The proteasome as a druggable target with multiple therapeutic potentialities: Cutting and non-cutting edges.

Ubiquitin Proteasome System (UPS) is an adaptable and finely tuned system that sustains proteostasis network under a large variety of physiopathological conditions. Its dysregulation is often associated with the onset and progression of human diseases; hence, UPS modulation has emerged as a promising new avenue for the development of treatments of several relevant pathologies, such as cancer and neurodegeneration. The clinical interest in proteasome inhibition has considerably increased after the FDA approval in 2003 of bortezomib for relapsed/refractory multiple myeloma, which is now used in the front-line setting. Thereafter, two other proteasome inhibitors (carfilzomib and ixazomib), designed to overcome resistance to bortezomib, have been approved for treatment-experienced patients, and a variety of novel inhibitors are currently under preclinical and clinical investigation not only for haematological malignancies but also for solid tumours. However, since UPS collapse leads to toxic misfolded proteins accumulation, proteasome is attracting even more interest as a target for the care of neurodegenerative diseases, which are sustained by UPS impairment. Thus, conceptually, proteasome activation represents an innovative and largely unexplored target for drug development. According to a multidisciplinary approach, spanning from chemistry, biochemistry, molecular biology to pharmacology, this review will summarize the most recent available literature regarding different aspects of proteasome biology, focusing on structure, function and regulation of proteasome in physiological and pathological processes, mostly cancer and neurodegenerative diseases, connecting biochemical features and clinical studies of proteasome targeting drugs.

Ubiquitin binds the amyloid ß peptide and interferes with its clearance pathways.

Several lines of evidence point to a compromised proteostasis associated with a reduction of the Ubiquitin Proteasome System (UPS) activity in patients affected by Alzheimer's Disease (AD) and suggest that the amyloid ß peptide (Aß) is an important player in the game. Inspired also by many reports, underlining the presence of ubiquitin (Ub) in the amyloid plaques of AD brains, here we set out to test whether Ub may bind the Aß peptide and have any effect on its clearance pathways. By using an integrated array of MALDI-TOF/UPLC-HRMS, fluorescence, NMR, SPR, Microscale Thermophoresis (MST) and molecular dynamics studies, we consistently demonstrated that Aß40 binds Ub with a 1 : 1 stoichiometry and K d in the high micromolar range. In particular, we show that the N-terminal domain of the Aß peptide (through residues D1, E3 and R5) interacts with the C-terminal tail of Ub (involving residues K63 and E64), inducing the central region of Aß (14HQKLVFFAEDVGSNK28) to adopt a mixed α-helix/ß-turn structure. ELISA assays, carried out in neuroblastoma cell lysates, suggest that Aß competitively binds Ub also in the presence of the entire pool of cytosolic Ub binding proteins. Ub-bound Aß has a lower tendency to aggregate into amyloid-like fibrils and is more slowly degraded by the Insulin Degrading Enzyme (IDE). Finally, we observe that the water soluble fragment Aß1-16 significantly inhibits Ub chain growth reactions. These results evidence how the non-covalent interaction between Aß peptides and Ub may have relevant effects on the regulation of the upstream events of the UPS and pave the way to future in vivo studies addressing the role played by Aß peptide in the malfunction of proteome maintenance occurring in AD.

Adiponectin profile at baseline is correlated to progression and severity of multiple sclerosis.

BACKGROUND AND PURPOSE: Adiponectin is a cytokine linking energy metabolism and immune system. After being assembled, adiponectin circulates as oligomers of different molecular weight, i.e. low, medium and high (HMW) molecular weight. These have the most potent biological effects. Multiple sclerosis (MS) is an autoimmune disease of the human central nervous system. The aim of this study was to characterize the expression levels of both total adiponectin and its oligomerization state in the serum from 99 patients with MS at baseline (i.e. not influenced by therapies). We also investigated the potential relationships between adiponectin and disease progression and severity. METHODS: Adiponectin was quantified and visualized by enzyme-linked immunosorbent assay, western blotting and fast protein liquid chromatography. During the follow-up (3.6 ± 2.20 years), the patients were evaluated using total annualized relapse rate and Expanded Disability Status Scale score. RESULTS: Total adiponectin is statistically higher in patients with MS compared with matched controls (12.18 vs. 10.02 µg/mL, P = 0.001). Interestingly, the adiponectin oligomerization state is altered in MS, with an increase of HMW oligomers. In addition, patients with MS with higher levels of adiponectin at baseline have significantly higher risk of progression and severity (Multiple Sclerosis Severity Score, 3.84 vs. 2.44, P = 0.001). No statistical difference in adiponectin expression was found between active and inactive patients with MS and among the different forms of disease. CONCLUSIONS: This study demonstrated that adiponectin and its HMW oligomers are greatly involved in MS autoimmune disorder representing a potential biomarker to predict worse MS prognosis and severity. Further studies are required to clarify the molecular mechanisms underlying the properties of adiponectin and HMW oligomers in MS.

The role of heavy metals and polychlorinated biphenyls (PCBs) in the oncogenesis of head and neck tumors and thyroid diseases: a pilot study.

Previous literature has highlighted the mechanisms of molecular toxicity induced by substances such as arsenic, cadmium, chromium, nickel, lead, barium and PCBs. The research was carried out on 20 volunteers, all the patients gave their consent to the research: the aim of the study was to evaluate the presence of metals and PCBs in these different matrices (blood and hair), correlating the biochemical data to pathological conditions present, and also to the area in which patients resided. Various quantitative determinations were carried out on samples of blood and hair for 14 heavy metals and on blood samples for 12 PCBs. For the 11 patients the results indicated that blood levels for half of the 14 displayed heavy metals measured considerably higher compared to the reference values, whilst the levels measured in hair evidenced some positive values significantly higher than the maximum reference. Of the 12 PCBs assayed in blood some showed higher positive values compared to the maximum tabular reference (although there is no clear reference quantified in the WHO-2005 report). In the 9 healthy patients heavy metals in the blood were within the expected target range, with those showing positive results (≤ 3 out of 14 heavy metals for each patient) having values only slightly higher than the reference maximum. The levels of 14 heavy metals measured in hair were below thresholds, and levels for the 12 PCBs measured in blood showed negativity or positivity with values close to the minimum benchmarks. The analyses carried out on biological matrices have uncovered important and significant differences between healthy and unhealthy subjects, both qualitative and quantitative differences with respect to heavy metals and PCBs. All patients with head and neck cancer enlisted for the study had heavy metal and PCB blood levels at least twice the maximum reference level. The levels of heavy metals in hair were at least double the maximum reference. In contrast, all healthy volunteers enrolled showed no significant levels for either metals or PCBs.

The natural history of gastro-esophageal reflux disease: a comprehensive review.

Gastroesophageal reflux disease (GERD) is a common disorder of the upper gastrointestinal tract which is typically characterized by heartburn and acid regurgitation. These symptoms are widespread in the community and range from 2.5% to more than 25%. Economic analyses showed an increase in direct and indirect costs related to the diagnosis, treatment and surveillance of GERD and its complications. The aim of this review is to provide current information regarding the natural history of GERD, taking into account the evolution of its definition and the worldwide gradual change of its epidemiology. Present knowledge shows that there are two main forms of GERD, that is erosive reflux disease (ERD) and non-erosive reflux disease (NERD) and the latter comprises the majority of patients (up to 70%). The major complication of GERD is the development of Barrett esophagus, which is considered as a pre-cancerous lesion. Although data from medical literature on the natural history of this disease are limited and mainly retrospective, they seem to indicate that both NERD and mild esophagitis tend to remain as such with time and the progression from NERD to ERD, from mild to severe ERD and from ERD to Barrett's esophagus may occur in a small proportion of patients, ranging from 0 to 30%, 10 to 22% and 1 to 13% of cases, respectively. It is necessary to stress that these data are strongly influenced by the use of powerful antisecretory drugs (PPIs). Further studies are needed to better elucidate this matter and overcome the present limitations represented by the lack of large prospective longitudinal investigations, absence of homogeneous definitions of the various forms of GERD, influence of different treatments, clear exclusion of patients with functional disorders of the esophagus.

Effect of cisplatin on proteasome activity.

Cisplatin is a widely used chemotherapy drug which exerts cytotoxic activity by affecting both nuclear and cytosolic pathways. Herewith, we report, for the first time, that cisplatin inhibits proteasome activity in vitro. Cisplatin induces a dose dependent inhibition of the three enzymatic activities of proteasome (i.e., the chymotrypsin-like activity, the trypsin-like activity and the caspase-like activity). Moreover, cisplatin administration to neuroblastoma cells brings about a fast loss of proteasome particle activity, which is followed by a de novo synthesis of proteasome. Lastly, we report that the simultaneous administration of lactacystin and cisplatin enhances the cytotoxicity of cisplatin alone. The overall bulk of data opens to an intriguing scenario, concerning the biological effects of cisplatin in the control of cellular life, which goes beyond the well established genotoxic effect.

Symptom-association probability between meal ingestion and abdominal pain in patients with irritable bowel syndrome. Does somatization play a role?

BACKGROUND: Patients with irritable bowel syndrome (IBS) complain of postprandial abdominal pain, but it is still unknown how much of this association is due to chance. Somatization enhances the perception of symptoms after a meal. We assessed: (i) the proportion of meal-related pain periods and the symptom-association probability (SAP) between the two variables in IBS patients; and (ii) how this association is affected by somatization. METHODS: Seventy IBS patients recorded the times of meals and abdominal pain in a 10-day diary card. The proportion of postmeal pain periods was calculated in relation to the total number of 90-min periods with pain. Fisher's exact test was used to calculate the probability (p) of an association within a time window of 90 min, and the SAP was calculated as (1 - p) × 100%. The IBS Symptom Severity Scale, the SCL90-R for psychological symptoms, and the SF-36 for the quality of life were completed. KEY RESULTS: The proportion of postmeal pain periods was 42 ± 27%. SAP was significant (p < 0.05) in 32 patients (45%). Somatization was altered in 30 patients (47%), who were younger and had more severe IBS and a poorer quality of life. Somatization did not influence the association between meal ingestion and abdominal pain. CONCLUSIONS & INFERENCES: Meal ingestion and abdominal pain are significantly associated in 45% of IBS patients. Somatization influences IBS severity and the patients' quality of life, but not the strength of the association between eating and pain.

Liver transplantation in neurological Wilson's Disease: is there indication? A case report.

Wilson's disease (WD) is an autosomal recessive disorder characterized by copper overload. In this disease, inadequate hepatic excretion leads to copper accumulation in the liver, brain, kidney, and cornea. Severe neurological symptoms can develop in patients with WD, often in the absence of relevant liver damage: it is unclear whether liver transplantation (LT) could reverse neurological symptoms, and at present LT is not recommended in this setting. We report a case of regression of neurological symptoms in a patient affected by WD with prevalent neurological involvement. A 19-year-old man with disabling neuropsychiatric symptoms from WD that included frontal ataxia, akinesia, dystonia, tremors, and behavioral disorders in the presence of preserved liver function (Model for End-Stage Liver Disease score=7; Child-Turcotte-Pugh score=A5) underwent LT in November 2009. At the time of LT, encephalic magnetic resonance imaging (MRI) indicated diffuse neurodegenerative alterations involving subtentorial and supratentorial structures; bilateral Kayser-Fleischer ring was present. Four years after LT, laboratory tests show normalized copper metabolism and excellent liver function test results. Encephalic MRI shows a substantial improvement of already-known signal alterations at nuclei thalamus and putamen, mesencephalon, and pons. Kayser-Fleischer ring disappeared from the right eye, but a little remnant is still visible in the left eye. At neurological examination, all of the previous symptoms and signs are no longer present and behavioral disorders are no longer present; psychosocial functions are completely restored. The present case provides some evidence that LT may be a valid therapeutic option for WD patients with marked neurological impairment, particularly in those no longer responsive to chelation therapy.

Is the subjective perception of lactose intolerance influenced by the psychological profile?

BACKGROUND: Symptoms of lactose intolerance are often attributed to lactose malabsorption but, as this relationship has not been demonstrated when a small dose of lactose similar to that contained in one cup of milk is ingested by intolerant patients, psychological factors may play a role in altered symptom perception. AIM: To assess the hypothesis that the psychological profile influences the symptoms of lactose intolerance. METHODS: One hundred and two consecutive patients underwent a 15 g lactose hydrogen breath test to assess lactose malabsorption. The patients recorded the presence and severity of symptoms of lactose intolerance during the breath test using visual analogue scales. The psychological profile was assessed using a psychological symptom checklist, and health-related quality of life by means of the short-form health survey. RESULTS: Lactose malabsorption and intolerance were diagnosed in, respectively, 18% and 29% of the patients. The two conditions were not associated, and the severity of intolerance was even less in the patients with malabsorption. Multivariate logistic analysis showed that a high somatisation t-score was significantly associated with lactose intolerance (odds ratio 4.184; 1.704-10.309); the effects of the other psychological variables and of lactose malabsorption were not statistically significant. Health-related quality of life was significantly reduced in the patients with somatisation, but not in those with lactose malabsorption. CONCLUSIONS: The symptoms of lactose intolerance during hydrogen breath testing at a low physiological lactose load, are unrelated to lactose malabsorption, but may reveal a tendency towards somatisation that could impair the quality of life.

Fluctuations and the rate-limiting step of peptide-induced membrane leakage.

Peptide-induced vesicle leakage is a common experimental test for the membrane-perturbing activity of antimicrobial peptides. The leakage kinetics is usually very slow, requiring minutes to hours for complete release of vesicle contents, and exhibits a biphasic behavior. We report here that, in the case of the peptaibol trichogin GA IV, all processes involved in peptide-membrane interaction, such as peptide-membrane association, peptide aggregation, and peptide translocation, take place on a timescale much shorter than the leakage kinetics. On the basis of these findings, we propose a stochastic model in which the leakage kinetics is determined by the discrete nature of a vesicle suspension: peptides are continuously exchanging among vesicles, producing significant fluctuations over time in the number of peptide molecules bound to each vesicle, and in the formation of pores. According to this model, the fast initial leakage is caused by vesicles that contain at least one pore after the peptides are randomly distributed among the liposomes, whereas the slower release is associated with the time needed to occasionally reach in an intact vesicle the critical number of bound peptides necessary for pore formation. Fluctuations due to peptide exchange among vesicles therefore represent the rate-limiting step of such a slow mechanism.

Discrepancy between recalled and recorded bowel habits in irritable bowel syndrome.

BACKGROUND: A discrepancy between recalled and recorded bowel habit subtypes has been reported in irritable bowel syndrome (IBS), but the reasons for it remain unclear. AIM: To assess the agreement between recalled and recorded bowel habit subtypes; to determine whether any discrepancy is related to stool form variability or psychological factors; and to test the correlations of recalled and recorded stool form with colonic transit time. METHODS: Bowel habit subtype was established in 54 IBS patients at the enrolment visit (recalled) and with the aid of diary cards (recorded). Colonic transit time, the variability of stool form and the patients' psychological profiles were also recorded. RESULTS: Recalled and recorded bowel habit subtypes agreed in only 54% of the patients (kappa = 0.28). Stool form variability was greater among the patients whose recalled and recorded bowel habit subtypes were discordant (P = 0.03), whereas the psychological profiles were not different. Colonic transit time significantly correlated with stool form only when it was recorded on diary cards. CONCLUSION: The discrepancy between recalled and recorded bowel habits in IBS patients is related more to stool form variability than an altered psychological profile. Diary cards should be used to ensure that stool form reflects colonic transit time.

Long-term results of laparoscopic adrenalectomy for primary aldosteronism.

BACKGROUND: The management of primary aldosteronism is currently achieved by both medical and surgical treatment. Laparoscopy has in recent years unquestionably become the gold standard in adrenal surgery for benign lesions. This study aims to evaluate our clinical results among patients who underwent laparoscopic adrenalectomy (LA) for primary aldosteronism. METHODS: From January 1994 to January 2006, amid LA series, 59 primary aldosteronism patients were treated in our institution. Patients were 33 males and 26 females with mean age 49.3 yr (19-78). The mean body mass index was 25.9 kg/m2 (20.5-33.3). The mean size of lesion was 2.9 cm (1-5.5). Clinical symptoms were as follows: hypertension and symptomatic/asymptomatic hypokalemia (54), hypokalemia (5). RESULTS: Thirty-five left and 24 right LA were performed. On the left side, 22 procedures were carried out by anterior approach, 9 by anterior submesocolic route, and 4 by means of flank approach. All right procedures were completed by the anterior supine approach. The mean operative time was 103.5 min for left and 92.8 min for right adrenalectomy. There was one major complication, a colonic post-operative fistula, regarding a left adrenalectomy case. The mean post-operative hospital stay was 3 days (1-9). The cure rate of hypertension and hypokalemia was similar to the current literature results. CONCLUSIONS: LA is a safe and effective option in the treatment of primary aldosteronism. Appropriate selection of patients, larger adrenal masses and duration of symptoms are determining factors in the success rate of hypertension management.

Electrothermal bipolar energy-based device in laparoscopic right colectomy: our experience.

AIM: Aim of the present study was to evaluate effectiveness of bipolar electrothermal energy in laparoscopic right colectomy, both for vascular pedicle ligature and dissecting manoeuvres. METHODS: Eighty-nine consecutive unselected patients underwent laparoscopic right colectomy between 2003 and 2006. All procedures were performed or supervised by the same surgical team (two staff of surgeons). Forty-four laparoscopic right colectomy were carried out with ultrasonic coagulating shears (UCS), (group 1) and forty-five by means of electrothermal bipolar atlas (EBA), (group 2). Intraoperative bleeding and postoperative blood loss, operating time, complication and hospital stay have been investigated within two groups. RESULTS: No mortality and no major intraoperative were reported in both series. A conversion to open surgery occurred in one case in both groups. A duodenal perforation occurred in UCS group as major postoperative complication. Comparing 1 and 2 series statistically significant differences were found concerning mean operating time (122.7 vs 98.4) and blood loss (220 vs 115 mL), with a P value <0.05. CONCLUSIONS: Both UCS and EBA devices were safe and effective in performing laparoscopic right colectomy. However, EBA did allow a statistically significant shorter operative time and bleeding/blood loss, tracing back its effectiveness in vessel sealing and dissection.

The learning curve in laparoscopic adrenalectomy.

BACKGROUND: Laparoscopic adrenalectomy (LA) is the procedure of choice for surgical management of most adrenal tumors. LA learning curve (LC) varies among surgeons and may be influenced by factors depending on surgeon, patient, and lesion peculiarities. The aim of this study was to evaluate the LC by multi-dimensional analysis. METHODS: Between August 1994 and August 2005, 241 LA were performed in our department. Data were prospectively collected. The pre-operative variables evaluated were patient-related (age, gender, body mass index, co-morbidities) and disease-related (histology, size, and side of lesion). Level of experience of surgical team and surgical approach (anterior, flank, submesocolic routes) were evaluated as well. Flank approached and bilateral procedures were excluded, while submesocolic LA, were collected separately. Operating time (OpT), conversion rate (CR), intra-operative and post-operative complications were evaluated. Patient, surgeon, and procedure-related factors involved in LC were investigated by a multi-factorial logistic regression analysis. RESULTS: Body mass index, side, size, histology, technology improvement, and experience of surgical team, evaluated through the progressive series of surgical procedures, were independent predictors of CR and OpT. The CR for right adrenalectomy was 3% (3 cases) compared to 4.2% for left side (6 cases). The submesocolic approach significantly influenced OpT, but not CR. Mean OpT for right and left LA was 83 and 109 min, respectively. Based on surgical experience increase, the OpT and CR flattened their curves, roughly at 30 and 40 procedures for right and left LA, respectively. Post-operative complications did not change considerably throughout the series. Readmission rate within 30 days was negligible. CONCLUSIONS: Manifold factors may affect LC and outcome in LA. Their knowledge may support teaching activities as well as reducing conversion and complication rates.

Increased release and activity of matrix metalloproteinase-9 in patients with mandibuloacral dysplasia type A, a rare premature ageing syndrome.

Mandibuloacral dysplasia type A (MADA; OMIM 248370), a rare disorder caused by mutation in the LMNA gene, is characterized by post-natal growth retardation, craniofacial and skeletal anomalies (mandibular and clavicular hypoplasia, acroosteolysis, delayed closure of cranial sutures, low bone mass and joint contractures), cutaneous changes and partial lipodystrophy. Little is known about the molecular mechanisms by which LMNA mutations produce bone alterations. An altered bone extracellular matrix (ECM) remodelling could play a pivotal role in this disorder and influence part of the typical bone phenotype observed in patients. Therefore, we have focused our investigation on matrix metalloproteinases (MMPs), which are degradative enzymes involved in ECM degradation and ECM remodelling, thus likely contributing to the altered bone mineral density and bone metabolism values seen in five MADA patients. We evaluated the serum levels of several MMPs involved in bone development, remodelling and homeostasis, such as MMP-9, -2, -3, -8 and -13, and found that only the 82 kDa active enzyme forms of MMP-9 are significantly higher in MADA sera compared with healthy controls (n = 16). The serum level of MMP-3 was instead lower in all patients. No significant differences were observed between controls and MADA patients for the serum levels of MMP-2, -8 and -13 and of tissue inhibitor of metalloproteinase 2, a natural inhibitor of MMP-9. Similarly, normal serum levels of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-6 and IL-1beta were detected. These data suggest a possible involvement of MMP-9 in MADA disease, underlying the potential use in diagnosis and therapy.

Experimental use of a new surface acoustic wave sensor for the rapid identification of bacteria and yeasts.

AIMS: Use of an electronic nose (zNose(TM)) to discriminate between volatile organic molecules delivered during bacterial/fungal growth on agar and in broth media. METHODS AND RESULTS: Cultures of bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli) and yeasts (two Candida albicans strains) were grown on agar and in broth media and incubated for 24 h at 37 degrees C. Headspace samples from microbial cultures were analysed by the zNose(TM), a fast gas chromatography-surface acoustic wave detector. Olfactory images of volatile production patterns were observed to be different for the various species tested after 24 h. Moreover, some strains (two K. pneumoniae, two C. albicans) did not show changes in volatile production patterns within our species. CONCLUSIONS: Our experiments demonstrate that the electronic nose system can recognize volatile production patterns of pathogens at species level. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results, although preliminary, promise exciting challenges for microbial diagnostics.