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Dopamine dyshomeostasis has been acknowledged among the determinants of nigrostriatal neuron degeneration in Parkinson's disease (PD). Several studies in experimental models and postmortem PD patients underlined increasing levels of the dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL has been shown to covalently modify the presynaptic protein αSynuclein (αSyn), whose misfolding and aggregation represent a major trait of PD pathology, triggering αSyn oligomerization in dopaminergic neurons. Here, we demonstrated that DOPAL elicits αSyn accumulation and hampers αSyn clearance in primary neurons. DOPAL-induced αSyn buildup lessens neuronal resilience, compromises synaptic integrity, and overwhelms protein quality control pathways in neurites. The progressive decline of neuronal homeostasis further leads to dopaminergic neuron loss and motor impairment, as showed in in vivo models. Finally, we developed a specific antibody which detected increased DOPAL-modified αSyn in human striatal tissues from idiopathic PD patients, corroborating the translational relevance of αSyn-DOPAL interplay in PD neurodegeneration.

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 1H/19F 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.

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.

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.

Though 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 non-specific 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 to 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.

Multiple 'overviews of reviews' conducted on the same topic ("overlapping overviews") represent a waste of research resources and can confuse clinicians making decisions amongst competing treatments. We aimed to assess the frequency and characteristics of overlapping overviews. MEDLINE, Epistemonikos and Cochrane Database of Systematic Reviews were searched for overviews that: synthesized reviews of health interventions and conducted systematic searches. Overlap was defined as: duplication of PICO eligibility criteria, and not reported as an update nor a replication. We categorized overview topics according to 22 WHO ICD-10 medical classifications, overviews as broad or narrow in scope, and overlap as identical, nearly identical, partial, or subsumed. Subsummation was defined as when broad overviews subsumed the populations, interventions and at least one outcome of another overview. Of 541 overviews included, 169 (31%) overlapped across similar PICO, fell within 13 WHO ICD-10 medical classifications, and 62 topics. 148/169 (88%) overlapping overviews were broad in scope. Fifteen overviews were classified as having nearly identical overlap (9%); 123 partial overlap (73%), and 31 subsumed (18%) others. One third of overviews overlapped in content and a majority covered broad topic areas. A multiplicity of overviews on the same topic adds to the ongoing waste of research resources, time, and effort across medical disciplines. Authors of overviews can use this study and the sample of overviews to identify gaps in the evidence for future analysis, and topics that are already studied, which do not need to be duplicated.

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 19F-containing FERM nanoemulsion encapsulates 89Zr 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 89Zr PET and 19F 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 19F and 89Zr 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 19F MRI. The FERM nanoemulsion has scalable production and is potentially useful for precise diagnosis, stratification, and treatment monitoring of inflammatory diseases.

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.

Overviews synthesising the results of multiple systematic reviews help inform evidence-based clinical practice. In this first of two companion papers, we evaluate the bibliometrics of overviews, including their prevalence and factors affecting citation rates and journal impact factor (JIF). We searched MEDLINE, Epistemonikos and Cochrane Database of Systematic Reviews (CDSR). We included overviews that: (1) synthesised reviews; (2) conducted a systematic search; (3) had a methods section; and (4) examined a healthcare intervention. Multivariable regression was conducted to determine the association between citation density, JIF and six predictor variables. We found 1218 overviews published from 2000 to 2020; the majority (73%) were published in the most recent 5-year period. We extracted a selection of these overviews (n = 541; 44%) dated from 2000 to 2018. The 541 overviews were published in 307 journals; "CDSR" (8%), "PLOS ONE" (3%) and "Sao Paulo Medical Journal" (2%) were the most prevalent. The majority (70%) were published in journals with impact factors between 0.05 and 3.97. We found a mean citation count of 10 overviews per year, published in journals with a mean JIF of 4.4. In multivariable analysis, overviews with a high number of citations and JIFs had more authors, larger sample sizes, were open access and reported the funding source. An eightfold increase in the number of overviews was found between 2009 and 2020. We identified 332 overviews published in 2020, which is equivalent to one overview published per day. Overviews perform above average for the journals in which they publish.

Communication between neurons relies on the release of diverse neurotransmitters, which represent a key-defining feature of a neuron's chemical and functional identity. Neurotransmitters are packaged into vesicles by specific vesicular transporters. However, tools for labeling and imaging synapses and synaptic vesicles based on their neurochemical identity remain limited. We developed a genetically encoded probe to identify glutamatergic synaptic vesicles at the levels of both light and electron microscopy (EM) by fusing the mini singlet oxygen generator (miniSOG) probe to an intralumenal loop of the vesicular glutamate transporter-2. We then used a 3D imaging method, serial block-face scanning EM, combined with a deep learning approach for automatic segmentation of labeled synaptic vesicles to assess the subcellular distribution of transporter-defined vesicles at nanometer scale. These tools represent a new resource for accessing the subcellular structure and molecular machinery of neurotransmission and for transmitter-defined tracing of neuronal connectivity.

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.

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.

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.

PurposeWe 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.ProceduresPerfluorinated 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.ResultsWe 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.ConclusionLipid-based nanoemulsion probes of perfluorocarbon synthesized with iron-binding fluorinated β-diketones can be formulated for intravenous delivery and inflammation detection in vivo.

PurposeA bottleneck in developing cell therapies for cancer is assaying cell biodistribution, persistence, and survival in vivo. Ex vivo cell labeling using perfluorocarbon (PFC) nanoemulsions, paired with 19 F MRI detection, is a non-invasive approach for cell product detection in vivo. Lymphocytes are small and weakly phagocytic limiting PFC labeling levels and MRI sensitivity. To boost labeling, we designed PFC nanoemulsion imaging probes displaying a cell-penetrating peptide, namely the transactivating transcription sequence (TAT) of the human immunodeficiency virus. We report optimized synthesis schemes for preparing TAT co-surfactant to complement the common surfactants used in PFC nanoemulsion preparations.MethodsWe performed ex vivo labeling of primary human chimeric antigen receptor (CAR) T cells with nanoemulsion. Intracellular labeling was validated using electron microscopy and confocal imaging. To detect signal enhancement in vivo, labeled CAR T cells were intra-tumorally injected into mice bearing flank glioma tumors.ResultsBy incorporating TAT into the nanoemulsion, a labeling efficiency of ~1012 fluorine atoms per CAR T cell was achieved that is a >8-fold increase compared to nanoemulsion without TAT while retaining high cell viability (~84%). Flow cytometry phenotypic assays show that CAR T cells are unaltered after labeling with TAT nanoemulsion, and in vitro tumor cell killing assays display intact cytotoxic function. The 19 F MRI signal detected from TAT-labeled CAR T cells was 8 times higher than cells labeled with PFC without TAT.ConclusionThe peptide-PFC nanoemulsion synthesis scheme presented can significantly enhance cell labeling and imaging sensitivity and is generalizable for other targeted imaging probes.

The most successful therapeutic strategies for locally advanced cancers continue to combine decades-old classical radiosensitizing chemotherapies with radiotherapy. Molecular targeted radiosensitizers offer the potential to improve the therapeutic ratio by increasing tumor-specific kill while minimizing drug delivery and toxicity to surrounding normal tissue. Auristatins are a potent class of anti-tubulins that sensitize cells to ionizing radiation damage and are chemically amenable to antibody conjugation. To achieve tumor-selective radiosensitization, we synthesized and tested anti-HER2 antibody-drug conjugates of two auristatin derivatives with ionizing radiation. Monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) were attached to the anti-HER2 antibodies trastuzumab and pertuzumab through a cleavable linker. While MMAE is cell permeable, MMAF has limited cell permeability as free drug resulting in diminished cytotoxicity and radiosensitization. However, when attached to trastuzumab or pertuzumab, MMAF was as efficacious as MMAE in blocking HER2-expressing tumor cells in G2-M. Moreover, MMAF anti-HER2 conjugates selectively killed and radiosensitized HER2-rich tumor cells. Importantly, when conjugated to targeting antibody, MMAF had the advantage of decreased bystander and off-target effects compared with MMAE. In murine xenograft models, MMAF anti-HER2 antibody conjugates had less drug accumulated in the normal tissue surrounding tumors compared with MMAE. Therapeutically, systemically injected MMAF anti-HER2 conjugates combined with focal ionizing radiation increased tumor control and improved survival of mice with HER2-rich tumor xenografts. In summary, our results demonstrate the potential of cell-impermeable radiosensitizing warheads to improve the therapeutic ratio of radiotherapy by leveraging antibody-drug conjugate technology.

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.

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.