Cryopreservation of equine mesenchymal stem cells in 95 % autologous serum and 5 % DMSO does not alter post-thaw growth or morphology in vitro compared to fetal bovine serum or allogeneic serum at 20 or 95 % and DMSO at 10 or 5 %

INTRODUCTION: Equine superficial digital flexor tendon injury is a well-accepted model of human tendon injury and is routinely treated with local injections of autologous mesenchymal stem cells (MSCs). Identification of a clinically safe medium for short-term cryopreservation of MSCs prior to cell implantation would streamline laboratory and clinical procedures for autologous regenerative therapies. Veterinary experience with short-term (MSCs prepared after the injury has occurred) cryopreserved MSCs in naturally occurring injury in the horse will be of value to human practitioners. METHODS: Equine bone marrow derived MSCs were cryopreserved in 6 different solutions consisting of 20 % serum, 10 % DMSO and 70 % media or 95 % serum and 5 % DMSO. Serum was autologous serum, commercially available pooled equine serum or fetal bovine serum (FBS). Cell survival, morphology and growth kinetics were assessed by total cell number, measurement of growth kinetics, colony-forming-unit-assay and morphology of MSCs after monolayer culture post-thaw. RESULTS: There were no significant differences in post-thaw viability, total cell number, morphology scores or growth kinetics among the 6 solutions. Post thaw viabilities from each group ranged from 80-90 %. In all solutions, there were significantly fewer MSCs and the majority (99 %) of MSCs remained in the original generation 24 hours post-thaw. Seventy two hours post-thaw, the majority of MSCs (50 %) were proliferating in the fourth generation. Mean colony count in the CFU-F assay ranged from 72 to 115 colonies. CONCLUSIONS: Each of the serum sources could be used for short-term cryopreservation of equine bone marrow derived MSCs. Prior to clinical use, clinicians may prefer autologous serum and a lower concentration of DMSO. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13287-015-0230-y) contains supplementary material, which is available to authorized users

The Covenant of Mayors for Climate and Energy Reporting Guidelines

The Covenant of Mayors for Climate and Energy brings together local and regional authorities voluntarily committing to implementing the European Union’s climate and energy objectives on their territory. Signatory local authorities share a vision for making cities decarbonised and resilient, where citizens have access to secure, sustainable and affordable energy. Signatories pledge to reduce CO2 emissions by at least 40% by 2030 and to increase their resilience to the impacts of climate change. The Covenant of Mayors helps local authorities to translate their greenhouse gas (GHG) emissions reduction ambitions into reality, while taking into account the immense diversity on the ground. it provides signatories with a harmonised data compilation and reporting framework which is unique in Europe which assists them to follow a systemic climate and energy planning and monitoring at the local level. The Sustainable Energy and Climate Action Plan (SECAP) template constitutes the standard reporting framework for Covenant Signatories. The SECAP template forms the skeleton of the individual action plans. The SECAP and its monitoring part allow signatories to collect and analyse data in a structured and systematic manner, serve as a basis for good climate and energy management and for tracking progress in implementation. This guide has been developed to assist signatories in understanding the Covenant reporting framework. It seeks to provide signatories with step-by-step guidelines throughout the reporting process. Step I is dedicated to guide signatories through the process of filling in the templates, namely Section I for the SECAP template and Section II for the monitoring template. Step II addresses the upload of documents such as the SECAP, while Step III is focused on the integrated checking system developed for the climate mitigation part of the template and official submission. The guide is enriched with some practical recommendations and concrete examples. Link to the SECAP Template and other technical material: http://www.covenantofmayors.eu/Covenant-technical-materials.html (selectable in URL below).JRC.C.2-Energy Efficiency and Renewable

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Additional file 2: of Cryopreservation of equine mesenchymal stem cells in 95 % autologous serum and 5 % DMSO does not alter post-thaw growth or morphology in vitro compared to fetal bovine serum or allogeneic serum at 20 or 95 % and DMSO at 10 or 5 %

Is a figure showing flow histograms of CellTrace™ dye in MSCs cryopreserved in six different freezing solutions, 24 hours post thaw and monolayer expansion. (JPEG 285 kb

Serine-129 phosphorylation of α-synuclein is a trigger for physiologic protein-protein interactions and synaptic function

&lt;p&gt;Phosphorylation of &alpha;-synuclein at the Serine-129 site (&alpha;-syn Ser129P) is an established pathologic hallmark of synucleinopathies, and also a therapeutic target. In physiologic states, only a small fraction of &alpha;-syn is phosphorylated at this site, and most studies have focused on pathologic roles of this post-translational modification. We found that unlike wild-type (WT) &alpha;-syn that is widely expressed throughout the brain, the overall pattern of &alpha;-syn Ser129P is restricted, suggesting intrinsic regulation. Surprisingly, preventing Ser129P blocked activity-dependent synaptic attenuation by &alpha;-syn &ndash; thought to reflect its normal function. Exploring mechanisms, we found that neuronal activity augments Ser129P, which is a trigger for protein-protein interactions that are necessary for mediating &alpha;-syn function at the synapse. AlphaFold2-driven modeling and membrane-binding simulations suggest a scenario where Ser129P induces conformational changes that facilitates interactions with binding partners. Our experiments offer a new conceptual platform for investigating the role of Ser129 in synucleinopathies, with implications for drug-development.&lt;/p&gt

Acupuncture and Cardiovascular Disease: Focus on Heart Failure

Symptomatic heart failure is managed with interdisciplinary approaches to reduce acute exacerbations and to improve mortality. Acupuncture is a standardized treatment of Traditional Chinese Medicine that has been shown to have beneficial effects on the cardiovascular system via a neurohumoral pathway known as the long-loop pathway. This article serves to examine recent evidence supporting the long-loop pathway as the physiologic mechanism of acupuncture and the sympatholytic, vasodilatory, and cardioprotective effects of acupuncture that could specifically improve cardiac function and quality of life measures in the management of congestive heart failure