Sporadic pediatric vestibular schwannoma: a case report in a 4-year-old boy.

Sporadic vestibular schwannomas (VSs) are rare in children. When occurred in the pediatric population, they usually appear bilaterally and are related to neurofibromatosis type 2 (NF2). The current study reports a 4-year-old boy without family history of VS or NF2 who presented with a large (5.7-cm) VS involving the right cerebellopontine angle and internal auditory canal. Through seven-staged surgical interventions and two stereotactic γ­knife radiosurgery, the disease was stabilized. At 2-year follow-up, the child had right ear hearing loss, grade IV facial palsy, and normal motor function and gait. No definite evidence of gene mutation regarding NF2 can be identified after sequence analysis and deletion/duplication testing. This case highlights the significance of considering the possibility of sporadic VSs, even in very young children. It emphasizes the importance of not overlooking initial symptoms, as they may indicate the presence of a large tumor and could potentially result in delayed diagnosis.

A Proof-of-Concept Inhibitor of Endothelial Lipase Suppresses Triple-Negative Breast Cancer Cells by Hijacking the Mitochondrial Function.

Triple-negative breast cancer (TNBC) cells reprogram their metabolism to provide metabolic flexibility for tumor cell growth and survival in the tumor microenvironment. While our previous findings indicated that endothelial lipase (EL/LIPG) is a hallmark of TNBC, the precise mechanism through which LIPG instigates TNBC metabolism remains undefined. Here, we report that the expression of LIPG is associated with long non-coding RNA DANCR and positively correlates with gene signatures of mitochondrial metabolism-oxidative phosphorylation (OXPHOS). DANCR binds to LIPG, enabling tumor cells to maintain LIPG protein stability and OXPHOS. As one mechanism of LIPG in the regulation of tumor cell oxidative metabolism, LIPG mediates histone deacetylase 6 (HDAC6) and histone acetylation, which contribute to changes in IL-6 and fatty acid synthesis gene expression. Finally, aided by a relaxed docking approach, we discovered a new LIPG inhibitor, cynaroside, that effectively suppressed the enzyme activity and DANCR in TNBC cells. Treatment with cynaroside inhibited the OXPHOS phenotype of TNBC cells, which severely impaired tumor formation. Taken together, our study provides mechanistic insights into the LIPG modulation of mitochondrial metabolism in TNBC and a proof-of-concept that targeting LIPG is a promising new therapeutic strategy for the treatment of TNBC.

Role of Glycogen Synthase Kinase-3 in Interferon-γ-Mediated Immune Hepatitis.

Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase, is a vital glycogen synthase regulator controlling glycogen synthesis, glucose metabolism, and insulin signaling. GSK-3 is widely expressed in different types of cells, and its abundant roles in cellular bioregulation have been speculated. Abnormal GSK-3 activation and inactivation may affect its original bioactivity. Moreover, active and inactive GSK-3 can regulate several cytosolic factors and modulate their diverse cellular functional roles. Studies in experimental liver disease models have illustrated the possible pathological role of GSK-3 in facilitating acute hepatic injury. Pharmacologically targeting GSK-3 is therefore suggested as a therapeutic strategy for liver protection. Furthermore, while the signaling transduction of GSK-3 facilitates proinflammatory interferon (IFN)-γ in vitro and in vivo, the blockade of GSK-3 can be protective, as shown by an IFN-γ-induced immune hepatitis model. In this study, we explored the possible regulation of GSK-3 and the potential relevance of GSK-3 blockade in IFN-γ-mediated immune hepatitis.

Pharmacologically Inhibiting Glycogen Synthase Kinase-3ß Ameliorates Renal Inflammation and Nephrotoxicity in an Animal Model of Cisplatin-Induced Acute Kidney Injury.

The adverse effect of cisplatin administration causes acute kidney injury (AKI) following renal inflammation and nephrotoxicity, characterized by proximal tubular cell apoptosis and necrosis. Pro-apoptotic and pro-inflammatory roles of glycogen synthase kinase (GSK)-3ß have been reported. This study investigated the therapeutic blockade of GSK-3ß in cisplatin-induced AKI. A renal cisplatin nephrotoxicity model showed activation of GSK-3ß in vivo, particularly in proximal tubular epithelial cells. Pharmacologically inhibiting GSK-3ß abolished cisplatin nephrotoxicity, including proximal tubular injury, cell cytotoxicity, and biochemical dysfunction. Additionally, GSK-3ß inhibitor treatment ameliorated renal inflammation by reducing immune cell infiltration, cell adhesion molecule expression, and pro-inflammatory cytokine/chemokine production. Cisplatin treatment caused GSK-3ß activation in vitro in the human renal proximal tubular epithelial cell line HK-2, whereas either pharmacological administration of GSK-3ß inhibitors or genetic transduction of GSK-3ß short-hairpin RNA impeded cisplatin-induced cytotoxicity. These results indicate that cisplatin activates GSK-3ß followed by GSK-3ß-mediated renal inflammation and nephrotoxicity, contributing to AKI.

Mutant glucocorticoid receptor binding elements on the interleukin-6 promoter regulate dexamethasone effects.

BACKGROUND: Glucocorticoids (GCs) have been extensively used as essential modulators in clinical infectious and inflammatory diseases. The GC receptor (GR) is a transcription factor belonging to the nuclear receptor family that regulates anti-inflammatory processes and releases pro-inflammatory cytokines, such as interleukin (IL)-6. RESULTS: Five putative GR binding sites and other transcriptional factor binding sites were identified on theIL-6 promoter, and dexamethasone (DEX) was noted to reduce the lipopolysaccharide (LPS)-induced IL-6 production. Among mutant transcriptional factor binding sites, nuclear factor-kappa B (NF-κB), activator protein (AP)-1, and specificity protein (Sp)1-2 sites reduced basal and LPS-induced IL-6 promoter activities through various responses. The second GR binding site (GR2) was noted to play a crucial role in both basal and inducible promoter activities in LPS-induced inflammation. CONCLUSIONS: We concluded that selective GR2 modulator might exert agonistic and antagonistic effects and could activate crucial signaling pathways during the LPS-stimulated inflammatory process.

Glycogen Synthase Kinase-3ß Facilitates Cytokine Production in 12-O-Tetradecanoylphorbol-13-Acetate/Ionomycin-Activated Human CD4+ T Lymphocytes.

Cytokines are the major immune regulators secreted from activated CD4+ T lymphocytes that activate adaptive immunity to eradicate nonself cells, including pathogens, tumors, and allografts. The regulation of glycogen synthase kinase (GSK)-3ß, a serine/threonine kinase, controls cytokine production by regulating transcription factors. The artificial in vitro activation of CD4+ T lymphocytes by a combination of 12-O-tetradecanoylphorbol-13-acetate and ionomycin, the so-called T/I model, led to an inducible production of cytokines, such as interferon-γ, tumor necrosis factor-α, and interleukin-2. As demonstrated by the approaches of pharmacological targeting and genetic knockdown of GSK-3ß, T/I treatment effectively caused GSK-3ß activation followed by GSK-3ß-regulated cytokine production. In contrast, pharmacological inhibition of the proline-rich tyrosine kinase 2 and calcineurin signaling pathways blocked cytokine production, probably by deactivating GSK-3ß. The blockade of GSK-3ß led to the inhibition of the nuclear translocation of T-bet, a vital transcription factor of T lymphocyte cytokines. In a mouse model, treatment with the GSK-3ß inhibitor 6-bromoindirubin-3'-oxime significantly inhibited T/I-induced mortality and serum cytokine levels. In summary, targeting GSK-3ß effectively inhibits CD4+ T lymphocyte activation and cytokine production.

Polyelectrolyte in Electric Field: Disparate Conformational Behavior along an Aminopolysaccharide Chain.

Electrical signals are increasingly used in fabrication of hydrogels (e.g., based on aminopolysaccharide chitosan) to guide the emergence of complex and anisotropic structure; however, how an imposed electric field affects the polymer chain conformation and orientation during the self-assembly process is not understood. Here, we applied nonequilibrium all-atom molecular dynamics simulations to explore the response of a charged chitosan chain comprising 5- or 20-monomer units to a constant uniform electric field in water and salt solution. While no conformational or orientational response was observed for the polyelectrolyte (PE) chains under the small electric fields within the simulation time, a field strength of 400 mV/nm induced significant changes. In water, a 5-mer chain is found to be slightly bent and oriented parallel to the field; however, surprisingly, a 20-mer chain displays candy-cane-like conformations whereby one half of the chain is collapsed and flexible, while the other half of the chain is stretched along the electric field. In salt solution, the disparity remains between the two halves of the 20-mer chain, although the backbone is extremely flexible with multiple bent regions and non-native conformations occur near the chain center in one of the three trajectories. The disparate conformational response along the polyelectrolyte chain may be attributed to the balancing forces between chain dynamics, electric polarization, counterion binding, and hydrodynamic pressure as well as friction. These findings reconcile existing experiments and theoretical studies and represent an important step toward understanding the complex roles of electric field and salt in controlling the structure and properties of soft matter.

How Electrostatic Coupling Enables Conformational Plasticity in a Tyrosine Kinase.

Protein kinases are important cellular signaling molecules involved in cancer and a multitude of other diseases. It is well-known that inactive kinases display a remarkable conformational plasticity; however, the molecular mechanisms remain poorly understood. Conformational heterogeneity presents an opportunity but also a challenge in kinase drug discovery. The ability to predictively model various conformational states could accelerate selective inhibitor design. Here we performed a proton-coupled molecular dynamics study to explore the conformational landscape of a c-Src kinase. Starting from a completely inactive structure, the simulations captured all major types of conformational states without the use of a target structure, mutation, or bias. The simulations allowed us to test the experimental hypotheses regarding the mechanism of DFG flip, its coupling to the αC-helix movement, and the formation of regulatory spine. Perhaps the most significant finding is how key titratable residues, such as DFG-Asp, αC-Glu, and HRD-Asp, change protonation states dependent on the DFG, αC, and activation loop conformations. Our data offer direct evidence to support a long-standing hypothesis that protonation of Asp favors the DFG-out state and explain why DFG flip is also possible in simulations with deprotonated Asp. The simulations also revealed intermediate states, among which a unique DFG-out/α-C state formed as DFG-Asp is moved into a back pocket forming a salt bridge with catalytic Lys, which can be tested in selective inhibitor design. Our finding of how proton coupling enables the remarkable conformational plasticity may shift the paradigm of computational studies of kinases which assume fixed protonation states. Understanding proton-coupled conformational dynamics may hold a key to further innovation in kinase drug discovery.

Assessing Lysine and Cysteine Reactivities for Designing Targeted Covalent Kinase Inhibitors.

Targeted covalent inhibitor design is gaining increasing interest and acceptance. A typical covalent kinase inhibitor design targets a reactive cysteine; however, this strategy is limited by the low abundance of cysteine and acquired drug resistance from point mutations. Inspired by the recent development of lysine-targeted chemical probes, we asked if nucleophilic (reactive) catalytic lysines are common on the basis of the published crystal structures of the human kinome. Using a newly developed p Ka prediction tool based on continuous constant pH molecular dynamics, the catalytic lysines of eight unique kinases from various human kinase groups were retrospectively and prospectively predicted to be nucleophilic, when kinase is in the rare DFG-out/αC-out type of conformation. Importantly, other reactive lysines as well as cysteines at various locations were also identified. On the basis of the findings, we proposed a new strategy in which selective type II reversible kinase inhibitors are modified to design highly selective, lysine-targeted covalent inhibitors. Traditional covalent drugs were discovered serendipitously; the presented tool, which can assess the reactivities of any potentially targetable residues, may accelerate the rational discovery of new covalent inhibitors. Another significant finding of the work is that lysines and cysteines in kinases may adopt neutral and charged states at physiological pH, respectively. This finding may shift the current paradigm of computational studies of kinases, which assume fixed solution protonation states.

Considering Relationship of Proteins for Radiotherapy Prognosis of Bladder Cancer Cells in Small Data Set.

BACKGROUND: Radiotherapy has serious side effects and thus requires prudent and cautious evaluation. However, obtaining protein expression profiles is expensive and timeconsuming, making it necessary to develop a theoretical and rational procedure for predicting the radiotherapy outcome for bladder cancer when working with limited data. OBJECTIVE: A procedure for estimating the performance of radiotherapy is proposed in this research. The population domain (range of the population) of proteins and the relationships among proteins are considered to increase prediction accuracy. METHODS: This research uses modified extreme value theory (MEVT), which is used to estimate the population domain of proteins, and correlation coefficients and prediction intervals to overcome the lack of knowledge regarding relationships among proteins. RESULTS: When the size of the training data set was 5 samples, the mean absolute percentage error rate (MAPE) was 31.6200%; MAPE fell to 13.5505% when the number of samples was increased to 30. The standard deviation (SD) of forecasting error fell from 3.0609% for 5 samples to 1.2415% for 30 samples. These results show that the proposed procedure yields accurate and stable results, and is suitable for use with small data sets. CONCLUSIONS: The results show that considering the relationships among proteins is necessary when predicting the outcome of radiotherapy.

How Ligand Protonation State Controls Water in Protein-Ligand Binding.

The role of water in protein-ligand binding has been an intensely studied topic in recent years; however, how ligand protonation state change perturbs water has not been considered. Here we show that water dynamics and interactions can be controlled by the protonation state of ligand using continuous constant pH molecular dynamics simulations of two closely related model systems, ß-secretase 1 and 2 (BACE1 and BACE2), in complex with a small-molecule inhibitor. Simulations revealed that, upon binding, the inhibitor pyrimidine ring remains deprotonated in BACE1 but becomes protonated in BACE2. Pyrimidine protonation results in water displacement, rigidification of the binding pocket, and shift in the ligand binding mode from water-mediated to direct hydrogen bonding. These findings not only support but also rationalize the most recent structure-selectivity data in BACE1 drug design. Binding-induced protonation state changes are likely common; our work offers a glimpse at how modeling protein-ligand binding while allowing ligand titration can further advance the understanding of water and structure-based drug design.

Predicting Catalytic Proton Donors and Nucleophiles in Enzymes: How Adding Dynamics Helps Elucidate the Structure-Function Relationships.

Despite the relevance of understanding structure-function relationships, robust prediction of proton donors and nucleophiles in enzyme active sites remains challenging. Here we tested three types of state-of-the-art computational methods to calculate the p Ka's of the buried and hydrogen bonded catalytic dyads in five enzymes. We asked the question what determines the p Ka order, i.e., what makes a residue proton donor vs a nucleophile. The continuous constant pH molecular dynamics simulations captured the experimental p Ka orders and revealed that the negative nucleophile is stabilized by increased hydrogen bonding and solvent exposure as compared to the proton donor. Surprisingly, this simple trend is not apparent from crystal structures and the static structure-based calculations. While the generality of the findings awaits further testing via a larger set of data, they underscore the role of dynamics in bridging enzyme structures and functions.

Electrical Programming of Soft Matter: Using Temporally Varying Electrical Inputs To Spatially Control Self Assembly.

The growing importance of hydrogels in translational medicine has stimulated the development of top-down fabrication methods, yet often these methods lack the capabilities to generate the complex matrix architectures observed in biology. Here we show that temporally varying electrical signals can cue a self-assembling polysaccharide to controllably form a hydrogel with complex internal patterns. Evidence from theory and experiment indicate that internal structure emerges through a subtle interplay between the electrical current that triggers self-assembly and the electrical potential (or electric field) that recruits and appears to orient the polysaccharide chains at the growing gel front. These studies demonstrate that short sequences (minutes) of low-power (∼1 V) electrical inputs can provide the program to guide self-assembly that yields hydrogels with stable, complex, and spatially varying structure and properties.

Proton-Coupled Conformational Allostery Modulates the Inhibitor Selectivity for ß-Secretase.

Many important pharmaceutical targets, such as aspartyl proteases and kinases, exhibit pH-dependent dynamics, functions and inhibition. Accurate prediction of their binding free energies is challenging because current computational techniques neglect the effects of pH. Here we combine free energy perturbation calculations with continuous constant pH molecular dynamics to explore the selectivity of a small-molecule inhibitor for ß-secretase (BACE1), an important drug target for Alzheimer's disease. The calculations predicted identical affinity for BACE1 and the closely related cathepsin D at high pH; however, at pH 4.6 the inhibitor is selective for BACE1 by 1.3 kcal/mol, in excellent agreement with experiment. Surprisingly, the pH-dependent selectivity can be attributed to the protonation of His45, which allosterically modulates a loop-inhibitor interaction. Allosteric regulation induced by proton binding is likely common in biology; considering such allosteric sites could lead to exciting new opportunities in drug design.

Blockade of dengue virus infection and viral cytotoxicity in neuronal cells in vitro and in vivo by targeting endocytic pathways.

Dengue virus (DENV) infection in neuronal cells was speculated to trigger neuropathy. Herein, we determined the blockade of DENV infection by targeting endocytic pathways in vitro and in vivo. In DENV-infected mouse brains, we previously showed that viral proteins are expressed in neuronal cells around the hippocampus with accompanying neurotoxicity. DENV caused infection, including entry, double-stranded (ds)RNA replication, protein expression, and virus release, followed by cytotoxicity in the mouse neuronal Neuro-2a cell line. Pharmacologically blocking clathrin-mediated endocytosis of the DENV retarded viral replication. Targeting vacuolar-type H+-ATPase (V-ATPase)-based endosomal acidification effectively blocked the DENV replication process, but had no direct effect on viral translation. Blockade of the clathrin- and V-ATPase-based endocytic pathways also attenuated DENV-induced neurotoxicity. Inhibiting endosomal acidification effectively retarded DENV infection, acute viral encephalitis, and mortality. These results demonstrate that clathrin mediated endocytosis of DENV followed by endosomal acidification-dependent viral replication in neuronal cells, which can lead to neurotoxicity.

Targeting heat shock factor 1 as an antiviral strategy against dengue virus replication in vitro and in vivo.

Fever onset is correlated with viremia in dengue virus (DENV) patients. Heat shock factor 1 (HSF1), a heat stress response host transcription factor, plays a crucial role in regulating multiple cellular functions, as well as the onset of infectious diseases. This study evaluated the role of HSF1 in DENV replication as a means of regulating DENV infection in vitro and in vivo. DENV infection activated HSF1 in both Ca2+ and protein kinase A-dependent manners. Inhibiting HSF1 effectively reduced DENV replication, not only in THP-1 cells but also in primary human monocytes. Activated HSF1 contributed to DENV replication by upregulating autophagy-related protein (Atg) 7, as autophagy is crucial for virus replication. Heat stress also activated HSF1, which in turn facilitated DENV replication. Activated HSF1, the increased Atg7, and autophagic induction were founded in the DENV-infected brains and pharmacologically inhibiting HSF1 reduced autophagy, viral protein expression, neuropathy, and mortality. These results provide new insight into HSF1 as a novel host factor for DENV infection through its role in facilitating autophagy-regulated viral replication in the brains.

Trowaglerix Venom Polypeptides As a Novel Antithrombotic Agent by Targeting Immunoglobulin-Like Domains of Glycoprotein VI in Platelet.

OBJECTIVE: Currently prescribed antiplatelet drugs have 1 common side effect-an increased risk of hemorrhage and thrombocytopenia. On the contrary, bleeding defects associated with glycoprotein VI (GPVI) expression deficiency are usually slightly prolonged bleeding times. However, GPVI antagonists are lacking in clinic. APPROACH AND RESULTS: Using reverse-phase high-performance liquid chromatography and sequencing, we revealed the partial sequence of trowaglerix α subunit, a potent specific GPVI-targeting snaclec (snake venom C-type lectin protein). Hexapeptide (Troα6 [trowaglerix a chain hexapeptide, CKWMNV]) and decapeptide (Troα10) derived from trowaglerix specifically inhibited collagen-induced platelet aggregation through blocking platelet GPVI receptor. Computational peptide design helped to design a series of Troα6/Troα10 peptides. Protein docking studies on these decapeptides and GPVI suggest that Troα10 was bound at the lower surface of D1 domain and outer surface of D2 domain, which was at the different place of the collagen-binding site and the scFv (single-chain variable fragment) D2-binding site. The newly discovered site was confirmed by inhibitory effects of polyclonal antibodies on collagen-induced platelet aggregation. This indicates that D2 domain of GPVI is a novel and important binding epitope on GPVI-mediated platelet aggregation. Troα6/Troα10 displayed prominent inhibitory effect of thrombus formation in fluorescein sodium-induced platelet thrombus formation of mesenteric venules and ferric chloride-induced carotid artery injury thrombosis model without prolonging the in vivo bleeding time. CONCLUSIONS: We develop a novel antithrombotic peptides derived from trowaglerix that acts through GPVI antagonism with greater safety-no severe bleeding. The binding epitope of polypeptides on GPVI is novel and important. These hexa/decapeptides have therapeutic potential for developing ideal small-mass GPVI antagonists for arterial thrombogenic diseases.

Conformational dynamics of cathepsin D and binding to a small-molecule BACE1 inhibitor.

BACE1 is a major therapeutic target for prevention and treatment of Alzheimer's disease. Developing inhibitors that can selectively target BACE1 in favor of other proteases, especially cathepsin D (CatD), has presented significant challenges. Here, we investigate the conformational dynamics and protonation states of BACE1 and CatD using continuous constant pH molecular dynamics with pH replica-exchange sampling protocol. Despite similar structure, BACE1 and CatD exhibit markedly different active site dynamics. BACE1 displays pH-dependent flap dynamics that controls substrate accessibility, while the CatD flap is relatively rigid and remains open in the pH range 2.5-6. Interestingly, although each protease hydrolyzes peptide bonds, the protonation states of the catalytic dyads are different within the active pH range. The acidic and basic components of the BACE1 catalytic dyad are clear, while either aspartic acid of the CatD catalytic dyad could play the role of acid or base. Finally, we investigate binding of the inhibitor LY2811376 developed by Eli Lilly to BACE1 and CatD. Surprisingly, in the enzyme active pH range, LY2811376 forms a stronger salt bridge with the catalytic dyad in CatD than in BACE1, which might explain the retinal toxicity of the inhibitor related to off-target inhibition of CatD. This work highlights the complexity and challenge in structure-based drug design where receptor-ligand binding induces protonation state change in both the protein and the inhibitor. © 2017 Wiley Periodicals, Inc.

Dengue virus infection increases microglial cell migration.

Activated microglial cells are present in dengue virus (DENV)-infected brains; however, the possible effects of DENV on microglia remain unclear. Here, we demonstrated DENV caused infection, including viral entry, RNA replication, viral protein expression, and virus release, in the murine microglial cell line BV2. DENV infection caused an increase in the formation of the multipolar phenotype in vitro and in vivo without affecting cell growth and cytotoxicity. DENV infection considerably increased cell motility and disrupting either actin filaments or clathrin retarded such effect. Increase in cell migration was only occurred by DENV infection following a clathrin-regulated endocytosis of DENV entry. Ultraviolet-inactivated DENV did not affect cell migration, and pharmacologically blocking toll-like receptor (TLR) 3 and TLR3-related signaling pathways reduced the DENV-induced increase in cell migration. These results demonstrate an advanced effect of DENV infection on microglial migration via a mechanism involving viral entry, RNA release, and TLR3 signal activation.