Formalin-induced infidelity in PCR-amplified DNA fragments

A 643-nucleotide-long fragment of rDNA gene was amplified by PCR in the nematode worm Caenorhabditis. When the experiments were performed by using samples fixed in formalin, artefacts were detected. While the size of the amplified fragment resulted unaffected, very striking differences were seen in the nucleotide sequences of the amplified fragments. Furthermore, in many cases, the PCR reaction failed completely. The results obtained might warn of potential problems, especially when the amount of DNA to be amplified scarce.     

Study protocol: The DOse REsponse Multicentre International collaborative initiative (DO-RE-MI)

Introduction

Current practices for renal replacement therapy in intensive care units (ICUs) remain poorly defined. The DOse REsponse Multicentre International collaborative initiative (DO-RE-MI) will address the issue of how the different modes of renal replacement therapy are currently chosen and performed. Here, we describe the study protocol, which was approved by the Scientific and Steering Committees.

Methods

DO-RE-MI is an observational, multicentre study conducted in ICUs. The primary end-point will be the delivered dose of dialysis, which will be compared with ICU mortality, 28-day mortality, hospital mortality, ICU length of stay and number of days of mechanical ventilation. The secondary end-point will be the haemodynamic response to renal replacement therapy, expressed as percentage reduction in noradrenaline (norepinephrine) requirement. Based on the the sample analysis calculation, at least 162 patients must be recruited. Anonymized patient data will be entered online in electronic case report forms and uploaded to an internet website. Each participating centre will have 2 months to become acquainted with the electronic case report forms. After this period official recruitment will begin. Patient data belong to the respective centre, which may use the database for its own needs. However, all centres have agreed to participate in a joint effort to achieve the sample size needed for statistical analysis.

Conclusion

The study will hopefully help to collect useful information on the current practice of renal replacement therapy in ICUs. It will also provide a centre-based collection of data that will be useful for monitoring all aspects of extracorporeal support, such as incidence, frequency, and duration.     

Diagnostic value of changes in the Q wave induced by exertion

In order to evaluate the diagnostic value of exercise-induced Q wave changes and its relationship with the extent of coronary involvement and presence and location of a previous myocardial infarction, we examined the stress electrocardiograms of 188 consecutive patients with chest pain. Coronary arteriography shoved single vessel disease (SV) in 28 patients and multivessel disease (MV) in 130 patients; a previous myocardial infarction was present in 64 patients. The Q wave amplitude was measured as average of ten values in CM5 at rest and at peak exercise; a Delta-Q less than 0, i.e. reduction or no change of Q wave at peak exercise, was considered a positive response for coronary artery disease. The Delta-Q criterion shoved a significantly better sensitivity than ST depression, as a whole, but this improvement was nullified when patients with anterior myocardial infarction were excluded; as well specificity of Delta-Q although better than ST, did not allow a significant improvement for the diagnostic value of stress test. We also evaluated the diagnostic accuracy for multivessel coronary artery disease of both criteria positive was 78% whereas the negative predictive value of both criteria negative was 91%. We concluded that the exercise-induced Delta-Q less than 0 is a good indicator of coronary artery disease, although not superior to ST depression; the negativity of both criteria seems to be highly reliable for the exclusion of multivessel coronary artery disease.     

The Ubiquitin–Proteasome System Regulates the Stability of Neuronal Nicotinic Acetylcholine Receptors

Ubiquitination is a key event for protein degradation by the proteasome system, membrane protein internalization, and protein trafficking among cellular compartments. Few data are available on the role of the ubiquitin–proteasome system (UPS) in the trafficking of neuronal nicotinic acetylcholine receptors (nAChRs). Experiments conducted in neuron-like differentiated rat pheochromocytoma cells (PC12 cells) show that the α3, β2, and β4 nAChR subunits are ubiquitinated and that their ubiquitination is necessary for degradation. A 24-h treatment with the proteasome inhibitor PS-341 increased the total levels of α3 and the two β subunits in both whole cell lysates and fractions enriched for the ER/Golgi compartment. nAChR subunit upregulation was also detected in plasma membrane-enriched fractions. Inhibition of the lysosomal degradation machinery by E-64 had a significantly smaller effect on nAChR turnover. The present data, together with previous results showing that the α7 nAChR subunit is a target of the UPS, point to a prominent role of the proteasome in nAChR trafficking.     

Cytomegalovirus Infection After Liver Transplantation: Prophylaxis and Preemptive Treatment—A Single-Center Experience

Background

Cytomegalovirus (CMV) infection represents one of the most frequent opportunistic infections following solid-organ transplantation. The incidence and severity of CMV infection depend on the immunosuppressive regimen, the CMV serostatus of donor and recipient, and the type of transplant.

Methods

We evaluated CMV infection rates during the last 2 years in our center: March 2007 to March 2009. We enrolled 55 patients—13 females and 42 males—who underwent liver transplantation (OLT) due to hepatitis C virus (HCV) cirrhosis (n = 9), hepatitis B virus (HBV) cirrhosis (n = 5) HCC both on HCV and HBV cirrhosis (n = 37), or autoimmune disease (n = 4). Fifty percent of the patients received tacrolimus (TRL) and the others cyclosporine (CsA), both dosed according to weight. All patients received oral acyclovir (400 mg/td or less, adapted to renal function) as herpes simplex prophylaxis for 6 months. CMV prophylaxis prescribed CMV- hyperimmunoglobulin on postoperative days 1 and 7. CMV infection was monitored using polymerase chain reaction (PCR <1000 IU/mL) according to the following schedule: every week for the first month, every 2 weeks from month 2 to 3 and monthly from month 4 to 6. Patients were treated when three positive PCR results not affected by immunosuppressive dose reduction or when the PCR showed DNA greater than three times the limit of detection. CMV treatment stipulated valgancyclovir (900 mg twice daily) until three consecutive PCRs were negative or for 3 months dosed according to renal function. PCR was measured every 2 weeks during treatment.

Results

Among the patients who were all D⁺/R⁺ (CMV-Immunoglobulin G [IgG]⁺/IgG⁺). 10 required treatment (18%) within 3 months from OLT. There subjects were prescribed TRL (n = 4) or CsA (n = 6). No renal impairment was observed among treated patients. Of those having the infection, one died due to other causes—sepsis from candida at 5 months after OLT.

Conclusion

CMV-hyperimmunoglobulin on postoperative days 1 and 7 did not confer protection for CMV among OLT patients. Preemptive treatment with intravenous gancyclovir plus valgancyclovir per os seemed to be useful and safe in infected patients requiring treatment.     

Characterization and evaluation of chitosan nanoparticles for dopamine brain delivery

The aim of this study was to characterize nanoparticles (NPs) composed of chitosan (CS) and evaluate their potential for brain delivery of the neurotransmitter Dopamine (DA). For this purpose, CS based NPs were incubated with DA at two different concentrations giving rise to nanocarriers denoted as DA/CSNPs (1) and DA/CSNPs (5), respectively. X-ray Photoelectron Spectroscopy (XPS) analysis confirmed that DA was adsorbed onto the external surface of such NPs. The cytotoxic effect of the CSNPs and DA/CSNPs was assessed using the MTT test and it was found that the nanovectors are less cytotoxic than the neurotransmitter DA after 3 h of incubation time. Transport studies across MDCKII-MDR1 cell line showed that DA/CSNPs (5) give rise to a significant transport enhancing effect compared with the control and greater than the corresponding DA/CSNPs (1). Measurement of reactive oxygen species (ROS) suggested a low DA/CSNPs neurotoxicity after 3 h. In vivo brain microdialysis experiments in rat showed that intraperitoneal acute administration of DA/CSNPs (5) (6-12 mg/kg) induced a dose-dependent increase in striatal DA output. Thus, these CS nanoparticles represent an interesting technological platform for DA brain delivery and, hence, may be useful for Parkinson's disease treatment.     

Extraction, characterization and in vivo neuromodulatory activity of phytosterols from microalga Dunaliella tertiolecta

In recent years, a great deal of research has been devoted to identify new natural sources of phytosterols and to improve methods for their recovery and purification. In this regard, unexplored natural sources of bioactive ingredients are gaining much attention since they can lead to the isolation of new compounds or bioactivities. The field of available natural sources has been further increased by including algae and, even more interestingly, microalgae. In the present study, a multidisciplinary approach has been used considering, in an integrated view, extraction, chemical composition and bioactivity of phytosterols from the microalga Dunaliella tertiolecta. A novel methodology to extract, separate and characterize microalgal-derived phytosterols has been developed. In addition, recoverable and reusable eluents have been selected in order to reduce the quantities of employed organic solvents. Finally, we addressed the question whether orally administered phytosterols reach the brain and if those interfere with the major neurotransmitter systems, such as the dopaminergic, serotoninergic and noradrenergic ones, in several brain areas of rats. Flash Liquid Chromatography has been used to separate the Total Sterol (TS) fraction, composed of twelve sterols, with a purity of 97.87% and a recovery percentage of 98%, while the "flash version" of Silver Ion Liquid Chromatography has been used to purify the most abundant phytosterols in TS, (22E,24R)- methylcholesta-5,7,22-trien-3β-ol (ergosterol) and (22E,24R)-ethylcholesta-5,7,22-trien-3β-ol (7-dehydroporiferasterol), with a purity of 97.4%. These two combined methods did not need sophisticated technologies but only cheap laboratory supplies. Moreover, the possibility of recovering and recycling the solvents used as eluents made it a cleaner process. Finally, for the first time, a neuromodulatory action of Dunaliella tertiolecta-derived phytosterols has been found in selective brain areas of rats.     

A comparison between the early and mid-term results of surgical as opposed to percutaneous closure of defects in the oval fossa in children aged less than 6 years

OBJECTIVES: To compare surgical as opposed to percutaneous interventional closure of isolated atrial septal defects in the oval fossa in terms of hospital stay, efficacy, and complications, and to study the respective role of the two techniques in current practice. METHODS: Between January 1998 and April 2004, 126 out of 1210 patients treated at our institution for closure of an isolated defect in the oval fossa were aged less than 6 years. The mean age of these 126 patients at procedure was 4.2 plus or minus 1 year. The ratio of females to males was 74 to 52. RESULTS: Of the patients, 62% were treated successfully using a percutaneous approach. The groups treated surgically or percutaneously did not differ for age, gender, or indications for treatment. No deaths occurred. The rates of total and major complications were higher in the group undergoing surgical closure, at 34% versus 9%, p less than 0.0001, and 10.5% versus 1%, p equal to 0.01, respectively. Embolisation of the device requiring subsequent surgery occurred in 1% of patients. The stay in hospital was shorter in those closed percutaneously, at 3.2 plus or minus 0.9 days versus 6.8 plus or minus 2.8 days, p equal to 0.0001. During a mean follow-up of 3.4 plus or minus 1.9 years, no major complications occurred in either group, and symptoms improved significantly in both groups. Additional sequels occurred in 2 patients who had major complications subsequent to surgical closure. CONCLUSIONS: Even in young children, it is both feasible and safe to close defects in the oval fossa percutaneously. Compared to surgical closure, the transcatheter approach allows a shorter stay in hospital, and has a lower rate of complications. Early and mid-term follow-up has confirmed the safety and efficacy of both techniques.