Molecular mechanisms of neutron radiation dose effects on M1 generation peas.

The dose effect of radiation has long been a topic of concern, but the molecular mechanism behind it is still unclear. In this study, dried pea seeds were irradiated with 252Cf fission neutron source. Through analyzing the transcriptome and proteome of M1 generation pea (Pisum sativum L.) leaves, we studied the molecular rule and mechanism of neutron dose effect. Our results showed three important rules of global gene expression in the studied dose range. The rule closely related to the neutron absorbed dose at the transcription and translation levels is: the greater the difference in neutron absorbed dose between two radiation treatment groups, the greater the difference in differential expression between the two groups and the control group. We also obtained important sensitive metabolic pathways of neutron radiation, as well as related key genes. Furthermore, the overall molecular regulation mechanism of dose effect was revealed based on the main functional items obtained. Our research results can be applied to appropriate radiation dose estimation and agricultural production practice.

AI-assisted mass spectrometry imaging with in situ image segmentation for subcellular metabolomics analysis.

Subcellular metabolomics analysis is crucial for understanding intracellular heterogeneity and accurate drug-cell interactions. Unfortunately, the ultra-small size and complex microenvironment inside the cell pose a great challenge to achieving this goal. To address this challenge, we propose an artificial intelligence-assisted subcellular mass spectrometry imaging (AI-SMSI) strategy with in situ image segmentation. Based on the nanometer-resolution MSI technique, the protonated guanine and threonine ions were respectively employed as the nucleus and cytoplasmic markers to complete image segmentation at the subcellular level, avoiding mutual interference of signals from various compartments in the cell. With advanced AI models, the metabolites within the different regions could be further integrated and profiled. Through this method, we decrypted the distinct action mechanism of isomeric drugs, doxorubicin (DOX) and epirubicin (EPI), only with a stereochemical inversion at C-4'. Within the cytoplasmic region, fifteen specific metabolites were discovered as biomarkers for distinguishing the drug action difference between DOX and EPI. Moreover, we identified that the downregulations of glutamate and aspartate in the malate-aspartate shuttle pathway may contribute to the higher paratoxicity of DOX. Our current AI-SMSI approach has promising applications for subcellular metabolomics analysis and thus opens new opportunities to further explore drug-cell specific interactions for the long-term pursuit of precision medicine.

Ligation-Based High-Performance Mimetic Enzyme Sensing Platform for Nucleic Acid Detection.

G-quadruplex (G4)/hemin DNAzyme is a promising candidate to substitute horseradish peroxidase in biosensing systems, especially for the detection of nucleic acids. However, the relatively suboptimal catalytic capacity limits its potential applications. This makes it imperative to develop an ideal signal for the construction of highly sensitive biosensing platforms. Herein, we integrated a novel chimeric peptide-DNAzyme (CPDzyme) with the ligase chain reaction (LCR) for the cost-efficient and highly sensitive detection of nucleic acids. By employing microRNA (miRNA) and single-nucleotide polymorphism detection as the model, we designed a G4-forming sequence on the LCR probe with a terminally labeled amino group. Subsequently, asymmetric hemin with carboxylic arms allowed assembly with the LCR products and peptide to form CPDzyme, followed by the magnetic separation of the extraneous components and chemiluminescence detection. Compared with the conventional G4/hemin signaling-based method, the LCR-CPDzyme system demonstrated 3 orders of magnitude improved sensitivity, with accurate quantification of as low as 25 aM miRNA and differentiation of 0.1% of mutant DNA from the pool containing a large amount of wild-type DNA. The proposed LCR-CPDzyme strategy is a potentially powerful method for in vitro diagnostics and serves as a reference for the development of other ligation- or hybridization-based nucleic acid amplification assays.

Mycorrhizal effects on crop yield and soil ecosystem functions in a long-term tillage and fertilization experiment.

It is well understood that agricultural management influences arbuscular mycorrhizal (AM) fungi, but there is controversy about whether farmers should manage for AM symbiosis. We assessed AM fungal communities colonizing wheat roots for three consecutive years in a long-term (> 14 yr) tillage and fertilization experiment. Relationships among mycorrhizas, crop performance, and soil ecosystem functions were quantified. Tillage, fertilizers and continuous monoculture all reduced AM fungal richness and shifted community composition toward dominance of a few ruderal taxa. Rhizophagus and Dominikia were depressed by tillage and/or fertilization, and their abundances as well as AM fungal richness correlated positively with soil aggregate stability and nutrient cycling functions across all or no-tilled samples. In the field, wheat yield was unrelated to AM fungal abundance and correlated negatively with AM fungal richness. In a complementary glasshouse study, wheat biomass was enhanced by soil inoculum from unfertilized, no-till plots while neutral to depressed growth was observed in wheat inoculated with soils from fertilized and conventionally tilled plots. This study demonstrates contrasting impacts of low-input and conventional agricultural practices on AM symbiosis and highlights the importance of considering both crop yield and soil ecosystem functions when managing mycorrhizas for more sustainable agroecosystems.

Real-Time Monitoring of Air Discharge in a Switchgear by an Intelligent NO2 Sensor Module.

An air-insulated power equipment adopts air as the insulating medium and is widely implemented in power systems. When discharge faults occur, the air produces decomposition products represented by NO2. The efficient NO2 sensor enables the identification of electrical equipment faults. However, single-sensor-dependent NO2 detection is vulnerable to interfering gases. Implementing the sensor array could reduce the interference and improve detection efficiency. In the field of NO2 detection, In2O3 sensors have exhibited tremendous advantages. In our work, four composites based on In2O3 are integrated into sensor arrays, which could detect 250 ppb of NO2 and exhibit excellent selectivity when simultaneously exposed to CO. To further reduce the impact of humidity on gas-sensing performance, a convolutional neural network and a long short-term memory model equipped with an attention mechanism are proposed to evaluate NO2 concentration within 1 ppm, and the detection error is 63.69 ppb. In addition, the NO2 concentration estimation platform based on a microgas sensor is established to detect air discharge faults. The average concentration of NO2 generated by 10 consecutive discharge faults at 15 kV is 726.58 ppb, which indicates severe discharge in the switchgear. Our NO2 estimation method has great potential for large-scale deployment in low- and medium-voltage switchgears.

Nanoconfined Cathodic Electrochemiluminescence for Self-Sensitized Bioimaging of Membrane Protein.

Self-enhanced electrochemiluminescence (ECL) can be achieved via the confinement of coreactants and ECL emitters in a single nanostructure. This strategy has been used for the design of anodic ECL systems with amine compounds as coreactants. In this work, a novel confinement system was proposed by codoping positively charged ECL emitter tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)32+) and negatively charged coreactant peroxydisulfate (S2O82-) in silica nanoparticles. The codoping process could be performed by introducing S2O82- in cationic poly(diallyldimethylammonium chloride) (PDDA) to form PDDA@S2O82- and then encapsulating it and Ru(bpy)32+ in the Triton X-100 vesicle followed by the hydrolysis of tetraethyl ortosilicate, surface modification, and demulsification. The obtained RuSSNs exhibited good homogeneity, excellent monodispersity, acceptable biocompatibility, and 2.9-fold stronger ECL emission than Ru(bpy)32+-doped silica nanoparticles at an equal amount of nanoparticles in the presence of 0.1 M K2S2O8. Thus, an in situ self-sensitized cathodic ECL imaging method was designed for the monitoring of glycoprotein on living cell membranes. This work provides a new way for the modification, enhancement, and application of nano-ECL emitters in biological analysis.

Nanometer Resolution Mass Spectro-Microtomography for In-Depth Anatomical Profiling of Single Cells.

Visually identifying the molecular changes in single cells is of great importance for unraveling fundamental cellular functions as well as disease mechanisms. Herein, we demonstrated a mass spectro-microtomography with an optimal voxel resolution of ∼300 × 300 × 25 nm3, which enables three-dimensional tomography of chemical substances in single cells. This mass imaging method allows for the distinguishment of abundant endogenous and exogenous molecules in subcellular structures. Combined with statistical analysis, we demonstrated this method for spatial metabolomics analysis of drug distribution and subsequent molecular damages caused by intracellular drug action. More interestingly, thanks to the nanoprecision ablation depth (∼12 nm), we realized metabolomics profiling of cell membrane without the interference of cytoplasm and improved the distinction of cancer cells from normal cells. Our current method holds great potential to be a powerful tool for spatially resolved single-cell metabolomics analysis of chemical components during complex biological processes.

Research progress and prospects on gas-sensitive mechanisms of semiconductor sensors.

Semiconductor materials with wide bandgaps are extensively employed for gas detection due to their advantages of low cost, high sensitivity, fast speed, excellent stability, and distinctive selectivity. Previous studies have reported on different kinds of semiconductor materials and their complex synthesis procedures. However, the research progress on gas-sensitive mechanisms seriously lags behind the performance improvement. The research route of the gas-sensing mechanism is not clear, resulting in an unclear development direction of novel sensitive materials. This review aims to summarize existing approaches and their progress on the interpretation of gas-sensing mechanisms in semiconductors, such as the calculations based on density functional theory, semiconductor physics, and in situ experiments. Ultimately, a reasonable route for the mechanism investigation has been proposed. It guides the development direction of novel materials and reduces the cost of screening highly selective materials. Overall, this review can provide helpful guidance concerning the gas-sensitive mechanism for scholars.

Array electrochemiluminescence device with ultra-high sensitivity and selectivity for rapid visualized monitoring of trace radon in environment.

The World Health Organization has reported radioactive Rn gas as the second leading cause of lung cancer and gives an extreme limit to indoor Radon (Rn) concentration as 100 Bq/m3. To realize rapid and accurate Rn monitoring, we report an efficient visualized electrochemiluminescence (ECL) device for Rn detection with the lowest limit of detection (0.9 Bq/m3/3.6 Bq h m-3) compared to known Rn detection methods and the shortest measurement time (less than 5 h) among non-pump methods. In detail, an efficient Rn probe is prepared by Au nanoparticles, Pb2+ aptamer, as well as NH2-ssDNA co-reactant and then modified on ITO electrodes to obtain Rn detection devices. With tris(2,2'-bipyridyl)ruthenium(II)chloride (Ru(bpy)3Cl2) as an ECL emitter, the devices can exhibit ultra-high sensitivity and selectivity to trace Rn in environment via the ECL quenching caused by 210Pb, the relatively stable decay product of Rn. Furthermore, ECL imaging technology can be applied to realize the visualized Rn detection. An efficient up-response ECL detector was also invented to support this detection device to achieve accurate Rn detection in environment. This work reports noble gas ECL detection for the first time and provides an efficient strategy for rapid and accurate monitoring of trace Rn in environment.

Competitive interactions in two different plant species: Do grassland mycorrhizal communities and nitrogen addition play the same game?

In the Tibetan Plateau grassland ecosystems, nitrogen (N) availability is rising dramatically; however, the influence of higher N on the arbuscular mycorrhizal fungi (AMF) might impact on plant competitive interactions. Therefore, understanding the part played by AMF in the competition between Vicia faba and Brassica napus and its dependence on the N-addition status is necessary. To address this, a glasshouse experiment was conducted to examine whether the grassland AMF community's inocula (AMF and NAMF) and N-addition levels (N-0 and N-15) alter plant competition between V. faba and B. napus. Two harvests took day 45 (1st harvest) and day 90 (2nd harvest), respectively. The findings showed that compared to B. napus, AMF inoculation significantly improved the competitive potential of the V. faba. In the occurrence of AMF, V. faba was the strongest competitor being facilitated by B. napus in both harvests. While under N-15, AMF significantly enhanced tissue N:P ratio in B. napus mixed-culture at 1st harvest, the opposite trend was observed in 2nd harvest. The mycorrhizal growth dependency slightly negatively affected mixed-culture compared to monoculture under both N-addition treatments. The aggressivity index of AMF plants was higher than NAMF plants with both N-addition and harvests. Our observation highlights that mycorrhizal associations might facilitate host plant species in mixed-culture with non-host plant species. Additionally, interacting with N-addition, AMF could impact the competitive ability of the host plant not only directly but also indirectly, thereby changing the growth and nutrient uptake of competing plant species.

Electrochemiluminescence Imaging at a Single Nanoparticle Scale to Elucidate Diffusion-Accelerated Charge Transfer and Monitor Cell Permeability.

Accelerating the charge transfer between electroactive species and the electrode is always a hot topic. Here, we report a finding of Ru(bpy)33+ diffusion-induced acceleration of charge transfer from Ru(bpy)32+-doped silica nanoparticles (RDSNs) to the electrode via electrochemiluminescence (ECL) imaging at a single nanoparticle scale. Ru(bpy)32+ in the electrolyte can act as an enhancer of RDSN ECL emission in the presence of coreactant tripropylamine, which amplifies the RDSN ECL by 478 times at 10 µM free Ru(bpy)32+. According to percolation theory, the diffusion of electro-generated Ru(bpy)33+ near a single RDSN brings much quicker charge transfer to the electrode than electron hopping in RDSN, which is demonstrated by spatial and temporal interaction imaging of the RDSN and the Ru(III) diffusion layer. Taking advantage of this new mechanism, a real-time ECL imaging method has been constructed to monitor the rapid change of cell permeability during surfactant treatment.

SARS-CoV-2 monitoring by automated target-driven molecular machine-based engineering.

Biosensors based on nucleic acid-structured electrochemiluminescence are rapidly developing for medical diagnostics. Here, we build an automated DNA molecular machine on Ti3C2/polyethyleneimine-Ru(dcbpy)3 2+@Au composite, which alters the situation that a DNA molecular machine requires laying down motion tracks. We use this DNA molecular machine to transduce the target concentration information to enhance the electrochemiluminescence signal based on DNA hybridization calculations. Complex bioanalytical processes are centralized in a single nucleic acid probe unit, thus eliminating the tedious steps of laying down motion tracks required by the traditional molecular machine. We found a detection limit of 0.68 pM and a range of 1 pM to 1 nM for the analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific DNA target. Recoveries range between 96.4 and 104.8% for the analysis of SARS-CoV-2 in human saliva. Supplementary Information: The online version contains supplementary material available at 10.1007/s10311-022-01434-9.

Experimental Warming Has Not Affected the Changes in Soil Organic Carbon During the Growing Season in an Alpine Meadow Ecosystem on the Qinghai-Tibet Plateau.

The effects of climate warming and season on soil organic carbon (SOC) have received widespread attention, but how climate warming affects the seasonal changes of SOC remains unclear. Here, we established a gradient warming experiment to investigate plant attributes and soil physicochemical and microbial properties that were potentially associated with changes in SOC at the beginning (May) and end (August) of the growing season in an alpine meadow ecosystem on the Qinghai-Tibet Plateau. The SOC of August was lower than that of May, and the storage of SOC in August decreased by an average of 18.53 million grams of carbon per hectare. Warming not only failed to alter the content of SOC regardless of the season but also did not affect the change in SOC during the growing season. Among all the variables measured, microbial biomass carbon was highly coupled to the change in SOC. These findings indicate that alpine meadow soil is a source of carbon during the growing season, but climate warming has no significant impact on it. This study highlights that in the regulation of carbon source or pool in alpine meadow ecosystem, more attention should be paid to changes in SOC during the growing season, rather than climate warming.

Bipolar Electrode Array for Multiplexed Detection of Prostate Cancer Biomarkers.

Owing to the characteristics of high throughput, high flexibility, and convenient separation of the sensing and reporting reactions, the bipolar electrode (BPE) shows great potential in clinical analysis. However, there are some difficulties in the combination of BPEs and multiplex electrochemiluminescence (ECL) biosensing, such as the need for small sample consumption, multistep operations, and separated sample loading. In this paper, a microfluidic BPE array chip was fabricated toward multiplex detection of cancer biomarkers. With a special channel structure and the difference in flow resistance of channels of different sizes, the direction of liquid flow was successfully controlled. In this way, rapid and automatic multiplex sampling was achieved on the array, which would help improve the sensing efficiency and reduce the reagent consumption. The ECL BPE array chip served as an immunosensor for multiple prostate cancer biomarkers including prostate-specific antigen (PSA), interleukin-6 (IL-6), and prostate-specific membrane antigen (PSMA). The microfluidic BPE chip shows good reproducibility and high sensitivity. The limits of detection for PSA, IL-6, and PSMA are 0.093 ng/mL, 0.061 pg/mL, and 0.059 ng/mL, respectively. It also exhibits excellent performance in real sample analysis. The integrated ECL BPE array shows a good application prospect in clinical sensing of cancer biomarkers, as well as point-of-care testing.

Slope aspect determines the abundance and composition of nitrogen-cycling microbial communities in an alpine ecosystem.

Slope aspect is an important topographic feature that can influence local environmental conditions. While strong effects of slope aspect on aboveground and belowground communities have been frequently elucidated, how slope aspect affects soil nitrogen (N) cycling microbes remains unclear. Here, we characterized the communities of soil N-cycling microbes on south- and north-facing slopes in an alpine ecosystem, by quantifying (qPCR) and high-throughput sequencing six genes involved in N-fixation (nifH), nitrification (archaeal and bacterial amoA) and denitrification (nirK, nirS and nosZ). We found that the abundance, diversity and community composition of major N-cycling microbes differed dramatically between the two slope aspects, and these variances could be well explained by the aspect-driven differences in environmental conditions, especially soil temperature and moisture. The response patterns of different N-cycling groups to slope aspect were much inconsistent, especially for those with similar functions (i.e. ammonia-oxidizing archaea vs. bacteria, nirK- vs. nirS-reducers), indicating strong niche differentiation between these counterparts. We also observed strong preferences and distinct co-occurrence patterns of N-cycling microbial taxa for the two slope aspects. These findings highlight the importance of slope aspect in determining the abundance, species distribution and community structure of N-cycling microbes, and consequently influencing N-cycling processes and ecosystem functioning.

Label-Free Electrochemiluminescence Imaging of Single-Cell Adhesions by Using Bipolar Nanoelectrode Array.

A label-free and fast approach for positive electrochemiluminescence (ECL) imaging of single cells by bipolar nanoelectrode array is proposed. The reduction of oxygen at a platinized gold nanoelectrode array in a closed bipolar electrochemical system is coupled with an oxidative ECL process at the anodic side. For elevating the ECL imaging contrast of single cells, a driving voltage of -2.0 V is applied to in situ generate oxygen confined beneath cells that is subsequently used for ECL imaging at 1.1 V. High oxygen concentration in the confined space resulting from steric hindrance generates prominent oxygen reduction current at the cathodic side and higher ECL intensity at the anodic side, allowing positive ECL imaging of the cells adhesion region with excellent contrast. Cell morphology and adhesion strength can be successfully imaged with high image acquisition rate. This approach opens a new avenue for label-free imaging of single cells.

2D/3D Copper-Based Metal-Organic Frameworks for Electrochemical Detection of Hydrogen Peroxide.

Metal-organic frameworks (MOFs) have been extensively used as modified materials of electrochemical sensors in the food industry and agricultural system. In this work, two kinds of copper-based MOFs (Cu-MOFs) with a two dimensional (2D) sheet-like structure and three dimensional (3D) octahedral structure for H2O2 detection were synthesized and compared. The synthesized 2D and 3D Cu-MOFs were modified on the glassy carbon electrode to fabricate electrochemical sensors, respectively. The sensor with 3D Cu-MOF modification (HKUST-1/GCE) presented better electrocatalytic performance than the 2D Cu-MOF modified sensor in H2O2 reduction. Under optimal conditions, the prepared sensor displayed two wide linear ranges of 2 µM-3 mM and 3-25 mM and a low detection limit of 0.68 µM. In addition, the 3D Cu-MOF sensor exhibited good selectivity and stability. Furthermore, the prepared HKUST-1/GCE was used for the detection of H2O2 in milk samples with a high recovery rate, indicating great potential and applicability for the detection of substances in food samples. This work provides a convenient, practical, and low-cost route for analysis and extends the application range of MOFs in the food industry, agricultural and environmental systems, and even in the medical field.

Ultrasensitive Nucleic Acid Assay Based on AIE-Active Polymer Dots with Excellent Electrochemiluminescence Stability.

Aggregation-induced emission (AIE) active Pdots are attractive nanomaterials applied in electrochemiluminescence (ECL) fields, while the irreversible redox reaction of these Pdots is a prevailing problem, resulting in instability of ECL emission. Herein, we first designed and synthesized an AIE-active Pdot with reversible redox property, which contains a tetraphenylethene derivate and benzothiadiazole (BT) to achieve stable ECL emission. BT has a good rigid structure with excellent electrochemical behaviors, which is beneficial for avoiding the destruction of the conjugated structure as much as possible during the preparation of Pdots, thus maintaining good redox property. The tetraphenylethene derivate, as a typical AIE-active moiety, provides a channel for highly efficient luminescence in the aggregated states. The Pdots exhibited reversible and quasi-reversible electrochemical behaviors during cathodic and anodic scanning, respectively. The stable annihilation, reductive-oxidative, and oxidative-reductive ECL signals were observed. Subsequently, we constructed an ultrasensitive ECL biosensor based on the oxidative-reductive ECL mode for the detection of miRNA-21 with a detection limit of 32 aM. This work provides some inspiration for the future design of ECL materials featuring AIE-active property and stable ECL emission.

Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection.

The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV-vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.

Exploring the trans-cleavage activity of CRISPR-Cas12a for the development of a Mxene based electrochemiluminescence biosensor for the detection of Siglec-5.

Sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs) is a type I transmembrane receptor on the cell surface. Siglec-5, as one of the Siglecs family, play an important role as an inhibitory receptor for leukocytes in the human body. The development of novel siglec-5 assays can help to study the pathogenesis of related diseases as well as to develop novel therapeutic drugs. We use catalytic hairpin assembly (CHA) amplification strategy combined with CRISPR-Cas12a's side-cutting feature to build a 2D ultra-thin Ti3C2Tx (MXene) based electrochemiluminescence (ECL) biosensor for the detection of Siglec-5. By using this ECL biosensor, the cleavage of CRISPR-Cas12a is reasonably combined with CHA-mediated isothermal amplification, thereby realizing the sensitive amplification assay Siglec-5 with 20.22 fM sensitivity. By introducing pairs of sites that are not in the same double-stranded DNA into the DNA duplex, the hybridization sequence of CRISPR-Cas12a complements the targeting mechanism to enhance indirect Siglec-5 amplification assay. Also, the double-strand DNA (dsDNA) design based on CRISPR-Cas12a amplification allows the same CRISPR RNA (crRNA, also known as guide RNA (gRNA)) to detect the output of DNA duplexes from different intermediate DNAs, which provides a common way for biomarker detection based on the conversion of protein analytes to intermediate DNA strategy. This work extends the application scope of CRISPR-Cas12a to the construction of ECL biosensors, evaluates the role of lectins, which can be used for the biochemical research and clinical diagnosis of protein markers. This is the first investigative work exploring the Trans-Cleavage activity of CRISPR-Cas12a for Mxene-based ECL biosensor establishment to the best of our knowledge.