Simulating the land use change effects on non-point source pollution in the Duliujian River Basin.

The uncertainty in the generation and formation of non-point source pollution makes it challenging to monitor and control this type of pollution. The SWAT model is frequently used to simulate non-point source pollution in watersheds and is mainly applied to natural watersheds that are less affected by human activities. This study focuses on the Duliujian River Basin (Xiqing section), which is characterized by a dense population and rapid urbanization. Based on the calibrated SWAT model, this study analyzed the effects of land use change on non-point source pollution both temporally and spatially. It was found that nitrogen and phosphorus non-point source pollution load losses were closely related to land use type, with agricultural land and high-density urban land (including rural settlements) being the main contributors to riverine nitrogen and phosphorus pollution. This indicates the necessity of analyzing the impact of land use changes on non-point source pollution loads by identifying critical source areas and altering the land use types that contribute heavily to pollution in these areas. The simulation results of land use type changes in these critical source areas showed that the reduction effect on non-point source pollution load is in the order of forest land > grassland > low-density residential area. To effectively curb surface source pollution in the study area, strategies such as modifying urban land use types, increasing vegetation cover and ground infiltration rate, and strictly controlling the discharge of domestic waste and sewage from urban areas can be implemented.

Spatially Decoupled H2O2 Activation Pathways and Multi-Enzyme Activities in Rod-Shaped CeO2 with Implications for Facet Distribution.

CeO2, particularly in the shape of rod, has recently gained considerable attention for its ability to mimic peroxidase (POD) and haloperoxidase (HPO). However, this multi-enzyme activities unavoidably compete for H2O2 affecting its performance in relevant applications. The lack of consensus on facet distribution in rod-shaped CeO2 further complicates the establishment of structure-activity correlations, presenting challenges for progress in the field. In this study, the HPO-like activity of rod-shaped CeO2 is successfully enhanced while maintaining its POD-like activity through a facile post-calcination method. By studying the spatial distribution of these two activities and their exclusive H2O2 activation pathways on CeO2 surfaces, this study finds that the increased HPO-like activity originated from the newly exposed (111) surface at the tip of the shortened rods after calcination, while the unchanged POD-like activity is attributed to the retained (110) surface in their lateral area. These findings not only address facet distribution discrepancies commonly reported in the literature for rod-shaped CeO2 but also offer a simple approach to enhance its antibacterial performance. This work is expected to provide atomic insights into catalytic correlations and guide the design of nanozymes with improved activity and reaction specificity.

Identification of Hedyotis diffusa Willd-specific mRNA-miRNA-lncRNA network in rheumatoid arthritis based on network pharmacology, bioinformatics analysis, and experimental verification.

Hedyotis diffusa Willd (HDW) possesses heat-clearing, detoxification, anti-cancer, and anti-inflammatory properties. However, its effects on rheumatoid arthritis (RA) remain under-researched. In this study, we identified potential targets of HDW and collected differentially expressed genes of RA from the GEO dataset GSE77298, leading to the construction of a drug-component-target-disease regulatory network. The intersecting genes underwent GO and KEGG analysis. A PPI protein interaction network was established in the STRING database. Through LASSO, RF, and SVM-RFE algorithms, we identified the core gene MMP9. Subsequent analyses, including ROC, GSEA enrichment, and immune cell infiltration, correlated core genes with RA. mRNA-miRNA-lncRNA regulatory networks were predicted using databases like TargetScan, miRTarBase, miRWalk, starBase, lncBase, and the GEO dataset GSE122616. Experimental verification in RA-FLS cells confirmed HDW's regulatory impact on core genes and their ceRNA expression. We obtained 11 main active ingredients of HDW and 180 corresponding targets, 2150 RA-related genes, and 36 drug-disease intersection targets. The PPI network diagram and three machine learning methods screened to obtain MMP9, and further analysis showed that MMP9 had high diagnostic significance and was significantly correlated with the main infiltrated immune cells, and the molecular docking verification also showed that MMP9 and the main active components of HDW were well combined. Next, we predicted 6 miRNAs and 314 lncRNAs acting on MMP9, and two ceRNA regulatory axes were obtained according to the screening. Cellular assays indicated HDW inhibits RA-FLS cell proliferation and MMP9 protein expression dose-dependently, suggesting HDW might influence RA's progression by regulating the MMP9/miR-204-5p/MIAT axis. This innovative analytical thinking provides guidance and reference for the future research on the ceRNA mechanism of traditional Chinese medicine in the treatment of RA.

Enzyme-Directed and Organelle-Specific Sphere-to-Fiber Nanotransformation Enhances Photodynamic Therapy in Cancer Cells.

Employing responsive nanoplatforms as carriers for photosensitizers represents an effective strategy to overcome the challenges associated with photodynamic therapy (PDT), including poor solubility, low bioavailability, and high systemic toxicity. Drawing inspiration from the morphology transitions in biological systems, a general approach to enhance PDT that utilizes enzyme-responsive nanoplatforms is developed. The transformation of phosphopeptide/photosensitizer co-assembled nanoparticles is first demonstrated into nanofibers when exposed to cytoplasmic enzyme alkaline phosphatase. This transition is primarily driven by alkaline phosphatase-induced changes of the nanoparticles in the hydrophilic and hydrophobic balance, and intermolecular electrostatic interactions within the nanoparticles. The resulting nanofibers exhibit improved ability of generating reactive oxygen species (ROS), intracellular accumulation, and retention in cancer cells. Furthermore, the enzyme-responsive nanoplatform is expanded to selectively target mitochondria by mitochondria-specific enzyme sirtuin 5 (SIRT5). Under the catalysis of SIRT5, the succinylated peptide/photosensitizer co-assembled nanoparticles can be transformed into nanofibers specifically within the mitochondria. The resulting nanofibers exhibit excellent capability of modulating mitochondrial activity, enhanced ROS formation, and significant anticancer efficacy via PDT. Consequently, the enzyme-instructed in situ fibrillar transformation of peptide/photosensitizers co-assembled nanoparticles provides an efficient pathway to address the challenges associated with photosensitizers. It is envisaged that this approach will further expand the toolbox for enzyme-responsive biomaterials for cancer therapy.

Inhibition of resurgent Na+ currents by rufinamide.

Na+ channels are essential for the genesis of action potentials in most neurons. After opening by membrane depolarization, Na+ channels enter a series of inactivated states (e.g. the fast, intermediate, and slow inactivated states; or If, Ii, and Is). The inactivated Na+ channel may recover via the open state upon membrane repolarization, giving rise to "resurgent" Na+ currents which could be critical for densely repetitive or burst discharges. We incubated CHO-K1 cells transfected with human NaV1.7 cDNA and measured resurgent currents with whole-cell patch recordings. We found Ii is the major inactivated state responsible for the genesis of resurgent currents. Rufinamide, in therapeutic concentrations, could selectively bind to Ii to slow the recovery process and dose-dependently inhibit resurgent currents. The other Na+ channel-inhibiting antiseizure medications (ASM), such as phenytoin and lacosamide (selectively binds to If and Is, separately), fail to show a similar inhibitory effect in clinically relevant concentrations. Resurgent currents are decreased with lengthening of the prepulse, presumably because of redistribution of the channel from Ii to If. Rufinamide could accentuate the decrease to mimic a use-dependent inhibitory effect. The molecular action of slowing of recovery from inactivation by binding to Ii also explains the highly correlative inhibitory effect of rufinamide on both transient and resurgent Na+ currents. The modest but correlative inhibition of both currents may make a novel synergistic effect and thus strong-enough suppression of pathological repetitive and especially burst discharges. Rufinamide may thus have a unique spectrum of therapeutic applications for disorders with excessive neural excitabilities.

Association of Chronic Kidney Disease with Cardiovascular Disease in Cancer Patients: A Cross-Sectional Study.

INTRODUCTION: Due to the cardiotoxicity of cancer treatment and traditional risk factors for cardiovascular disease (CVD) such as obesity, diabetes, dyslipidemia, and hypertension, cancer patients are at higher risk of developing CVD. However, limited research exists on the correlation between chronic kidney disease (CKD) and CVD risk in cancer patients. METHODS: This cross-sectional study selected cancer patients aged ≥20 years from the National Health and Nutrition Examination Survey (NHANES) conducted from 2015 to 2020. Multivariable logistic regression was used to assess the association between CKD and CVD in cancer patients. Additionally, subgroup analyses were conducted to investigate the association among different groups of cancer patients. RESULTS: We included 1,700 adult cancer patients (52.53% were females). After multivariable adjustment for covariates including traditional CVD factors, CKD was significantly associated with CVD, with an odds ratio (95% confidence interval) and p value of 1.61 (1.18, 2.19) and 0.004. Subgroup analyses after multivariable adjustment showed a significant correlation between CKD and increased CVD risk in the following cancer patients: age ≥60 years, males, white ethnicity, and individuals with or without traditional CVD factors (obesity, diabetes, dyslipidemia, and hypertension). CONCLUSIONS: CKD remains a significant factor in the higher risk of CVD among adult cancer patients in the United States, even after adjustment for traditional CVD risk factors. Therefore, to reduce the risk of CVD in cancer patients, it is important to treat CKD as a non-traditional risk factor for CVD and actively manage it.

Regulating the H2O2 Activation Pathway on a Well-Defined CeO2 Nanozyme Allows the Entire Steering of Its Specificity between Associated Enzymatic Reactions.

Nanozymes are promising alternatives to natural enzymes, but their use remains limited owing to poor specificity. For example, CeO2 activates H2O2 and displays peroxidase (POD)-like, catalase (CAT)-like, and haloperoxidase (HPO)-like activities. Since they unavoidably compete for H2O2, affecting its utilization in the target application, the precise manipulation of reaction specificity is thus imperative. Herein, we showed that one can simply achieve this by manipulating the H2O2 activation pathway on pristine CeO2 in well-defined shapes. This is because the coordination and electronic structures of Ce sites vary with CeO2 surfaces, wherein the (100) and (111) surfaces display nearly 100% specificity toward POD-/CAT-like and HPO-like activities, respectively. The antibacterial results suggest that the latter surface can well-utilize H2O2 to kill bacteria (cf., the former), which is promising for anti-biofouling applications. This work provides atomic insights into the synthesis of nanozymes with improved activity, reaction specificity, and H2O2 utilization.

Teleost skin microbiome: An intimate interplay between the environment and the host immunity.

The mucosal microbiome plays a role in regulating host health. The research conducted in humans and mice has governed and detailed the information on microbiome-host immunity interactions. Teleost fish, different from humans and mice, lives in and relies on the aquatic environment and is subjected to environmental variation. The growth of teleost mucosal microbiome studies, the majority in the gastrointestinal tract, has emphasized the essential role of the teleost microbiome in growth and health. However, research in the teleost external surface microbiome, as the skin microbiome, has just started. In this review, we examine the general findings in the colonization of the skin microbiome, how the skin microbiome is subjected to environmental change and the reciprocal regulation with the host immune system, and the current challenges that potential study models can address. The information collected from teleost skin microbiome-host immunity research would help future teleost culturing from the potential parasitic infestation and bacterial infection as foreseeing growing threats.

A Dye-Assisted Paper-Based Assay to Rapidly Differentiate the Stress of Chlorophenols and Heavy Metals on Enterococcus faecalis and Escherichia coli.

Biological toxicity testing plays an essential role in identifying the possible negative effects induced by substances such as organic pollutants or heavy metals. As an alternative to conventional methods of toxicity detection, paper-based analytical device (PAD) offers advantages in terms of convenience, quick results, environmental friendliness, and cost-effectiveness. However, detecting the toxicity of both organic pollutants and heavy metals is challenging for a PAD. Here, we show the evaluation of biotoxicity testing for chlorophenols (pentachlorophenol, 2,4-dichlorophenol, and 4-chlorophenol) and heavy metals (Cu2+, Zn2+, and Pb2+) by a resazurin-integrated PAD. The results were achieved by observing the colourimetric response of bacteria (Enterococcus faecalis and Escherichia coli) to resazurin reduction on the PAD. The toxicity responses of E. faecalis-PAD and E. coli-PAD to chlorophenols and heavy metals can be read within 10 min and 40 min, respectively. Compared to the traditional growth inhibition experiments for toxicity measuring which takes at least 3 h, the resazurin-integrated PAD can recognize toxicity differences between studied chlorophenols and between studied heavy metals within 40 min.

De Novo Designed Self-Assembling Rhodamine Probe for Real-Time, Long-Term and Quantitative Live-Cell Nanoscopy.

Super-resolution imaging provides a powerful approach to image dynamic biomolecule events at nanoscale resolution. An ingenious method involving tuning intramolecular spirocyclization in rhodamine offers an appealing strategy to design cell-permeable fluorogenic probes for super-resolution imaging. Nevertheless, precise control of rhodamine spirocyclization presents a significant challenge. Through detailed study of the structure-activity relationship, we identified that multiple key factors control rhodamime spirocyclization. The findings provide opportunities to create fluorogenic probes with tailored properties. On the basis of our findings, we constructed self-assembling rhodamine probes for no-wash live-cell confocal and super-resolution imaging. The designed self-assembling probe Rho-2CF3 specifically labeled its target proteins and displayed high ring-opening ability, fast labeling kinetics (<1 min), and large turn-on fold (>80 folds), which is very difficult to be realized by the existing methods. Using the probe, we achieved high-contrast super-resolution imaging of nuclei and mitochondria with a spatial resolution of up to 42 nm. The probe also showed excellent photostability and proved ideal for real-time and long-term tracking of mitochondrial fission and fusion events with high spatiotemporal resolution. Furthermore, Rho-2CF3 could resolve the ultrastructure of mitochondrial cristae and quantify their morphological changes under drug treatment at nanoscale. Our strategy thus demonstrates its usefulness in designing self-assembling probes for super-resolution imaging.

Patent mining on soil pollution remediation technology from the perspective of technological trajectory.

Recent years have seen a marked growth in soil environmental problems, however, the research & development (R&D) direction of soil pollution remediation technology (SPRT) for addressing related challenges to the global ecosystem is still unclear. Patent is the most effective carrier of technological information. Therefore, this study investigates the status and future direction of SPRT through the analysis and mining of 14,475 patents from 1971 to 2020. In 2006-2020, 14,435 SPRT patents (79% of the total) were published, which is in the development stage. By measuring the proportion of high-value patents, determined by the ratio of the number of patent families containing two or more patents (PF2) to that containing at least one patent (PF1), we found that United States (PF2/PF1 = 0.711), Japan (PF2/PF1 = 0.500), and South Korea (PF2/PF1 = 0.431) hold a monopoly. International patent organizations serve as a bridge for technology transfer. Patent CN101947539-A measured by structural hole index (Effective size = 98.194, Efficiency = 0.926) has the most significant technological influence. Therefore, in order to accomplish the technological transition and improve the soil remediation capacity, more attention should be paid to the microbial-assisted phytoremediation technology related to inorganic pollutants, hyperaccumulators and stabilizers. Additionally, patents CN102834190-A (Effective size = 23.930, Efficiency = 0.855, Constraint = 0.141, Hierarchy = 0.089) and CN105855289 (Effective size = 21.453, Efficiency = 0.795 Constraint = 0.149, Hierarchy = 0.086) are both at the location of structural holes. So, more research should be carried out on green and cost-effective solutions for reducing organic pollutants in soil remediation. The current study identifies opportunities for innovations and breakthroughs in SPRT and offers relevant information on technological development prospects.

A Graphene Oxide-based Covalent Resorufin-Conjugated Fluorescence "OFF-ON" Probe for Detection of Hydrazine.

The unique properties of graphene oxide (GO), such as good water solubility, non-toxicity, biocompatibility and cell permeability, make GO well suited for a number of biological applications. In this study, we explore the application of GO as an efficient fluorescent quencher for a highly fluorescent organic dye, resorufin (RF). The quenching effect of GO on resorufin is studied by noncovalent and covalent bonding of resorufin, respectively. The fluorescence is completely quenched when resorufin is covalently linked to GO; while resorufin's fluorescence could be still observed through noncovalent bonding to GO sheet. Interestingly, addition of hydrazine into RF-GO complex causes the fluorescence recovered, providing a potential ''OFF-ON'' fluorescence responsive probe for detecting hydrazine in vitro and in living cells.

Cannabidiol Selectively Binds to the Voltage-Gated Sodium Channel Nav1.4 in Its Slow-Inactivated State and Inhibits Sodium Current.

Cannabidiol (CBD), one of the cannabinoids from the cannabis plant, can relieve the myotonia resulting from sodium channelopathy, which manifests as repetitive discharges of muscle membrane. We investigated the binding kinetics of CBD to Nav1.4 channels on the muscle membrane. The binding affinity of CBD to the channel was evaluated using whole-cell recording. The CDOCKER program was employed to model CBD docking onto the Nav1.4 channel to determine its binding sites. Our results revealed no differential inhibition of sodium current by CBD when the channels were in activation or fast inactivation status. However, differential inhibition was observed with a dose-dependent manner after a prolonged period of depolarization, leaving the channel in a slow-inactivated state. Moreover, CBD binds selectively to the slow-inactivated state with a significantly faster binding kinetics (>64,000 M-1 s-1) and a higher affinity (Kd of fast inactivation vs. slow-inactivation: >117.42 µM vs. 51.48 µM), compared to the fast inactivation state. Five proposed CBD binding sites in a bundle crossing region of the Nav1.4 channels pore was identified as Val793, Leu794, Phe797, and Cys759 in domain I/S6, and Ile1279 in domain II/S6. Our findings imply that CBD favorably binds to the Nav1.4 channel in its slow-inactivated state.

Electronic-State Manipulation of Surface Titanium Activates Dephosphorylation Over TiO2 Near Room Temperature.

Dephosphorylation that removes a phosphate group from substrates is an important reaction for living organisms and environmental protection. Although CeO2 has been shown to catalyze this reaction, cerium is low in natural abundance and has a narrow global distribution (>90 % of these reserves are located within six countries). It is thus imperative to find another element/material with high worldwide abundance that can also efficiently extract the phosphate out of agricultural waste for phosphorus recycle. Using para-nitrophenyl phosphate (p-NPP) as a model compound, we demonstrate that TiO2 with a F-modified (001) surface can activate p-NPP dephosphorylation at temperatures as low as 40 °C. By probe-assisted nuclear magnetic resonance (NMR), it was revealed that the strong electron-withdrawing effect of fluorine makes Ti atoms (the active sites) on the (001) surface very acidic. The bidentate adsorption of p-NPP on this surface further promotes its subsequent activation with a barrier ≈20 kJ mol-1 lower than that of the pristine (001) and (101) surfaces, allowing the activation of this reaction near room temperature (from >80 °C).

[Effect of scalp acupuncture on cognitive function and self-care ability of daily life in patients with traumatic brain injury].

OBJECTIVE: To observe the therapeutic effect of scalp acupuncture on cognitive dysfunction of traumatic brain injury. METHODS: Seventy patients with cognitive dysfunction of traumatic brain injury were randomly divided into an observation group and a control group, 35 cases in each group. After treatment, 5 cases dropped off in each group. The patients in the control group were treated with cognitive training; the patients in the observation group were treated with cognitive training and scalp acupuncture at Baihui (GV 20), Sishencong (EX-HN 1), Zhisanzhen and Niesanzhen, and the needles were retained for 6 h. The two groups were treated once a day, 6 times a week; one-month treatment was taken as one course, and 3 continuous courses were given. The scores of mini-mental state examination (MMSE), Montreal cognitive assessment (MoCA), activity of daily living (ADL) and functional independence measure (FIM) were compared between the two groups before and after treatment. RESULTS: Compared before treatment, the MMSE and MoCA scores in the observation group, and ADL and FIM scores in the two groups were significantly increased after treatment (P<0.05), and the improvement of each index in the observation group was more significant than those in the control group (P<0.05). CONCLUSION: Scalp acupuncture could improve cognitive function and self-care ability of daily life in patients with traumatic brain injury.

A simple paper-based colorimetric analytical device for rapid detection of Enterococcus faecalis under the stress of chlorophenols.

Bacteria detection and toxicity measurement are essential in many aspects. Becoming increasingly popular in recent years, paper-based analytical devices (PADs) have proven to be cost-effective, portable and eco-friendly with quantitative diagnostic results. In this work, by a straightforward soaking-drying method, a resazurin-deposited PAD has been developed for rapid bacteria detection and biotoxicity measurement. The colorimetric response on the PAD was generated from metabolic reduction of resazurin by Enterococcus faecalis, a facultative anaerobic bacterial strain. After recording and quantifying the colorimetric response with Hue value by a smartphone, the bioassay on PAD enables the detection of resazurin reduction kinetics difference among bacteria at various densities in 10 min. Thereby, the bioassay on PAD was applied to study the toxicity of two chlorophenols, i.e. pentachlorophenol (PCP) and 4-chlorophenol (4-CP), to E. faecalis. Compared to growth-based inhibition test, which takes 5 h, this assay shows higher efficiency, i.e. in 30 min, the biotoxicity difference between PCP and 4-CP can be identified.

Single-step fluorescent probes to detect decrotonylation activity of HDACs through intramolecular reactions.

Lysine crotonylation plays vital roles in gene transcription and cellular metabolism. Nevertheless, methods for dissecting the molecular mechanisms of decrotonyaltion remains limited. So far, there is no single-step fluorescent method developed for enzymatic decrotonylation activity detection. The major difficulty is that the aliphatic crotonylated lysine doesn't allow π-conjugation to a fluorophore and decrotonylation can not modulate the electronic state directly. Herein, we have designed and synthesized two activity-based single-step fluorogenic probes KTcr-I and KTcr-II for detecting enzymatic decrotonylation activity. These two probes can be recognized by histone deacetylases and undergo intramolecular nucleophilic exchange reaction to generate fluorescence signal. Notably, peptide sequence-dependent effect was observed. KTcr-I can be recognized by Sirt2 more effectively, while KTcr-II with LGKcr peptide sequence preferentially reacted with HDAC3. Compared to other methods of studying enzymatic decrotonylation activity, our single-step fluorescent method has a number of advantages, such as facileness, high sensitivity, cheap facility and little material consumed. We envision that the probes developed in this study will provide useful tools to screen inhibitors which suppress the decrotonylation activity of HDACs. Such probes will be useful for further delineating the roles of decrotonylation enzyme and aid in biomarker identification and drug discovery.

Inhibition of neuronal Na+ currents by lacosamide: Differential binding affinity and kinetics to different inactivated states.

Lacosamide is a new-generation anticonvulsant acting on Na+ channels. Compared to the classic anticonvulsants targeting Na+ channels, lacosamide is unique in structure and in its molecular action requiring longer membrane depolarization. Selective binding to the slow inactivated state of Na+ channels was then advocated for lacosamide, although slow binding to the fast inactivated state was alternatively proposed recently. In addition, quantitative characterization of lacosamide action has been deficient. We investigated the interactions between lacosamide and Na+ channels in native mammalian neurons, and found that the apparent dissociation constant (~13.7 µM) of lacosamide to the slow inactivated state is well within the therapeutic concentration range and is much (>15-fold) lower than the dissociation constant of lacosamide to the fast inactivated state. Besides, lacosamide has extremely slow binding rates (<400 M-1sec-1) to the fast but much faster binding rates (>3000 M-1sec-1) to the slow inactivated Na+ channels. Consistent with these biophysical characters, we further demonstrated that lacosamide is much more effective against the repetitive burst discharges with interburst intervals at -60 mV than -80 mV. With preponderant binding to the slow inactivation state in therapeutic concentrations and thus less propensity to affect normal discharges, lacosamide could be a drug of choice for seizure discharges characterized by relatively depolarized interburst intervals, during which more slow inactivated states could be generated and more binding of lacosamide would ensue.

Progress and Perspectives in Point of Care Testing for Urogenital Chlamydia trachomatis Infection: A Review.

Worldwide, genital infection with Chlamydia trachomatis (C. trachomatis) is one of the most common sexually transmitted infections. Most infections are asymptomatic. However, particularly in women, untreated infection with C. trachomatis can lead to complications that include pelvic inflammatory disease, infertility, and tubal ectopic pregnancy. Rapid methods for early and accurate diagnosis for infection with C. trachomatis that can be performed in the clinic would allow for earlier treatment to prevent complications. Traditional laboratory-based tests for C. trachomatis infection include culture, enzyme immunoassay, direct immunofluorescence, nucleic acid hybridization, and nucleic acid amplification tests, which take time but have high diagnostic sensitivity. Novel and rapid diagnostic tests include extraordinary optical transmission (EOT), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and microwave-accelerated metal-enhanced fluorescence (MAMEF). Although these new tests offer the promise of rapid screening and diagnosis, they may have lower diagnostic sensitivity. This review aims to provide an overview of traditional methods for the diagnosis of urogenital infection with C. trachomatis, the current status of POC testing for urogenital C. trachomatis infection and discusses recent progress and perspectives.

Identification of virion-associated transcriptional transactivator (VATT) of SGIV ICP46 promoter and their binding site on promoter.

BACKGROUND: Iridoviruses are large DNA viruses that cause diseases in fish, amphibians and insects. Singapore grouper iridovirus (SGIV) is isolated from cultured grouper and characterized as a ranavirus. ICP46 is defined to be a core gene of the family Iridoviridae and SGIV ICP46 was demonstrated to be an immediate-early (IE) gene associated with cell growth control and could contribute to virus replication in previous research. METHODS: The transcription start site (TSS) and 5'-untranslated region (5'-UTR) of SGIV ICP46 were determined using 5' RACE. The core promoter elements of ICP46s were analyzed by bioinformatics analysis. The core promoter region and the regulation model of SGIV ICP46 promoter were revealed by the construction of serially deleted promoter plasmids, transfections, drug treat and luciferase reporter assays. The identification of virion-associated transcriptional transactivator (VATT) that interact with SGIV ICP46 promoter and their binding site on promoter were performed by electrophoretic mobility shift assays (EMSA), DNA pull-down assays and mass spectrometry (MS). RESULTS: SGIV ICP46 was found to have short 5'-UTR and a presumptive downstream promoter element (DPE), AGACA, which locates at + 36 to + 39 nt downstream of the TSS. The core promoter region of SGIV ICP46 located from - 22 to + 42 nt relative to the TSS. VATTs were involved in the promoter activation of SGIV ICP46 and further identified to be VP12, VP39, VP57 and MCP. A 10-base DNA sequence "ATGGCTTTCG" between the TSS and presumptive DPE was determined to be the binding site of the VATTs. CONCLUSION: Our study showed that four VAATs (VP12, VP39, VP57 and MCP) might bind with the SGIV ICP46 promoter and be involved in the promoter activation. Further, the binding site of the VATTs on promoter was a 10-base DNA sequence between the TSS and presumptive DPE.