A Coupling-Induced Assembly Strategy for Constructing Artificial Shell on Mitochondria in Living Cells.

The strategy of in vivo self-assembly has been developed for improved enrichment and long-term retention of anticancer drug in tumor tissues. However, most self-assemblies with non-covalent bonding interactions are susceptible to complex physiological environments, leading to weak stability and loss of biological function. Here, we develop a coupling-induced assembly (CIA) strategy to generate covalently crosslinked nanofibers, which is applied for in situ constructing artificial shell on mitochondria. The oxidation-responsive peptide-porphyrin conjugate P1 is synthesized, which self-assemble into nanoparticles. Under the oxidative microenvironment of mitochondria, the coupling of thiols in P1 causes the formation of dimers, which is further ordered and stacked into crosslinked nanofibers. As a result, the artificial shell is constructed on the mitochondria efficiently through multivalent cooperative interactions due to the increased binding sites. Under ultrasound (US) irradiation, the porphyrin molecules in the shell produce a large amount of reactive oxygen species (ROS) that act on the adjacent mitochondrial membrane, exhibiting ~2-fold higher antitumor activity than nanoparticles in vitro and in vivo. Therefore, the mitochondria-targeted CIA strategy provides a novel perspective on improved sonodynamic therapy (SDT) and shows potential applications in antitumor therapies.

Sonoactivated Cascade Fenton Reaction Enhanced by Synergistic Modulation of Electron-Hole Separation for Improved Tumor Therapy.

Chemodynamic therapy (CDT) is an emerging targeted treatment technique for tumors via the generation of highly cytotoxic hydroxyl radical (·OH) governed by tumor microenvironment-assisted Fenton reaction. Despite high effectiveness, it faces limitations like low reaction efficiency and limited endogenous H2 O2 , compromising its therapeutic efficacy. This study reports a novel platform with enhanced CDT performance by in situ sono-activated cascade Fenton reaction. A piezoelectric g-C3 N4 (Au-Fe-g-C3 N4 ) nanosheet is developed via sono-activated synergistic effect/H2 O2 self-supply mediated cascade Fenton reaction, realizing in situ ultrasound activated cascade Fenton reaction kinetics by synergistic modulation of electron-hole separation. The nanosheets consist of piezoelectric g-C3 N4 nanosheet oxidizing H2 O to highly reactive H2 O2 from the valence band, Fe3+ /Fe2+ cycling activated by conduction band to generate ·OH, and Au nanoparticles that lower the bandgap and further adopt electrons to generate more 1 O2 , resulting in improved CDT and sonodynamic therapy (SDT). Moreover, the Au-Fe-g-C3 N4 nanosheet is further modified by the targeted peptide to obtain P-Au-Fe-g-C3 N4 , which inhibits tumor growth in vivo effectively by generating reactive oxygen species (ROS). These results demonstrated that the sono-activated modulation translates into a high-efficiency CDT with a synergistic effect using SDT for improved anti-tumor therapy.

Cell membrane-specific self-assembly of peptide nanomedicine induces tumor immunogenic death to enhance cancer therapy.

Immunogenic cell death (ICD), as an unusual cell death pattern, mediates cancer cells to release a series of damage-associated molecular patterns (DAMPs), and is widely used in the field of cancer immunotherapy. Injuring the cell membrane can serve as a novel ICD initiation strategy. In this study, a peptide nanomedicine (PNpC) is designed using the fragment CM11 of cecropin, which is effective in disrupting cell membranes because of its α-helical structure. PNpC self-assembles in situ in the presence of high levels of alkaline phosphatase (ALP) on the tumor cell membrane, transforming from nanoparticles to nanofibers, which reduces the cellular internalization of the nanomedicine and increases the interaction between CM11 and tumor cell membranes. Both in vitro and in vivo results indicate that PNpC plays a significant role in killing tumor cells by triggering ICD. The ICD induced by the destruction of the cancer cell membrane is accompanied by the release of DAMPs, which promotes the maturation of DCs and facilitates the presentation of tumor-associated antigens (TAA), resulting in the infiltration of CD8+ T cells. We believe that PNpC can trigger ICD while killing cancer cells, providing a new reference for cancer immunotherapy.

Biomimetic nanoparticles for DC vaccination: a versatile approach to boost cancer immunotherapy.

Cancer immunotherapy, which harnesses the immune system to fight cancer, has begun to make a breakthrough in clinical applications. Dendritic cells (DCs) are the bridge linking innate and adaptive immunity and the trigger of tumor immune response. Considering the cumbersome process and poor efficacy of classic DC vaccines, there has been interest in transferring the field of in vitro-generated DC vaccines to nanovaccines. Conventional nanoparticles have insufficient targeting ability and are easily cleared by the reticuloendothelial system. Biological components have evolved very specific functions, which are difficult to fully reproduce with synthetic materials, making people interested in using the further understanding of biological systems to prepare nanoparticles with new and enhanced functions. Biomimetic nanoparticles are semi-biological or nature-derived delivery systems comprising one or more natural materials, which have a long circulation time in vivo and excellent performance of targeting DCs, and can mimic the antigen-presenting behavior of DCs. In this review, we introduce the classification, design, preparation, and challenges of different biomimetic nanoparticles, and discuss their application in activating DCs in vivo and stimulating T cell antitumor immunity. Incorporating biomimetic nanoparticles into cancer immunotherapy has shown outstanding advantages in precisely coaxing the immune system against cancer.

Exploiting autophagy-regulative nanomaterials for activation of dendritic cells enables reinforced cancer immunotherapy.

Dendritic cells (DCs), as the most powerful antigen presenting cells, play a critical role in regulating immune response and anti-tumor process. However, the immunosuppressive cells and factors resided in the tumor microenvironment (TME) pose various challenges that can subvert competent DC function comprising antigen presentation and immune initiation. In this setting, developing potent strategies to improve the function of DCs is critically required for improving the efficacy of tumor immunotherapy. Autophagy is found to be closely associated to the various functions of DCs under physiological and pathological conditions. Especially, nanomaterials (NMs) can engage in the disorder and regularity of autophagy to modulate their metabolism and function of DCs. Reasonable design of nanomaterials with autophagy regulation is of great significance to activate DCs and enhance its immunological functions, provoking robust and durable antitumor immunity. In this review, we study the design and optimization of nanomaterials with the function of regulating DCs autophagy, discuss the main mechanism of DCs autophagy induced by nanomaterials and its application in tumor immunotherapy, promoting the progress and development of cancer immunotherapy strategies in the future.

LRF maintains genome integrity by regulating the non-homologous end joining pathway of DNA repair.

Leukemia/lymphoma-related factor (LRF) is a POZ/BTB and Krüppel (POK) transcriptional repressor characterized by context-dependent key roles in cell fate decision and tumorigenesis. Here we demonstrate an unexpected transcription-independent function for LRF in the classical non-homologous end joining (cNHEJ) pathway of double-strand break (DSB) repair. We find that LRF loss in cell lines and mouse tissues results in defective cNHEJ, genomic instability and hypersensitivity to ionizing radiation. Mechanistically, we show that LRF binds and stabilizes DNA-PKcs on DSBs, in turn favouring DNA-PK activity. Importantly, LRF loss restores ionizing radiation sensitivity to p53 null cells, making LRF an attractive biomarker to direct p53-null LRF-deficient tumours towards therapeutic treatments based on genotoxic agents or PARP inhibitors following a synthetic lethal strategy.

Real-Time Monitoring of Alzheimer's-Related Amyloid Aggregation via Probe Enhancement-Fluorescence Correlation Spectroscopy.

This work describes the use of fluorescence correlation spectroscopy (FCS) and a novel amyloid-binding fluorescent probe, ARCAM 1, to monitor the aggregation of the Alzheimer's disease-associated amyloid ß-peptide (Aß). ARCAM 1 exhibits a large increase in fluorescence emission upon binding to Aß assemblies, making it an excellent candidate for probe enhancement FCS (PE-FCS). ARCAM 1 binding does not change Aß aggregation kinetics. It also exhibits greater dynamic range as a probe in reporting aggregate size by FCS in Aß, when compared to thioflavin T (ThT) or an Aß peptide modified with a fluorophore. Using fluorescent burst analysis (via PE-FCS) to follow aggregation of Aß, we detected soluble aggregates at significantly earlier time points compared to typical bulk fluorescence measurements. Autocorrelation analysis revealed the size of these early Aß assemblies. These results indicate that PE-FCS/ARCAM 1 based assays can detect and provide size characterization of small Aß aggregation intermediates during the assembly process, which could enable monitoring and study of such aggregates that transiently accumulate in biofluids of patients with Alzheimer's and other neurodegenerative diseases.

Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.

We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein-protein interactions. We also use MPE-FCCS to detect drug-protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

High-affinity accumulation of a maytansinoid in cells via weak tubulin interaction.

The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4-6 × 10(7) copies). Efflux of 3[H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.

Prion-like proteins sequester and suppress the toxicity of huntingtin exon 1.

Expansions of preexisting polyglutamine (polyQ) tracts in at least nine different proteins cause devastating neurodegenerative diseases. There are many unique features to these pathologies, but there must also be unifying mechanisms underlying polyQ toxicity. Using a polyQ-expanded fragment of huntingtin exon-1 (Htt103Q), the causal protein in Huntington disease, we and others have created tractable models for investigating polyQ toxicity in yeast cells. These models recapitulate key pathological features of human diseases and provide access to an unrivalled genetic toolbox. To identify toxicity modifiers, we performed an unbiased overexpression screen of virtually every protein encoded by the yeast genome. Surprisingly, there was no overlap between our modifiers and those from a conceptually identical screen reported recently, a discrepancy we attribute to an artifact of their overexpression plasmid. The suppressors of Htt103Q toxicity recovered in our screen were strongly enriched for glutamine- and asparagine-rich prion-like proteins. Separated from the rest of the protein, the prion-like sequences of these proteins were themselves potent suppressors of polyQ-expanded huntingtin exon-1 toxicity, in both yeast and human cells. Replacing the glutamines in these sequences with asparagines abolished suppression and converted them to enhancers of toxicity. Replacing asparagines with glutamines created stronger suppressors. The suppressors (but not the enhancers) coaggregated with Htt103Q, forming large foci at the insoluble protein deposit in which proteins were highly immobile. Cells possessing foci had fewer (if any) small diffusible oligomers of Htt103Q. Until such foci were lost, cells were protected from death. We discuss the therapeutic implications of these findings.

Monoalkoxy BODIPYs--a fluorophore class for bioimaging.

Small molecule fluorophores are indispensable tools for modern biomedical imaging techniques. In this report, we present the development of a new class of BODIPY dyes based on an alkoxy-fluoro-boron-dipyrromethene core. These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties. MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, and allow for facile modification with functional groups of relevance to bioconjugate chemistry and bioorthogonal labeling. Biological profiling in living cells demonstrates excellent membrane permeability, low nonspecific binding, and lack of cytotoxicity.

Dephosphorylation enables the recruitment of 53BP1 to double-strand DNA breaks.

Excluding 53BP1 from chromatin is required to attenuate the DNA damage response during mitosis, yet the functional relevance and regulation of this exclusion are unclear. Here we show that 53BP1 is phosphorylated during mitosis on two residues, T1609 and S1618, located in its well-conserved ubiquitination-dependent recruitment (UDR) motif. Phosphorylating these sites blocks the interaction of the UDR motif with mononuclesomes containing ubiquitinated histone H2A and impedes binding of 53BP1 to mitotic chromatin. Ectopic recruitment of 53BP1-T1609A/S1618A to mitotic DNA lesions was associated with significant mitotic defects that could be reversed by inhibiting nonhomologous end-joining. We also reveal that protein phosphatase complex PP4C/R3ß dephosphorylates T1609 and S1618 to allow the recruitment of 53BP1 to chromatin in G1 phase. Our results identify key sites of 53BP1 phosphorylation during mitosis, identify the counteracting phosphatase complex that restores the potential for DDR during interphase, and establish the physiological importance of this regulation.

Activation and control of p53 tetramerization in individual living cells.

Homo-oligomerization is found in many biological systems and has been extensively studied in vitro. However, our ability to quantify and understand oligomerization processes in cells is still limited. We used fluorescence correlation spectroscopy and mathematical modeling to measure the dynamics of the tetramers formed by the tumor suppressor protein p53 in single living cells. Previous in vitro studies suggested that in basal conditions all p53 molecules are bound in dimers. We found that in resting cells p53 is present in a mix of oligomeric states with a large cell-to-cell variation. After DNA damage, p53 molecules in all cells rapidly assemble into tetramers before p53 protein levels increase. We developed a model to understand the connection between p53 accumulation and tetramerization. We found that the rapid increase in p53 tetramers requires a combination of active tetramerization and protein stabilization, however tetramerization alone is sufficient to activate p53 transcriptional targets. This suggests triggering tetramerization as a mechanism for activating the p53 pathway in cancer cells. Many other transcription factors homo-oligomerize, and our approach provides a unique way for probing the dynamics and functional consequences of oligomerization.

Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks.

Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.

Noise reduction in the intracellular pom1p gradient by a dynamic clustering mechanism.

Chemical gradients can generate pattern formation in biological systems. In the fission yeast Schizosaccharomyces pombe, a cortical gradient of pom1p (a DYRK-type protein kinase) functions to position sites of cytokinesis and cell polarity and to control cell length. Here, using quantitative imaging, fluorescence correlation spectroscopy, and mathematical modeling, we study how its gradient distribution is formed. Pom1p gradients exhibit large cell-to-cell variability, as well as dynamic fluctuations in each individual gradient. Our data lead to a two-state model for gradient formation in which pom1p molecules associate with the plasma membrane at cell tips and then diffuse on the membrane while aggregating into and fragmenting from clusters, before disassociating from the membrane. In contrast to a classical one-component gradient, this two-state gradient buffers against cell-to-cell variations in protein concentration. This buffering mechanism, together with time averaging to reduce intrinsic noise, allows the pom1p gradient to specify positional information in a robust manner.

[Effective analysis of the posterior vertebral pedicle screw fixation, vertebral body removal, decompression and titanium mesh reconstruction for the treatment of the lower lumbar fractures].

OBJECTIVE: To evaluate the effect of the treatment of the lower lumbar fractures by posterior vertebral pedicle screw fixation, vertebral canal decompression,bone graft and titanium mesh reconstruction. METHODS: From January 2006 to December 2008, 22 patients with lower lumbar fractures were treated by posterior vertebral pedicle screw fixation, vertebral canal decompression, bone graft and titanium mesh reconstruction at same period. There were 18 males and 4 females with an average age of 43.8 years ranging from 22 to 63 years old. The injured vertebrae were L3 in 11 cases, L4, in 8 cases, and L5 in 3 cases. The operative time, blood loss, the preoperative and postoperative vertebral height,sagittal index, and the lumbar lordosis angle were recorded and evaluated. RESULTS: The operative time was 3 to 4.2 hours (means 3.6 h). The blood loss averaged 1300 ml (900 to 1500 ml). The preoperative and postoperative sagittal index were (57.5 +/- 7.6)% and (93.5 +/- 8.1)%, respectively. The preoperative and postoperative lumbar lordosis angle were (34.3 +/- 7.3) degrees and (38.5 +/- 9.8) degrees, respectively. All patients were followed up for 10 months to 3 years (means 2.6 years). No fixation were failed,the segment of titanium mesh reconstruction obtained bone healing, no pseudoarticulation formation. At the last time of followed-up, 15 patients with nerve injuries were evaluated according to Frankel grade, there were 10 cases in grade E, 4 in D, 1 in C. According to the low back outcome scores (LBOS), the results were excellent in 20 cases, good in 1, fair in 1. CONCLUSION: The stability of the lower lumbar spine can be reconstructed by bone graft and titanium mesh combined with transpedicular screw fixation through a posterior approach. The decompression and vertebral body removal can also be performed in this approach. The recovery of the vertebral height and lumbar lordosis can prevent the delayed neurological deficit and traumatic kyphosis.

Identification of signaling pathways regulating primary cilium length and flow-mediated adaptation.

The primary cilium acts as a transducer of extracellular stimuli into intracellular signaling [1, 2]. Its regulation, particularly with respect to length, has been defined primarily by genetic experiments and human disease states in which molecular components that are necessary for its proper construction have been mutated or deleted [1]. However, dynamic modulation of cilium length, a phenomenon observed in ciliated protists [3, 4], has not been well-characterized in vertebrates. Here we demonstrate that decreased intracellular calcium (Ca(2+)) or increased cyclic AMP (cAMP), and subsequent protein kinase A activation, increases primary cilium length in mammalian epithelial and mesenchymal cells. Anterograde intraflagellar transport is sped up in lengthened cilia, potentially increasing delivery flux of cilium components. The cilium length response creates a negative feedback loop whereby fluid shear-mediated deflection of the primary cilium, which decreases intracellular cAMP, leads to cilium shortening and thus decreases mechanotransductive signaling. This adaptive response is blocked when the autosomal-dominant polycystic kidney disease (ADPKD) gene products, polycystin-1 or -2, are reduced. Dynamic regulation of cilium length is thus intertwined with cilium-mediated signaling and provides a natural braking mechanism in response to external stimuli that may be compromised in PKD.

Real-time detection of alpha1A-AR movement stimulated by phenylephrine in single living cells.

AIM: To investigate the movement of alpha(1A)-adrenergic receptors(alpha(1A)-AR) stimulated by agonist, phenylephrine (PE), and the dynamics of receptor movement in real time in single living cells with millisecond resolution. METHODS: We labeled alpha(1A)-AR using the monoclonal, anti-FLAG (a kind of tag) antibody and Cy3-conjugated goat anti-mouse IgG and recorded the trajectory of their transport process in living HEK293A cells stimulated by agonist, PE, and then analyzed their dynamic properties. RESULTS: The specific detection of alpha(1A)-AR on the surface of living HEK293A-alpha(1A) cells was achieved. alpha(1A)-AR internalize under the stimulation of PE. After the cells were stimulated with PE for 20 min, apparent colocalization was found between alpha(1A)-AR and F-actins. After 40 min stimulation of PE, trajectories of approximate linear motion in HEK293A-alpha(1A) cells were recorded, and their velocity was calculated. CONCLUSION: The specific labeling method on the living cell surface provides a convenient means of real-time detection of the behavior of surface receptors. By this method we were able to specifically detect alpha(1A)-AR and record the behavior of individual particles of receptors with 50 ms exposure time in real time in single living cells.

Heterogeneous transportation of alpha1B-adrenoceptor in living cells.

The heterogeneous motion of alpha(1B)-adrenoceptor (alpha(1B)-AR) was visualized in living cells with BODIPY-labeled antagonist of AR by single molecule fluorescence microscopy at high spatial resolution. The moving trajectory was reconstructed by precise localization (better than 20 nm) with a least-square fit of a two-dimensional Gaussian point spread function to each single spot. Trajectory analysis revealed two apparent groups of movements: directed motion and hindered motion. The directed motion had speeds higher than 0.1 mum/s. The histogram of diffusion coefficients of the hindered motion showed distinction between the cell membrane and the cytoplasm: the diffusion coefficient was lower near the cell membrane than in the internal cytoplasm, suggesting that alpha(1B)-AR was located or trapped in different networks, which was consistent with the natural distribution of cytoskeleton in living cells. These results suggested that the heterogeneity in the motion of alpha(1B)-AR in living cell might be associated with different localizations of cell skeleton proteins in the cell, which could provide molecular insight of AR regulation in living cells.

The transport of alpha(1A)-adrenergic receptor with 33-nm step size in live cells.

We used the technique of single particle tracking (SPT) with high tempo-spatial resolution to efficiently explore the route and mechanism for the transport of alpha(1A)-adrenergic receptor (alpha(1A)-AR) in real time in living cells. We found that the initial transport of alpha(1A)-AR in cells depended on actin filaments with the velocity of 0.2 microm/s and exhibited discrete 33-nm steps. It was noted that the step size, the rate constant, and the velocities were in accordance with the character of single myosin in vitro, implying that while transporting each endosome myosins did not work in the "tug-of-war" mode and that they did not adopt the strategy to boost up transporting speed by working coordinately. These results provided insight into the mechanism of GPCR transport in vivo.