3D-printed silicate porous bioceramics promoted the polarization of M2-macrophages that enhanced the angiogenesis in bone regeneration.

The failure of bone regeneration has been considered as a serious problem that troubling patients for decades, most of which was resulted by the poor angiogenesis and chronic inflammation after surgery. Among multiple materials applied in the repair of bone defect, silicate bioceramics attracted researchers because of its excellent bioactivity. The purpose of this study was to detect the effect of specific bioactive glass ceramic (AP40, based on crystalline phases of apatite and wollastonite) on angiogenesis and the subsequent bone growth through the modulation of macrophages. Two groups were included in this study: control group (macrophages without any stimulation, denominated as Control) and AP40 group (macrophages incubated on AP40). This study investigated the effect of AP40 on macrophages polarization (RAW264.7) and angiogenesis in vitro and in vivo. Additionally, the changes of angiogenic ability regulated by macrophages were explored. AP40 showed excellent angiogenesis potential and the expression of CD31 was promoted through the modulation of macrophages toward M2 subtype. Additionally, the macrophages incubated on AP40 synthesized more PDGF-BB comparing to macrophages without any stimulation, which contributed to the improved angiogenetic ability of human umbilical vein endothelial cells (HUVECs). Results of in vivo studies indicated increased bone ingrowth along the implants, which indicated the potential of bioceramics for bone defect repair clinically.

Self-assembled branched polypeptides as amelogenin mimics for enamel repair.

The regeneration of demineralized enamel holds great significance in the treatment of dental caries. Amelogenin (Ame), an essential protein for mediating natural enamel growth, is no longer secreted after enamel has fully matured in childhood. Although biomimetic mineralization based on peptides or proteins has made significant progress, easily accessible, low-cost, biocompatible and highly effective Ame mimics are still lacking. Herein, we construct a series of amphiphilic branched polypeptides (CAMPs) by facile coupling of the Ame's C-terminal segment and poly(γ-benzyl-L-glutamate), which serves to simulate the Ame's hydrophobic N-terminal segment. Among them, CAMP15 is the best biomimetic mineralization template with great self-assembly performance to guide the oriented crystallization of hydroxyapatite and is capable of inhibiting the adhesion of Streptococcus mutans and Staphylococcus aureus on the enamel surfaces. This work highlights the potential application of amphiphilic branched polypeptide as Ame mimics in repairing defected enamel, providing a promising strategy for prevention and treatment of dental caries.

Phosphorylation-regulated phase separation of syndecan-4 and syntenin promotes the biogenesis of exosomes.

The biogenesis of exosomes that mediate cell-to-cell communication by transporting numerous biomolecules to neighbouring cells is an essential cellular process. The interaction between the transmembrane protein syndecan-4 (SDC4) and cytosolic protein syntenin plays a key role in the biogenesis of exosomes. However, how the relatively weak binding of syntenin to SDC4 efficiently enables syntenin sorting for packaging into exosomes remains unclear. Here, we demonstrate for the first time that SDC4 can undergo liquid-liquid phase separation (LLPS) to form condensates both in vitro and in the cell membrane and that, the SDC4 cytoplasmic domain (SDC4-CD) is a key contributor to this process. The phase separation of SDC4 greatly enhances the recruitment of syntenin to the plasma membrane (PM) despite the weak SDC4-syntenin interaction, facilitating syntenin sorting for inclusion in exosomes. Interestingly, phosphorylation at the only serine (179) in the SDC4-CD (Ser179) disrupts SDC4 LLPS, and inhibited phosphorylation or dephosphorylation restores the SDC4 LLPS to promote its recruitment of syntenin to the PM and syntenin inclusion into exosomes. This research reveals a novel phosphorylation-regulated phase separation property of SDC4 in the PM through which SDC4 efficiently recruits cytosolic syntenin and facilitates the biogenesis of exosomes, providing potential intervention targets for exosome-mediated biomedical events.

Pharmacological inhibition of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) induces ferroptosis in vascular smooth muscle cells.

MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a human paracaspase protein with proteolytic activity via its caspase-like domain. The pharmacological inhibition of MALT1 by MI-2, a specific chemical inhibitor, diminishes the response of endothelial cells to inflammatory stimuli. However, it is largely unknown how MALT1 regulates the functions of vascular smooth muscle cells (SMCs). This study aims to investigate the impact of MALT1 inhibition by MI-2 on the functions of vascular SMCs, both in vitro and in vivo. MI-2 treatment led to concentration- and time-dependent cell death of cultured aortic SMCs, which was rescued by the iron chelator deferoxamine (DFO) or ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, but not by inhibitors of apoptosis (Z-VAD-fmk), pyroptosis (Z-YVAD-fmk), or necrosis (Necrostatin-1, Nec-1). MI-2 treatment downregulated the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy polypeptide 1 (FTH1), which was prevented by pre-treatment with DFO or Fer-1. MI-2 treatment also activated autophagy, which was inhibited by Atg7 deficiency or bafilomycin A1 preventing MI-2-induced ferroptosis. MI-2 treatment reduced the cleavage of cylindromatosis (CYLD), a specific substrate of MALT1. Notably, MI-2 treatment led to a rapid loss of contractility in mouse aortas, which was prevented by co-incubation with Fer-1. Moreover, local application of MI-2 significantly reduced carotid neointima lesions and atherosclerosis in C57BL/6J mice and apolipoprotein-E knockout (ApoE-/-) mice, respectively, which were both ameliorated by co-treatment with Fer-1. In conclusion, the present study demonstrated that MALT1 inhibition induces ferroptosis of vascular SMCs, likely contributing to its amelioration of proliferative vascular diseases.

Synthesis, Enzymatic Peptide Incorporation, and Applications of Diazirine-Containing Isoprenoid Diphosphate Analogues.

Prenylated proteins contain C15 or C20 isoprenoids linked to cysteine residues positioned near their C-termini. Here we describe the preparation of isoprenoid diphosphate analogues incorporating diazirine groups that can be used to probe interactions between prenylated proteins and other proteins that interact with them. Studies using synthetic peptides and whole proteins demonstrate that these diazirine analogues are efficient substrates for prenyltransferases. Photo-cross-linking experiments using peptides incorporating the diazirine-functionalized isoprenoids selectively cross-link to several different proteins. These new isoprenoid analogues should be broadly useful in the studies of protein prenylation.

Exogenous H2S alleviates senescence of glomerular mesangial cells through up-regulating mitophagy by activation of AMPK-ULK1-PINK1-parkin pathway in mice.

Hydrogen sulfide (H2S) is the third gas signaling molecule that has been shown to be involved in the regulating vital activities in the body, including inhibition of aging. However, it is unknown whether H2S alleviates aging in the kidney and glomerular mesangial cells (GMCs) by modulating their mitophagy. Here, results of experiments in vivo and in vitro showed that compared with control group, the renal function of mice and GMCs viability were decreased in D-gal (D-galactose) group, while the activity of SA-ß-gal and p21 expression were increased, Cyclin D1 and Klotho expressions were decreased; H2S content and CSE expression were lower; ROS and MDA contents and mitochondrial permeability transition pore (mPTP) opening were risedose; ATP production and mitochondrial membrane potential (Δψm) were reduced; Apoptotic rate, the expression of Cleaved caspase-9 and -3, Cyt c, p62 and Drp1 were enhanced and the expression of Bcl-2, Mfn2, Beclin-1, LC3 II/I, PINK1 and parkin were decreased. In addition, phospho-AMPK/AMPK and phospho-ULK1/ULK1 were also decreased significantly. Compared with the D-gal group, the changes of above indexes were reversed in the D-gal + NaHS (Sodium hydrosulfide, an exogenous H2S donor) group. The reverse effects of NaHS were similar to that of AICAR (an AMPK agonist) and kinetin (a PINK1 agonist), respectively. Taken together, these results suggest that exogenous H2S increases mitophagy and inhibits apoptosis as well as oxidative stress through up-regulation of AMPK-ULK1-PINK1-parkin pathway, which delays kidney senescence in mice.

Increased Ca2+ Transient Underlies RyR2-Related Left Ventricular Noncompaction.

BACKGROUND: A loss-of-function cardiac ryanodine receptor (RyR2) mutation, I4855M+/-, has recently been linked to a new cardiac disorder termed RyR2 Ca2+ release deficiency syndrome (CRDS) as well as left ventricular noncompaction (LVNC). The mechanism by which RyR2 loss-of-function causes CRDS has been extensively studied, but the mechanism underlying RyR2 loss-of-function-associated LVNC is unknown. Here, we determined the impact of a CRDS-LVNC-associated RyR2-I4855M+/- loss-of-function mutation on cardiac structure and function. METHODS: We generated a mouse model expressing the CRDS-LVNC-associated RyR2-I4855M+/- mutation. Histological analysis, echocardiography, ECG recording, and intact heart Ca2+ imaging were performed to characterize the structural and functional consequences of the RyR2-I4855M+/- mutation. RESULTS: As in humans, RyR2-I4855M+/- mice displayed LVNC characterized by cardiac hypertrabeculation and noncompaction. RyR2-I4855M+/- mice were highly susceptible to electrical stimulation-induced ventricular arrhythmias but protected from stress-induced ventricular arrhythmias. Unexpectedly, the RyR2-I4855M+/- mutation increased the peak Ca2+ transient but did not alter the L-type Ca2+ current, suggesting an increase in Ca2+-induced Ca2+ release gain. The RyR2-I4855M+/- mutation abolished sarcoplasmic reticulum store overload-induced Ca2+ release or Ca2+ leak, elevated sarcoplasmic reticulum Ca2+ load, prolonged Ca2+ transient decay, and elevated end-diastolic Ca2+ level upon rapid pacing. Immunoblotting revealed increased level of phosphorylated CaMKII (Ca2+-calmodulin dependent protein kinases II) but unchanged levels of CaMKII, calcineurin, and other Ca2+ handling proteins in the RyR2-I4855M+/- mutant compared with wild type. CONCLUSIONS: The RyR2-I4855M+/- mutant mice represent the first RyR2-associated LVNC animal model that recapitulates the CRDS-LVNC overlapping phenotype in humans. The RyR2-I4855M+/- mutation increases the peak Ca2+ transient by increasing the Ca2+-induced Ca2+ release gain and the end-diastolic Ca2+ level by prolonging Ca2+ transient decay. Our data suggest that the increased peak-systolic and end-diastolic Ca2+ levels may underlie RyR2-associated LVNC.

14-3-3ζ Participates in the Phase Separation of Phosphorylated and Glycated Tau and Modulates the Physiological and Pathological Functions of Tau.

Tau plays a major role in Alzheimer's disease (AD) and several other neurodegenerative diseases. Tau undergoing liquid-liquid phase separation (LLPS) performs specific physiological functions, induces pathological processes, and contributes to neurodegeneration. Regulating Tau phase separation helps maintain physiological functions of Tau and inhibits pathological aggregation. Here, we show that the 14-3-3 zeta isoform (14-3-3ζ) participates in Tau LLPS. 14-3-3ζ can undergo co-phase separation with WT Tau, participate in and stabilize Tau droplets, and inhibit Tau droplet-driven tubulin assembly. On the other hand, 14-3-3ζ disrupts the LLPS of phosphorylated and glycated Tau, thereby inhibiting the amyloid aggregation initiated by LLPS.

Polo-like kinase 4 inhibitor CFI-400945 inhibits carotid arterial neointima formation but increases atherosclerosis.

Neointima lesion and atherosclerosis are proliferative vascular diseases associated with deregulated proliferation of vascular smooth muscle cells (SMCs). CFI-400945 is a novel, highly effective anticancer drug that inhibits polo-like kinase 4 (PLK4) and targets mitosis. In this study, we aim to investigate how CFI-400945 affects the development of proliferative vascular diseases. In C57BL/6 mice, neointima formation was generated by complete carotid ligation. In apolipoprotein E knockout (ApoE-/-) mice fed a high-fat diet, atherosclerosis was induced by partial carotid ligation. CFI-400945 was directly applied to carotid arteries via a perivascular collar. Our results showed that CFI-400945 drastically inhibited neointima formation but significantly accelerated atherosclerosis. In vitro studies showed that CFI-400945 treatment induced SMC polyploidization and arrested cells in the G2/M phase. CFI-400945 treatment upregulated p53 and p27 expression but decreased p21 and cyclin B1 expression. CFI-400945 also induced SMC apoptosis, which was inhibited by hydroxyurea, a DNA synthesis inhibitor that inhibits polyploidization. Furthermore, CFI-400945 caused supernumerary centrosomes, leading to mitotic failure, resulting in polyploidization. In conclusion, CFI-400945 prevents carotid arterial neointima formation in C57BL/6 mice but accelerates atherosclerosis in ApoE-/- mice, likely through mitotic arrest and subsequent induction of polyploidization and apoptosis.

Clinical outcomes of a novel porcine small intestinal submucosa patch for full-thickness hand skin defects: a retrospective investigation.

OBJECTIVE: To investigate the clinical outcomes of a novel soft tissue repair patch (porcine small intestinal submucosa patch, SIS patch) in the treatment of full-thickness hand skin defects. METHODS: From January 2017 to July 2019, 80 patients with hand soft tissue defects, who met the inclusion criteria, were retrospectively reviewed and divided into two groups. After debridement, patients in group A were treated with the novel SIS patch to cover the wound, and patients in group B were treated with autologous skin graft. The dimensions of skin defect area and healing outcome were evaluated and recorded. Scar assessment was carried out using Scar Cosmesis Assessment and Rating Scale (SCAR scale) at the last follow-up postoperation, and the recovery of wound sensation was assessed at the same time using British Medical Research Council (BMRC) grading of sensorimotor recovery. All the data were collected and statistically analyzed. RESULTS: A total of 80 patients were enrolled in the study with 40 patients in each group. Four patients in group A and 5 patients in group B were excluded due to wound infection and lost to follow-up. There were 36 patients in group A and 35 patients in group B finally got follow-up postoperation with mean interval of 12.75 ± 5.61 months in group A and 14.11 ± 5.42 months in group B. The dimensions of skin defect area in group A ranged from 7.5 to 87.5 cm2 (mean 25.97 ± 18.66 cm2) and in group B ranged from 7.5 to 86.25 cm2 (mean 33.61 ± 19.27 cm2) which have no significant difference (P > 0.05). SCAR scale results of group A and group B were 10.98 ± 0.33 and 9.49 ± 0.35, respectively, and the difference was statistically significant (P < 0.05). BMRC grading results showed 6 cases of S4, 11 cases of S3+, 5 cases of S3, 6 cases of S2, 6 cases of S1 and 2 cases of S0 in group A, and 8 cases of S4, 10 cases of S3+, 7 cases of S3, 4 cases of S2, 5 cases of S1, and 1 case of S0 in group B, which had no significant difference between them (P > 0.05). CONCLUSIONS: The novel SIS patch is an applicable biological material in the treatment of hand skin defect, which could achieve a better cosmetic appearance of the newborn skin tissue.

Polo-like kinase 1 (PLK1) O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma.

The O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification. O-GlcNAcylation occurs on Ser/Thr residues and is important for numerous physiological processes. OGT is essential for dividing mammalian cells and is involved in many human diseases; however, many of its fundamental substrates during cell division remain unknown. Here, we focus on the effect of OGT on polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation, and mitotic exit. We show that PLK1 interacts with OGT and is O-GlcNAcylated. By utilizing stepped collisional energy/higher-energy collisional dissociation mass spectrometry, we found a peptide fragment of PLK1 that is modified by O-GlcNAc. Further mutation analysis of PLK1 shows that the T291A mutant decreases O-GlcNAcylation. Interestingly, T291N is a uterine carcinoma mutant in The Cancer Genome Atlas. Our biochemical assays demonstrate that T291A and T291N both increase PLK1 stability. Using stable H2B-GFP cells, we found that PLK1-T291A and PLK1-T291N mutants display chromosome segregation defects and result in misaligned and lagging chromosomes. In mouse xenograft models, we demonstrate that the O-GlcNAc-deficient PLK1-T291A and PLK1-T291N mutants enhance uterine carcinoma in animals. Hence, we propose that OGT partially exerts its mitotic function through O-GlcNAcylation of PLK1, which might be one mechanism by which elevated levels of O-GlcNAc promote tumorigenesis.

RyR2 Serine-2030 PKA Site Governs Ca2+ Release Termination and Ca2+ Alternans.

BACKGROUND: PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. METHODS: We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. RESULTS: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.

Customized synthesis of phosphoprotein bearing phosphoserine or its nonhydrolyzable analog.

Studies on the mechanism of protein phosphorylation and therapeutic interventions of its related molecular processes are limited by the difficulty in the production of purpose-built phosphoproteins harboring site-specific phosphorylated amino acids or their nonhydrolyzable analogs. Here we address this limitation by customizing the cell-free protein synthesis (CFPS) machinery via chassis strain selection and orthogonal translation system (OTS) reconfiguration screening. The suited chassis strains and reconfigured OTS combinations with high orthogonality were consequently picked out for individualized phosphoprotein synthesis. Specifically, we synthesized the sfGFP protein and MEK1 protein with site-specific phosphoserine (O-pSer) or its nonhydrolyzable analog, 2-amino-4-phosphonobutyric acid (C-pSer). This study successfully realized building cell-free systems for site-specific incorporation of phosphonate mimics into the target protein. Our work lays the foundation for developing a highly expansible CFPS platform and the streamlined production of user-defined phosphoproteins, which can facilitate research on the physiological mechanism and potential interference tools toward protein phosphorylation.

Genetically Encoded Phosphine Ligand for Metalloprotein Design.

Phosphine ligands are the most important class of ligands for cross-coupling reactions due to their unique electronic and steric properties. However, metalloproteins generally rely on nitrogen, sulfur, or oxygen ligands. Here, we report the genetic incorporation of P3BF, which contains a biocompatible borane-protected phosphine, into proteins. This step is followed by a straightforward one-pot strategy to perform deboronation and palladium coordination in aqueous and aerobic conditions. The genetically encoded phosphine ligand P3BF should significantly expand our ability to design functional metalloproteins.

Intrinsically Disordered Protein Condensate-Modified Surface for Mitigation of Biofouling and Foreign Body Response.

Mitigation of biofouling and the host's foreign body response (FBR) is a critical challenge with biomedical implants. The surface coating with various anti-fouling materials provides a solution to overcome it, but limited options in clinic and their potential immunogenicity drive the development of more alternative coating materials. Herein, inspired by liquid-liquid phase separation of intrinsically disordered proteins (IDPs) to form separated condensates in physiological conditions, we develop a new type of low-fouling biomaterial based on flexible IDP of FUS protein containing rich hydrophilic residues. A chemical structure-defined FUS IDP sequence tagged with a tetra-cysteine motif (IDPFUS) was engineered and applied for covalent immobilization on various surfaces to form a uniform layer of protein tangles, which boosted strong hydration on surfaces, as revealed by molecular dynamics simulation. The IDPFUS-coated surfaces displayed excellent performance in resisting adsorption of various proteins and adhesion of different cells, platelets, and bacteria. Moreover, the IDPFUS-coated implants largely mitigated the host's FBR compared with bare implants and particularly outperformed PEG-coated implants in reducing collagen encapsulation. Thus, this novel low-fouling and anti-FBR strategy provides a potential surface coating material for biomedical implants, which will also shed light on exploring similar applications of other IDP proteins.

A gain-of-function mutation in the ITPR1 gating domain causes male infertility in mice.

Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an intracellular Ca2+ release channel critical for numerous cellular processes. Despite its ubiquitous physiological significance, ITPR1 mutations have thus far been linked to primarily movement disorders. Surprisingly, most disease-associated ITPR1 mutations generate a loss of function. This leaves our understanding of ITPR1-associated pathology oddly one-sided, as little is known about the pathological consequences of ITPR1 gain of function (GOF). To this end, we generated an ITPR1 gating domain mutation (D2594K) that substantially enhanced the inositol trisphosphate (IP3 )-sensitivity of ITPR1, and a mouse model expressing this ITPR1-D2594K+/- GOF mutation. We found that heterozygous ITPR1-D2594K+/- mutant mice exhibited male infertility, azoospermia, and acrosome loss. Furthermore, we functionally characterized a human ITPR1 variant V494I identified in the UK Biobank database as potentially associated with disorders of the testis. We found that the ITPR1-V494I variant significantly enhanced IP3 -induced Ca2+ release in HEK293 cells. Thus, ITPR1 hyperactivity may increase the risk of testicular dysfunction.

Recent Advances in Post-polymerization Modifications on Polypeptides: Synthesis and Applications.

Polypeptides, a kind of very promising biomaterial, have shown a wide range of applications due to their excellent biocompatibility, easy accessibility, and structural variability. To synthesize polypeptides with desired functions, post-polymerization modification (PPM) plays an important role in introducing novel chemical structure on their side-chains. The key to PPM strategy is to develop highly selective and efficient reactions that can couple the additional functional moieties with pre-installed side-chain functionalities on polypeptides. In this minireview, classic PPM approaches and their recent progresses are summarized and categorized into five sections, including various reactions on unsaturated alkyl, oxygen-, nitrogen-, sulfur-containing and other special functional groups. In addition, we also highlight the applications of the resultant structure-diversified polypeptides in the field of biomaterial.

Phosphorylated and Phosphonated Low-Complexity Protein Segments for Biomimetic Mineralization and Repair of Tooth Enamel.

Biomimetic mineralization based on self-assembly has made great progress, providing bottom-up strategies for the construction of new organic-inorganic hybrid materials applied in the treatment of hard tissue defects. Herein, inspired by the cooperative effects of key components in biomineralization microenvironments, a new type of biocompatible peptide scaffold based on flexibly self-assembling low-complexity protein segments (LCPSs) containing phosphate or phosphonate groups is developed. These LCPSs can retard the transformation of amorphous calcium phosphate into hydroxyapatite (HAP), leading to merged mineralization structures. Moreover, the application of phosphonated LCPS over phosphorylated LCPS can prevent hydrolysis by phosphatases that are enriched in extracellular mineralization microenvironments. After being coated on the etched tooth enamel, these LCPSs facilitate the growth of HAP to generate new enamel layers comparable to the natural layers and mitigate the adhesion of Streptococcus mutans. In addition, they can effectively stimulate the differentiation pathways of osteoblasts. These results shed light on the potential biomedical applications of two LCPSs in hard tissue repair.

Accumulation of Plasma-Derived Lipids in the Lipid Core and Necrotic Core of Human Atheroma: Imaging Mass Spectrometry and Histopathological Analyses.

Objective: To clarify the pathogenesis of human atheroma, the origin of deposited lipids, the developmental mechanism of liponecrotic tissue, and the significance of the oxidation of phospholipids were investigated using mass spectrometry-aided imaging and immunohistochemistry.Atherosclerotic lesions in human coronary arteries were divided into 3 groups: pathologic intimal thickening with lipid pool, atheroma with lipid core, and atheroma with necrotic core. The lipid pool and lipid core were characterized by the deposition of extracellular lipids. The necrotic core comprised extracellular lipids and liponecrotic tissue. The proportion of cholesteryl linoleate in cholesteryl linoleate+cholesteryl oleate fraction in the extracellular lipid and liponecrotic regions differed significantly from that of the macrophage foam cell-dominant region, and the plasma-derived components (apolipoprotein B and fibrinogen) were localized in the regions. The liponecrotic region was devoid of elastic and collagen fibers and accompanied by macrophage infiltration in the surrounding tissue. Non-oxidized phospholipid (Non-OxPL), OxPL, and Mox macrophages were detected in the three lesions. In the atheroma with lipid core and atheroma with necrotic core, non-OxPL tended to localize in the superficial layer, whereas OxPL was distributed evenly. Mox macrophages were colocalized with OxPL epitopes.In human atherosclerosis, plasma-derived lipids accumulate to form the lipid pool of pathologic intimal thickening, lipid core of atheroma with lipid core, and necrotic core of atheroma with necrotic core. The liponecrotic tissue in the necrotic core appears to be developed by the loss of elastic and collagen fibers. Non-OxPL in the accumulated lipids is oxidized to form OxPL, which may contribute to the lesion development through Mox macrophages.

Different phosphorylation and farnesylation patterns tune Rnd3-14-3-3 interaction in distinct mechanisms.

Protein posttranslational modifications (PTMs) are often involved in the mediation or inhibition of protein-protein interactions (PPIs) within many cellular signaling pathways. Uncovering the molecular mechanism of PTM-induced multivalent PPIs is vital to understand the regulatory factors to promote inhibitor development. Herein, Rnd3 peptides with different PTM patterns as the binding epitopes and 14-3-3ζ protein were used as models to elucidate the influences of phosphorylation and farnesylation on binding thermodynamics and kinetics and their molecular mechanism. The quantitative thermodynamic results indicate that phosphorylated residues S210 and S218 (pS210 and pS218) and farnesylated C241 (fC241) enhance Rnd3-14-3-3ζ interactions in the presence of the essential pS240. However, distinct PTM patterns greatly affect the binding process. Initial association of pS240 with the phosphate-binding pocket of one monomer of the 14-3-3ζ dimer triggers the binding of pS210 or pS218 to another monomer, whereas the binding of fC241 to the hydrophobic groove on one 14-3-3ζ monomer induces the subsequent binding of pS240 to the adjacent pocket on the same monomer. Based on the experimental and molecular simulation results, we estimate that pS210/pS218 and pS240 mediate the multivalent interaction through an additive mechanism, whereas fC241 and pS240 follow an induced fit mechanism, in which the cooperativity of these two adjacent PTMs is reflected by the index ε described in our established thermodynamic binding model. Besides, these proposed binding models have been further used for describing the interaction between 14-3-3ζ and other substrates containing adjacent phosphorylation and lipidation groups, indicating their potential in general applications. These mechanistic insights are significant for understanding the regulatory factors and the design of PPI modulators.