A novel procedure for absolute real-time quantification of gene expression patterns

<p>Abstract</p> <p>Background</p> <p>Temporal and tissue-specific patterns of gene expression play important roles in functionality of a biological system. Real-time quantitative polymerase chain reaction (qPCR) technique has been widely applied to single gene expressions, but its potential has not been fully released as most results have been obtained as fold changes relative to control conditions. Absolute quantification of transcripts as an alternative method has yet to gain popularity because of unresolved issues.</p> <p>Results</p> <p>We propose a solution here with a novel procedure, which may accurately quantify the total cDNA conventionally prepared from a biological sample at the resolution of ~70 pg/μl, and reliably estimate the absolute numbers of transcripts in a picogram of cDNA. In comparison to the relative quantification, cDNA-based absolute (CBA) qPCR method is found to be more sensitive to gene expression variations caused by factors such as developmental and environmental variations. If the number of target transcript copies is further normalized by reference transcripts, cell-level variation pattern of the target gene expression may also be detectable during a developmental process, as observed here in cases across species (<it>Ipomoea purpurea, Nicotiana benthamiana</it>) and tissues (petals and leaves).</p> <p>Conclusion</p> <p>By allowing direct comparisons of results across experiments, the new procedure opens a window to make inferences of gene expression patterns across a broad spectrum of living systems and tissues. Such comparisons are urgently needed for biological interpretations of gene expression variations in diverse cells.</p

Design, fabrication, and cold test of a high frequency system for an H-band sheet beam travelling wave tube

The design, fabrication and cold test of a high frequency system (HFS) for an H-band (220-325GHz) sheet beam travelling wave tube is presented in this article. The HFS was composed of a 90-period double-staggered grating waveguide and a pair of identical L-shaped couplers with Bragg reflectors and matching steps. The HFS was manufactured by nano-computer numerical control machining and its electromagnetic properties were measured by using a vector network analyzer. The measured S-parameters were in good agreement with the simulated ones, which predicted a 3 dB bandwidth of ∼47.0 GHz. The maximum value of the measured transmission coefficient S 21 was -4.9 dB and the in-band port reflection S 11 was around -15.0 dB. Based on the cold testing-based HFS, simulations of the beam wave interaction predicted a stable output power of over 55.1 W in the frequency range of 230-280 GHz. In addition, the instabilities of the beam wave interaction were investigated in the simulation

Identification and validation of a novel glycolysis-related ceRNA network for sepsis-induced cardiomyopathy

PurposeSepsis-induced cardiomyopathy (SIC) is a major life-threatening condition in critically infected patients. Early diagnosis and intervention are important to improve patient prognosis. Recognizing the pivotal involvement of the glycolytic pathway in SIC, this study aims to establish a glycolysis-related ceRNA network and explore novel diagnostic avenues.Materials and methodsSIC-related datasets were carefully filtered from the GEO database. CytoHubba was used to identify differentially expressed genes (DEGs) associated with glycolysis. A predictive method was then used to construct an lncRNA-miRNA-mRNA network. Dual-luciferase reporter assays validated gene interactions, and the specificity of this ceRNA network was confirmed in peripheral blood mononuclear cells (PBMCs) from SIC patients. Logistic analysis was used to examine the correlation between the ceRNA network and SIC. Diagnostic potential was assessed using receiver operating characteristic (ROC) curves, and correlation analysis investigated any associations between gene expression and clinical indicators.ResultsIER3 was identified as glycolysis-related DEG in SIC, and a ceRNA network (SNHG17/miR-214-3p/IER3) was established by prediction. Dual luciferase reporter gene assay confirmed the presence of mutual binding between IER3, miR-214-3p and SNHG17. RT-qPCR verified the specific expression of this ceRNA network in SIC patients. Multivariate logistic analysis established the correlation between the ceRNA network and SIC. ROC analysis demonstrated its high diagnostic specificity (AUC &gt; 0.8). Correlation analysis revealed a negative association between IER3 expression and oxygenation index in SIC patients (p &lt; 0.05). Furthermore, miR-214-3p expression showed a negative correlation with NT-proBNP (p &lt; 0.05).ConclusionIn this study, we identified and validated a ceRNA network associated with glycolysis in SIC: SNHG17/miR-214-3p/IER3. This ceRNA network may play a critical role in the onset and development of SIC. This finding is important to further our understanding of the pathophysiological mechanisms underlying SIC and to explore potential diagnostic and therapeutic targets for SIC

Conservation and diversification of the miR166 family in soybean and potential roles of newly identified miR166s

Identity between pre-miR166s in soybean. (TIF 5906 kb

Tumor cell network integration in glioma represents a stemness feature

BACKGROUND: Malignant gliomas including glioblastomas are characterized by a striking cellular heterogeneity, which includes a subpopulation of glioma cells that becomes highly resistant by integration into tumor microtube (TM)-connected multicellular networks. METHODS: A novel functional approach to detect, isolate, and characterize glioma cell subpopulations with respect to in vivo network integration is established, combining a dye staining method with intravital two-photon microscopy, Fluorescence-Activated Cell Sorting (FACS), molecular profiling, and gene reporter studies. RESULTS: Glioblastoma cells that are part of the TM-connected tumor network show activated neurodevelopmental and glioma progression gene expression pathways. Importantly, many of them revealed profiles indicative of increased cellular stemness, including high expression of nestin. TM-connected glioblastoma cells also had a higher potential for reinitiation of brain tumor growth. Long-term tracking of tumor cell nestin expression in vivo revealed a stronger TM network integration and higher radioresistance of the nestin-high subpopulation. Glioblastoma cells that were both nestin-high and network-integrated were particularly able to adapt to radiotherapy with increased TM formation. CONCLUSION: Multiple stem-like features are strongly enriched in a fraction of network-integrated glioma cells, explaining their particular resilience

U-Shaped Relationship Between Functional Outcome and Serum Uric Acid in Ischemic Stroke

Background/Aims: We sought to assess a consecutive number of patients with first-ever acute ischemic stroke (AIS), the clinical relevance in regard to functional outcome of the serum uric acid (SUA) measured at admission. Methods: In 2 prospective centers for observational study, serum concentrations of SUA were measured on admission in the serum of 710 consecutive patients with AIS. SUA concentrations were determined by high-performance liquid chromatography. SUA, NIH stroke scale (NIHSS), and conventional risk factors were evaluated to determine their value to predict functional outcome within 3 months. Results: During the follow-up, an unfavorable functional outcome (defined as a mRS score &#x3e; 2) was found in 219 (30.8%) patients. The unfavorable functional outcome distribution across the SUA quartiles ranged between 12.4% (third quartile) and 50.6% (first quartile). After adjusting for all other significant outcome predictors, SUA concentration remained an independent unfavorable outcome predictor with an adjusted OR of 0.996 (95% CI, 0.993-0.998; P&#x3c; 0.001). Conclusions: The data show that the U-shaped nature of the exposure-risk relationship was more prominent when the data were assessed in deciles (based on the SUA values). This model predicted the lowest relative risk of unfavorable outcome in the 67th percentile (corresponding to 309 µmol/L). SUA was significantly associated with the risk of poor functional outcomes in Chinese patients with stroke

Study of the mass balance, biotransformation and safety of [14C]SHR8554, a novel μ-opioid receptor injection, in healthy Chinese subjects

Background: SHR8554 is a novel μ-opioid receptor-biased agonist. It has analgesic effects by selectively activating the G protein-coupled pathway. Additionally, it can weakly activate the ß-arrestin-2 pathway, resulting in a limited number of side effects, such as gastrointestinal inhibition. Previous studies have shown that SHR8554 has good analgesic effects, safety and tolerability, but the pharmacokinetic characteristics of SHR8554 in humans have not been reported. This study was designed to investigate the pharmacokinetics and safety of SHR8554 in healthy Chinese male subjects.Methods: A single 1 mg/41.3 μCi intravenous dose of [14C]SHR8554 was administered to six healthy male subjects. Blood, urine and faecal samples were collected at continuous time points to analyse SHR8554 parent drug levels and their metabolites. The total radioactivity in blood, plasma, urine and faeces was detected by using a liquid scintillation counter. The dynamic changes of SHR8554 and its metabolite concentration were by liquid chromatography-tandem mass spectrometry (LC/MS), and then pharmacokinetic analysis. The safety of the drug on the subjects was also observed after a single intravenous injection.Results: The total recovery of radioactivity in urine and faeces was 99.68% ± 0.79% in 216 h, including 76.22% ± 1.12% in urine and 23.46% ± 1.36% in faeces. Seventeen major metabolites in blood, urine and faeces were analysed and identified. The main metabolic pathways of SHR8554 in the human body involve 1) N-dealkylation; 2) O-deethylation; 3) mono-oxidation; 4) glucuronidation, etc. The primary mechanism of SHR8554 clearance in the human body is through urinary excretion, primarily in its parent drug and metabolite forms. The drug has good safety, and no serious adverse effects were observed.Conclusion: SHR8554 showed favourable pharmacokinetic characteristics and safety profiles in this study. SHR8554 is extensively metabolized in human body. The main metabolic pathways include N-dealkylation and O-deethylation, as well as mono-oxidation and glucuronidation. The main excretion route of SHR8554 and its metabolites is through urine.Clinical Trial Registration:http://www.chinadrugtrials.org.cn/, identifier CTR2022045