Complete Remission of BCG-Refractory High-grade Bladder CIS with Pharmacologic Ascorbate and Mistletoe.

Context: Bladder cancer is the fourth-most-common cancer in males in the U.S., who develop about 90% of the high-grade, carcinoma in situ (CIS) of non-muscle involved disease (NMIBC). Smoking and occupational carcinogens are well-known causes. For females without known risk factors, bladder cancer can be regarded as a sentinel environmental cancer. It's also one of the costliest to treat due to its high rate of recurrence. No treatment innovations have occurred in nearly two decades; intravesical instillation of Bacillus Calmette-Guerin (BCG), an agent in short supply globally, or Mitomycin-C (MIT-C) is effective in about 60% of cases. Cases refractory to BCG and MIT-C often undergo cystectomy, a procedure with numerous impacts on life styles and potential complications. The recent completion of a small Phase I trial of mistletoe in cancer patients that have exhausted known treatments at Johns Hopkins provides corroboration of its safety, with 25 % showing no disease progression. Objective: The study examined the benefits of pharmacologic ascorbate (PA) and mistletoe for a nonsmoking female patient with an environmental history of NMIBC refractory to BCG, in a non-smoking female with exposures in childhood and early adult life to several known carcinogens, including ultrafine particulate air pollution, benzene, toluene, and other organic solvents, aromatic amines and engine exhausts, and possibly arsenic in water. Design: The research team performed an integrative oncology case study on pharmacologic ascorbate (PA) and mistletoe, both agents shown to activate NK cells, enhance growth and maturation of T-cells, and induce dose-dependent pro-apoptotic cell death, suggesting shared and potentially synergistic mechanisms. Setting: The study began at the University of Ottawa Medical Center in Canada with treatment continuing over six years at St. Johns Hospital Center in Jackson, Wyoming, and George Washington University Medical Center for Integrative Medicine, with surgical, cytological, and pathological evaluations at University of California San Francisco Medical Center. Participant: The patient in the case study was a 76-year-old, well-nourished, athletic, nonsmoking female with high-grade CIS of the bladder. Her cancer was considered to be a sentinel environmental cancer. Intervention: Intravenous pharmacologic ascorbate (PA) and subcutaneous mistletoe (three times weekly) and intravenous and intravesical mistletoe (once weekly) were employed for an 8-week induction treatment, using a dose-escalation protocol as detailed below. Maintenance therapy was carried out with the same protocol for three weeks every three months for two years. Results: The patient has experienced a cancer-free outcome following 78 months of treatments that incorporated intravesical, intravenous, and subcutaneous mistletoe; intravenous PA; a program of selected nutraceuticals; exercise; and other supplementary treatments. Conclusions: This study is the first reported instance of combined treatments to achieve complete remission for high-grade NMIBC refractory to BCG and MIT-C, using intravesical, subcutaneous, and intravenous mistletoe and intravenous PA. It includes pharmacological information on possible mechanisms. In light of the global shortage of BCG, the high proportion of cases refractory to BCG and MIT-C, the unproven use of costly off-label pharmaceuticals, such as gemcitabine, and the relative cost-effectiveness of mistletoe and PA, clinicians should give serious consideration to employing these combined functional medicine treatments for BCG- and MIT-C-refractory NMIBC. Further research is needed with additional patients that can advance our understanding, including standardization of methods for systematically evaluating combined therapies-blinded and non-blinded, nomenclature regarding mistletoe preparation, doses, concentrations, regimes of administration, lengths of treatment, targeted cancer types, and other aspects.

4'-Fluoro-nucleosides and nucleotides: from nucleocidin to an emerging class of therapeutics.

The history and development of 4'-fluoro-nucleosides is discussed in this review. This is a class of nucleosides which have their origin in the discovery of the rare fluorine containing natural product nucleocidin. Nucleocidin contains a fluorine atom located at the 4'-position of its ribose ring. From its early isolation as an unexpected natural product, to its total synthesis and bioactivity assessment, nucleocidin has played a role in inspiring the exploration of 4'-fluoro-nucleosides as a privileged motif for nucleoside-based therapeutics.

Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro.

The fluorinase enzyme represents the only biological mechanism capable of forming stable C-F bonds characterized in nature thus far, offering a biotechnological route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis constant (KM ) for S-adenosyl-l-methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-l-methionine affinity points to a long-range effect of hexamerization on substrate binding - likely decreasing substrate dissociation and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system.

Small herbaria contribute unique biogeographic records to county, locality, and temporal scales.

PREMISE: With digitization and data sharing initiatives underway over the last 15 years, an important need has been prioritizing specimens to digitize. Because duplicate specimens are shared among herbaria in exchange and gift programs, we investigated the extent to which unique biogeographic data are held in small herbaria vs. these data being redundant with those held by larger institutions. We evaluated the unique specimen contributions that small herbaria make to biogeographic understanding at county, locality, and temporal scales. METHODS: We sampled herbarium specimens of 40 plant taxa from each of eight states of the United States of America in four broad status categories: extremely rare, very rare, common native, and introduced. We gathered geographic information from specimens held by large (≥100,000 specimens) and small (<100,000 specimens) herbaria. We built generalized linear mixed models to assess which features of the collections may best predict unique contributions of herbaria and used an Akaike information criterion-based information-theoretic approach for our model selection to choose the best model for each scale. RESULTS: Small herbaria contributed unique specimens at all scales in proportion with their contribution of specimens to our data set. The best models for all scales were the full models that included the factors of species status and herbarium size when accounting for state as a random variable. CONCLUSIONS: We demonstrated that small herbaria contribute unique information for research. It is clear that unique contributions cannot be predicted based on herbarium size alone. We must prioritize digitization and data sharing from herbaria of all sizes.

A fluoride-responsive genetic circuit enables in vivo biofluorination in engineered Pseudomonas putida.

Fluorine is a key element in the synthesis of molecules broadly used in medicine, agriculture and materials. Addition of fluorine to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to integrate fluorometabolites into the biochemistry of living cells are scarce. In this work, synthetic gene circuits for organofluorine biosynthesis are implemented in the platform bacterium Pseudomonas putida. By harnessing fluoride-responsive riboswitches and the orthogonal T7 RNA polymerase, biochemical reactions needed for in vivo biofluorination are wired to the presence of fluoride (i.e. circumventing the need of feeding expensive additives). Biosynthesis of fluoronucleotides and fluorosugars in engineered P. putida is demonstrated with mineral fluoride both as only fluorine source (i.e. substrate of the pathway) and as inducer of the synthetic circuit. This approach expands the chemical landscape of cell factories by providing alternative biosynthetic strategies towards fluorinated building-blocks.

An Engineered E.†coli Strain for Direct in Vivo Fluorination.

Selectively fluorinated compounds are found frequently in pharmaceutical and agrochemical products where currently 25-30 % of optimised compounds emerge from development containing at least one fluorine atom. There are many methods for the site-specific introduction of fluorine, but all are chemical and they often use environmentally challenging reagents. Biochemical processes for C-F bond formation are attractive, but they are extremely rare. In this work, the fluorinase enzyme, originally identified from the actinomycete bacterium Streptomyces cattleya, is engineered into Escherichia coli in such a manner that the organism is able to produce 5'-fluorodeoxyadenosine (5'-FDA) from S-adenosyl-l-methionine (SAM) and fluoride in live E. coli cells. Success required the introduction of a SAM transporter and deletion of the endogenous fluoride efflux capacity in order to generate an E. coli host that has the potential for future engineering of more elaborate fluorometabolites.

An enzymatic Finkelstein reaction: fluorinase catalyses direct halogen exchange.

The fluorinase enzyme from Streptomyces cattleya is shown to catalyse a direct displacement of bromide and iodide by fluoride ion from 5'-bromodeoxyadenosine (5'-BrDA) and 5'-iododeoxyadenosine (5'-IDA) respectively to form 5'-fluorodeoxyadenosine (5'-FDA) in the absence of l-methionine (l-Met) or S-adenosyl-l-methionine (SAM). 5'-BrDA is the most efficient substrate for this enzyme catalysed Finkelstein reaction.

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.

Two 3'-O-ß-glucosylated nucleoside fluorometabolites related to nucleocidin in Streptomyces calvus.

The antibiotic nucleocidin is a product of the soil bacterium Streptomyces calvus T-3018. It is among the very rare fluorine containing natural products but is distinct from the other fluorometabolites in that it is not biosynthesised from 5'-fluorodeoxyadenosine via the fluorinase. It seems to have a unique enzymatic fluorination process. We disclose here the structures of two 4'-fluoro-3'-O-ß-glucosylated metabolites (F-Mets I and II) which appear and then disappear before nucleocidin production in batch cultures of S. calvus. Full genome sequencing of S. calvus T-3018 and an analysis of the putative biosynthetic gene cluster for nucleocidin identified UDP-glucose dependent glucosyl transferase (nucGT) and glucosidase (nucGS) genes within the cluster. We demonstrate that these genes express enzymes that have the capacity to attach and remove glucose from the 3'-O-position of adenosine analogues. In the case of F-Met II, deglucosylation with the NucGS glucosidase generates nucleocidin suggesting a role in its biosynthesis. Gene knockouts of nucGT abolished nucelocidin production.

Enzymatic Fluorination of Biotin and Tetrazine Conjugates for Pretargeting Approaches to Positron Emission Tomography Imaging.

The use of radiolabelled antibodies and antibody-derived recombinant constructs has shown promise for both imaging and therapeutic use. In this context, the biotin-avidin/streptavidin pairing, along with the inverse-electron-demand Diels-Alder (iEDDA) reaction, have found application in pretargeting approaches for positron emission tomography (PET). This study reports the fluorinase-mediated transhalogenation [5'-chloro-5'-deoxyadenosine (ClDA) substrates to 5'-fluoro-5'-deoxyadenosine (FDA) products] of two antibody pretargeting tools, a FDA-PEG-tetrazine and a [18 F]FDA-PEG-biotin, and each is assessed either for its compatibility towards iEDDA ligation to trans-cyclooctene or for its affinity to avidin. A protocol to avoid radiolytically promoted oxidation of biotin during the synthesis of [18 F]FDA-PEG-biotin was developed. The study adds to the repertoire of conjugates for use in fluorinase-catalysed radiosynthesis for PET and shows that the fluorinase will accept a wide range of ClDA substrates tethered at C-2 of the adenine ring with a PEGylated cargo. The method is exceptional because the nucleophilic reaction with [18 F]fluoride takes place in water at neutral pH and at ambient temperature.

A New Class of Fluorinated A2A Adenosine Receptor Agonist with Application to Last-Step Enzymatic [18 F]Fluorination for PET Imaging.

The A2A adenosine receptor belongs to a family of G-coupled protein receptors that have been subjected to extensive investigation over the last few decades. Due to their prominent role in the biological functions of the heart, lungs, CNS and brain, they have become a target for the treatment of illnesses ranging from cancer immunotherapy to Parkinson's disease. The imaging of such receptors by using positron emission tomography (PET) has also been of interest, potentially providing a valuable tool for analysing and diagnosing various myocardial and neurodegenerative disorders, as well as offering support to drug discovery trials. Reported herein are the design, synthesis and evaluation of two new 5'-fluorodeoxy-adenosine (FDA)-based receptor agonists (FDA-PP1 and FDA-PP2), each substituted at the C-2 position with a terminally functionalised ethynyl unit. The structures enable a synthesis of 18 F-labelled analogues by direct, last-step radiosynthesis from chlorinated precursors using the fluorinase enzyme (5'-fluoro-5'-deoxyadenosine synthase), which catalyses a transhalogenation reaction. This delivers a new class of A2A adenosine receptor agonist that can be directly radiolabelled for exploration in PET studies.

Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch.

Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ1 riboswitch from Bacillus subtilis into an orthogonal OFF-switch. In this case, the evaluation of just six synthetic compounds with seven riboswitch mutants led to the identification of an orthogonal riboswitch-ligand pairing that effectively repressed the transcription of selected genes in B. subtilis. The streamlining of the re-engineering approach, and its extension to a second class of riboswitches, provides a methodological platform for the creation of new orthogonal regulatory components for biotechnological applications including gene functional analysis and antimicrobial target validation and screening.

Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species.

Ligand-dependent control of gene expression is essential for gene functional analysis, target validation, protein production, and metabolic engineering. However, the expression tools currently available are difficult to transfer between species and exhibit limited mechanistic diversity. Here we demonstrate how the modular architecture of purine riboswitches can be exploited to develop orthogonal and chimeric switches that are transferable across diverse bacterial species, modulating either transcription or translation, to provide tunable activation or repression of target gene expression, in response to synthetic non-natural effector molecules. Our novel riboswitch-ligand pairings are shown to regulate physiologically important genes required for bacterial motility in Escherichia coli and cell morphology in Bacillus subtilis. These findings are relevant for future gene function studies and antimicrobial target validation, while providing new modular and orthogonal regulatory components for deployment in synthetic biology regimes.

Empirical models of transitions between coral reef states: effects of region, protection, and environmental change.

There has been substantial recent change in coral reef communities. To date, most analyses have focussed on static patterns or changes in single variables such as coral cover. However, little is known about how community-level changes occur at large spatial scales. Here, we develop Markov models of annual changes in coral and macroalgal cover in the Caribbean and Great Barrier Reef (GBR) regions. We analyzed reef surveys from the Caribbean and GBR (1996-2006). We defined a set of reef states distinguished by coral and macroalgal cover, and obtained Bayesian estimates of the annual probabilities of transitions between these states. The Caribbean and GBR had different transition probabilities, and therefore different rates of change in reef condition. This could be due to differences in species composition, management or the nature and extent of disturbances between these regions. We then estimated equilibrium probability distributions for reef states, and coral and macroalgal cover under constant environmental conditions. In both regions, the current distributions are close to equilibrium. In the Caribbean, coral cover is much lower and macroalgal cover is higher at equilibrium than in the GBR. We found no evidence for differences in transition probabilities between the first and second halves of our survey period, or between Caribbean reefs inside and outside marine protected areas. However, our power to detect such differences may have been low. We also examined the effects of altering transition probabilities on the community state equilibrium, along a continuum from unfavourable (e.g., increased sea surface temperature) to favourable (e.g., improved management) conditions. Both regions showed similar qualitative responses, but different patterns of uncertainty. In the Caribbean, uncertainty was greatest about effects of favourable changes, while in the GBR, we are most uncertain about effects of unfavourable changes. Our approach could be extended to provide risk analysis for management decisions.

Applications of tailored ferrocenyl molecules as electrochemical probes of biochemical interactions.

The development of electrochemical probes useful for investigating the occupancy by other molecules of sites on complex proteins such as human serum albumin (HSA) is described. Ferrocenyl-(oxoethylene)-fatty acid compounds of different fatty acid chain length probed different binding sites on HSA. The interaction could be changed from one primarily with a drug binding site, when the probe was ferrocene methanol, to one predominantly with medium-chain fatty acid binding sites, by adding an (oxoethylene)-fatty acid substituents. Finally, the interaction could be changed to one interacting primarily with high-affinity long-chain fatty acid binding sites, as the fatty acid chain length in ferrocene-(oxoethylene)-fatty acid molecules increased. These results strongly implied that the binding could be further tailored by relatively simple modifications to the probe, for example, by changing the balance of hydrophobicity and hydrophilicity. The possibility of a procedure using mass-produced electrochemical cells to determine the fractional occupancy of different sites on HSA is demonstrated.

Design and synthesis of ferrocene probe molecules for detection by electrochemical methods.

A series of ferrocenyl conjugates to fatty acids have been designed and synthesized to establish the key properties required for use in biomolecular binding studies. Amperometric detection of the ferrocene conjugates was sought in the region of 0.3 V (vs Ag/AgCl) for use in protein/blood solutions. Different linkers and solubilizing moieties were incorporated to produce a conjugate with optimal electrochemical properties. In electrochemical studies, the linker directly attached to the ferrocene was found to affect significantly the E(1/2) value and the stability of the ferrocenium cation. Ester-linked ferrocene conjugates had E(1/2) ranging from +400 to +410 mV, while amide-linked compounds ranged from +350 to +370 mV and the amines +260 to +270 mV. Folding of long-chain substituents around the ferrocene, also significantly affected by the choice of linker, was inferred as a secondary effect that increased E(1/2). The stability of the ferrocenium cation decreased systematically as E(1/2) increased. Disubstituted ferrocene ester and amide conjugates, with oxidation potentials of +640 and +570 mV, respectively, showed only a barely discernible reduction wave in cyclic voltammetry at 50 mV/s. Electrochemical measurements identified two lead compounds with the common structural characteristics of an amide and carbamate linker (compounds 17 and 21) with a C(11) fatty acid chain attached. It is envisaged that such molecules can be used to mimic and study the biomolecular binding interaction between fatty acids and molecules such as human serum albumin.

A new method to estimate earthquake liquefaction damage to buried lifelines

A new method that relates soil movement to buried lifeline damage state has been developed and applied during a Federal Emergency Management Agency-sponsored study to evaluate the impacts of lifeline proximity during earthquakes. The method considers landslides and liquefaction as a continum of ground displacement; it uses similar analysis methods for estimating the impacts of such motions on buried lifelines. For landslides, a critical acceleration parameter is used to estimate the lifeline damage state, which is also related to a displacement scaling parameter. Similary, a Liquefaction Severity Index (LSI) parameter is used to estimate the lifeline damage state for lateral spread or liquefactions conditions. The LSI can also be related to a displacement scaling parameter. The relationship of the LSI parameter to the earthquake intensity and lifeline damage state is new and is based on empirical data and expert judgement.(AU)