Reconstruction of the Allerød vegetation of the Neuwied Basin, western Germany, and its surroundings at 12,900 cal b.p.

During the late-glacial eruption of the Laacher See volcano 12,900 b.p. the whole area of the Neuwied Basin, middle Rhine region, was covered by several metres of the Laacher See Tephra (LST) damaging but also partly conserving the former vegetation. This gave the unique opportunity to study plant remains in situ and to estimate the spatial biodiversity of plant taxa within the area investigated at the end of the last glacial as if in a snapshot of 12,900 years ago. Different kinds of plant material can be found because of different preservation conditions namely imprints, charcoal and waterlogged remains. Investigations of this material from several places within the Neuwied Basin and the Brohl valley north of it allowed the reconstruction of the former vegetation at different scales from local to regional. This resulted in a large scale draft of a vegetation map of the studied area also based on the distribution and the stage of soil development at that time. As to whether thermophilous trees were already present is discussed but largely rejected. Electronic Supplementary Material Supplementary material is available for this article at     

Vegetation changes and human action from the Neolithic to the Bronze Age (7000–4000 B.P.) in Alicante,Spain, based on charcoal analysis

Charcoal analysis reveals various palaeo-ecological phases from the Neolithic to the Bronze Age. Agriculture starts about 7000 B.P. in favourable ecological conditions. Most of the charcoal spectra from sites on the coast represent thermomediterranean holm-oak forest; those from the inland mountains represent mesome-diterranean holm-oak forest. The Neolithic I Impressed Ware people were the first to clear the forest to plant their crops. This clearance of primary woodland resulted in the development of secondary vegetation of pine woods or scrub. The scrub reached its maximum during the Bell Beaker phase and Bronze Age in the Cova de les Cendres. In the Neolithic II open air sites, the percentages of Quercus ilex/coccifera remain high. This may be the result of a different exploitation of the land, or suitable conditions for the growth and survival of the vegetation.     

Can the Study of Natural Vegetation Succession Assist in the Control of Soil Erosion on Abandoned Croplands on the Loess Plateau,China?

In the Loess Plateau, China, arable cultivation of slope lands is common and associated with serious soil erosion. Planting trees or grass may control erosion, but planted species may consume more soil water and can threaten long‐term ecosystem sustainability. Natural vegetation succession is an alternative ecological solution to restore degraded land, but there is a time cost, given that the establishment of natural vegetation, adequate to prevent soil erosion, is a longer process than planting. The aims of this study were to identify the environmental factors controlling the type of vegetation established on abandoned cropland and to identify candidate species that might be sown soon after abandonment to accelerate vegetation succession and establishment of natural vegetation to prevent soil erosion. A field survey of thirty‐three 2 × 2–m plots was carried out in July 2003, recording age since abandonment, vegetation cover, and frequency of species together with major environmental and soil variables. Data were analyzed using correspondence analysis, classification tree analysis, and species response curves. Four vegetation types were identified and the data analysis confirmed the importance of time since abandonment, total P, and soil water in controlling the type of vegetation established. Among the dominant species in the three late‐successional vegetation types, the most appropriate candidates for accelerating and directing vegetation succession were King Ranch bluestem (Bothriochloa ischaemum) and Lespedeza davurica (Leguminosae). These species possess combinations of the following characteristics: tolerance of low water and nutrient availability, fibrous root system and strong lateral vegetative spread, and a persistent seed bank.     

Prehistory of plant growing and collecting in northern Italy, based on seed remains from the early Neolithic to the Chalcolithic (c. 5600–2100 cal b.c.)

The stages of the early Neolithic and the spread of agriculture in northern Italy are difficult to determine and basically still unclear, since this region was influenced by deeply different cultures coming from both the Mediterranean coasts and the Balkans. The complex interrelations due to the contributions from both cultures are reinterpreted here thanks to recent data, modifying a picture which 15 years ago was believed to be definite. According to radiocarbon chronology, the appearance of the earliest farming communities in northern Italy should be dated around 5600–5500 cal b.c. Early farmers cultivated several cereal and pulse taxa, of which the more important were Hordeum vulgare/distichum, Triticum dicoccum, T. monococcum, T. aestivum/durum/turgidum, Lens culinaris and Pisum sp. In addition they gathered many wild plants. The spread of agriculture was a rapid phenomenon and within a few centuries agriculture was established into the Alps. Little is known about the middle and late Neolithic, with the Square-mouthed pottery culture “Bocca Quadrata”, from c. 5100 cal b.c. onwards, since most of the archaeological features discovered up to the present have produced only a few plant remains. We demonstrate the introduction of poppy and a few other innovations like a slightly increased cultivation of free-threshing cereals and flax. Archaeobotanical analyses from Chalcolithic or Copper Age settlements, from c. 3500 cal b.c. onwards, are even scarcer and a comparison with the earlier Neolithic settlements does not yet seem possible.     

Molecular phylogeny based on the κ-casein and cytochrome b sequences in the mammalian suborder Ruminantia

Nucleotide sequences for the -casein precursor proteins have been determined from the genomic DNAs or hair roots of the Ruminantia. The coding regions, exons 2, 3, and 4, were amplified separately via the three kinds of PCRs and then directly sequenced. The primers were designed from the sequence of bovine -casein gene; they were applicable for the amplification of the -casein genes from the 13 species in the Ruminantia except exon 2 of the lesser mouse deer. These results permitted an easy phylogenetic analysis based on the sequences of an autosomal gene. A phylogenetic tree was constructed from the mature K-casein sequences and compared with the tree of the cytochrome b genes which were sequenced from the same individuals. The Cervidae (sika deer, Cervus nippon) were separated from the branch of the Bovidae on the tree of -casein genes with a relatively high confidence level of the bootstrap analysis, but included in the branch of the Bovidae on the tree of cytochrome b genes. The -casein tree indicated a monophyly of the subfamily Caprinae, although the internal branches were uncertain in the Caprinae. The tree based on the nucleotide sequences of cytochrome b genes clearly showed the relationships of the closely related species in the genus Capricornis consisting of serow (C. smatorensis), Japanese serow (C. crispus), and Formosan serow (C. swinhoei). These results would be explained by the difference of resolving power between the -casein and the cytochrome b sequences. Correspondence to: K. Chikuni     

Effect of activated charcoal and pruning of the taproot on the in vitro mycorrhization of Hevea brasiliensis Müll. Arg.

In vitro mycorrhization of Hevea brasiliensis under autotrophic culture conditions is a promising methodology to produce plantlets adapted to overcome stresses during acclimatization. However, to succeed in the in vitro production of mycorrhizal plantlets, root production and subsequent colonization by the mycorrhizal fungus need to be optimized. Plantlets of H. brasiliensis clone PB 260 were grown in contact with the extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833. Addition of activated charcoal to the medium and pruning of the taproot were evaluated for their effects on root growth and colonization. None of the treatments stimulated the early formation of new roots. However, total root length, total root colonization, and production of arbuscules and intraradical spores/vesicles were significantly higher in plantlets grown in the presence of activated charcoal (especially after 13 wk of culture). In contrast, total root colonization was significantly lower in the pruned plantlets, while total root length and arbuscule formation were not affected. None of the treatments affected activities of succinate dehydrogenase and alkaline phosphatase measured in the extraradical mycelium of the fungus. It appeared that the addition of activated charcoal to the culture medium favored root growth and mycorrhization of rubber plantlets under in vitro culture conditions, while taproot pruning did not favor these parameters.     

Vegetation history and landscape management from 6500 to 1500 cal. b.p. at Lac d’Antre, Gallo-Roman sanctuary of Villards d’Héria, Jura, France

The lake sediment record was used to reconstruct past vegetation dynamics and human impacts from the middle Neolithic (6500 cal. b.p.) to the Middle Ages (1500 cal. b.p.) around Lac d’Antre in the southern Jura mountains of France. This lake was surrounded by the Gallo-Roman sanctuary of Villards d’Héria, which has been widely investigated by archaeologists and enables a comparison between palaeoenvironmental proxies and archaeological data. Pollen and microscopic charcoal analyses were conducted on a 500 cm sediment core with eleven radiocarbon dates providing the chronological control. In a mixed oak woodland context, the successive development of Taxus, Fagus and Abies were mainly caused by climatic variations during the Neolithic, in which there was weak human impact. The first significant signs of human activity were detected during the Bronze Age from 3900 cal. b.p., followed by an increase of human pressure and woodland clearances during the Iron Age, from 2700 cal. b.p. The occupation of the Gallo-Roman sanctuary was continuous with the Iron Age occupation. All the analysed palaeoenvironmental data indicate that the strongest human impact occurred during the Gallo-Roman period, which matches the occupation of Villards d’Héria previously dated by archaeologists from 2000 to 1700 cal. b.p., 1st to 3rd century a.d. Moreover, there appears to have been a new period of human settlement close to the lake at the beginning of the Middle Ages. The low charcoal accumulation rate (CHAR) recorded during the Bronze and Iron Ages suggests that fire was not the main agent used to clear the dense woods to create new cultivated fields and pastures. High CHAR values recorded during the Roman period may represent fire use for domestic and agro-pastoral activities.     

How many species of Apodemus and Rattus occur in China? A survey based on mitochondrial cyt b and morphological analyses

Apodemus(mice) and Rattus(rats) are the top rodent reservoirs for zoonoses in China, yet little is known about their diversity. We reexamined the alpha diversity of these two genera based on a new collection of specimens from China and their cyt b sequences in Gen Bank. We also tested whether species could be identified using external and craniodental measurements exclusively. Measurements from 147 specimens of Apodemus and 233 specimens of Rattus were used for morphological comparisons. We analysed 74 cyt b sequences of Apodemus and 100 cyt b sequences of Rattus to facilitate phylogenetic estimations. Results demonstrated that nine species of Apodemus and seven species of Rattus, plus a new subspecies of Rattus nitidus, are distributed in China. Principal component analysis using external and craniodental measurements revealed that measurements alone could not separate the recognized species. The occurrence of Rattus pyctoris in China remains uncertain.     

Does superoxide channel between the copper centers in peptidylglycine monooxygenase? A new mechanism based on carbon monoxide reactivity

Peptidylglycine monooxygenase (PHM) carries out the hydroxylation of the alpha-C atom of glycine-extended propeptides, the first step in the amidation of peptide hormones by the bifunctional enzyme peptidyl-alpha-amidating monooxygenase (PAM). Since PHM is a copper-containing monooxygenase, a study of the interaction between the reduced enzyme and carbon monoxide has been carried out as a probe of the interaction of the Cu(I) sites with O(2). The results show that, in the absence of peptide substrate, reduced PHM binds CO with a stoichiometry of 0.5 CO/Cu(I), indicating that only one of the two copper centers, Cu(B), forms a Cu(I)-carbonyl. FTIR spectroscopy shows a single band in the 2200-1950 cm(-)(1) energy region with nu(CO) = 2093 cm(-)(1) assigned to the intraligand C-O stretch via isotopic labeling with (13)CO. A His242Ala mutant of PHM, which deletes the Cu(B) site by replacing one of its histidine ligands, completely eliminates CO binding. EXAFS spectroscopy is consistent with binding of a single CO ligand with a Cu-C distance of 1.82 +/- 0.03 A. The Cu-S(met) distance increases from 2.23 +/- 0. 02 A in the reduced unliganded enzyme to 2.33 +/- 0.01 A in the carbonylated enzyme, suggesting that the methionine-containing Cu(B) center is the site of CO binding. The binding of the peptide substrate N-Ac-tyr-val-gly perturbs the CO ligand environment, eliciting an IR band at 2062 cm(-)(1) in addition to the 2093 cm(-)(1) band. (13)CO isotopic substitution assigns both frequencies as C-O stretching bands. The CO:Cu binding stoichiometry and peptide/CO FTIR titrations indicate that the 2062 cm(-)(1) band is due to binding of CO at a second site, most likely at the Cu(A) center. This suggests that peptide binding may activate the Cu(A) center toward O(2) binding and reduction to superoxide. As a result of these findings, a new mechanism is proposed involving channeling of superoxide across the 11 A distance between the two copper centers.     

Apoptosis in Parkinson's disease: is p53 the missing link between genetic and sporadic Parkinsonism?

Parkinson's disease (PD) is a major age-related neurodegenerative disorder characterized by a massive and specific loss of dopaminergic neurons of the substantia nigra pars compacta. The cellular alterations are clinically translated into an invalidating movement disability associated to three canonical symptoms that are bradykinesia, resting tremor and rigidity. The exact causes of this neuronal loss are unknown, but a network of evidences indicates a major contribution of orchestrated cell death processes, also known as apoptosis. Apoptotic cell death is a normal process, the alteration of which triggers several pathologies including cancer and neurodegenerative disorders. Exhaustive work has been done to delineate the cellular mechanisms responsible for the exacerbated cell death of dopaminergic neurons observed in PD. Overall, the oncogene p53 has been identified as a key effector protein.This review will focus on the clues linking p53 to the etiology of PD and the evidences that this protein may be at the center of multiple signaling cascades not only altered by mutations of various proteins responsible for familial cases of PD but also on more general sporadic cases of this devastating disease.     

Ethers and esters derived from apocynin avoid the interaction between p47phox and p22phox subunits of NADPH oxidase: evaluation in?vitro and in silico

NOX (NADPH oxidase) plays an important role during several pathologies because it produces the superoxide anion (O₂^•−), which reacts with NO (nitric oxide), diminishing its vasodilator effect. Although different isoforms of NOX are expressed in ECs (endothelial cells) of blood vessels, the NOX2 isoform has been considered the principal therapeutic target for vascular diseases because it can be up-regulated by inhibiting the interaction between its p47phox (cytosolic protein) and p22phox (transmembrane protein) subunits. In this research, two ethers, 4-(4-acetyl-2-methoxy-phenoxy)-acetic acid (1) and 4-(4-acetyl-2-methoxy-phenoxy)-butyric acid (2) and two esters, pentanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester (3) and heptanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester (4), which are apocynin derivatives were designed, synthesized and evaluated as NOX inhibitors by quantifying O₂^•− production using EPR (electron paramagnetic resonance) measurements. In addition, the antioxidant activity of apocynin and its derivatives were determined. A docking study was used to identify the interactions between the NOX2′s p47phox subunit and apocynin or its derivatives. The results showed that all of the compounds exhibit inhibitory activity on NOX, being 4 the best derivative. However, neither apocynin nor its derivatives were free radical scavengers. On the other hand, the in silico studies demonstrated that the apocynin and its derivatives were recognized by the polybasic SH3A and SH3B domains, which are regions of p47phox that interact with p22phox. Therefore this experimental and theoretical study suggests that compound 4 could prevent the formation of the complex between p47phox and p22phox without needing to be activated by MPO (myeloperoxidase), this being an advantage over apocynin.     

New combination in Veronica （Scrophulariaceae s.l.） based on morphological characters and the seed storage protein polymorphism

Veronica erciyasdagi （M. A. Fischer） C. Vural comb. ＆ stat. nov., previously regarded as a variety, is proposed as a new combination, based on the morphological characters and seed storage protein polymorphism presented in this study. In addition, information about the ecology and conservation status of Veronica erciyasdagi was reported. This taxon is endemic to central Anatolia, Turkey and is critically endangered.     

Discordant Phylogeographic Patterns between the Y Chromosome and Mitochondrial DNA in the House Mouse: Selection on the Y Chromosome?

We have compared patterns of geographic variation and molecular divergence of mitochondrial DNA (mtDNA) and Y chromosome over the range of the different subspecies of Mus musculus. MtDNA was typed for 305 nucleotides in the control region, the Y chromosome for 834 base pairs (bp) in Zfy introns and 242 bp in Sry, a Zfy2 18-bp deletion, and two microsatellites. Apparent discrepancies exist between the distributions of the lineages of mtDNA and of the two major Y-chromosome lineages thus defined: some subspecies share the same mtDNA lineage but have different Y-chromosome lineages or vice versa. One microsatellite reveals a geographically clustered variation inside the distribution of each Y-chromosome lineage, showing that new Y-chromosome variants can rapidly spread locally. The two major Y-chromosome lineages have a divergence time only about one fourth of that between mtDNA lineages. Although this recent coalescence of the Y chromosomes between subspecies could partly be due to a lower ancestral polymorphism of the Y chromosome, it suggests that secondary introgression after the radiation of the subspecies might have occurred. There is evidence that the differentiation of the Y-chromosome lineages contributes to partial reproductive isolation between subspecies, and patterns of molecular evolution suggest that selection has played a role in the rapid spread across subspecies.     

Interaction between fast and ultra-slow inactivation in the voltage-gated sodium channel. Does the inactivation gate stabilize the channel structure?

Recently, we reported that mutation A1529D in the domain (D) IV P-loop of the rat skeletal muscle Na(+) channel mu(1) (DIV-A1529D) enhanced entry to an inactivated state from which the channels recovered with an abnormally slow time constant on the order of approximately 100 s. Transition to this "ultra-slow" inactivated state (USI) was substantially reduced by binding to the outer pore of a mutant mu-conotoxin GIIIA. This indicated that USI reflected a structural rearrangement of the outer channel vestibule and that binding to the pore of a peptide could stabilize the pore structure (Hilber, K., Sandtner, W., Kudlacek, O., Glaaser, I. W., Weisz, E., Kyle, J. W., French, R. J., Fozzard, H. A., Dudley, S. C., and Todt, H. (2001) J. Biol. Chem. 276, 27831-27839). Here, we tested the hypothesis that occlusion of the inner vestibule of the Na(+) channel by the fast inactivation gate inhibits ultra-slow inactivation. Stabilization of the fast inactivated state (FI) by coexpression of the rat brain beta(1) subunit in Xenopus oocytes significantly prolonged the time course of entry to the USI. A reduction in USI was also observed when the FI was stabilized in the absence of the beta(1) subunit, suggesting a causal relation between the occurrence of the FI and inhibition of USI. This finding was further confirmed in experiments where the FI was destabilized by introducing the mutations I1303Q/F1304Q/M1305Q. In DIV-A1529D + I1303Q/F1304Q/M1305Q channels, occurrence of USI was enhanced at strongly depolarized potentials and could not be prevented by coexpression of the beta(1) subunit. These results strongly suggest that FI inhibits USI in DIV-A1529D channels. Binding to the inner pore of the fast inactivation gate may stabilize the channel structure and thereby prevent USI. Some of the data have been published previously in abstract form (Hilber, K., Sandtner, W., Kudlacek, O., Singer, E., and Todt, H. (2002) Soc. Neurosci. Abstr. 27, program number 46.12).     

Gravitropic responses of the Avena coleoptile in space and on clinostats. II. Is reciprocity valid?

Experiments were undertaken to determine if the reciprocity rule is valid for gravitropic responses of oat coleoptiles in the acceleration region below 1 g. The rule predicts that the gravitropic response should be proportional to the product of the applied acceleration and the stimulation time.
Seedlings were cultivated on 1 g centrifuges and transferred to test centrifuges to apply a transverse g-stimulation. Since responses occurred in microgravity, the uncertainties about the validity of clinostat simulation of weightlessness was avoided Plants at two stages of coleoptile development were tested. Plant responses were obtained using time-lapse video recordings that were analyzed after the flight. Stimulus intensities and durations were varied and ranged from 0.1 to 1.0 g and from 2 to 130 min, respectively. For threshold g-doses the reciprocity rule was obeyed. The threshold dose was of the order of 55 g s and 120 g s, respectively, for two groups of plants investigated. Reciprocity was studied also at bending responses which are from just above the detectable level to about 10 degrees. The validity of the rule could not be confirmed for higher g-doses, chiefly because the data were more variable.
It was investigated whether the uniformity of the overall response data increased when the gravitropic dose was defined as (g^m× 1), with m-values different from unity. This was not the case and the reciprocity concept is, therefore, valid also in the hypogravity region. The concept of gravitropic dose, the product of the transverse acceleration and the stimulation time, is also well-defined in the acceleration region studied. With the same hardware, tests were done on earth where responses occurred on clinostats. The results did not contradict the reciprocity rule but scatter in the data was large.     

Lithostratigraphy of the Lower Cambrian metaclastics and their age based on trace fossils in the Sand?kl? region, southwestern Turkey

In the Sand?kl? region of the Taurus Range of Turkey, greater than 3000 m in thickness metamorphosed siliciclastics and volcanics (Kocayayla Group) underlies the trilobite-and conodont-bearing Middle-Late Cambrian Hudai Quartzite and Çaltepe Formation.The Kocayayla Group, previously regarded as Infracambrian or Precambrian, is dated for the first time as Early Cambrian on the basis of trace fossils. Cruziana ?fasciculata, C. ?salomonis, ?Cruziana isp., ?Diplichnites isp., Monomorphichnus isp., Petalichnus isp., Rusophycus ?avalonensis, R. ?latus, Arenicolites isp., cf. Altichnus foeyni, Planolites isp., Skolithos isp., and ?Treptichnus isp. have been recognised. These trace fossils are considered Tommotian or younger in age but older than the overlying, trilobite and conodont bearing Middle Cambrian limestones of the Çaltepe Formation. The trace fossils were likely produced by trilobites, suspension feeding annelids and deposit feeding “worms”, probably polychaetes. Sections bearing abundant Skolithos represent the Skolithos ichnofacies, which is typical of high energy environments with loose sandy, well sorted to slightly muddy substrates in intertidal to shallow subtidal zones. The other trace fossils represent the Cruziana ichnofacies, which is typical of subtidal, poorly sorted and soft substrates, from moderate energy to low energy environments between the fairweather and storm wave base.The Kocayayla Group was deposited at an early stage in a shallow marine stable shelf condition. The shelf subsided in a later stage and was affected by normal faults along which mafic and felsic volcanic rocks erupted. The volcanic activity had ceased and a shallow marine clastic sedimentation took place in the final stage of the shelf development. The Kocayayla Group was deformed and metamorphosed before the deposition of the trilobite-bearing Middle-Upper Cambrian succession.     

Intensification of agriculture in southwestern Germany between the Bronze Age and Medieval period,based on archaeobotanical data from Baden-Württemberg

Vegetation History and Archaeobotany - A system of farming with an alternation of land use between being cultivated or left fallow as grassland (Feldgraswirtschaft) developed in southwestern...     

Differences in arthropods found in flowers versus trapped in plant resins on Haplopappus platylepis Phil. (Asteraceae): Can the plant discriminate between pollinators and herbivores?

Plants produce secondary metabolites related to ecologically relevant processes. These compounds include surface secretions such as latex, mucilage and resins that help plants face abiotic and biotic environmental threats such as drought, nutrient deficiency, extreme temperatures and UV radiation, as well as herbivory, pathogenic microorganisms and other natural enemies. We studied the resinous coating found on involucral bracts of Haplopappus platylepis Phil. (Asteraceae). This plant belongs to a speciose genus widely distributed in South America (Lane and Hartman in Am J Bot 83:356, 1996). H. platylepis is characterized by resinous fragrant leaves. In this species, resins cover the involucral bracts as well as young leaves and are also secreted on reproductive stalks in smaller amounts. We carried out chemical analysis and natural history observations in order to identify whether arthropods caught in inflorescence resin differed from the ones freely visiting floral disks. Regarding bracteal adhesive’s chemistry, we identified a mixture of diterpenoids and flavonoids; these compounds form a dense surface resin layer, especially over inflorescence’s bracts. In relation to associated arthropods, we found a marked difference in the main organisms captured by bracteal resin compared to insects we observed foraging on disk florets; Arthrobracus (Coleoptera: Melyridae) and Linepithema (Hymenoptera: Formicidae) were the predominant insects “trapped in resin”, while Diadasia (Hymenoptera: Apidae) was the most frequent “floral visitor”. We propose that bracteal resin of H. platylepis may function as a selective trap for non-mutualistic insects reaching reproductive structures of this plant and discuss other multiple possible roles for this secretion, including protocarnivory.     

The significance of climate fluctuations for lake level changes and shifts in subsistence economy during the late Neolithic (4300–2400 b.c.) in central Europe

In the last decades, data on the economy and environment of the Neolithic period of lake dwellings (4300–2400 b.c.) in central Europe has increased considerably and also palaeoecological data on lake level fluctuations has been thoroughly elaborated. Lake shores were mainly settled during warm and rather dry climate periods which caused a fall in the lake levels. Nevertheless, there were strong and partly very short-term shifts in the economy during the lake-dwelling period. These can be recognised only because the settlement layers can be very precisely dated by dendrochronology. In this article we discuss in an interdisciplinary way the possible interrelations between climatic and economic changes. To explain the latter, we assume crop failures as the main reason, which caused intensified hunting and gathering. There are three different possibilities which might explain this: cold and wet summers, severe droughts during spring and summer, or local over-exploitation of soils in densely settled areas.