We describe a method for protein quantification and for mRNA quantification in small sample quantities of human induced pluripotent stem-cell-derived-cardiomyocytes (hiPSC-CMs). Demonstrated here is how the capillary-based protein detection system Wes^TM by ProteinSimple and the Power SYBR^TM Green Cells-to-CT^TM Kit by Invitrogen can be applied to individual samples in a 96-well microplate format and thus made compatible with high-throughput (HT) cardiomyocyte assays. As an example of the usage, we illustrate that Cx43 protein and GJA1 mRNA levels in hiPSC-CMs are enhanced when the optogenetic actuator, channelrodopsin-2 (ChR2), is genetically expressed in them. Instructions are presented for cell culture and lysate preparations from hiPSC-CMs, along with optimized parameter settings and experimental protocol steps. Strategies to optimize primary antibody concentrations as well as ways for signal normalization are discussed, i.e. antibody multiplexing and total protein assay. The sensitivity of both the Wes and Cells-to-CT kit enables protein and mRNA quantification in a HT format, which is important when dealing with precious small samples. In addition to being able to handle small cardiomyocyte samples, these streamlined and semi-automated processes enable quick mechanistic analysis.

Astrocytes have been implicated in a wide range of neurodegenerative diseases, with the literature indicating harmful or beneficial roles on a case-by-case basis. On such conditions, these cells are capable of secreting several inflammatory factors and also promote synapse elimination and remodeling. These responses are possible because they sense their surroundings via several receptors, including metabotropic glutamate receptor (mGluR5). However, mGluR5 activation in astrocytes under neuroinflammatory conditions can be neuroprotective or have the opposite effect. In this work, we investigated the role of mGluR5 in hiPSC-derived astrocytes subjected to pro-inflammatory stimulation by recombinant TNF-α (rTNF-α). Our results show that mGluR5 blockade by CTEP is capable of reducing pro-inflammatory cytokines (IL-6, IL-8 and TNF-α) release in rTNF-α-stimulated astrocytes, but, in contrast, leads to an augmented expression of the pan-reactive marker SERPINA3. Additionally, CTEP enhances synaptoneurosomes phagocytosis by astrocytes in both non-stimulated and TNF-α-stimulated conditions, indicating that mGluR5 blockade alone is enough to drive synaptic material engulfment. Finally, mGluR5 antagonism as well as rTNF-α stimulation leads to a reduction in the expression of synaptogenic molecules. Altogether, these data suggest a complex role for mGluR5 in human astrocytes, since its blockage may have beneficial and detrimental effects under inflammatory conditions.

A number of research projects and a rich literature have dealt with the theme of abandoned medieval villages in Sardinia since the end of the 60s of the last century. Some more or less precise catalogues and reviews of villages in limited territories have been published. Only recently, however, this subject is being addressed in an interdisciplinary manner, combining traditional historical research with the results of archaeological surveys and excavation campaigns, geo-archaeology, toponymy, paleoclimatology. This allows us to have a picture of the landscape and human settlement evolution with its historical changes, conditioned not only by institutional superstructures but also by human and natural traumatic events. Particular attention will be given to the sudden changes that occurred between the thirteenth and fourteenth centuries. To carry out this survey, it is possible to use some very powerful IT tools which, through the aggregation, organization, correlation and management of information, allow the geo-localization of abandoned villages as proven by the documentary evidence. Thus, on this documentation, existing and acquired in the future, is founded the construction of the related information system. The most easy and suitable tools for this purpose are the CMSs (Content Management Systems) which, in association with GIS (Geographic Information Systems) engines, allow spatial and contextual analysis of the settlements, as they were inserted in their territory. This type of tools aggregates different peculiarities of the object of study, supporting a multidisciplinary reading on the argument. The computerized tools, integrated as a system, offer the possibility to implement it, feeding it and correcting it continuously, basing on new acquisitions. In this study, we will examine a historical areal, for which we have a sufficient number of sources available: Sarrabus, Colostrai and Quirra, adding to the geographical visual information, the temporal visual evolution.

Proximal tubular epithelial cells (PTEC) are constantly exposed to potentially toxic metabolites and xenobiotics. The regenerative potential of the kidney enables the replacement of damaged cells either via the differentiation of stem cells or the regain of proliferative properties of the PTEC. Nevertheless, it is known that renal function declines, suggesting that the deteriorated cells are not replaced by fully functional cells. To understand the possible causes of this loss of kidney cell function, it would be beneficial to understand the role of toxins during the regeneration process. Therefore, we investigated the sensitivity and function of human induced pluripotent stem cells (hiPSC), hiPSC differentiating, and hiPSC differentiated into proximal tubular epithelial-like cells (PTELC) to known nephrotoxins. hiPSC were differentiated into PTELC, exhibiting a similar morphology to PTEC, expressing prototypical PTEC markers and performing albumin endocytosis. When treated with two nephrotoxins, hiPSC and differentiating hiPSC were more sensitive to cisplatin than differentiated PTELC, whereas all stages were equally sensitive to cyclosporin A. Both toxins also had an inhibitory effect on albumin uptake. Our results suggest a high sensitivity of differentiating cells towards toxins, which could have an unfavorable effect on regenerative processes. To study this, our model of hiPSC differentiating into PTELC appears suitable.

In 2014, a whistleblower reported that many U.S. veterans died while waiting for care at the Phoenix VHA. Problems with veteran’s care through 2018 reveal ongoing and systematic problem. In March 2018, the VA Inspector General identified critical deficiencies at the Washington, DC VA Medical Center including failures to track patient safety events accurately, ineffective sterile processing, and more than 10 thousand open or pending prosthetic / sensory aid consults. The VHA clearly has problems with access and quality in a budget-constrained environment. In this policy analysis, four separate interventions that address the gap between the magnitude as well as the use of the VHA’s fixed budget versus access and cost expectations are explored. These policy interventions include maintaining the status quo, returning to a “VHA-only” option, transitioning to a CMS central payer system, and consolidating care under the DoD TRICARE insurance plans. An objective evaluation suggests that extending TRICARE to veterans while phasing out the VHA’s care responsibilities, while politically unpalatable, would likely provide the best of four possible solutions under various criterion weighting schemes. A central payer solution under the CMS would also be viable consideration. A Friedman’s test with Wilcoxon rank sum post-hoc tests suggests that TRICARE patient perceptions of quality are superior to VHA and non-VHA / non-DoD (p<.001), that access provided by the TRICARE program is ranked second in terms of venue acceptance only to the CMS solution set based on primary provider acceptance, and that the cost per beneficiary of a TRICARE solution ($6.5K / beneficiary) is far better than a VHA-only solution ($14.0 K / beneficiary), the CMS central payer solution ($12.2K / beneficiary), or the status quo (between $12.2K and $14.0K / beneficiary). The intent of this paper is to provoke thoughtful consideration of solutions for providing access to high-quality healthcare for veterans within our outside of the VHA. In this policy analysis, separate interventions that address the gaps between cost, quality, and access are explored. These policy interventions include maintaining the status quo, returning to a VHA-only option, transitioning to a CMS central payer system, and consolidating care under TRICARE.

The process of identifying and approving a new drug is a time-consuming and expensive procedure. One of the biggest issues to overcome is the risk of hepatotoxicity, which is one of the main reasons for drug withdrawal from the market. While animal models are the gold standard in preclinical drug testing, the translation of results into therapeutic intervention is often ambiguous due to interspecies differences in hepatic metabolism. The discovery of human induced Pluripotent Stem Cells (hiPSCs) and their derivatives has opened new possibilities for drug testing. We used mesenchymal stem cells and hepatocytes both derived from hiPSC, together with endothelial cells, to miniaturize the process of generating hepatic organoids. These organoids were then cultivated in vitro using both static and dynamic cultures. Additionally, we tested spheroids composed solely by induced hepatocytes. By miniaturizing the system, we demonstrated the possibility of maintaining the organoids, but not the spheroids, in culture for up to 15 days. This timeframe may be sufficient to carry out a hypothetical pharmacological test or screening. In conclusion, we propose that the hiPSC-derived liver organoids model could complement or, in the near future, replace the pharmacological and toxicological tests conducted on animals.

Human induced pluripotent stem cells (hiPSCs) have two characteristic abilities 1) self-renewal and 2) differentiation into all cell types in the human body. It is this ability of iPSCs to differentiate into any desired cell type that makes them amenable to disease modeling. iPSCs can further be engineered to carry a disease-specific mutation or be derived from patients to phenocopy the disease. To model neurodegenerative/neurodevelopmental disorders iPSCs are differentiated into neurons. This chapter describes protocols for two different approaches to generate neurons from iPSCs, first using dual SMAD inhibition to generate neural progenitor cells which are then differentiated into neurons, and second using single transcription factor (NGN2) over-expression to drive differentiation of iPSCs directly into neurons.

Saiga (Saiga tatarica) was extirpated in China. Since Mid-1980s, attempts have been made for revival the species in the country, however, only a breeding herd of Saiga was successfully established at Wuwei, Gansu, China. The reintroduced Saiga population experienced a bumpy growth. Then, the population collapsed following the catastrophe die-off in the Saiga ranging countries in Central Asia, then population started to rebound when 6 new lams were born in 2019. After reviewing the population trend and conservation breeding of Saiga in China, we concluded that to establish a migratory species that needs vast range size like Saiga on central Asia steppe, an international collaboration is needed for introducing new genes. We recommend China to ratify the CMS in order to facilitate international conservation efforts to restoring the species in its former range.

This paper presents an extension for principles of Differential Geometry on Surfaces (re-hashed through the budding field of CMS, the Calculus of Moving Surfaces). It analyzes mostly 2D Hypersurfaces with Riemannian Geometry and proposes the construction of a 3D Static Frame combining the Surface Basis Vectors with the Orthogonal Normal Field as a 3D Orthogonal Vector Frame. The paper introduces conventions for manipulating Tensors defined using this 3D Orthogonal Vector Frame as well as Curvature Connections associated with this Vector Frame. It then finally introduces Symbols and Tensors to describe Inner Products and Variance within the 3D Vector Frame and then extends all the above concepts to a surface which is Dynamic utilizing principles from CMS. This formulation has potential to extend identities and concepts from CMS and from Differential Geometry in a compact Tensorial Framework, which agrees with the new Framework proposed by CMS.