Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column.

In addition to the importance of sample preparation and extract separation, MS detection is a key factor in the sensitive quantification of large undigested peptides. In this article, a linear ion trap MS (LIT-MS) and a triple quadrupole MS (TQ-MS) have been compared in the detection of large peptides at subnanomolar concentrations. Natural brain natriuretic peptide, C-peptide, substance P and D-Junk-inhibitor peptide, a full D-amino acid therapeutic peptide, were chosen. They were detected by ESI and simultaneous MS(1) and MS(2) acquisitions. With direct peptide infusion, MS(2) spectra revealed that fragmentation was peptide dependent, milder on the LIT-MS and required high collision energies on the TQ-MS to obtain high-intensity product ions. Peptide adsorption on surfaces was overcome and peptide dilutions ranging from 0.1 to 25 nM were injected onto an ultra high-pressure LC system with a 1 mm id analytical column and coupled with the MS instruments. No difference was observed between the two instruments when recording in LC-MS(1) acquisitions. However, in LC-MS(2) acquisitions, a better sensitivity in the detection of large peptides was observed with the LIT-MS. Indeed, with the three longer peptides, the typical fragmentation in the TQ-MS resulted in a dramatic loss of sensitivity (> or = 10x).

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating.

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.

Transapical aortic valve implantation following a left pneumonectomy.

Transapical transcatheter aortic valve implantation is an emerging technique for high-risk patients with symptomatic aortic valve stenosis, peripheral vascular disease, and severe concomitant comorbidities. However, a previous major surgical intervention involving the left hemithorax and the lung has always been considered a technical surgical challenge or even a potential contraindication for this minimally invasive procedure. With this report, we demonstrate, for the first time, that a previous left pneumonectomy followed by mediastinal radiotherapy does not affect the feasibility of transapical transcatheter aortic valve implantation, and we discuss the preoperative workup and the peculiar intraoperative cardiac imaging and surgical assessment.

Selective ion changes during spontaneous mitochondrial transients in intact astrocytes.

The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na(+) concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na(+) concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca(2+) concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg(2+) concentration accompanying mitochondrial Na(+) spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na(+)-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication.

International union of basic and clinical pharmacology. XCI. structure, function, and pharmacology of acid-sensing ion channels and the epithelial Na+ channel.

The epithelial Na(+) channel (ENaC) and the acid-sensing ion channels (ASICs) form subfamilies within the ENaC/degenerin family of Na(+) channels. ENaC mediates transepithelial Na(+) transport, thereby contributing to Na(+) homeostasis and the maintenance of blood pressure and the airway surface liquid level. ASICs are H(+)-activated channels found in central and peripheral neurons, where their activation induces neuronal depolarization. ASICs are involved in pain sensation, the expression of fear, and neurodegeneration after ischemia, making them potentially interesting drug targets. This review summarizes the biophysical properties, cellular functions, and physiologic and pathologic roles of the ASIC and ENaC subfamilies. The analysis of the homologies between ENaC and ASICs and the relation between functional and structural information shows many parallels between these channels, suggesting that some mechanisms that control channel activity are shared between ASICs and ENaC. The available crystal structures and the discovery of animal toxins acting on ASICs provide a unique opportunity to address the molecular mechanisms of ENaC and ASIC function to identify novel strategies for the modulation of these channels by pharmacologic ligands.

Short-term clinical outcomes among patients undergoing transcatheter aortic valve implantation in Switzerland: the Swiss TAVI registry.

AIMS: To evaluate short-term clinical outcomes following transcatheter aortic valve implantation (TAVI) using CE-mark approved devices in Switzerland. METHODS AND RESULTS: The Swiss TAVI registry is a national, prospective, multicentre, monitored cohort study evaluating clinical outcomes in consecutive patients undergoing TAVI at cardiovascular centres in Switzerland. From February 2011 to March 2013, a total of 697 patients underwent TAVI for native aortic valve stenosis (98.1%), degenerative aortic bioprosthesis (1.6%) or severe aortic regurgitation (0.3%). Patients were elderly (82.4±6 years), 52% were females, and the majority highly symptomatic (73.1% NYHA III/IV). Patients with severe aortic stenosis (mean gradient 44.8±17 mmHg, aortic valve area 0.7±0.3 cm²) were either deemed inoperable or at high risk for conventional surgery (STS 8.2%±7). The transfemoral access was the most frequently used (79.1%), followed by transapical (18.1%), direct aortic (1.7%) and subclavian access (1.1%). At 30 days, rates of all-cause mortality, cerebrovascular events and myocardial infarction were 4.8%, 3.3% and 0.4%, respectively. The most frequently observed adverse events were access-related complications (11.8%), permanent pacemaker implantation (20.5%) and bleeding complications (16.6%). The Swiss TAVI registry is registered at ClinicalTrials.gov (NCT01368250). CONCLUSIONS: The Swiss TAVI registry is a national cohort study evaluating consecutive TAVI procedures in Switzerland. This first outcome report provides favourable short-term clinical outcomes in unselected TAVI patients.

Modulatory effects of acid-sensing ion channels on action potential generation in hippocampal neurons.

Extracellular acidification has been shown to generate action potentials (APs) in several types of neurons. In this study, we investigated the role of acid-sensing ion channels (ASICs) in acid-induced AP generation in brain neurons. ASICs are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family and are transiently activated by a rapid drop in extracellular pH. We compared the pharmacological and biophysical properties of acid-induced AP generation with those of ASIC currents in cultured hippocampal neurons. Our results show that acid-induced AP generation in these neurons is essentially due to ASIC activation. We demonstrate for the first time that the probability of inducing APs correlates with current entry through ASICs. We also show that ASIC activation in combination with other excitatory stimuli can either facilitate AP generation or inhibit AP bursts, depending on the conditions. ASIC-mediated generation and modulation of APs can be induced by extracellular pH changes from 7.4 to slightly <7. Such local extracellular pH values may be reached by pH fluctuations due to normal neuronal activity. Furthermore, in the plasma membrane, ASICs are localized in close proximity to voltage-gated Na(+) and K(+) channels, providing the conditions necessary for the transduction of local pH changes into electrical signals.

Evaluation gériatrique des patients agés candidats a un remplacement valvulaire aortique percutané [Geriatric assessment in old patients candidate to transcatheter aortic implantation].

Aortic stenosis mostly occurs among old-old patients. Once symptoms appear, prognosis is guarded, with 2-year mortality as high as 50%. Transcatheter Aortic Valve Implantation (TAVI) is a new therapeutic option in patients at very high surgical risk, who are mostly older persons. However, TAVI is associated with some complications, and patient selection remains a challenge. Comprehensive geriatric assessment (CGA) identifies patients with medical and functional problems likely to affect the TAVI post-operative course. Collaboration between cardiologists and geriatricians will likely become a standard approach to enhance the assessment of these frail patients and identify those most likely to benefit from TAVI.

Surgical outcomes of deep sclerectomy versus Baerveldt aqueous shunt implantation following initial failed deep sclerectomy

Purpose: To examine the efficacy and safety of repeat deep sclerectomy (DS) versus Baerveldt shunt (BS) implantation as second line surgery following failed primary DS. Methods: Fifty one patients were prospectively recruited to undergo BS implantation following failed DS and 51 patients underwent repeat DS, for which data was collected retrospectively. All eyes had at least one failed DS. Surgical success was defined as IOP≤21mmHg and 20% reduction in IOP from baseline. Success rates, number of glaucoma medications (GMs), IOP, and complication rates were compared between the two groups at year 1, post-operatively. Results: Mean age, sex and the proportion of glaucoma subtypes were similar between groups. Preoperatively IOP was significantly lower in DS group vs BS group (18.8mmHg vs 23.8mmHg, p<0.01, two sample t-test). Postoperatively IOP was significantly higher in DS group than BS group (14.6mmHg vs 12.0mmHg, p<0.01, two-sample t-test). In the DS group, 47% of eyes did not achieve 20% reduction in IOP from baseline, as a result the success rates were significantly lower in eyes with DS (51%) than in eyes with BS (88%) (p=0.02, log-rank test). Preoperatively the number of GMs used in DS and BS groups were similar (2.2 vs 2.7 p=0.02, two sample t-test). Postoperatively there remained no significant difference in GMs between groups (0.9 vs 1.1, p= 0.58, two sample t-test). Complication rates were similar between the two groups (12% vs 10%). Conclusions: Baerveldt tube implantation was more effective in lowering IOP than repeat deep sclerectomy in eyes with failed primary DS, at year one. Complications were minor and infrequent in both groups

Short-term stability of testosterone and epitestosterone conjugates in urine samples: quantification by liquid chromatography-linear ion trap mass spectrometry.

A simple method using liquid chromatography-linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1-3ng/mL), recovery (89-101%), intraday precision (2.0-6.8%), interday precision (3.4-9.6%) and accuracy (101-103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 degrees C during a time-storage of a week lead to the conclusion that addition of sodium azide (10mg/mL) is required for preservation of the analytes.