Nellix Device Failure Mechanisms Analysis on Explanted Grafts.

OBJECTIVE: To understand possible reasons for poor durability of the Nellix (Endologix Inc., Irvine, USA) endovascular aneurysm sealing (EVAS) device. MATERIALS AND METHODS: 21 Nellix endoprostheses explanted for endoleaks and migration underwent visual examinations of stent structures and instrumental examinations of the polymer endobags on 4 devices. We harvested 2.0-gram polymer slices out of each of them and tested the samples in an in vitro implantation replication that included wet and dry exposures. During the wet phase, we placed samples in a beaker with saline, mimicking the filling of the endobags during implantation. An exposure to a 37°C environment with 60% humidity during the dry phase replicated the postimplantation conditions inside the aneurysmal sac. RESULTS: Iatrogenic defects affected 16 (76%) metal stents and 20 (95%) endobags. The polymer was disintegrated owing to degradation in 15 (71%) cases. The polymer could lose more than 70% of its initial weight when partially dehydrated and regain 80% when placed in saline. We observed volume decrease and polymer fragmentation during these study phases. CONCLUSIONS: The polymer can lose weight and volume while it dehydrates. This structural degradation of the polymer could lead to the development of endoleaks and/or migration of the device. CLINICAL IMPACT: Based on the results of previous investigations, due to possible endovascular device degradation, patients with endografts should be offered life-long surveillance, and the Nellix device is no exception. Herein we suggest polymer degradation as one of the possible reasons for the device failure. Although Nellix has been withdrawn from the market, there are numerous patients with this type of endograft. Due to its unpredictable performance in the medium and long term, these patients should be recommended enhanced life-long surveillance every 6 months. Any suspicious conditions during the follow-up must be taken seriously and explantation should be considered.

Using Native Chemical Ligation for Site-Specific Synthesis of Hetero-bis-lanthanide Peptide Conjugates: Application to Ratiometric Visible or Near-Infrared Detection of Zn2.

The interest in ratiometric luminescent probes that detect and quantify a specific analyte is growing. Owing to their special luminescence properties, lanthanide(III) cations offer attractive opportunities for the design of dual-color ratiometric probes. Here, the design principle of hetero-bis-lanthanide peptide conjugates by using native chemical ligation is described for perfect control of the localization of each lanthanide cation within the molecule. Two zinc-responsive probes, r-LZF1Tb|Cs124|Eu and r-LZF1Eu|Cs124|Tb are described on the basis of a zinc finger peptide and two DOTA (DOTA=1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid) complexes of terbium and europium. Both display dual-color ratiometric emission in response to the presence of Zn2+ . By using a screening approach, anthracene was identified for the sensitization of the luminescence of two near-infrared-emitting lanthanides, Yb3+ and Nd3+ . Thus, two novel zinc-responsive hetero-bis-lanthanide probes, r-LZF3Yb|Anthra|Nd and r-LZF3Nd|Anthra|Yb were assembled, the former offering a neat ratiometric response to Zn2+ with emission in the near-infrared around 1000 nm, which is unprecedented.

N-Terminal Cu-Binding Motifs (Xxx-Zzz-His, Xxx-His) and Their Derivatives: Chemistry, Biology and Medicinal Applications.

Peptides and proteins with N-terminal amino acid sequences NH2 -Xxx-His (XH) and NH2 -Xxx-Zzz-His (XZH) form well-established high-affinity CuII -complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound healing factor. This opens a straightforward way to add a high-affinity CuII -binding site to almost any peptide or protein, by chemical or recombinant approaches. Thus, these motifs, NH2 -Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation properties, useful in anticancer or antimicrobial drugs. More recent research suggests novel biological functions, mainly based on the redox inertness of CuII in XZH, like PET imaging (with 64 Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters and activation of biological functions by strong CuII binding. This Review gives an overview of the chemical properties of Cu-XH and -XZH motifs and discusses the pros and cons of the vastly different biological applications, and how they could be improved depending on the application.

Luminescent Zinc Fingers: Zn-Responsive Neodymium Near-Infrared Emission in Water.

Responsive luminescent probes emitting in the near-infrared (NIR) are in high demand today for biological applications as they allow for the easy and unambiguous discrimination of autofluorescence. Due to their luminescence properties, lanthanide ions offer an interesting alternative to classical organic fluorescent dyes. This has stimulated the development of lanthanide-based responsive probes. Nevertheless, responsive probes that can operate in water with NIR-emitting lanthanide ions are scarce. In this communication, zinc fingers are shown to be versatile scaffolds to elaborate a variety of Zn2+ -responsive probes based on lanthanide emission and featuring desirable properties for the selective detection of Zn2+ in experimental conditions close to cellular. Of special interest is a NIR-emitting probe relying on Nd3+ emission.

Solid phase protein chemical synthesis.

The chemical synthesis of peptides or small proteins is often an important step in many research projects and has stimulated the development of numerous chemical methodologies. The aim of this review is to give a substantial overview of the solid phase methods developed for the production or purification of polypeptides. The solid phase peptide synthesis (SPPS) technique has facilitated considerably the access to short peptides (<50 amino acids). However, its limitations for producing large homogeneous peptides have stimulated the development of solid phase covalent or non-covalent capture purification methods. The power of the native chemical ligation (NCL) reaction for protein synthesis in aqueous solution has also been adapted to the solid phase by the combination of novel linker technologies, cysteine protection strategies and thioester or N,S-acyl shift thioester surrogate chemistries. This review details pioneering studies and the most recent publications related to the solid phase chemical synthesis of large peptides and proteins.

Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters.

Protein total chemical synthesis enables the atom-by-atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C-terminal peptide thioesters with N-terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc-SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2-sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C-terminal bis(2-sulfanylethyl)amido peptides and of peptide thioesters derived from 3-mercaptopropionic acid.

Restoring cellular copper homeostasis in Alzheimer disease: a novel peptide shuttle is internalized by an ATP-dependent endocytosis pathway involving Rab5- and Rab14-endosomes.

CPPs, or Cell-Penetrating Peptides, offer invaluable utility in disease treatment due to their ability to transport various therapeutic molecules across cellular membranes. Their unique characteristics, such as biocompatibility and low immunogenicity, make them ideal candidates for delivering drugs, genes, or imaging agents directly into cells. This targeted delivery enhances treatment efficacy while minimizing systemic side effects. CPPs exhibit versatility, crossing biological barriers and reaching intracellular targets that conventional drugs struggle to access. This capability holds promise in treating a wide array of diseases, including cancer, neurodegenerative disorders, and infectious diseases, offering a potent avenue for innovative and targeted therapies, yet their precise mechanism of cell entry is far from being fully understood. In order to correct Cu dysregulation found in various pathologies such as Alzheimer disease, we have recently conceived a peptide Cu(II) shuttle, based on the αR5W4 CPP, which, when bound to Cu(II), is able to readily enter a neurosecretory cell model, and release bioavailable Cu in cells. Furthermore, this shuttle has the capacity to protect cells in culture against oxidative stress-induced damage which occurs when Cu binds to the Aß peptide. The aim of this study was therefore to characterize the cell entry route used by this shuttle and determine in which compartment Cu is released. Pharmacological treatments, siRNA silencing and colocalization experiments with GFP-Rab fusion proteins, indicate that the shuttle is internalized by an ATP-dependent endocytosis pathway involving both Rab5 and Rab14 endosomes route and suggest an early release of Cu from the shuttle.

Quest for a stable Cu-ligand complex with a high catalytic activity to produce reactive oxygen species.

Metal ion-catalyzed overproduction of reactive oxygen species (ROS) is believed to contribute significantly to oxidative stress and be involved in several biological processes, from immune defense to development of diseases. Among the essential metal ions, copper is one of the most efficient catalysts in ROS production in the presence of O2 and a physiological reducing agent such as ascorbate. To control this chemistry, Cu ions are tightly coordinated to biomolecules. Free or loosely bound Cu ions are generally avoided to prevent their toxicity. In the present report, we aim to find stable Cu-ligand complexes (Cu-L) that can efficiently catalyze the production of ROS in the presence of ascorbate under aerobic conditions. Thermodynamic stability would be needed to avoid dissociation in the biological environment, and high ROS catalysis is of interest for applications as antimicrobial or anticancer agents. A series of Cu complexes with the well-known tripodal and tetradentate ligands containing a central amine linked to three pyridyl-alkyl arms of different lengths were investigated. Two of them with mixed arm length showed a higher catalytic activity in the oxidation of ascorbate and subsequent ROS production than Cu salts in buffer, which is an unprecedented result. Despite these high catalytic activities, no increased antimicrobial activity toward Escherichia coli or cytotoxicity against eukaryotic AGS cells in culture related to Cu-L-based ROS production could be observed. The potential reasons for discrepancy between in vitro and in cell data are discussed.

Total synthesis of biotinylated N domain of human hepatocyte growth factor.

Hepatocyte growth factor/scatter factor (HGF/SF) is the high affinity ligand of MET tyrosine kinase receptor. We report here the total synthesis of a biotinylated analogue of human HGF/SF N domain. Functionally, N domain is part of the HGF/SF high affinity binding site for MET and also the main HGF/SF binding site for heparin. The 97 Aa linear chain featuring a C-terminal biotin group was assembled in high yield using an N-to-C one-pot three segments assembly strategy relying on a sequential Native Chemical Ligation (NCL)/bis(2-sulfanylethyl)amido (SEA) native peptide ligation process. The folded protein displayed the native disulfide bond pattern and showed the ability to bind heparin.

Sequential native peptide ligation strategies for total chemical protein synthesis.

Total chemical synthesis of proteins is usually achieved by assembling unprotected peptide segments using site-specific and chemoselective native peptide ligation methods. Access to large proteins often requires the assembly of at least three segments due to the current limits of solid phase synthesis of individual peptide segments. The aim of this tutorial review is to present the basic concepts and challenges underlying the design of sequential peptide ligation strategies using solution or solid phase chemistry. A special emphasis is given to C-to-N and N-to-C three-segment assembly strategies, which potentially give access to proteins composed of up to 150 amino acid residues.

Evaluation of Candidate Theranostics for 227Th/89Zr Paired Radioimmunotherapy of Lymphoma.

227Th is a promising radioisotope for targeted α-particle therapy. It produces 5 α-particles through its decay, with the clinically approved 223Ra as its first daughter. There is an ample supply of 227Th, allowing for clinical use; however, the chemical challenges of chelating this large tetravalent f-block cation are considerable. Using the CD20-targeting antibody ofatumumab, we evaluated chelation of 227Th4+ for α-particle-emitting and radiotheranostic applications. Methods: We compared 4 bifunctional chelators for thorium radiopharmaceutical preparation: S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 2-(4-isothicyanatobenzyl)-1,2,7,10,13-hexaazacyclooctadecane-1,4,7,10,13,16-hexaacetic acid (p-SCN-Bn-HEHA), p-isothiacyanatophenyl-1-hydroxy-2-oxopiperidine-desferrioxamine (DFOcyclo*-p-Phe-NCS), and macrocyclic 1,2-HOPO N-hydroxysuccinimide (L804-NHS). Immunoconstructs were evaluated for yield, purity, and stability in vitro and in vivo. Tumor targeting of the lead 227Th-labeled compound in vivo was performed in CD20-expressing models and compared with a companion 89Zr-labeled PET agent. Results: 227Th-labeled ofatumumab-chelator constructs were synthesized to a radiochemical purity of more than 95%, excepting HEHA. 227Th-HEHA-ofatumumab showed moderate in vitro stability. 227Th-DFOcyclo*-ofatumumab presented excellent 227Th labeling efficiency; however, high liver and spleen uptake was revealed in vivo, indicative of aggregation. 227Th-DOTA-ofatumumab labeled poorly, yielding no more than 5%, with low specific activity (0.08 GBq/g) and modest long-term in vitro stability (<80%). 227Th-L804-ofatumumab coordinated 227Th rapidly and efficiently at high yields, purity, and specific activity (8 GBq/g) and demonstrated extended stability. In vivo tumor targeting confirmed the utility of this chelator, and the diagnostic analog, 89Zr-L804-ofatumumab, showed organ distribution matching that of 227Th to delineate SU-DHL-6 tumors. Conclusion: Commercially available and novel chelators for 227Th showed a range of performances. The L804 chelator can be used with potent radiotheranostic capabilities for 89Zr/227Th quantitative imaging and α-particle therapy.

Development of Cu(ii)-specific peptide shuttles capable of preventing Cu-amyloid beta toxicity and importing bioavailable Cu into cells.

Copper (Cu) in its ionic forms is an essential element for mammals and its homeostasis is tightly controlled. Accordingly, Cu-dyshomeostasis can be lethal as is the case in the well-established genetic Wilson's and Menkes diseases. In Alzheimer's disease (AD), Cu-accumulation occurs in amyloid plaques, where it is bound to the amyloid-beta peptide (Aß). In vitro, Cu-Aß is competent to catalyze the production of reactive oxygen species (ROS) in the presence of ascorbate under aerobic conditions, and hence Cu-Aß is believed to contribute to the oxidative stress in AD. Several molecules that can recover extracellular Cu from Aß and transport it back into cells with beneficial effects in cell culture and transgenic AD models were identified. However, all the Cu-shuttles currently available are not satisfactory due to various potential limitations including ion selectivity and toxicity. Hence, we designed a novel peptide-based Cu shuttle with the following properties: (i) it contains a Cu(ii)-binding motif that is very selective to Cu(ii) over all other essential metal ions; (ii) it is tagged with a fluorophore sensitive to Cu(ii)-binding and release; (iii) it is made of a peptide platform, which is very versatile to add new functions. The work presented here reports on the characterization of AKH-αR5W4NBD, which is able to transport Cu ions selectively into PC12 cells and the imported Cu appeared bioavailable, likely via reductive release induced by glutathione. Moreover, AKH-αR5W4NBD was able to withdraw Cu from the Aß1-16 peptide and consequently inhibited the Cu-Aß based reactive oxygen species production and related cell toxicity. Hence, AKH-αR5W4NBD could be a valuable new tool for Cu-transport into cells and suitable for mechanistic studies in cell culture, with potential applications in restoring Cu-homeostasis in Cu-related diseases such as AD.

A luminescent ATCUN peptide variant with enhanced properties for copper(II) sensing in biological media.

The measurement of labile CuII in biological samples is fundamental for understanding Cu metabolism and has been emerging as a promising diagnostic marker for Cu-related pathologies such as Wilson's and Alzheimer's diseases. The use of fluorescent chelators may be useful to circumvent separation steps employed by current methods. For this purpose, we recently designed a selective and suited-affinity turn-off luminescent probe based on a peptide bearing the CuII-binding Xxx-Zzz-His (Amino-Terminal CuII- and NiII-binding, ATCUN) motif and a TbIII-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) complex. Here, we present an analogue probe bearing the ATCUN motif variant Xxx-His-His. This probe showed much faster response in biologically-relevant media and higher stability than the previous motif at low pH. These features could be beneficial to the measurement of dynamic CuII fluctuations and the application in slightly acidic media, such as urine.

A terbium(iii) luminescent ATCUN-based peptide sensor for selective and reversible detection of copper(ii) in biological media.

The measurement of exchangeable Cu2+ levels in biological samples is gaining interest in the context of copper-related pathologies. Here, we report a Tb3+ luminescent turn-off sensor for Cu2+ based on the specific and suitable-affinity Xxx-Zzz-His (ATCUN) peptide motif, enabling Cu2+ detection in the presence of a biological fluorescent background.

A novel DOTA-like building block with a picolinate arm for the synthesis of lanthanide complex-peptide conjugates with improved luminescence properties.

Combination of complexes of lanthanide cations (Ln3+) for their luminescent properties and peptides for their recognition properties is interesting in view of designing responsive luminescent probes. The octadentate DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelate is the most popular chelate to design Ln3+ complex-peptide conjugates. We describe a novel building block, DO3Apic-tris(allyl)ester, which provides access to peptides with a conjugated nonadentate chelate, namely DO3Apic, featuring a picolinate arm in place of one of the acetate arms compared to DOTA, for improved luminescence properties. This building block, with allyl protecting groups, is readily obtained by solid phase synthesis. We show that it is superior to its analogue with tBu protecting groups for the preparation of peptide conjugates because of the difficult removal of the tBu protecting groups for the latter. Then, we compare two luminescent zinc fingers (LZF) comprising (i) a zinc finger peptide for selective Zn2+ binding, (ii) a Eu3+ complex and (iii) an acridone antenna (ACD) for long-wavelength sensitization of Eu3+ luminescence. The first one, LZF3ACD|Eu, incorporates a DOTA chelate for Eu3+ whereas the other, LZF4ACD|Eu, incorporates a DO3Apic chelate. Both act as Zn2+-responsive luminescent probes but we show that changing DOTA for DO3Apic results in a higher Eu3+ luminescence lifetime and in a doubling of the quantum yield, confirming the interest of the DO3Apic chelate and the DO3Apic(tris(allyl)ester building block for the preparation of Ln3+ complex-peptide conjugates. Additionally, the DO3Apic chelate provides self-calibration for LZF4ACD|Eu luminescence upon excitation of its picolinamide chromophore, making LZF4ACD|Eu a ratiometric sensor for Zn2+.

How trimerization of CTR1 N-terminal model peptides tunes Cu-binding and redox-chemistry.

Employing peptide-based models of copper transporter 1 (CTR1), we show that the trimeric arrangement of its N-terminus tunes its reactivity with Cu, promoting Cu(ii) reduction and stabilizing Cu(i). Hence, the employed multimeric models of CTR1 provide an important contribution to studies on early steps of Cu uptake by cells.

Copper-binding motifs Xxx-His or Xxx-Zzz-His (ATCUN) linked to an antimicrobial peptide: Cu-binding, antimicrobial activity and ROS production.

Depending on the coordination, copper ions can have a very high activity in catalyzing the production of reactive oxygen species. Thus interest arose in increasing the activity of antimicrobial peptides (AMPs) by equipping them with a Cu-binding unit. Several examples, native and engineered, have been investigated with the motif Xxx-Zzz-His, called Amino Terminal Cu(II)- and Ni(II)-binding (ATCUN) motif. Here we investigate a short AMP that was equipped either with Xxx-Zzz-His or Xxx-His. Xxx-His is a shorter motif and yields a more redox active copper complex. The control AMP, Xxx-His-AMP and Xxx-Zzz-His-AMP were investigated toward Cu-binding, Reactive Oxygen Species (ROS) production and antimicrobial activity in E. coli. The data indicate that these Cu-binding motifs have very limited impact on antimicrobial activity and low ROS production capability.

Reversible turn-on fluorescent Cu(ii) sensors: rather dream than reality?

Reversible turn-on fluorescent sensors of Cu(ii) are of high interest for biological studies. We re-investigate a reported sensor, showing that turn-on occurs via irreversible Cu(ii)-induced sensor oxidation only in the presence of acetonitrile. This prevents its application in biological studies and highlights the challenge of establishing a reversible Cu(ii) turn-on sensor.

Low catalytic activity of the Cu(ii)-binding motif (Xxx-Zzz-His; ATCUN) in reactive oxygen species production and inhibition by the Cu(i)-chelator BCS.

The catalytic redox activity of Cu(ii) bound to the motif NH2-Xxx-Zzz-His (ATCUN) with ascorbate and H2O2/O2 is very low and can be stopped via Cu(i)-chelation. This impacts its application as an artificial Cu-enzyme to degrade biomolecules via production of reactive oxygen species in a Cu(i)-chelator rich environment like the cytosol.

Design of a synthetic luminescent probe from a biomolecule binding domain: selective detection of AU-rich mRNA sequences.

We report the design of a luminescent sensor based upon the zinc finger (ZF) protein TIS11d, that allows for the selective time-resolved detection of the UUAUUUAUU sequence of the 3'-untranslated region of messenger RNA. This sensor is composed of the tandem ZF RNA binding domain of TIS11d functionalized with a luminescent Tb3+ complex on one of the ZFs and a sensitizing antenna on the other. This work provides the proof of principle that an RNA binding protein can be re-engineered as an RNA sensor and, more generally, that tunable synthetic luminescent probes for biomolecules can be obtained by modifying biomolecule-binding domains.