One-pot wet-chemical fabrication of 3D urchin-like core-shell Au@PdCu nanocrystals for electrochemical breast cancer immunoassay.

Carbohydrate antigen 15-3 (CA15-3) is an important biomarker for early diagnosis of breast cancer. Herein, a label-free electrochemical immunosensor was built based on three-dimensional (3D) urchin-like core-shell Au@PdCu nanocrystals (labeled Au@PdCu NCs) for highly sensitive detection of CA15-3, where K3[Fe(CN)6] behaved as an electroactive probe. The Au@PdCu NCs were synthesized by a simple one-pot wet-chemical approach and the morphology, structures, and electrocatalytic property were investigated by several techniques. The Au@PdCu NCs prepared worked as electrode material to anchor more antibodies and as signal magnification material by virtue of its exceptional catalytic property. The developed biosensor exhibited a wide linear detection range from 0.1 to 300 U mL-1 and a low limit of detection (0.011 U mL-1, S/N = 3) for determination of CA15-3 under the optimal conditions. The established biosensing platform exhibits some insights for detecting other tumor biomarkers in clinical assays and early diagnosis.

Bimetallic AuPt alloy/rod-like CeO2 nanojunctions with high peroxidase-like activity for colorimetric sensing of organophosphorus pesticides.

Organophosphorus pesticides (OP) have extensive applications in agriculture, while their overuse causes inevitable residues in food, soil, and water, ultimately being harmful to human health and even causing diverse dysfunctions. Herein, a novel colorimetric platform was established for quantitative determination of malathion based on peroxidase mimic AuPt alloy decorated on CeO2 nanorods (CeO2@AuPt NRs). The synthesized nanozyme oxidized colorless 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. Besides, the oxidized TMB was inversely reduced by ascorbic acid (AA), which were originated from hydrolysis of L-ascorbic acid-2-phosphate (AA2P) with the assistance of acid phosphatase (ACP). Based upon this observation ACP analysis was explored by colorimetry, showing a wid linear range of 0.2 ~ 3.5 U L-1 and a low limit of detection (LOD = 0.085 U L-1, S/N = 3). Furthermore, malathion present in the colorimetric system inhibited the activity of ACP and simultaneously affected the generation of AA, in turn promoting the recovery of the chromogenic reaction. Based on this, the LOD was decreased to 1.5 nM (S/N = 3) for the assay of malathion with a wide linear range of 6 ~ 100 nM. This simple colorimetric platform provides some informative guidelines for determination of other pesticides and disease markers.

Dual II-scheme nanosheet-like Bi2S3/Bi2O3/Ag2S heterostructures for ultrasensitive PEC aptasensing of aflatoxin B1 coupled with catalytic signal amplification by dendritic nanorod-like Au@Pd@Pt nanozyme.

As one of the most toxic chemical substances, aflatoxin B1 (AFB1) has a strong carcinogenic effect even at a trace level in human and animal, which severely threatens human health and even causes cancers. Therefore, ultrasensitive detection of AFB1 is of significant importance. For such analysis, dual II-scheme sheet-like Bi2S3/Bi2O3/Ag2S heterostructures were prepared by the in-situ growth method, which exhibited high separation efficiency for the electron-hole (e--h+) pairs, prominent stability, and high photoactivity. Moreover, the dendritic nanorod-like Au@Pd@Pt (Au@Pd@Pt DNRs) nanozyme was homely synthesized, whose peroxidase-like activity was scrupulously investigated by catalytical oxidation of diaminobenzidine (DAB) in the presence of H2O2. Integration by the aptasensing strategy, a photoelectrochemical (PEC) "signal-on" aptasensor was prepared, which exhibited a broader linear range of 0.5 pg mL-1-100 ng mL-1 with a lower limit of detection (LOD = 0.09 pg mL-1, S/N = 3). This work provides a feasible strategy to develop advanced PEC biosensors for actual analysis of environmental pollutants.

Electrochemical label-free immunoassay of HE4 using 3D PtNi nanocubes assemblies as biosensing interfaces.

Human epididymis protein 4 (HE4) is a vital biomarker for early diagnosis of epithelial ovarian cancer (EOC). Herein, a new label-free biosensor was developed using K3[Fe(CN)6] as the electrochemical probe for ultrasensitive immunoassay of HE4 based on PtNi nanocubes assemblies (NCAs) as efficient biosensing interfaces. The PtNi NCAs were synthesized by a simple solvothermal approach, where N-hexadecyltrimethylammonium chloride (HTAC) and 2,2'-bis(4,5-dimethylimidazole) (BDMM) behaved as co-structuring directors. Under the optimal conditions, the obtained HE4 immunosensor displayed a wide detection range from 0.001 to 100 ng mL-1 and a low detection limit (0.11 pg mL-1, S/N = 3). As a result, the current sensing platform would serve as a useful reference for detecting cancer biomarkers in the clinical assay and diagnosis.

Ultrasensitive label-free electrochemical immunosensor of NT-proBNP biomarker based on branched AuPd nanocrystals/N-doped honeycombed porous carbon.

N-terminal pro-B-type natriuretic peptide is a major cardiac biomarker for early diagnosis and prognosis of heart failure. Herein, a novel label-free electrochemical immunosensor was developed based on home-made branched AuPd nanocrystals/N-doped porous carbon (AuPd NCS/NPC) for ultrasensitive and high-selective detection of N-terminal pro-B-type natriuretic peptide. Specifically, the AuPd NCS/NPC was prepared by a one-pot wet-chemical strategy by using thymine as a green structural directing agent, whose morphology, structures, and properties were strictly examined, showing high-efficiency catalysis towards electro-reduction of hydrogen peroxide. Under the optimized conditions, the fabricated sensor exhibited a dynamic linear range of 0.001 âˆ¼ 10 ng mL-1 and a low limit of detection (0.34 pg mL-1, S/N = 3) for immunoassay of N-terminal pro-B-type natriuretic peptide. Furthermore, this platform was explored for detection of the biomarker in human serum sample with satisfactory results. Thus, the built biosensor can render valuable guidance for prospective clinical diagnostic applications.

A sandwich-type electrochemical immunosensor for CYFRA 21-1 based on probe-confined in PtPd/polydopamine/hollow carbon spheres coupled with dendritic Au@Rh nanocrystals.

A signal-on sandwich-like electrochemical immunosensor was built for determination of cytokeratin 19 fragments 21-1 (CYFRA 21-1) in non-small cell lung cancer (NSCLC) by confining electroactive dye (e.g., methylene blue, MB) as a probe for amplifying signals. Specifically, core-shell gold@rhodium dendritic nanocrystals (Au@Rh DNCs) behaved as a substrate for primary antibody and accelerate interfacial electron transfer. Besides, hollow carbon spheres (HCSs) were subsequently modified with polydopamine (PDA) and PtPd nanoparticles for sequential integration of the secondary antibody and confinement of MB as a label, termed as MB/PtPd/PDA/HCSs for clarity. The built sensors showed a broad linear range (100 fg mL-1 ~ 100 ng mL-1) for detection of CYFRA 21-1 with an ultra-low detection limit (31.72 fg mL-1, S/N = 3), coupled with satisfactory performance in human serum samples. This work can be explored for assays of other proteins and provides some constructive insights for early and accurate diagnosis of NSCLC.

Ancient Mitogenomes Reveal the Origins and Genetic Structure of the Neolithic Shimao Population in Northern China.

Shimao City is considered an important political and religious center during the Late Neolithic Longshan period of the Middle Yellow River basin. The genetic history and population dynamics among the Shimao and other ancient populations, especially the Taosi-related populations, remain unknown. Here, we sequenced 172 complete mitochondrial genomes, ranging from the Yangshao to Longshan period, from individuals related to the Shimao culture in northern Shaanxi Province and Taosi culture in southern Shanxi Province, Middle Yellow River basin. Our results show that the populations inhabiting Shimao City had close genetic connections with an earlier population in the Middle Neolithic Yangshao period of northern Shaanxi Province, revealing a mostly local origin for the Shimao Society. In addition, among the populations in other regions of the Yellow River basin, the Shimao-related populations had the closest maternal affinity with the contemporaneous Taosi populations from the Longshan period. The Shimao-related populations also shared more affinity with present-day northern Han populations than with the minorities and southern Han in China. Our study provides a new perspective on the genetic origins and structure of the Shimao people and the population dynamics in the Middle Yellow River basin during the Neolithic period.

Comparing Smartphone Apps for Traditional Chinese Medicine and Modern Medicine in China: Systematic Search and Content Analysis.

BACKGROUND: Traditional Chinese medicine (TCM) is an integral part of mainstream medicine in China, with theories and practices that are completely different from modern medicine. TCM should not be ignored or confused with modern medicine in the analysis of the Chinese health care system, including the analysis of mobile health (mHealth) apps. To date, differences between TCM apps and modern medicine apps have not be systematically investigated. OBJECTIVE: The aim of this study was to systematically compare the quality of apps for TCM and modern medicine in China. METHODS: In December 2020, we searched iOS (iTunes) and Android (Tencent, Oppo, and Huawei app stores) platforms for all mHealth apps and then categorized them as TCM or modern medicine apps if they were included in the final analysis. The included apps were downloaded on smartphones and assessed by 2 reviewers on the following 4 aspects: (1) data in the app stores, including user ratings, download counts, cost, target users, and year of last update; (2) functionality; (3) quality of the app content as determined by the Mobile App Rating Scale (MARS); and (4) analysis of the app privacy and security. RESULTS: In total, 658 apps were analyzed, including 261 TCM medicine apps and 397 modern medicine apps. The average download count of modern medicine apps (approximately 5 million) was more than 10 times that of TCM apps (approximately 400,000). Regarding functionalities, 64.7% (257/397) of modern medicine apps provided telemedicine (74/261, 28.4% in TCM apps), 62.7% (249/397) provided registration (70/261, 26.8% in TCM apps), and 45.6% (181/397) provided communication (38/261, 14.6% in TCM apps). A larger proportion of TCM apps provided prescription and medication management (144/261, 55.2% in TCM apps versus 168/397, 42.3% in modern medicine apps). The majority of modern medicine apps (329/397, 82.9%) combined ≥3 functionalities compared with one-third of TCM apps (93/261, 34.6%). We then selected 81 top apps for quality and safety assessment (41 TCM apps and 40 modern medicine apps). Of these, the mean overall MARS score of TCM apps (2.7, SD 0.5) was significantly lower than modern medicine apps (3.6, SD 0.4). Almost all modern medicine apps (38/40, 95%) addressed privacy and security by providing a privacy policy and describing how to protect personal data, but less than half of the TCM apps (18/41, 44%) described this information (P<.001). CONCLUSIONS: The different functionalities reflect the distinct innate characteristics of these two medical systems. Although great progress has been made and the Chinese mHealth market size is large, there still exist many opportunities for future development, especially for TCM.

An undiscovered facet of hydraulic redistribution driven by evaporation-a study from a Populus tomentosa plantation.

Maintaining the activity and function of the shallow root system of plants is essential for withstanding drought stress, but the associated mechanism is poorly understood. By investigating sap flow in 14 lateral roots (LRs) randomly selected from trees of a Chinese white poplar (Populus tomentosa) plantation receiving three levels of irrigation, an unknown root water transport mode of simultaneous daytime bi-directional water flow was discovered. This mode existed in five LRs confined to the surface soil without attached sinker roots. In the longer term, the bi-directional water flow was correlated with the soil water content. However, within the day, it was associated with transpiration. Our data demonstrated that bi-directional root sap flow occurred during the day, and was driven by evaporative demand, further suggesting the existence of circumferential water movement in the LR xylem. We named this phenomenon evaporation-driven hydraulic redistribution (EDHR). A soil-root water transport model was proposed to encapsulate this water movement mode. EDHR may be a crucial drought-tolerance mechanism that allows plants to maintain shallow root survival and activity by promoting root water recharge under extremely dry conditions.

The origins of specialized pottery and diverse alcohol fermentation techniques in Early Neolithic China.

In China, pottery containers first appeared about 20000 cal. BP, and became diverse in form during the Early Neolithic (9000-7000 cal. BP), signaling the emergence of functionally specialized vessels. China is also well-known for its early development of alcohol production. However, few studies have focused on the connections between the two technologies. Based on the analysis of residues (starch, phytolith, and fungus) adhering to pottery from two Early Neolithic sites in north China, here we demonstrate that three material changes occurring in the Early Neolithic signal innovation of specialized alcoholic making known in north China: (i) the spread of cereal domestication (millet and rice), (ii) the emergence of dedicated pottery types, particularly globular jars as liquid storage vessels, and (iii) the development of cereal-based alcohol production with at least two fermentation methods: the use of cereal malts and the use of moldy grain and herbs (qu and caoqu) as starters. The latter method was arguably a unique invention initiated in China, and our findings account for the earliest known examples of this technique. The major ingredients include broomcorn millet, Triticeae grasses, Job's tears, rice, beans, snake gourd root, ginger, possible yam and lily, and other plants, some probably with medicinal properties (e.g., ginger). Alcoholic beverages made with these methods were named li, jiu, and chang in ancient texts, first recorded in the Shang oracle-bone inscriptions (ca. 3200 cal. BP); our findings have revealed a much deeper history of these diverse fermentation technologies in China.

Intron-specific shRNA-mediated downregulation of survivin and promotion of apoptosis in HeLa cells.

Overexpression of the survivin gene contributes to tumorigenesis; it has been recognized as an important target for cancer therapy. In the present study, survivin expression was suppressed using recombinant plasmid mediated short hairpin RNAs (shRNAs) that were constructed to target exonic or intronic sequences of the survivin gene. In addition, a negative control shRNA was constructed. HeLa cells were transfected with specific shRNA constructs and the blocking efficiency of each shRNA was assessed at the mRNA and protein levels; and the five shRNA constructs with higher blocking efficiency were selected. Cell apoptosis was assessed by flow cytometry (FCM) following Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Hoechst staining was used to detect the morphological diversity of the nuclei in apoptotic cells. The results demonstrated that survivin expression was effectively reduced by the transfection of shRNAs in HeLa cells. In addition, the apoptotic rates of the shRNA-treated groups were significantly increased compared with the negative control group according to the FCM results. The nuclei of HeLa cells exhibited apoptotic characteristics in the shRNA-treated groups as identified by Hoechst staining. Survivin-targeting shRNAs effectively downregulated the expression of the gene and markedly increased the apoptotic rate of HeLa cells. Data from the present study also indicated that the intron-specific shRNA demonstrate a high efficiency of inhibition of survivin expression and were able to induce cell apoptosis of HeLa cells through RNAi, potentially providing novel target sites for tumor therapy. In conclusion, the present study suggests that intron-specific blocking of survivin by RNAi may provide a tool for anticancer therapy.

Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α.

The aim of the present study was to construct a fast-acting, eukaryotic expression vector in eukaryotic cells based on transmembrane-tumor necrosis factor­α (TM­TNF­α) structure. Two types of recombinant eukaryotic expression vectors were constructed, pcDNA3.1-TM-enterokinase-TNF­α and pcDNA3.1­TM­Factor Xa­TNF­α, according to the TNF­α transmembrane segments. Following the generation of these vectors, mouse embryonic 3T3 fibroblasts were transfected and reverse transcription­polymerase chain reaction and western blotting analyses were used to analyze mTNF­α mRNA and protein expression levels, respectively, in total cellular protein extracts and extracellular fluid. The biological activity of TNF-α in the extracellular fluid was then measured using an MTT assay. The vectors were successfully constructed, and mRNA and fusion proteins were detected in the 3T3 cells. Among the fusion proteins, the one observed in pcDNA3.1-TM-FactorXa-TNF-α-transfected 3T3 cells remained as a transmembrane protein. In addition, treatment of L929 cells with TNF­α derived extracellular fluid samples from pcDNA3.1­TM­FactorXa­TNF­α­transfected 3T3 cells was associated with a dose­dependent reduction in in cell­specific activity. The results indicate that proteins expressed using pcDNA3.1­TM­FactorXa­TNF­α vectors form transmembrane proteins. In addition, the results indicate that, only when coupled with FactorXa activity, the extracellular region of TM­TNF­α forms s­TNF­α, and the controlled expression of the fusion protein is initiated.

Construction of the POT1 promoter report gene vector, and the effect and underlying mechanism of the POT1 promoter in regulating telomerase and telomere length.

By using human genomic DNA as a template to clone protection of telomere 1 (POT1) promoter gene segments and construct the POT1 promoter luciferase report gene vector (pGL3-Control-POT1-promoter), the association between POT1, and the regulation of telomerase and telomere length was investigated. In the present study, two recombinant luciferase report gene vectors were constructed, which included different regions of the POT1 promoter. The plasmids were transformed into DH5α and the positive clones were obtained. The two plasmids termed as pGL3-Control-POT1-promoter-1 and pGL3-Control-POT1-promoter-2, were confirmed using restriction enzyme analysis and sequencing. They were separately and transiently transfected into four types of human tumor cells (A549, H460, HepG2 and HeLa). The transcriptional activities of the POT1 promoter were verified using the dual-luciferase assay. The relative expression of POT1 and human telomerase reverse transcriptase (hTERT), and telomere length were analyzed using quantitative polymerase chain reaction in the four types of non-transfected tumor cells. Using SPSS software, correlations between POT1 promoter activity, and POT1 expression, hTERT expression and telomere length were analyzed. Two POT1 promoter fragments (POT1-promoter-1 and -2) were successfully constructed into the pGL3-Control luciferase report gene vector. POT1-promoter-1 exhibited significantly stronger transcription activity compared with POT1-promoter-2. The results of the partial correlation and linear regression analyses were similar: POT1 promoter activity was identified to be significantly and positively correlated with POT1 expression and telomere length (partial correlation coefficients, both P<0.05; linear regression, both P<0.01). However, POT1 promoter activity and hTERT expression were significantly negatively correlated (both P<0.05). The results obtained in the present study suggest that the POT1 promoter influences telomere length. Furthermore, these data indicated that POT1 promoter activity and POT1, as well as telomere length, may be a useful biomarker for tumor detection and future patient prognosis.

Application of Nucleic Acid Aptamers in Polypeptides Researches / 分析化学

Polypeptides play a vital role in physiological processes of life. The pharmacological and medical value of polypeptides has attracted the attention of researchers in recent years. Aptamers are short, single stranded DNA or RNA which developed by an in vitro process called systematic evolution of ligands by exponential enrichment ( SELEX ) . Aptamers can bind targets with high affinity and specificity. Hence, aptamer is also called "chemical antibody" or "chemist's antibody". To date, there are two main application aspects for polypeptides-targeted aptamers. First, aptamer can be used as specific affinitive elements based on their ability of recognition, which would be applied to polypeptides detection or imaging. The other one is that aptamer can also be used as antagonists based on their ability of inhibiting, which can restrict the activity of polypeptides and block the downstream signaling pathways in vivo, thus can be used to treat the disease associated with polypeptides. In this review, we summarize the numbers of polypeptides-targeted aptamers and the related applications in vitro and in vivo. Current issues and development trends throughout the screening, characterizing and applying of polypeptides-targeted aptamers are also discussed.

Matrix metalloproteinase-mediation of tumor targeting human recombinant tumor necrosis factor-α fusion protein.

The aim of the present study was to use genetic engineering in order to establish an efficient tumor necrosis factor (TNF)-α fusion protein with low toxicity, which may be used to target tumors. Four types of matrix metalloproteinase (MMP)-mediated tumor targeting human recombinant TNF-α (rhTNF-α) fusion protein vectors were constructed. These were subsequently introduced into Escherichia coli. rhTNF-α fusion protein with a glutathione S-transferase (GST)-tag was purified using GST resin affinity chromatography, and GST-tags were digested using factor Xa. The cytotoxic effects of the fusion protein on L929 cells were determined using MTT assays. At a concentration of 1 pM, the GST-tagged fusion protein exerted no cytotoxic effects on the cells, compared with the negative control cells (P=0.975>0.05). However, at a concentration of 1000 pM, the deblocking fusion protein exerted greater cytotoxic effects on L929 cells, compared with positive control cells (P<0.05). Treatment with the fusion protein also induced cell apoptosis in the nasopharyngeal cancer cell line, CNE-2Z, which secretes high levels of MMP-1. In conclusion, the results of the present study suggested that MMP-mediated rhTNF-α fusion protein induces CNE-2Z cells apoptosis. rhTNF-α exhibits high efficacy and tumor cell targeting capability, with low toxicity effects on healthy cells.

Antitumor effect and mechanism of action of a tumor-targeting recombinant human tumor necrosis factor-α fusion protein mediated by urokinase.

The aim of this study was to investigate the effect of the tumor­targeting recombinant human tumor necrosis factor (rhTNF)­α fusion protein mediated by urokinase on Sl80 tumor­bearing mice, as well as to explore its mechanisms of action. Furthermore, the study aimed to observe the effect of the protein on liver and kidney function. rhTNF­α fusion protein prokaryotic expression vectors were constructed using genetic engineering techniques, and were introduced into Escherichia coli. Expression of the fusion protein was induced, and it was then separated and purified in order to determine its cytotoxic activity on L929 cells. Kunming mice were randomly divided into four groups after being inoculated with S180 tumor cells. The groups were then injected with saline (control group, group S), or saline with 0.1 µg/ml fusion protein (low dose group, group L), 0.2 µg/ml fusion protein (middle dose group, group M) or 0.3 µg/ml (high dose group, group H). The mice were sacrificed after 12 days and liver [mg/kg; (liver weight/body weight) x 1,000] and kidney [mg/kg; (kidney weight/body weight) x 1,000] indices, tumor weight, the percentage reduction in mean tumor size, and the levels of alanine transaminase (ALT), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in each group of mice were determined. In addition, the levels of urokinase­type plasminogen activator (uPA), the expression of bcl­2, bax and vascular endothelial growth factor (VEGF), and the percentage of apoptotic cells were measured with an enzyme­linked immunosorbent assay, streptavidin­biotin complex of immunohistochemistry and terminal deoxynucleotidyl transferase­mediated dUTP nick end labeling, respectively. The fusion protein significantly inhibited the growth of S180 tumor cells in vivo in a dose­dependent manner. With an increase in the dose of fusion protein, ALT, uPA, bcl­2 and VEGF levels decreased, and ALB levels increased. However, liver and kidney indices and bax expression were not significantly altered. Cr and BUN levels did not change significantly in the low and middle dose groups, but did increase in the high dose group. Compared with the control group, the percentage of apoptotic cells in the high­dose group was significantly higher. In conclusion, the fusion protein significantly inhibited S180 tumor growth in a mouse model, possibly by reducing the levels of uPA, bcl­2 and VEGF. There was a mildly toxic effect on the kidneys with the high dose, but a protective effect in the liver.

Targeting antitumor effect of rhTNF-α fusion protein mediated by matrix metalloproteinase-2.

The aim of this study was to examine the tumor therapy, targeting effects and side effects of tumor-targeting rhTNF-α fusion protein mediated by matrix metalloproteinase-2 in an animal model in order to provide experimental data for future development of drugs. The median lethal dose (LD50) was obtained from acute toxicity experiments. The A549 lung cancer xenograft model was established, and then randomly divided into the saline, standard substance, and low-, middle- and high-dose fusion protein experiment groups. Each group was administered drugs for 18 days. The length and width of the xenografts were measured every three days, after which the xenograft growth curve was drawn. The mice were sacrificed in each group following treatment and the tumor volume and weight were measured. The targeting, effectiveness and toxicity of the transformed fusion protein, and pathological changes of tumor and organ tissues were examined by hematoxylin and eosin (H&E) staining. Additionally, biochemical markers were used to detect damage of various organs after protein processing. Cell apoptosis and angiogenesis were determined using terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) testing and immunohistochemistry, respectively, in different dose groups. Tumor growth was markedly retarded in the high-dose experimental and standard hTNF-α groups with antitumor rates of 85.91 and 72.25%, respectively, as compared with the control group. Furthermore, the tumor tissue showed obvious apoptosis (the apoptotic index was 78.78 and 66.65%, respectively) and pathological changes in the high-dose experimental and standard hTNF-α groups. Tumor angiogenesis in each fusion protein group was inhibited (P<0.01) and the biochemical markers of various organs were greatly reduced in the high-dose experimental group (P<0.05). This finding indicated that slight toxic effects of fusion proteins were evident for the heart, liver and kidney. The reforming fusion protein can therefore target tumor tissues and efficiently kill tumor cells, with few side effects.

Fused polypeptide with DEF induces apoptosis of lung adenocarcinoma cells.

To analyze the effects of a new unknown peptide DEF on the growth of tumor cells, a fused polypeptide TAT-DV1-DEF was designed and synthesized. The lung adenocarcinoma cell line GLC-82 treated with TAT- DV1-DEF was analyzed with a cell counting kit 8, and the location of polypeptides in cells was observed under laser confocal microscopy. The efficiency of polypeptide transfection and changes in nuclear morphology were analyzed by flow cytometry and fluorescence microscopy, respectively. Finally, the mechanism of tumor cell growth inhibition was evaluated by Western blotting. We found that TAT-DV1-DEF could significantly inhibit the growth of the lung adenocarcinoma cell line GLC-82, but not the normal human embryonic kidney cell line HEK-293. Polypeptides were found to be mostly localized in the cytoplasm and some mitochondria. The efficiency of polypeptide transfection in the two cell types was approximately 99%. Apoptotic nuclei were observed under fluorescence microscopy upon treatment with polypeptides and DAPI staining. Western blot analyses indicated that the polypeptide inhibition of tumor cell growth was apoptosis dependent. In the present study, we demonstrated that fused polypeptides could induce apoptosis of the lung adenocarcinoma cell line GLC-82, indicating that the new unknown peptide DEF has antitumor effects.

Expression of hPOT1 in HeLa cells and the probability of gene variation of hpot1 Exon14 in endometrial cancer are much higher than in other cancers.

To investigate the expression of hPOT1 in the HeLa cell line and screen point mutations of hpot1 in different tumor tissues a two step osmotic method was used to extract nuclear proteins. EMSA was performed to determine the expression of hPOT1 in the HeLa cell line. PCR was also employed to amplify the exon14 sequence of the hpot1 gene in various of cancer tissues. A SV gel and PCR clean-up system was performed to enrich PCR products. DNAStar was used to analyse the exon14 sequence of the hpot1 gene. hPOT1 was expressed in the HeLa cell line and the signal was gradually enhanced as the amount of extracted nuclear proteins increased. The DNA fragment of exon14 of hpot1 was successfully amplified in the HeLa cell line and all cancer tissues, point mutations being observed in 2 out of 3 cases of endometrial cancer (66.7%) despite the hpot1 sequence being highly conserved. However, the sequence of hpot1 exon14 do not demonstrate point mutations in most cancer tissues. Since hPOT1 was expressed in HeLa cell and the probability of gene point variants was obviously higher in endometrial cancer than other cancers, it may be involved in the pathogenesis of gynecological cancers, especially in cervix and endometrium.