Biomedical subjects

Ling Fu

Publications and source records attributed to Ling Fu.

At least 19 recordsLinked to original sources

PubMed2006

Double-clad photonic crystal fiber coupler for compact nonlinear optical microscopy imaging.

A 1 x 2 double-clad photonic crystal fiber coupler is fabricated by the fused tapered method, showing a low excess loss of 1.1 dB and a splitting ratio of 97/3 over the entire visible and near-infrared wavelength range. In addition to the property of splitting the laser power, the double-clad feature of the coupler facilitates the separation of a near-infrared single-mode beam from a visible multimode beam, which is ideal for nonlinear optical microscopy imaging. In conjunction with a gradient-index lens, this coupler is used to construct a miniaturized microscope based on two-photon fluorescence and second-harmonic generation. Three-dimensional nonlinear optical images demonstrate potential applications of the coupler to compact all-fiber and nonlinear optical microscopy and endoscopy.

Journal Article↗

PubMed2006

Carambola optics for recycling of light.

Recycling of light allows the luminance (radiance) emitted by a light source to be increased at the cost of reducing the total luminous flux (radiant power). Recycling of light means returning part of the emitted light to the source, where part of it will escape absorption. An optical design that is suitable for multiple and controlled recycling is described. Carambola optics is named for its resemblance to star fruit. Several pairs of mirrors or prisms redirect light repeatedly onto the source, thus achieving multiple transits of the light through the source. This recycled light exits the carambola in the same phase space as light directly emitted and not recycled.

Journal Article↗

PubMed2006

The 2beta2-2beta3 loop of anthrax protective antigen contains a dominant neutralizing epitope.

Anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. PA is the major component in the current anthrax vaccine, but the antigenic epitopes on it are not well-defined. We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) to analyze the neutralizing epitopes of PA. Nine toxin-neutralizing MAbs obtained were found bound to three different domains of PA respectively, among which three MAbs with the strongest toxin-neutralizing activity recognized the same epitope within domain 2. This epitope was fine mapped to the chymotrypsin-sensitive site, (312)SFFD(315), in the 2beta(2)-2beta(3) loop of PA, using phage-displayed random peptide libraries and mutation analysis. The result demonstrated for the first time that the 2beta(2)-2beta(3) loop, which is involved in the transition of PA oligomers from prepore to pore, contains a dominant neutralizing epitope. This work contributes to the immunological and functional analysis of PA and offers perspective for the development of a new epitope vaccine against anthrax.

Animals↗

PubMed2006

Somatic mutations lead to an oncogenic deletion of met in lung cancer.

Activating mutations in receptor tyrosine kinases play a critical role in oncogenesis. Despite evidence that Met kinase is deregulated in human cancer, the role of activating mutations in cancers other than renal papillary carcinoma has not been well defined. Here we report the identification of somatic intronic mutations of Met kinase that lead to an alternatively spliced transcript in lung cancer, which encodes a deletion of the juxtamembrane domain resulting in the loss of Cbl E3-ligase binding. The mutant receptor exhibits decreased ubiquitination and delayed down-regulation correlating with elevated, distinct Met expression in primary tumors harboring the deleted receptor. As a consequence, phospho-Met and downstream mitogen-activated protein kinase activation is sustained on ligand stimulation. Cells expressing the Met deletion reveal enhanced ligand-mediated proliferation and significant in vivo tumor growth. A hepatocyte growth factor competitive Met antagonist inhibits receptor activation and proliferation in tumor cells harboring the Met deletion, suggesting the important role played by ligand-dependent Met activation and the potential for anticancer therapy. These results support a critical role for Met in lung cancer and somatic mutation-driven splicing of an oncogene that leads to a different mechanism for tyrosine kinase activation through altered receptor down-regulation in human cancer.

Alternative Splicing↗

PubMed2006

[Preliminary study of LFn-MAGE3 fusion protein expression and its biological function].

AIM: To explore the possibility of using the nontoxic form of anthrax toxin in cancer immunotherapy by LFn-MAGE3 fusion protein expression. METHODS: A fusion expression vector named PET21a-LFn was constructed by inserting LFn coding senquence into PET21a. PET21a-LFn-MAGE3 fusion protein expression vector was constructed by cloning the whole MAGE-3 gene into plasmid PET21a-LFn. Q sepharose FF and Phe HP columns were employed to purify the fusion protein. The biological activity of LFn-MAGE3 was determined by cell test of repressing the cytotoxity of LF and the tests of immunofluscence of mouse macrophage. RESULTS: The resulting plasmid expressed fusion protein LFn-MAGE3 in the soluble form in E.coli BL21, the cell tests showed that purified LFn-MAGE fusion protein was delivered into macrophage effectively with the help of PA(anthrax protective antigen). CONCLUSION: The successful delivery of fusion protein into macrophages coordinated by PA may lay the foundation for its further use in cancer immunotherapy in animal experiments.

Animals↗

PubMed2006

[Detection of serum interleukin-6 level in patients with multiple myeloma of Vighur nationality and Han nationality and its clinical significance].

To investigate the difference of serum interleukin-6 level in the patients with multiple myeloma (MM) of Vighur nationality and Han nationality and its clinical significance, the activity of serum interleukin-6 was detected in 80 patients with MM (33 Vighur patients, 47 Han patients) and 30 normal subjects as control by using sandwich ELISA with double antibody. The results showed that there was significant difference in serum interleukin-6 level between Vighur patients and Han patients (P < 0.01). The serum interleukin-6 level of patients with multiple myeloma was significantly higher than that of nomal control (P < 0.01). The serum interleukin-6 level of MM patients in second and third stage were also signifivantly higher than that of patients in first stage (P < 0.05). It is concluded that there was significant difference in the serum interleukin-6 level between Vighur patients and Han patients. The serum interleukin-6 level can be considered as one of indexes to judge the patient severity and the prognosis, also as one of comprehensive multiparameters to evaluate the curative efficiency and guide the clinical application of drugs.

Adult↗

PubMed2005

Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients.

Significant improvements in the outcome of non-small cell lung carcinoma (NSCLC) have been reported in patients treated with the epidermal growth factor receptor (EGFR) inhibitor, erlotinib. To discover biomarkers for the enrichment of patients who might benefit from treatment, a pharmacogenomic approach was used to identify gene signatures that may predict erlotinib activity using in vitro model systems. Erlotinib sensitivity in a panel of 42 NSCLC cell lines was determined by EGFR-mediated proliferative potential, EGFR mutations, and/or EGFR gene amplification, thus supporting an underlying biological mechanism of receptor activation. A strong multigene signature indicative of an epithelial to mesenchymal transition (EMT) was identified as a determinant of insensitivity to erlotinib through both supervised and unsupervised gene expression approaches. This observation was further supported by expression analysis of classic EMT marker proteins, including E-cadherin and vimentin. To investigate the clinical relevance of these findings, we examined expression of the epithelial marker E-cadherin by immunohistochemistry on primary tumor samples from subjects enrolled in a randomized NSCLC clinical trial in which erlotinib in combination with chemotherapy previously failed to show clinical activity. The majority (75%) of the 87 subjects tested showed strong E-cadherin staining and exhibited a significantly longer time to progression (hazard ratio, 0.37; log rank P=0.0028) and a nonsignificant trend toward longer survival with erlotinib plus chemotherapy treatment versus chemotherapy alone. These data support a potential role for EMT as a determinant of EGFR activity in NSCLC tumor cells and E-cadherin expression as a novel biomarker predicting clinical activity of the EGFR inhibitor erlotinib in NSCLC patients.

Antineoplastic Agents↗

PubMed2005

Continuous wave-based multiphoton excitation fluorescence for capillary electrophoresis.

It was reported that a novel detection method, continuous wave (CW)-based multiphoton excitation (MPE) fluorescence detection with diode laser (DL), has been firstly proposed for capillary electrophoresis (CE). Special design of end-column detection configuration proved to be superior to on-column type, considering the detection sensitivity. Three different kinds of fluorescent tags that were widely used as molecular label in bio-analysis, such as small-molecule dye, fluorescent protein and nano particle or also referred to as quantum dot (QD), have been evaluated as samples for the constructed detection scheme. Quantitative analyses were also performed using rhodamine species as tests, which revealed dynamic linear range over two orders of magnitude, with detection limit down to zeptomole-level. Simultaneous detection of fluorescent dyestuffs with divergent excitation and emission wavelengths in a broad range showed advantage of this scheme over conventional laser-induced fluorescence (LIF) detection. Further investigations on CW-MPE fluorescence detection with diode laser for capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations of fluorescein isothiocyanate (FITC) labeled amino acids indicated good prospect of this detection approach in various micro or nano-column liquid phase separation technologies.

Electrophoresis, Capillary↗

PubMed2005

Characterization of gradient-index lens-fiber spacing toward applications in two-photon fluorescence endoscopy.

We report on the experimental investigation into the characterization of two-photon fluorescence microscopy based on the separation distance of a single-mode optical fiber coupler and a gradient-index (GRIN) rod lens. The collected two-photon fluorescence signal exhibits a maximum intensity at a defined separation distance (gap length) where the increasing effective excitation numerical aperture is balanced by the decreasing confocal emission collection. A maximum signal is found at gap lengths of approximately 2, 1.25, and 1.75 mm for GRIN lenses with pitches of 0.23, 0.25, and 0.29 wavelength at 830 nm. The maximum two-photon fluorescence signal collected corresponds to a threefold reduction of axial resolution (38.5 microm at 1.25 mm), compared with the maximum resolution (11.6 microm at 5.5 mm), as shown by the three-dimensional imaging of 10 microm beads. These results demonstrate an intrinsic trade-off between signal collection and axial resolution.

Journal Article↗

PubMed2005

Cross-species vascular endothelial growth factor (VEGF)-blocking antibodies completely inhibit the growth of human tumor xenografts and measure the contribution of stromal VEGF.

To fully assess the role of VEGF-A in tumor angiogenesis, antibodies that can block all sources of vascular endothelial growth factor (VEGF) are desired. Selectively targeting tumor-derived VEGF overlooks the contribution of host stromal VEGF. Other strategies, such as targeting VEGF receptors directly or using receptor decoys, result in inhibiting not only VEGF-A but also VEGF homologues (e.g. placental growth factor, VEGF-B, and VEGF-C), which may play a role in angiogenesis. Here we report the identification of novel anti-VEGF antibodies, B20 and G6, from synthetic antibody phage libraries, which block both human and murine VEGF action in vitro. Their affinity-improved variants completely inhibit three human tumor xenografts in mice of skeletal muscle, colorectal, and pancreatic origins (A673, HM-7, and HPAC). Avastin, which only inhibits the tumor-derived human VEGF, is approximately 90% effective at inhibiting HM-7 and A673 growth but is <50% effective at inhibiting HPAC growth. Indeed, HPAC tumors contain more host stroma invasion and stroma-derived VEGF than other tumors. Thus, the functional contribution of stromal VEGF varies greatly among tumors, and systemic blockade of both tumor and stroma-derived VEGF is sufficient for inhibiting the growth of tumor xenografts.

Amino Acid Sequence↗

PubMed2005

Use of a single-mode fiber coupler for second-harmonic-generation microscopy.

We present a compact second-harmonic-generation (SHG) microscope based on a three-port single-mode fiber coupler. The fiber coupler is used to deliver a near-infrared ultrashort-pulsed laser beam as well as to collect the SHG signal in the visible wavelength range. The SHG microscope exhibits an axial resolution of 1.8 microm, which shows a slight enhancement of the optical sectioning effect compared with that for two-photon excitation at the same excitation wavelength. It is also demonstrated that SHG and two-photon fluorescence images under parallel and perpendicular laser excitation polarization can be simultaneously obtained.

Journal Article↗

PubMed2005

Plasmalemmal vesicle-associated protein (PLVAP) is expressed by tumour endothelium and is upregulated by vascular endothelial growth factor-A (VEGF).

Vascular endothelial growth factor-A (VEGF) is an important regulator of vascular permeability. In preclinical studies, VEGF induces endothelial fenestrations in pre-existing and neo-vasculature, while inhibition of VEGF leads to a reduction in endothelial fenestrations. Recently, vascular regression in response to VEGF inhibition has been shown to correlate with the presence of endothelial fenestrations. Plasmalemmal vesicle-associated protein (PLVAP) is believed to be a component of diaphragmed endothelial fenestrations, but a direct relationship with VEGF signalling has not been established. The aim of this study was to characterize the expression pattern of PLVAP and investigate whether PLVAP is a transcriptional target of VEGF signal transduction. The expression pattern of PLVAP was characterized in normal and neoplastic human tissues by in situ hybridization and/or immunohistochemistry. The role of VEGF signal transduction in the regulation of PLVAP expression was investigated in vitro using receptor-selective engineered forms of VEGF, a neutralizing monoclonal antibody against VEGF, and inhibitors of downstream signalling pathways. PLVAP mRNA and protein were widely expressed in the endothelium of normal and neoplastic tissues. In cultured endothelial cells, VEGF signalling through receptor 2 stimulated expression of PLVAP total RNA and protein. This induction could be blocked with an anti-VEGF monoclonal antibody and by inhibitors of phosphatidylinositol 3-kinase (LY294002) or p38 mitogen-activated protein kinase (SB203580), but not by PD98059, a mitogen-activated protein/extracellular signal-regulated kinase 1 inhibitor. These data show that PLVAP is more widely expressed in the vasculature of normal tissues than previously thought and that it is expressed in the vasculature of most human tumours. We suggest that PLVAP is a downstream target of VEGF signalling. This work solidifies the association between VEGF and the appearance and maintenance of fenestrations by providing a potential mechanistic link.

Carrier Proteins↗

PubMed2005

Expression of connective tissue growth factor in renal tubulointerstitial fibrosis in rats and its pathogenic role.

In order to explore the role of connective tissue growth factor (CTGF) in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction (UUO) group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor-beta1 (TGF-beta1), collagen I (col I), and plasminogen activator inhibitor-1 (PAI-1) were detected using reverse transcriptional-polymerase chain reaction (RT-PCR). Immunohistochemistry was performed to evaluate the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF-beta1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO (P < 0.01). With the progression of the disease, the mRNA expression of CTGF, col I and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index (r = 0.62, P < 0.01), the expression of TGF-beta1 (r = 0.85, P < 0.01), col I (r = 0.78, P < 0.01), and PAI-1 (r = 0.76, P < 0.01). Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course (P < 0.01, as compared with sham-operated group). It is concluded that CTGF can be induced by TGF-beta and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix (ECM) synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis.

Animals↗

PubMed2005

B-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the S2 domain of the surface spike glycoprotein.

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel strain of coronavirus. Examination of the immune responses of patients who have recovered from SARS should provide important information for design of a safe and effective vaccine. We determined the continuous viral epitopes targeted by antibodies in plasma samples from convalescent SARS patients through biopanning with a vast M13 phage display dodecapeptide library. These epitopes converged to very short peptide fragments, one on each of the structural proteins spike and nucleocapsid and the nonstructural proteins 3a, 9b, and nsp 3. Immunoassays found that most of the patients who had recovered from SARS developed complementary antibodies to the epitope-rich region on the spike S2 protein, indicating that this is an immunodominant site on the viral envelope comprising the spike, matrix, and small envelope glycoproteins. These S2-targeting antibodies were shown to effectively neutralize the coronavirus, indicating that they provided protective immunity to help the patients recover from the viral infection. These results suggest that the SARS coronavirus might have an antigenic profile distinct from those of other human or animal coronaviruses. Due to the tested safety and protective effects of the convalescent-phase serological antibodies, identification of their complementary antigens may enable the design of an epitope-based vaccine to prevent potential antibody-mediated immunopathology.

Adult↗

PubMed2005

[Expression and analysis of biological activity of the recombination anthrax edema factor].

Anthrax toxin consists of three separate proteins, protective antigen (PA), lethal factor (LF) and edema factor (EF). EF is bacterial adenylate cyclase which, upon activation by its eukaryotic cofactor, calmodulin, causes a rapid increase in the intracellular cAMP level of host cells. EF can reduce the protective ability of host animal. In order to further research the mechanism of anthrax toxin, the expression plasmid was constructed and the structural gene for anthrax edema factor (EF) was expressed in Escherichia coli. Recombinant EF (rEF) was purified to homogeneity by a three-step procedure involving metal chelating affinity chromatography, cation-exchange chromatography and gel chromatography. From 1 liter of culture, 5mg of biologically active EF was easily purified. Rabbits were immuned with rEF, anti-EF antibodies were prepared and can neutralize rEF in cells. Tests in vitro proved rEF have good biological activity. rEF can competed the binding regions of PA with rLF in J774A.1 and CHO cells. rEF and rLF can restrain each other by competition. The successful expression of rEF has placed a solid foundation for the research on toxicity mechanism of EF, and screening for inhibitors against EF.

Adenylyl Cyclases↗

PubMed2004

Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

Introns are present in some human pre-tRNAs. They are spliced out during the maturation processes of pre-tRNAs in a way that is irrelevant to their specific nucleotide sequences. This unique characteristic of tRNA splicing can be used for generation of small antisense RNAs by replacing the intron sequences with corresponding antisense sequences. In this work, the intron sequence of human pre-tRNAtyr gene was replaced with a 20 bp antisense sequence targeted to the 5' coding region of cyclin D1, a molecule that was over-expressed in many malignant proliferating cells. Under the control of U6 SnRNA promoter to further enhance transcription efficiency of the modified pre-tRNAtyr gene and subsequent antisense generation, the antisense RNA exhibited obvious suppression of cyclin D1 expression in H22 hepatoma cells. The growth of H22-transplanted tumors in mice was significantly inhibited when treated with naked plasmid DNA harboring the cyclin D1 antisense RNA generating cassette. Such tumor growth inhibition might be due to apoptosis caused by reduced cyclin D1 expression as revealed by immunohistochemical analysis of tumor samples.

Alternative Splicing↗

PubMed2004

Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference.

The severe acute respiratory syndrome (SARS) has been one of the most epidemic diseases threatening human health all over the world. Based on clinical studies, SARS-CoV (the SARS-associated coronavirus), a novel coronavirus, is reported as the pathogen responsible for the disease. To date, no effective and specific therapeutic method can be used to treat patients suffering from SARS-CoV infection. RNA interference (RNAi) is a process by which the introduced small interfering RNA (siRNA) could cause the degradation of mRNA with identical sequence specificity. The RNAi methodology has been used as a tool to silence genes in cultured cells and in animals. Recently, this technique was employed in anti-virus infections in human immunodeficiency virus and hepatitis C/B virus. In this study, RNAi technology has been applied to explore the possibility for prevention of SARS-CoV infection. We constructed specific siRNAs targeting the S gene in SARS-CoV. We demonstrated that the siRNAs could effectively and specifically inhibit gene expression of Spike protein in SARS-CoV-infected cells. Our study provided evidence that RNAi could be a tool for inhibition of SARS-CoV.

Animals↗

PubMed2004

Fibroblast growth factor 19 increases metabolic rate and reverses dietary and leptin-deficient diabetes.

Hormonal control of metabolic rate can be important in regulating the imbalance between energy intake and expenditure that underlies the development of obesity. In mice fed a high-fat diet, human fibroblast growth factor 19 (FGF19) increased metabolic rate [1.53 +/- 0.06 liters O(2)/h.kg(0.75) (vehicle) vs. 1.93 +/- 0.05 liters O(2)/h.kg(0.75) (FGF19); P < 0.001] and decreased respiratory quotient [0.82 +/- 0.01 (vehicle) vs. 0.80 +/- 0.01 (FGF19); P < 0.05]. In contrast to the vehicle-treated mice that gained weight (0.14 +/- 0.05 g/mouse.d), FGF19-treated mice lost weight (-0.13 +/- 0.03 g/mouse.d; P < 0.001) without a significant change in food intake. Furthermore, in addition to a reduction in weight gain, treatment with FGF19 prevented or reversed the diabetes that develops in mice made obese by genetic ablation of brown adipose tissue or genetic absence of leptin. To explore the mechanisms underlying the FGF19-mediated increase in metabolic rate, we profiled the FGF19-induced gene expression changes in the liver and brown fat. In brown adipose tissue, chronic exposure to FGF19 led to a gene expression profile that is consistent with activation of this tissue. We also found that FGF19 acutely increased liver expression of the leptin receptor (1.8-fold; P < 0.05) and decreased the expression of acetyl coenzyme A carboxylase 2 (0.6-fold; P < 0.05). The gene expression changes were consistent with the experimentally determined increase in fat oxidation and decrease in liver triglycerides. Thus, FGF19 is able to increase metabolic rate concurrently with an increase in fatty acid oxidation.

Acetyl-CoA Carboxylase↗