Detection Of mRNA Research Articles

A New Link to Primate Heart Development.

Long non-coding RNAs (lncRNAs) are important regulators of development. In this issue of Developmental Cell, Wilson etal. utilize pluripotent stem cell models to demonstrate that a primate lncRNA, BANCR, is primarily expressed in fetal cardiomyocytes and promotes cell migration.

Identification of Clinically Significant Prostate Cancer by Combined PCA3 and AMACR mRNA Detection in Urine Samples.

PurposePreclinical evaluation of PCA3 and AMACR transcript simultaneous detection in urine to diagnose clinical significant prostate cancer (prostate cancer with Gleason score ≥7) in a Russian cohort.Patients and MethodsWe analyzed urine samples of patients with a total serum PSA ≥2 ng/mL: 31 men with prostate cancer scheduled for radical prostatectomy, 128 men scheduled for first diagnostic biopsy (prebiopsy cohort). PCA3, AMACR, PSA and GPI transcripts were detected by multiplex reverse transcription quantitative polymerase chain reaction, and the results were used for scores for calculation and statistical analysis.ResultsThere was no significant difference between clinically significant and nonsignificant prostate cancer PCA3 scores. However, there was a significant difference in the AMACR score (patients scheduled for radical prostatectomy p=0.0088, prebiopsy cohort p=0.029). We estimated AUCs, optimal cutoffs, sensitivities and specificities for PCa and csPCa detection in the prebiopsy cohort by tPSA, PCA3 score, PCPT Risk Calculator and classification models based on tPSA, PCA3 score and AMACR score. In the clinically significant prostate cancer ROC analysis, the PCA3 score AUC was 0.632 (95%CI: 0.511–0.752), the AMACR score AUC was 0.711 (95%CI: 0.617–0.806) and AUC of classification model based on the PCA3 score, the AMACR score and total PSA was 0.72 (95%CI: 0.58–0.83). In addition, the correlation of the AMACR score with the ratio of total RNA and RNA of prostate cells in urine was shown (tau=0.347, p=6.542e–09). Significant amounts of nonprostate RNA in urine may be a limitation for the AMACR score use.ConclusionThe AMACR score is a good predictor of clinically significant prostate cancer. Significant amounts of nonprostate RNA in urine may be a limitation for the AMACR score use. Evaluation of the AMACR score and classification models based on it for clinically significant prostate cancer detection with larger samples and a follow-up analysis is promising.

Development of cell-free platform-based toehold switch system for detection of IP-10 mRNA, an indicator for acute kidney allograft rejection diagnosis

Quantitative PCR and droplet digital PCR were elucidated as non-invasive methods for quantifying the level of signaling markers, such as CD3ɛ and IP-10 mRNAs from urine samples, for the diagnosis of acute rejection in the kidney allograft recipients. Although the sensitivity and accuracy make PCR as the gold standard for diagnosis, a point-of-care (POC) testing is required for the rapid and low-cost preliminary prognosis and diagnosis. In this study, the applicability of the cell-free platform-based toehold switch system was preliminary demonstrated for the detection of synthetic IP-10 mRNA, one of indicators of acute kidney allograft rejection. For POC applications, the colorimetric output was utilized for direct recognition by naked eyes. A total of 5 switches was screened from 289 putative toehold switches. Among these, the toehold switch 4 illustrated the highest fold change after a 45-min incubation with relatively high specificity. The sensitivity of the toehold switch 4 was also demonstrated with the cognate IP-10 mRNA. The results in this study showed the feasibility of the synthetic system of RNA toehold switches in combination with the cell-free platform as a preliminary prognostic and diagnostic method for acute kidney allograft rejection.

Enzyme-free electrochemical biosensor based on amplification of proximity-dependent surface hybridization chain reaction for ultrasensitive mRNA detection

The ability to recognize mRNA with high efficiency in cells would greatly facilitate the elucidation of mRNA-mediated cellular cascades and their disease associations. However, most traditional electrochemical strategies targeting nucleotides are always confronted with cumbersome interface operation and washing procedures, as well as the high cost of labeling and the strict reaction conditions of tool enzymes, limiting their potential applications. To address these issues, herein we reported, for the first time, a simple label-free, isothermal, non-enzymatic, and ultrasensitive homogeneous electrochemical biosensor based on autonomous proximity-dependent surface hybridization chain reaction (HCR), for sensitive signal amplification and highly specific detection of target survivin mRNA with a detection limit of 3 fM. The target triggers hybridization chain reaction and mRNA-fueled surface hybridization of ferrocene-tagged metastable DNA hairpin probes on proximity-dependent surface hybridization, resulting in the formation of multiple long-range duplex DNA chains which are immobilized onto the gold electrodes with a substantially stable ferrocene-mediated redox current. Thus, a significant electrochemical signal increase is observed dependent on the concentration of the target RNA, with a very low detection limit. Mo-reover, this molecular biosensor also exhibits excellent specificity to distinguish even single base mismatched, with strong reliability. The developed biosensor provides a novel promising tool for ultra-sensitive and selective detection, and it has great potential to be applied in mRNA-related biochemical research and clinical cancer diagnostics in more detail.

BUB1B Promotes Proliferation of Prostate Cancer via Transcriptional Regulation of MELK.

Prostate cancer remains one of the most common and deadliest forms of cancer, generally respond well to radical prostatectomy and associated interventions, up to 30% of individuals will suffer disease relapse. Although BUB1B was found to be essential for cell growth and proliferation, even in several kinds of tumor cells, the specific importance and mechanistic role of BUB1B in prostate cancer remain unclear. Quantitative Real-Time PCR and Western-blot were used in the detection of mRNA and protein expression. Lentivirus infection was used to overexpression or knock down the target gene. Flow cytometry analysis was performed to test protein expression and apoptosis level. Immunohistochemistry was used to identify protein expression in tissue. Statistical differences between the two groups are evaluated by two-tailed t-tests. The comparison among multiple groups is performed by one-way Analysis of Variance (ANOVA) followed by Dunnett's posttest. The statistical significance of the Kaplan-Meier survival plot is determined by log-rank analysis. In the present report, we found BUB1B expression to be highly increased in prostate cancer tissues relative to normal controls. We further found BUB1B to be essential for efficient tumor cell proliferation, and to correlate with poorer prostate cancer patient outcomes. From a mechanistic perspective, the ability of BUB1B to regulate MELK was found to be essential for its ability to promote prostate cancer cell proliferation. Altogether, our data suggest that BUB1B is up-regulated in prostate cancer, suggesting that the growth of cancer cells may depend on BUB1B-dependent regulation of MELK transcription. BUB1B may serve as a clinical prognostic factor and a druggable target for prostate cancer.

Northern Blotting Technique for Detection and Expression Analysis of mRNAs and Small RNAs.

Northern analysis is a conventional but gold standard method for detection and quantification of gene expression changes. It not only detects the presence of a transcript but also indicates size and relative comparison of transcript abundance on a single membrane. In recent years it has been aptly adapted to validate and study the size and expression of small noncoding RNAs. Here, we describe protocols employed in our laboratory for conventional northern analysis with total RNA/mRNA to study gene expression and validation of small noncoding RNAs using low molecular weight fraction of RNAs. A brief account on the recent advancements for improving the sensitivity and efficiency of northern blot detection is also included in this chapter.

Selection and validation of reference genes for qPCR analysis of miRNAs and their targets during somatic embryogenesis in tamarillo (Solanum betaceum Cav.)

Quantitative reverse transcriptase PCR is a powerful tool for precise gene expression quantification. Using suitable reference genes is crucial for a proper normalization and interpretation of results, particularly in dynamic transcriptional networks with the involvement of several miRNAs and their targets, such as somatic embryogenesis (SE). Here, we aimed to identify genes to be used as internal controls for quantitative detection of miRNAs and mRNAs during the SE process in Solanum betaceum. The expression of EF1α, GAPDH, ACT, UBQ10, Fe-SOD, U6, snoR14, miR159a, miR162 and miR166a was tested in nine time-points of the SE process, particularly during induction, embryo development and conversion. Analyses of transcriptional stability were performed using RefFinder tool that integrates geNorm, NormFinder, BestKeeper and Delta-Ct algorithms. Among these candidates, Fe-SOD, ACT and U6 emerged consistently as the most suitable to normalize gene expression, whereas miR166a was the most appropriate for normalization in calli samples. In order to validate these results, expression of miR396 and GRF1 was quantified and normalized with the most and the least stable genes and also with the most stable miRNA. Fe-SOD + ACT revealed to be the optimal combination for precise quantification of miRNA-mediated regulation of gene expression during SE in tamarillo. Fe-SOD + ACT is the most suitable combination of reference genes for RT-qPCR normalization and accurate quantification of miRNAs and their targets during tamarillo somatic embryogenesis induction, embryo development and conversion stages.

Abstract 760: miRNA and mRNA detection in plasma-derived extracellular vesicles (EVs) using the nCounter NanoString platform

Abstract Introduction: Although genetic and transcriptomic analysis of tumor tissue can provide useful information for prognosis and treatment decision making, 5-20% of advanced-stage lung cancer patients cannot be biopsied, or the amount of tumor tissue is insufficient for successful analysis. In addition, repeated sampling is often not possible. Liquid biopsies have shown potential to be used as minimally invasive, safe and sensitive alternative for tissue biopsies, but lack of standardized protocols is hampering implementation in the clinic. The nCounter platform could provide the solution for this problem, with an easy-to-use technical workflow and straightforward data analysis. Extracellular vesicles (EVs) are mediators of intercellular communication and may play a role in early cancer development. Therefore, RNA found within EVs can be used as a biomarker for cancer development and progression. In addition, the lipid bilayer of EVs makes their cargo particularly stable and allows the use of biobank stored samples. Methods: EVs were isolated from 600 μL plasma of 19 cancer patients and 10 healthy donors, using the miRCURY® Exosome Serum/Plasma Kit (Qiagen), and total RNA was extracted using TRI Reagent® (MRC Inc) or the automated QIAsymphony® System (Qiagen) with the DSP Virus/Pathogen Kit, after RNAse A (Sigma-Aldrich) treatment to remove plasma cell-free RNA. The Human Immune V2 panel (NanoString Technologies), including 600 mRNA targets, was used to analyze EV-derived mRNA after a pre-amplification (pre-amp) step with the Low RNA Input Amplification Kit. In addition, the Human V3 miRNA panel (NanoString Technologies), including 800 miRNA targets, was used to analyze the same EV samples without pre-amp. Results: Total amount of RNA isolated from EVs was found to be significantly higher using TRI Reagent®, versus automated RNA isolation. In addition, the conditions for the pre-amp step were tested and optimized. A pre-amp of 10 cycles for the mRNA panel was shown to be sufficient to detect mRNA targets in EVs without saturation, and the NanoString retrotranscription (RT) enzyme outperformed the other RT enzyme tested. In addition, supernatant collected during EV isolation was also analyzed, and results showed that the RNA targets were derived from within the EVs. On average, 337 mRNA targets were detected within the EVs, while 157 miRNA targets were detected in the same samples without pre-amp, with no significant differences between cancer patients and healthy donors. Interestingly, most differentially expressed (DE) mRNAs were shown to be lower expressed in cancer patients, while most DE miRNAs were found to be higher expressed in cancer patients. Conclusion: Our results demonstrate that the nCounter NanoString platform can be used for miRNA and mRNA detection in plasma-derived EVs from cancer patients and healthy donors. Further studies will focus on specific mRNA and miRNA expression differences between these two cohorts. Citation Format: Jillian Wilhelmina Bracht, Ana Gimenez-Capitan, Chung-Ying Huang, Carlos Pedraz-Valdunciel, Joselyn Valarezo, Sarah Warren, Rafael Rosell, Miguel Angel Molina-Vila. miRNA and mRNA detection in plasma-derived extracellular vesicles (EVs) using the nCounter NanoString platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 760.

Abstract 5237: High-throughput slide preparation for spatially-resolved, multiplexed quantification of protein or mRNA in tumor tissues with automation of GeoMx™ DSP Assays on Leica Biosystems BOND RX

Abstract Characterization of the spatial distribution and abundance of proteins and mRNAs with morphological context within tissues enables a better understanding of biological systems in many research areas, including immunology and oncology. The GeoMxTM Digital Spatial Profiler (DSP) enables the highly-multiplexed detection of mRNA or protein targets within distinct regions of tissues, including FFPE. This assay utilizes in situ hybridization (ISH) probes or antibodies linked to indexing oligo barcodes via a photo-cleavable linker. The GeoMx DSP enables profiling of RNA or protein targets by releasing and collecting these barcodes from user-defined regions-of-interest. Manual preparation of FFPE tissue uses conventional immuno-fluorescence (IF) and ISH reagents, however it requires greater than two hours of hands-on-time over 2 days. Protocols have been developed to enable the automation of these tissue preparation protocols using the Leica Biosystems BOND RX and BOND RXm Fully Automated Research Stainers. Here we describe protocols for the semi-automated preparation of GeoMx RNA and protein assays as well as the fully-automated preparation of GeoMx RNA assays. These protocols reduce the hands-on time of performing the assay to 72 minutes and 35 minutes for the semi- and fully-automated protocols, respectively. Signal-to-noise ratios (SNR) are highly concordant between serial sections processed manually and processed on the Leica BOND RXm using the GeoMx Immune Cell Profiling Panel for protein (58-plex) and GeoMx Immune Pathways Panel for RNA (84-plex). Furthermore, automated assays show high concordance between the BOND RX and BOND RXm instruments. Lastly, we demonstrate the use of the fully automated slide preparation protocol with the GeoMx Cancer Transcriptome Atlas, a panel that targets >1,600 key cancer mRNAs that are read out by next-generation sequencing. GeoMx Assays are for RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Daniel R. Zollinger, Sarah Ketting, Kristina Sorg, Oliver Appelbe, Danielle Aaker, Damian Cockfield, Chris Merritt, Joseph M. Beechem. High-throughput slide preparation for spatially-resolved, multiplexed quantification of protein or mRNA in tumor tissues with automation of GeoMx™ DSP Assays on Leica Biosystems BOND RX [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5237.

Urinary exosomal mRNA detection using novel isothermal gene amplification method based on three-way junction

Exosomal messenger RNA (mRNA) has emerged as a valuable biomarker for liquid biopsy-based disease diagnosis and prognosis due to its stability in body fluids and its biological regulatory function. Here, we report a rapid one-step isothermal gene amplification reaction based on three-way junction (3WJ) formation and the successful detection of urinary exosomal mRNA from tumor-bearing mice. The 3WJ structure can be formed by the association of 3WJ probes (3WJ-template and 3WJ-primer) in the presence of target RNA. After 3WJ structure formation, the 3WJ primer is repeatedly extended and cleaved by a combination of DNA polymerase and nicking endonuclease, producing multiple signal primers. Subsequently, the signal primers promote a specially designed network reaction pathway to produce G-quadruplex probes under isothermal conditions. Finally, G-quadruplex structure produces highly enhanced fluorescence signal upon binding to thioflavin T. This method provides a detection limit of 1.23 pM (24.6 amol) with high selectivity for the target RNA. More importantly, this method can be useful for the sensing of various kinds of mRNA, including breast cancer cellular mRNA, breast cancer exosomal mRNA, and even urinary exosomal mRNA from breast cancer mice. We anticipate that the developed RNA detection assay can be used for various biomedical applications, such as disease diagnosis, prognosis, and treatment monitoring.

P16/ki67 and E6/E7 mRNA Accuracy and Prognostic Value in Triaging HPV DNA-Positive Women.

The study presents cross-sectional accuracy of E6 and E7 (E6/E7) mRNA detection and p16/ki67 dual staining, alone or in combination with cytology and human papillomavirus (HPV)16/18 genotyping, as a triage test in HPV DNA-positive women and their impact on cervical intraepithelial neoplasia (CIN2+) overdiagnosis. Women aged 25-64 years were recruited. HPV DNA-positive women were triaged with cytology and tested for E6/E7 mRNA and p16/ki67. Cytology positive women were referred to colposcopy, and negatives were randomly assigned to immediate colposcopy or to 1-year HPV retesting. Lesions found within 24 months since recruitment were included. All P values were 2-sided. 40 509 women were recruited, and 3147 (7.8%) tested HPV DNA positive; 174 CIN2+ were found: sensitivity was 61.0% (95% confidence interval [CI] = 53.6 to 68.0), 94.4% (95% CI = 89.1 to 97.3), and 75.2% (95% CI = 68.1 to 81.6) for cytology, E6/E7 mRNA, and p16/ki67, respectively. Immediate referral was 25.6%, 66.8%, and 28.3%, respectively. Overall referral was 65.3%, 78.3%, and 63.3%, respectively. Cytology or p16/ki67, when combined with HPV16/18 typing, reached higher sensitivity with a small impact on referral. Among the 2306 HPV DNA-positive and cytology-negative women, relative CIN2+ detection in those randomly assigned at 1-year retesting vs immediate colposcopy suggests a -28% CIN2+ regression (95% CI = -57% to +20%); regression was higher in E6/E7 mRNA-negatives (Pinteraction = .29). HPV clearance at 1 year in E6/E7 mRNA and in p16/ki67 negative women was about 2 times higher than in positive women (Pinteraction < .001 for both). p16/ki67 showed good performance as a triage test. E6/E7 mRNA showed the highest sensitivity, at the price of too high a positivity rate to be efficient for triage. However, when negative, it showed a good prognostic value for clearance and CIN2+ regression.

Study on the effect of superparamagnetic γ-Fe2O3 combined with carmustine on cervical cancer

This study aims to investigate the killing effect of γ-Fe2O3 combined with carmustine on cervical carcinoma cells, as well as the mechanism of drug resistance of this combined treatment. RT-PCR was used to detect the expression of Multidrug Resistance Gene (MDR-l) gene in all the samples. Results showed that the combination of γ-Fe2O3 and carmustine drugs was significantly better than the control group (P &lt; 0.05), and the tumor showed different degrees of tolerance in combination with the drug. The results of an in vitro drug sensitivity test were consistent with those of mRNA expression of MDR-l gene (P &lt; 0.04). The combination of superparamagnetic γ-Fe2O3 and carmustine derivatives has a better therapeutic effect on cervical carcinoma than either treatment alone. The drug sensitivity test and MDR-l mRNA detection in vitro can improve the accuracy and predictability of cervical carcinoma chemotherapy, providing a scientific basis for individual tumor chemotherapy.

FOXM1-Dependent Transcriptional Regulation of EZH2 Induces Proliferation and Progression in Prostate Cancer.

Prostate cancer is one of the most commonly diagnosed cancers and one of the most common causes of cancer-related deaths among men worldwide. Patients who are diagnosed with localized prostate cancer and treated with radical prostatectomy often respond well to therapy. The current standard therapy for prostate cancer involves maximal surgical resection, followed by radiotherapy and chemotherapy. Clarifying the molecular mechanism of tumor proliferation and recurrence becomes more and more important for clinical therapies of prostate cancer. Quantitative Real-Time PCR and Western-blot were used in the detection of mRNA and protein expression. Lentivirus infection was used to overexpress or knockdown the target gene. Flow cytometry analysis was performed to test protein expression and apoptosis level. Immunohistochemistry was used to identify protein expression in tissue. Statistical differences between the two groups are evaluated by two-tailed t-tests. The comparison among multiple groups is performed by one-way Analysis of Variance (ANOVA) followed by Dunnett's posttest. The statistical significance of the Kaplan-Meier survival plot is determined by log-rank analysis. In this study, we identified that FOXM1 expression was significantly enriched in prostate cancer compared with normal tissue. Additionally, FOXM1 was functionally required for tumor proliferation and its expression was associated with poor prognosis in prostate cancer patients. Mechanically, FOXM1-dependent regulation of EZH2 is essential for proliferation and progression in prostate cancer. Taken together, our data suggest that oncogenic transcription factor FoxM1 is up-regulated in prostate cancer, suggesting that the growth of cancer cells may depend on FOXM1 activity. FOXM1 may serve as a clinical prognostic factor and a therapeutic target for prostate cancer.

COMPARATIVE ANALYSIS OF qPCR MEASUREMENT OF HIV VIRAL LOAD AND ELISA DETECTION OF p24 ANTIGEN AFTER HYPERBARIC OXYGEN EXPOSURE.

Background:A decrease in the number of viruses or viral nucleic acid components will determine whether a therapy successfully eradicates the virus. Sensitivity and specificity are needed to enable easy, precise and efficient diagnosis and evaluation of therapy. This study examined the sensitivity of quantitative PCR (qPCR) for detecting viral nucleic acids as compared with enzyme-linked immunosorbent assay (ELISA) for detecting the human immunodeficiency virus 1 (HIV-1) p24 antigen after hyperbaric oxygen therapy.Materials and Methods:In this study, peripheral blood mononuclear cells (PBMCs) from healthy whole blood and inoculated HIV-1/MT4 virus in PBMC cultures were isolated. The cultures were exposed to hyperbaric oxygen at 2.4 ATA with 100% O2 for 3 × 30 minutes for 5 days. ELISA and qPCR were used to measure the p24 antigen and HIV-1 mRNA, respectively, in the treatment and control groups.Result:The amounts of p24 antigen and HIV-1 mRNA were significantly different (p = 0.001, p < α). The two examination methods were significantly different. Hyperbaric oxygen therapy can reduce virus numbers, as observed from the p24 antigen and HIV-1 mRNA levels. The treatment group had significantly lower virus numbers than the control group. HIV-1 mRNA detection is more sensitive than p24 antigen detection.Conclusion:Both qPCR and ELISA have their advantages, depending on whether the goal is to establish, diagnose or monitor antiretroviral therapy or to evaluate disease progression.

An Effective Method for Quantifying RNA Expression of IbsC-SibC, a Type I Toxin-Antitoxin System in Escherichia coli.

Toxin and antitoxin (TA) systems are small genetic modules consisting of a toxin protein and an RNA or protein antitoxin. It is difficult to study their functions in a large part due to the lack of effective methods to study toxin RNAs, which usually exist at exceptionally low levels. Herein, we describe a sensitive reverse transcription quantitative PCR (RT-qPCR) method that is able to quantitate such RNA species. The method was directed at detection of the toxin mRNA of the ibsC-sibC TA pair, and its high specificity was validated by sequencing. The approach was used to determine relative expression of the IbsC and SibC RNAs at different cell-growth phases; this revealed an expression pattern that cannot be explained by the prevailing notion of growth stasis by the toxin and rescue by the antitoxin. The usefulness of the method was further showcased by the determination of average cellular copy numbers of the IbsC-SibC RNAs in wild-type E. coli cells and RNA abundance in E. coli cells engineered with extra copies of the ibsC-sibC genes. With a robust method to quantitate cellular small RNAs at very low concentrations, we are now equipped to study the expression of TA systems under different conditions to gain useful insights about their functions.

Early posttherapy clearance of human papillomavirus and treatment response in cervical carcinoma.

Among patients with cervical cancer, little is known about the significance of persistent human papillomavirus (HPV) expression after chemoradiation (CRT). This study evaluated associations between early posttreatment HPV clearance and patient outcomes with an added focus on the value of posttherapy positron emission tomography (PET) imaging. Included patients underwent pretreatment testing indicating a high-risk HPV infection and posttreatment testing with a messenger RNA (mRNA)-based genital swab after CRT. Posttherapy responses were stratified on the basis of HPV mRNA detection into an early clearance (EC) group (no mRNA) and a persistent expression (PE) group (detectable mRNA) on the basis of an evaluation at a median of 6weeks after therapy. The Kaplan-Meier method was used to compare outcomes, and multivariable analysis was used to identify predictors of outcomes. Seventy-two of the 97 eligible patients (74.2%) had EC. The mean follow-up time was 25months (range, 4-56months), and 2-year pelvic control (76.9% vs 50.2%; P=.01) and overall survival (OS; 80.9% vs 52.2%; P<.01) were superior among EC patients. In the multivariable analysis, EC predicted for improved survival (hazard ratio [HR] for mortality, 0.46; 95% confidence interval [CI], 0.21-0.96; P=.047), as did a complete response (CR) on posttherapy PET (HR for less than a CR on PET, 6.17; 95% CI, 2.58-14.72; P<.01). In a subset analysis of patients with a posttherapy PET CR, HPV clearance retained prognostic significance (2-year OS, 95.6% with EC vs 66.7% with PE; P=.04), whereas PE patients without a PET CR had the worst survival (35.9%; P<.01 for trend). Early posttherapy clearance of HPV is associated with improved survival in cervical cancer. Evaluating HPV expression at this 6-week time point provides prognostic information beyond posttherapy PET imaging and may aid in risk stratification and decisions for treatment escalation.

Enhancement of a biotechnological platform for the purification and delivery of a human papillomavirus supercoiled plasmid DNA vaccine

New biotechnological strategies are being explored, aimed at rapid and economic manufacture of large quantities of DNA vaccines with the required purity for therapeutic applications, as well as their correct delivery as biopharmaceuticals to target cells. This report describes the purification of supercoiled (sc) HPV-16 E6/E7 plasmid DNA (pDNA) vaccine from a bacterial lysate, using an arginine-based monolith, presenting a spacer arm in its configuration. To enhance the performance of the purification process, monolith modification with the spacer arm can improve accessibility of the arginine ligand. By using a low NaCl concentration at pH 7.0, a condition to eliminate the RNA impurity directly in the flow through was established. The pH increase to 7.5 allowed the elimination of non-functional pDNA isoforms, the sc pDNA being recovered by increasing the ionic strength. As well as a binding capacity of 2.53 mg/mL obtained with a pre-purified sc pDNA sample, the column also purified sc pDNA from high lysate loading, with capacities above 1 mg/mL. Due to the sample displacement phenomena, non-functional pDNA isoforms were eliminated throughout column loading, favoring the degree of purity of final sc pDNA of 93.3%–98.5%. Thereafter, purified sc pDNA was successfully encapsulated into CaCO3-gelatin nano-complexes. Delivery of the pDNA-carriers to THP-1 cells was assessed through pDNA cellular uptake evaluation and correct E6 expression was verified by mRNA and protein detection. A biotechnological platform was established for sc pDNA purification and delivery to dendritic cells, stimulating further in vivo studies.

Cancer Biomarkers to Improve Performance of Xpert HPV for Cervical Cancer Screening

PURPOSE We evaluated whether cancer biomarkers improve the performance of Xpert human papillomavirus (HPV) testing as screening for cervical intraepithelial neoplasia grade 2 or greater (CIN2+). METHODS In a clinical study in Cape Town, South Africa, 418 HIV-negative and 426 HIV-positive women, age 30 to 65 years, were recruited. All women had cervical samples collected and underwent colposcopy and histologic sampling with consensus pathology review. Cervical samples were tested onsite using Xpert HPV, which detects 14 high-risk HPV types in 5 channels: HPV 16; HPV 18 and -45; HPV 31, -33, -35, -52, and -58; HPV 51 and -59; and HPV 39, -56, -66, and -68. Cycle threshold (Ct) values are outputted for each channel. Samples were also tested using real-time polymerase chain reaction to detect mRNA for CDKN2A, TOP2A, and MKi-67. Results were reported as ΔCt values relative to internal controls. Logistic regression and receiver operating characteristic curves were used to evaluate associations between Ct values for HPV and biomarkers with CIN2+. RESULTS Ct values from channels detecting HPV 16, -18, -45, -33, -35, -52, and -58 were informative to predict CIN2+. Area under the curve was 0.906 in HIV-negative and 0.870 in HIV-positive women. When sensitivity was set at 80%, specificity was 94.0% in HIV-negative and 82.7% in HIV-positive women. ΔCt values for detection of mRNA for CDKN2A and MKi-67 added significant improvement to the prediction of CIN2+ when included with the Ct values from the 3 informative Xpert channels. Including both HPV and biomarker parameters, area under the curve was 0.944 in HIV-negative and 0.913 in HIV-positive women. When sensitivity was set at 80%, specificity was 97.3% in HIV-negative and 89.6% in HIV-positive women. CONCLUSION Improvements in specificity of a round of screening using Xpert HPV can be achieved with the addition of cancer biomarkers.

Astrocytic histone deacetylase 2 facilitates delayed depression and memory impairment after subarachnoid hemorrhage by negatively regulating glutamate transporter-1.

BackgroundDelayed cognitive impairment (DCI) after subarachnoid hemorrhage (SAH) is one of the most common sequelae in patients. This study aimed to investigate the characteristics of the course and glutamatergic pathogenesis of DCI after SAH in mice.MethodsA SAH mouse model of internal carotid puncture was used. Depressive and cognitive behaviors were detected by forced swimming and sucrose preference tests and Morris water maze test, respectively. Microdialysis and high-performance liquid chromatography (HPLC) were used to detect the interstitial glutamate. The expressions of histone deacetylases (HDACs), glutamate transporters, and glutamate receptors were examined. Primary astrocytes magnetically sorted from adult mice were cultured for glutamate uptake assay and protein and mRNA detection. Selective HDAC2 inhibitor and glutamate transporter-1 (GLT-1) inhibitor administered via were intraperitoneal injection to evaluate their effects on DCI in SAH mice.ResultsDepression and memory impairment lasted for more than 12 weeks and peaked at 8 weeks after SAH. Interstitial glutamate accumulation in the hippocampus and impaired glutamate uptake in astrocytes of the SAH mice were found during DCI, which could be explained by there being a significant decrease in GLT-1 expression but not in glutamate and aspartate transporter (GLAST) in hippocampal astrocytes. Meanwhile, the phosphorylation level of excitatory glutamate receptors (GluN2B and GluA1) in the hippocampus was significantly reduced, although there was no significant change in the expression of the receptors. Importantly, the expression of HDAC2 increased most significantly in astrocytes after SAH compared with that of other subtypes of HDACs. Inhibition of HDAC2 markedly rescued the decrease in GLT-1 expression after SAH through transcriptional regulation. Behavioral results showed that a selective HDAC2 inhibitor effectively improved DCI in SAH mice, but this effect could be weakened by GLT-1 inhibition.ConclusionsIn summary, our study suggests that the dysfunction of GLT-1-mediated glutamate uptake in astrocytes may be a key pathological mechanism of DCI after SAH, and that a specific inhibitor of HDAC2 may exert a potential therapy.

Fiber Optic Particle Plasmon Resonance Biosensor for Label-Free Detection of Nucleic Acids and Its Application to HLA-B27 mRNA Detection in Patients with Ankylosing Spondylitis.

We developed a label-free, real-time, and highly sensitive nucleic acid biosensor based on fiber optic particle plasmon resonance (FOPPR). The biosensor employs a single-strand deoxyoligonucleotides (ssDNA) probe, conjugated to immobilized gold nanoparticles on the core surface of an optical fiber. We explore the steric effects on hybridization affinity and limit of detection (LOD), by using different ssDNA probe designs and surface chemistries, including diluent molecules of different lengths in mixed self-assembled monolayers, ssDNA probes of different oligonucleotide lengths, ssDNA probes in different orientations to accommodate target oligonucleotides with a hybridization region located unevenly in the strand. Based on the optimized ssDNA probe design and surface chemistry, we achieved LOD at sub-nM level, which makes detection of target oligonucleotides as low as 1 fmol possible in the 10-μL sensor chip. Additionally, the FOPPR biosensor shows a good correlation in determining HLA-B27 mRNA, in extracted blood samples from patients with ankylosing spondylitis (AS), with the clinically accepted real-time reverse transcription-polymerase chain reaction (RT-PCR) method. The results from this fundamental study should guide the design of ssDNA probe for anti-sense sensing. Further results through application to HLA-B27 mRNA detection illustrate the feasibility in detecting various nucleic acids of chemical and biological relevance.