Preclinical Evaluation of a Fluorescent Probe Targeting Receptor CDCP1 for Identification of Ovarian Cancer.

Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.

Genomic mosaicism underlies the adaptation of marine Synechococcus ecotypes to distinct oceanic iron niches.

Phytoplankton are limited by iron (Fe) in ~40% of the world's oceans including high-nutrient low-chlorophyll (HNLC) regions. While low-Fe adaptation has been well-studied in large eukaryotic diatoms, less is known for small, prokaryotic marine picocyanobacteria. This study reveals key physiological and genomic differences underlying Fe adaptation in marine picocyanobacteria. HNLC ecotype CRD1 strains have greater physiological tolerance to low Fe congruent with their expanded repertoire of Fe transporter, storage and regulatory genes compared to other ecotypes. From metagenomic analysis, genes encoding ferritin, flavodoxin, Fe transporters and siderophore uptake genes were more abundant in low-Fe waters, mirroring paradigms of low-Fe adaptation in diatoms. Distinct Fe-related gene repertories of HNLC ecotypes CRD1 and CRD2 also highlight how coexisting ecotypes have evolved independent approaches to life in low-Fe habitats. Synechococcus and Prochlorococcus HNLC ecotypes likewise exhibit independent, genome-wide reductions of predicted Fe-requiring genes. HNLC ecotype CRD1 interestingly was most similar to coastal ecotype I in Fe physiology and Fe-related gene content, suggesting populations from these different biomes experience similar Fe-selective conditions. This work supports an improved perspective that phytoplankton are shaped by more nuanced Fe niches in the oceans than previously implied from mostly binary comparisons of low- versus high-Fe habitats and populations.

Dual Site-Specific Labeling of an Antibody Fragment through Sortase A and π-Clamp Conjugation.

The functionalization of proteins with different cargo molecules is highly desirable for a broad range of applications. However, the reproducible production of defined conjugates with multiple functionalities is a significant challenge. Herein, we report the dual site-specific labeling of an antibody fragment, utilizing the orthogonal Sortase A and π-clamp conjugation methods, and demonstrate that binding of the antibody fragment to its target receptor is retained after dual labeling.

Small neutral Gd(iii) tags for distance measurements in proteins by double electron-electron resonance experiments.

Spin labels containing a Gd(iii) ion have become important for measuring nanometer distances in proteins by double electron-electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(iii) tag used. Here we report the performance of two Gd(iii) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B-NS3 protease than previously established Gd(iii) tags. The improved performance is consistent with the absence of specific hydrophobic or charge-charge interactions with the protein. In the case of the Zika virus NS2B-NS3 protease, unexpectedly broad Gd(iii)-Gd(iii) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B-NS3 protease, both in the presence and absence of an inhibitor.

Jellyfish Life Stages Shape Associated Microbial Communities, While a Core Microbiome Is Maintained Across All.

The key to 650 million years of evolutionary success in jellyfish is adaptability: with alternating benthic and pelagic generations, sexual and asexual reproductive modes, multitudes of body forms and a cosmopolitan distribution, jellyfish are likely to have established a plenitude of microbial associations. Here we explored bacterial assemblages in the scyphozoan jellyfish Chrysaora plocamia (Lesson 1832). Life stages involved in propagation through cyst formation, i.e., the mother polyp, its dormant cysts (podocysts), and polyps recently excysted (excysts) from podocysts - were investigated. Associated bacterial assemblages were assessed using MiSeq Illumina paired-end tag sequencing of the V1V2 region of the 16S rRNA gene. A microbial core-community was identified as present through all investigated life stages, including bacteria with closest relatives known to be key drivers of carbon, nitrogen, phosphorus, and sulfur cycling. Moreover, the fact that half of C. plocamia's core bacteria were also present in life stages of the jellyfish Aurelia aurita, suggests that this bacterial community might represent an intrinsic characteristic of scyphozoan jellyfish, contributing to their evolutionary success.

Small Gd(III) Tags for Gd(III)-Gd(III) Distance Measurements in Proteins by EPR Spectroscopy.

The C7-Gd and C8-Gd tags are compact hydrophilic cyclen-based lanthanide tags for conjugation to cysteine residues in proteins. The tags are enantiomers, which differ in the configuration of the 2-hydroxylpropyl pendant arms coordinating the lanthanide ion. Here, we report the electron paramagnetic resonance (EPR) performance of the C7-Gd ( S configuration) and C8-Gd ( R configuration) tags loaded with Gd(III) on two mutants of the homodimeric ERp29 protein. The W-band EPR spectra were found to differ between the tags in the free state and after conjugation to the protein. In addition, the spectra were sensitive to the labeling position, which may originate from an environment-dependent charge density on the Gd(III)-coordinating oxygens. This is in agreement with previous NMR experiments with different lanthanide ions, which suggested sensitivity to H-bonding. W-band 1H-ENDOR (electron-electron double resonance) experiments detected effects from orientation selection in the central transition, due to a relatively narrow distribution in the ZFS parameters as indicated by simulations. In contrast, the distance distributions derived from DEER (double electron-electron resonance) measurements were insensitive to the R or S configuration of the tags and did not exhibit any orientation selection effects. The DEER measurements faithfully reflected the different widths of the distance distributions at the different protein sites in agreement with previous DEER measurements using other Gd(III) tags. Due to their small size, short tether to the protein, and a broad central EPR transition, the C7-Gd and C8-Gd tags are attractive Gd(III) tags for measurements of relatively short (<4 nm) distances by EPR spectroscopy.

Double-Arm Lanthanide Tags Deliver Narrow Gd3+ -Gd3+ Distance Distributions in Double Electron-Electron Resonance (DEER) Measurements.

Double-arm cyclen-based Gd3+ tags are shown to produce accurate nanometer scale Gd3+ -Gd3+ distance measurements in double electron-electron resonance (DEER) experiments by confining the space accessible to the metal ion. The results show excellent agreement with predictions both for the maximum and width of the measured distance distributions. For distance measurements in proteins, the tags can be attached to two cysteine residues located in positions i and i+4, or i and i+8, of an α-helix. In the latter case, an additional mutation introducing an aspartic acid at position i+4 achieves particularly narrow distribution widths. The concept is demonstrated with cysteine mutants of T4 lysozyme and maltose binding protein. We report the narrowest Gd3+ -Gd3+ distance distributions observed to date for a protein. By limiting the contribution of tag mobility to the distances measured, double-arm Gd3+ tags open new opportunities to study the conformational landscape of proteins in solution with high sensitivity.

New Lanthanide Tag for the Generation of Pseudocontact Shifts in DNA by Site-Specific Ligation to a Phosphorothioate Group.

Pseudocontact shifts (PCS) generated by paramagnetic lanthanides provide a rich source of long-range structural restraints that can readily be measured by nuclear magnetic resonance (NMR) spectroscopy. Many different lanthanide-binding tags have been designed for site-specific tagging of proteins, but established routes for tagging DNA with a single metal ion rely on difficult chemical synthesis. Here we present a simple and practical strategy for site-specific tagging of inexpensive phosphorothioate (PT) oligonucleotides. Commercially available PT oligonucleotides are diastereomers with S and R stereoconfiguration at the backbone PT site. The respective SP and RP diastereomers can readily be separated by HPLC. A new alkylating lanthanide-binding tag, C10, was synthesized that delivered quantitative tagging yields with both diastereomers. PCSs were observed following ligation with the complementary DNA strand to form double-stranded DNA duplexes. The PCSs were larger for the SP than the RP oligonucleotide and good correlation between back-calculated and experimental PCSs was observed. The C10 tag can also be attached to cysteine residues in proteins, where it generates a stable thioether bond. Ligated to the A28C mutant of ubiquitin, the tag produced excellent fits of magnetic susceptibility anisotropy (Δχ) tensors, with larger tensors than for the tagged PT oligonucleotides, indicating that the tag is not completely immobilized after ligation with a PT group.

Mechanisms of increased Trichodesmium fitness under iron and phosphorus co-limitation in the present and future ocean.

Nitrogen fixation by cyanobacteria supplies critical bioavailable nitrogen to marine ecosystems worldwide; however, field and lab data have demonstrated it to be limited by iron, phosphorus and/or CO2. To address unknown future interactions among these factors, we grew the nitrogen-fixing cyanobacterium Trichodesmium for 1 year under Fe/P co-limitation following 7 years of both low and high CO2 selection. Fe/P co-limited cell lines demonstrated a complex cellular response including increased growth rates, broad proteome restructuring and cell size reductions relative to steady-state growth limited by either Fe or P alone. Fe/P co-limitation increased abundance of a protein containing a conserved domain previously implicated in cell size regulation, suggesting a similar role in Trichodesmium. Increased CO2 further induced nutrient-limited proteome shifts in widespread core metabolisms. Our results thus suggest that N2-fixing microbes may be significantly impacted by interactions between elevated CO2 and nutrient limitation, with broad implications for global biogeochemical cycles in the future ocean.

Installation of a Rigid EDTA-Like Motif into a Protein α-Helix for Paramagnetic NMR Spectroscopy with Cobalt(II) Ions.

Coupling two copies of an iminodiacetic acid-cysteine hybrid ligand to a pair of cysteine residues positioned in an i, i+4 arrangement within a protein α-helix leads to generation of an EDTA-like metal ion-binding motif. Rigid binding of a Co(II) ion by this motif produces pseudo-contact shifts suitable for paramagnetic NMR structural studies.

Asynchronous presentation of follicle center lymphoma and reactive lymphoid hyperplasia of the ocular adnexa.

Although ocular lymphoproliferative diseases may be rarely encountered by dermatopathologists, the frequency may increase particularly as more and more subspecialties rely on dermatopathology services. Emerging data suggest that there are some similarities between ocular and cutaneous lymphoproliferative diseases with respect to their clinical behavior, prognosis, and cytogenetics. In this case report, a patient with ocular follicle center lymphoma who subsequently developed ocular reactive lymphoid hyperplasia is presented with an accompanying review of the literature on the subjects. The encounter of both follicular center lymphoma and reactive lymphoid hyperplasia in the same patient provides a rare opportunity to compare and contrast the clinical, histological, and immunohistochemical findings of the respective lymphoproliferative diseases situated at opposite ends of the spectrum.

Overexpression of fetuin-a counteracts ectopic mineralization in a mouse model of pseudoxanthoma elasticum (abcc6(-/-)).

The pathologic hallmark of pseudoxanthoma elasticum (PXE) is ectopic mineralization of soft connective tissues. Recent studies have suggested that PXE is a metabolic disease, and perturbations in a number of circulatory factors have been postulated. One of them is fetuin-A, a 60-kDa glycoprotein synthesized in the liver and secreted into blood. Observations in targeted mutant mice (Ahsg(-/-)) and in cell culture model systems have shown that fetuin-A is a powerful anti-mineralization factor in circulation, and the serum levels of fetuin-A in patients with PXE as well as in a mouse model of PXE (Abcc6(-/-)) have been shown to be reduced by up to 30%. In this study, we tested the hypothesis that overexpression of fetuin-A in Abcc6(-/-) mice counteracts the ectopic mineralization. Delivery of an expression construct containing full-length mouse fetuin-A complementary DNA (cDNA), linked to a His-tag, to the liver of these mice resulted in elevated serum levels of this protein. As a consequence, soft tissue mineralization, which is a characteristic of Abcc6(-/-) mice, was reduced by approximately 70% at 12 weeks of age, but the effect was transient when examined 4 weeks later. The results suggest that normalization of serum fetuin-A, either through gene therapy approaches or by direct protein delivery to the circulation, may offer strategies for treating PXE and perhaps other heritable disorders of soft tissue mineralization.