The human ion channel TRPM2 modulates migration and invasion in neuroblastoma through regulation of integrin expression.

Transient receptor potential channel TRPM2 is highly expressed in many cancers and involved in regulation of key physiological processes including mitochondrial function, bioenergetics, and oxidative stress. In Stage 4 non-MYCN amplified neuroblastoma patients, high TRPM2 expression is associated with worse outcome. Here, neuroblastoma cells with high TRPM2 expression demonstrated increased migration and invasion capability. RNA sequencing, RT-qPCR, and Western blotting demonstrated that the mechanism involved significantly greater expression of integrins α1, αv, ß1, and ß5 in cells with high TRPM2 expression. Transcription factors HIF-1α, E2F1, and FOXM1, which bind promoter/enhancer regions of these integrins, were increased in cells with high TRPM2 expression. Subcellular fractionation confirmed high levels of α1, αv, and ß1 membrane localization and co-immunoprecipitation confirmed the presence of α1ß1, αvß1, and αvß5 complexes. Inhibitors of α1ß1, αvß1, and αvß5 complexes significantly reduced migration and invasion in cells highly expressing TRPM2, confirming their functional role. Increased pAktSer473 and pERKThr202/Tyr204, which promote migration through mechanisms including integrin activation, were found in cells highly expressing TRPM2. TRPM2 promotes migration and invasion in neuroblastoma cells with high TRPM2 expression through modulation of integrins together with enhancing cell survival, negatively affecting patient outcome and providing rationale for TRPM2 inhibition in anti-neoplastic therapy.

Reflections of Nursing Students Following Hurricane Michael.

Background: Students in the southeastern United States were affected by Hurricane Michael in 2018. The storm's devastation led to communication and education issues that transformed students' paths to completing their nursing degree program. Climate change will cause increased natural disasters, and educators must be prepared. Method: This qualitative study used a descriptive phenomenological design. A purposive sample of 10 graduate nursing students were recruited and interviewed. The data gathering ended when data saturation was achieved. Results: Five themes were generated: Devastation and no communication, survival mode, emotional impact, before and after, and forever changed. The rich data documented the resiliency of the students as they described events during and after the hurricane in relation to their personal and educational experiences. Conclusion: Universities and students need to be prepared for natural disasters; understanding graduate nursing students' prior experiences can benefit educators.

The human ion channel TRPM2 modulates cell survival in neuroblastoma through E2F1 and FOXM1.

Transient receptor potential channel melastatin 2 (TRPM2) is highly expressed in cancer and has an essential function in preserving viability through maintenance of mitochondrial function and antioxidant response. Here, the role of TRPM2 in cell survival was examined in neuroblastoma cells with TRPM2 deletion with CRISPR technology. Viability was significantly decreased in TRPM2 knockout after doxorubicin treatment. RNA sequence analysis and RT-qPCR revealed reduced RNAs encoding master transcription regulators FOXM1 and E2F1/2 and downstream cell cycle targets including Cyclin B1, CDK1, PLK1, and CKS1. CHIP analysis demonstrated decreased FOXM1 binding to their promoters. Western blotting confirmed decreased expression, and increased expression of CDK inhibitor p21, a CKS1 target. In cells with TRPM2 deletion, cell cycle progression to S and G2/M phases was reduced after treatment with doxorubicin. RNA sequencing also identified decreased DNA repair proteins in cells with TRPM2 deletion after doxorubicin treatment, and DNA damage was increased. Wild type TRPM2, but not Ca2+-impermeable mutant E960D, restored live cell number and reconstituted expression of E2F1, FOXM1, and cell cycle/DNA repair proteins. FOXM1 expression alone restored viability. TRPM2 is a potential therapeutic target to reduce tumor proliferation and increase doxorubicin sensitivity through modulation of FOXM1, E2F1, and cell cycle/DNA repair proteins.

Expression Patterns of Immune Genes Reveal Heterogeneous Subtypes of High-Risk Neuroblastoma.

High risk neuroblastoma (HR-NB) remains difficult to treat, and its overall survival (OS) is still below 50%. Although HR-NB is a heterogeneous disease, HR-NB patients are currently treated in a similar fashion. Through unsupervised biclustering, we further stratified HR-NB patients into two reproducible and clinically distinct subtypes, including an ultra-high risk neuroblastoma (UHR-NB) and high risk neuroblastoma (HR-NB). The UHR-NB subtype consistently had the worst OS in multiple independent cohorts ( P < 0 . 008 ). Out of 283 neuroblastoma-specific immune genes that were used for stratification, 39 of them were differentiated in UHR-NB, including four upregulated and 35 downregulated, as compared to HR-NB. The four UHR-NB upregulated genes (ADAM22, GAL, KLHL13 and TWIST1) were all upregulated in MYCN amplified neuroblastoma in 5 additional cohorts. TWIST1 and ADAM22 were also positively correlated with cancer stage, while GAL was an independent OS predictor in addition to MYCN and age. Furthermore, we identified 26 commonly upregulated and 311 downregulated genes in UHR-NB from all 4723 immune-related genes. While 43 KEGG pathways with molecular functions were enriched in the downregulated immune-related genes, only the P53 signaling pathway was enriched in the upregulated ones, which suggested that UHR-NB was a TP53 related subtype with reduced immune activities.

Glucocorticoids enhance the antileukemic activity of FLT3 inhibitors in FLT3-mutant acute myeloid leukemia.

FLT3 is a frequently mutated gene that is highly associated with a poor prognosis in acute myeloid leukemia (AML). Despite initially responding to FLT3 inhibitors, most patients eventually relapse with drug resistance. The mechanism by which resistance arises and the initial response to drug treatment that promotes cell survival is unknown. Recent studies show that a transiently maintained subpopulation of drug-sensitive cells, so-called drug-tolerant "persisters" (DTPs), can survive cytotoxic drug exposure despite lacking resistance-conferring mutations. Using RNA sequencing and drug screening, we find that treatment of FLT3 internal tandem duplication AML cells with quizartinib, a selective FLT3 inhibitor, upregulates inflammatory genes in DTPs and thereby confers susceptibility to anti-inflammatory glucocorticoids (GCs). Mechanistically, the combination of FLT3 inhibitors and GCs enhances cell death of FLT3 mutant, but not wild-type, cells through GC-receptor-dependent upregulation of the proapoptotic protein BIM and proteasomal degradation of the antiapoptotic protein MCL-1. Moreover, the enhanced antileukemic activity by quizartinib and dexamethasone combination has been validated using primary AML patient samples and xenograft mouse models. Collectively, our study indicates that the combination of FLT3 inhibitors and GCs has the potential to eliminate DTPs and therefore prevent minimal residual disease, mutational drug resistance, and relapse in FLT3-mutant AML.

Transient receptor potential ion channel TRPM2 promotes AML proliferation and survival through modulation of mitochondrial function, ROS, and autophagy.

Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in maintaining cell survival following oxidant injury. Here, we show that TRPM2 is highly expressed in acute myeloid leukemia (AML). The role of TRPM2 in AML was studied following depletion with CRISPR/Cas9 technology in U937 cells. In in vitro experiments and in xenografts, depletion of TRPM2 in AML inhibited leukemia proliferation, and doxorubicin sensitivity was increased. Mitochondrial function including oxygen consumption rate and ATP production was reduced, impairing cellular bioenergetics. Mitochondrial membrane potential and mitochondrial calcium uptake were significantly decreased in depleted cells. Mitochondrial reactive oxygen species (ROS) were significantly increased, and Nrf2 was decreased, reducing the antioxidant response. In TRPM2-depleted cells, ULK1, Atg7, and Atg5 protein levels were decreased, leading to autophagy inhibition. Consistently, ATF4 and CREB, two master transcription factors for autophagosome biogenesis, were reduced in TRPM2-depleted cells. In addition, Atg13 and FIP200, which are known to stabilize ULK1 protein, were decreased. Reconstitution with TRPM2 fully restored proliferation, viability, and autophagy; ATF4 and CREB fully restored proliferation and viability but only partially restored autophagy. TRPM2 expression reduced the elevated ROS found in depleted cells. These data show that TRPM2 has an important role in AML proliferation and survival through regulation of key transcription factors and target genes involved in mitochondrial function, bioenergetics, the antioxidant response, and autophagy. Targeting TRPM2 may represent a novel therapeutic approach to inhibit myeloid leukemia growth and enhance susceptibility to chemotherapeutic agents through multiple pathways.

The Human Transient Receptor Potential Melastatin 2 Ion Channel Modulates ROS Through Nrf2.

Transient receptor potential melastatin channel subfamily member 2 (TRPM2) has an essential role in protecting cell viability through modulation of oxidative stress. TRPM2 is highly expressed in cancer. When TRPM2 is inhibited, mitochondria are dysfunctional, ROS levels are increased, and cell viability is reduced. Here, the importance of NF-E2-related factor (Nrf2) in TRPM2-mediated suppression of oxidant stress was explored. In TRPM2 depleted cells, antioxidant cofactors glutathione, NADPH, and NADH were significantly reduced. Cytoplasmic and nuclear expression of Nrf2 and of IQGAP1, a modulator of Nrf2 stability regulated by intracellular calcium, were decreased. Antioxidant enzymes transcriptionally regulated by Nrf2 and involved in GSH, NADPH, and NADH generation were significantly lower including PRX1 and PRX3, GPX4, GSTP1, GCLC, and MTHFD2. The glutamine pathway leading to GSH production was suppressed, and ATP and GTP levels were impaired. Reconstitution with wild type TRPM2 or Nrf2, but not TRPM2 pore mutant E960D, rescued expression of enzymes downstream of Nrf2 and restored GSH and GTP. Cell viability, ROS, NADPH, NADH, and ATP levels were fully rescued by TRPM2 and partially by Nrf2. These data show that TRPM2 maintains cell survival following oxidative stress through modulation of antioxidant pathways and cofactors regulated by Nrf2.

Infrared spectroscopy of C3-(H2O)n and C3-(D2O)n complexes in helium droplets.

The C3 molecule is an important species with implications in combustion and astrochemistry, and much of the interest in this molecule is related to its interactions with other species found in these environments. We have utilized helium droplet beam techniques along with a recently developed carbon cluster evaporation source to assemble C3-(H2O)n and C3-(D2O)n complexes with n = 1-2 and to record their rovibrational spectra. We observe only a single isomer of the n = 1 complex, in agreement with theoretical predictions as well as data from earlier matrix isolation studies. The spectra of the n = 1 complex are consistent with the ab initio structure, which involves a nearly linear arrangement of CCC-HO atoms in the complex. The C3-H2O spectrum we obtain exhibits slight differences from the analogous C3-D2O spectrum, which we assign to a difference in linewidth between the two spectra. We have also examined the n = 2 species and obtained a structure that appears to be distinct from those observed in matrix isolation studies and, to our knowledge, has not been previously observed.

TRPM2 in Cancer.

The TRP ion channel TRPM2 has an essential function in cell survival and protects the viability of a number of cell types after oxidative stress. It is highly expressed in many cancers including breast, prostate, and pancreatic cancer, melanoma, leukemia, and neuroblastoma, suggesting it promotes cancer cell survival. TRPM2 is activated by production of ADP-ribose (ADPR) following oxidative stress, which binds to the C-terminus of TRPM2, resulting in channel opening. In a number of cancers including neuroblastoma, TRPM2 has been shown to preserve viability and mechanisms have been identified. Activation of TRPM2 results in expression of transcription factors and kinases important in cell proliferation and survival including HIF-1/2α, CREB, nuclear factor (erythroid-derived 2)-related factor-2 (Nrf2), and Pyk2, and Src phosphorylation. Together, HIF-1/2α and CREB regulate expression of genes encoding proteins with roles in mitochondrial function including members of the electron transport complex involved in ATP production. These contribute to lower mitochondrial ROS production while expression of antioxidants regulated by HIF-1/2α, FOXO3a, CREB, and Nrf2 is maintained. CREB is also important in control of expression of key proteins involved in autophagy. When TRPM2-mediated calcium influx is inhibited, mitochondria are dysfunctional, cellular bioenergetics are reduced, production of ROS is increased, and autophagy and DNA repair are impaired, decreasing tumor growth and increasing chemotherapy sensitivity. Inhibition of TRPM2 expression or function results in decreased tumor proliferation and/or viability in many malignancies including breast, gastric, pancreatic, prostate, head and neck cancers, melanoma, neuroblastoma, and T-cell and acute myelogenous leukemia. However, in a small number of malignancies, activation of TRPM2 rather than inhibition has been reported to reduce tumor cell survival. Here, TRPM2-mediated Ca2+ signaling and mechanisms of regulation of cancer cell growth and survival are reviewed and controversies discussed. Evidence suggests that targeting TRPM2 may be a novel therapeutic approach in many cancers.

Trpm2 enhances physiological bioenergetics and protects against pathological oxidative cardiac injury: Role of Pyk2 phosphorylation.

The mechanisms by which Trpm2 channels enhance mitochondrial bioenergetics and protect against oxidative stress-induced cardiac injury remain unclear. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in Trpm2 signaling is explored. Activation of Trpm2 in adult myocytes with H2 O2 resulted in 10- to 21-fold increases in Pyk2 phosphorylation in wild-type (WT) myocytes which was significantly lower (~40%) in Trpm2 knockout (KO) myocytes. Pyk2 phosphorylation was inhibited (~54%) by the Trpm2 blocker clotrimazole. Buffering Trpm2-mediated Ca2+ increase with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) resulted in significantly reduced pPyk2 in WT but not in KO myocytes, indicating Ca2+ influx through activated Trpm2 channels phosphorylated Pyk2. Part of phosphorylated Pyk2 translocated from cytosol to mitochondria which has been previously shown to augment mitochondrial Ca2+ uptake and enhance adenosine triphosphate generation. Although Trpm2-mediated Ca2+ influx phosphorylated Ca2+ -calmodulin kinase II (CaMKII), the CaMKII inhibitor KN93 did not significantly affect Pyk2 phosphorylation in H2 O2 -treated WT myocytes. After ischemia/reperfusion (I/R), Pyk2 phosphorylation and its downstream prosurvival signaling molecules (pERK1/2 and pAkt) were significantly lower in KO-I/R when compared with WT-I/R hearts. After hypoxia/reoxygenation, mitochondrial membrane potential was lower and superoxide level was higher in KO myocytes, and were restored to WT values by the mitochondria-targeted superoxide scavenger MitoTempo. Our results suggested that Ca2+ influx via tonically activated Trpm2 phosphorylated Pyk2, part of which translocated to mitochondria, resulting in better mitochondrial bioenergetics to maintain cardiac health. After I/R, Pyk2 activated prosurvival signaling molecules and prevented excessive increases in reactive oxygen species, thereby affording protection from I/R injury.

Targeted Inhibition of ULK1 Promotes Apoptosis and Suppresses Tumor Growth and Metastasis in Neuroblastoma.

Neuroblastoma is the most common extracranial solid malignancy in the pediatric population, accounting for over 9% of all cancer-related deaths in children. Autophagy is a cell self-protective mechanism that promotes tumor cell growth and survival, making it an attractive target for treating cancer. However, the role of autophagy in neuroblastoma tumor growth and metastasis is largely undefined. Here we demonstrate that targeted inhibition of an essential autophagy kinase, unc-51 like autophagy kinase 1 (ULK1), with a recently developed small-molecule inhibitor of ULK1, SBI-0206965, significantly reduces cell growth and promotes apoptosis in SK-N-AS, SH-SY5Y, and SK-N-DZ neuroblastoma cell lines. Furthermore, inhibition of ULK1 by a dominant-negative mutant of ULK1 (dnULK1K46N) significantly reduces growth and metastatic disease and prolongs survival of mice bearing SK-N-AS xenograft tumors. We also show that SBI-0206965 sensitizes SK-N-AS cells to TRAIL treatment, but not to mTOR inhibitors (INK128, Torin1) or topoisomerase inhibitors (doxorubicin, topotecan). Collectively, these findings demonstrate that ULK1 is a viable drug target and suggest that inhibitors of ULK1 may provide a novel therapeutic option for the treatment of neuroblastoma. Mol Cancer Ther; 17(11); 2365-76. ©2018 AACR.

The human ion channel TRPM2 modulates neuroblastoma cell survival and mitochondrial function through Pyk2, CREB, and MCU activation.

Transient receptor potential melastatin channel subfamily member 2 (TRPM2) has an essential function in cell survival and is highly expressed in many cancers. Inhibition of TRPM2 in neuroblastoma by depletion with CRISPR technology or expression of dominant negative TRPM2-S has been shown to significantly reduce cell viability. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in TRPM2 modulation of neuroblastoma viability was explored. In TRPM2-depleted cells, phosphorylation and expression of Pyk2 and cAMP-responsive element-binding protein (CREB), a downstream target, were significantly reduced after application of the chemotherapeutic agent doxorubicin. Overexpression of wild-type Pyk2 rescued cell viability. Reduction of Pyk2 expression with shRNA decreased cell viability and CREB phosphorylation and expression, demonstrating Pyk2 modulates CREB activation. TRPM2 depletion impaired phosphorylation of Src, an activator of Pyk2, and this may be a mechanism to reduce Pyk2 phosphorylation. TRPM2 inhibition was previously demonstrated to decrease mitochondrial function. Here, CREB, Pyk2, and phosphorylated Src were reduced in mitochondria of TRPM2-depleted cells, consistent with their role in modulating expression and activation of mitochondrial proteins. Phosphorylated Src and phosphorylated and total CREB were reduced in TRPM2-depleted nuclei. Expression and function of mitochondrial calcium uniporter (MCU), a target of phosphorylated Pyk2 and CREB, were significantly reduced. Wild-type TRPM2 but not Ca2+-impermeable mutant E960D reconstituted phosphorylation and expression of Pyk2 and CREB in TRPM2-depleted cells exposed to doxorubicin. Results demonstrate that TRPM2 expression protects the viability of neuroblastoma through Src, Pyk2, CREB, and MCU activation, which play key roles in maintaining mitochondrial function and cellular bioenergetics.

Transient Receptor Potential-Melastatin Channel Family Member 2: Friend or Foe.

Transient receptor potential melastatin 2 (Trpm2) channels are nonvoltage-activated channels permeable to monovalent and divalent cations, and are expressed in heart, brain, kidney, vasculature, and hematopoietic cells. Trpm2 is overexpressed in bladder, lung, breast, liver, head, and neck cancers. Classically, Trpm2 activation induces cell injury and death by Ca2+ overload or enhanced inflammatory response. Recent studies show that Trpm2 protects lungs from endotoxin-induced injury by reducing reactive oxygen species production in phagocytes; and improves cardiac function after ischemia-reperfusion injury by preserving mitochondrial respiration and cellular adenosine triphosphate levels while decreasing reactive oxygen species levels. In neuroblastoma xenografts, Trpm2 overexpression promotes tumor growth through modulation of hypoxia-inducible transcription factor expression and cellular bioenergetics; whereas Trpm2 inhibition results in enhanced sensitivity to doxorubicin. The robust expression in cancer cells and its pro-survival and proliferative properties make Trpm2 a rational target for cancer therapy. Indiscriminate Trpm2 inhibition, however, may engender serious untoward side effects in other vital organs.

Predicting Racial and Ethnic Disparities in Advance Care Planning Using the Integrated Behavioral Model.

Purpose To explain and predict racial or ethnic disparities in advance care planning (ACP) behaviors among American adults by using the Integrated Behavioral Model (IBM) and the Precaution Adoption Process Model. Methods A randomized, observational, nonexperimental, cross-sectional study design was used to survey American adults between 40 and 80 years of age ( n = 386). Results The majority of respondents (75%) had not completed ACP. Significant differences were found by race or ethnicity: 33% of Whites had completed ACP versus Hispanics (18%) and Blacks (8%). Whites had statistically significantly higher levels of most IBM constructs compared with Blacks and Hispanics. The IBM predicted 28% of the variance in behavioral intention. After controlling for sociodemographic variables, direct attitudes, indirect attitudes, and indirect perceived norms were significant predictors of behavioral intention. Conclusion The IBM and the Precaution Adoption Process Model are useful frameworks for interventions designed to increase ACP among racial or ethnic minorities in the United States.

Note: A simple detection method for helium droplet spectroscopy experiments.

Helium droplet methods are currently established as a premier experimental technique for the production and spectroscopic study of novel clusters and complexes. Unfortunately, some of the essential equipment required to perform the experiments, such as the detector used to monitor photon-induced depletion of the helium droplet beam, can be relatively large, complex, and expensive. Most often this detector is a quadrupole mass spectrometer (QMS). In this report, we describe the development and evaluation of an extremely simple, straightforward, small, and inexpensive droplet beam detector for use in helium droplet spectroscopy experiments and compare its performance to that of a QMS by recording the infrared spectra of helium droplets doped with either 13CO2 or CD4.

Depletion of the Human Ion Channel TRPM2 in Neuroblastoma Demonstrates Its Key Role in Cell Survival through Modulation of Mitochondrial Reactive Oxygen Species and Bioenergetics.

Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in modulating cell survival following oxidant injury and is highly expressed in many cancers including neuroblastoma. Here, in xenografts generated from neuroblastoma cells in which TRPM2 was depleted with CRISPR/Cas9 technology and in in vitro experiments, tumor growth was significantly inhibited and doxorubicin sensitivity increased. The hypoxia-inducible transcription factor 1/2α (HIF-1/2α) signaling cascade including proteins involved in oxidant stress, glycolysis, and mitochondrial function was suppressed by TRPM2 depletion. TRPM2-depleted SH-SY5Y neuroblastoma cells demonstrated reduced oxygen consumption and ATP production after doxorubicin, confirming impaired cellular bioenergetics. In cells in which TRPM2 was depleted, mitochondrial superoxide production was significantly increased, particularly following doxorubicin. Ectopic expression of superoxide dismutase 2 (SOD2) reduced ROS and preserved viability of TRPM2-depleted cells, however, failed to restore ATP levels. Mitochondrial reactive oxygen species (ROS) were also significantly increased in cells in which TRPM2 function was inhibited by TRPM2-S, and pretreatment of these cells with the antioxidant MitoTEMPO significantly reduced ROS levels in response to doxorubicin and protected cell viability. Expression of the TRPM2 pore mutant E960D, in which calcium entry through TRPM2 is abolished, also resulted in significantly increased mitochondrial ROS following doxorubicin treatment, showing the critical role of TRPM2-mediated calcium entry. These findings demonstrate the important function of TRPM2 in modulation of cell survival through mitochondrial ROS, and the potential of targeted inhibition of TRPM2 as a therapeutic approach to reduce cellular bioenergetics, tumor growth, and enhance susceptibility to chemotherapeutic agents.

NLRP3 inflammasome signaling is activated by low-level lysosome disruption but inhibited by extensive lysosome disruption: roles for K+ efflux and Ca2+ influx.

Nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) is a cytosolic protein that nucleates assembly of inflammasome signaling platforms, which facilitate caspase-1-mediated IL-1ß release and other inflammatory responses in myeloid leukocytes. NLRP3 inflammasomes are assembled in response to multiple pathogen- or environmental stress-induced changes in basic cell physiology, including the destabilization of lysosome integrity and activation of K(+)-permeable channels/transporters in the plasma membrane (PM). However, the quantitative relationships between lysosome membrane permeabilization (LMP), induction of increased PM K(+) permeability, and activation of NLRP3 signaling are incompletely characterized. We used Leu-Leu-O-methyl ester (LLME), a soluble lysosomotropic agent, to quantitatively track the kinetics and extent of LMP in relation to NLRP3 inflammasome signaling responses (ASC oligomerization, caspase-1 activation, IL-1ß release) and PM cation fluxes in murine bone marrow-derived dendritic cells (BMDCs). Treatment of BMDCs with submillimolar (≤1 mM) LLME induced slower and partial increases in LMP that correlated with robust NLRP3 inflammasome activation and K(+) efflux. In contrast, supramillimolar (≥2 mM) LLME elicited extremely rapid and complete collapse of lysosome integrity that was correlated with suppression of inflammasome signaling. Supramillimolar LLME also induced dominant negative effects on inflammasome activation by the canonical NLRP3 agonist nigericin; this inhibition correlated with an increase in NLRP3 ubiquitination. LMP elicited rapid BMDC death by both inflammasome-dependent pyroptosis and inflammasome-independent necrosis. LMP also triggered Ca(2+) influx, which attenuated LLME-stimulated NLRP3 inflammasome signaling but potentiated LLME-induced necrosis. Taken together, these studies reveal a previously unappreciated signaling network that defines the coupling between LMP, changes in PM cation fluxes, cell death, and NLRP3 inflammasome activation.

Enhanced Fluorescence Properties of Carbon Dots in Polymer Films.

Carbon dots of small carbon nanoparticles surface-functionalized with 2,2'-(ethylenedioxy)bis(ethylamine) (EDA) were synthesized, and the as-synthesized sample was separated on an aqueous gel column to obtain fractions of the EDA-carbon dots with different fluorescence quantum yields. As already discussed in the literature, the variations in fluorescence performance among the fractions were attributed to the different levels and/or effectiveness of the surface functionalization-passivation in the carbon dots. These fractions, as well as carbon nanoparticles without any deliberate surface functionalization, were dispersed into poly(vinyl alcohol) (PVA) for composite films. In the PVA film matrix, the carbon dots and nanoparticles exhibited much enhanced fluorescence emissions in comparison with their corresponding aqueous solutions. The increased fluorescence quantum yields in the films were determined quantitatively by using a specifically designed and constructed film sample holder in the emission spectrometer. The observed fluorescence decays of the EDA-carbon dots in film and in solution were essentially the same, suggesting that the significant enhancement in fluorescence quantum yields from solution to film is static in nature. Mechanistic implications of the results, including a rationalization in terms of the compression effect on the surface passivation layer (similar to a soft corona) in carbon dots when embedded in the more restrictive film environment resulting in more favorable radiative recombinations of the carbon particle surface-trapped electrons and holes, and also potential technological applications of the brightly fluorescent composite films are highlighted and discussed.

TRPM2 protects against tissue damage following oxidative stress and ischaemia-reperfusion.

TRPM channels are a subgroup of the transient receptor potential (TRP) channel superfamily whose members have important roles in cell proliferation and survival. TRPM2, the second subfamily member to be cloned, is expressed in many tissues including brain, heart, vasculature and haematopoietic cells. TRPM2 is activated by oxidative stress and several other extracellular signals including tumour necrosis factor α (TNF-α) and amyloid ß-peptide, which increase production of ADP-ribose (ADPR). ADPR binds to the TRPM2 C-terminal NUDT9-H domain, activating the channel. Early studies support the paradigm that TRPM2 activation induces cell death by sustained Ca(2+) influx or by enhancing cytokine production, aggravating inflammation and tissue injury. However, more recent data show that for a number of physiological processes, TRPM2 is protective. TRPM2 protects lungs from endotoxin-induced injury by reducing reactive oxygen species (ROS) production by phagocytes. It protects hearts from oxidative damage after ischaemia-reperfusion or hypoxia-reoxygenation by maintaining better mitochondrial bioenergetics and by decreasing ROS. Sustained Ca(2+) entry through TRPM2 is required to maintain cellular bioenergetics and protect against hypoxia-reoxygenation injury. TRPM2 also protects neuroblastoma from moderate oxidative stress by decreasing ROS through increased levels of forkhead box transcription factor 3a (FOXO3a) and a downstream effector, superoxide dismutase 2. TRPM2 is important for tumour growth and cell survival through modulation of hypoxia-inducible transcription factor expression, mitochondrial function and mitophagy. These findings in cardiac ischaemia and in neuroblastoma suggest that TRPM2 has a basic role in sustaining mitochondrial function and in cell survival that applies to a number of physiological systems and pathophysiological processes including ischaemia-reperfusion injury.

Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity.

As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca(2+) entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 and 0.015 pm/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) influx in response to ionomycin, as a result of ionophore H2O2 sensitivity. Exogenously added H2O2 or H2O2 generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), actin polymerization, and IL-6 production are reversed in TRPM2-null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca(2+) signaling and limit host immune function.