Table_1_Predicting the prognosis in patients with sepsis by a pyroptosis-related gene signature.docx

BackgroundSepsis remains a life-threatening disease with a high mortality rate that causes millions of deaths worldwide every year. Many studies have suggested that pyroptosis plays an important role in the development and progression of sepsis. However, the potential prognostic and diagnostic value of pyroptosis-related genes in sepsis remains unknown.MethodsThe GSE65682 and GSE95233 datasets were obtained from Gene Expression Omnibus (GEO) database and pyroptosis-related genes were obtained from previous literature and Molecular Signature Database. Univariate cox analysis and least absolute shrinkage and selection operator (LASSO) cox regression analysis were used to select prognostic differentially expressed pyroptosis-related genes and constructed a prognostic risk score. Functional analysis and immune infiltration analysis were used to investigate the biological characteristics and immune cell enrichment in sepsis patients who were classified as low- or high-risk based on their risk score. Then the correlation between pyroptosis-related genes and immune cells was analyzed and the diagnostic value of the selected genes was assessed using the receiver operating characteristic curve.ResultsA total of 16 pyroptosis-related differentially expressed genes were identified between sepsis patients and healthy individuals. A six-gene-based (GZMB, CHMP7, NLRP1, MYD88, ELANE, and AIM2) prognostic risk score was developed. Based on the risk score, sepsis patients were divided into low- and high-risk groups, and patients in the low-risk group had a better prognosis. Functional enrichment analysis found that NOD-like receptor signaling pathway, hematopoietic cell lineage, and other immune-related pathways were enriched. Immune infiltration analysis showed that some innate and adaptive immune cells were significantly different between low- and high-risk groups, and correlation analysis revealed that all six genes were significantly correlated with neutrophils. Four out of six genes (GZMB, CHMP7, NLRP1, and AIM2) also have potential diagnostic value in sepsis diagnosis.ConclusionWe developed and validated a novel prognostic predictive risk score for sepsis based on six pyroptosis-related genes. Four out of the six genes also have potential diagnostic value in sepsis diagnosis.</p

Table_3_Predicting the prognosis in patients with sepsis by a pyroptosis-related gene signature.xlsx

BackgroundSepsis remains a life-threatening disease with a high mortality rate that causes millions of deaths worldwide every year. Many studies have suggested that pyroptosis plays an important role in the development and progression of sepsis. However, the potential prognostic and diagnostic value of pyroptosis-related genes in sepsis remains unknown.MethodsThe GSE65682 and GSE95233 datasets were obtained from Gene Expression Omnibus (GEO) database and pyroptosis-related genes were obtained from previous literature and Molecular Signature Database. Univariate cox analysis and least absolute shrinkage and selection operator (LASSO) cox regression analysis were used to select prognostic differentially expressed pyroptosis-related genes and constructed a prognostic risk score. Functional analysis and immune infiltration analysis were used to investigate the biological characteristics and immune cell enrichment in sepsis patients who were classified as low- or high-risk based on their risk score. Then the correlation between pyroptosis-related genes and immune cells was analyzed and the diagnostic value of the selected genes was assessed using the receiver operating characteristic curve.ResultsA total of 16 pyroptosis-related differentially expressed genes were identified between sepsis patients and healthy individuals. A six-gene-based (GZMB, CHMP7, NLRP1, MYD88, ELANE, and AIM2) prognostic risk score was developed. Based on the risk score, sepsis patients were divided into low- and high-risk groups, and patients in the low-risk group had a better prognosis. Functional enrichment analysis found that NOD-like receptor signaling pathway, hematopoietic cell lineage, and other immune-related pathways were enriched. Immune infiltration analysis showed that some innate and adaptive immune cells were significantly different between low- and high-risk groups, and correlation analysis revealed that all six genes were significantly correlated with neutrophils. Four out of six genes (GZMB, CHMP7, NLRP1, and AIM2) also have potential diagnostic value in sepsis diagnosis.ConclusionWe developed and validated a novel prognostic predictive risk score for sepsis based on six pyroptosis-related genes. Four out of the six genes also have potential diagnostic value in sepsis diagnosis.</p

Table_2_Predicting the prognosis in patients with sepsis by a pyroptosis-related gene signature.xlsx

BackgroundSepsis remains a life-threatening disease with a high mortality rate that causes millions of deaths worldwide every year. Many studies have suggested that pyroptosis plays an important role in the development and progression of sepsis. However, the potential prognostic and diagnostic value of pyroptosis-related genes in sepsis remains unknown.MethodsThe GSE65682 and GSE95233 datasets were obtained from Gene Expression Omnibus (GEO) database and pyroptosis-related genes were obtained from previous literature and Molecular Signature Database. Univariate cox analysis and least absolute shrinkage and selection operator (LASSO) cox regression analysis were used to select prognostic differentially expressed pyroptosis-related genes and constructed a prognostic risk score. Functional analysis and immune infiltration analysis were used to investigate the biological characteristics and immune cell enrichment in sepsis patients who were classified as low- or high-risk based on their risk score. Then the correlation between pyroptosis-related genes and immune cells was analyzed and the diagnostic value of the selected genes was assessed using the receiver operating characteristic curve.ResultsA total of 16 pyroptosis-related differentially expressed genes were identified between sepsis patients and healthy individuals. A six-gene-based (GZMB, CHMP7, NLRP1, MYD88, ELANE, and AIM2) prognostic risk score was developed. Based on the risk score, sepsis patients were divided into low- and high-risk groups, and patients in the low-risk group had a better prognosis. Functional enrichment analysis found that NOD-like receptor signaling pathway, hematopoietic cell lineage, and other immune-related pathways were enriched. Immune infiltration analysis showed that some innate and adaptive immune cells were significantly different between low- and high-risk groups, and correlation analysis revealed that all six genes were significantly correlated with neutrophils. Four out of six genes (GZMB, CHMP7, NLRP1, and AIM2) also have potential diagnostic value in sepsis diagnosis.ConclusionWe developed and validated a novel prognostic predictive risk score for sepsis based on six pyroptosis-related genes. Four out of the six genes also have potential diagnostic value in sepsis diagnosis.</p

Immunohistochemical analysis of fascin expression in the spinal cord.

<p>Fascin staining was more intense in the chronic morphine exposure group than in the control group (A, D). In the LV-shPKCã group (C, F), fascin staining was significantly attenuated compared with the LV-NC group (B, E). Magnification: A, B, C 100×; D, E, F 200×.</p

Proteomic Analysis of PKCγ-Related Proteins in the Spinal Cord of Morphine-Tolerant Rats

<div><h3>Background</h3><p>Morphine tolerance is a common drawback of chronic morphine exposure, hindering use of this drug. Studies have shown that PKCã may play a key role in the development of morphine tolerance, although the mechanisms are not fully known.</p> <h3>Methodology/Principal Findings</h3><p>In a rat model of morphine tolerance, PKCã knockdown in the spinal cord was successfully carried out using RNA interference (RNAi) with lentiviral vector-mediated short hairpin RNA of PKCã (LV-shPKCã). Spinal cords (L4-L5) were obtained surgically from morphine-tolerant (MT) rats with and without PKCã knockdown, for comparative proteomic analysis. Total proteins from the spinal cords (L4-L5) were extracted and separated using two-dimensional gel electrophoresis (2DGE); 2D gel images were analyzed with PDQuest software. Seven differential gel-spots were observed with increased spot volume, and 18 spots observed with decreased spot volume. Among these, 13 differentially expressed proteins (DEPs) were identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), comparing between MT rats with and without PKCã knockdown. The DEPs identified have roles in the cytoskeleton, as neurotrophic factors, in oxidative stress, in ion metabolism, in cell signaling, and as chaperones. Three DEPs (GFAP, FSCN and GDNF) were validated with Western blot analysis, confirming the DEP data. Furthermore, using immunohistochemical analysis, we reveal for the first time that FSCN is involved in the development of morphine tolerance.</p> <h3>Conclusions/Significance</h3><p>These data cast light on the proteins associated with the PKCã activity during morphine tolerance, and hence may contribute to clarification of the mechanisms by which PKCã influences MT.</p> </div

Differentially expressed proteins in the spinal cords of PKCã-knockdown morphine tolerant rats, identified with 2DGE-based comparative proteomics.

<p>Up-arrows  =  up-regulated. Down-arrows  =  down-regulated. The % coverage of the analyzed peptides, the score from Mascot searches, the mass and pI (from the database) and Swiss-Prot accession number are shown for each protein. Proteins displaying up-regulation or down-regulation, with an average fold-difference (<i>P</i><0.05) of ≥2-fold between pairs of conditions, are marked (+) and (−), respectively. MALDI-TOF MS result for % coverage of analyzed peptide and the score from Mascot searches.</p

Effect of LV-PKCã treatment on expression of proteins in the spinal cords of morphine-tolerant rats.

<p>(A) Western blot detection of PKCã and other differentially expressed proteins. (B) The expression level for each protein was estimated by densitometry, and is shown as a ratio to the loading control, â-actin. Data are presented as mean ± SD. *<i>P</i><0.05, compared with the corresponding value in the LV-NC group.</p

Effects of morphine injections on thermal paw withdrawal latencies (PWLs).

<p>The anti-nociceptive effect was measured 30 min after morphine injection. The baseline PWLs were determined before chronic morphine exposure (10 µg i.t., twice daily for 6 days). On day 7, rats were injected intrathecally with LV-NC or LV-shPKCã. Data are presented as mean ± SD. *<i>P</i><0.05, compared with the corresponding value in the saline control group.</p

Experimental data-based diagram that rationalizes each DEP in the biological system that PKCã down-regulation inhibits morphine tolerance.

<p>AINX, alpha-internexin; RHOB, Rho-related GTP-binding protein RhoB; FSCN, fascin; GDNF, glial cell line-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; GLRX, glutaredoxin; ADPRH, Protein ADP-ribosylarginine hydrolase; PPIA, peptidyl-prolyl cis-trans isomerase A; GAMT, guanidinoacetate N-methyltransferase; TRFE, serotransferrin; HPT, haptoglobin; HBB1, hemoglobin subunit beta-1; S10A9, protein S100-A9.</p

2DE images of the intumescentia lumbaris from the spinal cords of LV-NC- (A) or LV-shPKCã- (B) treated morphine-tolerant rats.

<p>Proteins were separated on a pH 4-7 IPG strip in the first dimension, and on an SDS-polyacrylamide (12%) gel in the second dimension. (C) Close-up image of partial differential expression protein spots, between LV-NC and LV-shPKCã.</p