Molecular and Cellular Probes最新文献

Human Toll-like receptor (TLR) family plays a crucial role in immunity and cancer progression. However, the specific role of human Toll-like receptor 4 (TLR4) in kidney renal clear cell carcinoma (KIRC) remains obscure. Thus, we used single-cell RNA sequencing (RNA-seq) and bulk RNA-seq data combined with in vitro studies to evaluate the expression and prognostic value of TLR4 in KIRC. In our study, we observed that TLR4 was over expressed in KIRC tissues compared to normal renal tissues. And the expression of TLR4 was higher in macrophages/monocytes than other cell types. Besides, there is a close association between TLR4 expression and immune cell infiltration (Neutrophils, Macrophages, T cells and B cells) in KIRC. Immunohistochemical staining also showed that TLR4 was overexpressed in inflammatory infiltration renal tissue compared with normal tissue. Meanwhile, high expression of TLR4 exhibited correlations with improved survival, lower tumor grade and stage. Interestingly, the protective significance of TLR4 only showed in female patients (HR = 0.37, P < 0.01), other than male patients (HR = 0.71, P = 0.08) with KIRC. Consistently, KIRC samples with lymph node metastasis showed lower expression of TLR4. Knockdown of TLR4 in 786-O cell line increased cell proliferation and clonogenic capacity. In summary, this study found TLR4 could inhibit the progression of kidney cancer and was associated with improved survival in KIRC. The overexpression of TLR4 in macrophages and the close association between TLR4 and immune cell infiltration also underline the critical role of TLR4 in building the immune microenvironment for kidney cancer. These results may offer insights into the mechanism and immune microenvironment of kidney cancer.

Objective

The effects of mesenchymal stem cells (MSCs) and MSC-derived exosomes (MSC-exos) on serum metabolites and intestinal microbiota in rats after liver trauma were discussed.

Methods

Adult Wistar Albino rats were assigned into control, model (liver trauma), MSCs, and MSC-exos groups (n = 6). The study examined changes in the inflammatory environment in liver tissues were analyzed by histological examination and analysis of macrophage phenotypes. Alterations in serum metabolites were determined by untargeted metabonomics, and gut microbiota composition was characterized by 16S rDNA sequencing. Correlations between specific gut microbiota, metabolites, and inflammatory response were calculated using Spearman correlation analysis.

Results

Rats with liver trauma after MSCs and MSC-exos treatment exhibited attenuated inflammatory infiltration and necrosis in liver tissues. MSCs and MSC-exos treatment reduced the proportion of M1 macrophages, accompanied by a decrease in inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) levels. Furthermore, MSCs and MSC-exos treatment expanded the proportion of M2 macrophages, accompanied by an increase in arginase-1 (Arg-1) and interleukin-10 (IL-10) levels. The beneficial effects of MSC-exo treatment on rats with liver trauma were superior to those of MSC treatment. The composition and abundance of the gut microbiota and metabolites were altered in pathological rats, whereas MSC and MSC-exo intervention partially restored specific gut microbiota and metabolite alterations. At the phylum level, alterations in Bacteroidota, Proteobacteria, and Verrucomicrobiota were observed after MSC and MSC-exo intervention. At the genus level, Intestinimonas, Alistipes, Aerococcus, Faecalibaculum, and Lachnospiraceae_ND3007_group were the main differential microbiota. 6-Methylnicotinamide, N-Methylnicotinamide, Glutathione, oxidized, ISOBUTYRATE, ASCORBATE, EICOSAPENTAENOATE, GLYCEROL 3-PHOSPHATE, and Ascorbate radical were selected as important differential metabolites. There was a clear correlation between Ascorbate, Intestinimonas/Faecalibaculum and inflammatory cytokines.

Conclusion

MSC-exos promoted the repair of tissue damage in rats with liver trauma by regulating serum metabolites and intestinal microbiota, providing new insights into how MSC-exos reduced inflammation in rats with liver trauma.

With rising society stress, depression-induced osteoporosis is increasing. However, the mechanism involved is unclear. In this study, we explored the effect of plasma exosomal miRNAs on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation in a chronic unpredictable mild stress (CUMS)-induced depression rat model. After 12 weeks of CUMS-induced depression, the pathological changes in the bone tissue and markers of osteogenic differentiation were tested by micro-computed tomography, hematoxylin-eosin staining, and quantitative real-time reverse transcription PCR (qRT-PCR). Plasma exosomes from rats were isolated and co-incubated with BMSCs for 14 d to detect the effect on osteogenic markers. Next-generation sequencing identified the miRNAs in the plasma exosomes, and the differential miRNAs were analyzed and verified by qRT-PCR. BMSCs were infected with lentivirus to upregulate miRNA-30a-5p and incubated in a medium that induced osteogenic differentiation for 14 d. The effect of miR-30a-5p on osteogenic differentiation was determined by qPCR and alizarin red staining. CUMS-induced depression rat model was established successfully, and exhibited reduced bone mass and damaged bone microstructure compared to that of the controls. The observed pathological changes suggested the occurrence of osteoporosis in the CUMS group, and the mRNA expression of osteogenic markers was also significantly reduced. Incubation of BMSCs with plasma exosomes from the CUMS group for 14 d resulted in a significant decrease in the expression of osteogenic markers. Twenty-five differentially expressed miRNAs in plasma exosomes were identified and upregulation of miR-30a-5p was observed to significantly inhibit the expression of osteogenic markers in BMSCs. Our findings contributed to a comprehensive understanding of the mechanism of osteoporosis caused by depression, and demonstrated the potential of miR-30a-5p as a novel biomarker or therapeutic target for the treatment of osteoporosis.

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFP_Ft that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

Recurrent implantation failure (RIF) is a condition with a multifactorial basis. Recent research has focused on the role of genetic factors in the pathophysiology of RIF. Of particular note, miRNAs have been found to contribute to the pathogenesis of RIF. Several miRNA polymorphisms have been investigated in this context. Moreover, dysregulation of expression of a number of miRNAs, including miR-374a-5p, miR-145-5p, miR-30b-5p, miR-196b-5p, miR-22, miR-181 and miR-145 has been found in RIF. This review concentrates on the role of miRNAs in RIF to help in identification of the molecular basis for this condition and design of more effective methods for management of RIF, especially in a personalized manner that relies on the expression profiles of miRNAs in the peripheral blood or endometrium.

Sepsis as a severe systemic inflammation leads oftentimes to organ dysfunction and subsequently to death. In polytrauma patients, septic complications represent with 45% the predominant cause of late death and are responsible for extremely high costs in the healthcare system. Therefore, clinicians have to detect as early as possible the begin of sepsis to improve the patient's outcome. One new promising diagnostic tool to diagnose septic complications in polytraumatized patients are exosomes.

Plasma samples from polytraumatized patients (Injury Severity Score (ISS) ≥16) which developed sepsis (n = 10) and without sepsis (n = 10), were collected at emergency room (ER), 24h and 5 days after trauma. The EVs subpopulations were investigated by a bead-based multiplex flow cytometry measurement of surface epitopes and were compared with plasma EVs from healthy controls (n = 10). Moreover, exosomal cytokine concentrations were measured via high-sensitive ELISA and were correlated with systemic concentrations. For miRNA cargo analysis, we analysed the miRNAs miR-1298-5p, miR-1262, miR-125b-5p, miR-92a-3p, miR-93-5p, miR-155-5p and miR-21-5p and compared their exosomal concentrations by means of RT-qPCR.

CD62p + exosomes were significantly increased in septic polytrauma-patients (p ≤ 0.05), while CD40+exosomes, as well as CD49e + exosomes were diminished (p ≤ 0.05). Furthermore, we observed that the exosomal IL-6 concentration reflects the systemic IL-6 concentration (r² = 0.63) and did not significantly alter between patients with and without sepsis. The exosomal IL-10 concentration seemed to be constant in all patients and healthy controls. We observed that a decrease of miR-21-5p in exosomes was associated with the development of sepsis (p ≤ 0.05), while exosomal miR-93-5p, miR-155-5p and miR-92a-3p were not specifically altered in septic patients.

Taken together, the present study in polytraumatized patients demonstrated that the development of sepsis is associated with an increase of CD62p + exosomes. Furthermore, the exosomal cargo was changed in septic patients: miR-21-5p was diminished.

Introduction

Estrogen hormones and their metabolites are implicated in the maintenance of healthy pregnancy and adequate fetal development. Abnormal levels were related to increased risk of pregnancy complications, particularly preeclampsia. Our aims were (1) to develop a methodological platform for the comprehensive assessment of estrogen metabolome in pregnancy; (2) to collect healthy reference data for relevant elements of estrogen metabolome in each trimester; (3) to assess unconjugated fractions of the estrogen metabolome, (4) to assess the dominant metabolic pathways of estrogen compounds.

Methods

We enrolled healthy pregnant mothers between gestational week 5–15 (on the confirmation of pregnancy; 79 samples), gestational weeks 19–27 (70 samples), and gestational week 34–39 (54 samples). A method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to assess estrone, 17-beta-estradiol, estriol levels, and their metabolites as conjugated and unconjugated forms. Descriptive statistics were used to characterize the level of compounds in each trimester.

Results

Estrone, 17-beta-estradiol and estriol levels are dramatically increasing with the advancement of pregnancy. Measured levels were in a very wide range. 17-beta-estradiol is neither glucuronated nor sulphated. To the contrary, estriol and estrone are significantly conjugated; unconjugated fraction is <15% of total hormone levels in any trimester. Regarding metabolism, 4-methoxy-estradiol and 17-epiestriol were not detected.

Conclusion

We concluded that (1) the levels of estrogen compounds and metabolites increase with advancing gestational age; (2) the wide ranges of levels challenge the establishment of a healthy reference range for clinical purposes; (3) 17-beta-estradiol is not conjugated significantly; (4) 4-methylation and 17-epimerization pathways of estrogens are negligible with our LC-MS/MS method.

Background

Rickets occurs in infants and children (aged 2 months to 3 years), compromising their skeletal development and damaging nervous, hematopoietic, immune, and other system functions. This study aimed to explore the significance of CD38 in rickets.

Methods

The microarray dataset GSE22523 was analyzed to obtain differentially expressed genes in rickets patients. A total of 36 rickets patients and healthy controls were recruited for the study, and their blood samples were collected, followed by detecting mRNA levels of CD38 using quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, the significance of CD38 in rickets patients was analyzed by receiver operating characteristic (ROC) analysis, while the correlation between CD38 and 25-hydroxy-vitamin D (25OHD)/parathyroid hormone (PTH) was analyzed with Pearson's correlation.

Results

Results showed that CD38 mRNA levels and PTH contents were significantly increased in the rickets patients while 25OHD contents were decreased. Correlation analysis indicated that CD38 was positively correlated with PTH and negatively correlated with 25OHD in both serum and plasma samples of rickets patients. Moreover, ROC analysis showed that serum CD38 was 0.9005 (95 % CI: 0.8313–0.9696), and the AUCs of plasma CD38 was 0.7215 (95 % CI: 0.6031–0.8398) in differentiating rickets patients from healthy persons, advocating serum CD38 had better diagnostic value.

Conclusion

CD38 mRNA levels were upregulated in rickets patients and closely correlated with PTH and 25OHD contents, indicating CD38 might be a diagnostic marker of rickets patients. Further research on the diagnostic utility of CD38 is necessary for the diagnosis and treatment of ricketsin rickets in the future.

Cholangiocarcinoma (CCA) is a rare malignancy originating from the biliary tree and is anatomically categorized as intrahepatic (iCCA), perihilar, and extrahepatic or distal. iCCA, the second most prevalent hepatobiliary cancer following hepatocellular carcinoma (HCC), constitutes 5–20 % of all liver malignancies, with an increasing incidence. The challenging nature of iCCA, combined with nonspecific symptoms, often leads to late diagnoses, resulting in unfavorable outcomes. The advanced phase of this neoplasm is difficult to treat with dismal results. Early diagnosis could significantly reduce mortality attributed to iCCA but remains an elusive goal. The identification of biomarkers specific to iCCA and their translation into clinical practice could facilitate diagnosis, monitor therapy response, and potentially reveal novel interventions and personalized medicine. In this review, we present the current landscape of biomarkers in each of these contexts. In addition to CA19.9, a widely recognized biomarker for iCCA, others such as A1BG, CYFRA 21–1, FAM19A5, MMP-7, RBAK, SSP411, TuM2-PK, WFA, etc., as well as circulating tumor DNA, RNA, cells, and exosomes, are under investigation. Advancing our knowledge and monitoring of biomarkers may enable us to improve diagnosis, prognostication, and apply treatments dynamically and in a more personalized manner.

Background

There is increasing evidence that platelet-derived extracellular vesicles (PEVs) may be involved in the mechanisms of inflammatory storm and organ damage in sepsis. However, there are no available studies on PEVs and renal injury in patients with urosepsis.

Methods

We analyzed the concentration and ratio of PEVs in plasma by flow cytometry and measured plasma IL-1β/IL-6/TNF-α/NGAL levels by ELISA. Correlation analysis was also used to examine the concentration of PEVs in relation to levels of inflammatory factors and indicators of kidney damage, as well as the severity of the disease. Finally, the receiver operating characteristic curves were produced for PEVs concentrations as a diagnosis of S-AKI/AKI.

Results

We found significantly higher levels of IL-1β/IL-6/TNF-α/NGAL in patients with urogenital sepsis. Furthermore, the concentrations of PEVs in plasma were significantly elevated in patients with urosepsis, especially in patients with Gram-negative bacterial infections, which were significantly and positively correlated with IL-1β/IL-6/TNF-α/NGAL levels. The area under the curve for PEVs diagnosing S-AKI and AKI was 0.746 [0.484, 1.000] and 0.943 [0.874, 1.000] respectively.

Conclusion

Overall, the present study suggested that PEVs may mediate the release of inflammatory mediators in patients with urosepsis and participate in the mechanism of acute kidney injury, as well as having potential as diagnostic indicators of S-AKI and AKI and as early warning indicators of the severity of patients with urosepsis.