Patterning of the neural tube establishes midbrain and hindbrain structures that coordinate motor movement, process sensory input and integrate cognitive functions. Cellular impairment within these structures underlies diverse neurological disorders, and in vitro organoid models promise inroads to understanding development and modeling disease. Here, we use paired single-cell transcriptome and accessible chromatin sequencing to map cell composition and regulatory mechanisms in organoid models of midbrain and hindbrain. We find that existing midbrain organoid protocols generate ventral and dorsal cell types, covering regions including floor plate, dorsal and ventral midbrain and adjacent hindbrain regions. Gene regulatory network inference and transcription factor perturbation resolve mechanisms underlying neuronal differentiation. A single-cell multiplexed patterning screen identifies morphogen concentrations that expand existing organoid models, including conditions generating medulla glycinergic neurons and cerebellum glutamatergic subtypes. Together, the multi-omic atlas and morphogen screen reveal morphogen-regulon relationships guiding region-specific progenitor differentiation towards diverse neuron types of the posterior brain.

Defining the spatiotemporal expression pattern of Rap1 in the chicken embryonic spinal cord.

dorsoventral polarity of the neural tube is based on molecular patterning established during neurulation and is suggested to be induced by fine-tuned interpretation of opposing morphogen gradients in the neural tube: a ventral-to-dorsal gradient is formed by shh secreted from floor plate and notochord whereas a dorsal-to-ventral gradient is created by BMP and Wnt originating from the roof plate. Recent results revealed an unexpected early induction of Shh in the chick prospective floor plate prior to notochord formation. Hence, we addressed the questions as to whether key components of dorsoventral patterning show 'precocious' expression in chick and Xenopus as well and whether hedgehog signaling contributes to this early patterning. mRNA expression of key dorsoventral markers was analysed by in-situ-hybridisation between late gastrulation and neurulation stages of chick and Xenopus laevis embryos. Involvement of hedgehog signaling was analysed by expression of selected marker genes after experimental inhibition and activation using Cyclopamine and SAG respectively, in the chick. The study revealed mediolateral pattern emerging during chick gastrulation in that ventral markers Nkx6.1 and Nkx6.2 display in the emerging neural plate a medial and bilateral paramedian expression, respectively, while Nkx2.2 expression is also confined medially, but is delayed until the beginning of the neural tube closure. The dorsal markers Pax7 and Pax3, associated with both dorsal neural tube and neural crest, display bilateral lateral expression during gastrulation while Pax3, is expressed, in addition, in the posterior primitive streak region. In Xenopus laevis, Nkx6.2 and Pax3 show a reciprocal mediolateral pattern at the beginning of gastrulation while Nkx6.1 is expressed medially starting at late gastrulation. After activation and inhibition of hedgehog signaling the newly defined mediolateral Nkx6.2 and Pax7 domains appeared to be unchanged. dorsoventral gene expression is preceded by topographically "homologous" mediolateral expression of some key components. The initial functional analysis of early hedgehog signaling in the chick points to as yet unknown mediolateral interactions during gastrulation which lead to prospective dorsoventral differentiation of the neural plate.

Dynein axonemal assembly factors (DNAAF) are essential for the assembly and transport of dynein motor complexes, which are crucial for the movement of cilia. Mutations in genes encoding these proteins often lead to motile ciliopathies called primary ciliary dyskinesia (PCD). In humans, loss-of-function mutations of Cilia and flagella-associated protein 300 (CFAP300, also known as DNAAF17) have been reported to cause PCD.The function of CFAP300 during embryogenesis, however, has not been reported. We carried out functional studies using zebrafish to understand its function during vertebrate development. The expression of cfap300 mRNA during zebrafish embryogenesis was identified using whole-mount in situ hybridization (WISH). A knockout zebrafish line was created using TALEN and the function of cfap300 during embryogenesis was studied. cfap300 mRNA is highly expressed in the zebrafish pronephros. Hence, the effect of cfap300 knock-out on zebrafish pronephros segmentation, multiciliated cell (MCC) and Corpuscle of Stannius (CS) development was studied by analysing the expression of marker genes in these segments and cells using WISH. The gene expression during CS gland development was analysed using RT-qPCR and digital PCR. In zebrafish embryos,cfap300is expressed in multiple ciliated organs, beginning at 10 hpf in Kupffer's vesicle and later in the notochord, pronephros, otic placode, floor plate, brain, and tail bud. cfap300 mutants develop normally and show no nephron segmentation defects, but exhibit impaired CS development. The CS gland is an endocrine gland in teleost fish, including zebrafish, that secretes Stanniocalcin 1 (Stc1) hormone and regulates calcium homeostasis. The CS gland originates by transdifferentiation of pronephric tubule epithelial cells at the distal early (DE) and distal late (DL) boundary. Mechanistically, hnf1ba expression is increased in the cfap300 mutants, which in turn drives transcription of cdh17 and leads to impaired CS precursor transdifferentiation. In cfap300 mutants, knockdown of hnf1ba or knockout of cdh17 restores CS gland formation. These results uncover a Cfap300-Hnf1ba-Cdh17 pathway that regulates epithelial-to-endocrine transdifferentiation, revealing a novel, lineage-specific role for a DNAAF protein in endocrine organogenesis.

During neural tube patterning, a gradient of Sonic hedgehog (Shh) signaling specifies ventral progenitor fates. The cellular response to Shh is processed through a genetic regulatory network (GRN) to specify distinct fate decisions. This process integrates Shh response level, duration, and other inputs and is affected by noise in signaling and cell position. How reliably the Shh response profile predicts the fate choice of a single cell remains unclear. Here, we use live imaging to track neural progenitors in developing zebrafish and quantify both Shh and fate reporters in single cells over time. We found that there is significant heterogeneity between Shh response and fate choice in single cells. We quantitatively modeled reporter intensities to obtain single-cell response levels over time and systematically evaluated their correlation with cell fate specification. Motor neuron progenitors (pMNs) exhibit a high degree of variability in their Shh responses, which is particularly prominent in the posterior neural tube where the Shh response dynamics are similar to those of the more ventrally fated lateral floor plate cells (LFPs). Our results highlight the precision limit of morphogen-interpretation GRNs in small and dynamic target cell fields.

C-mannosyltransferase Dpy19l1l regulates body axis formation via secretion of SCO-spondin in zebrafish.

Dorsoventral (DV) patterning of the spinal cord (SC) is orchestrated by morphogen gradients that specify distinct neural progenitor domains, the dorsal roof plate (RP), ventral floor plate (FP), and delaminating neural crest cells (NCCs). While foundational insights into SC patterning have been gained from animal models, key aspects - such as the role of retinoic acid (RA), dynamics of NCC lineage development, and human-specific features - remain poorly understood due to limitations in existing in vivo and in vitro models. Here, we present a human pluripotent stem cell (hPSC)-derived SC model, termed microfluidic SC-like structures (µSCLSs), generated by applying bioengineered, antiparallel morphogen gradients via a microfluidic platform over micropatterned hPSC colonies. The µSCLS robustly recapitulates complete DV patterning with human specific transcriptional signatures. Using this platform, we uncover a previously unrecognized RA-BMP signaling crosstalk that could explain conflicting reports on the role of RA in SC DV patterning. We further demonstrate lineage-specific ventral migration of NCCs in response to chemoattractant cues, enabling direct visualization and mechanistic dissection of sensory vs. other fate trajectories. This controllable, reproducible, and human-relevant model provides a powerful system for probing human SC development, neural crest biology, and disease modeling with unprecedented resolution.

Smart lighting has moved from a niche building automation feature to a strategic infrastructure layer for high-performance buildings, campuses, and public-realm developments. In Saudi Arabia, this shift is particularly significant because Vision 2030 links digital transformation, energy productivity, sustainable urbanism, and quality-of-life improvements to the delivery of new districts, giga-projects, and retrofit programs. This review examines how smart lighting control systems can support energy conservation across Saudi Vision 2030 developments, including residential communities, commercial buildings, institutional campuses, mixed-use districts, public space, and transport corridors. The paper is structured as a PRISMA-guided review and synthesizes peer-reviewed and institutional sources published from 2020 to early 2026. The literature screened for the review covered LED efficacy, occupancy and vacancy sensing, daylight harvesting, scheduling, addressable controls, wireless communication, Internet of Things integration, building energy management systems, digital twins, artificial intelligence, public-lighting analytics, Saudi policy frameworks, and implementation challenges in hot-arid climates. The synthesis shows that the strongest savings do not come from any single technology alone. Rather, the most credible energy-conservation pathway combines high-efficacy LED luminaires, robust commissioning, zone-level sensing, daylight-responsive dimming, adaptive scheduling, and integration with broader building or city-management platforms. For Saudi developments, climate responsiveness matters: deep floor plates, high solar exposure, glare control, dust, maintenance cycles, and the interaction between lighting, cooling loads, and occupant comfort all affect system performance. The review also finds that the value proposition of smart lighting is widening beyond operational electricity savings to include demand flexibility, maintenance intelligence, user wellbeing, urban safety, and carbon-aware asset management. Based on the evidence, the paper proposes a Saudi-oriented implementation framework that aligns lighting design, controls architecture, governance, procurement, and post-occupancy verification. The paper concludes that smart lighting is one of the most scalable and near-term opportunities for energy conservation in Vision 2030 developments, but its full impact depends on standards alignment, interoperable controls, data governance, commissioning discipline, and life-cycle decision making.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are among the most common genetic causes of Parkinson's disease (PD), yet substantial heterogeneity exists among pathogenic variants. How mutations in distinct functional domains of LRRK2 differentially perturb cellular homeostasis remains incompletely understood. Here, we compared two pathogenic LRRK2 mutations-G2019S in the kinase domain and I1371V in the GTPase domain-across multiple cellular models, including SH-SY5Y and U87 cells, and healthy human iPSC-derived floor plate cells. We demonstrate that the I1371V mutation induces markedly more severe cellular dysfunction than G2019S. I1371V-expressing cells exhibited elevated LRRK2 autophosphorylation at S1292 and robust hyperphosphorylation of Rab8A and Rab10, indicating enhanced downstream signaling. These alterations impaired sterol trafficking, leading to selective depletion of membrane cholesterol without changes in total cellular cholesterol. Consequently, I1371V cells displayed increased membrane fluidity, disrupted microdomain organization, altered membrane topology, reduced caveolin-1 expression, and impaired dopamine transporter surface expression and dopamine uptake. Lipidomic profiling further revealed a broad disruption of lipid homeostasis, including reductions in cholesteryl esters, sterols, sphingolipids, and glycerophospholipids, whereas G2019S cells showed comparatively modest changes. Pharmacological intervention revealed mutation-specific responses, with the non-selective LRRK2 modulator GW5074 outperforming the kinase-selective inhibitor MLi-2 in restoring Rab8A phosphorylation, membrane integrity, and dopaminergic function. Collectively, these findings identify membrane lipid dysregulation as a central cell biological mechanism in LRRK2-associated PD and underscore the importance of variant-specific therapeutic strategies.

Hedgehog signaling is a conserved developmental pathway that patterns diverse tissues during vertebrate embryogenesis. In zebrafish, disruptions to the hedgehog pathway cause well-characterized defects in specific cell types including neurons and glia derived from the ventral neural tube. We inhibited hedgehog signaling by overexpressing the Gli3 repressor ubiquitously and performed bulk RNA sequencing of 30 h postfertilization zebrafish embryos. Consistent with known roles of hedgehog signaling, we observed reduced expression of genes marking lateral floor plate, motor neurons, Kolmer-Agduhr cells, dopaminergic neurons, slow muscle cells, and anterior pituitary. Gene set enrichment analysis using marker genes derived from the Daniocell atlas also revealed downregulation of genes marking H+-ATPase-rich ionocytes, which are located in the embryonic skin and are responsible for osmotic homeostasis. Reduced expression of ionocyte-specific transporter genes and the transcription factor foxi3a suggests that Gli activity may play a previously unrecognized role in the specification of this cell type.

The potassium voltage-gated channel Kv2.1 plays a crucial role in the development of the brain's ventricular system. Defects in the development of this system affect the formation of the Reissner fiber, a rope-like structure produced by the flexural and subcommissural organs that secrete Scospondin. The development of the Reissner fiber has been studied during normal development and in zebrafish mutants deficient in activity of the two Kv2.1 subunits - Kcnb1 and Kcng4b using a combination of immunohistochemistry and transgenic lines expressing EGFP in the subcommissural organ and floor plate. The Reissner fiber develops in stages. First, the midline floor plate cells, originating from the embryonic organizer, secrete Scospondin, forming the posterior Reissner fiber. This allows us to define the posterior Reissner fiber as the acellular derivative of the embryonic organizer. The fiber separates from the floor plate, beginning in the hindbrain and extends through the neural tube, from the most anterior floor plate (i.e. the flexural organ) anteriorly to the ampulla terminalis. Second, the subcommissural organ, which is derived from the anterior roof plate, begins secreting Scospondin. This forms the anterior Reissner fiber, which spans the cerebral aqueduct. Third, the anterior Reissner fiber connects to the flexural organ, where the two fibers fuse. Fourth, after the floor plate ceases to express Scospondin, the Reissner fiber derived from the subcommissural organ replaces the transient posterior fiber derived from the floor plate. Like the subcommissural organ, the flexural organ is an attachment point for the Reissner fiber. Reissner fiber assembly involves the formation of individual microfilaments that fuse in several steps to form the single fiber. Analysis of zebrafish mutants of Kv2.1 subunits (Kcnb1 and Kcng4b) revealed that Kv2.1 negatively regulates Scospondin production at several levels. These mutations have opposing effects on the transcript levels of several genes involved in Reissner fiber development (sspo, lgals2, and chl1a/camel), affect the subcommissural organ and microfilament formation, and impact Reissner fiber assembly.

Unbiased, scalable behavioral phenotyping that captures multi-animal interactions in home-cage settings is increasingly needed. Here we present ‘IntelliProfiler’, a research workflow consisting of data processing scripts that extract locomotor activity and pairwise proximity from a commercially available, previously validated, high-resolution radio frequency identification floor plate. IntelliProfiler is not a standalone system; it operates on data acquired with the Phenovance floor plate and is not yet validated with other hardware configurations. The workflow reconstructs individual trajectories and positions of multiple mice, enabling long-term assessment of locomotor activity and social spacing. In proof-of-concept analyses, male mice placed in a novel cage environment maintained greater inter-animal distances than female mice, an effect that strengthened as group size increased. Aging reduced locomotor activity in a group size-dependent manner and altered proximity patterns. In addition, offspring of aged fathers (a paternal-aging autism spectrum disorder model) exhibited hyperactivity and increased social distance relative to controls, consistent with autism spectrum disorder-related phenotypes and motivating further investigations. Together, these findings demonstrate that IntelliProfiler workflow provides a practical and versatile approach for screening group dynamics and quantifying complex social behaviors in neuroscience research.

This pragmatic review addresses those protocols containing innovative steps for differentiating human inducible Pluripotent Stem Cells (iPSCs) into mature midbrain dopaminergic (mDA) neurons, leading to successful implementation in other articles of in vitro models for neurodegenerative or psychiatric disorders and for pharmacological tests, in the period 2004-2025. Article were selected according to the presence of protocols that allowed the iPSC differentiation into mDA neurons as defined by the expression of DA neural lineage markers and Thyrosine Hydroxylase, by the evidence of neuronal morphology, electrophysiology, neurochemistry, biomarkers and intracellular depositions that recapitulate various stages of differentiation and maturation of the mammalian brain mDA neuron. The critical innovative aspects that allowed the article selection was the presence of a significative methodological step in increasing the protocol performance and the useful replicability in term the number of peer-reviewed articles implementing this procedure. Out of 108 articles of protocols based on stem cells-derived mDA neurons, we identified 12 articles regarding 2-dimensional (2D) cultures and 11 articles regarding 3-dimensional (3D)/organoid cultures. For 2D cultures, the most effective protocol would include a floor plate induction with dual-SMAD inhibition improved by a biphasic WNT activation process and by a pre-differentiation step of neural precursor expansion. Regarding the more recent protocols for 3D midbrain-like organoids, less disease model articles were generated, requiring more time for assessing of the most performing protocols. 3D/organoids were found superior to 2D cultures since they better represent the original midbrain neuronal circuitry, allowing better personalized disease modelling.

Failure to close the cranial neural tube, known as exencephaly/anencephaly, is a lethal congenital defect. However, the mechanisms driving patterning and reshaping of the broad cranial neural folds are poorly understood. Loss of the primary cilium-localized G protein-coupled receptor GPR161 causes ectopic, excessive hedgehog signaling in the mouse neural tube and fully penetrant exencephaly. GPR161 promotes GLI3 transcriptional repressor (GLI3R) formation while preventing GLI2 transcriptional activator formation. Here, we studied the mechanisms underlying cranial closure in mice using a Gpr161 mutant allelic series, epistasis between Gpr161 knockout and GLI effectors, and in toto imaging of cell behavior. A functional non-ciliary Gpr161 knock-in implicated GPR161 ciliary localization directly in initiation and maintenance of cranial closure. Furthermore, Gli3R expression, but not Gli2 loss, rescued exencephaly in Gpr161 knockout mice. GLI3R specifically restricted forebrain ventral floor plate expansion and mediated apical constriction in the lateral midbrain neural folds prior to closure. These results reveal metamere-specific, cilia-dependent hedgehog repression thresholds in control of spatially restricted gene expression and dynamic cell behavior during cranial closure. Targeted interventions increasing hedgehog repression could ameliorate regional cranial defects.

Abstract New discoveries prompt reinterpretations of one of the most enigmatic echinoderm classes, the Cyclocystoidea. Exceptionally well-preserved cyclocystoids have been discovered in the Upper Ordovician (Katian) Kirkfield and Verulam formations in the Lake Simcoe region of southern Ontario, Canada. Four different cyclocystoid taxa are present, including Cyclocystoides cf. C. scammaphoris , Nicholsodiscus cf. N. anticostiensis , Zygocycloides marstoni , and Brechincycloides stanhynei new genus new species. These specimens reveal that the central disk of cyclocystoids consists largely of perforate extraxial plates and that the ever-present sutural pores are epispires surrounded by annular plates that may or may not be in lateral contact with each other. The specimens of B. stanhynei n. gen. n. sp. are significant because they are the first cyclocystoid fossils to reveal that the hydropore/gonopore and periproct are positioned on the aboral side of the central disk. The suggestively complex marginal ring, previously of undetermined origin, represents articulated floor plates in a circular arrangement supporting a circumferential water vascular system. Furthermore, the marginal ring reveals growth zones that correspond to axes of pentaradial symmetry. In the new interpretation, cupules are tube foot basins, and channels through the marginal ossicles can be explained best in the context of the axial origin as floor plates accompanied by a cover plate system. Application of the Extraxial–Axial Theory (EAT) compels a revised understanding of key anatomies comparable with other early radial echinoderms. We document evidence for cyclocystoid orientation in life with epispires facing upward and the tube foot basins in the floor plates directed towards the substrate. The new family Brechincycloididae is proposed for all cyclocystoids whose apomorphy consists of a flat, attenuated shelf tapering away from the proximal side of each floor plate. UUID: http://zoobank.org/605fdf27-b38b-4d41-824b-d10b139f90f5

Structural work, especially in the casting process, plays an important role. One of the methods that can be used is casting with ready mix concrete, because of the better time efficiency than conventional methods. The purpose of this study is to find out about the stages, specifications and technical requirements on the 2nd floor plate. A case study on the construction of the Tabanan Public Service Mall located in Banjar Anyar, Kediri District, Tabanan Regency. The research method used is a quantitative method, the data used is the Cost Budget Plan (RAB) analyzed to obtain the description of the work and volume, the plan drawing is used for the creation of work drawings, the time schedule is used as a reference in the duration of the work, the Unit Price Analysis of Work is used to obtain the resource coefficient and resource price. From the job description data, the stages of the activity are obtained, the plan drawings produce work drawings, and the time schedule produces the duration of the work. The results of the study obtained that in the stages of casting work with ready mix on the 2nd floor plate in scaffolding work there are 5 stages of activities, bodeman work 3 stages of activities, B1 beam formwork 30 x 50 cm and B2 beam formwork 25 x 35 cm there are 4 stages of activities, B1 beam and B2 beam ironing work there are 3 stages of activities, Formwork with bondek plates has 5 stages of activities, wiremesh ironing work has 3 stages of activities, casting work with ready mix on the 2nd floor plate has 3 stages of activities.

Neuromesodermal progenitors (NMPs) are a vertebrate cell type that contribute descendants to both the spinal cord and the mesoderm. The undifferentiated bipotential NMP state is maintained when both Wnt signaling is active and Sox2 is present. We used transgenic zebrafish reporter lines to live-image both Wnt activity and Sox2 levels in NMPs and observed a unique cellular ratio in NMPs compared to NMP-derived mesoderm or neural tissue. We used this unique signature to identify the previously unknown anatomical position of a progenitor population that gives rise to midline tissues of the floor plate of the spinal cord and the mesodermal notochord. Thus, quantification of the active Wnt signaling to Sox2 ratio can be used to predict and identify cells with neuromesodermal potential. We also developed the auxin-inducible 2 degron system for use in zebrafish to test the temporal role that Sox2 plays during midline formation. We found that ectopic Sox2 in the presence of Wnt activity holds cells in the undifferentiated floor plate/notochord progenitor state, and that degradation of the ectopic Sox2 is required for cells to adopt a notochord fate.

SummaryIn vitro modelling of highly vulnerable nigral dopaminergic (DA) neuronal subtypes in Parkinson’s disease (PD), is necessary for studying disease mechanisms. Here, we optimized a new approach by expressing the pioneer neurogenic transcription factor, Achaete-scute-like 1 (Ascl1), implicated in determining dopaminergic fate. Sequential small-molecule patterning of iPSCs into early floor plate mesencephalic progenitors, followed by inducible Ascl1 expression, rapidly differentiates midbrain DA neurons. Immunocytochemistry and transcriptomic analysis of these patterned Ascl1-driven DA neurons (PA-DANs) confirmed midbrain-lineage specificity. Importantly, we found an enrichment of DA subpopulations that corresponded to the adult human ventral SOX6-positive A9 DA subtypes vulnerable in PD. Furthermore, we combined these ventral A9-like PA-DANs with human iPSC-derived midbrain astrocytes and microglia in defined ratios to generate mature 3D A9-like assembled organoids that display characteristic spontaneous neuronal activity and electrical propagation along the axon. Our method efficiently generates a mature and functional A9-like DA neuronal platform to study PD.

Abstract On the component level, floor plates are one of the most carbon intensive building elements with a disproportionally high global warming potential (GWP) compared to their built volume. The challenge they pose to engineers and architects is the high mass needed to ensure acoustic comfort while spanning long distances. Mass timber solutions lack economic competitiveness due to their inefficient use of material, enormous content of adhesives, and poor acoustic performance. The market for sustainable solutions has hence shifted towards timber-concrete composite floor plates that improve material and economic efficiency at the cost of an increased environmental footprint and reduced circularity. This paper introduces the development of a timber-earth floor plate based on the application of large-scale earth blocks as a carbon negative alternative. Earth blocks have recently undergone a renaissance in Europe, with building regulations allowing their inclusion as load-bearing masonry units, and manufacturers increasingly developing scaled solutions to produce them at a competitive price point. Through their research, the authors have designed, prototyped, and evaluated a composite floor plate that capitalises on the scaled production capacity of earth blocks, and designed a fully circular assembly. The proposed system fully substitutes hydraulically bound materials and improves the effectiveness of timber. Besides presenting the development, testing, and comparative life-cycle assessment (LCA) of the building solution, the paper documents the process of industrially scaling the production of the earth blocks. Through the repurposing of the existing production infrastructure of a brick factory, the researchers were able to produce the product at an attractive price point. Ultimately, the authors demonstrate that their nature-based floor slab assembly invites for decentralised, global replication, advocates for the repurposing of existing infrastructure, and promotes a material culture within planetary boundaries.

Spinal commissural neurons play a fundamental role in motor control and sensory perception. Robo3, a receptor expressed on pre-crossing commissural axons, is required for midline crossing. Its downregulation in post-crossing axons is essential for forming synapses with contralateral CNS targets. We demonstrate that, at the transcriptional level, the dynamic expression of Robo3 in pre-crossing neurons is controlled by incoherent feed-forward loops (iFFLs) and negative feedback loops (NFLs) that precisely regulate its transience and thereby determine midline crossing. Lhx2 and Lhx9 activate Robo3 while also inducing its repressor, Barhl2. Additionally, negative feedback loops fine-tune the transience of Robo3 expression by adjusting the relative strength of the activation and repression modules. Our genomic analysis reveals that this regulatory circuitry converges on an enhancer element of the Robo3 gene. These findings imply that diverse iFFLs, together with NFLs, are essential for regulating the extent of midline crossing across different spinal commissural subtypes.