bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein/Protein Interactions in the Human Cytochrome P450 System
Authors: Burris-Hiday Sarah Danyelle , Scott Emily Elizabeth 
Published: April 2020

Cytochrome P450 enzymes are the major superfamily of enzymes involved in xenobiotic metabolism and steroid biosynthesis. In vivo, human P450 enzymes are embedded in the lipid membrane and require a membrane‐bound redox partner protein for catalysis. This study focuses on P450 enzyme interactions with their redox partner, cytochrome P450 reductase. This protein is the required redox partner for 50 of the 57 human P450 enzymes. It accepts electrons from NADPH via its FAD and then FMN domains and then transfers these electrons one at a time to the P450 heme to support P450 catalysis. The FMN domain binds directly to the proximal face of P450 enzymes. However, this interaction is transient, and little is known about the details of the interactions of the FMN domain and different human P450 enzymes. Thus, this study sought to better define the structural interactions and the functional implications of the reductase FMN domain binding to multiple human P450 enzymes: steroidogenic CYP17A1 and drug‐metabolizing CYP3A4, CYP2A6, and CYP2D6.To facilitate interactions of the reductase FMN domain and the different P450 enzymes, artificial fusion proteins were generated. The fusion proteins were successfully expressed in E. coli and purified. Binding studies and substrate metabolism assays with typical ligands were used to compare the functions of these fusion proteins with the respective isolated P450 enzyme. These fusions are also being used to generate X‐ray structures to reveal the specific interactions between the reductase FMN domain and each different P450 enzyme. While the P450 proximal surface is thought to be the general binding surface for the reductase FMN domain, we expect to observe variations in the specific FMN domain/P450 interactions that may correlate with functional differences between the enzymes.Support or Funding InformationResearch supported by NIH R37 GM 076343 and T32 GM007767.
Potential Novel Role of UDP Glucuronosyltransferases 2B11 and 2B28 in Crosstalk Between Androgen and Lipid Signalling
Authors: Mackenzie Peter , Meyers Jai , Hulin Julie-Ann , Hu Dong-Gui , Mubarokah Siti , McKinnon Ross , Meech Robyn 
Published: April 2020

Androgens play critical roles in both prostate and breast cancer progression. Recent work has revealed extensive crosstalk between androgen and lipid signalling, including the ability of the androgen receptor to control lipid synthesis and uptake to fuel prostate cancer growth
[1,
2]. Members of the UDP‐glucuronosyltransferase (UGT) enzyme family can metabolically inactivate multiple lipophilic small molecules including steroids such as androgens, which modulates both prostate and breast cancer progression
[3,
4]. Moreover, transcriptional induction of UGTs by steroid‐liganded nuclear receptors (e.g. AR and ER) can create a feedback loop to control steroid signalling
[3].The UGT2B11 and UGT2B28 enzymes are dramatically induced by androgens in prostate and breast cancer cells (up to 200‐fold); however, unlike some other UGTs they have little or no activity towards androgens and hence their functions have been enigmatic. We now have evidence that UGT2B11 and UGT2B28 function via two novel mechanisms that together indirectly drive cancer cell growth downstream of androgen signals. First, UGT2B11 and UGT2B28 control the activity of the SREBP lipid‐sensing and signalling pathway leading to increased expression of target genes involved in lipid synthesis/metabolism. They also appear to influence feedback regulation of nuclear SREBP via proteolytic turnover. Second, UGT2B11 and UGT2B28 heterodimerize with and inhibit known androgen‐metabolizing UGT enzymes, thus preventing androgen inactivation and clearance. These findings suggest a new mechanism for crosstalk between androgen and lipid signalling pathways in breast and prostate cancer cells, and prompt further investigation of UGTs as therapeutic targets.Support or Funding InformationNational Health and Medical Research Council of Australia, Flinders Foundation
Global Examination of the Effects of Methamphetamine on Histone Post‐Translational Modifications in the Rat Striatum
Authors: Radosavljevic Adriana , Silverwood Tyler , Madore Victoria , Charntikov Sergios , Chu Feixia 
Published: April 2020

Methamphetamine (METH) is a highly addictive psychostimulant that has been associated with deleterious physical and social consequences. A variety of factors ranging from life experiences to genetics are believed to play a role in the addiction process. Post‐translational modifications (PTMs) on histone proteins can lead to alterations in the organization of nucleosomal arrays and the accessibility of transcriptional factors to DNA as a form of epigenetic regulation. This study investigated the effects of chronic METH self‐administration in rats on PTMs of histones H3 and H4 in the striatum using liquid chromatography‐mass spectrometry. Chronic METH use induced group downregulation of the following PTMs on histone H3: K9 dimethylation with unmodified S10 and T11 and K14 acetylation; K18 methylation with K23 acetylation; and K79 dimethylation with T80 phosphorylation. Individual economic demand for METH was positively correlated with H3K79 methylation, H3K14 acetylation, H4K5 acetylation, H4K8 acetylation, H4K12 acetylation, and H4K16 acetylation. The significant decrease of H3T80 phosphorylation supports a downregulation of neural progenitor cell proliferation and neurogenesis, which is consistent with previously discovered inhibition of astrocyte and neural progenitor propagation as a result of METH use. Our novel findings, showing correlation between individual demand and the abundance of post‐translational modifications, offer a novel insight into the epigenetic regulation associated with METH use and further the current understanding of the impact of substance use on epigenome remodeling, while also elucidating potential therapeutic targets for future studies.Support or Funding InformationThis project was funded by the Hamel Center for Undergraduate Research whose financial support made this research possible.
Exploring the copper resistance mechanism of the lipoprotein DcrB in Salmonella enterica
Authors: Gonske Sara J. , May John F. 
Published: April 2020

Salmonella enterica is a major bacterial cause of foodborne illness and invasive disease. Salmonella establishes infections by evading host macrophages; these host immune cells engulf the bacteria into a phagosome and use mechanisms to destroy the foreign body. Within the phagosome, bacteria are subjected to many harsh conditions, one possibly being toxic amounts of copper metal ions. We have previously demonstrated that the periplasmic‐anchored lipoprotein DcrB (possessed by S. enterica serovar Typhimurium) conveys resistance to toxic concentrations of copper ions, and thereby may play a role in Salmonella’s survival within the host. To gain a better understanding of the protein’s function, we have explored gene expression affected by DcrB and possible interactions between the protein and small, cellular metabolites. Using reverse‐transcriptase quantitative PCR, we have shown that DcrB plays a role in the upregulation of some cell envelope stress response genes. DcrB, however, does not significantly affect regulation of copper ion transport genes or other copper ion resistance genes. Through thermofluor assays, we have shown that purified DcrB protein has increased stability in toxic copper ion concentrations when extracellular amino acids are present in solution. This result suggests that DcrB has copper‐dependent interactions with free amino acids. Overall, these findings advance our understanding of how DcrB confers resistance to copper ions. This knowledge could be used to develop new antibiotics targeting bacterial copper resistance, ultimately decreasing the number of invasive illnesses caused by S. enterica and other gram‐negative bacteria.Support or Funding InformationThis research was supported by a UWL Faculty Research Grant to JFM.
Isorhamnetin ameliorates sleep deprivation induced cognitive dysfunction by reducing oxidative stress, inflammation and neuronal apoptosis in mice
Authors: Zhang Xiuli , Wang Xiuhua , Yu Yan , Zhang Jing , Sun Hongliu , Han Fang , Lv Changjun 
Published: April 2020

Sleep deprivation (SD) could cause a sequence of neurobehavioral deficits and physiological consequences. Isorhamnetin (ISOR) has been verified to have neuronal protective, anti‐inflammatory and anti‐oxidative effects. This article aimed to explore the mechanism under the neuroprotective effect of ISOR against 72h SD‐induced anxiety like behavior, oxidative stress, neuroinflammation in mice. The ameliorating effect of ISOR on SD‐induced learning and memory impairment was evaluated by Open field test (OFT) and Morris water maze tests (MWM). After the behavioral tests, antioxidant effects were detected by malondialdehyde (MDA), superoxide dismutase (SOD), reduced glutathione (GSH) and catalase (CAT) assays. The level of tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) both in serum and brain homogenates were detected by ELISA. Expressions of Bcl‐2, Bax were also detected along with JNK, P38 and ERK phosphorylation by western blot. HE staining and TUNEL assays were used to observe the neuron pathologic morphology and apoptosis caused by SD. The results suggest that ISOR could improve cognitive functions induced by 72h SD by reducing the anxiety like behavior, oxidative stress, neuroinflammation and the neuronal apoptosis.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China (Grant No.81102828, 81273037) and the Natural Science Foundation of Shandong province of China (No.2015ZRB14548)
Computer‐Aided Discovery of Novel Human RORγ Inverse Agonists
Authors: Huang Mian , Bolin Shelby , Miller Hannah , Dykeman Anna , Ng Ho Leung 
Published: April 2020

Overexpression of pro‐inflammatory cytokine IL‐17 can lead to various autoimmune disorders, including psoriasis, Crohn’s disease, and Ankylosing Spondylitis. However, when an inverse agonist binds to the ligand‐binding domain (LBD) of human retinoic acid receptor‐related orphan receptor γ (RORγ), the transcriptional function of RORγ is down‐regulated, resulting in diminished IL‐17 expression. It makes RORγ an ideal drug target for treating autoimmune diseases. This has been studied for over ten years, but only several RORγinverse agonists are under clinical trials. New methods may be needed to develop new drugs efficiently. In this project, we aim to discover potential RORγ inverse agonists by developing a structure‐based virtual screening method that effectively screens millions of compounds in a shorter time scale. We here applied the in silico program iDock to screen a ligand‐like compound library by docking every compound into the binding pocket of RORγ LBD. iDock evaluated the performance of each compound in the pocket and outputted the top 1000 hits. Through excluding the hits structurally similar to the known RORγ inverse agonists, we focused on the ones with unique chemical scaffolds and tested their bioactivity via luciferase reporter assay. A family of compounds sharing the same backbone was found. Two members in this family, RG14 and RG14‐2, inhibited the RORγ transcriptional function with IC50 values at 3.2 μM and 1.5 μM, respectively. To further study the interactions between the compounds and the protein, binding affinities of RG14 and RG14‐2 to the LBD are under investigation by thermal shift assay. RORγ LBD has been successfully co‐crystallized with RG14 and RG14‐2. With solving crystal structures of the protein‐ligand complexes, we will obtain more information to interpret the interactions structurally. A lead compound of a novel class of RORγ inverse agonists is expected to develop, basing on the chemical structures of RG14 and RG14‐2 and their binding actions to RORγ LBD.Support or Funding InformationNSF 1833181
Endothelial Small GTPase RhoA: A Potential Target for Anti‐angiogenic Therapy
Authors: Zahra Fatema Tuz , Sajib Md Sanaullah , Akwii Racheal , Tullar Paul , Lionakis Mihalis S. , Mikelis Constantinos M. 
Published: April 2020

ObjectiveTumor vessels display abnormal structure and function due to imbalanced angiogenesis. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the best known pro‐angiogenic factors in this process. The goal of antiangiogenic therapy is to inhibit tumor growth by oxygen and nutrient deprivation. Current antiangiogenic therapies target VEGF/VEGFR signaling pathways. However development of resistance due to upregulation of bFGF remains a serious disadvantage of current approaches, leading to efforts of simultaneous targeting of both VEGF and FGF pathways. A small GTPase RhoA has been reported to regulate VEGF‐induced angiogenesis. In the present study we investigate the role of endothelial RhoA in bFGF‐induced angiogenesis to identify whether RhoA can be considered a target for both VEGF‐ and FGF‐induced angiogenesis.MethodsWe performed RhoA pulldown assays to identify RhoA activation downstream of bFGF treatment. We used Boyden chamber cell migration assay, 2D tube formation assay using matrigel and 3D sprouting assay with collagen to assess angiogenic potential. Endothelial RhoA was knocked down by siRNAs or blocked by pharmacological inhibitors. Biochemical assays were performed to identify the molecular mechanism of bFGF‐induced RhoA activation. In vivo, endothelial‐specific inducible RhoA‐deficient mice with a fluorescent reporter were used in a newly modified matrigel plug angiogenesis model allowing identification of endothelial RhoA excision efficiency.ResultsbFGF treatment activates RhoA in human umbilical vein endothelial Cells (HUVEC). RhoA inhibition with C3 toxin or RhoA knockdown abrogated bFGF‐induced endothelial cell migration and tube formation in vitro. In vivo, endothelial RhoA deficiency blocked bFGF‐induced angiogenesis in matrigel plug assay. Among the bFGF receptors, FGFR1 is predominantly expressed in HUVECs and FGFR1 knockdown abrogated bFGF‐induced RhoA activation and angiogenesis. RhoA inhibition partially blocked bFGF‐induced JNK phosphorylation but did not affect ERK and p38 activation. Ongoing experiments are aiming to delineate the precise role of endothelial RhoA in the FGF downstream pathway.ConclusionCollectively, our data suggest that RhoA pathway participates in bFGF‐induced angiogenesis and can be a potential target for anti angiogenic therapy.
Engineering and Structural Analysis of a Covalent HRas Dimer
Authors: Volkman Lauren E. , Packer Morgan R. , Mattos Carla 
Published: April 2020

Ras is part of the major signaling cascade Ras/Raf/MEK/ERK which regulates cell proliferation. The dysregulation of this pathway through the mutation of Ras is highly implicated in up to 30% of human cancers. Pathway activation is dependent on the binding of Ras to Raf‐RBD and the subsequent dimerization of the Ras/Raf complex. This complex, however, is only robust in the presence of the membrane. In order to create a form of the Ras/Raf‐RBD dimer that is more stable in solution, we engineered mutations to promote covalent bonding at the dimer interface. Our data reveal the structure of a covalent HRas dimer in the absence of Raf‐RBD, and we present an analysis of the disulfide bonded dimer interface and its impact on allosteric connections to the active site. Going forward we plan to use crosslinking as a means to characterize the Ras/Raf‐RBD dimer biochemically, including affinity of the Ras dimer for Raf‐RBD and hydrolysis rate constants in the presence and absence of ligand binding at an allosteric site near the dimer interface.Support or Funding InformationNSFCHE‐1757078
Subchondral and Trabecular Bone Respond Differently to Exercise in Juvenile Sheep
Authors: Polk John D. , Song Hyunggwi , Kersh Mariana E. 
Published: April 2020

Evaluating the responses of subchondral and underlying trabecular bone properties to exercise interventions have implications for understanding normal developmental processes as well as the etiology of diseases affecting these tissues. We hypothesized that exercise would result in (1) increased subchondral and trabecular thickness and density, and increased bone volume fraction, (2) the distribution of increased bone properties would reflect experimentally induced postural differences, and (3) shallower trabecular bone would show a greater response to exercise than deeper trabecular bone. To evaluate these hypotheses we randomly assigned thirty juvenile sheep to flat exercise (n=11), inclined exercise (15% grade; n=11) which used more flexed knee postures, and non‐exercised control (n=8) groups. Exercise was conducted on motorized treadmills twice daily for 20 min at 1.12m/s for 60 days. Distal femora were micro‐CT‐scanned at 50μm resolution. Trabecular thickness, apparent density, bone volume fraction, anisotropy, as well as subchondral thickness and apparent density were measured using custom algorithms in AMIRA, ImageJ and MATLAB. Subchondral thickness showed significant and predictable response to exercise and with approximately 20% thicker bone in both exercise groups compared to controls, and postural changes with increased thickness toward the posterior side of the condyle in the incline group. Subchondral density did not increase substantially with exercise. In contrast trabecular density was increased slightly with exercise (~5%) but the response did not correspond to incline‐exercised and control groups. These results suggest a greater responsiveness to exercise in subchondral than trabecular bone, and imply that increases in stiffness in these tissues may be achieved through modulation of different tissue properties.Support or Funding InformationNational Science Foundation 1638756
Characterization of Constitutively Activating Eel Follicle‐Stimulating Hormone Receptor
Authors: Min Kwan-Sik , Byambaragchaa Munkhzaya 
Published: April 2020

This study aimed to investigate the function of the constitutively activating mutation D540G on eel FSHR activity by in vitro functional studies. Site‐directed mutagenesis was carried out to generate the D‐to‐G mutation at position 540 of the pcDNA3‐eel FSHR construct. Vectors expressing either wild type or mutant receptor were transfected into Chinese hamster ovary (CHO‐K1) cells. The functional characteristics of both the wild type and mutant receptors were analyzed by a cAMP assay. cAMP accumulation was highly increased in cells transfected with the D540G mutant receptor in a dose‐dependent manner. Of note, basal cAMP levels were remarkably increased (□13.1‐fold) with expression of this mutant when compared to wild type receptor. We also analyzed inactivation mutants (A193V, N195I, R546C and A548V). The mutant (R546V) indicated a little response and the other 3 mutants was completely flat in the cAMP responsiveness. These findings suggest that the D540G mutation in the eel FSHR may contribute to ovulation during eel sex maturation as well as play a pivotal role in inducing FSHR activity.Support or Funding InformationThis work was financially supported by Korean Research Foundation Program (2018R1A2B6007794), Republic of Korea
A Cadaveric Analysis of Morphological Variations of the Anterior Belly of the Digastric Muscle
Authors: Anderson Hana , Tucker Richard P. 
Published: April 2020

The digastric is a suprahyoid muscle characterized by two distinct muscle bellies, anterior and posterior, linked by an intermediate tendon. The anterior belly of the digastric muscle (ABDM) displays a wide array of anatomical variations and there is rich literature documenting its variations, going back as early as the 18th century. A wide range of prevalence of ABDM variations is found in the literature, ranging from as low as ~5% to as high as ~70% of the examined cadavers in each study. Morphologies of ABDM variations are also fairly complex, including fusion of the right and left ABDM, addition of a unilateral or bilateral accessory muscle, and composites of multiple accessory muscles. Reflecting the common embryological origin in the first pharyngeal arch and shared innervation of the mandibular branch of the trigeminal nerve, ABDM variations frequently also involve the mylohyoid muscle.In the current study, we sought to improve our understanding of ABDM variations in the ethnically diverse whole‐body donor population in Northern California. Specific aims were: (1) To determine the frequency of ABDM variations in cadavers donated to the UC Davis Body Donation Program, (2) To classify the observed ABDM variations in this population using the nomenclatures proposed by Zlabek (1933) and Yamada (1935), and (3) To investigate the innervation and arterial supply to ABDM accessory muscles.Forty‐eight cadavers (male 23, female 25, average age 75) were examined over a two‐year period and ABDM variations were identified in 15 donors (31.2%). These variations were classified as: (1) Atavistic (Atavique) type – 2 cadavers, (2) Origin (Antérieur) type – 5 cadavers, (3) Insertion (Postérieur) type – 5 cadavers, (4) Mixed (Mixte) type – 1 cadaver, and (5) Composite (Composé) type – 2 cadavers. Innervation and arterial supply were determined for one of the atavistic types and one of the origin types. It was found that innervation was from the nerve to mylohyoid and arterial supply was from the submental artery originating from the facial artery, which fit to the typical innervation and blood supply to the ABDM. In summary, on average we found ABDM variations in 1 in 3 individuals in the local Northern California population. Knowledge of a high prevalence of ABDM variations in the general population as reported here would be valuable information when an operation or examination is performed in the submental region.
Protease Activities of Proteins Secreted by Fusarium sp. in Aerobic and Anaerobic Conditions
Authors: Mercado Benjamin , Bayman Paul 
Published: April 2020

A broad spectrum of infections has been associated with opportunistic pathogens in the genus Fusarium, ranging from the superficially and locally invasive to disseminated infections. Proteases secreted by filamentous fungi like Fusarium help the pathogen colonize the mucosa and penetrate tissues during dissemination, causing disease. When Fusarium infections are not treated correctly, the host can develop an abscess, necrosis or ischemia resulting in severe damage to the infected tissue or even death. Fungal infection may occur in low oxygen in the lungs and there are study cases of immunocompromised patient dying after a disseminated fungal infection developing necrosis and abscess. There are no studies on growth of Fusarium in infected tissues with low oxygen. The main aim of our study is to determine and compare the activities of proteases secreted by Fusarium in aerobic and anaerobic conditions. A strain of Fusarium was grown in media culture enriched with soybean grains for 3 weeks in aerobics and anaerobic conditions. Extracts from cultures were filtered and tested at pH ranging from 8–11 at 40° 50° 60° and 70° C to determine protease activity against azocasein as measured by absorbance at 436 nm. Results showed higher activity of proteases at 70° C for enzymes secreted in both aerobic and anaerobic conditions. Protease activity was highest at pH 9 for enzymes secreted in aerobic conditions but was highest at pH 10 for enzymes secreted in anaerobic conditions. These results confirm the secretion of proteases by filamentous fungi in anaerobic conditions. The differences in pH maxima could suggest that a different protein profile is secreted under low oxygen availability. Also, the secretion of proteases in anaerobic conditions by Fusarium could suggest an active role in the process of tissue damage during anaerobic infections.Support or Funding InformationG‐RISE program: 5R25GM061151‐18.