Housekeeping (HK) genes are constitutively expressed genes that are required for the maintenance of basic cellular functions. Despite their importance in the calibration of gene expression, as well as the understanding of many genomic and evolutionary features, important discrepancies have been observed in studies that previously identified these genes. Here, we present Housekeeping and Reference Transcript Atlas (HRT Atlas v1.0, www.housekeeping.unicamp.br) a web-based database which addresses some of the previously observed limitations in the identification of these genes, and offers a more accurate database of human and mouse HK genes and transcripts. The database was generated by mining massive human and mouse RNA-seq data sets, including 11 281 and 507 high-quality RNA-seq samples from 52 human non-disease tissues/cells and 14 healthy tissues/cells of C57BL/6 wild type mouse, respectively. User can visualize the expression and download lists of 2158 human HK transcripts from 2176 HK genes and 3024 mouse HK transcripts from 3277 mouse HK genes. HRT Atlas also offers the most stable and suitable tissue selective candidate reference transcripts for normalization of qPCR experiments. Specific primers and predicted modifiers of gene expression for some of these HK transcripts are also proposed. HRT Atlas has also been integrated with a regulatory elements resource from Epiregio server.

Free extracellular heme has been shown to activate several compartments of innate immunity, acting as a danger-associated molecular pattern (DAMP) in hemolytic diseases. Although localized endothelial barrier (EB) disruption is an important part of inflammation that allows circulating leukocytes to reach inflamed tissues, non-localized/deregulated disruption of the EB can lead to widespread microvascular hyperpermeability and secondary tissue damage. In mouse models of sickle cell disease (SCD), EB disruption has been associated with the development of a form of acute lung injury that closely resembles acute chest syndrome (ACS), and that can be elicited by acute heme infusion. Here we explored the effect of heme on EB integrity using human endothelial cell monolayers, in experimental conditions that include elements that more closely resemble in vivo conditions. EB integrity was assessed by electric cell-substrate impedance sensing in the presence of varying concentrations of heme and sera from SCD patients or healthy volunteers. Heme caused a dose-dependent decrease of the electrical resistance of cell monolayers, consistent with EB disruption, which was confirmed by staining of junction protein VE-cadherin. In addition, sera from SCD patients, but not from healthy volunteers, were also capable to induce EB disruption. Interestingly, these effects were not associated with total heme levels in serum. However, when heme was added to sera from SCD patients, but not from healthy volunteers, EB disruption could be elicited, and this effect was associated with hemopexin serum levels. Together our in vitro studies provide additional support to the concept of heme as a DAMP in hemolytic conditions.

Background Recent evidence suggests that generation of neutrophil extracellular traps (NETosis), one of the components of immunothrombosis, is associated with the pathogenesis of both venous thromboembolism and sickle cell disease (SCD). NETosis is a complex process regulated by several proteins such as peptidyl arginine deaminase 4 (PADI4), neutrophil elastase (ELANE), and myeloperoxidase (MPO). Among these regulators, PADI4 is responsible of histone citrullination, an essential step for NETosis. Accordingly, its inhibition has been recently cited as a promising therapeutic strategy for diseases such as SCD. Although attractive, this strategy requires supportive evidence of its role in the pathogenesis of SCD. Patients and Methods Patients from two independent cohorts were enrolled in this study. Samples were obtained at steady state (53 patients) or during acute episodes of vaso‐occlusive crisis (VOC; 28 patients) in patients from cohort 1. mRNA was extracted from granulocytes to analyze PADI4 , ELANE , and MPO expression by qPCR. Furthermore, plasma activity of PADI4 was assessed from an independent cohort in 15 patients, within 24 hours from admission for VOC. Race‐matched healthy individuals from the same geographic regions were used as controls for each cohort. Results and Conclusions Higher levels of gene expression of PADI4 and ELANE were observed during VOC. Furthermore, plasma activity of PADI4 was higher in acute VOC when compared to healthy individuals. These results demonstrate that NETosis regulators are modulated during acute VOC, and pave the way for studies of PADI4 inhibition as a therapeutic strategy for acute VOC in SCD.

Objective/Background: Sickle cell anemia (SCA) is associated with increased levels of extracellular heme, which is a key mediator of inflammation in this condition. Despite abundant evidence supporting this concept in cell and animal models, few studies addressed the association between heme levels and the development and severity of acute vasoocclusive crises (VOC) in humans. Methods: A cross-sectional study was conducted in patients with acute VOC. Total extracellular heme levels were measured in both plasma and serum at admission and after convalescence, and correlated with other clinical and laboratory markers of SCA severity. Results: A total of 28 episodes of VOC in 25 patients were included. Heme levels were similar between admission and convalescence, and correlated with the difference between pre and post hemoglobin, and SCA severity estimated by a composite score of clinical and laboratory markers. Heme levels were neither associated with VOC severity nor with markers of hemostasis activation, and were similar to those reported in an independent population of SCA patients at steady state. Discussion: Acute VOC are not characterized by significant increases in total extracellular heme levels. Studies measuring the fraction of free extracellular heme unbound to proteins are warranted to further refine our understanding of the role of heme in acute VOC.

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