A method for evaluating and verifying biochemical methane potential test completion performed with landfilled municipal solid waste.

The biochemical methane potential (BMP) test is significant for the landfill industry as it provides a means to evaluate the gas potential, and therefore potential degradability, of both incoming and in-place municipal solid waste (MSW). However, the BMP test is not standardized making comparison of BMP results across sites problematic. For example, the BMP test duration has historically ranged from 20 days to several months with most current BMP tests lasting 60 days. However, the gas generation data can potentially be modelled for any of those durations to produce a prediction of the ultimate BMP value (BMPULT). Currently, the predicted BMPULT values of 23 long-duration (115-150 days) BMP tests were used to determine the required quantity of data (i.e. number of days) needed to produce an accurate BMPULT prediction. Results showed that no single test duration produced both accurate and efficient results, so a novel performance-based endpoint was proposed. The relative change in predicted BMPULT values with respect to time (dBMPULT/dt) was chosen as a potential performance-based completion metric. Results indicate that once the absolute normalized dBMPULT/dt value is within <2.5, <1.5 and <0.6% day-1 that the predicted BMPULT is within 20, 10 and 5% of the true BMPULT, respectively. Overall, the use of performance-based metrics for determining BMP test completion will allow for the collection of partial data sets, reduced experimental times and verification of results.

Binding of active Ras and its mutants to the Ras binding domain of PI-3-kinase: A quantitative approach to KD measurements.

Ras family GTPases (H/K/N-Ras) modulate numerous effectors, including the lipid kinase PI3K (phosphatidylinositol-3-kinase) that generates growth signal lipid PIP3 (phosphatidylinositol-3,4,5-triphosphate). Active GTP-Ras binds PI3K with high affinity, thereby stimulating PIP3 production. We hypothesize the affinity of this binding interaction could be significantly increased or decreased by Ras mutations at PI3K contact positions, with clinical implications since some Ras mutations at PI3K contact positions are disease-linked. To enable tests of this hypothesis, we have developed an approach combining UV spectral deconvolution, HPLC, and microscale thermophoresis to quantify the KD for binding. The approach measures the total Ras concentration, the fraction of Ras in the active state, and the affinity of active Ras binding to its docking site on PI3K Ras binding domain (RBD) in solution. The approach is illustrated by KD measurements for the binding of active H-Ras and representative mutants, each loaded with GTP or GMPPNP, to PI3Kγ RBD. The findings demonstrate that quantitation of the Ras activation state increases the precision of KD measurements, while also revealing that Ras mutations can increase (Q25L), decrease (D38E, Y40C), or have no effect (G13R) on PI3K binding affinity. Significant Ras affinity changes are predicted to alter PI3K regulation and PIP3 growth signals.

Genomic evidence of recent European introgression into North American farmed and wild Atlantic salmon.

Gene flow between wild and domestic populations has been repeatedly demonstrated across a diverse range of taxa. Ultimately, the genetic impacts of gene flow from domestic into wild populations depend both on the degree of domestication and the original source of the domesticated population. Atlantic salmon, Salmo salar, used in North American aquaculture are ostensibly of North American origin. However, evidence of European introgression into North American aquaculture salmon has accumulated in recent decades, even though the use of diploid European salmon has never been approved in Canada. The full extent of such introgression as well as the potential impacts on wild salmon in the Northwest Atlantic remains uncertain. Here, we extend previous work comparing North American and European wild salmon (n = 5799) using a 220 K SNP array to quantify levels of recent European introgression into samples of domestic salmon, aquaculture escapees, and wild salmon collected throughout Atlantic Canada. Analysis of North American farmed salmon (n = 403) and escapees (n = 289) displayed significantly elevated levels of European ancestry by comparison with wild individuals (p < 0.001). Of North American farmed salmon sampled between 2011 and 2018, ~17% had more than 10% European ancestry and several individuals exceeded 40% European ancestry. Samples of escaped farmed salmon similarly displayed elevated levels of European ancestry, with two individuals classified as 100% European. Analysis of juvenile salmon collected in rivers proximate to aquaculture locations also revealed evidence of elevated European ancestry and larger admixture tract in comparison to individuals collected at distance from aquaculture. Overall, our results demonstrate that even though diploid European salmon have never been approved for use in Canada, individuals of full and partial European ancestry have been in use over the last decade, and that some of these individuals have escaped and hybridized in the wild.

Distinct early life stage gene expression effects of hybridization among European and North American farmed and wild Atlantic salmon populations.

Due to multigeneration domestication selection, farmed and wild Atlantic salmon diverge genetically, which raises concerns about potential genetic interactions among escaped farmed and wild populations and disruption of local adaptation through introgression. When farmed strains of distant geographic origin are used, it is unknown whether the genetic consequences posed by escaped farmed fish will be greater than if more locally derived strains are used. Quantifying gene transcript expression differences among divergent farmed, wild and F1  hybrids under controlled conditions is one of the ways to explore the consequences of hybridization. We compared the transcriptomes of fry at the end of yolk sac absorption of a European (EO) farmed ("StofnFiskur", Norwegian strain), a North American (NA) farmed (Saint John River, NB strain), a Newfoundland (NF) wild population with EO ancestry, and related F1  hybrids using 44 K microarrays. Our findings indicate that the wild population showed greater transcriptome differences from the EO farmed strain than that of the NA farmed strain. We also found the largest differences in global gene expression between the two farmed strains. We detected the fewest differentially expressed transcripts between F1  hybrids and domesticated/wild maternal strains. We also found that the differentially expressed genes between cross types over-represented GO terms associated with metabolism, development, growth, immune response, and redox homeostasis processes. These findings suggest that the interbreeding of escaped EO/NA farmed and NF wild population would alter gene transcription, and the consequences of hybridization would be greater from escaped EO farmed than NA farmed salmon, resulting in potential effects on the wild populations.

Characterization of ADAT2/3 molecules in Trypanosoma cruzi and regulation of mucin gene expression by tRNA editing.

Adenosine-to-inosine conversion at position 34 (A34-to-I) of certain tRNAs is essential for expanding their decoding capacity. This reaction is catalyzed by the adenosine deaminase acting on tRNA (ADAT) complex, which in Eukarya is formed by two subunits: ADAT2 and ADAT3. We herein identified and thoroughly characterized the ADAT molecules from the protozoan pathogen Trypanosoma cruzi, the causative agent of Chagas Disease. TcADAT2 and TcADAT3 spontaneously form a catalytically active complex, as shown by expression in engineered bacteria and/or by the increased ex vivo tRNA A-to-I deamination activity of T. cruzi epimastigotes overexpressing TcADAT subunits. Importantly, enhanced TcADAT2/3 activity in transgenic parasites caused a shift in their in vivo tRNAThrAGU signature, which correlated with significant changes in the expression of the Thr-rich TcSMUG proteins. To our knowledge, this is the first evidence indicating that T. cruzi tRNA editing can be modulated in vivo, in turn post-transcriptionally changing the expression of specific genes. Our findings suggest tRNA editing/availability as a forcible step in controlling gene expression and driving codon adaptation in T. cruzi. Moreover, we unveil certain differences between parasite and mammalian host tRNA editing and processing, such as cytosine-to-uridine conversion at position 32 of tRNAThrAGU in T. cruzi, that may be exploited for the identification of novel druggable targets of intervention.

Enhancing the utility of virtual surgical pre-assessment.

During the COVID-19 pandemic, virtual pre-assessment was introduced for all elective and semi-urgent surgeries to maintain surgical clinical services in the authors' Trust. This mainly involved telephone pre-assessments, although occasionally video technology was used. This had to be managed and maintained at a distance with little or no training or established method. This article includes experiences of staff involved in a single tertiary centre, an assessment of the pros and cons of virtual pre-assessment and concludes with a set of recommendations to enhance the utility of the service for the future.

Growth-enhanced salmon modify stream ecosystem functioning.

Use of fast-growing domesticated and/or genetically modified strains of fish is becoming increasingly common in aquaculture, increasing the likelihood of deliberate or accidental introductions into the wild. To date, their ecological impacts on ecosystems remain to be quantified. Here, using a controlled phenotype manipulation by implanting growth hormone in juvenile Atlantic salmon (Salmo salar), we found that growth-enhanced fish display changes in several phenotypic traits known to be important for ecosystem functioning, such as habitat use, morphology and excretion rate. Furthermore, these phenotypic changes were associated with significant impacts on the invertebrate community and key stream ecosystem functions such as primary production and leaf-litter decomposition. These findings provide novel evidence that introductions of growth-enhanced fish into the wild can affect the functioning of natural ecosystems and represent a form of intraspecific invasion. Consequently, environmental impact assessments of growth-enhanced organisms need to explicitly consider ecosystem-level effects.

Clinical value of 3'-deoxy-3'-[18F]fluorothymidine-positron emission tomography for diagnosis, staging and assessing therapy response in lung cancer.

Lung cancer has the highest mortality rate of any tumour type. The main driver of lung tumour growth and development is uncontrolled cellular proliferation. Poor patient outcomes are partly the result of the limited range of effective anti-cancer therapies available and partly due to the limited accuracy of biomarkers to report on cell proliferation rates in patients. Accordingly, accurate methods of diagnosing, staging and assessing response to therapy are crucial to improve patient outcomes. One effective way of assessing cell proliferation is to employ non-invasive evaluation using 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) positron emission tomography [18F]FLT-PET. [18F]FLT, unlike the most commonly used PET tracer [18F]fluorodeoxyglucose ([18F]FDG), can specifically report on cell proliferation and does not accumulate in inflammatory cells. Therefore, this radiotracer could exhibit higher specificity in diagnosis and staging, along with more accurate monitoring of therapy response at early stages in the treatment cycle. This review summarises and evaluates published studies on the clinical use of [18F]FLT to diagnose, stage and assess response to therapy in lung cancer.

Widespread tissue hypoxia dysregulates cell and metabolic pathways in SMA.

OBJECTIVE: The purpose of the study was to determine the extent and role of systemic hypoxia in the pathogenesis of spinal muscular atrophy (SMA). METHODS: Hypoxia was assayed in vivo in early-symptomatic (postnatal day 5) SMA-model mice by pimonidazole and [18 F]-Fluoroazomycin arabinoside injections, which accumulate in hypoxic cells, followed by immunohistochemistry and tracer biodistribution evaluation. Glucose uptake in hypoxic cells was assayed by [18 F]-Fluorodeoxyglucose labeling. In vitro knockdown of Survival Motor Neuron (SMN) was performed on motor neurons and lactate metabolism measured biochemically, whereas cell cycle progression and cell death were assayed by flow cytometry. RESULTS: All assays found significant levels of hypoxia in multiple organ systems in early symptomatic SMA mouse pups, except aerated tissues such as skin and lungs. This was accompanied by significantly increased glucose uptake in many affected organs, consistent with a metabolic hypoxia response. SMN protein levels were shown to vary widely between motor neuron precursors in vitro, and those with lower levels were most susceptible to cell death. In addition, SMA-model motor neurons were particularly sensitive to hypoxia, with reduced ability to transport lactate out of the cell in hypoxic culture, and a failure in normal cell cycle progression. INTERPRETATION: Not only is there widespread tissue hypoxia and multi-organ cellular hypoxic response in SMA model mice, but SMA-model motor neurons are especially susceptible to that hypoxia. The data support the hypothesis that vascular defects leading to hypoxia are a significant contributor to disease progression in SMA, and offer a route for combinatorial, non-SMN related therapy.

Evaluating the effect of dorsal muscle biopsies on adult Atlantic salmon growth and marine return rates.

Increasing conservation and animal-welfare concerns have driven the development of non-lethal sampling of fish populations, with the use of muscle tissue biopsies now being routinely applied as a sampling method in the wild. Crucial to the success of non-lethal sampling, however, is an evaluation of the short- and long-term consequences of the treatment and ultimately the determination of how these may affect organism mortality and other fitness-related traits. The current study evaluated the use of a dorsal muscle biopsies on post-spawned Atlantic salmon emigrating to sea and undertaking a 2-month long-feeding migration before returning to spawn. Using mark-recapture, return rates and growth were compared between fish that were biopsied and externally tagged, and a control group tagged only with external tags. The biopsy treatment showed no lasting effects on fish as estimated from the two key fitness-related parameters. Results, therefore, suggest the technique can be more widely applied to gather information on marine migrating Atlantic salmon and other anadromous fishes that can be intercepted as they descend and ascend rivers during seasonal migrations. Coupled with modern tagging technologies, the use of biopsies may facilitate an improved understanding of movement and its consequences in terms of feeding patterns and growth.

A role for spinal cord hypoxia in neurodegeneration.

The vascular system of the spinal cord is particularly complex and vulnerable. Damage to the main vessels or alterations to the regulation of blood flow will result in a reduction or temporary cessation of blood supply. The resulting tissue hypoxia may be brief: acute, or long lasting: chronic. Damage to the vascular system of the spinal cord will develop after a traumatic event or as a result of pathology. Traumatic events such as road traffic accidents, serious falls and surgical procedures, including aortic cross-clamping, will lead to an immediate cessation of perfusion, the result of which may not be evident for several days, but may have long-term consequences including neurodegeneration. Pathological events such as arterial sclerosis, venous occlusion and spinal cord compression will result in a progressive reduction of blood flow, leading to chronic hypoxia. While in some situations the initial pathology is exclusively vascular, recent research in neurodegenerative disease has drawn attention to concomitant vascular anomalies in disorders, including amyotrophic lateral sclerosis, spinal muscular atrophy and muscular sclerosis. Understanding the role of, and tissue response to, chronic hypoxia is particularly important in these cases, where inherent neural damage exacerbates the vulnerability of the nervous system to stressors including hypoxia.

Estimating the relative fitness of escaped farmed salmon offspring in the wild and modelling the consequences of invasion for wild populations.

Throughout their native range, wild Atlantic salmon populations are threatened by hybridization and introgression with escapees from net-pen salmon aquaculture. Although domestic-wild hybrid offspring have shown reduced fitness in laboratory and field experiments, consequential impacts on population abundance and genetic integrity remain difficult to predict in the field, in part because the strength of selection against domestic offspring is often unknown and context-dependent. Here, we follow a single large escape event of farmed Atlantic salmon in southern Newfoundland and monitor changes in the in-river proportions of hybrids and feral individuals over time using genetically based hybrid identification. Over a three-year period following the escape, the overall proportion of wild parr increased consistently (total wild proportion of 71.6%, 75.1% and 87.5% each year, respectively), with subsequent declines in feral (genetically pure farmed individuals originating from escaped, farmed adults) and hybrid parr. We quantify the strength of selection against parr of aquaculture ancestry and explore the genetic and demographic consequences for populations in the region. Within-cohort changes in the relative proportions of feral and F1 parr suggest reduced relative survival compared to wild individuals over the first (0.15 and 0.81 for feral and F1, respectively) and second years of life (0.26, 0.83). These relative survivorship estimates were used to inform an individual-based salmon eco-genetic model to project changes in adult abundance and overall allele frequency across three invasion scenarios ranging from short-term to long-term invasion and three relative survival scenarios. Modelling results indicate that total population abundance and time to recovery were greatly affected by relative survivorship and predict significant declines in wild population abundance under continued large escape events and calculated survivorship. Overall, this work demonstrates the importance of estimating the strength of selection against domestic offspring in the wild to predict the long-term impact of farmed salmon escape events on wild populations.

Can variation in standard metabolic rate explain context-dependent performance of farmed Atlantic salmon offspring?

Escaped farmed Atlantic salmon interbreed with wild Atlantic salmon, leaving offspring that often have lower success in nature than pure wild salmon. On top of this, presence of farmed salmon descendants can impair production of wild-type recruits. We hypothesize that both these effects connect with farmed salmon having acquired higher standard metabolic rates (SMR, the energetic cost of self-maintenance) during domestication. Fitness-related advantages of phenotypic traits associated with both high SMR and farmed salmon (e.g., social dominance) depend on environmental conditions, such as food availability. We hypothesize that farmed offspring have an advantage at high food availability due to, for example, dominance behavior but suffer increased risks of starvation when food is scarce because this behavior is energy-demanding. To test these hypotheses, we first compare embryo SMR of pure farmed, farmed-wild hybrids and pure wild offspring. Next, we test early-life performance (in terms of survival and growth) of hybrids relative to that of their wild half-siblings, as well as their competitive abilities, in semi-natural conditions of high and low food availability. Finally, we test how SMR affects early-life performance at high and low food availability. We find inconclusive support for the hypothesis that domestication has induced increased SMR. Further, wild and hybrid juveniles had similar survival and growth in the semi-natural streams. Yet, the presence of hybrids led to decreased survival of their wild half-siblings. Contrary to our hypothesis about context-dependency, these effects were not modified by food availability. However, wild juveniles with high SMR had decreased survival when food was scarce, but there was no such effect at high food availability. This study provides further proof that farmed salmon introgression may compromise the viability of wild salmon populations. We cannot, however, conclude that this is connected to alterations in the metabolic phenotype of farmed salmon.

Enzymatic radiosynthesis of a 18F-Glu-Ureido-Lys ligand for the prostate-specific membrane antigen (PSMA).

Prostate cancer represents a major public health threat as it is one of the most common male cancers worldwide. The prostate-specific membrane antigen (PSMA) is highly over-expressed in prostatic cancer cells in a manner that correlates with both tumour stage and clinical outcome. As such, PSMA has been identified as an attractive target for both imaging and treatment of prostate cancer. In recent years the focus on urea-based peptidomimetic inhibitors of the PSMA (representing low molecular weight/high affinity binders) has intensified as they have found use in the clinical imaging of prostate tumours. Reported herein are the design, synthesis and evaluation of a new fluorinated PSMA targeting small-molecule, FDA-PEG-GUL, which possesses the Glu-NH-CO-NH-Lys pharmacophore conjugated to a 5'-fluorodeoxy-adenosine unit. Inhibition assays were performed with FDA-PEG-GUL which revealed that it inhibits the PSMA in the nanomolar range. Additionally, it has been purposely designed so that it can be produced using the fluorinase enzyme from its chlorinated precursor, allowing for the enzymatic synthesis of radiolabelled [18F]FDA-PEG-GUL via a nucleophilic reaction that takes place in experimentally advantageous conditions (in water at neutral pH and at ambient temperature). Specific binding of [18F]FDA-PEG-GUL to PSMA expressing cancer cells was demonstrated, validating it as a promising PSMA diagnostic tool. This work establishes a successful substrate scope expansion for the fluorinase and demonstrates its first application towards targeting the PSMA.

Planning for spectator thermal comfort and health in the face of extreme heat: The Tokyo 2020 Olympic marathons.

The 2020 Olympic Games marathon will be run through the streets of Tokyo on the mornings of August 2nd and 9th, a time of year that is typically hot, sunny, and humid. Few studies have assessed the potential impact of extreme heat along the marathon course to understand the multiple factors (e.g., radiation, wind flow) affecting human thermal comfort (TC) as influenced by urban design and vegetation. The current research establishes a baseline of microclimate conditions and scenarios to estimate the projected TC along the marathon route for spectators. Mobile microclimate data (air and surface temperatures, solar radiation, humidity, wind speed) were collected along the marathon course over 15 periods in the summer of 2016 and aligned with sky view factors (SVF). Human energy budget modeling was applied to provide spatially-explicit heat budget and TC information along the route. Conditions are expected to create the most discomfort along open, sun-exposed locations, with ~50% of the area along the second half of the course resulting in 'Hot' (budget > 200 W m-2) or 'Very hot' (budget > 295 W m-2) conditions. The heat strain index frequently rises above 80% in these locations, with high humidity and low wind flow exacerbating discomfort. Buildings and trees producing a low SVF over roads and sidewalks protect spectators from the morning radiant heat, but such locations should be balanced with wind flow to optimize comfort. The modeling and spatial information can aid in preparing for and mitigating heat stress during the Olympics. Potential solutions can be implemented in collaboration with local organizers and government. This 'research through design' strategy can aid in preparing for and mitigating heat illness during the Olympics. Knowledge gained can be extended to other areas of Tokyo to reduce urban heat, and further provide targeted guidance for effective environmental cooling techniques for human health.

Extensive hybridization following a large escape of domesticated Atlantic salmon in the Northwest Atlantic.

Domestication is rife with episodes of interbreeding between cultured and wild populations, potentially challenging adaptive variation in the wild. In Atlantic salmon, Salmo salar, the number of domesticated individuals far exceeds wild individuals, and escape events occur regularly, yet evidence of the magnitude and geographic scale of interbreeding resulting from individual escape events is lacking. We screened juvenile Atlantic salmon using 95 single nucleotide polymorphisms following a single, large aquaculture escape in the Northwest Atlantic and report the landscape-scale detection of hybrid and feral salmon (27.1%, 17/18 rivers). Hybrids were reproductively viable, and observed at higher frequency in smaller wild populations. Repeated annual sampling of this cohort revealed decreases in the presence of hybrid and feral offspring over time. These results link previous observations of escaped salmon in rivers with reports of population genetic change, and demonstrate the potential negative consequences of escapes from net-pen aquaculture on wild populations.

Characterization of Athabasca lean oil sands and mixed surficial materials: Comparison of capillary electrophoresis/low-resolution mass spectrometry and high-resolution mass spectrometry.

RATIONALE: Oil sands mining in Alberta, Canada, requires removal and stockpiling of considerable volumes of near-surface overburden material. This overburden includes lean oil sands (LOS) which cannot be processed economically but contain sparingly soluble petroleum hydrocarbons and naphthenic acids, which can leach into environmental waters. In order to measure and track the leaching of dissolved constituents and distinguish industrially derived organics from naturally occurring organics in local waters, practical methods were developed for characterizing multiple sources of contaminated water leakage. METHODS: Capillary electrophoresis/positive-ion electrospray ionization low-resolution time-of-flight mass spectrometry (CE/LRMS), high-resolution negative-ion electrospray ionization Orbitrap mass spectrometry (HRMS) and conventional gas chromatography/flame ionization detection (GC/FID) were used to characterize porewater samples collected from within Athabasca LOS and mixed surficial materials. GC/FID was used to measure total petroleum hydrocarbon and HRMS was used to measure total naphthenic acid fraction components (NAFCs). HRMS and CE/LRMS were used to characterize samples according to source. RESULTS: The amounts of total petroleum hydrocarbon in each sample as measured by GC/FID ranged from 0.1 to 15.1 mg/L while the amounts of NAFCs as measured by HRMS ranged from 5.3 to 82.3 mg/L. Factors analysis (FA) on HRMS data visually demonstrated clustering according to sample source and was correlated to molecular formula. LRMS coupled to capillary electrophoresis separation (CE/LRMS) provides important information on NAFC isomers by adding analyte migration time data to m/z and peak intensity. CONCLUSIONS: Differences in measured amounts of total petroleum hydrocarbons by GC/FID and NAFCs by HRMS indicate that the two methods provide complementary information about the nature of dissolved organic species in a soil or water leachate samples. NAFC molecule class Ox Sy is a possible tracer for LOS seepage. CE/LRMS provides complementary information and is a feasible and practical option for source evaluation of NAFCs in water.

Binding of αvß3 Integrin-Specific Radiotracers Is Modulated by Both Integrin Expression Level and Activation Status.

PURPOSE: Molecular imaging of αvß3 integrin has exhibited real potential to guide the appropriate use of anti-angiogenic therapies. However, an incomplete understanding of the factors that influence binding of αvß3 integrin-specific radiotracers currently limits their use for assessing response to therapy in cancer patients. This study identifies two fundamental factors that modulate uptake of these radiotracers. Procedures Experiments were performed in prostate cancer (PC3) and glioblastoma (U87MG) cells, which differentially express αvß3 integrin. αvß3 integrin-specific radiotracers were used to investigate the effect of manipulating αvß3 integrin expression or activation in cellular binding assays. ß3 integrin and αvß3 integrin expression were measured by western blotting and flow cytometry, respectively. The effect of select pharmacological inhibitors on αvß3 integrin activation and expression was also determined. RESULTS: Radiotracer binding was proportional to αvß3 integrin expression when it was decreased (ß3 knock-down cells) or increased, either using pharmacological inhibitors of cell signalling or by culturing cells for different times. Studies with both small molecule and arginine-glycine-aspartic acid (RGD)-based radiotracers revealed increased radiotracer binding after activation of αvß3 integrin with Mn2+ or talin head domain. Moreover, inhibition of fundamental signalling pathways (mitogen-activated protein kinase kinase (MEK), Src and VEGFR2) decreased radiotracer binding, reflecting reduced αvß3 integrin activity. CONCLUSION: Binding of small molecule ligands and radiolabelled RGD peptides is modulated by expression and activation status of αvß3 integrin. αvß3 integrin-specific radiotracers can provide otherwise inaccessible information of the effect of signalling pathways on αvß3 integrin. This has significant implications for assessing response to anti-angiogenic therapies in clinical studies.

KEMTUB012-NI2, a novel potent tubulysin analog that selectively targets hypoxic cancer cells and is potentiated by cytochrome p450 reductase downregulation.

PURPOSE: There is an urgent need to develop effective therapies and treatment strategies to treat hypoxic tumors, which have a very poor prognosis and do not respond well to existing therapies. METHODS: A novel hypoxia-targeting agent, KEMTUB012-NI2, was synthesized by conjugating a 2-nitroimidazole hypoxia-targeting moiety to a synthetic tubulysin, a very potent antimitotic. Its hypoxic selectivity and mode of action were studied in breast cancer cell lines. RESULTS: KEMTUB012-NI2 exhibited a similar selectivity for hypoxic cells to that of tirapazamine, a well-established hypoxia-targeting agent, but was >1,000 times more potent in cell cytotoxicity assays. The hypoxia-targeting mechanism for both KEMTUB012-NI2 and tirapazamine was selective and mediated by one-electron reductases. However, while cytochrome p450 reductase (POR) downregulation could inhibit tirapazamine cytotoxicity, it actually sensitized hypoxic cells to KEMTUB012-NI2. CONCLUSION: KEMTUB012-NI2 is a potent new agent that can selectively target hypoxic cancer cells. The hypoxia selectivity of KEMTUB012-NI2 and tirapazamine appears to be differentially activated by reductases. Since reductases are heterogeneously expressed in tumors, the different activation mechanisms will allow these agents to complement each other. Combining POR downregulation with KEMTUB012-NI2 treatment could be a new treatment strategy that maximizes efficacy toward hypoxic tumor cells while limiting systemic toxicity.

High-Affinity "Click" RGD Peptidomimetics as Radiolabeled Probes for Imaging αv ß3 Integrin.

Nonpeptidic Arg-Gly-Asp (RGD)-mimic ligands were designed and synthesized by click chemistry between an arginine-azide mimic and an aspartic acid-alkyne mimic. Some of these molecules combine excellent in vitro properties (high αv ß3 affinity, selectivity, drug-like logD, high metabolic stability) with a variety of radiolabeling options (e.g., tritium and fluorine-18, plus compatibility with radio-iodination), not requiring the use of chelators or prosthetic groups. The binding mode of the resulting triazole RGD mimics to αv ß3 or αIIb ß3 receptors was investigated by molecular modeling simulations. Lead compound 12 was successfully radiofluorinated and used for in vivo positron emission tomography/computed tomography (PET/CT) studies in U87 tumor models, which showed only modest tumor uptake and retention, owing to rapid excretion. These results demonstrate that the novel click RGD mimics are excellent radiolabeled probes for in vitro and cell-based studies on αv ß3 integrin, whereas further optimization of their pharmacokinetic and dynamic profiles is necessary for successful use in in vivo imaging.