Mutations In Sequence Research Articles

Design of Vif-Derived Peptide Inhibitors with Anti-HIV-1 Activity by Interrupting Vif-CBFβ Interaction.

One of the major functions of the accessory protein Vif of human immunodeficiency virus type 1 (HIV-1) is to induce the degradation of APOBEC3 (A3) family proteins by recruiting a Cullin5-ElonginB/C-CBFβ E3 ubiquitin ligase complex to facilitate viral replication. Therefore, the interactions between Vif and the E3 complex proteins are promising targets for the development of novel anti-HIV-1 drugs. Here, peptides are designed for the Vif-CBFβ interaction based on the sequences of Vif mutants with higher affinity for CBFβ screened by a yeast surface display platform. We identified two peptides, VMP-63 and VMP-108, that could reduce the infectivity of HIV-1 produced from A3G-positive cells with IC50 values of 49.4 μM and 55.1 μM, respectively. They protected intracellular A3G from Vif-mediated degradation in HEK293T cells, consequently increasing A3G encapsulation into the progeny virions. The peptides could rapidly enter cells after addition to HEK293T cells and competitively inhibit the binding of Vif to CBFβ. Homology modeling analysis demonstrated the binding advantages of VMP-63 and VMP-108 with CBFβ over their corresponding wild-type peptides. However, only VMP-108 effectively restricted long-term HIV-1 replication and protected A3 functions in non-permissive T lymphocytes. Our findings suggest that competitive Vif-derived peptides targeting the Vif-CBFβ interaction are promising for the development of novel therapeutic strategies for acquired immune deficiency syndrome.

Switching from combination therapy with entecavir hydrate plus tenofovir alafenamide fumarate to tenofovir alafenamide fumarate monotherapy in patients with chronic hepatitisB based on nucleotide sequences of hepatitisB virus pregenome RNA.

Patients with chronic hepatitisB virus (HBV) infection experiencing viral breakthrough (BTH) or partial response (PR) during lamivudine (LAM) or entecavir hydrate (ETV) administration often took ETV plus tenofovir alafenamide fumarate (TAF) due to the emergence of a drug-resistance mutation. However, in patients lacking drug-resistance mutation against TAF, sufficient antiviral effects may be achievable with TAF monotherapy. We assessed the drug-resistance profile through nucleotide sequences of HBV pregenome RNA, and subsequently changed to TAF monotherapy from ETV plus TAF. This prospective study included 25 patients with serum HBV-DNA below 20IU/mL under ETV plus TAF administration. Pregenome RNA nucleotide sequences of HBV in the sera were analyzed using direct sequencing and deep sequencing. ETV was discontinued in patients without rtA194T and rtS106C+rtH126Y +rtD134E +rtL269I quadruple mutations in direct sequencing. LAM-PR, LAM-BTH, ETV-PR, and ETV-BTH were observed in 1, 16, 7, and 1 patient(s), respectively. Pregenome RNA nucleotide sequences were analyzable in 20 patients. Among the 12 patients classified as LAM-BTH, six patients showed rtL180M+rtM204V/I in direct sequencing, and one patient showed minor clones containing rtL180M+rtM204V+A194T in deep sequencing at a frequency of 0.3%. In the six patients classified as ETV-PR, one patient harbored rtM204I. No clones showing rtS106C+rtH126Y+rtD134E+rtL269I quadruple mutation were detected in deep sequencing. Subsequently, ETV was discontinued, and serum HBV-DNA remained undetectable up to 48weeks in all patients. Patients receiving ETV plus TAF due to partial response or BTH during initial LAM or ETV administration were able to safely transition to TAF monotherapy based on nucleotide sequences of HBV pregenome RNA in the sera.

Improving somatic exome sequencing performance by biological replicates

BackgroundNext-generation sequencing (NGS) technologies offer fast and inexpensive identification of DNA sequences. Somatic sequencing is among the primary applications of NGS, where acquired (non-inherited) variants are based on comparing diseased and healthy tissues from the same individual. Somatic mutations in genetic diseases such as cancer are tightly associated with genomic instability. Genomic instability increases heterogenity, complicating sequencing efforts further, a task already challenged by the presence of short reads and repetitions in human DNA. This leads to low concordance among studies and limits reproducibility. This limitation is a significant problem since identified mutations in somatic sequencing are major biomarkers for diagnosis and the primary input of targeted therapies. Benchmarking studies were conducted to assess the error rates and increase reproducibility. Unfortunately, the number of somatic benchmarking sets is very limited due to difficulties in validating true somatic variants. Moreover, most NGS benchmarking studies are based on relatively simpler germline (inherited) sequencing. Recently, a comprehensive somatic sequencing benchmarking set was published by Sequencing Quality Control Phase 2 (SEQC2). We chose this dataset for our experiments because it is a well-validated, cancer-focused dataset that includes many tumor/normal biological replicates. Our study has two primary goals. First goal is to determine how replicate-based consensus approaches can improve the accuracy of somatic variant detection systems. Second goal is to develop highly predictive machine learning (ML) models by employing replicate-based consensus variants as labels during the training phase.ResultsEnsemble approaches that combine alternative algorithms are relatively common; here, as an alternative, we study the performance enhancement potential of biological replicates. We first developed replicate-based consensus approaches that utilize the biological replicates available in this study to improve variant calling performance. Subsequently, we trained ML models using these biological replicates and achieved performance comparable to optimal ML models, those trained using high-confidence variants identified in advance.ConclusionsOur replicate-based consensus approach can be used to improve variant calling performance and develop efficient ML models. Given the relative ease of obtaining biological replicates, this strategy allows for the development of efficient ML models tailored to specific datasets or scenarios.

Abstract 4629: QClampTM Plex VEXAS syndrome UBA1 mutation detection assay: A noval XNA based assay to simultaneously detect eight UBA1 somatic mutations associated with VEXAS syndrome

Abstract VEXAS syndrome (acronym for Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) is a monogenic disease discovered in December 2020. It is an adult-onset systemic inflammatory disorder genetically characterized by somatic mutation of the UBA1 gene which encodes the ubiquitin like modifier activating enzyme 1. VEXAS patients have overlapping rheumatologic and hematologic manifestations, including increased risk for myelodysplastic syndrome (MDS). It is critically important to diagnose VEXAS syndrome, but UBA1 mutation test is still not commercially available. The three most frequent mutations of UBA1 gene are p.M41T(122T>C), p.M41V (c.121A>G), and p.M41L (c.121A>C) in codon 41 of exon 3. Other mutations including splice site mutations at exon 3 (c.118-2A>C, c.118-1G>C and c.118- 9_118-2del), p.S56F (c.167C>T) in codon 56 of exon 3, and p.S621C (c.1861A>T) in codon 621 of exon 16 have been reported. Based on XNA technology, we developed a QClampTM Plex platform to detect all the eight mutations in a single reaction with Luminex system. XNA technology enriches mutant target sequence in a PCR reaction by using synthetic DNA analog XNA (xenonucleic acid) to block wild type DNA. With spiking synthetic mutant DNA in GM24385 cell line DNA, we determined that this assay can detect mutations with a detection limit of variant allele frequency (VAF) for M41T at 0.1%, M41V at 0.1%, M41L at 0.25%, c.118-2A>C at 1%, c.118-1G>C at 1%, c.118- 9_118-2del at 1%, S56F at 0.25% and S621C at 1%, respectively, and specificity of 100% for all mutations. We tested saved DNA samples extracted from whole blood previously collected from suspected VEXAS patients and tested at the National Cancer Institute by Sanger sequencing, including 13 positive and 20 negative samples. Our assay detected all corresponding UBA1 mutations in the 13 positive patient samples, and did not detect UBA1 mutation in 18 of the 20 negative patient samples. Two of the 20 negative samples were also tested positive for UBA1 M41T and M41L on our assay, respectively, due to its significantly higher sensitivity than the Sanger sequencing platform, which has a detection limit of 15-20% VAF. Citation Format: Yunqing Ma. QClampTM Plex VEXAS syndrome UBA1 mutation detection assay: A noval XNA based assay to simultaneously detect eight UBA1 somatic mutations associated with VEXAS syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4629.

The effects of electromagnetic field radiation of extremely low frequency on growth parameters and nucleotide substitutions in L. minor clones

Current technologies have become a source of electromagnetic pollution resulting from artificially generated electromagnetic radiation (EMR). To understand the influence of the EMR on living organisms, we investigated the long-term effects of EMR of 50 Hz frequency on duckweed (Lemna minor) clones. Experimental groups of duckweed were treated directly and indirectly by changing EMR generating magnetic flux (MF) starting from 2 µT (0–11 weeks from the beginning of the experiment) and switching to 300 µT (12–48 weeks) MF density during the second part of the experiment. The growth parameters (plant growth, frond area, and frond number) and the point mutations appearing at the antioxidant genes DNA sequences [ascorbate peroxidase (APx), glutathione peroxidase (GPx), and catalase (Cat)] were analyzed. The significantly enhanced number of nucleotide substitutions in DNA sequences of L. minor clones directly affected by LF EMR in comparison to indirectly affected clones was revealed at the introns of APx, GPx, and Cat genes starting from the 10th week of the experiment. The results indicate that even low-dose chronic electromagnetic radiation may contribute to the changes in growth parameters and generation of point mutations in antioxidant gene sequences, especially in the intron regions.

Mesoscale DNA features impact APOBEC3A and APOBEC3B deaminase activity and shape tumor mutational landscapes

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Cytoplasmic retention of the DNA/RNA-binding protein FUS ameliorates organ fibrosis in mice.

Uncontrolled accumulation of extracellular matrix leads to tissue fibrosis and loss of organ function. We previously demonstrated in vitro that the DNA/RNA-binding protein fused in sarcoma (FUS) promotes fibrotic responses by translocating to the nucleus, where it initiates collagen gene transcription. However, it is still not known whether FUS is profibrotic in vivo and whether preventing its nuclear translocation might inhibit development of fibrosis following injury. We now demonstrate that levels of nuclear FUS are significantly increased in mouse models of kidney and liver fibrosis. To evaluate the direct role of FUS nuclear translocation in fibrosis, we used mice that carry a mutation in the FUS nuclear localization sequence (FUSR521G) and the cell-penetrating peptide CP-FUS-NLS that we previously showed inhibits FUS nuclear translocation in vitro. We provide evidence that FUSR521G mice or CP-FUS-NLS-treated mice showed reduced nuclear FUS and fibrosis following injury. Finally, differential gene expression analysis and immunohistochemistry of tissues from individuals with focal segmental glomerulosclerosis or nonalcoholic steatohepatitis revealed significant upregulation of FUS and/or collagen genes and FUS protein nuclear localization in diseased organs. These results demonstrate that injury-induced nuclear translocation of FUS contributes to fibrosis and highlight CP-FUS-NLS as a promising therapeutic option for organ fibrosis.

Resolving the Nanoscale Structure of β-Sheet Peptide Self-Assemblies Using Single-Molecule Orientation-Localization Microscopy.

Synthetic peptides that self-assemble into cross-β fibrils are versatile building blocks for engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarities to amyloid species have been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize by using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize Nile red (NR), an amyloidophilic fluorogenic probe, and single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers KFE8L and KFE8D and the pathological amyloid-beta peptide Aβ42. Importantly, NR SMOLM reveals the helical (bilayer) ribbon structure of both KFE8 and Aβ42 and quantifies the precise tilt of the fibrils' inner and outer backbones in relevant buffer conditions without the need for covalent labeling or sequence mutations. SMOLM also distinguishes polymorphic branched and curved morphologies of KFE8, whose backbones exhibit much more heterogeneity than those of typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross-β-rich fibrils.

Appropriate whole genome amplification and pathogenic loci detection can improve the accuracy of preimplantation genetic diagnosis for deletional α-thalassemia.

To improve the accuracy of preimplantation genetic testing (PGT) in deletional α-thalassemia patients. Article. fifty-two deletional α-thalassemia couples. Whole genome amplification (WGA), Next-generation sequencing (NGS) and PCR mutation loci detection. WGA, Single nucleotide polymorphism (SNP) and PCR mutation loci detection results; Analysis of embryo chromosome copy number variation (CNV). Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) methods for PGT for deletional α-thalassemia. Blastocyst biopsy samples (n = 253) were obtained from 52 deletional α-thalassemia couples. The results of the comparison of experimental data between groups MALBAC and MDA are as follows: (i) The average allele drop-out (ADO) rate, MALBAC vs. MDA = 2.27% ± 3.57% vs. 0.97% ± 1.4%, P=0.451); (ii) WGA success rate, MALBAC vs. MDA = 98.61% vs. 98.89%, P=0.851; (iii) SNP haplotype success rate, MALBAC vs. MDA = 94.44% vs. 96.68%, P=0.409; (iv) The result of SNP haplotype analysis is consistent with that of Gap-PCR/Sanger sequencing results, MALBAC vs. MDA = 36(36/72, 50%) vs. 151(151/181, 83.43%), P=0; (v) Valid SNP loci, MALBAC vs. MDA = 30 ± 9 vs. 34 ± 10, P=0.02; (vi) The mean CV values, MALBAC vs. MDA = 0.12 ± 0.263 vs. 0.09 ± 0.40, P=0.916; (vii) The average number of raw reads, MALBAC vs. MDA =3244259 ± 999124 vs. 3713146 ± 1028721, P=0; (viii) The coverage of genome (%), MALBAC vs. MDA = 5.02 ± 1.09 vs. 5.55 ± 1.49, P=0.008. Our findings indicate that MDA is superior to MALBAC for PGT of deletional α-thalassemia. Furthermore, SNP haplotype analysis combined with PCR loci detection can improve the accuracy and detection rate of deletional α-thalassemia.

Chloroplast Genome-Wide Analysis Reveals New Single-Nucleotide Polymorphism Resources for the ARMS-qPCR Identification of Dendrobium brymerianum

Dendrobium brymerianum Rchb. f. is a species of orchid with pharmacological interest for its potential to inhibit the growth of human lung cancer cells. The identification of the Dendrobium species is a notable problem due to morphological similarities and the limitations of universal DNA barcodes. To overcome these difficulties, this study employed complete chloroplast (cp) genome sequences as useful resources for the identification of D. brymerianum. Based on Illumina sequencing, the complete cp genomes of five D. brymerianum individuals were assembled. These genomes were in the quadripartite structure, diverse in length between 151,832 and 152,189 bp, and comprised 126 genes. Moreover, significant differences were found in the Small Single-Copy (SSC) and Large Single-Copy (LSC) regions in comparison to the Inverted Repeat (IR) regions. This study recognized hotspot regions and simple sequence repeat (SSR) loci, providing valuable insights into genetic markers. The phylogenetic relationship of Dendrobium species was discovered, highlighting the need for more precise differentiation practices. To address this, ARMS-specific primers, mainly AAob1/AAob2, confirmed strong specificity, permitting the accurate identification of D. brymerianum from other species through ARMS-qPCR. Overall, this study of D. brymerianum chloroplast genomes has generated valuable data about sequence variations, phylogenetics, and mutation dynamics. These perceptions will be valuable in future research on population genetics, taxonomy, and species identification within the Dendrobium genus.

Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methods.

Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles. Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis.

Machine learning to predict continuous protein properties from binary cell sorting data and map unseen sequence space

Proteins are a diverse class of biomolecules responsible for wide-ranging cellular functions, from catalyzing reactions to recognizing pathogens. The ability to evolve proteins rapidly and inexpensively toward improved properties is a common objective for protein engineers. Powerful high-throughput methods like fluorescent activated cell sorting and next-generation sequencing have dramatically improved directed evolution experiments. However, it is unclear how to best leverage these data to characterize protein fitness landscapes more completely and identify lead candidates. In this work, we develop a simple yet powerful framework to improve protein optimization by predicting continuous protein properties from simple directed evolution experiments using interpretable, linear machine learning models. Importantly, we find that these models, which use data from simple but imprecise experimental estimates of protein fitness, have predictive capabilities that approach more precise but expensive data. Evaluated across five diverse protein engineering tasks, continuous properties are consistently predicted from readily available deep sequencing data, demonstrating that protein fitness space can be reasonably well modeled by linear relationships among sequence mutations. To prospectively test the utility of this approach, we generated a library of stapled peptides and applied the framework to predict affinity and specificity from simple cell sorting data. We then coupled integer linear programming, a method to optimize protein fitness from linear weights, with mutation scores from machine learning to identify variants in unseen sequence space that have improved and co-optimal properties. This approach represents a versatile tool for improved analysis and identification of protein variants across many domains of protein engineering.

Abstract IA021: Novel genomic features of POLE-mutant tumors: the utility for tumor classification and identification of new driver alleles

Abstract Alterations in the exonuclease domain of POLE drive the development of ultramutated endometrial tumors with better prognosis. A hallmark of these tumors is the accumulation of G>T mutations in AGA sequences; however, understanding of their genomic features beyond the trinucleotide motifs is limited. We applied a novel computational framework to the whole-exome sequencing data of 524 endometrial tumors reported by The Cancer Genome Atlas network to analyze the extended DNA sequence context of ultramutation. We found that the presence of POLE driver alleles is associated with an increased frequency of G>T mutations in polypurine tracts. Sequences containing three consecutive purines, including the classic AGA context, are only moderately mutable, but the mutability increased abruptly as the tract length reached six or more purines. Moreover, mutations showed a strong preference for certain positions within the tracts, creating a characteristic quantifiable pattern. Using this signature, we developed a machine learning classifier to identify tumors with hitherto unknown POLE drivers and validated two new drivers, POLE-E978G and POLE-S461L, by functional assays in yeast. Unlike previously known pathogenic variants, the E978G substitution affects the DNA polymerase rather than exonuclease domain of POLE. We further show that the extended genomic signature of POLE ultramutation is shared by tumors with POLD1 drivers. These findings uncover new mechanisms of ultramutation and highlight the limitations of the current focus on POLE exonuclease domain variants when stratifying patients. Citation Format: Daria Ostroverkhova, Kathryn Tyryshkin, Annette K. Beach, Elizabeth A. Moore, Yosef Masoudi-Sobhanzadeh, Stephanie R. Barbari, Igor B Rogozin, Konstantin V Shaitan, Anna R Panchenko, Polina V. Shcherbakova. Novel genomic features of POLE-mutant tumors: the utility for tumor classification and identification of new driver alleles [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA021.

Integrating Fréchet distance and AI reveals the evolutionary trajectory and origin of SARS-CoV-2.

A genome, composed of a precisely ordered sequence of four nucleotides (ATCG), encompasses a multitude of specific genome features like AAA motif. Mutations occurring within a genome disrupt the sequential order and composition of these features, thereby influencing the evolutionary trajectories and yielding variants. The evolutionary relatedness between a variant and its ancestor can be estimated by assessing evolutionary distances across a spectrum of genome features. This study develops a novel, alignment-free algorithm that considers both the sequential order and composition of genome features, enabling computation of the Fréchet distance (Fr) across multiple genome features to quantify the evolutionary status of a variant. Integrating this algorithm with an artificial recurrent neural network (RNN) reveals the quantitative evolutionary trajectory and origin of SARS-CoV-2, a puzzle unsolved by alignment-based phylogenetics. The RNN generates the evolutionary trajectory from Fr data at two levels: genome sequence mutations and organism variants. At the genome sequence level, SARS-CoV-2 evolutionarily shortens its genome to enhance its infectious capacity. Mutating signature features, such as TTA and GCT, increases its infectious potential and drives its evolution. At the organism level, variants mutating a single biomarker possess low infectious potential. However, mutating multiple markers dramatically increases their infectious capacity, propelling the COVID-19 pandemic. SARS-CoV-2 likely originates from mink coronavirus variants, with its origin trajectory traced as follows: mink, cat, tiger, mouse, hamster, dog, lion, gorilla, leopard, bat, and pangolin. Together, mutating multiple signature features and biomarkers delineates the evolutionary trajectory of mink-origin SARS-CoV-2, leading to the COVID-19 pandemic.

NAT10-mediated ac4C-modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear-cell renal cell carcinoma by attenuating YWHAE-driven cytoplasmic retention of YAP1.

Lymphatic metastasis is one of the most common metastatic routes and indicates a poor prognosis in clear-cell renal cell carcinoma (ccRCC). N-acetyltransferase 10 (NAT10) is known to catalyze N4-acetylcytidine (ac4C) modification of mRNA and participate in many cellular processes. However, its role in the lymphangiogenic process of ccRCC has not been reported. This study aimed to elucidate the role of NAT10 in ccRCC lymphangiogenesis, providing valuable insights into potential therapeutic targets for intervention. ac4C modification and NAT10 expression levels in ccRCC were assessed using public databases and clinical samples. Functional investigations involved manipulating NAT10 expression in cellular and mouse models to study its role in ccRCC. Mechanistic insights were gained through a combination of RNA sequencing, mass spectrometry, co-immunoprecipitation, RNA immunoprecipitation, immunofluorescence, and site-specific mutation analyses. We found that ac4C modification and NAT10 expression levels increased in ccRCC. NAT10 promoted tumor progression and lymphangiogenesis of ccRCC by enhancing the nuclear import of Yes1-associated transcriptional regulator (YAP1). Subsequently, we identified ankyrin repeat and zinc finger peptidyl tRNA hydrolase 1 (ANKZF1) as the functional target of NAT10, and its upregulation in ccRCC was caused by NAT10-mediated ac4C modification. Mechanistic analyses demonstrated that ANKZF1 interacted with tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE) to competitively inhibit cytoplasmic retention of YAP1, leading to transcriptional activation of pro-lymphangiogenic factors. These results suggested a pro-cancer role of NAT10-mediated acetylation in ccRCC and identified the NAT10/ANKZF1/YAP1 axis as an under-reported pathway involving tumor progression and lymphangiogenesis in ccRCC.

Tumor location matters, next generation sequencing mutation profiling of left-sided, rectal, and right-sided colorectal tumors in 552 patients

Despite the introduction of new molecular classifications, advanced colorectal cancer (CRC) is treated with chemotherapy supplemented with anti-EGFR and anti-VEGF targeted therapy. In this study, 552 CRC cases with different primary tumor locations (250 left side, 190 rectum, and 112 right side) were retrospectively analyzed by next generation sequencing for mutations in 50 genes. The most frequently mutated genes were TP53 in left-sided tumors compared to right-sided tumors and BRAF in right-sided tumors compared to left-sided tumors. Mutations in KRAS, NRAS, and BRAF were not detected in 45% of patients with left-sided tumors and in 28.6% of patients with right-sided tumors. Liver metastases were more common in patients with left-sided tumors. Tumors on the right side were larger at diagnosis and had a higher grade (G3) than tumors on the left. Rectal tumors exhibit distinctive biological characteristics when compared to left-sided tumors, including a higher absence rate of KRAS, NRAS, and BRAF mutations (47.4% in rectal versus 42.8% in left-sided tumors). These rectal tumors are also unique in their primary metastasis site, which is predominantly the lungs, and they have varying mutation rates, particularly in genes such as BRAF, FBXW7, and TP53, that distinguish them from tumors found in other locations. Primary tumor location has implications for the potential treatment of CRC with anti-EGFR therapy.

The possible effects of the MTOR polymorphisms on preeclampsia susceptibility, severity, and onset: a case-control study and in silico analysis.

Preeclampsia (PE) is a gestational complication with developed hypertension and proteinuria. Evidence showed the role of mTOR in various cellular processes. Therefore, this study aimed to evaluate the effects of MTOR polymorphisms on susceptibility, severity, and onset of Preeclampsia (PE). A total of 250 PE pregnant women and 258 age-matched control subjects were recruited in this study. To genotype MTOR polymorphisms, the PCR-RFLP method was used. The SpliceAid 2 and PROMO tools were used for in silico analysis. The maternal MTOR rs17036508T/C polymorphism was associated with PE risk in various genetic models. There was no relationship between rs2536T/C and rs2295080T/G polymorphisms and PE. The TTC and TGC haplotypes of rs2536/ rs2295080/ rs17036508 polymorphisms were significantly higher in PE women. Subgroup analysis revealed the association between the MTOR rs2295080 variant and an increased risk of Early-onset PE (EOPE). However, the MTOR rs17036508 was associated with a higher risk of EOPE and Late- Onset PE. In addition, the MTOR rs2295080 could increase the risk of severe PE. The results of the in silico analysis showed that rs17036508 disrupted several binding motifs in the mutant sequence. The PROMO database revealed that the T to C substitution leads to the loss of the TFII-I binding site in the mutant allele. The MTOR rs17036508T/C polymorphism was associated with PE risk. There was an association between the MTOR rs2295080 variant and an increased risk of EOPE. The MTOR rs17036508T/C and rs2295080T/C variants could disrupt several binding motifs and TFII-I binding respectively.

Improving the Sensitivity of a Mn(II)-Specific DNAzyme for Cellular Imaging Sensor through Sequence Mutations.

Detection of Mn2+ in living cells is important in understanding the roles of Mn2+ in cellular processes and investigating its potential implications in various diseases and disorders. Toward this goal, we have previously selected a Mn2+-specific 11-5 DNAzyme through an in vitro selection method and converted it into a fluorescence sensor for intracellular Mn2+ sensing. Despite the progress, the nucleotides responsible for the activity are unclear, and the performance of the DNAzyme needs to be improved in order for more effective applications in biological systems. To address these issues, we herein report site-specific mutations within the catalytic domain of the selected 11-5 DNAzyme. As a result, we successfully identified a variant DNAzyme, designated as Mn5V, which exhibited a twofold increase in activity compared to the original 11-5 DNAzyme. Importantly, Mn5V DNAzyme maintained its high selectivity for Mn2+ over other competing metal ions. Upon the addition of Mn2+, Mn5V DNAzyme exhibited a higher fluorescence signal within the tumor cells compared to that of the 11-5 DNAzyme. This study therefore provides a better understanding of how the DNAzyme functions and a more sensitive probe for investigating Mn2+ in biological systems.

Entropic Dynamics of Mutations in SARS-CoV-2 Genomic Sequences.

In this paper, we investigate a certain class of mutations in genomic sequences by studying the evolution of the entropy and relative entropy associated with the base frequencies of a given genomic sequence. Even if the method is, in principle, applicable to every sequence which varies randomly, the case of SARS-CoV-2 RNA genome is particularly interesting to analyze, due to the richness of the available sequence database containing more than a million sequences. Our model is able to track known features of the mutation dynamics like the Cytosine-Thymine bias, but also to reveal new features of the virus mutation dynamics. We show that these new findings can be studied using an approach that combines the mean field approximation of a Markov dynamics within a stochastic thermodynamics framework.

Association of polymorphisms of IGF1 promoter with growth and fertility performance in PB1 parent line of broiler chicken variety

Blood samples from 180 birds pertaining to a single generation of PB1 parent line were collected for present study. The birds were raised under standard management and different growth variables were recorded up to 20 weeks of age. Age at first egg and egg production till 40 weeks of age was recorded in females. PCR-RFLP analysis was used to screen individuals with polymorphisms in IGF1 promoter region and three genotypes AA, AC and CC were identified at frequencies of 0.79, 0.18 and 0.03, respectively. CC homozygotes were lower with respect to their performance in growth and fertility traits. Sequencing results of both alleles revealed T244G transversion mutation in the C allele. Gene regulation analysis confirmed that such transversion resulted in non-binding of Oct-1 transcription factor at 241 to 250 bp in C allele, causing down regulation of the gene. The mutations in the promoter sequence affected the transcriptional gene regulation affecting growth and fertility performance.