• The objective of this study was to evaluate the effects of cold storage (4°C) on osteoblast viability and interleukin-6 (IL-6) production. Osteoblasts were harvested from murine calvaria utilizing sequential collagenase digestion and the Ficoll-Paque technique. Twelve-well plates were then inoculated with 2 X 104 cells/ml and placed in cold storage for up to 14 days. The cells were then incubated at 37°C for up to 20 days. During the incubation periods supernatants were collected daily and the cells were evaluated for alkaline phosphatase activity. We developed an enzyme immunoassay (EIA) for IL-6 which was sensitive to 50 pg/ml (standard curve r~ .996) with no cross reactivity with other recombinant murine cytokines. The EIA was th~n employed to assay the supernatants for IL-6 production. Storage at 4 oc for up to 48 hours resulted in a pre9ipitous drop in IL-6 production. After 48 hours in cold storage there was no bone cell via~ility.

      Bisch, Frederic C.; Department of Oral Biology (Augusta University, 1994-08)
      The objective of this study was to evaluate the effects of cold storage (4°C) on osteoblast viability and interleukin-6 (IL-6) production. Osteoblasts were harvested from murine calvaria utilizing sequential collagenase digestion and the Ficoll-Paque technique. Twelve-well plates were then inoculated with 2 X 104 cells/ml and placed in cold storage for up to 14 days. The cells were then incubated at 37°C for up to 20 days. During the incubation periods supernatants were collected daily and the cells were evaluated for alkaline phosphatase activity. We developed an enzyme immunoassay (EIA) for IL-6 which was sensitive to 50 pg/ml (standard curve r~ .996) with no cross reactivity with other recombinant murine cytokines. The EIA was th~n employed to assay the supernatants for IL-6 production. Storage at 4 oc for up to 48 hours resulted in a pre9ipitous drop in IL-6 production. After 48 hours in cold storage there was no bone cell via~ility.
    • ole conflict and ambiguity experienced by nurse administrators in hospital organizations

      Sleeth, Carolyn Cunningham; School of NUrsing (1986-06)
      The purpose of. this study was to e~amine the relationship between the degree and type of role conflict and rol~ ambiguity experienced by top, mid, and first level nurse administrat~rs and the nurse administrator's hierarchical level in hospital organizationso A descriptive correlational design was employedo Th~ subj~tts consisted of 119 nurse administrators who participated in an administrative staff development program in 4 local hospitals Demographic data and role conflict and ambiguity scores were obtained· from information on self-report questionnaires tha~ were administered prior to imple~entation of the program. Role conflict and role ambiguit~ were calculated from.the · Role Conflict and Ambiguity Scale devised by Rizzo, House and Lirtzman (1970). Analy~is of variance techniques were utilized to determine differences in the administrative group scores on role conflict and role ambiguity. Descriptive statistics were employed in additional analysis . to describe different types of role conflict the subjects were experiencing. No significant relationship was foun~ between the degree and _type· of role conflict experienced by the nurse administrators and their hierarchical level in thehospital organizations. Mid level nurse administrators reported significantly less role· a~biguity thari did top and first level administrators. All groups demonstrated distinct patterns ot role conflict type experiences.

      Rashid, Mohammad Harun; Department of Biochemistry and Molecular Biology (Augusta University, 2019-07)
      Exosomes are critical mediators of intercellular crosstalk and regulators of the cellular/tumor microenvironment. Exosomes have great prospects for clinical application as a theranostic and prognostic probe. Nevertheless, the advancement of exosome research has been thwarted by our limited knowledge of the most efficient isolation method and the in vivo trafficking. Here we have shown that a combination of two size-based methods using a 0.20 μm syringe filter and 100k centrifuge membrane filter followed by ultracentrifugation yields a greater number of uniform exosomes compared to other available methods. We demonstrated the visual representation and quantification of the differential in vivo distribution of radioisotope 131I-labeled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments, myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the exosomal protein/cytokine contents. Further, we also generated engineered exosomes expressing precision peptide for targeting CD206 positive M2-macrophages. M2-macrophages participate in immune suppression, epithelial to mesenchymal transition, invasion, angiogenesis, tumor progression and subsequent metastasis foci formation. Given their pro-tumorigenic function and prevalence in most malignant tumors with lower survival, early in vivo detection and intervention of M2-macrophages may boost the clinical outcome. To determine in vivo distribution of M2-macrophages, we adopted 111In-oxine based radiolabeling of the targeted exosomes and SPECT. When injected these radiolabeled targeted exosomes into 4T1 breast tumor-bearing mice, exosomes accumulated at the periphery of the primary tumor, metastatic foci in the lungs, in the spleen, and liver. Ex vivo quantification of radioactivity also showed similar distribution. Injected DiI dye-labeled exosomes into the same mice showed the adherence of exosomes to the CD206 positive macrophages on ex vivo fluorescent microscopy imaging, confirming the targeting efficacy of the exosomes. In addition, we utilized these engineered exosomes to carry the Fc portion of mouse IgG2b with the intention of augmenting antibody-dependent cell-mediated cytotoxicity. We have auspiciously demonstrated that M2-macrophage targeting therapeutic exosomes deplete M2-macrophages both in vitro and in vivo, and reduce tumor burden in a metastatic breast cancer model. The applied in vivo imaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.
    • Orthodontic movement with continuous and interrupted electrical stimulation

      Waugh, Robert L., JR.; Department of Oral Biology (1987-04)
      It has been reporteq th.at exoge.nous application of dir~ct currents can enhance the rate pf orthodontic tooth movement. In the present study externally applied constant direct current was ·delivered either continuously (24 hr/day) or discontinuously (8 hr/day) to, feline maxillary ( segments fitted with orthodontic app 1 i ances in order to ·compare the ·rates·· of in vivo orthodontic tooth movement that resulted from these two different modes_ of stimulation. The maxillary canines of 8 adult female· cats were tipped distally with bilateral coil springs attached between the canine and third premolar. · Curreni generators were incorporated into custom made appl ia·nces· that were· secured in the palate. These generators:_ delivered 15±1 ~A aGross the alveolus of the canines undergoing orthodontic movement~ Independent circuits established for the left. and right ~ani.nes in the s_ame cat allowed us· to compare the effects- of 24 h'r/day stimulation of one canine with the effects of 8 hr/day stimulation of the oppos.ite canine. These two periods of stimulation emulated full-time and overnight treatment periods,. respectively. ·oirect measurements of tooth movement were made using Vernier calipers in the following 3 areas: (1) canine to third incisor; (2) canine to second premolar; and (3) third incisor· to second premolar. Continuous (24 hr/day) application· of· electrical stimulation to orthodontically treated teeth enhanced the rate of tooth movement· when compared with orthodontically treated controls·. Nostatistically significant differenc.es ·(P<05) were found between continuous (24 hr/day) and interrupted (8 hr/day) electrical stimulation. Histological analyses of the treated tissues were employed in an attempt to correlate clinically measured tooth movement with ultrast~uctural events. Microscopic cell counts of··asteoclasts were made along the inner wall of canihe alveoli fa~ regions of orthodontic compressibn. Values· were determined for numbers of osteoc 1 asts per mil J imeter of 1 ami ria dura. · No significant differences (P<~OS) were found when comparisons were performed for teeth treated with continuous (24 hr/day), interrupted (8 hr/day) ~and control (.O hr/day) periods of electrical stimulation. · The immature bone area- analysis was then performed for· zones · of canine 1 am·; na dura corresponding to orthodontic tension. Polarized light photomicrographs allowed for mature and imm·ature bone to be distinguished and quantified using histomorphometric-. techniques. Significantly (.P<.OS). less immature· bone was found in _teeth· treated 8 hrs/day when compared with contrb 1 s. , .· Teeth treated with _interrupted applications of electricity (8 hrs/day) a.lso _showed significantly less immature bone when compared with-the continuously (24 hr/day) stj~ulat~d group~

      Klement, John; Department of Biochemistry and Molecular Biology (Augusta University, 2020-05)
      The host adaptive immune system functions to discriminate self from non-self, eliminating threats from viral infection to tumors. Cytotoxic lymphocytes (CTLs) are the primary effector arm of adaptive immunity. To prevent aberrant activation and autoimmunity, immune checkpoints function physiologically to restrain the CTL response. Tumors pathologically express these checkpoints, preventing immune-driven tumor clearance. Accordingly, immune checkpoint inhibitors (ICIs) have shown remarkable clinical success. However, many types of malignancies, as well as many individual patients with responsive tumor types, fail to benefit from current ICI immunotherapies. This conundrum suggests that as-yet undiscovered immune checkpoints exist. We observed that mice deficient in the transcription factor interferon regulatory factor eight (IRF8) tolerated allogenic tumor grafts and demonstrated impaired CTL activation with an accumulation of CD44hi memory-like CTLs. We sought to investigate the mechanism of this immunosuppression. Conditional deletion of IRF8 in T cells, as well as a mixed chimera model, demonstrated that IRF8 did not directly control CTL activation or differentiation into a CD44hi population. Instead, global loss of IRF8 lead to an expansion of an immature myeloid CD11b+Ly6G+Ly6Clo population which highly expressed osteopontin (OPN), a physiological ligand for CD44. Elevated levels of OPN were shown to suppress murine CTL activation and proliferation. A similar IRF8-OPN-CD44 axis was observed in murine and human colorectal cancer, which is refractory to current ICI therapies. Malignant cells and human patients displayed enhanced OPN levels relative to healthy donor controls. This was shown to be mediated by loss of IRF8 expression, which directly bound to the OPN promoter to repress its transcription. Elevated levels of OPN similarly prevented human CTL activation, and higher levels of OPN were correlated with decreased survival in human patients. We have shown that the IRF8-OPN-CD44 axis functions as a novel immune checkpoint in both myeloid and tumor cells. Blockade of OPN may have potent anti-tumor activity, expanding the pool of patients responsive to ICI therapy.
    • Oxidation of Dietary Amino Acids Disrupts their Anabolic Effects on Bone Marrow-Derived Mesenchymal Stem Cells

      El Refaey, Mona M.; Department of Cellular Biology and Anatomy (2016-07)
      Age-dependent bone loss has been well documented in both human and animal models. Since it has been proposed that aging is associated with an increase in the generation of damaging reactive oxygen species (ROS), our hypothesis was that the oxidized products of dietary amino acids could play a role in age-induced bone loss by altering osteoprogenitor cell differentiation and function or activating osteoclastic activity. We first examined the effects of the oxidized nutrients on the bone marrow-derived mesenchymal stem cells and our data showed a decrease in the protein and gene expression of osteogenic markers normally stimulated by nutrients. Aromatic amino acids activated signaling pathways involved in protein synthesis in vitro, and thus, in contrast, the oxidized metabolites of these aromatic amino acids had no effect on the activation of these anabolic pathways. We then examined the bone marrow concentration of the oxidized aromatic amino acids in mature (12 months) vs. aged (24 months) C57BL/6 mice and found that kynurenine, the oxidized product of the aromatic amino acid tryptophan, was found in the highest concentration in 12 months mice. Thus, we tested the effects of kynurenine, fed as a dietary supplement, on the bone mass of twelve-month-old C57BL/6 mice compared to a normal protein diet to see if the oxidized amino acid would induce a pattern consistent with age-related bone loss. Twelve-month-old, male C57BL/6 mice were fed one of four diets; 18% protein diet (normal protein diet); 8% protein diet + tryptophan; 8% protein diet + kynurenine (50 μM) and 8% protein diet + kynurenine (100 μM) for 8 wks. Bone densitometry and micro-CT analyses demonstrated bone loss following the kynurenine diet. Histological and histomorphometric studies showed a decreased bone formation and an increased MONA M. EL REFAEY Oxidation of Dietary Amino Acids Disrupts Their Anabolic Effects on Bone Marrow-Derived Mesenchymal Stem Cells (Under the direction of DR. CARLOS M. ISALES) osteoclastic activity in the kynurenine groups; these animals also exhibited an increase in serum pyridinoline, a marker of bone breakdown. Thus, these data demonstrate that feeding an oxidized product of an essential amino acid induces bone loss in a pattern consistent with accelerated aging, and we propose that one of the mechanisms involved in age-induced bone loss may be from alterations of dietary nutrients by the increased generation of ROS associated with aging.
    • p21B, a variant of p21wAF1/Cip1, is induced by the p53 family

      Nozell, Susan; School of Graduate Studies (2001-12)
    • ParaDIME: Genome-wide differential DNA methylation analyses using next generation sequencing

      Pabla, Sarabjot; Institute of Molecular Medicine and Genetics (12/27/2016)
      Epigenetic modifications are key players in the regulation of a plethora of cellular and physiological processes. DNA methylation is one of the most widely studied epigenetic modification. Genomic abnormalities in DNA methylation have been implicated in various complex diseases including cancer and autoimmunity. With advent of next generation sequencing, investigating DNA methylation patterns at genome-wide scale has become increasingly feasible. However, the pace of developing appropriate statistical methods to analyze large scale DNA methylation data has been slower. This can be attributed to both statistical and computational challenges faced by current methods. In order to overcome these statistical and computational shortcomings, we developed ParaDIME, a web application for differential DNA methylation analysis. ParaDIME tests CpG dinucleotide sites or pre-defined regions of CpG sites for differential DNA methylation using Rao-Scott chi squared test. ParaDIME not only uses a nonparametric test that accounts for differential sequencing coverage but also uses permutation testing to compute exact p values. In order to overcome computation challenges of large amount of permutations, we use parallel computing to share the workload and decrease execution time significantly. To test ParaDIME in-silico, we initially simulated bisulfitesequencing data and tested it against two most widely used methods: MethylSig and MethylKit. It performed equal or better at accurately detecting differentially methylated regions than both the methods. Especially, at important, low differences of percent methylation, ParaDIME performed better than existing tools. In order to test ParaDIME’s ability to detect biologically relevant differentially methylation regions (DMRs), it was then tested on publically available methylation data from chronic lymphocytic leukemia patients. Our method was able to detect previously known and experimentally verified DMR in CLL, especially DMRs located in Nfatc1 and FOXA2 genes. Additionally, it was able to detect other DMRs in genes present in caner related pathways. Due to ParaDIME’s ability to detect biologically relevant DMRs, we employed it in an integrative analysis study to identify epigenetically regulated genes in Sjogren’s syndrome mouse model, B6.NOD aec1/aec2. We performed reduced representation bisulfite sequencing and RNA sequencing on salivary glands of four and eighteen weeks old B6.NOD aec1/aec2 compared to age and gender matched C57BL/6 mice. After removing age and mouse model effect, we discovered 89 differentially expressed as well as differentially methylated genes. Spearman rank order correlation analysis found a significant correlation between DNA methylation and gene expression. Autoimmunity related genes Klf9 and Nfkbid showed significant negative correlation whereas, other genes like Fgf12 and Coll11a2 genes showed significant positive correlation. Subnetwork enrichment using MATISSE showed three jointly active connected subnetworks that were highly enriched in Immune system related pathways, especially, T cell and B cell activation along with cytokine signaling and endocrine system development. Evidence presented in this report presents a novel and a robust differential DNA methylation analysis method with high accuracy to detect disease-relevant DMRs. ParaDIME is a user-friendly and scalable web application with appropriate test statistic to analyze large-scale DNA methylation studies.
    • Parenting a child in the hospital : factors influencing maternal role alteration stress

      Bennett, Nancy R.; School of Nursing (Augusta University, 1993-03)
      The purpose of this descriptive correlational study was to investigate factors influencing parental role alteration stress of mothers with a child hospitalized on a general care pediatric unit. The subjects consisted of 30 mothers whose children were 7 to 59 months (4 years 11 months) and had no previous hospitalizations. The relationship of parent role alteration stress to perception of competency with normal child rearing tasks, the child's acuity of illness, and whether the child's admission was expected, not planned, or unexpected was investigated. The stress scores were low for the subjects' (mean 35.27) out of a possible range of 16-80. No significant relationship was obtained between parent role alteration stress and either competency or acuity. There were no significant differences among the stress scores when the children's admissions were expected, not planned or unexpected.
    • Partial Elucidation of the Primary Structure of the Heavy Chain of a Myeloma Protein: igG GAR

      Apelgren, Lynn David; Department of Cell and Molecular Biology (1977-06)
    • Partial elucidation of the structure of IgG 1 heavy chain disease protein BAZ

      Chang, Lebe S.; Department of Cell and Molecular Biology (1974-09)
    • Partnering With a Formal Program: Expanding the Boundaries of Family Caregiving for Frail Older Adults

      Poole, Deborah K.; Department of Biobehavioral Nursing (1999-12)
      Caring for frail older adults at home is an increasingly common lifestyle among American families. A growing array of community-based programs has been developed to assist family caregivers in this endeavor. Certain of these programs are comprehensive in nature and require a particularly close working relationship between the program’s health professionals and the lay caregiver at home. A paucity of literature exists that can act as a guide to formal and informal caregivers within such a context as they strive to develop an effective working relationship. This study used grounded theory methodology to develop a substantive theory of the process by which family caregivers of frail older adults establish and maintain a working relationship with a comprehensive formal caregiving system. The context of the study was a program belonging to the Program of All-inclusive Care for the Elderly (PACE) network. An initial sample of six primary caregivers of PACE participants was selected. The primary means of data collection was in-depth individual interviews with documents review also being used as a data source. An additional 13 primary caregivers were chosen via theoretical sampling for a total sample size of 19 informants. The method of constant analysis was employed to direct data acquisition and analysis until saturation was complete and the core variable was identified. The basic social-psychological problem identified by informants was termed Helplessness, defines by them as “needing additional help with caregiving.” Partnering with the Program was the basic social-psychological process informants used to relieve their helplessness in caregiving. Partnering with the Program was comprised of three phases: Connecting, Discovering Self, and Transcending Self. The first phase of Connecting represented “the honeymoon phase” of the relationship with the program and was made up of three stages: finding out, “joining up”, and adjusting. Discovering Self, the second phase, had three stages: communicating concerns, evaluating the program’s response, and expecting more. Informants in this phase related with the program in a conflicted manner, wanting to assert their autonomy but realizing their dependence on the program. The final phase, Transcending Self, was also made up of three stages. These stages were monitoring, advocating, and choosing to work it out. The hallmark of the final phase was that informants chose to have a positive, family-like personal relationship with the program staff rather than perpetuate conflict over unmet desires about service provision. This substantive theory provided information heretofore unavailable regarding the trajectory of close healthcare relationships from the perspective of the family caregiver. Implications of the theory related to health and social policy, clinical practice with older adults, and nursing knowledge are made explicit in the final chapter of the report.