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The kinetic and dissociation constants for FIAsH, a membrane-permanent fluorescein derivative with two As(III) substituents, binding to model tetracysteine peptides are investigated. Affinities in vitro and detection limits in living cells suggest that the preferred peptide conformation is a hairpin instead of alpha-helix.

Fluorescence is enhanced by cloning and transcribing malachite green (MG) aptamer sequences in tandem, revealing concatenate aptamers result in increased levels of fluorescence without quenching one another. The specificity of MG and Sulforhodamine B aptamers for different sets of dyes suggests that it could be possible t discriminate multiple RNAs simultaneously.

A number of international agreements exist which provide for a single regional patent office to grant patents on behalf of its member states. The most well-known is the European Patent Office, but similar organizations exist, in working or embryonic form, in most parts of the world. Procedures at the offices covering the former Soviet Union, Africa, the Middle East and South America are described. The Patent Cooperation Treaty is not a true regional patent system, but represents a very significant move towards greater international harmonization in patent granting procedures. [ABSTRACT FROM AUTHOR]

The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques. [ABSTRACT FROM AUTHOR]

Highlights the activities of the American Meteorological Society's Northern Nevada chapter last September and October 1998. Barbecue and potluck at the Hidden Valley Regional Park; Planning for additional field trips; Tour of the Fallon Naval Air Station.

The technique of colour EM that was recently developed enabled localisation of specific macromolecules/proteins of interest by the targeted deposition of diaminobenzidine (DAB) conjugated to lanthanide chelates. By acquiring lanthanide elemental maps by energy‐filtered transmission electron microscopy (EFTEM) and overlaying them in pseudo‐colour over the conventional greyscale TEM image, a colour EM image is generated. This provides a powerful tool for visualising subcellular component/s, by the ability to clearly distinguish them from the general staining of the endogenous cellular material. Previously, the lanthanide elemental maps were acquired at the high‐loss M4,5 edge (excitation of 3d electrons), where the characteristic signal is extremely low and required considerably long exposures. In this paper, we explore the possibility of acquiring the elemental maps of lanthanides at their N4,5 edge (excitation of 4d electrons), which occurring at a much lower energy‐loss regime, thereby contains significantly greater total characteristic signal owing to the higher inelastic scattering cross‐sections at the N4,5 edge. Acquiring EFTEM lanthanide elemental maps at the N4,5 edge instead of the M4,5 edge, provides ∼4× increase in signal‐to‐noise and ∼2× increase in resolution. However, the interpretation of the lanthanide maps acquired at the N4,5 edge by the traditional 3‐window method, is complicated due to the broad shape of the edge profile and the lower signal‐above‐background ratio. Most of these problems can be circumvented by the acquisition of elemental maps with the more sophisticated technique of EFTEM Spectrum Imaging (EFTEM SI). Here, we also report the chemical synthesis of novel second‐generation DAB lanthanide metal chelate conjugates that contain 2 lanthanide ions per DAB molecule in comparison with 0.5 lanthanide ion per DAB in the first generation. Thereby, fourfold more Ln3+ per oxidised DAB would be deposited providing significant amplification of signal. This paper applies the colour EM technique at the intermediate‐loss energy‐loss regime to three different cellular targets, namely using mitochondrial matrix‐directed APEX2, histone H2B‐Nucleosome and EdU‐DNA. All the examples shown in the paper are single colour EM images only. [ABSTRACT FROM AUTHOR]

The upper 50-kDa region of myosin may be critical for coupling between the nucleotide- and actin-binding regions. We introduced a tetracysteine motif in the upper 50-kDa domain (residues 292–297) of myosin V containing a single IQ domain (MV 11Q), allowing us to label this site with the fluorescein biarscenical hairpin-binding dye (FlAsH) (MV 1IQ FlAsH). The enzymatic properties of MV 1IQ FlAsH were similar to those of unlabeled MV 1IQ except for a 3-fold reduced ADP-release rate. MV 1IQ FlAsH was also capable of moving actin filaments in the in vitro motility assay. To examine rotation of the upper 50-kDa region, we determined the difference in the degree of energy transfer from Nomethylanthraniloyl (mant)-labeled nucleotides to FlAsH in both steady-state and transient kinetic experiments. The energy transfer efficiency was higher with mant-ATP (0.65 ± 0.02) compared with mant-ADP (0.55 ± 0.02) in the absence of actin. Stopped-flow measurements suggested that the energy transfer efficiency decreased with phosphate release (0.04 s-1) in the absence of actin. In contrast, upon mixing MV 1IQ FlAsH in the ADP-Pi state with actin, a decrease in the energy transfer signal was observed at a rate of 13s-1, similar to the ADP release rate. Our results demonstrate there was no change in the energy transfer signal upon actin-activated phosphate release and suggest that actin binding alters the dynamics of the upper 50-kDa region, which may be critical for the ability of myosin to bind tightly to both ADP and actin. [ABSTRACT FROM AUTHOR]

Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H 2 O 2 . Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O 2 instead of toxic H 2 O 2 , and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.

Purpose: We explore the use of intravenously delivered fluorescent perfluorocarbon (PFC) nanoemulsion tracers and multi-spectral cryo-fluorescence tomography (CFT) for whole-body tracer imaging in murine inflammation models. CFT is an emerging technique that provides high-resolution, three-dimensional mapping of probe localization in intact animals and tissue samples, enabling unbiased validation of probe biodistribution and minimizes reliance on laborious histological methods employing discrete tissue panels, where disseminated populations of PFC-labeled cells may be overlooked. This methodology can be used to streamline the development of new generations of non-invasive, cellular-molecular imaging probes for in vivo imaging., Procedures: Mixtures of nanoemulsions with different fluorescent emission wavelengths were administered intravenously to naïve mice and models of acute inflammation, colitis, and solid tumor. Mice were euthanized 24 h post-injection, frozen en bloc, and imaged at high resolution (~ 50 µm voxels) using CFT at multiple wavelengths., Results: PFC nanoemulsions were visualized using CFT within tissues of the reticuloendothelial system and inflammatory lesions, consistent with immune cell (macrophage) labeling, as previously reported in in vivo magnetic resonance and nuclear imaging studies. The CFT signals show pronounced differences among fluorescence wavelengths and tissues, presumably due to autofluorescence, differential fluorescence quenching, and scattering of incident and emitted light., Conclusions: CFT is an effective and complementary methodology to in vivo imaging for validating PFC nanoemulsion biodistribution at high spatial localization, bridging the resolution gap between in vivo imaging and histology., (© 2024. The Author(s), under exclusive licence to World Molecular Imaging Society.)

Locally advanced cancers remain therapeutically challenging to eradicate. The most successful treatments continue to combine decades old non-targeted chemotherapies with radiotherapy that unfortunately increase normal tissue damage in the irradiated field and have systemic toxicities precluding further treatment intensification. Therefore, alternative molecularly guided systemic therapies are needed to improve patient outcomes when applied with radiotherapy. In this work, we report a trimodal precision cytotoxic chemo-radio-immunotherapy paradigm using spatially targeted auristatin warheads. Tumor-directed antibodies and peptides conjugated to radiosensitizing monomethyl auristatin E (MMAE) specifically produce CD8 T cell dependent durable tumor control of irradiated tumors and immunologic memory. In combination with ionizing radiation, MMAE sculpts the tumor immune infiltrate to potentiate immune checkpoint inhibition. Here, we report therapeutic synergies of targeted cytotoxic auristatin radiosensitization to stimulate anti-tumor immune responses providing a rationale for clinical translational of auristatin antibody drug conjugates with radio-immunotherapy combinations to improve tumor control., (© 2022. The Author(s).)

We describe an in vivo imaging probe platform that is readily modifiable to accommodate binding of different molecular targeting moieties and payloads for multimodal image generation. In this work, we demonstrate the utility of perfluorocarbon (PFC) nanoemulsions incorporating dibenzocyclooctyne (DBCO) by enabling postemulsification functionalization via a click reaction with azide-containing ligands. The addition of DBCO-lipid to the surfactant in PFC nanoemulsions did not affect nanoemulsion size or nanoemulsion stability. As proof-of-concept, fluorescent dye-azides were conjugated to PFC nanoemulsions, demonstrating the feasibility of functionalization the by click reaction. Uptake of the fluorescent PFC by macrophages was demonstrated both in vitro in cultured macrophages and in situ in an acute inflammation mouse model, where fluorescence imaging and 1 H/ 19 F magnetic resonance imaging (MRI) were used for in vivo detection. Overall, these data demonstrate the potential of PFC nanoemulsions incorporating DBCO as a versatile platform for generating functionalized probes., Competing Interests: The authors declare the following competing financial interest(s): E.T.A. is a founder and shareholder of Celsense, Inc. The other authors have nothing to disclose., (© 2021 The Authors. Published by American Chemical Society.)

Although WNT signaling is frequently dysregulated in solid tumors, drugging this pathway has been challenging due to off-tumor effects. Current clinical pan-WNT inhibitors are nonspecific and lead to adverse effects, highlighting the urgent need for more specific WNT pathway-targeting strategies. We identified elevated expression of the WNT receptor Frizzled class receptor 7 ( FZD7 ) in multiple solid cancers in The Cancer Genome Atlas, particularly in the mesenchymal and proliferative subtypes of ovarian serous cystadenocarcinoma, which correlate with poorer median patient survival. Moreover, we observed increased FZD7 protein expression in ovarian tumors compared with normal ovarian tissue, indicating that FZD7 may be a tumor-specific antigen. We therefore developed a novel antibody-drug conjugate, septuximab vedotin (F7-ADC), which is composed of a chimeric human-mouse antibody to human FZD7 conjugated to the microtubule-inhibiting drug monomethyl auristatin E (MMAE). F7-ADC selectively binds human FZD7, potently kills ovarian cancer cells in vitro , and induces regression of ovarian tumor xenografts in murine models. To evaluate F7-ADC toxicity in vivo , we generated mice harboring a modified Fzd7 gene where the resulting Fzd7 protein is reactive with the human-targeting F7-ADC. F7-ADC treatment of these mice did not induce acute toxicities, indicating a potentially favorable safety profile in patients. Overall, our data suggest that the antibody-drug conjugate approach may be a powerful strategy to combat FZD7-expressing ovarian cancers in the clinic., (©2021 The Authors; Published by the American Association for Cancer Research.)

Inflammation is associated with a range of serious human conditions, including autoimmune and cardiovascular diseases and cancer. The ability to image active inflammatory processes greatly enhances our ability to diagnose and treat these diseases at an early stage. We describe molecular compositions enabling sensitive and precise imaging of inflammatory hotspots in vivo. Methods: A functionalized nanoemulsion with a fluorocarbon-encapsulated radiometal chelate (FERM) was developed to serve as a platform for multimodal imaging probe development. The 19 F-containing FERM nanoemulsion encapsulates 89 Zr in the fluorous oil via a fluorinated hydroxamic acid chelate. Simple mixing of the radiometal with the preformed aqueous nanoemulsion before use yields FERM, a stable in vivo cell tracer, enabling whole-body 89 Zr PET and 19 F MRI after a single intravenous injection. Results: The FERM nanoemulsion was intrinsically taken up by phagocytic immune cells, particularly macrophages, with high specificity. FERM stability was demonstrated by a high correlation between the 19 F and 89 Zr content in the blood (correlation coefficient > 0.99). Image sensitivity at a low dose (37 kBq) was observed in a rodent model of acute infection. The versatility of FERM was further demonstrated in models of inflammatory bowel disease and 4T1 tumor. Conclusion: Multimodal detection using FERM yields robust whole-body lesion detection and leverages the strengths of combined PET and 19 F MRI. The FERM nanoemulsion has scalable production and is potentially useful for precise diagnosis, stratification, and treatment monitoring of inflammatory diseases., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)