validated to mimic human anatomy. Without the addition of padded clothing, both wearing (abrasion) and tearing (laceration) of the synthetic tissue surrogate were seen. The addition of padded clothing saw no sign of stud-induced injury, even after six repeated trials of the same product, showing padded clothing can prevent or lessen the severity of lacerations and abrasions. The developed testing protocols could be used to assess the safety of any sports stud designs in relation to skin injury as well as the effectiveness of various protective clothing products across the sports industry. [ABSTRACT FROM AUTHOR]

The safety of aquatic ecosystems has been compromised by numerous anthropogenic activities, especially leachates from non-point source toxicants, leaching into aquatic systems. This study evaluated the toxicity of the water-soluble fractions (WSFs) of burnt tire ash (BTA) on Clarias gariepinus via a battery of integrated biomarkers. Juvenile C. gariepinus were exposed to sublethal (0.56, 1.12, and 2.24 g/L) concentrations of BTA, derived from 11.2 g/L median lethal concentration (96 LC50), at duration intervals of 1, 14, and 28 days, followed by a recovery trial that lasted for 14 days. Serum biochemical parameters, antioxidant enzyme activities of the gill and liver, lysozymes activity and erythron profile were assessed. The findings of the present study revealed that BTA-WSF induced prominent alterations on biochemical parameters, lysozymes activity and antioxidant enzymes activities in the exposed fish. Furthermore, toxicant exposure promoted oxidative stress, cellular damage and genotoxicity (erythrocytic nuclear and cellular abnormalities) in the exposed fish. In general, a post-exposure trial showed partial recovery from the exposure effects of the toxicant, following the evident modifications of serum enzymes and erythron pathopathology in the experimental model. Biomonitoring of BTA, using sentinel aquatic species such as C. gariepinus, provides insights into the ecotoxicological potency of this toxicant. [ABSTRACT FROM AUTHOR]

Migrating sea trout Salmo trutta L. in the waters of the Shetland Islands (UK) were once found in abundant numbers but they have been in decline since the mid-1980s. Therefore, it is becoming ever more critical to investigate sea trout behaviour with respect to the prevailing environmental conditions in the unique Shetland habitat so that we can determine how this prized and important species might be re-established to more sustainable levels. Here, during a period of four months (July to October) in 2015, environmental and fish catch rate data were collected for a small sample size of sea trout caught at six Shetland locations. We then looked for trends and correlations in the data that might help account for the migratory behaviour of the fish with respect to the environmental conditions at the time of capture. Our data showed that wind speed and pH correlated to the catch rate. However, no significant correlation was found between the water temperature, air temperature, barometric pressure, or humidity, and catch rate. Multivariate regression showed that wind speed and pH explained 26.3% of the variance in the catch rate. The sex ratio of the sea trout caught was skewed towards females at a ratio of 4.7 : 1.0 and the largest trout caught were all female. Recording biological data and understanding the migration patterns and behaviours of the remaining wild populations of sea trout are important for optimizing management plans to support current efforts to increase the numbers of this valuable anadromous species in Shetland waters. [ABSTRACT FROM AUTHOR]

This study explores the dynamic effects of patent policy on innovation and income inequality in a Schumpeterian growth model with endogenous market structure and heterogeneous households. We find that strengthening patent protection has a positive effect on economic growth and a positive or an inverted-U effect on income inequality when the number of differentiated products is fixed in the short run. However, when the number of products adjusts endogenously, the effects of patent protection on growth and inequality become negative in the long run. We also calibrate the model to US data to perform a quantitative analysis and find that the long-run negative effect of patent policy on inequality is much larger than its short-run positive effect. This result remains consistent with our empirical finding from a panel vector autoregression. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionisation-imaging mass spectrometry (MALDI-IMS) is now an established imaging modality with particular utility in the study of biological, biomedical and pathological processes. In the first instance, the use of stable isotopically labelled (SIL) compounds in MALDI-IMS has addressed technical barriers to increase the accuracy and versatility of this technique. This has undoubtedly enhanced our ability to interpret the two-dimensional ion intensity distributions produced from biological tissue sections. Furthermore, studies using delivery of SIL compounds to live tissues have begun to decipher cell, tissue and inter-tissue metabolism while maintaining spatial resolution. Here, we review both the technical and biological applications of SIL compounds in MALDI-IMS, before using the uptake and metabolism of glucose in bovine ocular lens tissue to illustrate the current limitations of SIL compound use in MALDI-IMS. Finally, we highlight recent instrumentation advances that may further enhance our ability to use SIL compounds in MALDI-IMS to understand biological and pathological processes. [ABSTRACT FROM AUTHOR]

This study provides a growth-theoretic analysis of the effects of intellectual property rights on the take-off of an economy from an era of stagnation to a state of sustained economic growth. We incorporate patent protection into a Schumpeterian growth model in which take-off occurs when the population size crosses an endogenous threshold. We find that strengthening patent protection has contrasting effects on economic growth at different stages of development. Specifically, it leads to an earlier take-off but also reduces economic growth in the long run. [ABSTRACT FROM AUTHOR]

Meeting Report on the 9th Annual Symposium of the Colorado Alphaherpesvirus Latency Society (CALS) held on May 8–11, 2019, in Vail, CO. [ABSTRACT FROM AUTHOR]

We assessed total mercury (THg) concentrations in breast feathers of diurnal North American raptors collected at migration monitoring stations. For 9 species in the Pacific Flyway, we found species and age influenced feather THg concentrations whereas sex did not. Feather THg concentrations µg/g dry weight (dw) averaged (least squares mean ± standard error) higher for raptors that generally consume > 75% avian prey (sharp-shinned hawk Accipiter striatus: n = 113; 4.35 ± 0.45 µg/g dw, peregrine falcon Falco peregrinus: n = 12; 3.93 ± 1.11 µg/g dw, Cooper's hawk Accipiter cooperii: n = 20; 2.35 ± 0.50 µg/g dw, and merlin Falco columbarius: n = 59; 1.75 ± 0.28 µg/g dw) than for raptors that generally consume < 75% avian prey (northern harrier Circus hudsonius: n = 112; 0.75 ± 0.10 µg/g dw, red-tailed hawk Buteo jamaicensis: n = 109; 0.56 ± 0.06 µg/g dw, American kestrel Falco sparverius: n = 16; 0.57 ± 0.14 µg/g dw, prairie falcon Falco mexicanus: n = 10; 0.41 ± 0.13 µg/g dw) except for red-shouldered hawks Buteo lineatus: n = 10; 1.94 ± 0.61 µg/g dw. Feather THg concentrations spanning 13-years (2002–2014) in the Pacific Flyway differed among 3 species, where THg increased for juvenile northern harrier, decreased for adult red-tailed hawk, and showed no trend for adult sharp-shinned hawk. Mean feather THg concentrations in juvenile merlin were greater in the Mississippi Flyway (n = 56; 2.14 ± 0.18 µg/g dw) than those in the Pacific Flyway (n = 49; 1.15 ± 0.11 µg/g dw) and Intermountain Flyway (n = 23; 1.14 ± 0.16 µg/g dw), and Atlantic Flyway (n = 38; 1.75 ± 0.19 µg/g dw) averaged greater than the Pacific Flyway. Our results indicate that raptor migration monitoring stations provide a cost-effective sampling opportunity for biomonitoring environmental contaminants within and between distinct migration corridors and across time. [ABSTRACT FROM AUTHOR]

Rationale: Environmental stimuli, or cues, associated with the use of drugs such as cocaine are one of the primary drivers of relapse. Thus, identifying mechanisms to reduce the motivational properties of drug cues is an important research goal.Objectives: The purpose of this study was to identify cellular signaling events in the nucleus accumbens (NAc) that are induced when a cocaine cue memory is either extinguished through repeated cue presentation in the absence of drug, or when the memory is reactivated and reconsolidated by a brief cue re-exposure. Signaling events specific to extinction or reconsolidation represent potential targets for pharmacotherapeutics that may enhance extinction or disrupt reconsolidation to reduce the likelihood of relapse.Methods: Male Sprague-Dawley rats were trained to self-administer cocaine paired with an audiovisual cue. Following a period of self-administration, the memory for the cocaine-associated cue was either extinguished, reactivated, or not manipulated (control) 15 min before sacrifice. Tissue from the NAc was subsequently analyzed using mass spectrometry based phosphoproteomics to identify cellular signaling events induced by each condition.Results: Extinction and reconsolidation of the cocaine cue memory produced both common and distinct changes in protein phosphorylation. Notably, there were no significant changes in protein phosphorylation that were modulated in the opposite direction by the two behavioral conditions. Comparison of NAc phosphoproteomic changes to previously identified changes in the basolateral amygdala (BLA) revealed that cue extinction increases phosphorylation at serine (S) 883 of the GABAB receptor subunit 2 and on S14 of syntaxin 1a in both regions, while no common regional signaling events were identified in the reconsolidation group.Conclusions: Phosphoproteomics is a useful tool for identifying signaling cascades involved in different memory processes and revealed novel potential targets for selectively targeting extinction versus reconsolidation of a cocaine cue memory. Furthermore, cross region analysis suggests that cue extinction may produce unique signaling events associated with increased inhibitory signaling. [ABSTRACT FROM AUTHOR]

NMDA receptor signaling is critical for the development of synaptic plasticity, learning, and memory, and dysregulation of NMDAR signaling is implicated in a number of neurological disorders including schizophrenia (SZ). Previous work has demonstrated that the STriatal-Enriched protein tyrosine Phosphatase 61 kDa (STEP61) is elevated in human SZ postmortem cortical samples and after administration of psychotomimetics to cultures or mice. Here, we report that activation of synaptic NMDAR by bicuculline or D-serine results in the ubiquitination and proteasomal degradation of STEP61, and increased surface localization of GluN1/GluN2B receptors. Moreover, bicuculline or D-serine treatments rescue the motor and cognitive deficits in MK-801-treated mice and reduce STEP61 in mouse frontal cortex. These results suggest that STEP61 may contribute to the therapeutic effects of D-serine. [ABSTRACT FROM AUTHOR]

An increase in amyloid-β (Aβ) production is a major pathogenic mechanism associated with Alzheimer's disease (AD), but little is known about possible homeostatic control of the amyloidogenic pathway. Here we report that the amyloid precursor protein (APP) intracellular domain (AICD) downregulates Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1 (WAVE1 or WASF1) as part of a negative feedback mechanism to limit Aβ production. The AICD binds to the Wasf1 promoter, negatively regulates its transcription and downregulates Wasf1 mRNA and protein expression in Neuro 2a (N2a) cells. WAVE1 interacts and colocalizes with APP in the Golgi apparatus. Experimentally reducing WAVE1 in N2a cells decreased the budding of APP-containing vesicles and reduced cell-surface APP, thereby reducing the production of Aβ. WAVE1 downregulation was observed in mouse models of AD. Reduction of Wasf1 gene expression dramatically reduced Aβ levels and restored memory deficits in a mouse model of AD. A decrease in amounts of WASF1 mRNA was also observed in human AD brains, suggesting clinical relevance of the negative feedback circuit involved in homeostatic regulation of Aβ production. [ABSTRACT FROM AUTHOR]

The cAMP and cAMP-dependent protein kinase A (PKA) signaling cascade is a ubiquitous pathway acting downstream of multiple neuromodulators. We found that the phosphorylation of phosphodiesterase-4 (PDE4) by cyclin-dependent protein kinase 5 (Cdk5) facilitated cAMP degradation and homeostasis of cAMP/PKA signaling. In mice, loss of Cdk5 throughout the forebrain elevated cAMP levels and increased PKA activity in striatal neurons, and altered behavioral responses to acute or chronic stressors. Ventral striatum- or D1 dopamine receptor-specific conditional knockout of Cdk5, or ventral striatum infusion of a small interfering peptide that selectively targeted the regulation of PDE4 by Cdk5, produced analogous effects on stress-induced behavioral responses. Together, our results demonstrate that altering cAMP signaling in medium spiny neurons of the ventral striatum can effectively modulate stress-induced behavioral states. We propose that targeting the Cdk5 regulation of PDE4 could be a new therapeutic approach for clinical conditions associated with stress, such as depression. [ABSTRACT FROM AUTHOR]

The immense intercellular and intracellular heterogeneity of the CNS presents major challenges for high-throughput omic analyses. Transcriptional, translational and post-translational regulatory events are localized to specific neuronal cell types or subcellular compartments, resulting in discrete patterns of protein expression and activity. A spatial and quantitative knowledge of the neuroproteome is therefore critical to understanding both normal and pathological aspects of the functional genomics and anatomy of the CNS. Improvements in mass spectrometry allow the profiling of proteins at a sufficient depth to complement results from high-throughput genomic and transcriptomic assays. However, there are challenges in integrating proteomic data with other data modalities and even greater challenges in obtaining comprehensive neuroproteomic data with cell-type specificity. Here we discuss how proteomics should be exploited to enhance high-throughput functional genomic analysis by tighter integration of data analyses. We also discuss experimental strategies to achieve finer cellular and subcellular resolution in transcriptomic and proteomic studies of neural tissues. [ABSTRACT FROM AUTHOR]

The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets. PP1 associates with ≥200 regulatory proteins to form highly specific holoenzymes. These regulatory proteins target PP1 to its point of action within the cell and prime its enzymatic specificity for particular substrates. However, how they direct PP1's specificity is not understood. Here we show that spinophilin, a neuronal PP1 regulator, is entirely unstructured in its unbound form, and it binds PP1 through a folding-upon-binding mechanism in an elongated fashion, blocking one of PP1's three putative substrate binding sites without altering its active site. This mode of binding is sufficient for spinophilin to restrict PP1's activity toward a model substrate in vitro without affecting its ability to dephosphorylate its neuronal substrate, glutamate receptor 1 (GluR1). Thus, our work provides the molecular basis for the ability of spinophilin to dictate PP1 substrate specificity. [ABSTRACT FROM AUTHOR]

This paper develops an R&D-based growth model and calibrates the model to aggregate data of the US economy to quantify a structural relationship between patent length, R&D and consumption. Under parameter values that match the empirical flow-profit depreciation rate of patents and other key features of the US economy, extending the patent length beyond 20 years leads to a negligible increase in R&D despite equilibrium R&D underinvestment. In contrast, shortening the patent length leads to a significant reduction in R&D and consumption. Finally, this paper also analytically derives and quantifies a dynamic distortionary effect of patent length on capital investment. [ABSTRACT FROM AUTHOR]

Loss of cell division cycle 2 (Cdc2, also known as Cdk1) activity after cyclin B degradation is necessary, but not sufficient, for mitotic exit. Proteins phosphorylated by Cdc2 and downstream mitotic kinases must be dephosphorylated. We report here that protein phosphatase-1 (PP1) is the main catalyst of mitotic phosphoprotein dephosphorylation. Suppression of PP1 during early mitosis is maintained through dual inhibition by Cdc2 phosphorylation and the binding of inhibitor-1. Protein kinase A (PKA) phosphorylates inhibitor-1, mediating binding to PP1. As Cdc2 levels drop after cyclin B degradation, auto-dephosphorylation of PP1 at its Cdc2 phosphorylation site (Thr 320) allows partial PP1 activation. This promotes PP1-regulated dephosphorylation at the activating site of inhibitor-1 (Thr 35) followed by dissociation of the inhibitor-1–PP1 complex and then full PP1 activation to promote mitotic exit. Thus, Cdc2 both phosphorylates multiple mitotic substrates and inhibits their PP1-mediated dephosphorylation. [ABSTRACT FROM AUTHOR]

Abnormalities of striatal function have been implicated in several major neurological and psychiatric disorders, including Parkinson's disease, schizophrenia and depression. Adenosine, via activation of A2A receptors, antagonizes dopamine signaling at D2 receptors and A2A receptor antagonists have been tested as therapeutic agents for Parkinson's disease. We found a direct physical interaction between the G protein–coupled A2A receptor (A2AR) and the receptor tyrosine kinase fibroblast growth factor receptor (FGFR). Concomitant activation of these two classes of receptors, but not individual activation of either one alone, caused a robust activation of the MAPK/ERK pathway, differentiation and neurite extension of PC12 cells, spine morphogenesis in primary neuronal cultures, and cortico-striatal plasticity that was induced by a previously unknown A2AR/FGFR-dependent mechanism. The discovery of a direct physical interaction between the A2A and FGF receptors and the robust physiological consequences of this association shed light on the mechanism underlying FGF functions as a co-transmitter and open new avenues for therapeutic interventions. [ABSTRACT FROM AUTHOR]

Dopamine orchestrates motor behaviour and reward-driven learning. Perturbations of dopamine signalling have been implicated in several neurological and psychiatric disorders, and in drug addiction. The actions of dopamine are mediated in part by the regulation of gene expression in the striatum, through mechanisms that are not fully understood. Here we show that drugs of abuse, as well as food reinforcement learning, promote the nuclear accumulation of 32-kDa dopamine-regulated and cyclic-AMP-regulated phosphoprotein (DARPP-32). This accumulation is mediated through a signalling cascade involving dopamine D1 receptors, cAMP-dependent activation of protein phosphatase-2A, dephosphorylation of DARPP-32 at Ser 97 and inhibition of its nuclear export. The nuclear accumulation of DARPP-32, a potent inhibitor of protein phosphatase-1, increases the phosphorylation of histone H3, an important component of nucleosomal response. Mutation of Ser 97 profoundly alters behavioural effects of drugs of abuse and decreases motivation for food, underlining the functional importance of this signalling cascade. [ABSTRACT FROM AUTHOR]

The heterozygous reeler mouse has been proposed as a genetic mouse model of schizophrenia based on several neuroanatomical and behavioral similarities between these mice and patients with schizophrenia. However, the effect of reelin haploinsufficiency on one of the cardinal symptoms of schizophrenia, the impairment of prefrontal-cortex-dependent cognitive function, has yet to be determined. Here, we investigated multiple aspects of cognitive function in heterozygous reeler mice that are known to be impaired in schizophrenic patients. Heterozygous reeler mice were assessed for (1) cognitive flexibility in an instrumental reversal learning task, (2) impulsivity in an inhibitory control task, (3) attentional function in a three-choice serial reaction time task, and (4) working memory in a delayed matching-to-position task. No differences were found between heterozygous reeler mice and wild-type littermate controls in any prefrontal-related cognitive measures. However, heterozygous reeler mice showed deficits in the acquisition of two operant tasks, consistent with a role for reelin in certain forms of learning. These findings suggest that heterozygous reeler mice may not be an appropriate model for the core prefrontal-dependent cognitive deficits observed in schizophrenia, but may model more general learning deficits that are associated with many psychiatric disorders. [ABSTRACT FROM AUTHOR]

WAVE1—the Wiskott–Aldrich syndrome protein (WASP)-family verprolin homologous protein 1—is a key regulator of actin-dependent morphological processes in mammals, through its ability to activate the actin-related protein (Arp2/3) complex. Here we show that WAVE1 is phosphorylated at multiple sites by cyclin-dependent kinase 5 (Cdk5) both in vitro and in intact mouse neurons. Phosphorylation of WAVE1 by Cdk5 inhibits its ability to regulate Arp2/3 complex-dependent actin polymerization. Loss of WAVE1 function in vivo or in cultured neurons results in a decrease in mature dendritic spines. Expression of a dephosphorylation-mimic mutant of WAVE1 reverses this loss of WAVE1 function in spine morphology, but expression of a phosphorylation-mimic mutant does not. Cyclic AMP (cAMP) signalling reduces phosphorylation of the Cdk5 sites in WAVE1, and increases spine density in a WAVE1-dependent manner. Our data suggest that phosphorylation/dephosphorylation of WAVE1 in neurons has an important role in the formation of the filamentous actin cytoskeleton, and thus in the regulation of dendritic spine morphology. [ABSTRACT FROM AUTHOR]

Mice lacking DARPP-32, a striatal-enriched phosphoprotein, show abnormal behavioral and biochemical responses to cocaine, but the role of individual phosphorylation sites in DARPP-32 in these responses is unknown. We show here that mutation of Thr-34 in DARPP-32 mimicked the behavioral phenotype of the constitutive DARPP-32 knockout in cocaine-induced place conditioning, locomotor activity, and sensitization paradigms. In contrast, mutations of Thr75 did not affect conditioned place preference or the acute locomotor response to cocaine, but DARPP-32 Thr-75 mutants showed no locomotor sensitization in response to repeated cocaine administration. Consistent with these behavioral findings, we found that cocaine regulation of gene expression in striatum, including the acute induction of the immediate early genes c-fos and arc (activity-regulated cytoskeletal-associated gene), was abolished in DARPP-32 Thr-34 mutants, but not in Thr-75 mutants. Similarly, induction of the transcription factor ΔFosB in the ventral striatum (nucleus accumbens) by chronic cocaine was diminished by the Thr-34, but not the Thr-75, mutation. These findings highlight distinct roles of the Thr-34 and Thr-75 phosphorylation sites of DARPP-32 in mediating short- and long-term behavioral and biochemical actions of cocaine.Neuropsychopharmacology (2006) 31, 555–562. doi:10.1038/sj.npp.1300832; published online 3 August 2005 [ABSTRACT FROM AUTHOR]

Amyloid-β peptide is elevated in the brains of patients with Alzheimer disease and is believed to be causative in the disease process. Amyloid-β reduces glutamatergic transmission and inhibits synaptic plasticity, although the underlying mechanisms are unknown. We found that application of amyloid-β promoted endocytosis of NMDA receptors in cortical neurons. In addition, neurons from a genetic mouse model of Alzheimer disease expressed reduced amounts of surface NMDA receptors. Reducing amyloid-β by treating neurons with a γ-secretase inhibitor restored surface expression of NMDA receptors. Consistent with these data, amyloid-β application produced a rapid and persistent depression of NMDA-evoked currents in cortical neurons. Amyloid-β–dependent endocytosis of NMDA receptors required the α-7 nicotinic receptor, protein phosphatase 2B (PP2B) and the tyrosine phosphatase STEP. Dephosphorylation of the NMDA receptor subunit NR2B at Tyr1472 correlated with receptor endocytosis. These data indicate a new mechanism by which amyloid-β can cause synaptic dysfunction and contribute to Alzheimer disease pathology. [ABSTRACT FROM AUTHOR]

Abstract The total ash and acid-insoluble ash contents of Chinese materia medica (CMM) have been widely used as one of the indices to illustrate the quality as well as purity of herbal medicines. Such an approach has also been adopted by many official organizations in writing monographs for pharmacopoeia worldwide. In the present study, a full validation on the methodology was conducted with the inclusion of various commonly encountered matrices in herbs, namely, leaf, flower, fruit, stem and root. The practice ensures method suitability for a wider scope of matrix variation in CMM. Moreover, in order to comply with the technical requirement of International Organization of Standardization 17025 quality assurance system, the concept of measurement uncertainty has also been incorporated in the present study. Measurement uncertainties for total ash and acid-insoluble ash contents have been estimated. [ABSTRACT FROM AUTHOR]

ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR's two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2). Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP, and hydrolysis of the ATP causes dimer dissociation. Here, using single-channel recording methods on intact CFTR molecules, we directly follow opening and closing of the channel gates, and relate these occurrences to ATP-mediated events in the NBDs. We find that energetic coupling between two CFTR residues, expected to lie on opposite sides of its predicted NBD1-NBD2 dimer interface, changes in concert with channel gating status. The two monitored side chains are independent of each other in closed channels but become coupled as the channels open. The results directly link ATP-driven tight dimerization of CFTR's cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains. This establishes a molecular mechanism, involving dynamic restructuring of the NBD dimer interface, that is probably common to all members of the ABC protein superfamily. [ABSTRACT FROM AUTHOR]

The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-a as a fundamental regulator of cardiac contractility and Ca2+ handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-a into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-a activity affects dephosphorylation of the sarcoplasmic reticulum Ca2+ ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca2+ loading and the Ca2+ transient. PKC-a directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-a on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-a functions as a nodal integrator of cardiac contractility by sensing intracellular Ca2+ and signal transduction events, which can profoundly affect propensity toward heart failure. [ABSTRACT FROM AUTHOR]

The intracellular mechanism(s) by which a cell determines the duration of extracellular signalregulated kinase (ERK) activation is not well understood. We have investigated the role of STEP, a striatal-enriched tyrosine phosphatase, in the regulation of ERK activity in rat neurons. Glutamatemediated activation of NMDA receptors leads to the rapid but transient phosphorylation of ERK in cultured neurons. Here we show that activation of NMDA receptors led to activation of STEP, which limited the duration of ERK activity as well as its translocation to the nucleus and its subsequent downstream nuclear signaling. In neurons, STEP is phosphorylated and inactive under basal conditions. NMDA-mediated influx of Ca[sup 2+], but not increased intracellular Ca[sup 2+] from other sources, leads to activation of the Ca[sup 2+]-dependent phosphatase calcineurin and the dephosphorylation and activation of STEP. We have identified an important mechanism involved in the regulation of ERK activity in neurons that highlights the complex interplay between serine/threonine and tyrosine kinases and phosphatases. [ABSTRACT FROM AUTHOR]

Shows that the stimulatory effect of caffeine on motor activity in mice was greatly reduced following genetic deletion of dopamine- and cyclic adenosine monophosphate-regulated phosphoprotein of relative molecular mass (DARPP-32). Involvement of DARPP-32 in the behavioral response to blockade of adenosine A[sub 2A] receptors; Expression of striato-pallidal neurons; Determination of adenosine A[sub 1] receptor binding.

Presents an experiment to determine the effect of chronic exposure to cocaine. Methods; Results; Conclusion that changes in cyclin-dependent protein kinase 5 (Cdk5) levels and resulting alterations in signalling involving dopamine receptors contribute to adaptive changes in the brain related to cocaine addiction.

We demonstrate a rapid and complex effect of N-methyl-D-aspartate receptor (NMDAR) activation on synaptic protein synthesis in the superior colliculi of young rats. Within minutes of receptor activation, translation of alpha Ca[sup 2+]/calmodulin dependent kinase II (αCamK II) was increased, whereas total protein synthesis was reduced. NMDAR activation also increased phosphorylation of eukaryotic elongation factor 2 (eEF2), a process known to inhibit protein translation by reducing peptide chain elongation. Low doses of cycloheximide, which reduce elongation rate independently of eEF2 phosphorylation, decreased overall protein synthesis but increased αCaMK II synthesis. These observations suggest that regulation of peptide elongation via eEF2 phosphorylation can link NMDAR activation to local increases in the synthesis of specific proteins during activity-dependent synaptic change. [ABSTRACT FROM AUTHOR]

Modulation of AMPA-type glutamate channels is important for synaptic plasticity. Here we provide physiological evidence that the activity of AMPA channels is regulated by protein phosphatase 1 (PP-1) in neostriatal neurons and identify two distinct molecular mechanisms of this regulation. One mechanism involves control of PP-1 catalytic activity by DARPP-32, a dopamine- and cAMP-regulated phosphoprotein highly enriched in neostriatum. The other involves binding of PP-1 to spinophilin, a protein that colocalizes PP-1 with AMPA receptors in postsynaptic densities. The results suggest that regulation of anchored PP-1 is important for AMPA-receptor-mediated synaptic transmission and plasticity. [ABSTRACT FROM AUTHOR]

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, here we profile m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen. Adenovirus transcripts contain N6-methyladenosine (m6A) modification. Here the authors profile m6A modification sites on adenovirus mRNAs using Illumina meRIP-Seq and nanopore direct RNA sequencing, and showcase a role for m6A in splicing of viral late mRNAs. [ABSTRACT FROM AUTHOR]