bluuebunny/crossref_metadata_2025 huggingface dataset:
Increase of myogenic tone in the cerebral arteries of dyslipidemic mice is not due to a vessel wall remodelling
Authors: Bolduc Virginie , Drouin Annick , Robillard Isabelle F. , Des Rosiers Christine , Thorin Éric
Published: June 2021

The role of dyslipidemia in mediating changes in vascular structure, mechanic and function is poorly understood. Isolated pressurized cerebral arteries of LDLr‐/‐hApoB+/+ (ATX) mice showed an increase of myogenic tone (MT) compared to WT at 3‐mo‐old (22 ± 2 % vs 14 ±1 % at 80 mmHg, p<0.05). We hypothesized that the increased MT was associated with vessel wall remodelling. Cerebral compliance, measured as pressure‐induced dilatation in the presence of potassium cyanide (7.7 mM), showed no difference between ATX and WT mice. Because ATX mice present a 7 fold increase in circulating cholesterol and triglyceride, we studied the phospholipids (PL) bilayer composition. There was no variation between groups in the amounts of C16:0, C18:0, C18:1n9, C18:1n7, C18:2, C20:4 and C22:6 contained in the PL and free fatty acids fractions extracted from total brain vessels. A quantitation kit measured similar level of free cholesterol in brain vessel between groups. Western blot showed no difference in collagen III deposition in the vessel wall. In conclusion, our data suggest that increase in MT in cerebral arteries of ATX mice is not due to a vessel wall remodelling.

This work is financed by the Canadian Institutes of Health Research.
Quantitative Proteomic Analysis of SILAC‐Labeled Proteins from High and Low Glucose Treated Vascular Endothelial Cells Using Spectral Counting
Authors: Parker Sarah J , Halligan Brian , Greene Andrew S
Published: June 2021

A major goal of mass‐spectrometry based proteomics is the relative quantification of a large number of differentially expressed proteins between two or more biological samples. Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) is a widely used quantitative technique in proteomics, though well‐known limitations in its analytical power exist (i.e., throughput, signal‐to‐noise). Spectral counting is a high throughput label‐free quantification technique that bypasses the limitations of SILAC data analysis, however samples are analyzed in independent mass spectrometry runs and thus are subject to high technical variability. We report a hybrid approach that analyzed SILAC data sets by counting heavy and light peptide spectra from proteins detected within the same LC MS/MS run. We show that SILAC spectral counting is an accurate and sensitive technique for quantitative analysis. Further, we applied SILAC spectral counting to compare vascular endothelial cell protein expression between high (20mM) and low (11mM) glucose culture conditions. Among the 153 proteins quantified, we detected a network of differentially expressed proteins linked to a transcription factor, NRF2, which is activated by oxidative stress. We concluded that SILAC spectral counting is a high throughput technique capable of detecting biologically relevant differences in protein expression.
SPIRE: An Innovative Approach to Postdoctoral Training
Authors: Uno Jennifer K , Rybarczyk Brian , Lund P K , Lerea Leslie S , Dykstra Linda
Published: June 2021

Seeding Postdoctoral Innovators in Research and Education (SPIRE) is an integrative postdoctoral fellowship program that combines research training, professional development and hands‐on teaching experience. This NIH‐funded program involves a partnership between the University of North Carolina at Chapel Hill (UNC Chapel Hill) and Minority Serving Institutions (MSIs) in North Carolina. The objective of this program is to prepare SPIRE scholars for academic careers in the biomedical sciences and to have a lasting impact at the MSIs through the exchange that takes place as the postdoctoral scholars teach science courses and interact with faculty and students. During the first two years of the program, SPIRE scholars are focused on cutting‐edge research at UNC Chapel Hill. They also receive exposure to core professional skills, pedagogical training from education professionals, and workshops to bring technology into the classroom. During the third year each SPIRE scholar is involved in a teaching internship where they bring active learning techniques into the classroom, as well as develop advanced‐level courses at the MSIs. The combined research, professional development, and hands‐on teaching prepares SPIRE scholars with the skills required to succeed as independent researchers, research‐oriented educators and committed mentors for undergraduate students in the sciences.
APS Archive of Teaching Resources expands to create a multi‐society, collaborative digital library
Authors: Matyas Marsha Lakes , Lowy Melinda E , Feuerstein Amy , Pressley Thomas A.
Published: June 2021

The American Physiological Society (APS) Archive of Teaching Resources provides a venue for colleague‐to‐colleague sharing. This study examines the Archive's transformation into a collaborative digital library with resources catalogued by a growing list of scientific societies (APS, Human Anatomy and Physiology Society, Society for Developmental Biology, and National Association for Health and Science Education Programs). Partners customize their own submissions and reviews, and create/manage partner‐specific discipline lists, keywords, review criteria and review forms. Users can search via keyword and advanced search and browse by disciplines, learning resource type and grade level. The personalized "My Archive" feature retains previous functions (saving and sharing searches and resources) while also providing a listing of users' submissions and allowing users to track submissions through the review process. The Archive is open to external search engines (e.g., Google). Measures of the transformation impact include rates of submission by societies and individuals, resources accessed and downloaded, and new user registrations as well as use of new browse features and highlighted resources (featured items, research "bytes," historical item links, and news items).
Persistent Pulmonary Hypertension of the Newborn is Associated with Remodeling of the Umbilical Vasculature
Authors: Swartz Daniel D , Lakshminrusimha Satyan , Nielsen Lori C , Arter Alison
Published: June 2021

Introduction

Persistent Pulmonary Hypertension of the Newborn (PPHN) has unknown consequences on systemic vasculature. Soluble elastin peptides derived from vascular remodeling have been shown to enhance or elicit a systemic inflammatory response. We studied the association between pulmonary vascular remodeling in PPHN and umbilical vessel remodeling.

Methods

Pregnant ewes underwent fetal ductal ligation at 126‐128d gestation (term 145d) to induce PPHN (Morin et al, 1989). The lambs, 8‐9 days later, were then delivered by cesarean section and the umbilical cord was clamped and cut. Cord blood plasma was collected for assay of soluble elastin fragments. Immunohistochemistry and proteomics were performed with paraffin‐embedded and quick frozen umbilical vessels (artery ‐ UA, vein ‐ UV) samples respectively. Umbilical vessel sections were analyzed for cell density, MMP‐2, MMP‐9, and elastin content.

Results

Cell density, as well, MMP‐9 and MMP‐2 staining increased in UA and UV of PPHN animals compared to controls. Quick frozen umbilical vascular tissue demonstrated a trend decrease in elastin content. There was an increase in soluble elastin fragments in plasma from PPHN lambs.

Conclusion

The umbilical vessels of a surgically created model of PPHN demonstrate vascular remodeling, possibly secondary to soluble factors released from the pulmonary vasculature.
PATHOLOGICAL ANGIOGENESIS IN VASCULAR TUMORS IS DRIVEN BY AKT1 AND INHIBITED BY TOPICAL RAPAMYCIN
Authors: PHUNG THUY L , Antonello Zeus , Gerald Damien , Perruzzi Carole , Rocuts Alexander , Hochman Marcelo , Cunningham Michael , Mihm Martin , Benjamin Laura E
Published: June 2021

Vascular tumors are lesions of dysregulated angiogenesis of neoplastic endothelial cells (EC). To date, the mechanisms leading to EC tumorigenesis are poorly understood. We found increased endothelial Akt activation in benign and malignant human vascular tumors such as hemangioma and angiosarcoma. Expression of constitutively active Akt1 in vascular EC in mice resulted in hemangioma formation with recruitment of bone marrow‐derived precursor cells. Loss of Akt1 significantly inhibited sprouting angiogenesis, cell migration and proliferation in response to VEGF‐A in primary human hemangioma EC. Furthermore, loss of Akt1 but not Akt2 and Akt3 isoforms significantly reduced the growth of mouse hemangioendothelioma cells in vivo. Inhibition of mTOR activity downstream of Akt with rapamycin blocked VEGF‐induced angiogenesis in hemangioma EC. Rapamycin, given i.p. or topically, strongly inhibited the growth of subcutaneous vascular tumors in vivo. In neoplastic EC, rapamycin inhibited both TORC1 activity, which activates S6 kinase downstream of mTOR, and TORC2 activity, which activates Akt in a feedback regulatory loop. In summary, these studies show that endothelial Akt1 activation is important in vascular tumor formation, and demonstrate the possible clinical utility of topical rapamycin or other Akt/mTOR pathway inhibitors in the treatment of cutaneous vascular tumors. Funding source: NHLBI.
TGF‐Beta is required for retinal vascular barrier function, endothelial cell survival and homeostasis of the adult retina
Authors: Walshe Tony E , Saint‐Geniez Magali , Maharaj Arindel , Sekiyama Eiichi , Maldonado Angel , D'Amore Patricia
Published: June 2021

Cell‐cell interactions are critical to both vascular development and vessel stability in the adult and TGF‐β signaling between endothelial and underlying mural cells is a potent stabilization signal for the quiescent vasculature. Here we describe the effects of inhibition of TGF‐β signaling in the retinal vasculature by overexpressing soluble endoglin (sEng). TGF‐β inhibition increased vascular and neural cell apoptosis in the inner layers of the retina as determined by TUNEL staining, transmission electron microscopy and western blotting for cleaved caspase 3, concomitant with a decrease in inner retinal visual function as measured by electroretinogram (ERG). We further demonstrate impaired inner retinal vascular perfusion, retinal capillary collapse and an impaired ability to autoregulate peripheral blood flow in response to acetylcholine (ACh). Fundus angiography and evans blue permeability tests revealed increased circulating sEng causes breakdown of the blood‐retinal‐barrier due to a decrease in association of tight junction proteins zo‐1 and occludin. In vitro, inhibition of TGF‐β signaling leads to endothelial but not 10T1/2 cell death and dissociation of endothelial and 10T1/2 cells, concomitant with a breakdown of endothelial barrier function. Our results demonstrate a potent role for endogenous TGF‐β signaling in the quiescent retinal vasculature.

Grant Funding Source

NIH
Upregulated placenta growth factor expression in human myocardium is associated with cardiomyocyte damage
Authors: Torry Ronald J. , Labarrere Carlos A. , Baldridge Lee Ann , Miller Steven J. , Torry Donald S.
Published: June 2021

The usefulness of plasma PlGF as a biomarker of acute coronary syndrome patient outcomes has been described, however little is known regarding its expression and localization in human myocardium.

Right endomyocardial biopsies were obtained from donor hearts and cardiac allograft recipients. Samples were grouped according to the presence or absence of myocardial fibrin and changes in PlGF mRNA expression, protein localization, cellular infiltrates, cardiac Tn‐I, time since transplant and ISHLT rejection grades were compared between groups.

Biopsies (n = 7) with fibrin expressed 1.8‐fold more PlGF mRNA than biopsies without fibrin (n=10; p < 0.03). PlGF protein was localized in cardiomyocytes, extracellular matrix and microvessels in areas of fibrin deposition and not in fibrin‐negative areas. Fibrin‐positive biopsies were also associated with increased cellular infiltrates (p < 0.05) and elevated cardiac Tn‐I titers (p < 0.03). There was no correlation between PlGF mRNA expression and degree of macrophage, T‐cell, or neutrophil infiltrates or rejection grade. PlGF mRNA expression decreased with time since transplantation (R = ‐ 0.792; p < 0.001).

Our results show human myocardium expresses PlGF mRNA/protein and its expression increases with cellular and biochemical indices of myocyte damage. These data provide new support that endogenous PlGF could promote cardiac healing. Support: R01‐HD36830 (DT)
Alcohol Augments Fetal Skeletal Malformations in Diabetic Pregnancy
Authors: Rengasamy Padmanabhan , Shafiullah Mohamed , Nagelkerke Nico
Published: June 2021

Maternal alcohol consumption and pregnancy in diabetes mellitus (DM) are independent risk factors for offspring maldevelopment. DM women might consume alcohol during pregnancy. We aimed to determine the effects of potential interaction between acute doses of alcohol and maternal DM during gestation on fetal skeletal development. Mice were made diabetic with streptozotocin on gestation day (GD) 2. Single doses of 25%v/v of absolute alcohol in saline was administered (0.03 ml/g body weight) to groups of DM and non‐DM animals on GD 7 or 8. One group of DM animals were treated with insulin. Skeletons of GD 18 fetuses were stained with Alizarin red‐S and Alcian blue. Both ethanol alone and DM alone induced a low incidence of skull bones hypoplasia and vertebral defects. Ethanol injection of DM mice on GD 7 resulted in accentuation of DM‐related skeletal defects. Treatment on GD 8 resulted in defects of axial and appendicular skeletons differentially. Combination treatment induced markedly poor ossification in about 60% of animals. Alcohol also augmented frequency of supernumerary ribs and sternal anomalies and induced poor ossification of bones of paws. These data suggest that alcohol affects developmental processes of mesoderm of embryos of diabetic mouse resulting in augmentation of skeletal malformations. (Support: ETSU & UAEU)
SURFACE MODIFICATION OF METALLIC IMPLANTS TO ENHANCE OSTEOGENESIS
Authors: Patel Shraddha , Mills David Keith , Howard Jason , Lvov Yuri
Published: June 2021

Surface topology and chemistry play a role in effecting osseointegration of dental and orthopedic implants with surrounding bone. Titanium dioxide (TiO2) nanofilms, functionalized with BMP‐2, was investigated as an approach to fabricate an instructional surface coating. Six, nine and twelve‐layered TiO2 substrates were also studied to investigate the additive effect of surface roughness. Osteoblast growth, functionality and matrix mineralization on nanofilms were evaluated. Cell proliferation decreased with increasing surface roughness on both uncoated and BMP‐2 coated TiO2 substrates as indicated by MTT results. A significant reduction in cell proliferation was also observed on BMP‐2 coated substrates in comparison with uncoated substrates. Osteonectin and osteopontin expression on BMP‐2 coated substrates was significantly greater than that on uncoated substrates suggesting an enhanced osteogenic effect by BMP‐2 on osteoblast protein expression. Varying layers of TiO2 did not measurably affect osteonectin and osteopontin expression by osteoblasts seeded on both uncoated and BMP‐2 coated substrates. A significant enhancement in calcium production was observed with increasing layers of TiO2 on both sets of experimental substrates. Increase in calcium levels and deposition on BMP‐2 immobilized substrates showed an enhanced osteogenic effect resulting in increased osteoblast mineralization. This study demonstrated that BMP‐2 could be immobilized onto TiO2 substrates via nanoassembly and that osteoblast osteogenic activity can be directly influenced by a bioactive substrate. This study suggests that functionalized TiO2 nanofilms may lead to the development of implants with better osseointegration and enhanced durability.
A comparison of midline and lateral measures of the middle cranial fossa in hominoid primates
Authors: Ritzman Terrence Bradley , Copes Lynn Erin , Lewton Kristi Lynn
Published: June 2021

Research has shown that, in primates, cranial base morphology is important to the overall architecture of the cranium, and particularly affects the position of the facial skeleton. In addition, recent studies demonstrate that the morphology of the lateral and midline basicranium in Homo sapiens are not tightly integrated and the lateral basicranium is a more critical determinant of the anatomy of the splanchnocranium. The relationship between the midline and lateral basicranium has not, however, been explored in taxonomically diverse samples of primates. Thus, the current study tests for correlations between midline and lateral measures of the middle cranial fossa (MCF) in a radiographic sample (N=96) of hominoid primates. As the midline MCF houses the pituitary gland and the lateral MCF houses the temporal lobes, this study also tests for interspecific correlations between temporal lobe size and measures of the midline and lateral MCF. Results indicate that midline and lateral measures of the MCF are not tightly correlated and the lateral measures predict temporal lobe size better than midline measures. These results suggest that studies of the relationship between basicranial morphology and facial positioning in primates, including those of fossil hominins, should employ lateral measures of the MCF, rather than those of the midline MCF.

Grant Funding Source

n/a
Morphometry of pedicle and vertebral body in a Mexican population by CT and Fluroscopy
Authors: Guzmán López Santos , Urritua Vega Edgar , De la Garza Castro Oscar , Elizondo Omaña Rodrigo Enrique
Published: June 2021

To quantify the morphometric characteristics of the pedicles of the lumbar spine in Mexican population. There are not reports about the morphometry of lumbar pedicle in a Mexican population exist.

60 cadavers were evaluated by fluoroscopy and CT from L1 to L5. Each vertebral pedicle was measured in the axial, sagittal and coronal planes.

Wide variation in pedicle morphology between specimens at each vertebral level was found in the population. Pedicles from the lumbar vertebrae attained dimensions acceptable for 5.5 ‐ 6.5 mm screw diameters. The data in this study indicates that pedicle screws may be used in the lumbar region.
A mitotic role for GSK‐3β kinase in Drosophila
Authors: Wojcik Edward
Published: June 2021

Glycogen synthase kinase‐3ß (GSK‐3ß) is involved in a wide variety of cellular processes, and implicated in a growing list of human diseases. Recent drug inhibition studies have suggested a role for GSK‐3ß in mitosis in animals. Here, we take an alternative approach to understanding GSK‐3ß function in mitosis by genetic mutational analysis in Drosophila. GSK‐3ß function is well conserved between Drosophila (Zw3) and humans, frequently operating similarly in pathways, as diverse as the Wnt signaling and circadian rhythm pathways, and sharing a key role in the development of the neuromuscular junction. Unlike drug inhibitor studies, we find that loss of function mutations of zw3 result in markedly curved, or bent, metaphase spindles that exhibit metaphase delay. These defects do not routinely result in mitotic catastrophe, and argue that Zw3 plays a role in the maintenance of the mitotic spindle, rather than an essential role in spindle morphogenesis. Consistent with a mitotic function, we observe a complex and dynamic localization of Zw3 during cell division. These studies provide genetic data that validate and extend drug inhibition studies on a novel mitotic role for glycogen synthase kinase in the maintenance of the mitotic spindle. This work was supported by NIH grant # GM066328.
Identifying molecular mechanisms regulating the enzymatic activity of TFIID subunit TAF1
Authors: Kloet Susan L , Wang Edith H
Published: June 2021

The largest subunit of transcription factor IID (TFIID) complex, TBP associated factor 1 (TAF1), possesses two serine/threonine kinase domains and intrinsic histone acetyltransferase (HAT) activity. Both TAF1 kinase and HAT activity are essential for transcription from a subset of genes and G1 progression in mammalian cells. TAF7, another TFIID subunit, directly binds TAF1 and inhibits TAF1 HAT activity. This interaction is disrupted by TAF1 phosphorylation. These data suggest a role for TAF1 kinase activity in mediating TAF1/TAF7 binding, transcriptional regulation and cell cycle progression. We have demonstrated that the N‐terminal kinase (NTK) and C‐terminal kinase (CTK) domains of TAF1 are capable of autophosphorylation and TAF7 trans‐phosphorylation in vitro. In vivo and in vitro methods identified Serine‐264 of TAF7 as a putative phosphosite for TAF1 NTK and CTK. Titration of TAF7 in a TAF1 kinase assay produced a biphasic dose‐response curve. This curve shape suggests TAF1 autophosphorylation levels affect TAF1 kinase activity or alternatively, TAF7 has two substrate sites for TAF1. The overall objective of these studies is to elucidate the molecular mechanism regulating the enzymatic activities of TAF1 and to understand their contribution to transcriptional regulation and cell cycle control. This work was supported by PHS NRSA 2T32 GM007270 from NIGMS and RSG‐04‐234‐01‐GMC from ACS.
Targeting G‐quadruplex DNA and associated proteins in cell chromatin using specific oligonucleotide probes
Authors: Zhang Huiping , Haegele Joe , Lin Brian , Wang Yuexi , Quinones Gerri , Ligon Lee A , McGown Linda B
Published: June 2021

Recent bioinformatics studies have suggested strong ties between G‐quadruplex (G4) forming DNA sequences and oncogene expression. Although there is not yet a direct proof that these sequences form G4 structures in vivo, numerous in vitro studies have suggested so.

We use G‐rich insulin‐linked polymorphic region (ILPR) located upstream of insulin gene as the model system to tackle this issue. If ILPR's G‐rich strand does form G4 in vivo during certain nuclear processes such as transcription, its complementary C‐rich strand would turn into an open loop in the process. By hybridizing the C‐rich loop with biotinylated oligonucleotides, we are able to capture ILPR's G4 structures and associated proteins directly from cells. Enzyme‐linked immuno‐sorbent assays are then utilized to identify the proteins.

This newly developed direct chromatin precipitation method is an important addition to the chromatin analysis toolbox, and should provide new directions in understanding the regulation of oncogenes at molecular level.
Cathepsin L Participates in the Production of the Dynorphin Opioid Peptide Neurotransmitter
Authors: Minokadeh Ardalan , Toneff Thomas , Funkelstein Lydiane , Reinheckel Thomas , Peters Christoph , Zadina James , Hook Vivian
Published: June 2021

Dynorphins, as well as other opioid neuropeptides, have an established role in analgesia and pain processing among other central nervous system functions. Dynorphin neuropeptides, as well as numerous peptide neurotransmitters, are generated by proteolytic processing of respective proneuropeptide precursors. Processing of proneuropeptides has recently been found to be mediated by the recently discovered cathepsin L cysteine protease in secretory vesicles. To assess the role of secretory vesicle cathepsin L in dynorphin production, studies in cathepsin L gene knockout mice and with cathepsin L protease expression were conducted. In brains of cathepsin L gene knockout mice, substantial reduction of prodynorphin‐derived neuropeptides was observed. Brain levels of dynorphin A, dynorphin B, and a‐neoendorphin were decreased by more than 70% in cathepsin L knockout mice compared to wild‐type. Expression of cathepsin L with prodynorphin in neuroendocrine PC12 cells resulted in increased production of dynorphin A, indicating cathepsin L mediated conversion of prodynorphin to the dynorphin peptide product. Moreover, colocalization of dynorphins with cathepsin L in secretory vesicles of neuronal cells was demonstrated by immunofluorescence confocal microscopy. These results demonstrate a significant role for secretory vesicle cathepsin L in the produciton of dynorphin opioid neuropeptides.
How Do Small Heat Shock Proteins Function?
Authors: Rajagopal Ponni , Jehle Stefan , Caputo Christine , Cho Seung‐Hee , Oschkinat Hartmut , Klevit Rachel
Published: June 2021

We are investigating the structure of human αB‐Crystallin (αB), an archetypal member of the small Heat Shock Protein (sHSP) family using an hybrid solid‐solution state NMR approach and probing functional questions with the α‐crystallin domain that's common to all sHSPs. The polydisperse, ~580 kDa αB is assembled from 20 kDa subunits and has an 89‐residue α‐crystallin domain. We have isolated the α‐crystallin domain (αB10.1) for solution NMR studies and we are using solid‐state NMR to investigate the structure of αB. The dimeric α‐crystallin domain has an unique structure that's maintained in both the isolated and oligomeric forms. The unusual architecture may provide key answers to the functional mechanisms of αB. αB binds cardiac muscle proteins such as desmin and titin more tightly at acidic pH values presumably to maintain filament integrity of these proteins during cardiac ischemia which is accompanied by acidosis. A mutant, R120G‐αB, is associated with desmin‐related cardio‐myopathy. αB10.1 undergoes a sharp pH‐dependent dimer‐monomer transition in contrast to the mutant R120G‐αB10.1. pH‐dependent dissociation of the α‐crystallin domain can lead to disruption of the oligomer assembly, an event that's postulated to be essential for sHSP function. The absence of this dissociation in the mutant abrogates its protective role, thereby explaining its disease association.

Funding from NEI, 1R01EY017370
Analogues of cysmethynil that demonstrate improved isoprenylcysteine carboxymethyltransferase (Icmt) inhibition activity and antiproliferative activity in MDA‐MB‐231 breast cancer cells
Authors: Leow Jo Lene , Gorla Suresh Kumar , Wang Mei , Casey Patrick J , Go Mei‐Lin
Published: June 2021

Many eukaryotic proteins, such as the Ras family of GTPases, contain a C‐terminal CAAX motif which directs the proteins through a three‐step post‐translational process termed prenylation. Icmt catalyzes the methylation of the C‐terminal prenylcysteine found on the prenylated protein, the last step in this process. Many studies have shown that inhibition of Icmt results in mislocalization and impaired function of the prenylated proteins, significantly impacts oncogenic transformation. Cysmethynil, an indole core small molecule inhibitor of Icmt, has been shown to inhibit cancer cell growth in an Icmt‐dependent fashion. Further evaluation of its mechanism of cell death in prostate cancer cells has revealed that the induction of autophagy is a major contributor.

In an attempt to optimize activity, lead optimization of the indole core structure of cysmethynil was carried out and a series of structural analogues were synthesized. Evaluation of their enzyme inhibition and antiproliferative activities identified two analogues that show improvement of these properties of cysmethynil while maintaining a favourable drug‐like profile. These findings confirm and enhance our understanding that indole core Icmt inhibitors have significant impact on cancer cell growth and survival, and these new analogues may prove to be better suited for preclinical study of Icmt inhibitors as cancer therapeutics.
Monitoring calcium‐deficient calmodulin in mouse embryonic stem cells before and after cellular differentiation
Authors: Israel Odisho Koma , Jervis Eric , Guillemette Joseph Guy
Published: June 2021

Calmodulin (CaM) is a calcium‐sensing protein that binds and regulates over 300 cellular proteins. Cellular localization and trafficking of calcium‐free CaM prior and after Ca2+ stimulation is not well understood. It has been reported that 95% of total endogenous CaM is bound to target proteins in the cytoplasm, and that the influx of Ca2+ results in its translocation to the nucleus. The present study was designed to investigate the Ca2+‐ dependent and Ca2+‐independent protein trafficking of CaM during cellular differentiation of mouse embryonic stem cell (MESC). Several CaM mutants were generated and labeled with a fluorescent marker for our investigation. In addition, a FITC labeled cell penetrating peptide (CPP) was used as a tool to introduce these CaM mutants into the cytoplasm of MESCs while maintaining high cell viability. The effects of these modifications on protein trafficking in MESCs during cellular differentiation are monitored via live fluorescent imaging and confocal microscopy. This research is supported by NSERC (J.G.G)
Evidence that caveolin‐1 negatively modulates HIV‐1 replication
Authors: Simmons Glenn , Hildreth James E.K.
Published: June 2021

Caveolin‐1 is an integral membrane protein expressed in a number of cell types including macrophages. Caveolins are primarily responsible for the formation of intracellular vesicles known as caveolae, which are involved in protein trafficking and cholesterol homeostasis. In recent years cholesterol has been demonstrated to have an essential function in both HIV infectivity and replication. It has been demonstrated that overexpression of caveolin‐1 in cells has a suppressive effect on HIV‐1 protein expression. We find that upon cotransfecting 293T cells with HIV‐LTR reporter and caveolin‐1 expression constructs there is a marked decrease in the level of HIV‐1 transcription. This finding suggests that the suppressive effects of caveolin‐1 are imposed upstream of HIV‐1 protein translation and independent of protein half‐life. Reduction of endogenous caveolin‐1, via siRNA‐mediated activity in HIV‐1 expressing 293T cells, resulted in an increase in the levels of viral proteins and virus release. Taken together, these data support caveolin‐1 as a novel host restriction factor against HIV‐1 replication. The work was supported by NIH (5 T32 AI007281).
How falsely believing you are in control can shape brain responses to aversive stimuli using functional magnetic resonance imaging
Authors: Bickart Kevin , Sauder Colin , Kim Dae‐Shik , Putnam Katherine
Published: June 2021

How does falsely believing you are in control affect the brain's response to aversive stimuli? The goal of this study was to use functional magnetic resonance imaging to identify brain regions that mediate the effect of perceived control on emotional experience. Placebo research suggests that a cingulofrontal brain network might mediate the effect of thoughts or beliefs on aversive experience. Thinking more positively about aversive stimuli might modulate brain regions such as the amygdala which encode the emotional salience of stimuli. In our study, participants were lead to believe they had control over the duration of time that aversive photographs were visible by performing a button‐pressing task during brain imaging. In conditions when participants believed they had control, they exhibited significant increases in functional activity within a dorsal cingulofrontal brain network and significantly reduced activation within both amygdalae. Participants also reported less aversion to the stimuli they believed to be controllable. From our findings, we suggest that even the illusion of control might be sufficient to recruit brain regions implicated in the cognitive control of emotion.
Measurement of TNF induced oxidative stress in the paraventricular nucleus using electron paramagnetic resonance spectroscopy
Authors: Mariappan Nithya , Francis Joseph
Published: June 2021

Recent findings from our lab suggest that TNF plays an important role in sympathoexcitation in heart failure. We have demonstrated that TNF treatment induces oxidative stress and contributes to end organ damage. In this study, we used a novel EPR method to determine whether chronic TNF treatment induces oxidative stress in the paraventricular nucleus of the hypothalamus.

Adult male Sprague‐Dawley rats were divided into four groups: control; TNF treatment; TNF + apocynin (APO; 200 μmol/kg); and TNF + TEMPOL (TEMP; 300 μmol/kg). TNF (40 μg/kg) was injected daily for five days. Rats were sacrificed in a CO2 chamber. We used EPR, the most sensitive and definitive method for quantification of free radicals, to determine reactive oxygen species levels in the PVN. EPR is superior to other free radical detection methods in that it allows for the direct measurement of specific free radicals using specific spin probes. The PVN was micro punched and treated with the specific spin probes 1‐hydroxy‐3‐methoxycarbonyl‐2, 2, 5, 5‐tetramethylpyrrolidine (CMH), for the measurement of total reactive oxygen species and superoxide by EPR. Treatment with TNF increased total ROS and superoxide levels in the PVN (p<0.05). In contrast, treatment with the NADPH oxidase inhibitor apocynin or with tempol attenuated the TNF induced oxidative stress. These findings suggest that EPR is a novel and sensitive method for the measurement of free radicals in brain nuclei.
Enhanced Microdialysis Sampling in vitro Recovery of Heparin‐Binding Cytokines
Authors: Newhart William Henry , Stenken Julie , Herbaugh Anthony , Duo Jia
Published: June 2021

Microdialysis sampling, a well known diffusion‐based separation method, has been used to collect solutes from various tissues in selected organisms. Cytokines have low aqueous diffusion coefficients and can frequently be in low (fM to pM) concentrations. The purpose of this study was to analyze heparin for its ability to increase the RR of selected cytokines. Heparin is a naturally‐occurring glycosaminoglycan and has high binding affinity to many types of cytokines. Two cytokines, monocyte chemoattractant protein‐1 (MCP‐1) and RANTES were chosen since we have confirmed their binding to heparin using surface plasmon resonance (SPR) to be KD = 5.08 ± 1.63 nM and 474 ± 110 nM respectively. For each cytokine, it was important to ensure that heparin did not affect the chemical analysis. Heparin concentrations in the range of 10 µM to 0.001 µM did not interfere with ELISA analysis. In this study, the relative recovery of the cytokine MCP‐1 was shown to increase with heparin concentrations 1 µM (18.34%) and 10 µM (21.20%), but possible interference by heparin of the ELISA analysis was observed. Cytokine RANTES was suspected to cause high dose hook effect with a modified ELISA protocol.
The Effect of Atrazine on the Hypothalamic Cell Line
Authors: Chakraborty Tandra R. , Chu Philemon , Chakraborty Sanjoy
Published: June 2021

Atrazine is a widely used herbicide. It has been shown to exhibit endocrine disrupting properties. A few studies demonstrate that atrazine enhances estrogenic activity by stimulating aromatase or by inhibits phosphodiesterase causes elevation of cAMP in cell lines which leads to increase in estrogen production. Although evidence supports that atrazine affects the hypothalamic pituitary gonadal axis, the specific molecular mechanism of atrazine‐induced endocrine disruption is not completely understood. The goal of this study is to analyze the in vitro effects and cellular mechanisms of atrazine‐induced endocrine disruption using N38 mouse hypothalamic cell lines. Initial observation has shown that exposure of the hypothalamic cells within the EPA recommended (0.014μM) limits causes 50% cell death within 12 hours. There was about 20% cell death within 10 minutes of exposure to atrazine. Trypan Blue cell counting analysis and 3‐(4,5‐ Dimethyl‐2‐thiazolyl)‐2,5‐diphrnyltetrazolium bromide (MTT) assay revealed a similar trend. Neuronal cell death could lead to apoptotic cell death causing activation of caspases. Analyses of caspases‐3 would confer the atrazine‐induced cell death and would be done using immunocytochemical techniques. Further, aromatase (CYP19) activity would be quantitated in order to access the signaling pathway and the mechanism of action.
The Structural Basis For the Dual Effects of Fatty Acids on Kv4 Channel Function
Authors: Timoney Gabriela Garcia , Spicer Lori M , Boland Linda M
Published: June 2021

Polyunsaturated fatty acids (PUFAs), including arachidonic acid (AA) and docosahexanoic acid (DHA), modulate fast‐inactivating Kv4/KChIP potassium channels, speeding inactivation by inhibiting the outward potassium current. These channels are localized to post‐synaptic regions of neurons and are linked to synaptic plasticity in the brain. This research is designed to identify structural determinants and a molecular mechanism PUFA inhibition of Kv4/KChIP channel. Recombinant channel subunits are expressed in frog oocytes and studied by two electrode voltage clamp electrophysiology. Using site‐directed mutagenesis, we substituted serine or alanine for a highly conserved threonine in the pore region. The T370S mutation largely prevented PUFA inhibition of outward potassium current, greatly reduced the kinetic effect of PUFAs on channels co‐expressed with KChIPs, and induced an unexpected leftward shift in the activation curve of the macroscopic current. To test the impact of this change in activation gating, a mutation in a conserved arginine of the S4 voltage sensor region of Kv4 channels was tested, inducing a similar leftward shift in activation gating, but failing to prevent inhibitory effects of PUFAs on outward current, when co‐expressed with KChIPs. This suggests that the ability of the T370S pore mutant to prevent PUFA inhibition of the current is not a result of the change in activation gating.
Effects of bFGF and EGF on expression of β1 integrin in mouse neuroepithelial cells
Authors: Suzuki Yusuke , Yanagisawa Makoto , Nakatani Yoshihiko , Yu Robert K
Published: June 2021

Integrins are cell adhesion and signaling molecules formed by two transmembrane subunits, α and β. In neuroepithelial cells (NECs) which consist of proliferative immature neural precursor cells capable of differentiating into glial or neuronal cells, β1 integrin is highly expressed as one of the subunits. The β1 integrin is known as a ligand for extracellular matrix and have been shown to play important roles in self renewal, proliferation, migration, and differentiation of NECs. Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) are also known to promote the proliferation and self renewal of NECs. In this study, we analyzed the effects of bFGF and EGF on β1 integrin expression in NECs and found that β1 integrin expression in NECs was increased by these growth factors in a dose‐dependent manner. These results suggest that bFGF/EGF and β1 integrin modulate NEC fates such as proliferation and self renewal via their crosstalk. (This work was supported by USPHS grants NS11853, AG027199 and a grant from the Childrens′ Medical Research Foundation, Chicago, IL.)
Structures and Binding Specificities of the human CHD family double chromodomains
Authors: Wiggins Kimberly , Kim Daesung , Khorasanizadeh Sepideh
Published: June 2021

CHD (Chromo Helicase DNA‐binding) polypeptides contain a pair of tandem chromodomains adjacent to a SNF2‐type helicase domain. Nine distinct genes in human encode CHD proteins all implicated in chromatin remodelling. Our group has established the structure and specificity of CHD1 which binds to a lysine 4 methylated histone H3 tail. Sequences in the CHD chromodomain regions suggest the family has expanded to acquire novel target selectivity. We expect these differences contribute to distinct epigenetic signalling during differentiation. Therefore, we are elucidating the structures and functions of uncharacterized human CHD chromodomains. Using in‐vitro fluorescence binding assays, we have characterized the binding specificities of CHD4, CHD7 and CHD9 chromodomains. While CHD7 and CHD9 prefer interactions with methylated histone tails, CHD4 prefers binding to DNA. The molecular bases for specificity in these complexes are being investigated by Nuclear Magnetic Resonance Spectroscopy and X‐ray Crystallography. Structural determinants of peptide and DNA binding will provide the knowledge necessary to design point mutations that could be tested in tissue culture studies to further elucidate the role of chromodomains during differentiation. NIH NIGMS funding is acknowledged through a Ruth L. Kirschstein NRSA and RO1.
Multiple populations of pluripotent stem cells exist in adult mammals, including humans
Authors: Young Henry E. , Black Asa C.
Published: June 2021

Previous studies from our laboratory noted two populations of pluripotent stem cells in adult mammals, including humans: blastomere‐like stem cells (BLSCs) and epiblast‐like stem cells (ELSCs) [Minerva Biotech 17:55‐63, 2005]. These two populations of stem cells were determined to be different, based on unique characteristics. The current study was designed to test whether any additional populations of pluripotent stem cells existed within adult mammals, including humans. Adult rats were euthanized following the guidelines of MU‐IACUC. Adult humans had their blood withdrawn by venipuncture following the guidelines of MU / MCCG‐IRB. Potential stem cells were isolated, cultivated and characterized utilizing previously established procedures. Two additional populations of pluripotent stem cells were discovered. This was based on their inherent size, Trypan blue staining, contact inhibition at confluence, growth requirements, activity in serum‐free defined media, lifespan, cell surface markers, and differentiation potential. Because of shared and unique characteristics, we designated these two populations of pluripotent stem cells as Tr‐BLSCs and Tr‐ELSCs. A full characterization of the four populations of adult‐derived pluripotent stem cells will be given as well as proposals for their potential to heal and/or repair damaged tissues. Funding provided by LM & HO Young Estate Trust and Dragonfly Foundation.
Role of MAPK/AP‐1 Signaling in the protection of 2‐choloroethyl ethyl sulfide (CEES) ‐ induced lung injury by antioxidant liposome
Authors: Mukhopadhyay Sutapa , Mukherjee Shyamali , Smith Milton , Das Salil K
Published: June 2021

We reported earlier that antioxidant liposomes containing N‐acetyl cysteine (NAC) and vitamin E protect against CEES‐induced lung injury. Our previous studies also indicated a role of MAPK/AP‐1 signaling in lung injury. The present study examines whether the protection of lung injury by antioxidant liposomes is mediated by a control of MAPKs/AP‐1 signaling. A single dose of the antioxidant liposome (200 μl) was administered intratracheally 5 min after CEES exposure. The animals were sacrificed either 1 h or 30 days after CEES exposure. The treatment caused a down regulation of CEES‐induced activation of TNF‐α. Although, no change was observed in the activation of any member of the MAPKs family, the activation of AP‐1 transcription factors was down‐regulated by the treatment. In addition, the antioxidant liposome treatment caused a decrease in the protein levels of two cell cycle proteins, cyclin D1 and PCNA. This suggests that NAC in combination with vitamin E as a liposome can be used as an effective antidote against lung injury. (Supported by US Army Grant W81XWH‐06‐2‐0044)
Fungal endophytes: an untapped natural products source of cytotoxic agents
Authors: Lee Kyung‐Seon , Portman Jonathan L , Sweeney Colleen A , Cheung Anthony TW
Published: June 2021

Endophytes are microorganisms that live within the intercellular space of almost all vascular plants. Endophytes benefit their hosts by producing bioactive secondary metabolites that prevent invasion by pathogenic agents. Endophytic metabolites that specifically kill plant‐pathogenic or other competing microorganisms might also have bioactive or cytotoxic effects in humans, and thus represent a potential source of medicinal compounds. It is estimated that 95% of fungal endophytes remain undiscovered and unexplored. In the present study, fungal endophytes were isolated from medicinal plants collected from locations within the central valley area of California. The plants were plated onto potato dextrose agar plates under aseptic conditions and incubated at 28°C in the dark until endophyte isolates emerged. Each endophyte was then grown in potato dextrose broth at 28°C until sufficient fungal mass was obtained. Cytotoxicity of the endophyte culture media was then assessed by formazan precipitation assay in Hep3B cells (human hepatocellular carcinoma) and rat hepatocytes. Among 25 endophyte cultures tested, 15TWA01 from blue gum (Eucalyptus globules) showed 60‐92% and 20‐60% cytotoxicity in Hep3B cells and rat hepatocytes, respectively. Future studies will determine the cytotoxic component(s) of the 15TWA01 culture, as well as the mechanism of cytotoxicity.
TYLCV Influences Biomolecules in Plant Tissues
Authors: Montasser Magdy S. , Al‐Own Fadaá Duaij , Afzal Mohamed A.
Published: June 2021

Tomato yellow leaf curl virus (TYLCV) was characterized as a whitefly‐transmitted monopartite Geminivirus. The viral genome DNA fragment of 1.1 kb was successfully amplified from infected tissues using polymerase chain reaction (PCR) technology. Chemical constituents of infected tomato leave tissues were severely influenced. Photosynthetic pigments, chlorophyll a and b, total lipids, saturated and unsaturated fatty acids, sugar and protein contents were significantly decreased compared with healthy tissues of test plants. On the other hand, total phenols accumulated to a greater extent in infected leaves. The concentrations of ascorbic acid, as an antioxidant, leaf extract pH were not dramatically affected by the virus. The extent of increase or decrease of such constituents was varied based on the plant developmental stages at the time of infection. It was concluded from this study that some of the biochemical constituents in tomato leaves were changed in response to TYLCV infection.
Flavonoids protect against endothelial dysfunction by induction of heme oxygenase‐1
Authors: Zheng Yuanyuan , Oesterling Elizabeth , Toborek Michal , Hennig Bernhard
Published: June 2021

Flavonoids have been suggested to protect against atherosclerosis by their anti‐oxidant and anti‐inflammatory properties. Little is known about the mechanisms and flavonoids structures required for prevention against endothelial cell dysfunction in the development of atherosclerosis. Heme oxygenase (HO‐1) is an enzyme that plays an important role in protecting against endothelial cell oxidative stress and inflammation. We hypothesize that flavonoids with specific structures protect against endothelial cell inflammation by induction of HO‐1. We found that the 2, 3‐double bond, 4‐keto group and 3‐hydroxyl group of the C‐ring are the main structure requirements for the induction of HO‐1. Flavonoids with these chemical structures, such as epigallocatechin gallate (EGCG), quercetin, and galangin up‐regulated the expression of HO‐1, and protected against hydrogen peroxide‐induced oxidative stress as well as TNF‐α‐induced prostaglandin PGE‐2 expression. These protective effects were partially blocked by pre‐treatment with tin‐protoporphyrin IX, the inhibitor of HO‐1. These data suggested that the protective effects of specific flavonoids against oxidative stress and inflammation are regulated through induction of HO‐1. These data add to nutritional recommendations for optimal cardiovascular health. (Supported by grants from NIEHS, NIH (P42ES07380) and the University of Kentucky AES).