Revealing atomic-scale molecular diffusion of a plant-transcription factor WRKY domain protein along DNA.

Transcription factor (TF) target search on genome is highly essential for gene expression and regulation. High-resolution determination of TF diffusion along DNA remains technically challenging. Here, we constructed a TF model system using the plant WRKY domain protein in complex with DNA from crystallography and demonstrated microsecond diffusion dynamics of WRKY on DNA by employing all-atom molecular-dynamics (MD) simulations. Notably, we found that WRKY preferentially binds to one strand of DNA with significant energetic bias compared with the other, or nonpreferred strand. The preferential DNA-strand binding becomes most prominent in the static process, from nonspecific to specific DNA binding, but less distinct during diffusive movements of the domain protein on the DNA. Remarkably, without employing acceleration forces or bias, we captured a complete one-base-pair stepping cycle of the protein tracking along major groove of DNA with a homogeneous poly-adenosine sequence, as individual hydrogen bonds break and reform at the protein-DNA binding interface. Further DNA-groove tracking motions of the protein forward or backward, with occasional sliding as well as strand crossing to minor groove of DNA, were also captured. The processive diffusion of WRKY along DNA has been further sampled via coarse-grained MD simulations. The study thus provides structural dynamics details on diffusion of a small TF domain protein, suggests how the protein approaches a specific recognition site on DNA, and supports further high-precision experimental detection. The stochastic movements revealed in the TF diffusion also provide general clues about how other protein walkers step and slide along DNA.

Energetic vs. entropic stabilization between a Remdesivir analogue and cognate ATP upon binding and insertion into the active site of SARS-CoV-2 RNA dependent RNA polymerase.

SARS-CoV-2 RNA dependent RNA polymerase (RdRp) serves as a highly promising antiviral drug target such as for a Remdesivir nucleotide analogue (RDV-TP or RTP). In this work, we mainly used alchemical all-atom simulations to characterize relative binding free energetics between the nucleotide analogue RTP and natural cognate substrate ATP upon initial binding and pre-catalytic insertion into the active site of SARS-CoV-2 RdRp. Natural non-cognate substrate dATP and mismatched GTP were also examined for computation control. We first identified significant differences in dynamical responses between nucleotide initial binding and subsequent insertion configurations to the open and closed active sites of the RdRp, respectively, though the RdRp protein conformational changes between the active site's open and closed states are subtle. Our alchemical simulations indicated that upon initial binding (active site open), RTP and ATP show similar binding free energies to the active sites while in the insertion state (active site closed), ATP is more stabilized (∼-2.4 kcal mol-1) than RTP in free energetics. Additional analyses show, however, that RTP is more stabilized in binding energetics than ATP, in both the insertion and initial binding states, with RTP more stabilized due to the electrostatic energy in the insertion state and due to vdW energy in the initial binding state. Hence, it appears that natural cognate ATP still excels at association stability with the RdRp active site due to that ATP maintains sufficient flexibilities e.g., in base pairing with the template, which exemplifies an entropic contribution to the cognate substrate stabilization. These findings highlight the importance of substrate flexibilities in addition to energetic stabilization in antiviral nucleotide analogue design.

Switching promotor recognition of phage RNA polymerase in silico along lab-directed evolution path.

In this work, we computationally investigated how a viral RNA polymerase (RNAP) from bacteriophage T7 evolves into RNAP variants under lab-directed evolution to switch recognition from T7 promoter to T3 promoter in transcription initiation. We first constructed a closed initiation complex for the wild-type T7 RNAP and then for six mutant RNAPs discovered from phage-assisted continuous evolution experiments. All-atom molecular dynamics simulations up to 1 µs each were conducted on these RNAPs in a complex with the T7 and T3 promoters. Our simulations show notably that protein-DNA electrostatic interactions or stabilities at the RNAP-DNA promoter interface well dictate the promoter recognition preference of the RNAP and variants. Key residues and structural elements that contribute significantly to switching the promoter recognition were identified. Followed by a first point mutation N748D on the specificity loop to slightly disengage the RNAP from the promoter to hinder the original recognition, we found an auxiliary helix (206-225) that takes over switching the promoter recognition upon further mutations (E222K and E207K) by forming additional charge interactions with the promoter DNA and reorientating differently on the T7 and T3 promoters. Further mutations on the AT-rich loop and the specificity loop can fully switch the RNAP-promoter recognition to the T3 promoter. Overall, our studies reveal energetics and structural dynamics details along an exemplary directed evolutionary path of the phage RNAP variants for a rewired promoter recognition function. The findings demonstrate underlying physical mechanisms and are expected to assist knowledge and data learning or rational redesign of the protein enzyme structure function.

Allosteric regulation in CRISPR/Cas1-Cas2 protospacer acquisition mediated by DNA and Cas2.

Cas1 and Cas2 are highly conserved proteins across clustered-regularly-interspaced-short-palindromic-repeat-Cas systems and play a significant role in protospacer acquisition. Based on crystal structure of twofold symmetric Cas1-Cas2 in complex with dual-forked protospacer DNA (psDNA), we conducted all-atom molecular dynamics simulations to study the psDNA binding, recognition, and response to cleavage on the protospacer-adjacent-motif complementary sequence, or PAMc, of Cas1-Cas2. In the simulation, we noticed that two active sites of Cas1 and Cas1' bind asymmetrically to two identical PAMc on the psDNA captured from the crystal structure. For the modified psDNA containing only one PAMc, as that to be recognized by Cas1-Cas2 in general, our simulations show that the non-PAMc association site of Cas1-Cas2 remains destabilized until after the stably bound PAMc being cleaved at the corresponding association site. Thus, long-range correlation appears to exist upon the PAMc cleavage between the two active sites (∼10 nm apart) on Cas1-Cas2, which can be allosterically mediated by psDNA and Cas2 and Cas2' in bridging. To substantiate such findings, we conducted repeated runs and further simulated Cas1-Cas2 in complex with synthesized psDNA sequences psL and psH, which have been measured with low and high frequency in acquisition, respectively. Notably, such intersite correlation becomes even more pronounced for the Cas1-Cas2 in complex with psH but remains low for the Cas1-Cas2 in complex with psL. Hence, our studies demonstrate that PAMc recognition and cleavage at one active site of Cas1-Cas2 may allosterically regulate non-PAMc association or even cleavage at the other site, and such regulation can be mediated by noncatalytic Cas2 and DNA protospacer to possibly support the ensued psDNA acquisition.

Inchworm stepping of Myc-Max heterodimer protein diffusion along DNA.

Oncogenic protein Myc serves as a transcription factor to control cell metabolisms. Myc dimerizes via leucine zipper with its associated partner protein Max to form a heterodimer structure, which then binds target DNA sequences to regulate gene transcription. The regulation depends on Myc-Max binding to DNA and searching for target sequences via diffusional motions along DNA. Here, we conduct structure-based molecular dynamics (MD) simulations to investigate the diffusion dynamics of the Myc-Max heterodimer along DNA. We found that the heterodimer protein slides on the DNA in a rotation-uncoupled manner in coarse-grained simulations, as its two helical DNA binding basic regions (BRs) alternate between open and closed conformations via inchworm stepping motions. In such motions, the two BRs of the heterodimer step across the DNA strand one by one, with step sizes reaching about half of a DNA helical pitch length. Atomic MD simulations of the Myc-Max heterodimer in complex with DNA have also been conducted. Hydrogen bond interactions are revealed between the two BRs and two complementary DNA strands, respectively. In the non-specific DNA binding, the BR from Myc shows an onset of stepping on one association DNA strand and starts detaching from the other strand. Overall, our simulation studies suggest that the inchworm stepping motions of the Myc-Max heterodimer can be achieved during the protein diffusion along DNA.

Inspecting fluctuation and coordination around chromophore inside green fluorescent protein from water to nonpolar solvent.

Green fluorescent protein (GFP) is a widely used biomarker that demands systematical rational approaches to its structure function redesign. In this work, we mainly utilized atomistic molecular dynamics simulations to inspect and visualize internal fluctuation and coordination around chromophore inside GFP, from water to nonpolar octane solvent. We found that GFP not only maintains its ß-barrel structure well into the octane, but also sustains internal residue and water coordination to position the chromophore stably while suppress dihedral fluctuations of the chromophore, so that functional robustness of GFP is achieved. Our accompanied fluorescence microscope measurements accordingly confirmed the GFP functioning into the octane. Furthermore, we identified that crucial water sites inside GFP along with permeable pores on the ß-barrel of the protein are largely preserved from the water to the octane solvent, which allows sufficiently fast exchanges of internal water with the bulk or with the water layer kept on the surface of the protein. By additionally pulling GFP from bulk water to octane, we suggest that the GFP function can be well maintained into the nonpolar solvent as long as, first, the protein does not denature in the nonpolar solvent nor across the polar-nonpolar solvent interface; second, a minimal set of water molecules are in accompany with the protein; third, the nonpolar solvent molecules may need to be large enough to be nonpermeable via the water pores on the ß-barrel.

Deciphering Intrinsic Inter-subunit Couplings that Lead to Sequential Hydrolysis of F1-ATPase Ring.

Rotary sequential hydrolysis of the metabolic machine F1-ATPase is a prominent manifestation of high coordination among multiple chemical sites in ring-shaped molecular machines, and it is also functionally essential for F1 to tightly couple chemical reactions and central γ-shaft rotation. High-speed AFM experiments have identified that sequential hydrolysis is maintained in the F1 stator ring even in the absence of the γ-rotor. To explore the origins of intrinsic sequential performance, we computationally investigated essential inter-subunit couplings on the hexameric ring of mitochondrial and bacterial F1. We first reproduced in stochastic Monte Carlo simulations the experimentally determined sequential hydrolysis schemes by kinetically imposing inter-subunit couplings and following subsequent tri-site ATP hydrolysis cycles on the F1 ring. We found that the key couplings to support the sequential hydrolysis are those that accelerate neighbor-site ADP and Pi release upon a certain ATP binding or hydrolysis reaction. The kinetically identified couplings were then examined in atomistic molecular dynamics simulations at a coarse-grained level to reveal the underlying structural mechanisms. To do that, we enforced targeted conformational changes of ATP binding or hydrolysis to one chemical site on the F1 ring and monitored the ensuing conformational responses of the neighboring sites using structure-based simulations. Notably, we found asymmetrical neighbor-site opening that facilitates ADP release upon enforced ATP binding. We also captured a complete charge-hopping process of the Pi release subsequent to enforced ATP hydrolysis in the neighbor site, confirming recent single-molecule analyses with regard to the role of ATP hydrolysis in F1. Our studies therefore elucidate both the coordinated chemical kinetics and structural dynamics mechanisms underpinning the sequential operation of the F1 ring.

Function of chalcone reductase gene CHR1 in soybean.

To verify the function of chalcone reductase gene (CHR1) in soybean Daidzein synthesis, CHR1 gene in soybean was cloned, and an RNAi expression vector pCPB-CHR1-RNAi was constructed. Four transformed plants in T0 generation and thirteen transformed plants in T1 generation of soybean "Jinong28" were obtained by agrobacterium-mediated genetic transformation, in which transcriptions of CHR1 gene were depressed. Southern blotting showed the functional fragment of pCPB-CHR1-RNAi was integrated into the genome of recipient soybean in the form of a single copy. Detection of the transcription of CHR1 gene using quantitative real-time PCR (qRT-PCR) showed that the expression of CHR1 gene in transformed plants decreased 60%-99% compared to the recipient soybean, while the content of isoliquiritigenin, the precursors of daidzein, decreased 38.7%. These results indicate that RNA interference can suppress the transcription of CHR1 gene expression successfully.

Dissecting the CRISPR Cas1-Cas2 Protospacer Binding and Selection Mechanism by Using Molecular Dynamics Simulations.

Cas1 and Cas2 are highly conserved proteins among the clustered regularly interspaced short palindromic repeat Cas (CRISPR-Cas) systems and play a crucial role in protospacer selection and integration. According to the double-forked CRISPR Cas1-Cas2 complex, we conducted extensive all-atom molecular dynamics simulations to investigate the protospacer DNA binding and recognition. Our findings revealed that single-point amino acid mutations in Cas1 or in Cas2 had little impact on the binding of the protospacer, both in the binding and precatalytic states. In contrast, multiple-point amino acid mutations, particularly G74A, P80L, and V89A mutations on Cas2 and Cas2' proteins (m-multiple1 system), significantly affected the protospacer binding and selection. Notably, mutations on Cas2 and Cas2' led to an increased number of hydrogen bonds (#HBs) between Cas2&Cas2' and the dsDNA in the m-multiple1 system compared with the wild-type system. And the strong electrostatic interactions between Cas1-Cas2 and the protospacer DNA (psDNA) in the m-multiple1 system again suggested the increase in the binding affinity of protospacer acquisition. Specifically, mutations in Cas2 and Cas2' can remotely make the protospacer adjacent motif complementary (PAMc) sequences better in recognition by the two active sites, while multiple mutations K211E, P202Q, P212L, R138L, V134A, A286T, P282H, and P294H on Cas1a/Cas1b&Cas1a'/Cas1b' (m-multiple2 system) decrease the #HBs and the electrostatic interactions and make the PAMc worse in recognition compared with the wild-type system.

Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion.

One-dimensional (1-D) sliding of transcription factor (TF) protein along DNA is essential for facilitated diffusion of the TF to locate target DNA site for genetic regulation. Detecting base-pair (bp) resolution of the TF sliding or stepping on the DNA is still experimentally challenging. We have recently performed all-atom molecular dynamics (MD) simulations capturing spontaneous 1-bp stepping of a small WRKY domain TF protein along DNA. Based on the 10 µs WRKY stepping path obtained from such simulations, the protocol here shows how to conduct more extensive conformational samplings of the TF-DNA systems, by constructing the Markov state model (MSM) for the 1-bp protein stepping, with various numbers of micro- and macro-states tested for the MSM construction. In order to examine processive 1-D diffusional search of the TF protein along DNA with structural basis, the protocol further shows how to conduct coarse-grained (CG) MD simulations to sample long-time scale dynamics of the system. Such CG modeling and simulations are particularly useful to reveal the protein-DNA electrostatic impacts on the processive diffusional motions of the TF protein above tens of microseconds, in comparison with sub-microseconds to microseconds protein stepping motions revealed from the all-atom simulations.

Genetic mapping of northern corn leaf blight-resistant quantitative trait loci in maize.

Northern corn leaf blight (NCLB), a corn disease infected by Exserohilum turcicum, can cause loss of harvest and economy. Identification or evaluation of NCLB-resistant quantitative trait loci (QTL) and genes could improve maize breeds. This study aimed to identify novel QTLs for NCLB-resistance.Two maize strains (BB and BC) were utilized to generate B73 × B97 and B73 × CML322 and constructed the genetic linkage using high-throughput single nucleotide polymorphism (SNP) linkage map analysis of 170 (BB) and 163(BC) recombinant inbred line (RIL) genomic DNA samples. NCLB-resistant QTL was associated with phenotypic data from the field trial of 170 BB and 163 BC strains over two years using these 1100 SNPs to identify high-density NCLB-resistant QTLs.In BB, QTL of the NCLB resistance was on chromosome 1 and 3 (LOD scores between 2.74 and 5.44); in BC, QTL of NCLB resistance was on chromosome 1, 2, 4, 8, and 9 (LOD scores between 2.52 and 8.53). A number of genes or genetic information related to NCLB resistance in both BB and BC were identified with the maximum number of genes/NCLB resistance-related QTL on chromosome 3 for BB and on chromosome 1 for BC.This study successfully mapped and identified NCLB-resistant QTL and genes for these 2 different maize strains, which provides insightful information for future study of NCLB-resistance and selection of NCLB-resistant maize variants.

Effects of moderate soil salinity on osmotic adjustment and energy strategy in soybean under drought stress.

Under drought and soil salinity, plants usually respond to accumulate inorganic and organic osmolytes for adaptation, that would induce changes in energy consumption strategy of plants. Moderate soil salinity would enable plants to lower energy consumption for osmotic adjustment by passively absorbing more Na+. This action would keep more energies for growth of drought-stressed plants. Thus, Na+ accumulation might be an energy-efficient strategy for plants to cope with drought was speculated. To support this speculation, we assessed the effects of soil salinity on osmotic adjustment and energy utilization under drought in this study. Our results indicated that the ratio and content of inorganic osmolytes was significantly higher under drought-saline stress (D + S) than those under single drought stress (D), while the osmolarity and contents of organic osmolytes of D + S were significantly lower than those of D. This indicated that moderate soil salinity could enable soybean seedlings to consume relatively lower energies to produce less organic osmolytes and accumulate more inorganic ions for osmotic adjustment coping with drought. Meanwhile the water content, cell turgor, ash content, and specific leaf area and biomass of D + S were significantly higher than those of D, but the leaf construction cost of D + S was significantly lower than those of D. This suggested that moderate soil salinity could enhance water retention, and reduce the photoassimilate and energy consumption of droughty soybean seedlings. This work would help to understand the positive effects of moderate soil salinity on plant growth on the level of osmotic adjustment and energy consumption strategy.

Postmarketing surveillance study of benazepril in chinese patients with hypertension: An open-label, experimental, epidemiologic study.

BACKGROUND: Benazepril hydrochloride is an angiotensin-converting enzyme inhibitor. Previous clinical trials show that antihypertensive treatment with benazepril provides effective blood pressure (BP) control and is generally well tolerated by patients with hypertension. However, the long-term antihypertensive effects and tolerability of benazepril remain to be established in Chinese patients with hypertension. OBJECTIVE: The aim of this study was to investigate the long-term efficacy and tolerability of benazepril in Chinese patients with essential hypertension. METHODS: This 36-month, community-based, open-label, postmarketing surveillance study was conducted in the Nanshi District (Shanghai, China). Chinese patients with essential hypertension were to receive 1 or more benazepril tablets PO QD in the morning for 36 months. Data for BP and pulse pressure (PP) were collected at baseline (month 0) and throughout the surveillance period. The rate of patients achieving BP targets (systolic BP [SBP]/diastolic BP [DBP], <140/<90 mm Hg) was determined, as was the rate of decrease in BP. Subanalyses by sex and age group also were conducted. RESULTS: A total of 1831 patients (1090 men, 741 women; mean [SD] age, 55.8 [10.1] years [range, 35-88 years]) entered the study. After the 36-month treatment period, 75.7% of patients receiving benazepril as prescribed (1289 patients) had achieved the SBP target, 87.4% achieved the DBP target, and 71.5% achieved both targets. After 36 months of treatment, the mean (SD) decreases in SBP, DBP, and PP were 15.1 (0.4) mm Hg, 11.0 (0.3) mm Hg, and 4.2 (0.4) mm Hg, respectively, among compliers. In general, the rate of BP decrease slowed over time. No serious adverse drug reactions (ADRs) were detected during the 36-month follow-up period. All ADRs except cough (19.9%) occurred at a relatively low incidence rate (<3.0%). The cumulative incidence of benazepril related cough was statistically significantly higher in women than in men (23.6% vs 18.8%, respectively; P = 0.007). Of the 1831 patients studied, 1360 patients (74.3%) persisted in taking benazepril and were considered optimally compliant at 36-month follow-up. CONCLUSION: In this study of Chinese patients with hypertension, benazepril was associated with prolonged, stable efficacy in lowering BP and relatively low incidence of ADRs.

[Relationship between socioeconomic status and the prevalence of cardiovascular disease among retired residents living in a community, Shanghai].

OBJECTIVE: To explore the relationship between socioeconomic status and the risk factors of cardiovascular diseases in retirees from a community in Shanghai. METHODS: Observational study involved 9 943 retirees aged 50 and over in Shanghai. Both single factor and multi-factor analyses methods were used to describe the correlation between factors as:educational level, marital status, annual household income and risk of hypertension, coronary heart disease, stroke etc. A new defined compound index was used to assess the relevance of socioeconomic status on the risk of cardiovascular diseases, based on logistic regression model. RESULTS: After adjusted for age, the risk of cardiovascular diseases in these retirees was influenced by socioeconomic status. In general, opponent correlations in education levels and prevalence of hypertension were found between female and male. Compared with those having received college or higher education, the risk of hypertension increased in females when the education level declined, with OR as 1.08 (95% CI:0.89-1.30). For those having had senior high school junior high school or elementary education, the risks of hypertension were 1.26 (95%CI:1.05-1.51), 1.34 (95%CI:1.08-1.65), 0.72 (95%CI:0.59-0.87),0.78 (95%CI:0.64-0.94), and 0.70 (95%CI:0.52-0.92) for males, respectively. The risk of cardiovascular diseases increased with annual household income. Compared with high level of socioeconomic status, lower socioeconomic status might decline the risk of cardiovascular diseases in males by approximately 30%, with OR for medium being 0.72 (95%CI:0.61-0.84) and for lower ones it was 0.70 (95% CI:0.57-0.87). However, similar correlations were not found in females. No significant relationship was found between marital status and the prevalence of cardiovascular diseases in this study. CONCLUSION: The risks of cardiovascular diseases varied with different socioeconomic status, indicating that tailored interventions should be conducted in different socioeconomic groups.

[Relationship between the clustering of risk factors and the prevalence of hypertension in the community residents living in Shanghai].

OBJECTIVE: To study the association between the clustering manifestation of factors as overweight and central obesity, family heredity, immoderate alcohol drinking, tobacco smoking, hyperlipidemia, hyperglycemia and the prevalence of hypertension. METHODS: Data was from a program related to the comprehensive prevention and control strategies on cardiac-cerebral vascular disease carried out in the communities of Shanghai, to describe the relationship between the clustering of risk factors and hypertension. This program included 15 158 people with complete data at the age of 35 - 74, from 2008 - 2011. Both single factor and multi-factor analysis were used and longitudinal study was performed to further explore the causal relationship. RESULTS: The overall prevalence of hypertension at the baseline survey was 41.9%. The associated ORs (age-adjusted) of hypertension parallelly increased with the number of risk factors under clustering. The associated OR of the males with 1, 2, 3, 4 as well as 5 and above risk factors were 3.157 [95% confidence interval (CI): 2.152 - 4.630], 6.428 (95%CI: 4.435 - 9.319), 11.797 (95%CI: 8.135 - 17.105), 19.723 (95%CI: 13.414 - 29.000), 33.051 (95%CI: 21.449 - 50.930) respectively. In females with 1, 2, 3 as well as 4 risk factors, the associated ORs were 2.917 (95%CI: 2.374 - 3.585), 6.499 (95%CI: 5.307 - 7.959), 15.717 (95%CI: 12.609 - 19.591) and 31.719 (95%CI: 21.744 - 46.270), respectively. For longitudinal study, the 2-year incidence of hypertension in males and females were 1.9% and 1.6%, respectively. Compared to those people without risk factors, the incidence was higher in the people with a larger number of clustering. When the clustering number reaching 2 or 3 in females, the relative risk (RR) were 2.111 (95%CI: 1.024 - 4.350) and 3.000 (95%CI: 1.287 - 6.995) respectively, with statistically significant difference. CONCLUSION: The risk of hypertension parallelly increased with the clustering number of relevant risk factors. Comprehensive prevention and control on related risk factors was required.

[Prevalence and relevant factors on echocardiographic left ventricular hypertrophy among community-based hypertensive patients in Shanghai].

OBJECTIVE: To investigate the prevalence and relevant factors on the echocardiographic left ventricular hypertrophy (LVH). METHOD: A cross-sectional study was conducted among the hypertensive patients in an urban community. RESULTS: The prevalence of LVH was 29.2% in 1 686 hypertensive patients, with 25.4% in males and 34.5% in females, respectively. The prevalence was significantly higher in females than in males (chi(2) = 16.17, P < 0.01). The rate was increasing with age and significantly higher prevalence was observed in 45-, 55-, 65- age groups of females (P < 0.05). Moreover, elevated systolic blood pressure and higher BMI were related to the LVH in hypertensive patients, while higher education level seemed a protective factor. CONCLUSION: These results implied that a comprehensive intervention should be taken in the prevention of LVH.

[An analysis on the cost of hypertensive outpatient in the community hospital in Shanghai].

OBJECTIVE: To study the cost of the hypertensive outpatients. METHODS: The study randomly selected 460 insured patients with hypertension and investigated their cost on each case in the out-patient department through 2002, based on the electronic system of medical insurance. RESULTS: As a whole, the distribution of hypertensive outpatient expenditure takes on the positively skewed, with the median of 1 567.9 Yuan RMB. With the increase of age, the average expenses in each age group increased accordingly. In the study, the average number of outpatient attendances per patient was 19.5, the average expenses per visit was 115.4 Yuan RMB. In age groups 40 - 49 and 50 - 59, expenses of outpatient in male and female groups are obviously different in 2002 (Wilcoxon W(40 - 49) = 36, P(40 - 49) = 0.037; Wilcoxon W(50 - 59) = 374, P(50 - 59) = 0.023), as well as the number of out-patients (Wilcoxon W(40 - 49) = 52.5, P(40 - 49) = 0.007; Wilcoxon W(50 - 59) = 379, P(50 - 59) = 0.028). When considering the factors of gender and age at one time, the outpatient expenditures in the male group were significantly different between the different age groups (chi(2) = 22.3, P < 0.001), as well as the number of outpatients (chi(2) = 25.4, P < 0.001). In addition, the expenditure of drugs, which took a large proportion of the total expenditure of hypertensive outpatients (about 83.6 percent), was divided into three parts according to the degree of correlation with hypertension: direct expenses related to the with disease, the indirect expenses and the irrespective. The proportions of each part were 19.9 percent, 32.3 percent and 47.8 percent respectively. CONCLUSION: When economic evaluation of community prevention is carried out, the cost and cost-benefit analysis based on the analysis of outpatient expenditure and the proportion of expenses on hypertension should be taken into account. Additionally, to provide appropriate mode of medicare, to impact the behaviors and expenditure of patients, and to provide low-cost but good effective drug are also essential and important factors.