bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mapping Purinosome Protein‐Protein Interactions Using In Vivo Photocrosslinking
Authors: He Jingxuan , Zou Ling-Nan , Anand Ganesh S. , Benkovic Stephen J. 
Published: April 2020

Efficient de novo purine biosynthesis in higher eukaryotes is hypothesized to depend on the existence of a multienzyme complex — the purinosome. However, there is as yet no compelling identification of specific protein‐protein interactions between purine biosynthetic enzymes. Similarly, functionally important interactions between purine biosynthetic enzymes and other proteins have yet to be elucidated. Inspired by the highly symmetric oligomeric structure of PAICS, a de novo purine biosynthetic enzyme, we identified several putative interaction surfaces. Using genetically encoded unnatural amino acid incorporation, we inserted photocrosslinking amino acid analogs into these surfaces at specific residues. Upon UV irradiation, we were able to detect multiple crosslinked products between PAICS and other proteins, formed in their native context inside living cells. Distinct sites of photocrosslinker incorporation yielded distinct products; this suggests they are the result of specific protein‐protein interactions. Using mass spectrometry, we are in the process of identifying PAICS’s crosslinked partners, as well as their interaction interface with PAICS. We will also extend this approach to study other pathway enzymes as we seek to identify functionally important protein‐protein interactions in de novo purine biosynthesis.Support or Funding InformationThe National Institutes of Health (GM024129 to S.J.B.)
Endothelial Cell Bulge Occludes the Vasa Vasorum in the Cerebellar Vascular Hyalinosis Lesions
Authors: Shults Nataliia V. , Marcocci Lucia , Rybka Vladyslava , Suzuki Yuichiro , Gychka Sergiy G. , Gavrish Alexander S. 
Published: April 2020

BackgroundThe remodeling of the cerebral vascular wall contributes to the occurrence of ischemic stroke in systemic hypertension patients, representing a leading cause of death in the U.S. and worldwide. Cerebral vascular remodeling in systemic hypertension is characterized by progressive degradation of structural components of each layer of the vessel wall with the subsequent transformation to hyaline material leading to the thickening vascular wall and lumen narrowing. The vasa vasorum as a network of small blood vessels supplying the blood to the vascular wall tissues contributes to the damage vascular structure and results in the formation of hyalinosis. The occlusion of vasa vasorum contributes to development of cerebral vascular hyalinization in systemic hypertension. However, mechanisms of occlusion of vasa vasorum that contribute to the brain vascular remodeling events have not been well defined. Lack of such knowledge interferes with the development of therapeutic strategies to reduce the occurrence of ischemic stroke.Methods & ResultsLight microscopy and transmission electron microscopy (TEM) of hyalinized cerebral vessels from human patients who suffered from systemic hypertension and died by ischemic stroke visualized the occlusion of the vasa vasorum due to the formation of the “bulge” structure that protrude from the endothelial cells. Treatment of microvascular endothelial cell with adrenaline (a major biogenic amine that are increased in the blood due to disbalance of neurohumoral homeostasis in systemic hypertension) resulted in a cytoskeletal rearrangement that contributes to the formation of the bulge‐like structure in the vasa vasorum of the brain vessels with hyalinosis lesion. The adrenaline activated clathrin‐mediated endocytosis of endothelial cells that in turn elicited SNX9/N‐WASP‐dependent disruption of cytoskeleton. siRNA knockdown of SNX9 inhibited endocytosis and prevented the adrenaline‐induced bulge formation in human microvascular endothelial cells. TEM visualized the adrenaline‐induced an increase endocytosis of the endothelial cells. Immunofluorescence staining showed the actin filament structural reorganization and TEM confirmed myofilament damage within endothelial cells. Adrenaline treatment induced post‐translational modifications of N‐WASp as a regulator of actin polymerization.ConclusionsThis is the first demonstration that adrenaline contributes to the occlusion of vasa vasorum in the hyalinized cerebral vascular wall that will allow to the development new strategies that can be used to prevent ischemic stroke.Support or Funding InformationSupported by NIH
Capsaicin changes the pattern of brain rhythms in sleeping rat
Authors: Tian Yuhua , Du Guangxu , Jin Jingyu , Yang Zhihong , Hu Fang , Lu Wei , Wang KeWei , Zheng Jie 
Published: April 2020

The heat and capsaicin sensor TRPV1 ion channels were originally discovered from sensory neurons in dorsal root ganglia, and later found in many other tissues and organs. However, whether TRPV1 channels are present in brain regions other than hypothalamus has been a subject of debate. Here we addressed this issue with an unbiased functional test, by recording electroencephalogram (EEG) to examine whether capsaicin injection directly into the rat lateral ventricle could alter brain electrical activity. We observed that EEG during the sleep stage could be significantly perturbed by capsaicin, whereas EEG during the awake stage did not show a detectable change. Our results are consistent with TRPV1 expression in selective brain regions whose activities are dominative during the sleep stage.Support or Funding InformationThis work is supported by funding from the Qingdao Postdoctoral Research Project to YHT.
The Howl of Rancho La Brea: the functional morphology of Pleistocene canid hyoids
Authors: Flores Mary Deanna , Eldridge Emma , Elminowski Erin , Dickinson Edwin , Hartstone-Rose Adam 
Published: April 2020

The Rancho La Brea (RLB) fossils housed at the La Brea Tar Pits and Museum in Los Angeles, California represent one of the richest collections of carnivoran fossils in the world. The collection is particularly well known for exceptional preservation, including that of of rare and understudied bones. Among these, the RLB collection houses hyoids from several extinct species, including relatively large samples of specimens from the dire wolf (Canis dirus) and the coyote (Canis latrans). In this study, we compare fossilized hyoid elements from these two fossil samples to modern specimens: the gray wolf (C. lupus), red wolf (C. rufus), and modern coyote (C. latrans). Seven measurements were taken on each long bone of the hyoid apparatus and eight measurements of the u‐shaped basihyoid. Principal component analysis was performed to determine statistical differences between species and the drivers of those morphological differences. For most of the bones, the majority of the variation was driven by size. Red wolves, modern coyotes, and RLB coyotes could not be differentiated from each other based on any of the hyoid elements, but dire wolves could be clearly differentiated from all other canids for all elements. The hyoid apparatus of C. dirus is larger and more robust than that of C. lupus, its closest modern analogue. As larger hyoids have been associated with a lower vocal frequency, this distinction would likely result in the vocalizations of C. dirus occurring at a lower frequency than those produced by C. lupus.Support or Funding InformationNorth Carolina State University Office of Undergraduate Research Grant
Capsaicin enhances the pro‐apoptotic activity of Camptothecin in human small cell lung cancer
Authors: Dasgupta Piyali , Friedman Jamie R , Perry Haley E , Colclough Kate W , Stevenson Cathryn D , Hurley John D , Akers Austin T , Nolan Nick A , Brown Kathleen C , Chen Yi Charlie , Denning Krista L 
Published: April 2020

Small cell lung cancer (SCLC) is characterized by early dissemination, aggressive clinical course and a dismal survival rate. The first line therapy for SCLC patients is cisplatin‐based combination chemotherapy along with radiation. Initially, cisplatin shows excellent results causing remission in 60–80% of the SCLC patients but within a year the tumor inevitably relapses and this relapsed tumor is resistant to cisplatin. A subset of SCLC tumors are unresponsive to cisplatin from the outset. These patients are said to have platinum‐refractory SCLC tumors. Patients with platinum resistant or refractory disease have very limited options, as the only standard chemotherapy with an FDA‐approved drug, camptothecin (and its derivations), has an objective response rate of approximately 3% and little or no survival benefit. Such sobering statistics define the arena where there is an urgent need for drugs which enhance the pro‐apoptotic activity of camptothecin in human SCLC. The present study tests the hypothesis that capsaicin can sensitize human SCLC cells to the apoptotic effects of camptothecin. Caspase‐3 activity assays reveal that the combination of camptothecin and capsaicin displayed greater apoptotic activity than the drugs used alone. We verified these results using a second apoptosis assay, namely the Cell Death ELISA and obtained similar results. Chou‐Talalay isobologram analysis showed the interaction between camptothecin and capsaicin is synergistic. Chicken chorioallantoic membrane (CAM) assays confirmed the combinatorial apoptotic effects of capsaicin and camptothecin in human SCLC. The co‐operative effects of capsaicin and camptothecin were also confirmed in human SCLCs xenografted on athymic mice. The results of our experiments pave the way to novel combination therapies for human small cell lung cancer.Support or Funding InformationSupport or Funding Information Funding for our study was supported by a NIH R15‐AREA Grant (2R15CA161491‐02). Furthermore, this study was supported in part by a Center for Natural Products pilot grant from Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH).
Circulating Irisin Level as an Early Biomarker of Decline of Metabolic Health
Authors: Romanelli Massimiliano Marco Corsi , Vianello Elena , Sitzia Clementina , Benedini Stefano , Gorini Silvia , Rampoldi Benedetta , Rigolini Roberta , Tacchini Lorenza , Ambrogi Federico , Dozio Elena 
Published: April 2020

A decline in metabolic health may take place before observing an alteration in the levels of the traditional metabolic markers. New indicators of metabolic derangement are therefore compelling. Irisin is a myokine which exerts important metabolic functions: it increases energy expenditure, improves insulin resistance, reduces glucose level and advanced glycation end products (AGE)‐induced inflammation. The optimal level for irisin in human and its potential role as a metabolic biomarker have not been fully established yet.To this aim, we quantified plasma irisin level in 106 healthy subjects (age 43 ± 11.72 years, 58 males/48 females) and we performed a cluster analysis to evaluate the association between irisin and subject characteristics [age, total cholesterol, LDL, HDL, tryglicerides, NEFA (non‐esterified free fatty acids), glucose, HbA1c (glycated hemoglobin), insulin, GA (glycated albumin), uric acid, creatinine, irisin, sRAGE (total soluble receptor for AGE), esRAGE (endogenously secreted receptor for AGE), cRAGE (membrane‐cleaved receptor for AGE) and cRAGE/esRAGE ratio]. ELISA tests were used for the quantification of parameters different from routine biochemical markers. Glycated albumin (GA) was meausured by the enzymatic method Lucica®GA‐L. The study was conducted in accordance with the Declaration of Helsinki.Irisin median level was 7.89 ng/ml. Although the mean levels of the traditional metabolic blood markers were within the ranges of normality, subjects in cluster 1 displayed a metabolic profile shifted toward the highest reference values. In fact, we observed higher concentrations of total cholesterol, LDL, triglycerides, NEFA, glucose, HbA1c, insulin, uric acid and creatinine. The reduced levels of the anti‐AGE molecules sRAGE and esRAGE, the increased levels of cRAGE/esRAGE (a surrogate marker of inflammation) and GA (an AGE product), and a greater percentage of subjects with visceral obesity in cluster 1 confirmed an increased metabolic stress and a pro‐inflammatory profile in this group. Irisin concentration was higher in this group too.The traditional metabolic blood tests seem to be not enough to early identify a metabolic decline. The increase of irisin level may reflect a metabolic derangement at the beginning of its development. The role of irisin as an early biomarker of decline in metabolic health seems an interest topic to be further explored.Support or Funding InformationThe study was supported by funding from Fondazione E. A. Fiera Internazionale di Milano to Università degli Studi di Milano and Ricerca Corrente funding from Italian Ministry of Health to IRCCS Policlinico San Donato.
The Metabolic Woes of the Alveolar Macrophage in HIV
Authors: Yeligar Samantha M. , Auld Sara C. , Staitieh Bashar S. 
Published: April 2020

ObjectiveDespite anti‐retroviral therapy, people with human immunodeficiency virus (HIV) are still at high risk for respiratory infections and non‐infectious pulmonary diseases. Alveolar macrophages (AM), which act as a viral reservoir, are critical immune effectors in the lung that phagocytize and clear pathogens. We have previously shown that oxidative stress impairs AM phagocytic function. Therefore, we hypothesized that HIV causes AM immune dysfunction by increasing oxidative stress by (1) up‐regulating NADPH oxidases (Noxes), which are the primary sources of cellular oxidative stress in the AM, and (2) causing mitochondrial derangements and consequent increases in mitochondria‐derived oxidative stress.MethodsAM were isolated from the bronchoalveolar lavage fluid of HIV transgenic rats that express the HIV proteins, trans‐activator of transcription and glycoprotein‐120, or littermate control rats. AM were also isolated from the bronchoalveolar lavage fluid of people with HIV or otherwise healthy control subjects. mRNA and protein levels of Noxes were assessed by qRT‐PCR and cytoimmunostaining, respectively. Mitochondrial oxygen consumption rate and bioenergetics were measured by Cell Mito Stress Test assay using an extracellular flux analyzer. AM phagocytosis was evaluated by pHrodo‐labelled S. aureus internalization.ResultsCompared with controls, AM from HIV transgenic rats and people with HIV exhibited: (a) increased mRNA levels of Nox1, Nox2, and Nox4; (b) increased protein expression of Nox1, Nox2, and Nox4; (c) decreased mitochondrial respiration and bioenergetics; and (d) decreased AM phagocytic function.ConclusionsHIV increases cellular sources of oxidative stress via upregulation of Noxes and mitochondrial sources of oxidative stress via derangements in mitochondrial function, which may contribute to impaired AM phagocytosis. Therapeutic targeting of oxidative stress pathways could provide a novel approach to improving lung immune defense in people with HIV.Support or Funding InformationResearch Funding Sources: NIAAA R00AA021803 and R01AA026086 to SMY, NIAID K23AI134182 to SCA, and NIAAA K08AA024512 to BSS.
Differential inhibition of target gene expression by human microRNAs
Authors: Zeng Yan , Li Peng , Chen Yi , Juma Conslata , Yang Chengyong , Zhang Xiaoxiao 
Published: April 2020

microRNA (miRNA) target genes are commonly computationally predicted, yet how dependable predictions are remains unclear, as most predicted target genes have not been rigorously tested experimentally. In addition, the degree to which a miRNA differentially inhibits the expression of its targets is underappreciated. We selected human miR‐1, miR‐122, and miR‐124 as representatives to investigate the reliability of miRNA target predictions and to examine how miRNAs suppress their target genes. We first constructed target gene reporter libraries for the miRNAs based on three prediction programs: TargetScan, miRanda, and PicTar, and used reporter assays in cell cultures to directly evaluate whether as well as how strongly a predicted target gene is repressed by its cognate miRNA. We found that all three programs have approximately 72–85% success rates in predicting genuine miRNA targets, and that miRNAs inhibit different targets to various degrees. We then performed statistical and correlation analyses to examine how well parameters offered by the online programs and features in target mRNAs might predict the degrees of repression by miRNAs. To show that endogenous miRNAs act through the same mechanism, we correlated our reporter assay results with publically available mRNA expression data in vivo. Overexpression of miR‐1, miR‐122, or miR‐124 further led to differential reduction of target mRNA levels in cell cultures. Our studies systematically investigated hundreds of miRNA target genes, shed light onto the performance of miRNA target gene prediction programs, and suggested a new mechanism by which differential target repression by miRNAs regulates gene expression in vivo.Support or Funding InformationThis work was supported in part by the National Natural Science Foundation of China [grant number 31570843].
Cell Toxicity Studies of Tremetone Isolated From Rayless Goldenrod (Isocoma pluriflora)
Authors: Green Benedict , Lee Stephen , Davis Zane , Welch Kevin 
Published: April 2020

This research compared the cytotoxic actions of the benzofuran ketone tremetone in B16 murine melanoma cells, TE‐671 human rhabdomyosarcoma cells, and SH‐SY5Y human neuroblastoma cells using a MTT assay, with and without exposure to rat liver microsomes (RLM). Tremetone is of interest because it is a putative toxin in white snakeroot and rayless goldenrod. Identifying the toxic principal(s) of white snakeroot is important because in addition to poisoning livestock by causing “trembles,” it also causes “milk sickness” in humans that drink the milk from dairy animals that have eaten these toxic plants. Tremetone was not cytotoxic in B16 cells. In TE‐671 and SH‐SY5Y cells, concentration‐dependent tremetone cytotoxicity occurred without microsomal activation. In SH‐SY5Y cells the best fit values were; tremetone IC50 = 490 μM, tremetone and RLM IC50 = 505 μM. In TE‐671 cells the best fit values were; tremetone IC50 = 2.5 μM, tremetone and RLM IC50 = 2.2 μM. These results suggest that in B16 cells, tremetone is not cytotoxic. The results from experiments with SH‐SY5Y and TE‐671 cells suggest that tremetone does not require microsomal activation to be cytotoxic in a concentration‐dependent manner. This study demonstrates that tremetone is cytotoxic and plays a role in the disease caused by white snakeroot and rayless goldenrod.Support or Funding InformationThis research was supported by USDA.
Tracing the Route of the Dorsal Nerve of the Clitoris
Authors: Gordon Victoria , Rowe Joanna , Grubb Lauren , Segars Larry , Surek Chris C. , McCumber Travis , Willett Gilbert , Olinger Anthony 
Published: April 2020

OBJECTIVELiterature indicates an increasing number of complications following the labiaplasty procedure which is a surgery with a known problem of underreported and understudied long‐term outcomes and complications such as loss of sensation in the female pudendum. The aim of this study was to determine the course and any variations of the dorsal nerve of the clitoris (DNC) after it pierces the perineal membrane to generate a surgical safe zone for procedures involving the female pudendum.METHODSCadavers from the University of Nebraska Medical Center, Creighton University, and Kansas City University anatomy labs were dissected for this study. A shallow vertical incision was made from the pudendal cleft superiorly through the mons pubis. The glans clitoris was identified and used as a landmark while the fascia surrounding the body of the clitoris was carefully removed. The DNC was located where it pierces the perineal membrane and traced distally. The following measurements were taken: 1. from the point where the DNC pierces the perineal membrane to the urethra, 2. from the point where the DNC pierces the perineal membrane to the pubic bone, 3. from the angle of the clitoris to where the nerve branches on the dorsum of the body of the clitoris and 4. from that branch point to the distal most portion of the glans clitoris. Any anomalous branching patterns were noted.RESULTSSpecific branching patterns were identified, mapped out and characterized into individual types. In addition to the patterns, the measurements described above were used to generate a surgical safe zone which can be used to avoid injury to the DNC during procedures involving the female pudendum. The variance was also calculated for each point of measurement to describe how much variation there is within a specific measurement to identify where the most caution is needed during surgery. Furthermore, it was determined if there were statistically significant differences between the right and left sides with regards to the nerve.CONCLUSIONSThe findings of this study have allowed the investigators to map out a surgical safe zone for procedures involving the female pudendum to avoid injury to the DNC. Furthermore, multiple anomalous branching patterns were identified and the frequency of those patterns were reported. This information should be useful to physicians performing procedures near the DNC. Some of the specimens displayed anomalous branching patterns which did not fit any of the other types identified and physicians performing procedures should be aware of those patterns as well.Support or Funding InformationThere is no support or funding information.