, 2010). However, many geologists have argued from the perspective of their own subdiscipline that uniformitarian approaches are relevant and that ‘the present is the key to the past’ (e.g., Windley, 1993, Retallack, 1998 and Racki and Cordey, 2000). A more nuanced view is that ‘the basic physical laws appear to apply to all of geologic time as well as the present’ (Garner, 1974, pp. 41–42). As such, it is useful to distinguish www.selleckchem.com/products/ly2109761.html between ‘strong’ and ‘weak’ interpretations of uniformitarianism (Balashov, 1994). ‘Strong’ uniformitarianism refers to the application of the classical Principle of Uniformitarianism, as outlined above

(see Table 1). ‘Weak’ uniformitarianism (lowercase letter u) refers to the methodological and interpretive approach undertaken in many studies GDC-0449 mouse in physical geography, geomorphology, sedimentology and stratigraphy, whereby understanding of processes and environments in the past (or present) are informed by those of the present (or past). Such disconnected, circular reasoning is common in all types of palaeo studies (Edwards et al., 2007), and is the context in which we consider uniformitarianism

in this paper. The changing dynamics of Earth systems in the Anthropocene, and the explicit involvement of human activity in Earth system processes and feedbacks in ways that have not been experienced throughout Earth’s previous history, mean that the applicability of the viewpoint that ‘the present is the key to the past’ should now be reviewed. The Anthropocene is now an era of post-normal science (Funtowicz and Ravetz, 1993 and Funtowicz and Ravetz, 1994), in which scientific uncertainty has increased and traditional modes of scientific reasoning have become increasing limited in their capacity to interpret the past based on observations from the present, and vice versa. In this paper we argue that geographic and geologic viewpoints of the Anthropocene Reverse transcriptase cannot be seen through the lens of past behaviour(s) of Earth systems. Instead, the Anthropocene

probably has no analogue in Earth’s geological past and thus neither the ‘natural laws’ expounded by Principle of Uniformitarianism nor reference to high-CO2 periods of the past can be used as guides to Earth system behaviour in the Anthropocene. Earth system behaviour can be measured as the functional relationship between forcing and response, including the magnitude of response relative to forcing, the time lag(s) involved, and any other associated system feedbacks. This relationship is described by the concept of geomorphological sensitivity, which is the equilibrium Earth system response to climate forcing (Knight and Harrison, 2013a). Geomorphological sensitivity is of relevance to evaluating the Principle of Uniformitarianism because it is a representation of the different ways in which the land surface responds to climate forcing.

Stirring was continued for another hour after complete addition. The resulting white suspension was washed three times with water by centrifugation and twice with acetone. Finally, the precipitate was dried in an oven at 37 °C for 2 days. For the final dispersion, 0.56 g of the intermediate was dissolved in 15 ml 1 M HCl and filtered over a Minisart disposable cellulose acetate filter (0.2 μm pore size, 16534-K). The solution was injected into 35 ml 0.39 M NaOH solution while stirring vigorously with a magnetic stirrer. The turbid white dispersion was stirred for another 10 min after injection, the pH of the final dispersion was 7. The sample was

GW786034 nmr washed twice by centrifugation and redispersed in a final volume of 50 ml water. It has been shown previously that the stability of metal-pyrophosphate dispersions is strongly dependent on the ionic strength

of the solution (van Leeuwen et al., 2012a). Therefore, mixed systems at a fixed concentration of pyrophosphate were prepared as this set the concentration of the counterions. Mixed systems were prepared by substituting part of the iron in the precursor solution with calcium or magnesium (together referred to as M2+), the LY2109761 amounts of Fe3+ and M2+ in the mixture are then determined in stoichiometry with the concentration of PPi. This resulted in the following Fe:M2+ ratios: Fe10M2+PPi8 (10:1 ratio), Fe16 M2+2PPi13 (8:1), Fe8 M2+2PPi7 (4:1), Fe4M2+4PPi5 (1:1), Fe2M2+11PPi7 (1:5) or Fe2M2+19PPi11 (1:10). Here complete precipitation without inclusion of the Na+ and Cl− from the reactants was assumed. Iron pyrophosphate prepared without any substitution was referred to

as ‘pure FePPi’. Full substitution of iron results in the pure M2+ pyrophosphate, M2+PPi. For Fe:Na, the following ratios were prepared: Fe22Na2PPi17 (10:1), Fe32Na4PPi25 (8:1), Fe16Na4PPi13 (4:1). Samples containing a lower iron content remained clear and no particles were pheromone formed. All samples were stored in plastic (Teflon™) bottles. Mixed systems prepared using the pH dependent precipitation method only resulted in stable dispersions when prepared using Magnesium. Colloidal (mixed) iron pyrophosphates were coated with zein protein through an antisolvent precipitation method (Velikov & Pelan, 2008). As colloidal iron pyrophosphate aggregates over time in water (van Leeuwen et al., 2012a), the nanoparticles were prepared either immediately before (in case of the NP-Z system) or simultaneously with the zein precipitation. The concentrations were also lowered: the final dispersion contained 2 mM iron and 1.5 mM pyrophosphate, in order to prevent aggregation during the addition of zein. After complete precipitation of the iron pyrophosphate, the 30 ml dispersion was removed and 40 ml zein solution (1 g zein in 80 vol.% ethanol) was slowly poured into the dispersion, which turned more turbid and slightly yellow. Some aggregates were formed, which were filtered out of the dispersion before further analysis.

4%), wheat (2.7%) and corn (1.7%) (Alvarez-Jubete, Arendt, et al., 2010). This is in particular important for celiac disease patients, where the intake of fiber in the gluten-free diet is considered to be inadequate, and thus the incorporation of quinoa seeds in their diets should help alleviate, at least in part, their deficit in fiber intake (Alvarez-Jubete,

Arendt, et al., 2010). The polysaccharides that compose the dietary fiber of quinoa have attracted our attention, and there is no report in the literature about their structures. Polysaccharides have beneficial effects on health and are ubiquitous in plant foods. To better understand the bio-functionality of polysaccharides scientific elucidation of the structures responsible for the beneficial effect is very important (Yamada, Kiyohara, & Matsumoto, 2003). Thus, in this work the chemical composition, structural features

and gastroprotective AZD5363 nmr activity of arabinan and arabinan-rich pectic polysaccharides isolated from the seeds of INCB024360 clinical trial quinoa (C. quinoa) have been described. Seeds of C. quinoa were purchased at local market (QUINUA REAL®). The total lipid quantitation was performed by the method of Bligh and Dyer (1959). Fractions were carboxy-reduced by the carbodiimide method (Taylor & Conrad, 1972), using NaBH4 as the reducing agent, giving products with the –COOH groups of its uronic acid residues reduced to –CH2OH. Seeds of quinoa (466.6 g) were milled and then deffated with acetone, in order to remove lipids,

pigments and other hydrophobic material. The polysaccharides were extracted from the residue with water at 60 °C for 4 h (8×, 1 l each). The aqueous extracts were obtained by centrifugation (3860g, 20 min at 25 °C), joined and concentrated under reduced pressure. The polysaccharides were precipitated with EtOH (3 vol.) and freeze-dried, giving fraction QW. The remaining residue was then extracted twice (1 l each) with aq. 10% KOH, at 100 °C for 4 h and the alkaline extracts were neutralized with acetic acid, dialyzed for 48 h with tap water, concentrated under reduced pressure and freeze-dried, originating fractions QK1 and QK2. In order to remove starch, fractions QW, QK1 and QK2 were extensively 4-Aminobutyrate aminotransferase treated with α-amylase (from Bacillus licheniformis, Sigma A3403) and dialyzed. Moreover, to remove proteins, they were treated with 10% aqueous trichloroacetic acid and/or Pronase (Roche) and newly dialyzed. Then, a freeze–thaw treatment was applied in these fractions, to give cold-water soluble fractions SQW, SQK1 and SQK2. In this procedure, the sample was frozen and then thaw at room temperature. Insoluble polysaccharides were recovered by centrifugation. The cold-water soluble polysaccharides were purified by sequential ultrafiltration through membranes (Millipore) with cut-offs of 100 kDa (PLHK04710-Ultracel), 30 kDa (PLTK04710-Ultracel) and 10 kDa (PLGC04710-Ultracel).

UN biome definitions were used in this instance to calculate carbon storage, and Olson et al. (2001) biome codes are supplemented in parentheses to aid comparison see more with other data. Total carbon storage is defined in this instance as carbon in above- and below-ground biomass, litter and soil organic matter to 1 m depth. Uncertainty exists in each of these estimates and the carbon content of peatland soils in particular may be under accounted. For example, the boreal forest

biome has ~ 500 Pg C attributed to soil storage alone at 1 m depth, according to the estimate by Tarnocai et al. (2009). The authors are grateful to the NERC for supporting this research (grant NE/H023690/1). We would also like to thank Dr. Sara Rassner at Aberystwyth University for ARC-GIS assistance. “
“Reliable human exposure models are critical for understanding human health risks from chemicals. The U.S. EPA has developed, refined, applied, and evaluated the probabilistic SHEDS-Multimedia model to improve estimates of human exposure to multimedia, multipathway chemicals

to support both aggregate and cumulative assessments (Zartarian et al., 2006, Zartarian et al., 2012, Xue et al., 2006 and Xue et al., 2010a; http://www.epa.gov/heasd/research/sheds/). SHEDS-Multimedia is a physically-based (simulates human contact with chemicals), probabilistic model that can simulate aggregate or cumulative exposures over time via dietary and residential routes of exposure for a variety of multimedia,

check details multipathway environmental chemicals. SHEDS-Multimedia can be linked with physiologically-based pharmacokinetic (PBPK) models to characterize variability and uncertainty Bay 11-7085 in risk assessments. It is important to evaluate model estimates with available biomarker data. Pyrethroids are the latest class of insecticides in global use and are replacing organophosphates in agricultural and consumer applications (Nishi et al., 2006). Pyrethroids are used in agricultural, forest, textile, and public health programs worldwide (Heudorf and Angerer, 2001). With the passage of the Food Quality Protection Act of 1996 (FQPA), EPA is required to consider available information concerning the cumulative effects on human health resulting from exposure to multiple chemicals that have a common mechanism of toxicity when making decisions related to pesticide tolerances (EPA OPP, 2011). In their review of 22 rodent studies, Shafer et al. (2005) reported that pyrethroids exert their neurotoxicity by slowing the opening and closing of voltage-gated sodium channels in insect and mammalian nerve cells and associations between in utero exposures and persistent changes in neurochemistry, motor activity, behavior, and learning. Zartarian et al.

Any recovery from interruptions involves a switch in task (albeit not necessarily from a competing task) and on the basis of the current results alone we cannot rule out that this switch is somehow responsible for the cost asymmetry. However, in Bryck and Mayr (2008) we did find a cost asymmetry even after 5 s unfilled delays between trials and in the absence of any switches in tasks. As we would argue, such delays increase the probability of loosing the current task set, which then triggers working

memory updating. The LTM-interference/updating account can explain both the clearly LTM-induced cost asymmetry studied here and the traditional switch cost asymmetry (see also, Mayr, 2008, 2009; Waszak et al., 2003). Therefore, according to Occam’s razor any inferences about an additional process (such as carry-over) should be based on strong evidence that the trial-to-trial switching situation is in

some way unique BMS-754807 mw and cannot be handled by the LTM-interference/updating Smad inhibitor account alone. One result that at first sight seems to support the carry-over model comes from Yeung, Nystrom, Aronson, and Cohen (2006). Participants switched between tasks that required subjects to attend either to word or to face stimuli. After task switches there was greater activity in neural areas associated with the currently irrelevant task than after non-switch trials. Also, the degree of this irrelevant-task activity predicted RT switch costs. These results are consistent with the carry-over account, but not necessarily inconsistent with the LTM interference account. With only two tasks it is impossible to tell to what degree there would have been also heightened activity on no-switch trials––only to a lesser degree than on switch trials. It is not at all incompatible Adenosine with an LTM model when interference is greater for a more recently used alternate task (e.g., on switch trials) than for a less recently used task (e.g., on no-switch trials; for

related findings see Bryck & Mayr, 2008). In fact, Wylie, Javitt, and Foxe (2004) looked for activation in neural areas associated with task dimensions that had been performed in a previous block of trials, but were not relevant on the current single-task block. Consistent with the LTM model, they found increased task-irrelevant activity (compared to a control situation in which the irrelevant task had not yet been experienced), even though no immediate switching between tasks was involved. We are aware of one set of studies that proposed a specific version of a carry-over model, and also made an explicit effort of ruling out an LTM account of the cost asymmetry. Yeung and Monsell (2003a) had presented a connectionist model that explains different patterns of cost asymmetry as resulting from the combination of carry-over of task activation and the relative amounts of control activation required when tasks either do or do not directly compete with each other.

, 2005), possibly indicating differences between the shelterwood examined by Harmer et al. (2005) and the more extensive clearfells that we considered. The determination of any relationship between vascular plant cover and regeneration density was complicated by the constantly changing nature of ground flora – the current vegetation structure does not necessarily reflect that present when the seedlings first started growing. Indeed, the

only significant correlation between regeneration density and vascular plant cover was the negative correlation found for birch seedlings (shorter learn more than 0.5 m). The small size of a birch seed means that its food reserve is only sufficient to grow to 2 cm in height (Miles and Kinnaird, 1979), before it must be able to support itself through photosynthesis. This results this website in birch’s difficulty in establishing itself

in thick vegetation. Scarification (exposure of mineral soil) can increase seedling density in birch spp. (Kinnaird, 1974 and Karlsson, 1996). The ground disturbance and lack of ground vegetation after clear felling provides opportunities for seedlings to become established in bare ground before it is covered with vegetation. In contrast, the lack of regeneration seen on the unplanted upland moorland and unplanted improved farmland sites is likely due to the dense flora coverage (120% and 142% respectively) in Suplatast tosilate combination with the lack of any ground disturbance. The rate of tree growth was slow, with regenerating trees achieving a median height of 104 cm

after 10 years of growth post-felling. These growth rates are markedly poorer than those recorded by Harmer and Morgan (2009) in lowland England or by Worrell et al. (2000) in upland NE Scotland. We found that the height distribution of the regenerating trees changed with time since clearfelling (Fig. 3), with large numbers of small trees 4 years post-felling changing to a more even distribution of heights 10 years post-felling. This indicates that the recruitment of new trees is most prolific in the first few years following felling, with fewer seedlings 10 years post-felling indicating a slowdown in this process. This decline is likely to be driven by the increase in herbaceous cover following clearfelling combined with the negative correlation between birch regeneration and herbaceous cover. The weighting of seedling recruitment to the years immediately following clearfelling may also contribute to the observed site to site variability in regenerating tree number since any temporal fluctuations in the ability of trees to regenerate will have substantial effects on the resulting density.

brasiliensis mycelia. Since this compound presented a potential anti-HSV activity, its mechanism of action was also evaluated. The fruiting bodies of Agaricus brasiliensis Wasser strain UFSC 51 (syn A. subrufescens, A. blazei) were collected in Biguaçu, Santa Catarina State, Southern Brazil. The characterization BTK inhibitor of the species

was performed by Dr. Maria Alice Neves, and a voucher specimen (FLOR 11 797) was deposited in the FLOR Herbarium (Universidade Federal de Santa Catarina). The mycelium of A. brasiliensis was isolated and cultivated on potato dextrose agar (PDA) (Oxoid, UK) at 25 °C during 7 days. The liquid inoculum was produced by transference of mycelial disks to flasks containing Melin-Norkrans Modified medium (MNM) ( Marx, 1969) and cultivated at 25 °C during 10 days. Mycelia were filtered and fragmented in 300 mL of NaCl 0.8%. The inoculum was then added to MNM in an airlift bioreactor (5 L) and cultivated during 7 days at 26 °C. The liquid culture was centrifuged and the mycelial biomass was dehydrated at 55 °C until constant weight. Agaricus brasiliensis

polysaccharide was this website isolated as previously described ( Camelini et al., 2005), with minor modifications. Fifty grams of dried mycelial biomass were blended twice with five volumes of distilled water and refluxed at 100 °C for 3 h. The material was filtered under vacuum through a Whatman n°42 filter paper. Three volumes of ethanol were added to the filtrate. The mixture was maintained PDK4 at 4 °C for 24 h and centrifuged (1100 g, 10 min). The mycelial polysaccharide was freeze-dried and designated as MI. To produce the sulfated derivative, MI was sulfated using the pyridine-chlorosulfonic acid reagent as described by Zhang et al. (2003). After sulfation, resulting polysaccharides were dialyzed through

a 5 kDa molecular weight cut-off membrane (Spectrum Laboratories, Rancho Dominguez, CA) against distilled water and freeze-dried yielding the sulfated derivative (MI-S). MI and MI-S were characterized by spectroscopic methods [Fourier transform infrared (FTIR) and 13C Nuclear magnetic resonance (13C NMR)] and elemental analyses (C, H, O, S). Determination of homogeneity and molecular weight (Mw) was carried out by high-performance gel filtration chromatography (HPGFC) using a Perkin Elmer series 200 equipment coupled with a RI detector, using a gel filtration column (TSK-Gel 5000 PW 7.8 × 300 mm connected to a TSK PWH 5 × 7 mm guard column; Tosoh, Japan). Samples were eluted with 0.2 M NaCl mobile phase at a flow rate of 1 mL/min. Mean Mw was estimated by comparison with retention times of standard dextrans.

Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to manufacturer instructions. Viral load was determined using bDNA method (Versant 3.0 Siemens, Germany) and CD4 + T-cells were measured by flow cytometer (FACS Calibur, BD, USA) during regular clinical follow up at the local laboratory. The study was approved by

the ethical committees of the institutions involved. Polymerase genotyping was performed using TRUGENE® HIV-1 Genotyping Assay or OpenGene® DNA System (Siemens, USA) and a one step RT-PCR using proof reading enzyme, adapted from Van Laethem et al. 2008, followed by a nested PCR to amplify the complete integrase gene. The PCR product was then submitted to direct sequencing using BigDye® v3.1 Cycle Sequencing kit (Applied Biosystems, USA), resolved in an ABI3130XL (Applied Biosystems, USA). Three independent replicate integrase sequences were obtained from each sample. The sequences click here were assembled and edited using Sequencher 4.7 (GeneCodes, USA). Sequences Accession numbers: JQ797715 to JQ797734. Resistance mutations and susceptibility to antiretroviral drugs were analyzed according to Stanford Resistance Database (Supplementary data 1, SD-1),

selleckchem Geno2pheno[resistance], IAS 2011 mutation list (Johnson et al., 2011) and the ANRS algorithm. Sequences were aligned with HXB2 reference sequence using BioEdit v.7.0.9. Subtype screening was done at NCBI Genotyping and REGA BioAfrica websites, confirmed by phylogenetic reconstruction of Neighbor Joining and Maximum Likelihood trees using Paup∗ 4.10b (SD-2). Viral load, CD4, antiretroviral treatment, resistance mutations and sampling time points are depicted in SD-3. Polymerase genotyping (see SD-1) prior to raltegravir exposure predicted a high-level resistance profile to all NNRTI and NRTI except for etravirine, which showed a potential low-level resistance score according to Stanford Database (G190A). As the patient had no prior exposure to

the drug and did not use other NNRTI in the year preceding this sampling, the drug was considered here as fully active. The virus had high-level resistance to all PI drugs except for darunavir/r, which exhibited an intermediate resistance Farnesyltransferase profile (I47V, I50V, I84V, L89V). Therefore, the patient started raltegravir regimens at best with one additional active drug (etravirine) and one partially active drug, darunavir/r. This fact may have been determinant for the virological failure within a few weeks. Samples weeks 40 and 88 showed high resistance to etravirine (E138Q, Y181C and G190A). Therefore, after 40 week of exposure the regimen contained only a partially active darunavir. On the first available sample obtained after raltegravir introduction on the regimen (week 32) the substitution F121Y was observed on all replicate sequences. Alongside this mutation, the emergence of L74I, T97A, Q137H and V151I was observed, as well as synonymous polymorphisms in codon 167.

3B), whereas the phosphorylation of TBK1, the phosphorylating enzyme of IRF-3 [4], was suppressed (Fig. 3C). These results seem to imply that MKK4, MKK6, MKK7, and TBK1 upstream kinase could be directly targeted by this fraction. However, this website we did not observe any inhibitory effect of PPD-SF in a direct enzyme assay

performed with purified MKK4, MKK7, and MKK6, indicating that these enzymes are not targets of PPD-SF. Moreover, we could not test the upstream TBK1-phosphorylating enzyme, because the TBK1-phosphorylating enzymes have not yet been identified [36]. Therefore, we will continue to identify targets specifically inhibited by PPD-SF for the suppression of AP-1 and IRF-3 pathways. Meanwhile, the inhibitory activities of SP600125, a JNK inhibitor, and BX795, a TBK1 inhibitor, on the production of PGE2 (Fig. 3E) strongly suggested the critical involvement of these enzymes in the inflammatory process. Other research groups have also found that the enzymes, JNK and TBK1, play important pathological

roles in many different inflammatory responses and symptoms, such as colitis [37], [38] and [39]. To develop a strong and safe anti-inflammatory remedy, determining whether the preparation is orally active in an in vivo model is critical. selleck products Although orally administered KRG-water extract is reported to have anti-inflammatory activity in a mouse inflammation model with allergic rhinitis [40], whether PPD-SF is able to ameliorate in vivo inflammatory symptoms was examined using a HCl/ethanol-induced mouse gastritis model. As Fig. 4A shows, PPD-SF strongly suppressed the formation of gastric ulcer triggered by Tolmetin HCl/ethanol. In particular, it was also revealed that the level of phospho-JNK2 was markedly decreased by PPD-SF, according to immunoblotting analysis with stomach lysates ( Fig. 4B). Therefore, these results also strongly suggest that PPD-SF can be an orally effective anti-inflammatory preparation

with JNK inhibitory properties. In summary, we found that PPD-SF is capable of diminishing in vitro inflammatory responses mediated by macrophage-like RAW264.7 cells treated with LPS and suppressing in vivo gastritis symptoms induced by HCl/ethanol in mice. Through the analysis of transcription factors and their upstream signaling enzymes, it was demonstrated that c-Jun, ATF-2, and IRF-3 and their upstream activation pathways including p38, JNK, and TBK1 could be targeted by PPD-SF, as summarized in Fig. 5. Therefore, our results strongly suggest that PPD-SF can be developed as a KRG-derived anti-inflammatory remedy. The authors declare that there is no conflict of interests regarding the publication of this paper. This work was supported by a grant (2012-2013) from the Korean Society of Ginseng.

Appreciation

is also extended to Dr. Stephanie from Colorado University at Boulder, for her help in refining the language usage. “
“Eleven years after Crutzen (2002) suggested the term Anthropocene as a new epoch of geological time (Zalasiewicz et Selleckchem Navitoclax al., 2011a), the magnitude and timing of human-induced change on climate and environment have been widely debated, culminating in the establishment of this new journal. Debate has centred around whether to use the industrial revolution as the start of the Anthropocene as suggested by Crutzen, or to include earlier anthropogenic effects on landscape, the environment (Ellis et al., 2013), and possibly climate (Ruddiman, 2003 and Ruddiman, 2013), thus backdating it to the Neolithic revolution and possibly beyond Pleistocene megafauna extinctions

around 50,000 years ago (Koch and Barnosky, 2006). Here, we appeal for leaving the beginning of the Anthropocene at around 1780 AD; this time marks the beginning of immense rises in human population and carbon emissions as well as atmospheric CO2 levels, the so-called “great acceleration”. This also anchors the Anthropocene on the first measurements of atmospheric CO2, confirming the maximum level of around 280 ppm recognized from ice cores to be typical for the centuries preceding the Anthropocene (Lüthi et al., 2008). The cause of the great acceleration was the SCH727965 purchase increase in burning of fossil fuels: this did not begin in the 18th century, indeed coal was used 800 years earlier in China and already during

Roman times in Britain ( Hartwell, 1962 and Dearne and Branigan, 1996), but the effects on atmospheric CO2 are thought to have been less than 4 ppm until 1850 ( Stocker et al., 2010). The Anthropocene marks the displacement of agriculture as the world’s leading industry ( Steffen et al., 2011). However, the beginning of the Anthropocene is more controversial than its existence, and if we consider anthropogenic effects on the environment rather than on climate, there is abundant evidence for earlier events linked to human activities, including land use changes associated with the spread of agriculture, Plasmin controlled fire, deforestation, changes in species distributions, and extinctions (Smith and Zeder, 2013). The further one goes back in time, the more tenuous the links to human activities become, and the more uncertain it is that they caused any lasting effect. The proposition of the Anthropocene as a geological epoch raises the question of what defines an epoch. To some extent this is a thought experiment applied to a time in the far future – the boundary needs to be recognizable in the geological record millions of years in the future, just as past boundaries are recognized.