Microarray Method Research Articles

The role of miRNAs as biomarkers in cancer

MicroRNAs (miRNAs), a class of non-coding RNAs, play a critical role in regulating gene expression and have demonstrated significant potential as biomarkers in cancer research. This review explores the role of miRNAs in tumorigenesis, invasion, and metastasis, highlighting their altered regulation in various cancers, including lung, breast, liver, colorectal, and prostate cancer. miRNA expression patterns analysis helps clinicians in early cancer diagnosis, classification, and therapeutic monitoring. The stability of miRNAs in body fluids makes them ideal candidates for liquid biopsy, offering a non-invasive tool for cancer detection and prognosis assessment. Despite the promising clinical applications, challenges remain in the standardization of detection methods and integration of multi-omics data. Results are variable because different detection platforms, including qPCR, microarray and sequencing methods which have varying sensitivity and specificity. However, integrating multi-omics data comes with additional technological challenges because it calls for sophisticated bioinformatics tools to manage intricate and huge datasets. Further advancements are expected to establish miRNAs as a robust foundation for personalized cancer therapy.

Analysis of Genetic Characteristics of Patients with Thalassemia in the Chengdu Region, Sichuan Province

To analyze the gene mutation types and composition characteristics of patients with thalassemia in Chengdu Region, Sichuan Province. 6 649 suspected thalassemia patients with positive screening results who visited Chengdu Women's and Children's Center Hospital from January 2017 to December 2020 were selected as the study subjects. Among them, there were 2 273 males and 4 376 females. The frequency and distribution of α and β genotypes of thalassemia in this cohort was analyzed by Luminex liquid-phase microarray method. Among the 6 649 samples, 3 787 were genetically diagnosed as thalassemia, with a total positive rate of 56.96%; in which, 2 063 (31.03%) cases were β-thalassemia, 1 629 (24.50%) cases were α-thalassemia, and 95 (1.43%) cases were α combined with β thalassemia. The types of β-thalassemia gene mutation were mainly CD17/N (36.45%, 752/2 063), CD41-42/N (25.30%, 522/2 063), and IVS-II-654/N (24.72%, 510/2 063); and 2 037 cases of simple heterozygous mutations were identified, accounting for 98.74% of β-thalassemia patients. The types of α-thalassemia gene mutation were mainly -- SEA/αα (79.01%, 1 287/1 629), -α3.7/αα (10.62%, 173/1 629), -α3.7/-- SEA (2.95%, 48/1 629), and -α4.2/αα (2.15%, 35/1 629). The α combined with β thalassemia was dominated by -α3.7/αα; CD17/N and -α3.7/αα; IVS-II-654/N, both accounting for 14.74% (14/95) of patients with α combined with β thalassemia. In Chengdu region, Sichuan province, β thalassemia is more common than α thalassemia, the main type of β thalassemia mutation is CD17/N, and the main type of α thalassemia mutation is -- SEA/αα, with regional characteristics.

Immunohistochemical analysis of the immune checkpoint molecule Galectin-9 in meningiomas

Purpose: Most meningiomas can be treated by surgical resection. However, depending on the location of the lesion, incomplete resection or high-grade meningiomas may have a poor prognosis. The new methods such as immunotherapy may improve our options for effective, patient-specific treatment of meningiomas. We aim to contribute to the development of new personalized treatment strategies by investigating the status of Gal-9 in meningiomas. Materials and Methods: Four hundred two cases diagnosed in our laboratory between 2007 and 2020 were used for the study. New blocks of multiple tissues were prepared for immunohistochemistry using the tissue microarray method. Immunohistochemical staining of Gal-9 antibody was evaluated using the H-score method. Results: Of the 402 cases studied, 289 were female and 113 were male. Two hundred and seventy-one (67.4%) cases were WHO grade 1; 121 (30.1%) were grade 2 and 10 (2.5%) were grade 3. A high H-score was observed in grade 1 and 2 tumors (H-score: 93.38 and 93.91) and a low H-score in grade 3 tumors (H-score: 59.40). There was no significant correlation between brain invasion and Gal-9 expression. No significant correlation was found between Gal-9 expression and minor criteria used in tumor grading. Conclusion: A statistically significant difference was found between Gal-9 H-score and tumor grade. Gal-9 had a lower H-score in high-grade meningiomas and its expression level decreased. Therefore, Gal-9 with different expression levels can be used as a prognostic and predictive biomarker as well as an important molecule for treatment.

Investigation of the status of immune checkpoint molecules (PD-L1 and PD-1) in meningiomas by immunohistochemistry.

Meningiomas are the most common primary brain tumors of the central nervous system. Immunotherapy is a promising treatment method applied in many types of cancer. There is no standard and effective medical treatment to reduce recurrence and mortality in cases of incomplete resection of meningiomas and in high-grade cases. In order to investigate medical treatments in addition to surgery and radiotherapy, in this study, the status of immune checkpoint molecules (PD-L1/PD-1), which are the target of immunotherapy, in meningiomas was investigated. Four hundred two cases of meningioma diagnosed between 2007 and 2020 at our institution were used. New blocks were prepared from the appropriate blocks of the cases using the tissue microarray method. Sections obtained from these blocks were immunohistochemically stained with PD-1 and PD-L1 antibodies. Obtained data were interpreted with statistical analysis. Expression of PD-L1 was observed in 28.4% of meningiomas. Staining rates are higher in high-grade tumors. The staining rate of PD-L1 in the tumor increased significantly with pattern loss. PD-L1 expression in immune cells is 19.9%. Immune cell expression and the number of expressing immune cells correlate with spontaneous necrosis. Immune cell expression and the number of expressing immune cells are increased in high-grade meningiomas. PD-1 expression in immune cells is 9.0%, and this correlates with brain invasion. With these data, it was observed that the expression of immune checkpoint molecules PD-L1 and PD-1 increased especially in high-grade meningiomas. It may be the subject of research that these molecules may be targets of immunotherapy in the treatment of meningiomas.

Simultaneous detection of five mycotoxins in traditional Chinese medicines (TCMs) by visual protein microarray

The pollution of mycotoxins to crops such as traditional Chinese medicines (TCMs) is an established problem throughout the world. Thus, mycotoxin determination in TCMs during production and processing is significantly necessary, which means rapid, sensitive and accurate analytical methods are needed. In this work, a new method of visual protein microarray based on a 96-well microtiter plate was proposed. Combined with a colorimetric method, five mycotoxins (ochratoxin A, zearalenone, deoxynivalenol, aflatoxin B1 and fumonisin B1) in 90 samples (TCMs) could be detected simultaneously within 30 minutes. The detection limits for the five mycotoxins are 0.25 μg/kg, 0.33 μg/kg, 11.84 μg/kg, 0.06 μg/kg, and 3.58 μg/kg, which can satisfy specified requirements of mycotoxins in the Chinese Pharmacopoeia (2020 edition) adequately. Under repeated conditions, experiments were carried out on actual samples to verify the feasibility of the method. The results showed that the recoveries of all analytes were between 70 % and 120 %, and the relative standard deviations were less than 15 %. In comparison to LC-MS/MS, this method significantly reduces the required time, and the colorimetric technique offers more direct results compared to fluorescence-based screening assays. This method exhibits substantial potential for the rapid and sensitive on-site detection of TCMs for quality control.

A Review of Next-Generation Sequencing Technologies and Their Impact on Clinical Research: Assessing Clinical Efficacy and Cost-Effectiveness

In contrast to microarray methods, sequence-based technologies directly determine the nucleic acid sequence. A number of modern sequencing technologies are referred to collectively as "next-generation sequencing" (NGS), often known as "high-throughput sequencing." Compared to conventional Sanger sequencing, NGS gives orders of magnitude more data at a much lower ongoing cost. These new technologies allow for much faster and more affordable sequencing of DNA and RNA, revolutionizing the study of genomics and molecular biology. Technical improvements in NGS sequencing methods have rapidly increased sequencing volume to several billion nucleotides within a short period and at a reasonable cost. Currently, NGS is developing into a molecular microscope that is permeating almost all areas of biological research. The last ten years have seen the development of NGS platforms and methodologies, and the quality of the sequences has increased to the point where NGS is now utilized in human clinical diagnosis. Due to significant cost reductions and greater community acceptance of NGS, the utilization of NGS techniques in studying clinical trials has significantly increased. NGS is a useful tool for detecting mutations in people with cancer and genetic abnormalities. To ascertain whether NGS can cost-effectively improve patient outcomes, more thorough cost-effectiveness studies of NGS applied to patient care management are required.

Insights into the enzymatic synthesis of alcoholic flavor esters with molecular docking analysis

The enzymatic synthesis is essential for the flavor esters in the food and fragrance industries. This paper introduces a novel preparation method for lipase microarrays (CALB@PMHOS-TEOS) with loadings up to 229 ± 1.4 mg/g. Using surfactant-free hydrophobic silica-hybridized mesoporous materials and Candida antarctica lipase, this resulted in the effective synthesis of flavor esters. Using CALB@PMHOS-TEOS a Pickering emulsion system was formed at the oil-water interface for the sustainable synthesis of flavor esters. This resulted in a 93.5 ± 0.5 % conversion of hexanoic acid within 2 h at an optimal temperature of 35 °C, which is the highest level recorded in the literature to date. Furthermore, the conversion of hexanoic acid was maintained at 63.9 ± 1.2 % after 9 cycles of CALB@PMHOS-TEOS reuse. The application of the enzyme to the synthesis in a variety of flavor esters achieved a new benchmark in the existing literature. A molecular docking model was evaluated to understand the molecular mechanism underpinning the immobilized lipase. This work introduces a novel method for the eco-friendly and efficient synthesis of flavor esters for applications across various fields including food and cosmetics.

Integrated Analysis of Genomic and Genome-Wide Association Studies Identified Candidate Genes for Nutrigenetic Studies in Flavonoids and Vascular Health: Path to Precision Nutrition for (Poly)phenols.

Flavonoids exert vasculoprotective effects in humans, but interindividual variability in their action has also been reported. This study aims to identify genes that are associated with vascular health effects of flavonoids and whose polymorphisms could explain interindividual variability in response to their intake. Applying the predetermined literature search criteria, we identified five human intervention studies reporting positive effects of flavonoids on vascular function together with global genomic changes analyzed using microarray methods. Genes involved in vascular dysfunction were identified from genome-wide association studies (GWAS). By extracting data from the eligible human intervention studies, we obtained 5807 differentially expressed genes (DEGs). The number of identified upstream regulators (URs) varied across the studies, from 227 to 1407. The search of the GWAS Catalog revealed 493 genes associated with vascular dysfunction. An integrative analysis of transcriptomic data with GWAS genes identified 106 candidate DEGs and 42 candidate URs, while subsequent functional analyses and a search of the literature identified 20 top priority candidate genes: ALDH2, APOE, CAPZA1, CYP11B2, GNA13, IL6, IRF5, LDLR, LPL, LSP1, MKNK1, MMP3, MTHFR, MYO6, NCR3, PPARG, SARM1, TCF20, TCF7L2, and TNF. In conclusion, this integrated analysis identifies important genes to design future nutrigenetic studies for development of precision nutrition for polyphenols.

Unveiling Prognostic RNA Biomarkers through a Multi-Cohort Study in Colorectal Cancer.

Because cancer is a leading cause of death and is thought to be caused by genetic errors or genomic instability in many circumstances, there have been studies exploring cancer's genetic basis using microarray and RNA-seq methods, linking gene expression data to patient survival. This research introduces a methodological framework, combining heterogeneous gene expression data, random forest selection, and pathway analysis, alongside clinical information and Cox regression analysis, to discover prognostic biomarkers. Heterogeneous gene expression data for colorectal cancer were collected from TCGA-COAD (RNA-seq), and GSE17536 and GSE39582 (microarray), and were integrated with Entrez Gene IDs. Using Cox regression analysis and random forest, genes with consistent hazard ratios and significantly affecting patient survivability were chosen. Predictive accuracy was evaluated using ROC curves. Pathway analysis identified potential RNA biomarkers. The authors identified 28 RNA biomarkers. Pathway analysis revealed enrichment in cancer-related pathways, notably EGFR downstream signaling and IGF1R signaling. Three RNA biomarkers (ZEB1-AS1, PI4K2A, and ITGB8-AS1) and two clinical biomarkers (stage and age) were chosen for a prognostic model, improving predictive performance compared to using clinical biomarkers alone. Despite biomarker identification challenges, this study underscores integration of heterogenous gene expression data for discovery.

Knocking down GRAMD1C expression reduces 6-OHDA-induced apoptosis in PC12 cells.

To explore the differential genes in Parkinson's disease (PD) through a preliminary GEO database, and to investigate the possible mechanisms. The PD differentially expressed genes (DEGs) were analyzed by the microarray method. Then, these DEGs were applied to KEGG and GO analyses to predict the related signaling pathways and molecular functions. Comparison of GRAMD1C expression levels in the putamen of normal and Parkinson's patients by bioinformatic analysis. PC12 cells were cultured to construct a 6-hydroxydopamine (6-OHDA)-induced Parkinson's cell model. RT-qPCR was performed to detect the efficiency of GRAMD1C siRNA. MTT assay was conducted to examine the proliferation of cells. Then, the apoptosis of each group of cells was measured by flow cytometry. Western blot was carried out to determine the expression of apoptosis-related proteins. Through bioinformatics, GRAMD1C was confirmed to be one of the most significantly upregulated genes in PD. Furthermore, GRAMD1C was notably enhanced in the PD patients and 6-OHDA-induced PC12 cells. Besides, 6-OHDA stimulation significantly reduced PC12 cell proliferation, and it reverted with the GRAMD1C siRNA. Moreover, the flow cytometry results showed that knockdown of GRAMD1C could effectively reduce the high apoptosis rate of PC12 cells induced by 6-OHDA treatment. Similarly, western blot results found that 6-OHDA stimulation markedly increased the expression levels of Bax and Caspase 3Caspase 3 and decreased the Bcl-2 expression in PC12 cells, and GRAMD1C knockdown reversed these changes. GRAMD1C is upregulated in PD, and may affect the PD process through the apoptotic pathway.

Kinin Receptors and Kinin-Related Gene Expression in Astrocytic Brain Tumors.

Kinins are a set of peptides present in tissues that are involved in the inflammatory response and cancer progression. However, studies showing the expression of kinin receptors in human glioma samples are still incomplete and contradictory. The aim of the present study was to ascertain the expression of BDKRB1 and BDKRB2 genes, as well as the level of B1R and B2R proteins in human gliomas, depending on the degree of malignancy. Additionally, representative kinin-dependent genes with altered expression were indicated. The expression profile of kinin-dependent genes was determined using oligonucleotide microarray technique. In addition, RT-qPCR was used to assess the expression level of selected differentiating genes. The location of kinin receptors in brain gliomas was assessed using immunohistochemical methods. The oligonucleotide microarray method was used to identify 12 mRNA IDs of kinin-related genes whose expression was upregulated or downregulated in gliomas of different grades. In immunohistochemically stained samples, the concentrations of BR1 and BR2 proteins, measured by optical density, were statistically significantly higher in grade G3 vs. G2 and G4 vs. G3. Increased expression of kinin receptors BDKRB1 and BDKRB2 in brain gliomas, depending on the degree of malignancy, suggests the involvement of kinins and their receptors in the disease's pathogenesis. Quantitative assessment of mRNA BDKRB1, PRKAR1A, MAP2K, and EGFR in patients with brain tumors may hold diagnostic and therapeutic significance.

Protective effects of galacto-oligosaccharide and polydextrose on Caco-2 intestinal cell barrier injury model

To explore the protective effect of different ratios of galactose oligosaccharide(GOS) and polydextrose(PDX) on intestinal cell barrier damage model of Caco-2. The same batch of Caco-2 cells were cultured to form a cell barrier model and randomly divided into damaged model group without calcium, calcium-containing blank control group(1.8 mmol/L Ca~(2+)), low-ratio/low-dose group(1.8 mmol/L Ca~(2+)+2 mg/mL GOS+2 mg/mL PDX) and low-ratio/medium-dose group(1.8 mmol/L Ca~(2+)+4 mg/mL GOS+4 mg/mL PDX), low-ratio/high-dose group(1.8 mmol/L Ca~(2+)+8 mg/mL GOS+8 mg/mL PDX) and high-ratio/low-dose group(1.8 mmol/L Ca~(2+)+0.8 mg/mL GOS+3.2mg/mL PDX), high-ratio/medium-dose group(1.8 mmol/L Ca~(2+)+1.6 mg/mL GOS+6.4 mg/mL PDX), high-ratio/high-dose group(1.8 mmol/L Ca~(2+)+3.2mg/mL GOS+12.8 mg/mL PDX), a total of 8 groups, three parallel groups were performed in each group. The Trans Epithelial Electrical Resistance value and apparent permeability coefficient value of each group were determined after 4 d culture, and the morphology of tight junction proteins ZO-1, Occludin and Claudin-1 were observed by immunofluorescence method, and the expression levels of inflammatory related factors in each group were determined by protein microarray method. Compared with damaged model group, TEER ratio in calcium-containing blank control group was significantly increased(P<0.05), while Papp value was significantly decreased(P<0.05);Compared with calcium-containing blank control group, TEER ratio in low-ratio/medium-dose group and high-ratio/high-dose group was significantly increased(P<0.05) while Papp value was significantly decreased(P<0.05), and they could significantly down-regulate some inflammatory response related cytokines. The cell barrier was intact in all groups except for the compact junction protein structure in the model group. Compared with Ca~(2+) alone, the combination of two prebiotics can enhance the density of Caco-2 cell barrier and reduced the permeability of cell bypass. And it can significantly reduce the expression level of some inflammatory cytokines and effectively protect the intestinal cell barrier.

Analysis of plant expression profiles revealed that aphid attack triggered dynamic defense responses in sorghum plant.

Sorghum [Sorghum bicolor (L.) Moench] is one of the most important cereal crops grown worldwide but is often attacked by greenbug (aphid). In response to aphid attack, host plant initiates a large transcriptional reorganization, leading to activation of the host defense genes in aphid-attacked plants. In this study, our objective was to analyze defensive responses of sorghum against aphid and identify aphid resistance genes in sorghum. For the experiments, seedlings developed from an aphid resistant germplasm line (PI 550607) were divided into two groups, then, one group was infested with greenbug ((Schizaphis graminum Rondani) and the other group was used as control (un-infested). In addition, seedlings of sorghum cultivar Tx 7000, a susceptible genotype, prepared under the same conditions, were used as a genetic control. Those plant samples were used to develop transcriptional profiles using the microarray method, from which 26.1% of the 1,761 cDNA sequences spotted on the microarray showed altered expression between two treatments at 4days after infestation. Sequence annotation and molecular analysis revealed that many differentially expressed genes (DEGs) were related to direct host defense or signal transduction pathways, which regulate host defense. In addition to common responsive genes, unique transcripts were identified in response to greenbug infestation specifically. Later, a similar transcriptional profiling was conducted using the RNA-seq method, resulted in the identification of 2,856 DEGs in the resistant line with a comparison between infested and non-infested at 4days and 4,354 DEGs in the resistant genotype compared to the susceptible genotype at 4days. Based on the comparative analysis, the data of RNA-seq provided a support for the results from the microarray study as it was noticed that many of the DEGs are common in both platforms. Analysis of the two differential expression profiles indicate that aphid triggered dynamic defense responses in sorghum plants and sorghum plant defense against aphid is a complex process involving both general defense systems and specific resistance mechanisms. Finally, the results of the study provide new insights into the mechanisms underlying host plant defense against aphids and will help us design better strategies for effectively controlling aphid pest.

Evaluation of annexin A1 expression in lung, breast, colon, and prostatic adenocarcinomas and in tumor microenvironment

Objectives: Annexin A1 (ANXA1) which plays a role in tumor development and metastasis has been reported to be an effective regulator for tumor stroma and interacts with different components in the tumor microenvironment. The role of ANXA1 in tumorigenesis has not been fully understood. One of the main reasons for this is the great variability of ANXA1 expression in malignant tumors across different tumor types. Materials and Methods: Archived hematoxylin-eosin stained preparations of lung adenocarcinoma, breast invasive ductal carcinoma, colonic adenocarcinoma, and prostatic acinar carcinoma were re-evaluated and tumor regions to be analyzed with the tissue microarray method were determined. The ANXA1 expressions between the tumors and tumor microenvironment were evaluated immunohistochemically. Results: ANXA1 expression was decreased in the lung, breast, colon, and prostate adenocarcinomas. The most prominent staining was seen in lung adenocarcinoma cases. There was no statistically significant difference between the tumors in terms of ANXA1 staining (P > 0.05). ANXA1 was shown to be a more stained tumor microenvironment than in the tumor. Statistically significant staining with ANXA1 between within tumor and tumor microenvironment was observed in breast adenocarcinomas (P < 0.05). Our study showed differences between ANXA1 expression in different cancers, in tumor cells, and tumor microenvironment. Conclusion: Considering the effects of ANXA1 on tumor development and metastasis, a potential use as a biomarker may be suggested. Particularly, in breast adenocarcinomas, the high expression of ANXA1 in the tumor microenvironment supports the notion that it could induce the tumor stroma response.

Construction of lncRNA- and circRNA-associated ceRNA networks in the prostatic urethra of rats after simulating transurethral laser prostatectomy (TULP)

Non-coding RNA appears to be involved in wound repair. Competing endogenous RNA (ceRNA) appears to be an important post-transcriptional mechanism, it means that long noncoding RNA (lncRNA) or circular RNA (circRNA) acts as a microRNA (miRNA) sponge to further regulate mRNA. However, ceRNA network related to wound repair after prostatectomy has yet been constructed. TULP is the main surgical method of prostatectomy, but there have been no reports of TULP rat models in the past. We simulated TULP on rats, and observed the whole process of wound injury and repair after operation through pathological examination of wound tissue. Next, we discovered 732 differentially expressed lncRNAs (DElncRNAs), 47 differentially expressed circRNAs (DEcircRNAs), 17 differentially expressed miRNAs (DEmiRNAs), and 1892 differentially expressed mRNAs (DEmRNAs) related to wound repair after TULP through full transcriptome microarray and bioinformatics methods, and confirmed the reliability of transcriptome data by quantitative Reverse Transcription PCR (qRT-PCR), and immunohistochemistry. Then, we constructed the lncRNA- and circRNA-associated ceRNA regulatory networks related to wound repair after TULP in rats. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that molecules in these networks were mainly involved in inflammatory infiltration, cell differentiation, and intercellular interactions and involved signal pathways such as the PI3K-Akt signaling pathway. Thus, this study successfully established the TULP model in rats, revealed potentially important biomarkers and ceRNA networks after prostatectomy in rats, and provided theoretical support for the repair of post-prostatectomy wound.

Advancements in COVID-19 Testing: An In-depth Overview.

COVID-19 rapidly evolved as a pandemic, killing and hospitalising millions of people, and creating unprecedented hurdles for communities and health care systems worldwide. The rapidly evolving pandemic prompted the head of the World Health Organisation to deliver a critical message: "test, test, test." The response from the diagnostic industry and researchers worldwide was overwhelming, resulting in more than a thousand commercial tests being available worldwide. Several sampling approaches and diagnostic techniques have been employed from the early stages of the pandemic, such as SARS-CoV-2 detection by targeting the viral RNA or protein indirectly via antibody testing, biochemical estimation, and various imaging techniques, and many are still in the various stages of development and yet to be marketed. Accurate testing techniques and appropriate sampling are the need of the hour to manage, diagnose and treat the pandemic, especially in the current crisis where SARS-CoV-2 undergoes constant mutation, evolving into various strains, which are pretty challenging. The article discusses various testing techniques as well as screening methods for detection, treatment, and management of COVID-19 transmissions, such as NAAT, PCR, isothermal detection including RT-LAMP, RPA, NASBA, RCA, SDA, NEAR, and TMA, CRISPR strategy, nanotechnology approach, metagenomic profiling, point of care tests, virus neutralization test, ELISA, biomarker estimation, utilization of imaging techniques such as CT, ultrasonography, brain MRI in COVID-19 complications, and other novel strategies including microarray methods, microfluidic methods and artificial intelligence with an emphasis on advancements in the testing strategies for the diagnosis, management, and prevention of COVID-19.

USP51/PD-L1/ITGB1-deployed juxtacrine interaction plays a cell-intrinsic role in promoting chemoresistant phenotypes in non-small cell lung cancer.

Programmed death ligand 1 (PD-L1) has been demonstrated to facilitate tumor progression and therapeutic resistance in an immune-independent manner. Nevertheless, the function and underlying signaling network(s) of cancer cell-intrinsic PD-L1 action remain largely unknown. Herein, we sought to better understand how ubiquitin-specific peptidase 51 (USP51)/PD-L1/integrin beta-1 (ITGB1) signaling performs a cell-intrinsic role in mediating chemotherapeutic resistance in non-small cell lung cancer (NSCLC). Western blotting and flow cytometry were employed for PD-L1 detection in NSCLC cell lines. Coimmunoprecipitation and pulldown analyses, protein deubiquitination assay, tissue microarray, bioinformatic analysis and molecular biology methods were then used to determine the significance of PD-L1 in NSCLC chemoresistance and associated signaling pathways in several different cell lines, mouse models and patient tissue samples. Ubiquitin-7-amido-4-methylcoumarin (Ub-AMC)-based deubiquitinase activity, cellular thermal shift and surface plasmon resonance (SPR) analyses were performed to investigate the activity of USP51 inhibitors. We provided evidence that cancer cell-intrinsic PD-L1 conferred the development of chemoresistance by directly binding to its membrane-bound receptor ITGB1 in NSCLC. At the molecular level, PD-L1/ITGB1 interaction subsequently activated the nuclear factor-kappa B (NF-κB) axis to elicit poor response to chemotherapy. We further determined USP51 as a bona fide deubiquitinase that targeted the deubiquitination and stabilization of the PD-L1 protein in chemoresistant NSCLC cells. Clinically, we found a significant direct relationship between the USP51, PD-L1 and ITGB1 contents in NSCLC patients with chemoresistant potency. The elevated USP51, PD-L1 and ITGB1 levels were strongly associated with worse patient prognosis. Of note, we identified that a flavonoid compound dihydromyricetin (DHM) acted as a potential USP51 inhibitor and rendered NSCLC cells more sensitive to chemotherapy by targeting USP51-dependent PD-L1 ubiquitination and degradation in vitro and in vivo. Together, our results demonstrated that the USP51/PD-L1/ITGB1 network potentially contributes to the malignant progression and therapeutic resistance in NSCLC. This knowledge is beneficial to the future design of advanced cancer therapy.

Prenatal detection of copy number variants in fetuses with detected congenital devolpmental disordes, from 2015 to 2020 by Multiplex Ligation-Dependent Probe Amplification and microarray analysis.

Analysis of prenatal samples from 2015 to 2020. Comparison detection rates of clinically relevant variants by cytogenetic karyotype analysis and cytogenomic MLPA (Multiplex Ligation-Depent Probe Amplification) and microarray methods (CMA - chromosomal microarray). 1,029 prenatal samples were analyzed by cytogenetic karyotyping (N = 1,029), cytogenomic methods - MLPA (N = 144) and CMA (N = 111). All unbalanced changes were confirmed by MLPA or CMA. From the analyzed set of fetuses, after subtraction of aneuploidies - 107 (10.40%, N = 1,029), 22 structural aberrations (2.39%, N = 922) - nine unbalanced changes (0.98%), 10 balanced changes (1.08%), one case of unclear mosaicism (0.09%), one case of presence of a marker chromosome (0.09%) and one case of sex discordance (0.09%) - were detected by karyotype analysis. A total of eight (7.21%, N = 111) pathological variants were detected by CMA in 255 samples with physiological karyotype indicated for cytogenomic examination. Five (3.47%, N = 144) of eight pathogenic variants were detected by MLPA method. The total capture of pathogenic variants by MLPA and CMA methods was 14 (5.14%) and 17 (6.25%) (N = 272), including confirmatory pathological karyotype testing. Detection of pathological variants in the isolated disorders group was lower than in the multiple disorders group (5.08 vs. 21.42%). A higher success rate for the detection of pathological copy number variation variants by the microarray method than by the MLPA method was confirmed.

Local radiotherapy for murine breast cancer increases risk of metastasis by promoting the recruitment of M-MDSCs in lung

BackgroundRadiotherapy is one of the effective methods for treatment of breast cancer; however, controversies still exist with respect to radiotherapy for patients with TNBC. Here, we intend to explore the mechanism by which local radiotherapy promotes the recruitment of M-MDSCs in the lung and increases the risk of lung metastasis in TNBC tumor-bearing mice.MethodsA single dose of 20 Gy X-ray was used to locally irradiate the primary tumor of 4T1 tumor-bearing mice. Tumor growth, the number of pulmonary metastatic nodules, and the frequency of MDSCs were monitored in the mice. Antibody microarray and ELISA methods were used to analyze the cytokines in exosomes released by irradiated (IR) or non-IR 4T1 cells. The effects of the exosomes on recruitment of MDSCs and colonization of 4T1 cells in the lung of normal BALB/c mice were observed with the methods of FCM and pathological section staining. T lymphocytes or 4T1 cells co-cultured with MDSCs were performed to demonstrate the inhibitory effect on T lymphocytes or accelerative migration effect on 4T1 cells. Finally, a series of in vitro experiments demonstrated how the exosomes promote the recruitment of M-MDSCs in lung of mice.ResultsEven though radiotherapy reduced the burden of primary tumors and larger lung metastatic nodules (≥ 0.4 mm2), the number of smaller metastases (< 0.4 mm2) significantly increased. Consistently, radiotherapy markedly potentiated M-MDSCs and decreased PMN-MDSCs recruitment to lung of tumor-bearing mice. Moreover, the frequency of M-MDSCs of lung was positively correlated with the number of lung metastatic nodules. Further, M-MDSCs markedly inhibited T cell function, while there was no difference between M-MDSCs and PMN-MDSCs in promoting 4T1 cell migration. X-ray irradiation promoted the release of G-CSF, GM-CSF and CXCl1-rich exosomes, and facilitated the migration of M-MDSCs and PMN-MDSCs into the lung through CXCL1/CXCR2 signaling. While irradiated mouse lung extracts or ir/4T1-exo treated macrophage culture medium showed obvious selective chemotaxis to M-MDSCs. Mechanistically, ir/4T1-exo induce macrophage to produce GM-CSF, which further promoted CCL2 release in an autocrine manner to recruit M-MDSCs via CCL2/CCR2 axis.ConclusionsOur work has identified an undesired effect of radiotherapy that may promote immunosuppressive premetastatic niches formation by recruiting M-MDSCs to lung. Further studies on radiotherapy combined CXCR2 or CCR2 signals inhibitors were necessary.

Microarray Chip and Method for Simultaneous and Highly Consistent Electroporation of Multiple Cells of Different Sizes.

Cell electroporation is an important cell manipulation technology to artificially transfer specific extracellular components into cells. However, the consistency of substance transport during the electroporation process is still an issue due to the wide size distribution of the natural cells. In this study, a cell electroporation microfluidic chip based on a microtrap array is proposed. The microtrap structure was optimized for single-cell capture and electric field focusing. The effects of the cell size on the cell electroporation in the microchip were investigated through simulation and experiment methods using the giant unilamellar vesicle as the simplified cell model, and a numerical model of a uniform electric field was used as a comparison. Compared with the uniform electric field, a lower threshold electric field is required to induce electroporation and produces a higher transmembrane voltage on the cell under a specific electric field in the microchip, showing an improvement in cell viability and electroporation efficiency. The larger perforated area produced on the cells in the microchip under a specific electric field allows a higher substance transfer efficiency, and the electroporation results are less affected by the cell size, which is beneficial for improving substance transfer consistency. Furthermore, the relative perforation area increases with the decrease of the cell diameter in the microchip, which is exactly opposite to that in a uniform electric field. By manipulating the electric field applied to the microtrap individually, a consistent proportion of substance transfer during electroporation of cells with different sizes can be achieved.