Biology subjects

Makrigiorgos, G. M.

Publications and source records attributed to Makrigiorgos, G. M..

7 recordsLinked to original sources

bioRxiv2025

MEDUSA: Maintaining Entire DNA Duplexes for Utmost Sequencing Accuracy

Highly accurate DNA sequencing is essential for many applications. Duplex sequencing-based methods achieve unparalleled accuracy by requiring reads from both strands of the original DNA duplex to match. Yet, methods to prepare dsDNA for sequencing may resynthesize portions of each DNA duplex and cause base damage errors on one strand to become indistinguishable from true mutations on both strands. Here, we report MEDUSA (Maintaining Entire DNA Duplexes for Utmost Sequencing Accuracy) which minimizes dsDNA resynthesis to maximize duplex sequencing accuracy and yield. MEDUSA carefully repairs and blunts fragmented dsDNA, then employs apyrase to digest residual dNTPs, followed by restricted dA-tailing to prevent resynthesis. MEDUSA affords full genome coverage in a simplified protocol that is broadly compatible with dsDNA fragmentation and library preparation kits. We benchmarked MEDUSA on sheared genomic DNA and cell-free DNA and found a residual SNV frequency within a median 1.23-fold (range 0.92-1.88; p < 0.001) of what was expected if resynthesis was mostly blocked, but with full genome coverage and duplex yields within a median 1.02-fold (range 0.33 - 1.46; p = 0.258) of traditional methods that do not limit resynthesis. In all, MEDUSA could enable high breadth or depth of duplex sequencing while limiting false discovery. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/687202v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1ddbc77org.highwire.dtl.DTLVardef@802ca9org.highwire.dtl.DTLVardef@f443ecorg.highwire.dtl.DTLVardef@974bdb_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Genome-wide extraction of differentially methylated DNA regions using adapter-anchored proximity primers

The epigenetic deregulation of CpG islands (CGIs) plays a crucial role in cancer initiation and progression. CGIs comprise 1-2% of the human genome and are rich in differentially methylated regions (DMRs) that can serve as cancer biomarkers in clinical samples and liquid biopsies. Focusing epigenetic sequencing on CpG-rich sequences, including CGIs and avoiding non-informative regions, offers an efficient and sensitive approach for cancer identification and tracking, especially within samples containing excess of unaltered, normal DNA. To this end, we have developed Adaptor-anchored Methylation amplification via Proximity Primers (aMAPP), a versatile PCR-based enrichment method. aMAPP employs specially designed primers to selectively enrich either methylated or unmethylated CpGs, depending on the upstream methylation conversion method employed. aMAPP achieves high coverage of genome-wide CGIs and detects hundreds of DMRs in tumor samples compared to adjacent normal tissue using ultra-low depth sequencing ([~]300,000 reads). It enables tracing of aberrant methylation down to allelic frequency 0.01% in dilutions of tumor DNA and in cell-free DNA samples, can be applied using picogram amounts of DNA, and can be adapted to enrich either small panels of cancer-specific DMRs, or the majority (>90%) of genomic CGIs and CpGs. aMAPP offers a simple, cost-effective, and highly sensitive approach for capturing the epigenetic footprint of genome-wide CpGs and identifying aberrantly methylated or un-methylated genomic regions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/660377v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@b8b303org.highwire.dtl.DTLVardef@1f0a70corg.highwire.dtl.DTLVardef@67391dorg.highwire.dtl.DTLVardef@d6ac75_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Methyl-CODEC enables simultaneous methylation and duplex sequencing

DNA mutations and methylation often contribute to disease development in a synergistic manner. While duplex sequencing is the most accurate method for detecting DNA mutations, it typically lacks the ability to simultaneously assess methylation or requires many reads. Here, we developed Methyl-CODEC to enable simultaneous methylation sequencing and duplex sequencing using single read pairs. To achieve this, Methyl-CODEC links an enzymatically deaminated sense strand to the reverse complement of the antisense strand, which is protected from conversion by using conversion-resistant dCTPs in the strand linking step of CODEC. Methyl-CODEC shows high concordance with standard enzymatic or bisulfite based whole genome methylation sequencing, while also uniquely preserving the original DNA sequence. This improves genetic sequencing accuracy, enables better alignment for next-generation sequencing (NGS), and distinguishes C>T mutations from unmethylated Cs. It also identifies rare mutations including those producing methylated Cs, which are enriched in CpG contexts. Methyl-CODEC opens new horizons for enhanced detection of biomarkers in cancer and molecular medicine.

genomics↗

bioRxiv2024

Aerosol delivery of immunotherapy and Hesperetin-loaded nanoparticles increases survival in a murine lung cancer model

PurposeStudies have shown that flavonoids like Hesperetin, an ACE2 receptor agonist with antioxidant and pro-apoptotic activity, can induce apoptosis in cancer cells. ACE2 receptors are abundant in lung cancer cells. Here, we explored the application of Hesperetin bound to PLGA-coated nanoparticles (Hesperetin-nanoparticles, HNPs), and anti-CD40 antibody as an aerosol treatment for lung tumor-bearing mice. MethodsIn-vitro and in-vivo studies were performed in human A549 (ATCC) and murine LLC1 (ATCC) lung cancer cell lines. Hesperetin Nanoparticles (HNP) of about 60nm diameter were engineered using a nano-formulation microfluidic technique. A syngeneic orthotopic murine model of lung adenoma was generated in wild (+/+) C57/BL6 background mice with luciferase-positive cell line LLC1 cells. Lung tumor-bearing mice were treated via aerosol inhalation with HNP, anti-CD40 antibody, or both. Survival was used to analyze the efficacy of aerosol treatment. Cohorts were also analyzed for body condition score, weight, and liver and kidney function. ResultsAnalysis of an orthotopic murine lung cancer model demonstrates a differential uptake of the HNP and anti-CD40 by cancer cells relative to normal cells. A higher survival rate, relative to untreated controls, was observed when aerosol treatment with HNP was added to treatment via anti-CD40 (p<0.001), as compared to CD40 alone (p<0.01). Moreover, 2 out of 9 tumor-bearing mice survived long term, and their tumors diminished. These 2 mice were shown to be refractory to subsequent development of subcutaneous tumors, indicating systemic resilience to tumor formation. ConclusionWe successfully established increased therapeutic efficacy of anti-CD40 and HNP in an orthotopic murine lung cancer model using inhalation-based administration. Our findings open the possibility of improved lung cancer treatment using flavonoids and immuno-adjuvants.

bioengineering↗

bioRxiv2021

CODEC enables 'single duplex' sequencing

Detecting mutations as rare as a single molecule is crucial in many fields such as cancer diagnostics and aging research but remains challenging. Third generation sequencers can read a double-stranded DNA molecule (a single duplex) in whole to identify true mutations on both strands apart from false mutations on either strand but with limited accuracy and throughput. Although next generation sequencing (NGS) can track dissociated strands with Duplex Sequencing, the need to sequence each strand independently severely diminishes its throughput. Here, we developed a hybrid method called Concatenating Original Duplex for Error Correction (CODEC) that combines the massively parallel nature of NGS with the single-molecule capability of third generation sequencing. CODEC physically links both strands to enable NGS to sequence a single duplex with a single read pair. By comparing CODEC and Duplex Sequencing, we showed that CODEC achieved a similar error rate (10-6) with 100 times fewer reads and conferred single duplex resolution to most major NGS workflows.

bioengineering↗

bioRxiv2021

MAESTRO affords 'breadth and depth' for mutation testing

The ability to assay large numbers of low-abundance mutations is crucial in biomedicine. Yet, the technical hurdles of sequencing multiple mutations at extremely high depth and accuracy remain daunting. For sequencing low-level mutations, its either depth or breadth but not both. Here, we report a simple and powerful approach to accurately track thousands of distinct mutations with minimal reads. Our technique called MAESTRO (minor allele enriched sequencing through recognition oligonucleotides) employs massively-parallel mutation enrichment to empower duplex sequencing--one of the most accurate methods--to track up to 10,000 low-frequency mutations with up to 100-fold less sequencing. In example use cases, we show that MAESTRO could enable mutation validation from cancer genome sequencing studies. We also show that it could track thousands of mutations from a patients tumor in cell-free DNA, which may improve detection of minimal residual disease from liquid biopsies. In all, MAESTRO improves the breadth, depth, accuracy, and efficiency of mutation testing.

molecular biology↗

bioRxiv2018

Targeted Drug Delivery by Radiation-Induced Tumor Vascular Modulation

Effective drug delivery is severely restricted by the presence of complex pathophysiological barriers in solid tumors. In human pancreatic adenocarcinoma, mature and hypopermeable tumor blood vessels limit the permeation and penetration of chemo or nanotherapeutics to cancer cells and substantially reduce the treatment efficacy. New, clinically-viable strategies are therefore sought to breach the neoplastic barriers that prevent optimal tumor-specific drug delivery. Here, we present an original idea to boost targeted drug delivery by selectively knocking down the tumor vascular barrier in a poorly permeable human pancreatic cancer model. For the first time, we demonstrate that clinical irradiation (10 Gy, 6 MV) can induce tumor vascular modulation when combined with tumor endothelial-targeting gold nanoparticles. Active disruption of tumor blood vessels by nanoparticle-combined radiotherapy led to increased vessel permeability and improved tumor uptake of two prototypical model nanodrugs: i) a short-circulating nanocarrier with MR-sensitive gadolinium (Gad-NC; 8 kDa; t1/2=1.5 h) and ii) a long-circulating nanocarrier with fluorescence-sensitive NIR dye (FL-NC; 30 kDa; t1/2=25 h). Functional changes in the altered tumor vessel dynamics, measured by relative changes in permeability (Ktrans), flux rate (Kep) and extracellular interstitial volume (Ve) were consistent with the concomitant increase in nanodrug delivery. This combination of radiation-induced antivascular and nanodrug-mediated anti-tumor treatment offers high therapeutic benefit for tumors with pathophysiology that restricts efficient drug delivery.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC=\"FIGDIR/small/268714_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (84K):\norg.highwire.dtl.DTLVardef@a6df8borg.highwire.dtl.DTLVardef@19e3179org.highwire.dtl.DTLVardef@c223aaorg.highwire.dtl.DTLVardef@1b3a08d_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT\n\nC_FIG

bioengineering↗