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Research Square May 2024Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complimentary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 hr treatment of 10μM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS affected neurons.
PubMed: 38826483
DOI: 10.21203/rs.3.rs-4439430/v1 -
Nature Communications Aug 2023Cellular glycosylation is characterized by chemical complexity and heterogeneity, which is challenging to reproduce synthetically. Here we show chemoenzymatic synthesis...
Cellular glycosylation is characterized by chemical complexity and heterogeneity, which is challenging to reproduce synthetically. Here we show chemoenzymatic synthesis on phage to produce a genetically-encoded liquid glycan array (LiGA) of complex type N-glycans. Implementing the approach involved by ligating an azide-containing sialylglycosyl-asparagine to phage functionalized with 50-1000 copies of dibenzocyclooctyne. The resulting intermediate can be trimmed by glycosidases and extended by glycosyltransferases yielding a phage library with different N-glycans. Post-reaction analysis by MALDI-TOF MS allows rigorous characterization of N-glycan structure and mean density, which are both encoded in the phage DNA. Use of this LiGA with fifteen glycan-binding proteins, including CD22 or DC-SIGN on cells, reveals optimal structure/density combinations for recognition. Injection of the LiGA into mice identifies glycoconjugates with structures and avidity necessary for enrichment in specific organs. This work provides a quantitative evaluation of the interaction of complex N-glycans with GBPs in vitro and in vivo.
Topics: Animals; Mice; Glycosylation; Asparagine; Azides; Bacteriophages; Gene Library
PubMed: 37640713
DOI: 10.1038/s41467-023-40900-y -
Frontiers in Oncology 2024In clinical oncology, cell-free DNA (cfDNA) has shown immense potential in its ability to noninvasively detect cancer at various stages and monitor the progression of... (Review)
Review
In clinical oncology, cell-free DNA (cfDNA) has shown immense potential in its ability to noninvasively detect cancer at various stages and monitor the progression of therapy. Despite the rapid improvements in cfDNA liquid biopsy approaches, achieving the required sensitivity to detect rare tumor-derived cfDNA still remains a challenge. For next-generation sequencing, the perceived presentation of cfDNA is strongly linked to the extraction and library preparation protocols. Conventional double-stranded DNA library preparation (dsDNA-LP) focuses on assessing ~167bp double-stranded mononucleosomal (mncfDNA) and its other oligonucleosomal cell-free DNA counterparts in plasma. However, dsDNA-LP methods fail to include short, single-stranded, or nicked DNA in the final library preparation, biasing the representation of the actual cfDNA populations in plasma. The emergence of single-stranded library preparation (ssDNA-LP) strategies over the past decade has now allowed these other populations of cfDNA to be studied from plasma. With the use of ssDNA-LP, single-stranded, nicked, and ultrashort cfDNA can be comprehensively assessed for its molecular characteristics and clinical potential. In this review, we overview the current literature on applications of ssDNA-LP on plasma cfDNA from a potential cancer liquid biopsy perspective. To this end, we discuss the molecular principles of single-stranded DNA adapter ligation, how library preparation contributes to the understanding of native cfDNA characteristics, and the potential for ssDNA-LP to improve the sensitivity of circulating tumor DNA detection. Additionally, we review the current literature on the newly reported species of plasma ultrashort single-stranded cell-free DNA plasma, which appear biologically distinct from mncfDNA. We conclude with a discussion of future perspectives of ssDNA-LP for liquid biopsy endeavors.
PubMed: 38511142
DOI: 10.3389/fonc.2024.1332004 -
ACS Central Science Aug 2023Encoded combinatorial library technologies have dramatically expanded the chemical space for screening but are usually only analyzed by affinity selection binding. It...
Encoded combinatorial library technologies have dramatically expanded the chemical space for screening but are usually only analyzed by affinity selection binding. It would be highly advantageous to reformat selection outputs to "one-bead-one-compound" solid-phase libraries, unlocking activity-based and cellular screening capabilities. Here, we describe hydrogel-encapsulated magnetic beads that enable such a transformation. Bulk emulsion polymerization of polyacrylamide hydrogel shells around magnetic microbeads yielded uniform particles (7 ± 2 μm diameter) that are compatible with diverse in-gel functionalization (amine, alkyne, oligonucleotides) and transformations associated with DNA-encoded library synthesis (acylation, enzymatic DNA ligation). In a case study of reformatting mRNA display libraries, transcription from DNA-templated magnetic beads encapsulated in gel particles colocalized both RNA synthesis via hybridization with copolymerized complementary DNA and translation via puromycin labeling. Two control epitope templates (V5, HA) were successfully enriched (50- and 99-fold, respectively) from an NNK library bead screen via FACS. Proximity-driven library synthesis in concert with magnetic sample manipulation provides a plausible means for reformatting encoded combinatorial libraries at scale.
PubMed: 37637732
DOI: 10.1021/acscentsci.3c00316 -
MedRxiv : the Preprint Server For... Aug 2023mutations are the most common cause of young onset and autosomal recessive Parkinson's disease (PD). is located in FRA6E which is one of the common fragile sites in...
BACKGROUND
mutations are the most common cause of young onset and autosomal recessive Parkinson's disease (PD). is located in FRA6E which is one of the common fragile sites in the human genome, making this region prone to structural variants. However, complex structural variants such as inversions of are seldom reported, suggesting that there are potentially unrevealed complex pathogenic structural variants.
OBJECTIVES
To identify complex structural variants in using long-read sequencing.
METHODS
We investigated the genetic cause of monozygotic twins presenting with a young onset dystonia-parkinsonism using targeted sequencing, whole exome sequencing, multiple ligation probe amplification, and long-read. We assessed the presence and frequency of complex inversions overlapping using whole-genome sequencing data of AMP-PD and UK-Biobank datasets.
RESULTS
Multiple ligation probe amplification identified a heterozygous exon 3 deletion in and long-read sequencing identified a large novel inversion spanning over 7Mb, including a large part of the coding DNA sequence of . We could diagnose the affected subjects as compound heterozygous carriers of . We analyzed whole genome sequencing data of 43,538 participants of the UK-Biobank and 4,941 participants of the AMP-PD datasets. Nine inversions in the UK-Biobank and two in AMP PD were identified and were considered potentially damaging and likely to affect isoforms.
CONCLUSIONS
This is the first report describing a large 7Mb inversion involving breakpoints outside of . This study highlights the importance of using long-read whole genome sequencing for structural variant analysis in unresolved young-onset PD cases.
PubMed: 37790330
DOI: 10.1101/2023.08.14.23293948 -
STAR Protocols Jun 2024Dinoflagellate genomes often are very large and difficult to assemble, which has until recently precluded their analysis with modern functional genomic tools. Here, we...
Dinoflagellate genomes often are very large and difficult to assemble, which has until recently precluded their analysis with modern functional genomic tools. Here, we present a protocol for mapping three-dimensional (3D) genome organization in dinoflagellates and using it for scaffolding their genome assemblies. We describe steps for crosslinking, nuclear lysis, denaturation, restriction digest, ligation, and DNA shearing and purification. We then detail procedures sequencing library generation and computational analysis, including initial Hi-C read mapping and 3D-DNA scaffolding/assembly correction. For complete details on the use and execution of this protocol, please refer to Marinov et al..
Topics: Dinoflagellida; Genome, Protozoan; Genomics; Chromosome Mapping; Sequence Analysis, DNA
PubMed: 38483898
DOI: 10.1016/j.xpro.2024.102941 -
Extremophiles : Life Under Extreme... Sep 2023Bacterial NAD-dependent DNA ligases (LigAs) are enzymes involved in replication, recombination, and DNA-repair processes by catalyzing the formation of phosphodiester...
Bacterial NAD-dependent DNA ligases (LigAs) are enzymes involved in replication, recombination, and DNA-repair processes by catalyzing the formation of phosphodiester bonds in the backbone of DNA. These multidomain proteins exhibit four modular domains, that are highly conserved across species, with the BRCT (breast cancer type 1 C-terminus) domain on the C-terminus of the enzyme. In this study, we expressed and purified both recombinant full-length and a C-terminally truncated LigA from Deinococcus radiodurans (DrLigA and DrLigA∆BRCT) and characterized them using biochemical and X-ray crystallography techniques. Using seeds of DrLigA spherulites, we obtained ≤ 100 µm plate crystals of DrLigA∆BRCT. The crystal structure of the truncated protein was obtained at 3.4 Å resolution, revealing DrLigA∆BRCT in a non-adenylated state. Using molecular beacon-based activity assays, we demonstrated that DNA ligation via nick sealing remains unaffected in the truncated DrLigA∆BRCT. However, DNA-binding assays revealed a reduction in the affinity of DrLigA∆BRCT for dsDNA. Thus, we conclude that the flexible BRCT domain, while not critical for DNA nick-joining, plays a role in the DNA binding process, which may be a conserved function of the BRCT domain in LigA-type DNA ligases.
Topics: DNA Ligases; Deinococcus; Extremophiles; NAD; DNA Repair
PubMed: 37712998
DOI: 10.1007/s00792-023-01309-z -
International Journal of Molecular... Mar 2024A better understanding of the function of neutrophil extracellular traps (NETs) may facilitate the development of interventions for sepsis. The study aims to investigate...
A better understanding of the function of neutrophil extracellular traps (NETs) may facilitate the development of interventions for sepsis. The study aims to investigate the formation and degradation of NETs in three murine sepsis models and to analyze the production of reactive oxygen species (ROS) during NET formation. Murine sepsis was induced by midgut volvulus (720° for 15 min), cecal ligation and puncture (CLP), or the application of lipopolysaccharide (LPS) (10 mg/kg body weight i.p.). NET formation and degradation was modulated using mice that were genetically deficient for -KO) or and -DKO). After 48 h, mice were killed. Plasma levels of circulating free DNA (cfDNA) and neutrophil elastase (NE) were quantified to assess NET formation and degradation. Plasma deoxyribonuclease1 (DNase1) protein levels, as well as tissue malondialdehyde (MDA) activity and glutathione peroxidase (GPx) activity, were quantified. DNase1 and DNase1L3 in liver, intestine, spleen, and lung tissues were assessed. The applied sepsis models resulted in a simultaneous increase in NET formation and oxidative stress. NET formation and survival differed in the three models. In contrast to LPS and Volvulus, CLP-induced sepsis showed a decreased and increased 48 h survival in -KO and -DKO mice, when compared to WT mice, respectively. -KO mice showed decreased formation of NETs and ROS, while -DKO mice with impaired NET degradation accumulated ROS and chronicled the septic state. The findings indicate a dual role for NET formation and degradation in sepsis and ischemia-reperfusion (I/R) injury: NETs seem to exhibit a protective capacity in certain sepsis paradigms (CLP model), whereas, collectively, they seem to contribute adversely to scenarios where sepsis is combined with ischemia-reperfusion (volvulus).
Topics: Animals; Mice; Disease Models, Animal; Extracellular Traps; Intestinal Volvulus; Lipopolysaccharides; Reactive Oxygen Species; Reperfusion Injury; Sepsis; Blood Group Antigens; Cell-Free Nucleic Acids; Protons; Ischemia
PubMed: 38612596
DOI: 10.3390/ijms25073787 -
Cancers Jan 2024CIC-DUX4-rearranged sarcoma (CDS) is a rare and aggressive soft tissue tumor that occurs most frequently in young adults. The key oncogenic driver of this disease is the...
CIC-DUX4-rearranged sarcoma (CDS) is a rare and aggressive soft tissue tumor that occurs most frequently in young adults. The key oncogenic driver of this disease is the expression of the CIC-DUX4 fusion protein as a result of chromosomal rearrangements. CIC-DUX4 displays chromatin binding properties, and is therefore believed to function as an aberrant transcription factor. However, the chromatin remodeling events induced by CIC-DUX4 are not well understood, limiting our ability to identify new mechanism-based therapeutic strategies for these patients. Here, we generated a genome-wide profile of CIC-DUX4 DNA occupancy and associated chromatin states in human CDS cell models and primary tumors. Combining chromatin profiling, proximity ligation assays, as well as genetic and pharmacological perturbations, we show that CIC-DUX4 operates as a potent transcriptional activator at its binding sites. This property is in contrast with the repressive function of the wild-type CIC protein, and is mainly mediated through the direct interaction of CIC-DUX4 with the acetyltransferase p300. In keeping with this, we show p300 to be essential for CDS tumor cell proliferation; additionally, we find its pharmacological inhibition to significantly impact tumor growth in vitro and in vivo. Taken together, our study elucidates the mechanisms underpinning CIC-DUX4-mediated transcriptional regulation.
PubMed: 38275898
DOI: 10.3390/cancers16020457 -
Scientific Reports Aug 2023Although methods for sequencing library preparation from double-stranded DNA are well established, those from single-stranded DNA (ssDNA) have not been well studied....
Although methods for sequencing library preparation from double-stranded DNA are well established, those from single-stranded DNA (ssDNA) have not been well studied. Further, the existing methods have limitations in efficiency and yield. Therefore, we developed a highly efficient procedure for sequencing library preparation from ssDNA. In this method, the first adaptor tagging of ssDNA is performed using terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation, which we reported recently. After complementary strand synthesis using the adaptor-tagged ssDNA, second adaptor tagging via Vaccinia virus topoisomerase I (VTopoI or TOPO)-based adaptor ligation is performed. With additional steps for degradation, repression, and removal of the adaptor dimer, the proposed TACS-TOPO scheme realizes adaptor dimer-free sequencing library preparation from ssDNA samples of 24 pg. The TACS-TOPO scheme was successfully applied to cell-free DNA analysis with amplification-free library preparation from 50 µL of human serum. A modified TACS-TOPO scheme was also applied to DNA extracted from ancient human bones, bringing two to eight times more library yields than those using a conventional library preparation protocol. The procedures for preparing VTopoI and its complex with a double-stranded oligonucleotide adaptor are also described. Overall, the proposed TACS-TOPO scheme can facilitate practical and sensitive sequencing analysis of ssDNA.
Topics: Humans; DNA, Single-Stranded; Gene Library; Oligonucleotides; Cell-Free Nucleic Acids; DNA Nucleotidylexotransferase; Neoplasms, Squamous Cell
PubMed: 37626096
DOI: 10.1038/s41598-023-40890-3