Stanford Advisors

All Publications

  • Short Tandem Repeat DNA Profiling Using Perylene-Oligonucleotide Fluorescence Assay. Analytical chemistry Hernandez Bustos, A., Martiny, E., Bom Pedersen, N., Parvathaneni, R. P., Hansen, J., Ji, H. P., Astakhova, K. 2023


    We report an amplification-free genotyping method to determine the number of human short tandem repeats (STRs). DNA-based STR profiling is a robust method for genetic identification purposes such as forensics and biobanking and for identifying specific molecular subtypes of cancer. STR detection requires polymerase amplification, which introduces errors that obscure the correct genotype. We developed a new method that requires no polymerase. First, we synthesized perylene-nucleoside reagents and incorporated them into oligonucleotide probes that recognize five common human STRs. Using these probes and a bead-based hybridization approach, accurate STR detection was achieved in only 1.5 h, including DNA preparation steps, with up to a 1000-fold target DNA enrichment. This method was comparable to PCR-based assays. Using standard fluorometry, the limit of detection was 2.00 ± 0.07 pM for a given target. We used this assay to accurately identify STRs from 50 human subjects, achieving >98% consensus with sequencing data for STR genotyping.

    View details for DOI 10.1021/acs.analchem.3c00063

    View details for PubMedID 37183373

  • p38γ and p38δ as biomarkers in the interplay of colon cancer and inflammatory bowel diseases. Cancer communications (London, England) Fajardo, P., Taskova, M., Martín-Serrano, M. A., Hansen, J., Slott, S., Jakobsen, A. K., Wibom, M. L., Salegi, B., Muñoz, A., Barbachano, A., Sharma, A., Gubatan, J. M., Habtezion, A., Sanz-Ezquerro, J. J., Astakhova, K., Cuenda, A. 2022

    View details for DOI 10.1002/cac2.12331

    View details for PubMedID 35796643

  • Carbon Nanotubes-Potent Carriers for Targeted Drug Delivery in Rheumatoid Arthritis. Pharmaceutics Kofoed Andersen, C., Khatri, S., Hansen, J., Slott, S., Pavan Parvathaneni, R., Mendes, A. C., Chronakis, I. S., Hung, S., Rajasekaran, N., Ma, Z., Zhu, S., Dai, H., Mellins, E. D., Astakhova, K. 2021; 13 (4)


    Two types of single-walled carbon nanotubes (SWCNTs), HiPco- and carboxyl-SWCNT, are evaluated as drug carriers for the traditional anti-inflammatory drug methotrexate (MTX) and a small interfering RNA (siRNA) targeting NOTCH1 gene. The nanotubes are solubilized by PEGylation and covalently loaded with MTX. The coupling efficiency (CE%) of MTX is 77-79% for HiPco-SWCNT and 71-83% for carboxyl-SWCNT. siRNA is noncovalently attached to the nanotubes with efficiency of 90-97% for HiPco-SWCNT and 87-98% for carboxyl-SWCNT. Through whole body imaging in the second near-infrared window (NIR-II window, 1000-1700 nm), SWCNTs were found to be selectively accumulated in inflamed joints in a serum transfer mouse model. We further investigated the interactions of the siRNA/MTX loaded nanotubes with human blood and mice bone marrow cells. In human blood, both types of unloaded SWCNTs were associated with B cells, monocytes and neutrophils. Interestingly, loading with MTX suppressed SWCNTs targeting specificity to immune cells, especially B cells; in contrast, loading siRNA alone enhanced the targeting specificity. Loading both MTX and siRNA to carboxyl-SWCNT enhanced targeting specificity to neutrophils and monocytes but not B cells. The targeting specificity of SWCNTs can potentially be adjusted by altering the ratio of MTX and siRNA loaded. The combined results show that carbon nanotubes have the potential for delivery of cargo drugs specifically to immune cells involved in rheumatoid arthritis.

    View details for DOI 10.3390/pharmaceutics13040453

    View details for PubMedID 33801590

  • A First in Class Therapeutic Nanoparticle for Specific Targeting of Anti-citrullinated Protein Antibody Ameliorates Serum Transfer and Collagen Induced Arthritis Khatri, S., Hansen, J., Clausen, M., Kragstrup, T., Hung, S., Mellins, E., Astakhova, K. WILEY. 2020
  • Ultra-fast detection and quantification of nucleic acids by amplification-free fluorescence assay. The Analyst Uhd, J. n., Miotke, L. n., Ji, H. P., Dunaeva, M. n., Pruijn, G. J., Jørgensen, C. D., Kristoffersen, E. L., Birkedal, V. n., Yde, C. W., Nielsen, F. C., Hansen, J. n., Astakhova, K. n. 2020


    Two types of clinically important nucleic acid biomarkers, microRNA (miRNA) and circulating tumor DNA (ctDNA) were detected and quantified from human serum using an amplification-free fluorescence hybridization assay. Specifically, miRNAs hsa-miR-223-3p and hsa-miR-486-5p with relevance for rheumatoid arthritis and cancer related mutations BRAF and KRAS of ctDNA were directly measured. The required oligonucleotide probes for the assay were rationally designed and synthesized through a novel "clickable" approach which is time and cost-effective. With no need for isolating nucleic acid components from serum, the fluoresence-based assay took only 1 hour. Detection and absolute quantification of targets was successfully achieved despite their notoriously low abundance, with a precision down to individual nucleotides. Obtained miRNA and ctDNA amounts showed overall a good correlation with current techniques. With appropriate probes, our novel assay and signal boosting approach could become a useful tool for point-of-care measuring other low abundance nucleic acid biomarkers.

    View details for DOI 10.1039/d0an00676a

    View details for PubMedID 32648858

  • Citrullinated Peptide Epitope Targets Therapeutic Nanoparticles to Human Neutrophils. Bioconjugate chemistry Khatri, S. n., Hansen, J. n., Mendes, A. C., Chronakis, I. S., Hung, S. C., Mellins, E. D., Astakhova, K. n. 2019


    Multiple drugs have been proposed for reducing harsh symptoms of human rheumatic diseases. However, a targeted therapy with mild to no side effects is still missing. In this study, we have prepared and tested a series of therapeutic nanoparticles for specific targeting of human neutrophils associated with rheumatoid arthritis. In doing this, a series of citrullinated peptide epitopes derived from human proteins, fibrinogen, vimentin, and histone 3, were screened with regard to specific recognition of neutrophils. The most potent epitope proved to be a mutated fragment of an alpha chain in human fibrinogen. Next, a straightforward synthetic strategy was developed for nanoparticles decorated with this citrullinated peptide epitope and an antisense oligonucleotide targeting disease associated microRNA miR-125b-5p. Our study shows that the nanoparticles specifically recognize neutrophils and knock down miR-125b-5p, with no apparent toxicity to human cells. In contrast to organic dendrimers, chitosan-hyaluronic acid formulations do not activate human innate immune response. Our data proves that the strategy we report herein is effective in developing peptide epitopes for decorating delivery vehicles bearing biological drugs, targeted to a specific cell type.

    View details for DOI 10.1021/acs.bioconjchem.9b00518

    View details for PubMedID 31524379