Extended bottom-up proteomics with secreted aspartic protease Sap9.

We investigate the benefits and experimental feasibility of approaches enabling the shift from short (1.7kDa on average) peptides in bottom-up proteomics to about twice longer (~3.2kDa on average) peptides in the so-called extended bottom-up proteomics. Candida albicans secreted aspartic protease Sap9 has been selected for evaluation as an extended bottom-up proteomic-grade enzyme due to its suggested dibasic cleavage specificity and ease of production. We report the extensive characterization of Sap9 specificity and selectivity revealing that protein cleavage by Sap9 most often occurs in the vicinity of proximal basic amino acids, and in select cases also at basic and hydrophobic residues. Sap9 is found to cleave a large variety of proteins in a relatively short, ~1h, period of time and it is efficient in a broad pH range, including slightly acidic, e. g., pH5.5, conditions. Importantly, the resulting peptide mixtures contain representative peptides primarily in the target 3-7kDa range. The utility and advantages of this enzyme in routine analysis of protein mixtures are demonstrated and the limitations are discussed. Overall, Sap9 has a potential to become an enzyme of choice in an extended bottom-up proteomics, which is technically ready to complement the traditional bottom-up proteomics for improved targeted protein structural analysis and expanded proteome coverage. BIOLOGICAL SIGNIFICANCE: Advances in biological applications of mass spectrometry-based bottom-up proteomics are oftentimes limited by the extreme complexity of biological samples, e.g., proteomes or protein complexes. One of the reasons for it is in the complexity of the mixtures of enzymatically (most often using trypsin) produced short (<3kDa) peptides, which may exceed the analytical capabilities of liquid chromatography and mass spectrometry. Information on localization of protein modifications may also be affected by the small size of typically produced peptides. On the other hand, advances in high-resolution mass spectrometry and liquid chromatography have created an intriguing opportunity of improving proteome analysis by gradually increasing the size of enzymatically-derived peptides in MS-based bottom-up proteomics. Bioinformatics has already confirmed the envisioned advantages of such approach. The remaining bottle-neck is an enzyme that could produce longer peptides. Here, we report on the characterization of a possible candidate enzyme, Sap9, which may be considered for producing longer, e.g., 3-7kDa, peptides and lead to a development of extended bottom-up proteomics.

Is it worth treating fever in intensive care unit patients? Preliminary results from a randomized trial of the effect of external cooling.

BACKGROUND: Antipyresis is a common clinical practice in intensive care, although it is unknown if fever is harmful, beneficial, or a negligible adverse effect of infection and inflammation. METHODS: In a randomized study, rectal temperature and discomfort were assessed in 38 surgical intensive care unit patients without neurotrauma or severe hypoxemia and with fever (temperature >/=38.5 degrees C) and systemic inflammatory response syndrome. Eighteen patients received external cooling while 20 received no antipyretic treatment. RESULTS: Temperature and discomfort decreased similarly in both groups after 24 hours. No significant differences in recurrence of fever, incidence of infection, antibiotic therapy, intensive care unit and hospital length of stay, or mortality were noted between the groups. CONCLUSIONS: These results suggest that the systematic suppression of fever may not be useful in patients without severe cranial trauma or significant hypoxemia. Letting fever take its natural course does not seem to harm patients with systemic inflammatory response syndrome or influence the discomfort level and may save costs.

Abaisser la pression arterielle: urgent ou pas urgent? [How urgent is it to decrease high blood pressure?]

Severe hypertension represents a frequent problem for the general practitioner. One has to decide if the blood pressure needs to be decreased immediately (hypertensive emergency), or if the blood pressure maybe progressively decreased in a few hours and normalized in a few days (hypertensive crisis). Thus it is crucial to identify on the basis of the clinical history and a careful physical examination, the patients for whom the arterial blood pressure elevation represents an acute danger for organ damage or a vital threat in the absence of immediate blood pressure control. In the case of hypertensive crisis, oral medication is usually sufficient (slow release or GITS nifedipine, nitroglycerin, labetalol, captopril). The hypertensive emergency sometimes requires an oral medication before the admission to the emergency room, then followed by intravenous drug administration (sodium nitroprussiate, nitroglycerin, labetalol).

A novel technique using echocardiography to evaluate venous cannula performance perioperatively in CPB cardiac surgery.

OBJECTIVE: Transthoracic echocardiography (TTE) has been used clinically to disobstruct venous drainage cannula and to optimise placement of venous cannulae in the vena cava but it has never been used to evaluate performance capabilities. Also, little progress has been made in venous cannula design in order to optimise venous return to the heart lung machine. We designed a self-expandable Smartcanula (SC) and analysed its performance capability using echocardiography. METHODS: An epicardial echocardiography probe was placed over the SC or control cannula (CTRL) and a Doppler image was obtained. Mean (V(m)) and maximum (V(max)) velocities, flow and diameter were obtained. Also, pressure drop (DeltaP(CPB)) was obtained between the central venous pressure and inlet to venous reservoir. LDH and Free Hb were also compared in 30 patients. Comparison was made between the two groups using the student's t-test with statistical significance established when p<0.05. RESULTS: Age for the SC and CC groups were 61.6+/-17.6 years and 64.6+/-13.1 years, respectively. Weight was 70.3+/-11.6 kg and 72.8+/-14.4 kg, respectively. BSA was 1.80+/-0.2 m(2) and 1.82+/-0.2 m(2), respectively. CPB times were 114+/-53 min and 108+/-44 min, respectively. Cross-clamp time was 59+/-15 min and 76+/-29 min, respectively (p=NS). Free-Hb was 568+/-142 U/l versus 549+/-271 U/l post-CPB for the SC and CC, respectively (p=NS). LDH was 335+/-73 mg/l versus 354+/-116 mg/l for the SC and CC, respectively (p=NS). V(m) was 89+/-10 cm/s (SC) versus 63+/-3 cm/s (CC), V(max) was 139+/-23 cm/s (SC) versus 93+/-11 cm/s (CC) (both p<0.01). DeltaP(CPB) was 30+/-10 mmHg (SC) versus 43+/-13 mmHg (CC) (p<0.05). A Bland-Altman test showed good agreement between the two devices used concerning flow rate calculations between CPB and TTE (bias 300 ml+/-700 ml standard deviation). CONCLUSIONS: This novel Smartcanula design, due to its self-expanding principle, provides superior flow characteristics compared to classic two stage venous cannula used for adult CPB surgery. No detrimental effects were observed concerning blood damage. Echocardiography was effective in analysing venous cannula performance and velocity patterns.

The differentiation of fibre- and drug type Cannabis seedlings by gas chromatography/mass spectrometry and chemometric tools

Cannabis cultivation in order to produce drugs is forbidden in Switzerland. Thus, law enforcement authorities regularly ask forensic laboratories to determinate cannabis plant's chemotype from seized material in order to ascertain that the plantation is legal or not. As required by the EU official analysis protocol the THC rate of cannabis is measured from the flowers at maturity. When laboratories are confronted to seedlings, they have to lead the plant to maturity, meaning a time consuming and costly procedure. This study investigated the discrimination of fibre type from drug type Cannabis seedlings by analysing the compounds found in their leaves and using chemometrics tools. 11 legal varieties allowed by the Swiss Federal Office for Agriculture and 13 illegal ones were greenhouse grown and analysed using a gas chromatograph interfaced with a mass spectrometer. Compounds that show high discrimination capabilities in the seedlings have been identified and a support vector machines (SVMs) analysis was used to classify the cannabis samples. The overall set of samples shows a classification rate above 99% with false positive rates less than 2%. This model allows then discrimination between fibre and drug type Cannabis at an early stage of growth. Therefore it is not necessary to wait plants' maturity to quantify their amount of THC in order to determine their chemotype. This procedure could be used for the control of legal (fibre type) and illegal (drug type) Cannabis production.

Genetik der essentiellen arteriellen Hypertonie [Essential hypertension - which genes can be held responsible for it?].

Hypertension is a common heritable cardiovascular risk factor. Some rare monogenic forms of hypertension have been described, but the majority of patients suffer from essential hypertension, for whom the underlying genetic mechanisms are not clear. Essential hypertension is a complex trait, involving multiple genes and environmental factors. Recently, progress in the identification of common genetic variants associated with essential hypertension has been made due to large-scale international collaborative projects. In this article we review the new research methods used as well as selected recent findings in this field.

High elevation Plantago lanceolata plants are less resistant to herbivory than their low elevation conspecifics: is it just temperature?

Traits that mediate species interactions are evolutionarily shaped by biotic and abiotic drivers, yet we know relatively little about the relative importance of these factors. Herbivore pressure, along with resource availability and third-party' mutualists, are hypothesized to play a major role in the evolution of plant defence traits. Here, we used the model system Plantago lanceolata, which grows along steep elevation gradients in the Swiss Alps, to investigate the effect of elevation, herbivore pressure, mycorrhizal inoculation and temperature on plant resistance. Over a 1200 m elevation gradient, the levels of herbivory and iridoid glycosides (IGs) declined with increasing elevation. By planting seedlings at three different elevations, we further showed that both low-elevation growing conditions and mycorrhizal inoculation resulted in increased plant resistance to herbivores. Finally, using a temperature-controlled experiment comparing high- and low-elevation ecotypes, we showed that high-elevation ecotypes are less resistant to herbivory, and that lower temperatures impair IGs deployment after herbivore attack. We thus propose that both lower herbivore pressure, and colder temperatures relax the defense syndrome of high elevation plants.

Low density lipoprotein signaling and pancreatic beta cells protection

SummaryLow-density lipoproteins (LDLs) have an important physiological role in organism transporting cholesterol and other fatty substances to target tissues. However, elevated LDL levels in the blood are associated with the formation of arterial plaques and consequently atherosclerosis. It is therefore important to characterize the intracellular pathways induced upon LDL stimulation as they might be involved in the pathological properties of these lipoproteins. It has been previously found that LDL stimulation of mouse embryonic fibroblasts activates p38 mitogen activated protein kinases (MAPKs). This leads to cell spreading and increase in the wound healing capabilities of the cells. These two responses might occur within atherosclerotic plaques.The aim of this project is to reveal the missing links between LDL particle and activation of p38 MAPK kinase. As previously shown in our lab activation of p38 MAPK kinase by the LDL particles occur independently of classical LDL receptor (LDLR). In this study we have shown that scavenger receptor type Β class I (SR-BI) is responsible for the signal transduction from the LDLs to the p38 MAPK. We have also shown that Mitogen activated kinase kinases (MKKs) that can directly activate ρ 38 MAPK in these conditions are MKK3 and MKK6 but not MKK4. We have also tested some of the intermediate components of the pathway like Ras and PI3 kinase but found that they do not play a role.The data obtained in this study showed a part of molecular mechanism responsible for p38 MAPK activation and subsequent wound healing and can contribute to our knowledge on function of the fibroblasts in the development of the atherosclerotic plaques.Diabetes Mellitus is a condition caused by disordered metabolism of blood glucose level. It is one of the most commonly spread disease in the western world, with the incidence reaching 8% of population in United States. Two most common types of diabetes are type 1 and 2 that differs slightly in the mechanism of the development. However in the basis of both types lies the cell death of pancreatic beta cells. The aim of this work is to improve beta cells survival in different pathophysiological settings. This could be extrapolated to the conditions in which Diabetes develops in humans. We decided to use RasGAP- derived fragment Ν with its strong antiapoptotic effect in beta cells. In our lab we have demonstrated that in the mild stress conditions RasGAP can be cleaved by caspases at the position 455 producing two fragments, fragment Ν and fragment C. Fragment Ν exerts

Clients of sex workers in Switzerland: it makes sense to counsel and propose rapid test for HIV on the street, a preliminary report.

BACKGROUND: Clients of street sex workers may be at higher risk for HIV infection than the general population. Furthermore, there is a lack of knowledge regarding HIV testing of clients of sex workers in developed countries. METHOD: This pilot study assessed the feasibility and acceptance of rapid HIV testing by the clients of street-based sex workers in Lausanne, Switzerland. For 5 evenings, clients in cars were stopped by trained field staff for face-to-face interviews focusing on sex-related HIV risk behaviors and HIV testing history. The clients were then offered a free anonymous rapid HIV test in a bus parked nearby. Rapid HIV testing and counselling were performed by experienced nurse practitioners. Clients with reactive tests were offered confirmatory testing, medical evaluation, and care in our HIV clinic. RESULT: We intercepted 144 men, 112 (77.8%) agreed to be interviewed. Among them, 50 (46.6%) had never been tested for HIV. A total of 31 (27.7%) rapid HIV tests were performed, 16 (51.6%) in clients who had not previously been tested. None were reactive. Initially, 19 (16.9%) additional clients agreed to HIV testing but later declined due to the 40-minute queue for testing. CONCLUSION: This pilot study showed that rapid HIV testing in the red light district of Lausanne was feasible, and that the clients of sex workers accepted testing at an unexpectedly high rate. This setting seems particularly appropriate for targeted HIV screening, since more than 40% of the clients had not previously been tested for HIV even though they engaged in sex-related HIV risk behaviour.

L'erreur en médecine ambulatoire: comment l'aborder. [Error in ambulatory medicine: how to deal with it?]

Medical errors compromise patient safety in ambulatory practice. These errors must be faced in a framework that reduces to a minimum their consequences for the patients. This approach relies on the implementation of a new culture without stigmatization and where errors are disclosed to the patients; this culture implies the build up of a system for reporting errors associated to an in-depth analysis of the system, looking for root causes and insufficient barriers with the aim to fix them. A useful education tool is the "critical situations" meeting during which physicians are encouraged to openly present adverse events and "near misses". Their analysis, with supportive attitude towards involved staff members, allows to reveal systems failures within the institution or the private practice.

Teaching cardiac auscultation to trainees in internal medicine and family practice: does it work?

BACKGROUND: The general proficiency in physical diagnostic skills seems to be declining in relation to the development of new technologies. The few studies that have examined this question have invariably used recordings of cardiac events obtained from patients. However, this type of evaluation may not correlate particularly well with bedside skills. Our objectives were 1) To compare the cardiac auscultatory skills of physicians in training with those of experienced cardiologists by using real patients to test bedside diagnostic skills. 2) To evaluate the impact of a five-month bedside cardiac auscultation training program. METHODS: 1) In an academic primary care center, 20 physicians (trainees in internal medicine and family practice) and two skilled academic cardiologists listened to 33 cardiac events in 13 patients directly at bedside and identified the cardiac events by completing an open questionnaire. Heart sounds, murmurs and diagnosis were determined beforehand by an independent skilled cardiologist and were validated by echocardiography. Thirteen primary cardiologic diagnoses were possible.2) Ten of the physicians agreed to participate in a course of 45-minute sessions once a week for 5 months. After the course they listened again to the same patients (pre/post-interventional study). RESULTS: 1) The experts were the most skillful, achieving 69% recognition of heart sounds and murmurs and correct diagnoses in 62% of cases. They also heard all of the diastolic murmurs. The residents heard only 40% of the extra heart sounds and made a correct diagnosis in 24% of cases. 2) After the weekly training sessions, their mean percentage for correct diagnosis was 35% [an increase of 66% (p < 0.05)]. CONCLUSIONS: The level of bedside diagnostic skills in this relatively small group of physicians in training is indeed low, but can be improved by a course focusing on realistic bedside teaching.