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Single molecule fluorescence in situ hybridization (smFISH) and in situ hybridization chain reaction (HCR) have become powerful tools to visualize gene expression in many different animal species. We show here that smFISH and in situ HCR can be put to effective use in the satellite nematode model organism Pristionchus pacificus . Examining the expression of a homeobox gene ( Ppa-unc-30) , we found that HCR is more sensitive than smFISH. We confirmed the robustness of HCR by visualization of the expression of several genes involved in neurotransmitter synthesis or transport ( Ppa-unc-25 /GAD, Ppa-unc-17/VAChT, Ppa-eat-4 /VGLUT). Combined with its relative cost-effectiveness compared to smFISH analysis, in situ HCR constitutes a useful addition to the toolbox for P. pacificus research ., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

This study aimed to evaluate trends in the prevalence of dental caries in preschool children and associated factors considering different time variations. This is a time series study performed using data from three cross-sectional studies with pre-school children from southern Brazil in 2008, 2013 and 2019. This children group was born between the years of 2003 to 2018. Dental caries was evaluated by decayed, missing and filled deciduous teeth (dmft index). Demographic, socioeconomic, behavioural and psychosocial variables were also collected. Chi-square test for trends and a hierarchical age-period-cohort (HAPC) analysis using multilevel Poisson regression model for testing the associations between predictor variables and dental caries experience were used. A total of 1,644 pre-school children participated in all surveys. There was a significant difference in caries experience considering all APC effects. The prevalence of dental caries was 25.0% in 2008, 16.3% in 2013, and 19.4% in 2019 (p < 0.01) and no statistical difference was observed. An age effect showed that older children were more likely to experience dental caries. Considering the cohort effect, there is a significant difference between the generations, mainly between 2003 and 2018. Household income, use of dental services, and parent's perception of child oral health were associated with dental caries experience no matter the time variation. Despite recent declines in dental caries prevalence among preschool children, caries levels increased with age and social inequalities persisted through the years, indicating a need of reviewing the policies to reduce the burden of this oral disease.

This study aimed to explore the pathways that can influence cigarette smoking among adolescents. This population-based cohort followed a random sample of 12-year-old adolescents from southern Brazil for 6 years. Regular cigarette smoking was assessed through a self-reported question, previously used in the Brazilian National Survey of Scholar Health. We also gathered data on bullying, dental caries at baseline, incidence of caries, sex, friend network, and Sense of Coherence (SOC). Socioeconomic and demographic characteristics were also collected. Structural equation modeling was used to evaluate the pathways. Of the 1,134 adolescents examined at baseline, 768 were re-evaluated (67.7% retention rate). The prevalence of smoking was 37.6%. This prevalence was directly affected by low SOC (SC: -0.14, p < 0.01), low household income (SC: -0.12, p < 0.01), and male sex (SC: 0.15, p < 0.01). Presence of dental caries at baseline indirectly influenced the occurrence of dental bullying at follow-up via the incidence of dental caries (SC: 0.01, p < 0.05). Dental bullying indirectly influenced cigarettes consumption via SOC (SC: 0.62, p < 0.05). Friend network also indirectly influenced the consumption of cigarettes via SOC (SC: 0.32, p < 0.05). Psychosocial factors influence adolescent cigarette consumption through its higher direct and indirect effects (via bullying). In addition, behavioral, sociodemographic, and clinical factors also influence the occurrence of smoking.

Objective: To verify the relations between sense of coherence (SOC) and dental caries on oral health-related quality of life (OHRQoL) of adolescents., Subject and Methods: A cross-sectional sample of 768 adolescents nested in a cohort study was evaluated. SOC was measured using Antonovsky's scale. Dental caries was collected considering the decayed, missing, and filled teeth index (DMFT). OHRQoL was collected through the Child Perception Questionnaire. Demographic, socioeconomic, and use of service variables were also collected. The mediation effects between variables were tested by structural equation modeling., Results: The average age of the individuals was 17.5 years. Individuals with high SOC scores had a positive impact on OHRQoL (β-coefficient = -0.573, p < 0.00). Individuals with high DMFT (β-coefficient = 0.080, p = 0.034) and dental pain (β-coefficient = 0.079, p = 0.039) negatively impacted OHRQoL. Still, adolescents who had dental pain (β-coefficient = 0.112, p = 0.005), mothers with lower education levels (β-coefficient = -0.114, p = 0.004), lower household income (β-coefficient = -0.131, p = 0.003), and those who visited the dentist (β-coefficient = 0.109, p = 0.005) exhibited a higher DMFT index., Conclusions: SOC and dental caries had a direct effect on OHRQoL, indicating that oral conditions and psychosocial characteristics are important contributors to OHRQoL., (© 2021 Wiley Periodicals LLC.)

Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C. elegans nervous system. To probe homeobox function, we make use of a number of reporter gene tools, including a novel multicolor reporter transgene, NeuroPAL, which permits simultaneous monitoring of the execution of multiple differentiation programs throughout the entire nervous system. Using these tools, we add to the previous characterization of homeobox gene function by identifying neuronal differentiation defects for 14 homeobox genes in 24 distinct neuron classes that are mostly unrelated by location, function and lineage history. 12 of these 24 neuron classes had no homeobox gene function ascribed to them before, while in the other 12 neuron classes, we extend the combinatorial code of transcription factors required for specifying terminal differentiation programs. Furthermore, we demonstrate that in a particular lineage, homeotic identity transformations occur upon loss of a homeobox gene and we show that these transformations are the result of changes in homeobox codes. Combining the present with past analyses, 113 of the 118 neuron classes of C. elegans are now known to require a homeobox gene for proper execution of terminal differentiation programs. Such broad deployment indicates that homeobox function in neuronal identity specification may be an ancestral feature of animal nervous systems., Competing Interests: The authors have declared that no competing interests exist.

This study evaluated the influence of Oral Health-Related Quality of Life (OHRQoL) on the consumption of alcohol and cigarette use in adolescents. This prospective cohort began in 2012 (T1) with an initial random sample of 1134 12-year-old adolescents followed for 6 years in Santa Maria, southern Brazil. The present study comprised data from the two cohort reassessments that took place in 2014 (T2) and 2018 (T3). OHRQoL was measured with the Brazilian short version of the Child Perceptions Questionnaire (CPQ11-14) at T2. Socioeconomic, demographic, and oral health measures were also collected during this period. Alcohol and cigarette consumption (regular use) in the past 30 days was evaluated at T3 through questions in the of the National School Health Survey (PeNSE) questionnaire. A multilevel Poisson regression model was used to evaluate the influence of predictor variables on substance use. Of the 770 adolescents at T2, 575 and 576 adolescents were reassessed at T3 for alcohol and cigarette consumption, respectively. Adolescents with higher overall CPQ11-14 scores were at higher risk for regular consumption of alcohol (IRR 1.01; 95% CI 1.01-1.02) and cigarette (IRR 1.04; 95% CI 1.03-1.05). Non-white adolescents with low socioeconomic status, untreated dental caries, and who had not been to the dentist (last 6 months) were also associated with increased regular consumption of licit substances. Adolescents with worse OHRQoL presented a higher consumption of alcohol and cigarette. These findings are useful for planning public health strategies to improve adolescent OHRQoL and reduce the harmful substance use.

Many neuronal identity regulators are expressed in distinct populations of cells in the nervous system, but their function is often analyzed only in specific isolated cellular contexts, thereby potentially leaving overarching themes in gene function undiscovered. We show here that the Caenorhabditis elegans Prop1-like homeobox gene unc-42 is expressed in 15 distinct sensory, inter- and motor neuron classes throughout the entire C. elegans nervous system. Strikingly, all 15 neuron classes expressing unc-42 are synaptically interconnected, prompting us to investigate whether unc-42 controls the functional properties of this circuit and perhaps also the assembly of these neurons into functional circuitry. We found that unc-42 defines the routes of communication between these interconnected neurons by controlling the expression of neurotransmitter pathway genes, neurotransmitter receptors, neuropeptides, and neuropeptide receptors. Anatomical analysis of unc-42 mutant animals reveals defects in axon pathfinding and synaptic connectivity, paralleled by expression defects of molecules involved in axon pathfinding, cell-cell recognition, and synaptic connectivity. We conclude that unc-42 establishes functional circuitry by acting as a terminal selector of functionally connected neuron types. We identify a number of additional transcription factors that are also expressed in synaptically connected neurons and propose that terminal selectors may also function as 'circuit organizer transcription factors' to control the assembly of functional circuitry throughout the nervous system. We hypothesize that such organizational properties of transcription factors may be reflective of not only ontogenetic, but perhaps also phylogenetic trajectories of neuronal circuit establishment., Competing Interests: EB, LG, AB, HS, EV, NM, AA, YF, KN, SC, JW, NM, CC, YR, DF, DH No competing interests declared, OH Reviewing editor, eLife, (© 2021, Berghoff et al.)

Microtubules (MTs) are cytoskeletal elements that provide structural support and act as roadways for intracellular transport in cells. MTs are also needed for neurons to extend and maintain long axons and dendrites that establish connectivity to transmit information through the nervous system. Therefore, in neurons, the ability to independently regulate cytoskeletal stability and MT-based transport in different cellular compartments is essential. Posttranslational modification of MTs is one mechanism by which neurons regulate the cytoskeleton. The carboxypeptidase CCP1 negatively regulates posttranslational polyglutamylation of MTs. In mammals, loss of CCP1, and the resulting hyperglutamylation of MTs, causes neurodegeneration. It has also long been known that CCP1 expression is activated by neuronal injury; however, whether CCP1 plays a neuroprotective role after injury is unknown. Using shRNA-mediated knock-down of CCP1 in embryonic rat spinal cord cultures, we demonstrate that CCP1 protects spinal cord neurons from excitotoxic death. Unexpectedly, excitotoxic injury reduced CCP1 expression in our system. We previously demonstrated that the CCP1 homolog in Caenorhabditis elegans is important for maintenance of neuronal cilia. Although cilia enhance neuronal survival in some contexts, it is not yet clear whether CCP1 maintains cilia in mammalian spinal cord neurons. We found that knock-down of CCP1 did not result in loss or shortening of cilia in cultured spinal cord neurons, suggesting that its effect on survival of excitotoxicity is independent of cilia. Our results support the idea that enzyme regulators of MT polyglutamylation might be therapeutically targeted to prevent excitotoxic death after spinal cord injuries., (Copyright © 2021 Ramadan et al.)

Ciliary microtubules are subject to post-translational modifications that act as a "Tubulin Code" to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in a tubulin tyrosine ligase-like glutamylase gene. We show that mutation in the ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of ciliary dye filling and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 localizes to the ciliary base but does not localize to cilia, suggesting an indirect role in ciliary processes. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into the roles of the tubulin code in human ciliopathies and neurodegenerative diseases., Competing Interests: The authors have declared that no competing interests exist.

Religiosity has been associated with a better dental condition and positive self-perception of health, but there are no studies on the relationship between religious practice and oral health-related quality of life (OHRQoL), especially among schoolchildren. Thus, the aim of this study was to evaluate the association between family religiosity and OHRQoL in 12-year-old schoolchildren. We conducted a cross-sectional study in a representative sample of 12-year-old schoolchildren from Santa Maria, a city in southern Brazil. A total of 1,134 schoolchildren were assessed in a randomly selected in 20 public schools in the city. Participants were examined by 4 calibrated dentists (minimum Kappa-value for intra and inter-examiner agreement were 0.79 and 0.77, respectively) according to dental caries (Decayed, Missed, Filled Teeth Index), and gingival bleeding (Community Periodontal Index criteria). OHRQoL was assessed by the Brazilian short version of Child Perceptions for Questionnaire (CPQ11-14). Parents or guardians answered a structured questionnaire regarding their socioeconomic status and religious practice. Data were analyzed using multilevel Poisson regression analysis to assess the association of religiosity and overall and domain-specific CPQ 11-14 scores. Religious practice less than once a week was associated with higher mean symptoms domain scores and higher mean CPQ 11-14 overall scores in schoolchildren. In conclusion, our findings demonstrate that family religiosity was positively associated with schoolchildren's OHRQoL.

Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs), such as glutamylation of tubulin tails. PTMs and tubulin isotype diversity act as a "tubulin code" that regulates cytoskeletal stability and the activity of MT-associated proteins such as kinesins. We previously showed that, in C. elegans cilia, the deglutamylase CCPP-1 affects ciliary ultrastructure, localization of the TRP channel PKD-2 and the kinesin-3 KLP-6, and velocity of the kinesin-2 OSM-3/KIF17, whereas a cell-specific α-tubulin isotype regulates ciliary ultrastructure, intraflagellar transport, and ciliary functions of extracellular vesicle (EV)-releasing neurons. Here we examine the role of PTMs and the tubulin code in the ciliary specialization of EV-releasing neurons using genetics, fluorescence microscopy, kymography, electron microscopy, and sensory behavioral assays. Although the C. elegans genome encodes five tubulin tyrosine ligase-like (TTLL) glutamylases, only ttll-11 specifically regulates PKD-2 localization in EV-releasing neurons. In EV-releasing cephalic male (CEM) cilia, TTLL-11 and the deglutamylase CCPP-1 regulate remodeling of 9+0 MT doublets into 18 singlet MTs. Balanced TTLL-11 and CCPP-1 activity fine-tunes glutamylation to control the velocity of the kinesin-2 OSM-3/KIF17 and kinesin-3 KLP-6 without affecting the intraflagellar transport (IFT) kinesin-II. TTLL-11 is transported by ciliary motors. TTLL-11 and CCPP-1 are also required for the ciliary function of releasing bioactive EVs, and TTLL-11 is itself a novel EV cargo. Therefore, MT glutamylation, as part of the tubulin code, controls ciliary specialization, ciliary motor-based transport, and ciliary EV release in a living animal. We suggest that cell-specific control of MT glutamylation may be a conserved mechanism to specialize the form and function of cilia., (Copyright © 2017 Elsevier Ltd. All rights reserved.)