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Journal of Clinical Immunology Jun 2024A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid...
A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3' end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.
Topics: Female; Humans; Male; DNA Ligase ATP; Fibroblasts; Homozygote; Molecular Dynamics Simulation; Mutation; Severe Combined Immunodeficiency; Infant
PubMed: 38896336
DOI: 10.1007/s10875-024-01754-1 -
BioRxiv : the Preprint Server For... Jun 2024Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes . In...
Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes . In general, this strategy necessitates a protein folding step following ligation of the unstructured fragments, a requirement that constrains the types of systems amenable to the approach. Here, we report an strategy that allows internal regions of target proteins to be replaced in a single operation. Conceptually, our system is analogous to a DNA transposition reaction, but employs orthogonal pairs of split inteins to swap out a designated region of a host protein with an exogenous molecular cassette. We show using isotopic labeling experiments that this 'protein transposition' reaction is concerted when the kinetics for the embedded intein pairs are suitably matched. Critically, this feature allows for efficient manipulation of protein primary structure in the context of a native fold. The utility of this method is illustrated using several protein systems including the multisubunit chromatin remodeling complex, ACF, where we also show protein transposition can occur within the cell nucleus. By carrying out a molecular 'cut and paste' on a protein or protein complex under native folding conditions, our approach dramatically expands the scope of protein semisynthesis.
PubMed: 38895383
DOI: 10.1101/2024.06.03.597171 -
BioRxiv : the Preprint Server For... Jun 2024Among dozens of known epigenetic marks, naturally occurring phosphorothioate (PT) DNA modifications are unique in replacing a non-bridging phosphate oxygen with...
Among dozens of known epigenetic marks, naturally occurring phosphorothioate (PT) DNA modifications are unique in replacing a non-bridging phosphate oxygen with redox-active sulfur and function in prokaryotic restriction-modification and transcriptional regulation. Interest in PTs has grown due to the widespread distribution of the , and genes among bacteria and archaea, as well as the discovery of PTs in 5-10% of gut microbes. Efforts to map PTs in complex microbiomes using existing next-generation and direct sequencing technologies have failed due to poor sensitivity. Here we developed PT-seq as a high-sensitivity method to quantitatively map PTs across genomes and metagenomically identify PT-containing microbes in complex genomic mixtures. Like other methods for mapping PTs in individual genomes, PT-seq exploits targeted DNA strand cleavage at PTs by iodine, followed by sequencing library construction using ligation or template switching approaches. However, PT-specific sequencing reads are dramatically increased by adding steps to heat denature the DNA, block pre-existing 3'-ends, fragment DNA after T-tailing, and enrich iodine-induced breaks using biotin-labeling and streptavidin beads capture. Iterative optimization of the sensitivity and specificity of PT-seq is demonstrated with individual bacteria and human fecal DNA.
PubMed: 38895297
DOI: 10.1101/2024.06.03.597111 -
Nature Protocols Jun 2024Microbial split-pool ligation transcriptomics (microSPLiT) is a high-throughput single-cell RNA sequencing method for bacteria. With four combinatorial barcoding rounds,... (Review)
Review
Microbial split-pool ligation transcriptomics (microSPLiT) is a high-throughput single-cell RNA sequencing method for bacteria. With four combinatorial barcoding rounds, microSPLiT can profile transcriptional states in hundreds of thousands of Gram-negative and Gram-positive bacteria in a single experiment without specialized equipment. As bacterial samples are fixed and permeabilized before barcoding, they can be collected and stored ahead of time. During the first barcoding round, the fixed and permeabilized bacteria are distributed into a 96-well plate, where their transcripts are reverse transcribed into cDNA and labeled with the first well-specific barcode inside the cells. The cells are mixed and redistributed two more times into new 96-well plates, where the second and third barcodes are appended to the cDNA via in-cell ligation reactions. Finally, the cells are mixed and divided into aliquot sub-libraries, which can be stored until future use or prepared for sequencing with the addition of a fourth barcode. It takes 4 days to generate sequencing-ready libraries, including 1 day for collection and overnight fixation of samples. The standard plate setup enables single-cell transcriptional profiling of up to 1 million bacterial cells and up to 96 samples in a single barcoding experiment, with the possibility of expansion by adding barcoding rounds. The protocol requires experience in basic molecular biology techniques, handling of bacterial samples and preparation of DNA libraries for next-generation sequencing. It can be performed by experienced undergraduate or graduate students. Data analysis requires access to computing resources, familiarity with Unix command line and basic experience with Python or R.
PubMed: 38886529
DOI: 10.1038/s41596-024-01007-w -
Journal of Microbiology and... Jun 2024The accurate and rapid detection of methicillin-resistant (MRSA) holds significant clinical importance. This work presents a new method for detecting...
The accurate and rapid detection of methicillin-resistant (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant () in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.
Topics: DNA, Catalytic; Colorimetry; Methicillin-Resistant Staphylococcus aureus; Aptamers, Nucleotide; Staphylococcal Infections; Humans; Biosensing Techniques; Bacterial Proteins; Staphylococcus aureus; Sensitivity and Specificity; Methicillin Resistance; Penicillin-Binding Proteins
PubMed: 38881169
DOI: 10.4014/jmb.2404.04012 -
Molecular Biology Reports Jun 2024The diagnosis of neonatal diabetes can be problematic in preterm infants with fetal growth restriction (FGR). Growth restricted fetuses may have impaired insulin...
BACKGROUND
The diagnosis of neonatal diabetes can be problematic in preterm infants with fetal growth restriction (FGR). Growth restricted fetuses may have impaired insulin production and secretion; low birthweight infants may have a reduced response to insulin. We report a novel missense ABCC8 variant associated with a clinical phenotype compatible with transient neonatal diabetes mellitus (TNDM) in a fetal growth restricted preterm infant.
METHODS AND RESULTS
A preterm growth restricted infant experienced hyperglycemia from the first day of life, requiring insulin therapy on the 13th and 15th day of life and leading to the diagnosis of TNDM. Glycemic values normalized from the 35th day of life onwards. Genetic screening was performed by next generation sequencing, using a Clinical Exon panel of 4800 genes, filtered for those associated with the clinical presentation and by means of methylation-specific multiplex ligation-dependent probe amplification analysis to identify chromosomal aberrations at 6q24. Genetic tests excluded defects at 6q24 and were negative for KCNJ11, SLC2A2 (GLUT-2) and HNF1B, but revealed the presence of the heterozygous missense variant c.2959T > C (p.Ser987Pro) in ABCC8 gene. The presence of the variant was excluded in parents' DNA and the proband variant was then considered de novo.
CONCLUSIONS
In our infant, the persistence of hyperglycemia beyond 3 weeks of life led us to the diagnosis of TNDM and to hypothesize a possible genetic cause. The genetic variant we found could be, most likely, the main cause of both FGR and TNDM.
Topics: Humans; Fetal Growth Retardation; Mutation, Missense; Sulfonylurea Receptors; Infant, Newborn; Diabetes Mellitus; Female; Male; Infant, Premature; Insulin; Infant, Newborn, Diseases
PubMed: 38874636
DOI: 10.1007/s11033-024-09668-2 -
Practical Laboratory Medicine May 2024While recent studies have demonstrated several genetic alterations are associated with pathogenesis of RCC, the significance of cyclin-dependent kinase inhibitor 2A and...
OBJECTIVES
While recent studies have demonstrated several genetic alterations are associated with pathogenesis of RCC, the significance of cyclin-dependent kinase inhibitor 2A and cyclin-dependent kinase inhibitor 2B in tumorigenesis of RCC is less clear. We investigate the distribution of and mutations in patients with RCC and analyze the impact of and mutations on RCC.
METHODS
A pathological examination was conducted using thirty fresh renal tissue samples with renal masses that had undergone partial or radical nephrectomy. Multiplex ligation-dependent probe amplification (MLPA) was used to detect genetic aberrations of and in genomic DNA isolated from samples. Subsequently, and mutations were confirmed using chromosomal microarray technique.
RESULTS
Twenty-one patients were diagnosed with RCC, eight with benign diseases, including angiomyolipoma (AML) and oncocytoma, and one with mucinous adenocarcinoma of renal pelvis. Two of twenty-one patients (9.5 %) with clear-cell RCC were positive for and gene deletions. Interestingly, patients with and mutations were associated with sarcomatoid patterns of RCC (2 out of 4, 50 %). In contrast, no or deletions were detected in samples from benign renal tumors, papillary RCC, or other kidney cancers.
CONCLUSIONS
This study demonstrated the potential use of and as biomarkers for the prognostic and molecular classification of renal cancer. and mutations may be associated with RCC development and sarcomatoid changes. Further research is needed to understand the underlying molecular mechanisms of and in the pathogenesis of RCC.
PubMed: 38867760
DOI: 10.1016/j.plabm.2024.e00410 -
Cold Spring Harbor Protocols Jun 2024In the nearly 50 years since the neuromuscular junction (NMJ) was first established as a model synapse, its molecular composition has been extensively characterized....
In the nearly 50 years since the neuromuscular junction (NMJ) was first established as a model synapse, its molecular composition has been extensively characterized. Early work relied on fluorescent signals to determine whether proteins localized to the pre- and postsynaptic regions. As more synaptic molecules were identified, determining the localization of these proteins relative to each other became important. Conventional microscopy lacks the resolving power to assess whether two proteins are within an appropriate distance to bind directly or be part of a larger complex. Super-resolution and immunoelectron microscopies can improve spatial resolution, but these techniques can be difficult to execute and troubleshoot, and access to these instruments is limiting. However, another approach, proximity labeling, overcomes many of these limitations by using a DNA secondary label that can only be amplified if the two proteins of interest are within 40 nm of each other, which is ∼5× greater than the resolving power of conventional microscopy. In this protocol, we describe the use of the proximity ligation assay, which combines immunohistochemistry with DNA amplification, to reveal protein colocalization in the NMJ.
PubMed: 38866544
DOI: 10.1101/pdb.prot108502 -
Tissue & Cell May 2024Varicocele (VC) refers to expansion and tortuosity of spreading venous plexus in spermatic cord due to poor blood flow. This study aimed to investigate effects of Shugan...
Varicocele (VC) refers to expansion and tortuosity of spreading venous plexus in spermatic cord due to poor blood flow. This study aimed to investigate effects of Shugan Tongluo Qiangjing recipe (SGTL) on sperm DNA damage and oxidative stress in experimental VC (EVC) rats. EVC model was established by partial ligation of left renal vein. Spermatic vein diameter, testicular weight, sperm DNA fragmentation index (DFI) were evaluated. Telomere reverse transcriptase (TERT) expression, telomere gene transcription, and testicular tissue morphology were determined·HO, catalase, SOD, T-AOC were measured with colorimetry. SGTL significantly decreased spermatic vein diameter (P=0.000) and increased testicular weight (P=0.013) of rats compared those of EVC rats. SGTL maintained testicular tissue morphology in EVC rats. SGTL markedly reduced sperm DFI value in sperm of rats compared to EVC rats (P=0.000). SGTL significantly enhanced TERT expression and telomere gene transcription (P=0.028) in testis of rats compared to EVC rats. SGTL reduced HO levels (P=0.001) and promoted CAT activity (P=0.016), SOD activity (P=0.049), and T-AOC activity (P=0.047) of rats, compared to EVC rats. In conclusion, SGTL could reduce pathogenic process of EVC by reducing sperm DNA damage and regulating telomere length in EVC rats, which may be related to oxidative stress regulation.
PubMed: 38865824
DOI: 10.1016/j.tice.2024.102414 -
Scientific Reports Jun 2024Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) suppress production of pro-inflammatory cytokines and chemokines in...
Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) suppress production of pro-inflammatory cytokines and chemokines in human smooth muscle cells (SMCs) through sequestration of RelA in the NF-κB complex, but additional mechanisms are likely involved. The cGAS-STING pathway is activated by double-stranded DNA in the cytosolic compartment and acts through TANK-binding kinase 1 (TBK1) to spark inflammation. The present study tested if MRTFs suppress inflammation also by targeting cGAS-STING signaling. Interrogation of a transcriptomic dataset where myocardin was overexpressed using a panel of 56 cGAS-STING cytokines showed the panel to be repressed. Moreover, MYOCD, MRTFA, and SRF associated negatively with the panel in human arteries. RT-qPCR in human bronchial SMCs showed that all MRTFs reduced pro-inflammatory cytokines on the panel. MRTFs diminished phosphorylation of TBK1, while STING phosphorylation was marginally affected. The TBK1 inhibitor amlexanox, but not the STING inhibitor H-151, reduced the anti-inflammatory effect of MRTF-A. Co-immunoprecipitation and proximity ligation assays supported binding between MRTF-A and TBK1 in SMCs. MRTFs thus appear to suppress cellular inflammation in part by acting on the kinase TBK1. This may defend SMCs against pro-inflammatory insults in disease.
Topics: Humans; Protein Serine-Threonine Kinases; Nuclear Proteins; Myocytes, Smooth Muscle; Trans-Activators; Inflammation; Signal Transduction; Cytokines; Phosphorylation; Transcription Factors; Membrane Proteins; Cells, Cultured
PubMed: 38858497
DOI: 10.1038/s41598-024-63901-3