Confirmation of oral squamous cell cancer (OSCC) currently relies on histological analysis, which does not provide clear indication of cancer development from precancerous lesions. In the present study, whole saliva proteins of patients with OSCC (n = 12) and healthy subjects (n = 12) were separated by 2DE to identify potential candidate biomarkers that are much needed to improve detection of the cancer. The OSCC patients' 2DE saliva protein profiles appeared unique and different from those obtained from the healthy subjects. The patients' saliva α1-antitrypsin (AAT) and haptoglobin (HAP) β chains were resolved into polypeptide spots with increased microheterogeneity, although these were not apparent in their sera. Their 2DE protein profiles also showed presence of hemopexin and α-1B glycoprotein, which were not detected in the profiles of the control saliva. When subjected to densitometry analysis, significant altered levels of AAT, complement C3, transferrin, transthyretin, and β chains of fibrinogen and HAP were detected. The increased levels of saliva AAT, HAP, complement C3, hemopexin, and transthyretin in the OSCC patients were validated by ELISA. The strong association of AAT and HAP with OSCC was further supported by immunohistochemical staining of cancer tissues. The differently expressed saliva proteins may be useful complementary biomarkers for the early detection and/or monitoring of OSCC, although this requires validation in clinically representative populations.
Diagnosis of prostate cancer (PCa) is currently much reliant on the invasive and time-consuming transrectal ultrasound-guided biopsy of the prostate gland, particularly in light of the inefficient use of prostate-specific antigen as its biomarker. In the present study, we have profiled the sera of patients with PCa and benign prostatic hyperplasia (BPH) using the gel- and lectin-based proteomics methods and demonstrated the significant differential expression of apolipoprotein AII, complement C3 beta chain fragment, inter-alpha-trypsin inhibitor heavy chain 4 fragment, transthyretin, alpha-1-antitrypsin, and high molecular weight kininogen (light chain) between the two groups of patients' samples. Our data are suggestive of the potential use of the serum proteins as complementary biomarkers to effectively discriminate PCa from BPH, although this requires further extensive validation on clinically representative populations.
The use of lectin affinity chromatography prior to 2-DE separation forms an alternative method to unmask the expression of targeted glycoproteins of lower abundance in serum samples. Reduced expression of alpha-2 macroglobulin (AMG) and complement factor B (CFB) was detected in sera of patients with nasopharyngeal carcinoma (NPC) when pooled serum samples of the patients and those of healthy individuals were subjected to affinity isolation using immobilized champedak mannose-binding lectin and analyzed by 2-DE and densitometry. The AMG and CFB spots were not detected in the 2-DE protein profiles when the same pooled serum samples were subjected to albumin and IgG depletion and neither were they detected when the depleted samples were analyzed by western blotting and lectin detection. Together with other acute-phase response proteins that were previously reported to be altered in expression in NPC patients, AMG and CFB may serve as useful complementary biomarkers for NPC.
A 35 kDa glycoprotein whose abundance was previously demonstrated to be enhanced in sera of patients with endometrial adenocarcinoma (n = 12), was isolated from pooled sera of three of the cancer patients using champedak galactose-binding lectin affinity chromatography in the present study. Subjecting it to 2-DE and MS/MS, the glycoprotein was identified as the O-glycosylated fragment of inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4). When compared to control sera (n = 17), expression of the 35 kDa ITIH4 cleavage fragment was demonstrated to be significantly enhanced in sera of patients with breast carcinoma (n = 10), epithelial ovarian carcinoma (n = 10), and germ cell ovarian carcinoma (n = 10) but not in patients with nasopharyngeal carcinoma (n = 13) and osteosarcoma (n = 7). The lectin-based electrophoretic bioanalytical method adopted in the present study may be used to assess the physiological relevance of ITIH4 fragmentation and its correlation with different malignancies, their stages and progression.
The expression of high-abundance serum proteins in newly diagnosed patients with endometrial adenocarcinoma (EACa), squamous cell cervical carcinoma (SCCa) and cervical adenocarcinoma (ACCa), relative to control female subjects, was analyzed by subjecting serum samples to 2-DE followed by image analysis of the silver-stained protein profiles. The three cohorts of cancer patients demonstrated different altered expression of serum high-abundance proteins compared to negative control women. The expression of alpha1-antitrypsin, alpha1-B glycoprotein, cleaved high-molecular-weight kininogen (light chain) and antithrombin III were consistently altered in all the patients. However, clusterin was upregulated only in the patients with EACa, while those with SCCa and ACCa were typically characterized by the upregulated expression of zinc alpha-2-glycoprotein. The aberrant expression of selective serum proteins in the various cohorts of cancer patients was validated by competitive ELISA as well as by lectin detection. Analysis by using the champedak galactose binding lectin further highlighted an unidentified protein that may be differently glycosylated in the sera of the EACa patients that were studied.
The present study was aimed at the identification of proteins that are differentially expressed in the urine of patients with prostate cancer (PCa), those with benign prostatic hyperplasia (BPH) and age-matched healthy male control subjects. Using a combination of 2DE and MS/MS, significantly lower expression of urinary saposin B and two different fragments of inter-alpha-trypsin inhibitor light chain (ITIL) was demonstrated in the PCa patients compared to the controls. However, only one of the ITIL fragments was significantly different between the PCa and BPH patients. When image analysis was performed on urinary proteins that were transferred onto NC membranes and detected using a lectin that binds to O-glycans, a truncated fragment of inter-alpha-trypsin inhibitor heavy chain 4 was the sole protein found to be significantly enhanced in the PCa patients compared to the controls. Together, these urinary peptide fragments might be useful complementary biomarkers to indicate PCa as well as to distinguish it from BPH, although further epidemiological evidence on the specificity and sensitivity of the protein candidates is required.
We have analyzed unfractionated sera of newly diagnosed patients (n=10) with breast carcinoma (BC), prior to treatment, and patients (n=5) with fibrocystic disease of the breast (FDB) by two-dimensional gel electrophoresis (2-DE) and silver staining. The patients' 2-DE serum protein profiles obtained were then subjected to image analysis and compared to similar data generated from sera of normal healthy female controls (n=10) of the same range of age. The relative expression of alpha1-antichymotrypsin (ACT), clusterin, and complement factor B was significantly higher in all BC patients as compared to normal controls. However, the expression of alpha1-antitrypsin (AAT) in BC patients was apparently lower than that of the controls. Similar differential expression of ACT was detected in the FDB patients. The aberrant expression of the serum acute-phase proteins of patients with BC and FDB was confirmed by competitive enzyme-linked immunosorbent assay (ELISA). Similar altered proteins expression was also observed from immunohistochemical studies of malignant (n=5) and benign (n=5) breast lesions of the respective patients performed using antisera to the aberrantly expressed proteins. However, the malignant breast lesions were instead positively stained for AAT. The differential expression of the serum proteins was apparently abrogated when a six-month follow-up study was performed on nine of the BC patients subsequent to treatment.
The hypolipidemic effects of Tamarindus indica fruit pulp extract (Ti-FPE) have been earlier reported but the underlying molecular mechanisms are still uncertain. In this study, hamsters fed with Ti-FPE, both in the absence and presence of high-cholesterol diet, were shown to have significantly reduced levels of serum triglyceride, LDL-C and total cholesterol. The Ti-FPE-fed non-hypercholesterolemic hamsters also showed significant enhanced levels of serum apolipoprotein A1, antithrombin III, transferrin and vitamin D binding protein. In diet-induced hypercholesterolemic hamsters, apolipoprotein A1, antithrombin III and transferrin, which were relatively low in levels, became significantly enhanced when the hamsters were fed with Ti-FPE. These Ti-FPE-fed hypercholesterolemic hamsters also showed significant higher levels of serum vitamin D binding protein. When the different treated groups of hamsters were analyzed for the levels of the four serum proteins by ELISA, similar altered abundance were detected. Ingenuity Pathway Analysis of the Ti-FPE modulated serum proteins singled out "Lipid metabolism, molecular transport, small molecule biochemistry" as the top network. Our results suggest that the hypolipidemic effects of Ti-FPE are associated with alterations of serum proteins that are known to be cardioprotective and involved in the metabolism of lipids. The MS data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD010232.
Scale-up in droplet microfluidics achieved by increasing the number of devices running in parallel or increasing the droplet makers in the same device can compromise the narrow droplet-size distribution, or requires high fabrication cost, when glass- or polymer-based microdevices are used. This paper reports a novel way using parallelization of needle-based microfluidic systems to form highly monodispersed droplets with enhanced production rates yet in cost-effective way, even when forming higher order emulsions with complex inner structure. Parallelization of multiple needle-based devices could be realized by applying commercially available two-way connecters and 3D-printed four-way connectors. The production rates of droplets could be enhanced around fourfold (over 660 droplets/min) to eightfold (over 1300 droplets/min) by two-way connecters and four-way connectors, respectively, for the production of the same kind of droplets than a single droplet maker (160 droplets/min). Additionally, parallelization of four-needle sets with each needle specification ranging from 34G to 20G allows for simultaneous generation of four groups of PDMS microdroplets with each group having distinct size yet high monodispersity (CV < 3%). Up to six cores can be encapsulated in double emulsion using two parallelly connected devices via tuning the capillary number of middle phase in a range of 1.31 × 10-4 to 4.64 × 10-4 . This study leads to enhanced production yields of droplets and enables the formation of groups of droplets simultaneously to meet extensive needs of biomedical and environmental applications, such as microcapsules with variable dosages for drug delivery or drug screening, or microcapsules with wide range of absorbent loadings for water treatment.
Hepatitis B core antigen (HBcAg) expressed in Escherichia coli is able to self-assemble into large and small capsids comprising 240 (triangulation number T = 4) and 180 (triangulation number T = 3) subunits, respectively. Conventionally, sucrose density gradient ultracentrifugation and SEC have been used to separate these capsids. However, good separation of the large and small particles with these methods is never achieved. In the present study, we employed a simple, fast, and cost-effective method to separate the T = 3 and T = 4 HBcAg capsids by using native agarose gel electrophoresis followed by an electroelution method (NAGE-EE). This is a direct, fast, and economic method for isolating the large and small HBcAg particles homogenously based on the hydrodynamic radius of the spherical particles. Dynamic light scattering analysis demonstrated that the T = 3 and T = 4 HBcAg capsids prepared using the NAGE-EE method are monodisperse with polydispersity values of ∼15% and ∼13%, respectively. ELISA proved that the antigenicity of the capsids was not affected in the purification process. Overall, NAGE-EE produced T = 3 and T = 4 capsids with a purity above 90%, and the recovery was 34% and 50%, respectively (total recovery of HBcAg is ∼84%), and the operation time is 15 and 4 times lesser than that of the sucrose density gradient ultracentrifugation and SEC, respectively.
Matched MeSH terms: Electrophoresis, Agar Gel/methods*
Alternating-current electro-osmosis, a phenomenon of fluid transport due to the interaction between an electrical double layer and a tangential electric field, has been used both for inducing fluid movement and for the concentration of particles suspended in the fluid. This offers many advantages over other phenomena used to trap particles, such as placing particles at an electrode centre rather than an edge; benefits of scale, where electrodes hundreds of micrometers across can trap particles from the molecules to cells at the same rate; and a trapping volume limited by the vortex height, a phenomenon thus far unstudied. In this paper, the collection of particles due to alternating-current electro-osmosis driven collection is examined for a range of particle concentrations, inter-electrode gap widths, chamber heights and media viscosity and density. A model of collection behaviour is described where particle collection over time is governed by two processes, one driven by the vortices and the other by sedimentation, allowing the determination of the maximum height of vortex-driven collection, but also indicates how trapping is limited by high particle concentrations and fluid velocities. The results also indicate that viscosity, rather than density, is a significant governing factor in determining the trapping behaviour of particles.
Dielectrophoresis (DEP), the induced movement of dielectric particles placed in a nonuniform electric field, has been used as a potential technique for manipulation and separation of many biological samples without destructive consequences to the cell. Cells of the same genotype in different physiological and pathological states have unique morphological and structural features, therefore, it is possible to differentiate between them using their DEP responses. This paper reports the experimental discrimination of normal and dengue-infected human hepatic fetal epithelial cells (WRL-68 cells) based on their DEP crossover frequency, at which no resultant movement occurs in the cells in response to the DEP force. A microarray dot electrode was used to conduct the DEP experiments. The DEP forces applied to the cells were quantified by analyzing the light intensity shift within the electrode's dot region based on the Cumulative Modal Intensity Shift image analysis technique. The differences in dielectric properties between infected and uninfected cells were exploited by plotting a unique DEP spectrum for each set of cells. We observed that the crossover frequency decreased from 220 kHz for the normal WRL-68 cells to 140 kHz after infection with the dengue virus in a medium conductivity of 100 μS/cm. We conclude that the change in the DEP crossover frequency between dengue-infected cells and their healthy counterparts should allow direct characterization of these cell types by exploiting their electrophysiological properties.
This study aims to identify cancer-associated proteins in the secretome of oral cancer cell lines. We have successfully established four primary cell cultures of normal cells with a limited lifespan without human telomerase reverse transcriptase (hTERT) immortalization. The secretome of these primary cell cultures were compared with that of oral cancer cell lines using 2DE. Thirty five protein spots were found to have changed in abundance. Unambiguous identification of these proteins was achieved by MALDI TOF/TOF. In silico analysis predicted that 24 of these proteins were secreted via classical or nonclassical mechanisms. The mRNA expression of six genes was found to correlate with the corresponding protein abundance. Ingenuity Pathway Analysis (IPA) core analysis revealed that the identified proteins were relevant in, and related to, cancer development with likely involvements in tumor growth, metastasis, hyperproliferation, tumorigenesis, neoplasia, hyperplasia, and cell transformation. In conclusion, we have demonstrated that a comparative study of the secretome of cancer versus normal cell lines can be used to identify cancer-associated proteins.
Replicative senescence and stress-induced premature senescence (SIPS) cells are known to share certain traits. However, whether these cells are different at the protein level is unclear. Thus, this study has utilized proteomics to identify differences in the proteomes of replicative senescence and SIPS cells compared to normal cells. Replicative senescence was induced by serial passage of normal cells in culture. SIPS was established by exposure to H2 O2 at a subcytotoxic concentration of 20 μM for two weeks. Following 2DE, protein profiles were compared and protein spots that changed in abundance were identified by MALDI-TOF MS. Quantitative real-time RT-PCR was then performed to evaluate the transcript expression of selected altered proteins. A total of 24 and 10 proteins were found to have changed in abundance in replicative senescence and SIPS cells, respectively, when compared to young cells. Quantitative RT-PCR revealed that nine genes showed the same direction of change as observed in the proteomics analysis. Very little overlap was observed between proteins that changed in replicative senescence and SIPS cells, suggesting that although both SIPS and replicative senescence cells share hallmarks of cellular senescence, they were different in terms of proteins that changed in abundance.
The basidiomycete fungal pathogen Ganoderma boninense is the causative agent for the incurable basal stem rot (BSR) disease in oil palm. This disease causes significant annual crop losses in the oil palm industry. Currently, there is no effective method for disease control and elimination, nor is any molecular marker for early detection of the disease available. An understanding of how BSR affects protein expression in plants may help identify and/or assist in the development of an early detection protocol. Although the mode of infection of BSR disease is primarily via the root system, defense-related genes have been shown to be expressed in both the root and leafs. Thus, to provide an insight into the changes in the global protein expression profile in infected plants, comparative 2DE was performed on leaf tissues sampled from palms with and without artificial inoculation of the Ganoderma fungus. Comparative 2DE revealed that 54 protein spots changed in abundance. A total of 51 protein spots were successfully identified by LC-QTOF MS/MS. The majority of these proteins were those involved in photosynthesis, carbohydrate metabolism as well as immunity and defense.
Genetic variation of orosomucoid (ORM) in the genus Macaca was investigated. Plasma samples were subjected to isoelectric focusing in a pH range of 4-6.5, followed by immunoprinting with anti-human ORM antibodies. A total of 25 alleles were identified in 231 Asian macaques belonging to 13 species from 23 populations and 22 members belonging to a family of M. fascicularis. Family data presented evidence for a codominant mode of inheritance with multi-alleles at a single autosomal locus. A population study revealed enormous intra- and interspecies variations. The heterozygosity values varied from 0.855 in M. fascicularis (Malaysia) to 0.000 in M. radiata (India), M. silenus (India) and M. arctoides (Malaysia).
Combining both device and particle designs are the essential concepts to be considered in magnetophoretic system development. Researcher efforts are often dedicated to only one of these design aspects and neglecting the interplay between them. Herein, to bring out importance of the idea of integration between device and particle, we reviewed the working principle of magnetophoretic system (includes both device and particle design concepts). Since, the magnetophoretic force is influenced by both field gradient and magnetization volume, hence, accurate prediction of the magnetophoretic force is relying on the availability of information on both parameters. In device design, we focus on the different strategies used to create localized high-field gradient. For particle design, we emphasize on the scaling between hydrodynamic size and magnetization volume. Moreover, we also briefly discussed the importance of magnetoshape anisotropy related to particle design aspect of magnetophoretic systems. Next, we illustrated the need for integration between device and particle design using microscale applications of magnetophoretic systems, include magnetic tweezers and microfluidic systems, as our working example. On the basis of our discussion, we highlighted several promising examples of microscale magnetophoretic systems which greatly utilized the interplay between device and particle design. Further, we concluded the review with several factors that possibly resulted in the lack of research efforts related to device and particle design integration.
The spatiotemporal accuracy of microscale magnetophoresis has improved significantly over the course of several decades of development. However, most of the studies so far were using magnetic microbead composed of nanosphere particle for magnetophoretic actuation purpose. Here, we developed an in-house method for magnetic sample analysis called quadrupole magnetic steering control (QMSC). QMSC was used to study the magnetophoretic behavior of polystyrene microbeads decorated with iron oxide nanospheres-coated polystyrene microbeads (IONSs-PS) and iron oxide nanorods-coated polystyrene microbeads (IONRs-PS) under the influence of a quadrupole low field gradient. During a 4-s QMSC experiment, the IONSs-PS and IONRs-PS were navigated to perform 180° flip and 90° turn formations, and their kinematic results (2 s before and 2 s after the flip/turn) were measured and compared. The results showed that the IONRs-PS suffered from significant kinematic disproportion, translating a highly uneven amount of kinetic energy from the same magnitude of magnetic control. Combining the kinematic analysis, transmission electron microscopy micrographs, and vibrating sample magnetometry measurements, it was found that the IONRs-PS experienced higher fluid drag force and had lower consistency than the IONSs-PS due to its extensive open fractal nanorod structure on the bead surface and uneven magnetization, which was attributed to its ferrimagnetic nature.
Dielectrophoresis (DEP) is a technique to manipulate trajectories of polarisable particles in nonuniform electric fields by utilizing unique dielectric properties. The manipulation of a cell using DEP has been demonstrated in various modes, thereby indicating potential applications in the biomedical field. In this review, recent DEP applications in the biomedical field are discussed. This review is intended to highlight research work that shows significant approach related to DEP application in biomedical field reported between 2016 and 2020. First, single-shell model and multiple-shell model of cells are introduced. Current device structures and recently introduced electrode patterns for DEP applications are discussed. Second, the biomedical uses of DEP in liquid biopsies, stem cell-based therapies, and diagnosis of infectious diseases due to bacteria and viruses are presented. Finally, the challenges in DEP research are discussed, and the reported solutions are explained. DEP's potential research directions are mentioned.
Metabolic profiling of Glycyrrhiza glabra using comprehensive two-dimensional liquid chromatography (LC × LC) coupled with photodiode array (PDA) and mass spectrometry (MS) detection is described. The separation was conducted under reversed-phase conditions, using a combination of first dimension (1 D) 150 mm microbore cyano column utilising 2.7 μm diameter (dp ) particles, and second dimension (2 D) 50 mm superficially porous octadecylsilica column with 2.7 μm dp particles. A multi-segmented shift gradient (MSG) for the 2 D separation was developed, and the orthogonality achieved was compared with other modes of gradients, such as full in-fraction, and shift gradient systems. Results demonstrated a significant expansion of metabolic coverage using MSG in 2 D, providing the highest measure of orthogonality compared to other gradient modes. Compound identifications were performed by employing complementary data from PDA and MS detection, with reference to structural group-type distribution in 2D space. A total of ca. 120 compounds were detected, and among them 37 were tentatively identified, distributed over the chemical families of glycosylated flavanones, triterpene saponins, and others. In comparison with one-dimensional LC, the total number of compounds detected was ca. 2-fold greater when LC × LC was employed. To the best of our knowledge, this is the first demonstration of the MSG mode in LC × LC, representing a powerful strategy to expand the metabolic coverage for analysis of plant-derived extracts, containing a multitude of different phytochemical classes.