Accumulation Of Triphosphate Research Articles

Neurotoxicity by Hypoxia an Intermediate Between Alpha-synuclein and Mitochondrial Dysfunction in Parkinson’s Disease

This review article explores the relationship between alpha-synuclein and mitochondrial dysfunction in Parkinson’s disease (PD), focusing on the role of hypoxia as an intermediate factor. The interaction between alpha-synuclein and mitochondria, particularly through membranal lipids such as cardiolipins, is highlighted as a key factor in mitochondrial disruption and neurodegeneration. Hypoxia, caused by oxygen deprivation, is identified as a crucial link between alpha-synuclein and mitochondrial regulation, leading to neuronal death in PD. The article also discusses the involvement of other proteins, such as peroxisome proliferator-activated receptor gamma coactivator, Sirtuin-1, Sirtuin-3 and adenosine monophosphate-activated protein kinase, in maintaining mitochondrial biogenesis during hypoxia. The study emphasizes the need for further research to understand the complex molecular interactions causing Lewy body aggregation, improper mitochondrial functioning and neurodegeneration in PD, with a specific focus on the role of hypoxia. Alpha-synuclein aggregation disrupts mitochondrial respiration, leading to mitochondrial dysfunction and increased production of reactive oxygen species. Mitochondrial dysfunction, in turn, causes neurodegeneration in PD. Oligomeric alpha-synuclein results in mitochondrial dysfunction, lethal synaptic disruption and reduced adenosine triphosphate generation. Oligomeric alpha-synuclein also increases the accumulation of mitochondrial rho nucleotide guanosine triphosphate, leading to delayed mitophagy. Hypoxia, another factor in PD, alters both alpha-synuclein and mitochondria. Controlling hypoxia reduces the oligomerization of alpha-synuclein. The interaction between alpha-synuclein and mitochondria is complex, and determining the primary player in inducing the other is still debatable.

ATP-induced Cell Death Mechanism

"ATP-induced cell death" is a novel form of cellular demise identified since 1991, typically characterized by a substantial accumulation of adenosine triphosphate (ATP) and the activation of ATP membrane receptors during the cell death process. The perturbation of ATP homeostasis is intricately linked to mitochondrial function. In recent years, ATP-induced cell death (AICD) has emerged as a discernible pattern of cell demise attributed to heightened levels of extracellular ATP (eATP). This phenomenon is intimately associated with the pathophysiological mechanisms underpinning a plethora of maladies, including malignancies, neurodegenerative disorders, ischemia-reperfusion injury, renal impairment, and hematological conditions. The strategic modulation of ATP-induced cell death to mitigate the onset and progression of these related diseases has garnered significant attention and stands as a focal point in etiological research and therapeutic interventions. Nevertheless, the functional alterations and precise molecular mechanisms governing ATP-induced cell death demand further comprehensive exploration. This paper comprehensively synthesizes the most recent advancements in the understanding of ATP-induced cell death mechanisms, with the overarching objective of serving as a reference for a deeper comprehension of its pathogenesis and the proposition of novel therapeutic targets for the management of associated diseases.

An inosine triphosphate pyrophosphatase safeguards plant nucleic acids from aberrant purine nucleotides.

In plants, inosine is enzymatically introduced in some tRNAs, but not in other RNAs or DNA. Nonetheless, our data show that RNA and DNA from Arabidopsis thaliana contain (deoxy)inosine, probably derived from nonenzymatic adenosine deamination in nucleic acids and usage of (deoxy)inosine triphosphate (dITP and ITP) during nucleic acid synthesis. We combined biochemical approaches, LC-MS, as well as RNA-Seq to characterize a plant INOSINE TRIPHOSPHATE PYROPHOSPHATASE (ITPA) from A.thaliana, which is conserved in many organisms, and investigated the sources of deaminated purine nucleotides in plants. Inosine triphosphate pyrophosphatase dephosphorylates deaminated nucleoside di- and triphosphates to the respective monophosphates. ITPA loss-of-function causes inosine di- and triphosphate accumulation invivo and an elevated inosine and deoxyinosine content in RNA and DNA, respectively, as well as salicylic acid (SA) accumulation, early senescence, and upregulation of transcripts associated with immunity and senescence. Cadmium-induced oxidative stress and biochemical inhibition of the INOSINE MONOPHOSPHATE DEHYDROGENASE leads to more IDP and ITP in the wild-type (WT), and this effect is enhanced in itpa mutants, suggesting that ITP originates from ATP deamination and IMP phosphorylation. Inosine triphosphate pyrophosphatase is part of a molecular protection system in plants, preventing the accumulation of (d)ITP and its usage for nucleic acid synthesis.

Disease-associated inosine misincorporation into RNA hinders translation.

Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis.

Inositol triphosphate–triggered calcium release from the endoplasmic reticulum induces lysosome biogenesis via TFEB/TFE3

Lysosomes serve as dynamic regulators of cell and organismal physiology by integrating the degradation of macromolecules with receptor and nutrient signaling. Previous studies have established that activation of the transcription factor EB (TFEB) and transcription factor E3 (TFE3) induces the expression of lysosomal genes and proteins in signaling-inactive starved cells, that is, under conditions when activity of the master regulator of nutrient-sensing signaling mechanistic target of rapamycin complex 1 is repressed. How lysosome biogenesis is triggered in signaling-active cells is incompletely understood. Here, we identify a role for calcium release from the lumen of the endoplasmic reticulum in the control of lysosome biogenesis that is independent of mechanistic target of rapamycin complex 1. We show using functional imaging that calcium efflux from endoplasmic reticulum stores induced by inositol triphosphate accumulation upon depletion of inositol polyphosphate-5-phosphatase A, an inositol 5-phosphatase downregulated in cancer and defective in spinocerebellar ataxia, or receptor-mediated phospholipase C activation leads to the induction of lysosome biogenesis. This mechanism involves calcineurin and the nuclear translocation and elevated transcriptional activity of TFEB/TFE3. Our findings reveal a crucial function for inositol polyphosphate-5-phosphatase A–mediated triphosphate hydrolysis in the control of lysosome biogenesis via TFEB/TFE3, thereby contributing to our understanding how cells are able to maintain their lysosome content under conditions of active receptor and nutrient signaling.

Preparation of NIR-responsive, ROS-generating and antibacterial black phosphorus quantum dots for promoting the MRSA-infected wound healing in diabetic rats

Multidrug-resistant (MDR) bacteria-induced infection is becoming a huge challenge for clinical treatment, especially for non-healing diabetic wound infections, which increase patient mortality. MRSA infections and delayed wound healing (methicillin-resistant Staphylococcus aureus) accounted for a higher proportion. Although surgical debridement and continuous use of antibiotics are still the main clinical treatments, new multifunctional therapeutic nanoplatform are attractive for MIDW. Thus, in the present study, black phosphorus quantum dots (BPQDs) encapsulated in hydrogel (BPQDs@NH) were utilized as nanoplatforms for MIDW treatment to achieve the multifunctional properties of NIR (near infrared) responsiveness, ROS (reactive oxygen species) generation and antibacterial activity. Upon NIR irradiation, the temperature of the BPQDs@NH-treated MIDW area rapidly increased up to 55 °C for sterilization. In vitro experiments showed that BPQDs@NH exerted a synergistic effect on inhibiting MRSA by producing of ROS, lipid peroxidation, glutathione, adenosine triphosphate accumulation and bacterial membrane destruction upon NIR irradiation. The resulting BPQDs@NH achieved an effective sterilization rate of approximately 90% for MRSA. Furthermore, animal experiments revealed that BPQDs@NH achieved an effective closure rate of 95% for MIDW after 12 days by reducing the inflammatory response and regulating the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Meanwhile, intravenous circulation experiments showed good biocompatibility of BPQDs, and no obvious damage to rat major organs was observed. The obtained results indicated that BPQDs@NH achieved the synergistic functions of NIR-responsiveness, ROS generation, and antibacterial activity and promoted wound healing, suggesting that they are promising multifunctional nanoplatforms for MIDW healing. Statement of significance1. NIR-triggered ROS-generating and antibacterial nanoplatforms are attractive in the wound healing field. 2. In this work, black phosphorus quantum dots encapsulated in a hydrogel were used as a nanoplatform for treating MRSA infected wounds. 3. The obtained materials have achieved an effective sterilization rate for MRSA and effective wound closure rate.

MTH1 Inhibitors for the Treatment of Psoriasis

Inflammatory diseases, including psoriasis, are characterized by changes in redox regulation. The MTH1 prevents the incorporation of oxidized nucleotides during DNA replication. Using MTH1 small-molecule inhibitors, we found induced apoptosis through 8-oxodeoxyguanosine triphosphate accumulation and DNA double-strand breaks after oxidative stress in normal and malignant keratinocytes. In psoriasis, we detected increased MTH1 expression in lesional skin and PBMCs compared with that in the controls. Using the imiquimod psoriasis mouse model, we found that MTH1 inhibition diminished psoriatic histological characteristics and normalized the levels of neutrophils and T cells in the skin and skin-draining lymph nodes. The inhibition abolished the expression of T helper type 17‒associated cytokines in the skin, which was in line with decreased levels of IL-17-producing γδ T cells in lymph nodes. In human keratinocytes, MTH1 inhibition prevented the upregulation of IL-17‒downstream genes, which was independent of ROS-induced apoptosis. In conclusion, our data support MTH1 inhibition using small molecules suitable for topical application as a promising therapeutic approach to psoriasis.

Discovery of a Novel Inhibitor of Human Purine Nucleoside Phosphorylase by a Simple Hydrophilic Interaction Liquid Chromatography Enzymatic Assay.

Human purine nucleoside phosphorylase (HsPNP) belongs to the purine salvage pathway of nucleic acids. Genetic deficiency of this enzyme triggers apoptosis of activated T-cells due to the accumulation of deoxyguanosine triphosphate (dGTP). Therefore, potential chemotherapeutic applications of human PNP inhibitors include the treatment of T-cell leukemia, autoimmune diseases and transplant tissue rejection. In this report, we present the discovery of novel HsPNP inhibitors by coupling experimental and computational tools. A simple, inexpensive, direct and non-radioactive enzymatic assay coupled to hydrophilic interaction liquid chromatography and UV detection (LC-UV using HILIC as elution mode) was developed for screening HsPNP inhibitors. Enzymatic activity was assessed by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx) by LC-UV. A small library of 6- and 8-substituted nucleosides was synthesized and screened. The inhibition potency of the most promising compound, 8-aminoinosine (4), was quantified through Ki and IC50 determinations. The effect of HsPNP inhibition was also evaluated in vitro through the study of cytotoxicity on human T-cell leukemia cells (CCRF-CEM). Docking studies were also carried out for the most potent compound, allowing further insights into the inhibitor interaction at the HsPNP active site. This study provides both new tools and a new lead for developing novel HsPNP inhibitors.

Pentostatin antagonizes the antiviral activity of MBX-2168 by inhibiting the biosynthesis of the active compound

MBX-2168 has a mechanism of action similar to that of acyclovir (ACV) and ganciclovir (GCV), but two unique steps differentiate this drug from ACV/GCV. First, MBX-2168 is, at least partially, phosphorylated by the endogenous cellular kinase TAOK3 to a monophosphate. The second involves the removal of a moiety at the 6-position of MBX-2168-MP by adenosine deaminase like protein-1 (ADAL-1). It has been previously demonstrated that co-incubation with pentostatin (dCF), an ADAL-1 inhibitor, antagonizes the anti-viral activity of MBX-2168. We therefore hypothesize that inhibiting ADAL-1 results in a reduction of active compound produced in virus-infected cells. To test this, we examined the effect dCF has on the conversion of MBX-2168 to a triphosphate in HSV-1 and HCMV-infected cells. Our results demonstrate incubation of MBX-2168 alone or with dCF in HCMV-infected cells resulted in 53.1 ± 0.7 and 39.4 ± 1.5 pmol triphosphate/106 cells at 120 h, respectively. Incubation of MBX-2168 alone or with dCF in Vero cells resulted in 12.8 ± 0.1 and 6.7 ± 0.7 pmol triphosphate/106 cells at 24 h, respectively. HSV-1-infected Vero cells demonstrated no statistical difference in triphosphate accumulation at 24 h (13.1 ± 0.3 pmol triphosphate/106 cells). As expected, incubation with dCF resulted in the accumulation of MBX-2168-MP in both HFF (9.8 ± 0.9 pmol MBX-2168-MP/106 cells at 120 h) and Vero cells (4.7 ± 0.3 pmol MBX-2168-MP/106 cells at 24 h) while no detectable levels of monophosphate were observed in cultures not incubated with dCF. We conclude that dCF antagonizes the anti-viral effect of MBX-2168 by inhibiting the production of triphosphate, the active compound.

Exogenous phytosulfokine α (PSKα) application delays senescence and relieves decay in strawberry fruit during cold storage by triggering extracellular ATP signaling and improving ROS scavenging system activity

During postharvest life, strawberry fruit as a perishable commodity exhibiting short shelf life suffers from fungal decay that severely limits its marketability and nutritional value. Herein, we investigated the mechanisms activated by exogenous phytosulfokine α (PSKα) application at 150 nM to delay senescence and relieve decay in strawberry fruit during storage at 4 °C for 18 days. Our results showed higher extracellular adenosine triphosphate (ATP) accumulation arising from lower apyrase 1 (APY1) gene expression in strawberry fruit treated with 150 nM PSKα during storage at 4 °C for 6 days may serve as a signaling molecule to promote NADPH oxidase activity to trigger signaling H2O2 accumulation giving rise to higher SUMO E3 ligase (SIZ1) gene expression during 18 days of storage at 4 °C. Also, higher superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) genes expression and enzymes activity associated with higher methionine sulfoxide reductase (MSR) and peroxiredoxin (Prx) genes expression in strawberry fruit treated with 150 nM PSKα during storage at 4 °C for 18 days may be ascribed to extracellular ATP signaling. As a result, promoting ROS scavenging systems activity by triggering extracellular ATP signaling in strawberry fruit treated with 150 nM PSKα may be responsible for higher maintenance of membrane integrity as shown by lower malondialdehyde (MDA) accumulation during 18 days of storage at 4 °C. Besides, strawberry fruits treated with 150 nM PSKα exhibited lower weight loss, total soluble solids, and chroma value concurrent with higher firmness, titrable acidity, L* value, and hue angle as quality attributes, which indicated delayed fruit senescence. Based on our findings, exogenous PSKα application unveils its potential to be used as a promising signaling biopeptide for senescence delay and decay alleviation in strawberry fruit during its postharvest life.

INVOLVEMENT OF PURINERGIC SIGNALING AND LYMPHOID ORGANS IN THE BENEFICIAL EFFECTS OF AEROBIC TRAINING IN ASTHMA

PURPOSE: Asthma is a chronic airway inflammatory disease affecting more than 300 million people around the world. Purinergic signaling via purinergic receptors (mainly P2X7, P2Y2 and P2Y6) are thought play a key role in asthma pathogenesis and severity. High intensity aerobic exercise is known to trigger asthma attacks, while low to moderate intensity training reduces inflammation and improves asthma control. Therefore, this study investigated whether low intensity aerobic exercise reduces asthma phenotype by modulation of purinergic signaling. METHODS: Aerobic exercise (AE) was performed in a treadmill at low intensity, 5x/week, 1h/session, for 4 weeks, beginning 2 weeks after HDM administration. HDM (dermatophagoides pteronyssinus; 100mg/mouse) was administered 3x/week, for 6 weeks. RESULTS: The results demonstrated that AE reduced adenosine triphosphate (ATP) accumulation (p<0.001), IL-1beta, IL-4, IL-5, CXCL1/KC, IL-13, IL-17, IL-23, IL-33 and TNF-alpha (p<0.001), while increased IL-1ra, IL-2, IL-10 and IL-12p40 in bronchoalveolar lavage (BAL). Total number of leukocytes, eosinophils, lymphocytes and neutrophils in BAL and the number of eosinophils, neutrophils and lymphocytes in the airway wall (p<0.01) were reduced by AE. Airway remodeling (collagen, elastin, smooth muscle and mucus) were reduced by AE (p<0.01). TGF-beta, IGF-1 and VEGF levels was reduced by AE (p<0.001). Lung mechanics (Resistance, Elastance, GTIS, HTIS, RAW) and airway hyperresponsiveness (AHR) to methacholine was ameliorated by AE (p<0.01). IL-4, IL-5 and IL-13 production by re-stimulated mediastinal lymph nodes, splenocytes and bone marrow cells was also reduced by AE. The expression of P2X7, P2Y2 and P2Y6 by peribronchial leukocytes (p<0.01) and also by airway epithelial cells (p<0.01) were reduced by AE. CONCLUSIONS: Low intensity aerobic training reduces asthma phenotype by inhibiting purinergic signaling and lymphoid organs hyperactivation.

Biosynthesis and half-life of MBX-2168-triphosphate in herpes virus-infected cells

The third generation of methylenecyclopropane nucleoside analogs (MCPNAs) elicit an anti-viral effect against all three sub-classes of herpes viruses without inducing cytotoxicity in vitro. It has been previously established that the mechanism of action of MCPNAs is similar to that of ganciclovir (GCV) or acyclovir (ACV). However, the activation of MBX-2168, a third generation MCPNA, involves additional and unique enzymatic steps and this process has not been examined in virus-infected cells. To that end, herpes virus-infected cells were incubated with MBX-2168, synguanol, GCV, or ACV. Incubation of HCMV-infected cells with five times the EC50 of MBX-2168 (4.0 μM), synguanol (10.5 μM), or GCV (25 μM) resulted in a time-dependent increase in triphosphate accumulation reaching a maximum of 48.1 ± 5.5, 45.5 ± 2.5, and 42.6 ± 3.7 pmol/106 cells at 120 h, respectively. Additionally, half-lives of these compounds were similar in HCMV-infected cells (GCV-TP = 25.5 ± 2.7 h; MBX-2168-TP/synguanol-TP = 23.0 ± 1.4 h). HSV-1-infected cells incubated with five times the EC50 of MBX-2168 (33.5 μM) or ACV (5.0 μM) demonstrated a time-dependent increase in triphosphate levels reaching a maximum of 12.3 ± 1.5 and 11.6 ± 0.7 pmol/106 cells at 24 h, respectively. ACV-TP and MBX-2168-TP also had similar half-lives under these conditions (27.3 ± 4.8 h and 22.2 ± 2.2 h, respectively). We therefore conclude that although MBX-2168 does not follow the classical route of nucleoside analog activation, the metabolic profile of MBX-2168 is similar to other nucleoside analogs such as GCV and ACV that do.

8-Chloroadenosine Alters the Metabolic Profile and Downregulates Antioxidant and DNA Damage Repair Pathways in Macrophages.

The exposure of RNA and DNA nucleobases to the oxidant hypochlorous acid (HOCl) results in the generation of different stable chlorinated products. These chlorinated nucleobases are formed in vivo, particularly in chronic inflammatory pathologies, which are characterized by the overproduction of HOCl by myeloperoxidase. As such, chlorinated nucleosides are used as biomarkers of inflammation. However, these compounds have also attracted attention as potential chemotherapeutic agents with 8-chloro-adenosine (8ClA), for example, currently in clinical trials for the treatment of hematological cancers, including chronic lymphocytic leukemia. 8ClA has mainly RNA-directed effects in malignant cells, with exposure resulting in ATP depletion and apoptotic cell death. Whether 8ClA has significant reactivity with nonmalignant cells has not been widely studied. Here we show that prolonged incubation of J774A.1 macrophage-like cells with 8ClA results in the perturbation of cellular metabolism and apoptotic cell death. These effects are associated with an accumulation of 8-chloroadenosine triphosphate (8Cl-ATP), an effect not seen in experiments utilizing other chlorinated nucleosides. Exposure of the macrophages to 8ClA did not significantly change basal mitochondrial respiration or glycolysis but resulted in an increase in maximal mitochondrial respiration as well as spare respiratory capacity within these cells. Additionally, 8ClA exposure also altered the mRNA expression of a range of antioxidant and DNA damage repair genes in the macrophages in a manner consistent with a reduction in the capacity of the cells to cope with oxidative stress and repair DNA damage. Taken together, these results provide new insight into pathways by which the production of HOCl during chronic inflammation could perturb immune cell function and may also have implications for the use of 8ClA as a chemotherapeutic drug.

Inhibition of ubiquitin-specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts.

Ubiquitin‐specific protease 2 (USP2) is considered to participate in the differentiation of myoblasts to myotubes, however, its functions in myoblasts under growth conditions remain elusive. In this study, we analyzed the physiological roles of USP2 in myoblasts using Usp2 knockout (KO) C2C12 cells as well as a USP2 specific inhibitor. In addition to the disruption of differentiation, clustered regularly interspaced short palindromic repeats/Cas9‐generated Usp2KO cells exhibited inhibition of proliferation compared to parental C2C12 cells. Usp2KO cells reduced the accumulation of intracellular adenosine triphosphate (ATP) content and oxygen consumption. Moreover, Usp2KO cells had fragmented mitochondria, suggesting that mitochondrial respiration was inactive. The deficiency of Usp2 did not affect the enzymatic activities of respiratory chain complexes I, III, IV, and V. However, mitochondrial membrane permeability—evaluated using calcein AM‐cobalt staining—was increased in Usp2KO cells. The membrane potential of Usp2KO cells was clearly decreased. Usp2KO cells accumulated reactive oxygen species (ROS) in the mitochondria. The USP2‐selective inhibitor ML364 also increased the levels of mitochondrial ROS, and modulated the membrane potential and morphology of the mitochondria. These effects were followed by a decrement in the intracellular content of ATP. Based on these findings, we speculate that USP2 may be involved in maintaining the integrity of the mitochondrial membrane. This process ensures the supply of ATP in myoblasts, presumably leading to proliferation and differentiation.

Increased Cytotoxicity of Herpes Simplex Virus Thymidine Kinase Expression in Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a “thymidine block”, but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications.

Abnormal Regulation of Intracellular Calcium in Human Megakaryocytes Contributes to the Pathophysiology of Calr-Mutant Myeloproliferative Neoplasms

The BCR-ABL-negative Myeloproliferative Neoplasms (MPNs), are a group of clonal hematopoietic malignancies, that consist of three disorders: Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). Approximately one quarter of patients with ET or PMF carry a somatic mutation of CALR, the gene encoding for the endoplasmic reticulum (ER) chaperone calreticulin. A 52-bp deletion (Type I) and a 5-bp insertion (Type II mutation) are the most frequent lesions. Both generate a new carboxy-terminus of the mutant proteins, in which the ER retention signal KDEL is lost, causing abnormal interaction of the mutants with the thrombopoietin (TPO) receptor (MPL), thus constitutively activating its downstream signaling in megakaryocytes. CALR Type I mutation causes important changes in the charge of the carboxyl-terminal tail, which is responsible for calcium (Ca2+) binding activity. As a consequence, megakaryocytes from CALR Type I patients present enhanced activation of Store-Operated Ca2+ Entry (SOCE), the mechanisms that trigger extracellular Ca2+ inflow upon agonist-induced intracellular Ca2+ mobilization from the ER. A fine control of SOCE and overall Ca2+ homeostasis is fundamental to ensure proper megakaryocyte function. Despite this knowledge, the role of a crosstalk among MPL, CALR and SOCE in regulating megakaryopoiesis has never been hypothesised.Our research analysed the activation of MPL downstream pathways and Ca2+ signalling in human cord blood and peripheral blood-cultured megakaryocytes, upon stimulation with recombinant human TPO (rhTPO). Additionally, we cultured megakaryocytes from the peripheral blood of patients carrying the CALR Type I mutation to investigate whether CALR and SOCE alterations determine abnormal activation of the MPL downstream pathway in MPNs.We demonstrated that binding of rhTPO to MPL induces a series of downstream signaling events that lead to the activation of STAT5, AKT and ERK1/2 in human megakaryocytes. This activation relies on significant accumulation of inositol triphosphate (IP3), and consequent intracellular Ca2+ release from the ER followed by extracellular Ca2+ entry, indicative of activated SOCE. Pharmacologic inhibition of the IP3 receptor or SOCE, with 2-APB or BTP-2 respectively, resulted in the complete absence of rhTPO-induced MPL signaling activation. SOCE activation is thus dependent on the dissociation of CALR and STIM1, a protein of the SOCE machinery, which normally forms a complex in un-stimulated megakaryocytes. Importantly, we verified that in human megakaryocytes in rhTPO-starved conditions, CALR forms a molecular complex with its co-chaperone, ERp57, and STIM1, while they dissociate upon rhTPO stimulation. Conversely, we observed that CALR, ERp57 and STIM1 are constitutively dissociated in megakaryocytes from CALR Type I patients, even in complete absence of rhTPO. This dissociation resulted in markedly increased TPO-induced cytosolic Ca2+ flows with a consequent abnormal megakaryocyte proliferation that could be counteracted by pharmacological inhibition of SOCE with BTP-2.Our findings provide the first evidence that CARL Type I, in addition to MPL activation, concurs to the alteration of SOCE, which sustains the phosphorylation of STAT5, AKT and ERK1/2, and induces megakaryocyte proliferation. Further investigation is needed to understand whether this altered megakaryocyte function is determined by the decrease in CALR levels or by the presence of CALR mutants in the ER. Nevertheless, the abnormal regulation of Ca2+ flows may represent a potentially actionable target. DisclosuresNo relevant conflicts of interest to declare.

Mutational analysis of CCL20 reveals flexibility of N-terminal amino acid composition and length.

Chemokine-chemokine receptor (CKR) interactions are traditionally described by a two-step/two-site mechanism that details the major contact points between chemokine ligands and CKRs leading to ligand recognition and receptor activation. Chemokine recognition site 1 (CRS1) encompasses interactions between the CKR N-terminus and the globular chemokine core. Chemokine recognition site 2 (CRS2) includes interactions between the unstructured chemokine N-terminus and the binding pocket of the receptor. The two-step/two-site paradigm has been an adequate framework to study the intricacies of chemokine:CKR interactions, but emerging studies highlight the limitations of this model. Here, we present studies of CRS2 interactions between the chemokine CCL20 and its cognate receptor CCR6 driven by the hypothesis that CCL20 interacts with CCR6 as described by the two-step/two-site model. CCL20 is a chemokine with an unusually short N-terminus of 5 residues (NH2 -ASNFD), compared to the average length of 10 residues for chemokine ligands. We have investigated how well CCL20 tolerates manipulation of the N-terminus by monitoring binding affinity of variants and their ability to activate the receptor. We show the CCL20 N-terminus tolerates truncation of up to 3 residues, extension by up to 5 additional residues, and point mutations at 4 of 5 positions with minimal loss of binding affinity and minimal impairment in ability to stimulate calcium mobilization, inositol triphosphate accumulation, chemotaxis, and β-arrestin-2 recruitment. Mutation of the fifth residue, aspartate, to alanine or lysine has a dramatic impact on binding affinity for CCR6 and ligand potency. We postulate CCL20 does not activate CCR6 through the canonical two-step/two-site mechanism of CKR activation.

Effect of hydraulic retention time on nitrogen removal and functional gene quantity/transcription in biochar packed reactors at 5 °C: A control-strategy study

This study investigated the effect of hydraulic retention time (HRT = 8, 12, 16, and 24 h) on effluent dissolved organic nitrogen (DON), dissolved total nitrogen (DTN), and functional gene quantity/transcription in biochar packed reactors over a 125-day operation at 5 °C. The lowest effluent DON concentration (0.21 ± 0.14 mg/L) and DTN concentration (10.74 ± 0.41 mg/L) were in R12h and R24h, respectively. Adequate HRT (>12 h) was a necessary parameter for poly-β-hydroxybutyrate (PHB) accumulation (PHB/MLSS: 0.1181–0.1522), but higher HRT was detrimental to adenosine triphosphate (ATP) accumulation from 62.77–66.31 µg/g SS (R8h) to 48.21–48.39 µg/g SS (R24h). Effluent DON had a negative correlation relation with ATP and the relative abundance of amoA (p = 0.02), and effluent DTN had a negative correlation relation with PHB (p < 0.01), the relative abundance of napA (p = 0.01), and the transcriptional quantity of nirS (p = 0.14) and nxrA (p = 0.03).

Clofarabine: a next-generation deoxyadenosine analogue

Acute lymphoblastic leukaemia (ALL) is the most common of the paediatric leukaemias. It is estimated that the use of modern combination chemotherapy results in long-term remission in nearly 80% of children diagnosed with ALL. Despite therapy advances, approximately 20% of children with ALL, experience leukaemia relapse. Clofarabine (2-chloro-2’-fluoro-2’-deoxy-9-β-D-arabinofuranosyladenine) is a second-generation nucleoside analogue and is structurally related to fludarabine and cladribine which are widely used in the treatment of lymphoproliferative disorders. Clofarabine exhibits greater affinity to deoxycytidine kinase (dCyd kinase) and prolonged retention in leukaemic blasts compared to fludarabine and cladribine. Clofarabine inhibits both DNA polymerases and ribonucleotide reductase (RNR). This results in impaired DNA synthesis through inhibition of DNA elongation as well as depletion of deoxyribonucleotides. Accumulation of clofarabine triphosphate, in the blasts of patients with refractory leukemia has been demonstrated. Prolonged intracellular half-life of 24 hours for clofarabine triphosphate. Clofarabine is indicated for the treatment of pediatric patients 1 to 21 years old with relapsed or refractory acute lymphoblastic leukemia after at least two prior regimens.

Circadian ATP Release in Organotypic Cultures of the Rat Suprachiasmatic Nucleus Is Dependent on P2X7 and P2Y Receptors.

The circadian rhythms in physiological and behavioral functions are driven by a pacemaker located in the suprachiasmatic nucleus (SCN). The rhythms continue in constant darkness and depend on cell-cell communication between neurons and glia. The SCN astrocytes generate also a circadian rhythm in extracellular adenosine 5′-triphosphate (ATP) accumulation, but molecular mechanisms that regulate ATP release are poorly understood. Here, we tested the hypothesis that ATP is released via the plasma membrane purinergic P2X7 receptors (P2X7Rs) and P2Y receptors (P2YRs) which have been previously shown to be expressed in the SCN tissue at transcriptional level. We have investigated this hypothesis using SCN organotypic cultures, primary cultures of SCN astrocytes, ATP bioluminescent assays, immunohistochemistry, patch-clamping, and calcium imaging. We found that extracellular ATP accumulation in organotypic cultures followed a circadian rhythm, with a peak between 24:00 and 04:00 h, and the trough at ~12:00 h. ATP rhythm was inhibited by application of AZ10606120, A438079, and BBG, specific blockers of P2X7R, and potentiated by GW791343, a positive allosteric modulator of this receptor. Double-immunohistochemical staining revealed high expression of the P2X7R protein in astrocytes of SCN slices. PPADS, a non-specific P2 antagonist, and MRS2179, specific P2Y1R antagonist, also abolished ATP rhythm, whereas the specific P2X4R blocker 5-BDBD was not effective. The pannexin-1 hemichannel blocker carbenoxolone displayed a partial inhibitory effect. The P2Y1R agonist MRS2365, and the P2Y2R agonist MRS2768 potentiated ATP release in organotypic cultures and increase intracellular Ca2+ level in cultured astrocytes. Thus, SCN utilizes multiple purinergic receptor systems and pannexin-1 hemichannels to release ATP.