Bone Endosteal Mimics Regulates Breast Cancer Development and Phenotype.

Bone is a frequent site for metastatic development in various cancer types, including breast cancer, with a grim prognosis due to the distinct bone environment. Despite considerable advances, our understanding of the underlying processes leading to bone metastasis progression remains elusive. Here, we applied a bioactive three-dimensional (3D) model capable of mimicking the endosteal bone microenvironment. MDA-MB-231 and MCF7 breast cancer cells were cultured on the scaffolds, and their behaviors and the effects of the biomaterial on the cells were examined over time. We demonstrated that close interactions between the cells and the biomaterial affect their proliferation rates and the expression of c-Myc, cyclin D, and KI67, leading to cell cycle arrest. Moreover, invasion assays revealed increased invasiveness within this microenvironment. Our findings suggest a dual role for endosteal mimicking signals, influencing cell fate and potentially acting as a double-edged sword, shuttling between cell cycle arrest and more active, aggressive states.

Complexes of Cu-Polysaccharide of a Marine Red Microalga Produce Spikes with Antimicrobial Activity.

Metal-polysaccharides have recently raised significant interest due to their multifunctional bioactivities. The antimicrobial activity of a complex of Cu2O with the sulfated polysaccharide (PS) of the marine red microalga Porphyridium sp. was previously attributed to spikes formed on the complex surface (roughness). This hypothesis was further examined here using other Cu-PS complexes (i.e., monovalent-Cu2O, CuCl and divalent-CuO, CuCl2). The nanostructure parameters of the monovalent complexes, namely, longer spikes (1000 nm) and greater density (2000-5000 spikes/µm2) were found to be related to the superior inhibition of microbial growth and viability and biofilm formation. When Escherichia coli TV1061, used as a bioluminescent test organism, was exposed to the monovalent Cu-PS complexes, enhanced bioluminescence accumulation was observed, probably due to membrane perforation by the spikes on the surface of the complexes and consequent cytoplasmic leakage. In addition, differences were found in the surface chemistry of the monovalent and divalent Cu-PS complexes, with the monovalent Cu-PS complexes exhibiting greater stability (ζ-potential, FTIR spectra, and leaching out), which could be related to spike formation. This study thus supports our hypothesis that the spikes protruding from the monovalent Cu-PS surfaces, as characterized by their aspect ratio, are responsible for the antimicrobial and antibiofilm activities of the complexes.

Machine-Learning-Aided Quantification of Area Coverage of Adherent Cells from Phase-Contrast Images.

The advances in machine learning (ML) software availability, efficiency, and friendliness, combined with the increase in the computation power of personal computers, are harnessed to rapidly and (relatively) effortlessly analyze time-lapse image series of adherent cell cultures, taken with phase-contrast microscopy (PCM). Since PCM is arguably the most widely used technique to visualize adherent cells in a label-free, noninvasive, and nondisruptive manner, the ability to easily extract quantitative information on the area covered by cells, should provide a valuable tool for investigation. We demonstrate two cases, in one we monitor the shrinking of cells in response to a toxicant, and in the second we measure the proliferation curve of mesenchymal stem cells (MSCs).

Chemical Antibody Mimics Inhibit Cadherin-Mediated Cell-Cell Adhesion: A Promising Strategy for Cancer Therapy.

One of the most promising strategies to treat cancer is the use of therapeutic antibodies that disrupt cell-cell adhesion mediated by dysregulated cadherins. The principal site where cell-cell adhesion occurs encompasses Trp2 found at the N-terminal region of the protein. Herein, we employed the naturally exposed highly conserved peptide Asp1-Trp2-Val3-Ile4-Pro5-Pro6-Ile7, as epitope to prepare molecularly imprinted polymer nanoparticles (MIP-NPs) to recognize cadherins. Since MIP-NPs target the site responsible for adhesion, they were more potent than commercially available therapeutic antibodies for inhibiting cell-cell adhesion in cell aggregation assays, and for completely disrupting three-dimensional tumor spheroids as well as inhibiting invasion of HeLa cells. These biocompatible supramolecular anti-adhesives may potentially be used as immunotherapeutic or sensitizing agents to enhance antitumor effects of chemotherapy.

Trafficking of MHC molecules to the cell surface creates dynamic protein patches.

Major histocompatibility complex class I (MHC-I) molecules signal infection or transformation by engaging receptors on T lymphocytes. The spatial organization of MHC-I on the plasma membranes is important for this engagement. We and others have shown that MHC-I molecules, like other membrane proteins, are not uniformly distributed, but occur in patches in the plasma membrane. Here, we describe the temporal details of MHC-I patch formation and combine them with the spatial details, which we have described earlier, to yield a comprehensive quantitative description of patch formation. MHC-I is delivered to the plasma membrane in clathrin-coated vesicles, arriving at a rate of ∼2.5×10(-3) µm(-1) min(-1) (or about two arrivals per minute over the whole cell). The vesicles dock and fuse at non-random, apparently targeted, locations on the membrane and the newly delivered MHC-I molecules form patches that are a few hundred nanometers in diameter. The patches are maintained at steady state by a dynamic equilibrium between the rate of delivery and the rate of hindered diffusion of MHC-I molecules out of the patches (caused by components of the actin cytoskeleton).

Dextran-lipase conjugates as tools for low molecular weight ligand immobilization in microarray development.

The development of effective array biosensors relies heavily on careful control of the density of surface-immobilized ligands on the transducing platform. In this paper we describe the synthesis of new dextran-lipase conjugates for use in immobilizing low molecular weight haptens onto glass planar waveguides for immunosensor development. The conjugates were synthesized by immobilizing bacterial thermoalkalophilic lipases (Geobacillus thermocatenulatus lipase 2, BTL2) on agarose macroporous beads, followed by covalent coupling to dextran networks of variable molecular weight (1500-40000). The chimeras were immobilized via nonspecific hydrophobic interactions onto glass planar waveguides modified with 1,1,1,3,3,3-hexamethyldisilazane to obtain highly ordered and homogeneous molecular architectures as confirmed by atomic force microscopy. Microcystin LR (MCLR) was covalently bound to the dextran-BTL2 conjugates. The usefulness of this approach in immunosensor development was demonstrated by determining amounts of MCLR down to a few picograms per liter with an automated array biosensor and evanescent wave excitation for fluorescence measurements of attached DyLight649-labeled secondary antibody. Modifying BTL2 with dextrans of an increased molecular weight (>6000) provided surfaces with an increased loading capacity that was ascribed to the production of three-dimensional surfaces by the effect of analyte binding deep in the volume, leading to expanded dynamic ranges (0.09-136.56 ng L(-1)), lower limits of detection (0.007 ± 0.001 ng L(-1)), and lower IC50 values (4.4 ± 0.7 ng L(-1)). These results confirm the effectiveness of our approach for the development of high-performance biosensing platforms.

CD44 in Bone Metastasis Development: A Key Player in the Fate Decisions of the Invading Cells?

A participant in key developmental processes, the adhesion glycoprotein CD44 is also expressed in several types of malignancies and can promote metastasis. In addition, the expression of CD44 isoforms in different types of cancer such as prostate and breast cancers may facilitate bone metastases by enhancing tumorigenicity, osteomimicry, cell migration, homing to bone, and anchorage within the bone specialized domains. Moreover, there is evidence that the CD44-ICD fragments in breast cancer cells may promote the cells' osteolytic nature. Yet the mechanisms by which CD44 and its downstream effectors promote the establishment of these cells within the bone are not fully elucidated. In this review, we summarize the current data on the roles played by CD44 in cancer progression and bone metastasis and the possible effects of its interaction with the different components of the bone marrow milieu.

ZnT1 induces a crosstalk between T-type and L-type calcium channels through interactions with Raf-1 kinase and the calcium channel ß2 subunit.

ZnT1 is a major zinc transporter that regulates cellular zinc homeostasis. We have previously shown that ZnT1 has additional functions that are independent of its activity as a Zn2+ extruder. These include inhibition of the L-type calcium channel (LTCC) through interaction with the auxiliary ß-subunit of the LTCC and activation of the Raf-ERK signaling leading to augmented activity of the T-type calcium channel (TTCC). Our findings indicate that ZnT1 increases TTCC activity by enhancing the trafficking of the channel to the plasma membrane. LTCC and TTCC are co-expressed in many tissues and have different functions in a variety of tissues. In the current work, we investigated the effect of the voltage-gated calcium channel (VGCC) ß-subunit and ZnT1 on the crosstalk between LTCC and TTCC and their functions. Our results indicate that the ß-subunit inhibits the ZnT1-induced augmentation of TTCC function. This inhibition correlates with the VGCC ß-subunit-dependent reduction in ZnT1-induced activation of Ras-ERK signaling. The effect of ZnT1 is specific, as the presence of the ß-subunit did not change the effect of endothelin-1 (ET-1) on TTCC surface expression. These findings document a novel regulatory function of ZnT1 serving as a mediator in the crosstalk between TTCC and LTCC. Overall, we demonstrate that ZnT1 binds and regulates the activity of the ß-subunit of VGCC and Raf-1 kinase and modulates surface expression of the LTCC and TTCC catalytic subunits, consequently modulating the activity of these channels.

Lifetime of major histocompatibility complex class-I membrane clusters is controlled by the actin cytoskeleton.

Lateral heterogeneity of cell membranes has been demonstrated in numerous studies showing anomalous diffusion of membrane proteins; it has been explained by models and experiments suggesting dynamic barriers to free diffusion, that temporarily confine membrane proteins into microscopic patches. This picture, however, comes short of explaining a steady-state patchy distribution of proteins, in face of the transient opening of the barriers. In our previous work we directly imaged persistent clusters of MHC-I, a type I transmembrane protein, and proposed a model of a dynamic equilibrium between proteins newly delivered to the cell surface by vesicle traffic, temporary confinement by dynamic barriers to lateral diffusion, and dispersion of the clusters by diffusion over the dynamic barriers. Our model predicted that the clusters are dynamic, appearing when an exocytic vesicle fuses with the plasma membrane and dispersing with a typical lifetime that depends on lateral diffusion and the dynamics of barriers. In a subsequent work, we showed this to be the case. Here we test another prediction of the model, and show that changing the stability of actin barriers to lateral diffusion changes cluster lifetimes. We also develop a model for the distribution of cluster lifetimes, consistent with the function of barriers to lateral diffusion in maintaining MHC-I clusters.

ZnT-1 enhances the activity and surface expression of T-type calcium channels through activation of Ras-ERK signaling.

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.

Molecularly imprinted polymer nanogels targeting the HAV motif in cadherins inhibit cell-cell adhesion and migration.

Cadherins are cell-surface proteins that mediate cell-cell adhesion. By regulating their grip formation and strength, cadherins play a pivotal role during normal tissue morphogenesis and homeostasis of multicellular organisms. However, their dysfunction is associated with cell migration and proliferation, cancer progression and metastasis. The conserved amino acid sequence His-Ala-Val (HAV) in the extracellular domain of cadherins is implicated in cadherin-mediated adhesion and migration. Antagonists of cadherin adhesion such as monoclonal antibodies and small molecule inhibitors based on HAV peptides, are of high therapeutic value in cancer treatment. However, antibodies are not stable outside their natural environment and are expensive to produce, while peptides have certain limitations as a drug as they are prone to proteolysis. Herein, we propose as alternative, a synthetic antibody based on molecularly imprinted polymer nanogels (MIP-NGs) to target the HAV domain. The MIP-NGs are biocompatible, have high affinity for N-cadherin and inhibit cell adhesion and migration of human cervical adenocarcinoma (HeLa) cells, as demonstrated by cell aggregation and Matrigel invasion assays, respectively. The emergence of MIPs as therapeutics for fighting cancer is still in its infancy and this novel demonstration reinforces the fact that they have a rightful place in cancer treatment.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells.

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Patterning nanostructured, synthetic, polymeric receptors by simultaneous projection photolithography, nanomolding, and molecular imprinting.

Microscope projection photolithography is combined with nanomolding and molecular imprinting for the fast microfabrication of molecularly imprinted polymer (MIP) arrays in the form of micrometric islands of nanofilaments. Dot diameters from 70-90 µm are easily obtained using a 10× objective and a photomask carrying the desired pattern. The dots are composed of parallel nanofilaments of a high aspect ratio, 150 nm in diameter and several micrometers in length, which are obtained through a nanomolding procedure on porous alumina. The arrays are molecularly imprinted with the small molecule fluorescein or with the protein myoglobin. The fluorescein MIP arrays are able to specifically recognize their target, as demonstrated by fluorescence microscopy. A four-fold increase in binding capacity and imprinting factor (IF = 13) is obtained compared to non-nanostructured porous dots. Imprinting of the nanofilament arrays with the protein myoglobin as the template is also possible and allows for a high imprinting factor of 4.3. Such nanostructured microarrays of synthetic receptors obtained by projection photolithography have great potential in biosensor and biochip development.

Immobilization of molecularly imprinted polymer nanoparticles in electrospun poly(vinyl alcohol) nanofibers.

We describe the fabrication of polymer nanofibers with entrapped molecularly imprinted polymer (MIP) nanoparticles and study their possible use in a fluorescence-based biosensor application. The MIP was imprinted with the fluorescent amino acid derivative dansyl-L-phenylalanine. Poly(vinyl alcohol) was used as a support for MIP nanoparticles because it is water-soluble and can be spun into very thin fibers. The fibers were characterized by atomic force microscopy and optical microscopy, and fluorescence microscopy was used for the characterization of target binding to the MIP. The fibers show close to 100% recovery upon extraction and rebinding of the target molecule. The selectivity of the system has been demonstrated through competitive binding experiments with nonfluorescent analogues boc-L-phenylalanine and boc-D-phenylalanine.

Molecular basis for zinc transporter 1 action as an endogenous inhibitor of L-type calcium channels.

The L-type calcium channel (LTCC) has a variety of physiological roles that are critical for the proper function of many cell types and organs. Recently, a member of the zinc-regulating family of proteins, ZnT-1, was recognized as an endogenous inhibitor of the LTCC, but its mechanism of action has not been elucidated. In the present study, using two-electrode voltage clamp recordings in Xenopus oocytes, we demonstrate that ZnT-1-mediated inhibition of the LTCC critically depends on the presence of the LTCC regulatory beta-subunit. Moreover, the ZnT-1-induced inhibition of the LTCC current is also abolished by excess levels of the beta-subunit. An interaction between ZnT-1 and the beta-subunit, as demonstrated by co-immunoprecipitation and by fluorescence resonance energy transfer, is consistent with this result. Using surface biotinylation and total internal reflection fluorescence microscopy in HEK293 cells, we show a ZnT-1-dependent decrease in the surface expression of the pore-forming alpha(1)-subunit of the LTCC. Similarly, a decrease in the surface expression of the alpha(1)-subunit is observed following up-regulation of the expression of endogenous ZnT-1 in rapidly paced cultured cardiomyocytes. We conclude that ZnT-1-mediated inhibition of the LTCC is mediated through a functional interaction of ZnT-1 with the LTCC beta-subunit and that it involves a decrease in the trafficking of the LTCC alpha(1)-subunit to the surface membrane.

Cyclam-based "clickates": homogeneous and heterogeneous fluorescent sensors for Zn(II).

In an effort to improve upon the recently reported cyclam based zinc sensor 1, the "click"-generated 1,8-disubstituted analogue 2 has been prepared. The ligand shows a 2-fold increase in its fluorescence emission compared to 1 exclusively in the presence of Zn(II) that is typical of switch-on PET fluorescent sensors. Single crystal X-ray diffraction of complexes of model ligand 10 reveals that the configuration adopted by the macrocyclic framework is extremely sensitive to the metal ion to which it coordinates. For Zn(II), Mg(II), and Li(I) the metal ions adopt an octahedral geometry with a trans III configuration of the cyclam ring. In contrast for Ni(II) the ligand adopts the rare cis V configuration, while for Cu(II) a clear preference for five-coordinate geometry is displayed with a trans I configuration of the macrocyclic ring being observed in two essentially isostructural compounds prepared via different routes. The ligand displays an increased selectivity for Zn(II) compared to 1 in the majority of cases with excellent selectivity upheld over Na(I), Mg(II), Ca(II), Mn(II), Ni(II), Co(II), and Fe(III). In contrast for Cu(II) and Hg(II) little improvement was observed for 2 compared to 1 and for Cd(II) the selectivity of the new ligand was inferior. In the light of these findings and the slower response times for ligand 2, our original "click"-generated cyclam sensor system 1 was employed in a proof of concept study to prepare a heterogeneous sol-gel based material which retains its PET response to Zn(II). The versatile nature of the sol-gel process importantly allows the simple preparation of a variety of nanostructured materials displaying high surface area-volume ratio using fabrication methods such as soft lithography, electrospinning, and nanopipetting.

Writing droplets of molecularly imprinted polymers by nano fountain pen and detecting their molecular interactions by surface-enhanced Raman scattering.

Molecularly imprinted polymer (MIP) droplets were printed using a pipet or a nano fountain pen on surface-enhanced Raman scattering (SERS)-active surfaces, to directly monitor the uptake and release of a template molecule, the beta-blocking drug propranolol, by SERS. The monitored SERS bands can be related to the template, allowing for its detection but also identification in the MIP. This is an advantage if the technique is to be used during the development phase of MIPs as microstructures, but equally for the readout of MIP-based biochips.

Antitumor Effect of Lonidamine-Polypeptide-Peptide Nanoparticles in Breast Cancer Models.

Biomaterials folded into nanoparticles (NPs) can be utilized as targeted drug delivery systems for cancer therapy. NPs may provide a vehicle for the anticancer drug lonidamine (LND), which inhibits glycolysis but was suspended from use at the clinical trial stage because of its hepatotoxicity due to poor solubility and pharmacokinetic properties. The NPs prepared by coassembly of the anionic polypeptide poly gamma glutamic acid (γ-PGA) and a designed amphiphilic and positively charged peptide (designated as mPoP-NPs) delivered LND to the mitochondria in cell cultures. In this study, we demonstrate that LND-mPoP-NP effective drug concentrations can be increased to reach therapeutically relevant concentrations. The self-assembled NP solution was subjected to snap-freezing and lyophilization and the resultant powder was redissolved in a tenth of the original volume. The NP size and their ability to target the proximity of the mitochondria of breast cancer cells were both maintained in this new formulation, C-LND-mPoP-NPs. Furthermore, these NPs exhibited 40% better cytotoxicity, relative to the nonlyophilized LND-mPoP-NPs and led to tumor growth inhibition with no adverse side effects upon intravenous administration in a xenograft breast cancer murine model.

The forces applied by cilia depend linearly on their frequency due to constant geometry of the effective stroke.

Mucus propelling cilia are excitable by many stimulants, and have been shown to increase their beating frequency up to threefold, by physiological extracellular stimulants, such as adenosine-triphosphate, acetylcholine, and others. This is thought to represent the evolutionary adaptation of mucociliary systems to the need of rapid and efficient cleansing the airways of foreign particles. However, the mucus transport velocity depends not only on the beat frequency of the cilia, but on their beat pattern as well, especially in the case of mucus bearing cilia that beat in a complex, three-dimensional fashion. In this study, we directly measured the force applied by live ciliary tissues with an atomic force microscope, and found that it increases linearly with the beating frequency. This implies that the arc swept by the cilia during their effective stroke remains unchanged during frequency increase, thus leading to a linear dependence of transport velocity on the beat frequency. Combining the atomic force microscope measurements with optical measurements, we have indications that the recovery stroke is performed on a less inclined plane, leading to an effective shortening of the overall path traveled by the cilia tip during this nontransporting phase of their beat pattern. This effect is observed to be independent of the type of stimulant (temperature or chemical), chemical (adenosine-triphosphate or acetylcholine), or concentration (1 microM-100 microM), indicating that this behavior may result from internal details of the cilium mechanical structure.