bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific knockout of the Slc5a6 gene in mouse intestine via Cre/lox technology leads to impairment in biotin (and pantothenic acid) absorption
Authors: Ghosal Abhisek , Lambrecht Nils , Subramanya Sandeep B , Kapadia Rubina , Said Hamid M
Published: June 2021

The Slc5a6 gene expresses the sodium‐dependent multivitamin transporter (SMVT), a membrane protein that transports the water‐soluble vitamin biotin, as well as pantothenic acid and lipoate. The contribution of SMVT toward total carrier‐mediated absorption of biotin in native intestine in vivo is not known, and thus, was examined using intestine‐specific SMVT knockout (KO) generated by a Cre/lox technology. KO mice showed lack of expression of SMVT in the intestine compared to sex‐matched littermates but showed normal expression in other tissues. Almost 2/3rd of the KO mice died prematurely (ages of 6 and 10 weeks). Growth retardation, decreased bone density, decreased bone length and decreased biotin status were all observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice; the colon showed similar changes to the cecum but to a less extend. In vivo uptake studies demonstrated complete inhibition in carrier‐mediated biotin uptake in the intestine of the KO mice. These studies provide the first in vivo confirmation in native intestine that SMVT is the sole carrier for biotin absorption. The studies also provide evidence for a casual association between functionality of the SMVT and normal intestinal health/homeostasis. [Supported by DVA and NIH, (DK58057 and DK56061)].
Attenuated parasympathetic regulation of the heart in mice fed high fat diet
Authors: Li Yifan , Sample Alex
Published: June 2021

Altered autonomic regulation of the heart may contribute to the increased risk of cardiac diseases in obesity. The mechanisms behind the abnormal autonomic activities remain to be addressed. In this study, mice fed regular diet (RD) or high fat diet (HFD) for 6 weeks were used to test the effect of parasympathetic nervous system (PSNS) on the heart. The body weight, plasma insulin, and total cholesterols were significantly increased in HFD mice compared to RD mice. Functionally, the response in heart rate to vagal stimulation, but not the response to the direct stimulation of muscarinic receptors, was significantly attenuated in HFD mice compared with RD mice. These results suggest a reduced cholinergic activity at PSNS ganglia and/or terminals in HFD mice. Vesicular acetylcholine transporter (VAChT) is critical for acetylcholine storing and release at cholinergic neurons and terminals. Western blot showed that the protein level of VAChT was significantly reduced in atria samples from HFD mice compared with RD mice. Furthermore, in vitro experiments using a cholinergic cell line, SN56, showed that the treatment with insulin significantly increased VAChT ubiquitination. Prolonged treatment with insulin decreased VAChT protein level and VAChT trafficking. Together, these results suggest that reduced VAChT level, at least partially due to the insulin‐induced increase in ubiquitination‐mediated VAChT degradation, may contribute to the attenuated cholinergic activity at parasympathetic ganglia and/or terminals in the heart in HFD mice. This work was supported by AHA grant No.0835256N, NIH INBRE grant No. P20 RR016479, and the BBS bridge grant.
Integrating Quantitative Experiments into the Physiology Classroom
Authors: Anjur Sowmya
Published: June 2021

Physiology and Disease is a student led inquiry based class at the Illinois Mathematics and Science Academy, a teaching laboratory to encourage creative learning. Students in this course design and execute their own experiments and analyze their data based on the evidence they have gathered. Students with more of a Math or Physics background than Biology who take this course sometimes have more trouble visualizing the concepts behind their experimental designs than those with a Biology background. Some students also seem to have trouble measuring their blood pressure using the traditional sphygmomanometer. Student surveys have also indicated the need for more quantitation in lab experiments. To accommodate all these issues, several changes are being implemented into the course. One of the changes involves supplementing the existing sphygmomanometer measurements with blood pressure sensor measurements which allows students to test for accuracy and also graph their blood pressure measurements at the same time. Similar changes are planned for lung capacity and heart rate measurements, in an effort to allow students to identify the role of the neuronal controls for these parameters, and to understand better how they work in concert.
Jumpstarting STEM Careers
Authors: Sweazea Karen , Baluch Debra , Traynor Kirstin , Cease Arianne , Coulombe Margaret , Stout Valerie
Published: June 2021

A successful career in science, technology, engineering and mathematics (STEM) requires great education and research experiences as well as extensive training and preparation with dedicated mentors. Recent statistics show women received over 40% of all BA/BS degrees awarded by U.S. 4‐year colleges and universities in the life sciences and the proportion of women with doctorates has exceeded 40–60% in some fields. However, the proportion of women associate professors in the basic science departments of most universities is still below 30%, and the proportion with rank of full professor is only 20%. Therefore, the Central Arizona Chapter of the Association for Women in Science at ASU, in collaboration with colleagues from George Washington, Gallaudet and Ottawa Universities, has developed a program to help prepare graduate students, post docs and early faculty for careers in STEM. This NSF advance funded program tackles the problem of low career advancement of women and minorities by hosting career development seminars and workshops to provide training, mentoring and networking opportunities for graduate students and post docs. Such career training programs will help address the complex problem encountered by women and minorities and thus will aid in restoring a pipeline of diverse STEM professionals.
Can patient simulator scenarios be utilized to improve teamwork and communication skills in first year medical students taking Medical Physiology?
Authors: Harris David M
Published: June 2021

Communication (Com) skills, as well as decision making/clinical problem solving, are on the list of LCME “hot topics”. Additionally, there has been a recent push to improve patient safety which most commonly revolves around miscommunication. Development of these skills occurs most often during the clinical years of medical education and studies focused on incorporation of these skills during the preclinical years are few. The aim of the current study is to determine if Com and teamwork skills can be improved during the preclinical years by using patient simulator (SIM) scenarios based upon physiology concepts. We used a modified version of the Anesthetists’ Non‐Technical Skills Behavorial (ANTS) marker system. This focused on 4 major categories: task management(TM), team working (TW), situation awareness (SA), and decision making (DM). 100 1st medical students participated in two SIM activities (#1: heat exhaustion, #2: Heart failure) in which faculty rated 4 categories on a 1–4 scale (poor to good). Students were debriefed in 2–3 groups on basic science concepts, and teamwork/Com skills. Average scores decreased in all four categories between SIM 1 and 2. TM = 3.2 to 3.1, TW = 3.6 to 3.0, SA = 3.3 to 2.9, and DM = 3.4 to 3.00. Interestingly, verbal feedback and actions from the students as well as faculty observations of the students appears to show the opposite and that the students appear to be discussing and group assessing to a greater degree. Additionally, the complexity of the SIM may play a role in these skills as SIM 2 is a more complex case than SIM 1. In conclusion, more data is needed and additional analysis measurements are needed to address how to assess student behavior regarding Com and teamwork skills. However, the fact that these are vital life‐long skills which could improve patient safety long term makes it a noteworthy focus of medical institutions.
Factors contributing to recruiting students into health and science career paths
Authors: Cisek Karen L , Conway Pat , Wages Jennifer , Sens Don , Doze Van A , Olimb Sarah
Published: June 2021

This study examined factors contributing to students’ entrance into science and health careers based on Jolly, Campbell, & Perlman's (2004) theoretical framework (engagement, capacity, and continuity) and Bronfenbrenner's (1994) ecobiological theory. Engagement is students’ career motivation and interest; capacity is students’ ability or capacity to succeed in science and health careers; continuity factors are institutional and family supports. The ecological model adds influences at the individual, family, community and organizational levels. This qualitative study conducted focus groups with undergraduate students participating in summer research experiences at the University of North Dakota from 2009–2012, answering 3 research questions. 1) What role do engagement and capacity play in students’ career paths? 2) What role does academic continuity play? 3) How do families influence students’ career choices? Student comments were analyzed using NVIVO. Factors influencing students’ career choices were organized by six categories: Individual, Education, Family, Community, Chance, and Career. Individual comments were most common, demonstrating the importance of students’ level of engagement in science and their capacity. Chance emerged as a new category. Supported by National Center for Research Resources Grant P20 RR016471 and National Institute of General Medical Sciences Grant P20 GM103442 from the National Institutes of Health; and National Science Foundation REU Site Grant 0851869.
Human cortical bone maintenance: does mechanical loading compensate for bone loss associated with metabolic stress?
Authors: Eleazer Courtney D.
Published: June 2021

Cortical bone adapts to environmental factors at macroscopic and microscopic levels by altering mass and shape, and thus strength. As few studies have focused on how these factors interact in humans, researchers continue to investigate mechanical and metabolic influences on bone as if they act independently. This study utilizes an archaeological human juvenile sample from a medieval Lithuanian cemetery. Juvenile bone is favored over adult bone because it responds strongly to both mechanical and metabolic effects. Macroscopic and microscopic bone morphology were assessed in three skeletal elements under different levels of mechanical loading (i.e., femora, humeri, ribs) and compared among individuals under varying amounts of metabolic stress. Analyses evaluated whether, in the presence of bone loss due to metabolic stress, bone mass and strength were preferentially maintained in bones under the highest mechanical demands. Results indicate that such a preferential maintenance may occur, suggesting that systemic metabolic bone loss is compensated by high local loading demands. Results caution researchers against evoking solely mechanical or metabolic causes for variation in cortical bone morphology.

Grant Funding Source: NA
DNA intercalation impacts multiple steps in the recognition of damaged bases by a DNA glycosylase
Authors: Hendershot Jenna , O'Brien Patrick
Published: June 2021

To repair DNA, enzymes must overcome the problem of gaining access to bases that are normally embedded in the duplex. One solution to this problem is base flipping, the 180° rotation of a damaged base into an enzyme active site. Base flipping is ubiquitous for repair enzymes, but the mechanism is poorly understood. A dissection of the reaction mechanism will shed light on how repair enzymes can search the genome effectively to locate sites of DNA damage. Human alkyladenine DNA glycosylase (AAG) is a monomeric protein responsible for removing a diverse set of alkylated and oxidized purine bases. The crystal structure of AAG revealed the DNA is bent and a tyrosine (Y162) intercalates into the space left by the flipped‐out damaged nucleotide. In this study we have mutated this residue to smaller (Y162A) and larger (Y162W) amino acids and characterized the kinetic mechanism of these mutants using the naturally fluorescent 1,N6‐ethenoadenosine (εA) lesion. The Y162W mutant has robust glycosylase activity toward εA and the stably paired lesion hypoxanthine (Hx). Using stopped‐flow experiments, the binding of Y162W to εA‐DNA was measured monitoring both tryptophan fluorescence and εA fluorescence. Overall, the kinetic parameters for Y162W are almost identical to wild‐type AAG. In comparison, Y162A flips bases 60‐fold faster than wild‐type. This unexpected finding suggests Y162 in wild‐type acts as a selectivity filter and slows base flipping. The Y162A mutant also unflips very rapidly, so the overall equilibrium constant for base flipping is decreased by 300‐fold.

Supported by: Cellular Biotechnology Training Grant
A highly conserved GC‐rich element regulates alternative splicing of mRNA for the variant thyroid hormone receptor, TRα2
Authors: Munroe Stephen H.
Published: June 2021

In mammals the THRA gene is alternatively spliced and encodes two proteins. TRα1 is the α‐thyroid hormone receptor, which is widely expressed in all vertebrates. TRα2 is a non‐hormone‐binding variant, present only in eutherian mammals. Coding sequences unique to TRα2 share an antisense overlap with those of Rev‐erbα, a nuclear receptor and core component of the mammalian circadian clock. Intronic and exonic splicing enhancers for TRα2 have evolved in the context of sequences required expression of Rev‐erbα mRNA. Of particular interest is a GC‐rich region (G30) within the C‐terminal sequence of TRα2 mRNA and antisense to the 3'UTR of Rev‐erbα. Closely spaced deletions and substitutions within G30 have dramatically different effects on TRα2 splicing depending on their precise structure and position. For example, deletion of 12 nucleotides can lead to >;95% inhibition or >;2.5‐fold enhancement of TRα2 splicing. Substitutions of 5–8 bases within G30 also display a range of effects. Our results suggest that G30 is a highly structured sequence, possibly a G‐quadruplex, and part of a larger complex splicing regulatory element that exerts both positive and negative effects on TRα2 expression.
RNF4‐Dependent Hybrid SUMO‐Ubiquitin Chains are Signals for RAP80 and thereby Mediate the Recruitment of BRCA1 to Sites of DNA Damage
Authors: Guzzo Catherine Mary , Berndsen Christopher E , Zhu Jianmei , Gupta Vibhor , Datta Ajit , Greenberg Roger A , Wolberger Cynthia , Matunis Michael J
Published: June 2021

The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double strand breaks (DSBs) through recognition of K63‐linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. Here, we demonstrated that, in addition to having ubiquitin‐interacting motifs, RAP80 also contains a SUMO‐interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin‐binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO‐targeted ubiquitin E3 ligase that synthesizes hybrid SUMO‐ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin‐dependent and SUMO‐dependent DSB recognition, revealing that RNF4 synthesized hybrid SUMO‐ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.
Carbamazepine restores surface expression and function of trafficking‐impaired mutant ATP‐sensitive potassium channels identified in congenital hyperinsulinism
Authors: Chen Pei‐Chun , Olson Erik , Kryukova Yelena , Zhong Qing , Shyng Show‐Ling
Published: June 2021

ATP‐sensitive potassium (KATP) channels comprising the ABC transporter sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to â‐cell membrane potential. Mutations in SUR1 and Kir6.2 that disrupt protein folding and trafficking lead to loss of channel function and congenital hyperinsulinism. Carbamazepine is a voltage‐gated sodium channel inhibitor used clinically as an anticonvulsant; recently, it was shown to alleviate liver disease in mice expressing a á1 antitrypsin folding mutant by activating the autophagy pathway. We report here that carbamazepine corrects the folding and trafficking defects of mutant KATP channels and restores channel function in rat and human â‐cells. Carbamazepine rescues channel surface expression by improving mutant channel processing efficiency rather than stimulating autophagy. Interestingly, like the previously identified KATP channel chaperone drugs sulfonylureas, carbamazepine only rescues mutations located in the first transmembrane domain of SUR1 and requires subsequent removal to recover channel function. As carbamazepine retains its ability to rescue mutant channel expression when simultaneously applied with the KATP channel opener diazoxide, combination of the two drugs may be used to restore channel expression and function in a subset of congenital hyperinsulinism patients.
Characterization of a variant of ERGIC2 protein
Authors: Kwok Simon Chun Man , Kumar Sudhanshu , Dai Guoli
Published: June 2021

ERGIC2 is a gene identified in our laboratory as a potential tumor suppressor. Because of its high sequence homology with the yeast chaperone protein, Erv41p, it was suggested that ERGIC2 protein may be involved in protein trafficking between endoplasmic reticulum (ER) and cis‐Golgi. However, over‐expression of ERGIC2 in prostate cancer cell line, PC‐3 cells, induced cell growth arrest and senescence, suppressed colony formation in soft agar, and decreased invasive potential in invasive chamber assay. Therefore, ERGIC2 protein may also be involved in yet unknown signal transduction pathways. Recently, we isolated a cDNA encoding a variant of ERGIC2 that has a 4‐base deletion after codon #189. As a result of this frame‐shift mutation, a truncated protein of 215 residues is predicted as compared to the 377‐residue wild‐type protein. This effectively abrogates its function as ER‐Golgi transport shuttle. The objective of this study is to investigate the functions of this ERGIC2‐variant. Expression construct of the ERGIC2‐variant was transiently transfected into PC‐3 cells, and its effect on cell growth was studied. A protein with a size distinct from wild‐type ERGIC2 was detected by Western blot. However, when the cells were selected for stable expression, complete loss of the fusion‐protein was resulted, suggesting that the variant can also arrest cell growth as the wild‐type ERGIC2.
Programmed cell death protein 5 may exert its apoptotic function through CCT
Authors: Tracy Christopher Michael , Gray Amy J , Howlett Alyson C , Shaw Tanner Steven , Taylor Ryan , Makaju Aman , Prince John T. , Valpuesta José M. , Willardson Barry Matthew
Published: June 2021

Phosducin‐like protein 1 (PhLP1) was recently shown to be a co‐chaperone with the cytosolic chaperonin complex (CCT) in the folding and assembly of nascent G protein βγ dimers and Gβ5‐RGS proteins. However, other possible functions of PhLP1 warrant investigation. A co‐immunoprecipitation proteomics screen indicated PhLP1 interacts with a known tumor suppressor, programmed cell death 5 protein (PDCD5), which is implicated in apoptosis. Subsequent experiments showed PDCD5 associates with PhLP1 indirectly via a ternary complex with CCT and had no effect on PhLP1‐mediated Gβγ assembly. Previous studies suggested that PDCD5 translocates rapidly from the cytoplasm to the nucleus during the initial stages of apoptosis. However, our results indicate that PDCD5 is exclusively cytosolic in both resting and apoptotic cells, thus its apoptotic function is also cytosolic. Co‐immunoprecipitation proteomics screens show that PDCD5 interacts strongly with CCT and β‐tubulin. Cryo‐EM studies show a complex of PDCD5 and CCT, interacting at one of the apical domains. Moreover, biochemical analyses suggest that PDCD5 phosphorylation increases its binding to CCT during apoptosis and when bound to CCT, PDCD5 blocks β‐tubulin folding. We propose that PDCD5 binds to the apical domain of CCT as a co‐chaperone or inhibitor of CCT substrates involved in cell cycle progression or apoptosis. This research was funded by an NIH grant.
Ubiquitin chain editing mechanisms on the mammalian proteasome
Authors: Lee Byung‐Hoon , King Randall , Finley Daniel
Published: June 2021

The 26S proteasome is now accepted as a versatile and active player for fine‐tuning the substrate degradation cycle rather than a passive degradation machinery as once thought. However, ubiquitin conjugate processing by the mammalian proteasome remains poorly understood, partly due to the lack of specific tools. We recently developed a small‐molecule inhibitor selectively targeting USP14, a major deubiquitinating enzyme on the proteasome. By employing this inhibitor, we revealed that ubiquitin chain processing on the proteasome is surprisingly sophisticated, discriminating very sensitively among substrates on the basis of diverse configurations and architectures of the ubiquitin conjugates. Our data suggest a dynamic engagement of Usp14 in recognizing and processing the chain in the context of proteasome.
Quantifying the two populations of dual oriented P6 in nontypable Haemophilus influenzae
Authors: Schmidt Rachel Ann , Shaw Juliana , Novick Bethany , Pichichero Michael , Michel Lea Vacca
Published: June 2021

Nontypable Haemophilus influenzae (NTHi) causes otitis media (ear infections) and other respiratory illnesses. Currently, NTHi's outer membrane protein P6 is a leading vaccine candidate for the pathogenic bacteria. Previous work in our lab has demonstrated that P6 is not a transmembrane protein. However, P6 is also known to interact with peptidoglycan inside the cell. In light of other studies performed in our lab (flow cytometry and confocal microscopy), which demonstrate P6's surface exposure, we conclude that P6 exists in two orientations in the outer membrane of NTHi. To quantify the two populations of inside‐facing and outside‐facing P6, we utilized a biotinylating reagent (NHS‐LC‐LC‐ biotin) to isolate surface exposed proteins from internally localized proteins. Standard protein detection and immunoblotting techniques were utilized to quantify the two populations of P6. We conclude that the predominant population of P6 is located on the inside of NTHi (>;75%). Further elucidation of the dual orientation of P6 and other lipoproteins is certainly important for both the fields of bacteriology and vaccine development. This study was funded by the Rochester Institute of Technology and NIH NIDCD RO1 08671 (to MEP).
Different Dietary Sources of Omega‐3 Polyunsaturated Fatty Acids Influences Renal Fatty Acid Composition, Gene Expression, and Risk of Nephrocalcinosis in Female Rats
Authors: Oldaker Christopher Ryan , Gigliotti Joseph , Benedito Vagner , Tou Janet
Published: June 2021

Nephrocalcinosis, the accumulation of calcium (Ca) in the kidneys, is associated with multiple renal disorders including renal stones. Populations with low risk of renal stones have been shown to have a diet high in fish oil, a rich source of omega‐3 polyunsaturated fatty acids (n‐3 PUFAs). However, sources of n‐3 PUFAs differ in the amount, type, and structure. The study objective was to determine the effect of feeding different sources of n‐3 PUFAs on the development of nephrocalcinosis. Young (age 28 day) female Sprague‐Dawley rats were fed a casein diet supplemented with corn oil or omega‐3 rich oils consisting of flaxseed, krill, menhaden, salmon, or tuna oil for 12 wks. Gas chromatography showed that renal fatty acid composition reflected the fatty acid profile of the dietary oils. RT‐qPCR showed that rats fed fish oils had reduced inflammation as indicated by lower (P<0.05) COX‐2 and TGF‐β. Furthermore, rats fed salmon or tuna oil had increased (P<0.05) osteopontin (SPP1) gene expression, indicating antagonist action to renal Ca crystallization. Rats fed fish oils had lower (P<0.05) renal NFκB and prostaglandin E2 activity as determined by enzyme immunoassay. However, krill oil induced nephrocalcinosis due to the high phosphorous (P) content associated with phospholipids. Different sources of n‐3 PUFAs mitigated the effects of nephrocalcinosis, but failed to prevent nephrocalcinosis due to Ca:P imbalance.
Glia maturation factor activate mast cells and induce chemokine CCL2 release: Implication for neurodegenerative diseases
Authors: Kempuraj Duraisamy , Zaheer Asgar
Published: June 2021

We previously discovered, purified and sequenced Glia maturation factor (GMF) from bovine brain. We have also demonstrated increased expression of GMF in the central nervous system (CNS) of neurodegenerative diseases such as Alzheimer's disease and Multiple Sclerosis/Experimental Autoimmune Encephalomyelitis. GMF mediates neurodegeneration by releasing inflammatory mediators from glial cells. Although mast cells are known to secrete neuroactive histamine, serotonin, kinins, leukotriens, cytokine/chemokine and proteolytic enzymes, and mast cell activation has been previously reported in the CNS of neurodegenerative diseases, it is not known if GMF can activate mast cells to release inflammatory mediators. In the present study, mouse bone marrow‐derived mast cells (BMMCs) from Wild type (Wt) and GMF‐deficient (GMF‐KO) mice were incubated with GMF in vitro and the supernatant culture media were analyzed for CC ligand 2 (CCL2) release, which is implicated in neurodegenerative diseases. We demonstrate for the first time that GMF‐induced CCL2 release from BMMCs from Wt mice in a dose‐dependent and time‐dependent manner and that this release is lesser in mast cells derived from GMF‐KO mice. We suggest that GMF can activate mast cells and contribute to the neurodegeneration (supported by VA Merit Review award and by the National Institute of Neurological Disorders and Stroke grant NS47145).
In Silico and In Vitro Drug Screening for Novel Inhibitors of Human MDR1 P‐Glycoprotein
Authors: Brewer Frances Key , Follit Courtney A. , Rossi Isolina R. , Rossi Vincent J. , Vogel Pia D. , Wise John G.
Published: June 2021

Cancer chemotherapy failures often involve the over‐expression of ABC‐transporters like the MDR1 P‐glycoprotein (Pgp). These membrane transporters use ATP hydrolysis to move a wide variety of compounds including cytotoxins across the plasma membrane, a normally protective function for the cell. In chemotherapies, however, selection for cells over‐expressing transporters like Pgp results in cell populations that are resistant to the therapeutic agents. This results in cancer recurrence that is multidrug resistant and refractory to treatment. In recent work using targeted molecular dynamics methods, we uncovered some of the molecular details of the conformational changes Pgp undergoes during its catalytic transport mechanism (Biochemistry 2012 51:5125–41). The conformational changes elucidated in this study have been used in massively parallel in silico ligand docking studies employing a very large database of commercially‐available compounds (zinc.docking.org). Using novel screening procedures, we have been able to identify several compounds that differentially bind to ATP hydrolyzing power transducing structures and not to drug transporting structures within Pgp. These compounds are predicted to be effective inhibitors of Pgp. In vitro analysis of several of the identified leads demonstrated that a number of these compounds do inhibit Pgp. Supported by NIH 1R15GM094771–01A1 to PDV and JGW.
The role of Perilipin 5 in lipid droplet maturation
Authors: Saley Tara C. , Hardy Cara C , Tansey John T.
Published: June 2021

The perilipins are a family of 5 related proteins that have been shown to play important roles in the regulation of neutral lipid metabolism. These include the interaction of lipases and acyltransferases with the lipid storage droplet and dispersion, the fragmentation of the lipid storage droplet. Recent studies have shown that perilipin 5 exists in at least two cellular pools: the lipid storage droplet and the cytosol. We hypothesized that the cytosolic pool of perilipin 5 would translocate to the lipid storage droplet when the cells were presented with a lipid challenge. Immunofluorescence microscopy was utilized to assay movement of perilipin 5. Using a transfected CHO cell model, we were able to visualize fluorescently stained lipid storage droplets within 2 hours of incubation with oleic acid. Cells were pre‐treated with cycloheximide for 30 minutes then incubated for an additional 2 hours in oleic acid to promote lipid storage. Preliminary results indicate that the cytosolic pool of perilipin 5 translocated to the storage lipid droplets. These findings show that perilipin 5 plays a role in processing neutral lipid and development of the mature lipid storage droplet.
Mechanism of ATGL mediated changes in hepatic energy metabolism: role of LFABP
Authors: Ong Kuok Teong , Mashek Mara T. , Davidson Nicholas O. , Mashek Douglas G.
Published: June 2021

Adipose triglyceride lipase (ATGL) catalyzes the rate‐limiting step in triacylglycerol (TAG) hydrolysis in most tissues. We have previously shown that hepatic ATGL preferentially channels hydrolyzed fatty acids (FAs) to β‐oxidation and induces peroxisome proliferator‐activated receptor‐α (PPAR‐α) target gene expression. Nevertheless, the mechanisms that mediate this pathway are still unknown. We hypothesized that a FA carrier such as liver fatty acid binding protein (LFABP), which is known to influence FA oxidation and PPAR‐α, may be involved in transporting FAs from lipid droplets to mitochondria for β‐oxidation or the nucleus for ligand delivery. Thus, we employed the adenovirus‐mediated approach to suppress or overexpress hepatic ATGL expression in wild type (WT) or LFABP knockout (LFABP KO) mice. ATGL knockdown increased liver weight and TAG content and ATGL overexpression decreased liver TAG content to a similar extent in both strains of mice. As expected, the mRNA expression of PPAR‐α and its target genes were upregulated in WT mice injected with adenovirus overexpressing ATGL and decreased in mice treated with ATGL shRNA. Surprisingly, manipulating ATGL expression in LFABP KO mice resulted in nearly identical changes in PPAR‐α and its target genes compared to WT mice suggesting that LFABP is not required to carry FA ligands from ATGL to PPAR‐α. Furthermore, LFABP did not impair the effects of ATGL on TAG hydrolysis or oxidation of hydrolyzed FAs in primary hepatocyte cultures. Taken together, we conclude that LFABP, despite being a major intracellular FA carrier, does not mediate the effects of ATGL on PPAR‐α and is not required to channel ATGL‐hydrolyzed FAs to mitochondria for β‐oxidation. These data point to a more complex mechanism linking ATGL to downstream changes in hepatic energy metabolism.
Impact of high salt diet on Arylsulfatase B activity, glycosaminoglycans, kininogen, and bradykinin
Authors: Bhattacharyya Sumit , Kotlo Kumar , Feferman Leonid , Yang Bo , Linhardt Robert J. , Danziger Robert , Tobacman Joanne K.
Published: June 2021

Arylsulfatase B (ARSB; N‐acetylgalactosamine‐4‐sulfatase) removes sulfate groups from N‐acetylgalactosamine‐4‐sulfate residues at the non‐reducing end of chondroitin‐4‐sulfate (C4S). In this study, extending previous in vitro findings, we investigated the relationships among ARSB, C4S, kininogen, and bradykinin in Dahl salt‐sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets (total n=22). In renal tissue of SSH rats, ARSB activity was significantly less than in SSL rats, and C4S and total sulfated glycosaminoglycans were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the SSH renal tissue. In contrast, unsulfated, hyaluronan‐derived disaccharides were significantly increased in the SSL rats. In the SSH rats, with lower ARSB activity and higher C4S levels, cell‐bound, high‐molecular weight kininogen was significantly greater and urinary bradykinin was significantly lower (p<0.0001, unpaired t‐test, two‐tailed). ARSB activity in the SSH and SSL renal tissue and in normal rat kidney (NRK) cells declined with increasing exogenous sodium chloride concentration, but not with increased sodium acetate concentration. The effects of high chloride exposure in vivo on ARSB activity, chondroitin‐4‐sulfation, and kininogen‐C4S binding link dietary salt intake and regulation of bradykinin production from kininogen, although the mechanism whereby chloride regulates ARSB activity is not yet known.
Estrogen Receptor α Regulates ERK in MCF‐7 Cells
Authors: Rofelty Angela J. , Schmitt John M.
Published: June 2021

Estrogen (E2) signaling significantly affects breast tumorigenesis by enhancing cell growth and preventing apoptosis. The actions of estrogen in MCF‐7 breast cancer cells are mediated through a kinase pathway involving CaM KK, CaM KI, and ERK. Current research is examining the involvement of the Estrogen Receptor (ER) alpha (α) and beta (β) as well as G‐Protein Coupled Receptor GPR30 in cell growth and proliferation. ERα is suggested to be responsible for ERK and perhaps CaM Kinase activation. Our goal was to evaluate if ERα, rather than GPR30 or ERβ, mediates CaM KI and ERK activation upon treatment of MCF‐7 breast cancer cells with E2. E2 treatment of MCF‐7 cells led to a significant rise in CaM KI and ERK activation. This effect was blocked by pretreatment with the ERα antagonist MPP; however MPP did not block EGF stimulation of ERK. We then explored the role of ERβ in E2‐mediated ERK activation by pretreating cells with the ERβ inhibitor PHTPP. PHTPP did not reduce ERK phosphorylation, suggesting ERβ is not involved in activation of ERK. MCF‐7 cells were transfected with siRNA against ERα to further examine the role of ERα in ERK regulation. Knockout of ERα in MCF‐7 cells subsequently treated with E2 showed a significant decrease in ERK phosphorylation. To ensure the specificity of the siRNA for ERα, transfected cells were stimulated with EGF. ERK phosphorylation following EGF treatment was not inhibited in ERα‐knockout cells. Taken together our data suggests that ERα is capable of rapidly activating both CaM KI and ERK in MCF‐7 cells.
CaM KK Mediates MDM2 Activation in LNCaP Cells
Authors: Graber Philip , Schmitt John M.
Published: June 2021

Agonists and hormones that cause an influx of calcium in LNCaP prostate cancer cells activate the calcium/calmodulin‐dependent protein kinase (CaM Kinase) pathway and AKT phosphorylation. CaM KK and AKT are essential for promoting LNCaP cell survival. AKT phosphorylation of MDM2 protein may negatively regulate the tumor suppressor protein, p53. CaM KK and AKT have yet to be demonstrated as upstream regulators of MDM2 and p53 in LNCaP cells. Our goals were to examine the ability of carbachol and testosterone to stimulate MDM2 phosphorylation, its association with p53, and whether CaM KK is upstream of MDM2. Stimulation of LNCaP cells with carbachol increased MDM2 phosphorylation that was blocked by the CaM KK inhibitor, STO‐609 as well as the AKT inhibitor, AKT‐X. Similarly, testosterone also increased MDM2 phosphorylation through CaM KK and AKT. The requirement for CaM KK upstream of MDM2 was also examined using siRNA knockdown of CaM KK. Carbachol treatment of cells triggered phosphorylation of MDM2 that was blocked in cells transfected with siRNA against CaM KK. To assess the biochemical significance of signaling through CaM KK and MDM2 in LNCaP cells we assayed MDM2 association with p53. Carbachol stimulated MDM2 association with p53 in a manner that was dependent upon CaM KK. Our results suggest that carbachol treatment of LNCaP cells promoted phosphorylation of MDM2 and its association with p53 through CaM KK.
ARHGEF3.2 modulates Wnt‐PCP signaling in dorsal marginal zone of Xenopus embryos during early development
Authors: Yoon Jaeho , Heo Jee‐In , Kim Sung Chan , Park Jae‐Bong , Lee Jae‐Yong , Kim Jaebong
Published: June 2021

Rho guanine nucleotide exchange factor (RhoGEF) regulates various cellular processes including cell polarity, cytokinesis, and cell movements via activating small GTPase such as RhoA, Rac1 and CDC42. During gastrulation of Xenopus embryos, noncanonical Wnt signaling is the major signal pathway to regulate convergent and extension cell movements in DMZ region. In previous publications, activin induces dorsal mesoderm and noncanonical Wnt signaling is highly activated in dorsal mesoderm to promote cell movements. Although about 70 GEF distinct GEF homologues have been studied in humans, few GEFs of Xenopus are only identified and explored for its function during early embryogenesis.

In this study, we showed that EST gene (assesion No. XL.3374), which has been named xARHGEF3.2 was induced by activin treatment in ectodermal explants and expressed at dorsal marginal region in gastrula embryo. It was constructed to explore the role of xARHGEF3.2 during gastrulation. Over‐expression / knockdown of ARHGEF3.2 caused gastrulation defects. Furthermore, Biochemical analysis showed that xARHGEF3.2 activated RhoA through interaction with Dishevelled and Daam‐1.

Taken together, our results suggest that xARHGEF3.2 is induced by activin signaling and modulates convergent and extension cell movement via Wnt‐PCP signaling during the early Xenopus embryos development
A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1
Authors: Bar‐Peled Liron , Chantranupong Lynne , Cherniack Andrew , Chen Walter , Ottina Kathleen , Grabiner Brian , Spear Eric , Carter Scott , Meyerson Matthew , Sabatini David M
Published: June 2021

The mTOR Complex 1 (mTORC1) pathway promotes cell growth in response to many cues, including amino acids, which act through the Rag GTPases to promote mTORC1 translocation to the lysosomal surface, its site of activation. Although progress has been made in identifying positive regulators of the Rags, it is unknown if negative factors also exist and whether they are mutated in cancer. Here, we identify GATOR as a complex that interacts with the Rags. GATOR is composed of two subcomplexes we call GATOR1 and 2. Its GATOR1 subunits have been implicated as tumor suppressors and their inhibition makes mTORC1 signaling insensitive to amino acid deprivation. In contrast, inhibition of GATOR2 subunits suppresses mTORC1 signaling, and epistasis analysis reveals that GATOR2 is an upstream negative regulator of GATOR1. Furthermore, the GATOR1 subcomplex has GTPase activating protein (GAP) activity for RagA and RagB, consistent with its inhibitory role in mTORC1 signaling. In cancer cell lines containing GATOR1 inactivating mutations, mTORC1 is hyperactive and resistant to amino acid regulation, and these cells are hypersensitive to rapamycin, an FDA‐approved mTORC1 inhibitor. Thus, we identify the GATOR complex as a critical regulator of the pathway that signals amino acid sufficiency to mTORC1 and reveal that, like other mTORC1 regulators, the Rags can be deregulated in cancer.
Closing the conceptual gap between self‐perception and reality in obesity prevention
Authors: Song Hee‐Jung , Grutzmacher Stephanie K
Published: June 2021

Objective

The self‐perception of caloric intake and a healthy diet is an important indicator for lifestyle modification. The conceptual gap between the self‐perception and objective status attenuates the effectiveness of health programs. The purpose of this study is to present the conceptual gap between self‐perception and objective status to clarify problems and corresponding intervention strategies.

Design

Cross‐sectional study. 30–60 minute in‐person interviews (n=151) were conducted.

Setting

11 Supplemental Nutrition Assistance Program (SNAP) regional offices located in five counties in Maryland

Subjects

A convenience sample of 151 SNAP eligible adults

Results

Participants’ perceived adequacy of caloric intake was inconsistent with their weight status showing a significant conceptual gap. 65.1% had BMI ≥ 25, but only 40.8% perceived that their caloric intake is “too high” and 36.4% reported they have a weight problem. Also, a majority of people (range 67.1 – 96%) perceived that it is important to follow a healthy diet, but only 37.1% had adequate health literacy, implying that their perception could not be supported by their literacy level.

Conclusions

Broad consensus about the importance of following dietary guidelines has been established in the low‐income, SNAP eligible population, but significant conceptual and literacy gaps exist for translation. Exploring whether these gaps result from insufficient health literacy or a lack of internalization process is needed for future intervention strategies.
A survey of added sugars within the american food supply
Authors: Babula Katherine , Kavanagh Mary , Melanson Kathleen
Published: June 2021

Although consumers receive much advise on consumption of added sugars, little information is available on their presence in the food supply. Thus, we recorded the listing of added sweeteners in every packaged food item in two locations of a national supermarket chain in RI. Ingredient lists were read for High Fructose Corn Syrup(HFCS), other caloric sweeteners, non‐caloric sweeteners, and no added sweetener. Frequencies of each added sweetener classification were calculated for each of 25 main food groupings. Of note, ‘snack cakes’ had 33.9% of the items containing HFCS and 66.1% containing other caloric sweeteners, whereas ‘cookies’ had 62.1% of the items containing HFCS and 37.9% containing another caloric sweetener. These two food groups, which seem similar in nature, in reality have inverse sugar profiles. In the ‘Soda’ group, 40% contained HFCS, 17% other added sweeteners, 37% non caloric sweeteners, and 6% no added sweetener. Sports drinks where very low in HFCS with only 1.6%, and an even split between caloric sweeteners and noncaloric sweeteners at 44.3%, while 9.8% had no added sweeteners. The ‘condiments’ group had 48.4% containing HFCS, 30.3% other caloric sweeteners, 0.8% noncaloric sweeteners, and 20.5% no added sugars. The pronounced presence of sugars and sweeteners in the packaged food supply demonstrates the high degree of permeation that has occurred in the American food market.
Access to fruits, vegetables, low fat milk and low fat meat is limited in rural Oklahoma
Authors: Yuhas Jonathan , Betts Nancy , Hildebrand Deana
Published: June 2021

Oklahoma consistently has one of the nation's highest prevalence rates of overweight and obesity and this is expected to worsen. Recommendations to reduce obesity focus on increased fruits, vegetables, low fat milk and low fat meats. However, availability and cost of these foods are uncertain in many areas. The purpose of this project was to describe the availability and relative cost of recommended foods in a rural county of Oklahoma. Stores selling food were tagged by county sanitarians and the stores were located using GIS. Community members were trained to inventory stores using an adapted Nutrition Environment Measurement Survey. Among the 81 stores, 74% did not carry fresh fruit and 84% did not sell fresh vegetables. Nearly 90% did not sell frozen fruit or vegetables. Canned fruit was available in nearly 52% of stores and all of these sold fruit canned in juice or water. Vegetables were available at only 34% of stores with 7 stores having low sodium options. Nearly 50% did not sell low fat milk and 91% did not carry low fat ground beef. Among the stores that sold low fat milk, price comparisons of quarts and half gallons showed no significant differences. The average price of 90% lean ground beef was significantly higher than 80% lean ($4.30 and $3.53, respectively; p=.005). Attempts to promote food intake compatible with weight loss must include methods for increasing access to fruits, vegetables, low fat milk and low fat meat. Supported by Oklahoma Tobacco Settlement Endowment Trust.
Agriculture, Income and Nutrition Linkages in India: Findings from a Nationally Representative Survey
Authors: Kadiyala Suneetha , Bhagowalia Priya , Headey Derek
Published: June 2021

In India, the glacial reduction in the prevalence of undernutrition in the face of strong economic growth has raised the possibility that undernutrition is disconnected from economic growth in general and perhaps from agricultural growth in particular.

We utilize India Human and Development Survey (IHDS), a nationally representative survey covering 41,554 households in rura and urban areas. Using Ordinary Least Squares and Instrumental Variable regression techniques, we test if agricultural income, irrigation and land ownership determine agricultural production patterns, household dietary diversity and child anthropometry.

The income gradient for undernutrition is very weak, with only the richest quintile showing 0.5 HAZ gradient compared the poorest (p<0.001). Economic status and land ownership are negatively correlated with the budget shares of cereals, pulses and vegetables and positively correlated with milk. Irrigation and crop diversity predict household diet diversity, but neither the household dietary diversity score nor the food group budget shares significantly explain HAZ or WHZ. Access to safe water and sanitation, antenatal check‐ups and vaccinations predict child anthropometry.

Agricultural programs aimed at irrigation, crop and livestock diversification along with direct nutrion and health interventions can improve nutrition outcomes in India.
A label‐free photonic crystal biosensor for the assessment of iron status using ferritin
Authors: Peterson Ross David , Cunningham Brian , Andrade Juan , Engeseth Nicki
Published: June 2021

Identification of populations at risk of iron deficiency is of critical concern. We present an application of photonic crystal (PC) biosensors as a diagnostic test for ferritin, a biomarker for iron deficiency. PCs are periodic dielectric surface structures designed to reflect a narrow band of wavelengths when illuminated by a broadband light source. Adsorption of biomolecules causes the reflected wavelength to shift to greater values, providing a simple mechanism for label‐free detection using available detection instruments. PC surface was functionalized with silane for immobilization of primary ferritin antibodies, followed by a blocking step to inactivate remnant silane binding sites. Sensitivity was tested using serial dilutions of human liver ferritin (450 kDa) from 5.0 to 0.15 μg/ml; and a secondary antibody to amplify initial signal. Specificity of the assay was verified by evaluating biosensor binding to BSA. The PC sensor gave a concentration‐dependent response for ferritin (R2= 0.986). When compared to commercial ELISA kit, ferritin response provided comparable limits of detection. Antibodies showed high specificity to ferritin. A 40‐minute assay protocol for label‐free detection of ferritin provided dose‐dependent response in the physiologically relevant concentration range for ferritin in serum; a step towards a simple point‐of‐care assay for iron deficiency diagnosis.

Grant Funding Source: College of ACES DNS 20/20