Enhancing Tumor Immunotherapy by Multivalent Anti-PD-L1 Nanobody Assembled via Ferritin Nanocage.

Increasing immunotherapy response rate and durability can lead to significant improvements in cancer care. To address this challenge, a novel multivalent immune checkpoint therapeutic platform is constructed through site-specific ligation of anti-PD-L1 nanobody (Nb) on ferritin (Ftn) nanocage. Nb-Ftn blocks PD-1/PD-L1 interaction and downregulates PD-L1 levels via endocytosis-induced degradation. In addition, the cage structure of Ftn allows encapsulation of indocyanine green (ICG), an FDA-approved dye. Photothermal treatment with Nb-Ftn@ICG induces immunogenic death of tumor cells, which improves systemic immune response via maturation of dendritic cells and enhanced infiltration of T cells. Moreover, Nb-Ftn encapsulation significantly enhances cellular uptake, tumor accumulation and retention of ICG. In vivo assays showed that this nanoplatform ablates the primary tumor, suppresses abscopal tumors and inhibits tumor metastasis, leading to a prolonged survival rate. This work presents a novel strategy for improving cancer immunotherapy using multivalent nanobody-ferritin conjugates as immunological targeting and enhancing carriers.

Mutual promotion of co-condensation of KRAS G-quadruplex and a well-folded protein HMGB1.

Liquid-liquid phase separation (LLPS) of G-quadruplex (GQ) is involved in many crucial cellular processes, while the quadruplex-folding and their functions are typically modulated by specific DNA-binding proteins. However, the regulatory mechanism of binding proteins, particularly the well-folded proteins, on the LLPS of GQs is largely unknown. Here, we investigated the effect of HMGB1 on the condensation of a G-quadruplex of KRAS promoter (GQKRAS). The results show that these two rigid macro-biomolecules undergo co-condensation through a mutual promotion manner, while neither of them can form LLPS alone. Fluidity measurements confirm that the liquid-like droplets are highly dynamic. HMGB1 facilitates and stabilizes the quadruplex folding of GQKRAS, and this process enhances their co-condensation. The KRAS promoter DNA retains quadruplex folding in the droplets; interference with the GQ-folding disrupts the co-condensation of GQKRAS/HMGB1. Mechanistic studies reveal that electrostatic interaction is a key driving force of the interaction and co-condensation of GQKRAS/HMGB1; meanwhile, the recognition of two macro-biomolecules plays a crucial role in this process. This result indicates that the phase separation of GQs can be modulated by DNA binding proteins, and this process could also be an efficient way to recruit specific DNA binding proteins.

Peroxide-Simulating and GSH-Depleting Nanozyme for Enhanced Chemodynamic/Photodynamic Therapy via Induction of Multisource ROS.

Reactive oxygen species (ROS) generation, using photodynamic therapy (PDT) and chemodynamic therapy (CDT), is a promising strategy for cancer treatment. However, the production of ROS in tumor cells is often limited by hypoxia, insufficient substrates, and high level of ROS scavengers in a tumor microenvironment, which seriously affects the efficacy of ROS-related tumor therapies. Herein, we report a lipid-supported manganese oxide nanozyme, MLP@DHA&Ce6, by decorating a MnO2 nano-shell on the liposome loaded with dihydroartemisinin (DHA) and photosensitizer Ce6 for generating multisource ROS to enhance cancer therapy. MLP@DHA&Ce6 can be accumulated in tumors and can release active components, Mn2+ ions, and O2. The conjugate generates ROS via nanozyme-catalyzed CDT using DHA as a substrate, PDT through Ce6, and the Fenton reaction catalyzed by Mn2+ ions. The production of O2 from MnO2 enhanced Ce6-mediated PDT under near-infrared light irradiation. Meanwhile, MLP@DHA&Ce6 showed prominent glutathione depletion, which allowed ROS to retain high activity in tumor cells. In addition, the release of Mn2+ ions and DHA in tumor cells induced ferroptosis. This multisource ROS generation and ferroptosis effect of MLP@DHA&Ce6 led to enhanced therapeutic effects in vivo.

Cellular uptake of nickel by NikR is regulated by phase separation.

Bacterial cells were long thought to be "bags of enzymes" with minimal internal structures. In recent years, membrane-less organelles formed by liquid-liquid phase separation (LLPS) of proteins or nucleic acids have been found to be involved in many important biological processes, although most of them were studied on eukaryotic cells. Here, we report that NikR, a bacterial nickel-responsive regulatory protein, exhibits LLPS both in solution and inside cells. Analyses of cellular nickel uptake and cell growth of E. coli confirm that LLPS enhances the regulatory function of NikR, while disruption of LLPS in cells promotes the expression of nickel transporter (nik) genes, which are negatively regulated by NikR. Mechanistic study shows that Ni(II) ions induces the accumulation of nik promoter DNA into the condensates formed by NikR. This result suggests that the formation of membrane-less compartments can be a regulatory mechanism of metal transporter proteins in bacterial cells.

Cisplatin reacts with the RING finger domain of RNF11 and interferes with the protein functions.

Protein reactions play important roles in the mechanism of action of cisplatin. In this work, we found that cisplatin is highly reactive to the RING finger domain of RNF11, a key protein involved in tumorigenesis and metastasis. The results show that cisplatin binds to RNF11 at the zinc coordination site and leads to zinc ejection from the protein. The formation of S-Pt(II) coordination and Zn(II) ions release have been confirmed by UV-vis spectrometry using zinc dye and thiol agent, showing reducing the contents of thiol groups while forming S-Pt bonds and releasing zinc ions. Electrospray ionization-mass spectrometry measurement indicates that each RNF11 can bind up to three platinum atoms. Kinetical analysis shows a reasonable platination rate of RNF11 with t1/2 ∼ 3 h. CD, nuclear magnetic resonance, and gel electrophoresis measurements indicate that the cisplatin reaction causes protein unfolding and oligomerization of RNF11. Pull-down assay confirms that the platination of RNF11 interferes with the protein interaction of RNF11 with UBE2N, a key step of the functionalization of RNF11. Furthermore, Cu(I) was found to promote the platination of RNF11, which could lead to increased protein reactivity to cisplatin in tumor cells with high copper levels. These results indicate that the platination-induced zinc release of RNF11 disrupts the protein structure and interferes with its functions.

Metabolite profiling and identification in living cells by coupling stable isotope tracing and induced electrospray mass spectrometry.

Direct observation of metabolites in living cells by mass spectrometry offers a bright future for biological studies but also suffers a severe challenge to untargeted peak assignment to tentative metabolite candidates. In this study, we developed a method combining stable isotope tracing and induced electrospray mass spectrometry for living-cells metabolite measurement and identification. By using 13C6-glucose and ammonium chloride-15N as the sole carbon and nitrogen sources for cell culture, Escherichia coli synthesized metabolites with 15N and 13C elements. Tracing the number of carbon and nitrogen atoms could offer a complementary dimension for candidate peak searching. As a result, the identification confidence of metabolites achieved a universal improvement based on carbon/nitrogen labelling and filtration.

α-Synuclein as a Target for Metallo-Anti-Neurodegenerative Agents.

The unique thermodynamic and kinetic coordination chemistry of ruthenium allows it to modulate key adverse aggregation and membrane interactions of α-synuclein (α-syn) associated with Parkinson's disease. We show that the low-toxic RuIII complex trans-[ImH][RuCl4 (Me2 SO)(Im)] (NAMI-A) has dual inhibitory effects on both aggregation and membrane interactions of α-syn with submicromolar affinity, and disassembles pre-formed fibrils. NAMI-A abolishes the cytotoxicity of α-syn towards neuronal cells and mitigates neurodegeneration and motor impairments in a rat model of Parkinson's. Multinuclear NMR and MS analyses show that NAMI-A binds to residues involved in protein aggregation and membrane binding. NMR studies reveal the key steps in pro-drug activation and the effect of activated NAMI-A species on protein folding. Our findings provide a new basis for designing ruthenium complexes which could mitigate α-syn-induced Parkinson's pathology differently from organic agents.

Native Mass Spectrometry for Peptide-Metal Interaction in Picoliter Cell Lysate.

Native mass spectrometry, which takes a high concentration of ammonium acetate (NH4OAc) for ionization, coupled with tedious and solvent-consuming purification, which separates proteins from complicated environments, has shown great potential for proteins and their complexes. A high level of nonvolatile salts in the endogenous intracellular environment results in serious ion suppression and has been one of the bottlenecks for native mass spectrometry, especially for protein complexes. Herein, an integrated protocol utilizing the inner surface of a micropipette for rapid purification, desorption, and ionization of peptide-metal interaction at subfemtomole level in cell lysate was demonstrated for native mass spectrometry. The methods showed robust and reproducibility in protein measurement within 1 min from various buffers. The E. coli cells expressing with various proteins were lysed and used to test our method. The specific interaction between the peptide-metal complex in cell lysates could be reserved and distinguished by mass spectrometry.

Polymer Composite with Enhanced Thermal Conductivity and Insulation Properties through Aligned Al2O3 Fiber.

Thermoplastic polyolefins, such as polyethylene (PE), are traditionally one of the most widely used polymer classes with applications in the electric industry, and their nanocomposites have caught the interest of researchers. The linear filler is shown to be beneficial in decreasing the charge injection and hindering the formation of charge packs. So, we demonstrate a novel composite with excellent properties. The low-density polyethylene (LDPE) composite with aligned aluminum oxide (Al2O3) fiber has been prepared in electric field conditions. The direction of the Al2O3 fiber was parallel to the thickness direction of the LDPE composite. The breakdown strength of the Al2O3/LDPE composite with 0.2% aligned Al2O3 fiber was 498 kV/mm, which is higher than other fillers induced. The aligned Al2O3 fiber has effect on preventing accumulation of space charge and reducing the amount of free electron in the material. In addition, the thermal conductivity of the LDPE composite (0.22 W/m·K) was increased to 0.85 W/m·K when doped with 0.5 wt% aligned Al2O3 fiber. The present structure provides a new possibility for mass new nanocomposites with excellent microstructures and remarkable functionality.

Differentiation of renal cell carcinoma subtypes through MRI-based radiomics analysis.

OBJECTIVES: To explore whether clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC), and chromophobe renal cell carcinoma (cRCC) can be distinguished using radiomics features extracted from magnetic resonance (MR) images. METHODS: Seventy-seven patients (ccRCC = 32, pRCC = 23, cRCC = 22) underwent MRI before surgery between May 2013 and August 2018 in this retrospective study. Thirty-nine radiomics features were extracted from tumor volumes on three sequences (T2WI, EN-T1WI CMP, and EN-T1WI NP). The Kruskal-Wallis test with Bonferonni correction and variance threshold were used for feature selection among the three RCC subtypes. ROC curves for the three subtypes were generated based on radiomics features. AUC, accuracy, sensitivity, and specificity for subtype differentiation are reported. Linear discriminant analysis (LDA) was used to assess the discriminative ability of these radiomics features. RESULTS: Significant radiomics features among the three subtypes were identified, and ROC curves achieved excellent AUCs for T2WI, EN-T1WI CMP, EN-T1WI NP, and combined three MR sequences (0.631, 0.790, 0.959, and 0.959 between ccRCC and cRCC; 0.688, 0.854, 0.909, and 0.955 between pRCC and cRCC; 0.747, 0.810, 0.814, and 0.890 between ccRCC and pRCC). In addition, LDA demonstrated the three RCC subtypes were correctly classified by radiomics analysis (66.2% for EN-T1WI CMP, 71.4% for EN-T1WI NP, 55.8% for T2WI, and 71.4% for the combined three MR sequences). CONCLUSIONS: Radiomics analysis can be used to differentiate among ccRCC, pRCC, and cRCC based on radiomics features extracted from multiple-sequence MRI and may help diagnose and treat RCC patients in the future, while further study is still needed. KEY POINTS: • Radiomics features on multiple-sequence MRI can help differentiate the three subtypes of renal cell carcinoma (clear cell, papillary renal cell, and chromophobe renal cell carcinoma). • Radiomics features based on MRI indicate greater textural heterogeneity on ccRCCs than pRCCs and cRCCs (the highest AUCs on EN-T1WI NP are 0.814 for ccRCCs vs pRCCs and 0.959 for ccRCCs vs cRCCs, respectively). • There is a significant difference in the textural heterogeneity of radiomics features between pRCCs and cRCCs (the AUC is 0.909, 0.854, and 0.688 on EN-T1WI NP, EN-T1WI CMP, and T2WI, respectively).

Cisplatin binds to the MDM2 RING finger domain and inhibits the ubiquitination activity.

Cisplatin can directly bind to the RING finger domain of MDM2, leading to the zinc-release and protein unfolding. Consequently, cisplatin inhibits the MDM2-mediated ubiquitination, which is the molecular basis of p53 activation. This work provides insight into the cisplatin-induced p53-elevation that is involved in cell apoptosis.

Covalent versus Noncovalent Binding of Ruthenium η6 -p-Cymene Complexes to Zinc-Finger Protein NCp7.

Ruthenium-arene complexes are a unique class of organometallic compounds that have been shown to have prominent therapeutic potencies. Here, we have investigated the interactions of Ru-cymene complexes with a zinc-finger protein NCp7, aiming to understand the effects of various ligands on the reaction. Five different binding modes were observed on selected Ru-complexes. Ru-cymene complex can bind to proteins through either noncovalent binding alone or through a combination of covalent and noncovalent binding modes. Moreover, the noncovalent interaction can promote the coordination of RuII to NCp7, resulting synergistic effects of the different ligands. The binding of Ru(Cym) complexes leads to dysfunction of NCp7 through zinc-ejection and structural perturbation. These results indicate that the reactivity of Ru-complexes can be modulated by ligands through different approaches, which could be closely correlated to their different therapeutic effects.

Computed tomographic characteristics of gastric schwannoma.

OBJECTIVE: This study aimed to characterize the computed tomographic (CT) features of gastric schwannoma (GS). METHODS: We retrospectively reviewed CT images of 19 cases of histologically proven GS between January 2010 and December 2015. Tumor location, size, contour, margin, growth pattern, and degree and pattern of enhancement, perigastric lymph nodes, ulceration, necrosis, and calcification were evaluated. RESULTS: GS was located in the gastric body (73.7%), gastric antrum (15.8%), and gastric fundus (10.5%), with a mean maximum diameter of 4.5 ± 1.8 cm. All tumors presented as oval, well-defined solid masses, with exophytic (36.8%), endoluminal (15.8%), or mixed (47.4%) growth patterns. Ulcers (57.9%) and perigastric lymph nodes (47.4%) were observed. Moderate enhancement (87.5%) was observed in the portal phase. Eighteen (94.7%) cases showed homogeneous enhancement. CONCLUSIONS: GS typically presents as a mass in the stomach with an exophytic or mixed growth pattern, moderate homogeneous enhancement, and is prone to be accompanied by perigastric lymph node inflammatory reactive swelling. Larger GSs are more likely to be associated with ulcers.

Charge-Selective Delivery of Proteins Using Mesoporous Silica Nanoparticles Fused with Lipid Bilayers.

Efficient and safe intracellular delivery of proteins is highly desired in the development of protein therapeutics. Current methods of protein delivery commonly suffer from low loading efficiency, low stability in serum, and lack of versatility for different proteins. Here, we developed a platform for efficient protein delivery using mesoporous silica nanoparticles (MSN) with lipid fusion. By different surface modifications on MSN, the positively charged MSN (MSN+) and the negatively charged MSN (MSN-), were generated for loading different proteins. The cargo proteins, based on the surface charges, can be selectively loaded in very high efficiency. The protein-loaded MSNs were fused with liposomes to form a protocell-like delivery system (MSN-LP) in order to prevent burst release of proteins. The lipid fusion significantly increases the stability of the nanosystem in physiological conditions, and the MSN-LP protocell can efficiently deliver proteins into cells. The cargo proteins can be released in cells in a sustained manner. Fifteen different proteins, including two protein complexes, were tested using this delivery system. Further analyses indicate that the proteins can maintain their functions after delivery into cells. Fluorescent proteins, GFP, and KillerRed show fluorescence in cells, indicating the correct folding of proteins during encapsulation and delivery. Protein activity analysis shows that KillerRed protein can generate ROS in cells, while SOD can eliminate ROS in cells. Hence, the proteins delivered by this system remain their structure and function in cells. This work provides a versatile strategy for charge-selective delivery of proteins with high loading efficiency and high stability.

A dual functional ruthenium arene complex induces differentiation and apoptosis of acute promyelocytic leukemia cells.

Human acute promyelocytic leukemia (APL) is the most malignant form of acute leukemia. The fusion of PML and RARα genes is responsible for over 98% of cases of APL. In this work, we found that a Ru(ii) arene complex, [(η6-p-bip)Ru(en)Cl][PF6] (Ru-1), can selectively react with PML, leading to zinc-release and protein unfolding. Consequently, the degradation of the fusion protein PML-RARα occurs, which causes the differentiation of APL cells. In addition, Ru-1 can also bind to DNA and trigger apoptosis of APL cells. Therefore, Ru-1 acts as a dual functional agent that inhibits the growth of APL cells and induces cell differentiation. In contrast, the other non-selective Ru(ii) compound, though also highly reactive to PML, does not exhibit anti-APL activity. The selectivity of Ru-1 to PML suggests a new strategy for the development of anti-APL drugs using ruthenium agents.

Selective Targeting of the Zinc Finger Domain of HIV Nucleocapsid Protein NCp7 with Ruthenium Complexes.

Nucleocapsid protein 7 (NCp7) is an attractive target for anti-HIV drug development. Here we found that ruthenium complexes are reactive to NCp7 and various Ru-agents exhibit significantly different reactivity. Interestingly, the zinc-finger domains of NCp7 also demonstrate different affinity to Ru-complexes; the C-terminal domain is much more reactive than the N-terminal domain. Each zinc-finger domain of NCp7 binds up to three Ru-motifs, and the ruthenium binding causes zinc-ejection from NCp7 and disrupts the protein folding. Therefore, ruthenium complexes interfere with the DNA binding of NCp7 and interrupt the protein function. The different reactivity of Ru-agents suggests a feasible strategy for improving the targeting of NCp7 by ligand design. This work provides an insight into the mechanism of ruthenium complex with NCp7, and suggests more potential application of ruthenium drugs.

TSA1 interacts with CSN1/CSN and may be functionally involved in Arabidopsis seedling development in darkness.

The COP9 signalosome (CSN) is a multiprotein complex which participates in diverse cellular and developmental processes. CSN1, one of the subunits of CSN, is essential for assembly of the multiprotein complex via PCI (proteasome, COP9 signalosome and initiation factor 3) domain in the C-terminal half of CSN1. However, the role of the N-terminal domain (NTD) of CSN1, which is critical for the function of CSN, is not completely understood. Using a yeast two-hybrid (Y2H) screen, we found that the NTD of CSN1 interacts with TSK-associating protein 1 (TSA1), a reported Ca(2+)-binding protein. The interaction between CSN1 and TSA1 was confirmed by co-immunoprecipitation in Arabidopsis. tsa1 mutants exhibited a short hypocotyl phenotype in darkness but were similar to wild-type Arabidopsis under white light, which suggested that TSA1 might regulate Arabidopsis hypocotyl development in the dark. Furthermore, the expression of TSA1 was significantly lower in a csn1 null mutant (fus6), while CSN1 expression did not change in a tsa1 mutant with weak TSA1 expression. Together, these findings suggest a functional relationship between TSA1 and CSN1 in seedling development.

Endogenous protein mono-ADP-ribosylation in Arabidopsis thaliana.

Protein mono-ADP-ribosylation post-translationally transfers the ADP-ribose moiety from the ß-NAD+ donor to various protein acceptors. This type of modification has been widely characterized and shown to regulate protein activities in animals, yeast and prokaryotes, but has never been reported in plants. In this study, using [³²P]NAD+ as the substrate, ADP-ribosylated proteins in Arabidopsis were investigated. One protein substrate of 32 kDa in adult rosette leaves was found to be radiolabeled. Heat treatment, protease sensitivity and nucleotide derivative competition assays suggested a covalent reaction of NAD+ with the 32 kDa protein. [carbonyl-¹4C]NAD+ could not label the 32 kDa protein, confirming that the modification was ADP-ribosylation. Poly (ADP-ribose) polymerase inhibitor failed to suppress the reaction, but chemicals that destroy mono-ADP-ribosylation on specific amino acid residues could break up the linkage, suggesting that the reaction was not a poly-ADP-ribosylation but rather a mono-ADP-ribosylation. This modification mainly existed in leaves and was enhanced by oxidative stresses. In young seedlings, two more protein substrates with the size of 45 kDa and over 130 kDa, respectively, were observed in addition to the 32 kDa protein, indicating that different proteins were modified at different developmental stages. Although the substrate proteins remain to be identified, this is the first report on the characterization of endogenously mono-ADP-ribosylated proteins in plants.

MULTIPOLAR SPINDLE 1 (MPS1), a novel coiled-coil protein of Arabidopsis thaliana, is required for meiotic spindle organization.

The spindle is essential for chromosome segregation during meiosis, but the molecular mechanism of meiotic spindle organization in higher plants is still not well understood. Here, we report on the identification and characterization of a plant-specific protein, MULTIPOLAR SPINDLE 1 (MPS1), which is involved in spindle organization in meiocytes of Arabidopsis thaliana. The homozygous mps1 mutant exhibits male and female sterility. Light microscopy showed that mps1 mutants produced multiple uneven spores during anther development, most of which aborted in later stages. Cytological analysis showed that chromosome segregation was abnormal in mps1 meiocytes. Immunolocalization showed unequal bipolar or multipolar spindles in mps1 meiocytes, which indicated that aberrant spindles resulted in disordered chromosome segregation. MPS1 encodes a 377-amino-acid protein with putative coiled-coil motifs. In situ hybridization analysis showed that MPS1 is strongly expressed in meiocytes.

Regulation of COP1 nuclear localization by the COP9 signalosome via direct interaction with CSN1.

COP1 and COP9 signalosome (CSN) are key regulators of plant light responses and development. Deficiency in either COP1 or CSN causes a constitutive photomorphogenic phenotype. Through coordinated actions of nuclear- and cytoplasmic-localization signals, COP1 can respond to light signals by differentially partitions between nuclear and cytoplasmic compartments. Previous genetic analysis in Arabidopsis indicated that the nuclear localization of COP1 requires CSN, an eight-subunit heteromeric complex. However the mechanism underlying the functional relationship between COP1 and CSN is unknown. We report here that COP1 weakly associates with CSN in vivo. Furthermore, we report on the direct interaction involving the coiled-coil domain of COP1 and the N-terminal domain of the CSN1 subunit. In onion epidermal cells, expression of CSN1 can stimulate nuclear localization of GUS-COP1, and the N-terminal domain of CSN1 is necessary and sufficient for this function. Moreover, CSN1-induced COP1 nuclear localization requires the nuclear-localization sequences of COP1, as well as its coiled-coil domain, which contains both the cytoplasmic localization sequences and the CSN1 interacting domain. We also provide genetic evidence that the CSN1 N-terminal domain is specifically required for COP1 nuclear localization in Arabidopsis hypocotyl cells. This study advances our understanding of COP1 localization, and the molecular interactions between COP1 and CSN.