Thio-conjugation of substituted benzofurazans to peptides: molecular sieves catalyze nucleophilic attack on unsaturated fused rings

Bioconjugates of 2,1,3-benzoxadiazole (benzofurazan) and its derivatives have attracted considerable interest due to their biological activities and applications as fluorescent tags. A high-yield, chemoselective, and mild procedure for the S-alkylation of cysteine containing peptides by benzofurazan halogenides is reported. The key feature of this procedure is the use of activated molecular sieves (MS) to catalyze thiol activation for nucleophilic substitution under very mild conditions (room temperature and no need for added bases). To the best of our knowledge, this is the first report about thiol nucleophilic substitution performed on unsaturated and annelated systems catalyzed by activated molecular sieves. Reaction yields were remarkable even with benzofurazans having weakly activating groups or no activating groups at all. The potential of the new methodology was explored by synthesizing fluorescent, hydrophilic benzofurazan/peptide conjugates, also with peptides containing unprotected lysine residues

A Late-Stage Synthetic Approach to Lanthionine-Containing Peptides via S-Alkylation on Cyclic Sulfamidates Promoted by Molecular Sieves

A one-pot, high-yield procedure for synthesizing lanthionine-containing peptides was developed. It relies on the S-alkylation of cysteine-containing peptides with chiral cyclic sulfamidates. The key feature of this approach is the use of mild reaction conditions (only activated molecular sieves are employed as the catalyst), leading to good chemoselectivity and excellent stereochemical control. The potential of the new methodology has been investigated by synthesizing the thioether ring of a natural lantibiotic, Haloduracin \u3b2

Biodegradable microparticles as scaffolds for cell therapy

Cell therapy is promising strategy that has attracted a lot of attention recently regarding regeneration of diverse tissues and treatment of various pathological conditions. Despite its great potential, several issues still need to be addressed. Among them administration route and dose, microenvironment conditions and host immune response are recognized as a major causes which lead to cells transplantation failure. In this work it is presented novel microstructural system based on biodegradable polymer poly(lactide-co-glycolide) (PLGA) and combination of biocompatible polyvinyl alcohol (PVA) and chitosan, as a scaffold for human mesenchymal stem cells (hMSCs) growth. The obtained microparticles with diameter 200-600 μm showed full biocompatibility with human hMSCs. Besides serving as a solid support, polymeric particles provided controlled release of contrast agent - gadolinium fluoride nanoparticles (Gd-NP) up to 5 weeks. The release of Gd-NP is enhanced by acidic conditions. Magnetic Resonance Imaging (MRI) of the samples embedded in 1% agar showed that contrast enhancement in T1-weighted (T1w) MR images is influenced by the amount of released Gd-NP. Based on these preliminary results, presented theranostic system could be considered for cells grafting

BS43 A new collagen III-specific MRI imaging probe to assess cardiac fibrosis

Heart failure (HF) has reached epidemic proportions, affecting approximately 64 million people globally and is the main cause of death and disability.1 Myocardial fibrosis, characterised by changes in the amount and/or distribution of collagen I and III, impairs cardiac function and relates to adverse outcomes of HF.2 3 Clinically, we rely on indirect or surrogate measurements of collagen in the myocardium and current targeted molecular imaging probes are limited to collagen I. Here, we report the discovery of a peptide selective for collagen III and a strategy to develop an imaging probe with superior properties for in vivo molecular magnetic resonance imaging (MRI) applications. A small peptide was screened and selected from a library of peptides with potential to bind to collagen identified based on protein-protein interaction studies. The peptide was conjugated to a DOTA-chelator and labelled with Europium [Eu(III)] for in vitro binding assays; gallium (68Ga) for in vivo PET/CT biodistribution; and gadolinium [Gd(III)] for in vivo MRI studies. The probe was further modified to increase the number of Gd(III) per peptide (from one to four) to amplify and prolong the MRI signal. The probe was validated using a surgical mouse model of myocardial infarction (MI). In vivo MRI was performed at days 10 and 21 post-MI (n=7). Imaging findings were validated with tissue analysis. A negative control probe, carrying a scrambled peptide sequence was used. All MRI experiments were performed at a 3 Tesla clinical MRI scanner. In vitro binding assays showed that the probe has a good affinity towards collagen III (Kd= 5.2±1.3µM) that is in the ideal range for a molecular imaging probe.4 Lack of binding of the scrambled probe (negative control) proved the specificity our probe (figure 1A). In vivo PET/CT biodistribution showed favourable pharmacokinetics with fast blood clearance and no unspecific binding (figure 1B). In vivo cardiac MRI showed selective late gadolinium enhancement (LGE) of the fibrotic scar at day 10 which decreased by day 21. This observation is expected as collagen III naturally gets replaced by collagen I at the later stages of cardiac fibrosis. The imaging data are validated histologically showing co-localisation of the MRI signal with collagen III (green colour) at day 10 and reduction of collagen III at day 21 (figure 2). Importantly, no enhancement was observed using the negative control probe and a clinically approved non-collagen targeting probe (Gadovist). We have developed a new molecular imaging probe specific for collagen type III. Using this probe, we have successfully imaged - previously undetectable - collagen III in cardiac fibrosis. This approach may enable early detection and characterisation of cardiac fibrosis in vivo allowing staging of disease and monitoring of therapie

Mutations in KCNK4 that Affect Gating Cause a Recognizable Neurodevelopmental Syndrome

Aberrant activation or inhibition of potassium (K+) currents across the plasma membrane of cells has been causally linked to altered neurotransmission, cardiac arrhythmias, endocrine dysfunction, and (more rarely) perturbed developmental processes. The K+ channel subfamily K member 4 (KCNK4), also known as TRAAK (TWIK-related arachidonic acid-stimulated K+ channel), belongs to the mechano-gated ion channels of the TRAAK/TREK subfamily of two-pore-domain (K2P) K+ channels. While K2P channels are well known to contribute to the resting membrane potential and cellular excitability, their involvement in pathophysiological processes remains largely uncharacterized. We report that de novo missense mutations in KCNK4 cause a recognizable syndrome with a distinctive facial gestalt, for which we propose the acronym FHEIG (facial dysmorphism, hypertrichosis, epilepsy, intellectual disability/developmental delay, and gingival overgrowth). Patch-clamp analyses documented a significant gain of function of the identified KCNK4 channel mutants basally and impaired sensitivity to mechanical stimulation and arachidonic acid. Co-expression experiments indicated a dominant behavior of the disease-causing mutations. Molecular dynamics simulations consistently indicated that mutations favor sealing of the lateral intramembrane fenestration that has been proposed to negatively control K+ flow by allowing lipid access to the central cavity of the channel. Overall, our findings illustrate the pleiotropic effect of dysregulated KCNK4 function and provide support to the hypothesis of a gating mechanism based on the lateral fenestrations of K2P channels

Endogenous glutamine decrease is associated with pancreatic cancer progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is becoming the second leading cause of cancer-related death in the Western world. The mortality is very high, which emphasizes the need to identify biomarkers for early detection. As glutamine metabolism alteration is a feature of PDAC, its in vivo evaluation may provide a useful tool for biomarker identification. Our aim was to identify a handy method to evaluate blood glutamine consumption in mouse models of PDAC. We quantified the in vitro glutamine uptake by Mass Spectrometry (MS) in tumor cell supernatants and showed that it was higher in PDAC compared to non-PDAC tumor and pancreatic control human cells. The increased glutamine uptake was paralleled by higher activity of most glutamine pathway-related enzymes supporting nucleotide and ATP production. Free glutamine blood levels were evaluated in orthotopic and \u202

Deep phenotyping of the neuroimaging and skeletal features in KBG syndrome: a study of 53 patients and review of the literature

Background: KBG syndrome is caused by haploinsufficiency of ANKRD11and is characterised by macrodontia of upper central incisors, distinctive facial features, short stature, skeletal anomalies, developmental delay, brain malformations and seizures. The central nervous system (CNS) and skeletal features remain poorly defined. Methods: CNS and/or skeletal imaging were collected from molecularly confirmed individuals with KBG syndrome through an international network. We evaluated the original imaging and compared our results with data in the literature. Results: We identified 53 individuals, 44 with CNS and 40 with skeletal imaging. Common CNS findings included incomplete hippocampal inversion and posterior fossa malformations; these were significantly more common than previously reported (63.4% and 65.9% vs 1.1% and 24.7%, respectively). Additional features included patulous internal auditory canal, never described before in KBG syndrome, and the recurrence of ventriculomegaly, encephalic cysts, empty sella and low-lying conus medullaris. We found no correlation between these structural anomalies and epilepsy or intellectual disability. Prevalent skeletal findings comprised abnormalities of the spine including scoliosis, coccygeal anomalies and cervical ribs. Hand X-rays revealed frequent abnormalities of carpal bone morphology and maturation, including a greater delay in ossification compared with metacarpal/phalanx bones. Conclusion: This cohort enabled us to describe the prevalence of very heterogeneous neuroradiological and skeletal anomalies in KBG syndrome. Knowledge of the spectrum of such anomalies will aid diagnostic accuracy, improve patient care and provide a reference for future research on the effects ofANKRD11variants in skeletal and brain development