Objective To investigate the role of ribosomal protein S7(rpS7) in the defense of Anopheles dirus against infection. Methods rpS7 was amplified from Anopheles dirus hemocytes with degenerated primers designed according to the conservative region of S7, rpS7 was then cloned using T/A cloning kit and the inserted fragment was sequenced. The difference of the transcript abundance of rpS7 from Anopheles dirus hemocyte among non-blood-fed (N), normal-blood-fed (B) and Plasmodium yoelii infected groups (I) was also analyzed by RT-PCR and gel scanning system at d1, d2, d3, d4, d7 and d11 after blood feeding. Results There is no significant difference of rpS7 signal between the three groups. Conclusion Anopheles dirus S7 can be used as an internal control to study the role of Anopheles dirus related immune factors in Plasmodium infection.

Objective To observe the effect of blood feeding and plasmodium yoelii infection on the transcript abundance of ribosomal protein S7 from Anopheles dirus hemocyte Methods Anopheles dirus of 3～5 days old were divided into normal group (N), blood feeding group(B) and Plasmodium yoelii infection group(I) Hemocytes of 50 Anopheles dirus from each group were collected by centrifuge method on days 1, 2, 3, 4, 7 and 11 after blood feeding, respectively Then, all hemocyte samples were used for total RNA isolation and analyzed by RT PCR Finally, the same volume(10 ?l) of all the PCR products from each group was used for agarose gel electrophoresis and the data obtained were analyzed statistically Results There was no significant difference in ribosomal protein S7 signal between the three groups Conclusion Similar to Anopheles gambiae and human rpS7, Anopheles dirus rpS7 might be also used as an internal control for the studies of the role of Anopheles dirus related immune factors in attacking Plasmodium infection

Objective To construct cDNA library from adults of Anopheles dirus for cloning the immune genes or related genes for malaria parasites development. Methods The mRNA of adult Anopheles dirus was isolated. The library was constructed by using the Zap Express vector(Stratagene) and the quality was evaluated. Results The efficiency of the library was 2.1?10 6 pfu/ml with 98% clones positive. The average length of the insert fragment was over 1 kb. Conclusion cDNA library of adult Anopheles dirus with high efficiency can be constructed by using the Zap Express library construction Kit (Stratagene).

Objective To investigate the expression patterns of serine proteases Adsp1 and Adsp3 in the main tissue of Anopheles dirus and the effects of blood feeding and Plasmodium infection on the transcript abundance of Adsp1 and Adsp3 from Anopheles dirus haemocytes. Methods Haemocytes were collected by centrifugation. The midguts and salivary glands were dissected from 50 adult female Anopheles dirus of 3-5 d old for the extraction of total RNA for amplification by RT PCR. Anopheles dirus of 3-5 d old were divided into normal group (N), blood feeding group (B), and Plasmodium yoelii infection group (I). Haemocytes of 50 adult female Anopheles dirus from each group after blood feeding were also collected, and the total RNA was isolated at 1, 2, 3, 4, 7, and 11 d, respectively. Then, the same volume (10 ?l) of total RNA was analyzed by semi quantitative RT PCR with Ads7 as the internal control. Agarose gel analysis was performed on PCR products from each group. Results Expression of both Adsp1 and Adsp3 mRNA was found in the haemocytes and salivary glands, but not in the midguts. Semi quantitative RT PCR indicated that transcript abundance of AdsP1 and AdsP3 from Anopheles dirus haemocytes was enhanced after blood feeding and Plasmodium yoelii infection, especially during melanotic encapsulation of Plasmodium yoelii . Conclusion AdsP1 and AdsP3 may be involved in the melanotic encapsulation response of Plasmodium yoelii by Anopheles dirus , and may be the prophenoloxidase activating enzyme of Anopheles dirus .

Objective To study the change of intracellular free Ca2+ in the oocyst when it melanized and to find out the relationship between the melanized oocyst and its intracellular level of free Ca2+ in a Plasmodium-refractory strain of Anopheles dirus. Methods The distribution and experimental condition of the intracellular free Ca2+ in oocyst of Plasmodium yoelii was measured with Ca2+ sensitive dye Fluo-3/AM and Plutonic F-127 under confocal laser scanning microscope (CLSM) at different time. Results The best load condition was that the oocysts were incubated in 3 ?mol/ml Fluo-3/AM adding 1 ?l/ml 25% Pluronic F-127 for 60 min at 37 ℃ . Fluorescent imaging of oocysts was affected by an increase or decrease of the concentration of Fluo-3/Am and incubation time. The distribution of intracellular free Ca2+ was heterogeneous in the oocysts. The mean value of Ca2 + in the mature oocysts was (137.15 ?7.02) nmol/L(X?S) but was (18.44? 1.75) nmol/L in melanized oocysts with Ca2+ sedimentation in the wall of oocyst. Conclusion The results suggest that the level of the intracellular free Ca2+ in oocyst decreased and excreted during its melanization in a Plasmodium-reiractory anopheline mosquito species.

Objective To analyze the relationship between the TEP1 gene of Anopheles stephensi and melanotic encapsulation of Plasmodium yoelii induced by anti-malaria drug nitroquine. Methods Haemolymph samples from three groups of An. stephensi fed with sucrose solution, Plasmodium-infected blood and nitroquine, respectively, were collected at the 1st, 2nd, 3rd and 4th day after drug adminstration. Degenerate primers were designed according to the conserved amino acid sequence within TEPs of the mosquitoes. Fluorescent quantitation PCR was used to detect the variation of TEP1 gene transcript induced by nitroquine. The melanization of oocysts was observed by light microcopy. Results TEP1 gene was cloned, the predicted amino acid residues harbored a highly conserved canonical thioester motif GCGEQ. The fluorescent quantitation PCR revealed that nitroquine induced an up-regulation of TEP1 activity. The transcription of TEP1 gene in nitroquine treated group (2.423) was significantly higher than that of the infected blood-fed group(1.036) at the 3rd day after nitroquine treatment (P

Objective　To explore the relationship between hemolymph phenol oxidase and the melanization of Plasmodium yoelii oocysts in Anopheles dirus. Methods　An Anopheles dirus-Plasmodium yoelii system was used Anopheles dirus were divided into 3 groups, that is, non-blood-fedding (N), normal-blood-fedding (B) and infected-blood-fedding (I). The activities of MPO and o-DPO in hemolymph from 3 groups were determined with native polyacrylamide gel electrophoresis (PAGE) and density scanning at 5, 7, 11 and 15 d after blood feeding. Results　Both MPO and o-DPO activity were significantly higher in group I than group N and B (P<0.05). But with the melanization of Plasmodium yoelii oocysts, both MPO and o-DPO activity in group I were decreased in comparison with group N, especially on the 15 th day after infected-blood feeding. MPO and o-DPO activity in group B were significantly stronger than those of group N. Conclusion　Blood feeding and infection of Plasmodium yoelii both can activate the cascade. The heamolymph phenol oxidase may play an important role in the melanization of Plasmodium yoelii oocysts in Anopheles dirus.

Objective:To analyze the dosimetric differences between gamma knife stereotactic body radiation therapy (SBRT) and linear accelerator-based SBRT for lung tumors by comparison to provide a theoretical basis for the selection of treatment strategies.Methods:Seven patients who underwent SBRT for lung tumors in the Cancer Center of Affiliated Zhongshan Hospital of Dalian University from January 2022 to May 2023 were enrolled. Plans of gamma knife SBRT (γ_SBRT) or linear accelerator-based SBRT plans (X_SBRT) were designed for the 13 lesions in the patients, with adjacent lesions in the same patient sharing one plan. As a result, 10 γ_SBRT plans and 10 X_SBRT plans were obtained. All lesions received 30-50 Gy of radiation in 5-10 fractions. Then, dosimetric parameters were analyzed and compared between γ_SBRT and X_SBRT plans, including the target coverage, gradient index (GI), conformity index (CI), maximum dose ( Dmax); mean dose ( Dmean), and minimum dose ( Dmin) of planning target volumes (PTVs); lung volumes receiving 20 Gy or more ( V20), 10 Gy or more ( V10), 5 Gy or more ( V5), 100% of the prescription dose ( V100%), and 50% of the prescription dose ( V50%); Dmean and the percentages of lung volume receiving doses of 20 Gy or more (Lung_ V20) and 5 Gy or more (Lung_ V5) of ipsilateral lung; Dmean and Lung_ V5 of contralateral lung; and Dmax values of the esophagus, spinal cord, and heart. Results:Compared to X_SBRT plans, γ_SBRT plans exhibited superior GI, V20, V10, V5, V50%, the Dmean, Lung_ V20, and Lung_ V5 of ipsilateral lung, the Dmean and Lung_ V5 of the contralateral lung, and the Dmax of esophageal and heart ( z = -2.81 to -1.99, P < 0.05), higher Dmax and Dmean of PTVs ( z = -2.80, -2.80, P < 0.05), and longer delivery time ( z=-2.70, P<0.05). Meanwhile, there was no significant difference in target coverage, CI, and Dmax of the spinal cord ( P > 0.05). Conclusions:Gamma knife SBRT plans can achieve sharper dose falloff outside target volumes than linear accelerator-based SBRT plans. Gamma knife radiosurgery is expected to reduce the radiation dose to low-dose areas around PTVs and normal lung tissue in SBRT for lung tumors. However, it significantly prolongs the delivery time.

OBJECTIVE To obtain stronger cytotoxic activity of panaxadiol derivatives. METHODS The 3-amino panaxadiol was prepared by the bioelectronic isosteric principle, and then 18 derivatives of cinnamic acid, NO donor and other types of panaxadiol derivatives were synthesized, among them, 12 compounds had not been reported in the literature, and their structures had been confirmed by 1H-NMR, 13C-NMR and mass spectrometry. These compounds were evaluated for their cytotoxic activity by MTS assay against human leukemia cell line HL-60, liver cancer cell line SMMC-7721, lung cancer cell line A-549, breast cancer cell line MCF-7, and colon cancer cell line SW480. RESULTS These results showed that compounds 6c, 7 as well as 7j exhibited potent inhibitory activities against all five tumor cells, especially the IC50 values of compound 7 against HL-60 and SMMC-7721cells were 3.41 and 4.51 μmol·L−1, respectively. It was significantly superior to panaxadiol in cytotoxicity. CONCLUSION These results show that 7 and 7j can be used as promising lead compounds for further research.