Data_Sheet_1_Exploring perceived restoration, landscape perception, and place attachment in historical districts: insights from diverse visitors.docx

Improving the quality of the built environment to enhance people’s mental health is gaining traction across various fields, precipitating valuable actions on the wave of “Healthy China 2030” initiative. While ample studies have confirmed the benefits of interaction with natural or green spaces, the investigation into the restorative potential in urban built settings remains notably underexplored. In this study, we focused on historical districts, conducting a questionnaire survey to evaluate the restorative experiences of individuals visiting these sites. We used Partial Least Square-Structural Equation Modelling (PLS-SEM) to analyze a conceptual model that encompasses landscape perception, place attachment, and perceived restoration, with a specific focus on detecting the mediating role of place attachment and the moderating influence of visitor groups. The results showed that landscape perception significantly influenced the perceived restoration, which contained the indirect effect pathway through place dependence and place identity, as well as the potent direct impact of landscape perception. Moreover, employing a multi-group analysis (MGA), we discerned that different visitor groups partially moderate the relationship between landscape perception, place attachment, and perceived restoration. This study validates the restorative features in historic districts and highlights the importance of cognitive-emotional bond in promoting psychological restoration.</p

Summary of well-expressed uORFs that have significantly different TE relative to the downstream CDSs or zero RPF coverage in each sample.

Summary of well-expressed uORFs that have significantly different TE relative to the downstream CDSs or zero RPF coverage in each sample.</p

Genome-wide maps of ribosomal occupancy provide insights into adaptive evolution and regulatory roles of uORFs during <i>Drosophila</i> development

<div><p>Upstream open reading frames (uORFs) play important roles in regulating the main coding DNA sequences (CDSs) via translational repression. Despite their prevalence in the genomes, uORFs are overall discriminated against by natural selection. However, it remains unclear why in the genomes there are so many uORFs more conserved than expected under the assumption of neutral evolution. Here, we generated genome-wide maps of translational efficiency (TE) at the codon level throughout the life cycle of <i>Drosophila melanogaster</i>. We identified 35,735 uORFs that were expressed, and 32,224 (90.2%) of them showed evidence of ribosome occupancy during <i>Drosophila</i> development. The ribosome occupancy of uORFs is determined by genomic features, such as optimized sequence contexts around their start codons, a shorter distance to CDSs, and higher coding potentials. Our population genomic analysis suggests the segregating mutations that create or disrupt uORFs are overall deleterious in <i>D</i>. <i>melanogaster</i>. However, we found for the first time that many (68.3% of) newly fixed uORFs that are associated with ribosomes in <i>D</i>. <i>melanogaster</i> are driven by positive Darwinian selection. Our findings also suggest that uORFs play a vital role in controlling the translational program in <i>Drosophila</i>. Moreover, we found that many uORFs are transcribed or translated in a developmental stage-, sex-, or tissue-specific manner, suggesting that selective transcription or translation of uORFs could potentially modulate the TE of the downstream CDSs during <i>Drosophila</i> development.</p></div

uORFs are prevalent translational repressors during <i>Drosophila</i> development.

(A) The ratio of median TE for genes with single or multiple ribosome-associated uORFs, relative to the median TE for genes that do not have uORFs in each sample (only genes with mRNA RPKM ≥ 1 were included in analysis). WRSTs were performed to test the differences in each sample (**, P P S4 Data. (B) The relative importance (relative proportion of variance explained by each predictor, x-axis) of different features in 5′ UTRs on log2(TE) of CDSs. For each feature (y-axis), the 25%, 50%, and 75% quantiles of the relative importance across the 12 samples are manifested in the box plots. Only genes with at least 1 ribosome-associated uORF were included in the analysis. The raw data can be found in S12 Table. (C) Spearman’s correlation between the TE of CDSs and the features of uORFs (y-axis) across samples (x-axis). For each cell in the matrix, the genes were grouped into 50 bins of equal size based on the corresponding feature, and Spearman’s correlations were calculated using median log2(TE) and the median value of the feature in each bin. The raw data can be found in S1 Data. (D) The relative importance (x-axis) of various uORF features (y-axis) in the multiple linear regression on log2(TE) of CDSs across the 12 samples. The raw data can be found in S13 Table. BLS, branch length score; cAUG, AUG start codon of CDS; CDS, coding DNA sequence; DMSO, dimethyl sulfoxide; MFE, minimum free energy; RPF, ribosome-protected mRNA fragment; RPKM, reads per kilobase of transcript per million mapped reads; TE, translational efficiency; uAUG, AUG start codon of uORF; uORF, upstream open reading frame; UTR, untranslated region; WRST, Wilcoxon rank-sum test.</p

The number of genes that switched the major transcripts between neighboring stages or tissues.

The number of genes that switched the major transcripts between neighboring stages or tissues.</p

Evolutionary analysis of uAUGs.

(A) The phyloP score (y-axis) of uAUGs and flanking triplets in uORFs in Classes I, II, III, and IV. The position of each triplet relative to uAUGs is shown in the x-axis. The mean and 95% CI (by bootstrapping) of the phyloP score are shown for each uORF class. The dashed line indicates the average phyloP score of positions 8–30 nt of short introns (neutral controls). The raw data can be found in S1 Data. (B) The derived allele frequency of uAUGs (from Classes I–IV) that are polymorphic in the GDL of D. melanogaster. Mutations from Class I, II, and III were combined to increase the statistical power (**, P S1 Data. (C) Frequencies of the derived mutations that cause the gain or loss of uORFs in the 5′ UTR, the remaining derived mutations in the 5′ UTR, and the derived mutations in positions 8–30 nt of short introns in the GDL of D. melanogaster (***, P S3 Data. (D) Examples of uAUGs (uORFs) that are newly created in D. melanogaster (fixed: blue; polymorphic: red) after divergence from D. sechellia or that are lost in D. sechellia (orange). The phylogenetic tree of D. yakuba, D. sechellia, and D. melanogaster (ISO-1 strain and other strains) is shown in the top panel, and the triplet sequences corresponding to each species or strains in the tree above are shown in the lower panel. For both the polymorphic and newly fixed uAUGs, only the ones present in the ISO-1 strain were considered in the analysis. (E) The α values of MK tests on the newly fixed mutations in uAUGs using AUGs in 8–30 nt of short introns as the neutral control for both GDL (blue) and DGRP (orange) data. Three different methods were used: the original MK test, DFE-alpha, and AsymptoticMK. The mutations in all the strains of D. melanogaster were used in the analysis. The error bars indicate 95% CI of αdfe and αasym. The exact values can be found in S1 Data. (F) The α values of MK tests with randomly resampled mutations for both GDL (blue) and DGRP (orange) data. For both newly fixed and polymorphic AUGs in 5′ UTR and 8–30 nt of short introns, the same number of triplets were randomly sampled with replacement and used to perform the original MK and the AsymptoticMK tests. The median (points) and the 2.5% and 97.5% quantile (error bars) of αori and αasym in 1,000 replicates were given. The raw data can be found in S1 Data. (G) The αori for mutations in uAUGs of Classes I and II (combined), III, and IV in GDL data. Only mutations present in the ISO-1 strain of D. melanogaster were used in the analysis. The raw data can be found in S11 Table. (H) The αasym for mutations in uAUGs of Classes I and II (combined), III, and IV in GDL data. AUGs in 8–30 nt of short introns were used as the neutral control. Only mutations present in the ISO-1 strain of D. melanogaster were used. The error bars indicate 95% CI of αasym. The exact values can be found in S1 Data. The raw data for panels (A-E) can be found in S1 Data. DFE, distribution of fitness effects; DGRP, Drosophila Genetic Reference Panel; GDL, Global Diversity Lines; MK test, McDonald-Kreitman test; uAUG, AUG start codon of uORF; uORF, upstream open reading frame; UTR, untranslated region</p

Genome-wide translational events on CDSs and uORFs during <i>Drosophila</i> development.

<p>(A) Heat map showing log₂(TE) of CDSs for 13,917 protein-coding genes (column) in the 12 samples (rows). The numbers of CEGs and NCEGs are presented. TE values smaller than 0.1 or larger than 10 are displayed as 0.1 and 10, respectively. (B) Heat map showing log₂(TE) of Class I–IV uORFs in the 12 samples. For each category, the total number of uORFs and the number of uORFs in CEGs or NCEGs are presented, respectively. TE values smaller than 0.1 or larger than 10 are displayed as 0.1 and 10, respectively. (C) The tissue specificity index <i>H</i>_<i>g</i> of uORFs (y-axis) against that for CDSs (x-axis) in mRNA-Seq (left) and Ribo-Seq (right) experiments. The exact values can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003903#pbio.2003903.s067" target="_blank">S1 Data</a>. CDS, coding DNA sequence; CEG, constitutively expressed gene; DMSO, dimethyl sulfoxide; NCEG, nonconstitutively expressed gene; TE, translational efficiency; RPF, ribosome-protected mRNA fragment; uORF, upstream open reading frame</p

A model of uORF evolution.

<p>Mutations frequently generate novel uORFs (uAUGs) in the 5′ UTRs. A newly emerged uORF in the population might be deleterious, neutral, or advantageous. The highly detrimental uORFs are removed by natural selection or persist in the population at low frequencies, whereas the neutral or slightly deleterious ones might randomly drift in the population. The beneficial new uORFs, which often have a higher tendency to be associated with ribosomes, are favored by natural selection and become fixed in the population very rapidly. The newly fixed uORFs, which regulate the translation of their downstream CDSs, are maintained by natural selection during evolution. The fitness effect of uORF-mediated translational repression is represented with arrows: gray, neutral; red, deleterious; green, beneficial. CDS, coding DNA sequence; uAUG, AUG start codon of uORF; uORF, upstream open reading frame; UTR, untranslated region.</p

Validating the translation initiation of uORFs.

<p>(A) The proportions of canonical uORFs (beginning with AUG, red) and the other 60 kinds of hypothetical uORFs (each beginning with a distinct non-stop-codon triplet, blue) that are bound with ribosomes in at least 11 out of 12 samples (only uORFs with mRNA RPKM ≥ 1 and TE ≥ 0.5 were considered). (B) Signal-to-noise ratio at different cutoffs of minimum TE_uORF. For each cutoff of minimum TE_uORF, the proportion of canonical uORFs that are bound with ribosomes at this cutoff is divided by those for the other 60 kinds of hypothetical uORFs. The average of those ratios is used as signal-to-noise ratio at this cutoff. (C) The relative mRNA and RPF coverage around cAUGs in S2 cells. For each codon downstream (or triplet upstream) the cAUG of a gene (x-axis), the sequencing coverage was calculated, and the relative coverage of that codon (triplet) was calculated by normalization with the median coverage of CDS of this gene. And then, the relative sequencing coverage was averaged across all the genes (y-axis). Red and blue lines represent mRNA-Seq and Ribo-Seq, respectively, of S2 cells treated with DMSO for 30 min. The green line represents Ribo-Seq of S2 cells treated with harringtonine for 30 min. (D) The relative mRNA and RPF coverage around uAUGs in S2 cells. The data normalization and line colors are the same as those in (C). (E) The proportions of CDSs and uORFs (y-axis) with start codons that showed peaks in Ribo-Seq of S2 cells treated with harringtonine for 30 min. CDSs and uORFs with RPF RPKM > 10 in Ribo-Seq of S2 cells are stratified into 10 bins of equal size based on increasing RPF RPKM (x-axis). Spearman’s rank correlation between the proportion of CDS (or uORFs) with peaks of ribosome occupancy, and the median RPKM of CDSs (or uORFs) in each bin, is displayed in the plot. The raw data for panels (A-E) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003903#pbio.2003903.s067" target="_blank">S1 Data</a>. cAUG, AUG start codon of CDS; CDS, coding DNA sequence; DMSO, dimethyl sulfoxide; RPF, ribosome-protected mRNA fragment; RPKM, reads per kilobase of transcript per million mapped reads; TE, translational efficiency; uAUG, AUG start codon of uORF; uORF, upstream open reading frame</p

Features of uORFs with different translational breadths and efficiencies.

<p>(A-C) Box plots for Kozak score around uAUGs (A), the distance from a uAUG to the downstream cAUG (B), and the phyloCSF score (C) for each class of uORFs (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001). The raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003903#pbio.2003903.s067" target="_blank">S1 Data</a>. (D) The relationship between phyloCSF (x-axis) and log₂(TE) (y-axis) of ribosome-associated uORFs in each of the 12 samples. The ribosome-associated uORFs were ranked with increasing phyloCSF and divided into 200 bins of equal size. Median phyloCSF and median log₂(TE) in each bin were displayed in the plot and used to calculate Spearman’s correlation coefficient. In each sample, only uORFs with mRNA RPKM ≥ 1 and TE ≥ 0.5 were used in the analysis. The raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003903#pbio.2003903.s068" target="_blank">S2 Data</a>. The raw data for panels (A-E) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003903#pbio.2003903.s067" target="_blank">S1 Data</a>. cAUG, AUG start codon of CDS; DMSO, dimethyl sulfoxide; RPKM, reads per kilobase of transcript per million mapped reads; TE, translational efficiency; uAUG, AUG start codon of uORF; uORF, upstream open reading frame</p