The folding and aggregation properties of a single KH-domain protein: Ribosome binding factor A (RbfA) from Pseudomonas aeruginosa.

BACKGROUND: Ribosome-binding factor A from the pathogenic bacterium Pseudomonas aeruginosa (PaRbfA) is a small ribosome assembly factor, composed by a single KH domain, involved in the maturation of the 30S subunit. These domains are characterized by the ability to bind RNA or ssDNA and are often located in proteins involved in a variety of cellular functions. However, although the ability of proteins to fold properly, to misfold or to aggregate is of paramount importance for their cellular functions, limited information is available on these dynamic properties in the case of KH domains. METHODS: PaRbfA thermodynamic stability and folding mechanism: Far-UV CD and fluorescence spectroscopy, stopped-flow kinetics and chevron plot analysis, site-directed mutagenesis. Fibrils characterization: FT-IR spectroscopy, Thioflavin T fluorescence, Transmission Electron Microscopy (TEM) and X-ray fibrils diffraction. RESULTS: Quantitative analysis of the (un)folding kinetics of PaRbfA show that, in vitro, the protein folds via a 3-states mechanism involving a transiently populated folding intermediate. We also provide experimental evidences that PaRbfA can form ordered fibrils endowed with cross-ß structure even in mild conditions. CONCLUSION: These results lead to the hypothesis that the folding intermediate of PaRbfA may expose (some of) the predicted amyloidogenic regions, which could act as aggregation nuclei in the fibrillogenesis. GENERAL SIGNIFICANCE: The methodological approach presented herein could be readily adapted to verify the ability of other KH domain proteins to form cross-ß structured fibrils and to transiently populate a folding intermediate.

YAP/TAZ mechano-transduction as the underlying mechanism of neuronal differentiation induced by reduced graphene oxide.

AIM: The aim of this work is the dissection of the molecular pathways underlying the differentiation effect of reduced graphene oxide (GO) materials in the absence of differentiation agents. MATERIALS & METHODS: Reduced GO is obtained either by drop casting method and heat-treated or biological reduction by the interaction between GO and wtPrxI. Cells were grown on both materials and the differentiation process studied by immunological and morphological detection. RESULTS & CONCLUSION: The results obtained indicate that both reduction methods of GO can determine the modulation of pathway involved in mechano-transduction and differentiation, by affecting YAP/TAZ localization outside the nuclei and increasing neuronal differentiation markers. This suggests that the mechano-transduction pathways are responsible for the differentiation process.

Management della retinopatia diabetica e dell'edema maculare diabetico: linee guida "Euretina 2017".

Si prevede che la malattia diabetica con tutte le sue complicanze avrà un forte aumento di incidenza con un grosso impatto socioeconomico nei prossimi decenni in tutto il mondo. Pertanto ben si comprende l'importanza di individuare attraverso una fine diagnostica quanto più precocemente la comparsa dei sintomi diabetici, migliorare lo stile di vita ed impostare cure efficienti. Riportiamo la serie di raccomandazioni EURETINA 2017, dei maggiori esperti in Europa per la gestione della malattia diabetica e delle complicanze della retina. Per combattere questa "pestilenza" occorre un team medico preparato. Il trattamento laser è stato considerato sino a non molto tempo fa il Gold standard della retinopatia diabetica e dell'edema diabetico (RD e EMD). Recenti studi hanno dimostrato, invece, che si possono raggiungere risultati migliori mediante l'iniezione diretta di farmaci nella cavità vitreale. In particolare è emerso terapia di prima linea, molecole in grado di inibire il fattore di crescita endoteliale vascolare (anti VEGF) mentre non è più raccomandata la fotocoagulazione laser per il trattamento del DME. Nell'ambito delle molecole farmacologiche gli steroidi hanno mantenuto un ruolo nella gestione del DME cronicamente persistente.

Infections in hospital departments. What is Hospital Responsibility?

Infections in hospitals still have a high incidence and many of them could be avoided through better welfare standards. To try to overcome them, a strategy based on prevention is needed, but cleaning, disinfection and sterilization procedures are also a key tool. It is important to provide for all healthcare professionals a constant update and the creation of protocols that take into account the technical, scientific and economic aspects, but also specific operational needs, so that the proposed solutions can be applied in daily routines. The authors outline the mandatory duties to the doctors and hospital and underline the need to document in the clinical record the treatments performed. In case of infections occurred in hospital environment, the patient must demonstrate the guilty nature of the hospital's conduct, the existence of a harm and the causal connection. The hospital must demonstrate that asepsis measures were adopted according to the actual scientific knowledge and they must cover not only the treatment but also the diagnosis, all the activities prior to surgery and the postoperative phase. The sentences examined show that hospitals can avoid being accused of negligence and imprudence only if they can prove that they have implemented all prophylaxis measures contained in the guidelines and protocols. They must demonstrate that the infection was caused by an unforeseeable event. While some initiatives to improve the quality of hospital care have already allowed a decrease in the incidence and cost of these infections, much remains to be done.

Physiological effect of olfactory stimuli inhalation in humans: an overview.

The importance of odorants in human life has long been recognized. Literature contains different approaches of physiological and psychological effects of odorant compounds, fragrances and essential oils. This work discusses odorants inhalation effect, based on an overview of major studies in humans. Beneficial effect of fragrances is mainly related to human behaviour. Studies document odorants influence in sympathetic and parasympathetic nervous systems, and neurophysiological brain activity. Moreover, odours compounds can act on the neuroendocrine system, neurotransmitters and neuromodulators, influencing psychological behaviour as well as body function. Odorant inhalation modulates physiological pathways, and in some cases, results in skin function regulation. The mechanism is incompletely elucidated. These findings suggest that olfactory system plays a role in central nervous system function beyond that of smell. In this overview, it was observed that odour compounds influenced stress biomarkers, dehydroepiandrosterone, oxidative stress, estradiol, dopamine, cutaneous barrier, sebum secretion and cutaneous immune system in humans. Some can be related with skin function. As the skin is associated with an extensive biochemical cascade and has complex mechanisms, studies have far to go, as there are processes not yet investigated related to skin that may be affected through olfaction. Future researches are needed to further understand and describe the mechanisms of action of physiological effects in fragrance compounds.

Switching between the alternative structures and functions of a 2-Cys peroxiredoxin, by site-directed mutagenesis.

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.

BDNF Val66Met polymorphism is associated with aggressive behavior in schizophrenia.

Brain-derived neurotrophic factor (BDNF) gene variants may potentially influence behaviour. In order to test this hypothesis, we investigated the relationship between BDNF Val66Met polymorphism and aggressive behaviour in a population of schizophrenic patients. Our results showed that increased number of BDNF Met alleles was associated with increased aggressive behaviour.

BDNF plasma levels in acute stroke.

The transport of brain derived neurotrophic factor (BDNF) across the blood-brain barrier (BBB) is negligible in normal conditions. However, BDNF might pass through the BBB when BBB is disrupted by a pathological condition such as stroke. This migration of BDNF through the BBB might be important during post-ischemic phase since BDNF can exert a protective action in the site of lesion. This study aimed to investigate plasma levels of BDNF in the acute phase of stroke in humans. Serial venous blood samples were taken in ten patients with acute stroke at the admission to the Stroke Unit and on the following 4 days. In the same samples we also evaluated the plasma levels of S100beta protein, a marker of BBB disruption. There was no significant change in BDNF plasma levels in our patients, even in the presence of a pronounced BBB dysfunction, as demonstrated by a significant increase of S100beta protein concentrations at days 2 and 3 after stroke. Our data, though indirectly, suggest that there is no significant increase in endogenous extracellular BDNF after a stroke in humans.

The anti-schistosomal drug praziquantel is an adenosine antagonist.

The mechanism of action of praziquantel (PZQ), the drug of choice against schistosomiasis, is still unclear. Since exposure of schistosomes to the drug is associated with calcium influx and muscular contraction, calcium channels have been suggested as the target, although direct combination of PZQ with their subunits was never demonstrated. We report a hitherto unknown effect of PZQ, namely the inhibition of nucleoside uptake, as observed in living worms using radio-isotope labelled adenosine and uridine. This effect is clearly seen in schistosomes but is absent in mammalian cells in culture. Moreover it is a specific pharmacological effect seen exclusively with the active levo-R(-)stereo isomer of the drug, and is shared by at least one benzodiazepine having antischistosomal activity. This novel effect acquires significance given that schistosomes cannot synthesize purine nucleosides de novo. A possible relationship between this novel effect and the known action of PZQ on calcium channels is discussed, since adenosine is known to bind to specific receptors and to behave as an indirect antagonist of calcium release in mammalian cells. If calcium channels were correlated with adenosine receptors also in schistosomes, as they are in mammals, this would support the hypothesis that PZQ-induced calcium influx may be correlated to adenosine receptor blockade.

Neurotrophic factors and clinical recovery in relapsing-remitting multiple sclerosis.

Pathogenic autoimmune cells are demonstrated to be able to produce neurotrophic factors during acute phase of multiple sclerosis (MS). In this study, we determined the production of various neurotrophins [brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin 3 (NT3) and neurotrophin 4 (NT4)] and some pro-inflammatory cytokines [tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] by unstimulated peripheral blood mononuclear cells (PBMC) in 21 relapsing-remitting MS patients during different phases of disease (stable, relapse and post-relapse). During acute phase of disease, we detected a considerable increase of BDNF, TNF-alpha and IFN-gamma production, while significantly higher levels of GDNF, NGF, NT3 and NT4 were found in post-relapse phase. When neurotrophin production was correlated with clinical outcome (complete or partial recovery from new symptoms), we found a significantly higher BDNF production in relapse phase followed by increased GDNF, NGF, NT3 and NT4 levels during post-relapse phase in subjects with complete remission only. During relapse phase, we detected a significant increase of pro-inflammatory cytokines, that was more evident in patients with partial recovery. The neuroprotective potential of immune cells seems to be inversely correlated with disease duration and with the age of patients.

Clinical characteristics, course and prognosis of spinal multiple sclerosis.

STUDY DESIGN: Retrospective examination. OBJECTIVE: To define the clinical characteristics and response to therapy of spinal multiple sclerosis (MS). SETTING: Italy. METHODS: Retrospective review was performed on 563 patients with clinical definite MS. Selection criteria were two or more spinal cord lesions in the presence of normal magnetic resonance imaging of the brain. RESULTS: Spinal MS was diagnosed in 13 patients (2.3%) out of 563 with clinical definite MS. There were seven female and six male patients; nine had a relapsing-remitting (RR) and four, a primary progressive (PP) course. All patients were treated with immunosoppressive or immunomodulatory therapy. Mean disease duration in patients with RR-MS was 13.1+/-10.1 years with a mean age at onset of 29.5+/-14.3 years; the mean Expanded Disability Status Scale (EDSS) at the time of the study was 3.5+/-2.5 with a progression index of 0.28. Mean disease duration in patients with PP course was 7+/-6.2 years with a mean age at onset of 56.7+/-10.4 years; the mean EDSS at the time of the study was 6.2+/-2.0 with a progression index of 1.48. CONCLUSIONS: Patients with spinal RR-MS are characterised by an early disease onset with minimal or moderate disability progression; patients with spinal PP-MS show a late disease onset and more rapid disability progression. In our series of spinal MS patients, disability progression seems to be mainly due to the disease course and age at onset rather than to the site of lesion.

BDNF in schizophrenia, depression and corresponding animal models.

Understanding the etiology and pathogenesis schizophrenia and depression is a major challenge facing psychiatry. One hypothesis is that these disorders are secondary to a malfunction of neurotrophic factors. Inappropriate neurotrophic support during brain development could lead to structural disorganisation in which neuronal networks are established in a nonoptimal manner. Inadequate neurotrophic support in adult individuals could ultimately be an underlying mechanism leading to decreased capacity of brain to adaptive changes and increased vulnerability to neurotoxic damage. Brain-derived neurotrophic factor (BDNF) is a mediator involved in neuronal survival and plasticity of dopaminergic, cholinergic, and serotonergic neurons in the central nervous system (CNS). In this review, we summarize findings regarding altered BDNF in schizophrenia and depression and animal models, as well as the effects of antipsychotic and antidepressive treatments on the expression of BDNF.

Identification of the bioactive peptide PEC-60 in brain.

PEC-60 is a 60-residue peptide originally isolated from pig intestine. It inhibits glucose-induced insulin secretion from perfused pancreas in a hormonal manner and also has biological activity in the immune system. PEC-60-like immunoreactive material has been reported in catecholamine neurons of the central and peripheral nervous systems, but the peptide has not been identified from that material. We have now isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is expressed in the gastrointestinal tract and the central nervous system. The specific regional brain distribution and interaction with classical neurotransmitters raise the possibility that PEC-60 may play a role in the central nervous system disorders involving dopamine dysregulation.

NGF, BDNF, leptin, and mast cells in human coronary atherosclerosis and metabolic syndrome.

While multiple growth factor, cytokines, and immune cells are identified in atherosclerotic lesions, as well as an essential nonneuronal function of neurotrophins implicated in cardiovascular tissue development and in lipid and glucose metabolism, the role of the neurotrophins NGF and BDNF and also the adipokine leptin in human coronary atherosclerosis and related disorders, such as metabolic syndrome, remains unclear. Here we report that (i) both the amount and the immunoreactivity of NGF was reduced and the expression of p75NGF receptor and the number of mast cell increased in human atherosclerotic coronary arteries (n = 12) compared with control specimens (n = 9) obtained from autopsy cases, and (ii) NGF and BDNF plasma levels were reduced in patients with metabolic syndrome (n = 23) compared with control subjects (n = 10). Also, in metabolic syndrome patients, a positive correlation between the plasma leptin levels and the number of adipose tissue mast cells was found, suggesting that leptin may be a novel adipoimmune mediator. Altogether, the results provide the first correlative evidence for the potential involvement of NGF, BDNF, leptin, and mast cells in human coronary atherosclerosis and metabolic syndrome, implying neuroimmune and adipoimmune pathways in the pathobiology of these cardiovascular disorders.

A pilot study of rat brain regional distribution of calcitonin, katacalcin and calcitonin gene-related peptide before and after antipsychotic treatment.

In contrast to extensive determinations of calcitonin gene-related peptide (CGRP) in neural tissues, calcitonin and its carboxyl-terminal flanking peptide katacalcin (in human PDN-21) have not been systematically measured by radioimmunoassay (RIA) in discrete brain structures. Using microwave irradiation (MW), a procedure that increases the recovery of neuropeptides, we investigated by radioimmunoassay (RIA) the rat brain regional distribution of CGRP like- immunoreactivity (-LI), calcitonin-LI, and katacalcin-LI. Calcitonin-LI and katacalcin-LI were found in low concentrations in frontal cortex, occipital cortex, striatum and hippocampus. Moreover, a 4-week treatment with antipsychotic drugs altered the concentrations of the calcitonin-gene family peptides in the frontal cortex, occipital cortex, and hippocampus; the magnitude of these changes, however, was only moderate. Lastly, calcitonin-LI and katacalcin-LI baseline concentrations as well as after antipsychotic treatment were highly correlated in the frontal cortex, striatum, and hippocampus. The possible regulatory role of calcitonin gene family peptides in the central nervous system (CNS) needs to be further explored.

Studies in animal models and humans suggesting a role of nerve growth factor in schizophrenia-like disorders.

Neurotrophic factors, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), are known to play a crucial role in growth, differentiation and function in a variety of brain neurons during development and in adult life. We have recently shown that environmental changes, aggressive behavior and anxiety-like responses alter both circulating and brain basal NGF levels. In the present review, we present data obtained using animal models which suggest that neurotrophic factors, particularly NGF and BDNF, might be implicated in mechanism(s) leading to a condition associated with schizophrenic-like behaviors. The hypothesis that neurotrophins of the NGF family can be implicated in some maldevelopmental aspects of schizophrenia is supported by findings indicating that the constitutive levels of NGF and BDNF are affected in schizophrenic patients.

Data and hypotheses on the role of nerve growth factor and other neurotrophins in psychiatric disorders.

Nerve growth factor (NGF) was discovered and characterized for its role on the growth, differentiation and maintenance of specific neurons of the peripheral nervous system. Subsequent studies revealed that NGF is synthesized and released within the central nervous system and exerts a trophic and functional role on basal forebrain cholinergic neurons; it is involved in a protective role following brain insults induced by an epileptic status, seizure, as well as surgical and chemical lesions.More recently our collaborative studies provided evidence that NGF is implicated in neurobehavioral response including cerebral alterations associated with psychiatric disorders. In this brief review, ongoing and emerging data are presented and discussed.

Learning performances, brain NGF distribution and NPY levels in transgenic mice expressing TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine involved in a variety of neurobiological activities including changing behavior and regulation of both neurotrophin and neuropeptide levels. In this study we used two lines of transgenic mice overexpressing brain TNF-alpha characterized by neurological deficits (line Tg6074) or phenotypically normal (line TgK3). We analyzed whether or not impairments in learning and memory processes due to TNF-alpha overexpression were associated with changes in endogenous brain NGF, NPY and beta-amyloid. The results indicate that full TNF-alpha transgene expression disrupted the learning capabilities of transgenic mice (both Tg6074 and TgK3). NGF decreased in the hippocampus of both transgenic mice whereas hippocampal NPY slightly potentiated in Tg6074. The decrease in NGF is correlated with deficits in spatial learning and memory whereas inflammation in the brain of Tg6074 could be responsible of the hippocampal increase in NPY. As a whole, these results show that transgenic mice overexpressing TNF-alpha in the brain represent a useful model for studying neuronal degeneration and brain inflammatory processes.

Brain-derived neurotrophic factor and tyrosine kinase receptor TrkB in rat brain are significantly altered after haloperidol and risperidone administration.

The antipsychotics haloperidol and risperidone are widely used in the therapy of schizophrenia. The former drug mainly acts on the dopamine (DA) D(2) receptor whereas risperidone binds to both DA and serotonin (5HT) receptors, particularly in the neurons of striatal and limbic structures. Recent evidence suggests that neurotrophins might also be involved in antipsychotic action in the central nervous system (CNS). We have previously reported that haloperidol and risperidone significantly affect brain nerve growth factor (NGF) level suggesting that these drugs influence the turnover of endogenous growth factors. Brain-derived neurotrophic factor (BDNF) supports survival and differentiation of developing and mature brain DA neurons. We hypothesized that treatments with haloperidol or risperidone will affect synthesis/release of brain BDNF and tested this hypothesis by measuring BDNF and TrkB in rat brain regions after a 29-day-treatment with haloperidol or risperidone added to chow. Drug treatments had no effects on weight of brain regions. Chronic administration of these drugs, however, altered BDNF synthesis or release and expression of TrkB-immunoreactivity within the brain. Both haloperidol and risperidone significantly decreased BDNF concentrations in frontal cortex, occipital cortex and hippocampus and decreased or increased TrkB receptors in selected brain structures. Because BDNF can act on a variety of CNS neurons, it is reasonable to hypothesize that alteration of brain level of this neurotrophin could constitute one of the mechanisms of action of antipsychotic drugs. These observations also support the possibility that neurotrophic factors play a role in altered brain function in schizophrenic disorders.

Mapping the differences in the brain concentration of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in an animal model of depression.

Antidepressant drugs as well as electroconvulsive stimuli can significantly influence brain concentrations of neurotrophic factors. However, it is not known whether the baseline brain concentrations of neurotrophic factors are altered in human subjects suffering from affective disorders or whether there are sex differences in concentrations of neurotrophins in human brain. In order to elucidate some of these questions, we measured by ELISA brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in an animal model of depression, the Flinders Sensitive Line (FSL) rats and their controls, the Flinders Resistant Line (FRL). Altered BDNF and NGF concentrations were found in frontal cortex, occipital cortex, and hypothalamus of depressed FSL compared to FRL control rats. Furthermore, different levels of these neurotrophins were also found in the male and female brain. Cumulatively these observations suggest that BDNF and NGF may play a role in depression and, hypothetically, different brain regional concentrations of BDNF and NGF in male and female animals may be relevant to gender differences in vulnerability to depression.