Retrospective Evaluation of Platelet-Leukocyte Indices and Cardiac Surgical Outcomes in Acyanotic Heart Disease Patients with Pulmonary Hypertension (REPLICA-PH)

Abstract Introduction: Acyanotic congenital heart disease (ACHD) patients with pulmonary hypertension (PH) are prone to postoperative complications, and characterization of the risk profile continues to fail in identifying inflammatory predilection. Our objective is to investigate the role of platelet-leukocyte indices (neutrophil-lymphocyte ratio [NLR], platelet-lymphocyte ratio [PLR], and systemic immune-inflammation index [SII] [neutrophil × platelet/lymphocyte]) in predicting poor outcomes following cardiac surgery in ACHD cohort with preoperative PH. Methods: This single-center, retrospective risk-predictive study included ACHD patients undergoing surgical correction at our tertiary cardiac center between January 2015 and December 2019. Standard institutional perioperative management protocol was followed, and poor postoperative outcome was defined as ≥ 1 of: low cardiac output syndrome, new-onset renal failure, prolonged mechanical ventilation (MV > 24 hours), stroke, sepsis, and/or death. Results: One hundred eighty patients out of 1,040 (17.3%) presented poor outcome. On univariate analysis, preoperative factors including right ventricular systolic pressure (RVSP) (PH-severity marker), congestive heart failure, albumin, NLR, PLR, SII, and aortic cross-clamping (ACC) and cardiopulmonary bypass (CPB) times predicted poor outcome. However, on multivariate analysis, RVSP, NLR, SII, and ACC and CPB times emerged as independent predictors. An NLR, SII prognostic cutoff of 3.33 and 860.6×103/mm3 was derived (sensitivity: 77.8%, 78.9%; specificity: 91.7%, 82.2%; area under the curve: 0.871, 0.833). NLR and SII values significantly correlated with postoperative MV duration, mean vasoactive-inotropic scores, and length of intensive care unit and hospital stay (P<0.001). Conclusion: Novel parsimonious, reproducible plateletleukocyte indices present the potential of stratifying the risk in congenital cardiac surgical patients with pre-existing PH.

A dual-antigen self-amplifying RNA SARS-CoV-2 vaccine induces potent humoral and cellular immune responses and protects against SARS-CoV-2 variants through T cell-mediated immunity.

Self-amplifying RNA vaccines may induce equivalent or more potent immune responses at lower doses compared to non-replicating mRNA vaccines via amplified antigen expression. In this paper, we demonstrate that 1 µg of an LNP-formulated dual-antigen self-amplifying RNA vaccine (ZIP1642), encoding both the S-RBD and N antigen, elicits considerably higher neutralizing antibody titers against Wuhan-like Beta B.1.351 and Delta B.1.617.2 SARS-CoV-2 variants compared to those of convalescent patients. In addition, ZIP1642 vaccination in mice expanded both S- and N-specific CD3+CD4+ and CD3+CD8+ T cells and caused a Th1 shifted cytokine response. We demonstrate that the induction of such dual antigen-targeted cell-mediated immune response may provide better protection against variants displaying highly mutated Spike proteins, as infectious viral loads of both Wuhan-like and Beta variants were decreased after challenge of ZIP1642 vaccinated hamsters. Supported by these results, we encourage redirecting focus toward the induction of multiple antigen-targeted cell-mediated immunity in addition to neutralizing antibody responses to bypass waning antibody responses and attenuate infectious breakthrough and disease severity of future SARS-CoV-2 variants.

RUNX1 and TGF-ß signaling cross talk regulates Ca2+ ion channels expression and activity during megakaryocyte development.

Thrombocytopenia is characterized by low platelet count and is typically observed among all preterm and low birthweight neonates admitted to the neonatal intensive care unit. Although the underlying cause for this predisposition is unclear, recent studies have proposed that the intrinsic inability of neonatal hematopoietic stem/progenitor cells to produce mature polyploid megakaryocytes (MKs) may result in delayed platelet engraftment. The developmental and molecular differences between neonatal and adult MKs are not yet fully understood. Previously, we had reported that the key MK transcription factor RUNX1, which is crucial for the regulation of MK specification and maturation, is down-regulated in neonatal MKs when compared with adult MKs. In humans, loss-of-function mutations in RUNX1 cause familial platelet disorder, which is characterized by thrombocytopenia, indicating its crucial role in MK development. However, information about its cross talk with developmentally regulated signaling pathways in MKs is lacking. In this study, we performed a differential gene expression analysis in MKs derived from human cord blood (CB) and adult peripheral blood (PB) CD34+ cells. Further, validation and correlation studies between RUNX1 and transforming growth factor beta (TGF-ß) were performed in a differentiating megakaryocytic cell line model. The analysis revealed that TGF-ß pathway was the main pathway affected between CB- and PB-MKs. RUNX1 is reported to be a modulator of TGF-ß signaling in several studies. The correlation between RUNX1 and TGF-ß pathway was analyzed in the PMA-induced megakaryocytic differentiating K562 cells, which exhibit mature megakaryocytic features. The RT2 profiler PCR array analysis revealed that TGF-ß pathway components were up-regulated in the PMA-induced megakaryocytic differentiating cells. Furthermore, our study indicated that human TGF-ß1 promotes cytosolic calcium (Ca2+ ) activity and MK maturation. We noticed that TGF-ß1 increased intracellular free Ca2+ ([Ca2+ ]i) via reactive oxygen species-mediated activation of transient receptor potential (TRP) ion channels. Moreover, we observed that decreased cytosolic Ca2+ activity in the siRUNX1-transfected cells was associated with down-regulation of TRP ion channel expression. Finally, we demonstrated that TGF-ß/SMAD signaling augments the development of MKs derived from CB-CD34+ . Present data suggest that RUNX1/TGF-ß pathway cross talk is crucial for MK maturation.

Beta-Glycerophosphate-Induced ORAI1 Expression and Store Operated Ca2+ Entry in Megakaryocytes.

Impairment of renal phosphate elimination in chronic kidney disease (CKD) leads to enhanced plasma and tissue phosphate concentration, which in turn up-regulates transcription factor NFAT5 and serum & glucocorticoid-inducible kinase SGK1. The kinase upregulates ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by Ca2+-sensors STIM1 and/or STIM2. In megakaryocytes and blood platelets SOCE and thus ORAI1 are powerful regulators of activity. The present study explored whether the phosphate-donor ß-glycerophosphate augments NFAT5, ORAI1,2,3 and/or STIM1,2 expressions and thus SOCE in megakaryocytes. Human megakaryocytic Meg01cells were exposed to 2 mM of phosphate-donor ß-glycerophosphate for 24 hours. Platelets were isolated from blood samples of patients with impaired kidney function or control volunteers. Transcript levels were estimated utilizing q-RT-PCR, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). NFAT5 and ORAI1 protein abundance was estimated with Western blots. As a result, ß-glycerophosphate increased NFAT5, ORAI1/2/3, STIM1/2 transcript levels, as well as SOCE. Transcript levels of NFAT5, SGK1, ORAI1/2/3, and STIM1/2 as well as NFAT5 and ORAI1 protein abundance were significantly higher in platelets isolated from patients with impaired kidney function than in platelets from control volunteers. In conclusion, phosphate-donor ß-glycerophosphate triggers a signaling cascade of NFAT5/SGK1/ORAI/STIM, thus up-regulating store-operated Ca2+-entry.

Recent Developments in mRNA-Based Protein Supplementation Therapy to Target Lung Diseases.

Protein supplementation therapy using in vitro-transcribed (IVT) mRNA for genetic diseases contains huge potential as a new class of therapy. From the early ages of synthetic mRNA discovery, a great number of studies showed the versatile use of IVT mRNA as a novel approach to supplement faulty or absent protein and also as a vaccine. Many modifications have been made to produce high expressions of mRNA causing less immunogenicity and more stability. Recent advancements in the in vivo lung delivery of mRNA complexed with various carriers encouraged the whole mRNA community to tackle various genetic lung diseases. This review gives a comprehensive overview of cells associated with various lung diseases and recent advancements in mRNA-based protein replacement therapy. This review also covers a brief summary of developments in mRNA modifications and nanocarriers toward clinical translation.

MicroRNA function in megakaryocytes.

Megakaryocytes (MKs), the largest cells in the bone marrow, are generated from hematopoietic stem cells (HSCs) in a sequential process called megakaryocytopoiesis in which HSCs undergo MK-progenitor (MP) commitment and maturation to terminally differentiated MK. Megakaryocytopoiesis is controlled by a complex network of bone marrow niche factors. Traditionally, the studies on megakaryocytopoiesis were focused on different cytokines, growth factors and transcription factors as the regulators of megakaryocytopoiesis. Over the past two decades many research groups have uncovered the key role of microRNAs (miRNAs) in megakaryocytopoiesis. miRNAs are a class of small length non-coding RNAs which play key regulatory role in cellular processes such as proliferation, differentiation and development and are also known to be involved in disease development. This review summarizes the current state of knowledge of miRNAs which have changed expression during megakaryocytopoiesis, also focuses on miRNAs which are differentially regulated during developmental maturation of MKs. Further, we aimed to discuss potential mechanisms of miRNAs-mediated regulation underlying megakaryocytopoiesis and developmental maturation of MKs.

Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin.

BACKGROUND/AIMS: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. METHODS: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. RESULTS: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). CONCLUSION: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition.

Sonidegib, a Novel Inhibitor of Suicidal Erythrocyte Death.

BACKGROUND/AIMS: The Hedgehog pathway disrupting drug sonidegib is used in the treatment of basal cell carcinoma. Side effects of sonidegib include anemia, which could result either from impaired erythropoiesis or from loss of erythrocytes e.g. due to suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is stimulated by cell stress, including energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether sonidegib exerts an effect on eryptosis. METHODS: Human erythrocytes have been treated with energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of sonidegib (2-6 µg/ ml). After treatment flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter. Hemolysis was estimated from the hemoglobin concentration in the supernatant. RESULTS: In the absence of cell stress exposure to sonidegib did not significantly modify annexin-V-binding or forward scatter, but triggered hemolysis. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Sonidegib significantly blunted the effect of energy depletion, hyperosmotic shock, and oxidative stress, but not of ionomycin on annexin-V-binding. Sonidegib further significantly blunted the effect of energy depletion, but not of hyperosmotic shock, oxidative stress, and ionomycin on forward scatter. CONCLUSIONS: Sonidegib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion, hyperosmotic shock and oxidative stress.

Systems biology approach to study the role of miRNA in promoter targeting during megakaryopoiesis.

The distinct process of megakaryopoiesis requires occurrence of endomitosis for polyploidization of the megakaryocytes. Although, Cyclins, CDKs and have been described to regulate endomitosis, the exact mechanism still remains an enigma. miRNA which were otherwise known as post transcriptional gene silencers are now emerging with various non-canonical functions including gene regulation at pre-transcriptional level by miRNA binding at promoter region. Out of the many processes they regulate, miRNA have been manifested to play a role in megakaryocyte differentiation. In this study an attempt has been made to identify miRNA that could regulate cell cycle genes (Cyclins and CDKs) by targeting their promoters, during megakaryopoiesis. A new computational algorithm was implemented using Perl programming to identify putative targets of miRNA in CDK and Cyclin promoters. Perl script was also used to check nuclear localizing miRNA based on the presence of a consensus sequence. Real-time PCR was performed to analyze the expression of miRNA and their predicted targets in Dami vs. PMA treated Dami cells. Putative targets of miRNAs with longest, high complementarity matches in CDK/Cyclin promoters were obtained. We identified two significant miRNA, miR-1273g-3p and miR-619-5p with longest seed sequence matches. We further identified three main targets (CDK10, CDK11, Cyclin F) through which these two miRNA could regulate cell cycle during megakaryopoiesis. Our results reinforce the role of promoting targeting miRNA in regulation of cell cycle through certain CDK/Cyclins to support the process of endomitosis during megakaryopoiesis.

Epigenetic Mechanisms: Role in Hematopoietic Stem Cell Lineage Commitment and Differentiation.

Major breakthroughs in the last several decades have contributed to our knowledge of the genetic regulation in development. Although epigenetics is not a new concept, unfortunately, the role of epigenetics has not come to fruition in the past. But the field of epigenetics has exploded within the past decade. Now, growing evidences show a complex network of epigenetic regulation in development. The epigenetic makeup of a cell, tissue or individual is much more complex than their genetic complement. Epigenetic modifications are more important for normal development by maintaining the gene expression pattern in tissue- and context-specific manner. Deregulation of epigenetic mechanism can lead to altered gene expression and its function, which result in altered tissue specific function of cells and malignant transformation. Epigenetic modifications directly shape Hematopoietic Stem Cell (HSC) developmental cascades, including their maintenance of self-renewal and multilineage potential, lineage commitment, and aging. Hence, there is a growing admiration for epigenetic players and their regulatory function in haematopoiesis. Epigenetic mechanisms underlying these modifications in mammalian genome are still not completely understood. This review mainly explains 3 key epigenetics mechanisms including DNA methylation, histone modifications and non-coding RNAs inference in hematopoietic lineage commitment and differentiation.

Inhibition of Suicidal Erythrocyte Death by Volasertib.

BACKGROUND/AIMS: The Polo-like kinase 1 (Plk1) inhibitor volasertib is used in the treatment of malignancy. Volasertib is partially effective by triggering suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal cell death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether volasertib impacts on eryptosis. METHODS: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of volasertib (0.5-1.5 µg/ml) and flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3 fluorescence, reactive oxygen species from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing antibodies. For comparison, annexin-V-binding and forward scatter were determined following a 48 hours exposure of human leukemic K562 cells in RPMI-1640 medium to volasertib. RESULTS: Treatment with volasertib alone did not significantly modify annexin-V-binding or forward scatter in mature erythrocytes. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Volasertib significantly blunted the effect of energy depletion and hyperosmotic shock, but not of oxidative stress and ionomycin on annexin-V-binding. Volasertib did not significantly influence the effect of any maneuver on forward scatter. In K562 cells, volasertib enhanced annexin-V-binding and decreased the forward scatter. CONCLUSIONS: Volasertib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and hyperosmotic shock, effects contrasting the stimulation of K562 cell apoptosis.

Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients.

BACKGROUND: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. METHODS: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. RESULTS: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. CONCLUSIONS: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment.

P38 Kinase, SGK1 and NF-κB Dependent Up-Regulation of Na+/Ca2+ Exchanger Expression and Activity Following TGFß1 Treatment of Megakaryocytes.

BACKGROUND: TGFß1, a decisive regulator of megakaryocyte maturation and platelet formation, has previously been shown to up-regulate both, store operated Ca2+ entry (SOCE) and Ca2+ extrusion by Na+/Ca2+ exchange. The growth factor thus augments the increase of cytosolic Ca2+ activity ([Ca2+]i) following release of Ca2+ from intracellular stores and accelerates the subsequent decline of [Ca2+]i. The effect on SOCE is dependent on a signaling cascade including p38 kinase, serum & glucocorticoid inducible kinase SGK1, and nuclear factor NFκB. The specific Na+/Ca2+ exchanger isoforms involved and the signalling regulating the Na+/Ca2+ exchangers remained, however elusive. The present study explored, whether TGFß1 influences the expression and function of K+ insensitive (NCX) and K+ sensitive (NCKX) Na+/Ca2+ exchangers, and aimed to shed light on the signalling involved. METHODS: In human megakaryocytic cells (MEG01) RT-PCR was performed to quantify NCX/NCKX isoform transcript levels, [Ca2+]i was determined by Fura-2 fluorescence, and Na+/Ca2+ exchanger activity was estimated from the increase of [Ca2+]i following switch from an extracellular solution with 130 or 90 mM Na+ and 0 mM Ca2+ to an extracellular solution with 0 Na+ and 2 mM Ca2+. K+ concentration was 0 mM for analysis of NCX and 40 mM for analysis of NCKX. RESULTS: TGFß1 (60 ng/ml, 24 h) significantly increased the transcript levels of NCX1, NCKX1, NCKX2 and NCKX5. Moreover, TGFß1 (60 ng/ml, 24 h) significantly increased the activity of both, NCX and NCKX. The effect of TGFß1 on NCX and NCKX transcript levels and activity was significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), the effect on NCX and NCKX activity further by SGK1 inhibitor GSK-650394 (10 µM) and NFκB inhibitor Wogonin (100 µM). CONCLUSIONS: TGFß1 markedly up-regulates transcription of NCX1, NCKX1, NCKX2, and NCKX5 and thus Na+/Ca2+ exchanger activity, an effect requiring p38 kinase, SGK1 and NFκB.

Lithium Sensitive ORAI1 Expression, Store Operated Ca2+ Entry and Suicidal Death of Neurons in Chorea-Acanthocytosis.

Chorea-Acanthocytosis (ChAc), a neurodegenerative disorder, results from loss-of-function-mutations of chorein-encoding gene VPS13A. In tumour cells chorein up-regulates ORAI1, a Ca2+-channel accomplishing store operated Ca2+-entry (SOCE) upon stimulation by STIM1. Furthermore SOCE could be up-regulated by lithium. The present study explored whether SOCE impacts on neuron apoptosis. Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. ORAI1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, as well as apoptosis from annexin-V-binding and propidium-iodide uptake determined by flow cytometry. As a result, ORAI1 and STIM1 transcript levels and protein abundance and SOCE were significantly smaller and the percentage apoptotic cells significantly higher in ChAc neurons than in control neurons. Lithium treatment (2 mM, 24 hours) increased significantly ORAI1 and STIM1 transcript levels and protein abundance, an effect reversed by inhibition of Serum & Glucocorticoid inducible Kinase 1. ORAI1 blocker 2-APB (50 µM, 24 hours) significantly decreased SOCE, markedly increased apoptosis and abrogated the anti-apoptotic effect of lithium. In conclusion, enhanced neuronal apoptosis in ChAc at least partially results from decreased ORAI1 expression and SOCE, which could be reversed by lithium treatment.

Role of Na+/Ca2+ Exchangers in Therapy Resistance of Medulloblastoma Cells.

BACKGROUND/AIMS: Alterations of cytosolic Ca2+-activity ([Ca2+]i) are decisive in the regulation of tumor cell proliferation, migration and survival. Transport processes participating in the regulation of [Ca2+]i include Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether medulloblastoma cells express Na+/Ca2+-exchangers, whether expression differs between therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells, and whether Na+/Ca2+-exchangers participate in the regulation of cell survival. METHODS: In therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells transcript levels were estimated by RT-PCR, protein abundance by Western blotting, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/ Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free by Na+ free and Ca2+ containing (2 mM) extracellular perfusate as well as cell death from PI -staining and annexin-V binding in flow cytometry. RESULTS: The transcript levels of NCX3, NCKX2, and NCKX5, protein abundance of NCX3, slope and peak of Δ[Ca2+]i as well as Ica were significantly lower in therapy sensitive D283 than in therapy resistant UW228-3 medulloblastoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i, and augmented the ionizing radiation-induced apoptosis but did not significantly modify clonogenicity of medulloblastoma cells. Apoptosis was further enhanced by NCX3 silencing. CONCLUSIONS: Na+/Ca2+-exchanger activity significantly counteracts apoptosis but does not significantly affect clonogenicity after radiation of medulloblastoma cells.

NFAT5-sensitive Orai1 expression and store-operated Ca2+ entry in megakaryocytes.

The transcription factor nuclear factor of activated T cells 5 (NFAT5) is up-regulated in several clinical disorders, including dehydration. NFAT5-sensitive genes include serum and glucocorticoid-inducible kinase 1 (SGK1). The kinase is a powerful regulator of Orai1, a Ca2+ channel accomplishing store-operated Ca2+ entry (SOCE). Orai1 is stimulated after intracellular store depletion by the Ca2+ sensors stromal interaction molecule 1 (STIM1), or STIM2, or both. In the present study, we explored whether nuclear factor of activated T cell (NFAT)-5 influences Ca2+ signaling in megakaryocytes. To this end, human megakaryocytic (MEG-01) cells were transfected with NFAT5 or with siNFAT5. Platelets and megakaryocytes were isolated from wild-type mice with either access to water ad libitum or dehydration by 36 h of water deprivation. Transcript levels were determined with quantitative RT-PCR and protein abundance by Western blot analysis and flow cytometry, cytosolic (intracellular) Ca2+ concentration ([Ca2+]i) by fura-2-fluorescence. SOCE was estimated from the increase of [Ca2+]i following readdition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). Platelet degranulation was estimated from P-selectin abundance and integrin activation from αIIbß3 integrin abundance determined by flow cytometry. As a result, NFAT5 transfection or exposure to hypertonicity (+40 mM NaCl) of MEG-01 cells increased Orai1, Orai2, STIM1, and STIM2 transcript levels. Orai1 transcript levels were decreased by NFAT5 silencing. NFAT5 transfection and IκB inhibitor BMS 345541 (5 µM) increased SOCE, whereas NFAT5 silencing and SGK1 inhibitor GSK650394 (10 µM) decreased SOCE. In the mice, dehydration increased NFAT5 and Orai1 protein abundance in megakaryocytes and NFAT5, Orai1, and Orai2 abundance in platelets. Dehydration further augmented the degranulation and integrin activation by thrombin and collagen-related peptide. In summary, NFAT5 is a powerful regulator of Orai1-expression and SOCE in megakaryocytes.-Sahu, I., Pelzl, L., Sukkar, B., Fakhri, H., al-Maghout, T., Cao, H., Hauser, S., Gutti, R., Gawaz, M., Lang, F. NFAT5-sensitive Orai1 expression and store-operated Ca2+ entry in megakaryocytes.

Evaluation of ischemia-modified albumin, oxidative stress, and antioxidant status in acute ischemic stroke patients.

BACKGROUND: Oxidative stress is characterized by increased production of reactive oxygen species resulting in the generation of lipid peroxides such as malondialdehyde (MDA). The studies have shown that ischemia-modified albumin (IMA), which has widely been studied as a marker of ischemia, also increases as result of oxidative stress. Hence, the current study was done to evaluate the serum MDA, IMA along with serum uric acid, and albumin, which are important metabolic antioxidants. MATERIALS AND METHODS: Fifty patients with acute ischemic stroke were taken as cases and compared with 50 age- and sex-matched controls. Serum MDA, IMA, uric acid, and albumin were estimated both in cases and controls. Serum MDA was estimated by the method of Satoh and IMA by Bar-Or et al. The results were analyzed statistically. RESULTS: Serum MDA and IMA values were significantly increased in cases (P < 0.0001), whereas serum uric acid and albumin values were significantly decreased (P < 0.05) in comparison to controls. There was also highly significant positive correlation between serum IMA and MDA (r = 0.843,P < 0.0001), whereas there were significant negative correlations between serum IMA and uric acid (r = -0.237,P < 0.05), and albumin (r = -0.326,P < 0.05). CONCLUSION: Hence, we conclude the oxidative stress plays a major role in the etiopathogenesis of acute ischemic stroke, and the deranged oxidant-antioxidant balance further contributes to its severity.

Erythropoietin and thrombopoietin mimetics: Natural alternatives to erythrocyte and platelet disorders.

Erythropoietin (EPO) and thrombopoietin (TPO) plays a major role in the regulation of hematopoietic development. Though, blood transfusion was the most widely used method to treat low blood count, over the years with advancements in recombinant technology and drug designing, the EPO and TPO mimetics are dominating the therapeutics industry. On the other hand, the recombinant human EPO and TPO are associated either with reduced half-life or immune reactions. The restoration of alternate medicine in recent years has the hope to overcome limitations associated with recombinant technology, to treat various disorder including blood diseases, with low to no side effects. The work in recent years on plant derived mimetics suggests a paradigm shift in the way diseases are treated. Here, we are providing a comprehensive review on the EPO and TPO recombinant counterparts and synthetic mimetics studied till date with a focus on the need for more natural alternatives.

Wnt Signaling: Role in Regulation of Haematopoiesis.

Hematopoietic stem cells (HSCs) are a unique population of bone marrow cells which are responsible for the generation of various blood cell lineages. One of the significant characteristics of these HSCs is to self-renew, while producing differentiating cells for normal hematopoiesis. Deregulation of self-renewal and differentiation leads to the hematological malignancies. Several pathways are known to be involved in the maintenance of HSC fate among which Wnt signaling is a crucial pathway which controls development and cell fate determination. Wnt signaling also plays a major role in differentiation, self-renewal and maintenance of HSCs. Wnt ligands activate three major pathways including planar cell polarity, Wnt/ß-catenin and Wnt/Ca(2+). It has been shown that Wnt/ß-catenin or canonical pathway regulates cell proliferation, survival and differentiation in HSCs, deregulation of this pathway leads to hematological malignancies. Wnt non-canonical pathway regulates calcium signaling and planar cell polarity. In this review, we discuss various signaling pathways induced by Wnt ligands and their potential role in hematopoiesis.

Understanding thrombocytopenia: physiological role of microRNA in survival of neonatal megakaryocytes.

Neonates are predisposed to developing thrombocytopenia and neonates are affected by megakaryocytic disorders such as thrombocytopenia with absent radius syndrome and transient myeloproliferative disorder. Small double stranded non-coding microRNAs (miRNAs) have been shown to crucially involve in the regulation of stem-cell differentiation in normal as well as malignant haematopoiesis. The regulatory mechanism in developmental megakaryocytopoiesis and role of miRNAs in biological differences between adult and neonatal megakaryopoiesis is unknown. Here in we compared miR-99a levels in megakaryocytes (MKs) derived from cord blood (CB) and peripheral blood using qRT-PCR. CTDSPL is predicted as potential target of miR-99a and was confirmed by western blot. CTDSPL is shown to involve in regulation of cell growth and differentiation and exhibits tumor suppressor activity. We believe that miR-99a regulates CTDSPL, which induces the G1/S transition by increasing Cyclin expression and play a significant role in proliferation of CB-MKs.