Tissue factor pathway inhibitors disrupt structures of rhabdovirus/ranairidovirus and inhibit viral infection in Chinese perch, Siniperca chuatsi.

Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.

A Pyroglutamate Aminopeptidase 1 Responsive Fluorescence Imaging Probe for Real-Time Rapid Differentiation between Thyroiditis and Thyroid Cancer.

Current diagnostic methods for thyroid diseases, including blood tests, ultrasound, and biopsy, always have difficulty diagnosing thyroiditis accurately, occasionally mistaking it for thyroid cancer. To address this clinical challenge, we developed Ox-PGP1, a novel fluorescent probe realizing rapid, noninvasive, and real-time diagnostic techniques. This is the first imaging tool capable of noninvasively distinguishing between thyroiditis and thyroid cancer. Ox-PGP1 was introduced as a fluorescent probe custom-built for the specific detection and quantification of pyroglutamate aminopeptidase 1 (PGP-1), a known pivotal biomarker of inflammation. Ox-PGP1 overcame the disadvantages of traditional enzyme-responsive fluorescent probes that relied on the intramolecular charge transfer (ICT) mechanism, including the issue of high background fluorescence, while offering exceptional photostability under laser irradiation. The spectral properties of Ox-PGP1 were meticulously optimized to enhance its biocompatibility. Furthermore, the low limit of detection (LOD) of Ox-PGP1 was determined to be 0.09 µg/mL, which demonstrated its remarkable sensitivity and precision. Both cellular and in vivo experiments validated the capacity of Ox-PGP1 for accurate differentiation between normal, inflammatory, and cancerous thyroid cells. Furthermore, Ox-PGP1 showed the potential to rapidly and sensitively differentiate between autoimmune thyroiditis and anaplastic thyroid carcinoma in a mouse model, achieving results in just 5 min. The successful design and application of Ox-PGP1 represent a substantial advancement in technology over traditional diagnostic approaches, potentially enabling earlier interventions for thyroid diseases.

A Dual-Mode Fluorescent Nanoprobe for the Detection and Visual Screening of Pathogenic Bacterial Spores.

Bacterial vegetative cells turn into metabolically dormant spores in certain environmental situations. Once suitable conditions trigger the germination of spores belonging to the pathogenic bacterial category, public safety and environmental hygiene will be threatened, and lives will even be endangered when encountering fatal ones. Instant identification of pathogenic bacterial spores remains a challenging task, since most current approaches belonging to complicated biological methods unsuitable for onsite sensing or emerging alternative chemical techniques are still inseparable from professional instruments. Here we developed a polychromatic fluorescent nanoprobe for ratiometric detection and visual inspection of the pathogenic bacterial spore biomarker, dipicolinic acid (DPA), realizing rapidly accurate screening of pathogenic bacterial spores such as Bacillus anthracis spores. The nanoprobe is made of aminoclay-coated silicon nanoparticles and functionalized with europium ions, exhibiting selective and sensitive response toward DPA and Bacillus subtilis spores (simulants for Bacillus anthracis spores) with excellent linearity. The proposed sensing strategy allowing spore determination of as few as 0.3 × 105 CFU/mL within 10 s was further applied to real environmental sample detection with good accuracy and reliability. Visual quantitative determination can be achieved by analyzing the RGB values of the corresponding test solution color via a color recognition APP on a smartphone. Different test samples can be photographed at the same time, hence the efficient accomplishment of examining bulk samples within minutes. Potentially employed in various on-site sensing occasions, this strategy may develop into a powerful means for distinguishing hazardous pathogens to facilitate timely and proper actions of dealing with multifarious security issues.

Synthesis of in-situ magnetized MOF-cellulose membranes for high-efficiency enrichment of diamide insecticides in vegetables and determination by LC-MS/MS.

This study presents a novel, eco-friendly composite adsorbent material designed for the magnetic solid-phase extraction of diamide insecticides from vegetable samples. The membrane, denoted as Fe-MMm, was incorporated with a cellulose framework embedded with Metal-Organic Frameworks (MOFs) and Multi-Walled Carbon Nanotubes (MWCNTs) magnetized with Fe3O4. This innovative material streamlined the conventional solid-phase extraction process, simplifying the sample pre-treatment. By combining it with liquid chromatography tandem mass spectrometry (LC-MS/MS), the method achieves significantly enhanced extraction efficiency through systematic optimization of experimental parameters, including adsorbent selection, pH, ionic strength, adsorption time, and elution time. The method had a wide linear range of 0.1-1000 ng/mL and an exceptionally low detection limit ranging from 0.023 to 0.035 ng/mL. The successful identification of diamide insecticides in vegetable samples underscores the potential of Fe-MMm as a robust material for sample pretreatment in analytical applications.

Visualization of production and remediation of acetaminophen-induced liver injury by a carboxylesterase-2 enzyme-activatable near-infrared fluorescent probe.

Acetaminophen (APAP) overdose, also known as APAP poisoning, may directly result in hepatic injury, acute liver failure and even death. Nowadays, APAP-induced liver injury (AILI) has become an urgent public health issue in the developing world so the early accurate diagnosis and the revelation of underlying molecular mechanism of AILI are of great significance. As a major detoxifying organ, liver is responsible for metabolizing chemical substances, in which human carboxylesterase-2 (CES2) is present. Hence, we chose CES2 as an effective biomarker for evaluating AILI. By developing a CES2-activatable and water-soluble fluorescent probe PFQ-E with superior affinity (Km = 5.9 µM), great sensitivity (limit of detection = 1.05 ng/mL), near-infrared emission (655 nm) and large Stokes shift (135 nm), activity and distribution of CES2 in cells were determined or imaged effectively. More importantly, the APAP-induced hepatotoxicity and the underlying molecular mechanism of pathogenesis of AILI were investigated by measuring the "light-up" response of PFQ-E towards endogenous CES2 in vivo for the first time. Based on the superior performance of the probe PFQ-E for sensing CES2, we believe that it has broad potential in clinical diagnosis and therapy response evaluation of AILI.

Visualizing Dipeptidyl Peptidase-IV with an Advanced Non-π-Conjugated Fluorescent Probe for Early Thyroid Disease Diagnosis.

Early detection and effective treatment of thyroid cancer are vital due to the aggressiveness and high mortality rate of the cancer. Nevertheless, the exploration of dipeptidyl peptidase-IV (DPP-IV) as a biomarker for thyroid diseases has not been widely conducted. In this study, we developed a novel non-π-conjugated near-infrared fluorescent probe, MB-DPP4, specifically designed to visualize and detect endogenous DPP-IV. Traditional DPP-IV-specific fluorescent probes rely primarily on the intramolecular charge transfer mechanism. For this reason, these probes are often hampered by high background levels that can inhibit their ability to achieve a fluorescence turn-on effect. MB-DPP4 successfully surmounts several drawbacks of traditional DPP-IV probes, boasting unique features such as exceptional selectivity, ultrahigh sensitivity (0.29 ng/mL), innovative structure, low background, and long-wavelength fluorescence. MB-DPP4 is an "off-on" chemosensor that exhibits strong fluorescence at 715 nm and releases a methylene blue (MB) fluorophore upon interacting with DPP-IV, resulting in a visible color change from colorless to blue. Given these remarkable attributes, MB-DPP4 shows great promise as a versatile tool for advancing research on biological processes and for evaluating the physiological roles of DPP-IV in living systems. Finally, we conducted a comprehensive investigation of DPP-IV expression in human serum, urine, thyroid cells, and mouse thyroid tumor models. Our findings could potentially establish a foundation for the early diagnosis and treatment of thyroid diseases.

Spindle Monitor: A Tool for Real-Time Assessment and Concurrent Treatment of Postoperative Tumor Prognosis.

Cancer surgery remains a mainstay in clinical treatment. However, the efficacy of subsequent therapies largely depends on the precise evaluation of postoperative prognoses, underscoring the critical need for a comprehensive and accurate assessment of surgical outcomes. Nanoprobes targeting tumors offer a promising solution for visual prognostic assessment. In this study, we developed a "Spindle Monitor" system, designated as APPADs (Au NBPs@PDA-pep-AS1411-Dox), composed of core-shell nanoparticles. The core was made up of gold nanobipyramids (Au NBPs), coated with polydopamine (PDA), and subsequently loaded with peptide chains, AS1411, and doxorubicin (Dox). Upon deployment in the acidic tumor microenvironment (TME), APPADs released substantial amounts of Dox, initiating the apoptotic process. This triggered the activity of caspase-3, which is a crucial executor in the apoptotic pathway. Consequently, DEVD, a specific recognition site for caspase-3, was cleaved, enabling the disconnection of FITC-conjugated peptide chains and the recovery of fluorescence. Through assessing this fluorescence imaging effect, local laser irradiation could be precisely guided to the postoperative site, facilitating a synergistic combination of photothermal therapy and chemotherapy. Specifically, our "Spindle Monitor" APPADs had been validated to achieve accurate fluorescence imaging in vitro and in vivo, which demonstrated its potential value as a versatile tool for evaluating postoperative prognosis in surgical treatments, such as thyroid cancer, and assessing chemotherapy efficacy in difficult cases, like late-stage osteosarcoma. This promising tool lays a good foundation for development in visual prognosis evaluation after tumor surgery.

Customized fluorescent probe for peering into the expression of butyrylcholinesterase in thyroid cancer.

BACKGROUND: Thyroid cancer has been increasingly prevalent in recent years. The main diagnostic methods for thyroid are B-ultrasound scan, serum detection and puncture detection. However, these methods are invasive and complex. It is a pressing need to develop non-invasive or minimally invasive methods for thyroid cancer diagnosis. Fluorescence method as a non-invasive detection method has attracted much attention. Butyrylcholinesterase (BChE) is a common enzyme in the human body, and many diseases affect its reduction. We found that BChE is also a marker for thyroid cancer. Therefore, it is of certain clinical value to explore the expression of BChE in thyroid cancer cells through a customized fluorescent probe to provide valuable experimental data and clues for studying the expression of thyroid cancer marker to reflect thyroid status. RESULTS: In this study, we customized a fluorescent probe named Kang-BChE, which is easy to synthesize with a high yield. The experimental results show that the probe Kang-BChE can detect BChE in the linear range of 0-900 U L-1 (R2 = 0.9963), and the detection limit is as low as 3.93 U L-1 (λex/em = 550/689 nm). In addition, Kang-BChE probes have low cytotoxicity, good specificity, and can completely eliminate interference from acetylcholinesterase (AChE). Kang-BChE showed excellent stability in the detection of complex biological samples in serum recovery experiments (95.64-103.12 %). This study was the first time using Kang-BChE to study the low expression of BChE in thyroid cancer cells (Tpc-1 cells). In addition, we observed that H2O2 concentration in Tpc-1 cells was positively correlated with BChE activity. SIGNIFICANCE: Kang-BChE is expected to be an important tool for monitoring the change of BChE content in complex biological environments due to its excellent performance. Kang-BChE can also be used to explore the influence of molecules in more organisms on the change of BChE content due to its excellent anti-interference ability. We expect that Kang-BChE can play a significant role in the clinical diagnosis and treatment of thyroid cancer.

Leucine Aminopeptidase-Mediated Multifunctional Molecular Imaging Tool for Diagnosis, Drug Evaluation, and Surgical Guidance of Liver-Related Diseases.

Traditional molecular imaging tools used for detecting liver diseases own several drawbacks, such as poor optical performance and limited applicability. Monitoring the concentration of leucine aminopeptidase (LAP), which is closely related to liver diseases such as liver cancer and liver injury, and analyzing it in diagnosis, drug evaluation, and surgical treatment is still a challenging task. Herein, we construct an intramolecular charge-transfer mechanism-based, ultrasensitive, near-infrared fluorescent probe (LAN-lap) for dynamic monitoring of LAP fluctuations in living systems. LAN-lap, with high specificity, stability, sensitivity, and water solubility, can achieve in vitro monitoring of LAP through both fluorescence and colorimetric methods. Moreover, LAN-lap can successfully be used for the localization imaging of endogenous LAP, confirming the upregulation of LAP expression in liver cancer and liver injury cells. In addition, LAN-lap can realize the imaging of liver tumors in living organisms. Meanwhile, it can intuitively present the degree of drug-induced liver injury, achieving semi-quantitative imaging evaluation of the hepatotoxicity of two drugs. Furthermore, LAN-lap can track liver cancer tumors in mice with peritoneal metastasis and can assist in fluorescence-guided surgical resection of liver cancer tumors. This multifunctional LAN-lap probe could play an important role in facilitating simultaneous diagnoses, imaging, and synergistic surgical navigation to achieve better point-of-care therapeutic efficacy.

A quasi-experimental analysis on the causal effects of COVID-19 on urban park visits: The role of park features and the surrounding built environment.

Although many studies have explored the correlations between mobility intervention policies and park use during COVID-19, only a few have used causal inference approaches to assessing the policy's treatment effects and how such effects vary across park features and surrounding built environments. In this study, we develop an interrupted time-series quasi-experimental design based on three-month mobile phone big data to infer the causal effects of mobility intervention policies on park visits in Shenzhen, including the first-level response (FLR) and return-to-work (RTW) order. The results show that the FLR caused an abrupt decline of 2.21 daily visits per park, with a gradual reduction rate of 0.54 per day, whereas the RTW order helped recover park visits with an immediate increase of 2.20 daily visits and a gradual growth rate of 0.94 visits per day. The results also show that the impact of COVID-19 on park visits exhibited social and spatial heterogeneities: the mobility-reduction effect was smaller in low-level parks (e.g., community-level parks) with small sizes but without sports facilities and water scenes, whereas parks surrounded by compact neighborhoods and land use were more impacted by the pandemic. These findings provide planners with important insights into resilient green space and sustainable neighborhood planning for the post-COVID era.

A Strategy for the Determination of Alkaline Phosphatase Based on the Self-Triggered Degradation of Metal-Organic Frameworks by Phosphate.

Alkaline phosphatase (ALP) is widely present in the human body and is an important biomarker. Numerous ALP detection studies have been carried out, and ascorbic acid (AA) is often used as the reducing component in the sensors to monitor ALP levels since it can be produced from ascorbic acid 2-phosphate (AA2P) hydrolysis in the presence of ALP. However, it is well-known that AA is a strong reducing agent and can be easily oxidized. The disproportion between oxidized AA and reduced AA reactions results in the generation of AA free radicals with single electrons that may lead to inaccurate results in assays. To solve this problem, we synthesized a core-shell metal-organic framework sensor (PATP-Au@ZIF-8 NP) and used it as a sensitive and accurate ALP detection sensor with self-triggered control of phosphate ions (Pi) to avoid the potential inaccuracy of the method that uses AA as the reducing component. By establishing a physical shell on the surface of the gold nanoparticles (Au NPs), the sensor not only can eliminate the random assembly of metal nanoparticles caused by plasma exposure but also can generate self-triggering of Pi caused by ALP. Pi can decompose ZIF-8 through coordination with Zn2+ and thus can destroy the ZIF-8 shell structure of the prepared PAZ NPs. Au NPs are released and then become aggregated, in turn causing the SERS "hot spot" area to increase. The enhancement of the SERS signals was found to be directly associated with the level of Pi released from ALP-triggered hydrolysis. The response of the strategy was linear at ALP concentrations ranging from 0.1 to 150 mU/mL (r = 0.996) with a detection limit of 0.03 mU/mL. Lastly, the developed strategy was employed in the evaluation of ALP inhibitors, and the possibility to implement the developed SERS strategy for rapid and selective analysis of ALP in human serum was demonstrated.

Rapid and sensitive detection of pathogenic Elizabethkingia miricola in black spotted frog by RPA-LFD and fluorescent probe-based RPA.

Elizabethkingia miricola is a highly infectious pathogen, which causes high mortality rate in frog farming. Therefore, it is urgent to develop a rapid and sensitive detection method. In this study, two rapid and specific methods including recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) and fluorescent probe-based recombinase polymerase amplification (exo RPA) were established to effectively detect E. miricola, which can accomplish the examination at 38 °C within 30 min. The limiting sensitivity of RPA-LFD and exo RPA (102 copies/µL) was ten-fold higher than that in generic PCR assay. The specificities of the two methods were verified by detecting multiple DNA samples (E. miricola, Staphylococcus aureus, Aeromonas hydrophila, Aeromonas veronii, CyHV-2 and Edwardsiella ictaluri), and the result showed that the single band was displayed in E. miricola DNA only. By tissue bacterial load and qRT-PCR assays, brain is the most sensitive tissue. Random 24 black spotted frog brain samples from farms were tested by generic PCR, basic RPA, RPA-LFD and exo RPA assays, and the results showed that RPA-LFD and exo RPA methods were able to detect E. miricola accurately and rapidly. In summary, the methods of RPA-LFD and exo RPA were able to detect E. miricola conveniently, rapidly, accurately and sensitively. This study provides prospective methods to detect E. miricola infection in frog culture.

Extraction of parabens by melamine sponge with determination by high-performance liquid chromatography.

In the present study, we propose a novel method for the extraction of parabens in personal care products. A new, simple adsorptive material was obtained by combining metal-organic frameworks and melamine sponges using the adhesive property of polyvinylidene fluoride. This new material, metal-organic frameworks/melamine sponges, was found to be particularly suitable for solid-phase extraction. The structural characteristics of metal-organic frameworks/melamine sponges were first analyzed by scanning electron microscopy. Subsequently, solid-phase extraction was performed on sample solutions, and the extracted substances were then analyzed by high-performance liquid chromatography. Following optimization of important experimental conditions, excellent recovery rates were obtained. Our novel method was then applied to the extraction of four parabens (methylparahydroxybenzoates, ethylparahydroxybenzoates, propylparahydroxybenzoates, and butylparahydroxybenzoates) from real samples. The results yielded limits of detection of 0.26-0.41 ng/mL. The inter- and intra-day recoveries were 104.0-109.7% and 91.2-98.1%, respectively (relative standard deviation, <13.8%).

A semi-automatic solid phase extraction system based on MIL-101(Cr) foam-filled syringe for detection of triazines in vegetable oils.

In this study, several metal-organic framework-melamine foam columns were first developed and used as a laboratory-made semi-automatic solid phase extraction packed in syringe adsorber for the extraction of six triazine herbicides from vegetable oil samples coupled to high-performance liquid chromatography-tandem mass spectrometry. The metal-organic framework-foam columns were prepared using a simple approach by embedding the solid particles in melamine foam using polyvinylidene difluoride physical encapsulation. The method was applicable to a wide variety of metal-organic framework materials, and the incorporated materials retained their unique properties. Key factors that affect the extraction efficiency, including the MIL-101(Cr) amount, sample flow rate, type and volume of the eluting solvent, and flow rate of eluting solvent, were investigated. Under optimum conditions, the proposed method exhibited low limits of detection (0.017-0.096 ng/mL, S/N = 3) for six triazines. The relative standard deviations calculated for all herbicides ranged from 0.2 to 14.9%. This study demonstrated that the MIL-101(Cr)-foam column can be used as a high-quality adsorption material for the detection of triazines in vegetable oils.

Vortex-assisted solid-phase extraction based on metal-organic framework/chitosan-functionalized hydrophilic sponge column for determination of triazine herbicides in environmental water by liquid chromatography-tandem mass spectrometry.

In the presented work, MIL-101(Cr) and chitosan were directly embedded on the skeleton of melamine sponge material using a simple and environmentally friendly method. Chitosan acts not only as an adhesive during the preparation of functionalized sponges, but also as an adsorption adjuvant in herbicide detection. Unlike other polymers, chitosan has excellent hydrophilicity and contains numerous adsorption sites; thus, it enables the sponge material to be used for determination of contaminants in an aqueous phase. Scanning electron microscopic (SEM) analysis showed that the coating material was uniformly distributed on the skeleton of melamine sponge. The prepared material was used as a sorbent in a vortex-assisted solid-phase extraction and combined with high performance liquid phase tandem mass spectrometry for the extraction and trace determination of six triazines in water samples (Atraton, Desmetryn, Prometon, Ametryn, Prometryn and Dimethametryn). Several parameters that affect the extraction efficiencies were investigated. Under the optimal conditions (MIL-101(Cr) loading, 150 mg; sample pH, 7; salt concentration, 0%; adsorption time, 3 min; desorption solvent, 1.5 mL acetonitrile; desorption time, 4 min), the proposed method was successfully used in the determination of trace triazines in five real water samples (drinking water, tap water, lake waters and river water), satisfactory recoveries were obtained in the range of 78.9%-118.6%. The limits of detection of the proposed method in detecting triazine herbicides in spiked water samples ranged from 0.014 to 0.045 ng mL-1.

One-step fabrication of hydrophilic MIL-68(Al)/Chitosan-coated melamine sponge for vortex-assisted solid-phase extraction of parabens in water samples.

A novel vortex-assisted solid-phase extraction method based on MIL-68(Al)/Chitosan-melamine sponge column (or V-MIL-68(Al)/CS-SC-SPE) is presented in this paper. The MIL-68(Al)/Chitosan-sponge column was prepared by a simple infiltration method and a preparation process that does not consume organic solvents. Scanning electron microscopy was used to characterize the functionalized sponge columns, and the skeleton and pores of the melamine sponge were successfully modified with the coating material (MIL-68(Al) and chitosan). Chitosan was used successfully not only as an adsorption adjuvant material, but also as an adhesive in the preparation of MIL-68(Al)/CS coating sponge materials. The presented method was further combined with high performance liquid chromatography (HPLC) and used in the determination of trace parabens (including methyl-, ethyl-, propyl- and butyl-parabens) in environmental water samples. Several important factors that affect the extraction performance were investigated. Under the optimum conditions, the method was successfully used to detect the four kinds of parabens in different water samples (domestic water, lake water and river water). The limits of detection ranged between 0.21 and 0.50 ng/mL, and the relative standard deviations for all parabens were below 10.8%.

MIL-101(Cr)/MWCNTs-functionalized melamine sponges for solid-phase extraction of triazines from corn samples, and their subsequent determination by HPLC-MS/MS.

In the presented work, we proposed the use of polyvinylidene difluoride (PVDF) as a coupling agent to load MIL-101(Cr) and carboxylated multi-walled carbon nanotubes (MWCNTs) on the skeleton of melamine sponge (MeS) for the preparation of novel extraction media with high-efficiency. The morphology and structure of this composite were characterized by scanning electron microscopy (SEM) analyses. The obtained MIL-101(Cr)/MWCNTs functionalized MeS(MIL/M-MeS) cube was used as a sorbent for the solid-phase extraction (SPE) of six triazines (Atraton, Simetryne, Prometon, Ametryn, Propazine, and Prometryn) spiked corn samples. Several parameters that may affect the extraction efficiencies, including type and volume of extraction solvent, times of ultrasonic extraction, adsorption, and desorption; type and volume of desorption solvent, were optimized. This was followed by elution containing the herbicides, which were quantified using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Under optimum conditions, the analytical method for the preparation of MIL/M-MeS cube produced satisfactory recoveries and repeatability. The LODs of the method when applied to spiked corn samples were in the range of 0.01-0.04 ng g-1 (LODs = 3δ/k). The relative intra- and inter-day recoveries ranged from 90.30 to 116.50% and 92.38-116.24%, respectively, and relative standard deviations ranged from 1.08 to 12.32% and 5.36-16.23%, respectively.

Vector promoters used in Klebsiella pneumoniae.

Much effort has been devoted to the metabolic engineering of Klebsiella pneumoniae; however, our knowledge of the actual expression level of promoters used in K. pneumoniae is limited. In this study, the expression levels of three promoters were compared systematically by using the lacZ reporter gene with different carbon sources in K. pneumoniae. The results showed that, although promoters PT5 and Ptac designed for Escherichia coli were functional, PT5 appeared more efficient and the induction/repression ratio of Ptac was decreased extremely in K. pneumoniae. The basal level of Ptac for lacZ expression reached 396.5 U/mg, which was 9.5-fold higher compared with PT5 in LB medium, indicating Ptac can be used as an efficient "constitutive" promoter as well as an efficient induced promoter in K. pneumoniae. In different carbon sources medium, a newly constructed endogenous constitutive Pbud proved to be a stable and weak promoter. On the basis of our data, a set of Pbud and Ptac promoters could meet the broad range (about 1,000 orders of magnitude) of gene expression needed for engineered K. pneumoniae in glycerol-based medium.