Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry as Internship Teaching Content in Laboratory Medicine.

BACKGROUND: Matrix-assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) is one of the preferred detection techniques for identification of clinical microorganisms and it has the characteristics of rapid identification, simple operation, low cost, and updatable databases. For laboratory medicine undergraduates, clinical internship is an important stage for the connection of basic theoretical knowledge and clinical practice. Internship teaching choosing MALDI-TOF MS as the content will greatly increase the popularity and applicability of the new technology in the clinical microbiology laboratory. METHODS: With the help of electronic databases on the network, we conducted a systematic review. According to the purpose of research, we singled out forty papers. Latest studies on history, basic principles, clinical features, and applications of MALDI-TOF MS and the internship teaching contents introducing new technologies are summarized and focused on. In internship teaching, firstly we explain the historical development, basic principle and widespread applications of MALDI-TOF MS in the identification of clinical pathogenic microorganisms and the detection of antibiotic resistance. Subsequently, we instruct the students to perform the experimental operations, analyze the common problems, and find solutions. Finally, we highlight quality control and laboratory biosafety. RESULTS: Most of the reviews published previously report the clinical features and applications of MALDI-TOF MS and the internship teaching contents choosing other new technologies. It is the first study selecting MALDI-TOF MS technology as an internship teaching content creatively. Primary outcome is that the students understand the theoretical knowledge in detail, master the operation skills of MALDI-TOF MS quickly, and obtain excellent internship performances in the clinical internship through the internship teaching. Secondary outcome is that it is a help to cultivate medical students' train of thought for scientific research and to understand the application of the new technology in clinical testing and scientific research. CONCLUSIONS: Laboratory medicine undergraduates should cherish the opportunity to learn the new technology during the internship period and should master basic principle and operation. As internship teachers, it is necessary to introduce the new technology to students during the internship and encourage undergraduates to cultivate creative and innovative thinking of scientific research.

INT-HA induces M2-like macrophage differentiation of human monocytes via TLR4-miR-935 pathway.

As a major component of the microenvironment of solid tumors, tumor-associated macrophages (TAMs) facilitate tumor progression. Intermediate-sized hyaluronan (INT-HA) fragments have an immunological function in cell differentiation; however, their role in promoting the polarization of non-activated macrophages to an M2-like TAM phenotype has not been characterized, and the underlying mechanisms remain unclear. Here, we used a miRNA microarray to find that some miRNAs (especially miR-935) were differentially regulated in INT-HA-induced M2-like macrophages. According to RT-qPCR and Western blot, there was an association between miR-935 and C/EBPß, that control the polarization of macrophages. Moreover, we found that INT-HA induced an M2-like phenotype via the TLR4 receptor. In our study, there was a negative correlation between plasma HA and miR-935 in monocytes from the peripheral blood of patients with solid tumors. There was also a negative correlation between miR-935 and M2-like macrophage markers in monocytes. These findings suggest that HA fragments interact with TLR4 and educate macrophage polarization to an M2-like phenotype via miR-935. Therefore, this study provides new insight into the role of miR-935 in INT-HA-induced M2-like polarization, and suggests a potential therapeutic target for antitumor treatment.

Increased circulating M2-like monocytes in patients with breast cancer.

M2-like tumor-associated macrophages promote breast tumor growth and survival and may migrate into the peripheral blood. However, the frequency of circulating M2-like monocytes in the peripheral blood of breast cancer patients has not been clarified. The objective of this study was to determine the percentages of circulating M2-like monocytes in patients with breast cancer. Immunofluorescence staining for CD68 and CD163 was performed to detect M2-like macrophages in pathological tissues. Flow cytometry was used to assess the frequencies of circulating CD14+CD163+/CD14+CD204+/CD14+CD163+CD204+ M2-like monocytes in 99 breast cancer patients, 56 patients with benign breast disease, and 60 healthy controls. Receiver operating characteristic curve analysis was used to compare the diagnostic values of circulating M2-like monocytes, carcinoembryonic antigen, and cancer antigen 15-3. The associations among circulating M2-like monocytes and clinical breast cancer parameters were analyzed. The number of CD68+CD163+ M2-like macrophages was significantly higher in breast cancer tissues than in benign tissues. In the peripheral blood, CD14+CD163+/CD14+CD204+/CD14+CD163+CD204+ M2-like monocytes were elevated in breast cancer patients compared with normal controls and patients with benign breast disease. The area under the receiver operating curve for circulating CD14+CD163+CD204+ M2-like monocytes was 0.888 (95% confidence interval: 0.839-0.936), a value higher than those for carcinoembryonic antigen and cancer antigen 15-3. High frequencies of circulating CD14+CD204+ and CD14+CD163+CD204+ M2-like monocytes were associated with tumor-node-metastasis stage, lymph node metastasis, histological differentiation, and estrogen receptor expression. Circulating M2-like monocytes may serve as a diagnostic biomarker in breast cancer and have a potential role in reflecting breast cancer progression.

Detection of respiratory pathogenic bacterial nucleic acid detection by Loop-mediated Isothermal Amplification in patients with bacterial pulmonary infections.

Objective: Nucleic acid testing can accurately and rapidly identify the presence of pathogenic bacteria. In this study, we analyzed respiratory pathogenic bacteria nucleic acids by LAMP (Loop-mediated isothermal amplification) to clarify the clinical application in patients with bacterial pulmonary infections. Methods: Clinical data and specimens were collected from 99 patients with bacterial pulmonary infections from June 2021 to April 2023. We compared the differences between nucleic acid detection of LAMP and sputum culture. The correlation between inflammation manifestations of pulmonary imaging and the nucleic acid detection of LAMP was compared and analyzed. And the relationship between LAMP and blood inflammatory markers were analyzed. Results: The positive rate of LAMP using sputum specimens was significantly higher than that of sputum culture (P < 0.05). Pathogenic bacteria in sputum samples are more likely to be detected by LAMP in patients with inflammatory on lung imaging examination. The coincidence rate of elevated PCT and CRP expression with positive LAMP results were 83.87 % and 88.71 %, respectively. Moreover, PCT, CRP and WBC were significantly higher in LAMP positive group than those in negative group (P < 0.05). Conclusion: Nucleic acid testing of sputum specimens for pathogenic bacteria by LAMP on the basis of imaging examination can provide a rapid and accurate experimental basis for clinical diagnosis of bacterial pulmonary infections.

Identification of ACHE as the hub gene targeting solasonine associated with non-small cell lung cancer (NSCLC) using integrated bioinformatics analysis.

Background: Solasonine, as a major biological component of Solanum nigrum L., has demonstrated anticancer effects against several malignancies. However, little is understood regarding its biological target and mechanism in non-small cell lung cancer (NSCLC). Methods: We conducted an analysis on transcriptomic data to identify differentially expressed genes (DEGs), and employed an artificial intelligence (AI) strategy to predict the target protein for solasonine. Subsequently, genetic dependency analysis and molecular docking were performed, with Acetylcholinesterase (ACHE) selected as a pivotal marker for solasonine. We then employed a range of bioinformatic approaches to explore the relationship between ACHE and solasonine. Furthermore, we investigated the impact of solasonine on A549 cells, a human lung cancer cell line. Cell inhibition of A549 cells following solasonine treatment was analyzed using the CCK8 assay. Additionally, we assessed the protein expression of ACHE, as well as markers associated with apoptosis and inflammation, using western blotting. To investigate their functions, we employed a plasmid-based ACHE overexpression system. Finally, we performed dynamics simulations to simulate the interaction mode between solasonine and ACHE. Results: The results of the genetic dependency analysis revealed that ACHE could be identified as the pivotal target with the highest docking affinity. The cell experiments yielded significant findings, as evidenced by the negative regulatory effect of solasonine treatment on tumor cells, as demonstrated by the CCK8 assay. Western blotting analysis revealed that solasonine treatment resulted in the downregulation of the Bcl-2/Bax ratio and upregulation of cleaved caspase-3 protein expression levels. Moreover, we observed that ACHE overexpression promoted the expression of the Bcl-2/Bax ratio and decreased cleaved caspase-3 expression in the OE-ACHE group. Notably, solasonine treatment rescued the Bcl-2/Bax ratio and cleaved caspase-3 expression in OE-ACHE cells compared to OE-ACHE cells without solasonine treatment, suggesting that solasonine induces apoptosis. Besides, solasonine exhibited its anti-inflammatory effects by inhibiting P38 MAPK. This was supported by the decline in protein levels of IL-1ß and TNF-α, as well as the phosphorylated forms of JNK and P38 MAPK. The results from the molecular docking and dynamics simulations further confirmed the potent binding affinity and effective inhibitory action between solasonine and ACHE. Conclusions: The findings of the current investigation show that solasonine exerts its pro-apoptosis and anti-inflammatory effects by suppressing the expression of ACHE.

Krebs von den Lungen-6 (KL-6) as a diagnostic marker for pulmonary fibrosis: A systematic review and meta-analysis.

BACKGROUND: Pulmonary fibrosis (PF) is a respiratory disease with end-stage pathological changes of interstitial lung disease that severely affects the survival of patients. Among the many biomarkers that have been identified, serum Krebs von den Lungen-6 (KL-6) is by far the most frequent marker for detecting pulmonary fibrosis. METHODS: We searched Medline (Pubmed), Embase, Web of science, and Cochrane databases for articles published between inception and August 2022 in order to explore the association between KL-6 and pulmonary fibrosis. Characteristics of patients and studies included in the articles were extracted by two independent investigators according to the inclusion and exclusion criteria. We reflected the accuracy of KL-6 in distinguishing between PF and non-PF by calculating sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and area under the curve by SROC curves. The presence of heterogeneity was reflected by I2 in the forest plot, and then the source of heterogeneity was investigated by meta-regression. RESULTS: We searched for 939 research papers, of which 16 met the inclusion criteria. Meta-analysis showed a sensitivity of 0.87 (95 % CI: 0.78-0.92), specificity of 0.91 (95 % CI: 0.86-0.95), positive likelihood ratio of 10.2 (95 % CI: 6.1, 17.0) and negative likelihood ratio of 0.14 (95 % CI: 0.08, 0.25) for KL-6 in diagnosing pulmonary fibrosis. The area under the receiver operating characteristic curve was 0.95 (95 % CI: 0.93-0.97). The results showed significant heterogeneity in both sensitivity and specificity (I2 = 94.55 and 91.52, respectively). Meta-regression analysis identified race as the cause of sensitivity heterogeneity and assay methodology as the cause of specificity heterogeneity. CONCLUSIONS: The analysis of this study suggests that serum KL-6 is a better tool for the diagnosis of pulmonary fibrosis when factors such as disease cause and control group category are not specifically considered.

CEACAM1 Is a Prognostic Biomarker and Correlated with Immune Cell Infiltration in Clear Cell Renal Cell Carcinoma.

Background: CEACAM1 has been shown to be aberrantly expressed in a variety of tumors, and modulation of CEACAM1-related signaling pathways has been suggested as a novel approach for cancer immunotherapy in recent years. However, its role in clear cell renal cell carcinoma (ccRCC) is unclear. Methods: The relationship between CEACAM1 and ccRCC was demonstrated based on data from TCGA, GEO, and HPA databases. And the relationship between clinicopathological features and CEACAM1 expression was also assessed. Survival curve analysis was performed to analyze the prognostic relationship between CEACAM1 expression and ccRCC. Protein interaction network analysis was used to analyze the relationship between CEACAM1 and microenvironment-related proteins. In addition, the immunomodulatory role of CEACAM1 in ccRCC was assessed by analyzing CEACAM1 and immune cell infiltration. Results: The expression of CEACAM1 was lower in ccRCC tissues than in adjacent normal tissues, and its expression level was negatively correlated with tumor size status (P < 0.001), metastasis status (P = 0.009), pathological stage (P = 0.002), gender (P < 0.001), histological grade (P < 0.001), and primary therapy outcome (P = 0.045) of ccRCC. Survival curve analysis showed that ccRCC patients with lower CEACAM1 expression exhibited shorter overall survival (P < 0.001), and CEACAM1 interacted with microenvironmental molecules such as fibronectin and integrins. Furthermore, immune infiltration analysis showed that CEACAM1 expression correlated with CD8+ and CD4+ T cells, macrophage, neutrophil, and dendritic cell infiltration in ccRCC. Conclusions: CEACAM1 expression correlates with progression, prognosis, and immune cell infiltration in ccRCC patients, and it may be a promising prognostic biomarker and therapeutic target for ccRCC.

Increased SPHK1 and HAS2 Expressions Correlate to Poor Prognosis in Pancreatic Cancer.

OBJECTIVE: SPHK1 and HAS2 have been reported to play important roles in tumorigenesis and development. However, their expression and prognostic value in pancreatic cancer (PC) remain unclear. This study is aimed at investigating the expression of SPHK1 and HAS2 on the prognosis of pancreatic cancer. MATERIALS AND METHODS: The expression of SPHK1 and HAS2 in pancreatic cancer tissues was analyzed through TCGA and GTEx databases and validated by qRT-PCR and Western blot in pancreatic cancer cell lines. χ 2 test was used to explore the correlation of the SPHK1 and HAS2 expressions with clinical characteristics. Kaplan-Meier survival analysis and ROC curve were used to evaluate the prognostic and diagnostic roles of SPHK1 and HAS2 in pancreatic cancer. Additionally, Spearman correlation analysis was applied to assess the correlation between the SPHK1 and HAS2 in pancreatic cancer. GO analysis and KEGG analysis were applied to explore the possible signaling pathway that SPHK1 and HAS2 coregulated genes mediated. RESULTS: The expression of SPHK1 and HAS2 was markedly upregulated in pancreatic cancer tissue and cell lines, respectively. Furthermore, there was a significant positive correlation between SPHK1 and HAS2 expressions. ROC curves showed that SPHK1 combine with HAS2 has good diagnostic value in pancreatic cancer patients with 85% sensitivity and 99.4% specificity. Kaplan-Meier analysis showed that increased expression of SPHK1 and HAS2 was significantly associated with short overall survival (OS) of pancreatic cancer patients. GO and KEGG results revealed that SPHK1 and HAS2 mainly involved cell proliferation and invasion mediated by extracellular matrix- (ECM-) receptor interaction, focal adhesion, and PI3K-AKT signaling pathways. CONCLUSIONS: Overexpression of SPHK1 and HAS2 could be important markers for the prognosis of pancreatic cancer.

Identification of prognostic biomarkers and drug target prediction for colon cancer according to a competitive endogenous RNA network.

Colorectal cancer is one of the commoner digestive tract malignant tumor types, and its incidence and mortality rate are high. Accumulating evidence indicates that long­chain non­coding RNAs (lncRNAs) and protein­coding RNAs interact with each other by competing with the same micro(mi)RNA response element (MREs) and serve an important role in the regulation of gene expression in a variety of tumor types. However, the regulatory mechanism and prognostic role of lncRNA­mediated competing endogenous (ce)RNA networks in colon cancer have yet to be elucidated. The expression profiles of mRNAs, lncRNAs and miRNAs from 471 colon cancer and 41 paracancerous tissue samples were downloaded from The Cancer Genome Atlas database. A lncRNA­miRNA­mRNA ceRNA network in colon cancer was constructed and comprised 17 hub lncRNAs, 87 hub miRNA and 144 hub mRNAs. The topological properties of the network were analyzed, and the random walk algorithm was used to identify the nodes significantly associated with colon cancer. Survival analysis using the UALCAN database indicated that 2/17 lncRNAs identified [metastasis­associated lung adenocarcinoma transcript (MALAT1) and maternally expressed gene 3 (MEG3)] and 5/144 mRNAs [FES upstream region (FURIN), nuclear factor of activated T­cells 5 (NFAT5), RNA Binding Motif Protein 12B (RBM12B), Ras related GTP binding A (RRAGA) and WD repeat domain phosphoinositide­interacting protein 2 (WIPI2)] were significantly associated with the overall survival of patients with colon cancer, and may therefore be used as potential prognostic biomarkers of colon cancer. According to extracted lncRNA­miRNA­mRNA interaction pairs, the GSE26334 dataset was used to confirm that the lncRNA MALAT1/miR­129­5p/NFAT5 axis may represent a novel regulatory mechanism concerning the progression of colon cancer. The clusterProfiler package was used to analyze Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in colon cancer. Finally, drugs that significantly interact with the core genes identified in colon cancer were predicted using a hypergeometric test. Of these, fostamatinib was identified to be a targeted drug for colon cancer therapy. The present findings provide a novel perspective for improved understanding of the lncRNA­associated ceRNA network and may facilitate the development of novel targeted therapeutics in colon cancer.

Long noncoding RNA OR3A4 promotes the proliferation and invasion of osteosarcoma cells by sponging miR-1227-5p.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been identified as key players in promoting tumourigenesis in osteosarcoma. LncRNA OR3A4 (OR3A4) has been reported as an oncogene in a number of tumours. However, the clinical value of OR3A4 in osteosarcoma and the role of OR3A4 in osteosarcoma progression are still unknown. METHODS: The expression levels of OR3A4 in the tumour tissue of osteosarcoma patients and osteosarcoma cell lines were detected by RT-PCR. Kaplan-Meier analysis and log-rank test were performed to evaluate the relationship between the level of OR3A4 expression and the prognosis of osteosarcoma patients. We investigated the association between the tissue expression levels of OR3A4 and different clinicopathological characteristics of osteosarcoma patients by χ2 tests. Bioinformatic databases and luciferase reporter assays were used to predict and validate the target microRNA of OR3A4. Finally, the role of OR3A4 in the proliferation and invasion of osteosarcoma cells was tested by MTT and Transwell assays, respectively. RESULTS: We observed that the expression level of OR3A4 was upregulated in the tumour tissue of osteosarcoma patients (p < 0.001) and osteosarcoma cell lines (p < 0.01) compared with the normal adjacent tissue and a normal human foetal osteoblastic cell line, respectively. The survival curve revealed that patients with high expression levels of OR3A4 had lower overall survival. Increased OR3A4 expression in osteosarcoma patients was associated with distant metastasis (p = 0.02) and advanced clinical stage (p < 0.001). In addition, bioinformatics analysis and luciferase reporter assays verified the complementary binding between OR3A4 and miR-1227-5p. Furthermore, we found that OR3A4 acted as a miR-1227-5p "sponge" to modulate osteosarcoma cell proliferation and invasion via downregulation of miR-1227-5p. CONCLUSION: OR3A4 promotes osteosarcoma cell proliferation and invasion by sponging miR-1227-5p, which might be related to the metastasis of osteosarcoma and could be used as a potential prognostic biomarker and therapeutic target in osteosarcoma.

Combination of plasma HA and circulating M2-like monocytes may serve as a diagnostic marker for breast cancer.

Background: Breast cancer (BC)-derived hyaluronan (HA) can induce the formation of M2-like tumor-associated macrophages (TAMs) in tumor context. However, little is known about the correlation between circulating M2-like monocytes and plasma HA in BC patients. This study focused on evaluating the relationship between circulating M2-like monocytes and plasma HA, and further appraised the diagnostic value of them in BC. Methods: The expression of M2-like TAMs and HA was determined in pathological tissues by immunohistochemistry. Flow cytometry was used to detect the levels of circulating CD14+CD204+ M2-like monocytes in 81 BC patients, 45 patients with breast benign diseases, and 46 healthy subjects. The levels of HA, CEA, and CA15-3 were measured in plasma samples using chemiluminescence method. Results: M2-like TAMs and HA expressions were elevated in BC tissues compared with benign tissues. In correspondence, the frequency of circulating CD14+CD204+ M2-like monocytes and the plasma HA levels were significantly higher in patients with BC than those in control groups. Importantly, there was a positive correlation between circulating M2-like monocytes and the plasma HA (Spearman r = 0.404, p < 0.001). Area under receiver operating characteristic curve (ROC) for the combination of circulating M2-like monocytes and HA was 0.899 (95% CI: 0.853-0.946), which was higher than the panel of CEA and CA15-3. Conclusions: The frequency of circulating CD14+CD204+ M2-like monocytes was positively correlated to plasma HA levels. The combination of circulating CD14+CD204+ M2-like monocytes and plasma HA could provide considerable diagnostic value in BC.