Real-time Polymerase Chain Reaction Data Research Articles

Prevention of premature birth in female patients with bacterial vaginosis using a complex of natural antimicrobial peptides and cytokines

Introduction. The prevalence of bacterial vaginosis (BV) comprises 23–29 %, which in pregnant women is a known risk factor for premature birth (PB) that rates increases by 2.9-fold. BV treatment with antibiotics has no effect PB incidence, therefore stressing a need to search for alternative remedies.Aim: to evaluate the effectiveness of treatment, including antibacterial therapy and a complex preparation containing natural antimicrobial peptides and cytokines, to reduce the incidence of birth defects in pregnant women with BV.Materials and Methods. Design: a prospective open comparative cohort study in parallel groups was conducted with 101 pregnant women: Group I (n = 69) received the antibiotic Metronidazole, 500 mg tablets orally twice a day for 7 days, and a complex preparation containing exogenous natural antimicrobial peptides and cytokines (Superlymph®) suppositories per 25 IU once a day vaginally in the evening for 20 days; Group II (n = 32) received Metronidazole alone (the same regimen). Patient examination was carried out using approaches included clinical methods, accepted in obstetrics, and laboratory tests – microscopy of vaginal content smears, real-time polymerase chain reaction (PCR).Results. The PB (within 240–366 weeks) incidence in Group I was significantly lower than in Group II and comprised 2.9 % vs. 21.9 %, respectively (p = 0.004), with an 8-fold decline in developing PB risk (relative risk (RR) = 0.13; 95 % confidence interval (CI) = 0.03–0.60), whereas inter-group percentage of pregnant women with high risk (PB history) was comparable (p = 0.39) so that PB incidence did not differ from pregnant women without former PB. Microbiological recovery after treatment for BV was achieved in 85.5 % of patients from Group I vs. 56.3 % in Group II (RR = 1.52; 95 % CI = 1.10–2.10; p = 0.002) based on real-time PCR data. The persistence of anaerobic flora after treatment was significantly lower in Group I vs. Group II reaching 7.2 and 34.4 % (p < 0.001), respectively, with a 5-fold lower PB risk (RR = 0.21; 95 % CI = 0.08–0.56). Cessation of viral shedding compared to the number of patients with initial viral shedding was achieved in 94.6 % vs. 8.3 % of patients, respectively, with a 50-fold decline in risk (RR = 0.02; 95 % CI = 0.005–0.08; p < 0.001). The number of newborns weighing less than 2500 g was significantly lower from paired mothers who received Superlymph® + Metronidazole comprising 2.9 % vs. 15.6 % treated with Metronidazole alone (p = 0.03), whereas a risk of low birth weight neonates was decreased by 6-fold (RR = 0.16; 95 % CI = 0.03–0.88). The condition of the neonates assessed by birth Apgar score was comparable.Conclusion. The use of a complex preparation Superlymph® (suppositories per 25 IU once an day vaginally, for 20 days) along with oral antibiotic Мetronidazole in pregnant women with BV facilitates a decline in PB incidence down to 2.9 % at gestational age of 240–366 weeks lowering a risk of PB exceeding that of antibacterial therapy by 8-fold, including patients with former PB.

Prevention of premature birth in female patients with bacterial vaginosis using a complex of natural antimicrobial peptides and cytokines

Introduction. The prevalence of bacterial vaginosis (BV) comprises 23–29 %, which in pregnant women is a known risk factor for premature birth (PB) that rates increases by 2.9-fold. BV treatment with antibiotics has no effect PB incidence, therefore stressing a need to search for alternative remedies.Aim: to evaluate the effectiveness of treatment, including antibacterial therapy and a complex preparation containing natural antimicrobial peptides and cytokines, to reduce the incidence of birth defects in pregnant women with BV.Materials and Methods. Design: a prospective open comparative cohort study in parallel groups was conducted with 101 pregnant women: Group I (n = 69) received the antibiotic Metronidazole, 500 mg tablets orally twice a day for 7 days, and a complex preparation containing exogenous natural antimicrobial peptides and cytokines (Superlymph®) suppositories per 25 IU once a day vaginally in the evening for 20 days; Group II (n = 32) received Metronidazole alone (the same regimen). Patient examination was carried out using approaches included clinical methods, accepted in obstetrics, and laboratory tests – microscopy of vaginal content smears, real-time polymerase chain reaction (PCR).Results. The PB (within 240–366 weeks) incidence in Group I was significantly lower than in Group II and comprised 2.9 % vs. 21.9 %, respectively (p = 0.004), with an 8-fold decline in developing PB risk (relative risk (RR) = 0.13; 95 % confidence interval (CI) = 0.03–0.60), whereas inter-group percentage of pregnant women with high risk (PB history) was comparable (p = 0.39) so that PB incidence did not differ from pregnant women without former PB. Microbiological recovery after treatment for BV was achieved in 85.5 % of patients from Group I vs. 56.3 % in Group II (RR = 1.52; 95 % CI = 1.10–2.10; p = 0.002) based on real-time PCR data. The persistence of anaerobic flora after treatment was significantly lower in Group I vs. Group II reaching 7.2 and 34.4 % (p < 0.001), respectively, with a 5-fold lower PB risk (RR = 0.21; 95 % CI = 0.08–0.56). Cessation of viral shedding compared to the number of patients with initial viral shedding was achieved in 94.6 % vs. 8.3 % of patients, respectively, with a 50-fold decline in risk (RR = 0.02; 95 % CI = 0.005–0.08; p < 0.001). The number of newborns weighing less than 2500 g was significantly lower from paired mothers who received Superlymph® + Metronidazole comprising 2.9 % vs. 15.6 % treated with Metronidazole alone (p = 0.03), whereas a risk of low birth weight neonates was decreased by 6-fold (RR = 0.16; 95 % CI = 0.03–0.88). The condition of the neonates assessed by birth Apgar score was comparable.Conclusion. The use of a complex preparation Superlymph® (suppositories per 25 IU once an day vaginally, for 20 days) along with oral antibiotic Мetronidazole in pregnant women with BV facilitates a decline in PB incidence down to 2.9 % at gestational age of 240–366 weeks lowering a risk of PB exceeding that of antibacterial therapy by 8-fold, including patients with former PB.

Low Expression of SCN4B Predicts Poor Prognosis in Non-Small Cell Lung Cancer.

Sodium voltage-gated channel beta subunit 4 (SCN4B) plays a suppressive role in various tumors. However, the role of SCN4B in non-small cell lung cancer (NSCLC) is not yet clear. This study aims to investigate the expression of SCN4B in NSCLC patients and its correlation with prognosis. Firstly, the expression of SCN4B in non-small cell lung cancer (NSCLC) was analyzed using The Cancer Genome Atlas (TCGA) database. Then, differential expression genes (DEGs) were identified using R software. Next, DEG enrichment pathways were analyzed using the R package clusterProfiler. Protein-protein interaction networks were revealed through STRING analysis. A heatmap showed significant differential expression of SCN4B. Further analysis included examining SCN4B expression in a pan-cancer context and its correlation with 24 types of immune cells in NSCLC. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR), Western Blot, immunohistochemistry, and clinical data were used to validate SCN4B expression and prognostic value in NSCLC patients. SCN4B mRNA expression in non-small cell lung cancer tissues was significantly lower than in adjacent normal tissues (p < 0.001). Clinical correlation analysis confirmed its association with clinical pathology. Gene set enrichment analysis (GSEA) and tumor immune-related analyses indicated that SCN4B is involved in NSCLC-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and participates in immune infiltration. qRT-PCR, Western Blot, and immunohistochemistry also confirmed that SCN4B is downregulated in NSCLC patients and is associated with poor prognosis. SCN4B is downregulated in tumor tissues of NSCLC patients and is associated with a poor prognosis.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

BACKGROUND Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. AIM To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. METHODS We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0 ), TATA-binding protein (TBP ), GAPDH , actin beta (ACTB ), tubulin (TUB ), aminolevulinic acid synthase 1 (ALAS1 ), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ ), eukaryotic translational elongation factor 1 alpha (EF1a ), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA ), and beta-2-microglobulin (B2M )] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms’ two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. CONCLUSION For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

The Effects of Convalescent Plasma Transfusion on Serum Levels of Macrophage-Associated Inflammatory Biomarkers in Patients with Severe COVID-19.

As an antibody-based therapy, plasma therapy has been used as an emergency therapeutic strategy against severe acute respiratory syndrome coronavirus type 2 infection. Due to the critical role of macrophages in coronavirus disease-19 (COVID-19)-associated hyperinflammation, the main objective of this study was to assess the effect of plasma transfusion on the expression levels of the inflammatory biomarkers involved in activation and pulmonary infiltration of macrophages. The target population included 50 severe hospitalized COVID-19 patients who were randomly assigned into 2 groups, including intervention and control. Serum levels of chemokine (C-C motif) ligand (CCL)-2, CCL-3, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were measured by enzyme-linked immunosorbent assay. Moreover, quantitative real-time polymerase chain reaction (PCR) was carried out to assess the relative expression of nuclear factor (NF)-κB1, NF-κB2, nuclear factor erythroid 2 p45-related factor 2 (NRF-2), and thioredoxin-interacting protein genes. Sampling was done at baseline and 72 h after receiving plasma. The intervention group demonstrated significantly lower serum levels of IL-6, TNF-α, and CCL-3. In addition, real-time PCR data analyses showed that the relative expression of NF-κB2 was significantly declined in the patients who received plasma. The use of convalescent plasma probably has a significant inhibitory effect on the cytokines, chemokines, and inflammatory genes related to macrophage activation, which are closely associated with the worsening of clinical outcomes in severe COVID-19.

Identification and Analysis of Expression Patterns of the Caleosin Genes in Hickory (Carya cathayensis Sarg.)

The deciduous tree hickory (Carya cathayensis) holds economic significance in China due to its high oil content, particularly in unsaturated fatty acids. Oil bodies are crucial for storing triacylglycerol (TAG), with caleosin serving as a predominant oil body protein that aids in oil body formation and stability maintenance. Our study utilized bioinformatics techniques to identify caleosin genes within Carya cathayensis, Carya illinoinensis, and Juglans regia. Three caleosin genes were discovered in the genomes of Carya cathayensis, Carya illi-noinensis, and Juglans regia. These genes encode hydrophilic proteins. Additionally, all caleosin proteins feature a single Ca2+-binding EF-hand, a conserved “proline knot” motif, and a C-terminal hydrophilic region with four potential phosphorylation sites. The caleosin proteins in Carya cathayensis consist of α-helix, β-corner, extended chain, and random curl structures. Cis-acting elements related to stress response and hormone signaling were identified in Carya cathayensis, Carya illinoinensis, and Juglans regia, with distinct cis-acting elements implicated in seed-specific regulation in Carya cathayensis. Additionally, subcellular localization analysis confirmed that CcaCLO1 and CcaCLO2 were localized within oil bodies. Transcriptome analysis and quantitative real-time polymerase chain reaction (qRT-PCR) data demonstrated a significant up-regulation of CcaCLO1 expression during the developmental stages of the Carya cathayensis embryo. Furthermore, qPCR findings indicated that caleosins from Carya cathayensis were responsive to salt stress, with a significant up-regulation of CcaCLO1 following exposure to salt stress treatment. Consequently, caleosin genes in Carya cathayensis, Carya illinoinensis, and Juglans regia share similar physicochemical characteristics and conserved motifs. Specifically, CcaCLO1 in Carya cathayensis primarily responds to embryo development and salt stress. These findings offer foundational insights for future investigations into the regulatory mechanisms of oil accumulation and response to salt stress in hickory.

Abstract 530: The effect of cardamonin on PD-L1 expression in triple-negative breast cancer cells

Abstract Triple-negative breast cancer (TNBC) accounts for about 10% of all breast cancers and does not express estrogen, progesterone, or HER2 receptors. It has an aggressive nature, it is highly metastatic, and because of the lack of therapies, it continues to be a challenge. These cancer cells employ mechanisms to escape the immune system that induce the upregulation of PD-L1 expression, a ligand encoded by the CD274 gene. Because cancer cells tend to produce higher levels of PD-L1 compared to normal cells, therapies that inhibit PD-L1 may be helpful. Evidence also shows that high levels of oxidative stress and inflammation may participate in both the initiation and progression of cancer. Cardamonin, an aromatic enone flavonoid, has displayed an array of pharmacological activities, including modulation of different signaling molecules involved in the development and progression of cancer. This work investigated the cardamonin’s ability to modulate PD-L1 expression and NF-KB in genetically distinct MDA-MB-231 (Caucasian) and MDA-MB-468 (African American) TNBC cell lines. The methodology included cytotoxic assays, ELISA, Real Time-Polymerase Chain Reaction (RT-PCR) assays, and Wes analysis. The results showed that cardamonin treatment caused a dose-dependent decrease in cell viability in both cell lines, ranging from 3.12 µM to 200 µM. The RT-PCR and ELISA data showed that even though IFN-γ stimulated MDA-MB-231 cells showed a higher expression of PD-L1, cardamonin reduced mRNA and protein expression of PD-L1 in both cell lines. As a possible molecular mechanism for PD-L1 inhibition, cardamonin modulated MUC-1, STAT3, and JAK, which are genes that activate the PD1/PD-L1 mechanism. Moreover, data also show that cardamonin modulated NF-KB1 and NF-KB2 genes involved in activating NF-KB signaling. NF-KB is a transcription factor of inflammation and immunity, emerging as a key positive regulator of PD-L1 expression in cancer. Therefore, cardamonin may have the potential utilization as a checkpoint inhibitor that acts by blocking PD-L1 protein, allowing the immune system to recognize and attack the TNBC cancer cells, providing a novel cancer therapy. (Supported by a grant from NIH-NIMHD U54 MD 007582) Citation Format: Patricia Mendonca, Bhonessa Kirpal, Sukhmandeep Kaur, Karam F. A. Soliman. The effect of cardamonin on PD-L1 expression in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 530.

Extracellular proteins enhance Cupriavidus pauculus nickel tolerance and cell aggregate formation

Heavy metal-resistant bacteria secrete extracellular proteins (e-PNs). However, the role of e-PNs in heavy metal resistance remains elusive. Here Fourier Transform Infrared Spectroscopy implied that N-H, C = O and NH2-R played a crucial role in the adsorption and resistance of Ni2+ in the model organism Cuprividus pauculus 1490 (C. pauculus). Proteinase K treatment reduced Ni2+ resistance of C. pauculus underlining the essential role of e-PNs. Further three-dimension excitation-emission matrix fluorescence spectroscopy analysis demonstrated that tryptophan proteins as part of the e-PNs increased significantly with Ni2+ treatment. Proteomic and quantitative real-time polymerase chain reaction data indicated that major changes were induced in the metabolism of C. pauculus in response to Ni2+. Among those lipopolysaccharide biosynthesis, general secretion pathways, Ni2+-affiliated transporters and multidrug efflux play an essential role in Ni2+ resistance. Altogether the results provide a conceptual model for comprehending how e-PNs contribute to bacterial resistance and adsorption of Ni2+.

In vitro protective effect of recombinant prominin-1 combined with microRNA-29b on N-methyl-D-aspartate-induced excitotoxicity in retinal ganglion cells.

To determine the in vitro protective effect of recombinant prominin-1 (Prominin-1)+microRNA-29b (P1M29) on N-methyl-D-aspartate (NMDA)-induced excitotoxicity in retinal ganglion cells (RGCs). RGC-5 cells were cultured, and NMDA-induced excitotoxicity at the range of 100-800 µmol/L was assessed using the MTT assay. NMDA (800 µmol/L) was selected as the appropriate concentration for preparing the cell model. To evaluate the protective effect of P1M29 on the cell model, Prominin-1 was added at the concentration of 1-6 ng/mL for 48h, and the cell survival was investigated with/without microRNA-29b. After obtaining the appropriate concentration and time of P1M29 at 48h, real-time polymerase chain reaction (PCR) was utilized to detect the relative mRNA expression of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β2. Western blot detection was applied to measure the phosphorylation levels of protein kinase B (AKT) and extracellular regulated protein kinases (ERK) in RGC-5 cells after treatment with Prominin-1. Apoptosis study of the cell model was conducted by flow cytometry for estimating the anti-apoptotic effect of P1M29. Immunofluorescence analysis was used to analyze the expression levels of VEGF and TGF-β2. MTT cytotoxicity assays demonstrated that P1M29 group had significantly higher cell survival rate than Prominin-1 group (P<0.05). Real-time PCR data indicated that the expression levels of VEGF were significantly increased in both Prominin-1 and P1M29 groups compared NMDA and microRNA-29b group (P<0.05), while TGF-β2 were significantly decreased in both microRNA-29b and P1M29 groups compared NMDA and Prominin-1 group (P<0.05). Western blot results showed that both Prominin-1 and P1M29 groups significantly increased the phosphorylation levels of AKT and ERK compared to NMDA and microRNA-29b groups (P<0.05). Flow cytometry analysis revealed that P1M29 could prevent RGC-5 cell apoptosis in the early stage of apoptosis, while immunofluorescence results showed that P1M29 group had higher expression of VEGF and lower expression of TGF-β2 with a stronger green fluorescence than NMDA group. Prominin-1 combined with microRNA-29b can provide a suitable therapeutic option for ameliorating NMDA-induced excitotoxicity in RGC-5 cells.

Screening microRNAs as potential prognostic biomarkers for lung adenocarcinoma

Objective To screen and identify microRNAs (miRNAs) associated with the prognosis of lung adenocarcinoma (LUAD) using clinical samples and construct a prediction model for the prognosis of LUAD. Methods 160 patient samples were used to screen and identify miRNAs associated with the prognosis of LUAD. Differentially expressed miRNAs were analyzed using gene chip technology. The selected miRNAs were validated using samples from the validation sample group. Cox proportional hazards regression was used to construct the model and Kaplan-Meier was used to plot survival curves. Model power was assessed by testing the prognosis of the constructed model using real-time polymerase chain reaction (RT-PCR) data. Results The data showed that miR-1260b, miR-21-3p and miR-92a-3p were highly expressed in the early recurrence and metastasis group, while miR-2467-3p, miR-4659a-3p, miR-4514, miR-1471 and miR-3621 were lowly expressed. It was further confirmed that miR-21-3p was significantly highly expressed in the early recurrence and metastasis group (p = 0.02). Receiver operating characteristic (ROC) curve results showed cut-off point value of 0.0172, sensitivity of 88.2% and specificity of 100%. The predictive results of the constructed model were in good agreement with the actual prognosis of patients by using the validation sample test (Kappa = 0.426, p < 0.001), with a model sensitivity of 74.4%, a specificity of 68.3%, and an accuracy of 71.3%. Conclusion miRNAs associated with the prognosis of patients with stage I LUAD were screened and validated, and a risk model for predicting the prognosis of patients was constructed. This model has good consistency with the actual prognosis of patients.

Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System.

DNA repair pathways trigger robust downstream responses, making it challenging to select suitable reference genes for comparative studies. In this study, our goal was to identify the most suitable housekeeping genes to perform comparable molecular analyses for DNA damage-related studies. Choosing the most applicable reference genes is important in any kind of target gene expression-related quantitative study, since using the housekeeping genes improperly may result in false data interpretation and inaccurate conclusions. We evaluated the expressional changes of eight well-known housekeeping genes (i.e., 18S rRNA, B2M, eEF1α1, GAPDH, GUSB, HPRT1, PPIA, and TBP) following treatment with the DNA-damaging agents that are most frequently used: ultraviolet B (UVB) non-ionizing irradiation, neocarzinostatin (NCS), and actinomycin D (ActD). To reveal the significant changes in the expression of each gene and to determine which appear to be the most acceptable ones for normalization of real-time quantitative polymerase chain reaction (RT-qPCR) data, comparative and statistical algorithms (such as absolute quantification, Wilcoxon Rank Sum Test, and independent samples T-test) were conducted. Our findings clearly demonstrate that the genes commonly employed as reference candidates exhibit substantial expression variability, and therefore, careful consideration must be taken when designing the experimental setup for an accurate and reproducible normalization of RT-qPCR data. We used the U2OS cell line since it is generally accepted and used in the field of DNA repair to study DNA damage-induced cellular responses. Based on our current data in U2OS cells, we suggest using 18S rRNA, eEF1α1, GAPDH, GUSB, and HPRT1 genes for UVB-induced DNA damage-related studies. B2M, HPRT1, and TBP genes are recommended for NCS treatment, while 18S rRNA, B2M, and PPIA genes can be used as suitable internal controls in RT-qPCR experiments for ActD treatment. In summary, this is the first systematic study using a U2OS cell culture system that offers convincing evidence for housekeeping gene selection following treatment with various DNA-damaging agents. Here, we unravel an indispensable issue for performing and assessing trustworthy DNA damage-related differential gene expressional analyses, and we create a "zero set" of potential reference gene candidates.

Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy has shown unprecedented results in patients with B cell relapsed/refractory acute lymphoblastic leukemia (R/R-ALL) and B cell non-Hodgkin lymphomas where no other curative options are available. In vivo monitoring of CAR-T cell kinetics is fundamental to understand the correlation between CAR-T cells expansion and persistence with treatment response and toxicity development. The aim of this study was to define a robust, sensitive, and universal method for CAR-T cell detection using flow cytometry. We set up and compared with each other three assays for CD19 CAR-T cell detection, all based on commercially available reagents. All methods used a recombinant human CD19 protein fragment recognized by the single-chain variable fragment of the CAR construct. The two indirect staining assays (CD19his + APC-conjugated antihistidine antibody and CD19bio + APC-conjugated antibiotin antibody) showed better sensitivity and specificity compared with the direct staining with CD19-FITC, and CD19his had a better cost-effective profile. We validated CAR detection with CD19his with parallel quantitative real-time polymerase chain reaction data and we could demonstrate a strong positive correlation. We also showed that CD19his staining can be easily included in a multicolor flow cytometry panel to achieve additional information about the cell phenotype of CAR-T cell positive subpopulations. Finally, this method can be used for different anti-CD19 CAR-T cell products and for different sample sources. These data demonstrate that detection of CAR-T cells by CD19his flow cytometry staining is a reliable, robust, and broadly applicable tool for in vivo monitoring of CAR-T cells.

Key Molecules in Bladder Cancer Affect Patient Prognosis and Immunotherapy Efficacy: Further Exploration for CNTN1 and EMP1.

Immunotherapy has been widely used in bladder cancer (BCa) in recent years and has significantly improved the prognosis of patients with BCa. However, further identification of immunotherapy-sensitive individuals to improve the efficacy of immunotherapy remains an important unmet need. The key genes were screened and identified from Gene Expression Omnibus database and The Cancer Genome Atlas database to construct the risk prediction function (risk scores). Real-time polymerase chain reaction, immunohistochemistry, and IMvigor210 data sets were used to verify the roles of key molecules and efficacy of risk scores. The biologic function of CNTN1 and EMP1 was further explored through cell proliferation experiments. Five key genes, CNTN1, MAP1A, EMP1, MFAP5, and PTGIS, which were significantly related to the prognosis and immune checkpoint molecules of patients, were screened out. CNTN1 and EMP1 were further experimentally confirmed for their significant tumor-promoting effects. Besides, the constructed risk scores on the basis of these five key genes can accurately predict the prognosis and immunotherapy efficacy of patients with BCa. Interestingly, the high-risk patients identified by the risk scores have significantly worse prognosis and immunotherapy effects than low-risk patients. The key genes we screened can affect the prognosis of BCa, tumor microenvironment immune infiltration, and the efficacy of immunotherapy. The risk scores tool we constructed will contribute to the development of individualized treatment for BCa.

Relative and Quantitative Characterization of the Bovine Bacterial Ocular Surface Microbiome in the Context of Suspected Ocular Squamous Cell Carcinoma.

The ocular surface microbiome is altered in certain disease states. The aim of this study was to characterize the bovine bacterial ocular surface microbiome (BBOSM) in the context of ocular squamous cell carcinoma (OSCC). The conjunctiva of normal (n = 28) and OSCC (n = 10) eyes of cows aged 2 to 13 years from two farms in Louisiana and Wyoming were sampled using individual sterile swabs. DNA extraction followed by 16S ribosomal ribonucleic acid (rRNA) gene sequencing and real-time polymerase chain reaction (RT-PCR) were performed to, respectively, assess the relative and absolute BBOSM. Discriminant analysis (DA) was performed using RT-PCR data, and relative abundance analysis was performed using 16S rRNA gene sequencing data. The 11 most abundant phyla in both normal and OSCC-affected cows were identified using 16S rRNA gene sequencing analysis. The relative abundance of Euryarchaeota was found to be significantly lower (p = 0.0372) in OSCC eyes compared to normal eyes. Relative abundance differences within and between geographic locations were also identified. Quadratic DA categorized samples as OSCC or normal with 100% sensitivity and 83.3-100% specificity. Relative abundance analysis identified relative BBOSM phylum alterations in OSCC. Quadratic DA can be used to accurately categorize BBOSM from normal and OSCC ocular surface samples.

Vitamin E Enhances Cell Viability and the Osteogenic Differentiation of Cell Spheroids Made of Gingiva-Derived Stem Cells.

Background and Objectives: Vitamin E is reported to expedite new bone formation in animal models, and this has led to a decrease in the time needed for treatment. In this study, human gingiva-derived stem cell-derived spheroids were examined to determine the effects of vitamin E on cell survival, osteogenic differentiation, and mineralization. Materials and Methods: Human gingiva-derived stem cells were used to create spheroids, which were then cultivated with vitamin E at doses of 0, 0.1, 1, 10, and 100 ng/mL. The morphological examination and the qualitative and quantitative vitality of the cells were assessed. Alizarin Red S staining and alkaline phosphatase activity assays were performed on days 7 and 14 to evaluate the osteogenic differentiation. The expression levels of RUNX2 and COL1A1 were assessed using a real-time polymerase chain reaction. Results: The addition of vitamin E did not appear to alter the spheroid's shape at the measured quantities without altering the diameter. During the culture time, the majority of the cells in the spheroids fluoresced green. Regardless of concentration, there were substantial increases in cell viability in the vitamin E-loaded groups on day 7 (p < 0.05). On day 14, the Alizarin Red S staining was statistically higher in the 1 ng/mL group compared to the unloaded control (p < 0.05). The addition of vitamin E to the culture enhanced the mRNA expression levels of RUNX2, OCN, and COL1A1 based on the real-time polymerase chain reaction data. Conclusions: We draw the conclusion that vitamin E may be used to promote the osteogenic differentiation of stem cell spheroids in light of these data.

Expression of microRNA-590 in patients with chronic coronary heart disease, atrial fibrillation, and their combination

Background. The role of microRNAs in the processes of remodeling, proliferation, and fibrogenesis of myocardial cells is not clear enough.&#x0D; Aim. Analysis of the microRNA-590 expression level in patients with chronic coronary heart disease, atrial fibrillation, as well as their combination to assess the potential prognostic significance of the expression level.&#x0D; Material and methods. The study included 94 patients divided into three clinical groups: the first with non-valvular atrial fibrillation without coronary heart disease (39 people); the second with non-valvular atrial fibrillation and coronary heart disease (22 patients); the third with ischemic heart disease without atrial fibrillation (23 patients). The comparison group consisted of 10 people without atrial fibrillation and coronary heart disease. Venous blood was taken from all subjects, from the plasma of which microRNA was isolated. The relative level of microRNA expression was estimated based on real-time polymerase chain reaction data obtained during the reaction on a cycler using commercial TaqMan probes and primers. The statistical significance of differences between groups was determined using one-way analysis of variance, followed by post-hoc analysis using Tukey's contrasts, differences were considered statistically significant at p 0.05. The ShapiroWilk test was used to assess the normality of the distribution of residuals.&#x0D; Results. A statistically significant decrease in the expression level of microRNA-590 was registered in the third (p=0.0104) and in the second (p=0.0046) groups compared with the control group, as well as in patients with atrial fibrillation and left atrial dilatation (p=0.0313), with recurrent arrhythmias after radiofrequency ablation (p=0.0083) and with permanent atrial fibrillation (p=0.0242).&#x0D; Conclusion. Ischemic heart disease, including when combined with atrial fibrillation, and aggravating factors, such as left atrial dilatation, permanent atrial fibrillation, recurrence of arrhythmia after radiofrequency ablation, lead to a decrease in the expression level of microRNA-590.

Selection and validation of reference genes for quantitative real-time polymerase chain reaction analyses of Serratia ureilytica DW2

Background: Serratia ureilytica DW2 is a highly efficient phosphate-solubilizing bacteria isolated from Codonopsis pilosula rhizosphere soil that can promote the growth of C. pilosula; nonetheless, until now, no validated reference genes from the genus Serratia have been reported that can be used for the normalization of quantitative real-time polymerase chain reaction (RT–qPCR) data. Methods: To screen stable reference genes of S. ureilytica DW2, the expression of its eight candidate reference genes (16S rRNA, ftsZ, ftsA, mreB, recA, slyD, thiC, and zipA) under different treatment conditions (pH, temperature, culture time, and salt content) was assayed by RT–qPCR. The expression stability of these genes was analyzed using different algorithms (geNorm, NormFinder, and BestKeeper). To verify the reliability of the data, the expression of the glucose dehydrogenase (gdh) gene under different soluble phosphate levels was quantified using the most stably expressed reference gene. Results: The results showed that the zipA and 16S rRNA genes were the most stable reference genes, and the least stable genes were thiC and recA. The expression of gdh was consistent with the phosphate solubilization ability on plates containing the National Botanical Research Institute phosphate growth medium. Conclusion: Therefore, this study provides a stable and reliable reference gene of Serratia for the accurate quantification of functional gene expression in future studies.

Effects of MRI on stemness properties of Wharton's jelly-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs), derived from various tissues, are served as a promising source of cells in clinic and regenerative medicine. Umbilical cord-Wharton's jelly (WJ-MSCs)-derived MSCs exhibit advantages over those from adult tissues, such as no ethical concerns, shorter population doubling time, broad differentiation potential, readily available non-invasive source, prolonged maintenance of stemness properties. The aim of this study was to evaluate the effect of MRI (1.5T, 10min) on stemness gene expression patterns (OCT-4, SOX-2, NANOG) of WJ-MSCs. Additionally, we assessed cell viability, growth kinetics and apoptosis of WJ-MSCs after MRI treatment. The quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) data showed that transcript levels of SOX-2, NANOG in MRI-treated WJ-MSCs were increased 32- and 213-fold, respectively. MTT assay was performed at 24, 48, and 72h post-treatment and the viability was not significantly different between the two groups. The doubling time of the MRI group was markedly higher than the control group. In addition, the colony formation ability of WJ-MSCs after MRI treatment significantly increased. Furthermore, no change in apoptosis was seen before or after MRI treatment. Our results suggest that the use of MRI can improve the quality of MSCs and enhance the efficacy of mesenchymal stem cell-based therapies.

Genome-wide identification and expression analysis of SBP-box gene family reveal their involvement in hormone response and abiotic stresses in Chrysanthemum nankingense.

SQUAMOSA promoter-binding-protein (SBP)-box family proteins are a class of plant-specific transcription factors, and widely regulate the development of floral and leaf morphology in plant growth and involve in environment and hormone signal response. In this study, we isolated and identified 21 non-redundant SBP-box genes in Chrysanthemum nankingense with bioinformatics analysis. Sequence alignments of 21 CnSBP proteins discovered a highly conserved SBP domain including two zinc finger-like structures and a nuclear localization signal region. According to the amino acid sequence alignments, 67 SBP-box genes from Arabidopsis thaliana, rice, Artemisia annua and C. nankingense were clustered into eight groups, and the motif and gene structure analysis also sustained this classification. The gene evolution analysis indicated the CnSBP genes experienced a duplication event about 10 million years ago (Mya), and the CnSBP and AtSPL genes occurred a divergence at 24 Mya. Transcriptome data provided valuable information for tissue-specific expression profiles of the CnSBPs, which highly expressed in floral tissues and differentially expressed in leaf, root and stem organs. Quantitative Real-time Polymerase Chain Reaction data showed expression patterns of the CnSBPs under exogenous hormone and abiotic stress treatments, separately abscisic acid, salicylic acid, gibberellin A3, methyl jasmonate and ethylene spraying as well as salt and drought stresses, indicating that the candidate CnSBP genes showed differentiated spatiotemporal expression patterns in response to hormone and abiotic stresses. Our study provides a systematic genome-wide analysis of the SBP-box gene family in C. nankingense. In general, it provides a fundamental theoretical basis that SBP-box genes may regulate the resistance of stress physiology in chrysanthemum via exogenous hormone pathways.

The Treatment of Keloid Scars via Modulating Heterogeneous Gelatin-Structured Composite Microneedles to Control Transdermal Dual-Drug Release.

Keloid scarring is an abnormal scar disease characterised by excessive proliferation of fibroblasts and over-deposition of collagen during wound healing. Although various treatments for keloid scars have been developed, preventive medicine is believed to be a promising strategy. The skin barrier limits the gentle topical administration of medicaments such as creams and hydrogel dressings, resulting in reduced therapeutic efficacy. In recent years, microneedles (MNs) have been regarded as an appreciable device for topical administration without inducing side effects, and they are painless and do not cause bleeding. In this study, an MN patch with controlled transdermal dual-drug release was developed to achieve combinatory treatment of keloid scars using a heterogeneous gelatin-structured composite MN. Gelatin hydrogel was used as a substrate to load gallic acid (GA) and quercetin-loaded amphiphilic gelatin nanoparticles to fabricate dual-drug heterogeneous composite MNs. The results of the insertion test and mechanical properties of the MNs showed that the heterogeneous composite MN patches could be self-pressed into the stratum corneum and control dual-drug release at different time periods. GA was released at an earlier stage to retard the proliferation of fibroblasts, and quercetin was released at a later stage as a strong antioxidant to erase the generation of reactive oxygen species. Furthermore, real-time quantitative polymerase chain reaction data indicated that the gene expression of fibroblasts (such as Col I and III) was downregulated in the dual-drug system. The above results demonstrate that using heterogeneous composite MNs with the combination of dual-drug pharmacology is beneficial for preventing keloid scar formation.