Various solution studies were conducted to address the discrepanc

Various solution studies were conducted to address the discrepancy in the quaternary structure of AK which revealed that the formation of the cooperative tetramer is possible upon effector binding [25] and [38]. Despite the fact that the enzyme had been crystallized in the absence of lysine, the structure reveals lysine bound form of CaAK which enable us to identify the key elements which are responsible for the large conformational changes associated with the inhibitor binding. The DynDom analysis clearly indentified the bending residues at the domain crossover regions (D208–L213

and E237–I250) in order to support the domain motion between MAPK inhibitor the regulatory and catalytic domains of CaAK ( Fig. 4A and B). The analysis provides the rotation angle of monomers B, D, E, I as 7.3°; 8.2°; 7.3° and 3.7°, respectively whereas no rotational angle was detected for the monomers C, F, G, H, J, K and

L when monomer A was used as the reference structure. Further rotational analysis on all combinations of monomers showed the rotational angle and the value lies between 4° to 8° between the monomers. The domain reorientation is mainly controlled by interaction between the residues K232, R235, E236, S238, Y239, H246 and E247 of catalytic domain and E303, L306, N308, V335, D336 and S337 of regulatory domains. The varied interaction is induced by either lysine binding at the homodimeric interface or nucleotide binding/release at the domain crossover regions. In order to support this observation, the relative reorientation of the domains is observed in different MjAK complex structures (PDB Ids 3C1N, 3C20 and 3C1M). The rotational angle varies between 6.3° and 18.9° and demonstrates the inhibitor, substrate and cofactor binding to mjAK induces the conformational changes

between the domains. Both the CaAK and MjAK structures have shortened latch loop regions (CaAK: E343–D348 and MjAK: S366–V370) and do not appear to play a role in conformational arrangements. In contrast, the crystal structures of EcAKIII solved in both R- and T-state conformation (PDB Ids 2J0X and 2J0W) demonstrated the largest rotation (∼36.3°) between the catalytic and regulatory domain. The critical latch loop (D354–T364) leading Bumetanide to the transition from R- to T-state and tetramer formation that undergoes major rotational rearrangements. The latch loop is well conserved in the structure of AtAK (D387–I397) appears to play a role in conformational rearrangements and tetermer formation similar to EcAKIII. The superposition of four ACT domains of CaAK dimer on the corresponding four ACT domains of dimeric structures of EcAKIII (PDB 2J0X and 2J0W with rmsd of 1.3 Å and 1.5 Å, respectively), AtAK (PDB 2CDQ with rmsd of 4 Å), MjAK (PDB 3 C1 M, 3 C1 N and 3C20 with rmsd of 2 Å; 1.9 Å and 1.8 Å, respectively) revealed that ACT domains adopt a similar conformation.

Values were reported as mean ± standard

error of mean (SE

Values were reported as mean ± standard

error of mean (SEM). Statistical significance was set as P < 0.05. Ang II injection induced a slight but consistent constriction in isolated mesenteric venules (Fig. 1A). No significant differences were observed between the responses of Wistar rats (10.6 ± 1.1 mmHg; n = 6) and SHR (10.6 ± 1.3 mmHg; n = 8). Basal perfusion pressure in mesenteric venous bed was not modified by pre-incubation with different antagonists. In SHR preparations, the constriction induced by Ang II was nearly abolished (P < 0.05) by perfusion with losartan (0.8 ± 0.2 mmHg; n = 7), while PD123319 and L-NAME had no effect at all. In contrast, Ang II venoconstriction increased (P < 0.05) after B2R blockade with HOE 140 (15.7 ± 1.6 mmHg; n = 8), and also after COX inhibition with indomethacin (16.8 ± 1.5 mmHg,

n = 6) or celecoxib (18.8 ± 1.4 mmHg, n = 5). The results are shown in Fig. 1B. Starting at 1 nmol/L, Ang II contracted rings of portal vein in a concentration-dependent manner. The Emax was reached at 50 nmol/L. At concentrations selleck compound higher than 100 nmol/L, Ang II induces rapid desensitization (tachyphylaxis) in this preparation (Fig. 2A). Fig. 2B shows the CCRCs to Ang II in both Wistar and SHR portal vein preparations. The Emax to Ang II was significantly reduced (P < 0.05) in SHR (0.62 ± 0.09; n = 6) compared to Wistar rats (1.00 ± 0.15; n = 6). No changes were detected in response to KCl (Wistar: 0.43 ± 0.07 g; n = 7 versus SHR: 0.31 ± 0.06 g; n = 8). Pre-incubation

of portal vein rings from SHR with losartan shifted to the right the CCRC to Ang II [Control: pEC50: 8.62 ± 0.05 mol/L; n = 6 versus Losartan: 7.95 ± 0.06 mol/L; n = 4 (P < 0.05)], whereas PD 123319 treatment had no effect ( Fig. 2C). Pre-incubation with indomethacin and HOE 140 increased the Emax to Ang II [Control: 0.57 ± 0.09 g, n = 8 versus Indomethacin: 1.21 ± 0.14 g, n = 7 and HOE 140: 1.01 ± 0.08 g, n = 11 (P < 0.05)], as demonstrated in Fig. DOCK10 2D. L-NAME and celecoxib did not alter the Ang II response (data not shown). To investigate a possible alteration in angiotensin receptor expression between SHR and Wistar rats, we quantified the levels of AT1R and AT2R mRNA in samples from portal veins. The results are shown in Fig. 3. While no differences were detected in AT1R expression, AT2R mRNA levels in the portal vein samples were significantly reduced in SHR [0.34 ± 0.13 arbitrary units (a.u.); n = 7; P < 0.05] compared to Wistar rats (1.05 ± 0.19 a.u.; n = 4) ( Fig. 3). Immunohistochemical assays revealed similar results. Fig. 4 contains representative images of immunohistochemical staining for AT1R and AT2R in SHR and Wistar rats. AT1R and AT2R were present in the endothelium, vascular smooth muscle cells, and adventitial layer. There was no difference in AT1R expression in SHR and Wistar rats, while AT2R expression was reduced in the portal veins of SHR (6.85 ± 0.50 a.u.; n = 5; P < 0.05) compared to Wistar rats (9.

The correct caption to Fig  7 should read: Double-label fluoresce

The correct caption to Fig. 7 should read: Double-label fluorescent immunohistochemistry of fos and tyrosine hydroxylase in the LC after IVth ventricular infusion of Vehicle (A, B, C), 6FNE 30 nmoles (D, ABT-199 manufacturer E, F), or TER 10 nmoles (G, H, I) in representative animals. Left column, TH images; middle column, fos images; right column merged images. Note in B some nonspecific

staining of cytoplasm of adjacent mesencephalic trigeminal nucleus neurons by fos antibody. Bar is 200 µ. “
“This corrigendum relates to the Results, Section 2.2 Rac1 association with long-term synaptic plasticity (page 82) as well as Fig. 5 (page 87). In this figure, the concentration of the drug was erroneous, and the controls were similarly published in a previous manuscript (Martinez and Tejada-Simon, 2011). It was indicated that analogous control points cannot be duplicated herein. Thus, two panels from

the original Fig. 5 have been removed and the previous publication referenced to support reported findings. Accordingly, the Experimental Procedures Section 4.9 Electrophysiology (page 93) has been also corrected. Results Section 2.2 Rac1 association with long-term synaptic plasticity (page 82) should read: Besides dendritic spine morphology, long-term plasticity has been shown to be also altered in FXS. CP-868596 cost We and others have suggested that Rac1 might be critical for these two phenomena (Haditsch et al., 2009; Martinez and Tejada-Simon, 2011). Thus, searching for a connection between Rac1 and FXS, we next studied whether Rac1 is involved not only in LTP but also in LTD, and whether Rac1 inhibition alters this type of plasticity. This is very relevant since an exaggerated LTD is one of the strongest phenotypes observed in Fmr1 knockout mice. In previous work by our laboratory, LTD was induced in hippocampal slices from wild-type mice treated with a Rac1 inhibitor, NSC23766 (Gao

et al., 2004). LTD was induced either with low frequency stimulation (LFS) delivered at 1 Hz for 15 min (Huber et al., 2001), or by treating the hippocampal slices with 100 μM DHPG for 5 min in the presence of the N-methyl-d-aspartate (NMDA) receptor antagonist d,l-2-amino-5-phosphonovalerate (d,l-AP5, 100 μM; Nosyreva and Huber, 2006). Thiamet G Application of NSC23766 to hippocampal slices of wild-type mice inhibited LTD regardless of the induction protocol (Martinez and Tejada-Simon, 2011). Herein, to confirm the involvement of Rac1, LTD was also induced in hippocampal slices from Rac1 mutant mice. Control slices derived from wild-type mice showed a significant lasting decrease in the fEPSP slope. However, slices derived from the Rac1 mutant mice were unable to sustain this response (Fig. 5). These results further suggest that Rac1 is required and also important for LTD. Experimental Procedures Section 4.9 Electrophysiology (page 93) should read: Transverse hippocampal slices (400 μm) were prepared from age-matched animals as described before.

This explains the poor knowledge of prey characteristics and seab

This explains the poor knowledge of prey characteristics and seabird diving behaviour within these habitats. Below, several methods that could provide these data are discussed. As hydroacoustic sonar methods can record both prey behaviour [92] seabird dives [103], [104] and [107] and predator–prey interactions [103] and [104] at fine spatiotemporal scales, a single deployment could provide much of the data needed to answer fundamental questions (Section

4.3). They also have several other benefits. Firstly, hydroacoustic sonar methods are unaffected by low light and high turbidity and therefore have advantages over others that can record underwater behaviours, such as video cameras. Secondly, they are also flexible in their application and can be deployed from vessels to target several micro-habitats within a survey [104], or from static moorings Selleck Pictilisib to monitor single micro-habitats over extended time periods [108], [109] and [110]. Having said this, hydroacoustic methods do have

some shortcomings when recording seabird dives as they cannot discriminate between species underwater. Moreover, the narrowness of sonar beams often makes collecting whole dive profiles difficult. However, having observers on vessels or alongside moorings during hydroacoustic sonar surveys can help to overcome identification problems [103], [104] and [107] whereas estimating dive depths is often possible by using trails of air bubbles that persist behind diving seabirds to trace their movements [104]. Combining several sonar beams to increase the overall coverage could also overcome these issues. In addition to the development of GPS loggers (see 2.4.3 and 3.4.4), there have also been developments

in time-depth recorders Nintedanib (BIBF 1120) (TDR) that record individuals′ subsurface movements. When GPS loggers and TDR devices are used in combination, they have the ability to record the location, depths and durations of foraging dives [55]. As devices are attached directly onto individuals at the nest site, dive profiles can also be attributed to species. The major limitation is that these methods are most suitable for Black Guillemots and Cormorants that usually forage within a few kilometres of their nest sites (see Section 3.4.4). As these species generally exploit benthic prey items [8], their dive depths are perhaps more predictable than those exploiting pelagic prey [8].

Of these, the NCEP-CFSR model detects only Lake Ladoga, presumabl

Of these, the NCEP-CFSR model detects only Lake Ladoga, presumably because of the sparser resolution of the model. The diurnal evolution of PW, with a 6-hour time step, is shown in Figure 3. At night, from 00 to 06 UTC, there is no change

in PW above the sea, but a decrease above the land is detectable. In the morning, from 06 to 12 UTC, PW decreases above the sea, but increases above the land, especially to the east of the Baltic Sea. In the afternoon, from 12 to 18 UTC, PW still decreases slightly above the water, except in the Gulf of Finland and on Lake Ladoga, where PW is already increasing, as is the case to the west of the Baltic Sea. In the evening, from 18 to 00 UTC, PW is increasing above the water, but PD-0332991 ic50 is mostly decreasing above the land. For the sake of comparison with previous studies (Bouma and Stoew, 2001 and Jakobson et al., 2009), shorter periods were also processed, but because of the insufficient number of data, the diurnal differences remained mostly insignificant (not shown), without any justifiable opportunity for making comparisons. To estimate the influence of different atmospheric layers on PW diurnal variation, the PW difference

between 18 and 06 UTC (dPW = PW18 UTC − PW06 UTC) was calculated, as this time interval usually gives the largest differences in PW. After that, the contributions to dPW from vertical intervals 900–1000 hPa, 800–900 hPa and 800–1000 hPa were calculated (Figure 4). Lower 100 hPa humidity diurnal variations affect PW diurnal variability more above the water than the land, while the 800 to 900 hPa interval affects it more above land than the water. Relatively speaking, ITF2357 nmr the regional average contribution to dPW was 25% in the interval 900–1000 hPa and 45% in the next 100 hPa layer. The 800–1000 hPa interval holds 70% of the dPW with a ca 20% larger contribution above the land than over the sea. Specific humidity

and temperature at 00, 06, 12 and 18 UTC differ from their diurnal average values at different vertical pressure levels and exhibit fundamental differences for the sea and the land (Figure 5). The results for BaltAn65 + and NCEP-CFSR (not Resveratrol shown) were similar at all vertical levels with respect to both specific humidity and temperature. The behaviour of specific humidity above 950 hPa is the reverse of that above the sea and the land. Above the sea there is less humidity at 12 and 18 UTC, while above the land the humidity is lower at 00 and 06 UTC. The situation regarding the specific humidity below 950 hPa is more complicated and will be analysed in the Discussion. Over land, temperatures are higher at 18 and 12 UTC and lower at 06 and 00 UTC. Diurnal variability in the temperature above the water is delayed for about 6 hours, compared to the variability above the land, with higher temperatures at 18 and 00 UTC and lower temperatures at 06 and 12 UTC, although the delay fades out above 850 hPa.

Such an account is congruent with recent evidence in rodents that

Such an account is congruent with recent evidence in rodents that stimulus-selective cells in medial OFC, unlike in lateral OFC, show a small but significant increase in firing to odours associated LDK378 cost with the least valuable option in a delay/reward decision task [56]. Lesions to an adjacent structure — prelimbic cortex — also

cause rats to fail to downregulate attention to a novel cue in a blocking paradigm even though it provides no new information to guide predictions and choice [57]. It will be interesting to determine whether a similar process of competition by mutual inhibition, which can successfully account for VMPFC value comparison signals and even the paradoxical effects of a distracting alternative 39•• and 52••], might be extended to generally

predict such a function. In this brief review, we have outlined ideas that suggest that OFC and VMPFC have key complimentary roles in selecting the appropriate information to allow appropriate value learning and value comparison to occur. OFC, through interactions with sensory cortex, can use stimulus-reward associations to enhance attention towards specific, task-relevant environmental PF-562271 molecular weight information, which in turn can allow rapid contingent learning when new information is acquired; VMPFC, with access to information about the current motivational goals, can help suppress irrelevant value information impinging on an ongoing decision. These regions clearly do not perform these functions in isolation (cf. [52••]) and it will be critical in the coming years 4-Aminobutyrate aminotransferase to investigate how these two networks cooperate to promote selection. This will also require a comparison between

OFC and VMPFC signals with interconnected brain areas 14, 24•, 48 and 52••], examining interactions between structures 52••, 58 and 59], and particularly looking at how interference in one part of the network affects coding elsewhere 27 and 60]. Moreover, understanding the way in which these or other regions determine current task relevance and gather information in a dynamic setting is of primary importance 61 and 62]. Nothing declared. Papers of particular interest, published within the period of review, have been highlighted as: • of special interest MEW is supported by a Wellcome Trust Research Career Development Fellowship (090051) and SWK by a Wellcome Trust New Investigator Award (096689). Many of the ideas in this article were initiated through work with Matthew Rushworth, Jonathan Wallis, Tim Behrens and MaryAnn Noonan, as well as from lengthy discussions with Laurence Hunt, Erie Boorman, and Nils Kolling. “
“Current Opinion in Behavioral Sciences 2015, 1:86–93 At the core of most vision research is implicitly or explicitly a hierarchical and feedforward model, in which visual processing proceeds from the analysis of basic features to more and more complex ones (e.g. [1••]).

Patient studies, too, would appear to support this interpretation

Patient studies, too, would appear to support this interpretation. Deficits for processing tool concepts and words are associated with frontoparietal sensorimotor systems (Gainotti, 2004 and Gainotti et al., 1995) and deficits for animals with occipitotemporal regions (Hart and Gordon,

1992 and Tranel et al., 1997). These dissociations appear to be underpinned by the dissociation between action- and perception-related knowledge, with manipulability and other action-features most relevant for tools, and visual-features such as colour and form most relevant for animals. More recent work with stringent psycholinguistic Selleck MAPK inhibitor matching has revealed relative impairments for action-word processing in a range of neurological diseases and disorders characterised by motor impairment (Bak et al., 2001, Bak et al., 2006, Boulenger et al., Cobimetinib in vitro 2008, Cappa et al., 1998, Cotelli et al., 2006 and Moseley et al., 2013). Importantly, deficits in processing action language, associated with lesions to inferior frontal and motor systems, are accompanied by concordant deficits in semantic processing of actions in nonverbal tasks ( Bak et al. 2006). This pattern of deficits provides further evidence for a semantic rather than grammatical basis of category-specific semantic and conceptual disorders, a position reached by two recent reviews of the literature ( Kemmerer et al., 2012 and Kiefer

and Pulvermüller, 2012). The conclusions drawn in the present paper are consistent with previous works but avoid some of the methodological pitfalls evident in the same. Vigliocco et al. (2006), as in the current paper, reported brain dissociations between sensory and motor words but no distinctions on the basis of lexical category. Problematically, this study used Italian nouns and verbs sharing the same stem but differing in Pregnenolone their affixes, which immediately inform the reader of the word’s lexical category. The co-occurrence of verb affixes with verb stems (used to speak

about actions) and the co-presence of noun affixes with nouns (related to objects) appears to indirectly load the neuronal circuit of affixes with semantic links (Pulvermüller & Shtyrov, 2009). The study also suffered from poor stimulus matching, such that apparent dissociation between motor and sensory words might also be explained by differences in familiarity, imageability and age of acquisition (see, for example, Hauk et al., 2008). Other electrocortical dissociations on the basis of both lexical and semantic distinctions were reported by Kellenbach et al. (2002) and Barber, Kousta, Otten, and Vigliocco (2010). Whilst these could not be localised to specific brain regions in the former, the latter argued that, as both differences showed the same N400 topography, they might both best be explained in terms of a semantic effect ( Barber et al., 2010).

Por outro lado, os níveis séricos de vitamina B12 não foram um pr

Por outro lado, os níveis séricos de vitamina B12 não foram um preditor da presença de hHcys. É importante sublinhar que as reservas de vitamina B12 são geralmente suficientes

para 3‐4 anos, mesmo que todas a fontes desta vitamina sejam suprimidas o que poderá explicar, em parte, o reduzido número de doentes com défice de vitamina B12 no presente estudo. Uma associação entre a presença de hHcys e os níveis elevados de proteína C reativa foi previamente reportada numa série de 106 doentes com DII30. No entanto, no nosso estudo não foi encontrada nenhuma associação entre a hHcys e este marcador bioquímico de inflamação, sendo este achado corroborado por outros estudos31. Os aminossalicilatos têm sido implicados na má absorção de ácido fólico e hHcys em doentes com DII32. No presente estudo não se observou qualquer efeito selleckchem do tratamento (aminossalicilatos, corticosteroides, azatioprina, biológico) nos níveis de homocisteína. No nosso estudo, a idade jovem foi apenas um preditor marginalmente significativo da presença de hHcys. A relação entre a idade e os níveis de homocisteína foi previamente reportada see more em

outros estudos20 and 33, no entanto, o verdadeiro mecanismo subjacente a esta alteração não se encontra definido na literatura. Vários fatores poderão estar subjacentes a estes achados, nomeadamente o consumo de álcool, o tabagismo e os diferentes padrões de ingestão alimentar. O tabagismo tem uma conhecida associação com níveis elevados de homocisteína séria34 and 35 e com a ocorrência de eventos tromboembólicos. No presente estudo, o tabagismo foi um fator associado com a presença de hHcys (p < 0,001). Vários mecanismos poderiam explicar o aumento do risco tromboembólico

GNA12 em fumadores com hHcys. Fumar interfere com múltiplos mecanismos vaso‐oclusivos, tais como a agregação plaquetária, a viscosidade do plasma e os níveis de fibrinogénio36. Também a hHcys tem sido associada com alterações da função endotelial e do fluxo sanguíneo37 and 38. O fato de ambos os fatores de risco poderem exercer efeitos semelhantes, sugere um forte potencial de interação entre eles no sentido de produzir dano vascular. Estudos retrospetivos demonstraram 1,3‐6,4% de complicações tromboembólicas em doentes com DII1 and 23. No nosso estudo encontramos uma alta prevalência (5/47; 10,6%) de eventos tromboembólicos neste grupo de doentes, no entanto, não foi observada uma correlação estatisticamente significativa entre a presença de complicações tromboembólicas e os níveis séricos de homocisteína. Apesar da elevada prevalência de eventos tromboembólicos na nossa população em estudo, o número de casos foi ainda pequeno para fornecer conclusões seguras, embora se encontre descrito que elevados níveis de homocisteína podem predispor os doentes com DII para complicações tromboembólicas em combinação com outros fatores de risco circunstanciais ou permanentemente existentes39.

The test section of this tunnel is rectangular with a length of 2

The test section of this tunnel is rectangular with a length of 2.6 m, a width of 0.6 m and a height Selleckchem PD0325901 of 0.6 m. The maximum flow speed is 12 m/s, and the pressure can vary from 10 to 200 kPa. A schematic diagram of the MOERI medium-sized tunnel is shown in Fig. 7. A wake screen composed of a brass wire mesh was made to reproduce the nominal wake flow measured

behind the model ship in the MOERI towing tank. The propeller configurations and the nominal wake distributions measured at the propeller plane inside the cavitation tunnel are shown in Fig. 8. The pressure fluctuation is measured on a flat plate above the model propeller. The flat plate is away from the model propeller tip, which corresponds to the vertical clearance selleck chemical of the hull. Pressure transducers, model XTM-190-25A, were used to measure the pressure values. The computation and the five measured positions on the plate are shown in Fig. 9. Using the method recommended by ITTC (1987), the full-scale pressure fluctuation amplitudes can be predicted from the model scale measurement according to the following formula. equation(9) PS=PM×ρSρM(nSnM)2(DSDM)2fSfM=nSnMwhere ρρ is the density, nn is the rotational speed, and D is the diameter; suffix S represents the ship, and M represents the model.

The cavitation patterns of the model propellers are obtained for the selected blade′s angular position, and the corresponding numerical flow analysis results are shown in Fig. 10, Fig. 11 and Fig. 12. The angular positions of a key blade shown in these figures are measured from the vertically upward position in a clockwise direction when the propeller is viewed from behind. Fig. 13 shows the computed sheet cavitation volume variations. Fig. 14, Fig. 15 and Fig. 16 are the comparison results. The experimental result, the potential-based prediction results, and the results of the newly developed time domain prediction method are compared at positions ‘P’, ‘C’, and ‘S’. As shown Table 4 and Fig. 17, the maximum value of the pressure fluctuation is experimentally measured

and numerically predicted at a slightly starboard side of the propeller. There are two reasons. The first reason is that sheet cavitation volume is occurred analogously symmetric shape whose maximum volume is located slightly starboard side as shown in Fig. 13. The second reason is Bumetanide the source movement. Because sources are moving from port side to starboard side, induced pressure fluctuation at the starboard side is higher than that of port side. The newly developed time domain prediction results show good agreement with the experimental results, and the developed method is qualitatively and quantitatively superior to the potential-based prediction method. A new time domain prediction method has been presented with the aim of computing the pressure fluctuation induced by a propeller sheet cavitation. Modern acoustic theory is applied to the source modeling of the pressure fluctuation.

11); third, solute interactions To reduce the dimensionality of

11); third, solute interactions. To reduce the dimensionality of the model, we allowed an increase in L to include solute interactions ( Suppl. Section 2.12). We combined these effects by allowing σ and L to vary, modeled in Fig. 2c, and fitted multiple peaks to each dataset ( Suppl. Section 2.13). Overlapping peaks in our results could represent TCEP; non-helical peptide; N- to C-terminal cyclic cross-linked non-helical peptide monomer; cross-linked non-helical peptide dimers; cross-linked cyclic dimeric peptide; peptide triple helix; cross-linked triple helix dimers; small groups of (∼3–5) cross-linked triple helices; and larger groups of (6+) cross-linked triple helices. The last four classes are heterogeneous

NVP-BEZ235 research buy and could not be fully resolved, so it was decided to fit a peak representing a variety of molecule sizes, for instance, grouping all 3–5 helix aggregates under one fitted peak. The deconvolution was used to present data giving the percentages of peptide in each peptide form (Fig. 3, Fig. 4 and Fig. 5). Peptide samples were desalted by adsorbing to a preconditioned

μC18 Ziptip (Millipore). For electrospray, they were eluted with 70% MeOH/0.2% formic acid, and delivered to the mass spectrometer by direct infusion at 4 μL min−1 using 70% MeOH/0.2% formic acid as mobile phase, with a capillary temperature of 80 °C. Internal calibration data was also collected using either ubiquitin or myoglobin. For MALDI (Waters MicroMX), they were washed with 0.1% trifluoroacetic acid and eluted with matrix solution, mixed with ferulic acid matrix (10 mg mL−1 in 50% acetonitrile, 0.1% trifluoroacetic acid), dried and washed with 0.1% trifluoroacetic acid. To confirm

the redox state of peptide samples and next a TCEP-reduced negative control, peptides were alkylated using 120 mM iodoacetamide (Sigma I6125), pH 8, for 30 min at room temperature before analysis [31]. Blood from healthy volunteers was collected into 40 μM d-phe-pro-arg-chloromethylketone (PPACK, Cambridge Bioscience, UK), and supplemented hourly with 10 μM PPACK. It was incubated with 1 μM 3,3′-dihexyloxacarbocyanine iodide (DIOC6, Sigma–Aldrich, UK) for 15 min before use. Acid-cleaned coverslips (Menzel-Glazer, Germany) were washed in a solution of 1 M HCl in 50% ethanol, followed by two washes with 300 mM NaCl and a final wash with water. Base-treated coverslips were washed finally with 1 M NaOH. These coverslips were incubated with a mixture of two peptides (100 μg mL−1 each) in a humidity chamber overnight. The peptide mixture was either CRPcys and GFOGERcys or the cysteine-lacking CRP and GFOGER (Table 1). After removal of excess fluid, coverslips were blocked with 1% BSA in HEPES buffer (36 mM NaCl, 2.7 mM KCl, 5 mM HEPES, 10 mM glucose, 2 mM MgCl2, 2 mM CaCl2, pH 7.4) for 15 min. Individual coverslips were loaded into a 125 μm deep flow chamber mounted on an FV300 laser-scanning confocal microscope (Olympus, UK) and washed for 1 min with HEPES buffer.