Although human cardiomyocytes (CMs) are capable of some cell division, this response is neither sufficient to repair damaged cardiac tissue nor efficient to compensate for pathological stress. Danio rerio (zebrafish) CMs have been shown to have high proliferative capability to completely repair hearts after injury; however, no reports have focused on their physiological and cellular response to cardiac overload stress. We hypothesized that forced excessive long-term cardiac overload stress would elicit a proliferative response similar to regenerative cardiac repair in zebrafish. We completed a 10-week forced fast-speed swimming exercise regimen, comparing exercised hearts to nonexercised controls for physiological function and histological evidence of cell proliferation. Our results indicate that exercised heart ventricles are 33% larger, yet exhibit no significant changes in cardiac physiological function as evaluated by the heart rate and the percent shortening fraction. We found 8% more CM nuclei per cross-sectional area within exercised ventricular tissue, indicating that cardiomegaly was not due to individual cell hypertrophy, but due to hyperplasia. This novel zebrafish cardiac stress model may be used to identify genes or proteins with therapeutic potential for treating cardiac stress pathologies, as well as molecules that could be used as initiators of cardiac cell proliferation in humans.

Transmission of plant viruses by phytophagous hemipteran insects encompasses complex interactions underlying a continuum of processes involved in virus acquisition, retention and inoculation combined with vector feeding behavior. Here, we investigated the effects of dietary pH on whitefly (Bemisia tabaci) feeding behavior and release of Lettuce infectious yellows virus (LIYV) virions retained in the vector's foregut. Electrical penetration graph analysis revealed that variables associated with whitefly probing and ingestion did not differ significantly in pH (4, 7.4, and 9) adjusted artificial diets. To investigate virus retention and release, whiteflies allowed to acquire LIYV virions in a pH 7.4 artificial diet were fed pH 4, 7.4, or 9 virion-free artificial (clearing) diets. Immunofluorescent localization analyses indicated that virions remained bound to the foreguts of approximately 20%-24% of vectors after they fed on each of the 3 pHadjusted clearing diets. When RNA preparations from the clearing diets were analyzed by reverse transcription (RT) nested-PCR and, in some cases, real-time qPCR, successful amplification of LIYV-specific sequence was infrequent but consistently repeatable for the pH 7.4 diet but never observed for the pH 4 and 9 diets, suggesting a weak pH-dependent effect for virion release. Viruliferous vectors that fed on each of the 3 pH-adjusted clearing diets transmitted LIYV to virus-free plants. These results suggest that changes in pH values alone in artificial diet do not result in observable changes in whitefly feeding behaviors, an observation that marks a first in the feeding of artificial diet by whitefly vectors; and that there is a potential causal and contingent relationship between the pH in artificial diet and the release/inoculation of foregut bound virions.

Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, chronic hemolysis, and progressive organ damage. This disease affects ~100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2α and ATF4 trans-activation in the γ-globin gene promoter. Sickle erythroid progenitors treated with 24µM SAL increased F-cells levels 1.4-fold (p=0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p=0.0441), along with dose-dependent increases of p-eIF2α and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p=0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.

Sickle cell disease (SCD) is a genetic disorder caused by a mutation in the adult β-globin gene, affecting ~100,000 people in the United States and millions worldwide. Clinical symptoms of SCD include anemia, pain, and progressive organ damage creating a great burden to annual healthcare costs. An effective therapeutic intervention for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents to ameliorate clinical symptoms. Hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD, however, it is not effective in all people. Therefore, the goal of this study is to determine the ability of Salubrinal (SAL), to induce HbF. Salubrinal is a selective inhibitor of protein phosphatase 1 leading to increased levels of p-eIF2α (phosphorylated eukaryotic initiation factor 2α) and inhibition of global protein synthesis. Activating transcription factor 4 (ATF4) is a downstream target of p-eIF2α activated during oxidative stress. The main function of these signaling events is to attenuate stress to the endoplasmic reticulum. Previously we identified a Gγ-globin cAMP response element (G-CRE) that binds ATF2, a binding partner of ATF4 involved in HbF induction (Sangerman J et al. Blood 2006). Furthermore, ENCODE analysis showed ATF4 sites at -822Gγ and β-globin gene second intron. Thus, studies were performed to determine if p-eIF2α-ATF4 signaling is involved in mechanisms of HbF induction by SAL. Initial experiments involved the use of day 8 erythroid progenitors generated from human CD34+ stem cells; treatments included SAL 12, 18 and 24 µM, and 0.5% DMSO (vehicle control) for 48 h; cell viability remained >90% in all drug conditions. The level of γ-globin mRNA increased 1.2-fold and 1.3-fold at SAL 18 and 24 µM respectively (p<0.05). Comparable, HbF was induced by SAL 24 µM alone and combined SAL/HU treatments to 1.8-fold. To gain insights into mechanisms of HbF induction by SAL, we next quantified the level of p-eIF2α. We observed a 1.7-fold increase in p-eIF2α with SAL 12 and 24 µM and parallel increase in ATF4 (4.8-fold). Flow cytometry revealed SAL increased F-cells (HbF positive cells) from 30.9% (DMSO treated) to 90.6%. Similarly, studies were performed using sickle erythroid progenitors generated from peripheral blood mononuclear cells. On day 8, SAL (9, 18, 24 µM) dissolved in water was added for 48 h; cell viability remained >90% for all drug conditions. SAL (18 μM) increased γ-globin mRNA 3.2-fold and F-cells 2.5-fold (p<0.05) compared to the untreated control. We used mean fluorescence intensity (MFI) to quantify HbF protein per cells, which showed a dose-dependent increase with SAL treatment. Since sickle red blood cells are under oxidative stress, we measured the levels of reactive oxygen species (ROS) by flow cytometry. SAL 12, 18, 24 µM decreased ROS levels in a dose-dependent manner by 7.6%, 8.7% and 10% respectively. Interestingly, SAL/HU treatment decreased ROS levels by 10.2% compared to a 4.3% mediated by the nitric oxide donor HU. Western blot analysis showed a dose-dependent increase in HbF and a 3.3-fold increase in p-eIF2α (p<0.05) and ATF4, without changing HbS expression. To generate data for clinical development, we utilized the Townes SCD mouse model. SCD mice (n=5 per group) were treated with SAL (3 and 5mg/kg), HU (100mg/kg; positive control) or water control (vehicle), 5 days a week for 4 weeks. Blood was drawn at week 0 (baseline), 2 and 4 at treatment completion. All data were normalized for each group and treatment response at week 2 and 4 compared to week 0 using a paired t-test and ANOVA to compare across treatment groups; statistical significance set at p<0.05. All groups showed normal weight gain and no significant changes in complete blood counts, differential or reticulocyte counts. Flow cytometry of peripheral blood showed that SAL (3mg/kg) produced a 2-fold increase in F-cells by 2 and 4 weeks while SAL (5mg/kg) produced a further 3.1-fold increase in F-cells by week 4 (p<0.05) without toxicity. Our initial in vitro findings, supports HbF induction by SAL involving p-eIF2α-ATF4 signaling. The interaction of ATF4 in the G-CRE and/or other predicted binding sites will be investigated. To support clinical trials, studies in the SCD preclinical model support the ability of SAL to induce HbF in vivo; additional studies are underway. Defining the mechanism of HbF induction by SAL has the potential to impact treatment for SCD. Disclosures No relevant conflicts of interest to declare.

We identified fresh human leukocytes as an abundant source of the candidate epithelial tumor suppressor gene, Ecrg4; an epigenetically regulated gene that, unlike other tumor suppressor genes, encodes an orphan secreted ligand‐like protein. In cultured human cell lines, Ecrg4 gene expression was low, the Ecrg4‐ encoded protein undetectable and Ecrg4 promoter hypermethylation was high (45–90%). Low gene expression was also reversible by incubation with the methylation inhibitor 5‐Aza‐ Cytidine. In contrast, Ecrg4 gene expression in fresh normal human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) was 600–800 times higher than cultured cells, the protein was readily detected in cell lysates and Ecrg4 promoter hyper‐methylation in normal leukocytes was low (<3%). Immunoblotting, cell surface biotinylation, immunofluorescent staining and cell flow analyses also established that the Ecrg4‐encoded protein was not constitutively secreted but instead exists as an extracellular, membrane‐anchored protein on the surface leukocytes. Its presence on the surface however is dynamic and upon leukocyte activation by 5 min incubations with fMLP, cell surface Ecrg4 protein is shed in concordance with fMLP‐activation of neutrophils and polarization. In light of these findings, we propose that the pathological sequelae of losing Ecrg4 expression in cancer may reflect a more global physiological response that links Ecrg4 to the biology of leukocytes in cancer surveillance, innate immunity, injury and inflammation.