Rational Design of Dual-Functionalized Gd@C82 Nanoparticles to Relieve Neuronal Cytotoxicity in Alzheimer's Disease via Inhibition of Aß Aggregation.

The accumulation of amyloid-ß (Aß) peptides is a major hallmark of Alzheimer's disease (AD) and plays a crucial role in its pathogenesis. Particularly, the structured oligomeric species rich in ß-sheet formations were implicated in neuronal organelle damage. Addressing this formidable challenge requires identifying candidates capable of inhibiting peptide aggregation or disaggregating preformed oligomers for effective antiaggregation-based AD therapy. Here, we present a dual-functional nanoinhibitor meticulously designed to target the aggregation driving force and amyloid fibril spatial structure. Leveraging the exceptional structural stability and facile tailoring capability of endohedral metallofullerene Gd@C82, we introduce desired hydrogen-binding sites and charged groups, which are abundant on its surface for specific designs. Impressively, these designs endow the resultant functionalized-Gd@C82 nanoparticles (f-Gd@C82 NPs) with high capability of redirecting peptide self-assembly toward disordered, off-pathway species, obstructing the early growth of protofibrils, and disaggregating the preformed well-ordered protofibrils or even mature Aß fibrils. This results in considerable alleviation of Aß peptide-induced neuronal cytotoxicity, rescuing neuronal death and synaptic loss in primary neuron models. Notably, these modifications significantly improved the dispersibility of f-Gd@C82 NPs, thus substantially enhancing its bioavailability. Moreover, f-Gd@C82 NPs demonstrate excellent cytocompatibility with various cell lines and possess the ability to penetrate the blood-brain barrier in mice. Large-scale molecular dynamics simulations illuminate the inhibition and disaggregation mechanisms. Our design successfully overcomes the limitations of other nanocandidates, which often overly rely on hydrophobic interactions or photothermal conversion properties, and offers a viable direction for developing anti-AD agents through the inhibition and even reversal of Aß aggregation.

C3N nanodots inhibits Aß peptides aggregation pathogenic path in Alzheimer's disease.

Despite the accumulating evidence linking the development of Alzheimer's disease (AD) to the aggregation of Aß peptides and the emergence of Aß oligomers, the FDA has approved very few anti-aggregation-based therapies over the past several decades. Here, we report the discovery of an Aß peptide aggregation inhibitor: an ultra-small nanodot called C3N. C3N nanodots alleviate aggregation-induced neuron cytotoxicity, rescue neuronal death, and prevent neurite damage in vitro. Importantly, they reduce the global cerebral Aß peptides levels, particularly in fibrillar amyloid plaques, and restore synaptic loss in AD mice. Consequently, these C3N nanodots significantly ameliorate behavioral deficits of APP/PS1 double transgenic male AD mice. Moreover, analysis of critical tissues (e.g., heart, liver, spleen, lung, and kidney) display no obvious pathological damage, suggesting C3N nanodots are biologically safe. Finally, molecular dynamics simulations also reveal the inhibitory mechanisms of C3N nanodots in Aß peptides aggregation and its potential application against AD.

[Differentiated embryonic chondrocyte gene 2 (DEC2) inhibits transdifferentiation of mouse glomerular endothelial cells and renal fibrosis by blocking TGF-ß/ROCK1 signaling pathway].

Objective To explore the protective mechanism of transdifferentiation of glomerular endothelial cells based on the differentiated embryonic chondrocyte gene 2 (DEC2) via the TGF-ß/ROCK1 signaling pathway. Methods The 24 mice were randomly divided into sham group, UUO group, UUO combined with vector group and UUO combined with DEC2 group, with 6 mice in each group. A unilateral ureteral obstruction (UUO) model was established in each group, except for the sham group. In the UUO combined with vector group and UUO combined with DEC2 group, 10 µL (108 PFU) of vector or DEC2 was injected into each kidney on day 0 (immediately after UUO) under the guidance of the ultrasound system. The mice were sacrificed 14 days after the operation, and the kidneys were collected for histological examination and Western blot analysis: HE staining was used to observe the histological changes of kidneys, Masson staining to observe the renal fibrosis, and Western blot analysis to detect the protein expression. In vitro, normal human glomerular endothelial cells (GEnCs) was selected as the research objects. GEnCs stimulated with TGF-ß were treated with ROCK1 inhibitor Y-27632 or DEC2 transfection. Western blot analysis was used to detect the expression of ROCK1, α-SMA, DEC2 and E-cadherin in GEnC exposed to transforming growth factor ß (TGF-ß). The localization of ROCK1 and DEC2 in GEnCs cells was detected by immunofluorescence cytochemistry. The relationship between the ROCK1 and DEC2 was confirmed by co-immunoprecipitation. Results Compared with the sham group, the UUO groups showed significant renal fibrosis and collagen accumulation on the 14th day. In the UUO groups, the expression of DEC2 and E-cadherin in the kidney tissue of the mice was significantly reduced, and the expression of α-SMA significantly increased. Compared with the UUO combined with vector group, the kidney fibrosis and collagen accumulation in the UUO combined with DEC2 group decreased, and the expression of ROCK1 and α-SMA decreased and the expression of DEC2 and E-cadherin increased in the kidney tissue. TGF-ß enhanced the expression of ROCK1 and α-SMA in GEnCs cells in a time-dependent manner, and the levels of DEC2 and E-cadherin decreased. Treatment with the ROCK1 inhibitor Y-27632 partially abrogated the TGF-ß-induced increase in the expression of ROCK1 and α-SMA and decrease in the expression of DEC2 and E-cadherin. In addition, transfection of GEnCs cells with DEC2 before TGF-ß stimulation reduced the expression of ROCK1 and α-SMA, and increased the expression of DEC2 and E-cadherin. Immunofluorescence cytochemical staining showed that DEC2 co-localized with ROCK1 in GEnCs, and the co-immunoprecipitation showed that DEC2 and ROCK1 pulled down each other. Conclusions DEC2 is down-regulated in fibrotic renal tissue, while up-regulated DEC2 inhibits epithelial myofibroblast transdifferentiation and renal fibrosis of GEnC by blocking TGF-ß/ROCK1 signaling pathway.

Cytocompatibility of Ti3C2Tx MXene with Red Blood Cells and Human Umbilical Vein Endothelial Cells and the Underlying Mechanisms.

Two-dimensional (2D) nanomaterials have been widely used in biomedical applications because of their biocompatibility. Considering the high risk of exposure of the circulatory system to Ti3C2Tx, we studied the cytocompatibility of Ti3C2Tx MXene with red blood cells (RBCs) and human umbilical vein endothelial cells (HUVECs) and showed that Ti3C2Tx had excellent compatibility with the two cell lines. Ti3C2Tx at a concentration as high as 200 µg/mL caused a negligible percent hemolysis of 0.8%. By contrast, at the same treatment concentration, graphene oxide (GO) caused a high percent hemolysis of 50.8%. Scanning electron microscopy revealed that RBC structures remained intact in the Ti3C2Tx treatment group, whereas those in the GO group completely deformed, sunk, and shrunk, which resulted in the release of cell contents. This difference can be largely ascribed to the distinct surficial properties of the two nanosheets. In specific, the fully covered surface-terminating -O and -OH groups leading to Ti3C2Tx had a very hydrophilic surface, thereby hindering its penetration into the highly hydrophobic interior of the cell membrane. However, the strong direct van der Waals attractions coordinated with hydrophobic interactions between the unoxidized regions of GO and the lipid hydrophobic tails can still damage the integrity of the cell membranes. In addition, the sharp and keen-edged corners of GO may also facilitate its relatively strong cell membrane damage effects than Ti3C2Tx. Thus, the excellent cell membrane compatibility of Ti3C2Tx nanosheets and their ultraweak capacity to provoke excessive ROS generation endowed them with much better compatibility with HUVECs than GO nanosheets. These results indicate that Ti3C2Tx has much better cytocompatibility than GO and provide a valuable reference for the future biomedical applications of Ti3C2Tx.

Nonmonotonic Relationship between the Oxidation State of Graphene-Based Materials and Its Cell Membrane Damage Effects.

With the rapid development of carbon-based two-dimensional nanomaterials in biomedical applications, growing concern has emerged regarding their biocompatibility and especially their interactions with cell membranes. Our experimental studies found that the oxidation state, as one of the most important chemical parameters of graphene derivatives, regulates the hemolysis effect on human red blood cells in a nonmonotonic manner. Scanning electron microscopy and optical microscopy observations suggested that graphene oxides with medium oxygen content have the most serious destructive effects on the cell membranes. Molecular dynamics simulations and potential of mean force calculations revealed that, on the one hand, with the decrease in the surface oxygenated groups, more sp2 carbon area of graphene-based materials will be exposed, playing a facilitating role in the damage of cell membranes; on the other hand, fewer oxygenated groups also lead to the accumulation of graphene-based nanosheets in solutions. The formation of the multilayer structure of graphene-based nanosheets reduces the exposed sp2 carbon area, prevents the collective extraction of lipid molecules, and eventually results in a weakened extraction effect on cell membranes. Together, these factors generate a nonmonotonic relationship between the oxidation state of graphene oxides and their destructive effects on cell membranes.

Different platinum crystal surfaces show very distinct protein denaturation capabilities.

Different platinum (Pt) surfaces of nanocrystals usually exhibit significant distinctions with regard to various biological, physical, and chemical characteristics, such as bio-recognition, surface wetting, and catalytic activities. In this study, we report for the first time that two shape-controlled Pt nanocrystals with the most common low-index surfaces, Pt(100) and Pt(111), show very dissimilar protein denaturation capabilities based on all-atom molecular dynamics simulations employing the widely used model protein, villin headpiece (HP35). We demonstrate that HP35 is well preserved on the Pt(100) crystal surface, whereas it is severely disrupted on the Pt(111) crystal surface. This surprising difference originates from the distinct water behavior in the first solvation shell (FSS) of the two Pt crystal surfaces. Within the FSS of the Pt(100) crystal surface, water molecules form a very compact and stable monolayer through a highly uniform rhombic hydrogen-bond network. This water monolayer prefers the adsorption of acidic residues (such as Glu and Asp) and acts as a shield to prevent other residues from directly coming into contact with the metal surface. On the other hand, the hydrogen bond network in the water monolayer in the FSS of the Pt(111) crystal surface is very sparse and quite defective, which makes it more vulnerable to the penetration of various residues, particularly those with planar side chains such as Phe, Trp and Arg due to strong dispersion interactions, leading to subsequent protein unfolding. The binding free energy calculations for some key amino acids on the two different crystal surfaces further uncover the molecular origin behind their distinct protein denaturation capability. Our study reveals the vital importance of interfacial water in determining the structure of proteins when binding to different metal crystal surfaces. The discovered molecular mechanisms may be helpful for the future development of a bio-assisted programmable synthetic strategy of sophisticated Pt nanostructures for biomedical applications.

Different protonated states at the C-terminal of the amyloid-ß peptide modulate the stability of S-shaped protofibril.

Studies have found strong correlations between polymorphism and structural variations in amyloid-ß (Aß) fibrils and the diverse clinical subtypes of Alzheimer's disease (AD). Thus, a detailed understanding of the conformational behavior of Aß fibrils may be an aid to elucidate the pathological mechanisms involved in AD. However, a key point that has been inadvertently underestimated or dismissed is the role of the protonated state at the C-terminal residue of amyloid-ß peptides, which can give rise to intrinsic differences in the morphology and stability of the fibrils. For instance, the effects of the salt bridge formed between the C-terminal residue A42 and the residue K28 on the S-shaped Aß protofibril structure remain unknown and may be different from those in the U-shaped Aß protofibril structures. To address this effect, we explore the stability of the S-shaped protofibrils capped with different C-terminal modifications, including carboxyl group in its deprotonated (COO-) and protonated (COOH) states, by using molecular dynamics simulations. Our findings indicated that the C-terminal deprotonated protofibril is significantly more stable than its C-terminal protonated counterpart due to a well-defined and highly stable zipper-like salt-bridge-chain formed by the ε-NH3 + groups on the sidechain of residue K28 and the C-terminal COO- group at the A42 residue. The revealed underlying molecular mechanism for the different stability of the protofibrils provides insights into the diversity of polymorphism in Aß fibrils.

Molecular Origin of the Stability Difference in Four Shark IgNAR Constant Domains.

Improving the stability of antibodies for manufacture and shelf life is one of the main focuses of antibody engineering. One stabilization strategy is to perform specific mutations in human antibodies based on highly stable antibodies in other species. To identify the key residues for mutagenesis, it is necessary to understand the roles of these residues in stabilizing the antibody. Here, we use molecular dynamics simulations to study the molecular origin of the four shark immunoglobulin new antigen receptors constant domains (C1-C4). According to the unfolding pathways and the conformational free energy surfaces in 8 M urea at 380 K, the C2 domain is the most stable, followed by C4, C1, and C3, which agrees with the experimental findings. The C1 and C3 domains follow a common unfolding pathway in which the unfolding starts from the edge strands, particularly strand g, and then gradually progresses to the inner strands. Detailed structural analysis of the C2 domain reveals a "sandwich-like" R339-E322-R341 salt-bridge cluster on strand g, which grants ultrahigh stability to the C2 domain. We further design two sets of mutations by mutating E322 to alanine or setting all atomic charges in E322 to zero to break the salt-bridge cluster in the C2 domain, which confirms the importance of the salt bridges in stability. In the C4 domain, the D80-K104 salt bridge on strand g also strengthens the stability. On the other hand, in the C1 and C3 domains, there is no salt bridge on strand g. In addition to the salt bridges, the overall hydrophobicity score of the hydrophobic core is also positively correlated with the domain stability. Our findings provide a detailed microscopic picture of the molecular origin of the four shark immunoglobulin new antigen receptors constant domains that not only explains the differences in their structural stability but also provides important insights into future antibody design.

Effect of the surface curvature on amyloid-ß peptide adsorption for graphene.

The adsorption of amyloid-ß peptide (Aß) onto graphene nanosheets with curvature at a neutral pH has been studied by using molecular dynamics simulations in combination with umbrella sampling. We found that Aß adsorbed onto graphene with distinct characteristics, causing the breakage of hydrogen bonds which leads to its conformational change. Interestingly, the adsorption capacity of graphene's surface varies significantly depending on its curvature, namely, the surface with negative curvature has a higher probability to adsorb the Aß than the one with positive curvature. This phenomenon is further evidenced by the binding energy between the complex of graphene and Aß derived from the potential of mean force (PMF). The hydrophobic interactions and the direct dispersion interactions between the graphene nanosheet and the Aß play a dominant role in the adsorption process. These findings indicate that not only is the chemical composition an important factor but also the shape of the nanoparticle is important in determining its interaction with proteins: the contacting surface curvature can lead to different adsorption capability.

Girdin locates in centrosome and midbody and plays an important role in cell division.

Girdin is a downstream effector of epidermal growth factor receptor (EGFR)-AKT and interacts with actin and microtubule. Increasing evidence confirmed that Girdin played an important role in cell migration. Here we report that Girdin also regulates cell division. Overexpression or suppression of Girdin leads to attenuated cell proliferation. Imaging of mitotic cells revealed that Girdin is located in the cell division apparatus such as centrosome and midbody. The sub-cellular localization of Girdin was dependent on the domains, which interacted with actin or microtubules. Overexpression of Girdin lead to increased centrosome splitting and amplification. In addition, data show that pAKT also locates in both the centrosome and midbody, indicating the regulating role of AKT in Girdin-mediated cell division. To elucidate the effect of Girdin on tumor growth in vivo, HeLa cells infected with retrovirus harboring either control or Girdin shRNAs were injected subcutaneously into the immunocompromised nude mice. Downregulation of Girdin by shRNA markedly inhibited the cell growth of subcutaneously transplanted tumors in nude mice. These data demonstrate that Girdin is important for efficient cell division. Taking our previous data into consideration, we speculate that Girdin regulates both cell division and cell migration through cytoskeletal molecules.

[Effects of honokiol on proliferation and apoptosis of human cervical carcinoma cell line Hela in vitro].

OBJECTIVE: To assess the effects of honokiol on proliferation and apoptosis of human cervical carcinoma cell line Hela in vitro. METHODS: Cultured HeLa cells were treated with different concentrations of honokiol for the varieties of period (24, 48, 72, 96 h). Cell proliferation was assessed by MTT colorimetric assay. Cell apoptosis was determined by flow cytometry (FCM), Hoechst 33258 fluorescent staining and DNA ladder respectively. RESULTS: MTT assay demonstrated that the proliferation of Hela cells were suppressed significantly by honokiol in dose-and time-dependent manner. FCM analysis showed that the apoptosis rates of Hela cells treated with 10 microg/mL and 20 microg/mL honokiol for 24 h were 22.5% and 62.2%, respectively, while that of the control group cells was 8.7%. After treatment with honokiol, typically morphologic changes of apoptosis were observed by Hoechst 33258 fluorescence staining; Genomic DNA from Hela cells treated with honokiol displayed a characteristic ladder pattern on agarose gel electrophoresis. CONCLUSION: honokiol can inhibit the proliferation and induce apoptosis of human cervical carcinoma cell line Hela.