Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α.

Exercise benefits the human body in many ways. Irisin is secreted by muscle, increased with exercise, and conveys physiological benefits, including improved cognition and resistance to neurodegeneration. Irisin acts via αV integrins; however, a mechanistic understanding of how small polypeptides like irisin can signal through integrins is poorly understood. Using mass spectrometry and cryo-EM, we demonstrate that the extracellular heat shock protein 90α (eHsp90α) is secreted by muscle with exercise and activates integrin αVß5. This allows for high-affinity irisin binding and signaling through an Hsp90α/αV/ß5 complex. By including hydrogen/deuterium exchange data, we generate and experimentally validate a 2.98 Å RMSD irisin/αVß5 complex docking model. Irisin binds very tightly to an alternative interface on αVß5 distinct from that used by known ligands. These data elucidate a non-canonical mechanism by which a small polypeptide hormone like irisin can function through an integrin receptor.

PLCL/SF/NGF nerve conduit loaded with RGD-TA-PPY hydrogel promotes regeneration of sciatic nerve defects in rats through PI3K/AKT signalling pathways.

Peripheral nerve defect are common clinical problem caused by trauma or other diseases, often leading to the loss of sensory and motor function in patients. Autologous nerve transplantation has been the gold standard for repairing peripheral nerve defects, but its clinical application is limited due to insufficient donor tissue. In recent years, the application of tissue engineering methods to synthesize nerve conduits for treating peripheral nerve defect has become a current research focus. This study introduces a novel approach for treating peripheral nerve defects using a tissue-engineered PLCL/SF/NGF@TA-PPy-RGD conduit. The conduit was fabricated by combining electrospun PLCL/SF with an NGF-loaded conductive TA-PPy-RGD gel. The gel, synthesized from RGD-modified tannic acid (TA) and polypyrrole (PPy), provides growth anchor points for nerve cells. In vitro results showed that this hybrid conduit could enhance PC12 cell proliferation, migration, and reduce apoptosis under oxidative stress. Furthermore, the conduit activated the PI3K/AKT signalling pathway in PC12 cells. In a rat model of sciatic nerve defect, the PLCL/SF/NGF@TA-PPy-RGD conduit significantly improved motor function, gastrocnemius muscle function, and myelin sheath axon thickness, comparable to autologous nerve transplantation. It also promoted angiogenesis around the nerve defect. This study suggests that PLCL/SF/NGF@TA-PPy-RGD conduits provide a conducive environment for nerve regeneration, offering a new strategy for peripheral nerve defect treatment, this study provided theoretical basis and new strategies for the research and treatment of peripheral nerve defect.

Substrate adhesion determines migration during mesenchymal cell condensation in chondrogenesis.

Mesenchymal condensation is a prevalent morphogenetic transition that is essential in chondrogenesis. However, the current understanding of condensation mechanisms is limited. In vivo, progenitor cells directionally migrate from the surrounding loose mesenchyme towards regions of increasing matrix adherence (the condensation centers), which is accompanied by the upregulation of fibronectin. Here, we focused on the mechanisms of cell migration during mesenchymal cell condensation and the effects of matrix adherence. Dendrimer-based nanopatterns of the cell-adhesive peptide arginine-glycine-aspartic acid (RGD), which is present in fibronectin, were used to regulate substrate adhesion. We recorded collective and single-cell migration of mesenchymal stem cells, under chondrogenic induction, using live-cell imaging. Our results show that the cell migration mode of single cells depends on substrate adhesiveness, and that cell directionality controls cell condensation and the fusion of condensates. Inhibition experiments revealed that cell-cell interactions mediated by N-cadherin (also known as CDH2) are also pivotal for directional migration of cell condensates by maintaining cell-cell cohesion, thus suggesting a fine interplay between cell-matrix and cell-cell adhesions. Our results shed light on the role of cell interactions with a fibronectin-depositing matrix during chondrogenesis in vitro, with possible applications in regenerative medicine. This article has an associated First Person interview with the first author of the paper.

Combination of STING agonist with anti-vascular RGD-(KLAKLAK)2 peptide as a novel anti-tumor therapy.

Immunotherapy is one of the most promising anti-cancer treatment. It involves activating the host's own immune system to eliminate cancer cells. Activation of cGAS-STING pathway is promising therapeutic approach for cancer immunotherapy. However, in human clinical trials, targeting cGAS-STING pathway results in insufficient or unsustainable anti-tumor response. To enhance its effectiveness, combination with other anti-cancer therapies seems essential to achieve synergistic systemic anti-tumor response.The aim of this study was to evaluate whether the combination of STING agonist-cGAMP with anti-vascular RGD-(KLAKLAK)2 peptide results in a better anti-tumor response in poorly immunogenic tumors with various STING protein and αvß3 integrin status.Combination therapy inhibited growth of murine breast carcinoma more effectively than melanoma. In melanoma, the administration of STING agonist alone was sufficient to obtain a satisfactory therapeutic effect. In both tumor models we have noted stimulation of innate immune response following cGAMP administration alone or in combination. The largest population of immune cells infiltrating the TME after therapy were activated NK cells. Increased infiltration of cytotoxic CD8+ T lymphocytes within the TME was only observed in melanoma tumors. However, they also expressed the "exhaustion" PD-1 receptor. In contrast, in breast carcinoma tumors each therapy caused the drop in the number of infiltrating CD8+ T cells.The obtained results indicate an additional therapeutic benefit from combining STING agonist with an anti-vascular agent. However, this effect depends on the type of tumor, the status of its microenvironment and the expression of specific proteins such as STING and αvß3 family integrin.

Multimaterial 3D Laser Printing of Cell-Adhesive and Cell-Repellent Hydrogels.

Here, a straightforward method is reported for manufacturing 3D microstructured cell-adhesive and cell-repellent multimaterials using two-photon laser printing. Compared to existing strategies, this approach offers bottom-up molecular control, high customizability, and rapid and precise 3D fabrication. The printable cell-adhesive polyethylene glycol (PEG) based material includes an Arg-Gly-Asp (RGD) containing peptide synthesized through solid-phase peptide synthesis, allowing for precise control of the peptide design. Remarkably, minimal amounts of RGD peptide (< 0.1 wt%) suffice for imparting cell-adhesiveness, while maintaining identical mechanical properties in the 3D printed microstructures to those of the cell-repellent, PEG-based material. Fluorescent labeling of the RGD peptide facilitates visualization of its presence in cell-adhesive areas. To demonstrate the broad applicability of the system, the fabrication of cell-adhesive 2.5D and 3D structures is shown, fostering the adhesion of fibroblast cells within these architectures. Thus, this approach allows for the printing of high-resolution, true 3D structures suitable for diverse applications, including cellular studies in complex environments.

Dorsoventral hippocampus distinctly modulates visceral sensitivity and anxiety behaviors in male IBS-like rats.

Accumulating evidences suggest dysfunctions in the hippocampus are associated with chronic pain. Nevertheless, the role of hippocampal circuitry in pain memories and emotional responses is not yet fully understood. In this study, we utilized a comprehensive approach that combined electromyography (EMG), photochemical genetic techniques, and anxiety-related behavioral paradigms to investigate the involvement of dorsal hippocampus (DH) and ventral hippocampus (VH) in visceral sensitivity and anxiety behaviors in male rats. Our results demonstrated that IBS-like rats exhibited comorbid visceral hypersensitivity and anxiety, along with the number of activated neurons in the VH was higher than that in the DH. Manipulation of glutamatergic neurons in the hippocampus was identified as a crucial mechanism underlying the mediation of both visceral sensitivity and anxiety behaviors. Specifically, optogenetic activation of the DH induced both visceral hypersensitivity and anxiety, while activation of the VH induced anxiety but did not affect visceral sensitivity. Conversely, chemogenetic inhibition of the DH reduced both visceral hypersensitivity and anxiety, whereas inhibition of the VH alleviated anxiety but did not alleviate visceral hypersensitivity in IBS-like rats. Our study highlights the important role of early life stress in inducing visceral hypersensitivity and anxiety, and further elucidates the distinct functional contributions of the DH and VH to these behavioral changes. These findings provide a theoretical basis for the diagnosis and treatment of IBS, and suggest that targeting specific hippocampal neuron subtypes may represent a promising therapeutic approach.

Integrin Facilitates the Internalization of TAT Peptide Conjugated to RGD Motif in Model Lipid Membranes.

In recent years, targeted drug delivery has attracted a great interest for enhanced therapeutic efficiency, with diminished side effects, especially in cancer therapy. Cell penetrating peptides (CPPs) like HIV1-TAT peptides, appear to be the perfect vectors for translocating drugs or other cargoes across the plasma membrane, but their application is limited mostly due to insufficient specificity for intended targets. Although these molecules were successfully used, the mechanism by which the peptides enter the cell interior still needs to be clarified. The tripeptide motif RGD (arginine-glycine-aspartate), found in extracellular matrix proteins has high affinity for integrin receptors overexpressed in cancer and it is involved in different phases of disease progression, including proliferation, invasion and migration. Discovery of new peptides with high binding affinity for disease receptors and permeability of plasma membranes is desirable for both, development of targeted drug delivery systems and early detection and diagnosis. To complement the TAT peptide with specific targeting ability, we conjugated it with an integrin-binding RGD motif. Although the idea of RGD-CPPs conjugates is not entirely new,[1] here we describe the permeability abilities and specificity of integrin receptors of RGD-TAT peptides in model membranes. Our findings reveal that this novel RGD sequence based on TAT peptide maintains its ability to permeate lipid membranes and exhibits specificity for integrin receptors embedded in giant unilamellar vesicles. This promising outcome suggests that the RGD-TAT peptide has significant potential for applications in the field of targeted drug delivery systems.

Development of probes for radiotheranostics with albumin binding moiety to increase the therapeutic effects of astatine-211 (211At).

PURPOSE: We have developed probes for multiradionuclides radiotheranostics using RGD peptide ([67Ga]Ga-DOTA-c[RGDf(4-I)K] ([67Ga]1) and Ga-DOTA-[211At]c[RGDf(4-At)K] ([211At]2)) for clinical applications. The introduction of an albumin binding moiety (ABM), such as 4-(4-iodophenyl)-butyric acid (IPBA), that has high affinity with the blood albumin and prolongs the circulation half-life can improve the pharmacokinetics of drugs. To perform more effective targeted alpha therapy (TAT), we designed and synthesized Ga-DOTA-K([211At]APBA)-c(RGDfK) ([211At]5) with 4-(4-astatophenyl)-butyric acid (APBA), which has an astato group instead of an iodo group in IPBA. We evaluated whether APBA functions as ABM and [211At]5 is effective for TAT. In addition, we prepared 67Ga-labeled RGD peptide without ABM, [67Ga]Ga-DOTA-K-c(RGDfK) ([67Ga]3), and 125I-labeled RGD peptide with ABM, Ga-DOTA-K([125I]IPBA)-c(RGDfK) ([125I]4), to compare with [211At]5. METHODS: Biodistribution experiments of [67Ga]3 without ABM, [125I]4 and [211At]5 with ABM were conducted in normal mice and U-87 MG tumor-bearing mice. In addition, two doses of [211At]5 (370 or 925 kBq) were administered to U-87 MG tumor-bearing mice to confirm the therapeutic effects. RESULTS: The blood retention of [125I]4 and [211At]5 was remarkably increased compared to [67Ga]3. Also, [125I]4 and [211At]5 showed similar biodistribution and significantly greater tumor accumulation and retention compared to [67Ga]3. In addition, [211At]5 inhibited tumor growth in a dose-dependent manner. CONCLUSION: The functionality of APBA as ABM like IPBA, and the usefulness of [211At]5 as the radionuclide therapy agent for TAT was revealed.

Optimizing the pharmacokinetics of an 211At-labeled RGD peptide with an albumin-binding moiety via the administration of an albumin-binding inhibitor.

PURPOSE: A probe for targeted alpha therapy (TAT) using the RGD peptide (Ga-DOTA-K([211At]APBA)-c(RGDfK) ([211At]1)) with albumin-binding moiety (ABM) was recently developed. [211At]1 highly accumulated in tumors and significantly inhibited tumor growth in U-87 MG tumor-bearing mice. However, high [211At]1 retention in blood may cause critical adverse events, such as hematotoxicity. Therefore, we attempted to accelerate the blood clearance of [211At]1 by competitively inhibiting the binding of [211At]1 to albumin to modulate the pharmacokinetics of the former. METHODS: To evaluate the effects of albumin-binding inhibitors in normal mice, sodium 4-(4-iodophenyl)butanoate at 2, 5, or 10 molar equivalents of blood albumin was administered at 1-h postinjection of [211At]1. The biodistribution of [211At]1, SPECT/CT imaging of [67Ga]Ga-DOTA-K(IPBA)-c(RGDfK) ([67Ga]2), and the therapeutic effects of [211At]1 were compared with or without IPBA administration in U-87 MG tumor-bearing mice. RESULTS: Blood radioactivity of [211At]1 was decreased in a dose-dependent manner with IPBA in normal mice. In U-87 MG tumor-bearing mice, the blood radioactivity and accumulation in nontarget tissues of [211At]1 were decreased by IPBA. Meanwhile, tumor [211At]1 accumulation was not changed at 3-h postinjection of IPBA. In SPECT/CT imaging of [67Ga]2, IPBA administration dramatically decreased radioactivity in nontarget tissues, and only tumor tissue was visualized. In therapeutic experiments, [211At]1 with IPBA injected-group significantly inhibited tumor growth compared to the control group. CONCLUSION: IPBA administration (as an albumin-binding inhibitor) could modulate the pharmacokinetics and enhance the therapeutic effects of [211At]1.

A comparison of [18F]AlF- and 68Ga-labeled dual targeting heterodimer FAPI-RGD in malignant tumor: preclinical evaluation and pilot clinical PET/CT imaging.

Due to the heterogeneity of tumors, strategies to improve the effectiveness of dual-targeting tracers in tumor diagnostics have been intensively practiced. In this study, the radiolabeled [18F]AlF-NOTA-FAPI-RGD (denoted as [18F]AlF-LNC1007), a dual-targeting heterodimer tracer targeting both fibroblast activation protein (FAP) and integrin αvß3 to enhance specific tumor uptake and retention, was synthesized and evaluated. The tracer was compared with [68Ga]Ga-LNC1007 in preclinical and clinical settings. METHODS: The preparation of [18F]AlF- and 68Ga-labeled FAPI-RGD was carried out with an optimized protocol. The stability was tested in PBS and fetal bovine serum (FBS). Cellular uptake and in vivo distribution of the two products were compared and carried out on the U87MG cell line and its xenograft model. The safety and dosimetry of [18F]AlF-LNC1007 PET/CT scan were evaluated in six patients with malignant tumors. RESULTS: Two radiolabeling protocols of [18F]AlF-/[68Ga]Ga-LNC1007 were developed and optimized to give a high yield of tracers with good stability. In vivo microPET images showed that the two tracers exhibited comparable pharmacokinetic characteristics, with high tumor uptake and prolonged tumor retention. In vivo distribution data showed that the target-to-non-target ratios of [18F]AlF-LNC1007 were similar to[68Ga]Ga-LNC1007. A total of six patients underwent [18F]AlF-LNC1007 PET/CT evaluation while two had head-to-head [18F]FDG PET/CT scans. The total body effective dose was 9.94E-03 mSv/MBq. The biodistribution curve showed optimal normal organ uptake with high tumor uptake and long retention of up to 3h p.i., and notably, the tumor-to-background ratio increased over time. CONCLUSION: We successfully prepared an [18F]AlF-LNC1007 dual-targeting PET probe with comparable performances as [68Ga]Ga-LNC1007. With prolonged tumor retention and tumor specificity, it produced good imaging quality in preclinical and clinical translational studies, indicating that [18F]AlF-LNC1007 is a promising non-invasive tracer for detecting tumors expressing FAP and/or integrin avß3, with the prospect of clinical implementation.

Optimization of 3D Synthetic Scaffolds for Neuronal Tissue Engineering Applications.

The increasing prevalence of neurodegenerative diseases has spurred researchers to develop advanced 3D models that accurately mimic neural tissues. Hydrogels stand out as ideal candidates as their properties closely resemble those of the extracellular matrix. A critical challenge in this regard is to comprehend the influence of the scaffold's mechanical properties on cell growth and differentiation, thus enabling targeted modifications. In light of this, a synthesis and comprehensive analysis of acrylamide-based hydrogels incorporating a peptide has been conducted. Adequate cell adhesion and development is achieved due to their bioactive nature and specific interactions with cellular receptors. The integration of a precisely controlled physicochemical hydrogel matrix and inclusion of the arginine-glycine-aspartic acid peptide sequence has endowed this system with an optimal structure, thus providing a unique ability to interact effectively with biomolecules. The analysis fully examined essential properties governing cell behavior, including pore size, mechanical characteristics, and swelling ability. Cell-viability experiments were performed to assess the hydrogel's biocompatibility, while the incorporation of grow factors aimed to promote the differentiation of neuroblastoma cells. The results underscore the hydrogel's ability to stimulate cell viability and differentiation in the presence of the peptide within the matrix.

Engineering a 3D Biomimetic Peptides Functionalized-Polyethylene Glycol Hydrogel Model Cocultured with Endothelial Cells and Astrocytes: Enhancing In Vitro Blood-Brain Barrier Biomimicry.

The blood-brain barrier (BBB) poses a significant challenge for drug delivery and is linked to various neurovascular disorders. In vitro BBB models provide a tool to investigate drug permeation across the BBB and the barrier's response to external injury events. Yet, existing models lack fidelity in replicating the BBB's complexity, hindering a comprehensive understanding of its functions. This study introduces a three-dimensional (3D) model using polyethylene glycol (PEG) hydrogels modified with biomimetic peptides that represent recognition sequences of key proteins in the brain. Hydrogels were functionalized with recognition sequences for laminin (IKVAV) and fibronectin peptides (RGD) and chemically cross-linked with matrix metalloprotease-sensitive peptides (MMPs) to mimic the extracellular matrix of the BBB. Astrocytes and endothelial cells were seeded within and on the surface of the hydrogels, respectively. The barrier integrity was assessed through different tests including transendothelial electrical resistance (TEER), the permeability of sodium fluorescence (Na-F), the permeability of Evan's blue bound to albumin (EBA), and the expression of zonula occluden-1 (ZO-1) in seeded endothelial cells. Hydrogels with a combination of RGD and IKVAV peptides displayed superior performance, exhibiting significantly higher TEER values (55.33 ± 1.47 Ω·cm2) at day 5 compared to other 2D controls including HAECs-monoculture and HAECs-cocultured with NHAs seeded on well inserts and 3D controls including RGD hydrogel and RGD-IKVAV monoculture with HAECs and RGD hydrogel cocultured with HAECs and NHAs. The designed 3D system resulted in the lowest Evan's blue permeability at 120 min (0.215 ± 0.055 µg/mL) compared to controls. ZO-1 expression was significantly higher and formed a relatively larger network in the functionalized hydrogel cocultured with astrocytes and endothelial cells compared to the controls. Thus, the designed 3D model effectively recapitulates the main BBB structure and function in vitro and is expected to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.

[Forensic characteristic of damages caused by offensive grenades detonation]. / Sudebno-meditsinskaya kharakteristika povrezhdenii, prichinennykh pri vzryve nastupatel'nykh granat.

OBJECTIVE: To determine the specific morphological features of damages on the cloth and biological simulator of the human body in the detonation of RGD-5 and RGN fragmentation hand offensive grenades. MATERIAL AND METHODS: The study was carried out on the 12 targets from a biological simulator of the human body wrapped in cotton cloth by detonation of examined grenades at a distance of 50 cm and 1 m from the target. RESULTS: The character and features of damages of cloth and human body biological simulator, features of soot deposition made it possible to determine specific signs for each of examined offensive grenades types. CONCLUSIONS: The complex of obtained data allows to determine with high accuracy the detonation distance and the type of explosive device.

Extracellular cathepsin Z signals through the α5 integrin and augments NLRP3 inflammasome activation.

Respiratory silicosis is a preventable occupational disease that develops secondary to the aspiration of crystalline silicon dioxide (silica) into the lungs, activation of the NLRP3 inflammasome, and IL-1ß production. Cathepsin Z has been associated with the development of inflammation and IL-1ß production; however, the mechanism of how cathepsin Z leads to IL-1ß production is unknown. Here, the requirement for cathepsin Z in silicosis was determined using WT mice and mice deficient in cathepsin Z. The activation of the NLRP3 inflammasome in macrophages was studied using WT and cathepsin Z-deficient bone marrow-derived murine dendritic cells and the human monocytic cell line THP-1. The cells were activated with silica, and IL-1ß release was determined using enzyme-linked immunosorbent assay or IL-1ß bioassays. The relative contribution of the active domain or integrin-binding domain of cathepsin Z was studied using recombinant cathepsin Z constructs and the α5 integrin neutralizing antibody. We report that the lysosomal cysteine protease cathepsin Z potentiates the development of inflammation associated with respiratory silicosis by augmenting NLRP3 inflammasome-derived IL-1ß expression in response to silica. The secreted cathepsin Z functions nonproteolytically via the internal integrin-binding domain to impact caspase-1 activation and the production of active IL-1ß through integrin α5 without affecting the transcription levels of NLRP3 inflammasome components. This work reveals a regulatory pathway for the NLRP3 inflammasome that occurs in an outside-in fashion and provides a link between extracellular cathepsin Z and inflammation. Furthermore, it reveals a level of NLRP3 inflammasome regulation that has previously only been found downstream of extracellular pathogens.

New opportunities for RGD-engineered metal nanoparticles in cancer.

The advent of nanotechnology has opened new possibilities for bioimaging. Metal nanoparticles (such as gold, silver, iron, copper, etc.) hold tremendous potential and offer enormous opportunities for imaging and diagnostics due to their broad optical characteristics, ease of manufacturing technique, and simple surface modification. The arginine-glycine-aspartate (RGD) peptide is a three-amino acid sequence that seems to have a considerably greater ability to adhere to integrin adhesion molecules that exclusively express on tumour cells. RGD peptides act as the efficient tailoring ligand with a variety of benefits including non-toxicity, greater precision, rapid clearance, etc. This review focuses on the possibility of non-invasive cancer imaging using metal nanoparticles with RGD assistance.

Toward a theranostic device for gliomas.

BACKGROUND: In the surgical management of glioblastoma, a highly aggressive and incurable type of brain cancer, identification and treatment of residual tissue is the most common site of disease recurrence. Monitoring and localized treatment are achieved with engineered microbubbles (MBs) by combining ultrasound and fluorescence imaging with actively targeted temozolomide (TMZ) delivery. METHODS: The MBs were conjugated with a near-infrared fluorescence probe CF790, cyclic pentapeptide bearing the RGD sequence and a carboxyl-temozolomide, TMZA. The efficiency of adhesion to HUVEC cells was assessed in vitro in realistic physiological conditions of shear rate and vascular dimensions. Cytotoxicity of TMZA-loaded MBs on U87 MG cells and IC50 were assessed by MTT tests. RESULTS: We report on the design of injectable poly(vinyl alcohol) echogenic MBs designed as a platform with active targeting ability to tumor tissues, by tethering on the surface a ligand having the tripeptide sequence, RGD. The biorecognition of RGD-MBs onto HUVEC cells is quantitatively proved. Efficient NIR emission from the CF790-decorated MBs was successfully detected. The conjugation on the MBs surface of a specific drug as TMZ is achieved. The pharmacological activity of the coupled-to-surface drug is preserved by controlling the reaction conditions. CONCLUSIONS: We present an improved formulation of PVA-MBs to achieve a multifunctional device with adhesion ability, cytotoxicity on glioblastoma cells and supporting imaging.

SARS-CoV-2 Omicron subvariant spike N405 unlikely to rapidly deamidate.

The RGD motif on the SARS-CoV-2 spike protein has been suggested to interact with RGD-binding integrins αVß3 and α5ß1 to enhance viral cell entry and alter downstream signaling cascades. The D405N mutation on the Omicron subvariant spike proteins, resulting in an RGN motif, has recently been shown to inhibit binding to integrin αVß3. Deamidation of asparagines in protein ligand RGN motifs has been demonstrated to generate RGD and RGisoD motifs that permit binding to RGD-binding integrins. Two asparagines, N481 and N501, on the Wild-type spike receptor-binding domain have been previously shown to have deamidation half-lives of 16.5 and 123 days, respectively, which may occur during the viral life cycle. Deamidation of Omicron subvariant N405 may recover the ability to interact with RGD-binding integrins. Thus, herein, all-atom molecular dynamics simulations of the Wild-type and Omicron subvariant spike protein receptor-binding domains were conducted to investigate the potential for asparagines, the Omicron subvariant N405 in particular, to assume the optimized geometry for deamidation to occur. In summary, the Omicron subvariant N405 was primarily found to be stabilized in a state unfavourable for deamidation after hydrogen bonding with downstream E406. Nevertheless, a small number of RGD or RGisoD motifs on the Omicron subvariant spike proteins may restore the ability to interact with RGD-binding integrins. The simulations also provided structural clarification regarding the deamidation rates of Wild-type N481 and N501 and highlighted the utility of tertiary structure dynamics information in predicting asparagine deamidation. Further work is needed to characterize the effects of deamidation on spike-integrin interactions.

Extracellular Vesicle-Conjugated Functional Matrix Hydrogels Prevent Senescence by Exosomal miR-3594-5p-Targeted HIPK2/p53 Pathway for Disc Regeneration.

Nucleus pulposus stem cells (NPSCs) senescence plays a critical role in the progression of intervertebral disc degeneration (IDD). Stem cell-derived extracellular vesicles (EV) alleviate cellular senescence. Whereas, the underlying mechanism remains unclear. Low stability largely limited the administration of EV in vivo. RGD, an arginine-glycine-aspartic acid tripeptide, strongly binds integrins expressed on the EV membranes, allowing RGD to anchor EV and prolong their bioavailability. An RGD-complexed nucleus pulposus matrix hydrogel (RGD-DNP) is developed to enhance the therapeutic effects of small EV (sEV). RGD-DNP prolonged sEV retention in vitro and ex vivo. sEV-RGD-DNP promoted NPSCs migration, decreased the number of SA-ß-gal-positive cells, alleviated cell cycle arrest, and reduced p16, p21, and p53 activation. Small RNA-seq showed that miR-3594-5p is enriched in sEV, and targets the homeodomain-interacting protein kinase 2 (HIPK2)/p53 pathway. The HIPK2 knockdown rescues the impaired therapeutic effects of sEV with downregulated miR-3594-5p. RGD-DNP conjugate with lower amounts of sEV achieved similar disc regeneration with free sEV of higher concentrations in DNP. In conclusion, sEV-RGD-DNP increases sEV bioavailability and relieves NPSCs senescence by targeting the HIPK2/p53 pathway, thereby alleviating IDD. This work achieves better regenerative effects with fewer sEV and consolidates the theoretical basis for sEV application for IDD treatment.

Intrahippocampal injection of IL-1ß upregulates Siah1-mediated degradation of synaptophysin by activation of the ERK signaling in male rat.

Interleukin-1ß (IL-1ß) has been described to exert important effect on synapses in the brain. Here, we explored if the synapses in the hippocampus would be adversely affected following intracerebral IL-1ß injection and, if so, to clarify the underlying molecular mechanisms. Adult male Sprague-Dawley rats were divided into control, IL-1ß, IL-1ß + PD98059, and IL-1ß + MG132 groups and then sacrificed for detection of synaptophysin (syn) protein level, synaptosome glutamate release, and synapse ultrastructure by western blotting, glutamate kit and electron microscopy, respectively. These rats were tested by Morris water maze for learning and memory ability. It was determined by western blotting whether IL-1ß exerted the effect of on syn and siah1 expression in primary neurons via extracellular regulated protein kinases (ERK) signaling pathway. Intrahippocampal injection of IL-1ß in male rats and sacrificed at 8d resulted in a significant decrease in syn protein, damage of synapse structure, and abnormal release of neurotransmitters glutamate. ERK inhibitor and proteosome inhibitor treatment reversed the above changes induced by IL-1ß both in vivo and in vitro. In primary cultured neurons incubated with IL-1ß, the expression level of synaptophysin was significantly downregulated coupled with abnormal glutamate release. Furthermore, use of PD98059 had confirmed that ERK signaling pathway was implicated in synaptic disorders caused by IL-1ß treatment. The present results suggest that exogenous IL-1ß can suppress syn protein level and glutamate release. A possible mechanism for this is that IL-1ß induces syn degradation that is regulated by the E3 ligase siah1 via the ERK signaling pathway.

Enzymatic Late-Stage Halogenation of Peptides.

The late-stage site-selective derivatisation of peptides has many potential applications in structure-activity relationship studies and postsynthetic modification or conjugation of bioactive compounds. The development of orthogonal methods for C-H functionalisation is crucial for such peptide derivatisation. Among them, biocatalytic methods are increasingly attracting attention. Tryptophan halogenases emerged as valuable catalysts to functionalise tryptophan (Trp), while direct enzyme-catalysed halogenation of synthetic peptides is yet unprecedented. Here, it is reported that the Trp 6-halogenase Thal accepts a wide range of amides and peptides containing a Trp moiety. Increasing the sequence length and reaction optimisation made bromination of pentapeptides feasible with good turnovers and a broad sequence scope, while regioselectivity turned out to be sequence dependent. Comparison of X-ray single crystal structures of Thal in complex with d-Trp and a dipeptide revealed a significantly altered binding mode for the peptide. The viability of this bioorthogonal approach was exemplified by halogenation of a cyclic RGD peptide.