Miracle-dz/UniFAR-FTUs huggingface dataset:
Interaction of catechol and non‐catechol substrates with externally or internally facing dopamine transporters

Our previous work suggested that collapsing the Na+ gradient and membrane potential converts the dopamine (DA) transporter (DAT) to an inward‐facing conformation with a different substrate binding profile. Here, DAT expressing human embryonic kidney 293 cells were permeabilized with digitonin, disrupting ion/voltage gradients and allowing passage of DAT substrates. The potency of p‐tyramine and other non‐catechols (d‐amphetamine, β‐phenethylamine, MPP+) in inhibiting cocaine analog binding to DAT in digitonin‐treated cells was markedly weakened to a level similar to that observed in cell‐free membranes. In contrast, the potency of DA and another catechol, norepinephrine, was not significantly changed by the same treatment, whereas epinephrine showed only a modest reduction. These findings suggest that catechol substrates interact symmetrically with both sides of DAT and non‐catechol substrates, favoring binding to outward‐facing transporter. In the cocaine analog binding assay, the mutant W84L displayed enhanced intrinsic binding affinity for substrates in interacting with both outward‐ and inward‐facing states; D313N showed wild‐type‐like symmetric binding; but D267L and E428Q showed an apparent improvement in the permeation pathway from the external face towards the substrate site. Thus, the structure of both substrate and transporter play a role in the sidedness and mode of interaction between them.
Tumor necrosis factor‐α‐induced neutral sphingomyelinase‐2 modulates synaptic plasticity by controlling the membrane insertion of NMDA receptors

The insertion and removal of NMDA receptors from the synapse are critical events that modulate synaptic plasticity. While a great deal of progress has been made on understanding the mechanisms that modulate trafficking of NMDA receptors, we do not currently understand the molecular events required for the fusion of receptor containing vesicles with the plasma membrane. Here, we show that sphingomyelin phosphodiesterase 3 (also known as neutral sphingomyelinase‐2) is critical for tumor necrosis factor (TNF) α‐induced trafficking of NMDA receptors and synaptic plasticity. TNFα initiated a rapid increase in ceramide that was associated with increased surface localization of NMDA receptor NR1 subunits and a specific clustering of NR1 phosphorylated on serines 896 and 897 into lipid rafts. Brief applications of TNFα increased the rate and amplitude of NMDA‐evoked calcium bursts and enhanced excitatory post‐synaptic currents. Pharmacological inhibition or genetic mutation of neutral sphingomyelinase‐2 prevented TNFα‐induced generation of ceramide, phosphorylation of NR1 subunits, clustering of NR1, enhancement of NMDA‐evoked calcium flux and excitatory post‐synaptic currents.
Late endocytic dysfunction as a putative cause of amyloid fibril formation in Alzheimer’s disease

The assembly of amyloid β‐protein to amyloid fibrils is a critical event in Alzheimer’s disease. Evidence exists that endocytic pathway abnormalities, including the enlargement of early endosomes, precede the extraneuronal amyloid fibril deposition in the brain. We determined whether endocytic dysfunction potently promotes the assembly of amyloid β‐protein on the surface of cultured cells. Blocking the early endocytic pathway by clathrin suppression, inactivation of small GTPases, removal of membrane cholesterol, and Rab5 knockdown did not result in amyloid fibril formation on the cell surface from exogenously added soluble amyloid β‐protein. In contrast, blocking the late endocytic pathway by Rab7 suppression markedly induced the amyloid fibril formation in addition to the enlargement of early endosomes. Notably, a monoclonal antibody specific to GM1‐ganglioside‐bound amyloid β‐protein, an endogenous seed for Alzheimer amyloid, completely blocks the amyloid fibril formation. Our results suggest that late but not early endocytic dysfunction contributes to the amyloid fibril formation by facilitating the generation of amyloid seed in the Alzheimer’s brain.
A Medical Education Model for Collaborative Chronic Disease Management

Purpose: The purpose of this study was to evaluate the impact of a curriculum enhancement project on physician assistant (PA) students' abilities, attitudes, and preparedness to provide collaborative disease management. Method: The authors identified competencies needed for collaborative chronic disease management and developed curriculum interventions. The interventions were piloted with a single cohort of PA students at the Jefferson College of Health Sciences. Full implementation and evaluation was conducted with a separate cohort of students. Students self‐reported their (1) previous exposure to chronic disease; (2) abilities related to collaborative disease management; (3) attitudes towards chronic disease patients and collaborative disease management; (4) preparedness to provide chronic disease care; (5) perceptions of the benefit of the community medicine rotation and; (6) self‐efficacy to support patient self‐management. Results: Significant improvements (p < .05) were noted in all 10 ability areas. Few significant changes in attitudes occurred though many attitudes were positively maintained. Significant changes were found for students' preparedness for chronic disease care. Students also reported a high level of self‐efficacy to provide patient self‐management support. Conclusions: Findings indicate that curriculum interventions can positively affect PA students' collaborative disease management abilities and sense of preparedness to manage patients with chronic disease. While curriculum enhancement may not significantly improve attitudes, the study suggests that interventions may be supportive of already favorable attitudes. Further study is needed to determine the most time‐ and cost‐effective methods for facilitating improvement in both ability and attitude.
Enhanced antinociceptive response to intracerebroventricular kyotorphin in Pept2 null mice

l‐Kyotorphin (l‐KTP), an endogenous analgesic neuropeptide, is a substrate for aminopeptidases and a proton‐coupled oligopeptide transporter, PEPT2. This study examined the CSF efflux, antinociceptive response, and hydrolysis kinetics in brain of l‐KTP and its synthetic diastereomer d‐kyotorphin (d‐KTP) in wild‐type and Pept2 null mice. CSF clearance of l‐KTP was slower in Pept2 null mice than in wild‐type animals, and this difference was reflected in greater l‐KTP‐induced analgesia in Pept2 null mice. Moreover, dose‐response analyses showed that the ED50 of l‐KTP in Pept2‐deficient animals was one‐fifth of the value observed in Pept2‐competent animals (4 vs. 21 nmol for null vs. wild‐type mice, respectively). In contrast, the ED50 of d‐KTP was very similar between the two genotypes (9–10 nmol). Likewise, there was little difference between genotypes in slope factor or baseline effects of l‐KTP and d‐KTP. The enhanced antinociceptive response to l‐KTP in Pept2 null mice could not be explained by differences in neuropeptide degradation as Vmax and Km values did not differ between genotypes. Our results demonstrate that PEPT2 can significantly impact the analgesic response to an endogenous neuropeptide by altering CSF (and presumably brain interstitial fluid) concentrations and that it may influence the disposition and response to exogenous peptide/mimetic substrates.
Normal protein composition of synapses in Ts65Dn mice: a mouse model of Down syndrome

Down syndrome (DS) is the most prevalent form of intellectual disability caused by the triplication of ∼230 genes on chromosome 21. Recent data in Ts65Dn mice, the foremost mouse model of DS, strongly suggest that cognitive impairment in individuals with DS is a consequence of reduced synaptic plasticity because of chronic over‐inhibition. It remains unclear however whether changes in plasticity are tied to global molecular changes at synapses, or are due to regional changes in the functional properties of synaptic circuits. One interesting framework for evaluating the activity state of the DS brain comes from in vitro studies showing that chronic pharmacological silencing of neuronal excitability orchestrates stereotyped changes in the protein composition of synaptic junctions. In the present study, we use proteomic strategies to evaluate whether synapses from the Ts65Dn cerebrum carry signatures characteristic of inactive cortical neurons. Our data reveal that synaptic junctions do not exhibit overt alterations in protein composition. Only modest changes in the levels of synaptic proteins and in their phosphorylation are observed. This suggests that subtle changes in the functional properties of specific synaptic circuits rather than large‐scale homeostatic shifts in the expression of synaptic molecules contribute to cognitive impairment in people with DS.
Diffuse Myocardial Fibrosis in Children After Heart Transplantations: A Magnetic Resonance T1 Mapping Study

Background It is unclear whether the myocardium undergoes accelerated fibrotic remodeling in children after heart transplantation (HTx). Methods In this prospective study, cardiac magnetic resonance (CMR) studies in 17 patients 1.3 years (median, range 0.03-12.6 years) after HTx (mean age, 9.8 ± 6.2 years; 8 girls) were compared to CMR studies in 9 healthy controls (mean age, 12.4 ± 2.4 years; 4 girls). T1 measurements were performed at a midventricular short axis slice before (ie, native T1 times) and after the application of 0.2 mmol/kg gadopentetate dimeglumine in the interventricular septum, left ventricular (LV) free wall and encompassing the entire LV myocardium. The tissue-blood partition coefficient (TBPC), reflecting the degree of diffuse myocardial fibrosis, was calculated as a function of the ratio of T1 change of myocardium compared to blood. Native T1 times and TBPC were correlated with echocardiographic parameters of diastolic function. Results Native T1 times were significantly higher in HTx patients compared to controls in all regions assessed (LV free wall 973 ± 42 vs 923 ± 12 ms; P < 0.005; interventricular septum 1003 ± 31 vs 974 ± 21 ms, P < 0.05; entire LV myocardium 987 ± 33 vs 951 ± 16 ms; P < 0.005) and correlated with LV E/e' as an echocardiographic marker of diastolic dysfunction (r = 0.54, P < 0.05). The TBPC was elevated in the LV free wall (0.45 ± 0.06 vs 0.40 ± 0.03, P < 0.005) and the entire LV myocardium (0.47 ± 0.06 vs 0.43 ± 0.03, P < 0.05). Conclusions Evidence of diffuse myocardial fibrosis and is already present in children after HTx. It appears to be associated with diastolic dysfunction.
Atlastin‐1, the dynamin‐like GTPase responsible for spastic paraplegia SPG3A, remodels lipid membranes and may form tubules and vesicles in the endoplasmic reticulum

We examined the effects of wild‐type and mutant atlastin‐1 on vesicle transport in the endoplasmic reticulum (ER)‐Golgi interface and vesicle budding from ER‐derived microsomes using the temperature‐sensitive reporter vesicular stomatitis virus glycoprotein (VSV‐G), and the ability of purified atlastin‐1 to form tubules or vesicles from protein‐free phosphatidylserine liposomes. A GTPase domain mutation (T162P) altered the cellular distribution of the ER, but none of the mutations studied significantly affected transport from the ER to the Golgi apparatus. The mutations also had no significant effect on the incorporation of VSV‐G into vesicles formed from ER microsomes. Atlastin‐1, however, was also incorporated into microsome‐derived vesicles, suggesting that it might be implicated in vesicle formation. Purified atlastin‐1 transformed phosphatidylserine liposomes into branched tubules and polygonal networks of tubules and vesicles, an action inhibited by GDP and the synthetic dynamin inhibitor dynasore. The GTPase mutations T162P and R217C decreased but did not totally prevent this action; the C‐terminal transmembrane domain mutation R495W was as active as the wild‐type enzyme. Similar effects were observed in human embryonic kidney cells over‐expressing mutant atlastin‐1. We concluded that atlastin‐1, like dynamin, might be implicated in membrane tubulation and vesiculation and participated in the formation as well as the function of the ER.
Neuroprotective mesenchymal stem cells are endowed with a potent antioxidant effect in vivo

Experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis, is characterized by demyelination, inflammation and neurodegeneration of CNS in which free radicals play a role. Recently, the efficacy of murine mesenchimal stem cells (MSCs) as treatment of EAE induced in mice by the encephalitogenic peptide MOG(35–55) was demonstrated. The present study analyzed some markers of oxidative stress, inflammation/degeneration and apoptosis such as metallothioneins (MTs), antioxidant enzymes (superoxide dismutase, catalase and glutathione‐S‐transferase), poly(ADP‐ribose) polymerase‐1 and p53 during EAE progression and following MSC treatment. Expression of the three brain MT isoforms increased significantly in EAE mice compared with healthy controls, but while expression of MT‐1 and MT‐3 increased along EAE course, MT‐2 was up‐regulated at the onset, but returned to levels similar to those of controls in chronic phase. The changes in the transcription and activity of the antioxidant enzymes and in expression of poly(ADP‐ribose) polymerase‐1 and p53 showed the same kinetics observed for MT‐1 and MT‐3 during EAE. Interestingly, i.v. administration of MSCs reduced the EAE‐induced increases in levels/activities of all these proteins. These results support an antioxidant and neuroprotective activity for MSCs that was also confirmed in vitro on neuroblastoma cells exposed to an oxidative insult.
Minimal Detectable Change of the London Chest Activity of Daily Living Scale in Patients With COPD

BACKGROUND: Dyspnea and fatigue frequently impair the ability to perform activities of daily living (ADL) in patients with chronic obstructive pulmonary disease. Questionnaires and scales have been used to assess limitations in ADL. The London Chest Activity of Daily Living (LCADL) scale is responsive to intervention to a higher extent when compared with other tools. However, the minimal detectable change (MDC) for this scale remains unknown. The aim of this study was to determine the MDC for functional status improvement measured by the LCADL scale in patients with chronic obstructive pulmonary disease. METHODS: Forty patients (20 men, 66 ± 7 years, forced expired volume in 1 second 44 ± 16% predicted) participated in a 3-month high-intensity exercise training program. Before and after the protocol, participants complete the LCADL scale and the Saint George Respiratory Questionnaire. To calculate the MDC, we used the standard error of measurement and the effect size (distribution-based estimates). RESULTS: There was improvement in the LCADL self-care, domestic and leisure domains, and total score after the training program, with a strong trend for improvement in the physical activity domain. The MDC estimated for the LCADL were 0.89, 2.60, 0.44, 0.58, and 3.88 points for self-care, domestic, physical, and leisure domains, and total score, respectively. Correlations between changes in LCADL and in the Saint George Respiratory Questionnaire were weak (r < 0.4 for all). CONCLUSION: This study provided the MDC for the domains and total score of the LCADL. A change of 4 points in the total score of the LCADL can be interpreted as a meaningful change.
Identification and characterization of a novel amphioxus dopamine D1‐like receptor

Dopamine receptors function to control many aspects of motor control and other forms of behaviour in both vertebrates and invertebrates. They can be divided into two main groups (D1 and D2) based on sequence similarity, ligand affinity and effector coupling. However, little is known about the pharmacology and functionality of dopamine receptors in the deuterostomian invertebrates, such as the cephalochordate amphioxus (Branchiostoma floridae) which has recently been placed as the most basal of all the chordates. A bioinformatic study shows that amphioxus has at least three dopamine D1‐like receptor sequences. One of these receptors, AmphiD1/β, was found to have high levels of sequence similarity to both vertebrate D1 receptors and to β‐adrenergic receptors. Here, we report on the cloning of AmphiD1/β from an adult amphioxus cDNA library, and its pharmacological characterization subsequent to its expression in both mammalian cell lines and Xenopus oocytes. It was found that AmphiD1/β has a similar pharmacology to vertebrate D1 receptors, including responding to benzodiazepine ligands. The pharmacology of the receptor exhibits ‘agonist‐specific coupling’ depending upon the second messenger pathway to which it is linked. Moreover, no pharmacological characteristics were observed to suggest that AmphiD1/β may be an amphioxus orthologue of vertebrate β‐adrenergic receptors.
Regulation of major efflux transporters under inflammatory conditions at the blood‐brain barrier in vitro

ATP‐driven efflux transport proteins at the blood‐brain barrier protect the healthy brain but impede pharmacotherapy of the disordered CNS. To investigate the question how ATP‐binding cassette (ABC)‐transporters are regulated during inflammation or infection we analysed the effects of the cytokines tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) on the expression of brain multidrug resistance proteins in primary cultures of porcine brain capillary endothelial cells. We found that TNF‐α and IL‐1β rapidly decrease Abcg2 (BMDP/BCRP) mRNA expression within 6 h. After 24 and 48 h the mRNA level came back to control values. The mRNA reduction at 6 h was counter‐regulated by the anti‐inflammatory glucocorticoid hydrocortisone. Abcg2 protein levels were suppressed at prolonged stimulations but not after 6 h of stimulation which correlates with Abcg2 specific substrate uptake measurements. Abcb1 (p‐glycoprotein) protein expression was transiently increased after TNF‐α addition within 6 h of incubation followed by a reduction after 24 and 48 h whereas the Abcb1 mRNA levels were not changed. IL‐1β caused a continuous decrease in protein expression of both ABC‐transporters. Long‐term treatment with an assumed TNF‐α‐downstream agent, the vasoconstrictor endothelin‐1, induced Abcg2 protein expression but suppressed Abcb1. Other efflux pumps like multidrug resistance‐associated proteins/Abcc were rarely affected. The present results imply a complex regulation of the two most abundant ABC‐transporters at the blood‐brain barrier during early inflammation stages suggesting that Abcb1 (p‐glycoprotein) is an early target of TNF‐α‐signalling counterbalanced by Abcg2.
Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain‐derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson’s disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole‐cell patch‐clamp recordings, l‐glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration‐response plots of differentiated NPCs yielded an EC50 of 2.2 μM for glutamate and an EC50 of 36 μM for NMDA. Glutamate‐induced currents were markedly inhibited by memantine in contrast to 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) suggesting a higher density of functional NMDA than alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptors. NMDA‐evoked currents and calcium signals were blocked by the NR2B‐subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage‐gated calcium channels by verapamil abolished AMPA‐induced calcium responses but only partially reduced NMDA‐evoked transients suggesting the expression of calcium‐impermeable, GluR2‐containing AMPA receptors. Quantitative real‐time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 μM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase‐immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.
Translation of Incremental Talk Test Responses to Steady-State Exercise Training Intensity

PURPOSE: The Talk Test (TT) is a submaximal, incremental exercise test that has been shown to be useful in prescribing exercise training intensity. It is based on a subject's ability to speak comfortably during exercise. This study defined the amount of reduction in absolute workload intensity from an incremental exercise test using the TT to give appropriate absolute training intensity for cardiac rehabilitation patients. METHODS: Patients in an outpatient rehabilitation program (N = 30) performed an incremental exercise test with the TT given every 2-minute stage. Patients rated their speech comfort after reciting a standardized paragraph. Anything other than a “yes” response was considered the “equivocal” stage, while all preceding stages were “positive” stages. The last stage with the unequivocally positive ability to speak was the Last Positive (LP), and the preceding stages were (LP-1 and LP-2). Subsequently, three 20-minute steady-state training bouts were performed in random order at the absolute workload at the LP, LP-1, and LP-2 stages of the incremental test. Speech comfort, heart rate (HR), and rating of perceived exertion (RPE) were recorded every 5 minutes. RESULTS: The 20-minute exercise training bout was completed fully by LP (n = 19), LP-1 (n = 28), and LP-2 (n = 30). Heart rate, RPE, and speech comfort were similar through the LP-1 and LP-2 tests, but the LP stage was markedly more difficult. CONCLUSION: Steady-state exercise training intensity was easily and appropriately prescribed at intensity associated with the LP-1 and LP-2 stages of the TT. The LP stage may be too difficult for patients in a cardiac rehabilitation program.
Diminished hippocalcin expression in Huntington’s disease brain does not account for increased striatal neuron vulnerability as assessed in primary neurons

Hippocalcin is a neuronal calcium sensor protein previously implicated in regulating neuronal viability and plasticity. Hippocalcin is the most highly expressed neuronal calcium sensor in the medium spiny striatal output neurons that degenerate selectively in Huntington’s disease (HD). We have previously shown that decreased hippocalcin expression occurs in parallel with the onset of disease phenotype in mouse models of HD. Here we show by in situ hybridization histochemistry that hippocalcin RNA is also diminished by 63% in human HD brain. These findings lead us to hypothesize that diminished hippocalcin expression might contribute to striatal neurodegeneration in HD. We tested this hypothesis by assessing whether restoration of hippocalcin expression would decrease striatal neurodegeneration in cellular models of HD comprising primary striatal neurons exposed to mutant huntingtin, the mitochondrial toxin 3‐nitropropionic acid or an excitotoxic concentration of glutamate. Counter to our hypothesis, hippocalcin expression did not improve the survival of striatal neurons under these conditions. Likewise, expression of hippocalcin together with interactor proteins including the neuronal apoptosis inhibitory protein did not increase the survival of striatal cells in cellular models of HD. These results indicate that diminished hippocalcin expression does not contribute to HD‐related neurodegeneration.