Zero-length Cross-linker Research Articles

Highly conductive, stretchable, and biocompatible graphene oxide biocomposite hydrogel for advanced tissue engineering

The importance of hydrogels in tissue engineering cannot be overemphasized due to their resemblance to the native extracellular matrix. However, natural hydrogels with satisfactory biocompatibility exhibit poor mechanical behavior, which hampers their application in stress-bearing soft tissue engineering. Here, we describe the fabrication of a double methacrylated gelatin bioink covalently linked to graphene oxide (GO) via a zero-length crosslinker, digitally light-processed (DLP) printable into 3D complex structures with high fidelity. The resultant natural hydrogel (GelGOMA) exhibits a conductivity of 15.0 S m-1as a result of the delocalization of theπ-orbital from the covalently linked GO. Furthermore, the hydrogel shows a compressive strength of 1.6 MPa, and a 2.0 mm thick GelGOMA can withstand a 1.0 kg ms-1momentum. The printability and mechanical strengths of GelGOMAs were demonstrated by printing a fish heart with a functional fluid pumping mechanism and tricuspid valves. Its biocompatibility, electroconductivity, and physiological relevance enhanced the proliferation and differentiation of myoblasts and neuroblasts and the contraction of human-induced pluripotent stem cell-derived cardiomyocytes. GelGOMA demonstrates the potential for the tissue engineering of functional hearts and wearable electronic devices.

Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence.

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit fromfunctional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (kcat). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

Boron-doped diamond nanosheet volume-enriched screen-printed carbon electrodes: a platform for electroanalytical and impedimetric biosensor applications

This paper focuses on the development of a novel electrode based on boron-doped diamond nanosheet full-volume-enriched screen-printed carbon electrodes (BDDPE) for use as an impedimetric biosensor. Impedimetric biosensors offer high sensitivity and selectivity for virus detection, but their use as point-of-care devices is limited by the complexity of nanomaterials’ architecture and the receptor immobilisation procedures. The study presents a two-step modification process involving the electroreduction of diazonium salt at the BDDPE and the immobilisation of antibodies using zero-length cross-linkers for a selective impedimetric biosensor of Haemophilus influenzae (Hi). The incorporation of diamond nanosheets into BDDPE leads to enhanced charge transfer and electrochemical behaviour, demonstrating greatly improved electrochemically active surface area compared with unmodified screen-printed electrodes (by 44% and 10% on average for [Ru(NH3)6]Cl2 and K3[Fe(CN)6], respectively). The presented sensing system shows high specificity towards protein D in Hi bacteria, as confirmed by negative controls against potential interference from other pathogens, with an estimated tolerance limit for interference under 12%. The Hi limit of detection by electrochemical impedance spectroscopy was 1 CFU/mL (measured at − 0.13 V vs BDDPE pseudo-reference), which was achieved in under 10 min, including 5 min sample incubation in the presence of the analyte.Graphical abstract

Determination of HER2 binding domain in antigen-antibody complexes based on chemical crosslinking mass spectrometry

Chemical crosslinking (XL) of non-covalent antigen-antibody complexes followed by mass spectrometric identification (MS) of inter-protein crosslinks can provide spatial constraints between relevant residues, which are valuable structural information associated with the molecular binding interface. To highlight the potential of XL/MS in the biopharmaceutical industry, we herein developed and validated an XL/MS workflow that employed a zero-length linker, 1,1′‑carbonyldiimidazole (CDI), and a widely used medium-length linker, disuccinimidyl sulfoxide (DSSO), for fast, accurate determination of antigen domains targeted by therapeutic antibodies. To avoid false identification, system suitability samples and negative samples were designed for all experiments, and all tandem mass spectra were manually examined. To validate the proposed XL/MS workflow, two complexes involving human epidermal growth factor receptor 2 Fc fusion protein (HER2Fc) with known crystal structures, including HER2Fc-pertuzumab and HER2Fc-trastuzumab, have been subjected to CDI and DSSO crosslinking. Crosslinks established by CDI and DSSO between HER2Fc and pertuzumab accurately revealed their interaction interface. CDI crosslinking contributes more than DSSO because of its short spacer arm and high reactivity towards hydroxyl groups, demonstrating its capacity in protein interaction analysis. The correct binding domain cannot be revealed solely based on DSSO in the HER2Fc-trastuzumab complex, because domain proximity revealed by this 7-atom spacer linker cannot be directly translated as binding interfaces. As the first successful XL/MS application in early-stage therapeutic antibody discovery, we analyzed the molecular binding interface between HER2Fc and H-mab, an innovant drug candidate whose paratopes have not been studied yet. We predict that H-mab probably targets HER2 Domain I. The proposed XL/MS workflow can serve as an accurate, fast, and low-cost method to study the interaction between antibodies and large multi-domain antigens. SignificanceThis article described a fast, low-consumption approach based on chemical crosslinking mass spectrometry (XL/MS) using two linkers for binding domain determination in multidomain antigen-antibody complexes. Our results highlighted the higher importance of zero-length crosslinks established by CDI than 7-atom DSSO crosslinks, as residue proximity revealed by zero-length crosslinks is closely related to epitope-paratope interaction surfaces. Furthermore, the higher reactivity of CDI towards hydroxyl groups broadens the ranges of possible crosslinks, despite the necessity of delicate operation in CDI crosslinking. We suggest that all established CDI and DSSO crosslinks should be comprehensively considered for correct binding domain analysis because predictions solely based on DSSO might be ambiguous. We have determined the binding interface in the HER2-H-mab using CDI and DSSO, which is the first successful application of XL/MS in real-world early-stage biopharmaceutical development.

Electrospun Nanofibers including Organic/Inorganic Nanohybrids: Polystyrene- and Clay-Based Architectures in Immunosensor Preparation for Serum Amyloid A.

Diagnostic techniques based on biomolecules have application potential that can be realized in many fields, such as disease diagnosis, bioprocess imaging, food/beverage industries, and environmental pollutant imaging. Successful surface immobilization of biomolecules is critical to increasing the stabilization, sensitivity, and selectivity of biomolecules used in bioassay systems. Nanofibers are good candidates for the immobilization of biomolecules owing to many advantages such as morphology and pore size. In this study, montmorillonite (MMT) clay is modified with poly(amidoamine) (PAMAM) generation 3 (PAMAMG3) and added to polystyrene (PS) solutions, following which PS/MMT-PAMAMG3 nanofibers are obtained using the electrospinning method. The nanofibers are obtained by testing PS% (wt%) and MMT-PAMAMG3% (wt%) ratios and characterized with scanning electron microscopy. Antiserum amyloid A antibody (Anti-SAA) is then conjugated to the nanofibers on the electrode surface via covalent bonds using a zero-length cross linker. Finally, the obtained selective surface is used for electrochemical determination of serum amyloid A (SAA) levels. The linear range of PS/MMT-PAMAM/Anti-SAA is between 1 and 200 ng/mL SAA, and the detection limit is 0.57 ng/mL SAA. The applicability of PS/MMT-PAMAMG3/Anti-SAA is investigated by taking measurements in synthetic saliva and serum both containing SAA.

Ynamide Coupling Reagent for the Chemical Cross-Linking of Proteins in Live Cells.

Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) is a powerful method for the study of protein structure and protein-protein interactions (PPIs). However, the chemical probes used in the CXMS are limited to bidentate reactive warheads, and the available zero-length cross-linkers are restricted to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). To alleviate this issue, an efficient coupling reagent, sulfonyl ynamide, was developed as a new zero-length cross-linker that can connect high-abundance carboxyl residues (D/E) with lysine (K) to form amide bonds in the absence of any catalyst. Significant improvement in the cross-linking efficiency and specificity in comparison with traditional EDC/NHS was achieved with model proteins, which includes inter- and intramolecular conjugations. The cross-linked structures were validated by X-ray crystallography. Importantly, this coupling reagent can be successfully used to capture interacting proteins in the whole proteome and can be a useful reagent for probing potential protein-protein interactions in situ.

Direct Electron Transfer of Glucose Oxidase on Pre-Anodized Paper/Carbon Electrodes Modified through Zero-Length Cross-Linkers for Glucose Biosensors.

A disposable paper-based glucose biosensor with direct electron transfer (DET) of glucose oxidase (GOX) was developed through simple covalent immobilization of GOX on a carbon electrode surface using zero-length cross-linkers. This glucose biosensor exhibited a high electron transfer rate (ks, 3.363 s-1) as well as good affinity (km, 0.03 mM) for GOX while keeping innate enzymatic activities. Furthermore, the DET-based glucose detection was accomplished by employing both square wave voltammetry and chronoamperometric techniques, and it achieved a glucose detection range from 5.4 mg/dL to 900 mg/dL, which is wider than most commercially available glucometers. This low-cost DET glucose biosensor showed remarkable selectivity, and the use of the negative operating potential avoided interference from other common electroactive compounds. It has great potential to monitor different stages of diabetes from hypoglycemic to hyperglycemic states, especially for self-monitoring of blood glucose.

Study on the Crosslinking Modification of Collagen-Based Materials by DMTMM

In order to explore the mechanism of interaction between DMTMM and collagen-based materials, gelatin was used as a skin simulant and prepare DMTMM crosslinked modified gelatin materials. FT-IR and Zeta potential measurements were used to analyze and characterize DMTMM crosslinking modified gelatin materials; revealing the mechanism of interaction between DMTMM and collagen-based materials. Firstly, the “zero-length” crosslinking agent DMTMM activated the carboxyl groups on the collagen-based materials and promoted the formation of amide bonds between the collagen-based materials. At the same time, the hydrogen bond formed between the hydroxyl group and its active group causes zero-length crosslinking modification of collagen-based material under the joint action of these two kinds of bonding. DSC, TG, swelling properties, SEM and other analytical methods were used to test the thermal stability and microstructure of DMTMM crosslinked modified gelatin materials. It was found that DMTMM can effectively improve the stability of collagen-based materials, increase the thermal denaturation temperature and humidity and reduce the decomposition rate.

Abstract 2802: Rare earth albumin nanoparticles engineered to target cytotoxic T cells to evaluate response to immunotherapy

Abstract Checkpoint immunotherapy, through the reversal of tumor-mediated inactivation of the immune system, has shown promise in the treatment of several types of cancer. This has culminated in the approval of seven immune checkpoint inhibitors (ICIs). However, only a small population of patients respond to these drugs. Because of the physical and economic burden of ICIs on the patient, there is a critical need to identify biomarkers that can inform on the potential response to ICIs. The presence of tumor infiltrating lymphocytes (TILs) has demonstrated good prognostic value in determining if a patient should receive ICIs. Current clinical methods to assess TILs involve invasive biopsies and immunohistochemistry, which suffer from intratumoral heterogeneity, observer variability, and a lack of real-time feedback. Here, we report on near infrared light excitable rare earth metal-based nanoparticles, termed rare earth albumin nanocomposites (ReANCs), that emit shortwave infrared (SWIR) light, allowing for deep tissue imaging and high signal-to-noise ratios compared to visible or near infrared fluorescence probes. Tumor-targeted ReANCs have been previously employed to monitor tumor progression and response to chemotherapy in mouse models of breast cancer metastasis. In this study, to target CD3+ T cells, ReANCs were conjugated using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to a peptide derived from the sequence of the CD3-ϵ receptor sub-unit. Target specific binding was validated by flow cytometry as a measure of increased uptake of peptide-conjugated ReANCs by Jurkat cells. To specifically target cytotoxic T lymphocytes, we employed the fragment antigen binding (Fab) derived from enzymatic digestion of a CD8 antibody (clone 53-6.7) with papain. The Fab fragments were conjugated to ReANCs with sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). Conjugation was confirmed by non-reducing gel electrophoresis and high performance liquid chromatography (HPLC). A loading efficiency of approximately 60% was achieved. Target specific binding was validated by flow cytometry as a measure of increased uptake of Fab-conjugated ReANCs by T cells isolated from splenocytes. We generated a metric for measuring immune burden around tumor spheroids by pre-labeling T cells with ReANCs and co-culturing them with tumor cell spheroids in vitro. Imaging of T cells with CD3 and CD8-targeted ReANCs provides a basis for future in vivo small animal imaging studies where we will investigate the potential of this technology to track immune cells in relation to a tumor in real time. Metrics of immune cell imaging will then inform on the potential of immunotherapy and monitor response to treatment in a longitudinal study. Citation Format: Jay V. Shah, Jake N. Siebert, Amber Gonda, Rahul Pemmaraju, Shashank Kosuri, Carolina Bobadilla Mendez, Xinyu Zhao, Shuqing He, Richard E. Riman, Mei Chee Tan, Mark C. Pierce, Edmund C. Lattime, Prabhas V. Moghe, Vidya Ganapathy. Rare earth albumin nanoparticles engineered to target cytotoxic T cells to evaluate response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2802.

Electrochemical Immunosensor for the Quantification of S100B at Clinically Relevant Levels Using a Cysteamine Modified Surface.

Neuronal damage secondary to traumatic brain injury (TBI) is a rapidly evolving condition, which requires therapeutic decisions based on the timely identification of clinical deterioration. Changes in S100B biomarker levels are associated with TBI severity and patient outcome. The S100B quantification is often difficult since standard immunoassays are time-consuming, costly, and require extensive expertise. A zero-length cross-linking approach on a cysteamine self-assembled monolayer (SAM) was performed to immobilize anti-S100B monoclonal antibodies onto both planar (AuEs) and interdigitated (AuIDEs) gold electrodes via carbonyl-bond. Surface characterization was performed by atomic force microscopy (AFM) and specular-reflectance FTIR for each functionalization step. Biosensor response was studied using the change in charge-transfer resistance (Rct) from electrochemical impedance spectroscopy (EIS) in potassium ferrocyanide, with [S100B] ranging 10–1000 pg/mL. A single-frequency analysis for capacitances was also performed in AuIDEs. Full factorial designs were applied to assess biosensor sensitivity, specificity, and limit-of-detection (LOD). Higher Rct values were found with increased S100B concentration in both platforms. LODs were 18 pg/mL(AuES) and 6 pg/mL(AuIDEs). AuIDEs provide a simpler manufacturing protocol, with reduced fabrication time and possibly costs, simpler electrochemical response analysis, and could be used for single-frequency analysis for monitoring capacitance changes related to S100B levels.

Exploring the effects of the crosslink density on the physicochemical properties of collagen-based scaffolds

The optimization of collagen-based scaffolds for tissue engineering goes through the careful selection of the crosslinking method(s), which should impart the prerequisite mechanical and degradation properties without impairing the cell/tissue response. Here, we investigated the chemically effective (ρxch) and the elastically effective (ρxel) crosslink density of collagen-based scaffolds, induced by various crosslinking methods. The aim was to get a deeper insight into the influence of intramolecular and intermolecular crosslinks on several scaffold properties. Freeze-dried collagen matrices were crosslinked via a dehydrothermal treatment (DHT), and then treated with different chemical agents, including carbodiimide (EDC), glutaraldehyde (GTA), formaldehyde (FA), genipin (GP) and dimethyl suberimidate (DMS). Quantification of primary amines and stress-relaxation compressive tests were performed to evaluate ρxch and ρxel, respectively. Scaffolds were then assessed for their water uptake, thermal stability and in vitro resistance to enzymatic degradation. Interestingly, for the various crosslinking treatments ρxch was found to increase in the order DHT < DHT + GP < DHT + DMS < DHT + GTA < DHT + FA < DHT + EDC, while ρxel increased according to this slightly different trend: DHT < DHT + GP < DHT + DMS < DHT + EDC < DHT + GTA < DHT + FA. Indeed, treatment DHT + EDC induced a higher ρxch but a lower ρxel than aldehyde-based ones. This finding, together with the higher denaturation temperature (Td) of EDC-treated samples compared to others, suggested that zero-length EDC crosslinking promoted intramolecular crosslinks, along with intermolecular ones. Accordingly, the increase of Td was correlated with the increase of ρxch rather than ρxel, whereas the decrease in water uptake was consistent with the increase of ρxel, as expected. An exponential relationship between ρxel and the in vitro half-life was also determined.

Catalase-conjugated surfaces: H2O2 detection based on quenching of tryptophan fluorescence on conducting polymers

Conducting polymers are promising materials used to prepare electrochemical and optical detection platforms for bioanalytical systems. After conjugation of biomolecules onto electropolymerized monomers for preparation of multifunctional surfaces, they can be used to easily monitor small molecules, macromolecules, and cells. In this study, multifunctional monomer with indole (necessary for fluorescence detection of H2O2) and carboxyl functional groups (necessary for covalent immobilization of biological material) was synthesized, electropolymerized, modified with biomolecules (catalase) and then applied for the selective detection of target analyte (H2O2). Tryptophan sequestered dithione [3,2-b:2′,3′-d] pyrrole (DTP-Trp) was synthesized and electropolymerized on indium tin oxide (ITO) coated polyethylene terephthalate (PET) substrates, which is a transparent support. Afterward, catalase (CAT) was immobilized on the Poly(DTP-Trp) using a zero-length crosslinker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), via covalent bonds between carboxyl groups of CAT and amino groups of Poly(DTP-Trp). Surface characterization of Poly(DTP-Trp)/CAT was done by scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDX). The presence of Poly(DTP-Trp)/CAT on ITO-PET surfaces were also confirmed by electrochemical methods to show the success of support modification. Determination of H2O2 was investigated using fluorescence spectrometry based on quenching mechanism of H2O2 on the fluorescence of tryptophan. After optimization of working conditions of Poly(DTP-Trp)/CAT, H2O2 detection in synthetic samples was done without any interference of the matrices.

Non-lytic M13 phage-based highly sensitive impedimetric cytosensor for detection of coliforms

A highly sensitive and selective non-lytic M13 phage-based electrochemical impedance spectroscopy (EIS) cytosensor for early detection of coliforms is introduced for the first time. Gold nanoparticles were electrochemically deposited on the surface of glassy carbon electrode, and the M13 phage particles were immobilized on them using 3-mercaptopropionic acid linker and zero-length crosslinking chemistry (EDC/NHS). Next, the sensor surface was blocked to avoid non-specific binding. The M13-EIS cytosensor was tested for detection of F+ pili Escherichia coli species, using XL1-Blue and K12 strains, as examples of coliforms. The selectivity against non-host strains was demonstrated using Pseudomonas Chlororaphis. The binding of E. coli to the M13 phage on the cytosensor surface increased the charge transfer resistance, enabling detection of coliforms. The biosensor achieved a limit of detection (LOD) of 14 CFU/mL, the lowest reported to-date using EIS-phage sensors, and exhibited a high selectivity towards the tested coliforms. The SEM micrographs confirmed the successful capturing of E. coli on the M13-based EIS cytosensor. Moreover, the sensor showed almost the same sensitivity in the simulated river water samples as in phosphate buffer, reflecting its applicability to real samples. On the other hand, this sensor system exhibited high stability under harsh environmental conditions of pH (3.0–10.0) and temperature as high as 45 °C for up to two weeks. Overall, this sensor system has excellent potential for real field detection of fecal coliforms.

Magnetic Nanoparticles As a Biological Sensors for Early Diagnosis and Treatment of Cancer Cells

Recently, Nano-bio interactions become very attractive due to their early disease diagnosis and therapy. Furthermore, studies of these nano-particles and their interactions with healthy tissues and organs have enabled new approaches for the precise control of off-target nano-particle clearance, which is equally important as disease targeting. For this reason, coating them with bio-compatible agents became necessary for in vitro and in vivo applications. In this research, magnetic nano-particles synthesized by chemical decomposition method and coated with the bio-compatible agents around them. Besides, as-synthesized RMNPs powders were characterized by X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM/EDAX), Fourier transforms infrared spectroscopy (FT-IR), Vibrating sample magnetometry (VSM), and Zeta Potential analyser. In order to conjugate several Biological agents, zero-length crosslinkers such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or dicyclohexylcarbodiimide (DCC) which makes use of carbodiimide chemistry to covalently bond a primary or secondary amine to the alpha carbon of a carboxylate group has been used to forming an amide linkage. On the surface of these nano-particles, fluorescent agents have been used in order to early diagnosing of cancer cells. For this purpose, Bio-compatible peptide-based Nano-systems proposed to demonstrate the fluorescence. On the one hand, due to the magnetic nature of these nano-particles with high magnetization (~80 emu/g) succeeded to guide them to the exact location of tumors and also we used hyperthermia cancer therapy. On the other hand, by mounting the photosensitizer agents on the surface of these nano-particles, activated these agents by stimulating them in the specific wavelengths (450 nm), so, this help us to damage tumor cells by a photodynamic method. Also, these nano-particles were able to use as a cargo for anti-cancer drugs. As, Arginylglycylaspartic acid (RGD) peptides, are designed, fabricated, and experimentally validated for drug delivery and imaging they were a good candidate for this purpose. So that, these nano-particles used to encapsulate anti-cancer drugs such as Cisplatin, Doxorubicin and, 5fluorouracil. Multi-functionalized MNPs not only have the ability of early detection of illnesses, including tumor cells, but also, by engineering their surface, we succeeded to damage the tumor cells in the Multilateral way. 1-Du B, Yu M, Zheng J. Transport and interactions of nanoparticles in the kidneys. Nature Reviews Materials. 2018 Aug 3:1. 2- Bagherpour AR, Kashanian F, Ebrahimi SS, Habibi-Rezaei M. L-arginine modified magnetic nanoparticles: green synthesis and characterization. Nanotechnology. 2018 Jan 15;29(7):075706. 3- Shi D, Sadat ME, Dunn AW, Mast DB. Photo-fluorescent and magnetic properties of iron oxide nanoparticles for biomedical applications. Nanoscale. 2015;7(18):8209-32. 4- Jin D, Xi P, Wang B, Zhang L, Enderlein J, van Oijen AM. Nanoparticles for super-resolution microscopy and single-molecule tracking. Nature methods. 2018 May 28:1. 5- Fan Z, Chang Y, Cui C, Sun L, Wang DH, Pan Z, Zhang M. Near infrared fluorescent peptide nanoparticles for enhancing esophageal cancer therapeutic efficacy. Nature communications. 2018 Jul 4;9(1):2605. 6- Zhang J, Jiang C, Longo JP, Azevedo RB, Zhang H, Muehlmann LA. An updated overview on the development of new photosensitizers for anticancer photodynamic therapy. Acta pharmaceutica sinica B. 2018 Mar 1;8(2):137-46. Figure 1

The distribution of cell-penetrating peptides on polymeric nanoparticles prepared using microfluidics and elucidated with small angle X-ray scattering

HypothesisThe distribution of three cell-penetrating peptides (CPPs) with different architectures (short, long linear and branched) on poly(lactic-co-glycolic) acid (PLGA) nanoparticles depends on the conjugation approach. Here, we explore the utilization of a zero-length crosslinking reaction for the covalent attachment of CPPs to PLGA nanoparticles and the translation of the reaction into a microfluidic platform. ExperimentsA microfluidic device with a staggered herringbone mixer was used for the formulation of CPP-tagged PLGA nanoparticles. CPP-tagged PLGA nanoparticles were labeled with gold nanoparticles (AuNPs) and transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS) were used to elucidate the distribution of CPPs. FindingsThe SAXS scattering profiles for the CPP-tagged PLGA nanoparticles prepared with the in situ microfluidics conjugation approach indicated a distribution of the Au-labeled CPPs throughout the PLGA nanoparticles. For the post-microfluidics conjugation approach, the SAXS scattering profiles did not show the feature of the Au-labeled CPPs distributed throughout the PLGA nanoparticles and an arrangement of the Au-labeled CPP on the surface was support by TEM micrographs. The distribution of the CPPs was highly dependent on the conjugation approach and was not influenced by the architecture of the CPPs. The results provided insight for the rational design of CPP-tagged PLGA nanoparticles using microfluidics.

Apolipoprotein A1 Forms 5/5 and 5/4 Antiparallel Dimers in Human High-density Lipoprotein

Apolipoprotein A1 (APOA1), the major protein of high-density lipoprotein (HDL), contains 10 helical repeats that play key roles in protein-protein and protein-lipid interactions. The current structural model for HDL proposes that APOA1 forms an antiparallel dimer in which helix 5 in monomer 1 associates with helix 5 in monomer 2 along a left-left (LL5/5) interface, forming a protein complex with a 2-fold axis of symmetry centered on helix 5. However, computational studies suggest that other orientations are possible. To test this idea, we used a zero-length chemical cross-linking reagent that forms covalent bonds between closely apposed basic and acidic residues. Using proteolytic digestion and tandem mass spectrometry, we identified amino acids in the central region of the antiparallel APOA1 dimer of HDL that were in close contact. As predicted by the current model, we found six intermolecular cross-links that were consistent with the antiparallel LL5/5 registry. However, we also identified three intermolecular cross-links that were consistent with the antiparallel LL5/4 registry. The LL5/5 is the major structural conformation of the two complexes in both reconstituted discoidal HDL particles and in spherical HDL from human plasma. Molecular dynamic simulations suggest that that LL5/5 and LL5/4 APOA1 dimers possess similar free energies of dimerization, with LL5/5 having the lowest free energy. Our observations indicate that phospholipidated APOA1 in HDL forms different antiparallel dimers that could play distinct roles in enzyme regulation, assembly of specific protein complexes, and the functional properties of HDL in humans.

The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1.

The protein folding capacity of the endoplasmic reticulum (ER) is tightly regulated by a network of signaling pathways, known as the unfolded protein response (UPR). UPR sensors monitor the ER folding status to adjust ER folding capacity according to need. To understand how the UPR sensor IRE1 maintains ER homeostasis, we identified zero-length crosslinks of RNA to IRE1 with single nucleotide precision in vivo. We found that IRE1 specifically crosslinks to a subset of ER-targeted mRNAs, SRP RNA, ribosomal and transfer RNAs. Crosslink sites cluster in a discrete region of the ribosome surface spanning from the A-site to the polypeptide exit tunnel. Moreover, IRE1 binds to purified 80S ribosomes with high affinity, indicating association with ER-bound ribosomes. Our results suggest that the ER protein translocation and targeting machineries work together with the UPR to tune the ER's protein folding load.

Fibril bending stiffness of 3D collagen matrices instructs spreading and clustering of invasive and non-invasive breast cancer cells

Extracellular matrix stiffening of breast tissues has been clinically correlated with malignant transformation and poor prognosis. An increase of collagen fibril diameter and lysyl-oxidase mediated crosslinking has been observed in advanced tumor stages. Many current reports suggest that the local mechanical properties of single fibrillar components dominantly regulate cancer cell behavior. Here, we demonstrate by an independent control of fibril diameter and intrafibrillar crosslinking of three-dimensional (3D) collagen matrices that fibril bending stiffness instructs cell behavior of invasive and non-invasive breast cancer cells. Two types of collagen matrices with fibril diameter of either 650 nm or 800 nm at a similar pore size of 10 μm were reconstituted and further modified with the zero-length crosslinker 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) at concentrations of 0, 20, 100 and 500 mM. This approach yields two sets of collagen matrices with overlapping variation of matrix elasticity. With these matrices we could prove the common assumption that matrix elasticity of collagen networks is bending dominated with a linear dependence on fibril bending stiffness. We derive that the measured variation of matrix elasticity is directly correlated to the variation of fibril bending stiffness, being independently controlled either by fibril diameter or by intrafibrillar crosslinking. We use these defined matrices to demonstrate that the adjustment of fibril bending stiffness allows to instruct the behavior of two different breast cancer cell lines, invasive MDA-MB-231 (human breast carcinoma) and non-invasive MCF-7 cells (human breast adenocarcinoma). Invasiveness and spreading of invasive MDA-MB-231 cells as well as clustering of non-invasive MCF-7 cells is thereby investigated over a broad parameter range. Our results demonstrate and quantify the direct dependence of cancer cell phenotypes on the matrix mechanical properties on the scale of single fibrils.

Mechanistic Insight into Species‐specific Redox Partner Interactions in the Vitamin D Carbon‐24 Hydroxylase CYP24A1

The mitochondrial cytochrome P450 24A1 (CYP24A1) is responsible for side chain hydroxylation of vitamin D, the initial step in deactivation of the bioactive hormone. Mutations in CYP24A1 lead to incomplete clearance of active vitamin D and correlate with idiopathic infantile hypercalcemia, or elevated calcium in blood serum, and can lead to loss of appetite, nausea, vomiting, and failure to thrive in newborns. The regiospecific hydroxylation of the vitamin D side chain at either carbon‐23 (C23) or carbon‐24 (C24) relies in part on differences between species; while human isoforms are both C23 or C24 hydroxylases, other species are either C23 or C24 pure hydroxylases, despite an overall sequence identity of 80% between isoforms. Recent work from our group using solution NMR to evaluate the interaction between CYP24A1 and the redox accessory protein adrenodoxin (from bovine) suggests that species displaying distinct regioselectivity for vitamin D also form distinct P450‐redox partner complexes. Evidence of species‐specific complexes combined with the fact that mix‐species P450‐redox complexes are widely used to examine P450 function, highlights the need to examine this redox complex using proteins from the same species. Therefore, in this work we have used a combined approach of site‐directed mutagenesis, protein NMR, chemical cross‐linking, and vitamin D hydroxylation assays to evaluate the CYP24A1‐adrenodoxin complex from rat (a C24 hydroxylase). Differences in the 2D NMR spectra between bovine and rat adrenodoxin led to the complete re‐assignment of the protein backbone for rat adrenodoxin. Nonetheless, differential broadening of the NMR signal when titrated with rat CYP24A1 bound to 1α‐hydroxyvitamin D suggests the complex is mediated by surface charges. Dependence on surface charges was also verified by site‐directed mutagenesis of both proteins as well as zero‐length chemical cross‐linking using 1‐Ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide. While preliminary, this works demonstrates the combined use of biophysical and biochemical approaches to evaluate nuanced differences in a P450‐redox complex that is critical to the maintenance of proper levels of bioactive vitamin D.Support or Funding InformationNIGMSR00GM112862 (DFE)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Flagellar-associated Protein FAP85 Is a Microtubule Inner Protein That Stabilizes Microtubules.

Genomics and proteomics studies in Chlamydomonas have revealed that an axoneme is composed of 200-600 types of proteins, including uncharacterized proteins collectively named flagellar-associated proteins (FAPs). Nine FAPs contain the EF-hand motif; however, they have not yet been well characterized. To find components responsible for Chlamydomonas-specific waveform changes coupled with intracellular Ca2+ concentrations, we focused on FAP85, an EF-hand motif-containing FAP specific to Chlamydomonas and its relatives. We cloned the cDNA encoding FAP85, expressed it in Escherichia coli cells, and generated a polyclonal antibody against the expressed protein. Immunoblotting showed that FAP85 was present in every axoneme of several flagellar mutants lacking major axonemal components. Immuno-electron microscopy revealed that anti-FAP85 antibodies were found only on the inner wall of A-tubules of the doublets exposed by N-lauroylsarcosine (Sarkosyl) treatment. The zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied to 0.6 M KCl-extracted axonemes generated a 75-kDa complex containing β-tubulin and FAP85. Further characterization of FAP85 and its effects on microtubule dynamics showed that FAP85 binds to tubulin and stabilized microtubules. According to these results, we conclude that FAP85 is a novel member of microtubule-binding proteins, localizing on the inner wall of the A-tubule and stabilizing microtubules.Key words: Chlamydomonas, flagella, doublet microtubule, microtubule inner proteins.