Inhibitor affinity chromatography: profiling the specific reactivity of the proteome with immobilized molecules

An inhibitor affinity chromatography (IAC) method has been developed for the analysis of inhibitor-protein interactions as a complementary approach to two-dimensional electrophoresis for functional proteomics studies. The procedure was developed utilizing a cyclin-dependent kinase 2 (Cdk2) inhibitor coupled to a polymeric resin and validated using a number of proteins interacting with the inhibitor with different specificities. Cdk2 and the other kinases bound and eluted from the resin in accordance with the relative in vitro potency of the inhibitor for each enzyme. Molecular interactions with the Cdk2 inhibitor were compared for HCT116 cancer cells versus rat pancreatic acinar cells. Proteins interacting with the ligand on the IAC matrix were identified by mass spectrometry. Isothermal calorimetry was used to confirm and quantitatively evaluate the binding affinity of some of the interacting proteins. Heat-shock protein (Hsp) 70 and Hsp27 were the strongest interactors with the inhibitor, displaying binding affinities comparable to those of Cdk2. These results support the use of IAC as a general method for the rapid identification and qualitative evaluation of the in vivo targets and potential side effects of a given drug.     

Prenatal Stress Impairs Spinal Cord Oligodendrocyte Maturation via BDNF Signaling in the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis

One of the most substantial and established environmental risk factors for neurological and psychiatric disorders is stress exposure, whose detrimental consequences hinge on several variables including time. In this regard the gestational period is known to present an intrinsic vulnerability to environmental insults and thus stressful events during pregnancy can lead to severe consequences on the offspring’s brain development with long-term repercussions throughout adulthood. On this basis, we investigated the long-lasting impact of prenatal stress exposure on the susceptibility to the experimental autoimmune encephalomyelitis (EAE), a well-established murine model of multiple sclerosis. Although stress is considered a triggering factor for this chronic, progressive, autoimmune disease, little is known about the underlying mechanisms. To this end, EAE was induced by immunization with MOG35-55/CFA and pertussis toxin administration in adult female C57BL/6 mice born from control or stressed dams exposed to restraint stress during the last days of gestation. Our results demonstrate that gestational stress induces a marked increase in the severity of EAE symptoms in adulthood. Further, we highlight an altered maturation of oligodendrocytes in the spinal cord of prenatally stressed EAE mice, as indicated by the higher levels of GPR17, a marker of immature oligodendrocyte precursor cells. These behavioral and molecular alterations are paralleled by changes in the expression and signaling of the neurotrophin BDNF, an important mediator of neural plasticity that may contribute to stress-induced impaired remyelination. Since several already marketed drugs are able to modulate BDNF levels, these results pave the way to the possibility of repositioning these drugs in multiple sclerosis.

     

Contrasting life-history traits of two sympatric smooth-hound species: implication for vulnerability

In this study, life-history traits (maximum and average size, size at maturity and fecundity) of two congeneric smooth-hounds, Mustelus mustelus and Mustelus punctulatus, which share a geographical distribution and experience a similar fishing exploitation, were estimated and compared between species. The results indicated a lower maximum and average size, a lower size at maturity and a higher fecundity in M. punctulatus compared with those in M. mustelus. Considering that these two species co-occur in the same areas and are caught by the same fishing gears, the results indicate a higher vulnerability to exploitation of M. mustelus compared with that of M. punctulatus.     

In vitro activities of tachyplesin III against Pseudomonas aeruginosa

The in vitro activities of tachyplesin III were investigated against 20 multidrug-resistant Pseudomonas aeruginosa clinical isolates. Methods included minimal inhibitory concentrations, minimal bactericidal concentrations, time-kill studies, checkerboard titration method, endotoxin-binding activity and cytotoxicity assay. Overall the organisms were susceptible to the peptide at concentrations of 0.50-4 mg/l. Tachyplesin III completely inhibits the endotoxin procoagulant activity at 22.36 mg/l concentration. Fractional inhibitory concentration indexes demonstrated synergy between the peptide and betalactams or colistin. In conclusion, the intrinsic antibacterial and antiendotoxin activities and the synergistic interactions demonstrated with clinically used antibiotics make tachyplesin III valuable as potential candidate for new therapeutic strategies aimed to treat P. aeruginosa infection.     

18F-fluoro-deoxy-glucose focal uptake in very small pulmonary nodules: fact or artifact? Case reports

ABSTRACT: BACKGROUND: F-fluoro-deoxy-glucose (18F-FDG) positron emission tomography integrated/combined with computed tomography (PET-CT) provides the best diagnostic results in the metabolic characterization of undetermined solid pulmonary nodules. The diagnostic performance of 18F-FDG is similar for nodules measuring at least 1 cm and for larger masses, but few data exist for nodules smaller than 1 cm. CASE PRESENTATION: We report five cases of oncologic patients showing focal lung 18F-FDG uptake on PET-CT in nodules smaller than 1 cm. We also discuss the most common causes of 18F-FDG false-positive and false-negative results in the pulmonary parenchyma. In patient 1, contrast-enhanced CT performed 10 days before PET-CT did not show any abnormality in the site of uptake; in patient 2, high-resolution CT performed 1 month after PET showed a bronchiole filled with dense material interpreted as a mucoid impaction; in patient 3, contrast-enhanced CT performed 15 days before PET-CT did not identify any nodules; in patients 4 and 5, contrast-enhanced CT revealed a nodule smaller than 1 cm which could not be characterized. The 18F-FDG uptake at follow-up confirmed the malignant nature of pulmonary nodules smaller than 1 cm which were undetectable, misinterpreted, not recognized or undetermined at contrast-enhanced CT. CONCLUSION: In all five oncologic patients, 18F-FDG was able to metabolically characterize as malignant those nodules smaller than 1 cm, underlining that: 18F-FDG uptake is not only a function of tumor size but it is strongly related to the tumor biology; functional alterations may precede morphologic abnormalities. In the oncologic population, especially in higher-risk patients, PET can be performed even when the nodules are smaller than 1 cm, because it might give an earlier characterization and, sometimes, could guide in the identification of alterations missed on CT.     

Chronic exposure to agmatine results in the selection of agmatine-resistant hepatoma cells

During our study of the cytostatic effect of agmatine, we were able to isolate an agmatine resistant clone from a parental hepatoma cell line, HTC. These cells, called Agres, had slower growth rate than the parental cells when cultured in normal medium. The modification in polyamine content induced by agmatine was much lower in these cells and ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine/spermine acetyltransferase activities were much less affected. By investigating the mechanism responsible for these modifications, it was shown that agmatine and polyamines were not taken up by Agres cells. Their resistance to the antiproliferative effects of agmatine may thus arise from a lack of the polyamine transport system. Moreover, Agres cells were able to take up both glutamic acid and arginine at a rate significantly higher than that detected for HTC cells, most likely to provide components for compensatory increase of PA synthesis. These results emphasize the importance of polyamine transport for cell growth.     

Altered chromatin organization and SUN2 localization in mandibuloacral dysplasia are rescued by drug treatment

Mandibuloacral dysplasia type A (MADA) is a rare laminopathy characterized by growth retardation, craniofacial anomalies, bone resorption at specific sites including clavicles, phalanges and mandibula, mottled cutaneous pigmentation, skin rigidity, partial lipodystrophy, and insulin resistance. The disorder is caused by recessive mutations of the LMNA gene encoding for A-type lamins. The molecular feature of MADA consists in the accumulation of the unprocessed lamin A precursor, which is detected at the nuclear rim and in intranuclear aggregates. Here, we report the characterization of prelamin A post-translational modifications in MADA cells that induce alterations in the chromatin arrangement and dislocation of nuclear envelope-associated proteins involved in correct nucleo-cytoskeleton relationships. We show that protein post-translational modifications change depending on the passage number, suggesting the onset of a feedback mechanism. Moreover, we show that treatment of MADA cells with the farnesyltransferase inhibitors is effective in the recovery of the chromatin phenotype, altered in MADA, provided that the cells are at low passage number, while at high passage number, the treatment results ineffective. Moreover, the distribution of the lamin A interaction partner SUN2, a constituent of the nuclear envelope, is altered by MADA mutations, as argued by the formation of a highly disorganized lattice. Treatment with statins partially rescues proper SUN2 organization, indicating that its alteration is caused by farnesylated prelamin A accumulation. Given the major role of SUN1 and SUN2 in the nucleo-cytoskeleton interactions and in regulation of nuclear positioning in differentiating cells, we hypothesise that mechanisms regulating nuclear membrane-centrosome interplay and nuclear movement may be affected in MADA fibroblasts.     

Celiac disease‐associated Neisseria flavescens decreases mitochondrial respiration in CaCo‐2 epithelial cells: Impact of Lactobacillus paracasei CBA L74 on bacterial‐induced cellular imbalance

We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo‐2 cells. We also found that vesicular trafficking was delayed after the CD‐immunogenic P31‐43 gliadin peptide‐entered CaCo‐2 cells and that Lactobacillus paracasei CBA L74 (L. paracasei‐CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD‐N. flavescens‐infected CaCo‐2 cells and if any alteration could be mitigated by pretreating cells with L. paracasei‐CBA supernatant, despite the presence of P31‐43. We measured CaCo‐2 bioenergetics by an extracellular flux analyser, N. flavescens and P31‐43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD‐N. flavescens colocalised more than control N. flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31‐43 increased colocalisation of N. flavescens with early vesicles. Mitochondrial respiration was lower (P < .05) in CD‐N. flavescens‐infected cells versus not‐treated CaCo‐2 cells, whereas pretreatment with L. paracasei‐CBA reduced CD‐N. flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD‐N. flavescens induces metabolic imbalance in CaCo‐2 cells, and the L. paracasei‐CBA probiotic could be used to correct CD‐associated dysbiosis.     

An update on magnesium and bone health

In 2009 EFSA Panel concludes that a cause and effect relationship has been established between the dietary intake of magnesium (Mg) and maintenance of normal bone. After 2009, numerous studies have been published, but no reviews have made an update on this topic. So, the aim of this narrative review was to consider the state of the art since 2009 on relationship between Mg blood levels, Mg dietary intake and Mg dietary supplementation (alone or with other micronutrients; this last topic has been considered since 1990, because it is not included in the EFSA claims) and bone health in humans. This review included 28 eligible studies: nine studies concern Mg blood, 12 studies concern Mg intake and seven studies concern Mg supplementation, alone or in combination with other nutrients. From the various studies carried out on the serum concentration of Mg and its relationship with the bone, it has been shown that lower values are related to the presence of osteoporosis, and that about 30–40% of the subjects analyzed (mainly menopausal women) have hypomagnesaemia. Various dietetic investigations have shown that many people (about 20%) constantly consume lower quantities of Mg than recommended; moreover, in this category, a lower bone mineral density and a higher fracturing risk have been found. Considering the intervention studies published to date on supplementation with Mg, most have used this mineral in the form of citrate, carbonate or oxide, with a dosage varying between 250 and 1800 mg. In all studies there was a benefit both in terms of bone mineral density and fracture risk.