6 research outputs found
SARS-CoV-2 detection with aptamer-functionalized gold nanoparticles
A rapid detection test for SARS-CoV-2 is urgently required to monitor virus spread and containment. Here, we describe a test that uses nanoprobes, which are gold nanoparticles functionalized with an aptamer specific to the spike membrane protein of SARS-CoV-2. An enzyme-linked immunosorbent assay confirms aptamer binding with the spike protein on gold surfaces. Protein recognition occurs by adding a coagulant, where nanoprobes with no bound protein agglomerate while those with sufficient bound protein do not. Using plasmon absorbance spectra, the nanoprobes detect 16 nM and higher concentrations of spike protein in phosphate-buffered saline. The time-varying light absorbance is examined at 540 nm to determine the critical coagulant concentration required to agglomerates the nanoprobes, which depends on the protein concentration. This approach detects 3540 genome copies/ÎĽl of inactivated SARS-CoV-2
Xrs2 C-terminus mediates Tel1-dependent meiotic double-strand break interference
During meiosis, the programmed formation of DNA double-strand breaks (DSBs) by Spo11, a conserved topoisomerase-like protein, initiates homologous recombination that leads to crossovers between homologous chromosomes, essential for accurate segregation and genome evolution. Because DSBs are a threat to genome integrity, their number, distribution and timing of formation are regulated during the meiotic program. In S. cerevisiae, DSB interference prevents the coincident formation of DNA double-strand breaks (DSBs) in neighboring hotspots through a Tel1/ATM dependent mechanism that remains unexplored. Here, we demonstrate that Tel1 is recruited to meiotic DSBs hotspots in response to Spo11-DSB formation. Tel1 also localizes to chromosomal axis sites in a DSB-dependent manner, thus supporting the TLAC model that postulates meiotic DSBs are being formed within the chromosome axis environment. Tel1 recruitment to meiotic DSBs, DSB interference and the meiotic DNA damage checkpoint are dependent on both the Tel1-FATC domain and the Cterminal moiety of Xrs2, known to mediate Tel1-Xrs2 interaction in somatic cells. However, in a Xrs2 (FxF/Y) mutant DSBs interference remains functional despite Tel1 binding to DSB sites being significantly reduced and the Tel1-dependent DNA damage checkpoint abolished. Altogether, this work highlights the complex regulation of Tel1 multiple functions in meiotic cells, and fine-tuning through interaction with Xrs2
Fibroblasts Accelerate Formation and Improve Reproducibility of 3D Cellular Structures Printed with Magnetic Assistance
Fibroblasts (mouse, NIH/3T3) are combined with MDA-MB-231 cells to accelerate the formation and improve the reproducibility of 3D cellular structures printed with magnetic assistance. Fibroblasts and MDA-MB-231 cells are cocultured to produce 12.5 : 87.5, 25 : 75, and 50 : 50 total population mixtures. These mixtures are suspended in a cell medium containing a paramagnetic salt, Gd-DTPA, which increases the magnetic susceptibility of the medium with respect to the cells. A 3D monotypic MDA-MB-231 cellular structure is printed within 24 hours with magnetic assistance, whereas it takes 48 hours to form a similar structure through gravitational settling alone. The maximum projected areas and circularities, and cellular ATP levels of the printed structures are measured for 336 hours. Increasing the relative amounts of the fibroblasts mixed with the MDA-MB-231 cells decreases the time taken to form the structures and improves their reproducibility. Structures produced through gravitational settling have larger maximum projected areas and cellular ATP, but are deemed less reproducible. The distribution of individual cell lines in the cocultured 3D cellular structures shows that printing with magnetic assistance yields 3D cellular structures that resemble in vivo tumors more closely than those formed through gravitational settling. The results validate our hypothesis that (1) fibroblasts act as a “glue” that supports the formation of 3D cellular structures, and (2) the structures are produced more rapidly and with greater reproducibility with magnetically assisted printing than through gravitational settling alone. Printing of 3D cellular structures with magnetic assistance has applications relevant to drug discovery, lab-on-chip devices, and tissue engineering
PRDM9 interactions with other proteins provide a link between recombination hotspots and the chromosomal axis in meiosis.
In mammals, meiotic recombination occurs at 1- to 2-kb genomic regions termed hotspots, whose positions and activities are determined by PRDM9, a DNA-binding histone methyltransferase. We show that the KRAB domain of PRDM9 forms complexes with additional proteins to allow hotspots to proceed into the next phase of recombination. By a combination of yeast-two hybrid assay, in vitro binding, and coimmunoprecipitation from mouse spermatocytes, we identified four proteins that directly interact with PRDM9\u27s KRAB domain, namely CXXC1, EWSR1, EHMT2, and CDYL. These proteins are coexpressed in spermatocytes at the early stages of meiotic prophase I, the limited period when PRDM9 is expressed. We also detected association of PRDM9-bound complexes with the meiotic cohesin REC8 and the synaptonemal complex proteins SYCP3 and SYCP1. Our results suggest a model in which PRDM9-bound hotspot DNA is brought to the chromosomal axis by the action of these proteins, ensuring the proper chromatin and spatial environment for subsequent recombination events. Mol Biol Cell 2017 Feb 1; 28(3):488-499
Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies
Abstract Aberrantly expressed onco-mucin 16 (MUC16) and its post-cleavage generated surface tethered carboxy-terminal (MUC16-Cter) domain are strongly associated with poor prognosis and lethality of pancreatic (PC) and non-small cell lung cancer (NSCLC). To date, most anti-MUC16 antibodies are directed towards the extracellular domain of MUC16 (CA125), which is usually cleaved and shed in the circulation hence obscuring antibody accessibility to the cancer cells. Herein, we establish the utility of targeting a post-cleavage generated, surface-tethered oncogenic MUC16 carboxy-terminal (MUC16-Cter) domain by using a novel chimeric antibody in human IgG1 format, ch5E6, whose epitope expression directly correlates with disease severity in both cancers. ch5E6 binds and interferes with MUC16-associated oncogenesis, suppresses the downstream signaling pFAK(Y397)/p-p70S6K(T389)/N-cadherin axis and exert antiproliferative effects in cancer cells, 3D organoids, and tumor xenografts of both PC and NSCLC. The robust clinical correlations observed between MUC16 and N-cadherin in patient tumors and metastatic samples imply ch5E6 potential in targeting a complex and significantly occurring phenomenon of epithelial to mesenchymal transition (EMT) associated with disease aggressiveness. Our study supports evaluating ch5E6 with standard-of-care drugs, to potentially augment treatment outcomes in malignancies inflicted with MUC16-associated poor prognosis