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Conventional liquid crystal (LC)-based biosensors utilize predominantly thermotropic LCs as the signal-transducing media, which are less environmentally sustainable compared with lyotropic counterparts. In this study, the nematic phase of the anionic azo dye sunset yellow (SSY), a type of lyotropic chromonic liquid crystals (LCLCs), was employed in the optical and electrical biosensing of bovine serum albumin (BSA) and the cancer biomarker CA125. The optical response observed under a polarizing optical microscope was quantified by image analysis, taking advantage of the specific absorption of SSY. The electrical response derived from the dielectric spectra of SSY provided a new alternative for quantitative bioassay based on nematic LCLCs. The limit of detection (LOD) of the optical and electrical protein assay was ∼10

Mutations of SPINT2, the gene encoding the integral membrane, Kunitz-type serine inhibitor HAI-2, primarily affect the intestine, while sparing many other HAI-2-expressing tissues, causing sodium loss in patients with syndromic congenital sodium diarrhea. The membrane-bound serine protease prostasin was previously identified as a HAI-2 target protease in intestinal tissues but not in the skin. In both tissues, the highly related inhibitor HAI-1 is, however, the default inhibitor for prostasin and the type 2 transmembrane serine protease matriptase. This cell-type selective functional linkage may contribute to the organ-selective damage associated with SPINT 2 mutations. To this end, the impact of HAI-2 deletion on matriptase and prostasin proteolysis was, here, compared using Caco-2 human colorectal adenocarcinoma cells and HaCaT human keratinocytes. Greatly enhanced prostasin proteolytic activity with a prolonged half-life and significant depletion of HAI-1 monomer were observed with HAI-2 loss in Caco-2 cells but not HaCaT cells. The constitutive, high level prostasin zymogen activation observed in Caco-2 cells, but not in HaCaT cells, also contributes to the excessive prostasin proteolytic activity caused by HAI-2 loss. HAI-2 deletion also caused increased matriptase zymogen activation, likely as an indirect result of increased prostasin proteolysis. This increase in activated matriptase, however, only had a negligible role in depletion of HAI-1 monomer. Our study suggests that the constitutive, high level of prostasin zymogen activation and the cell-type selective functional relationship between HAI-2 and prostasin renders Caco-2 cells more susceptible than HaCaT cells to the loss of HAI-2, causing a severe imbalance favoring prostasin proteolysis.

The pathophysiological functions of matriptase, a type 2 transmembrane serine protease, rely primarily on its enzymatic activity, which is under tight control through multiple mechanisms. Among those regulatory mechanisms, the control of zymogen activation is arguably the most important. Matriptase zymogen activation not only generates the mature active enzyme but also initiates suppressive mechanisms, such as rapid inhibition by HAI-1, and matriptase shedding. These tightly coupled events allow the potent matriptase tryptic activity to fulfill its biological functions at the same time as limiting undesired hazards. Matriptase is converted to the active enzyme via a process of autoactivation, in which the activational cleavage is thought to rely on the interactions of matriptase zymogen molecules and other as yet identified proteins. Matriptase autoactivation can occur spontaneously and is rapidly followed by the formation and then shedding of matriptase-HAI-1 complexes, resulting in the presence of relatively low levels of the complex on cells. Activation can also be induced by several non-protease factors, such as the exposure of cells to a mildly acidic buffer, which rapidly causes high-level matriptase zymogen activation in almost all cell lines tested. In the current study, the structural requirements for this acid-induced zymogen activation are compared with those required for spontaneous activation through a systematic analysis of the impact of 18 different mutations in various structural domains and motifs on matriptase zymogen activation. Our study reveals that both acid-induced matriptase activation and spontaneous activation depend on the maintenance of the structural integrity of the serine protease domain, non-catalytic domains, and posttranslational modifications. The common requirements of both modes of activation suggest that acid-induced matriptase activation may function as a physiological mechanism to induce pericellular proteolysis by accelerating matriptase autoactivation.

Our research was designed to evaluate the effect on bone regeneration with 3-dimensional (3D) printed polylactic acid (PLA) and 3D printed polycaprolactone (PCL) scaffolds, determine the more effective option for enhancing bone regeneration, and offer tentative evidence for further research and clinical application. Employing the 3D printing technique, the PLA and PCL scaffolds showed similar morphologies, as confirmed via scanning electron microscopy (SEM). Mechanical strength was significantly higher in the PLA group (63.4 MPa) than in the PCL group (29.1 MPa) (p &lt, 0.01). Average porosity, swelling ratio, and degeneration rate in the PCL scaffold were higher than those in the PLA scaffold. SEM observation after cell coculture showed improved cell attachment and activity in the PCL scaffolds. A functional study revealed the best outcome in the 3D printed PCL-TGF-β1 scaffold compared with the 3D printed PCL and the 3D printed PCL-Polydopamine (PDA) scaffold (p &lt, 0.001). As confirmed via SEM, the 3D printed PCL- transforming growth factor beta 1 (TGF-β1) scaffold also exhibited improved cell adhesion after 6 h of cell coculture. The 3D printed PCL scaffold showed better physical properties and biocompatibility than the 3D printed PLA scaffold. Based on the data of TGF-β1, this study confirms that the 3D printed PCL scaffold may offer stronger osteogenesis.

A liquid crystal (LC)-based single-substrate biosensor was developed by spin-coating an LC thin film on a dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAP)-decorated glass slide. Compared with the conventional sandwiched cell configuration, the simplified procedure for the preparation of an LC film allows the film thickness to be precisely controlled by adjusting the spin rate, thus eliminating personal errors involved in LC cell assembly. The limit of detection (LOD) for bovine serum albumin (BSA) was lowered from 10−5 g/mL with a 4.2-μm-thick sandwiched cell of the commercial LC E7 to 10−7 g/mL with a 4.2-μm-thick spin-coated E7 film and further to 10−8 g/mL by reducing the E7 film thickness to 3.4 μm. Moreover, by exploiting the LC film of the highly birefringent nematic LC HDN in the immunodetection of the cancer biomarker CA125, an LOD comparable to that determined with a sandwiched HDN cell was achieved at 10−8 g/mL CA125 using a capture antibody concentration an order of magnitude lower than that in the LC cell. Our results suggest that employing spin-coated LC film instead of conventional sandwiched LC cell provides a more reliable, reproducible, and cost-effective single-substrate platform, allowing simple fabrication of an LC-based biosensor for sensitive and label-free protein detection and immunoassay.

Background Previously, we found that (−)-epigallocatechin-3-gallate (EGCG) enhanced osteogenic differentiation of murine bone marrow mesenchymal stem cells by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and eventually mineralization. We further found EGCG supplementation preserved bone mass and microarchitecture in female rats during estrogen deficiency in the proximal tibia and lumbar spine at least in part by increasing bone morphogenetic protein-2 (BMP2). BMP2 can enhance de novo bone formation. Purpose In this study, we evaluate the effect of local EGCG application in de novo bone formation in bone defect healing. Methods Twenty-four rats aged 4 months were weight-matched and randomly allocated to 2 groups: defect control with vehicle treatment (control) and defect with 10 µM EGCG treatment (EGCG). Daily vehicle and EGCG were applied locally by percutaneous local injection 2 days after defect creation for 2 weeks. Four weeks after treatment, animals were sacrificed for micro-computed tomography (μ-CT) and biomechanical analysis. Results Local EGCG at femoral defect can enhance de novo bone formation by increasing bone volume and subsequently improve mechanical properties including max load, break point, stiffness, area under the max load curve, area under the break point curve and ultimate stress. Conclusions Local EGCG may enhance bone defect healing via at least partly by the de novo bone formation of BMP-2.

A dual-frequency-liquid-crystal (DFLC)-based biosensor was developed and its frequency-dependent dielectric properties were exploited to detect and quantitate a protein standard, bovine serum albumin (BSA). By analyzing the dielectric spectra of DFLC in the presence of BSA at various concentrations, we found that the difference in dielectric permittivity between the high- and low-frequency regimes is correlated to BSA concentration, thereby permitting a DFLC-based protein quantitative method. The dielectric properties of DFLCs are fundamental in the design of liquid crystal displays and fast-switching devices. Results from this study demonstrate the extended potential of the frequency-revertible dielectric anisotropy of DFLC in biosensing and protein quantitation.

Label-free qualitative and quantitative protein assays were established with a single-substrate biosensing platform based on cholesteric liquid crystal (CLC) films with different ratios of the thickness d to the helical pitch P. By adjusting the amount of chiral dopant incorporated in CLC, the d/P ratio or the center wavelength of Bragg’s reflection band can be fine-tuned to achieve signal amplification or to improve detection sensitivity. The single-substrate platform eliminates the need for LC cell assembly, which requires a pair of glass substrates, and can be conveniently integrated with the format of most clinical assays performed on a single solid surface.

Human bone marrow stem cells (HBMSCs) are isolated from the bone marrow. Stem cells can self-renew and differentiate into various types of cells. They are able to regenerate kinds of tissue that are potentially used for tissue engineering. To maintain and expand these cells under culture conditions is difficult—they are easily triggered for differentiation or death. In this study, we describe a new culture formula to culture isolated HBMSCs. This new formula was modified from NCDB 153, a medium with low calcium, supplied with 5% FBS, extra growth factor added to it, and supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate to maintain the cells in a steady stage. The cells retain these characteristics as primarily isolated HBMSCs. Moreover, our new formula keeps HBMSCs with high proliferation rate and multiple linage differentiation ability, such as osteoblastogenesis, chondrogenesis, and adipogenesis. It also retains HBMSCs with stable chromosome, DNA, telomere length, and telomerase activity, even after long-term culture. Senescence can be minimized under this new formulation and carcinogenesis of stem cells can also be prevented. These modifications greatly enhance the survival rate, growth rate, and basal characteristics of isolated HBMSCs, which will be very helpful in stem cell research.

The pathophysiological functions of matriptase, a type 2 transmembrane serine protease, rely primarily on its enzymatic activity, which is under tight control through multiple mechanisms. Among those regulatory mechanisms, the control of zymogen activation is arguably the most important. Matriptase zymogen activation not only generates the mature active enzyme but also initiates suppressive mechanisms, such as rapid inhibition by HAI-1, and matriptase shedding. These tightly coupled events allow the potent matriptase tryptic activity to fulfill its biological functions at the same time as limiting undesired hazards. Matriptase is converted to the active enzyme via a process of autoactivation, in which the activational cleavage is thought to rely on the interactions of matriptase zymogen molecules and other as yet identified proteins. Matriptase autoactivation can occur spontaneously and is rapidly followed by the formation and then shedding of matriptase-HAI-1 complexes, resulting in the presence of relatively low levels of the complex on cells. Activation can also be induced by several non-protease factors, such as the exposure of cells to a mildly acidic buffer, which rapidly causes high-level matriptase zymogen activation in almost all cell lines tested. In the current study, the structural requirements for this acid-induced zymogen activation are compared with those required for spontaneous activation through a systematic analysis of the impact of 18 different mutations in various structural domains and motifs on matriptase zymogen activation. Our study reveals that both acid-induced matriptase activation and spontaneous activation depend on the maintenance of the structural integrity of the serine protease domain, non-catalytic domains, and posttranslational modifications. The common requirements of both modes of activation suggest that acid-induced matriptase activation may function as a physiological mechanism to induce pericellular proteolysis by accelerating matriptase autoactivation.

Conventional liquid crystal (LC)-based biosensing at the LC–glass interface requires the assembly of an LC cell formed by two glass substrates with an LC film sandwiched in between. As most biochemical and clinical assays are performed on a single solid substrate, the feasibility of a single-substrate biodetection platform based on a thin film of LC–photopolymer composite was explored in this study. The LC mixture, consisting of nematic LC, E7 or AY40-006, doped with a small amount (≤ 5 wt%) of a photocurable prepolymer was spin-coated on a glass substrate modified with dimethyloctadecyl[3-trimethoxysilyl)propyl] ammonium chloride (DMOAP), a vertical alignment reagent, followed by irradiation with ultraviolet (UV) light. During the photopolymerization process, the accumulated and polymerized NOA65 at the LC–glass interface weakened the anchoring strength of DMOAP, resulting in a decrease in the pretilt angle of LC and allowing the LC molecules to be more easily disturbed in the presence of biomolecules, compared with vertically aligned LC in the absence of polymerized NOA65. Incorporating NOA65 in the LC film therefore provides a means for signal amplification. When an LC–photopolymer composite film consisting of AY40-006 and 4-wt% NOA65 was exposed to UV at 15 mW/cm2 for 30 s and utilized as the biosensing mesogen, the limits of detection were 1.6 × 10−12 g/ml for the direct detection of bovine serum albumin (BSA) and 2.1 × 10−8 g/ml for the immunoassay of the cancer biomarker CA125, significantly lower than those detected with AY40-006 alone or AY40-006/NOA65 mixture without UV irradiation. The results from this study offer a compelling implication on the biomedical application of LC–photopolymer composites in label-free and single-substrate biodetection.

A quantitative protein assay and label-free immunodetection based on dye-doped liquid crystal (DDLC) was developed in this study. The black dye incorporated as the dopant in the DDLC mixture features a high dichroic ratio and wide visible-light absorption band so that the level of disturbance in DDLC orientation in the presence of biomolecules can be readily observed with the naked eye. Due to the absorption and anisotropic features of DDLC, transmission spectrometry was exploited to establish a quantitative method for DDLC-based biosensing. Our results indicate that the transmittance of DDLC decreased with increasing concentration of bovine serum albumin (BSA), a common protein standard, and the detection sensitivity can be improved by using linearly polarized light during spectral measurement. A linear correlation between the spectral parameters of DDLC and BSA concentration was thus derived to facilitate protein quantitation. As a demonstration of clinical relevance, a label-free DDLC-based immunoassay for the cancer biomarker CA125 was manifested by reacting CA125 with immobilized anti-CA125 antibody. The transmittance of DDLC decreased with increasing concentration of CA125, but when a nonspecific protein such as BSA was reacted with the anti-CA125 antibody, the transmission spectrum was similar to that in the absence of CA125, suggesting that immunodetection via the DDLC-based biosensing platform was specific. It is evident from this study that with its unique dichroic and optical properties, DDLC is a potential biosensing material with capabilities of both qualitative detection as required in rapid screening as well as quantitative bioassay as in cancer screening.

Dye liquid crystals (DLCs) have been extensively investigated in the field of liquid crystal display, but their potential in biosensing has not been explored. To provide insights into the potential of DLCs as a means of biosensing material, a DLC-based biosensor was developed for protein detection and quantitation. Unlike dye-doped LCs, the DLC used in this study contains an azobenzene LC molecule chemically modified with two azo groups as chromophores, enabling the DLC to exhibit unique optical anisotropy and dichroic absorption at 470 nm, which were exploited in the quantitation of bovine serum albumin (BSA). The change in orientation of DLC molecules from the homeotropic to a disrupted state by BSA immobilized on the aligning-layer surface was evaluated in terms of the relative loss in transmittance, expressed by the standard parameter S%, due to the absorption as well as scattering of light. The DLC-based protein assay was capable of determining the BSA concentration, with which S% varied linearly between 1 μg/ml and 7.5 μg/ml. When the DLC molecules were reoriented from a BSA-disrupted state to the homeotropic alignment by an externally applied electric field, a linear correlation between S% and BSA concentration could be also derived from the voltage–transmittance plots. The unique electro-optical properties of the intrinsically dichroic DLC are promising toward the development of a simple and reliable sensing platform for the quantitative analysis of biomolecules.

Azobenzene-doped liquid crystals (ADLCs) have found widespread applications in fields such as holographic technology, photoactuators, and smart windows, but their potential in biosensing has not been explored. In this study, ADLCs were prepared by doping the nematic liquid crystal (LC) host E7 with a photochromic azobenzene dye ChAD-3C-S, which, upon exposure to ultraviolet (UV) light, underwent trans - cis photoisomerization and induced the phase transition of ADLCs from the cholesteric to the nematic state. The isomerization and phase transition are reversed when the nematic ADLCs are irradiated with green light. A biosensing platform based on the photoresponsive ADLCs for the detection and quantitation of bovine serum albumin (BSA) at the LC-glass interface was constructed by assembling a sandwiched LC cell with a pair of glass substrates modified with the homeotropic alignment reagent dimethyloctadecyl[3-(trimethoxysilyl)propyl] ammonium chloride (DMOAP). By immobilizing BSA on one of the DMOAP-coated glass substrates, the concentration-dependent effect of the biomolecule on the physical properties of ADLCs was analyzed quantitatively through multiple modes of detection including optical texture observation under a polarizing optical microscope as well as transmission spectrometry and capacitance measurements. In the photocontrolled ADLC-based capacitive biosensor, BSA was quantitated by measuring the time-evolved capacitance of an ADLC during the trans-cis-trans isomerization of ChAD-3C-S by alternating the wavelength of incident light, which improved the limit of detection (LOD) to 8.8 × 10−3 μg/ml, an order of magnitude lower than that obtained by spectrometric analysis. Results from this study suggest that the photocontrollable and fast-response features unique to ADLCs may create new possibilities for the development of innovative LC-based biosensing technologies.

The fundamental principle of liquid-crystal (LC)-based biosensing is the sensitive response of LC orientation to external stimuli. Biomolecules such as proteins or DNAs immobilized on the glass substrate of a LC cell are detected through disrupting the LC alignment and, in turn, altering the birefringence, resulting in changes in the optical texture that can be readily observed under a polarizing optical microscope. With an additional weak electric field across a sandwiched LC cell, we demonstrate in this study a novel label-free biodetection technique with amplified signal and improved detection limit. By applying the binarization analysis as the quantitative approach, the increase in the light leakage area in the optical texture of LCs with increasing amount of biomolecules can be quantitated with a bright-area-ratio (BAR)-versus-concentration curve. The reported biosensing technique exploits both the optical and electrical properties of LCs and is potentially applicable to other LC-based rapid screening and bioassays.

A novel aptamer-based competitive drug screening platform for osteoporosis was devised in which fluorescence-labeled, sclerostin-specific aptamers compete with compounds from selected chemical libraries for the binding of immobilized recombinant human sclerostin to achieve high-throughput screening for potential small-molecule sclerostin inhibitors and to facilitate drug repurposing and drug discovery. Of the 96 selected inhibitors and FDA-approved drugs, six were shown to result in a significant decrease in the fluorescence intensity of the aptamer, suggesting a higher affinity toward sclerostin compared with that of the aptamer. The targets of these potential sclerostin inhibitors were correlated to lipid or bone metabolism, and several of the compounds have already been shown to be potential osteogenic activators, indicating that the aptamer-based competitive drug screening assay offered a potentially reliable strategy for the discovery of target-specific new drugs. The six potential sclerostin inhibitors suppressed the level of both intracellular and/or extracellular sclerostin in mouse osteocyte IDG-SW3 and increased alkaline phosphatase activity in IDG-SW3 cells, human bone marrow-derived mesenchymal stem cells and human fetal osteoblasts hFOB1.19. Potential small-molecule drug candidates obtained in this study are expected to provide new therapeutics for osteoporosis as well as insights into the structure–activity relationship of sclerostin inhibitors for rational drug design.

Cholesteric liquid crystal (CLC) is characterized by the selective reflection in the Bragg condition for constructive interference. Instead of assembling a LC cell with a pair of glass substrates, only a single glass substrate modified with vertical alignment reagent and layered with a CLC film of 3.4 ± 0.2 μm in thickness was utilized in this study for the detection of bovine serum albumin (BSA). By fine-tuning the ratio of the thickness d to the helical pitch P of the CLC film, both qualitative and quantitative biosensing capabilities were demonstrated with an E7/R811 CLC (d/P close to but smaller than unity) and an E7/R5011 CLC (d/P > > 1) film, respectively. The E7/R811 film was in the unwound homeotropic state in the absence of BSA, but transitioned to the fingerprint state at 10−9 to 10−6-g/ml BSA and to the planar state between 10−5 and 10−2-g/ml BSA. Quantitative protein assay based on E7/R5011 CLCs was established through transmission spectrometric analysis, and detection sensitivity was optimized by adjusting the Bragg reflection wavelength. A limit of detection of 5.9 × 10−9 g/ml was achieved for the CLC film consisting of 2.0-wt% R5011 in the nematic host E7 and exhibiting a Bragg reflection wavelength of 700 nm. As a majority of clinical assays are performed on a single solid substrate, the CLC-based, single-substrate biosensing platform allows for better integration with current technologies. Besides, the protein array employed in this work can be further miniaturized and converted to a protein microarray format to facilitate high-throughput analysis.

Cyclophosphamide is indicated for the treatment of cancerous diseases such as breast cancer and cervical cancer. Recent studies have shown that cyclophosphamide may induce cancer metastasis, but the cause of this unexpected adverse effect is not fully understood. In this study, we investigate the effect of cyclophosphamide on cancer cell migration and its correlation to chemokine (C-X-C motif) receptor 4 (CXCR4), a biomarker for cancer metastasis. Two human cancer cell lines with significant difference in endogenous CXCR4 expression, the breast cancer cell line, MDA-MB-231, and the melanoma cell line, MDA-MB-435S, were treated with various concentrations of cyclophosphamide, followed by the assessment of CXCR4 expression and cell migration. We found that the migration ability of MDA-MB-231 cells was enhanced with increasing concentrations of cyclophosphamide, which induced the cell-surface expression of CXCR4, but had no effect on the overall amount of CXCR4. In MDA-MB-435S cells, in which CXCR4 was barely detectable, cyclophosphamide was unable to activate cell-surface CXCR4, and did not promote cell migration. Studies on the mRNA expression profile of matrix metalloproteinases (MMPs) in MDA-MB-231 cells further indicate that MMP9 and MMP13 may be involved in the action of cyclophosphamide. The protein expression of both MMP9 and MMP13 was increased in the presence of cyclophosphamide. Results from this study provide the molecular basis for the possible pathway of cyclophosphamide to induce cancer metastasis.

Liquid crystals (LCs) have been suggested to have a place in biological sensing for detection and quantitation of biomolecules. Through many years, texture observation has long been the core technique in LC-based bioassays. Its principle stems from the texture change of LCs due to the interruption of the initially homeotropic alignment in nematic bulks or the radial-to-bipolar configuration transition in LC droplets in the presence of biomolecules. Biodetection through optical texture observation is convenient but remains a qualitative method, and alternative approaches of detection and quantitative analysis are necessary for the development of a practical LC-based biosensor. In this study, we explore the potential of two dye-enhanced LCs, a dye LC (DLC) and a dye-doped LC (DDLC), in biosensing and protein quantitation. Depending on the chromophore or dye incorporated in DLC and DDLC, the dichroic features of these LCs enable us to analyze the change in their orientation in the presence of biomolecules by transmission spectrometry, from which spectral parameters are derived to establish novel LC-based protein quantitative methods.

In comparison with the conventional approach entailing sandwiched cells for LC-based biosensing, here we demonstrate a simpler platform for label-free biodetection. This unique LC- on-single-substrate technique is capable of rapid screening and bioassay of DNA hybridization.

Polyamines are naturally occurring organic polycations that are ubiquitous in all organisms, and are essential for cell proliferation and differentiation. Although polyamines are involved in various cellular processes, their roles in stem cell differentiation are relatively unexplored. In this study, we found that exogenous polyamines, putrescine, spermidine, and spermine, promoted osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) without inducing cell death or apoptosis. Alkaline phosphatase (ALP) activity and the mRNA level of osteogenic genes, including Runx2, ALP, osteopontin, and osteocalcin, were up-regulated by exogenous polyamines. When hBMSCs were cultured at high cell density favoring adipocyte formation, exogenous polyamines resulted in down-regulation of adipogenic genes such as PPARγ, aP2, and adipsin. Extracellular matrix mineralization, a marker for osteoblast maturation, was enhanced in the presence of exogenous polyamines, while lipid accumulation, an indication of adipogenic differentiation, was attenuated. Exogenous polyamines increased the mRNA expression of polyamine-modulated factor 1 (PMF-1) and its downstream effector, spermidine/spermine N(1)-acetyltransferase (SSAT), while that of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was suppressed. These results lead to possible connections between polyamine metabolism and osteogenic differentiation pathways. To summarize, this study provides evidence for the involvement of polyamines in osteogenic differentiation of hBMSCs, and is the first to demonstrate that osteogenic and adipogenic differentiation are reciprocally regulated by exogenous polyamines.

Elevated cathepsin S (Cat S) level is correlated with higher migration ability in tumor cells. This study investigates the inhibitory effect of novel synthetic α-ketoamide compounds on cathepsin activity and cancer cell migration. The effect of several α-ketoamide compounds on the activity of recombinant cathepsins (Cat S, Cat L and Cat K) was examined. Two highly metastatic cancer cell lines were incubated with three Cat S-specific compounds (6n, 6 w and 6r) to analyze their effect on cellular Cat S activity and cell migration. At a 100 nM concentration, compounds 6n, 6r and 6 w effectively inhibited Cat S activity. Cat S activity and cell migration were significantly reduced in CL1-3 cells after treatment with either 6n or 6 w at 5 μM. Similar results were also obtained when A2058 cells were treated with 6n. These results highlight the therapeutic potential of α-ketoamide compounds, especially 6n and 6 w, to prevent or delay cancer metastasis.

Peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis, and has been indicated as a potential therapeutic target to promote osteoblast differentiation. However, recent studies suggest that suppression of PPARγ inhibits adipogenesis, but does not promote osteogenic differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs). It was reasoned that the osteogenic effect of PPARγ suppression may be masked by the strong osteogenesis-inducing condition commonly used, resulting in a high degree of matrix mineralization in both control and experimental groups. This study investigates the role of PPARγ in the lineage commitment of human adipose-derived mesenchymal stem cells (hADSCs) by interfering with the function of PPARγ mRNA through small interfering RNAs (siRNAs) specific for PPARγ2. By applying an osteogenic induction condition less potent than that used conventionally, we found that PPARγ silencing led to retardation of adipogenesis and stimulated a higher level of matrix mineralization. The mRNA level of PPARγ decreased to 47% of control 2 days after treatment with 50 nmol/l PPARγ2 siRNA, while its protein expression was 60% of mock control. In the meantime, osteogenic marker genes, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OC), were up-regulated under PPARγ silencing. Our results suggest that transient suppression of PPARγ promotes the onset of osteogenesis, and may be considered a new strategy to stimulate bone formation in bone tissue engineering using hADSCs.

The texture observation has long been the core technique in liquid crystal (LC)-based bioassays. Its working principle stems from the dark-to-bright texture change induced by the interruption of the initially homeotropic alignment in nematic bulks or from the radial-to-bipolar configuration transition in LC droplets in the presence of biomolecules. One of the drawbacks of this observational scheme, which requires a polarizing optical microscope, is the difficulty in quantitative analysis. In this invited paper, we report on our recent development of alternative optical biosensing techniques based on cholesteric LCs (CLCs) without the use of a polarizer. The increase in structural order in a vertically anchored CLC cell in the quasi-planar state provides a means to allow detection and quantification of the concentration of biomolecules immobilized on the interface between the mesophase and the surfactant DMOAP for LC vertical alignment.

Cyclophosphamide is indicated for the treatment of cancerous diseases such as breast cancer and cervical cancer. Recent studies have shown that cyclophosphamide may induce cancer metastasis, but the cause of this unexpected adverse effect is not fully understood. In this study, we investigate the effect of cyclophosphamide on cancer cell migration and its correlation to chemokine (C-X-C motif) receptor 4 (CXCR4), a biomarker for cancer metastasis. Two human cancer cell lines with significant difference in endogenous CXCR4 expression, the breast cancer cell line, MDA-MB-231, and the melanoma cell line, MDA-MB-435S, were treated with various concentrations of cyclophosphamide, followed by the assessment of CXCR4 expression and cell migration. We found that the migration ability of MDA-MB-231 cells was enhanced with increasing concentrations of cyclophosphamide, which induced the cell-surface expression of CXCR4, but had no effect on the overall amount of CXCR4. In MDA-MB-435S cells, in which CXCR4 was barely detectable, cyclophosphamide was unable to activate cell-surface CXCR4, and did not promote cell migration. Studies on the mRNA expression profile of matrix metalloproteinases (MMPs) in MDA-MB-231 cells further indicate that MMP9 and MMP13 may be involved in the action of cyclophosphamide. The protein expression of both MMP9 and MMP13 was increased in the presence of cyclophosphamide. Results from this study provide the molecular basis for the possible pathway of cyclophosphamide to induce cancer metastasis.

The carbon-based nanomaterial graphene can be chemically modified to associate with various molecules such as chemicals and biomolecules and developed as novel carriers for drug and gene delivery. In this study, a nonviral gene transfection reagent was produced by functionalizing graphene oxide (GO) with a polycationic polymer, polyethylenimine (PEI), to increase the biocompatibility of GO and to transfect small interfering RNA (siRNA) against C-X-C chemokine receptor type 4 (CXCR4), a biomarker associated with cancer metastasis, into invasive breast cancer cells. PEI-functionalized GO (PEI-GO) was a homogeneous aqueous solution that remained in suspension during storage at 4 °C for at least 6 months. The particle size of PEI-GO was 172 ± 4.58 and 188 ± 5.00 nm at 4 and 25 °C, respectively, and increased slightly to 262 ± 17.6 nm at 37 °C, but remained unaltered with time. Binding affinity of PEI-GO toward siRNA was assessed by electrophoretic mobility shift assay (EMSA), in which PEI-GO and siRNA were completely associated at a PEI-GO:siRNA weight ratio of 2:1 and above. The invasive breast cancer cell line, MDA-MB-231, was transfected with PEI-GO in complex with siRNAs against CXCR4 (siCXCR4). Suppression of the mRNA and protein expression of CXCR4 by the PEI-GO/siCXCR4 complex was confirmed by real-time PCR and western blot analysis. In addition, the metastatic potential of MDA-MB-231 cells was attenuated by the PEI-GO/siCXCR4 complex as demonstrated in wound healing assay. Our results suggest that PEI-GO is effective in the delivery of siRNA and may contribute to targeted gene therapy to suppress cancer metastasis.

Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), and plays a crucial role in cell proliferation and differentiation. The gatekeeping loop identified in the structure of spermidine synthase was predicted to contain residues important for substrate binding, but its correlation with enzyme catalysis has not been fully understood. In this study, recombinant Escherichia coli spermidine synthase (EcSPDS) was produced and its enzyme kinetics was characterized. Site-directed mutants of EcSPDS were obtained to demonstrate the importance of the amino acid residues in the gatekeeping loop. Substitution of Asp158 and Asp161 with alanine completely abolished EcSPDS activity, suggesting that these residues are absolutely required for substrate interaction. Reduction in enzyme activity was observed in the C159A, T160A, and P165Q variants, indicating that hydrophobic interactions contributed by Cys159, Thr160, and Pro165 are important for enzyme catalysis as well. On the other hand, replacement of Pro162 and Ile163 had no influence on EcSDPS activity. These results indicate that residues in the gatekeeping loop of spermidine synthase are indispensable for the catalytic reaction of EcSPDS. To the best of our knowledge, this is the first functional study on the gatekeeping loop of EcSPDS by site-directed mutagenesis.

Aging has less effect on adipose-derived mesenchymal stem cells (ADSCs) than on bone marrow-derived mesenchymal stem cells (BMSCs), but whether the fact holds true in stem cells from elderly patients with osteoporotic fractures is unknown. In this study, ADSCs and BMSCs of the same donor were harvested and divided into two age groups. Group A consisted of 14 young patients (36.4 ± 11.8 years old), and group B consisted of eight elderly patients (71.4 ± 3.6 years old) with osteoporotic fractures. We found that the doubling time of ADSCs from both age groups was maintained below 70 hrs, while that of BMSCs increased significantly with the number of passage. When ADSCs and BMSCs from the same patient were compared, there was a significant increase in the doubling time of BMSCs in each individual from passages 3 to 6. On osteogenic induction, the level of matrix mineralization of ADSCs from group B was comparable to that of ADSCs from group A, whereas BMSCs from group B produced least amount of mineral deposits and had a lower expression level of osteogenic genes. The p21 gene expression and senescence-associated β-galactosidase activity were lower in ADSCs compared to BMSCs, which may be partly responsible for the greater proliferation and differentiation potential of ADSCs. It is concluded that the proliferation and osteogenic differentiation of ADSCs were less affected by age and multiple passage than BMSCs, suggesting that ADSCs may become a potentially effective therapeutic option for cell-based therapy, especially in elderly patients with osteoporosis.

Background Previous reports have indicated that statins could prevent bone loss in ovariectomized (OVX) rats and increase the expressions of osteogenic genes in cultured osteoblasts. In this study, we hypothesized that simvastatin might increase osteoblast number and protein expressions of osteogenic markers localized in bones in concomitance with the prevention of bone loss in OVX rats. Materials and methods Fifty-four 3-month-old OVX and sham-operated (SHAM) female Sprague–Dawley rats were used. Simvastatin (10–20 mg kg−1 day−1) was administrated orally for 6 weeks. Trabecular volume, osteoblast number and osteogenic proteins including BMP2, collagen type I and osteocalcin on bone sections obtained from lumbar vertebral body, distal femur and proximal tibia were measured. Results The results showed that SHAM rats had significantly less trabecular bone volume and osteoblast number than that of OVX rats 6 weeks after operation. Oral simvastatin treatment (10–20 mg kg−1 day−1) increased bone volume and osteoblast number in the distal femurs, proximal tibiae and vertebrae of OVX rats. Furthermore, the osteoblastic cells with immuno-stained BMP2, collagen type I and osteocalcin in vertebral bones were significantly increased by simvastatin treatment (20 mg kg−1 day−1) in OVX rats. Conclusions This study demonstrates that simvastatin enhances the production of osteogenic proteins in bone and this effect may contribute to the prevention of bone loss in OVX rats.

The inorganic pyrophosphatase from the human pathogen Helicobacter pylori (HpPPase) is a family I PPase. It is a homohexamer consisting of identical 20-kDa subunits. Hydrolysis of inorganic pyrophosphate (PP(i)) by HpPPase relied on the presence of magnesium and followed Michaelis-Menten kinetics, with k (cat) being 344 s(-1) and K (m) being 83 microM at pH 8.0, which was the optimal pH for catalysis. HpPPase was activated by both thiol and non-thiol reductants, distinct from the previously suggested inactivation/reactivation process involving formation and breakage of disulfide bonds. Substitution of Cys16 of HpPPase, which was neither located at the active site nor evolutionarily conserved, resulted in a loss of 50% activity and a reduction in sensitivity to reductants and oxidized glutathione. In addition, the C16S replacement caused a considerable disruption in thermostability, which exceeded that resulted from active-site mutations such as Y140F HpPPase and those of Escherichia coli. Although Cys16 was not located at the subunit interface of the hexameric HpPPase, sedimentation analysis results suggested that the C16S substitution destabilized HpPPase through impairing trimer-trimer interactions. This study provided the first evidences that the single cysteine residue of HpPPase was involved in enzyme activation, thermostability, and stabilization of quaternary structure.

The texture observation has long been the core technique in liquid crystal (LC)-based biosensing. One of the drawbacks of this observational means is the difficulty in quantitative analysis. In this invited paper, we report on our recent attempt to improve the LC-based biosensing technique through the analysis of bovine serum albumin (BSA), a protein standard commonly used in the assay of protein concentration. We propose to overcome the technical limitations in the analysis of LC-based biosensors by considering alternative measuring schemes. By means of the induced changes in electro-optical properties of LCs in the presence of different concentrations of BSA, novel quantitative techniques specific for LC-based biosensors are developed.

Liquid crystals (LCs) can be employed in biological sensing and applied to label-free immunodetection owing to their unique birefringent, anchoring, alignment and collective properties. Like all different kinds of immunoassays, both sensitivity and specificity are universally the most important key points of concern. In this study, we developed various approaches toward ultrahigh sensitivity in LC-based immunoassays for potential clinical applications. The LC-based immunodetection technique was demonstrated with the cancer biomarker CA125, which is a mucin-like glycoprotein commonly present in the serum of patients with ovarian and other types of cancer. By using LCs with larger birefringence, such as HDN, the sensitivity of immunodetection was drastically enhanced compared to 5CB, which has a relatively lower birefringence and is commonly used in LC biosensing studies. In addition, UV modification of the monolayer of dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAP), which functions as the alignment layer for LCs, is suggested to increase functional groups suitable for covalent binding of biomolecules, stabilizing the immobilized anti-CA125 antibody and the immunocomplex thus formed, and contributing to the lowered detection limit. Finally, we show that it is possible to directly identify the formation of CA125 immunocomplex with HDN in a mixture of antigen and antibody without the need to eliminate unbound or unreacted biomolecules through washing, thereby creating a simplified procedure for faster LC-based immunoassay. It is evident from our results that label-free immunodetection based on birefringent LCs represents a novel biosensing technique with potentials to detect a wide range of biomolecules, providing an alternative to conventional label-based immunoassays.