Objective To determine sequence of sporogony stage-specific (S type) 18S ribosomal RNA gene of Plasmodium yoelii (P.y) By265 strain, and by using it to detect the malaria parasites within vector mosquito. Methods A pair of conserved DNA primers, universe primer (Pu) and reverse transcription one (Pr), was designed and synthesized according to sequence of the 18S rRNA gene of Plasmodium berghei (P.b). The segment of the S type 18S rDNA of P.y was amplified by reverse transcript-polymerase chain reaction (RT-PCR) from dissected midguts of Anopheles stephensi infected with P.y on the 7th day after infective blood-meal, and its sequence was then determined. One P.y sporogony stage-specific primer (Pys) was selected according to the sequence. Using this primer and Pr, the parasites within mosquitoes were semi-quantitatively detected through RT-PCR between 1-7 d post-infection. Results The length of the amplified segment was 920 bp. Alignment in match region of the 18S rDNA among S type of P.y (PyS), S type of P.b (PbS) and asexual blood stage-specific one of P.y (PyA) revealed that the similarity between the former and the latter two reached 95\^3% and 94\^0% respectively. The density of amplified band was significantly concordance with the intensity of oocyst in the midgut. Sensitivity of RT-PCR method was higher than that of the traditional dissection and oocyst observation also. The assay could detect the 18S rRNA molecule of the parasites on the third day post-infection while their oocysts were difficult to be recognized under an optical microscope at that time. Conclusion This S type 18S rDNA sequence in P.y species was first reported (AF266261). As a molecular marker, it could be applied to monitoring the parasite development in its vector at an earlier stage semi-quantitatively with an adequate sensitivity and specificity.

ObjectiveTo explore the characteristics of traditional Chinese medicine (TCM) syndrome differentiation in chronic complications of type 2 diabetes mellitus (DM).Methods124 patients with chronic complications of type 2 DM were scored by 5 grades according as the severities of their symptoms. There were 5 kinds of patterns such as deficiency of Qi, deficiency of Yin, deficiency of Yang, blood stasis and retention of phlegm and fluid by which the TCM syndrome differentiation was generalized.ResultsThe sequence of TCM patterns was deficiency of Yin, blood stasis, deficiency of Qi, deficiency of Yang and retention of phlegm and fluid, and the syndrome of the two formers were greater than 50%. The proportion of unity of deficient and excessive pattern was 80.5%. Three larger syndrome types were deficiency of both Yin and Yang combined with blood stasis (17.7%), Qi-Yin deficiency with blood stasis ( 16.9 %) and Yin deficiency with blood stasis (16.9%). There was a statistically significant difference in TCM syndromes which were divided into different groups by course of diseases (P<0.05). At onset of DM, the typical symptoms were less observed in the group whose course of disease was less than 5 years, and only 39.1% of patients had the typical symptoms. But at the same time, the prevalences of hypertension and hyperlipidemia were higher in this group than in the others, respectively 63.0% and 87.0%.ConclusionThe primary syndrome is unity of deficient and excessive pattern in chronic complications of type 2 DM and deficiency of Qi and blood stasis are the commonest patterns in course of DM.

Objective To optimize technology of three can group dynamic countercurrent extraction process of ferulic acid from Angelicae Sinensis Radix.Methods The content of ferulic acid was determined by HPLC. With content of ferulic acid as index, comprehensive test was used to investigate effect of extraction solvent and extraction time on extraction efficiency.Results The optimum process parameters were as follows:extraction solvent with 10 times of water;20 minutes for each extraction time.Conclusion The process which uses method of three can group of dynamic countercurrent extraction of ferulic acid from Angelicae Sinensis Radix is reliable, highly efficient and energy saving.

Objective To study the effects of ultrafiltration process on activating blood and removing blood stasis efficacy ofShentong Zhuyu Decoction; To investigate the feasibility of applying ultrafiltration technology in purifying Shentong Zhuyu Decoction.Methods The mice micro artery and vein diameter, clotting time and opening capillary of auricle microcirculation of mice were used as indexes to observe the effects of different ultrafiltration process on activating blood and removing blood stasis efficacy ofShentong Zhuyu Decoction.ResultsShentong Zhuyu Decoction showed satisfying efficacy of activating blood and removing blood stasis. There was no significant difference between the non-ultrafiltration process and ultrafiltration processed by 20 and 50 nm ultrafiltration membranes.Conclusion Ultrafiltration technology can be applied to purifying Shentong Zhuyu Decoction, and the membrane pore size must be more than 20 nm.

Objective To establish the reference value ranges of specific thyroid function indexes among pregnant women in the Longgang District Maternity and Child Healthcare Hospital for avoiding the misdiagnosis and missed diagnosis of pregnant thyroid diseases .Methods A total of 637 normal pregnant women with establishing the card in the outpatient department of this hospital and 201 non‐pregnant women undergoing the normal physical .The fasting venous blood was collected .The values of FT3 ,FT4 ,T3 , T4 and TSH were detected by adopting the chemiluminescence immunoassay .The median and 95% credibility interval statistical method was used to establish the normal reference value ranges of specific serum free triiodothyronine(FT3) ,free thyroxine(FT4) , three icdine thyroid gland(T3) ,total thyroid hormone(T4) and thyroid stimulating hormone(TSH) in pregnancy .Results The me‐dian(M value) and double‐side limiting value(P2 .5 and P97 .5 ) were used to express the reference value ranges of thyroid hormones in early ,middle and late pregnant women and non‐pregnant women .FT3 and FT4 were significantly increased at early pregnancy ,then gradually increased with the pregnant period progression ,began to decrease from middle pregnancy and were higher than the levels before pregnancy .FT3 and FT4 were slightly increased ,then progressively decreased .TSH was significantly decreased in early pregnancy ,began to increase at middle pregnancy and was higher than the level in non‐pregnant women until late pregnancy .Conclu‐sion The establishment of reference value ranges of specific thyroid hormones during various stages of pregnancy ,which provides the clinical data for the diagnosis and treatment of thyroid disease during pregnant period;the change rule of thyroid hormones in pregnant women provides a theoretical foundation for the prevention of thyroid disease during pregnant period .

Objective To isolate and identify genes related to malaria parasite infection in vector mosquito, and to explore the mechanisms. Methods Anopheles stephensi infected with Plasmodium yoelii was used as tester (T) group, while uninfected but normal blood fed as driver (D) one. Engorged female mosquitoes of two groups were collected separately at 24 hours after biting. An enriched subtractive cDNA pool was generated through the course of suppression subtractive hybridization (SSH) and selective PCR amplification. The subtracted library was screened by hybridization using T and D cDNA mixture as probes, respectively. The positive clones, which produced stronger signal when probed with T than with D, were sequenced and their sequence homologues in GenBank database were searched with BLAST by internet. Results The analysis of subtraction efficiency showed that the differentially expressed genes in T comparing to in D were enriched significantly. In dot blot screening, 24 of 58 randomly selected clones (41.4%) were shown up regulation in malaria infected mosquitoes. The BLAST search of 23 genes revealed that 12 were homologous to functionally known genes, 4 were homologous to functionally unknown entries, and 7 were novel without any relatives. Nine of the 23 genes (39.1%) also hit homologous sequences in the An. gambiae EST database generated from an immune competent cell line treated with lipopolysaccharide (LPS). Conclusion An enriched cDNA pool of the mosquito genes which up regulated responsively at the early stage of malaria parasite infection was obtained. Expression screening against the pool indicated that various biochemical processes and mechanisms might be involved in the response of mosquito to parasite infection, especially those related with the innate immune system and energy metabolism.

Objective To investigate the effect of unfractionated heparin (UFH) on the expression of heme oxygenase-1 (HO-1) in intestinal mucosa of mice with sepsis.Methods Thirty-six male C57BL/6J mice were randomly divided into sham group,cecal ligation and puncture (CLP) group and UHF group,n =12 in each group.Model of intestinal injury in sepsis was induced by CLP.In sham group,the mice were exposed without ligation of cecum.In UFH group,the mice were treated intravenously with 8 U of UFH via the tail vein half an hour before the operation and 12 hours after the surgery respectively.Six mice in each group were randomly chosen at 4 hours and 24 hours after operation to collect inferior vena venous blood samples and terminalileum tissues.The serum levels of interleukins (IL-1 β,IL-6),and tumor necrosis factor-α (TNF-α) were determined by enzyme linked immunosorbent assay (ELISA).The serum level of D-lactate was determined by colorimetry.Pathological changes of ileum tissue and Chiu score were observed after hematoxylin eosin (HE) staining.The HO-1 expression was detected immunohistochemically.Results In sham group,no significant changes in the serum levels of IL-1 β,IL-6,TNF-α and D-lactate were observed.Twenty-four hours after the operation,the structure of intestinal mucosa was basically normal without obvious pathology change and no HO-1 positive cells were found.The serum levels of IL-1 β,IL-6,TNF-α,and D-lactate in CLP group were gradually increased,and they were significantly increased as compared with sham group [IL-1 β (ng/L):40.87±2.88 vs.22.60±2.05 at 4 hours,113.73±3.96 vs.22.07±2.74 at 24 hours;IL-6 (ng/L):63.89±3.26 vs.44.89±3.38 at 4 hours,176.56±5.45 vs.45.76±4.02 at 24 hours;TNF-α (ng/L):194.62± 14.13 vs.152.05±6.22 at 4 hours,599.62± 10.20 vs.155.90± 14.18 at 24 hours;D-lactate (mmol/L):0.24± 0.02 vs.0.19 ± 0.01 at 4 hours,0.33 ± 0.04 vs.0.20 ± 0.02 at 24 hours,all P ＜ 0.05].Twenty-four hours after the operation,edema and inflammation in ileal mucosa,intestinal villi structural damage were observed,the Chiu score was significantly higher than those in the sham group [4.5 (3.0-5.0) vs.0 (0-1.0),P ＜ 0.05],and a small amount of HO-1 positive cells were localized in the intestinal mucosa.Compared with CLP group,the serum levels of IL-1 β,IL-6,TNF-α,and D-lactate of UFH group were significantly decreased [IL-1 β (ng/L):31.53 ± 2.90 vs.40.87 ± 2.88 at 4 hours,61.13 ± 2.80 vs.113.73 ± 3.96 at 24 hours;IL-6 (ng/L):51.16 ± 5.68 vs.63.89 ± 3.26 at 4 hours,81.16 ± 4.54 vs.176.56 ± 5.45 at 24 hours;TNF-α (ng/L):171.76± 5.60 vs.194.62± 14.13 at 4 hours,328.48 ± 10.79 vs.599.62± 10.20 at 24 hours;D-lactate (mmol/L):0.21 ±0.01 vs.0.24±0.02 at 4 hours,0.24±0.02 vs.0.33±0.04 at 24 hours,all P ＜ 0.05].Twenty-four hours after the operation,intestinal injury was ameliorated,the Chiu score was significantly lower [1.5 (1.0-5.0) vs.4.5 (3.0-5.0),P ＜ 0.05],and HO-1 positive cells in the intestinal mucosa was remarkably increased.Conclusion UFH can enhance the expression of HO-1 in intestinal mucosa,reduce the release of inflammatory factors,ameliorate the intestinal inflammatory response,and thus play a protective role in intestinal tissue in mice with sepsis.

Objective To investigate the etiological and antibiotic resistance profile in the old patients with bloodstream infection (BSI).Methods Microbiological and clinical data were collected and reviewed retrospectively for the patients with confirmed bloodstream infection and at least 65 years of age who were treated as inpatients in Tongling People′s Hospital from January to December 2015.Results A total of 107 strains of pathogen were isolated from the blood samples of 107 patients with bloodstream infections, of which community-acquired BSI accounted for 57.9 % (62/107), and hospital-acquired BSI 42.1 % (45/107). Gram negative bacilli accounted for 67.7 % in the pathogens of community-acquired BSI and gram positive cocci accounted for 55.5 % in the pathogens of hospital-acquired BSI. More male BSI patients were secondary to respiratory tract infection than female patients (P<0.001), while more female BSI patients were secondary to urinary tract infection than male patients (P<0.001). Of the 107 isolates, gram negative bacilli, gram positive cocci and fungi accounted for 55.1 % (59/107), 42.1 % (45/107) and 2.8 % (3/107), respectively. The top six pathogens were E. coli (30.9 %), coagulase negativeStaphylococcus (CNS) (20.6 %), S. aureus (10.3 %),K. pneumoniae (6.5 %),Enterococcusspp. (6.5 %) and Acinetobacter spp. (4.7 %). About 51.5 % of the E. coli isolates and 28.6 % of the K. pneumoniae isolates produced extended-spectrum β-lactamases (ESBLs).E. coli isolates showed low resistance rate (< 10 %) to amikacin,cefoxitin and piperacillin-tazobactam. No E. coli isolate was found resistant to carbapenem. About 14.3 % to 28.6 % of K. pneumoniae isolates were resistant to carbapenems. No tigecycline-resistant K. pneumoniae was found. The prevalence of MRSA and MRCNS was 36.4 % and 72.7 %, respectively. No staphylococcal isolates were found resistant to vancomycin, teicoplanin or linezolid. One strain of E. faecium was identified as resistant to vancomycin (VRE).Conclusions This surveillance data indicate that gram negative bacilli play an important role in the BSI of old patients. E. coli and CNS are the most common pathogens. We should pay more attention to the effect of gender and site of infection on the BSI in old patients.

In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene (Sj26), and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into T vector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac, then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells. The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro. By using Western blot, the expression of 26 ku glutathione S-transferase (GST) was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24 x 10(8). Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells. It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.

Objective To investigate the relationship between nerve conduction velocity(NCV)and syndrome differentiation of traditional Chinese medicine(TCM)in patients with diabetic peripheral neuropathy(DPN).Methods 172 patients with DPN were scored by 5 grades according to their symptoms which were classified by 5 kinds of patterns such as Qi-deficiency,Yin-deficiency,Yang-deficiency,Blood-stasis,Phlegm-dampness.NCV of the patients were also tested.Results The frequency of Yin-deficiency and Blood-stasis was more than 50%.The proportion and scores were significantly higher in the elder-age group than in the younger-age group(P<0.05).The scores of Blood-stasis and Yang-deficiency increased in the patients with longer-course(P<0.01).The correlation analysis showed that scores of nervous system were correlated positively with scores of Yin-deficiency,Yang-deficiency and Blood-stasis.There were positive correlation between scores of Qi-deficiency and fasting blood glucose(FBG)as well as between scores of Yin-deficiency and a-lipoprotein(LPa),and between scores of Yang-deficiency and homocysteine(Hcy).The proportion of Yin-deficiency and blood stasis in abnormal NCV group were higher than the normal while the results of Qi-deficiency and Phlegm-dampness were contrary.But the consequence had no statistical significance(P>0.05).Abnormality of NCV was aggravated with the high scores of Yang-deficiency and blood stasis.The analysis with logistic stepwise regression showed that diabetic course,levels of FBG and systolic blood pressure(SBP),be complicated by DN and DR,scores of nervous system sign were the independent risk factors for NCV.Conclusion Yin-deficiency and Blood-stasis are the major TCM syndromes in patients with DPN.The degree of Yang-deficiency and Blood-stasis influenced on the parameters of NCV.Abnormality of NCV can be aggravated with long diabetic course,high levels of FBG and SBP,be complicated by DN and DR,high scores of nerve system sign.