Magnetic Bead-based Separation Research Articles

Interacting Characteristics of Chrysene with Free DNA in vitro

Deoxyribonucleic acid (DNA), an important material in life science, is considered a natural and highly specific functional biopolymer. Various analytical techniques were used to analyze the interaction of chrysene with free DNA in vitro, and based on this interaction, we studied the removal of chrysene from contaminated water by DNA via magnetic beads-based separation. The results of UV-visible spectroscopy, DNA thermal denaturation measurement, circular dichroism (CD) spectroscopy, and fluorescence microscopy showed that chrysene interacted with DNA through an intercalating mode. The results of fluorescence spectroscopy demonstrated that DNA had a static quenching effect on chrysene, indicating the formation of a stable complex between chrysene and DNA. Thermodynamic studies revealed that the interaction of chrysene with free DNA was spontaneous in vitro. In addition, chrysene could be efficiently removed from contaminated water by DNA via DNA-intercalation and magnetic bead-based separation. The chrysene removal efficiency of DNA was better than that of activated carbon even when the amount of activated carbon was 400 times more than that of DNA. The results of this study provide insights into the development of new chrysene elimination processes based on DNA-intercalation in water pollution.

A biosensor using semi-DNA walker and CHA -FRET loop for ultrasensitive detection of single nucleotide polymorphism

Single nucleotide polymorphisms (SNPs) are emerging as important biomarkers for disease diagnosis, prognostics and disease pathogenesis. However, it is still challenging to make highly specific and sensitive SNPs detection to distinguish target DNA with single-base difference, so that flap endonuclease 1 (FEN 1) manipulated semi-DNA walker reaction and catalytic hairpin assembly (CHA)-FRET loop for ultrasensitive SNP detection has been proposed. Herein, FEN 1 was employed to specifically recognize and cut the 5′ flap of three-base overlapping structure formed by only hybridizing with mutant target (MT). Simultaneously, FEN 1 drove MT hybridization to autonomously walk along downstream probe (DP) and the cut sequence (CS) from semi-DNA walker reaction was collected by magnetic bead-based separation to avoid false-positive signal. Next, the cleaved CS triggered CHA and generated fluorescence resonance energy transfer (FRET) via Cy3 and Cy5. FEN 1 manipulated semi-DNA walker reaction to specifically recognize the target DNA and combined CHA-FRET loop to dually amplify signal. Thus, the SNP biosensor demonstrated superior performance for ultrasensitive detection of mutant KRAS gene with a low detection limit as low as 80 aM and was successfully applied to monitor the expression of mutant KRAS gene in human cancer cell lysates. This strategy provides an ultrasensitive way for the detection of biomolecules and reveals an effective avenue for diseases diagnosis.

A FEN 1-driven DNA walker-like reaction coupling with magnetic bead-based separation for specific SNP detection.

Single-nucleotide polymorphism (SNP) plays a key role in the carcinogenesis of the human genome, and understanding the intrinsic relationship between individual genetic variations and carcinogenesis lies heavily in the establishment of a precise and sensitive SNP detection platform. Given this, a powerful and reliable SNP detection platform is proposed by a flap endonuclease 1 (FEN 1)-driven DNA walker-like reaction coupling with a magnetic bead (MB)-based separation. A carboxyfluorescein (FAM)-labeled downstream probe (DP) was decorated on a streptavidin magnetic bead (SMB). The target DNA, as a walker strand, was captured by hybridization with DP and an upstream probe (UP) to form a three-base overlapping structure and execute the walking function on the surface of SMB. FEN 1 was employed to specifically recognize the three-base overlapping structure and cut the 5'flap at the SNP site to report the walking event and signal amplification. Considering the fact that the fluorescence was labeled on the cleavage and uncleavage sequences of DP and the target DNA-triggered walking event was undistinguishable from the mixtures, magnetic separation came in handy for cleavage probe (CP) isolation and discrimination of the amplified signal from the background signal. In comparison with the conventional DNA walker reaction, this strategy was coupling with SMB-based separation, thus promising a powerful and reliable method for SNP detection and signal amplification.

Separation mixed semen of two individuals using magnetic beads coupled ABH blood group antibody.

In sexual assault cases, one of the most common samples collected is a mixed semen stain, which is often found on the vagina, female underwear, or bed sheets. However, it is usually difficult to identify the perpetrator based on this sample alone. One technique that has been developed to address this issue is magnetic bead-based separation. This method involves using modified magnetic microspheres to capture and enrich specific target cells, in this case, sperm cells. In this study, we utilized magnetic beads coupled with ABH blood group antibody to isolate sperm cells from an individual of a single ABO blood type. Subsequently, polymerase chain reaction amplification and capillary electrophoresis were employed to perform the genotyping the short tandem repeat (STR) loci. This approach allows for the identification of different individuals in a mixed seminal stain sample from two individuals, by first separating sperm cells based on ABH antigen differences and subsequently utilizing autosomal STR typing on the enriched single blood group cells.

Magnetic bead-based separation of pneumococcal serotypes

The separation of pneumococcal serotypes from a complex polymicrobial mixture may be required for different applications. For instance, a minority strain could be present at a low frequency in a clinical sample, making it difficult to identify and isolate by traditional culture-based methods. We therefore developed anassay to separate mixed pneumococcal samples using serotype-specific antiserum and a magnetic bead-based separation method. Using qPCR and colony counting methods, we first show that serotypes (12F, 23F, 3, 14, 19A, and 15A) present at ∼0.1% of a dual serotype mixture can be enriched to between 10% and 90% of the final sample. We demonstrate two applications for this method: extraction of known pneumococcal serotypes from saliva samples and efficient purification of capsule switch variants from experimental transformation experiments. This method may have further laboratory or clinical applications when the selection of specific serotypes is required.

Curcumin Induces Ferroptosis in A549 CD133+ Cells through the GSH-GPX4 and FSP1-CoQ10-NAPH Pathways.

Cancer stem cells (CSCs) are characterized by an ability for unlimited proliferation and efficiency of self-renewal. The targeting of lung CSCs (LCSCs)-related signaling pathways represent a promising therapeutic strategy for treatment of lung cancer. Ferroptosis a potential strategy for LCSCs treatment, and curcumin cloud induce ferroptosis. In this study, we aimed to observe the effects of curcumin on LCSCs via ferroptosis-related pathways. In this study, A549 cluster of differentiation (CD)133+ and A549 CD133- cells were isolated using magnetic bead-based separation. Colony formation and sphere formation assays, as well as cells injection in non-obese diabetes/severe combined immune deficiency (NOD/SCID) mice, were used to analyze the tumorigenic ability of cells differentially expressing CD133. A549 CD133+ cells were treated with different doses of curcumin (0, 10, 20, 40, 80 μM). Cell viability, glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) expressions were measured. The 50% inhibitory concentration (IC50) of curcumin, two ferroptosis inducers, inhibitor of GPX4 (RSL3) and inhibitor of FSP1 (iFSP1), and a ferroptosis inhibitor, ferrostatin-1 (Fer-1), were used to investigate the mechanism underlying the effect of curcumin on ferroptosis in A549 CD133+ cells. A549 CD133+ cells had greater tumorigenic ability than A549 cells. Curcumin treatment suppressed the expressions of GPX4 (glutathione peroxidase 4) and FSP1 in A549 CD133+ cells, thereby inducing ferroptosis. RSL3 and iFSP1 respectively suppressed the GSH (glutathione)-GPX4 and FSP1 (ferroptosis suppressor protein 1)-CoQ10 (coenzyme Q10)-nicotinamide adenine dinucleotide (NADH) pathways in A549 CD133+ cells. However, the roles of curcumin were blocked by Fer-1 treatment. In this study, curcumin induced ferroptosis through inhibiting the GSH-GPX4 and FSP1-CoQ10-NADH pathways in A549 CD133+ cells, resulting in the inhibition of their self-renewal potential.

Magnetic bead-based separation of sperm cells from semen-vaginal fluid mixed stains using an anti-ACRBP antibody.

Forensic DNA analysis of semen-vaginal fluid mixed stains is essential and necessary in sexual assault cases. Here, we used a magnetic bead conjugated acrosin binding protein (ACRBP) antibody to separate and enrich sperm cells from mixed stains. Previously, western blotting indicated that ACRBP was specifically expressed in sperm cells, but not in female blood and epithelial cells, while immunofluorescence data showed ACRBP was localized to the acrosome in sperm cells. In our study, sperm were separated from mixed samples at three sperm cell/female buccal epithelial cell ratios (103:103; 103:104; and 103:105) using a magnetic bead conjugated ACRBP antibody. Subsequently, 23 autosomal short tandem repeat (STR) loci were amplified using the Huaxia™ Platinum PCR Amplification System and genotyped using capillary electrophoresis. The genotyping success rate for STR loci was 90% when the sperm to female buccal epithelial cell ratio was > 1:100 in mixed samples. Our results suggest that the magnetic bead conjugated ACRBP antibody is effective for isolating sperm cells in sexual assault cases.

Construction of a Polarity-Switchable Photoelectrochemical Biosensor for Ultrasensitive Detection of miRNA-141.

As an early-stage tumor biomarker, microRNA (miRNA) has clinical application potential and its sensitive and accurate detection is significant for early tumor diagnosis. In this study, a photoelectrochemical (PEC) biosensing platform was fabricated for ultrasensitive miRNA-141 detection, which is based on a photocurrent polarity-switchable system using CdS quantum dots (QDs) in the presence of a 5,10,15,20-tetrakis (4-aminophenyl)-21H,23H-porphine (Tph-2H)-coated glassy carbon electrode (GCE). As an excellent photoactive material, Tph-2H has a narrow band gap that effectively gathers photoelectrons under visible light irradiation and improves the transfer ability of photogenerated electrons. Further, the detection sensitivity of miRNA-141 could be significantly improved by combining an enzyme-assisted recycle amplification reaction and a magnetic bead-based separation strategy. The proposed photocurrent polarity-switchable PEC biosensor could efficiently eliminate the false-positive or false-negative signals and achieve a wide linear response range from 1 fM to 1 nM with a low detection limit of 0.33 fM for miRNA-141, providing a potentially alternative solution for detecting other biomarkers in bioanalysis and clinical diagnosis.

Paper-Based Multiplex Surface-Enhanced Raman Scattering Detection Using Polymerase Chain Reaction Probe Codification

We construct a multiplex surface-enhanced Raman scattering (SERS) platform based on a plasmonic paper substrate and a double-labeled probe for the detection of multiple fluorescent dyes at high sensitivity in a single-wavelength light source system. Plasmonic paper, made of silver nanodots on three-dimensional cellulose fibers, enables highly sensitive SERS biosensing based on localized surface plasmon resonance (LSPR). The proposed method enables the identification and quantification of a range of fluorescent dyes ranging from picomolar to millimolar concentrations. The use of 5' fluorescent dyes and 3' biotin-modified probes as SERS-coded probes renders possible the separation of fluorescent dyes with streptavidin-coated magnetic beads (SMBs) and the sensitive detection of multiple dyes after the reverse transcription polymerase chain reaction (RT-PCR). This experimental study reveals the multiplex detection capability of PCR-based SERS under existing PCR conditions without modifying primer and probe sequences. The combination of magnetic bead-based separation and paper SERS platform is efficient, economical, and can be used for the simultaneous detection of two or more pathogens.

Removal of 1,2-benzanthracene via the intercalation of 1,2-benzanthracene with DNA and magnetic bead-based separation

In this study, DNA-functionalize-magnetic beads were investigated as sorbent materials for effective removing 1,2-benzanthracene (BaA) from water. In order to reveal the removal mechanism, the interaction mode between BaA and DNA was evaluated by using various characterization tools such as UV-visible and circular dichroism spectroscopy, fluorescence and resonance scattering spectroscopy, and agarose gel electrophoresis. In the presence of BaA, the melting temperature of DNA increased from 76.2 °C to 82.3 °C, which closely related to the intercalating of BaA. It was found that a part of the ethidium bromide (EB) binding sites to DNA were occupied by BaA in EB competing study. The results indicated that a new complex appeared between hsDNA and BaA, and the number of the binding sites (n) and the binding constants (KA) at different temperatures were obtained. DNA binding saturation value (≈0.80) was obtained by resonance scattering spectra study. BaA could be enriched and removed by DNA-functionalize-magnetic beads via the intercalation, and the removal efficiency was 97.73% when the initial concentration was 2.45 x10−6 mol·L−1 (559.31 μg/L).

Portable Resource-Independent Blood-Plasma Separator.

The centrifuge is the gold standard for lab-based sample processing. While extremely efficient and robust, centrifuges are seldom used in the field due to the high-power requirements, size, and operational complexity. The lack of viable alternatives for remote sample collection has crippled the ability for mobile practitioners in human and animal medicine to reliably collect blood samples from their patients. There is no truly resource-independent solution that is able to perform highly efficient blood-plasma separation. Here, we describe our initial efforts in developing the High Efficiency Rapid Magnetic Erythrocyte Separator (H.E.R.M.E.S) sleeve, an apparatus that uses a magnetic bead-based separation assay in a scaled-up form factor to achieve highly efficient separation of erythrocytes from plasma within a short amount of time. The sleeve is easy-to-use, is completely resource independent, and achieves highly efficient separation in sample volumes as large as 1 mL by means of a unique mixing scheme. We demonstrate the performance of the sleeve with human blood samples and compare it against conventional end-over-end mixing.

Adsorptive removal of patulin from apple juice via sulfhydryl-terminated magnetic bead-based separation.

Patulin is a naturally produced toxin having potential carcinogenic properties. It is mainly produced by species of Penicillium growing on fruits, especially on apples. Detoxification of patulin, therefore, is very important for providing food safety. In this study, sulfhydryl-terminated, silica coated iron oxide beads were investigated as a multi-use sorbents for effective recovery of patulin from apple juice. The effect of beads amount, contact time, pH and temperature were evaluated for high adsorption capacity and the results showed maximum adsorption capacity at a reaction condition of 1.5 mg of beads for 4 h incubation at pH 7.2 and 25 °C. These sorbents were highly effective both in aqueous solution and apple juice with adsorption efficiencies of 99% and 71.25%, respectively. Adsorption process was explained by Langmuir isotherm model with pseudo-second order kinetic model. Thermodynamic parameters described spontaneous adsorption of patulin onto beads with high feasibility and preferences. The reusability of sulfhydryl coated magnetic beads was shown for at least four times without any significant decreases in efficiency. Results showed that this sorbent had potential for removal of patulin from apple juice without any negative effects on final quality parameters, Brix, color, clarity, total sugar and titratable acidity.

Establishment of oral squamous cell carcinoma cell line and magnetic bead-based isolation and characterization of its CD90/CD44 subpopulations.

In this study, we describe the establishment of the human papillomavirus 18-positive, stage II, grade 1, T2N0M0 head and neck tumor primary cell line derived from oral squamous cell carcinoma of a non-smoking patient by using two different protocols. Furthermore, a preparation of subpopulations derived from this primary cell line according to the cluster of differentiation molecules CD44/CD90 status using magnetic bead-based separation and their characterization was performed. Impedance-based real-time cell analysis, enzyme-linked immunsorbant assay (ELISA), wound-healing assay, flow-cytometry, gene expression analysis, and MTT assay were used to characterize these four subpopulations (CD44+/CD90−, CD44−/CD90−, CD44+/CD90+, CD44−/CD90−). We optimised methodics for establishement of primary cell lines derived from oral squamous cell carcinoma tissue samples and subsequent separation of mesenchymal (CD90+) and epithelial (CD90−) types of tumorous cells. Primary cell line prepared by using trypsin proteolysis was more viable than the one prepared by using collagenase. According to our results, CD90 separation is a necessary step in preparation of permanent tumor-tissue derived cell lines. Based on the wound-healing assay, CD44+ cells exhibited stronger migratory capacity than CD44− subpopulations. CD44+ subpopulations had also significantly higher expression of BIRC5 and SOX2, lower expression of FLT1 and IL6, and higher levels of basal autophagy compared to CD44− subpopulations. Furthermore, co-cultivation experiments revealed that CD44−/CD90+ cells supported growth of epithelial tumor cells (CD44+/CD90−). On the contrary, factors released by CD44+/CD90+ type of cells seem to have rather inhibiting effect. The most cisplatin-resistant subpopulation with the shortest doubling time was CD44−/CD90+, but this subpopulation had a low migratory capacity.

Development of GMP-Compatible Protocols for Thymus-Derived Regulatory T Cell Expansion

Introduction: Animal and early clinical studies have shown that regulatory T cell (Treg) therapy can prevent graft-versus-host disease (GVHD) following hematopoietic stem cell transplant. Despite these promising results, challenges remain in administering Treg therapy including the difficulty in isolating pure Tregs and expanding them to a therapeutic dose. We have previously found that the thymus, which is routinely removed during pediatric cardiac surgery, is a plentiful source of Tregs. These Tregs can be easily isolated, and can prevent xenoGVHD in mice. However, to use this therapy in humans, standard GMP-compliant methods for isolation and large-scale expansion of thymic Tregs must be developed. Methods: CD4+CD8-CD25+ thymic Tregs were isolated from pediatric thymuses using GMP-compatible magnetic bead-based separation. Tregs were expanded using artificial antigen-presenting cells or GMP-compatible antibody tetramer complexes that stimulate CD3 and CD28, with or without additional activation of CD2. Cells were cultured with rapamycin and IL-2, and were restimulated after 7 days without rapamycin. Results: Thymic Tregs expand in vitro when cultured with tetrameric antibody complexes. About 50-fold expansion was achieved using the anti-CD3/CD28/CD2 antibody tetramers, which represents the expansion that will be necessary to reach a clinically relevant dose. A higher expansion rate was obtained using the anti-CD3/CD28/CD2 antibody tetramer complexes than the anti-CD3/CD28. Cells cultured with these antibody tetramers express high levels of FOXP3, similar levels of Treg-characteristic markers as those expanded with artificial antigen-presenting cells, and do not produce inflammatory cytokines. Conclusion: Thymic Tregs can be isolated and expanded with GMP-compliant methods. Once the protocol is optimized, we can transfer our protocols to a GMP facility for further process development and to generate data required for a clinical trial application. We are the first group to pursue clinical application using allogeneic thymic Tregs, which provide an opportunity for an “off-the-shelf” therapy to treat GVHD. Canadian National Transplant Research Program.

Identification of potential serum proteomic biomarkers for clear cell renal cell carcinoma.

ObjectiveTo investigate discriminating protein patterns and serum biomarkers between clear cell renal cell carcinoma (ccRCC) patients and healthy controls, as well as between paired pre- and post-operative ccRCC patients.MethodsWe used magnetic bead-based separation followed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to identify patients with ccRCC. A total of 162 serum samples were analyzed in this study, among which there were 58 serum samples from ccRCC patients, 40 from additional paired pre- and post-operative ccRCC patients (n = 20), and 64 from healthy volunteers as healthy controls. ClinProTools software identified several distinct markers between ccRCC patients and healthy controls, as well as between pre- and post-operative patients.ResultsPatients with ccRCC could be identified with a mean sensitivity of 88.38% and a mean specificity of 91.67%. Of 67 m/z peaks that differed among the ccRCC, healthy controls, pre- and post-operative ccRCC patients, 24 were significantly different (P<0.05). Three candidate peaks, which were upregulated in ccRCC group and showed a tendency to return to healthy control values after surgery, were identified as peptide regions of RNA-binding protein 6 (RBP6), tubulin beta chain (TUBB), and zinc finger protein 3 (ZFP3) with the m/z values of 1466.98, 1618.22, and 5905.23, respectively.ConclusionMB-MALDI-TOF-MS method could generate serum peptidome profiles of ccRCC, and provide a new approach to identify potential biomarkers for diagnosis as well as prognosis of this malignancy.

Magnetic bead-based separation of sperm from buccal epithelial cells using a monoclonal antibody against MOSPD3

Forensic DNA analysis of sexual assault evidence requires unambiguous differentiation of DNA profiles in mixed samples. To investigate the feasibility of magnetic bead-based separation of sperm from cell mixtures using a monoclonal antibody against MOSPD3 (motile sperm domain-containing protein 3), 30 cell samples were prepared by mixing 10(4) female buccal epithelial cells with sperm cells of varying densities (10(3), 10(4), or 10(5) cells/mL). Western blot and immunofluorescence assays showed that MOSPD3 was detectable on the membrane of sperm cells, but not in buccal epithelial cells. After biotinylated MOSPD3 antibody was incubated successively with the prepared cell mixtures and avidin-coated magnetic beads, microscopic observation revealed that each sperm cell was bound by two or more magnetic beads, in the head, neck, mid-piece, or flagellum. A full single-source short tandem repeat profile could be obtained in 80% of mixed samples containing 10(3) sperm cells/mL and in all samples containing ≥10(4) sperm cells/mL. For dried vaginal swab specimens, the rate of successful detection was 100% in both flocked and cotton swabs preserved for 1 day, 87.5% in flocked swabs and 40% in cotton swabs preserved for 3 days, and 40% in flocked swabs and 16.67% in cotton swabs preserved for 10 days. Our findings suggest that immunomagnetic bead-based separation is potentially a promising alternative to conventional methods for isolating sperm cells from mixed forensic samples.

Identification of Novel Low Molecular Weight Serum Peptidome Biomarkers for Non‐Small Cell Lung Cancer (NSCLC)

To identify discriminating protein patterns in serum samples among non-small cell lung cancer (NSCLC), chronic obstructive pulmonary disease (COPD), pneumonia, and healthy controls. To discover specific low molecular weight (LMW) serum peptidome biomarkers and establish a diagnostic pattern for NSCLCby using proteomic technology. We used magnetic bead-based separation followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify patients with NSCLC, COPD, and pneumonia. A total of 154 serum samples were analyzed in this study, among which there were 60 serum samples from NSCLC patients, 30 from patients with other lung-related diseases (16 pneumonia patients and 14 patients with COPD) as disease controls, and 64 from healthy volunteers as healthy control. The mass spectra, analyzed using ClinProTools software, distinguished between cancer patients and healthy individuals based on GA algorithm model. In this study, we generated numerous discriminating m/z peaks as well as disease-specific discrimination peaks. A set of five potential biomarkers (m/z: 7,763.24, 1,012.61, 4,153.16, 1,450.55, and 2,878.89) could be used as the diagnostic biomarkers to distinguish NSCLCpatients from healthy controls. In the training set, patients with NSCLC could be identified with sensitivity of 97.5% and specificity of 98.8%. Similar results were obtained in the testing set, showing 80.7% sensitivity and 91.2% specificity. Our study demonstrated that a combined application of magnetic beads with MALDI-TOF MS technique was suitable for identification of serum biomarkers for NSCLC.

Serum peptidome profiling in patients with gastric cancer

To identify discriminating protein patterns in serum samples between gastric cancer patients (early and advanced stages) and healthy controls. We used magnetic bead-based separation followed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to identify patients with gastric cancer. In total, serum samples from 62 gastric cancer patients (32 in the training set and 30 in the test set; 19 of which had early-stage tumors and 43 of which had advanced-stage tumors) and 64 healthy controls (32 in the training set and 32 in the test set) were analyzed. The mass spectra, analyzed using ClinProTools software, distinguished between cancer patients and healthy individuals based on three different algorithm models. In the training set, patients with gastric cancer could be identified with a mean sensitivity of 94.7% and a mean specificity of 99%. Similar results were obtained with the test set, showing 79.3% sensitivity and 86.5% specificity. Our study demonstrates the high sensitivity and specificity of screening serum protein patterns using MALDI-TOF MS for the identification of patients with gastric cancer.

Efficient magnetic bead-based separation of HIV-1-infected cells using an improved reporter virus system reveals that p53 up-regulation occurs exclusively in the virus-expressing cell population

HIV-1 infection in cell lines is very efficient, since the target population is clonal and highly dividing. However, infection of primary cells such as CD4 T lymphocytes and monocyte-derived macrophages is much more difficult, resulting in a very small percentage of infected cells. In order to study events occurring in productively infected primary cells, we determined that a way to isolate this population from bystander cells was needed. We engineered a novel HIV-1-based reporter virus called NL4-3-IRES-HSA that allows for the magnetic separation of cells infected with fully competent virions. This X4-using virus encodes for the heat-stable antigen (HSA/murine CD24) without the deletion of any viral genes by introducing an IRES sequence between HSA and the auxiliary gene Nef. Using commercial magnetic beads, we achieved efficient purification of HIV-1-infected cells (i.e. purity >85% and recovery >90%) from diverse primary cell types at early time points following infection. We used this system to accurately quantify p53 protein levels in both virus-infected and uninfected bystander primary CD4(+) T cells. We show that p53 up-regulation occurs exclusively in the infected population. We devised a strategy that allows for an efficient separation of HIV-1 infected cells from bystanders. We believe that this new reporter virus system will be of great help to study in depth how HIV-1 interacts with its host in a primary cells context.

Applying Magnetic Bead Separation / MALDI-TOF Mass Spectrometry to Human Tear Fluid Proteome Analysis

The proteins and peptides in tears play an important role in preserving the integrity and stability of the ocular surface. Proteomic analysis of tear films will enable us to detect early biological markers of eye diseases, however, it is often hampered by the small amount of tear volume and the low protein concentration. Here we adopted magnetic bead-based purification (ClinProt system) followed by matrix-assisted laser desorption/ionization timeof- flight mass spectrometry (MALDI-TOF-MS) to profile human tear proteins. Basal and reflex tear fluids were collected from normal healthy volunteers using glass microcapillary tubes. Reversed phase (C8) and weak cation exchange (WCX) magnetic beads were applied to obtain multiple components detected as clear signals. Principal component analysis showed a clear differentiation between basal and reflex tears. Among the key alterations, two markedly increased peaks in the reflex tear fluids at m/z 2422.12 and m/z 2721.29 were subsequently analyzed by tandem MS analysis and their source to be proline-rich protein 4 (PRP4). We conclude that magnetic beadbased separation combined with MALDI-TOF-MS (ClinProt MALDI-TOF) appears to be ideally suited for the first-line screening of peptides and proteins in tears.