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1.  Long-acting antipsychotic drugs for the treatment of schizophrenia: use in daily practice from naturalistic observations 
BMC Psychiatry  2012;12:122.
Background
Current guidelines suggest specific criteria for oral or long-acting injectable antipsychotic drugs (LAIs). This review aims to describe the demographic and clinical characteristics of the ideal profile of the patient with schizophrenia treated with LAIs, through the analysis of nonrandomized studies.
Methods
A systematic review of nonrandomized studies in English was performed attempting to analyze the factors related to the choice and use of LAIs in daily practice. The contents were outlined using the Cochrane methods for nonrandomized studies and the variables included demographic as well as clinical characteristics. The available literature did not allow any statistical analysis that could be used to identify the ideal profile of patients with schizophrenia to be treated with LAIs.
Results
Eighty publications were selected and reviewed. Prevalence of LAI use ranged from 4.8% to 66%. The only demographic characteristics that were consistently assessed through retrieved studies were age (38.5 years in the 1970’s, 35.8 years in the 1980’s, 39.3 years in the 1990’s, to 39.5 years in the 2000’s) and gender (male > female).
Efficacy was assessed through the use of various symptom scales and other indirect measurements; safety was assessed through extrapyramidal symptoms and the use of anticholinergic drugs, but these data were inconsistent and impossible to pool. Efficacy and safety results reported in the different studies yielded a good therapeutic profile with a maximum of 74% decrease in hospital admissions and the prevalence of extrapyramidal symptoms with LAIs consistently increased at 6, 12, 18, and 24 months (35.4%, 37.1%, 36.9%, and 41.3%, respectively).
Conclusions
This analysis of the available literature strongly suggests that further observational studies on patients with schizophrenia treated with LAIs are needed to systematically assess their demographic and clinical characteristics and the relationships between them and patient outcome.
Besides the good efficacy and safety profile of LAIs, health care staff must also take into account the importance of establishing a therapeutic alliance with the patient and his/her relatives when selecting the most appropriate treatment. LAIs seem to be a good choice not only because of their good safety and efficacy profile, but also because they improve compliance, a key factor to improving adherence and to establishing a therapeutic alliance between patients with schizophrenia, their relatives, and their health care providers.
doi:10.1186/1471-244X-12-122
PMCID: PMC3573926  PMID: 22909285
Delayed-action preparations; Antipsychotic agents; Schizophrenia; Patients; Review
2.  Electrophysiological study of the bulbocavernosus reflex: normative data 
Functional Neurology  2014;28(4):293-295.
Summary
In the clinical setting the bulbocavernosus reflex (BCR) is elicited by squeezing the glans penis and digitally palpating the contraction of the bulbocavernosus (BC) muscle. In neurophysiology the BCR is obtained by stimulating the dorsal nerve of the penis or clitoris and by recording the response from BC muscle and it should be performed in selected patients with suspected urinary, bowel, or sexual neurogenic dysfunction. The BCR is considered one of the sacral neurophysiological tests of the greatest clinical utility. Previous normative data were obtained on small samples. The aim of this study was to determine normative values for the BCR in a large sample of men.
We studied a large population (105 men; mean age 53 years, range 19–73 years) without central or peripheral neurological diseases. In each subject the sacral reflex was elicited by electrical stimulation of the base of the dorsum penis and recorded using a surface electrode from the BC muscle. We recorded the latency, calculated at onset, and the maximal amplitude of response, calculated peak to peak.
We were able to detect the BCR in all the men. No correlation between BCR latency and age was found (r=0.136; p=0.160). The mean onset latency value was 33.0±4.85 ms (mean±2SD, range 26.8–39.4). The mean amplitude value was 16.53±12.21 μV (mean±2SD, range 4.2–43.6). Our normative data on the BCR were similar to previously published data.
PMCID: PMC3951258  PMID: 24598398
bulbocavernosus reflex; normative data; sacral reflex testing
3.  The chemotherapy-induced peripheral neuropathy outcome measures standardization study: from consensus to the first validity and reliability findings 
Cavaletti, G. | Cornblath, D. R. | Merkies, I. S. J. | Postma, T. J. | Rossi, E. | Frigeni, B. | Alberti, P. | Bruna, J. | Velasco, R. | Argyriou, A. A. | Kalofonos, H. P. | Psimaras, D. | Ricard, D. | Pace, A. | Galiè, E. | Briani, C. | Dalla Torre, C. | Faber, C. G. | Lalisang, R. I. | Boogerd, W. | Brandsma, D. | Koeppen, S. | Hense, J. | Storey, D. | Kerrigan, S. | Schenone, A. | Fabbri, S. | Valsecchi, M. G. | Mazzeo, A. | Pace, A. | Pessino, A. | Schenone, A. | Toscano, A. | Argyriou, A.A. | Brouwer, B. | Frigeni, B. | Piras, B. | Briani, C. | Dalla Torre, C. | Dominguez Gonzalez, C. | Faber, C. G. | Tomasello, C. | Binda, D. | Brandsma, D. | Cortinovis, D. | Psimaras, D. | Ricard, D. | Storey, D. | Cornblath, D.R. | Galiè, E. | Lindeck Pozza, E. | Rossi, E. | Vanhoutte, E.K. | Lanzani, F. | Pastorelli, F. | Altavilla, G. | Cavaletti, G. | Granata, G. | Kalofonos, H.P. | Ghignotti, I. | Merkies, I.S.J. | Bruna, J. | Hense, J. | Heimans, J.J. | Mattavelli, L. | Padua, L. | Reni, L. | Bakkers, M. | Boogerd, M. | Campagnolo, M. | Cazzaniga, M. | Eurelings, M. | Leandri, M. | Lucchetta, M. | Penas Prado, M. | Russo, M. | Valsecchi, M.G. | Piatti, M.L. | Alberti, P. | Bidoli, P. | Grant, R. | Plasmati, R. | Velasco, R. | Lalisang, R.I. | Meijer, R.J. | Fabbri, S. | Dorsey, S. G. | Galimberti, S. | Kerrigan, S. | Koeppen, S. | Postma, T.J. | Boogerd, W. | Grisold, W.
Annals of Oncology  2012;24(2):454-462.
Background
Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating and dose-limiting complication of cancer treatment. Thus far, the impact of CIPN has not been studied in a systematic clinimetric manner. The objective of the study was to select outcome measures for CIPN evaluation and to establish their validity and reproducibility in a cross-sectional multicenter study.
Patients and methods
After literature review and a consensus meeting among experts, face/content validity were obtained for the following selected scales: the National Cancer Institute-Common Toxicity Criteria (NCI-CTC), the Total Neuropathy Score clinical version (TNSc), the modified Inflammatory Neuropathy Cause and Treatment (INCAT) group sensory sumscore (mISS), the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30, and CIPN20 quality-of-life measures. A total of 281 patients with stable CIPN were examined. Validity (correlation) and reliability studies were carried out.
Results
Good inter-/intra-observer scores were obtained for the TNSc, mISS, and NCI-CTC sensory/motor subscales. Test–retest values were also good for the EORTC QLQ-C30 and CIPN20. Acceptable validity scores were obtained through the correlation among the measures.
Conclusion
Good validity and reliability scores were demonstrated for the set of selected impairment and quality-of-life outcome measures in CIPN. Future studies are planned to investigate the responsiveness aspects of these measures.
doi:10.1093/annonc/mds329
PMCID: PMC3551481  PMID: 22910842
assessment; chemotherapy; clinimetrics; peripheral neuropathy; reliability; validity
4.  Psychotherapy versus usual care in pediatric migraine and tension-type headache: a single-blind controlled pilot study 
Background
Despite growing interest in psychotherapy in child and adolescent headache, efficacy studies in this research field have focused mainly on cognitive-behavioral therapies. Whereas relaxation and cognitive-behavioral techniques, in particular, have been found to reduce the intensity and frequency of headache in children and adolescents, data on psychodynamic psychotherapy in this population are lacking.
Our aim was to explore the effectiveness of a brief psychodynamic psychotherapy program in the treatment of idiopathic headache in childhood and adolescence.
Methods
Thirty-three newly diagnosed idiopathic headache sufferers aged 6–18 years, consecutively referred to our outpatient services, were randomized to receive either a brief cycle of psychodynamic psychotherapy (eight sessions administered at two-week intervals) or usual care (clinical interview, neurological examination, counselling, symptomatic therapy).
The two groups were evaluated at baseline (T0) and at six months (T1) to be assessed for headache characteristics (i.e. frequency, intensity and duration), quality of life (i.e. the EuroQoL score), patient’s global health status (i.e. the Clinical Global Impression score), and emotional-behavioral symptoms (i.e. Child Behavior Checklist scores).
Results
The two groups were fairly similar with reference to the main demographic and clinical variables. The T0/T1 comparison showed a statistically significant improvement in headache frequency (p = 0.005), intensity (p < 0.001) and duration (p = 0.002), a statistically significant improvement in the CGI score (p = 0.018), and a borderline improvement in the EuroQoL score (p = 0.053) in the group receiving psychotherapy.
Conclusions
According to our pilot findings, a brief psychodynamic psychotherapy program may be more effective than usual care in children and adolescents with idiopathic headache.
doi:10.1186/1824-7288-40-6
PMCID: PMC3899919  PMID: 24444140
5.  Technical Advance: Autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production 
Journal of Leukocyte Biology  2013;94(1):193-202.
A novel method to ultra-purify unstimulated neutrophils by flow sorting and subsequent demonstration of the neutrophils' ability to release IL-1β following inflammatory stimulus.
The technical limitations of isolating neutrophils without contaminating leukocytes, while concurrently minimizing neutrophil activation, is a barrier to determining specific neutrophil functions. We aimed to assess the use of FACS for generating highly pure quiescent neutrophil populations in an antibody-free environment. Peripheral blood human granulocytes and murine bone marrow-derived neutrophils were isolated by discontinuous Percoll gradient and flow-sorted using FSC/SSC profiles and differences in autofluorescence. Postsort purity was assessed by morphological analysis and flow cytometry. Neutrophil activation was measured in unstimulated-unsorted and sorted cells and in response to fMLF, LTB4, and PAF by measuring shape change, CD62L, and CD11b expression; intracellular calcium flux; and chemotaxis. Cytokine production by human neutrophils was also determined. Postsort human neutrophil purity was 99.95% (sem=0.03; n=11; morphological analysis), and 99.68% were CD16+ve (sem=0.06; n=11), with similar results achieved for murine neutrophils. Flow sorting did not alter neutrophil activation or chemotaxis, relative to presorted cells, and no differences in response to agonists were observed. Stimulated neutrophils produced IL-1β, although to a lesser degree than CXCL8/IL-8. The exploitation of the difference in autofluorescence between neutrophils and eosinophils by FACS is a quick and effective method for generating highly purified populations for subsequent in vitro study.
doi:10.1189/jlb.0113040
PMCID: PMC3685014  PMID: 23667167
granulocyte; FACS; interleukin-1β
6.  Transcription of N- and O-linked mannosyltransferase genes is modulated by the pacC gene in the human dermatophyte Trichophyton rubrum 
FEBS Open Bio  2012;2:294-297.
In fungi, ambient pH sensing involves the activation of the Pal/PacC signalling pathway. In the dermatophyte Trichophyton rubrum, pH-dependent secretion of keratinases, which are major virulence determinants, is affected by disruption of the pacC gene. Here, the transcription profiling of the genes coding for N- and O-linked mannosyltransferases, enzymes involved in protein glycosylation, was evaluated in T. rubrum in response to disruption of the pacC gene and growth in keratin, glucose, and glucose plus glycine. We show that transcription of these mannosyltransferase genes is affected by nutrients at acidic pH and by PacC.
Highlights
▸ The PacC/Pal transduction pathway mediates the metabolic response to pH sensing. ▸ pH-dependent secretion of keratinases is modulated by the pacC gene in T. rubrum. ▸ The pacC gene modulates transcription of O- and N-mannosyltransferase genes at acidic pH. ▸ An O-mannosyltransferase gene is preferentially transcribed in keratin at pH 5.0. ▸ An N-mannosyltransferase gene is preferentially transcribed in non-buffered glucose at pH 5.0.
doi:10.1016/j.fob.2012.09.005
PMCID: PMC3678131  PMID: 23772361
Glycosylation; Enzyme secretion; pH regulation; Transcription profiling
7.  Comparative Genome Analysis of Trichophyton rubrum and Related Dermatophytes Reveals Candidate Genes Involved in Infection 
mBio  2012;3(5):e00259-12.
ABSTRACT
The major cause of athlete’s foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response.
IMPORTANCE
Athlete’s foot, jock itch, ringworm, and nail infections are common fungal infections, all caused by fungi known as dermatophytes (fungi that infect skin). This report presents the genome sequences of Trichophyton rubrum, the most frequent cause of athlete’s foot, as well as four other common dermatophytes. Dermatophyte genomes are enriched for four gene classes that may contribute to the ability of these fungi to cause disease. These include (i) proteases secreted to degrade skin; (ii) kinases, including pseudokinases, that are involved in signaling necessary for adapting to skin; (iii) secondary metabolites, compounds that act as toxins or signals in the interactions between fungus and host; and (iv) a class of proteins (LysM) that appear to bind and mask cell wall components and carbohydrates, thus avoiding the host’s immune response to the fungi. These genome sequences provide a strong foundation for future work in understanding how dermatophytes cause disease.
doi:10.1128/mBio.00259-12
PMCID: PMC3445971  PMID: 22951933
8.  Evaluation of a Novel Hexavalent Humanized Anti-IGF-1R Antibody and Its Bivalent Parental IgG in Diverse Cancer Cell Lines 
PLoS ONE  2012;7(8):e44235.
A major mechanism of monoclonal antibodies that selectively target the insulin-like growth factor type 1 receptor (IGF-1R) to inhibit tumor growth is by downregulating the receptor, regardless whether they are capable (antagonistic) or incapable (agonistic) of blocking the binding of cognate ligands. We have developed and characterized a novel agonistic anti-IGF-1R humanized antibody, hR1, and used the Dock-and-Lock (DNL) method to construct Hex-hR1, the first multivalent antibody comprising 6 functional Fabs of hR1, with the aim of enhancing potency of hR1. Based on cross-blocking experiments, hR1 recognizes a region of cysteine-rich domain on the α-subunit, different from the epitopes mapped for existing anti-IGF-1R antibodies, yet hR1 is similar to other anti-IGF-1R antibodies in downregulating IGF-1R and inhibiting proliferation, colony formation, or invasion of selected cancer cell lines in vitro, as well as suppressing growth of the RH-30 rhabdomyosarcoma xenograft in nude mice when combined with the mTOR inhibitor, rapamycin. Hex-hR1 and hR1 are generally comparable in their bioactivities under the in-intro and in-vivo conditions investigated. Nevertheless, in selective experiments involving a direct comparison of potency, Hex-hR1 demonstrated a stronger effect on inhibiting cell proliferation stimulated by IGF-1 and could effectively downregulate IGF-1R at a concentration as low as 20 pM.
doi:10.1371/journal.pone.0044235
PMCID: PMC3432068  PMID: 22952934
9.  Diving Into the Lipid Bilayer to Investigate the Transmembrane Organization and Conformational State Transitions of P-type Ion ATPases 
Current chemical biology  2011;5(2):118-129.
Although membrane proteins constitute more than 20% of the total proteins, the structures of only a few are known in detail. An important group of integral membrane proteins are ion-transporting ATPases of the P-type family, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. There are several crystal structures of the sarcoplasmic reticulum Ca2+ pump (SERCA) revealing different conformations, and recently, crystal structures of the H+-ATPase and the Na+/K+-ATPase were reported as well. However, there are no atomic resolution structures for other P-type ATPases including the plasma membrane calcium pump (PMCA), which is integral to cellular Ca2+ signaling. Crystallization of these proteins is challenging because there is often no natural source from which the protein can be obtained in large quantities, and the presence of multiple isoforms in the same tissue further complicates efforts to obtain homogeneous samples suitable for crystallization. Alternative techniques to study structural aspects and conformational transitions in the PMCAs (and other P-type ATPases) have therefore been developed. Specifically, information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid–protein interactions. Here, we review recent efforts using different hydrophobic photo-labeling methods to study the non-covalent interactions between the PMCA and surrounding phospholipids under different experimental conditions, and discuss how the use of these lipid probes can reveal valuable information on the membrane organization and conformational state transitions in the PMCA, Na+/K+-ATPase, and other P-type ATPases.
doi:10.2174/187231311795243319
PMCID: PMC3117579  PMID: 21691422
Ca2+-ATPase; hydrophobic photo-labeling; lipid-protein interaction; membrane protein; Na+/K+-ATPase; PMCA; P-type ATPase
10.  Transcription of the Hsp30, Hsp70, and Hsp90 heat shock protein genes is modulated by the PalA protein in response to acid pH-sensing in the fungus Aspergillus nidulans 
Cell Stress & Chaperones  2011;16(5):565-572.
Heat shock proteins are molecular chaperones linked to a myriad of physiological functions in both prokaryotes and eukaryotes. In this study, we show that the Aspergillus nidulans hsp30 (ANID_03555.1), hsp70 (ANID_05129.1), and hsp90 (ANID_08269.1) genes are preferentially expressed in an acidic milieu, whose expression is dependent on the palA+ background under optimal temperature for fungal growth. Heat shock induction of these three hsp genes showed different patterns in response to extracellular pH changes in the palA+ background. However, their accumulation upon heating for 2 h was almost unaffected by ambient pH changes in the palA− background. The PalA protein is a member of a conserved signaling cascade that is involved in the pH-mediated regulation of gene expression. Moreover, we identified several genes whose expression at pH 5.0 is also dependent on the palA+ background. These results reveal novel aspects of the heat- and pH-sensing networks of A. nidulans.
doi:10.1007/s12192-011-0267-5
PMCID: PMC3156257  PMID: 21553327
Aspergillus nidulans; pH sensing; pal signaling pathway; HSP
11.  50 The Allergic March Resolved at Allergen Component Level 
Background
The allergic march is well known at the level of pattern of sensitisation, but there is no information of its evolution in term of sensitzation to single allergenic molecules. We investigated the evolution of the IgE repertoire by means of a microarray allergen assay.
Methods
Serum samples from allergic patients of a wide age range were analyzed by a micorarray chip, which allow to identify in a single assay the presence of specific IgE towards 103 allergenic molecules. Total IgE were also evaluated as an internal control. Patients were stratified in 6 groups according to their age (0–2; 3–5; 6–9; 10–13; 14–17 and >17 years).
Results
Samples from 609 patients were analysed. The behaviour of total IgE according to age strictly paralleled that of the sum of specific IgE. Food-related components were the more frequently recognized in the first ages, whereas specific IgE to plant allergens appeared later. Nonetheless, mite-specific IgE were the most represented in all age classes. Specific IgE against cross-reacting allergens were virtually absent in the first years and tended to appear after the age of 6.
Conclusions
The molecular pattern of allergen recognition according to age well reflects the clinical characteristics of the allergic march.
doi:10.1097/01.WOX.0000411795.30118.b9
PMCID: PMC3512585
12.  Reverse left ventricular remodeling is more likely in non ischemic cardiomyopathy patients upgraded to biventricular stimulation after chronic right ventricular pacing 
Background
Chronic right ventricular (RV) apical pacing may lead to left ventricular (LV) dyssynchrony and LV dysfunction. In heart failure due to RV pacing, upgrading to biventricular stimulation (CRT) can improve NYHA Class and LV function. A proportion of patients do not respond to upgrading. Aim was to assess whether etiology of LV dysfunction accounts for responses to CRT in RV-paced patients.
Methods
Sixty-two patients treated by CRT, under RV pacing from 50.2 ± 5.4 months, were studied. Cause of LV dysfunction was non-ischemic (NIC) in 28 and ischemic cardiomyopathy (IC) in 34 patients. Clinical and conventional echocardiographic parameters were available within 1 month before RV pacing, within 1 month before CRT and at 12 ± 2 months of follow-up (FU).
Results
Decreased LVEF (from 37.0 ± 8.8 to 25.6 ± 6.1%, p <0.001), increased LV end-systolic dimensions (LVESD) (from 48.1 ± 8.6 to 55.2 ± 7.9 mm, p <0.001) and worsened NYHA Class (from 1.9 ± 1.1 to 3.2 ± .6, p < 0.005) were found before CRT, compared to pre RV-pacing. After CRT, 44/62 patients showed a ≥ 1 NYHA Class improvement; >10% decrease in LVESD was observed in 24 patients: 5 with IC, 19 with NIC (p < .0.001). The association between cause of LV dysfunction with >10% decrease in LVESD remained highly significant (p < 0.001) adjusting for pre-CRT QRS duration, NYHA Class, LVEF, LVESD, treatment or RV pacing duration.
Conclusions
CRT improves functional class even after long-lasting pacing. Reverse remodeling is evident in a small population, more likely with NIC.
doi:10.1186/1476-7120-9-41
PMCID: PMC3265428  PMID: 22177469
congestive heart failure; biventricular stimulation; non-ischemic cardiomyopathy; ischemic cardiomyopathy
13.  Cortico-Cortical Connectivity between Right Parietal and Bilateral Primary Motor Cortices during Imagined and Observed Actions: A Combined TMS/tDCS Study 
Previous transcranial magnetic stimulation (TMS) studies showed functional connections between the parietal cortex (PC) and the primary motor cortex (M1) during tasks of different reaching-to-grasp movements. Here, we tested whether the same network is involved in cognitive processes such as imagined or observed actions. Single pulse TMS of the right and left M1 during rest and during a motor imagery and an action observation task (i.e., an index–thumb pinch grip in both cases) was used to measure corticospinal excitability changes before and after conditioning of the right PC by 10 min of cathodal, anodal, or sham transcranial direct current stimulation (tDCS). Corticospinal excitability was indexed by the size of motor-evoked potentials (MEPs) from the contralateral first dorsal interosseous (FDI; target) and abductor digiti minimi muscle (control) muscles. Results showed selective ipsilateral effects on the M1 excitability, exclusively for motor imagery processes: anodal tDCS enhanced the MEPs’ size from the FDI muscle, whereas cathodal tDCS decreased it. Only cathodal tDCS impacted corticospinal facilitation induced by action observation. Sham stimulation was always uneffective. These results suggest that motor imagery, differently from action observation, is sustained by a strictly ipsilateral parieto-motor cortex circuits. Results might have implication for neuromodulatory rehabilitative purposes.
doi:10.3389/fncir.2011.00010
PMCID: PMC3163809  PMID: 21909322
TMS; tDCS; motor imagery; action observation; connectivity; M1; right PC
14.  Transcription of the Neurospora crassa 70-kDa class heat shock protein genes is modulated in response to extracellular pH changes 
Cell Stress & Chaperones  2009;15(2):225-231.
Heat shock proteins belong to a conserved superfamily of molecular chaperones found in prokaryotes and eukaryotes. These proteins are linked to a myriad of physiological functions. In this study, we show that the N. crassa hsp70-1 (NCU09602.3) and hsp70-2 (NCU08693.3) genes are preferentially expressed in an acidic milieu after 15 h of cell growth in sufficient phosphate at 30°C. No significant accumulation of these transcripts was detected at alkaline pH values. Both genes accumulated to a high level in mycelia that were incubated for 1 h at 45°C, regardless of the phosphate concentration and extracellular pH changes. Transcription of the hsp70-1 and hsp70-2 genes was dependent on the pacC+ background in mycelia cultured under optimal growth conditions or at 45°C. The pacC gene encodes a Zn-finger transcription factor that is involved in the regulation of gene expression by pH. Heat shock induction of these two hsp genes in mycelia incubated in low-phosphate medium was almost not altered in the nuc-1− background under both acidic and alkaline pH conditions. The NUC-1 transcriptional regulator is involved in the derepression of nucleases, phosphatases, and transporters that are necessary for fulfilling the cell's phosphate requirements. Transcription of the hsp70-3 (NCU01499.3) gene followed a different pattern of induction—the gene was depressed under insufficient phosphate conditions but was apparently unaffected by alkalinization of the culture medium. Moreover, this gene was not induced by heat shock. These results reveal novel aspects of the heat-sensing network of N. crassa.
doi:10.1007/s12192-009-0131-z
PMCID: PMC2866986  PMID: 19618296
Neurospora crassa; hsp70; Heat shock; Pi sensing; pH regulation; nuc-1; pacC
15.  Lack of neo-sensitization to Pen a 1 in patients treated with mite sublingual immunotherapy 
Background
Some studies reported the possible induction of food allergy, caused by neo-sensitization to cross-reacting allergens, during immunotherapy with aeroallergens, while other studies ruled out such possibility.
Objectives
The aim of this study was to evaluate the development of neo-sensitization to Pen a 1 (tropomyosin) as well as the appearance of reactions after ingestion of foods containing tropomyosin as a consequence of sublingual mite immunization.
Materials and methods
Specific IgE to Tropomyosin (rPen a 1) before and after mite sublingual immunotherapy in 134 subjects were measured. IgE-specific antibodies for mite extract and recombinant allergen Pen a 1 were evaluated using the immunoenzymatic CAP system (Phadia Diagnostics, Milan, Italy).
Results
All patients had rPen a 1 IgE negative results before and after mite SLIT and did not show positive shrimp extract skin reactivity and serological rPen a 1 IgE conversion after treatment. More important, no patient showed systemic reactions to crustacean ingestion.
Conclusions
Patients did not show neo-sensitization to tropomyosin, a component of the extract (namely mite group 10) administered. An assessment of a patient's possible pre-existing sensitisation to tropomyosin by skin test and/or specific IgE prior to start mite extract immunotherapy is recommended.
Trial Registration
This trial is registered in EudraCT, with the ID number of 2010-02035531.
doi:10.1186/1476-7961-8-4
PMCID: PMC2859740  PMID: 20230633
16.  Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: mean fluorescence intensity T/B ratio versus percentage of positive cells 
Background
ZAP-70 is an independent negative prognostic marker in chronic lymphocytic leukemia (CLL). Usually, its expression is investigated by flow cytometric protocols in which the percentage of ZAP-70 positive CLL cells is determined in respect to isotypic control (ISO-method) or residual ZAP-70 positive T cells (T-method). These methods, however, beside suffering of an inherent subjectivity in their application, may give discordant results in some cases. The aim of this study was to assess the prognostic significance of these methods in comparison with another in which ZAP-70 expression was evaluated as a Mean-Fluorescence-Intensity Ratio between gated T and CLL cells (T/B Ratio-method).
Methods
Cytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set).
Results
The optimal prognostic cut-offs for ZAP-70 expression were selected at 11% (ISO-method) or 20% of positive cells (T-method), as well as at 3.0 (T/B Ratio-method) in the test set; these cut-offs yielded 66, 60 and 73 ZAP-70+ cases, respectively. Univariate analyses resulted in a better separation of ZAP-70+ vs. ZAP-70- CLL patients utilizing the T/B Ratio, compared to T- or ISO-methods. In multivariate analyses which included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio method was applied. These findings were confirmed in the validation set, in which ZAP-70 expression, evaluated by the T- (cut-off = 20%) or T/B Ratio- (cut-off = 3.0) methods, yielded 180 or 127 ZAP-70+ cases, respectively. ZAP-70+ patients according to the T/B Ratio-method had shorter TTT, both if compared to ZAP-70- CLL, and to cases classified ZAP-70+ by the T-method only.
Conclusions
We suggest to evaluate ZAP-70 expression in routine settings using the T/B Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B Ratio value as optimal cut-off to discriminate ZAP-70+ (T/B Ratio less than 3.0) from ZAP-70- (T/B Ratio more/equal than 3.0) cases.
doi:10.1186/1479-5876-8-23
PMCID: PMC2846891  PMID: 20211015
17.  Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum 
BMC Microbiology  2010;10:39.
Background
Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries.
Results
The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases.
Conclusion
In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.
doi:10.1186/1471-2180-10-39
PMCID: PMC2831883  PMID: 20144196
18.  Autism and classification systems: a study of 84 children 
Background
A number of studies have shown that current classification systems (ICD 10, DSM IV TR) have limitation when applied to autistic children and the category PDD NOS (DSM IV TR) has in particular been criticized. To check the possible usefulness of other classification systems to better describe patient's functioning, we retrospectively studied 84 patients, seen consecutively in our Child Neurology and Psychiatry Department (excluding only those presenting for another disease even if with clinical signs of a PDD).
Methods
We tried to classify them according to ICD 10, DSM IV TR, CFTMEA-R, "operational classification" (Manzano and Palacio) and de Ajuriaguerra's classification.
Results
We found a good correspondence between DSM IV TR and ICD 10 and the use of psychodynamic classification systems (in particular CFTMEA-R) was useful to differentiate clinical subtypes collected under the PDD NOS etiquette according to DSM IV TR.
Conclusions
To rationalize research efforts and find better tailored therapies, we need to improve PDD classification systems, using contributions coming from every field of child psychiatry and neurology: it's possible that 0-3 Classification could help this.
doi:10.1186/1824-7288-36-10
PMCID: PMC2824795  PMID: 20205822
19.  Osteopontin Is Associated with Increased Arterial Stiffness in Rheumatoid Arthritis 
Molecular Medicine  2009;15(11-12):402-406.
Rheumatoid arthritis (RA) patients are characterized by increased arterial stiffness, an independent predictor of cardiovascular risk. It has been suggested that osteopontin (OPN), a cytokine involved in RA pathogenesis, might have vascular effects. To study a possible relationship between OPN and arterial stiffness, aortic pulse wave velocity (PWV) was measured by tonometry in 69 patients (41 with RA, 28 with systemic sclerosis [SSc]) and 18 healthy controls. Plasma OPN levels, oxidative stress markers, and endothelin 1 (ET-1) were assessed. OPN levels were significantly (P < 0.05) higher in RA (median 9.93, range 4.36–47.80 ng/mL) than in SSc (4.3, 2.1–19.7 ng/mL) or controls (5.2, 4.1–9.4 ng/mL). In RA patients, log-OPN was related to log–C-reactive protein (log-CRP) (r = 0.30, P < 0.05), age (r = 0.38, P < 0.01), Health Assessment Questionnaire (HAQ) (r = 0.58, P < 0.0001), and inversely related to total cholesterol (r = −0.33, P < 0.05) and apolipoprotein A (apoA) (r = −0.58, P < 0.001), but not to oxidative stress markers and ET-1. PWV was similar in RA (median 8.1, range 4.7–16.4 m/s) and SSc (median 8.7, range 7.1–13.1 m/s), but significantly greater (P < 0.01) than controls (median 7.5, range 4.1–10.4 m/s). Aortic PWV was related to log-OPN (r = 0.40, P < 0.01) only in RA patients. It also was related to age (r = 0.34, P < 0.05), mean blood pressure (r = 0.44, P < 0.001), and HAQ (r = 0.48, P < 0.001). In multiple regression analysis (r2 = 0.36), including confounders, log-OPN remained a significant predictor (P < 0.05) of PWV in RA. Elevated plasma OPN levels are associated with increased arterial stiffness in RA patients, suggesting that this protein might represent a bridge protein between inflammation and the consequent joint damage and cardiovascular risk in RA patients.
doi:10.2119/molmed.2009.00052
PMCID: PMC2710292  PMID: 19603104
20.  Rationale and design of an independent randomised controlled trial evaluating the effectiveness of aripiprazole or haloperidol in combination with clozapine for treatment-resistant schizophrenia 
Nosè, Michela | Accordini, Simone | Artioli, Paola | Barale, Francesco | Barbui, Corrado | Beneduce, Rossella | Berardi, Domenico | Bertolazzi, Gerardo | Biancosino, Bruno | Bisogno, Alfredo | Bivi, Raffaella | Bogetto, Filippo | Boso, Marianna | Bozzani, Alberto | Bucolo, Piera | Casale, Marcello | Cascone, Liliana | Ciammella, Luisa | Cicolini, Alessia | Cipresso, Gabriele | Cipriani, Andrea | Colombo, Paola | Dal Santo, Barbara | De Francesco, Michele | Di Lorenzo, Giorgio | Di Munzio, Walter | Ducci, Giuseppe | Erlicher, Arcadio | Esposito, Eleonora | Ferrannini, Luigi | Ferrato, Farida | Ferro, Antonio | Fragomeno, Nicoletta | Parise, Vincenzo Fricchione | Frova, Maria | Gardellin, Francesco | Garzotto, Nicola | Giambartolomei, Andrea | Giupponi, Giancarlo | Grassi, Luigi | Grazian, Natalia | Grecu, Lorella | Guerrini, Gualtiero | Laddomada, Francesco | Lazzarin, Ermanna | Lintas, Camilla | Malchiodi, Francesca | Malvini, Lara | Marchiaro, Livio | Marsilio, Alessandra | Mauri, Massimo Carlo | Mautone, Antonio | Menchetti, Marco | Migliorini, Giuseppe | Mollica, Marco | Moretti, Daniele | Mulè, Serena | Nicholau, Stylianos | Nosè, Flavio | Occhionero, Guglielmo | Pacilli, Anna Maria | Pecchioli, Stefania | Percudani, Mauro | Piantato, Ennio | Piazza, Carlo | Pontarollo, Francesco | Pycha, Roger | Quartesan, Roberto | Rillosi, Luciana | Risso, Francesco | Rizzo, Raffella | Rocca, Paola | Roma, Stefania | Rossattini, Matteo | Rossi, Giuseppe | Rossi, Giovanni | Sala, Alessandra | Santilli, Claudio | Saraò, Giuseppe | Sarnicola, Antonio | Sartore, Francesca | Scarone, Silvio | Sciarma, Tiziana | Siracusano, Alberto | Strizzolo, Stefania | Tansella, Michele | Targa, Gino | Tasser, Annamarie | Tomasi, Rodolfo | Travaglini, Rossana | Veronese, Antonio | Ziero, Simona
Trials  2009;10:31.
Background
One third to two thirds of people with schizophrenia have persistent psychotic symptoms despite clozapine treatment. Under real-world circumstances, the need to provide effective therapeutic interventions to patients who do not have an optimal response to clozapine has been cited as the most common reason for simultaneously prescribing a second antipsychotic drug in combination treatment strategies. In a clinical area where the pressing need of providing therapeutic answers has progressively increased the occurrence of antipsychotic polypharmacy, despite the lack of robust evidence of its efficacy, we sought to implement a pre-planned protocol where two alternative therapeutic answers are systematically provided and evaluated within the context of a pragmatic, multicentre, independent randomised study.
Methods/Design
The principal clinical question to be answered by the present project is the relative efficacy and tolerability of combination treatment with clozapine plus aripiprazole compared with combination treatment with clozapine plus haloperidol in patients with an incomplete response to treatment with clozapine over an appropriate period of time. This project is a prospective, multicentre, randomized, parallel-group, superiority trial that follow patients over a period of 12 months. Withdrawal from allocated treatment within 3 months is the primary outcome.
Discussion
The implementation of the protocol presented here shows that it is possible to create a network of community psychiatric services that accept the idea of using their everyday clinical practice to produce randomised knowledge. The employed pragmatic attitude allowed to randomly allocate more than 100 individuals, which means that this study is the largest antipsychotic combination trial conducted so far in Western countries. We expect that the current project, by generating evidence on whether it is clinically useful to combine clozapine with aripiprazole rather than with haloperidol, provides physicians with a solid evidence base to be directly applied in the routine care of patients with schizophrenia.
Trial Registration
Clincaltrials.gov Identifier: NCT00395915
doi:10.1186/1745-6215-10-31
PMCID: PMC2689216  PMID: 19445659
21.  Muscle modifications in fibromyalgic patients revealed by surface electromyography (SEMG) analysis 
Background
Several studies have been carried out in order to investigate surface electromyography (SEMG) response on fibromyalgic (FM) patients. Some studies failed to demonstrate differences between FM patients and healthy individuals while others found differences in SEMG parameters. Different muscular region have been analyzed in FM patients and heterogeneity is also produced because of the different ways in which the SEMG technique is used.
The aims of this study were to evaluate muscle modifications by SEMG analysis in FM women with respect to a sample of healthy controls and to investigate the relationships between SEMG parameters and the clinical aspects of the disease.
Methods
SEMG was recorded in 100 FM women (48.10 ± 11.96 yr) and in 50 healthy women (48.60 ± 11.18 yr), from the tibialis anterior and the distal part of vastus medialis muscle during isometric contraction. Initial values and rate of change of median spectral frequency (MDF) and conduction velocity (CV) of the SEMG signal were calculated.
The clinical parameters "Fibromyalgia Impact Questionnaire", pain, tender points, tiredness were evaluated and the relationships between these data and the SEMG results were also studied. For the statistical analysis Mann-Whitney test, chi-square test and Spearman correlation were used.
Results
MDF absolute values and the so-called Fatigue Index (FI) were significantly lower (p < 0.001) in both muscles studied in FM patients (MDF: 93.2 μV; FI: 1.10, 0.89) with respect to healthy controls (MDF: 138.2 μV; FI: 2.41, 1.66) and a smaller reduction in the percentage values of MDF was observed in FM patients vs controls (22% vs 38%). A significant correlation was found between the SEMG parameter decrement of normalized median frequency (MNF) (%) and seriousness of FM (evaluated by means of tender points).
Conclusion
We have found some interesting muscle modifications in FM patients with respect to healthy controls, regarding MDF, CV and FI values which resulted significantly lower in FM. Patients might have a different fiber recruitment or a possible atrophy of type II fibers suggesting that they are not able to reach muscle relaxation.
doi:10.1186/1471-2474-10-36
PMCID: PMC2678091  PMID: 19368705
22.  Titanium and Ruthenium Phthalocyanines for NO2 Sensors: A Mini-Review 
Sensors (Basel, Switzerland)  2009;9(7):5277-5297.
This review presents studies devoted to the description and comprehension of phenomena connected with the sensing behaviour towards NO2 of films of two phthalocyanines, titanium bis-phthalocyanine and ruthenium phthalocyanine. Spectroscopic, conductometric, and morphological features recorded during exposure to the gas are explained and the mechanisms of gas-molecule interaction are also elucidated. The review also shows how X-ray reflectivity can be a useful tool for monitoring morphological parameters such as thickness and roughness that are demonstrated to be sensitive variables for monitoring the exposure of thin films of sensor materials to NO2 gas.
doi:10.3390/s90705277
PMCID: PMC3274156  PMID: 22346697
phthalocyanine; X-ray reflectivity; NO2; sensors
23.  Epstein–Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-κB pathway 
Nucleic Acids Research  2008;36(20):6608-6619.
The Epstein–Barr virus (EBV)-encoded latent membrane protein-1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, substantially contributes to EBV's oncogenic potential by activating nuclear factor-κB (NF-κB). miR-155 is an oncogenic miRNA critical for B-cell maturation and immunoglobulin production in response to antigen. We report that miR-155 expression is much higher in EBV-immortalized B cells than in EBV-negative B cells. LMP1, but not LMP2, up-regulated the expression of miR-155, when transfected in EBV-negative B cells. We analyzed two putative NF-κB binding sites in the miR-155 promoter; both sites recruited NF-κB complex, in nuclear extract from EBV-immortalized cells. The exogenous expression of LMP1, in EBV-negative background, is temporally correlated to induction of p65 with binding on both NF-κB sites and with miR-155 overexpression. The induction of p65 binding together with increased RNA polymerase II binding, confirms that LMP1-mediated activation of miR-155 occurs transcriptionally. In reporter assays, miR-155 promoter lacking NF-κB binding sites was no longer activated by LMP1 expression and an intact AP1 site is needed to attain maximum activation. Finally, we demonstrate that LMP1-mediated activation of miR-155 in an EBV-negative background correlates with reduction of protein PU.1, which is a possible miR target.
doi:10.1093/nar/gkn666
PMCID: PMC2582607  PMID: 18940871
24.  Plasma membrane calcium pump activity is affected by the membrane protein concentration. Evidence for the involvement of the actin cytoskeleton 
Biochimica et biophysica acta  2007;1768(6):1641-1649.
Plasma membrane calcium pumps (PMCAs) are integral membrane proteins that actively expel Ca2+ from the cell. Specific Ca2+-ATPase activity of erythrocyte membranes increased steeply up to 1.5–5 times when the membrane protein concentration decreased from 50 μg/ml to 1 μg/ml. The activation by dilution was also observed for ATP-dependent Ca2+ uptake into vesicles from Sf9 over-expressing the PMCA 4b isoform, confirming that it is a property of the PMCA. Dilution of the protein did not modify the activation by ATP, Ca2+ or Ca2+-calmodulin. Treatment with non-ionic detergents did not abolish the dilution effect, suggesting that it was not due to resealing of the membrane vesicles. Pre-incubation of erythrocyte membranes with Cytochalasin D under conditions that promote actin polymerization abolished the dilution effect. Highly-purified, micellar PMCA showed no dilution effect and was not affected by Cytochalasin D. Taken together, these results suggest that the concentration-dependent behavior of the PMCA activity was due to interactions with cytoskeletal proteins. The dilution effect was also observed with different PMCA isoforms, indicating that this is a general phenomenon for all PMCAs.
doi:10.1016/j.bbamem.2007.03.012
PMCID: PMC2041878  PMID: 17481573
PMCA; calmodulin; calcium; membrane; cytoskeleton; Cytochalasin D
25.  Strategies for the transfusion of subjects with complex red cell immunisation: the Bank of rare blood donors of the Region of Lombardy 
Blood Transfusion  2007;5(4):217-226.
Introduction
Selecting units of rare blood for transfusion to patients with complex immunisation is one of the most critical processes of a Transfusion Centre. In January 2005 the ‘Rare Blood Components Bank – Reference Centre of the Region of Lombardy’ w as established with the following goals: 1) identifying regional rare blood donors; 2) creating a regional registry of rare donors; 3) organising a regional bank of liquid and frozen rare blood units; 4) setting up a regional Immunohaematology Reference Laboratory (IRL) to type donors and resolve complex cases.
Methods
The key elements in establishing the Bank were periodic meetings organised by the directors and representatives of the regional Departments of Transfusion Medicine and Haematology (DTMH) and the institution of three working groups (informatics, regulations, finance).
Results
The regional IRL was set up, the relevant operating procedures were distributed region-wide, software features were defined and later validated upon activation, and the funds assigned were allocated to various cost items. The number and characteristics of the donors to be typed were identified and 14 regional DTMHs started to send samples. Overall, 20,714 donors were typed, for a total of 258,003 typings, and 2,880 rare donors were identified. Of these, 97% were rare donors because of combinations of antigens (2,139 negative for the S antigen and 659 negative for the s antigen) and 3% (n=82) because they were negative for high-frequency antigens. In the first 2 years of activity, the IRL carried out investigations of 140 complex cases referred from other Centres and distributed 2,024 units with rare phenotypes to 142 patients.
Conclusions
The main goal achieved in the first 24 months from the start of the project was to set up a regional network able to meet the transfusion needs of patients with complex immunisation.
doi:10.2450/2007.0016-07
PMCID: PMC2581908  PMID: 19204778
complex red cell immunisation; transfusion support; banks and registries of rare donors

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