Detection of water quality changes through optimal tests and reliability of tests

Submitted to Office of Water Research and Technology.Bibliography: page 77.OWRT Project no. B-186-COLO; Grant agreement no. 14-34-0001-8069

Scaled-up expansion of equine cord blood mesenchymal stem cells (MSCs) from stirred suspension bioreactors to 100mL computer controlled stirred suspension bioreactors using computational fluid dynamic modeling

Musculoskeletal injuries are the leading cause of lameness and loss of performance in horses and conventional treatments are often associated with high rates of re-injury. Mesenchymal Stem Cells (MSCs) have shown promise for the treatment of such injuries in horses. Currently, the majority of studies are focused on the use of either bone-marrow derived or adipose-derived MSCs. However, equine cord-blood derived MSCs (eCB-MSCs) also provide a promising alternative, due to their high proliferation potential, ability to differentiate towards the chondrogenic lineage, and comparable immune-modulatory properties. Static adherent culture of eCB-MSCs has limited potential to produce sufficient cell numbers for large-scale research studies and possible commercial distribution. Expansion of cells in stirred suspension bioreactors using microcarriers as a scaffold has the potential to generate a large number of cells, using a significantly smaller space, under highly controlled conditions, with reduced time, labour, and monetary requirements. A robust protocol is required for the expansion of eCB-MSCs for use in large research studies and commercial applications. Initially, the hydrodynamic environment in the 10mL and the 100mL bioreactors was modeled using COMSOL Multiphysics software. The volume distributions of shear stress and energy dissipation rate in the bioreactors were calculated and used to determine the operating conditions that would create similar conditions within both scales of bioreactors. Next, eCB-MSCs were expanded in 10mL stirred suspension bioreactors and run at 60rpm and 80rpm with two different impeller geometries: paddles and rounded edges. The bioreactors were loaded at 4500 cells/cm2, and 2g/L microcarriers. The cells at different operating conditions in the 10mL bioreactors achieved varying population doubling times ranging from 0.8d to 1.1d with an average of 0.9d and initial cell attachment ranging from 5000 cells/cm2 to 7700 cells/cm2. The different speeds and geometries produced varying results with maximum attached cell densities from 35,000 to 50,000 cells/cm2 in the bioreactors, compared to maximum cell densities of 44,000 cells/cm2 achieved instatic growth. The expansion of eCB-MSCs was then scaled up in 100mL stirred suspension bioreactors with no direct pH or dissolved oxygen control, using 4500 cells/cm2 and 2g/L microcarriers, with a speed of 40rpm. At this larger scale, the initial cell attachment was 6900 cells/cm2 compared to 6300 cells/cm2 for the 10mL bioreactor. With respect to initial cell attachment, the 100mL bioreactor at 40rpm was most similar to the condition of 80rpm with round edge impeller geometry. The highest attached cell density in the 100 mL vessel was 70,000 cells/cm2. The 100mL uncontrolled bioreactor at 40rpm achieved the most similar results to the 10mL bioreactor run at 60rpm with paddled geometry, with respect to population doubling time with a doubling time of 0.93d for the 10mL bioreactor compared to 0.92d for the 100mL bioreactor. Finally, the eCB-MSCs were expanded in 100mL stirred suspension bioreactors at 4500 cells/cm2, 2g/L and 40rpm with pH and oxygen controlled at 7.4 and 21% DO, respectively, using the DASGIP bioreactor control system. This series of experiments revealed that eCB-MSCs can be expanded in stirred suspension bioreactors

Temporal localized states and square-waves in semiconductor micro-resonators with strong time-delayed feedback

In this paper we study the dynamics of a vertically-emitting micro-cavity operated in the Gires-Tournois regime that contains a semiconductor quantum-well and that is subjected to strong time-delayed optical feedback and detuned optical injection. Using a first principle time-delay model for the optical response, we disclose sets of multistable dark and bright temporal localized states coexisting on their respective bistable homogeneous backgrounds. In the case of anti-resonant optical feedback, we disclose square-waves with a periodic of twice the round-trip in the external cavity. Finally, we perform a multiple time-scale analysis in the good cavity limit. The resulting normal form is in good agreement with the original time-delayed model

Pepducin-mediated cardioprotection via β-arrestin-biased β2-adrenergic receptor-specific signaling

Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. β-arrestin (βarr)-biased β-adrenergic receptor (βAR) activation promotes survival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte death at the time of reperfusion to better preserve function. However, a lack of efficacious βarr-biased orthosteric small molecules has prevented investigation into whether this pathway relays protection against ischemic injury in vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of β2AR, allosterically engaged pro-survival signaling cascades in a βarr-dependent manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against ischemia/reperfusion (I/R)-induced injury in vivo. Methods: Wild-type (WT) C57BL/6, β2AR knockout (KO), βarr1KO and βarr2KO mice received intracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min) followed by reperfusion for either 24 h, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor the impact of ICL1-9 on long-term cardiac structure and function. Neonatal rat ventricular myocytes (NRVM) were used to assess the impact of ICL1-9 versus Scr pepducin on cardiomyocyte survival and mitochondrial superoxide formation in response to either serum deprivation or hypoxia/reoxygenation (H/R) in vitro and to investigate the associated mechanism(s). Results: Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and improved cardiac function in a β2AR- and βarr-dependent manner, which led to improved contractile function early and less fibrotic remodeling over time. Mechanistically, ICL1-9 attenuated mitochondrial superoxide production and promoted cardiomyocyte survival in a RhoA/ROCK-dependent manner. RhoA activation could be detected in cardiomyocytes and whole heart up to 24 h post-treatment, demonstrating the stability of ICL1-9 effects on βarr-dependent β2AR signaling. Conclusion: Pepducin-based allosteric modulation of βarr-dependent β2AR signaling represents a novel therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term cardiac remodeling benefits

Isolation of mesenchymal stem cells from equine umbilical cord blood

<p>Abstract</p> <p>Background</p> <p>There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC) have been isolated from fresh umbilical cord blood of humans collected non-invasively at the time of birth and from sheep cord blood collected invasively by a surgical intrauterine approach. Mesenchymal stem cells isolation percentage from frozen-thawed human cord blood is low and the future isolation percentage of MSCs from cryopreserved equine cord blood is therefore expectedly low. The hypothesis of this study was that equine MSCs could be isolated from fresh whole equine cord blood.</p> <p>Results</p> <p>Cord blood was collected from 7 foals immediately after foaling. The mononuclear cell fraction was isolated by Ficoll density centrifugation and cultured in a DMEM low glucose based media at 38.5°C in humidified atmosphere containing 5% CO₂. In 4 out of 7 samples colonies with MSC morphology were observed. Cellular morphology varied between monolayers of elongated spindle-shaped cells to layered cell clusters of cuboidal cells with shorter cytoplasmic extensions. Positive Alizarin Red and von Kossa staining as well as significant calcium deposition and alkaline phosphatase activity confirmed osteogenesis. Histology and positive Safranin O staining of matrix glycosaminoglycans illustrated chondrogenesis. Oil Red O staining of lipid droplets confirmed adipogenesis.</p> <p>Conclusion</p> <p>We here report, for the first time, the isolation of mesenchymal-like stem cells from fresh equine cord blood and their differentiation into osteocytes, chondrocytes and adipocytes. This novel isolation of equine cord blood MSCs and their preliminary <it>in vitro </it>differentiation positions the horse as the ideal pre-clinical animal model for proof-of-principle studies of cord blood derived MSCs.</p

Long-term insect herbivory slows soil development in an arid ecosystem

Although herbivores are well known to alter litter inputs and soil nutrient fluxes, their long-term influences on soil development are largely unknown because of the difficulty of detecting and attributing changes in carbon and nutrient pools against large background levels. The early phase of primary succession reduces this signal-to-noise problem, particularly in arid systems where individual plants can form islands of fertility. We used natural variation in tree-resistance to herbivory, and a 15 year herbivore-removal experiment in an Arizona piñon-juniper woodland that was established on cinder soils following a volcanic eruption, to quantify how herbivory shapes the development of soil carbon (C) and nitrogen (N) over 36–54 years (i.e., the ages of the trees used in our study). In this semi-arid ecosystem, trees are widely spaced on the landscape, which allows direct examination of herbivore impacts on the nutrient-poor cinder soils. Although chronic insect herbivory increased annual litterfall N per unit area by 50% in this woodland, it slowed annual tree-level soil C and N accumulation by 111% and 96%, respectively. Despite the reduction in soil C accumulation, short-term litterfall-C inputs and soil C-efflux rates per unit soil surface were not impacted by herbivory. Our results demonstrate that the effects of herbivores on soil C and N fluxes and soil C and N accumulation are not necessarily congruent: herbivores can increase N in litterfall, but over time their impact on plant growth and development can slow soil development. In sum, because herbivores slow tree growth, they slow soil development on the landscape. http://dx.doi.org/10.1890/ES12-00411.

The use of induced pluripotent stem cells in domestic animals: a narrative review

Induced pluripotent stem cells (iPSCs) are undifferentiated stem cells characterized by the ability to differentiate into any cell type in the body. iPSCs are a relatively new and rapidly developing technology in many fields of biology, including developmental anatomy and physiology, pathology, and toxicology. These cells have great potential in research as they are self-renewing and pluripotent with minimal ethical concerns. Protocols for their production have been developed for many domestic animal species, which have since been used to further our knowledge in the progression and treatment of diseases. This research is valuable both for veterinary medicine as well as for the prospect of translation to human medicine. Safety, cost, and feasibility are potential barriers for this technology that must be considered before widespread clinical adoption. This review will analyze the literature pertaining to iPSCs derived from various domestic species with a focus on iPSC production and characterization, applications for tissue and disease research, and applications for disease treatment

Characterization and immunomodulatory effects of canine adipose tissue- and bone marrow-derived mesenchymal stromal cells

Background Mesenchymal stromal cells (MSC) hold promise for both cell replacement and immune modulation strategies owing to their progenitor and non-progenitor functions, respectively. Characterization of MSC from different sources is an important and necessary step before clinical use of these cells is widely adopted. Little is known about the biology and function of canine MSC compared to their mouse or human counterparts. This knowledge-gap impedes development of canine evidence-based MSC technologies. Hypothesis and Objectives We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC (derived from the same dogs) will have similar differentiation and immune modulatory profiles. Our objectives were to evaluate progenitor and non-progenitor functions as well as other characteristics of AT- and BM-MSC including 1) proliferation rate, 2) cell surface marker expression, 3) DNA methylation levels, 4) potential for trilineage differentiation towards osteogenic, adipogenic, and chondrogenic cell fates, and 5) immunomodulatory potency in vitro. Results 1) AT-MSC proliferated at more than double the rate of BM-MSC (population doubling times in days) for passage (P) 2, AT: 1.69, BM: 3.81; P3, AT: 1.80, BM: 4.06; P4, AT: 2.37, BM: 5.34; P5, AT: 3.20, BM: 7.21). 2) Canine MSC, regardless of source, strongly expressed cell surface markers MHC I, CD29, CD44, and CD90, and were negative for MHC II and CD45. They also showed moderate expression of CD8 and CD73 and mild expression of CD14. Minor differences were found in expression of CD4 and CD34. 3) Global DNA methylation levels were significantly lower in BM-MSC compared to AT-MSC. 4) Little difference was found between AT- and BM-MSC in their potential for adipogenesis and osteogenesis. Chondrogenesis was poor to absent for both sources in spite of adding varying levels of bone-morphogenic protein to our standard transforming growth factor (TGF-β3)-based induction medium. 5) Immunomodulatory capacity was equal regardless of cell source when tested in mitogen-stimulated lymphocyte reactions. Priming of MSC with pro-inflammatory factors interferon-gamma and/or tumour necrosis factor did not increase the lymphocyte suppressive properties of the MSC compared to untreated MSC. Conclusions/Significance No significant differences were found between AT- and BM-MSC with regard to their immunophenotype, progenitor, and non-progenitor functions. Both MSC populations showed strong adipogenic and osteogenic potential and poor chondrogenic potential. Both significantly suppressed stimulated peripheral blood mononuclear cells. The most significant differences found were the higher isolation success and proliferation rate of AT-MSC, which could be realized as notable benefits of their use over BM-MSC