bluuebunny/crossref_metadata_2025 huggingface dataset:
Antioxidant Activity of Aster scaber Thunb. With Different Cooking Methods (LB409)
Authors: Kim Gun‐Hee , Choi Hye‐Young , Park Hee Sook
Published: June 2021

Aster scaber Thunb. is a perennial herb of the compositae family and grows wild in the dry mountain regions of Korea. Aster scaber Thunb. possess many biological activities owing to their potential anticancer, anti‐inflammatory and antioxidant properties. This study investigated the antioxidant effect of Aster scaber Thunb.by various cooking methods and times. The Aster scaber Thunb. cooked by blanching, pan‐frying (1, 3, 5 and 10min) and microwaving (0.5, 1, 2 and 3min). The 70% ethanol extract of Aster scaber Thunb. was performed with ultrasound for 40 min and evaporated. Aster scaber Thunb. evaluated antioxidant activity based on protein degradation by reactive oxygen species including hypochlorite ion (CLO), hydroxyl radical (OH), peroxyl radical (AAPH) and peroxynitrite (ONOO). Our results showed that blanching of Aster scaber Thunb. against hypochlorite ion (CLO) has the highest protective effect by 95.08% at 3min. Antioxidant activity of blanced Aster scaber Thunb. against hydroxyl radical (OH) showed high myoglobin protective effects by 24.30% at 1min. Pan‐frying of Aster scaber Thunb. was showed the highest antioxidant activity against peroxyl radical (AAPH) by 31.98% at 1min. In case of peroxynitrite (ONOO), microwaved Aster scaber Thunb. showed the highest antioxidant effect by 73.87% at 0.5 min. With these results, we identified that the antioxidant capacity of Aster scaber Thunb. was affected by cooking method and time condition. Thus, Aster scaber Thunb. would be a useful dietary food to predict physiological effects.

Grant Funding Source: This work was supported by a National
Hydrolytic properties of the four small intestinal mucosal α‐glucosidases on disaccharides with different linkages and compositions (LB433)
Authors: Lee Byung‐Hoo , Nichols Buford , Hamaker Bruce
Published: June 2021

The mucosal α‐glucosidases in the small intestine, consisting of maltase, glucoamylase, sucrase, and isomaltase, are the major enzymes that digest carbohydrates to monosaccharides (e.g., glucose and fructose). In this study, disaccharides (maltose isomers, sucrose isomers) with various possible α‐linkages and compositional types (between glucose‐glucose and glucose‐fructose) were hydrolyzed by each recombinant enzyme to determine their specific α‐hydrolytic kinetic properties. The individual α‐glucosidases had various digestion activities on the disaccharides, however maltase showed the most versatile α‐hydrolytic activity on them. The results indicate that the major hydrolysis activity differs based on linkages, structural conformations of disaccharides, and their composition. This study can be applied to establish a strategy for developing new carbohydrates for extended postprandial glucose response with low glycemic response profile. These findings also suggest that all of the mammalian mucosal α‐glucosidases are necessary relevant enzymes to be used for in vitro digestibility assays when applied to testing various digestible carbohydrates and to accurately characterize their hydrolysis properties.
Iron supplementation and iron deficiency anemia impact malaria pathogenesis (LB472)
Authors: Clark Martha , Goheen Morgan , Kasthuri Raj , Cerami Carla
Published: June 2021

Iron deficiency is the most widespread nutritional deficiencies in the developing world and iron is commonly administered compounds through mass distribution campaigns. However, recent evidence suggests that iron deficiency may protect against malaria and that iron supplementation may increase susceptibility to malaria, complicating recommendations for universal supplementation in malaria‐endemic regions. It has been previously hypothesized that iron deficiency inhibited malaria growth through iron deprivation, as is the case with other pathogens. However, our work has generated a novel hypothesis for the effect of iron deficiency and iron supplementation on parasite growth ‐ namely that changes in RBC physiology and RBC age population structure drive resistance of RBCs from IDA donors and increased susceptibility of RBCs from iron supplemented IDA donors to malaria Using an in vitro system with RBCs from donors with well‐defined physiological iron states, we have investigated the cellular mechanisms associated with malaria protection by iron‐deficiency anemia (IDA). We described multiple IDA impacts on parasites, including attenuated invasion of IDA red blood cells (RBCs) and reduced production of parasite progeny. We demonstrate that as iron deficient individuals increase their rate of erythropoiesis in response to iron supplementation, their RBCs becomes more susceptible to infection. Additionally, we show that the replacement of iron deficient RBCs with young normocytic iron‐replete RBCs increases susceptibility to P. falciparum infection. Our results provide a cautionary message to well‐intentioned public health workers who follow the current World Health Organization recommendation to routinely provide iron supplements to iron‐deficient children.

Grant Funding Source: National Institute of Child Health and Human Development under award number U01HD061235
A Preliminary Consumer Survey for Aesthetic Design and Development of a Mobile Health Application for DIY Diagnosis of 21 Common Acute Diseases (LB478)
Authors: Stewart Kennedy , Skibsted Michele , Federico Niksha , Federico Tessya , Federico Cybele , Esmaeli‐Azad Babak
Published: June 2021

The myriad shortcomings of the healthcare system are self‐evident. Issues ranging from lack of access, to high cost of services, to widely variable quality of care are endemic not only in the US but also worldwide. Efforts to address these issues have met with limited success. To effect real change, fundamental movement in current practice, technologies generated by a synergy of fresh ideas, emerging technologies, and “disruptive” integration and approaches is necessary. Key to widespread adoption will be a focus on the user rather than the devices and technical capabilities. Technology by itself is not transformative ‐ it requires framing and usability that provides value to individuals and groups of people.

We are currently developing a mobile health solution for self diagnosis of 21 indications as a solution for the Qualcomm Medical Tricorder X Prize Competition: team “SMART McCoy”. Medical testing generally evokes fear and discomfort, presenting significant barriers to be overcome before an instrument is adopted for regular at‐home use. A key element to our solution lies in the user experience and interface engaging people to medically test themselves. This unique capability is brought to bear on the challenge of making the Tricorder technical capabilities friendly, engaging and even fun using virtual human and avatar interface. Through collaboration our team has close ties with the entertainment industry, especially in games and narrative, and these “soft skills” will be applied to bringing digital health to analog humans.

To determine consumer experience and refine our design and development strategies, we have devised a consumer survey based on questions and answers that attempt to engage consumers to test their own ailments using our mobile health solutions. Results of this preliminary survey, our conclusions, and modifications to the mobile health solution for the Qualcomm Medical Tricorder X Prize Competition will be presented.
Tungsten Carbide‐Cobalt Nanoparticle induced miR‐21 Expression and PDCD4 Inhibition in JB6 Cells Involves a ROS‐ Mediated ERK Pathway (LB486)
Authors: Ding Min
Published: June 2021

Tungsten Carbide‐Cobalt Nanoparticle induced miR‐21 Expression Involves ROS‐ Mediated ERK Pathway

Min Ding1, Lichao Hou1,, Linda Bowman1, Terence G. Meighan1, Jun Liu2 Xianglin Shi3, 1Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV 26505, 2Department of Physiology & Pharmacology, West Virginia University, Morgantown WV 26505 3Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40503

Abstract Tungsten Carbide‐Cobalt (WC‐Co) nanoparticle composites have wide applications because of their hardness and toughness. WC‐Co has been classified as ‘probably carcinogenic’ to humans by the IARC. Earlier studies demonstrated that the tumor suppressor gene (PDCD4 and its upstream regulator miR‐21 may be considered as oncogenes for novel cancer prevention or anti‐cancer therapies. The present study examined the alterations of miR‐21‐PDCD4 signaling in JB6 cells after exposure to WC‐Co nanoparticles. The results showed that (1) WC‐Co caused PDCD4 inhibition in JB6 cells; (2) Exposure of cells to WC‐Co caused a significant increase of miR‐21, the upstream regulator of PDCD4, expression; (3) Inhibition of ERKs with U0126 reversed WC‐Co‐induced PDCD4 inhibition, but inhibition of p38 with SB203580 did not; and (4) ROS scavengers, N‐acetyl‐L‐cysteine and catalase, reversed the inhibitory effect of WC‐Co on PDCD4 expression, while superoxide dismutase promoted the inhibition. These findings demonstrate that WC‐Co nanoparticles induce miR‐21 expression and PDCD4 inhibition, which may be mediated through ROS, especially endogenous H2O2 and ERK pathways. Unraveling the complex mechanisms associated with these events may provide insights into the initiation and progression of WC‐Co‐induced carcinogenesis.

Disclaimer The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for occupational Safety and Health.

Grant Funding Source: US Government
Role of ROS Generation in Tungsten Carbide‐Cobalt Nanoparticles Induced Apoptosis in JB6 Cells (LB487)
Authors: Ding Min
Published: June 2021

Role of ROS Generation in Tungsten Carbide‐Cobalt Nanoparticles Induced Apoptosis in JB6 Cells

Min Ding1, Jinshun

Zhao2, Linda Bowman1, Stephen Leonard1, Jun

Liu3 Vincent Castranova1

1Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV

2Department of Preventive Medicine, Ningbo University, Ningbo, Zhejiang 315211

3Department of Physiology & Pharmacology, West Virginia University, Morgantown WV 26505

Tungsten Carbide‐Cobalt (WC‐Co) nanoparticle composites have wide applications because of their hardness and toughness. In this study, apoptosis and related signaling induced by WC‐Co were investigated. Electron spin resonance (ESR) and fluorescent staining indicated that both WC‐Co nano‐ and fine particles stimulated reactive oxygen species (ROS) generation. Catalase inhibits WC‐Co‐induced ROS as well as mitochondrial membrane permeability damage, indicating that H2O2 may play an important role in the cytotoxicity induced by WC‐Co. Further studies indicated that WC‐Co nanoparticles elicited a higher cytotoxicity and apoptotic induction than fine particles. Western‐blot analysis showed an activation of pro‐apoptotic factors including Fas, FADD, caspase 3, 8 and 9, BID, and BAX. Lamin A and beta‐actin were cleaved. Interestingly, WC‐Co particles also induced Bcl‐2, an anti‐apoptotic factor, up‐regulation. In addition, both cytochrome c and AIF were up‐regulated and released from mitochondria to the cytoplasm. The results demonstrate that apoptosis induced by WC‐Co involve both 'extrinsic' and 'intrinsic' apoptotic pathways and nanoparticles exhibit higher cytotoxicity and apoptotic induction than fine particles.

Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the National Institute for occupational Safety and Health.

Grant Funding Source: US Government
The male gender bias in autism may be due to preferential depletions of oxytocin and arginine‐vasopressin receptors positive neurons exposed to certain fragrances during fetal development (LB502)
Authors: Sealey Leanna , Bagasra omar
Published: June 2021

The male gender bias in autism may be due to preferential depletions of oxytocin and arginine‐vasopressin receptors positive neurons exposed to certain fragrances during fetal development Leanna Sealey and Omar Bagasra M.D. Ph.D.

Autism spectrum disorders (ASDs) are developmental conditions characterized by deficits in social interaction, verbal and nonverbal communication, and obsessive/stereotyped patterns of behavior. There is also evidence of impoverished language and empathy, and a profound inability to adopt another's viewpoint ‐ a failure to construct a “theory of mind” for interpreting another person's thoughts and intentions. In developed countries, it is now reported that 1%–1.5% of children have ASD, and in the US 2013 CDC report, approximately one in 80 children suffer from ASD. Although there is no reliable neurophysiological marker associated with ASDs, low levels of plasma oxytocin (OXY) and arginine‐vasopressin (AVP) have been reported. We have hypothesized that synthetic fragrances, which many of us are exposed to on a daily have potential to cause neurological damage to developing fetuses.Notably, the alarming rise in ASD parallels the rise in production of synthetic fragrances from petrochemicals and carcinogenic benzene‐based compounds. Numerous studies have shown that children with autism have significantly lower levels of OXY in plasma samples than their typical peers. Furthermore, in normal children, lower concentrations of OXY in plasma are associated with lower social and cognitive functioning. Here we show that male neuroblastoma cell lines, but not the female, exposure to fentomoles concentrations of certain fragments results in depletions of both OXY and AVP receptor + neurons. Our studies are the first to reveal a direct connection between fragrance exposures as the potential neuropathic agent that may cause autism and explains the gender bias.

Grant Funding Source: Department of Education
Development of a quantitative assay for detecting pro‐inflammatory bacteria genes directly in human stool samples by real‐time Polymerase Chain Reaction (rtPCR) (LB515)
Authors: Gomez‐Moreno, BS Ramon , Baerga‐Ortiz, PhD Abel
Published: June 2021

The human intestinal microbiota plays a key role in the maintenance of health and also in the development of intestinal disorders. Numerous studies have established the definitive association between the presence of certain intestinal bacteria in the flora and the development of gastrointestinal disorders. In this work, we developed an rtPCR assay for the detection of intestinal bacteria pro‐inflammatory genes directly from human stool samples. As a test case, we chose the pks genomic island genes that are present in some strains of intestinal Escherichia coli, and have been shown to induce, megalocytosis (figure 1), DNA damage, and aberrant inflammatory response. Metagenomic DNA was extracted from anonymous stool samples and used as the template for a rtPCR reaction. The fluorophore SYBR Green I was used to visualize the real time amplification response. The linearity for the SYBR Green I response with increasing amounts of input DNA was recorded in a standard curve with a correlation value of 0.9856. The fluorescence signal was detected in all pks+ samples after 18.97 cycles for HUO33, 19.24 cycles for HUO37, 16.61 cycles for HUO38 and 24.39 cycles for HUO47. As for the pks‐ samples, three of the samples the fluorescence signal was detected at cycle 9.65 for HUO31, 10.83 cycles for HUO39 and cycle 16.02 for HUO50. The fluorescence signal detected in pks‐ sample could be attributed to contamination between sample and non‐specific amplification. However, no fluorescence signal was detected in the negative control and the blank. Work is underway for the optimization of this assay towards the screening of patient samples.

Grant Funding Source: This work is supported by the National Science Foundation (NSF) grant CHE0953254 to ABO and the National Institute of Health (NIH) grant R25GM061838 (MBRS RISE Program) to RGM
A high sensitive Homocysteine (hsHCY) assay improves the clinical usefulness of this cardiovascular and cancer predictive biomarker (LB520)
Authors: Liang Genqing , Jaffe Anna , Larkin Jamie , Jaffe Russell
Published: June 2021

Elevated plasma Homocysteine (HCY) levels predict accelerated chronic diseases. A clinical limitation has been leakage into plasma of red cell HCY starting shortly after phlebotomy. A more stable specimen is needed especially at lower values.

Specimens (N=20) were collected in EDTA tubes (current preferred specimen) and a novel cell preservative. Specimens were stored at 4±1°C until analysis. Plasma was isolated and analyzed at 0.5, 4, 8, 24, 48, 72, and 96 hours after phlebotomy using a Diazyme™ Analyzer 700 and statistically analyzed by one‐way ANOVA.

HCY levels started to increase within 30 minutes in EDTA specimens while the new cell preservative sample values were stable for at least 48 hours. Indeed initial HCY levels in EDTA samples were typically 32% higher the new preservative (p<0.0001). This suggests that HCY is released to plasma from red blood cells in EDTA samples starting minutes after phlebotomy and less so in EAB cell preservative.

Initial and more precise HCY can be measured and maintained for at least 48 hours when the EAB cell preservative is used as specimen anticoagulant. EDTA in contrast allows HCY increase that starts within minutes after blood draw. The reported cell preservative allows a more stable and accurate HCY assay for this predictive biomarker. More studies are needed to confirm the clinical and global predictive significance of this biomarker.
A cell adhesion molecule (Ig‐SF/hemolin) from caterpillar regulates cell survival and induces extracellular matrix components in human osteoarthritic chondrocytes (LB528)
Authors: Alvarez‐Flores Miryam , Bosch Rosemary , Antonioli Eliane , Pereira‐Junior Edgard , Ferretti Mario , Cohen Moises , Chudzinski‐Tavassi Ana
Published: June 2021

Background: Losac is a hemolin from Lonomia obliqua caterpillar that promotes blood coagulation through proteolytic activation of factor X. In HUVECs, Losac increased cell proliferation and inhibited the apoptosis induced by starvation. Additionally, we have observed the cytoprotective effect of rLosac in other cell types, such as neurons and fibroblasts. Recombinant Losac (rLosac) was obtained from bacteria system. This report aims to investigate the potential of rLosac as a chondroprotective agent in human articular chondrocytes and to analyze its capacity to stimulate the production of extracellular matrix molecules and to reduce senescence. Methods: Primary cultures of human chondrocytes were prepared from cartilage of osteoarthritic knee joints obtained from 4 donors (age range: 68‐83) undergoing total knee replacement surgery. The study was carried out in full accordance with local ethics guidelines, and cartilage samples were collected after obtaining written informed consent of the donors. Chondrocytes were treated with rLosac and several parameters were measured: specific senescence‐associated beta‐galactosidase activity, cell viability by the MTT assay, production of extracellular matrix molecules by western blot and immunofluorescence microscopy, and cell cycle by flow cytometry. Results: rLosac was found to be an effective anti‐apoptotic agent, as evidenced by the strong inhibition of cellular death. The cytoprotective effect was observed specially starting from 48h and 72h of treatment, even in the presence of IL‐1β (50 ng/ml) to induce an inflammatory environment. Moreover, rLosac was able to reduce the expression of β‐galactosidade after 6 days of treatment. In addition, rLosac stimulated the production of collagen type I, laminin and fibronectin, and to increase the expression of the receptor of collagen type I, and to decrease the expression of IL‐1β. Conclusion: rLosac has the potential to become a therapeutic agent for protection of articular cartilage against the progression of osteoarthritic diseases.

Grant Funding Source: CETICs/FAPESP (Processo No. 2013/07467‐1), CAPES, INCTTox‐CNPq
Biosensor‐based identification and characterization of three distinct calmodulin‐binding domains in the human angiotensin II receptor type 1A (LB569)
Authors: Ehlers Kevin , VerMeer Mark , Tran Quang‐Kim
Published: June 2021

The angiotensin II receptor type 1A (AT1R) is responsible for many effects of angiotensin II (AngII). Calmodulin (CaM) is essential for many cell functions due to its interactions with many proteins. We tested the idea that signaling via AT1R involves CaM at the receptor level via multiple binding domains. AT1R coimmunoprecipitates with CaM in vascular smooth muscle under resting conditions, association that is enhanced by stimulation with AngII or thapsigargin. To identify all CaM‐binding domains in AT1R and characterize their binding properties, we generated FRET biosensors in which each the 4 sub‐membrane domains (SMDs) in AT1R is flanked by enhanced variants of yellow and cyan fluorescence protein. We named these biosensors BSAT1Rx, with x denoting the amino acid numbering of the SMD sequence. In response to purified Ca2+‐saturated CaM, BSAT1R125‐141, BSAT1R215‐242 and BSAT1R309‐327, corresponding to SMD2, SMD3 and the juxta membranous segment SMD4 in human AT1R, display drastic responses that are characteristic of conformational changes caused by direct binding of CaM. BSAT1R53‐64, corresponding to SMD1, does not response to Ca2+‐CaM. Titrations of biosensor responses with purified CaM yielded Kd values of 44.7±1.00, 0.36± 0.05, and 0.51±0.01 microM, respectively for SMD2, 3, and the juxta‐membranous segment of SMD4. The respective EC50(Ca2+) values of these interactions were 4.1±0.11, 0.13 ± 0.006, and 1.26 ± 0.09 microM, determined by monitoring concurrent responses of the respective BSAT1Rx and a suitable Ca2+ indicator. The entire SMD3 is involved in interaction with CaM, as a truncated domain (a.a. 215‐232) displays substantial decreases in affinity and Ca2+ sensitivity. These data demonstrate that CaM is involved in AngII signaling via direct interactions with multiple domains in AT1R and allow prediction of interactions between CaM and individual SMDs under distinct physiological scenarios, including resting and stimulated conditions.

Grant Funding Source: National Institutes of Health
Anti‐depressant, cognitive, anxiolytic and anti‐convulsant evaluation of Lophira alata (Ochnaceae) stem bark in mice (LB629)
Authors: Iniaghe Loretta , Igbe Ighodaro , Magaji Mohammed , Tabot Tabot , Maduka Ijeoma
Published: June 2021

Background: Stem bark and leaves of Lophira alata (Family: Ochnaceae) have been used traditionally for their anti‐psychotic, anti‐convulsant, analgesic and sedative properties.

Objective/Aim: The study was carried out to evaluate some neuropharmacological properties of the aqueous extract of the stem bark of Lophira alata in animal models.

Methods: Anxiolysis, depression, anti‐convulsion, memory and cognition were evaluated using animal models of these central nervous system indices.

Results: Oral administration of Lophira alata extract (200‐800mg/kg) caused a significant (p<0.05) non‐dose dependent increase in the period of vigorous activity in both the forced swim and tail suspension tests. In the novel object recognition test, administration of the extract resulted in a positive discrimination index score; spontaneous alteration performance was slightly enhanced in the Y‐maze test. While significant decrease (p<0.05) in upwards climbs in the staircase test was recorded, number of head dips were not significantly reduced (p>0.05) in the hole board test. In the Pentylenetetrazole induced convulsion, a non‐dose dependent increase in onset of tonic‐clonic seizures and modest protection from death was recorded. These effects were comparable to standard drugs‐Imipramine, Diazepam, Scopolamine and Phenobarbitone.

Conclusion: The results obtained suggest that the aqueous stem bark leaf extract of Lophira alata possesses neuropharmacological properties which may account for its use in Ethnomedicine.

Grant Funding Source: Supported by personal funds
Role of human CYP2A13 and CYP2F1 in naphthalene bioactivation and toxicity in the lung and nasal mucosa of a CYP2A13/2F1‐humanized Mouse (LB636)
Authors: Li Lei , Jia Kunzhi , Hartog Matthew , Wang Yanan , Van Winkle Laura , Ding Xinxin
Published: June 2021

The aim of this study is to characterize the expression and activity of CYP2A13 and CYP2F1, two human P450 enzymes expressed preferentially in the respiratory tract. CYP2A and CYP2F enzymes have been reported to metabolize many lung toxicants, including naphthalene (NA). The function of human CYP2A13 and CYP2F1 in NA bioactivation and respiratory tract toxicity was examined in this study using CYP2A13/2F1‐humanized mice and CYP2A13(only)‐humanized mice (on Cyp2abfgs‐null background). In vitro studies indicated that, while the activity in the nasal olfactory mucosa (OM) of the CYP2A13/2F1‐humanized mice was primarily contributed by CYP2A13, the activity in the lung was mainly contributed by CYP2F1. Further in vivo studies were conducted, in order to assess the capabilities of CYP2A13 and CYP2F1 to mediate acute inhalation toxicity of NA, at an NA dose relevant to occupational exposure (10 ppm for 4 h). CYP2A13/2F1‐humanized mice showed greater sensitivity than Cyp2abfgs‐null mice did, to NA‐induced depletion of non‐protein sulfhydryl and occurrence of cytotoxicity (observable by routine histology) in the OM (but not lung), when examined at 2 or 24 h after termination of NA exposure. However, preliminary results showed that the lungs of NA‐treated CYP2A13/2F1‐humanized mice had greater numbers of dye‐permeable (injured) cells than that of NA‐treated Cyp2abfgs‐null mice, in both proximal and distal bronchioles, when examined using an ethidium homodimer incorporation assay, which is more sensitive than routine histology examination. These results demonstrate that both CYP2A13 and CYP2F1 are active toward NA in the lung and OM of the CYP2A13/2F1‐humanized mice, and they play a significant role in NA‐induced toxicity.
Mutagenic modification effect of pomegranate juice in nitrite treated aminopyrene using Ames test (LB640)
Authors: Tongyonk Linna , Siritientong Tippawan , Tansawat Rossarin
Published: June 2021

This study evaluated the direct mutagenicity of fresh, and sterilized pomegranate juices and their modification effect on mutagenesis induced by nitrite treated 1‐aminopyrene in the absenceof metabolic activation on Salmonella typhimurium strains TA 98 and TA100. It was found that fresh juice was not mutagenic on both strains either with or without nitrite treatment. But the highest amount (24.67 mg/plate) of sterilized sample after being treated with nitrite exhibited slight mutagenicity on TA 98. Fresh pomegranate juice did not show antimutagenic activity against nitrite treated 1‐aminopyrene. In addition, it presented co‐mutagenic effect. Sterilized pomegranate juices kept for 0, 30 and 90 days were also evaluated for their antimutagenicity. It was found that sterilized sample kept for 90 days exhibited weak antimutagenic activity against nitrite treated 1‐ aminopyrene on TA 98. The results obtained from the present experimental condition revealed that antimutagenic activity of pomegranate juice is not clear.Further studies will be needed to investigate the antimutagenicity potential of pomegranate juice.
Lumbriculus variegatus as a model organism for studying angiogenesis during tissue regeneration (LB696)
Authors: Henry Charmaine
Published: June 2021

Lumbriculus variegatus is an aquatic oligochaete that is often used to study epimorphic regeneration following removal of head or tail segments. In this pattern of regeneration, the cut surface forms a blastema in which undifferentiated cells begin to regenerate the lost tissues. L. variegatus fully regenerates lost tail segments within two to three weeks. While several studies have investigated the regeneration of neural networks during epimorphosis in L. variegatus, little is known about its pattern of angiogenesis during regeneration. We used L. variegatus cultures along with light microscopy and image capturing and analysis for daily in vivo documentation of the structural changes that occur during angiogenesis in regenerating tail segments. The transparent cuticle, closed circulatory system and erythrocruorin pigment in the plasma made the living vasculature visible for microscopic analysis. The branching pattern of new vasculature was easily observed. Pulsations of the dorsal blood vessel allowed tracking of blood flow to determine when new vessels became patent. A brief application of the anesthetic Tricaine reduced locomotion and facilitated the capturing of images. We suggest that this organism is a useful model to study the effects of angiogenic stimulators or inhibitors during wound healing and tissue regeneration.

Grant Funding Source: Supported by Baker University Faculty Development Fund
Correlative super‐resolution light microscopy and electron microscopy determines spatial Ryanodine receptor type 2 distribution in mouse ventricular myocytes (LB707)
Authors: Das Tapaswini , Hakozaki Hiroyuki , Nitu Florentin , Bers Donald , Ellisman Mark , Cornea Razvan , Hoshijima Masahiko
Published: June 2021

Couplons are uniquely formed microdomains which utilize calcium (Ca) as a primary messenger to trigger Ca release from the sarcoplasmic reticulum (SR) via SR‐localized ryanodine receptors (RyRs), leading to cell contraction. Despite the critical role these sites play in excitation‐contraction coupling, our quantitative understanding of couplon biology remains limited largely due to the fact that their size falls between the most practical performance limits of light and electron microscopic methods, rendering them difficult to visualize. Here we present cutting‐edge correlated microscopic imaging technologies for detailing the multi‐scale organization and distribution of RyRs in ventricular cardiomyocytes. Electron microscope (EM) tomography provides the unique ability to resolve organelle geometries and to quantify the 3D ultrastructure of couplons at an isotropic resolution of 3‐5 nm. To map RyR distribution, instead of relying on typical immunostaining techniques that are limited in resolution by the size of the antibodies, we used fluorophore‐tagged FKBP12.6 recombinant protein in light microscopy (LM). This probe provides the advantage of stoichiometrically labeling RyR2, which also makes it ideal for super resolution LM imaging. Thus, the molecular details attained by super resolution LM are correlated with high‐resolution 3D geometric maps reconstructed using EM tomography to determine the spatial distribution of RyR tetramers.

Grant Funding Source: Supported by Human Frontier Science Program
Automated workflows for the morphological characterization of organelles in electron microscopy image stacks (LB72)
Authors: Perez Alex , Seyedhosseini Mojtaba , Tasdizen Tolga , Ellisman Mark
Published: June 2021

Advances in three‐dimensional electron microscopy (EM) have facilitated the collection of image stacks with a field‐of‐view that is large enough to cover a significant percentage of anatomical subdivisions at nano‐resolution. When coupled with enhanced staining protocols, such techniques produce data that can be mined to establish the morphologies of all organelles across hundreds of whole cells in their in situ environments. Although instrument throughputs are approaching terabytes of data per day, image segmentation and analysis remain significant bottlenecks in achieving quantitative descriptions of whole cell organellomes. Here we describe computational workflows that achieve the automatic segmentation of organelles from regions of the central nervous system by applying supervised machine learning algorithms to slices of serial block‐face scanning EM (SBEM) datasets. We also demonstrate that our workflows can be parallelized on supercomputing resources, resulting in a dramatic reduction of their run times. These methods significantly expedite the development of anatomical models at the subcellular scale and facilitate the study of how these models may be perturbed following pathological insults.

Grant Funding Source: Supported by NIGMS under awards number P41 GM103412‐25 and P41 GM103426, the NINDS under award number 1R01NS07531, and NIDA under award number 5T32DA007315‐10.
NDFIP1: the missing adaptor for aquaporin‐2 regulation by NEDD4 and NEDD4L (LB723)
Authors: de Groot Theun , Trimpert Christiane , Wesche Daniel , Wong Victoria , van den Berg Dennis , Stagljar Igor , Deen Peter
Published: June 2021

The aquaporin 2 (AQP2) water channel in collecting duct principal cells is essential for renal water reabsorption. Fine‐tuning of its expression in the apical membrane is critical for reabsorption of the proper amount. This fine‐tuning is the result of signals regulating AQP2 exocytosis and endocytosis. Whereas vasopressin‐induced cAMP signaling causes AQP2 translocation to the apical membrane, protein kinase C‐activating hormones stimulate ubiquitination‐dependent internalization of AQP2. The involved E3‐ubiquitin ligase has not been identified yet.

HEK293 cells (from human embryonic kidney) and mpkCCD cells (from murine cortical collecting duct) were used to study ubiquitination and degradation of AQP2.

SiRNA knockdown of NEDD4 and NEDD4L in mpkCCD cells increased AQP2 abundance, while both proteins did not interact with AQP2. Yeast Two‐Hybrid assay using full‐length AQP2 as bait identified NEDD4 family interacting protein 2 (NDFIP2). NDFIP2 and its homologue NDFIP1 are adaptor proteins, which bind NEDD4 ubiquitin ligases via their PY‐motifs and bring them close to target proteins. In HEK293 cells, NDFIP1/2 bound AQP2 and were essential for NEDD4/NEDD4L‐mediated ubiquitination and degradation of AQP2, an effect not observed with PY‐lacking NDFIP1/2 proteins. In mpkCCD cells, downregulation of NDFIP1, NEDD4 and NEDD4L, but not NDFIP2, increased AQP2 abundance, but did not affect the extent of AQP2 internalization. In mouse kidney, NDFIP1, but not NDFIP2, co‐localized and co‐immuno‐precipated with AQP2.

Our results reveal NDFIP1 as a potent and rate‐limiting regulator of NEDD4/NEDD4L‐mediated ubiquitination and degradation of AQP2.

Grant Funding Source: Supported by a Marie Curie Intra European Fellowship grant for CT and a VICI grant for PD.
Voluntary wheel running is necessary for hepatic PGC‐1α adenovirus overexpression to increase hepatic mitochondria in LCR rats (LB741)
Authors: Meers Grace , Morris E. Matthew , Koch Lauren , Britton Steve , Thyfault John
Published: June 2021

The hepatic adenoviral overexpression (o/e) of peroxisome proliferator‐activated receptor‐γ coactivator‐1α adenovirus (PGC‐1α) in sedentary (SED) low capacity running (LCR) rats increases PGC‐1α mRNA, but does not induce increased PGC‐1α protein, increased mitochondrial content or fatty acid oxidation, as we have previously observed in SED Sprague‐Dawley rats. It is well known that PGC‐1α expression is highly regulated post‐transcriptionally leading us to believe that LCR possess an unknown “brake” that blocks the impact of increased PGC‐1α mRNA. We hypothesized that increased energy expenditure via voluntary wheel running (VWR) would result in increased hepatic PGC‐1α protein in the LCR following PGC‐1α o/e. LCR rats (age=20 wks; n=4‐8) were assigned to SED+PGC‐1α or VWR+PGC‐1α for 4 weeks while β‐galactosidase adenovirus groups served as controls. VWR reduced fat pad mass (~ 50%; p<0.05) but not body mass compared to SED. Both the SED+PGC‐1α and the VWR+PGC‐1α increased hepatic PGC‐1α mRNA, but only the VWR group displayed increased citrate synthase activity and complete palmitate oxidation (p<0.05). The lack of an increase in PGC‐1α protein and mitochondrial function following PGC‐1α o/e cannot be explained by known regulators of PGC‐1α transcriptional activity, SIRT3 or GCN5, as they did not differ between groups. The micro RNA, MiR‐23A, is known to lower PGC‐1α mRNA and protein content in muscle. We observed that MiR‐23A was unexpectedly higher in VWR vs. SED suggesting that this was also not a brake on PGC‐1α function. In conclusion, hepatic adenoviral o/e of PGC‐1α only increases mitochondrial function in running but not SED LCR rats, and the improved function is independent of increased PGC‐1α protein content.

Grant Funding Source: Supported by: NIH DK‐088940, NIH T32 AR‐048523, and HSTMVA resources and facilities
Glia maturation factor regulates 1‐methyl‐4‐phenylpyridinium‐induced neuron loss and oxidative damage in primary neuron‐glia cells (LB75)
Authors: Zaheer Smita , Khan Mohammad , Nehman John , Zaheer Asgar
Published: June 2021

Inflammation mediated by glial activation appears to play critical role in the pathogenesis of Parkinson disease (PD). Glia maturation factor (GMF), a proinflammatory protein localized in the central nervous system, was isolated, sequenced and cloned in our laboratory. In the present investigation, we demonstrate that altered expression of GMF has direct consequences on the survival of dopaminergic neurons and the production of reactive oxygen species (ROS) and NF‐κB mediated production of inflammatory mediators by 1‐methyl‐4‐phenylpyridinium (MPP+). We examined MPP+‐ induced dopaminergic neuronal loss in primary cultures of mouse mesencephalic neuron/glia obtained from GMF‐deficient (GMF‐KO) and GMF‐containing wild type (Wt) mice. Our results demonstrate that overexpression of GMF induced dopaminergic neurodegeneration, whereas GMF downregulation by GMF‐specific shRNA protected dopaminergic neurons from MPP‐induced toxicity. Additionally, deficiency of GMF in GMF‐KO neuron/glia led to decrease production of ROS and downregulation of NF‐κB mediated production of TNF‐α and IL‐1β as compare to Wt neuron/glia. GMF deficiency ameliorates antioxidant balance, as evidenced by the decreased level of lipid peroxidation, less ROS production along with increased level of glutathione; and attenuated the dopaminergic neuronal loss via the downregulation of NF‐κB mediated inflammatory responses. In conclusion, our overall data indicate that GMF modulates oxidative stress and release of deleterious agents by MPP+ leading to loss of dopaminergic neurons. Our study provides new insights into the potential role of GMF and identifies targets for therapeutic interventions in neurodegenerative diseases.

Grant Funding Source: Supported by the NINDS grants NS073670
Differential response to cold pressor test in normal weight versus overweight college students is blunted by palatable reward (LB767)
Authors: Arnhold Davies Michelle
Published: June 2021

There is increasing evidence that obesity contributes to dysregulation of the hypothalamic pituitary adrenal (HPA) axis; however, the magnitude and direction of dysfunction remains unclear. The objectives of this experiment were to identify differences in non‐stress and stress salivary cortisol concentrations in normal weight (NW) and overweight/obese (OW) college students. Furthermore, we aimed to determine if access to palatable reward differentially blunts activation of the HPA axis in response to a physical stressor. Body mass index (BMI) was calculated and participants were placed in a reward or no reward group. Participants underwent a cold presser test (CPT), placement of the non‐dominant hand into ice water for 90 seconds. The reward group had free access to candy (reward) during the CPT; the control group was not offered candy. Salivary samples were collected before CPT (Pre) and 15 minutes after the start of the CPT (Post). Cortisol was measured by enzyme linked immunoassay (Salimetrics). Pre cortisol was elevated in OW versus NW. CPT without reward increased cortisol in both NW and OW, with OW having a greater increase from Pre than NW. Reward blunted the CPT‐induced cortisol increase similarly in NW and OW. These data suggest that overweight individuals have higher baseline HPA activity and an exaggerated response to physical stress than normal weight individuals; however, effects of reward were similar in both groups.
Gestational high‐fat feeding impairs female offspring energy metabolism in mice (LB777)
Authors: Guidotti Stefano , Qiao Liping , Sun Yoo Hyung , Shao Jianhua
Published: June 2021

Maternal obesity and excessive gestational weight gain increase the risk of offspring to the development of obesity and metabolic defects later in life. The present study aimed to elucidate the effects of gestational high‐fat (HF) feeding on offspring energy metabolism and its underlying mechanisms. Pregnant C57BL/6J mice received HF diet (60% calories from fat) or chow from conception until parturition. After delivery, all dams and offspring were fed with chow. Body composition was monitored by echoMRI throughout the study.

At 3 months of age, significantly increased body adiposity and reduced lean body mass (LBM) were observed in female offspring from HF‐fed dams without changes in body weight. Female offspring displayed similar energy intake with reduced energy expenditure (EE). When normalized by LBM, EE was no different between groups. No difference was found in UCP‐1 protein levels in brown adipose tissue and in expression of mitochondrial markers in skeletal muscle. Investigating the molecular mechanism underlying this metabolic phenotype, we found a significant reduction in β‐catenin protein levels in skeletal muscle and white adipose tissue. We also observed a reduction in GSK3‐β phosphorylation and other downstream targets of wnt signaling.

Together our data indicate that gestational HF‐feeding increases body adiposity in expense of LBM in female offspring. Suppression of the wnt/ β‐catenin signaling in skeletal muscle could potentially lead to deranged muscle development and hence be related to the reduction in LBM. This defect in muscle development may ultimately lead to reduced energy needs, supporting a mechanism aimed at energy conservation that favors the storage of fat.
Theobromine, the primary methylxanthine found in Theobroma cacao, inhibits malignant glioblastoma cell growth by negatively regulating Akt/mammalian target of rapamycin kinase (LB836)
Authors: Sugimoto Naotoshi , Miwa Shinji , Katakura Masanori , Matsuzaki Kentaro , Shido Osamu , Tsuchiya Hiroyuki , Yachie Akihiro
Published: June 2021

Theobromine, a caffeine derivative, is the primary methylxanthine produced by Theobroma cacao. We previously showed that methylxanthines, including caffeine and theophylline, have anti‐tumor and anti‐inflammatory effects, which are in part mediated by their inhibition of phosphodiesterase (PDE) [1‐3]. A member of the PDE family, PDE4, is widely expressed in and promotes the growth of glioblastoma, the most common type of brain tumor. Here, we investigate whether theobromine could exert growth inhibitory effects on U87‐MG, a cell line derived from human malignant glioma.

We show that theobromine treatment elevates intracellular cAMP levels, indicating the prevention of PDE4 by theobromine, whereas it attenuates the Akt/mammalian target of rapamycin kinase pathway. Theobromine also inhibits cell growth. These results suggest that theobromine might be extremely effective in preventing human glioblastoma.

[1] Miwa S, Sugimoto N et al. Int J Oncol., 2011; 39: 465‐472.

[2] Miwa S, Sugimoto N et al. Anticancer Res., 2012; 32: 3643‐3649.

[3] Saito T, Sugimoto N et al. ScientificWorldJournal, 2012; 2012:748572 (8pages).
Membrane trafficking and PIP2‐dependent regulation of Kir2.2 by LPS in THP‐1 human monocyte (LB839)
Authors: Kim Kyung Soo , Jang Ji Hyun , Shin Dong Hoon , Choi Sung Woo , Zhang Yin Hua , Kim Sung Joon
Published: June 2021

Lipopolysaccharide (LPS) is an effective agonist for Toll‐like receptor 4 (TLR4) in monocyte/macrophage. However, investigation on ion channels affected by LPS is not completely understood. In THP‐1 human monocyte cells, we observed decrease in voltage‐gated K+ channel current (51 %) and increase in Ca2+‐activated K+ channel (235 %) at 6 h after LPS application (1 μg/ml). Interestingly, marked increase of inwardly rectifying K+ channel (Kir) current was observed from 1‐2 h (IKir‐LPS), which peaked at 4 h and spontaneously decayed to 27 % of peak at 24 h. Single channel conductance of the IKir‐LPS was 38 pS, and their Ba2+‐sensitivity was relatively high (IC50, 1.42 μM), which were consistent with Kir2.2 isotype. Functional upregulation of IKir‐LPS was also found in primary human peripheral blood monocytes. While total Kir2.2 protein was not changed, its membrane fraction was increased time‐dependently and confocal microscopy confirmed the increased plasma membrane expression from 2 h. Also, IKir‐LPS was partially attenuated by Exo‐1 (vesicular trafficking inhibitor). However, the spontaneous decay of IKir‐LPS after 4 h was not prevented by dynamin inhibitor (Dynasore). Interestingly, the decayed IKir‐LPS at 24 h was rapidly reversed by PI3 kinase (PI3K) inhibitors (Wortmannin and LY294002). In contrast, IKir‐LPS was suppressed by bpV(Phen), an inhibitor for PTEN (PIP3 phosphatase). IKir‐LPS was not affected by Akt inhibitors, suggesting that the decay of IKir‐LPS was not due to PIP3‐dependent signaling but to decreased availability of PIP2 (conversion to PIP3 by PI3K) that is known to be critical for Kir2.2 activity. Store‐operated Ca2+ entry significantly increased in 4 h LPS treated THP‐1, which was prevented by KCl‐induced depolarization. Taken together, we firstly report that Kir2.2 is functionally upregulated by LPS via membrane trafficking in human monocytes. Subsequent hyperpolarization enhanced Ca2+ influx, implying a physiological role of K+ channels in the innate immunity.
Myoglobin: O2 or not O2襃 that is the question (LB90)
Authors: Bord Evan , Laughlin Robert , LeMense Andrew , Marable Chad , Mielke Suzie , Poniewaz Brett , Tuncel Virginia , Wade Gina , Weeden Mike , Wisth Ryan , Sampe David , Wagie Hannah , Geissinger Peter
Published: June 2021

Free divers can’t hold their breath as long as whales, but they train their bodies to maximize their oxygen (O2) storing potential using the protein myoglobin. Myoglobin’s structure has been known for decades, but researchers are still trying to determine just how myoglobin functions. Found in muscle tissue, myoglobin stores O2, a molecule needed to produce chemical energy. Toxic ligands, such as carbon monoxide (CO) and cyanide, also bind to myoglobin. When CO binds to a free heme group, the heme's binding affinity for CO is 20,000 times that for O2. When heme is surrounded by myoglobin, that binding affinity ratio drops to only 25. The decrease was thought to be due to steric interactions which prevented CO from occupying the same space as His64. Recent evidence suggests that electrostatic interactions and hydrogen bonds play a more important role. The O2 is stabilized as opposed to the CO being pushed out. Several amino acids (His64, Val68, Phe43, Phe46, and Leu29,) seem to stabilize the ligand. With 3D printing technology, the Brown Deer SMART (Students Modeling a Research Topic) Team created a model of myoglobin. If researchers can fully understand ligand discrimination by heme proteins, not only will divers be able to hold their breath longer, but we may be able to cure diseases like anemia where there is a lack of O2 in the blood.

Grant Funding Source: The SMART team program is supported by a grant from NIH‐CTSA.