Predictive Value of Combined HbA1c and Neutrophil-to-Lymphocyte Ratio for Diabetic Peripheral Neuropathy in Type 2 Diabetes.

BACKGROUND Diabetic peripheral neuropathy (DPN) is a prevalent complication affecting over 60% of type 2 diabetes patients. Early diagnosis is challenging, leading to irreversible impacts on quality of life. This study explores the predictive value of combining HbA1c and Neutrophil-to-Lymphocyte Ratio (NLR) for early DPN detection. MATERIAL AND METHODS An observational study was conducted at the First People's Hospital of Linping District, Hangzhou spanning from May 2019 to July 2020. Data on sex, age, biochemical measurements were collected from electronic medical records and analyzed. Employing multivariate logistic regression analysis, we sought to comprehend the factors influencing the development of DPN. To assess the predictive value of individual and combined testing for DPN, a receiver operating characteristic (ROC) curve was plotted. The data analysis was executed using R software (Version: 4.1.0). RESULTS The univariate and multivariate logistic regression analysis identified the level of glycated hemoglobin (HbA1C) (OR=1.94, 95% CI: 1.27-3.14) and neutrophil-to-lymphocyte ratio (NLR) (OR=4.60, 95% CI: 1.15-22.62, P=0.04) as significant risk factors for the development of DPN. Receiver operating characteristic (ROC) curve analysis demonstrated that HbA1c, NLR, and their combined detection exhibited high sensitivity in predicting the development of DPN (71.60%, 90.00%, and 97.2%, respectively), with moderate specificity (63.8%, 45.00%, and 50.00%, respectively). The area under the curve (AUC) for these predictors was 0.703, 0.661, and 0.733, respectively. CONCLUSIONS HbA1c and NLR emerge as noteworthy risk indicators associated with the manifestation of DPN in patients with type 2 diabetes. The combined detection of HbA1c and NLR exhibits a heightened predictive value for the development of DPN.

Elevation and phylogeny shape herbaceous seed dormancy in a biodiversity hotspot of southwest China.

Seed dormancy contributes greatly to successful establishment and community stability and shows large variation over a continuous status scale in mountain ecosystems. Although empirical studies have shown that seed dormancy status (SDS) is shaped by elevation and phylogenetic history in mountain ecosystems, few studies have quantified their combined effects on SDS. Here, we collected mature seeds from 51 populations of 11 Impatiens species (Balsaminaceae) along an elevational gradient in the Gaoligong Mountains of southwest China and estimated SDS using mean dormancy percentage of fresh seeds germinated at three constant temperatures (15, 20, and 25°C). We downloaded 19 bioclimatic variables from WorldClim v.2.1 for each Impatiens population and used internal transcribed spacer (ITS), atpB-rbcL, and trnL-F molecular sequences from the GenBank nucleotide database to construct a phylogenetic tree of the 11 species of Impatiens. Logistic regression model analysis was performed to quantify the effects of phylogeny and environment on SDS. Results identified a significant phylogenetic SDS signal in the Impatiens species. Furthermore, elevation and phylogeny accounted for 63.629% of the total variation in SDS among the Impatiens populations. The best logistic model indicated that temperature was the main factor influencing variation in SDS among the Impatiens species, and model residuals were significantly correlated with phylogeny, but not with elevation. Our results indicated that seed dormancy is phylogenetically conserved, and climate drives elevational patterns of SDS variation in mountain ecosystems. This study provides new insights into the response of seed plant diversity to climate change.

[Effect of proximal fibula osteotomy on tension of lateral knee soft tissue in patients with knee osteoarthritis].

OBJECTIVE: To evaluate the short-term efficacy of proximal fibula osteotomy in the treatment of knee osteoarthritis, and to analyze the effect of osteotomy on the tension of the lateral knee soft tissue of patients and verify the reliability of the Arch string theory. METHODS: A total of 71 patients with varus knee osteoarthritis from December 2019 to March 2022 were included, 3 patients dropped out, and 68 patients completed all trials, collected 27 males and 41 females, aged from 51 to 79 years old, with an average of (68.0±7.0 ) years old. The follow-up time ranged from 4 to 12 weeks, with an average of (3.76±1.94) weeks. After admission, the patient underwent Proximal fibula osteotomy, and the tension of lateral knee soft tissue, visual analogue scale (VAS) of pain, the western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and other indicators were recorded before surgery and 1 month after surgery in the weight-bearing state. RESULTS: According to the VAS, the curative effect of a single index was evaluated by referring to the score before and after treatment by Bao Zongzhao. Thirty seven cases were markedly effective, 27 cases were effective, and 4 cases were ineffective. After surgery, 3 patients presented with weakness of dorsalis pedis extension and 1 presented with paresthesia of dorsalis pedis, which disappeared after symptomatic treatment . The VAS and WOMAC score at 1 month after operation were lower than those before operation, and the differences were statistically significant(P<0.001). The tension of lateral knee soft tissue 1 month after operation was lower than that before operation, and the difference had statistical significance(P<0.001). CONCLUSION: Proximal fibula osteotomy is safe and effective in the treatment of varus knee osteoarthritis in the short term. One month after osteotomy, the tension of lateral knee soft tissue increases under weight-bearing state, but the long-term changes still need further observation and follow-up.

Hypoxia-induced FOXO4/LDHA axis modulates gastric cancer cell glycolysis and progression.

BACKGROUND AND AIM: We previously identified forkhead box (FOX) O4 mRNA as a predictor in gastric cancer (GC). However, the underlying mechanism has yet to be elucidated. We aimed to illustrate the mechanism by which FOXO4 regulated glycolysis under hypoxia in GC. METHODS: FOXO4 protein expression was investigated by immunohistochemical staining of 252 GC and their normal adjacent tissues. We restored or silenced FOXO4 expression in GC cell lines to explore the underlying mechanisms. RESULTS: FOXO4 was downregulated in GC. Loss of FOXO4 expression was validated in univariate and multivariate survival analysis as an independent prognostic predictor for overall survival (P < 0.05) and disease-free survival (P<0.05). Restored FOXO4 expression significantly impaired the glycolysis rate in GC cells, while silencing FOXO4 expression enhanced glycolysis rate. FOXO4 expression was inversely associated with maximum standardized uptake value in mice models and patient samples. Mechanistically, FOXO4 bound to the glycolytic enzyme lactate dehydrogenase (LDH)A promoter and inactivated its activity in a dose-dependent manner (P < 0.05). Finally, we determined that FOXO4 was a transcriptional target of hypoxia-inducible factor (HIF) -1α, which is central in response to hypoxia. CONCLUSIONS: Our data suggested that FOXO4 plays a key role in the regulation of glycolysis in GC, and disrupting the HIF-1α-FOXO4-LDHA axis might be a promising therapeutic strategy for GC.

[Study on the activity against influenza A virus with traditional Chinese medicine Kanggan granules].

OBJECTIVE: To study the anti-influenza A virus effects of traditional Chinese medicine Kanggan granules in chicken embryo and BALB/c mice. METHODS: The influenza A virus (H(1)N(1), FM1) was used in the experiments. FM1 was cultured in chicken embryo and the anti-FM1 activity of Kanggan granules was evaluated through the post-medication hemagglutination titer of FM1. In animal test, 120 healthy BALB/c mice were randomly divided into six groups, normal control, virus control, ribavirin, high-dosage, middle-dosage and low-dosage. The FM1 infection model was established by dripping FM1 into nasal cavity and then the appropriate treatments were prescribed. The effective anti- FM1 indices of Kanggan granules included survival status, protective percentage of death and life elongation percentage of mice infected with FM1. RESULTS: The high and middle doses of Kanggan granules could inhibit the replication of FM1 remarkably in chicken embryo, and could reduced hemagglutination titer to 5 and 3 times. In animal experiments, all mice treated with Kanggan granules could improve the general status of infected mice, the protective percentages of death were 35.0% to 55.0%, the life elongation percentages were 73.0% to 88.9% and the minimal effective dose was 3.00 g/kg. CONCLUSION: Kanggan granules can inhibit the replication of influenza A virus and protect the mice infected with FM1.

[Effect of danshensu on activation of JNK pathway in hepatic stellate cells(HSCs) induced by IL-1 beta].

OBJECTIVES: To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1beta. METHODS: The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expression and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen I were detected by the quantitative immunocytochemical assay and ELISA. RESULTS: Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-1beta (P < 0.05). Synthesis and secretion of Type I collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1beta was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. CONCLUSION: Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type I collagen, possibly via the repression of the JNK signal transduction.

[Construction and expression characteristics in vitro of human papillomavirus type 16 E7 suicidal DNA vaccine].

OBJECTIVE: To construct the suicidal DNA vaccine of human papillomavirus type 16 E7 gene (HPV16), and explore the DNA vaccine expression characteristics in vitro and capacity of inducing the transfected cells into apoptosis. METHODS: HPV16 E7 gene cloned by PCR from pET32/E7 was inserted into the plasmid pSCA1 to construct the recombinant plasmid pSCA/E7, followed by identification with PCR, BamH I and Sma I digestion and sequencing. pSCA/E7 was then used to transfect BHK-21 cell line. The transient expression of HPV16 E7 gene was confirmed by immuno-fluorescent staining, and the apoptosis induced by pSCA/E7 was checked with TDT-mediated dUTP nick end-labeling (TUNEL). RESULTS: The cloned E7 gene fragment was about 400 bp in length. PCR, restriction endonuclease digestion and sequence analysis revealed that the HPV16 E7 gene was cloned into the eukaryotic expression plasmid pSCA1 successfully. Immunofluorescent staining confirmed that the E7 gene could express in BHK-21 cell line. The BHK-21 cells transfected with pSCA/E7 could be induced into apoptosis which was confirmed by TUNEL. CONCLUSION: The results show that HPV16 E7 suicidal DNA vaccine can express in BHK-21 cell line, and induce the pSCA/E7 transfected cells into apoptosis. These findings may provide the foundation for exploring the therapeutic vaccine against HPV16-associated cervical cancer.

[Effects of ST6Gal I antisense oligonucleotide-mediated gene silencing on cell adhesion and invasiveness of hela cells].

OBJECTIVE: To study the effects of antisense oligonucleotide (ASODN) targeting ST6Gal I on cell adhesion and invasiveness of human cervical carcinoma cell line HeLa which over-expressed ST6Gal I . METHODS: ASODN and sense oligonucleotide (SODN) targeting ST6Gal I were designed and constructed, and transfected into a cervical cancer cell line, HeLa, by lipofectmine 2000. HeLa cells were cultured and divided into 4 groups: blank control group, liposome group, SODN group and ASODN group. RT-PCR was used to examine the ST6Gal I mRNA expression. Flow cytometry was used to examine the amount of alpha2, 6-sialylation on the HeLa cell surface. The HeLa cell adhesion and invasiveness to extracellular matrix ( ECM) were analyzed by using CytoMatrixTM kit and cell invasion assay kit, respectively. RESULTS: The expression of ST6Gal I mRNA in HeLa cells at 48 hrs after transfection in the ASODN group was significantly decreased in comparison with that in the blank control group, liposome group, and SODN group(P <0. 01). The amount of alpha2,6-sialylation on cell surface in ASODN group was significantly lower than that of the other 3 groups ( P <0. 05). The adhesion and invasiveness of the cells in the ASODN group decreased remarkably, both significantly lower than those of the other 3 groups ( all P < 0. 05). CONCLUSION: Specific ASODN targeting ST6Gal I effectively inhibits HeLa cell ST6Gal I expression, decreases the amount of alpha2,6-sialylation on cell surface and leads to a decline of cell adhesion and invasiveness to ECM. This result also established a fine base for further studying on anti-tumor treatment with antisense oligonucleotide.

[Combinatorial effects of ST6Gal I siRNA and antisense oligonucleotide-mediated gene silence on metastasis ability of cervical carcinoma cells].

OBJECTIVE: To study the combinatorial effects of small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) targeting alpha 2,6 sialyltransferase (ST6Gal I) on the cell adhesion and invasiveness of human cervical carcinoma cell line, HeLa with over-expressing ST6Gal I. METHODS: The siRNA and ASOs targeting to ST6Gal I were designed and synthesized chemically, and then with lipofectamine 2000, transferred into HeLa cells, which were cultured and divided into 7 groups: blank control group, liposome group, siRNA group transfected with ST6Gal I siRNA, ASO1 group transfected with ST6Gal I ASO whose target site is same as siRNA, ASO2 group transfected with ST6Gal I ASO whose target site is different with siRNA, siRNA+ASO1 group transfected with siRNA and its homologous ASO1, siRNA+ASO2 group transfected with siRNA and its nonhomologous ASO2. RT-PCR was used to examine the ST6Gal I mRNA expression. Flow cytometry was used to examine the amount of alpha2, 6-sialylation on the HeLa cell surface. The HeLa cell adhesion or invasiveness to extracellular matrix (ECM) was analyzed by using CytoMatrix kit or cell invasion assay kit, respectively. RESULTS: The expression of ST6Gal I mRNA, the amount of a2,6-sialylation of cell surface, the cell adhesion and invasion to ECM were remarkably decreased in siRNA group, ASO1 group, ASO2 group, siRNA+ASO, group and siRNA+ASO2 group, and all significantly lower than those in the blank control group and liposome group (all P < 0.05), especially in siRNA+ASO2 group. The difference was significant between siRNA+ASO2 group and siRNA group (all P < 0.05). No significant difference was observed between siRNA+ASO1 group and siRNA group, neither between blank control group and liposome group (all P > 0.05). CONCLUSION: The synthesized chemically specific siRNA targeting to ST6Gal I can effectively downregulate the ST6Gal I expression in HeLa cell and further lead to the decline of cell adhesion and invasiveness to ECM, and use of associating siRNA with ASO targeting to same gene but different oligonucleotide location possesses the synergy and additive effect on targeted gene expression knocked down.

[Steady expression and activity determination of recombinant signal peptide of mBD2 in SiHa cell].

OBJECTIVE: To observe the expression pattern and effect of recombinant murine beta defensin 2 (rmBD2) on the proliferation of cell transfected with pcDNA3. 1 (+)/rmBD2. METHODS: The recombinant plasmid pcDNA3. 1 (+)/rmBD2 was transferred into SiHa cells. The transfected SiHa cells were selected by G418 with 100 microg/mL for over 20 days. The steady expressions of rmBD2 protein and rmBD2 mRNA were detected by immunofluorescence and RT-PCR. The effects of rmBD2 on SiHa cell growth and reproduction were measured by MTT. RESULTS: The SiHa cells which could stably express the rmBD2 protein were harvested with 100 microg/mL of G418. The rmBD2 protein expression was, by immunofluorescence, confirmed and its mRNA in SiHa with rmBD2 could be detected at 4 weeks, 6 weeks, 8 weeks, and 10 weeks after cell transfected. A 220 bp segment encoding rmBD2 was amplified by RT-PCR and proved by sequencing. The SiHa/K cells transfected pcDNA3. 1 (+) and SiHa had no expression of rmBD2 protein. The SiHa with rmBD2 expression grew slowly than SiHa/K and SiHa control (P < 0.05). CONCLUSION: The screening for SiHa with rmBD2 expressing stably is successful. The cell growth curve shows that rmBD2 can inhibit the proliferation of tumor cells. The above results establish a solid foundation for further studying the biological properties and the anti-tumor mechanism of beta defensins.

[The research on construction and expression of eukaryotic expression plasmid for a recombinant immunotoxin mMIP-1alpha-DT390].

OBJECTIVE: To construct a novel recombinant immunotoxin (IT) expression vector by fusing mouse macrophage inflammatory protein-1alpha gene (mMIP-1alpha) into a truncated diphtheria toxin gene (DT390), and examine the expression of mMIP-1alpha-DT390 fusion protein in NIH3T3 cells. METHODS: mMIP-1alpha cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction (RT-PCR), and inserted in the expression plasmid SRalpha, that includes the DT390 gene, to form the recombinant vector SRa-mMIP-1alpha-DT390. The positive recombinant plasmid was identified by PCR, the restriction endonucleases digestion and DNA sequencing, and then by liposome protocol, the identified positive plasmid was transferred into NIH3T3 cells for observing the fusion protein expression by immunofluorescence, with detecting the activities of the immunotoxin in vitro through MTT. RESULTS: We have successfully constructed a recombinant immunotoxin expression vector named as SRalpha-mMIP-1alpha-DT390. The immunofluorescence photograph sourced from fluorescence immunocytochemical method showed that the fusion gene could express in the cytomembrane and cytoplasm of NIH3T3 cells. In the bioactivity detection assay, the supernatant of the transfected cell culture was observed to have the obvious cytotoxic activity to activated T-lymphocyte. CONCLUSION: The SRalpha-mMIP-1alpha-DT390 recombinant immunotoxin expression vector will provide the basis of studying the targeted cytotoxic activity to inflammatory cells, and may have some potential value for clinical application.

[Analysis of protein expression feature and construction of procaryotic expression system for human papillomavirus type 16 (HPV-16) E4 gene].

OBJECTIVE: To clone the E4 gene (E4) from human papillomavirus type 16(HPV-16), construct the engineering bacteria of prokaryotic expression, and explore the expression conditions and the characters of expression product. METHODS: The complete E4 gene was cloned by PCR from the sample cell extract of clinical cervical disease that was the positive HPV-16 confirmed by Real-PCR. The E4 DNA fragment was inserted into the pET32a(+) to construct a prokaryotic expression plasmid, called as pET32/E4. Then the expression plasmids were transferred into competent E. coli BL21 (DE3). Recombinant DNA was identified by Bgl II and Hind III digestion, and then sequencing. The recombine bacterium, BL21/E4, was induced with different IPTG concentrations at different temperatures. The expressed proteins were checked and analyzed by SDS-PAGE and Gel-Pro Analyzer 4. His-tag of BL21/E4 expression protein was hybridized to McAb. RESULTS: The E4 gene cloned by PCR was about 342 bp. The blasted result showed that the E4 gene had 99% homology of HVP-16 DNA sequence, the cloned E4 gene expression frame was the same as HVP-16 East Asia strain's. Compared with other HPV-16 strains in GenBank, the homology of E4 gene was above 97%. pET32/E4 could express recombinant E4 (rE4) in BL21. The highest expression, which was 12.2% or 12.8% of total bacterial proteins respectively, was gotten when BL21/E4 was induced by 0.1 mmol/L IPTG at 28 degrees C or 37 degrees C for 18 hours. The results of SDS-PAGE and Western blot showed the rE4 was expressed mainly to form the inclusion body, and to fuse with his-tag (rE4/His), that was soluble and had a molecular weight as about 34 KDa. CONCLUSION: We cloned successfully the E4 gene from HPV-16 and constructed the prokaryotic expression E. coli BL21/E4, which could expression rE4 protein fused with his-tag (rE4/His), effectively. The fused protein could react to McAb recognizing His-tag, which was convenience purified by affinity chromatography. The above research results built a good foundation for preparing the high grade of purity E4 protein and developing the relative study.

[Construction of eukaryotic expressing plasmids for HA and HA1 of influenza A virus and their transient expression in HEK293 cells].

OBJECTIVE: To construct influenza A virus (A/PR/8/34) HA and HA1 eukaryotic expressing plasmids and study their expression in HEK293 cells. METHODS: HA and HA1 genes were cloned by RT-PCR and then inserted into pcDNA3. 1 (+). After identification of restriction enzyme digestion, PCR and sequencing analysis, HA and HA1 eukaryotic expressing plasmids were transfected into HEK293 cells with PolyFect Transfection Reagent. Immunofluorescence assay was used to observe the transient expressing result. RESULTS: It was confirmed that the construction of HA and HA1 eukaryotic expressing plasmids was made successfully. The stronger fluorescence signals were detected in transfected HEK293 cells with these two kinds of plasmids by immunofluorescence assay. CONCLUSION: The experiment is a success in the construction of eukaryotic expressing plasmids for HA and HA1, thus providing a basis for further probing into the mechanism of virus infection and exploring DNA vaccine.

[Prokaryotic expression and identification of human papillomavirus type 16 E7 protein encoding gene].

OBJECTIVE: To construct the prokaryotic expression plasmid pET32/E7 and express the human papillomavirus type 16 E7 protein in E. coli. METHODS: HPV16 E7 gene was amplified by PCR. The amplified E7 fragment was inserted into the plasmid pET32a (+) that was digested with BamH I and Hind III. The recombinant plasmid pET32/E7 was transformed into E. coli JM109 which was selected with ampicillin. The positive clones containing recombinant plasmid pET32/E7 were verified by BamH I and Xho I digestion, and then sequenced. HPV16 E7-TRX recombinant protein expression in the E. coli BL21(ED3) was identified by SDS-PAGE and Western blot. RESULTS: The prokaryotic recombinant plasmid pET32/E7 was successfully constructed. The BL21(DE3) transformed recombinant plasmid pET32/E7 had expressed HPV16 E7-TRX recombinant protein effectively. Under the conditions of 1 mmol/L IPTG and 30 degrees C, the amount of HPV16 E7-TRX recombinant protein was about 30% of bacterial total proteins. CONCLUSION: The construction of the prokaryotic recombinant plasmid pET32/E7 and the successful expression of the recombinant protein HPV16 E7-TRX would strongly promote the research of the biological properties and the transformational mechanism of the HPV16 E7 protein on the specific cells.

[Construction and functional analysis of a novel eukaryotic expression plasmid of recombinant immunotoxin DT390-Rantes].

OBJECTIVE: To construct a novel eukaryotic expression plasmid for recombinant immunotoxin DT390-Rantes and perform preliminary analysis of its function. METHODS: The gene fragment coding for Rantes was obtained from the liver tissues of C57BL/6 mice using RT-PCR, and inserted into the eukaryotic expression plasmid SRalpha containing DT390 gene to construct the recombinant plasmid DT390-Rantes-SRalpha, which was transformed into E. coli JM109, followed by selection of the positive clones containing the target inserts. The eukaryotic expression plasmid was analysed by PCR, restriction endonuclease digestion and DNA sequencing. The recombinance plasmid DT390-Rantes-SRalpha was transfected into NIH3T3 cells and its expression was observed by immunofluorescence detection. The activity of the expressed DT390-Rantes in vitro was evaluated by MTT assay. RESULTS: The gene fragment of Rantes was correctly inserted into the eukaryotic expression plasmid SRalpha as verified by restriction endonuclease digestion and DNA sequencing, and could be expressed in NIH3T3 cells. MTT assay confirmed that the expression product DT390-Rantes could kill activated T cells in vitro. CONCLUSIONS: The recombinant eukaryotic expression plasmid DT390-Rantes-SRalpha is successfully constructed and expressed in eukaryotic cells. The expressed product can specifically kill activated T cells in vitro.

[Construction of interleukin-18-PE38 fusion gene eukaryotic expression vector and its expression in the. chondrocyte].

OBJECTIVE: To construct the interleukin-18-PE38 fusion gene expression vector and explore the expression of the fusion gene in the chondrocyte. METHODS: The recombinant eukaryotic expression vector PsecTag2B-IL-18-PE38 was constructed by inserting interleukin-18-PE38 fusion gene into eukaryotic expression vector PsecTag2B with molecular cloning technique. It was confirmed by restrictive enzymes (EcoR I) digestion assay and PCR. The vector was transfected into primary chrondrocyte by liposome protocol, and the transient expression was identified by fluorescence immunocytochemical assay. RESULTS: Restrictive enzymes digestion analysis and PCR revealed that the interleukin-18-PE38 fusion gene was cloned into the eukaryotic expression vector PsecTag2B successfully. Immunofluorescence photograph of fluorescence immunocytochemical method confirmed that the fusion gene can be expressed in the cytomembrane and cytoplasm. CONCLUSION: The results confirmed that PsecTag2B-IL-18-PE38 fusion gene can be expressed in the chondrocyte, which could serve as a foundation for the study on rheumatoid arthritis therapy.

[Construction and prokaryotic expression of a recombinant immunotoxin fused with mouse interleukin 18 and truncated Pseudomonas exotoxin].

OBJECTIVE: To construct a new recombinant immunotoxin expression vector by fusion of mouse interleukin 18 (mIL-18) gene and a truncated form of A (PE38) gene, and examine the expression of IL-18-PE38 fusion protein in Escherichia coli. METHODS: mIL-18 cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction (RT-PCR). The mIL-18 cDNA was subcloned into a PE38 gene inserted fusion protein expression plasmid. The recombinant vector was identified by restriction endonucleases digestion, PCR and DNA sequencing. After transformed into E. coli BL21 and induced by IPTG, the expressed product was obtained and detected the molecular weight and specificity by SDS-PAGE and Western-blotting. RESULTS: The new recombinant immunotoxin expression vector was constructed successfully. The IL-18-PE38 fusion protein was expressed in E. coli BL21, and the molecular weight of the expression product was identical to the expected value. In addition, the protein expressed could react with the specific antibody against mIL-18. CONCLUSION: IL-18-PE38 recombinant immunotoxin expression vector will provide the basis for study on the targeted cytotoxic activity to Th1 cell and may have some potential value in the treatment of autoimmune disease and T cell leukemias.

[Induction of apoptosis of cultured hepatocarcinoma cell by essential oil of Artemisia Annul L].

OBJECTIVE: To know whether the essential oil of Artemisia annul L. can induce apoptosis of cultured hepatocarcinoma cell SMMC-7721. METHODS: Hepatocarcinoma cells were treated by the essential oil of Artemisia annul L. while positive control was treated by hydroxycamptothecine (HPT) and negative or mock control was treated by normal saline (NS). Induction of the apoptosis was analyzed by using flow cytometry (FCM), light microscope and electronic microscope, Giemsa's stain and DNA pattern after drug treatment. RESULTS: After the treatment of the cells with 100 micrograms/ml essential oil of Artemisia annul L. for 24 hours, the morphological changes of classic apoptosis such as condensation of cytoplasm, fragmentation of nuclear chromatin, and apoptosis body were seen. The sub-G1 peak was exhibited by FCM, and the DNA ladder pattern was observed. CONCLUSION: These findings demonstrated that the essential oil of Artemisia annul L. could induce apoptosis of cultured SMMC-7721.

miR-99 inhibits cervical carcinoma cell proliferation by targeting TRIB2.

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3'-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics.

The associations of single nucleotide polymorphisms in miR-146a, miR-196a and miR-499 with breast cancer susceptibility.

BACKGROUND: Previous studies have investigated the association between single nucleotide polymorphisms (SNPs) located in microRNAs (miRNAs) and breast cancer susceptibility; however, because of their limited statistical power, many discrepancies are revealed in these studies. The meta-analysis presented here aimed to identify and characterize the roles of miRNA SNPs in breast cancer risk, and evaluate the associations of polymorphisms in miR-146a rs2910164, miR-196a rs11614913 and miR-499 rs3746444 with breast cancer susceptibility, respectively. METHODOLOGY/PRINCIPAL FINDINGS: The PubMed and Embases databases were searched updated to 31(st) December, 2012. The complete data of polymorphisms in miR-146a rs2910164, miR-196a rs11614913 and miR-499 rs3746444 from case-control studies for breast cancer were analyzed by odds ratios (ORs) with 95% confidence intervals (CIs) to reveal the associations of SNPs in miRNAs with breast cancer susceptibility. Totally, six studies for rs2910164 in miR-146a, involving 4225 cases and 4469 controls; eight studies for rs11614913 in miR-196a, involving 4110 cases and 5100 controls; and three studies of rs3746444 in miR-499, involving 2588 cases and 3260 controls, were investigated in the meta-analysis. The rs11614913 (TT+CT) genotype of miR-196a2 was revealed to be associated with a decreased breast cancer susceptibility compared with the CC genotypes (OR = 0.906, 95% CI: 0.825-0.995, P = 0.039); however, no significant associations were observed between rs2910164 in miR-146a (or rs3746444 in miR-499) and breast cancer susceptibility. CONCLUSIONS: This meta-analysis demonstrates the compelling evidence that the rs11614913 CC genotype in miR-196a2 increases breast cancer risk, which provides useful information for the early diagnosis and prevention of breast cancer.