Studying multiprotein complexes by multisignal sedimentation velocity analytical ultracentrifugation

Protein interactions can promote the reversible assembly of multiprotein complexes, which have been identified as critical elements in many regulatory processes in cells. The biophysical characterization of assembly products, their number and stoichiometry, and the dynamics of their interactions in solution can be very difficult. A classical first-principle approach for the study of purified proteins and their interactions is sedimentation velocity analytical ultracentrifugation. This approach allows one to distinguish different protein complexes based on their migration in the centrifugal field without isolating reversibly formed complexes from the individual components. An important existing limitation for systems with multiple components and assembly products is the identification of the species associated with the observed sedimentation rates. We developed a computational approach for integrating multiple optical signals into the sedimentation coefficient distribution analysis of components, which combines the size-dependent hydrodynamic separation with discrimination of the extinction properties of the sedimenting species. This approach allows one to deduce the stoichiometry and to assign the identity of the assembly products without prior assumptions of the number of species and the nature of their interaction. Although chromophoric labels may be used to enhance the spectral resolution, we demonstrate the ability to work label-free for three-component protein mixtures. We observed that the spectral discrimination can synergistically enhance the hydrodynamic resolution. This method can take advantage of differences in the absorbance spectra of interacting solution components, for example, for the study of protein-protein, protein-nucleic acid or protein-small molecule interactions, and can determine the size, hydrodynamic shape, and stoichiometry of multiple complexes in solution.     

Natural history and risk stratification of patients undergoing non-invasive ventilation in a non-ICU setting for severe COPD exacerbations

Non-invasive ventilation (NIV) delivered in an intensive care unit (ICU) has become the cornerstone in the treatment of patients with severe chronic obstructive pulmonary disease (COPD) exacerbations. A trend towards managing these patients in non-ICU setting has emerged in recent years, although out-of-hospital survival by this approach and how to prognosticate it is unknown. We aimed to investigate these issues. We consecutively recruited 100 patients (49 males; median age 82 years) who received NIV treatment for acute respiratory failure due to COPD exacerbation in non-ICU medical wards of our hospital, between November 2008 and July 2012. We assessed survival (both in-hospital and out-of-hospital) of all these patients, and analyzed baseline parameters in a Cox proportional hazards model to develop a prognostic score. The median survival in the study population was 383 days (240–980). Overall survival rates were 71.0, 65.3, and 52.7 % at 1, 3, and 12 months, respectively. Age >85 years, a history of heart disorders and a neutrophil count ≥10 × 10⁹ were associated with higher mortality at Cox’s analysis (χ ² = 35.766, p = 0.0001), and were used to build a prognostic score (NC85). The presence of two or more factors determined the deepest drop in survival (when NC85 ≥2, mortality at 1, 3, and 12 was 60.7, 70.4, and 77.2 %, respectively, while when NC85 = 0 were 4.0, 4.0, and 14.0 %). A simple model, based on three variables (age, neutrophil count and history of heart disease), accurately predicts survival of COPD patients receiving NIV in a non-ICU setting.     

Analysis of a series of cases of liver metastasis of colorectal carcinoma

The Authors present a six years case-report concerning 135 patients operated on colo-rectal carcinoma (82 of them are still followed up). The incidence of hepatic metastases (synchronous and metachronous) has been 30.37% in all. The preoperative ultrasonography has shown an 11% about margin mistake to visualize secondary lesions of liver. They have acknowledged the intraoperative ultrasonography very useful to visualize occult metastases and to guide an exeresis operation. The resectability of hepatic metastases from colo-rectal cancer has been of 12.19%. Synchronous forms have been rarely operable (5.88%) because they are constituted by multiple and disseminated lesions. They have found an higher incidence of operable forms among the metachronous metastases (42.85%), whose average time of appearance from the primary operation has been about 13 months. The operative mortality and the complications following hepatectomy have been null. The average survival time from operation has been over 22 months. Therefore they recognized the importance of a careful follow-up to find precociously operable lesions.     

Increased MCP-1 and MIP-1beta in bronchoalveolar lavage fluid of chronic bronchitics.

CC-chemokines are chemotactic factors expressed in a wide range of cell types and tissues. The aim of this study was to evaluate the involvement of CC-chemokines in the airways inflammation of patients affected by chronic bronchitis. The study evaluated, with an immunoassay, the concentrations of monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and macrophage inflammatory protein-1beta (MIP-1beta), in the bronchoalveolar lavage fluid (BALF) of 12 smokers affected by chronic bronchitis and 14 smoking, 15 nonsmoking and six exsmoking healthy subjects. MCP-1 was significantly increased in patients with chronic bronchitis ((mean+/-SD) 10.75+/-4.04 pg x mL(-1)) and in the smoker control group (12.39+/-5.87 pg x mL(-1)) compared with healthy exsmokers: (7.12+/-1.60 pg x mL(-1), p=0.035 and p=0.045, respectively) and nonsmokers (6.41+/-3.87 pg x mL(-1), p=0.003 and p=0.006, respectively). MIP-1alpha concentrations were undetectable. A significant difference was observed in MIP-1-beta levels in BALF of chronic bronchitics (8.11+/-5.97 pg x mL(-1)) compared to smoker (3.57+/-2.90 pg x mL(-1), p=0.018), exsmoker (3.43+/-0.68 pg x mL(-1), p=0.025) and nonsmoker (3.39+/-3.73 pg x mL(-1), p=0.008) control groups. A negative correlation was observed between MIP-1beta levels and forced expiratory volume in one second values (p=-0.64, p=0.035) in chronic bronchitics. An increase of monocyte chemotactic protein-1 is related to smoking habit and seems consistent with a lung inflammatory reaction. On the contrary, an increase in macrophage inflammatory protein-1beta levels is restricted to smokers developing chronic obstructive pulmonary disease. These data suggest a role of CC-chemokines in the pathogenesis of chronic bronchitis.     

Pancreatic somatostatinoma

The Authors describe a case of somatostatinoma in the pancreatic tail, characterized by the absence of a specific symptomatology, with the exception of a slight hyperglycaemia. The patient underwent a surgical operation of pancreatic resection and splenectomy, and now enjoys very good health. They have examined literature from 1977 to 1988, and overall the 30 cases of pancreatic localization, describing the symptomatology, the pathological anatomy, the therapy, and evaluating the malignity degree. Then the Authors could notice that the prognosis of these neoplasms is often fatal, both for the frequent tumor metastatization, and for the difficulties in making an early diagnosis. The therapy is essentially surgical.     

Platelet gel for the treatment of traumatic loss of finger substance

Background

Autologous or allogenic platelet gel is a blood component that exploits the effects of the cytokines contained in platelet α granules to stimulate repair processes. The properties of platelet gel were first tested on chronic ulcers to accelerate healing and later in orthopaedic, dental, vascular and cardiothoracic surgery.In our centre, we have been using platelet gel for 5 years, first for surgical patients with difficult wounds, then for orthopaedic patients undergoing osteosynthesis surgery and patients with ulcers not responding to traditional therapies. Subsequently we decided to extend the use of platelet gel also to amputations or traumatic loss of tissue of fingers.

Materials and methods

In this article we present the results obtained over 5 years concerning 115 patients with finger amputations or wounds treated with platelet gel in our Service of Transfusion Medicine. Platelets were obtained fom allogeneic buffy coats (10 mL) and the gel was produced by adding thrombin to concentrated platelets.The decision to use homologous platelet gel was based on its limited cost, ease of preparation, almost unlimited availability, the fact that the number of platelets that can be collected is much higher than the therapeutic range and so able to replace the losses due to secondary medication, and last, but not least, it causes no discomfort to patients. The safety of the product was ensured by virology tests including molecular biology studies.

Results

The recovery of soft tissue in all patients ranged from 80 to 100%; the median time for this recovery was 3 weeks (range, 10 days - 6 weeks). Approximately 60% of the patients complained of local hypoaesthesia for some weeks; 30% of the patients developed hyperaesthesia, which resolved completely within 6–8 weeks from starting treatment. Loss of bone tissue represented an obstacle to total tissue recovery, but the aesthetic results were satisfactory in nearly all cases.

Conclusion

All patients showed good compliance, both because of the low frequency of medications (at most, twice a week) and because of the painless platelet gel applications. The only negative aspect was abnormal nail growth in a case of distal partial amputation of a finger.In conclusion, we believe that platelet gel can be very useful in patients with traumatic or surgical loss of finger tissue, since it can resolve critical situations thus avoiding amputation of residual tissue and compromised joint function.     

Lhermitte's sign during lateral cervical puncture: survey of possible accidents in the lateral C1-C2 puncture and report of 2 cases of spinal cord penetration

Lhermitte's sign was referred by two patients during lateral cervical puncture (LCP) due to accidental puncture of the spinal cord; no sequelae were observed in these patients. A brief review of the literature is presented, concerning: 1. Lhermitte's sign; 2. reported accidents during LCP. Avoidance of neural and/or vascular injuries is possible by employing the posterior approach to LCP (puncture of the dorsal spinal subarachnoid space at C1-C2). The lateral suboccipital puncture (at the atlanto-occipital space) of the cisterna magna is also recommended.     

Regeneration of perivascular adrenergic innervation in rat tibial nerve after nerve crush

Summary Adrenergic innervation of blood vessels in the rat tibial nerve during degeneration and regeneration was studied using the formaldehyde-induced fluorescence method. The left sciatic nerve was crushed with suture threads to produce a 4-mm length of crushed nerve. At 1, 3, 7, 14, 28, 56 and 84 days after nerve crush, degenerative and regenerative changes in the nerve were verified using light microscopy. At each time point, adrenergic innervation was examined in epi-perincurial whole mount and nerve cross-section preparations. One day after nerve crush, fluorescence of adrenergic nerve fibers in the endoneurium was absent. Fluorescent adrenergic nerve fibers reappeared in the endoneurium at day 56 and reached the control density by 84 days. In the epi-perineurium, adrenergic innervation of small and medium-size arterioles was absent at 3 days, in large arterioles at 7 days. At 56 days, all epi-perineurial arterioles were reinnervated by a faint, sparse adrenergic network, which reached the control density at 84 days. The results suggest that adrenergic innvervation in the rat peripheral nerve is lost during nerve degeneration, but recovers when the nerve has regenerated.