This study presents a viable strategy using fmoc-protected peptides hydrogels, to encapsulate and stretch mesenchymal stem cells (MSC). To explore the peptide hydrogel potential, a custom mechanical stretching device with polydimethylsiloxane (PDMS) chambers were used to stretch MSCs encapsulated in Fmoc hydrogels. We investigated the impact of fmoc- FF prepared in dimethyl sulfoxide (DMSO), 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and deionizied water in the self-assembly, and mechanical properties of the gels. The peptide hydrogel is formed through molecular self-assembly of peptide sequence into β-sheets that are connected with the π-π aromatic stacking of F-F groups. The hydrogels provided a stiff, hydrated gel with round nanofiber morphology representing an elastic modulus of 174-266 KPa. MSCs cultured on peptide hydrogels undergo viability, morphology, and alignment evaluations using MTT, live/dead, and phalloidin (F-actin) staining. The F-actins of 3D- cultured MSCs in Fmoc-FF/HFP, and Fmoc-FF/DMSO followed by mechanical stretching showed elongated morphology with defined microfilament fibers compared to the round and spherical F-actin shape of the control cells. Peptide gels with 5mM concentration preserved 100% viability of MSC. Results reveals the feasibility and conditions for successful cell encapsulation and alignment within peptide hydrogels. Encapsulation of MSC in peptide nanofiber followed by a stretching process present a promising tissue engineering platform. By enhancing our understanding of MSC-peptide hydrogel interactions, this research con- tributes to the development of biomaterials tailored for regenerative medicine.

Coccidia are obligate intracellular parasites belonging to the phylum Apicomplexa. Many coccidia are of medical and veterinary importance such as Cystoisospora species and Toxoplasma gondii. The need to discover new anticoccidial therapies has increased due to development of resistance by the parasite or toxicity issues in the patient. The goals of this work were to develop a model for canine coccidiosis while proving that Cystoisospora canis is a true primary pathogen in dogs and to determine the efficacy of a new anticoccidial agent. A canine coccidiosis model would be useful in evaluating new anticoccidial treatments. Oral infections with 5 X 104 (n=2) and 1 X 105 (n=20) sporulated C. canis oocysts were attempted in 22 purpose bred beagle puppies. Clinical signs associated with disease were observed in all dogs. Bacterial and viral pathogens were ruled out by transmission electron microscopy (TEM) and bacterial growth assays. Development of C. canis in cell culture was also evaluated. The efficacy of ponazuril, a new anticoccidial drug, was examined in T. gondii. In vitro studies were conducted to determine the activity of ponazuril on tachyzoites and how this agent affects development of apicomplexcan parasites. The tachyzoite production assay was conducted. Ponazuril at a dose of 1.0 µg/ml had a significant affect on tachyzoite reproduction. Comparisons were made on how ponazuril affects T. gondii and Neospora caninum. Inhibition of T. gondii tachyzoites occurred after the second round of replication and with N. caninum tachyzoites after 4 rounds of replication. Results of TEM revealed ponazuril affects replication of T. gondii and N. caninum differently. The efficacy of ponazuril to prevent and treat acute and chronic toxoplasmosis was investigated. Mice treated prophylactically with ponazuril were completely protected from developing an acute T. gondii infection. Fatal toxoplasmosis was prevented in mice starting treatment 3 and 6 days post infection at a dose of 20 mg/kg. Immunohistochemistry was used to evaluate ponazuril's effect on chronic toxoplasmosis. Sections of brain were scored according to the number of tissue cysts present. Ponazuril also proved to be highly active against toxoplasmic encephalitis in an interferon-gamma knockout mouse model. / Ph. D.

Lipid oxidation is one of the leading causes of food quality degradation. Manufacturers typically add antioxidants or purge a product's package of oxygen to inhibit oxidation and the resulting off-flavors. Synthetic antioxidants (e.g. BHT, BHA) and some natural antioxidants (e.g. α-tocopherol) have found widespread use in this application. Unfortunately, the public views synthetic additives in a negative light and the current natural antioxidants have been unable to match the protection afforded by the synthetic antioxidants. The search for underutilized and natural antioxidants has led scientists to investigate many different plant-based extracts for use in food and in the treatment and prevention of disease. The objectives of this research were (1) to use ORAChromatography to identify peanut root extract fractions with high antioxidant capacity, (2) identification of compounds in peanut root extracts using HPLC and mass spectrometry, (3) test for the presence of aflatoxins in the extracts, (4) test peanut root extract in food model system for oxidation reduction capabilities, and (5) Testing peanut root extract's ability to decrease protein oxidation in cell culture. Crude peanut root extracts have high antioxidant activities that do not vary by cultivar. The ORAC activities of the peanut root fractions separated by HPLC with a C18 column varied (600.3 – 6564.4 μM TE/g dry extract), as did the total phenolic contents (23.1 – 79.6 mg GAE/g dry extract). Peanut root fractions had aflatoxins contamination well above the 20 ppb limit. Peanut root extracts and the known antioxidants tested were found to have no significant effect in inhibiting oxidation of peanut paste or HBMEC. Peanut root extracts were not shown to have any positive effects, but further research is necessary to eliminate peanut root extracts as a possible food ingredient and health supplement. / Ph. D.

No / There is evidence that both the herb Sanicle and the cytokine TGF- β3 can be beneficial in enhancing wound repair. In this study 3T3 fibroblast cells were cultured and the confluent monolayers were wounded (scarred) using a disposable plastic pipette. Various amounts of TGF-β3 (a growth factor) and Sanicle extract were applied to the cell monolayers. TGF-β3 was applied at concentrations of 50ng/ml, 5ng/ml, 500pg/ml, 50pg/ml and 5pg/ml to five different culture flasks with one additional flask acting as control. Sanicle was applied at concentrations of 100μg/ml, 10μg/ml, 1μg/ml, 100ng/ml and 10ng/ml with one additional flask as a control. The cells were imaged over a period of 20 hours with or without presence of TGF-β3 and Sanicle. The results indicated that although there were no significant increases in the rate of wound closure in relation to application of TGF-β3, there is an indication that TGF-β3 may enhance model wound closure at optimum working concentration between 5ng/ml and 50ng/ml. However, the sanicle extract did not stimulate enhanced repair of the model in vitro wound, but instead seemed to promote cell death along the wound margin. These results indicate that sanicle may be used in the care of wounds, but not as a growth promoter, but because it acts as an antibiotic agent, and possibly because it aids wound debridement.

Yes / Protocols which permit the extraction of primary astrocytes from either embryonic or postnatal mice are well established however astrocytes in culture are different to those in the mature CNS. Three dimensional (3D) cultures, using a variety of scaffolds may enable better phenotypic properties to be developed in culture. We present data from embryonic (E15) and postnatal (P4) murine primary cortical astrocytes grown on coated coverslips or a 3D polystyrene scaffold, Alvetex. Growth of both embryonic and postnatal primary astrocytes in the 3D scaffold changed astrocyte morphology to a mature, protoplasmic phenotype. Embryonic-derived astrocytes in 3D expressed markers of mature astrocytes, namely the glutamate transporter GLT-1 with low levels of the chondroitin sulphate proteoglycans, NG2 and SMC3. Embroynic astrocytes derived in 3D show lower levels of markers of reactive astrocytes, namely GFAP and mRNA levels of LCN2, PTX3, Serpina3n and Cx43. Postnatal-derived astrocytes show few protein changes between 2D and 3D conditions. Our data shows that Alvetex is a suitable scaffold for growth of astrocytes, and with appropriate choice of cells allows the maintenance of astrocytes with the properties of mature cells and a non-reactive phenotype. / BBSRC

Yes / The extraction of bioactive compounds, produced by plant cell cultures, directly from their culture medium, which contains other by-products, is a great challenge. Resveratrol extraction from its grapevine cell cultures is considered here as an example to improve the extraction processes from plant cell cultures using solubility parameters. Successive liquid-liquid extraction (LLE) processes were exploited to extract resveratrol from the culture medium with an extraction ratio approaching 100%, high selectivity and minimum amounts of solvents. The calculations of partition coefficients as a function of solubility parameters demonstrated that benzyl benzoate is the most suitable intermediate solvent to extract resveratrol from its aqueous medium. The calculations also illustrated the high ability of methanol and ethanol to extract resveratrol from benzyl benzoate. The physicochemical properties of benzyl benzoate and processing conditions were exploited to separate it from aqueous media and organic solvents. The agitation method, component ratios and extraction time were studied to maximize the extraction yield. Under the best studied conditions, the recovery of resveratrol from different culture media approached ∼100% with a selectivity of ∼92%. Ultimately, the improved extraction processes of resveratrol are markedly efficient, selective, rapid and economical. / Mohammad Amin Mohammad gratefully acknowledges CARA (The Council for At-Risk Academics, Stephen Wordsworth and Ryan Mundy) for providing the financial support for an academic fellowship.

Yes / The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variabil-ity and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmis-sion, we sought to determine if decellularisation and/or c-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5 M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without c-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, DNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and c-irradiation did not significantly affect the LSC phe-notype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p < 0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a c-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer.

Yes / Cytokines are extremely potent biomolecules that regulate cellular functions and play multiple roles in initiation and inhibition of disease. These highly specialised macromolecules are actively involved in control of cellular proliferation, apoptosis, cell migration and adhesion. This work, investigates the effect of transforming growth factor-beta2 (TGF-β2) on the biological regulation of chondrocyte and the repair of a created model wound on a multilayer culture system. Also the effect of this cytokine on cell length, proliferation, and cell adhesion has been investigated. Chondrocytes isolated from knee joint of rats and cultured at 4 layers. Each layer consisted of 2 × 105 cells/ml with and without TGF-β2. The expression of mRNA and protein levels of TGF-β receptors and Smad1, 3, 4, and 7 have been analysed by RT-PCR and western blot analysis. The effect of different supplementations in chondrocyte cell proliferation, cell length, adhesion, and wound repair was statistically analysed by One-way ANOVA test. Our results showed that the TGFβ2 regulates mRNA levels of its own receptors, and of Smad3 and Smad7. Also the TGF-β2 caused an increase in chondrocyte cell length, but decreased its proliferation rate and the wound healing process. TGF-β2 also decreased cell adhesion ability to the surface of the culture flask. Since, TGF-β2 increased the cell size, but showed negative effect on cell proliferation and adhesion of CHC, the effect of manipulated TGF-β2 with other growth factors and/or proteins needs to be investigated to finalize the utilization of this growth factor and design of scaffolding in treatment of different types of arthritis.

No / The development of therapeutics to treat cancer is conceptually more difficult than for nonlife-threatening diseases for several reasons, including its complex pathophysiological nature, the molecular individuality of each tumor, and the robustness and predictability of preclinical models toward determining efficacy and safety. A major limitation to development of a “blockbuster” therapeutic strategy is the infinite combination of cellular and molecular perturbations and associated heterogeneity of causative genetic factors driving disease progression. Although challenging, the diversity of drug targets, coupled with the lethality of the disease, has encouraged studies of a vast array of approaches and opportunities for disease treatment over the years.

BACKGROUND: Regulatory T cells (Tregs) are an essential subset of T cells that despite over 10 years of research have yet to be fully characterized. These suppressive immune cells, derived from the thymus, express high levels of interleukin-2 receptor alpha-chain (CD25). Tregs are needed to maintain self-tolerance and to control responses to non-self-antigen. The mechanism of Treg repression is unknown. The direct cell-to-cell contact through binding of cell surface molecules as well as secretion of suppressive cytokines has been shown to suppress the proliferation of Thl and Th2 cells against auto, allo and foreign antigens. The role of Tregs in regulating B cell response is also uncertain. The objective of this study is to determine how the removal of T regulatory cells can increase B cell responses in vitro. METHOD: This study focuses on the effect Tregs have on the generation of an antigen specific immune response in vitro. T cells with and without Tregs were co-cultured with monocyte derived dendrtic cells. The antigen specific activation was determined by analyzing cytokine production using intracellular cytokine staining, a flow based assay. Next, the effects of Tregs on both recall and naive B cell responses was analyzed using a co-culture of B cells, CD4 T cells with and without Tregs and monocyte-derived dendritic cells. Analysis of lymphoproliferation, activation, and antibody production was analyzed by using flow cytometry, Elispot and ELISA assays. RESULTS: An antigen specific response against gp120 was generated in naive T cell culture. Tregs were shown to inhibit antigen specific cytokine production in CD4 T cell culture to de novo antigens. The activation in the absence of Tregs was superior to the addition of exogenous factors of IL-2 and IL-7 with a third less non-specific background activation. When analyzed in a TT recall B cell assay, however, the removal of Tregs proved to have an inhibitory effect on antigen secreting cells detected by Elispot. This inhibition appeared at both a 1: 1 and 1 :4 T to B cell ratios though was slightly decreased at the 1 :4 ratio. The same was true for na1ve B cell assay showing a decrease in the generation ofMSPl-42 IgM antigen secreting cells. ELISA assays also confirmed the results showing a nearly 2.5 fold decrease in the amount of MSP 1-42 specific IgM Ab in the L TE cell culture supernatant. Conclusion: While the removal of T regulatory cells is beneficial for the activation of na1ve T cells, the removal of Tregs seems to be inhibitory to B cell activation in the LTE. This inhibitory effect maybe due to T cells becoming too stimulatory before culture with B cells. Studies involving a wider range of T to B cell ratios and culture times may be beneficial to determine if the depletion of Tregs would benefit this culture method.