bluuebunny/crossref_metadata_2025 huggingface dataset:
The Structurally Unusual Retaining β‐Kdo Glycosyltransferase WbbB Uses a Double‐Displacement Mechanism with an Intermediate Adduct Rearrangement Step
Authors: Kimber Matthew S. , Forrester Taylor J.B. , Mallette Evan , Ovchinnikova Olga G. , Nothof Jeremy T. , Koizumi Akihiko , Lowary Todd L. , Whitfield Chris
Published: April 2020

WbbB is a modular, trifunctional glycosyltransferase (GT) which synthesizes lipopolysaccharide O‐antigen in Raoultella terrigena. The two C‐terminal WbbB GT domains build the polysaccharide repeat, while the N‐terminal GT domain acts as a terminator, adding a β‐Kdo (3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid) residue to O3 to rhamnose as required for O‐antigen export. This N‐terminal GT domain is highly unusual in sequence and was not recognized as a GT by bioinformatics tools. The structure shows the dual Rossmann‐fold motifs characteristic of GT‐Bs, but with extensive deletions, insertions and rearrangements result in a unique architecture. The CMP‐binding site, however, retains motifs homologous to GT‐B sialyltransferases. WbbB is a retaining GT, transferring Kdo from the CMP‐®‐Kdo donor with net retention of anomeric configuration. All well‐characterized retaining GTs seem to employ a front‐side SNi substitution mechanism. This is somewhat surprising, as analogous retaining glycosyl hydrolases use a double‐displacement mechanism; here an essential catalytic acid residue attacks the donor saccharide to form a covalently bonded intermediate, with a second acidic residue acting as a general base to hydrolyse this adduct. We show, using mass spectrometry, that WbbB forms a Kdo adduct with Asp232 and its variants (D232N or D232C), while a D232A variant is both wholly inactive and forms no adduct. The x‐ray structure of D232N CMP‐®‐Kdo donor complex shows that the anomeric carbon of the donor is inaccessible to any potential acceptor but is instead positioned immediately in contact with, and in line with, the carboxylate of Asp232. Structures of D232N‐ and D232C‐Kdo adducts show that the Kdo adduct is rearranged into a second half‐site, interacting with a distinct set of catalytic residues. A ternary complex of D232C‐Kdo plus a synthetic acceptor shows that Glu158 forms direct hydrogen bonds with O3 of rhamnose, positioned adjacent to and in‐line with the anomeric carbon of Kdo, activating it for attack. Glu158 is also essential, with variants being wholly inactive. Together, this shows that WbbB uses a glycosyl hydrolase‐like double‐displacement mechanism. We propose that crowding around the anomeric carbon by the carboxylate group likely precludes retaining ulosonic acid transferases using the more straightforward SNi mechanism, while adduct rearrangement avoids having the acceptor compete with the leaving nucleotide for access to the anomeric carbon of the donor.

Support or Funding Information

This work was funded by an NSERC Discovery grant (04045‐2015) to MSK and support from the Canadian Glycomics Network (SD‐1) to TLL; TJBF is the recipient of a University of Guelph Graduate Excellence Entrance Scholarship and an Ontario Graduate Scholarship.
The Herpes Simplex Viruses Utilize a Recombination‐Dependent Replication Mechanism to Replicate Viral Genomes
Authors: Weerasooriya Savithri , DiScipio Katherine A. , Peterson Jaliyah , Weller Sandra K.
Published: April 2020

Although it has been known for decades that HSV replication results in the formation head‐to‐tail concatermers, the mechanism by which concatemers form is not understood. One long‐standing model involves circularization of incoming viral genomes followed by rolling circle replication. However, several lines of evidence suggest that HSV DNA replication may be more complex, relying instead on a recombination‐dependent replication mechanism. The HSV single‐strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a single strand annealing protein (SSAP). HSV encodes a conserved alkaline nuclease (UL12) that interacts with ICP8 to form a complex that is reminiscent of evolutionarily conserved phage recombinases, such as lambda red a/ß. We hypothesize that ICP8 and UL12 interact to promote recombination‐dependent DNA replication to generate head‐to‐tail concatemers necessary for the production of infectious virus. Due to the multifunctional nature of ICP8, it has been difficult to tea1se out the role of ICP8 as a SSAP from the various roles it plays during viral replication as a classical SSB. We have recently identified a novel ICP8 mutant, Q706A/F707A, that lacks annealing activity but retains many of the other functions characteristic of classical SSBs. Interestingly, this mutant is unable to synthesize viral DNA, indicating that the annealing function of ICP8 is essential for viral DNA synthesis (Weerasooriya, DiScipio et al. PNAS 2018)

Annealing is a complex process requiring many types of molecular interactions. We hypothesize that it involves both ICP8‐DNA and ICP8‐ICP8 interactions as well as conformational changes induced by the recognition of complementary DNA. Based on their proximity to Q706A/F707A, we suggest that annealing may be mediated by a cluster of conserved residues (residues 701 to 719) in the shoulder domain of ICP8. We have identified five conserved arginine residues (R701, R704, R712, R713 and R714) in this region. Alanine mutagenesis of these residues showed that all mutants, except R704A are unable to complement the growth of a null mutant suggesting that these residues are important for viral growth. Biochemical characterization of purified mutant and WT proteins will be presented along with models for how ICP8 promotes efficient annealing during DNA synthesis.

Support or Funding Information

NIH funding AI069136 and AI021747 to S.K.W and F30AI143125 to K.A.D
PGRMC1 Stabilizes Cytochromes P450 And Is Required for Activity In Vivo
Authors: McGuire Meredith R. , Mosher Eric , Myers Stephanie , Bumpus Namandje , Espenshade Peter
Published: April 2020

Progesterone receptor membrane component 1 (PGRMC1) is a membrane‐bound hemoprotein with many ascribed functions that binds cytochromes P450 in yeast and human cells. In this study, we generated a whole‐body Pgrmc1 KO mouse to examine the function of PGRMC1 in cytochrome P450 biology. Here, we report that Pgrmc1 is a novel post‐translational stabilizer of cytochromes P450 required for cytochrome P450 activity in vivo. In mouse liver, Pgrmc1 bound 13 cytochromes P450 of diverse families (Cyp1, 2, 3, 8, and 51) and functioned to maintain liver cytochrome P450 levels post‐transcriptionally. In the absence of Pgrmc1, cytochrome P450 protein levels were 22–70% of wild‐type. These reductions in P450 enzymes were shown to be physiologically important. Pgrmc1 promoted Cyp1a2 activity in vitro as the Vmax for the Cyp1a2‐dependent O‐deethylation of 7‐ethoxycoumarin was 40% lower in the absence of Pgrmc1 in liver membranes. Mechanistically, PGRMC1 stabilized CYP1A2 post‐translationally, doubling the half‐life of the cytochrome P450 in transfected human cells. The stabilization of cytochromes P450 and promotion of cytochrome P450 activity by Pgrmc1 were the result of a protein‐protein interaction between Pgrmc1 and the cytochrome P450 that did not require Pgrmc1 to bind heme. Reductions in Cyp2e1 protein in Pgrmc1 KO mice protected animals against hepatotoxicity during Cyp2e1‐dependent acetaminophen overdose, the leading cause of acute liver failure in the U.S. This study establishes PGRMC1 as a central component controlling cytochrome P450 activity thus making PGRMC1 the first new player in the liver cytochrome P450 system in 50 years as it joins cytochrome P450 oxidoreductase and cytochrome b5. This study also establishes PGRMC1 as a clinically relevant effector of cytochrome P450 activity, which has direct clinical implications related to the efficacy of pharmacotherapies and will be of interest to pharmacologists, clinicians, and the pharmaceutical industry.

Support or Funding Information

M.M. was supported by NIH 5T32GM007445, NIH F31HL131185, and American Heart Association 15PRE25250008. P.E. was supported by NIH R01HL077588 and NIH R21HL094774.
Novel Control Mechanism of the Neurovascular Coupling in an Alzheimer’s Disease Model
Authors: Puebla Mariela , Fritz Elsa , Álvarez Alejandra , Figueroa Xavier
Published: April 2020

Alzheimer’s disease (AD) is a type of dementia related to age characterized by a progressive and irreversible deterioration of memory and general cognitive abilities. There is a consensus that the production of β‐amyloid protein (Aβ) aggregates correlates with neuronal alterations, triggering the characteristic neurodegeneration process of this disease. Furthermore, disease progression has been closely associated with cerebral vascular alterations, such as reduction in cerebral blood flow observed in patients and animal models of AD.

It has been documented that Aβ not only has harmful effects at neuronal level but has also been associated with the alteration of astrocytes normal activity. One of the events modulated by astrocytes at cerebral level is its key role in the neurovascular coupling (NVC), mechanism that ensures nutrients and oxygen supply in proportion to the increase in neuronal activity. The NVC functions controlled by the astrocytes is mediated by vasodilator signals, a process that is dependent of intracellular calcium increase in astrocytes, which is propagated and coordinated by the release of paracrine and autocrine adenosine triphosphate (ATP).

There is no consensus about the way of ATP release from astrocytes during NVC, but the description of a new channel, Calcium Homeostasis Modulator 1 (CALHM1) which permit the ATP release in some type of neurons, could be an important element in astrocyte signaling during this mechanism. In previous results we observed that ATP is released through CALHM1 in an astrocyte primary culture model stimulated with glutamate (a stimulus validated in NVC studies) and also was dependent of the CALHM1 activation by S‐nitrosylation through nitric oxide (NO) produced by the astrocytes.

Considering that it has been reported that NVC is altered in AD, which contribute to the pathophysiological mechanisms of this disease, our main aim was to evaluate the ATP release in astrocytes primary cultures from a validated murine model of AD: B6C3‐Tg(APPswe,PSEN1dE9)85Dbo/Mmjax.

Our results show that ATP release, and CALHM1 relative levels and expression are significatively reduced in astrocytes primary cultures from AD animals in comparing with cultures from wild type animals. These results suggest that calcium signaling could be diminished in the astrocytes in AD and could explicate the relation between the alteration of NVC in AD.

Support or Funding Information

Universidad del Desarrollo Intern Proyect 23.400.163
Therapeutic Vulnerabilities in Acute Myeloid Leukemia (AML) with TP53 Alterations
Authors: Ogunsan Oluwateniayo , Antony Marie Lue , Noble-Orcutt Klara E. , He Fiona , Sachs Zohar
Published: April 2020

Acute myeloid leukemia (AML) is a cancer of the blood and bone marrow with a low two‐year survival rate. Standard chemotherapy achieves complete remission in 60–80% of patients, but 20–30% patients relapse due to leukemia stem cells (LSCs) that can self‐renew and recapitulate disease. AML with TP53 alterations has a lower prognosis with survival rates of 0–10% at one year. In vitro drug screens have identified drugs to target AML, but none have studied the effect of these therapies on LSCs with TP53 alterations. These in vitro drug screens include Crizotinib, Elesclomol, AZD1480, GW2580, Venetoclax, and Entrectinib. Crizotinib, AZD1480, GW2580, Venetoclax, and Entrectinib are signaling pathway inhibitors. Elesclomol induces oxidative stress and apoptosis in cancer cells. The goal of this research is to understand the molecular mechanisms of self‐renewal and therapeutic vulnerabilities in LSCs of AML with TP53 alterations. Specifically, we investigate whether these agents target LSCs from AML with TP53 alterations. We performed in vitro viability and in vitro colony forming assays (CFAs) on primary human AML samples with TP53 alterations, plating them with drugs or vehicle control. Furthermore, we used CYTOF to assess the effects of each drug on signaling within leukemia subpopulations. Crizotinib proved to be effective in reducing in vitro viability, in vitro CFAs, and affecting the expression of immunophenotypic markers, such as CD34 and CD123, which are markers for leukemia stem cell populations. Our data reveals cell‐type specific effects of Crizotinib in human AML with TP53 alterations. Our findings suggest that Crizotinib may be an effective therapy for patients with AML with TP53 alterations.

Support or Funding Information

This research was supported and funded by: NIH/NIGMS MARC U* STAR T34 HHS 00026 National Research Service Award to UMBC, Summer Research at the University of Minnesota Medical School NIH HLBI 2R25HL088728‐11A1, Dr. Colin Campbell, Dr. Craig Henke, American Cancer Society Mentored Research Scholar Grant (MSRG‐16‐195‐01‐DDC), Frederick A. Deluca Foundation, CTSI K to R01 Award, NIH/NCATS, Lois and Richard King Assistant Professorship in Medicine, Division of Hematology, Oncology, Transplantation, Dept. of Medicine, University of Minnesota University of Minnesota Foundation Donors
Identification and characterization of a novel Anoctamin 1 inhibitor and its anticancer effects
Authors: Jo Sungwoo , Seo Yohan , Namkung Wan
Published: April 2020

Anoctamin 1 (ANO1 also known as TMEM16A) is a calcium‐activated chloride channel that is amplified in various carcinomas including prostate cancer, esophageal cancer, breast cancer, and pancreatic cancer, and its overexpression promotes cell proliferation and cell migration in various cancer cells. A cell base‐screening revealed that (E)‐1‐(7,7‐dimethyl‐7Hfuro[2,3‐f]chromen‐2‐yl)‐3‐(1H‐pyrrol‐2‐yl)prop‐2‐en‐1‐one (Ani‐E1) is a novel, potent and selective ANO1 inhibitor with an IC50 value of 1.23 μM. Ani‐E1 showed 144 times stronger activity on ANO1 inhibition than ANO2 inhibition and did not alter the chloride channel activity of CFTR and the intracellular calcium signaling. Notably, Ani‐E1 strongly decreased cell viability of PC‐3 and FaDu cells expressing high levels of ANO1 with a decrease in ANO1 protein levels. In addition, Ani‐E1 significantly enhanced apoptosis via activation of caspase 3 and cleavage of PARP in PC‐3 and FaDu cells. This study shows that a novel ANO1 inhibitor, Ani‐E1, can be a potential candidate for the treatment of cancers overexpressing ANO1, such as prostate cancer and esophageal cancer.

Support or Funding Information

We thank the National Research Foundation of Korea (NRF‐2017R1A2A2A05069364 and 53 NRF‐2018R1A6A1A03023718, NRF‐2019R1I1A1A01061117) for generous financial support. This research was supported (in part) by Yonsei University Research Fund (Post Doc. Researcher Supporting Program) of 2019 (project no.: 2019‐12‐0013).
Proximal tubule albumin uptake – potential role for endothelin system in sickle cell disease mice
Authors: Kasztan Malgorzata , Alrefai Hasan , Taylor Crystal , Pollock David M.
Published: April 2020

Elevated plasma endothelin‐1 (ET‐1) reported in sickle cell disease (SCD) patients correlate with microalbuminuria, a major mortality risk factor in SCD. ET‐1 contributes to glomerular and tubular injury in SCD, as evidenced by increased glomerular permeability to albumin and urinary excretion of tubular injury markers. Although selective ETA receptor antagonism reduces albuminuria in humanized sickle cell (HbSS) mice, the mechanism of this action remains unclear. The aim of the study was to determine whether selective ETA receptor antagonism prevents albuminuria by preserving the expression of tubular albumin‐associated transporters in proximal tubule (PT) cells. Male C57BL/6 or HbSS and genetic control (HbAA) mice were used to determine the effect of ET‐1 on the expression of proximal tubule albumin trafficking mediators, such as megalin and NHE‐3. Exposure of primary mouse PT cells to ET‐1 (50nM) for 8h decreased megalin (53% reduction) and doubled NHE‐3 expression. Pre‐treatment with the ETA antagonist, BQ123 (1 μM), preserved megalin expression and had no effect on NHE‐3. Moreover, selective ETB receptor blockade (BQ788, 1 μM) prevented ET‐1‐mediated increase in NHE‐3 expression. Primary PT cells isolated from HbSS mice showed decreased megalin mRNA expression as well as protein abundance relative to HbAA PT controls. Ten‐week treatment with selective ETA receptor antagonist (10mg/kg/day) preserved expression of megalin at control levels. There were no differences in NHE‐3 mRNA expression in HbSS PT cells regardless the treatment. Interestingly, PT cells from HbSS cultured with HbSS plasma and ET‐1 (10nM) had decreased NHE‐3 protein abundance compared to non‐treated cells. These results potentially uncover a novel role for ET‐1 in PT albumin handling and suggest that PT ET‐1 receptor signaling contributes to albuminuria in SCD.

Support or Funding Information

Support provided by an NIH U01 HL117684 to D.M.P, AHA 19CDA34660073 and NIH‐NHLBI K99HL144817 to M.K.
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 Information

Research supported by NIH R37 GM 076343 and T32 GM007767.
The Interactive Influence of Climate and Energetic Factors on Human Nasal Morphology
Authors: Kelly Alexa P. , Maddux Scott D. , Ocobock Cara
Published: April 2020

Cold‐dry environments require considerable transfers of heat and moisture from the respiratory tract to inspired air in order to prevent thermal damage and desiccation of the lungs. Accordingly, humans indigenous to cold‐dry environments typically exhibit narrow nasal passages, as these enhance intranasal air‐conditioning by increasing contact between respired air and the heat/moisture‐rich nasal mucosa. Yet, cold‐dry environments are also metabolically expensive due to thermoregulatory demands, requiring greater oxygen intake than in tropical environments. Thus, humans from colder environments are also predicted to possess larger nasal passages (i.e., cross‐sectional areas) in order to transmit larger volumes of air during each breath. Still, despite substantial evidence that both climate and metabolism likely influence nasal size and shape, virtually no research has investigated the interplay of these two factors on nasal anatomy. Here it is hypothesized that climate‐mediated nasal narrowing may necessitate a compensatory increase in nasal height to ensure the airways remain large enough to transmit a metabolically adequate volume of oxygen. To test this, we collected 17 linear measurements from the nasal skeleton of 119 modern humans from 10 climatically diverse geographic areas (Arctic Circle, Europe, Iran, Australia, North Africa, Khoisan, South African Bantu, East Africa, West Africa, Papua New Guinea). Measurements of associated postcranial elements were then used to estimate body mass and basal metabolic rate (BMR) for each individual. Climatic data (i.e., monthly air temperature and absolute humidity) were similarly collected for the geographic provenance of each individual. These morphological, energetic, and climatic data were subsequently employed in multivariate analyses to assess potential interactive influences of climate and metabolism on nasal morphology. Our results indicate that most measurements of nasal complex breadth are significantly correlated with climate (all r‐values >0.45, all p‐values <0.009), but not BMR. Conversely, nasal height is more strongly correlated with BMR (r=0.47, p=0.02) than climate. Additionally, nasal passage cross‐sectional area demonstrates a positive association with BMR (r=0.74, p=0.0007), while passage cross‐sectional shape exhibits a significant relationship with climate (r=0.52, p=0.0017) with taller/narrower airways found in colder‐drier environments. Our results support assertions that nasal narrowing in colder climates necessitates a concomitant increase in nasal height to maintain an overall airway size capable of meeting energetic demands for oxygen intake. Moreover, these patterns of nasal morphology appear consistent with well‐established ecogeographic distributions in human body size/shape attributed to Bergmann’s and Allen’s Rules. Thus, future research employing larger and more diverse samples appears poised to provide far‐reaching insights regarding climatic adaptation during human evolution.
Engineering protein therapeutics for cancer based on the natural matrix metalloproteinase inhibitor TIMP‐1
Authors: Raeeszadeh-Sarmazdeh Maryam , Coban Matt , Sankaran Banumathi , Radisky Evette
Published: April 2020

Enzymes of the matrix metalloproteinase (MMP) family have been shown to play critical roles in cancer progression and metastasis, making them ideal targets for developing cancer therapeutics. MMP‐9 plays a significant role in tumor progression and metastasis of triple negative breast cancer (TNBC), while MMP‐3, the biological activator of MMP‐9, can also promote mammary carcinoma through inducing epithelial‐mesenchymal‐transition (EMT) in tumor cells. These enzymes are attractive as therapeutic targets; however, broad spectrum MMP inhibitors have proven ineffective, so inhibitors with greater selectivity are desired. Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) is a natural inhibitor of MMP‐3 and MMP‐9 with picomolar affinity, and offers a scaffold for engineering of selective MMP‐targeted therapeutics. To overcome the challenges of wide multispecificity of TIMP‐1 for different classes of MMPs, we used state‐of‐the‐art directed evolution and yeast surface display techniques recruiting high‐throughput library screening technology to fully evolve novel protein‐based drugs, based on the TIMP‐1 scaffold, with high selectivity for MMP‐3 or MMP‐9. We have developed a counter‐selection strategy to screen TIMP‐1 mutants that bind selectively to MMP‐3 in the presence of MMP‐10, an MMP with the greatest sequence and structural similarity to MMP‐3. The isolated TIMP‐1 mutants after five rounds of competitive screening showed up to 20‐fold improvement in binding selectivity, highlighting the significant potential of this approach for development of inhibitors with single‐MMP selectivity. In ongoing studies, I have identified some of the key TIMP‐1 mutations responsible for improvements in MMP‐3 affinity and selectivity. I have co‐crystallized the top affinity and selectivity mutants with the catalytic domain of MMP‐3, and have solved the structure. These structures provide insights into the structural basis for improvements in binding affinity and selectivity. These studies and methodology developed in this project, will lay the foundation for developing novel therapeutic strategies for MMP‐related diseases and understanding protein‐protein interaction between MMPs and their inhibitors.
Behavioral Effects of Nitrite on Goldfish (Carassius auratus)
Authors: Trujillo Isaac Atticus , Martinez Melody , Huertas Mar
Published: April 2020

As the human population grows the demand for food products grows, increasing the need for high agricultural output. This increase in agriculture leads to the frequent use of inorganic fertilizers which can pollute freshwater systems with nitrogenous compounds. Nitrite, a byproduct of fertilizer pollution, can accumulate and reach concentrations between 0.5 μM and 1.0 mM in aquatic ecosystems. Nitrite is known to disrupt physiological processes such as ion homeostasis, respiration, and neural and cardiovascular function. Nitrite is also known to cause methemoglobinemia, the irreversible oxidation of red blood cells. The effect of nitrite on internal organs and functions is extensively studied. However, little is known about the effects of nitrites on external sensitive organs in aquatic animals, where the pollutant is in continuous contact with surface epithelia. The olfactory system is critical for coordination reproductive events, predator avoidance, and food detection in fish. To detect odors, the olfactory system must be continuously exposed to the environment, therefore it is sensitive to pollutants. There is a need to determine the effects of nitrite on the olfactory system in fish. Behavioral trials, using Goldfish (Carassius auratus), were conducted to establish the effects of nitrite on food detection via olfactory system. In order to conduct the behavior trials a two‐choice maze was built. The two‐choice maze had a food odor released in one channel while a control odor was released in the second channel. Each behavior trial lasted twenty minutes and consisted of an acclimation period, pre‐cue exploratory period, re‐acclimation, and a cue exposure period. Time spent in each channel during the pre‐cue exploratory period was recorded to establish preference bias. Time spent in each channel during the cue exposure period was also recorded. To study the effects of nitrite on goldfish feeding behavior, fish were exposed to four different sublethal nitrite concentrations (0 mM, 0.01mM, 0.3 mM, and 1.0 mM), for a week before the behavior trials began. Weekly behavior trials were then conducted for four weeks. A Wilcox rank test was used to determine the preference for food odor, after four weeks of nitrite exposure. The goldfish exposed to nitrite were significantly less attracted to food odor than the control group (p < .05). Food preference decreased as nitrite concentration increased. The results indicate that nitrite negatively affects the olfactory system and it’s ability to detect food odor, therefore showing that sublethal concentrations of nitrite negatively affects food detection before any other physiological function is affected.
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 Information

National 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 Information

This 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 Information

This 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 Information

This 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 Information

NSF 1833181
Student Perceptions and Successes in Various Levels of Undergraduate Chemistry Education
Authors: Parsons Julie , Schlosser Sarah
Published: April 2020

Perceptions affect the way in which people engage in the world and with one another. By evaluating student perception in relation to Chemistry, valuable insight can be gained as to the manner in which students engage with and understand the nature of Chemistry. Student’s perceptions and success within Chemistry courses affords insight into student’s current motivation and understanding. Utilization of the Colorado Learning Attitudes about Science Survey‐Chemistry (CLASS‐Chem) administered at the beginning and middle of the semester provides insight into changes in student’s motivations and perceptions of self‐efficacy throughout their involvement in various chemistry courses. Students ranging from introductory nursing chemistry courses to biochemistry were surveyed. Comparison with student data including current class grade, male/female, GPA, and college year (freshman, sophomore, junior, or senior) allows for a broader understanding of student engagement with Chemistry.
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

Objective

Tumor 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.

Methods

We 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.

Results

bFGF 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.

Conclusion

Collectively, 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 Information

NSF

CHE‐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 Information

National 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 Information

This work was financially supported by Korean Research Foundation Program (2018R1A2B6007794), Republic of Korea
Biophysical characterization of a series of novel non‐RAFT copolymers with improved buffer behavior
Authors: Esmaili Mansoore , Overduin Michael
Published: April 2020

Synthetic amphipathic polymers present an invaluable tool for excision and liberation of superstructures of membrane proteins (MPs) and their surrounding annular membrane bilayer in donut‐shaped nanoparticles or discs. Styrene‐co‐maleic acid polymers (SMAs) have demonstrated most efficiency for the structural characterization of membrane proteins. However, some drawbacks such as sensitivity to acidic pH and high concentration of divalent cations, interference with the spectroscopic profiles of proteins, high polydispersity index (PDI) and nonspecific interaction with protein surface have hindered the ultimate optimal utilization of SMA polymers for functional characterization of MPs in lipid bilayer and hence for drug discovery purposes. We have developed multiple series of novel non‐RAFT amphipathic polymers that demonstrate improved behaviors in acidic pH and high concentration of calcium. Some of these unique amphipathic polymers display exemplary polydispersity (PDI ~1), strict alternation (a well‐defined sequence of comonomers) and distinct spectrophotometric profiles. We further characterized the application of these novel polymeric detergents for purification of a model integral beta‐barrel MP, PagP, from the outer membrane of E. coli.