Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4+ Tissue-resident Memory T cells in Crohn's Disease.

BACKGROUND & AIMS: As the most abundant memory T cells and major source of tumor necrosis factor α in the intestinal mucosa of Crohn's disease (CD) patients, CD4+ tissue-resident memory T (TRM) cells play a critical role in CD pathogenesis. We investigated the role of metabolic reprogramming in the regulation of proinflammatory and apoptosis-resistant phenotype for CD4+ TRM cells. METHODS: CD4+ TRM cells were collected from intestinal resection tissues from control and CD patients. Transcriptomic and metabolomic analysis were performed to identify metabolic characteristics of CD4+ TRM cells. Enzyme-linked immunosorbent assay and quantitative polymerase chain reaction experiments were used to assess cytokines level in CD4+ TRM cells; activation-induced cell apoptosis rate was evaluated by flow cytometry. Transwell assay and wound healing assay were performed to detect the effect of CD4+ TRM cells on the migration of normal intestinal epithelial cells. RESULTS: Transcriptomic data combined with unbiased metabolomic analysis revealed an increased fatty acid oxidation (FAO) phenotype existed in CD4+ TRM cells from CD patients. The lipidomic data and stable isotope tracer experiments demonstrated that CD4+ TRM cells up-regulated their lipid lipolysis and fatty acid uptake to fuel FAO in CD patients. Mechanistically, the activated nuclear factor kappa B signaling increased transcription of genes involved in lipid lipolysis, fatty acid uptake, and oxidation in CD4+ TRM cells from CD patients. Targeting FAO of CD4+ TRM cells reversed their apoptosis-resistant and proinflammatory phenotype in CD patients. CONCLUSIONS: CD4+ TRM cells process an accelerated FAO mediated by activated nuclear factor kappa B signaling in CD patients; targeting FAO could reverse their apoptosis-resistant and proinflammatory phenotype. These findings shed a new light on the pathogenic mechanism investigation and novel therapy development in CD patients.

The m7G Reader NCBP2 Promotes Pancreatic Cancer Progression by Upregulating MAPK/ERK Signaling.

PDAC is one of the most common malignant tumors worldwide. The difficulty of early diagnosis and lack of effective treatment are the main reasons for its poor prognosis. Therefore, it is urgent to identify novel diagnostic and therapeutic targets for PDAC patients. The m7G methylation is a common type of RNA modification that plays a pivotal role in regulating tumor development. However, the correlation between m7G regulatory genes and PDAC progression remains unclear. By integrating gene expression and related clinical information of PDAC patients from TCGA and GEO cohorts, m7G binding protein NCBP2 was found to be highly expressed in PDAC patients. More importantly, PDAC patients with high NCBP2 expression had a worse prognosis. Stable NCBP2-knockdown and overexpression PDAC cell lines were constructed to further perform in-vitro and in-vivo experiments. NCBP2-knockdown significantly inhibited PDAC cell proliferation, while overexpression of NCBP2 dramatically promoted PDAC cell growth. Mechanistically, NCBP2 enhanced the translation of c-JUN, which in turn activated MEK/ERK signaling to promote PDAC progression. In conclusion, our study reveals that m7G reader NCBP2 promotes PDAC progression by activating MEK/ERK pathway, which could serve as a novel therapeutic target for PDAC patients.

Novel potential biomarkers for severe alcoholic liver disease.

Background: Alcoholic liver disease (ALD) is a leading cause of advanced liver disease; however, minor clinical symptoms in the early stage frequently result in delayed diagnosis and therapy. Invasive liver biopsy, the gold standard for diagnosing ALD, is unsuitable for repetitive analysis. This study aims to identify potential serum biomarkers that could contribute to non-invasive disease screening and monitoring. Methods: Label-free LC-MS/MS quantitative proteomics analysis was performed to identify differentially expressed proteins in the discovery cohort, followed by bioinformatics analysis based on the KEGG, GO, and String databases. Prioritized proteins were validated subsequently by quantitative assays. The area under the receiver operating characteristic curve (AUROC) was used to assess the diagnosis performance of potential biomarkers. Results: A total of 161 differentially expressed proteins were identified in the discovery cohort, of which 123 were up-regulated and 38 were down-regulated. B2M, IGFALS, and IGFBP3 were evaluated, and all demonstrated excellent diagnosis performance with AUROCs of over 0.9 when distinguishing patients with severe ALD from healthy controls. The AUROC values of B2M, IGFBP3, and IGFALS were 0.7131, 0.8877, and 0.9896 for differentiating severe ALD from non-severe ALD to indicate disease severity. B2M could distinguish patients with non-severe ALD and HC participants with an AUROC value of 0.8985. The efficiency of multiple combinations of these biomarkers was superior to that of the existing liver fibrosis evaluation indices used to monitor disease progression, with AUROC values of over 0.9. IGFALS showed a positive correlation with ALT/AST (r=0.4648, P=0.0009) and may be developed as a therapeutic target. Conclusion: This proteomic study identified three novel candidate proteins as promising circulating biomarkers for clinical diagnosis and disease progression and also provided the proteomic atlas for ALD pathophysiological mechanisms.

A Novel m7G-Related Gene Signature Predicts the Prognosis of Colon Cancer.

Colon cancer (CC), one of the most common malignancies worldwide, lacks an effective prognostic prediction biomarker. N7-methylguanosine (m7G) methylation is a common RNA modification type and has been proven to influence tumorigenesis. However, the correlation between m7G-related genes and CC remains unclear. The gene expression levels and clinical information of CC patients were downloaded from public databases. Twenty-nine m7G-related genes were obtained from the published literature. Via unsupervised clustering based on the expression levels of m7G-related genes, CC patients were divided into three m7G clusters. Based on differentially expressed genes (DEGs) from the above three groups, CC patients were further divided into three gene clusters. The m7G score, a prognostic model, was established using principal component analysis (PCA) based on 15 prognosis-associated m7G genes. KM curve analysis demonstrated that the overall survival rate was remarkably higher in the high-m7G score group, which was much more significant in advanced CC patients as confirmed by subgroup analysis. Correlation analysis indicated that the m7G score was associated with tumor mutational burden (TMB), PD-L1 expression, immune infiltration, and drug sensitivity. The expression level of prognosis-related m7G genes was further confirmed in human CC cell lines and samples. This study established an m7G gene-based prognostic model (m7G score), which demonstrated the important roles of m7G-related genes during CC initiation and progression. The m7G score could be a practical biomarker to predict immunotherapy response and prognosis in CC patients.

Identification of proteomic markers for prediction of the response to 5-Fluorouracil based neoadjuvant chemoradiotherapy in locally advanced rectal cancer patients.

BACKGROUND: Neoadjuvant chemoradiotherapy (nCRT) prior to surgery is the standard treatment for patients with locally advanced rectal cancer (LARC), while parts of them show poor therapeutic response accompanied by therapy adverse effects. Predictive biomarkers for nCRT response could facilitate the guidance on treatment decisions but are still insufficient until now, which limits the clinical applications of nCRT in LARC patients. METHODS: In our study, 37 formalin-fixed paraffin-embedded tumor biopsies were obtained from patients with LARC before receiving 5-fluorouracil based nCRT. Proteomics analyses were conducted to identify the differentially expressed proteins (DEPs) between total responders (TR) and poor responders (PR). The DEPs were validated via ROC plotter web tool and their predictive performance was evaluated by receiver operating characteristic analysis. Functional enrichment analyses were performed to further explore the potential mechanisms underlying nCRT response. RESULTS: Among 3,998 total proteins, 91 DEPs between TR and PR were screened out. HSPA4, NIPSNAP1, and SPTB all with areas under the curve (AUC) ~ 0.8 in the internal discovery cohort were independently validated by the external mRNA datasets (AUC ~ 0.7), and their protein levels were linearly correlated with the graded responses to nCRT in the internal cohort. The combination of HSPA4 and SPTB could distinctly discriminate the TR and PR groups (AUC = 0.980, p < 0.0001). Moreover, multiple combinations of the three proteins realized increased specificity and/or sensitivity, while achieving favorable predictive value when moderate responders were introduced into the ROC analysis. Pathways including DNA damage repair, cell cycle, and epithelial mesenchymal transition were involved in nCRT response according to the enrichment analysis results. CONCLUSIONS: HSPA4, SPTB and NIPSNAP1 in tumor biopsies and/or their optional combinations might be potential predictive markers for nCRT response in patients with LARC. The DEPs and their related functions have implications for the potential mechanisms of treatment response to nCRT in patients with LARC.

PGAM5-Mediated PHB2 Dephosphorylation Contributes to Diabetic Cardiomyopathy by Disrupting Mitochondrial Quality Surveillance.

Disruption of the mitochondrial quality surveillance (MQS) system contributes to mitochondrial dysfunction in diabetic cardiomyopathy (DCM). In this study, we observed that cardiac expression of phosphoglycerate mutase 5 (PGAM5), a mitochondrial Ser/Thr protein phosphatase, is upregulated in mice with streptozotocin-induced DCM. Notably, DCM-related cardiac structural and functional deficits were negated in cardiomyocyte-specific Pgam5 knockout (Pgam5CKO ) mice. Hyperglycemic stress impaired adenosine triphosphate production, reduced respiratory activity, and prolonged mitochondrial permeability transition pore opening in acutely isolated neonatal cardiomyocytes from control Pgam5f/f mice, and these effects were markedly prevented in cardiomyocytes from Pgam5CKO mice. Likewise, three main MQS-governed processes-namely, mitochondrial fission/fusion cycling, mitophagy, and biogenesis-were disrupted by hyperglycemia in Pgam5f/f , but not in Pgam5CKO , cardiomyocytes. On the basis of bioinformatics prediction of interaction between PGAM5 and prohibitin 2 (PHB2), an inner mitochondrial membrane-associated scaffolding protein, co-immunoprecipitation, and immunoblot assays demonstrated that PGAM5 dephosphorylates PHB2 on Ser91. Transfection of cardiomyocytes with phosphodefective or phosphomimetic Ser91 mutants of PHB2 confirmed a critical role for PGAM5-mediated dephosphorylation of PHB2 in mitochondrial dysfunction associated with hyperglycemic stress. Furthermore, knockin mice expressing phosphomimetic PHB2S91D were resistant to diabetes-induced cardiac dysfunction. Our findings highlight the PGAM-PHB2 axis as a novel and critical regulator of mitochondrial dysfunction in DCM.

Magnetic Resonance Neurography for Evaluation of Dorsal Root Ganglion Morphology.

BACKGROUND: In this study, the morphologic characteristics and anatomic position of the dorsal root ganglion (DRG) were measured and analyzed in healthy people using magnetic resonance neurography (MRN), which provided an anatomical reference for minimally invasive spinal surgery. METHODS: From January 2018 to December 2019, 20 healthy adult volunteers (10 male and 10 female volunteers between 20 and 65 years old) were scanned and imaged by 3.0 T magnetic resonance imaging combined with neuroimaging technology. Here, the position of the DRG was located, and the shape and size of the DRG, as well as its distance to the upper pedicle, were measured. RESULTS: All volunteers provided satisfactory MRN scans of the L1-S1 lumbar DRG. According to the spatial position of the DRG, the morphology of the DRG can be divided into the intervertebral foramen type (81.01%), intraspinal type (16.01%), extraforaminal type (0.8%), and mixed type (2.0%). CONCLUSIONS: The intervertebral foramen type and Intraspinal type were observed to be the main distribution forms of lumbar DRG. Due to the downward movement of lumbar segments, the position of the DRG was noted to gradually move to the spinal canal while its volume gradually increased. In addition, the distance from the upper pedicle was found to decrease gradually. MRN imaging can clearly show the shape, location, and adjacent relationship of the DRG, providing effective imaging guidance for the minimally invasive lumbar techniques.

Fibronectin and colorectal cancer: signaling pathways and clinical implications.

Colorectal cancer (CRC) is the fourth leading cause of cancer deaths worldwide, with poor prognosis mainly related to metastasis. Fibronectin (FN), a vital component of the extracellular matrix (ECM), has been found involved in tumorigenesis and malignant progression in different types of malignancy. Numerous studies have indicated the distinct expression of FN in various cancers and demonstrated the different functions of FN in the proliferation, migration, and invasion of cancers. Meanwhile, FN isoforms have been extensively used for targeted drug delivery and imaging for tumors. Although a growing number of studies on FN in CRC have been reported, integrated reviews on the relationship between FN and CRC are rare. In this review, we will summarize the association between FN and CRC, including the signaling pathways and molecules involved in, as well as potential diagnostic and therapeutic values of FN for patients with CRC.

ITIH4, as an inflammation biomarker, mainly increases in bacterial bloodstream infection.

Bloodstream infection (BSI) is usually accompanied with the changes of varieties of inflammation proteins. In our previous study, we identified that inter-α-trypsin inhibitor heavy chain H4 (ITIH4) was highly expressed in the infection arms than the normal control arm. However, the correlated verification and mechanism remain obscure. Escherichia coli infected mice model and clinical serum samples were used to validate the concentration of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), as well as ITIH4, in ELISA method. Cytokines (IL-6, TNF-α, IL-10 and lipopolysaccharide (LPS)) were used to stimulate the HepG2 cell model to explore which cytokines influence the expression of ITIH4. JAK/STAT inhibitor was treated before IL-6 and LPS stimulation. Westernblot, as well as real-time PCR were performed to detect the expression of ITIH4 in liver tissue from protein and transcription levels. Immunohistochemistry analysis was used to observe the expression of ITIH4 in mice liver tissue. In mice model, IL-6, TNF-α, as well as IL-10 increased in the infection arms than the normal control arm. ITIH4 in serum and liver tissue of mice model increased from 1 h to 128 h, which were remarkably different from that of the normal control arm. Besides, ITIH4 increased in the bacterial infection arm greatly than the fungemia arm, mycoplasma pneumoniae (MP) arm and febrile arm in clinical serum samples. Furthermore, using the HepG2 cell line, we demonstrated that ITIH4 was up-regulated at both protein and mRNA levels upon dose- and time- response treatments with IL-6, as well as LPS. Moreover, IL-6 or LPS mediated induction of ITIH4 expression could be significantly decreased by treatment with an JAK/STAT inhibitor in protein or mRNA level. No changes were observed after TNF-α or IL-10 stimulation. ITIH4 might be a critical inflammatory biomarker which correlated with the development of BSI, especially with bacterial bloodstream infection. It is expected that this study would provide some insights into potential functional mechanisms underlying BSI.

Oxycodone protects cardiomyocytes from ischemia-reperfusion-induced apoptosis via PI3K/Akt pathway.

Ischemia/reperfusion (I/R) cause secondary myocardial damage following a blood reflow after myocardial infarction. This study aimed to explore oxycodone as a myocardial protector after an I/R injury in rats. Oxycodone reduced myocardial infarction volume, an I/R-induced apoptosis of the cardiomyocytes, the serum levels of CK-MB and LDH. The ejection fraction and fraction shortening in the I/R rats also increased. From the molecular mechanism, it was evident that oxycodone not only decreased the expression levels of Bax, active-caspase 3 protein but also increased the expression levels of Bcl2, p-PI3K, and p-Akt protein in heart tissue of the I/R rats. In vitro, oxycodone induced anti-H9c2 cell apoptosis after hypoxia/reoxygenation (H/R). However, its ability to act as a myocardial protector deteriorated in the presence of a PI3K/Akt pathway inhibitor.

Two novel mutations of COL1A1 in fetal genetic skeletal dysplasia of Chinese.

BACKGROUND: Skeletal disorders, which have great genotypic and phenotypic varieties, are a considerable challenge to differentiate these diseases and provide a definitive prenatal diagnosis or pre-implantation. The present study aims to identify the causative mutation in two unrelated outbred Han-Chinese families. METHOD: Two short-limb fetuses were referred to our hospital. Genomic DNA was extracted from the amniotic fluid of the short-limb fetuses and from peripheral blood of their parents. To identify the causative gene, next-generation-based target capture sequencing was performed on these two fetuses, followed by Sanger Sequencing in unrelated healthy controls. Segregation analysis of the candidate variant was performed in parents by using Sanger sequencing. The mutations were analyzed by SIFT, PolyPhen and Provean. RESULTS: We found that fetal genetic skeletal dysplasia was confirmed according to the correlations between genetic mutations and phenotypes in two Chinese families. Targeted next generation sequencing was performed to screen causative mutations in patients. Two novel heterozygous mutations COL1A1 c.1706 G > C (p. G569A) and c.3307 G > A (p. G1103S) were respectively identified. The results suggested that COL1A1 novel mutations were in highly conserved glycine residues present in the Gly-X-Y sequence repeats of the triple helical region of the collagen type I α chain, which was responsible for Osteogenesis Imperfecta. The presence of the missense mutation was also confirmed with the Sanger sequence. These two mutations were predicted to be pathogenic by SIFT, PolyPhen and Provean. CONCLUSION: Our findings showed that the mutations of COL1A1 may play important roles in fetal genetic skeletal dysplasia in Chinese patients. Exome sequencing enhances the accurate diagnosis in utero then provides appropriate genetic counseling.

Noninvasive prenatal test for FGFR3-related skeletal dysplasia based on next-generation sequencing and plasma cell-free DNA: Test performance analysis and feasibility exploration.

OBJECTIVE: To explore the feasibility and accuracy of a noninvasive prenatal test for fibroblast growth factor receptor 3 (FGFR3)-related skeletal dysplasia based on next-generation sequencing (NGS) of plasma cell-free DNA. METHOD: Fragmented genome DNA (gDNA) of fetuses with achondroplasia (ACH) and thanatophoric dysplasia type I (TD I) was mixed with postdelivery maternal plasma cell-free DNA to generate spiked samples of different modeled fetal fractions. Multiplex polymerase chain reaction was used to amplify the 19 FGFR3 loci, and the amplification products were then sequenced by NGS to detect the fetal mutant alleles. Then, maternal plasma samples of pregnant women carrying ACH (n = 4) and TD I fetuses (n = 2), as well as healthy controls (n = 15), were tested by NGS, and the test performance was evaluated. RESULTS: Fetal FGFR3 mutations were detected in all artificial mixtures with fetal gDNA concentrations above 3%. In clinical validation, our method identified all fetal FGFR3 mutant alleles from maternal plasma, with no false positive results. The sensitivity and specificity of our method were 100% (95% CI, 54.1%-100%) and 100% (78.2%-100%), respectively. CONCLUSION: Our method had a favorable performance for noninvasively detecting fetal FGFR3 mutations in maternal plasma, highlighting its promising value in developing a noninvasive prenatal test for de novo and paternally inherited disorders.

Protective effects of edaravone combined puerarin on inhalation lung injury induced by black gunpowder smog.

OBJECTIVE: The present study aimed to investigate the combined effects of puerarin with edaravone on inhalation lung injury induced by black gunpowder smog. MATERIALS AND METHODS: Male Wistar rats were divided into five groups (control group, edaravone group, puerarin group, edaravone combined with puerarin group and inhalation group). The severity of pulmonary injuries was evaluated after inducing acute lung injury. Arterial blood gas, inflammatory cytokines, biochemical, parameters, cell counting, W/D weight ratio and histopathology were analyzed. Results in lung tissues, either edaravone or puerarin treatment alone showed significant protective effects against neutrophil infiltration and tissue injury, as demonstrated by myeloperoxidase activity and histopathological analysis (all p<0.05). In addition, combined treatment with both edaravone and puerarin demonstrated additive protective effects on smog-induced lung injury, compared with single treatment. CONCLUSIONS: Combination of edaravone and puerarin shows promise as a new treatment option for acute lung injury/acute respiratory distress syndrome patients.