Calcium as a reliable marker for the quantitative assessment of endoplasmic reticulum stress in live cells.

Calcium (Ca2+) is an essential mineral of endoplasmic reticulum (ER) luminal biochemistry because of the Ca2+ dependence of ER-resident chaperones charged with folding de novo proteins that transit this cellular compartment. ER Ca2+ depletion reduces the ability of chaperones to properly fold the proteins entering the ER, thus leading to an accumulation of misfolded proteins and the onset of a state known as ER stress. However, not all conditions that cause ER stress do so in a manner dependent on ER Ca2+ depletion. Agents such as tunicamycin inhibit the glycosylation of de novo polypeptides, a key step in the maturation process of newly synthesized proteins. Despite this established effect of tunicamycin, our understanding of how such conditions modulate ER Ca2+ levels is still limited. In the present study, we report that a variety of ER stress-inducing agents that have not been known to directly alter ER Ca2+ homeostasis can also cause a marked reduction in ER Ca2+ levels. Consistent with these observations, protecting against ER stress using small chemical chaperones, such as 4-phenylbutyrate and tauroursodeoxycholic acid, also attenuated ER Ca2+ depletion caused by these agents. We also describe a novel high-throughput and low-cost assay for the rapid quantification of ER stress using ER Ca2+ levels as a surrogate marker. This report builds on our understanding of ER Ca2+ levels in the context of ER stress and also provides the scientific community with a new, reliable tool to study this important cellular process in vitro.

Estimating the annual economic burden for the management of patients with transthyretin amyloid polyneuropathy in Spain.

Background: Transthyretin amyloid polyneuropathy (ATTR-PN) is a fatal disease associated with substantial burden of illness. Three therapies are approved by the European Medicines Agency for the management of this rare disease. The aim of this study was to compare the total annual treatment specific cost per-patient associated with ATTR-PN in Spain.Methods: An Excel-based patient burden and cost estimator tool was developed to itemize direct and indirect costs related to treatment with inotersen, patisiran, and tafamidis in the context of ATTR-PN. The product labels and feedback from five Spanish ATTR-PN experts were used to inform resource use and cost inputs.Results: Marked differences in costs were observed between the three therapies. The need for patisiran- and inotersen-treated patients to visit hospitals for pre-treatment, administration, and monitoring was associated with increased patient burden and costs compared to those treated with tafamidis. Drug acquisition costs per-patient per-year were 291,076€ (inotersen), 427,250€ (patisiran) and 129,737€ (tafamidis) and accounted for the majority of total costs. Overall, the total annual per-patient costs were lowest for patients treated with tafamidis (137,954€), followed by inotersen (308,358€), and patisiran (458,771€).Conclusions: Treating patients with tafamidis leads to substantially lower costs and patient burden than with inotersen or patisiran.

The trypan blue cellular debris assay: a novel low-cost method for the rapid quantification of cell death.

Cell death is a common driver of human disease and is frequently studied in a variety of in vitro settings. There currently exists a range of commercially available assays to examine cell death, however, most are costly and require assay-specific experimental conditions that may not be suitable for many cell types. Here, we show that cellular debris occurring as a result of cell death can be used to quantify cell death using trypan blue. Furthermore, we demonstrate that the data generated using this technique are comparable to the widely-used lactate dehydrogenase (LDH) assay. Overall, we describe a novel application for trypan blue, a stain found in most biology laboratories, as a novel and cost-effective method for the quantification of cell death via staining of cell debris.