A carbon-based fluorescent probe (N-CDs) encapsulated in a zeolite matrix (NaFZ) for ultrasensitive detection of Hg (II) in fish.

In this work, a novel strategy was addressed to fabricate new sensing probe (N-CDs@NaFZ) from nitrogen doped carbon dots (N-CDs) confined in Al-free ferrisilicates zeolite (NaFZ) by hydrothermal/solvothermal method. The probe was systematically characterized by HR-TEM, FTIR, energy dispersive X-ray (EDX), powder X-ray diffraction, and UV-Vis absorption and fluorescence spectrophotometers. Characterization of the designed nanocomposite template N-CDs@NaFZ by fluorescence spectrum demonstrates a variety of important conducts as stability improvements, reasonable dispersibility in water, highly emission intensity enhancement at 435 nm when excited at 340 nm, excitation independent fluorescence behaviors, great quantum yield percentage of 91.2%, and narrow size distribution 12 nm, as a nano-space confinement effect of zeolite effectively increase the rigidity of N-CDs. Based on the fluorescence quenching mechanism, the designed approach exhibits an excellent selectivity and good sensitive response to the presence of Hg(II) ions under ambient temperature, with a wide linear range of 0.1-1500 nM and lower detection limits of 5.5 pM. Influences of variables pH and incubation time were optimized. The N-CDs@NaFZ sensor was effectively applied for the detection of Hg(II) ions in the farmed and wild rainbow trout fishes, and the results are in reasonable agreement when compared with that obtained by the cold vapor atomic absorption method.

Directed remodeling of the mouse gut microbiome inhibits the development of atherosclerosis.

The gut microbiome is a malleable microbial community that can remodel in response to various factors, including diet, and contribute to the development of several chronic diseases, including atherosclerosis. We devised an in vitro screening protocol of the mouse gut microbiome to discover molecules that can selectively modify bacterial growth. This approach was used to identify cyclic D,L-α-peptides that remodeled the Western diet (WD) gut microbiome toward the low-fat-diet microbiome state. Daily oral administration of the peptides in WD-fed LDLr-/- mice reduced plasma total cholesterol levels and atherosclerotic plaques. Depletion of the microbiome with antibiotics abrogated these effects. Peptide treatment reprogrammed the microbiome transcriptome, suppressed the production of pro-inflammatory cytokines (including interleukin-6, tumor necrosis factor-α and interleukin-1ß), rebalanced levels of short-chain fatty acids and bile acids, improved gut barrier integrity and increased intestinal T regulatory cells. Directed chemical manipulation provides an additional tool for deciphering the chemical biology of the gut microbiome and might advance microbiome-targeted therapeutics.

Design of a DNA-Programmed Plasminogen Activator.

Although the functional specificity and catalytic versatility of enzymes have been exploited in numerous settings, controlling the spatial and temporal activity of enzymes remains challenging. Here we describe an approach for programming the function of streptokinase (SK), a protein that is clinically used as a blood "clot buster" therapeutic. We show that the fibrinolytic activity resulting from the binding of SK to the plasma proenzyme plasminogen (Pg) can be effectively regulated (turned "OFF" and "ON") by installing an intrasteric regulatory feature using a DNA-linked protease inhibitor modification. We describe the design rationale, synthetic approach, and functional characterization of two generations of intrasterically regulated SK-Pg constructs and demonstrate dose-dependent and sequence-specific temporal control in fibrinolytic activity in response to short predesignated DNA inputs. The studies described establish the feasibility of a new enzyme-programming approach and serves as a step toward advancing a new generation of programmable enzyme therapeutics.

Synthesis of stabilized hydroxyapatite nanosuspensions for enamel caries remineralization.

BACKGROUND: The aim of this study was to develop and evaluate a method for synthesizing a stable suspension of hydroxyapatite nanoparticles and to test its efficacy for remineralizing carious enamel lesions. METHODS: Hydroxyapatite (HA) particles were synthesized using wet chemistry. Synthesized particles were introduced into a high-pressure homogenizer (5-10 homogenization passes at 15 000 psi) in the presence of different stabilizers. Size and distribution of the resultant particles were determined using dynamic light scattering (DLS). The morphology and composition of the nanoparticles were determined using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Subsequently, artificial lesions were treated with HA nanosuspension plus artificial saliva or a fluoride-containing artificial saliva only. Visual analysis and quantification of the lesion mineral density before and after remineralization were performed using microcomputed tomography. RESULTS: DLS and SEM results confirmed the formation of nonagglomerated HA nanoparticles (20-40 nm) following high-pressure homogenization treatment. Quantitative evaluation of the lesions showed that remineralization of the lesion with hydroxyapatite nanosuspension led to a significantly higher level of mineral gain compared to the control group (P < 0.05). CONCLUSION: High-pressure homogenization is an effective method for facile preparation of a stable suspension of hydroxyapatite nanoparticles. Treatment of artificial lesions with nonagglomerated spherical HA nanoparticles improves the remineralization of enamel lesion.

A kinetically controlled, isothermal method for the detection of single nucleotide mismatches.

We describe an isothermal, enzyme-free method to detect single nucleotide differences between oligonucleotides of close homology. The approach exploits kinetic differences in toe-hold-mediated, nucleic acid strand-displacement reactions to detect single nucleotide polymorphisms (SNPs) with essentially "digital" precision. The theoretical underpinning, experimental analyses, predictability, and accuracy of this new method are reported. We demonstrate detection of biologically relevant SNPs and single nucleotide differences in the let-7 family of microRNAs. The method is adaptable to microarray formats, as demonstrated with on-chip detection of SNP variants involved in susceptibility to the therapeutic agents abacavir, Herceptin, and simvastatin.

Evaluation of a new dispersion technique for assessing triboelectric charging of powders.

In a number of applications, especially in pharmaceutical drug development, there is often a very small powder quantity available for evaluating the manufacturability of new drugs. However, it is highly desirable to be able to quickly evaluate processing issues, and where possible using the smallest powder quantity. In the present work, a proprietary commercial powder dispersion device (the disperser of Malvern© Morphologi G3) is adapted to evaluate the triboelectric charging tendency. A very small powder quantity (as small as 0.1 mg) is dispersed by a pressure pulse of compressed gas such as air or nitrogen. This causes the particles to become air borne and collide with the containing walls, resulting in dispersion and leading to triboelectric charge transfer between the particles and the walls. In this work, the charging propensity of a number of materials is evaluated and the effect of particle surface functional groups on the tribo-electric charge transfer is analysed. Model materials with a well-defined shape (glass ballotini) but with different silane groups deposited on their surfaces as well as a number of organic crystalline particles (such as aspirin, α-lactose monohydrate and paracetamol) are tested. Following dispersion the particles move immediately to a Faraday cup placed directly underneath the disperser. Therefore, particle charge is measured with no decay. The method can differentiate charging of different polymorphs of the same material, different silane groups on the surfaces of glass ballotini and different crystal morphologies obtained from crystallisation from various solvents.

Impact breakage of pharmaceutical tablets.

Tablets are the most common solid dosage form of pharmaceutical active ingredients due to their ease of use. Their dissolution behaviour depends on the particle size distribution and physicochemical properties of the formulation, and the compression process, which need to be optimised for producing consistently robust tablets, as weaker tablets are often prone to breakage during production, transport and end use. Tablet strength is typically determined by diametric compression and friability tests. The former gives rise to propagation of a crack on a plane along the compression axis, whilst the latter, carried out in a rotating drum, incurs surface damage and produces chips and debris. These tests produce different measures of strength, neither of which have been correlated with mechanical properties that are accountable for breakage, i.e. hardness, elastic modulus and fracture toughness. We propose a new method based on single tablet impact testing, following the work of Ghadiri and Zhang, 2002, who analysed particle damage by propagation of sub-surface lateral cracks and identified the fundamental form accountable for impact surface damage to be a lumped parameter related to hardness and fracture toughness. Microindentation, carried out separately, to determine fracture toughness led to complete failure of the tablets, hence an unreliable measurement of fracture toughness and no correlation with the experimental trend. In addition, by assuming the fracture toughness to be proportional to the square root of Young's modulus, the indentation measurements do not correlate well with the impact breakage. The discrepancy between the impact and indentation methods is expected to be due to mechanical property variation across the tablet surface, and with strain rate. The impact method is a more suitable test to describe tablet propensity for attrition as it directly represents the failure mode tablets may experience during processing under well-defined conditions. In contrast, the friability test subjects tablets to a similar breakage mechanism but under less well-defined conditions, whilst the compression test represents a different failure mode that is not representative of stresses incurred during processing.

Templated Self-Assembly of Dynamic Peptide Nucleic Acids.

Template-directed macromolecule synthesis is a hallmark of living systems. Inspired by this natural process, several fundamentally novel mechanisms for template-directed assembly of nucleic acid analogues have been developed. Although these approaches have broad significance, including potential applications in biotechnology and implications for the origins of life, there are unresolved challenges in how to characterize in detail the complex assembly equilibria associated with dynamic templated reactions. Here we describe mechanistic studies of template-directed dynamic assembly for thioester peptide nucleic acid (tPNA), an informational polymer that responds to selection pressures under enzyme-free conditions. To overcome some of the inherent challenges of mechanistic studies of dynamic oligomers, we designed, synthesized, and implemented tPNA-DNA conjugates. The DNA primer region affords a high level of control over the location and register of the tPNA backbone in relation to the template strand. We characterized the degree and kinetics of dynamic nucleobase mismatch correction at defined backbone positions. Furthermore, we report the fidelity of dynamic assembly in tPNA as a function of position along the peptide backbone. Finally, we present theoretical studies that explore the level of fidelity that can be expected for an oligomer having a given hybridization affinity in dynamic templated reactions and provide guidance for the future development of sequence self-editing polymers and materials. As our results demonstrate, the use of molecular conjugates of constitutionally static and dynamic polymers establishes a new methodology for expediting the characterization of the complex chemical equilibria that underlie the assembly of dynamic informational polymers.

Self-Assembling Cyclic d,l-α-Peptides as Modulators of Plasma HDL Function. A Supramolecular Approach toward Antiatherosclerotic Agents.

There is great interest in developing new modes of therapy for atherosclerosis to treat coronary heart disease and stroke, particularly ones that involve modulation of high-density lipoproteins (HDLs). Here, we describe a new supramolecular chemotype for altering HDL morphology and function. Guided by rational design and SAR-driven peptide sequence enumerations, we have synthesized and determined the HDL remodeling activities of over 80 cyclic d,l-α-peptides. We have identified a few distinct sequence motifs that are effective in vitro in remodeling human and mouse plasma HDLs to increase the concentration of lipid-poor pre-beta HDLs, which are key initial acceptors of cholesterol in the reverse cholesterol transport (RCT) process, and concomitantly promote cholesterol efflux from macrophage cells. Functional assays with various control peptides, such as scrambled sequences, linear and enantiomeric cyclic peptide variants, and backbone-modified structures that limit peptide self-assembly, provide strong support for the supramolecular mode of action. Importantly, when the lead cyclic peptide c[wLwReQeR] was administered to mice (ip), it also promoted the formation of small, lipid-poor HDLs in vivo, displayed good plasma half-life (∼6 h), did not appear to have adverse side effects, and exerted potent anti-inflammatory effects in an acute in vivo inflammation assay. Given that previously reported HDL remodeling peptides have been based on α-helical apoA-I mimetic architectures, the present study, involving a new structural class, represents a promising step toward new potential therapeutics to combat atherosclerosis.

Cyclic tetrapeptide HDAC inhibitors as potential therapeutics for spinal muscular atrophy: Screening with iPSC-derived neuronal cells.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that is caused by inactivating mutations in the Survival of motor neuron 1 (SMN1) gene, resulting in decreased SMN protein expression. Humans possess a paralog gene, SMN2, which contains a splicing defect in exon 7 leading to diminished expression of full-length, fully functional SMN protein. Increasing SMN2 expression has been a focus of therapeutic development for SMA. Multiple studies have reported the efficacy of histone deacetylase inhibitors (HDACi) in this regard. However, clinical trials involving HDACi have been unsatisfactory, possibly because previous efforts to identify HDACi to treat SMA have employed non-neuronal cells as the screening platform. To address this issue, we generated an SMA-patient specific, induced pluripotent stem cell (iPSC) derived neuronal cell line that contains homogenous Tuj1+neurons. We screened a small library of cyclic tetrapeptide HDACi using this SMA neuronal platform and discovered compounds that elevate SMN2 expression by an impressive twofold or higher. These candidates are also capable of forming gems intranuclearly in SMA neurons, demonstrating biological activity. Our study identifies new potential HDACi therapeutics for SMA screened using a disease-relevant SMA neuronal cellular model.

Peptide Macrocyclization Inspired by Non-Ribosomal Imine Natural Products.

A thermodynamic approach to peptide macrocyclization inspired by the cyclization of non-ribosomal peptide aldehydes is presented. The method provides access to structurally diverse macrocycles by exploiting the reactivity of transient macrocyclic peptide imines toward inter- and intramolecular nucleophiles. Reactions are performed in aqueous media, in the absence of side chain protecting groups, and are tolerant of all proteinogenic functional groups. Macrocyclic products bearing non-native and rigidifying structural motifs, isotopic labels, and a variety of bioorthogonal handles are prepared, along with analogues of four distinct natural products. Structural interrogation of the linear and macrocyclic peptides using variable-temperature NMR and circular dichroism suggests that preorganization of linear substrates is not a prerequisite for macrocyclization.

Natural and Synthetic Macrocyclic Inhibitors of the Histone Deacetylase Enzymes.

Inhibition of histone deacetylase (HDAC) enzymes has emerged as a target for development of cancer chemotherapy. Four compounds have gained approval for clinical use by the Food and Drug Administration in the US, and several are currently in clinical trials. However, none of these compounds possesses particularly good isozyme selectivity, which would be a highly desirable feature in a tool compound. Whether selective inhibition of individual HDAC isozymes will provide improved drug candidates remains to be seen. Nevertheless, it has been speculated that using macrocyclic compounds to target HDAC enzymes might hold an advantage over the use of traditional hydroxamic-acid-containing inhibitors, which rely on chelation to the conserved active-site zinc ion. Here we review the literature on macrocyclic HDAC inhibitors obtained from natural sources and on structure-activity relationship studies inspired by these molecules, as well as on efforts aimed at fully synthetic macrocyclic HDAC inhibitors.

Peptide Bond Formation in Water Mediated by Carbon Disulfide.

Demonstrating plausible nonenzymatic polymerization mechanisms for prebiotic monomers represents a fundamental goal in prebiotic chemistry. While a great deal is now known about the potentially prebiotic synthesis of amino acids, our understanding of abiogenic polymerization processes to form polypeptides is less well developed. Here, we show that carbon disulfide (CS2), a component of volcanic emission and sulfide mineral weathering, and a widely used synthetic reagent and solvent, promotes peptide bond formation in modest yields (up to ∼20%) from α-amino acids under mild aqueous conditions. Exposure of a variety of α-amino acids to CS2 initially yields aminoacyl dithiocarbamates, which in turn generate reactive 2-thiono-5-oxazolidone intermediates, the thio analogues of N-carboxyanhydrides. Along with peptides, thiourea and thiohydantoin species are produced. Amino acid stereochemistry was preserved in the formation of peptides. Our findings reveal that CS2 could contribute to peptide bond formation, and possibly other condensation reactions, in abiogenic settings.

Altered inhibition in the hippocampal neural networks after spreading depression.

Prolonged neuronal depression after spreading depression (SD) is followed by a late cellular and synaptic hyperexcitability. Intra- and extracellular recordings of bioelectrical activities were performed in the rodent hippocampus to investigate the role of γ-aminobutyric acid (GABA)-mediated inhibition in the late hyperexcitable state of SD. The effect of KCl-induced negative DC potential shifts was investigated on extracellularly recorded paired-pulse depression (PPD) and bicuculline-induced afterdischarges as well as intracellularly recorded inhibitory post synaptic potentials (IPSPs) in the hippocampal CA1 area. The results revealed that SD decreased the degree of PPD, enhanced the number and duration of bicuculline-induced afterdischarges, and reduced the amplitude and duration of IPSPs. Application of low concentrations of bicuculline before the induction of SD enhanced the inhibitory effect of SD on IPSPs. Data indicate the contribution of GABA-mediated inhibition to SD-induced delayed hyperexcitability. Modulation of GABA function in the late hyperexcitability phase of SD may play a role in therapeutic management of SD-related neurological disorders.

In vivo efficacy of HDL-like nanolipid particles containing multivalent peptide mimetics of apolipoprotein A-I.

We have observed that molecular constructs based on multiple apoA-I mimetic peptides attached to a branched scaffold display promising anti-atherosclerosis functions in vitro. Building on these promising results, we now describe chronic in vivo studies to assess anti-atherosclerotic efficacy of HDL-like nanoparticles assembled from a trimeric construct, administered over 10 weeks either ip or orally to LDL receptor-null mice. When dosed ip, the trimer-based nanolipids markedly reduced plasma LDL-cholesterol levels by 40%, unlike many other apoA-I mimetic peptides, and were substantially atheroprotective. Surprisingly, these nanoparticles were also effective when administered orally at a dose of 75 mg/kg, despite the peptide construct being composed of l-amino acids and being undetectable in the plasma. The orally administered nanoparticles reduced whole aorta lesion areas by 55% and aortic sinus lesion volumes by 71%. Reductions in plasma cholesterol were due to the loss of non-HDL lipoproteins, while plasma HDL-cholesterol levels were increased. At a 10-fold lower oral dose, the nanoparticles were marginally effective in reducing atherosclerotic lesions. Intriguingly, analogous results were obtained with nanolipids of the corresponding monomeric peptide. These nanolipid formulations provide an avenue for developing orally efficacious therapeutic agents to manage atherosclerosis.

Antibiotic eluting clay mineral (Laponite®) for wound healing application: an in vitro study.

Different materials in form of sponge, hydrogel and film have been developed and formulated for treating and dressing burn wounds. In this study, the potential of Laponite, a gel forming clay, in combination with an antimicrobial agent (mafenide), as a wound dressing material was tested in vitro. Laponite/mafenide (Lap/Maf) hydrogel was formulated in three different ratios of Lap/Maf 1:1, 1:2, 1:3. Laponite/mafenide/alginate (Lap/Maf/Alg) film was also formulated by combining Lap/Maf gel (1:1) with alginate. Intercalation rate of mafenide into the layers of Laponite nanoparticles and physico-chemical properties, including wound dressing characteristics of materials were studied using various analytical methods. Furthermore, the degradation of materials and the release profile of mafenide were investigated in simulated wound exudates fluid and antibacterial effectiveness of the eluted mafenide was tested on a range of bacterial species. The cytotoxicity of materials was also evaluated in skin fibroblast culture. The results showed that mafenide molecules were intercalated between the nano-sized layers of Laponite. The eluted mafenide showed active antibacterial effects against all three tested bacteria. All intercalated mafenide released from Lap/Maf 1:1 and 1:2 gel formulations and nearly 80% release from 1:3 formulation during test period. No significant difference was observed in release profile of mafenide between Lap/Maf/Alg film and Lap/Maf formulations. Wound dressing tests on Lap/Maf/Alg film showed it is a breathable dressing and has capacity to absorb wound exudates. The study showed that prepared Lap/Maf composite has the potential to be used as an antibiotic eluting gel or film for wound healing application. Additionally, Laponite has shown benefits in wound healing processes by releasing Mg(2+) ions and thereby reducing the cytotoxic effect of mafenide on fibroblast cells.

Cortical spreading depression modulates the caudate nucleus activity.

Cortical spreading depression (CSD) plays an important role in migraine with aura. The caudate nucleus has crucial functional interactions with brain regions likely to be important in migraine. The aim of the present in vitro study was to investigate the effect of CSD on the neuronal activity of the caudate. Intracellular recording was performed in the head of the caudate nucleus alongside of extracellular recording in Wistar rat somatosensory cortex. CSD was induced by local KCl injection. Changes in the membrane potentials of the caudate neurons began 1.2±0.2min after CSD. The neurons of the caudate nucleus depolarized first gradually and slightly then it depolarized abruptly at nearly the same point of time of the recovery of the cortical DC potential. Action potentials (APs) reappeared after the cortical DC shift returned to the baseline. Forty-five minutes after CSD, the caudate neurons showed lower frequency of APs and larger amplitude of depolarization prior to APs. The firing pattern of the caudate neurons evoked by injection of intracellular current pulses changed from slow adapting to fast adapting after CSD. Reduced neuronal activity in the caudate after CSD may be assumed to contribute to pain as well as changes in cognition and behavior in patients with migraine.

A mini-review on novel intraperiodontal pocket drug delivery materials for the treatment of periodontal diseases.

Periodontal disease is defined as chronic inflammatory condition characterized by the destruction of the periodontal tissues causing loss of connective tissue attachment, loss of alveolar bone, and the formation of pathological pockets around the diseased teeth. The use of systemic antibiotics has been advocated for its treatment, but concerns emerged with respect to adverse drug reactions and its contribution to bacterial resistance. Thus local drug delivery devices have been developed that aim to deliver a high concentration of antimicrobial drugs directly to the affected site, while minimizing drug's systemic exposure. A burst release of antimicrobial agent from carrier, resulting in a short and inadequate exposure of bacteria residing in periodontal pocket to the agent, remains the main challenge of current local delivery systems for the treatment of periodontal disease. This review aims to investigate and compare different local antimicrobial delivery systems with regard to the treatment of periodontal disease.

Conventional and advanced imaging in neuromyelitis optica.

Myelitis and optic neuritis are prototypic clinical presentations of both multiple sclerosis and neuromyelitis optica. Once considered a subtype of multiple sclerosis, neuromyelitis optica, is now known to have a discrete pathogenesis in which antibodies to the water channel, aquaporin 4, play a critical role. Timely differentiation of neuromyelitis optica from MS is imperative, determining both prognosis and treatment strategy. Early, aggressive immunosuppression is required to prevent the accrual of severe disability in neuromyelitis optica; conversely, MS-specific therapies may exacerbate the disease. The diagnosis of neuromyelitis optica requires the integration of clinical, MR imaging, and laboratory data, but current criteria are insensitive and exclude patients with limited clinical syndromes. Failure to recognize the expanding spectrum of cerebral MR imaging patterns associated with aquaporin 4 antibody seropositivity adds to diagnostic uncertainty in some patients. We present the state of the art in conventional and nonconventional MR imaging in neuromyelitis optica and review the place of neuroimaging in the diagnosis, management, and research of the condition.

Molecules that mimic apolipoprotein A-I: potential agents for treating atherosclerosis.

Certain amphipathic α-helical peptides can functionally mimic many of the properties of full-length apolipoproteins, thereby offering an approach to modulate high-density lipoprotein (HDL) for combating atherosclerosis. In this Perspective, we summarize the key findings and advances over the past 25 years in the development of peptides that mimic apolipoproteins, especially apolipoprotein A-I (apoA-I). This assemblage of information provides a reasonably clear picture of the state of the art in the apolipoprotein mimetic field, an appreciation of the potential for such agents in pharmacotherapy, and a sense of the opportunities for optimizing the functional properties of HDL.