CycLS: Accurate,whole-library sequencing of cyclic peptides using tandem mass spectrometry

Cyclic peptides are of great interest as therapeutic compounds due to their potential for specificity and intracellular activity, but specific compounds can be difficult to identify from large libraries without resorting to molecular encoding techniques. Large libraries of cyclic peptides are often DNA-encoded or linearized before sequencing, but both of those deconvolution strategies constrain the chemistry, assays, and quantification methods which can be used. We developed an automated sequencing program, CycLS, to identify cyclic peptides contained within large synthetic libraries. CycLS facilitates quick and easy identification of all library-members via tandem mass spectrometry data without requiring any specific chemical moieties or modifications within the library. Validation of CycLS against a library of 400 cyclic hexapeptide peptoid hybrids (peptomers) of unique mass yielded a result of 95% accuracy when compared against a simulated library size of 234,256 compounds. CycLS was also evaluated by resynthesizing pure compounds from a separate 1800-member library of cyclic hexapeptides and hexapeptomers with high mass redundancy. Of 22 peptides resynthesized, 17 recapitulated the retention times and fragmentation patterns assigned to them from the whole-library bulk assay results. Implementing a database-matching approach, CycLS is fast and provides a robust method for sequencing cyclic peptides that is particularly applicable to the deconvolution of synthetic libraries.     

Ribosomally-synthesised cyclic peptides from plants as drug leads and pharmaceutical scaffolds

Owing to their exceptional stability and favourable pharmacokinetic properties, plant-derived cyclic peptides have recently attracted significant attention in the field of peptide-based drug design. This article describes the three major classes of ribosomally-synthesised plant peptides – the cyclotides, the PawS-derived peptides and the orbitides – and reviews their applications as leads or scaffolds in drug design. These ribosomally-produced peptides have a range of biological activities, including anti-HIV, cytotoxic and immunomodulatory activity. In addition, recent interest has focused on their use as scaffolds to stabilise bioactive peptide sequences, thereby enhancing their biopharmaceutical properties. There are now more than 30 published papers on such ‘grafting’ applications, most of which have been reported only in the last few years, and several such studies have reported in vivo activity of orally delivered cyclic peptides. In this article, we describe approaches to the synthesis of cyclic peptides and their pharmaceutically-grafted derivatives as well as outlining their biosynthetic routes. Finally, we describe possible bioproduction routes for pharmaceutically active cyclic peptides, involving plants and plant suspension cultures.     

Effects of charge and hydrophobicity on the oligomerization of peptides derived from IAPP

Changes in pH resulting in modifications of charge can dramatically alter the folding and interaction of proteins. This article probes the effects of charge and hydrophobicity on the oligomerization of macrocyclic β-sheet peptides derived from residues 11–17 of IAPP (RLANFLV). Previous studies have shown that a macrocyclic β-sheet peptide containing this IAPP sequence (peptide 1_Arg) does not form oligomers in aqueous solution at low millimolar concentrations. Replacing arginine with the uncharged isostere citrulline generates a homologue (peptide 1_Cit) that forms a tetramer consisting of a sandwich of hydrogen-bonded dimers. The current study probes the role of charge and hydrophobicity by changing residue 11 to glutamic acid (peptide 1_Glu) and leucine (peptide 1_Leu). Diffusion-ordered spectroscopy (DOSY) studies show that peptides 1_Glu and 1_Leu form tetramers in solution. NOESY studies confirm that both peptides form the same sandwich-like tetramer as peptide 1_Cit. ¹H NMR spectroscopy at various concentrations reveals that peptide 1_Leu has the highest propensity to form tetramers. The effects of pH and charge on oligomerization are further probed by incorporating histidine at position 11 (peptide 1_His). DOSY studies show that peptide 1_His forms a tetramer at high pH. At low pH, peptide 1_His forms a new species that has not been previously observed by our research group—a dimer. These studies demonstrate the importance of charge and hydrophobicity in the oligomerization of IAPP-derived peptides.     

Synthesis and antimicrobial studies of novel derivatives of 4-(4-formyl-3-phenyl-1H-pyrazol-1-yl)benzoic acid as potent anti-Acinetobacter baumannii agents

Microbial resistance to antibiotics is a global concern. The World Health Organization (WHO) has identified antimicrobial resistance as one the three greatest threats for human beings in the 21st century. Without urgent and coordinated action, the world is moving toward a post-antibiotic era, in which normal infections or minor injuries may become fatal. In an effort to find new agents, we report the synthesis and antimicrobial activities of 40 novel 1,3-diphenyl pyrazole derivatives. These compounds have shown zones of growth inhibition up to 85 mm against Acinetobacter baumannii. We tested the active compounds against this Gram-negative bacterium in minimum inhibitory concentration (MIC) tests and found activity with concentration as low as 4 μg/mL.     

BET bromodomain ligands: Probing the WPF shelf to improve BRD4 bromodomain affinity and metabolic stability

Ligands for the bromodomain and extra-terminal domain (BET) family of bromodomains have shown promise as useful therapeutic agents for treating a range of cancers and inflammation. Here we report that our previously developed 3,5-dimethylisoxazole-based BET bromodomain ligand (OXFBD02) inhibits interactions of BRD4(1) with the RelA subunit of NF-κB, in addition to histone H4. This ligand shows a promising profile in a screen of the NCI-60 panel but was rapidly metabolised (t_½?=?39.8?min). Structure-guided optimisation of compound properties led to the development of the 3-pyridyl-derived OXFBD04. Molecular dynamics simulations assisted our understanding of the role played by an internal hydrogen bond in altering the affinity of this series of molecules for BRD4(1). OXFBD04 shows improved BRD4(1) affinity (IC₅₀?=?166?nM), optimised physicochemical properties (LE?=?0.43; LLE?=?5.74; SFI?=?5.96), and greater metabolic stability (t_½?=?388?min).     

Prosthetic groups for radioiodination and astatination of peptides and proteins: A comparative study of five potential bioorthogonal labeling strategies

¹²⁵I- and ²¹¹At-labeled azide and tetrazine based prosthetic groups for bioorthogonal conjugation were designed and tested in a comparative study of five bioorthogonal systems. All five bioconjugation reactions conducted on a model clickable peptide led to quantitative yields within less than a minute to several hours depending on the system used. Transferability to the labeling of an IgG was demonstrated with one of the bioorthogonal system. This study provides several new alternatives to the conventional and suboptimal approach currently in use for radioiodination and astatination of biomolecules and should accelerate the development of new probes with these radionuclides for applications in nuclear imaging and targeted alpha-therapy.     

Drosophila ubiquitin E3 ligase dSmurf is required for synapse remodeling and axon pruning by glia

正Animal behaviors and higher-order functions rely on complex neural circuits built by synaptic connections(synapses)to deliver messages among different brain cells.As the major mediator in the nervous system,neurons communicate via synapses,which undergo constant structural remodeling with strict regulation.It is     

Disruptor of telomeric silencing 1-like (DOT1L) is involved in breast cancer metastasis via transcriptional regulation of MALAT1 and ZEB2

<正>Epigenetics refers to how chromatin-associated factors and reversible chromatin modifications maintain DNA-based programs by regulating the chromatin structure (Strahl and Allis,2000).In recent years,genome sequencing studies of cancer have shown that genes encoding epigenetic factors are commonly mutated in cancer(Garraway and Lander,2013).Dys regulated,or mutant,epigenetic factors influence tumorigenesis progression (Dawson and Kouzarides,     

Protozoal sequences may reveal additional isoforms of the 14-3-3 protein family

The phylogenetic position of eleven 14-3-3 proteins from five protozoal species was tested relative to other eukaryotic 14-3-3 versions representing many of the previously described isoforms. The protozoal proteins, four from Entodinium caudatum, three from Entameoba histolytica and four from apicomplexan parasites formed clusters closer to the plant and animal epsilon isoforms than to the animal beta, gamma/eta, sigma/theta, and zeta isoforms. This extends the preliminary findings of Wang and Shakes (1996) but data from a wider range of genera are still required to strengthen our hypothesis that the protozoan isoforms may constitute novel isoforms of the 14-3-3 family.     

Epigallocatechin-3-gallate inhibits bacterial virulence and invasion of host cells

Increasing antibiotic resistance and beneficial effects of host microbiota has motivated the search for anti-infective agents that attenuate bacterial virulence rather than growth. For example, we discovered that specific flavonoids such as baicalein and quercetin from traditional medicinal plant extracts could attenuate Salmonella enterica serovar Typhimurium type III protein secretion and invasion of host cells. Here, we show epigallocatechin-3-gallate from green tea extracts also inhibits the activity of S. Typhimurium type III protein effectors and significantly reduces bacterial invasion into host cells. These results reveal additional dietary plant metabolites that can attenuate bacterial virulence and infection of host cells.     

The synergy between RSC,Nap1 and adjacent nucleosome in nucleosome remodeling

Eukaryotes have evolved a specific strategy to package DNA. The nucleosome is a 147-base-pair DNA segment wrapped around histone core proteins that plays important roles regulating DNA-dependent biosynthesis and gene expression. Chromatin remodeling complexes (RSC, Remodel the Structure of Chromatin) hydrolyze ATP to perturb DNA-histone contacts, leading to nucleosome sliding and ejection. Here, we utilized tethered particle motion (TPM) experiments to investigate the mechanism of RSC-mediated nucleosome remodeling in detail. We observed ATP-dependent RSC-mediated DNA looping and nucleosome ejection along individual mononucleosomes and dinucleosomes. We found that nucleosome assembly protein 1 (Nap1) enhanced RSC-mediated nucleosome ejection in a two-step disassembly manner from dinucleosomes but not from mononucleosomes. Based on this work, we provide an entire reaction scheme for the RSC-mediated nucleosome remodeling process that includes DNA looping, nucleosome ejection, the influence of adjacent nucleosomes, and the coordinated action between Nap1 and RSC.     

Multiple abnormalities in the feet and associated changes elsewhere in the skeleton: The case of 3A-7 from a Capsian Site in Algeria

A skeleton with a number of abnormalities is described with full discussion of alternative diagnoses. In this complex case, the primary diagnosis is of avulsion of the stem of the bifurcate ligament causing a fracture of the anterior process of the calcaneus. The bilateral fracture identified in Skeleton 3A-7 from Site 12, a Capsian site in Algeria, is a result of the feet being inverted and plantar flexed: the fracture is prone to non-union, which is asymmetrical here. There is also a separate anomaly of the feet, 3rd cuneiform and 3rd metatarsal coalition, which was not the cause of trauma. The bifurcate ligament is a major stabilizer of the lateral Chopart (transverse talar) joint, and the trauma could lead to further issues: however, multiple other traumatic changes in 3A-7 most likely occurred at the same time, rather than as the result of pre-existing foot trauma. The asymmetry of the calcaneal condition and asymmetry of the sequelae of the original trauma led to long bone asymmetry, the result of locomotor difficulties.     

Mesoporous silica templated-albumin nanoparticles with high doxorubicin payload for drug delivery assessed with a 3-D tumor cell model

Human serum albumin (HSA) nanoparticles emerge as promising carriers for drug delivery. Among challenges, one important issue is the design of HSA nanoparticles with a low mean size of ca. 50?nm and having a high drug payload. The original strategy developed here is to use sacrificial mesoporous nanosilica templates having a diameter close to 30?nm to drive the protein nanocapsule formation. This new approach ensures first an efficient high drug loading (ca. 30%) of Doxorubicin (DOX) in the porous silica by functionalizing silica with an aminosiloxane layer and then allows the one-step adsorption and the physical cross-linking of HSA by modifying the silica surface with isobutyramide (IBAM) groups. After silica template removal, homogenous DOX-loaded HSA nanocapsules (30–60?nm size) with high drug loading capacity (ca. 88%) are thus formed. Such nanocapsules are shown efficient in multicellular tumor spheroid models (MCTS) of human hepatocarcinoma cells by their significant growth inhibition with respect to controls. Such a new synthesis approach paves the way toward new protein based nanocarriers for drug delivery.     

Development of a novel NURR1/NOT agonist from hit to lead and candidate for the potential treatment of Parkinson's disease

In the course of a programme aimed at identifying Nurr1/NOT agonists for potential treatment of Parkinson’s disease, a few hits from high throughput screening were identified and characterized. A combined optimization pointed to a very narrow and stringent structure activity relationship. A comprehensive program of optimization led to a potent and safe candidate drug displaying neuroprotective and anti-inflammatory activity in several in vitro and in vivo models.     

Overexpression of the nuclear protein gene AtDUF4 increases organ size in Arabidopsis thaliana and Brassica napus

正Organ size is an important trait of many crops that is influenced by internal and environmental signals and controlled by a combination of factors during organogenesis(Krizek,2009).The final size of plant organs is determined by two successive but overlapping processes:cell division,which increases cell number,and cell expansion,which determines final cell size(Anastasiou and Lenhard,     

Effect of insulin on alpha1-adrenergic actions in hepatocytes from euthyroid and hypothyroid rats. Possible involvement of two pathways in alpha1-adrenergic actions

The effect of insulin on the alpha1-adrenergic stimulation of glycogenolysis and ureogenesis, which is very small or undetectable in hepatocytes from control animals, is marked in hepatocytes from hypothyroid rats; the metabolic actions due to alpha1-adrenergic activation, but not those due to glucagon, were nearly blocked by insulin in cells from hypothyroid rats. The alpha1-adrenergic-mediated stimulation of phosphatidylinositol labelling was not affected by insulin in cells from either control or hypothyroid rats. The data suggest that the alpha1-adrenergic action proceeds through two pathways, one of which is very sensitive to insulin and predominates in cells from hypothyroid rats.