bluuebunny/crossref_metadata_2025 huggingface dataset:
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 Information

Supported 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 Information

Supported 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 Information

NIH R01 DK61578 and R01 DK117495
TRPM2 channel‐STAT3 complex regulates the polarity of tumor‐associated macrophage and tumor angiogenesis
Authors: Mohamed Tarek
Published: April 2020

The tumor microenvironment is a complex tissue composed of various stromal cells including immune cells. Especially, tumor‐associated macrophages (TAM) are one of the major components of tumor tissues, and they play a pivotal role in prompting the various tumor growths by producing growth factors and reactive oxygen species (ROS). Previously, we have reported that TRPM2 channel, a ROS‐sensitive Ca2+ channel, is abundantly expressed in macrophages and is tuning gene expression via transcription factor NF‐κB. Here, we report the significance of TRPM2 channel in the regulation of pro‐inflammatory M1 and pro‐angiogenic M2 phenotype of TAM. In TRPM2 knockout mice, TAMs around s.c.‐injected B16F10 melanoma tumor showed strong expression of M2 phenotypic markers and pro‐angiogenic factor VEGF according to the enhanced activity of STAT3. Interestingly, blood vessels in TRPM2 knockout mice tumor were so‐called non‐productive, which is characterized by an increase in vascular density and decrease in tissue perfusion and thus the tumor progression was suppressed. We also found that the activation of TRPM2 channel induced by hydrogen peroxide (H2O2) suppress the activity of STAT3 in HEK293 cells and macrophage‐like RAW 264.7 cells. Importantly, TRPM2 protein showed physical interaction with STAT3 protein, and their complex was degraded gradually in the presence of H2O2. Therefore, our data suggest that TRPM2‐STAT3 complex is critical for handling the phenotypes of macrophages depending on the environmental oxygen/redox conditions.
The Anti‐Leishmanial Effects of Compounds Derived from the Fungus Geosmithia langdonii on Visceral Leishmaniasis
Authors: Atencio Nicole , Milller Kenny , Cheney Austin , Benally B. Andre
Published: April 2020

Leishmaniasis is among seven of the most prominent tropical diseases and is the second most prevalent parasitic killer after malaria. Existing treatments are often highly toxic to infected patients and must be taken intravenously in hospitalization. These current treatments also have lost viability due to increasing drug resistance. A more cost‐efficient and less harmful treatment urgently needs to be identified. Diarylmethanes have been shown to have many benefits including HIV treatment, cancer treatment, antibacterial activity, and antioxidant activity. A diarylmethane isolated from the fungus Geosmithia langdonii has shown activity against Leishmaniasis. We plan to synthesize this metabolite and similar compounds with varying moieties at different positions to test the significance of these functional groups against Leishmaniasis and to identify an understanding of how these compounds inhibit this parasites’ growth. We were able to successfully grow and propagate Leishmaniasis donovani in M199 media. We also were able to successfully infect human THP1 macrophages and screen several commercially available diarylmethanes, bis(2‐hydroxyphenyl) methane and bis(4‐hydroxyphenyl) methane, by staining for A2, an amastigote specific protein, by indirect immunofluorescence. Results of these two diarylmethanes were compared to a positive control, Amphotericin B, and were shown to have ineffective inhibition of the parasite. Current work is focused on the synthesis of the fungal metabolite and other derivatives. This work will allow us to determine how the structure of these compounds is related to antiparasitic activity. We will utilize what we learn in these experiments to develop more effective treatments for Leishmaniasis.
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 Information

Funding 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 Information

Acknowledgements

Funded 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 Information

National 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)
Characterizing the Role of the Na+‐H+ Exchanger Isoform 1 (NHE1) in Pulmonary Fibrosis
Authors: Withers Emily , Wallert Mark A.
Published: April 2020

Pulmonary fibrosis is a chronic, progressive lung disease characterized by stiffening and thickening of the lung tissue leading to reduced respiratory function. When lung tissue becomes damaged, the response is to increase recruitment and proliferation of fibroblasts and increase deposition of extracellular matrix (ECM) proteins such as collagen. This initiates the release of transforming growth factor beta 1 (TGF‐β1), a cytokine known to enhance fibrosis. TGF‐β1 induces myofibroblast differentiation and further stiffening of the ECM by stimulating formation of cytoplasmic microfilament bundles and increasing expression of alpha‐smooth muscle actin (α‐SMA). Signal molecules such as serotonin (5‐HT), and lysophosphatidic acid (LPA) contribute to this by binding to G‐protein coupled receptors that support increased release of TGF‐β1. Fibrosis ultimately results in a state of low blood flow and hypoxia in the lung tissue, a microenvironment associated with upregulation of the sodium‐hydrogen exchanger isoform 1 (NHE1). NHE1 is a ubiquitously expressed protein that regulates intracellular pH. Upregulation of NHE1 supports increased cell proliferation and migration, indicating that it may play a role in the behavior of fibroblasts in pulmonary fibrosis. The proliferation, collagen deposition, and myofibroblast differentiation of two fibroblast cell lines will be characterized. CCL39, Chinese hamster lung fibroblasts, and WI‐38, normal human fibroblast cells, will both be studied. CCL39 cells treated with EIPA, a chemical inhibitor of NHE1, show reduced proliferative rates. This suggests that proliferation in these cells is NHE1‐dependent. In addition to proliferation, it is hypothesized that the collagen deposition and myofibroblast differentiation of this cell line will be NHE1‐dependent. Furthermore, it is hypothesized that WI‐38 cells will display similar NHE1‐dependent proliferation, collagen deposition and myofibroblast differentiation as the CCL39 cells. This suggests that CCL39 cells can serve as a predictor of human fibroblast cell behaviors.
Cerebral Blood Flow in the Internal Carotid Artery Throughout the Menstrual Cycle in Young, Healthy Women
Authors: Favre Michelle E. , Serrador Jorge M.
Published: April 2020

Background

Previous reports suggest female sex hormones may affect cerebral blood flow and cerebral flow velocity in women. The purpose of this study was to examine whether changes in cerebral blood flow occur throughout the menstrual cycle phases that are associated with fluctuations in sex hormones in young, healthy women.

Methods

A subset of eight female participants (mean age 25 yrs) from our previously published study (Favre & Serrador, 2019) underwent one minute of supine steady‐state cerebral blood flow measurements in the internal carotid artery. Women were studied at three different time points across the menstrual cycle: the early‐follicular (EF) phase associated with low levels of estrogen and progesterone, the late‐follicular (LF) phase associated with high levels of estrogen and low levels of progesterone, and the mid‐luteal (ML) phase associated with high levels of both estrogen and progesterone. Cerebral blood flow in the internal carotid artery was measured using duplex ultrasonography. Beat‐by‐beat blood pressure, end‐tidal CO2, ECG, and middle cerebral flow velocity (MCAv) using transcranial Doppler ultrasonography were also obtained.

Results

There were no statistically significant changes (P=0.269) in internal carotid blood flow across the menstrual cycle phases (EF=203.9±42.2, LF=227.0±53.7, ML=203.2±47.8 ml/min). When examined as a percent of flow from the early follicular phase, there were also no significant changes (P=0.207) across the menstrual cycle (EF=100.0±0.0, LF=111.6±14.5, ML=100.8±17.0 %). Additionally, there were no significant changes (P=0.488) in MCAv (EF=84.7±19.9, LF=89.5±21.2, ML=87.9±24.1 cm/s). End‐tidal CO2 significantly decreased (P=0.025) during the mid‐luteal phase compared to the late follicular phase (EF=38.0±3.0, LF=39.3±2.8, ML=37.0±3.1 mmHg). There were no significant changes (P=0.174) in internal carotid velocity (EF=41.5±7.0, LF=44.8±7.7, ML=40.1±9.2 cm/s) or internal carotid diameter (P=0.327) across the menstrual cycle phases (EF=0.45±0.02, LF=0.46±0.03, ML=0.46±0.01 cm).

Conclusion

Although we did not observe statistically significant changes in internal carotid blood flow across the menstrual cycle phases, we recognize that women demonstrated a 11% increase in flow during the late follicular phase, corresponding to a large effect size (Partial Eta Squared= 0.201). Because of our small sample size, we cannot rule out the possibility that the fluctuations in female sex hormones affect cerebral blood flow. Additionally, it is possible that the decreased end‐tidal CO2 in the mid‐luteal phase hid an underlying increase in cerebral blood flow. However, a larger sample of women is needed to conclude whether cerebral blood flow is affected throughout the menstrual cycle phases.

Support or Funding Information

This work was supported by the Pharmacology, Physiology and Neuroscience Department at Rutgers Biomedical and Health Sciences.
Overcoming multidrug resistance in cancer cells: Biochemical evaluation of new lead drug candidates
Authors: de Oliveira Maisa C. , Okwuone Dakota D.D. , Jensen Madeline K. , Wise John G. , Vogel Pia D.
Published: April 2020

P‐glycoprotein (P‐gp) overexpression is often correlated with multidrug‐resistance (MDR) of cancer cells due to its role in the transport of chemotherapeutic drugs out of the cells in an ATP‐dependent process. To search for P‐gp inhibitors as co‐therapeutics to combat MDR, we previously used high‐throughput in silico ligand docking studies to identify drug‐like compounds. To assess functionality of the hit compounds we measured their effects on P‐gp ATPase hydrolysis activity. Some of the compounds inhibited ATP hydrolysis of purified P‐gp and successfully reversed the MDR in prostate and ovarian cancer cell lines. We subsequently used computational approaches and structure‐based design to produce variants of one of the hit compounds with predicted higher affinity to P‐gp. The efficacy of these variants in reversing MDR in cell culture as well as the biochemical evaluation of inhibition was assessed. High expression of human P‐gp in the yeast Pichia pastoris in a biologically active form was previously reported and the purified P‐gp was used on our recent assays. Currently, reconstitution of P‐gp into nanodiscs allowed for human P‐gp to be solubilized in its active form with ATPase activity enhanced by 2–3 fold as well as increased stability. Here we report the evaluation of the potential of our previously discovered drug‐like compounds to serve as inhibitors of human P‐gp ATP hydrolysis activity.

Support or Funding Information

This work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D. of Naples Florida. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.
Resolvin E1 is a pro‐repair mediator that promotes intestinal epithelial wound healing
Authors: Quiros Miguel , Feier Darius , Birkl Dorothee , Agarwal Rachit , Zhou Dennis W. , Parkos Charles A. , Garcia Andres J. , Nusrat Asma
Published: April 2020

Resolution of inflammation and epithelial repair are active processes mediated by bioactive lipids known as specialized pro‐resolving mediators (SPMs). Resolvin E1 (RvE1), a SPM derived from omega‐3 fatty acid, has been reported to mediate anti‐inflammatory response in different tissues. Nevertheless, the role of RvE1 in mediating intestinal epithelial wound healing is not well understood.

We identified increased RvE1 in healing colonic wounds. RvE1 treatments enhanced intestinal epithelial wound repair in‐vitro. Analysis of the signaling pathways revealed activation of pathways such as CREB, mTOR and Src‐FAK in response to RvE1 exposure. Additionally, RvE1 increased activation of the small GTPase Rac1, leading to increased intracellular reactive oxygen species (ROS) production, cell‐matrix adhesion and cellular protrusions at the leading edge of migrating cells. To harness this mechanism of repair, we performed intramucosal injection of synthetic nanoparticles containing RvE1 in injured murine colon. RvE1 nanoparticles increased wound closure compared to naked RvE1 and empty nanoparticles. In summary, the above findings provide important insight on mechanisms of intestinal mucosal wound repair driven by RvE1 and determine its potential as a therapeutic agent aimed at facilitating mucosal wound closure and epithelial barrier recovery in the intestine

Support or Funding Information

This work was supported by a Crohn’s and Colitis Foundation Career Development Award (544599, to MQ) and NIH grants (HL127236 to AJG; DK055679, DK089763, DK059888, to AN; and DK61739, DK72564, DK79392, to CAP).
Low Birth Weight Pigs Exhibit Altered Cerebral Hemodynamics and Vasomotor Control
Authors: El Karsh Zeyad , Morton Jude , Jarque Maria T. , Patton Breanna , Rodrigues Lucas , Illing Kate , Chicoine Al , Sands Jade , Wellette-Hunsucker Austin , Ferguson David , Columbus Daniel A. , Weber Lynn , Olver T. Dylan
Published: April 2020

Low birth weight (LBW) offspring have a higher prevalence of long‐term cognitive impairments, possibly the result of altered blood flow control in the brain. Using a pig model, we tested the hypothesis that LBW progeny exhibit abnormal cerebral hemodynamics and vasomotor control during early developmental stages of growth. At birth, littermate pigs were identified as normal (NBW=1.7±0.1 kg) or low birth weight (LBW=1.2±0.2 kg). Measurements were obtained at 28 d (weaning) and 56 d of age (n=8/group, 4M/4F per group) : i) 28‐d old NBW (28‐NBW; body mass=10.0±0.4 kg); ii) 28‐d old LBW (28‐LBW; body mass=8.0±0.4 kg); iii) 56‐d old NBW (56‐NBW; body mass=23.0±1.0 kg); and iv) 56‐d old LBW (56‐LBW; body mass=17.0±0.8 kg). Cerebral blood flow velocity was measured using Transcranial Doppler ultrasound. Thereafter, pigs were euthanatized, their brains were harvested, weighed and their pial arteries were dissected for pressure myography experiments. Briefly, pial artery vasomotor control was studied in response to a Ca2+‐activated potassium (BK) channel α‐subunit agonist NS1619 (elicits vasodilation; 1e‐10‐1e‐4 M) and sympathetic co‐transmitter neuropeptide Y (NPY; potent vasoconstrictor; 1e‐10‐1e‐4 M). Brain mass and body mass were significantly lower in LBW vs. NBW pigs (p<0.01). Mean cerebral blood flow velocities were similar between LBW and NBW pigs (p≥0.20). Peak systolic velocities were greater (main effect of BW, p<0.01) and reductions in end diastolic velocities approached significance in LBW vs. NBW pigs (main effect of BW, p=0.08). Accordingly, the resistive index was greater in LBW vs. NBW pigs (main effect of BW, p<0.01). The maximum vasodilator response to NS1619 was not significantly different between LBW and NBW pigs (p=0.12). However, the overall magnitude of dilation (area under the curve) was lower in LBW vs. NBW pigs (main effect of BW, p<0.05). The maximum vasoconstrictor response to NPY was greater in LBW vs. NBW groups (main effect of BW, p<0.05). Similarly, the overall magnitude of constriction was greater in LBW vs. NBW pigs (main effect of BW, p<0.01). With respect to cerebral hemodynamics and vasomotor control, no main effects of age were observed (p≥0.50). Results from this study show that LBW pigs display alterations in cerebral hemodynamics as well as cerebral vasomotor control that persist for at least 56 days. Impaired BK channel function and enhanced vasoreactivity to sympathetic co‐transmitter NPY may underlie altered cerebrovascular hemodynamics in LBW pigs.

Support or Funding Information

SHRF Establishment grant (#4522) and NSERC
‐The Influence of Histone Variant H2A.Z and Linker Histone H1 on Saccharomyces cerevisiae Meiosis
Authors: Chigweshe Lorencia , MacQueen Amy , Holmes Scott
Published: April 2020

Meiosis in Saccharomyces cerevisiae is associated with unique chromosome choreography and chromatin reorganization. In vegetative cells, the histone variant H2A.Z, has a significant role in gene expression, DNA repair, chromosome segregation and maintenance of euchromatin‐heterochromatin boundaries, however, its function in meiosis has not been thoroughly investigated. The linker histone H1 has a role in global gene repression and genome compaction. We have shown that the loss of H1 suppresses some of the defects associated with the absence of H2A.Z. We investigated the impact of histones H2A.Z and H1 on budding yeast meiosis. We found that strains lacking either H2A.Z, H1, or both proteins exhibit wild type levels of sporulation efficiency. H1 does not influence spore viability, whereas the absence of H2A.Z reduces spore viability. Interestingly, the deletion of HHO1 can partially suppress the spore germination defect observed in the absence of H2A.Z. Our results indicate that strains lacking H2A.Z, H1 or both histones have recombination efficiencies similar to those of the wild type. We observe a chromosome axis defect in a subset of cells lacking H2A.Z. Our results suggest that histones H2A.Z and H1 collaborate to influence meiosis in budding yeast.
Transient Reductions in Serotonin Transporter Expression and Function Elicited by Blast‐Induced Traumatic Brain Injury
Authors: Benton Avery A. , Collins Sean M. , Robson Matthew J.
Published: April 2020

Traumatic brain injury (TBI) is a leading cause of disability among civilians and the hallmark injury associated with modern military conflicts around the globe. TBI is associated with enduring psychiatric comorbidities such as post‐traumatic stress disorder (PTSD), major depressive disorder (MDD), anxiety and social dysfunction. The molecular mechanisms by which TBI results in the generation of neuropsychiatric disorders are currently not well understood, however these disorders have classically been linked to serotonin (5‐HT) signaling. The single largest determinant of the cessation of 5‐HT signaling is the serotonin transporter (SERT), a Na+/Cl−‐dependent transporter that is the target of clinically utilized serotonin reuptake inhibitors (SRI’s) and is highly expressed on 5‐HT neurons located within the raphe nucleus of the mammalian CNS. We hypothesized that TBI results in alterations in the regional expression and/or function of SERT within the CNS. To test this hypothesis, we utilized a combination of a model for blast‐induced TBI, scaled in intensity and duration for murine subjects in combination with adult, wildtype C57Bl/6J mice. Subjects received a single, blast‐induced TBI or sham treatment followed by sample collection at 3, 10 and 30 days post‐injury (dpi). Righting reflex time (RRT) was recorded for all subjects immediately following injury. TBI was found to significantly increase RRT as compared to sham treatment, indicative of a loss of consciousness in rodent subjects. Western blot analysis or [3H] 5‐HT synaptosomal uptake assays were utilized to determine SERT protein expression levels and SERT‐mediated 5‐HT uptake post‐injury, respectively. Reductions in SERT protein expression were found in the dorsal raphe nucleus (DRN) 3 dpi. Concomitant reductions in SERT‐mediated midbrain synaptosomal [3H] 5‐HT uptake were found 3 dpi. These effects were found to be time dependent as SERT protein expression normalized by 10 dpi. Further, no post‐injury differences in SERT expression or [3H] 5‐HT uptake in the prefrontal cortex (PFC), a region of dense 5‐HT innervation were found. Data contained here are the first to show that blast‐induced TBI results in transient and regional alterations in SERT expression/function. A more thorough understanding of the temporal regulation of SERT function following neurotrauma may allow for better utilization of currently available pharmacotherapies for the treatment of TBI‐elicited neuropsychiatric disorders.

Support or Funding Information

Studies were supported by the PhRMA Foundation, the University of Cincinnati ASPET SURF Program and the University of Cincinnati.
Impaired Endothelial Caveolin‐1‐TRPV4 Channel Signaling Contributes to Endothelial Dysfunction in Pulmonary Hypertension
Authors: Daneva Zdravka , Marziano Corina , Ottolini Matteo , Cope Eric , Chen Yen-Li , Sonkusare Swapnil
Published: April 2020

Impaired endothelium‐dependent vasodilation is a key contributor to elevated pulmonary arterial pressure (PAP) in pulmonary hypertension (PH). While the mechanisms of pathogenesis of PH remain elusive, recent studies show that spatially localized Ca2+ influx via endothelial transient receptor potential vanilloid 4 (TRPV4EC) channels promotes vasodilation in resistance‐sized pulmonary arteries (PAs) by activating endothelial nitric oxide synthase (eNOS) and releasing nitric oxide (NO). Caveolin‐1 (Cav‐1EC) is an essential structural protein within the pulmonary endothelium, where it has been found to associate with TRPV4EC channels in cultured endothelial cells. We hypothesized that Cav‐1EC regulates TRPV4EC channel activity and PAP, and that impaired Cav‐1EC‐TRPV4EC signaling contributes to elevated PAP in PH. To test these hypotheses, we used right ventricular systolic pressure (RVSP) and PAP measurements in normoxic or chronic hypoxic Cav‐1EC−/− and TRPV4EC−/− mice, and pressure myography and high‐speed Ca2+ imaging studies in PAs from these mice. TRPV4EC channel activity and TRPV4EC‐mediated vasodilation were significantly impaired in PAs from Cav‐1EC−/− mice, suggesting that Cav‐1EC enhances TRPV4EC channel activity in PAs. Purinergic signaling plays an important role in pulmonary circulation, and we recently identified adenosine triphosphate (ATP) as a novel physiological activator of TRPV4EC channels in PAs. ATP activation of TRPV4EC channels was abolished by protein kinase C (PKC) inhibition, and was absent in the arteries from Cav‐1EC−/− mice, supporting a Cav‐1EC‐PKC‐TRPV4EC signaling that enables ATP activation of TRPV4‐EC channels. Resting RVSP and PAP were elevated in Cav‐1EC−/− and TRPV4EC−/− mice supporting the concept that Cav‐1EC‐TRPV4EC signaling lowers resting PAP. Moreover, exposure to chronic hypoxia (CH, 3 weeks) resulted in higher increases in RVSPs in Cav‐1EC−/− and TRPV4EC−/− mice when compared to the respective wild‐type control mice, suggesting that Cav‐1EC‐TRPV4EC channel signaling is protective against CH‐induced PH. We further used two well‐established models of PH— CH and sugen 5416 injection + CH. TRPV4EC channel activity, and ATP‐ and PKC‐activation of TRPV4EC channels were impaired in PAs from both the models of PH, suggesting that Cav‐1EC‐PKC‐TRPV4EC signaling may be impaired in PH. Taken together, these results suggest that Cav‐1EC‐PKC‐TRPV4EC signaling lowers resting PAP, and is impaired in PH. Deciphering the mechanisms that impair the Cav‐1EC‐PKC‐TRPV4EC signaling in PH may lead to novel therapeutic strategies to restore endothelium‐dependent vasodilation in PH.

Support or Funding Information

HL121484‐01, HL‐138496
Quantifying over expression of FoxP2 in the brain of budgerigars using immunohistochemistry
Authors: McGee Ryan Michelle
Published: April 2020

The gene FoxP2 is believed to have a significant role in the vocal circuit pathway in humans and other vocal learning taxa. We wanted to understand the role that FoxP2 plays in the ability to have vocal plasticity in parrots. We hypothesized that manipulating levels of expression of the FoxP2 gene in the brain of a Budgerigar would affect the degree of vocal plasticity in the parrots. Specifically, we predicted that inserting a viral vector containing FoxP2 into the vocal learning center MMst would result in over Expression of FoxP2 and a decrease in vocal plasticity relative to birds that received a control vector containing GFP. One crucial step is to assess the efficacy of our experimental is measuring FoxP2 levels on the targeted region of the Budgerigar brain. We used immunohistochemistry to attach antibodies with fluorescence, that then bonded to cells expressing FoxP2 in the experimental birds. Our results show that manipulations were successful and that both viral vectors showed uptake in the brain cells, with increased FoxP2 expression in the MMst of treated animals relative to surrounding untreated striatal tissue. These results lend credence to behavioral measures showing that the FoxP2 manipulated birds had lower levels of call sharing than controls. Our results shed light on the neurogenetic mechanisms underlying the impressive vocal plasticity seen in parrots and have the potential to contribute to therapies for the speech deficits seen in some patients with PTSD, stroke or cochlear implants.

Support or Funding Information

National Institute of Health, Howard Hughes Medical Institute, and the Discovery Scholars program at NMSU