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Chemical & Pharmaceutical Bulletin 2024DNA-encoded libraries (DELs) are attracting attention as a screening tool in the early stages of drug discovery. In the development of DELs, drug candidate compounds are...
DNA-encoded libraries (DELs) are attracting attention as a screening tool in the early stages of drug discovery. In the development of DELs, drug candidate compounds are chemically synthesized on barcode DNA. Therefore, it is important to perform the synthesis under mild conditions so as to not damage the DNA. On the other hand, coumarins are gaining increasing research focus not only because they possess excellent fluorescence properties, but also because many medicines contain a coumarin skeleton. Among the various reactions developed for the synthesis of coumarins thus far, Knoevenagel condensation followed by intramolecular cyclization under mild conditions can yield coumarins. In this study, we developed a new synthetic method for preparing a coumarin-conjugated oligonucleotide library via Knoevenagel condensation. The results showed that coumarins substituted at the 5-, 6-, 7-, or 8-positions could be constructed on DNA to afford a total of 26 coumarin-conjugated DNAs. Moreover, this method was compatible with enzymatic ligation, demonstrating its utility in DEL synthesis. The developed strategy for the construction of coumarin scaffolds based on Knoevenagel condensation may contribute to the use of DELs in drug discovery and medicinal chemistry.
Topics: Oligonucleotides; Coumarins; DNA; Cyclization
PubMed: 38296555
DOI: 10.1248/cpb.c23-00295 -
JCI Insight Mar 2024Pseudohypoparathyroidism type 1B (PHP1B) results from aberrant genomic imprinting at the GNAS gene. Defining the underlying genetic cause in new patients is challenging...
Pseudohypoparathyroidism type 1B (PHP1B) results from aberrant genomic imprinting at the GNAS gene. Defining the underlying genetic cause in new patients is challenging because various genetic alterations (e.g., deletions, insertions) within the GNAS genomic region, including the neighboring STX16 gene, can cause PHP1B, and the genotype-epigenotype correlation has not been clearly established. Here, by analyzing patients with PHP1B with a wide variety of genotypes and epigenotypes, we identified a GNAS differentially methylated region (DMR) of distinct diagnostic value. This region, GNAS AS2, was hypomethylated in patients with genetic alterations located centromeric but not telomeric of this DMR. The AS2 methylation status was captured by a single probe of the methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) assay utilized to diagnose PHP1B. In human embryonic stem cells, where NESP55 transcription regulates GNAS methylation status on the maternal allele, AS2 methylation depended on 2 imprinting control regions (STX16-ICR and NESP-ICR) essential for NESP55 transcription. These results suggest that the AS2 methylation status in patients with PHP1B reflects the position at which the genetic alteration affects NESP55 transcription during an early embryonic period. Therefore, AS2 methylation levels can enable mechanistic PHP1B categorization based on genotype-epigenotype correlation and, thus, help identify the underlying molecular defect in patients.
Topics: Humans; GTP-Binding Protein alpha Subunits, Gs; DNA Methylation; Pseudohypoparathyroidism; Genomic Imprinting; Alleles; Chromogranins
PubMed: 38290008
DOI: 10.1172/jci.insight.177190 -
Human Genetics Feb 2024The purpose of this study was to screen Copy Number Variations (CNVs) in 35 unsolved Inherited Retinal Dystrophy (IRD) families. Initially, next generation sequencing,...
The purpose of this study was to screen Copy Number Variations (CNVs) in 35 unsolved Inherited Retinal Dystrophy (IRD) families. Initially, next generation sequencing, including a specific Hereditary Eye Disease Enrichment Panel or Whole exome sequencing, was employed to screen (likely) pathogenic Single-nucleotide Variants (SNVs) and small Insertions and Deletions (indels) for these cases. All available SNVs and indels were further validated and co-segregation analyses were performed in available family members by Sanger sequencing. If not, after excluding deep intronic variants, Multiplex ligation-dependent probe amplification (MLPA), quantitative fluorescence PCR (QF-PCR) and Sanger sequencing were employed to screen CNVs. We determined that 18 probands who had heterozygous SNVs/indels or whose parents were not consanguineous but had homozygous SNVs/indels in autosomal recessive IRDs genes had CNVs in another allele of these genes, 11 families had disease-causing hemizygous CNVs in X-linked IRD genes, 6 families had (likely) pathogenic heterozygous CNVs in PRPF31 gene. Of 35 families, 33 different CNVs in 16 IRD-associated genes were detected, with PRPF31, EYS and USH2A the most common disease-causing gene in CNVs. Twenty-six and 7 of them were deletion and duplication CNVs, respectively. Among them, 14 CNVs were first reported in this study. Our research indicates that CNVs contribute a lot to IRDs, and screening of CNVs substantially increases the diagnostic rate of IRD. Our results emphasize that MLPA and QF-PCR are ideal methods to validate CNVs, and the novel CNVs reported herein expand the mutational spectrums of IRDs.
Topics: Humans; DNA Copy Number Variations; Mutation; Usher Syndromes; Retinal Dystrophies; Heterozygote; Eye Proteins
PubMed: 38282009
DOI: 10.1007/s00439-023-02631-4 -
BMC Pediatrics Jan 2024Severe neonatal hyperbilirubinemia could lead to kernicterus and neonatal death. This study aimed to analyze the association between single nucleotide polymorphisms in...
BACKGROUND
Severe neonatal hyperbilirubinemia could lead to kernicterus and neonatal death. This study aimed to analyze the association between single nucleotide polymorphisms in genes involved in bilirubin metabolism and the incidence of severe hyperbilirubinemia.
METHODS
A total of 144 neonates with severe hyperbilirubinemia and 50 neonates without or mild hyperbilirubinemia were enrolled in 3 institutions between 2019 and 2020. Twelve polymorphisms of 5 genes (UGT1A1, SLCO1B1, SLCO1B3, BLVRA, and HMOX1) were analyzed by PCR amplification of genomic DNA. Genotyping was performed using an improved multiplex ligation detection reaction technique based on ligase detection reaction.
RESULTS
The frequencies of the A allele in UGT1A1-rs4148323 and the C allele in SLCO1B3-rs2417940 in the severe hyperbilirubinemia group (30.2% and 90.6%, respectively) were significantly higher than those in the controls (30.2% vs.13.0%, 90.6% vs. 78.0%, respectively, both p < 0.05). Haplotype analysis showed the ACG haplotype of UGT1A1 were associated with an increased hyperbilirubinemia risk (OR 3.122, p = 0.001), whereas the GCG haplotype was related to a reduced risk (OR 0.523, p = 0.018).
CONCLUSION
The frequencies of the A allele in rs4148323 and the C allele in rs2417940 are highly associated with the incidence of severe hyperbilirubinemia in Chinese Han neonates.
TRIAL REGISTRATION
Trial registration number:ChiCTR1800020424; Date of registration:2018-12-29.
Topics: Infant, Newborn; Humans; Liver-Specific Organic Anion Transporter 1; Polymorphism, Single Nucleotide; Alleles; Hyperbilirubinemia, Neonatal; Glucuronosyltransferase; China; Solute Carrier Organic Anion Transporter Family Member 1B3; Heme Oxygenase-1
PubMed: 38279097
DOI: 10.1186/s12887-024-04537-0 -
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 -
Molecular Cancer Jan 2024The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity...
BACKGROUND
The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity for the p53 mRNA secondary structure. Disruption of this interaction prevents the activation of the nascent p53. The link of the MDM2 protein-p53 mRNA interaction with the upstream DNA damage sensor ATM kinase and the role of the p53 mRNA in the DNA damage sensing mechanism, are still highly anticipated.
METHODS
The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells.
RESULTS
This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm.
CONCLUSION
The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.
Topics: Humans; Polynucleotide 5'-Hydroxyl-Kinase; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53; DNA Damage; DNA Repair; Ataxia Telangiectasia Mutated Proteins
PubMed: 38263180
DOI: 10.1186/s12943-024-01933-z -
Life (Basel, Switzerland) Jan 2024Deciphering the origins of life on a molecular level includes unravelling the numerous interactions that could occur between the most important biomolecules being the...
From the RNA-Peptide World: Prebiotic Reaction Conditions Compatible with Lipid Membranes for the Formation of Lipophilic Random Peptides in the Presence of Short Oligonucleotides, and More.
Deciphering the origins of life on a molecular level includes unravelling the numerous interactions that could occur between the most important biomolecules being the lipids, peptides and nucleotides. They were likely all present on the early Earth and all necessary for the emergence of cellular life. In this study, we intended to explore conditions that were at the same time conducive to chemical reactions critical for the origins of life (peptide-oligonucleotide couplings and templated ligation of oligonucleotides) and compatible with the presence of prebiotic lipid vesicles. For that, random peptides were generated from activated amino acids and analysed using NMR and MS, whereas short oligonucleotides were produced through solid-support synthesis, manually deprotected and purified using HPLC. After chemical activation in prebiotic conditions, the resulting mixtures were analysed using LC-MS. Vesicles could be produced through gentle hydration in similar conditions and observed using epifluorescence microscopy. Despite the absence of coupling or ligation, our results help to pave the way for future investigations on the origins of life that may gather all three types of biomolecules rather than studying them separately, as it is still too often the case.
PubMed: 38255723
DOI: 10.3390/life14010108 -
G3 (Bethesda, Md.) Mar 2024The Ecuadorian brown-headed spider monkey (Ateles fusciceps fusciceps) is currently considered one of the most endangered primates in the world and is classified as...
First whole-genome sequence and assembly of the Ecuadorian brown-headed spider monkey (Ateles fusciceps fusciceps), a critically endangered species, using Oxford Nanopore Technologies.
The Ecuadorian brown-headed spider monkey (Ateles fusciceps fusciceps) is currently considered one of the most endangered primates in the world and is classified as critically endangered [International union for conservation of nature (IUCN)]. It faces multiple threats, the most significant one being habitat loss due to deforestation in western Ecuador. Genomic tools are keys for the management of endangered species, but this requires a reference genome, which until now was unavailable for A. f. fusciceps. The present study reports the first whole-genome sequence and assembly of A. f. fusciceps generated using Oxford Nanopore long reads. DNA was extracted from a subadult male, and libraries were prepared for sequencing following the Ligation Sequencing Kit SQK-LSK112 workflow. Sequencing was performed using a MinION Mk1C sequencer. The sequencing reads were processed to generate a genome assembly. Two different assemblers were used to obtain draft genomes using raw reads, of which the Flye assembly was found to be superior. The final assembly has a total length of 2.63 Gb and contains 3,861 contigs, with an N50 of 7,560,531 bp. The assembly was analyzed for annotation completeness based on primate ortholog prediction using a high-resolution database, and was found to be 84.3% complete, with a low number of duplicated genes indicating a precise assembly. The annotation of the assembly predicted 31,417 protein-coding genes, comparable with other mammal assemblies. A reference genome for this critically endangered species will allow researchers to gain insight into the genetics of its populations and thus aid conservation and management efforts of this vulnerable species.
Topics: Male; Animals; Ecuador; Endangered Species; Nanopores; Atelinae; Sequence Analysis, DNA; High-Throughput Nucleotide Sequencing; Mammals
PubMed: 38244218
DOI: 10.1093/g3journal/jkae014 -
PLoS Pathogens Jan 2024Rosellinia necatrix is a prevalent soil-borne plant-pathogenic fungus that is the causal agent of white root rot disease in a broad range of host plants. The limited...
Rosellinia necatrix is a prevalent soil-borne plant-pathogenic fungus that is the causal agent of white root rot disease in a broad range of host plants. The limited availability of genomic resources for R. necatrix has complicated a thorough understanding of its infection biology. Here, we sequenced nine R. necatrix strains with Oxford Nanopore sequencing technology, and with DNA proximity ligation we generated a gapless assembly of one of the genomes into ten chromosomes. Whereas many filamentous pathogens display a so-called two-speed genome with more dynamic and more conserved compartments, the R. necatrix genome does not display such genome compartmentalization. It has recently been proposed that fungal plant pathogens may employ effectors with antimicrobial activity to manipulate the host microbiota to promote infection. In the predicted secretome of R. necatrix, 26 putative antimicrobial effector proteins were identified, nine of which are expressed during plant colonization. Two of the candidates were tested, both of which were found to possess selective antimicrobial activity. Intriguingly, some of the inhibited bacteria are antagonists of R. necatrix growth in vitro and can alleviate R. necatrix infection on cotton plants. Collectively, our data show that R. necatrix encodes antimicrobials that are expressed during host colonization and that may contribute to modulation of host-associated microbiota to stimulate disease development.
Topics: Ascomycota; Plants; Anti-Infective Agents
PubMed: 38236788
DOI: 10.1371/journal.ppat.1011866 -
Saudi Journal of Biological Sciences Feb 2024This study aimed to identify thermo-stable pullulanase-producing bacteria in soil samples of potato fields and food-producing companies. Pullulan agar medium was used to...
This study aimed to identify thermo-stable pullulanase-producing bacteria in soil samples of potato fields and food-producing companies. Pullulan agar medium was used to screen 17 bacterial strains, which were incubated at 65 °C. The isolate with the maximum activity (375U/ml) was selected and recognized as ADM-11 by morphological, biochemical characterization, and 16S rRNA gene sequencing. The pullulanase production required optimum pH of 7 and temperature of 75 °C, respectively. The electrophoresis of purified pullulanase on SDS-polyacrylamide gel revealed 83 kDa of a molecular weight that is active at 70 °C and pH 7.0. It was also stable at 90 °C but its activity was decreased by 10 % at 100 °C. The action of pullulanase was increased and stabilized by Ca among the metal ions. Beta and gamma-cyclodextrins inhibited enzyme activity while ethylenediaminetetraacetate (EDTA) and phenylmethylsulfonyl fluoride (PMSF) have no significant effect on pullulanase activity. A full-length pullulanase gene was amplified from . ADM-11 using genomic DNA 2.1 kb of PCR product which was then purified and ligated in the cloning vector pTZ57R using the TA cloning technique. Colony PCR confirmed cloning on the positive clones after the pullulanase gene had been ligated and subjected to restriction digestion. It revealed 74 % similarity with the reported pullulanase gene from sp. 44C. The thermostability of pullulanase and its ability to degrade raw pullulan may therefore have wide-scale applications in starch processing, the detergent business, and new biotechnological applications.
PubMed: 38234990
DOI: 10.1016/j.sjbs.2023.103901