Routine sensor-augmented pump therapy in type 1 diabetes: the INTERPRET study.

BACKGROUND: Sensor-augmented pump (SAP) therapy can improve glycemic control, compared with multiple daily insulin injections or with insulin pump therapy alone, without increasing the risk of hypoglycemia. SUBJECTS AND METHODS: A 12-month observational study in patients with type 1 diabetes treated with continuous subcutaneous insulin infusion (CSII), upon the introduction of continuous glucose monitoring (CGM), was conducted in 15 countries (in Europe and in Israel) to document the real-life use of SAP and assess which variables are associated with improvement in type 1 diabetes management. RESULTS: Data from 263 patients (38% male; mean age, 28.0 ± 15.7 years [range, 1-69 years]; body mass index, 23.3 ± 4.9 kg/m(2); diabetes duration, 13.9 ± 10.7 years; CSII duration, 2.6 ± 3 years) were collected. Baseline mean glycated hemoglobin A1c (HbA1c) was 8.1 ± 1.4%; 82% had suboptimal HbA1c (≥ 7%). The average sensor use for 12 months was 30% (range, 0-94%), and sensor use decreased with time (first 3 months, 37%; last 3 months, 27%). Factors associated with improvement in HbA1c after 12 months in patients with baseline HbA1c ≥ 7% were high baseline HbA1c (P<0.001), older age group (P<0.001), and more frequent sensor use (P = 0.047). Significantly less hospitalization, increased treatment satisfaction, and reduced fear of hypoglycemia were reported after 12 months of SAP. CONCLUSIONS: This is the largest and longest multicenter prospective observational study providing real-life data on SAP. These results are consistent with those of controlled trials showing the effectiveness of CGM in pump users.

Age-related proteome analysis of the mouse brain: a 2-DE study.

2-DE remains the most popular and versatile protein separation method among a rapidly growing array of various proteomics technologies. However, variability in sample processing, experimental design and data analyses results in a limited cross-validation between studies performed in different laboratories. One of the goals of the Human Proteome Organization (HUPO) is to establish standards and guidelines for proteomics studies. We contributed to the HUPO Brain Proteome Project by analyzing brains from neonatal and adult mice using 2-DE. Here we propose a standard workflow to analyze 2-DE images and extract statistically significant differences. After differential analysis and identification by MALDI-TOF/TOF, dihydropyrimidinase-related proteins, brain FABP, stathmin, isocitrate dehydrogenase, gamma enolase, annexin V, glutamine synthetase, creatine kinase B chain, triosephosphate dehydrogenase, and malate dehydrogenase were found differentially expressed between the two groups. The functions and potential mechanisms underlying the variation observed for these proteins are discussed.

HUPO Brain Proteome Project: summary of the pilot phase and introduction of a comprehensive data reprocessing strategy.

The Human Proteome Organisation (HUPO) initiated several projects focusing on the proteome analysis of distinct human organs. The Brain Proteome Project (BPP) is the initiative dedicated to the brain, its development and correlated diseases. Two pilot studies have been performed aiming at the comparison of techniques, laboratories and approaches. With the help of the results gained, objective data submission, storage and reprocessing workflow have been established. The biological relevance of the data will be drawn from the inter-laboratory comparisons as well as from the re-calculation of all data sets submitted by the different groups. In the following, results of the single groups as well as the centralised reprocessing effort will be summarised and compared, showing the added value of this concerted work.

State-of-the-art two-dimensional gel electrophoresis: a key tool of proteomics research.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the most popular and versatile method of protein separation among a rapidly growing array of proteomics technologies. Based on two distinct procedures, it combines isoelectric focusing (IEF), which separates proteins according to their isoelectric point (pI), and SDS-PAGE, which separates them further according to their molecular mass. At present, 2D-PAGE is capable of simultaneously detecting and quantifying up to several thousand protein spots in the same gel image. Here we provide comprehensive step-by-step instructions for the application of a standardized 2D-PAGE protocol to a sample of human plasma or cerebrospinal fluid (CSF). The method can be easily adapted to any type of sample. This four-day protocol provides detailed information on how to apply complex biological fluids to an immobilized dry strip gel, cast home-made gradient acrylamide gels, run the gels, and perform standard staining methods. A troubleshooting guide is also included.

Truncated cystatin C in cerebrospiral fluid: Technical [corrected] artefact or biological process?

Cystatin C, a low molecular weight cysteine proteinase inhibitor present in human body fluids at physiological concentrations, is more expressed in cerebrospinal fluid (CSF) than in plasma. Mass spectrometric characterization showed that after 3 months of storage of human CSF at -20 degrees C, cystatin C was cleaved in the peptide bond between R8 and L9 and lost its eight N-termini amino acids, whereas this cleavage did not occur when stored at -80 degrees C. This truncation occurred in all CSF samples studied irrespective of the underlying neurological status, indicating a storage-related artefact rather than a physiological or pathological processing of the protein. These results stress the importance of optimal preanalytical storage conditions of any sample prior to proteomics studies.

Cystatin C as a potential cerebrospinal fluid marker for the diagnosis of Creutzfeldt-Jakob disease.

The definite diagnosis of Creutzfeldt-Jakob disease (CJD), the most common form of human prion diseases, relies upon neuropathological data usually obtained at autopsy. In living patients, the diagnosis, based on suggestive clinical features and EEG abnormalities, can be aided by the detection of altered levels of isoforms of the 14-3-3 protein in the cerebrospinal fluid (CSF). However, the validity of this test has been recently challenged and the search for other, more reliable biomarkers for CJD remains highly desirable. The present study describes the identification of a new potential surrogate marker in the CSF of CJD-affected patients. A preliminary study employing surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) technology highlighted a protein at 13.4 kDa in a small group (n = 8) of CJD-affected patients. Further analysis aimed at identifying this protein using cationic exchange chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed it to be cystatin C. Additional immunoblot assays confirmed that the level of cystatin C was significantly increased (p

Mining mass spectra for diagnosis and biomarker discovery of cerebral accidents.

In this paper we try to identify potential biomarkers for early stroke diagnosis using surface-enhanced laser desorption/ionization mass spectrometry coupled with analysis tools from machine learning and data mining. Data consist of 42 specimen samples, i.e., mass spectra divided in two big categories, stroke and control specimens. Among the stroke specimens two further categories exist that correspond to ischemic and hemorrhagic stroke; in this paper we limit our data analysis to discriminating between control and stroke specimens. We performed two suites of experiments. In the first one we simply applied a number of different machine learning algorithms; in the second one we have chosen the best performing algorithm as it was determined from the first phase and coupled it with a number of different feature selection methods. The reason for this was 2-fold, first to establish whether feature selection can indeed improve performance, which in our case it did not seem to confirm, but more importantly to acquire a small list of potentially interesting biomarkers. Of the different methods explored the most promising one was support vector machines which gave us high levels of sensitivity and specificity. Finally, by analyzing the models constructed by support vector machines we produced a small set of 13 features that could be used as potential biomarkers, and which exhibited good performance both in terms of sensitivity, specificity and model stability.

A panel of cerebrospinal fluid potential biomarkers for the diagnosis of Alzheimer's disease.

The diagnosis of Alzheimer's disease (AD), the most common form of dementia in the general population, usually relies upon the presence of typical clinical features and structural changes on brain magnetic resonance imaging. Over the last decade, a number of biological abnormalities have been reported in the cerebrospinal fluid (CSF) of AD patients, in particular altered levels of the tau protein and the 1-42 fragment of the amyloid precursor protein. These, however, have not yet proved sensitive and specific enough to be included in the diagnostic criteria for AD, leaving plenty of room for the search of novel biomarkers. The present study describes the analysis of CSF polypeptides by a protein-chip array technology called surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS). Using this approach, we detected statistically significant quantitative differences (p < 0.05) regarding four overexpressed and one underexpressed polypeptides in the CSF of AD patients as compared to healthy controls. Four of them were further purified by strong anionic exchange chromatography (SAX) and identified by MS analysis as cystatin C, two beta-2-microglobulin isoforms, an unknown 7.7 kDa polypeptide, and a 4.8 kDa VGF polypeptide. The combination of the five polypeptides for the diagnosis of AD allowed to classified six AD patients out of the nine included in this study and all the ten controls, which means in this small cohort that the specificity and sensitivity are 100% and 66%, respectively. This study, based on the protein-chip array technology, demonstrates the presence in the CSF of novel potential biomarkers for AD, which may be used for the diagnosis and perhaps the assessment of the severity and progression of the disease.

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session.

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.

Involvement of nitric oxide and the ovarian blood follicle barrier in murine follicular cyst development.

OBJECTIVE: To test the involvement of nitric oxide in murine ovarian follicular cysts. DESIGN: Controlled animal study. SETTING: Academic research environment. ANIMAL(S): Immature female B6D2F1 mice at 23 +/- 2 days old. Ovarian cysts were induced by implanting miniosmotic pumps that delivered and maintained constant levels of hCG. Nitric oxide studies included the delivery of nitric oxide synthase (NOS) inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME), or N(G)-nitro-D-arginine methyl ester, by the same method. Ovulation assays measured cumulus oocyte complexes and blood follicle barrier (BFB) function. RESULT(S): Chronic treatment with hCG induced enlarged ovaries containing multiple follicular cysts, which were approximately double the size of follicles in sham-operated mice. These cysts enclosed few, if any granulosa cells, secreted high levels of testosterone, and had impaired ovarian BFB function. Inhibition of NOS by L-NAME during ovarian cyst formation reduced the size of follicular cysts, sustained normal testosterone levels, and maintained hormonal BFB reactivity in cystic follicles. CONCLUSION(S): Nitric oxide was found to be involved in the formation of hCG-induced murine follicular cysts and complications associated with these cysts were ameliorated by the NOS inhibitor L-NAME.