bluuebunny/crossref_metadata_2025 huggingface dataset:
Lymphotoxin‐alpha Concentrations at Sites of Periodontitis and Gingival Overgrowth
Authors: Johnson Roger B , Lester S. Reid , Bain Jennifer L , Serio Francis G
Published: June 2021

Objective

There are no studies available comparing lymphotoxin‐alpha (LT‐alpha) gingival concentrations between sites of periodontal disease and gingival overgrowth (OG).

Methods

Gingiva was obtained from human donors prior to extraction of the adjacent teeth. The tissue was grouped based on adjacent pocket depth and bleeding on probing (BOP). Gingiva adjacent to a = 3 mm sulcus without BOP were classified as "normal" (N; n=26); Gingiva adjacent to a 4‐6 mm sulcus featuring BOP were classified as "diseased, moderate" (DM; n=23) and gingiva adjacent to > 6 mm sulci were classified as "diseased, severe" (DSev; n=28). OG gingiva was obtained following gingivectomy from patients receiving nifedipine/cyclosporine treatment (n=24). Tissues were solubilized and IL‐6, IL‐1‐β and LT‐alpha concentrations were assessed by ELISA. Data were compared by factorial ANOVA and a post‐hoc Tukey test. Groups were defined as significantly different when p<0.05.

Results

Gingival concentrations of IL‐6 were significantly greater at DSev than at N sites (p<0.001) and significantly greater within OG than within gingiva adjacent to N, DM, and DSev sites (p<0.001). In contrast, IL‐1‐β concentrations within OG tissues were significantly lower than within gingiva adjacent to N, DM, and DSev sites (p<0.001). LT‐alpha concentrations were significantly greater within OG than within gingiva adjacent to N, DM, and DSev sites (p<0.001) and at DM compared to N and DSev sites (p<0.05).

Conclusion

Our data suggest that LT‐alpha may have an important role in the etiology of OG gingival inflammation and could be a target for therapeutic intervention for OG.
EXPRESSION OF PROINFLAMMATORY CHEMOKINES AS BIOCOMPATIBILITY MARKERS OF DENTAL SEALERS
Authors: SOLIS‐SOTO JUAN MANUEL , SOUSA‐MORENO VANESSA
Published: June 2021

Biocompatibility of new dental sealers should be evaluated before their clinical application.

Objective

We studied the in situ production of chemokines in order to evaluate the biocompatibility of five endodontic sealers of different composition (Sealer 26, Tubli‐Seal, MTA, Silverfree AH 26, and Endorez).

Methods

BALB/c mice were subcutaneously implanted with the different dental sealers. Then the tissue was prepared for histopathological and immunohistochemical analysis.

Results

The proinflammatory chemokines CCL3 and CCL4 participated in both, acute and chronic inflammatory response induced by each dental sealer studied. Immunoreactive cells to both chemokines, were observed abundantly in the dermis, especially in the inflamed tissue around the dental sealers. Higher amounts were observed in the acute inflammatory response. According to morphometrical analysis, the order from more to less biocompatible was: MTA, Sealer 26, Tubli‐Seal, Endorez, Silverfree AH26. There were more immunoreactive cells to CCL4 than CCL3 in response to dental sealers (p<0.05).

Conclusions

Expression of the proinflammatory chemokines CCL3 and CCL4 is an excellent in vivo model to study the biocompatibility of dental materials. MTA and Sealer 26 are the most biocompatible dental sealers.
EGFR Mediated mTOR Activation Attenuates UVB‐Induced Apoptosis
Authors: Lu Shan , Cao Cong , Kivlin Rebecca , Wallin Brittany , Sarkisian Simon , Tamakloe Tyrone , Bagdasarian Andrew , Chu Wen‐ming , Wan Yinsheng
Published: June 2021

mTOR acts as an important integrator of several upstream signals, including growth factors, nutrients, energy levels, and stresses. We found in this study that UVB radiation induces mTOR activation in human skin keratinocytes and both cultured mouse dendritic cells and mouse monocyte derived dendritic cells. Using various specific inhibitors and gene manipulation methods, we observed that UVB‐induced mTOR activation is dependent on EGFR‐mediated AKT activation. We also found that TSC2/mTOR, downstream of AKT, is involved in UVB‐induced S6K activation. Inhibition of EGFR, AKT and mTOR enhances UVB‐induced apoptosis. Collectively, our data suggest that EGFR mediated mTOR activation, together with its upstream signals, serve as a novel survival signal against UVB‐induced cell apoptosis.
Crystal structure of E. coli RecE exonuclease reveals a toroidal tetramer and a conserved architecture for processive DNA digestion.
Authors: Zhang Jinjin , Xing Xu , Herr Andrew B. , Bell Charles E.
Published: June 2021

The E. coli RecET recombination system is a classic model for understanding the repair of double strand DNA breaks by the single strand annealing pathway. RecE protein, one of the two key components of this system, is a processive ATP‐independent exonuclease that specifically binds to double strand DNA ends and degrades the 5′‐terminated strand. The 3′‐ended ssDNA tail that is generated serves as a substrate for RecT recombinase, the other key component of RecET system, which promotes homologous recombination. In this study, a 2.8 Åresolution crystal structure of the C‐terminal nuclease domain of RecE, residues 606 ‐ 866, was determined. RecE forms a toroidal tetramer with a central channel that is wide enough to bind double strand DNA at one end, but is partially plugged at the other end by the C‐terminal segment of the protein. Four narrow tunnels, one within each subunit of the tetramer, lead from the central channel to the four active sites, which lie about 15 Åfrom the channel. The structure suggests a mechanism in which DNA binds to the open end of the central channel, the 5′‐ended strand passes through a tunnel to access and be degraded by one of the four active sites, and the 3′‐ended strand passes through the plugged end of the channel at the back of the tetramer.
Scavenger function of liver sinusoidal endothelial cells
Authors: Smedsrod Bard
Published: June 2021

The lining of the liver sinusoid is made up of a specialized endothelium referred to as liver sinusoidal endothelial cells (LSECs). These cells are the major site of clearance of an array of blood borne physiologic and foreign waste macromolecules. The LSECs are equipped with specific endocytosis receptors and an endocytic machinery that enables the cells to carry out endocytosis at a speed and capacity that surpass that of any other cell type in the body. By means of 3 major types of endocytosis receptors (scavenger receptor/stabilin, mannose receptor, and Fc gamma receptor) the cells are able to effectively clear most waste materials of colloidal and soluble macromoleculear nature from the blood. Although the liver macrophage was previously believed to make up the liver reticuloendothelial system (RES), we have established that the LSEC is as important. There is a clear division of work between the two cell types: the liver macrophages remove blood borne particles (roughly 200 nm and above) by phagocytosis, and the LSECs eliminate soluble macromolecules of size less than 200 nm. This share of work between macrophages and scavenger endothelial cells is the same in all vertebrates.
Protein Interactions and Structure Studies of the Transcriptional Modulator Ashwin
Authors: Uzman Akif , Lou Chih‐Hong , Fryszczyn Bartlomiej , Pereida Norma , Sater Amy
Published: June 2021

Ashwin is a novel modulator of canonical wnt‐dependent transcription, which regulates Organizer gene expression in Xenopus embryos. Coimmunoprecipitation studies demonstrate that Ashwin interacts with TCF3 in vivo, and that increasing amounts of Ashwin disrupt the interaction between beta‐catenin and TCF3. Chromatin immunoprecipitation studies indicate that overexpression of Ashwin reduces occupancy of the siamois promoter by beta‐catenin. Ashwin also interacts with Polycomb1 (Pc1), a member of the Polycomb Repressor Complex1 (PRC1), and our results suggest that Pc1 acts with Ashwin to inhibit beta‐catenin‐dependent gene expression. To learn more about the structure of Ashwin, we have used the Studier method of auto‐induction of recombinant protein under the control of lacI to obtain recombinant protein for crystallization and structure determination via X‐ray diffraction. Further, we present bioinformatic evidence that ashwin is a novel and highly conserved vertebrate gene that may have evolved from previously undescribed open reading frames
Alterations in proteasome plasticity and biogenesis with aging and dietary restriction
Authors: Dasuri Kalavathi , Keller Jeffrey N.
Published: June 2021

The proteasome is an essential enzyme that appears to be inhibited with aging, although the basis for age‐related impairments in proteasome function remain poorly defined. In the present study we report that aging increases oxidative inactivation, and decreases the stability of the 20S proteasome, with dietary restriction (DR) ameliorating both of these aspects of aging in the liver. Additionally, we have found increased levels of 20S proteasome complexes and evidence for increased levels of proteasome biogenesis in the aging liver, suggesting that during aging tissues may attempt to overcome age‐related increases in oxidative stress via elevating the levels of 20S complexes. Lastly, our studies demonstrate that increased levels of Hsp90 within the 20S proteasome fraction are observed with aging of the liver, which may be an additionally mechanism by which the aging liver attempts to preserve proteasome function during aging. Taken together, these data suggest that the proteasome complex exhibits plasticity in response to aging and DR with regards to proteasome levels, proteasome stability, proteasome biogenesis, and proteasome associated heat shock proteins. These factors may play an important role in regulating the function of the proteasome during aging and in response to beneficial stimuli such as DR.
Plasmin is a chemoattractant for immature dendritic cells acting through Akt2‐dependent mechanisms
Authors: Syrovets Tatiana , Li Xuehua , Genze Felicitas , Pitterle Kai , Simmet Thomas
Published: June 2021

Pivotal roles have been proposed for dendritic cells (DCs) and the adaptive immune response in atherosclerosis. Here we demonstrate that plasmin is a potent activator of human DCs. Stimulation of immature DCs with plasmin, but not with catalytically inactivated plasmin, elicits actin polymerization and a chemotactic cell migration comparable to that triggered by the standard chemoattractant FMLP. Plasmin‐stimulated DCs show rapid activation of Akt, ERK1/2 and p38 MAP kinases, followed by phosphorylation of the regulatory myosin light chain and chemotaxis. DCs express Akt1 and Akt2, yet plasmin activates exclusively Akt2 via a p38 MAPK‐dependent pathway. Knockdown of Akt2, but not of Akt1 by shRNA inhibited the plasmin‐induced ERK1/2 activation and the chemotaxis. Using immunohistochemistry we show that both, plasmin and DCs, are abundant in human atherosclerotic lesions, where they colocalize. Therefore, this novel link between protease activity and the adaptive immune response may be of particular relevance in atherogenesis.
Cellular Nucleic Acid Binding Protein (CNBP) in Aging and Disease
Authors: Murphy Michael Paul , Beckett Tina , Webb Robin , Holler Chris , Niedowicz Dana
Published: June 2021

Type II Myotonic Dystrophy is caused by a tetranucleotide expansion in the ZNF9 gene, which encodes the single strand RNA binding protein CNBP (Cellular Nucleic Acid Binding Protein). CNBP also regulates the activity of â‐secretase (BACE1), the rate limiting enzyme in the production of the amyloid‐â peptide (Aâ), and also instrumental in the development of Alzheimer's disease (AD). The activity and protein levels of BACE1 increase in Alzheimer's disease, in normal aging, and in Inclusion Body Myositis, an age‐related, degenerative disease of the skeletal musculature. In this study, we sought to characterize CNBP in both age and disease state. We examined levels of CNBP protein and BACE1 and protein and activity across the normal life span of the mouse, and in individuals with AD and age matched controls. A fluorometric â‐secretase assay was developed to measure both â‐secretase activity and protein levels. Using this and related techniques, enzymatic activity and protein levels were measured in aging mice, and in human tissue samples. Cell culture studies demonstrated that CNBP regulates BACE1 protein and activity, most likely at the mRNA level. â‐secretase increases in the disease state, and CNBP also increased slightly with age. These data suggest CNBP may regulate â‐secretase in vivo and in vitro, and may contribute to the development of AD and other age‐related degenerative diseases. Supported by NIH NS058382.
Investigating Molecular Chaperone Activity Associated with Human TorsinA
Authors: Burdette Alexander Justin , Churchill Perry F. , Caldwell Guy A , Caldwell Kim A
Published: June 2021

TorsinA is a member of the AAA+ ATPase family of proteins and, interestingly, is the only such ATPase localized to the ER lumen. TorsinA has been suggested to act as a molecular chaperone while a deletion mutant form associated with early‐onset torsion dystonia, a dominantly inherited movement disorder, appears to result in a net loss‐of‐function in vivo. Thus far, no studies have examined the chaperone activity of torsinA in vitro. Here we expressed and purified both WT and mutant torsinA fusion proteins (to maltose binding protein) in bacteria and examined their ability to function as molecular chaperones by monitoring suppression of luciferase and citrate synthase aggregation, as well as luciferase reactivation. As measured by light scattering, both WT and mutant torsin effectively and similarly suppressed protein aggregation compared to controls. Further, both forms of torsinA demonstrated the capacity to hold luciferase in an intermediate state for refolding and reactivation by other chaperones. These data not only provide conclusive evidence that torsinA acts as a molecular chaperone in vitro, but also suggest that dystonia is likely not a consequence of a loss in torsinA chaperone activity.

Research support: Dystonia Medical Research Foundation
Activation of the stress protein response inhibits the STAT1 signaling pathway and iNOS function in alveolar macrophages: role of Hsp90 and Hsp70
Authors: Howard Marybeth , Roux Jeremie , Lee Hyon , Miyazawa Byron , Lee Jae‐Woo , Gartland Brandi , Howard Amanda , Matthay Michael , Pittet Jean‐Francois
Published: June 2021

Alveolar fluid clearance is impaired by iNOS/NO‐dependent mechanisms in ALI/ARDS. We investigated whether activation of the stress protein response (SPR) by heat, or using an Hsp90 inhibitor inhibits the STAT1 signaling pathway, which is activated in ALI/ARDS, and thus inhibits iNOS/NO production. Inhibition of STAT1 activation after heat stress results in detergent insolubilization of the STAT1 protein and its proteasomal degradation which is reversed with the pretreatment of cells with glycerol, a chemical chaperone that reduces the extent of heat‐induced protein denaturation. This early effect of SPR is the result of the disruption of the Hsp90 binding to the STAT1 protein and confirmed using an Hsp90 inhibitor. Recovery of STAT1 activation and iNOS synthesis occurs by 12 hours post‐SPR induction, however, NO production did not occur until 48 hours later. These results demonstrate a late effect of SPR activation involving the regulation of iNOS function by inducible Hsp70 (Hsp70i). Inhibiting Hsp70i expression after heat stress recovers iNOS function whereas overexpressing Hsp70i in the absence of heat stress inhibits iNOS function. These results represent novel mechanisms by which the activation of SPR inhibits STAT1 signaling in alveolar macrophages and highlight a potential clinical application for Hsp90 inhibitors in modulating NO signaling during the early phase of acute lung injury. [Research support: UCSF academic senate grant (MH); NIH GM62188 (JFP)]
Hydrogen‐Deuterium Exchange Reveals Antibiotic Resistance Enzyme Aminoglycoside Phosphotransferase 3′‐IIIa as Intrinsically Unstructured Protein
Authors: Norris Adrianne Lee , Serpersu Engin Halit
Published: June 2021

Hydrogen deuterium (H/D) exchange detected by NMR spectroscopy is used to determine the dynamic properties of the aminoglycoside phosphotransferase 3′‐IIIa (APH), which confer resistance to aminoglycoside antibiotics. This work represents the first characterization of dynamic properties of an aminoglycoside modifying enzyme. H/D exchange experiments performed with apo, binary and ternary complexes of APH with kanamycin A, neomycin B, and metal‐nucleotide showed that regions of APH in different complexes that are superimposable in crystal structures show remarkably different dynamic behavior. A complete exchange of backbone amides is observed within the first 15 hours of exposure to D2O in the apo form of this 31 kDa protein. Binding of aminoglycosides to the enzyme induces significant protection against exchange and ~35% of the amides remain unexchanged up to 90 hours after exposure to D2O. Our data also indicate that neomycin creates greater solvent protection and overall enhanced structural stability to APH than kanamycin. Surprisingly, nucleotide binding to enzyme‐aminoglycoside complex increases solvent accessibility of a number of amides and is responsible for destabilization of a nearby β‐sheet thus providing a rational explanation for previously observed global thermodynamic parameters. Our data also provide molecular basis for broad substrate selectivity of APH.
SpH‐protease exists in Caco‐2 cells
Authors: Morin Violeta Marilyn , Dufey Estefanie , Jaña Claudia , Soliz Carlos , Rivas Coralia , Puchi Marcia , Imschenetzky Maria
Published: June 2021

We have described a 60 kDa nuclear cysteine protease that degrades sperm histones during male chromatin remodeling after fertilization in sea urchins (SpH protease). This enzyme was shown to be localized in the mitotic furrow and to be involved in cell cycle control in sea urchin embryos. The N terminal sequence and the gene encoding this protease have homology with human cathepsin L family; therefore we investigated with antibodies raised against the sea urchins protease if a similar protease is present in Caco‐2 cells. Among Caco 2 chromosomal proteins obtained from confluent cell cultures, we found by Western blots, two bands migrating at 60 and 100 kDa in SDS/PAGE. We have also used these anti SpH protease antibodies to immunolocalize this protease in the intracellular context. This protease in mitotic cells localize on the mitotic furrow and remains in the nucleus in binuclear cells. On the other hand, when inhibitors of cysteine‐proteasas were used in different phases of the cell cycle we observed a disorganization of the mitotic spindle and an alteration in the localization of the protease during the S phase affecting DNA replication. Our results demonstrate that in Caco 2 cells exist a variant of cathepsin L protease that exhibits a similar localization to that was described for embryos of sea urchin, indicating a putative rol in cell proliferation. FONDECYT 11070067.
Matrix metalloproteinase (MMP)‐9 deletion slows cardiac aging
Authors: Chiao Ying Ann , Zamilpa Rogelio , Dai Qiuxia , Lindsey Merry L
Published: June 2021

Cardiac aging is associated with decreased function of the left ventricle (LV) and higher prevalence of cardiovascular diseases, but the mechanisms of cardiac aging are not fully understood. We have shown that matrix metalloproteinase (MMP)‐9 regulates cardiac remodeling after MI, and MMP‐9 LV levels increase from middle‐aged to old mice. Accordingly, we tested the hypothesis that the age‐related increase in MMP‐9 regulates cardiac aging. As mice do not develop hypertension with age, the mouse is a good model to study cardiac aging without the complication of increased vascular stiffness. We compared young (8 month old) and old (18‐23 month old) wild type and MMP‐9 null mice (n=3‐7 per group). By dobutamine stress echocardiography, the increase in relative wall thickness by dobutamine was higher in old WT mice compared to young controls, indicating a lower tolerance to stress. Interestingly, this increase in stress response was not observed in old MMP‐9 null mice. Old MMP‐9 null mice had an LV to body weight ratio of 3.5±0.3, which was higher than young null (2.8±0.2) and old WT (2.8±0.2) mice (both p<0.05), suggesting that compensatory hypertrophy persists for a longer period in the absence of MMP‐9. Together, these data indicate that blocking MMP‐9 function may slow the kinetics of cardiac aging. NIH (R01 HL‐75360) and American Heart Association grant (0855119F) to M.L.L. and T32 (HL07446) to R.Z.
Biocompatible Peptide‐nanoparticle Constructs for Molecular Imaging and Therapy
Authors: Pan Hua , Myerson Jacob W , Soman Neelesh R , Schlesinger Paul H , Lanza Gregory M , Wickline Samuel A
Published: June 2021

Our recent work on melittin delivery demonstrates that melittin tightly inserts into the perfluorocarbon (PFC) nanoparticles. Here, we hypothesize that altering the melittin sequence can convert melittin from a therapeutic agent to a cargo loading linker for PFC nanoparticles. Our results illustrate that mutated melittin, D1‐7 (Ac‐ VLTTGLPALISWIKRKRQQ‐NH2), reduces the melittin lytic activity by about 50‐fold (A). While it still insertes tightly into the PFC nanoparticles (Kd=0.4 μM) without affecting their integrity (B). We also show that the linker peptide, D1‐7, is capable of integrating cargos into PFC nanoparticles by a simple mixing step. As an example, VCAM‐1 targeted PFC nanoparticles are generated through this linker peptide. 2F2B cells, treated with the VCAM‐1 targeted PFC nanoparticles, co‐labeled with Alexa Fluor 488, manifest stronger fluorescent signals (C) than those, treated with non‐targeted ones (D). VCAM‐1 targeting dramatically increases cellular PFC nanoparticle delivery (E). Moreover, targeting efficacy is controllable by amount of peptide loading (F). We conclude that this linker peptide strategy provides extended flexibility for multiplexing targeting ligands and/or drug payloads, which can be selected from a stock of possible active agents, without the need of base nanoparticle reformulation to behoove personalized molecular imaging and therapy.
INFLUENCE OF INSATURATION DEGREE OF Saccharomyces cerevisiae MEMBRANE LIPIDS OVER THE EFFECTS OF Fe2+ ION ON MITOCHONDRIAL FUNCTION AND CELL INTEGRITY
Authors: Cortés‐Rojo Christian , Manzo‐Ávalos Salvador , Clemente‐Guerrero Mónica , Saavedra‐Molina Alfredo
Published: June 2021

In cells, the mitochondrial electron transport chain (ETC) is one of the most important sources of reactive oxygen species (ROS). ROS generation in ETC may be promoted in vitro through addition of certain inhibitors of respiratory complexes, whereas some pathological situations, such as cardiomyopathy, hyperglycaemia, calcium overload or genetic defects in proteins that constitute respiratory complexes may increase the in vivo rate of mitochondrial ROS production. The goal of the present work was to determinate if a lower susceptibility to lipid peroxidation may confer resistance to mitochondrial functioning and cell integrity towards damaging effects of oxidative stress. To achieve this goal, unsaturation degree of cell membranes from Saccharomyces cerevisiae was augmented through addition of linolenic acid (C18:3) and spheroplasts were obtained to evaluate in situ mitochondrial function. Lipid peroxidation levels, stimulated by the treatment with Fe2+, increased only in spheroplasts with C18:3, while mitochondrial function, measured as cyanide‐sensitive oxygen consumption, and cell integrity, determinated by trypan blue exclusion, were affected significatively only in spheroplasts with C18:3. Taken together, these results show that lipid peroxidation sensitizes cell integrity and mitochondrial function towards damaging effects of oxidative stress.
The effect of Epoxyeicosatrienoic acids on oxidative stress‐induced apoptosis in mesencephalic dopaminergic neuronal cells.
Authors: Terashvili Maia , Narayanan Jayashree , Harder David R
Published: June 2021

Oxidative stress and apoptotic cell death have been implicated in the dopaminergic cell loss that characterizes Parkinson's disease. While factors contributing to apoptotic cell death are not well characterized, oxidative stress is known to activate an array of cell signaling molecules that participate in apoptotic cell death mechanisms. Furthermore, epoxyeicosatrienoic acids (EETs) such as 14,15‐EET have been recognized for their protective effects on the cardiovascular system. In this study, we are investigating the effect of EETs on oxidative stress‐induced cytotoxicity by hydrogen peroxide (H2O2) in the dopaminergic mesencephalon‐derived N27 cell line. N27 cells stimulated with different doses of H202 (0.01 to 5mM for 4h) produce dose‐dependent oxidative stress‐induced apoptotic cell death. N27 cells stimulated with H2O2 increase the oxidation of PTEN and increased the phosphorylation of Akt. However, N27 cells stimulated with a high dose of 14,15‐EET (3μM) did not oxidize PTEN, but showed a decrease in Akt phosphorylation. These data make it essential to understand the role of EETs in protecting dopaminergic neurons from oxidative stress, which may help in understanding deficits seen in Parkinson's disease. Support: NIH/NHLBI PO1 HL59996‐05; RO1 HL33833‐17; RO1 HL092105‐01A1.
Modulation of cytochrome P450 1A2 and 3A4 by Morinda citrifolia (noni), Euterpe oleracea (acai), Lycium barbarum (goji) and Garcinia mangostana (mangosteen)
Authors: Eck Philip Ofosu , Shah Pranjal A , Nerurkar Pratibha V
Published: June 2021

The use of complementary and alternative medicine (CAM) including herbal products, for health maintenance and treatment of chronic diseases is continuously rising in the United States. Although considered safe due to their "natural" origin, herbal products are prone to herb‐drug interactions. Cytochrome (CYP) P450 enzymes are responsible for metabolizing xenobiotics including herbal products. The aim of our study was to investigate the effects of Morinda citrifolia (noni), Euterpe oleracea (açai), Lycium barbarum (goji) and Garcinia mangostana (mangosteen) on CYP1A2 and CYP3A4 activity using human cDNA‐expressed P450 enzymes and human hepatocytes. Açai and mangosteen significantly inhibited cDNA‐expressed CYP‐1A2 and ‐3A4 activities with IC50 values between 0.341% and 1.387% (v/v). In HepG2 cells, CYP‐1A2 and ‐3A4 activities were significantly inhibited after a four hour treatment with all juices, suggestive of direct effects on P450 protein. In contrast, CYP1A2 activity was maximally induced after a 10 h treatment, while CYP3A4 activity was inhibited after a 24 h treatment, suggesting effects at gene expression level. In primary human hepatocytes, CYP‐1A2 and ‐3A4 activities were maximally inhibited by açai and mangosteen. Our results indicate possible drug interactions with noni, açai, goji and mangosteen. USDA‐CREES (2004‐34135‐15182), NIH/NCMHD (P20MD000173‐06).
A mechanism linking lipid metabolism and longevity
Authors: Titorenko Vladimir I , Goldberg Alexander , Gregg Christopher , Boukh‐Viner Tatiana , Kyryakov Pavlo , Bourque Simon , Beach Adam , Burstein Michelle , Richard Vincent , Rampersad Sonia , Milijevic Svetlana
Published: June 2021

We use the yeast Saccharomyces cerevisiae as a model to study the mechanisms linking lipid metabolism and longevity. Yeast aging can be slowed down by calorie restriction (CR), a low‐calorie diet that extends life span and delays age‐related disorders in a wide spectrum of organisms. We assessed the effect of CR and numerous mutations extending yeast life span on the spatiotemporal dynamics of the proteomes and lipidomes of organelles involved in lipid metabolism. These organelles include the endoplasmic reticulum (ER), peroxisomes and lipid bodies. Our findings imply that a calorie‐rich diet suppresses peroxisomal oxidation of free fatty acids (FFA) that originate from neutral lipids synthesized in the ER and deposited within lipid bodies. The resulting accumulation of FFA initiates several negative feedback loops regulating the metabolism of neutral lipids, ultimately leading to the accumulation of diacylglycerol (DAG). The buildup of FFA promotes necrotic cell death, whereas the accumulation of DAG hampers a stress response‐related signal transduction network. Implementing our knowledge, we identified five groups of novel anti‐aging small molecules that greatly extend yeast longevity by remodelling lipid metabolism in the ER, peroxisomes and lipid bodies and by promoting "mitohormesis" through the activation of a distinct set of stress response‐related processes in mitochondria.
A novel approach for investigating glycan involvement in embryonic cellular interactions
Authors: Le Christine , Oppenheimer Steven B.
Published: June 2021

We use the NIH sea urchin embryo model to investigate molecular mechanisms of cellular interactions. Twenty‐four hour Strongylocentrotus purpuratus embryos were incubated with 14 different sugars in low calcium seawater (that speeds molecular entry into the living embryo blastocoel) using a microplate assay. Three experiments with 10 replicate wells, and hundreds of embryos for each sugar concentration and controls without sugar showed that the most effective sugars were alpha cyclodextrin, fructose, and maltotriose in inhibiting archenteron elongation/attachment at 3 mM concentration. Cyclodextrin and maltotriose are polymers of glucose monosaccharide that was a less effective inhibitor. In all cases, unpaired t‐tests revealed p values of less than 0.05, when comparing archenteron attachment versus no attachment in the sugar samples vs controls. In these experiments, sugars were tested at concentrations of 0.6 mM ‐ 330 mM. In new experiments lower sugar concentrations are being tested to more definitively identify those sugars that are most effective at the lowest concentrations. The working hypothesis is that specific sugars enter the embryo blastocoel and bind to receptors involved in the cellular interaction. Information on lowest effective sugar concentrations should help pin down those sugars that might be more likely to be involved in the cellular interaction under study. The use of sugars, low calcium sea water and a quantitative microplate assay, that easily identifies specific embryo morphologies, is a novel approach for helping to elucidate the nature of glycan‐involved cellular interactions in many different systems (supported by NIH NIGMS SCORE (S0648680), RISE, MARC, the Joseph Drown Foundation and the Sidney Stern Memorial Trust).
Using Next Generation Solexa Sequencing to Identify Genes that Regulate Stem Cell Proliferation in the Caenorhabditis elegans Germline
Authors: Jones Vovanti , Fox Paul , Mardis Elaine , Schedl Tim
Published: June 2021

Multicellular organisms depend on cell‐cell interactions to coordinate their development. In C. elegans, germline stem cell fate depends on the activity of the Notch receptor homolog, glp‐1. The loss of glp‐1 function prevents germ cell proliferation and produces sterile hermaphrodites. To identify new genes that regulate germline proliferation, suppressors of the temperature sensitive allele glp‐1(q231) have been isolated. These mutants, sog (suppressor of glp‐1), rescue sterility of q231 at 20°C. The sog‐1 gene was previously mapped to a 2 Mbp region on chrI. To [LL1]identify the gene responsible for the sog‐1 phenotype, we used Solexa sequencing to re‐sequence the entire genome of two independently isolated sog‐1 mutants. In the critical region, we identified 62 SNPs between the two strains and the reference genome. We performed RNAi screens of candidate genes to test whether they phenocopy sog‐1 suppression of glp‐1. RNAi screens of pbrm‐1 displayed an embryonic lethal phenotype for all strains, while unc‐14 and F57B10.3 potentially phenocopy the germline suppression of q231 by sog‐1. [LL2]Screens of other potential candidates were negative. These results may help identify new genes in germline proliferation and determine if Solexa sequencing will be useful in identifying genes defined by mutation.

This work was funded by NIH/HIGMS MARC U*STAR T34 08663 National Research Service Award to UMBC, HHMI and BioMedRAP.
Understanding the effects of chronic cadmium exposure on breast cancer
Authors: Vegas Esera , Louie Maggie C.
Published: June 2021

Cadmium is a heavy metal that is found in the environment and enters our body through either dietary sources or cigarette smoke. Heavy metals, like cadmium tend to bioaccumulate and induce toxicities by interacting with organic compounds inside the cell. Recent studies have suggested that cadmium may play a role in breast cancer by activating the estrogen receptor (ER) (Antila1996, Stoica et al. 2000, Martin et al. 2003, Johnson et al. 2003). Data from our lab also suggest that acute exposure of cadmium promotes ER‐dependent cell proliferation and induced ER target gene expression (i.e. cyclin D1, Cathepsin D, and c‐myc). However the effects of chronic cadmium exposure are unclear and require further investigation. The current focus of this project is to understand how chronic exposure of cadmium affects breast cancer cell growth. To accomplish this, we have established a cadmium‐breast cancer cell line (MCF‐7 Cd) by maintaining parental ER+ MCF‐7 cells in 10‐7 M cadmium for over 3 months. By comparing the growth and gene expression patterns of MCF7‐Cd and MCF7 cells, we hope to establish an understanding of how chronic cadmium exposure may contribute to the development of breast cancer.

This project is supported by NIH R15 CA121983‐01A1
Recent Advances in Imaging of the Lung
Authors: Singh Baljit
Published: June 2021

Lung has a complex cellular, physical and physiological architecture. To fully appreciate the normal and the altered cellular and physiological phenotype of the lung, it is critical to acquire cell‐specific and quantifiable information in intact lungs or isolated lung cells using relevant imaging methods. The integration of sound principles of morphometry with advanced light and ultrastructural imaging systems has provided quantification of cellular and molecular alterations in the lung. Recent use of intravital imaging has provided novel data on the interactions of leukocytes with lung microvascular endothelium and the responses of microvascular endothelium. Synchrotron radiation is also emerging as a tool to image changes in the living lung. This talk will provide an overview of the established and newly emerging imaging methods and their applications to the lung.
Investigation of Ppt1 and other lysosomal storage disease genes during Drosophila neurogenesis
Authors: Blanchette Cassandra , Chu‐LaGraff Quynh
Published: June 2021

Infantile Neuronal Ceroid Lipofuscinoses (INCL) is a lysosomal storage disease caused by a defect in the Palmitoyl Protein Thioesterase 1 (PPT1) protein. Studies showed that the Drosophila ortholog, Ppt1, is essential for proper embryonic neurogenesis. Without Ppt1, flies exhibit defects in neuronal precursor cell fates, axon misrouting and fasciculation. This study extends the analysis by examining the role of other lysosomal storage disease genes during Drosophila neurogenesis, namely Palmitoyl Protein Thioesterase 2, Blue Cheese, Spinster, and Cathepsin D. Additionally, flies carrying mutations in these genes were individually crossed into the Ppt1 knockout background in order to investigate whether they genetically interact with Ppt1. Results from these experiments with regards to cell fate specification, axon guidance and fasciculation will be presented.
Phosphorylation and protein expression and dynamics at synapses in the central nervous system
Authors: Trinidad Jonathan C , Thalhammer Agnes , Schoepfer Ralf , Burlingame Alma L
Published: June 2021

The neurotransmitter glutamate is detected by ligand‐gated ion channels at synapses in the brain. These ion channels reside within a complex protein network known as the postsynaptic density (PSD), which contains kinases/phosphatases, scaffolding molecules, and structural molecules. Activation of these ion channels results in cellular excitation and initiates signal‐transduction networks regulating PSD structure.

We used mass spectrometry to identify 1700 proteins and 1500 sites of phosphorylation from PSD preparations. These were then quantified with stable isotope labeling using iTRAQ. We examined differential protein and phosphorylation patterns across synapses isolated from the cortex, midbrain, cerebellum, and hippocampus. NMDA‐type glutamate receptors had higher levels of phosphorylation compared to AMPA‐type receptors. We also examined temporal dynamics of synaptic changes resulting from chemically induced seizures. We observed multiple patterns of phosphorylation dynamics, presumably mediated by distinct kinase/phosphatase pathways.

Funding by the Wellcome Trust and BBSRC (to RS) and NIH grant RR 01614 (to ALB).
Structural basis of filamin A‐filGAP interaction and its impairment in congenital anomalies associated with filamin A mutations
Authors: Nakamura Fumihiko , Heikkinen Outi , Pentikainen Olli T. , Osborn Teresia M. , Kasza Karen E. , Weitz David A. , Kupiainen Olga , Permi Perttu , Kilpelainen Ilkka , Ylanne Jari , Hartwig John H. , Stossel Thomas P.
Published: June 2021

Filamin A (FLNa) is an essential cellular protein that interacts with numerous binding partners and controls architecture and mechanics of the actin cytoskeleton. FLNa mutations lead to developmental anomalies in humans. We report here on the structural details of the interface between the C and D β‐strands of the 23rd Ig repeat of FLNa and the C‐terminal 32 residues of FilGAP, a Rac‐specific GTPase‐activating protein and regulator of cell polarity and movement. NMR structure and interaction analysis and in silico modeling informed a mutagenesis strategy confirming the binding interactions and providing specific reagents for functional analysis of FLNa‐FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa‐FilGAP interaction perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc, explaining, at least in part, FLNa specific phenotypes in Flna‐null mice and patients with FLNa mutations. Tight binding requires dimerization of both partners and alignment of the binding surfaces by a flexible hinge on FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction. Our findings represent the first functional validation of FLNa‐partner interactions in cell physiology and human disease using specific reagents informed by structural analysis.
Domain requirements for the dynamic targeting of p130Cas to cell/ECM adhesions
Authors: Donato Dominique M. , Ryzhova Larisa , Meenderink Leslie M. , Kaverina Irina , Hanks Steven K.
Published: June 2021

P130Cas is a major Src substrate in cell‐ECM adhesions implicated in cell motility. The objective of this study was to measure the dynamics of p130Cas localization to adhesions, while also assessing domain requirements for this targeting. Variants of YFP‐tagged p130Cas were stably expressed in p130Cas‐null fibroblasts. In addition to full‐length p130Cas, deletion mutants lacking either the SH3 domain or the highly conserved "Cas‐family C‐terminal homology" (CCH) domain were analyzed. Total internal reflection fluorescence microscopy of live cells revealed that p130Cas localizes to adhesions with a kinetic profile similar to paxillin ‐ a standard marker for monitoring adhesion dynamics. Variants lacking either the SH3 or CCH domain localized poorly to adhesions, while deletion of both domains completely abrogated the localization. The CCH domain was also found to be sufficient for adhesion targeting. Deletion of the CCH domain was further linked to defects in p130Cas tyrosine phosphorylation and wound healing cell migration. Thus both the SH3 and CCH domains play important roles in p130Cas adhesion site targeting and this localization is critical for p130Cas signaling leading to cell motility.
The novel membrane protein SccA affects cell wall integrity in Aspergillus nidulans
Authors: Musgrove John , Ogburn Erinn , Sartain Caroline , Jackson‐Hayes Loretta , Hill Terry , Loprete Darlene
Published: June 2021

This research describes a novel gene designated SccA, which affects cell wall integrity in the filamentous fungus Aspergillus nidulans. Plasmid‐borne extra copies of SccA can suppress the calC2 mutation in the A. nidulans orthologue of protein kinase C (PkcA), which results in hypersensitivity to the chitin‐binding agent Calcofluor White (CFW). In filamentous fungi, as in yeasts, hypersensitivity to CFW correlates with defects in cell wall integrity. The hypothetical translated product of SccA is an ST‐rich 271‐aa protein (42% S/T) of 27.4 kDa mass (unprocessed), with a cleavable N‐terminal ER‐targeting domain and a probable internal membrane anchor. No known homologies have been identified in the sequence databases. A SccA‐GFP hybrid localizes to the plasma membrane of vegetative hyphae and is especially enriched in septa. When SccA is placed under the control of the regulatable AlcA promoter, vegetative growth is normal under both high expression and low expression conditions (growth on glycerol or glucose containing medium, respectively), but asexual sporulation is suppressed during growth on glycerol. Taking into consideration the protein's cell surface location and its influence on the function of PkcA, we hypothesize that SccA plays a role in signal transduction as part of a cell wall integrity pathway.
Epigenetic control of RGS5 expression following femoral artery injury in mice
Authors: Gu Steven , Al‐Sabeq Basil , Chis Roxy , Heximer Scott
Published: June 2021

RGS5 is involved in blood pressure regulation and tumor vascularization. Its expression is dynamic varying with age and disease, however, the specific pathways of regulation are unknown. Thus, the objective of this study is to identify the key modulators that regulate RGS5 expression. To assess the activity of the RGS5 promoter, which like other VSMC genes contains degenerate CArG boxes [CC(A/T)6GG], we generated a construct with luciferase under the control of the RGS5 promoter. This construct is 3‐fold more active in the presence of myocardin than with YFP control. Previous work in mouse carotid arteries show that degenerate CArGs are important in the post‐injury down‐regulation of several genes. In a model of femoral artery injury, we show that RGS5 expression decreases and remains so for up to 4 weeks. To understand pathways involved in this loss of expression, we looked at possible epigenetic changes. Using pyro‐sequencing and clonal analysis, we show that there is hypermethylation at 3 CpG sites in injured arteries compared to control. Interestingly, one site is within a degenerate CArG box suggesting that methylation could impede SRF binding and transcription activation. Thus, DNA methylation at sites important for promoter activity can be an effective way to silence RGS5 expression. This work was supported by the Canadian Institute of Health Research and the Heart and Stroke Foundation of Ontario.
Neurogenesis permissive chromatin in neural crest cells as novel epigenetic marks during mouse neural tube closure
Authors: Ichi Shunsuke , Shen Yueh‐Wei , Nakazaki Hiromichi , Sapru Sidanth , Mania‐Farnell Barbara , Tomita Tadanori , McLone David G , Mayanil Chandra S. K
Published: June 2021

Although we have shown recently that Pax3 regulates Hes1 and Ngn2 by directly interacting with the cis‐regulatory regions within their promoters during caudal neural tube closure (Dev. Biol. 316, 510‐523, 2008), nothing is known about the chromatin remodeling at these candidate Pax3 target promoters that lead to premature neurogenesis. Recently Lan et al., (Nature 449, 689‐694, 2007) showed that histone H3 lysine27 (H3K27) demethylase regulates animal posterior development. Here we try to elucidate how chromatin remodeling at these Pax3 target gene promoters may affect premature neurogenesis in Pax3 mutant (Splotch) mouse embryo. By using neural tube explant culture from E9.5 mouse embryos (wild type and Splotch homozygous) and allowing the migratory neural crest cells to differentiate, we observed that H3K4me2 and H3K27me2 methylation to be significantly increased in Splotch mouse embryos. Additionally, chromatin immunoprecipitation (ChIP) assays using H3K4me2, H3K27me2, RNA polymerase II, UTX and JMJD3 antibodies revealed novel chromatin marks on Hes1 and Ngn2 promoters during premature neurogenesis. Thus these studies identified novel "neurogenesis permissive chromatin" in neural crest cells during neural tube closure.
Relaxin activation of PPAR[gamma] is ligand independent
Authors: Singh Sudhir , Bennett Robert G
Published: June 2021

Peroxisome proliferator‐activated receptor gamma (PPARγ) has many antifibrotic effects similar to those of relaxin (Rlx), raising the possibility that Rlx's antifibrotic effects may involve activation of the PPARγ pathway. To test this possibility, the Rlx effect on PPAR activity was assessed using a luciferase reporter vector fused to the PPAR response element (PPRE). Rlx increased the PPRE activity in a dose dependent manner. The PPARγ agonists increased the PPRE activity. The combined effect of Rlx and PPARγ agonists on PPRE activity was more than additive. Rlx increased the expression of PPARγ target genes, and when Rlx was combined with PPARγ agonists the effect was synergistic. PPARγ knockdown reduced the Rlx effect on PPRE activity, suggesting that part of Rlx effect is mediated through PPARγ. A PPARγ ligand blocker reduced PPRE activation and PPARγ target genes expression in response to PPARγ agonists but not Rlx, suggesting that Rlx stimulates ligand‐independent PPAR activation. Moreover, Rlx further increased the PPRE activity in cells overexpressing PPARγ without exogenous PPARγ ligands. PPARγ has diverse biological implications in broad range of diseases and the synergistic effects of PPARγ agonist with Rlx and better understanding of the signaling pathway will help to develop new therapies targeting PPARγ.

Funding: NIAAA

Department of Veterans Affairs
Characterization of human epidermal neural crest stem cell (hEPI‐NCSC) candidate cells
Authors: Gillinder Kevin , Rajan Neil , Loughney Andrew , Murdoch Alison , Sieber‐Blum Maya
Published: June 2021

We provide the first comprehensive characterization of hEPI‐NCSC candidate cells. Mouse EPI‐NCSC are multipotent remnants of the embryonic neural crest in a postnatal location, the bulge of hair follicles. Here, the bulge region of human anagen hair follicles was micro‐dissected from pubic skin that was procured with informed consent after elective Caesarean section. Emigrating cells were multipotent, as progeny of clone‐forming cells differentiated into smooth muscle cells, neuroblasts, and bone/cartilage cells. Gene expression profiling using the Illumina platform suggested that 17/18 mouse neural crest molecular signature genes identifiable in humans were also highly abundant in hEPI‐NCSC (PCBP4, MSX2, H1FX, PYGO2, THOP1, MYO10, ETS1, PYGO2, ADAM12, BEX1, VDAC1, CALR, CRYAB, PEG10 AGPAT6, CRMP1, UBE4B). Expression of eight of those genes, nestin and the neural crest marker SOX10, was validated by immunocytochemistry. Additional key neural crest‐characteristic genes included WNT1, SNAI2, MSX1, PAX9 and TNFRSF1B (p75NTR). The iPS genes MYC, KLF4, LIN28, and SOX2 were present at highly significant levels. Taken together, the data suggest that hEPI‐NCSC are multipotent cells of neural crest origin.