Single probe shear scanning method for on-machine measurement of an optical profile.

This study presents a new (to the best of our knowledge) error separation method with a single displacement probe, named as single probe shear scanning (SPSS) method, for the on-machine optical profile measurement to overcome the problems of the existing multiprobe method like the large deviation of probe spacing and the probes' performance difference. The confocal sensor with superior dynamic range, high lateral resolution, and large measurement angle to surface is applied in this study to fulfill the measurement of the optical aspheric surface. The single probe measurement system, in which the probe fixed on a flexure hinge is driven straight within a millimeter-level travel range, is established to realize the function of the multiprobe. For the established system, a new exact profile reconstruction algorithm is built to eliminate the influences of straightness errors of the scanning stage and the systemic errors of shear stage, and to reduce the effect of the sensor drift. The reconstruction algorithms by difference measurement with two shears are studied to build the bidirectional segment stitching reconstruction method, which reduces the error accumulation and improves the reconstruction accuracy under the condition of measuring errors. A profile reconstruction method with three shears measurement is proposed to make a further improvement on the reconstruction accuracy. The proposed reconstruction method with three shears measurement is successfully employed for the on-machine measurement of an aspheric surface profile, and the evaluation results agree well with those from the Taylor profiler.

The Effects of Transcription Directions of Transgenes and the gypsy Insulators on the Transcript Levels of Transgenes in Transgenic Arabidopsis.

Manipulation of a single abiotic stress-related gene could improve plant performance under abiotic stress conditions. To simultaneously increase plant tolerance to multiple stresses, it is usually required to overexpress two (or more) genes in transgenic plants. The common strategy is to assemble two or more expression cassettes, where each gene has its own promoter and terminator, within the same T-DNA. Does the arrangement of the two expression cassettes affect expression of the two transgenes? Can we use the Drosophila gypsy insulator sequence to increase the expression of the two transgenes? Answers to these questions would contribute to design better transformation vectors to maximize the effects of multi-gene transformation. Two Arabidopsis genes, PP2A-C5 and AVP1, and the gypsy insulator sequence were used to construct six transformation vectors with or without the gypsy insulator bracketing the two expression cassettes: uni-directional transcription, divergent transcription, and convergent transcription. Total RNAs were isolated for reverse transcription- quantitative real-time polymerase chain reaction (RT-qPCR) assays and a thorough statistical analysis was conducted for the RT-qPCR data. The results showed that the gypsy insulator does promote the expression of two transgenes in transgenic plants. Besides, the plants containing the divergent transcription cassettes tend to have more correlated expression of both genes.

Defining the enzymatic pathway for polymorphic O-glycosylation of the pneumococcal serine-rich repeat protein PsrP.

Protein O-glycosylation is an important post-translational modification in all organisms, but deciphering the specific functions of these glycans is difficult due to their structural complexity. Understanding the glycosylation of mucin-like proteins presents a particular challenge as they are modified numerous times with both the enzymes involved and the glycosylation patterns being poorly understood. Here we systematically explored the O-glycosylation pathway of a mucin-like serine-rich repeat protein PsrP from the human pathogen Streptococcus pneumoniae TIGR4. Previous works have assigned the function of 3 of the 10 glycosyltransferases thought to modify PsrP, GtfA/B, and Gtf3 as catalyzing the first two reactions to form a unified disaccharide core structure. We now use in vivo and in vitro glycosylation assays combined with hydrolytic activity assays to identify the glycosyltransferases capable of decorating this core structure in the third and fourth steps of glycosylation. Specifically, the full-length GlyE and GlyG proteins and the GlyD DUF1792 domain participate in both steps, whereas full-length GlyA and the GlyD GT8 domain catalyze only the fourth step. Incorporation of different sugars to the disaccharide core structure at multiple sites along the serine-rich repeats results in a highly polymorphic product. Furthermore, crystal structures of apo- and UDP-complexed GlyE combined with structural analyses reveal a novel Rossmann-fold "add-on" domain that we speculate to function as a universal module shared by GlyD, GlyE, and GlyA to forward the peptide acceptor from one enzyme to another. These findings define the complete glycosylation pathway of a bacterial glycoprotein and offer a testable hypothesis of how glycosyltransferase coordination facilitates glycan assembly.

Ex vivo and in vivo capture and deactivation of circulating tumor cells by dual-antibody-coated nanomaterials.

Circulating tumor cells (CTCs) have been detected by us and others in cancer patient blood. However, little is known about how to specifically capture and deactivate CTCs in vivo, which may lead to successful metastasis prevention in asymptomatic cancer survivors after surgery. We hypothesize that the dual antibody conjugates may have the advantage of capturing CTCs specifically over their single antibody counterparts. Here we show that the surface-functionalized dendrimers can be sequentially coated with two antibodies directed to surface biomarkers (EpCAM and Slex) of human colorectal CTCs. The dual antibody-coated dendrimers exhibit a significantly enhanced specificity in capturing CTCs in the presence of interfering blood cells, and in both eight-patient bloods and nude mice administered with the labeled CTCs in comparison to their single antibody-coated counterparts. The dual antibody-coated conjugates down-regulate the captured CTCs. This study provides the first conceptual evidence that two antibodies can be biocompatibly conjugated to a nanomaterial to capture and down-regulate CTCs in vivo with the enhanced specificity.

Multivalent conjugation of antibody to dendrimers for the enhanced capture and regulation on colon cancer cells.

Circulation tumor cells (CTCs) in the bloodstream of early-stage cancer patients carry the important information about valuable biomarkers and biological properties of primary tumor. However, detection and capture of CTCs are challenging owing to their low concentrations. Traditional technologies have the limited detection sensitivity and the low capture efficiency. We, herein, report an effective approach to specifically bind and capture colon cancer HT29 cells by using multiple Sialyl Lewis X antibodies (aSlex)-conjugated PAMAM dendrimers. The conjugation was characterized by using atom force microscope, UV and fluorescence measurements. The capturing and regulating HT29 cells by the aSlex-coated dendrimer conjugate were analyzed by microscopy and flow cytometry. The results indicated that the conjugate showed the enhanced capture of HT29 cells in a concentration-dependent manner and the maximum capture efficiency of 77.88% was obtained within 1 h-exposure. G6-5aSlex-FITC conjugate showed capture efficiency better than FITC-G6-COOH-5aSlex conjugate. G6-5aSlex-FITC conjugate could specifically capture HT29 cells even when the target HT29 cells were diluted with the interfering cells (e.g., RBCs) to a low concentration. The capture resulted in a concentration-dependent restraint of the cell activity. In conclusion, the aSlex-coated dendrimer conjugate displayed the great potential in capturing and restraining colorectal CTCs in blood.

Exploring cancer metastasis prevention strategy: interrupting adhesion of cancer cells to vascular endothelia of potential metastatic tissues by antibody-coated nanomaterial.

BACKGROUND: Cancer metastasis caused by circulating tumor cells (CTCs) accounts for 90% cancer-related death worldwide. Blocking the circulation of CTCs in bloodstream and their hetero-adhesion to vascular endothelia of the distant metastatic organs may prevent cancer metastasis. Nanomaterial-based intervention with adhesion between CTCs and endothelia has not been reported. Driven by the novel idea that multivalent conjugation of EpCAM and Slex antibodies to dendrimer surface may enhance the capacity and specificity of the nanomaterial conjugates for capturing and down-regulating colorectal CTCs, we conjugated the dendrimer nanomaterial with the EpCAM and Slex antibodies, and examined the capacity of the dual antibody-coated nanomaterial for their roles in interrupting CTCs-related cancer metastasis. RESULTS: The antibody-coated nanomaterial was synthesized and characterized. The conjugates specifically bound and captured colon cancer cells SW620. The conjugate inhibited the cells' viability and their adhesion to fibronectin (Fn)-coated substrate or human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. In comparison with SW480 and LoVo cell lines, the activity and adhesion of SW620 to Fn-coated substrate and HUVECs were more specifically inhibited by the dual antibody conjugate because of the higher levels of EpCAM and Slex on SW620 cell surface. The hetero-adhesion between SW620 and Fn-coated substrate, or HUVECs was inhibited by about 60-70%. The dual conjugate showed the inhibition capacity more significant than its corresponding single antibody conjugates. CONCLUSIONS: The present study provides the new evidence that coating nanomaterials with more than one antibody against CTCs may effectively interfere with the interaction between SW620 and HUVECs.

The architecture and biological function of dual antibody-coated dendrimers: enhanced control of circulating tumor cells and their hetero-adhesion to endothelial cells for metastasis prevention.

Dissemination of circulating tumor cells (CTCs) in blood and their hetero-adhesion to vascular endothelial bed of distant metastatic secondary organs are the critical steps to initiate cancer metastasis. The rarity of CTCs made their in vivo capture technically challenging. Current techniques by virtue of nanostructured scaffolds monovalently conjugated with a single antibody and/or drug seem less efficient and specific in capturing CTCs. Here, we report a novel platform developed to re-engineer nanoscale dendrimers for capturing CTCs in blood and interfering their adhesion to vascular endothelial bed to form micrometastatic foci. The nanoscale dendrimers were spatiotemporally accommodated with dual antibodies to target two surface biomarkers of colorectal CTCs. Physiochemical characterization, including spectra, fluorescence, electron microscope, dynamic light scattering, electrophoresis, and chromatography analyses, was conducted to demonstrate the successful conjugation of dual antibodies to dendrimer surface. The dual antibody conjugates were able to specifically recognize and bind CTCs, moderately down-regulate the activity of the captured CTCs by arresting them in S phase. The related adhesion assay displayed that the dual antibody conjugates interfered the hetero-adhesion of CTCs to fibronectin (Fn)-coated substrates and human umbilical vein endothelial cells (HUVECs). The dual antibody conjugates also showed the enhanced specificity and efficiency in vitro and in vivo in restraining CTCs in comparison with their single antibody counterparts. The present study showed a novel means to effectively prevent cancer metastatic initiation by binding, restraining CTCs and inhibiting their hetero-adhesion to blood vessels, not by traditional cytotoxic-killing of cancer cells.

Nanotechnology-based intelligent drug design for cancer metastasis treatment.

Traditional chemotherapy used today at clinics is mainly inherited from the thinking and designs made four decades ago when the Cancer War was declared. The potency of those chemotherapy drugs on in-vitro cancer cells is clearly demonstrated at even nanomolar levels. However, due to their non-specific effects in the body on normal tissues, these drugs cause toxicity, deteriorate patient's life quality, weaken the host immunosurveillance system, and result in an irreversible damage to human's own recovery power. Owing to their unique physical and biological properties, nanotechnology-based chemotherapies seem to have an ability to specifically and safely reach tumor foci with enhanced efficacy and low toxicity. Herein, we comprehensively examine the current nanotechnology-based pharmaceutical platforms and strategies for intelligent design of new nanomedicines based on targeted drug delivery system (TDDS) for cancer metastasis treatment, analyze the pros and cons of nanomedicines versus traditional chemotherapy, and evaluate the importance that nanomaterials can bring in to significantly improve cancer metastasis treatment.

Distribution of CCR5-Delta32, CCR5m303A, CCR2-64I and SDF1-3'A in HIV-1 infected and uninfected high-risk Uighurs in Xinjiang, China.

BACKGROUND: Genetic variants of the genes encoding HIV-1 co-receptors and their ligands, CCR5-Delta32, CCR5m303A, CCR2-64I and SDF1-3'A, are implicated in the susceptibility to HIV-1 infection, and the prevalence of these mutations varies by ethnicity. However, little is known about their distribution in Uighurs. OBJECTIVES: This study aimed at characterizing the frequency of these HIV-related gene variants in a high-risk Uighur population. STUDY DESIGNS: A total of 251 HIV-1 seropositive and 238 seronegative high-risk Uighurs were recruited and their genotypes of CCR5-Delta32, CCR5m303A, CCR2-64I and SDF1-3'A were analyzed by PCR and PCR-ligase detection reaction (PCR-LDR). RESULTS: The allelic frequency of CCR5-Delta32, CCR5m303A, CCR2-64I and SDF1-3'A was 4.40%, 2.66%, 25.66% and 57.36%, respectively, in this population. Apparently, the Uighur population has low frequency of CCR5-Delta32 and CCR5m303A, but high frequency of CCR2-64I and SDF1-3'A. While there was no significant difference in the frequency of CCR5-Delta32, CCR2-64I and SDF1-3' A between HIV-1 seropositive and seronegative groups the frequency of CCR5m303A in HIV-1 seropositive group was significantly higher than that in seronegative group (P=0.006, OR=3.982 and 95%CI 1.514-10.476). CONCLUSIONS: Our data suggest that the CCR5-Delta32, CCR2-64I and SDF1-3'A variants may have limited effect on protecting from HIV-1 infection in Uighurs. Rather, the CCR5m303A may be associated with the risk for HIV-1 infection in high-risk Uighurs.

[Effect of several penetration enharcers on transdermal permeation and skin accumulation of artemether].

OBJECTIVE: To investigate penetration characteristics of artemether and the effect of different permeation enhancer on transdermal permeation of artemether through rat skin. METHOD: The permeation experiments were performed using rat skin on modified Franz diffusion cells in vitro. The concentrations of artemether in receptor compartment at specified time points were determined by HPLC. RESULT: The permeating ratio through human skin of artemether solution was Js (2.78 +/- 0.78) microg x cm(-2) x h(-1), the quantity of drug penetrated through and accumulated in the skin by the end of the experiment were (69.07 +/- 3.01) microg x cm(-2), (58.93 +/- 3.56) microg x cm(-2) respectively. Four different permeation enhancers can improve the transdermal permeation of artemether. CONCLUSION: Artemether have the potential to be developed to new transdermal preparation.

Dual agents loaded PLGA nanoparticles: systematic study of particle size and drug entrapment efficiency.

PLGA nanoparticles simultaneously loaded with vincristine sulfate (VCR) and quercetin (QC) were prepared via O/W emulsion solvent evaporation. Six independent processing parameters and PLGA characteristics were assessed systematically to enhance the incorporation of the dual agents with different properties (VCR and QC, hydrophilic and hydrophobic molecule, respectively) into PLGA nanoparticles and control particle size. Approaches investigated for the enhancement of drug entrapment efficiencies and the controlling of particle size included the influence of the molecular weight (MW) of PLGA and the lactide-to-glycolide (L:G) ratio of PLGA, PLGA concentration, PVA concentration, initial QC content, acetone-to-dichloromethane (A/D) volume ratio, aqueous phase pH and aqueous to organic phase (W/O) volume ratio. The nanoparticles produced by optimal formulation were submicron size (139.5+/-4.3 nm, n=3) with low polydispersity index (0.095+/-0.031, n=3). Nanoparticles observed by transmission electron microscopy (TEM) showed extremely spherical shape. The entrapment efficiencies determined by high performance liquid chromatography (HPLC) by ultracentrifuge method were 92.84+/-3.37% for VCR and 32.66+/-2.92% for QC (n=3). The drug loadings were 0.0037+/-0.0001% for VCR and 1.36+/-0.12% for QC (n=3).

[Comparison of kinetic behavior in both plasma and tissue after intravenous administration of alpha-asarone in lipid emulsion and aqueous solution in rats and mice].

OBJECTIVE: To compare the pharmacokinetics and tissue distribution of alpha-asarone in lipid emulsion and aqueous solution for injection and study the feasibility of lipid emulsion of alpha-asarone as the parenteral drug delivery system. METHOD: HPLC was used to determine the drug concentration in rat plasma and mice tissues after intravenous (i.v.) administration of lipid emulsion and aqueous solution of alpha-asarone at a single dose (40 mg x kg(-1)), respectively. RESULT: The plasma concentration-time profiles of lipid emulsion and aqueous solution of alpha-asarone after intravenous administration of them are similar and the drug concentration-time data were fitted to a two-compartment open model. The results of tissues distribution showed that distribution contents of alpha-asarone from lipid emulsion and aqueous solution in vivo are similar in lungs but lipid emulsion increased the uptake in livers and spleens, and decreased the uptake in hearts and kidneys for alpha-asarone. CONCLUSION: The plasma concentration-time profiles of alpha-asarone in lipid emulsion and aqueous solution are similar, but lipid emulsion significantly altered the tissue distribution of alpha-asarone, which may be beneficial to decrease its potential toxicity to heart and kidney.