PIF4 Directly Binds to the Promoter Region of <i>YUC8</i>.

<p>(A) Illustration of the <i>YUC5</i>, <i>YUC8</i>, <i>YUC9</i> and <i>YUC10</i> promoter regions showing the presence of G-box DNA motifs. The arrows indicate positions of primers used for ChIP-PCR experiment. Shown are 2-kb upstream sequences of the <i>YUC</i> genes. The translational start site (ATG) is shown at position +1. (B) Gel photographs showing the amplified products from the ChIP assay. The ChIP assays were performed using 6-d-old seedlings expressing the PIF4-HA fusion protein untreated or treated with 29°C for 6 h. Antibody to the HA tag was used to immunoprecipitate PIF4-HA and associated DNA fragments. DNA was amplified by using primers specific to the region containing the G-box element or control regions in <i>ACT2</i> promoter as indicated. Shown are representative data from one biological replicate; this experiment was conducted for three biological replicates, yielding similar results. (C) EMSA assay showing that PIF4 binds the G-box motifs present in the <i>YUC8</i> promoter <i>in vitro</i>. The <i>YUC8</i> promoter fragments containing the G-box motifs were incubated with <i>in vitro</i> TNT-expressed PIF4 protein as indicated. Competition for PIF4 binding was performed with 10×, 20× and 50× cold <i>YUC8</i> probes containing G-box (G-wt, CACGTG) or mutated G-box (G-mut, CACGGG), respectively. FP, free probe. TnT indicates <i>in vitro</i>-expressed luciferase proteins used as a control.</p

The <i>yuc8</i> Mutation Reduces the Induction of Hypocotyl Elongation by High Temperature and <i>PIF4</i> Overexpression.

<p>(A) Hypocotyl length showing that the <i>yuc8</i> mutation reduces high temperature-induced hypocotyl elongation. Four-d-old seedlings grown at 22°C were transferred to 29°C in continuous light for additional 2 d before hypocotyl length measurement. Data shown are average±SD. Asterisks represent Student's <i>t</i>-test significance between 29°C and 22°C grown plants for each genotype or between pairs indicated with brackets (**, P<0.01). Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results. (B) Hypocotyl length showing that the <i>yuc8</i> mutation partially suppresses the long-hypocotyl phenotype of <i>35S-PIF4</i> plants. The hypocotyl length of 6-d-old seedlings of the indicated genotypes grown at 22°C was measured. Data shown are average±SD. Asterisks represent Student's <i>t</i>-test significance between transgenic/mutant and wild-type plants or between pairs indicated with brackets (*, P<0.05; **, P<0.01). Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results.</p

Overexpression of <i>PIF4</i> Increases the Expression of <i>YUC8</i> and Elevates Endogenous Free IAA Levels.

<p>(A) <i>YUC8</i> expression in wild type (Col-0) and <i>35S-PIF4</i> plants. Six-d-old Col-0 and <i>35S-PIF4</i> seedlings grown in normal growth conditions (22°C) were harvested at the same time for RNA extraction and qRT-PCR analyses. Transcript levels of <i>YUC8</i> were normalized to the <i>ACTIN7</i> expression and then were relative to those of Col-0 seedlings. Data shown are average and SD of triplicate reactions. Student's <i>t</i>-test between Col-0 and <i>35S-PIF4</i> seedlings was performed (**, P<0.01). Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results. (B–C) <i>PIF4</i> and <i>YUC8</i> expression in transgenic plants containing a chemical-inducible construct <i>pMDC7:PIF4</i>. Eight-d-old <i>pMDC7:PIF4</i> seedlings were untreated or treated with 10 µM estradiol for 3 h before harvest for RNA extraction and qRT-PCR analyses. Transcript levels of target genes were normalized to the <i>ACTIN7</i> expression and then were relative to those of untreated seedlings (0 h). Data shown are average and SD of triplicate reactions. Student's <i>t</i>-test between estradiol-treated and untreated plants was performed (**, P<0.01). Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results. (D) Overexpression of <i>PIF4</i> leads to increased free IAA levels. Eight-d-old seedlings of wild type and <i>35S-PIF4</i> seedlings grown in normal growth conditions (22°C) were harvested at the same time for free IAA measurement. Data shown are average±SD. Student's <i>t</i>-test between wild-type and <i>35S-PIF4</i> plants was performed (**, P<0.01). Shown are representative data from one biological replicate; this experiment was conducted for three biological replicates, yielding similar results.</p

The <i>shy2-2</i> Mutation Suppresses the Long-Hypocotyl Phenotype of <i>35S-PIF4</i> Seedlings.

<p>(A) Representative images showing that <i>shy2-2</i> suppresses the long-hypocotyl phenotype of <i>35S-PIF4</i>. Shown are 6-d-old seedlings of Col-0, L<i>er</i>, <i>shy2-2</i>, <i>35S-PIF4</i> and <i>35S-PIF4/shy2-2</i> grown at 22°C. (B) Hypocotyl length showing that <i>shy2-2</i> suppresses the long-hypocotyl phenotype of <i>35S-PIF4</i>. Hypocotyl length of six-d-old seedlings of the indicated genotypes grown at 22°C was measured. Data shown are average±SD. Student's <i>t</i>-test between mutant/transgenic and wild-type seedlings was performed (**, P<0.01). Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results.</p

PIF4 Activates <i>YUC8</i> Expression, as Revealed by Transient Assays of <i>N. benthamiana</i> Leaves.

<p>(A) Transient expression assays showing that PIF4 activates the expression of <i>YUC8</i>. Representative images of <i>N. benthamiana</i> leaves 72 h after infiltration are shown. The right panel indicates the infiltrated constructs. (B) Quantitative analysis of luminescence intensity in (A). Five independent determinations were assessed. Error bars represent SD. Asterisks denote Student's <i>t</i>-test significance compared with control plants: ***, P<0.001. (C) qRT-PCR analysis of <i>PIF4</i> expression in the infiltrated leaf areas shown in (A). Total RNAs were extracted from leaves of <i>N. benthamiana</i> infiltrated with the constructs. Five independent determinations were assessed. Error bars represent SD.</p

Loss of PIF4 Function Disrupts the High Temperature–Induced Elevation of <i>YUC8</i> Transcripts and Free IAA Levels.

<p>(A–B) High temperature-induced expression patterns of <i>PIF4</i> and <i>YUC8</i> in wild type (Col-0) or the <i>pif4</i> mutant. Six-d-old Col-0 and <i>pif4</i> seedlings grown at 22°C in continuous light were transferred to 29°C in continuous light or were continually placed at 22°C for a 24 h time course, respectively. The 22°C-grown and 29°C-grown seedlings for each time point were harvested at the same time for RNA extraction and qRT-PCR analyses. Transcript levels of target genes were normalized to the <i>ACTIN7</i> expression and were relative to those of untreated Col-0 seedlings (0 h). Data shown are average and SD of triplicate reactions. Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results. (C) High temperature-induced elevation of free IAA levels in hypocotyls of Col-0 and <i>pif4</i>. The hypocotyls of 6-d-old wild-type and <i>pif4</i> mutant seedlings grown at 22°C and 29°C in continuous light, respectively, were harvested for free IAA measurement. Data shown are average±SD. Student's <i>t</i>-test between 22°C and 29°C grown plants for each genotype was performed (**, P<0.01). Shown are representative data from one biological replicate; This experiment was conducted for three biological replicates, yielding similar results.</p

Table_1_Correlation between lung cancer probability and number of pulmonary nodules in baseline computed tomography lung cancer screening: A retrospective study based on the Chinese population.docx

BackgroundScreening for lung cancer with LDCT detects a large number of nodules. However, it is unclear whether nodule number influences lung cancer probability. This study aimed to acquire deeply insight into the distribution characteristics of nodule number in the Chinese population and to reveal the association between the nodule number and the probability of lung cancer (LC).Methods10,167 asymptomatic participants who underwent LDCT LC screening were collected. Noncalcified nodules larger than 4 mm were included. The nodule number per participant was determined. We defined five categories according to the number of nodules (based on nodule type and size): one, two, three, four, and more than four nodules. We stratified the nodules as groups A, B, and C and participants as Amax, Bmax, and Cmax groups, and explored the association between nodule number and the probability of LC on nodule and participant levels.Results97 participants were confirmed to have LC. The probabilities of LC were 49/1719, 22/689, 11/327, 6/166, and 9/175 in participants with one, two, three, four, and more than four nodules (p>0.05), respectively. In the Bmax group, the probability of LC was significantly higher in participants with one nodule than those with >4 nodules (p4 nodules (pConclusionLC probability does not significantly change with the number of nodules. However, when stratified by the nodule size, the effect of nodule number on LC probability was nodule-size dependent, and greater attention and active follow-up are required for solitary nodules especially SNs/solid component of PSNs measuring 6-15 mm or NSNs measuring 8-15 mm. Assessing the nodule number in conjunction with nodule size in baseline LDCT LC screening is considered beneficial.</p

DataSheet_1_Impaired meningeal lymphatic drainage in Listeria monocytogenes infection.pdf

Previous studies have demonstrated an association between lymphatic vessels and diseases caused by bacterial infections. Listeria monocytogenes (LM) bacterial infection can affect multiple organs, including the intestine, brain, liver and spleen, which can be fatal. However, the impacts of LM infection on morphological and functional changes of lymphatic vessels remain unexplored. In this study, we found that LM infection not only induces meningeal and mesenteric lymphangiogenesis in mice, but also impairs meningeal lymphatic vessels (MLVs)-mediated macromolecules drainage. Interestingly, we found that the genes associated with lymphatic vessel development and function, such as Gata2 and Foxc2, were downregulated, suggesting that LM infection may affect cellular polarization and valve development. On the other hand, photodynamic ablation of MLVs exacerbated inflammation and bacterial load in the brain of mice with LM infection. Overall, our findings indicate that LM infection induces lymphangiogenesis and may affect cell polarization, cavity formation, and valve development during lymphangiogenesis, ultimately impairing MLVs drainage.</p