Comparative transcriptomic analysis primarily explores the molecular mechanism of compound eye formation in Neocaridina denticulata sinensis.

Compound eyes formation in decapod crustaceans occurs after the nauplius stage. However, the key genes and regulatory mechanisms of compound eye development during crustacean embryonic development have not yet been clarified. In this study, RNA-seq was used to investigate the gene expression profiles of Neocaridina denticulata sinensis from nauplius to zoea stage. Based on RNA-seq data analysis, the phototransduction and insect hormone biosynthesis pathways were enriched, and molting-related neuropeptides were highly expressed. There was strong cell proliferation in the embryo prior to compound eye development. The formation of the visual system and the hormonal regulation of hatching were the dominant biological events during compound eye development. The functional analysis of DEGs across all four developmental stages showed that cuticle formation, muscle growth and the establishment of immune system occurred from nauplius to zoea stage. Key genes related to eye development were discovered, including those involved in the determination and differentiation of the eye field, eye-color formation, and visual signal transduction. In conclusion, the results increase the understanding of the molecular mechanism of eye formation in crustacean embryonic stage.

A morphological basis for path-dependent evolution of visual systems.

Path dependence influences macroevolutionary predictability by constraining potential outcomes after critical evolutionary junctions. Although it has been demonstrated in laboratory experiments, path dependence is difficult to demonstrate in natural systems because of a lack of independent replicates. Here, we show that two types of distributed visual systems recently evolved twice within chitons, demonstrating rapid and path-dependent evolution of a complex trait. The type of visual system that a chiton lineage can evolve is constrained by the number of openings for sensory nerves in its shell plates. Lineages with more openings evolve visual systems with thousands of eyespots, whereas those with fewer openings evolve visual systems with hundreds of shell eyes. These macroevolutionary outcomes shaped by path dependence are both deterministic and stochastic because possibilities are restricted yet not entirely predictable.

The effects of physical activity on pediatric eyes: A systematic review and meta-analysis.

INTRODUCTION: Examining the retina represents a non-invasive method to evaluate abnormalities pertaining to the nervous and cardiovascular systems. Evidence indicates that physical activity is a non-pharmacological intervention to enhance the nervous and cardiovascular systems. However, little is unknown about its effects on ocular characteristics in children and adolescents. The purpose of this study was to examine the effects of physical activity interventions on ocular characteristics in children and adolescents. METHOD: The electronic bases Web of Science, Embase, Cochrane Library, PubMed, SPORTDiscus, CINAHL, and ERIC were searched from inception to May 2023. Incorporated were randomized controlled trials or quasi-experimental designs that had implemented acute or chronic physical activity interventions among children and adolescents to evaluate various eye-related attributes via clinical examinations or surveys. Two authors independently performed the data extraction and risk of bias assessment, utilizing the Physiotherapy Evidence Database checklist. RESULTS: A total of 474 articles were identified, of which eight articles underwent a systematic review, and six were chosen for meta-analysis. Chronic physical activity interventions positively impacted central retinal artery equivalent (CRAE) with a small to moderate effect (SMD = 0.21; 95% CI 0.04 to 0.39, p = 0.034, I2 = 0%) and central retinal venular equivalent (CRVE) with a small effect (SMD = 0.098; 95% CI 0.08 to 0.11; p = 0.008, I2 = 0%). Intraocular pressure, kinetic visual acuity, and eye strain also improved significantly after physical activity interventions. DISCUSSION: Participating in chronic physical activity programs appear to impact children and adolescents' eye-related attributes positively.

Agreement between Lea Symbols and Patti Pics visual acuity in children and adults

Background Patti Pics (PP) and Lea Symbols (LS) are commonly used by eye care practitioners worldwide. Although the relationship between the two tests is fairly well understood, the availability of different chart designs (single optotypes, multiple optotypes, multiple optotypes with crowding box) merits futher understanding. The purpose of this study is to explore the agreement between the acuity measures obtained with Patti Pics and Lea Symbols in children and adults and compare their performance with the Sloan Letter (SL) chart in adults. Methods Monocular visual acuity was obtained from ninety-three 3 to 5-year-old children using Patti Pics and Lea Symbols. Acuities were also obtained from 113 adults using the same tests under identical conditions. Acuity results obtained with the pediatric tests were compared with the gold-standard Sloan Letter chart in adults. The Bland-Altman method was implemented to compare the level of agreement between tests. Results Patti Pics yielded worse visual acuity than the Lea Symbols by approximately half a logMAR line in both children (mean difference: -0.07 ± 0.07 logMAR, p <0.01) and adults (Mean difference: -0.05 ± 0.06 logMAR, p <0.01). The 95% limits of agreement between Lea Symbol acuity and Patti pics acuity in children was ± 0.14 logMAR. Mean difference between the Sloan Letter chart and Lea Symbols acuity was not statistically significant (p = 0.08) in adults but the difference was statistically significant between PP and SL (p<0.001). The 95% limits of agreement between LS and SL and between PP and SL was ± 0.19 logMAR and ± 0.22 logMAR, respectively. Conclusion Patti Pics consistently underestimated visual acuity as compared to Lea Symbols both in children and adults although the differences were not clinically significant. The LS and PP did not yield clinically significant differences in acuities when compared with Sloan letters in adults. (AU)

Distribution of Axial Length in Australians of Different Age Groups, Ethnicities, and Refractive Errors.

Purpose: Treatments are available to slow myopic axial elongation. Understanding normal axial length (AL) distributions will assist clinicians in choosing appropriate treatment for myopia. We report the distribution of AL in Australians of different age groups and refractive errors. Methods: Retrospectively collected spherical equivalent refraction (SER) and AL data of 5938 individuals aged 5 to 89 years from 8 Australian studies were included. Based on the SER, participants were classified as emmetropes, myopes, and hyperopes. Two regression model parameterizations (piece-wise and restricted cubic splines [RCS]) were applied to the cross-sectional data to analyze the association between age and AL. These results were compared with longitudinal data from the Raine Study where the AL was measured at age 20 (baseline) and 28 years. Results: A piece-wise regression model (with 1 knot) showed that myopes had a greater increase in AL before 18 years by 0.119 mm/year (P < 0.001) and after 18 years by 0.011 mm/year (P < 0.001) compared to emmetropes and hyperopes, with the RCS model (with 3 knots) showing similar results. The longitudinal data from the Raine Study revealed that, when compared to emmetropes, only myopes showed a significant change in the AL in young adulthood (by 0.016 mm/year, P < 0.001). Conclusions: The AL of myopic eyes increases more rapidly in childhood and slightly in early adulthood. Further studies of longitudinal changes in AL, particularly in childhood, are required to guide myopia interventions. Translational Relevance: The axial length of myopic eyes increases rapidly in childhood, and there is a minimal increase in the axial length in non-myopic eyes after 18 years of age.

Integrator complex subunit 15 controls mRNA splicing and is critical for eye development.

The eye and brain are composed of elaborately organized tissues, development of which is supported by spatiotemporally precise expression of a number of transcription factors and developmental regulators. Here we report the molecular and genetic characterization of Integrator complex subunit 15 (INTS15). INTS15 was identified in search for the causative gene(s) for an autosomal-dominant eye disease with variable individual manifestation found in a large pedigree. While homozygous Ints15 knockout mice are embryonic lethal, mutant mice lacking a small C-terminal region of Ints15 show ocular malformations similar to the human patients. INTS15 is highly expressed in the eye and brain during embryogenesis and stably interacts with the Integrator complex to support small nuclear RNA 3' end processing. Its knockdown resulted in missplicing of a large number of genes, probably as a secondary consequence, and substantially affected genes associated with eye and brain development. Moreover, studies using human iPS cells-derived neural progenitor cells showed that INTS15 is critical for axonal outgrowth in retinal ganglion cells. This study suggests a new link between general transcription machinery and a highly specific hereditary disease.

Systematic mapping of rRNA 2'-O methylation during frog development and involvement of the methyltransferase Fibrillarin in eye and craniofacial development in Xenopus laevis.

Ribosomes are essential nanomachines responsible for protein production. Although ribosomes are present in every living cell, ribosome biogenesis dysfunction diseases, called ribosomopathies, impact particular tissues specifically. Here, we evaluate the importance of the box C/D snoRNA-associated ribosomal RNA methyltransferase fibrillarin (Fbl) in the early embryonic development of Xenopus laevis. We report that in developing embryos, the neural plate, neural crest cells (NCCs), and NCC derivatives are rich in fbl transcripts. Fbl knockdown leads to striking morphological defects affecting the eyes and craniofacial skeleton, due to lack of NCC survival caused by massive p53-dependent apoptosis. Fbl is required for efficient pre-rRNA processing and 18S rRNA production, which explains the early developmental defects. Using RiboMethSeq, we systematically reinvestigated ribosomal RNA 2'-O methylation in X. laevis, confirming all 89 previously mapped sites and identifying 15 novel putative positions in 18S and 28S rRNA. Twenty-three positions, including 10 of the new ones, were validated orthogonally by low dNTP primer extension. Bioinformatic screening of the X. laevis transcriptome revealed candidate box C/D snoRNAs for all methylated positions. Mapping of 2'-O methylation at six developmental stages in individual embryos indicated a trend towards reduced methylation at specific positions during development. We conclude that fibrillarin knockdown in early Xenopus embryos causes reduced production of functional ribosomal subunits, thus impairing NCC formation and migration.

Biometry of the human cornea and globe: An evaluation by age, gender and population.

The purpose of this study was to examine the effects of age, gender and population origin on human globe and corneal dimensions and to explore the relationships between the dimensions. Human post-mortem eyes were obtained in Hyderabad (n = 223; range, 0-85 years) and Miami (n = 486; range, 6-103 years). The eyes were freed of extraneous tissues and globe antero-posterior length (GAPL), mean globe diameter (MGD) (average of horizontal and vertical), and corneal horizontal (HCD) and vertical (VCD) diameters were measured using digital calipers. The relationships of age, gender and population origin with globe and corneal dimensions and the relationships between the dimensions were assessed by bivalent and multiple regression analyses. Globe and cornea dimensions increase asymptotically with age until around the late teens but do not change thereafter. Bivariate and multivariate regression analyses of the >20-year-old eyes showed that population was significantly correlated with GAPL, MGD, HCD and VCD. Male globes and corneas were larger than those from females, but the difference did not appear to be statistically significant. All Hyderabad dimensions were significantly larger than those from the Miami. Neither GAPL nor MGD were correlated with the corneal dimensions. GAPL was significantly correlated with MGD as was HCD with VCD.

PAX6-positive microglia evolve locally in hiPSC-derived ocular organoids.

Microglia are the resident immune cells of the central nervous system (CNS). They govern the immunogenicity of the retina, which is considered to be part of the CNS; however, it is not known how microglia develop in the eye. Here, we studied human-induced pluripotent stem cells (hiPSCs) that had been expanded into a self-formed ectodermal autonomous multi-zone (SEAM) of cells that partially mimics human eye development. Our results indicated that microglia-like cells, which have characteristics of yolk-sac-like linage cells, naturally develop in 2D eye-like SEAM organoids, which lack any vascular components. These cells are unique in that they are paired box protein 6 (PAX6)-positive, yet they possess some characteristics of mesoderm. Collectively, the data support the notion of the existence of an isolated, locally developing immune system in the eye, which is independent of the body's vasculature and general immune system.

Extrinsic and Intrinsic Factors Regulating Juvenile Refractive Development and Eye Growth.

Purpose: Peripheral refraction and accommodation are intrinsic factors that were once hypothesized to trigger myopia but are now controversial. Previously, home nearwork environment (i.e., extrinsic factor) was reported to be associated with myopia progression. In this study, we aimed to evaluate the potential interaction between extrinsic and intrinsic factors with juvenile refractive development. Methods: Nearwork environmental parameters were measured for 50 children (aged 9.3 ± 1.2 years), including net amount and dispersion of defocus. Refraction was measured at near distances and in central field (±30° horizontal) at 3m. The relative peripheral refraction (RPRE) was obtained and presented in a vectoral approach. The linear regression coefficient was extracted (mAcc) from the accommodative stimulus-response curve. RPRE was quadratically regressed against field eccentricity, and the first coefficients (aM, aJ0, aP90, and aP180) were extracted. Relationships between RPRE, baseline accommodation, and 1-year myopia progression (∆M), controlled for the nearwork environment, were evaluated. Results: Coefficients of RPRE were independent of ∆M. However, additional nearwork environmental parameters significantly improved the variance in ∆M explained by aM and aP180 (P < 0.03). The relationship between intrinsic factor and ∆M was stronger when the extrinsic risk was low (P ≤ 0.01), whereas the relationship was abolished when extrinsic risk was high. For mAcc, it also significantly improved the variance in ∆M explained by nearwork environmental parameters. Conclusions: The interaction between extrinsic (environment) and intrinsic (RPRE and accommodation) factors is speculated to contribute to juvenile myopia progression. Our findings may also explain the inconsistencies of such intrinsic factors in the literature.

Alteration of organ size and allometric scaling by organ-specific targeting of IGF signaling.

The size of an organ is proportional to the other body parts or the whole body. This relationship is known as allometry. Understanding how allometry is determined is a fundamental question in biology. Here we tested the hypothesis that local insulin-like growth factor (Igf) signaling is critical in regulating organ size and its allometric scaling by organ-specific expression of Igf binding protein (Igfbp). Overexpression of Igfbp2a or 5b in the developing zebrafish eye, heart, and inner ear resulted in a disproportional reduction in their growth relative to the body. Stable transgenic zebrafish with lens-specific Igfbp5b expression selectively reduced adult eye size. The action is Igf-dependent because an Igf-binding deficient Igfbp5b mutant had no effect. Targeted expression of a dominant-negative Igf1 receptor (dnIgf1r) in the lens caused a similar reduction in relative eye growth. Furthermore, co-expression of IGF-1 with an Igfbp restored the eye size. Finally, co-expression of a constitutively active form of Akt with Igfbp or dnIgf1r restored the relative eye growth. These data suggest that local Igf availability and Igf signaling activity are critical determinants of organ size and allometric scaling in zebrafish.

Elastic Instabilities Govern the Morphogenesis of the Optic Cup.

Because the normal operation of the eye depends on sensitive morphogenetic processes for its eventual shape, developmental flaws can lead to wide-ranging ocular defects. However, the physical processes and mechanisms governing ocular morphogenesis are not well understood. Here, using analytical theory and nonlinear shell finite-element simulations, we show, for optic vesicles experiencing matrix-constrained growth, that elastic instabilities govern the optic cup morphogenesis. By capturing the stress amplification owing to mass increase during growth, we show that the morphogenesis is driven by two elastic instabilities analogous to the snap through in spherical shells, where the second instability is sensitive to the optic cup geometry. In particular, if the optic vesicle is too slender, it will buckle and break axisymmetry, thus, preventing normal development. Our results shed light on the morphogenetic mechanisms governing the formation of a functional biological system and the role of elastic instabilities in the shape selection of soft biological structures.

Visually induced changes in cytokine production in the chick choroid.

Postnatal ocular growth is regulated by a vision-dependent mechanism that acts to minimize refractive error through coordinated growth of the ocular tissues. Of great interest is the identification of the chemical signals that control visually guided ocular growth. Here, we provide evidence that the pro-inflammatory cytokine, interleukin-6 (IL-6), may play a pivotal role in the control of ocular growth using a chicken model of myopia. Microarray, real-time RT-qPCR, and ELISA analyses identified IL-6 upregulation in the choroids of chick eyes under two visual conditions that introduce myopic defocus and slow the rate of ocular elongation (recovery from induced myopia and compensation for positive lenses). Intraocular administration of atropine, an agent known to slow ocular elongation, also resulted in an increase in choroidal IL-6 gene expression. Nitric oxide appears to directly or indirectly upregulate choroidal IL-6 gene expression, as administration of the non-specific nitric oxide synthase inhibitor, L-NAME, inhibited choroidal IL-6 gene expression, and application of a nitric oxide donor stimulated IL-6 gene and protein expression in isolated chick choroids. Considering the pleiotropic nature of IL-6 and its involvement in many biological processes, these results suggest that IL-6 may mediate many aspects of the choroidal response in the control of ocular growth.

Eye morphogenesis in the blind Mexican cavefish.

The morphogenesis of the vertebrate eye consists of a complex choreography of cell movements, tightly coupled to axial regionalization and cell type specification processes. Disturbances in these events can lead to developmental defects and blindness. Here, we have deciphered the sequence of defective events leading to coloboma in the embryonic eye of the blind cavefish of the species Astyanax mexicanus. Using comparative live imaging on targeted enhancer-trap Zic1:hsp70:GFP reporter lines of both the normal, river-dwelling morph and the cave morph of the species, we identified defects in migratory cell behaviours during evagination that participate in the reduced optic vesicle size in cavefish, without proliferation defect. Further, impaired optic cup invagination shifts the relative position of the lens and contributes to coloboma in cavefish. Based on these results, we propose a developmental scenario to explain the cavefish phenotype and discuss developmental constraints to morphological evolution. The cavefish eye appears as an outstanding natural mutant model to study molecular and cellular processes involved in optic region morphogenesis.

Early Pediatric Benefit of Lutein for Maturing Eyes and Brain-An Overview.

Lutein is a dietary carotenoid preferentially accumulated in the eye and the brain in early life and throughout the life span. Lutein accumulation in areas of high metabolism and oxidative stress such as the eye and the brain suggest a unique role of this ingredient during the development and maturation of these organs of common embryological origin. Lutein is naturally provided to the developing baby via the cord blood, breast milk and then infant diet. The presence of this carotenoid depends on fruit and vegetable intakes and its bioavailability is higher in breastmilk. This paper aims to review the anatomical development of the eye and the brain, explore the presence and selective deposition of lutein in these organs during pregnancy and infancy and, based on its functional characteristics, present the latest available research on the beneficial role of lutein in the pediatric population. The potential effects of lutein in ameliorating conditions associated with increase oxidative stress such as in prematurity will be also addressed. Since consumption of lutein rich foods falls short of government guidelines and in most region of the world infant formulas lack this bioactive, dietary recommendations for pregnant and breastfeeding women and their child can help to bridge the gap.

Transcriptome-based insights into gene networks controlling myopia prevention.

Myopia (short-sightedness), usually caused by excessive elongation of the eye during development, has reached epidemic proportions worldwide. In animal systems including the chicken model, several treatments have been shown to inhibit ocular elongation and experimental myopia. Although diverse in their apparent mechanism of action, each one leads to a reduction in the rate of ocular growth. We hypothesize that a defined set of retinal molecular changes may underlie growth inhibition, irrespective of the treatment agent used. Accordingly, across five well-established but diverse methods of inhibiting myopia, significant overlap is seen in the retinal transcriptome profile (transcript levels and alternative splicing events) in chicks when analyzed by RNA-seq. Within the two major pathway networks enriched during growth inhibition, that of cell signaling and circadian entrainment, transcription factors form the largest functional grouping. Importantly, a large percentage of those genes forming the defined retinal response are downstream targets of the transcription factor EGR1 which itself shows a universal response to all five growth-inhibitory treatments. This supports EGR1's previously implicated role in ocular growth regulation. Finally, by contrasting our data with human linkage and GWAS studies on refractive error, we confirm the applicability of our study to the human condition. Together, these findings suggest that a universal set of transcriptome changes, which sit within a well-defined retinal network that cannot be bypassed, is fundamental to growth regulation, thus paving a way for designing novel targets for myopia therapies.

The Eyes Absent proteins in development and in developmental disorders.

The Eyes Absent (EYA) transactivator-phosphatase proteins are important contributors to cell-fate determination processes and to the development of multiple organs. The transcriptional regulatory activity as well as the protein tyrosine phosphatase activities of the EYA proteins can independently contribute to proliferation, differentiation, morphogenesis and tissue homeostasis in different contexts. Aberrant EYA levels or activity are associated with numerous syndromic and non-syndromic developmental disorders, as well as cancers. Commensurate with the multiplicity of biochemical activities carried out by the EYA proteins, they impact upon a range of cellular signaling pathways. Here, we provide a broad overview of the roles played by EYA proteins in development, and highlight the molecular signaling pathways known to be linked with EYA-associated organ development and developmental disorders.

Relationship of the cornea and globe dimensions to the changes in adult human crystalline lens diameter, thickness and power with age.

It is well known that human crystalline lens shape, dimensions and optical properties change throughout life and influence whole eye refraction. However, it is not clear if lens properties are associated with other ocular parameters. The purpose of the present study was to investigate the relationship of corneal and external globe dimensions with adult lens diameter (LD), lens thickness (LT) and lens power (LP) in order to determine if external factors influence lens properties. Postmortem human eyes (n = 66, age = 20-78 years) were obtained from the Ramayamma International Eye Bank, Hyderabad, India. Globe antero-posterior length (GAPL) and mean (average of horizontal and vertical) diameters of cornea (MCD) and globe (MGD) were measured using digital calipers. Eyes were dissected to produce ocular structures that contain the lens maintained in its accommodating framework, including intact zonules, ciliary body and sections of sclera. Specimens were mounted in a mechanical lens stretching system. LD, LT and LP were measured using high magnification retro-illumination photography, slit illumination photography and Scheiner principle-based optical system respectively in the unstretched (accommodated) state. Relationships between external globe and corneal dimensions and LD, LT or LP were assessed by multiple regression analysis. Age (0.012 ± 0.003 mm/year; p<0.001) and GAPL (0.185 ± 0.045 mm/mm; p<0.001) were significant (p<0.0001) predictors of LD. After adjusting for age-related increases, LD appears to be positively correlated with GAPL. Age (0.010 ± 0.004 mm/year; p = 0.009) and GAPL (-0.143 ± 0.060 mm/mm; p = 0.02) were significant (p = 0.001) predictors of LT. After adjusting for the age-related increase, LT appears to be negatively correlated with GAPL. Only age was a significant predictor of LP (-0.26 ± 0.04 D/year; p<0.001). The results suggest that, apart from aging, lens diameter and thickness are dependent on the anteroposterior length of the eye globe. Lens power is not influenced by globe dimensions.

Crumbs and the apical spectrin cytoskeleton regulate R8 cell fate in the Drosophila eye.

The Hippo pathway is an important regulator of organ growth and cell fate. In the R8 photoreceptor cells of the Drosophila melanogaster eye, the Hippo pathway controls the fate choice between one of two subtypes that express either the blue light-sensitive Rhodopsin 5 (Hippo inactive R8 subtype) or the green light-sensitive Rhodopsin 6 (Hippo active R8 subtype). The degree to which the mechanism of Hippo signal transduction and the proteins that mediate it are conserved in organ growth and R8 cell fate choice is currently unclear. Here, we identify Crumbs and the apical spectrin cytoskeleton as regulators of R8 cell fate. By contrast, other proteins that influence Hippo-dependent organ growth, such as the basolateral spectrin cytoskeleton and Ajuba, are dispensable for the R8 cell fate choice. Surprisingly, Crumbs promotes the Rhodopsin 5 cell fate, which is driven by Yorkie, rather than the Rhodopsin 6 cell fate, which is driven by Warts and the Hippo pathway, which contrasts with its impact on Hippo activity in organ growth. Furthermore, neither the apical spectrin cytoskeleton nor Crumbs appear to regulate the Hippo pathway through mechanisms that have been observed in growing organs. Together, these results show that only a subset of Hippo pathway proteins regulate the R8 binary cell fate decision and that aspects of Hippo signalling differ between growing organs and post-mitotic R8 cells.

Comprehensive phenotypic and functional analysis of dominant and recessive FOXE3 alleles in ocular developmental disorders.

The forkhead transcription factor FOXE3 is critical for vertebrate eye development. Recessive and dominant variants cause human ocular disease but the full range of phenotypes and mechanisms of action for the two classes of variants are unknown. We identified FOXE3 variants in individuals with congenital eye malformations and carried out in vitro functional analysis on selected alleles. Sixteen new recessive and dominant families, including six novel variants, were identified. Analysis of new and previously reported genetic and clinical data demonstrated a broad phenotypic range with an overlap between recessive and dominant disease. Most families with recessive alleles, composed of truncating and forkhead-domain missense variants, had severe corneal opacity (90%; sclerocornea in 47%), aphakia (83%) and microphthalmia (80%), but some had milder features including isolated cataract. The phenotype was most variable for recessive missense variants, suggesting that the functional consequences may be highly dependent on the type of amino acid substitution and its position. When assessed, aniridia or iris hypoplasia were noted in 89% and optic nerve anomalies in 60% of recessive cases, indicating that these defects are also common and may be underrecognized. In dominant pedigrees, caused by extension variants, normal eye size (96%), cataracts (99%) and variable anterior segment anomalies were seen in most, but some individuals had microphthalmia, aphakia or sclerocornea, more typical of recessive disease. Functional studies identified variable effects on the protein stability, DNA binding, nuclear localization and transcriptional activity for recessive FOXE3 variants, whereas dominant alleles showed severe impairment in all areas and dominant-negative characteristics.