[Results and analysis of pulmonary function examination in 76 698 cases of physical examination crowd in Henain Province].

Objective: Pulmonary function testing (PFT) and electrocardiograph (ECG) are the vital components of the cardiopulmonary exercise test (CPET). This study is to investigate clinical characteristics of abnormal PFT as pulmonary ventilation dysfunction, small airway dysfunction and gas exchange (diffusion) dysfunction. Methods: Across-sectional study was conducted The 76 698 outpatient subjects who received health examination from December 2016 to February 2019 in Henan Provincial People's Hospital were recruited. The results of the ECG, PFT were compared among different sex and age sub-groups. Then the severity of their impaired PFT were analyzed. Results: Among 76 698 subjects, 39 237 subjects were male and 37 461 subjects were female. There were total 71.04% patients with abnormal ECG. There were total 28 273 (36.86%) patients with abnormal pulmonary ventilation function. The 17 570 patients (44.78%) (17 570/39 237) were male, 10 703 patients (28.57%) (10 703/37 461) were female, both the number and percentage of abnormal pulmonary ventilation function in male was significantly more than these in female (P<0.01). The percentage detectable rates of male were significant higher than that of female in all the different age sub-groups: 20~29, 30~39, 40~49, 50~59, 60~69 and ≥70 year (P<0.01). The total detectable abnormal rate of small airway dysfunction were 43 160 and 56.26% (43 160/76 698). The 57.73% (22 661/39 237) in male was significantly higher than 54.72% (20 499/37 461) in female (x2=74.87, P<0.01). The detectable abnormal rate of small airway dysfunction in male were lower than female in 30~39 year and 40~49year sub-groups (P<0.05), but were significantly higher in 20~29, 50~59, 60~69, and ≥70 yr sub-groups (P<0.05). Abnormal gas exchange (diffusion) dysfunction were detected in 28.54% (12 940/45 107) subjects. They were 7 433 (30.55%) in male,and 5 507 (26.50%)in female with significant gender difference (P<0.05). The abnormal diffusion detectable rate in 30~39 year sub-group was significant higher in female than in male (P<0.05), and were slightly higher without significant difference in 20~29 and 40~49 year sub-groups (P>0.05), but were significant lower in female than male in 50~59, 60~69 and ≥70 year sub-groups (P<0.05). Conclusion: The abnormal detectable rates in ECG, pulmonary ventilation dysfunction, gas exchange dysfunction and small airway dysfunction were higher in male than female, and higher in elder ≥70 year subgroup than all other younger age subgroups.

PIFA-Mediated Dearomatizative Spirocyclization of Phenolic Biarylic Ketones via Oxidation and C-C Bond Cleavage.

The dearomatizing spirocyclization of phenolic biarylic ketones using PhI(OCOCF3)2 as oxidant is presented. The reaction affords various cyclohexadienones through C-C bond cleavage under mild conditions. Mechanistic investigations reveal that an exocyclic enol ether acts as the key intermediate in the transformation.

Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction.

Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat's small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5-2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

Highly enhanced adsorption of Congo red by functionalized finger-citron-leaf-based porous carbon.

A novel high-performance porous carbon material, lanthanum(III)-doped finger-citron-leaf-based porous carbon (La/FPC), has been synthesized and used as an adsorbent for anion dye Congo red (CR). The La/FPC was characterized by nitrogen adsorption and desorption isotherms, scanning electron microscopy, transmission electron microscopy and X-ray photoelectron spectroscopy. The adsorption performance of CR by the FPC and La/FPC composites with different contents of lanthanum(III) were evaluated in fixed-bed breakthrough experiments and batch tests at room temperature (298 K). The La/FPC had a high CR uptake capacity, which was superior to those previously reported for other adsorbents. The La/FPC sorbents can be easily regenerated using an ethanol elution technique, and after five cycles the reused La/FPC maintained about 98% of its original CR adsorption capacity. The adsorption kinetics of CR onto the lanthanum(III)-doped FPCs followed a pseudo-second-order kinetic model and fitted well with a Langmuir adsorption isotherm. La/FPC is a promising adsorbent for the removal of the anionic dyes from wastewater.

Construction of C35 gene bait recombinants and T47D cell cDNA library.

C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C351-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMARTTM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 107 pfu/mL with high transformation efficiency of 1.4 × 106, and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

Characteristics of Imported Malaria and Species of Plasmodium Involved in Shandong Province, China (2012-2014).

Malaria remains a serious public health problem in Shandong Province, China; therefore, it is important to explore the characteristics of the current malaria prevalence situation in the province. In this study, data of malaria cases reported in Shandong during 2012-2014 were analyzed, and Plasmodium species were confirmed by smear microscopy and nested-PCR. A total of 374 malaria cases were reported, 80.8% of which were reported from 6 prefectures. Of all cases, P. falciparum was dominant (81.3%), followed by P. vivax (11.8%); P. ovale and P. malariae together accounted for 6.4% of cases. Notably, for the first time since 2012, no indigenous case had been reported in Shandong Province, a situation that continued through 2014. Total 95.2% of cases were imported from Africa. The ratio of male/female was 92.5:1, and 96.8% of cases occurred in people 20-54 years of age. Farmers or laborers represented 77.5% of cases. No significant trends of monthly pattern were found in the reported cases. All patients were in good condition after treatment, except for 3 who died. These results indicate that imported malaria has increased significantly since 2012 in Shandong Province, especially for P. falciparum, and there is an emergence of species diversity.

Epidemiological Investigation of Asymptomatic Dogs with Leishmania Infection in Southwestern China Where Visceral Leishmaniasis is Intractable.

Heishui county, located in northwest Sichuan province, southwestern China, is an endemic area of zoonotic visceral leishmaniasis (VL) and is the most intractable area. VL is never destroyed in it. Asymptomatic dogs (Leishmania parasites have been diagnosed but clinically healthy) are considered to be a potential reservoir host in zoonotic VL area, and most can lead to infection of individuals, that is a new challenge for controlling VL in humans. The present study aimed to assess the Leishmania infection rate of asymptomatic dogs in Heishui county. Total 105 asymptomatic domestic dogs were gathered from 4 districts in Heishui county to investigate the infection rate with serological and molecular methods based on ELISA and kinetoplast minicircle DNA(kDNA) PCR, respectively. Out of 105 dogs, 44 (41.9%) were positive by more than 1 method; 21 (20.0%) were positive by ELISA, and 30 (28.6%) were positive by kDNA-PCR. Our study showed that Leishmania infection of domestic dogs which is clinically healthy is prevalent in the studied district, and the asymptomatic dogs infected by Leishmania may be the primary reason for the prevalence of visceral leishmaniasis in the area.

[Cloning, prokaryotic expression and immunological identification of Toxoplasma surface antigen IMP1].

OBJECTIVE: To subelone, express and identify the immune mapped protein 1 (IMP1) which encodes a surface antigen of Toxoplasma gondii. METHODS: The cDNA of T. gondii RH strain was synthesized by reverse transcription PCR, the IMP1 open reading frame (ORF) was amplified by PCR using the T. gondii RH strain cDNA as template, the PCR products were identified by TA-cloning and sequencing, then the IMPI ORF was subcloned into the Nde I and Xho I sites of the vector pET28b, and the positive recombinant pET28b-IMP1 was identified by double-digesting and sequencing. The protein of 6 x His tagged IMP1 was inducibly expressed in E. coli strain BL21 (DE3) with isopropyl ß-D-1-thiogalactopyranoside (IPTG), and the induction time, concentration of IPTG and temperature gradients to optimize protein expression conditions were determined. After the cells carried IMP1 were induced by the optimized conditions and harvested, the resulting bacteria were suspended in resuspension buffer and lysed by sonication, and the supernatants were loaded onto the Ni2+ Chelating Sepharose Fast Flow column for affinity chromatography of the N-terminal 6 x His tagged IMP1 protein. Finally, the fusion IMP1 proteins were identified by Western blotting. RESULTS: The ORF sequence of IMP1 was successfully subcloned from the cDNA of Toxoplasma Gondii RH strain, and the amplified product was sequenced and identified, based on which the IMP1 ORF gene was inserted into the prokaryotic expression vector pET28b, and the recombinant pET28b-IMP1 was constructed successfully. The double-digesting and sequencing results indicated the validity of the recombinant vector. And the optimized conditions for the expression of IMP1 was determined, namely 0.3 mmol/L IPTG induction for 9 h at 20 °C. Furthermore, IMP1 protein was expressed solubly and chelated on Ni2 sepharose beads with high affinity, thus this protein could be purified efficiently by affinity chromatography. The pure fusion protein was confirmed with fine immunocompetence by SDS-PAGE and Western blotting. CONCLUSIONS: IMP1 protein can be high efficiently expressed by the E. coli prokaryotic expression systems, the protein of IMP1 is soluble and has stable characters. The study may lay a useful foundation for the following works including in vivo expression of IMP1, crystal structure study of IMP1 and anti-toxoplasmosis subunit vaccine development.

Construction and identification of Complex DNA vaccine of hepatitis B and Toxoplasma gondii.

OBJECTIVE: To construct and identify multi-gene recombinant expression vector pcDNA3-HBsAg-p30-ROP2. METHOD: Primers were designed according to the gene sequences of restriction enzyme cutting site of recombinant pcDNA3-p30-ROP2 and hepatitis B surface antigen (HBsAg). The target fragment of HBsAg was amplified and cloned to expression vector pcDNA3-p30-ROP2 by restriction enzyme digestion and ligation. The recombinant expression vector pcDNA3-HBsAg-p30-ROP2 was identified by PCR detection, followed by enzyme restriction and sequencing. RESULTS: The target fragment of HBsAg was successfully amplified, and the multi-gene eukaryotic expression vector pcDNA3-HBsAg-p30-ROP2 was established. PCR detection and restriction enzyme digestion showed that the length of the target fragment was consistent with the theoretical value. The recombinant expression vector contained the complete sequences of p30-ROP2 compound gene and HBsAg. CONCLUSION: Multi-gene recombinant expression vector pcDNA3-HBsAg-p30-ROP2 was successfully established. The constructed expression vector could be used to develop multi-gene nucleic acid vaccines.

[Application of Nested PCR in the Diagnosis of Imported Plasmodium Ovale Infection].

OBJECTIVE: To identity Plasmodium ovale infection by 18S rRNA gene nested PCR. METHODS: Whole blood and filter paper blood samples of malaria patients in Shandong Province were collected during 2012-2013. The parasites were observed under a microscope with Giemsa staining. The genome DNA of blood samples were extracted as PCR templates. Genus- and species-specific primers were designed according to the Plasmodium 18S rRNA gene sequences. Plasmodium ovale-positive specimens were identified by nested PCR as well as verified by sequencing. RESULTS: There were 7 imported cases of P. ovale infection in the province during 2012-2013. Nested PCR results showed that the P. ovale specific band (800 bp) was amplified in all the 7 specimens. Blast results indicated that the PCR products were consistent with the Plasmodium ovale reference sequence in GenBank. CONCLUSION: Seven imported cases of ovale malaria in Shandong Province in 2012-2013 are confirmed by nested PCR.

[Development and application of rapid molecular method for detection of asymptomatic infection of Leishmania].

OBJECTIVE: To develop a rapid molecular biological method for detection of the asymptomatic infection of Leishmania. METHODS: Two pairs of primers named RV1-RV2 and K13A-K13B were selected to be the fast diagnosis primers since they were designed according to the conserved region of Leishmania kinetoplast DNA (kDNA) minicircles. The PCR amplification products of Leishmania donovani promastigote from Shandong Province were sequenced to compare their conservatism. The method was applied to detect 105 venous blood samples from healthy home canine and 7 venous blood samples from home canine suffered from Kala-azar in Heishui County of Sichuan Province, and 75 venous blood samples from susceptible population (no leishmaniasis symptoms) and 7 venous blood samples from patients in Xinjiang Kashi area in order to verify the feasibility and accuracy of the method. RESULTS: The size of PCR products was consistent with the expected fragments with high conservative among Leishmania species. The positive rates of 105 home canine samples and 75 susceptible population samples were 37.14% (39/105) and 82.67% (62/75) rspectively, and the positive rates of home canine suffered from Kala-azar and patients were all 100%(7/7). CONCLUSION: This rapid diagnosis method is suitable for detection of asymptomatic infection of Leishmania in Kalaazar endemic areas of China with high sensitive and specific, thus it has bright perspective to be used.

[Extraction Method of Malaria Parasite DNA from Preserved Positive Blood Smears].

OBJECTIVE: To develop a method for DNA extraction from malaria parasites on preserved blood smears, to provide basis for research on malaria genetic traceability. METHODS: The improved DNA extraction kit (QIAamp DNA Mini Kit) was used to extract plasmodium DNA from 41 giemsa-stained blood smears, and the extraction was compared with that using the Chelex-100 and Na(2)HPO(4) methods. Nested PCR was used to amplify small subunit ribosomal RNA to identify Plasmodium parasite. The PCR products underwent sequencing and sequence alignment, to analyze the difference in PCR positive rates between blood smears prepared in the 1980s and in recent 10 years, between blood smears with and without deoil/decoloration, and between blood smears with different qualities. RESULTS: The total PCR positive rate for the improved kit method was 70.7% (29/41). The PCR positive rate for blood smears prepared in the 1980s and in recent 10 years was 78.6% (11/14) and 66.7% (18/27) respectively, with no significant difference (W=0.63, P>0.05). The PCR positive rate for blood smears with and with- out deoil/decoloration was 62.5% (15/24) and 82.4% (14/17) respectively, also with no significant difference (χ(2)= 1.89, P>0.05). However, the PCR positive rate was significantly higher in blood smears with high quality [93.3% (28/30)] than those with low quality [9.1%(1/1l)](=27.59, P<0.01). Sequence alignment showed that the PCR products were consistent with the target DNA fragments. However, DNA extracted using the Chelex-100 and Na(2)HPO(4) methods showed negative PCR results. CONCLUSIONS: DNA extracted from blood smears prepared in the 1980s using the improved Kit (QIAamp DNA Mini Kit) shows a high PCR positive rate. Besides, blood smear staining and use of oil for microscopic examination do not affect DNA extraction.

[Research progress of interaction between Toxoplasma gondii rhoptry proteins and host cells].

Rhoptry proteins are the major virulence factors of Toxoplasma gondii. They locate in different parts of the host cells, and can affect the membrane, cytoskeleton structure and active factors of the host cells, so as to block the cell intrinsic defense mechanisms of the host, and let T. gondii invade, parasitize and proliferate in the host successfully. The function and ac- tion mode of rhoptry proteins reflect the pathogenic mechanism of T. gondii, which holds great significance to looking for toxoplasmosis drug targets and developing molecule vaccines. This paper reviews the research progess of the interaction between rhoptry proteins of T. gondii and host cells.

[Impact of TDZ and NAA on adventitious bud induction and cluster bud multiplication in Tulipa edulis].

To explore the method of explants directly induced bud and establish the tissue culture system of mutiple shoot by means of direct organogenesis, core bud and daughter bulbs (the top of bud stem expanded to form daughter bulb) of T. edulis were used as explants and treated with thidiazuron (TDZ) and 1-naphthlcetic acid (NAA). The results showed that the optimal medium for bud inducted form core bud and daughter bulb were MS + TDZ 2.0 mg x L(-1) + NAA 4.0 mg x L(-1) and MS +TDZ 2.0 mg x L(-1) + NAA 2.0 mg x L(-1) respectively, both of them had a bud induction rate of 72.92%, 79.22%. The optimal medium for cluster buds multiplication was MS + TDZ 0.2 mg x L(-1) + NAA 0.2 mg x L(-1), and proliferation coefficient was 2.23. After proliferation, cluster buds rooting occurred on MS medium with IBA 1.0 mg x L(-1) and the rooting rate was 52.6%, three to five seedlings in each plant. Using core bud and daughter bulb of T. edulis, the optimum medium for adventitious bud directly inducted from daughter bulb, core bud and cluster bud multiplication were screened out and the tissue culture system of multiple shoot by means of direct organogenesis was established.

[Therapeutic effect of 138 imported falciparum malaria patients].

OBJECTIVES: To summarize the clinical characteristics of imported falciparum malaria patients and the treatment, so as to provide the evidences for improving the diagnosis and treatment of the disease. METHODS: A total of 138 imported falciparum malaria patients who received the treatment in Shandong Institute of Parasitic Diseases from January 2007 to February 2013 were adopted as the observation subjects, and their clinical data were collected and analyzed. RESULTS: All the 138 patients were back from African countries. The main manifestations were fever, headache, asthenia, and hepatosplenomegaly, and most of them were with decreased RBC, PLT levels and increased LDH levels, and 36.96% of them were misdiagnosed as respiratory diseases, nephritis, hepatitis and so on. Through antimalarial treatment of artemether or artesunate or dihydroartemisinin and primaquine, or dihydroartemisinin and piperaquine, and symptomatic treatment, the short-term and long-term cure rates were 98.55% and 94.93% respectively, with 1 case unrecovered and 1 died. CONCLUSIONS: Artemisinins are still the most effective antimalarial drugs for falciparum malaria. However, some patients recrudesce as the Plasmodium in their body is resistant or insensitive to these drugs. We should pay more attention to the antimalarial and symptomatic treatments in the early stage of severe malaria so as to improve the cure rate.

Study of the anti-sapstain fungus activity of Bacillus amyloliquefaciens CGMCC 5569 associated with Ginkgo biloba and identification of its active components.

An endophytic bacterium, designated strain Bacillus amyloliquefaciens CGMCC 5569 was isolated from Chinese medicinal Ginkgo biloba collected from Xuzhou, China. Both the filtrate and the ethyl acetate extract of strain CGMCC 5569 showed growth inhibition activity against the sapstain fungi Lasiodiplodia rubropurpurea, L. crassispora, and L. theobromae obviously (>65%) based on the comparison of the length of zones on the petri dish. From the ethyl acetate extract of the filtrate, the antifungal compounds were obtained as a series of lipopeptides, which including series of fengycin, surfactin and bacillomycin. It showed strong growth inhibition activity in vitro against the L. rubropurpurea, L. crassispora and L. theobromae by about 70.22%, 69.53% and 78.76%, respectively. The strong anti-sapstain fungus activity indicated that the endophytic B. amyloliquefaciens CGMCC 5569 and its bioactive components might provide an alternative bio-resource for the bio-control of sapstain.

[Identification of six Trichinella isolates from China by multiplex PCR].

OBJECTIVE: To identify and classify six isolates of swine-originated Trichinella from China. METHODS: Five specific pairs of primers were synthesized based on DNA sequence of expansion segment V region and internal transcribed spacers (ITS1 and ITS2) of ribosomal DNA repeat from Trichinella. International reference strains of five Trichinella species [Trichinella spiralis (T1), T. nativa (T2), T. britovi (T3), T. pseudospiralis (T4) and T. nelsoni (T7)] were used as control. Six swine Trichinella isolates from Henan, Yunnan, Harbin, Tongjiang of Heilongjiang, Hubei and Tianjin were identified by multiplex PCR and its effecting factors of PCR amplification were observed. RESULTS: Electrophoresis results of multiplex PCR products of Trichinella larvae showed that the band (173 bp) of the six isolates was the same as T. spiralis (T1). The specific band (173 bp) was detected by multiplex PCR through amplification from issues of single T. spiralis larva, the larvae conserved in 80% ethanol for 6 months, the larvae stored in 10% formaldehyde, in 0.05% formaldehyde, 0.2% sodium azide or 0.05% merthiotate for 2 weeks, or fresh mouse muscle with larvae. CONCLUSION: All the six swine Trichinella isolates are identified as T. spiralis (T1) by multiplex PCR.