Assessment of inhalation toxicity of cigarette smoke and aerosols from flavor mixtures: 5-week study in A/J mice.

Most flavors used in e-liquids are generally recognized as safe for oral consumption, but their potential effects when inhaled are not well characterized. In vivo inhalation studies of flavor ingredients in e-liquids are scarce. A structure-based grouping approach was used to select 38 flavor group representatives (FGR) on the basis of known and in silico-predicted toxicological data. These FGRs were combined to create prototype e-liquid formulations and tested against cigarette smoke (CS) in a 5-week inhalation study. Female A/J mice were whole-body exposed for 6 h/day, 5 days/week, for 5 weeks to air, mainstream CS, or aerosols from (1) test formulations containing propylene glycol (PG), vegetable glycerol (VG), nicotine (N; 2% w/w), and flavor (F) mixtures at low (4.6% w/w), medium (9.3% w/w), or high (18.6% w/w) concentration or (2) base formulation (PG/VG/N). Male A/J mice were exposed to air, PG/VG/N, or PG/VG/N/F-high under the same exposure regimen. There were no significant mortality or in-life clinical findings in the treatment groups, with only transient weight loss during the early exposure adaptation period. While exposure to flavor aerosols did not cause notable lung inflammation, it caused only minimal adaptive changes in the larynx and nasal epithelia. In contrast, exposure to CS resulted in lung inflammation and moderate-to-severe changes in the epithelia of the nose, larynx, and trachea. In summary, the study evaluates an approach for assessing the inhalation toxicity potential of flavor mixtures, thereby informing the selection of flavor exposure concentrations (up to 18.6%) for a future chronic inhalation study.

A 6-month inhalation toxicology study in Apoe-/- mice demonstrates substantially lower effects of e-vapor aerosol compared with cigarette smoke in the respiratory tract.

Cigarette smoking is the major cause of chronic obstructive pulmonary disease. Considerable attention has been paid to the reduced harm potential of nicotine-containing inhalable products such as electronic cigarettes (e-cigarettes). We investigated the effects of mainstream cigarette smoke (CS) and e-vapor aerosols (containing nicotine and flavor) generated by a capillary aerosol generator on emphysematous changes, lung function, and molecular alterations in the respiratory system of female Apoe-/- mice. Mice were exposed daily (3 h/day, 5 days/week) for 6 months to aerosols from three different e-vapor formulations-(1) carrier (propylene glycol and vegetable glycerol), (2) base (carrier and nicotine), or (3) test (base and flavor)-or to CS from 3R4F reference cigarettes. The CS and base/test aerosol concentrations were matched at 35 µg nicotine/L. CS exposure, but not e-vapor exposure, led to impairment of lung function (pressure-volume loop area, A and K parameters, quasi-static elastance and compliance) and caused marked lung inflammation and emphysematous changes, which were confirmed histopathologically and morphometrically. CS exposure caused lung transcriptome (activation of oxidative stress and inflammatory responses), lipidome, and proteome dysregulation and changes in DNA methylation; in contrast, these effects were substantially reduced in response to the e-vapor aerosol exposure. Compared with sham, aerosol exposure (carrier, base, and test) caused a slight impact on lung inflammation and epithelia irritation. Our results demonstrated that, in comparison with CS, e-vapor aerosols induced substantially lower biological and pathological changes in the respiratory tract associated with chronic inflammation and emphysema.

Impact of whole-body versus nose-only inhalation exposure systems on systemic, respiratory, and cardiovascular endpoints in a 2-month cigarette smoke exposure study in the ApoE-/- mouse model.

Cigarette smoking is one major modifiable risk factor in the development and progression of chronic obstructive pulmonary disease and cardiovascular disease. To characterize and compare cigarette smoke (CS)-induced disease endpoints after exposure in either whole-body (WB) or nose-only (NO) exposure systems, we exposed apolipoprotein E-deficient mice to filtered air (Sham) or to the same total particulate matter (TPM) concentration of mainstream smoke from 3R4F reference cigarettes in NO or WB exposure chambers (EC) for 2 months. At matching TPM concentrations, we observed similar concentrations of carbon monoxide, acetaldehyde, and acrolein, but higher concentrations of nicotine and formaldehyde in NOEC than in WBEC. In both exposure systems, CS exposure led to the expected adaptive changes in nasal epithelia, altered lung function, lung inflammation, and pronounced changes in the nasal epithelial transcriptome and lung proteome. Exposure in the NOEC caused generally more severe histopathological changes in the nasal epithelia and a higher stress response as indicated by body weight decrease and lower blood lymphocyte counts compared with WB exposed mice. Erythropoiesis, and increases in total plasma triglyceride levels and atherosclerotic plaque area were observed only in CS-exposed mice in the WBEC group but not in the NOEC group. Although the composition of CS in the breathing zone is not completely comparable in the two exposure systems, the CS-induced respiratory disease endpoints were largely confirmed in both systems, with a higher magnitude of severity after NO exposure. CS-accelerated atherosclerosis and other pro-atherosclerotic factors were only significant in WBEC.

A 6-month systems toxicology inhalation study in ApoE-/- mice demonstrates reduced cardiovascular effects of E-vapor aerosols compared with cigarette smoke.

Smoking cigarettes is harmful to the cardiovascular system. Considerable attention has been paid to the reduced harm potential of alternative nicotine-containing inhalable products such as e-cigarettes. We investigated the effects of E-vapor aerosols or cigarette smoke (CS) on atherosclerosis progression, cardiovascular function, and molecular changes in the heart and aorta of female apolipoprotein E-deficient (ApoE-/-) mice. The mice were exposed to aerosols from three different E-vapor formulations: 1) carrier (propylene glycol and vegetable glycerol), 2) base (carrier and nicotine), or 3) test (base and flavor) or to CS from 3R4F reference cigarettes for up to 6 mo. Concentrations of CS and base or test aerosols were matched at 35 µg nicotine/L. Exposure to CS, compared with sham-exposed fresh air controls, accelerated atherosclerotic plaque formation, whereas no such effect was seen for any of the three E-vapor aerosols. Molecular changes indicated disease mechanisms related to oxidative stress and inflammation in general, plus changes in calcium regulation, and altered cytoskeletal organization and microtubule dynamics in the left ventricle. While ejection fraction, fractional shortening, cardiac output, and isovolumic contraction time remained unchanged following E-vapor aerosols exposure, the nicotine-containing base and test aerosols caused an increase in isovolumic relaxation time similar to CS. A nicotine-related increase in pulse wave velocity and arterial stiffness was also observed, but it was significantly lower for base and test aerosols than for CS. These results demonstrate that in comparison with CS, E-vapor aerosols induce substantially lower biological responses associated with smoking-related cardiovascular diseases.NEW & NOTEWORTHY Analysis of key urinary oxidative stress markers and proinflammatory cytokines showed an absence of oxidative stress and inflammation in the animals exposed to E-vapor aerosols. Conversely, animals exposed to conventional cigarette smoke had high urinary levels of these markers. When compared with conventional cigarette smoke, E-vapor aerosols induced smaller atherosclerotic plaque surface area and volume. Systolic and diastolic cardiac function, as well as endothelial function, were further significantly less affected by electronic cigarette aerosols than conventional cigarette smoke. Molecular analysis demonstrated that E-vapor aerosols induce significantly smaller transcriptomic dysregulation in the heart and aorta compared with conventional cigarette smoke.

E-vapor aerosols do not compromise bone integrity relative to cigarette smoke after 6-month inhalation in an ApoE-/- mouse model.

Cigarette smoke (CS) exposure is one of the leading risk factors for human health. Nicotine-containing inhalable products, such as e-cigarettes, can effectively support tobacco harm reduction approaches. However, there are limited comparative data on the effects of the aerosols generated from electronic vapor products (e-vapor) and CS on bone. Here, we report the effects of e-vapor aerosols and CS on bone morphology, structure, and strength in a 6-month inhalation study. Eight-week-old ApoE-/- mice were exposed to aerosols from three different e-vapor formulations-CARRIER (propylene glycol and vegetable glycerol), BASE (CARRIER and nicotine), TEST (BASE and flavor)-to CS from 3R4F reference cigarettes at matched nicotine concentrations (35 µg/L) or to fresh air (Sham) (N = 10 per group). Tibiae were analyzed for bone morphology by µCT imaging, biomechanics by three-point bending, and by histological analysis. CS inhalation caused a significant decrease in cortical and total bone volume fraction and bone density relative to e-vapor aerosols. Additionally, CS exposure caused a decrease in ultimate load and stiffness. In contrast, bone structural and biomechanical parameters were not significantly affected by e-vapor aerosol or Sham exposure. At the dissection time point, there was no significant difference in body weight or tibia bone weight or length among the groups. Histological findings revealed microcracks in cortical bone areas among all exposed groups compared to Sham control. In conclusion, because of the bone-preserving effect of e-vapor aerosols relative to CS exposure, e-vapor products could potentially constitute less harmful alternatives to cigarettes in situations in which bone health is of importance.

ATM- and NEMO-dependent ELKS ubiquitination coordinates TAK1-mediated IKK activation in response to genotoxic stress.

Activation of the transcription factor NF-κB by multiple genotoxic stimuli modulates cancer cell survival. This response is mediated by a conserved pathway involving the nuclear ATM kinase and cytoplasmic IκB kinase (IKK); however, the molecular link between them remains incompletely understood. Here we show that ATM activates the IKK kinase TAK1 in a manner dependent on IKKγ/NEMO and ELKS (a protein rich in glutamate, leucine, lysine, and serine). K63-linked polyubiquitination of ELKS, dependent on the ubiquitin ligase XIAP and the conjugating enzyme UBC13, allows ELKS association with TAK1 via its ubiquitin-binding subunits TAB2/3. Although NEMO mutants defective in ubiquitin binding permit ATM-dependent TAK1 activation, they block NEMO association with ELKS and IKK activation. Thus, ATM- and NEMO-dependent ubiquitination of ELKS leads to the ubiquitin-dependent assembly of TAK1/TAB2/3 and NEMO/IKK complexes, resulting in IKK and NF-κB activation following genotoxic stimuli.

Evaluation of the Tobacco Heating System 2.2 (THS2.2). Part 5: microRNA expression from a 90-day rat inhalation study indicates that exposure to THS2.2 aerosol causes reduced effects on lung tissue compared with cigarette smoke.

Modified-risk tobacco products (MRTP) are designed to reduce the individual risk of tobacco-related disease as well as population harm compared to smoking cigarettes. Experimental proof of their benefit needs to be provided at multiple levels in research fields. Here, we examined microRNA (miRNA) levels in the lungs of rats exposed to a candidate modified-risk tobacco product, the Tobacco Heating System 2.2 (THS2.2) in a 90-day OECD TG-413 inhalation study. Our aim was to assess the miRNA response to THS2.2 aerosol compared with the response to combustible cigarettes (CC) smoke from the reference cigarette 3R4F. CC smoke exposure, but not THS2.2 aerosol exposure, caused global miRNA downregulation, which may be explained by the interference of CC smoke constituents with the miRNA processing machinery. Upregulation of specific miRNA species, such as miR-146a/b and miR-182, indicated that they are causal elements in the inflammatory response in CC-exposed lungs, but they were reduced after THS2.2 aerosol exposure. Transforming transcriptomic data into protein activity based on corresponding downstream gene expression, we identified potential mechanisms for miR-146a/b and miR-182 that were activated by CC smoke but not by THS2.2 aerosol and possibly involved in the regulation of those miRNAs. The inclusion of miRNA profiling in systems toxicology approaches increases the mechanistic understanding of the complex exposure responses.

Evaluation of the Tobacco Heating System 2.2. Part 4: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects compared with cigarette smoke.

The objective of the study was to characterize the toxicity from sub-chronic inhalation of test atmospheres from the candidate modified risk tobacco product (MRTP), Tobacco Heating System version 2.2 (THS2.2), and to compare it with that of the 3R4F reference cigarette. A 90-day nose-only inhalation study on Sprague-Dawley rats was performed, combining classical and systems toxicology approaches. Reduction in respiratory minute volume, degree of lung inflammation, and histopathological findings in the respiratory tract organs were significantly less pronounced in THS2.2-exposed groups compared with 3R4F-exposed groups. Transcriptomics data obtained from nasal epithelium and lung parenchyma showed concentration-dependent differential gene expression following 3R4F exposure that was less pronounced in the THS2.2-exposed groups. Molecular network analysis showed that inflammatory processes were the most affected by 3R4F, while the extent of THS2.2 impact was much lower. Most other toxicological endpoints evaluated did not show exposure-related effects. Where findings were observed, the effects were similar in 3R4F- and THS2.2-exposed animals. In summary, toxicological changes observed in the respiratory tract organs of THS2.2 aerosol-exposed rats were much less pronounced than in 3R4F-exposed rats while other toxicological endpoints either showed no exposure-related effects or were comparable to what was observed in the 3R4F-exposed rats.

Deconvolution of Systemic Pharmacokinetics Predicts Inhaled Aerosol Dosimetry of Nicotine.

Absorption of inhaled compounds can occur from multiple sites based on upper and lower respiratory tract deposition, and clearance mechanisms leading to differential local and systemic pharmacokinetics. Deriving inhaled aerosol dosimetry and local tissue concentrations for nose-only exposure in rodents and inhaled products in humans is challenging. In this study we use inhaled nicotine as an example to identify regional respiratory tract deposition, absorption fractions, and their contribution toward systemic pharmacokinetics in rodents and humans. A physiologically based pharmacokinetic (PBPK) model was constructed to describe the disposition of nicotine and its major metabolite, cotinine. The model description for the lungs was simplified to include an upper respiratory tract region with active mucociliary clearance and a lower respiratory tract region. The PBPK model parameters such as rate of oral absorption, metabolism and clearance were fitted to the published nicotine and cotinine plasma concentrations post systemic administration and oral dosing. The fractional deposition of inhaled aerosol in the upper and lower respiratory tract regions was estimated by fitting the plasma concentrations. The model predicted upper respiratory tract deposition was 63.9% for nose-only exposure to nicotine containing nebulized aqueous aerosol in rats and 60.2% for orally inhaled electronic vapor product in humans. A marked absorption of nicotine from the upper respiratory tract and the gastrointestinal tract for inhaled aqueous aerosol contributed to the differential systemic pharmacokinetics in rats and humans. The PBPK model derived dosimetry shows that the current aerosol dosimetry models with their posteriori application using independent aerosol physicochemical characterization to predict aerosol deposition are insufficient and will need to consider complex interplay of inhaled aerosol evolutionary process. While the study highlights the needs for future research, it provides a preliminary framework for interpreting pharmacokinetics of inhaled aerosols to facilitate the analysis of in vivo exposure-responses for pharmacological and toxicological assessments.

Discriminating Spontaneous From Cigarette Smoke and THS 2.2 Aerosol Exposure-Related Proliferative Lung Lesions in A/J Mice by Using Gene Expression and Mutation Spectrum Data.

Mice, especially A/J mice, have been widely employed to elucidate the underlying mechanisms of lung tumor formation and progression and to derive human-relevant modes of action. Cigarette smoke (CS) exposure induces tumors in the lungs; but, non-exposed A/J mice will also develop lung tumors spontaneously with age, which raises the question of discriminating CS-related lung tumors from spontaneous ones. However, the challenge is that spontaneous tumors are histologically indistinguishable from the tumors occurring in CS-exposed mice. We conducted an 18-month inhalation study in A/J mice to assess the impact of lifetime exposure to Tobacco Heating System (THS) 2.2 aerosol relative to exposure to 3R4F cigarette smoke (CS) on toxicity and carcinogenicity endpoints. To tackle the above challenge, a 13-gene gene signature was developed based on an independent A/J mouse CS exposure study, following by a one-class classifier development based on the current study. Identifying gene signature in one data set and building classifier in another data set addresses the feature/gene selection bias which is a well-known problem in literature. Applied to data from this study, this gene signature classifier distinguished tumors in CS-exposed animals from spontaneous tumors. Lung tumors from THS 2.2 aerosol-exposed mice were significantly different from those of CS-exposed mice but not from spontaneous tumors. The signature was also applied to human lung adenocarcinoma gene expression data (from The Cancer Genome Atlas) and discriminated cancers in never-smokers from those in ever-smokers, suggesting translatability of our signature genes from mice to humans. A possible application of this gene signature is to discriminate lung cancer patients who may benefit from specific treatments (i.e., EGFR tyrosine kinase inhibitors). Mutational spectra from a subset of samples were also utilized for tumor classification, yielding similar results. "Landscaping" the molecular features of A/J mouse lung tumors highlighted, for the first time, a number of events that are also known to play a role in human lung tumorigenesis, such as Lrp1b mutation and Ros1 overexpression. This study shows that omics and computational tools provide useful means of tumor classification where histopathological evaluation alone may be unsatisfactory to distinguish between age- and exposure-related lung tumors.

Reduced Chronic Toxicity and Carcinogenicity in A/J Mice in Response to Life-Time Exposure to Aerosol From a Heated Tobacco Product Compared With Cigarette Smoke.

We conducted an inhalation study, in accordance with Organisation for Economic Co-operation and Development Test Guideline 453, exposing A/J mice to tobacco heating system (THS) 2.2 aerosol or 3R4F reference cigarette smoke (CS) for up to 18 months to evaluate chronic toxicity and carcinogenicity. All exposed mice showed lower thymus and spleen weight, blood lymphocyte counts, and serum lipid concentrations than sham mice, most likely because of stress and/or nicotine effects. Unlike THS 2.2 aerosol-exposed mice, CS-exposed mice showed increased heart weight, changes in red blood cell profiles and serum liver function parameters. Similarly, increased pulmonary inflammation, altered lung function, and emphysematous changes were observed only in CS-exposed mice. Histopathological changes in other respiratory tract organs were significantly lower in the THS 2.2 aerosol-exposed groups than in the CS-exposed group. Chronic exposure to THS 2.2 aerosol also did not increase the incidence or multiplicity of bronchioloalveolar adenomas or carcinomas relative to sham, whereas CS exposure did. Male THS 2.2 aerosol-exposed mice had a lower survival rate than sham mice, related to an increased incidence of urogenital issues that appears to be related to congenital factors rather than test item exposure. The lower impact of THS 2.2 aerosol exposure on tumor development and chronic toxicity is consistent with the significantly reduced levels of harmful and potentially harmful constituents in THS 2.2 aerosol relative to CS. The totality of the evidence from this study further supports the risk reduction potential of THS 2.2 for lung diseases in comparison with cigarettes.

Comparing the preclinical risk profile of inhalable candidate and potential candidate modified risk tobacco products: A bridging use case.

Cigarette smoking causes major preventable diseases, morbidity, and mortality worldwide. Smoking cessation and prevention of smoking initiation are the preferred means for reducing these risks. Less harmful tobacco products, termed modified-risk tobacco products (MRTP), are being developed as a potential alternative for current adult smokers who would otherwise continue smoking. According to a regulatory framework issued by the US Food and Drug Administration, a manufacturer must provide comprehensive scientific evidence that the product significantly reduces harm and the risk of tobacco-related diseases, in order to obtain marketing authorization for a new MRTP. For new tobacco products similar to an already approved predicate product, the FDA has foreseen a simplified procedure for assessing "substantial equivalence". In this article, we present a use case that bridges the nonclinical evidence from previous studies demonstrating the relatively reduced harm potential of two heat-not-burn products based on different tobacco heating principles. The nonclinical evidence was collected along a "causal chain of events leading to disease" (CELSD) to systematically follow the consequences of reduced exposure to toxicants (relative to cigarette smoke) through increasing levels of biological complexity up to disease manifestation in animal models of human disease. This approach leverages the principles of systems biology and toxicology as a basis for further extrapolation to human studies. The experimental results demonstrate a similarly reduced impact of both products on apical and molecular endpoints, no novel effects not seen with cigarette smoke exposure, and an effect of switching from cigarettes to either MRTP that is comparable to that of complete smoking cessation. Ideally, a subset of representative assays from the presented sequence along the CELSD could be sufficient for predicting similarity or substantial equivalence in the nonclinical impact of novel products; this would require further validation, for which the present use case could serve as a starting point.

Respiratory Effects of Exposure to Aerosol From the Candidate Modified-Risk Tobacco Product THS 2.2 in an 18-Month Systems Toxicology Study With A/J Mice.

Smoking cessation is the most effective measure for reducing the risk of smoking-related diseases. However, switching to less harmful products (modified-risk tobacco products [MRTP]) can be an alternative to help reduce the risk for adult smokers who would otherwise continue to smoke. In an 18-month chronic carcinogenicity/toxicity study in A/J mice (OECD Test Guideline 453), we assessed the aerosol of Tobacco Heating System 2.2 (THS 2.2), a candidate MRTP based on the heat-not-burn principle, compared with 3R4F cigarette smoke (CS). To capture toxicity- and disease-relevant mechanisms, we complemented standard toxicology endpoints with in-depth systems toxicology analyses. In this part of our publication series, we report on integrative assessment of the apical and molecular exposure effects on the respiratory tract (nose, larynx, and lungs). Across the respiratory tract, we found changes in inflammatory response following 3R4F CS exposure (eg, antimicrobial peptide response in the nose), with both shared and distinct oxidative and xenobiotic responses. Compared with 3R4F CS, THS 2.2 aerosol exerted far fewer effects on respiratory tract histology, including adaptive tissue changes in nasal and laryngeal epithelium and inflammation and emphysematous changes in the lungs. Integrative analysis of molecular changes confirmed the substantially lower impact of THS 2.2 aerosol than 3R4F CS on toxicologically and disease-relevant molecular processes such as inflammation, oxidative stress responses, and xenobiotic metabolism. In summary, this work exemplifies how apical and molecular endpoints can be combined effectively for toxicology assessment and further supports findings on the reduced respiratory health risks of THS 2.2 aerosol.

Roles of NF-kappaB in health and disease: mechanisms and therapeutic potential.

The NF-kappaB (nuclear factor kappaB) family of transcription factors are involved in a myriad of activities, including the regulation of immune responses, maturation of immune cells, development of secondary lymphoid organs and osteoclastogenesis. Fine tuning by positive and negative regulators keeps the NF-kappaB signalling pathway in check. Microbial products and genetic alterations in NF-kappaB and other signalling pathway components can lead to deregulation of NF-kappaB signalling in several human diseases, including cancers and chronic inflammatory disorders. NF-kappaB-pathway-specific therapies are being actively investigated, and these hold promises as interventions of NF-kappaB-related ailments.

Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange.

Comparative analysis of mutants using transfection is complicated by clones exhibiting variable levels of gene expression due to copy number differences and genomic position effects. Recombinase-mediated cassette exchange (RMCE) can overcome these problems by introducing the target gene into pre-determined chromosomal loci, but recombination between the available recombinase targeting sites can reduce the efficiency of targeted integration. We developed a new LoxP site (designated L3), which when used with the original LoxP site (designated L2), allows highly efficient and directional replacement of chromosomal DNA with incoming DNA. A total of six independent LoxP integration sites introduced either by homologous recombination or retroviral delivery were analyzed; 70-80% of the clones analyzed in hamster and human cells were correct recombinants. We combined the RMCE strategy with a new, tightly regulated tetracycline induction system to produce a robust, highly reliable system for inducible transgene expression. We observed stable inducible expression for over 1 month, with uniform expression in the cell population and between clones derived from the same integration site. This system described should find significant applications for studies requiring high level and regulated transgene expression and for determining the effects of various stresses or oncogenic conditions in vivo and in vitro.

Therapeutic strategies for hepatitis B virus-associated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a one of the most prevalent cancers worldwide, especially in the Asia Pacific region. At present, the five-year survival of individuals with HCC is low, mainly due to the late presentation of the disease, and limited therapeutic options. The major risk factors for HCC in the Asia Pacific region include hepatitis B and C viral infection. Removing or reducing these risk factors, such as by immunizing against the hepatitis B virus, may reduce the incidence of hepatitis B-associated HCC in the distant future. However, immunization does not decrease the risk of HCC in individuals who are currently infected with HBV worldwide highlighting the continued importance of examining strategies for the treatment of HCC. Current treatment strategies include surgical resection, liver transplantation, chemotherapy, transcatheter arterial chemoembolism and percutaneous injection. Except for surgical resection and liver transplantation, which represent the most viable treatment options, most of the other present treatments are mainly for palliation. Hence novel treatment modalities continue to be investigated and several of these are currently in clinical trials. One of the more promising novel approaches for HCC treatment is gene therapy. Potential promising gene therapeutic approaches for the treatment of HCC include augmentation of tumor suppressor genes, inhibition of abnormally over-expressed oncogenes as well as specifically inducing death of cancer cells either via "suicide" gene therapy, conditional replicative adenovirus strategy, immunomodulation or inhibiting tumor angiogenesis. Nonetheless, successful implementation of these gene therapeutic approaches is dependent on overcoming current practical and technical hurdles underscoring the need for a better understanding of the basic aspects of gene therapy.

A versatile platform to analyze low-affinity and transient protein-protein interactions in living cells in real time.

Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL's ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL's ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms.

NUCKS is a positive transcriptional regulator of insulin signaling.

Although much is known about the molecular players in insulin signaling, there is scant information about transcriptional regulation of its key components. We now find that NUCKS is a transcriptional regulator of the insulin signaling components, including the insulin receptor (IR). Knockdown of NUCKS leads to impaired insulin signaling in endocrine cells. NUCKS knockout mice exhibit decreased insulin signaling and increased body weight/fat mass along with impaired glucose tolerance and reduced insulin sensitivity, all of which are further exacerbated by a high-fat diet (HFD). Genome-wide ChIP-seq identifies metabolism and insulin signaling as NUCKS targets. Importantly, NUCKS is downregulated in individuals with a high body mass index and in HFD-fed mice, and conversely, its levels increase upon starvation. Altogether, NUCKS is a physiological regulator of energy homeostasis and glucose metabolism that works by regulating chromatin accessibility and RNA polymerase II recruitment to the promoters of IR and other insulin pathway modulators.

The POPX2 phosphatase regulates cancer cell motility and invasiveness.

Rho GTP ases play major roles in the regulation of actin cytoskeleton, cell movement and cell cycle. PAK, one of the effector kinases of these small GTP ases, has long been associated with different types of cancer. Therefore, it is likely that deregulation of PAK activity or expression may contribute to the development of cancer. POP X2, a PP 2C serine/threonine phosphatase, is known to dephosphorylate PAK and downregulate its activity. We find that POPX2 is expressed in a wide variety of tumour cell lines, the levels being highest in the more invasive MDA-MB-231 and lowest in the non-invasive MCF7 breast cancer lines. We show that silencing of POPX2 reduces the amount of stress fibers and focal adhesions in both cells lines. Interestingly, POPX2 deficiency dramatically reduces cell motility and invasiveness in MDA-MB-231 cells, and cell motility in MCF7 cells. Conversely, overexpression of POP X2 in MDA-MB-231 and MCF7 cells increased their motility. The silencing of POP X2 also inhibits the expression of beta1 integrin implying that POP X2 may modulate cell attachment to the extra-cellular matrix, as reflected in diminished initial colonization of POPX2 knockdown cells in nude mice. Based on these results, we propose a mechanism by which POP X2 regulates the invasive behavior of the cells.

Telomere-independent Rap1 is an IKK adaptor and regulates NF-kappaB-dependent gene expression.

We describe a genome-wide gain-of-function screen for regulators of NF-kappaB, and identify Rap1 (Trf2IP), as an essential modulator of NF-kappaB-mediated pathways. NF-kappaB is induced by ectopic expression of Rap1, whereas its activity is inhibited by Rap1 depletion. In addition to localizing on telomeres, mammalian Rap1 forms a complex with IKKs (IkappaB kinases), and is crucial for the ability of IKKs to be recruited to, and phosphorylate, the p65 subunit of NF-kappaB to make it transcriptionally competent. Rap1-mutant mice display defective NF-kappaB activation and are resistant to endotoxic shock. Furthermore, levels of Rap1 are positively regulated by NF-kappaB, and human breast cancers with NF-kappaB hyperactivity show elevated levels of cytoplasmic Rap1. Similar to inhibiting NF-kappaB, knockdown of Rap1 sensitizes breast cancer cells to apoptosis. These results identify the first cytoplasmic role of Rap1 and provide a mechanism through which it regulates an important signalling cascade in mammals, independent of its ability to regulate telomere function.