, 2008) we also observed high CK serum levels in both strains wit

, 2008) we also observed high CK serum levels in both strains within 3 h of injury. Furthermore, these histomorphometric and sacolermal permeability analysis after 24 h of injury confirms that the delay in muscular regeneration between mouse strains was not due to acute tissue damage induced by B. jararacussu venom. Endogenous danger signals activate Toll-like receptors (TLR 2, 4, and 9) and induce homeostatic or harmful responses, depending on the physiological context, thus explaining contradictory reports showing that TLR4-deficient mice develop harmful noninfectious lung inflammation (Zhao et al., 2010), but not in the model of cardiac ischemia (Zhao et al., 2009) or brain injury (Caso et al., 2007).

In the Selleck Alpelisib skeletal muscle injury model with cardiotoxin it was suggested that

TLR3 may exert a protective role in muscle regeneration (Mathes and Lafyatis, 2011). MyD88 is utilized by most TLRs with exception of TLR3 that learn more utilizes TRIF to activate the NF-κB pathway and IRF3 pathway. TLR4 utilizes MyD88 adapter molecule to activate the NF-κB pathway and TRIF adapter molecule to activate the IRF3 pathway inducing production of proinflammatory cytokines (McGettrick and O’Neill, 2010). In the noninfectious lung inflammation, the TLR4 anti-inflammatory signaling is dependent upon a MyD88-independent pathway (Zhao et al., 2010). C3H/HeJ mice used in the present study have a mutation in the cytoplasmic domain caused by substitution of a proline residue for histidine at position 712 in the TLR4 polypeptide chain that halts the activation of both signaling pathways (Poltorak et al., 1998). TLR4-deficient mice showed 10-fold more F4/80-positive macrophages in the injury site in comparison with wild-type mice in 10 DPI, suggesting that such persistence is associated with delayed transition to the early differentiation stage of myogenesis. Delayed muscle repair observed in our study suggests

that TLR4 plays a protective role in muscle regeneration although further studies with knockout mice (MyD88−/− and TRIF−/−) are necessary to determine main signaling pathway involved in the skeletal injury induced by intramuscular injection of B. jararacussu venom. Edema formation and influx of inflammatory cells with subsequent loss of muscle Fossariinae mass during later stages of tissue regeneration is regarded as a critical event of venom poisoning caused by snakes of the Bothrops genus (Barbosa et al., 2008; Doin-Silva et al., 2009). The edematogenic effect is related to widespread damage in the local microvasculature due to release of venom proteases (Escalante et al., 2011; Neto and Marques, 2005). Edema formation as evidenced by increased muscle mass was consistently observed in both TLR-deficient and wild-type mice up to 3 days after venom extract injection. Nonetheless, TLR4-deficient mice showed a significant increase in edema formation comparing to TLR4 wild-type mice, which was an indication that TLR4 probably control mechanisms related to edematogenic effect.

, 2003) and the automated anatomical labelling (AAL) atlas ( Tzou

, 2003) and the automated anatomical labelling (AAL) atlas ( Tzourio-Mazoyer et al., 2002) of SMA, angular gyrus, insula, superior frontal medial cortex and precuneus. The resulting statistical maps were overlaid onto a normalized T1-weighted MNI template (colin27) and the coordinates reported correspond to the MNI coordinate system. The repeated-measures ANOVA revealed a significant main effect for agency [F(1,17) = 5.96, p < .05] with participants giving significantly shorter interval estimates in the active compared to the passive condition on the judgement error. There was

an unsurprising significant main effect of delay [F(2,34) = 16.49, p < .001] with more pronounced underestimation of the action–effect interval at longer delays. There was also a significant interaction of both factors [F(2,34) = 6.48, Alectinib purchase p < .01] ( Fig. 1). This interaction arose because selleck chemical the difference in judgement error between active and passive conditions increased with action-tone delay. The interval estimation task was analysed by parametrically modulating the action onset

with the judgement error of the action-tone interval. We then contrasted the active with the passive condition based on the fact that the active condition should involve a shortening of the awareness of the interval, whereas the passive condition should not. This analysis identifies brain regions that correlate with the compression of the action-tone interval more strongly in the active than in the passive condition. The previous literature gave strong predictions about involvement of specific regions in the experience of agency: the angular gyrus and the SMA (see Etofibrate Introduction) but also insula, frontomedian cortex and precuneus. Therefore we used a small volume correction within anatomically defined mask of these structures of interest. We identified a significant cluster within the SMA ROI family-wise error corrected p < .05 after small volume correction at −11, −8, 74 (cluster

size = 7 voxels) ( Fig. 2). Closer inspection confirmed that the cluster was located in left BA6, effectively on the margin between the lateral portion of BA6 comprising the dorsal premotor cortex, and the medial portion comprising the SMA proper. We applied a similar small volume correction to the bilateral angular gyrus, insula, frontomedian cortex and precuneus ROI, but found no significant difference between the parametric regressors for the active and passive conditions (nor in the reverse contrast) surviving correction within these areas. A whole brain analysis of the contrast between the parametric regressors for the active and passive condition with a statistical criterion of p < .001 uncorrected for multiple comparisons again revealed the same SMA cluster at −11, −8, 74, but no other significant clusters. Further, no significant clusters of activation were found in the reverse contrast.

Broad-scale maps exist for some taxa such as fish (e g , Froese a

Broad-scale maps exist for some taxa such as fish (e.g., Froese and Pauly, 2013), but coverage is generally poor for seamounts. Seamounts are visited by large pelagic vertebrates like tunas, billfishes, sharks, marine mammals, turtles, and seabirds (see Pitcher et al., 2007), and are important spawning areas for deep-water fishes (Clark, 2008). Fisheries data are often available at national or regional scales, and will likely be useful for evaluating this criterion. This criterion defines crucial habitats for endangered, threatened or declining species, or areas with significant assemblages

of such species; conservation of these habitats supports restoration or recovery of threatened species (CBD, 2009a). The primary data source for evaluating this criterion is the IUCN Red List (http://www.iucnredlist.org/), OSI-744 with additional data provided by national lists (e.g., Freeman et al., 2010 for New Zealand species). While these lists often Ribociclib supplier do not include location information, they serve to identify records in global or national databases that contain geo-referenced species records (e.g., OBIS www.iobis.org, Seamounts Online seamounts.sdsc.edu/). This criterion defines areas that contain a relatively high proportion of sensitive habitats, biotopes or species that are functionally fragile or with slow recovery (CBD, 2009a). Maps of vulnerable species

and habitats are Cediranib (AZD2171) the primary data for evaluating this criterion. Cold-water corals are particularly fragile and recover very slowly, and maps exist that either show the known distribution of such corals (Rogers et al., 2007), or the distribution of suitable coral habitat predicted by models (e.g., Davies and Guinotte, 2011 and Yesson et al., 2012). Other data sources include FAO or RFMO records of taxa that may characterise Vulnerable Marine Ecosystems (VMEs) (which often include corals as defining species) (FAO, 2013), and the sensitivity of corals to aragonite saturation depth (e.g., Tittensor et al., 2010). Habitat

suitability models for corals have been used with specific reference to seamounts (Tittensor et al., 2009) and for assessments of the vulnerability of seamounts to fishing impacts (Clark and Tittensor, 2010). This criterion defines areas containing species, populations or communities with comparatively higher productivity (CBD, 2009a). Oceanographic conditions, depth, and topography can play important roles in determining the location and magnitude of productivity. Areas of current mixing (e.g., frontal zones) and upwelling can increase surface productivity (Rivas, 2006), as can particular topographic features that may alter circulation characteristics locally, trap plankton, and attract predators (e.g., Genin and Dower, 2007, Kaschner, 2007 and Thompson, 2007).

Expressing snail or slug also suppressed E-cadherin but up-regula

Expressing snail or slug also suppressed E-cadherin but up-regulated

Etoposide mw fascin ( Figure 3D and Supplementary 6A). Knockdown of slug reduced fascin expression ( Supplementary Figure 6B), and stable expression of twist ( Figure 3C and D and Supplementary Figure 6A) or transient knockdown of zeb1, zeb2, or E-cadherin, did not change fascin levels ( Supplementary Figure 6C). Knockdown of fascin did not affect slug expression ( Supplementary Figure 6C). These observations were confirmed in 061843 PDAC cells ( Supplementary Figure 6D and E). Slug mediates fascin expression in PDAC cells. In addition, expression of slug or snail in human pancreatic cancer cells PANC-1 and human colon cancer cells HT29 induced fascin expression ( Supplementary Figure 6F), suggesting a general effect of slug and BAY 73-4506 chemical structure snail on fascin expression in both mouse and human cancer cells. We next investigated expression of fascin and slug during EMT changes

in KPC PDAC tumors. Interestingly, fascin and slug were both absent from ductal and acinar cells in normal pancreas and PanIN1/2 lesions (Figure 4A). Slug was expressed in fascin-positive (but not negative) PanIN3 lesions ( Figure 4A), indicating a correlation between early markers of EMT and fascin expression during PDAC progression. Fascin and slug were present in all PDACs, regardless of E-cadherin staining or differentiation status ( Figure 4B). In addition, fascin expression significantly correlated with slug expression in 3 independent cohorts of pancreatic cancer patients ( Supplementary Figure 7). We propose that slug-induced EMT is an important regulator of fascin expression in pancreatic cancer. Given the induction of fascin by slug and their tight association in human and mouse pancreatic cancer, we set out to determine whether fascin is a direct transcriptional target of slug. We screened the promoter and first intron region of mouse fascin for slug-binding E-box sequences (CACCTG or CAGGTG).26 We found a potential E-box sequence CACCTG located within the first intron of the

mouse fascin gene at +2470 to +2475 bp (Figure 5A). This consensus E-box sequence is highly conserved among mammalian fascins ( Figure 5A). We designed 3 sets of primers around the putative E-box sequence: primer ID-8 set 1 targets the identified E-box, while primer sets 2 and 3 target adjacent regions ( Figure 5A). Slug co-precipitated with the putative fascin E-box element ( Figure 5B). Cotransfection of the +2345 to +2600 region of the fascin first intron in a luciferase reporter plasmid with a plasmid expressing slug into 070669 PDAC cells drove a significant increase in luciferase activity ( Figure 5C). Mutagenesis of the E-box sequence eliminated the ability of slug to induce luciferase activity ( Figure 5C). We propose that fascin is a direct transcriptional target of slug. We next explored the hypothesis that fascin was a driver of invasion and metastasis in PDAC.

The plasma NO levels were evaluated by NO derivatives nitrate and

The plasma NO levels were evaluated by NO derivatives nitrate and nitrite,

as previously described [28]. Blood samples were collected into EDTA-coated tubes and plasma was immediately separated by low-speed centrifugation (1500 × g). The concentration of nitrate in blood was determined by chemiluminescence, elicited by the reaction of NO with ozone after nitrate reduction with VCl3 saturated solution in 1 mol/L HCl, at 90 °C, using a NO analyzer (NOA™280 Sievers Instruments Inc., Boulder, CO, USA). Nitrite was determined after reduction with 1% KCl solution in glacial acetic acid to convert nitrite to NO. Basal NO in mesenteric arterioles was determined by using a fluorescent cell permeable dye for NO, 4,5 diaminofluorescein diacetate (DAF-2 DA, Alexis, USA), as previously described [10].

Once inside the cell, DAF-2 DA is hydrolyzed by cytosolic Bleomycin esterases thus releasing DAF-2. The reaction between DAF-2 and NO yields the corresponding bright green-fluorescent triazolofluoresceins (DAF-2T). The mesenteric arterioles were dissected, immersed in medium for cryosectioning and cut AZD6738 molecular weight into 10 μm thick sections (Leica CM 1850 cryostat, Leica Instruments, Germany). In order to stimulate NOS activation and provide optimal levels of substrate, slices were pre-incubated with phosphate buffer (PB) solution containing CaCl2 (0.45 μmol/L) and l-arginine (100 μmol/L) during 30 min at 37 °C. Slices were washed, incubated with PB containing DAF-2 DA (10 μmol/L) for 30 min at 37 °C and observed on a microscope (Axiovert 100 M – Carl Zeiss SMT, Germany) equipped with fluorescein filter (excitation at 488 nm and measuring emission at 515 nm). Fluorescence emitted in response to NO production was quantified

through optic densitometry (arbitrary units, a.u.) using the AxioVision 4.8. digital images analysis software (Carl Zeiss). The semi-quantitative analysis of basal NO production was determined, at least, in three slices from each animal. Significant auto-fluorescence Vitamin B12 was discarded by experiments performed in the absence of DAF-2DA. NOS activity was measured by the biochemical conversion of l-[3H] arginine to l-[3H] citrulline according to the method described by Rees et al. [33]. Mesenteric vessels were dissected, washed, homogenized in ice-cold buffer and stored at −80°C. On the day of assay, homogenates were incubated (37 °C/60 min) in a buffer containing FMN and FAD 4 μmol/L, calmodulin 10 μg/mL, Ca2+ 1.25 mmol/L, NADPH 1 mmol/L, l-arginine 120 nmol/L, l-[3H] arginine 50 nmol/L (NEN Life Science Products, USA) and BH4 10 μmol/L. For the determination of iNOS activity, experiments were performed in the absence of Ca2+. Reaction was terminated by the addition of cation-exchange resin (Dowex 50WX8-400) to remove the excess of substrate.

For north-westerly and south-easterly winds states of stagnation

For north-westerly and south-easterly winds states of stagnation appear. In the process of evolution the ecosystem of the North Sea has become adapted to these current regimes. Climate change could, in turn, disturb the marine ecosystem. The wind further controls the spectrum of sea waves in the North Sea, and storms can lead to heavy and http://www.selleckchem.com/products/CAL-101.html dangerous storm surges. The atmosphere influences the heat budget of the North Sea via the heat fluxes and their variability. A

thermal stratification is generated in the northern and central parts during early summer (see Figure 5) and remains up to early autumn, when stronger winds mix the water again. There is no thermocline in southern coastal waters throughout the year as a result of strong tidal mixing. Precipitation on the north-west European shelf influences the salinity of the North Sea and its seasonal variability directly or via continental discharge. Temperature and salinity determine

the density of the sea water and the structure Maraviroc cell line of the water masses. The corresponding thermohaline circulation exhibits a cyclonal current pattern as well. The open connection with the Atlantic (mainly through the northern entrance, less so through the English Channel) allows the free exchange of momentum, energy and matter between the two seas. Planetary waves generated by astronomical and atmospheric Tyrosine-protein kinase BLK forces in the ocean penetrate over the shelf break into the North Sea, where they produce tides and water mass transports. In contrast, continental fresh water discharges (specifically the Baltic outflow) influence the current system of the North Atlantic. Figure 6 depicts a 40-year time series of net outflows from the North Sea with an average of about 2 Sverdrups. The decadal variability of the Atlantic, mainly the North Atlantic Oscillation (NAO), is transferred to the North Sea. Figure 7 illustrates the wind- and thermohaline driven circulation in the North Sea for two different NAO indices (NAOI) as a result of a model simulation. It

is obvious that a positive winter NAOI causes a significantly stronger flow than a negative one. The transfer of NAO variability to the North Sea happens mainly through the atmosphere, less through the direct exchange of water masses. This can be inferred from the correlation pattern of the NAOI and SST anomalies in the North Sea (see Figure 8). The high values in the central North Sea indicate this interrelation. The dynamics of the North Sea is significantly influenced by astronomical tides. These are co-oscillations with the autonomous tidal waves of the Atlantic (the North Sea is too small for a direct effect of the tidal potential). The specific geometry of the North Sea basin implies eigen-periods and hence resonance in the semidiurnal spectral range (see Figure 2).

The first page of the intervention is entitled “Test Your Knowled

The first page of the intervention is entitled “Test Your Knowledge” and consists of four true or false questions on the use of the benzodiazepines. The second page lists the correct answers. Elements of constructivist learning theory are incorporated into the answers to create selleck products cognitive dissonance and challenge the patient’s beliefs for each incorrect answer. The third page incorporates self-assessment and education about potential inappropriate

use, side effects, drug-drug interactions and information about physiologic changes that occur with age that affect drug metabolism. The fourth and fifth pages present evidence-based risks associated with benzodiazepine use in the elderly and suggestions for equally or more effective therapeutic substitutes. The sixth page describes a case scenario highlighting one woman’s success at weaning herself off benzodiazepines. The last page outlines a simple 21-week tapering program. The reader is encouraged on four occasions and is warned

in large, red lettering to “Please Consult your Doctor or Pharmacist Before Stopping Any Medication. The tool was field-tested with a convenience sample of older adults to determine selleck chemical the readability and comprehension of the information. Six focus-groups (n = 60 adults) were conducted. Based on the focus group discussions, the wording, ordering of the material and visual presentation of the intervention was changed in an iterative process until acceptability was reached. The final educational intervention consisted of a seven-page

letter-size paper brochure written in 14-point font. The educational tool was mailed to the study participants within six months of the initial assessment. The primary Astemizole outcome was a self-reported change in perception of risk associated with benzodiazepine use one week post-intervention. Participants were asked whether they perceived the same, increased, or no risk from consumption of their benzodiazepine following the intervention. A common idea in models of risk perception is that risk is perceived from two dimensions: the first being knowledge about the risk, and the second, beliefs about that risk [20]. To explain changes in perception of risk we therefore measured changes in knowledge and beliefs about medications as a mechanism through which cognitive dissonance could occur. Change in knowledge was measured by comparing the pre-intervention and post-intervention answers from the four-item true or false questions listed in the “Test Your Knowledge” section of the questionnaire. The first statement on the safety of long-term benzodiazepine was “(Example: Ativan®)…is a mild tranquilizer that is safe when taken for long periods of time”. The second statement focused on side effects and was worded, “The dose of Ativan® that I am taking causes no side effects.

Using observations of the densest waters found within the fjord d

Using observations of the densest waters found within the fjord during 1981 to 2002 ( Skogseth et al., 2005b) we vary the inflow salinity Apoptosis Compound Library cell assay S from 34.75 to 35.81. The flow rate Q is varied from 0.01 to 0.08 Sv, based on observations at the sill of a mean volume transport of 0.05 to 0.08 Sv ( Schauer and Fahrbach, 1999, Skogseth et al., 2005a and Geyer et al., 2009). In the present study we do not attempt to model the dense water formation process itself. The flow rate Q and the salinity S of the simulated overflow waters are intended to capture the parameters of the SFOW behind and at the sill. We employ the NEMO-SHELF model (O’Dea et al., 2012) at 1 km resolution

with a 109×109109×109 grid in the horizontal and 42 levels in the vertical. The baroclinic time step is 40s with time splitting for the barotropic component every 20 steps. O’Dea et al. (2012) describe in detail the modifications to NEMO (Madec, 2008) for use in shelf seas and regional studies. We include here only a brief summary of the differences as well as its configuration specific to this study and SCH772984 mouse our own modifications to the NEMO-SHELF code. A key departure of the NEMO shelf code from the open ocean is the use of a terrain-following s  -coordinate discretisation in the vertical instead of z  -coordinates.

The s  -coordinate system is well suited to the modelling of density currents (see e.g. Wobus et al., 2011), but the horizontal boundaries

between ambient layers ( Fig. 2(b)) would suffer numerical diffusion over areas of sloping topography where s  -levels intersect the isopycnals at an angle. We therefore modify the vertical coordinate system because neither the traditional s  -coordinate nor z  -coordinate systems suit our scenario where strong gradients are orientated vertically (in the ambient water) and also normal to the slope (at the upper plume boundary). The approach of blending s  - and z  -coordinates in this study can be traced back to Enriquez et al. (2005) who used a traditional s  -coordinate stretching function ( Song and Haidvogel, 1994) but achieved horizontal s  -levels over the interior of a basin by capping its bathymetry. Ivanov (2011) changed the traditional s  -coordinate formulation O-methylated flavonoid by introducing virtual seabeds at certain depth levels to maintain horizontal s  -levels closer to the slope. The levels designated as virtual seabeds (here called “shsh-levels”) follow the terrain only at shallower depths, while maintaining a prescribed depth over deep bathymetry. Our modified shsh-coordinate system1 refines the Ivanov (2011) approach by smoothing the transition between horizontal and terrain-following s-levels ( Fig. 3). The smoothing reduces errors in the calculation of the second derivative of the s-level slope. In this study we reserve 16 out of the 42 levels for a bottom layer of constant thickness (60 m).

Six studies [19], [32], [33], [35], [38] and [42] reported on die

Six studies [19], [32], [33], [35], [38] and [42] reported on dietary

outcomes; two [33] and [38] had positive effects. Venetoclax Thirty-six intervention features were included in the analysis, of which 11 were associated with a positive rate difference (see Table 2). Refer to the online supplemental data for more information on percent success rate differences (Table 3) and analysis of features within each individual outcome (Tables 4–7). DSME programs are complex interventions with various content and delivery components necessary for the education and skills building required for diabetes self-management. However, limited efforts have been made to investigate which intervention features are associated with a positive outcome, specifically for women of diverse ethnic backgrounds. Studies mainly concentrated on glycemic control (i.e., HbA1c levels) (10 studies) or anthropometric outcomes (11 studies), as opposed to behavioral outcomes such as diet (5 studies) and physical Selleckchem CHIR 99021 activity (5 studies). Since

behavioral outcomes strongly reflect the lifestyle changes needed to achieve the desirable metabolic outcomes [18] and [44], it is imperative to understand how intervention features affect these intermediary outcomes as well. Only five (of 38) intervention features had positive success rate differences for at least three of the outcomes examined in this review: hospital-based intervention setting; group intervention format; situational problem-solving; high intensity (10 or more intervention sessions); and incorporating dietitians as interventionists. Because of their broad influence, we recommend the features

that demonstrate success across multiple outcomes in DSME programming for the populations of interest. Many of these features are also recommended in DSME programming for the general population by the American Diabetes Association (ADA) and the Canadian Diabetes Association (CDA). Specifically, group programming and situational PJ34 HCl problem-solving are recommended by both national organizations [45] and [45], as these features are shown to be effective in improving HbA1c outcomes [46]. Furthermore, the CDA recommends nutritional counseling of clients with diabetes by a dietitian, either one-on-one or in small group settings, to lower HbA1c levels [45]. A recent study supports this recommendation; it found that visits by a dietitian are associated with lower hospitalization rates and charges in persons of varied cultural backgrounds compared to diabetes classes and one-on-one visits from non-dietitian health professionals [47]. Our analysis suggests that incorporating dietitians has positive success rate differences on anthropometrics, and physical activity, in addition to HbA1c. We are unsure why hospital-based interventions appear more successful across outcomes.

However, the complexity and asymmetry of multiplet structures due

However, the complexity and asymmetry of multiplet structures due to proton–proton scalar/dipolar couplings may render the accurate definition of peak positions difficult or even impossible. A breakthrough in the removal of unwanted line-splittings is offered by the Afatinib cell line use of broadband homonuclear decoupling methods that have been reported in the last few years [22], [23], [24], [25], [26], [27], [28], [29], [30] and [31]. Such experiments can be classified into two groups,

depending on the decoupling approach employed. The first group [22], [23], [25], [26], [28] and [30] utilizes the Zangger–Sterk approach [22], which achieves broadband homonuclear decoupling by combining a hard 180° and a selective 180° proton pulse, the latter applied under the action of a weak gradient field pulse to give an effect that is both spatially- and frequency-selective. As a result, in a given slice of the sample the on-resonance magnetization experiences no net effect, whereas all other proton magnetizations are inverted, refocusing any homonuclear scalar couplings to the observed spin. The second group [24], [27], [29] and [31] of experiments performs broadband homonuclear decoupling with a bilinear rotation decoupling (BIRD) module [32], utilizing isotope selection instead of the slice/chemical

shift filtering of the Zangger–Sterk approach. Depending on the phases Epigenetic inhibitors library of BIRD pulse elements, either the direct or the remote protons attached to 13C/15N isotopes can be independently and selectively inverted. The BIRD approach is used in the variants of the gradient enhanced CLIP/CLAP-HSQC experiments presented here, and yields

spectra with simple, pure absorptive in- or anti-phase F2 doublets displaying only the desired 1JXH splitting in isotropic or (1JXH + 1DXH) many splitting in anisotropic solution, respectively and allowing high spectral resolution along the F2 dimension. The one exception is that because the BIRD module does not distinguish between methylene protons, geminal 1H–1H couplings are not suppressed. In the modified CLIP/CLAP-HSQC experiments reported here, broadband proton decoupling in the 1H dimension is achieved by replacing the conventional data acquisition of a free induction decay (FID) s  (t  2) at the end of the HSQC pulse sequence with a second evolution time, t  2, at the centre of which a hard 180° proton pulse and a BIRD pulse sequence element are applied in succession, followed by acquisition of a FID s  (t  3). The BIRD(d) pulse selectively inverts all proton magnetization directly attached to the X nuclei, but leaves the magnetizations of remotely bound protons and X nuclei unperturbed. In combination with the non-selective 180° proton pulse, therefore, the net effect is for the 1H chemical shift and the heteronuclear one-bond coupling to continue to evolve throughout t  2.