BACKGROUND: Extraction of fungal DNA plays an important role in fungal genetic engineering and molecular biology research.The result of experiment is affected seriously by the efficiency of extracting DNA especially the quality of DNA. OBJECTIVE: To develop a method for extracting genomic DNA of pathogenic fungi and discuss the optimal combination of components in simple sequence repeat PCR (SSR-PCR) system. DESIGN, TIME AND SETTING: A comparative analysis of DNA extraction methods and an orthogonal experiment were conducted in the Mycology Research Lab of Department of Pathogenobiology in Norman Bethune College of Medicine, Jilin University from July 2004 to December 2006.MATERIALS: Clinical specimens were inoculated on Potato dextrose agar, Potato dextrose broth and Yeast extract peptone dextrose and cultivated under the temperature of 28 ℃ for 3-7 days, after which suspectable colonies were selected to be isolated and purified.METHODS: Three kinds of methods of extracting DNA(beading-salt fractionation method, CTAB method and Gene TLE~(TM) extraction method ) were compared in terms of their effects on DNA quality; Experiment was performed with orthogonal design to four factors (Taq DNA polymerase, template DNA, dNTP, primers) in three levels on the basis of L9 (3~4) orthogonal table, The appropriate annealing temperature and cycles were determined through PCR.MAIN OUTCOME MEASURES: The optimal reaction system determined according to the polymorphism and specificity of amplification of banding pattern.RESULTS: The objective fragments were all amplified by Gene TLE~(TM) extraction method, and the banding patterns obtained were clearer and brighter compared with the other two methods. The result of orthogonal experiment on SSR-PCR system showed that,according to the value of R, the significance of factors followed by ascending were template DNA (2.67), Taq DNA polymerase (2.00), dNTP (0.67) and primers (0.33). According to the value of ki, the optimal level of each factor combination was 30 mg/L template DNA, 1U Taq DNA polymerase, 150 μmol/L dNTP, 0.5 μmol/L primer. However, because primers were nonsignificant factors, which was presented by their small R value, we took A level of primer as 0.25 μmol/L. The best reaction condition was 55 ℃ annealing temperature and 35 cycles.CONCLUSION: The Gene TLE~(TM) method shows higher efficiency of extracting DNA and its operation is fast and simplel According to the results of orthogonat experiment, the optimal SSR-PCR system was 30 mg/L template DNA, 1U Taq DNA polymerase, 150 μmol/L dNTP and 0.25 μmol/L primer. The best reaction condition was 55℃ annealing temperature and 35 cycles.

Background: Because of a severe shortage of clinical researchers in Japan, training clinical physicians to perform clinical research is an important issue in medical education. Although education has started to provide a foundation for clinical research, it is unclear whether clinicians, who should play a central role in a clinical research, are interested in performing clinical research and participating in a training program for clinical research.1) We performed a cross-sectional Internet survey to determine the interest of clinicians' interest in performing a clinical research and participating in a clinical-research training program.2) A total of 2176 clinicians were sent emails requesting their participation in this survey, and 310 responded (response rate, 14.6%). Eighty-five percent of the respondents were interested in conducting clinical research, and 78% were willing to participate in a clinical-research training program.3) Most respondents were willing to participate in a training program as part of an educational seminar or a training course after a few years of clinical practice. The respondents desired an educational system that would allow them to learn about clinical research while continuing their clinical practice.4) Although the rate of willingness to participate in a training program was highest (90%) among respondents who wanted to earn a doctorate, the rates were also high among those who did not want to earn a doctorate (76%) and those who had already earned a doctorate (74%).5) An educational system for clinical research should allow graduate schools to play leading role in training and should be flexible enough for clinicians who do not want to earn a doctorate.

Objective To investigate the etiological and epidemiological characteristics of fungal keratitis in Jilin province of China and to establish a rapid and specific method for molecular identification of the prevalent fungal pathogens.Methods Corneal scrapings were collected from 225 patients with suspected fungal keratitis.Fungal strains were isolated and identified based on their morphology and physiological characteristics.The epidemiological characteristics of all isolated strains causing fungal keratitis were statistically analyzed.Species-specific primers of Fusarium solani (F.solani) were designed and used together with the universal fungal primers to establish a multiplex PCR assay for identification of F.solani in corneal scrapings.Results 156 out of 225 patients (69.3％) were diagnosed as fungal keratitis by fungal culture followed by the examination of morphological and physiological characteristics.A total of 168 pathogenic fungi strains were isolated,most of which were Fasarium spp.(49.4％),followed by Aspergillus spp.(17.9％) and Candida spp.(14.3％).F.solani was the predominant pathogen accounting for 34.5％ in all patients.Most of the patients (87.5％) were farmer and male patients (57.1％) accounted for the majority of 156 patients as well.Corneal trauma (38.5％) was considered as the main predisposing factor.The established multiplex PCR could specifically amplify a 300 bp nucleotide fragment of F.solani.It could be used for a rapid identification of F.solani in corneal scrapings.Conclusion Fusarium genus,particularly the species of F.solani,was the predominant pathogen for fungal keratitis in Jilin province of China.Corneal trauma was the most important predisposing factor.The established multiplex PCR could identify fungal infection from corneal scrapings rapidly and specifically.These findings are very important for the early diagnosis and treatment of fungal keratitis.

A survey has been under way on the incidence of cerebral apoplexy among the inhabitants (pop. 105, 185, National Census, 1970) of the Saku district of Nagano Prefecture since April 1972.
This survey, which constitutes a WHO cooperative study, has been under way with cooperation rendered by the local Medical Association, Association of Public Health Nurses in Saku District and Saku Public Health Center with the Saku Central Hospital acting as the survey center.
In the period of two years till March 1974, 585 persons were registered as having been seized with cerebral apoplexy. The annual incidence rate stood at 265 per 100, 000 population. Classified by types of cerebral apoplexy, cerebral hemorrhage accounted for 26%, cerebral thrombosis for 57%, cerebral embolism for 3%, subarachnoid hemorrhage for 9%, and other types for 5%.
By sexes, the incidence rate among males was 1.3 times higher than among females. By ages, the incidence was highest in the seventies.
When the prognosis of cerebral apoplexy is viewed in terms of deaths in the early period of less than three weeks after the onset, the mortality rate stood at 45%.
The hospitalization rate of patients seized with cerebral apoplexy was 55%. There was a significant difference in the mortality rate between inpatients (with 28%) and outpatients (65%).
The ratio of concurrence of autopsy and clinical diagnosis stood at 83% with Okinaka's criteria and 79% with Ikeda's CVD index.
The incidence of hypertension before the onset of cerebral apoplexy was extremely high among the patients seized with cerebral hemorrhage and cerebral infarction.With respect to the treatment of hypertension, the discontinuance of treatment and the failure to undergo it are high in percentage among the patients seized with cerebral hemorrhage. With reference to cerebral infarction patients of 70 years and older in age, there is little defference between the group who discontinued or failed to undergo treatment and the group who underwent it.
The recurrence of cerebral apoplexy stood at 11% for cerebral hemorrhage, 19% for cerebral infarction, and 11% for subarachnoid hemorrhage.

BACKGROUND/AIMS: Weekly granulocyte/monocyte adsorption (GMA) to deplete elevated and activated leucocytes should serve as a non-pharmacological intervention to induce remission in patients with ulcerative colitis (UC). This trial assessed the efficacy of monthly GMA as a maintenance therapy to suppress UC relapse. METHODS: Thirty-three corticosteroid refractory patients with active UC received 10 weekly GMA sessions as a remission induction therapy. They were then randomized to receive one GMA session every 4 weeks (True, n=11), extracorporeal circulation without the GMA column every 4 weeks (Sham, n=11), or no additional intervention (Control, n=11). The primary endpoint was the rate of avoiding relapse (AR) over 48 weeks. RESULTS: At week 48, the AR rates in the True, Sham, and Control groups were 40.0%, 9.1%, and 18.2%, respectively. All patients were steroid-free, but no statistically significant difference was seen among the three arms. However, in patients who could taper their prednisolone dose to <20 mg/day during the remission induction therapy, the AR in the True group was better than in the Sham (p<0.03) or Control (p<0.05) groups. CONCLUSIONS: Monthly GMA may potentially prevent UC relapse in patients who have achieved remission through weekly GMA, especially in patients on <20 mg/day PSL at the start of the maintenance therapy.
Adsorption ; Arm ; Blood Component Removal ; Colitis, Ulcerative ; Extracorporeal Circulation ; Humans ; Inflammatory Bowel Diseases ; Prednisolone ; Prospective Studies ; Recurrence ; Remission Induction ; Salicylamides ; Ulcer

Fecal microbiota transplantation (FMT) is a treatment designed to correct gut dysbiosis by administration of feces from a healthy volunteer. It is still unclear whether FMT for children with ulcerative colitis (UC) is effective or hazardous. Here we describe a young patient to have received FMT for UC. A three-year-old girl was admitted to our hospital with severe active UC, and treated with aminosalicylates and various immunosuppressive drugs. As remission was not achieved, we decided to try FMT before colectomy. We administered donor fecal material a total of six times by retention enema (×2) and via a nasoduodenal tube (×4) within 10 days. The patient developed abdominal pain and pyrexia after each FMT session. Analyses revealed the transferred donor fecal microbiota had not been retained by the patient, who ultimately underwent colectomy. The severity of the UC and/or timing of FMT may have partly accounted for the poor outcome.
Abdominal Pain ; Child* ; Colectomy ; Colitis, Ulcerative* ; Dysbiosis ; Enema ; Fecal Microbiota Transplantation* ; Feces ; Female ; Fever ; Gastrointestinal Microbiome ; Healthy Volunteers ; Humans ; Inflammatory Bowel Diseases ; Microbiota ; Tissue Donors ; Ulcer*

The promotion of clinical research in Japan requires the establishment of a formal and systematic education and training program for clinicians to ensure they become effective clinician investigators. The first of its kind in Japan, a formal 1-year masters-degree-level training program (MCR course) was started at Kyoto University School of Medicine and Public Health. The first 28 students graduated in 2008, with most returning to their original clinical institutions.
1) As follow-up, we conducted a self-administered questionnaire survey of all 28 graduates (response rate, 86%) concerning the current status of clinical research and problems encountered at their institutions.
2) Almost 40% of respondents (n=24) reported "no time" or "no research collaborators" for clinical research.
3) Twenty respondents (83%) have attempted to promote clinical research at their hospital or workplace, but only 1 has received institutional support.
4) Over half of the respondents (54%) would like to be working in both clinical research and clinical practice at their hospital in the future (10-year timescale). Forty-two percent of respondents had a concrete image of the clinical researcher's career path.
5) Although open to improvement, the MCR program presents a concrete model for the education of clinical researchers. These findings suggest that promoting the conduct of clinical research requires the implementation of a support system and adjustment of personal and physical infrastructure.

BACKGROUND/AIMS: Infliximab (IFX), an antibody to tumor necrosis factor, (TNF)-alpha has efficacy in treating Crohn's disease (CD). However, knowledge of the potential effects of IFX on patients' immune profiles is lacking. The purpose of this study was to reveal the immunological effects of IFX. METHODS: Twenty-two patients with a CD activity index (CDAI) of 194.2+/-92.9 and an average duration of disease of 3.26 months and 21 healthy controls were included. Patients were to have their first IFX remission induction therapy with 3 infusions (5 mg/kg) at weeks 0, 2, and 6. Oral 5-aminosalicylic acid was the only ongoing medication in the patient population. Blood samples at baseline, 12 hours after the first infusion and at week 14 were labeled with anti-CD4/CD25 antibodies for immunohistochemical measurement of regulatory T-cells (Treg). Serum cytokines and chemokines were measured by suspension array and ELISA. RESULTS: CDAI significantly decreased prior to the second IFX infusion (p<0.001). Clinical remission rates were 77.3% and 91% by the second and third infusions, respectively. At baseline, interleukin (IL)-6 (p<0.03), IL-8 (p<0.03), IL-10 (p=0.050), IL-13 (p<0.01), transforming growth factor-beta1 (p<0.01), and 'regulated on activation, normal T cell expressed and secreted' (RANTES) (p<0.01) were elevated in patients. After the initial IFX infusion, TNF-alpha (p<0.04), IL-6 (p<0.03), interferon (IFN)-gamma (p<0.04), IFN-gamma-inducible protein-10 (p<0.01), monocyte chemoattractant protein-1 (p<0.01), macrophage inflammatory protein-1beta (p<0.01), and RANTES (p<0.01) were decreased. IFX infusion was associated with an increase in Treg (p<0.01) and a decrease in the Th1 (IFN-gamma)/Th2 (IL-4) ratio (p<0.03). CONCLUSIONS: IFX use was associated with restoration of the Th1/Th2 balance after a single infusion and seemed to promote induction of naive Th0 lymphocytes to Treg. This knowledge should have clinical relevance.
Antibodies ; Antibodies, Monoclonal ; Chemokine CCL2 ; Chemokine CCL5 ; Chemokines ; Crohn Disease ; Cytokines ; Humans ; Immune System ; Interferons ; Interleukin-10 ; Interleukin-13 ; Interleukin-6 ; Interleukin-8 ; Interleukins ; Lymphocytes ; Macrophages ; Mesalamine ; Remission Induction ; T-Lymphocytes, Regulatory ; Tumor Necrosis Factor-alpha ; Infliximab