bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of the Regulation of JNK2 through Oligomerization
Authors: Kaoud Tamer S , Riggs Austin F. , Dalby Kevin N.
Published: June 2021

c‐Jun N‐terminal kinases (JNKs) are mediators of cellular signals associated with stress and inflammation. Activation of the JNKs is mediated cooperatively by MKK4 & MKK7. Uniquely, JNK2 isoforms can self‐activate, which underlies the constitutive activation of JNK2 in primary glial tumors. Herein, we investigate the regulation of JNK2 self‐activation in both cell‐free and cell‐based systems. Size exclusion chromatography & light scattering analysis (SEC‐LS) suggests that unphosphorylated, recombinant JNK2α2 exists in solution as a mixture of monomers, dimers and tetramers. JNK2α2 self‐phosphorylation occurs rapidly at low concentration leading to its activation. At higher concentrations of JNK2, where the formation of a JNK2 tetramer is favored, self‐phosphorylation is suppressed in vitro. Transfection of pcDNA‐JNK2α2 into HEK293 cells suppresses constitutive activation of JNK2 at higher levels of expression in a dose‐dependent manner. The F170R mutant of JNK2, which is predicted to favor an inactive conformation, stabilizes the tetramer as determined by SEC‐LS. When HEK293 cells are incubated with BIRB796 (which binds an inactive conformation of JNK2) and the cell‐permeable crosslinker disuccinimidyl suberate, a crosslinked JNK2 tetramer is detectable by SDS PAGE. We conclude that oligomerization of JNK2 has potential to contribute to the regulation of its self‐activation in mammalian cells.
3D geometric morphometric analysis of phenotypic plasticity in the pectoral girdle of a basal actinopterygiian fish
Authors: Du Trina Y , Standen Emily M , Larsson Hans CE
Published: June 2021

Polypterids are a basal group of actinopterygian fishes that represent a possible living analogue for the evolutionary transition from fish to tetrapods for number of reasons, including their ability to walk on land using robustly, muscled pectoral fins. We examined the phenotypic plasticity in the pectoral anatomy of Polypterus senegalus induced by habitual terrestrial locomotion. Fish were reared in a fully terrestrial environment where they were forced to live and locomote over land for up to two years. MicroCT scans were taken of the experimental walking group and a swimming control group. 3D reconstructions of the pectoral anatomy were described using spherical harmonics, a 3D extension of elliptic Fourier analysis. Here we present a geometric morphometric analysis of morphological differences in the pectoral anatomy between swimming and walking fish, and discuss potential small‐scale morphological changes in response to the functional demands of terrestrial locomotion during the fin‐to‐limb transition.

Grant Funding Source: NSERC, Canada Research Chair, Tomlinson Postdoctoral Fellowship
Investigations of the determinants of 5‐formyltetrahydrofolate binding in the active site of 5,10‐methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae
Authors: Johann Timothy , Wojtera Casey , Bryant Matthew
Published: June 2021

The pathways for the synthesis of tetrahydrofolates form the core of one carbon metabolism and are an excellent source of targets in the treatment of cancer. 5‐formyltetrahydrofolate (5‐formylTHF) is administered in these treatments, either as a rescue agent or as an enhancer. The only enzyme known to use 5‐formylTHF as a substrate is 5,10‐methenyltetrahydrofolate synthetase (MTHFS). This enzyme catalyzes the conversion of 5‐formylTHF to 5,10‐ methenyltetrahydrofolate in conjunction with the hydrolysis of ATP to ADP. We have investigated the determinants of 5‐ formylTHF binding in the active site of the enzyme through site‐directed mutagenesis of wild type residues to alanine followed by a circular dichroism spectroscopy and kinetics. Replacement of glutamate at position 55 (E55A) or tyrosine at position 123 (Y123A) produced mutants with no activity. Mutant proteins F118A and Y122A demonstrated higher Michaelis constants for 5‐ formylTHF and lower turnover numbers compared to wild type. Curiously, mutant Q144A was found to have a higher Michaelis constant for 5‐formylTHF and a higher turnover number. Circular dichroism data were consistent with all mutants being folded.
The role of ketone body‐utilizing enzyme, acetoacetyl‐CoA synthetase, in lipogenic tissue
Authors: Hasegawa Shinya , Yamasaki Masahiro , Fukui Tetsuya
Published: June 2021

Acetoacetyl‐CoA synthetase (AACS) is a ketone body‐utilizing enzyme for the synthesis of cholesterol and fatty acids and is highly expressed in the lipogenic tissues. In this study, we investigated the regulation of AACS in liver, adipocyte and neuron to clarify the physiological role of AACS. ChIP assay and knockdown experiments showed that AACS is mainly regulated by SREBP‐2 in the neuron and liver, and by C/EBP alpha in adipocytes. In 3T3‐L1 adipocytes, treatment with shRNA against AACS resulted in inhibition of lipid storage and reduction of adipocyte markers, such as C/EBP alpha and PPAR gamma. In the liver, knockdown of AACS using hydrodynamics method decreased total serum cholesterol. Moreover, suppression of AACS by shRNA in primary neurons caused decreases in the expression of MAP‐2 and NeuN, which are markers of neuronal differentiation, as well as synaptopodin, a marker of spine apparatus. These results suggest that ketone body utilization via AACS is an important reaction for lipogenic cellular function.
Circulating Lymphocyte T cell levels following radical nonsmall cell tumor surgery
Authors: TULP ORIEN LEE , Einstein George P , Kowalski Janusz , Dancewicz Maciej
Published: June 2021

T lymphocytes play an important role in mechanisms of immunological tolerance, where they contribute to tumor suppression activities. To determine the effects of surgical intervention on T cell status, a group of 21 patients with small cell lung cancer [NDRP], 13 of whom had confirmed lesions and 8 without who served as controls. The size and mass of all tumors were confirmed by MRI and PET scan imaging with Glu18 isotope enrichment. Measures of CD4/CD25 T cells before and after therapy resulted in a return toward normal of circulating T cell concentrations, consistent with a normalization of the expression and function of T lymphocytes after tumor reduction. These results suggest that the monitoring of circulating T cell populations may serve as viable indicators of treatment effectiveness for NDRP treatment. Institutional Support.
A new therapeuthical approach to block cancer cachexia: focusing inhibition of STAT3
Authors: Silva Kleiton Augusto Santos , Dong Yanlan , Dong Yanjun , Tweardy David J. , Mitch William E. , Zhang Liping
Published: June 2021

Cachexia is a devastating complication of cancer. The mechanisms by which cancers cause cachexia are unclear and there are few successful therapeutic options. In muscles from mouse models of cancer cachexia, we found increased STAT3 phosphorylation associated with muscle atrophy. In muscle cells (C2C12 myotubes) incubated with media from cultured Lewis lung carcinoma (LLC) or Colon 26 (C26) tumor cells, we found rapid phosphorylation of STAT3 and progressive myotube atrophy. We created mice with muscle‐specific STAT3 knockout (KO) with implanted tumor cells. We found a prevention of body and muscle weight loss in KO mice compared to control mice injected with LLC tumors. We treated C2C12 cells and mice with a small molecule inhibitor of STAT3 (C188–9). C2C12 myotubes were incubated with C188–9 and conditioned media from C26 colon tumors and after 24 hours, the STAT3 phosphorylation was suppressed and the wasting of myotubes was blocked. In CD2F1 mice bearing C26 xenografts, daily i.p. administration of C188–9 (6.25mg/kg) over 14 days significantly reduced loss of body weight and muscle mass compared to tumor‐bearing mice injected with the diluent. The increase in muscle mass of C188–9‐treated mice was due to increased protein synthesis and decreased protein degradation. Thus, small‐molecule inhibition of STAT3 represents a novel therapeutic strategy for preventing cachexia‐induced muscle atrophy.
Generation and characterization of a tamoxifen‐inducible MGAT2 knockout mouse
Authors: Banh Taylor , Gao Yu , Nelson David W , Yen Mei‐I , Yen Chi‐Liang Eric
Published: June 2021

Acyl CoA: monoacylglycerol acyltransferase (MGAT) 2 catalyzes the resynthesis of triacylglycerol during intestinal fat absorption. Mice lacking the encoding gene (Mogat2−/−) are protected from obesity and associated metabolic disorders induced by high‐fat feeding, despite absorbing normal quantities of fat. To determine if MGAT2 inactivation in adulthood is a feasible strategy for managing metabolic disorders, we generated and characterized a tamoxifen‐inducible knockout model using the Cre/loxP system, which enables inactivation of MGAT2 during different life stages. Mice with loxP sites flanking exon 2 of the Mogat2 gene (floxed mice) exhibited expression and activity similar to wildtypes. Floxed mice expressing the inducible‐Cre (UBC‐Cre mice) also had similar MGAT2 expression and showed no differences in growth. After tamoxifen treatment at 2–3 months of age, these UBC‐Cre mice had no MGAT2 expression like Mogat2−/− mice (which do not have a functional Mogat2 gene since fertilization), confirming the efficiency of MGAT2 inactivation. In preliminary short‐term feeding studies, UBC‐Cre mice grew similarly to wildtype mice on low‐fat diets but are protected from weight‐gain induced by high‐fat feeding. These data suggest that inhibiting MGAT2 in adulthood may prove useful in treating obesity. Funding: NIH 5T32DK007665.
Soluble oligomers of amyloid β cause oxidative stress and alter redox/methylation‐linked gene transcription by disrupting EAAT3‐mediated cysteine uptake
Authors: Hodgson Nathaniel , Trivedi Malav , Deth Richard
Published: June 2021

Depletion of GSH, oxidative stress and hyperhomocysteinemia have been linked to Alzheimer's disease. Recent work has shown that soluble oligomers of amyloid β, the pathological protein of Alzheimer's disease, disrupts glutamate uptake via EAAT3. EAAT3 is the transporter responsible for neuronal cysteine uptake. Cysteine is the rate‐limiting precursor of the antioxidant glutathione (GSH) which establishes the redox potential of a cell. Changes in redox state modify the activity of redox‐sensitive enzymes such as methionine synthase, the enzyme that methylates homocysteine to methionine. Since methionine is subsequently adenosylated to S‐adenosylmethionine (SAM), the major methyl donor of biological reactions including DNA methylation, methionine synthase links redox status to epigenetic regulation of gene expression. We examined the influence of soluble Aβ oligomers on [35S]‐labeled cysteine uptake, intracellular thiol and thioester metabolism, global DNA methylation and transcription of a panel of select redox and methylation‐linked genes in SH‐SY5Y cells, a human neuroblastoma cell line. Soluble Aβ oligomers significantly decreased cysteine uptake, caused oxidative stress, and decreased the ratio of SAM to SAH which was accompanied by a decrease in global DNA methylation. Finally, soluble Aβ oligomers were shown to change the transcription of a number of redox and methylation‐linked genes.
Inhibition of Isw2‐mediated chromatin remodeling by calorie restriction extends lifespan by potentiating stress response
Authors: Dang Weiwei , Berger Shelley
Published: June 2021

ATP‐dependent chromatin remodeling is a highly conserved process involved in all aspects of DNA transactions and linked to a range of human diseases and developmental disorders. Here we explored possible functions of chromatin remodelers during cellular aging, finding that deletion of ISW2 or mutations inactivating the Isw2 enzyme complex extend yeast replicative lifespan. This extension by ISW2 deletion is epistatic to the longevity effect of calorie restriction (CR) and this mechanism is parallel to the suppression of TOR signaling by CR. We find that CR both lowers the intracellular ATP concentration and inhibits Isw2 enzymatic activity. Transcriptome analysis indicates that isw2Δ; mimics an up‐regulated stress response in CR cells. This study represents the first evidence that ATP‐dependent chromatin remodeling plays an important role during aging and in calorie restriction.
The nature of sequence and structure divergence in the Haloalkanoate Dehalogenase SuperFamily (HADSF)
Authors: Pandya Chetanya , Brown Shoshana D , Dunaway‐Mariano Debra , Babbitt Patricia C , Xia Yu , Allen Karen N
Published: June 2021

Though the number of protein folds is predicted to be finite, the universe of protein structures is vast and diverse. Thus understanding evolution of protein folds is a fundamental research challenge. The HADSF serves as an ideal model as it has a modular architecture where the conserved, Rossmann fold core domain is accessorized by a variable cap domain. This work aims to determine the impact of the cap domain on the sequence and structure divergence of the core domain. By performing quantitative analysis on a dataset of core‐domain‐only and cap‐domain‐ only structures, we have uncovered some basic protein design principles. We find that the relationship between sequence and structure divergence is robust, non‐linear (ρ=0.69) and, independent of the corresponding cap type. Core domains with the same cap type share greater similarity (; = 0.76) at the sequence and structure level than core domains with different cap types (; = 0.64), consistent with coevolution of the cap and core domains. Moreover, this similarity between cores is a global phenomenon with contributions from all residues of the Rossmann fold. Together, these results are consistent with coevolution of the cap domain in the context of a thermodynamically stable core domain, a strategy that may be active in other multi‐domain families. This project was supported by National Institute of General Medical Sciences (U54GM093342).
Unraveling the complexity of the Skull: An evo‐devo approach
Authors: Franz‐Odendaal Tamara
Published: June 2021

The complexity of the vertebrate skull has intrigued scientists for decades. This complexity largely exists because the bones and cartilages that make up the skull have different embryonic origins, develop at different times and ossify via different mechanisms. Understanding both the evolutionary history of skeletal elements and their developmental pathways can provide novel insight into this complexity. I will discuss our work on the skeletal elements associated with the eye, specifically the sclerotic ring. The sclerotic ring is composed of neural crest derived intramembranous bones and is situated in the eyeball (sclera) of many vertebrates, including reptiles and bony fish. Our comparative approach in different organisms and our phylogenetic analyses has enabled us to understand the diversity of this part of the skull. Manipulating the embryo results in multiple effects and provides important clues to underlying the mechanisms governing craniofacial plasticity and constraint at the cellular and tissue level. Since evolutionary and developmental trajectories operate at very different temporal scales, understanding both will provide a more comprehensive view of the vertebrate skull and its complexities.
Synthesis, Characterization and Anticancer Activities of New Cu(II) Complexes
Authors: Lin Han Xing , Foreman David R. , Fox Kristin M. , Tanski Joseph M. , Tyler Laurie A.
Published: June 2021

Popular treatments of cancer such as Cisplatin have many drawbacks, including the development of chemoresistance in cancer cells and serious side effects such as nephrotoxicity and neurotoxicity. Therefore, there is a need for development of more efficient, target‐specific, and less toxic cancer drugs. Copper complexes have shown to be a promising alternative, partly because copper, an essential element for most aerobic organisms, is involved in many biological pathways as structural and catalytic cofactor. Additionally, studies have shown a correlation between serum copper level and different aspects of cancers. We have synthesized and evaluated the biological activities of Cu‐quinoline thiazole (CuQ(oBt)) and Cu‐pyridine thiazole (Cu(Py(oBt))2). The nuclease activities of these complexes were visualized via gel electrophoresis. We found that Cu(Py(oBt))2 and CuQ(oBt), in the presence of a reductant, convert supercoil (SC) DNA to the single‐nicked (SN) and double‐nicked (DN) forms with high but different efficiencies. The binding of complexes to DNA was further investigated using electronic absorption and fluorescence spectroscopy. Fluorescence spectroscopy with bovin serum albumin (BSA) showed that the complexes can bind to serum proteins. The comparison of results provides insights for aspects of the ligand frames that affect the DNA‐binding, protein‐binding, and nuclease activities for copper complexes.
Single clone expression cloning and protein microarray platforms for novel extracellular receptor‐ligand identification
Authors: Tom Irene , Quinones Gabriel , Estevez Alberto , Paluch Maciej , Bowman Krista , Franke Yvonne , Billeci Karen , Hass Phil , Tang Jerry , Lewin‐Koh Nicholas , Eaton Dan , Stephan Jean‐Philippe , Gonzalez Lino
Published: June 2021

Here we describe the development of two complementary high‐throughput screening technologies consisting of single clone expression cloning and protein microarray platforms that permit genome‐wide screening of a protein of interest against thousands of interactions either displayed on a cell surface or on a microarray. The single clone expression cloning platform is fully automated and capable of efficient transfection, expression, and binding analysis of a bait protein to cells transfected with individual clones from the clone library. For low affinity interactions, we generated bait protein with an Fc fusion to increase the avidity. Our secreted protein microarray has over 1500 secreted or extracellular domain of single transmembrane human proteins represented to enable simultaneous systematic probing of a bait protein against proteins on the microarray. To enhance detection of low affinity interactions, we assembled bait Fc fusion proteins into multivalent complexes with protein A microbeads. As proof of concept, we evaluated well‐characterized extracellular receptor‐ligand interactions with diverse binding affinities on the single clone expression cloning and protein microarray platforms. Overall, we found that the interactions identified using both of our screening methodologies were highly specific and displayed minimal off‐target binding. Furthermore, we demonstrate the utility of both technologies for novel receptor‐ligand discovery.
Using the Structurally Aligned Local Sites of Activity (SALSAs) computational method to determine biochemical function of structural genomics proteins
Authors: Lee Joslynn Shannon , Ondrechen Mary Jo
Published: June 2021

In the Protein Data Bank, there are over 84,600 deposited structures and 11,800 of those are structural genomics (SG) proteins. The functional annotations of large number of solved SG structures are uncertain or incorrect, due to assignments based on sequence or overall structure comparison. The present method to predict function, Structurally Aligned Local Sites of Activity (SALSAs), uses functional active site predictors, Theoretical Microscopic Anomalous Titration Curve Shapes (THEMATICS) and Partial Order Optimum Likelihood (POOL), to first determine the important active site residues from the 3D structure. Second, these local active site structures are aligned to compare proteins of known function with SG proteins. Third, those matches are assigned scores with a similarity scoring matrix to assign correct annotation. We apply this method to the ribulose‐phosphate binding barrel (RPBB) superfamily and annotate twenty‐eight SG proteins. The superfamily was divided into eight functional subclasses and their active sites were differentiated along the eight beta‐sheets. The location of the active site and residues were correctly predicted for all protein structures.
Label‐free Spectroscopic Imaging of Lipids in Live Cells and Intact Tissues
Authors: Cheng Ji‐Xin
Published: June 2021

Lipids are commonly visualized with the aid of fluorescent lipid analogs. These analogs do not provide compositional information about the organelles. Moreover, the labeling often perturbs the molecular properties due to the bulkiness of the tag. New platforms are being developed to overcome such barriers through using intrinsic molecular signals as contrast. In this presentation, label‐free imaging of lipids using signals arising from chemical bond vibrations will be demonstrated. By making use of the strong signal from C‐H stretch vibration, coherent Raman scattering microscopy allowed fast imaging of lipid droplet dynamics in live cells. Furthermore, by coupling fast coherent Raman imaging with spontaneous Raman spectral analysis of the fingerprint region, the composition of lipid droplets and the phase of lipid membranes are unveiled. These capabilities reveal previously underappreciated roles of lipids in health and diseases such as human cancers.
Structural Basis of FTY720 (Gilenya) Phosphate Selectivity of the Sphingosine 1‐Phosphate Receptor Subtype 1
Authors: Tigyi Gabor , Valentine William , Osborne Daniel , Fujiwara Yuko , Liu Jianxiong , Godwin Virginia , Parrill Abby , Bittman Robert , Van Brocklyn James
Published: June 2021

FTY720 phosphate (FTY720P) is a high potency agonist for all the EDG‐family S1P receptors except S1P2. To map the distinguishing features of S1P2 ligand recognition we applied a computational modeling‐guided mutagenesis strategy that was based on the high degree of sequence homology between S1P1 and S1P2. S1P2 point mutants of the ligand‐binding pocket were characterized. The residues R3.28, E3.29, and K7.34 were essential for activation. Mutation of residues A3.32, L3.36, V5.41, F6.44, W6.48, S7.42, and S7.46, predicted to interact with the S1P hydrophobic tail, impaired activation by S1P. Replacing individual or multiple residues in the ligand‐binding pocket of S1P2 with S1P1 sequence did not impart activation by FTY720P. Chimeric S1P1/S1P2 receptors were generated and characterized. The S1P2 chimera with S1P1 sequence from the N‐terminus to TM2 was activated by FTY720P and the S1P2(IC1‐TM2)S1P1 domain‐insertion chimera showed S1P1‐like activation. Twelve residues in this domain differ between S1P1 and S1P2, distributed in four motifs a–d. Insertion of 78RPMYY in motif b alone or simultaneous swapping of five other residues in motifs c and d from S1P1 into S1P2 introduced FTY720P responsiveness. Molecular dynamics calculations indicate that FTY720P binding selectivity is a function of the entropic contribution to the binding free energy rather than enthalpic contributions and that preferred agonists retain substantial flexibility when bound. Supported by NCI grant CA‐092160 and the Van Vleet Endowment.
Determining the Mechanism of Sphingosine‐1‐Phosphate Transport Between Membranes
Authors: Massa Mitterand Nformi , Wattenberg Binks
Published: June 2021

Sphingosine‐1‐phosphate (S1P) is synthesized from sphingosine by the enzyme sphingosine kinase (SK). The main pathways of S1P degradation occur via the action of two important enzymes; S1P phosphatase (SPP) and S1P lyase (SPL) which are both bound to the endoplasmic reticulum (ER). So the ER plays a vital role in the metabolism of S1P.

The goal of these studies is to determine the mechanism of S1P transport between membranes. We are hypothesizing that this is a protein mediated transfer. Initially we have defined the parameters of S1P extraction from membranes, which is the first step in intermembrane transfer. We have used a model lipid‐binding protein, bovine serum albumin (BSA). To measure S1P extraction from membranes, membranes were first loaded with 33P‐S1P by using membranes ectopically expressing a membrane‐anchored SK construct (Lck‐SK). Extraction of S1P was measured by incubating pre‐labeled membranes in the presence or absence of fatty acid‐free BSA for various lengths of time. With this system we find that extraction of S1P is protein‐dependent, but very rapid. We then tested the ability of cytosol harvested from Hela cells to replace BSA in extracting S1P from membranes. Interestingly, results indicated the presence of an S1P transport factor(s) in the cytosol. Size‐exclusion chromatography was then used to further fractionate the cytosol and various fractions were assayed for activity. S1P extraction activity was detected to be higher in higher molecular weight fractions away from bulk protein. Concentrates of these fractions demonstrated enhanced S1P extraction activity from membranes over whole cytosol.
Mechanisms of cholesterol transport by Niemann Pick C2 protein (NPC2) and cyclodextrin
Authors: McCauliff Leslie , Storch Judith
Published: June 2021

Niemann‐Pick C (NPC) disease is characterized by the accumulation of cholesterol and other lipids in the late endo/lysosome, due to defects in endo/lysosomal cholesterol binding proteins NPC1 or soluble NPC2. Using novel fluorescence‐based in vitro kinetic assays, we showed that NPC2 rapidly transports cholesterol to phospholipid vesicles via direct interaction with the membranes, and rates are greatly enhanced by lyso‐bisphosphatidic acid (LBPA). Site‐directed mutagenesis studies suggest that at least two surface membrane interacting domains on NPC2 may be involved in its cholesterol transport properties. This hypothesis is supported by results from a light scattering assay, used to determine whether NPC2 can interact with more than one membrane simultaneously; a dose‐dependent increase in scattering upon addition of NPC2 to vesicles indicates that NPC2 can cause vesicle‐vesicle interaction. We have also examined the cholesterol transport mechanism of cyclodextrins (CD), shown recently to have therapeutic potential in npc1−/− and npc2−/− mice. Results show that CD enhances sterol transfer rates in a manner similar to NPC2, though higher concentrations of CD are required and LBPA had no effect on sterol transfer by CD. Thus CD can substitute for NPC2 as a cholesterol transporter, but with lower sensitivity and specificity. Ara Parseghain Medical Research Foundation, American Heart Association
Steroid composition in wound healing preparations from the skin of the Arabian Gulf catfish (Arius Bilineatus, Val.)
Authors: Al‐Hassan Jassim Mohammad , Oommen Sosamma , Al‐Khamis Abdul Aziz , Afzal Mohammad
Published: June 2021

The Arabian Gulf catfish elaborates viscous proteinaceous material when threatened or injured. The viscous material contains 84% proteins 13.4% lipids and 2.6% nucleic acid components of its dry weight. Both lipids and proteins contain highly biologically active components. Preparations from the skin of the catfish were shown to accelerate wound healing in animals and humans. In order to understand the role of the preparations in inflammatory as well as anti‐inflammatory actions in the healing processes, the steroidal contents in the preparations were studied. The following steroids were found in catfish skin preparations (CSP) by separation on column chromatography and TLC, then identification by the use of GC/MS: (1) Cholest‐2,4‐diene; (2) Cholesta‐4,6‐diene‐3‐ol (3β); (3) Cholest‐3,5‐diene; (4) Cholest‐5,22‐dien‐3‐ol (3β); (5) Cholesterol; (6) 3β‐hydroxy‐cholest‐5‐en‐7‐one; (7) Cholest‐4‐en‐3‐one; (8) Cholest‐5‐en‐3β‐nonanoate; (9) Cholest‐4,6‐dien‐3‐ one; (10) Progesterone; (11) Ergosta‐5,24‐dien‐3β‐ol. Squalene and tocopherol were also discovered in the neutral lipids fraction. The roles of these steroids in the fish skin as well as in the wound healing processes are not yet known, but they may contribute to the anti‐inflammatory activities of the preparations. (This project is supported by Kuwait University Grant No. SL04/09)
Retinoic acid modifies lipid metabolism in isolated adult and aged rat cerebellum nuclei
Authors: Gaveglio Virginia Lucía , Giusto Norma María , Pasquaré Susana Juana
Published: June 2021

Nuclear lipid metabolism gives rise to several lipid second messengers that seem to be involved in the regulation of nuclear structure and gene expression. The purpose of the present research was to study the metabolic pathways involved in the metabolism of phosphatidic acid (PA) and its regulation in isolated nuclei from the central nervous system. This study was also conducted in isolated nuclei from aged animals, thus allowing us to analyze the effects of neurodegeneration processes on PA metabolism as a result of aging. Adult (4 mo) and aged (28 mo) rat cerebellums were homogenized and highly purified nuclei were isolated by sucrose‐density ultracentrifugation. Using radiolabelled substrates we demonstrated lipid phosphate phosphatase, diacylglycerol lipase (DAGL), monoacylglycerol lipase (MAGL), lysophosphatidate phosphohydrolase and phospholipase as well as phospholipase type A activities. We further studied their regulation by retinoic acid (RA) which was observed to decrease DAGL, MAGL and PA‐PLA activities. Significant aged‐related changes in the above‐mentioned enzymatic activities as well as in their regulation by RA were observed. Taken together, our results demonstrate a RA‐regulated PA metabolism in rat cerebellum nuclei which could be involved in neurodegeneration processes.

This research was funded by: CONICET, FONCyT, SCyT‐UNS.
TDX, a galectin‐1 and galectin‐3‐specific inhibitor, mitigates VEGF‐A‐induced angiogenesis
Authors: Chen Wei‐Sheng , Leffler Hakon , Nilsson Ulf J , Panjwani Noorjahan
Published: June 2021

Both galectin‐1 (Gal‐1) and galectin‐3 (Gal‐3) are vital modulators for vascular endothelial growth factors receptor‐2 (VEGFR‐2) signaling pathway. To date, TDX is the only compound that specifically targets both galectins with high affinity. Our goal is to study TDX's efficacy against VEGF‐A‐mediated pathological angiogenesis.

Here we showed that Gal‐1 and Gal‐3 interact with VEGFR‐2 in a carbohydrate‐dependent manner and that TDX: (i) reduces cautery‐induced corneal neovascularization in vivo, (ii) reduces VEGF‐A‐induced sprouting of human umbilical vein endothelial cells (HUVECs) as assessed by a three‐dimensional sprouting assay, (iii) efficiently abolishes VEGF‐A‐induced HUVEC chemotaxis, (iv) has little effect on HUVEC viability, and (v) does not affect total protein expression of VEGFR‐2 in HUVECs.

Our data provide proof of principle that targeting Gal‐1 and Gal‐3 by the novel, small molecule inhibitor, TDX, ameliorates pathological angiogenesis. The findings provide a new therapeutic strategy for various disease conditions, including inflammation, diabetes, cancer metastasis, age‐related macular degeneration and corneal neovascularization.

This work is supported by the NIH Grant R01EY007088 to N. Panjwani.
Insight into the involvement of Gβγ in nuclear G protein‐coupled receptor signaling
Authors: Spiegelberg Bryan David , Cymerman Anna , Ripley Lucas , Enalls Brandon
Published: June 2021

Recent data have expanded on well‐established roles for G protein‐coupled receptors by demonstrating the presence of GPCRs and the activities of their cognate pathways within certain cellular nuclei. As other groups have shown, sphingosine 1‐phosphate receptor 1 (S1P1), for example, relocalizes upon stimulation to endothelial and immune cell nuclei where it modulates transcription of specific genes in a direct manner. While biological consequences of this novel regulatory mechanism are being uncovered, the mechanisms by which nuclear GPCRs impact gene transcription remain to be elucidated in many cases. We, and others, have shown that Gβγ interacts with and modulates the activity of histone deacetylases and other transcriptional regulators; therefore, we propose that Gβγ plays an important role in the control of transcription by nuclear GPCRs. In support of this hypothesis, our data confirm the presence of a population of Gβγ in the nuclei of normally cycling mammalian cells. We also demonstrate, with microscopy and cellular dissection, that Gβγ enters the nucleus following cell stimulation in a time‐and temperature‐dependence consistent with a co‐translocation with S1P1. The dynamics of Gβγ and its activation by nuclearized S1P1 have a significant impact on Gβγ/HDAC5 complex formation. Clarification of the nuclear entry of Gβγ, its interaction with reversible protein acetylation, and its quantitative impact on transcription will shed new light on the functions of S1P1 and other ubiquitous membrane‐bound receptors.
Study of the effect of Ca(OAc)2 concentration on the state of the Allosteric Switch in Ras GTPase
Authors: Johnson Christian William , Davis Kathleen , Mattos Carla
Published: June 2021

Ras GTPase cycles between GDP inactive and GTP active forms and is of great importance in signal transduction that regulates cell proliferation, differentiation and survival. Although signaling is largely attenuated by the presence of GTPase activating proteins (GAPs), our group has recently discovered and allosteric mechanism that may promote hydrolysis of GTP in the presence of Raf and possibly other tight binding effectors, demonstrating subtle levels of regulation of the GTP‐bound protein. The allosteric switch is ON, with complete ordering of the active site, in crystals soaked in 100mM Ca(OAc)2, which binds to an allosteric site remote from the catalytic center. In the presence of CaCl2 the allosteric switch is OFF. Ca(OAc)2 binding is mediated by arginine 97 (R97) through salt bridge interactions with acetate. Here we show that R97A, which cannot bind acetate, is in the OFF state in the presence of 245 mM Ca(OAc)2 but switches to the ON state at 365 mM Ca(OAc)2. The R97F mutant is observed in the OFF state in the presence of 200mM Ca(OAc)2. It is clear that the bulk solvent composition has an important effect in the state of the allosteric switch in R97A. The soaking of crystals in 365 mM Ca(OAc)2 will test whether this is also the case for R97F and other allosteric site mutants.
Thyroid hormones and their associations with cognitive function: moderation by sex, race and depressive symptoms
Authors: Beydoun May A , Beydoun Hind A. , Kitner‐Triolo Melissa H. , Kaufman Jay S. , Evans Michele K. , Zonderman Alan B.
Published: June 2021

Thryroid hormone fluctuations’ influence on cognition is under‐studied, particularly within normal ranges and in population subgroups. We describe associations between thyroid hormones within reference ranges and cognitive performance, stratifying by sex, race and depressive symptoms (CES‐D). This is a cross‐sectional analysis of n=1,776–2,127 adults (age:30–64y) from the Healthy Aging in Neighborhoods of Diversity Across the Life Span study, Baltimore City, MD. Outcomes included thirteen test scores spanning various cognitive domains. Exposures included serum total and free T4 (tT4; fT4), thyroid stimulating hormone (TSH) and T3(uptake). After familywise Bonferroni correction for multiple testing (p<0.004), higher T4 was associated with better performance on animal fluency (tT4,fT4) and identical pictures(tT4). Stratum‐specific associations that remained statistically significant included: Higher tT4 and better performance on the Benton Visual Retention Test (BVRT) among White and depressed individuals, higher tT4 and better performance on animal fluency among men, Whites and non‐depressed individuals, higher fT4 and better performance on animal fluency among men, higher T3(uptake) and poorer performance on animal fluency among depressed individuals. In sum, thyroid function and cognition are closely linked, with associations varying by sex, race and depressive symptoms.
Altered hippocampal activation during memory encoding in type 2 diabetes
Authors: Meusel Liesel‐Ann Catharine , Anderson Nicole , Tchistiakova Ekaterina , MacIntosh Brad , Greenwood Carol
Published: June 2021

Type 2 diabetes (T2DM) increases risk for cognitive decline and dementia. While much is known about T2DM‐associated cognitive changes and brain atrophy, the impact of T2DM on brain function is underexplored. Blood oxygenation level dependent (BOLD) signal changes were compared in older adults with T2DM and hypertension (DO, n = 9) and older adults with hypertension only (HO, n = 10) during a cerebrovascular reactivity (CVR) task and a word‐list learning task. We hypothesized that memory deficits and altered BOLD signaling would be apparent in DO compared to HO but that CVR, obtained during a breath‐hold task, would be comparable. There were no group differences in performance on neuropsychological memory tests nor on the word‐list learning task. A region‐of‐interest analysis on hippocampal (HC) activation during word‐list encoding showed stronger right HC activation in the DO compared to the HO group after accounting for CVR (p < .01). Across groups, there was a significant negative correlation between right HC activation and number of words learned during encoding, controlling for performance on the first trial (r = −0.61, p < .01). Collectively these findings suggest that 1) T2DM elevates memory‐related activation of right HC independent of vascular reactivity, and 2) increases in right HC activation parallel decreased learning success and may signal efforts to compensate for cognitive decline (CIHR – MOP111244).

Grant Funding Source: Canadian Institutes of Health Research
Developmental and Gender Differences in Cognitive Responses to Caffeine
Authors: Graczyk Adam , Ziegler Amanda M , Bendlin Ashley , Sion Teresa , Vattana Karina , Temple Jennifer L.
Published: June 2021

Background

Caffeine is the most widely used stimulant in the world. Previous studies have shown that caffeine can enhance cognitive functioning.

Objective

To measure developmental and gender difference in cognitive responses to caffeine in children and adolescents.

Methods

Pre‐ and post‐pubertal children were given a cognitive test battery using the Automated Neurological Assessment Metrics (ANAM) before and 60 minutes after administration of placebo or caffeine (1mg/kg or 2mg/kg).

Results

Caffeine increased the number correct in the memory search task and decreased the standard deviation in the go/no‐go task. Females had more correct responses than males in simple reaction time and the Stroop color‐word task. Also in post‐pubertal females, there was an interaction between menstrual cycle phase and caffeine treatment in simple reaction time and the go/no‐go task.

Conclusions

These results are consistent with previous studies, primarily in adults, showing caffeine facilitates performance on some cognitive tasks and not others. In addition, these results are similar to previous studies showing sex differences on performance on some cognitive tasks. This study is the first to report sex differences in responses to caffeine on cognitive tasks as well as differences across the menstrual cycle in children and adolescents. This study was funded by RO1DA030386 to JLT.
Factors influencing Latino participation in community‐based diabetes education
Authors: Francis Sarah L , Noterman Amber , Litchfield Ruth
Published: June 2021

The growing Latino diabetes epidemic merits the creation of culturally‐sensitive community diabetes education programs. Dining with Diabetes (DWD) was revised as an Extension program for Iowa‐residing Latinos based on focus group (FG) discussions of Latinos with diabetes. DWD (4 weeks) was taught in Spanish, used interactive strategies and taste‐testing. Despite initial interest attendance was insufficient. Five Latino adults interested in DWD participated in a Spanish‐speaking FG examining factors influencing participation. Transcripts were analyzed for common themes. FG results suggest multi‐session, local group classes led by an engaging Spanish speaking instructor, during the summer is ideal. Participants learned of DWD through friends and Extension staff; preferred recruitment methods were Spanish radio, local health clinics, and Spanish‐print media. Barriers to DWD attendance were family and work conflicts. Results suggest that scheduling conflicts and limited use of culturally preferred recruitment methods were barriers to DWD attendance. Future nutrition programs for Latinos may be more successful if offered during summer months and if culturally‐preferred marketing strategies are utilized.
Patterns of food source usage among low‐income African Americans are associated with dietary intake
Authors: Cuccia Alison , Budd Nadine , Liu Debra , Jock Brittany , Jeffries Jayne , Powell Lisa , Gittelsohn Joel
Published: June 2021

Obesity rates among Baltimore City adult residents are 30% higher than national averages, and are highest among minorities living in low‐income areas. While food availability has been linked to obesity and nutritional adequacy, little research has examined patterns of food source usage and dietary quality. 362 low‐income African American adults recalled their 24‐hour dietary intake and past 30 day frequency of food source usage. Parallel Analysis was used to extract three significant factors (using varimax rotation and factor loadings greater than 0.30 to interpret factors). Public markets, corner stores, carryouts, and fast food restaurants loaded high on the Prepared Foods Factor; street food vendors, food pantries, and community centers loaded high on the Food Giveaway Factor; and farmers markets, horse drawn produce carts (arabbers), and specialty food stores load high on Whole Food Factor. The Prepared Food Factor was associated with decreased diet quality in women, and the Food Giveaway Factor and the Specialty Food Factor was associated with decreased diet quality in men. The Food Giveaway Factor (p=0.022) and the Whole Food Factor (p=0.003) were positively associated with education. These results may inform how food policy in Baltimore City can target low‐income men and women differently.

Funded by National Health, Lung & Blood Institute, Heart Disease (NHLBIDHV), Grant #1R21HL102812–01A1.
Brain dynamics as a function of iron status: Relating electroencephalographic (EEG) patterns and body iron measures in Indian adolescents
Authors: Wenger Michael J , Scott Samuel P , Murray‐Kolb Laura E , Ghugre Padmini , Udipi Shobha , Haas Jere D.
Published: June 2021

World‐wide, iron deficiency (ID) is the most common micronutrient deficiency, and has been linked in both human and animal models to deficits in perceptual, cognitive, and globally adaptive behaviors. In addition, studies using electroenchelography (EEG) have suggested abnormal patterns of brain activity (lower amplitudes and longer latencies of EEG components) in ID vs. non‐ID populations. The present study was one part of a randomized, double‐blind feeding trial testing the efficacy of iron‐biofortified pearl millet as a mechanism to improve iron status. Subjects (n = 75, 12–16 y, 38 females) were from two residential schools in Maharashtra, India. Blood measures included hemoglobin, serum ferritin, and serum transferrin receptor. Brain functioning was assessed using EEG while subjects completed five tests of perceptual and cognitive performance, selected to assess perceptual and cognitive components of schoolwork and for comparability to previous work in adults. We found relationships between baseline iron measures and the magnitude (positive) and latency (negative) of a set of EEG components relevant to the behavioral expression of selective attention and episodic memory, suggesting a functional role for iron in determining healthy brain dynamics. Supported by HarvestPlus/IFPRI.
Anti‐inflammatory effects of açai (Euterpe oleracea Martius) extract on normal intestinal CCD‐18Co cells
Authors: dos Santos Dias Manoela Maciel , Roque‐Andrade Andrea , Talcott Susanne
Published: June 2021

Açai (Euterpe oleracea Mart.) has a growing demand due to its health benefits and antioxidant capacity. Another property of the fruit is its ability to potentially reduce inflammation leading to irritable bowel disease. This study evaluated the anti‐inflammatory effects of açai polyphenolic extract in non‐cancer human colon fibroblasts cells (CCD‐18). The polyphenolic profile of açai extract was analyzed by HPLC‐MS and it was assessed in CCD‐18 cells using the cell proliferation assay, the dichlorofluorescein diacetate assay for reactive oxygen species (ROS), qRT‐PCR for mRNA, and Western Blot for protein levels. The major compounds present in açai extract were cyanidin‐rutinoside (1395.3 mg/L) and cyanidin‐O‐glucoside (451.5 mg/L). The anti‐inflammatory and antiproliferative effects of the single compound cyanidin‐3‐O‐glucoside were evaluated to determine if the beneficial effects of açaí can be attributed to this compound. The extract (1–5 mg gallic acid equivalent/L) did not reduce cell growth significantly, but reduced ROS induced by lipopolysaccharide (LPS 0.53‐fold). Cyanidin‐3‐O‐glucoside (1–5 mg/L) did not inhibit cell proliferation in CCD‐18Co. Açai extract (5 mg GAE/L) downregulated the LPS‐induced expression of TNF‐α (to 0.42‐ fold), COX‐2 (to 0.61‐fold), TLR‐4 (to 0.52‐fold), TRAF‐6 (to 0.64‐fold), NF‐kB (to 0.76‐fold), VCAM‐1 (to 0.71‐fold) and ICAM‐1 (to 0.68‐fold). The protein levels of COX‐2, TLR‐4, p‐ NF‐kB and ICAM‐1 stimulated by LPS were also downregulated by the extract in a dose‐dependent manner. Cyanidin‐3‐O‐glucoside showed similar anti‐inflammatory effects. These results suggest the anti‐inflammatory effect of açai polyphenolic extract in intestinal cells, involving the inhibition of toll‐like receptor‐4 (TLR‐4) and factor nuclear kappa‐B (NF‐kB). Our results indicate the potential for acai polyphenolics in the prevention of intestinal inflammation.
Contribution of the ECS to COX expression and activity in myoblast cultures
Authors: Kim Jeffrey , Li Yong , Watkins Bruce A
Published: June 2021

Cyclooxygenase (COX) possesses substrate affinity to the endocannabinoids (EC) anandamide (AEA) and 2‐ arachidonylglycerol (2‐AG). Moreover, the relationship between EC signaling (ECS) and the COX pathway is not known therefore we investigated agonist, antagonists and PUFA effects on ECS genes in myoblasts. At 50% confluency, C2C12 were pretreated with 5 μM of the CB2 antagonist, AM630, for 2 h and one with both AM630 and 1 μM of CB1 antagonist, NESS0327. Cells were then treated with 25 μM of either AA (20:4n6), EPA (20:5n3), AEA, or BSA (vehicle control) for 24 h. Western blot analysis of COX and enzyme activity were measured. qPCR analyses were performed on ECS relevant genes. Total COX activity was greater in the AM630 + AEA treated cells compared to all other cells. Further analysis revealed that COX‐1 and COX‐2 proteins were greater in the AM630 + AEA treated cells compared to all others. mRNA for the AEA synthesis enzyme, NAPE‐PLD, and 2‐AG synthesis enzymes, DAGLα and DAGLβ, were higher in AM630 + EPA treated cells compared to all other groups. The mRNA levels of CB1 and CB2 were both highest in the AM630 + EPA group. These findings suggest that COX activity and protein levels are influenced by the ECS, particularly by the activation of CB1 receptors. However, treatments of either AA or EPA did not alter COX status in C2C12 cells under testing condition.

Grant Funding Source: Diet and Health Initiative, UConn, Storrs, CT, USA