Simultaneous EEG-fMRI Investigation of Rhythm-Dependent Thalamo-Cortical Circuits Alteration in Schizophrenia.

Schizophrenia is accompanied by aberrant interactions of intrinsic brain networks. However, the modulatory effect of electroencephalography (EEG) rhythms on the functional connectivity (FC) in schizophrenia remains unclear. This study aims to provide new insight into network communication in schizophrenia by integrating FC and EEG rhythm information. After collecting simultaneous resting-state EEG-functional magnetic resonance imaging data, the effect of rhythm modulations on FC was explored using what we term "dynamic rhythm information." We also investigated the synergistic relationships among three networks under rhythm modulation conditions, where this relationship presents the coupling between two brain networks with other networks as the center by the rhythm modulation. This study found FC between the thalamus and cortical network regions was rhythm-specific. Further, the effects of the thalamus on the default mode network (DMN) and salience network (SN) were less similar under alpha rhythm modulation in schizophrenia patients than in controls ([Formula: see text]). However, the similarity between the effects of the central executive network (CEN) on the DMN and SN under gamma modulation was greater ([Formula: see text]), and the degree of coupling was negatively correlated with the duration of disease ([Formula: see text], [Formula: see text]). Moreover, schizophrenia patients exhibited less coupling with the thalamus as the center and greater coupling with the CEN as the center. These results indicate that modulations in dynamic rhythms might contribute to the disordered functional interactions seen in schizophrenia.

A generalizable reactive blending strategy to construct flame-retardant, mechanically-strong and toughened poly(L-lactic acid) bioplastics.

Poly(L-lactic acid) (PLA) is an environmentally-friendly bioplastic with high mechanical strength, but suffers from inherent flammability and poor toughness. Many tougheners have been reported for PLA, but their synthesis usually involves organic solvents, and they tend to dramatically reduce the mechanical strength and cannot settle the flammability matter. Herein, we develop strong, tough, and flame-retardant PLA composites by reactive blending PLA, 6-((double (2-hydroxyethyl) amino) methyl) dibenzo [c, e] [1,2] oxyphosphate acid 6-oxide (DHDP) and diphenylmethane diisocyanate (MDI) and define it PLA/xGH, where x indicates that the molar ratio of -NCO group in MDI to -OH group in PLA and DHDP is 1.0x: 1. This fabrication requires no solvents. PLA/2GH with a -NCO/-OH molar ratio of 1.02: 1 maintains high tensile strength of 63.0 MPa and achieves a 23.4 % increase in impact strength compared to PLA due to the incorporation of rigid polyurethane chain segment. The vertical combustion (UL-94) classification and limiting oxygen index (LOI) of PLA/2GH reaches V-0 and 29.8 %, respectively, because DHDP and MDI function in gas and condensed phases. This study displays a generalizable strategy to create flame-retardant bioplastics with great mechanical performances by the in-situ formation of P/N-containing polyurethane segment within PLA.

Flame-retardant poly(L-lactic acid) with enhanced UV protection and well-preserved mechanical properties by a furan-containing polyphosphoramide.

Despite good biodegradability and mechanical strength, the intrinsic flammability of poly(L-lactic acid) (PLA) impede its practical application. Introducing phosphoramide is an effective method to enhance the flame retardancy of PLA. However, most of the reported phosphoramides derive from petroleum resources, and their addition tends to deteriorate the mechanical properties, especially toughness, of PLA. Herein, a bio-based, furan-containing polyphosphoramide (DFDP) with high flame-retardant efficiency was synthesized for PLA. Our study found that 2 wt% DFDP enabled PLA to pass a UL-94 V-0 rating, and 4 wt% DFDP increased the limiting oxygen index (LOI) to 30.8 %. DFDP effectively maintained the mechanical strength and toughness of PLA. The tensile strength of PLA with 2 wt% DFDP reached 59.9 MPa, and its elongation at break and impact strength were increased by 15.8 % and 34.3 %, respectively, relative to those of virgin PLA. The UV protection of PLA was significantly enhanced by introducing DFDP. Hence, this work provides a sustainable and comprehensive strategy for the creation of flame-retardant biomaterials with improved UV protection and well-preserved mechanical properties, which possess a broad prospect in industrial application.

Synthesis and Biological Evaluation of 5'-O-Fatty Acyl Ester Derivatives of 3'-Fluoro-2',3'-dideoxythymidine as Potential Anti-HIV Microbicides.

A number of 5'-O-fatty acyl derivatives of 3'-fluoro-2',3'-dideoxythymidine (FLT, 1) were synthesized. These conjugates were evaluated for their potential as topical microbicides with anti-HIV activity against cell-free (X4 and R5), cell-associated, and multidrug-resistant viruses. Compared to FLT and 3'-azido-2',3'-dideoxythymidine (AZT), 5'-O-(12-azidododecanoyl) (5), 5'-O-myristoyl (6), and 5'-O-(12-thioethyldodecanoyl) (8) derivatives of FLT were found to be more active against both cell-free viruses (lymphocytotropic and monocytotropic strains) with EC50 values of 0.4 µM, 1.1 µM, and <0.2 µM, respectively, as well as cell-associated virus with EC50 values of 12.6, 6.4, and 2.3 µM, respectively. Conjugates 5, 6, and 8 exhibited >4 and >30 times better antiviral index than FLT and AZT, respectively. Conjugates 5 and 8 were significantly more potent than FLT against many multidrug-resistant strains. A comparison of the anti-HIV activity with the corresponding non-hydrolyzable ether conjugates suggested that ester hydrolysis to FLT and fatty acids is critical to enable anti-HIV activity. Cellular uptake studies were conducted using fluorescent derivatives of FLT attached with 5(6)-carboxyfluorescein through either ß-alanine (23) or 12-aminododecanoic acid (24) spacers. The lipophilic fluorescent analog with a long chain (24) showed more than 12 times higher cellular uptake profile than the fluorescent analog with a short chain (23). These studies further confirmed that the attachment of fatty acids improved the cellular uptake of nucleoside conjugates. In addition, 5, 6, and 8 were the least cytotoxic and did not alter vaginal cell and sperm viability compared to the positive control, a commercial topical spermicide (N-9), which significantly decreased sperm and vaginal cell viability inducing the generation of proinflammatory cytokines.

Self-Assembled Surfactant Cyclic Peptide Nanostructures as Stabilizing Agents.

A number of cyclic peptides including [FR]4, [FK]4, [WR]4, [CR]4, [AK]4, and [WK]n (n = 3-5) containing L-amino acids were produced using solid-phase peptide synthesis. We hypothesized that an optimal balance of hydrophobicity and charge could generate self-assembled nanostructures in aqueous solution by intramolecular and/or intermolecular interactions. Among all the designed peptides, [WR]n (n = 3-5) generated self-assembled vesicle-like nanostructures at room temperature as shown by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and/or dynamic light scattering (DLS). This class of peptides represents the first report of surfactant-like cyclic peptides that self-assemble into nanostructures. A plausible mechanistic insight into the self-assembly of [WR]5 was obtained by molecular modeling studies. Modified [WR]5 analogues, such as [WMeR]5, [WR(Me)2]5, [WMeR(Me)2]5, and [WdR]5, exhibited different morphologies to [WR]5 as shown by TEM observations. [WR]5 exhibited a significant stabilizing effect for generated silver nanoparticles and glyceraldehyde-3-phosphate dehydrogenase activity. These studies established a new class of surfactant-like cyclic peptides that self-assembled into nanostructures and could have potential applications for the stabilization of silver nanoparticles and protein biomolecules.

Synthesis and biological evaluation of fatty acyl ester derivatives of (-)-2',3'-dideoxy-3'-thiacytidine.

A number of fatty acyl derivatives of (-)-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC, 1) were synthesized and evaluated for their anti-HIV activity. The monosubstituted 5'-O-fatty acyl derivatives of 3TC (EC(50) = 0.2-2.3 µM) were more potent than the corresponding monosubstituted N(4)-fatty acyl (EC(50) = 0.4-29.4 µM) and 5'-O-N(4)-disubstituted (EC(50) = 72.6 to >154.0 µM) derivatives of the nucleoside. 5'-O-Myristoyl (16) and 5'-O-12-azidododecanoyl derivatives (17) were found to be the most potent compounds (EC(50) = 0.2-0.9 µM) exhibiting at least 16-36-fold higher anti-HIV activity against cell-free virus than 1 (EC(50) = 11.4-32.7 µM). The EC(90) values for 16 against B-subtype and C-subtype clinical isolates were several folds lower than those of 1. The cellular uptake studies confirmed that compound 16 accumulated intracellularly after 1 h of incubation with CCRF-CEM cells and underwent intracellular hydrolysis. 5'-O-Fatty acyl derivatives of 1 showed significantly higher anti-HIV activity than the corresponding physical mixtures against the B-subtype virus.

The use of strong anion-exchange (SAX) magnetic particles for the extraction of therapeutic siRNA and their analysis by liquid chromatography/mass spectrometry.

Traditional methods for extracting oligonucleotides from serum and other biological fluids are often time-consuming and require multiple steps. Magnetic particle based separation of oligonucleotides has gained importance recently due to the advantages of simplicity and high efficiency. Here we report the development and optimization of commercially available strong anion-exchange (SAX) magnetic beads for the extraction of siRNA from human serum. The beads allowed for rapid extraction of siRNA from human serum in 100-200 µL of liquid chromatography/mass spectrometry (LC/MS)-compatible buffer in less than 1 h for a 96-well plate with no further drying steps. Due to the strong cation-binding properties of oligonucleotides, volatile ammonium salts such as triethylammonium bicarbonate (TEAB), ammonium bicarbonate, and NH(4) Cl were used to elute the siRNA from the beads. For more hydrophobic siRNA sequences, the addition of 5-10% organic solvent was required for elution. The recovery of chemically modified siRNA from human serum was around 80% for two types of beads examined; however, the recovery for highly modified sequences differed greatly between the two types of beads. In addition to extracting highly modified oligonucleotides, the SAX beads were also able to extract liposomal formulated siRNAs from serum with no interference from the lipid formulation. The extraction of siRNA from human serum was linear over the tested range of 50 ng/mL to 10 µg/mL. Using this extraction methodology, we have created a workflow to monitor siRNA serum stability by LC/MS. Initial observations confirm that RNase A type degradation with strand cleavage on the 3' side of uridine or cytosine is the dominant cleavage pattern in serum. This finding has implications for the selection and modification of therapeutic siRNAs and demonstrates the utility of magnetic beads as a simple and rapid extraction technique for siRNA.

Bilayer disruption and liposome restructuring by a homologous series of small Arg-rich synthetic peptides.

The effects of a series of low molecular weight water-soluble cationic linear peptide analogs (LPAs, <1000 MW) with increasing hydrophobic/hydrophilic balance on lipid bilayer phase behavior and permeability were examined using liposomes composed of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and mixed zwitterionic/anionic DPPC/dipalmitoylphosphatidylglycerol (DPPG) lipid bilayers. LPAs were synthesized using a previously reported alkyl linkage strategy as Arg-C(n)-Arg-C(n)-Lys, where C(n) represents the saturated alkyl linkage separating the cationic residues (n=4, 7, or 11) (Ye et al., 2007 [1]). Differential scanning calorimetry results show that the cationic LPAs bound to and disrupted DPPC and, to a greater extent, DPPC/DPPG phase behavior. When added to preformed unilamellar liposomes, the LPAs led to significant structural changes based on cryogenic transmission electron microscopy (cryo-TEM). Coupling cryo-TEM with carboxyfluorescein leakage studies indicate that the LPAs induced permeabilization through bilayer expansion, which caused membrane thinning. The effects were inconsistent with increasing LPA hydrophobicity, which suggests that a cooperative effect between electrostatic binding and hydrophobic insertion determined the location of LPAs within the bilayer and their membrane activity. Our results for LPA-induced membrane disruption correlate with previous breast cancer cell uptake studies that showed minimal LPA-C(4) uptake, but high LPA-C(11) uptake through a non-endocytic mechanism.

Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain.

Phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) has been introduced as an optimal Src SH2 domain ligand. Peptides, Ac-K(IDA)pYEEIEK(IDA) (1), Ac-KpYEEIEK (2), Ac-K(IDA)pYEEIEK (3), and Ac-KpYEEIEK(IDA) (4), containing 0-2 iminodiacetate (IDA) groups at the N- and C-terminal lysine residues were synthesized and evaluated as the Src SH2 domain binding ligands. Fluorescence polarization assays showed that peptide 1 had a higher binding affinity (K(d) = 0.6 microM) to the Src SH2 domain when compared with Ac-pYEEI (K(d) = 1.7 microM), an optimal Src SH2 domain ligand, and peptides 2-4 (K(d) = 2.9-52.7 microM). The binding affinity of peptide 1 to the SH2 domain was reduced by more than 2-fold (K(d) = 1.6 microM) upon addition of Ni(2+) (300 microM), possibly due to modest structural effect of Ni(2+) on the protein as shown by circular dichroism experimental results. The binding affinity of 1 was restored in the presence of EDTA (300 microM) (K(d) = 0.79 microM). These studies suggest that peptides containing IDA groups may be used for designing novel SH2 domain binding ligands.

Development of Src tyrosine kinase substrate binding site inhibitors.

The majority of marketed drugs or drug candidates that target protein kinases and which are currently undergoing clinical trials are ATP binding site inhibitors. The process of designing a selective inhibitor as an ATP mimic is challenging, mainly because of the presence of a large number of protein kinases that show a conserved ATP binding site. The substrate binding site of protein kinases is less conserved than the ATP binding site, and provides an opportunity to design valuable chemical tools which can be utilized to understand the catalytic mechanism of the enzyme, or to develop inhibitors with enhanced specificity. In this review, the latest developments of four classes of substrate binding site inhibitors of Src tyrosine kinase are discussed.

Synthesis and evaluation of tripodal peptide analogues for cellular delivery of phosphopeptides.

Tripodal peptide analogues were designed on the basis of the phosphotyrosine binding pocket of the Src SH2 domain and assayed for their ability to bind to fluorescein-labeled phosphopeptides. Fluorescence polarization assays showed that a number of amphipathic linear peptide analogues (LPAs), such as LPA4, bind to fluorescein-labeled GpYEEI (F-GpYEEI). LPA4 was evaluated for potential application in cellular delivery of phosphopeptides. Fluorescence microimaging cellular uptake studies with fluorescein-attached LPA4 (F-LPA4) alone or with the mixture of LPA4 and F-GpYEEI in BT-20 cells showed dramatic increase of the fluorescence intensity in cytosol of cells, indicating that LPA4 can function as a delivery tool of F-GpYEEI across the cell membrane. Fluorescent flow cytometry studies showed the cellular uptake of F-LPA4 in an energy-independent pathway and confirmed the cellular uptake of F-GpYEEI in the presence of LPA4. These studies suggest that amphipathic tripodal peptide analogues, such as LPA4, can be used for cellular delivery of phosphopeptides.

Design and evaluation of hydroxamate derivatives as metal-mediated inhibitors of a protein tyrosine kinase.

Protein tyrosine kinases use two Mg(2+) ions as cofactors in catalysis, one as the ATP-Mg complex (M1) and the other as an essential activator (M2). The M2-binding site has high affinity for transition metal cations such as cobalt and zinc. Taking advantage of this high affinity, we examined hydroxamates as metal-mediated inhibitors against C-terminal Src kinase (Csk), a protein tyrosine kinase. Of a small group of amino acid hydroxamates, tyrosine and phenylalanine hydroxamates inhibited Csk activity only in the presence of Co(2+). Four classes of phenylalanine and tyrosine hydroxamate derivatives were synthesized and evaluated as metal-mediated inhibitors of Csk, leading to improved inhibition and a better understanding of the structure-activity relationships. This study suggests that hydroxamates may serve as a general scaffold for developing metal-mediated inhibitors against protein tyrosine kinases. To the best of our knowledge, this is the first report of designing metal-mediated inhibitors against a protein tyrosine kinase by targeting a metal binding site.

Synthesis of polymer-bound 4-acetoxy-3-phenylbenzaldehyde derivatives: applications in solid-phase organic synthesis.

Aminomethyl polystyrene resin was reacted with 4-(5'-formyl-2'-hydroxyphenyl)benzoic acid and 4-(5'-formyl-2'-hydroxyphenyl)phenyl propionic acid, respectively, in the presence of 1-hydroxybenzotriazole and 1,3-diisopropylcarbodiimide to yield polymer-bound benzaldehydes. The phenolic group in resins was acetylated with acetic anhydride to afford two polymer-bound 4-acetoxybenzaldehydes. The reductive amination of polymer-bound linkers by amines and sodium triacetoxyborohydride, followed by sulfonylation with arylsulfonyl chloride derivatives in the presence of pyridine and the cleavage with TFA/DCM/H2O, produced pure sulfonamides.

Synthesis and structure-activity relationships of linear and conformationally constrained peptide analogues of CIYKYY as Src tyrosine kinase inhibitors.

A series of peptide analogues of Ac-CIYKYY (1) were synthesized by functional group modifications in peptide side chains or by introducing conformational constraints, to improve the inhibitory potency against active Src kinase. Ac-CIYKF(4-NO2)Y (2, IC50 = 0.53 microM) and conformationally constrained peptide 31 (IC50 = 0.28 microM) exhibited 750- and 1400-fold higher inhibitory activities, respectively, versus that of 1 (IC50 = 400 microM). Compound 2 exhibited a partial competitive inhibition pattern against ATP.

Solid-phase binding assays of peptides using EGFP-Src SH2 domain fusion protein and biotinylated Src SH2 domain.

Two solid-phase binding assays were designed and evaluated for their potential use in comparing the affinity of peptides to the Src SH2 domain. Resin beads attached to peptides were incubated with the enhanced green fluorescence protein(EGFP)-Src SH2 domain fusion protein or the biotinylated Src SH2 domain and extensively washed. The beads-attached tetrapeptides with high affinities to the EGFP-Src SH2 domain showed more fluorescence intensity than those beads containing tetrapeptides with weak binding affinities, as shown by fluorescence microscopy and fluorescence imaging system. Only the beads attached to pYEEI produced a dark purple color on incubation of the beads, respectively, with the biotinylated Src kinases SH2 domain, alkaline phosphatase-coupled streptavidin, and BCIP/NBT. These solid-phase binding assays may have potential applications for the screening of peptides for the Src kinases SH2 domains.

Functional diversity of Csk, Chk, and Src SH2 domains due to a single residue variation.

The C-terminal Src kinase (Csk) family of protein tyrosine kinases contains two members: Csk and Csk homologous kinase (Chk). Both phosphorylate and inactivate Src family kinases. Recent reports suggest that the Src homology (SH) 2 domains of Csk and Chk may bind to different phosphoproteins, which provides a basis for different cellular functions for Csk and Chk. To verify and characterize such a functional divergence, we compared the binding properties of the Csk, Chk, and Src SH2 domains and investigated the structural basis for the functional divergence. First, the study demonstrated striking functional differences between the Csk and Chk SH2 domains and revealed functional similarities between the Chk and Src SH2 domains. Second, structural analysis and mutagenic studies revealed that the functional differences among the three SH2 domains were largely controlled by one residue, Glu127 in Csk, Ile167 in Chk, and Lys200 in Src. Mutating these residues in the Csk or Chk SH2 domain to the Src counterpart resulted in dramatic gain of function similar to Src SH2 domain, whereas mutating Lys200 in Src SH2 domain to Glu (the Csk counterpart) resulted in loss of Src SH2 function. Third, a single point mutation of E127K rendered Csk responsive to activation by a Src SH2 domain ligand. Finally, the optimal phosphopeptide sequence for the Chk SH2 domain was determined. These results provide a compelling explanation for the functional differences between two homologous protein tyrosine kinases and reveal a new structure-function relationship for the SH2 domains.

ATP-phosphopeptide conjugates as inhibitors of Src tyrosine kinases.

A number of Src SH2 domain inhibitors enhance the kinase catalytic activity by switching the closed inactive to the open active conformation. ATP-phosphopeptide conjugates were designed and synthesized as Src tyrosine kinase inhibitors based on a tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI) and ATP to block the SH2 domain signaling and substrate phosphorylation by ATP, respectively. In general, ATP-phosphopeptide conjugates with optimal linkers such as compounds 5 and 7 (K(i) = 1.7-2.6 microM) showed higher binding affinities to the ATP-binding site relative to the other ATP-phosphopeptide conjugates having short or long linkers, 1-4 and 6, (K(i) = 10.1-16.1 microM) and ATP (K(m) = 74 microM). These ATP-phosphopeptide conjugates may serve as novel templates for designing protein tyrosine kinase inhibitors to block SH2 mediated protein-protein interactions and to counter the activation of enzyme that resulted from the SH2 inhibition.

Copper dipicolinates as peptidomimetic ligands for the Src SH2 domain.

The introduction of copper chelates into peptide mimetics creates the Src SH2 binding ligands and paramagnetic complexes suitable for EPR studies of peptide protein interactions. The dipicolinic acid was attached to SH2 domain targeting fragments by two different linkers.

Conformationally constrained peptide analogues of pTyr-Glu-Glu-Ile as inhibitors of the Src SH2 domain binding.

A series of conformationally constrained peptides were designed and synthesized as the Src SH2 domain ligands based on a tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI). In general, the constrained peptides such as compounds 6, 7, and 11 (IC(50) = 1.1-1.5 microM) showed higher binding affinities to the Src SH2 domain relative to the corresponding linear peptides 8a, 9a, and 13a, respectively (IC(50) > 100 microM), and pYEEI (IC(50) = 6.5 microM), as evaluated by a fluorescence polarization assay. Molecular modeling studies revealed that in constrained peptides, the isoleucine side chain penetrates very deeply into the hydrophobic binding pocket (P + 3 site) of the Src SH2 domain. These constrained peptides can serve as novel templates for the design of small and nonpeptidic inhibitors of the Src SH2 domain.