Semipermeable Research Articles

Design of Cell‐Penetrating Domain Antibodies via a Genetically Encoded β‐Lactam Amino Acid

AbstractDomain antibodies such as monobodies provide an attractive immunoglobin fold for evolving high‐affinity protein binders targeting the intracellular proteins implicated in cell signalling. However, it remains a challenge to endow cell permeability to these small and versatile protein binders. Here, we report a streamlined approach combining orthogonal crosslinking afforded by a genetically encoded β‐lactam‐lysine (BeLaK) and genetic supercharging to generate cell‐penetrating monobodies. When introduced to the N‐terminal β‐strand of a series of supercharged monobodies, BeLaK enabled efficient inter‐strand crosslinking with the neighbouring lysine. Compared to its non‐crosslinked counterpart, a BeLaK‐crosslinked, +18‐charged monobody exhibited enhanced thermostability and greater cellular uptake at 40 nM. Moreover, this structurally rigidified, supercharged monobody inhibited ERK1/2 phosphorylation in KYSE‐520 esophageal cancer cell line at sub‐micromolar concentration, indicating significant endosomal escape after endocytosis. Together, the discovery of this BeLaK‐encoded, rigidified immunoglobin fold should facilitate the design of cell‐penetrating monobodies targeting intracellular signalling proteins.

Endothelial Damage in JAK2V617F Myeloproliferative Neoplasms with Splanchnic Vein Thrombosis.

JAK2V617F-mutated myeloproliferative neoplasms (MPN) exhibit abnormal proliferation of bone marrow progenitors and increased risk of thrombosis, specifically in splanchnic veins (SVT). The contribution of the endothelium to the development of the prothrombotic phenotype was explored. Plasma and serum samples from JAK2V617F MPN patients with (n=26) or without (n=7) thrombotic debut and different treatments, were obtained (n=33). Cultured endothelial cells (ECs) were exposed to serum samples from these patients and from healthy donors as controls. Changes in markers of inflammation (VCAM-1, ICAM-1), cell permeability (VE-cadherin), production of VWF, extracellular matrix (ECM) reactivity, and activation of intracellular signaling pathways related to stress, proliferation, inflammation (Akt, p44/42, IkBa), and JAK2/STAT3 pathway, were assessed by immunofluorescence, flow adhesion, SDS-PAGE and immunoblot. Additionally, circulating markers of endothelial activation and damage (VWF, sVCAM-1, sTNFRI, thrombomodulin, angiopoietin-2, a2-antiplasmin activity, PAI-1) were evaluated in Patients' plasma. The in vitro studies showed that EC exposure to MPN thrombotic patients' sera resulted in increased VCAM-1 and ICAM-1, and reduced VE-cadherin expression (p<0.05) at the cell surface. Production and release of VWF to the ECM were higher (p<0.05), with increased platelet adhesion after perfusing whole blood, being more noticeable in response to sera from non-treated patients. Furthermore, intracellular activation of Akt, p44/42, IkBa and JAK2/STAT3 was observed. Moreover, plasma levels of VWF, TNF-R1, VCAM-1, thrombomodulin, and angiopoietin-2 were higher in JAK2V617F+ MPN patients with thrombosis. The present findings suggest that circulating factors in MPNs with SVT debut induce endothelial proinflammatory and prothrombotic phenotypes, which are modulated in vitro with MPN treatment.

Dietary Compounds in the Prevention of Arsenic Induced Intestinal Toxicity In Vitro.

Recent studies show that inorganic arsenic (As) exerts a toxic effect on the intestinal epithelium, causing a significant increase in its permeability. This disruption of the epithelial barrier may favor the entry of contaminants or toxins into the systemic circulation, thus causing toxicity not only at the intestinal level but possibly also at the systemic level. The present study conducts an in vitro evaluation of the protective effect of various dietary supplements and plant extracts against the intestinal toxicity of inorganic As. Some of these compounds were found to exert a protective effect. A significant decrease was observed in intracellular reactive oxygen/nitrogen species (10-31%), as well as a lower secretion of the pro-inflammatory cytokine IL-8 (25-41%) in the intestinal monolayers treated with the supplements and extracts, compared with those exposed only to As(III). The most effective supplements (glutathione/cysteine/vitamin C and lipoic acid) also normalized the distribution of tight junction protein zonula occludens-1, with partial restoration of the paracellular permeability and cell regeneration capacity of the intestinal epithelial cells. The results obtained show that dietary supplements and plant extracts can reduce the intestinal barrier disruption caused by inorganic As, and this may have a positive impact at both local and systemic levels.

Determination of Osmotic Flow in Water Transport in an Illitic Clay

Experimental studies have shown that osmosis could be one of the mechanisms of water transport in porous materials that act, to a certain extent, as semipermeable membranes. In this paper, an experimental apparatus and the corresponding model to measure and determine the osmotic efficiency, σ, of bulk porous materials are described. Both the apparatus and model to interpret water transport in samples are modifications of those of Sherwood and Craster. In addition to σ, the transport parameters of the model include Darcy permeability and water and salt diffusivity. These parameters are used to calculate the ratio of the individual components of the total molar flow. We used the apparatus to measure cylindrical samples made from an illitic clay with a diameter of 45 mm and thickness of 5 mm. The measured transport coefficients were then used to estimate the relative importance of the individual contributions to the total flow of water through the samples. Our results show that the contribution of the osmosis is 82–88%, while the diffusion contributes only 11–13% and the Darcy flow caused by the pressure difference contributes only 1–5%. Even after considering the uncertainties in the measurement of the transport coefficients, which are estimated to be up to 22%, the results show that osmosis makes an important contribution to the total water flow and should not be neglected in general.

Pterostilbene protects against lipopolysaccharide-induced inflammation and blood–brain barrier disruption in immortalized brain endothelial cell lines in vitro

Brain microvascular endothelial cells are connected by tight junction (TJ) proteins and interacted by adhesion molecules, which participate in the selective permeability of the blood–brain barrier (BBB). The disruption of BBB is associated with the progression of cerebral diseases. Pterostilbene is a natural compound found in blueberries and grapes with a wide range of biological activities, including anti-inflammatory, antioxidant, and anti-diabetic effects. In this study, we investigated the protective effects of pterostilbene on LPS-stimulated mouse brain endothelial (bEnd.3) cells and underlying mechanisms. The results showed that pterostilbene effectively upregulated the expressions of tight junction (TJ) proteins such as zonula occludens (ZO)-1 and claudin-5 and downregulated the expression of adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, preventing BBB damage under LPS stimulation. Pterostilbene decreased the LPS-triggered expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 as well as the levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and nitric oxide (NO). Meanwhile, we found that pterostilbene exerted an inhibitory effect on nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in bEnd.3 cells upon LPS stimulation. Additionally, pterostilbene exhibited antioxidant effects by activating heme oxygenase 1 (HO-1). These findings indicated that pterostilbene protected against lipopolysaccharide (LPS)-induced inflammation, oxidative stress and blood–brain barrier (BBB) disruption in bEnd.3 cells.

Design of Cell-Penetrating Domain Antibodies via a Genetically Encoded β-Lactam Amino Acid.

Domain antibodies such as monobodies provide an attractive immunoglobin fold for evolving high-affinity protein binders targeting the intracellular proteins implicated in cell signalling. However, it remains a challenge to endow cell permeability to these small and versatile protein binders. Here, we report a streamlined approach combining orthogonal crosslinking afforded by a genetically encoded β-lactam-lysine (BeLaK) and genetic supercharging to generate cell-penetrating monobodies. When introduced to the N-terminal β-strand of a series of supercharged monobodies, BeLaK enabled efficient inter-strand crosslinking with the neighbouring lysine. Compared to its non-crosslinked counterpart, a BeLaK-crosslinked, +18-charged monobody exhibited enhanced thermostability and greater cellular uptake at 40 nM. Moreover, this structurally rigidified, supercharged monobody inhibited ERK1/2 phosphorylation in KYSE-520 esophageal cancer cell line at sub-micromolar concentration, indicating significant endosomal escape after endocytosis. Together, the discovery of this BeLaK-encoded, rigidified immunoglobin fold should facilitate the design of cell-penetrating monobodies targeting intracellular signalling proteins.

Oxygen Transport in Nanoporous SiN Membrane Compared to PDMS and Polypropylene for Microfluidic ECMO.

Extracorporeal Membrane Oxygenation (ECMO) serves as a crucial intervention for patients with severe pulmonary dysfunction by facilitating oxygenation and carbon dioxide removal. While traditional ECMO systems are effective, their large priming volumes and significant blood-contacting surface areas can lead to complications, particularly in neonates and pediatric patients. Microfluidic ECMO systems offer a promising alternative by miniaturizing the ECMO technology, reducing blood volume requirements, and minimizing device surface area to improve safety and efficiency. This study investigates the oxygen transport performance of three membrane types- polydimethylsiloxane (PDMS), polypropylene, and a novel nanoporous silicon nitride (SiN) membrane-in a microfluidic ECMO platform. While nanoporous membranes rely on pore-mediated diffusion and PDMS on polymer lattice diffusion, results show no significant differences in device oxygenation efficiency (p > 0.05). Blood-side factors, including the diffusion rate of oxygen through the red blood cell (RBC) membrane, RBC residence time, and hemoglobin binding kinetics, were identified as primary bottlenecks. Even computational models of a hypothetical infinitely permeable membrane corroborate the limited impact of membrane material. These findings suggest a shift in ECMO design priorities from membrane material to blood-side enhancements. This research provides a foundation for optimizing ECMO systems.

SPH-FE coupling for the simulation of confined flow through permeable deformable membranes

AbstractWe present an extension of smoothed particle hydrodynamics (SPH) toward fluid flows involving the interaction with permeable deformable membranes. For this purpose, a coupled SPH-FE method based on a variational formulation of the immersed boundary (IB) method is developed. In the proposed method, weakly compressible SPH is used for the discretization of the fluid and a finite element (FE) method for thin structures for the discretization of the membrane. We consider confined flow in a two-dimensional fluid domain, with the membrane being represented as an elastic beam. Adopting the framework available for the IB method, the flux through the permeable membrane as described by Darcy’s law is considered. Finally, the proposed SPH-FE method is applied to two benchmark problems, i.e., the contraction of a circular membrane and the deformation of a membrane in a channel flow, comparing the numerical results with available analytical solutions.

Interleukin 8 exacerbates age-related hearing loss through regulating perivascular-resident macrophage-like melanocytes viability and the permeability of the endothelial cells.

The etiology and mechanism causing Age-related hearing loss (ARHL) are not understood. This study aimed to investigate the molecular mechanism of interleukin 8 (IL-8) associated with ARHL. Sera content of IL-8 was significantly higher in patients with ARHL than normal volunteers and had a positive association with disease severity of ARHL. Human IL-8 (hIL-8) could exacerbate the progressive ARHL with time increase and promoted apoptosis of hair cells in cochlea. As the important component in maintaining the integrity of the blood-labyrinth barrier (BLB) and hearing function, cell viability of perivascular-resident macrophage-like melanocytes (PVM/Ms) was restrained while apoptosis of PVM/Ms was enhanced in the presence of hIL-8. Using a cell culture-based in vitro model, the permeability of the endothelial cells (ECs) monolayer increased markedly in the presence of IL-8-treated PVM/Ms or PVM/Ms-derived from LV5-hIL-8 mice as compared with the presence of PVM/Ms-derived from wild type (WT) mice or 12-months WT mice. Mechanistically, IL-8 exposure enhanced the expression of histone deacetylase 3 (HDAC3) in PVM/Ms and HDAC3 inhibitor significantly blocked the permeability of the ECs in the presence of IL-8-treated PVM/Ms. Besides, HDAC3 inhibitor had a protective effect on hIL-8-launched ARHL in mice. Collectively, the elevated of serum IL-8 in ARHL patients activated the activity of HDAC3 in PVM/Ms, subsequently increased the permeability of the ECs, resulting in the impairments of the BLB and hair cells in cochlea. Possibly, IL-8 could be used in the diagnosis of ARHL and these findings might help to identify the clinical therapeutic direction for ARHL.

The effect of platelet-derived growth factor-aa (PDGFA) conjugated with low-molecular-weight protamine (LMWP) on hair loss improvement effect

Platelet-derived growth factor-AA (PDGFA) is known to play an important role in hair loss and hair growth by involving in the anagen phase of the hair follicle growth cycle. In this study, we synthesized skin-permeable recombinant low-molecular-weight protamine (LMWP)-conjugated PDGFA (LMWP-PDGFA) by linking LMWP to the N terminus of PDGFA. We evaluated the hair loss improvement effect, wound healing efficacy, and skin permeability of LMWP-PDGFA. LMWP-PDGFA showed higher cell proliferation, cell permeability, 5α-reductase inhibition activity and IGF-1 activity than PDGFA in human hair follicle dermal papilla cells (HFDPC). We also found that LMWP-PDGFA increased cell migration and collagen synthesis better than PDGFA. These results indicate that LMWP-PDGFA can be a good candidate as a hair treatment agent with superior cell permeability than PDGFA. Additionally, it is expected that the efficacy of the protein can be maximized by combining LMWP with other functional proteins.

Design and optimization of hybrid seawater reverse osmosis-solar-driven desalination-pressure retarded osmosis system for energy efficient desalination maximizing economic potential.

Seawater reverse osmosis (SWRO)-pressure retarded osmosis (PRO) hybrid desalination system is being actively researched to reduce energy consumption by generating energy in the PRO. However, the SWRO-PRO hybrid system still faces the following challenges: low freshwater recovery and low energy generation. To resolve these challenges, this study first proposes a novel SWRO-Solar-driven desalination (SD)-PRO hybrid system for energy-efficient desalination. The proposed system comprises three major processes: SWRO for freshwater recovery, SD for freshwater recovery, and PRO for energy generation. First, the pressurized seawater passes through a semi-permeable SWRO membrane to produce freshwater, and the remaining concentrated brine enters the SD system. Second, an evaporator, that absorbs solar energy and quickly evaporates water floats on the SD system to recover additional freshwater. Third, the highly concentrated brine that remains unevaporated is used as a draw solution in PRO to generate energy. Consequently, the total freshwater recovery is increased by 14.54%, the specific energy consumption is reduced by 38.86%, and the levelized cost of the freshwater is reduced by 16.67% compared with the conventional SWRO-PRO system. Furthermore, the life cycle assessment results demonstrate that the proposed system is environmentally friendly. These results indicate that the proposed system is a feasible solution for sustainable desalination.

Development of continuous fabric phase sorptive extraction imidacloprid and acetamiprid from fruit juice samples using covalent organic frameworks prior to HPLC-MS/MS analysis

Herein, a fabric phase sorptive extraction procedure followed by high-performance liquid chromatography-tandem mass spectrometry was employed as an environmentally friendly and rapid sample preparation method for the simultaneous extraction of imidacloprid and acetamiprid residues in fruit juices. In this work, the covalent organic framework coated on cotton was introduced as the permeable fabric phase sorptive extraction membrane. By the means of approach, the unique characteristics of covalent organic framework sorbent along with the high contact area of the cotton substrate resulted in the formation of an efficient microextraction system that provided high extraction recovery of the desired pesticides from complicated samples. The effective factors on the efficiency of the developed method such as the sorbent amount, flow rate, ionic strength, and the kind and volume of elution solvent were evaluated and optimized through one parameter at a time method. Notably, the current procedure did not require clean-up, centrifugation step, and large number of organic solvents that is complies with green analytical chemistry principles. The coefficient of determination ≥ 0.9928 demonstrates the good linearity of the developed method the range of 17.3–500 ng.mL−1. The extraction recovery was 57 % for imidacloprid and 49 % for acetamiprid. The intra- and inter-day precisions, expressed as relative standard deviations, were ≤ 6.5 % and ≤ 7.1 %, respectively. For both pesticides, the limits of detection and quantification were less than 5.2 and 17 ng.mL−1, respectively.

Current status of cyclopropane fatty acids on bacterial cell membranes characteristics and physiological functions.

Wide-ranging sophisticated physiological activities of cell membranes are associated with changes in fatty acid structure and composition. The cfa gene is a core regulator of cell membrane fatty acid cyclopropanation reaction. Its encoded cyclopropane fatty acid synthase (CFA synthase) catalyzes the binding of unsaturated fatty acid (UFA) to methylene groups, which undergoes cyclopropanation modification to produce cyclopropane fatty acids (CFAs). Compelling evidence suggests a large role for the cfa gene and CFAs in bacterial adaptive responses. This review provides an overview of the relationship of CFAs with bacterial cell membrane properties and physiological functions, including the roles of cell membrane fluidity, stability, and permeability to protons, bacterial growth, acid resistance, and especially in bacterial antibiotic resistance and pathogenicity. The dysregulation and inhibition of the cfa gene may serve as potential therapeutic targets against bacterial drug resistance and pathogenicity. Therefore, elucidating the biological function of CFAs during the stationary growth phase therefore provides invaluable insights into the bacterial pathogenesis and the development of novel antimicrobial agents.

An environment-responsive platform based on acid-resistant metal organic framework for efficient oral insulin delivery

Oral insulin delivery is considered a revolutionary alternative to daily subcutaneous injections in terms of compliance and convenience. However, significant challenges remain in terms of inactivation in gastrointestinal environment and limited permeation across the intestinal epithelium. Herein, we used acid-resistant metal-organic framework (PCN-222) to load insulin and modified the exterior with sodium dodecyl sulfate (SDS) to achieve efficient oral insulin delivery. The PCN-222 nanocarrier with ordered mesoporous cage structure and suitable pore size achieved a high insulin loading of 75 %. The SDS on the surface of nanocarrier reduces its hydrophilicity while reversibly altering cell morphology and increasing epithelial cell permeability, thereby promoting intestinal epithelial absorption. The constructed particle (I@P@S) was encapsulated in sodium alginate (SA) microspheres to protect it from gastric acid degradation and releases it upon entry into the intestinal tract. Through an uptake pathway dominated by clathrin-mediated endocytosis, the released I@P@S realized efficient intestinal permeability and controlled insulin release under physiological conditions due to the phosphate sensitivity of PCN-222, leading to an in vivo bioavailability of 12.9 %. This work provides a valuable reference for the design of oral insulin delivery systems.

Immunosuppressant drug tacrolimus inhibits HUVEC angiogenesis and production of placental growth factor.

Tacrolimus is a cornerstone of immunosuppression in solid organ transplants, but its use is linked with the development of endothelial dysfunction. Pregnant solid organ transplant recipients are four to six times more likely to develop preeclampsia, which is also associated with endothelial dysfunction. Therefore, this in vitro study investigated the acute effects of tacrolimus on the expression of common angiogenic factors related to preeclampsia, and effects on angiogeneis in primary human tissues. Primary human umbilical vein endothelial cells (HUVECs) were exposed to tacrolimus (0, 5, 20, 50ng/mL) for 24h alone, or in combination with tumour necrosis factor (TNF, 10ng/mL) and high dose glucose (25mM). Cell culture concentrations of sFlt-1, PlGF and activin A were measured. In addition, the effect of tacrolimus on markers of endothelial dysfunction and permeability were assessed, as were the effect of tacrolimus on tube formation. Angiogenic factors and mRNA markers of oxidative stress and inflammation were also assessed in primary placental tissue after an acute 24h exposure to tacrolimus. Tacrolimus exposure significantly reduced HUVEC secretion of PlGF, increased production of activin A, andreduced tubular structure formation without impacting cell permeability or viability. There was no change in ICAM1 or VCAM1 expression in HUVECs treated with tacrolimus treatment alone, however co-culture with TNF significantly increased expression of ICAM1 and VCAM1. In placental explants tacrolimus did not change angiogenic factor production or markers of inflammation or oxidative stress. An acute tacrolimus exposure reduced PlGF secretion and impaired angiogenesis in primary endothelial cells, without affecting. These findings provide a potential mechanistic basis for tacrolimus to contribute to the endothelial dysfunction contributing to preeclampsia.

Oral multistage nanomedicine for synergistic chemo/chemodynamic/near-infrared-II photothermal cancer therapy.

The oral administration of drugs for cancer therapy can maintain optimal blood concentrations, is biologically safe and simple, and is preferred by many patients. However, the complex lumen environment, mucus layer, and intestinal epithelial cells are biological barriers that hinder the absorption of orally administered drugs. In this study, sea urchin-like manganese-doped copper selenide nanoparticles (Mn-Cu2-xSe NPs) were designed using an anion exchange method and coated with calcium alginate and chitosan (AC) to form Mn-Cu2-xSe@AC capsules. The pH-responsive swelling behavior of the AC protective layer aided doxorubicin (DOX)-loaded Mn-Cu2-xSe NPs in overcoming multiple biological barriers, maintained their stability in gastric acid, and facilitated the release of the NPs in the small intestine. The intestinal epithelial cell permeability of DOX/Mn-Cu2-xSe NPs was confirmed using a monolayer absorption model involving Caco-2 human epithelial cells. The released DOX/Mn-Cu2-xSe NPs smoothly passed through the mucus layer, and were absorbed by intestinal epithelial cells. In mice, the NPs circulated in the blood and passively targeted the tumors through blood circulation by enhancing the permeability and retention effect to achieve significant tumor suppression and reduce damage to normal tissues. In addition, the unique sea urchin-like morphology of Mn-Cu2-xSe NPs enhanced the absorption in the near-infrared-II (NIR-II) window for photothermal therapy, realized the near-infrared-stimulated response release of DOX for increased chemotherapy, and promoted the Fenton-like effect because of the doping of manganese ions for chemodynamic therapy. These effects could permit the development of various synergistic cancer treatments. The use of DOX/Mn-Cu2-xSe@AC capsules as a multistage oral drug delivery system may overcome the sequential absorption barriers that currently hinder chemotherapy, chemodynamic, and photothermal therapies.

Chemoselective, regioselective, and positionally selective fluorogenic stapling of unprotected peptides for cellular uptake and direct cell imaging.

Peptide stapling reactions represent powerful methods for structuring native α-helices to improve their bioactivity in targeting protein-protein interactions (PPIs). In light of a growing need for regio- and positionally selective stapling methods involving natural amino acid residues in their unprotected states, we report a rapid, mild, and highly chemoselective three-component stapling reation using a class of molecular linchpins based on 2-arylketobenzaldehydes (ArKBCHOs) that create a fluorescent staple, hereafter referred to as a Fluorescent Isoindole Crosslink (FlICk). This methodology offers positional selectivity favouring i, i + 4 helical staples comprising a lysine and cysteine, in the presence of competing nucleophiles on unprotected peptides. In our efforts to further validate this chemistry, we have successfully shown in vitro cytotoxicity of a FlICk-ed peptide (IC50 = 5.10 ± 1.27 μM), equipotent to an olefin-stapled congener. In harnessing the innate fluorescence of the thiol-isoindole, we report new blue-green fluorophores, which arise as a consequence of stapling, with appreciable quantum yields that enable direct cellular imaging in the assessment of cell permeability, thus bridging therapeutic potential with cytological probe development.

A rolling circle amplification-based DNAzyme walker against intracellular degradation for imaging tumor cells' microRNA.

We present a novel DNA molecular machine (RCA-D-Walker) that integrates a DNAzyme-based molecular beacon with RCA-based vectors for miRNA imaging in tumor cells. It can accurately target tumor cells through the sgc8 aptamer. The target miRNA can restore the DNAzyme's ability to cleave the substrate, which in turn produces an amplified fluorescent signal. The RCA-D-Walker exhibits enhanced tumor cell targeting, improved cell permeability, and greater resistance to nuclease degradation. Utilizing this strategy, we achieved accurate and efficient imaging of miRNA-21 in tumor cells.

ST6GAL1-Mediated Sialylation of PECAM-1 Promotes a Transcellular Diapedesis-Like Process that Directs Lung Tropism of Metastatic Breast Cancer.

Metastasis is the leading cause of mortality in breast cancer, with lung metastasis being particularly detrimental. Identification of the processes determining metastatic organotropism could enable the development of approaches to prevent and treat breast cancer metastasis. Here, we found that lung-tropic and non-lung-tropic breast cancer cells differ in their response to sialic acids, affecting the sialylation of surface proteins. Lung-tropic cells showed higher levels of ST6GAL1, while non-lung-tropic cells had more ST3GAL1. ST6GAL1-mediated α-2,6-sialylation, unlike ST3GAL1-mediated α-2,3-sialylation, increased lung metastasis by promoting cancer cell migration through pulmonary endothelial layers and reducing junction protein levels. α-2,6-sialylated PECAM-1 on breast cancer cells facilitated extravasation through the pulmonary endothelium, a critical step in lung metastasis. Knockdown of ST6GAL1 or PECAM-1 significantly reduced lung metastasis. Human pulmonary endothelium displayed high PECAM-1 levels. Through transhomophilic interaction with pulmonary PECAM-1, α-2,6-sialylated PECAM-1 on ST6GAL1-positive cancer cells increased pulmonary extravasation in a diapedesis-like, cell-autonomous manner. Additionally, lung-tropic cells and their exosomes increased the permeability of pulmonary endothelial cells, promoting metastasis in a non-cell-autonomous manner. Analysis of human breast cancer samples showed a correlation between elevated ST6GAL1/PECAM-1 expression and lung metastasis. These results suggest that targeting ST6GAL1-mediated α-2,6-sialylation could be a potential therapeutic strategy to prevent lung metastasis in breast cancer patients.

Nucleic Acid Specificity, Cellular Localization and Reduced Toxicities of Thiazole Orange-Neomycin Conjugates.

Selective binding of small molecule ligands to nucleic acids with high affinity and limited toxicity remains an important goal in the development of compounds that can probe DNA or RNA in cells. Thiazole orange is a cell semi-permeant, fluorescent cyanine dye, with low background noise, that binds several forms of nucleic acids. However, thiazole orange can exhibit cytotoxicity when used at high concentration and/or with prolonged exposure. Neomycin is a non-fluorescent antibiotic with affinity for several forms of nucleic acids, but does not readily enter mammalian cells. Conjugation of neomycin with thiazole orange can exploit the properties of each individual compound, yielding a small molecule that could be used for nontoxic application in cellular analysis by microscopic imaging. We demonstrate that conjugation of neomycin with thiazole orange increases the cell permeability of neomycin, decreases the cytotoxicity of thiazole orange, and exhibits a greater degree of intracellular RNA targeted localization in the nucleolus, when compared to thiazole orange. Relative to thiazole orange, the conjugated compounds showed a much higher degree of stabilization of the nucleic acids as reflected in a greater denaturation temperature. Ultimately, our studies indicate that the conjugated thiazole orange-neomycin compounds can be used as an RNA targeted, less cytotoxic alternative for cellular labeling.