Nanoarchitectonics of g-C3N4 Nanosheets with a AuCu Enhancement Effect for Superior Photo- and Electrochemical Activity.

AuCu alloy nanoparticles (NPs) were embedded in superior thin g-C3N4 nanosheets by a mechanochemical pre-reaction and subsequent thermal polymerization at high temperature. The introduction of AuCu NPs increased conductivity, decreased the band gap, expended light absorption, and improved the separation and transfer efficiencies of photogenerated electrons and holes. Moreover, the uniform distribution of AuCu NPs in g-C3N4 nanosheets is ascribed to the pre-reaction of bulk g-C3N4 and metal salts to create activity cites. The adsorption ability in the visible light region was improved due to the plasma effect of Au. AuCu/g-C3N4 composites (AuCu/CN-1%) with optimized component ratios revealed the highest transient photocurrent responses, the lowest electrochemical impedance arc radius, and the best photocatalytic H2 evolution rate of 930.2 µmol g-1 h-1. These findings exhibited that loading AuCu bimetallic NPs could efficiently offset some disadvantages of g-C3N4 and improve its photocatalytic performances.

Side-chain modification of collagen-targeting peptide prevents dye aggregation for improved molecular imaging of arthritic joints.

Near-infrared (NIR) dye-peptide conjugates are widely used for tissue-targeted molecular fluorescence imaging of pathophysiologic conditions. However, the significant contribution of both dye and peptide to the net mass of these bioconjugates implies that small changes in either component could alter their photophysical and biological properties. Here, we synthesized and conjugated a type I collagen targeted peptide, RRANAALKAGELYKCILY, to either a hydrophobic (LS1000) or hydrophilic (LS1006) NIR fluorescent dye. Spectroscopic analysis revealed rapid self-assembly of both LS1000 and LS1006 in aqueous media to form stable dimeric/H aggregates, regardless of the free dye's solubility in water. We discovered that replacing the cysteine residue in LS1000 and LS1006 with acetamidomethyl cysteine to afford LS1001 and LS1107, respectively, disrupted the peptide's self-assembly and activated the previously quenched dye's fluorescence in aqueous conditions. These results highlight the dominant role of the octadecapeptide, but not the dye molecules, in controlling the photophysical properties of these conjugates by likely sequestering or extruding the hydrophobic or hydrophilic dyes, respectively. Application of the compounds for imaging collagen-rich tissue in an animal model of inflammatory arthritis showed enhanced uptake of all four conjugates, which retained high collagen-binding affinity, in inflamed joints. Moreover, LS1001 and LS1107 improved the arthritic joint-to-background contrast, suggesting that reduced aggregation enhanced the clearance of these compounds from non-target tissues. Our results highlight a peptide-driven strategy to alter the aggregation states of molecular probes in aqueous solutions, irrespective of the water-solubilizing properties of the dye molecules. The interplay between the monomeric and aggregated forms of the conjugates using simple thiol-modifiers lends the peptide-driven approach to diverse applications, including the effective imaging of inflammatory arthritis joints.

Synthesis of dye conjugates to visualize the cancer cells using fluorescence microscopy.

The clinical diagnosis of most cancers is based on evaluation of histology microscopic slides to view the size and shape of cellular nuclei and morphological structure of tissue. To achieve this goal for in vivo and in-deep tissues, near infrared dyes-bovine serum albumin and immunoglobulin G conjugates were synthesized. The spectral study shows that the absorption and fluorescence of the dye conjugates are in the "tissue optical window" spectral ranges between 650 and 900 nm. The internalization and pinocytosis of the synthesized compounds were investigated at cell level using fluorescence microscopy to obtain the optimal concentration and staining time.

Evaluation of a triple-helical peptide with quenched FluorSophores for optical imaging of MMP-2 and MMP-9 proteolytic activity.

Matrix metalloproteinases (MMP) 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand the molecular role of the gelatinases in tumor development. Recently, a self-assembling homotrimeric triple-helical peptide (THP), incorporating a sequence from type V collagen, with high substrate specificity to the gelatinases has been developed. To determine whether this THP would be suitable for imaging protease activity, 5-carboxyfluorescein (5FAM) was conjugated, resulting in 5FAM3-THP and 5FAM6-THP, which were quenched up to 50%. 5FAM6-THP hydrolysis by MMP-2 and MMP-9 displayed kcat/KM values of 1.5 × 104 and 5.4 × 103 M-1 s-1, respectively. Additionally 5FAM6-THP visualized gelatinase activity in gelatinase positive HT-1080 cells, but not in gelatinase negative MCF-7 cells. Furthermore, the fluorescence in the HT-1080 cells was greatly attenuated by the addition of a MMP-2 and MMP-9 inhibitor, SB-3CT, indicating that the observed fluorescence release was mediated by gelatinase proteolysis and not non-specific proteolysis of the THPs. These results demonstrate that THPs fully substituted with fluorophores maintain their substrate specificity to the gelatinases in human cancer cells and may be useful in in vivo molecular imaging of gelatinase activity.

Probing distance-dependent plasmon-enhanced near-infrared fluorescence using polyelectrolyte multilayers as dielectric spacers.

Owing to their applications in biodetection and molecular bioimaging, near-infrared (NIR) fluorescent dyes are being extensively investigated. Most of the existing NIR dyes exhibit poor quantum yield, which hinders their translation to preclinical and clinical settings. Plasmonic nanostructures are known to act as tiny antennae for efficiently focusing the electromagnetic field into nanoscale volumes. The fluorescence emission from NIR dyes can be enhanced by more than thousand times by precisely placing them in proximity to gold nanorods. We have employed polyelectrolyte multilayers fabricated using layer-by-layer assembly as dielectric spacers for precisely tuning the distance between gold nanorods and NIR dyes. The aspect ratio of the gold nanorods was tuned to match the longitudinal localized surface plasmon resonance wavelength with the absorption maximum of the NIR dye to maximize the plasmonically enhanced fluorescence. The design criteria derived from this study lays the groundwork for ultrabright fluorescence bullets for in vitro and in vivo molecular bioimaging.

Development of New CD38 Targeted Peptides for Cancer Imaging.

PURPOSE: Multiple myeloma (MM) affects over 35,000 patients each year in the US. There remains a need for versatile Positron Emission Tomography (PET) tracers for the detection, accurate staging, and monitoring of treatment response of MM that have optimal specificity and translational attributes. CD38 is uniformly overexpressed in MM and thus represents an ideal target to develop CD38-targeted small molecule PET radiopharmaceuticals to address these challenges. PROCEDURES: Using phage display peptide libraries and pioneering algorithms, we identified novel CD38 specific peptides. Imaging bioconjugates were synthesized using solid phase peptide chemistry, and systematically analyzed in vitro and in vivo in relevant MM systems. RESULTS: The CD38-targeted bioconjugates were radiolabeled with copper-64 (64Cu) with100% radiochemical purity and an average specific activity of 3.3 - 6.6 MBq/nmol. The analog NODAGA-PEG4-SL022-GGS (SL022: Thr-His-Tyr-Pro-Ile-Val-Ile) had a Kd of 7.55 ± 0.291 nM and was chosen as the lead candidate. 64Cu-NODAGA-PEG4-SL022-GGS demonstrated high binding affinity to CD38 expressing human myeloma MM.1S-CBR-GFP-WT cells, which was blocked by the non-radiolabeled version of the peptide analog and anti-CD38 clinical antibodies, daratumumab and isatuximab, by 58%, 73%, and 78%, respectively. The CD38 positive MM.1S-CBR-GFP-WT cells had > 68% enhanced cellular binding when compared to MM.1S-CBR-GFP-KO cells devoid of CD38. Furthermore, our new CD38-targeted radiopharmaceutical allowed visualization of tumors located in marrow rich bones, remaining there for up to 4 h. Clearance from non-target organs occurred within 60 min. Quantitative PET data from a murine disseminated tumor model showed significantly higher accumulation in the bones of tumor-bearing animals compared to tumor-naïve animals (SUVmax 2.06 ± 0.4 versus 1.24 ± 0.4, P = 0.02). Independently, tumor uptake of the target compound was significantly higher (P = 0.003) compared to the scrambled peptide, 64Cu-NODAGA-PEG4-SL041-GGS (SL041: Thr-Tyr-His-Ile-Pro-Ile-Val). The subcutaneous MM model demonstrated significantly higher accumulation in tumors compared to muscle at 1 and 4 h after tracer administration (SUVmax 0.8 ± 0.2 and 0.14 ± 0.04, P = 0.04 at 1 h; SUVmax 0.89 ± 0.01 and 0.09 ± 0.01, P = 0.0002 at 4 h). CONCLUSIONS: The novel CD38-targeted, radiolabeled bioconjugates were specific and allowed visualization of MM, providing a starting point for the clinical translation of such tracers for the detection of MM.

Induction of complementary immunogenic necroptosis and apoptosis cell death pathways inhibits cancer metastasis and relapse.

Interactions of light-sensitive drugs and materials with Cerenkov radiation-emitting radiopharmaceuticals generate cytotoxic reactive oxygen species (ROS) to inhibit localized and disseminated cancer progression, but the cell death mechanisms underlying this radionuclide stimulated dynamic therapy (RaST) remain elusive. Using ROS-regenerative nanophotosensitizers coated with a tumor-targeting transferrin-titanocene complex (TiO2-TC-Tf) and radiolabeled 2-fluorodeoxyglucose (18FDG), we found that adherent dying cells maintained metabolic activity with increased membrane permeabilization. Mechanistic assessment of these cells revealed that RaST activated the expression of RIPK-1 and RIPK-3, which mediate necroptosis cell death. Subsequent recruitment of the nuclear factors kappa B and the executioner mixed lineage kinase domain-like pseudo kinase (MLKL) triggered plasma membrane permeabilization and pore formation, respectively, followed by the release of cytokines and immunogenic damage-associated molecular patterns (DAMPs). In immune-deficient breast cancer models with adequate stroma and growth factors that recapitulate the human tumor microenvironment, RaST failed to inhibit tumor progression and the ensuing lung metastasis. A similar aggressive tumor model in immunocompetent mice responded to RaST, achieving a remarkable partial response (PR) and complete response (CR) with no evidence of lung metastasis, suggesting active immune system engagement. RaST recruited antitumor CD11b+, CD11c+, and CD8b+ effector immune cells after initiating dual immunogenic apoptosis and necroptosis cell death pathways in responding tumors in vivo. Over time, cancer cells upregulated the expression of negative immune regulating cytokine (TGF-ß) and soluble immune checkpoints (sICP) to challenge RaST effect in the CR mice. Using a signal-amplifying cancer-imaging agent, LS301, we identified latent minimal residual disseminated tumors in the lymph nodes (LNs) of the CR group. Despite increased protumor immunogens in the CR mice, RaST prevented cancer relapse and metastasis through dynamic redistribution of ROS-regenerative TiO2 from bones at the early treatment stage to the spleen and LNs, maintaining active immunity against cancer progression and migration. This study reveals the immune-mechanistic underpinnings of RaST-mediated antitumor immune response and highlights immunogenic reprogramming of tumors in response to RaST. Overcoming apoptosis resistance through complementary necroptosis activation paves the way for strategic drug combinations to improve cancer treatment.

Detection of MMP-2 and MMP-9 activity in vivo with a triple-helical peptide optical probe.

We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (k(cat)/K(M) = 30,000 s(-1) M(-1)) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than that of muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.

Near-infrared fluorescent divalent RGD ligand for integrin αvß3-targeted optical imaging.

A new near-infrared fluorescent compound containing two cyclic RGD motifs, cypate-[c(RGDfK)](2) (1), was synthesized based on a carbocyanine fluorophore bearing two carboxylic acid groups (cypate) for integrin α(v)ß(3)-targeting. Compared with its monovalent counterpart cypate-c(RGDfK) (2), 1 exhibited remarkable improvements in integrin α(v)ß(3) binding affinity and tumor uptake in nude mice of A549. The results suggest that cypate-linked divalent ligands can serve as an important molecular platform for exploring receptor-targeted optical imaging and treatment of various diseases.

Exploring new near-infrared fluorescent disulfide-based cyclic RGD peptide analogs for potential integrin-targeted optical imaging.

We synthesized disulfide-based cyclic RGD pentapeptides bearing a near-infrared fluorescent dye (cypate), represented by cypate-c(CRGDC) (1) for integrin-targeted optical imaging. These compounds were compared with the traditional lactam-based cyclic RGD counterpart, cypate-c(RGDfK) (2). Molecular modeling suggests that the binding affinity of 2 to integrin α(v)ß(3) is an order of magnitude higher than that of 1. This was confirmed experimentally, which further showed that substitution of Gly with Pro, Val and Tyr in 1 remarkably hampered the α(v)ß(3) binding. Interestingly, cell microscopy with A549 cells showed that 1 exhibited higher cellular staining than 2. These results indicate that factors other than receptor binding affinity to α(v)ß(3) dimeric proteins mediate cellular uptake. Consequently, 1 and its analogs may serve as valuable molecular probes for investigating the selectivity and specificity of integrin targeting by optical imaging.

Identification of early intestinal neoplasia protein biomarkers using laser capture microdissection and MALDI MS.

Obtaining protein profiles from a homogeneous cell population in tissues can significantly improve our capability in protein biomarker discovery. In this study, unique protein profiles from the top and bottom sections of mouse crypts and Apc(Min+/-) adenomas were obtained using laser capture microdissection (LCM) combined with MALDI MS. Statistically significant protein peaks with differential expression were selected, and a set of novel protein biomarkers were identified. Immunohistochemistry was performed to confirm the differentially expressed protein biomarkers found by LCM combined with MALDI MS. To validate the relevance of the findings in human colorectal cancer (CRC), S100A8 was further confirmed in human CRC using immunohistochemistry. In addition, S100A8 was found to have an increased expression at different human CRC stages (Duke's A-D) compared with controls at both protein (n = 168 cases) and RNA (n = 215 cases) levels. Overall LCM combined with MALDI MS is a promising method to identify intestinal protein biomarkers from minute amounts of tissue. The novel protein biomarkers identified from the top and bottom crypts will increase our knowledge of the specific protein changes taking place during cell migration from the crypt bottom to top. In addition, the identified cancer protein biomarkers will aid in the exploration of colorectal tumorigenesis mechanisms as well as in the advancement of molecularly based diagnosis of colorectal cancer.

Orthogonal targeting of osteoclasts and myeloma cells for radionuclide stimulated dynamic therapy induces multidimensional cell death pathways.

Rationale: Multiple myeloma (MM) is a multifocal malignancy of bone marrow plasma cells, characterized by vicious cycles of remission and relapse that eventually culminate in death. The disease remains mostly incurable largely due to the complex interactions between the bone microenvironment (BME) and MM cells (MMC). In the "vicious cycle" of bone disease, abnormal activation of osteoclasts (OCs) by MMC causes severe osteolysis, promotes immune evasion, and stimulates the growth of MMC. Disrupting these cancer-stroma interactions would enhance treatment response. Methods: To disrupt this cycle, we orthogonally targeted nanomicelles (NM) loaded with non-therapeutic doses of a photosensitizer, titanocene (TC), to VLA-4 (α4ß1, CD49d/CD29) expressing MMC (MM1.S) and αvß3 (CD51/CD61) expressing OC. Concurrently, a non-lethal dose of a radiopharmaceutical, 18F-fluorodeoxyglucose ([18F]FDG) administered systemically interacted with TC (radionuclide stimulated therapy, RaST) to generate cytotoxic reactive oxygen species (ROS). The in vitro and in vivo effects of RaST were characterized in MM1.S cell line, as well as in xenograft and isograft MM animal models. Results: Our data revealed that RaST induced non-enzymatic hydroperoxidation of cellular lipids culminating in mitochondrial dysfunction, DNA fragmentation, and caspase-dependent apoptosis of MMC using VLA-4 avid TC-NMs. RaST upregulated the expression of BAX, Bcl-2, and p53, highlighting the induction of apoptosis via the BAK-independent pathway. The enhancement of multicopper oxidase enzyme F5 expression, which inhibits lipid hydroperoxidation and Fenton reaction, was not sufficient to overcome RaST-induced increase in the accumulation of irreversible function-perturbing α,ß-aldehydes that exerted significant and long-lasting damage to both DNA and proteins. In vivo, either VLA-4-TC-NM or αvß3-TC-NMs RaST induced a significant therapeutic effect on immunocompromised but not immunocompetent MM-bearing mouse models. Combined treatment with both VLA-4-TC-NM and αvß3-TC-NMs synergistically inhibited osteolysis, reduced tumor burden, and prevented rapid relapse in both in vivo models of MM. Conclusions: By targeting MM and bone cells simultaneously, combination RaST suppressed MM disease progression through a multi-prong action on the vicious cycle of bone cancer. Instead of using the standard multidrug approach, our work reveals a unique photophysical treatment paradigm that uses nontoxic doses of a single light-sensitive drug directed orthogonally to cancer and bone cells, followed by radionuclide-stimulated generation of ROS to inhibit tumor progression and minimize osteolysis in both immunocompetent murine and immunocompromised human MM models.

Quantitative analysis of the secretome of TGF-beta signaling-deficient mammary fibroblasts.

Transforming growth factor beta (TGF-beta) is a master regulator of autocrine and paracrine signaling pathways between a tumor and its microenvironment. Decreased expression of TGF-beta type II receptor (TbetaRII) in stromal cells is associated with increased tumor metastasis and shorter patient survival. In this study, SILAC quantitative proteomics was used to identify differentially externalized proteins in the conditioned media from the mammary fibroblasts with or without intact TbetaRII. Over 1000 proteins were identified and their relative differential levels were quantified. Immunoassays were used to further validate identification and quantification of the proteomic results. Differential expression was detected for various extracellular proteins, including proteases and their inhibitors, growth factors, cytokines, and extracellular matrix proteins. CXCL10, a cytokine found to be up-regulated in the TbetaRII knockout mammary fibroblasts, is shown to directly stimulate breast tumor cell proliferation and migration. Overall, this study revealed hundreds of specific extracellular protein changes modulated by deletion of TbetaRII in mammary fibroblasts, which may play important roles in the tumor microenvironment. These results warrant further investigation into the effects of inhibiting the TGF-beta signaling pathway in fibroblasts because systemic inhibition of TGF-beta signaling pathways is being considered as a potential cancer therapy.

A novel comprehensive wave-form MS data processing method.

MOTIVATION: Mass spectrometry (MS) can generate high-throughput protein profiles for biomedical research to discover biologically related protein patterns/biomarkers. The noisy functional MS data collected by current technologies, however, require consistent, sensitive and robust data-processing techniques for successful biomedical application. Therefore, it is important to detect features precisely for each spectrum, quantify them well and assign a unique label to features from the same protein/peptide across spectra. RESULTS: In this article, we propose a new comprehensive MS data preprocessing package, Wave-spec, which includes several novel algorithms. It can overcome several conventional difficulties. Wave-spec can be applied to multiple types of MS data generated with different MS technologies. Results from this new package were evaluated and compared to several existing approaches based on a MALDI-TOF MS dataset. AVAILABILITY: An example of MATLAB scripts used to implement the methods described in this article, along with Supplementary Figures, can be found at http://www.vicc.org/biostatistics/supp.php. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ultrabright fluorescent nanoscale labels for the femtomolar detection of analytes with standard bioassays.

The detection and quantification of low-abundance molecular biomarkers in biological samples is challenging. Here, we show that a plasmonic nanoscale construct serving as an 'add-on' label for a broad range of bioassays improves their signal-to-noise ratio and dynamic range without altering their workflow and readout devices. The plasmonic construct consists of a bovine serum albumin scaffold with approximately 210 IRDye 800CW fluorophores (with a fluorescence intensity approximately 6,700-fold that of a single 800CW fluorophore), a polymer-coated gold nanorod acting as a plasmonic antenna and biotin as a high-affinity biorecognition element. Its emission wavelength can be tuned over the visible and near-infrared spectral regions by modifying its size, shape and composition. It improves the limit of detection in fluorescence-linked immunosorbent assays by up to 4,750-fold and is compatible with multiplexed bead-based immunoassays, immunomicroarrays, flow cytometry and immunocytochemistry methods, and it shortens overall assay times (to 20 min) and lowers sample volumes, as shown for the detection of a pro-inflammatory cytokine in mouse interstitial fluid and of urinary biomarkers in patient samples.

Selective imaging of solid tumours via the calcium-dependent high-affinity binding of a cyclic octapeptide to phosphorylated Annexin A2.

The heterogeneity and continuous genetic adaptation of tumours complicate their detection and treatment via the targeting of genetic mutations. However, hallmarks of cancer such as aberrant protein phosphorylation and calcium-mediated cell signalling provide broadly conserved molecular targets. Here, we show that, for a range of solid tumours, a cyclic octapeptide labelled with a near-infrared dye selectively binds to phosphorylated Annexin A2 (pANXA2), with high affinity at high levels of calcium. Because of cancer-cell-induced pANXA2 expression in tumour-associated stromal cells, the octapeptide preferentially binds to the invasive edges of tumours and then traffics within macrophages to the tumour's necrotic core. As proof-of-concept applications, we used the octapeptide to detect tumour xenografts and metastatic lesions, and to perform fluorescence-guided surgical tumour resection, in mice. Our findings suggest that high levels of pANXA2 in association with elevated calcium are present in the microenvironment of most solid cancers. The octapeptide might be broadly useful for selective tumour imaging and for delivering drugs to the edges and to the core of solid tumours.

Electrospray Functionalization of Titanium Dioxide Nanoparticles with Transferrin for Cerenkov Radiation Induced Cancer Therapy.

Titanium dioxide (TiO2) nanoparticles have shown success as photosensitizers in the form of light-based cancer therapy called Cerenkov radiation induced therapy (CRIT). While TiO2 nanoparticles have been reported to be an effective therapeutic agent, there has been little work to control their functionalization and stability in aqueous suspension. In this work, the controlled coating of 25 nm diameter TiO2 nanoparticles with the glycoprotein transferrin (Tf) for application in CRIT was demonstrated using an electrospray system. Monodisperse nanoscale droplets containing TiO2 and Tf were dried during flight, coating the proteins on the surface of the metal oxide nanoparticles. Real-time scanning mobility particle sizing, dynamic light scattering, and transmission electron microscopy show efficient control of the Tf coating thickness when varying the droplet size and the ratio of Tf to TiO2 in the electrospray precursor suspension. Further, the functionality of Tf-coated TiO2 nanoparticles was demonstrated, and these particles were found to have enhanced targeting activity of Tf to the Tf receptor after electrospray processing. The electrospray-coated Tf/TiO2 particles were also found to be more effective at killing the multiple myeloma cell line MM1.S than that of nanoparticles prepared by other reported functionalization methods. In summary, this investigation not only provides a single-step functionalization technique for nanomaterials used in Cerenkov radiation induced therapy but also elucidates an electrospray coating technique for nanomaterials that can be used for a wide range of drug design and delivery purposes.

Dural lymphatics regulate clearance of extracellular tau from the CNS.

BACKGROUND: Alzheimer's disease is characterized by two main neuropathological hallmarks: extracellular plaques of amyloid-ß (Aß) protein and intracellular aggregates of tau protein. Although tau is normally a soluble monomer that bind microtubules, in disease it forms insoluble, hyperphosphorylated aggregates in the cell body. Aside from its role in AD, tau is also involved in several other neurodegenerative disorders collectively called tauopathies, such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), some forms of frontotemporal dementia, and argyrophilic grain disease (AGD). The prion hypothesis suggests that after an initial trigger event, misfolded forms of tau are released into the extracellular space, where they spread through different brain regions, enter cells, and seeding previously normal forms. Thus understanding mechanisms regulating the clearance of extracellular tau from the CNS is important. The discovery of a true lymphatic system in the dura and its potential role in mediating Aß pathology prompted us to investigate its role in regulating extracellular tau clearance. METHODS: To study clearance of extracellular tau from the brain, we conjugated monomeric human tau with a near-infrared dye cypate, and injected this labeled tau in the parenchyma of both wild-type and K14-VEGFR3-Ig transgenic mice, which lack a functional CNS lymphatic system. Following injection we performed longitudinal imaging using fluorescence molecular tomography (FMT) and quantified fluorescence to calculate clearance of tau from the brain. To complement this, we also measured tau clearance to the periphery by measuring plasma tau in both groups of mice. RESULTS: Our results show that a significantly higher amount of tau is retained in the brains of K14-VEGFR3-Ig vs. wild type mice at 48 and 72 h post-injection and its subsequent clearance to the periphery is delayed. We found that clearance of reference tracer human serum albumin (HSA) was also significantly delayed in the K14-VEGFR3-Ig mice. CONCLUSIONS: The dural lymphatic system appears to play an important role in clearance of extracellular tau, since tau clearance is impaired in the absence of functional lymphatics. Based on our baseline characterization of extracellular tau clearance, future studies are warranted to look at the interaction between tau pathology and efficiency of lymphatic function.

Variability of in situ proteomic profiling and implications for study design in colorectal tumors.

Knowledge of intrinsic tumor heterogeneity is vital for understanding of tumor progression mechanisms as well as for providing efficient treatments. In situ proteomic profiling of tumors is a powerful technology with potential to enhance our understanding of tumor biology, but sources of variability due to patient and tumor heterogeneity are poorly understood and are the topic of this investigation. Clarification of variability within case and between cases is also important for designing future studies. Direct protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a sensitive and powerful technology for obtaining hundreds of protein expression peaks from a thin tissue section. By combining robotic microspotting and laser capture microdissection with MALDI MS, we acquired multiple spectra per case to evaluate inter- and intra-case variability in human colorectal tumor and murine cecal carcinoma. We detected 256 peaks from 164 samples of 111 patients, which consisted of 55 normal colorectal mucosal samples, 24 adenomas, 71 primary carcinomas, and 14 hepatic metastases. In addition, we detected 291 peptide/protein peaks from 34 orthotopically transplanted murine cecal carcinomas and 14 hepatic metastases. In human colorectal samples, we observed that proteomic profiling in adenomas was more homogeneous across patients than in normal mucosa specimens (p=0.0008), but primary carcinoma exhibited greater heterogeneity than normal mucosa and adenomas (both p<0.0001). Murine cecal carcinomas were homogeneous within and between carcinomas, while their hepatic metastases tended toward greater intra-tumor differences (p<0.0001). Inter- and intra-case variability was approximately equal for many protein peaks. Acquiring up to 5 subsamples per case could reduce the total number of cases required, but further reduction from additional subsampling was modest unless intra-case variability comprises a greater proportion of total variation (e.g. >70%). In summary, this study characterizes intra- and inter-case variability of high-throughput protein expression in colorectal tumors, and provides guidance for the sample numbers required for in situ proteomic studies.

Design, synthesis, and evaluation of near infrared fluorescent multimeric RGD peptides for targeting tumors.

Molecular interactions between RGD peptides and integrins are known to mediate many biological and pathological processes. This has led to an increased interest in the development of RGD compounds with high affinity and improved selectivity for integrin receptors. In this study, we synthesized and evaluated a series of multimeric RGD compounds constructed on a dicarboxylic acid-containing near-infrared (NIR) fluorescent dye (cypate) for tumor targeting. An array of NIR fluorescent RGD compounds was prepared efficiently, including one RGD monomer (cypate-(RGD)(2)-NH(2)), two RGD dimers (cypate-(RGD)(2)-NH(2) and cypate-(RGD-NH(2))(2)), one trimer (cypate-(RGD)(3)-NH(2)), two tetramers (cypate-(RGD)(4)-NH(2) and cypate-[(RGD)(2)-NH(2)](2)), one hexamer (cypate-[(RGD)(3)-NH(2)](2)), and one octamer (cypate-[(RGD)(4)-NH(2)](2)). The binding affinity of the multimeric RGD compounds for alpha(v)beta(3) integrin receptor (ABIR) showed a remarkable increase relative to the monomer cypate-RGD-NH(2). Generally, the divalent linear arrays of the multimeric RGD units bound the ABIR with slightly higher affinity than their monovalent analogues. These results suggest that the receptor binding affinity was not only dependent on the number of RGD moieties but also on the spatial alignments of the pendant peptides. Internalization of the compounds by ABIR-positive tumor cells (A549) was monitored by NIR fluorescence microscopy. The data showed that endocytosis of the octameric RGD derivative was significantly higher by comparison to other compounds in this study. In vivo noninvasive optical imaging and biodistribution data showed that the compounds were retained in A549 tumor tissue. These results clearly demonstrated that an array of simple RGD tripeptides on a NIR fluorescent dye core can be recognized by ABIR. Optimization of the spatial alignment of the RGD moieties through careful molecular design and library construction could induce multivalent ligand-receptor interactions useful for in vivo tumor imaging and tumor-targeted therapy.