Distribution of Mycobacterium tuberculosis lineages in South America

Molecular genotyping of Mycobacterium tuberculosis allows for the identification of circulating lineages and sublineages in the population and their relationship with migratory movements. The purpose of this review is to describe the phylogeography of Mycobacterium tuberculosis reported in South American countries that was analyzed using genotyping tools, analyze the Tuberculosis hotspots for the region and determine the impact of the COVID-19 pandemic on the Tuberculosis control program. The Latin American Mediterranean (LAM) sublineage belonging to the Euro-American lineage (Lineage 4) presents the highest prevalence in South America and is followed by the Beijing sublineage belonging to the East Asian lineage (Lineage 2). The Beijing sublineage is considered of worldwide interest because of its association with multidrug-resistant tuberculosis (MDR-TB), which is almost entirely distributed in South America, with Peru being the country with the highest prevalence for this sublineage. On the other hand, the Indo-Oceanic (Lineage 1), India-East Asia (Lineage 3) and West- African 2 (Lineage 6) sublineages have been reported with lower prevalence in South America. The molecular techniques used in the genotyping studies for Mycobacterium tuberculosis in South America were as follows: typing by complementary oligonucleotide spacer sequences (Spoligotyping), restriction-hybridization patterns (IS6110-RFLP, PGRS-RFLP), mycobacterial interspaced repeat units-variable number tandem repeats (MIRU-VNTR) and whole genome sequencing (WGS). At present, Brazil and Peru are the hotspots for tuberculosis and MDR-TB in South America, where the control of tuberculosis wholly affected by the COVID-19 pandemic. Thus, there have been significant impacts on containment programs and possible post-pandemic scenarios such that scientific contributions will need to be evaluated and implemented with new strategies for prevention, diagnosis, treatment and control of Tuberculosis.

Characterization of BisI Homologs

BisI is a sequence-specific and 5-methylcytosine (m5C)-dependent restriction endonuclease (REase), that cleaves the modified DNA sequence Gm5CNGC (G indicates that the cytosine opposite to G is modified). We expressed and purified a number of BisI homologs from sequenced bacterial genomes and used Illumina sequencing to determine the Pam7902I (Esp638I-like) cleavage sites in phage Xp12 DNA. One BisI homolog KpnW2I is EcoBLMcrX-like, cleaving GCNGC/RCNGY/RCNRC sites with m5C. We also cloned and expressed three BisI homologs from metagenome sequences derived from thermophilic sources. One enzyme EsaTMI is active at 37 to 65°C. EsaHLI cleaves GCNGC sites with three to four m5C and is active up to 50°C. In addition, we determined the number and position of m5C in BisI sites for efficient cleavage. BisI cleavage efficiency of GCNGC site is as following: Gm5CNGC (two internal m5C) > Gm5CNGC (one internal m5C) > GCNGm5C (one external m5C) > > GCNGC (unmodified). Three or four m5C in GCNGC site also supports BisI cleavage although partial inhibition was observed on duplex oligos with four m5C. BisI can be used to partially cleave a desired GCNGC site targeted with a complementary oligonucleotide (hemi-methylated). The m5C-dependent BisI variants will be useful for epigenetic research.

A Timely Review of Cross-Kingdom Regulation of Plant-Derived MicroRNAs

MicroRNAs (miRNAs) belong to a class of non-coding RNAs that suppress gene expression by complementary oligonucleotide binding to the sites in target messenger RNAs. Numerous studies have demonstrated that miRNAs play crucial role in virtually all cellular processes of both plants and animals, such as cell growth, cell division, differentiation, proliferation and apoptosis. The study of rice MIR168a has demonstrated for the first time that exogenous plant MIR168a influences cholesterol transport in mice by inhibiting low-density lipoprotein receptor adapter protein 1 expression. Inspired by this finding, the cross-kingdom regulation of plant-derived miRNAs has drawn a lot of attention because of its capability to provide novel therapeutic agents in the treatment of miRNA deregulation-related diseases. Notably, unlike mRNA, some plant miRNAs are robust because of their 3′ end modification, high G, C content, and the protection by microvesicles, miRNAs protein cofactors or plant ingredients. The stability of these small molecules guarantees the reliability of plant miRNAs in clinical application. Although the function of endogenous miRNAs has been widely investigated, the cross-kingdom regulation of plant-derived miRNAs is still in its infancy. Herein, this review summarizes the current knowledge regarding the anti-virus, anti-tumor, anti-inflammatory, anti-apoptosis, immune modulation, and intestinal function regulation effects of plant-derived miRNAs in mammals. It is expected that exploring the versatile role of plant-derived miRNAs may lay the foundation for further study and application of these newly recognized, non-toxic, and inexpensive plant active ingredients.

A DNA sensor based on upconversion nanoparticles and two-dimensional dichalcogenide materials

We demonstrate the fabrication of a new DNA sensor that is based on the optical interactions occurring between oligonucleotide-coated NaYF4:Yb3+;Er3+ upconversion nanoparticles and the two-dimensional dichalcogenide materials, MoS2 and WS2. Monodisperse upconversion nanoparticles were functionalized with single-stranded DNA endowing the nanoparticles with the ability to interact with the surface of the two-dimensional materials via van der Waals interactions leading to subsequent quenching of the upconversion fluorescence. By contrast, in the presence of a complementary oligonucleotide target and the formation of double-stranded DNA, the upconversion nanoparticles could not interact with MoS2 and WS2, thus retaining their inherent fluorescence properties. Utilizing this sensor we were able to detect target oligonucleotides with high sensitivity and specificity whilst reaching a concentration detection limit as low as 5 mol·L−1, within minutes.

P01.05 Development of signal amplification for spatially-resolved, highly multiplexed biomarker analysis of human tumor tissues

BackgroundCharacterizing the complexities of the tumor microenvironment is fundamental to understanding cancer. Spatial relationships between infiltrating immune cells and the existing cellular matrix are now recognized as key determinants of tumor heterogeneity. Nevertheless, most available technologies for studying cells within the context of their tissue microenvironment, like traditional immunofluorescence (IF) and immunohistochemistry (IHC), are limited—allowing the visualization of only a few markers at a time.Materials and MethodsCO-Detection by indEXing (CODEX®) technology has overcome this limitation through a DNA-based labeling strategy, involving the sequential addition and removal of dye-labeled oligonucleotide reporters to antibodies equipped with complementary oligonucleotide tags. In this manner, it is possible to visualize tens of antibodies in the same tissue, in situ and at cellular resolution. Additionally, CODEX® interfaces with existing inverted microscopes and provides a cost-effective, fully automated platform for ultra-high plex immunofluorescence imaging. We have expanded the CODEX® platform to include Tyramide Signal Amplification of weak fluorescent signals, i.e. from low-expression biomarkers. This approach was tested with key biomarkers used in routine analyses of the tumor microenvironment, including PD-L1, PD-1 and FOXP3.ResultsWe demonstrate >50X amplification of PD-L1, PD-1 and FOXP3 signals when compared to control tissues. Moreover, we successfully included our amplification step in the CODEX® labeling/imaging workflow, so that it was possible to analyze amplified PD-L1, PD-1 and FOXP3 signals concurrently with a panel of 20+ additional antibodies. Analysis of our data also generated unique biological insights, including increased PD-L1 expression in Treg cells and other tumor and stromal regions.ConclusionsOur findings demonstrate the feasibility of amplifying weak biomarker signals in the CODEX® workflow. Furthermore, our experiments were conducted on human formalin fixed paraffin embedded tumor tissues, thereby demonstrating the applicability of CODEX® analyses for clinical and translational research agendas.Disclosure InformationO. Braubach: A. Employment (full or part-time); Significant; Akoya Biosciences. S. Mistry: A. Employment (full or part-time); Significant; Akoya Biosciences. G. Dakshinamoorthy: A. Employment (full or part-time); Significant; Akoya Biosciences. J. Yuan: A. Employment (full or part-time); Significant; Akoya Biosciences. P. Noordam: A. Employment (full or part-time); Significant; Akoya Biosciences. J. Kim: A. Employment (full or part-time); Significant; Akoya Biosciences. W. Lee: A. Employment (full or part-time); Significant; Akoya Biosciences. J. Kennedy-Darling: A. Employment (full or part-time); Significant; Akoya Biosciences.

Generation and Characterization of a DNA-GCN4 Oligonucleotide-Peptide Conjugate: The Impact DNA/Protein Interactions on the Sensitization of DNA

Radiotherapy, the most common therapy for the treatment of solid tumors, exerts its effects by inducing DNA damage. To fully understand the extent and nature of this damage, DNA models that mimic the in vivo situation should be utilized. In a cellular context, genomic DNA constantly interacts with proteins and these interactions could influence both the primary radical processes (triggered by ionizing radiation) and secondary reactions, ultimately leading to DNA damage. However, this is seldom addressed in the literature. In this work, we propose a general approach to tackle these shortcomings. We synthesized a protein-DNA complex that more closely represents DNA in the physiological environment than oligonucleotides solution itself, while being sufficiently simple to permit further chemical analyses. Using click chemistry, we obtained an oligonucleotide-peptide conjugate, which, if annealed with the complementary oligonucleotide strand, forms a complex that mimics the specific interactions between the GCN4 protein and DNA. The covalent bond connecting the oligonucleotide and peptide constitutes a part of substituted triazole, which forms due to the click reaction between the short peptide corresponding to the specific amino acid sequence of GCN4 protein (yeast transcription factor) and a DNA fragment that is recognized by the protein. DNAse footprinting demonstrated that the part of the DNA fragment that specifically interacts with the peptide in the complex is protected from DNAse activity. Moreover, the thermodynamic characteristics obtained using differential scanning calorimetry (DSC) are consistent with the interaction energies calculated at the level of metadynamics. Thus, we present an efficient approach to generate a well-defined DNA-peptide conjugate that mimics a real DNA-peptide complex. These complexes can be used to investigate DNA damage under conditions very similar to those present in the cell.

Alpha-l-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce Efficient Splice Modulation In Vitro

Alpha-l-Locked nucleic acid (α-l-LNA) is a stereoisomeric analogue of locked nucleic acid (LNA), which possesses excellent biophysical properties and also exhibits high target binding affinity to complementary oligonucleotide sequences and resistance to nuclease degradations. Therefore, α-l-LNA nucleotides could be utilised to develop stable antisense oligonucleotides (AO), which can be truncated without compromising the integrity and efficacy of the AO. In this study, we explored the potential of α-l-LNA nucleotides-modified antisense oligonucleotides to modulate splicing by inducing Dmd exon-23 skipping in mdx mouse myoblasts in vitro. For this purpose, we have synthesised and systematically evaluated the efficacy of α-l-LNA-modified 2′-O-methyl phosphorothioate (2′-OMePS) AOs of three different sizes including 20mer, 18mer and 16mer AOs in parallel to fully-modified 2′-OMePS control AOs. Our results demonstrated that the 18mer and 16mer truncated AO variants showed slightly better exon-skipping efficacy when compared with the fully-23 modified 2′-OMePS control AOs, in addition to showing low cytotoxicity. As there was no previous report on using α-l-LNA-modified AOs in splice modulation, we firmly believe that this initial study could be beneficial to further explore and expand the scope of α-l-LNA-modified AO therapeutic molecules.

Graphene Oxide-Based Nanostructured DNA Sensor

Quick detection of DNA sequence is vital for many fields, especially, early-stage diagnosis. Here, we develop a graphene oxide-based fluorescence quenching sensor to quickly and accurately detect small amounts of a single strand of DNA. In this paper, fluorescent magnetic nanoparticles (FMNPs) modified with target DNA sequence (DNA-t) were bound onto the modified graphene oxide acting as the fluorescence quenching element. FMNPs are made of iron oxide (Fe3O4) core and fluorescent silica (SiO2) shell. The average particle size of FMNPs was 74 ± 6 nm and the average thickness of the silica shell, estimated from TEM results, was 30 ± 4 nm. The photoluminescence and magnetic properties of FMNPs have been investigated. Target oligonucleotide (DNA-t) was conjugated onto FMNPs through glutaraldehyde crosslinking. Meanwhile, graphene oxide (GO) nanosheets were produced by a modified Hummers method. A complementary oligonucleotide (DNA-c) was designed to interact with GO. In the presence of GO-modified with DNA-c, the fluorescence intensity of FMNPs modified with DNA-t was quenched through a FRET quenching mechanism. Our study indicates that FMNPs can not only act as a FRET donor, but also enhance the sensor accuracy by magnetically separating the sensing system from free DNA and non-hybridized GO. Results indicate that this sensing system is ideal to detect small amounts of DNA-t with limitation detection at 0.12 µM.

A tunable assay for modulators of genome-destabilizing DNA structures

Regions of genomic instability are not random and often co-localize with DNA sequences that can adopt alternative DNA structures (i.e. non-B DNA, such as H-DNA). Non-B DNA-forming sequences are highly enriched at translocation breakpoints in human cancer genomes, representing an endogenous source of genetic instability. However, a further understanding of the mechanisms involved in non-B DNA-induced genetic instability is needed. Small molecules that can modulate the formation/stability of non-B DNA structures, and therefore the subsequent mutagenic outcome, represent valuable tools to study DNA structure-induced genetic instability. To this end, we have developed a tunable Förster resonance energy transfer (FRET)-based assay to detect triplex/H-DNA-destabilizing and -stabilizing ligands. The assay was designed by incorporating a fluorophore-quencher pair in a naturally-occurring H-DNA-forming sequence from a chromosomal breakpoint hotspot in the human c-MYC oncogene. By tuning triplex stability via buffer composition, the assay functions as a dual-reporter that can identify stabilizers and destabilizers, simultaneously. The assay principle was demonstrated using known triplex stabilizers, BePI and coralyne, and a complementary oligonucleotide to mimic a destabilizer, MCRa2. The potential of the assay was validated in a 384-well plate with 320 custom-assembled compounds. The discovery of novel triplex stabilizers/destabilizers may allow the regulation of genetic instability in human genomes.