Research Progress of Graphene and Its Derivatives towards Exhaled Breath Analysis.

The metabolic process of the human body produces a large number of gaseous biomarkers. The tracking and monitoring of certain diseases can be achieved through the detection of these markers. Due to the superior specific surface area, large functional groups, good optical transparency, conductivity and interlayer spacing, graphene, and its derivatives are widely used in gas sensing. Herein, the development of graphene and its derivatives in gas-phase biomarker detection was reviewed in terms of the detection principle and the latest detection methods and applications in several common gases, etc. Finally, we summarized the commonly used materials, preparation methods, response mechanisms for NO, NH3, H2S, and volatile organic gas VOCs, and other gas detection, and proposed the challenges and prospective applications in this field.

Combining combretastatin A4 phosphate with ginsenoside Rd synergistically inhibited hepatocellular carcinoma by reducing HIF-1α via PI3K/AKT/mTOR signalling pathway.

OBJECTIVES: Combretastatin A4 phosphate (CA4P), a vascular disrupting agent (VDA), can cause rapid tumour vessel occlusion. Subsequently, extensive necrosis is discovered in the tumour center, which induces widespread hypoxia and the rise of the α subunit of hypoxia-inducible factor-1 (HIF-1α). The aim of this study was to evaluate the inhibition of hepatocellular carcinoma growth by combining CA4P with HIF-1 α inhibitor and investigate the mechanism of this combination. METHODS: Ginsenoside Rd (Rd) was used in combination with CA4P to estimate the inhibition effect in HepG2 cells and HepG2 xenograft mouse model. The efficacy of anti-tumour was evaluated by tumour growth curve. The protein expression of HIF-1α and PI3K/AKT/mTOR signalling pathway were analysed by western blot. KEY FINDINGS: Combination of CA4P and Rd inhibited HepG2 cell proliferation and induced apoptosis in vivo and in vitro. It also increased the necrotic area of the tumour and delayed the tumour growth. Moreover, Rd down-regulated HIF-1α protein expression by inhibiting PI3K/AKT/mTOR signalling pathway. CONCLUSIONS: Combination of CA4P and Rd had synergistic anti-tumour effects. The mechanism may be related to the inhibition of HIF-1α by PI3K/AKT/mTOR signalling pathway. This strategy provides a new thought for the combinative therapy of VDAs.

Ultrasensitive fluorescence detection of sequence-specific DNA via labeling hairpin DNA probes for fluorescein o-acrylate polymers.

Sensitive detection of DNA is conducive to enhance the accuracy of diseases diagnosis and risk prediction. In this work, we report the use of activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP) as a novel on-chip amplification strategy for the fluorescence detection of DNA. More specifically, the target DNA was captured by the on-chip immobilized hairpin DNA probes. Upon hybridization, exposed 3'-N3 of the hairpin was used to attach AGET ATRP initiators onto the silicon surface by click chemistry. Then, numerous fluorescent labeling linked to the end of the probes via the formation of long chain polymers of fluorescein o-acrylate, which in turn amplified the fluorescence signal for DNA detection. Under optimal conditions, it showed a good linear range from 100 fM to 1 µM in DNA detection, with the limit of detection as low as 4.3 fM. Moreover, this strategy showed good detection performance in complex real serum samples, the fluorescence intensity of 0.1 nM tDNA in 1% fetal bovine serum samples was 97.6% of that in Tris-EDTA buffer. Based on its high sensitivity, reduced cost and simplicity, the proposed signal amplification strategy displays translational potential in clinical application.

Preparation and preliminary application of monoclonal antibodies against Trichokirin-S1, a small ribosome-inactivating peptide from the seeds of Trichosanthes kirilowii.

Trichokirin-S1, a small ribosome-inactivating peptide recently purified from the seeds of Trichosanthes kirilowii, has potential clinical applications because of its small molecular mass. Two stable strains of hybridomas (1F11 and 2A5) that can secrete highly specific monoclonal antibodies (mAbs) against Trichokirin-S1 have been developed using the hybridoma technique. The isotypes of these two mAbs, 1F11 and 2A5, were determined to be IgG2a and IgG1, respectively. The affinity constants, which were measured by non-competitive ELISA, were found to be 2.3 x10(8) M(-1) and 2.8 x 10(8) M(-1), respectively. An immunoaffinity method using 2A5-coupled Sepharose 4B was successfully developed to purify Trichokirin-S1. These two antibodies have also been used to detect Trichokirin-S1 in Western blot.

Gene cloning, bacterial expression, in vitro refolding, and characterization of a single-chain Fv antibody against PreS1(21-47) fragment of HBsAg.

The murine monoclonal antibody 125E11 is an IgG which recognizes PreS1(21-47) fragment of large hepatitis B surface antigen. It has been successfully used for clinical detection of HBV virion in serum of hepatitis B patients. In present study, the genes of variable region in heavy chain (VH) and light chain (VL) of 125E11 have been cloned. Sequence analysis of cloned VH gene and VL gene showed that they had general characterization of immunoglobin variable region genes. According to Kabat classification, VH gene and VL gene belong to VH10 family, subgroup IIID and Vkappa family subgroup I, respectively. An expression vector of 125E11 single-chain Fv antibody fusion protein, in which VH and VL peptide were connected by a flexible linker (Gly(4)Ser)(3), was constructed. The scFv fusion protein was highly expressed in Escherichia coli mainly in inclusion body form. Using urea and pH gradient gel filtration method, the refolding of scFv was efficiently achieved. The refolding efficiency reached about 11% and 2.7 mg refolded scFv was obtained from 1L of culture. The binding activity and specificity of 125E11 scFv against PreS1(21-47)-containing antigen were also analyzed.

Identification of the immunogenic domains in HBsAg preS1 region using overlapping preS1 fragment fusion proteins.

AIM: The incorporation of hepatitis B virus (HBV) preS1 region into epitope-based vaccines against HBV has been accepted widely, but the incorporate site and size of preS1 sequence is controversial. Therefore our purpose was to further investigate its immunogenic domains for the epitope-based hepatitis B vaccine design. METHODS: Eight GST fusion proteins containing overlapping preS1 fragments in preS1 (21-119) region were expressed in E.coli. Using these purified fusion proteins, the immunogenic domains in preS1 region were identified in detail in mice and humans by Western blot analysis and ELISA. RESULTS: The results in mice showed that the immu-nogenic domains mainly existed in preS1 (21-59) and preS1 (95-109). Similarly, these fragments had strong immunogenicity in humans; whereas the other parts except for preS1 (60-70) also had some immunogenicity. More importantly, a major immunogenic domain, preS1 (34-59), which has much stronger immunogenicity, was identified. Additionally, the antibodies against some preS1 fragments, especially preS1 (34-59), were speculated to be virus-neutralizing. CONCLUSION: Eight GST fusion proteins containing overlapping preS1 fragments were prepared successfully. They were used for the study on the immunogenic dom-ains in preS1 (21-119) region. The preS1 (34-59) fragments were the major immunogenic domains in the preS1 region, and the antibodies against these fragments were speculated to be virus-neutralizing. Therefore, the incorporation of preS1 (34-59) fragments into epitope-based HBV vaccines may be efficient for enhancement of immune response. Additionally, the results also imply that there are more complex immune responses to preS1 region and more abundant immunogenic domains in humans.

Expression of overlapping PreS1 fragment recombinant proteins for the determination of immunogenic domains in HBsAg PreS1 region.

In this study, eight preS1 fragments overlapped in preS1 (21-119) region of HBV adr subtype, i.e. preS1 (21-47), preS1 (34-59), preS1 (48-70), preS1 (60-85), preS1 (71-94), preS1 (86-109), preS1 (95-119) and preS1 (21-119), were cloned by PCR, and expressed as GST fusion proteins. These GSTpreS1 fusion proteins were highly expressed in soluble form in E. coli, and about 50 to 90 mg soluble fusion proteins were purified from 1 L culture. Using these fusion proteins, the immunogenic domains in preS1 (21-119) region were identified by Western blot analysis and competitive ELISA. The results showed that the immunogenic domains mainly existed in preS1 (21-59) in N-terminus and preS1 (95-109) in C-terminus, and more importantly, a major immunogenic domain preS1 (34-59), which has much stronger immunogenicity, was identified. It was also supported by the predictions of secondary structure and immunological property in the preS1 (21-119) region. The results here would be helpful for the design of new vaccines against HBV.

Preparation and primary application of monoclonal antibodies against a novel ribosome-inactivating protein Moschatin from pumpkin seeds.

Plant ribosome-inactivating proteins (RIPs) have multiple biological functions, and have been widely used in the studies on biomedical and agronomic applications. Moschatin is a novel single-chain RIP recently purified from pumpkin seeds, and it has been successfully applied to construct the immunotoxin that can selectively kill the cultured human melanoma cells. Six stable strains of hybridomas (2H8, 4A8, 5B6, 6F8, 4H10 and 6C2) that can secrete high specific monoclonal antibodies against Moschatin have been successfully prepared using hybridoma technique. The isotypes of these monoclonal antibodies are IgG1, IgG1, IgG1, IgG1, IgG2a and IgGM. Their affinity constants were determined to be 1.42x10(8), 2.71x10(8), 8.72x10(7), 2.06x10(8), 1.36x10(8) and 1.51x10(8) M(-1) in a sequent order, measured by non-competitive ELISA. The monoclonal antibody 4A8 has been used to detect Moschatin in Western blot. An immunoaffinity gel, which consisted of a monoclonal antibody 4H10 and Sepharose 4B, was prepared and used to purify Moschatin from pumpkin seeds crude extract.

[Purification and characterization of trichokirin-S1, a novel ribosome-inactivating peptide from seeds of Trichosanthes kirilowii].

A novel peptide from the seeds of Trichosanthes kirilowii, trichokirin-S1, was purified by extraction of protein body, ammonia sulfate precipitation, Blue-gel affinity chromatography, FPLC Mono S ion exchange chromatography and Superose12 gel filtration chromatography. Its molecular weight was determined to be 11,426 by MALDI-TOF MS analysis. Its reaction mechanism to inactive ribosome was the same as that of the ribosome-inactivating protein trichosanthin, a rRNA N-glycosidase. The purified trichokirin-S1 showed a strong inhibitory activity on protein synthesis in cell-free rabbit reticulocyte lysate system, with IC(50) of 0.7 nmol/L. Therefore, trichokirin-S1 may be a promising and efficient toxin moiety of immunotoxins.

[Purification and partial characterization of luffin P1, a peptide with translational inhibitory activity and trypsin inhibitory activity, from seeds of Luffa cylindrica].

A peptide, luffin P1, from seeds of Luffa cylindrica, was purified by ammonia sulfate precipitation, CM-52 ion exchange chromatography, Blue-gel affinity chromatography and FPLC Mono S ion exchange chromatography. Its molecular weight was 5226.5 as determined by MALDI-TOF-MS analysis. The sequence of N-terminal 11 amino acids of luffin P1 was identical with the partial N-terminal sequence (from G3 to R13) of 6.5K Arg/Glu rich peptide, which was also isolated from the seeds of Luffa cylindrica. Besides, luffin P1 had a very high homology with a trypsin inhibitor, named C2 peptide, from pumpkin seeds. Interestingly, the purified luffin P1 not only showed a strong inhibitory activity on protein synthesis in rabbit reticulocyte lysate cell-free translation system with IC(50) of 0.6 nmol/L, but also had trypsin inhibitory activity with IC(50) of 22 micromol/L.

Purification and characterization of Luffin P1, a ribosome-inactivating peptide from the seeds of Luffa cylindrica.

A peptide designated Luffin P1 was purified from the seeds of Luffa cylindrica. Its molecular mass was determined to be 5226.1 Da by MALDI-TOF MS analysis. The purified Luffin P1 shows a strong inhibitory activity on protein synthesis in the cell-free rabbit reticulocyte lysate with IC(50) of 0.88 nM. Its reaction mechanism is the same as that of the ribosome-inactivating protein trichosanthin, which is an rRNA N-glycosidase. Besides, the results of gel filtration chromatography suggested the existence of polymers of Luffin P1 and polymerization of Luffin P1 enhanced its rRNA N-glycosidase activity. Luffin P1 was the smallest peptide yet reported that has translational inhibitory activity. The cDNA and deduced amino acid sequence of Luffin P1 has also been determined.

Cloning and Expression of Luffin-b cDNA from the Seeds of Luffa cylindrica.

Luffin-b with Mr. 28 kD, isolated from the seeds of Luffa cylindrica,is one of the most toxic single chain plant ribosome inactivating proteins. The cDNA sequence of luffin-b was already reported by Kataoka in 1992. In this work, the luffin-b gene(lufB) coding sequence was cloned from the fresh seeds of Luffa cylindrica by RT-PCR, and the coding sequence of the gene was shown to be identical with that determined by Kataoka. The lufB expression plasmid was constructed by inserting the lufB cDNA fragment into vector pET24a( ), and the pET24a( )- lufB vector was expressed in E.coli by 0.5 mmol/L IPTG induction. The recombinant product, which mainly existed in inclusion bodies, was identified by SDS-PAGE and Western blotting. The recombinant luffin-b, which was renatured by dialysis with step by step decreasing concentration of urea, showed high activity of ribosome inactivation.

Preparation and Use of a Monoclonal Antibodyagainst Luffin b.

Luffin b is one of the most toxic single chain plant ribosome inactivating proteins. It has been successfully used to prepare an immunotoxin against human melanoma cells. Two strains of hybridomas (1E5 and 2E1) were screened out using cell fusion technique which steadily secreted monoclonal antibodies against luffin b. These antibodies specifically reacted with luffin b. The affinity constants of 1E5 and 2E1 monoclonal antibodies were determined to be 1.1x10(9) mol(-1).L and 7.5x10(8) mol(-)1.L, by RIA,respectively. An immunoaffinity gel composed with the 1E5 monoclonal antibody and Sepharose 4B was prepared. The luffin b was successfully purified by one step immunoaffinity chromatography from the crude extract of Luffa cylindrica seeds. An immunoconjugate 1E5-HRP was also prepared and it was successfully used in Western blotting for detection of recombinant luffin b from E.coli total proteins.