bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthetic Cannabinoids: Rapidly Emerging Drugs of Abuse
Authors: Radominska‐Pandya Anna , Moran Jeff , Fantegrossi William , Prather Paul 
Published: June 2021

K2, also called “Spice” or “synthetic marijuana,” is a rapidly emerging drug of abuse that possesses psychoactive properties similar to those of Δ9‐tetrahydrocannabinol (Δ9‐THC). K2 use has exploded in many sections of the population including teenagers and first time drug users. Use of K2 can result in extreme agitation, hallucinations, supraventricular tachycardia, syncope, and seizures. The presence of more than 20 different K2‐aminoalkyl indoles (AAIs) have been reported in various K2s, but the two most commonly observed are JWH‐018 and JWH‐073; however, new generations of structurally related compounds are constantly being produced. Our studies demonstrate for the first time that the native K2s undergoes extensive metabolism by cytochrome P450s and UDP glucuronosyltransferases. Due to the activity of these enzymes, a variety of hydroxylated metabolites, have been biosythesized and excreted in human urine primarly as glucuronidated conjugates. These metabolites were identified and characterized using LC‐MS/MS and HPLC‐UV/Vis, and steady state kinetic analyses were also investigated. Consequently, we hypothesize that the severe effects observed for some K2 users could be related to a defect in their metabolism. We have also shown that these K2 products cause psychoactive effects similar to those of Δ9‐THC by activating cannabinoid receptors (CBRs) in the central nervous system. Moreover, CBRs were able to bind several hydroxylated and glucuronidated K2‐AAI metabolites with an affinity similar to that of the parent compound. The new generation of K2s like AB‐Pinaca exhibit higher affinity, potency, and efficacy at CBRs relative to THC. Furthermore, phase one metabolites of AB‐Pinaca retain affinity and full agonist activity at CBRs. Chronic treatment of cells with AB‐Pinaca produces greater desensitization of CBRs (e.g., tolerance) than similar exposure to THC. The higher affinity and activity of AB‐Pinaca at CBRs relative to THC might be expected to produce enhanced psychotropic and adverse effects, contributing to the reported abuse and marked toxicity of this novel SCB. Finally, our in vivo data demonstrates that K2 metabolites retain biological activity in mice. Therefore, we postulate that synthetic cannabinoids and their derivatives could act as ligands for CBRs. The fact that some hydroxylated derivatives and their glucuronides can retain their biological activity makes the study of these compounds essential for understanding their severe toxicity and pharmacokinetics/dynamics.Support or Funding Information[DoD‐W81XWH1110795, NIH/NIDA DA039143 to ARP, PLP, WF and JM]
Tissue Inflammatory Responses in an Anesthetized Rat Hemorrhage Model Associated with Endothelial Dysfunction
Authors: Dubick Michael A , Torres Luciana N , Barr Johnny L , Torres Filho Ivo P 
Published: June 2021

We have demonstrated endothelial dysfunction such as reduction in glycocalyx thickness and increased permeability in venules in cremaster muscle following hemorrhage and fluid resuscitation with a crystalloid or a colloid in a rat model. The present study further characterizes the model by measuring indices of inflammation and oxidative and nitrosative stress in tissues from these animals. Anesthetized Sprague‐Dawley rats (n=4–5/group) were bled 40% of their blood volume, subjected to a 30 min shock period and resuscitated with limited volumes of normal saline (NS) or 5% albumin (A), or no treatment (Hem). At 1hr after resuscitation, rats were euthanized and liver, kidney, lung and skeletal muscle were collected and assayed for cytokines and select indices of nitrosative and oxidative stress. In liver and kidney, IL‐1β, IL‐6 and TNF‐α levels were 2–3‐fold higher in the NS than the A group and about 2‐fold higher than the Hem group. IL‐10 levels were also significantly higher in the NS group than the other groups. Similar responses were observed in lung from these groups, while in muscle, only IL‐1β and IL‐6 levels were significantly higher. In contrast, no significant differences were observed in all tissues in nitric oxide, total antioxidant or reduced glutathione concentrations nor xanthine oxidase activity among the groups. In this model of hemorrhagic shock, NS induced the highest inflammatory response, while 5% albumin was associated with the least response. Interestingly, these current observations were related to the degree of endothelial dysfunction observed previously. Taken together these data suggest that the endothelial dysfunction observed in hemorrhagic shock and fluid resuscitation is associated with an inflammatory response without a major nitrosative or oxidative stress component.Support or Funding InformationFunded by the US Army Medical Research Materiel Command
GABA and Glycine are Co‐released into Dorsal Motor Nucleus of the Vagus Neurons following a Perinatal High Fat Diet
Authors: Clyburn Courtney Elizabeth , Browning Kirsteen N. 
Published: June 2021

Efferent motoneurons of the adult dorsal motor nucleus of the vagus (DMV) have previously been shown to only receive GABAergic inhibitory synaptic inputs. In contrast, it has been demonstrated that, at early postnatal time points, motoneurons of the DMV receive both GABAergic and glycinergic inhibitory synaptic inputs. The developmental decline in glycinergic input has been determined to occur in the second postnatal week. Our previous work has suggested that rats that are fed a perinatal high fat diet (pHFD) do not undergo this developmental shift and maintain a glycinergic input into adulthood. The purpose of this study was to test the hypothesis that glycine and GABA are co‐released from the same synaptic terminal in pHFD rats.Whole cell patch clamp recordings were made from DMV neurons in pHFD or control fed Sprague‐Dawley rats 4–6 weeks of age. These rats were fed either a control chow or HFD (14 or 60%kcal from fat, respectively) from embryonic day 13. The effects of the benzodiazepines (BZD) clonazepam (100nM), an allosteric modulator of the a2/3 subunit containing GABAA receptor, and zolpidem (1μM) an allosteric modulator of the a1 subunit containing GABAA receptor, on miniature postsynaptic inhibitory currents (mIPSCs) were examined. Application of BZDs is known to modulate the open probability of ligand‐gated GABAA, but not glycine, ion channels, thus increasing the decay of the slower GABAergic, but not the faster glycinergic mIPSCs. This allows for the differentiation of mixed concurrent glycinergic and GABAergic currents through the identification of two distinct rates of decay.Application of clonazepam significantly increased the proportion of two‐phased mIPSCs in the DMV of pHFD rats (76/200 vs 14/213 n=6 neurons, p<0.05), but not in controls (10/156 vs 11/164, n=6 neurons, p>0.05). Similarly, application of zolpidem significantly increased the proportion of two‐phased mIPSCs in the DMV of pHFD rats (60/179 vs and 14/153, n=6, p<0.05) but not in controls (27/150 vs and 8/138, n=5, p>0.05).This study confirms our previous studies suggesting that exposure to a pHFD prevents the developmental decline in glycinergic currents onto the DMV neurons and further suggests that glycine and GABA are co‐released from the same synaptic terminal. Furthermore, significant differences were not apparent between the effects of zolpidem and clonazepam, suggesting that a1, 2 and 3 receptor subunits form functional GABAA receptors in pHFD DMV neurons. These developmental alternations in inhibitory signaling in brainstem motoneurons may result in the dysregulation of gastrointestinal reflexes and gastric motility observed in these rats.Support or Funding InformationFunded by NIH 078364 and NSF IOS1148978
Phrenic Motor Neuron Survival Caudal to C2 Hemisection
Authors: Allen Latoya Lynn , Santiago Juliet V , Asa Zachary A , Holland Ashley E , Ciesla Marissa , Satriotomo Irawan S , Mitchell Gordon S , Gonzalez‐Rothi Elisa Janine 
Published: June 2021

Cervical spinal injury (cSCI) severs descending brainstem projections to spinal respiratory motor neurons, paralyzing respiratory muscles caudal to injury. Spinal hemisection at C2 (C2Hx) has frequently been used as an experimental model of cSCI to study both spontaneous and induced functional recovery of both respiratory and non‐respiratory somatic motor function after cSCI. One assumption implicit in these studies is that C2Hx dennervates motor neurons below the injury, but does not cause significant motor neuron pathology or cell death. However, one recent study reported substantial, bilateral motor neuron death caudal to C2Hx (Satkunendrarajah et al, 2016); in this study, motor neurons were identified by ChAT immunoreactivity in the spinal ventral horn. We have also observed that retrograde labeling with Cholera toxin B fragment (CtB) is diminished in phrenic motor neurons following C2Hx (unpublished data). However, these observations are potentially misleading since: 1) C2Hx may diminish ChAT immunoreactivity without motor neuron cell death; and 2) retrograde transport of CtB may be diminished when administered following an injury, due to the relative inactivity of motor neurons below the injury, giving the false impression that phrenic motor neurons had died. Because phrenic motor neuron cell death following cSCI would have profound implications for studies of spontaneous and/or induced phrenic motor plasticity after C2Hx, we tested the hypothesis that C2Hx has minimal impact on phrenic motor neuron cell counts if phrenic motor neurons are retrogradely labeled with CtB PRIOR to spinal injury. Phrenic motor neurons were retrogradely labeled in intact adult Sprague Dawley rats via intrapleural injections of CtB. Rats were randomly assigned to one of four groups: 1) 2 week C2Hx, 2) 8 week C2Hx, or 3) age matched, spinal intact controls. One week following the CtB injection, rats in the C2Hx groups underwent a left lateral C2Hx injury under isoflurane anesthesia. Rats were then perfused at 2 or 8 weeks post‐injury, and longitudinal sections (40 μm) from C3 to C8 were processed to reveal CtB, ChAT and NeuN. CtB labeled phrenic motor neurons were quantified using a custom MATLAB code. Preliminary analyses suggest minimal loss of phrenic motor neurons at either time post‐C2Hx relative to spinal intact rats. Assessment of ChAT and NeuN immunoreactivity in phrenic motor neurons after C2Hx is ongoing. Although we found little evidence for phrenic motor neuron cell death caudal to C2Hx, we cannot rule out greater cell loss in other motor neuron pools. These findings have important implications for studies concerning motor plasticity following cervical spinal injury.Support or Funding InformationSupported by: T32HD043730, K12HD055929 (EGR), RO1HL69084 (GSM), and grants from the DoD (GSM), and the McKnight Brain Institute (GSM, MCC)
Characterization of the Receptors Associated with the Differing Responses to the Neuropeptide, AST‐C, by the Cardiac Ganglion of the American Lobster, Homarus americanus
Authors: Walsh Patrick , Pong Sovannarath , Armstrong Matthew , Christie Andrew E , Dickinson Patsy S 
Published: June 2021

Central pattern generators (CPGs) are neural networks that produce consistent, rhythmic patterned outputs to reliably activate specific muscles, resulting in repeated and rhythmic movements. The cardiac ganglion (CG) is a CPG in the American lobster, and is responsible for producing a consistent rhythmic heartbeat. Flexibility of rhythmic motor outputs arises from modulation of CPGs, largely by neuropeptides. The lobster heart is modulated by C‐type allatostatins (AST‐C), two of which [pQIRYHQCYFNPISCF, now AST‐C I, and SYWKQCAFNAVSCFamide, now AST‐C II] were previously known; both have been shown to modulate the lobster heart. Using transcriptomes generated from nervous system tissues, we identified a third isoform of AST‐C, GNGDGRLYWRCYFNAVSCF, or AST‐C III, in the lobster. All three AST‐C isoforms consistently lead to decreases in contraction frequency when perfused through an isolated lobster heart; in contrast, all can elicit variable responses in contraction amplitude, with amplitude increasing in some lobsters and decreasing in others. The responses to AST‐C I and AST‐C III are more similar to one another than to AST‐C II in any given lobster.We are currently testing the hypothesis that differences in AST‐C receptor expression among individuals underlie these varying responses. Mining of a Homarus americanus transcriptome has led to the identification of three full‐length and one partial putative AST‐C receptors. We are currently conducting experiments to determine whether and to what extent the four putative AST‐C receptors bind the three AST‐C isoforms. Understanding these receptor‐peptide relationships will allow us to elucidate the role the receptors play in the observed differential response to AST‐C.Support or Funding InformationNSF (IOS‐1353023, IOS‐1354567), NIH (8P20GM103423‐12), Cades Foundation, Doherty Foundation gift to Bowdoin College
Linking morphology and motion: Testing multibody simulations against in vivo cranial kinematics in suction feeding fishes using XROMM
Authors: Olsen Aaron , Hernandez Patricia , Camp Ariel , Brainerd Elizabeth 
Published: June 2021

The heads of ray‐finned fishes contain over 20 mobile skeletal elements, interconnected by a morphologically and functionally varied set of articulations that allow a high degree of mobility. Many fish use this mobility to expand the buccal cavity during feeding to create suction and draw in prey items. Due to this high degree of mobility, a varied set of articulations, and the diversity of cranial forms among fishes, fish skulls provide an excellent system for relating skeletal and articular morphology to skeletal motion. The motion of fish skulls is traditionally simulated using planar mechanical linkage models, such as the 4‐bar linkage. While linkage models accurately predict the in vivo kinematics for particular subsets of bones within the skull, there are other aspects of cranial motion for which planar 4‐bar linkage models have proven insufficient. Additionally, these models have rarely been tested against three‐dimensional kinematics. We used CT data and the R package linkR to build multibody simulations of cranial motion in three species of ray‐finned fishes: largemouth bass (Micropterus salmoides), channel catfish (Ictalurus punctatus), and common carp (Cyprinus carpio). For each species, we created a series of simulations with varying numbers of parameters and degrees of freedom. We then tested the accuracy of these simulations against three‐dimensional in vivo kinematics, collected using X‐ray Reconstruction of Moving Morphology (XROMM). In evaluating model accuracy, we apply a new approach that measures how model error changes as a function of model complexity. We demonstrate the potential, and limitations, of using morphology to predict in vivo kinematics and ultimately to test how the evolution of organismal form influences the evolution of organismal motion and performance.Support or Funding InformationThis work was funded by NSF DBI‐1612230.
DNA Polymerase η Is an &[Iota]mportant &[Beta]ypass DNA Polymerase in the Repair of DNA Interstrand Cross‐Links
Authors: Zhao Linlin 
Published: June 2021

DNA interstrand cross‐links (ICLs) are cytotoxic DNA lesions derived from reactions of DNA with a number of anti‐cancer reagents as well as endogenous bifunctional electrophiles. Deciphering the DNA repair mechanism of ICLs is important for understanding the toxicity of DNA cross‐linking agents and for the development of effective chemotherapies. The repair of ICLs in mammalian cells involves sequential endonucleolytic excision of the lesion from one strand and then the other, and translesion synthesis (TLS) is a critical step for bypassing the unhooked intermediates from the nontemplate strand or for conducting postreplication gap‐filling DNA synthesis. A number of TLS DNA polymerases in humans have diverse bypass specificities and replication fidelities. To illustrate the important role of a certain TLS in ICL repair, we used model ICLs induced by oxidized abasic lesions and those‐derived from O6‐2′‐deoxyguanosine. We investigated the DNA replication‐blocking and mutagenic properties of these ICLs during TLS using primer extension assays and steady‐state kinetic analysis of single nucleotide incorporation prior to, opposite and past the cross‐linked template nucleotide. Liquid chromatography‐mass spectrometry‐based oligonucleotide sequencing was also used to determine the relative yield of the mutagenic bypass products. Our results demonstrated that among the several human TLS DNA polymerases tested, pol η exhibits the relatively high bypass activity; however, a significant portion of bypass products is mutagenic and contains substitution or deletion mutations. The importance of pol η in ICL bypass is demonstrated using whole cell extracts of Xeroderma pigmentosum variant patient cells and those complemented with pol η. Together, this body of work provides in vitro evidence of the important role of DNA polymerase η in ICL repair and underscores the importance of further evaluating of the role of pol η in ICL repair pathways in biological systems.Support or Funding InformationThis work was supported by in part by Central Michigan University start‐up funds, Central Michigan University Faculty Research and Creative Endeavors Award and a grant from U.S. Army Research Office (W911NF‐15‐1‐0140).
Interactions and Dynamics Within CHIP/Hsp70 Complexes
Authors: Mannion Matthew M , Zhang Huaqun , VanPelt Jamie , Cottingim Kelsey , Tsutakawa Susan , Page Richard C 
Published: June 2021

Interactions between the E3 ubiquitin ligase CHIP (C‐terminus of Hsp70 interacting protein) and the chaperone Hsp70 represent the physical intersection of two competing protein quality control efforts: refolding, and degradation. Interaction with CHIP allows for the formation of a productive encounter complex that facilitates ubiquitination of Hsp70‐bound substrates, thereby targeting those proteins for proteasomal degradation. Central to the CHIP/Hsp70 interaction are changes in structure and dynamics that occur upon binding. Although recruitment of Hsp70 to CHIP via interaction with the CHIP tetratricopeptide repeat (TPR) domain is well established, alterations in structure and dynamics of CHIP and Hsp70 upon binding are not well understood. Using NMR we describe residue‐level changes in dynamics to the CHIP‐TPR domain that occur upon binding Hsp70. We also utilize small angle X‐ray scattering (SAXS) and electron paramagnetic resonance (EPR) to identify motions with the CHIP/Hsp70 complex. Independent SAXS and EPR experiments both point to a mobile Hsp70 substrate binding domain that is only partially constrained through interactions with the CHIP‐TPR. Building upon static models of the full‐length CHIP/Hsp70 complex, afforded by our recent crystal structure of the CHIP‐TPR/Hsp70‐lid‐tail complex, we have assembled a dynamic model of the CHIP‐Hsp70 complex. This dynamic model sheds light on the function of the CHIP/Hsp70 chaperoned ubiquitination complex and how CHIP harnesses dynamics of Hsp70 to overcome significant distance separations identified in earlier static models of the CHIP/Hsp70 complex.Support or Funding InformationThe authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the American Heart Association (Award No. 16SDG26960000 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.
The Mechanism of Inhibition of Botulinum Neurotoxin Type A by Two Quinolinol Compounds
Authors: Minnow Yacoba Vroom Teschemaker , Goldberg Ronald , Tummalapalli Sreedhar R. , Rotella David P , Goodey Nina M 
Published: June 2021

The potential of Botulinum neurotoxins (BoNTs) to be used as biological weapons makes the need to identify small molecule inhibitors critical but it is a challenging target due to the large substrate‐enzyme interface. BoNTs are produced by the bacterium Clostridium botulinum in seven antigenically distinct serotypes (A‐G), with serotype A (BoNT/A) being the most potent toxin known to man. Recently, quinolinol‐based compounds have been identified as a promising starting point for effective BoNT/A inhibitors. Understanding the mechanism of inhibition by quinolinol compounds would facilitate inhibitor optimization studies. In this study, a Foster resonance energy transfer (FRET) based substrate of BoNT/A, SNAPtide, was used to study the mechanism of inhibition of BoNT/A endopeptidase activity by two new quinolinol compounds, MSU58 and MSU84, with IC50 values of 3.3 μM and 5.8 μM, respectively. Kinetic analysis and model discrimination analysis revealed both compounds to be competitive inhibitors of BoNT/A endopeptidase activity. Inhibition constants (KI) were found to be 3.2 μM for MSU58 and 6.2 μM for MSU84. The kinetic rate constant for the binding and release of substrate and inhibitor were also determined. These data indicate that these inhibitors bind in the active site of BoNT/A and that binding is mutually exclusive with the binding of the substrate. This study, is the first to report on the competitive inhibition of BoNT/A by a quinolinol group of compounds. These data help define the inhibitor binding pocket and, along with structure activity relationship studies, provide immediate direction for further compound synthesis.Support or Funding InformationNovartis Graduate Scholarship Award and US Army Medical Research (grant number W81XWH‐14‐C‐0090 S)
Phosphoproteins in Extracellular Vesicles as Candidate Markers for Breast Cancer
Authors: Tao Weiguo Andy , Chen I‐Hsuan , Iliuk Anton 
Published: June 2021

The state of protein phosphorylation can be a key determinant of cellular physiology such as early stage cancer, but the development of phosphoproteins in biofluids for disease diagnosis remains elusive. Here we demonstrate, for the first time, a strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs isolated from small volumes of plasma samples. Using label‐free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer as compared to healthy controls. Several novel biomarkers were validated in individual patients using Paralleled Reaction Monitoring for targeted quantitation. This study demonstrates that the development of phosphoproteins in plasma EV as disease biomarkers is highly feasible and may transform cancer screening and monitoring.Support or Funding InformationThis study was supported partially by NIH grants 5R41GM109626, 1R01GM111788, and 1R41CA210772 and by NSF grant 1506752.