Microarray Results Research Articles

Myofibroblast-derived extracellular vesicles facilitate cancer stemness of hepatocellular carcinoma via transferring ITGA5 to tumor cells

BackgroundMyofibroblasts constitute a significant component of the tumor microenvironment (TME) and play a pivotal role in the progression of hepatocellular carcinoma (HCC). Integrin α5 (ITGA5) is a crucial regulator in myofibroblasts of malignant tumors. Therefore, the potential of ITGA5 as a novel target for the therapeutic strategy of HCC should be investigated.MethodsDigital scanning and analysis of the HCC tissue microarray were performed to locate the distribution of ITGA5 and conduct the prognosis analysis. CRISPR Cas9-mediated ITGA5 knockout was performed to establish the ITGA5-KO myofibroblast cell line. Extracellular vesicles (EVs) derived from LX2 were extracted for the treatment of HCC cells. Subsequently, the sphere-forming ability and the stemness markers expression of the treated HCC cells were examined. An orthotopic HCC mouse model with fibrotic injury was constructed to test the outcomes of ITGA5-targeting therapy and its efficacy in the programmed death-ligand 1 (PD-L1) treatment. Co-immunoprecipitation/mass spectrometry and transcriptome data were integrated to delve into the mechanism.ResultsThe tissue microarray results revealed that ITGA5 was highly enriched in the stromal myofibroblasts of HCC tissues and contributed to enhanced tumor progression and poor prognosis. Notably, ITGA5 transmission via extracellular vesicles (EVs) from myofibroblasts to HCC cells induced the acquisition of cancer stem cell-like properties. Mechanistically, ITGA5 directly bind to YES1, facilitating the activation of YES1 and its downstream pathways, thereby enhancing the stemness of HCC cells. Furthermore, the blockade of ITGA5 impeded tumor progression driven by ITGA5+ myofibroblasts and enhanced the efficacy of treatment with PD-L1 in a mouse model of HCC.ConclusionsOur findings elucidated a novel mechanism by which the EV-mediated transfer of ITGA5 from myofibroblasts to tumor cells augmented HCC stemness. ITGA5-targeting therapy helped prevent the progression of HCC and improved the efficacy of PD-L1 treatment.Graphical

Possible Role of Endothelial-Derived Cellular and Exosomal-miRNAs in Lipid-Mediated Diabetic Retinopathy: Microarray Studies.

Diabetic retinopathy (DR) is a salient cause of blindness worldwide. There is still an immense need to understand the pathophysiology of DR to discover better diagnostic and therapeutic modalities. Human retinal endothelial cells (HRECs) were treated with 15-HETE or D-glucose, then miRNAs were isolated, and a microarray was performed. MirWALK 2 and Ingenuity Pathway Analysis (IPA) were used to analyze the microarray results. Exosomal miRNAs from 15-HETE-treated HRECs were isolated, microarrayed, and then imported into IPA for further analysis. The microarray results showed that 15-HETE downregulated 343 miRNAs and upregulated 297 miRNAs in HRECs. High glucose treatment induced a differential expression of HREC-miRNAs where 185 miRNAs were downregulated and 244 were upregulated. Comparing the impact of 15-HETE versus DG or diabetic mouse retina elaborated commonly changing miRNAs. Pathway and target analysis for miRNAs changed in 15-HETE-treated HRECs revealed multiple targets and pathways that may be involved in 15-HETE-induced retinal endothelial dysfunction. The HREC-exosomal miRNAs were differentially expressed after 15-HETE treatment, with 34 miRNAs downregulated and 45 miRNAs upregulated, impacting different cellular pathways. Here, we show that 15-HETE induces various changes in the cellular and exosomal miRNA profile of HRECs, highlighting the importance of targeting the 12/15 lipoxygenase pathway in DR.

Genetic Alterations in Chromatin Regulatory Genes in Upper Tract Urothelial Carcinoma and Urothelial Bladder Cancer.

Upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) share histomorphological and therapeutic features but distinct epidemiologic and clinicopathologic characteristics. We examined alterations of chromatin regulatory genes in molecular subtypes, clonal relatedness, and T-cell receptor (TCR) diversity in UTUC and UCB. Targeted next-generation sequencing or whole-exome DNA sequencing and TCR sequencing were conducted with 34 UTUC and 49 UCB specimens from 63 patients. Tumors were subtyped based on the expression of CK5 and GATA3. Results of tissue microarray of 78 muscle-invasive bladder cancer (MIBC) samples were used as prognostic factors of different subtypes of MIBC. Chromatin regulatory genes were frequently mutated in both UTUC and UCB. Rapid relapse and progression of non-MIBC are correlated with alterations of KMT2C and EP300. Frequency of alterations in chromatin regulatory genes is higher in UTUC patients with SBS22 and SBS2 signatures and lower in UCB patients with SBS2 and SBS6 signatures. GATA3 and CK5 double-positive patients with higher frequencies of SMARCA4, ARID1A, and EP300 mutations have better prognoses than patients with basal subtypes. Although UTUC and UCB in the same patient can be either clonally related or developed independently, mutated genes in chromatin pathway were enriched in the related clones. Compared to UTUC, UCB had more deleterious mutations in DNA damage repair (DDR) genes, higher levels of tumor mutation burden (TMB) and copy number variations (CNVs), as well as higher TCR clonality and lower TCR diversity. Since genetic alterations of the chromatin pathway genes are important in both UTUC and UCB, they could serve as potential biomarkers for predicting disease progression and therapeutic targets. Differences in mutation frequencies of DDR pathway, TMB, CNV, and TCR might be the contributing factors for the distinct responses to immune checkpoint inhibitor (ICI) between UTUC and UCB.

Identification of Specific microRNAs in Adipose Tissue Affected by Lipedema.

Lipedema is a chronic disorder affecting women with a 10% incidence worldwide. It is often confused with obesity. This study was undertaken to study microRNAs in lipedema tissue assessed by direct hybridization using the robust n-counter flex DX CE-IVD platform. The mean age of the subjects participating in the study was 40.29 (±12.17). The mean body weight and BMI were 67.37 (±10.02) and 25.75 (±4.10), respectively. The lipedema stages included were I and II. The differential expressed human (hsa)-miRNAs were determined according to a log2 fold-change (LFC) of 0.5 and p value < 0.05. To these, increased expression of hsa-let-7g-5p was evident, as well as reduced levels of hsa-miR-371a-5p, -4454+7975, -365a+b-3p, -205-5p, -196a-5p, -4488, -2116-5p, -141-3p, -208a-3p, -302b-3p, 374a-5p, and -1297. Then, several bioinformatics tools were used to analyze microarray data focusing on validated target genes in silico. KEGG and Gene Ontology (GO) pathway enrichment analysis was conducted. Furthermore, the protein-protein interaction and co-expression network were analyzed using STRING and Cytoscape, respectively. The most upregulated miRNA mainly affected genes related to cell cycle, oocyte meiosis, and inflammatory bowel disease. The downregulated microRNAs were related to endocrine resistance, insulin resistance, hypersensitivity to AGE-RAGEs, and focal adhesion. Finally, we validated by RT-PCR the upregulated hsa-let-7g-5p and two down-regulated ones, hsa-miR-205-5p and hsa-miR-302b-3p, confirming microarray results. In addition, three mRNA target miRNAs were monitored, SMAD2, the target of the hsa-let-7g-5p, and ESR1 and VEGFA, the target of hsa-miR-205-5p and hsa-miR-302b-3p, respectively. Our results open a new direction for comprehending biochemical mechanisms related with the pathogenesis of lipedema, shedding light on this intricate pathophysiological condition that could bring to light possible biomarkers in the future.

Fast and Economic Microarray-Based Detection of Species-, Resistance-, and Virulence-Associated Genes in Clinical Strains of Vancomycin-Resistant Enterococci (VRE)

Today, there is a continuous worldwide battle against antimicrobial resistance (AMR) and that includes vancomycin-resistant enterococci (VRE). Methods that can adequately and quickly detect transmission chains in outbreaks are needed to trace and manage this problem fast and cost-effectively. In this study, DNA-microarray-based technology was developed for this purpose. It commenced with the bioinformatic design of specific oligonucleotide sequences to obtain amplification primers and hybridization probes. Microarrays were manufactured using these synthesized oligonucleotides. A highly parallel and stringent labeling and hybridization protocol was developed and employed using isolated genomic DNA from previously sequenced (referenced) clinical VRE strains for optimal sensitivity and specificity. Microarray results showed the detection of virulence, resistance, and species-specific genes in the VRE strains. Theoretical predictions of the microarray results were also derived from the sequences of the same VRE strain and were compared to array results while optimizing protocols until the microarray result and theoretical predictions were a match. The study concludes that DNA microarray technology can be used to quickly, accurately, and economically detect specifically and massively parallel target genes in enterococci.

Application of Whole-Exome Sequencing in the Prenatal Diagnosis of Foetuses With Central Nervous System Abnormalities.

To investigate the clinical value of whole-exome sequencing (WES) in the diagnosis of foetuses with central nervous system (CNS) abnormalities but having a normal karyotyping and chromosomal microarray result. During the period of 2016-2022, there were a total of 149 foetuses with CNS abnormalities but having negative karyotyping and chromosomal microarray analysis results; WES was performed on these foetuses and their parents. Variants were classified according to ACMG guidelines, and the association of pathogenic variants with specific types of CNS abnormalities was explored. Among these 149 foetuses, three categories of abnormalities, namely, single CNS abnormality, multiple CNS abnormalities, CNS abnormalities along with other organ system abnormalities were identified, for which the detection rate of P/LP variants is 17.4% (12/69), 28.6% (14/49) and 54.8% (17/31), respectively. WES brought about an increase of 28.9% in diagnostic yield in the prenatal evaluation of foetuses with CNS abnormalities but having negative karyotyping and chromosome array results. WES may also be of benefit for the diagnosis of foetuses with isolated CNS abnormalities, as well as for making more informed interpretations of imaging findings and for providing better genetic counselling.

Distributions of MICA and MICB Alleles Typed by Amplicon-Based Next-Generation Sequencing in South Koreans.

Major histocompatibility complex class I chain-related genes A and B (MICA and MICB) play a role as ligands in activating the NKG2D receptor expressed in natural killer cells, γδ T-cells and αβ CD8 T-cells and have been defined in human diseases and haematopoietic stem cell transplantation (HSCT). MICA and MICB alleles were genotyped at the three-field level by amplicon-based next-generation sequencing (NGS) using a MiSeqDx system and compared with the results from previous studies in healthy South Korean donors. Exons 2-5 of MICA and exons 2-4 of MICB were amplified using a multiplex polymerase chain reaction (PCR). Sequence reads of ≥ 51 depth counts were consistently obtained for each sample exon, and target exons were determined to match reference sequences contained in the IPD-IMGT/HLA database. MICA and MICB alleles were tested using exon combinations. The program was designed to recognise specific sequences and discriminate between the MICA*008:01:01/*027 alleles. A total of 22 alleles were found in MICA and MICB. We observed 1 HLA-C ~ HLA-B ~ MICA ~ MICB ~ HLA-DRB1 haplotype with significant linkage disequilibrium between alleles at all neighbouring HLA loci. These results are consistent with previous microarray results. Genotyping of MICA and MICB was possible using 11-loci HLA genes. We updated the distribution of MICA and MICB based on three-field allele and haplotype frequencies containing linkage disequilibrium in South Koreans using amplicon-based NGS. These data suggest that high-resolution MICA and MICB typing data obtained using NGS may aid in performing HSCT and disease association studies.

Association of prenatal Cleft Lip and Palate ultrasound abnormalities with copy number variants at a single Chinese tertiary center

BackgroudA systematic analysis was conducted to investigate the molecular etiology of fetal cleft lip and/or palate (CL/P) and the association between various types of CL/P and copy number variations (CNVs), as well as their impact on birth outcomes.MethodsIn this retrospective study conducted between January 2016 and July 2022, a cohort of pregnancies diagnosed with fetal CL/P was enrolled and comprehensive clinical data for all cases were extracted from our medical record database, including demographic data about the pregnancies, ultrasound findings, results of Chromosomal microarray (CMA), as well as relevant pregnant and perinatal outcomes.ResultsAmong the 358 cases, 32 clinically significant variants in 29 (8.1%) fetuses with CL/P were detected by CMA. In 338 singleton pregnancies, the diagnostic yield of CMA in the context of CL/P fetuses was determined to be 7.7% (26/338). CP cases exhibited a relatively higher prevalence of pathogenic/likely pathogenic CNVs at a rate of 25% (3/12), followed by CLP cases at 8.0% (23/288). Notably, the CL group did not demonstrate any pathogenic/likely pathogenic CNV findings among the examined cases (0/38). The diagnostic rate of clinically significant variants was notably higher in the non-isolated CL/P group than in the isolated CL/P group (11/33, 33.3% vs. 15/305, 4.9%, p < 0.001). Within the remaining 20 twin pregnancies, three clinically significant variants (15%) were observed.ConclusionsThis study provides powerful evidence supporting the efficacy of CMA as a valuable tool for facilitating the prenatal genetic diagnosis of fetal CL/P. The presence of CP and CLP in fetal cases demonstrated a relatively higher incidence of pathogenic/likely pathogenic CNVs. Moreover, when these cases were accompanied by additional ultrasound abnormalities, the likelihood of identifying diagnostic CNVs significantly increased. Conversely, cases of CL alone might not be associated with positive CNVs. The present data may significantly enhance prenatal diagnosis accuracy and facilitate informed genetic counseling for cases of fetal CL/P.

Transdermal characteristic study of bovine sialoglycoproteins with anti-skin aging and accelerating skin wound healing.

Sialoglycoproteins play important roles in various biological processes, including cell adhesion, immune response, and cell signaling. Our previous studies indicated that the bovine sialoglycoproteins could be developed as a reagent against skin aging and as a new candidate for accelerating skin wound healing as well as inhibiting scar formation. However, transdermal characteristic of the bovine sialoglycoproteins is still unknown. This study investigated the transdermal permeation of the bovine sialoglycoproteins through porcine skin using the Franz diffusion cell method. Our study showed that the bovine sialoglycoproteins could penetrate through the porcine skin with a linear permeation pattern described by the regression equation N% = 11.49 t-3.858, with a high coefficient of determination (R2 = 0.9903). The histochemical results demonstrated the widespread distribution of the bovine sialoglycoproteins between the epidermal and dermal layers, which suggesting parts of the bovine sialoglycoproteins had ability to traverse the epidermal barrier. The results of the lectin microarrays indicated highly enriched glycopatterns on the bovine sialoglycoproteins, which also appeared in permeated porcine skin. The LC-MS/MS analysis further showed that the bovine sialoglycoproteins were composed of approximately 100 proteins with molecular weight ranging from 748.4 kDa to 10 kDa, and there were 23 specific bovine sialoglycoproteins with molecular weight ranging from 69.2 kDa to 10 kDa to be characterized in permeated porcine skin. Parts of the bovine sialoglycoproteins with molecular weight less than 69.2 kDa had ability to traverse the epidermal barrier. Understanding the permeation characteristics of the bovine sialoglycoproteins for developing innovative formulations with therapeutic benefits, contributing to advancements in cosmetic and dermatological fields.

Expression profile of circular RNAs in blood samples of Northern Chinese males with intracerebral hemorrhage shows downregulation of hsa-circ-0090829

Circular RNAs (circRNAs) are involved in several neurological disorders; however, the mechanisms underlying their involvement remain to be clarified. We attempted to explore the expression profiles of circRNAs and their potential functions and mechanisms in the pathogenesis of intracerebral hemorrhage (ICH) in Northern Chinese males. The microarray results showed that 50 circRNAs were significantly upregulated, while 194 circRNAs were significantly downregulated in ICH patients compared with healthy controls (p < 0.05). After bioinformatics analysis, a circRNA–microRNA–messenger RNA network and a protein–protein interaction network were constructed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that the neurotrophin signaling pathway, long-term potentiation, and the mitogen-activated protein kinase pathway are potentially implicated in ICH pathophysiology. The quantitative real-time polymerase chain reaction results revealed that hsa-circ-0090829 was significantly downregulated in ICH. The receiver operating characteristic curve analysis showed that the area under the curve of hsa-circ-0090829 between ICH and healthy controls was 0.807. Furthermore, the dual-luciferase assay showed that hsa-circ-0090829 sponged miR-526b-5p. This study reports the altered expression of circRNAs and identifies the potential functions of these circRNAs in ICH. Our results may facilitate further mechanistic research on circRNAs in ICH and provide probable novel diagnostic biomarkers and therapeutic targets for ICH.

Synergistic Effects of Glutamine Deprivation and Metformin in Acute Myeloid Leukemia.

The metabolic reprogramming of acute myeloid leukemia (AML) cells is a compensatory adaptation to meet energy requirements for rapid proliferation. This study aimed to examine the synergistic effects of glutamine deprivation and metformin exposure on AML cells. SKM-1 cells (an AML cell line) were subjected to glutamine deprivation and/or treatment with metformin or bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES, a glutaminase inhibitor) or cytarabine. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, and cell apoptosis and reactive oxygen species (ROS) by flow cytometry. Western blotting was conducted to examine the levels of apoptotic proteins, including cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP). Moreover, the human long noncoding RNA (lncRNA) microarray was used to analyze gene expression after glutamine deprivation, and results were confirmed with quantitative RT-PCR (qRT-PCR). The expression of metallothionein 2A (MT2A) was suppressed using siRNA. Cell growth and apoptosis were further detected by CCK-8 assay and flow cytometry, respectively, in cells with MT2A knockdown. Glutamine deprivation or treatment with BPTES inhibited cell growth and induced apoptosis in SKM-1 cells. The lncRNA microarray result showed that the expression of MT family genes was significantly upregulated after glutamine deprivation. MT2A knockdown increased apoptosis, while proliferation was not affected in SKM-1 cells. In addition, metformin inhibited cell growth and induced apoptosis in SKM-1 cells. Both glutamine deprivation and metformin enhanced the sensitivity of SKM-1 cells to cytarabine. Furthermore, the combination of glutamine deprivation with metformin exhibited synergistic antileukemia effects on SKM-1 cells. Targeting glutamine metabolism in combination with metformin is a promising new therapeutic strategy for AML.

BOC targets SMO to regulate the Hedgehog pathway and promote proliferation, migration, and invasion of glioma cells

The purpose of this study was to investigate the effects of BOC on glioblastoma cells and its underlying mechanisms. In vitro, BOC-knockdown was performed in glioma cell lines. CCK-8 and Transwell were used to assess the impact of BOC on the viability, invasion, and migration of gliobma cells. RNA-seq technology was employed to analyze the differential gene expression between BOC-knockdown glioma cells and the control group, and qRT-PCR was used to validate the expression of downstream differential genes. SMO-overexpression was performed to investigate the effects of SMO on glioma cells. A BOC-knockdown mouse subcutaneous tumor model was to verify the effects of BOC on mouse tumors. Tissue microarray technology was used to detect the expression of BOC and SMO in samples of normal human brain tissue and glioma tissue. In vitro, BOC-knockdown inhibited the viability, invasion, and migration of glioma cells, as well as downregulated the expression of downstream differential genes SMO, EGFR, HRAS, and MRAS. Conversely, SMO-overexpression upregulated the viability, invasion, and migration abilities of BOC-knockdown cells. In vivo, BOC-knockdown suppressed tumor growth in mice and downregulated the expression of downstream differential genes SMO, EGFR, HRAS, and MRAS. Tissue microarray results showed that both BOC and SMO were highly expressed in glioma tissues. BOC is aberrantly overexpressed in glioma patients and promotes glioma development. Mechanistically, BOC activates the Hedgehog (Hh) and RAS signaling pathways by upregulating the expression of SMO, EGFR, HRAS, and MRAS, thereby facilitating the Proliferation, invasion and migration of glioma cells.

Cell-Free DNA Analysis of Fetal Aneuploidies in Early Pregnancy Loss.

Background: Products of conception samples are often collected and analyzed to try to determine the cause of an early pregnancy loss. However, sample collection may not always be possible, and maternal cell contamination and culture failure can affect the analysis. Cell-free DNA-based analysis of a blood sample could be used as an alternative method in early pregnancy loss cases to detect if aneuploidies were present in the fetus. Methods: In this prospective study, blood samples from early pregnancy loss patients were analyzed for the presence of fetal aneuploidies using a modified version of a noninvasive prenatal testing assay for cell-free DNA analysis. Results from cell-free DNA analysis were compared against the gold standard, microarray analysis of products of conception samples. This study was registered with ClinicalTrials.gov, identifier: NCT04935138. Results: Of the 76 patient samples included in the final study cohort, 11 were excluded from performance calculations. The 65 patient samples included in the final analysis included 49 with an abnormal microarray result and 16 with a normal microarray result. Based on results from these 65 samples, the study found that genome-wide cell-free DNA analysis had a sensitivity of 73.5% with a specificity of 100% for the detection of fetal aneuploidies in early pregnancy loss cases. Conclusions: This prospective study provides further support for the utility of cell-free DNA analysis in detecting fetal aneuploidies in early pregnancy loss cases. This approach could allow for a noninvasive method of investigating the etiology of miscarriages to be made available clinically.

The effect of opting-in versus opting-out forming on the rate of reported variants of questionable significance in prenatal microarray.

To examine the effect of patient-selected opt-in versus opt-out option on the rate of reported variants of uncertain clinical significance (VOUS) and high-frequency low-penetrant (HFLP) findings in prenatal microarray testing. A standard microarray consent form in Israel includes a requirement to note patient choice to be or not to be informed about the presence of VOUS and HFLP variants. The original form was designed as an opting-out method, in which the women had to actively mark if they did not want to be informed about questionable findings. In the authors' Genetic Institute, the form was changed for an opting-in option in October 2019. In this study we have compared the rates of reported VOUS and HFLP variants between the opt-in and opt-out periods. Of the 1014 prenatal CMA tests, 590 (58.2%) were performed in the opt-out period. A significant decrease in the rate of women requesting to be informed of VOUS findings was noted (66.8% in opt-out period vs 34.0% in opt-in period), yielding a relative risk (RR) of 0.46 (95% confidence interval [CI] 0.39-0.53). Rate of women preferring to be informed of HFLP variants decreased from 75.3% to 48.1% (RR 0.52, 95% CI 0.45-0.60). We present a simple and effective method to decrease the rate of reported findings of questionable significance in the prenatal setting. These results are important not only for microarray results, but also for next-generation sequencing techniques, such as whole exome or genome sequencing.

P-387 The use of cell-free DNA analysis to identify fetal aneuploidies in early pregnancy loss

Abstract Study question Can cell-free (cf) DNA-based analysis of a maternal blood sample be used to identify presence of fetal aneuploidies in early pregnancy loss (EPL) cases? Summary answer CfDNA analysis was able to detect fetal aneuploidies in EPL cases with 73.5% sensitivity and 100% specificity. What is known already Miscarriages occur in about 10%–20% of pregnancies, and the majority occur in the first trimester. About 50% of EPL cases result from the presence of fetal aneuploidy. Knowing the etiology of a miscarriage can be helpful for future reproductive planning along with providing a psychological benefit for patients. Although products of conception (POC) samples can be collected and assessed for presence of fetal aneuploidies, this is not always possible. Recent studies have shown that a noninvasive approach using cfDNA analysis of a blood sample can identify fetal aneuploidies in patients that experience EPL. Study design, size, duration In this prospective study, samples were collected from 78 patients across the United States from June 2021–December 2022. Blood specimens were collected for cfDNA analysis and maternal cell contamination analysis, and POC samples were collected following surgical evacuation for microarray analysis. Performance of the cfDNA assay was determined by comparing results from the cfDNA analysis against the gold standard of microarray results. Participants/materials, setting, methods Patients were recruited from six reproductive endocrinology/infertility practices. Eligible participants were between 5–20 weeks of gestation with miscarriage of a singleton pregnancy diagnosed by ultrasound and presence of pregnancy tissue in utero. Limited clinical information was collected, including demographics and expected clinical gestational age (GA) at sample collection. Blood samples underwent cfDNA analysis using an adapted version of a noninvasive prenatal test, which included establishment and use of pregnancy loss-specific log likelihood ratio thresholds. Main results and the role of chance Seventy-eight patients were recruited, of which two were excluded due to blood sample collection issues. Eleven of the 76 patients that underwent cfDNA analysis were excluded from performance calculations for several reasons including maternal cell contamination of POC samples and detection of triploidy by microarray. The final 65 samples included 49 affected and 16 unaffected samples based on POC microarray results; this included 42 with one fetal aneuploidy, six with two fetal aneuploidies, and one with three fetal aneuploidies. Average fetal fraction of these 65 samples was 4.9% (range: 0.6%–13.9%) and average expected clinical GA at time of sample collection was 8.7 weeks (range: 5.9–12.1 weeks). Performance of the cfDNA assay was determined by comparing cfDNA analysis results with POC microarray results using either a full concordance approach (correct classification of all affected chromosomes in an affected sample) or a partial concordance approach (correct classification of at least one affected chromosome in an affected sample). Based on the full concordance approach, sensitivity of the cfDNA assay was 69.4% and specificity was 100%. For the partial concordance approach, sensitivity of the cfDNA assay increased to 73.5% whilst specificity remained at 100%. The cfDNA assay failure rate was 0%. Limitations, reasons for caution Study limitations include a small study cohort, the inability of the cfDNA assay to detect triploidy or partial deletions/duplications &amp;lt;7 Mb and possibly mosaicism, and that this cfDNA analysis approach can only be used for EPL cases that are identified before complete expulsion of the products of conception. Wider implications of the findings This study provides further evidence of the ability of cfDNA analysis to detect fetal aneuploidies in EPL cases. A clinically available cost-effective noninvasive approach to determine the etiology of a miscarriage could help inform medical management of patients for future reproductive planning and could provide psychological benefits to patients. Trial registration number NCT04935138

SMP30 Interacts with ROCK1 to Inhibit HCC Invasiveness by Effecting the Epithelial-Mesenchymal Transition.

The senescence marker protein 30 (SMP30) is a calcium-binding protein whose expression decreases with age, and is closely associated with hepatocellular carcinoma (HCC) development. The primary goal of this study was to examine the mechanistic effect of SMP30 on HCC migration and invasion. Bioinformatic and immunohistochemical approaches were used to examine the expression of SMP30 in HCC tissues and its relationship to patient survival. We investigated the effects of SMP30 expression on HCC cell proliferation, migration, invasion, and cell cycle dynamics. cDNA microarray technology was used to determine the gene expression profile of SK-Hep-1 cells following recombinant SMP30 overexpression to identify genes downstream of SMP30 that regulate HCC cell migration and invasion. We identified SMP30 interacting proteins by affinity purification-mass spectrometry (AP-MS) and co-immunoprecipitation/western blotting (COIP-WB). SMP30 expression was lower in HCC tissues compared with normal liver tissues, and its expression positively correlated with overall survival in HCC patients. Additionally, SMP30 overexpression effectively blocked the migratory and invasive properties of SK-Hep-1 cells, but did not affect either proliferation rates or cell cycle. cDNA microarray results confirmed that many of the differentially expressed genes identified are involved in the process of epithelial-mesenchymal transition (EMT). AP-MS and COIP-WB experiments confirmed that Rho-associated protein kinase 1 (ROCK1) interacts with SMP30 in SK-Hep-1 cells, and ROCK1 is known to intimately regulate the EMT process. SMP30 inhibits HCC metastasis by influencing the expression of EMT-related proteins after interacting with ROCK1.

Detection of Sulfur Oxidizing Bacteria to Oxidize Hydrogen Sulfide in Biogas from Pig Farm by NGS and DNA Microarray Technique

A high concentration of hydrogen sulfide (H2S) released from pig farming is one of the major environmental problems affecting surrounding communities. In modern pig farms, the bioscrubber is used to eliminate H2S, which is found to be driven mainly by the sulfur-oxidizing bacteria (SOB) community. Therefore, in this study, molecular biology techniques such as next-generation sequencing (NGS) and DNA microarray are proposed to study the linkage between enzyme activity and the abundance of the SOB community. The starting sludge (SFP1) and recirculating sludge (SFP2) samples were collected from the bioscrubber reactor in the pig farm. The abundance of microbial populations between the two sampling sites was considered together with the gene expression results of both soxABXYZ and fccAB. Based on the NGS analysis, the members of phylum Proteobacteria such as Halothiobacillus, Acidithiobacillus, Thiothrix, Novosphingobium, Sulfuricurvum, Sulfurovum, Sulfurimonas, Acinetobacter, Thiobacillus, Magnetospirillum, Arcobacter, and Paracoccus were predominantly found in SFP2. The presence of Cyanobacteria in SFP pig farms is associated with increased biogas yields. The microarray results showed that the expression of soxAXBYZ and fccAB genes involved in the oxidation of sulfide to sulfate was increased in Halothiobacillus, Paracoccus, Acidithiobacillus, Magnetospirillum, Sphingobium, Thiobacillus, Sulfuricurvum, Sulfuricurvum, Arcobacter, and Thiothrix. Both NGS and DNA microarray data supported the functional roles of SOB in odor elimination and the oxidation of H2S through the function of soxABXYZ and fccAB. The results also identified the key microbes for H2S odor treatment, which can be utilized to monitor the stability of biological treatment systems and the toxicity of sulfide minerals by oxidation.

A functional variant rs10409772 in FUT6 promoter regulates colorectal cancer progression through PKA/CREB signaling

Fucosyltransferase 6 (FUT6) is overexpressed in colorectal cancer tissue according to TCGA samples and immunohistochemistry results of a tissue microarray. FUT6 effects cell migration, tumor formation and proliferation of colorectal cancer cells in different essays. FUT6 promotes cancer cell proliferation in vitro and colorectal tumorigenesis in vivo by upregulating PKA/CREB pathway activation. Moreover, FUT6 expression is regulated by rs10409772 shown in the luciferase essays, a single nucleotide polymorphism in the promoter of FUT6. Our study suggests that elevated expression of FUT6 promotes PKA/CREB signaling, which in turn augments colorectal carcinogenesis, indicating a potential therapeutic target for colorectal cancer patients with increased FUT6 expression.

Overexpression of PLK1 Molecule Following Incomplete Thermal Ablation Promotes the Proliferation and Invasion of Residual Hepatocellular Carcinoma.

TAT, a widely used treatment for HCC, can exacerbate the progression of residual HCC. The present study investigated the mechanism of action of PLK1 following ITA of HCC. The PLK1 levels in HCC were determined using qRT-PCR from clinical patient samples, IHC from tissue microarray, and data from globally high-throughput data and microarrays. The PLK1 levels and their effect on the biological phenotype of heat-stress HCC cells were evaluated through in vitro experiments. We detected PLK1 abnormal expression in HCC models of nude mice subjected to ITA. We detected the effects of different PLK1 expression levels on EMT pathway proteins. PLK1 exhibited an overexpression in HCC tissues with an SMD of 1.19 (3414 HCC and 3036 non-HCC tissues were included), distinguishing HCC from non-HCC effectively (AUC = 0.9). The qRT-PCR data from clinical HCC patient samples and IHC from HCC tissue microarray results also indicated an overexpressed level. In the incomplete ablation models, an increased PLK1 expression was found in both heat-stress cells and subcutaneous tumors. The upregulation of PLK1 following ITA was found to enhance the malignancy of HCC and exacerbate the proliferation, migration, and invasion of residual HCC cells, whereas PLK1 knockdown suppressed the biological malignancy of HCC cells. Meanwhile, PLK1 has different regulatory effects on various EMT pathway proteins. PLK1 promotes the progression of residual HCC by activating EMT pathway after ITA, which might provide a novel idea for the treatment and prognosis of residual HCC.

Irbesartan ameliorates diabetic kidney injury in db/db mice by restoring circadian rhythm and cell cycle.

Irbesartan has been widely used in the clinical treatment of diabetic kidney disease (DKD). However, the molecular mechanism of its delay of DKD disease progression has not been fully elucidated. The aim of the present study was to investigate the mechanism of irbesartan in the treatment of DKD. C57BL/KsJ db/db mice were randomly divided into the model group and irbesartan-treated group. After treatment with irbesartan for 12 weeks, the effects on blood glucose, body weight, 24-h urinary albumin, and renal injuries were evaluated. Microarray was used to determine the differentially expressed genes (DEGs) in the renal cortex of mice. |Log FC| <0.5 and false discovery rate (FDR) <0.25 were set as the screening criteria. Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), protein-protein interaction (PPI) network and modules, and microRNA (miRNA)-DEGs network analysis were applied to analyze the DEGs. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the results of microarray. The present study demonstrated irbesartan could significantly improve the renal function in db/db mice through decreasing 24-h urinary albumin and alleviating the pathological injury of kidney. Irbesartan may affect the expression of numerous kidney genes involved in circadian rhythm, cell cycle, micoRNAs in cancer, and PI3K-AKT signaling pathway. In the miRNA-DEGs network, miR-1970, miR-703, miR-466f, miR-5135, and miR-132-3p were the potential targets for irbesartan treatment. The validation test confirmed that key genes regulating circadian rhythm (Arntl, Per3, and Dbp) and cell cycle (Prc1, Ccna2, and Ccnb2) were restored in db/db mice on treatment with Irbesartan. Generally, irbesartan can effectively treat DKD by regulating the circadian rhythm and cell cycle. The DEGs and pathways identified in the study will provide new insights into the potential mechanisms of irbesartan in the treatment of DKD.