bluuebunny/crossref_metadata_2025 huggingface dataset:
Regional Patterns of Pulsatility Index and Wall Shear Stress Across Cerebral Circulation of Adolescents with High Insulin Resistance
Authors: Carter Katrina J. , Kellawan J. Mikhail , Peltonen Garrett L. , Ward Aaron T. , Carrel Aaron L. , Wieben Oliver , Bendlin Barbara B. , Schrage William G.
Published: June 2021

Background

Insulin resistance (IR) is associated with altered arterial stiffness, blood flow, and shear stress patterns in the peripheral circulation of middle‐aged and older diabetic and non‐diabetic hypertensive populations. Both altered blood flow and shear stress patterns may contribute to the elevated risk of cerebrovascular disease in IR populations. The aim of this study was to determine if adolescents (12–18yrs) with high IR (indexed as High Homeostatic Model Assessment of IR, HOMA‐IR) display early signs of cerebrovascular dysfunction. We hypothesized that young adolescents with a high HOMA‐IR would exhibit increased stiffness and decreased shear patterns in cerebral conduit arteries.

Methods

2 healthy controls (17±3) and 3 IR subjects (14±1) underwent a single 4D Flow MRI scan (scan time ~ 5min, no contrast given) to quantify cerebral blood flow (CBF), pulsatility index (PI) and wall shear stress (WSS) in extracranial and intracranial conduit arteries. 11 arteries were analyzed: middle cerebral (MCA) x2, anterior cerebral (ACA) x2, posterior cerebral (PCA) x2, vertebral (VA) x2, internal carotid (ICA) x2, and basilar. PI was calculated for each artery as [(CBFmax − CBFmin)/CBFmean]. WSS was measured by segmenting the vascular tree from PC angiograms and calculating the velocity gradient at the vessel wall using 4D flow MRI data over ~3 voxel. Due to voxel limitations, the PCA value is exploratory. Total CBF was calculated as the sum of both ICA and both VA. Significance was determined using a two‐tail Welch's t‐test.

Results

Individuals with IR displayed higher HOMA‐IR (IR 5.3±0.2, Ctrl, 3.3±0.3, p=0.02), BMI (kg•m2; IR 28±2, Ctrl 19±4, p=0.03) and fasting insulin (μU/mL; IR 25±4, Ctrl 13±0, p=0.04), but fasting glucose was not different (mg/dL; IR 79±4, Ctrl 93±5, p=0.07). PI was not different between groups among the 11 conduit arteries. However, there was a trend for higher PI in the IR group in the left PCA and right VA. Resting WSS did not differ between Ctrl and IR among the 11 conduit arteries. Similarly, total resting CBF was not different between Ctrl and IR (mL/min; IR 811±115, Ctrl 735±79, p=0.64).

Conclusion

PI or WSS were similar in 11 conduit arteries of cerebral circulation in adolescent controls compared to IR. These data indicate IR does not cause measurable arterial stiffening or altered total cerebral blood flow patterns. Therefore, the pathogenic arterial characteristics observed in middle‐aged adults with IR may be linked to the duration and/or severity of IR in association with other known vascular pathological processes linked to aging.

Support or Funding Information

This research was made possible by the National Science Foundation Grant #HRD‐1500138 (UW‐Madison WiscAMP‐BD program) and UW Graduate School Pilot Funding.
The Effects of 12 Days Exposure to 4,300M Altitude on the Sympathetic Neural and Cardiovascular Responses to Head‐Up Tilt
Authors: Bradbury Karleigh Eleanor , Mitchell Katherine M , Beidleman Beth A , Fulco Charles S , Muza Stephen R , Posch Alexander M , Charkoudian Nisha
Published: June 2021

Musclesympathetic nerve activity (MSNA) increases with acute and chronic altitude exposure. However, the effects of altitude exposure on the neural and hemodynamic responses to orthostasis are not clear. The purpose of the current study was to evaluate the effects of altitude exposure (12 days; 4,300 m, Pikes Peak, CO) on MSNA, heart rate (HR) and systolic (SBP) and diastolic (DBP) blood pressure during supine rest and 30° and 45°head up tilt (HUT). We hypothesized that: (1) MSNA would be elevated upon return to sea level (SL, 50 m) from Pikes Peak and (2) increased resting MSNA would be associated with decreased responses to HUT due to a potential ‘ceiling effect’. Supine MSNA, HR, SBP and DBP were measured in 9 healthy subjects (7 M, 2 F; 24 ± 8 years)prior to‐, and within 24 hr of return from altitude. MSNA was measured via peroneal microneurography. Subjects rested supine for 10–15 min to collect baseline data, then were tilted to 30° HUT for 2–3min, followed by 45° HUT for 2–3 min. Supine MSNA was increased within 24 hr return from altitude (27 ± 5 vs 17 ±6 bursts/min [SL], p < 0.05, ANOVA). MSNA at 30° HUT was higher within 24 hr return from altitude (36 ± 8bursts/min vs 25 ± 7 [SL], p< 0.05). MSNA tended to be higher at 45° HUT (39 ± 8 bursts/min vs 30 ± 9 [SL], p = 0.07). There was no difference in the extent of change in MSNA from supine to 30° HUT orsupine to 45° HUT between SL and post‐altitude (p > 0.05). There were no changes in HR, SBP or DBP supine, 30° HUT, and 45° HUT post‐Pikes Peak compared to sea level(p > 0.05 for all). Our data suggest that 12 days of altitude exposure increases resting (supine) MSNA but does not alter the ability of sympathetic vasoconstrictor nerves to increase activity in response to a baroreflex stimulus (HUT).

Support or Funding Information

Funded by USAMRMC; author views not official US Army or DOD policy.
Peptidergic Modulation in the Lobster Cardiac Neuromuscular System: A Transcriptomic Analysis of Peptides and Peptide Receptors in Cardiac Ganglion and Muscle
Authors: Gandler Helen , Stanhope Meredith , Shea Devlin , Christie Andrew , Yu Andy , LaMeyer Tess , Roncalli Vittoria , Cieslak Matthew , Dickinson Patsy
Published: June 2021

Central pattern generator (CPG)‐effector systems can produce flexible rhythmic motor output via modulation by both intrinsically‐ and extrinsically‐derived neuropeptides. In the lobster, Homarus americanus, the cardiac neuromuscular system, which consists of the cardiac ganglion (CG) and cardiac muscle (CM), controls the rhythmic motor output of the heart. This is a well‐studied, but simple, CPG‐effector system that has been used extensively for investigating peptidergic control of rhythmic behavior. Here, transcriptomes were generated for the two components that comprise the cardiac neuromuscular system, the cardiac ganglion (CG) and cardiac muscle (CM). Transcripts encoding precursor proteins for approximately ten peptide families, including allatostatin‐C (AST‐C), diuretic hormone 31 (DH31), diuretic hormone 44, myosuppressin, and proctolin, were identified from the CG transcriptome, suggesting that these peptide groups may be produced by neurons in the CG. Transcripts encoding receptors for many of the peptides identified from the CG, including those for all four of the above‐mentioned peptide families, were also identified from the CG and/or CM transcriptomes, suggesting that these peptides serve as intrinsic modulators of the cardiac neuromuscular system. To investigate hormonal modulation in the cardiac neuromuscular system, the transcriptomes were mined for the receptors of peptides known to be present in the sinus gland and/or pericardial organs, two neuroendocrine organs. Receptors for seven peptide families were identified, with some receptors restricted either to the CG (crustacean cardioactive peptide, red pigment concentrating hormone, and tachykinin‐related peptide) or CM (pigment dispersing hormone), and others found in both tissues (AST‐C, myosuppressin, and proctolin). These data suggest that some peptides may have different sites of action within the cardiac neuromuscular system. Immunohistochemistry confirmed that DH31 is produced by the motor neurons of the CG, and suggested that AST‐C is present in the CG. Immunohistochemical confirmation of the other putative intrinsic CG modulators is ongoing.

Support or Funding Information

NSF (IOS‐1353023, IOS‐1354567, OCE‐1459235), NIH (8P20GM103423‐12), Cades Fdn., University of Hawaii at Manoa's Undergrad Res. Opp. Prog., APS Undergrad Res. Fellowship, Doherty Fdn./Bowdoin College
Water Restrictions and Macronutrient Level Intake of Locust
Authors: Diaz Mildred Isabel , Harrison Jon , Cease Arianne
Published: June 2021

Locust are grasshoppers that tend to migrate in swarms that can cause major damage to agriculture. Chortoicetes terminifera are locust native to Australian deserts, where they can potentially experience water restrictions. I hypothesized that water restriction would slow growth; and that high‐protein diets will be more deleterious under water‐restricted conditions because nitrogenous wastes require water to eliminate. Locusts were collected on the day they molted into the fifth (final) instar, weighed and placed in an individual plastic cage. Locusts received ad lib quantities of two different dry, granular diets: 35% protein:7% carbohydrate or 14% protein:28% carbohydrate. Locusts either had unlimited water or had ad lib access to water during every third day. All locust were given water on the day they molted for 24 hours. Then the locust on the restricted water treatment had no water for two days, followed by 24 hours with water, then 48 hours without water, continuing this pattern until the locust molted. After the locusts molted into adults, which took approximately 10 – 15 days, their final body mass (wet and dry) was recorded. Consumption rate was calculated by the loss in mass of the food pile over the entire instar, and their growth rates from the change in wet mass and development time (measured with a precision of ±1 day). Water restriction did not affect body water content or many measures of growth and size, suggesting this was a mild stress. Sexes differed in their responses, with water restriction reducing growth in males but not females. However, size and growth of females were much more affected by the diet than males; with the high‐protein diet strongly lowering body mass, food consumption and growth rate in females. Males consumed less of the high‐protein diet, but their adult mass and growth rate were unaffected by diet. High protein diets increased body water content for both sexes, possibly because high carbohydrate diets were associated with an increase in fat storage. There were few interactions between diet and water restriction, and contrary to our hypothesis, no consistent evidence that high‐protein diets were more deleterious under water restricted conditions. Future studies should examine the sex‐specific growth and metabolic responses to dietary protein:carbohydrate content. This research project was made possible thanks supported by the American Physiological Society and a grant from the National Science Foundation Integrative Organismal Systems IOS‐1238831 as well as the DEB 1313696, and CHE 1313958.

Support or Funding Information

This research project was made possible thanks supported by the American Physiological Society and a grant from the National Science Foundation Integrative Organismal Systems IOS‐1238831 as well as the DEB 1313696 and CHE 1313958.
> π* Interaction in Designed Peptides
Authors: Wenzell Nicole , Ganguly Himal , Pandey Anil , Yap Glenn P.A , Zondlo Neal J
Published: June 2021

An n‐‐>π* interaction is a non‐covalent interaction that orders two consecutive residues in a peptide/protein by delocalizing the lone pair of electrons from the oxygen of a donor carbonyl to the antibonding orbital of an acceptor carbonyl carbon. This interaction gives insight into the inherent biases that drive protein folding, as it stabilizes secondary structures such as α‐helices and polyproline helices.

We synthesized a series of hydroxyproline‐based peptides with variable N‐terminal substituents, modulating the electron‐donating capabilities on the donor carbonyl. By changing the electronics of each donor carbonyl we can tune the strength of this n‐‐>π* interaction and explore its structural implications. The n‐‐>π* interaction stabilizes the trans conformation of a peptide bond. Because proline can readily populate both trans and cis conformations, the ratio of the two species (Ktrans/cis) indicates the strength of the n‐‐>π* interaction.

The series of derivatives synthesized were selected to compare the electron‐donating capabilities of their donor carbonyl to its n‐‐>π* interaction strength. The derivatives synthesized for analysis were, X‐Hyp(4‐NO2Bz)‐OMe where X = Boc, pivaloyl, formyl, acetyl, propionyl, monochloroacetyl, monofluoroacetyl, dichloroacetyl, difluoroacetyl, trichloroacetyl and trifluoroacetyl. These derivatives provide a range of electronic properties to be analyzed for the strength of the nàπ* interaction in each compound.

Combined analysis via 1H and 13C NMR and X‐ray crystallography reveals the characteristics of the n‐‐>π* interaction. Cis and trans species can be differentiated by 1H NMR, providing the Ktrans/cis for each derivative. Electron donation involved in the n‐‐>π* interaction is seen via the carbonyl chemical shifts using 13C NMR. The chemical shift of the acceptor carbonyl moves downfield with stronger electron‐donating substituents, while carbonyls uninvolved in the interaction remain constant. X‐ray crystallography reveals the distances and angles of the n‐‐>π* interaction, including torsion angles seen in protein secondary structures. Specifically, the propionyl donor carbonyl results in the crystallographic observation of an α‐helical conformation, inducing compact torsion angles (φ = −58.6° and ψ = −37.8°), without the presence of a hydrogen bond. An electron rich donor results in the strongest interaction. Our comparison of this series of donor‐acceptor carbonyl pairs reveals the energetics and geometry of the n‐‐>π* interaction, granting new and unique insights into the problem of protein folding.

Support or Funding Information

I'd like to extend my gratitude towards David Plastino and the NSF for providing funding for this research.
How the Loss of a Single Hydroxyl Group Affects the Proteolytic Activity of the Model Serine Protease, Trypsin
Authors: Huang Jennifer , Batt Anna R. , St. Germain Commodore P. , Baird Teaster
Published: June 2021

Trypsin is used as a model for the study of serine proteases, for its fold is commonly observed in this family of enzymes. The overall aim of this study is to discern whether the Y39F trypsin variant will act on a macromolecular substrate similar to the way it interacts with macromolecular inhibitors, specifically focusing on the S prime (S') side. In wild‐type trypsin, the hydroxyl group in tyrosine at position 39 serves to stabilize the interaction with macromolecular inhibitors via the formation of hydrogen bonds at the P prime (P') subsites. A Y39F substitution was made to reduce the affinity between the enzyme variant and inhibitor. A phenylalanine was used in place of the tyrosine because the two amino acid residues are chemically similar with the exception of the hydroxyl functional group missing from the phenylalanine. The absence of the hydroxyl functional group serves to prevent the formation of hydrogen bonds between macromolecular substrates that interact with the S' site of the enzyme, presumably weakening of the enzyme‐inhibitor interaction. However, previous research from our group demonstrated that the Y39F trypsin variant was more sensitive to inhibition from both bovine pancreatic trypsin inhibitor (BPTI) and ecotin compared to wild‐type trypsin. Molecular modeling suggests that the tighter binding is a consequence of the formation of a hydrogen bond between the P2′ site and the histidine side chain at position 40 not seen in wild type trypsin. The presence of the newly formed hydrogen bond in the Y39F trypsin variant helps stabilize the enzyme‐inhibitor complex. Still, Y39F trypsin retained similar catalytic activity against N‐α‐benzyloxycarbonyl‐glycylprolylarginine p‐nitroanilide (Z‐GPR‐pNA). However, Z‐GPR‐pNA physically cannot interact with the modified S' subsites as the macromolecular inhibitors, and likely macromolecular substrates, do. Therefore, the activity observed with Z‐GPR‐pNA may not accurately reflect the activity of the Y39F variant towards macromolecular substrates. In this study, we used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and gel imaging to monitor and compare the progression of macromolecular substrate hydrolysis of Y39F and wild‐type trypsin. Based on the observations made with BPTI and ecotin, we would expect that the Y39F variant would bind more tightly to a macromolecular substrate as well, likely increasing the efficiency of cleavage relative to wild‐type.

Support or Funding Information

NSF Award MCB‐0643988‐02
Effect of ZNT7 on the CD40 signaling pathway in B lymphocytes: a possible mechanism for a regulatory role of zinc in immune function
Authors: Huang Liping , Tepaamorndech Surapun , Oort Pieter , Kirschke Catherine P. , Cai Yimeng
Published: June 2021

Zinc deficiency impairs the immune system leading to frequent infections. Although it is known that zinc plays critical roles in maintaining healthy immune function, the underlying molecular targets are largely unknown. In this study, we demonstrate that zinc is important for the CD154‐CD40‐mediated activation of downstream signaling pathways in human B lymphocytes. CD40 is a receptor localized on the cell surface of many immune cells, including B lymphocytes. When the T helper lymphocyte (Th) is activated by antigens, it expresses a membrane protein, CD154 that allows Th to bind to B cells via CD154‐CD40 interaction leading to B cell activation. We show that cellular zinc deficiency impaired the CD154‐CD40‐mediated p38 MAPK phosphorylation. Most importantly, we demonstrate that ZNT7 interacted with CD40. ZNT7 knockdown in B lymphocytes had a negative effect on the cell surface expression of CD40. Consequently, the CD40‐mediated p38 MAPK signaling transduction was down‐regulated in ZNT7KD B lymphocytes. Conversely, this p38 MAPK signaling activity was up‐regulated by over‐expression of ZNT7 in B lymphocytes. We conclude that prevention of zinc deficiency is a key to maintain an optimal function of immune system and CD40 is the molecule target for ZNT7 in regulation of immune function of B lymphocytes.

Support or Funding Information

This work was supported by the United States Department of Agriculture, ARS CRIS project 2032‐51000‐004‐00D.
Skin‐specific stearoyl‐CoA desaturase 1 deficiency protects against adiposity by enhancing IL‐6 expression
Authors: Dumas Sabrina N , Guo Chang‐an , Ntambi James M
Published: June 2021

Stearoyl‐CoA desaturase 1 (SCD1), a lipogenic enzyme that adds a double bond at the delta 9 position of stearate (C18:0) and palmitate (C16:0) has been proven to be important in the development of obesity. Skin‐specific deficiency of SCD1 (SKO) provides protection against adiposity from a high‐fat diet, with improvements in glucose clearance, insulin sensitivity and hepatic steatosis. Curiously, these mice also display elevated levels of plasma interleukin‐6 (IL‐6) and increased white adipose tissue lipolysis. IL‐6 is a known mediator of white adipose tissue lipolysis. In the SKO mouse model, IL‐6 expression is evident in the skin sebaceous glands and around the hair follicles. Recently, IL‐6 has been connected to increased gluconeogenesis. While gluconeogenesis is paradoxical to the increased glucose clearance seen in SKO mice, a pyruvate tolerance test in the SKO mice confirmed a transient increase in glucose production. Therefore, we propose that skin‐derived IL‐6 leads to white adipose tissue lipolysis, which decreases the size of fat pads leading to decreased adiposity. In addition the release of triglycerides in adipose tissue provides the liver with acetyl‐CoA, NADH and glycerol necessary for hepatic glucose production.

Support or Funding Information

Supported by USDA Hatch W2005
Dolicholphosphate Mannosyltransferase: A Glycosyltransferase with Unity in Molecular Diversities
Authors: Baksi Krishna , Zhang Zhenbo , Banerjee Dipak K
Published: June 2021

N‐glycan provides structural and functional stability to glycoproteins and adds to their flexibility. Glycan biosynthesis is elaborative, multi‐compartmental and uses glycosyltransferases. Failure to assemble N‐glycan leads to phenotypic changes developing infection, cancer, congenital disorders of glycosylation (CDGs) among others. The biosynthetic process begins at the endoplasmic reticulum (ER) with the assembly of dolichol‐linked tetradecasaccharide Glc3Man9GlcNAc2‐PP‐Dol where DPMS plays a critical role. DPMS is also essential for GPI anchor biosynthesis, protein O‐mannosylation in yeast as well as O‐ and C‐mannosylation in eukaryotes. DPMS has been purified from several species and its gene has been cloned from 40 species (protozoan parasite to human). The sequence alignment indicates the presence of a cAMP‐dependent protein phosphorylation motif (PKA motif) in DPMS from 39 species. Hydrophobicity analysis of the DPMS sequence from bovine, human, S. crevisiae and A. thaliana all show the PKA motif each preceded and succeeded by a sequence of hydrophobic amino acid residues, i.e., the presence of a transmembrane domain. Irrespective of these similarities, the amino acid sequence at the C‐terminus and the kinetic behavior have classified DPMS as Type I and Type II enzyme. DPMS belonging to Type II sub‐class has been proposed to be a multimeric protein requiring DPM2 and DPM3 to anchor down DPM1 to the ER membrane. Based on the hydrophobicity as well as the successful expression of bovine DPM1 gene in E. coli we conclude that the logic proposing the above model needs to be revisited. Because of inherent diversities, DPMS has acquired sensing ability of cellular microenvironment and can serve as an excellent biosensor. In addition, DPMS activity is down regulated during aging but when upregulated in capillary endothelial cells due to activation of β‐adrenoreceptor angiogenesis, a hallmark of tumor growth is enhanced. Furthermore, DPMS from rat liver, rat parotid acinar cells, hen oviduct, bovine brain as well as from S. cerevisiae all showed increased activity upon phosphorylation in vitro.

Support or Funding Information

The work was partly supported by funds from NIH/NIMHD G12MD007583 (KB), NIH U54‐CA096297, Susan G. Komen for the Cure BCTR0600582, and NSF EPS‐1002410 (DKB).
Mannose moieties exhibit self‐adhesive interactions
Authors: Perera Komitige Hashanthi , Chandran Preethi L.
Published: June 2021

Carbohydrate‐Carbohydrate interactions (CCIs) are self‐adhesive interactions, generally observed among proteoglycans and glycolipids expressed on the cell surfaces. Many biological processes such as embryogenesis, fertilization and metastasis are driven by CCIs. Mannoses are biologically‐significant carbohydrate moieties, that are extensively present on the surfaces of fungi and viruses, and known to mediate host‐pathogen interactions. We studied the adhesive interactions between mannobiose (disaccharides of mannose sugars)‐mannobiose molecules using atomic force spectroscopy (AFS).

Bifunctional linkers were synthesized with thiol functional groups on one end and aryl‐azide functional group on the other. The linkers were self‐assembled on gold substrates (gold coated AFM sample disks and gold coated AFM probes) via the thiol end, and mannobiose was photochemically linked to the free aryl‐azide end of the surface‐anchored linkers. The mannobiose attachment on the substrates was verified via its selective binding to the lectin, concanavalin‐A.

Mannobiose‐mannobiose molecules exhibited strong adhesive forces when probed with AFS in an aqueous medium. The force‐distance curves exhibited peak adhesive forces that subsequently ruptured in ‘step’ like patterns, indicating that the adhesive forces were due to well‐defined molecular interactions. The peak adhesive forces between mannobiose‐mannobiose molecules ranged from 0.3 to 5.7 nN. The final ‘step’ or rupture forces ranged from 0.1 to 1nN, with forces clustering at regular intervals which were multiples of 25 +/− 3pN. The latter suggests the likely magnitude of the single mannose‐mannose unbinding force to be 25 +/− 3pN. Lactose, a diastereoisomer of mannobiose, did not show self‐adhesive interactions. Free mannobiose reduced the mannobiose‐mannobiose adhesive forces in a concentration‐dependent manner; whereas free lactose was less effective in reducing the forces. Also, the mannobiosylated surface displayed ‘water structuring’ effects that extended tens of nanometers beyond the surface, and which were disrupted by increasing the salt concentration.

This is the first study to report self‐adhesive function between mannobiosylated surfaces. The majority of fungi and virulent viruses such as HIV, Ebola, and Marburg have mannose patches on their surfaces, and this fundamental study might shed light on the biophysical role of these pathogen surfaces. The water‐structuring ability of clustered mannose molecules may manifest as a dense water shell around pathogens, to keep them hydrated and protected from host immune molecules.

Support or Funding Information

This work is supported by National Science Foundation under grant no. 1407891 awarded to Dr. Preethi Chandran and by a mini‐grant awarded to Dr. Preethi Chandran under NSF grant no. 1208880 (PI: Dr. Sonya Smith)
GRP78 is Neither Expressed on ER−/PR−/Her2− Human Breast Cancer Cell Surface nor Secreted
Authors: Serrano Jesus E , Romero‐Nutz Eva C , Sanchez Neysharie , Banerjee Aditi , Baksi Krishna , Banerjee Dipak K
Published: June 2021

GRP78 (Mr78 kDa) is a glucose regulated protein. It is a member of the heat‐shock protein (HSP) family but located in the lumen of the endoplasmic reticulum (ER). Its function as ER chaperone translocating protein across the ER membrane that needs to be glycosylated at the asparagine residue present in the sequeon Asn‐X‐Ser/Thr (N‐linked glycosylation) is well recognized. This important biochemical event is essential for glycoprotein folding and function. For example, when the protein N‐glycosylation is impaired with tunicamycin a protein N‐glycosylation inhibitor, angiogenesis, a hallmark for tumor progression and metastasis is inhibited due to an induction of ER stress. Under such condition, GRP78 is overexpressed in microvasculature and in tumor tissue, concluding that GRP78 is a master regulator in ER stress induced unfolded protein response (upr)‐mediated apoptosis in tumor microvasculature (J. Biol. Chem. 286, 29127–29138, 2011; Pure Appl. Chem. 84, 1907–1918, 2012). This contradicts the current dogma that supports GRP78 expression on the tumor cell surface interfering with the therapeutic(s) and making them as tumor promoters instead. To evaluate the GRP78 localization, we have used ER−/PR−/Her2− (i.e., triple negative) human breast cancer cell line MDA‐MB‐231 (Caucasian) cultured normally or without serum as well as before or after inducing the ER stress. After establishing the expression of GRP78 mRNA by qPCR and protein by western blotting, we focused on its cell surface expression. Unfixed cells were stained with Concanavalin A (Con A; Specificity = α‐D‐Mannose, α‐D‐Glucose, branched mannose), wheat germ agglutinin (WGA; Specificity = (GlcNAc‐β‐(1,4)‐GlcNAc)1‐4>β‐GlcNAc‐NeuAc) as well as with anti‐GRP78 antibody followed by their detection by immunofluorescence microscopy using either Rhodamin or Alexa–conjugated secondary antibody. The fluorescence images were captured in a Zeiss microscope with Axiocam camera. Con A and WGA staining provided images of N‐glycans on the cell surface and supported the intactness of the cell membrane. On the other hand, GRP78 fluorescence was absent from the surface of these cancer cells. Similar results were also obtained irrespective of cells cultured in the absence of serum and/or in the presence of 1μg/mL of tunicamycin. GRP78 fluorescence however was detected in cells after either fixing them with ice‐cold methanol or after permeabilization with digitonin. In addition, western blotting also failed to detect GRP78 in the culture media of MDA‐MB‐231 cells. We, therefore, conclude that GRP78 is neither expressed on the outer‐leaflet of the cell surface of ER−/PR−/Her2− human breast cancer cells MDA‐MB‐231 nor secreted in the culture media.

Support or Funding Information

Supported in part by NSF EPS‐1002410 (DKB) and NIH/NIMHD G12MD007583 (KB).
Acculturation and Hispanic‐Heritage Mothers' Perception of Child Weight as Related to Child Feeding Practices
Authors: Luna Viridiana , Villegas Elizabeth , Hannon Bridget , Hammons Amber , Cruz‐Santiago Michelle , Wiley Angela , Teran‐Garcia Margarita
Published: June 2021

Hispanic children in the United States are disproportionately affected by obesity. Previous analysis revealed that mothers participating in the Abriendo Caminos intervention, aimed at preventing childhood obesity in Hispanic‐heritage families, incorrectly classified their overweight/obese child as normal weight. Effective obesity prevention efforts must consider mothers' roles in child feeding practices and their impact on child weight status over the life course. Acculturation and mother perception of weight may explain some child feeding practices. The aim of this study was to determine whether mothers' acculturation and perception of children's weight was associated with mothers' rationale for offering children snacks. Mother‐child dyads (n=53) of Mexican or Puerto Rican descent were recruited as part of the Abriendo Caminos randomized control/workshop multi‐state intervention. Here we report on the cross‐sectional baseline data acquired to date from participating mothers in the Illinois and California cohorts. The Brief Acculturation Rating Scale for Mexican Americans‐II (ARSMA‐II) was used for assessment of acculturation and a set of questions previously developed by Blaine and colleagues (Nutrients, 2015) was used to assess the rationale behind offering snacks. Anthropometric measures were collected from all participants. One‐way ANOVA and Fisher's LSD were used to compare differences in mean times per week that snacks were offered. At baseline, 64% of children were overweight or obese, while only 24% of mothers categorized their child as such. Over half of the mothers (66%) were categorized with low levels of acculturation (very Hispanic oriented). There were no significant associations between child BMI, mother perception of child weight and reasons mothers offered snacks. Compared to mothers who were the least acculturated, those who were the most acculturated reported offering their child more snacks per week because their child was hungry (4.00, p < 0.05). A further understanding of how weight perceptions and acculturation influence health and feeding practices can equip professionals to provide culturally relevant tools for mothers to enact positive changes in their families.

Support or Funding Information

This research was supported by the Agriculture and Food Research Initiative Competitive Grant no. 2015‐68001‐23248 from the USDA National Institute of Food and Agriculture.
Dietary Long‐chain PUFAs Enrich Porcine Alveolar Macrophages and Alter Prostaglandin E2 Production Following LPS Challenge
Authors: Walter Kathleen R , Xi Lin , Jacobi Sheila K , Esposito Debora , Odle Jack
Published: June 2021

The US swine industry experiences severe production and profit losses from respiratory infections despite the wide spread use of vaccines and antibiotics. Long‐chain PUFAs can modulate immune cell function through the production of eicosanoids, such as prostaglandin E2 (PGE2) which is capable of stimulating both inflammation and inflammatory resolution upon infection. The aim of this study was to determine the effects of dietary arachidonic acid (ARA) and eicosapentaenoic acid (EPA) on cultured porcine alveolar macrophages stimulated with LPS. Day‐old pigs (n=60) were fed milk replacer for 21d. Diets contained 0.5% ARA (control), 2.5% ARA or 3.5% EPA of total fatty acids. Alveolar macrophages were isolated via bronchoalveolar lavage, cultured and stimulated with LPS for 24h. Total fatty acids in alveolar macrophages and lung tissue were analyzed via gas chromatography‐mass spectrometry (GC‐MS). Alveolar macrophages were cultured and stimulated with 10ng/mL LPS or unstimulated (control). Concentrations of PGE2 in tissue culture media were measured via competitive enzyme‐linked immunosorbent assay (ELISA) kits. Average body weight and clinical hematology were unaffected by diet treatments (P >0.5). Concentration of ARA (% of fatty acids, w/w) in alveolar macrophages from pigs fed 2.5% ARA had an 82% increase in ARA compared to controls, and the concentration was decreased 21% in pigs fed 3.5% EPA compared to control (P < 0.01). Lung parenchymal tissue showed a similar enrichment pattern; tissue was enriched by 39% in pigs fed 2.5% ARA, while pigs fed the 3.5% EPA diet had a 45% decrease in ARA compared to control (P < 0.002). Media concentration of PGE2 was increased following LPS stimulation of alveolar macrophages. The increase in PGE2 concentration was greater in macrophages enriched in ARA, exceeding that from control cells by 187% and that from EPA‐enriched cells by 191% (P < 0.05). These data demonstrate that dietary supplementation of LC‐PUFAs can effectively alter lung tissue and alveolar macrophage fatty acid composition. Furthermore, these data validate that increased dietary ARA is an effective means to increase eicosanoid production by LPS‐stimulated immune cells.

Support or Funding Information

USDA Animal Health Funds
In vitro Hypoallergenic Potential of Peanut Protein‐Polyphenol Aggregate Particles
Authors: Plundrich Nathalie J. , Bansode Rishipal R. , Williams Leonard L. , Lila Mary Ann
Published: June 2021

The single most effective management of peanut (PN) allergy is the strict avoidance of the allergenic food. However, PN proteins are found in a variety of food products. A lot of effort has focused on mediating PN allergy either through attempted modulation of the human immune system (e.g. oral immunotherapy) or the allergenic food proteins (by processing techniques or genetic modification). In this study, we took advantage of the natural binding properties of plant‐derived compounds (i.e. polyphenols) to proteins. We created stable aggregate particles comprised of PN proteins and cranberry pomace or PN skin polyphenols which were investigated for their IgE binding potential. Their capacity to modulate mast cell degranulation and cell signaling involving phosphorylation of MAPK and p‐38 was investigated in vitro using RBL‐2H3 cells.

PN protein‐cranberry pomace and PN protein‐PN skin polyphenol aggregate particles were created by complexing cranberry (Vaccinium macrocarpon Ait.) pomace or PN skin polyphenols with roasted PN flour. Particles containing 5 to 40% polyphenols were created. For the detection of PN‐specific IgE binding by PN proteins, immunoblotting was performed with pooled plasma from seven PN‐allergic individuals. RBL‐2H3 mast cells were exposed to aggregate particles or PN flour and evaluated for their inhibitory activity on ionomycin‐induced degranulation (β‐hexosaminidase and histamine).

Results showed that PN protein‐bound polyphenols were able to decrease the IgE binding capacity to PN proteins in aggregate particles and the highest reduction was observed in the 40% polyphenol samples, a 22% and a 38% decrease in the PN protein‐PN skin and cranberry pomace polyphenol aggregate particles, respectively. This was significant when compared to IgE binding capacity of unmodified PN proteins (p<0.05; n=4 or 5). Anti‐DNP IgE‐sensitized RBL‐2H3 cells challenged with DNP‐BSA and ionomycin in the presence of aggregate particles were evaluated for their release of β‐hexosaminidase and histamine. PN protein‐PN skin and cranberry pomace polyphenol aggregate particles appeared to reduce ionomycin‐induced β‐hexosaminidase release (60 and 61%, respectively) and histamine release (64 and 66%, respectively). PN protein‐PN skin and cranberry pomace polyphenol aggregate particles when exposed to ionomycin‐induced RBL‐2H3 cells, inhibited phosphorylation of MAPK and induced the phosphorylation of p‐38 in a dose dependent manner.

Results suggest the modification of PN proteins with cranberry pomace and PN skin polyphenols led to the formation of PN protein‐polyphenol aggregate particles with reduced allergenic potential. Future trials are warranted to investigate the immunomodulatory mechanisms of these aggregate particles.

Support or Funding Information

This study was funded through USDA NIFA
US and Canada Joint Effort for Dietary Reference Intake Updates
Authors: de Jesus Janet , Klurfeld David M , Lynch Christopher J. , Brooks Stephen P
Published: June 2021

Representatives from the US and Canadian Government will present updates from the Joint US and Canada Dietary Reference Intake (DRI) Working Group on activities conducted to work toward updating the sodium and potassium DRIs. The Governments of the United States and Canada have jointly undertaken the development of the Dietary Reference Intakes since the mid‐1990s. Federal DRI committees from each country work collaboratively to identify DRI needs, prioritize nutrient reviews and advance work to resolve any methodological issues that could impede new reviews. The commission of a systematic review for nutrients under review is now an integral part of the DRI process. Recently the DRI Working Group sponsored an evidence review by the Agency for Health Care Research and Quality (AHRQ) on sodium and potassium to inform the update of the sodium and potassium DRIs by the National Academy of Medicine (NAM). The AHRQ review will be completed in 2017. Additionally, the DRI steering committees jointly decided that prior to undertaking a nutrient review, the scientific framework for setting DRIs needed to be adapted to include a framework for including data on chronic disease risk reduction. Thus, a scientific expert panel was convened to review and critically evaluate evidentiary, dose response, and process issues related to the use of chronic disease endpoints and develop options for their incorporation into future DRI reviews. The Joint US/Canadian‐sponsored Working Group's report ‘Options for Basing DRIs on Chronic Disease Endpoints’ will inform the NAM committee that will develop guiding principles for inclusion of chronic disease endpoints to be used by committees setting future DRIs. In summary, the US and Canadian Governments are working collaboratively to adapt the methods for updating the DRIs utilizing rigorous systematic evidence reviews and a chronic disease endpoint framework.

Support or Funding Information

United States Department of Health and Human Services, United States Department of Agriculture, and Health Canada.
Dietary Prebiotics and Arachidonic Acid Alter Intestinal Phospholipid Composition and Time‐Dependently Change Fecal Microbiome in Formula‐Fed Piglets
Authors: Thorpe Makenzie K , Xi Lin , Maltecca Christian , Walters Kathleen R. , Smith Andrew , Odle Jack , Jacobi Sheila K.
Published: June 2021

Establishment of a homeostatic gut microbiome early in life could have long term outcomes for health of adults. Neonatal nutrition plays a critical role in establishment of the gut microbiome. Milk oligosaccharides (MO) serve as nutrients involved in establishing the gut microbiome, and long‐chain PUFA (LCPUFA) may be involved in modulation of microbiome but are critically important in neonatal development. The objective of this experiment was to investigate the effect of supplemental LCPUFA and prebiotics on establishment of the intestinal microbiome in early life. Day‐old pigs were randomized among 4 diets (0.5% arachidonic acid (ARA; control); 4g/L galactooligosaccharide + 4 g/L polydextrose (PRE); 2.5% ARA, and PRE+ARA; n=24 per group) and fed 3 times per d for 21 d. Fecal swabs were collected weekly and microbiome analysis was performed via 16S rDNA Illumina sequencing. Pigs were euthanized on d22 and fatty acid composition of colonic mucosa was analyzed via GC‐MS. Diet did not affect growth. Microbial taxa significantly changed from d0 to 21 in a diet dependent manner with diets containing prebiotics clustering together by d7 post‐feeding and becoming more closely associated with longer feeding times (P<0.05). Specifically, an increase in bacterial taxa belonging to the phyla Bacteroidetes was observed (P<0.05). Colonic mucosal ARA% (w/w) increased from 3.83 in controls to 6.67 in pigs fed diets enriched in ARA (P < .001), with reciprocal reductions in linoleate concentration. Dietary prebiotics increased ARA and linoleate concentration but reduced stearate concentration in colonic mucosa P<.002). In conclusion supplementation of formula with prebiotics significantly shifts microbial taxa from d7 to d21 of age and also altered colonic fatty acid composition. Dietary LCPUFA (arachidonic acid) has minimal impact on microbial taxa but predictably enriched colonic mucosa in ARA.

Support or Funding Information

This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2014‐67017‐21750 from the USDA National Institute of Food and Agriculture.
Anti‐Atherogenic Potential of Edible Mushrooms
Authors: Kim Sharon , Thomas Michael , Meydani Mohsen
Published: June 2021

For centuries, mushrooms have been used as medicinal plants, and, more recently, have been a growing component of a healthy diet. Mushrooms are low in sodium and are good sources of fiber, bioactive compounds, selenium, potassium and vitamin D, all which may contribute to a healthy heart through anti‐inflammatory and anti‐atherogenic activities. While several types of mushrooms have been studied for their effects on serum lipid profiles, few studies have demonstrated edible mushrooms' effects on atherogenesis. The specific objective of this study was to determine the effectiveness of two commonly consumed edible mushrooms, portobello mushroom (PBM) and shiitake mushroom (SHM), on the prevention of atherosclerosis and associated inflammation. Ldlr−/− mice were fed with one of 5 dietary compositions for 16 weeks: low‐fat (4% fat) control diet (LF‐C), high fat (8% fat) control (HF‐C) diet, 10% of HF‐C diet replaced with PBM powder (HF‐PBM), or with SHM powder, (HF‐SHM), or with a “control mixture”, best matched to the average nutrient levels of the mushroom powders (MIX‐C). Mice fed with HF‐PBM or HF‐SHM diet displayed lower body weight gains (p<0.05) compared to the other dietary groups. Among the two mushrooms fed mice, HF‐SHM fed mice had significantly lower average body weight gain compared to HF‐PBM group (p<0.01). Despite the low body weight gains, EchoMRI™ analysis of body composition revealed that the overall lean mass was not affected as significantly as fat mass, indicating a plausible positive effect of mushrooms on fat metabolism and lipid profiles. Mice fed HF‐SHM displayed the lowest fat mass (p<0.0001) and lean mass (p<0.01), relative to other groups, whereas mice fed HF‐PBM had a lower body fat mass (p<0.001) but not lean mass (p=0.24) compared to other groups. Ex‐vivo adhesion assay of monocytes to luminal surface of aorta showed that mice fed HF‐C exhibited a trend of a higher number of splenocyte attachment, whereas feeding mice with LF‐C and HF‐SHM exhibited a lower number of splenocyte adhesion. Further, quantitative en‐face analysis of aortic lesions using Oil Red O staining revealed that only mice fed HF‐SHM had significantly fewer aortic lesions compared to those of mice fed HF‐C (p<0.01) and MIX‐C (p<0.01). These results further support the potential role of high levels of bioactive compounds such as ergothioneine, a strong antioxidant in SHM, on suppression of dietary fat induced atherosclerosis, an inflammatory disease of arteries.

Support or Funding Information

Supported by USDA contract #58‐1950‐4‐003 and Mushroom Council.
Glioma Cell Proliferation Promoted by AK4 Regulated mTORC1 Pathway Activation
Authors: Silva Joshua , Lanning Nathan
Published: June 2021

A hallmark of cancer cell biology is the dysregulation of signaling pathways that manage cancer‐associated phenotypes such as proliferation and survival. One dysfunctional pathway in cancer cells is the mechanistic target of rapamycin (mTOR). The mTOR pathway, involving both mTOR Complex 1 (mTORC1) and mTORC2, functions as a central regulator of cell metabolism, growth, proliferation, and survival. mTORC1 kinase signaling allows for transitions between metabolic states by responding to environmental signals and controlling processes that regulate energy. Recently, the expression of an Adenylate Kinase 4 (AK4) protein has been identified as modulating cellular bioenergetics through the activation of pathways such as mTORC1, resulting in both enhanced proliferation and activation of stress responses. Additionally, reduced AK4 expression has been observed in primary human glioma cells, while the mTORC1 signaling pathway is highly activated in identical primary glioma cells. We hypothesize that AK4 expression regulates mTORC1 signaling and induces downstream effects that promote glioma cell biology. To determine if AK4 expression affects mTORC1 signaling, we knocked down AK4 expression using siRNA in U87‐MG glioma cell lines. Phospho‐specific modifications of activators and inhibitors upstream of the mTORC1 pathway and its downstream targets were assessed using Western blot (WB). AK4 knockdown (KD) and WB analysis revealed over‐active mTORC1 phosphorylation and inactivation of the downstream translational repressor, 4E‐BP1, compared to control glioma cell lines. Likewise, WB analysis displayed increased mTORC1 phosphorylation and activation of the downstream protein ribosomal S6, a regulator of protein translation. Reduced phosphorylation of upstream inhibitors and regulatory proteins of mTORC1, Tuberous Sclerosis Complex 2 (TSC2) and Regulatory‐associated protein of mTOR (RPTOR), was observed when compared to controls. Additionally, reduced mTORC1 phosphorylation of the autophagy inducing serine/threonine protein kinase ULK1 was evident by WB. Furthermore, bioluminescence analysis of AK4 KD U87‐MG glioma cells shows an increase in cell proliferation under nutrient stressed conditions compared to control. Overall, these data suggest that AK4 reduction may contribute to over‐active mTORC1 kinase signaling in glioma cells. Studies characterizing the role of AK4 reduction on glioma cell proliferation could further explain the contribution of endogenously reduced AK4 expression in glioma cell biology. By identifying the regulatory role of AK4, we hope to elucidate the therapeutic potential of modulating AK4 levels in cancer cells.

Support or Funding Information

NSF# HRD‐1463889.