Background

Rifampicin or rifampin (R) is a common drug used to treat inactive meningitis, cholestatic pruritus and tuberculosis (TB), and it is generally prescribed for long-term administration under regulated dosages. Constant monitoring of rifampicin is important for controlling the side effects and preventing overdose caused by chronic medication. In this study, we present an easy to use, effective and less costly method for detecting residual rifampicin in urine samples using protein (bovine serum albumin, BSA)-stabilized gold nanoclusters (BSA-Au NCs) adsorbed on a paper substrate in which the concentration of rifampicin in urine can be detected via fluorescence quenching. The intensity of the colorimetric assay performed on the paper-based platforms can be easily captured using a digital camera and subsequently analyzed.

Results

The decreased fluorescence intensity of BSA-Au NCs in the presence of rifampicin allows for the sensitive detection of rifampicin in a range from 0.5 to 823 µg/mL. The detection limit for rifampicin was measured as 70 ng/mL. The BSA-Au NCs were immobilized on a wax-printed paper-based platform and used to conduct real-time monitoring of rifampicin in urine.

Conclusion

We have developed a robust, cost-effective, and portable point-of-care medical diagnostic platform for the detection of rifampicin in urine based on the ability of rifampicin to quench the fluorescence of immobilized BSA-Au NCs on wax-printed papers. The paper-based assay can be further used for the detection of other specific analytes via surface modification of the BSA in BSA-Au NCs and offers a useful tool for monitoring other diseases.

Background

Cells explore the surfaces of materials through membrane-bound receptors, such as the integrins, and use them to interact with extracellular matrix molecules adsorbed on the substrate surfaces, resulting in the formation of focal adhesions. With recent advances in nanotechnology, biosensors and bioelectronics are being fabricated with ever decreasing feature sizes. The performances of these devices depend on how cells interact with nanostructures on the device surfaces. However, the behavior of cells on nanostructures is not yet fully understood. Here we present a systematic study of cell-nanostructure interaction using polymeric nanopillars with various diameters.

Results

We first checked the viability of cells grown on nanopillars with diameters ranging from 200 nm to 700 nm. It was observed that when cells were cultured on the nanopillars, the apoptosis rate slightly increased as the size of the nanopillar decreased. We then calculated the average size of the focal adhesions and the cell-spreading area for focal adhesions using confocal microscopy. The size of focal adhesions formed on the nanopillars was found to decrease as the size of the nanopillars decreased, resembling the formations of nascent focal complexes. However, when the size of nanopillars decreased to 200 nm, the size of the focal adhesions increased. Further study revealed that cells interacted very strongly with the nanopillars with a diameter of 200 nm and exerted sufficient forces to bend the nanopillars together, resulting in the formation of larger focal adhesions.

Conclusions

We have developed a simple approach to systematically study cell-substrate interactions on physically well-defined substrates using size-tunable polymeric nanopillars. From this study, we conclude that cells can survive on nanostructures with a slight increase in apoptosis rate and that cells interact very strongly with smaller nanostructures. In contrast to previous observations on flat substrates that cells interacted weakly with softer substrates, we observed strong cell-substrate interactions on the softer nanopillars with smaller diameters. Our results indicate that in addition to substrate rigidity, nanostructure dimensions are additional important physical parameters that can be used to regulate behaviour of cells.

Replication forks are vulnerable to wayward nuclease activities. We report here our discovery of a new member in guarding genome stability at replication forks. We previously isolated a Drosophila mutation, wuho (wh, no progeny), characterized by a severe fertility defect and affecting expression of a protein (WH) in a family of conserved proteins with multiple WD40 repeats. Knockdown of WH by siRNA in Drosophila, mouse, and human cultured cells results in DNA damage with strand breaks and apoptosis through ATM/Chk2/p53 signaling pathway. Mice with mWh knockout are early embryonic lethal and display DNA damage. We identify that the flap endonuclease 1 (FEN1) is one of the interacting proteins. Fluorescence microscopy showed the localization of WH at the site of nascent DNA synthesis along with other replication proteins, including FEN1 and PCNA. We show that WH is able to modulate FEN1's endonucleolytic activities depending on the substrate DNA structure. The stimulatory or inhibitory effects of WH on FEN1's flap versus gap endonuclease activities are consistent with the proposed WH's functions in protecting the integrity of replication fork. These results suggest that wh is a new member of the guardians of genome stability because it regulates FEN1's potential DNA cleavage threat near the site of replication.

Galectin-3 has been reported to regulate the functions of a number of immune cell types. We previously reported that galectin-3 is translocated to immunological synapses in T cells upon T-cell receptor engagement, where it associates with ALG-2-interacting protein X (Alix). Alix is known to coordinate with the endosomal sorting complex required for transport (ESCRT) to promote human immunodeficiency virus (HIV)-1 virion release. We hypothesized that galectin-3 plays a role in HIV-1 viral budding. Cotransfection of cells of the Jurkat T line with galectin-3 and HIV-1 plasmids resulted in increased HIV-1 budding, and suppression of galectin-3 expression by RNAi in Hut78 and primary CD4+ T cells led to reduced HIV-1 budding. We used immunofluorescence microscopy to observe the partial colocalization of galectin-3, Alix and Gag in HIV-1-infected cells. Results from co-immunoprecipitation experiments indicate that galectin-3 expression promotes Alix-Gag p6 association, whereas the results of Alix knockdown suggest that galectin-3 promotes HIV-1 budding through Alix. HIV-1 particles released from galectin-3-expressing cells acquire the galectin-3 protein in an Alix-dependent manner, with proteins primarily residing inside the virions. We also found that the galectin-3 N-terminal domain interacts with the proline-rich region of Alix. Collectively, these results suggest that endogenous galectin-3 facilitates HIV-1 budding by promoting the Alix-Gag p6 association.

Circulating fetal nucleated cells (CFNCs) in maternal blood offer an ideal source of fetal genomic DNA for noninvasive prenatal diagnostics (NIPD). We developed a class of nanoVelcro microchips to effectively enrich a subcategory of CFNCs, i.e., circulating trophoblasts (cTBs) from maternal blood, which can then be isolated with single-cell resolution by a laser capture microdissection (LCM) technique for downstream genetic testing. We first established a nanoimprinting fabrication process to prepare the LCM-compatible nanoVelcro substrates. Using an optimized cTB-capture condition and an immunocytochemistry protocol, we were able to identify and isolate single cTBs (Hoechst+/CK7+/HLA-G+/CD45-, 20 μm > sizes > 12 μm) on the imprinted nanoVelcro microchips. Three cTBs were polled to ensure reproducible whole genome amplification on the cTB-derived DNA, paving the way for cTB-based array comparative genomic hybridization (aCGH) and short tandem repeats analysis. Using maternal blood samples collected from expectant mothers carrying a single fetus, the cTB-derived aCGH data were able to detect fetal genders and chromosomal aberrations, which had been confirmed by standard clinical practice. Our results support the use of nanoVelcro microchips for cTB-based noninvasive prenatal genetic testing, which holds potential for further development toward future NIPD solution.

A glycan-stimulated and poly(3,4-ethylene-dioxythiophene)s (PEDOT)-based nanomaterial platform is fabricated to purify circulating tumor cells (CTCs) from blood samples of prostate cancer (PCa) patients. This new platform, phenylboronic acid (PBA)-grafted PEDOT NanoVelcro, combines the 3D PEDOT nanosubstrate, which greatly enhances CTC capturing efficiency, with a poly(EDOT-PBA-co-EDOT-EG3) interfacial layer, which not only provides high specificity for CTC capture upon antibody conjugation but also enables competitive binding of sorbitol to gently release the captured cells. CTCs purified by this PEDOT NanoVelcro chip provide well-preserved RNA transcripts for the analysis of the expression level of several PCa-specific RNA biomarkers, which may provide clinical insights into the disease.