Medicine and Health Sciences Commons^™

Characterization Of Cerebrospinal Fluid (Csf) Microbiota At The Time Of Initial Surgical Intervention For Children With Hydrocephalus, Shailly Pandey, Kathryn B. Whitlock, Matthew R. Test, Paul Hodor, Christopher E. Pope, David D. Limbrick, Patrick J. Mcdonald, Jason S. Hauptman, Lucas R. Hoffman, Tamara D. Simon, Cerebrospinal Fluid Microbiota In Shunts (Climb) Study Group

2020-Current year OA Pubs

OBJECTIVE: To characterize the microbiota of the cerebrospinal fluid (CSF) from children with hydrocephalus at the time of initial surgical intervention.

STUDY DESIGN: CSF was obtained at initial surgical intervention. One aliquot was stored in skim milk-tryptone-glucose-glycerol (STGG) medium and the second was unprocessed; both were then stored at -70°C. Bacterial growth for CSF samples stored in STGG were subsequently characterized using aerobic and anaerobic culture on blood agar and MALDI-TOF sequencing. All unprocessed CSF samples underwent 16S quantitative polymerase chain reaction (qPCR) sequencing, and a subset underwent standard clinical microbiological culture. CSF with culture growth (either after storage in …

Homozygous Mtap Deletion In Primary Human Glioblastoma Is Not Associated With Elevation Of Methylthioadenosine., Yasaman Barekatain, Jeffrey J Ackroyd, Victoria C Yan, Sunada Khadka, Lin Wang, Ko-Chien Chen, Anton H Poral, Theresa Tran, Dimitra K Georgiou, Kenisha Arthur, Yu-Hsi Lin, Nikunj Satani, Elliot S Ballato, Eliot I Behr, Ana C Decarvalho, Roel G W Verhaak, John De Groot, Jason T Huse, John M Asara, Raghu Kalluri, Florian L Muller

Faculty Research 2021

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP's substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate …

Growth, Detection, Quantification, And Inactivation Of Sars-Cov-2, James Brett Case, Adam L. Bailey, Arthur S. Kim, Rita E. Chen, Michael S. Diamond

2020-Current year OA Pubs

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. We have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious …

Reactive Oxygen Species And Nitric Oxide Signaling In Bystander Cells, Kishore Kumar Jella, Roisin Moriarty, Brendan Mcclean, Hugh Byrne, Fiona Lyng

Articles

It is now well accepted that radiation induced bystander effects can occur in cells exposed to media from irradiated cells. The aim of this study was to follow the bystander cells in real time following addition of media from irradiated cells and to determine the effect of inhibiting these signals. A human keratinocyte cell line, HaCaT cells, was irradiated (0.005, 0.05 and 0.5 Gy) with γ irradiation, conditioned medium was harvested after one hour and added to recipient bystander cells. Reactive oxygen species, nitric oxide, Glutathione levels, caspase activation, cytotoxicity and cell viability was measured after the addition of irradiated …

Adiponectin Partially Rescues High Glucose/High Fat-Induced Impairment Of Mitochondrial Biogenesis And Function In A Pgc-1Α Dependent Manner., H. Wang, W.-J. Yan, J.-L. Zhang, F.-Y. Zhang, C. Gao, Y.-J. Wang, W.B. Lau, L. Tao

Department of Emergency Medicine Faculty Papers

OBJECTIVE: Plasma adiponectin (APN) levels are decreased in diabetic patients. Dysfunctional mitochondrial biogenesis is involved in type 2 diabetes (T2DM) pathogenesis, by unclear mechanisms. The present study determined (1) whether myocardial mitochondrial biogenesis was impaired in cardiomyocytes exposed to a high glucose/high fat (HGHF) medium (a T2DM in vitro model), (2) the effects of APN administration upon mitochondrial biogenesis in cardiomyocytes affected by HGHF incubation, and 3) the involved underlying mechanisms.

MATERIALS AND METHODS: Neonatal rat ventricular myocytes (NRVMs) were isolated and incubated in HGHF medium. Mitochondrial function was assessed by ATP content, and fluorescent microscopic analysis of myocardial apoptosis …

Staphylococcus Aureus Biofilms Induce Macrophage Dysfunction Through Leukocidin Ab And Alpha-Toxin., Tyler D. Scherr, Mark L. Hanke, Ouwen Huang, David B.A. James, Alexander R. Horswill, Kenneth W. Bayles, Paul D. Fey, Victor J. Torres, Tammy Kielian

Journal Articles: Pathology and Microbiology

UNLABELLED: The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla …

Increased Production Of Interleukin-8 In Primary Human Monocytes And In Human Epithelial And Endothelial Cell Lines After Dengue Virus Challenge, Irene Bosch, Kris Xhaja, Luis Estevez, Gregory Raines, Heather Melichar, Rajas Warke, Marcia Fournier, Francis Ennis, Alan Rothman

Alan Rothman

The more severe form of dengue virus infection, dengue hemorrhagic fever, is characterized by plasma leakage and derangements in hemostasis. As elevated interleukin-8 (IL-8) levels have been observed in sera from patients with more severe disease manifestations, a study was initiated to look at the effect of dengue virus infection in vitro on proinflammatory cytokine secretion and expression. A significant increase in IL-8 levels in the culture supernatant of primary human monocytes infected with dengue 2 virus (D2V) New Guinea C (NGC) was found by enzyme-linked immunosorbent assay. Additionally, by reverse transcriptase PCR, the mRNA was also augmented. Among the …

Transfection Of C6 Glioma Cells With Glia Maturation Factor Upregulates Brain-Derived Neurotrophic Factor And Nerve Growth Factor: Trophic Effects And Protection Against Ethanol Toxicity In Cerebellar Granule Cells, N. Pantazis, A. Zaheer, Donghai Dai, S. Zaheer, S. Green, R. Lim

Donghai Dai

Glial cells play active roles in neuronal survival, as well as neuroprotection against toxic insult. Recent studies suggest that the brain protein glia maturation factor (GMF) is involved in intracellular signaling in glia. This study investigated whether or not GMF plays a role in the survival-promoting and neuroprotective functions of glia. C6 glioma cells were transfected in vitro with GMF utilizing an adenovirus vector. The transfected cells overexpressed GMF intracellularly, but did not secrete the protein. The conditioned medium (CM) was obtained from the GMF-transfected cells (CM-GMF) and tested on primary neuronal cultures, consisting of cerebellar granule cells (CGC). The …

Non-Typeable Haemophilus Influenzae Decreases Cilia Beating Via Protein Kinase Cε., Kristina L. Bailey, Tricia D. Levan, Daniel A. Yanov, Jacqueline A. Pavlik, Jane M. Devasure, Joseph H. Sisson, Todd A. Wyatt

Journal Articles: Pulmonary & Critical Care Med

BACKGROUND: Haemophilus influenzae infection of the nasal epithelium has long been associated with observations of decreased nasal ciliary beat frequency (CBF) and injury to the ciliated epithelium. Previously, we have reported that several agents that slow CBF also have the effect of activating protein kinase C epsilon (PKCε) activity in bronchial epithelial cells. The subsequent auto-downregulation of PKCε or the direct inhibition of PKCε leads to the specific detachment of the ciliated cells.

METHODS: Primary cultures of ciliated bovine bronchial epithelial cells were exposed to filtered conditioned media supernatants from non-typeable H. influenzae (NTHi) cultures. CBF and motile points were …

Culture Medium, Gas Atmosphere And Mapk Inhibition Affect Regulation Of Rna-Binding Protein Targets During Mouse Preimplantation Development., Michele D Calder, Patricia H Watson, Andrew J Watson

Obstetrics & Gynaecology Publications

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos …

Mitogen-Activated Protein Kinase (Mapk) Pathways Mediate Embryonic Responses To Culture Medium Osmolarity By Regulating Aquaporin 3 And 9 Expression And Localization, As Well As Embryonic Apoptosis., Christine E Bell, Nathalie M K Larivière, Patricia H Watson, Andrew J Watson

Obstetrics & Gynaecology Publications

BACKGROUND: In order to advance the development of culture conditions and increase the potential for supporting normal preimplantation embryo development in vitro, it is critical to define the mechanisms that early embryos utilize to survive in culture. We investigated the mechanisms that embryos employ in response to culture medium osmolarity. We hypothesized that mitogen-activated protein kinase (MAPK) pathways mediate responses to hyperosmotic stress by regulating Aquaporin (AQP) 3 and 9 expression as well as embryonic apoptosis.

METHODS: Real-time reverse transcription and polymerase chain reaction and whole-mount immunofluorescence were used to determine the relative mRNA levels and protein localization patterns of …

Mouse Preimplantation Embryo Responses To Culture Medium Osmolarity Include Increased Expression Of Ccm2 And P38 Mapk Activation., Barry Fong, Patricia H Watson, Andrew J Watson

Obstetrics & Gynaecology Publications

BACKGROUND: Mechanisms that confer an ability to respond positively to environmental osmolarity are fundamental to ensuring embryo survival during the preimplantation period. Activation of p38 mitogen-activated protein kinase (MAPK) occurs following exposure to hyperosmotic treatment. Recently, a novel scaffolding protein called Osmosensing Scaffold for MEKK3 (OSM) was linked to p38 MAPK activation in response to sorbitol-induced hypertonicity. The human ortholog of OSM is cerebral cavernous malformation 2 (CCM2). The present study was conducted to investigate whether CCM2 is expressed during mouse preimplantation development and to determine whether this scaffolding protein is associated with p38 MAPK activation following exposure of preimplantation …

Ligand-Signaled Upregulation Of Enterococcus Faecalis Ace Transcription, A Mechanism For Modulating Host-E Faecalis Interaction, Sreedhar R Nallapareddy, Barbara E Murray

Journal Articles

Enterococcus faecalis, the third most frequent cause of bacterial endocarditis, appears to be equipped with diverse surface-associated proteins showing structural-fold similarity to the immunoglobulin-fold family of staphylococcal adhesins. Among the putative E. faecalis surface proteins, the previously characterized adhesin Ace, which shows specific binding to collagen and laminin, was detectable in surface protein preparations only after growth at 46 degrees C, mirroring the finding that adherence was observed in 46 degrees C, but not 37 degrees C, grown E. faecalis cultures. To elucidate the influence of different growth and host parameters on ace expression, we investigated ace expression using E. …

Building New Culture Media For Assisted Reproductive Technology., Mary N. Egbuniwe, H. Lee Higdon Iii, William R. Boone

Journal of the South Carolina Academy of Science

No abstract provided.

Responsiveness Of Bovine Cumulus-Oocyte-Complexes (Coc) To Porcine And Recombinant Human Fsh, And The Effect Of Coc Quality On Gonadotropin Receptor And Cx43 Marker Gene Mrnas During Maturation In Vitro., Michele D Calder, Anita N Caveney, Lawrence C Smith, Andrew J Watson

Obstetrics & Gynaecology Publications

Substantially less development to the blastocyst stage occurs in vitro than in vivo and this may be due to deficiencies in oocyte competence. Although a large proportion of bovine oocytes undergo spontaneous nuclear maturation, less is known about requirements for proper cytoplasmic maturation. Commonly, supraphysiological concentrations of FSH and LH are added to maturation media to improve cumulus expansion, fertilization and embryonic development. Therefore, various concentrations of porcine FSH (pFSH) and recombinant human FSH (rhFSH) were investigated for their effect on bovine cumulus expansion in vitro. Expression of FSHr, LHr and Cx43 mRNAs was determined in cumulus-oocyte complexes to determine …

Sensitivity Of Bovine Blastocyst Gene Expression Patterns To Culture Environments Assessed By Differential Display Rt-Pcr., D R Natale, P A De Sousa, M E Westhusin, A J Watson

Obstetrics & Gynaecology Publications

The use of culture media to support the development of preimplantation embryos to the blastocyst stage is often associated with detrimental effects on normal development. These effects have been uncovered largely by investigating the phenotypic abnormalities displayed by fetuses and newborns derived from cultured preimplantation embryos. Research to understand the impact of culture on the embryonic developmental programme has focused on embryo metabolism, gene expression and genomic imprinting. We have used differential display RT-PCR to examine culture influences on global transcript pools in bovine embryos. Others have examined culture influences on candidate "marker genes" in cultured murine, ovine and bovine …

Amino Acid Concentrations In Fluids From The Bovine Oviduct And Uterus And In Ksom-Based Culture Media., Y M Elhassan, G Wu, A C Leanez, R J Tasca, A J Watson, M E Westhusin

Obstetrics & Gynaecology Publications

Amino acids in bovine oviductal and uterine fluids were measured and compared with those in modified simplex optimized medium (KSOM) supplemented with either fetal calf serum or Minimum Essential Medium amino acids in addition to bovine serum albumin, fetal calf serum or polyvinyl alcohol. Concentrations of cysteine, threonine, tryptophan, alanine, aspartate, glycine, glutamate, proline, beta-alanine, and citrulline were higher in oviductal fluids than in KSOM-based culture media. Nonessential and essential amino acids were present in ratios of 5:1 and 2:1 in oviductal and uterine fluids, respectively. Concentrations of alanine (3.7 mM), glycine (14.1 mM) and glutamate (5.5 mM) were high …

Impact Of Bovine Oocyte Maturation Media On Oocyte Transcript Levels, Blastocyst Development, Cell Number, And Apoptosis., A J Watson, P De Sousa, A Caveney, L C Barcroft, D Natale, J Urquhart, M E Westhusin

Obstetrics & Gynaecology Publications

The objectives were 1) to investigate the effects of oocyte maturation in serum-free and amino acid-supplemented defined media on oocyte transcript levels, blastocyst cell number, and apoptosis; 2) to investigate the influence of oocyte maturation culture atmosphere on blastocyst development, total cell number, and apoptosis; and 3) to examine the influence of epidermal growth factor (EGF) during oocyte maturation on blastocyst cell number and apoptosis. The results demonstrate that blastocysts derived from in vitro maturation, fertilization, and embryo culture protocols undergo apoptosis but that apoptotic levels are not greatly influenced by the oocyte maturation environment. Amino acid supplementation of oocyte …

Glycerol With Fetal Calf Serum - A Better Cryoprotectant For Blastocystis Hominis, Init Ithoi

Init Ithoi

In recent years, a growing number of reports have sug- gested that the protozoan parasite, Blastocystis hominis (Brumpt 1912), is a pathogenic cause of diarrhoea. It is imperative, however, that more studies are conducted on the various human isolates of the parasite before in- criminating it is a pathogen. In our laboratory we maintain human and animal isolates of Blastocystis using the monophasic medium Iscove's Modi®ed Dul- becco Medium (IMDM) as established by Ho et al. (1993), but maintaining continuous cultures is tedious, time consuming and expensive, and the parasites may be prone to genetic drift. There is therefore a …

Bovine Oviductal And Embryonic Insulin-Like Growth Factor Binding Proteins: Possible Regulators Of "Embryotrophic" Insulin-Like Growth Factor Circuits., Q A Winger, P De Los Rios, V K Han, D T Armstrong, D J Hill, A J Watson

Obstetrics & Gynaecology Publications

Bovine oviductal monolayer and vesicle primary cultures express insulin-like growth factor (IGF)-I and -II mRNAs and polypeptides. Early bovine embryos also express IGF-I, IGF-II, IGF-I receptor, IGF-II receptor, and insulin receptor mRNAs. This study reports the expression of IGF binding protein (IGFBP) mRNAs and polypeptides in bovine oviduct primary cultures and IGFBP mRNAs in preattachment embryos. Release of immunoreactive IGF-I and IGF-II by oviduct cultures and bovine blastocysts was also determined. IGFBP-2, -3, -4, and -5 transcripts were observed in oviduct primary cultures throughout an 8-day interval. IGFBP-1 and -6 mRNAs were consistently not detected in the oviduct. Messenger RNAs …

Removal Of Extracellular Chloride Suppresses Transmitter Release From Photoreceptor Terminals In The Mudpuppy Retina., Wallace B. Thoreson, R . F. Miller

Journal Articles: Ophthalmology

Removal of extracellular Cl- has been shown to suppress light-evoked voltage responses of ON bipolar and horizontal cells, but not photoreceptors or OFF bipolar cells, in the amphibian retina. A substantial amount of experimental evidence has demonstrated that the photoreceptor transmitter, L-glutamate, activates cation, not Cl-, channels in these cells. The mechanism for Cl-free effects was therefore reexamined in a superfused retinal slice preparation from the mudpuppy (Necturus maculosus) using whole-cell voltage and current clamp techniques. In a Cl-free medium, light-evoked currents were maintained in rod and cone photoreceptors but suppressed in horizontal, ON bipolar, and OFF bipolar cells. Changes …

Preimplantation Development Of In Vitro-Matured And In Vitro-Fertilized Ovine Zygotes: Comparison Between Coculture On Oviduct Epithelial Cell Monolayers And Culture Under Low Oxygen Atmosphere., A J Watson, P H Watson, D Warnes, S K Walker, D T Armstrong, R F Seamark

Obstetrics & Gynaecology Publications

The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.

Influence Of Temperature Of Incubation And Type Of Growth Medium On Pigmentation In Serratia Marcescens., Robert P. Williams, Cora L. Gott, S M. Qadri, Randolph H. Scott

Journal Articles

Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize …