Bridging Molecules Research Articles

The role of secreted proteins in efferocytosis.

The clearance of apoptotic cells known as efferocytosis is the final stage of apoptosis, and includes the recognition, phagocytosis, and degradation of apoptotic cells. The maintenance of tissue homeostasis requires the daily elimination of billions of apoptotic cells from the human body via the process of efferocytosis. Accordingly, aberrations in efferocytosis underlie a growing list of diseases, including atherosclerosis, cancer, and infections. During the initial phase of apoptosis, "Eat-Me" signals are exposed and recognized by phagocytes either directly through phagocyte receptors or indirectly through secreted proteins that function as bridge molecules that cross-link dying cells to phagocytes. Here, we set out to provide a comprehensive review of the molecular mechanisms and biological significance of secreted proteins in apoptotic cell clearance. Specifically, it focuses on how these secreted proteins act as bridging molecules to facilitate the clearance process.

Ladder-like heteropolynuclear assemblies via cyanido bridges and platinum(II)-thallium(I) bonds: structural and photophysical properties.

We describe the mononuclear anionic cyanido-pentafluorophenyl complexes, (NBu4)[Pt(C^N)(C6F5)(CN)] [C^N = 7,8-benzoquinolate (bzq) 1, 2-(2,4-difluorophenyl)pyridinate (dfppy) 2] and the heteropolynuclear derivatives [{Pt(C^N)(C6F5)(CN)}Tl] (C^N = bzq 3, dfppy 4). These complexes were synthesized via a two-step modular synthesis by reaction of the corresponding potassium salts K[Pt(C^N)(C6F5)(CN)], prepared in situ from [Pt(C^N)(C6F5)(DMSO)] and KCN in acetone/H2O, with TlPF6. The structures of {[Pt(bzq)(C6F5)(CN)Tl]·THF}n (3·THF)n and [{Pt(dfppy)(C6F5)(CN)}Tl]4·dioxane [4]4·dioxane, determined by X-ray crystallography, confirm the presence of Pt(II)-Tl(I) bonds [2.9795(6)-3.0736(3) Å], but in the dfppy complex, the incorporation of dioxane, causes a significant structural change. Thus, whereas [3·THF]n achieves a bent-ladder shape extended double chain Tl⋯[Pt⋯Tl]n⋯Pt supported by lateral bridging [Pt](μ-CN)[Tl] ligands, [4]4·dioxane is formed by discrete Pt4Tl4 rectangular aggregates stabilized by [Pt](μ-CN)[Tl] and Pt⋯Tl bonds, which are connected by dioxane bridging molecules through Tl⋯O(dioxane) additional contacts. Solid state emissions are redshifted compared with the mononuclear derivatives 1 and 2 and have been assigned, with the support of theoretical calculations on Pt4Tl4 models, to metal-metal'-to-ligand charge transfer (3MM'LCT [d/s σ*(Pt, Tl) → π*(C^N)]) for 3 and mixed 3MM'LCT/3IL for 4. In fluid THF solution, the complexes are not emissive. At 77 K, 3 and 4 exhibit bright emissions attributed to the formation of bimetallic [{Pt(C^N)(C6F5)(CN)}Tl(THF)x], and anionic [Pt(C^N)(C6F5)(CN)]- fragments. Furthermore, both 3 and 4 exhibit a reversible mechanochromism with a red shift of the emissions upon crushing, suggesting some degree of shortening of metal-metal separation. Finally, complex 3 shows solvatochromic behavior with color/luminescence changes by treatment with a drop of MeOH, CH2Cl2, THF or Et2O, with shifts from 583 in 3-MeOH to 639 nm in 3-THF. However, 4 only demonstrates a bathochromic response to MeOH.

Mesoporous Poly(3,4-ethylenedioxythiophene):Poly(styrenesulfonate) as Efficient Iodine Host for High-Performance Zinc-Iodine Batteries.

Here, by introducing polystyrenesulfonate (PSS) as a multifunctional bridging molecule to synchronously coordinate the interaction between the precursor and the structure-directing agent, we developed a mesoporous conductive polymer of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) featuring adjustable size in the range of 105-1836 nm, open nanochannels, large specific surface area (105.5 m2 g-1), and high electrical conductivity (172.9 S cm-1). Moreover, a large-area ultrathin PEDOT:PSS thin film with well-defined mesopores can also be obtained by controllable growth on various functional interfaces. As an example, we demonstrated that the iodine-loaded mesoporous PEDOT:PSS nanospheres can serve as a promising cathode for aqueous zinc-iodine batteries with high specific capacity (241 mAh g-1), excellent rate performance, and superlong 20,000 cycle life. In-depth theoretical calculations and systematic experimental results together reveal that the exposed sulfur- and oxygen-containing functional groups hold strong interactions with iodine species, resulting in effectively anchoring iodine species and inhibiting the shuttling of polyiodide intermediates, thus ensuring the long-term stability of the batteries. This work introduces a member to the family of mesoporous materials as well as porous polymers with versatile applications.

Abstract B045: The effect of metastasis-associated in colon cancer 1 (MACC1) on therapy-induced senescence and its impact on post-senescence-driven metastasis

Abstract Senescence is considered a safeguard program against cancer progression. However, recent studies revealed that these cells, reprogrammed in a senescence-associated fashion to express stemness transcripts, may occasionally re-enter the cell cycle, thereby executing their increased tumorigenicity. Although the effects of senescence, its associated secretome (SASP) and release from senescence on tumor progression have been elucidated before, the role of post-senescent cells in metastasis remains elusive. To enlighten the relationship between senescence, stemness, and metastasis, we employed a key bridging molecule between metastasis and cancer stemness, metastasis-associated in colon cancer 1 (MACC1). Numerous research groups have acquainted its importance as a metastasis inducer, prognostic, and predictive biomarker for more than 20 different tumor entities, including breast and colorectal cancer (CRC). We initially used CRC cell lines with genetically engineered MACC1 expression for our study. Senescence was induced with different chemotherapeutic agents such as 5-FU, mafosfamid (a cyclophosphamide analogue active in vitro), and doxorubicin. Cells with high MACC1 expression were more prone to senesce after treatment. These findings were further validated in CRC patient- and mouse-derived organoids. Across these models, we found MACC1 expression to increase therapy-induced senescence (TIS). Moreover, senescence induction was accompanied by increased MACC1 expression in conjunction with elevated expression of cancer stemness genes like CD44 and LGR5; this increase was more prominently detectable in MACC1 high-expressing cells. Furthermore, senescent cells were forced to exit from senescence through genetic manipulations of independent senescence-essential factors such as overexpression of JMJD2C, which demethylates the senescence-essential H3K9me3 histone mark. Post-senescent cells showed enhanced migration, colony formation, wound healing, proliferation, and increased cancer stemness-related gene expression, especially when compared to never-senescent cells in which we overexpressed JMJD2C prior to chemo-exposure. Our studies were complemented by in vivo work, which showed that metastasis-incapable CRC cells formed metastasis in the liver and lung after their release from senescence. We further used MACC1 inhibitors such as atorvastatin and fluvastatin to interfere with MACC1-induced TIS. The co-treatment of a MACC1 inhibitor with conventional chemotherapeutics reduced senescence entry in cells with high MACC1 expression. These data, consistent with our findings in primary patient material, uncover a hitherto unknown relationship between MACC1 and TIS. Furthermore, senescence release increases the tumorgenicity of the cells, which is more prominent in the cells with high MACC1 expression. In particular, we were able to reduce MACC1-induced senescence by using MACC1 inhibitors, aiming to reduce the senescence release-induced metastasis. Citation Format: Belma Nazli Güllü, Dorothy N. Y. Fan, Dennis Kobelt, Margarita Mokrizkij, Janice Smith, Clemens A. Schmitt, Ulrike Stein. The effect of metastasis-associated in colon cancer 1 (MACC1) on therapy-induced senescence and its impact on post-senescence-driven metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B045.

Invasion-Associated Reorganization of Laminin 332 in Oral Squamous Cell Carcinomas: The Role of the Laminin γ2 Chain in Tumor Biology, Diagnosis, and Therapy.

Simple SummaryThe destructive growth of carcinomas is associated with crossing the border between the epithelial and the connective tissue parts of an organ. One component of this borderline, the basement membrane, is the heterotrimeric laminin 332, which mediates the adhesion of basal epithelial cells. This protein, in particular its gamma 2 chain, is fundamentally reorganized during tumor cell invasion. Specific deposition patterns of laminin 332 are also present in oral squamous cell carcinomas and have been shown to be of high diagnostic and predictive value. Furthermore, laminin 332 restructuring is associated with important tumor biological processes, e.g., stromal activation, the development of a motile phenotype, and tumor spreading. In this review, current knowledge in the field is summarized and the recommendation to consider laminin 332 as a promising grading and monitoring parameter and as a potential therapeutic target is discussed.Invasion of the connective tissue by carcinoma cells is accompanied by disintegration and reorganization of the hemidesmosomes, which connect the basement membrane to the basal epithelial cells. In terms of mediating the basement membrane, i.e., basal cell interactions, the heterotrimeric laminin 332 is the most important bridging molecule. Due to this distinct function, laminin 332, especially its gamma 2 chain, came into the focus of cancer research. Specific de novo synthesis and deposition patterns of laminin 332 are evident upon development and progression of oral squamous cell carcinomas (OSCCs). Loss from the basement membrane, cytoplasmic accumulation, and extracellular deposition are associated with crucial processes such as stromal activation and immune response, epithelial to mesenchymal transition, and tumor cell budding. In networks with components of the tumor microenvironment, altered expression of laminin 332 chains, proteolytic processing, and interaction with integrin receptors seem to promote cancer cell migration. Indeed, reorganization patterns are shown to have a high diagnostic and prognostic value. Here, we summarize the current knowledge on laminin 332 reorganization in OSCCs with special focus on its gamma 2 chain and provide, based on the current literature, evidence on its promising role as a grading and monitoring parameter and as a potential therapeutic target.

Mechanistic study on DNA mutation of the cytosine methylation reaction at C5 position

DNA methylation is a crucial epigenetic mark connected to conventionally changing the DNA bases, typically by adding methyl groups into DNA bases. Methylation of cytosine at the C5 position (5-methylcytosine) occurs mostly in the context of cytosine-phosphate-guanine dinucleotides, the methylation of which has important impacts on gene regulation and expression. However, the mechanistic details of this reaction are still debatable concerning the concertedness of the key reaction steps and the roles played by the base that abstracts the proton in the β-elimination and water molecules at the active site. To gain a deeper insight into the formation of 5-mehtylcytosine, an extensive density functional theory (DFT) study was performed with the B3LYP functional in conjunction with different basis sets. Our study has clearly established the mechanistic details of this methylation approach, based on which the roles of conserved active site residues, such as glutamic acid and waters, are well understood. Our results show that the reaction of 5-methylcytosine follows a concerted mechanism in which water molecules are critically involved. Moreover, arginine and alanine give more significant catalytic effects than glutamic acid on the 5-methylcytosine process. Considering the effect of Alanine, Arginine, and one water bridging molecule, the activation energy is 31 kJ mol −1 calculated at B3LYP/6-31G(d) level of theory.

Serum bridging molecules drive candidal invasion of human but not mouse endothelial cells.

During hematogenously disseminated candidiasis, blood borne fungi must invade the endothelial cells that line the blood vessels to infect the deep tissues. Although Candida albicans, which forms hyphae, readily invades endothelial cells, other medically important species of Candida are poorly invasive in standard in vitro assays and have low virulence in immunocompetent mouse models of disseminated infection. Here, we show that Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei can bind to vitronectin and high molecular weight kininogen present in human serum. Acting as bridging molecules, vitronectin and kininogen bind to αv integrins and the globular C1q receptor (gC1qR), inducing human endothelial cells to endocytose the fungus. This mechanism of endothelial cell invasion is poorly supported by mouse endothelial cells but can be restored when mouse endothelial cells are engineered to express human gC1qR or αv integrin. Overall, these data indicate that bridging molecule-mediated endocytosis is a common pathogenic strategy used by many medically important Candida spp. to invade human vascular endothelial cells.

Cover Picture: [FeFe]‐Hydrogenase: Defined Lysate‐Free Maturation Reveals a Key Role for Lipoyl‐H‐Protein in DTMA Ligand Biosynthesis (Angew. Chem. Int. Ed. 22/2022)

The generation of an active [FeFe]-hydrogenase , the most efficient enzyme for H2 production, requires the synthesis of a complex metal center, the H-cluster, which is responsible for the reversible reduction of protons to H2. In their Communication (e202203413), Joan B. Broderick and co-workers show that the last step of this synthesis, which consists of the assembly of the azadithiolate bridging molecule, involves the aminomethyl-lipoyl-H-protein from the glycine cleavage system.

The Research Progress in Physiological and Pathological Functions of TRAF4.

Tumour necrosis factor receptor-associated factor 4 (TRAF4) is a member of the TRAF protein family, a cytoplasmic bridging molecule closely associated with various immune functions. The physiological processes of TRAF4 are mainly involved in embryonic development, cell polarity, cell proliferation, apoptosis, regulation of reactive oxygen species production. TRAF4 is overexpressed in a variety of tumors and regulates the formation and development of a variety of tumors. In this review, we summarize the physiological and pathological regulatory functions of TRAF4 and focus on understanding the biological processes involved in this gene, to provide a reference for further studies on the role of this gene in tumorigenesis and development.

Efficient Human Acute Myeloid Leukemia Targeting By Universal Chimeric Antigen Receptor T-Cells Via Combinatorial Use of Linkers

Chimeric antigen receptor (CAR) T-cells are genetically engineered T-cells with potent biocidal activity against respective target-expressing cells. Recently, CAR T-cells have been successfully used clinically to eradicate B cell-derived malignancies by targeting B-cell lineage specific surface antigens (e.g. CD19, BCMA). However, several limitations of current single-antigen targeting CAR T-cell therapies are becoming evident: a) low target tumor-antigen expression might lead to low CAR T-cell targeting efficacy; b) single antigen-targeting might lead to rapid selection of tumor cells with low or loss of antigen expression; c) single antigen-targeting does frequently not generate a high tumor-selectivity as tumor-antigens are frequently also expressed on healthy tissues; d) production of single-antigen CAR T-cells is time- and resource-consuming but results in effectors that target only one antigen; e) on-target off-tumor as well as off-target side-effects are difficult to control without terminally eliminating CAR T-cells in the recipient. While a-c) might be overcome by combinatorial tumor-antigen targeting, d-e) might be addressed by production of Universal CAR T-cells that recognize a specific tag on selective bridging molecules with short half-life.To address some of these limitations in principle, we have here developed universal CAR T-cells targeting fluorescein, as well as fluorescein-labeled antibody-constructs directed against several cell surface antigens, that would serve as versatile, combinatorial selective linkers and, upon target binding, also CAR T-cells activators. We then tested the system on human acute myeloid leukemia (AML) cell lines and primary patient AML blasts. Specifically, we engineered CAR T-cells, termed FluA-CAR T-cells, to display the anti-fluorescein engineered lipocalin FluA, which mediated recognition of randomly or site-specifically fluorescein-labeled antibodies in IgG or short half-life diabody (Db) format, directed against the frequently AML expressed antigens CD33, CD117 and CD371. Site-specific chemical modification methods and cysteine-tagged Db mediated the strongest AML killing results in vitro over a broad range of antibody concentrations.We then hypothesized that FluA-CAR T-cells, targeting AML cells via combinatorial use of linker molecules adapted to specific AML antigen-expression profiles, would allow to most efficiently eliminate AML cells in a dose- and timely-regulated fashion. To this end, we tested single and combinatorial use of fluoresceinated anti-CD117 Db and anti-CD371 Db on MOLM13 AML cell lines, engineered to be either CD117 highCD371 neg, CD117 negCD371 high, or CD117 highCD371 high. Indeed, combination of anti-CD117 and anti-CD371 Db-linkers, resulted in significantly improved MOLM13 cell lysis compared to equimolar concentrations of single agents in vitro (example figure). We then tested the same approach, targeting CD117 +CD371 + primary AML cells from two different patients, using either patient-derived or allogeneic FluA CAR T-cells. Again, the combinatorial use of linkers generated a significantly higher AML cell lysis than the use of single Db linkers.We thus here provide proof-of-concept for the generation of highly potent universal targeting FluA CAR T-cells from healthy donors and AML patients. By choosing suitable CAR-adaptors with respect to their conjugation chemistry and size, it is possible to tightly regulate CAR-T cell activity against CD33, CD117 and CD371 expressing AML cells and likely any tumor cell expressed antigen. Short half-life small molecule linkers will allow to control FluA CAR T-cell on-off activity and combinatorial use of linkers will allow to maximize anti-tumor activity and to minimize on-target off-tumor toxicity. [Display omitted] DisclosuresMyburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.

Variations of structures on changing the ratios of metal ions in rare Ca(II)–Zn(II) hetero-metallic self-assembled coordination polymers of hexamethylenetetramine and benzoate

Two rare hetero-metallic calcium(II)-zinc(II) complexes [CaZn4(OBz)10(μ2-hmt)2]n (1) and [Ca2Zn4(OBz)12(μ2-hmt)2]n (2) have been synthesized using basic zinc carbonate, benzoic acid (HOBz), hydrated calcium chloride and hexamethylenetetramine (hmt) by varying the molar ratio of the reactants. Both the complexes have been analyzed by elemental analysis, IR spectroscopy and X-ray crystallography. The complex 1 is a 1D polymer which contains one calcium ion and four zinc atoms in the asymmetric unit together with ten benzoates and two hmts. The polymer has been constructed by the alternate joining of paddle-wheel Zn2(OBz)4 units and Zn2Ca trinuclear species by μ2-hmt bridging molecules connecting Zn2+ ions. Zinc atoms have five coordinate square pyramidal geometries and four coordinate tetrahedral geometries in Zn2(OBz)4 and Zn2Ca moieties, respectively, whereas calcium atoms have six-coordinate distorted octahedral geometry. Complex 2 is also a 1D polymer but unlike complex 1, it contains four independent zinc and two independent calcium atoms in the asymmetric unit together with twelve benzoates and two hmts. By contrast, the polymeric structure of complex 2 has been formed by the connection of Zn2Ca trinuclear species via μ2 hmt bridging molecules at Zn centers. Complex 2 is also a 1D polymer but unlike complex 1, it contains four independent zinc and three independent calcium atoms in the asymmetric unit together with twelve benzoates and two hmts. All four zinc atoms are four coordinate with tetrahedral environments and the calcium atoms are six coordinated (two are located on a center of symmetry) exhibiting a distorted octahedral geometry.

MFG-E8 regulated by miR-99b-5p protects against osteoarthritis by targeting chondrocyte senescence and macrophage reprogramming via the NF-\u03baB pathway

Milk fat globule-epidermal growth factor (EGF) factor 8 (MFG-E8), as a necessary bridging molecule between apoptotic cells and phagocytic cells, has been widely studied in various organs and diseases, while the effect of MFG-E8 in osteoarthritis (OA) remains unclear. Here, we identified MFG-E8 as a key factor mediating chondrocyte senescence and macrophage polarization and revealed its role in the pathology of OA. We found that MFG-E8 expression was downregulated both locally and systemically as OA advanced in patients with OA and in mice after destabilization of the medial meniscus surgery (DMM) to induce OA. MFG-E8 loss caused striking progressive articular cartilage damage, synovial hyperplasia, and massive osteophyte formation in OA mice, which was relieved by intra-articular administration of recombinant mouse MFG-E8 (rmMFG-E8). Moreover, MFG-E8 restored chondrocyte homeostasis, deferred chondrocyte senescence and reprogrammed macrophages to the M2 subtype to alleviate OA. Further studies showed that MFG-E8 was inhibited by miR-99b-5p, expression of which was significantly upregulated in OA cartilage, leading to exacerbation of experimental OA partially through activation of NF-κB signaling in chondrocytes. Our findings established an essential role of MFG-E8 in chondrocyte senescence and macrophage reprogramming during OA, and identified intra-articular injection of MFG-E8 as a potential therapeutic target for OA prevention and treatment.

FOXA3 induction under endoplasmic reticulum stress contributes to non-alcoholic fatty liver disease

Chronic endoplasmic reticulum (ER) stress in the liver has been shown to play a causative role in non-alcoholic fatty liver disease (NAFLD) progression, yet the underlying molecular mechanisms remain to be elucidated. Forkhead box A3 (FOXA3), a member of the FOX family, plays critical roles in metabolic homeostasis, although its possible functions in ER stress and fatty liver progression are unknown. Adenoviral delivery, siRNA delivery, and genetic knockout mice were used to crease FOXA3 gain- or loss-of-function models. Tunicamycin (TM) and a high-fat diet (HFD) were used to induce acute or chronic ER stress in mice. Chromatin immunoprecipiation (ChIP)-seq, luciferase assay, and adenoviral-mediated downstream gene manipulations were performed to reveal the transcriptional axis involved. Key axis protein levels in livers from healthy donors and patients with NAFLD were assessed via immunohistochemical staining. FOXA3 transcription is specifically induced by XBP1s upon ER stress. FOXA3 exacerbates the excessive lipid accumulation caused by the acute ER-inducer TM, whereas FOXA3 deficiency in hepatocytes and mice alleviates it. Importantly, FOXA3 deficiency in mice reduced diet-induced chronic ER stress, fatty liver, and insulin resistance. In addition, FOXA3 suppression via siRNA or adeno-associated virus delivery ameliorated the fatty liver phenotype in HFD-fed and db/db mice. Mechanistically, ChIP-Seq analysis revealed that FOXA3 directly regulates Period1 (Per1) transcription, which in turn promotes the expression of lipogenic genes, including Srebp1c, thus enhancing lipid synthesis. Of pathophysiological significance, FOXA3, PER1, and SREBP1c levels were increased in livers of obese mice and patients with NAFLD. The present study identified FOXA3 as the bridging molecule that links ER stress and NAFLD progression. Our results highlighted the role of the XBP1s-FOXA3-PER1/Srebp1c transcriptional axis in the development of NAFLD and identified FOXA3 as a potential therapeutic target for fatty liver disease. The molecular mechanisms linking endoplasmic reticulum stress to non-alcoholic fatty liver disease (NAFLD) progression remain undefined. Herein, via invitro and invivo analysis, we identified Forkhead box A3 (FOXA3) as a key bridging molecule. Of pathophysiological significance, FOXA3 protein levels were increased in livers of obese mice and patients with NAFLD, indicating that FOXA3 could be a potential therapeutic target in fatty liver disease.

Unfolding of the chromatin fiber driven by overexpression of noninteracting bridging factors

Nuclear molecules control the functional properties of the chromatin fiber by shaping its morphological properties. The biophysical mechanisms controlling how bridging molecules compactify chromatin are a matter of debate. On the one side, bridging molecules could cross-link faraway sites and fold the fiber through the formation of loops. Interacting bridging molecules could also mediate long-range attractions by first tagging different locations of the fiber and then undergoing microphase separation. Using a coarse-grained model and Monte Carlo simulations, we study the conditions leading to compact configurations both for interacting and noninteracting bridging molecules. In the second case, we report on an unfolding transition at high densities of the bridging molecules. We clarify how this transition, which disappears for interacting bridging molecules, is universal and controlled by entropic terms. In general, chains are more compact in the case of interacting bridging molecules because interactions are not valence limited. However, this result is conditional on the ability of our simulation methodology to relax the system toward its ground state. In particular, we clarify how, unless using reaction dynamics that change the length of a loop in a single step, the system is prone to remain trapped in metastable, compact configurations featuring long loops.

Ultra-thin metal-organic framework nanosheets for chemo-photodynamic synergistic therapy.

Synergistic therapies, such as chemo-photodynamic therapy, have been growing fast because of their efficacy against cancers. Although metal-organic frameworks have been widely studied in the field of drug delivery, the metal-organic frameworks (MOFs) with a two-dimensional (2D) structure integrated by photosensitizers are rarely reported. However, chemo-photodynamic therapy still has limitations such as the inhibitory effect from intracellular glutathione (GSH). In this work, a simple bottom-up synthesis method was used to synthesize a pH-responsive drug delivery system with a 2D MOF structure. In particular, tetracarboxyporphyrin (TCPP) derivatives were coordinated with bivalent copper ions as organic bridging molecules in a polyvinylpyrrolidone (PVP) solution, and copper porphyrin MOFs (Cu-TCPP nanosheets) were synthesized by a hydrothermal method from bottom to top. DOX was loaded onto Cu-TCPP nanosheets by π-π stacking with a high drug loading rate of 33%. DOX@Cu-TCPP nanosheets showed pH-responsive DOX releasing behaviour and significant GSH scavenging ability. In addition, the evaluation of in vitro and in vivo treatment showed that DOX@Cu-TCPP nanosheets had high anti-tumor activity and excellent biocompatibility. Therefore, this study opens a new idea for the application of MOF nanosheets in tumor therapy and provides a supporting basis for the treatment of cancers by chemo-photodynamic synergistic therapy.

Advances in Understanding TKS4 and TKS5: Molecular Scaffolds Regulating Cellular Processes from Podosome and Invadopodium Formation to Differentiation and Tissue Homeostasis.

Scaffold proteins are typically thought of as multi-domain “bridging molecules.” They serve as crucial regulators of key signaling events by simultaneously binding multiple participants involved in specific signaling pathways. In the case of epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR) binding, the activated EGFR contacts cytosolic SRC tyrosine-kinase, which then becomes activated. This process leads to the phosphorylation of SRC-substrates, including the tyrosine kinase substrates (TKS) scaffold proteins. The TKS proteins serve as a platform for the recruitment of key players in EGFR signal transduction, promoting cell spreading and migration. The TKS4 and the TKS5 scaffold proteins are tyrosine kinase substrates with four or five SH3 domains, respectively. Their structural features allow them to recruit and bind a variety of signaling proteins and to anchor them to the cytoplasmic surface of the cell membrane. Until recently, TKS4 and TKS5 had been recognized for their involvement in cellular motility, reactive oxygen species-dependent processes, and embryonic development, among others. However, a number of novel functions have been discovered for these molecules in recent years. In this review, we attempt to cover the diverse nature of the TKS molecules by discussing their structure, regulation by SRC kinase, relevant signaling pathways, and interaction partners, as well as their involvement in cellular processes, including migration, invasion, differentiation, and adipose tissue and bone homeostasis. We also describe related pathologies and the established mouse models.

Diversification of polyphosphate end-labeling viabridging molecules.

Investigation of the biological roles of inorganic polyphosphate has been facilitated by our previous development of a carbodiimide-based method for covalently coupling primary amine-containing molecules to the terminal phosphates of polyphosphate. We now extend that approach by optimizing the reaction conditions and using readily available “bridging molecules” containing a primary amine and an additional reactive moiety, including another primary amine, a thiol or a click chemistry reagent such as dibenzocyclooctyne. This two-step labeling method is used to covalently attach commercially available derivatives of biotin, peptide epitope tags, and fluorescent dyes to the terminal phosphates of polyphosphate. Additionally, we report three facile methods for purifying conjugated polyphosphate from excess reactants.

Role of Metal Ion-Linked Multilayer Thickness and Substrate Porosity in Surface Loading, Diffusion, and Solar Energy Conversion.

Metal ion-linked multilayers offer an easily prepared and modular architecture for controlling energy and electron transfer events on nanoparticle, metal oxide films. However, unlike with planar electrodes, the mesoporous nature of the films inherently limits both the thickness of the multilayer and subsequent diffusion through the pores. Here, we systematically investigated the role of TiO2 nanoparticle film porosity and metal ion-linked multilayer thickness in surface loading, through-pore diffusion, and overall device performance. The TiO2 porosity was controlled by varying TiO2 sintering times. Molecular multilayer thickness was controlled through assembling ZnII-linked bridging molecules (B = p-terphenyl diphosphonic acid) between the metal oxide and the Ru(bpy)2((4,4'-PO3H2)2bpy)]Cl2 dye (RuP), thus producing TiO2-(Bn)-RuP films. Using attenuated total reflectance infrared absorption and UV-vis spectroscopy, we observed that at least two molecular layers (i.e., TiO2-B2 or TiO2-B1-RuP) could be formed on all films but subsequent loading was dependent on the porosity of the TiO2. Rough estimates indicate that in a film with 34 nm average pore diameter, the maximum multilayer film thickness is on the order of 4.6-6 nm, which decreases with decreasing pore size. These films were then incorporated as the photoanodes in dye-sensitized solar cells with cobalt(II/III)tris(4,4'-di-tert-butyl-2,2'-bipyridine) as a redox mediator. In agreement with the surface-loading studies, electrochemical impedance spectroscopy measurements indicate that mediator diffusion is significantly hindered in films with thicker multilayers and less porous TiO2. Collectively, these results show that care must be taken to balance multilayer thickness, substrate porosity, and size of the mediator in designing and maximizing the performance of new multilayer energy and electron management architectures.

Synthesis of CaCO3-Based Nanomedicine for Enhanced Sonodynamic Therapy via Amplification of Tumor Oxidative Stress

Summary Development of multifunctional stimuli-responsive nanomedicine is appealing for effective cancer treatment. Herein, we utilize the biocompatible CaCO3 nanoparticles as the template to guide the formation of pH-dissociable hollow coordination nanostructures, in which meso-tetra-(4-carboxyphenyl)porphine (TCPP), a sonosensitizer, acts as the organic bridging molecule and ferric ion serves as the metallic center. L-buthionine sulfoximine (BSO), an inhibitor for glutathione (GSH) biosynthesis, can be simultaneously loaded during the preparation of TCPP-Fe@CaCO3, obtaining BSO-TCPP-Fe@CaCO3 with pH-responsive dissociation to endow fast release of Ca2+ and BSO under an acidic tumor microenvironment. Such BSO-TCPP-Fe@CaCO3 confers synergistic oxidative stress amplification via intracellular Ca2+-overloading-induced mitochondria damage, BSO-mediated GSH depletion, and TCPP-mediated sonodynamic therapy (SDT), leading to remarkable cell death. Consequently, tumors on the mice treated with BSO-TCPP-Fe@CaCO3 administration and subsequent ultrasound exposure are effectively suppressed. Our work thus highlights a facile strategy to prepare pH-dissociable nanomedicine for effective SDT treatment of tumors via triple amplification of tumor oxidative stress.

Understanding the Spatial Topology of Artificial Immunological Synapses Assembled in T Cell-Redirecting Strategies: A Major Issue in Cancer Immunotherapy.

T cell-redirection strategies aim to selectively eliminate cancer cells by physically linking T lymphocytes with cancer cells using tumor-targeted cell-cell bridging (CCB) molecules, such as membrane-anchored chimeric antigen receptors (CARs) or soluble bispecific antibodies (bsAbs) that specifically recognize a cell-surface tumor-associated antigen (TAA) (Blanco et al., 2019). In the CAR approach, a TAA-specific antibody is genetically fused to intracellular T cell signaling domains. CARs have evolved greatly since their initial description, as single-chain antibody fragment (scFv)-based receptors containing the signaling domain of the CD3ζ chain (CD247) of the T cell receptor (TCR) (Eshhar et al., 1993). Subsequently, constructs incorporating signaling domains of costimulatory molecules (e.g., CD28 or 4-1BB) in tandem with the CD3ζ signaling domain were generated (Finney et al., 1998). Engrafting T-cells with such receptors, termed second-generation CARs, enables sustained proliferation and increased cytokine secretion. Third-generation CARs contain two costimulatory domains, in addition to the CD3ζ signaling domain (Carpenito et al., 2009; Milone et al., 2009). Current CAR-T cell therapy involves the isolation of autologous T cells using leukapheresis, followed by in vitro stimulation, genetic modification to express the TAA-specific CAR, and expansion to infuse back into the patient (Blanco et al., 2019). The bsAbs are designed to simultaneously bind to the TAA in the surface of tumor cells and the CD3e chain of the TCR/CD3 complex in the surface of T cells (Blanco et al., 2019). More than a 100 different bsAb formats have been reported, including small bsAbs composed only by two antigen-binding sites, IgG-like bsAbs and larger and non-IgG bsAbs formed by different antigen-binding moieties, often combined with oligomerization modules (Nunez-Prado et al., 2015; Brinkmann and Kontermann, 2017). By connecting CD3 signaling molecules with a recognition process independent of the TCR variable domains, T cells can be hot-wired to recognize a user-defined cell-surface TAA that is not associated with the major histocompatibility complex (MHC) to activate effector cell responses and kill cancer cells (Blanco et al., 2019). Nonetheless, the precise molecular mechanisms by which T cells are activated through these CCB molecules are poorly understood. The administration of bsAbs and CAR-T cells has achieved remarkable clinical outcomes in hematological tumors, and several products have been approved by regulatory agencies for clinical use. Blinatumomab, an anti-CD19xanti-CD3 bsAb designed in the BiTE (bispecific T cell-engager) format, was approved by the US Food and Drug Administration (FDA) for the treatment of relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL) (Przepiorka et al., 2015). Two CD19-specific second generation CAR-T cell products, tisagenlecleucel and axicabtagene ciloleucel (axi-cel), have been approved by the US FDA for the treatment of pediatric and young adult patients with relapsed or refractory B-ALL (Maude et al., 2018) and adult patients with relapsed or refractory diffuse large B cell lymphomas (Neelapu et al., 2017), respectively. However, the utility of these approaches in the treatment of solid tumors targeting TAAs has been limited by organ toxicities related to activation of T cell effector functions by non-tumor cells expressing low levels of the TAA, as well as systemic cytokine-associated toxicities (Alonso-Camino et al., 2016).