bluuebunny/crossref_metadata_2025 huggingface dataset:
Implementing the Dietary Guidelines for Americans Using New Technologies as a Channel for Nutrition Education: Evidence From Research and Best Practices
Authors: Blum-Kemelor Donna Michele , Spahn Joanne M. , Obbagy Julie E. , Fungwe Thomas V. , Olson Sara L. , Samuels Sarah E. , Yoshida Sallie , Shen Ted
Published: June 2021

A systematic review documented the potential value of using nutrition education delivered via digital media and technology to improve dietary behaviors among youth. This project was designed to complement the systematic review with qualitative research describing the use of and opportunities for leveraging new technologies in schools and communities for child‐focused nutrition education. Systematic review conclusions were based on review of the peer reviewed literature. Qualitative research data was collected via in‐depth searches to find pertinent unpublished and published articles with outcomes other than diet‐related behavior. A market research review and environmental scans found that while new technologies have been integrated into school curricula, their use in nutrition education is limited. Some technological nutrition education applications found were interactive websites and lessons, nutrition blogs, and smart phone programs. Ethnographic research, in which 30 semi‐structured interviews and 6 site visits with nutrition education experts are conducted, will yield best practices for maximizing the use of technologies to educate children about nutrition in settings such as school gardens and cafeterias. These research findings will highlight strategies that practitioners can use to creatively communicate Dietary Guidelines principles using new technologies.

Supported by USDA.
Regulation of Jaw Size During Development and Evolution
Authors: Ealba Erin L , Yu Jane , Jheon Andrew H , Butcher Kristin D , Schneider Richard A
Published: June 2021

The jaw shows great evolutionary variation in size. Our recent work demonstrates that skeletal cells derived from neural crest mesenchyme (NCM) control jaw size. Yet mechanisms by which NCM accomplishes this task remain unclear. One scenario is that interactions between NCM and adjacent cells, such as osteoclasts, play a critical role in determining jaw size. Unlike NCM‐derived osteoblasts that make bone, osteoclasts come from the hematopoietic lineage and resorb bone. A finely tuned balance between osteoblast and osteoclast activities may ultimately establish jaw size. Factors known to mediate these interactions include members and targets of the TGFÎ2 signaling pathway. To test if NCM controls jaw size through TGFÎ2 signaling and osteoclasts, we employ a quail‐duck chimeric transplant system. Previously, we have shown that quail donor NCM makes bone by executing autonomous molecular programs within duck hosts. Here, we perform transplants and gain‐ and loss‐of‐function experiments. We find that NCM controls jaw size through TGFÎ2 signaling and by regulating osteoclasts. Funded by NIDCR T32 DE007306 and K08 DE021705A to ELE; NSF DGE‐0648991 to JY; CIHR Fellowship to AHJ; and NIDCR R01 DE016402 to RAS.

Grant Funding Source : NIH
Using the S. cerevisiae Stn1 telomere capping protein to dissect downstream responses of the S phase checkpoint
Authors: Gasparyan Hovik John , Nugent Connie
Published: June 2021

In the budding yeast S. cerevisiae, Stn1 is an essential protein that caps chromosome ends, and facilitates telomere replication. Previous work in our lab has shown that increasing the levels of Stn1 makes cells extremely sensitive to the replication inhibitors hydroxyurea (HU) and methyl‐methane sulfonate (MMS). Remarkably, this sensitivity corresponds with overproduced Stn1 abrogating most aspects of the S phase checkpoint, including destabilization of replication forks, de‐repression of late replication origins, and causing cells to arrest with elongated mitotic spindles. The Rad53 checkpoint kinase is activated normally in Stn1 overproducing cells, indicating Stn1 is impacting checkpoint responses downstream of the checkpoint signal transduction pathway. These observations indicate that Stn1 overproduction provides a unique reagent to probe downstream aspects of Rad53 function, and we are therefore analyzing the mechanism and genetic requirements by which Stn1 shuts down execution of the S phase checkpoint pathway. This work was funded in part by the NSF, grant number 1024792 awarded to Connie Nugent.
Enhancing Student Engagement in an Advanced Protein Structure, Function & Biophysics Class
Authors: Bell Ellis
Published: June 2021

Junior and Senior majors in a biochemistry and molecular biology major take advanced courses in the discipline yet have often only been exposed to traditional lecture based courses. As a result they are often ill‐prepared to think about foundational concepts or apply them to new situations and are not adept at the various skill sets necessary for them to compete in graduate school or future professions. To counter these effects a dynamic and active classroom engages students more fully in their own education. In an upper level protein structure, function and biophysics course which revolves around three major themes, Protein Structure and Flexibility, Energy Barriers and ways to overcome them, and Function and Regulation, a variety of active learning strategies including small group discussion and peer teaching, literature discussion and presentation, original summary synthesis of topics from the literature, challenging problem sets containing real data, and a fully integrated laboratory experience where students have to design their own experiments and interprete data replace traditional lectures and labs. Although students take a while at the start of the course to adapt to such a different teaching and learning style, pre and post testing using tools designed to assess foundational knowledge and skills shows significant gains in student understanding.

This work is supported by NSF Grant MCB 0448905 to EB
Temporal changes in serum hepcidin concentrations and iron status in male Soldiers during military training
Authors: McClung James P. , Lutz Laura J. , Karl J. Philip , Rood Jennifer C. , Cable Sonya J. , Williams Kelly W. , Pasiakos Stefan M. , Young Andrew J.
Published: June 2021

Iron is a nutritionally essential trace element known to affect physical and cognitive performance in military personnel. Previous studies indicate reductions in iron status in Soldiers during basic combat training (BCT). As the mechanism conferring declines in iron status during BCT has not been elucidated, the objective of this longitudinal study was to investigate the temporal effects of a 10‐week BCT course on indicators of iron status and serum hepcidin concentrations in male Soldiers. Blood biomarkers were assessed in fasted volunteers (n = 99, age [mean ± SD] = 23 ± 5 yrs) at four timepoints (wks 0, 3, 6, and 9) during BCT. Iron status declined during BCT, as serum ferritin, transferrin saturation, and hemoglobin levels decreased (P < 0.05), and soluble transferrin receptor levels increased (P < 0.05) at wk 9 as compared to wk 0. Serum hepcidin levels decreased (P < 0.05) from wk 0 to wk 6 ([mean ± SEM] 35.2 ± 2.5 vs. 31.1 ± 2.0 ng/mL) and increased (P < 0.05) from wk 6 to wk 9 (31.1 ± 2.0 vs. 37.4 ± 2.4). Temporal changes in serum hepcidin observed during BCT may have an affect on iron status, however future studies will be required to determine the direct relationship between serum hepcidin and declines in iron status during military training. Research supported by MRMC.
Effects of whole grain phytochemicals on biomarkers of postprandial metabolic dysregulation in overweight/obese adults following an oral glucose challenge
Authors: McKay Diane L. , Chen C-Y. Oliver , Blumberg Jeffrey B.
Published: June 2021

The phytochemicals of whole grains may contribute substantially to their health benefits, but studies examining their bioactivity in humans are limited. To determine the degree to which whole grain oat and barley phytochemicals affect postprandial glucoregulation and inflammation, we conducted a controlled, 3‐way crossover trial in 13 healthy subjects, age 40–70 y with BMI 27–35.9. After a 2‐d run‐in period following a diet low in phytochemicals, subjects were randomized to receive muffins made with 48 g whole oat, barley, or refined wheat flour + fiber (control), with a 1 wk washout period between each intervention. Blood and urine samples were collected at specified time points before and for 3 h following the consumption of each test muffin concomitant with a 75 g glucose bolus to perturb homeostasis. Biomarkers of glucoregulation were better controlled with barley and oats compared to the control (Matsuda index: oats>barley>control; HOMA‐IR: oats<barley<control; AUCins/glu: barley<oats<control), although between group differences were not statistically significant. In a preliminary analysis of other outcome markers (n=5), changes in leptin:adiponectin and IL‐6 following the control were attenuated following oat and barley consumption. These preliminary data suggest that barley and/or oats may positively affect glucoregulation and select postprandial biomarkers. (Supported by USDA and Kellogg Co.)
Group III metabotropic glutamate receptors activate selectively pancreas‐projecting neurons in the dorsal motor nucleus of the vagus that control endocrine secretions
Authors: Babic Tanja , Travagli Renato Alberto
Published: June 2021

We have shown that pancreas‐projecting vagal preganglionic neurons (DMV) can be distinguished as exocrine vs endocrine‐controlling based on their response to either cholecystokinin (CCK) and pancreatic polypeptide (PP) or glucagon‐like peptide‐1 (GLP‐1), respectively. We have also shown that the group III metabotropic glutamate receptors (mGluR) agonist L‐AP4 decreases synaptic inputs to pancreas‐projecting DMV neurons. However, in an in vivo preparation, microinjections of L‐AP4 into the DMV have no effect on pancreatic exocrine secretion.

Here we aim to determine whether L‐AP4 can be used to distinguish central vagal circuits controlling pancreatic exocrine vs endocrine secretions.

Whole‐cell patch‐clamp recordings in identified pancreas‐projecting rat DMV neurons show that perfusion with L‐AP4 (100μM) decreased the frequency of miniature excitatory postsynaptic currents in 8/9 neurons tested. None of the L‐AP4‐responsive neurons responded to CCK (100 nM) and 1/5 neurons tested responded to PP (100 nM). In contrast, 4/5 L‐AP4‐responsive neurons also responded to GLP‐1 (100 nM).

Since CCK and PP regulate exocrine‐, whereas GLP‐1 regulates endocrine‐pancreatic secretions, our findings support the hypothesis that distinct central vagal circuits control exocrine vs endocrine secretions and suggest that activation of group III mGluRs in the DMV regulates selectively endocrine functions.

Supported by: NSF IBN‐104‐9618 to AT
Arrangement of the 4.5 Myb domain repeats of SNAP190 on the U1 gene promoter
Authors: Stumph William E , Doherty Matthew , Kang Yoon Soon
Published: June 2021

The small nuclear RNA activating protein complex (SNAPc) is a transcription factor essential for expression of genes coding for the spliceosomal snRNAs. In D. melanogaster, DmSNAPc recognizes a 21 base‐pair DNA sequence, the PSEA, located approximately 40–60 base pairs upstream of the transcription start site. DmSNAPc is a heterotrimer of subunits named DmSNAP190, DmSNAP50, and DmSNAP43. All three subunits are required for DNA binding activity, but only DmSNAP190 contains a “canonical” DNA binding domain that consists of 4.5 Myb repeats. Myb repeats are each ~50 amino acid residues in length and were first identified in the DNA binding domain of the Myb oncoprotein. By using a site‐specific protein‐DNA photo‐cross‐linking assay combined with site‐specific protein digestion, we have been able to determine where each of the 4.5 Myb repeats of DmSNAP190 contact the DNA of the U1 PSEA. The protein is arranged with the more C‐terminal Myb repeats contacting the upstream region of the PSEA and the N‐terminal repeats contacting the PSEA closer to the start site. Moreover, the C‐terminal repeats appear to contact the 5′ half of the PSEA in a manner consistent with those observed for Myb‐DNA complexes, whereas the N‐terminal repeats appear to contact the 3′ half of the PSEA (where DmSNAP50 and DmSNAP43 also bind to the PSEA) in a non‐canonical manner. (Supported by NSF and in part by the California Metabolic Research Foundation.)
Self‐aggregating mutant TRAPPC6A from partial exon 1 gene deletion activates caspases, binds TIAF1, and generates amyloid beta in hippocampus
Authors: Chang Nan-Shan , Chang Jean-Yun
Published: June 2021

TGF‐β‐induced factor TIAF1 forms aggregates in the hippocampus of normal individuals, and this may lead to the generation of amyloid beta (Aβ) and plaques as shown in Alzheimer's disease (AD). Here, we determined that TRAPPC6A gene deletion occurs at approximately a 3‐time higher incidence in AD than in normal controls. TRAPPC6A (TPC6A), a component of transport protein particle complex, forms aggregates with TIAF1 in the normal hippocampus, and the TIAF1/TPC6A complexes possess increasing amounts of Aβ in the AD hippocampus. TGF‐β induces aggregation of wild type TPC6A (TPC6Awt) and an N‐terminal deletion mutant (TPC6AΔ). Aggregated TPC6AΔ, but not TPC6Awt, upregulates p53 expression, causes caspase activation and Aβ superproduction in vitro. Tumor suppressor WWOX or WOX1 is frequently downregulated in AD. TPC6A physically interacts with WOX1, and TGF‐β1 dissociates the complex for causing TPC6A aggregation. Wwox−/− MEF cells were generated and prone to possess TPC6A and TIAF1 aggregates. Thus, downregulation of WOX1 is likely to elicit a sequential aggregation of TPC6A and TIAF1, respectively, for leading to the formation of Aβ and plaques in the brain. (Supported by NSC and NHRI, Taiwan, and DoD, USA)
Protein splicing facilitated by highly similar inteins from two extreme thermophiles
Authors: Williams Jennie E. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational event by which an intervening polypeptide, the intein, facilitates its own excision from the flanking polypeptides, the exteins, and the ligation of the exteins. Splicing of the Pyrococcus abyssi (Pab) PolII intein is initiated by an increase in temperature, which allows us to measure the rate of individual steps of the splicing reaction in vitro. We explored the rate of the first step of splicing for versions of the Pab PolII intein modified by mutation at conserved residues Asp74 and Ser166. We found no influence on the rate of the first step of splicing, but mutations at Ser166 influence the efficiency of the overall splicing reaction. The Pyrococcus horikoshii PolII intein has 71% sequence identity with the Pab PolII intein, but lacks an unstructured loop found in the Pab PolII intein. We hypothesize that this loop plays a role either in protein stability or in allowing for a mechanism‐linked conformational change to coordinate the steps of splicing. We will compare the temperature dependence of the rate and extent of splicing reactions and of the overall fold for both inteins.

This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.
Do the Inter‐Nucleotide Domain loops act as an Entropy Sink in the Catalytic Activity and Regulation of 3‐phosphoglycerate Dehydrogenase?
Authors: Meehan Cristina , Falk Bradley , Bell Ellis
Published: June 2021

Proteins can take on an array of conformations determined by regional flexibility of the protein. This may play critical roles in catalysis and allosteric regulation. The relationship between structure and function was studied to determine the contributions particular amino acids in 3phospho‐glycerate dehydrogenase. (3pGDH), may play in flexibility and the protein's function. Based upon available crystal structures a number of amino acids were identified by analysis of the temperature factors that may show altered flexibility in response to serine binding. In addition in conjunction with analysis of the temperature factors of bound cofactor (NAD(H)) we observe that two of the 4 central loops in the tetramer structure are more flexible than the other two and that this correlates with increased affinity for the cofactor. This creates an entropy sink that can be used to harness flexibility changes for catalysis and allosteric regulation. Various point mutations of specific amino acid residues involved in this “entropy sink” were created and the expressed proteins purified using Ni‐NTA affinity chromatography. Preliminary results suggest significant effects on activity. Our observations suggest the presence of an entropy sink which contributes to cofactor binding cooperativity and possibly catalysis.

This work is supported by NSF Grant MCB 0448905 to EB
Probing Subunit Interactions in 3‐Phosphoglycerate Dehydrogenase
Authors: Falk Bradley , Bell Ellis
Published: June 2021

3‐Phosphoglycerate Dehydrogenase is a homotetramer with three types of subunit interfaces; between adjacent regulatory domains, cofactor domains, and across the central cavity of the tetramer. A single tryptophan per subunit lies at the cofactor interface and crosses over between adjacent subunits. Collisional quenching approaches using steady‐state and lifetime measurements with both charged and neutral quenchers have been used to investigate the effects of ligands on exposure and asymmetry of this interface in native enzyme and a truncation mutant lacking the serine binding domain. NADH and á‐ketoglutarate decrease overall accessibility of the tryptophan possibly through the rotation of an interface‐spanning loop while serine primarily affects the charge environment around the tryptophan. Rotation of this loop may orient residues at the interface to form cross interface interactions necessary for communication. A K141Q/L mutations shows a decrease in acrylamide quenching as well as a reversal in iodide and cesium quenching. Ligand binding has less effect than with native. Thermal melts indicate a decrease in stability in these mutants while the minimal effects of ligands suggests changes in flexibility. Lifetime measurements indicate upward curvature seen in some Stern‐Volmer plots results from static quenching.

This work is supported by NSF Grant MCB 0448905 to EB
Sensory‐Motor Integration of Gill Lateral Cilia in the Bivalve Mollusc, Crassostrea virginica
Authors: Akande Patrick , Bandaogo Zeekanapi , Carroll Margaret A. , Catapane Edward J.
Published: June 2021

Gill lateral cilia of Crassostrea virginica are controlled by serotonin (HT) and dopamine (DA) innervation from their ganglia. Most bivalves that have been studied have lateral cilia that respond to HT and DA indicating a neuro or endocrine mechanism. While the motor aspects have been studied over the years, the sensory side has not. We examined effects of sensory cues on beating of lateral cilia of gill of C. virginica. Cilia beating was measured in whole animal preparations. Irritating the mantle rim with a metal probe caused a 70% drop in beating rates lasting at least 15 min. Shining a light on mantle rim decreased beating by 50%. Applying crab extract reduced beating to zero. When the branchial nerve, was cut crab extract had no effect on the cilia. When the cerebrovisceral connective was cut the basal rate was lower than controls and the crab extract was effective in slowing beating rate. Since the mantle rim is a major site of sensory cells in the animal, we excised the rim and applied crab extract. The absence of the mantle rim prevented the crab extract from affecting beating. The study shows a sensory motor integration of the beating of lateral cilia which involves the mantle sensory rim, and the visceral and cerebral ganglia. This work was supported by grants 2R25GM0600309 of NIGMS, 0516041071 of NYSDOE and 0622197 of NSF.
Studying the differential gene expression patterns of Hippocampus and Amygdala associated with social stress in mouse model
Authors: srinivasan seshamalini , Sowe Bintu , Miller Stacy-Ann , Muhie Seid , Chakraborty Nabarun , Hammamieh Rasha , Meyerhoff James , Jett Marti
Published: June 2021

The interaction of hippocampus (HC) with amygdala (AY) during the encoding of emotional memories apprises threat‐related stimuli and initiates fear conditioning, a process that is highly relevant in establishing proper care management for post‐traumatic stress disorder (PTSD). Our objective is to carry out global gene expression profiling of these brain regions in order to elucidate the molecular mechanisms underlying stress‐induced plasticity in fear circuits. We developed a social stress (SS) mouse model which parallels the classic symptoms of PTSD such as avoidance and retarded locomotion. This model was established by housing a male C57B1/6 naïve subject mouse with an aggressive (AGG) mouse for 6h/d × 5d or 10d and briefly pairing with the AGG for 3x/ day. Controls were housed without food/liquid and no direct physical contact with AGG. RNA samples from HC and AY were collected immediately (1d) after the SS session and at a delayed time points (1.5wks for 5d and 6wks for 10d). Genes related to cell mediated immune response, cell morphology and cell death, neurological disease were differentially expressed. Further comprehensive analysis can potentially lead to identifying diagnostic and therapeutic biomarkers for stress vulnerability and stress resilience, thereby aiding in early diagnosis and treatment of PTSD. This work was supported by the Defense Threat Reduction Agency, contract TMTI0029_09_WR_T.
TIAF1 self‐aggregation causes spontaneous activation of SMAD‐responsive promoter in p53‐deficient environment and cell death
Authors: Chang Nan-Shan
Published: June 2021

Self‐aggregation of TGF‐beta‐induced antiapoptotic factor (TIAF1) is known in the nondemented human hippocampus, and the aggregating process may lead to generation of amyloid beta (Abeta) for causing neurodegeneration. Here, we determined that solid tumors frequently overexpress TIAF1, which exhibits as aggregates together with Smad4 and Abeta in the cancer stroma and peritumor capsules. Also, TIAF1/Abeta aggregates are shown on the interface between neural cells and the metastatic cancer cell mass. In vitro induction of TIAF1 self‐association upregulated the expression of tumor suppressors Smad4 and WOX1 (or WWOX), and WOX1 in turn increased the TIAF1 expression. TIAF1/Smad4 interaction further generated Abeta. TIAF1 suppresses SMAD‐regulated promoter activation. Intriguingly, without p53, TIAF1 self‐association spontaneously activated the SMAD‐regulated promoter. TIAF1 was shown to be essential for p53‐, WOX1‐ and dominant negative JNK1‐induced cell death. Indeed, TIAF1, p53 and WOX1 acted synergistically in suppressing anchorage‐independent growth and causing apoptosis. Together, TIAF1 shows an aggregation‐dependent control of tumor progression and metastasis, and regulation of cell death. (supported by NSC and NHRI, Taiwan and DoD, USA)
Macronutrient absorption from almonds: the measured energy value of almonds in the human diet
Authors: Gebauer Sarah K , Novotny Janet A , Baer David J
Published: June 2021

Epidemiological studies have demonstrated an inverse association between frequent nut consumption and body mass index. Clinical studies have shown that lipid from nuts may be more poorly absorbed than from other food sources; thus, the metabolizable energy (ME) contained in nuts may be less than that predicted by Atwater general factors. Data from human feeding studies are lacking. The ME value currently used for food labeling, based on Atwater general factors, is 5.7 kcal/g (160 kcal/1 oz svg). The objective of the current study is to empirically determine the measured energy value of almonds utilizing a randomized, crossover study in healthy adults (n = 18). Almonds were fed in 3 doses as part of a controlled diet: 0 g/d (control), 1.5 oz/d (42 g/d), and 3 oz/d (84 g/d). Urine and fecal samples were collected for 7 to 10‐day periods during each 3‐week diet period. Diet and feces were analyzed for nitrogen, fat, total dietary fiber, ash, and combustible energy; urine was analyzed for nitrogen and combustible energy. The energy value of almonds was calculated from differences in energy excretion during the dietary treatments. The measured ME density of almonds was 4.56 kcal/g (128 kcal/1 oz svg) – 20% less than the current value reported on the food label. Accurate information about ME content of foods is important for reliable food labeling so consumers can make informed dietary choices.

Support: USDA & Almond Board of California
Mechanical deformation decreases activation time for neutrophils
Authors: Burdette Aaron Jeffrey , Rogers Steven , Tees David F.J.
Published: June 2021

Activation of polymorphonuclear leukocytes (PMN) can be induced by mechanical deformation. When a PMN is aspirated into a micropipette of a smaller diameter than of the cell, the cell experiences mechanical deformation. Activation is assumed to occur when pseudopod projection is observed, along with an influx of intracellular calcium. We hypothesize that increased deformation of PMNs leads to faster activation. The size of micropipettes (~5–7 μm) used mimicked the conditions inside lung capillaries. We measured the elapsed time required for activation following aspiration into various pipette diameters. The cell‐penetrating calcium‐sensitive dye Fura‐2AM was loaded on to the PMN to detect increases in the intracellular calcium. The ratios of intensity among the two excitation wavelengths, 340 nm and 380 nm, give calcium concentration. A cooled intensified CCD camera was used to measure the intensities every 15 s. From observations of pseudopod projection, the activation time of the PMN was inversely proportional to the amount of mechanical deformation as characterized by the deformation ratio of cell diameter to the diameter of the micropipette. This supported our hypothesis that increased deformation would decrease activation time. This work was supported by grant BES‐0547165 from the National Science Foundation.
Regulation of myo‐inositol in tilapia (Oreochromis mossambicus) brain during hyperosmotic stress
Authors: Gardell Alison M. , Sacchi Romina , Kültz Dietmar
Published: June 2021

Myo‐inositol (MI) is a compatible organic osmolyte that is accumulated in cells exposed to hyperosmotic stress via various biochemical pathways. We and others have shown that the de novo biosynthetic pathway, which synthesizes MI from glucose‐6‐phosphate, is especially important for osmoregulation in Mozambique tilapia (Oreochromis mossambicus). Additional pathways of MI accumulation utilize membrane transporters, such as sodium/myo‐inositol cotransporter and proton/myo‐inositol cotransporter, to move exogenous MI into the cell. In order to investigate mechanisms of the osmotic stress response, an immortalized cell line was established from primary explant cultures of tilapia brain tissue. To validate this cell line, we studied the effects of acute and chronic osmotic stress on MI regulation in tilapia brain. Myo‐inositol phosphate synthase (MIPS) mRNA expression data were compared from in vitro (tilapia brain cell line) and in vivo (tilapia whole brain) approaches. MIPS, which is one of two key enzymes of the biosynthetic pathway, was highly upregulated both in vivo and in vitro after acute exposure to osmotic stress (25ppt seawater or 700mOsm medium). Blood plasma osmolality, sodium, chloride, and potassium concentrations were also measured in vivo to complement mRNA expression data and relate changes in osmotic blood chemistry to changes in cell culture medium. Supported by NSF grant IOS‐1049780.
Estradiol and Metabotropic Glutamate Receptors interaction within the anxiety neural circuitry at behavioral and protein levels
Authors: De Jesus-Burgos Maria Ines , Gonzalez-Garcia Stephanie , Cruz Yanira , Gonzalez Gabriela , Gonzalez Beatriz , Portela Lucila , Pérez-Acevedo Nivia L
Published: June 2021

Anxiety is the most common psychiatric disorders affecting females twice more than males. In hippocampal cells, estradiol regulates group I/II metabotropic glutamate receptors (mGluR1a/5 and mGluR2/3, respectively) activation. Thus, estradiol might modulate female anxiety within the basolateral amygdala (BLA) through mGluRs. We hypothesized that: 1) BLA activation of mGluRs might produce anxiolytic‐like effects; and 2) mGluR protein expression will be up‐regulated, in an estradiol dependent manner. Ovariectomized female rats, with (OVX+EB) and without (OVX) estradiol treatment were compared to male rats. We infused (S)‐3,5‐dihydroxyphenylglycin (DHPG), a group I mGluR agonist into the BLA. Anxiety was analyzed with the elevated plus maze and risk assessment behaviors (RABs); and protein expression by western blots analysis. DHPG (1.0 μM) increased the time and entries in open arms; and reduced RABs in OVX+EB (p<0.05), but not OVX and/or male rats. In the amygdala, protein expression of mGluR1a and mGluR2/3 were up‐regulated in OVX+EB (p<0.05) than OVX and/or male rats. No differences were found for mGluR5. Thus, activation of group I mGluRs, within the BLA, depends upon estradiol to modulate female anxiety. This effect might be underlined by region‐specific estradiol genomic and nongenomic effects.

Grant Funding Source: NIH‐EARDA 1G11H046326, MBRS‐RISE GM61838 and NSF DBI‐0932955
The β‐Catenin/Tcf‐4 Pathway Regulates High Mobility Group A1 (hmga1) Expression in Colorectal Cancers
Authors: Deng Jiayue Amy , Brock Ashton T. , Bush Bethany M. , Sumter Takita Felder
Published: June 2021

High Mobility Group A1 (HMGA1) encodes proteins that act as mediators in viral integration, chromatin modification, neoplastic transformation, and metastatic progression. Because hmga1 expression is overexpressed in most cancers and has known interactions with several Wnt/β‐catenin‐responsive genes, we explored the involvement of HMGA1 in colorectal cancer. Apc mutations occur in most colorectal cancers and are associated with the mobilization of the β‐catenin/Tcf‐4 transcriptional complex. In adenomatous polyposis coli (Apc)(Min/+) mice expressing truncated Apc, we observed 9‐fold greater HMGA1 mRNA levels relative to mice bearing wild‐type Apc. HMGA1 protein levels were also significantly elevated in Apc(Min/+)intestinal tumors. Sequence analysis of the 5′‐flanking sequence of hmga1 identified a functional Tcf‐4 binding element (TBEs) that specifically binds Tcf‐4 in vitro and in vivo suggesting that hmga1 is a downstream target of the β‐catenin/Tcf‐4 signaling pathway. These findings will be further substantiated by transcription assays to evaluate the impact of Tcf‐4 and β‐catenin expression on hmga1 promoter activity. The results confirm previous work identifying hmga1 as a downstream target of the β‐catenin/Tcf‐4 complex and provide a new target for the development of therapies aimed at treating colorectal cancer. This research is supported by NIH 2P20 RR016461‐05, 1R15CA137520‐01 and NSF MCB055810542242.
A Systematic Approach to Transcriptional Regulator Function Discovery: Function Discovery for Bxe_B3018
Authors: Martí-Arbona Ricardo , Unkefer Pat J , Unkefer Clifford J
Published: June 2021

Transcriptional Regulators (TR) regulate and control the physiological processes that dictate phenotypic traits. Regulation occurs in response to stimuli, this response manifests itself in the binding of a effector molecule by the TR. Function prediction and possible effector library formulation are key factors in the discovery of the biological function of TR. Potential effectors are predicted by the identification of functional patterns within the proteins found in the genomic proximity of the TR of interest. These patterns can suggest involvement of the TR on the regulation of specific metabolic pathways. Utilizing Frontal Affinity Chromatography coupled to Mass Spectrometry detection (FAC‐MS), Anisotropy and Electrophoretic Mobility Shift Assay we have developed a systematic approach for the screening, characterization and validation of small‐molecules that may modulate TR binding to DNA operator sequences (DOS). Utilizing this approach, we have predicted the metabolic pathway regulated by the previously unknown function TRs Bxe_3018 from Burkholderia xenovorans. Aminoacetone disrupts the affinity of Bxe_B3018 for its DOS (5’‐AGAANNNNNNTTCT‐3’). These findings validate the effectiveness of our systematic approach to TRE discovery and open the door to a new era of protein function discovery and greater understanding of metabolic regulation. This work was supported by the LDRD program at LANL.
Reciprocal telomerase inhibition by human and Xenopus PinX1
Authors: Shampay Janis , Constant David , Gaubatz Jayne , Fink Patrick , Bazilevsky Gleb , Cylinder Isabel , Pires Jacqueline
Published: June 2021

The human TRF1/Pin2 interacting protein, hPinX1, has been shown to inhibit telomerase both in vitro and in vivo. Telomerase regulation is particularly important in the frog Xenopus, which expresses abundant telomerase in all adult somatic tissues. We have cloned and characterized the ortholog of PinX1 from X. laevis (xPinX1). The N‐terminal half of xPinX1 is 76% identical to hPinX1, while the C‐terminal half, containing hPinX1ˈs telomerase inhibition domain, is far more diverged. Quantitative RT‐PCR demonstrated that the xPinX1 transcript is relatively weakly expressed in high‐telomerase spleen, although there was no clear inverse correlation between telomerase activity and xPinX1 transcription in tissues with more modest levels of telomerase. To assess function in vitro, both xPinX1 and hPinX1 were purified from E. coli as fusions to His8‐GFP. Both fusion proteins inhibited telomerase in a PCR‐based assay, whereas His8‐GFP did not. Furthermore, each PinX1 was able to inhibit telomerase activity from both species. This suggests structural constraints on the inhibition mechanism, despite the rapid divergence in primary sequence between the two proteins. Further characterization of in vivo and in vitro functions are ongoing. Supported by grant 0642104 to JS from the National Science Foundation, a Betty Liu Research Fellowship to DAC, and a grant to Reed College by the James F. and Marion L. Miller Foundation.
Thermodynamic investigation of the dinucleotide bulge of domain V of group II intron
Authors: Mhlanga Siphelele Leslie , Grover Neena
Published: June 2021

Group II introns are self‐splicing ribozymes, found in RNA of organelles in fungi, plants, and protists, as well as in bacterial mRNA. Group II introns are composed of six structural domains, which are designated domain I to domain VI. Domain V (DV) is a short stem loop that contains a dinucleotide AC bulge; it is implicated in the catalytic function of the intron, as it is expected to bind the catalytic Mg2+ ion(s). A crystal structure of a group II intron from Oceanobacillus iheyensis shows inner sphere interactions between two Mg2+ ions and the phosphate oxygens of the cytosine of the dinucleotide bulge. In this study, we are examining the role of the dinucleotide sequence on RNA stability along with quantifying the contribution of RNA‐magnesium interactions. Various RNA and DNA constructs of the dinucleotide bulge were designed and thermal denaturation experiments were performed on the wild type and the modified constructs in 1 M KCl and in varying concentrations of Mg2+ ions. Comparative thermodynamic data will be presented on various dinucleotide RNA and DNA constructs and their ion interactions. This work was funded by NSF Grant MCB‐0950582 to NG.
Thermodynamic Examination of Bulge Sequence, Ion Binding, and Helical Context of 2X2 and 2X1 Bulges in RNA
Authors: Mahajan Priyam , Grover Neena
Published: June 2021

Bulges are non‐helical regions in RNA that provide sites for protein and ligand interactions. The interactions in and around the bulge region are non‐canonical and often not well understood. In crystal structures, bulges and non‐canonical base pairs are often the sites for interactions with metal ions. The pyrophosphate sensor helix of the thiamine pyrophosphate (TPP) riboswitch contains a 2×2 bulge that binds the pyrophosphate group along with Mg2+ ions. The 1×2 internal stem loop of the U6 spliceosomal RNA changes conformation upon the formation of A+•C base pair and Mg2+ binding. In this project, we are investigating the non‐canonical base pairs in the bulge regions, the helical context in which these are found, and the stabilization of these RNA in the presence of Mg2+ ions. Wild type and modified RNA constructs were designed to study RNA stability using thermal denaturation experiments in 1 M KCl and in varying concentrations of Mg2+, in pH 5.5 and 7.5. In 1 M KCl, the wild type U6 2X1 and TPP 2X2 constructs showed a greater stability at pH 5.5 over pH 7.5 likely due to the formation of A+•C base pair. All TPP 2X2 constructs, with one exception, are more stable in 9.5 mM Mg2+ over 1 M KCl. Comparative thermodynamic data will be presented on various constructs to better elucidate the differences and similarities of 2X2 and 1×2 bulge regions. This work was funded by NSF Grant MCB‐0950582 to NG.
Intein‐mediated peptide bond cleavage adjacent to aspargine or glutamine
Authors: Chin Stacy L. , Nadelson Adam C. , Urbanski Laura M. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational, self‐catalyzed process in which an intervening polypeptide, the intein, is self‐excised from the flanking polypeptides, or exteins. The intein that interrupts the DNA polymerase of Pyrococcus abyssi can facilitate protein splicing in a temperature‐dependent fashion. This intein has a Cterminal Gln, which facilitates the third step of splicing, side chain cyclization coupled to peptide bond cleavage. We plan to induce C‐terminal cleavage of an intein (uncoupled from splicing) to facilitate purification from a solid affinity resin. We have fused the intein with an N‐terminal affinity domain and a C‐terminal target protein for purification, such that C‐terminal cleavage of the intein via cyclization of Asn or Gln liberates the C‐terminal domain with an N‐terminal Cys. This fragment will serve as a substrate for expressed protein ligation. In addition, we will compare the rate of the cyclization between glutamine and asparagine in model peptides with an N‐terminal Abz fluorophore and a C‐terminal Lys‐DNP quencher, using a fluorescence assay to measure cleavage. We will determine the rate of cleavage as a function of temperature and pH.

This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.
Kinetic characterization of calmodulin‐troponin C chimeras binding to endothelial and neuronal nitric oxide synthases
Authors: Brown Christopher C. , Helms Katy A. , Hill Stephanie A. , Ghosh Dipak , Salerno John C. , McMurry Jonathan L.
Published: June 2021

Endothelial and neuronal nitric oxide synthases (eNOS and nNOS) are signal‐generating enzymes with numerous physiological effects including angiogenesis and signaling at synapses. Calmodulin (CaM) is an important regulator of NOS activity. Following on our recent report (McMurry, et al. FEBS J. 2011) describing the kinetics of CaM‐NOS binding, present results describe binding of nNOS and eNOS to Calmodulin‐Troponin C (CaM, TnC) chimeras differing in EF hand domain composition. CaM and TnC have similar structures consisting of two globular domains linked by a central helix with each domain consisting of two EF hands. Chimeras examined included each of the four domains of TnC exchanged for the analogous CaM domain. Chimeras exhibited a wide range of rate and affinity constants as determined by optical biosensing, generally reflecting their ability to activate eNOS and nNOS. Affinities ranged from subnanomolar to micromolar. Binding was rapidly dissociable in the presence of EDTA, confirming that observed binding was calcium‐dependent. This work was supported by NIH grants GM080701 and GM083317 and NSF grants 0950920 and 0922699 and a KSU Mentor‐Protégé Grant.
Ribosomal S‐6 kinase and Rho‐associated kinase phosphorylation of NHE1
Authors: Dornbusch Andrew , Berthelsen Nicholas , Anderson Kristina , Provost Joseph , Wallert Mark
Published: June 2021

Regulation of the Sodium‐Hydrogen Exchanger isoform‐1 (NHE1) is critical in the control of intracellular pH and cell volume. NHE1 also provides a focal point between the cytoskeleton and the plasma membrane resulting in the coordination of directed cellular migration. Both RhoA Kinase (ROCK) and Ribosomal S‐6 kinase (Rsk) regulate these NHE1 functions, however the site of NHE1 phosphorylated by ROCK and it's specific impact on NHE1 function has not been determined. Using recombinant c‐terminus NHE1 we show site‐specific ROCK phosphorylation of NHE1. To understand the role of both kinases on NHE1 we have created stable cell lines that express full‐length tagged human wild‐type NHE1 with Ser/Thr to Ala mutations. The expression level and location of recombinant NHE1 was assessed by western blot and confocal microscopy. Loss of either NHE1 phosphorylation site altered NHE1 activity following intracellular acidosis. The impact of NHE1 phosphorylation showed that phosphorylation of ROCK and RSK are both critical in formation of stress fibers and migration, however loss of either phosphorylation site poorly impacts proliferation. Our data demonstrates that NHE1 is a substrate for ROCK phosphorylation and that both ROCK and Rsk sites play a critical role in NHE1 function. This work was supported with funds from NSF MCB‐0817784
Acute bioavailability and pharmacokinetics of avenanthramides (AV) from “false malted” oat bran high in endogenous AV
Authors: McKay Diane L. , Chen C-Y. Oliver , Collins F. William , Blumberg Jeffrey B.
Published: June 2021

Avenanthramides (AV), hydroxycinnamoyl alkaloids unique to oats, have antioxidant and anti‐inflammatory activities in vitro and in vivo. Concentrated AV mixtures extracted from oat are bioavailable in hamsters and humans. We examined the bioavailability of AV from the bran of oats subjected to “false malting,” a process that enriches the AV content of native kernels up to 40‐fold, in a placebo controlled, crossover study of 10 healthy adults (age 50–70 y, BMI 18.5–29.9). After a 2‐d run‐in period following a diet low in polyphenols and absent oats/oat products, subjects consumed either a muffin made with 20 g AV‐enriched oat bran (containing 45 mg AV) or a placebo muffin absent oats, with a 1 wk washout period. Blood samples were collected before and up to 24 h after consumption for determination of AV isomers, A, B, C, O, P, and Q. Following consumption of the AV‐enriched oat bran muffin, maximal plasma concentration (Cmax) was highest for AV‐O at 74.8±41.3 ng/mL and lowest for AV‐C at 2.9±1.8 ng/mL. Time to reach Cmax (Tmax) for all 6 AV ranged from 1.4±0.6 to 2.3±0.8 h. There was an apparent bimodal distribution for AV‐O, P, and Q. The second peak for each was nearly equal in concentration to their respective Cmax and appeared at ~4.0 h. No AV were detected following the placebo. These data indicate AV from this oat bran matrix are bioavailable in older adults. The bimodal pattern suggests reabsorption of some AV isomers through enterohepatic circulation. (Supported by USDA and AAFC)
Probing the Effects of Cofactor and Substrate on Conformation and Flexibility of Human pGDH
Authors: Roach Courtney , Bell Ellis
Published: June 2021

3Phosphoglycerate dehydrogenase is a tetramic enzyme found in higher eukaryotes including humans and in prokaryotes such as E. coli, and plays a role in catalyzing the first step of L‐serine biosynthesis. Long (human and certain prokaryotes such as M. tuberculosis) and short (E. coli) forms of 3‐phosphoglycerate dehydrogenase exist, differing in the presence of an added domain between the substrate binding domain and the serine regulatory domain. We have compared the E coli and human forms using NSD mutants containing the cofactor and substrate binding domains and the critical cofactor domain dimerization interface where a conserved tryptophan crosses the interface. Using multiwavelength collisional quenching with differently charged quenchers, ANS Fluorescence and Circular Dichroism and thermal melts, we have examined the effects of cofactor and substrate individually and in combination on the local environments of the two tryptophans in human pGDH as well as overall exposure of hydrophobic residues and conformational stability. The two tryptophans in the human NSD appear to reside in multiple conformations suggesting significant conformational flexibility, and enhance ANS fluorescence more than than E coli NSD. While cofactor produces some conformational effects, alpha‐ketoglutarate appears to enhance indicators of conformational flexibility. Supported by NSF Grant MCB 0448905 to EB
Observation of TonB motion using fluorescence anisotropy
Authors: Jordan Lorne D , Zhou Yongyao , Smallwood Chuck R , Lill Yoriko , Ritchie Ken P , Yip Wai Tak , Newton Salete M , Klebba Phillip E
Published: June 2021

Using fluorescent hybrid proteins, we determined the anisotropy of TonB in the cytoplasmic membrane (CM) of Escherichia coli. With the aim of understanding the bioenergetics of outer membrane (OM) iron transport, we monitored the dependence of TonB motion on the electrochemical gradient and the CM proteins ExbB & ExbD. The native E. coli siderophore enterobactin chelates Fe+3 in the environment and ferric enterobactin (FeEnt) enters the cell by energy‐ and TonB‐dependent uptake through FepA, its OM transporter. The TonB‐ExbBD complex in the CM is hypothesized to transfer energy to OM metal transporters such as FepA. We addressed two main experimental goals: determination of the effect of proton‐motive force (PMF) on the natural motion of TonB, and the role of ExbB and ExbD in this phenomenon. We observed the polarization of GFP‐TonB hybrid proteins and used metabolic inhibitors (CCCP, azide and dinitrophenol) and chromosomal deletions of exbBD to study these questions. The results showed higher anisotropy (R) values for GFP‐TonB in energy‐depleted cells, and lower R‐values in bacteria lacking ExbB and ExbD. Metabolic inhibitors did not change the anisotropy of GFP‐TonB in ΔexbBD cells. These findings suggest that TonB undergoes constant, energized motion in the bacterial CM, and that ExbBD mediates its coupling to the electrochemical gradient. This research was funded by NSF award MCB09522999 to PEK and SMN, and the NSF Bridge to Doctorate program to LDJ.
RhoA Kinase Phosphorylation of NHE1 impact on Growth Factor and G Protein Coupled Receptor Signaling
Authors: Berthelsen Nick , Pantera Harrison , Sauer Jon , Lawrence Jim , Wallert Mark , Provost Joseph
Published: June 2021

The sodium hydrogen exchanger (NHE1) plays a role in intracellular pH homeostasis and acts as scaffolding anchor for a diverse set of proteins. The extended carboxyl terminus is phosphorylated by seven known protein kinases. Phosphorylation site of NHE1 by RhoA Kinase (Rock) was recently defined and found critical for a number of cellular functions. However, the impact of Rock phosphorylation on NHE1‐related signaling for a range of agonists has not been determined. Here we examine three different signaling pathways and the role Rock phosphorylation of NHE1. Using stably expressing tagged NHE1 we identify proteins co‐immunoprecipitating with NHE1 in control and agonist stimulated cells. Cellular function of adhesion and proliferation is also presented. Finally the influence of Rock phosphorylation of NHE1 on cellular migration was determined for three signaling pathways using an impedance‐based assay. This work was supported with funds from NSF MCB‐0817784