bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the redox potential in the endocytic pathway of different cell types by imaging live cells via FRET
Authors: Zilikana Sandrine , Lee Juhee , Provoda Chester , Lee Kyung-Dall
Published: June 2021

Sub‐cellular compartments have different redox environments and these electrochemical differences play a vital role in their distinct functions. Although much research has been conducted on the mitrochondria, nuclei, secretory pathway, and extracellular space, much remains to be elucidated regarding the redox potential in the endocytic pathway. Additionally, although some cells have been studied in this regard such as macrophages and dendritic cells, there exists no real comparison across different cell as to whether there is any variance in the redox potential across the endocytic pathway. We used fluorescence resonance energy transfer (FRET)‐ based redox‐sensitive fusion protein probes to compare FRET signals across cancer cells (PC3 and BT549 cell line), fibroblasts, and bone marrow‐derived dendritic cells in order to draw conclusions about the redox potential in the endocytic pathways. We found that the redox‐sensitive reporter encapsulated inside pHsensitive liposomes could be taken up by the different cells and tracked live by using FRET. Our results show that all the endosomes have a reducing environment although to a different degree depending upon the cell tested. In comparing the redox data with previously obtained Bone Marrow Macrophage (BMMO) data, our preliminary results show that all the cells tested were less reducing than the BMMOs but further study is needed to determine any statistical differences in comparison to each other. These findings may have implications for drug delivery via the endocytic pathway across different types of cells.
An Automated Reinvention of the Western Blot ‐ A Simple Western Analysis of the AKT Pathway Signaling Cascade
Authors: Boge Annegret , Ramirez Francisco , Nguyen Uyen , Kazakova Irina , Dermody Jessica , Yang Tom , Gavin Robert
Published: June 2021

Protein kinase AKT is a central player for a variety of cell stimuli affecting downstream events such as the inhibition of apoptosis, regulation of glycogen synthesis, and cell cycle regulation. Many of these events are routinely assessed by Western blot analysis which exploits high sensitivity of enzyme amplification as well as specificity of antibody binding. The process itself, however, is very labor intensive and has not significantly changed since it was first introduced. Simple Western assays utilize capillary electrophoresis, a powerful separation technique with high resolution and reproducibility, and incorporate sample separation performed in capillaries followed by immobilization of the proteins directly onto the capillary walls, followed by immunodetection. Data comparing analysis of key targets of the AKT signaling cascade via Western blot and Simple Western highlighting workflow, biological response, sensitivity, and resolution is presented.

Conclusions

Simple Western assays are a reinvention of the Western blot, automating all the manual steps associated with the process. Highly reproducible, quantitative, reliable and sensitive assessments of key targets in the AKT signaling cascade were observed. Additionally, total time to results was dramatically pared down to 3–5 hours in a walk‐away mode with a total hands‐on time of less than an hour.
Improved antibody immobilization using aniline‐catalyzed aldehyde‐hydrazide chemistry for the study of protein‐protein interactions
Authors: Meier Scott M , Bomgarden Ryan , Etienne Chris , Opperman Kay , Kaboord Barb
Published: June 2021

Covalent attachment of an antibody to a beaded support before immunoprecipitation prevents antibody contamination upon antigen elution and allows for the detection of antigens with similar molecular weights as antibody heavy and light chains. Common methods of antibody immobilization include direct coupling of antibody amino groups to aldehyde supports followed by reduction and indirect coupling of antibodies to Protein A/G‐coated beads followed by amine‐reactive crosslinking. Drawbacks of these methods include the loss of antibody functionality due to chemical modification or obstruction of the antigen binding site. An alternative approach for the covalent attachment of antibody to a beaded support utilizes oxidization of sugars in the antibody Fc region to aldehydes, which react with a hydrazide‐activated support. This method allows for unobstructed and oriented antibody conjugation without adverse modifications to antigen binding sites. However, compared to amine‐reactive antibody crosslinking, aldehyde‐hydrazide coupling often has lower efficiency and requires significantly longer incubation times. In order to improve this conjugation chemistry, we developed new aldehyde‐hydrazide immobilization and immunoprecipitation kits. These kits include aniline as a catalyst in a buffer with optimized pH for increased antibody coupling efficiency. The addition of aniline results in an increase of coupling efficiency from 50% to 90% for most antibody subtypes, and shortens reaction times to less than 4 hours. In addition, we demonstrate improved immunoprecipitation of Epitheleal Growth Factor Receptor (EGFR) and Signal Transducers and Activator of Transcription 1 Protein (STAT1), as well as the co‐immunoprecipitation of endogenous Cyclin B1/Cyclin Dependant Kinase 1 (Cdk1) complex from cell lysates using 2–10μg of immobilized antibody.
Robust and sensitive control of nitrogen sequestration in Escherichia coli
Authors: Kim Minsu , Zhang Zhongge , Yan Dalai , Groisman Alex , Hwa Terry
Published: June 2021

The sequestration of essential nutrients is vital for the growth and survival of both prokaryotes and eukaryotes. Some nutrients, in particular gas molecules such as CO2 and NH3, are readily diffusible across the cell membrane. The large membrane permeability of these molecules is both a blessing and a curse: It obviates the need of transporters when the ambient nutrient level is high; however, when the ambient level is low, maintaining a high intracellular nutrient level against passive back diffusion is both challenging and costly. Here we study the delicate management of ammonium sequestration by exponentially growing E. coli cells, using microfluidic chambers which provide steady, low ammonium concentrations not attainable in batch or continuous culture. Ammonium is the preferred nitrogen source for many microorganisms including E. coli, and the membrane protein AmtB is crucial for maintaining rapid cell growth at low ambient ammonium levels. Using a combination of quantitative gene expression and flux analysis, we show that E. coli cells sharply activate ammonium transport and maintain the internal ammonium concentrations at a steady level barely above the minimum needed for rapid cell growth in ammonium‐poor conditions. The integration of known molecular interactions suggests that these can be implemented molecularly by a simple integral feedback scheme without the need of cooperative molecular interactions. The exquisite control of AmtB activity ensures that the expensive cost of upholding the internal ammonia level be kept at a minimum, a survival strategy possibly employed by a variety of organisms in dealing with essential but rapidly diffusible substances.
A Mitochondrial Lipid Phosphatase in Cell Metabolism and Membrane Organization
Authors: Zhang Ji , Guan Ziqiang , Murphy Anne , Wiley Sandra , Perkins Guy , Worby Carolyn , Engel James , Raetz Christian R.H. , Dowhan William , Dixon Jack
Published: June 2021

Cardiolipin, the signature phospholipid of mitochondria, plays pleiotropic roles in regulating the membrane integrity and activities of the organelle. The cardiolipin biosynthetic pathway is well characterized, but the mammalian enzyme that catalyzes the dephosphorylation of phosphatidylglycerophosphate (PGP) to phosphatidylglycerol (PG) has been long sought. Herein we demonstrated that PTPMT1, the first protein tyrosine phosphatase localized to the mitochondrion, is the elusive PGP phosphatase. Whole body deletion of Ptpmt1 in mice led to embryonic lethality, suggesting an indispensable role for PTPMT1 during mammalian development. Surprisingly, lipid analysis of Ptpmt1‐deficient fibroblasts revealed an accumulation of PGP along with a concomitant decrease in PG content. We further demonstrated that PTPMT1 specifically dephosphorylated PGP in vitro. Moreover, loss of PTPMT1 led to dramatic diminution of cardiolipin, inhibition of mitochondrial respiration and mitochondrial fragmentation. Together, our study identifies PTPMT1 as a major mammalian PGP phosphatase and points to its role as a novel player in cardiolipin biosynthesis.

This work was supported by grants from the National Institutes of Health. (DK18024 and NIDDK DK18849 to J.E.D.), and the Larry L. Hillblom Foundation (2007‐D‐016‐FEL) to J.Z.
Identification of a novel phosphatidate phosphatase gene PAH2 from Saccharomyces cerevisiae
Authors: Chae Minjung , Carman George M.
Published: June 2021

Phosphatidate (PA) phosphatase catalyzes the dephosphorylation of PA to yield diacylglycerol (DAG) and Pi. In the yeast Sacchromyces cerevisiae, the enzyme activity is encoded by the PAH1, DPP1, and LPP1 genes. However, cells lacking these genes still exhibit PA phosphatase activity indicating the presence of another gene(s) encoding the enzyme. In this study, we purified this activity with an aim to identify the unknown gene(s) for PA phosphatase. We purified PA phosphatase activity from membranes of the pah1Δ dpp1Δ lpp1Δ triple mutant by a nine‐step purification scheme, which included chromatography with phosphocellulose, Affi‐gel blue, phenyl Sepharose, Mono Q, and Superdex 200. The purification scheme resulted in an 8,300‐fold enrichment of PA phosphatase activity. Mass spectrometry analysis of proteins from SDS‐polyacrylamide gels revealed multiple proteins that contained putative phosphatase motifs. The analysis of PA phosphatase activity in available mutants lacking genes encoding proteins with the phosphatase motifs led to the identification of a novel PA phosphatase gene that we named PAH2. Supported by NIH grant GM‐28140.
Human Lysyl Oxidase‐like 2 in Breast Cancer Metastasis
Authors: Mure Minae , Xu Li , Rebecchi Kathryn
Published: June 2021

Recently, human lysyl oxidase‐like 2 (HLOXL2) has gained much attention as a novel therapeutic target for metastatic breast cancer. Currently, there are no crystal structures available for HLOXL2 and its function at the molecular level has not been defined. We characterized the extent of post‐translational modifications (PTMs) and catalytic activity of recombinant HLOXL2s as well as native HLOXL2 in MDA‐MB‐231. HLOXL2 is N‐glycosylated, mostly secreted in the extracellular matrix (ECM), and undergoes proteolytic activation. In addition to the secreted HLOXL2, we also detected cytosolic HLOXL2 that is not glycosylated. We prepared MCF‐7 cells stably expressing the wild‐type and a series of mutant forms of recombinant HLOXL2, and examine the effect of PTMs and catalytic activity on epithelial‐to‐mesenchymal transition (EMT). We found that the extracellular HLOXL2 is N‐glycosylated (Asn288, Asn455 and Asn644), and the latter two glycosylation is essential for secretion and proteolytic activation. Unglycosylated HLOXL2 accumulates in the cytosol, mostly in the peri‐nuclear region. The cytosolic and peri‐nuclear HLOXL2 are catalytically competent comparable to the secreted HLOXL2. We propose that the peri‐nuclear localized HLOXL2 has the prominent effect in EMT where LOX catalytic activity is essential.
Antioxidant protein regulates ionizing radiation‐induced apoptosis
Authors: Park Jeen-Woo , Kim Sung Youl , Yang Eun Sun
Published: June 2021

Organisms exposed to ionizing radiation (IR) undergo increases in the production of reactive oxygen species (ROS), which are determinant components in the induction of apoptosis. Sensitive to apoptosis gene (SAG) encodes a redox‐inducible and apoptosis‐protective antioxidant protein. This report demonstrates that the modulation of SAG expression in cultured cells regulates IR‐induced apoptosis. A protective role for SAG against IR‐induced apoptosis was found in U937 cells transfected with SAG cDNA. A significant decrease in the endogenous production of ROS was also observed in SAG over‐expressing cells, compared to control cells, exposed to 2 Gy gamma‐irradiation. These results suggest that SAG plays an important role in regulating IR‐induced apoptosis, presumably by maintaining the cellular redox status. Because SAG is over‐expressed in many human cancers, targeting SAG expression may have therapeutic value in cancer treatment. Transfection of the pancreatic cancer cell line PC3 with SAG small interfering RNA markedly attenuated the expression of SAG, augmenting their susceptibility to IR‐induced apoptosis. The knockdown of SAG expression by RNA interference combined with radiotherapy may be a potential method for radiosensitization.
Differential apoptotic response in normal and virus‐transformed human cells
Authors: Maczis Melissa Ann , Birckbichler Paul J
Published: June 2021

Human fibroblasts (WI‐38) and their simian virus‐transformed counterparts (VA13a), a paired cell system, represent a unique model for experimentation. WI‐38 cells demonstrated contact inhibition of growth, whereas the VA13a counterpart continued to proliferate forming multiple cell layers. The objective of the study was the response of WI‐38 and VA13a cells to exposure of sodium butyrate, calcium ionophore (A23187), or a combination of sodium butyrate and calcium ionophore. We hypothesize that exposure of the cells to these chemicals will elicit a differential response in apoptosis. The VA13a cells and WI‐38 cells underwent apoptosis within different time periods of exposure to the chemicals. In addition, sodium butyrate‐treated VA13a cells showed an altered “normal” morphology, whereas the treatment of sodium butyrate on WI‐38 cells had little to no effect on morphology. The combination treatment of calcium ionophore and sodium butyrate had the same affects on the WI‐38 and VA13a cells as the calcium alone. The results suggest calcium ionophore is a stronger apoptosis inducer. Applied clinically, this approach has the potential to produce a differential chemical response in the transformed (cancer) cells. Sponsored by College of Health, Environment, and Science and Slippery Rock University.
Ghrelin Prevents TBI‐induced Neuronal Apoptosis via Induction of Mitochondrial Uncoupling Protein 2 (UCP2)
Authors: Gaston Lindsey Sara , Lopez Nicole
Published: June 2021

Traumatic brain injury (TBI) is a significant cause of morbidity and mortality in humans. Presently, no pharmacologic agent exists to prevent neuronal loss; however, the second wave of cellular death that follows TBI occurs within a timeframe amenable to therapeutic intervention. Previous research has demonstrated that transient mitochondrial uncoupling may help prevent initiation of apoptosis via the intrinsic pathway. Among the several endogenous mitochondrial uncoupling proteins identified, UCP2 has been shown to be expressed throughout the central nervous system and to provide a protective role in a variety of neural injury models. Moreover, production of UCP2 is upregulated when levels of ghrelin, a protein with anti‐inflammatory and anti‐apoptotic properties, are elevated. Our study sought to correlate these two observations in an experimental model of TBI. We hypothesized that ghrelin administration would inhibit neuronal apoptosis via induction of UCP2. BALB/c mice were divided into sham, TBI, and TBI/ghrelin treatment groups, and TBI was induced with a weight drop model following anesthesia. Neuronal apoptosis was characterized by staining coronal brain sections with the TUNEL method, and relative expression levels of UCP2 and the apoptotic protein caspase‐3 were analyzed with immunohistochemistry. We found that increased UCP2 expression in response to TBI/ghrelin treatment correlated with decreased neuronal apoptosis, suggesting that ghrelin treatment may provide an amenable therapeutic target for reducing neurodegeneration following TBI.
N‐Glycans in cell survival and death: cross‐talk between glycosyltransferases
Authors: Banerjee Dipak K , Banerjee Aditi , Zhang Zhenbo , Espinoza Lydia A , Torres Marina , Placido Jenny S , Baksi Krishna
Published: June 2021

Asparagine‐linked (N‐linked) protein glycosylation is one of the most important protein modifications. Its importance has been realized in cellular processes including neo‐vascularization (i.e., angiogenesis), a process essential for breast tumor progression. Mannosylphopsho dolichol synthase (DPMS) a positive regulator of N‐acetylglucosamiyl 1‐phosphate transferase (GPT) of the N‐glycan pathway is catalytically upregulated by phosphorylation. Conserved phosphorylation motif has been found in Dpm gene cloned from 35 different species. As a result, triggering of intracellular cAMP activates DPMS and increases protein N‐glycosylation which gets translated in increased angiogenesis. This is reproducible in cells overexpressing DPMS. Interestingly, when ER stress is induced with a GPT inhibitor, tunicamycin cells undergo apoptosis due to unfolded protein response (upr). The DPMS activity is abolished at a rate faster than the cell cycle arrest and the induction of apoptosis. DPMS protein or the mRNA remained higher. Additionally, the DPMS mRNA is synthesized and processed. We then hypothesized that DPMS expression would remain high in a double negative breast tumor microvasculature treated with tunicamycin. Analysis of DPMS expression in tumor tissue sections confirms the hypothesis. Supported by Komen for the Cure BCTR06582; and NIH/NCRR/RCMI G12‐RR03035 (KB) grants.
Leptin Reduces hypoxia‐reoxygenation‐induced Fas/FasL gene overexpression and apoptosis in human L02 cells
Authors: Zhou Shaoli , Chen Guihua , Li Xiaoyun , Li Shangrong , Guo Na , Irwin Michael G , Xia Zhengyuan , Hei Ziqing
Published: June 2021

Fas/FasL activation induces caspase‐3 activation that may exacerbate hepatic ischemia‐reperfusion injury (IRI) by increasing apoptotic cell death. Leptin, an endogenous cytokine, facilitates cell growth and attenuates hypoxic brain injury (Stroke, 2007;8:2329–2336). We postulated that leptin may attenuate hypoxia/reoxygenation‐induced hepatocyte injury by reducing apoptosis and suppressing Fas/FasL gene expression. Human hepatic L02 cell IRI was induced by exposing the cell to hypoxia for 12 hours followed by 12 hours reoxygenation in the absence or presence of different concentrations of leptin (100 μg/L, 200 μg/L, 400 μg/L, 800 μg/L and 1600 μg/L). Cell survival rates were significantly reduced in cells subjected to IRI compared to control cells receiving normoxia accompanied with increased apoptotic index as measured by flow cytometry and terminal dUTP nick‐end labeling, which was coincident with increased gene expressions of Fas and FasL (all P<0.01, group IRI vs. control). All the above changes were significantly attenuated by treatments with various concentrations of leptin (all P<0.05, vs. group IRI), while the optimal effects were seen at leptin concentration of 400 μg/L. It is concluded that leptin may have reduced hypoxia/reoxygenation‐induced hepatic L02 cell apoptosis by down‐regulating Fas/FasL gene expression.

Supported by a Doctoral Start‐up Foundation grant in (NO. 10451008901004797)
Psoralidin, a dual inhibitor of COX‐2 and 5‐LOX, regulates ionizing radiation (IR)‐induced pulmonary inflammation
Authors: Yang Hee Jung , Youn HyeSook , Seong Ki Moon , Kim Eun Gi , Youn BuHyun
Published: June 2021

Radiotherapy is the most significant non‐surgical cancer treatment, however it is restricted by two major problems: radioresistance and normal tissue damage. We focused on radiation‐induced normal cell damage, and are concerned with inflammation as a main limiting factor in the radiotherapy. Psoralidin, a coumestan derivative isolated from the seed of Psoralea corylifolia, has been studied for various biological properties. However, little is known regarding its effects on IR‐induced pulmonary inflammation. The aim of this study is to investigate mechanisms of IR‐induced inflammation and to examine therapeutic mechanisms of psoralidin. We demonstrated that IR‐induced ROS activated COX‐2 and 5‐LOX pathway in HFL‐1 and MRC‐5 cells. Psoralidin inhibited the IR‐induced COX‐2 expression and PGE2 production through regulation of PI3K/Akt and NF‐κB signaling. Also, psoralidin blocked IR‐induced LTB4 production through a direct interaction of psoralidin and FLAP. IR‐induced fibroblast migration was notably attenuated by psoralidin. Moreover, psoralidin suppressed IR‐induced expression of pro‐inflammatory cytokines and ICAM‐1 in vivo. Taken together, our findings reveal a regulatory mechanism of IR‐induced pulmonary inflammation in human normal lung fibroblast and mice, and suggest that psoralidin may be useful as a potential radiopreventive agent against radiation‐induced normal tissue injury.
The Role of the President in Guiding the University: Learners, Lawyers and Legislators
Authors: Kelley Robert Otis
Published: June 2021

The university president sets the tone for the entire academic institution. The emphasis given to each portion of the university's mission, teaching and learning, research and scholarship, and community engagement, is detected quickly in the activities and attitudes of faculty, staff and students. Rhetoric, personal commitment, and individual dedication to the mission, communicated by the president, are central to the morale and success of the institution. In this regard, it becomes important for the president to prepare for such a role by understanding, to some extent through personal and professional experience, the major components of the institution being served. Direct experience in teaching, research, scholarship, and creative work, and engagement with the several stakeholders that support the university may contribute to the president's ability to manage and lead through the multitude of challenges that face universities and colleges today. These challenges include working with legislators for appropriations, if the institution is state supported; working with governing boards to assure policy compliance and productive working relationships with institutions within a university/college system; dealing with a plethora of local, state and federal legal issues; and defending the institution through legal avenues when necessary. In addition to understanding the many moving parts of a large institution, the president must continue to be mentored by individuals who share common goals and the desire to advance the standing of the university. Furthermore, these mentors may no longer be in the higher education sector, depending upon the specific challenges facing the university. Determination of a successful presidency will be measured against outcomes that include development of faculty and staff, facilities that serve the contemporary mission of the institution, and an advanced learning experience that prepares students for success in a global environment.
Peripheral nerve morphometry: From neurosciences to clinical applications
Authors: Fazan Valeria Paula S.
Published: June 2021

Quantitative methods are important tools for experimental neuropathies studies. Morphometry has been used to define normal parameters of human nerves and also for laboratory animals, since obtained data are useful for comparisons with injured nerves. Morphometry can be performed manually or with the aid of semi‐automated or fully automated computer‐assisted programs. Manual morphometry has been usually described as tedious, time‐consuming, difficult to perform correctly and subject to many sources of errors. The above considerations might suggest that fully automated, computadorized image analysis systems could be ideally programmed to analyze myelinated fibers. However, literature reports indicated that operator intervention is necessary to manually eliminate dark tissue elements such as pericytes and Schwann cell nuclei. Thus, semi‐automated methods are widely used for peripheral nerve morphometry. The present talk will discuss the advantages of each technique available for nerve morphometry and the clinical applications for their use.
The Association of Leishmania RNA Viruses with metastatic or mucocutaneous Leishmaniasis in South America
Authors: Beverley Stephen M. , Lye Lon-Fye , Owens Katherine , Hickerson Suzanne M. , Kuhlmann F. Matt , Acino Erin , Dobson Deborah , Akopyants Natalia , Pratlong Francine , Bastien Patrick , Fasel Nicolas
Published: June 2021

Leishmaniasis is a spectral disease caused by the Trypanosomatid protozoan parasite Leishmania. One manifestation is a disfiguring mucocutaneous form (MCL) involving dissemination to nasopharyngeal areas and tissue destruction, caused by species from the South American Leishmania subgenus Viannia. We reported previously that metastatic L. guyanensis bear high levels of a novel dsRNA totivirus (LRV1), while non‐metastatic lines have low levels or lack LRV1. LRV1‐infected Leishmania were associated with a hyper‐inflammatory response dependent on the host Toll‐like receptor 3 (TLR3) signaling. Thus, LRV in metastatic parasites subverts the host immune response and promotes dissemination. This is the first parasite factor implicated causally in phenotypes relevant to metastatic mucocutaneous disease, with LRV acting as a ‘parasite within a parasite’. The closely related species L. braziliensis is responsible for the great majority of human MCL. Thus we are surveying L. braziliensis strains to establish the distribution and potential for clinical association(s) of LRVs in parasites taken from humans. Several novel LRVs were identified; these are currently being characterized by virus and genome sequencing, classical virological approaches, and infectivity tests of the LRV‐bearing L. braziliensis in animal models.
A reversible acetylation system in mycobacteria
Authors: Xu Hua , Hegde Subray , Blanchard John S.
Published: June 2021

Recent proteomics studies have revealed that protein acetylation is an abundant and evolutionarily conserved post‐translational modification from prokaryotes to eukaryotes. Although an astonishing number of acetylated proteins have been identified, the acetyltransferases that target these proteins remain largely unknown. Here we characterized a reversible acetylation system in Mycobacterium smegmatis, which includes MSMEG_5458 and MSMEG_5175. We show that MSMEG_5458 is a protein acetyltransferase (MsPat) that specifically acetylates the ε‐amino group of a highly conserved lysine residue in acetyl‐CoA synthetase (ACS). This acetylation results in the inactivation of ACS activity. Interestingly, acetylation by MsPat is dependent on 3′,5′‐cyclic adenosine monophosphate (cAMP), indicating that MsPat is a downstream target of the intracellular cAMP signaling pathway. In addition, we show that MSMEG_5175, a sirtuin‐like deacetylase, as well as its orthologue in M. tuberculosis Rv1151c, reactivates acetylated ACS through an NAD+‐dependent deacetylation. Therefore, Pat and the sirtuin‐like deacetylase in mycobacteria constitute a reversible acetylation system that regulates the activity of ACS. Currently we are searching for other targets of this system.
Solution Structures and Models Describing the Thioredoxin System from Mycobacterium tuberculosis
Authors: Neumann Terrence S , Olson Andrew , Cai Sheng , Sem Daniel
Published: June 2021

Mycobacterium tuberculosis (M. tb) resists oxidative killing in part by using the thioredoxin (Trx) system. Trx's catalyzes thioldisulfide exchange reactions using redox active cysteine thiols to reduce disulfides of other essential proteins, including metabolically essential enzymes. Oxidized Trx is reduced by thioredoxin reductase (TrxR) in an NADPH dependant reaction. The M. tb Trx system consists of three Trx's (TrxA, TrxB, and TrxC) and one Trx reductase (TrxR). TrxB and TrxC are known substrates of TrxR, while TrxA has been reported to not bind to TrxR, and to possibly be “cryptic.” The M. tb Trx system is dissimilar to the human Trx system (25–35% identity) indicating inhibitor specificity for the M. tb Trx system is achievable. The M. tb Trx system appears to be a viable anti‐tuberculosis drug target.

This study's objective was to calculate NMR solution structures of oxidized and reduced Trx's from M. tb. Structures were calculated using standard NMR methods and indicate that TrxA is wellfolded in oxidized and reduced states. Structures of the Trx's and binding models of the TrxN(N=A,B, or C)‐TrxR, constructed from NMR titrations of each 15N enriched TrxN and TrxR, will be presented. These binding models show a pocket between the Trx and the TrxR which are being used to identify uncompetitive inhibitors that would be effective as drugs even at saturating substrate (Trx) levels. Supported by NIH GM085739.
Investigating the cytotoxic effects of mycobacteriophage Vix Gene 80
Authors: Goodman Danielle , McDonough Virginia , Stukey Joseph
Published: June 2021

A bacteriophage is a virus that infects and replicates in bacteria. To reproduce, the phage must convert the host cell metabolism from that suited for cell survival to one favorable for producing new phage progeny. One mechanism by which phage can cause host cell conversion is by expressing ‘cytotoxic’ phage gene products that interact with and inhibit the function of specific protein targets of key metabolic pathways of the bacterial host. Identifying ‘cytotoxic’ phage genes and understanding more about how phage exploit their host's weaknesses could lead to new and important therapies for many bacterial illnesses. In this work, a cytotoxic gene from mycobacteriophage Vix, gene 80, was studied. The protein of the Vix80 gene is 68% identical to the product of gene 77 of mycobacteriophage L5, a gene that has been shown to be cytotoxic to Mycobacterium. We confirmed that the Vix80 gene product is also cytotoxic when expressed in Mycobacterium smegmatis. Our hypothesis is that a physical interaction between the Vix80 gene product and a specific host cell protein affects cell metabolism and causes growth inhibition. A 6xHis tagged Vix80 protein was created and expressed in E. coli, and then purified by column chromatography. SDS‐PAGE and staining verified that we had expressed and purified the Vix80 protein of correct size. We are using the His‐tagged Vix80 protein in a biochemical approach to identify the interacting host cell protein. Supported by an ASBMB Undergraduate Research Award, and a Summer Research Scholar Award from Hope College Biology Department
Dimensions of workplace breastfeeding support and the duration of breastfeeding
Authors: Bai Yeon , Wunderlich Shahla M , Fly Alyce D
Published: June 2021

The purpose of this study was to examine the relationship between breastfeeding duration and perception of workplace support. Working mothers (N=113) from education, health care, and retails organizations in New Jersey completed a 29‐item questionnaire. Four dimensions (break time, climate, technical support, and workplace policy) of the scale were examined to delineate the dimension most in need of improvement. Participants breastfed their babies for 5.3 ±1.5 months, were predominantly white (89%), married (92%), and had no lactation room in the workplace (82.6%). “Fear of losing my job,” “being made fun of by co‐workers” and “breastfeeding favorable remarks by supervisors” were the attributes of climate that were associated with the breastfeeding duration (p=0.01). Climate was perceived as more positive when the supervisors were female (p<0.05). These results indicate that breastfeeding promotion tactics addressing climate factors and targeting co‐workers and supervisors could improve duration of breastfeeding in working mothers.
Early parenting correlates of 9 mo formula amount of predominantly breastfed US 3 mo infants
Authors: Valtr Tabitha , Hubbs-Tait Laura , Grant Stephanie , Aubuchon-Endsley Nicki , Kennedy Tay Seacord , Thomas David
Published: June 2021

Purpose was to identify correlates of amount of formula fed to 9 mo infants who had been predominantly breastfed at 3 mo. Participants were 117 mothers and infants (68 female, 49 male) weighed and measured at 3, 6, 9 mo. Exclusion criteria for study enrollment were birthweight < 2.95 kg or > 4.32 kg. At 3 mo, infant weight ranged from 4.78 to 8.31 kg; at 9 mo, infant weight ranged from 5.22 to 11.73 kg. Mothers were: 87% white; 66% college graduate or postgraduate; 91% married. At 9 mo, 41 infants were fed some formula; amount ranged from .86 oz to 42 oz. All but one infant were eating solids (range of start time: 3 mo to 8.5 mo; Mdn = 6 mo); 66% were fed some table food by 9 mo. Maternal correlates of 9 mo formula amount were: mother's weight at 9mo‐visit, r = .188, p = .04; mother's positive affect at 6 mo‐visit, r = −.176, p = .07; mother's permissive parenting style at 3 mo‐visit, r = .199, p = .05. Maternal parenting stress was not related to formula amount. Amount of 9 mo formula was also related to infant 6 to 9 mo kg weight gain, p = .252, p = .01. Results extend findings on correlates of formula to predominantly breastfed infants. Funding: USDA‐NIFA NRI 2008‐35200‐18779.
Postoperative vitamin deficiencies in patients undergoing laparoscopic roux‐en‐y gastric bypass
Authors: Lodhia Nayna A , Almario Leanne V , Eltorai Adam , Kattan Jaffer , Nkansah Margaret M , Kerolus Matthew , Kiely Kate , Morton John
Published: June 2021

BACKGROUND

Nutritional abnormalities are common in severely obese patients and can be worsened following laparoscopic Roux‐en‐Y Gastric Bypass (LRYGB). This study aimed to determine the relationship between postoperative vitamin B12, folate and iron deficiency to LRYGB outcomes.

METHODS

630 patients underwent LRYGB by three surgeons at a single academic institution. Patient records were reviewed for vitamin levels, weight loss, and cardiac risk factors postoperatively.

RESULTS

Overall patient demographics were: mean age 44.4 years, mean BMI 46.3, and 89% female. At 3, 6 and 12 months postoperative, 0.2, 1.2 and 1.6% had Vitamin B12 deficiency; 0.3, 0.7 and 0% had folate deficiency; and 11.3, 4.5, and 7.3% had iron deficiency, respectively. Men had higher rates than women of vitamin deficiencies at all time points (p<0.001). Patients who had a known deficiency at 3 months had significantly higher weights preoperative (278lbs vs 311lbs, p<0.001), at 3 months (227lbs vs 254lbs, p=0.01) and at 12 months (185lbs vs 208lbs, p=0.01).

CONCLUSIONS

Male patients and patients with higher preoperative weight should be monitored closely for nutritional deficiencies following LRYGB.

Grant Funding Source: NIH Clinical and Translational Science Award
Weight loss, long‐term weight maintenance, and health status of members of Food Addicts in Recovery Anonymous (FA)
Authors: Stookey Jodi
Published: June 2021

This study describes the experience of a convenience sample of members of Food Addicts in Recovery Anonymous (FA), who consumed a portion‐controlled, low glycemic diet for 0–30 years. FA is a free 12 step recovery program that includes a strong supportive fellowship. In 2010, 4,044 FA members were surveyed. Of 3,624 respondents in the USA, 3,340 reported weighing and measuring their meals, and abstinence from flour and sugar at the time of the survey. Of these, 98% reported weight loss; 80% reported a weight loss of more than 25lbs. Of those who reported weight loss, 50% were at their goal weight. Longer duration of “abstinence” was associated with significantly greater likelihood of being at the goal weight, independent of age, sex, race/ethnicity, marital status, and education. 87% of those who had been abstinent for 1 year or longer were at their goal weight. Of those at their goal weight, 70% had already maintained their goal weight for 1 year or more; Abstinence and losing weight reportedly helped with high blood pressure, high cholesterol, joint pain, depression, anxiety, overeating, body image, compulsive dieting, and/or food craving. 41% of those at their goal weight no longer needed prescription medicine. For this sample of FA members, longer‐term abstinence was associated with attainment and maintenance of the goal weight, and improved health status.
A qualitative analysis of exercise and health practices in Korean American vs. American adults in Southern California
Authors: Wright Alely , Yu Kyeong Hee , Hong Mee Young
Published: June 2021

Korean Americans are one of the fastest growing Asian populations in the US; more than 1.4 million Koreans live in the US. The purpose of this study was to compare health practices of Korean American and non‐Asian American adults (30 years and older). Cross‐sectional surveys using a validated food frequency and general health questionnaire including exercise habits, dietary practices, and body image was carried out in 242 Korean Americans and 261 Americans in San Diego and Los Angeles. Korean Americans reported lower body mass indexes (BMI) (22.35 vs. 25.31), body weights, and healthier body images than Americans (p < 0.001). Paradoxically, more Americans participated in structured exercise (p < 0.001). Compared to the Korean Americans who did participate in structured exercise, Americans exercised for longer bouts (p < 0.001). Korean American adults still prefer traditional Korean foods. Americans may feel they can eat more because they exercise. Additionally, poor dietary choices may negate the benefits of exercise. This study points to important discrepancies in health advocacy. Further research examining the importance of dietary components and overall energy intake is warranted.
Formative Evaluation of a Community‐Based Nutrition Education Curriculum for Hispanics in South Carolina
Authors: Aragon M. Catalina , Cason Katherine L. , Hernandez-Garbanzo Yenory , Ugalde-Gonzalez Maciel , Rossi Maria Mercedes , Senior-Angulo Joyce
Published: June 2021

Objective

To assess through formative evaluation an Expanded Food and Nutrition Program (EFNEP) curriculum designed for South Carolina Hispanics.

Methods

The formative evaluation process consisted of a 4‐step approach: 1) curriculum initial pilot testing (n=31), 2) curriculum review/refinement, 3) pilot testing of the refined curriculum (n=16) and 4) focus groups with Hispanic participants (n=14).

Results

Cultural Appropriateness. Recipes and vocabulary were appropriate for Mexican participants; however, some terminology used was unfamiliar to other Latin American audiences. Educational Strategies. The first curriculum delivered lessons through storytelling with visual aids. In the second version, storytelling was eliminated and more individual and group activities were included. Accuracy. The curriculum was updated with the 2010 Dietary Guidelines and MyPlate. The subjects of physical activity, food security and food shopping practices were incorporated to meet EFNEP's goals. Theory adherence. Goal setting, environmental strategies and selfmonitoring were included after the review.

Conclusions

The 4‐step‐formative‐evaluation approach used in this study revealed important strengths, limitations and opportunities for curriculum improvement. Overall, activities, language and recipes used were improved to meet the specific needs of the audience.
Integrating sustainable agriculture into professional practice: a survey of dietetic professionals in Arkansas
Authors: Hardin Anna , Gonzales Dana , Carroll Polly , Hakkak Reza
Published: June 2021

The American Dietetic Association promotes “environmentally responsible practices that conserve natural resources, minimize the quantity of waste generated, and support the ecological sustainability of the food system.” The objective of this study was to assess the perceptions and beliefs of dietetic professionals in Arkansas about applying issues of sustainable agriculture into professional practice. A descriptive, cross‐sectional study was conducted by survey and e‐mailed to a representative sample of Arkansas Dietetic Association members to evaluate attitudes and intentions. We used independent t tests, one‐way ANOVA, correlations, and multiple regression analyses (P<.05). Perceived behavioral control, subjective norm, and rated importance of knowing about agricultural practices independently predicted intention. Dietitians that completed the survey (N=61) were knowledgeable about sustainable agriculture and believed that it was important to consume foods produced with sustainable practices (P<.05). Most of the dietitians participating in the study felt that it would be likely (2.98+0.24) they would be educating clients and patients about principles that coincide with their values. In conclusion, dietitians surveyed were interested in learning more but most were not integrating these issues into practice because of perceived barriers (Supported by Dept Dietetics & Nutrition).
Skipping Breakfast and Having Snacks of Middle School Students in Incheon and Factors Affecting Their Dietary Habits
Authors: Chung Hyun-Jung , Park Ju-Il
Published: June 2021

This study was to investigate habits of skipping breakfast and having snacks of middle school students in Incheon, Korea and to identify factors that affect their dietary habits, to help them take the right nutrients and form the right dietary habits, and to provide basic data for dietary life education to improve their health. The subjects were 392 boys and girls attending a middle school in Incheon. Of the middle school students in Incheon, 46.7% had breakfast every day. Having breakfast had not much influence on having snacks. They preferred taste(66.6%) and price(13%) to nutrition(8.4%) and hygiene(10.2%) when choosing snacks. There were small differences in the kinds of snacks preferred between boys and girls; while the girls often had “flour‐based foods and street foods”(26%), “confectionery”(41%) and “candy and chocolate”(42.7%), the boys had “ices”(32.5%) and “soft drinks”(30%) more often. As for dietary habits and attitudes, there were significant differences between the boys and the girls in that the former drinks milk more often (34.5%) and finish foods faster(81.5%) than the girls. BMI, sleeping hours, stress, and mother's educational background affected having breakfast and snacks, dietary habits and attitudes. Those who did not experience stress(56.3%) had more snacks than those who suffered a lot of stress(39.4%). The subjects with normal weight (34.1%) had meals regularly than the underweight(21.3%) and overweight(28.6%) subjects. The less stress they had, the more likely they were to have all three meals, have meals on scheduled time, and have meals slowly with more leisure. Most of the middle school students in Incheon were aware of the importance of breakfast but failed to have breakfast every morning due to various reasons. These results indicate that there are needs for education and research about the criteria of choosing snacks so that the students can have desirable and nutritious snacks on their own.

Grant Funding Source : Inha University
POSTPRANDIAL RESPONSE OF BEAN CONSUMPTION ON INFLAMMATION, OXIDATIVE STRESS, GLUCOSE, AND INSULIN IN ADULTS WITH METABOLIC SYNDROME
Authors: Reverri Elizabeth J. , Randolph Jody M. , Steinberg Francene M. , Kappagoda C. Tissa , Edirisinghe Indika , Burton-Freeman Britt
Published: June 2021

Metabolic Syndrome (MS) is a condition associated with inflammatory and oxidative stress. Consumption of a high carbohydrate/fat meal induces postprandial increases in markers of oxidative stress and insulin resistance. The present investigation aimed to determine whether inclusion of beans with a high fat meal would modify these postprandial responses. After an overnight fast, 12 adults with MS consumed a nutrient‐matched standard breakfast with either black beans (BB), fiber matched (FM), or antioxidant matched (AM) in a randomized crossover design. Postprandial responses (triglycerides (TG), cholesterol, glucose, insulin, and markers of oxidative stress and inflammation) were measured for 5 hours. Baseline characteristics include BMI 32.2±5.7 kg/m2, HOMA‐IR 2.4±1.0, and HOMA‐β cell function 92.6±32.0%. Preliminary results show that postprandial TG (mg/dL) was significantly lower after BB (162.5±17.3), compared to AM (176.8±17.3 P=0.0208) and FM (179.4±17.3 P=0.0091). Postprandial insulin (μIU/mL) was significantly lower after BB (33.5±4.3), compared to FM (38.4±4.3 P=0.0310) and AM (50.3±4.3 P=<0.0001). Postprandial oxLDL was significantly higher 5 hours after FM, compared to BB (50.7±3.2 vs 43.7±3.2 P=0.0438). Current results suggest that black beans may transiently attenuate exaggerated postprandial TG and insulin responses in adults with MS.

Grant Funding Source : UC Davis Departmental Funds and Dry Bean Council
Dietary obesity impairs regulation of hepatic AMP‐activated kinase in rats
Authors: Harper Marianna A. , Kuo Yu-Fu , Clarke Steven D. , Axen Kenneth , Axen Kathleen
Published: June 2021

Hepatic AMP‐activated kinase (AMPK), activated under conditions of energy depletion such as fasting, increases fatty acid oxidation and inhibits fatty acid synthesis and lipogenic gene expression. Our hypothesis is that regulation of AMPK activity by fasting and feeding may be impaired in obesity and hepatic steatosis.

Method

Male Sprague‐Dawley rats were fed either a low‐fat (LF, n=12) – 15% fat, 55% carbohydrate – or a high‐fat (HF, n=8) – 55% fat, 19% carbohydrate diet for 8 wk. After a 20 h fast rats were either dissected in the fasted state (D) or 3 h after an oral glucose gavage (G); liver samples were flash‐frozen and homogenized. AMPK activity was measured by degree of density.

Results

HF‐fed rats developed obesity and hepatic steatosis. AMPK activity was greater in the deprived state in LF vs. HF rats. The ability of glucose to reduce AMPK phosphorylation was also greater in LF vs. HF rats.

Conclusions

HF induced obesity decreased the ability of hepatic AMPK to act as an energy sensor, suggesting impaired regulation of fat mobilization and synthesis in response to nutritional state. Grant number: 1 SC3 GM086298‐01A1

Grant Funding Source: 1 SC3 GM086298
Effect of dietary fibers on fecal fat excretion in overweight and obese humans
Authors: Park Sonhee , Scott Robert , Schlottmann Rick , Striegel Don , Maki Kevin , Rain Tia , Witchger MarySue
Published: June 2021

The aim of this study was to examine the effects of dietary fibers, alpha‐cyclodextrin (ACD) or water soluble modified cellulose (WSMC), on fecal fat excretion in overweight and obese humans as compared to positive control (orlistat) or negative control (placebo). This was a randomized, controlled, crossover study with three, double‐blind 7‐d treatment periods followed by an open label, 7‐d orlistat period (180 mg/d). ACD (30 g/d), WSMC (6 g/d), or isocaloric placebo drink (35 kcal/355 ml; 2 servings/d) was consumed in the clinic as part of breakfast and dinner. All foods were provided during the treatment periods. Feces collected during the last 4 days of each treatment were analyzed for fecal fat excretion by nuclear magnetic resonance spectroscopy. Repeated measures of analysis of variance were used to compare responses. Among 31 subjects randomized (mean age: 45.3 ± 2.3 years; BMI: 31.0 ± 0.6 kg/m2), 21 were eligible for efficacy evaluation. There were no significant differences between the ACD or WSMC and placebo in total fecal fat excretion (g fecal fat/4‐d), daily fecal fat (g/d) or % fecal fat. Fecal fat excretion was significantly increased during orlistat period vs. 3 other periods. In conclusion, the results of the present study do not provide evidence that either 30 g/d of ACD or 6 g/d of WSMC, delivered in beverage form, increases fecal fat excretion in overweight and obese humans. Funded by Coca‐Cola Company.