NO• /RUNX3/kynurenine metabolic signaling enhances disease aggressiveness in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy and is refractory to available treatments. Delineating the regulatory mechanisms of metabolic reprogramming, a key event in pancreatic cancer progression, may identify candidate targets with potential therapeutic significance. We hypothesized that inflammatory signaling pathways regulate metabolic adaptations in pancreatic cancer. Metabolic profiling of tumors from PDAC patients with a high- (>median, n = 31) and low-NOS2 (inducible nitric oxide synthase;

The impact of volatiles on tick-host interaction and vector competence.

Ticks are obligatory hematophagous arachnids, serving as vectors for a wide array of pathogens that can be transmitted to humans or animals. The ability of tick-borne pathogens to maintain within natural reservoirs is intricately influenced by the attractiveness of ticks to their animal hosts, including humans. However, the complex dynamics of tick behavior and host-seeking strategies remain understudied. This review aims to summarize the impact of volatiles or odors on tick behavior and vector competence. Our literature review has identified a selection of compounds, such as 1-octen-3-ol, hexanal, heptanal, nonanal, 6-methyl-5-hepten-2-one, acetone, and octanal, as having the potential to impact both ticks' and mosquitos' behaviors. In addition, carbon dioxide (CO2) is a universal attractant for hematophagous arthropods. Moreover, we have gathered some clues indicating that volatiles emitted by infected animal hosts might play a role in the transmission of tick-borne pathogens. Nonetheless, our understanding of this phenomenon remains largely inadequate, particularly with regarding to whether the tick microbiome or the skin microbiota of the feeding mammals, including humans, can actively modulate tick-host-seeking behavior. Further investigations in this emerging field hold immense promise for the development of innovative strategies aimed at controlling vectors and curtailing the spread of tick-borne diseases.

Ecoclimate drivers shape virome diversity in a globally invasive tick species.

Spillovers of viruses from animals to humans occur more frequently under warmer conditions, particularly arboviruses. The invasive tick species Haemaphysalis longicornis, the Asian longhorned tick, poses a significant public health threat due to its global expansion and its potential to carry a wide range of pathogens. We analyzed meta-transcriptomic data from 3595 adult H. longicornis ticks collected between 2016 and 2019 in 22 provinces across China encompassing diverse ecological conditions. Generalized additive modeling revealed that climate factors exerted a stronger influence on the virome of H. longicornis than other ecological factors, such as ecotypes, distance to coastline, animal host, tick gender, and antiviral immunity. To understand how climate changes drive the tick virome, we performed a mechanistic investigation using causality inference with emphasis on the significance of this process for public health. Our findings demonstrated that higher temperatures and lower relative humidity/precipitation contribute to variations in animal host diversity, leading to increased diversity of the tick virome, particularly the evenness of vertebrate-associated viruses. These findings may explain the evolution of tick-borne viruses into generalists across multiple hosts, thereby increasing the probability of spillover events involving tick-borne pathogens. Deep learning projections have indicated that the diversity of the H. longicornis virome is expected to increase in 81.9% of regions under the SSP8.5 scenario from 2019 to 2030. Extension of surveillance should be implemented to avert the spread of tick-borne diseases.

Macrophage migration inhibitory factor induces epithelial to mesenchymal transition, enhances tumor aggressiveness and predicts clinical outcome in resected pancreatic ductal adenocarcinoma.

MIF is a proinflammatory cytokine and is implicated in cancer. A higher MIF level is found in many human cancer and cancer-prone inflammatory diseases, including chronic pancreatitis and pancreatic cancer. We tested the hypothesis that MIF contributes to pancreatic cancer aggressiveness and predicts disease outcome in resected cases. Consistent with our hypothesis we found that an elevated MIF mRNA expression in tumors was significantly associated with poor outcome in resected cases. Multivariate Cox-regression analysis further showed that MIF is independently associated with patients' survival (HR = 2.26, 95% CI = 1.17-4.37, p = 0.015). Mechanistic analyses revealed that MIF overexpression decreased E-cadherin and increased vimentin mRNA and protein levels in pancreatic cancer cell lines, consistent with the features of epithelial-to-mesenchymal transition (EMT). Furthermore, MIF-overexpression significantly increased ZEB1/2 and decreased miR-200b expression, while shRNA-mediated inhibition of MIF increased E-cadherin and miR-200b expression, and reduced the expression of ZEB1/2 in Panc1 cells. Re-expression of miR-200b in MIF overexpressing cells restored the epithelial characteristics, as indicated by an increase in E-cadherin and decrease in ZEB1/2 and vimentin expression. A reduced sensitivity to the chemotherapeutic drug, gemcitabine, occurred in MIF-overexpressing cells. Indicative of an increased malignant potential, MIF over-expressing cells showed significant increase in their invasion ability in vitro, and tumor growth and metastasis in an orthotopic xenograft mouse model. These results support a role of MIF in disease aggressiveness, indicating its potential usefulness as a candidate target for designing improved treatment in pancreatic cancer.

Inflammation regulates microRNA expression in cooperation with p53 and nitric oxide.

microRNA (miRNA) are small non-coding RNA targeting mRNAs leading to their instability and diminished translation. Altered expression of miRNA is associated with cancer. Inflammation and nitric oxide modulates the development of lymphomas in p53 knockout mice and there exists a negative feedback loop between p53 and NOS2. Using a genetic strategy, we tested the hypothesis that inflammation-induced oxidative and nitrosative stress modulates miRNA expression in mouse model deficient in either p53 or NOS2. Mice treated with Corynebacterium parvum (C. parvum), to induce inflammation, clearly separated from controls by their miRNA profiles in wild-type, p53- and NOS2-knockout genetic backgrounds. C. parvum-induced inflammation significantly (p < 0.005) increased miR-21, miR-29b and miR-34a/b/c and decreased (p < 0.005) mir-29c and mir-181a/c expression in the spleen of C57BL mice. However, p53-knockout C57BL mice did not show a significant increase in the mir-34b/c or a decrease in mir-29c expression following C. parvum-induced inflammation. Expression of mir-21, mir-29b and mir-181a was independent of p53-status. NOS2-knockout C57BL mice showed a significant increase in miR-21 and miR-34a/b/c and decrease in miR-181a similar to the wild-type (WT) mice following C. parvum-induced inflammation. However, in contrast to the WT mice, miR-29b/c expression was not affected following C. parvum-induced inflammation in NOS2 knockout mice. N-acetyl cysteine, an anti-oxidant, reduced the expression of miR-21 and miR-29b in C. parvum-treated WT mice (p < 0.005) as compared with control C. parvum-treated mice. These data are consistent with the hypothesis that inflammation modulates miRNA expression in vivo and the alteration in specific miRNA under an inflammatory microenvironment, can be influenced by p53 (miR-34b/c) and NO(•) (29b/c).

Optimization of flow path parameters for enhanced sensitivity lateral flow devices.

Lateral flow devices (LFDs) or lateral flow tests (LFTs) are one of the most widely used biosensor platforms for point-of-care (POC) diagnostics. The basic LFD design has remained largely unchanged since its first appearance, and this has limited LFD use in clinical applications due to a general lack of analytical sensitivity. We report here a comprehensive study of the use of laser-patterned geometric control barriers that influence the flow dynamics within an LFD, with the specific aim of enhancing LFD sensitivity and lowering the limit of detection (LOD). This control of sample flow produces an increase in the time available for optimizing the binding kinetics of the implemented assay. The geometric modification to the flow path is in the form of a constriction that is produced by depositing a photo-sensitive polymer onto the nitrocellulose membrane which when polymerized, creates impermeable barrier walls through the depth of the membrane. Both the position of the constriction within the flow path and the number of constrictions allow for an increase in the sensitivity because of a slower overall flow rate within the test and a larger volume of sample per unit width of the test line. For these high sensitivity LFDs (HS-LFD), through optimization of the constriction position and addition of a second constriction we attained a 62% increase in test line color intensity for the detection of procalcitonin (PCT) and were also able to lower the LOD from 10 ng/mL to 1 ng/mL. In addition, of relevance for future commercial exploitation, this also significantly decreases the antibody consumption per device leading to reduced costs for test production. We have further tested our HS-LFD with contrived human samples, validating its application for future clinical use.

MicroRNAs-A Promising Tool for Asthma Diagnosis and Severity Assessment: A Systematic Review.

Micro RNAs (miRNAs) are short, non-coding RNAs (Ribonucleic acids) with regulatory functions that could prove useful as biomarkers for asthma diagnosis and asthma severity-risk stratification. The objective of this systematic review is to identify panels of miRNAs that can be used to support asthma diagnosis and severity-risk assessment. Three databases (Medline, Embase, and SCOPUS) were searched up to 15 September 2020 to identify studies reporting differential expression of specific miRNAs in the tissues of adults and children with asthma. Studies reporting miRNAs associations in animal models that were also studied in humans were included in this review. We identified 75 studies that met our search criteria. Of these, 66 studies reported more than 200 miRNAs that are differentially expressed in asthma patients when compared to non-asthmatic controls. In addition, 16 studies reported 17 miRNAs that are differentially expressed with differences in asthma severity. We were able to construct two panels of miRNAs that are expressed in blood and can serve as core panels to further investigate the practicality and efficiency of using miRNAs as non-invasive biomarkers for asthma diagnosis and severity-risk assessment, respectively.

Semi-quantitative detection of inflammatory biomarkers using a laser-patterned multiplexed lateral flow device.

Inflammatory markers including C-reactive protein (CRP) and procalcitonin (PCT) have been shown to be useful biomarkers to improve triage speed and prevent the inappropriate use of antibiotics for infections such as pneumonia. Here, we present a novel and exciting solution to guide the administration of antibiotic treatment via rapid, semi-quantitative and multiplexed detection of CRP and PCT using an advanced lateral flow device (LFD) designed to have multiple parallel flow-paths, produced via the precise laser-based partitioning of the single flow-path of a standard LFD. Each flow-path within this multiplexed LFD has a unique detection capability which permits tailored detection of CRP within a predefined cut-off range (20 µg/mL - 100 µg/mL) and PCT above a pre-defined threshold (0.5 ng/mL). We demonstrate the use of this LFD in the successful detection of CRP and PCT semi-quantitatively within spiked human serum samples. This multiplexed near-patient assay has potential for development into a rapid triage and treatment of patients with suspected pneumonia.

A SARS-CoV-2 nucleocapsid ELISA represents a low-cost alternative to lateral flow testing for community screening in LMI countries.

Background Controlling the spread of SARS-CoV-2 is problematic because of transmission driven by asymptomatic and pre-symptomatic individuals. Community screening can help identify these individuals but is often too expensive for countries with limited health care resources. Low-cost ELISA assays may address this problem, but their use has not yet been widely reported. Methods We developed a SARS-CoV-2 nucleocapsid ELISA and assessed its diagnostic performance on nose and throat swab samples from UK hospitalised patients and sputum samples from patients in Ghana. Results The ELISA had a limit of detection of 8.4 pg/ml antigen and 16 pfu/ml virus. When tested on UK samples (128 positive and 10 negative patients), sensitivity was 58.6% (49.6-67.2) rising to 78.3% (66.7-87.3) if real-time PCR Ct values > 30 were excluded, while specificity was 100% (69.2-100). In a second trial using the Ghanaian samples (121 positive, 96 negative), sensitivity was 52% (42.8-61.2) rising to 72.6% (61.8-81.2) when a > 30 Ct cut-off was applied, while specificity was 100% (96.2-100). Conclusions: Our data show that nucleocapsid ELISAs can test a variety of patient sample types while achieving levels of sensitivity and specificity required for effective community screening. Further investigations into the opportunities that this provides are warranted.

Circulating miRNAs-A potential tool to identify severe asthma risk?

BACKGROUND: Identifying patients at risk of severe asthma is vitally important given the disproportionate burden of disease imposed by that state. However, biomarkers to support such needs remain elusive. METHODS: In this letter, we assessed whether specific panels of circulating miRNAs (microRNAs) can differentiate between mild and severe asthma patients as well as between healthy subjects and severe asthma patients. RESULTS: To our knowledge, the miRNAs identified in our work such as miR-28-3p, miR-16-2-3p, and miR-210-3p have not been previously reported as differentially expressed in the serum of severe asthma patients. CONCLUSION: Our findings suggest that miRNA expression profiles may have the capability as potential biomarkers that signal the risk of having severe asthma. As such, these findings have significant novelty and merit wider dissemination to facilitate further work in this field.

Capillary-based reverse transcriptase loop-mediated isothermal amplification for cost-effective and rapid point-of-care COVID-19 testing.

As the SARS-CoV-2 pandemic continues to spread, the necessity for rapid, easy diagnostic capabilities could never have been more crucial. With this aim in mind, we have developed a cost-effective and time-saving testing methodology/strategy that implements a sensitive reverse transcriptase loop-mediated amplification (RT-LAMP) assay within narrow, commercially available and cheap, glass capillaries for detection of the SARS-CoV-2 viral RNA. The methodology is compatible with widely used laboratory-based molecular testing protocols and currently available infrastructure. It employs a simple rapid extraction protocol that lyses the virus, releasing sufficient genetic material for amplification. This extracted viral RNA is then amplified using a SARS-CoV-2 RT-LAMP kit, at a constant temperature and the resulting amplified product produces a colour change which can be visually interpreted. This testing protocol, in conjunction with the RT-LAMP assay, has a sensitivity of ∼100 viral copies per reaction of a sample and provides results in a little over 30 min. As the assay is carried out in a water bath, commonly available within most testing laboratories, it eliminates the need for specialised instruments and associated skills. In addition, our testing pathway requires a significantly reduced quantity of reagents per test while providing comparable sensitivity and specificity to the RT-LAMP kit used in this study. While the conventional technique requires 25 µl of reagent, our test only utilises less than half the quantity (10 µl). Thus, with its minimalistic approach, this capillary-based assay could be a promising alternative to the conventional testing, owing to the fact that it can be performed in resource-limited settings, using readily available apparatus, and has the potential of increasing the overall testing capacity, while also reducing the burden on supply chains for mass testing.

Laser-patterned paper-based sensors for rapid point-of-care detection and antibiotic-resistance testing of bacterial infections.

Antimicrobial resistance (AMR) has been identified by the World Health Organisation as a global threat that currently claims at least 25,000 deaths each year in Europe and 700,000 globally; the number is projected to reach 10 million per year between 2015 and 2050. Therefore, there is an urgent need for low-cost but reliable point-of-care diagnostics for early screening of infections especially in developing countries lacking in basic infrastructure and trained personnel. This work is aimed at developing such a device, a paper-based microfluidic device for infection testing by an unskilled user in a low resource setting. Here, we present our work relating to the use of our laser-patterned paper-based devices for detection and susceptibility testing of Escherichia coli, via a simple visually observable colour change. The results indicate the suitability of our integrated paper devices for timely identification of bacterial infections at the point-of-care and their usefulness in providing a hugely beneficial pathway for accurate antibiotic prescribing and thus a novel route to tackling the global challenge of AMR.

Effects of Pulsed Electric Field on the Cell Wall and Infection Activity of Rhizoctonia solani.

This paper adopts the Design-Expert software to design an orthogonal experiment with a pulse voltage amplitude of 30 kV, processing time of three minutes, and a pulse width of 45 µs as the center points, in order to study the effects of the pulsed electric field on the cell wall and infection activity of Rhizoctonia solani. High-voltage pulse power was used to treat the bacteria solution with the pulsed electric field. Untreated Rhizoctonia solani were used as the control group. Transmission electron microscope images were used to analyze the cell wall damage. ANOVA was performed on the experimental results and the fitting degree of the model was good (F>>1). Response surface analysis was used to optimize the parameters based on chitin content and polygalacturonase activity. The optimal treatment conditions were obtained as a pulse voltage amplitude of 25 kV, processing time of 2.54 min, and a pulse width of 34.35 µs. On this basis, experiments were designed to verify the optimized conditions. The results demonstrated that, under the optimal processing conditions, the damage index of the cell wall of Rhizoctonia solani was 9.59% lower in chitin content and 83.05% lower in polygalacturonase activity compared with those of the control group. All indexes were significantly different (P < 0.001), which is consistent with the parameter optimization results. The results provide a theoretical basis for the pulsed electric field assisted sterilization and reference for the design of plant protection machinery in the latter stage.

Regulation of human nitric oxide synthase 2 expression by Wnt beta-catenin signaling.

Nitric oxide (NO.), an important mediator of inflammation, and beta-catenin, a component of the Wnt-adenomatous polyposis coli signaling pathway, contribute to the development of cancer. We have identified two T-cell factor 4 (Tcf-4)-binding elements (TBE1 and TBE2) in the promoter of human inducible NO synthase 2 (NOS2). We tested the hypothesis that beta-catenin regulates human NOS2 gene. Mutation in either of the two TBE sites decreased the basal and cytokine-induced NOS2 promoter activity in different cell lines. The promoter activity was significantly reduced when both TBE1 and TBE2 sites were mutated (P < 0.01). Nuclear extract from HCT116, HepG2, or DLD1 cells bound to NOS2 TBE1 or TBE2 oligonucleotides in electrophoretic mobility shift assays and the specific protein-DNA complexes were supershifted with anti-beta-catenin or anti-Tcf-4 antibody. Overexpression of beta-catenin and Tcf-4 significantly increased both basal and cytokine-induced NOS2 promoter activity (P < 0.01), and the induction was dependent on intact TBE sites. Overexpression of beta-catenin or Tcf-4 increased NOS2 mRNA and protein expression in HCT116 cells. Lithium chloride (LiCl), an inhibitor of glycogen synthase kinase-3beta, increased cytosolic and nuclear beta-catenin level, NOS2 expression, and NO. production in primary human and rat hepatocytes and cancer cell lines. Treatment with Wnt-3A-conditioned medium increased beta-catenin and NOS2 expression in fetal human hepatocytes. When administered in vivo, LiCl increased hepatic beta-catenin level in a dose-dependent manner with simultaneous increase in NOS2 expression. These data are consistent with the hypothesis that beta-catenin up-regulates NOS2 and suggest a novel mechanism by which the Wnt/beta-catenin signaling pathway may contribute to cancer by increasing NO. production.

Rapid Multiplexed Detection on Lateral-Flow Devices Using a Laser Direct-Write Technique.

Paper-based lateral flow devices (LFDs) are regarded as ideal low-cost diagnostic solutions for point-of-care (POC) scenarios that allow rapid detection of a single analyte within a fluidic sample, and have been in common use for a decade. In recent years, there has been an increasing need for rapid and simultaneous detection of multiple analytes present within a single sample and to facilitate this, we report here a novel solution-detection using a multi-path LFD created via the precise partitioning of the single flow-path of a standard LFD using our previously reported laser direct-write (LDW) technique. The multiple flow-paths allow the simultaneous detection of the different analytes individually within each of the parallel channels without any cross-reactivity. The appearance of coloured test lines in individual channels indicates the presence of the different analytes within a sample. We successfully present the use of a LDW-patterned multi-path LFD for multiplexed detection of a biomarker panel comprising C-reactive protein (CRP) and Serum amyloid A-1 (SAA1), used for the diagnosis of bacterial infections. Overall, we demonstrate the use of our LDW technique in the creation of a novel LFD that enables multiplexed detection of two inflammation markers within a single LFD providing a detection protocol that is comparatively more efficient than the standard sequential multiplexing procedure.

Improved sensitivity and limit-of-detection of lateral flow devices using spatial constrictions of the flow-path.

We report on the use of a laser-direct write (LDW) technique that allows the fabrication of lateral flow devices with enhanced sensitivity and limit of detection. This manufacturing technique comprises the dispensing of a liquid photopolymer at specific regions of a nitrocellulose membrane and its subsequent photopolymerisation to create impermeable walls inside the volume of the membrane. These polymerised structures are intentionally designed to create fluidic channels which are constricted over a specific length that spans the test zone within which the sample interacts with pre-deposited reagents. Experiments were conducted to show how these constrictions alter the fluid flow rate and the test zone area within the constricted channel geometries. The slower flow rate and smaller test zone area result in the increased sensitivity and lowered limit of detection for these devices. We have quantified these via the improved performance of a C-Reactive Protein (CRP) sandwich assay on our lateral flow devices with constricted flow paths which demonstrate an improvement in its sensitivity by 62x and in its limit of detection by 30x when compared to a standard lateral flow CRP device.

Regulation of cyclooxygenase-2 expression by the Wnt and ras pathways.

Mutations in the adenomatous polyposis coli (APC) gene and K-ras occur in the majority of human colorectal cancers. Loss of functional APC protein activates the Wnt signal transduction pathway, allowing the nuclear accumulation of beta-catenin, which then binds to T-cell factor-4 (Tcf-4), causing increased transcriptional activation of downstream target genes. We investigated the hypothesis that the activation of the WNT pathway regulates cyclooxygenase-2 (COX-2). COX-2 was down-regulated after the induction of full-length APC in the HT29-APC cell line. We identified a Tcf-4-binding element (TBE) in the COX-2 promoter that specifically bound to Tcf-4 in an electrophoretic mobility shift assay. COX-2 promoter luciferase activity is down-regulated by APC in a promoter reporter construct containing the, TBE but not with mutant TBE. Mutant beta-catenin expression up-regulated the COX-2 promoter activity and the endogenous COX-2 mRNA expression in HuH7, hepatocellular carcinoma cell line, which is partially abrogated by cotransfection with a dominant-negative Tcf-4 expression vector. Although beta-catenin alone did not increase COX-2 protein to detectable levels in HuH7 cells, coexpression of both mutant beta-catenin and mutant K-ras increased COX-2 protein expression, which is consistent with the previous reports that K-ras can stabilize COX-2 mRNA. Taken together, our data support the hypothesis that COX-2 is down-regulated by APC and up-regulated by nuclear beta-catenin accumulation, and additionally implicate the Wnt signal transduction pathway in colon and liver carcinogenesis.

p53-induced up-regulation of MnSOD and GPx but not catalase increases oxidative stress and apoptosis.

p53-mediated apoptosis may involve the induction of redox-controlling genes, resulting in the production of reactive oxygen species. Microarray expression analysis of doxorubicin exposed, related human lymphoblasts, p53 wild-type (WT) Tk6, and p53 mutant WTK1 identified the p53-dependent up-regulation of manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx). Consensus p53 binding sequences were identified in human MnSOD and GPx promoter regions. A 3-fold increase in the MnSOD promoter activity was observed after the induction of p53 in Li-Fraumeni syndrome (LFS) fibroblast, TR9-7, expressing p53 under the control of a tetracycline-regulated promoter. An increased protein expression of endogenous MnSOD and GPx also positively correlated with the level of p53 induction in TR9-7 cells. However, catalase (CAT) protein expression remained unaltered after p53 induction. We also examined the expression of MnSOD, GPx, and CAT in a panel of normal or LFS fibroblasts, containing either WT or mutant p53. We found increased MnSOD enzymatic activity, MnSOD mRNA expression, and MnSOD and GPx protein in LFS fibroblasts carrying a WT p53 allele when compared with homozygous mutant p53 isogenic cells. The CAT protein level was unchanged in these cells. We observed both the release of cytochrome C and Ca(2+) from the mitochondria into the cytoplasm and an increased frequency of apoptotic cells after p53 induction in the TR9-7 cells that coincided with an increased expression of MnSOD and GPx, and the level of reactive oxygen species. The increase in apoptosis was reduced by the antioxidant N-acetylcysteine. These results identify a novel mechanism of p53-dependent apoptosis in which p53-mediated up-regulation of MnSOD and GPx, but not CAT, produces an imbalance in antioxidant enzymes and oxidative stress.

Nitric oxide, a mediator of inflammation, suppresses tumorigenesis.

Inflammation influences the development of cancer. The nitric oxide synthase (NOS2) is induced by inflammatory cytokines, e.g., tumor necrosis factor alpha and interleukin 1beta, and produces nitric oxide (NO*), a critical mediator of the inflammatory response. Because p53 governs NO* production by transcriptionally transrepressing NOS2, we used a genetic strategy to determine whether NO* and p53 cooperatively regulate tumorigenesis. Lymphomas developed more rapidly in p53-/-NOS2-/- or p53-/-NOS2+/- mice than in p53-/-NOS2+/+ mice that were cross-bred into a >95% C57BL6 background and maintained in a pathogen-free condition. Likewise, sarcomas and lymphomas developed faster in p53+/-NOS2-/- or p53+/-NOS2+/- than in p53+/-NOS2+/+ mice. When compared with the double knockout mice, p53-/-NOS2+/+ mice showed a higher apoptotic index and a decreased proliferation index with an increased expression of death receptor ligands, CD95-L and tumor necrosis factor-related apoptosis-inducing ligand, and the cell cycle checkpoint protein, p21(waf1), in the spleen and thymus before tumor development. Furthermore, mice deficient in both p53 and NOS2 produced a high level of anti-inflammatory interleukin 10 when compared with p53-deficient mice. These studies provide genetic and mechanistic evidence that NO* can suppress tumorigenesis.

Inducible nitric oxide synthase enhances disease aggressiveness in pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies and is refractory to the available treatments. Pancreatic ductal adenocarcinoma (PDAC) expresses high level of inducible nitric oxide synthase (NOS2), which causes sustained production of nitric oxide (NO). We tested the hypothesis that an aberrantly increased NO-release enhances the development and progression of PDAC. Enhanced NOS2 expression in tumors significantly associated with poor survival in PDAC patients (N = 107) with validation in independent cohorts. We then genetically targeted NOS2 in an autochthonous mouse model of PDAC to examine the effect of NOS2-deficiency on disease progression and survival. Genetic ablation of NOS2 significantly prolonged survival and reduced tumor severity in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice. Primary tumor cells isolated from NOS2-deficient KPC (NKPC) mice showed decreased proliferation and invasiveness as compared to those from KPC mice. Furthermore, NKPC tumors showed reduced expression of pERK, a diminished inactivation of Forkhead box transcription factor O (FOXO3), a tumor suppressor, and a decrease in the expression of oncomir-21, when compared with tumors in KPC mice. Taken together, these findings showed that NOS2 is a predictor of prognosis in early stage, resected PDAC patients, and provide proof-of-principle that targeting NOS2 may have potential therapeutic value in this lethal malignancy.