Differentially expressed genes in the head of the 2^nd instar pre-molting larvae of the <i>nm2</i> mutant of the silkworm, <i>Bombyx mori</i> - Fig 6

<p>Synthesis pathways of juvenile hormone ^A and ecdysone ^B. Genes with green background represent their downregulation and red for upregulation in <i>nm2</i> mutant, and yellow background represent no significant differences between C603 and <i>nm2</i>. Three key genes in the juvenile hormone biosynthesis pathway, <i>JHDH</i>, <i>JHDK</i> and <i>JHAMT</i>, were without any significant difference in the <i>nm2</i> mutant. In the ecdysone synthesis pathway, <i>neverland</i>, <i>spook</i> and <i>sad</i> were upregulated in the <i>nm2</i> mutant, and <i>CYP314A1</i> and <i>CYP18A1</i> were downregulated. There were no significant difference for <i>Phm</i> and <i>Dib</i>.</p

Analysis of read data.

<p>(A) Percentage composition of bases in the reads. The four types of base and unknown bases (N) were uniform from the 10^th base. (B) Distribution of quality. High quality was observed from the 6^th base to the last and even for the first 6 bases.</p

Statistics of top 20 pathway enrichment of differentially expressed genes in each pairwise.

<p>Rich factor is the ratio of differentially expressed gene numbers annotated in this pathway term to all gene numbers annotated in this pathway term. Greater rich factor means greater intensiveness. Q-value is corrected P-value ranging from 0~1, and less Q-value means greater intensiveness.</p

The ecdysone-induced signaling pathway.

<p>The 20E titer in the <i>nm2</i> mutant was lower than in the wildtype, leading to upregulation of ecdysone biosynthesis genes and downregulation of <i>CYP314A1</i>, which takes part in the conversion from ecdysone to 20E. Two nuclear receptor genes, <i>HR3</i> and <i>HR4</i>, were markedly downregulated, whereas the key nuclear receptor gene <i>βFTZ-F1</i> was significantly upregulated. <i>BmCPG10</i> might act as an EPDFP monitor to regulate the molting process by controlling the biosynthesis of ecdysone.</p

Scatter plots of all expressed genes in each pairwise.

<p>Blue blots mean down-regulation genes, orange blots mean up-regulation genes and brown blots mean non-regulation genes. The screening threshold was on top legend.</p

Differentially expressed genes in the head of the 2^nd instar pre-molting larvae of the <i>nm2</i> mutant of the silkworm, <i>Bombyx mori</i>

<div><p>Molting is an important physiological process in the larval stage of <i>Bombyx mori</i> and is controlled by various hormones and peptides. The silkworm mutant that exhibits the phenotype of non-molting in the 2^nd instar (<i>nm2</i>) is incapable of molting in the 2^nd instar and dies after seven or more days. The ecdysone titer in the <i>nm2</i> mutant is lower than that in the wildtype, and the mutant can be rescued by feeding with 20E and cholesterol. The results of positional cloning indicated that structural alteration of <i>BmCPG10</i> is responsible for the phenotype of the <i>nm2</i> mutant. To explore the possible relationship between <i>BmCPG10</i> and the ecdysone titer as well as the genes affected by <i>BmCPG10</i>, digital gene expression (DGE) profile analysis was conducted in the <i>nm2</i> mutant, with the wildtype strain C603 serving as the control. The results revealed 1727 differentially expressed genes, among which 651 genes were upregulated and 1076 were downregulated in <i>nm2</i>. BLASTGO analysis showed that these differentially expressed genes were involved in various biological processes, cellular components and molecular functions. KEGG analysis indicated an enrichment of these differentially expressed genes in 240 pathways, including metabolic pathways, pancreatic secretion, protein digestion and absorption, fat digestion and absorption and glycerolipid metabolism. To verify the accuracy of the DGE results, quantitative reverse transcription PCR (qRT-PCR) was performed, focusing on key genes in several related pathways, and the results were highly consistent with the DGE results. Our findings indicated significant differences in cuticular protein genes, ecdysone biosynthesis genes and ecdysone-related nuclear receptors genes, but no significant difference in juvenile hormone and chitin biosynthesis genes was detected. Our research findings lay the foundation for further research on the formation mechanism of the <i>nm2</i> mutant.</p></div

Expression of cuticle protein genes.

<p>Many <i>CPG</i>s were expressed differentially between <i>nm2</i> and C603, with more than 20 differentially expressed genes showing a fold-change of 10 or greater.</p

Comparison of FPKM and qRT-PCR results.

<p>Comparison of FPKM and qRT-PCR results.</p

GO enrichment of biological processes, cellular components and molecular functions.

<p>The differentially expressed genes were mainly related to biological processes such as metabolic processes, cellular processes, single-organism processes, localization, the response to stimulus and biological regulation; cellular components such as the cell, cell parts, membranes and organelles; and molecular functions such as catalytic activity, binding and transporter activity.</p

Deep-Learning-Enhanced Diffusion Imaging Assay for Resolving Local-Density Effects on Membrane Receptors

G-protein-coupled receptor (GPCR) density at the cell surface is thought to regulate receptor function. Spatially resolved measurements of local-density effects on GPCRs are needed but technically limited by density heterogeneity and mobility of membrane receptors. We now develop a deep-learning (DL)-enhanced diffusion imaging assay that can measure local-density effects on ligand–receptor interactions in the plasma membrane of live cells. In this method, the DL algorithm allows the transformation of 100 ms exposure images to density maps that report receptor numbers over any specified region with ∼95% accuracy by 1 s exposure images as ground truth. With the density maps, a diffusion assay is further established for spatially resolved measurements of receptor diffusion coefficient as well as to express relationships between receptor diffusivity and local density. By this assay, we scrutinize local-density effects on chemokine receptor CXCR4 interactions with various ligands, which reveals that an agonist prefers to act with CXCR4 at low density while an inverse agonist dominates at high density. This work suggests a new insight into density-dependent receptor regulation as well as provides an unprecedented assay that can be applicable to a wide variety of receptors in live cells