EDN3 is associated with decreased levels of RA receptor mRNA and RA metabolic enzyme mRNA.

<p>p75^NTR+ cells were grown for 72 hours in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. Relative <i>Rarb</i> (A), <i>Rarg</i> (B), <i>Raldh2</i> (C), and <i>Cyp26a1</i> (D) mRNA levels were measured by quantitative RT-PCR. Levels are normalized to β-actin and the -RA/-EDN3 condition was standardized to a value of 1. Solid lines denote a statistically significant difference (p<0.05) between “No EDN3” and “EDN3”, and broken lines indicate a p value >0.05. Asterisk denotes a statistically significant difference (p<0.05) between “No RA” and “RA”. EDN3 was associated with a net decrease in <i>Rarb</i> (A) and <i>Rarg</i> (B) mRNA transcripts in the presence of exogenous RA. In the absence of exogenous RA, EDN3 did not have an effect on RA receptor levels. (C) EDN3 was also associated with a decrease in <i>Raldh2</i> abundance, but not when RA was present. (D) EDN3 also reduced <i>Cyp26a1</i> mRNA levels, but only in the presence of RA. Consistent with autoregulation, RA treatment was responsible for a reduction in <i>Raldh2</i> levels (C) and an increase in <i>Cyp26a1</i> (D), in the absence of exogenous EDN3 However, associations of RA with <i>Raldh2</i> and <i>Cyp26a1</i> levels were lost when EDN3 was simultaneously present. There was a statistically significant interaction (ⓧ; p<0.001) between EDN3 and RA in their effect on <i>Cyp26a1</i> levels at 3 days.</p

Effects of RA and EDN3 on the terminal culture morphology of ENS precursors.

<p>p75^NTR immunoselected cells were grown for 14 days in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. Culture morphology was evaluated by immunofluorescence microscopy. Peripherin expression (red) is consistent with neuronal morphology and myofibroblasts are indicated by expression of α-SMA (green). The third and fourth columns include DAPI staining for nuclei. The first three columns were photographed at 10x (Scale bar = 100 µm) and the fourth column was photographed at 40x (Scale bar = 50 µm). In the absence of EDN3 and RA (EDN3-RA-), SMA+ myofibroblasts predominated, but sparse neurons were also seen. EDN3+RA- treated cultures formed a homogeneous sheet of SMA+ myofibroblasts. In EDN3-RA+ cultures, neurons formed a plexus punctuated by large multicellular ganglia, abundant peripherin+ neurites and cell bodies, and non-myofibroblast-like SMA+ cells. SMA+ myofibroblasts were also present beneath the plexus. With EDN3+RA+ treatment, many peripherin+ neurons and long complex neurites were seen, without forming a plexus. Myofibroblasts, while excluded from neuronal regions, were still present in large number.</p

RA decreases <i>EDN3</i> and <i>Ece1</i> mRNA levels, depending on whether exogenous EDN3 is present.

<p>p75^NTR+ cells were grown for 72 hours in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. Relative <i>EDN3</i> (A-B) and <i>Ece1</i> (C-D) mRNA levels were measured by quantitative RT-PCR after 3 (A, C) and 14 (B, D) days. Levels are normalized to β-actin and the -RA/-EDN3 condition was standardized to a value of 1. Solid lines denote a statistically significant difference (p<0.05) between “No RA” and “RA”, and broken lines indicate a p value >0.05. Asterisk denotes a statistically significant difference (p<0.05) between “No EDN3” and “EDN3”. (A) At 3 days, RA was associated with a net decrease in the level of <i>EDN3</i> in the absence and presence of exogenous EDN3 (C) Similarly, RA was associated with a decrease in the relative abundance of <i>Ece1</i>, albeit only in the presence of EDN3 After 14 days, RA still decreased the relative abundance of <i>EDN3</i> (B) and <i>Ece1</i> (D) mRNA.</p

Culture in the presence of RA increases <i>Sox10</i>, but EDN3 suppresses this effect.

<p>p75^NTR+ cells were grown for 72 hours in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. Relative <i>Sox10</i> (A–B) and <i>Ret</i> (C–D) mRNA levels were measured by quantitative RT-PCR after 3 (A, C) and 14 (B, D) days. Expression is normalized to β-actin and the -RA/-EDN3 condition was standardized to a value of 1. Solid lines denote a statistically significant difference (p<0.05) between “No RA” and “RA”, and broken lines indicate a p value >0.05. Asterisk denotes a statistically significant difference (p<0.05) between “No EDN3” and “EDN3”. (A) In the absence of EDN3, RA increased <i>Sox10</i> levels; however, the concurrent addition of EDN3 abolished this phenomenon. There was a statistically significant interaction (ⓧ; p=0.019) between EDN3 and RA in their effect on <i>Sox10</i> levels at 3 days. This interaction is specific to <i>Sox10</i> expression and was not observed with <i>Ret</i>. (B) RA treatment was associated with increased <i>Sox10</i> levels but EDN3 had no effect at 14 days. (C) RA decreased <i>Ret</i> levels at 3 days regardless of whether EDN3 was present, but EDN3 did not affect <i>Ret</i> gene expression. (D) Decreased <i>Ret</i> levels in response to RA persisted at 14 days, but only in the presence of EDN3</p

RA increases S100β+ and decreases peripherin+ cell prevalence while EDN3 decreases S100β+ cell prevalence.

<p>p75^NTR+ cells were grown for 72 hours in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. The proportion (A and C) and proliferating fraction (B and D) of peripherin- and S100β-immunoreactive cells were quantified. Solid lines denote a statistically significant difference (p<0.05) between “No RA” and “RA”, and broken lines indicate a p value >0.05. Asterisk denotes a statistically significant difference (p<0.05) between “No EDN3” and “EDN3”. (A) The proportion of S100β+ cells was enriched by RA and decreased in response to EDN3 (B) RA increased, and EDN3 decreased the fraction of proliferating S100β+ cells. (C) the proportion of peripherin+ cells decreased in response to RA treatment, but EDN3 did not have an effect. (D) Neither RA nor EDN3 had an effect on proliferation of peripherin+ cells.</p

A hypothetical model depicting the bidirectional relationship of RA and EDN3 signaling in ENS precursors.

<p>The above pictogram, based on our data, summarizes and integrates the observed RNA expression profiles and the culture morphological data. Solid green arrows indicate increases in mRNA levels and solid red arrows with flat arrowheads denote decreases in mRNA levels. Although RA and EDN3 levels were not quantified in our culture model, dashed green and red arrows denote putative RA synthesis and degradation, respectively. We hypothesize that RA inhibits EDN signaling in ENS precursors by modulating the gene expression of <i>EDN3</i> and <i>Ece1</i>, and EDN3 regulates RA signaling by inhibiting RA receptor expression, thereby decreasing RA responsiveness, and fine-tuning RA metabolic enzyme expression, putatively altering RA availability. We propose that RA perpetuates glia by decreasing <i>Ret</i> and enhancing <i>Sox10</i> expression, and that EDN3 prevents the proliferation of glia by decreasing <i>Sox10</i>. The persistence of glia is conducive to the formation of a heterocellular ganglionated plexus. This model suggests that control of <i>Sox10</i> marks a key developmental decision point - a switch - that sustains multipotent progenitors in culture and ultimately depends on the balance of RA and EDN3</p

Table_1_SR-BI Interactome Analysis Reveals a Proviral Role for UGGT1 in Hepatitis C Virus Entry.DOCX

Hepatitis C virus (HCV) entry is mediated by multiple co-receptors including scavenger receptor class B, type I (SR-BI). To elucidate the interactome of human SR-BI, we performed immunoprecipitation (IP) experiment coupled with mass spectrometry (MS) analysis. UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), a key component of calnexin cycle involved in protein glycosylation, was identified as a SR-BI-interacting protein. Silencing UGGT1 or N-glycosylation inhibitor treatment reduced SR-BI protein level. Further study demonstrated that human SR-BI was N-glycosylated at nine asparagines. Moreover, HCV entry and infection were reduced by the absence of UGGT1. Interestingly, silencing SR-BI reduced protein stability of UGGT1 and protein quality control function mediated by UGGT1. Our finding not only identified UGGT1 as a HCV host factor, but also identified a UGGT1-mediated protein folding function for SR-BI.</p

Additional file 2: of Associations of mood symptoms with NYHA functional classes in angina pectoris patients: a cross-sectional study

Table S2. Characteristics of patients stratified by anxiety severity. (DOCX 22 kb

Effects of RA and EDN3 on the terminal culture morphology of ENS precursors.

<p>p75^NTR immunoselected cells were grown for 14 days in the presence of RA, EDN3, RA with EDN3, or neither compound. When EDN3 was absent, the EDN signaling inhibitor BQ-788 was added to inhibit endogenous EDNRB signaling. Culture morphology was evaluated by immunofluorescence microscopy. Peripherin expression (red) is consistent with neuronal morphology and glia are indicated by expression of S100β (green). The third and fourth columns include DAPI staining for nuclei. The first three columns were photographed at 10x (Scale bar = 100 µm) and the fourth column was photographed at 40x (Scale bar = 50 µm). Without EDN3 and RA (EDN3-RA-) sparse neurons developed, but S100β+ glia were more numerous and more brightly staining. EDN3+RA- cultures did not contain glia or neurons. EDN3-RA+ cultures formed discrete heterocellular ganglia with abundant peripherin+ cell bodies and S100β+ glia, linked by thick peripherin+ neurites in a plexus pattern. EDN3+RA+ treatment also contained many peripherin+ neurons and long complex neurites, but these neurons were disorganized, and did not form a plexus. Weakly staining S100β+ cells with glial cell morphology were also found amidst the neurons. The glia in these cultures were more fusiform and were mainly associated with neurons.</p

DataSheet_2_Integrative Analysis From Multicenter Studies Identifies a WGCNA-Derived Cancer-Associated Fibroblast Signature for Ovarian Cancer.pdf

Cancer-associated fibroblasts (CAFs) are a major contributor to tumor stromal crosstalk in the tumor microenvironment (TME) and boost tumor progression by promoting angiogenesis and lymphangiogenesis. This study aimed to identify prognostic genes associated with CAFs that lead to high morbidity and mortality in ovarian cancer (OC) patients. We performed bioinformatics analysis in 16 multicenter studies (2,742 patients) and identified CAF-associated hub genes using the weighted gene co-expression network analysis (WGCNA). A machine learning methodology was used to identify COL16A1, COL5A2, GREM1, LUM, SRPX, and TIMP3 and construct a prognostic signature. Subsequently, a series of bioinformatics algorithms indicated risk stratification based on the above signature, suggesting that high-risk patients have a worse prognosis, weaker immune response, and lower tumor mutational burden (TMB) status but may be more sensitive to routine chemotherapeutic agents. Finally, we characterized prognostic markers using cell lines, immunohistochemistry, and single-cell sequencing. In conclusion, these results suggest that the CAF-related signature may be a novel pretreatment guide for anti-CAFs, and prognostic markers in CAFs may be potential therapeutic targets to inhibit OC progression.</p