An optimal growth law for RNA composition and its partial implementation through ribosomal and tRNA gene locations in bacterial genomes.

The distribution of cellular resources across bacterial proteins has been quantified through phenomenological growth laws. Here, we describe a complementary bacterial growth law for RNA composition, emerging from optimal cellular resource allocation into ribosomes and ternary complexes. The predicted decline of the tRNA/rRNA ratio with growth rate agrees quantitatively with experimental data. Its regulation appears to be implemented in part through chromosomal localization, as rRNA genes are typically closer to the origin of replication than tRNA genes and thus have increasingly higher gene dosage at faster growth. At the highest growth rates in E. coli, the tRNA/rRNA gene dosage ratio based on chromosomal positions is almost identical to the observed and theoretically optimal tRNA/rRNA expression ratio, indicating that the chromosomal arrangement has evolved to favor maximal transcription of both types of genes at this condition.

Effects of Tactical Periodization on Workload, Physical Fitness, and Well-Being in Professional Rugby Union Players During a Preseason Period.

ABSTRACT: Hu, X, Boisbluche, S, Philippe, K, Maurelli, O, Li, S, Xu, B, and Prioux, J. Effects of tactical periodization on workload, physical fitness, and well-being in professional rugby union players during a preseason period. J Strength Cond Res 38(1): 105-115, 2024-Tactical periodization (TP) emerged approximately 30 years ago and has recently gained considerable attention in rugby union (RU). It aims to develop specific physical fitness components with 3 acquisition days (strength, endurance, and speed). However, no study has investigated the effects of TP on workload, physical fitness, and well-being across an RU preseason. This study aimed to determine how RU players' workload response to TP focusing on positional differences, observe the influence of a TP preseason training program on aerobic fitness and neuromuscular performance between positions, and analyze the variation of well-being reported by forwards and backs from the 3 acquisition days. Thirty-two male players completed a 6-week TP protocol. External and internal workload variables were recorded through global positioning systems and session rating of perceived exertion (s-RPE) separately. Fitness assessments included Bronco and countermovement jump (CMJ) tests. The sum of well-being indices was measured using the Hooper index. Kruskal-Wallis H tests revealed that the highest values of PlayerLoad slow, PlayerLoad slow percentage, and s-RPE were found on endurance day and the lowest on speed day. Mann-Whitney U tests showed that 15 external workload parameters were higher in backs than forwards for each acquisition day. Small improvements were observed on the Bronco test. No differences were observed in CMJ performance during the preseason period and well-being values between acquisition days. This study provides unique insights into external and internal workload variables during each acquisition day. Furthermore, it highlights TP as an efficient theoretical concept to use in an RU context.

An association analysis for genetic factors for dental caries susceptibility in a cohort of Chinese children.

OBJECTIVE: To comprehensively investigate the effects of 25 variants in 15 genes on dental caries susceptibility in a cohort of Chinese children. METHODS: A total of 25 variants in 15 genes were genotyped with MassARRAY iPLEX system and analyzed in 265 healthy controls and 254 children affected by dental caries with different dmft scores. The children with dental caries were stratified into "mild group" (scores from 1 to 3), "moderate group" (scores from 4 to 6), and "severe group" (scores from 7 to 14). RESULTS: The association analysis revealed that rs11362 of defensin ß1 (DEFB1) was significantly associated with dental caries susceptibility (OR = 2.447, p = 1.165E-04). Furthermore, rs11362 was positively correlated with the severity of dental caries. For another selected variant of DEFB1, rs1799946 was significantly associated with dental caries susceptibility in the severe group (OR = 0.473, p = 3.70E-03) and also significant in the group consisted of moderate and severe subjects (OR = 0.623, p = .033). The results from logistic regression in additive, dominant, and recessive models also exhibited the similar patterns. CONCLUSION: Out of 25 selected variants, only 2 of DEFB1 gene (rs11362 and rs1799946) were significantly associated with dental caries susceptibility in children.

Are protein hubs faster folders? Exploration based on Escherichia coli proteome.

Protein hubs in protein-protein interaction network are especially important due to their central roles in the entire network. Despite of their importance, the folding kinetics of hub proteins in comparison with non-hubs is still unknown. In this work, the folding rates for protein hubs and non-hubs were predicted and compared for the interactome of Escherichia coli K12, and the results showed that hub proteins fold faster than non-hub proteins. A possible explanation might be that protein hubs have more and fast-folding structural conformations than non-hubs, which leads to the notion of "hub of hubs" in the protein conformation space. It was found that the sequence and structure features relevant to protein folding rates are also different between hub and non-hub proteins. Moreover, the interacting proteins tend to have similar folding rates. These results gave insightful implications for understanding the interplay between the mechanisms of protein folding and interaction.

[Microbial Community Structure on the Root Surface of Patients with Periodontitis.]

OBJECTIVES: To study the microbial community structure on the root surface of patients with periodontitis. METHODS: Bacterial plaque and tissues from the root neck (RN group),root middle (RM group) and root tine (RT group) of six teeth with mobility 3 in one patient with periodontitis were sampled.The V3V4 region of 16S rRNA was sequenced on the Illumina MiSeq platform.The microbial community structure was analyzed by Mothur,Qiime and SPSS software. RESULTS: The principal component analysis (PCoA) results indicated that the RM samples had a similar microbial community structure as that of the RT samples,which was significant different from that of the RN samples.Thirteen phyla were detected in the three groups of samples,which included 7 dominant phyla.29 dominant genera were detected in 184 genera.The abundance of Bacteroidetes_[G-6] and Peptostre ptococcaceae_[XI][G-4] had a positive correlation with the depth of the collection site of samples (P<0.05),while the abundance of Prevotella,Selenomonas,Corynebacterium and Olsenella had a negative correlation with the depth of the collection site of samples (P<0.05). CONCLUSIONS: There is region-specificity of microbial community structure on the root surface of patients with periodontitis.

Position-specific workload of professional rugby union players during tactical periodization training.

The positional workload characteristics in rugby union on three acquisition days (i.e. strength, endurance, and speed days) of tactical periodization are still relatively unknown. Therefore, the primary aim of this study was to shed light on the positional external workload variables (10 Hz Global Positioning System and accelerometer microtechnology) and internal workload indicators (the session rating of perceived exertion) of players in a professional rugby union team by utilizing and comparing two tactical periodization models. Twenty-six male players (15 forwards and 11 backs) were recruited from a French second-division rugby club. Data were obtained over 10 weeks of in-season home games: a total of 780 observations were analyzed. Student's t-test observed different external workload profiles between positions among acquisition days. Mean external workload values, except PlayerLoadslow, were significantly higher (p≤0.01; effect size: 0.41-1.93) for backs than forwards for all acquisition days. Moreover, forwards perceived a higher internal workload than backs on the strength day of both models. The findings demonstrate that applying these two tactical periodization models could result in effective rugby union training. Validating external and internal workload characteristics on tactical periodization acquisition days enables extensive analysis of training load monitoring data; these data can be utilized to discover the unique characteristics of each position and design position-specific acquisition days to improve performance.

Proteome efficiency of metabolic pathways in Escherichia coli increases along the nutrient flow.

IMPORTANCE: Protein translation is the most expensive cellular process in fast-growing bacteria, and efficient proteome usage should thus be under strong natural selection. However, recent studies show that a considerable part of the proteome is unneeded for instantaneous cell growth in Escherichia coli. We still lack a systematic understanding of how this excess proteome is distributed across different pathways as a function of the growth conditions. We estimated the minimal required proteome across growth conditions in E. coli and compared the predictions with experimental data. We found that the proteome allocated to the most expensive internal pathways, including translation and the synthesis of amino acids and cofactors, is near the minimally required levels. In contrast, transporters and central carbon metabolism show much higher proteome levels than the predicted minimal abundance. Our analyses show that the proteome fraction unneeded for instantaneous cell growth decreases along the nutrient flow in E. coli.

Turnover number predictions for kinetically uncharacterized enzymes using machine and deep learning.

The turnover number kcat, a measure of enzyme efficiency, is central to understanding cellular physiology and resource allocation. As experimental kcat estimates are unavailable for the vast majority of enzymatic reactions, the development of accurate computational prediction methods is highly desirable. However, existing machine learning models are limited to a single, well-studied organism, or they provide inaccurate predictions except for enzymes that are highly similar to proteins in the training set. Here, we present TurNuP, a general and organism-independent model that successfully predicts turnover numbers for natural reactions of wild-type enzymes. We constructed model inputs by representing complete chemical reactions through differential reaction fingerprints and by representing enzymes through a modified and re-trained Transformer Network model for protein sequences. TurNuP outperforms previous models and generalizes well even to enzymes that are not similar to proteins in the training set. Parameterizing metabolic models with TurNuP-predicted kcat values leads to improved proteome allocation predictions. To provide a powerful and convenient tool for the study of molecular biochemistry and physiology, we implemented a TurNuP web server.

Effects of preseason training on body composition, running performance, biochemical markers and workload variation in professional rugby union players.

Few studies have examined the impact of a preseason training intervention through systematic measures in Pro D2 rugby union (RU). Therefore, this study aimed to describe the effects of 12 weeks of preseason training (three blocks) on body composition, running performance, biochemical markers, and workload (WL) variation in professional RU players. Physiological (physical and biochemical) responses to preseason WL were analyzed by examining changes in anthropometric characteristics, Yo-Yo intermittent recovery level 1 (Yo-Yo IR1) test, blood samples (BS), Hooper index (1-7), the 10-Hz global positioning system (GPS), and session rating of perceived exertion (s-RPE) in nineteen elite male players. Changes throughout the preseason were analyzed using the one-way and mixed-model analysis of variance. Significant (p < 0.01) improvements occurred in anthropometry and Yo-Yo IR1 running performance in forwards and backs. Total distance (p < 0.01) and impact (p < 0.05) during the second block were meaningfully higher than the other two blocks, with backs showing higher values than forwards. As expected, WL decreased significantly (p < 0.01) during the last training block. The WL variations were correlated with changes in biochemical markers over the preseason period. The collected data can be used for i) profiling French Pro D2 rugby championships players, ii) establishing effective training strategies, and iii) setting preseason WL expectations.

The Spatial Organization of Bacterial Transcriptional Regulatory Networks.

The transcriptional regulatory network (TRN) is the central pivot of a prokaryotic organism to receive, process and respond to internal and external environmental information. However, little is known about its spatial organization so far. In recent years, chromatin interaction data of bacteria such as Escherichia coli and Bacillus subtilis have been published, making it possible to study the spatial organization of bacterial transcriptional regulatory networks. By combining TRNs and chromatin interaction data of E. coli and B. subtilis, we explored the spatial organization characteristics of bacterial TRNs in many aspects such as regulation directions (positive and negative), central nodes (hubs, bottlenecks), hierarchical levels (top, middle, bottom) and network motifs (feed-forward loops and single input modules) of the TRNs and found that the bacterial TRNs have a variety of stable spatial organization features under different physiological conditions that may be closely related with biological functions. Our findings provided new insights into the connection between transcriptional regulation and the spatial organization of chromosome in bacteria and might serve as a factual foundation for trying spatial-distance-based gene circuit design in synthetic biology.

Aerobic fitness and isokinetic knee strength of semi-professional rugby union players: a comparison between backs and forwards.

BACKGROUND: This study aimed to describe the physical fitness characteristics of semi-professional rugby union (RU) players using VO2max (mL. min-1. kg-1), knee peak torque (PT), mean power (MP), hamstring: quadriceps (H:Q) ratio and basic anthropometrics characteristics and to compare backs and forwards across these same characteristics. METHODS: VO2max and ventilatory threshold (VT) as a percentage of VO2max were measured using the Bruce protocol maximal graded test. Knee strength test PT, MP and H:Q were analyzed using the isokinetic dynamometer test. Basic anthropometrics measures were obtained by measures of body mass, height (from Tanita Body Composition Analysis; Tanita Corp., Tokyo, Japan). While fat mass was measured using skinfold thicknesses taking from different body parts. Twenty-five semi-professional players aged (26.3±0.9 years) from the same team were recruited for the study. Mean experimental values from maximal graded test and isokinetic dynamometer test were analyzed using t-tests from the SigmaStat 3.11 program (Systat Software Inc., San Jose, CA, USA). RESULTS: The results showed that forwards were significant taller (P=0.036), heavier (P=0.01), with greater fat mass (P=0.001) than backs. Backs had a greater VO2max (P=0.02) than forwards. With forwards having a greater (P=0.01) eccentric H PT. CONCLUSIONS: This study presents important information for players and athletic coordinators as to the differences between backs and forwards across physiological and strength measures. This information can greatly inform the individualized training protocol and preparation of current and future players across different positions and different levels.

The protein translation machinery is expressed for maximal efficiency in Escherichia coli.

Protein synthesis is the most expensive process in fast-growing bacteria. Experimentally observed growth rate dependencies of the translation machinery form the basis of powerful phenomenological growth laws; however, a quantitative theory on the basis of biochemical and biophysical constraints is lacking. Here, we show that the growth rate-dependence of the concentrations of ribosomes, tRNAs, mRNA, and elongation factors observed in Escherichia coli can be predicted accurately from a minimization of cellular costs in a mechanistic model of protein translation. The model is constrained only by the physicochemical properties of the molecules and has no adjustable parameters. The costs of individual components (made of protein and RNA parts) can be approximated through molecular masses, which correlate strongly with alternative cost measures such as the molecules' carbon content or the requirement of energy or enzymes for their biosynthesis. Analogous cost minimization approaches may facilitate similar quantitative insights also for other cellular subsystems.

Lack of associations between lactoferrin (LTF) and mannose-binding lectin 2 (MBL2) gene polymorphism and dental caries susceptibility.

OBJECTIVE: With the development of human genomics, the genetic factors associated with dental caries have receiving increasing attention. This study was performed to evaluate the relationship between lactoferrin (LTF) and mannose-binding lectin 2 (MBL2) gene single nucleotide polymorphisms (SNPs) and dental caries susceptibility in Chinese children. METHODS: This prospective case-control study included 360 unrelated children (aged 12-15 years) who received oral health examinations and questionnaire surveys. The children were divided into two groups by counting the numbers of decayed, missing, and filled teeth (DMFT/dmft): case group (n = 162, DMFT/dmft ≥ 1) and control group (n = 198, DMFT/dmft = 0); non-invasive saliva samples were collected to extract genomic DNA. Six SNPs (rs2073495C/G, rs1042073C/T, rs10865941C/T, and rs1126477A/G in LTF; rs7096206C/G and rs7095891G/A in MBL2) were tested by mass spectrometry. RESULTS: The study included 360 individuals with (85 boys and 77 girls) and without a history of caries (96 boys and 102 girls). There were no statistically significant differences in alleles and genotypes among the six SNPs between the two groups. CONCLUSION: There is no evidence that polymorphisms of LTF and MBL2 genes are associated with dental caries susceptibility in populations from northwest China; further confirmation is needed with larger sample sizes.

Association of ENAM, TUFT1, MMP13, IL1B, IL10 and IL1RN gene polymorphism and dental caries susceptibility in Chinese children.

OBJECTIVE: To investigate the association between single nucleotide polymorphisms (SNPs) in six candidate genes (enamelin [ ENAM]; tuftelin 1 [ TUFT1]; matrix metallopeptidase 13 [ MMP13]; interleukin 1 beta [ IL1B]; interleukin 10 [ IL10]; interleukin 1 receptor antagonist [ IL1RN]) and dental caries in children from northwest China. METHODS: This case-control study enrolled children (12-15 years) who underwent routine dental examinations. The children were divided into two groups based on the presence of dental caries. A saliva sample was collected and seven SNPs (rs3806804A/G in ENAM, rs3811411T/G in TUFT1, rs2252070A/G and rs597315A/T in MMP13, rs1143627C/T in IL1B, rs1800872A/C in IL10 and rs956730G/A in IL1RN) were genotyped. RESULTS: A total of 357 children were enrolled in the study: 161 with dental caries and 196 without dental caries. No significant difference was found in the alleles and genotypes of five genes ( ENAM, TUFT1, MMP13, IL10 and IL1RN) between those with and without dental caries. A significant relationship was found between the IL1B rs1143627C/T polymorphism and dental caries susceptibility with those carrying the rs1143627CT genotype having a lower risk of dental caries compared with those carrying the CC genotype (odds ratio 0.557; 95% confidence interval 0.326, 0.952). CONCLUSION: The IL1B rs1143627C/T polymorphism may be associated with dental caries susceptibility in children from northwest China.

Construction and simulation of the Bradyrhizobium diazoefficiens USDA110 metabolic network: a comparison between free-living and symbiotic states.

Bradyrhizobium diazoefficiens is a rhizobium able to convert atmospheric nitrogen into ammonium by establishing mutualistic symbiosis with soybean. It has been recognized as an important parent strain for microbial agents and is widely applied in agricultural and environmental fields. In order to study the metabolic properties of symbiotic nitrogen fixation and the differences between a free-living cell and a symbiotic bacteroid, a genome-scale metabolic network of B. diazoefficiens USDA110 was constructed and analyzed. The metabolic network, iYY1101, contains 1031 reactions, 661 metabolites, and 1101 genes in total. Metabolic models reflecting free-living and symbiotic states were determined by defining the corresponding objective functions and substrate input sets, and were further constrained by high-throughput transcriptomic and proteomic data. Constraint-based flux analysis was used to compare the metabolic capacities and the effects on the metabolic targets of genes and reactions between the two physiological states. The results showed that a free-living rhizobium possesses a steady state flux distribution for sustaining a complex supply of biomass precursors while a symbiotic bacteroid maintains a relatively condensed one adapted to nitrogen-fixation. Our metabolic models may serve as a promising platform for better understanding the symbiotic nitrogen fixation of this species.

Relationship between Gingival Crevicular Fluid Microbiota and Cytokine Profile in Periodontal Host Homeostasis.

As potential biomarkers in periodontitis, microbiome, and cytokines have recently been extensively investigated, but combined analyses of the variations between the microbial structure and cytokine composition are rare. The present study aimed to investigate whether there are differences in the combined profile of microbiome and cytokines in individuals with or without periodontitis. The microbiome and cytokine composition in gingival crevicular fluid (GCF) from 16 patients and 15 controls from Jishi Shan (Gansu, China) were analyzed using 454 pyrosequencing and RayBio Quantibody Arrays. The results showed that a higher co-occurrence of genera in periodontitis group compared with the healthy group, as evaluated by Schoener's abundance-based co-occurrence index. C-reactive protein (CRP) was significantly (P < 0.05) higher in the GCF of the periodontitis group while interleukin (IL)-8 was significantly (P < 0.01) higher in the GCF of the healthy group. The Mantel test revealed a significant concordance between cytokines and microbiota, in the healthy group (Mantel statistic r = 0.36, P ≤ 0.05) but not in the periodontitis group (Mantel statistic r = 0.013, P = 0.434). The results were further confirmed by the Procrustes test. Matrix metalloproteinase (MMP)-9, osteoactivin, IL-8, and macrophage inflammatory protein (MIP)-1a were significantly associated with bacterial composition at the phylum, class, order, family, and genus levels. CRP was also associated with bacterial composition at the species level. In conclusion, alterations in the polymicrobial community structure leads to disruption in the healthy correlation between cytokines and microbiomes. This dysbiosis between the microbiota and the immune response could be one of the major etiological mechanisms underlying periodontitis.

Exploration of Human Salivary Microbiomes--Insights into the Novel Characteristics of Microbial Community Structure in Caries and Caries-Free Subjects.

Recently, high-throughput sequencing has improved the understanding of the microbiological etiology of caries, but the characteristics of the microbial community structure in the human oral cavity with and without caries are not completely clear. To better understand these characteristics, Illumina MiSeq high-throughput sequencing was utilized to analyze 20 salivary samples (10 caries-free and 10 caries) from subjects from the same town in Dongxiang, Gansu, China. A total of 5,113 OTUs (Operational Taxonomic Units, 97% cutoff) were characterized in all of the salivary samples obtained from the 20 subjects. A comparison of the two groups revealed that (i) the predominant phyla were constant between the two groups; (ii) the relative abundance of the genera Veillonella, Bifidobacterium, Selenomonas, Olsenella, Parascardovia, Scardovia, Chryseobacterium, Terrimonas, Burkholderia and Sporobacter was significantly higher in the group with caries (P < 0.05); and (iii) four genera with low relative abundance (< 0.01% on average), including two characteristic genera in caries (Chryseobacterium and Scardovia), significantly influenced the microbial community structure at the genus and OTU levels. Moreover, via co-occurrence and principal component analyses, the co-prevalence of the pathogenic genera was detected in the caries samples, but in the caries-free samples, the function of clustered genera was more random. This result suggests that a synergistic effect may be influencing the assembly of the caries microbial community, whereas competition may play a more dominant role in governing the microbial community in the caries-free group. Our findings regarding the characteristics of the microbial communities of the groups with and without caries might improve the understanding of the microbiological etiology of caries and might improve the prevention and cure of caries in the future.

Amino Acid Flux from Metabolic Network Benefits Protein Translation: the Role of Resource Availability.

Protein translation is a central step in gene expression and affected by many factors such as codon usage bias, mRNA folding energy and tRNA abundance. Despite intensive previous studies, how metabolic amino acid supply correlates with protein translation efficiency remains unknown. In this work, we estimated the amino acid flux from metabolic network for each protein in Escherichia coli and Saccharomyces cerevisiae by using Flux Balance Analysis. Integrated with the mRNA expression level, protein abundance and ribosome profiling data, we provided a detailed description of the role of amino acid supply in protein translation. Our results showed that amino acid supply positively correlates with translation efficiency and ribosome density. Moreover, with the rank-based regression model, we found that metabolic amino acid supply facilitates ribosome utilization. Based on the fact that the ribosome density change of well-amino-acid-supplied genes is smaller than poorly-amino-acid-supply genes under amino acid starvation, we reached the conclusion that amino acid supply may buffer ribosome density change against amino acid starvation and benefit maintaining a relatively stable translation environment. Our work provided new insights into the connection between metabolic amino acid supply and protein translation process by revealing a new regulation strategy that is dependent on resource availability.

Sequence determinants of prokaryotic gene expression level under heat stress.

Prokaryotic gene expression is environment-dependent and temperature plays an important role in shaping the gene expression profile. Revealing the regulation mechanisms of gene expression pertaining to temperature has attracted tremendous efforts in recent years particularly owning to the yielding of transcriptome and proteome data by high-throughput techniques. However, most of the previous works concentrated on the characterization of the gene expression profile of individual organism and little effort has been made to disclose the commonality among organisms, especially for the gene sequence features. In this report, we collected the transcriptome and proteome data measured under heat stress condition from recently published literature and studied the sequence determinants for the expression level of heat-responsive genes on multiple layers. Our results showed that there indeed exist commonness and consistent patterns of the sequence features among organisms for the differentially expressed genes under heat stress condition. Some features are attributed to the requirement of thermostability while some are dominated by gene function. The revealed sequence determinants of bacterial gene expression level under heat stress complement the knowledge about the regulation factors of prokaryotic gene expression responding to the change of environmental conditions. Furthermore, comparisons to thermophilic adaption have been performed to reveal the similarity and dissimilarity of the sequence determinants for the response to heat stress and for the adaption to high habitat temperature, which elucidates the complex landscape of gene expression related to the same physical factor of temperature.

[Oral health status and its correlation with oral health knowledge among middle-aged people in Dongxiang, Bonan, and Yugur].

OBJECTIVE: To provide basic data for the prevention of oral diseases in minorities by investigating the oral health status and behavior related to oral health knowledge of individuals aged 35 to 44 years in Dongxiang, Bonan, and Yugur. METHODS: The caries and periodontal health of 445 individuals aged 35 to 44 years were examined according to the method and criterion prescribed by the World Health Organization and the Third National Oral Health Epidemiologic Investigation. A questionnaire survey on related oral health knowledge and behavior was conducted. RESULTS: The crown caries prevalence rate in Dongxiang, Baoan, and Yugur were 48.28%, 79.47%, and 67.11%, respectively; the root caries prevalence rates were 38.62%, 69.54%, and 42.95%, respectively. The rates of gum bleeding in Dongxiang, Bonan, and Yugur were 86.90%, 90.07%, and 65.77%, respectively. The rates of dental calculus in Dongxiang, Bonan, and Yugur were 99.31%, 100.00%, 99.33%, respectively, and the rates of periodontal bags were 68.97%, 67.55%, and 43.62%, respectively. Only 69.84% of the respondents brush their teeth every day; 94.90% of the respondents do not floss. Only 20.19% of the respondents contact a doctor for a toothache, and 42.23% of the respondents have never seen a dentist. CONCLUSION: Caries morbidity is high among the respondents aged 35 to 44 years from Dongxiang, Bonan, and Yugur. The periodontal health status and oral hygiene of the respondents are poor, and behavior related to oral health knowledge is insufficient. Thus, more attention must be provided to the prevention and control of caries and periodontal diseases among middle-aged people in the area.