Nicolaos Avlonitis

CD133+ cancer stem-like cells in small cell lung cancer are highly tumorigenic and chemoresistant but sensitive to a novel neuropeptide antagonist

Small cell lung cancer (SCLC) is a highly aggressive malignancy with poor survival rates, with initial responses nearly invariably followed by rapid recurrence of therapy-resistant disease. Drug resistance in SCLC may be attributable to the persistence of a subpopulation of cancer stem-like cells (CSC) that exhibit multiple drug resistance. In this study, we characterized the expression of CD133, one important marker of CSC in other cancers, in SCLC cancer cells. CD133 expression correlated with chemoresistance and increased tumorigenicity in vitro and in vivo accompanied by increased expression of Akt/PKB and Bcl-2. CD133 expression was increased in mouse and human SCLC after chemotherapy, an observation confirmed in clinical specimens isolated longitudinally from a patient receiving chemotherapy. We discovered in CD133(+) SCLC cells, an increased expression of the mitogenic neuropeptide receptors for gastrin-releasing peptide and arginine vasopressin. Notably, these cells exhibited increased sensitivity to the growth inhibitory and proapoptotic effects of a novel broad spectrum neuropeptide antagonist (related to SP-G), which has completed a phase I clinical trial for SCLC. Our results offer evidence that this agent can preferentially target chemoresistant CD133(+) cells with CSC character in SCLC, emphasizing its potential utility for improving therapy in this setting.

G protein-associated, specific membrane binding sites mediate the neuroprotective effect of dehydroepiandrosterone

The neurosteroid dehydroepiandrosterone (DHEA) at 1 nM protects NMDA‐/GABAA‐receptor negative neural crest‐derived PC12 cells from apoptosis. We now report that membrane‐impermeable DHEA‐BSA conjugate replaces unconjugated DHEA in protecting serum‐deprived PC12 cells from apoptosis (IC50=1.5 nM). Protection involves phosphorylation of the prosurvival factor Src and induction of the anti‐apoptotic protein Bcl‐2 and is sensitive to pertussis toxin. Binding assays of [3H]DHEA on isolated PC12 cell membranes revealed saturation within 30 min and binding of DHEA with a Kd of 0.9 nM. A similar binding activity was detectable in isolated membranes from rat hippocampus and from normal human adrenal chromaffin cells. The presence of DHEA‐specific membrane binding sites was confirmed by flow cytometry and confocal laser microscopy of DHEA‐BSA‐FITC stained cells. In contrast to estrogens and progestins, glucocorticoids and androgens displaced DHEA from its membrane binding sites but with a 10‐fold lower affinity than DHEA (IC50=9.3 and 13.6 nM, respectively). These agents acted as pure antagonists, blocking the antiapoptotic effect of DHEA as well as the induction of Bcl‐2 proteins and Src kinase activation. In conclusion, our findings suggest that neural crest‐derived cells possess specific DHEA membrane binding sites coupled to G proteins. Binding to these sites confers neuroprotection.

Neurosteroid Dehydroepiandrosterone Interacts with Nerve Growth Factor (NGF) Receptors, Preventing Neuronal Apoptosis

The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75NTR membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [3H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75NTR receptors (KD: 7.4±1.75 nM and 5.6±0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75NTR receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75NTR receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor.

Highly specific, multi-branched fluorescent reporters for analysis of human neutrophil elastase

Human neutrophil elastase (HNE) is a serine protease implicated in the pathogenesis of acute and chronic inflammatory disease. Here a series of, internally quenched, single fluorophore fluorescent reporters were synthesised that allowed the rapid, highly specific and sensitive analysis of HNE activity over closely related proteases.

Neuroprotective effects of steroid analogues on P19-N neurons.

Naturally occurring neurosteroids are potent allosteric modulators of gamma-aminobutyric acid(A) receptor and through augmentation of gamma-aminobutyric acid(A) receptor function, can protect neuronal cells against N-methyl-d-aspartate receptor over-activation, ischemia and traumatic brain injury. In this study, mouse P19 cells were induced to differentiate into post-mitotic neurons and were subjected to excitotoxicity in the presence of N-methyl-d-aspartate. Novel synthetic analogues of the endogenous neurosteroids allopregnanolone and dehydroepiandrostrone, inhibited excitotoxic cell death of P19-N neurons, by directly maintaining the activation of PKB/Akt kinase and interfering with the intrinsic mitochondrial apoptotic pathway, preserving cytochrome c in the mitochondria and Bax in the cytoplasm. The efficiency and the potency of these neurosteroids were similar to those of allopregnanolone and dehydroepiandrostrone. Their effects were gamma-aminobutyric acid(A) receptor mediated, since they were abolished in the presence of bicuculline, an antagonist of receptors function. In addition, the synthetic compounds retained the ability to alter gamma-aminobutyric acid(A) receptor subunit gene expression, but their effects on transcriptional activity were less pronounced than those of allopregnanolone and dehydroepiandrostrone. These results suggest that synthetic neurosteroids may serve as potent, membrane acting, neuroprotectants against N-methyl-d-aspartate receptor neurotoxicity on neuronal cells.

Methylene blue not ferrocene: Optimal reporters for electrochemical detection of protease activity

Electrochemical peptide-based biosensors are attracting significant attention for the detection and analysis of proteins. Here we report the optimisation and evaluation of an electrochemical biosensor for the detection of protease activity using self-assembled monolayers (SAMs) on gold surfaces, using trypsin as a model protease. The principle of detection was the specific proteolytic cleavage of redox-tagged peptides by trypsin, which causes the release of the redox reporter, resulting in a decrease of the peak current as measured by square wave voltammetry. A systematic enhancement of detection was achieved through optimisation of the properties of the redox-tagged peptide; this included for the first time a side-by-side study of the applicability of two of the most commonly applied redox reporters used for developing electrochemical biosensors, ferrocene and methylene blue, along with the effect of changing both the nature of the spacer and the composition of the SAM. Methylene blue-tagged peptides combined with a polyethylene-glycol (PEG) based spacer were shown to be the best platform for trypsin detection, leading to the highest fidelity signals (characterised by the highest sensitivity (signal gain) and a much more stable background than that registered when using ferrocene as a reporter). A ternary SAM (T-SAM) configuration, which included a PEG-based dithiol, minimised the non-specific adsorption of other proteins and was sensitive towards trypsin in the clinically relevant range, with a Limit of Detection (LoD) of 250pM. Kinetic analysis of the electrochemical response with time showed a good fit to a Michaelis-Menten surface cleavage model, enabling the extraction of values for kcat and KM. Fitting to this model enabled quantitative determination of the solution concentration of trypsin across the entire measurement range. Studies using an enzyme inhibitor and a range of real world possible interferents demonstrated a selective response to trypsin cleavage. This indicates that a PEG-based peptide, employing methylene blue as redox reporter, and deposited on an electrode as a ternary SAM configuration, is a suitable platform to develop clinically-relevant and quantitative electrochemical peptide-based protease biosensing.

FeCl3-catalysed ultrasonic-assisted, solvent-free synthesis of 4-substituted coumarins. A useful complement to the Pechmann reaction

The catalytic activity of FeCl3 for the synthesis of a variety of 4-substituted coumarins using high energy techniques has been investigated. The ultrasonic-assisted conditions provide a useful complement to the Pechmann reaction, affording the coumarin derivatives in excellent yields, under solvent-free conditions, in short reaction times using an inexpensive, mild and benign Lewis acid catalyst.

Structural modifications of the antimicrobial peptide ubiquicidin for pulmonary imaging of bacteria in the alveolar space

Abstract Background The direct visualisation of bacteria in the distal lung would increase the spatiotemporal understanding of pulmonary infection and be a powerful tool to stratify patients with suspected pneumonia. In critically ill patients, the diagnostic dilemma of pulmonary opacities leads to overprescribing of antimicrobial agents while waiting for culture results from bronchoalveolar lavage. Ubiquicidin (UBI) is an innate cytosolic antimicrobial peptide with a twelve aminoacid portion (UBI 29–41 ) that specifically binds bacteria. We aimed to modify the chemical structure of UBI 29–41 so that in-situ bacterial imaging with optical endomicroscopy (OEM) could be achieved. Methods UBI 29-41 compounds were labelled with the environmentally sensitive fluorophore NBD (NBD-UBI), with incorporation of synthetic aminoacids (NBD-UBI nma ) and alteration of the secondary structure of the native peptide on a dendrimeric scaffold (NBD-UBI dend ). These compounds were assessed in vitro and delivered endobronchially in an ex-vivo sheep lung model and then OEM applied to allow alveolar imaging. The NBD-UBI dend –OEM platform was also evaluated in explanted whole cystic fibrosis lungs. Findings NBD-UBI selectively labelled bacteria over mammalian cells but remained susceptible to proteolytic degradation and poor affinity. NBD-UBI nma improved stability but not affinity. NBD-UBI dend remained structurally stable and exhibited high affinity for bacteria in vitro. It retained bacterial selectivity over mononuclear cells (p=0·0015), neutrophils (p=0·0034), bronchoalveolar lavage macrophages (p=0·0169), and labelled Escherichia coli (p=0·0035), Klebsiella pneumoniae (p=0·0003), Pseudomonas aeruginosa (p=0·0009), and meticillin-sensitive Staphylococcus aureus (p 5 colony-forming units per mL on lavage. NBD-UBI dend also detected bacteria in situ in ex-vivo explanted human cystic fibrosis lungs (p=0·0027 compared with peptide and fluorophore control segments). Interpretation We describe an OEM strategy that can immediately detect bacteria in size-relevant preclinical models, with crucial requirements for pulmonary molecular imaging of peptide stability and affinity. This method has the potential to stratify pulmonary opacities in the intensive care unit when pneumonia is suspected and offers the substantial advantage of real-time detection, therefore allowing immediate decision making about antimicrobial treatment. This imaging strategy is now undergoing first-in-man translation. Funding Wellcome Trust, Department of Health, Engineering and Physical Sciences Research Council.

Super-silent FRET Sensor Enables Live Cell Imaging and Flow Cytometric Stratification of Intracellular Serine Protease Activity in Neutrophils

Serine proteases are released by neutrophils to act primarily as antimicrobial proteins but excessive and unbalanced serine protease activity results in serious host tissue damage. Here the synthesis of a novel chemical sensor based on a multi-branched fluorescence quencher is reported. It is super-silent, exhibiting no fluorescence until de-quenched by the exemplar serine protease human neutrophil elastase, rapidly enters human neutrophils, and is inhibited by serine protease inhibitors. This sensor allows live imaging of intracellular serine protease activity within human neutrophils and demonstrates that the unique combination of a multivalent scaffold combined with a FRET peptide represents a novel and efficient strategy to generate super-silent sensors that permit the visualisation of intracellular proteases and may enable point of care whole blood profiling of neutrophils.

In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A

A topically administered fluorescently labeled peptide targeting lipid A permits rapid, real-time visualization of bacteria in the distal human lung. Lung infection in real time Lung infections are frequent causes of complications in mechanically ventilated and immunosuppressed patients. However, the diagnosis is challenging, requires risky procedures, and is time consuming. Now, Akram et al. have developed an imaging method that is able to detect Gram-negative bacteria in real time in the distal part of the human lung. Using a fluorescent probe binding to lipid A, a molecule expressed on Gram-negative bacterial membranes, in combination with an optical endomicroscope, the researchers rapidly detected Gram-negative infections in distal airways in hospitalized individuals. The results suggest that the approach could accelerate the diagnosis of bacterial lung infection and facilitate the evaluation of antibiotic treatment efficacy. Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time. We used optical endomicroscopy to visualize a water-soluble optical imaging probe based on the antimicrobial peptide polymyxin conjugated to an environmentally sensitive fluorophore. The probe was chemically stable and nontoxic and, after in-human intrapulmonary microdosing, enabled the specific detection of Gram-negative bacteria in distal human airways and alveoli within minutes. The results suggest that pulmonary molecular imaging using a topically administered fluorescent probe targeting bacterial lipid A is safe and practical, enabling rapid in situ identification of Gram-negative bacteria in humans.