Measuring Digital PCR Quality: Performance Parameters and Their Optimization.

Digital PCR is rapidly being adopted in the field of DNA-based food analysis. The direct, absolute quantification it offers makes it an attractive technology for routine analysis of food and feed samples for their composition, possible GMO content, and compliance with labelling requirements. However, assessing the performance of dPCR assays is not yet well established. This article introduces three straightforward parameters based on statistical principles that allow users to evaluate if their assays are robust. In addition, we present post-run evaluation criteria to check if quantification was accurate. Finally, we evaluate the usefulness of Poisson confidence intervals and present an alternative strategy to better capture the variability in the analytical chain.

Inhibition of Notch1 signaling overcomes resistance to the death ligand Trail by specificity protein 1-dependent upregulation of death receptor 5.

The Notch1 signaling pathway contributes to tumorigenesis by influencing differentiation, proliferation and apoptosis. Here, we demonstrate that inhibition of the Notch1 signaling pathway sensitizes glioblastoma cell lines and glioblastoma initiating cells to apoptosis induced by the death ligand TRAIL. This sensitization occurs through transcriptional upregulation of the death receptor 5 (DR5, TRAIL-R2). The increase in DR5 expression is abrogated by concomitant repression of the transcription factor Sp1, which directly binds to the DR5 promoter in the absence of Notch1 as revealed by chromatin immunoprecipitation. Consistent with these findings, Notch1 inhibition resulted in increased DR5 promoter activity, which was impaired by mutation of one out of two Sp1-binding sites within the proximal DR5 promoter. Moreover, we demonstrate that JNK signaling contributes to the regulation of DR5 expression by Notch1. Taken together, our results identify Notch1 as key driver for TRAIL resistance and suggest Notch1 as a promising target for anti-glioblastoma therapy.

Use of pJANUS-02-001 as a calibrator plasmid for Roundup Ready soybean event GTS-40-3-2 detection: an interlaboratory trial assessment.

Owing to the labelling requirements of food and feed products containing materials derived from genetically modified organisms, quantitative detection methods have to be developed for this purpose, including the necessary certified reference materials and calibrator standards. To date, for most genetically modified organisms authorized in the European Union, certified reference materials derived from seed powders are being developed. Here, an assessment has been made on the feasibility of using plasmid DNA as an alternative calibrator for the quantitative detection of genetically modified organisms. For this, a dual-target plasmid, designated as pJANUS-02-001, comprising part of a junction region of genetically modified soybean event GTS-40-3-2 and the endogenous soybean-specific lectin gene was constructed. The dynamic range, efficiency and limit of detection for the soybean event GTS-40-3-2 real-time quantitative polymerase chain reaction (Q-PCR) system described by Terry et al. (J AOAC Int 85(4):938-944, 2002) were shown to be similar for in house produced homozygous genomic DNA from leaf tissue of soybean event GTS-40-3-2 and for plasmid pJANUS-02-001 DNA backgrounds. The performance of this real-time Q-PCR system using both types of DNA templates as calibrator standards in quantitative DNA analysis was further assessed in an interlaboratory trial. Statistical analysis and fuzzy-logic-based interpretation were performed on critical method parameters (as defined by the European Network of GMO Laboratories and the Community Reference Laboratory for GM Food and Feed guidelines) and demonstrated that the plasmid pJANUS-02-001 DNA represents a valuable alternative to genomic DNA as a calibrator for the quantification of soybean event GTS-40-3-2 in food and feed products.

Cardiac transplantation from a carbon monoxide intoxicated donor.

Heart transplantation is a demonstrated successful and life-saving treatment for an increasing number of patients. The growth of heart transplantation surgery is limited by the relative lack of suitable donors, and the increasing demand has lead to the expansion of acceptance criteria. Patients succumbing to carbon monoxide (CO) poisoning are usually considered not suitable organ donors and they are routinely rejected in many centers. Although organs from CO poisoning donors have been occasionally used, cardiac transplantation in this scenario remains very uncommon. We report the successful heart transplantation from a CO intoxicated donor, who was previously refused by two other transplantation teams. Standard donor evaluation criteria, transplantation techniques and management were used. Limited cases are described in literature. The present case may increase awareness among emergency department physicians, as well as transplantations teams, that patients dying of CO exposure may be acceptable cardiac donors.

Effect of sulodexide on albuminuria, NAG excretion and glomerular filtration response to dopamine in diabetic patients.

BACKGROUND: Albuminuria is the best and most readily available marker for glomerular damage and progressive renal function loss in patients with diabetic nephropathy. Recently, administration of the oral glycosaminoglycan sulodexide (a mixture of 80% fast-moving heparin and 20% dermatan sulphate) was shown to effectively decrease albumin excretion rate in diabetics with nephropathy. AIMS: To evaluate whether the hypoalbuminuric effect of sulodexide is associated with improvement of the renal vascular or tubule function. METHODS: Forty-five type 1 diabetic patients, affected by diabetic nephropathy with albuminuria for at least 5 years, were randomly allocated to sulodexide or untreated. Those allocated to sulodexide were given 100 mg of sulodexide daily for 120 days. Renal vascular function (DIR) and N-acetyl-beta-D-glucosaminidase (NAG) excretion were estimated before and at the end of the study, the former in thesulodexide group only. DIR was measured as two Cr(cl) lasting 120 min (before and during 2 mug/kg b.w. i.v. dopamine). RESULTS: The analysis of trends during the study demonstrated a marked reduction of albuminuria in the sulodexide group (from 126.1 +/- 15.41 to 93.6 +/- 13.7 mg/day). DIR rose from 13.2 +/- 2.1% to 15.44 +/- 1.9% (relative increase: +16.9%), and NAG excretion showed a decreasing trend decreased in the sulodexide group only (from 5.1 +/- 0.62 to 4.7 +/- 0.40 U/g(creat)). CONCLUSION: The findings presented in this study indicate for the first time that orally available sulodexide may favorably affect the renal vascular function in type 1 diabetic patients with nephropathy and microalbuminuria. The effect of sulodexide on NAG is strongly influenced by the baseline NAG values, with a significant NAG reduction in the patients with the highest baseline NAG values.

Myocardial protection using HTK solution in minimally invasive mitral valve surgery.

BACKGROUND: Minimally invasive cardiac surgery (MICS) is a safe and satisfactory approach used mainly in mitral valve surgery with excellent results in many centers. Cardioplegia administration can be still a problem, especially when an endoaortic clamp is used. We retrospectively analyzed our early results with histidine-triptophane-ketoglutarate (HTK) solution used for myocardial protection in MICS. METHODS: Between February 2003 and February 2004, 8 patients underwent mitral valve surgery using an endo- cardiopulmonary bypass (CPB) system and HTK solution as myocardial protection. The mean patient age was 67.7 +/- 9.2 years, and the preoperative ejection fraction was normal in all patients. Three patients had valve repair and 5 had valve replacement. Mean CPB time was 129.2 +/- 19.4 minutes, and aortic cross-clamp duration was 88.5 +/- 15.4 minutes. RESULTS: In every case HTK solution was used for only a single dose for cardioplegia at the beginning of the procedure, without any recalls. The heart restarted spontaneously at reperfusion in 6 of 8 cases (75%), and there were no significant modifications in electrocardiogram results or myocardial cytonecrosis enzymes (creatine kinase and its MB fraction) during the postoperative period. CONCLUSIONS: HTK solution is a cold crystalloid cardioplegia solution that has demonstrated its utility in MICS because it provides a safe long cardioplegic arrest time and it reduces the risk of inadequate coronary perfusion due to dislodgement of the endoaortic clamp.

Surgery using median sternotomy for diffuse aneurysmal disease of the thoracic aorta.

We present our experience using an anterior approach for the replacement of an extensive aneurysm of the thoracic aorta. In recent years we have performed surgery on 20 patients by means of a median sternotomy for aneurysms of the ascending aorta, aortic arch, or descending thoracic aorta. In all but 1 of the patients, a procedure in the ascending aorta was also performed. In some patients a small anterior left thoracotomy at the 4th intercostal space was required to allow the replacement of the aorta as for the diaphragm. Antegrade selective cerebral perfusion (ASCP) according to Kazui's technique was used as a brain protection method. All procedures were performed successfully and the aneurysm was completely resected. No neurologic complications or other major complications occurred. We believe that the anterior approach for extensive thoracic aorta replacement is feasible, allows the use of ASCP, and has shown encouraging results.

Evidence of hypertriglyceridemia in managing HIV patients on efavirenz.

Of the recently approved antiretroviral agents, efavirenz has become a popular medication for the treatment of HIV infection. The efficacy of an efavirenz-based regimen has allowed the use of a combination of this medication with other antiretroviral agents in all levels of HIV disease, resulting in decreased viral replication and positive clinical results. Efavirenz is also proposed as an acceptable agent to switch to from a protease inhibitor-based regimen to reduce the risk of metabolic complications. In addition, the favorable side-effect profile, diminished pill burden for clients, and daily dosing have contributed to its popularity. Primary care practitioners should be cognizant of the possible side-effect profile of antiretroviral therapy combinations and enter into a collaborative relationship with the infectious disease clinician to ensure safe and effective patient care.

Antegrade selective cerebral perfusion during surgery of the thoracic aorta: risk analysis.

OBJECTIVE: To determine independent predictors of neurologic outcome and hospital mortality after surgery of the thoracic aorta using moderate hypothermic circulatory arrest and antegrade selective cerebral perfusion. METHODS: Between November 1996 and June 2000, 96 consecutive patients (69 men, 27 women; mean age 63+/-10 years) underwent operations on the thoracic aorta with the aid of moderate hypothermic circulatory arrest and antegrade selective cerebral perfusion. Sixty-four patients were operated on electively (66.7%), 32 emergently (33.3%). Indications for surgery were: type A acute dissection in 30 patients (31.3%), chronic aneurysm in 66 (68.8%). Seventeen patients (17.7%) had undergone previous aortic/cardiac surgical procedures. The mean selective cerebral perfusion time was 52.2+/-31.9 min (range, 18-220 min). Preoperative, intraoperative, and postoperative factors were analyzed by univariate and multivariate analysis to identify predictors of hospital mortality and neurologic outcome. RESULTS: There were no operative deaths; the hospital mortality rate was 11.5% (11/96). Stepwise logistic regression revealed preoperative renal dysfunction (P=0.021), type A acute dissection (P=0.053), coronary artery bypass grafting (P=0.058), post-operative pulmonary complications (P=0.000) and repeat thoracotomy for bleeding (P=0.027) as independent predictors of hospital mortality. One patient sustained a permanent neurologic deficit (1%). Transient neurologic deficit occurred in eight patients (8.3%). Coronary artery bypass grafting (P=0.013), and postoperative cardiac complications (P=0.049) were statistically associated with an increased risk of any (transient and permanent) neurologic dysfunction on univariate analysis. Stepwise logistic regression indicated coronary artery bypass grafting as independent factor for any neurologic dysfunction. CONCLUSION: This study confirmed that selective cerebral perfusion is an effective method of cerebral protection allowing complex thoracic aorta operations to be performed with low risk of hospital mortality and adverse neurologic outcome. We didn't find that the duration of selective cerebral perfusion time influence hospital mortality and any neurologic deficit.

Effect on rats and mice of a diet with low tryptophan content (maize flour).

Female Wistar rats when crowded together become aggressive if fed a diet with low tryptophan (Trp) content (maize, flour, Mf). When isolated during 30 days and fed Mf they show a decrease of weight, of total plasma Trp, and of Trp and 5-hydroxytryptamine (5-HT) in brain stem, but 5-hydroxyindolacetic acid (5-HIAA) of the same cerebral area does not change. These results partially agree with other authors observations. Mice isolated during 47 days and fed with Mf do not show any significant weight variation, the hypermotility of isolated mice on normal diet persists, but 5-HT and 5-HIAA significantly decreases in total brain, an effect that has been observed by another author. The effect on 5-HIAA does not depend on the diet, either balanced or with low Trp content.

Effect on rats and mice of a diet with low tryptophan content (maize flour)

Female Wistar rats when crowded together become aggressive if fed a diet with low tryptophan (Trp) content (maize, flour, Mf). When isolated during 30 days and fed Mf they show a decrease of weight, of total plasma Trp, and of Trp and 5-hydroxytryptamine (5-HT) in brain stem, but 5-hydroxyindolacetic acid (5-HIAA) of the same cerebral area does not change. These results partially agree with other authors observations. Mice isolated during 47 days and fed with Mf do not show any significant weight variation, the hypermotility of isolated mice on normal diet persists, but 5-HT and 5-HIAA significantly decreases in total brain, an effect that has been observed by another author. The effect on 5-HIAA does not depend on the diet, either balanced or with low Trp content.

Effect on rats and mice of a diet with low tryptophan content (maize flour).

Female Wistar rats when crowded together become aggressive if fed a diet with low tryptophan (Trp) content (maize, flour, Mf). When isolated during 30 days and fed Mf they show a decrease of weight, of total plasma Trp, and of Trp and 5-hydroxytryptamine (5-HT) in brain stem, but 5-hydroxyindolacetic acid (5-HIAA) of the same cerebral area does not change. These results partially agree with other authors observations. Mice isolated during 47 days and fed with Mf do not show any significant weight variation, the hypermotility of isolated mice on normal diet persists, but 5-HT and 5-HIAA significantly decreases in total brain, an effect that has been observed by another author. The effect on 5-HIAA does not depend on the diet, either balanced or with low Trp content.

Role of ACTH on the effect of medroxyprogesterone in brain stem serotonin.

PIP: A clinical study with 361 female rats was conducted to elucidate the mechanism whereby MPA (medroxyprogesterone acetate) lowers 5-HT/5-HIAA ratio in the brain area and the possible role of serotoniergic mechanisms. In addition, the participation of MAO (monoamino oxidase) system and the effects of some steroids were studied in order to establish a relationship between chemical structure and activity. The effects of the following steroids were studied: MPA (medroxyprogesterone acetate), melengestrol acetate, chlormadinone, pregnenolone, -methyl pregnenolone, DOCA, acetoxi-progesterone, and ACTH (synacthen). Effects of these substances on LTP (liver tryptophan pyrrolase) activity, total and free plasma and brain stem Trp (tryptophan), and the 5HT and 5HTAA content in brain stem are tabulated. Of all the substances, only MPA and melengestrol acetate significantly raised LTP activity and both also lowered 5-HT content of brain stem. The high levels of ACTH in the blood of the adrenalectomized rats, as in those under fasting conditions, antagonized MPA effects. To further test this seeming result, ACTH and ACTH-MPA were injected into another group of animals. The ACTH not only increased plasma corticosterone but also antagonized the effect of MPA on the 5-HT content of brain stem. The study did not identify a relationship between chemical structure of the steroids studied and effects observed.^ieng