Exploitation of proteomics strategies in protein structure-function studies

Mass spectrometry plays a central role in structural proteomics, particularly in highly intensive structural genomics projects. This review paper reports some examples taken from recent work from the authors' laboratory and is aimed at showing that modem proteomics strategies are instrumental in the integration of structural genomic projects in fields such as: (i) protein-protein interactions, (ii) protein-DNA interactions, (iii) protein-ligand interactions, and (iv) protein-folding intermediates

CoQ10 and vitamin A supplementation support voice rehabilitation. A double-blind, randomized, controlled, three-period cross-over pilot study

Objectives: To evaluate the effectiveness of an adjuvant therapy (CoQ10 in its watersoluble form and vitamin A) in supporting voice rehabilitation in a large group of patients with muscle tension dysphonia (MTD). Study Design: Twelve-week, double-blind, randomized, controlled, three-period crossover pilot study. The primary endpoint was the change in the Dysphonia Severity Index (DSI) over the 12-week study period. Secondary endpoints were the changes in the subcomponents of DSI, including MPT, F0-high, I-low, and jitter. Exploratory endpoints were the changes in the Shimmer and in Voice Handicap Index (VHI). Methods: Patients were randomly assigned in a 1:1 ratio to two counter-balanced arms. Group A (ADJ-PLA) patients were administered QTer 300 mg and Vit A acetate 500.000 Ul/g 1 mg twice daily for a 4-week intervention period, followed by a 4-week period of wash-out, and then were submitted to a last 4-week period of placebo. Patients in Group B (PLB-ADJ) were given the treatment period in reverse order. Both groups received a 45-min voice therapy in a group format once a day for 4 weeks during the first and the second active periods. The therapy was held during the wash-out period. Results: The analysis of main time effect indicated a trend toward recovery of vocal function regardless of group assignment. A significant time by group effect was found on DSI [F = 3.4 (2.5, 80.5), p = 0.03], F0-high [F = 4.5 (2.6, 82.9), p = 0.008] and Shimmer [F = 3.6 (1.5, 46.9), p = 0.048], under CoQ10 and Vit A treatment, with a small effect size. There was no significant time by group effect on the other study measures, namely MPT, I-low, VHI. Conclusions: A trend toward recovery of vocal function was observed in all the patients, likely due to voice rehabilitation. The improvement of DSI was greater under CoQ10 and Vitamin treatment, indicating a more pronounced improvement of vocal quality under adjuvant therapy. The study protocol was reviewed and approved by the Ethics Committee of Policlinico Umberto I Hospital, Rome, Italy Rif. 3069/13.02.2014

ATTRITION OF BED MATERIALS AND FUEL PELLETS FOR FLUIDIZED BED GASIFICATION APPLICATION

This paper reports on a study of the attrition/fragmentation behavior of different bed materials and fuel pellets for application in fluidized bed gasification. Three different bed materials displaying catalytic activity, namely fresh and sintered dolomite and a Ni-alumina catalyst, were tested for their resistance to fragmentation and attrition in fluidized bed. The fresh dolomite displayed extensive particle breakage upon calcination and a large production of attrited fines during fluidized bed operation. The other two materials were much more resistant to attrition and appeared to be suitable for further long-term operation testing. The attrition/fragmentation resistance of three pelletized fuels, one based on wood and the other two on a mixture of wood and coal, was also characterized under both inert and gasification conditions. Pellet breakage by primary fragmentation upon devolatilization appeared to be rather limited for all fuels. On the contrary, attrition of carbon fines from the char particles during gasification was extensive, due to a gasification-assisted attrition mechanism

Molecular analysis has allowed the definitive diagnosis of multiple acyl-CoA dehydrogenase deficiency (MADD)

Multiple acyl-CoA dehydrogenation deficiency (MADD) is a rare autosomal recessive disorder due to defects in the electron transfer flavoprotein (ETF) or in the electron transfer flavoprotein dehydrogenase (ETFDH) enzymes, involved in the mitochondrial electron transport chain. Patients with MADD fall into different clinical phenotypes, ranging from a severe neonatal presentation, with metabolic acidosis, cardiomyopathy and liver disease to a mild childhood/adult disease, with episodic metabolic decompensation, muscle weakness and respiratory failure.Nowadays, the MADD diagnosis is established by the presence of dicarboxylic organic acids and acylglycine derivatives in the urine and increased levels of medium-and long-chain acylcarnitines in the blood. Mutations in ETFA, ETFB, ETFDH genes, encoding for alpha and beta subunits of ETF and for ETF-dehydrogenase are associated with MADD. We report the case of a three years old child, affected by lethargy and asthenia associated with anorexia. Biochemical analyses showed hypoketotic hypoglycemia with remarkable increments in transaminases, lactic dehydrogenase, aldolase and creatine kinase. The chromatographic layout of urinary organic acids showed a typical dicarboxylic aciduria. Thus, based on these features, MADD was suspected. Fifteen years later, at the age of 19, MADD diagnosis was confirmed by molecular analysis, showing a compound heterozygosity for the mutations c.1074G>C (p.R358S; HGMD: CM031670 in HGMD database) and c.1073G>A (p.R358K) in the ETFDH gene. The c.1073G>A (p.R358K; rs796051959) mutation is reported in ClinVar database as pathogenic allele, although lacking link to a specific clinical condition. However, familial segregation study and in silico analysis, performed by bioinformatics tools, confirmed that this substitution is likely pathogenetic. Her parents were healthy carriers of one of the two mutations. It is known that the severity of the clinical phenotype of MADD may be related to the type of mutation in the ETFA/ETFB/ETFDH genes. Particularly, missense mutations in the ETFDH gene, leaving a detectable residual enzyme activity, may account for the milder form of the disease, as is the case here. In conclusion we suggest that molecular analysis is essential to the definitive diagnosis of MADD and to direct the adequate therapeutic management. Thus, through a close nutritional follow up, a few months ago the patient gave birth to a healthy boy. References Olsen et al. Clear relationship between ETF/ETFDH genotype and phenotype in patients with multiple acyl-CoA dehydrogenation deficiency. Hum Mutat. 2003; 22:12–23

Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein.

UN1 is a membrane glycoprotein that is expressed in immature human thymocytes, a subpopulation of peripheral T lymphocytes, the HPB acute lymphoblastic leukemia (ALL) T-cell line and fetal thymus. We previously reported the isolation of a monoclonal antibody (UN1 mAb) recognizing the UN1 protein that was classified as "unclustered" at the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. UN1 was highly expressed in breast cancer tissues and was undetected in non-proliferative lesions and in normal breast tissues, indicating a role for UN1 in the development of a tumorigenic phenotype of breast cancer cells. In this study, we report a partial purification of the UN1 protein from HPB-ALL T cells by anion-exchange chromatography followed by immunoprecipitation with the UN1 mAb and MALDI-TOF MS analysis. This analysis should assist in identifying the amino acid sequence of UN

Hierarchical formation of disulphide bonds in the immunoglobulin Fc-Fragment is assisted by Protein Disulphide Isomerase

Antibodies provide an excellent system to study the folding and assembly of all β-sheet proteins and to elucidate the hierarchy of intra/inter chain disulfide bonds formation during the folding process of multimeric and multidomain proteins. Here, the folding process of the Fc fragment of the heavy chain of the antibody MAK33 was investigated. The Fc fragment consists of the CH3 and CH2 domains of the immunoglobulin heavy chain, both containing a single S-S bond. The folding process was investigated both in the absence and presence of the folding catalyst protein-disulfide isomerase (PDI), monitoring the evolution of intermediates by electrospray mass spectrometry. Moreover, the disulfide bonds present at different times in the folding mixture were identified by mass mapping to determine the hierarchy of disulfide bond formation. The analysis of the uncatalyzed folding showed that the species containing one intramolecular disulfide predominated throughout the entire process, whereas the fully oxidized Fc fragment never accumulated in significant amounts. This result suggests the presence of a kinetic trap during the Fc folding, preventing the one-disulfide-containing species (1S2H) to reach the fully oxidized protein (2S). The assignment of disulfide bonds revealed that 1S2H is a homogeneous species characterized by the presence of a single disulfide bond (Cys-130-Cys-188) belonging to the CH3 domain. When the folding experiments were carried out in the presence of PDI, the completely oxidized species accumulated and predominated at later stages of the process. This species contained the two native S-S bonds of the Fc protein. Our results indicate that the two domains of the Fc fragment fold independently, with a precise hierarchy of disulfide formation in which the disulfide bond, especially, of the C H2 domain requires catalysis by PD

Cross-cultural adaptation and validation of the voice handicap index into Italian

Objectives. To evaluate the internal consistency, reliability, and clinical validity of the Italian version of the Voice Handicap Index (VHI). Study Design. Cross-sectional survey study was carried out. Methods. One hundred and seventy-five patients with voice disorders, divided in four groups according to the etiology of the disease (neurogenic, structural, functional, and inflammatory), and 84 asymptomatic subjects were included in the study. Internal consistency was analyzed through Cronbach alpha coefficient. For the VHI test-retest reliability analysis, the Italian VHI was filled twice by 56 patients and 56 control subjects. The test-retest reliability was assessed through the Pearson correlation test. For the clinical validity assessment, the scores obtained in the pathological group were compared with those found in asymptomatic individuals through the Kruskal-Wallis test. Also, the correlation between VHI and the grade of voice disorder was assessed. Finally, the effect of age and gender on overall VHI and its three subscales was analyzed. Results. Optimal internal consistency was found (alpha = 0.93); the test-retest reliability in both groups was high (r > 0.86). Nonparametric Kruskal-Wallis analysis of variance for the overall VHI score and its three domains revealed a significant main effect for group (P = 0.000). The control group scored significantly lower than the four groups of voice-disordered patients. The overall VHI score positively correlated with the grade of voice disorder (r = 0.43). In the voice-disorder group, age and gender were not correlated to the overall VHI score and to their three domains. Conclusion. The Italian VHI is highly reproducible, and exhibits excellent clinical validity

Il Pooling-score (P-score): Variabilit\ue0 inter- e intra-individuale nella valutazione endoscopica della gravit\ue0 della disfagia

This study evaluated the intra- and inter-rater reliability of the Pooling score (P-score) in clinical endoscopic evaluation of severity of swal- lowing disorder, considering excess residue in the pharynx and larynx. The score (minimum 4 - maximum 11) is obtained by the sum of the scores given to the site of the bolus, the amount and ability to control residue/bolus pooling, the latter assessed on the basis of cough, raclage, number of dry voluntary or re ex swallowing acts ( 5). Four judges evaluated 30 short lms of pharyngeal transit of 10 solid (1/4 of a cracker), 11 creamy (1 tablespoon of jam) and 9 liquid (1 tablespoon of 5 cc of water coloured with methlyene blue, 1 ml in 100 ml) boluses in 23 subjects (10 M/13 F, age from 31 to 76 yrs, mean age 58.56\ub111.76 years) with different pathologies. The lms were randomly distributed on two CDs, which differed in terms of the sequence of the lms, and were given to judges (after an explanatory ses- sion) at time 0, 24 hours later (time 1) and after 7 days (time 2). The inter- and intra-rater reliability of the P-score was calculated using the intra-class correlation coef cient (ICC; 3,k). The possibility that consistency of boluses could affect the scoring of the lms was considered. The ICC for site, amount, management and the P-score total was found to be, respectively, 0.999, 0.997, 1.00 and 0.999. Clinical evaluation of a criterion of severity of a swallowing disorder remains a crucial point in the management of patients with pathologies that predispose to complications. The P-score, derived from static and dynamic parameters, yielded a very high correlation among the scores attributed by the four judges during observations carried out at different times. Bolus consistencies did not affect the outcome of the test: the analysis of variance, performed to verify if the scores attributed by the four judges to the parameters selected, might be in uenced by the different consistencies of the boluses, was not signi cant. These initial data validate the clinical use of the P-score in the management of patients with deglutition disorders by a multidisciplinary team

A new case of Congenital Hyperinsulinemic Hypoglycemia due to M/SCHAD deficiency: the contribution of metabolic and molecular diagnosis for the management

Congenital Hyperinsulinemic Hypoglycemia (CHH) is a rare metabolic disease (prevalence <1/1.000.000) characterized by a persistent hypoglycemia and high secretion of insulin in the neonatal and infancy period. An early management of patients with CHH is mandatory to avoid brain damage. Recent advances in molecular analysis have linked CHH to mutations in nine genes: ABCC8, KCNJ11, GCK causing either diazoxide-responsive or diazoxide-unresponsive Hyperinsulinemic Hypoglycemia, and GLUD1, HADH, SLC16A1, UCP2, HNF4A and HNF1A, causing generally diazoxide-responsive CHH. However, HADH defect is the most common form in presence of consanguinity and diazoxide-responsiveness. The HADH gene codifies the M/SCHAD mitochondrial enzyme, which catalyses the penultimate reaction in the β-oxidation of medium and short-chain fatty acids, causing in some affected individuals an elevated plasmatic hydroxybutyrylcarnitine and urinary medium-chain dicarboxylic, and 3-hydroxydicarboxylic metabolites. To date about 40 cases of M/SCHAD defect have been reported in literature.We report here a new case of CHH due to M/SCHAD deficiency. The index case was a Pakistan infant, born from consanguineous parents, showing a diazoxide-responsive hyperinsulinism and organic aciduria. The M/SCHAD deficiency was confirmed by the molecular diagnosis performed by sequencing of HADH gene, which revealed the presence of the nonsense mutation c.706C>T (p.R236*) in HADH gene, at homozygous state, while both parents were heterozygous for the mutated allele. The patient started diazoxide treatment at the maximum dose of 10 mg/kg/day, which resulted in adverse drug reactions (hypertrichosis, peripheral edemas and persistent hypertension) gradually solved with antihypertensive regimen. Diazoxide was progressively titrated to 2 mg/kg/ day with good results in glycemic control and no hypertensive crisis. Low organic aciduria was followed.In conclusion, when the metabolic profile suggests a CHH disorder, the molecular analysis is necessary for the precise diagnosis and the appropriate counseling to the parents, also for the possibility of a prenatal diagnosis. In this setting, the definitive diagnosis of CHH, due to M/SCHAD deficiency, may suggest also the most appropriate therapeutic intervention to avoid both risk of worsening or adverse drug effect

Improved production of succinic acid from Basfia succiniciproducens growing on A-donax and process evaluation through material flow analysis

BackgroundDue to its wide range of applications in the food, pharmaceutical and chemical fields, microbial synthesis of succinic acid is receiving growing attention, generating already relevant industrial results, as well as fueling constant research for improvements. In order to develop a sustainable process, a special focus is now set on the exploitation and conversion of lignocellulosic biomasses into platform chemicals.ResultsIn the present work we used Basfia succiniciproducens BPP7 in separated hydrolysis and fermentation experiments with Arundo donax as starting material. Fed-batch strategies showed a maximal production of about 37g/L of succinic acid after 43h of growth and a productivity of 0.9g/Lh on the pilot scale. Global mass balance calculations demonstrated a hydrolysis and fermentation efficiency of about 75%. Moreover, the application of a material flow analysis showed the obtainment of 88.5 and 52 % of succinic acid, per kg of virgin biomass and on the total generated output, respectively.ConclusionsThe use of fed-batch strategies for the growth of B. succiniciproducens on A. donax improved the titer and productivity of succinic acid on pre-pilot scale. Process evaluation through material flow analysis showed successful results and predicted a yield of succinic acid of about 30% in a fed-batch process that uses A. donax as only carbon source also in the feed. Preliminary considerations on the possibility to achieve an energetic valorization of the residual solid coming from the fermentation process were also carried out