bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cortisol as a Potential Biomarker for Suicidality
Authors: Nohr Maryssa , Mazzer Paula , Kelly Anne 
Published: April 2020

Cortisol levels have been identified as one potential risk factor for suicide (O’Connor, 2016) because they have been linked to stress. Fearlessness, including impulsivity and risk taking, contribute to suicidality and cortisol may be a biomarker to aid in diagnosis. If cortisol calms fears (de Quervain et al., 2011), for at least part of the population, and, if fearlessness heightens impulsivity and risk taking as demonstrated by previous research (Smith, 2008) then cortisol may contribute to suicidality and suicide attempts. The purpose of this research is to clarify the relationship between fear, impulsivity, and risk and the role cortisol may play. Thirty males and 30 females completed the Barratt Impulsiveness Scale (Patton, Stanford, & Barratt, 1995), Domain‐Specific Risk‐Taking Scale (Blais & Weber, 2006), Death Anxiety Scale (Templer, 2006), and Fear of Pain Questionnaire‐9 (McNeil & Rainwater, 1998). Cortisol is collected by swabbing the cheek and collecting saliva. The saliva samples were then kept in a −80° C freezer until tested using an ELISA detection technique. Preliminary results suggest levels of cortisol are negatively correlated with fear of death and fear of pain. Fearlessness is expected to be negatively correlated with impulsivity and risk taking, which, in turn, are expected to be positively correlated with each other and positively correlated with cortisol levels.Support or Funding InformationNIH P20GM103443
Cyclic Tetra‐Adenylate (cA4) Activates CRISPR Associated Transcription Factor Csa3, Providing Feedback Activation of Protospacer Acquisition and crRNA Expression
Authors: Lawrence C. Martin , Charbonneau Alexander , Gauvin Colin 
Published: April 2020

While there is extreme interest in CRISPR‐Cas gene editing technology, nature did not invent CRISPR‐Cas for this purpose. Rather, CRISPR‐Cas naturally serves as an adaptive immune system in single celled prokaryotic organisms, where it is found in more than 40% of Bacteria and 85% of Archaea. These systems are incredibly diverse, and classified by cas gene content into 2 major classes, six types and more than 20 different subtypes. In each case, however, they operate in the same three stage process; i) spacer acquisition, ii) crRNA expression and maturation and iii) target interference(Annu Rev Biochem, 82:237).The well‐known CRISPR‐Cas9 systems are class 2, type II systems and represent less than 10% of identified CRISPR‐Cas systems. Much more common are the Class 1 systems, especially type I and type III. Like class II systems, type I systems also target dsDNA in a PAM dependent process. Type III systems, however, do not require a PAM, and instead recognize nascent mRNAs emanating from transcriptionally active DNA. And upon recognition, they degrade both the RNA and DNA. Further, while complexed with the target RNA, the cyclase domain of the Cas10 subunit synthesizes cyclic oligo‐adenylate (cAn) signals (Science 357:605, Nature 548:543), which are in turn degraded by ring nucleases (Nature, 562:277).Sulfolobus solfataricus is a model archaeon utilizing both Type‐IA and Type‐IIIB CRISPR‐Cas systems. Structural studies suggest Csa3 is a transcription factor under the allosteric control of a 4‐base cyclic RNA signal that regulates expression of CRISPR‐Cas (J Mol Biol, 405:939, RNA Biol, 13:254). Here we show that Csa3 specifically recognizes a palindromic sequence present in the promoters for stage I acquisition genes (Cas1, Cas2, Csa1) and three CRISPR loci, that promoter recognition is enhanced by cyclic tetra‐adenylate (cA4), that over‐expression of Csa3 in S. solfataricus activates spacer acquisition, and that Csa3 lacks ring nuclease activity, suggesting long‐term potentiation of the cA4 signal. Further, we present the structure of Csa3 in complex with cA4, describe recognition of cA4 by Csa3 in atomic detail, and cA4 induced conformational changes that enhance promoter recognition. In all, this provides a molecular understanding for feedback activation of spacer acquisition and crRNA expression in cells struggling to clear viral infections. This feedback system is dependent on both type I Csa3 and type III Cas10, and thus represents a coordinated response by two different arms of CRISPR‐Cas. In this light, type I and type III CRISPR‐Cas in S. solfataricus are not independent systems, but instead represent two complementary arms of a coordinated anti‐viral response.Support or Funding InformationSupported by National Science Foundation grant MCB‐1413534.
Vascular Supply of Digit III and Foraging in the Aye‐Aye (Daubentonia madagascariensis)
Authors: Russell C. Kinley , Perry Jonathan M.G. 
Published: April 2020

The aye‐aye is an unusual primate that differs from other primates in several aspects of its anatomy and behavior. Its thin, elongated third digit serves as a tool for tap‐scanning and prey extraction during percussive foraging for wood‐boring insects. The surface temperature of digit III is higher than the other digits on the foraging hand; in contrast, digit III has a lower temperature than the other digits on the stabilizing hand and this difference has been hypothesized to relate to stenotic kinking of the palmar digital arteries during hyperextension at the metacarpophalangeal joint. Though previously described qualitatively, aye‐aye hand vasculature has never been photo‐documented or quantified. Here we describe and compare the arterial supply of the forearm and hand in Daubentonia madagascariensis and Lemur catta in the context of hand function during foraging.The forelimbs of two primates, one aye‐aye and one ring‐tailed lemur, were dissected and arterial morphology was documented and photographed. Measurements of palmar digital arteries were collected using a dissecting microscope equipped with an ocular reticle. Inter‐digit and inter‐species comparisons were made for the pattern and diameter of each vessel.Lemur catta and Daubentonia madagascariensis differ in several aspects of their forelimb vascular morphology. In L. catta, the brachial artery does not pass through the entepicondylar foramen and a superficial palmar arterial arch supplies the digits. By contrast, in D. madagascariensis the brachial artery does pass through the entepicondylar foramen and the palmar aspects of the digits are supplied by individual branches of the ulnar and radial arteries rather than an anastomotic arch. These two species also differ in terms of the diameter of the arteries leading to the digits relative to the diameter of the ulnar artery (as a way of assessing relative scale). In the aye‐aye, the arterial supply to digit III has the smallest relative diameter (digit IV has the largest). In the ring‐tailed lemur, digit V has the largest total relative diameter and digit I the smallest.These observed differences in the vascular supply of the forelimb and hand in Lemur catta and Daubentonia madagascariensis likely relate to differences in foraging and locomotor strategy. While it has been suggested that stenotic kinking is responsible for the relatively low temperature of digit III on the stabilizing hand in the aye‐aye, our study suggests that this temperature differential may instead be due to the relative under‐supply of this digit compared to the other digits of the hand.
Volumetric Muscle Loss Injury: Temporal Impact on the Neuromuscular Junction
Authors: Sorensen Jacob R. , Johnson Trevor K. , Hoffman Daniel B. , Vasdev Amrit K. , Corona Benjamin T. , Greising Sarah M. 
Published: April 2020

Volumetric muscle loss (VML) is the traumatic loss of muscle tissue that results in reduced regenerative capacity and long‐term functional deficits. In fact, in comparison to the mass of muscle removed from the VML injury, the functional deficits observed are disproportionally greater. Clinically, it is estimated that nearly 30% of VML injured patients are further impacted by some ongoing degree of peripheral nerve damage in the injured limb. These findings suggest that VML injury has a significant impact on the neuromuscular system that extends beyond the defect area. This is further supported by our recent finding that VML injury results in chronic motor neuron axotomy of ~75% through 21 days post‐VML; however, how this impacts individual muscle fibers is not yet clear. As such, the purpose of this study was to systematically quantify the neuromuscular junctions (NMJs) surrounding the VML injury site. We hypothesized that VML injury would result in a reduced NMJ area, altered innervation and an irregular morphology that ultimately impairs skeletal muscle function. A standard VML surgery was performed by removing approximately 20% of the middle third of the tibialis anterior (TA) muscle in adult male inbred Lewis rats (n=32). Full thickness injuries were performed unilaterally so that the non‐injured leg served as an intra‐animal control. The TA muscles were harvested at 0, 3, 7, 14, 21, and 48 days post injury and ~50 individual NMJs per muscle were evaluated. Pre‐ and post‐synaptic were identified immunohistochemically and quantitative confocal microscopy was used to evaluate 2‐ and 3‐D synaptic area, fiber denervation and morphologic characteristics. As expected, the mass of the TA muscle was 20–30% of the contralateral over the 48 days post‐VML; which is supported by an ~40% loss in contractile protein content over this same period. The muscle remaining after VML indicates a significant number of denervated (reduced overlap of pre‐ and post‐synaptic structures) NMJs after injury. Additionally, substantial changes in the NMJ morphological characteristics were identified in the muscle remaining after VML. Specifically, poly‐innervation and sprouting of individual NMJs, which suggest that motor unit reorganization may be occurring after VML injury. While the most obvious consequence of VML injury is the loss of contractile protein content and muscle fibers, the disorientate changes at both the motor neuron and NMJ may exacerbate the decline in muscle function following VML injury. This work is foundational to support future work to identify interventions to support maintenance of NMJ innervation following VML injury.Support or Funding InformationSupported by W81XWH‐19‐1‐0075.
Synergism between Angiotensin AT1 and AT2 Receptors function: Role of Angiotensin‐(1‐7) via AT1R in Modulating AT2R Agonist Response on Nitric Oxide in Kidney Cells
Authors: Hussain Tahir , Patel Sanket 
Published: April 2020

Angiotensin‐(1‐7) is evidently considered as an endogenous agonist for Mas receptor. Our recent studies show that MasR and AT2R physically interact and in response to their respective agonists Ang‐(1‐7) and C21 produce interdependent functional response in terms of nitric oxide (NO) generation and natriuresis. Moreover, recent studies suggest that Ang‐(1‐7) is cardio‐protective via AT1R stimulation. Considering the complex nature of Ang‐(1‐7) function, we tested as to how Ang‐(1‐7) affects AT2R function utilizing human kidney proximal tubule epithelial cells (HK‐2 cells). The agonists of MasR [(Ang‐(1‐7), AVE0991] or AT2R (C21), alone significantly stimulated NO formation at 10−6–10−5M (50–300%), but not at lower concentrations (10−12‐10−7M). Contrarily, Ang‐II alone reduced NO formation. In the interaction studies, the non‐responsive concentrations of Ang‐(1‐7) (10−12‐10−7M) synergized NO formation of C21 (10−12‐10−9M) or ang‐II (10−12‐10−9M) in HK‐2 cells (p<0.05 vs. either agonist alone). The synergy is observed only when the Ang‐(1‐7) was added to the HK‐2 cells 10 min prior to the C21/ang‐II, not when the agonists were added simultaneously. The synergy between Ang‐(1‐7) and C21/ang‐II was sensitive to AT2R antagonist (PD123319, 10−5M) and AT1R antagonist (candesartan, 10−5M), but not MasR antagonist (A779, 10−5M). Interestingly, pre‐stimulation with synthetic non‐peptide MasR selective agonist AVE0991 at any concentrations (10−10‐10−7M) did not show synergism with the AT2R agonist C21. Collectively, results show Ang‐(1‐7) at lower concentrations via AT1R, not MasR, synergies the AT2R function of NO formation and this phenomenon may have a physiological significance.Support or Funding InformationNIH R01 DK61578 and R01 DK117495
Repair of 1, N2‐ethenoguanine by Alkyladenine DNA Glycosylase
Authors: Thelen Adam , O’Brien Patrick 
Published: April 2020

Human DNA can be damaged by numerous endogenous processes as well as by diverse exogenous factors. The exocyclic amine groups of adenine, cytosine, and guanine can be alkylated by the reactive products of lipid peroxidation or by exposure to vinyl chloride pollutants, leading to the formation of harmful etheno adducts. These etheno lesions can be both mutagenic and cytotoxic, necessitating timely repair. Alkyladenine DNA glycosylase (AAG) has been proposed to initiate the base excision repair of both 1, N6‐ethenoadenine (ɛA) and 1, N2‐ethenoguanine (ɛG). However, examination of the structure of AAG in complex with ɛA‐DNA raises questions about how ɛG could be recognized in an active site optimized for recognition of ɛA. Systematic single and multiple turnover kinetic assays of ɛG excision by AAG reveal that ɛG is recognized far less readily than ɛA in both the natural ɛG:C context as well as in bulges and mispairs. Moreover, AAG is unable to repair ɛG in competition with a large excess of undamaged bases, calling into question how the lesion could be identified in genomic DNA. I hypothesized that the conserved active site residue, Asn169, may play a role in discriminating against the ɛG lesion. Indeed, mutation of Asn169 to Ser results in 7‐fold greater activity toward ɛG. Thus, the conserved active site residues are not optimized for recognition and repair of eG. Together these findings indicate that eG is not a preferred substrate of AAG, and other mechanisms are likely to contribute to eG repair in human cells.Support or Funding InformationFunding was provided by a graduate student award from the Department of Biological Chemistry at the University of Michigan, an NSF award MCB‐1615586 (P.J.O), and a fellowship from the Chemistry/Biology Interface Training Program supported by the NIGMS of the NIH under Grant T32GM008597 (A.Z.T.) and R01GM108022 (P.J.O.).
Discriminative Stimulus Effects of (R)(−)‐DOI and AM8936, a Synthetic Cannabinoid
Authors: Erwin Laura , Nikas Spyros , Makriyannis Alex , Paronis Carol 
Published: April 2020

Increased abuse of synthetic cannabinoids (SCBs) continues to be a public health concern. Preclinical studies have identified SCBs as being THC‐like; however, occurrences of emesis, hallucinations, and seizures have been reported with their usage. These studies examined the premise that the hallucinogenic effects of AM8936, a highly potent synthetic CB1 agonist, resemble the discriminative stimulus effects of another hallucinogen, the 5HT2A agonist (R)(−)2,5‐dimethoxy‐4‐iodoamphetamine (DOI). Male and female Sprague‐Dawley rats (n=6/group) were trained to discriminate either 0.18 mg/kg AM8936 or 0.56 mg/kg DOI from saline under a shock avoidance schedule of reinforcement. In both DOI and AM8936 trained subjects, their respective training dose produced full (>80%) substitution. In the DOI trained subjects, AM8936 (0.032 – 0.18 mg/kg), delta‐9‐tetrahydrocannabinol (Δ9‐THC) (1 – 10 mg/kg), and JWH‐018 (0.1 – 3.2 mg/kg) produced dose‐dependent increases in DOI‐lever responding, but only JWH‐018 and AM8936 produced full substitution. In the AM8936 trained subjects, DOI (0.56 – 3.2 m/k), Δ9‐THC (1 – 10 mg/kg), and JWH‐018 (0.1 – 10 mg/kg) produced dose‐dependent increases in AM8936‐lever responding, but only JWH‐018 produced full (>80%) substitution. Doses of DOI that produced AM8936‐appropriate responding (0.56 – 3.2 mg/kg) were higher than those that resulted in DOI‐appropriate responding (0.1 – 0.8 mg/kg). In addition, the mu‐opioid receptor agonist morphine (1 – 10 mg/kg) (negative control) engendered less than 20% DOI‐lever responding or AM8936‐lever responding up to doses that produced behavioral disruption. These studies indicate that there is bi‐directional overlap in the discriminative stimulus effects of SCBs and a 5‐HT2A agonist, suggesting similarities in their subjective effects.Support or Funding InformationAcknowledgementsFunded by NIH/NIDA: DA043700
Developing an E. coli model for investigating FET protein phase separation
Authors: Victor Rachel , Schwartz Jacob 
Published: April 2020

Some proteins in cells have the ability to bind RNA, rather than DNA, to regulate transcription in a manner comparable to traditional and DNA‐binding transcription factors. One such protein is FUS (FUsed in Sarcoma). FUS has prominent roles in the neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as well as sarcoma development. Nevertheless, the basic mechanism for FUS acting in normal biology, and disease, is unknown. The challenge in figuring out this mechanism arises because of the intrinsic properties of FUS as a mostly disordered protein that readily phase separates as part of its cellular functions. We have developed an E. coli model to investigate phase separation and granule formation for recombinantly expressed human FUS protein in the natural, highly crowded environment universally found in cells. We employ chemical crosslinking and chromatographic separation to observe FUS transitioning from a monomer to phase separated state. This E. coli model can provide a robust platform for large and parallel evaluation of protein phase separation, by offering rapid cell and protein production, sub‐cellular separation into small complexes and granules, and classification for structure/function relationships involving the low complexity amino acid sequences that are responsible for these unusual biophysical properties observed in FUS.Support or Funding InformationNational Institutes of Health, American Cancer Society, Alfred P. Sloan Foundation
A rapid and an efficient method to remove unreacted small‐molecules using a versatile resin packed in a convenient Spin‐and‐Go format.
Authors: Ganapathy Ramesh , Etnyre Deven , Nlend Marie , Etienne Chris , Deshpande Atul 
Published: April 2020

Removal of unreacted or residual compounds in the sample‐preparation workflow is a key success factor for downstream analysis and processing of biomolecules. For example, during sample preparation of biomolecules such as proteins or nucleic acids, there is often a need to label the biomolecules with dyes, affinity tags, radioactive labels, and mass tags. In other instances, there is a need to chemically modify biomolecules such as to reduce, oxidize, methylate, etc. During these treatments, some amount of the labeling or chemical agent remains in the sample as an unreacted label/chemical. These unreacted small molecules can cause several issues during downstream analysis or use of the biomolecule. For example, free unreacted fluorescent dyes after bioconjugation of protein cause non‐specific binding of free‐dye resulting in high background issues during fluorescent imaging. Commonly used clean‐up methods following sample preparation, such as dialysis, are tedious and lengthy with multiple buffer exchanges sometime resulting in loss of protein due to aggregation. Alternatively, size‐exclusion chromatography requires expensive instrumentation in addition to lengthy set up time.We have developed the Thermo Scientific™ Pierce™ Dye and Biotin Removal Spin Columns, a novel resin that is packed in an easy to use “Spin and Go” format of varying spin column sizes (0.5 mL, 2mL, 5 mL and 10 mL) to accommodate a range of sample volumes (100 uL–4 mLs). The resin is highly specialized to produce exceptional protein recovery and can be used effectively to remove 4 different class of small molecules: non‐conjugated fluorescent dyes, biotinylation reagents, reducing agents, and crosslinkers. It is a multimodal resin with size exclusion properties and proprietary surface chemistry that allows removal of the above‐mentioned small molecules, while the size exclusion property allows for the biomolecule to be efficiently recovered.Our results indicate efficient dye removal and protein recovery from fluorescent conjugated antibodies. The results also indicate removal of reducing agents such as TCEP at a concentration as high as 25 mM from protein samples, while recovering >90% of the added protein. Results also indicate >90% removal of biotinylated molecules and crosslinkers from Protein samples with >90% protein recovery. Our results show that the efficiency and binding capacity for removal of small molecules using this resin is better than any resin in the market. This is exemplified by immunofluorescent analysis of ZO‐1 monoclonal antibody conjugated DyLight™ 488 which was purified using Pierce™ Dye and Biotin Removal Spin columns and shows high intensity and lesser background in Caco‐2 cells than the non‐purified conjugate.
Annotating Contig 49 in F element of Drosophila takahashii
Authors: Gomez Austin , Fung Siaumin 
Published: April 2020

Two different chromatin structures can be found in eukaryotic nucleus, euchromatin and heterochromatin. Euchromatin is DNA loosely packed by histones and transcribed actively, while heterochromatin is densely packed DNA, often associated with gene silencing. The 4th chromosome in Drosophila, also known as the F element is heterochromatic, yet 80 genes reside in this chromosome are actively being transcribe. The goal of this project is to use comparative genomic to elucidate the regulatory motif of gene silencing by annotating genes on the 4th chromosomes and genes on the euchromatin 3rd chromosomes (control). By using genome browser, two features on contig 49 were predicted to have orthologs to D. melanogaster, unc13 and eIF4G1. FlyBase, and BLAST are used to align our predicted genes in contig 49 to the ortholog of Drosophila melanogaster to create a gene model. Several lines of evidence, including conservation of amino acid sequence to D. melanogaster, RNA‐Seq data and Top‐Hat splice‐junction predictor were used to produce the most parsimonious annotation of the coding sequence of unc13 and eIF4G1 in Drosophila takahashii.Support or Funding Information“This project was supported in part by grants from the Department of Education (Award #P031S150199), from Southern California Edison, and through Vanguard University’s SURP Program generously supported by the Office of the Provost and the Institute for Faculty Development.”Special thanks to the Genomics Education Partnership (GEP)