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The accurate and efficient quantification of nanodrug dosage is crucial for early anticancer therapy. The enzyme-linked immunosorbent assay (ELISA) has emerged as a robust tool for detecting anticancer nanodrug dosage; however, the development of sensing elements to quantify anticancer nanodrugs still poses a challenge. To overcome this problem, we utilize polysuccinimide-loaded curcumin (CUR @PSI OAm ) as a model to employ an ELISA based on peroxidase nanozyme Pt-SiO 2 Janus nanoparticles (Pt-SiO 2 JNPs) for the indirect quantitative analysis of intracellular anticancer nanodrug dosage. This novel approach employs an immunoassay to indirectly quantify the dosage of anticancer nanodrugs while preserving its structural integrity. The silica components of Pt-SiO 2 JNPs adsorb intermediates, while the Pt NP components exhibit high catalytic activity. Pt-SiO 2 JNPs are functionalized with anti-PSI OAm antibody (Pt-SiO 2 JNPs-Ab) to serve as an immunosensor capable of specific recognition of CUR @PSI OAm . Additionally, we employed cytotoxicity assays and confocal imaging techniques to demonstrate the excellent biocompatibility of CUR @PSI OAm , as well as its specific uptake by cancer cells. According to the experimental results, the limit of detection (LOD) for the immunoassay of Pt-SiO 2 JNPs as a marker for detecting CUR @PSI OAm is approximately 4.5-fold lower than that of horseradish peroxidase. Therefore, by optimizing the conditions, we established a direct competitive ELISA using Pt-SiO 2 JNPs as colorimetric indicators for the quantitative detection of intracellular CUR @PSI OAm . The LOD for this ELISA was determined to be 0.01 ng/mL, while the loaded CUR amount calculated from the drug loading capacity was found to be 0.22 pg/mL. Furthermore, the recoveries obtained from this established ELISA ranged between 94.0 and 108%, demonstrating excellent accuracy. Consequently, the peroxidase mimic Pt-SiO 2 JNPs-based ELISA exhibits significant potential for precise quantification of intracellular anticancer nanodrug dosages.

Charge variants are one of the most important quality attributes for protein therapeutics, including antibody drug conjugates (ADCs). ADCs are conjugation products between monoclonal antibodies (mAbs) and highly potent payloads. After attaching a payload, the charge profile of a mAb can be modified due to the change in net charge or surface charge. In this study, we present a unique challenge of charge assay development that arises from a desirable engineering of ADCs that incorporates the hydrolysis-prone succinimide-thioether conjugation chemistry. This engineered hydrolysis at conjugation sites is usually not complete during conjugation process and continuously progressing during mild stress. This hydrolysis also creates a carboxylic functional group, which manifests as acidic peaks in the ADC charge profiles. As a result, ion exchange chromatograms become sensitive measurements of this hydrolysis, which often masks the charge profile change due to other important post-translational modifications. In this study, two approaches were explored to address this unique challenge: to remove the hydrolysis heterogeneity by incubating ADCs under high pH conditions to drive complete hydrolysis; and to analyze charge variants at the subunit level after IdeS digestion. Acceptable charge profiles and quantitative integration results were successfully obtained by both approaches., Competing Interests: Declaration of competing interest All authors are employees of AstraZeneca and may hold stock and/or stock options., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Single-molecule Förster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their conformational changes over time. To monitor RNA dynamics with smFRET, we developed a method to covalently label RNAs at their termini with a FRET pair of fluorophores. This direct end-labeling strategy targets the 5'-phosphate by carbodiimide (EDC)/N-hydroxysuccinimide (NHS) activation and the 3'-ribose by periodate oxidation, which can be adapted to other RNAs regardless of their size and sequence to study them independently of artificial modifications. Furthermore, the 5'-EDC/NHS activation is of general interest to all nucleic acids with a 5'-phosphate. The use of commercially available chemicals eliminates the need to synthesize RNA-specific probes. Total Internal Reflection Fluorescence (TIRF) microscopy requires the surface-immobilized molecules of interest to be within the evanescent field to be illuminated. A sophisticated way of keeping the RNA molecules within the evanescent field is to encapsulate them in phospholipid vesicles. Encapsulation benefits from the best of both worlds, tethering the molecule to the surface while enabling free diffusion of the molecule. We ensure that each vesicle contains only a single RNA molecule, enabling single-molecule imaging. Upon dual-end labeling and encapsulation of the RNA of interest, smFRET measurements offer a dynamic and detailed view of RNA behavior.

Rationale: Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide-thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable., Methods: Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18 O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography-MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30., Results: A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18 O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker., Conclusions: Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody-drug conjugates., (© 2024 John Wiley & Sons Ltd.)

Age-related macular degeneration (AMD) is a well-recognized affliction among the elderly, causing vision impairment ranging from blurred vision to complete blindness. This underscores the critical need for accurate, precise, and early detection methods. Herein, we developed a noninvasive, label-free electrical biosensor, constructed on an economical printed circuit board (PCB) substrate, designed specifically for the precise quantification of AMD biomarker: complement component III (C3). The hydrothermally reduced graphene oxide (rGO) was deposited between gold-interdigitated microelectrodes, forming a conductive channel. The fabricated C3 biosensor exhibits a low detection limit of 0.4342 ng/mL and an impressive sensitivity of 9.238 ((Δ R / R )/ng.mL -1 )/cm 2 with a regression coefficient of 0.9815 calibrated within the clinical C3 range of 10-30 ng/mL. This excellent performance is ascribed to the synergistic effects of 1-pyrenebutanoic acid succinimidyl ester (PBASE) linker and conducting properties of rGO as they generate large active sites for higher anti-C3 antibody immobilization, thereby enhancing sensitivity and specificity. Furthermore, the performance of this proposed C3 sensor chip was validated with enzyme-linked immunosorbent assay (ELISA) using five human tear samples exhibiting an outstanding correlation of a regression value of 0.9774. The unparalleled merits of this newly crafted C3 biosensor transcend those of preceding platforms, boasting superior accuracy and precision in quantifying C3 levels in human tears, accelerated operational speed with results attainable within a mere 15 min, cost-effectiveness, and excellent sensitivity.

In this study, bovine serum albumin (BSA) is used as a globular protein model to examine the conformational changes that occur during the interaction of BSA with N-hydroxysulfo-succinimide (sodium salt)-functionalized gold nanourchins (GNUs), for which dynamic spectroscopic techniques are employed. The results showed that the absorbance of phosphate-buffered saline-BSA at 278 nm decreased when a GNU was added to the solution due to adsorption, and it decreased further when the GNU was increased. The intensity and width of the peak of local surface plasmon resonance increased, indicating the effect of corona formation. Dynamic UV-vis spectroscopy and scattering revealed a nonlinear behavior of BSA-GNU interaction. The bioplasmonic solution resulted in higher transmission and scattering than the BSA solution. Fourier transform-near-infrared spectra exhibited several bands due to overtones and combinations of the amide group and different proportions of α-helix and β-sheet components in BSA before and after the addition of the GNU. Time-resolved fluorescence spectroscopy demonstrated an initial increase in blueshifted emission, followed by a redshifted quenching of two major peaks of Tyr and tryptophan (Trp). The binding and dissociation constants were determined as Kb = 2.17 × 1010 M-1 and Kd = 4.6 × 10-11, respectively, using the Stern-Volmer relation. Both the dynamic CMOS-based imaging and the cadmium sulfide sensors demonstrated a nonlinear response of bioplasmonic solution. By increasing the GNU, the resistance of the solution decreased in the order of A > S1 > S3, where S3 exhibited the highest initial transmission with a longer desorption time. MATLAB modeling showed 80% surface coverage by the protein in 15 s at 0.05M, equivalent to a thickness of 1.7 nm, which was in agreement with the value determined by using the Stokes-Einstein relation., (2024 Published under an exclusive license by the AVS.)

The advent of nonfullerene acceptors (NFAs) has greatly improved the photovoltaic performance of organic solar cells (OSCs). However, to compete with other solar cell technologies, there is a pressing need for accelerated research and development of improved NFAs as well as their compatible wide bandgap polymer donors. In this study, a novel electron-withdrawing building block, succinimide-substituted thiophene (TS), is utilized for the first time to synthesize three wide bandgap polymer donors: PBDT-TS-C5, PBDT-TSBT-C12, and PBDTF-TSBT-C16. These polymers exhibit complementary bandgaps for efficient sunlight harvesting and suitable frontier energy levels for exciton dissociation when paired with the extensively studied NFA, Y6. Among these donors, PBDTF-TSBT-C16 demonstrates the highest hole mobility and a relatively low highest occupied molecular orbital (HOMO) energy level, attributed to the incorporation of thiophene spacers and electron-withdrawing fluorine substituents. OSC devices based on the blend of PBDTF-TSBT-C16:Y6 achieve the highest power conversion efficiency of 13.21%, with a short circuit current density (J sc ) of 26.83 mA cm -2 , an open circuit voltage (V oc ) of 0.80 V, and a fill factor of 0.62. Notably, the V oc × J sc product reaches 21.46 mW cm -2 , demonstrating the potential of TS as an electron acceptor building block for the development of high-performance wide bandgap polymer donors in OSCs., (© 2024 The Author(s). Macromolecular Rapid Communications published by Wiley‐VCH GmbH.)

Rationale: Isomerism can be an important aspect in pharmaceutical drug development. Identification of isomers can provide insights into drug pharmacology and contribute to better design of drug molecules. The general approaches to differentiate isomers include Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and circular dichroism. Additionally, a commonly used method to differentiate isomers is liquid chromatography coupled with mass spectrometry (LC-MS). Notably, LC-MS is routinely applied to leucine and isoleucine differentiation to facilitate protein sequencing. This work focuses on isomer differentiation of widely employed thio-succinimide structure bridging the antibody backbone and linker-payload of antibody-drug conjugates (ADCs). Thio-succinimide hydrolysis stabilizes the payload-protein structure while generating a pair of constitutional isomers: thio-aspartyl and thio-isoaspartyl., Methods: This paper introduces a hybrid method using ligand binding assay (LBA) and liquid chromatography coupled with tandem MS (LC-MS/MS) to reveal isomerization details of thio-succinimide hydrolysis over time in plasma samples incubated with ADC. Application of two orthogonal dissociation methods, collision-induced dissociation (CID) and electron-activated dissociation (EAD) revealed different MS/MS spectra for this pair of isomers. This observation enables a unique approach in distinguishing thio-succinimide hydrolysis isomers., Results: We observed signature [R 1  + Thio + 57 + H] + , [R 2  + Succ + H 2 O - 57 + H] + , and [R 2  + Succ + H 2 O - 44 + 2H] 2+ product ions (Succ = succinimide) that differentiated thio-aspartyl and thio-isoaspartyl isomers using EAD. A newly discovered [R 2  + ThioSucc + H 2 O - 44 + 2H] 2+ ion also served as additional evidence that further supported our findings., Conclusions: This study is a first-to-date identification of thio-succinimide hydrolysis isomers without using synthesized reference materials. This approach should be applicable to all thio-succinimide-linked molecules. Correct identification of thio-succinimide hydrolysis isomers may eventually benefit the development of ADCs in the future., (© 2024 The Author(s). Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Bi-functional N-Hydroxysuccinimide (NHS) linkers are widely used in the conjugation processes linking an immunogen with a carrier protein capable of boosting immunity. A potential vaccine candidate against HIV-1, called fusion peptide (FP), is covalently linked to the recombinant tetanus toxoid heavy-chain fragment C (rTTHC) via this type of linker. A reversed-phase liquid chromatography (RPLC-UV) method was used to monitor the linker's degradation kinetics in various buffers, mimicking the steps in the conjugation process. The kinetics of the reactivities of the linkers are revealed in this study and can provide a good guidance to help effective conjugation process before these linkers are completely hydrolyze to the inactive degradants. Three cross-linkers degradation pathways were evaluated: Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (Sulfo-SIAB), PEGylated SMCC (SM(PEG) 2 ), and N-γ-maleimidobutyryl-oxysulfosuccinimide ester (Sulfo-GMBS). We have reported kinetics for Sulfo-SIAB., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Published by Elsevier B.V.)

Cells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron-Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein-diacetate-succinimidyl esters (CFSE)-a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity-with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron-rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile-containing nutrients in live primary immune cell populations using flow cytometry and live-cell microscopy., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Synthesis of the pentasaccharide repeating unit of the cell O-polysaccharide produced by Salmonella milwaukee O:43 strain (group U) has been achieved in very good yield adopting a convergent stereoselective [3 + 2] block glycosylation strategy. Thioglycosides and glycosyl trichloroacetimidate derivative were used as glycosyl donors in the presence of a combination of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) as thiophilic activator and TMSOTf as trichloroacetimidate activator respectively. The stereochemical outcome of all glycosylation reactions was excellent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chemical post-translational protein-protein conjugation is an important technique with growing applications in biotechnology and pharmaceutical research. Maleimides represent one of the most widely employed bioconjugation reagents. However, challenges associated with the instability of first- and second-generation maleimide technologies are yet to be fully addressed. We report the development of a novel class of maleimide reagents that can undergo on-demand ring-opening hydrolysis of the resulting thio-succinimide. This strategy enables rapid post-translational assembly of protein-protein conjugates. Thio-succinimide hydrolysis, triggered upon application of chemical, photochemical, or enzymatic stimuli, allowed homobifunctional bis-maleimide reagents to be applied in the production of stable protein-protein conjugates, with complete temporal control. Bivalent and bispecific protein-protein dimers constructed from small binders targeting antigens of oncological importance, PD-L1 and HER2, were generated with high purity, stability, and improved functionality compared to monomeric building blocks. The modularity of the approach was demonstrated through elaboration of the linker moiety through a bioorthogonal propargyl handle to produce protein-protein-fluorophore conjugates. Furthermore, extending the functionality of the homobifunctional reagents by temporarily masking reactive thiols included in the linker allowed the assembly of higher order trimeric and tetrameric single-domain antibody conjugates. The potential for the approach to be extended to proteins of greater biochemical complexity was demonstrated in the production of immunoglobulin single-domain antibody conjugates. On-demand control of thio-succinimide hydrolysis combined with the facile assembly of chemically defined homo- and heterodimers constitutes an important expansion of the chemical methods available for generating stable protein-protein conjugates.

Background: Faced with the need to develop new herbicides with modes of action different to those observed for existing agrochemicals, one of the most promising strategies employed by synthetic chemists involves the structural modification of molecules found in natural products. Molecules containing amides, imides, and epoxides as functional groups are prevalent in nature and find extensive application in synthesizing more intricate compounds due to their biological properties. In this context, this paper delineates the synthesis of N-phenylnorbornenesuccinimide derivatives, conducts biological assays, and carries out in silico investigation of the protein target associated with the most potent compound in plant organisms. The phytotoxic effects of the synthesized compounds (2-29) were evaluated on Allium cepa, Bidens pilosa, Cucumis sativus, Sorghum bicolor, and Solanum lycopersicum., Results: Reaction of endo-bicyclo[2.2.1]hept-5-ene-3a,7a-dicarboxylic anhydride (1) with aromatic amines led to the N-phenylnorbornenesuccinic acids (2-11) with yields ranging from 75% to 90%. Cyclization of compounds (2-11) in the presence of acetic anhydride and sodium acetate afforded N-phenylnorbornenesuccinimides (12-20) with yields varying from 65% to 89%. Those imides were then subjected to epoxidation reaction to afford N-phenylepoxynorbornanesuccimides (21-29) with yields from 60% to 90%. All compounds inhibited the growth of seedlings of the plants evaluated. Substance 23 was the most active against the plants tested, inhibiting 100% the growth of all species in all concentrations. Cyclophilin was found to be the enzymatic target of compound 23., Conclusion: These findings suggest that derivatives of N-phenylnorbornenesuccinimide are promising compounds in the quest for more selective and stable agrochemicals. This perspective reinforces the significance of these derivatives as potential innovative herbicides and emphasizes the importance of further exploring their biological activity on weeds. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

3,4-disubstituted maleimides find wide applications in various pharmacologically active compounds. This study presents a highly effective approach for synthesizing derivatives of 3,4-disubstituted maleimides through the direct isomerization of α-succinimide-substituted allenoates, followed by a cascade γ'-addition and aryl imines using PR 3 as a catalyst. The resulting series of 3,4-disubstituted maleimides exhibited excellent stereoselectivities, achieving yields of up to 86%. To our knowledge, the phosphine-mediated γ'-addition reaction of allenoates is seldom reported.

Accurate structural determination of proteins is critical to understanding their biological functions and the impact of structural disruption on disease progression. Gas-phase cross-linking mass spectrometry (XL-MS) via ion/ion reactions between multiply charged protein cations and singly charged cross-linker anions has previously been developed to obtain low-resolution structural information on proteins. This method significantly shortens experimental time relative to conventional solution-phase XL-MS but has several technical limitations: (1) the singly deprotonated N -hydroxysulfosuccinimide (sulfo-NHS)-based cross-linker anions are restricted to attachment at neutral amine groups of basic amino acid residues and (2) analyzing terminal cross-linked fragment ions is insufficient to unambiguously localize sites of linker attachment. Herein, we demonstrate enhanced structural information for alcohol-denatured A-state ubiquitin obtained from an alternative gas-phase XL-MS approach. Briefly, singly sodiated ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) cross-linker anions enable covalent cross-linking at both ammonium and amine groups. Additionally, covalently modified internal fragment ions, along with terminal b-/y-type counterparts, improve the determination of linker attachment sites. Molecular dynamics simulations validate experimentally obtained gas-phase conformations of denatured ubiquitin. This method has identified four cross-linking sites across 8+ ubiquitin, including two new sites in the N-terminal region of the protein that were originally inaccessible in prior gas-phase XL approaches. The two N-terminal cross-linking sites suggest that the N-terminal half of ubiquitin is more compact in gas-phase conformations. By comparison, the two C-terminal linker sites indicate the signature transformation of this region of the protein from a native to a denatured conformation. Overall, the results suggest that the solution-phase secondary structures of the A-state ubiquitin are conserved in the gas phase. This method also provides sufficient sensitivity to differentiate between two gas-phase conformers of the same charge state with subtle structural variations.

A temperature/pH dual sensitive hydrogel with a semi-interpenetrating network (semi-IPN) structure was synthesized through an aqueous amino-succinimide reaction between water-soluble polysuccinimide and polyethyleneimine in the presence of thermosensitive cellulose derivatives. Single-factor experiments were carried out to optimize the preparation conditions of the semi-IPN hydrogel. The swelling behavior and cytotoxicity assay of the hydrogel were tested. Finally, taking 5- fluorouracil (5-Fu) as a model drug, the release performance of the 5-Fu-loaded hydrogel was investigated. The results indicated that the swelling ratio (SR) first decreased and then increased when the pH of the solutions ascended from 2 to 10. The SR decreased with the increase in temperature. In addition, the swelling behavior of the hydrogel was reversible and reproducible under different pH values and temperatures. The prepared hydrogels had good cytocompatibility. The release behavior of 5-Fu was most consistent with the Korsmeyer-Peppas model and followed the case II diffusion. The acidic environment was beneficial for the release of 5-Fu. The preparation process of the semi-IPN hydrogel is simple and the reaction can proceed quickly in water. The strategy introduced here has great potential for application in the preparation of drug carriers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Succinimide intermediates play the crucial role in the nucleation process for protein amyloid fibril formation, as they can usually induce a non-native conformation in a fraction of soluble proteins to render amyloidogenicity and neurotoxicity. Thus, in situ detection of succinimide intermediates during amyloid fibrillation kinetics is of considerable importance, albeit challenging, because these succinimides are generally unstable in physiological conditions. Here, we found an in situ Raman spectral fingerprint to trace the succinimide intermediates in amyloid fibril formation, wherein the carbonyl symmetric stretching of cyclic imide in the succinimide derivative is located at ca. 1790 cm -1 . Using its intensity as an indicator of succinimide intermediates, we have in situ detected and unravelled the role of succinimide intermediates during the oligomer formation from the Bz-Asp-Gly-NH 2 dipeptide or the amyloid fibrillation kinetics of lysozyme with thermal/acid treatment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiaoguo Zhou reports financial support was provided by National Natural Science Foundation of China., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Purpose: Succinimide formation and isomerization alter the chemical and physical properties of aspartic acid residues in a protein. Modification of aspartic acid residues within complementarity-determining regions (CDRs) of therapeutic monoclonal antibodies (mAbs) can be particularly detrimental to the efficacy of the molecule. The goal of this study was to characterize the site of succinimide accumulation in the CDR of a therapeutic mAb and understand its effects on potency. Furthermore, we aimed to mitigate succinimide accumulation through changes in formulation., Methods: Accumulation of succinimide was identified through intact and reduced LC-MS mass measurements. A low pH peptide mapping method was used for relative quantitation and localization of succinimide formation in the CDR. Statistical modeling was used to correlate levels of succinimide with basic variants and potency measurements., Results: Succinimide accumulation in Formulation A was accelerated when stored at elevated temperatures. A strong correlation between succinimide accumulation in the CDR, an increase in basic charge variants, and a decrease in potency was observed. Statistical modeling suggest that a combination of ion exchange chromatography and potency measurements can be used to predict succinimide levels in a given sample. Reformulation of the mAb to Formulation B mitigates succinimide accumulation even after extended storage at elevated temperatures., Conclusion: Succinimide formation in the CDR of a therapeutic mAb can have a strong negative impact on potency of the molecule. We demonstrate that thorough characterization of the molecule by LC-MS, ion exchange chromatography, and potency measurements can facilitate changes in formulation that mitigate succinimide formation and the corresponding detrimental changes in potency., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In this study, we report the isomerization of an aspartic acid residue in the complementarity-determining region (CDR) of crizanlizumab as a major degradation pathway. The succinimide intermediate and iso-aspartic acid degradation products were successfully isolated by ion exchange chromatography for characterization. The isomerization site was identified at a DG motif in the CDR by peptide mapping. The biological characterization of the isolated variants showed that the succinimide variant exhibited a loss in target binding and biological activity compared to the aspartic acid and iso-aspartic acid variants of the molecule. The influence of pH on this isomerization reaction was investigated using capillary zone electrophoresis. Below pH 6.3, the succinimide formation was predominant, whereas at pH values above 6.3, iso-aspartic acid was formed and the initial amounts of succinimide dropped to levels even lower than those observed in the starting material. Importantly, while the succinimide accumulated at long-term storage conditions of 2 to 8°C at pH values below 6.3, a complete hydrolysis of succinimide was observed at physiological conditions (pH 7.4, 37°C), resulting in full recovery of the biological activity. In this study, we demonstrate that the critical quality attribute succinimide with reduced potency has little or no impact on the efficacy of crizanlizumab due to the full recovery of the biological activity within a few hours under physiological conditions.

N-succinimidyl-4-[ 18 F]fluorobenzoate ([ 18 F]SFB), a widely used labeling agent to introduce the 4-[ 18 F]fluorobenzoyl-prosthetic group, is normally obtained in three consecutive steps from [ 18 F]fluoride ion. Here, we describe an efficient one-step labeling procedure of [ 18 F]SFB starting from a tin precursor. This method circumvents volatile radioactive side-products and simplifies automatization. [ 18 F]SFB was obtained after HPLC purification in a yield of 42 + 4% and a radiochemical purity (RCP) > 99% (n = 6). In addition, we investigate the automation of the coupling of [ 18 F]SFB to a nanobody (cAbBcII10, targeting β-lactamase enzyme) and purification by size exclusion chromatography (PD-10 desalting column) to remove unconjugated reagent. Production and use of [ 18 F]SFB were implemented on a radiosynthesis unit (Neptis ® ). The fully automated radiosynthesis process including purification and formulation required 160 min of synthesis time. [ 18 F]SFB-labeled nanobody was obtained in a yield of 21 + 2% (activity yield 12 + 1% non-decay corrected) and a radiochemical purity (RCP) of > 95% (n = 3). This approach simplifies [ 18 F]SFB synthesis to one-step, enhances the yield in comparison to the previous report and enables the production of radiolabeled nanobody on the same synthesis module., (© 2022. The Author(s).)

Isomerization of individual residues in long-lived proteins (LLPs) is a subject of growing interest in connection with many age-related human diseases. When isomerization occurs in LLPs, it can lead to deleterious changes in protein structure, function, and proteolytic degradation. Herein, we present a novel labeling technique for rapid identification of l-isoAsp using the enzyme protein l-isoaspartyl methyltransferase (PIMT) and Tris. The succinimide intermediate formed during reaction of l-isoAsp-containing peptides with PIMT and S -adenosyl methionine (SAM) is reactive with Tris base and results in a Tris-modified aspartic acid residue with a mass shift of +103 Da. Tris-modified aspartic acid exhibits prominent and repeated neutral loss of water when subjected to collisional activation. In addition, another dissociation pathway regenerates the original peptide following loss of a characteristic mass shift. Furthermore, it is demonstrated that Tris modification can be used to identify sites of isomerization in LLPs from biological samples such as the lens of the eye. This approach simplifies identification by labeling isomerization sites with a tag that causes a mass shift and provides characteristic loss during collisional activation.

An immunoaffinity magnetic beads (IMBs) based automatic pretreatment method was developed for the quantitative analysis of deoxynivalenol (DON) by ultra-performance liquid chromatography and ultraviolet detector (UPLC-UV). First, N-hydroxysuccinimide-terminated magnetic beads (NHS-MBs) with good magnetic responsivity and dispersibility were synthesized and characterized by optical microscopy, scanning electron microscopy (SEM), and laser diffraction-based particle size analyzer. Then, the amino groups of anti-DON monoclonal antibody (mAb) and the NHS groups of NHS-MBs were linked by covalent bonds to prepare IMB, without any activation reagent. The essential factors affecting the binding and elution of DON were meticulously tuned. Under optimal conditions, DON could be extracted from a real sample and eluted from IMB by water, enabling environmentally friendly and green analysis. Hence, there was no need for dilution or evaporation prior to UPLC-UV analysis. DON in 20 samples could be purified and concentrated within 30 min by the mycotoxin automated purification instrument (MAPI), allowing for automated, green, high-throughput and simple clean-up. Recoveries at four distinct spiking levels in corn and wheat ranged from 92.0% to 109.5% with good relative standard deviations (RSD, 2.1-7.0%). Comparing the test results of IAC and IMB in commercial samples demonstrated the reliability and superiority of IMB for quantitatively analyzing massive samples.

Postoperative adhesion not only causes severe complications for patients but also increases their economic burden. Injectable bioadhesives with adhesiveness to tissues can cover irregular wounds and stay stable in situ, which is a promising barrier for antiadhesion. However, the potential tissue adhesion caused by bioadhesives' indiscriminate adhesiveness between normal and wounded tissue is still a problem. Herein, by using poly(ethylene glycol) succinimidyl succinate (PEG-SS) and gelatin, a succinyl ester-based bioadhesive (SEgel) was fabricated with self-deactivating properties for postoperative antiadhesion. Because N -hydroxysuccinimide esters (NHS-esters) were used as the adhesive group, the bioadhesives' side in contact with the tissue built covalent anchors quickly to maintain the stability, but the superficial layer facing outward withstood fast hydrolysis and then lost its adhesion within minutes, avoiding the indiscriminate adhesiveness. In addition, because of the specific degradation behavior of succinyl ester, the SEgel with proper in vivo retention was achieved without the worry of causing foreign body reactions and unexpected tissue adhesion. Both the cecum-sidewall adhesion and hepatic adhesion models showed that the SEgel markedly reduced the severity of tissue adhesion. These results, together with the ease of the preparation process and well-proven biocompatibility of raw materials, revealed that the SEgel might be a promising solution for postoperative antiadhesion.

Human glutathione peroxidase1 (hGPx1) is a good antioxidant and potential drug, but the limited availability and poor stability of hGPx1 have affected its development and application. To solve this problem, we prepared a hGPx1 mutant (GPx1M) with high activity in an Escherichia coli BL21(DE3)cys auxotrophic strain using a single protein production (SPP) system. In this study, the GPx1M was conjugated with methoxypolyethylene glycol-succinimidyl succinate (SS-mPEG, M w = 5 kDa) chains to enhance its stability. SS-mPEG-GPx1M and GPx1M exhibited similar enzymatic activity and stability toward pH and temperature change, and in a few cases, SS-mPEG-GPx1M was discovered to widen the range of pH stability and increase the temperature stability. Lys 38 was confirmed as PEGylated site by liquid-mass spectrometry. H9c2 cardiomyoblast cells and Sprague-Dawley (SD) rats were used to evaluate the effects of GPx1M and SS-mPEG-GPx1M on preventing or alleviating adriamycin (ADR)-mediated cardiotoxicity, respectively. The results indicated that GPx1M and SS-mPEG-GPx1M had good antioxidant effects in vitro and in vivo , and the effect of SS-mPEG-GPx1M is more prominent than GPx1M in vivo . Thus, PEGylation might be a promising method for the application of GPx1M as an important antioxidant and potential drug.

The available treatment options include corneal transplantation for significant corneal defects and opacity. However, shortage of donor corneas and safety issues in performing corneal transplantation are the main limitations. Accordingly, we adopted the injectable in situ-forming hydrogels of collagen type I crosslinked via multifunctional polyethylene glycol (PEG)-N-hydroxysuccinimide (NHS) for treatment and evaluated in vivo biocompatibility. The New Zealand White rabbits (N = 20) were randomly grouped into the keratectomy-only and keratectomy with PEG-collagen hydrogel-treated groups. Samples were processed for immunohistochemical evaluation. In both clinical and histologic observations, epithelial cells were able to migrate and form multilayers over the PEG-collagen hydrogels at the site of the corneal stromal defect. There was no evidence of inflammatory or immunological reactions or increased IOP for PEG-collagen hydrogel-treated corneas during the four weeks of observation. Immunohistochemistry revealed the presence of α-smooth muscle actin (α-SMA) in the superior corneal stroma of the keratectomy-only group (indicative of fibrotic healing), whereas low stromal α-SMA expression was detected in the keratectomy with PEG-collagen hydrogel-treated group. Taken together, we suggest that PEG-collagen may be used as a safe and effective alternative in treating corneal defect in clinical setting., (© 2021. The Author(s).)

Targeted treatment of cerebral ischemia/reperfusion injury (CIRI) remains a problem due to the difficulty in drug delivery across the blood-brain barrier (BBB). In this study, we developed Bo-TSA-NP, a novel tanshinone IIA (TSA) loaded nanoparticles modified by borneol, which has long been proved with the ability to enhance other drugs' transport across the BBB. The Bo-TSA-NP, with a particle size of about 160 nm, drug loading of 3.6%, showed sustained release and P-glycoprotein (P-gp) inhibition property. It demonstrated a significantly higher uptake by 16HBE cells in vitro through the clathrin/caveolae-mediated endocytosis and micropinocytosis. Following intranasal (IN) administration, Bo-TSA-NP significantly improved the preventive effect on a rat model of CIRI with improved neurological scores, decreased cerebral infarction areas and a reduced content of malondialdehyde (MDA) and increased activity of superoxide dismutase (SOD) in rat brain. In conclusion, these results indicate that Bo-TSA-NP is a promising nose-to-brain delivery system that can enhance the prevention effect of TSA on CIRI.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious and concealed virus that causes pneumonia, severe acute respiratory syndrome, and even death. Although the epidemic has been controlled since the development of vaccines and quarantine measures, many people are still infected, particularly in third-world countries. Several methods have been developed for detection of SARS-CoV-2, but owing to its price and efficiency, the immune strip could be a better method for the third-world countries., Methods: In this study, two antibodies were linked to latex microspheres, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, as the bridge to decrease the cost further and improve the detection performance. The specificity of the lateral flow immunoassay strip (LFIA) was tested by several common viruses and respiratory bacterial infections. Besides, the reproducibility and stability of the LFIAs were tested on the same batch of test strips. Under optimal conditions, the sensitivity of LFIA was determined by testing different dilutions of the positive specimens., Results: The proposed LFIAs were highly specific, and the limit of detection was as low as 25 ng/mL for SARS-CoV-2 antigens. The clinical applicability was evaluated with 659 samples (230 positive and 429 negative samples) by using both LFIA and rRT-PCR. Youden's index (J) was used to assess the performance of these diagnostic tests. The sensitivity and specificity were 98.22% and 97.93%, respectively, and J is 0.9615. The sensitivity and specificity were 98.22% and 97.93%, respectively, and J is 0.9615. In addition, the consistency of our proposed LFIA was analyzed using Cohen's kappa coefficient (κ = 0.9620)., Conclusion: We found disease stage, age, gender, and clinical manifestations have only a slight influence on the diagnosis. Therefore, the lateral flow immunoassay SARS-CoV-2 antigen test strip is suitable for point-of-care detection and provides a great application for SARS-CoV-2 epidemic control in the third-world countries., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Successful imaging of atherosclerosis, one of the leading global causes of death, is crucial for diagnosis and intervention. Near-infrared fluorescence (NIRF) imaging has been widely adopted along with multimodal/hybrid imaging systems for plaque detection. We evaluate two macrophage-targeting fluorescent tracers for NIRF imaging (TLR4-ZW800-1C and Feraheme-Alexa Fluor 750) in an atherosclerotic murine cohort, where the left carotid artery (LCA) is ligated to cause stenosis, and the right carotid artery (RCA) is used as a control. Imaging performed on dissected tissues revealed that both tracers had high uptake in the diseased vessel compared to the control, which was readily visible even at short exposure times. In addition, ZW800-1C's renal clearance ability and Feraheme's FDA approval puts these two tracers in line with other NIRF tracers such as ICG. Continued investigation with these tracers using intravascular NIRF imaging and larger animal models is warranted for clinical translation.

An efficient synthesis of α-halo-α-arylseleno ketones has been developed via gem-difunctionalization of α-diazoarylketones with diaryldiselenides and N-halosuccinimides. With this multicomponent approach, a series of α-halo-α-arylseleno ketones were accessed in excellent yields and chemoselectivities under mild conditions. This transformation is proposed to proceed via the key intermediate arylselenenyl halide that generated from diaryldiselenides/N-halosuccinimides, followed by addition with α-diazoarylketones to give the desired gem-difunctionalization products., (© 2020. Springer Nature Switzerland AG.)

Cathepsins K and S are closely related papain-like cysteine peptidases and potential therapeutic targets for metabolic and inflammatory diseases such as osteoporosis and arthritis. Here we describe the reduction of a previously characterized succinimide (2,5-dioxopyrrolidine)-containing hyperbolic inhibitor of cathepsin K (methyl (RS)-N-[1-(4-methoxyphenyl)-2,5-dioxopyrrolidin-3-yl]glycinate), to obtain a better and more selective compound (compound 4a - methyl (2,5-dioxopyrrolidin-3-yl)glycinate), which acted as a hyperbolic mixed inhibitor/activator similar to already known allosteric effectors of cathepsin K. We then investigated the potential of the succinimide scaffold as inhibitors of cathepsins K and/or S and synthesized a library of such compounds by 1,4-addition of α-amino acid esters and related compounds to N-substituted maleimides. From the generated library, we identified the first small molecule hyperbolic inhibitors of cathepsin S (methyl ((R)-2,5-dioxopyrrolidin-3-yl)-l-threoninate (compound R-4c) and 3-{[(1S,2R,3'S)-2-hydroxycyclohexyl]amino}pyrrolidine-2,5-dione (compound (1S,2R,3'S-10)). The former acted via a similar mechanism to compound 4a, while the latter was a hyperbolic specific inhibitor of cathepsin S. Given the versatility of the scaffold, the identified compounds will be used as the basis for the development of high-affinity hyperbolic inhibitors of the individual peptidases and to explore the potential of hyperbolic inhibitors for the inhibition of cysteine cathepsins in in vitro models., (Copyright © 2021 Elsevier Inc. All rights reserved.)

N -Hydroxysuccinimide esters of small molecules are widely used to modify biomolecules such as antibodies or proteins. Primary amine groups preferably react with the ester to form covalent amide bonds. Currently, protocols strongly recommend replacing the buffer reagent tris(hydroxymethyl)aminomethane, and it has even been proposed as a stop reagent. Here, we show that TRIS indeed does not interfere with biotinylation of biomolecules with NHS chemistry.

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ., (© 2021 Shi et al.)

A novel Rh-catalyzed cascade C-H activation/annulation of 2-arylbenzimidazoles with maleimides is reported. Rapid chemoselective access to two structurally distinct succinimide-bearing benzoimidazoisoquinolinones is achieved, depending on the acidic and basic conditions. This atom- and step-economic strategy features a wide substrate scope, excellent functional group tolerance, and site-specific functionalization. Application of the methodology yields a novel benzimidazole-based probe as a fluorescent chemosensor for the nanomolar detection of Hg 2+ , Cu 2+ , and Fe 3+ ions.

Many emerging nanobiotechnologies rely on the proper function of proteins immobilized on gold nanoparticles. Often, the surface chemistry of the AuNP is engineered to control the orientation, surface coverage, and structure of the adsorbed protein to maximize conjugate function. Here, we chemically modified antibody to investigate the effect of protein surface chemistries on adsorption to AuNPs. A monoclonal anti-horseradish peroxidase IgG antibody (anti-HRP) was reacted with N-succinimidyl acrylate (NSA) or reduced dithiobissuccinimidyl propionate (DSP) to modify lysine residues. Zeta potential measurements confirmed that both chemical modifications reduced the localized regions of positive charge on the protein surface, while the DSP modification incorporated additional free thiols. Dynamic light scattering confirmed that native and chemically modified antibodies adsorbed onto AuNPs to form bioconjugates; however, adsorption kinetics revealed that the NSA-modified antibody required significantly more time to allow for the formation of a hard corona. Moreover, conjugates formed with the NSA-modified antibody lost antigen-binding function, whereas unmodified and DSP-modified antibodies adsorbed onto AuNPs to form functional conjugates. These results indicate that high-affinity functional groups are required to prevent protein unfolding and loss of function when adsorbed on the AuNP surface. The reduced protein charge and high-affinity thiol groups on the DSP-modified antibody enabled pH-dependent control of protein orientation and the formation of highly active conjugates at solution pHs (<7.5) that are inaccessible with unmodified antibody due to conjugate aggregation. This study establishes parameters for protein modification to facilitate the formation of highly functional and stable protein-AuNP conjugates.

Twelve double fatty chains and Aib 8 -Arg 34 -GLP-1 (7-37) were designed and obtained by microwave-assisted solid-phase synthesis. Then, twelve conjugates of Aib 8 -Arg 34 -GLP-1 (7-37) were synthesized in 1% triethylamine aqueous solution. Conjugates 2, 3, 6, 7, 10 and 11 showed better GLP-1 receptor activation potency than semaglutide. However, conjugates 2, 6 and 10 showed slightly worse glucose-lowering effects in vivo than semaglutide but better effects than conjugates 3, 7 and 11. The CD spectra of conjugates 2, 6 and 10 indicated that they had the same secondary structure as liraglutide and semaglutide. The receptor affinity results for conjugates 2, 6 and 10 measured by SPR (surface plasmon resonance) showed that conjugate 2 had higher receptor affinity than conjugates 6 and 10. In addition, albumin binding assays indicated that double fatty acid chains had obvious synergistic effects compared with single fatty acid chains. In conclusion, the structure-activity relationship of different side chains was summarized and one candidate, conjugate 2, was screened., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cyanobacteria have the capacity to use photosynthesis to fuel their metabolism, which makes them highly promising production systems for the sustainable production of chemicals. Yet, their dependency on visible light limits the cell-density, which is a challenge for the scale-up. Here, it was shown with the example of a light-dependent biotransformation that internal illumination in a bubble column reactor equipped with wireless light emitters (WLEs) could overcome this limitation. Cells of the cyanobacterium Synechocystis sp. PCC 6803 expressing the gene of the ene-reductase YqjM were used for the reduction of 2-methylmaleimide to (R)-2-methylsuccinimide with high optical purity (>99 % ee). Compared to external source of light, illumination by floating wireless light emitters allowed a more than two-fold rate increase. Under optimized conditions, product formation rates up to 3.7 mm h -1 and specific activities of up to 65.5 U g DCW -1 were obtained, allowing the reduction of 40 mm 2-methylmaleimide with 650 mg isolated enantiopure product (73 % yield). The results demonstrate the principle of internal illumination as a means to overcome the intrinsic cell density limitation of cyanobacterial biotransformations, obtaining high reaction rates in a scalable photobioreactor., (© 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH.)

The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry based method that is used to directly characterize and monitor many product quality attributes and impurities on biotherapeutics, most commonly at the peptide level. It utilizes high-resolution accurate mass spectral data which are analyzed in an automated fashion. MAM is a promising approach that is intended to replace or supplement several conventional assays with a single LC-MS analysis and can be implemented in a Current Good Manufacturing Practice environment. MAM provides accurate site-specific quantitation information on targeted attributes and the nontargeted new peak detection function allows to detect new peaks as impurities, modifications, or sequence variants when comparing to a reference sample. The high resolution MAM workflow was applied here for three independent case studies. First, to monitor the behavior of monoclonal antibody product quality attributes over the course of a 12-day cell culture experiment providing an insight into the behavior and dynamics of product attributes throughout the process. Second, the workflow was applied to test the purity and identity of a product through analysis of samples spiked with host cell proteins. Third, through the comparison of a drug product and a biosimilar with known sequence variants. The three case studies presented here, clearly demonstrate the robustness and accuracy of the MAM workflow that implies suitability for deployment in the regulated environment.

N, N'-Disuccinimidyl Carbonate (DSC) is a common reagent for the functionalization of polysaccharides for affinity chromatography. Difficulty in the removal of excess DSC after the functionalization reaction promoted the studies of its solubilities in organic solvents. Surprisingly, DSC has low solubility in several common organic solvents such as dichloromethane, ethyl acetate, tetrahydrofuran, and isopropanol. It is slightly soluble in acetone and acetonitrile, soluble in dimethylformamide, and very soluble in dimethyl sulfoxide. Solvent effect on DSC's use in the functionalization of the Sepharose-6-fastflow resin from GE Healthcare was also studied. While dimethyl sulfoxide did not improve the functionalization reaction, it proved to be an effective solvent removing excess DSC after the functionalization reaction., (Published by Elsevier Inc.)

Cerebrospinal fluid leakage is a frequent complication after cranial and spinal surgery. To prevent this complication and seal the dura watertight, we developed Liqoseal, a dural sealant patch comprising a watertight polyesterurethane layer and an adhesive layer consisting of poly(DL-lactide-co-ε-caprolactone) copolymer and multiarmed N-hydroxylsuccinimide functionalized polyethylene glycol. We compared acute burst pressure and resistance to physiological conditions for 72 h of Liqoseal, Adherus, Duraseal, Tachosil, and Tisseel using computer-assisted models and fresh porcine dura. The mean acute burst pressure of Liqoseal in the cranial model (145 ± 39 mmHg) was higher than that of Adherus (87 ± 47 mmHg), Duraseal (51 ± 42 mmHg) and Tachosil (71 ± 16 mmHg). Under physiological conditions, cranial model resistance test results showed that 2 of 3 Liqoseal sealants maintained dural attachment during 72 hours as opposed to 3 of 3 for Adherus and Duraseal and 0 of 3 for Tachosil. The mean burst pressure of Liqoseal in the spinal model (233 ± 81 mmHg) was higher than that of Tachosil (123 ± 63 mmHg) and Tisseel (23 ± 16 mmHg). Under physiological conditions, spinal model resistance test results showed that 2 of 3 Liqoseal sealants maintained dural attachment for 72 hours as opposed to 3 of 3 for Adherus and 0 of 3 for Duraseal and Tachosil. This novel study showed that Liqoseal is capable of achieving a strong watertight seal over a dural defect in ex vivo models., (© 2021. The Author(s).)

Fibrin has been used clinically for wound coverings, surgical glues, and cell delivery because of its affordability, cytocompatibility, and ability to modulate angiogenesis and inflammation. However, its rapid degradation rate has limited its usefulness as a scaffold for 3D cell culture and tissue engineering. Previous studies have sought to slow the degradation rate of fibrin with the addition of proteolysis inhibitors or synthetic crosslinkers that require multiple functionalization or polymerization steps. These strategies are difficult to implement in vivo and introduce increased complexity, both of which hinder the use of fibrin in research and medicine. Previously, we demonstrated that additional crosslinking of fibrin gels using bifunctionalized poly(ethylene glycol)-n-hydroxysuccinimide (PEG-NHS) slows the degradation rate of fibrin. In this study, we aimed to further improve the longevity of these PEG-fibrin gels such that they could be used for tissue engineering in vitro or in situ without the need for proteolysis inhibitors. It is well documented that increasing the salinity of fibrin precursor solutions affects the resulting gel morphology. Here, we investigated whether this altered morphology influences the fibrin degradation rate. Increasing the final sodium chloride (NaCl) concentration from 145 mM (physiologic level) to 250 mM resulted in fine, transparent high-salt (HS) fibrin gels that degrade 2-3 times slower than coarse, opaque physiologic-salt (PS) fibrin gels both in vitro (when treated with proteases and when seeded with amniotic fluid stem cells) and in vivo (when injected subcutaneously into mice). Increased salt concentrations did not affect the viability of encapsulated cells, the ability of encapsulated endothelial cells to form rudimentary capillary networks, or the ability of the gels to maintain induced pluripotent stem cells. Finally, when implanted subcutaneously, PS gels degraded completely within one week while HS gels remained stable and maintained viability of seeded dermal fibroblasts. To our knowledge, this is the simplest method reported for the fabrication of fibrin gels with tunable degradation properties and will be useful for implementing fibrin gels in a wide range of research and clinical applications., Competing Interests: The authors have declared that no competing interests exist.

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: Pretargeting radioimmunotherapy (PRIT) is a promising approach that can reduce long-time retention of blood radioactivity and consequently reduce hematotoxicity. Among the PRIT strategies, the combination of biotin-conjugated mAb and radiolabeled streptavidin (StAv) is a simple and convenient method because of its ease of preparation. This study performed three-step (3-step) PRIT using the sequential injection of (1) biotinylated bevacizumab (Bt-BV), (2) avidin, and (3) radiolabeled StAv for the treatment of triple-negative breast cancer (TNBC)., Methods: Four biodistribution studies were performed using 111 In in tumor-bearing mice to optimize each step of our PRIT methods. Further, a therapeutic study was performed with optimized 3-step PRIT using 90 Y-labeled StAv., Results: Based on the biodistribution studies, the protein dose of Bt-BV and avidin was optimized to 100 μg and 10 molar equivalent of BV, respectively. Succinylation of StAv significantly decreased the kidney accumulation level (with succinylation (6.96 ± 0.91) vs without succinylation (20.60 ± 1.47) at 1 h after injection, p < 0.0001) with little effect on the tumor accumulation level. In the therapeutic study, tumor growth was significantly suppressed in treatment groups with optimized 3-step PRIT using 90 Y-labeled succinylated StAv compared to that of the no-treatment group (p < 0.05)., Conclusions: The 3-step PRIT strategy of this study achieved fast blood clearance and low kidney uptake with little effect on the tumor accumulation level, and a certain degree of therapeutic effect was consequently observed. These results indicated that the pretargeting treatment of the current study may be effective for human TNBC treatment.

Macrophages are desirable targets for gene therapy of cancer and other diseases. Cationic diblock copolymers of polyethylene glycol (PEG) and poly-L-lysine (PLL) or poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (pAsp(DET)) are synthesized and used to form polyplexes with a plasmid DNA (pDNA) that are decorated with mannose moieties, serving as the targeting ligands for the C type lectin receptors displayed at the surface of macrophages. The PEG-b-PLL copolymers are known for its cytotoxicity, so PEG-b-PLL-based polyplexes are cross-linked using reducible reagent dithiobis(succinimidyl propionate) (DSP). The cross-linked polyplexes display low toxicity to both mouse embryonic fibroblasts NIH/3T3 cell line and mouse bone marrow-derived macrophages (BMMΦ). In macrophages mannose-decorated polyplexes demonstrate an ≈8 times higher transfection efficiency. The cross-linking of the polyplexes decrease the toxicity, but the transfection enhancement is moderate. The PEG-b-pAsp(DET) copolymers display low toxicity with respect to the IC-21 murine macrophage cell line and are used for the production of non-cross-linked pDNA-contained polyplexes. The obtained mannose modified polyplexes exhibit ca. 500-times greater transfection activity in IC-21 macrophages compared to the mannose-free polyplexes. This result greatly exceeds the targeting gene transfer effects previously described using mannose receptor targeted non-viral gene delivery systems. These results suggest that Man-PEG-b-pAsp(DET)/pDNA polyplex is a potential vector for immune cells-based gene therapy., (© 2021 Wiley-VCH GmbH.)

The cytokeratin fragment antigen 21-1 (CYFRA 21-1) protein is a critical tumor biomarker tightly related to non-small cell lung cancer (NSCLC). Herein, we prepared an effective electrochemiluminescence (ECL) immunosensor for CYFRA 21-1 detection using electrochemically mediated atom transfer radical polymerization (eATRP). The CYFRA 21-1 antigen was fixed on the electrode surface by constructing a sandwich type antibody-antigen-antibody immune system. The sensitivity of ECL was improved by using the eATRP reaction. In this method, eATRP was applied to CYFRA 21-1 detection antibody with N-acryloyloxysuccinimide as functional monomer. This is the first time that ECL and eATRP signal amplification technology had been combined. Under the optimized testing conditions, the immunosensor showed a good linear relation in the range from 1 fg mL -1 to 1 μg mL -1 at a limit of detection of 0.8 fg mL -1 (equivalent to ~ 134 molecules in a 10 μL sample). The ECL immunosensing system based on eATRP signal amplification technology provided a new way for rapid diagnosis of lung cancer by detecting CYFRA 21-1. The paper prepared an electrochemiluminescence biosensor for ultrasensitive detection of CYFRA 21-1 via eATRP signal amplification strategy, which had the advantages of high sensitivity, reproducibility, and held potential prospect for analysis of low-abundance.

Halogenated natural products number in the thousands, but only in rare cases are the evolutionary advantages conferred by the halogens understood. We set out to investigate the lissoclimide family of cytotoxins, which includes several chlorinated members, because of our long-standing interest in the synthesis of chlorinated secondary metabolites.Our initial success in this endeavor was a semisynthesis of chlorolissoclimide (CL) from the commercially available sesquiterpenoid sclareolide. Featuring a highly selective and efficient-and plausibly biomimetic-C-H chlorination, we were able to access enough CL for collaborative studies, including X-ray cocrystallography with the eukaryotic ribosome. Through this experiment, we learned that CL's chlorine atom engages in a novel halogen-π dispersion interaction with a neighboring nucleobase in the ribosome E-site.Owing to the limitations of our semisynthesis approach, we established an analogue-oriented approach to access numerous lissoclimide compounds to both improve our understanding of structure-activity relationships and to learn more about the halogen-π interaction. In the course of these studies, we made over a dozen lissoclimide-like compounds, the most interesting of which contained chlorine-bearing carbons with unnatural configurations. Rationalizing the retained potency of these compounds that appeared to be a poor fit for the lissoclimide binding pocket, we came to realize that the chlorine atoms would engage in these same halogen-π interactions even at the expense of a chair to twist-boat conformational change, which also permitted the compounds to fit in the binding site.Finally, because neither of the first two approaches could easily access the most potent natural lissoclimides, we designed a synthesis that took advantage of rarely used terminal epoxides to initiate polyene cyclizations. In this case, the chlorine atom was incorporated early and helped control the stereochemical outcome of the key step.Over the course of this project, three different synthesis approaches were designed and executed, and our ability to access numerous lissoclimides fueled a range of collaborative biological studies. Further, chlorine played impactful roles throughout various aspects of both synthesis and biology. We remain inspired to learn more about the mechanism of action of these compounds and to deeply investigate the potentially valuable halogen-π dispersion interaction in the context of small molecule/nucleic acid binding. In that context, our work offers an instance wherein we might have gained a rudimentary understanding of the evolutionary importance of the halogen in a halogenated natural product.

Succinimides are well recognized heterocyclic compounds in drug discovery which produce diverse therapeutically related applications in pharmacological practices. Researches in medicinal chemistry field have isolated and synthesized succinimide derivatives with multiple medicinal properties including anticonvulsant, anti-inflammatory, antitumor and antimicrobial agents, 5-HT receptor ligands and enzyme inhibitors. Simultaneously, SAR (Structure-Activity Relationship) analysis has been gradually possessed, along with a great deal of derivatives have been derived for potential targets. In this article, we comprehensively summarize the biological activities and SAR for succinimide derivatives, along with the featuring bioactive molecules reported in patents, wishing to provide an overall retrospect and prospect on the succinimide analogues., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Superporous poly(2-hydroxyethyl methacrylate- co -2-aminoethyl methacrylate) (P(HEMA-AEMA)) hydrogel scaffolds are designed for in vitro 3D culturing of leukemic B cells. Hydrogel porosity, which influences cell functions and growth, is introduced by adding ammonium oxalate needle-like crystals in the polymerization mixture. To improve cell vitality, cell-adhesive Arg-Gly-Asp-Ser (RGDS) peptide is immobilized on the N -(γ-maleimidobutyryloxy)succinimide-activated P(HEMA-AEMA) hydrogels via reaction of SH with maleimide groups. This modification is especially suitable for the survival of primary chronic lymphocytic leukemia cells (B-CLLs) in 3D cell culture. No other tested stimuli (interleukin-4, CD40 ligand, or shaking) can further improve B-CLL survival or metabolic activity. Both unmodified and RGDS-modified P(HEMA-AEMA) scaffolds serve as a long-term (70 days) 3D culture platforms for HS-5 and M2-10B4 bone marrow stromal cell lines and MEC-1 and HG-3 B-CLL cell lines, although the adherent cells retain their physiological morphologies, preferably on RGDS-modified hydrogels. Moreover, the porosity of hydrogels allows direct cell lysis, followed by efficient DNA isolation from the 3D-cultured cells. P(HEMA-AEMA)-RGDS thus serves as a suitable 3D in vitro leukemia model that enables molecular and metabolic assays and allows imaging of cell morphology, interactions, and migration by confocal microscopy. Such applications can prospectively assist in testing of drugs to treat this frequently recurring or refractory cancer.

Background: Infectious diseases are one of the main causes of morbidity and mortality worldwide. Nuclear molecular imaging would be of great help to non-invasively discriminate between septic and sterile inflammation through available radiopharmaceuticals, as none is currently available for clinical practice. Here, we describe the radiolabeling procedure and in vitro and in vivo studies of 99m Tc-polymyxin B sulfate (PMB) as a new single photon emission imaging agent for the characterization of infections due to Gram-negative bacteria., Results: Labeling efficiency was 97 ± 2% with an average molar activity of 29.5 ± 0.6 MBq/nmol. The product was highly stable in saline and serum up to 6 h. In vitro binding assay showed significant displaceable binding to Gram-negative bacteria but not to Gram-positive controls. In mice, 99m Tc-HYNIC-PMB was mainly taken up by liver and kidneys. Targeting studies confirmed the specificity of 99m Tc-HYNIC-PMB obtained in vitro, showing significantly higher T/B ratios for Gram-negative bacteria than Gram-positive controls., Conclusions: In vitro and in vivo results suggest that 99m Tc-HYNIC-PMB has a potential for in vivo identification of Gram-negative bacteria in patients with infections of unknown etiology. However, further investigations are needed to deeply understand the mechanism of action and behavior of 99m Tc-HYNIC-PMB in other animal models and in humans.

The biorecognition-based control of attachment/detachment of MCF-7 cancer cells from polymer-coated surfaces is demonstrated. A glass surface is coated with a thermoresponsive statistical copolymer of poly(N-isopropylacrylamide-co-acrylamide) [p(NIPAm-co-Am)], which is end-capped with the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide, and the hydrophilic polymer poly(ethylene glycol) (PEG). Below the lower critical solution temperature (LCST) of p(NIPAm-co-Am) (38 °C), the copolymers are in the extended conformation, allowing for accessibility of the GRGDS peptides to membrane-associated integrins thus enabling cell attachment. Above the LCST, the p(NIPAm-co-Am) polymers collapse into globular conformations, resulting in the shielding of the GRGDS peptides into the PEG brush with consequent inaccessibility to cell-surface integrins, causing cell detachment. The surface coating is carried out by a multi-step procedure that included: glass surface amination with 3-aminopropyltriethoxysilane; reaction of mPEG 5kDa -N-hydroxysuccinimide (NHS) and p(NIPam-co-Am) 15.1kDa -bis-NHS with the surface aminopropyl groups and conjugation of GRGDS to the carboxylic acid termini of p(NIPam-co-Am) 15.1kDa -COOH. A range of spectrophotometric, surface, and microscopy assays confirmed the identity of the polymer-coated substrates. Competition studies prove that MCF-7 cancer cells are attached via peptide recognition at the coated surfaces according to the mPEG 5kDa /p(NIPam-co-Am) 15.1kDa -GRGDS molar ratio. These data suggest the system can be exploited to modulate cell integrin/GRGDS binding for controlled cell capture and release., (© 2020 Wiley-VCH GmbH.)