A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed

Since its introduction a few years ago, the linear ion trap Orbitrap (LTQ Orbitrap) instrument has become a powerful tool in proteomics research. For high resolution mass spectrometry measurements ions are accumulated in the linear ion trap and passed on to the Orbitrap analyzer. Simultaneously with acquisition of this signal, the major peaks are isolated in turn, fragmented and recorded at high sensitivity in the linear ion trap, combining the strengths of both mass analyzer technologies. Here we describe a next generation LTQ Orbitrap system termed Velos, with significantly increased sensitivity and scan speed. This is achieved by a vacuum interface using a stacked ring radio frequency ion guide with 10-fold higher transfer efficiency in MS/MS mode and 3–5-fold in full scan spectra, by a dual pressure ion trap configuration, and by reduction of overhead times between scans. The first ion trap efficiently captures and fragments ions at relatively high pressure whereas the second ion trap realizes extremely fast scan speeds at reduced pressure. Ion injection times for MS/MS are predicted from full scans instead of performing automatic gain control scans. Together these improvements routinely enable acquisition of up to ten fragmentation spectra per second. Furthermore, an improved higher-energy collisional dissociation cell with increased ion extraction capabilities was implemented. Higher-collision energy dissociation with high mass accuracy Orbitrap readout is as sensitive as ion trap MS/MS scans in the previous generation of the instrument.Proteomics experiments typically involve the analysis of peptide mixtures obtained by the enzymatic digestion of proteomes that can be as complex as complete cell lysates (1, 2). Dynamic range of peptide abundances and the sheer number of peptides encountered in these mixtures require extremely sensitive and fast peptide detection and fragmentation (3). Although a first comprehensively identified and quantified proteome has recently been reported (4), further gains in instrumental performance are clearly needed to reduce overall measurement time, improve sequence coverage of identified proteins, and for the in-depth analysis of mammalian proteomes.Among many different instrumental formats (5), the combination of a linear ion trap (6) with a Fourier transform (FT)1 mass spectrometer has rapidly become a popular technological platform in proteomics because it combines the sensitivity, speed, and robustness of ion traps with the high resolution capabilities of FT instruments. The first implementation of this principle used an ion cyclotron resonance instrument with a 7T magnet as the high resolution device (7). Later, the Orbitrap^TM analyzer developed by Makarov was coupled to the LTQ, combining the linear ion trap with a very small and powerful analyzer (8–11).Here we describe a next generation linear ion trap-Orbitrap instrument with significant improvements in ion source transmission and with a new ion trap configuration. We show that this instrument, termed the LTQ Orbitrap Velos, is capable of much higher scan speeds compared with the current LTQ Orbitrap. Furthermore, we implemented more efficient ion extraction for the higher-energy collisional dissociation (HCD) cell (12). Due to this improvement and the 10-fold higher transmission of ions from atmosphere, high resolution and high mass accuracy MS/MS can now routinely be obtained at very high sensitivity and at scan speeds of up to 5 Hz acquisition rates. A related instrument, the LTQ-Velos, which does not contain the Orbitrap analyzer for high resolution measurements, has been described very recently (13).     

Increased intranuclear matrix metalloproteinase activity in neurons interferes with oxidative DNA repair in focal cerebral ischemia

Increased matrix metalloproteinase (MMP) activity is implicated in proteolysis of extracellular matrix in ischemic stroke. We recently observed intranuclear MMP activity in ischemic brain neurons at early reperfusion, suggesting a possible role in nuclear matrix proteolysis. Nuclear proteins, poly-ADP-ribose polymerase-1 (PARP-1) and X-ray cross-complementary factor 1 (XRCC1), as well as DNA repair enzymes, are important in DNA fragmentation and cell apoptosis. We hypothesized that intranuclear MMP activity facilitates oxidative injury in neurons during early ischemic insult by cleaving PARP-1 and XRCC1, interfering with DNA repair. We induced a 90-min middle cerebral artery occlusion in rats. Increase activity of MMP-2 and -9, detected in the ischemic neuronal nuclei at 3 h, was associated with DNA fragmentation at 24 and 48 h reperfusion. The intranuclear MMPs cleaved PARP-1. Treatment of the rats with a broad-spectrum MMP inhibitor, BB1101, significantly attenuated ischemia-induced PARP-1 cleavage, increasing its activity. Degradation of XRCC1 caused by ischemic insult in rat brain was also significantly attenuated by BB1101. We found elevation of oxidized DNA, apurinic/apyrimidinic sites, and 8-hydroxy-2'-deoxyguanosine, in ischemic brain cells at 3 h reperfusion. BB1101 markedly attenuated the early increase of oxidized DNA. Using tissue from stroke patients, we found increased intranuclear MMP expression. Our data suggest that intranuclear MMP activity cleaves PARP-1 and XRCC1, interfering with oxidative DNA repair. This novel role for MMPs could contribute to neuronal apoptosis in ischemic injuries.     

Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg²⁺. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K⁺-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K⁺-flux is convergent with the gating of PIP₂ because, at a saturating concentration, PIP₂ greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K⁺-flux gating.     

Seasonal variation in the diet of Redshank Tringa totanus in the Odiel Marshes,southwest Spain: a comparison of faecal and pellet analysis

Capsule Redshank diet from southern Europe during migration shows spatial and seasonal variations.

Aims To assess seasonal variation in Redshank diet at a major passage site, and to compare data derived from analysing pellets or faeces.

Methods At the Odiel Marshes in 2001, pellets from spring migration (39), autumn migration (121) and midwinter (15) were analysed, together with faecal samples from autumn (84).

Results The abundance of different invertebrate groups in pellets varied between seasons. In spring, Chironomus salinarius pupae and larvae dominated by volume, followed by Ephydridae larvae and the beetle Paracymus aenus. Polychaetes and molluscs dominated in autumn, and isopods in midwinter. In autumn, chironomid larvae, Mesembryanthemum nodiflorum seeds and Artemia cysts were relatively more abundant in faeces, whereas polychaetes, isopods, molluscs and cestode cysticercoids were more abundant in pellets. Harder and/or larger items were thus relatively more abundant in pellets than faeces. Pellet analysis gave more emphasis to mudflat prey, and faeces to saltpan prey.

Conclusion Pellet and faecal analysis give different results for wader diet, and it is useful to combine the two methods. However, they show significant correlations both in diet range and rank abundance of prey items. Redshank diet shows much seasonal and spatial variation in southern Europe.     

Multiple cytokine expression profiles reveal immune-based differences in occult hepatitis B genotype H-infected Mexican Nahua patients

A high prevalence of occult hepatitis B (OHB) genotype H infections has been observed in the native Mexican Nahua population. In addition, a low incidence of hepatitis B virus (HBV)-associated hepatocellular carcinoma has been described in Mexico. The immune response to infection among OHB-infected patients has been poorly evaluated in vivo. Therefore, we assessed the expression profiles of 23 cytokines in OHB genotype H-infected Nahua patients. A total of 41 sera samples from natives of the Nahua community were retrospectively analysed. Based on their HBV antibody profiles, patients were stratified into two groups: OHB patients (n = 21) and patients that had recovered from HBV infection (n = 20). Herein, we report distinctive cytokines profiles in OHB-infected individuals. Compared to healthy controls (n = 20) and patients who resolved HBV infection, OHB-infected patients displayed an increase in interleukin (IL)-2 secretion in addition to a characteristic inflammation profile (decrease in IL-8 and tumour necrosis factor-alpha levels and increased levels of tumour growth factor-beta). IL-15 and interferon-gamma levels were reduced in OHB-infected individuals when compared to those patients who resolved HBV infection. In contrast, OHB patients showed an increase in monocyte chemoattractant protein (MCP)-1 and MCP-2 compared to healthy controls and patients who resolved HBV infection. These findings suggest that cytokine expression can influence the severity of OHB disease and could lead to new investigation into the treatment of liver and other infectious diseases.     

Coagulation/flocculation-based removal of algal-bacterial biomass from piggery wastewater treatment

Two conventional chemical coagulants (FeCl₃ and Fe₂(SO₄)₃) and five commercial polymeric flocculants (Drewfloc 447, Flocudex CS/5000, Flocusol CM/78, Chemifloc CV/300 and Chitosan) were comparatively evaluated for their ability to remove algal-bacterial biomass from the effluent of a photosynthetically oxygenated piggery wastewater biodegradation process. Chlorella sorokiniana, Scenedesmus obliquus, Chlorococcum sp. and a wild type Chlorella, in symbiosis with a bacterial consortium, were used as model algal-bacterial consortia. While the highest biomass removals (66-98%) for the ferric salts were achieved at concentrations of 150-250 mg L⁻¹, dosages of 25-50 mg L⁻¹ were required for the polymer flocculants to support comparable removal efficiencies. Process efficiency declined when the polymer flocculant was overdosed. Biomass concentration did not show a significant impact on flocculation within the concentration range tested. The high flocculant requirements herein recorded might be due to the competition of colloidal organic for the flocculants and the stationary phase conditions of biomass.     

The APC/C subunit 10 plays an essential role in cell proliferation during leaf development

The largest E3 ubiquitin-ligase complex, known as anaphase-promoting complex/cyclosome (APC/C), regulates the proteolysis of cell cycle regulators such as CYCLIN B and SECURIN that are essential for sister-chromatid separation and exit from mitosis. Despite its importance, the role of APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, the Arabidopsis thaliana APC/C subunit APC10 was characterized and shown to functionally complement an apc10 yeast mutant. The APC10 protein was located in specific nuclear bodies, most probably resulting from its association with the proteasome complex. An apc10 Arabidopsis knockout mutant strongly impaired female gametogenesis. Surprisingly, constitutive overexpression of APC10 enhanced leaf size. Through kinematic analysis, the increased leaf size was found to be due to enhanced rates of cell division during the early stages of leaf development and, at the molecular level, by increased APC/C activity as measured by an amplification of the proteolysis rate of the mitotic cyclin, CYCB1;1.     

A Co-Adaptive Brain-Computer Interface for End Users with Severe Motor Impairment

Co-adaptive training paradigms for event-related desynchronization (ERD) based brain-computer interfaces (BCI) have proven effective for healthy users. As of yet, it is not clear whether co-adaptive training paradigms can also benefit users with severe motor impairment. The primary goal of our paper was to evaluate a novel cue-guided, co-adaptive BCI training paradigm with severely impaired volunteers. The co-adaptive BCI supports a non-control state, which is an important step toward intuitive, self-paced control. A secondary aim was to have the same participants operate a specifically designed self-paced BCI training paradigm based on the auto-calibrated classifier. The co-adaptive BCI analyzed the electroencephalogram from three bipolar derivations (C3, Cz, and C4) online, while the 22 end users alternately performed right hand movement imagery (MI), left hand MI and relax with eyes open (non-control state). After less than five minutes, the BCI auto-calibrated and proceeded to provide visual feedback for the MI task that could be classified better against the non-control state. The BCI continued to regularly recalibrate. In every calibration step, the system performed trial-based outlier rejection and trained a linear discriminant analysis classifier based on one auto-selected logarithmic band-power feature. In 24 minutes of training, the co-adaptive BCI worked significantly (p = 0.01) better than chance for 18 of 22 end users. The self-paced BCI training paradigm worked significantly (p = 0.01) better than chance in 11 of 20 end users. The presented co-adaptive BCI complements existing approaches in that it supports a non-control state, requires very little setup time, requires no BCI expert and works online based on only two electrodes. The preliminary results from the self-paced BCI paradigm compare favorably to previous studies and the collected data will allow to further improve self-paced BCI systems for disabled users.