Prodynorphin and Proenkephalin in Cerebrospinal Fluid of Sporadic Creutzfeldt–Jakob Disease

Proenkephalin (PENK) and prodynorphin (PDYN) are endogenous opioid peptides mainly produced in the striatum and, to a lesser extent, in the cerebral cortex. Dysregulated metabolism and altered cerebrospinal fluid (CSF) levels of PENK and PDYN have been described in several neurodegenerative diseases. However, no study to date investigated these peptides in the CSF of sporadic Creutzfeldt–Jakob disease (sCJD). Using liquid chromatography-multiple reaction monitoring mass spectrometry, we evaluated the CSF PDYN- and PENK-derived peptide levels in 25 controls and 63 patients with sCJD belonging to the most prevalent molecular subtypes (MM(V)1, VV2 and MV2K). One of the PENK-derived peptides was significantly decreased in each sCJD subtype compared to the controls without a difference among subtypes. Conversely, PDYN-derived peptides were selectively decreased in the CSF of sCJD MV2K, a subtype with a more widespread overall pathology compared to the sCJD MM(V)1 and the VV2 subtypes, which we confirmed by semiquantitative analysis of cortical and striatal neuronal loss and astrocytosis. In sCJD CSF PENK and PDYN were associated with CSF biomarkers of neurodegeneration but not with clinical variables and showed a poor diagnostic performance. CSF PDYN and PENK-derived peptides had no significant diagnostic and prognostic values in sCJD; however, the distinct marker levels between molecular subtypes might help to better understand the basis of phenotypic heterogeneity determined by divergent neuronal targeting.     

Intensification of biopolymeric microparticles production by ultrasonic assisted atomization

In this work ultrasonic atomization process is applied to produce biopolymer microparticles with potential applications in pharmaceutical and nutraceutical fields. Natural polymer (alginate)/water solution is atomized by ultrasonic assisted process and the droplets spray is reticulated using a solution of copper sulfate, where the Cu²⁺ ions cause the formation of a network structure (hard porous gel). Several operating parameters (solution concentration, flow rate, atomization power) are changed to study their effects on the produced microparticles. Literature correlations able to predict the features of the droplets as functions of process parameters are optimized using a statistical approach. Furthermore, the energy requirement for the drops production is compared with the energy required by traditional techniques to evaluate the intensification effect of the ultrasonic on the atomization process.     

Selective and pH-Dependent Binding of a Moth Pheromone to a Pheromone-Binding Protein

Fluorescence and circular dichroism (CD) data suggest that the major pheromone-binding protein (PBP) from the wild silkmoth, Antheraea polyphemus, ApolPBP1, undergoes a pH-dependent conformational change similar to that previously observed for the PBP from the silkworm moth, Bombyx mori, BmorPBP. All three constituents of the sex pheromone, E6,Z11-16Ac, E6,Z11-16Ald, and E4,Z9-14Ac, bound to ApolPBP1 with apparent high affinity at high pH, but reduced binding at low pH when tested individually in a “cold binding assay.” In competitive assays, however, ApolPBP1 showed considerable preference for the major constituent of the sex pheromone, E6,Z11-16Ac. These data suggest that specificity of PBPs contributes at least in part to the remarkable selectivity of moth's olfactory system.     

Synthesis and Biological Characterization of (3R,4R)‐4‐(2‐(Benzhydryloxy)ethyl)‐1‐((R)‐2‐hydroxy‐2‐phenylethyl)‐piperidin‐3‐ol and Its Stereoisomers for Activity toward Monoamine Transporters

A novel series of optically active molecules based on a 4‐(2‐(benzhydryloxy)ethyl)‐1‐((R)‐2‐hydroxy‐2‐phenylethyl)‐piperidin‐3‐ol template were developed. Depending on stereochemistry, the compounds exhibit various degrees of affinity for three dopamine, serotonin, and norepinephrine transporters. These molecules have the potential for treating several neurological disorders such as drug abuse, depression, and attention deficit hyperactivity disorder.

     

Truncation and Optimisation of Peptide Inhibitors of Cyclin‐Dependent Kinase 2‐Cyclin A Through Structure‐Guided Design

Peptides that inhibit cyclin‐dependent kinase 2 by blocking the macromolecular substrate recruitment site of cyclin A were simplified, for example, by replacement of dipeptide units with β‐amino acids. The smallest inhibitor retaining activity was a tripeptide, whose binding mode was confirmed by X‐ray crystallography. This result suggests that nonpeptidic cyclin groove inhibitors may be feasible therapeutic agents.

     

Acute Promyelocytic Leukemia in Children: A Model of Precision Medicine and Chemotherapy-Free Therapy

Acute promyelocytic leukemia (APL) represents a paradigm of precision medicine. Indeed, in the last decades, the introduction of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) completely revolutionized the therapeutic approach to this previously highly fatal disorder. This entirely chemotherapy-free treatment, which provided excellent survival rates, has been initially validated in adults and, recently, translated in the pediatric setting. This review summarizes currently available data on the use of ATRA and ATO combination in pediatric APL, providing a particular focus on peculiar issues and challenges, such as the occurrence of pseudotumor cerebri and death during induction (early death), as well as the advantage offered by the ATO/ATRA combination in sparing long-term sequelae.     

Functional Inactivation of Drosophila GCK Orthologs Causes Genomic Instability and Oxidative Stress in a Fly Model of MODY-2

Maturity-onset diabetes of the young (MODY) type 2 is caused by heterozygous inactivating mutations in the gene encoding glucokinase (GCK), a pivotal enzyme for glucose homeostasis. In the pancreas GCK regulates insulin secretion, while in the liver it promotes glucose utilization and storage. We showed that silencing the Drosophila GCK orthologs Hex-A and Hex-C results in a MODY-2-like hyperglycemia. Targeted knock-down revealed that Hex-A is expressed in insulin producing cells (IPCs) whereas Hex-C is specifically expressed in the fat body. We showed that Hex-A is essential for insulin secretion and it is required for Hex-C expression. Reduced levels of either Hex-A or Hex-C resulted in chromosome aberrations (CABs), together with an increased production of advanced glycation end-products (AGEs) and reactive oxygen species (ROS). This result suggests that CABs, in GCK depleted cells, are likely due to hyperglycemia, which produces oxidative stress through AGE metabolism. In agreement with this hypothesis, treating GCK-depleted larvae with the antioxidant vitamin B6 rescued CABs, whereas the treatment with a B6 inhibitor enhanced genomic instability. Although MODY-2 rarely produces complications, our data revealed the possibility that MODY-2 impacts genome integrity.     

The Age-Sensitive Efficacy of Calorie Restriction on Mitochondrial Biogenesis and mtDNA Damage in Rat Liver

Calorie restriction (CR) is the most efficacious treatment to delay the onset of age-related changes such as mitochondrial dysfunction. However, the sensitivity of mitochondrial markers to CR and the age-related boundaries of CR efficacy are not fully elucidated. We used liver samples from ad libitum-fed (AL) rats divided in: 18-month-old (AL-18), 28-month-old (AL-28), and 32-month-old (AL-32) groups, and from CR-treated (CR) 28-month-old (CR-28) and 32-month-old (CR-32) counterparts to assay the effect of CR on several mitochondrial markers. The age-related decreases in citrate synthase activity, in TFAM, MFN2, and DRP1 protein amounts and in the mtDNA content in the AL-28 group were prevented in CR-28 counterparts. Accordingly, CR reduced oxidative mtDNA damage assessed through the incidence of oxidized purines at specific mtDNA regions in CR-28 animals. These findings support the anti-aging effect of CR up to 28 months. Conversely, the protein amounts of LonP1, Cyt c, OGG1, and APE1 and the 4.8 Kb mtDNA deletion content were not affected in CR-28 rats. The absence of significant differences between the AL-32 values and the CR-32 counterparts suggests an age-related boundary of CR efficacy at this age. However, this only partially curtails the CR benefits in counteracting the generalized aging decline and the related mitochondrial involvement.     

The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential

Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the “Find Individual Motif Occurrences” (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.