Division Potential Research Articles

Controversy Over Non-Madhhab Practices

This study aims to thoroughly examine the dangers associated with not adhering to a specific madhhab in contemporary Islamic law from the perspective of Muhammad Sa'id Ramadhan Al-Buthy. In order to accurately achieve the study objective, a comprehensive examination of Al-Buthy’s critique concerning non-madhhab practices was carried out, with a primary focus on how these practices could disrupt legal interpretations and undermine the authority of Islamic jurisprudence. The analysis included the process of reviewing primary texts, scholarly commentaries, and secondary literature, all of which were carried out with the aim of accurately comparing the critique by Al-Buthy with other contemporary perspectives on the issue. The results showed that Al-Buthy strongly criticized non-madhhab movement for promoting subjective and inconsistent interpretations of Islamic law. The figure argued that this movement was capable of weakening the structured and scholarly tradition of Islamic jurisprudence. Furthermore, Al-Buthy emphasized that established madhhab played a very important role in preserving the integrity and continuity of Islamic legal principles, underscoring the dangers associated with abandoning these schools, including fragmentation and a lack of authoritative guidance. The critique of the observed figure typically emphasizes the risks of non-madhhab practices, such as the potential for division in Muslim community. This study makes a novel contribution by specifically focusing on Al-Buthy’s critique, which are often cited but not deeply analyzed in the context of non-madhhab movements. Specifically, the investigation situated the arguments of the observed figure in contemporary debates on the role of madhhab, thereby offering a fresh perspective on the relationship between traditional Islamic jurisprudence and modern legal challenges. The implications are particularly significant for scholars, legal practitioners, and policymakers in Islamic law, advocating for a renewed commitment to traditional legal frameworks to prevent the pitfalls of subjective interpretations associated with non-madhhab methodologies.

Functional omics of ORP7 in primary endothelial cells

BackgroundMany members of the oxysterol-binding protein-related protein (ORP) family have been characterized in detail over the past decades, but the lipid transport and other functions of ORP7 still remain elusive. What is known about ORP7 points toward an endoplasmic reticulum and plasma membrane-localized protein, which also interacts with GABA type A receptor-associated protein like 2 (GABARAPL2) and unlipidated Microtubule-associated proteins 1A/1B light chain 3B (LC3B), suggesting a further autophagosomal/lysosomal association. Functional roles of ORP7 have been suggested in cholesterol efflux, hypercholesterolemia, and macroautophagy. We performed a hypothesis-free multi-omics analysis of chemical ORP7 inhibition utilizing transcriptomics and lipidomics as well as proximity biotinylation interactomics to characterize ORP7 functions in a primary cell type, human umbilical vein endothelial cells (HUVECs). Moreover, assays on angiogenesis, cholesterol efflux, and lipid droplet quantification were conducted.ResultsPharmacological inhibition of ORP7 leads to an increase in gene expression related to lipid metabolism and inflammation, while genes associated with cell cycle and cell division were downregulated. Lipidomic analysis revealed increases in ceramides and lysophosphatidylcholines as well as saturated and monounsaturated triacylglycerols. Significant decreases were seen in all cholesteryl ester and in some unsaturated triacylglycerol species, compatible with the detected decrease of mean lipid droplet area. Along with the reduced lipid stores, ATP-binding cassette subfamily G member 1 (ABCG1)-mediated cholesterol efflux and angiogenesis decreased. Interactomics revealed an interaction of ORP7 with AKT1, a central metabolic regulator.ConclusionsThe transcriptomics results suggest an increase in prostanoid as well as oxysterol synthesis, which could be related to the observed upregulation of proinflammatory genes. We envision that the defective angiogenesis in HUVECs subjected to ORP7 inhibition could be the result of an unfavorable plasma membrane lipid composition and/or reduced potential for cell division. To conclude, the present study suggests multifaceted functions of ORP7 in lipid homeostasis, angiogenic tube formation, and gene expression of lipid metabolism, inflammation, and cell cycle in primary endothelial cells.

Urgensi Integritas Nasional Sebagai Salah Satu Parameter Persatuan dan Kesatuan Bangsa

Indonesia, an archipelago with rich culture and diverse ethnic groups, is faced with the challenge of maintaining national unity. National integrity, defined as a state in which all people have a sense of unity and love for the country, is an important parameter to achieve this goal. This paper discusses the urgency of national integration as one of the parameters of national unity. First, national integration can strengthen the sense of unity by fostering a sense of patriotism and nationalism among the people. Second, national integration can increase national stability by reducing the potential for conflict and division between community groups. Third, national integration can spur national development by creating a conducive and safe atmosphere for the implementation of development. Fourth, national integration can increase the nation's competitiveness by creating a society that is united and has a common goal to achieve national progress. Fifth, national integration can maintain territorial integrity by preventing the disintegration of the nation that can result in the division of the country.

Simulated lunar microgravity transiently arrests growth and induces osteocyte-chondrocyte lineage differentiation in human Wharton’s jelly stem cells

Human Wharton’s jelly stem cells (hWJSCs) are multipotent stem cells that are extensively employed in biotechnology applications. However, the impact of simulated lunar microgravity (sμG) on the growth, differentiation, and viability of this cell population is incompletely characterized. We aimed to determine whether acute (72 h) exposure to sμG elicited changes in growth and lineage differentiation in hWJSCs and if putative changes were maintained once exposure to terrestrial gravity (1.0 G) was restored. hWJSCs were cultured under standard 1.0 G conditions prior to being passaged and cultured under sμG (0.16 G) using a random positioning machine. Relative to control, hWJSCs cultured under sμG exhibited marked reductions in growth but not viability. Cell population expression of characteristic stemness markers (CD 73, 90, 105) was significantly reduced under sμG conditions. hWJSCs had 308 significantly upregulated and 328 significantly downregulated genes when compared to 1.0 G culture conditions. Key markers of cell replication, including MKI67, were inhibited. Significant upregulation of osteocyte–chondrocyte lineage markers, including SERPINI1, MSX2, TFPI2, BMP6, COMP, TMEM119, LUM, HGF, CHI3L1 and SPP1, and downregulation of cell fate regulators, including DNMT1 and EZH2, were detected in sμG-exposed hWJSCs. When returned to 1.0 G for 3 days, sμG-exposed hWJSCs had accelerated growth, and expression of stemness markers increased, approaching normal (i.e. 95%) levels. Our data support earlier findings that acute sμG significantly reduces the cell division potential of hWJSCs and suggest that acute sμG-exposure induces reversible changes in cell growth accompanied by osteocyte–chondrocyte changes in lineage differentiation.

Cultured Cells in the Aging Research, Exhibiting Cell Surface Component Functions, Intracellular Signaling, a Novel Adaptor Molecule, Aging Phenotype Expression and Various Aspects of the Cellular Physiology

Cellular aging is one of the most extraordinary phenomena that mammalian cells undergo in vivo and in vitro. We have been observing their behavior for approximately 4 decades and here would like to summarize some of our salient findings. Normal cells such as human diploid cells exhibit finite growth potential in vitro as well as a set of senescent cell phenotypes. Those changes appear probabilistic and irreversible. In the search of the factor(s) to evoke the features we have observed that cellular glycosaminoglycan molecules plays significant roles in the cell physiology. Besides, CCAAT-box binding transcription factor NF-Y relates to the aging-coupled changes in gene expression, and aging of gastric mucosal cells may relate to a decrease in cytoprotection. As to the intracellular signaling, we have confirmed that the breakdown of phosphatidylinositol bisphosphate is critical for mitogenesis by using micro-injection of its antibody. Subsequently, we have discovered a novel, pivotal adaptor protein Grb2/Ash, a missing link between the receptor tyrosine kinases and their downstream target Ras. The limiting factors for the cellular life span have been considered as telomere shortening and accumulation of cellular and genomic damages. We have observed that telomerase-expressing cells exhibit expanded division potential; yet oxidative stress similarly induces senescent cell phenotypes. Herein we have demonstrated that the treatment of senescent cells with nicotinamide or related reagents elicits unique cellular responses, which might indicate the capability of the cells to recover from the aging.

Ageing-associated changes in transcriptional elongation influence longevity

Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing1–4. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin–IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms5 and flies6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures.

Manifestation of Eastern Cultural Values by Re-arranging Norm on Insulting the President and Vice President

The draft criminal code (RKUHP) has again received a sandstone in its ratification. This is due to several articles that are judged by some people. One of them is about the re-entry of the Article of contempt for the head of state which again reaped polemics in the community. So that with the re-regulation of the article, it is feared by many circles to be a form of defiance of the constitution and violations of free speech which eventually enter into violations of human rights. With the emergence of the article, there has been a lot of polarization in the community between those who support the existence of the article and the contras to the article. To discuss this, normative legal research methods with a conceptual approach, a statutory approach and a historical approach are used. The results of the study show that the reappointment of the article of contempt for the head of the nation's values is not contradictory because the article falls into the category of rechtdelicten. This contempt clause is present because the President and Vice President are icons of the country that can have an impact on the potential for division of society and also harm to other countries.

Nuclear Prospero allows one-division potential to neural precursors and post-mitotic status to neurons via opposite regulation of Cyclin E.

In Drosophila embryonic CNS, the multipotential stem cells called neuroblasts (NBs) divide by self-renewing asymmetric division and generate bipotential precursors called ganglion mother cells (GMCs). GMCs divide only once to generate two distinct post-mitotic neurons. The genes and the pathways that confer a single division potential to precursor cells or how neurons become post-mitotic are unknown. It has been suggested that the homeodomain protein Prospero (Pros) when localized to the nucleus, limits the stem-cell potential of precursors. Here we show that nuclear Prospero is phosphorylated, where it binds to chromatin. In NB lineages such as MP2, or GMC lineages such as GMC4-2a, Pros allows the one-division potential, as well as the post-mitotic status of progeny neurons. These events are mediated by augmenting the expression of Cyclin E in the precursor and repressing the expression in post-mitotic neurons. Thus, in the absence of Pros, Cyclin E is downregulated in the MP2 cell. Consequently, MP2 fails to divide, instead, it differentiates into one of the two progeny neurons. In progeny cells, Pros reverses its role and augments the downregulation of Cyclin E, allowing neurons to exit the cell cycle. Thus, in older pros mutant embryos Cyclin E is upregulated in progeny cells. These results elucidate a long-standing problem of division potential of precursors and post-mitotic status of progeny cells and how fine-tuning cyclin E expression in the opposite direction controls these fundamental cellular events. This work also sheds light on the post-translational modification of Pros that determines its cytoplasmic versus nuclear localization.

Molecular and biochemical modifications of suspension-cultured tobacco cell walls after exposure to alternative gravity

The plant cell wall is a flexible physical barrier surrounding the cell which functions in growth and differentiation, signaling, and response to environmental stimuli including the Earth gravity force. In the present study, structural and molecular modifications of cell wall components of cultured tobacco (Nicotiana tabacum cv. Burley 21) cells under alternative gravity conditions induced by 7 days exposure to 2-D clinostat have been investigated. In comparison with the control group, clinorotation significantly increased biomass but reduced the total amounts of wall and the contents of cellulose, pectin, uronic acidic, and xyloglucan. Gene expression of H+-ATPase was not changed but of expansin A reduced in clinostat-treated cells. However, the gene expression and activity of xyloglucan endotransglycosylase/hydrolases (XTH; EC 2.4.1.207) and endo-(1,4)-β-D-glucanase (EGase; EC 3.2.1.4), the amount of arabinogalactan proteins (AGP), and the expression of wall-associated kinase (WAK) gene significantly increased by clinorotation. Altered gravity also reduced the activity of polyphenol oxidase and covalently bound peroxidase. The results suggest that altered gravity promoted orchestrated changes of wall-modifying genes and proteins which reduced its stiffness and enhanced cell expansion and division potential.

Diminished Auxin Signaling Triggers Cellular Reprogramming by Inducing a Regeneration Factor in the Liverwort Marchantia polymorpha.

Regeneration in land plants is accompanied by the establishment of new stem cells, which often involves reactivation of the cell division potential in differentiated cells. The phytohormone auxin plays pivotal roles in this process. In bryophytes, regeneration is enhanced by the removal of the apex and repressed by exogenously applied auxin, which has long been proposed as a form of apical dominance. However, the molecular basis behind these observations remains unexplored. Here, we demonstrate that in the liverwort Marchantia polymorpha, the level of endogenous auxin is transiently decreased in the cut surface of decapitated explants, and identify by transcriptome analysis a key transcription factor gene, LOW-AUXIN RESPONSIVE (MpLAXR), which is induced upon auxin reduction. Loss of MpLAXR function resulted in delayed cell cycle reactivation, and transient expression of MpLAXR was sufficient to overcome the inhibition of regeneration by exogenously applied auxin. Furthermore, ectopic expression of MpLAXR caused cell proliferation in normally quiescent tissues. Together, these data indicate that decapitation causes a reduction of auxin level at the cut surface, where, in response, MpLAXR is up-regulated to trigger cellular reprogramming. MpLAXR is an ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION 1/DORNRÖSCHEN, which has dual functions as a shoot regeneration factor and a regulator of axillary meristem initiation, the latter of which requires a low auxin level. Thus, our findings provide insights into stem cell regulation as well as apical dominance establishment in land plants.

Oil palm (Elaeis guineensis Jacq.) micropropagation via somatic embryogenesis from female inflorescences explants

The oil palm (Elaeis guineensis Jacq.) is a perennial woody oil crop in the Arecaceae family. Oil palm is well known for a long regeneration time, therefore, in vitro propagation received great enthusiasm from oil palm industries. Somatic embryogenesis (SE) has become one of the most promising clonal propagation techniques in recent times. This study was aimed to determine a protocol for micropropagation somatic embryogenesis from female inflorescences explants of oil palm. The explant used was obtained from the female inflorescences of the oil palm 2.5 years old Tenera variety. The basal media used was Y3 with the addition of the hormone 2,4-D with different concentrations (33,66,99 and 132 mg/L). Callus initiation was formed in 99 mg/L and 132 mg/L 2,4 – D concentration with the basal area and the percentage of callus formation is 31,25% and embryogenic callus was formed from primary callus development in the basal area of female florescence. Embryo somatic induction with cell suspension culture, because the liquid medium is more efficiently used in commercial-scale propagation. The embryogenic callus phase which is generally used in liquid medium is the nodular phase because it is still meristematic so that the potential for cell division is still high and can increase the percentage of embryogenic callus.

Visualization of individual cell division history in complex tissues using iCOUNT.

SummaryThe division potential of individual stem cells and the molecular consequences of successive rounds of proliferation remain largely unknown. Here, we developed an inducible cell division counter (iCOUNT) that reports cell division events in human and mouse tissues in vitro and in vivo. Analyzing cell division histories of neural stem/progenitor cells (NSPCs) in the developing and adult brain, we show that iCOUNT can provide novel insights into stem cell behavior. Further, we use single-cell RNA sequencing (scRNA-seq) of iCOUNT-labeled NSPCs and their progenies from the developing mouse cortex and forebrain-regionalized human organoids to identify functionally relevant molecular pathways that are commonly regulated between mouse and human cells, depending on individual cell division histories. Thus, we developed a tool to characterize the molecular consequences of repeated cell divisions of stem cells that allows an analysis of the cellular principles underlying tissue formation, homeostasis, and repair.

Fatty acids and its derivatives of Acorus calamus Linn. rhizome induce stem cell-mediated cell division in plants and animals

The phytochemicals from Acorus calamus Linn. medicinal plant rhizome were extracted with distilled water and followed by fractionation with 100% ethanol. Although the original aqueous extract inhibited the blastema development in amputated earthworms, the ethanol fraction partitioned from aqueous extract induced the blastema development. This ethanolic fraction was found to induce the cell division in onion root tips possessing stem cells in the root meristem. GC-MS analysis of ethanol fraction revealed the presence of putative 12 different fatty acids, 7 fatty aldehydes, one fatty alcohol and other hydrocarbon related compounds. Vero cell line was used to examine the cell division potential of ethanol fraction at in vitro conditions. The viability of Vero cells was around 84% even after 3 days with 1 mg/ml concentration of ethanol fraction compared to controls. It indicated that compounds present in the ethanol fraction were nontoxic. We propose that these fatty acids and their derivatives such as fatty aldehydes or fatty alcohol in the ethanol fraction are used as energy resources, in cell membrane biosynthesis or as signal molecules to facilitate the cell division.

Leukaemia: a model metastatic disease.

In contrast to solid cancers, which often require genetic modifications and complex cellular reprogramming for effective metastatic dissemination, leukaemic cells uniquely possess the innate ability for migration and invasion. Dedifferentiated, malignant leukocytes retain the benign leukocytes' capacity for cell motility and survival in the circulation, while acquiring the potential for rapid and uncontrolled cell division. For these reasons, leukaemias, although not traditionally considered as metastatic diseases, are in fact models of highly efficient metastatic spread. Accordingly, they are often aggressive and challenging diseases to treat. In this Perspective, we discuss the key molecular processes that facilitate metastasis in a variety of leukaemic subtypes, the clinical significance of leukaemic invasion into specific tissues and the current pipeline of treatments targeting leukaemia metastasis.

On the evolution of cellular senescence.

The idea that senescent cells are causally involved in aging has gained strong support from findings that the removal of such cells alleviates many age‐related diseases and extends the life span of mice. While efforts proceed to make therapeutic use of such discoveries, it is important to ask what evolutionary forces might have been behind the emergence of cellular senescence, in order better to understand the biology that we might seek to alter. Cellular senescence is often regarded as an anti‐cancer mechanism, since it limits the division potential of cells. However, many studies have shown that senescent cells often also have carcinogenic properties. This is difficult to reconcile with the simple idea of an anti‐cancer mechanism. Furthermore, other studies have shown that cellular senescence is involved in wound healing and tissue repair. Here, we bring these findings and ideas together and discuss the possibility that these functions might be the main reason for the evolution of cellular senescence. Furthermore, we discuss the idea that senescent cells might accumulate with age because the immune system had to strike a balance between false negatives (overlooking some senescent cells) and false positives (destroying healthy body cells).

Restriction on self-renewing asymmetric division is coupled to terminal asymmetric division in the Drosophila CNS.

Neuronal precursor cells undergo self-renewing and non-self-renewing asymmetric divisions to generate a large number of neurons of distinct identities. In Drosophila, primary precursor neuroblasts undergo a varying number of self-renewing asymmetric divisions, with one known exception, the MP2 lineage, which undergoes just one terminal asymmetric division similar to the secondary precursor cells. The mechanism and the genes that regulate the transition from self-renewing to non-self-renewing asymmetric division or the number of times a precursor divides is unknown. Here, we show that the T-box transcription factor, Midline (Mid), couples these events. We find that in mid loss of function mutants, MP2 undergoes additional self-renewing asymmetric divisions, the identity of progeny neurons generated dependent upon Numb localization in the parent MP2. MP2 expresses Mid transiently and an over-expression of mid in MP2 can block its division. The mechanism which directs the self-renewing asymmetric division of MP2 in mid involves an upregulation of Cyclin E. Our results indicate that Mid inhibits cyclin E gene expression by binding to a variant Mid-binding site in the cyclin E promoter and represses its expression without entirely abolishing it. Consistent with this, over-expression of cyclin E in MP2 causes its multiple self-renewing asymmetric division. These results reveal a Mid-regulated pathway that restricts the self-renewing asymmetric division potential of cells via inhibiting cyclin E and facilitating their exit from cell cycle.

Physiological, Metabolic, and Transcriptomic Analyses Reveal the Responses of Arabidopsis Seedlings to Carbon Nanohorns.

Carbon-based nanomaterials have potential applications in nanoenabled agriculture. However, the physiological and molecular mechanisms underlying single-walled carbon nanohorn (SWCNH)-mediated plant growth remain unclear. Here, we investigated the effects of SWCNHs on Arabidopsis grown in 1/4-strength Murashige and Skoog medium via physiological, genetic, and molecular analyses. Treatment with 0.1 mg/L SWCNHs promoted primary root (PR) growth and lateral root (LR) formation; 50 and 100 mg/L SWCNHs inhibited PR growth. Treatment with 0.1 mg/L SWCNHs increased the lengths of the meristematic and elongation zones, and transcriptomic and genetic analyses confirmed the positive effects of SWCNHs on root tip stem cell niche activity and meristematic cell division potential. Increased expression of YUC3 and YUC5 and increased PIN2 abundance improved PR growth and LR development in 0.1 mg/L SWCNH-treated seedlings. Metabolomic analyses revealed that SWCNHs altered the levels of sugars, amino acids, and organic acids, suggesting that SWCNHs reprogrammed carbon/nitrogen metabolism in plants. SWCNHs also regulate plant growth and development by increasing the levels of several secondary metabolites; transcriptomic analyses further supported these results. The present results are valuable for continued use of SWCNHs in agri-nanotechnology, and these molecular approaches could serve as examples for studies on the effects of nanomaterials in plants.

Evolution of vascular plants through redeployment of ancient developmental regulators

Vascular plants provide most of the biomass, food, and feed on earth, yet the molecular innovations that led to the evolution of their conductive tissues are unknown. Here, we reveal the evolutionary trajectory for the heterodimeric TMO5/LHW transcription factor complex, which is rate-limiting for vascular cell proliferation in Arabidopsis thaliana Both regulators have origins predating vascular tissue emergence, and even terrestrialization. We further show that TMO5 evolved its modern function, including dimerization with LHW, at the origin of land plants. A second innovation in LHW, coinciding with vascular plant emergence, conditioned obligate heterodimerization and generated the critical function in vascular development in Arabidopsis In summary, our results suggest that the division potential of vascular cells may have been an important factor contributing to the evolution of vascular plants.

A cell size threshold limits cell polarity and asymmetric division potential.

Reaction-diffusion networks underlie pattern formation in a range of biological contexts, from morphogenesis of organisms to the polarisation of individual cells. One requirement for such molecular networks is that output patterns be scaled to system size. At the same time, kinetic properties of constituent molecules constrain the ability of networks to adapt to size changes. Here we explore these constraints and the consequences thereof within the conserved PAR cell polarity network. Using the stem cell-like germ lineage of the C. elegans embryo as a model, we find that the behaviour of PAR proteins fails to scale with cell size. Theoretical analysis demonstrates that this lack of scaling results in a size threshold below which polarity is destabilized, yielding an unpolarized system. In empirically-constrained models, this threshold occurs near the size at which germ lineage cells normally switch between asymmetric and symmetric modes of division. Consistent with cell size limiting polarity and division asymmetry, genetic or physical reduction in germ lineage cell size is sufficient to trigger loss of polarity in normally polarizing cells at predicted size thresholds. Physical limits of polarity networks may be one mechanism by which cells read out geometrical features to inform cell fate decisions.

Cell competition in tumor evolution and heterogeneity: Merging past and present.

In many cases, cancers are difficult to eliminate because they develop resistance to a primary chemotherapy or targeted therapy. Tumors grow into diverse cell subpopulations, increasing the ability to resist elimination. The phenomenon of 'cell competition' describes our body's natural surveillance system to optimize tissue fitness by forcing viable but aberrant cells to undergo cell death. Cell competition is not simply comparison of cell division potential. Competition factors signal for 'loser' cell elimination and 'winner' cell dominance. New evidence demonstrates it is possible to restrict cancer growth by strengthening the cell fitness of surrounding healthy tissue via anti-apoptotic pathways. Hence, cell competition provides strong conceptual explanation for oncogenesis, tumor growth and suppression. Tumor heterogeneity is a hallmark of many cancers and establishes gradients in which competitive interactions are able to occur among tumor cell subpopulations as well as neighboring stromal tissue. Here we review cellular/molecular competition pathways in the context of tumor evolution, heterogeneity and response to interventions. We propose strategies to exploit these mediators and design novel broad-spectrum therapeutic approaches that eliminate cancer and enhance fitness of neighboring tissue to improve patient outcomes.