Wnt and Hedgehog Are Critical Mediators of Cigarette Smoke-Induced Lung Cancer

BACKGROUND: Lung cancer is the leading cause of cancer death in the world, and greater than 90% of lung cancers are cigarette smoke-related. Current treatment options are inadequate, because the molecular basis of cigarette-induced lung cancer is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that human primary or immortalized bronchial epithelial cells exposed to cigarette smoke for eight days in culture rapidly proliferate, show anchorage-independent growth, and form tumors in nude mice. Using this model of the early stages of smoke-induced tumorigenesis, we examined the molecular changes leading to lung cancer. We observed that the embryonic signaling pathways mediated by Hedgehog and Wnt are activated by smoke. Pharmacological inhibition of these pathways blocked the transformed phenotype. CONCLUSIONS/SIGNIFICANCE: These experiments provide a model in which the early stages of smoke-induced tumorigenesis can be elicited, and should permit us to identify molecular changes driving this process. Results obtained so far indicate that smoke-induced lung tumors are driven by activation of two embryonic regulatory pathways, Hedgehog (Hh) and Wnt. Based on the current and emerging availability of drugs to inhibit Hh and Wnt signaling, it is possible that an understanding of the role of Hh and Wnt in lung cancer pathogenesis will lead to the development of new therapies

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets ("apatopes") with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern Pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer-aptamer, aptamer-nonaptamer biomacromolecules (siRNAs, proteins) and aptamer-nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

There is no such thing as ‘undisturbed’ soil and sediment sampling: sampler-induced deformation of salt marsh sediments revealed by 3D X-ray computed tomography

Purpose: Within most environmental contexts, the collection of 'undisturbed' samples is widely relied-upon in studies of soil and sediment properties and structure. However, the impact of sampler-induced disturbance is rarely acknowledged, despite the potential significance of modification to sediment structure for the robustness of data interpretation. In this study, 3D-computed X-ray microtomography (μCT) is used to evaluate and compare the disturbance imparted by four commonly-used sediment sampling methods within a coastal salt-marsh. Materials and methods: Paired sediment core samples from a restored salt-marsh at Orplands Farm, Essex, UK were collected using four common sampling methods (push, cut, hammer and gouge methods). Sampling using two different area-ratio cores resulted in a total of 16 cores that were scanned using 3D X-Ray computed tomography, to identify and evaluate sediment structural properties of samples that can be attributed to sampling method. Results and discussion: 3D qualitative analysis identifies a suite of sampling-disturbance structures including gross-scale changes to sediment integrity and substantial modification of pore-space, structure and distribution, independent of sediment strength and stiffness. Quantitative assessment of changes to pore-space and sediment density arising from the four sampling methods offer a means of direct comparison between the impact of depth-sampling methods. Considerable disturbance to samples result from use of push, hammer and auguring samplers, whilst least disturbance is found in samples recovered by cutting and advanced trimming approaches. Conclusions: It is evident that with the small-bore tubes and samplers commonly used in environmental studies, all techniques result in disturbance to sediment structure to a far greater extent than previously reported, revealed by μCT. This work identifies and evaluates for the first time the full nature, extent and significance of internal sediment disturbance arising from common sampling methods

Single-dose administration and the influence of the timing of the booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooled analysis of four randomised trials.

BACKGROUND: The ChAdOx1 nCoV-19 (AZD1222) vaccine has been approved for emergency use by the UK regulatory authority, Medicines and Healthcare products Regulatory Agency, with a regimen of two standard doses given with an interval of 4-12 weeks. The planned roll-out in the UK will involve vaccinating people in high-risk categories with their first dose immediately, and delivering the second dose 12 weeks later. Here, we provide both a further prespecified pooled analysis of trials of ChAdOx1 nCoV-19 and exploratory analyses of the impact on immunogenicity and efficacy of extending the interval between priming and booster doses. In addition, we show the immunogenicity and protection afforded by the first dose, before a booster dose has been offered. METHODS: We present data from three single-blind randomised controlled trials-one phase 1/2 study in the UK (COV001), one phase 2/3 study in the UK (COV002), and a phase 3 study in Brazil (COV003)-and one double-blind phase 1/2 study in South Africa (COV005). As previously described, individuals 18 years and older were randomly assigned 1:1 to receive two standard doses of ChAdOx1 nCoV-19 (5 × 1010 viral particles) or a control vaccine or saline placebo. In the UK trial, a subset of participants received a lower dose (2·2 × 1010 viral particles) of the ChAdOx1 nCoV-19 for the first dose. The primary outcome was virologically confirmed symptomatic COVID-19 disease, defined as a nucleic acid amplification test (NAAT)-positive swab combined with at least one qualifying symptom (fever ≥37·8°C, cough, shortness of breath, or anosmia or ageusia) more than 14 days after the second dose. Secondary efficacy analyses included cases occuring at least 22 days after the first dose. Antibody responses measured by immunoassay and by pseudovirus neutralisation were exploratory outcomes. All cases of COVID-19 with a NAAT-positive swab were adjudicated for inclusion in the analysis by a masked independent endpoint review committee. The primary analysis included all participants who were SARS-CoV-2 N protein seronegative at baseline, had had at least 14 days of follow-up after the second dose, and had no evidence of previous SARS-CoV-2 infection from NAAT swabs. Safety was assessed in all participants who received at least one dose. The four trials are registered at ISRCTN89951424 (COV003) and ClinicalTrials.gov, NCT04324606 (COV001), NCT04400838 (COV002), and NCT04444674 (COV005). FINDINGS: Between April 23 and Dec 6, 2020, 24 422 participants were recruited and vaccinated across the four studies, of whom 17 178 were included in the primary analysis (8597 receiving ChAdOx1 nCoV-19 and 8581 receiving control vaccine). The data cutoff for these analyses was Dec 7, 2020. 332 NAAT-positive infections met the primary endpoint of symptomatic infection more than 14 days after the second dose. Overall vaccine efficacy more than 14 days after the second dose was 66·7% (95% CI 57·4-74·0), with 84 (1·0%) cases in the 8597 participants in the ChAdOx1 nCoV-19 group and 248 (2·9%) in the 8581 participants in the control group. There were no hospital admissions for COVID-19 in the ChAdOx1 nCoV-19 group after the initial 21-day exclusion period, and 15 in the control group. 108 (0·9%) of 12 282 participants in the ChAdOx1 nCoV-19 group and 127 (1·1%) of 11 962 participants in the control group had serious adverse events. There were seven deaths considered unrelated to vaccination (two in the ChAdOx1 nCov-19 group and five in the control group), including one COVID-19-related death in one participant in the control group. Exploratory analyses showed that vaccine efficacy after a single standard dose of vaccine from day 22 to day 90 after vaccination was 76·0% (59·3-85·9). Our modelling analysis indicated that protection did not wane during this initial 3-month period. Similarly, antibody levels were maintained during this period with minimal waning by day 90 (geometric mean ratio [GMR] 0·66 [95% CI 0·59-0·74]). In the participants who received two standard doses, after the second dose, efficacy was higher in those with a longer prime-boost interval (vaccine efficacy 81·3% [95% CI 60·3-91·2] at ≥12 weeks) than in those with a short interval (vaccine efficacy 55·1% [33·0-69·9] at <6 weeks). These observations are supported by immunogenicity data that showed binding antibody responses more than two-fold higher after an interval of 12 or more weeks compared with an interval of less than 6 weeks in those who were aged 18-55 years (GMR 2·32 [2·01-2·68]). INTERPRETATION: The results of this primary analysis of two doses of ChAdOx1 nCoV-19 were consistent with those seen in the interim analysis of the trials and confirm that the vaccine is efficacious, with results varying by dose interval in exploratory analyses. A 3-month dose interval might have advantages over a programme with a short dose interval for roll-out of a pandemic vaccine to protect the largest number of individuals in the population as early as possible when supplies are scarce, while also improving protection after receiving a second dose. FUNDING: UK Research and Innovation, National Institutes of Health Research (NIHR), The Coalition for Epidemic Preparedness Innovations, the Bill & Melinda Gates Foundation, the Lemann Foundation, Rede D'Or, the Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca