Simultaneous determination of four phytoecdysteroids by LC-MS/MS: application to a comparative pharmacokinetic study in normal and adjuvant arthritis rats after oral administration of C. officinalis Kuan phytoecdysteroids extract.

C. officinalis Kuan is the dry root of Cyathula officinalis Kuan. Clinically, it is used for fall and flutter injury, rheumatism and arthralgia. Phytoecdysteroids have significant anti-inflammatory effects, and the phytoecdysteroids present in C. officinalis Kuan exhibit potential for treating rheumatoid arthritis.This study first developed a selective, accurate and efficient LC-MS/MS method for 12-day pharmacokinetic studies regarding the simultaneous determination of cyasterone, 25-epi-28-epi-cyasterone, precyasterone and capitasterone from C. officinalis Kuan phytoecdysteroids extract in normal and adjuvant arthritis rats.An Agilent Eclipse Plus C18 RRHD column (1.8 µm, 50mm × 2.1 mm) with a gradient mobile phase consisting of water (A) and acetonitrile (B) was used for analysis. The mass analysis was performed in an Agilent 6430 QQQ-MS mass spectrometer with positive mode multiple reaction monitoring (MRM).The results indicated that the AUC0-t and AUC0-∞ values of the four phytoecdysteroids in adjuvant arthritis rats were different from those in normal rats on the first day, which could provide a helpful reference for pharmacological and toxicological studies, as well as clinical applications of C. officinalis Kuan in the treatment of rheumatoid arthritis.

1. C. officinalis Kuan is the dry root of Cyathula officinalis Kuan which has been used for the treatment of flapping injury, rheumatism arthralgia, foot flaccidity, and tendon contracture thousands of years in China, and has been officially included in the Chinese Pharmacopoeia.2. A highly accurate, stable, and sensitive ultra-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method was first established and validated for simultaneously determination four phytoecdysteroids: cyasterone, 25-epi-28-epi-cyasterone, precyasterone and capitasterone in normal and adjuvant arthritis rats plasma samples 12 days of continuous gavage of C. officinalis Kuan phytoecdysteroids extract.3. The phytoecdysteroids is the important component of C. officinalis Kuan, which is difficult to separated. And there is no report for the pharmacokinetic study of phytoecdysteroids from C. officinalis Kuan. And the method provides a good reference for the follow-up studies clinical medication of the phytoecdysteroids from C. officinalis Kuan.

Investigation of Kluyveromyces marxianus as a novel host for large-scale production of porcine parvovirus virus-like particles.

BACKGROUND: Porcine Parvovirus (PPV) is a Parvovirinae virus that can cause embryonic and fetal loss and death and mummification in affected fetal pigs. Unlike conventional vaccines, virus-like particles (VLPs) inherit the natural structure of their authentic virions and highly immunostimulatory that can induce strong humoral immune and T cell responses with no risk of pathogenicity. The production of PPV VLPs is still a challenge based on traditional expression platforms due to their low yields and high culture costs. Kluyveromyces marxianus is a safe and fast-growing eukaryote that can get high biomass with low-cost cultures. In this study, we investigated the expression and downstream processes of PPV VLPs in K. marxianus, and the potential for effective stand-alone vaccines. RESULTS: After optimization according to the codon bias of K. marxianus, the VP2 protein from Kresse strain was highly expressed. In a 5 L fermentator, the yield of PPV VLPs reached 2.5 g/L, quantified by HPLC, using a defined mineral medium after 48 h fermentation. Two strategies were established to purify intracellular PPV VLPs: (i) Using the cation exchange chromatography coupled with Sephacryl® S-500 HR chromatography to purify VLPs from the supernatants of pH adjusted cell lysates. (ii) Using anion exchange chromatography followed by cross-flow diafiltration to recover the VLPs precipitated in pH adjusted cell lysates. The purity of PPV VLPs reached about 95%, and total recovery was more than 60%. Vaccination of mice with the purified PPV VLPs induced high titers of specific IgG antibodies in sera, and showed hemagglutination inhibitions on both swine and guinea pig erythrocytes. Spleen lymphocyte proliferation and cytokines detection suggested the PPV VLPs produced by K. marxianus provoked the cellular immune and humoral immunity responses in mice. CONCLUSIONS: This is the highest production of recombinant PPV VLPs achieved to date. The superiorities, Generally Recognized As Safe (GRAS), high production, short lead time, and low cost, make K. marxianus a greatly competitive platform for bioproduction of PPV VLPs vaccine.

Efficient production of porcine circovirus virus-like particles using the nonconventional yeast Kluyveromyces marxianus.

Porcine circovirus type 2 (PCV2) is a ubiquitous virus with high pathogenicity closely associated with the postweaning multisystemic wasting syndrome (PMWS) and porcine circovirus diseases (PCVDs), which caused significant economic losses in the swine industry worldwide every year. The PCV2 virus-like particles (VLPs) are a powerful subunit vaccine that can elicit high immune response due to its native PCV2 virus morphology. The baculovirus expression system is the widely used platform for producing commercial PCV2 VLP vaccines, but its yield and cost limited the development of low-cost vaccines for veterinary applications. Here, we applied a nonconventional yeast Kluyveromyces marxianus to enhance the production of PCV2 VLPs. After codon optimization, the PCV2 Cap protein was expressed in K. marxianus and assemble spontaneously into VLPs. Using a chemically defined medium, we achieved approximately 1.91 g/L of PCV2 VLP antigen in a 5-L bioreactor after high cell density fermentation for 72 h. That yield greatly exceeded to recently reported PCV2 VLPs obtained by baculovirus-insect cell, Escherichia coli and Pichia pastoris. By the means of two-step chromatography, 652.8 mg of PCV2 VLP antigen was obtained from 1 L of the recombinant K. marxianus cell culture. The PCV2 VLPs induced high level of anti-PCV2 IgG antibody in mice serums and decreased the virus titers in both livers and spleens of the challenged mice. These results illustrated that K. marxianus is a powerful yeast for cost-effective production of PCV2 VLP vaccines.

Simultaneous Determination of Aesculin, Aesculetin, Fraxetin, Fraxin and Polydatin in Beagle Dog Plasma by UPLC-ESI-MS/MS and Its Application in a Pharmacokinetic Study after Oral Administration Extracts of Ledum palustre L.

A rapid, simple and sensitive ultra-performance liquid chromatography-electrospray-ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for the simultaneous determination of aesculin, aesculetin, fraxetin, fraxin and polydatin in beagle dog plasma for the first time. Plasma samples were pretreated by protein precipitation with methanol. Chromatographic separation was performed on an Acquity UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 µm) with gradient elution at a flow rate of 0.4 mL/min, using a mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). The analytes and IS were detected by multiple reaction monitoring (MRM) via negative ion mode with ion transitions of m/z 339.1⁻m/z 176.8 for aesculin, m/z 176.8⁻m/z 88.9 for aesculetin, m/z 206.8⁻m/z 192.1 for fraxetin, m/z 369.1⁻m/z 206.9 for fraxin, m/z 389.1⁻m/z 227.0 for polydatin and m/z 415.2⁻m/z 295.1 for puerarin. This method was validated according to the FDA guidelines and the results met the requirements of analysis. The calibration curves of analytes were linear with correlation coefficients more than 0.9980. The intra- and inter-day precisions were less than 15% and the accuracy was within ±15%. The maximum plasma concentration (Cmax) of aesculin, aesculetin, fraxetin, fraxin and polydatin was 46.75 ± 7.46, 209.9 ± 57.65, 369.7 ± 48.87, 67.04 ± 12.09 and 47.14 ± 12.04 ng/mL, respectively. The time to reach the maximum plasma concentration (Tmax) was 1.32 ± 0.38 h for aesculin, 1.03 ± 0.27 h for aesculetin, 0.94 ± 0.23 h for fraxetin, 0.83 ± 0.18 h for fraxin and 1.15 ± 0.15 h for polydatin. The results indicated that the absorption of aesculin might be slow in beagle dog plasma. This method was successfully applied for pharmacokinetics in beagle dog plasma after oral administration of the extracts of Ledum palustre L. at a dosage of 0.27 g/kg.

Obtaining source current density related to irregularly structured electromagnetic target field inside human body using hybrid inverse/FDTD method.

Inverse method is inherently suitable for calculating the distribution of source current density related with an irregularly structured electromagnetic target field. However, the present form of inverse method cannot calculate complex field-tissue interactions. A novel hybrid inverse/finite-difference time domain (FDTD) method that can calculate the complex field-tissue interactions for the inverse design of source current density related with an irregularly structured electromagnetic target field is proposed. A Huygens' equivalent surface is established as a bridge to combine the inverse and FDTD method. Distribution of the radiofrequency (RF) magnetic field on the Huygens' equivalent surface is obtained using the FDTD method by considering the complex field-tissue interactions within the human body model. The obtained magnetic field distributed on the Huygens' equivalent surface is regarded as the next target. The current density on the designated source surface is derived using the inverse method. The homogeneity of target magnetic field and specific energy absorption rate are calculated to verify the proposed method.

Study on the Hydrogen Effect and Interface/Border Traps of a Depletion-Mode AlGaN/GaN High-Electron-Mobility Transistor with a SiNx Gate Dielectric at Different Temperatures.

In this study, the electrical characteristics of depletion-mode AlGaN/GaN high-electron-mobility transistors (HEMTs) with a SiNx gate dielectric were tested under hydrogen exposure conditions. The experimental results are as follows: (1) After hydrogen treatment at room temperature, the threshold voltage VTH of the original device was positively shifted from -16.98 V to -11.53 V, and the positive bias of threshold was 5.45 V. When the VDS was swept from 0 to 1 V with VGS of 0 V, the IDS was reduced by 25% from 9.45 A to 7.08 A. (2) Another group of original devices with identical electrical performance, after the same duration of hydrogen treatment at 100 °C, exhibited a reverse shift in threshold voltage with a negative threshold shift of -0.91 V. The output characteristics were enhanced, and the saturation leakage current was increased. (3) The C-V method and the low-frequency noise method were used to investigate the effect of hydrogen effect on the device interface trap and border trap, respectively. It was found that high-temperature hydrogen conditions can passivate the interface/border traps of SiNx/AlGaN, reducing the density of interface/border traps and mitigating the trap capture effect. However, in the room-temperature hydrogen experiment, the concentration of interface/border traps increased. The research findings in this paper provide valuable references for the design and application of depletion-mode AlGaN/GaN HEMT devices.

Dichroa febrifuga Lour.: A review of its botany, traditional use, phytochemistry, pharmacological activities, toxicology, and progress in reducing toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Dichroa febrifuga Lour., a toxic but extensively used traditional Chinese medicine with a remarkable effect, is commonly called "Changshan" in China. It has been used to treat malaria and many other parasitic diseases. AIM OF THE REVIEW: The study aims to provide a current overview of the progress in the research on traditional use, phytochemistry, pharmacological activities, toxicology, and methods of toxicity reduction of D. febrifuga. Additionally, further research directions and development prospects for the plant were put forward. MATERIALS AND METHODS: The article uses "Dichroa febrifuga Lour." "D. febrifuga" as the keyword and all relevant information on D. febrifuga was collected from electronic searches (Elsevier, PubMed, ACS, CNKI, Google Scholar, and Baidu Scholar), doctoral and master's dissertations and classic books about Chinese herbs. RESULTS: 30 chemical compounds, including alkaloids, terpenoids, flavonoids and other kinds, were isolated and identified from D. febrifuga. Modern pharmacological studies have shown that these components have a variety of pharmacological activities, including anti-malarial activities, anti-inflammatory activities, anti-tumor activities, anti-parasitic activities and anti-oomycete activities. Meanwhile, alkaloids, as the material basis of its efficacy, are also the source of its toxicity. It can cause multiple organ damage, including liver, kidney and heart, and cause adverse reactions such as nausea and vomiting, abdominal pain and diarrhea. In the current study, the toxicity can be reduced by modifying the structure of the compound, processing and changing the dosage forms. CONCLUSIONS: There are few studies on the chemical constituents of D. febrifuga, so the components and their structure characterization contained in it can become the focus of future research. In view of the toxicity of D. febrifuga, there are many methods to reduce it, but the safety and rationality of these methods need further study.

A review on extraction, purification, structural characteristics, biological activities, applications of polysaccharides from Hovenia dulcis Thunb. (Guai Zao).

Hovenia dulcis Thunb. (H. dulcis) is a widely distributed plant with a long history of cultivation and consumption. As a common plant, it has economic, edible and medicinal value. H. dulcis polysaccharides are one of their main bioactive ingredients and have many health benefits, such as anti-diabetes, antioxidation, anti-glycosylation, anti-fatigue, immune regulation activities and alcoholic liver disease protection activity. In this paper, the research progress of H. dulcis polysaccharides in extraction, purification, structural characteristics, biological activities, existing and potential applications were reviewed, which could provide new valuable insights for future studies.

Prediction of Concrete Compressive Strength in Saline Soil Environments.

Saline soil in Western China contains high concentrations of chloride ions, sulfate ions, and other corrosive ions, and the performance of concrete will substantially deteriorate from exposure to this environment. Therefore, it is of great significance to study and predict the concrete compressive strength in saline soil environments. In this paper, the effects of corrosion on concrete were analyzed from the aspects of surface damage, damage depth, and X-ray diffraction (XRD) of the corrosion products. The effects of corrosion were quantified by damage depth and corrosion depth. Then, considering the corrosion effects combined with Fick's diffusion law, a time-dependent model of concrete compressive strength and a time-dependent model of damage depth were established. The results show that the deterioration of concrete gradually developed from the surface to the interior, and that the interface of the concrete specimen was equivalent to three parts: a failure zone, a filling zone, and an undisturbed zone. The results also showed that the time-varying model of concrete compressive strength proposed by the author was fully applicable, with an error of less than five percent. The service life of concrete predicted by the damage depth was found to be about 253 months (21.1 years), and the service life predicted by the time-varying compressive strength model was about 187 months (15.6 years). Both prediction results were far less than the normal concrete service life of 50 years. In addition, the long-term compressive strength of the corroded concrete was about 90% of that of the noncorroded concrete, which did not deteriorate with the corrosion time.

Fluorescence lifetime imaging of NAD(P)H upon oxidative stress in Kluyveromyces marxianus.

K. marxianus is a promising cell factory for producing heterologous proteins. Oxidative stresses were raised during overexpression of heterologous proteins, leading to the shift of the redox state. How to measure the redox state of live K. marxianus cells without perturbing their growth remains a big challenge. Here, a fluorescence lifetime imaging (FLIM)-based method was developed in live K. marxianus cells. During the early exponential growth, K. marxianus cells exhibited an increased mean fluorescence lifetime (τ-mean) of NAD(P)H compared with Saccharomyces cerevisiae cells, which was consistent with the preference for respiration in K. marxianus cells and that for fermentation in S. cerevisiae cells. Upon oxidative stresses induced by high temperature or H2O2, K. marxianus cells exhibited an increased τ-mean in company with decreased intracellular NAD(P)H/NAD(P)+, suggesting a correlation between an increased τ-mean and a more oxidized redox state. The relationship between τ-mean and the expression level of a heterologous protein was investigated. There was no difference between the τ-means of K. marxianus strains which were not producing a heterologous protein. The τ-mean of a strain yielding a high level of a heterologous protein was higher than that of a low-yielding strain. The results suggested the potential application of FLIM in the non-invasive screen of high-yielding cells.

A Single Vaccination of IBDV Subviral Particles Generated by Kluyveromyces marxianus Efficiently Protects Chickens against Novel Variant and Classical IBDV Strains.

Infectious bursal disease (IBD), caused by the infectious bursal disease virus (IBDV), is a highly contagious and immunosuppressive disease in chickens worldwide. The novel variant IBDV (nvIBDV) has been emerging in Chinese chicken farms since 2017, but there are no available vaccines that can provide effective protection. Herein, the capsid protein VP2 from nvIBDV strain FJ-18 was expressed in Kluyveromyces marxianus with the aim to produce nvIBDV subviral particles (SVPs). Two recombinant strains constructed for expression of nvIBDV VP2 (nvVP2) and His-tagged VP2 (nvHVP2) formed two types of nvIBDV subviral particles (SVPs), namely nvVP2-SVPs and nvHVP2-SVPs. TEM scans showed that both SVPs were about 25 nm in diameter, but there was a large portion of nvVP2-SVPs showing non-spherical particles. Molecular dynamics simulations indicate that an N-terminal His tag strengthened the interaction of the nvHVP2 monomer and contributed to the assembly of SVPs. Vaccination of chicks with the nvHVP2-SVPs provided 100% protection against novel variant IBDV infection when challenged with the FJ-18 strain, as well as the classical strain BC6/85. By contrast, vaccination with the nvVP2-SVPs only provided 60% protection against their parent FJ-18 strain, suggesting that the stable conformation of subviral particles posed a great impact on their protective efficacy. Our results showed that the nvHVP2-SVPs produced by the recombinant K. marxianus strain is an ideal vaccine candidate for IBDV eradication.

Integrated application of uniform design and least-squares support vector machines to transfection optimization.

BACKGROUND: Transfection in mammalian cells based on liposome presents great challenge for biological professionals. To protect themselves from exogenous insults, mammalian cells tend to manifest poor transfection efficiency. In order to gain high efficiency, we have to optimize several conditions of transfection, such as amount of liposome, amount of plasmid, and cell density at transfection. However, this process may be time-consuming and energy-consuming. Fortunately, several mathematical methods, developed in the past decades, may facilitate the resolution of this issue. This study investigates the possibility of optimizing transfection efficiency by using a method referred to as least-squares support vector machine, which requires only a few experiments and maintains fairly high accuracy. RESULTS: A protocol consists of 15 experiments was performed according to the principle of uniform design. In this protocol, amount of liposome, amount of plasmid, and the number of seeded cells 24 h before transfection were set as independent variables and transfection efficiency was set as dependent variable. A model was deduced from independent variables and their respective dependent variable. Another protocol made up by 10 experiments was performed to test the accuracy of the model. The model manifested a high accuracy. Compared to traditional method, the integrated application of uniform design and least-squares support vector machine greatly reduced the number of required experiments. What's more, higher transfection efficiency was achieved. CONCLUSION: The integrated application of uniform design and least-squares support vector machine is a simple technique for obtaining high transfection efficiency. Using this novel method, the number of required experiments would be greatly cut down while higher efficiency would be gained. Least-squares support vector machine may be applicable to many other problems that need to be optimized.

A broadband and low cross polarization antenna with a balun of microstrip line coupling to slot line.

In this paper, a wide-band low cross polarization antenna with a structure of microstrip line coupling to slot line as the balun is proposed. The radiation part of the antenna is fed by two pairs of parallel transmission line via a transition from a slot line which is coupled by a microstrip line. Because it is fed by parallel transmission lines, which is balanced-fed structure, the antenna can achieve an improved low cross-polarization performance. The height of the antenna is 0.146λ0 (λ0 is the wavelength of lowest frequency). The prototype antenna demonstrates a measured impedance bandwidth of 93.5% (2.7-7.44 GHz), a 3-dB-gain bandwidth of 77% (2.7-6.1 GHz), and a maximum gain of 10.5 dBi at 4.5 GHz.

Development of Monoclonal Antibodies Specifically Recognizing the Nonstructural Protein 12 of Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens that has caused tremendous economic losses to the swine industry worldwide. Although extensive research has been focused on PRRSV, little is known about the structure and biological functions of individual nonstructural viral proteins, especially the nonstructural protein 12 (Nsp12). In this study, we generated and identified the monoclonal antibody (mAb) against PRRSV Nsp12. Six strains of hybridoma cells named 2B10, 2B12, 5E1, 5G6, 5E7, and 8B2 secreting anti-Nsp12 mAbs were obtained by the hybridoma technique. All the mAbs were specifically reacted with PRRSV by indirect immunofluorescence assay and four of them (2B12, 5E1, 5G6, and 5E7) were specifically reacted by Western blot. Furthermore, the 5E7 specifically recognized multiple type 2 PRRSV strains, including highly pathogenic and classical PRRSV strains, but not type 1 PRRSV strain. Taken together, the mAbs against Nsp12 provide a valuable tool to specifically recognize type 2 PRRSV as a diagnostic reagent and study the biological function of Nsp12 in the future.

The emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus with additional 120aa deletion in Nsp2 region in Jiangxi, China.

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS), which emerged in China in 2006, was characterized by high fever, high morbidity and high mortality. The causative agent of the disease was a highly pathogenic variant of porcine reproductive and respiratory syndrome virus (also called HP-PRRSV), which has a discontinuous deletion of 1 + 29 amino acids (aa) in the Nsp2 coding region, compared to classical PRRSV. In 2014, fattened pigs on a pig farm in Jiangxi Province suffered from clinical symptoms of high fever, dyspnoea and death. A PRRSV, termed JX2014T2, was isolated from samples of the dead pigs. Genomic analysis of the isolated PRRSV indicated that the genome of the virus was 14,960 bp in length and belonged to the North American genotype. In the Nsp2-coding region, there was a discontinuous deletion of 1 + 29 aa, similar to HP-PRRSV; however, an additional continuous deletion of 120 amino acids between aa 628 and 747 was found. Further analysis of the pathogenicity of PRRSV JX2014T2 was performed in piglets, and the results indicated that all infected piglets suffered from typical clinical symptoms of PRRS, such as high fever, cough, mental depression, anorexia, dyspnoea and palpebral swelling and died within 15 days postinfection (dpi). This demonstrated that the newly isolated PRRSV JX2014T2 strain containing an additional deletion of 120 aa is highly pathogenic to piglets, suggesting that a highly pathogenic variant with new genetic features is circulating in China.

Multipole Modes Excitation of uncoupled dark Plasmons Resonators based on Frequency Selective Surface at X-band Frequency Regime.

In this report, we theoretically and experimentally demonstrates that multipole modes could be excited effectively in dark plasmonic resonators without introducing any other bright resonators at microwave range based on a two-dimensional frequency selective surface (FSS) structure. These excited multipole resonances are closely related to the coupling strength between adjacent S-LSPs resonators (the periodicity of the FSS). The modes splitting effects and resonance frequencies of the excited multipole modes are regulated by changing the numbers of grooves and inner disk radius, both of which play significant roles in the excitation of the dark S-LSPs disk resonator at normal incidence. Moreover, the multipole resonances characteristics of dark S-LSPs resonators in the case of oblique incidence are also presented. Observation of such multipole resonances in dark S-LSPs without introducing extra bright resonance at normal/oblique incidence would find more potential applications in microwave and terahertz based sensors, plasmonic resonance devices and metamaterial devices.

The Identification and Characterization of Two Novel Epitopes on the Nucleocapsid Protein of the Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea and death, particularly in neonatal piglets. The nucleocapsid protein (N protein) of PEDV presents strong immunogenicity and contributes to the cross-reactivity between PEDV and TGEV. However, the characterization of epitopes on the PEDV N protein remains largely unknown. Here, two monoclonal antibodies (MAbs) specific to the N protein of a PEDV strain, FJzz1/2011, were generated and screened against a partially overlapping library of 24 GST-fusion N protein-truncated constructs. We confirmed that residues 18-133 (designated NEP-D4) and residues 252-262 (designated NEP-D6) were the epitopes targeted by MAbs PN-D4 and PN-D6, respectively. Sequence analysis revealed that these two epitopes were highly conserved among PEDV strains but were significantly different from other members of the Coronavirinae subfamily. Western blot analysis showed that they could be specifically recognized by PEDV antisera but could not be recognized by TGEV hyperimmune antisera. Indirect immunofluorescence (IFA) assays confirmed no cross-reaction between these two MAbs and TGEV. In addition, the freeze-thaw cycle and protease treatment results indicated that NEP-D4 was intrinsically disordered. All these results suggest that these two novel epitopes and their cognate MAbs could serve as the basis for the development of precise diagnostic assays for PEDV.

[Bibliometrics study on dominant diseases in modern acupuncture clinic].

OBJECTIVE: To find out the dominant diseases in the clinic of modern acupuncture. METHODS: By means of bibliometrics, clinical acupuncture study literatures from 1978 to 2004, searched from CBM database, were sorted and counted to show the different clinical utilizing quantities and developing trends of different disease groups in the acupuncture clinic. RESULTS: Obviously dominant type: nervous system diseases; mature type: motor system diseases; developing type: 3 kind of diseases including psychosis; premature type: diseases related with surgery; steady type: 3 kind of diseases including digestive system diseases (diseases of liver and gallbladder are not included); pre-developing diseases: 5 kind of diseases including otorhinolaryngologic diseases. CONCLUSION: Among all these types, obvious advantage type and mature type are the most distinguishing. Developing type has the most significant ascending trend. Premature type has relatively strong developing potentiality.