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The years 2023–2024 bring up some significant anniversaries for a range of historical events in the development of modern intellectual property law. Many of them have implications for how patents are obtained and recorded in the public domain. The original documents are cited for further reading. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Legal status information is a critical component of many patent searches, particularly those conducted in industry. Although most major patenting authorities can provide reasonably comprehensive data on their own national legal status, this may not enable the user to obtain a complete picture of where patent rights are in force. Many smaller countries have a mechanism to recognise patent rights previously granted by other jurisdictions; however, the public information sources on these extensions are poorly controlled and difficult to access. A review is provided on the various means by which extra-territorial extensions may be obtained, and of the quality and quantity of public information which can be retrieved. [ABSTRACT FROM AUTHOR]

Efficient patentability searching demands that the searcher has the ability to identify efficiently the best sources of likely prior art for each invention being searched. This applies both within industry, at the point of drafting a patent application, and within patent offices conducting searches to support substantive examination. Industry 4.0 inventions are characterised by a particular nature and combination of technologies, notably the inclusion of aspects of Information and Communication Technologies (ICT). The default information sources currently used to establish patentability of 'conventional' inventions may be inappropriate for this new class of invention. This is partly due to the different structures of publication and methods of dissemination of research results found within computer science professions. It is suggested that searching in the secondary or tertiary literature may be a more fruitful approach to establish patentability in these areas of technology. However, expertise in the use of these types of source has been substantially lost, as the information industry has concentrated on production of full-text primary sources. Database production, database usage and search protocols all need to be reassessed if they are to meet the challenges of searching Industry 4.0 inventions. [ABSTRACT FROM AUTHOR]

Summary Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small-molecule probes, or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra of each lanthanide deposit via energy-filtered transmission electron microscopy. This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the postsynaptic membrane. [ABSTRACT FROM AUTHOR]

The year 2014 is the centenary of the filing of a key US patent in the development of the modern zip fastener. The named inventor for this patent was Gideon Sundback, who had successfully improved the original designs of Whitcomb Judson. In addition to his breakthrough invention, Sundback was responsible for many additional patents during his long working life with the Hookless Fastener Company. Some of his inventions were patented outside of the US as well, but despite retaining foreign rights to himself, he did not make a great fortune from inventing. His last patent was granted posthumously, in 1959. [ABSTRACT FROM AUTHOR]

Intracellular Ca2+ regulates numerous proteins and cellular functions and can vary substantially over submicron and submillisecond scales, so precisely localized fast detection is desirable. We have created a ∼1-kDa biarsenical Ca2+ indicator, called Calcium Green FlAsH (CaGF, 1), to probe [Ca2+] surrounding genetically targeted proteins. CaGF attached to a tetracysteine motif becomes ten-fold more fluorescent upon binding Ca2+, with a Kd of ∼100 μM, <1-ms kinetics and good Mg2+ rejection. In HeLa cells expressing tetracysteine-tagged connexin 43, CaGF labels gap junctions and reports Ca2+ waves after injury. Total internal reflection microscopy of tetracysteine-tagged, CaGF-labeled α1C L-type calcium channels shows fast-rising depolarization-evoked Ca2+ transients, whose lateral nonuniformity suggests that the probability of channel opening varies greatly over micron dimensions. With moderate Ca2+ buffering, these transients decay surprisingly slowly, probably because most of the CaGF signal comes from closed channels feeling Ca2+ from a tiny minority of clustered open channels. With high Ca2+ buffering, CaGF signals decay as rapidly as the calcium currents, as expected for submicron Ca2+ domains immediately surrounding active channels. Thus CaGF can report highly localized, rapid [Ca2+] dynamics. [ABSTRACT FROM AUTHOR]

Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent probes for imaging in cell biology. Here we review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small organic fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted. [ABSTRACT FROM AUTHOR]

Examines the synthesis of biarsenical ligands and tetracysteine motifs for protein labeling. Identification of the electron-microscopic localization of tetracysteine-tagged proteins; Genetic fusion of proteins to fluorescent proteins; Polymerization of diaminobenzidine.

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]

Investigates the dynamics of guanosine 3',5'-cyclic monophosphate (cGMP) in single living cells by constructing genetically encoded, fluorescent cGMP indicators by bracketing cGMP-dependent protein kinase between cyan and yellow mutants of green fluorescent protein. Emission ratio imaging of indicators; Basal activity of guanylate cyclase.

Perineuronal nets (PNNs), a specialized form of extra cellular matrix (ECM), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesize that PNNs serve as gatekeepers that guard and protect synaptic territory and thus may stabilize an engram circuit. We present high-resolution and 3D EM images of PNN-engulfed neurons in mice brains, showing that synapses occupy the PNN holes and that invasion of other cellular components is rare. PNN constituents in mice brains are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have a high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or matrix metalloproteases 9 (MMP9) knockout mice allowed normal fear memory acquisition but diminished long-term memory stabilization, supporting the above hypothesis. [ABSTRACT FROM AUTHOR]

Introduces a system that provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.

Roger Y. Tsien, Professor an der University of California in San Diego und Howard Hughes Medical Institute Investigator, ist am 24. August 2016 verstorben. Tsien hatte 2008 anteilig den Chemie ‐ Nobelpreis für die Entdeckung des grün fluoreszierenden Proteins erhalten. Darüber hinaus entwickelte er eine ganze Reihe farbig fluoreszierender Proteine sowie eine Vielzahl niedermolekuler Sonden für Ca2+ ‐ Ionen. [ABSTRACT FROM AUTHOR]

An obituary for Roger Y. Tsien, an American biochemist who won the 2008 Nobel Prize in Chemistry and developed colored flourescent proteins for biological and medical research, is presented.

Purpose: We aim to develop perfluorocarbon-based nanoemulsions with improved sensitivity for detection of inflammatory macrophages in situ using F-19 MRI. Towards this goal, we evaluate the feasibility of nanoemulsion formulation incorporating a metal chelate in the fluorous phase which shortens the F-19 longitudinal relaxation rate and image acquisition time.Procedures: Perfluorinated linear polymers were conjugated to metal-binding tris-diketonate, blended with unconjugated polymers, and emulsified in water. Phospholipid-based surfactant was used to stabilize nanoemulsion and provide biocompatibility. Nanoemulsions were metalated with the addition of ferric salt to the buffer. Physical stability of surfactant and nanoemulsion was evaluated by mass spectrometry and dynamic light scattering measurements. Nanoemulsions were injected intravenously into a murine granuloma inflammation model, and in vivo19F/1H MRI at 11.7 T was performed.Results: We demonstrated stability and biocompatibility of lipid-based paramagnetic nanoemulsions. We investigated potential oxidation of lipid in the presence of metal chelate. As a proof of concept, we performed non-invasive monitoring of macrophage burden in a murine inflammation model following intravenous injection of nanoemulsion using in vivo F-19 MRI.Conclusion: Lipid-based nanoemulsion probes of perfluorocarbon synthesized with iron-binding fluorinated β-diketones can be formulated for intravenous delivery and inflammation detection in vivo. [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]

Fluorescent reporters have revolutionized the study of biological systems by allowing exquisitely sensitive, real-time detection of tagged proteins both in vitro and in vivo. Ideally, the requisite tagging is performed in situ by genetic fusion, either to unusual fluorescent proteins from jellyfish or coral or to much shorter tetracysteine domains, which bind and enhance the fluorescence of membrane-permeant biarsenical dyes. However, comparable methods do not yet exist for nucleic acids. No sequence of natural, genetically encodable nucleotides is known to generate distinctive fluorescence by itself.

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.

Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concentrations of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to β-actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins. [ABSTRACT FROM AUTHOR]

Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of blarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ. [ABSTRACT FROM AUTHOR]

B Correction to: Mol Imaging Biol (2020) 22: 665-674 b https://doi.org/10.1007/s11307-019-01415-5 This article was updated to correct the funding information. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. The original article can be found online at https://doi.org/10.1007/s11307-019-01415-5. [Extracted from the article]

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]

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing. Aequorea victoria, the jellyfish that yielded GFP and transformed the study of living cells, held more secrets than first suspected. This study reveals that A. victoria and its Australian cousin encode a diverse set of unusual fluorescent proteins, including the brightest one ever described. [ABSTRACT FROM AUTHOR]

Despite the importance of understanding the regulation of microvascular blood flow in white matter, no data on subcortical capillary blood flow parameters are available, largely due to the lack of appropriate imaging methods. To address this knowledge gap, we employed two-photon microscopy using a far-red fluorophore Alexa680 and photon-counting detection to measure capillary red blood cell (RBC) flux in both cerebral gray and white matter, in isoflurane-anesthetized mice. We have found that in control animals, baseline capillary RBC flux in the white matter was significantly higher than in the adjacent cerebral gray matter. In response to mild hypercapnia, RBC flux in the white matter exhibited significantly smaller fractional increase than in the gray matter. Finally, during global cerebral hypoperfusion, RBC flux in the white matter was reduced significantly in comparison to the controls, while RBC flux in the gray matter was preserved. Our results suggest that blood flow in the white matter may be less efficiently regulated when challenged by physiological perturbations as compared to the gray matter. Importantly, the blood flow in the white matter may be more susceptible to hypoperfusion than in the gray matter, potentially exacerbating the white matter deterioration in brain conditions involving global cerebral hypoperfusion. [ABSTRACT FROM AUTHOR]

Objective Functional imaging of synovitis could improve both early detection of rheumatoid arthritis (RA) and long-term outcomes. Given the intersection of inflammation with coagulation protease activation, this study was undertaken to examine coagulation protease activities in arthritic mice with a dual-fluorescence ratiometric activatable cell-penetrating peptide (RACPP) that has a linker, norleucine (Nle)-TPRSFL, with a cleavage site for thrombin. Methods K/BxN-transgenic mice with chronic arthritis and mice with day 1 passive serum-transfer arthritis were imaged in vivo for Cy5:Cy7 emission ratiometric fluorescence from proteolytic cleavage and activation of RACPPNleTPRSFL. Joint thickness in mice with serum-transfer arthritis was measured from days 0 to 10. The cleavage-evoked release of Cy5-tagged tissue-adhesive fragments enabled microscopic correlation with immunohistochemistry for inflammatory markers. Thrombin dependence of ratiometric fluorescence was tested by ex vivo application of RACPPNleTPRSFL and argatroban to cryosections obtained from mouse hind paws on day 1 of serum-transfer arthritis. Results In chronic arthritis, RACPPNleTPRSFL fluorescence ratios of Cy5:Cy7 emission were significantly higher in diseased swollen ankles of K/BxN-transgenic mice than in normal mouse ankles. A high ratio of RACPPNleTPRSFL fluorescence in mouse ankles and toes on day 1 of serum-transfer arthritis correlated with subsequent joint swelling. Foci of high ratiometric fluorescence localized to inflammation, as demarcated by immune reactivity for citrullinated histones, macrophages, mast cells, and neutrophils, in soft tissue on day 1 of serum-transfer arthritis. Ex vivo application of RACPPNleTPRSFL to cryosections obtained from mice on day 1 of serum-transfer arthritis produced ratiometric fluorescence that was inhibited by argatroban. Conclusion RACPPNleTPRSFL activation detects established experimental arthritis, and the detection of inflammation by RACPPNleTPRSFL on day 1 of serum-transfer arthritis correlates with disease progression. [ABSTRACT FROM AUTHOR]

Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41's binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue-NP41-bound nerves, followed by biotin hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10-7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve "ghosts" months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures. [ABSTRACT FROM AUTHOR]

In Echtzeit und ratiometrisch kann die Thrombin ‐ Aktivierung in vivo mithilfe aktivierbarer zellgängiger Peptide (RACPPs) verfolgt werden. RACPPs verbinden die 1) Ermittlung des Verhältnisses zweier Emissionen, 2) In ‐ vivo ‐ Bildgebung in Säugern im fernen Rot ‐ bis Infrarotbereich und 3) spaltungsabhängige räumliche Lokalisierung. Das beste RACPP nutzt Norleucin(Nle) ‐ TPRSFL als Linker, was die Empfindlichkeit für Thrombin um das 90 ‐ Fache erhöht (siehe Bild). [ABSTRACT FROM AUTHOR]

In real time: Thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual‐emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage‐dependent spatial localization. The most advanced RACPP uses norleucine (Nle)‐TPRSFL as a linker that increases sensitivity to thrombin by about 90‐fold (see figure). [ABSTRACT FROM AUTHOR]

Summary: Fluorescent proteins have become valuable tools for biomedical research as protein tags, reporters of gene expression, biosensor components, and cell lineage tracers. However, applications of fluorescent proteins for deep tissue imaging in whole mammals have been constrained by the opacity of tissues to excitation light below 600 nm, because of absorbance by hemoglobin. Fluorescent proteins that excite efficiently in the “optical window” above 600 nm are therefore highly desirable. We report here the evolution of far-red fluorescent proteins with peak excitation at 600 nm or above. The brightest one of these, Neptune, performs well in imaging deep tissues in living mice. The crystal structure of Neptune reveals a novel mechanism for red-shifting involving the acquisition of a new hydrogen bond with the acylimine region of the chromophore. [Copyright &y& Elsevier]

The article highlights a research on the solution structure of 12-mer peptide FLNCCPGCCMEP with ReAsH fluorophore through nuclear magnetic resonance (NMR) spectroscopy. The study shows that the N-terminus of the 12-mer peptide is tightly constrained as suggested in the NMR results and solution structure calculations of the optimized ReAsH peptide complex. Moreover, while the fluorophore is bound, N- and C-terminal tripeptides may interact with each other.

Combinations of molecular tags visible in light and electron microscopes become particularly advantageous in the analysis of dynamic cellular components like the Golgi apparatus. This organelle disassembles at the onset of mitosis and, after a sequence of poorly understood events, reassembles after cytokinesis. The precise location of Golgi membranes and resident proteins during mitosis remains unclear, partly due to limitations of molecular markers and the resolution of light microscopy. We generated a fusion consisting of the first 117 residues of α-mannosidase II tagged with a fluorescent protein and a tetracysteine motif. The mannosidase component guarantees docking into the Golgi membrane, with the tags exposed in the lumen. The fluorescent protein is optically visible without further treatment, whereas the tetracysteine tag can be reduced acutely with a membrane-permeant phosphine, labeled with ReAsH, monitored in the light microscope, and used to trigger the photoconversion of diaminobenzidine, allowing 40 optical recording on live cells and correlated ultrastructural analysis by electron microscopy. These methods reveal that Golgi reassembly is preceded by the formation of four colinear clusters at telophase, two per daughter cell. Within each daughter, the smaller cluster near the midbody gradually migrates to rejoin the major cluster on the far side of the nucleus and asymmetrically reconstitutes a single Golgi apparatus, first in one daughter cell and then in the other. Our studies provide previously undescribed insights into Golgi disassociation and reassembly during mitosis and offer a powerful approach to follow recombinant protein distribution in 4D imaging and correlated high-resolution analysis. [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]

Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a well-established method to monitor protein-protein interactions or conformational changes of individual proteins. But protein functions can be perturbed by fusion of large tags such as CFP and YFP. Here we use G protein–coupled receptor (GPCR) activation in living cells as a model system to compare YFP with the small, membrane-permeant fluorescein derivative with two arsen-(III) substituents (fluorescein arsenical hairpin binder; FlAsH) targeted to a short tetracysteine sequence. Insertion of CFP and YFP into human adenosine A2A receptors allowed us to use FRET to monitor receptor activation but eliminated coupling to adenylyl cyclase. The CFP/FlAsH-tetracysteine system gave fivefold greater agonist-induced FRET signals, similar kinetics (time constant of 66–88 ms) and perfectly normal downstream signaling. Similar results were obtained for the mouse α2A-adrenergic receptor. Thus, FRET from CFP to FlAsH reports GPCR activation in living cells without disturbing receptor function and shows that the small size of the tetracysteine-biarsenical tag can be decisively advantageous. [ABSTRACT FROM AUTHOR]

Regulation of AMPA receptor (AMPAR) trafficking is important for neural plasticity. Here we examined the trafficking and synthesis of the GluR1 and GluR2 subunits using ReAsH-EDT2 and FlAsH-EDT2 staining. Activity blockade of rat cultured neurons increased dendritic GluR1, but not GluR2, levels. Examination of transected dendrites revealed that both AMPAR subunits were synthesized in dendrites and that activity blockade enhanced dendritic synthesis of GluR1 but not GluR2. In contrast, acute pharmacological manipulations increased dendritic synthesis of both subunits. AMPARs synthesized in dendrites were inserted into synaptic plasma membranes and, after activity blockade, the electrophysiological properties of native synaptic AMPARs changed in the manner predicted by the imaging experiments. In addition to providing a novel mechanism for synaptic modifications, these results point out the advantages of using FlAsH-EDT2 and ReAsH-EDT2 for studying the trafficking of newly synthesized proteins in local cellular compartments such as dendrites. [ABSTRACT FROM AUTHOR]

Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and a1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by ~90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or a1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected molecular specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen respectively inhibit or enhance CALI. [ABSTRACT FROM AUTHOR]

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described. [ABSTRACT FROM AUTHOR]

Recent advances in immunotherapy have revolutionized cancer therapy. Immunotherapies can engage the adaptive and innate arms of the immune system. Therapeutics targeting immune checkpoint inhibitors (i.e., CTLA-4; PD-1, and PD-L1) have shown efficacy for subsets of cancer patients by unleashing an adaptive antitumor immune response. Alternatively, small molecule immune modulators of the innate immune system such as toll-like receptor (TLR) agonists are being developed for cancer therapy. TLRs function as pattern recognition receptors to microbial products and are also involved in carcinogenesis. Reisquimod is a TLR 7/8 agonist that has antitumor efficacy. However, systemic delivery free resiquimod has proven to be challenging due to toxicity of nonspecific TLR 7/8 activation. Therefore, we developed a targeted peptide-drug conjugate strategy for systemic delivery of resiquimod. We designed an activatable cell penetrating peptide to deliver resiquimod specifically to the tumor tissue while avoiding normal tissues. The activatable cell penetrating peptide (ACPP) scaffold undergoes enzymatic cleavage by matrix metalloproteinases 2/9 in the extracellular matrix followed by intracellular lysosomal cathepsin B mediated release of the free resiquimod. Importantly, when conjugated to ACPP; the tumor tissue concentration of resiquimod was more than 1000-fold greater than that of surrounding non-cancerous tissue. Moreover, systemic ACPP-resiquimod delivery produced comparable therapeutic efficacy to localized free resiquimod in syngeneic murine tumors. These results highlight a precision peptide-drug conjugate delivery. [ABSTRACT FROM AUTHOR]

Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological systems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited. Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely used rhodamine scaffold and demonstrate their efficacy for chromophore-assisted light inactivation, cell ablation in culture and in vivo , and photopolymerization of diaminobenzidine for electron microscopy. These chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to high-resolution imaging. • Incorporation of iodine or sulfur into rhodamine dyes increases 1O 2 quantum yield • Rhodamine photosensitizers are compatible with live-cell-labeling strategies • Generating 1O 2 allows photoablation of entire cells in culture or in vivo • Photopolymerization of 3,3′-diaminobenzidine gives contrast in electron microscopy Binns et al. describe photosensitizers based on rhodamine dyes. Incorporation of halogen or sulfur atoms into the rhodamine structure substantially increases singlet oxygen quantum yield. Combined with the HaloTag-labeling system, these photosensitizers allow light-mediated destruction of proteins, ablation of entire cells, or deposition of electron microscopy contrast agents. [ABSTRACT FROM AUTHOR]

Patients with advanced cancers are treated with combined radiotherapy and chemotherapy, however curability is poor and treatment side effects severe. Drugs sensitizing tumors to radiotherapy have been developed to improve cell kill, but tumor specificity remains challenging. To achieve tumor selectivity of small molecule radiosensitizers, we tested as a strategy active tumor targeting using peptide-based drug conjugates. We attached an inhibitor of the DNA damage response to antibody or cell penetrating peptides. Antibody drug conjugates honed in on tumor overexpressed cell surface receptors with high specificity but lacked efficacy when conjugated to the DNA damage checkpoint kinase inhibitor AZD7762. As an alternative approach, we synthesized activatable cell penetrating peptide scaffolds that accumulated within tumors based on matrix metalloproteinase cleavage. While matrix metalloproteinases are integral to tumor progression, they have proven therapeutically elusive. We harnessed these pro-tumorigenic extracellular proteases to spatially guide radiosensitizer drug delivery using cleavable activatable cell penetrating peptides. Here, we tested the potential of these two drug delivery platforms targeting distinct tumor compartments in combination with radiotherapy and demonstrate the advantages of protease triggered cell penetrating peptide scaffolds over antibody drug conjugates to deliver small molecule amine radiosensitizers. Image 1 • Cell penetrating peptide (CPP) drug conjugates radiosensitize. • Activatable cell penetrating peptide (ACPP) engineered scaffolding cloaks CPPs. • Tumor overexpressed extracellular proteases cleave ACPPs and guide CPP delivery. • ACPP scaffolds increase tumor targeting and decreases normal tissue accumulation. • Radiosensitizers conjugated to ACPP given with radiotherapy increase tumor control. [ABSTRACT FROM AUTHOR]

A protein-fragment complementation assay (PCA) for detecting and localizing intracellular protein-protein interactions (PPIs) was built by bisection of miniSOG, a fluorescent flavoprotein derived from the light, oxygen, voltage (LOV)-2 domain of Arabidopsis phototropin. When brought together by interacting proteins, the fragments reconstitute a functional reporter that permits tagged protein complexes to be visualized by fluorescence light microscopy (LM), and then by standard as well as "multicolor" electron microscopy (EM) via the photooxidation of 3-3′-diaminobenzidine and its derivatives. • Split-miniSOG allows intracellular protein-protein interactions to be visualized by correlated fluorescence and electron microscopy • Split-miniSOG complementation is reversible • Reconstituted complexes are imaged by fluorescence microscopy and TEM/multicolor EM Boassa et al. describe the use of split-miniSOG for the visualization of protein aggregates associated with neurodegenerative diseases. This study shows the general utility of this reversible system for detection of spatial organization of molecular complexes in mammalian cells at nanometer resolution. [ABSTRACT FROM AUTHOR]