-
Cell Chemical Biology Jun 2024Velcrins are molecular glues that induce complex formation between PDE3A and SLFN12. The PDE3A-SLFN12 complex activates the SLFN12 RNase, resulting in cleavage of the...
Velcrins are molecular glues that induce complex formation between PDE3A and SLFN12. The PDE3A-SLFN12 complex activates the SLFN12 RNase, resulting in cleavage of the specific substrate, tRNA-Leu-TAA, global inhibition of translation, and death of cells expressing sufficient levels of both proteins. Here, unanswered questions about the mechanism of action and therapeutic promise of velcrin compounds are discussed.
Topics: Humans; Endoribonucleases; Bacterial Proteins; Endodeoxyribonucleases; CRISPR-Associated Proteins
PubMed: 38906108
DOI: 10.1016/j.chembiol.2024.05.012 -
The Journal of International Medical... Jun 2024Breast cancer, particularly triple-negative breast cancer (TNBC), poses a significant global health burden. Chemotherapy was the mainstay treatment for TNBC patients...
BACKGROUND
Breast cancer, particularly triple-negative breast cancer (TNBC), poses a significant global health burden. Chemotherapy was the mainstay treatment for TNBC patients until immunotherapy was introduced. Studies indicate a noteworthy prevalence (0.2% to 18.6%) of mismatch repair protein (MMRP) deficiency in TNBC, with recent research highlighting the potential of immunotherapy for MMRP-deficient metastatic breast cancer. This study aims to identify MMRP deficiency in TNBC patients using immunohistochemistry.
METHODS
A retrospective cohort study design was used and included TNBC patients treated between 2015 and 2021 at King Hussein Cancer Center. Immunohistochemistry was conducted to assess MMRP expression.
RESULTS
Among 152 patients, 14 (9.2%) exhibited deficient MMR (dMMR). Loss of PMS2 expression was observed in 13 patients, 5 of whom showed loss of MLH1 expression. Loss of MSH6 and MSH2 expression was observed in one patient. The median follow-up duration was 44 (3-102) months. Despite the higher survival rate (80.8%, 5 years) of dMMR patients than of proficient MMR patients (62.3%), overall survival did not significantly differ between the two groups.
CONCLUSION
Approximately 9% of TNBC patients exhibit dMMR. dMMR could be used to predict outcomes and identify patients with TNBC who may benefit from immunotherapy.
Topics: Humans; Female; Triple Negative Breast Neoplasms; Middle Aged; Adult; Retrospective Studies; Mismatch Repair Endonuclease PMS2; Aged; DNA-Binding Proteins; DNA Mismatch Repair; MutL Protein Homolog 1; MutS Homolog 2 Protein; Biomarkers, Tumor; Survival Rate; Immunohistochemistry; Aged, 80 and over; Prognosis
PubMed: 38902203
DOI: 10.1177/03000605241259747 -
Experimental Cell Research Jul 2024Mouse HORMAD1 is a phospho-protein involved in multiple functions during meiotic prophase I. To obtain insight into the significance of its phosphorylation, we generated...
Mouse HORMAD1 is a phospho-protein involved in multiple functions during meiotic prophase I. To obtain insight into the significance of its phosphorylation, we generated phospho-specific antibodies against two serine residues, Ser307 and Ser378, representing each of two serine clusters in mouse HORMAD1. The Ser307 phosphorylation is detectable from early leptotene substage in both wild-type and Spo11 spermatocytes, indicating that Ser307 is a primary and SPO11-independent phosphorylation site. In contrast, the Ser378 phosphorylation is negligible at earlier substages in wild-type and Spo11 spermatocytes. After mid-zygotene substage, the Ser378 phosphorylation is abundant on unsynapsed chromosome axes in wild-type spermatocytes and is detected only in a part of unsynapsed chromosome axes in Spo11 spermatocytes. We also generated a non-phosphorylated Ser307-specific antibody and found that Ser307 is phosphorylated on sex chromosome axes but is almost entirely unphosphorylated on desynapsed chromosome axes in diplotene spermatocytes. These results demonstrated a substage-specific phosphorylation status of mouse HORMAD1, which might be associated with multiple substage-specific functions.
Topics: Animals; Meiotic Prophase I; Phosphorylation; Male; Mice; Serine; Spermatocytes; Endodeoxyribonucleases; Mice, Inbred C57BL; Cell Cycle Proteins; Mice, Knockout; Sex Chromosomes
PubMed: 38897409
DOI: 10.1016/j.yexcr.2024.114133 -
Mikrochimica Acta Jun 2024CRISPR-Cas12a with robust trans-cleavage activity were employed to mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of...
CRISPR-Cas12a with robust trans-cleavage activity were employed to mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of trans-cleavage activity of Cas12a was achieved by utilizing cDNA as a trigger. Upon the presence of target miRNA-21, cDNA hybridizes with it forming a DNA/RNA double-stranded structure. Exonuclease III (ExoIII) facilitates the degradation of cDNA, releasing the target for subsequent cycles. Due to cDNA degradation, the trans-cleavage activity of Cas12a remains unactivated and does not disrupt the synthesis template of copper nanoparticles. Addition of Cu and AA leads to the formation of highly fluorescent copper nanoparticles. Conversely, in absence of miRNA-21, intact cDNA activates trans-cleavage activity of Cas12a, resulting in degradation of the synthesis template and failure in synthesizing fluorescent copper nanoparticles. This method exhibits excellent selectivity with a low limit of detection (LOD) at 5 pM. Furthermore, we successfully applied this approach to determine miRNA-21 in cell lysates and human serum samples, providing a new approach for sensitive determination of biomarkers in biochemical research and disease diagnosis.
Topics: Copper; Metal Nanoparticles; Humans; MicroRNAs; Limit of Detection; CRISPR-Cas Systems; Fluorometry; CRISPR-Associated Proteins; Biosensing Techniques; Bacterial Proteins; Endodeoxyribonucleases
PubMed: 38896292
DOI: 10.1007/s00604-024-06496-w -
International Journal of Molecular... May 2024Pathogenic variants in the gene lead to a systemic disease with karyomegalic interstitial nephritis (KIN) at the forefront clinically. The phenotypic-genotypic features...
Pathogenic variants in the gene lead to a systemic disease with karyomegalic interstitial nephritis (KIN) at the forefront clinically. The phenotypic-genotypic features of a mutation-related disease involving five members of a Hungarian Caucasian family are presented. Each had adult-onset chronic kidney disease of unknown cause treated with renal replacement therapy and elevated liver enzymes. Short stature, emaciation, latte-colored skin, freckles, and a hawk-like nose in four patients, a limited intellect in two patients, and chronic restrictive lung disease in one patient completed the phenotype. Severe infections occurred in four patients. All five patients had ceased. Four patients underwent autopsy. KIN and extrarenal karyomegaly were observed histologically; the livers showed no specific abnormality. The genotyping using formalin-fixed tissue samples detected a hitherto undescribed homozygous mutation (c.1673_1674insT/p.Met558lfs*4; exon 5) in three of these patients and a heterozygous mutation in one patient. The reason for the heterozygosity is discussed. In addition, 56 family members consented to the screening for mutation from which 17 individuals proved to be heterozygous carriers; a blood chemistry evaluation of their kidney and liver function did not find any abnormality. The clinical presentation of FAN1-related disease was multifaceted, and not yet described manifestations were observed besides kidney and liver disease. Mutation in this gene should be suspected in adults with small kidneys of unknown cause, elevated liver enzymes, and recurrent infections, even without a family history.
Topics: Humans; Male; Female; Hungary; Mutation; Adult; Phenotype; Pedigree; Middle Aged; Exodeoxyribonucleases; Multifunctional Enzymes; Endodeoxyribonucleases; Genotype; Renal Insufficiency, Chronic
PubMed: 38892095
DOI: 10.3390/ijms25115907 -
Journal of Translational Medicine Jun 2024Inherited variations in DNA double-strand break (DSB) repair pathway are known to influence ovarian cancer occurrence, progression and treatment response. Despite its...
BACKGROUND
Inherited variations in DNA double-strand break (DSB) repair pathway are known to influence ovarian cancer occurrence, progression and treatment response. Despite its significance, survival-associated genetic variants within the DSB pathway remain underexplored.
METHODS
In the present study, we performed a two-phase analysis of 19,290 single-nucleotide polymorphisms (SNPs) in 199 genes in the DSB repair pathway from a genome-wide association study (GWAS) dataset and explored their associations with overall survival (OS) in 1039 Han Chinese epithelial ovarian carcinoma (EOC) patients. After utilizing multivariate Cox regression analysis with bayesian false-discovery probability for multiple test correction, significant genetic variations were identified and subsequently underwent functional prediction and validation.
RESULTS
We discovered a significant association between poor overall survival and the functional variant GEN1 rs56070363 C > T (CT + TT vs. TT, adjusted hazard ratio (HR) = 2.50, P < 0.001). And the impact of GEN1 rs56070363 C > T on survival was attributed to its reduced binding affinity to hsa-miR-1287-5p and the resultant upregulation of GEN1 mRNA expression. Overexpression of GEN1 aggregated EOC cell proliferation, invasion and migration presumably by influencing the expression of immune inhibitory factors, thereby elevating the proportion of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and then constructing an immunosuppressive tumor microenvironment.
CONCLUSIONS
In conclusion, GEN1 rs56070363 variant could serve as a potential predictive biomarker and chemotherapeutic target for improving the survival of EOC patients.
Topics: Female; Humans; Middle Aged; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Cell Proliferation; China; East Asian People; Gene Expression Regulation, Neoplastic; Genome-Wide Association Study; Kaplan-Meier Estimate; MicroRNAs; Neoplasm Invasiveness; Ovarian Neoplasms; Polymorphism, Single Nucleotide; Prognosis; Survival Analysis; Holliday Junction Resolvases
PubMed: 38890669
DOI: 10.1186/s12967-024-05236-1 -
Mikrochimica Acta Jun 2024An efficient PEC biosensor is proposed for ATP detection based on exciton energy transfer from CdTe quantum dots (CdTe QDs) to Au nanoparticles (AuNPs), integrating...
An efficient PEC biosensor is proposed for ATP detection based on exciton energy transfer from CdTe quantum dots (CdTe QDs) to Au nanoparticles (AuNPs), integrating CRISPR/Cas12a trans-cleavage activity and specific recognition of ZIF-67 to ATP. Exciton energy transfer between CdTe QDs and AuNPs system is firstly constructed as photoelectrochemical (PEC) sensing substrate. Then, the activator DNAs, used to activate CRISPR/Cas12a, are absorbed on the surface of ZIF-67. In the presence of ATP, the activator DNAs are released due to more efficient adsorption of ZIF-67 to ATP. The released activator DNA activates trans-cleavage activity of CRISPR/Cas12a to degrade ssDNA on the electrode, leading to the recovery of photocurrent due to the interrupted energy transfer. Benefiting from the specific recognition of ZIF-67 to ATP and CRISPR/Cas12a-modulated amplification strategy, the sensor is endowed with excellent specificity and high sensitivity.
Topics: Biosensing Techniques; Adenosine Triphosphate; Electrochemical Techniques; Cadmium Compounds; Quantum Dots; Gold; CRISPR-Cas Systems; Metal Nanoparticles; Tellurium; Imidazoles; CRISPR-Associated Proteins; Limit of Detection; Zeolites; Endodeoxyribonucleases; Metal-Organic Frameworks; Photochemical Processes; Bacterial Proteins
PubMed: 38888689
DOI: 10.1007/s00604-024-06474-2 -
Nature Communications Jun 2024Holliday junction resolution is a crucial process in homologous recombination and DNA double-strand break repair. Complete Holliday junction resolution requires two...
Holliday junction resolution is a crucial process in homologous recombination and DNA double-strand break repair. Complete Holliday junction resolution requires two stepwise incisions across the center of the junction, but the precise mechanism of metal ion-catalyzed Holliday junction cleavage remains elusive. Here, we perform a metal ion-triggered catalysis in crystals to investigate the mechanism of Holliday junction cleavage by MOC1. We capture the structures of MOC1 in complex with a nicked Holliday junction at various catalytic states, including the ground state, the one-metal ion binding state, and the two-metal ion binding state. Moreover, we also identify a third metal ion that may aid in the nucleophilic attack on the scissile phosphate. Further structural and biochemical analyses reveal a metal ion-mediated allosteric regulation between the two active sites, contributing to the enhancement of the second strand cleavage following the first strand cleavage, as well as the precise symmetric cleavage across the Holliday junction. Our work provides insights into the mechanism of metal ion-catalyzed Holliday junction resolution by MOC1, with implications for understanding how cells preserve genome integrity during the Holliday junction resolution phase.
Topics: DNA, Cruciform; Metals; Holliday Junction Resolvases; Catalytic Domain; Saccharomyces cerevisiae Proteins; Crystallography, X-Ray; Ions; DNA Breaks, Double-Stranded; Models, Molecular; Allosteric Regulation
PubMed: 38886375
DOI: 10.1038/s41467-024-49490-9 -
Journal of Biotechnology Aug 2024The CRISPR/Cas12a system is emerging as a promising candidate for next-generation diagnostic biosensing platforms, with the discovery of new activation modes greatly...
The CRISPR/Cas12a system is emerging as a promising candidate for next-generation diagnostic biosensing platforms, with the discovery of new activation modes greatly expanding its applications. Here, we have identified two novel CRISPR/Cas12a system activation modes: PAM- and toehold-free DNA hairpins, and DNA-RNA hybrid strands. Utilizing a well-established real-time fluorescence method, we have demonstrated a strong correlation between DNA hairpin structures and Cas12a activation. Compared with previously reported activation modes involving single-stranded DNA and PAM-contained double-stranded DNA, the DNA hairpin activation way exhibits similar specificity and generality. Moreover, our findings indicate that increasing the number of RNA bases in DNA-RNA hybrid strands can decelerate the kinetics of Cas12a-triggered trans-cleavage of reporter probes. These newly discovered CRISPR/Cas12a activation ways hold significant potential for the development of high-performance biosensing strategies.
Topics: CRISPR-Cas Systems; RNA; DNA; CRISPR-Associated Proteins; Biosensing Techniques; DNA, Single-Stranded; Bacterial Proteins; Nucleic Acid Conformation; Endodeoxyribonucleases
PubMed: 38880387
DOI: 10.1016/j.jbiotec.2024.06.011 -
Analytica Chimica Acta Aug 2024Harmful algal blooms (HABs), caused by the rapid proliferation or aggregation of microorganisms, are catastrophic for the environment. The Prymnesium parvum is a...
BACKGROUND
Harmful algal blooms (HABs), caused by the rapid proliferation or aggregation of microorganisms, are catastrophic for the environment. The Prymnesium parvum is a haptophyte algal species that is found worldwide and is responsible for extensive blooms and death of larval amphibians and bivalves, causing serious negative impacts on the ecological environment. For the prevention and management of environmental pollution, it is crucial to explore and develop early detection strategies for HABs on-site using simple methods. The major challenge related to early detection is the accurate and sensitive detection of algae present in low abundance.
RESULTS
Herein, recombinase polymerase amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats and Cas12a protein (CRISPR-LbaCas12a) systems, and the lateral flow dipstick (LFD) was used for the first time for early detection of P. parvum. The internal transcribed spacer (ITS) of P. parvum was selected as the target sequence, and the concentration of single-strand DNA reporters, buffer liquid system, reaction time, and amount of gold particles were optimized. The RPA-CRISPR-LbaCas12a-LFD approach demonstrated highly specificity during experimental testing, with no cross-reaction against different microalgae used as controls. In addition, the lowest detection limit was 10,000 times better than the lowest detection limit of the standalone RPA approach. The feasibility and robustness of this approach were further verified by using the different environmental samples. It also observed that P. parvum are widely distributed in Chinese Sea, but the cell density of P. parvum is relatively low (<0.1 cells/mL).
SIGNIFICANCE
The developed approach has an excellent specificity and offers 10,000 times better sensitivity than the standalone RPA approach. These advantages make this approach suitable for early warning detection and prevention of HAB events in environmental water. Also, the outcomes of this study could promote a shift from traditional laboratory-based detection to on-site monitoring, facilitating early warning against HABs.
Topics: CRISPR-Cas Systems; Limit of Detection; Nucleic Acid Amplification Techniques; Recombinases; Harmful Algal Bloom; Gold; CRISPR-Associated Proteins; Endodeoxyribonucleases; Bacterial Proteins
PubMed: 38879209
DOI: 10.1016/j.aca.2024.342797