Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification.

The principal pathogen responsible for chronic urinary tract infections, immunocompromised hosts, and cystic fibrosis patients is Pseudomonas aeruginosa, which is difficult to eradicate. Due to the extensive use of antibiotics, multidrug-resistant P. aeruginosa has evolved, complicating clinical therapy. Therefore, a rapid and efficient approach for detecting P. aeruginosa strains and their resistance genes is necessary for early clinical diagnosis and appropriate treatment. This study combines recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats-association protein 13a (CRISPR-Cas13a) to establish a one-tube and two-step reaction systems for detecting the mexX gene in P. aeruginosa. The test times for one-tube and two-step RPA-Cas13a methods were 5 and 40 min (including a 30 min RPA amplification reaction), respectively. Both methods outperform Quantitative Real-time Polymerase Chain Reactions (qRT-PCR) and traditional PCR. The limit of detection (LoD) of P. aeruginosa genome in one-tube and two-step RPA-Cas13a is 10 aM and 1 aM, respectively. Meanwhile, the designed primers have a high specificity for P. aeruginosa mexX gene. These two methods were also verified with actual samples isolated from industrial settings and demonstrated great accuracy. Furthermore, the results of the two-step RPA-Cas13a assay could also be visualized using a commercial lateral flow dipstick with a LoD of 10 fM, which is a useful adjunt to the gold-standard qRT-PCR assay in field detection. Taken together, the procedure developed in this study using RPA and CRISPR-Cas13a provides a simple and fast way for detecting resistance genes.

Biotinylated Tn5 transposase-mediated CUT&Tag efficiently profiles transcription factor-DNA interactions in plants.

In contrast to CUT&Tag approaches for profiling bulk histone modifications, current CUT&Tag methods for analysing specific transcription factor (TF)-DNA interactions remain technically challenging due to TFs having relatively low abundance. Moreover, an efficient CUT&Tag strategy for plant TFs is not yet available. Here, we first applied biotinylated Tn5 transposase-mediated CUT&Tag (B-CUT&Tag) to produce high-quality libraries for interrogating TF-DNA interactions. B-CUT&Tag combines streptavidin-biotin-based DNA purification with routine CUT&Tag, optimizing the removal of large amounts of intact chromatin not targeted by specific TFs. The biotinylated chromatin fragments are then purified for construction of deep sequencing libraries or qPCR analysis. We applied B-CUT&Tag to probe genome-wide DNA targets of Squamosa promoter-binding-like protein 9 (SPL9), a well-established TF in Arabidopsis; the resulting profiles were efficient and consistent in demonstrating its well-established target genes in juvenile-adult transition/flowering, trichome development, flavonoid biosynthesis, wax synthesis and branching. Interestingly, our results indicate functions of AtSPL9 in modulating growth-defence trade-offs. In addition, we established a method for applying qPCR after CUT&Tag (B-CUT&Tag-qPCR) and successfully validated the binding of SPL9 in Arabidopsis and PHR2 in rice. Our study thus provides a convenient and highly efficient CUT&Tag strategy for profiling TF-chromatin interactions that is widely applicable to the annotation of cis-regulatory elements for crop improvement.

ompX contribute to biofilm formation, osmotic response and swimming motility in Citrobacter werkmanii.

Citrobacter werkmanii, an aerobe and mesophilic Proteobacterium, is universal in industrial putrefaction, coastal water, and human blood. Our previous studies have discovered that outer membrane protein X (OmpX) of C. werkmanii is involved in calcium response, but the underlying mechanisms and its molecular characteristics remain elusive. To that end, the ompX gene was deleted from the genome of C. werkmanii and its phenotypic variations were thoroughly investigated in conjunction with the wild type (WT) and complementary strains using biochemical and molecular techniques such as RNA-Seq, respectively. The results demonstrated that deleting ompX reduces biofilm formation on polystyrene and glass surfaces. Meanwhile, ΔompX's swimming ability but not for its twitching or swarming abilities, was also reduced on semi-solid plates compared with WT, which was caused by inhibition of flagellar assembly genes, such as flgC, flhB, and fliE, etc. Furthermore, ompX inactivation altered susceptibility to various bactericide classes, as well as responses to Ca2+ and Mg2+ stress. In addition, when compared to WT, ΔompX captures a total of 1,357 deferentially expressed genes (DEGs), of which 465 were up-regulated and 892 were down-regulated, which can be enriched into various GO ontology and KEGG pathway terms. Furthermore, ompX, as well as ompD and ompW, can be modulated at the transcriptional levels by rbsR and tdcA. Overall, the ompX gene contributed to a variety of biological functions in C. werkmanii and could be served as a targeted site for controlling biofilm formation and developing new bactericides.

Proteomic signatures of synergistic interactions in antimicrobials.

Mounting evidence has shown that antimicrobial agents can interfere synergistically with bacterial viability and proliferation when acting together at both the planktonic and biofilm levels without clear underlying molecular mechanisms. Here, multiplexed proteomics by iTRAQ was used to study the interplay between two biocides, the isothiazolone 1,2-benzisothiazolin-3-one (BIT) and the chelating agent disodium ethylenediaminetetraacetic acid (EDTA-2Na), employing the Citrobacter werkmanii as a model system. We first confirmed that these two biocides act synergistically on this bacterial species and then extracted the proteomic profiles of C. werkmanii cells in the presence of BIT, EDTA-2Na, and their combinations. In particular, we identified 43 core proteins that are differentially expressed in all three conditions simultaneously. Meanwhile, we found that these core proteins are consistently up-regulated when these two biocides are present, but not for single biocides, where we found a balanced mix of up- and down-regulation. Meanwhile, most of the deletion mutants of the core DEPs exhibited biofilm growth inhibition under joint biocide action, while their response was very heterogenous, with respect to the wild-type strain. Together, our results show that while BIT and EDTA-2Na act on multiple protein targets, they interact synergistically at the protein level in a very consistent manner. SIGNIFICANCE: Our preliminary experiments have demonstrated that a combination of 1,2-benzisothiazolin-3-one (BIT) and EDTA-2Na shows higher inhibitory effects on planktonic growth and biofilm formation in both C. werkmanii and Staphylococcus aureus than when these two biocides act alone. However, the mechanistic basis of such synergistic interaction is still unknown. Therefore, the key proteins involved in the above-mentioned enhanced antimicrobial synergy were elucidated using multiplexed proteomics analysis by isobaric tags for relative and absolute quantification (iTRAQ). Our results reveal that the joint action of BIT and EDTA-2Na induces consistent protein expression alteration in a set of core proteins of C. werkmanii, which underlies a strong synergistic antimicrobial effect, which increase our understanding of the action modes of BIT and EDTA-2Na as well as their combinations.

Outer membrane protein of OmpF contributes to swimming motility, biofilm formation, osmotic response as well as the transcription of maltose metabolic genes in Citrobacter werkmanii.

Bacterial outer membrane proteins (Omps) are essential for environmental sensing, stress responses, and substance transport. Our previous study discovered that OmpA contributes to planktonic growth, biocide resistance, biofilm formation, and swimming motility in Citrobacter werkmanii, whereas the molecular functions of OmpF in this strain are largely unknown. Thus, in this study, the ompF gene was firstly knocked out from the genome of C. werkmanii using a homologous recombination method, and its phenotypical alternations of ∆ompF were then thoroughly characterized using biochemical and molecular approaches with the parental wild type (WT) and complementary (∆ompF-com) strains. The results demonstrated that the swimming ability of ∆ompF on semi-solid plates was reduced compared to WT due to the down-regulation of flgC, flgH, fliK, and fliF. Meanwhile, ompF deletion reduces biofilm formation on both glass and polystyrene surfaces due to decreased cell aggregation. Furthermore, ompF inactivation induced different osmotic stress (carbon sources and metal ions) responses in its biofilms when compared to WT and ∆ompF-com. Finally, a total of 6 maltose metabolic genes of lamB, malE, malK, malG, malM, and malF were all up-regulated in ∆ompF. The gene knockout and HPLC results revealed that the MalEFGK2 cluster was primarily responsible for maltose transport in C. werkmanii. Furthermore, we discovered for the first time that the upstream promoter of OmpF and its transcription can be combined with and negatively regulated by MalT. Overall, OmpF plays a role in a variety of biochemical processes and molecular functions in C. werkmanii, and it may even act as a targeted site to inhibit biofilm formation.

Roles of ompA of Citrobacter werkmanii in bacterial growth, biocide resistance, biofilm formation and swimming motility.

The genus Citrobacter is commonly found in environmental and industrial settings, some members of which have been used for bioremediation of heavy metals owing to the absorption ability of their biofilms. Although our previous studies have found that the outer membrane protein A (OmpA) contributes to the process of Citrobacter werkmanii biofilm formation, the underlying mechanisms remain elusive. Therefore, we deleted ompA from the genome of C. werkmanii and investigated its phenotypes in comparison to the wild type strain (WT) and the complementary strain using biochemical and molecular techniques including RNA-Seq. Our results demonstrated that the deletion of ompA led to an increase in biofilm formation on both polystyrene and glass surfaces due to upregulation of some biofilm formation related genes. Meanwhile, swimming ability, which is mediated by activation of flagellar assembly genes, was increased on semi-solid plates in the ∆ompA strain when compared with WT. Additionally, inactivation of ompA also caused increased 1,2-benzisothiazolin-3-one (BIT) resistance, differential responses to Ca2+ stress, curli protein expression and cellulose production. Finally, ∆ompA caused differential expression of a total of 1470 genes when compared with WT, of which 146 were upregulated and 1324 were downregulated. These genes were classified into different Gene Ontology (GO) and KEGG pathways. In summary, ompA in C. werkmanii contributes to a variety of biological functions and may act as a target site to modulate biofilm formation. KEY POINTS: • ompA is a negative regulator for biofilm formation by C. werkmanii. • ompA inhibits swimming motility of C. werkmanii. • ompA deletion causes different expression profiles in C. werkmanii.

Chemical Components of Aqueous Extracts of Melia azedarach Fruits and Their Effects on The Transcriptome of Staphylococcus aureus.

Staphylococcus aureus is the causative agent of numerous and varied clinical infections. Crude aqueous extracts of Melia azedarach fruits inhibit the planktonic growth and initial biofilm formation of S. aureus in a dose-dependent manner. Moreover, the biofilm topologies became sparse and decreased as the concentration of the aqueous extracts increased. RNA-Seq analyses revealed 532 differentially expressed genes (DEGs) after S. aureus exposure to 0.25 g/ml extracts; 319 of them were upregulated, and 213 were downregulated. The majority of DEGs were categorized into abundant sub-groups in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Finally, untargeted UHPLC-MS/MS analyses of the aqueous extracts of M. azedarach fruits demonstrated a highly complex profile in positive and negative electrospray ionization modes. The extracts primarily consisted of lipids and lipid-like molecules, organic acids and their derivatives, phenylpropanoids, polyketides, organoheterocyclic compounds, and benzenoids annotated by abundant lipid maps and KEGG pathways. Overall, this study provides evidences that the aqueous extracts of M. azedarach fruits can control S. aureus infections and sought to understand the mode of action of these extracts on S. aureus.

Gold nanoparticle-based colorimetric method for the detection of prostate-specific antigen.

BACKGROUND: Prostate-specific antigen (PSA), a serine protease, is a biomarker for preoperative diagnosis and screening of prostate cancer and monitoring of its posttreatment. METHODS: In this work, we reported a colorimetric method for clinical detection of PSA using gold nanoparticles (AuNPs) as the reporters. The method is based on ascorbic acid (AA)-induced in situ formation of AuNPs and Cu2+-catalyzed oxidation of AA. Specifically, HAuCl4 can be reduced into AuNPs by AA; Cu2+ ion can catalyze the oxidation of AA by O2 to inhibit the formation of AuNPs. In the presence of the PSA-specific peptide (DAHSSKLQLAPP)-modified gold-coated magnetic microbeads (MMBs; denoted as DAHSSKLQLAPP-MMBs), complexation of Cu2+ by the MMBs through the DAH-Cu2+ interaction depressed the catalyzed oxidation of AA and thus allowed for the formation of red AuNPs. However, once the peptide immobilized on the MMB surface was cleaved by PSA, the DAHSSKLQ segment would be released. The resultant LAPP fragment remaining on the MMB surface could not sequestrate Cu2+ to depress its catalytic activity toward AA oxidation. Consequently, no or less AuNPs were generated. RESULTS: The linear range for PSA detection was found to be 0~0.8 ng/mL with a detection limit of 0.02 ng/mL. Because of the separation of cleavage step and measurement step, the interference of matrix components in biological samples was avoided. CONCLUSION: The high extinction coefficient of AuNPs facilitates the colorimetric analysis of PSA in serum samples. This work is helpful for designing of other protease biosensors by matching specific peptide substrates.

Prognostic significance of ischemia-modified albumin for severe acute pancreatitis.

Background: Severe acute pancreatitis (SAP) is characterized by the noxious combination of severe systemic inflammation and hypoperfusion and oxidative stress. Ischemia-modified albumin (IMA) was recognized as a novel marker of oxidative stress and ischemia. The purpose of this study was to evaluate serum IMA level in patients with SAP and analyze its prognostic significance. Methods: A total of 72 patients with SAP were enrolled. Serum IMA level was measured within 24 hours of the onset of SAP, and baseline characteristics were recorded. The BISAP, APACHE II and SOFA scores were calculated. Multivariate logistic regression and receiver operating characteristic curve analyses were used to evaluate predictive ability of LMA for in-hospital mortality of SAP. Kaplan-Meier analysis was further used to compare in-hospital mortality difference between high LMA and low LMA. Results: The overall in-hospital mortality rate of all 72 SAP patients was 23.6%. Non-survivor group had higher serum IMA (107.2±10.8 VS 88.4±11.9, P<0.05) than survivor group. Otherwise, the optimal cutoff levels for the IMA predicting in-hospital mortality of patients with SAP was 112 U/ml using a sensitivity of 77.4% and a specificity of 76.2% as optimal conditions (AUC, 0.734; 95% CI: 0.615-0.852; P=0.002). IMA level also was confirmed as an independent prognostic factor for SAP in multivariate analysis. Patient with high IMA level (≥112 U/ml) had poorer survival rate than low IMA (<112 U/ml) in log-rank test of Kaplan-Meier survival analysis (P<0.05). Conclusions: Serum IMA level can be considered as an independent predictor for in-hospital mortality of patients with SAP.

The Changes of Serum Leptin and Kisspeptin Levels in Chinese Children and Adolescents in Different Pubertal Stages.

The aim of the study is to investigate the changes of serum leptin and kisspeptin levels in children and adolescents with different pubertal stages and nutritional states. A total of 647 Chinese children and adolescents were recruited, and serum estradiol, testosterone, pituitary gonadotropins, leptin, and kisspeptin levels were measured. The results showed that serum leptin levels of boys in T2 stage were the highest among the five stages, while they showed a gradual increase from T1 to T5 stage in girls and reached the highest in T5 stage (P < 0.05). Conversely, serum kisspeptin levels of boys were higher in T4 and T5 stages than those in T1 stage, while its levels of girls were the highest in T2 stage, 21.4% higher than those in T1 stage (P < 0.05). Both leptin and kisspeptin levels were positively correlated with BMI, WC, and weight in all boys and girls (all P < 0.05). In conclusion, kisspeptin levels were firstly found to be notably changed in pubertal stages and nutritional status in Chinese children and adolescents with a significant sexual dimorphism. Obese/overweight girls had higher kisspeptin levels, and there was a positive correlation between kisspeptin and FSH and LH and obesity-related parameters in all boys and girls.

Parameters of diffusional kurtosis imaging for the diagnosis of acute cerebral infarction in different brain regions.

Diffusional kurtosis imaging (DKI) is a new type diffusion-weighted sequence which measures the non-Gaussianity of water diffusion. The present study aimed to investigate whether the parameters of DKI could distinguish between differences in water molecule diffusion in various brain regions under the conditions of acute infarction and to identify the optimal DKI parameter for locating ischemic lesions in each brain region. A total of 28 patients with acute ischemic stroke in different brain regions were recruited for the present study. The relative values of DKI parameters were selected as major assessment indices, and the homogeneity of background image and contrast of adjacent structures were used as minor assessment indices. According to the brain region involved in three DKI parametric maps, including mean kurtosis (MK), axial kurtosis (Ka) and radial kurtosis (Kr), 112 groups of regions of interest were outlined in the following regions: Corpus callosum (n=17); corona radiata (n=26); thalamus (n=21); subcortical white matter (n=24); and cerebral cortex (n=24). For ischemic lesions in the corpus callosum and corona radiata, significant increases in relative Ka were detected, as compared with the other parameters (P<0.05). For ischemic lesions in the thalamus, subcortical white matter and cerebral cortices, an increase in the three parameters was detected, however this difference was not significant. Minor assessment indices demonstrated that Ka lacked tissue contrast and the background of Kr was heterogeneous; thus, MK was the superior assessment parameter for ischemic lesions in these regions. In conclusion, Ka is better suited for the diagnosis of acute ischemic lesions in highly anisotropic brain regions, such as the corpus callosum and corona radiate. MK may be appropriate for the lesions in low anisotropic or isotropic brain regions, such as the thalamus, subcortical white matter and cerebral cortices.

Sonic hedgehog improves ischemia-induced neovascularization by enhancing endothelial progenitor cell function in type 1 diabetes.

The Sonic hedgehog (Shh) pathway is downregulated in type 1 diabetes, and it has been reported that augmentation of this pathway may alleviate diabetic complications. However, the cellular mechanisms underlying these protective effects are poorly understood. Recent studies indicate that impaired function of endothelial progenitor cells (EPCs) may contribute to cardiovascular problems in diabetes. We hypothesized that impaired Shh signaling contribute to endothelial progenitor cell dysfunction and that activating the Shh signaling pathway may rescue EPC function and promote diabetic neovascularization. Adult male C57/B6 mice and streptozotocin (STZ)-induced type 1 diabetic mice were used. Gli1 and Ptc1 protein levels were reduced in EPCs from diabetic mice, indicating inhibition of the Shh signaling pathway. EPC migration, tube formation ability, and mobilization were impaired in diabetic mice compared with non-diabetic controls (p < 0.05 vs control), and all were improved by in vivo administration of the Shh pathway receptor agonist SAG (p < 0.05 vs diabetes). SAG significantly increased capillary density and blood perfusion in the ischemic hindlimbs of diabetic mice (p < 0.05 vs diabetes). The AKT activity was lower in EPCs from diabetic mice than those from non-diabetic controls (p < 0.05 vs control). This decreased AKT activity led to an increased GSK-3ß activity and degradation of the Shh pathway transcription factor Gli1/Gli2. SAG significantly increased the activity of AKT in EPCs. Our data clearly demonstrate that an impaired Shh pathway mediated by the AKT/GSK-3ß pathway can contribute to EPC dysfunction in diabetes and thus activating the Shh signaling pathway can restore both the number and function of EPCs and increase neovascularization in type 1 diabetic mice.

[The role of JNK in apoptosis of renal tubular epithelial cells in diabetic rats with fluctuant high blood glucose].

OBJECTIVE: To explore the signal transduction mechanisms of apoptosis in renal tubular epithelial cells in diabetic rats with fluctuant high blood glucose. METHODS: Healthy SD rats were randomly divided into 3 groups: normal control group (A), stable high blood glucose group (B) and fluctuant high blood glucose group (C). Diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg), and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of ordinary insulin and glucose at different time point every day. The superoxide dismutase (SOD) activity and the content of malonaldehyde (MDA) in renal tissue homogenate were detected with colorimetry. The protein expression of Nox4 and JNK were examined by immunohistochemistry and Western blot. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). RESULTS: After 12 experimental weeks, significantly increased cell apoptosis, up-regulation of Nox4 and P-JNK expression in renal tubular epithelial cells were observed in B and C groups compared with those in A group. The MDA content increased and SOD activity decreased in renal tissue in B and C groups. Above effects were more obviously shown in C group. CONCLUSION: Fluctuant high blood glucose induced more apoptosis of renal tubular epithelial cell than stable high blood glucose in diabetic kidney, which might be related to the activation of JNK signal transduction pathway.

Inhibition of hepatitis C virus replication by single and dual small interfering RNA using an HCV-infected cell model.

Dual siRNA against different regions of gene in hepatitis C virus (HCV) synergistically inhibited replication of HCV RNA. An HCV-infected cell model was established, and HCV RNA and core protein were detected by RT-PCR and Western blot, respectively. Four HCV-specific siRNAs (siCore, siNS3, siNS4B, siNS5B) were designed and transfected into HCV-infected Huh7.5.1 cells. The antiviral efficacies of the siRNAs were compared using real time PCR and agarose gel electrophoresis. HCV replication in infected cells was inhibited by IFNα-2b in a dose-dependent manner. Synergistic inhibition effects were achieved with combination treatment of any two of the siRNAs (siCore, siNS3 and siNS5B) at low doses (0.1 and 10 nM), as compared to single siRNA treatment (P < 0.05). Furthermore, CCK-8 assay showed no toxicity of the siRNAs to Huh7.5.1 cells. These findings indicate a promising new therapeutic approach for treatment of HCV.

[Influence of miR-122 on IFN-α treatment for HCV infection].

OBJECTIVE: To investigate the influence of miR-122 on IFN-α treatment for HCV infection. METHODS: Huh7.5.1 cells infected with HCV were treated with miR-122 mimics (20 nmol/L, 100 nmol/L, 400 nmol/L) and/or IFN-α (1000 IU/ml). The relative expression of HCV RNA was detected by real-time polymerase chain reaction (PCR). Huh7.5.1 cells were treated with different amounts of HCV (107 copies, 106 copies and 105 copies) and/or IFN-α (1000 IU/ml). RESULTS: IFN-α suppressed the replication of HCV in a time-dependent manner, resulting in a ≊ 83% reduction of HCV at 48 h. MiR-122 mimics facilitated replication of HCV RNA in a dose-dependent manner (P<0.05). The antiviral effect of IFN-α was inverted to levels of miR-122 mimics (20 nmol/L, 100 nmol/L, 400 nmol/L), (73.3% ± 3.5% compared with 84% ± 4.5%, P>0.05; 64.67% ± 5.5% compared with 84% ± 4.5%, P>0.05; 56.33% ± 5.1% compared with 84% ± 4.5%, P<0.05). The antiviral effect of IFN-α was inverted to HCV load (105 copies group compared with 107 copies group, P<0.05). CONCLUSION: MiR-122 facilitates replication of HCV RNA in the cell culture system; and the expression of miR-122 may partly counteract the anti-HCV effect of IFN-α.

A nanochannel array-based electrochemical device for quantitative label-free DNA analysis.

A strategy for label-free oligonucleotide (DNA) analysis has been proposed by measuring the DNA-morpholino hybridization hindered diffusion flux of probe ions Fe(CN)(6)(3-) through nanochannels of a porous anodic alumina (PAA) membrane. The flux of Fe(CN)(6)(3-) passing through the PAA nanochannels is recorded using an Au film electrochemical detector sputtered at the end of the nanochannels. Hybridization of the end-tethered morpholino in the nanochannel with DNA forms a negatively charged DNA-morpholino complex, which hinders the diffusion of Fe(CN)(6)(3-) through the nanochannels and results in a decreased flux. This flux is strongly dependent on ionic strength, nanochannel aperture, and target DNA concentration, which indicates a synergetic effect of steric and electrostatic repulsion effects in the confined nanochannels. Further comparison of the probe flux with different charge passing through the nanochannels confirms that the electrostatic effect between the probe ions and DNA dominates the hindered diffusion process. Under optimal conditions, the present nanochannel array-based DNA biosensor gives a detection limit of 0.1 nM.

Real-time monitoring of mass-transport-related enzymatic reaction kinetics in a nanochannel-array reactor.

To understand the fundamentals of enzymatic reactions confined in micro-/nanosystems, the construction of a small enzyme reactor coupled with an integrated real-time detection system for monitoring the kinetic information is a significant challenge. Nano-enzyme array reactors were fabricated by covalently linking enzymes to the inner channels of a porous anodic alumina (PAA) membrane. The mechanical stability of this nanodevice enables us to integrate an electrochemical detector for the real-time monitoring of the formation of the enzyme reaction product by sputtering a thin Pt film on one side of the PAA membrane. Because the enzymatic reaction is confined in a limited nanospace, the mass transport of the substrate would influence the reaction kinetics considerably. Therefore, the oxidation of glucose by dissolved oxygen catalyzed by immobilized glucose oxidase was used as a model to investigate the mass-transport-related enzymatic reaction kinetics in confined nanospaces. The activity and stability of the enzyme immobilized in the nanochannels was enhanced. In this nano-enzyme reactor, the enzymatic reaction was controlled by mass transport if the flux was low. With an increase in the flux (e.g., >50 microL min(-1)), the enzymatic reaction kinetics became the rate-determining step. This change resulted in the decrease in the conversion efficiency of the nano-enzyme reactor and the apparent Michaelis-Menten constant with an increase in substrate flux. This nanodevice integrated with an electrochemical detector could help to understand the fundamentals of enzymatic reactions confined in nanospaces and provide a platform for the design of highly efficient enzyme reactors. In addition, we believe that such nanodevices will find widespread applications in biosensing, drug screening, and biochemical synthesis.

Study on the kinetics of homogeneous enzyme reactions in a micro/nanofluidics device.

In this paper, a micro/nanofluidic preconcentration device integrated with an electrochemical detector has been used to study the enrichment of enzymes and homogeneous enzyme reaction kinetics. The enzymes are first concentrated in front of a nanochannel via an exclusion-enrichment effect (EEE) mechanism of the nanochannel integrated in a microfluidics device. If a substrate is electrokinetically transported to the concentrated enzymes, homogeneous enzymatic reaction occurs. The enzymatic reaction product can penetrate through the nanochannel to be detected electrochemically. In this device, the enriched enzymes can be well retained and repeatedly used, thus, the enzymatic reaction occurs in a continuous-flow mode. For demonstration, Glucose oxidase (GOx) was chosen as the model enzyme to study the influence of enzyme concentration on its reaction kinetics. The different concentration of GOx in front of the nanochannel was simply achieved by using different enrichment time. When substrate glucose was introduced electrokinetically, a rapid electrochemical steady-state response could be obtained. It was found that the electrochemical response to a constant glucose concentration increased with the increase of enzyme enrichment time, which is expected for homogeneous enzymatic reactions. Under proper conditions, the electrochemical responds linearly to the glucose concentration ranging from 0 to 15 mM, and the Michaelis constants (K(m)) are relatively low, which indicates a more efficient complex formation between enzyme and substrate. These results suggest that the present micro/nanofluidics device is promising for the study of enzymatic reaction kinetics and other bioassays such as cell assays, drug discovery, and clinical diagnosis.

[CO2 flux and its correlation with soil temperature in winter wheat growth season under different tillage measures].

Based on a long-term experiment, this paper investigated the CO2 flux in winter wheat growth season under different tillage measures, and simulated the correlation between the flux and soil temperature by regression method. The results indicated that under different tillage measures, the average CO2 emission rate in winter wheat growth season was in the order of moldboard plow tillage (F) > minimum tillage (X) > no-tillage (M), with the value being 343.69, 337.54 and 190.47 mg x m(-2) x h(-1), respectively. There was a significant exponential correlation between CO2 flux and soil temperature, which was most significant for the soil temperature at the depth of 10 cm under F, and relatively significant for the soil temperature at the depth of 10 and 20 cm under X and M. Simulating with the soil temperature at the depth of 10 cm, the CO2 flux in winter wheat growth season was estimated as 1.88 kg x m(-2) under F, 1.89 kg x m(-2) under X, and 1.03 kg x m(-2) under M.