[Proposal and Analysis of "Ternary" Model of Sensitization for Proton Therapy].

In order to improve the biological effect of proton therapy, the authors first propose a new method of boron-based proton-enhanced radiotherapy in a " ternary " radiotherapy mode, based on the existing sensitizing effect of proton radiotherapy: i.e, Boron-based mediators (11B and 10B) induce the proton-hydrogen-boron fusion reaction of the low-energy protons arriving at the Bragg peak region of the tumor target area (p+11B→3α) and thermal neutron capture (10B+n→7Li3+(0.84 MeV)+4He2+(1.47 MeV)+γ(0.477 MeV)), which release low-energy α-particles with high LETs to enhance the biological effect of proton dose in the target area, thus improve the clinical effect of proton therapy. Then, the advantages and disadvantages of the "ternary" model were analyzed from the theoretical basis and current research status, and finally, the "ternary" model is summarized and prospected.

Empowering Photovoltaic Panel Anti-Icing: Superhydrophobic Organic Composite Coating with In Situ Photothermal and Transparency.

Solar energy is widely used in photovoltaic power generation as a kind of clean energy. However, the liquid film, frosting, and icing on the photovoltaic module seriously limit the efficiency of photovoltaic power generation. We developed a composite coating (Y6-NanoSH) by combining an in situ photothermal and transparent Y6 organic film with a nanosuperhydrophobic material. The Y6-NanoSH coated glass exhibited excellent optical clarity both indoors and outdoors, indicating that the coating holds great promise in anti-icing applications for photovoltaic panels. The Y6-NanoSH coating absorbs very little visible light but instead absorbs in the near-infrared region, thereby emitting heat. When exposed to sunlight, the Y6-NanoSH coated photovoltaic panel raises its surface temperature, inhibiting the growth and accumulation of ice and frost on its surface. This is achieved through a combination of photothermal emission and superhydrophobic repellency, which promotes the evaporation and rolling away of water droplets. This validates our success in developing a photothermal, transparent, and superhydrophobic coating with excellent anti-icing capabilities, suitable for use on photovoltaic panels, as well as potential applications in car windscreens, transmission lines, curtain walls, and weather radomes.

A Biomimetic Multifunctional Scaffold for Infectious Vertical Bone Augmentation.

The regenerative treatment of infectious vertical bone defects remains difficult and challenging today. Current clinical treatments are limited in their ability to control bacteria and infection, which is unfavorable for new bone formation and calls for a new type of material with excellent osteogenic and antibacterial properties. Here a multifunctional scaffold is synthesized that mimics natural bone nanostructures by incorporating silver nanowires into a hierarchical, intrafibrillar mineralized collagen matrix (IMC/AgNWs), to achieve the therapeutic goals of inhibiting bacterial activity and promoting infectious alveolar bone augmentation in rats and beagle dogs. An appropriate concentration of 0.5 mg mL-1 AgNWs is selected to balance biocompatibility and antibacterial properties. The achieved IMC/AgNWs exhibit a broad spectrum of antimicrobial properties against Gram-negative Porphyromonas gingivalis and Gram-positive Streptococcus mutans. When the IMC/AgNWs are cocultured with periodontal ligament stem cells, it possesses excellent osteoinductive activities under both non-inflammatory and inflammatory conditions. By constructing a rat mandibular infected periodontal defect model, the IMC/AgNWs achieve a near-complete healing through the canonical BMP/Smad signaling. Moreover, the IMC/AgNWs enhance vertical bone height and osseointegration in peri-implantitis in beagle dogs, indicating the clinical translational potential of IMC/AgNWs for infectious vertical bone augmentation.

Two pairs of bioactive cyclohexene alkaloid enantiomers from the roots of Piper nigrum.

Two pairs of cyclohexene amide alkaloid enantiomers were obtained from the root of Piper nigrum. Their plane structures were established by NMR and HRESIMS spectra. The absolute configurations of 1a/1b and 2a/2b were determined by the comparison between the experimental and calculated electronic circular dichroism (ECD) spectra. All identified compounds were tested for inhibitory effects on acetylcholinesterase (AChE) in vitro. Notably, compounds 1b and 2b showed strong inhibitory effects on AChE and the interaction between proteins and compounds was discussed by molecular docking studies.

Cost-effectiveness comparison of dalpiciclib and abemaciclib combined with an aromatase inhibitor as first-line treatment for HR+/HER2- advanced breast cancer.

OBJECTIVES: CDK4/6 inhibitors dalpiciclib and abemaciclib have been approved by the Chinese National Medical Products Administration as first-line treatment for postmenopausal females with hormone receptor-positive (HR+) and human epidermal growth factor receptor-2 negative (HER2-) advanced breast cancer (ABC). We aimed to assess the cost-effectiveness of dalpiciclib plus letrozole/anastrozole (non-steroidal aromatase inhibitor [NSAI]) compared with abemaciclib plus NSAI as a first-line treatment for HR+/HER2- ABC in China. METHODS: We constructed a Markov model with three health states to evaluate health and economic outcomes of first-line treatment with dalpiciclib plus NSAI and abemaciclib plus NSAI for HR+/HER2- ABC. Efficacy data was obtained from MONARCH3 and DAWNA-2 trials. Quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios (ICERs) were calculated. RESULTS: Compared with abemaciclib plus NSAI, dalpiciclib plus NSAI resulted in 4.27 additional QALYs, with an ICER of $14827.4/QALY. At a willingness-to-pay threshold of 3 times gross domestic product per capita in China for 2023 ($37721.5/QALY), the cost-effectiveness probability of dalpiciclib plus NSAI was 77.42%. CONCLUSIONS: From the perspective of Chinese payers, dalpiciclib plus NSAI appears to be a cost-effective strategy compared with abemaciclib plus NSAI for the first-line treatment of patients with HR+/HER2- ABC in China. CLINICAL TRIAL REGISTRATION: MONARCH3, www.clinicaltrials.gov, identifier is NCT02246621 and DAWNA-2, www.clinicaltrials.gov, identifier is NCT03966898.

[pMBRT: Principles and Design of Advanced Proton Radiotherapy Techniques].

The development history of pMBRT, the biological role of minibeam, the mechanism of minibeam protection of tissues, the generation of minibeam (collimator method and magnetic focusing method), and the analysis of advantages and disadvantages of proton minibeam matrix arrays is introduced with advanced proton minibeam arrays (pMBRT). It is proposed that the combination of proton minibeam arrays and magnetic resonance fields can help to exploit the normal tissue protective function of pMBRT, and improve the precision of proton therapy.

Expanded Application of Piper nigrum: Guided Isolation of Alkaloids with Inhibitory Activities of AChE/BuChE and Aß Aggregation.

Piper nigrum is a popular crop that can be used as seasoning or as an additive but its active ingredients also have an effect on the nervous system. Nineteen new amide alkaloids (1a/1b, 2-5, 6a/6b, 7, 8a/8b, 9, 10a/10b, 11a-11b, 12-14) were isolated from P. nigrum, guided by inhibitory activity of AChE and LC-MS/MS based on GNPS. The configurations were determined by extensive spectral analysis, Bulkiness rule, and NMR calculations. The inhibitory activities of AChE/BuChE and Aß aggregation were tested, and the results showed compounds 2, 7, and 12 had significant inhibitory activities. These components were identified in the crude fraction and their relative quantities were tested, which suggested that compound 2 was the index component in the active site from P. nigrum.

Integrated Network Pharmacology and In-silico Approaches to Decipher the Pharmacological Mechanism of Dioscorea septemloba Thunb in Treating Gout and Its Complications.

BACKGROUND: Dioscorea septemloba Thunb. (DST) has demonstrated therapeutic potential in the treatment of gout and its associated complications. However, the underlying mechanisms of DST's pharmacological activity remain unclear. This study aims to investigate the pharmacological substances and network regulatory mechanisms of DST in treating gout and its complications using network pharmacology. METHODS: According to ultra-high performance liquid chromatography coupled with hybrid quadrupole-Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap-MS) data and Lipinski's rule of five, 24 bioactive phytochemicals from DST were identified. The targets of gout were retrieved from Gene Expression Omnibus (GEO), GeneCards, and DisGeNET databases, followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG pathway) enrichment analysis. The Cytoscape network analysis was used to identify the primary pathological pathways and key targets. Finally, LeDock was used for molecular docking to verify the active components of DST and their core target proteins. RESULTS: DST contains several core active ingredients, such as tetrahydroimidazo[1,2-a]pyridine- 2,5-dione, diosgenin, beta-sitosterol, dioscorol B, montroumarin and 9,10-dihydro-5,7- dimethoxy-3,4-phenanthrenediol. Moreover, these active components were found to strongly bind to the key targets for treating gout and its complications, including HSP90AA1, STAT3, PTGS2, PPARG, MTOR, HIF1A, MMP9, ESR1, and TLR4. As a result, DST alleviates gout and its complications by inhibiting xanthine dehydrogenase (XDH) to reduce uric acid levels and regulating the HIF-1α, EZH2/STAT3, and COX-2/PPAR-γ pathways to reduce inflammation. Additionally, it also plays an analgesic role by regulating the neuroactive ligand-receptor interaction pathway and calcium ion signaling pathway. CONCLUSION: This study has provided insights into the underlying mechanisms of DST in the treatment of gout and its complications, which could serve as a scientific foundation for its clinical translation.

Precipitation pattern alters the effects of nitrogen deposition on the growth of alien species Robinia pseudoacacia.

Aims: Nitrogen (N) supply and precipitation pattern (amount and frequency) both affect plant growth. However, N deposition is increasing and precipitation regimes are changing in the context of global change. An experiment was conducted to access how the growth of Robinia pseudoacacia, a widely distributed and cultivated N2-fixing alien species, is affected by both the pattern of precipitation and N supplies. Methods: Seedlings were grown in a glasshouse at four different N levels combined with different precipitation regimes, including three precipitation amounts, and two precipitation frequencies. After treatment for 75 days, plant height, biomass allocation, leaf and soil nutrient concentrations were measured. Results: Plants under high precipitation frequency had greater biomass compared with plants lower precipitation frequency, despite receiving the same amount of precipitation. Higher N supply reduced biomass allocation to nodules. Under low precipitation level, nodule growth and N2 fixation of R. pseudoacacia was more inhibited by high N deposition compared with plants under higher precipitation level. Even slightly N deposition under higher precipitation inhibited N2 fixation but it was insufficient to meet the N needs of the plants. Conclusions: Even at low levels, N deposition might inhibit N2 fixation of plants but low N in soil cannot meet the N requirements of plants, and caused N2 fixation limitation in plants during seedling stage. There was likely a transition from N2 fixation to acquisition of N from soil directly with root when N supply was increased.

The combined effect of fire and nitrogen addition on biodiversity and herbaceous aboveground productivity in a coastal shrubland.

Introduction: Fire and nitrogen (N) deposition each impact biodiversity and ecosystem productivity. However, the effect of N deposition on ecosystem recovery after fire is still far from understood, especially in coastal wetlands. Methods: We selected a typical coastal shrubland to simulate three N deposition levels (0, 10, and 20 g N m-2 year-1) under two different burned conditions (unburned and burned) in the Yellow River Delta of North China. Soil properties, soil microbial biodiversity, shrub growth parameters, herbaceous biodiversity, and aboveground productivity were determined after experimental treatments for 1 year. Results: We found that fire had a stronger influence on the ecosystem than N addition. One year after the fire, shrub growth had significantly decreased, while soil pH, soil electrical conductivity, herbaceous biodiversity, soil microbial biodiversity, and herbaceous aboveground productivity significantly increased. Conversely, a single year of N addition only slightly increased herbaceous aboveground productivity. The combined effect of fire and N addition was only significant for fungus biodiversity and otherwise had minimal influence. Interestingly, we found that herbaceous aboveground productivity was positively associated with fungal community diversity under unburned conditions but not in burned shrublands. Fire showed a great impact on soil parameters and biodiversity in the coastal wetland ecosystem even after a full year of recovery. Discussion: Fire may also diminish the influence of several belowground factors on herbaceous aboveground productivity, which ultimately reduces recovery and stability. Appropriate N addition may be an effective way to improve the ecosystem productivity in a wetland dominated by shrub species.

Effects of drought hardening on the carbohydrate dynamics of Quercus acutissima seedlings under successional drought.

Introduction: As precipitation patterns are predicted to become increasingly erratic, the functional maintenance of warm-temperate forests constitutes a key challenge for forest managers. In this study, 2-year-old Quercus acutissima seedlings were selected to elucidate the mechanisms whereby they respond to soil water fluctuations and the drought hardening effects on plant carbohydrate dynamics. Methods: Seedlings were trained under different soil water conditions for 2 months: drought (D), well-watered (W), 1-month drought and then 1-month well-watered (D-W), and 1-month well-watered and then 1-month drought (W-D). The functional traits involved in water- and carbon-use strategies were explored at the end of the hardening period. Compared with seedlings in group W, seedlings in groups D, D-W, and W-D had increased potential for carbon uptake (i.e., light saturated point, maximum ribulose-1,5-bisphosphate (RuBP) saturated rate, and electron transport rate) and water uptake (i.e., fine root-to-coarse root ratio) and downregulated growth and mitochondrial respiration to decrease carbon consumption. After water fluctuation hardening, we performed a successional dry-down experiment for 1 month to detect carbohydrate dynamics and explore the acclimation caused by prior hardening. Results and discussion: Our results revealed that there were more soluble sugars allocated in the leaves and more starch allocated in the stems and roots of seedlings hardened in the D, W-D, and D-W treatments than that of seedlings hardened in the W treatment. No significant changes in total non-structural carbohydrates were found. In addition, we found near-zero (seedlings trained by D and D-W treatments) or negative (seedlings trained by W-D treatment) growth of structural biomass at the end of the dry-down experiment, which was significantly lower than that of W-hardened seedlings. This suggests that there was a shift in allocation patterns between carbon storage and growth under recurrent soil drought, which can be strengthened by drought memory. We conclude that Q. acutissima seedlings adjusted water- and carbon-use strategies in response to water fluctuations, whereas stress memory can enhance their overall performance in reoccurring drought. Therefore, taking advantage of stress memory is a promising management strategy in forest nurseries, and drought-trained seedlings might be more suitable for afforestation practices in sites characterized by fluctuating soil water content, considering the ongoing global climatic changes.

Downregulation of VRK1 Inhibits Progression of Lung Squamous Cell Carcinoma through DNA Damage.

Background: Lung squamous cell carcinoma (LUSC) is a common malignancy. And the antitumor effect of bovine pox virus-associated kinase 1 (VRK1) is becoming a hot research topic. Methods: VRK1 expression and prognosis in LUSC were analyzed using the GEPIA database. The expression of VRK1 mRNA was detected in 25 LUSC clinical tissue samples by RT-PCR. VRK1 shRNA was transfected into LUSC NCI-H520 and SK-MES-1 cell lines to interfere with VRK1 expression, and the efficiency of VRK1 shRNA interference was detected by the western blot. The effects of VRK1 downregulation on LUSC cell viability, migration, cell cycle, and apoptosis were analyzed by the CCK8 assay, scratch assay, transwell assay, and flow cytometry. The effect of VRK1 downregulation on DNA damage response (DDR) was examined by immunofluorescence staining and western blot assays and further validated by in vivo experiments. Results: VRK1 was highly expressed in both LUSC tissues and cells. Survival analysis showed that the overall survival of LUSC patients with high VRK1 expression was significantly lower than that of LUSC patients with low VRK1 expression (P=0.0026). The expression level of the VRK1 gene was significantly higher in cancer tissues of LUSC patients than in paracancerous tissues. After transfection of VRK1 shRNA in both LUSC cells, cell activity decreased (P < 0.001), migration ability started to be inhibited (P < 0.001), the ratio of G0/G1 phase cells increased (P < 0.001), and apoptosis rate increased (P < 0.001). Immunofluorescence and western blot results showed that shVRK1 increased the level of γ-H2A.X (P < 0.001) and promoted apoptosis of tumor cells (P < 0.001). In addition, the results of animal experiments showed that shVRK1 had antitumor effects (P < 0.001) and a combined effect with DOX (P < 0.001). Conclusion: The downregulation of VRK1 significantly affected the proliferation, apoptosis, migration, and cell cycle progression of LUSC cells via DDR, suggesting that VRK1 is a suitable target for potential LUSC therapy.

[Estimation of PM2.5 Hourly Concentration in Sichuan Province Based on GTWR-XGBoost Model].

Aerosol optical depths of satellites and meteorological factors have been widely used to estimate concentrations of surface particulate matter with an aerodynamic diameter ≤ 2.5 µm. Research on a high time resolution and high-precision PM2.5 concentration estimation method is of great significance for timely and accurate air quality prediction and air pollution prevention and mitigation. Himawari-8 AOD hour product and ERA5 meteorological reanalysis data were used as estimation variables, and a GTWR-XGBoost combined model was proposed to estimate hourly PM2.5 concentration in Sichuan Province. The results showed that:① the performance of the proposed combination model was better than that of the KNN, RF, AdaBoost, GTWR, GTWR-KNN, GTWR-RF, and GTWR-AdaBoost models in the full dataset; the fitting accuracy indexes R2, MAE, and RMSE were 0.96, 3.43 µg·m-3, and 5.52 µg·m-3, respectively; and the verification accuracy indexes R2, MAE, and RMSE were 0.9, 4.98 µg·m-3, and 7.92 µg·m-3, respectively. ② The model had a high goodness of fit (R2 of the whole dataset was 0.96, and R2 of different times ranged from 0.91 to 0.98) when applied to the estimation of PM2.5 concentration hour. It showed that the model had good time stability for hourly estimation and could provide accurate estimation information for regional air quality assessment. ③ In terms of time, the annual average PM2.5hourly concentration estimation showed an inverted U-shaped trend. It began to increase gradually at 09:00 am to a peak of 44.56 µg·m-3 at 11:00 and then gradually decreased. Moreover, the seasonal variation was very obvious, with winter>spring>autumn>summer. ④ In terms of spatial distribution, it showed the characteristics of high in the east and low in the west and a high degree of local pollution.

Enhancement of TKI sensitivity in lung adenocarcinoma through m6A-dependent translational repression of Wnt signaling by circ-FBXW7.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) that specifically target mutational points in the EGFR gene have significantly reduced suffering and provided greater relief to patients with lung adenocarcinoma (LUAD). The third-generation EGFR-TKI, Osimertinib, has been successfully employed in clinical treatments to overcome resistance to both original and acquired T790M and L858R mutational points. Nevertheless, the issue of treatment failure response has emerged as an insurmountable problem. METHODS: By employing a combination of multiple and integrated approaches, we successfully identified a distinct population within the tumor group that plays a significant role in carcinogenesis, resistance, and recurrence. Our research suggests that addressing TKI resistance may involve targeting the renewal and repopulation of stem-like cells. To investigate the underlying mechanisms, we conducted RNA Microarray and m6A Epi-Transcriptomic Microarray analyses, followed by assessment of transcription factors. Additionally, we specifically designed a tag to detect the polypeptide circRNA-AA, and its expression was confirmed through m6A regulations. RESULTS: We initially identified unique molecular signatures present in cancer stem cells that contributed to poor therapeutic responses. Activation of the alternative Wnt pathway was found to sustain the renewal and resistant status of these cells. Through bioinformatics analysis and array studies, we observed a significant decrease in the expression of circFBXW7 in Osimertinib-resistant cell lines. Notably, the abnormal expression pattern of circFBXW7 determined the cellular response to Osimertinib. Functional investigations revealed that circFBXW7 inhibits the renewal of cancer stem cells and resensitizes both resistant LUAD cells and stem cells to Osimertinib. In terms of the underlying mechanism, we discovered that circFBXW7 can be translated into short polypeptides known as circFBXW7-185AA. These polypeptides interact with ß-catenin in an m6A-dependent manner. This interaction leads to reduced stability of ß-catenin by inducing subsequent ubiquitination, thereby suppressing the activation of canonical Wnt signaling. Additionally, we predicted that the m6A reader, YTHDF3, shares common binding sites with hsa-Let-7d-5p. Enforced expression of Let-7d post-transcriptionally decreases the levels of YTHDF3. The repression of Let-7d by Wnt signaling releases the stimulation of m6A modification by YTHDF3, promoting the translation of circFBXW7-185AA. This creates a positive feedback loop contributing to the cascade of cancer initiation and promotion. CONCLUSIONS: Our bench study, in vivo experiments, and clinical validation have unequivocally shown that circFBXW7 effectively inhibits the abilities of LUAD stem cells and reverses resistance to TKIs by modulating Wnt pathway functions through the action of circFBXW7-185AA on ß-catenin ubiquitination and inhibition. The regulatory role of circRNA in Osimertinib treatment has been rarely reported, and our findings reveal that this process operates under the influence of m6A modification. These results highlight the tremendous potential of this approach in enhancing therapeutic strategies and overcoming resistance to multiple TKI treatments.

Construction of a Chiral Fluorescent Probe for Tryptophan Enantiomers/Ascorbic Acid Identification.

Chiral recognition of amino acid enantiomers is critical in enhancing drug efficacy, detecting disease markers, and understanding physiological processes. Enantioselective fluorescent identification has gained attention among researchers due to its nontoxicity, easy synthesis, and biocompatibility. In this work, chiral fluorescent carbon dots (CCDs) were produced through a hydrothermal reaction followed by chiral modification. The fluorescent probe, Fe3+-CCDs (F-CCDs), was constructed by complexing Fe3+ with CCDs to differentiate between the enantiomers of tryptophan (Trp) and determine ascorbic acid (AA) through an "on-off-on" response. It is worth noting that l-Trp can greatly enhance the fluorescence of F-CCDs with a blue shift, whereas d-Trp does not have any effect on the fluorescence of F-CCDs. F-CCDs showed a low limit of detection (LOD) for l-Trp and l-AA, with an LOD of 3.98 and 6.28 µM, respectively. The chiral recognition mechanism of tryptophan enantiomers using F-CCDs was proposed based on the interaction force between the enantiomers and F-CCDs, as confirmed by UV-vis absorption spectroscopy and density functional theory calculations. The determination of l-AA by F-CCDs was also confirmed through the binding of l-AA to Fe3+ to release CCDs, as seen in UV-vis absorption spectra and time-resolved fluorescence decays. In addition, AND and OR gates were constructed based on the different responses of CCDs to Fe3+ and Fe3+-CCDs to l-Trp/d-Trp, demonstrating the significance of molecular-level logic gates in drug detection and clinical diagnosis.

Insights into the Effect of SiO Particle Size on the Electrochemical Performance between Half and Full Cells for Li-Ion Batteries.

Silicon monoxide (SiO) has attracted growing attention as one of the most promising anodes for high-energy-density lithium-ion batteries (LIBs), benefiting from relatively low volume expansion and superior cycling performance compared to bare silicon (Si). However, the size of the SiO particle for commercial application remains uncertain. Besides, the materials and concepts developed on the laboratory level in half cells are quite different from what is necessary for practical operation in full cells. Herein, we investigate the electrochemical performance of SiO with different particle sizes between half cells and full cells. The SiO with larger particle size exhibits worse electrochemical performance in the half cell, whereas it demonstrates excellent cycling stability with a high capacity retention of 91.3% after 400 cycles in the full cell. The reasons for the differences in their electrochemical performance between half cells and full cells are further explored in detail. The SiO with larger particle size possessing superior electrochemical performance in full cells benefits from consuming less electrolyte and not being easier to aggregate. It indicates that the SiO with larger particle size is recommended for commercial application and part of the information provided from half cells may not be advocated to predict the cycling performances of the anode materials. The analysis based on the electrochemical performance of the SiO between half cells and full cells gives fundamental insight into further Si-based anode research.

RT-IVT method allows multiplex real-time quantification of in vitro transcriptional mRNA production.

For the past 30 years, in vitro transcription (IVT) technology has been extensively used for RNA production or for basic transcriptional mechanism research. However, methods for mRNA quantification still need to be improved. In this study, we designed a RT-IVT method using binary fluorescence quencher (BFQ) probes and the PBCV-1 DNA ligase to quantify mRNA production in real-time by fluorescence resonance energy transfer (FRET) and RNA-splinted DNA ligation. Compared with existing methods, the RT-IVT method is inexpensive and non-radioactive, and can detect mRNA production in unpurified systems in real-time and shows high sensitivity and selectivity. The activity of T7 RNA polymerase and Escherichia coli RNA polymerase holoenzyme was then characterized with this method. We then multiplexed the real-time mRNA quantification for three T7 promoters on a RT-PCR thermocycler by using BFQ probes with different colored fluorophores that were specific for each target. Ultimately, we created an inexpensive multiplexed method to quantify mRNA production in real-time, and future research could use these methods to measure the affinity of transcriptional repressors to their target DNA sequence.