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SCFSkp2/Cks1is an E3 ubiquitin ligase, whose substrate specificity is determined by the oncogenic F-box protein Skp2 and the adaptor protein Cks1. A principal target of SCFSkp2/Cks1is the cyclin-dependent kinase inhibitor p27. Elevated levels of Skp2 and reduced levels of p27 are common in a variety of cancers, and there is consequently a need to develop effective inhibitors of the Skp2-p27 interaction. However, conventional small-molecule approaches are challenging due to the extended bi-molecular interface that spans both Skp2 and Cks1, the lack of suitable binding pockets on this surface, and the intrinsically disordered nature of p27. Here, we develop macrocyclic peptides capable of binding to SCFSkp2/Cks1with nanomolar affinities, an enhancement of almost two orders of magnitude over the natural p27 peptide. We show that these macrocyclic peptides inhibit p27 ubiquitination in vitro, restore p27 levels in a breast cancer cell line, and reduce cell proliferation.

Aim: To investigate the potential of human growth hormone (hGH) to improve human hematopoietic reconstitution in humanized mice. Materials & methods: Immunodeficient mice were conditioned by total body irradiation and transplanted with human CD34+ fetal liver cells. Peripheral blood, spleen and bone marrow were harvested, and levels of human lymphohematopoietic cells were determined by flow cytometry. Results: Supplementation with hGH elevated human lymphohematopoietic chimerism by more than twofold. Treatment with hGH resulted in significantly increased reconstitution of human B cells and myeloid cells in lymphoid organs, enhanced human erythropoiesis in the bone morrow, and improved engraftment of human hematopoietic stem cells. Conclusion: hGH supplementation promotes human lymphohematopoietic reconstitution in humanized mice.

Some subjective methods should divide input values into local clusters before determining the ordered weighted averaging (OWA) operator weights based on the data distribution characteristics of input values. However, the process of clustering input values is complex. In this paper, a novel probability density based OWA (PDOWA) operator is put forward based on the data distribution characteristics of input values. To capture the local cluster structures of input values, the kernel density estimation (KDE) is used to estimate the probability density function (PDF), which fits to the input values. The derived PDF contains the density information of input values, which reflects the importance of input values. Therefore, the input values with high probability densities (PDs) should be assigned with large weights, while the ones with low PDs should be assigned with small weights. Afterwards, the desirable properties of the proposed PDOWA operator are investigated. Finally, the proposed PDOWA operator is applied to handle the multicriteria decision making problem concerning the evaluation of smart phones and it is compared with some existing OWA operators. The comparative analysis shows that the proposed PDOWA operator is simpler and more efficient than the existing OWA operators.

The normal electron-demand Diels-Alder (DA) cycloaddition is a classic transformation routinely used in synthesis; however, applications in biological systems are limited. Here, we report a spiro[2.4]hepta-4,6-diene-containing noncanonical amino acid (SCpHK) capable of efficient incorporation into antibodies and subsequent coupling with maleimide via a DA reaction. SCpHK was stable throughout protein expression in mammalian cells and enabled covalent attachment of maleimide drug-linkers yielding DA antibody-drug conjugates (DA-ADCs) with nearly quantitative conversion in a one-step process. The uncatalyzed DA reaction between SCpHK and maleimide in aqueous buffer was rapid (1.8-5.4 M-1 s-1), and the antibody-drug adduct was stable in rat serum for at least 1 week at 37 °C. Anti-EphA2 DA-ADCs containing AZ1508 or SG3249 maleimide drug-linkers were potent inhibitors of tumor growth in PC3 tumor models in vivo. The DA bioconjugation strategy described here represents a simple method to produce site-specific and stable ADCs with maleimide drug-linkers.

Little attention has been paid to the tolerance of osteoblasts to fluoride in distinct differentiation stages, and the role of TGF-β1 in fluoride-treated osteoblast differentiation of progenitors and precursors was rarely mentioned in previous studies. The present study aimed to clarify how fluoride affected different differentiation stages of osteoblasts, and to elucidate the role of TGF-β1 in this process. We assessed cell migration, proliferation, DNA damage, and apoptosis of early-differentiated osteoblasts derived from bone marrow stem cells (BMSCs) exposed to fluoride with or without TGF-β1. Subsequently, MC3T3-E1 cells cultured with mineral induction medium were treated with fluoride to test fluoride's effect on late-differentiated osteoblasts. The specific fluoride concentrations and treatment times were chosen to evaluate the role of TGF-β1 in fluoride-induced osteoblastic differentiation and function. Results showed early-differentiated osteoblasts treated with a low dose of fluoride grew and moved more rapidly. TGF-β1 promoted cell proliferation and inhibited cell apoptosis in early-differentiated osteoblasts exposed to a low fluoride dose, but enhanced apoptosis at higher fluoride conditions. In the late-differentiated osteoblasts, the fluorine dose range with anabolic effects was narrowed, and the fluoride range with catabolic effects was widened. Treatment with a low fluoride dose stimulated the alkaline phosphatase (ALP) expression. TGF-β1 treatment inhibited Runx2 expression but increased RANKL expression in late-differentiated osteoblasts exposed to fluoride. Meanwhile, TGF-β1 treatments activated Smad3 phosphorylation but blocked Wnt10b expression in osteoblasts. We conclude that TGF-β1 plays an essential role in fluoride-induced differentiation and osteoblast function via activation of Smad3 instead of Wnt10 signaling.

Analogs of the known inhibitor (peptide pDI) of the p53/MDM2 protein–protein interaction are reported, which are stapled by linkers bearing a photoisomerizable diarylethene moiety. The corresponding photoisomers possess significantly different affinities to the p53-interacting domain of the human MDM2. Apparent dissociation constants are in the picomolar-to-low nanomolar range for those isomers with diarylethene in the “open” configuration, but up to eight times larger for the corresponding “closed” isomers. Spectroscopic, structural, and computational studies showed that the stapling linkers of the peptides contribute to their binding. Calorimetry revealed that the binding of the “closed” isomers is mostly enthalpy-driven, whereas the “open” photoforms bind to the protein stronger due to their increased binding entropy. The results suggest that conformational dynamics of the protein-peptide complexes may explain the differences in the thermodynamic profiles of the binding.

Immunohistochemistry (IHC) and immunocytochemistry (ICC) are routinely employed for the microscopic identification and diagnosis of cancerous cells in histological tissues and cell cultures. The maximally attainable contrast of conventional histological staining techniques, however, is low. While the anti-Stokes emission of photon-upconversion nanoparticles (UCNP) can efficiently eliminate optical background interference, excluding non-specific interactions of the label with the histological sample is equally important for specific immunolabeling. To address both requirements, we have designed and characterized several UCNP-based nanoconjugates as labels for the highly specific detection of the cancer biomarker HER2 on various breast cancer cell lines. An optimized streptavidin-PEG-neridronate-UCNP conjugate provided an unsurpassed signal-to-background ratio of 319, which was 50-fold better than conventional fluorescent labeling under the same experimental conditions. In combination, the absence of optical interference and non-specific binding lays the foundation for computer-based data evaluation in digital pathology.

Individual selection, as an effective mechanism, is often used in spatial evolutionary games to promote cooperation. Previous research assumes that, individual selection usually occurs with people who fail to meet a certain criterion. However, individual selection is usually inevitable, regardless of whether players in population cooperate or defect. This paper studies the effects of wealth-based rule in costly public goods games when individual selection is inevitable. Specifically, we assume that only the top V individuals with relatively high cumulative payoffs in each group can be selected for costly PGG. The results show that when V is large, the increase of participation cost has slight inhibitory effects on the evolution of cooperation, but it alleviates the polarization of individuals. However, when V is small, the increase of participation cost within a certain range promotes cooperation prosperity, but it also causes an increase in the proportion of polarized individuals and a widening of the wealth gap between rich and poor individuals.

Emotion emerges along with individuals’ interactions, and numerous experimental studies have shown that emotion plays an important role in individual decision making. However, how emotion affects the evolution of cooperation in structured population is largely unknown. Here we introduce emotion into network reciprocity, where emotion is considered quantifiable and cumulative, and then divide individuals into two types, namely non-competitive individuals whose social value orientation is mutually beneficial and competitive individuals whose social value orientation is maximizing outcomes for the self. Here, we explore the effects of the proportion of non-competitive individuals and emotional cumulative length on the evolution of cooperation. Simulation results show that the existence of non-competitive individuals promotes cooperation in the system. Furthermore, we also find that emotional cumulative length plays a key role in promoting overall cooperation, but surprisingly, the cooperation rate of competitive individuals peaks at an intermediate value of emotional cumulative length.

Affected by geographical or social factors, human communities are organized with changeless groups for teamwork. Within groups, defection dominates because the payoffs of defectors are often higher than those of cooperators; among groups, higher cooperation level makes individuals wealthier. For the previous mechanism of evolutionary dynamics leading individuals to imitate the rich in their groups, defective strategy evolves. However, individuals who belong to different groups and have no direct interaction of interests actually could influence each other. Due to the similarity of environment, the strategies among groups have the channel and necessity of dissemination. To this end, we propose an evolutionary game model with inter-group selection of strategy. By simulations, the effects of groups’ selection preference, group size, multiplier factor, imitation intensity and competition among groups on cooperative evolution in populations are studied. We find that when the group makes selection without preference, the intensity of the choice positively affects the level of cooperation; the strategy selection with preference can effectively increase the group cooperation rate.

It is known that the vaccination is essential for suppressing the periodic prevalence of an infectious disease, while the vaccination decision is influenced by external factors. Whether or not to take vaccination depends not only on the expected benefit of individual, but also on the behavior of others. Nowadays, the speed of information transmission on the social media is fast with wide range, which extremely expands the scope of imitation, but it is unknown whether it is beneficial to vaccination. Inspired by this situation, a vaccination game model on two-layer network which increases the intensity of information sharing is proposed. The vaccination game layer and the information sharing layer are established to study the interaction between individual vaccination decisions and strategic exchanges. We find that although information sharing has inhibited individual vaccination decisions to some extent, the overall scale of disease spreading in society has decreased with increasing information sharing.

The Sondheimer dialkyne is extensively used in double strain-promoted azide-alkyne cycloadditions. This reagent suffers with poor water-solubility and rapidly decomposes in aqueous solutions. This intrinsically limits its application in biological systems, and no effective solutions are currently available. Herein, we report the development of novel highly water-soluble, stable, and azide-reactive strained dialkyne reagents. To demonstrate their extensive utility, we applied our novel dialkynes to a double strain-promoted macrocyclisation strategy to generate functionalised p53-based stapled peptides for inhibiting the oncogenic p53-MDM2 interaction. These functionalised stapled peptides bind MDM2 with low nanomolar affinity and show p53 activation in a cellular environment. Overall, our highly soluble, stable and azide-reactive dialkynes offer significant advantages over the currently used Sondheimer dialkyne, and could be utilised for numerous biological applications.

Excessive systemic uptake of inorganic fluorides causes disturbances of bone homeostasis. The mechanism of skeletal fluorosis is still uncertain. This study aimed to study the effect of fluoride on osteocyte-driven osteoclastogenesis and probe into the role of PTH in this process. IDG-SW3 cells seeded in collagen-coated constructs were developed into osteocyte-like cells through induction of mineral agents. Then, osteocyte-like cells were exposed to fluoride in the presence or absence of parathyroid hormone (PTH). Cell viability and their capacity to produce receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and sclerostin (SOST) were detected by MTT and Western blot assays, respectively. Finally, a transwell coculture system using osteocyte-like cells seeded in the low compartment, and osteoclast precursors added in the inserts was developed to observe the osteocyte-driven osteoclasogenesis response to fluoride with or without PTH, and the expression of molecules involved in this mechanism were measure by real time RT-PCR. Results showed that osteocytes withstood a toxic dose of fluoride, and yet PTH administration significantly reduced osteocytes viability. PTH amplified the effect of fluoride on the expression of osteoclastogenesis-related molecules in osteocyte, but did not enlarged the stimulating effect of fluoride on osteoclastogenesis drove by osteocyte coculture. Gene expression levels of TRAP, RANK, JNK and NFAtc1 significantly increased in fluoride affected osteoclast precursor cocultured with osteocyte-like cells. The impact of fluoride on osteocyte-driven osteoclast differentiation was stronger than that of PTH. In conclusion, osteocyte played a pivotal role on the mechanism underlying fluoride-affected osteoclastogenesis in which RANK-JNK-NFATc1 signaling pathway was involved, and PTH had a significant impact in this process.

Protein–protein interactions (PPIs) have traditionally been referred to as “undruggable” mainly due to the large contact surfaces involved and the lack of well-defined binding sites. Research in this area has led to the development of a number of effective strategies for inhibiting PPIs. This chapter describes one of these methodologies developed in the Spring group: two-component double-click peptide stapling. This involves constraining short linear peptides by covalently linking two of their non-proteogenic amino acid side chains with a dialkyne linker utilizing Cu-catalyzed click chemistry. The advantage of this technique is that the properties of the stapled peptide can be modified via the linker group. This technique was successfully applied to target the oncogenic p53–MDM2 interaction. An optimization of the peptide sequence in addition to the use of different functionalized linkers resulted in biologically active stapled peptides. This Cu-catalyzed stapling technique was extended to develop a metal-free stapling methodology utilizing strain-promoted click chemistry and hence eliminating the use of cytotoxic copper. Finally, the Cu-catalyzed double-click stapling technique was used to synthesize non-α-helical constrained peptides to target the substrate-recognition domain of tankyrase to antagonize Wnt signaling, and the transcription factor HNF1β/Importin α PPI.

Summary Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated., Highlights • Single-molecule FRET reveals previously hidden conformations of AnkyrinR • We show that ankyrin repeats of AnkyrinR switch between high- and low-FRET states • Switching is controlled by an unstructured “safety pin” from an adjacent subdomain • The results suggest that the order-disorder transition regulates AnkyrinR activity, Using single-molecule FRET, Tsytlonok et al. reveal previously hidden conformations of AnkyrinR. The ankyrin repeats of AnkyrinR switch between two FRET states, and the switching is controlled by an unstructured “safety pin” from an adjacent subdomain. Therefore, the order-disorder transition regulates AnkyrinR activity.

There is a lack of current treatment options for ovarian clear cell carcinoma (CCC) and the cancer is often resistant to platinum-based chemotherapy. Hence there is an urgent need for novel therapeutics. The transcription factor hepatocyte nuclear factor 1β (HNF1β) is ubiquitously overexpressed in CCC and is seen as an attractive therapeutic target. This was validated through shRNA-mediated knockdown of the target protein, HNF1β, in five high- and low-HNF1β-expressing CCC lines. To inhibit the protein function, cell-permeable, non-helical constrained proteomimetics to target the HNF1β–importin α protein–protein interaction were designed, guided by X-ray crystallographic data and molecular dynamics simulations. In this way, we developed the first reported series of constrained peptide nuclear import inhibitors. Importantly, this general approach may be extended to other transcription factors.

This paper studies the problem of robust H ∞ control for uncertain stochastic system with time-varying delay. The time delay is assumed to be of interval type. By constructing a new stochastic Lyapunov-Krasovskii functional, Ito's formula and introducing free weighting matrix, sufficient conditions for delay-dependent robust H ∞ control is obtained. The presented formulation is in the framework of linear matrix inequality and thus can be carried out conveniently. Finally, one numerical example is given to illustrate the effectiveness of the proposed approach.

Stapled peptides have arisen as a new class of chemical probe and potential therapeutic agents for modulating protein–protein interactions. Here, we report the first two‐component i,i+7 stapling methodology that makes use of two orthogonal, on‐resin stapling reactions to incorporate linkers bearing a chiral centre into a p53‐derived stapled peptide. Post‐stapling modifications to the chain were performed on‐resin and enabled rapid access to various peptide derivatives from a single staple. The stapled peptides have increased helicity, protease stability and in vitro binding affinities to MDM2 compared to the equivalent unstapled peptide. This approach can be used to generate a library of diverse stapled peptides with different properties starting from a single stapled peptide, with scope for much greater functional diversity than that provided by existing stapling methodologies.

Copper-catalyzed arylation of electron rich alkynes reveals stabilized trisubstituted vinyl cation equivalents that react with pendant arene nucleophiles to form all carbon tetrasubstituted alkenes. The new process streamlines the synthesis of important medicinally relevant molecules.

There is a lack of current treatment options for ovarian clear cell carcinoma (CCC) and the cancer is often resistant to platinum‐based chemotherapy. Hence there is an urgent need for novel therapeutics. The transcription factor hepatocyte nuclear factor 1β (HNF1β) is ubiquitously overexpressed in CCC and is seen as an attractive therapeutic target. This was validated through shRNA‐mediated knockdown of the target protein, HNF1β, in five high‐ and low‐HNF1β‐expressing CCC lines. To inhibit the protein function, cell‐permeable, non‐helical constrained proteomimetics to target the HNF1β–importin α protein–protein interaction were designed, guided by X‐ray crystallographic data and molecular dynamics simulations. In this way, we developed the first reported series of constrained peptide nuclear import inhibitors. Importantly, this general approach may be extended to other transcription factors.

Copper-catalyzed arylation of electron rich alkynes reveals stabilized trisubstituted vinyl cation equivalents that react with pendant arene nucleophiles to form all carbon tetrasubstituted alkenes. The new process streamlines the synthesis of important medicinally relevant molecules.