Artificial Peptide-Protein Necrosomes Promote Cell Death.

The presence of disordered region or large interacting surface within proteins significantly challenges the development of targeted drugs, commonly known as the "undruggable" issue. Here, we report a heterogeneous peptide-protein assembling strategy to selectively phosphorylate proteins, thereby activating the necroptotic signaling pathway and promoting cell necroptosis. Inspired by the structures of natural necrosomes formed by receptor interacting protein kinases (RIPK) 1 and 3, the kinase-biomimetic peptides are rationally designed by incorporating natural or D -amino acids, or connecting D -amino acids in a retro-inverso (DRI) manner, leading to one RIPK3-biomimetic peptide PR3 and three RIPK1-biomimetic peptides. Individual peptides undergo self-assembly into nanofibrils, whereas mixing RIPK1-biomimetic peptides with PR3 accelerates and enhances assembly of PR3. In particular, RIPK1-biomimetic peptide DRI-PR1 exhibits reliable binding affinity with protein RIPK3, resulting in specific cytotoxicity to colon cancer cells that overexpress RIPK3. Mechanistic studies reveal the increased phosphorylation of RIPK3 induced by RIPK1-biomimetic peptides, elucidating the activation of the necroptotic signaling pathway responsible for cell death without an obvious increase in secretion of inflammatory cytokines. Our findings highlight the potential of peptide-protein hybrid aggregation as a promising approach to address the "undruggable" issue and provide alternative strategies for overcoming cancer resistance in the future.

Co-assembly Behaviors of Flavonol Derivatives Induced by a Pyridine Derivative on HOPG via Hydrogen Bonding and Van der Waals Forces.

In this paper, two new flavonol derivatives, 2-(4-(dodecyloxy)phenyl)-3-hydroxyflavone (DHF) and 2-(3,5-bis(dodecyloxy)phenyl)-3-hydroxyflavone (BDHF), were synthesized to investigate the respective self-assembly behaviors at the liquid/solid interface by scanning tunneling microscopy. In addition, a linear pyridine derivative with acetylene groups called BisPy was added to regulate the assembly of DHF and BDHF, individually. However, only BDHF molecules successfully co-assembled into grid structures with BisPy molecules. Furthermore, the assembly and co-assembly behavior mechanism of flavonol derivatives and BisPy molecules were further studied by density functional theory calculations. This work will lay a foundation for investigating the self-assembly of flavonol derivatives and the co-assembly regulated by pyridine derivatives at the liquid-solid interface.

Antitumor Mechanism of Hydroxycamptothecin via the Metabolic Perturbation of Ribonucleotide and Deoxyribonucleotide in Human Colorectal Carcinoma Cells.

Hydroxycamptothecin (SN38) is a natural plant extract isolated from Camptotheca acuminate. It has a broad spectrum of anticancer activity through inhibition of DNA topoisomerase I, which could affect DNA synthesis and lead to DNA damage. Thus, the action of SN38 against cancers could inevitably affect endogenous levels of ribonucleotide (RNs) and deoxyribonucleotide (dRNs) that play critical roles in many biological processes, especially in DNA synthesis and repair. However, the exact impact of SN38 on RNs and dRNs is yet to be fully elucidated. In this study, we evaluated the anticancer effect and associated mechanism of SN38 in human colorectal carcinoma HCT 116 cells. As a result, SN38 could decrease the cell viability and induce DNA damage in a concentration-dependent manner. Furthermore, cell cycle arrest and intracellular nucleotide metabolism were perturbed due to DNA damage response, of which ATP, UTP, dATP, and TTP may be the critical metabolites during the whole process. Combined with the expression of deoxyribonucleoside triphosphates synthesis enzymes, our results demonstrated that the alteration and imbalance of deoxyribonucleoside triphosphates caused by SN38 was mainly due to the de novo nucleotide synthesis at 24 h, and subsequently the salvage pathways at 48 h. The unique features of SN38 suggested that it might be recommended as an effective supplementary drug with an anticancer effect.

Vitexin reduces epilepsy after hypoxic ischemia in the neonatal brain via inhibition of NKCC1.

BACKGROUND: Neonatal hypoxic-ischemic brain damage, characterized by tissue loss and neurologic dysfunction, is a leading cause of mortality and a devastating disease of the central nervous system. We have previously shown that vitexin has been attributed various medicinal properties and has been demonstrated to have neuroprotective roles in neonatal brain injury models. In the present study, we continued to reinforce and validate the basic understanding of vitexin (45 mg/kg) as a potential treatment for epilepsy and explored its possible underlying mechanisms. METHODS: P7 Sprague-Dawley (SD) rats that underwent right common carotid artery ligation and rat brain microvascular endothelial cells (RBMECs) were used for the assessment of Na+-K+-Cl- co-transporter1 (NKCC1) expression, BBB permeability, cytokine expression, and neutrophil infiltration by western blot, q-PCR, flow cytometry (FCM), and immunofluorescence respectively. Furthermore, brain electrical activity in freely moving rats was recorded by electroencephalography (EEG). RESULTS: Our data showed that NKCC1 expression was attenuated in vitexin-treated rats compared to the expression in the HI group in vivo. Oxygen glucose deprivation/reoxygenation (OGD) was performed on RBMECs to explore the role of NKCC1 and F-actin in cytoskeleton formation with confocal microscopy, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide, and FCM. Concomitantly, treatment with vitexin effectively alleviated OGD-induced NKCC1 expression, which downregulated F-actin expression in RBMECs. In addition, vitexin significantly ameliorated BBB leakage and rescued the expression of tight junction-related protein ZO-1. Furthermore, inflammatory cytokine and neutrophil infiltration were concurrently and progressively downregulated with decreasing BBB permeability in rats. Vitexin also significantly suppressed brain electrical activity in neonatal rats. CONCLUSIONS: Taken together, these results confirmed that vitexin effectively alleviates epilepsy susceptibility through inhibition of inflammation along with improved BBB integrity. Our study provides a strong rationale for the further development of vitexin as a promising therapeutic candidate treatment for epilepsy in the immature brain.

Engineering Ion Diffusion by CoS@SnS Heterojunction for Ultrahigh-Rate and Stable Potassium Batteries.

Transitional metal sulfides (TMSs) are considered as promising anode candidates for potassium storage because of their ultrahigh theoretical capacity and low cost. However, TMSs suffer from low electronic, ionic conductivity and significant volume expansion during potassium ion intercalation. Here, we construct a carbon-coated CoS@SnS heterojunction which effectively alleviates the volume change and improves the electrochemical performance of TMSs. The mechanism analysis and density functional theory (DFT) calculation prove the acceleration of K-ion diffusion by the built-in electric field in the CoS@SnS heterojunction. Specifically, the as-prepared material maintains 81% of its original capacity after 2000 cycles at 500 mA g-1. In addition, when the current density is set at 2000 mA g-1, it can still deliver a high discharge capacity of 210 mAh g-1. Moreover, the full cell can deliver a high capacity of 400 mAh g-1 even after 150 cycles when paired with a perylene-3,4,9,10-tetracarboxydiimide (PTCDI) cathode. This work is expected to provide a material design idea dealing with the unstable and low rate capability problems of potassium-ion batteries.

Minor adjustments in the chemical structures of pyridine derivatives induced different co-assemblies by O-H⋯N hydrogen bonds.

The co-adsorption behaviours of aromatic carboxylic acids with various pyridine derivatives were investigated with scanning tunneling microscopy and density functional theory. Surprisingly, minor adjustments in the chemical structures of the pyridine derivatives, such as the relative position of the nitrogen atom or the lengths of the side chains on the backbone would evidently affect the intermolecular O-H⋯N hydrogen bonds and further form various co-adsorption structures.

Heterochirality-Mediated Cross-Strand Nested Hydrophobic Interaction Effects Manifested in Surface-Bound Peptide Assembly Structures.

Amino acid chirality has been envisioned as an important strategy to regulate structure and function of peptide self-assembled architectures. However, the molecular mechanism of chirality effects in peptide assemblies remains largely elusive. Here, the assembly structures of l-peptide polyphenylalanine F10 (FFFFFFFFFF) and block heterochiral peptide F5f5 (FFFFFfffff) composed of two FFFFF repeat blocks with opposite chirality were characterized at the single-molecule level by using scanning tunneling microscopy. Each peptide formed two distinctively different assembly structures on the HOPG surface, in which peptide chains took parallel and antiparallel ß-sheet conformations, respectively. The molecular-level observations revealed that the staggered arrangement of cross-strand side chains achieved in the antiparallel ß-sheet structure of the block heterochiral peptide facilitated intimate packing of side chains and maximized inter-residue van der Waals interactions, which led to more residues participating in assembly and greatly stabilized the ß-sheet structure of the surface-bound peptide assembly, but such cross-strand nested interactions were not accessible in the heterochiral parallel ß-sheet structure and the enantiomerically pure assembly structures. This work could contribute to the molecular insights of stereochemical interactions in peptide assemblies and feasibility of extending this block heterochirality pattern to other peptides with various lengths and amino acid compositions for structural regulations.

Structural and Nanotribological Properties of a BODIPY Self-Assembly.

Boron-dipyrromethenes (BODIPY) are promising functional dyes, whose exceptional optical properties are closely related to their supramolecular assembly. Herein, the self-assembly of a BODIPY derivative functionalized with uracil groups is explicitly and thoroughly investigated by using scanning tunneling microscopy (STM). Based on the simulation and calculation by density functional theory (DFT) method, it can be concluded that the construction of ordered self-assembly structure is attributed to the formation of hydrogen bonds between uracil groups. Moreover, the nanotribological property of the self-assembly on HOPG surface is measured by using atomic force microscopy (AFM). The effort on self-assembly of the BODIPY derivative could enhance the understanding of surface assembly mechanism.

Light-Controlled Friction by Carboxylic Azobenzene Molecular Self-Assembly Layers.

Nowadays, reversible friction regulation has become the focus of scientists in terms of the flexible regulatory structure of photosensitive materials and theories since this facilitates rapid development in this field. Meanwhile, as an external stimulus, light possesses great potential and advantages in spatiotemporal control and remote triggering. In this work, we demonstrated two photo-isomerized organic molecular layers, tetra-carboxylic azobenzene (NN4A) and dicarboxylic azobenzene (NN2A), which were selected to construct template networks on the surface of the highly oriented pyrolytic graphite (HOPG) to study the friction properties, corresponding to the arrangement structure of self-assembled layers under light regulation. First of all, the morphology of the self-assembled layers were characterized by a scanning tunneling microscope (STM), then the nanotribological properties of the template networks were measured by atomic force microscope (AFM). Their friction coefficients are respectively changed by about 0.6 and 2.3 times under light control. The density functional theory (DFT) method was used to calculate the relationship between the force intensity and the friction characteristics of the self-assembled systems under light regulation. Herein, the use of external light stimulus plays a significant role in regulating the friction properties of the interface of the nanometer, hopefully serving as a fundamental basis for further light-controlling research for the future fabrication of advanced on-surface devices.

Insight Into the Superlubricity and Self-Assembly of Liquid Crystals.

Liquid crystals are promising molecular materials in the application of lubrication. Herein, the microscale solid superlubricity is accomplished by the construction of uniform and ordered self-assembly of several liquid crystals. The self-assembly structures on a highly oriented pyrolytic graphite (HOPG) surface are explicitly revealed by using scanning tunneling microscopy (STM). Meanwhile, the nanotribological performance of the self-assemblies are measured by using atomic force microscopy (AFM), revealing ultralow friction coefficients lower than 0.01. The interaction energies are calculated by density functional theory (DFT) method, indicating the positive correlation between friction coefficients and interaction strength. The effort on the self-assembly and superlubricity of liquid crystals could enhance the understanding of the nanotribological mechanism and benefit the further application of liquid crystals as lubricants.

Profiling Ribonucleotide and Deoxyribonucleotide Pools Perturbed by Remdesivir in Human Bronchial Epithelial Cells.

Remdesivir (RDV) has generated much anticipation for its moderate effect in treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the unsatisfactory survival rates of hospitalized patients limit its application to the treatment of coronavirus disease 2019 (COVID-19). Therefore, improvement of antiviral efficacy of RDV is urgently needed. As a typical nucleotide analog, the activation of RDV to bioactive triphosphate will affect the biosynthesis of endogenous ribonucleotides (RNs) and deoxyribonucleotides (dRNs), which are essential to RNA and DNA replication in host cells. The imbalance of RN pools will inhibit virus replication as well. In order to investigate the effects of RDV on cellular nucleotide pools and on RNA transcription and DNA replication, cellular RNs and dRNs concentrations were measured by the liquid chromatography-mass spectrometry method, and the synthesis of RNA and DNA was monitored using click chemistry. The results showed that the IC50 values for BEAS-2B cells at exposure durations of 48 and 72 h were 25.3 ± 2.6 and 9.6 ± 0.7 µM, respectively. Ten (10) µM RDV caused BEAS-2B arrest at S-phase and significant suppression of RNA and DNA synthesis after treatment for 24 h. In addition, a general increase in the abundance of nucleotides and an increase of specific nucleotides more than 2 folds were observed. However, the variation of pyrimidine ribonucleotides was relatively slight or even absent, resulting in an obvious imbalance between purine and pyrimidine ribonucleotides. Interestingly, the very marked disequilibrium between cytidine triphosphate (CTP) and cytidine monophosphate might result from the inhibition of CTP synthase. Due to nucleotides which are also precursors for the synthesis of viral nucleic acids, the perturbation of nucleotide pools would block viral RNA replication. Considering the metabolic vulnerability of endogenous nucleotides, exacerbating the imbalance of nucleotide pools imparts great promise to enhance the efficacy of RDV, which possibly has special implications for treatment of COVID-19.

Protective Effect of Thymidine on DNA Damage Induced by Hydrogen Peroxide in Human Hepatocellular Cancer Cells.

Intracellular ribonucleotide (RN) and deoxyribonucleotide (dRN) pool sizes are critical for the fidelity of DNA synthesis. They are likely to be severely perturbed by many factors which disrupt the integrity and stability of DNA, leading to DNA damage. Exogenously supplied nucleosides are able to increase the deoxynucleoside triphosphate pools, then reverse the DNA damage, and decrease the oncogene-induced transformation dramatically. In this study, the impact of thymidine on the hydrogen peroxide (H2O2)-induced DNA damage was investigated in HepG2 liver cancer cells. From the result of the comet assay, the tail length of cells in the thymidine 600 µM + H2O2 1.0 mM group was dramatically decreased from 42.1 ± 10.8 to 21.9 ± 2.4 µm compared to that exposed with 1.0 mM H2O2 (p < 0.05), suggesting that pretreatment of thymidine reduced the DNA damage of HepG2 cells. Although the RN and dRN contents decreased in the damage group, most of them presented increasing tendency when pretreated with thymidine, especially the key metabolites dCTP, which was mainly related with the decline in the rate of DNA synthesis. The restoration also showed a significant G0/G1 phase arrest of cell cycle progression from 44.6 ± 2.2 to 56.6 ± 0.4% after pretreated with thymidine (p < 0.05). In conclusion, our data demonstrated that the pretreatment with thymidine had a potential protective ability against oxidative damage for DNA in HepG2 cells through the perturbation of RN and dRN pools as well as cell cycle arrest, which should provide new insights into the molecular basis of preventing H2O2-induced oxidative DNA damage in mammalian cells.

Rapid and sensitive determination of four bisphosphonates in rat plasma after MTBSTFA derivatization using liquid chromatography-mass spectrometry.

Bisphosphonates (BPs) have broad medical applications against osteoporosis, bone metastasis and Paget's disease. The BP-related jaw osteonecrosis limits their use extensively and has a causal relationship with the process of drug disposition, such as deposition on bone and slow elimination rate. Thus it is imperative to accurately determine BP levels in either clinical or pharmacological/toxicological studies. The ability of trimethylsilyl diazomethane (TMSD) to alkylate the hydroxyls in phosphoric groups is an advantage in terms of decreasing polarity and enhancing mass response of BPs. There are, however, practical limitations to the cumbersome sample preparation procedure, the prolonged reaction time, the by-products and the obstacle to ionization. To overcome these disadvantages, a simplified and rapid precolumn derivatization method with N-(tert-Butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) to quantify etidronate, clodronate, alendronate and zoledronate BPs in rat plasma was established in this work. The derivatization reaction was conducted within 2 min at room temperature, and the unitary di-tert-butyldimethylsilyl (di-tBDMS) derivative was obtained for each BP. Derivatives were separated on a XTerra® MS C8 column (2.1 × 50 mm, 3.5 µm) with the mobile phase of 5 mM ammonium acetate buffer (pH 8.5) and acetonitrile, then detected using electrospray ionization tandem mass spectrometry in negative mode. An easy extraction process instead of the time-consuming solid-phase extraction (SPE) was employed for plasma treatment. The proposed method showed good linearity for BPs over the range of 2-500 ng/mL in 20 µL plasma and high sensitivity owing to the larger molecular ions, higher ionization capacity and more stable fragments of di-tBDMS derivatives. The intra- and inter-batch precision were <13.1 %, and the accuracy ranged within ±10 %. The extraction recovery varied from 75.4 to 88.0 %. The optimized method was successfully applied to characterize the pharmacokinetic profile of zoledronate in rats. Moreover, it is a promising approach for the determination of other phosphoric acid-containing metabolites.

Molecular recognition of human islet amyloid polypeptide assembly by selective oligomerization of thioflavin T.

Selective oligomerization is a common phenomenon existing widely in the formation of intricate biological structures in nature. The precise design of drug molecules with an oligomerization state that specifically recognizes its receptor, however, remains substantially challenging. Here, we used scanning tunneling microscopy (STM) to identify the oligomerization states of an amyloid probe thioflavin T (ThT) on hIAPP8-37 assembly to be exclusively even numbers. We demonstrate that both adhesive interactions between ThT and the protein substrate and cohesive interactions among ThT molecules govern the oligomerization state of the bounded ThT. Specifically, the work of the cohesive interaction between two head/tail ThTs is determined to be 6.4 k B T, around 50% larger than that of the cohesive interaction between two side-by-side ThTs (4.2 k B T). Overall, our STM imaging and theoretical understanding at the single-molecule level provide valuable insights into the design of drug compounds using the selective oligomerization of molecular probes to recognize protein self-assembly.

Peptide conformation and oligomerization characteristics of surface-mediated assemblies revealed by molecular dynamics simulations and scanning tunneling microscopy.

The characteristics of peptide conformations in both solution and surface-bound states, using poly-glycine as a model structure, are analyzed by using molecular dynamics (MD) simulations. The clustering analysis revealed significant linearization effect on the peptide conformations as a result of adsorption to surface, accompanied by varied adsorption kinetics and energetics. Depending on the inter-peptide interaction characteristics, distinctively different surface-mediated oligomerization modalities, such as antiparallel conformations, can be identified in MD and confirmed by scanning tunneling microscopy (STM) analysis of the assembly structures. These observations are beneficial for obtaining molecular insights of assembling propensity relating to peptide-surface and peptide-peptide interactions.

Bumetanide reduce the seizure susceptibility induced by pentylenetetrazol via inhibition of aberrant hippocampal neurogenesis in neonatal rats after hypoxia-ischemia.

Hypoxia-ischemia brain damage (HIBD) is one of prevalent causes of neonatal mortality and morbidity. Our data demonstrated that hypoxia-ischemia (HI) induced Na+-K+-Cl--co-transporter 1 (NKCC1) increasing in hippocampus. Previous studies demonstrated that NKCC1 regulates various stages of neurogenesis. In this study, we studied the role of increased NKCC1 in regulating of HI-induced neurogenesis. HIBD model was established in 7days old Sprague-Dawley rat pup, and the expression of NKCC1 was detected by western blot and qPCR. Brain electrical activity in freely rats was monitored by electroencephalography (EEG) recordings. HI-induced neurogenesis was detected by immunofluorescence staining. Neurobehavioral test was to investigate the neuro-protective role of bumetanide, an inhibitor of NKCC1, on neonatal rats after HI. The results showed that bumetanide treatment significantly reduced brain electrical activity and the seizure stage of epilepsy induced by pentylenetetrazol (PTZ) in vivo after HI. In addition, bumetanide restored aberrant hippocampal neurogenesis and associated cognitive function. Our data demonstrated that bumetanide reduces the susceptibility of epilepsy induced by PTZ in rats suffering from HI injury during neonatal period via restoring the ectopic newborn neurons in dentate gyrus (DG) and cognitive function.