Ubiquitin ligase SMURF2 enhances epidermal growth factor receptor stability and tyrosine-kinase inhibitor resistance.

The discovery of activating epidermal growth factor receptor (EGFR) mutations spurred the use of EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, as the first-line treatment of lung cancers. We previously reported that differential degradation of TKI-sensitive (e.g. L858R) and resistant (T790M) EGFR mutants upon erlotinib treatment correlates with drug sensitivity. We also reported that SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. However, the molecular mechanisms involved remain unclear. Here, using in vitro and in vivo ubiquitination assays, MS, and superresolution microscopy, we show SMURF2-EGFR functional interaction is important for EGFR stability and response to TKI. We demonstrate that L858R/T790M EGFR is preferentially stabilized by SMURF2-UBCH5 (an E3-E2)-mediated polyubiquitination. We identified four lysine residues as the sites of ubiquitination and showed that replacement of one of them with acetylation-mimicking glutamine increases the sensitivity of mutant EGFR to erlotinib-induced degradation. We show that SMURF2 extends membrane retention of EGF-bound EGFR, whereas SMURF2 knockdown increases receptor sorting to lysosomes. In lung cancer cell lines, SMURF2 overexpression increased EGFR levels, improving TKI tolerance, whereas SMURF2 knockdown decreased EGFR steady-state levels and sensitized lung cancer cells. Overall, we propose that SMURF2-mediated polyubiquitination of L858R/T790M EGFR competes with acetylation-mediated receptor internalization that correlates with enhanced receptor stability; therefore, disruption of the E3-E2 complex may be an attractive target to overcome TKI resistance.

The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity.

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle-dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.

Isoforms of RNF128 Regulate the Stability of Mutant P53 in Barrett's Esophageal Cells.

BACKGROUND & AIMS: Barrett's esophagus (BE) can progress to dysplasia and esophageal adenocarcinoma (EAC), accompanied by mutations in TP53 that increase the stability of its product, p53. We analyzed BE tissues for messenger RNAs (mRNAs) that associate with BE progression and identified one that affects the stabilization of p53. METHODS: We obtained 54 BE samples collected from patients with high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), from 1992 through 2015, and performed RNA sequence analyses, including isoform-specific analyses. We performed reverse-transcription polymerase chain reaction analyses of 166 samples and immunohistochemical analyses of tissue microarrays that contained BE tissues from 100 patients with HGD or EAC and normal esophageal squamous mucosa (controls). Proteins were expressed from transfected plasmids or knocked down with small interfering RNAs in BE cells and analyzed by immunoblots and in immunoprecipitation and ubiquitin ligase assays. Athymic nude mice bearing EAC xenograft tumors (grown from OE-33 cells) were given intraperitoneal injections of simvastatin; tumor growth was monitored and tumors were collected and analyzed by immunoblotting for levels of RNF128, p53, and acetylated p53. RESULTS: Progression of BE to HGD or EAC associated with changes in expression of mRNAs that encoded mucins and promoted inflammation and activation of ATM and the DNA damage response. As tissues progressed from BE to HGD to EAC, they increased expression of mRNAs encoding isoform 1 of RNF128 (Iso1) and decreased expression of Iso2 of RNF128. RNF128 is an E3 ubiquitin ligase that targets p53 for degradation. Incubation of BE cells with interferon gamma caused them to increase expression of Iso1 and reduce expression of Iso2. Iso1 was heavily glycosylated with limited ubiquitin ligase activity for p53, resulting in p53 stabilization. Knockdown of Iso1 in BE and EAC cells led to degradation of the mutant form of p53 and reduced clonogenic survival. In contrast, Iso2 was a potent ligase that reduced levels of the mutant form of p53 in BE cells. In BE cells, Iso2 was hypoglycosylated and degraded, via ATM and GSK3ß-mediated phosphorylation and activation of the beta-TrCP1-containing SCF ubiquitin ligase complex. Simvastatin, which degrades the mutant form of p53, also degraded RNF128 Iso1 protein in BE cells and slowed growth of EAC xenograft tumors in mice. CONCLUSIONS: We found that isoform 2 of RNF128 is decreased in BE cells, resulting in increased levels of mutant p53, whereas isoform 1 of RNF128 is increased in BE cells, further promoting the stabilization of mutant p53.

Constitutively Higher Level of GSTT2 in Esophageal Tissues From African Americans Protects Cells Against DNA Damage.

BACKGROUND & AIMS: African American and European American individuals have a similar prevalence of gastroesophageal reflux disease (GERD), yet esophageal adenocarcinoma (EAC) disproportionately affects European American individuals. We investigated whether the esophageal squamous mucosa of African American individuals has features that protect against GERD-induced damage, compared with European American individuals. METHODS: We performed transcriptional profile analysis of esophageal squamous mucosa tissues from 20 African American and 20 European American individuals (24 with no disease and 16 with Barrett's esophagus and/or EAC). We confirmed our findings in a cohort of 56 patients and analyzed DNA samples from patients to identify associated variants. Observations were validated using matched genomic sequence and expression data from lymphoblasts from the 1000 Genomes Project. A panel of esophageal samples from African American and European American subjects was used to confirm allele-related differences in protein levels. The esophageal squamous-derived cell line Het-1A and a rat esophagogastroduodenal anastomosis model for reflux-generated esophageal damage were used to investigate the effects of the DNA-damaging agent cumene-hydroperoxide (cum-OOH) and a chemopreventive cranberry proanthocyanidin (C-PAC) extract, respectively, on levels of protein and messenger RNA (mRNA). RESULTS: We found significantly higher levels of glutathione S-transferase theta 2 (GSTT2) mRNA in squamous mucosa from African American compared with European American individuals and associated these with variants within the GSTT2 locus in African American individuals. We confirmed that 2 previously identified genomic variants at the GSTT2 locus, a 37-kb deletion and a 17-bp promoter duplication, reduce expression of GSTT2 in tissues from European American individuals. The nonduplicated 17-bp promoter was more common in tissue samples from populations of African descendant. GSTT2 protected Het-1A esophageal squamous cells from cum-OOH-induced DNA damage. Addition of C-PAC increased GSTT2 expression in Het-1A cells incubated with cum-OOH and in rats with reflux-induced esophageal damage. C-PAC also reduced levels of DNA damage in reflux-exposed rat esophagi, as observed by reduced levels of phospho-H2A histone family member X. CONCLUSIONS: We found GSTT2 to protect esophageal squamous cells against DNA damage from genotoxic stress and that GSTT2 expression can be induced by C-PAC. Increased levels of GSTT2 in esophageal tissues of African American individuals might protect them from GERD-induced damage and contribute to the low incidence of EAC in this population.

Change of symptoms of schizophrenia across phases of menstrual cycle.

The relation between schizophrenia and the menstrual cycle has always been found attractive by researchers. It is still a question of debate whether the clinical picture changes during the menstrual cycle. Our study aimed to see whether there is any change of symptoms during different phases of menstrual cycle (premenstrual, menstrual, and postmenstrual) in patients suffering from schizophrenia. Over a period of 15 months, 40 female inpatients of a tertiary care psychiatric hospital with the diagnosis of schizophrenia were assessed by applying PANSS. Rating was done up to two menstrual cycles. Total scores, positive and negative subscale scores, and general psychopathology scores of PANSS in premenstrual, menstrual, and postmenstrual phases of those patients were compared with one another by applying paired t test. Symptoms in women suffering from schizophrenia frequently vary with the different phases of menstrual cycle. The positive symptoms improved significantly only during progesterone phase. Negative symptoms and general psychopathology subscale showed improvement on estrogen phases of menstrual cycle. So optimal treatment needs to be adjusted to the individual women suffering from schizophrenia.

In situ vibrational spectroscopy of adsorbed nitrogen in porous carbon materials.

This study uses in situ vibrational spectroscopy to probe nitrogen adsorption to porous carbon materials, including single-wall carbon nanotubes and Maxsorb super-activated carbon, demonstrating how the nitrogen Raman stretch mode is perturbed by adsorption. In all porous carbon samples upon N2 physisorption in the mesopore filling regime, the N2 Raman mode downshifts by ∼2 cm-1, a downshift comparable to liquid N2. The relative intensity of this mode increases as pressure is increased to saturation, and trends in the relative intensity parallel the volumetric gas adsorption isotherm. This mode with ∼2 cm-1 downshift is thus attributed to perturbations arising due to N2-N2 interactions in a condensed film. The mode is also observed for the activated carbon at 298 K, and the relative intensity once again parallels the gas adsorption isotherm. For select samples, a mode with a stronger downshift (>4 cm-1) is observed, and the stronger downshift is attributed to stronger N2-carbon surface interactions. Simulations for a N2 surface film support peak assignments. These results suggest that N2 vibrational spectroscopy could provide an indication of the presence or absence of porosity for very small quantities of samples.

CEH-28 activates dbl-1 expression and TGF-ß signaling in the C. elegans M4 neuron.

M4 is a multifunctional neuron in the Caenorhabditis elegans pharynx that can both stimulate peristaltic contractions of the muscles in the pharyngeal isthmus and function systemically to regulate an enhanced sensory response under hypoxic conditions. Here we identify a third function for M4 that depends on activation of the TGF-ß family gene dbl-1 by the homeodomain transcription factor CEH-28. dbl-1 is expressed in M4 and a subset of other neurons, and we show CEH-28 specifically activates dbl-1 expression in M4. Characterization of the dbl-1 promoter indicates that CEH-28 targets an M4-specific enhancer within the dbl-1 promoter region, while expression in other neurons is mediated by separate regulatory sequences. Unlike ceh-28 mutants, dbl-1 mutants do not exhibit M4 synaptic and signaling defects. Instead, both ceh-28 and dbl-1 mutants exhibit morphological defects in the g1 gland cells located adjacent to M4 in the pharynx, and these defects can be partially rescued by M4-specific expression of dbl-1 in these mutants. Identical gland cell defects are observed in sma-6 and daf-4 mutants defective in the receptor for DBL-1, but they are not observed in sma-2 and sma-3 mutants lacking the R-Smads functioning downstream of this receptor. Together these results identify a novel neuroendocrine function for M4 and provide evidence for an R-Smad-independent mechanism for DBL-1 signaling in C. elegans.

Influence of gas packing and orientation on FTIR activity for CO chemisorption to the Cu paddlewheel.

In situ Fourier-transform infrared (FTIR) spectroscopy is able to probe structural defects via site-specific adsorption of CO to the Cu-BTC (BTC = 1,3,5-benzenetricarboxylate) metal-organic framework (MOF). The temperature-programmed desorption (TPD) of CO chemisorbed to Cu-TDPAT (TDPAT = 2,4,6-tris(3,5-dicarboxylphenylamino)-1,3,5-triazine) is virtually identical to Cu-BTC, suggesting CO chemisorbs to the open metal site at the axial position of the copper paddlewheel that is the building unit of both MOFs. Yet, despite an increased gravimetric CO : Cu ratio, CO chemisorbed to Cu-TDPAT is FTIR inactive. We rule out the presence of residual solvent, thermal degradation, adsorption temperature, and ligand-induced electronic effects at the adsorption site. TPD at increased pressure suggests the multiple CO per Cu site rearrange in Cu-TDPAT as a dynamic function of temperature and pressure. Thus, the FTIR inactivity of CO chemisorbed to Cu-TDPAT is attributed to orientation and/or packing of the CO relative to the Cu binding site. The results suggest dynamic chemisorption complicate extension of a site-specific in situ FTIR probe of gas adsorption. For both Cu-BTC and Cu-TDPAT, the in situ FTIR probe is a less sensitive probe of defects than X-ray photoelectron spectroscopy and nitrogen adsorption.

ISG15-GRAIL1-CD3 axis influences survival of esophageal adenocarcinoma patients.

Immunosuppression is a common feature of esophageal adenocarcinoma (EAC) and has been linked to poor overall survival (OS). We hypothesized that upstream factors might negatively influence CD3 levels and T-cell activity, thus promoting immunosuppression and worse survival. We used clinical data and patient samples of those who progressed from Barrett's (BE) to dysplasia to EAC, investigated gene (RNAseq), protein (tissue microarray) expression and performed cell biology studies to delineate a pathway impacting CD3 protein stability that might influence EAC outcome. We show that the loss of both CD3-ε expression and CD3+ T-cell number are correlated with worse OS in EAC. The GRAIL (gene related to anergy in lymphocytes) isoform 1 (GRAIL1), which is the prominent isoform in EACs, degrades (ε, γ, δ) CD3s and inactivates T-cells. In contrast, isoform 2 (GRAIL2), which is reduced in EACs, stabilizes CD3s. Further, GRAIL1 mediated CD3 degradation is facilitated by interferon stimulated gene 15 (ISG15), a ubiquitin-like protein. Consequently, either the overexpression of a ligase-dead GRAIL1, ISG15 knockdown, or the overexpression of a conjugation-defective ISG15-LRAA mutant can increase CD3 levels. Together, we identified that an ISG15→GRAIL1→mutant p53 amplification loop negatively influencing CD3 levels and T-cell activity, thus promoting immunosuppression in EAC.

Interactions of cardiopulmonary bypass and erythrocyte transfusion in the pathogenesis of pulmonary dysfunction in Swine.

BACKGROUND: Allogeneic erythrocyte transfusion in cardiac surgical patients is associated with a fourfold increase in pulmonary complications. Our understanding of the processes underlying these observations is poor and there is no experimental model of transfusion-related acute lung injury that shows homology to cardiac surgical patients. Our objective was to develop a novel swine recovery model to determine how two clinical risk factors, allogenic erythrocyte transfusion and cardiopulmonary bypass, interact in the genesis of postcardiac surgery acute lung injury. METHODS: Thirty-six pigs were infused with allogeneic 14- or 42-day-old erythrocytes or they underwent cardiopulmonary bypass with or without transfusion of 42-day erythrocyte. Controls received saline. All pigs were recovered and assessed for pulmonary dysfunction, inflammation, and endothelial activation at 24 h. RESULTS: Transfusion of stored allogeneic erythrocytes in pigs compared with sham caused pulmonary dysfunction characterized by reduced lung compliance (mean difference -3.36 [95% CI, -5.31 to -1.42] ml/cm H2O), an increase in protein levels in bronchoalveolar lavage fluid, histological lung injury inflammation, and endothelial activation. Transfusion of blood stored for up to 42 days resulted in greater protein levels in bronchoalveolar lavage fluid, macrophage infiltration, platelet activation, and depletion of T-lymphocytes in recipient lungs versus 14-day-old blood. Transfusion interacted with cardiopulmonary bypass to increase lung injury in the absence of platelet activation. CONCLUSIONS: In this novel large animal model of allogeneic erythrocyte transfusion, pulmonary dysfunction occurs in the absence of any priming event, is increased when combined with other inflammatory stimuli, and is mediated by monocyte activation and T-lymphocyte depletion.

High fat feeding promotes obesity and renal inflammation and protects against post cardiopulmonary bypass acute kidney injury in swine.

INTRODUCTION: Obesity confers a survival advantage in the critically ill and in patients undergoing cardiac surgery. We explored whether an obesogenic high fat diet could confer protection against post cardiopulmonary bypass (CPB) acute kidney injury (AKI) in a swine model. METHODS: In this study, 28 anaesthetised adult female Landrace White swine (55 to 70 kg) were allocated into a 4 group design to either 2.5 hours of CPB or Sham operation with or without pre-procedural high fat (HF) feeding containing 15% lard, 1.5% cholesterol and 1% cholic acid for 12-weeks (Groups: Sham, CPB, CPB + HF and Sham + HF). Our primary endpoint was creatinine clearance measured at 1.5 and 24 hours post intervention. This is a validated index of the glomerular filtration rate (GFR) in swine and an endpoint used in our clinical studies. Secondary endpoints included measures of systemic and renal inflammation, endothelial homeostasis, tubular injury and dysfunction, and inflammatory cell signalling. Differences between groups were calculated using analysis of variance with adjustment for baseline differences for repeated measures. RESULTS: CPB in pigs fed a normal chow diet resulted in AKI. This was characterised by reductions in GFR sustained for up to 24 hours post injury relative to Sham operated pigs fed a normal diet; mean difference 50.2 ml/min (95% CI 5.9 to 94.4). Post CPB AKI was also characterised by renal inflammation, parallel activation of both pro-inflammatory (NF-kB, iNOS) and pro-survival pathways (pAkt, p70s6k, HIF-1α) and apoptosis. Pigs fed a 12-week high fat diet developed obesity and hyperlipidaemia. This was associated with increased redox sensitive pro-inflammatory and anti-apoptotic signalling, and tubular epithelial cell proliferation. High fat feeding also protected swine against post CPB AKI; mean difference in creatinine clearance CPB - CPB + HF -65.3 ml/min (95% CI -106.9 to -23.7), by preserving endothelial homeostasis and function, and preventing the reductions in GFR, loss of ATP and tubular apoptosis that characterise the extension phase of AKI in swine at 24 hours post injury. Reno-protection was not attributed to pAkt signaling. CONCLUSIONS: A high fat diet promoted obesity and renal inflammation and prevented post CPB AKI in swine. This study provides insights into the obesity paradox and the failure of anti-inflammatory interventions to improve clinical outcomes in patients at risk of post cardiac surgery AKI.

Secreted CXCL12 (SDF-1) forms dimers under physiological conditions.

Chemokine CXCL12 (CXC chemokine ligand 12) signalling through CXCR (CXC chemokine receptor) 4 and CXCR7 has essential functions in development and underlies diseases including cancer, atherosclerosis and autoimmunity. Chemokines may form homodimers that regulate receptor binding and signalling, but previous studies with synthetic CXCL12 have produced conflicting evidence for homodimerization. We used bioluminescence imaging with GL (Gaussia luciferase) fusions to investigate dimerization of CXCL12 secreted from mammalian cells. Using column chromatography and GL complementation, we established that CXCL12 was secreted from mammalian cells as both monomers and dimers. Secreted CXCL12 also formed homodimers in the extracellular space. Monomeric CXCL12 preferentially activated CXCR4 signalling through Gαi and Akt, whereas dimeric CXCL12 more effectively promoted recruitment of ß-arrestin 2 to CXCR4 and chemotaxis of CXCR4-expressing breast cancer cells. We also showed that CXCR7 preferentially sequestered monomeric CXCL12 from the extracellular space and had minimal effects on dimeric CXCL12 in cell-based assays and an orthotopic tumour xenograft model of human breast cancer. These studies establish that CXCL12 secreted from mammalian cells forms homodimers under physiological conditions. Since monomeric and dimeric CXCL12 have distinct effects on cell signalling and function, our results have important implications for ongoing efforts to target CXCL12 pathways for therapy.

3D Natural Mesoporous Biosilica-Embedded Polysulfone Made Ultrafiltration Membranes for Application in Separation Technology.

Diatoms are the most abundant photosynthetic microalgae found in all aquatic habitats. In the extant study, the spent biomass (after lipid extraction) of the centric marine diatom Thalassiosira lundiana CSIRCSMCRI 001 was subjected to acid digestion for the extraction of micro composite inorganic biosilica. Then, the resulting three-dimensional mesoporous biosilica material (diatomite) was used as a filler in polysulfone (PSF) membrane preparation by phase inversion. The fabricated PSF/diatomite composite membranes were characterized by SEM-EDX, TGA, and ATR-IR, and their performances were evaluated. The number of pores and pore size were increased on the membrane surface with increased diatomite in the composite membranes as compared to the control. The diatomite composite membranes had high hydrophilicity and thermal stability, lower surface roughness, and excellent water permeability. Membranes with high % diatomite, i.e., PSF/Dia0.5, had a maximum water flux of 806.8 LMH (Liter/m2/h) at 20 psi operating pressure. High-diatomite content membranes also exhibited the highest rejection of BSA protein (98.5%) and rhodamine 6G (94.8%). Similarly, in biomedical rejection tests, the PSF/Dia0.5 membrane exhibited a maximum rejection of ampicillin (75.84%) and neomycin (85.88%) at 20 Psi pressure. In conclusion, the mesoporous inorganic biosilica material was extracted from spent biomass of diatom and successfully used in filtration techniques. The results of this study could enhance the application of natural biogenic porous silica materials in wastewater treatment for water recycling.

UBCH5 Family Members Differentially Impact Stabilization of Mutant p53 via RNF128 Iso1 During Barrett's Progression to Esophageal Adenocarcinoma.

BACKGROUND & AIMS: TP53 mutations underlie Barrett's esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown. METHODS: Single-cell RNA sequencing of paired normal esophagus and BE tissues identified candidate E2s, further investigated in expression data from BE to esophageal adenocarcinoma (EAC) progression samples. Biochemical and cellular studies helped clarify the role of RNF128-E2 on mutant p53 stability. RESULTS: The UBE2D family member 2D3 (UBCH5C) is the most abundant E2 in normal esophagus. However, during BE to EAC progression, loss of UBE2D3 copy number and reduced expression of RNF128 Iso2 were noted, 2 known p53 degraders. In contrast, expression of UBE2D1 (UBCH5A) and RNF128 Iso1 in dysplastic BE and EAC forms an inactive E2-E3 complex, stabilizing mutant p53. To destabilize mutant p53, we targeted RNF128 Iso1 either by mutating asparagine (N48, 59, and 101) residues to block glycosylation to facilitate ß-TrCP1-mediated degradation or by mutating proline (P54 and 105) residues to restore p53 polyubiquitinating ability. In addition, either loss of UBCH5A catalytic activity, or disruption of the Iso1-UBCH5A interaction promoted Iso1 loss. Consequently, overexpression of either catalytically dead or Iso1-binding-deficient UBCH5A mutants destabilized Iso1 to degrade mutant p53, thus compromising the clonogenic survival of mutant p53-dependent BE cells. CONCLUSIONS: Loss of RNF128 Iso2-UBCH5C and persistence of the Iso1-UBCH5A complex favors mutant p53 stability to promote BE cell survival. Therefore, targeting of Iso1-UBCH5A may provide a novel therapeutic strategy to prevent BE progression.

Improved prediction of radiation pneumonitis by combining biological and radiobiological parameters using a data-driven Bayesian network analysis.

Grade 2 and higher radiation pneumonitis (RP2) is a potentially fatal toxicity that limits efficacy of radiation therapy (RT). We wished to identify a combined biomarker signature of circulating miRNAs and cytokines which, along with radiobiological and clinical parameters, may better predict a targetable RP2 pathway. In a prospective clinical trial of response-adapted RT for patients (n = 39) with locally advanced non-small cell lung cancer, we analyzed patients' plasma, collected pre- and during RT, for microRNAs (miRNAs) and cytokines using array and multiplex enzyme linked immunosorbent assay (ELISA), respectively. Interactions between candidate biomarkers, radiobiological, and clinical parameters were analyzed using data-driven Bayesian network (DD-BN) analysis. We identified alterations in specific miRNAs (miR-532, -99b and -495, let-7c, -451 and -139-3p) correlating with lung toxicity. High levels of soluble tumor necrosis factor alpha receptor 1 (sTNFR1) were detected in a majority of lung cancer patients. However, among RP patients, within 2 weeks of RT initiation, we noted a trend of temporary decline in sTNFR1 (a physiological scavenger of TNFα) and ADAM17 (a shedding protease that cleaves both membrane-bound TNFα and TNFR1) levels. Cytokine signature identified activation of inflammatory pathway. Using DD-BN we combined miRNA and cytokine data along with generalized equivalent uniform dose (gEUD) to identify pathways with better accuracy of predicting RP2 as compared to either miRNA or cytokines alone. This signature suggests that activation of the TNFα-NFκB inflammatory pathway plays a key role in RP which could be specifically ameliorated by etanercept rather than current therapy of non-specific leukotoxic corticosteroids.

Reversal of anemia with allogenic RBC transfusion prevents post-cardiopulmonary bypass acute kidney injury in swine.

Anemia during cardiopulmonary bypass (CPB) is strongly associated with acute kidney injury in clinical studies; however, reversal of anemia with red blood cell (RBC) transfusions is associated with further renal injury. To understand this paradox, we evaluated the effects of reversal of anemia during CPB with allogenic RBC transfusion in a novel large-animal model of post-cardiac surgery acute kidney injury with significant homology to that observed in cardiac surgery patients. Adult pigs undergoing general anesthesia were allocated to a Sham procedure, CPB alone, Sham+RBC transfusion, or CPB+RBC transfusion, with recovery and reassessment at 24 h. CPB was associated with dilutional anemia and caused acute kidney injury in swine characterized by renal endothelial dysfunction, loss of nitric oxide (NO) bioavailability, vasoconstriction, medullary hypoxia, cortical ATP depletion, glomerular sequestration of activated platelets and inflammatory cells, and proximal tubule epithelial cell stress. RBC transfusion in the absence of CPB also resulted in renal injury. This was characterized by endothelial injury, microvascular endothelial dysfunction, platelet activation, and equivalent cortical tubular epithelial phenotypic changes to those observed in CPB pigs, but occurred in the absence of severe intrarenal vasoconstriction, ATP depletion, or reductions in creatinine clearance. In contrast, reversal of anemia during CPB with RBC transfusion prevented the reductions in creatinine clearance, loss of NO bioavailability, platelet activation, inflammation, and epithelial cell injury attributable to CPB although it did not prevent the development of significant intrarenal vasoconstriction and endothelial dysfunction. In conclusion, contrary to the findings of observational studies in cardiac surgery, RBC transfusion during CPB protects pigs against acute kidney injury. Our study underlines the need for translational research into indications for transfusion and prevention strategies for acute kidney injury.

Prevention of post-cardiopulmonary bypass acute kidney injury by endothelin A receptor blockade.

OBJECTIVE: The aim of this study was to determine whether administration of a specific endothelin A receptor antagonist, sitaxsentan sodium, would prevent the development of post-cardiopulmonary bypass acute kidney injury in swine. DESIGN: Experimental study. SETTING: Cardiovascular Research Institute. INTERVENTIONS: Adult pigs (n = 8 per group) were randomized to undergo a sham procedure, cardiopulmonary bypass, or cardiopulmonary bypass plus administration of endothelin A receptor antagonist (RA), with recovery and reassessment at 24 hrs. MEASUREMENTS AND MAIN RESULTS: Cardiopulmonary bypass resulted in a significant reduction in creatinine clearance relative to sham pigs (mean difference for cardiopulmonary bypass vs. sham, -50.3 mL/min [95% confidence interval -89.2 to -11.4 mL/min], p = .008). This was reversed by the administration of endothelin A RA during cardiopulmonary bypass (mean difference for cardiopulmonary bypass + endothelin A RA vs. cardiopulmonary bypass, +43.3 mL/min [95% confidence interval +3.3 to +83.4 mL/min], p = .030). Cardiopulmonary bypass also resulted in a significant rise in the specific urinary biomarker of acute kidney injury interleukin-18 compared to sham procedures (mean difference +209 pg/mL [95% confidence interval +119 to +299 pg/mL], p < .001) that was reversed by endothelin A receptor antagonist administration. Post-cardiopulmonary bypass kidney injury was associated with vascular endothelial injury and dysfunction, reduced nitric oxide bioavailability, inflammation, and a significant increase in the expression of the paracrine vasoconstrictors adenosine and endothelin-1. In post-cardiopulmonary bypass kidneys at 24 hrs there was persistent hypoxia at the level of the outer medulla, cortical adenosine triphosphate depletion, and evidence of proximal tubule epithelial cell stress manifest as phenotypic change. There was no evidence of acute tubular necrosis. Administration of endothelin A RA to cardiopulmonary bypass pigs reversed endothelial dysfunction, regional hypoxia, inflammation, and tubular changes. CONCLUSION: In this model, post-cardiopulmonary bypass acute kidney injury is associated with endothelial dysfunction, regional tissue hypoxia, and proximal tubular epithelial cell stress but not acute tubular necrosis. Antagonism of the endothelin-1 A receptor reversed these changes and may represent a therapeutic target for the prevention of post-cardiac surgery acute kidney injury.

Ionic Liquid-Based Colloidal Formulations for the Synthesis of Nano-MOFs: Applications in Gas Adsorption and Water Desalination.

Microemulsions (MEs) comprising choline dioctylsulfosuccinate [Cho][AOT], a biobased ionic liquid (IL) surfactant as an emulsifier, (R)-(+)-limonene (RL) as a nonpolar phase, and ethylene glycol (EG)/ethanolammonium formate (EOAF) as an organic solvent/low-viscosity IL polar component were constructed. Spontaneous aggregation of [Cho][AOT] was observed with a negative ΔH form using isothermal titration calorimetry. The aggregates of [Cho][AOT] in RL showed a critical micellar concentration (cmc) of ∼5.49 mM, EG (cmc ∼3.99 mM), and EOAF (cmc ∼1.56 mM), and these are further characterized by various techniques. These novel IL-based MEs have been used as nanoreactors for the sustainable synthesis of uniform nanosized metal-organic frameworks (N-MOFs), such as MIL-53(Al), HKUST-1, UIO-66-NH2, and ZIF-8, with a precise control over size and morphology at room temperature. Characterization of N-MOFs has been performed using scanning electron microscopy, powder X-ray diffraction, and Fourier transform infrared spectroscopy. The synthesized N-MOFs have been used to prepare stable and uniform thin film nanocomposite nanofiltration membranes, suitable for desalination of brackish water with excellent flux (31.8 LMH/bar) and rejection (99.0%) of divalent salts.

Involvement of p38-ßTrCP-Tristetraprolin-TNFα axis in radiation pneumonitis.

Early release of tumor necrosis factor-alpha (TNF-α) during radiotherapy of thoracic cancers plays an important role in radiation pneumonitis, whose inhibition may provide lung radioprotection. We previously reported radiation inactivates Tristetraprolin (TTP), a negative regulator of TNF-α synthesis, which correlated with increased TNF-α release. However, the molecular events involved in radiation-induced TTP inactivation remain unclear. To determine if eliminating Ttp in mice resulted in a phenotypic response to radiation, Ttp-null mice lungs were exposed to a single dose of 15 Gy, and TNF-α release and lung inflammation were analyzed at different time points post-irradiation. Ttp-/- mice with elevated (9.5±0.6 fold) basal TNF-α showed further increase (12.2±0.9 fold, p<0.02) in TNF-α release and acute lung inflammation within a week post-irradiation. Further studies using mouse lung macrophage (MH-S), human lung fibroblast (MRC-5), and exogenous human TTP overexpressing U2OS and HEK293 cells upon irradiation (a single dose of 4 Gy) promoted p38-mediated TTP phosphorylation at the serine 186 position, which primed it to be recognized by an ubiquitin ligase (E3), beta transducing repeat containing protein (ß-TrCP), to promote polyubiquitination-mediated proteasomal degradation. Consequently, a serine 186 to alanine (SA) mutant of TTP was resistant to radiation-induced degradation. Similarly, either a p38 kinase inhibitor (SB203580), or siRNA-mediated ß-TrCP knockdown, or overexpression of dominant negative Cullin1 mutants protected TTP from radiation-induced degradation. Consequently, SB203580 pretreatment blocked radiation-induced TNF-α release and radioprotected macrophages. Together, these data establish the involvement of the p38-ßTrCP-TTP-TNFα signaling axis in radiation-induced lung inflammation and identified p38 inhibition as a possible lung radioprotection strategy.

Oncogenic Potential of CYP24A1 in Lung Adenocarcinoma.

INTRODUCTION: We have previously demonstrated that a subset of lung cancer cells express higher CYP24A1 mRNA, a metabolizing enzyme for 1,25-D3, compared to benign tumors or surrounding normal lung and that high CYP24A1 mRNA expression is associated with poor prognosis in resected lung adenocarcinoma (AC). We hypothesized that CYP24A1 has oncogenic potential and increased CYP24A1 expression may contribute to tumor growth, whereas, CYP24A1 targeting may reduce tumor burden. METHODS: Two low CYP24A1 expressing human lung cancer cell lines (SK-LU-1 and Calu-6) were stably transfected either with an empty lentiviral vector or with the CYP24A1 expressing vector. Over-expression of mRNA and protein levels of CYP24A1 in SK-LU-1 and Calu-6 were confirmed using qRT-PCR and immunoblotting respectively. Next, effects of targeting CYP24A1 were examined in lung cancer cells (A549 and H441), which express higher basal levels of CYP24A1. Finally, we studied the effects of stable knockdown of CYP24A1 in xenograft models. RESULTS: Over-expression of CYP24A1 correlated with accelerated cell growth and invasion compared to control vector-transfected cells. CYP24A1 over-expression also increased RAS protein expression. Knockdown of CYP24A1 using either si- or shRNA reduced CYP24A1 mRNA and protein expression and significantly decreased cell proliferation (30-60%) and reduced mitochondrial DNA content compared to non-targeting (NT) si-/shRNA transfected/transduced cells. Transfection with CYP24A1 siRNA also decreased total RAS protein, thus reducing phosphorylated AKT. Importantly, stable knockdown of CYP24A1 in A549 and H441 lung tumor xenograft models resulted in tumor growth delay and smaller tumor size as evident from tumor bioluminescence and tumor volume measurement studies. Such observations were correlated with decreased tumor cell proliferation as evidenced by reduced Ki67 and Cyclin D staining. CONCLUSIONS: Our data suggest that CYP24A1 has oncogenic properties mediated by increasing RAS signaling, targeting of which may provide an alternate strategy to treat a subset of lung AC.