bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigenetic inhibitors upregulate expression of MGAT3 in HepG2 cells and cause significant changes in glycosylation of secreted proteins (608.2)
Authors: Zoldos Vlatka , Horvat Tomislav , Korac Petra , Klasić Marija , Markulin Dora , Kristic Jasminka , Lauc Gordan
Published: June 2021

Protein glycosylation is a ubiquitous modification which affects protein structure and function. The majority of blood proteins are synthesized and glycosylated in the liver. Changes in plasma protein glycosylation are often associated with different types of liver disease, including hepatocellular carcinoma (HCC). Interestingly, secretomes of HCC patients and of HepG2 cells in culture are comparable, making this cell line an appropriate model to study how induced epigenetic changes can affect glycosylation of secreted proteins. We have correlated global changes in the expression of glyco‐genes with preferential appearance of particular glycan structures in the HepG2 secretome following the treatment of HepG2 cells with DNA methylation and histone deacetylation inhibitors. Using Human RT Profiler glycosylation qPCR array that contains 94 known glyco‐genes we identified that 20% of these genes are regulated by DNA methylation, and this regulation was reflected on glycan profile of secreted proteins from Hepg2 cells. The majority of changes in the glycosylation profile could be attributed to changed expression of Mgat3 responsible for addition of bisecting GlcNAc, which prevents further fucosylation and branching. We further confirmed its role in plasma glycome by targeted modulation of Mgat3 expression with epigenetic inhibitors. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, the presented work contributes to our understanding of their efficiency in altering the N‐glycan profiles of secreted proteins.

Grant Funding Source: Supported by European Commission grants IBD‐BIOM and IntegraLife
Using photo‐crosslinking to determine intracellular CD11b/CD18 signaling interactions (609.12)
Authors: Heidema Christy , Palmer Michelle , Korver Collin , Gupta Vineet , Stoub Darren
Published: June 2021

The leukocyte integrin, Mac‐1 (CD11b/CD18), plays an essential role in mediating both cell‐cell and cell‐extracellular matrix adhesion interactions during an inflammatory response. Historically, integrin signaling was understood to be both bidirectional, through inside‐out and outside‐in signaling, and involve either activation or deactivation of the integrin. However, more recent studies suggest a non‐binary activation pathway for Mac‐1, which results in a complex and differential signal transduction pattern. In order to understand the implications of a non‐binary activation of CD11b/CD18, direct protein interactions must be identified and quantified; however, determining direct interactions continues to be a challenge in the field of proteomics. Herein, we report our use of the unnatural amino acid (UAA), benzophenone, incorporated at several relevant sites in the cytoplasmic tails of Mac‐1 to photocrosslink proteins that directly interact with Mac‐1 under various physiological conditions. Plasmids coding for amber stop codons at the desired amino acid sites were prepared using PCR mediated site‐directed mutagenesis. HEK293 and CHO cell lines were transiently transfected and grown under appropriate physiological conditions in the presence of the unnatural amino acid, benzophenone. The cells were irradiated with UV light and the resulting photo‐crosslinked proteins were identified using western blot analysis. Our results demonstrate that photocrosslinking using UAA is a powerful tool for identifying protein interactions involved in signal transduction. Further analysis will enable us to more thoroughly understand the complex signaling process involved with the Mac‐1 integrin during leukocytic inflammation.
Rapamycin‐responsiveness and Gln3‐Tor1 interaction are associated with different target sites on the Gln3 protein (609.13)
Authors: Cooper Terrance , Rai Rajendra , Tate Jennifer
Published: June 2021

Nitrogen‐sensitive control is imposed by TorC1‐dependent and ‐independent regulatory pathways directing localization of the transcription activator, Gln3. In nitrogen excess Gln3 is cytoplasmic and Gln3‐mediated transcription is minimal. During nitrogen limitation, or after rapamycin (Rap, a specificTorC1 inhibitor) addition, Gln3 relocates to the nucleus and activates transcription. Alteration of a Gln3 α‐helix656‐662 abolishes Gln3‐Tor1 interaction, Rap‐responsiveness and diminishes cytoplasmic Gln3 sequestration in nitrogen excess. This prompts the question, are these characteristics inextricably linked to the Tor1 interacting site on Gln3? We demonstrate that they are not. Amino acid substitutions throughout Gln3510‐589, remote from the Gln3‐Tor1 interaction site, completely abolish Rap‐responsiveness, but leave cytoplasmic Gln3 sequestration, Gln3‐Tor1 interaction and nitrogen‐responsiveness of Gln3 localization untouched. These data identify a specific Gln3 region required for Rap‐responsiveness and suggest that events triggered by Rap inhibition of TorC1 resulting in nuclear Gln3 localization occur outside of the Gln3 region interacting with Tor1 or responding to nitrogen availability.

Grant Funding Source: Supported by NIH GM‐35642
RGS6 suppresses cellular transformation by oncogenic Ras through a Tip60‐Dnmt1‐apoptosis dependent mechanism (609.14)
Authors: Maity Biswanath , Huang Jie , Stewart Adele , Hagen Jussara , Fagan Rebecca , Yang Jianqi , Quelle Dawn , Brenner Charles , Fisher Rory
Published: June 2021

Silencing of tumor suppressor genes by DNA methyltransferase 1 (Dnmt1), dysregulated in human cancers, is essential for oncogenic transformation by the Ras protooncogene. Here, we provide evidence that Regulator of G protein Signaling 6 (RGS6) suppresses Ras‐induced cellular transformation by facilitating Tip60‐mediated Dnmt1 degradation and promoting apoptosis. Employing mouse embryonic fibroblasts (MEFs) from wild type (WT) and RGS6‐/‐ mice, we found that Ras induced up‐regulation of RGS6, which in turn blocked Ras‐induced cellular transformation. RGS6 suppresses cellular transformation by down regulating Dnmt1 leading to inhibition of Dnmt1‐mediated anti‐apoptotic activity. Further experiments showed that RGS6 is a scaffolding protein for Dnmt1 and Tip60 required for Tip60‐mediated acetylation of Dnmt1 and Dnmt1 ubiquitylation and degradation. Indeed, RGS6 loss promotes Dnmt1 up‐regulation in a mouse model of breast cancer possibly contributing to accelerated tumorigenesis observed in RGS6‐/‐ mice. This work demonstrates a novel signaling action for RGS6 in negative regulation of oncogenesis providing new insights into our understanding of the mechanisms regulating Ras‐induced cellular transformation and Dnmt1 expression. Importantly, these findings identify RGS6 as an essential cellular defender against oncogenic stress and a potential therapeutic target in cancer treatment.

Grant Funding Source: Supported by NCI CA161882 (RAF), HHSN261200433000C (CB), CA090367 (DEQ)
Human follicle stimulating hormone receptor signaling is membrane cholesterol and internalization dependent (609.16)
Authors: Dondarski Megan , Cohen Brian
Published: June 2021

Human follicle stimulating hormone (hFSH) is a dimeric glycoprotein secreted by the gonadotropic cells of the anterior pituitary that is essential for reproduction. Its receptor, (hFSHR), is a G protein‐coupled receptor expressed on the surface of granulosa cells in females and Sertoli cells in males. FSH stimulates the maturation of ovarian follicles in females and triggers sperm production in males. Based on previous research in our lab, we proposed that hFSHR resides in lipid rafts and/or caveolae on the cell membrane and that its signaling is cholesterol and internalization dependent. To test this hypothesis, we treated HEK‐293 cells stably transfected with human FSHR cDNA with the cholesterol depleting compound methyl‐beta‐cyclodextrin (MBCD) or the anti‐fungal compound Nystatin which alters the selective permeability of the cell membrane by sequestering cholesterol. Western blotting was used to measure the phosphorylation of map kinases p38 and p42/44 (ERK 1/2). MBCD did not alter FSH dependent activation of p44/42 or p38, but a decrease in activation of both signaling components was observed when the cells were treated with Nystatin. Both treatments decreased cAMP dependent activation of the cAMP response element‐binding protein. These results suggest that FSHR signaling is dependent not on cholesterol alone, but also on internalization, which is blocked by Nystatin. This adds to an increasing body of evidence for multivalent hFSHR signaling with the possibility of selective modulation of the pathways.
Eps8 protein participates in migration in Toll‐like receptor‐engaged macrophages (609.7)
Authors: Hsieh Ming Yu , Chen Yen‐Jen , Chang Miao Ying , Lee Chung Ta , Lee Jenq‐Chang , Maa Ming‐Chei , Leu Tzeng‐Horng
Published: June 2021

Previously, we demonstrated that iNOS/Src/FAK axis was a general mechanism of macrophage motility in response to various pathogen‐associated molecular patterns (PAMPs) and Eps8 participated in TLR4‐mediated signal transduction leading to enhanced phagocytosis and bacterial killing effect. However, the role of Eps8, an Src‐interacting protein, in PAMP‐mediated macrophage locomotion is still unclear. Here we observed that the expression of Eps8 was PAMP‐inducible, and the induction was iNOS/Src‐dependent. Attenuation of Eps8 simultaneously impaired Src activity and suppressed macrophage mobility. Remarkably, ectopic Eps8 could partly restore the reduced motility and Src activity in Src‐attenuated macrophages exposed to various PAMPs. Together, our findings indicated that Eps8 not only modulated TLR4‐mediated signal transduction but also participated in Src‐mediated cell migration in TLRs‐stimualted macrophages.
Organic solvent‐activated signaling pathways via cell surface sensors and transcription factors (613.1)
Authors: Kuroda Kouichi , Nishida Nao , Ueda Mitsuyoshi
Published: June 2021

Organic solvent‐tolerance of cells is an important characteristic in various ways such as bioconversion in the presence of organic solvent. In eukaryotes, cells with organic solvent tolerance have been hardly discovered except for yeast Saccharomyces cerevisiae KK‐211 strain. We have identified several factors such as Pdr1p and ABC transporters contributing to organic solvent tolerance, and found that one point mutation (R821S) of Pdr1p causes organic solvent tolerance [1, 2]. However, the mechanisms how yeast cells respond to organic solvent stress are unknown. Our findings suggest the possibility that pleiotropic drug resistance (PDR) pathway including up‐regulation of genes under the control of Pdr1p and Pdr3p (Pdr1p paralog) could be involved in organic solvent. In this study, the contribution of other representative components than Pdr1p in PDR pathway to organic solvent tolerance was examined. Furthermore, we investigated the activation of cell wall integrity (CWI) pathway in response to organic solvent. As a result, some components of the CWI pathway were induced and involved in organic solvent tolerance, indicating that yeast cells activated two kinds of signaling pathways (PDR pathway and CWI pathway) to overcome stresses from organic solvent [3].

[1] Matsui et al. (2008) Appl. Environ. Microbiol. 74, 4222‐4225 [2] Nishida et al. (2013) J. Biotechnol. 165, 145‐152 [3] Nishida et al. submitted.
Evaluating multiple signaling activities in LPS‐induced human peripheral blood mononuclear cells (613.6)
Authors: Martin Ashley , Kohlmeier Kayla , Maas Lindsay , Gautam Aarti , Jett Marti , Mendis Chanaka
Published: June 2021

Lipopolysaccharide (LPS) is an endotoxin that induces the secretion of pro‐inflammatory cytokines in many cells; and LPS has been well characterized to induce septic shock in humans. This project is aimed at characterizing the effects of LPS by utilizing signal transduction pathways induced by LPS in Peripheral Blood Mononuclear Cells (PBMC). A set of LPS induced genes were identified through microarray analysis and common signaling pathways were identified. Key pathway inter‐connectors such as JNK and p38 were blocked and were evaluated to the previously identified gene expression patterns. The main objective was to investigate the effect of two inhibitors on the time dependent gene expression pattern and then, the impact on the components that are indirectly associated with the inhibited signaling pathways. In addition, the specific inhibitors were evaluated by correlating the observed gene expression pattern to the protein expression pattern. We anticipate that the study will allow us to better understand the complex interactions of multiple signal transduction pathways induced by LPS as well as provide information about mitigating the deadly effects of LPS.
A novel pathway for bacterial nitric oxide signaling (614.3)
Authors: Rao Minxi , Marletta Michael
Published: June 2021

Nitric oxide (NO) serves dual biological roles as both a signaling molecule and cytotoxic agent. In higher animals including mammals, NO is synthesized by nitric oxide synthase (NOS), and NO signaling involves the heme‐nitric oxide/oxygen‐binding (H‐NOX) domain of soluble guanylate cyclase as a selective sensor. Bacterial NOS and H‐NOX proteins have been reported, but their functions are less well understood. We have recently identified two organisms that contain a predicted NOS, H‐NOX, and downstream histidine kinase, which are constituents in a two‐component signaling system, suggesting that these organisms are capable of both NO synthesis and sensing. In Silicibacter sp. TrichCH4B, we have shown that NO regulates cellular c‐di‐GMP levels and biofilm formation through two‐component signaling. Furthermore, this NOS is activated by a protein secreted by the algae Trichodesmium, which occupies the same habitat as Silicibacter. Therefore, NO could serve as a quorum sensing molecule that mediates Silicibacter biofilm behavior in a symbiotic relationship with Trichodesmium.

Grant Funding Source: Supported by the NSF Graduate Research Fellowship
The influence of endogenous protein complex on morpho‐functional activity of bone marrow cells (616.3)
Authors: Bakuradze Ekaterine , Modebadze Irina , Mosidze Giorgi , Peradze Eka , Dzidziguri Diana
Published: June 2021

Endogenous regulators of proliferation are considered as a main player of cell‐cycle regulatory system. Specific actions (stimulation or inhibition) on the target cell are the primary function of regulators. Stimulating growth factors are well described while Inhibitory factors are still less known. One of the growth inhibitory endogenous thermostabile complex is identified in various organs of phylogeneticaly distance organisms (from bacteria to human). The complex decreases the homologous cells proliferation by inhibiting of RNA synthesis. The complex is not species specific but it is characterized by organ specificity, which is revealed only on terminally differentiated cells.

The Goal Investigation of endogenous protein complex influence on morpho‐functional activity of bone marrow.

Objectives Investigations were carried out on growing rats and adult mice. Obtaining of endogenous thermostabile protein complex from various organs of rat with alcohol extraction was performed; The determination of mitotic index was used for estimation of cell proliferation activity.

Results We have found that mitotic index has not changed after the injection of endogenous thermostabile protein complex. Particularly colchicine mitotic index of bone marrow cells in adult mice of control group equals to 65±1.3, mitotic index is 63±2.5‰ after the injection of protein complex. The same picture is observed in growing rats. The value of mitotic index is not changed with compared to control group.

Conclusion On the bases of our results we can conclude that organ specificity of protein complex, is still maintained in case of bone marrow cells despite that fact that bone marrow contains the cells with different degrees of differentiation.

This results may be considered as new evidence about the action of endogenous protein complex and its possible usage in medicine for development of new therapeutic approaches in the treatment of cancer.
Arabidopsis CPL4 is an essential Ser2‐specific CTD‐phosphatase regulating general and xenobiotic responsive gene expression (617.2)
Authors: Fukudome Akihito , Aksoy Emre , Wu Xiaoqiang , Kumar Krishna , Jeong In Sil , May Kimberly , Russell William , Koiwa Hisashi
Published: June 2021

Phosphoregulation on C‐terminal domain (CTD) of RNA polymerase II (pol II) is a pivotal mechanism to control transcription. Pol II CTD kinases and phosphatases have been extensively studied in animal and yeast, but little is known in higher plants. Arabidopsis thaliana has four CTD phosphatase‐like (CPL) proteins and knock‐down of CPL4 (CPL4RNAi line) results in severe developmental and growth defects. In the current study, we dissect the molecular and physiological functions of CPL4.

Mass‐spectrometry analysis on tandem affinity purified CPL4 complex successfully identified pol II subunits. CPL4 exhibited CTD phosphatase activity with preference toward serine 2 residues in vitro and in vivo. A catalytically inactive form of CPL4 harboring an amino acid substitution in its catalytic motif strongly inhibited general gene expressions. The inhibitory activity was dependent on in BRCT, suggesting that both catalytic FCPH and BRCT domains are essential for proper function of CPL4. A set of xenobiotic stress responsive genes was up‐regulated in CPL4RNAi lines under normal condition, but upon exposure to xenobiotic stress, the expression level of those genes in wild type and CPL4RNAi was similar.

Taken together, we propose Arabidopsis CPL4 as an essential Ser2‐specific CTD phosphatase, which represses xenobiotic stress response genes under normal condition.
Designing 3D tactile protein models as an introduction to biology research (618.10)
Authors: Tirone Janine , Geritano Mariah , Solomon Irene , O'Neal Marvin
Published: June 2021

Technological advances in rapid prototyping provide an opportunity for students to understand structure‐function relationships in our three dimensional living world. We are currently examining the impact of a newly developed introductory biology laboratory (BIO207) in which undergraduates investigate Lyme disease by designing 3D tactile models of proteins. Assessments providing direct comparison between this new lab and the concurrent alternative introductory biology lab include CURE (Classroom Undergraduate Research Experience) pre/post survey data, matched exam questions between the two courses, and performance in future coursework. Success and growth of this course has led to the design of a BIO207 alternative for undergraduates interested in neuroscience. Using the cat pre‐Botzinger complex (which is the primary locus of respiratory rhythm generation) as a topical focus, students use high‐resolution structures from RCSB Protein Data Bank to print tactile models and research a structure‐function relationship in this CNS region.

Grant Funding Source: Supported by HHMI 52006940
A national comparison of learning objectives and outcome assessment of the biochemistry and molecular biology capstone experience: building the ASBMB Concept Inventory (618.13)
Authors: Aguanno Ann , Mertz Pamela , Martin Debra , Bell Ellis
Published: June 2021

Recognizing the increasingly integrative nature of the molecular life sciences, the American Society for Biochemistry and Molecular Biology (ASBMB) recommends that Biochemistry and Molecular Biology (BMB) programs develop curriculum based on concepts, content, topics and expected student outcomes, rather than courses. Several reports, including Vision and Change, endorse this “big idea” approach to undergraduate life science education. ASBMB recently developed an accreditation program to provide a national outcome‐based mechanism to accredit B.S and B.A. degrees and acknowledge undergraduate BMB programs which fulfill the expectations of the ASBMB recommended curriculum. Currently, ASBMB is a building a BMB Concept Inventory which will contain validated assessment tools based on foundational and discipline specific knowledge and essential skills for the molecular life science undergraduate. The capstone experience is an essential part of molecule life science undergraduate programs. Although capstone experiences are commonly defined as an attempt to measure student ability to synthesize and integrate acquired knowledge, the format, implementation, learning goals and approach to outcome assessment are quite varied across the nation. Here we report a national comparison of capstone experiences and align the results with both the ASBMB recommended BMB curriculum and the findings of the national workshops driving the development of the BMB Concept Inventory.

Grant Funding Source: NSF‐ 0957205 RCN‐UBE
The MACH model for explaining molecular mechanisms: themes across multiple disciplines (618.18)
Authors: Trujillo Caleb , Anderson Trevor , Pelaez Nancy
Published: June 2021

Constructing explanations is essential for both science and learning. This study addresses the question, “What are the essential aspects of biology experts’ explanations of cellular and molecular mechanisms within their domain?” To address this question we first built on previous work and our own thought experiments to derive an initial model about mechanistic explanations. Secondly, we tested the validity of the initial model by asking seven biologists from various sub‐disciplines, to explain a cellular mechanism of their choice. Data was collected from interviews, artifacts and drawings and subjected to thematic analysis. We found that biologists: use our initial mechanistic model of molecular explanation by focusing on entities, activities, and organization. But in addition, they highly contextualize and constrain their explanations according to biological and societal significance; integrate explanations with the methods, instruments, and measurements they use to investigate; and use narrative stories along with analogies to explain their systems. These themes informed a modified model of expert‐like explanations of cellular mechanisms. Expert explanations consider four components: the Methods (M), Analogies (A), Context (C), and How (H) the mechanism works. Our model will provide a foundation for future work in life science education research, as well as other science domains, and offers a way to teach explicitly about components of biological mechanisms.
The use of physical models of protein structures to probe for learning and reasoning difficulties (618.20)
Authors: Johnson Sara , Garratt Richard , Pelaez Nancy , Anderson Trevor
Published: June 2021

Biochemistry educators make extensive use of external representations (ERs) to help students visualize the submicroscopic world of biochemical structures, including proteins. Despite broad acceptance, little is understood about the effectiveness of ERs for promoting student learning about protein structure. In this study, we investigate the effectiveness of a physical model kit, Protein Folder, and a protein structure chart, The Protein Chart, at probing for student understanding of protein structure. Twenty‐six students, enrolled in a biochemistry course, participated in a two‐week unit, which made use of the ERs to teach about protein structure. To investigate the educational effectiveness of the ERs, we identified student difficulties resulting from worksheets, quizzes, tests and interviews. We classified difficulties using the conceptual‐reasoning‐mode (CRM) model. This work revealed four novel difficulties students encounter when learning about protein structure. We found that students have difficulties making distinctions between the linear sequence of a protein and its spatial sequence, building protein structures that reflect standard amino acid chirality, portraying correct spatial separation between amino acids in a protein model, and correctly orienting protein strands within a chain. Identification of these difficulties allows educators to anticipate obstacles their student can face and may lead to eventual improvements in the use of ERs for teaching about protein structure.
Outcomes of networking: RCN‐UBE: promoting concept‐driven teaching strategies in biochemistry and molecular biology through concept assessments (618.24)
Authors: Wright Ann , Bell Ellis , Bailey Cheryl , Caldwell Ben , Elliott Samantha , Sensibaugh Cheryl , Sirum Karen
Published: June 2021

Biochemistry and Molecular Biology instructors like all higher education faculty members, face challenges of becoming more effective instructors. They have very few opportunities or time to share their professional lives with others in education. However, when one collaborates in BMB research, there is always communication between scientists: exchanging information; collaborating on projects; and disseminating results. Therefore, why not use the same type of networking to share pedagogical knowledge? In addition to reporting outcomes of the RCN sessions at the Seattle Education meeting, this poster will describe an effective social networking which began in 2010 supported by a grant from the NSF (Undergraduate Biology Education track in the Research Coordination Network program) “Promoting Concept Driven Teaching Strategies in Biochemistry and Molecular Biology through Concept Assessments. “The social network is effective because the project steering committee monitor the way that individuals are situated in the network and support social ties so individuals can provide feedback and have access to valued resources (Coleman, 1990; Portes, 1998). By following network models used to create standard‐setting documents, such as Vision and Change and PCAST Report, grant administers use a network of selected experts to form Learning Goals and Objectives (Biochemistry and Molecular Biology Education, September/October 2013) and to build a website Toolkit for empirically proven best practices and learning activities. NSF Grant 0957205 to EB supported this work.

Grant Funding Source: Supported by RCN‐UBE NSF Grant 0957205
The “flipped” undergraduate research lab: teaching core molecular biology techniques with descriptive protocols and videos (618.30)
Authors: Beckman Matthew
Published: June 2021

Many researchers at primarily undergraduate institutions face a dilemma—how to balance the teaching, service and research loads. As the size of the research laboratory grows it becomes increasingly necessary to develop efficient strategies to train newer students coming into the lab. Historically, many lab heads have taken the approach where older, more seasoned students introduce new students to methods and techniques. In situations where the lab head is either starting a new laboratory, or is not able to recruit a steady stream of students, continuity between students or cohorts of students coming into the lab may not exist. I am developing two types of assistive tools to introduce undergraduate research students to the core methods and techniques in molecular biology using highly descriptive protocols that include images and narrated videos that demonstrate methods visually. While these tools will never serve as a replacement for mentorship, they will provide students with details about methods that can be reviewed at will and when the mentor is not available. These tools will be made freely available to other researchers through open‐access pipelines such as YouTube and our website.

Grant Funding Source: Supported by start up funds from the Dean's Office.
Undergraduate course centered on animal cell culture (618.31)
Authors: Carastro L. , Marks Jonathan , Witherow D.
Published: June 2021

Integration of biochemistry and molecular biology (BMB) concepts and principles into the curriculum of a laboratory‐based capstone course is an integral component of our experiential undergraduate program. This course enables students to develop deeper understandings through meaningful and exciting laboratory experiments. As part of our recently updated undergraduate biochemistry degree program, we describe a newly developed capstone course consisting of a unified series of laboratory experiments focused on animal cell culture. This course, Tissue Culture (CHE470), includes lectures on BMB aspects of culture media and additives, as well as cellular structure, metabolism and transformation. Laboratory exercises include sub‐culturing, cryopreservation, cell growth kinetics and quantitation techniques, transfection, and biochemical assays. The experimental modules are (1) electroporation transfection, (2) clonal selection, isolation and expansion, (3) induction of gene expression, (4) genomic DNA isolation and PCR screening, and (5) quantitation of gene expression. Course grades are derived from in‐class exams, a BMB peer‐review journal style report, an oral presentation, and laboratory performance. Student outcomes are measured by anonymous student feedback from an online survey using a numeric rating scale (5 = strongly agree; 1 = strongly disagree). Students reported as a result of taking this course they “know more about this subject” (mean = 4.86, dev. = 0.38), their “skills in this area have improved” (mean = 5.00, dev. = 0.00), their “appreciation of this subject increased” (mean = 4.71, dev. = 0.76), and that “learning objectives of the course were met” (mean = 4.86, dev. = 0.38). Survey data strongly suggests students found this course very engaging, informative and beneficial.
The Vetvip consortium of the EU‐Lifelong Learning Programme presents: “Death by Broken Heart” (618.4)
Authors: Küch Eva‐Maria , Branitzki‐Heinemann Katja , Brogden Graham , von Köckritz‐Blickwede Maren , Naim Hassan , ‐ Consortium Vetvip EU‐Lifelong Learning Programme
Published: June 2021

Teaching of basic sciences, such as biochemistry, is necessary for medical education and is crucial for supplying students with competent knowledge for professional practice. However, most often the teaching is limited to a presentation of facts with poor correlation to the clinical practice. A close integration between basic and clinical science would improve the teaching quality in veterinary medicine, especially with respect to memory effects and learning motivation of the students. Therefore, an interdisciplinary team of teachers and scientists of biochemistry from different veterinary faculties initiated the project “Use of virtual problems/virtual patients in veterinary basic sciences (Vetvip)” which is funded by the Lifelong Learning Programme of the European Commission. Innovative computer‐based case studies that teach basic scientific concepts with tangible clinical applications have been prepared.

The here presented case is about the deadly heart attack of a chimpanzee and incorporates several teaching aims such as the crucial role of oxygen for energy metabolism, the different energy metabolism pathways with particular emphasis on oxidative phosphorylation and the processes which trigger the damage of the myocardial tissue.

This project will help to improve veterinary education, boost student motivation and result in increased and long‐lasting learning success rates. www.vetvip.eu
Characterization of Thermotoga maritima maltose binding protein (618.43)
Authors: Masson Laura , Williams Ashley , Dattelbaum Jonathan
Published: June 2021

Thermotoga martima is a thermophilic, Gram‐negative bacteria found primarily in hot springs and hydrothermal vents. Its thermophilic characteristics mean that proteins found in this bacterium should be extremely stable, even at very high temperatures, making them ideal for manipulation and biosensor design. The protein of interest for our project is the T. maritima maltose binding protein (TmMBP), which is a member of the periplasmic binding protein superfamily. Using site‐directed mutagenesis of residues in and around the binding pocket, we constructed eight single cysteine mutants of TmMBP. The individual Cys residues we covalently modified with environmentally‐sensitive fluorophores to interrogate maltose binding to the protein. The altered fluorescence characteristics were used to determine a dissociation constant for maltose. We used circular dichroism to evaluate the stability of each mutant. Ultimately, we hope to design a TmMBP biosensor that is stable, can bind molecules other than maltose, and will fluoresce when the target molecule is bound.

Grant Funding Source: University of Richmond Arts and Sciences
Peptidomic profiling of human and bovine milk and across infant digestion (622.5)
Authors: Dallas David , Guerrero Andres , Khaldi Nora , Parker Evan , Castillo Patricia , Underwood Mark , Bevins Charles , Lebrilla Carlito , German J. , Barile Daniela
Published: June 2021

Milk co‐evolved with mammalian infants to be nourishing and immunoprotective. Many milk proteins have no known function intact. However, fragments of milk proteins (peptides) released by enzymatic degradation have actions, including immune system modulation and antimicrobial action.

The aims of this research were 3‐fold: 1) identify the extent of human and bovine milk protein proteolytic cleavage within the mammary gland and in the infant stomach; 2) identify the mechanisms for their release from the intact protein; and 3) identify peptide actions. Infant gastric aspirates were collected via a naso‐gastric tube 2 hours after feeding. Human milk, bovine milk and gastric aspirates were profiled for peptides by nano‐chip liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry (Chip‐Q‐TOF). Hundreds of milk peptides were identified in both human and bovine milks, most of which derived from the casein family. Most peptides were present in all human milks and all bovine milks sampled. Many low abundant milk proteins were the source of numerous peptides, whereas some high abundant proteins yielded no peptides. All peptides were derived from specific portions of the parent protein. These findings suggest a well‐regulated proteolytic system in the mammary gland of both cows and humans. Bioinformatic analysis of the cleavage sites suggested several proteases native to milk were active both within the mammary gland and the infant stomach. In the stomach, despite evidence that the main gastric enzyme, pepsin, is non‐functional in infants, many of the proteolytic sites were consistent with pepsin cleavage. Over 10% of peptides identified in milks and gastric aspirates had over 50% sequence homology with peptides previously annotated for antimicrobial or immunomodulatory function. As an ensemble, human milk peptides inhibited in vitro growth of attenuated pathogenic strains of Escherichia coli and Staphylococcus aureus.
Developing bovine milk and mammary gland functions during the first six month of lactation: insights from in‐depth quantitative proteomic analysis (622.6)
Authors: Lohe Kimberly , Maria Sarah , Cundiff Judy , McMahon Robert , Zhang Qiang
Published: June 2021

We applied quantitative bovine milk proteomic data as probes for the developing milk and mammary‐gland function (n = 5 cows with samples of colostrum at day 1 and milk at 1, 2, 3 and 6 months). 1913 protein were identified where 894 were quantified. The majority of proteins express constitutively during mature milk production. Main changes were observed between day 1 and 1 month with 66 and 139 upregulated (FDR‐adjusted p‐value < 0.05) in colostrum and milk, respectively. Proteolytically cleaved intercellular adhesion molecule (ICAM‐1) is upregulated in milk, suggesting that shedding of ICAM‐1 provide a means to suppress leukocyte recruitment into milk as lactation progresses. Shedding of E‐cadherin and catenin (adhesion complex) from mammary epithelium decreases in later milk, which suggests an increasing adherins junction barrier. Housekeeping proteins from blood serum are present at higher abundance in colostrum, which is consistent with the decreased mammary epithelial barrier during colostrum production. Abundance of enzymes in N‐glycoprotein biosynthesis decreases whereas abundance of enzymes in N‐glycoprotein degradation increases from colostrum to milk, indicating decreased N‐glycoprotein biosynthesis over lactation. Lysosomal enzymes are upregulated in milk (fold change > 3), implying higher lysosomal hydrolysis function during later milk production.
Transcriptomic profiling of intestinal epithelial cells in response to human, bovine and commercial bovine lactoferrins (623.2)
Authors: Jiang Rulan , Lönnerdal Bo
Published: June 2021

Lactoferrin (Lf) is an iron‐binding protein abundantly present in human milk. It is multifunctional and comparatively resistant to gastrointestinal proteolysis. Thus, Lf may play critical roles in intestinal development. Due to differences in sequences and isolation methods, Lfs from human and bovine milk as well as commercial available bovine Lf (CbLf) may differ functionally or exert functions diversely. The objective of the present study was to examine and compare effects of CbLf and Lfs isolated from human and bovine milk on intestinal epithelial cells and thus to provide a basis for applications of CbLf. Normal crypt‐like human intestinal epithelial crypt cells (HIEC) were used to evaluate effects of Lfs on intestinal cell proliferation and transcriptomic profiling. All Lfs (4‐50 µg/mL) significantly stimulated proliferation of HIEC and no differences were found among Lfs. Microarray assays (Illumina) were then conducted to investigate transcriptomic profiling of HIEC in response to Lfs. Genes markedly (>1.5 or < 0.5 fold) regulated by hLf, bLf, and CbLf were 152, 367, and 455, respectively. Although only few genes (16) were regulated by all Lfs, they remarkably stimulate signaling pathways tightly related to cellular development based on pathway analysis by IPA (Ingenuity). All Lfs triggered various signaling pathways, such as growth hormone signaling, cell cycle: G1/S checkpoint regulation, and IGF‐1 signaling. In summary, all Lfs may play important roles in intestinal development but through various mechanisms.
Simultaneous determination of nutritional elements in human milk by inductively coupled plasma‐mass spectrometry (623.4)
Authors: Gray Michael , Phillips Shay , McMahon Robert
Published: June 2021

Several factors, such as dietary intake and genetic background, influence the elemental composition of human milk. Recently, a simultaneous method was developed to rapidly and accurately determine the concentrations of P, Na, K, Ca, Mg, Mn, Cu, Zn, Fe, Mo, Cr, and Se in human milk. The method involves an efficient sample digestion using nitric acid in a microwave digestion system equipped with single reaction chamber technology. After digestion, the samples were diluted to volume and quantified by ICP‐MS. The method was validated by conducting experiments to determine precision, accuracy, linearity, limit of quantitation, and ruggedness. All external standard calibration curves had linearity correlation coefficients of 0.9999 or better and limits of quantitation as low as 0.2 ng/mL for several trace elements. A NIST Standard Reference Material 1849a ‐ Infant/Adult Nutritional Formula was utilized as a daily check sample and analyzed in conjunction with the human milk samples to confirm data acceptability and accuracy. The demonstrated performance indicates method suitability for the simultaneous elemental analysis of human milk. Utilization of this method is planned for the analysis of multiple samples from the Global Exploration of Human Milk (GEHM) study, a cohort with Cincinnati Children’s Hospital.
Exploring nutrition education resources, barriers, and nutrition knowledge in California public school teachers (625.4)
Authors: Jones Anna , Zidenberg‐Cherr Sheri
Published: June 2021

The study objective was to determine barriers to nutrition education, nutrition education resources used, and relationships between nutrition knowledge and whether or not nutrition is taught in the classroom by public school teachers in California. One hundred and two teachers participated in the web‐based survey about nutrition education barriers, resources used to plan nutrition lessons, and factors that would encourage inclusion of nutrition. A validated questionnaire was used to assess nutrition knowledge. Analyses included descriptive statistics and linear regression. Only cases that had data for all variables were included in the regression analysis, and one outlier was excluded (n=67). More than half of the sample indicated lack of instructional time (70%), lack of resources (61%), and unrelated subject matter (59%) were barriers to nutrition education. Teachers were unaware of many nutrition education resources, suggesting a need for promotion of existing resources. Mean nutrition knowledge score was 37.5 out of 58 for cases included in the regression analysis. Nutrition knowledge was not associated with nutrition lessons, but was associated with teaching high school, female gender, and identifying as Hispanic or Latino (p<.001; F(3) = 15.06). The proportion of variance explained by this model was 42%. Larger studies are needed to determine if these findings hold true in a broader sample.
Chronic administration of Artemisia annua L. attenuates oxidative stress induced by D‐galactose in mice (629.10)
Authors: Kim Mi Hye , Seo Ji Yeon , Han Mi Kyung , Kang Hye Ryung , Kim Seong Soon , Kim Bo Ram , Zakharova Alexandra , Woo Ji Eun , Kim Jong‐Sang
Published: June 2021

Artemisia annua L. (AA) is well known to contain a significant level of artemisinin which is an anti‐malarial agent and was also reported to have antitumor activity at high concentration. Our previous study, the ethanolic extract of Artemisia annua. L had in vitro antioxidant effect and induced antioxidant enzyme NQO1 activity in murine hepatoma Hepa1c1c7 cell line. In this study, we investigated whether or not AA attenuates oxidative stress and DNA damage induced by D‐galactose in mouse model. Our results indicated that D‐galactose treatment (6 weeks) caused cognitive impairment in Morris water maze test and produced oxidative stress as is evidenced by the increase of lipid peroxidation and high level of 8‐OH dG in plasma. Furthermore, we found that D‐galactose treatment caused several histological change in some tissues. The treatment for 6 weeks with AA extract attenuated cognitive impairment, lipid peroxidation and DNA damage, and induced some antioxidant enzyme activities in mice injected i.p. with D‐galactose. In conclusion, AA extract suppressed memory impairment induced by D‐galactose in mice, maybe through suppression of ROS generation by D‐galactose.

Grant Funding Source: Supported by NRF‐2010‐0027204 and 2011‐0009782 and IPET (High Value‐added Food TechnologyDevelopment Program, 2012, 112066‐3), S. Korea
Mexico’s fight against obesity: evidence, actions, results, and challenges (630.9)
Authors: Rivera Juan , Barquera Simón , Colchero Arantxa , Barrientos Tonatiuh , Hernández Mauricio
Published: June 2021

Mexico’s obesity prevalence is one of the highest in the world. The National Public Health Institute (INSP) has developed evidence based obesity prevention strategies, some of which were implemented by the Government during 2008‐2102, including recommendations on beverage intake and a multi‐faceted National Agreement for Healthy Nutrition (ANSA) which led to banning sodas and unhealthy food in schools. A main challenge of the ANSA was lack of harmonization between industry interests and public health objectives, and absence of effective accountability and monitoring mechanisms to assess implementation across government sectors. An evaluation of school regulations showed progress but also important implementation challenges which resulted in limited success. The current governmental obesity prevention strategy includes regulation of food marketing to children, implementation of a front‐of‐pack labeling system and nutrition and physical activity promotion. Estimations of own‐ and cross‐ price elasticity of the demand for SSB and models to estimate the effect of different tax levels on weight loss and diabetes prevention provided the evidence for congress to approve taxes to SSB and junk food. Civil society organizations have been key in the approval of initiatives such as taxing SSB. The Mexican experience in fighting obesity, the role of INSP, the challenges and lessons learned will be discussed.
Elementary school children’s consumption of lower calorie flavored milk, 2010‐2013 (632.1)
Authors: Yon Bethany , Johnson Rachel
Published: June 2021

USDA requires flavored milk (FM) in schools to be fat‐free. Added sugars (AS) were reduced from 15gm/8oz in 2009‐10 to 6gm/8oz in 2012‐13, reducing FM calories from 170 to 110 kcal/8oz. Milk is an important source of shortfall nutrients, thus it is important to know how well children consume reformulated FMs in school. Annual plate waste studies were conducted (April‐June 2010‐2013) in school districts (n=10) in the north and south as elementary schools moved to fat‐free, low added sugars FM. In 2010, children (grades 3‐5, n=497) consumed 5.8+ SE 0.12oz of the original 170 kcal/8oz FM. In subsequent years, as fat and AS were removed, children consumed 5.2+SE 0.12oz (n=388, 1277, 533). The data were highly skewed and converted to a binary variable. Overall, children in northern schools were more likely to consume >7oz FM (p<0.0001) than children in the south. Boys consumed more FM at lunch than girls (p<0.0001). Using linear mixed models adjusting for sex and region differences, the proportion of children consuming >7oz FM was stable over time (p=0.73). However, consumption patterns differed among schools (p=0.001). Changes to FM formulations to meet USDA standards appear to be successful in reducing fat and AS calories in children’s diets at school without impacting overall milk consumption. However, given the wide variations observed among schools, further monitoring of children’s dietary patterns is warranted.

Grant Funding Source: USDA Hatch and Dairy Research Institute
Perspectives of country actors on sustainability of food and nutrition security policies across presidential transitions (632.13)
Authors: Escobar Alegria Jessica , Frongillo Edward , Blake Christine , Walker Lee , McDermott Gerald
Published: June 2021

Developing countries with democratic governments periodically choose and replace presidents. Uncertainties in these transitions affect political and institutional decision‐making. Sustainability during transitions is important for food and nutrition security policies (FNSP), which require continuous political commitment, funding, capacity development, implementation, and collaboration among actors. The purpose of this study was to understand how country actors conceptualize sustainability of FNSP implementation across multiple presidential transitions. A case study in one Central‐American country was conducted using a qualitative, grounded‐theory approach. FNSP country actors described their understanding of sustainability in the context of specific actions. Sustainability was conceptualized differently depending on actors’ institution (government, international organization, private sector, civil society, research), level (central, departmental, municipal), and mode of job assignment (appointed, elected). Actors’ conceptualizations of sustainability were shaped by their background, work experience, mandate, and perception of what is important. This knowledge of the complex and diverse conceptualizations of FNSP sustainability by country actors will inform policy design, implementation, and monitoring to assist countries to more effectively reach long‐term goals