bluuebunny/crossref_metadata_2025 huggingface dataset:
Myocardin‐ related transcription factor A regulates myofibroblast formation and function
Authors: Crider Beverly Joy , Green Michael S. , Broiles JoSette L. , Howard Eric W. , Haaksma Carol J. , Tomasek James J.
Published: June 2021

Myofibroblasts (MFs) differentiate from fibroblasts and function to facilitate wound closure. Under pathological conditions, MFs fail to undergo apoptosis and continue to remodel the extracellular matrix, leading to increased contracture of connective tissue. MFs are characterized as having "super‐mature" focal adhesions and expression of smooth muscle α‐actin (SMAA). Myocardin‐related transcription factors A and B (MRTF‐A/MAL/MKL‐1 and MRTF‐B/MKL‐2) are putative mechanical stress‐induced co‐activators that activate expression of contractile proteins such as SMAA. Small interfering RNA (siRNA) has been used to study the effects of MRTF‐A and MRTF‐B reduction on the MF contractile phenotype in rat embryonic fibroblasts. Decreased SMAA levels, as determined by immunoblot analyses, and reduced contractile force generation, as determined by a wrinkling assay, were observed in cells transfected with MRTF‐A and ‐B‐targeted siRNA. Immunocytochemistry has shown a decrease in the size and number of focal adhesions in these cells when compared to control cells. Finally, transiently transfected myocardin, which acts similarly to constitutively‐active MRTF‐A, increased SMAA promoter activity and increased the contractile ability of cells plated on a deformable wrinkling substrate. These results suggest MRTFs play a critical role in regulating MF formation and function. (Funded by NIH 2R01GM60651)

Grant Funding Source

NIH
The minor groove binding drug berenil down‐regulates the expression of topoisomerase II alpha in zebrafish larvae
Authors: Daggett Melissa A. F. , Edwards Curtis , Showalter Miranda
Published: June 2021

Topoisomerase II α (topo II α) relieves supercoiling in the DNA helix during DNA replication. Topo II α is normally up‐regulated in rapidly proliferating cells. Minor groove binding drugs (MGBDs), such as Hoechst 33258, can down‐regulate the expression of topo II α in cancer cells grown in culture by interfering with transcription factor binding at specific A/T rich sequences in the topo II α promoter region. These results have indicated a clinical role for MGBDs in regulating effects of the MGBD berenil, on the expression of topo II α?in the rapidly dividing cells of zebrafish larvae. Semi‐quantitative PCR analysis of topo II α expression in 5 day old zebrafish larvae exposed to berenil indicated a down‐regulation of topo II α compared to the unexposed control embryos. These results merit further analysis of the effects of MGBDs, such as berenil, on topo II α expression in intact organisms as opposed to isolated cells in culture. Further characterization of berenil on cells in intact embryos, including its effects on cell proliferation, differential gene expression, and overall development of the zebrafish embryo will be presented.
Dissecting the role of an anatomy teaching assistant
Authors: Jacobs Sarah Marie , Chen Laura P , Camp Christopher L , Gregory Jeremy K , Alexander Cara J , Lachman Nirusha , Pawlina Wojciech
Published: June 2021

Gross anatomy courses often utilize teaching assistants (TAs) to serve a multifaceted purpose. Qualitative analysis of interviews with gross anatomy TAs reveals seven distinct roles: formal teaching during course lectures, informal teaching during dissection, prosecting a demonstration cadaver, orchestrating regular review sessions, coordinating formative feedback about academic progress and professionalism, tutoring struggling students outside of class time, and supporting students in crisis should the need arise. Analysis also finds that TAs did not anticipate the full scope of their duties prior to undertaking them, and felt particularly unprepared to address issues of student mental health. We report our findings and propose a model for proactively training TAs for their inherently complex role. This work was supported by the Mayo Clinic Department of Anatomy.

Grant Funding Source

Student/Postdoctoral Travel Award Student Award
Assessing Team‐Skills Using the Objective Structured Behavioural Exam
Authors: Keane Aisling , McCullough Stephen , Wilson David
Published: June 2021

The development of effective team‐skills is an essential component in modern day medical curricula and students regularly undertake group activities for this purpose. The quality of the end result is often used as a measure of team performance however it can say little about the team‐skills employed to achieve the goal. To encourage student to take the development of team‐skills seriously these need to be formatively assessed to give the process a sense of reality Student groups were given instructions and a set of histological images. As the task was time‐constrained teamskills were assessed on time management; organisation of group; communication; data processing; decision making and communication of the answer to examiner. The examiner also awarded marks for correct knowledge. The OSBE provided a total mark which contributed to a formative assessment mark based on team‐skills and knowledge. This mark was shared equally among individuals in the group. We believe that this is a valuable and reliable tool to test group skills within a subject‐based scenario.
A new course to bridge the gap between the laboratory and patient care: Pathology 777
Authors: Wakefield Dara N , Van Duyn Lauren B , Alexander C. Bruce
Published: June 2021

Graduate students can lose sight of the whole patient as a dynamic system during their studies of an individual protein or pathway. Clinicians become discouraged if there is nothing left in the treatment repertoire for their patients and hope there will be better future options. To bridge this gap between the bench and clinical practice, a new course was developed for Molecular and Cellular Pathology graduate students at UAB. The student requirements were to attend and participate in a full adult autopsy (from chart review, evisceration and organ dissection, Hematoxylin and Eosin slide review, and CNS examination) along side the pathology resident. Following the full autopsy, the students presented highlights of the links between their laboratory work and the disease processes occurring in the patient. We discuss a patient who presented to UAB for repeat right lower extremity bypass grafting who had many post operative complications that culminated in her death. We show the benefits of such a course for the pathology resident and the student. It provided a framework for the students and residents to discuss the needs in the clinic and the correlation to the student's work on wound healing. This course was offered within UAB Department of Pathology and thus received no research funding.
Using Tablet PCs and DyKnow Vision to Enhance Student Engagement in a Human Anatomy Class
Authors: Lafontant Pascal J
Published: June 2021

Keeping students engaged in human anatomy classes is a significant challenge. To increase students' active learning and facilitate understanding of material, Tablet PCs running the software DyKnow Vision were introduced in a human anatomy class. Tablet PCs and DyKnow Vision were specifically used to create a virtual blackboard for the identification and discussion of anatomical structures. Anatomical illustrations from the primary text used in the class were routinely imported and saved as DyKnow Vision files. Students were then provided with DyKnow panels of unlabeled structures to identify and label using digital ink. The labeled panels were submitted, reviewed, corrected with the class' participation, and appended to the class session. To assess students' level of engagement, their attitudes and perceptions of the effectiveness of the Tablet PCs/DyKnow as a pedagogical tool, an anonymous survey using a 5 point Likert scale was completed by students. Results strongly suggest that the Tablet PCs/DyKnow combination made the class enjoyable, enhanced students' comprehension of content, and had an overall positive impact on their learning of human anatomy.

Funding: DePauw University
The role of GATC flanking sequences in the methylation efficiency of Escherichia coli DNA adenine methyltransferase
Authors: Castillo Ana , Peterson Stacey
Published: June 2021

Escherichia coli DNA adenine methyltransferase (Dam) methylates the adenine in GATC sequences. The enzyme plays a crucial role in the timing of DNA replication, gene regulation, and mismatch repair. Many genes that Dam regulates are involved in pathogenesis. Previous in vitro studies showed that certain GATC sites were preferentially methylated depending upon the GATC flanking sequences. Sites rich in A/T base pairs tend to be less preferred than those rich in G/C. Poorly preferred GATC sites as determined by in vitro studies are also undermethylated in vivo. The lack of methylation at these sites is often important for the expression of pathogenic genes. Our goal is to determine if GATC flanking sequences affect Dam methylation efficiency at certain sites in vivo. We hypothesize that the same trend will hold true in vivo and G/C rich flanking sequences will be preferred. dam‐minus cells were transformed with a plasmid containing the dam gene under control of the arabinose promoter. After growth at low concentrations of Dam we isolated the plasmid and observed the methylation levels of GATC sites. We found that a GATC with G/C rich flanking sequences was preferentially methylated compared to a consecutive GATC with A/T rich flanking sequences. Our studies will help in understanding how Dam is able to discriminate between GATC sites in vivo which is important for its various roles, particularly gene regulation.
A dual‐kinase mechanism controls APC phosphorylation and dissociation from microtubules during mitosis
Authors: Qian Jiang , Trzepacz Chris , Phiel Christopher , Groden Joanna
Published: June 2021

Tumor suppressor gene APC mutations result in truncated protein products and initiate the development of most colorectal tumors, suggesting that proper APC function requires the presence of the deleted sequences. The APC C‐terminus is phosphorylated by the cyclin dependent kinase CDK1 during mitosis. To investigate the functional sequence of CDK1‐mediated phosphorylation of APC, a GFP‐tagged APC C‐terminus (GFP‐APCR1) containing amino acids 2167‐2843 and two mutants that respectively mimic CDK‐related unphosphorylated (GFP‐APCR1Ala) and phosphorylated (GFP‐APCR1Asp) forms of GFP‐APCR1 were expressed in cultured cells. Overexpressed GFP‐APCR1 associates with the microtubule and arrests cells at the G2/M phase. Bioinformatics analysis and in vitro kinase assays demonstrated that CDK1‐mediated phosphorylation primes APC phosphorylation by GSK3beta. Sequential phosphorylation of APCR1 by CDK1 and GSK3beta decreases interactions with microtubules in vitro and in vivo. Significantly, cotransfection of GFP‐APCR1Asp with wild‐type GSK3beta, but not cotransfection of GFP‐APCR1Asp with kinase‐negative GSK3beta or cotransfection of APCR1Ala with wild‐type GSK3beta, releases cell cycle arrest at the G2/M phase. These data suggest that the dynamic association of APC with the microtubule and consequent cell cycle control are regulated by a dual‐kinase mechanism during mitosis.
CSL ‐ DNA interactions, from thermodynamics to structure
Authors: Kovall Rhett Allen , Friedmann David Robert
Published: June 2021

Canonical Notch signaling ultimately results in changes in gene expression. The transcription factor CSL (CBF1, Su(H), Lag‐1) is a site‐specific DNA binding transcription factor that is central to the regulation of transcription from all Notch responsive genes. Additional regulation arises from DNA elements that contain a pair of CSL binding sites arranged in a head‐to‐head manner, as typified by the HES‐1 (Hairy and Enhancer of Split‐1) promoter. While the DNA consensus‐binding sequence of CSL has been identified and high‐resolution crystal structures of CSL bound to this site have been determined, there is a considerable gap in our understanding of the thermodynamics that define CSL‐DNA complexes. In particular, a quantitative description of the energetics that underlie CSL‐DNA interactions with the HES‐1 site is not available. The overall goal of this study is to thermodynamically and structurally characterize the interaction of CSL with DNA, including its interaction with the HES‐1 element, which contains both consensus and nonconsensus sites. Our calorimetry data suggests that CSL binding to DNA is an entropically driven reaction with a ~200 nM Kd. Our X‐ray structures of CSL bound to a nonconsensus DNA site reveal a number of structural differences when compared to CSL‐consensus DNA complexes. Research supported by a grant from the National Institutes of Health (R01 CA120199).
Mammalian Elongin A complex mediates DNA‐damage‐induced ubiquitylation and degradation of Rpb1
Authors: Aso Teijiro , Kamura Takumi , Kitajima Shigetaka , Conaway Ronald C , Conaway Joan W , Yasukawa Takashi
Published: June 2021

The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II (pol II) by suppressing transient pausing of the pol II at many sites along the DNA. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which can form an isolable Elongin BC subcomplex. Here, we have shown that both the ubiquitylation and proteasomal degradation of the largest subunit of pol II (Rpb1) following UV‐irradiation are significantly suppressed in Elongin A‐deficient cells; however, in both cases suppression is rescued by transfection of wild‐type Elongin A. Moreover, we have demonstrated that the Elongin A‐Elongin BC complex is capable of assembling with the Cul5/Rbx2 module, and that this hetero‐pentamer complex efficiently ubiquitylates Rpb1 in vitro. Mechanistic studies indicate that colocalization of Elongin A and Cul5 in cells and the interaction of Elongin A with the Ser5‐phosphorylated form of Rpb1 are strongly enhanced following UV‐irradiation. Taken together, our results suggest that mammalian Elongin A is directly involved in ubiquitylation and degradation of Rpb1 following DNA damage.
The Extended Loops of Ribosomal Proteins L4 and L22 are Not Essential for Ribosome Function, Cell Survival, or Peptide‐Mediated Translational Arrest
Authors: Lawrence Marlon , Lindahl Lasse , Zengel Janice
Published: June 2021

Ribosomes are the universal organelles responsible for polymerizing amino acids into proteins. Ribosome‐mediated peptide synthesis takes place within the peptidyl‐transferase center of the large subunit of the ribosome. Once synthesized, nascent peptides traverse the ribosome exit tunnel before reaching the extraribosomal environment. Several nascent peptides have been shown to interact with the exit tunnel to stall translation elongation at specific sites within their peptide chain. The exit tunnel is primarily lined by rRNA, but the extended loops of ribosomal proteins L4 and L22 contribute to the lining, forming the narrowest portion of the tunnel. This constriction has also been shown to be important for the action of and resistance to macrolide‐lincomycin‐streptogamin (MLS) antibiotics. Several mutations localized near the constriction of the tunnel have been shown to interfere with nascent peptide‐mediated pausing. Previous studies of MLS antibiotics led to the proposal that the extended loops of L4 and L22 provide a gating mechanism by which the ribosome exit tunnel is narrowed or widened. The ability of these two proteins to adjust tunnel width has been suggested to play important roles in both antibiotic resistance and in regulating the passage of newly formed peptides through the ribosome exit tunnel. Through mutation and the use of translational reporters we have obtained results which show that, contrary to the proposed model, the extended loops of these two proteins are not required for ribosomal function, cell survival, or several cases of peptide‐mediated translational arrest. We also present work showing that different classes of peptides known to stall translation respond differentially to mutations in L4 and L22.
SHAPE footprinting as complementary approach to structure‐based design of ribosomal antibiotics: Phenicol antibiotics prevent A2451 2′OH acylation
Authors: Skripkin Eugene , Franceschi Francois , Duffy Erin
Published: June 2021

Early biochemical experiments suggested that phenyl propanoid antibiotics, particularly chloramphenicol (CAM), had more than one binding site on ribosomes. Indeed recent X‐ray structures have shown two distinct, non‐overlapping, binding sites for CAM bound to 50S ribosomal subunits. The structure of CAM bound to the 50S ribosomal subunit of Haloarcula marismortui shows an overlap of the CAM binding site with that of macrolide antibiotics (e.g. erythromycin, etc). Whereas the structure of CAM bound to Deinococcus radiodurans 50S ribosomal subunits shows an overlaps between the binding site of CAM with that of puromycin or the CCA‐end of A‐site tRNA. We have used SHAPE footprinting to map the location, in solution, of antibiotics, including several novel phenicol analogs, onto 70S ribosomes from Escherichia coli and Staphylococcus aureus. Our data clearly indicate that phenyl propanoid antibiotics are able to simultaneously occupy both binding sites in E. coli as well as in S. aureus ribosomes. Nevertheless, "macrolide‐like" footprints were less reproducible than "puromycin‐like" (A‐site) footprints. We found that phenicol binding to the "puromycin" binding site blocks 2′‐OH hydroxyl modification of nucleotide A2451. Interestingly, this hydroxyl group has been shown to be critical in peptide bond formation, thus our results are shedding more light on the mechanism of phenyl propanoid antibiotic action.
DETERMINANTS OF THE ACTIVITY OF THE MAMMALIAN ANTIOXIDANT SELENOPROTEIN PLASMA GLUTATHIONE PEROXIDASE (GPx‐3)
Authors: Ottaviano Filomena Gabriella , Tang Shiow‐Shih , Handy Diane E. , Loscalzo Joseph
Published: June 2021

GPx‐3 scavenges reactive oxygen species in plasma by catalyzing the reduction of hydrogen peroxide and lipid hydroperoxides using glutathione (GSH) as a reducing co‐substrate. We compared the specific activity of wild type (WT) and Sec73Cys mutant recombinant GPx‐3 (rGPx‐3). rGPx‐3 WT, Sec73Cys, and empty vector cDNAs were transfected in Cos7 cells in the presence of sodium selenite. rGPx‐3 protein was isolated by His‐Gravi Trap purification, determined by Western blot under reducing and non‐reducing conditions, and compared to native plasma GPx‐3. GPx‐3 activity was assessed by a chromogenic assay, a redox‐sensitive fluorescence based endpoint assay, and an in‐gel assay. Western blot demonstrated a 28 kDa band under reducing conditions for WT and Sec73Cys rGPx‐3 and an additional ∼112 kDa band under non‐reducing conditions. An ∼2.5‐fold increase in tetramer‐to‐monomer ratio for Sec73Cys rGPx‐3 was observed compared to WT rGPx‐3. Native plasma GPx‐3 resulted in a tetramer‐to‐monomer ratio of 2.5. The enzyme activity of WT rGPx‐3 was ~3‐fold higher than Sec73Cys with GSH (ANOVA, p<0.01) using the chromogenic assay, and ~2‐fold higher with the redox‐sensitive fluorescence based endpoint assay (ANOVA, p<0.005). The in‐gel assay also showed activity. WT rGPx‐3 is more catalytically active than Sec73Cys. Quaternary structure does not seem to influence intrinsic activity.

Support: HL61795, HL28178, HL81587
Specific HIV‐1 integrase‐reverse transcriptase interactions as a mechanism for promoting reverse transcription
Authors: Chow Samson A. , Wilkinson Thomas A , Zhang Min , Januszyk Kurt , Phillips Martin L , Le Grice Stuart F. J. , Clubb Robert T
Published: June 2021

Integrase (IN) from human immunodeficiency virus‐type 1 (HIV‐1) exerts pleiotropic effects in the viral replication cycle, and IN mutations can impair reverse transcription. We showed previously that IN stimulates both the initiation and elongation modes of reverse transcription by increasing the processivity of reverse transcriptase (RT) and suppressing the formation of the pause products. IN and RT interact in vitro, and the IN C‐terminal domain (CTD) is both necessary and sufficient for binding RT. We used nuclear magnetic resonance spectroscopy to identify a putative RT‐binding surface on the IN CTD, and surface plasmon resonance to obtain kinetic parameters and the binding affinity for the IN‐RT interaction. An IN K258A substitution that disrupts reverse transcription in infected cells is located at the putative RT‐binding surface, and we find that this substitution substantially weakens IN CTD‐RT interactions. Other substitutions at the putative RT‐binding surface also rendered the virus replication‐incompetent. These results strengthen the notion that productive IN‐RT interactions are important for efficient reverse transcription during HIV‐1 replication, and provide insights into this interaction at the molecular level.

This research is supported by NIH R21 AI077386.
Testing the Shifting Specificity Model for Enzyme Catalysis
Authors: Britt Billy Mark
Published: June 2021

The Shifting Specificity model (SSM) for enzyme catalysis explains enzyme catalysis generally and addresses the deficiencies in the Haldane‐Pauling‐Koshland model (HPKM) of enzyme catalysis. The tenets of the SSM are outlined and the results of experiments to test the validity of each model are presented.

Strong ground state/enzyme interactions are positively correlated with catalytic efficiency. Larger enzymes are better catalysts than smaller enzymes. These data are explained by the SSM which calls for a direct role for the entire enzyme molecule in the catalytic event. The HPKM has no such role and maintains that weak ground state interactions are favorable for catalysis.

Many enzymes have two folded conformations. For bovine adenosine deaminase, the low‐temperature (< 30ºC) conformation appears to be better optimized for interaction with the reaction transition state than does the physiological conformation. The SSM requires two global conformations of the enzyme unlike the HPKM which requires only one.

It is argued that the SSM better explains the data than does the HPKM.
Autooxidation Properties of Mycobacterium tuberculosis DevS and Impact of the Y171F Mutation on Gas Binding Kinetics
Authors: Ioanoviciu Alexandra Smaranda , Meharenna Yergalem , Poulos Thomas , Ortiz de Montellano Paul
Published: June 2021

In Mycobacterium tuberculosis, DevS is responsible for DevR activation in conjunction with DosT. Exposure to unfavorable stimuli is translated via the DevS/DevR two‐component system into the expression of genes of the "dormancy regulon." The DevS kinase activity is modulated by the binding of nitric oxide, carbon monoxide, and oxygen to the heme iron. We have cloned and characterized both wild‐type DevS and the Y171F mutant. Mutation of tyrosine 171 to a phenyl alanine was previously shown to impact kinase activity.

In the present study we examine diatomic gas binding kinetics to wild‐type DevS and Y171F DevS using stopped‐flow combined with UV‐visible spectroscopy and laser flash photolysis. The Y171F mutation increases the on‐rates of carbon monoxide and oxygen, probably due to the requirement for displacement of a hydrogen bonded water molecule located in the distal heme cavity of the reduced wild‐type protein.

In addition, we examine the stability of the wild‐type DevS oxy‐complex in the presence of ubiquitous metal cations. Oxy‐DevS is stable to autooxidation, suggesting that it functions as a gas and not redox sensor in vivo.

This work was supported by the National Institutes of Health through grant AI074824.
Metabolism and metabolic effects of anaplerotic triheptanoin (TH) in normal rats
Authors: Gu Lei , Zhang Guofang , Kombu Rajan S. , Kutz Gerd , Brewer Wolf‐Ulrich , Roe Charles R. , Brunengraber Henri
Published: June 2021

Anaplerotic TH (glycerol triheptanoate) is used for the chronic oral treatment of long‐chain fatty acid oxidation disorders (FOD). To test whether TH could also be used for the acute intravenous treatment of decompensated FOD patients, we investigated the metabolism and metabolic effects of triheptanoin emulsion administered parenterally or enterally. Rats were infused with TH emulsion (intravenously or intragastrically). Stable isotope tracers were infused intravenously to measure the rates of appearance (Ra) of plasma glucose, glycerol and fatty acids. Surprisingly, intravenous infusion of triheptanoin markedly increased glycerol Ra but not oleate Ra. This indicated that massive lipolysis induced by intravenous TH was not accompanied by the release of long‐chain fatty acids from adipocytes. This probably results from the contribution of anaplerotic propionyl‐CoA moiety of heptanoate to glyceroneogenesis in adipocytes, generating sufficient amounts of glycerol‐3‐phosphate to re‐esterify the fatty acids derived from stimulated lipolysis. The intraduodenal infusion of TH did not induce lipolysis because TH was hydrolyzed in the gut. Our data support the use of intravenous TH emulsion for the acute treatment of FOD patients (for whom the release of fatty acids from adipocytes would be harmful). Supported by NIH.
Lutein Extraction and analysis from alfalfa germplasm
Authors: Lekies Brittany , Reddy Srinivasa M.S. , Whalen Dave , Temple Stephen J.
Published: June 2021

The leading cause of irreversible vision loss in Americans over 60 is age‐related macular degeneration (AMD). It is a degenerative disease resulting in the loss of central vision. Studies have shown that consumption of lutein can lead to a 40% reduction in AMD. Lutein is a lipid‐soluble member of the xanthophylls family of carotenoids. Lutein is found in the lens and the macula of the eye, but is not synthesized in the human body and must be obtained through one's diet. Currently, there are few sources available for commercial scale extraction, one being marigold petals. The current extraction method for obtaining lutein from marigolds is expensive and does not result in highly pure lutein. To overcome the disadvantages of current methods, this study was conducted to determine if alfalfa could be used as a potential source of lutein. An analytical scale method was established for lutein extraction and detection from alfalfa tissue. The field grown alfalfa plants were harvested and flash frozen in liquid nitrogen. The samples were ground to a powder, extracted, saponified, and analyzed for lutein content using HPLC. The lutein content of the samples ranged from 158 ppm to 285 ppm. Two alfalfa commercial varieties were found to contain statistically significantly higher lutein content than 25 other tested varieties. The method developed can be used as a selection tool in choosing alfalfa plants containing high levels of lutein.
Impaired anaplerosis in VLCAD (very long chain acyl‐CoA dehydrogenase) deficient mouse hearts
Authors: Gélinas Roselle , Bouchard Bertrand , McDuff Janie , Charron Guy , Labarthe François , Des Rosiers Christine
Published: June 2021

Current evidence suggests the benefit of a diet containing anaplerotic odd‐chain medium chain fatty acid (MCFAs) for cardiac symptoms in patients with long chain fatty acid oxidation defects (Roe & coll. JCI, 110(2):259‐69, 2002), although the underlying mechanism remains unclear. We conducted a study to evaluate the anaplerotic status of VLCAD−/− mouse hearts, using our working mouse heart model and 13C‐methodology. Furthermore, the expression of selected anaplerotic genes (using real time PCR), and the myocardial levels of CAC intermediates were assessed in fed and 24h fasted mice. Compared to controls VLCAD+/+, 3 month‐old VLCAD−/− hearts showed a 40% lower anaplertoic flux through pyruvate carboxylation (p<0.05). Furthermore, VLCAD−/− hearts displayed lower mRNA levels for enzymes involved in anaplerosis, namely pyruvate and propionyl‐CoA carboxylase, but not malic enzyme, when compared to their normal counterparts, changes that were however age‐ and condition‐dependent. Besides, two‐way Anova revealed a species effect on CAC intermediates levels, specifically succinate and malate, which were lower in VLCAD−/− hearts from 7 month‐old mice. Collectively, our data highlight, impaired anaplerosis in VLCAD−/− mouse hearts, suggesting a potential beneficial role for odd‐chain MCFAs. (Supported by NIH & CIHR)
The role of translational regulation in ultraviolet light‐induced cyclooxygenase‐2 expression
Authors: Carpenter Oliver Luke , László Csaba , Fayad Sherine , Wu Shiyong
Published: June 2021

Ultraviolet light (UV) induces a prolonged expression of Cyclooxygenase‐2 (COX‐2). While transcriptional regulation of COX‐2 expression is intensively studied, the role of translational regulation of COX‐2 synthesis upon UV‐irradiation is not yet clear. Through the use of Ingenuity Pathway Analysis(tm) software's Canonical Pathway Analysis, we proposed a novel alpha subunit of the eukaryotic initiation factor 2 (eIF2α) phosphorylation‐centered network for the regulation of COX‐2 expression after UV‐irradiation. Our data shows that UV light induces COX‐2 expression in wild‐type mouse embryo fibroblasts (MEFS/S) and that the inducibility is reduced in MEFA/A cells in which the phosphorylation site, Ser‐51 in the eIF2α, is replaced with a nonphosphorylatable Ala (S51A). Our data also shows that translational efficiency of COX‐2 is higher in MEFA/A cells than in MEFS/S cells, but not at the late stage of UV‐irradiation. This observation correlates to the translational regulation of COX‐2 binding protein T‐cell‐restricted intracellular antigen 1‐related protein (TIAR) expression, which is reduced in MEFS/S cells but not in MEFA/A cells at 24 hours post‐UV. In addition, our data indicates that newly synthesized COX‐2 protein is more stable in MEFA/A cells than in MEFS/S cells. These results suggest that translation initiation plays a role in a complex and dynamic regulation of COX‐2 expression. By utilizing Ingenuity Pathway Analysis(tm) software, we were able to analyze our results with respect to that of previously published research and deduce a pathway for the translational activation and regulation of COX‐2 expression. This work was supported by National Institutes of Health Grant RO1 CA86926 (to S. W.) and R56 CA086928 (to S. W.).
Insulin Enhances Posttranslational Processing of Nascent SREBP‐1c by Promoting its Phosphorylation and Association with COPII vesicles
Authors: Yellaturu Chandrahasa Reddy , Deng Xiong , Cagen Lauren M , Wilcox Henry , Park Edwards A , Raghow Rajendra , Elam Marshall B
Published: June 2021

The regulation of lipid homeostasis by insulin is mediated in part by the enhanced transcription of the gene encoding the sterol regulatory element‐binding protein‐1c (SREBP‐1c). Nascent SREBP‐1c is synthesized and embedded in the endoplasmic reticulum (ER) and must be transported to the Golgi in coatomer protein II (COPII) vesicles where two sequential cleavages generate the transcriptionally active N‐terminal fragment, nSREBP‐1c. There is limited indirect evidence to suggest that insulin may also regulate the posttranslational processing of the nascent SREBP‐1c protein. Therefore, we designed experiments to directly assess the action of insulin on the posttranslational processing of epitope‐tagged full‐length SREBP‐1c and SREBP‐2 proteins expressed in cultured hepatocytes. We demonstrate that insulin treatment led to enhanced phosphorylation of ER‐bound nascent SREBP‐1c protein that was associated with increased affinity of the SREBP‐1c cleavage‐activating protein (SCAP)/SREBP‐1c complex for the Sec23/24 proteins of the COPII vesicles. Furthermore, chemical and molecular inhibitors of the phosphoinositide 3‐kinase (PI3‐kinase) pathway and its downstream kinase PKB prevented both insulin‐mediated enhanced phosphorylation of the nascent SREBP‐1c protein and its posttranslational processing. Insulin had no effect on the proteolysis of the nascent SREBP‐2 under identical conditions. Thus, insulin selectively stimulates the processing of SREBP‐1c in rat hepatocytes by enhancing the association between the SCAP/SREBP‐1c complex and COPII proteins. The effect of insulin is tightly linked to PI3‐kinase and PKB‐dependent serine phosphorylation of the precursor SREBP‐1c protein.
Apoptosis of Breast Cancer Cells by D‐Threo‐PDMP and cis‐Platin: Translational and Transcriptional Regulation of Glycosyltransferase Genes
Authors: Basu Subhash , Loucks Audrey J , Dang Michelle E , Blasco Michael A , Kang Jennifer , Ma Rui , Basu Manju , Moskal Joseph R , Banerjee Sipra
Published: June 2021

The functions of glycosphingolipids on the eukaryotic cell membranes during the onset of oncogenic processes and cell death are not well understood. Inhibitors of glycosphingolipid biosynthesis (L‐PPMP and D‐PDMP) and DNA‐biosynthesis inhibitor (cis‐platin) were recently found to trigger apoptosis in human breast carcinoma cells (SKBR‐3, MCF‐7, and MDA‐468) through either intrinsic or extrinsic apoptotic pathways. These anti‐cancer inhibitors increase ceramide concentration. Apoptotic effects in these cells were determined by activationof caspases (‐3, ‐8, and ‐9) and fluorescent studies using PS‐binding PSS‐380 dye, and intracellular membrane‐binding AKS‐O dye. A time‐dependent scrambling of intracellular membranes was observed after treatment with D‐PDMP upto 6‐ hours. The neolacto‐series glycolipid (SA‐LeX) is distributed mostly on the outer lamella of plasma membranes of human metastatic breast cancer cells. The direct analyses of these glyco‐molecules suggest they alter their structures during the onset of oncogenic processes. Regulation of glycosyltransferase (GLTs) activities involved in the biosynthesis of SA‐LeX is reported in cells treated with L‐PPMP. DNA‐microarrays designed for screening over 340 Glyco‐related genes, transcriptional up‐regulation of several GLTs (in the biosyntheses of Sialo‐LeX and Sialo‐Lea ) was observed with L‐PPMP. GLT‐ activities and gene expressions will be compared with the cells treated with D‐PDMP. Down‐regulation of GLT activities and up‐regulation of some GLT mRNA suggest a tight regulation of these enzymes by signal transduction pathways. 11
Low Concentrations of Hydrogen Peroxide Induce Altered Physiology of Human Diploid Fibroblasts
Authors: Davis Willie , Brantley Eileen , Marks Brandy , Garispe Emily , Whitley Rebecca , Lucinian Tania , Amis Louisa
Published: June 2021

The objective of this study was to determine the physiological effects of low‐dose hydrogen peroxide (H2O2) treatments on the cellular physiology of GM00701, GM01354, GM01385, MRC‐5, and MRC‐9 human diploid fibroblast cell lines. A number of published reports have shown short‐term treatments of fibroblasts with H2O2 concentrations of 100 uM to 200 uM result in apoptosis. However, our preliminary studies show that 5.0 uM to 25.0 uM concentrations of H2O2 are capable of inducing apoptosis in the cell lines that were studied. One‐hour treatments of fibroblasts with these H2O2 concentrations were shown to result in statistically significant decreases in cell number. The relative cell numbers are measured 24 hours after treatment by Alamar Blue cytotoxicity assay. We also used flow cytometry to analyze the effects of H2O2 treatment on cell cycle progression in a time‐dependent manner 12 and 24 hours post‐treatment. We find a 1.5‐ to 3‐fold increase in sub G1 cells 24 hours after treatment when compared to untreated cells. The results are cell line specific. To confirm these findings, studies are currently underway to assess the effects of H2O2 treatment on intracellular reduced GSH concentrations and Poly (ADP‐ribose) polymerase (PARP) cleavage. We will assess the effects of sub‐lethal doses of H2O2 on the metabolism of low density lipoprotein (LDL) and oxidized low density lipoprotein (ox‐LDL) by flow cytometry. We will also assess the effects of sub‐lethal doses of H2O2 on cytokine‐induced IL‐6 and MCP‐1 secretion by ELISA. From our studies, we conclude that low levels of oxidative stress can have significant effects on the physiology of human fibroblasts.
Activated protein C protects against Pseudomonas aeruginosa‐induced pulmonary endothelial leak in vitro via PAR1 and the S1P1 pathway.
Authors: Bir Natasha Dinker , Howard Marybeth , Miyazawa Byron , Pittet Jean‐Francois
Published: June 2021

Ninety percent of Pseudomonas aeruginosa pneumonias culture strains produce Type III secretion proteins, and 40% produce both exotoxins S and T. PAK strain is a clinical isolate of P. aeruginosa that produces exoS and exoT (but not exoU) and causes endothelial leak in vitro via inhibition of the Rac1 and activation of the RhoA GTPases. Activated protein C (aPC), via binding to endothelial protein C receptor (EPCR), enhances endothelial monolayer integrity via a Rac‐1 dependent mechanism. We determine here whether pretreatment with aPC would protect against a P. aeruginosa‐mediated increase in lung endothelial permeability. Pulmonary artery endothelial monolayers were pretreated with aPC or vehicle for one hour prior to exposure to P. aeruginosa. The results showed that aPC confers protection from PAK‐induced leak, with maximal effect at 1ug/mL. APC's protective effect was mediated by protease activated receptor‐1 (PAR1) and sphingosine‐1‐phosphate‐1 (S1P1) pathway‐dependent mechanisms. Inhibition of the S1P1 receptor, sphingosine kinase 1, or phospho‐inositide‐3‐kinase resulted in a loss of aPC's protective effects. Furthermore, use of an S1P1 receptor agonist conferred protection comparable to that observed with aPC. These results represent the first experimental evidence that aPC protects against Pseudomonas‐induced endothelial leak via PAR1 and the S1P1 pathway. [Research support: NIH T32GM08258‐21 (NDB), NIH GM62188 (JFP)]
PAC‐1: A novel therapeutic approach to procaspase activation
Authors: Peterson Quinn Patrick , Hsu Danny , Goode David R , West Diana C , Ramsey Kara N , Hergenrother Paul J
Published: June 2021

The recent emergence of proapoptotic small molecules as powerful anti‐cancer agents allows for the direct induction of apoptotic pathways. Central to these pathways is the activation of executioner caspases responsible for the proteolysis of a host of cellular substrates. This vital role presents caspases as a promising new target in the search for new chemotherapeutic agents.

In a screen for small molecule activators of procaspase‐3, a novel compound, PAC‐1, was identified [Nature Chem. Biol. 2006, 2, 543]. The compound was shown to activate procaspase‐3 to caspase‐3 in vitro, kill cancer cells in culture and inhibit tumor growth in mouse xenograft models. Current work has elucidated the in vitro mechanism of action of PAC‐1 mediated activation of procaspase‐3, and has explored the effect of metal ions on procaspase activation. In addition, to further investigate the PAC‐1 mechanism of action we synthesized several derivatives to probe the structure‐activity relationship of this family of compounds. The ability of these derivatives to activate PAC‐1 sheds further light on the PAC‐1 mechanism of action and suggests means for the chemical optimization of this anti‐cancer agent.
Use of Artificial Nipple (Pacifier or Bottle) and Duration of Breastfeeding in Term Infants
Authors: Chung Mei , DellaValle Diane M , Yu Winifred W
Published: June 2021

Background

Breast milk is the natural nutrition for all infants. Evidence suggests that breastfeeding (BF) decreases various disease risks for infants and mothers.

Methods

Systematic review (SR) of all English‐language randomized controlled trials (RCTs), prospective cohorts (PCs), and SRs examining the use of artificial nipples and outcomes of BF duration in term infants (=35 weeks of gestation) published from 1985 to March 2008.

Data Synthesis

A total of 12 primary studies and 1 positive‐quality SR (with 9 PCs) were analyzed. Seven PCs consistently showed that use of pacifier was associated with shorter BF duration after controlling for potential confounders (adjusted rate ratio/hazard ratio of shorter BF duration ranged from 1.4 to 2.9 across studies), although the definitions of pacifier use varied across these studies. This is consistent with the findings from the prior SR. Additionally, the results from 2 RCTs and 3 cohort studies suggested that supplementary feedings (formula, water/tea, or expressed breast milk) using bottles or cups shorten BF duration compared to no supplementary feeding.

Conclusions

Evidence suggests a negative impact of artificial nipple on the duration of all types of BF (from partial to exclusive). Observational evidence consistently showed an association between use of pacifier before 3 months of age and shorter BF duration in healthy term infants after controlling for potential confounding. Whether increasing frequencies or introduction of artificial nipple use beyond 3 months of age has differential impacts on BF duration is still unclear. Well‐designed RCTs with blinded assessments of BF outcomes and specifying the amount and frequency of artificial nipple use are needed to further support the validity of these findings.

Grant Funding Source

ADA Evidence Analysis Library
Resting energy expenditure of female adolescents with phenylketonuria: agreement of predictive equations
Authors: Quirk Meghan Elise , Schmotzer Brian , Singh Rani H.
Published: June 2021

Objective

To determine the agreement of clinically‐used, predictive equations with measured resting energy expenditure (MREE) in a group of female adolescents with phenylketonuria (PKU).

Methods

MREE was measured by indirect calorimetry in 36 female adolescents (11.5‐18.7 y) with PKU. Eight predicted resting energy expenditure values were calculated from published equations for each subject and compared to MREE. The level of agreement was determined using Bland‐Altman and regression‐based methods.

Results

MREE was greater than predicted values, on average, for each equation (each p<0.005). The Schofield equation based on weight had the lowest mean underestimation of MREE (144 kcal), the largest concordance coefficient (0.626), and the most observations within +/‐10% of MREE (38.9%).

Conclusions

Predictive equations underestimate MREE in female adolescents with PKU. Of the eight equations evaluated, the Schofield equation based on weight had the highest level of agreement with MREE. Accuracy at the individual level was affected by systematic bias and interpersonal variability.

Funded by private donations and Emory University Hospital General Clinical Research Center.
Macronutrient composition of breakfast influences plasma glucose, satiety hormones and caloric intake in the next 24 h in adult men.
Authors: Ratliff Joseph C , Leite Jose O , DeOgburn Ryan , Puglisi Michael J , Ata Shymaa , Fernandez Maria Luz
Published: June 2021

We examined the effect of the macronutrient composition of breakfast on postprandial satiety and caloric intake throughout the day. Using a crossover design, 22 men aged 20‐70 y, consumed two isocaloric test breakfasts in a random order separated by one wk. The macronutrient composition of the breakfasts were: EGG: % CHO:fat: protein = 22:55:23 or BAGEL: % CHO:fat:protein = 72:12:16. Fasting blood samples were drawn at baseline before the test breakfast and at 30, 60, 120, and 180 min after breakfast. After 180 min, subjects were given a standard lunch and asked to eat until satisfied. Subjects filled out visual analog scales (VAS) during each blood draw and recorded food intake the day before and after test days. Plasma glucose and appetite hormones were analyzed at each time point. Subjects consumed fewer calories following the EGG breakfast compared to the BAGEL breakfast (P < 0.01). Additionally, subjects consumed more calories in the 24h period after the BAGEL breakfast compared to the EGG breakfast (P < 0.05). Based on VAS, subjects were hungrier and less satisfied 3h after the BAGEL breakfast compared to the EGG. In addition, subjects had lower plasma glucose 3h post BAGEL (P < 0.05) and higher plasma ghrelin concentrations 30 min post BAGEL (P < 0.05) compared to post EGG. These findings suggest that consumption of a protein‐rich breakfast (EGG) results in less variation in plasma glucose levels, lower acute ghrelin response at 30 min and a reduced caloric intake [Supported by the Egg Nutrition Center]
Red blood cell fatty acid profiles in risk prediction of nonfatal myocardial infarction: a case‐control study in Korea
Authors: Park Yongsoon , Lim Jeehyun , Lee Jaeung
Published: June 2021

The purpose of this study was to examine the hypothesis that lower in n‐3 fatty acids and higher in trans fatty acids in red blood cell (RBC) were associated with the increased risk of MI, and fatty acid profiles could discriminate MI cases from controls as compared with established risk factors. Fifty cases with a first nonfatal MI and fifty age‐ and sex matched controls without MI were recruited. The Omega‐3 Index (the sum of EPA and DHA in RBC) was significantly lower in cases than controls (9.57 ± 0.28% vs. 11.81 ± 0.35%, P < 0.001), while total trans fatty acids were significantly higher (1.01 ± 0.04% vs. 0.56 ± 0.03%, P < 0.001). Omega‐3 Index was associated with decreased risk (OR 59.0; 95% CI: 0.03, 0.09; P = 0.001), while total trans fatty acids were associated with increased risk (OR 59.0; 95% CI: 11.32, 307.51; P < 0.001). The area under the receiver operating characteristic curve of fatty acid profiles was larger than traditional factors, suggesting that fatty acid profiles contributed significantly to the discrimination of MI case from controls as compared with the modified Framingham risk factors. In conclusion, higher Omega‐3 Index and lower trans fatty acids of RBC were associated with decreased risk of nonfatal MI, and fatty acid profiles improved discrimination of nonfatal MI as compared with established risk factors.
Use of short duration breast milk pump‐expression to predict 24‐hr milk output
Authors: Aryeetey Richmond N O , Lartey Anna , Marquis Grace S
Published: June 2021

Test weighing (TW) is traditionally used to assess adequacy of infant breast milk (BM) intake. TW however intrudes on the breastfeeding process. We compared a brief BM expression procedure with 24‐hour TW among 20 rural and peri‐urban dwelling Ghanaian lactating women. TW was performed using standard procedures and milk intake was recorded by trained personnel in participants' homes. Within 48 hours of the TW, the brief procedure was carried out, involving hourly pump‐expression of BM over a 4‐hr duration. Total 24‐hr BM output in TW was compared to hourly expressed BM, multiplied by 24. Study participants were between 18 and 38 y, with a median age of 25 y. One half of the 20 participants were breastfeeding exclusively; the remaining practiced mixed breastfeeding. Average intake of 53 g (95% CI: 48, 58) per feed was recorded in TW with a range of 7 to 19 feeds per infant. BM intakes did not differ between feeds. Hourly pump‐expressed BM averaged 34 g (95% CI: 28, 41) and did not differ across breasts nor between women but decreased over time (p<0.01). Mean BM intake during 24‐hr TW was 598 + 230 g and did not differ by infant feeding. Mean 24‐hr TW intake was predicted by 4th hr pump‐expressed BM from the left breast only multiplied by 24 (95% CI: ‐39, 303 g) but not by the BM from the right breast (95% CI: 178, 465 g). Breast preference may affect the usefulness of the brief pumping method to estimate BM intake of young children.