Live applications of norbormide-based fluorescent probes in Drosophila melanogaster

In this study we investigated the performance of two norbormide (NRB)-derived fluorescent probes, NRBMC009 (green) and NRBZLW0047 (red), on dissected, living larvae of Drosophila, to verify their potential application in confocal microscopy imaging in vivo. To this end, larval tissues were exposed to NRB probes alone or in combination with other commercial dyes or GFP-tagged protein markers. Both probes were rapidly internalized by most tissues (except the central nervous system) allowing each organ in the microscope field to be readily distinguished at low magnification. At the cellular level, the probes showed a very similar distribution (except for fat bodies), defined by loss of signal in the nucleus and plasma membrane, and a preferential localization to endoplasmic reticulum (ER) and mitochondria. They also recognized ER and mitochondrial phenotypes in the skeletal muscles of fruit fly models that had loss of function mutations in the atlastin and mitofusin genes, suggesting NRBMC009 and NRBZLW0047 as potentially useful in vivo screening tools for characterizing ER and mitochondria morphological alterations. Feeding of larvae and adult Drosophilae with the NRB-derived dyes led to staining of the gut and its epithelial cells, revealing a potential role in food intake assays. In addition, when flies were exposed to either dye over their entire life cycle no apparent functional or morphological abnormalities were detected. Rapid internalization, a bright signal, a compatibility with other available fluorescent probes and GFP-tagged protein markers, and a lack of toxicity make NRBZLW0047 and, particularly, NRBMC009 one of the most highly performing fluorescent probes available for in vivo microscopy studies and food intake assay in Drosophila

Isolated cerebral sinovenous thrombosis: a rare case of neonatal antiphospholipid syndrome

We describe a case of neonatal cerebral sinovenous thrombosis associated with the presence of anti-phospholipid antibodies (aPL). We recommend that in all cases of neonatal thrombosis, the couple mother-infant should be extensively tested for the presence of both acquired (aPL) and congenital thrombophilia

DNA methylation patterns identify subgroups of pancreatic neuroendocrine tumors with clinical association

Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs

Traces of trauma – a multivariate pattern analysis of childhood trauma, brain structure and clinical phenotypes

Background: Childhood trauma (CT) is a major yet elusive psychiatric risk factor, whose multidimensional conceptualization and heterogeneous effects on brain morphology might demand advanced mathematical modeling. Therefore, we present an unsupervised machine learning approach to characterize the clinical and neuroanatomical complexity of CT in a larger, transdiagnostic context. Methods: We used a multicenter European cohort of 1076 female and male individuals (discovery: n = 649; replication: n = 427) comprising young, minimally medicated patients with clinical high-risk states for psychosis; patients with recent-onset depression or psychosis; and healthy volunteers. We employed multivariate sparse partial least squares analysis to detect parsimonious associations between combinations of items from the Childhood Trauma Questionnaire and gray matter volume and tested their generalizability via nested cross-validation as well as via external validation. We investigated the associations of these CT signatures with state (functioning, depressivity, quality of life), trait (personality), and sociodemographic levels. Results: We discovered signatures of age-dependent sexual abuse and sex-dependent physical and sexual abuse, as well as emotional trauma, which projected onto gray matter volume patterns in prefronto-cerebellar, limbic, and sensory networks. These signatures were associated with predominantly impaired clinical state- and trait-level phenotypes, while pointing toward an interaction between sexual abuse, age, urbanicity, and education. We validated the clinical profiles for all three CT signatures in the replication sample. Conclusions: Our results suggest distinct multilayered associations between partially age- and sex-dependent patterns of CT, distributed neuroanatomical networks, and clinical profiles. Hence, our study highlights how machine learning approaches can shape future, more fine-grained CT research

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Studio di sistemi supramolecolari per il delivery di proteine terapeutiche

Novel strategies for site-specific protein modification have been investigated by using low molecular weight linkers. In the first part of the study, glycoside-derivatives have been obtained by conjugation of alkyl chains and then activated with a maleimido function for selective derivatization of cysteines. The linkers were used to modify the free cysteins (Cys) of serum albumin (HSA) and recombinant human granulocyte colony-stimulating factor (rh-G-CSF). Hydrazide polyethylenglycole (PEG-Hz) was conjugated to the aldehydes groups generated by selective oxidation of the glycosides with periodate. This PEGylation strategy yielded "flag-type" bioconjugates as several PEG chains are attached to the glycosidic residues of the semi-synthetic neo-glycoprotein. The PEG-Hz conjugation to the oxidized glycosides yields pH-dependent cleavable hydrazone bonds which allow for PEG release. Therefore, un-PEGylated active protein can be released over time under physiological conditions. In the second part of the work preliminary studies of polyethylene methacrylate (PEGMA) radical polymerization from protein surface were conducted in aqueous solution using the Atom Transfer Radical Polymerization (ATRP) technique. Two different alkyl halide derivatives were synthesized: 2-bromo-isobutyryl-ethoxyethyl-maleimide and 2-bromo-isobutyryl-cadaverine. The linkers were used to modify selectively aminoacids of protein models: HSA, rh-G-CSF and recombinant human growth hormone (rh-GH) and to yield macro-initiators. The synthesized 2-bromo-isobutyryl- derivatives have been characterized by chromatographic and spectrometric techniques. However, preliminary studies of radical polymerization using ATRP from the protein surfaces did not give reasonable results. Therefore more detailed investigation will be worked up in order to set up a convenient polymerization protocol

Polysaccharide based anticancer prodrugs

So far, polysaccharides have been widely used in pharmaceutical technology as first choice excipients for production of traditional formulations. Nevertheless, in the recent years these natural and semisynthetic macromolecules have been addressed for new challenging applications as functional materials for innovative formulations. Their peculiar physicochemical and biological properties have been exploited to develop macromolecular prodrugs which can exhibit favourable biopharmaceutical properties and enforce the therapeutic performance of the parent drugs. These polymers display, in fact, high biocompatibility and biodegradability, multiple insertion points and biological properties that can be advantageously exploited in drug delivery. In particular, the multifunctional structure of these polymers are anchoring points for conjugation of several anticancer drugs, which usually suffer of poor physicochemical, biopharmaceutical and therapeutic properties that limit their therapeutic performance and proper use in anticancer treatment. Polysaccharide based anticancer prodrugs can be designed to endow derivatives with new bioresponse, targeting or environmental triggering properties or to combine molecules with synergistic therapeutic effect. Over the years, a variety of synthetic protocols have been set up to conjugate anticancer drugs to the polysaccharide backbone via specific linkages or through spacers which convey to the conjugates selective drug delivery properties. Furthermore, the intrinsic antitumor activity and cell targeting properties of few polysaccharides represent an additional value to the final therapeutic systems. Anticancer drugs with different pharmacodynamic, pharmacokinetic and physicochemical properties have been successfully conjugated to various natural and semisynthetic polysaccharides highlighting the interesting perspectives for exploitation of new promising anticancer polymer therapeutic

Corona-like (guanidyl)-oligosaccharidic derivatives as cell-penetrating enhancers for intracellular delivery of colloidal therapeutic systems

A novel class of cell-penetrating enhancers with unusual chemical structure is herein disclosed. Said cell-penetrating enhancers are non-linear and non peptidic (guanidyl)-oligosaccharidic derivatives, which can be easily obtained according to simple and reproducible synthetic steps. A wide array of cell penetration enhancers can be obtained by slight modification of the main structure oligosaccharidic backbone and in order to provide for their conjugation or physical combination with a variety of therapeutic systems. These molecules can be designed for conjugation to proteins or polymer therapeutics or for surface decoration of liposomes or nanoparticles. Finally, the cationic features and the penetration enhancer properties of the corona-like (guanidyl)-oligosaccharidic derivatives can be exploited for oligonucleotide, namely siRNA, or gene delivery