We have cloned a cDNA for a myocardial cGMP-inhibited cAMP phosphodiesterase (cGI PDE) from a human heart cDNA library in λ Zap II. The open reading frame 3.5 kilobases (kb) of cDNA clone n.13.2 (7.7 ...kb) encodes a protein of 125 kDa. In Northern blots of total human ventricle RNA, a single mRNA species (8.3 kb) hybridized with a 4-kb EcoRI restriction fragment of clone n.13.2 cDNA (containing the entire open reading frame). The carboxylterminal region of the deduced amino acid sequence of the cGI PDE contains the putative catalytic domain conserved among mammalian PDE families. A partial cDNA clone, n.2, encoding a truncated, 54-kDa cGI PDE containing the conserved domain was expressed as a catalytically active fusion protein in Escherichia coli. cAMP hydrolytic activity was inhibited by cGMP and OPC 3911 but not by rolipram. Thus, this report provides direct proof that the conserved domain contains the catalytic core of cGI PDEs.
Validation of predictive risk models for prolonged air leak (PAL) is essential to understand if they can help to reduce its incidence and complications. This study aimed to evaluate both the clinical ...and statistical performances of 4 existing models. We selected 4 predictive PAL risk models based on their scientific relevance. We referred to these models as Chicago, Bordeaux, Leeds and Pittsburgh model, respectively, according to the affiliation place of the first author. These predicting risk models were retrospectively applied to patients recorded on the second edition of the Italian Video-Assisted Thoracoscopic Surgery Group registry. Predictions for each patient were calculated based on the logistic regression coefficient values provided in the original manuscripts. All models were tested for their overall performance, discrimination, and calibration. We recalibrated the original models with the re-estimation of the model intercept and slope. We used curve decision analysis to describe and compare the clinical effects of the studied risk models. Better statistical metrics characterize the models developed on larger populations (Chicago and Bordeaux models). However, no model has a valid benefit for threshold probability greater than 0.30. The Net benefit of the most performing model (Bordeaux model) at the threshold probability of 0.11 is 23 of 1000 patients, burdened by 333 false positive cases. One of 1000 is the Net benefit at the threshold probability of 0.3. The use of PAL scores based on preoperative predictive factors cannot be currently used in a clinical setting because of a high false positive rate and low positive predictive value.
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Abstract
OBJECTIVES
This study compares the uniportal with the 3-portal video-assisted thoracic surgery (VATS) by examining the data collected in the Italian VATS Group Database. The primary end ...point was early postoperative pain; secondary end points were intraoperative and postoperative complications, surgical time, number of dissected lymph nodes and length of stay.
METHODS
This was an observational, retrospective, cohort, multicentre study on data collected by 49 Italian thoracic units. Inclusion criteria were clinical stage I–II non-small-cell lung cancer, uniportal or 3-portal VATS lobectomy and R0 resection. Exclusion criteria were cT3 disease, previous thoracic malignancy, induction therapy, significant comorbidities and conversion to other techniques. The pain parameter was dichotomized: the numeric rating scale ≤3 described mild pain, whereas the numeric rating scale score >3 described moderate/severe pain. The propensity score-adjusted generalized estimating equation was used to compare the uniportal with 3-portal lobectomy.
RESULTS
Among 4338 patients enrolled from January 2014 to July 2017, 1980 met the inclusion criteria; 1808 patients underwent 3-portal lobectomy and 172 uniportal surgery. The adjusted generalized estimating equation regression model using the propensity score showed that over time pain decreased in both groups (P < 0.001). There was a statistical difference on the second and third postoperative days; odds ratio (OR) 2.28 95% confidence interval (CI) 1.62–3.21; P < 0.001 and OR 2.58 (95% CI 1.74–3.83; P < 0.001), respectively. The uniportal-VATS group had higher operative time (P < 0.001), shorter chest drain permanence (P < 0.001) and shorter length of stay (P < 0.001).
CONCLUSIONS
Data from the Italian VATS Group Database showed that in clinical practice uniportal lobectomy seems to entail a higher risk of moderate/severe pain on second and third postoperative days.
Diaphragmatic hernias occurring during pregnancy are an uncommon event. In very rare occasions, the clinical situation can suddenly worsen due to obstruction, torsion or infarction of the herniated ...viscera. Here, we describe a challenging case of a post-partum diaphragmatic hiatus hernia complicated by intrathoracic gastric perforation. A 23-year old woman was admitted at our hospital with a syndrome characterized by epigastralgy, dyspnoea and fever. She had previously undergone a laparoscopic antireflux surgery for hiatus hernia (6 years before) and a recent (4 months) unremarkable vaginal delivery. Due to the persistence of a pelvic pain after the delivery, she had been taking pain-killers as a self-administered medication. A CT scan showed a massive left pleural effusion and a complete herniation of the stomach into the left hemithorax. After placing a chest drainage and removing up to 3000 ml of brownish purulent fluid, a repeat CT scan (with water soluble contrast swallow) showed a leak at the level of the stomach. At surgery, we observed a complete intrathoracic herniation through a large diaphragmatic hiatal defect and a small well-defined gastric ulcer. A primary repair of both the stomach and the diaphragm was performed. We take the opportunity presented by this report to briefly discuss the patho-physiological mechanisms underlying this unusual complication.
This study shows that sphingosine 1-phosphate (S1P) exerts an anti-migratory action in C2C12 myoblasts by reducing directional cell motility and fully abrogating the chemotactic response to ...insulin-like growth factor-1. The anti-migratory response to S1P required ligation to S1P
2, being attenuated in myoblasts where the receptor was down-regulated by specific antisense oligodeoxyribonucleotides or small interfering RNA (siRNA) and conversely potentiated in S1P
2-overexpressing myoblasts. The investigation of RhoA and Rac GTPases, critically implicated in cell motility regulation, demonstrated that RhoA was rapidly activated by S1P, while Rac1 was unaffected within the first 5 min but stimulated thereafter. RhoA, but not Rac activation, was identified as a S1P
2-dependent pathway in experiments in which receptor expression was attenuated by siRNA treatment or up-regulated by S1P
2-encoding plasmid transfection. Finally, by expression of the dominant negative mutant of RhoA, the GTPase was found implicated in the anti-migratory action of S1P, whereas modulation of Rac1 functionality unaffected the antichemotactic effect of S1P, ruling out a role for this protein in the biological response. Since S1P was previously shown to inhibit myoblast proliferation and stimulate myogenesis, the here identified novel biological activity is in favour of a complex physiological role of the sphingolipid in the process of muscle repair.
Rat adipocyte cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) appears to be dually regulated in intact cells by serine phosphorylations induced by isoprenaline and insulin, respectively (Degerman, ...E., Smith, C. J., Tornqvist, H., Vasta, V., Belfrage, P., and Manganiello, V. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,533-537; Smith, C. J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V. C. (1991) J. Biol. Chem. 266,13385-13390). Since cAMP-beta-dependent protein kinase (cAMP-PK) catalyzes the beta-adrenergic effects, the site in the isolated cGI-PDE phosphorylated by this kinase was explored. A peptide, L(RRSS)GASGLLTSEHHSR (P18), corresponding to the amino acid sequence Leu423-ARG440 in the putative regulatory domain of the rat adipocyte cGI-PDE was synthesized. It contains a consensus substrate sequence -RRXS- for cAMP-PK within two tryptic cleavage sites and was readily phosphorylated by cAMP-PK Two phosphopeptides, identified as RS-32PSGASGLLTSEHHSR and S-32PSGASGLLTSEHHSR, were obtained after stoichiometric phosphorylation and trypsinization of the peptide. These two peptides and the two main tryptic phosphopeptides obtained from immunoisolated 32PcGI-PDE phosphorylated with cAMP-PK in a solubilized crude adipocyte membrane fraction were immunoprecipitated by an affinity-purified polyclonal antibody raised against P18 and exhibited the same chromatographic and electrophoretic profiles in three different separation systems. Similar radiosequencing profiles indicated that the second most N-terminal serine, corresponding to Ser-427 in the intact cGI-PDE, was phosphorylated by cAMP-PK in both P18 and authentic cGI-PDE. It is concluded that serine 427 is the target for cAMP-PK phosphorylation of the rat adipocyte cGI-PDE in vitro
Sphingosine 1-phosphate (S1P) is a bioactive lipid that is abundantly present in the serum and mediates multiple biological responses. With the aim of extending our knowledge on the role played by ...S1P in the regulation of cytoskeletal reorganization, native as well as C2C12 myoblasts stably transfected with green fluorescent protein (GFP)-tagged alpha- and {szligbeta}-actin constructs were stimulated with S1P (1 microM) and observed under confocal and multiphoton microscopes. The addition of S1P induced the appearance of actin stress fibres and focal adhesions through Rho- and phospholipase D (PLD)-mediated pathways. The cytoskeletal response was dependent on the extracellular action of S1P through its specific surface receptors, since the intracellular delivery of the sphingolipid by microinjection was unable to modify the actin cytoskeletal assembly. Interestingly, it was revealed by whole-cell patch-clamp that S1P-induced stress fibre formation was associated with increased ion currents and conductance through stretch-activated channels (SACs), thereby suggesting a possible regulatory role for organized actin in channel sensitivity. Experiments aimed at stretching the plasma membrane of C2C12 cells, using the cantilever of an atomic force microscope, indicated that there was a Ca²⁺ influx through putative SACs. In conclusion, the present data suggest novel mechanisms of S1P signalling involving actin cytoskeletal reorganization and Ca²⁺ elevation through SACs that might influence myoblastic functions.
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