bluuebunny/crossref_metadata_2025 huggingface dataset:
Optogenetic Control of EphB Signaling in Dendrites
Authors: Locke Clifford , Yang Qingfen , Taslimi Amir , Tucker Chandra , Wu Yi , Yu Ji
Published: June 2021

EphrinB receptors (EphBs) are receptor tyrosine kinases that promote dendritic spine morphogenesis by altering local actin polymerization at axo‐dendritic contacts. Soluble ligands have been used with neuronal cultures to activate EphBs, though local stimulation is more desirable to model local contact and study downstream signaling dynamics. Since EphB signaling requires receptor clustering, we designed an optogenetic tool, named optoEphB, based on an optimized mutant of the plant protein cryptochrome 2 (Cry2olig, residues 1‐498 with E490G mutation), which shows blue light‐induced clustering. OptoEphB consists of the EphB2 cytoplasmic domain fused to Cry2olig and mCherry at the C‐terminus with myristoylation at the N‐terminus. Global and focal blue light illumination of HEK293 cells expressing optoEphB showed reversible optoEphB clustering and retraction of membrane protrusions, indicative of EphB2 signaling. In hippocampal neurons, focal illumination of dendrites produced transient, dynamic protrusions that resembled dendritic filopodia. We observed recruitment of the pleckstrin homology domain of Akt in illuminated regions, suggesting phosphoinositide 3‐kinase (PI3K) activation in this process. Our results demonstrate that optoEphB locally activates EphB signaling and thus represents a novel optogenetic method of Eph receptor activation. Further experiments will determine PI3K‐dependence of filopodial protrusions and use biosensors to measure the spatiotemporal dynamics of downstream Rho GTPase activity, implicated in EphB‐driven spine morphogenesis. These results will bring new insights into current models of dendritic spine development.
PCTAIRE Kinase 3/Cyclin Dependent Kinase 18 Is Activated through Association with Cyclin A and/or Phosphorylation by Protein Kinase A
Authors: Matsuda Shinya , Kominato Kyohei , Tsuji Akihiko , Yuasa Keizo
Published: June 2021

PCTAIRE kinase 3 (PCTK3)/cyclin dependent kinase 18 (CDK18) is a member of the CDK family. Although the PCTK3 is relatively more abundant in post‐mitotic cells, their enzymatic characteristic and physiological function have remained unknown because of unidentified its activator(s). Here, we describe the mechanisms of catalytic activation of PCTK3 by cyclin A2 and cAMP‐dependent protein kinase (PKA). Using a pull‐down experiment with HEK293T cells, cyclin A2 and cyclin E1 were identified as proteins that interacted with PCTK3. An in vitro kinase assay using retinoblastoma protein as the substrate showed that PCTK3 was specifically activated by cyclin A2 but not by cyclin E1. Furthermore, immunocytochemistry analysis showed that PCTK3 colocalized with cyclin A2 in the cytoplasm and regulated cyclin A2 stability. Amino acid sequence analysis revealed that PCTK3 contained four putative PKA phosphorylation sites. In vitro and in vivo kinase assays showed that PCTK3 was phosphorylated by PKA at Ser12, Ser66, and Ser109, and that PCTK3 activity significantly increased via phosphorylation at Ser12by PKA, even in the absence of cyclin A2. In the presence of cyclin A2, PCTK3 activity was comparable to CDK2 activity. We also found that PCTK3 knockdown in HEK293T cells induced morphological changes and polymerized actin accumulation in peripheral areas. In addition, we observed that cofilin phosphorylation and cell motility in PCTK3 knockdown HEK293T cells was markedly increased as compared with that in control cells. Taken together, our results provide the first evidence for the mechanisms of catalytic activation of PCTK3 by cyclin A2 and PKA, and a physiological function of PCTK3.
Defining the Tetramodular Dimer Structure of Laforin in Glycogen Metabolism and Lafora Disease
Authors: Brewer M Kathryn , Raththagala Madushi , Parker Matthew , Hellman Lance , Turner Ben , Auger Kyle , Chakravarthy Srinivas , Sanz Pascual , Vander Kooi Craig , Gentry Matthew
Published: June 2021

Glycogen is the major form of carbohydrate storage in mammals and a dynamic participant in brain energetics. Slight aberrations in glycogen metabolism induce neuronal apoptosis; thus glycogen must be highly regulated. Lafora disease (LD) is a recessive neurodegenerative disorder that manifests as a single seizure during adolescence followed by progressive central nervous system degeneration and death within ten years. Mutations in the glycogen phosphatase laforin lead to the accumulation of neurotoxic, hyperphosphorylated glycogen known as Lafora bodies, which cause LD. Our lab recently determined the crystal structure of dimeric laforin. Using a variety of biochemical and biophysical methods including size exclusion chromatography, analytical ultracentrifugation, and small angle X‐ray scattering we have demonstrated that laforin exists as a dimer in solution. We have probed the effects of mechanistic and LD‐associated mutations using a yeast two‐hybrid system and various in vitro techniques, and we have demonstrated that laforin dimerization plays a unique and critical role in glycogen binding and dephosphorylation. This work begins to define the mutation‐specific mechanisms of LD and sheds new light on the regulation of glycogen metabolism.
Erbin‐mediated Regulation of Colon Cancer
Authors: Stevens Payton , Wen Yang‐An , Li Xin , Gao Tianyan
Published: June 2021

The LAP (leucine‐rich repeat (LRR) and PDZ domain) family protein, ERBIN, is a basolateral‐localized scaffolding protein. Here, we have discovered that Erbin plays an important role in suppressing epithelial‐mesenchymal transition (EMT) of colorectal cancer (CRC) cells by controlling cell polarity as well as inhibiting CRC migration and metastasis. The proper establishment of epithelial polarity allows cells to sense and respond to signals that arise from the microenvironment in a spatiotemporally controlled manner. Increasing evidence has suggested that the disruption of this cellular polarity promotes the malignant progression of cancer cells. Here, we show that silencing Erbin results in the disruption of cell polarity in 3D cultures and mislocalization of EGF receptors in multiple CRC cell lines. As a consequence, activation of both Akt and ERK downstream of EGFR is enhanced as determined by Western blotting analysis. In addition to aberrant signaling, knockdown of Erbin induces an EMT‐like morphology and promotes migration in CRC cells, as observed using Transwell migration assays and live single‐cell microscopy. Similar to other LRR family members, the scaffolding function of Erbin could provide the required protein‐protein interactions to allow for spatial‐temporal control necessary to suppress oncogenic Akt and RAS/RAF signaling. Further supporting the role of Erbin as a tumor suppressor in CRC, the analysis of Erbin mRNA expression in two large sets of CRC patient samples revealed that Erbin is significantly downregulated in cancer specimens when compared to normal controls. Taken together, these data provide evidence for a novel role of Erbin in regulating colorectal cancer progression.
Spatiotemporal Analysis of the Role that Protein Kinase A Phosphorylation Plays In Gap Junction Assembly
Authors: Sosinsky Gina , Cavin Gabriel , Solan Joell , Kunkel Maya , Newton Alexandra , Lampe Paul , Cone Angela
Published: June 2021

Gap junction (GJ) trafficking and intercellular communication are regulated through interactions of the GJ protein, connexin, with several kinases culminating in phosphorylation on specific serines or tyrosines in the connexin C‐terminus. Connexin43 (Cx43) is an important, ubiquitous and highly phosphorylated isoform present in many vertebrate organs. Activation of Protein Kinase A (PKA) causes increased trafficking of Cx43 to the plasma membrane hypothesized to occur by direct phosphorylation on S364/S365. Here, we imaged Cx43 tagged with a PKA phosphorylation FRET biosensor (Cx43‐AKAR3). A strong Cx43‐AKAR3 FRET ratio signal was found in the Golgi apparatus and not in the GJs. Phosphorylation in the Golgi increased after stimulation with forskolin and IBMX, potent stimulators of PKA activity. However, we found no overlap of the PKA FRET ratio signal with antibody labeling for phospho‐S365 before or after PKA stimulation. Inhibition of PKA with H89 decreased but does not abrogate the effect of forskolin/IBMX stimulation. However, treatment with this inhibitor after stimulation did not result in rapid dephosphorylation. Previously published work indicated that direct PKA phosphorylation of Cx43 is low, especially as compared with PKC or MAPK. Thus, PKA activation and localization was not consistent with direct S365 phosphorylation. Most likely, PKA phosphorylates another kinase that increases Cx43 trafficking to the plasma membrane and GJ assembly.

Funding: GM072881 (GS), GM043154 & DK054441 (AN), GM055632 (PD) & GM103412 (NCMIR)
Expression modulation of different S6Ks isoforms in prostate cancer cell lines leads to alterations in proliferation, migration and chemotherapy resistance.
Authors: Freitas Lidia , Pavan Isadora , Amaral Camila , Bajgelman Marcio , Simabuco Fernando
Published: June 2021

The mTOR pathway is stimulated by high amounts of amino acids, ATP, and insulin and several studies have shown that mTOR is active in various biochemical processes involved in cell growth and metabolism. The S6Ks proteins have shown an important role in the mTOR signaling, acting as effectors of this pathway. Many works have demonstrated the participation of the mTOR pathway in cancer, but there is currently a demand to better characterize the S6Ks isoforms in this disease. The present work aimed to study the roles of p70‐S6K1, p85‐S6K1 and p54‐S6K2 isoforms in cancer, using DU145 and PC3 prostate cancer cell lines. Our results indicate that p85‐S6K1 isoform enhances proliferation and colony formation of DU145 and PC3 cells. Besides, overexpression of both S6K1 isoforms increased migration of PC3 cells in scratch assay. Moreover, all S6Ks isoforms when overexpressed increased docetaxel resistance of PC3 cells. Finally, knockdown experiments against S6K1 and S6K2 had complemented our data so far. In conclusion, we have identified different cellular effects of S6Ks isoforms in an in vitro model of prostate cancer.
Heart‐Specific Overexpression of FOXO Ameliorates Performance Decline through Enhanced UPS Processing in Aging Drosophila
Authors: Blice‐Baum A , Viswanathan M , Mullinax B , Zambon A , Kaushik G , Engler A , Bodmer R , Cammarato A
Published: June 2021

Heart performance declines with age. Reduced protein quality control due to decreased function of the ubiquitin/proteasome system (UPS) and autophagy is a likely contributor to age‐associated cardiac dysfunction. The transcription factor FOXO has been shown to be involved in the regulation of genes involved in both as well as a host of other processes. Here, the effect of overexpression (OE) of dFOXO was investigated in Drosophila melanogaster, a rapidly aging model in which the tissue‐specific UAS‐GAL4 expression system is often exploited. This system permitted different extents of heart‐restricted dFOXO OE. Excessive dFOXO doses proved fatal while mild levels appeared cardioprotective, ameliorating functional decline as determined by high‐speed video microscopy and motion analysis of aging hearts. OE of dFOXO in all muscle was previously shown to increase lifespan, likely caused by systemic expression of autophagy related proteins and reduced ubiquitin content. Similarly, in this study dFOXO‐mediated improvement in heart function with age was also accompanied by a significant decrease in ubiquitinated myocardial proteins as determined by quantitative western blot. However, microarray data suggested that this reduction was caused by increased expression of genes associated with UPS rather than autophagy, indicating FOXO may perform its function differently in the heart than in other striated muscles. FOXO gene candidates from the transcriptomic analysis will be probed to pinpoint how mild FOXO OE halts the natural decline in heart performance in Drosophila.
Investigation of Cysteamine as a Potential Antioxidant Supplement in TRX1 Yeast Mutants
Authors: Hendershot Allison , Mordan‐McCombs Sarah
Published: June 2021

In 1957, the free radical theory of aging proposed that cellular aging and death are the result of accumulating oxygen‐derived free radicals within the cell. These free radicals, also referred to as reactive oxidative species (ROS), accrue as a result of mitochondrial metabolism and can damage DNA, lipids, and proteins. Studies suggest that ROS‐related damage is associated with diseases such as Alzheimer's and cancer. Cells combat the detrimental effects of ROS by producing endogenous antioxidants, such as the thiol antioxidant glutathione and its associated enzymes, which reduce free radicals to a more stable state. This research seeks to determine the relationship between endogenous and dietary antioxidants, using Saccharomyces cerevisiae as a model system. Here we investigate the role of the TRX1 protein and its involvement in the thioredoxin and glutaredoxin pathways, and the ability of the exogenous antioxidant, cysteamine, to interact with disrupted antioxidant signaling. We hypothesized that, due to the structural similarities between cysteamine and glutathione, cysteamine may be able to act as a surrogate in the glutaredoxin and thioredoxin pathways in the absence of functional glutathione. ΔTRX1 yeast strains were treated with cysteamine, and analyzed for growth, gene expression, and protein expression changes under oxidative stress from hydrogen peroxide. Results indicate alterations in endogenous antioxidant pathways in gene and protein expression levels with deletion of the TRX1 gene and subsequent treatment with cysteamine.

This work is supported by the Biology Department at Franklin College and by a grant from the Indiana Academy of Science.
Western Blot Identification of Dopamine and GABA Receptors in Gill of the Bivalve Crassostrea viginica
Authors: Mondelus Fabienne , Boisette Beatrix , Bess Fiana , Catapane Edward , Carroll Margaret
Published: June 2021

Gill lateral cells of the bivalve Crassostrea virginica are innervated by serotonin and dopamine (DA) nerves from their ganglia and regulate cilia beating rates. DA slows down cilia beating and serotonin speeds them up. GABA a neurotransmitter in vertebrates and many invertebrates rarely has been reported in bivalves. We showed GABA is present in C. virginica and GABA acts as a ganglionic neurotransmitter modulating gill lateral cell cilia activity. We used immunohistofluorescence to localize GABA receptors in ganglia and gill and identify DA receptors in gill lateral cells as D2‐like (D2DR). We hypothesize Western Blot (WB) would verify D2DR and GABA receptors in gill. For WB gill cell lysate was prepared by in NP‐40 detergent buffer containing protease inhibitor, followed by centrifuging to obtain supernatant with solubilized membrane proteins. Up to 30 µg of solubilized protein was subjected to SDS‐PAGE with 10% acrylamide gels and electroblotted onto nitrocellulose. D2DR and GABA receptor immunoreactivity was revealed by 1̊ antibodies followed by HRP‐conjugated 2̊ antibodies. WB showed 70 ‐ 75 kD bands corresponding to DA D2DR and GABA RA1‐6 receptors in gill. The present project allows us to confirm our previous studies showing the presence of DA and GABA and furthers the understanding of their physiological roles in C. virginica.
Temporal Selectivity of Melanocortin‐4 Receptor Agonist to Modulate Signaling by Increased Intracellular cAMP
Authors: Cooney Kimberly , Molden Brent , West Kirk , Baldini Giulia
Published: June 2021

Melanocortin‐4 Receptor (MC4R) is a G‐protein coupled receptor expressed in the brain where it functions to regulate food intake and energy expenditure. MC4R signals in response to its natural agonist, α‐MSH, by increasing intracellular cAMP. It is not known whether synthetic MC4R agonists exhibit functional selectivity to modulate the cAMP signal. Effects by α‐MSH and two synthetic agonists, Melanotan II (MT‐II) and THIQ, were monitored using Neuro‐2A cells expressing both endogenous MC4R and exogenous tagged HA‐MC4R‐GFP, which can be visualized directly by fluorescence microscopy. By using Fluorescence Resonance Energy Transfer (FRET) we determined that α‐MSH induces a cAMP signal that disappears rapidly, within 15 min after agonist withdrawal. Conversely, MT‐II and THIQ induce a more prolonged cAMP signal, lasting for at least 40 min after agonist withdrawal. Internalization rate of ligand‐free MC4R and MC4R in a complex with a‐MSH and MT‐II, monitored by Fluorescence Recovery after Photobleaching (FRAP) occurred with the same half‐life of ~2.5 min. Together, these data indicate that MT‐II can induce a cAMP signal even after being internalized. Exposure to a‐MSH for 4 hours increased the distribution of MC4R to the intracellular localization more efficiently than MT‐II and THIQ, however desensitization of the receptor was the same for all agonists. We conclude that MC4R agonists have temporal selectivity to modulate the duration of the cAMP signal after agonist withdrawal, but not to change the desensitization properties of the receptor.

NIH Grants R01‐DK080424 (to G. B.) and Intramural Funding Support from the UAMS College of Medicine Research Council
Phosphorylation‐dependent Association of PDE3A1 with SERCA2 and its Regulation of SERCA2 Activity in Human Myocardium
Authors: Ahmad Faiyaz , Shen Weixing , Vandeput Fabrice , Szabo‐Fresnais Nicolas , Krall Judith , Degerman Eva , Goetz Frank , Klussmann Enno , Movsesian Matthew , Manganiello Vincent
Published: June 2021

PDE3 regulates cAMP‐mediated signaling in the heart. Studies in mice showed that PDE3A is associated with SERCA2, PLB and AKAP18 in a signaling complex in the SR that regulates inotropic responses. Immunohistochemical studies demonstrate that PDE3A is co‐localized in Z‐bands of human cardiomyocytes with desmin, SERCA2, PLB and AKAP18. In SR fractions, cAMP or rPKAc increased PLB phosphorylation and SERCA2 activity; this was potentiated specifically by PDE3 inhibition. During S6 chromatography of solubilized SR membranes, PDE3 activity was recovered in high‐ and low‐molecular‐weight (HMW, LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, while LMW contained PDE3A1, PDE3A2 and PDE3A3; PDE3A1 was the principal PKA‐phosphorylated isoform. Phosphorylation by rPKAc increased cAMP‐hydrolytic activity, shifted PDE3A from LMW to HMW peaks and increased the co‐IP of PDE3A with SERCA2 and AKAP18. Phosphorylation of rPDE3A by rPKAc increased co‐IP of rSERCA2 and rAKAP18. S292/S293, a site unique to PDE3A1, was identified by alanine substitutions as the principal site regulating this interaction. Phosphorylation of PDE3A1 at this site thus promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates contractility by modulating phosphorylation‐dependent protein‐protein interactions, PLB phosphorylation and SERCA2 activity. Agents capable of blocking this interaction and increasing myocardial contractility may constitute a novel therapeutic approach for heart failure.
Apoptotic and Inflammatory Role of TNF‐α in Adult Rat Testis upon Ethylene Dimethane Sulfonate Treatment
Authors: Singhal Barkha , Mills Nathaniel
Published: June 2021

Tumor necrosis factor (TNF‐ α) is multifunctional cytokine secreted in mammalian testis by germ cells, Sertoli cells and to some extent by macrophage. TNF‐α regulates different cellular processes pertinent to spermatogenesis including steroidogenesis, germ cell apoptosis and inflammation. Inflammatory marker, IL‐1b is secreted by activated macrophages. The Fas/FasL apoptotic pathway is the current model for depletion of Leydig cells with ethylene dimethane sulfonate (EDS) treated adult rats. As TNF‐α belongs to the same cytokine family, we are investigating the role of TNF‐α in Leydig cell loss at 6, 15 and 24 hr post‐EDS. Lhr and Insl3, primarily Leydig cells markers declined by more than 85% while Hsd3b2, Leydig cell enzyme marker declined by 96% at 24 hr post‐EDS. Using TUNEL, we found 5‐fold increase in interstitial apoptotic cells at 24 hr post‐EDS. Next, genes associated with macrophage and inflammation was assayed. We observed a 2.2 fold increase in Cd163/Cd68 at 15 hr‐post EDS, suggesting an increase in testicular macrophages. Tnfa was increased 2.7‐fold at 15 hr after EDS and Il1b increased 3.3 fold, 24 hr post‐EDS. ELISA detection of TNF‐α protein in the testicular tissue showed no change in expression at 6, 15 and 24 hr post‐EDS. Immunohistochemistry for 3β‐HSD and CD163 comply with q‐PCR results. Since no change was observed in Fas and Fasl over the course of time and both the receptors CD120a and CD120b changes, we are currently studying TNF‐ α inhibitors.
The N‐Terminal Atypical Region Regulates Non‐Mitochondrial Localization of Novel Hexokinase Isoform
Authors: Kumar Sujeet , Parameswaran Sreejit , Sharma Rajendra
Published: June 2021

Post‐translational modifications (PTM) constitute the core dynamic signalling pathways in diverse physiological states. The PTMs render unique functionalization to proteins mediated by specific interactions and are often involved in modular interactions. Hexokinase, the first enzyme in the glucose utilization pathway, is an integral player in modulation of signalling pathways in diverse states. An integral feature of hexokinase functional regulation is binding to VDAC located in the outer‐mitochondrial membrane. The mitochondrial attachment of hexokinase is mediated by 15‐20 amino acid long hydrophobic domain located at N‐terminus of the enzyme.We observe that, in a novel isoform lacking this hydrophobic domain, the membranous interaction is modulated by virtue of PTMs. We cloned the full‐length isoform of hexokinase under CMV promoter in fusion with red fluorescent protein (RFP). Comparisons in parallel were carried out with constructs encoding the normal hexokinase variant fused to green fluorescent protein. The constructs were analyzed for localization by confocal analysis after transfection into HEK293 cells. The localization of these two isoforms showed a clearly distinct behavior. Moreover, the localization of the novel isoform was perturbed when the putative residues involved in the PTMs were mutated to alanine. The PTMs in the atypical region seems to provide an unique functional module. It is imperative to understand the mechanisms of such re‐functionalization in regulation of protein functions. Further insights into the role of myristoylation on the process of ATP generation, if any, and on regulation of glycolytic pathway should improve our understanding of previously un‐described functions.
Serial Iron, Zinc and Copper Status in Adults with Pulmonary Tuberculosis in the Country of Georgia
Authors: Frediani Jennifer , Tukvadze Nestani , Sanikidze Ekaterina , Kipiani Maia , Hebbar Gautam , Millson Erin , Ramakrishnan Usha , Tangpricha Vin , Blumberg Henry , Ziegler Thomas
Published: June 2021

Objective

To determine serial dietary intake and serum concentrations of immune‐related micronutrients in patients with TB disease.

Methods: Estimated mean daily intake of iron (Fe), zinc (Zn), and copper (Cu) was determined in 192 adults with pulmonary TB within 7 days of diagnosis and at 16 weeks using a validated instrument and NDS‐R. Serum levels of Fe, Zn and Cu were determined by inductively coupled argon plasma spectrometry at baseline and week 16.

Results: Mean subject age was 34 y and 64% were male. Serum Fe levels were below normal in 25% at baseline (24±20 umol/L), and rose significantly by week 16 [to 26±15 umol/L (P< 0.01)]. Mean dietary Fe intake at baseline and week 16 was adequate. Serum Zn levels were below normal in 35% at baseline (11.6±2.8 umol/L) and 26% at week 16 (12.2±3.2 umol/L). Mean dietary Zn intake at baseline was 11.7±5.0 mg/day (121% of RDA)] and decreased at wk 16 (to 10±3.5 mg/day;P=0.03). Serum Cu levels were normal at baseline (26.5±0.6 umol/L) and fell significantly by week 16 to 20.5±0.5 umol/L (P<0.01). Mean dietary Cu intake at baseline and week 16 was adequate.

Conclusion

A significant proportion of adults with newly diagnosed TB exhibited low Fe and Zn levels, while Cu levels decreased over time. Changes during treatment may reflect diet intake, decreased inflammation and endogenous anti‐microbial processes.
A Novel Cell‐Based Screening Assay to Assess the Effects of Diverse Protein Sources on Muscle Protein Signaling
Authors: Roeseler David , McGraw Nancy , Butteiger Dustie , Napawan Nida , Hall‐Porter Janine , Krul Elaine
Published: June 2021

Protein sources that build or maintain muscle mass are of great interest to athletes, individuals on weight loss regimens and aging adults. Optimizing muscle health benefits derived from high quality proteins such as soy, dairy and egg may identify improved ingredients for sports nutrition and human health. The mammalian target of rapamycin (mTOR) is the central molecular pathway in muscle protein synthesis and is activated by dietary amino acids, specifically leucine. We used a cell‐based screening assay to investigate mTOR activation after incubation with proteins from diverse sources. Intact or partially hydrolyzed proteins were treated with pepsin and pancreatin to simulate gut digestion (SGD). Soluble fractions of SGD proteins were applied (1mg/mL) to C2C12 myotubes for 30 min. C2C12 cell lysates were then collected and mTOR activation was quantified by immunoassay of phosphorylated‐70S6KThr389, a critical downstream molecule in the mTOR signaling pathway. A range of mTOR activation was observed in response to the various proteins. The mean response for conventionally processed soy proteins, dairy, beef, and fish proteins were similar. mTOR activation was significantly higher for soy protein than for egg or pea proteins or soy whey preparations. These results suggest that most high quality proteins stimulate the mTOR pathway to a similar degree. Our data also confirm that insulin and protein stimulate mTOR via separate and additive upstream pathways and that these pathways are rapamycin‐sensitive. Our findings show this in vitro system to be a useful tool for comparing different proteins and other ingredients for their ability to impact muscle protein synthesis.
Vitamin D Correlation with Testosterone Concentration in US Army Special Operations Personnel
Authors: Wentz Laurel , Berry‐Cabán Cristóbal , Eldred Jerad , Wu Qiang
Published: June 2021

Vitamin D has been positively correlated with testosterone in older men, but these hormonal relationships have not been examined in younger, active military personnel. Therefore, the purpose of this study was to identify significant correlations between vitamin D and testosterone concentrations in male Special Operations Soldiers. This retrospective analysis examined unique cases of serum vitamin D assessments ordered at Womack Army Medical Center, Fort Bragg, NC, from January 2012 – September 2013. Inclusion criteria were male Special Operations soldiers who had a total testosterone assessment within 21 days of vitamin D assessment, yielding 312 service member records. Mean serum vitamin D concentrations were 29.9±9.6 ng/ml (range 10‐63), with 52% of subjects in the deficient range of <30 ng/ml according to the Army Medical Department guidelines. In vitamin D deficient subjects, serum vitamin D concentrations were a significant positive predictor of total testosterone when controlling for age and body mass index.

Independent Variable
Testosterone in Vitamin D Deficient Men (n=135)

β
Ρ

Vitamin D
8.429
0.008

Age
‐2.604
0.183

Body Mass Index
‐1.569
0.678

Model R2
0.062

These data indicate that deficient vitamin D concentrations may inhibit testosterone production and potentially limit human performance. Operational stress of military training has been shown to suppress testosterone concentrations in healthy men, causing muscle catabolism and fatigue, symptoms also characteristic of vitamin D deficiency. Given that more than half of male soldiers had insufficient vitamin D status in a southern latitude, measuring vitamin D concentrations in collaboration with testosterone assessments may be warranted.

No funding was used to support this work.
Variability of Portion Sizes within Popular Italian Restaurant Chains
Authors: Nickle Melissa , Pehrsson Pamela
Published: June 2021

As part of the Affordable Health Care Act, Congress passed a national law requiring restaurants with 20 or more outlets to post calorie information on menus, allowing consumers to make informed decisions when eating away from home. Nutrition information per serving is available in restaurants and online; however, serving size variability can affect overall nutrient profiles and subsequently, the amount of nutrients consumed. The objective is to assess the variability of serving sizes of popular Italian foods within restaurant chains. The USDA's Nutrient Data Laboratory conducted a 2013 nationwide sampling of select foods for nutrient analysis from leading Italian restaurant chains; serving size data was collected. Samples of ravioli, chicken parmesan, lasagna, and 2 types of spaghetti (meat sauce and pomodoro sauce) were collected from 12 (Restaurant A) and 6 (Restaurant B) locations and composited using USDA NFNAP protocols. Relative standard deviation (RSD) and ranges were assessed. Serving sizes for ravioli ranged from 359g‐541g (7‐9 pieces) and 296‐486g (8‐10 pieces) in Restaurant A (Rest A) and Restaurant B (Rest B) respectively. Serving sizes for chicken parmesan w/out noodles were 155g‐375g from Rest A and 261g‐383g from Rest B. Lasagna portions ranged from 355g‐527g (Rest A) and 233g‐508g (Rest B). Serving sizes for Rest A spaghetti were 367g‐643g (w/ meat sauce) and 397g‐571g (w/ pomodoro sauce) and Rest B spaghetti, 465g‐583g (w/ meat sauce) and 354g‐688g (w/ pomodoro sauce). The lowest RSD within restaurant chain was 9.2% for spaghetti w/ meat sauce from Rest B; the highest was 23.9% for spaghetti w/ pomodoro sauce from Rest B. Results show wide variation of portion sizes within restaurant chains, which affect nutrient profiles. Restaurant nutrition guides should be used as approximations due to the variability in portion sizes.
Inhibitory Activities of Medicinal Herbs against Lipid Accumulation in 3T3‐L1 Adipocyte
Authors: Ji Hyeon‐Kyeong , Seo Ji Yeon , Kim Min Sun , Kang Su Jin , Jo Gwang Rae , Erdenebileg Zolzaya , Jang Jin Ho , Kim Jae‐Sik , Suh Hwa Jin , Kim Jong‐Sang
Published: June 2021

Obesity is one of the metabolic diseases and is featured by excessive fat accumulation in the body and energy imbalance. Obesity is associated with metabolic diseases such as type 2 diabetes, hypertension, and cardiac disease. Recently, the incidence of obesity keeps increasing due to high caloric western‐style diet and lack of exercise. In order to explore food ingredient(s) with antiobesity activity, twenty kinds of fruits, vegetables, and medicinal plants were subjected to preliminary screening in which inhibitory activity against digestive enzymes and TG accumulation in 3T3‐L1 cells were measured. Among the samples tested, the seed extract of Oenothera biennis L. was found to have the highest inhibitory activity against TG accumulation in differentiated 3T3‐L1 adipocyte and regulate factors associated with adipogenesis.(Supported by a grant (R0002736) from Regional Industry R&D Assistance Program (2013) of Ministry of Trade, Industry and Energy, Republic of Korea)
Effect of Ezetimibe on Low‐and High‐Density Lipoprotein Subclasses in Sitosterolemia
Authors: Othman Rgia , Myrie Semone , Mymin David , Merkens Louise , Roullet Jean‐Baptiste , Steiner Robert , Jones Peter
Published: June 2021

Sitosterolemia (STSL) is a rare sterol disease manifested by very high plasma phytosterols (PS) with normal to high total cholesterol (TC) levels and increased atherosclerosis risk. Low density lipoprotein (LDL), intermediate density lipoprotein (IDL) and very lowdensity lipoprotein (VLDL) cholesterols are proatherogenic while high density lipoprotein (HDL) cholesterol is antiatherogenic. Ezetimibe (EZ), a sterol absorption inhibitor, can reduce plasma PS and TC levels in STSL but its effect on lipoprotein subclasses has not been studied. We evaluated the effect of EZ on lipoprotein subclasses in STSL patients (pts, n=8) taken off EZ for 14 wks then placed on EZ (10 mg/d) for 14 wks. Serum total lipids and subfractions were measured enzymatically or with the Lipoprint system. Data (mean±SEM) were analyzed by paired t‐test. EZ reduced serum TC (‐13±4%, p=0.02), VLDL (‐24±4%, p=0.002) and total LDL (‐17±6%, p=0.03) levels vs off EZ. Reduced LDL values were due to decreased IDLB and C (‐22±7 and ‐21±8%, p<0.05), not to large, buoyant LDL subclasses (LDL1: ‐8±8%, p=0.23 and LDL2: +35±55%, p=0.17). LDL size did not change with EZ (275±0 vs 274±1 Å, p=0.18). EZ increased HDL levels (26±8%, p=0.008) due to increased intermediate (34±14%, p=0.02) and large (33±16%, p=0.06) HDL subclasses. These data suggest EZ can favorably affect LDL and HDL subfractions distribution, thus providing potential clinical benefit in STSL beyond reducing TC and PS accrual.

Funded by NIH and CIHR
Differential Effects of Sphingomyelin Derived from Milk and Egg on Response to a High‐fat Diet
Authors: Norris Gregory , Farrell Nicholas , Blesso Christopher
Published: June 2021

Sphingomyelin (SM) has been shown to inhibit lipid absorption and is found in high levels in animal‐derived foods such as egg and milk. Egg and milk SM differ in their fatty acid and sphingoid base composition, and therefore may have different biological effects on lipid metabolism. We evaluated the effects of egg and milk‐derived SM on cholesterol metabolism in C57BL/6J mice. Mice were fed a high fat diet (45% kcal from fat, 21% milk fat by weight) (CTL, n = 10) or the same diet modified to contain 0.25% (wt/wt) of milk SM (MSM, n = 10), or 0.25% (wt/wt) of egg SM (ESM, n = 10). After 4 weeks, MSM had gained significantly less weight than CTL. ESM had significantly more epididymal adipose than the other groups. MSM significantly reduced serum cholesterol compared to both CTL and ESM groups, and reduced serum endotoxin compared to CTL. ESM had significantly higher levels of serum cholesterol, high‐density lipoprotein cholesterol, free cholesterol, and phospholipid compared with the other two groups. However, ESM did not display a greater increase in serum cholesterol from baseline compared with CTL. Both SM groups had significantly lower serum non‐esterified fatty acids and greater serum alanine transaminase compared with the CTL group. No differences were observed between groups for blood glucose and serum adiponectin. Overall, the addition of MSM significantly blunted increases in weight, serum cholesterol, and endotoxin observed with a 4‐week high fat diet. Differences in structure of the sphingoid bases may account for the differential effects observed between SM groups. Further research is warranted to examine molecular mechanisms of action of SM on cholesterol metabolism, and to explain the elevation in liver enzymes with SM feeding. [Supported by the University of Connecticut].
Fusing an open reading frame on either side of pfu polymerase does not affect its activity
Authors: Rashid Mohammad , Nayeem Syed
Published: June 2021

Fusion protein constructs based on genetic engineering could be useful in obtaining efficient fusion enzymes with dual function that is also cost effective. However, fusing two long open reading frames may be cumbersome in terms of producing a functional hybrid enzyme having two distinct domains. Here, we have been trying to fuse a pfu DNA polymerase enzyme of 90 kDa with a pfu dUTPase enzyme of 26 kDa. Fusing the dUTPase on either amino‐ or carboxyl‐terminal of DNA polymerase does not affect the polymerase activity of the enzyme in crude as well as in purified version of the hybrid enzyme. However, dUTPase function is yet to be analyzed in the fusion protein that would be the ultimate goal of this project.
Structural and FunctionalAnalysesof Carbohydrate Binding Module Family 36ofXylanase from Paenibacillus Campinasensis
Authors: Chang Hao‐Ting , Wang Yu‐Sheng , Yang Kai‐Jay , Chen Yu‐Jen , Ko Chun‐Han , Tzou Der‐Lii M.
Published: June 2021

Endo‐β‐1, 4‐xylanase (XylX), consisting of catalytic module and non‐catalytic module, was isolated from Paenibacillus campinasensis BL21 from black liquor. XylX displays a great potential for xylan‐degrading applications in biotechnological processing with a wide range of pH and thermal endurance. The catalytic module serves to degrade polysaccharide molecules into small molecules whereas the non‐catalytic module has a sequence similarity to CBM family 36 (denote as PcCBM36) that promotes the association of the enzyme with the carbohydrate substrates. To uncover the structural and functional relationship, we applied various techniques to PcCBM36, including solution NMR, ITC, and CD spectroscopy. The molecular structure of PcCBM36 has been determined by solution NMR. As revealed by the chemical shifts perturbation, we demonstrated that Glu10, Glu12, Asn29 and Asp119 are involved in one of the Ca+2 binding sites and Tyr34, Asp110, Trp114, and Asp115 involved the other, associated with the carbohydrate interaction. Here we demonstrate that binding of PcCBM36 to its substrate, xylohexaose, is Ca+2‐dependent, consistent with previous observations. And the biological roles with respect to these two Ca+2 binding sites and the underlying specific binding mechanism of PcCBM36/xylohexaose are to be elucidated.
Role of Nuclear Heme Oxygenase‐1 in Bortezomib Induced Cell Death
Authors: Li Volti Giovanni , Tibullo Daniele , Barbagallo Ignazio , Vanella Luca , Di Raimondo Francesco
Published: June 2021

Bortezomib, the first‐in‐class proteasome inhibitor, has displayed significant antitumor activity in Multiple myeloma (MM). Heme oxygenase‐1 (HO‐1) is an Nrf2 transcription factor‐regulated gene that is commonly induced following oxidative stress and cellular injury, functioning to decrease oxidative stress and inflammatory responses, protecting against apoptosis. Furthermore, we showed under some experimental conditions, HO‐1 might translocate into the nuclear compartment where it may play non‐canonical protein functions. In the present research we evaluated the role of Bortezomib on HO‐1 expression and cellular compartmentalization and how these effects are related to chemoresistance in U266, a MM cell line. Our results showed that Bortezomib (100nM) induces HO‐1 via ER stress induction as measured by PERK, IRE1 and Bip protein expression. HO activity inhibition by SnMP (10 μM) did not reverse such effects. We also showed that under basal conditions, HO‐1 is mainly localized into nuclear compartment whereas Bortezomib treatment resulted in a significant increase of the protein in the cytoplasm. Inhibition of HO‐1 nuclear translocation by E64d significantly increases Bortezomib mediated cell death. We also showed that nuclear HO‐1 localization is associated with increased genomic instability as measured my micronuclei test, nucleoplasmic bridge and tetranucleated cell formation. Our data suggest that cellular compartmentalization of HO‐1, rather than its enzymatic activity, plays a critical role in Bortezomib mediated cell death and resistance.
Molecular Mechanism of Action of 5‐Azacytidine and its Resistance in MDA‐MB 435 Cells
Authors: DiNatale Anthony , Hellreich Jaclyn , O'Donnell Robert
Published: June 2021

DNA methyltransferase inhibitors such as 5‐Azacytidine (5‐Aza) have shown promise in cancer treatment because of their ability to allow re‐expression of tumor suppressor genes in cancer cells. Preliminary studies showed that high doses of 5‐Aza are clearly cytotoxic to MDA‐MB 435 tumor cells. However, we have begun treating the MDA‐MB 435 cells with a low dose so that the majority of the cells survive. A resistant cell line has been developed through continuous growth in the presence of 5‐Aza. Tests were done to determine if the resistance inhibited growth in a methylcellulose assay. The number of colonies decreased in the 5‐Aza resistant cell line compared to the control. Control 435 cells were found to have an average colony count of 969±332 but when 5‐Aza was added the colony count dropped to 464±31. In order to look at the mechanism of 5‐Aza and its resistance, quantitative reverse transcriptase PCR was done to measure the expression of various genes. Between the control cells and the control cells plus 5‐Aza there was a five‐fold decreased expression of Mammaglobin B, a member of the secretoglobin family associated with chemoresistance, while the other genes tested did not have any change (TP53, RB1, BCL‐2, Myc). When the same tests were done on the resistant cell line the colony counts showed little change, 287±129 and 357±46, respectively, nor was there a difference between the resistant and resistant plus 5‐Aza in terms of Mammaglobin B expression. However, the resistant and the resistant plus 5‐aza both showed a six‐fold decrease in Mammaglobin B expression compared to the control cells. Future experiments will investigate a phenotypic role for this gene in the resistance.
Binding of Calcineurin Homologous Protein Isoforms 1 and 2 for the Na/H+ Exchanger Isoform 1 Using Recombinant Fluorescent Proteins
Authors: Fray Emily , Wallert Mark , Provost Joseph
Published: June 2021

The sodium hydrogen exchanger isoform 1 (NHE1) is a transmembrane transport protein critical for cell motility and intracellular pH regulation. NHE1 is regulated by a variety of cofactors and binding partners, including calcineurin homologous protein (CHP). Isoform 1 of CHP (CHP1) is expressed ubiquitously and is required for basal NHE1 activity; however, isoform 2 (CHP2) expression is only detected in transformed cancer cells and has been implicated as a putative target for cancer therapies. We have expressed and purified recombinant‐affinity tagged c‐terminal tail of NHE, RFP‐CHP1 and GFP‐CHP2 and used these for in vitro capture assays. This work demonstrates the affinity of each CHP isoform with NHE1. In vivo, we have stably expressed full‐length GST‐tagged NHE1 in lung fibroblast cells (PS120). Using pull‐down assays in cells transiently transfected with CHP1 or CHP2 we further demonstrate the affinity of CHP1 and CHP2 with NHE1. Follow‐up experiments with altered NHE1 amino acids are used to define potential sites of interaction of CHP2 and NHE1. Together this data illustrates the different affinity of CHP2 for NHE1 binding and points to a potential regulatory site for NHE1 function.
Generation of Superoxide and Toxicity of Amino Alcohols to K562 and HepG2 Cancer Cells
Authors: Shastri Anuradha , Boylan Mallory , Spallholz Julian
Published: June 2021

Purpose:

In our previous studies we have established that some water soluble aliphatic alcohols redox cycle generating superoxide. In the current study, we used the lucigenin chemiluminescence (CL) assay to determine if the amino alcohols (AA); 3‐Amino‐1‐propanol (3A1P), 4‐Amino‐1‐butanol (4A1B), 5‐Amino‐1‐pentanol (5A1P) and 6‐Amino‐1‐hexanol (6A1H) redox cycled in the presence of reduced glutathione. Cytotoxicity of the AA was assessed against the Chronic Myeloid Leukemia K562 and the Hepatocellular Carcinoma HepG2 cell lines.

Methods:

Stock solutions (5 mg/mL) of the AA corresponding to 62.5, 125, 250, 500 μg of the hydroxyl moiety were added to the CL cocktail. CL was integrated over 30 sec intervals at 36°C (n=20).

K562 and HepG2 cells were treated and incubated for 24, 48 and 96 hours with concentrations of 220, 240, 260, 280, 300 μg of the AA. Light microscopy and MTT assay were used to measure cell viability. Dihydroethidium (DHE) was used to detect intracellular superoxide generation.

Results:

The CL assays indicated that 5A1P redox cycled most, followed by 4A1B in a dose dependent manner. For both cell lines, the MTT assay showed that compared to control, AA treated cells showed reduced cell viability in a dose dependent manner. At 300 µg, at 48 hours 6A1H produced maximum cell death K562 (∼94%) and HepG2 (∼90%). The DHE assay indicated generation of superoxide in the cell.

Conclusions

All of the AA tested were shown to generate superoxide in vitro as determined by the CL. Both light microscopy of cell morphology and MTT cell viability assay showed that all the AA caused cell death, presumed to be due to their ability to redox cycle generating reactive oxygen species as in the CL assay in the absence of cells. The DHE assay confirmed the intracellular superoxide generation in treated cells.
Creating Change in Scientific Institutions through Subversion, Revolution (Title IX) & Climate Change
Authors: Rolison Debra
Published: June 2021

The slow crawl at which research universities diversify their faculty is a national disgrace in that these universities recruit for students that reflect the face of America, yet are still not yet fully incorporating that pool of talent onto their faculty. Similar difficulties are apparent among the scientific staff of national/federal laboratories. But how can one person change the world of science? Subvert the standard operating procedure. Create a microclimate that shows―over time―how new patterns of operation and inclusiveness yield productive, innovative science. Use the scientific capital and street credentials accrued over time, thanks to the humane microclimate and research productivity of one's team, to challenge the status quo with reasoned and bold arguments for change. Remember the importance of uppity behavior and applying “tipping point” mechanisms to move beyond initial reactions of dismissal to―over time―accepted inevitability (such as greeted my audacious suggestion in March 2000 to withhold federal funds from non‐diversified chemistry departments through application of Title IX). Ask the leaders of our S&T institutions the following: how good can American science, engineering, mathematics, and technology (STEM) be when we are missing more than two‐thirds of the talent? Learn to demand that our world of science be one that truly relishes the talent innate to all of humanity for science and discovery.
Phosphoinositide Signaling in the Control of Membrane Dynamics and Interactions
Authors: De Camilli Pietro
Published: June 2021

Phosphoinositides, the phosphorylated metabolites of phosphatidyl inositol, are minor components of cell membranes, but play key regulatory roles in cell physiology. The differentially phosphorylated inositol groups of these phospholipids control interactions of the bilayer with cytosolic proteins, regulate intrinsic membrane proteins and function as precursors of intracellular second messengers. As different phosphoinositides are preferentially concentrated on different membranes, they also function as major determinants of membrane identity. The talk will focus on the metabolism of PI4P and PI(4,5)P2 in the plasma membrane and on the mechanisms that control their dephosphorylation on endocytic membranes. I will also discuss the newly discovered role of these two phosphoinositides in the control of direct contacts between the endoplasmic reticulum (ER) and the plasma membrane. In this context I will focus on contacts mediated by the Extended‐Synaptotagmins (E‐Syts). The E‐Syts are ER‐localized proteins which that mediate PI(4,5)P2‐dependent and Ca2+‐regulated interactions of the ER with the plasma membrane and whose putative function is to mediate glycerophospholipids exchange (but not fusion) between the two membranes (Giordano, Saheki et al. Cell. 153: 1494‐1509, 2013; Schauder et al. Nature. 510: 552‐555, 2014).