Anatomical and ultrastructural analyses of in vitro organogenesis from root explants of commercial passion fruit (Passiflora edulis Sims)

This study aimed to characterize the anatomical events and ultrastructural aspects of direct and indirect in vitro organogenesis in Passiflora edulis. Root explants were cultured on induction medium, supplemented with 4.44 mu M 6-benzyladenine. Roots at different stages of development were collected and processed for observation by light microscopy and scanning and transmission electron microscopy. Patterns of direct and indirect regeneration were observed in the explants. During direct organogenesis, the organogenic buds and nodules, formed from meristemoids, originated from the pericycle regions distant from the cut surface. Completely differentiated buds were observed after 20 days of culture. During indirect organogenesis, bud formation occurred via meristemoids at the periphery of the calli, which differentiated from the cortical region of the initial explant. Regardless of the regeneration pattern, the meristemoids had similar ultrastructural characteristics; however, differences were reported in the nuclear shape of the cells of the meristemoids formed directly and indirectly. This study provides important information for enhancing the understanding and characterization of the organogenic process in non-meristematic explants and provides information on the use of roots as explants in genetic transformation protocols for this important tropical species.

Rhythms of anorexigenic neuropeptides in senegalese sole: Effects of light, feeding regimes and developmental stage.

Increasing knowledge on the endocrine mechanisms that regulate feeding and growth in cultured fish can contribute to make improvement in fish holding conditions and feeding strategies, supporting the development of new techniques that could ameliorate feeding, food conversion efficiency and growth in aquaculture practice. The main objective of this study was to investigate how daily mRNA expression of three specific anorexigenic hormones, i.e. the corticotropin-releasing hormone (CRH) and the paralogues α- and β- proopiomelanocortin (POMC), is modulated by different photoperiods, light spectra and feeding regimes, in both adult and larvae of Solea senegalensis. In addition, as Senegalese sole exhibits a shift from diurnal to nocturnal in locomotor activity and feeding habits during metamorphic process, we tried to elucidate if this shift is accompanied by relevant daily variations in the expression of these anorexigenic hormones before, during and after the completion of metamorphosis. In order to reach this main objective, three main experiments were developed. In a first experiment, adults were reared under LD (12 h light: 12h dark) cycle and fed at mid-light (ML), mid-dark (MD) and at random (RND). In a second experiment, adult specimens were reared in constant darkness (DD) and fed at subjective mid-light (sML) or at RND. Larvae of Senegalese sole were reared under LD cycle with white, blue or red light for 40 days. Our results show an independence of crh mRNA expression from the feeding time and suggest an endogenous control of crh expression in sole. Both pomc paralogues showed significant daily rhythms under LD conditions. The rhythms were maintained or were even more robust under DD conditions for pomc_a, but were completely abolished for pomc_b. Our results indicate an endogenous control of pomc_a expression by the molecular clock in telencephalon and diencephalon, but not in the pituitary gland. Our findings confirm for the first time the significant influence that ambient lighting has on larval growth and development in Senegalese sole, revealing an important effect of light spectra upon functional elements of this species. Our results also emphasize the importance of maintaining cycling light-dark conditions of the adequate wavelengths in aquaculture practices during early development of sole.

An investigation of phenolic glycoside and condensed tannin homeostasis in Populus by salicyl alcohol feeding to cell cultures and by transgenic manipulation of the sucrose transporter, PTSUT4, in planta

Secondary metabolites play an important role in plant protection against biotic and abiotic stress. In Populus, phenolic glycosides (PGs) and condensed tannins (CTs) are two such groups of compounds derived from the common phenylpropanoid pathway. The basal levels and the inducibility of PGs and CTs depend on genetic as well as environmental factors, such as soil nitrogen (N) level. Carbohydrate allocation, transport and sink strength also affect PG and CT levels. A negative correlation between the levels of PGs and CTs was observed in several studies. However, the molecular mechanism underlying such relation is not known. We used a cell culture system to understand negative correlation of PGs and CTs. Under normal culture conditions, neither salicin nor higher-order PGs accumulated in cell cultures. Several factors, such as hormones, light, organelles and precursors were discussed in the context of aspen suspension cells’ inability to synthesize PGs. Salicin and its isomer, isosalicin, were detected in cell cultures fed with salicyl alcohol, salicylaldehyde and helicin. At higher levels (5 mM) of salicyl alcohol feeding, accumulation of salicins led to reduced CT production in the cells. Based on metabolic and gene expression data, the CT reduction in salicin-accumulating cells is partly a result of regulatory changes at the transcriptional level affecting carbon partitioning between growth processes, and phenylpropanoid CT biosynthesis. Based on molecular studies, the glycosyltransferases, GT1-2 and GT1-246, may function in glycosylation of simple phenolics, such as salicyl alcohol in cell cultures. The uptake of such glycosides into vacuole may be mediated to some extent by tonoplast localized multidrug-resistance associated protein transporters, PtMRP1 and PtMRP6. In Populus, sucrose is the common transported carbohydrate and its transport is possibly regulated by sucrose transporters (SUTs). SUTs are also capable of transporting simple PGs, such as salicin. Therefore, we characterized the SUT gene family in Populus and investigated, by transgenic analysis, the possible role of the most abundantly expressed member, PtSUT4, in PG-CT homeostasis using plants grown under varying nitrogen regimes. PtSUT4 transgenic plants were phenotypically similar to the wildtype plants except that the leaf area-to-stem volume ratio was higher for transgenic plants. In SUT4 transgenics, levels of non-structural carbohydrates, such as sucrose and starch, were altered in mature leaves. The levels of PGs and CTs were lower in green tissues of transgenic plants under N-replete, but were higher under N-depleted conditions, compared to the levels in wildtype plants. Based on our results, SUT4 partly regulates N-level dependent PG-CT homeostasis by differential carbohydrate allocation.

Effects of pH, light and temperature on (1→3,1→4)-β-glucanase stability in wheat leaves

Changes in (1→3,1→4)-β-D-glucan endohydrolase (EC 3.2.1.73) protein levels were investigated in segments from second leaves of wheat (Triticum aestivum L.). The abundance of the enzyme protein markedly increased when leaf segments were incubated in the dark whereas the enzyme rapidly disappeared when dark-incubated segments were illuminated or fed with sucrose. Addition of cycloheximide (CHI) to the incubation medium led to the disappearance of previously synthesized (1→3,1→4)-β-glucanase and suppressed the dark-induced accumulation indicating that the enzyme was rather unstable. The degradation of (1→3,1→4)-β-glucanase was analyzed without the interference of de-novo synthesis in intercellular washing fluid (IWF). The loss of the enzyme protein during incubation of IWF (containing naturally present peptide hydrolases) indicated that the stability increased from pH 4 to pH 7 and that an increase in the temperature from 25 to 35 °C considerably decreased the stability. Chelating divalent cations in the IWF with o-phenanthroline also resulted in a lowered stability of the enzyme. A strong temperature effect in the range from 25 to 35 °C was also observed in wheat leaf segments. Diurnal changes in (1→3,1→4)-β-glucanase activity were followed in intact second leaves from young wheat plants. At the end of the dark period, the activity was high but constantly decreased during the light phase and remained low if the light period was extended. Activity returned to the initial level during a 10-h dark phase. During a diurnal cycle, changes in (1→3,1→4)-β-glucanase activity were associated with reciprocal changes in soluble carbohydrates. The results suggest that the synthesis and the proteolytic degradation of an apoplastic enzyme may rapidly respond to changing environmental conditions.

Regulation and transcript analysis of the ceroperon responsible for quorum sensing in Rhodobacter sphaeroides 2.4.1

Rhodobacter sphaeroides 2.4.1 is a Gram negative facultative photoheterotrophic bacterium that has been shown to have an N-acyl homoserine lactone-based quorum sensing system called cer for c&barbelow;ommunity e&barbelow;scape r&barbelow;esponse. The cer ORFs are cerR, the transcriptional regulator, cerI, the autoinducer synthase and cerA , whose function is unknown. The autoinducer molecule, 7,8- cis-N-(tetradecenoyl) homoserine lactone, has been characterized. The objective of this study was to identify an environmental stimulus that influences the regulation of cerRAI and, to characterize transcription of the cer operon. ^ A cerR::lacZ transcriptional fusion was made and β-Galactosidase assays were performed in R. sphaeroides 2.4.1 strains, wild type, AP3 (CerI−) and AP4 (CerR−). The cerR::lacZ β-Galactosidase assays were used as an initial survey of the mode of regulation of the Cer system. A cerA::lacZ translational fusion was created and was used to show that cerA can be translated. The presence of 7,8-cis-N-(tetradecenoyl) homoserine lactone was detected from R. sphaeroides strains wild type and AP4 (CerR−) using a lasR::lacZ translational fusion autoinducer bioassay. The cerR::lacZ transcriptional fusion in R. sphaeroides 2.4.1 wild type was tested under different environmental stimuli, such as various carbon sources, oxygen tensions, light intensities and culture media to determine if they influence transcription of the cer ORFs. Although lacZ assay data implicated high light intensity at 100 W/m2 to stimulate cer transcription, quantitative Northern RNA data of the cerR transcript showed that low light intensity at 3 W/m2 is at least one environmental stimulus that induces cer transcription. This finding was supported by DNA microarray analysis. Northern analysis of the cerRAI transcript provided evidence that the cer ORFs are co-transcribed, and that the cer operon contains two additional genes. Bioinformatics was used to identify genes that may be regulated by the Cer system by identifying putative lux box homologue sequences in the presumed promoter region of these genes. Genes that were identified were fliQ, celB and calsymin, all implicated in interacting with plants. Primer extension was used to help localize cis-elements in the promoter region. The cerR::lacZ transcriptional fusion was monitored in a subset of different global DNA binding transcriptional regulator mutant strains of R. sphaeroides 2.4.1. Those regulators involved in maintaining an anaerobic photosynthetic lifestyle appeared to have an effect. Collectively, the data imply that R. sphaeroides 2.4.1 activates the Cer system when grown anaerobic photosynthetically at low light intensity, 3 W/m2, and it may be involved in an interaction with plants. ^

Spatial community shift from hard to soft corals in acidified water

Anthropogenic increases in the partial pressure of CO2 (pCO2) cause ocean acidification, declining calcium carbonate saturation states, reduced coral reef calcification and changes in the compositions of marine communities. Most projected community changes due to ocean acidification describe transitions from hard coral to non-calcifying macroalgal communities; other organisms have received less attention, despite the biotic diversity of coral reef communities. We show that the spatial distributions of both hard and soft coral communities in volcanically acidified, semi-enclosed waters off Iwotorishima Island, Japan, are related to pCO2 levels. Hard corals are restricted to non-acidified low- pCO2 (225 µatm) zones, dense populations of the soft coral Sarcophyton elegans dominate medium- pCO2 (831 µatm) zones, and both hard and soft corals are absent from the highest- pCO2 (1,465 µatm) zone. In CO2-enriched culture experiments, high- pCO2 conditions benefited Sarcophyton elegans by enhancing photosynthesis rates and did not affect light calcification, but dark decalcification (negative net calcification) increased with increasing pCO2. These results suggest that reef communities may shift from reef-building hard corals to non-reef-building soft corals under pCO2 levels (550-970 µatm) predicted by the end of this century, and that higher pCO2 levels would challenge the survival of some reef organisms.

Red/far-red and blue light-responsive regions of maize rbcS-m3 are active in bundle sheath and mesophyll cells, respectively.

Leaves of the C4 plant maize have two major types of photosynthetic cells: a ring of five large bundle sheath cells (BSC) surrounds each vascular bundle and smaller mesophyll cells (MC) lie between the cylinders of bundle sheath cells. The enzyme ribulose bisphosphate carboxylase/oxygenase is encoded by nuclear rbcS and chloroplast rbcL genes. It is not present in MC but is abundant in adjacent BSC of green leaves. As reported previously, the separate regions of rbcS-m3, which are required for stimulating transcription of the gene in BSC and for suppressing expression of reporter genes in MC, were identified by an in situ expression assay; expression was not suppressed in MC until after leaves of dark-grown seedlings had been illuminated for 24 h. Now we have found that transient expression of rbcS-m3 reporter genes is stimulated in BSC via a red/far-red reversible phytochrome photoperception and signal transduction system but that blue light is required for suppressing rbcS-m3 reporter gene expression in MC. Blue light is also required for the suppression system to develop in MC. Thus, the maize gene rbcS-m3 contains certain sequences that are responsive to a phytochrome photoperception and signal transduction system and other regions that respond to a UVA/blue light photoperception and signal transduction system. Various models of "coaction" of plant photoreceptors have been advanced; these observations show the basis for one type of coaction.

Variation in Coral Photosynthesis, Respiration and Growth Characteristics in Contrasting Light Microhabitats: An Analogue to Plants in Forest Gaps and Understoreys?

1. The often complex architecture of coral reefs forms a diversity of light microhabitats. Analogous to patterns in forest plants, light variation may drive strategies for efficient light utilization and metabolism in corals. 2. We investigated the spatial distribution of light regimes in a spur-and-groove reef environment and examine the photophysiology of the coral Montipora monasteriata (Forskal 1775), a species with a wide habitat distribution. Specifically, we examined the variation in tissue and skeletal thickness, and photosynthetic and metabolic responses among contrasting light microhabitats. 3. Daily irradiances reaching corals in caves and under overhangs were 1-5 and 30-40% of those in open habitats at similar depth (3-5 m), respectively. Daily rates of net photosynthesis of corals in cave habitats approximated zero, suggesting more than two orders of magnitude variation in scope for growth across habitats. 4. Three mechanisms of photoadaptation or acclimation were observed in cave and overhang habitats: (1) a 20-50% thinner tissue layer and 40-60% thinner skeletal plates, maximizing light interception per unit mass; (2) a two- to threefold higher photosynthetic efficiency per unit biomass; and (3) low rates of dark respiration. 5. Specimens from open and cave habitats displayed a high capacity to acclimate to downshifts or upshifts in irradiance, respectively. However, specimens in caves displayed limited acclimation to further irradiance reduction, indicating that these live near their irradiance limit. 6. Analogous to patterns for some plant species in forest gaps, the morphological plasticity and physiological flexibility of M. monasteriata enable it to occupy light habitats that vary by more than two orders of magnitude.

Caracterização da fração de óleo de café extraido de grãos sadios, verdes e preto-verdes, e a influência na adição deste na sensorial de café liofilizado

The application of roasted coffee oil directly on freeze dried soluble coffee is used to minimize the formation of fine poder which adhere on the glass packaging, which results in a negative visual appearance, as well as contributes to the aromatic impact when opening the packaging. The coffee oil is considered a high cost product obtained from Arabica coffee beans, previosly selected and roasted, by mechanical press. In Brazil the coffee culture and marketing results in the selection of the beans by type of defects and beverage, the volume of exportation works with types of coffee beans with low quantity of defects resulting in a large volume of defective coffee beans trading on the domestic market. Nevertheless, coffees which present defective grains like green, black-green beans present differences in the final flavor of the roasted coffees. The aim of this study was to evaluate the chemical composition of the oils extracted from grains classified as normal, green and black-green, at natural and roasted conditions. The oil was obtained by cold extraction using solvents of different polarities, and yield was calculated as well as its fatty acid composition. The oil of the roasted defective coffee grains was also used to prepare drinks of lyophilized soluble coffee in order to verify if jugdes were able to differenciate the sensory caracteristics of the beverages, in comparison to the product prepared using commercial oil obtained by mechanical pressing. Samples of oil obtained from defective grains showed similar extraction yields compared to hot extraction. Cromatographs of oils of the deffective grains did not showed differences compared to normal grains. In relation to the sensory analysis of the soluble coffee beverages, it was verified that when applying oils of light roasted black-green beans or oils of medium and dark roasted green beans obtained with rapid process, judges had more difficulties to distinguish differences between samples. Economic viability demonstrated that with the actual prices of the coffee beans, the use of defective beans could reduce in 64% the costs of the raw beans.

Effects of light quality supplied by light emitting diodes (LEDs) on microalgal production

Dissertação de mestrado, Aquacultura, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Large scale laboratory cultures of Ankistrodesmus gracilis (Reisch) Korsikov (Chlorophyta) and Diaphanosoma biergei Korinek, 1981 (Cladocera)

O objetivo deste trabalho foi avaliar o cultivo em larga escala de Ankistrodesmus gracilis e Diaphanososma birgei em laboratório através do estudo da biologia das espécies, composição bioquímica e custo operacional de produção. A. gracilis apresentou um crescimento exponencial até o sexto dia, ao redor de 144 x 10(4) células mL-1. Logo em seguida, sofreu um brusco decréscimo apresentando 90 x 10(4) células mL-1 (oitavo dia). A partir do décimo primeiro dia, as células algais tenderam a crescer novamente, apresentando um máximo de 135 x 10(4) células mL-1 no 17º dia. No cultivo de D. birgei, foi observado o primeiro pico de crescimento no nono dia com 140 x 10² indivíduos L-1, aumentando novamente a partir do décimo segundo dia. A alga clorofícea A. gracilis e o zooplâncton D. birgei possuem aproximadamente 50 e 70% de proteína (PS), respectivamente, com teor de carboidrato acima de 5%. A eletricidade e mão de obra foram os itens mais dispendiosos e, de acordo com os dados obtidos, a temperatura, nutrientes, disponibilidade de luz e manejo do cultivo, foram fatores determinantes sobre a produtividade. Os resultados indicam que o meio NPK (20-5-20) pode ser utilizado diretamente como uma alternativa de cultivo em larga escala, considerando o baixo custo de produção, promovendo adequado crescimento e valor nutricional para A. gracilis e D. birgei.

Effects of dark or of red, blue or white light on germination of subterranean clover seeds

Dry or imbibed seeds of the negatively photoblastic burr burying subterranean clover cv. ‘Seaton Park’ were treated with dark or with red, blue or white light to evaluate the effects of light on seed germination. Dry seeds treated with constant white light, red light or blue light during 8 days and subsequently incubated in dark had final germination and duration of germination reduced, and the distribution of germination changed from highly asymmetric to symmetric respectively. Imbibing seeds incubated under constant blue or white light had final germination strongly reduced seven days after sowing (7.3% and 50.1% of the germination under dark) with significant differences between them. After transferral to dark, true complete recovery of germination of seeds treated with white light was observed 19 days after sowing, but only partial recovery in seeds treated with blue light. Results of dry and imbibed seeds are consistent with no activity of phytochromes, as expected in negatively photoblastic seeds. Results of dry seeds are seemingly contradictory because total germination data imply the inactivity of red and blue light photoreceptors, the opposite being implied by duration and shape of germination. A tentative hypothetical solution for the contradiction is presented. Results of imbibed seeds are fully consistent with cryptochromes but not with phototropins mediation of responses to light of seed germination in ‘Seaton Park’. The ecological and adaptive significance of such responses are discussed in the framework of light attenuation in soil and the requirement and ability of subterranean clover ‘Seaton Park’ to bury seeds.

The role of vision in obese and normal-weight children's gait control

Previous research has suggested that perceptual-motor difficulties may account for obese children's lower motor competence; however, specific evidence is currently lacking. Therefore, this study examined the effect of altered visual conditions on spatiotemporal and kinematic gait parameters in obese versus normal-weight children. Thirty-two obese and normal-weight children (11.2 ± 1.5 years) walked barefoot on an instrumented walkway at constant self-selected speed during LIGHT and DARK conditions. Three-dimensional motion analysis was performed to calculate spatiotemporal parameters, as well as sagittal trunk segment and lower extremity joint angles at heel-strike and toe-off. Self-selected speed did not significantly differ between groups. In the DARK condition, all participants walked at a significantly slower speed, decreased stride length, and increased stride width. Without normal vision, obese children had a more pronounced increase in relative double support time compared to the normal-weight group, resulting in a significantly greater percentage of the gait cycle spent in stance. Walking in the DARK, both groups showed greater forward tilt of the trunk and restricted hip movement. All participants had increased knee flexion at heel-strike, as well as decreased knee extension and ankle plantarflexion at toe-off in the DARK condition. The removal of normal vision affected obese children's temporal gait pattern to a larger extent than that of normal-weight peers. Results suggest an increased dependency on vision in obese children to control locomotion. Next to the mechanical problem of moving excess mass, a different coupling between perception and action appears to be governing obese children's motor coordination and control.

Dynamics of rod and cone photoreceptor interactions under mesopic light levels

Visual adaptation regulates contrast sensitivity during dynamically changing light conditions (Crawford, 1947; Hecht, Haig & Chase, 1937). These adaptation dynamics are unknown under dim (mesopic) light levels when the rod (R) and long (L), medium (M) and short (S) wavelength cone photoreceptor classes contribute to vision via interactions in shared non-opponent Magnocellular (MC), chromatically opponent Parvocellular (PC) and Koniocellular (KC) visual pathways (Dacey, 2000). This study investigated the time-course of adaptation and post-receptoral pathways mediating receptor specific rod and cone interactions under mesopic illumination. A four-primary photostimulator (Pokorny, Smithson & Quinlan, 2004) was used to independently control the activity of the four photoreceptor classes and their post-receptoral visual athways in human observers. In the first experiment, the contrast sensitivity and time-course of visual adaptation under mesopic illumination were measured for receptoral (L, S, R) and post-receptoral (LMS, LMSR, L-M) stimuli. An incremental (Rapid-ON) sawtooth conditioning pulse biased detection to ON-cells within the visual pathways and sensitivity was assayed relative to pulse onset using a briefly presented incremental probe that did not alter adaptation. Cone.Cone interactions with luminance stimuli (L cone, LMS, LMSR) reduced sensitivity by 15% and the time course of recovery was 25± 5ms-1 (μ ± SEM). PC mediated (+L-M) chromatic stimuli sensitivity loss was less (8%) than for luminance and recovery was slower (μ = 2.95 ± 0.05 ms-1), with KC mediated (S cone) chromatic stimuli showing a high sensitivity loss (38%) and the slowest recovery time (1.6 ± 0.2 ms-1). Rod-Rod interactions increased sensitivity by 20% and the time course of recovery was 0.7 ± 0.2 ms-1 (μ ± SD). Compared to these interaction types, Rod-Cone interactions reduced sensitivity to a lesser degree (5%) and showed the fastest recovery (μ = 43 ± 7 ms-1). In the second experiment, rod contribution to the magnocellular, parvocellular and koniocellular post-receptoral pathways under mesopic illumination was determined as a function of incremental stimulus duration and waveform (rectangular; sawtooth) using a rod colour match procedure (Cao, Pokorny & Smith, 2005; Cao, Pokorny, Smith & Zele, 2008a). For a 30% rod increment, a cone match required a decrease in [L/(L+M)] and an increase in [L+M] and [S/(L+M)], giving a greenish-blue and brighter appearance for probe durations of 75 ms or longer. Probe durations less than 75 ms showed an increase in [L+M] and no change in chromaticity [L/(L+M) or S/(L+M)], uggesting mediation by the MC pathway only for short duration rod stimuli. s We advance previous studies by determining the time-course and nature of photoreceptor specific retinal interactions in the three post-receptoral pathways under mesopic illumination. In the first experiment, the time-course of adaptation for ON cell processing was determined, revealing opponent cell facilitation in chromatic PC and KC pathways. The Rod-Rod and Rod-Cone data identify previously unknown interaction types that act to maintain contrast sensitivity during dynamically changing light conditions and improve the speed of light adaptation under mesopic light levels. The second experiment determined the degree of rod contribution to the inferred post-eceptoral pathways as a function of the temporal properties of the rod signal. r The understanding of the mechanisms underlying interactions between photoreceptors under mesopic illumination has implications for the study of retinal disease. Visual function has been shown to be reduced in persons with age-related maculopathy (ARM) risk genotypes prior to clinical signs of the disease (Feigl, Cao, Morris & Zele, 2011) and disturbances in rod-mediated adaptation have been shown in early phases of ARM (Dimitrov, Guymer, Zele, Anderson & Vingrys, 2008; Feigl, Brown, Lovie-Kitchin & Swann, 2005). Also, the understanding of retinal networks controlling vision enables the development of international lighting standards to optimise visual performance nder dim light levels (e.g. work-place environments, transportation).

Hybrid graphene/titania nanocomposite : interface charge transfer, hole doping, and sensitization for visible light response

We demonstrated for the first time by large-scale ab initio calculations that a graphene/titania interface in the ground electronic state forms a charge-transfer complex due to the large difference of work functions between graphene and titania, leading to substantial hole doping in graphene. Interestingly, electrons in the upper valence band can be directly excited from graphene to the conduction band, that is, the 3d orbitals of titania, under visible light irradiation. This should yield well-separated electron−hole pairs, with potentially high photocatalytic or photovoltaic performance in hybrid graphene and titania nanocomposites. Experimental wavelength-dependent photocurrent generation of the graphene/titania photoanode demonstrated noticeable visible light response and evidently verified our ab initio prediction.