Myricetin derivatives containing the benzoxazinone moiety discovered as potential anti-tobacco mosaic virus agents.

A series of myricetin derivatives containing benzoxazinone were designed and synthesized. The structures of all compounds were characterized by NMR and HRMS. The structure of Y4 had been confirmed by single-crystal X-ray diffraction analysis. The test results of EC50 values of tobacco mosaic virus (TMV) suggested that Y8 had the best curative and protective effects, with EC50 values of 236.8, 206.0 µg/mL, respectively, which were higher than that of ningnanmycin (372.4, 360.6 µg/mL). Microscale thermophoresis (MST) experiments demonstrated that Y8 possessed a strong binding affinity for tobacco mosaic virus coat protein (TMV-CP), with a dissociation constant (Kd) value of 0.045 µM, which was superior to the ningnanmycin (0.700 µM). The findings of molecular docking studies revealed that Y8 interacted with multiple amino acid residues of TMV-CP through the formation of non-covalent bonds, which had an effect on the self-assembly of TMV particles. The malondialdehyde (MDA) and superoxide dismutase assay (SOD) content assays also fully verified that Y8 could stimulate the plant immune system and enhance disease resistance by reducing MDA content and increasing SOD content. In summary, myricetin derivatives containing benzoxazinone could be considered to further research and development as novel antiviral agents.

Comparative volatiles profiling in milk-flavored white tea and traditional white tea Shoumei via HS-SPME-GC-TOFMS and OAV analyses.

White tea is a mildly fermented tea processed with withering and drying. Milk-flavored white tea has a unique milk flavor compared to the traditional white tea. Little is known about the aromas that make white tea taste milky. Here we conducted the volatile profiling via headspace solid-phase microextraction (HS-SPME)-gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and chemometrics to explore the key volatiles making milk-flavored white tea taste milky. Sixty-seven volatiles were identified, with 7 volatiles (OAV > 1 and VIP > 1) were characterized as the typical aromas. Green and light fruity scent volatiles, such as methyl salicylate, benzyl alcohol, and phenylethyl alcohol, were richer in TFs than MFs. Strong fruity and cheese aromas, such as dihydro-5-pentyl-2(3H)-furanone, 2-pentyl-furan, (E)-6,10-dimethyl-5,9-undecadien-2-one, and hexanal, were more abundant in MFs than TFs. Dihydro-5-pentyl-2(3H)-furanone, recognized as coconut and creamy aroma, should be the essential volatile for milky flavor. Also, (E)-6,10-dimethyl-5,9-undecadien-2-one and 2-pentyl-furan may contribute to the milk scent formation.

Integrated metabolomic and transcriptomic analysis reveal the effect of mechanical stress on sugar metabolism in tea leaves (Camellia sinensis) post-harvest.

Sugar metabolites not only act as the key compounds in tea plant response to stress but are also critical for tea quality formation during the post-harvest processing of tea leaves. However, the mechanisms by which sugar metabolites in post-harvest tea leaves respond to mechanical stress are unclear. In this study, we aimed to investigate the effects of mechanical stress on saccharide metabolites and related post-harvest tea genes. Withered (C15) and mechanically-stressed (V15) for 15 min Oolong tea leaves were used for metabolome and transcriptome sequencing analyses. We identified a total of 19 sugar metabolites, most of which increased in C15 and V15. A total of 69 genes related to sugar metabolism were identified using transcriptome analysis, most of which were down-regulated in C15 and V15. To further understand the relationship between the down-regulated genes and sugar metabolites, we analyzed the sucrose and starch, galactose, and glycolysis metabolic pathways, and found that several key genes of invertase (INV), α-amylase (AMY), ß-amylase (BMY), aldose 1-epimerase (AEP), and α-galactosidase (AGAL) were down-regulated. This inhibited the hydrolysis of sugars and might have contributed to the enrichment of galactose and D-mannose in V15. Additionally, galactinol synthase (Gols), raffinose synthase (RS), hexokinase (HXK), 6-phosphofructokinase 1 (PFK-1), and pyruvate kinase (PK) genes were significantly upregulated in V15, promoting the accumulation of D-fructose-6-phosphate (D-Fru-6P), D-glucose-6-phosphate (D-glu-6P), and D-glucose. Transcriptome and metabolome association analysis showed that the glycolysis pathway was enhanced and the hydrolysis rate of sugars related to hemicellulose synthesis slowed in response to mechanical stress. In this study, we explored the role of sugar in the response of post-harvest tea leaves to mechanical stress by analyzing differences in the expression of sugar metabolites and related genes. Our results improve the understanding of post-harvest tea's resistance to mechanical stress and the associated mechanism of sugar metabolism. The resulting treatment may be used to control the quality of Oolong tea.

Anaerobic Storage Completely Removes Suspected Fungal Pathogens but Increases Antibiotic Resistance Gene Levels in Swine Wastewater High in Sulfonamides.

Wastewater storage before reuse is regulated in some countries. Investigations of pathogens and antibiotic resistance genes (ARGs) during wastewater storage are necessary for lowering the risks for wastewater reuse but are still mostly lacking. This study aimed to investigate pathogens, including harmful plant pathogens, and ARGs during 180 d of swine wastewater (SWW) storage in an anaerobic storage experiment. The contents of total organic carbon and total nitrogen in SWW were found to consistently decrease with the extension of storage time. Bacterial abundance and fungal abundance significantly decreased with storage time, which may be mainly attributed to nutrient loss during storage and the long period of exposure to a high level (4653.2 µg/L) of sulfonamides in the SWW, which have an inhibitory effect. It was found that suspected bacterial pathogens (e.g., Escherichia-Shigella spp., Vibrio spp., Arcobacter spp., Clostridium_sensu_stricto_1 spp., and Pseudomonas spp.) and sulfonamide-resistant genes Sul1, Sul2, Sul3, and SulA tended to persist and even become enriched during SWW storage. Interestingly, some suspected plant fungal species (e.g., Fusarium spp., Ustilago spp. and Blumeria spp.) were detected in SWW. Fungi in the SWW, including threatening fungal pathogens, were completely removed after 60 d of anaerobic storage, indicating that storage could lower the risk of using SWW in crop production. The results clearly indicate that storage time is crucial for SWW properties, and long periods of anaerobic storage could lead to substantial nutrient loss and enrichment of bacterial pathogens and ARGs in SWW.

Temporal variations and trends prediction of water quality during 2010-2019 in the middle Yangtze River, China.

Water quality plays an important role in river habitats. This study revealed the annual and seasonal variations and trend prediction of water quality in the middle Yangtze River after the third impoundment stage of the Three Gorges Reservoir. Multivariate statistical methods including principal component analysis/factor analysis (PCA/FA), Mann-Kendall (M-K) tests, discriminant analysis (DA), rescaled range (R/S) analysis, and the Canadian Council of Ministers of the Environment Water Quality Index (CCME-WQI) were used. Herein, eight water quality constituents including pH, electrical conductivity (EC), chloride (Cl), dissolved oxygen (DO), ammonia nitrogen (NH3N), total phosphorus (TP), water temperature (T), and permanganate index (CODmn) were monthly monitored in the Jiujiang hydrological transaction during 2010-2019. The information of eight water quality constituents, related to salinity, nutrient status, and oxidation reactions efficiency, was extracted. Water quality status remained as fair-good during 2010-2019 based on the results of CCME-WQI, with the seasonal significance ranked as T > DO > Cl > pH > EC > TP > NH3N > CODmn. In the future decade, annual average T was predicted to continue to increase although it might decrease in the wet season. EC was predicted to continue increasing annually especially in the wet season while Cl might decrease. NH3N and TP might maintain a significant decreasing trend in the future wet and dry seasons. DO maintained significantly increasing especially in the future dry seasons, whereas CODmn will continue to decrease annually and seasonally. The continued alkalization trend of waterbody was predicted, which is more significant in the wet season. The results provide helpful references for the ecological protection of the middle Yangtze River.

Gas transport in landfill cover system: A critical appraisal.

Landfill gas (LFG) emission is gaining more attention from the scientific fraternity and policymakers recently due to its threat to the atmosphere and human health of the populace living in surrounding premises. Though landfill cover (LFC) (viz., daily, intermittent and final cover) is widely used by landfill operators to mitigate or reduce these emissions, their overall performance is still under question. A critical analysis of available literature, primarily pertaining to (i) the composition of the landfill gases and their migration in the LFC system, (ii) experimental and mathematical investigations of the transport mechanism of gas and (iii) the impact of additives to cover soils on transport and fate of gas, has been conducted and presented in this manuscript. Investigation of the efficiency of modified soil was mainly focused on laboratory test. More field tests and application of amended cover soils should be conducted and promoted further. Studies on nitrous oxide and emerging pollutants, including poly-fluoroalkyl substances transport in landfill cover system are limited and need further research. The transport mechanisms of these unconventional contaminants should be considered regarding the selection of LFC materials including geomembrane and geosynthetic clay liners. The existing analytical and numerical models can provide a basic understanding of LFG transport mechanisms and are able to predict the migration behaviour of LFG; however, there are still knowledge gaps concerning the interaction between different species of the gas molecule when modeling multi-component gas transport. Gas transport through fractured cover should also be considered when evaluating LFG emission in the future. Simplified design method for landfill cover system regarding LFG emission based on analytical models should be proposed. Overall, mathematical models combined with experiments can facilitate more visualized and intensive insights, which would be instrumental in devising climate adaptive landfill covers.

Temporal and spatial variation in water quality in the Yangtze Estuary from 2012 to 2018.

Water quality plays an important role in estuarine habitats and affects aquatic organisms. The focus of this study was to understand the temporal-spatial variation of water quality and reveal the natural and anthropogenic drivers by using multiple analysis approaches. Herein, during 2012-2018, six water quality constituents (pH, electrical conductivity (EC), dissolved oxygen (DO), ammonia nitrogen (NH3N), total nitrogen (TN), and total phosphorus (TP) were examined for eight sites in the Yangtze Estuary, and the hydro-sediment data, i.e., discharge (Q) and sediment transport rate (STR), was collected from the upstream hydrological station Datong. The cluster analysis (CA), principal component analysis (PCA)/factor analysis (FA), Canadian Council of Ministers of the Environment Water Quality Index (CCME-WQI), and the Mann-Kendall (MK) test were applied. The eight sampling sites were geographically grouped into cluster I, cluster II, and cluster III based on the characteristics of water quality changes, which are under the influence of the upstream runoff, the anthropogenic activities, and seawater. The results are as follows: (1) NH3N, TN, and DO were key constituents representing the water quality of cluster I and cluster III, and those were EC, TN, and DO for cluster II; (2) The monthly-average Q and STR of Datong were negatively correlated to water quality constituents with seasonal variation except for pH; (3) The impact of anthropogenic activities on water quality was especially reflected in cluster III which is close to the Huangpu River Estuary; upstream runoff was the natural driver of water quality changes for cluster I while that was seawater for cluster II. Seawater acts a key role in affecting the water quality of cluster II situated at the North Branch. Revealing the key drivers of water quality change in the Yangtze Estuary provides a reference for water quality management.

Design and Testing of a Separation and Desalination Device for Farmland Saline-Alkaline Water in Arid Areas.

To solve the problem of soil salination and to desalinate saline−alkaline water in arid areas, this study involved the design and testing of a separation and desalination device for farmland saline−alkaline water that is suitable for arid areas. The results of this study indicate that after the pretreatment of farmland saline−alkaline water, the water yielded by the pretreatment device had a mean turbidity of <1 and a mean silt density index (SDI) of <3, which met the working conditions of nanofiltration (NF) and reverse osmosis (RO) membranes. When used to filter saline−alkaline water, the composite NF−RO membrane system achieved a desalination rate of 97.06%, a total hardness removal rate of 97.83%, and a Cl− removal rate of 99.65%, which satisfied the standard for irrigation water quality. Some indicators of the yielded water reached the hygienic standard for drinking water, thus successfully reutilizing water resources. The circulating solar collector tube of the device was designed with a collection area of 6 m2, which could basically satisfy the heat demand of the flash tank for distillation. The design of the flash tank and the shell-and-tube circulating condenser met the requirements for vapor condensation. The crystals in the solar salt box precipitated under solar action. X-ray diffraction was used to identify the primary compound of the crystals as NaCl, suggesting that the precipitates have potential value as industrial salts. This study offers new technical references and helpful engineering guidance for arid saline−alkaline enrichment areas facing the problem of saline farmland irrigation water.

Patient Preference for Biologic Treatments of Psoriasis in the Chinese Setting.

Objective: Assessments of patients' preferences can support in clinical decision-making regarding biologic therapies for psoriasis. Our objective was to investigate patient preference for biologic treatments in patients with psoriasis in China. Methods: From October 2020 to January 2021, psoriasis patients were recruited for a survey that included demographic and disease-related questions, as well as a discrete choice experiment to measure their preferences for biologic therapy. A discrete-choice experiment was used in which respondents selected psoriasis treatments based on benefits (ie, early onset of efficacy, long-term efficacy, sustained efficacy) and treatment costs. We analyzed choice data using conditional logit model. Results: This study included 236 patients with moderate-to-severe psoriasis. The relative importance of the cost of biologic treatments, probability of keeping PASI100 at 5 years, probability of achieving PASI100 at 3 months and time to achieve PASI50 after initiation the biologic treatment were 0.593, 0.137, 0.185 and 0.085. Over 50% of patients regarded the cost of biologic treatments as the most important attribute. High-income and low-income subgroups had higher preference weight in probability of achieving PASI100 at 3-month and monthly cost. Conclusion: The cost of biologic treatments was found as the most important attribute for Chinese patients with psoriasis. Among efficacy attributes, the probability of achieving PASI100 at 3 months showed most sensitive. These results may be helpful to understand patient preference for biologic treatments used for psoriasis in China.

Adsorption behavior of bisphenol A on bentonite-loess mixtures.

The leakage of chemical compounds in landfill leachate led to serious environment pollution, especially, the compounds termed endocrine disruptors such as bisphenol A (BPA). It is very important to study the adsorption behavior of endocrine disruptors in modified soil for predicting and evaluating the potential harm of endocrine disruptors to the soil. Bentonite-amended Chinese loess mixtures, with various proportions of bentonite, were used for the removal of BPA from an aqueous solution. A batch test was used to investigate the adsorption properties of bisphenol A on bentonite and Chinese loess mixtures. The influences of bentonite proportion, temperature, reaction time, pH, and soil-water ratios on the adsorption process were considered. The Fourier transform infrared spectra (FTIR) was used to clarify the change of functional groups before and after the adsorption of BPA on soil. The adsorption mechanism of BPA on soil was discussed preliminary. The results show that the addition of bentonite to the loess can improve the adsorption rate of BPA. The adsorption of BPA was mainly a spontaneous, exothermic, entropy decreasing physical adsorption process. The interaction between bentonite content and reaction concentration had a beneficial effect on BPA adsorption. The linear relationship between bentonite content and adsorption capacity was obtained. The results indicate that bentonite amended loess can provide a good liner for BPA.

Preparation and Performance Evaluation of Cold Mix Asphalt Mixture.

Cold mix asphalt mixture has a low strength and poor water stability, hence to solve this challenge, a cold mix asphaltic liquid was prepared in this study based on microscopic properties. LB-10 gradation was adopted and basalt fiber was selected as a reinforcing agent. The optimum asphaltaggregate ratio was determined by the Marshall test, and mix proportion was also designed. The laboratory tests were used to systematically evaluate road performance of the cold mix asphalt mixture. From the test results, it was shown that the initial strength, molding strength, immersion Marshall strength, residual stability, freeze-thaw splitting ratio, residual rate and dynamic stability of self-made cold mix asphalt mixture were 2.42 kN, 4.87 kN, 6.79 kN, 92.8%, 82.05%, 99.5% and 632 times/mm respectively. The initial strength, molding strength and residual stability of mixture became 90.9%, 88.7%, and 96.2%, respectively of their former values after one-month storage, showing good workability. By analyzing its research data and comparing with its existing products, the developed cold mix asphalt mixture can not only meet the requirements for road performances of the cold mix asphalt mixture in terms of strength, workability and high temperature stability, but greatly improves the water stability, storage stability and cohesive performance of the mixture.

Role of satellite and reanalysis precipitation products in streamflow and sediment modeling over a typical alpine and gorge region in Southwest China.

Satellite and reanalysis precipitation products, as new and complementary data sources, are attractive for hydro-meteorological applications, especially in data-sparse areas. This study evaluates the accuracy of two satellite precipitation products (TMPA 3B42V7 and PERSIANN-CDR) and one reanalysis precipitation product (NCEP-CFSR) against gauge precipitation observations with four statistical indices over the upstream of the Lancang River Basin (ULRB), Southwest China. The reliability and applicability of these precipitation products as inputs to a hydrological model (Soil and Water Assessment Tool, SWAT) for streamflow and sediment simulations are also assessed. Furthermore, we compare the spatial plots of extreme water yield (99 percentiles) and suspended sediment yield (99 percentiles) driven by the four precipitation sources, and investigate the spatial and temporal variability of water yield and suspended sediment yield over the ULRB. Results show that for direct comparisons with gauge precipitation observations, monthly TMPA 3B42V7 precipitation product performs the best at the basin scale with the smallest error and bias, and the highest correlation, followed by NCEP-CFSR, and PERSIANN-CDR. For modeling-based indirect inference, TMPA 3B42V7 presents great capability for streamflow and sediment simulations in the SWAT model on a monthly time step at the basin outlet, and PERSIANN-CDR also performs well. NCEP-CFSR shows acceptable skills in modeling sediment but unacceptable skills in modeling streamflow. Extreme water yield presents moderate spatial variability over the ULRB while extreme suspended sediment yield presents strong spatial variability. Water yield of this basin shows a decreasing trend during 1998-2008 while there is no obvious trend in suspended sediment yield in this period.

Preparation and Performance Evaluation of Cold Mix Asphaltic Liquid.

High-performance cold mix asphalt was prepared based on suitable cold mix asphaltic liquid. First, the contents of raw materials were estimated. The optimum composition of asphaltic liquid was determined by the orthogonal experimental design method. The properties of evaporated residue, adhesiveness, and construction workability were determined. At the same time, the microscopic characteristics of cold mix asphaltic liquid were analyzed by scanning electron microscopy (SEM). The results indicated that Brookfield viscosity was greatly influenced by diluent content at 60 °C. Brookfield viscosity was decreased with the increase of diluent content. Brookfield viscosity was hardly influenced by contents of the additive and styreneic block copolymers (SBS) at 60 °C. Samples 2, 3, 5 and 6 met the requirement of viscosity. The penetration degree of the evaporated residue in asphaltic liquid met the requirement for roads. The Chinese Standard JTG F40-2004 was used to measure the ductility of asphaltic liquids at 5 and 15 °C. The softening points exceeded 50 °C, indicating the excellent high-temperature performance of cold mix asphaltic liquid. The adhesiveness between asphaltic liquid and aggregates was graded at Level 5 (Chinese Standard T0616-1993). The mixture containing samples 5 and 6 was slightly white in color, the fine aggregate was exposed completely. Changing the temperature of the mixture from 90 °C (testing temperature) to 70 °C (mixing temperature), the adhesiveness between aggregate and asphalt was reduced. The fine aggregates were not exposed in the mixture containing samples 2 and 3, and the corresponding mixing ratios can be applied for the production of cold mix asphaltic liquid. Modifiers and additives in cold mix asphaltic liquid were uniformly dispersed. At the same time, it demonstrated that the diluent showed a certain influence on the morphology of residue of cold mix asphaltic liquid, so that a number of folds and holes existed on the surface of the residue.

[Screening and establishment of human lung cancer cell lines with organ-specific metastasis potential].

BACKGROUND: Cancer metastasis is not only the malignant marker and characteristics, but also the main cause of failure to cure and lose their life in the patients with lung cancer. Lung cancer metastasis has organ-specific characteristics. The most common sites of lung cancer metastasis are mediastinal lymph node, brain, bone, liver and adrenal gland. The aim of this study is to screen and establish lung cancer cell model with organ-specific metastasis potential with human high-metastatic large cell lung cancer cell line L9981 established by our laboratory previously, and to provide cell models for studying the mechanisms and signal regulation of organ-specific metastasis of lung cancer. METHODS: The parent lung cancer cell line, L9981-Luc, was inoculated in the armpit of nude mice. The live animal imaging system, IVIS-200, was used to detect the lung cancer organ-specific metastasis every week. When the organ-specific metastasis were established, the nude mices bearing the lung cancer were sacrificed when they became moribund. Under sterile conditions, the organs (mediastinal lymph nodes, lung, spinal column and brain) with lung cancer organ-specific metastasis were removed and the metastasized nodules were dissected free of connective tissue and blood clots, and rinsed twice with medium. The metastasized nodules were finely minced using sterile scalpel blades in medium, and the cells were seeded in tissue culture dishes. Then, the cells with organ-specific metastasis potential were reinoculated into the armpit of nude mice, respectively. This processes were repeated to establish the organ-specific metastatic sublines of L9981-Luc cell line more than 10 times. Finally, the organ-specific metastasis sublines of L9981-Luc were screened and established, which the four cell lines have the characteristics only metastasized to brian, lung, bone and mediastinal lymph node. RESULTS: A group of organ-specific metastasis cell lines which only metastasized to brian, lung, bone and mediastinal lymph node were successfully established through repeating reinoculatation, live animal imaging in nude mice, and screening and identification in vitro. We named the four cell lines as L9981-BoM, L9981-LuM, L9981-BrM and L9981-LnM, respectively. The L9981-BoM cell was only metastasized to bone. The l9981-LuM cell was only metastasized to lung. The L9981-BrM only metastasized to brain. The L9981-LnM cell was only metastasized to midiastinal lymph nodes. CONCLUSIONS: A human large cell lung cancer cell model with bone, lung, brain and lymph node-specific metastasis potential was successfully established. It will be helpful to further study the molecular mechanisms and signal regulation of lung cancer organ- specific metastasis. It will be to also provide reliable cell model for developing new techniques and molecular targeting drugs of inhibiting or reversing lung cancer metastasis.

[Cloning, expression, purification and identification of multi-potent transcription factor CTCF recombinant and its segments].

OBJECTIVE: To clone ctc f cDNA, N, Zn and C fragments separately into expresstion vector, purify and identify the expressed proteins. METHODS: Using the recombinant plasmid pGEM7Zf (-)-ctc f as template for PCR, pGEX-4T-2-ctc f, pGEX-4T-2-ctc f-N, pGEX-4T-2- ctc f-Zn and pGEX-4T-2- ctc f-C recombinants were constructed successfully. After transformed into E. coli BL21 cells, the recombinants were confirmed by enzyme digestion and sequence analysis. After optimizing the IPTG inducing condition, the purified GST fusion proteins with affinity chromatography were conformed by Far-Western blotting. RESULTS: The recombinant plasmids pGEX-4T-2-ctc f, pGEX-4T-2- ctc f-N, pGEX-4T-2-ctc f-Zn and pGEX-4T-2-ctc f-C were confirmed by restriction enzyme assay and sequencing. All GST fusion proteins, CTCF, CTCF-N, CTCF-Zn and CTCF-C were successfully expressed at the optimal parameters and purified with affinity chromatography, and specifically recognized by anti-GST antibody. CONCLUSION: Ctc f, ctc f-n, ctc f-Zn and ctc f-c gene recombinants were constructed successfully and their corresponding fusion proteins were successfully purified with affinity chromatography and identified.

[Study of mental disorder due to brain damage].

OBJECTIVE: To study the incidence rate, pattern and affective factors of mental disorder due to brain damage. METHODS: According to CCMD-2-R, 388 subjects with traumatic brain damage in the Psychiatric Hospital of Huainan city within last 5 years were assessed by three psychiatrists 6 months to 1 year after brain injury. RESULTS: (1) 74.2% of the mental disordered due to brain injury have intellectual impairment, most of them is mild; (2) The intracranial hematoma, brain stem injury, brain injury extent, GCS, complicated mental disorder, and education have great effect on intellectual impairment; (3) There is an intimate relationship between the intellectual impairment and the brain stem injury, intracranial hematoma, GCS, brain injury extent, and unconsciousness time. There is an intimate relationship between the mental symptom and the brain injury extent, contusion and laceration of brain, frontal lobe injury, and intracranial hematoma. There is also an intimate relationship between the personality change and the frontal lobe injury, unfolding brain case treatment, and intracranial hematoma. CONCLUSION: To assess overall mental disorder should rely on the characteristics of craniocerebral injuries.

[Construction and application of a new prokaryotic expression vector derivative of pBV220].

A single-stranded oligonucleotides containing a 6 histidine sequence, a hydroxylamine cleavage site, a thrombin cleavage site, and stop codon TAA were inserted into the polylinker's downstream of prokaryotic expression vector pBV220 between BamHI and PstI. The resultant vector is named pBV223. Proteins expressed in this vector will have a 6 histidine tail as affinity handy fused to their C terminus and can be quickly purified by one step immobilized metal affinity chromatography (IMAC). This plasmid is verified by restriction map and DNA sequencing. Subsequently, the metastasis suppressor gene nm23-H1 cDNA (without the stop codon) was cloned into vector PBV223 in frame with the 6-histidine sequence, hydroxylamine and thrombin cleavage sites. The soluble nm23-H1 fusion protein was successfully induced in the bacterial DH5a and easily purified with Ni chromatograph. These results indicated that the strategy to clone the single-stranded oligonucleotides directly into the restriction sties between BamH I and Pst I in the pBV220 vector is the simplest and cost-effective method.

Hypomethylated and hypermethylated profiles of H19DMR are associated with the aberrant imprinting of IGF2 and H19 in human hepatocellular carcinoma.

In this study, 39 human hepatocellular carcinoma (HCC) tissues and 7 normal adult liver tissues were screened for heterozygous polymorphisms in IGF2, H19, and the differentially methylated region of H19 (H19DMR) using PCR-RFLP and PCR sequencing. The imprinting of IGF2 and H19 was examined by RT-PCR-RFLP, while the methylation profile of H19DMR was detected by bisulfite sequencing from every informative sample. Of the informative HCC samples 47.06% (8 of 17) demonstrated a gain of imprinting of IGF2, and 21.74% (5 of 23) of the informative HCC samples demonstrated a loss of imprinting of H19. Interestingly, we found three methylation profiles for H19DMR in the informative HCC samples: hyper-, medium-, and hypomethylated profiles. Furthermore, the hypomethylated and hypermethylated profiles were immediately associated with aberrant imprinting of IGF2 and H19.

Double mutant P96S/S120G of Nm23-H1 abrogates its NDPK activity and motility-suppressive ability.

The Nm23-H1 gene is a metastasis suppressor gene. However, its biochemical mechanism of suppressing the metastatic potential of cancer cells is still unknown. The previous hypothesis that a histidine protein kinase activity may contributes to the motility-suppressive effect of Nm23-H1 could not explain why the H118F mutant, a kinase-deficient mutant, still had motility-suppressive ability. We conducted a study on the double mutant P96S/S120G of Nm23-H1 and succeeded in introducing the RP-HPLC method in NDPK assay. The results showed that the double mutant P96S/S120G, when expressed in the bacteria, was completely aggregated in inclusion bodies; this mutant abrogated not only its motility-suppressive ability, but also its NDPK activity. Based on previous work and this study, we prompted that the deficiency of motility-suppressive function of S120G, P96S, and P96S/S120G mutants was due to their altered structure, which might deprive Nm23-H1 of most activities including kinase activity or interactions with other proteins.