High Circulating FGF-23 and Its Relationship with Severity of Spinal Involvement in Diffuse Idiopathic Skeletal Hyperostosis.

The primary objective of this study was to determine the role of fibroblast growth factor 23 (FGF-23) in the pathogenesis of diffuse idiopathic skeletal hyperostosis (DISH). A total of 61 patients with DISH and 61 age- and sex-matched control patients without DISH were included in this study. The serum FGF-23, creatinine, inorganic phosphate, calcium, albumin, albumin-adjusted calcium and alkaline phosphatase, and C-reactive protein were assessed in both groups. Based on the extent of ossification, DISH group was further divided into T-DISH and L-DISH subgroups. Data were comparatively analyzed between DISH and Non-DISH groups and among T-DISH, L-DISH, and Non-DISH groups, respectively. Besides, the number of ossification segments of all DISH patients was quantified and the correlation between the number of ossification segments and the serum concentration of FGF-23 was analyzed. The results revealed that serum FGF-23 was significantly higher in DISH group than in Non-DISH group, regardless of gender. Interestingly, serum Pi was significantly lower in DISH group than in Non-DISH group. Moreover, a significant difference in serum FGF-23 among T-DISH, L-DISH, and Non-DISH groups was also observed. In contrast to Non-DISH group, both T-DISH and L-DISH subgroups displayed significantly higher serum FGF-23 level. Although the mean value was relatively higher in L-DISH subgroup, no statistically significant difference was found between T-DISH and L-DISH subgroups. In addition, a moderately positive correlation was identified between the number of ossification segments and the serum level of FGF-23. It can be concluded that serum FGF-23 could serve as a positive biomarker for DISH and may play a significant role in ectopic ossification in DISH.

Clinical value of droplet digital PCR in the diagnosis and dynamic monitoring of suspected bacterial bloodstream infections.

BACKGROUND: Bloodstream infections (BSIs) represent a significant public health challenge due to their high morbidity and mortality. The clinical prognosis of BSIs is closely related to the timely and accurate diagnosis and the rational use of initial antimicrobials. We aimed to evaluate the clinical value of droplet digital PCR (ddPCR) in rapid diagnosis and dynamic monitoring of BSIs. METHODS: In this prospective study, using a ddPCR-based approach which detects 18 common pathogens, we compared the detection results and clinical concordance rates of ddPCR with blood culture (BC) in 211 patients with suspected BSIs. Further, the inflammatory profile of BSIs with Gram-negative bacteria was analyzed by Olink proteomics platform. RESULTS: Our data showed that the positive detection rate of ddPCR was 48.82%, which was higher than that of BC (9.48%). For BC-validated BSIs, ddPCR had a sensitivity of 90.00% and a specificity of 55.50%. When considering clinically-validated BSIs, the diagnostic value of ddPCR improved with a sensitivity of 92.59% and a specificity of 78.46%.The bacterial load detected by ddPCR was correlated with traditional clinical inflammatory indicators such as interleukin-6 (IL-6) and C-reactive protein (CRP). In addition, using Olink proteomics platform, we revealed that serological osteoprotegerin (OPG), interleukin-8 (IL-8), interleukin-18 receptor 1 (IL-18R1), C-C motif chemokine 20 (CCL20) and IL-6 were substantially elevated in Gram-negative bacteria-associated BSIs, which could serve as novel auxiliary diagnostic indicators for Gram-negative bacteria BSIs. CONCLUSION: ddPCR has the potential to provide early pathogen diagnosis, dynamic monitoring, and treatment regimen optimization for patients with BSIs.

Evaluation of reverse transcription-polymerase chain reaction and simultaneous amplification and testing for quantitative detection of serum hepatitis B virus RNA.

Background: Chronic hepatitis B virus (HBV) infection is one of the common infectious diseases in the world. HBV covalently closed circular DNA (cccDNA) is the initial template of HBV replication, which can exist in human hepatocytes for a long time and is difficult to be completely removed. It has been shown that HBV RNA can directly respond to the levels and transcriptional activity of cccDNA in hepatocytes and can be used as a surrogate marker of cccDNA transcriptional activity. At present, the detection techniques used for quantitative HBV RNA mainly include reverse transcription quantitative polymerase chain reaction (RT-qPCR) and simultaneous amplification and testing (SAT). Methods: In this study, we verified the performance of the SAT method for detecting HBV RNA and the clinical effectiveness of SAT and RT-qPCR, and compared the correlation and consistency of the two detection methods for HBV RNA detection. Results: The results showed that the limit of detection for HBV RNA by SAT method was 50 copies/mL, with a linear range of 1 × 102-1 × 108 copies/mL. There was no difference in HBV RNA levels detected by the two methods. The correlation and consistency of the results were good, with the coefficient of determination of 0.7787 in HBeAg positive group and 0.8235 in HBeAg negative group. Conclusions: Therefore, this study confirmed that the SAT method and RT-qPCR for detecting HBV RNA have good agreement, which are both reliable methods to detect HBV RNA and can replace each other.

CRISPR-Cas and catalytic hairpin assembly technology for target-initiated amplification detection of pancreatic cancer specific tsRNAs.

Transfer RNA-derived small RNAs (tsRNAs) tRF-LeuCAG-002 (ts3011a RNA) is a novel class of non-coding RNAs biomarker for pancreatic cancer (PC). Reverse transcription polymerase chain reaction (RT-qPCR) has been unfit for community hospitals that are short of specialized equipment or laboratory setups. It has not been reported whether isothermal technology can be used for detection, because the tsRNAs have rich modifications and secondary structures compared with other non-coding RNAs. Herein, we have employed a catalytic hairpin assembly (CHA) circuit and clustered regularly interspaced short palindromic repeats (CRISPR) to develop an isothermal and target-initiated amplification method for detecting ts3011a RNA. In the proposed assay, the presence of target tsRNA triggers the CHA circuit that transforms new DNA duplexes to activate collateral cleavage activity of CRISPR-associated proteins (CRISPR-Cas) 12a, achieving cascade signal amplification. This method showed a low detection limit of 88 aM at 37 °C within 2 h. Moreover, it was demonstrated for the first time that, this method is less likely to produce aerosol contamination than RT-qPCR by simulating aerosol leakage experiments. This method has good consistency with RT-qPCR in the detection of serum samples and showed great potential for PC-specific tsRNAs point-of-care testing (POCT).

Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins13a combined with magnetic beads, chemiluminescence and reverse transcription-recombinase aided amplification for detection of avian influenza a (H7N9) virus.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins (CRISPR-Cas) have promising prospects in the field of nucleic acid molecular diagnostics. However, Clustered Regularly Interspaced Short Palindromic Repeats-based fluorescence detection technology is mainly hindered by proteins with conjugated double bonds and autofluorescence, resulting in high fluorescence background, low sensitivity and incompatible reaction systems, which are not conducive to automatic clinical testing. Chemiluminescence (CL) detection technology has been applied mainly owing to its greatly high sensitivity, as well as low background and rapid response. Therefore, we developed a rapid, ultrasensitive and economical detection system based on Clustered Regularly Interspaced Short Palindromic Repeats-Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins 13a combined with magnetic beads (MBs) and chemiluminescence (CL) (Cas13a-MB-CL) to detect Influenza A (H7N9), an acute respiratory tract infectious disease. The carboxyl functionalized magnetic beads (MBs-COOH) were covalently coupled with aminated RNA probe while the other end of the RNA probe was modified with biotin. Alkaline phosphatase labeled streptavidin (SA-ALP) binds with biotin to form magnetic beads composites. In presence of target RNA, the collateral cleavage activity of Cas13a was activated to degrade the RNA probes on MBs and released Alkaline phosphatase from the composites. The composites were then magnetically separated followed by addition of ALP substrate Disodium 2-chloro-5-{4-methoxyspiro [1,2-dioxetane-3,2'-(5'-chloro) tricyclo (3.3.1.13,7) decan]-4-yl}-1-phenyl phosphate (CDP-star), to generate the chemiluminescence signal. The activity of Associated Proteins 13a and presence of target RNA was quantified by measuring the chemiluminescence intensity. The proposed method accomplished the detection of H7N9 within 30 min at 25°C. When combined with Reverse Transcription- Recombinase Aides Amplification (RT-RAA), the low detection limit limit of detection was as low as 19.7 fM (3S/N). Our proposed MB-Associated Proteins 13a-chemiluminescence was further evaluated to test H7N9 clinical samples, showing superior sensitivity and specificity.

Combined Detection of Exosome Concentration and Tumor Markers in Gastric Cancer.

It has been an established fact that exosomes act as a mediator in tumor microenvironment as well as participate actively in intercellular communication between cancer cells. Exosomes carry a variety of molecular cargoes that prevent cyclic degradation and represent the cells of their origin. In this study, the difference in expression levels of exosomes was measured for diagnosis of gastric cancer. We isolated exosomes from plasma by size-selective method. The morphology of the exosomes was characterized by transmission electron microscopy, and the particle size and concentration of the exosomes were detected by NanoSight's Nanoparticle Tracking Analysis. Results indicated that the expression level of exosomes in gastric cancer patients was higher than that in healthy individuals. The specificity and sensitivity were 65.2% and 73.1%, respectively. Currently, clinical tumor markers for gastric cancer detection mainly included Carbohydrate antigen 72-4 (CA72-4), Alpha-fetoprotein, Carbohydrate antigen 125, Carbohydrate antigen 19-9 (CA19-9), Carcinoembryonic Antigen, Carbohydrate antigen 242. When we combined positive rate for combined gastric cancer biomarkers, results showed that exosomes concentration +CA19-9 and exosomes concentration +CA72-4 in the two-combined test can provide enough positive rate. Therefore, it can be concluded that for gastric cancer, the concentration of exosomes may be regarded as a diagnostic indicator, eventually.

Shiga-like toxin I exerts specific and potent anti-tumour efficacy against gastric cancer cell proliferation when driven by tumour-preferential Frizzled-7 promoter.

OBJECTIVES: Tumour-targeted gene therapy is a promising approach for effective control of gastric cancer cell proliferation. Our study aims to develop a cancer therapy which combines tumour-targeting promoters with cytotoxins. METHODS: The expression of globotriaosylceramide (Gb3), which is a Shiga-like toxin I (Stx1) receptor, was verified in gastric cancer compared with normal stomach tissues as assessed by flow cytometry and immunohistochemical analysis. We therefore constructed the recombinant pFZD7-Stx1 plasmid vectors with tumour-preferential Frizzled-7 promoter and Stx1. pFZD7-Stx1 was used to treat gastric cancer in vitro and in vivo. The gastric cancer cell proliferation and tumour growth were identified after the transfection with the pFZD7-Stx1. RESULTS: Globotriaosylceramide was obviously increased in gastric cancer compared with normal stomach. The gastric cancer cell proliferation and tumour growth decreased significantly after the transfection with the pFZD7-Stx1. CONCLUSION: Frizzled-7 promoter is preferentially active, and Gb3 is abundant in gastric cancer cells. Frizzled-7 promoter and Stx1 may be used to determine a novel and relatively specific and potent gastric cancer therapeutic strategy.

A Sensitive Aptasensor Based on a Hemin/G-Quadruplex-Assisted Signal Amplification Strategy for Electrochemical Detection of Gastric Cancer Exosomes.

Emerging evidence indicates that exosomes derived from gastric cancer cells enhance tumor migration and invasion through the modulation of the tumor microenvironment. However, it remains a major problem to detect cancer-specific exosomes due to technical and biological challenges. Most of the methods reported could not achieve efficient detection of tumor-derived exosomes in the background of normal exosomes. Herein, a label-free electrochemical aptasensor is presented for specific detection of gastric cancer exosomes. This platform contains an anti-CD63 antibody modified gold electrode and a gastric cancer exosome specific aptamer. The aptamer is linked to a primer sequence that is complementary to a G-quadruplex circular template. The presence of target exosomes could trigger rolling circle amplification and produce multiple G-quadruplex units. This horseradish peroxidase mimicking DNAzyme could catalyze the reduction of H2 O2 and generate electrochemical signals. This aptasensor exhibits high selectivity and sensitivity toward gastric cancer exosomes with a detection limit of 9.54 × 102 mL-1 and a linear response range from 4.8 × 103 to 4.8 × 106 exosomes per milliliter. Therefore, this electrochemical aptasensor is expected to become a useful tool for the early diagnosis of gastric cancer.

CRISPR-Cas13a Nanomachine Based Simple Technology for Avian Influenza A (H7N9) Virus On-Site Detection.

It is urgent to find an avian influenza A H7N9 detection simple method which is suitable for on-site detection. The Cas13a protein just likes a nanomachine, when specifically bound to target RNA by single-stranded RNA (crRNA), changes its protein structure and produces RNase activity, which degrades RNA non-specifically. Harnessing Cas13a, the paper aims to establish an underlying on-site H7N9 virus nucleic acid detection method. LwCas13a protein nanomachine was expressed in a prokaryotic expression system and purified by nickel column. In vitro transcribed RNA of H7N9 HA gene has been used as a target, to design a specific crRNA. The activity of Cas13a was verified with a single-stranded RNA-bound fluorescent group and a quenching fluorophore as signals. Using Cas13a, a room temperature H7N9 detection system was established. Detection of 1 nm of single-stranded RNA can be done within 5 min. When combined with the RT-RPA and T7 transcription system at room temperature, the detection limits of HA and NA are 1 fM and the reaction time is 50 min. Excellent specificity was achieved by comparison with subtype viruses such as H1N1 and H5N1. The rapid detection method based on CRISPR-Cas13a nanomachine H7N9 has been successfully established, which can detect H7N9 quickly and specifically. In the future, it can be quickly detected in the field with portable fluorescence detector.

Golgi protein 73 and its diagnostic value in liver diseases.

Golgi protein 73 (GP73, also referred to as Golph 2) with 400 amino acids is a 73 kDa transmembrane glycoprotein typically found in the cis-Golg complex. It is primarily expressed in epithelial cells, which has been found upregulated in hepatocytes in patients suffering from both viral and non-viral liver diseases. GP73 has drawn increasing attention for its potential application in the diagnosis of liver diseases such as hepatitis, liver cirrhosis and liver cancer. Herein, we reviewed the discovery history of GP73 and summarized studies by many groups around the world, aiming at understanding its structure, expression, function, detection methods and the relationship between GP73 and liver diseases in various settings.

CRISPR-Cas13a mediated nanosystem for attomolar detection of canine parvovirus type 2.

Canine parvovirus type 2 (CPV-2) infection is the most lethal disease of dogs with higher mortality in puppies worldwide. In today's world, dogs are an integral part of our communities as well as dogs breeding and rearing has become a lucrative business. Therefore, a fast, accurate, portable, and cost-effective CPV-2 detection method with the ability for on-site detection is highly desired. In this study, we for the first time proposed a nanosystem for CPV-2 DNA detection with RNA-guided RNA endonuclease Cas13a, which upon activation results in collateral RNA degradation. We expressed LwCas13a in prokaryotic expression system and purified it through nickel column. Activity of Cas13a was verified by RNA-bound fluorescent group while using a quenched fluorescent probe as signals. Further Cas13a was combined with Recombinase polymerase amplification (RPA) and T7 transcription to establish molecular detection system termed specific high-sensitivity enzymatic reporter un-locking (SHERLOCK) for sensitive detection of CPV-2 DNA. This nanosystem can detect 100 amol/L CPV-2 DNA within 30 min. The proposed nanosystem exhibited high specificity when tested for CPV-2 and other dog viruses. This CRISPR-Cas13a mediated sensitive detection approach can be of formidable advantage during CPV-2 outbreaks because it is time-efficient, less laborious and does not involve the use of sophisticated instruments.

Magnetic Nanoparticle-Based Automatic Chemiluminescent Enzyme Immunoassay for Golgi Protein 73 and the Clinical Assessment.

Golgi protein 73 (GP73) is an independent diagnostic indicator of cirrhosis. However, it lacks automatic detection techniques to meet the large-scale clinical requirement in physical examination. In this paper, an automatic approach was established based on the ACL2800 automatic chemiluminescent analyzer, using magnetic nanoparticles (MNPs) and chemiluminescence. This method depended on sandwich strategy among biotin-labeled capture monoclonal antibody, target GP73 and acridinium ester (AE)-labeled reporter monoclonal antibody, conjugation of streptavidin-labeled MNPs to biotin-labeled capture monoclonal antibody, and chemiluminescent detection of AE-linked targets. Optimal conditions were investigated and clinical assessment was processed. Detection of GP73 demonstrated a high sensitivity of 1.19 ng/mL with a wide range from 1.34 ng/mL to 684.38 ng/mL. The quantitative detection was achieved with the repeatability of 2.69%, the coefficient of variation of 3.55% and the percent recovery of 93.46%-107.82%. Therefore, an automatic quantitative detection method was successfully developed, which was a potential screening method in physical examination.

Urine protein quantification in stacking gel by SDS-PAGE.

Urine total protein concentration is usually measured by the pyrogallol red-molybdate (PRM) assay in clinical laboratories, but it is often subject to sample interference. Here, we introduce a stacking gel-based method for accurate protein quantitation. In this method, the urine protein samples are run into the stacking gel by SDS-PAGE where it is concentrated into a single band, and then quickly stained by 0.001% Coomassie at high temperature. High correlations were found between the BSA and urine protein standards (R2 = 0.997 and 0.990, respectively). Addition of 80 ng urine protein standard into each of the ten clinical urine specimens with questionable PRM results yielded the expected increase in the results by this method. Comparison of the PRM method and with the gel quantitation approach on about 60 clinical urine samples demonstrated a general consistency between the results (R2 = 0.825), but in PRM samples with lower protein concentration showed more variations. Overall, the stacking gel method might be a good alternative for clinical urine samples with suspicious protein concentration results.

Stable Protein Gel Storage in Acetonitrile for Mass Spectrometric Analysis.

Long-term storage of protein samples for transportation is a challenge in the field of mass spectrometric analysis because the low temperature condition is not easy to be maintained. Here we introduce a simple method to preserve proteins at room temperature for at least one month. In this method, the protein sample is run shortly into a polyacrylamide gel which is then excised after Coomassie staining. The protein gel band is then dehydrated by 100% acetonitrile three times and kept in 100% acetonitrile for storage at room temperature. By the TMT 10-plex based quantitative proteomic analyses, we have found that these proteins are stable in their levels and modifications (phosphorylation, oxidation, and ubiquitination) for 30 days. Further analysis has revealed this storage method also well preserves proteins even at 45 °C. We therefore recommend to use acetonitrile to dehydrate and store protein gel pieces as an effective alternative for sample shipping over days. Therefore, it might facilitate worldwide collaborations in the mass spectrometry-based proteomic research.

A sensitive three monoclonal antibodies based automatic latex particle-enhanced turbidimetric immunoassay for Golgi protein 73 detection.

Golgi protein 73 (GP73) is a novel and potential marker for diagnosing hepatocellular carcinoma (HCC) that has been found to be abnormally elevated in liver disease. A latex particle-enhanced turbidimetric immunoassay (LTIA) was recently introduced and licensed for application in a variety of automated clinical chemistry analyzers. However, no studies have reported sufficient data on analytical performance of this method when using 3 monoclonal antibodies for GP73 measurement. The experimental conditions were firstly optimized and range of linearity, diagnostic potential, clinical relevance were compared with the LTIA based on polyclonal antibodies and ELISA. Dilution tests for the LTIA using 3 monoclonal antibodies produced a calibration curve from 10 to 350 ng/mL while the polyclonal antibodies produced the curve from 20 to 320 ng/mL. The detection limit was achieved at 1.82 ng/mL concentration. Within-run CV was obtained in the range of 1.5-2.9% and ROC curves indicated sensitivity and specificity of the LTIA based on 3 monoclonal antibodies were 96.7% and 93.3%, respectively, higher than for the polyclonal antibodies (94.6% and 72.4%) and ELISA (70.0% and 83.3%). Therefore, the LTIA assay based on 3 monoclonal antibodies is thus applicable in quantification of GP73 concentration in automated biochemistry analyzers.

Detection of Hepatocellular Carcinoma Biomarker Golgi Protein 73 Based on Magnetic Enzyme-Linked Immunoassay.

The magnetic enzyme-linked immunoassay (MEIA) based on magnetic nanoparticles as the solid phase was reported in this work. Magnetic nanoparticles (MNPs) have represented perfectly suitable materials for a variety of biomedical and biotechnological applications. Therefore, we used MEIA based on magnetic nanoparticles to provide a screening method for fast analysis of serum Golgi protein 73 (GP73) in hepatocellular carcinoma (HCC) and healthy subjects and comparison was made with the enzyme-linked immunosorbent assay (ELISA) method. Several relevant conditions, including the concentration of anti-GP73 monoclonal antibody and HRP-anti-human GP73 monoclonal antibody, amount of immunomagnetic beads, and the incubation time, were determined and optimized. Finally, the MEIA was successfully established and validated by 79 HCC and 64 healthy subjects. The results showed this method achieved a detection limit of 0.78 ng/mL, which was more sensitive than ELISA. Furthermore, the sensitivity and specificity of the MEIA were 78.43% and 91.47%, respectively, which were higher than ELISA. The MEIA based on MNPs proved to be simple, sensitive, specific and time-saving, therefore holds great potential for development of a commercial kit in the future.

Frizzled-7 promoter is highly active in tumors and promoter-driven Shiga-like toxin I inhibits hepatocellular carcinoma growth.

Frizzled-7 protein plays a significant role in the formation of several malignant tumors. Up regulation of the Frizzled-7 in cancer cell lines is associated with nuclear accumulation of wild-type ß-catenin from the Wnt/ß-catenin pathway which is frequently activated in tumors. To analyze activity of the Frizzled-7 promoter in tumor cells, we constructed two recombinant plasmid vectors in which the Frizzled-7 promoter was used to drive the expression of green fluorescent protein (GFP) and Shiga-like toxin I (Stx1) (pFZD7-GFP/Stx1) genes. The Frizzled-7 protein was found to be expressed in the cancer cell lines but not in the normal cell lines. The GFP expression was restricted to the cancer cell lines and xenografts in the BALB/C mice but not to normal cell lines. Moreover, cell proliferation and tumor growth decreased significantly after transfection with the pFZD7-Stx1. Results from this study will help determine a highly effective strategy for gene therapy of tumors.

Preparation and characterization of anti-GP73 monoclonal antibodies and development of double-antibody sandwich ELISA.

BACKGROUND: Serum Golgi protein 73 (GP73) as a novel and potential marker for diagnosing hepatocellular carcinoma (HCC) have been found to be elevated in HCC patients and associated with clinical variables representing tumor growth and invasiveness. The aim of this study was to prepare a pair of monoclonal antibodys (mAbs) against GP73 and develop a newly designed double-antibody sandwich enzyme-linked immunosorbent assay (s-ELISA), which would be used in the detection of serum GP73 (sGP73) as well as in the diagnosis of HCC. MATERIALS AND METHODS: Produced by prokaryotic expression, the purified recombinant GP73 (rGP73), produced by prokaryotic expression, was used to immunize the Balb/c mice. Two hybridoma cell lines against GP73 were obtained by fusing mouse Sp2/0 myeloma cells with spleen cells from the immunized mice. The titers of anti-GP73 mAb reached 1:243,000. Western blotting analysis and Immunohistochemistry staining revealed that anti-GP73 mAb could recognize GP73 protein. The double-antibody s-ELISA was successfully established and validated by 119 HCC and 103 normal serum samples. RESULTS: showed that the detection limit of this method could reach 1.56 ng/ml, and sGP73 levels in HCC group (mean=190.6 ng/ml) were much higher than those of in healthy controls (mean=70.92 ng/ml). CONCLUSIONS: Results of our study not only showed that sGP73 levels of HCC patients were significantly higher than those of healthy controls, but also indicated that the laboratory homemade anti-GP73 mAbs could be the optimal tool used in evaluating sGP73 levels, which would provide a solid foundation for subsequent clinical applications.

Gene therapy with biosynthetic nanoscale peptide Cecropin A driven by the survivin promoter for hepatocarcinoma cells.

The biosynthetic nanoscale peptide Cecropin A is postulated to disrupt microbial phospholipid membranes by forming stable or transient pores. We demonstrated previously that green fluorescent protein (GFP), driven by the survivin promoter, was expressed highly in HepG2 but not in LO2 cells when they were transfected with the recombinant plasmid reporter vector (pSURV-GFP). To investigate the selective killing effect of this survivin promoter-driven peptide toxin gene system on hepatocellular carcinoma (HCC) cells in vitro, the recombinant plasmid pSURV-Cecropin A was constructed. HepG2 and LO2 cells were then transfected with the recombinant plasmid, which was driven by the survivin promoter, and the effects of Cecropin A were evaluated. Forty-eight hours after transfection with pSURV-Cecropin A, the growth of HepG2 cells was inhibited significantly. This finding was confirmed further by immunoblotting, which revealed consistently suppressed expression of proliferating cell nuclear antigen (PCNA) and cysteinyl aspartate specific proteinase-3 (caspase-3). Data demonstrated that the plasmid carrying the gene for the Cecropin A fusion protein was constructed successfully, and that its specific expression in HepG2 cells could provide the basis for targeted gene therapy in HCC. The identification of novel gene therapies for cancer is highly desirable to reduce drug toxicity and improve therapeutic outcomes..

Nanocarriers in gene therapy: a review.

With its rapid development in the past few decades, gene therapy has shown potential for use as a standard clinical intervention for the treatment of several conditions, including cancers, infectious diseases, cardiovascular disorders, inner ear disorders, dermatological, ophthalmologic, and neurological pathologies. Current gene therapy is not limited to the delivery of DNA only. Other therapeutic nucleic acid materials such as small interfering RNA, antisense oligonucleotides, or microRNA have also been included into the protocols of gene therapy. The correct choice of vector is a key factor in the success of gene therapy, where both viral and non-viral vectors are commonly used. Viral vectors are associated with some severe side effects (e.g., immunologenicity and carcinogenicity). They show poor target cell specificity, are unable to transfer large-sized genes, and are costly. Therefore, non-viral vectors, especially nanocarriers, have become a realistic alternative to viral vectors for achieving better efficacy in gene therapy. Different types of nanocarriers such as liposomes, metallic and polymeric nanoparticles, dendrimers, gelatins, and quantum dots/rods have been developed, and each shows distinct characteristics. Nevertheless, a variety of new challenges should be properly addressed for ensuring the success of nanocarriers in clinical applications. In this review article, we first discuss the advances and applications of nanocarriers in gene therapy, and then describe the drawbacks and existing challenges of the emerging gene delivery methods based on the use of nanomaterials.