bluuebunny/crossref_metadata_2025 huggingface dataset:
Age‐related changes in the skeletal muscle DNA methylome and transcriptome
Authors: Thalacker-Mercer Anna Elizabeth , Day Kenneth , Cui Xiangqin , Waite Lindsay , Absher Devin , Merritt Edward , Bamman Marcas
Published: June 2021

Advances in technology may assist with unraveling mechanisms linked to characteristic skeletal muscle (SkM) aging. The objective of this study was to identify age‐related differences in the SkM DNA methylome and transcriptome. DNA and RNA were isolated from resting state, old and young male, human SkM. Human Methylation BeadChips and Agilent microarrays were used to identify age‐related differences in SkM DNA methylation (DM) and gene expression (GE), respectively. Differentially methylated (1596) and expressed genes (1179) were identified between old and young SkM (False Discovery Rate of p<0.05). 52 genes were common between the male, aging DM and GE. Gene networks and functional annotation were determined with Ingenuity Pathways Analysis. Overrepresented functional categories related to the top gene network, molecular and cellular functions, and diseases/disorders included cellular development, growth, proliferation, and morphology, gene expression, cell cycle, inflammation, and neurological disease. Interestingly, 50% (26) of the common genes were associated with neurological diseases/disorders. Of these 26, 12 were related to neuromuscular disorders. These data support the hypothesis that age‐related changes in GE and perhaps phenotypes stem from changes in DM and highlight the need to understand the role of DM in neuromuscular aging.

Support: 5R01AG017896, VA Merit, 5UL1 RR025777, UAB Center for Aging, P30DK056336
Mechanical Properties of Incisors in Rodents
Authors: Druzinsky Robert E , Naveh Gili , Weiner Steve , Brumfeld Vlad , Klein Ophir D. , Charles Cyril
Published: June 2021

Rodents sharpen their incisors by grinding the lingual and buccal surfaces of opposing teeth. Normally the buccal surface is covered with enamel, whereas the lingual surface is not. While grinding enamel against dentin, the dentin wears and a sharp tip is created.

We studied lower incisors in mice carrying mutations in sprouty genes, the protein products of which are antagonists of receptor‐tyrosine kinase signaling. Sprouty loss‐of‐function mutants (Spry2+/−;Spry4−/−) have phenotypic features that mimic the ancestral condition in rodents, which include second incisors and enamel on the lingual surfaces of the incisors. We found that in some individuals the phenotype was asymmetrical, with enamel on the lingual surface of one lower incisor but not on the other.

We compared the properties of mutated and regular lower incisors from sprouty mutated mice. Teeth were axially loaded and scanned within a micro‐CT (Xradia). Normal incisors, without lingual enamel, bent lingually primarily at the worn and sharp part near the tip, whereas the teeth with enamel on both sides bent at a more apical location, with the result that the entire tooth bent. Much smaller excursions of the loading device were required to fracture the incisors with enamel on both sides. Our results demonstrate that enamel deposited on the lingual surface lowers the extent to which teeth bend and elevates the brittleness of the teeth.
Longitudinal morphometric study of the cervical vagus nerve in young Wistar‐Kyoto rats
Authors: Fontanesi Lucas Baptista , Schiavoni Maria Cristina Lopes , Salgado Helio Cesar , Fazan Valéria Paula Sassoli
Published: June 2021

Spontaneously hypertensive rats (SHR) are widely used as a model for essential hypertension in humans and the vagus nerve morphology in this model was already investigated [1]. Nevertheless, there is no description of the vagus nerve histological characteristics in the SHR normotensive counterparts, the Wistar‐Kyoto rats (WKY), except for a study of the vagus vascularization [2]. We investigated the cervical vagus nerve in 30 days old WKY. Rats were systemically perfused with a fixative solution and had their cervical vagi nerves prepared for light microscopy study. Proximal, medium and distal segments of the left vagus nerve were morphometrically evaluated with the aid of computer software. Comparisons were made between segments in order to evaluate possible longitudinal changes along the nerve. Differences were considered significant when p < 0.05. Fascicle morphometric parameters evaluated were area, number and density of myelinated fibers and number and density of Schwann cell nuclei. None of these parameters were different between segments. This is the first description of vagus nerve morphometry in WKY and these data will provide morphological basis for further studies involving functional investigations of the efferent arm of the baroreflex in SHR.

Grant Funding Source: FAPESP, CNPq and FAEPA
The morphology and clinical significance of the meningovertebra ligaments in the lumbosacral epidural space
Authors: Ding Zihai , Shi Benchao
Published: June 2021

Introduction

To determine the morphology and morphometry of the dorsal meningovertebral lig. in the lumbosacral segments and to discuss its clinical significance.

Methods

30 adult embalmed cadavers were used. The vertebral canal was divided to expose the dural sac and the spinal nerve roots. The morphology, quantity and attachment of the dorsal meningovertebral lig. in lumbosacral region was observed, and the length, width or diameter and thickness of the lig. was measured with a vernier caliper.

Results

The dorsal meningovertebral lig. in lumbosacral region connected the dura to lig. flava or lamina. The attachment points on lig. flava were relatively larger, the frequency was 97% at L5/S1. The greatest thickness of lig. was at L5, S1 vertebrae. The length of lig. varied from 6.0 to 40.24 mm, and the orientation of lig. was craniocaudal. The shape of lig. was divided into five types.

Conclusion

The dorsal meningovertebral lig. are the anatomical factor of dura laceration and epidural hemorrhage in flavectomy and laminectomy, they should be identified and deal with carefully in operation.
Do anatomical axes and physiologic activity correlate in rat endopiriform nucleus and claustrum?
Authors: Orman Rena , Rozenberg Inna , Scalia Frank
Published: June 2021

The endopiriform nucleus and claustrum are brain regions that are anatomically and functionally situated between the insular cortex and subcortical structures such as striatum. Subregions of these structures have been defined with various anatomical tracing methods and suggest a crude topographic relation of subregions with neocortical or subcortical (thalamic) areas. We studied endopiriform and claustral cells staining and morphology in male Sprague Dawley rats. The patterns of dendritic branching by cells suggest an alternative view of the overall orientation of these brain regions. The main dendritic branches of these predominantly bipolar neurons tend to run in a rostro‐dorsal to caudo‐ventral axis, angled off the horizontal plane. Fewer dendrites extended in the plane orthogonal to this axis. We have followed the same planes of section in our electrophysiological studies of field potentials in acute brain slices. We find that anatomical and physiological data are consistent with segregation of activity along the angled rostro‐caudal axis described above and suggest a basis for integrating inputs to and distributing the output of endopiriform nucleus and claustrum.

Grant Funding Source: Multiple Sponsors
Cadaveric Research: Informing Current Heart Surgery Techniques
Authors: Losenno Katie L , Johnson Marjorie , Chu Michael
Published: June 2021

Cadavers have long been used in the anatomy lab as teaching tools however, they can also provide valuable research that addresses common surgical problems. Aortic root enlargement (ARE) procedures are employed to facilitate the implantation of larger valve prostheses however, published evidence remains largely anecdotal with respect to the specific increases achievable with these techniques. We sought to design a study using a cadaveric model that will allow for a controlled experiment to evaluate the efficacy of commonly employed ARE techniques. We harvested 20 adult hearts and stratified them into four groups based on aortic annular diameter. Each heart underwent a bileaflet mechanical valve implantation following four ARE techniques with the appropriate reversals between procedures. Annular diameters and implanted prosthesis sizes were recorded following each enlargement technique. We performed all four techniques on each heart with little disruption to the remaining aortic root tissue. In light of the study design, each heart served as its own control. We were also able to provide data as to the increases in implanted prosthesis size achieved with the four ARE techniques. Despite the limitations, cadaveric specimens can play a key role in cardiac surgical research. They provide important data for preoperative planning and allow for experimental research without patient risk.

Grant Funding Source : Queen Elizabeth II Graduate Scholarship in Science and Technology
Ultrastructure and immunohistochemistry of innervation in the regions of the mouse sinuatrial and atrioventricular nodes
Authors: Rysevaite Kristina , Jokubauskas Marius , Saburkina Inga , Pauziene Neringa , Pauza Dainius H.
Published: June 2021

The study was aimed to examine (1) the ultrastructure of innervation in regions of atrioventricular and sinuatrial nodes, and (2) the density of nerve fibers in these regions comparing images of fluorescent immunohistochemistry and transmission electron microscopy. The expression and distribution of adrenergic, cholinergic nerve fibers and conductive cardiac myocytes within the correspondent heart regions were respectively determined applying immunohistochemistry for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), and hyperpolarization activated cyclic nucleotide‐gated potassium channel 4. Both the electron microscopy and immunohistochemistry of the sampled conventional regions of the SA and AV nodes demonstrate the dense meshwork of ChAT(+) and TH(+) nerve fibers, but ChAT(+) nerve fibers are evidently predominant therein. The examined nerve samples involved exclusively the unmyelinated nerve fibers, in which many axons possessed varicosities with mediatory vesicles. Some conductive myocytes in atrioventricular node were in close contacts with axons. Electron microscopy and immunohistochemistry for adrenergic and cholinergic neuronal markers confirm the densest and extremely complex networks of nerve fibers in the regions of the sinuatrial and atrioventricular nodes compared with the adjacent cardiac zones. Such morphologic pattern of innervation implies a complex neural control of the mouse cardiac conduction system. This study was supported by the Research Council of Lithuania (No. MIP‐11184).
Structural and biophysical characterization of the coiled‐coil complex between MBD2 and p66α integral to MBD2‐NuRD mediated silencing
Authors: Walavalkar Ninad Maheshwar , Desai Megha , Ginder Gordon D , Williams David C
Published: June 2021

The MBD2‐NuRD complex recognizes methylated DNA and silences expression of the associated genes. Previous structural work has shown that a heterodimeric coiled‐coil interaction between MBD2 and p66α proteins is important for the assembly of the NuRD complex and necessary for MBD2 mediated silencing in vivo (Gnanapragasam et al.,Proc Natl Acad Sci, 2011, 108:7487). To further characterize this interaction, we have compared binding affinity and stability between MBD2 homologues, truncations of the p66α coiled‐coil peptide, and mutations of key residues in each domain. Analytical ultracentrifugation analysis shows that the individual peptides are largely monomeric in isolation. Circular dichroism analysis of the different mutations and homologues shows a direct correlation between helical propensity of the coiled‐coil domains in isolation and the binding affinity of the complex. Furthermore, specific charge‐charge interactions and a minimum length of each peptide are necessary to maintain high‐affinity association. These factors lead to a relative binding preference hierarchy of p66α for the different MBD2 homologues (MBD2 = MBD3 > MBD3L2 = MBD3L1). These studies define the minimum requirements for developing a small peptide inhibitor of methylation dependent gene silencing by the MBD2‐NuRD complex. Supported by ACS IRG‐73‐001 (DCW) and NIH R01‐DK029902 (GDG).
Bortezomib Inhibits Expression of Immunosuppressive Cytokines IL‐10 and TGFβ in Cutaneous T Cell Lymphoma
Authors: Chang Tzu-Pei , Sanacora Shannon , Manna Subrata , Singha Bipradeb , Ramaswami Sitharam , Vancurova Ivana
Published: June 2021

CTCL is characterized by the clonal expansion of helper T cells that release the immunosuppressive cytokines IL‐10 and TGFβ, thus suppressing the immune responses. NFκB is constitutively activated in CTCL and many other cancers, where it plays a crucial role in cell survival and resistance to apoptosis. While NFκB regulation of pro‐inflammatory and anti‐apoptotic genes has been studied extensively, the NFκB regulation of immunosuppressive genes is incompletely understood. Bortezomib is the 26S proteasome inhibitor, which has shown promising results in several forms of cancer, including CTCL. We have demonstrated that bortezomib inhibits NFκB activity and expression of anti‐apoptotic genes by inducing nuclear translocation of IκBα in CTCL Hut‐78 cells. In this study, we investigated whether bortezomib inhibits expression of IL‐10 and TGFβ in Hut‐78 cells, and we analyzed the underlying mechanisms. Our results show that bortezomib inhibits both IL‐10 and TGFβ expression in Hut‐78 cells. Chromatin immunoprecipitation analyses indicated that bortezomib inhibits NFκB recruitment to the promoter regions of IL‐10 and TGFβ genes. Since a variety of malignancies utilize the immunosuppressive cytokines IL‐10 and TGFβ to evade the immune surveillance and facilitate tumor growth, a better understanding of the mechanisms regulating transcription of these cytokines may lead to more effective cancer therapies.

Funded by: NIH grants GM079581 and AI085497 to I.V.
Asymmetry in activator ring opens up Sigma54 dependent transcription in bacteria
Authors: CHOWDHURY SAIKAT , De Carlo Sacha , Diaz-Avalos Ruben , Rice William , Nixon B Tracy
Published: June 2021

Bacterial transcription requires the recruitment of the core RNA‐polymerase (RNAP) to specific promoter by a σ factor. This usually leads to rapid isomerization from un‐melted DNA (closed) to a melted state (open), from which transcription can begin. Unlike this, σ54 cannot spontaneously isomerize from closed to open complex state. This requires the system to be remodeled by bacterial Enhancer Binding Proteins (bEBPs). They belong to AAA+ ATPases superfamily. These activators are known to act as rings, but how this architecture assists them to perform mechanical work on targets is unknown. Conformational changes occur in the ring during nucleotide binding and hydrolysis. Recent crystal structures have shown heterogeneous nucleotide occupancy in the ring and co‐operative extension of the surface loops, which are believed to contact and remodel the polymerase. Here we present variations in the bEBP structure in the context of closed complex, by transmission electron microscopy. They have shown persistence of ring asymmetry, in the form of a split ring and hints at two possible sequential hydrolysis mechanisms in which the gap propagates around the ring in a constant or varied position relative to the rest of the asymmetric complex. Thus, it helps us to advances our understanding of how bEBPs use ATP binding and hydrolysis to regulate gene expression in bacteria.

This work has been supported by NIH.
Yeast mRNA 5′triphosphatase, Cet1p, Represses the Accumulation of RNA Polymerase II Towards the 5′end of Active Coding Sequence in vivo: Implications for Promoter Proximal Pausing
Authors: Lahudkar Shweta , Durairaj Geetha , Bhaumik Sukesh R.
Published: June 2021

Yeast mRNA 5′‐triphosphatase, Cet1p, recognizes phosphorylated‐RNA polymerase II as a component of the capping machinery via Ceg1p for co‐transcriptional formation of mRNA cap structure that recruits cap‐binding complex (CBC) and protects mRNA from exonucleases. Intriguingly, we find here that the absence of Cet1p significantly enhances the association of RNA polymerase II toward the 5′‐end of the ADH1 coding sequence in Sacharomyces cerevisae. Cet1p is recruited to the 5′‐end of the ADH1 coding sequence, and its absence impairs mRNA capping, and hence, recruitment of CBC. However, such an impaired recruitment of CBC does not enhance the association of RNA polymerase II toward the 5′‐end of the ADH1 coding sequence. Thus, Cet1p specifically represses the accumulation of RNA polymerase II toward the 5′end of the ADH1 coding sequence independently of mRNA cap‐structure or CBC at ADH1 in vivo. Similar results are also found at several other genes. Collectively, our results demonstrate for the first time a significant enrichment of RNA polymerase II toward the 5′‐end of the coding sequence in the absence of Cet1p, thus implicating a new role of mRNA 5′‐triphosphatase in repressing promoter proximal pausing of RNA polymerase II in vivo. Funding: National Institutes of Health
Probing the Role of a “First Shell” Interaction in Trypsin‐Fold Serine Proteases
Authors: Sharma Abriti , Baird Teaster
Published: June 2021

Several co‐crystal structures of Trypsin complexed with macromolecular inhibitors show that the backbone carbonyl oxygen of Phe41 accepts a hydrogen from a backbone amide of inhibitor. This hydrogen bond interaction seems to be facilitated by the interaction between the phenyl ring of Phe41 and methylene groups of Lys60. The variant Phe41 –>Ala (F41A‐Tg) was generated to probe the role and/ or contribution of position 41 in substrate/ inhibitor binding in S1′ pocket of Trypsin. The variant was constructed using PCR mutagenesis, expressed in P. Pastoris and purified using hydrophobic interaction chromatography. It was expected that the smaller Ala41 would allow significant deviation from wild‐type structure and decrease the interaction with substrate/inhibitor resulting in weaker binding. The initial kinetic characterization suggest that the substitution does decrease the proteolytic activity of Trypsin and that the position may play an instrumental role in Trypsin activity. [This work was supported by Research Infrastructure at Minority Serving Institutions Fellowship, NIMHD Award P20MD000544 (AS)]
On the role of Cys150 in the mechanism of maleamate amidohydrolase (NicF)
Authors: Noel Jeff W. , Spittle Nicholas E. , Snider Mark J.
Published: June 2021

The oxidative degradation of nicotinate (vitamin B3) to fumarate is considered a model system for the bacterial degradation of N‐heterocyclic compounds, yet only recently has the complete set of genes corresponding to the enzymes involved in this pathway been identified. The identification of these six genes has enabled structural and functional analyses of the enzymes of this catabolic pathway to be undertaken. Maleamate amidohydrolase (NicF), catalyzes the penultimate reaction of the pathway ‐ the hydrolytic deamination of maleamate to maleate. The crystal structure of NicF, determined at 2.4 Å using molecular replacement, shows that the enzyme belongs to the cysteine hydrolase superfamily. The crystal structure offers insight into the roles of potential catalytically important residues, notably a conserved triad (Asp29, Lys117, Cys150) hypothesized to catalyze an addition‐elimination mechanism. In an effort to examine the functional importance of Cys150 in the mechanism iodoacetamide was discovered to be a potent inactivator. To test whether inhibition of NicF by sulfhydryl blocking reagents is reversible a study with methyl methanethiolsulfonate (MMTS) will be conducted. In addition, results of the kinetic analyses of NicF variants, Cys150Ser/Ala and Asp29Asn/Ala, are reported. This work was supported by a HHMI Undergraduate Science Education Program Award to the College of Wooster.
Oxygen activation in the Copper Amine Oxidases
Authors: Mills Stephen A , Choi Eunah , Chow Courtney , Bitsimis Alexandra , Sommer Dayn J
Published: June 2021

Oxygen‐utilizing enzymes must activate oxygen in a very controlled manner to avoid oxidative damage to themselves and surrounding molecules. By studying the mechanism of oxygen activation in enzymes, we can understand how these enzymes controllably activate oxygen. The copper amine oxidases (CAOs) are a family of enzymes that utilize oxygen two oxidize primary amines to aldehydes and ammonia. In this reaction, O2 is reduced to H2O2 using to cofactors: topaquinone (TPQ) and Cu(II). Two mechanisms have been proposed for oxygen reduction: an outer‐sphere mechanism, where O2 is directly reduced by TPQ forming superoxide, which is stabilized by Cu(II); and an inner‐sphere mechanism, where TPQ initially reduces Cu(II) to Cu(I) to which oxygen binds and is reduced to superoxide. We have evaluated the mechanism of oxygen activation in CAOs through kinetic analysis and metal substitution. Replacement of Cu(II) with Co(II) blocks the inner‐sphere mechanism, since Co(II) would not be reduced to Co(I) in a protein, and can distinguish between the two mechanisms. We have replaced Cu(II) with Co(II) in several CAOs, including the CAOs from pea seedlings (PSAO) and from E. coli (ECAO) and evaluated the effect on catalysis. These results allow us to determine which mechanism each enzyme uses for catalysis. This work was supported by the University of San Diego.
Improvement in blood lipid levels by Eryngium carlinae in diabetic rats
Authors: Noriega-Cisneros Ruth , Ortiz-Ávila Omar , Esquivel-Gutiérrez Edgar , Clemente-Guerrero Mónica , Manzo-Avalos Salvador , Salgado-Garciglia Rafael , Cortés-Rojo Christian , Saavedra-Molina Alfredo
Published: June 2021

Diabetes mellitus is a significant risk factor for the development of cardiovascular complications. This study was undertaken to investigate the effect of chronic administration of ethanolic extract of Eryngium carlinae on glucose, creatinine, uric acid, total cholesterol, and triglycerides levels in serum of streptozotocin (STZ)‐ induced diabetic rats. Triglycerides, total cholesterol, and uric acid levels increased in serum from diabetic rats. The treatment with E. carlinae prevented these changes. The administration of E. carlinae extract reduced the levels of creatinine, uric acid, total cholesterol, and triglycerides. Thus, administration of E. carlinae is able to reduce hyperlipidemia related to the cardiovascular risk in diabetes mellitus. Histopathological studies showed significant changes like necrosis and degeneration in pancreas of diabetic rats, these histopathological abnormalities were not normalized after treatment with the extract.
Extracts of purple sweet potato protect pancreatic β‐cells from oxidative stress
Authors: Lin Chia-Yu , Li Yi-Ying , Chiang Tzu-Yuan , Jeng Toong-Long
Published: June 2021

Pancreatic β‐cells are relatively low in the expression of antioxidant enzymes, which render β‐cells more susceptible to oxidative damage. It has been implied that purple sweet potato (PSP) have anti‐oxidative and anti‐inflammatory effects, as well as may improve blood glucose levels, suggesting the potential benefits of PSP on diabetes. Therefore, the purpose of this study was to investigate the effects of PSP on pancreatic β‐cell protection. INS‐1 cells were treated with PSP and/or H2O2 for different concentrations and different period of time. While H2O2 treatment alone results in a less than 50% viable cells, PSP (10 or 5mg/ml) significantly improves the cell survival in the presence of H2O2 (150μM) toxicity after 24 hours (p<0.05). This recovered cell populations could be due to the enhanced cell proliferation as indicated by BrdU assay. Moreover, the H2O2‐induced intracellular reactive oxygen species generation is partially inhibited by PSP extracts (p<0.05), suggesting the free radical scavenging activity of PSP. The attenuated insulin secretion after H2O2 toxicity is also slightly but significantly rescued by PSP treatment. In summary, these findings indicate that PSP extracts may protect INS‐1 cells from H2O2‐induced oxidative stress and therefore improve cell survival and function.
Global and local administration of BDNF regulate dendrite patterning via different pathways
Authors: Kwon Munjin , Firestein Bonnie L
Published: June 2021

Appropriate dendrite patterning determines how information is received and processed by a neuron. Abnormal changes in dendrite number result in improper signaling between neurons. Dendritogenesis is regulated by extracellular and intrinsic factors, many of which have been characterized recently. Brain‐derived neurotrophic factor (BDNF) is one of the most studied extrinsic factors regulating dendrite outgrowth and branching. Our laboratory has previously shown that cypin (cytosolic PSD‐95 interactor), a guanine deaminase, increases dendrite number when overexpressed in cultured hippocampal neurons. We also found that global stimulation of BDNF alters proximal dendrite branching by transcriptional regulation of cypin, and cAMP response element‐binding protein (CREB) and mitogen‐activated protein kinase (MAPK) signaling pathways are involved in this action of BDNF. Here we show that local BDNF application has different effects on the dendritic arbor by using BDNF‐coated beads. These studies suggest that global and local exposure to BDNF results in different effects on dendritic arborization and that these effects are due to different signaling pathways.
Examination of the reproductive effects of SirT1 expression in neurons
Authors: Rickert Emily Lynn , Olefsky Jerrold M. , Webster Nicholas J.
Published: June 2021

Caloric restriction (CR) in females often leads to a decrease in reproductive function and it is thought that SirT1 mediates the effects of CR at the cellular level. We hypothesize that SirT1 acts as a trigger for reproductive inhibition during CR. To test this hypothesis, we generated two mouse models. The first model overexpresses SirT1 (OX) to mimic times of CR and the second model expresses a deleted SirT1 lacking the catalytic domain (KO) rendering the protein inactive and as such should be resistant to CR. Both models are neuron specific through the use of a synapsin‐driven cre recombinase mouse to excise either a floxed‐STOP or the floxed‐catalytic domain. Modulation of SirT1 in neurons alters body weight in both the overexpressing and knockout models. We will present data on estrous cyclicity, endocrine and steroid hormone levels, and fertility in these animals and on the effects of CR on the knockout model. This research is supported by NIH grants U54HD12303 and T32 DK007494.
The studies of self‐assembled xylose derivative on gold nanoparticles
Authors: Chang Shu-Mei , Chang Chia-Ying , Lin Yung-Lun , Hwa KuoYuan
Published: June 2021

The fast development of metal nanoparticles capped with organic monolayer is offering the possibility to create a whole new variety of products with novel characteristics, functions and applications. Among these, the nanoparticles covered with carbohydrates (glyconanoparticles) constitute a good bio‐mimetic model of carbohydrate presentation at the cell surface and are currently centered on many glycobiological and biomedical applications. In this paper, a series of novel D‐xylose gold nanoparticles with the linkers of alkyl or polyethylene glycol have been synthesized via D‐xylosethiols formed self‐assembled monolayers with gold nanoparticles (circa 5 nm diameter). D‐xylosethiols were characterized by FTIR, NMR. The characterizations of Xylose‐modified gold nanoparticles by TEM, UV‐Vis, FTIR and colorimetry techniques and bioassay are investigated.

This work is supported by the National Taipei University to SMC and KYH (NTUT 100‐140‐01, NTUT 99‐140‐04)
Protein kinase A is required for resistance to the lysosomotropic drugs quinacrine and chloroquine in the fission yeast, Schizosaccharomyces pombe
Authors: Yuan Zezhi , Wang Yiwei , Marcus Stevan
Published: June 2021

Yeast vacuoles, the functionally diverse counterparts of mammalian lysosomes, are highly dynamic organelles that undergo morphological changes during the cell cycle and in response to various cellular stresses. Here, we report results of an investigation on the contribution of protein kinase A (PKA) to vacuole integrity and dynamics in the fission yeast, Schizosaccharomyces pombe. Under normal culturing conditions, vacuoles in S. pombe wild type cells and PKA null (pka1Δ) mutants appear similar in size, morphology, and distribution. Wild type and pka1Δ cells also exhibit similar intensities of staining with quinacrine, a fluorescent lysosomotropic drug that accumulates in acidic organelles, indicating that PKA is not required for vacuole acidification in S. pombe. However, whereas quinacrine induces coalescence of vacuoles in wild type S. pombe cells, it does not induce a similar response in pka1Δ cells. Moreover, pka1Δ mutants are hypersensitive to the growth inhibitory effects of both quinacrine and the lysosomotropic toxin, chloroquine. Interestingly, we also found that the toxin rotenone, while having no obvious affect on vacuole morphology in wild type S. pombe cells, induced formation of small vacuoles aggregated at the nuclear periphery in pka1Δ cells, which are hypersensitive to growth inhibitory effects of the toxin. Our findings implicate PKA as a mediator of vacuole stress response in S. pombe.
Myosin Light Chain Plays a Role in Cell Division in the Fungus Aspergillus nidulans
Authors: Wendt Kristen E. , Pluta Michael , Jackson-Hayes Loretta , Hill Terry W
Published: June 2021

Cell division in the filamentous fungi involves inward growth of the plasma membrane at predetermined sites, accompanied by synthesis of new cell wall material to create a partially closed barrier termed a septum. Research in budding and fission yeasts has established a role for the actin/myosin cytoskeleton in this process. The mechanisms of septation in the filamentous fungi are less well understood. We have GFP‐tagged a protein (product of gene AN6732) from the filamentous fungus Aspergillus nidulans, which shows sequence homology to myosin light chains (MLC), demonstrating its localization to sites of septum formation. Strains in which we have replaced AN6732 with the Aspergillus fumigatus gene encoding RiboA are still able to form septa at lower growth temperature (e.g., 30°C). However septation is blocked at 37°C, at which temperature wild type strains septate normally. Thus, MLC's role in septation must be viewed as one of fine‐tuning of the process rather than as a component necessary for its completion. We show that under conditions where septation is blocked in the AN6732‐deletion strain, both myosin II (MyoB) and the Septation Initiation Network Protein MobA successfully localize as rings at potential septation sites, while in strains where either of those proteins is deleted or suppressed MLC does not localize to pre‐septal rings.
Characterization of the Role Of A Novel Gene In Drosophila Oogenesis
Authors: Puplampu-Dove Yvonne Ayordeley , Manning Lathiena , Starz-Gaiano Michelle
Published: June 2021

There are various genes that regulate cellular migration. Mutations in these genes have the ability to alter proper movement, which provides insight into the normal function of these genes. We are taking advantage of the well‐characterized genetics of Drosophila melanogaster to identify new genes regulating cell movement. In Drosophila, Ecdysone steroid hormones influence development and serve as genetic regulators that control the expression of specific genes. In other organisms, steroid hormones have been shown to influence cancers such as ovarian and breast. In this study, our interest is the role of an uncharacterized but well‐conserved EF‐hand‐containing protein in the migration of the border cell cluster to the oocyte. We postulate that the encoded protein may regulate actin dynamics. Using immunofluorescence, live‐imaging and gene expression analysis, we have examined the function for this novel gene. RNA interference (RNAi) knock down of the gene specifically in the border cells disrupted their proper migration, suggesting its importance in ovarian cell migration in Drosophila and may play similar roles in cells of other organisms.
CB2 receptor‐mediated Regulation of Prostate Cancer Cell Migration: Involvement of RhoA and Stress fiber formation
Authors: Jha Shalini , Jones Victoria , Burridge Keith , Mukhopadhyay Somnath
Published: June 2021

One of the earliest responses of cancer cells to extracellular factors is a rapid re‐organization of their actin cytoskeleton. Two distinct types of actin cytoskeleton reorganization in relation to cell migration have been observed on addition of growth factors: i) changes in the number of actin stress fibers, and ii) accumulation of actin filaments at the plasma membrane forming membrane ruffles. These processes are regulated by small g proteins known as Rho family of GTPases. Rho family GTPases serve as molecular switches that transduce signals from the extracellular environment via transmembrane receptors to elicit a cascade of spatiotemporal signaling responses leading to cell migration.

In the current investigation, we have extended these studies to characterize the molecular mechanism of CB2 receptor‐mediated cell migration. We have found that in androgen sensitive human prostate cancer cell, CB2 receptor activation produced RhoA activation and stress‐fiber formation in a time‐dependent manner. Further, we also found that blockade of RhoA activation attenuated CB2 receptor‐mediated inhibition of cell migration in prostate cancer cells. Collectively these results suggest that CB2 receptor‐mediated signaling cross‐talks with RhoA activation pathway regulates prostate cancer cell migration.
Relative contribution of hepatic CYP3A and CYP2E1 to oxidative stress upon CHIP E3 ligase knockdown
Authors: Kim Sungmi , Patterson Cam , Correia Maria Almira
Published: June 2021

Liver microsomal cytochromes P450 (P450s) are responsible for drug‐metabolism. CYP3A4 and CYP2E1 incur ubiquitin (Ub)‐dependent proteasomal degradation, wherein CHIP is an active E3 Ub‐ligase. We found that upon shRNA‐mediated CHIP‐knockdown (CHIP‐KD; ≈ 80%), functional parent and inactive ubiquitinated CYP3A and CYP2E1 species are stabilized. CHIP knockout (CHIP‐KO) is associated with enhanced oxidative stress and premature ageing in mice (Min et al.). To determine whether CYPs 3A and 2E1, both prone to oxidative uncoupling/radical production, could contribute to oxidative stress upon CHIP‐KD we monitored the levels of hepatic 15‐F2t‐isoprostane (15‐IP), a lipid peroxidation indicator. Upon CHIP‐KD of cultured rat hepatocytes, a 2.5‐fold 15‐IP increase was observed. To determine the relative contribution of CYP3A and CYP2E1 to this increase, we employed relatively selective functional probes. CYP3A inhibition with ketoconazole (KTZ, 10 μM) decreased 15‐IP levels by ≈15%, whereas CYP2E1 inhibition with 4‐methylpyrazole (4MP, 2.5 mM) caused a corresponding ≈80% decrease. Similar 15‐IP increases were observed in cultured CHIP+/− relative to CHIP+/+ mouse hepatocytes that were also largely abolished by 4MP but not KTZ. These findings reveal that such P450 stabilization is also pathophysiologically relevant: CYP2E1 rather than CYP3A is the major contributor to hepatocellular oxidative stress.
Opi1p relocalizaton in response to inositol in scs2Δpct1Δ mutant
Authors: Chang Yu-Fang , Gaspar Maria L. , Henry Susan A.
Published: June 2021

Phosphatidic acid (PA) is a signaling lipid that plays an important role in regulating phospholipid biosynthetic genes such as INO1. PA levels in yeast can be manipulated by shifting cells from inositol‐containing medium to inositol‐lacking medium or deletion of enzymes involved in biosynthesis of phosphatidylcholine (PC). Scs2p, a homolog of VAP, is an integral membrane protein that targets to the ER and nuclear membranes. Scs2p contains a motif (FFAT) that binds to Opi1p, a repressor of phospholipid biosynthesis which binds to PA. The scs2Δ mutant exhibits weak inositol auxotrophy (Ino‐phenotype) at 30°C. Co‐deletion of enzymes in Kennedy pathway for PC biosynthesis had been shown to rescue this Ino‐phenotype (Kagiwada and Zen, 2003). We have now shown that pct1Δ not only rescues the Ino‐phenotype of scs2Δ, it also rescues the choline sensitivity of scs2Δ at both 30 °C and 37 °C. The de‐repression of INO1 gene in the scs2Δpct1Δ strain was accompanied by Opi1p‐GFP relocalization to punctate structures resembling lipid droplets when the cells were grown in the absence of inositol.
ACSL1 multi‐tissue knockout mice are resistant to diet‐and age‐induced obesity and insulin resistance
Authors: Grevengoed Trisha J , Paul David S , Li Lei O , Coleman Rosalind A
Published: June 2021

Long chain acyl‐CoA synthetase 1 (ACSL1) is the major activator of long‐chain fatty acids in heart and adipose tissue. In order to understand the functional role of ACSL1, we studied mice with a temporally‐induced knockout of ACSL1 in multiple tissues (Acsl1T−/−), including heart, skeletal muscle, kidney, and white adipose. Compared to littermate controls (tamoxifen‐injected ACSL1flox/flox), lack of ACSL1 caused 80–90% lower oxidation of palmitate in heart and adipose. Compared to controls, hearts that lacked ACSL1 took up 75% less [1‐14C]bromopalmitate and 10‐fold more 2‐[14C]deoxyglucose. Compared to controls, chow‐fed Acsl1T−/− mice had higher rates of glycogen degradation in liver and heart. When fed a high fat diet (HFD; 45% of kcal from lard and soybean oil), ACSL1T−/− male mice failed to become obese. HFD‐fed ACSL1T−/− and control mice had similar food intake, respiratory quotients and heat production, but ACSL1T−/− mice were significantly more active at night. HFD‐fed control mice had abnormal glucose and insulin tolerance tests, whereas HFD‐fed ACSL1T−/− mice maintained normal whole‐body insulin sensitivity. With aging, 14‐month old chow‐fed ACSL1T−/− female mice also had a lower fat mass and better glucose tolerance than controls. Thus, ACSL1T−/− mice were protected from HFD‐and age‐induced obesity and insulin resistance.
The role of phosphatidate phosphatase in the biosynthesis of phosphatidylcholine via the CDP‐choline pathway
Authors: Han Gil-Soo , Carman George M.
Published: June 2021

In Saccharomyces cerevisiae, phosphatidylcholine is generated by two different routes, the CDP‐diacylglycerol pathway and the CDP‐choline branch of the Kennedy pathway. The CDP‐choline pathway, which requires diacylglycerol in the last step, plays a critical role when the enzymes in the CDP‐diacylglycerol pathway are defective. In this study, we examined the role of phosphatidate phosphatase in the synthesis of phosphatidylcholine via the CDP‐choline pathway. Lipid analysis showed that diacylglycerol used for the synthesis of the choline‐derived phosphatidylcholine was mainly provided by the PAH1‐encoded phosphatidate phosphatase, which has been shown to play a major role in producing diacylglycerol used for the synthesis of the storage lipid triacylglycerol. The lack of PAH1 in the cho2Δ opi3Δ mutant, which is defective in the CDP‐diacylglycerol pathway, caused a > 50% reduction in the synthesis of phosphatidylcholine via the CDP‐choline pathway. The production of phosphatidylcholine in the cho2Δ opi3Δ pah1Δ mutant indicates the role of as yet unknown enzyme(s) in providing diacylglycerol for phosphatidylcholine synthesis. Supported by NIH grant GM 28140.
Structural and functional characterization of Glucosidase II N‐glycan binding domain
Authors: Dahms Nancy M. , Olson Linda J. , Alculumbre Solana G. , Stigliano Ivan D. , Peterson Francis C. , Caramelo Julio J. , Parodi Armando J. , D'Alessio Cecilia
Published: June 2021

Glucosidase II (GII) is a key player in glycoprotein biogenesis in the endoplasmic reticulum (ER). GII is composed of a catalytic GIIα subunit, which removes sequentially the two innermost Glc residues from the Glc3Man9GlcNAc2 glycan on nascent proteins, and a GIIβ regulatory subunit. GIIβ retains GIIα in the ER and mediates N‐glycan in vivo recognition by GII through its C‐terminal mannose 6‐phosphate receptor homology (MRH) domain. Here we report the expression, purification and characterization of the Schizosaccharomyces pombe GIIβ subunit and its MRH domain. Purified GIIβ was active in a functional complementation test, as mixing the microsomal fraction of a S. pombe mutant expressing only GIIα in the ER with the purified GIIβ subunit restored the ability of the catalytic subunit to efficiently hydrolyze the physiological substrate G1M9. MRH domain was unable to functionally complement isolated GIIα activity. However, an excess of purified MRH domain inhibited full length GIIβ‐mediated restoration of GIIα activity. These results show that the MRH domain cannot interact with GIIα, suggesting that GIIβ G2B domain is involved in GIIα‐GIIβ interaction. The results also show that the MRH domain competes with GIIβ for G1M9 binding, and thus is fully functional in binding N‐glycans. The NMR structure of the β‐strand‐containing MRH domain was determined. (NIH‐NIDDK, Mizutani Foundation for Glycoscience, CONICET)
Genome reduction in yeast involves programmed cell death
Authors: Hurton Matthew , Roblee Emily , DiBiasio Eric , Bennett Richard , Austriaco Nicanor
Published: June 2021

Genome reduction is often associated with cell death. For example, in the yeast, Candida albicans, tetraploid strains undergo significant cell death when cultured on media that induce them to reduce their genomes to the diploid state. To investigate the mechanism of death in these cells, we have examined them to determine if the manifest any of the markers associated with programmed cell death. Dying cells were stained with dihydrorhodamine 123 and the FLICA reagent and imaged with a Zeiss 700 LSM confocal microscope. Both assays revealed that these dying cells contained reactive oxygen species (ROS) and activated caspases ‐‐ two markers for apoptotic cell death in yeast ‐‐ suggesting that these dying tetraploid cells are undergoing programmed cell death. Similarly, we have discovered that diploid Saccharomyces cells undergoing sporulation also stain positive for increased ROS and caspase activation. To better understand the mechanism of death, we are currently testing the ability of mutants lacking several apoptosis‐related genes to die as they undergo genome reduction.

This research has been funded by the following grants to N. Austriaco: 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.
ATP release is altered in a mouse model for Duchenne muscular dystrophy and signals for proteins that promote cell death
Authors: Valladares Denisse , Almarza Gonzalo , Pavez Mario , Jaimovich Enrique
Published: June 2021

Several reports have described changes in ATP signaling in models for muscular dystrophy; nevertheless, none of them has been directly related with the dystrophy pathology. To study this pathway we used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. ATP was measured using luciferase, protein expression by Western blots and mRNA by qPCR. The basal ATP release occurs in bursts peaking every 5 minutes in fibers from both mice, being several fold more important in mdx fibers. We also studied the expression of different components of this pathway in isolated triads. We found that P2Y2 receptor and pannexin‐1 are increased and P2Y1 receptor is decreased in mdx fibers. Moreover, ‘selective’ dye uptake suggests that the activity of pannexin channels is elevated in dystrophic fibers. Upon stimulation with extracellular ATP, we found that a number of genes are differentially expressed between normal and dystrophic fibers. Among these genes, over expression of proapoptotic genes as Bax, Bim and PUMA in mdx fibers contrast to the repression of the same genes seen in normal fibers. This study suggests that ATP signaling is altered in mdx fibers and this pathway appears to be involved in the activation of a mechanism related with muscle cell death in muscular dystrophy.

FONDAP‐15010006, AFM‐14562, FONDECYT‐1110467, CONICYT‐PhD fellowship (DV)
The Pronociceptive Activity of Spinal Dynorphin A is not due to Sensitization of Wide Dynamic Range Neurons
Authors: Cai Alice , Bannister Kirsty , Rankin David , Dickenson Anthony , Lai Josephine
Published: June 2021

Intrathecal administration of dynorphin A to the lumbar spinal cord induces transient hypersensitivity in naïve rats; this effect is blocked by dynorphin A antiserum, suggesting the existence of an underlying non‐opioid, pronociceptive mechanism of dynorphin A. Here we examined whether dynorphin A may promote the sensitization of wide dynamic range (WDR) neurons. In vivo extracellular recordings were performed on the lumbar spinal cord of anesthetized rats in the presence of a dose range of dynorphin A(1–13) (high affinity for opioid receptors) or dynorphin A(2–13) (Ki > 10 uM for opioid receptors). WDR neurons’ response was attenuated in naïve rats by dynorphin A(1–13). This attenuation was of greater magnitude in spinal nerve ligated rats with lower intensity thermal and mechanical responses. Dynorphin A(2–13) did not potentiate WDR response to nociceptive inputs in naive rats. These findings suggest that dynorphin A activates opioid receptors at the WDR to attenuate nociceptive transduction under physiological conditions. Thus, WDR neuron sensitization is unlikely to be a mechanism of the pronociceptive actions of intrathecal dynorphin A. This study is supported in part by NIH grant T37 MD001427.
Identification of Host Factors to Block Vascular Leakage after Exposure to Staphylocococcal enterotoxin B using the siRNA Approach
Authors: Abegunrin Oladimeji O. , Hoke Allison , Gautam Aarti , Day Agnes , Hammamieh Rasha , Jett Marti
Published: June 2021

Staphylocococcal enterotoxin B (SEB) is a toxin that induces an intense and rapid release of cytokines such as interferon‐γ, interleukin‐6, and tumor necrosis factor‐α. The induced “cytokine storm” results in vascular leakage of the endothelium and multiorgan failure. In this study, human lung microvascular endothelial cells (HMVEC‐L) transfected with siRNA were treated with peripheral blood mononuclear cells (PBMC) infected with SEB at 4 hours and overnight incubation. Initially, viability of PBMC after infection with varying concentrations of SEB was performed. The results showed less than or exactly 20% cytotoxicity of SEB to PBMC regardless of SEB concentration after overnight incubation. PBMC without SEB treatment had very minimal, less than 20%, cytotoxic effect on HMVEC‐L at a 1:1 ratio of PBMC to HMVEC‐L. Vascular permeability of HMVEC‐L cell monolayer after treatment with PBMC shows a gradual increase at all concentrations of SEB. Real‐time PCR and statistical analysis showed significant knockdown of these genes. These studies indicate that siRNA transfection is a novel approach, with significant implications as a therapeutic treatment for vascular leakage induced by SEB. Research support is through DTRA, TMTI.DRUG.02.10.WR.023.

“The views, opinions, and/or findings contained in this report are those of the authors and should not be construed as official Department of the Army position, policy, or decision, unless so designated by other official documentation.”