bluuebunny/crossref_metadata_2025 huggingface dataset:
Receptor mediated signaling regulates subcellular gravin‐PKA distribution through calcium and PKC
Authors: Schott Micah , Grove Bryon
Published: June 2021

Gravin (AKAP12), a multivalent A‐kinase anchoring protein, anchors cAMP‐dependent protein kinase (PKA) and likely other signaling proteins to the plasma membrane, but undergoes redistribution to an intracellular compartment in response to PKC activation or increased [Ca2+]i. We postulate that Ca2+ mediated redistribution of gravin represents a novel cross‐talk mechanism between Ca2+ dependent and cAMP dependent signaling pathways. To assess this, we examined the effect of Ca2+‐elevating agents on gravin and gravin‐dependent PKA distribution in cells expressing EGFP‐ or EYFP‐tagged gravin constructs. Ionomycin and thapsigargin each initiated rapid gravin redistribution into the cytosol, an event which was inhibited by Ca2+ chelation using BAPTA‐AM. Treatment of cells coexpressing gravin‐EYFP and PKA RII‐ECFP with Ca2+‐elevating agents also resulted in PKA redistribution, but only when gravin was present. Consistent with this, treatment with ATP in a Ca2+ free medium also resulted in rapid gravin redistribution in concert with a rise in [Ca2+]i, suggesting that this change is mediated by purinergic P2Y receptors. ATP also caused PKA redistribution in cells coexpressing gravin and PKA constructs. However, inhibition studies using BAPTA‐AM and bisindolylmaleimide indicated that PKC activation also contributed to ATP mediated changes in gravin distribution. These studies confirm that receptor mediated signaling events involving changes in intracellular calcium and PKC activation alter gravin distribution and support the hypothesis that gravin mediates cross‐talk between cAMP dependent signaling events and both calcium and PKC dependent signaling events. Future studies are underway to assess the effect of gravin dynamics on cAMP‐dependent signaling events. This work was supported by funding from UNDSMHS and NIH P20RR017699.

Grant Funding Source: UNDSMHS and NIH P20RR017699
User experience and the influence on the evaluation of information presentation in an online brachial plexus module
Authors: Turgeon James G , Armstrong Kevin , Wilson Timothy D
Published: June 2021

E‐learning through computer modules is a growing trend amongst anatomical educators. However, little research has explored the translation of design components between users of different experience levels. This study explored the role of prior knowledge and its effect on user rating of an online learning module. Due to the brachial plexus’ complexity, difficulty in learning and the continued application within the medical field, a three‐dimensional model of the brachial plexus and surrounding structures was created. This model was integrated into two interactive interfaces demonstrating equivalent information in distinct presentation modes, guided and self‐guided. For a distribution of low (LK) and high (HK) prior knowledge participants, recruitment occurred from undergraduate and resident populations. Pre‐test scores verified participants as high or low prior knowledge. It is hypothesized that HK users prefer self‐guided module characteristics while LK users prefer the direction inherent in the guided module. It is expected that all users will show improvement between pre‐ and post‐test scores, and greater improvement will correspond with the higher rated module.

Grant Funding Source: Queen Elizabeth II Graduate Scholarship in Science and Technology
Teaching students to teach: a medical student teaching assistant perspective on the effectiveness of learning through apprenticeship
Authors: Erie Andrew Jay , Starkman Sidney , Pawlina Wojciech , Lachman Nirusha
Published: June 2021

Significant increase in literature regarding “residents as teachers” highlights the importance of providing opportunities and implementing guidelines for continuing medical education and professional growth. Structured teaching assistant (TA) experiences are not uncommon. Programs, both nationally and internationally, have implemented and continue to provide established opportunities for under‐ and post‐ graduate students to develop teaching skills. However, due to the nature of undergraduate medical curricula, there is neither sufficient time nor an abundance of opportunity for medical students to develop teaching skills. The TA program at Mayo Clinic incorporates learning through apprenticeship and mentoring during a 7‐week anatomy block. This study explores the impact of the TA experience and the effectiveness of its facilitation by faculty mentors. Structured questionnaires obtained feedback regarding improvement in teaching and communication skills, as well as, experiences in curriculum development and educational research. Preliminary data revealed that teaching opportunities, mentoring from course faculty, and exposure to educational research and curriculum development improved the TA's ability to teach and fostered an increased interest in teaching. Medical student TA's perceive learning through apprenticeship and mentoring as an effective way to learn how to teach.

Grant Funding Source : Department of Anatomy
Special K: regular diet of quizzes for knowledge in histology
Authors: Ettarh Raj , Jerrett David
Published: June 2021

Studies suggest that a regular regime of quizzes is useful in helping students retain and preserve information, and improves short term performance in summative assessments. Using multiple choice formatted quizzes administered prior to and immediately following each histology laboratory, medical students were assessed, over a 3‐week period, for recall of information relating to recognition of cell and tissue characteristics. Performance in practical and written components of a summative evaluation (block examination) was also compared in quizzed and un‐quizzed students. Results show an improvement in post‐lab quiz scores over pre‐lab performance in only 50% of quizzes. Quizzed students demonstrated better recall of information for identifying cell and tissue characteristics in the practical component of the summative assessment than they showed during quizzes but there was no significant difference in this ability or in recall of didactic information when compared to students who were not quizzed in lab during this period. These findings suggest that, in the short‐term, any improvements in identification skills and knowledge acquisition in histology as a result of regular non‐graded assessment may be limited.
Cigarette smoke induces DNA damage and inhibits cell cycle checkpoint and DNA repair pathways
Authors: Thai Philip , Chen Daniel , Wu Reen
Published: June 2021

Introduction

Tobacco smoke is a major cause of lung cancer. Smoke's many reactive chemicals can to lead to carcinogenesis through DNA damage and genomic instability. However, how it affects the DNA damage response (DDR) pathways has not been clearly delineated.

Objective

To determine the mechanism of cigarette smoke induced toxicity and carcinogenesis in airway epithelial cells.

Methods

In vitro exposure of airway epithelial cells (A549 and HBE1) to cigarette smoke extract (CSE) induces significant DNA strand breaks as assessed by Comet Assay. However this DNA damage induced by smoke doesn't activate key DDR pathways suggesting that smoke may actively inhibit these pathways. In particular, immunoblotting reveals minimal activation of ATM (ataxia telangiectasia, mutated) and ATR (ATM and Rad3‐related) protein kinases and corresponding downstream proteins BRCA‐1 and p53 following CSE exposure. In contrast, DNA damage induced by Cisplatin, a potent anti‐cancer drug that causes DNA crosslinks, activates all of these pathways in airway epithelial cells.

Conclusion

We propose that cigarette smoke has a unique effect in simultaneously inducing DNA damage but also inhibiting key DDR pathways in lung cells. This suggests that smoke induced carcinogenesis is not just due to direct damage of DNA, but also inhibition of cell cycle arrest and cell death pathways to enhance propagation cells with genomic instability.
In vitro characterization of ionizing radiation resistance protein YejH
Authors: Molzberger Eileen Therese , Byrne Rose T , Cox Michael M
Published: June 2021

DNA damage resulting from exposure to Ionizing Radiation (IR) is fatal unless it is repaired. Organisms have evolved mechanisms to maintain genomic integrity upon insult. In a screen for genes that contribute to IR resistance, gene of unknown function, yejH, was identified. Cells deleted for yejH are nearly a 100‐fold more sensitive to IR exposure than wild type. YejH is a predicted helicase; the primary amino acid sequence contains the seven conserved domains of Superfamily 2 helicases. To support our hypothesis that YejH is a DNA repair enzyme, both DNA binding and DNA‐dependent ATPase activity were assayed. We found that YejH is DNA‐independent ATPase, however it binds DNA in the presence of ATP and non‐hydrolyzable ATPγS. Using Tandem Affinity Purification, single‐stranded DNA binding protein (SSB) was identified as a binding partner of YejH. SSB binds single‐stranded DNA (ssDNA) and interacts with a growing list of proteins, thus localizing DNA metabolism proteins to replication forks and damaged DNA. This work aims to characterize YejH in vitro and determine how SSB affects YejH activity. As we understand more about the proteins that interact with YejH and how YejH activity is affected, we become closer to understanding the role of YejH in surviving IR exposure.
The role of chaperones in the initial steps of bacterial oxidoreductase maturation
Authors: Leach Thorin Gunnar Hallson , Turner Raymond
Published: June 2021

Bacterial oxidoreductases play a key role in anaerobic respiration and represent a group of enzymes with complex maturation pathways. A group of specific chaperones, or redox enzyme maturation proteins (REMP), have an essential role in the maturation of bioenergetics enzymes. While their role has yet to be defined, REMPs have been shown to interact tightly with the amino terminal signal peptide of oxidoreductase subunits. DmsD, the REMP associated with the oxidoreductase dimethyl sulfoxide (DMSO) reductase, has been putatively shown to interact with a number of proteins. These include the general chaperones DnaK, and Trigger Factor (TF), and also include MoeB, a protein involved in the molybdenum cofactor biosynthesis. An in vitro characterization of these protein‐protein interactions will improve our understanding of the early steps of oxidoreductase maturation, as well as provide further insight into the role of REMPs. Studies to date were done using an in vitro modified far‐western technique, a robust technique that quantitatively assesses protein‐protein interactions between folded and pure proteins. An interaction was found between MoeB and DmsD, but this technique failed to show an interaction between DnaK or TF and DmsD. Alternatively, both DnaK and TF were shown to interact with the DMSO reductase signal peptide. Size exclusion chromatography is used to confirm protein‐protein interactions as well as to determine if these proteins can interact concurrently or if binding excludes one or more proteins.
Integrated pathway‐level analysis of transcriptome and metabolome data for mouse model of social stress
Authors: Gautam Aarti , Muhie Seid , Chakraborty Nabarun , Hoke Allison , Hammamieh Rasha , Jett Marti
Published: June 2021

We have used a social defeat (SD) mouse model to simulate aspects of post‐traumatic stress disorder (PTSD) that is based on a brief exposure to an aggressor mouse. Metabolic profiling of mice plasma samples exposed to 5d or 10d of SD at both early (24h) and late (1.5 wk for 5d, and 4 wk for 10d) time points, revealed changes in neurotransmitters, neuronal damage and/or cell death, energetic and redox alterations. These metabolites when subjected to gene‐enzyme‐metabolite integrative network revealed enrichment of a number of signaling and biosynthetic pathways such as bile acid biosynthetic pathways in both 5d and 10d 24h groups. These changes are reflected in metabolic syndrome, the usual co‐morbid associate of PTSD. Although still in its infancy, the resulting high‐dimensional data has led to the emergence of networks that may have relevance in context to PTSD. Preliminary data from mouse neurotransmitter receptor and regulator array using hemi brain RNA samples enabled us to identify genes perturbed during this condition. Comparison of data to microarray profile revealed cholecystokinin B receptor gene (cckbr) common in both data sets. This gene was uniformly altered in 10d 24h and 4wk data sets. Detailed analysis is underway to compare the blood and brain profiles to identify common biomarkers for the diseased state.
Ceruloplasmin binds and inactivates matrix metalloproteinase‐2
Authors: Thompson Michael W.
Published: June 2021

An interaction between the iron‐binding protein lactoferrin and matrix metalloproteinase‐2 (MMP‐2) suggests that MMP‐2 may interact with other metal binding proteins. To examine this possibility, the copper binding protein ceruloplasmin was examined to determine its effect on MMP‐2 and pro‐MMP‐2. Apoceruloplasmin, and to a lesser extent, holoceruloplasmin, physically interacted with pro‐MMP‐2 in an immunoprecipitation assay. Furthermore, apoceruloplasmin inhibited the protease activity of active MMP‐2, suggesting that ceruloplasmin may regulate the activity of MMP‐2 in vivo.
Understanding Glycosaminoglycan – Protein Specificity Through Computational Approaches
Authors: Desai Umesh R , Mosier Philip D
Published: June 2021

Glycosaminoglycans – protein interactions modulate a number of physiological processes such as coagulation, immune response, angiogenesis, morphogenesis and pathogen infection. Yet, a majority of these interactions involve allosteric regulation of protein and are poorly understood. We have developed a genetic algorithm‐based combinatorial virtual screening approach to understand the nature of interaction of glycosaminoglycans with proteins. A dual‐filter strategy employing an ‘affinity’ filter followed by a ‘specificity’ filter rapidly sorted the combinatorial library into distinct groups of sequences. Application of this strategy on a library of 46,000 sequences resulted in identification of only five sequences that recognize heparin cofactor II with ‘high‐affinity and high‐specificity’. Likewise, the strategy led to the prediction of a novel geometry for dermatan sulfate hexasaccharide onto heparin cofactor II, which explained the formation of the ternary glycosaminoglycan – heparin cofactor II – protease complex. The strategy represents a general approach for understanding specificity of allosteric glycosaminoglycan – protein interactions and may lead to the design of novel allosteric agonists or antagonists of proteins.
Study on Mechanism of Cholesterol Metabolism in Leydig's Cells of Rats with Lower Serum Testosterone due to Intermittent Anaerobic Swimming Training
Authors: Yi Yan , Xie Minhao
Published: June 2021

The effect of cholesterol metabolic block of leydig's cell on the testosterone production during is still not well known.

Purpose

To compare the effect of different intermittent anaerobic swimming training time on the mRNA expression of HMG‐CoA reductase, SR‐BI, LDL‐R, StAR .

Methods

After the five‐week intermittent anaerobic swimming training(1 hour per day, with 5% body weight load, TA), TC group stopped training for one week, TD group continue one week training. TE group was trained 2 times per day. TF group was trained with 6% body‐weight load. The serum testosterone (T), the mRNA expression of HMG‐CoA reductase, LDL‐R, SR‐BI and StAR protein in Leydig's cells were examined by the real‐time PCR.

Result

The results showed that the serum T of TA group decreased significantly (1.44±0.42 vs. 8.83±4.57 ng/ml, p<0.01) due to the five‐week training, the LDL‐R mRNA in Leydig's cells increased significantly (p<0.05). Compared with TA group, TC group serum T (4.54±1.60 ng/ml, p<0.01) was increased significantly, but still under the normal level, the SR‐BI mRNA expression in Leydig's cells increased significantly too (p<0.01)l. After one week training, the serum T in TD and TF groups had a further decrease, but their were no significant difference between TA, TD (1.05±0.36 ng/ml) and TF(1.10±0.27, ng/ml) groups. In TE group, there was a further significant decrease in serum T (0.99±0. 29 ng/ml, p<0.05). The LDL‐R mRNA expression, in TD group, had a significantly decrease (p<0.05). In TE, TF groups, the mRNA expression of LDL‐R, SR‐BI, HMG‐CoA reductase in Leydig's cells, were also decreased significantly(p<0.01).

Conclusion

The cholesterol metabolic block in Leydig's cells was not the original reason of exercise inducing lower serum testosterone. The decrease of mRNA level of HMG‐CoA reductase, LDL‐R, SR‐BI and StAR in Leydig's cells would accelerate the decrease of serum T due to exercise.
Homogenous, luminescent HTS‐formatted platform technologies for cAMP‐, and cGMP‐dependent Phosphodiesterase (PDEs)
Authors: Goueli Said , Hsiao Kevin
Published: June 2021

Cyclic nucleotide phosphodiesterases (PDEs) represent eleven different gene families of enzymes that are encoded by at least 21 different genes and accounting for at least 55 isoforms. They have been implicated in controlling specific cellular functions including Cardiovascular, pulmonary, and inflammatory diseases and they play potential role as targets for treatment of cancer. Thus, the development of novel isoform‐specific inhibitors may be useful for the development of therapeutic agents against cancer. We have devised two novel approaches to monitor the activity of cAMP‐PDE and cGMP‐PDE and developed two homogenous, luminescent high through‐put screening (HTS) assays to screen for novel inhibitors of PDE. The first strategy relies on the activation of cAMP‐Dependent Protein Kinase (PKA) in the presence of low concentration of cAMP and also by moderately higher concentration of cGMP. The effect of compounds on the activity of PDE can be monitored by the cAMP‐dependent activation of PKA and measuring the active PKA using luminescent Kinase assay. The other strategy relies on the conversion of AMP generated in the presence of PDE into ATP and measuring the generated ATP using luminescent Kinase assay. Both strategies have been developed into HTS formatted assays and can be carried out in two simple steps in 384‐, and 1536‐well formatted plates. The Assays are luminescent and thus they encounter minimal interference from fluorescent compounds and the robustness of the assays are indicated by their remarkably high Z′ value (>0.8). In summary, both assays can be used to screen libraries of compounds targeting PDE that might be developed into potential drugs for treatment of cancer.
Lab‐on‐a‐Print: Desktop Microfabrication for Quantitative Biology
Authors: Pan Tingrui
Published: June 2021

Motivated by the growing demand for low‐cost, easy‐to‐use, compact‐size yet powerful micro‐nanofabrication technologies, we have been investigating engineering solutions to address emerging challenges of fundamental biology and translational medicine in regular laboratory settings. Recent advancements in the field benefit considerably from rapidly expanding material selections, ranging from inorganics to organics and from nanoparticles to self‐assembled molecules. Meanwhile a great number of novel methodologies, employing off‐the‐shelf consumer electronics, intriguing interfacial phenomena, bottom‐up self‐assembly principles, etc., have been implemented to transit micro‐nanofabrication from a cleanroom environment to a desktop setup. Furthermore, the latest application of micro‐nanofabrication to emerging biomedical research will be presented, which includes point‐of‐care diagnostics, on‐chip cell culture as well as bio‐manipulation.
A Mass Spectrometry Simulation for Biochemistry Education
Authors: Fisher Amanda , Craig Paul A.
Published: June 2021

The separations simulations team at RIT has been developing interactive simulations of biological separation processes for over 10 years. The JBioFramework collection of interactive simulations is now platform independent and runs equally well on all common operating systems. The updated 2D electrophoresis simulation utilizes multiple file types and Internet resources, and a new simulation has been added ‐ tandem mass spectrometry. The tandem MS simulation accepts data from the 2D electrophoresis simulation, allowing a student to select a protein from the gel and select a protease to digest it. Users can click on a peptide fragment on the first Mass Spectrometer readout, then see the b‐ and y‐fragments that give the peptide sequence in the second Mass Spectrometer readout. Support for this work has been provided by the RIT Colleges of Science and Computing and Information Science and by the Merck/AAAS Undergraduate Science Research Program.
Branched polyethyleneimine coating gold nanoparticle‐mediated gene delivery targeting nuclear protein in A549 cells
Authors: CHOI SEONYOUNG , JUNG SEROMI , RYU PANDONG , JOO SANGWOO , LEE SOYEONG
Published: June 2021

Recently, the regulation of gene transcription and protein abundance using nanotechnology is emerged field. Gold nanoparticles (AuNPs) have been shown to be of potential use in anticancer drugs/genes delivery systems. However, the targeted delivery of RNA‐based therapeutics for cancer therapy remains a challenge. We hypothesized that an AuNP complex with plasmid DNA inhibits the expression of target genes in the nucleus. In order to selectively knock‐down the protein expressed in the nucleus, 17 nm AuNPs coated with branched polyethyleneimine were bioconjugated with plasmid DNA expressed shRNA. For the study, MTT, quantitative real time PCR and western blot analysis were performed. As a result, the AuNP‐plasmid DNA complex successfully down‐regulated target genes expressed in the nucleus. The complex's gene silencing efficiency was high within the low cytotoxicity range of AuNPs. These results indicate that AuNPs may be used as nuclear‐targeting nanoparticles for gene delivery. This study received support from the National Research Foundation of Korea (2010‐0017216).
Stability and functionality of HL heteropolymer ferritins responsible for a hereditary ferritinopathy disorder
Authors: McNally Justin , Arosio Paolo , Bou-Abdallah Fadi
Published: June 2021

Mammalian ferritins typically consist of 24 structurally similar but functionally different subunit types named H and L capable of accommodating up to 4500 Fe atoms per molecule. Heteropolymer ferritins having mutations on the L‐subunit cause hereditary ferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin and iron in the central nervous system. Here, we characterize three pathogenic L‐ferritin mutants (L154fs, L167fs, and L148fs also known as Ln1, Ln2 and L442P) and one non‐pathogenic variant (L135P) for their iron uptake, oxidation and release properties. Overall, the data show iron loading capacity ranging between 1800–2400 Fe/shell for all HL samples with Ln2 holding the least amount of iron. The order of stability of the HL samples is HL > H‐chain > L135P > Ln2 > Ln1 > L442P. The rates of Fe oxidation were similar in all HL samples with a consistently noticeable higher rate with L442P. The mechanism of Fe oxidation appeared to proceed mainly via a ferroxidation reaction with about ~ 2:1 Fe:O2 ratio and slowly climbing to ~ 3:1 at higher iron loadings. Ln1 and L442P had a higher rate of Fe mobilization consistent with the onset of neuroferritinopathy.

This work is supported by a Kilmer Apprenticeship and a Cottrell College Science Award (ID # 7892) from Research Corporation.
Operation Invisible Dead: A Blind Study to Compare Molecular Fingerprints of Soil Samples between Crime Scene and Criminal's Shoe
Authors: Patel Niraj , Larson Sabreena , Freeman Victoria , Bailey Cheryl
Published: June 2021

Capillary electrophoresis can separate fragments of nucleic acids using single strand conformation polymorphism (SSCP) This method has been increasingly used in microbial ecology to develop fingerprints of the microbial community within soil. SSCP detects mutations in DNA fragments due to changes in the secondary structure of single‐stranded DNA fragments. Molecular fingerprinting can have a wide application in Forensics. It is important to find if a soil fingerprint from a criminal's shoe can be matched to the fingerprint found in the database. This is a blind study comparing the possible use of CESSCP in Forensics. The soil samples are collected from two locations by two people. Each individual acted as a “criminal” by digging the soil, walking over the soil and buried a small object. One shoe was removed at the “crime scene” and the other shoe was worn home. The soil was collected from the shovel and both of the shoes for each individual. All the soil samples were air dried for 48 hours before DNA was extracted. The samples were then run through polymerase chain reaction (PCR) to amplify and fluorescently tag the genes of interest. The samples will be processed in the Genetic Analyzer using the CE‐SSCP. The results can very well show that CE‐SSCP will not produce similar fingerprints for the shoe worn home and the database, thus inapplicable to Forensics.

Supported by National Institute of Justice (25‐6228‐0159‐001).
Ultrasensitive, background‐free single copy DNA detection: Benchmarking performance using HPV infected cell lines and tissue biopsies
Authors: Coleman Jack , Lebedeva Irina , Szczepanik Maciej , Pande Praveen , Liang Fred , Patton Wayne
Published: June 2021

In situ hybridization (ISH), immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) are common detection methods in biology. For high copy number targets, they perform well, but for single‐copy targets, sensitivity is often lacking. Chromosomal hybridizations require large probes (~100 kilobases) to obtain sufficient signal. At times, the desired target is not large enough for hybridization with probes of this size. We describe a method for single‐molecule detection wherein a probe contains multiple copies of a signaling moiety, like a fluorescent molecule, or an enzyme, like alkaline phosphatase, linked to a single binding molecule, like streptavidin. The small construct size allows efficient labeling of permeabilized cells. Cell lines and tissues infected with HPV, the disease‐causing agent of cervical cancer, were used to benchmark the method, employing biotinylated, probes specific to HPV subtypes. Using cell lines of known HPV DNA copy number, single copy detection per cell was demonstrated. The integrated copies of HPV16 in SiHa cells and chromosome spreads appear as punctate loci, as expected. Also, detection of the repressed p53 protein in HPV‐infected cells and tissue biopsies was demonstrated. The new method increases the overall sensitivity of FISH, ISH and IHC by roughly an order of magnitude and thus should prove applicable to the detection of cancer and infectious agents.
Alterations in ceramide in hyperglycemic and fatty acid supplemented macrophages
Authors: Albert Carolyn J. , Ford David A.
Published: June 2021

Ceramides are an important lipid pool that is used to store carbon energy substrates under high nutrient conditions. In the present study we examined the incorporation of fatty acids into ceramides of macrophages in the presence of euglycemic and hyperglycemic conditions. RAW 264.7 mouse macrophages were treated with selected fatty acids including stable isotope labeled fatty acids, and ceramides were detected by ESI‐MS/MS. Palmitic acid was incorporated into both the N‐fatty acyl position as well as the sphingosine backbone of ceramide during incubations with > 200 μM fatty acid. Using stable isotope labeled palmitic acid, fatty acyl elongation products were observed in the N‐acyl position. In comparison to stable isotope labeled palmitic acid, relatively less stable isotope labeled myristic acid was incorporated as elongated fatty acids into the ceramide pool. Palmitic acid elongation products were incorporated into ceramide at enhanced rates in the presence of high glucose. These studies revealed that not only are fatty acids stored in macrophage ceramide pools, but are also elongated and stored in ceramide pools under conditions of high saturated fatty acid incubations and/or hyperglycemic conditions.
A Multifaceted Investigation of the Localization of the Human Follicle Stimulating Hormone Receptor
Authors: Steinmetz Danielle , Cohen Brian D.
Published: June 2021

Follicle stimulating hormone (FSH) is responsible for follicular maturation in females and production of sperm‐stabilizing proteins in males. FSH receptor (hFSHR) is a G‐protein coupled receptor located in granulosa cells in the ovaries and Sertoli cells in the testes. It is believed that hFSHR is located in lipid raft microdomains of the plasma membrane. Lipid rafts are areas of the cell membrane that demonstrate increased rigidity, resistance to detergents, and have a high percent composition of cholesterol and sphingolipids. It is proposed that hFSHR is associated with a specific class of lipid rafts known as caveolae; hFSHR contains a caveolin binding motif in its 4th transmembrane domain. To verify if hFSHR resides in lipid rafts, sucrose gradient fractionation was performed and analyzed by western blot. hFSHR was observed to reside in the fractions associated with buoyant membrane components. To determine if hFSHR interacts with caveolin, four mutant receptors were created by altering the putative caveolin binding motif. Understanding hFSHR localization in the plasma membrane will lead to a greater understanding of its signaling pathways and the molecular basis of reproduction.
Extension of poly‐N‐acetyllactosamine chain in human cancer cell lines is under src regulation
Authors: Hwa KuoYuan , Liu Hsuan-Chen , Chou Teh-Ying
Published: June 2021

Alternation of glycan synthesis is an important process in cancer metastasis. And, the extent of poly‐N‐acetyllactosamine chains on tumor cells has been implicated for their metastatic potential. β1,3‐N‐acetylglucosamonyltransferase‐2 (β3GnT2) and β1,3‐N‐acetylglucosamonyltransferase‐8 (β3GnT8) are two of the glycosyltransferases responsible for the synthesis of poly‐N‐acetyllactosamine. Since Src is a well‐known oncogene, which has been implicated in pathways regulating cell proliferation, angiogenesis, invasion and metastasis, we had generated a stable mouse cell line expressing v‐Src. First, we had examined the expression of the two genes in stable transformed v‐Src and wild‐type NIH/3T3 cell lines. And, we found that the cell line with overexpressed Src had higher level β3GnT8 transcripts than the normal NIH/3T3 cell line. To further illustrate the regulatory role of Src on the two genes, we had investigated the expressions of β3GnT2 and β3GnT8 in SW620 and H23 by treating the cells with bosutinib, a Src inhibitor. We found that the decrease of the expression of β3GnT2 was not significant, but the expression of β3GnT8 was significantly decreased by the Src inhibitor. Furthermore, we had found that the surface expression of poly‐N‐acetyllactosamine was altered by the Src inhibitor, by FACS analysis.

This work is supported by the National Taipei University of Technology to KYH (NTUT 100‐140‐01, NTUT 99‐140‐04).
A sphingosine derivative recruits regulatory T cells to the kidney by nuclear translocation of NFKB and up‐regulation of CCL5
Authors: Ly Elina , Yong Kim-Chong , Lien Yeong-Hau H , Lai Li-Wen
Published: June 2021

A sphingosine‐derivative (SD) has been identified which ameliorates renal ischemic reperfusion injury and recruits regulatory (Treg) and effector T cells to the inflamed tissues. The underlying mechanisms of this protection were investigated. Depleting the Treg by anti‐CD25 antibodies abolished this protection, suggesting that Treg recruited by SD play a key role. Using real time qRT‐PCR, we found that SD rapidly increased CCL5 mRNA in mouse kidney (3–4 folds increase at 1 h), which is known to direct T cell migration. To examine if this up‐regulation is mediated by activation of NFKB, a transcription factor known to regulate CCL5, we measured NFKB protein in renal proximal tubular cells before and after SD treatment by immunofluorescence stain and ArrayScan® VTI HCS reader. SD increased NFKB nucleus/cytosol ratio up to 80%. This nuclear translocation is dose‐dependent (1–20 uM) and was observed within 15 min, reached a plateau at 30 min and remained stable for 2h. In conclusion, our findings are consistent with the hypothesis that SD triggers the cascade of NFKB activation, leading to NFKB nuclear translocation and up‐regulation of CCL5 in renal tubule cells to recruit T cells into the kidney. Studies are ongoing to further pinpoint SD's role in upstream signaling pathway of NFKB activation and to develop a novel Treg‐based therapy for inflammatory diseases. Funding: NIH RO1DK82718, ABRC, DCI.
Incorporating mathematics principles into high school biology experiments using GFP denaturation studies
Authors: Puccinelli Michael , Ogele Emmanuel , Kasper Sherry , West Lori
Published: June 2021

Increasing mathematics proficiency among high school students is a priority of our Federal and many state governments. Our goal was to develop a low‐cost experiment in which high school biology teachers with budget constraints could incorporate mathematics principles into their biology experiments in both an interesting and exciting manner. In our experiment, we wanted to determine if we could develop a protocol to measure a statistical difference in fluorescence between folded and fluorescing Green Fluorescent Protein (GFP) and the denatured protein. In order to denature GFP, we used various household chemicals which would be both accessible, cheap, and of which disposal would not be costly. Samples containing lysate obtained from cells expressing GFP in the absence or presence of denaturant were transferred to a microplate and exposed to UV light and photographed using the BlueView Transilluminator. The image was converted to black and white and then analyzed using ImageJ software. Student t tests were performed to determine if it was possible to quantify the difference between the folded and fluorescing and unfolded protein. We determined that statistically significant differences were observed between GFP in water (positive control) and GFP exposed to various denaturants (i.e. heat, vinegar, and others). Importantly, specialized equipment was not required as the transilluminator is both multifunctional and relatively inexpensive. In addition, ImageJ is downloadable free from the NIH and Excel can be used to conduct the t test.
Effect of the amount of dietary protein in the dam metabolism in rat liver and mammary gland during pregnancy and lactation
Authors: Velazquez Laura , Lopez Adriana , Tovar Armando R , Torres Nimbe
Published: June 2021

During pregnancy and lactation periods there are many metabolic adaptations in the liver, and mammary gland (MG) of the dam to use different metabolic fuels. The aim of the study was to analyze these adaptations on the expression of genes of glucose, lipid and amino acid metabolism in the liver and MG of pregnant and lactating rats fed graded concentrations of dietary protein (10, 20 or 30%). During pregnancy and lactation the expression of lipogenic (SREBP‐1, ACC and FAS), and glycolytic (PK) genes increased as the amount of dietary protein decreased, but hepatic lipogenesis was higher during lactation than pregnancy. The expression of amino acid degrading enzymes (SDH) and gluconeogenic (PEPCK) genes increased as the concentration of dietary increased during pregnancy. In the lactation period the expression of SDH did not increase with the amount of dietary protein. In contrast in the MG the expression of lipogenic, amino acid transport (SNAT2) and mTOR genes increased overtime independently of the amount of dietary protein whereas there was a reduced expression of lipolytic (HSL) and lipid oxidation (CPT‐1) genes. These results indicate that the excess of amino acids provide energy for the hepatic energy needs during gestation and lactation. The MG showed an increase fatty acids and protein synthesis to sustain the needs of the pups regardless of the amount of dietary protein during lactation.
Washing the milk fat globule minimizes cellular contamination without compromising mRNA quality
Authors: Nommsen-Rivers Laurie A , Ballard Olivia M , Horseman Nelson D , Morrow Ardythe L , Lemay Danielle G , Hughes Maria A
Published: June 2021

Milk fat globules (MFG) are a rich source of lactocyte mRNA. We examined if the lipid phase of centrifuged whole milk contains extraneous cells (presence of nucleic matter); if washing the MFG will reduce cell contamination; and factors associated with RNA quality. Early (E, day 2) and Mature (M, day 24–424) fresh human milk samples were centrifuged x 10 min to obtain MFG using 3 methods: Soft (300 g), Hard (15,000 g) and Washed (hard spin, re‐suspend MFG in buffer, repeat x 2). MFG (N=24 E and 31 M) were stored at −80 in TRIzol, RNA isolated, and assayed for RNA integrity number (RIN). The transcriptome was sequenced (1 Washed E and 6 Washed M to date; Illumina HiSeq2000). DAPI staining of the MFG confirmed cell presence in unwashed E and M. Washing dramatically reduced cells in E, and eliminated cells in M; Sequenced Washed E and M had no expression of CD45 (leukocyte marker) and Western blot revealed heavy bands of â‐Actin and no visible Lamin. RIN (N=55, Median=8.1) was not correlated with processing method, day postpartum, days in TRIzol, or days stored as isolated RNA. Among E samples (N=24), RIN was correlated with minutes from collection to TRIzol (range, 50–155; r=−0.48) and hours from collection until stage II lactogenesis (range, −14 to 44; r=−0.35). Overall, 31% were unsuitable for sequencing (RIN < 7.0), likely due to RNAase contact during sample collection. Funded by Cincinnati Diabetes and Obesity Center and HD1302.
Grape seed extract attenuates oxidative stress induced by high fat/carbohydrate meal in metabolic syndrome patients
Authors: Tadapaneni Ravi Kiran , Krishnankutty Sandhya , Alandete Lourdes , Randolph Jody M. , Cheema Mandeep , Park Eunyoung , Edirisinghe Indika , Freeman Britt Burton , Kappagoda C. Tissa
Published: June 2021

Metabolic syndrome (MS) is associated with increased oxidative stress. Consumption of a high carbohydrate moderate fat (HCMF) meal results in increased markers of postprandial oxidative stress. We hypothesized that grape seed extract (GSE‐Meganatural© Gold), rich in antioxidants would attenuate HCMF meal‐induced oxidative stress in MS. After an overnight fast, 12 subjects with MS (6 men/6 women) consumed a HCMF meal in a randomized cross‐over design. A GSE (300 mg) or placebo capsule was administrated 1 h before the meal. Postprandial changes in plasma lipophilic (LA), hydrophilic (HA) antioxidant status (Oxygen Radical Absorbance Capacity‐ORAC) and oxidized LDL (OX‐LDL) were measured hourly for 6 h. Postprandial plasma ORAC values (LA and HA) measured as area under the curve over 6 hr were significantly increased when the meal was preceded by GSE compared with placebo (P<0.05). Meal‐induced increases in OX‐LDL were attenuated with GSE (P<0.05). These data suggest that the antioxidant activity of GSE produces favorable effects in attenuating oxidative stress in subjects with the MS. Funding: Polyphenolic Inc. Madera, CA
High‐Oleic Acid Ground Beef and Risk Factors for Cardiovascular Disease in Men and Postmenopausal Women
Authors: Ghahramany Ghazal , Choi Seong-ho , Gilmore Linda Anne , Smith Stephen B.
Published: June 2021

Cardiovascular disease (CVD) risk can be influenced by diet, but optimal dietary content of fatty acids continues to be debated. The effects of fatty acid composition of ground beef on selected CVD risk indicators and peripheral blood mononuclear cells (PBMC) were evaluated. Five men and seven women over age of 45 completed a two‐way crossover design. Subjects consumed five, 114‐g ground beef patties per week for 5‐wk periods separated by a 3‐wk washout period. Patties contained MUFA:SFA of 0.8 and 1.1 for conventional ground beef (CGB) and premium ground beef (PGB) patties, respectively. Blood was collected from each subject before and at the end of each phase. PBMC were isolated and the expression of selected genes was quantified by real‐time PCR. Both ground beef interventions decreased total plasma cholesterol, triacylglycerol, and VLDL cholesterol. Plasma concentrations of HDL and LDL cholesterol decreased and increased, respectively, with PGB consumption. A significant increase was seen in stearoyl‐Coenzyme‐A desaturase 1 gene expression after the PGB intervention. LDL receptor and sterol regulatory element‐binding transcription factor 1 gene expression decreased with CGB consumption. Ground beef dietary interventions varying in fatty acid composition differentially affect lipoprotein concentrations and the expression of PBMC genes related to CVD.
Impact of a Functional Food Bar on Biomarkers of Heart Disease in Hypercholesterolemic Adults
Authors: Hutchins Andrea
Published: June 2021

This randomized crossover study examined the impact of consuming 2 functional food (FF) bars containing soy protein (2.4 grams(g)/bar), flaxseed (7 g/bar), oat bran (5 g/bar) and walnuts (8 g/bar) or 2 control bars on fasting total (TC), high‐density lipoprotein (HDL‐C), and low‐density lipoprotein cholesterol (LDL‐C) and triacylglycerol (TAG) concentrations in hypercholesterolemic adults. Use of functional foods or foods high in components that reduce cholesterol are recognized as options in dietary interventions to prevent or treat risk factors for CVD like hypercholesterolemia. Before and after each 8‐week intervention, 20 subjects provided a capillary blood sample for lipid assessment [CardioChek PA Professional blood test system (Polymer Technology Systems, Inc. Indianapolis, IN)]. Consumption of FF bars significantly lowered TAG concentrations (P=0.003) compared to control bars. There were no other statistically significant changes (p<0.05) with either intervention. These results demonstrate that consumption of FF bars may favorably impact lipid profiles in hypercholesterolemic adults. This study was funded by the University of Colorado Colorado Springs Committee on Research and Creative Works.
Internet‐Based Tools to Assess Diet and Provide Feedback in CKD IV
Authors: Arab Lenore , Murali Sameer , Rastogi Anjay , Ang Alfonso , Shetty Nidhi , Vargas Robert
Published: June 2021

Successfully changing patients’ dietary behavior is a challenging problem in the management of chronic kidney disease (CKD). To test the feasibility and acceptability of an internet‐based, self‐administered, dietary assessment tool equipped with instructional feedback, aimed at facilitating patient dietary change and reducing resource burdens on providers, 12 patients diagnosed with stage IV CKD (pre‐dialysis stage) were recruited. Multiple validated questionnaires, gauging nutritional education, importance in CKD management and patients’ compliance to required dietary guidelines were applied. Results show that 25–30% of the patients were severely non‐compliant to the requirement guidelines for each nutrient. Awareness about the role of diet in CKD management was found among 0%–58% of population varying over different nutrients. All of the patients successfully completed the web based dietary assessment. Patients and physicians differed considerably in their perception of the use of the report. Dietary knowledge and adherence to KDOQI guidelines is suboptimal among CKD patients, however, interest and motivation is high. Web based tools are well‐accepted among CKD patients as an aid to assessing dietary adherence to KDOQI guidelines and may pose a cost effective approach to bridging the adherence gap.
Changes in LDL particle size after aerobic training in CKD patients
Authors: Gregory Sara Michelle , McMahon Greg , Germain Michael , Wood Richard J. , Headley Samuel
Published: June 2021

Chronic kidney disease (CKD) is associated with dyslipidemia and increased risk of cardiovascular disease. We examined the effects of 48 wks of aerobic exercise training on LDL particle size in patients with CKD. Blood samples were obtained from 16 CKD patients (stage 3–4). The treatment group (TX; n = 7) performed supervised aerobic exercise 3X/week along with usual care. The control group (CG; n = 9) received usual care but did not exercise. LDL subfractionations (large = 1, medium = 2, and small = 3) were determined by polyacrylamide gel electrophoresis using the Lipoprint LDL system. Pattern A was a peak particle size ≥ 25.5 nm; pattern B: < 22.5 nm. VLDL decreased in both groups (p = 0.04; CG: 24.4 ± 9.1 to 20.8 ± 4.8; TX: 27.4 ± 0.5 to 24.0 ± 2.3 mg/dL). LDL‐C decreased (p = 0.02) in CG (102.2 ± 28.8 to 81.7 ± 11.1 mg/dL) but not for TX (95.6 ± 27.4 to 110.3 ± 31.5 mg/dL). LDL‐1 decreased in both groups (p = 0.04; CG: 36.4 ± 10.0 to 29.1 ± 5.7; TX: 32.3 ± 9.7 to 30.7 ± 7.9 mg/dL). LDL‐3 increased (p = 0.04) in TX (1.7 ± 2.1 to 3.6 ± 3.6 mg/dL) but not for CG (2.9 ± 3.7 to 1.7 ± 1.5 mg/dL). LDL‐3 increased in 4/7 patients in TX. Two subjects in the TX shifted from a pattern A to pattern B profile; no change occurred for control group subjects. We observed unfavorable changes in LDL subfractions after aerobic training in CKD patients, which warrant further investigation.
Monitoring of a Pilot Micronutrient Powder Home Fortification Program in the Kyrgyz Republic
Authors: Lundeen Elizabeth Ann
Published: June 2021

To address the high prevalence of anemia, in 2009, UNICEF launched a pilot micronutrient powder (MNP) home fortification program for children 6–24 months old in the Talas region of the Kyrgyz Republic. Children received 30 MNP sachets every 2 months from government health clinics. Two representative household surveys were conducted at 6‐months (n=636) and 12‐ months (n=543) following the launch of the program, to monitor program performance in rural Talas. Monitoring results were positive. Almost all caretakers had heard about the MNP product (100.0% at 6‐mo.; 99.6% at 12‐mo.) and had received ≥1 package of 30 MNP sachets (99.3% at 6‐mo.; 98.9% at 12‐mo.). The majority of caretakers reported that their child is currently taking MNP (84.7% at 6‐mo.; 71.2% at 12‐mo.). Self‐reported adherence to MNP was high. Out of the 30 sachets provided for 2 months, average consumption for the previous 2 months was 29.2 sachets at 6‐mo., and 24.4 sachets at 12‐mo. Complete adherence (use of all 30 sachets during the previous 2 months) was found among 60.4% of the children at 6‐mo. and among 49.5% at 12‐mo. Both surveys found that caretaker satisfaction and acceptance of MNP was high, and the caretakers’ knowledge of and practices pertaining to MNP use were found to be satisfactory. These survey results provided evidence to support the national scale‐up of this intervention. Source of research support: UNICEF.
Distribution of Sodium intake in Mexican adults from a national probabilistic survey
Authors: Angulo Juan Salomón , Quezada-Sanchez Amado David , Barquera Simon , Shamah-Levy Teresa
Published: June 2021

A model of Sodium intake by Food frequency questionnaire (FFQ) and socioeconomic characteristics in a sample representative of 59,029,340 Mexican adults were obtained from the Mexican National Survey (2006). A model for the shape and scale parameters of a Gamma distribution was fitted separately by sex, area of residence and age group. Estimation was performed by Maximum Likelihood taking into account the survey design and distribution fit assessed graphically Parameters were compared through formal tests and distributions were described through graphical analysis. The distribution of sodium moments were calculated from the estimated parameters and compared across groups. The scale parameter differed by sex (P=0.024) whereas the shape parameter of the distribution differed by area of residence (P<0.001) and was different in the oldest age group from the 20/29 year olds (P=0.024) and the group of 30/39 years of age (P=0.012). Men distribution is located to the right (high consumption) of Woman distribution, urban area distribution is located to the right of rural distribution, the distribution of the oldest age group is shifted to the left from the other age groups. The gamma model is useful to describe the sodium intake distribution and indicates that there are differences in its shape and scale by sex, area of residence and age group in Mexican adults.