bluuebunny/crossref_metadata_2025 huggingface dataset:
PAS Kinase Regulates Hepatic Lipid Metabolism through Activating SREBP‐1
Authors: Wu Xiaoying , Dorweiler Irene , Walkup Addie , Rutter Jared
Published: June 2021

PAS kinase (PASK) is a serine/threonine protein kinase conserved from yeast to human. PASK activity has been shown to be controlled by nutrient status, but its roles in regulating metabolism remain largely unclear. In order to study the function of PASK in mammals, we used PASK deficient (PASK−/−) mice as a model system. PASK−/− mice showed decreased liver accumulation of triglyceride induced by a high‐fat diet. This was accompanied by lower expression levels of glycerol‐3‐phosphate acyltransferase 1 (GPAT1), stearoyl‐CoA desaturase 1 (SCD1) and fatty acid elongase (FAE), three important enzymes responsible for biosynthesis of fatty acids and triglyceride. We also observed the down‐regulation of these genes upon PASK knockdown in cultured HepG2 (Hepatocellular carcinoma, human) cells. The expression of GPAT1, SCD1 and FAE are regulated by a family of transcription factors called sterol regulatory element binding proteins (SREBPs). Our data suggests that PASK promotes the activity of SREBP‐1, probably by enhancing the processing of the SREBP‐1 precursor to form the active nuclear form. We are now trying to determine the detailed mechanisms of how PASK regulates the processing step. These studies will further our understanding of the physiological roles of PASK, and hopefully provide a potential therapeutic target for metabolic diseases.
Structure‐based pharmacophore sampling of PPARγ
Authors: Garcia Zulma , Lewis Nikki , Bassaganya-Riera Josep , Bevan David
Published: June 2021

The nuclear receptor peroxisome proliferator‐activated receptor‐gamma (PPARγ) is a ligand‐activated transcription factor that plays an important role in many physiological processes, such as glucose homeostasis. Various ligand types affect the recruitment of accessory proteins, leading to differences in transcriptional regulation. The goal of this project was to compare PPARγ conformational differences induced by various ligands to identify interactions that are conserved or distinct among ligand types (e.g., full vs partial agonist). Two computational molecular docking tools were used to explore atomic interactions, binding geometry, and binding affinity between known ligands and PPARγ. AutoDock Vina was used to assess reproducibility of known ligand binding poses to assess prediction of binding geometry and affinity. Redocking results showed favorable pose prediction for 20 out of the 30 structures analyzed. AutoLigand was used to generate pharmacophore models by using a grid‐based representation of binding potentials. These pharmacophore “envelopes” represent potential atomic centers for ligand atoms. Overlapping envelopes allowed us to identify the most favorable binding features, which were then used to aid in a large‐scale screen for novel potential agonists. NIH‐NIDDK grant 1F31DK091186‐01A1 awarded to DRB and SNL.
Role of the β4 nicotinic receptor subunit in the anti‐addictive activity of 18‐methoxycoronaridine
Authors: Mukhida Samir , Arias Hugo R.
Published: June 2021

The ibogaine analog 18‐methoxycoronaridine (18‐MC) is known to reduce the self‐administration of several addictive drugs. It is proposed that this anti‐addictive activity is mediated by the noncompetitive inhibition of β4 nicotinic acetylcholine receptors (AChRs) expressed in the habenulo‐interpeduncular pathway, a brain reward circuit that is known to contribute to the process of drug addiction. To test this, we studied the role of the β4 nicotinic subunit in the process of drug addiction and in the anti‐addictive activity of 18‐MC by using forced swim tests on mutant mice. We first compared the results between cocaine and saline treatments between wild type (β4+/+) and mutant (β4−/−) male and female mice, and subsequently determined the anti‐addictive effect of 18‐ MC on cocaine‐induced locomotor sensitization between wild type and mutant mice from both sexes. The results indicate that the presence of the β4 subunit in males causes a lower cocaine induced total locomotor activity, but higher peak sensitization. Female mice expressing the β4 subunit have a higher peak activity 15 min after cocaine injections compared to male mice, indicating that the anti‐addictive effect of 18‐MC is more pronounced in male mice. In conclusion, the presence of the β4 subunit increases the anti‐cocaine effect of 18‐MC, confirming its modulatory role in drug addiction and the potential therapeutic relevance of the β4 subunit.
FK962 induces neurite elongation in cultured monkey trigeminal ganglion cells
Authors: Nakajima Emi , Walkup Ryan D , Shearer Thomas R , Azuma Mitsuyoshi
Published: June 2021

LASIK refractive surgery is widely accepted for correcting myopia. Creation of the corneal flap in LASIK surgery can lead to dry eye due to amputation of corneal sensory nerves. Previous studies in the hippocampus showed that a substituted fluorobenzamide drug, FK962, caused release of neurotrophic peptides that stimulate and control nerve activity. Our studies in rabbits also showed that FK962 accelerated extension of neuronal processes and recovery of corneal sensitivity. The purposes of the present study were to 1) determine if FK962 induces spouting and elongation of neurites in cultured monkey trigeminal ganglion (TG) cells, and 2) investigate involvement of the neurotrophic peptide GDNF in FK‐induced neurite elongation. Dissociated, cultured TG cells, containing neuronal cells and Schwann cells, were cultured for 48 hours with or without FK962. Neuronal length and elongation were evaluated by staining with antibody for neurofilament specific to neurons. Culture with GDNF, or with antibody against GDNF, was used to determine the role of GDNF in neurite elongation. Our results showed that FK962, or GDNF alone, significantly induced neurite elongation. GDNF antibody significantly inhibited neurite elongation induced by FK962 or GDNF. Our data suggested that GDNF was a mediator of FK962‐induced neurite elongation. FK962 might be a candidate drug for treatment of complications after LASIK.

Dr. Shearer receives consulting fees from, and Drs. Azuma and Nakajima are employees of, Senju Pharmaceutical Co. Ltd.
Regulation of activation of Rac1 and Cdc42 GTPases in CHRF‐288‐11 cells
Authors: Bhullar Rajinder Pal , Xu Bing
Published: June 2021

Rac1 and Cdc42 are members of the Rho family of small GTPases and have been shown to promote the formation of lamellipodia and filopodia at the leading edge of motile cells and thus affect cell migration. In the current study we have investigated the activation of Rac1 and Cdc42 by thrombin or collagen in the megakaryocytic cell line, CHRF‐288‐11. Maximal activation of Rac1 by thrombin or collagen was observed at 3 min and 1 min respectively. Similar results were obtained for the thrombin or collagen mediated activation of Cdc42. The calmodulin specific inhibitor, W7, abolished the thrombin or collagen mediated activation of Rac1 in CHRF‐288‐11 cells. However, W7 had no effect on the activation of Cdc42 by thrombin or collagen. The less potent calmodulin inhibitor, W5, did not have any effect on Rac1 or Cdc42 activation by thrombin or collagen. Transient over‐expression of calmodulin in CHRF‐288‐11 cells increased the basal and thrombin mediated activation of Rac1 when compared to control but had no effect on the basal and thrombin mediated activation of Cdc42 when compared to control. The results demonstrate a role for calmodulin in the activation step of Rac1 in CHRF‐288‐11 cells. This is similar to the results for Rac1 regulation in platelets and suggests that the CHRF cells can serve as a model for platelets. This work was supported by a grant to RPB from the Heart & Stroke Foundation of Manitoba.
Scalable Fluorescence Microscopic Assays of Organelle Transport and Mitochondrial Swelling
Authors: Gerencser Akos A , Nicholls David G , Brand Martin D
Published: June 2021

Fluorescence microscopic tools are widely used for assessing mitochondrial biology, however quantitative assays of parameters such as organelle dynamics are not trivial. High‐content imaging systems established the need for scalable assays that are robust and are able to evaluate biological parameters in an automated, unsupervised manner. We introduce here a software platform implementing novel image processing techniques allowing low light level but robust determination of mitochondrial swelling and organelle motion in cultured cells. Mitochondrial swelling is measured by the ratio of fluorescence intensities in high over low frequency spatial band pass filtered copies of the same image. This ratio is highly sensitive to mitochondrial swelling, but insensitive to fission or fusion, motion and overlaps of mitochondria. Organelle transport is assayed by optical flow, featuring instantaneous velocity determination from a pair of images by detecting motion of edges. Optical flow provides velocity vectors; therefore anterograde, retrograde transport and local, wiggling motion can be distinguished. Both assays are scalable, applicable for imaging in microplates and are accessible through a user friendly interface.
Optimizing peptide yields from human hair
Authors: Steenstra Joshua Adam , Clifford Patricia , Parker Glendon
Published: June 2021

Hair is physically robust and persists in the environment for long periods. It is a common component of crime scenes, yet it is not often analyzed because the DNA information is absent or difficult to obtain. Hair, a complex biological connection between the crime scene and the suspect, is now only rarely used in crime scene investigation. This project seeks to examine the use of protein in developing a measure of identity, we currently have a power of discrimination of 1 in 15,500. The degree of identity between an individual and a hair sample is a function of proteomic information obtained.

The purpose and question of our research is to determine if there is a more efficient way to optimize protein yields from hair. To maximize the surface area to volume ratio, a hair is ground into powder. The sample is then digested and a fraction of the sample is then loaded onto a HPLC and a profile is measured, both total absorbance and peak complexity. This corresponds to how many different polypeptides and how much polypeptide is present in the sample. A range of conditions will be tested to optimize the protocol. We have already determined that mass spectrophotometry compatible detergents increase peptide yield. We anticipate developing a robust protocol that optimizes peptide yields from hair samples. This will maximize the amount of information from each sample to achieve the greatest attainable degree of identity that is consistently reliable. This methodology will prove useful in optimizing the degree of information extracted from forensically relevant material.
High‐Throughput, Single‐Step Purification of Affinity‐Tagged Protein Complexes
Authors: LaCava John , Hakhverdyan Zhanna , Fenyo David , Domanski Michal , Oroskar Asha , Oroskar Anil , Hough Loren , Jensen Torben Heick , Chait Brian T. , Rout Michael P.
Published: June 2021

Most strategies for the capture of protein‐protein interactions by affinity chromatography still follow a trial and error approach to identify best conditions for interaction partner co‐precipitation. Moreover, in the case of some model systems (e.g. mammalian cell culture, animal tissue, etc.), the cost of obtaining enough material for extensive testing can be prohibitively expensive. Our lab has developed and successfully applied a high‐throughput, 96‐well protein affinity chromatography protocol to the yeast S. cerevisiae. We also demonstrate that this protocol is compatible with human cell culture. Our procedure requires a minimal amount of cell material per purification (~10–50 x less than typically used in a bench‐scale experiment), producing quantities of purified protein complexes that can be visualized by Coomassie blue stain even in the case of proteins of modest abundance (~1000 copies/cell). By testing 96 extraction conditions at a time we are able to greatly expand the number of known parameters affecting protein‐protein interactions. This approach increases the chances and reduces the time to find multiple instances of distinct protein complexes containing the tagged protein of interest, and should prove a useful tool for revealing protein‐protein interaction networks. The procedure is performed in a microtiter plate format, and so, is amenable to automation. NCRR ‐ 2U54 RR022220‐07
AP‐3‐dependent peroxiredoxin 6 trafficking to lamellar bodies in pearl mice
Authors: Sorokina Elena M. , Dodia Chandra , Yu Kevin , Hong NanKang , Zhang Peggy , Guttentag Susan , Fisher Aron B.
Published: June 2021

Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A2 (aiPLA2) activities, participates in lung surfactant phospholipid (PL) turnover and protects lung epithelium from oxidative stress. Prdx6 has been localized to cytosolic and acidic compartments (lamellar bodies and lysosomes) in lung epithelium. Prdx6 null mice accumulate PL in lung tissue and have enlarged lamellar bodies, compatible with the role of PLA2 activity in lung phospholipid turnover. Western blot analysis indicated near total absence of Prdx6 in lamellar bodies isolated from Pearl mice, a naturally‐occurring model of Hermansky Pudlak syndrome (HPS), suggesting that the Pearl mutation (AP‐3) abolishes Prdx6 organellar targeting. Immunofluoresce of isolated alveolar type II cells showed co‐localization of Prdx6 with LAMP1, a marker of lysosome‐related organelles, in wild type but not in Pearl cells. Immunogold electron microscopy confirmed the absence of Prdx6 expression in lamellar bodies of Pearl mouse lung. These findings indicate that Prdx6 targeting to lysosome‐related organelles in mouse lung epithelium requires AP‐3 and suggest that absence of Prdx6 in lamellar bodies may be responsible for phospholipid accumulation associated with HPS. [HL 19737]
Myosin‐II is Necessary for Septum Formation in Aspergillus nidulans
Authors: Wang Xiao , Du Wenbin , Hoge Brianna L. , Jackson-Hayes Loretta , Hill Terry W.
Published: June 2021

Cytokinesis in filamentous fungi involves the progressive inward growth of the cell wall and plasma membrane, resulting in a series of functionally connected multinucleate compartments, delimited by crosswalls termed septa. The process of septation, despite intensive study, is still incompletely understood. By screening chemically mutagenized strains of the model fungus Aspergillus nidulans, we have identified several isolates with impaired septum formation, which complementation studies using previously‐identified Aspergillus nidulans “sep” mutations show to be mutations in novel loci. Here we report that the mutation in one of these strains (RCH‐2) occurs in the gene that encodes the fungal homologue of mammalian myosin II (MyoB, AN4706). Sequencing of the MyoB allele in the RCH‐2 mutant reveals a point mutation that is predicted to result in a glycine‐to‐aspartate amino acid substitution at residue 843 in the myosin‐II converter domain. This residue is conserved in all fungal, plant, and animal myosin‐II sequences that we have examined. Down‐regulation of wild‐type MyoB expression under control of the A. nidulans AlcA promoter replicates the septation‐minus phenotype of the RCH‐2 mutation. A GFP‐tagged version of MyoB localizes to constricting septal rings in a manner consistent with the protein's involvement in the process of septation.
Interaction between NFATc1 and STAT3 is required for thrombin‐induced cyclin D1 expression in vascular smooth muscle cells
Authors: Kundumani-Sridharan Venkatesh , Van Quyen Dong , Subramani Jaganathan , Singh Nikhlesh K , Rao Gadiparthi N
Published: June 2021

Towards understanding the mechanisms by which nuclear factors of activated T cells (NFATs) mediate cell proliferation and migration, here we demonstrate that NFATc1 exists in complex with signal transducer and activator of transcription‐3 (STAT‐3) in quiescent vascular smooth muscle cells (VSMCs). In response to thrombin, a G protein‐coupled receptor agonist, both NFATc1 and STAT‐3 bound to cyclin D1 promoter and mediated its activity in a cooperative manner. Furthermore, while transposing their binding sites in the promoter region did not affect either their binding to the promoter or the promoter activity, moving these sites closer to each other negated these effects. In addition blockade of either NFATc1 or STAT‐3 activation signaling events inhibited cyclin D1 expression in VSMCs as well as the migration and proliferation of these cells. Thus, we demonstrate for the first time a crosstalk between NFATc1 and STAT‐3, whose activation mechanisms are dependent on opposite posttranslational modifications, in the induction of expression of cyclin D1 in VSMCs and thereby in enhancing the migration and proliferation of these cells.
Insecticide Toxicity and Characterization of Cytochrome P450 CYP6BB1 and CYP6P10 in Aedes sollicitans Larvae
Authors: Suwanchaichinda Chansak , Brattsten Lena B
Published: June 2021

Cytochrome P450 monooxygenases (P450s) are involved in biotransformation of endogenous and exogenous compounds, including insecticides, which often result in insecticide resistance in insects. Two P450‐encoding genes, CYP6BB1 and CYP6P10, were identified in Aedes sollicitans, an Eastern Equine Encephalitis vector. We examined mosquitoes collected in Atlantic County, New Jersey, where multiple insecticides have been used. We found that pre‐exposure of the mosquito larvae to pentamethylbenzene (PMB) significantly decreased the toxicity of carbaryl, but not that of spinosad. Gene expression of CYP6BB1 was slightly up‐regulated upon induction by PMB, whereas CYP6P10 remained unchanged. We also compared the Atlantic mosquitoes to another population collected from a location on Long Island where insecticides have not been used. We found that the gene expression level of CYP6P10 in the Long Island population was considerably higher than that in the Atlantic population. The gene expression level of CYP6BB1 was also higher in the Long Island mosquitoes. We concluded that PMB induced P450s that detoxified carbaryl. Whether these two genes are involved in the insecticide detoxification in the mosquitoes will require further studies. (Supported by Rutgers Hatch Fund)
Peripheral Ligand Binding and Allostery in Cytochrome P450 3A4
Authors: Davydov Dmitri R , Rumfeldt Jessica A. O. , Sineva Elena V , Davydova Nadezhda Y. , Halpert James R.
Published: June 2021

The mechanisms of ligand binding and allostery in the major human drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, Fluorol‐7GA (F7GA), as a model substrate. Incorporation into the enzyme of an SH‐reactive FRET donor, pyrene iodoacetamide (PIA), allowed us to use FRET from the pyrene donor to the F7GA acceptor to detect its binding to CYP3A4. Cooperativity of the interactions detected by FRET indicates that the enzyme possess at least two F7GA‐binding sites that have different FRET efficiencies and are therefore widely separated. To probe spatial localization of the sites we studied FRET in a series of mutants bearing PIA at different positions, and measured the distances from each of the two binding sites to the donor. Our results demonstrate the presence of a high‐affinity binding site at the periphery of the enzyme. Molecular modeling and docking show that the most probable location of this site is at the distal surface of the enzyme, similar to the site of peripheral binding of progesterone in a prior X‐ray crystal structure. The binding of F7GA to this site causes a substantial spin shift and is a prerequisite for subsequent binding of F7GA in the active site. Our results demonstrate the importance of peripheral ligand binding and substrate‐induced conformational changes in substrate recognition. (Supported by GM054995).
Assessment of intracellular O2 gradient on a microplate reader
Authors: Dmitriev Ruslan I , Zhdanov Alexander V , Jasionek Greg , Papkovsky Dmitri B
Published: June 2021

The diffusion of oxygen (O2) into tissue, cell and mitochondria regulates metabolism, gene expression and cell fate. Depending on the cell type and mitochondrial function the gradient between extracellular and intracellular O2 may vary and is hypothesized to play a physiological role, i.e. stabilization of HIF protein. With numerous approaches proposed, there is still no good method to analyze it in a simple and high‐throughput manner. We present a new method which uses Pt‐porphyrin based probes directed to extracellular (EC), pericellular (PC) and intracellular (IC) compartments of the cell, enabling to probe iO2 gradients in static cell culture by phosphorescence quenching method with the help of time‐resolved fluorescent reader. A new probe targeted to plasma membrane was developed and combined with the IC nanoparticle‐based and EC MitoXpress probes for the assessment of EC, PC and IC O2. MEF cells stained with these probes on the same microplate and exposed to graded hypoxia demonstrated different levels of EC, PC and IC O2. The diffusion nature of these gradients was confirmed by the experiments with reduced cell number and cells with activated and inhibited mitochondrial respiration. These results demonstrate that in a model of adherent mammalian cells iO2 gradients exist and can be conveniently measured by this technique. Supported by SFI.
Targeting Angeogenesis for Bone Repair
Authors: Clemens Thomas L.
Published: June 2021

Impaired bone healing following trauma, surgery, and/or infections continues to be challenging clinical problems. A major goal of our work is to identify the cellular and molecular mechanisms that couple angiogenesis and osteogenesis during bone regeneration, and to use this information to develop strategies to enhance bone repair. Neoangiogenesis is critical for bone regeneration and is dependent of hypoxic stimuli, which upregulate pro‐angiogenic factors including VEGF and BMP‐2. Using both genetic and pharmacologic strategies we have manipulated the VEGF and BMP‐2 pathways in mouse osteoblasts to drive angiogenesis in an attempt to enhance osteogenesis. Mice with constitutive VEGF or BMP expression in osteoblasts had markedly increased vascularity and bone regeneration, which were inhibited by concomitant administration of VEGF receptor antibodies. Administration of synthetic inhibitors of HIF prolylhydroxylation stabilized VEGF production and increased angiogenesis and bone regeneration. Additional strategies developed to increase angiogenesis involve delivering mesenchymal stromal cells transduced with a recombinant adeno‐associated viruses encoding VEGF or BMP‐2. Finally, high throughput screening approaches identified several small molecules capable activating Smad‐1 signaling downstream of the BMP receptor. These stable BMP‐activating molecules manufactured into nanoparticles can be readily delivered to sites of bone injury and are proving useful in animal models for accelerating bone repair.

Supported by NIAMS R01AR49410
Molecular Fingerprinting of Hsp27 Anti‐Apoptotic Activity
Authors: Tossey Justin , Voss Oliver , Doseff Andrea
Published: June 2021

Apoptosis is an evolutionarily conserved mechanism crucial to the homeostasis of cell numbers, tissue development, and immune response. Several checkpoints regulate the activation of apoptosis, including heat shock protein 27 (Hsp27), an anti‐apoptotic regulator that blocks several steps in the apoptotic cascade. However, the structural basis for this protective effect remains unknown.

Increased cardiomyocyte (CM) apoptosis contributes to cardiovascular disease (CVD) in doxorubicin (dox)‐treated cancer patients. CM apoptosis requires the activation of caspase‐3, a key evolutionarily conserved protease. We showed that Hsp27 associates with and inhibits caspase‐3, but the exact mechanisms remain unknown. The goal of this study was to determine the structural‐functional relationships responsible for the anti‐apoptotic activity of Hsp27. Serial deletion clones of Hsp27 were used to fingerprint the Hsp27 domains responsible for the association with caspase‐3 and inhibition of apoptosis. We identified different molecular signatures responsible for caspase‐3 association and the regulation of cytochrome c. These findings contribute to the understanding of Hsp27 anti‐apoptotic function and will aid in the development of anti‐apoptotic peptide therapies for the prevention of CVD in cancer patients.

This research was funded by AHA SURF and ASC URS to JCT and RO1 HL075040 and NSF‐MCB 0542244 to AID
Genetic characterization of the mechanism of action of sulforaphane in the yeast, S. cerevisiae
Authors: Tucker Douglass , Murphy Michael , Wilcox Alexander , Austriaco Nicanor
Published: June 2021

The isothiocyanate, sulforaphane (SFN), isolated from broccoli and other cruciferous vegetables is a potential chemotherapeutic agent. Recent studies that investigated the mechanism of action of SFN in mammalian cells have suggested that SFN works by causing cell cycle arrest and/or initiating programmed cell death (PCD). In order to identify genes that may be involved in the cell's response to SFN, we have initiated a genetic screen using the yeast knockout (YKO) library of the budding yeast, Saccharomyces cerevisiae, to identify loss of function mutants that are sensitive to SFN. At the time of this writing, we have screened the entire YKO collection twice, and we have isolated 54 mutants that were SFNS in both screenings of the library. We also have 174 single hits that need to be tested again. Significantly, we have discovered that mutations in several genes required for autophagy including VAM3 and VAM7 result in SFN sensitivity. This appears to confirm an earlier finding from our lab that yeast cells lacking ATG1, a kinase essential for autophagy, are also sensitive to sulforaphane, suggesting a possible role for autophagy in the mechanism of SFN‐induced programmed cell death in yeast.

This research has been funded by a faculty research (CAFR) award from Providence College, a faculty development award from the Rhode Island‐INBRE program, and by grant R15 GM094712 from the NIGMS.
Temperature as a cellular stress of Chinook salmon embryo cells
Authors: Reese Amy L , Williams E. Eugene
Published: June 2021

In this study, we used Chinook salmon embryo cells (CHSE‐214) to assess the relationship between the timing of the expression of heat shock mRNAs and the timing of heat‐induced cell death. We first examined the timing of cell death [lactate dehydrogenase (LDH) release] after a 24hr exposure to 22, 30, 37, 45, and 55°C. As expected, cells did not release LDH after 24 hrs of exposure to their acclimation temperature (22°C). At 30°C LDH release was detected beginning at 6, and all of the cells were dead by 24hrs. Exposure to 37°C, LDH released at 3hrs; all cells were dead by 6hrs. At 45°C the cells released LDH within the first hour, and were all dead by 3hrs of exposure. At 55°C the cells began to release LDH within the first 15mins, and were all dead within 1hr of exposure. mRNAs for heat shock proteins were assessed at 37°C and it was found that the constitutive heat shock protein (Hsc70) was present at all time points, and there was no significant difference in the expression of the message for the inducible form of the heat shock protein (Hsp70) over a 6 hour period. These data will help us to further understand the molecular meaning of the critical thermal max of fish. This project is supported by the Department of Biological Sciences, and the Henson School of Science and Technology of Salisbury University.
Adherence analysis between human erythroleukemia cells and human lung fibroblasts
Authors: Dover Cheri Denise
Published: June 2021

Initially, human erythroluekemia (HEL) cells were mixed with human lung fibroblasts (WI‐38 cells) to create a simulated in vivo environment. Normally, HEL cells grow in suspension while WI‐38 cells grow attached. When the two cell types grow together, HEL cells adhere to the WI‐38 cells (80%). This mixed cellular environment was analyzed for viability, growth, and adherence via trypan blue exclusion and cell enumeration. The viability of the HEL cells that grew attached and unattached to the WI‐38 cells was significantly elevated and depressed, respectively. Growth remains constant for HEL cells with and without WI‐38 cells. To determine adherence, HEL cells were grown in chamber slides coated with and without fibronectin (FN). Approximately 57% of HEL cells adhered to the fibronectin‐coated plate suggesting that FN is the extracellular matrix protein that is responsible for HEL adhesion. Breakage of disulfide bonds with dithiothreitol did not disrupt the attachment between the two cell types, indicating the presence of an additional linkage. Most of the WI‐38 cells that detached were nonviable suggesting that HEL cells reroute WI‐38 cell nutrients to increase their own viability. Support from College of Health, Environment and Science and Slippery Rock University.
Ultrastructural analysis of experimental tenoplasty using bovine peritoneum
Authors: Masuko Telma Sumie
Published: June 2021

Frequent lesions to tendons and ligaments bring lots of concern to orthopedists and veterinaries. Rupture of the calcaneal tendon is one of the most common tendon injury observed in the dog. The calcaneal tendon, also known as Achilles tendon or occasionally Achilles’ tendon is a tendon of the posterior leg and it is the strongest tendon in the structure of the musculoskeletal system in the dog. In dogs the calcaneal tendon originates from different muscle‐tendon units, the gastrocnemius tendon, the superficial digital flexor tendon, and the common tendon of the biceps femoris, gracilis and semitendinosus muscles. The aim of this study is to examine by scanning electron microscopy 98%‐glycerin‐fixed bovine peritoneum (GFP) used to fix lesions in dog's calcaneal tendon.

Twenty one mixed bread healthy adult dogs, weighting between 10 and 15 kg were submitted to the implantation of a 1 cm‐GFP graft and sacrified 2, 7, 15, 30, 60, 90 and 120 days after the surgery. Fragments from 3 different segments, proximal, middle and distal, of the implanted graft were processed for the scanning electron microscopy analysis. The samples were fixed in 2% glutaraldehyde, post‐fixed in 1% Osmium Tetroxide, dehydrated in a series of alcohol, sputtered and observed in the scanning electron microscope.

In all studied dogs, the peritoneum graft has been easily found without significant macroscopic derangements. Fifty five days after surgery, dogs were able to walk normally. The analysis under electron microscopy showed a presence of collagen tissue formation after 30 days post‐operative In this period we could observed the formation of neotendon involving peritoneum and nylon thread implanted in the segment. With 120 days post‐operative surgery can observe the presence of abundant collagen fibers arranged longitudinally, with morphology similar to the calcaneous tendon.

Putting all into a nutshell, it is possible to conclude that glycerin‐fixed bovine peritoneum would be an interesting alternative for fixing tendon lesions as it seems to stimulate connective tissue deposition in order to form a good matrix for new tissue development.
Tetracycline Therapy Against Phytoplasma Causing Yellowing Disease of Date Palms
Authors: Montasser Magdy Shaban , Hanif Asma M. , Al_Awadhi Husain A. , Suleman Patrice
Published: June 2021

An outbreak of phytoplasma microorganisms was observed in date palms grown in Kuwait. Phytoplasma are widespread prokaryotic microorganisms that infect palm trees, ornamentals and some horticultural crops. It sometimes causes lethal yellowing diseases in palm trees, which also multiplies in the host plant grown in tissue culture and spread infections by insect vectors. The presence of phytoplasma was detected by using both transmission and scanning electron microscopy. Fluorescence microscopy was used to detect DNA contents of the causal microorganism. Tetracycline therapy was carried out as a further evidence for the presence of the phytoplasma and as an attempt to control the disease. Infected young palms treated with tetracycline‐HCl at early stages of the phytoplasmal infection showed remission of the yellowing symptoms. We show in this work that by injecting the infected young palm trees with tetracycline antibiotic the treated plants were recovered. The use of antibiotic treatment is valuable for the control of yellows disease in date palms especially in the areas where the pathogen is endemic and causes extreme crop losses. Companies producing date palm trees in tissue culture and growers will especially benefit from this knowledge in the development of control strategies for yellow diseases that are caused by phytoplasma.
Thrombospondin‐1 binding protein on the surface of Trypanosoma cruzi enhances cellular infection
Authors: Johnson Candice A. , Cardenas Tatiana C. , Pratap Siddharth , Duquette Mark A. , Lima Maria F. , Lawler Jack , Villalta Fernando , Nde Pius N.
Published: June 2021

The mechanism by which Trypanosoma cruzi enhances cellular infection remains to be well elucidated. We have reported that T. cruzi up‐regulates the expression of host thrombospondin‐1 (TSP‐ 1) to enhance cellular invasion. Here we characterize a novel TSP‐ 1 interaction with T. cruzi that enhances cellular infection. Labeled TSP‐1 and the N‐terminal domain of TSP‐1 (NTSP) interact specifically with the surface of T. cruzi trypomastigotes. TSP‐1 and NTSP pulled down a novel TSP‐1‐interacting parasite surface protein in a protein‐protein interaction assay. We also observed that pre‐exposure of rNTSP to T. cruzi significantly enhances parasite infection of wild type mouse embryo fibroblast (MEF) in contrast to the C‐terminal domain of TSP‐1 (E3T3C1). Our findings indicate that TSP‐1 interacts with a novel protein on the surface of T. cruzi through the NTSP domain and that this amplifies the infection process.

(Supported by NIH grants SC2AI083925 to NPN, 1 U54 RR026140‐03, AI007281‐23)
Zinc and copper status are associated with bone mineral measures in lactating adolescent mothers
Authors: Bezerra Flávia F. , Maia Patricia A. , Anastácio Alexandra , Mendonça Laura M.C. , Donangelo Carmen M.
Published: June 2021

We investigated the relationship between bone mass and copper and zinc status in lactating (LA, n=33, 8.7±3.4wk postpartum) and non‐lactating (NL, n=14) adolescents (14–19y). Total bone mineral content (TBMC), total (TBMD) and lumbar spine (L2‐L4, LSBMD) bone mineral density, dietary intake, copper (CuP), zinc (ZnP) and ceruloplasmin (Cp) in plasma, and zinc (ZnE) and metallothionein (MT‐E) in erythrocytes were assessed. Over 70% of adolescents in each group had zinc and copper intakes lower than RDA. Biochemical indices were similar between groups except for ZnE and MT‐E that were 26% and 123% higher, respectively, in LA than in NL (P<0.05). LSBMD z score was lower in LA than in NL (−1.09 ± 1.5 vs. 0.04 ± 1.23, P<0.05). In LA, zinc intake was correlated with TBMC (r=0.38), TBMD z score (r=0.37) and LSBMD z score (r=0.37); ZnP was correlated with TBMC (r=0.44); and Cp with TBMD (r=0.50), LSBMD (r=0.42) and LSBMD z score (r=0.39) (P<0.05). In NL, no significant correlations between bone mass indices and zinc or copper status were observed. Our results suggest that a poor copper and zinc nutritional status may exacerbate the magnitude of bone loss during lactation in adolescent mothers.

Financial support: CNPq, FAPERJ (Brazil).
Introduction of complementary feeding for infants: a dilemma for parents and caregivers
Authors: Friel James Kenneth , Qasem Wafaa , Fenton Tanis
Published: June 2021

Data are scarce with respect to infant iron status, but at least in developing country settings where newborn iron stores may be suboptimal, exclusive breastfeeding without iron supplementation through 6 months may compromise hematologic status (WHO 2002). Our objective was to systematically review the scientific literature regarding iron and hematologic status among infants breastfed exclusively to 6 months, stratified by developed versus developing countries.

Methods

An electronic search of the MEDLINE database was conducted for studies of iron deficiency (ID) and (IDA) in infants exclusively breastfed in developed and developing countries. Prevalence estimates were calculated as weighted averages, and compared using a 2‐sided test of proportions.

Results

Twenty nine observational studies and three of eight RCTs met the inclusion criteria. All RCTs were conducted in developing countries. The prevalence estimates for healthy exclusively breastfed infants living in developed (n=769) and developing (n=642) countries, respectively, was 21.9% and 14.1% for ID (p = 0.0001), and 12.2% and 18.2% (p= 0.0019).

Conclusion

A substantial proportion of exclusively breastfed infants develop ID, IDA, and compromise hematologic status in both developed and developing countries. Data to support the current recommendation of solid commencement at six months is limited regarding irons status. Larger randomized controlled trials, perhaps in multi centre design, are needed to investigate the advantages and disadvantages of introduction of solids before and after six months of age.
The relationship between the use of blood pressure medication, a low‐sodium diet, and health and nutrition literacy among Community‐dwelling hypertensive Older Adults
Authors: Jones Samira , Young Heather , Miller Lisa
Published: June 2021

More than 90 million U.S. adults have low health literacy, which is the ability to obtain and understand health information in order to make appropriate health decisions. Nutrition literacy is a component of health literacy pertaining to nutrition information. Nearly 68 million adults have hypertension, a preventable risk factor for cardiovascular disease and stroke; and persons with limited knowledge and understanding of diet and health concepts such as hypertension, generally have low health literacy and poor medication adherence. Community‐dwelling seniors in Sacramento County completed a modified health and nutrition knowledge survey, and two validated health literacy assessments: 1) the short Test for Functional Health Literacy (s‐ToFHLA) and 2) the Newest Vital Signs (NVS) to measure health or nutrition literacy. The goal was to determine the relationship between health and nutrition ˈknowledgeˈ and ˈliteracyˈ among hypertensive older adults (≥ 65 years). The sample included 69 older adults with an average age of 72 (SD= 7.3), and 26% African American, 9% Hispanic, and 58% White participants. Study findings included a modest correlation between the total literacy score and total knowledge score (r=0.58, p = 0.04), and significantly lower literacy scores for participants on blood pressure medication compared to those who were not (n=27) (95% CI: −83.04, −18.49%, p ≤ 0.01). No significant differences in scores were found for those on a low‐sodium diet compared to those who were not (p= 0.17). Future studies are needed to understand the relationship of health literacy to health outcomes to facilitate the development of tailored, effective interventions for older adults with chronic conditions.

*Funding for this study was provided by the UC Davis Betty Irene Moore School of Nursing
Effects of dietary calcium intake on osteopenia and osteoporosis in middle aged and old men
Authors: Yang Yoon Jung
Published: June 2021

Studies investigating the effect of dietary calcium intake on bone mineral density in middle aged and old men are limited. The aim of this study was to determine the effects of dietary calcium intake on the risk of osteopenia and osteoporosis in middle aged and old men. It was a cross‐sectional study. A total of 1,217 male subjects aged 50–79 years from the 2009 Korean National Health and Nutrition Examination Survey participated in the study. Bone mineral density was measured using dual energy X‐ray absorptiometry. Calcium intake was estimated by 24‐hour dietary recall method. Prevalence of osteopenia at total femur, femoral neck, and lumbar spine was 13.5%, 40.0%, and 35.3%, respectively. Prevalence of osteoporosis at total femur, femoral neck, and lumbar spine was 0.5%, 3.5%, and 6.6%, respectively. The mean of calcium intake was 531.4±363.7 mg/d and 21.8% of the subjects fulfilled the Recommended Intake (RI) of calcium, 700mg/d. Dietary calcium intake was inversely associated with the risk of osteopenia and osteoporosis after adjusting for potential confounders at total femur (5th vs. 1st quintile, OR = 0.52, 95% CI = 0.27–0.99, P for trend = 0.010), femoral neck (5th vs. 1st quintile, OR = 0.47, 95% CI = 0.30–0.73, P for trend <0.0001), and lumbar spine (5th vs. 1st quintile, OR = 0.69, 95% CI = 0.46–1.05, P for trend = 0.037). These results suggest that adequate calcium intake is necessary to prevent osteopenia and osteoporosis in middle aged and old men.
Metabolic assessment of pediatric patients with chronic disease
Authors: Leung Randy B. , Schaub John R. , Klein Daniel S. , Borum Peggy R.
Published: June 2021

Background

Chronic disease can exacerbate obesity, type II diabetes, high blood pressure, and problems with children's growth. Changes in diet can impact these health issues.

Objective

Develop a program for a comprehensive metabolic assessment of patients seen in pediatric clinics for chronic disease.

Methods

Patient interviews conducted at a weekly clinic for pediatric HIV+ patients included a 24‐hour diet recall; activity recall; height, weight, anthropometric skinfold and circumference measurements; and bioelectrical impedance analysis. Tools were created and refined to better define the above concerns, in order to better evaluate health issues.

Results

Tools were developed for the following concerns and tested at a weekly clinic:

Assessment Concerns

Patient's MyPlate education

Patient's Solid Fats and Added Sugars intakes

Caloric requirements due to chronic disease

Quantitative analysis of medication load

Analysis of body growth and fat distribution

Risk for developing insulin resistance

Trends of abnormal blood lipids

Risk for hypertension

Conclusion

The program facilitates the assessment of potential comorbidities associated with nutrition in chronic disease and increases clinicians’ awareness of opportunities to improve patient care through education about lifestyle changes.
Comparison of Parent and Child Reported Fruit and Vegetable Preferences
Authors: Nanney Kristin , Carraway-Stage Virginia , Cox Tiffany , Spangler Hillary , Martin Alexa , Borges Michelle , Worf Aly , Goodell L. Suzanne
Published: June 2021

Research has demonstrated health habits formed in early childhood persist into adulthood, especially food preferences. Parents can play an influential role in the development of preferences, due to their involvement in shaping early dietary behaviors. The purpose of this study was to compare fruit and vegetable (FV) food preferences of 3–5 year old children enrolled in a Head Start program as reported by parents and the children themselves. Ten target fruits (n=6) and vegetables (n=4) were chosen based on published accounts of commonly consumed FV in this age group. Parents were asked to report their child's FV preference on a 5‐ point Likert scale (strongly likes to strongly dislikes). A researcher developed electronic tool was utilized to assess each child's FV preference. The tool assessed preference using a 5‐point Likert scale (very yucky to very yummy) anchored with stars increasing in size. Using iPads, children were shown images of each FV in a random order. Information concerning children's exposure to FV in the home and school environment was also assessed through parent report and school menus. This study serves to establish baseline data regarding parental opinion of their child's FV preference compared to the child's reported FV preference. Information obtained from this study can be used to develop future nutrition education materials and interventions for preschool‐aged children and their parents.
Relationship between obesity markers and blood pressure among African Americans with and without T2D
Authors: Duenas Carla , Podesta Cinthia , Zarini Gustavo G. , Huffman Fatma G.
Published: June 2021

This study investigated the association between anthropometric measurements of obesity with blood pressure as well as whether these associations vary by diabetes status and gender among African‐Americans (AA). This cross‐sectional study included AA (n=249) with (n=129) and without (n=120) type 2 diabetes (T2D). Participants were recruited from multiple sources from Miami‐ Dade and Broward Counties, Florida. Analyses included t‐test, chisquare, Pearson's correlations and linear regression. Subjects with T2D had a higher BMI (P<.001), WC (P<.001), WtHR (P<.001), and SBP (P<.001) compared to subjects without T2D. Males without T2D had significant associations between WC and WtHR with SBP (β=.392, SE= .147, P=.005; β=.389, SE= 25.96, P=.004, respectively); whereas DBP was associated with BMI (females: β=.450, SE= .250, P=.006; males: β=.516, SE=.239, P<.001), WC (males: β=.513, SE=.093, P<.001) and WtHR (females: β=.597, SE= 19.08, P=.001; males: β=.501, SE=16.43, P<.001) in subjects without T2D. Among AA, the ability of anthropometric measures to predict SBP and DBP varies by diabetes status and gender. Funding for this research was provided through an NIH/NIDDK sponsored grant.
Bioavailability, glucose disposal, and anti‐inflammatory properties of grape products in high fat‐fed obese mice
Authors: Chuang Chia-Chi , Shen Wan , Xie Guoxiang , Jia Wei , McIntosh Michael
Published: June 2021

The objective of this study was to determine which polyphenols in grape powder (GP) extract (GPE) were absorbed and the effect of GPE and GP on attenuating insulin resistance and inflammation in obese mice. We found that serum levels of quercetin 3‐glucoside (Q3G) and rutin were increased after 1 h post‐gavage of GPE (1 g/kg body weight) and decreased to baseline levels within 3–8 h. Next, C57BL/6J mice were fed a high fat diet supplemented for 18 wk with 0.02% GPE or 3% GP. Obese mice supplemented with GP, but not GPE, had better glucose tolerance acutely (e.g., at 5 wk), but not chronically (e.g., at 10 or 15 wk) compared to controls. Notably, obese mice supplemented with GP, but not GPE, had lower inflammatory cytokine (e.g., tumor necrosis factor alpha or TNFa) and chemokine (e.g., monocyte chemoattractant protein‐ 1 or MCP‐1) levels in circulation and mRNA levels of inflammatory‐related genes (e.g., TNFa, interleukin‐6, MCP‐1, interferon gamma‐induced protein‐10, F4/80, CD11c) in white adipose tissue compared to controls at 18 wk. Consistent with these data, Q3G and rutin decreased MCP‐1 gene expression in primary human adipocytes treated with TNFa. Overall, we demonstrate that several grape polyphenols (i.e., quercetin metabolites) are bioavailable and quercetin‐rich GP improves glucose tolerance acutely and attenuates markers of inflammation chronically in high fat‐fed obese mice.

Grant Funding Source: NCARS02288 and UNCG127131