Mutant RNA Research Articles

CNSC-12. IMMUNOLOGICALLY TARGETING U1 MUTANT SHH MEDULLOBLASTOMA

Abstract OBJECTIVES Medulloblastoma (MB) is the most common malignant pediatric brain tumor representing a significant burden of morbidity and mortality in the US. MB is comprised of four subgroups: Wnt, Shh, Group 3, and Group 4. Shh tumors represent 25% of cases and subdivides into Shh-beta and Shh-gamma, Shh-alpha, and Shh-delta. Half of Shh MB carry an identical somatic point mutation in a non-coding small nuclear RNA (snRNA) called U1 (r.3A>G) which is found in 97% of Shh-d tumors, and in most Shh-alpha tumors with TP53 mutations. Current therapies for patients with TP53 and U1 mutant Shh-alpha MB observe rare survivors, and adult Shh-delta patients continue to experience significant morbidity and mortality calling for urgent prioritization of these tumors for targeted therapy. METHODS Cryptic exons were identified in both Shh-delta U1 snRNA mutant samples and Shh-delta U1 wildtype (WT) samples using CryEx pipeline. In-house scripts were utilized for selecting for cryptic exons that are uniquely expressed in Shh-delta U1 snRNA mutant compared to wildtype (WT) samples. RESULTS Analyzing 180 Shh MB RNA-seq samples, we identified 23% Shh-alpha, no Shh-beta, 97% of Shh-delta and 3% Shh-gamma harbored the U1 mutation. The splicing landscape was then interrogated comparing Shh-delta U1 snRNA mutant samples to WT samples. Expressed exons were filtered to exclude known exons to identify novel or cryptic exons. To select for Shh-delta U1 snRNA mutant induced cryptic exons, CryEx arising from introns, not identified in Shh-delta U1 WT and included >10% of their inclusion rates measured by percent spliced in (PSI) in Shh-delta U1 snRNA mutant samples were filtered. Of the middle CryEx, we identified 43,188 that were U1 mutant induced. Further filtering for cell surface Middle CryEx, three of the 75 middle CryEx overlapped. CONCLUSION The PTCH1 neoantigen formed from the CryEx insertion translates a protein that is unique to the tumor cells (i.e., not in normal tissue) was identified as a juxtamembrane for therapeutic drug discovery.

Assessing expression patterns of PTGR1, a potential biomarker for acylfulven sensitivity in urothelial carcinoma

BackgroundMetastatic urothelial carcinoma (mUC) has a poor prognosis, despite recent therapeutic advancements. Prostaglandin Reductase 1 (PTGR1) is essential for activating acylfulvens, a promising class of drugs for treating a subset of urothelial carcinoma (UC) patients. The efficacy of acylfulvens depends on PTGR1 activity and defects in the nucleotide excision repair (NER) pathway, notably ERCC2 mutations present in 10–15% of bladder tumors. This study identifies patients eligible to be included in acylfulven clinical trials based on the presence of PTGR-1 by immunohistochemistry (IHC) staining, RNA expression level and mutations in the NER pathway. Additionally, it evaluates PTGR-1 expression as a prognostic biomarker.MethodsThree PTGR-1 antibodies were tested in a kidney cancer cell line A498 and in tissues with known PTGR1 expression (liver, tonsils, pancreas, small intestine, etc.). Patients with untreated mUC receiving 1st line platinum from Dec. 2019 to Dec. 2021 in the Capital Region, Denmark, were included retrospectively. FFPE tumor samples were collected, and tissue microarrays (TMAs) were constructed. TMAs were stained with the best-performing PTGR1 antibody, RNA expression was analyzed using Nanostring nCounter PanCancer panel and gene mutations were assessed using a targeted genomic panel (TSO-500). Kaplan-Meier and multivariate Cox regression assessed overall survival and covariate impacts.ResultsThe AB181131 PTGR1 antibody was the most reliable in validation tissues. Tumors from 71 mUC patients were used to construct the TMA, and 40% of tumors scored positive for PTGR1 by IHC staining. A normalized PTGR1 RNA cutoff at 2,550 normalized counts achieved an AUC of 0.9, defining 35 samples as positive with a sensitivity of 96% and specificity of 85% in relation to IHC-positivity. Differential expression showed a significant upregulation of PTGR1 RNA in PTGR1 IHC-positive cases. NER-deficiency and PTGR1 positivity (mutations in ERCC1, ERCC2, ERCC3, ERCC4) was seen in 9 patients (13%). Median overall survival was 16 months in the cohort. Overall survival (OS) analysis indicates that overexpression of PTGR1 RNA was associated with a reduced median OS (12 months vs. 25 months, p = 0.039, log-rank).ConclusionPTGR1 IHC staining pattern using the Abcam AB181131 antibody is highly correlated with PTGR1 RNA expression. 13% in our cohort were identified as NER deficient and PTGR1 positive. Lower levels of PTGR1 indicates better outcome in this cohort.

High-resolution and programmable RNA-IN and RNA-OUT genetic circuit in living mammalian cells

RNAs and their encoded proteins intricately regulate diverse cell types and states within the human body. Dysregulated RNA expressions or mutations can lead to various diseased cell states, including tumorigenesis. Detecting and manipulating these endogenous RNAs offers significant promise for restoring healthy cell states and targeting tumors both in research and clinical contexts. This study presents an RNA-IN and RNA-OUT genetic circuit capable dynamically sensing and manipulating any RNA target in a programmable manner. The RNA-IN module employes a programmable CRISPR-associated protease (CASP) complex for RNA detection, while the RNA-OUT module utilizes an engineered protease-responsive dCas9-VPR activator. Additionally, the CASP module can detect point mutations by harnessing an uncovered dual-nucleotide synergistic switching effect within the CASP complex, resulting in the amplification of point-mutation signals from initially undetectable levels (1.5-fold) to a remarkable 94-fold. We successfully showcase the circuit’s ability to rewire endogenous RNA-IN signals to activate endogenous progesterone biosynthesis pathway, dynamically monitor adipogenic differentiation of mesenchymal stem cells (MSCs) and the epithelial-to-mesenchmal trans-differentiation, as well as selective killing of tumor cells. The programmable RNA-IN and RNA-OUT circuit exhibits tremendous potential for applications in gene therapy, biosensing and design of synthetic regulatory networks.

Distinct regulatory mechanisms by the nuclear Argonautes HRDE-1 and NRDE-3 in the soma of Caenorhabditis elegans.

This study examines the transcriptional consequences during the disruption of the nuclear RNAi silencing mechanism in C. elegans . Through microscopy and bioinformatic work, we demonstrate that although nuclear RNAi mutants exhibit significantly decondensed X chromosomes, chromosome-wide transcriptional de-repression is not detectable. Downstream analyses further explore the global influence of the nuclear RNAi pathway, indicating that the nuclear Argonautes HRDE-1 and NRDE-3 function through two distinct mechanisms.

Requirement of the N-terminal region of nonstructural protein 1 in cis for SARS-CoV-2 defective RNA replication.

SARS-CoV-2 belongs to the family Coronaviridae and carries a single-stranded positive-sense RNA genome. During coronavirus (CoV) replication, defective or defective interfering RNAs that lack a large portion of the genome often emerge. These defective RNAs typically carry the necessary RNA elements that are required for replication and packaging. We identified the minimum requirement of the 5' proximal region necessary for viral RNA replication by using artificially generated SARS-CoV-2 minigenomes. The minigenomes consist of the 5'-proximal region, an open reading frame (ORF) that encodes a fusion protein consisting of the N-terminal of viral NSP1 and a reporter gene, and the 3' untranslated region of the SARS-CoV-2 genome. We used a modified SARS-CoV-2 variant to support replication of the minigenomes. A minigenome carrying the 5' proximal 634 nucleotides replicated, whereas those carrying shorter than 634 nucleotides did not, demonstrating that the entire 265 nt-long 5' untranslated region and N-terminal portion of the NSP1 coding region are required for the minigenome replication. Minigenome RNAs carrying a specific amino acid substitution or frame shift insertions in the partial NSP1 coding sequence abrogated minigenome replication. Introduction of synonymous mutations in the minigenome RNAs also affected the replication efficiency of the minigenomes. These data suggest that the expression of the N-terminal portion of NSP1 and the primary sequence of the 5' proximal 634 nucleotides are important for minigenome replication.IMPORTANCESARS-CoV-2, the causative agent of COVID-19, is highly transmissible and continues to have a significant impact on public health and the global economy. While several vaccines mitigate the severe consequences of SARS-CoV-2 infection, mutant viruses with reduced reactivity to current vaccines continue to emerge and circulate. This study aimed to identify the minimal 5' proximal region of SARS-CoV-2 genomic RNA required for SARS-CoV-2 defective RNA replication and investigate the importance of an ORF encoded in these defective RNAs. Identifying cis-acting replication signals of SARS-CoV-2 genomic RNA is critical for the development of antivirals that target these signals. Additionally, replication-competent defective RNAs can serve as therapeutic reagents to interfere with SARS-CoV-2 replication. Our findings provide valuable insights into the mechanisms of SARS-CoV-2 RNA replication and the development of reagents that suppress SARS-CoV-2 replication.

RNA language models predict mutations that improve RNA function.

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. While advances in deep learning enable the prediction of accurate protein structural models, RNA structure prediction is not possible at present due to a lack of abundant high-quality reference data 1 . Furthermore, available sequence data are generally not associated with organismal phenotypes that could inform RNA function 2-4 . We created GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB) 5 . GARNET links RNA sequences derived from GTDB genomes to experimental and predicted optimal growth temperatures of GTDB reference organisms. This enables construction of deep and diverse RNA sequence alignments to be used for machine learning. Using GARNET, we define the minimal requirements for a sequence- and structure-aware RNA generative model. We also develop a GPT-like language model for RNA in which overlapping triplet tokenization provides optimal encoding. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identified mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB- derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

Sialylation-associated long non-coding RNA signature predicts the prognosis, tumor microenvironment, and immunotherapy and chemotherapy options in uterine corpus endometrial carcinoma

BackgroundSialylation in uterine corpus endometrial carcinoma (UCEC) differs significantly from apoptotic and ferroptosis pathways. It plays a crucial role in cancer progression and immune response modulation. Exploring how sialylation affects tumor behavior and its link with long non-coding RNAs (lncRNAs) may provide new insights into UCEC prognosis and treatment.MethodsWe obtained RNA transcriptome, clinical, and mutation data of UCEC samples from the TCGA database. Our approach involved developing a risk model based on the co-expression patterns of sialylation genes and lncRNAs. Prognostic lncRNAs were identified through Cox regression and further refined using LASSO analysis. To understand the biological functions and pathways of model-associated differentially expressed genes (MADEGs), we conducted enrichment analyses. We also assessed the immune infiltration status of MADEGs using eight different algorithms, which helped in evaluating the potential for immunotherapy. Additionally, we validated the expression of these lncRNAs in UCEC using cell lines and clinical samples.ResultsWe developed a UCEC risk model using five sialylation-related lncRNAs (AC004884.2, AC026202.2, LINC01579, LINC00942, SLC16A1-AS1). This model, confirmed through Cox analysis and clinical evaluation, effectively predicted patient outcomes. Survival data analysis across entire cohort, as well as within training and test groups, indicated better survival in low-risk UCEC patients. Enrichment analyses linked MADEGs to sialylation functions and cancer pathways. High-risk patients showed increased responsiveness to immune checkpoint inhibitors (ICIs), as indicated by immunological assessments. Subgroup C2 patients showed superior outcomes and a robust response to immunotherapy and chemotherapy. Notably, LINC01579, LINC00942, and SLC16A1-AS1 were significantly overexpressed in UCEC clinical tumor samples as well as in Ishikawa and HEC-1-B cell lines, compared to the normal groups.ConclusionsThis lncRNA signature associated with sialylation could guide prognosis, enhance the understanding of molecular mechanisms, and inform treatment strategies in UCEC. It highlights the potential for the use of ICIs and chemotherapy.

LpY3IP1 Enhances the drought and salt tolerance of perennial ryegrass by protecting the photosynthetic apparatus

Perennial ryegrass (Lolium perenne L.), one of the main turfgrass species widely planted, is often subjected to drought and salt stresses due to its perennial nature and worldwide distribution. However, the molecular mechanisms and key genes involved in the adaptation of perennial ryegrass to these environmental stresses are largely unknown. Ycf3-interacting protein 1 (Y3IP1), an auxiliary factor of photosystem I (PSI), has recently been shown to enhance stress tolerance. However, the detailed mechanisms through which Y3IP1 enhances stress resistance remain poorly understood. In this study, we discovered that LpY3IP1 in perennial ryegrass positively regulates the drought and salt tolerance by protecting the photosynthetic apparatus under adverse conditions. Seedlings overexpressing LpY3IP1 exhibited improved photosynthetic performance and survival rates under drought and salt stresses, whereas RNAi lines were more vulnerable. Under drought and salt stresses, overexpression lines maintained higher levels of PSI core subunits, while the RNAi mutants showed a reduction in these subunits. LpY3IP1 promoted cyclic electron flow (CEF) at PSI and inhibited the accumulation of ROS under stress. Chloroplasts in RNAi lines were more disorganized and degraded, exhibiting shrunken structures, low staining vesicles, protrusions, and more unstacked and swollen thylakoids under drought or salt treatment. Nevertheless, these changes were less pronounced in the overexpression lines. Therefore, our results reveal that LpY3IP1 enhances the drought and salt tolerance of perennial ryegrass by promoting CEF and mitigating oxidative damage to chloroplasts under stressful conditions.

Quantitative assessment and genomic profiling of Campylobacter dynamics in poultry processing: a case study in the United Arab Emirates integrated abattoir system.

In the United Arab Emirates, no previous research has investigated the dynamics of the foodborne pathogen Campylobacter in broiler abattoir processing. This study conducted in one of the largest poultry producers in the UAE, following each key slaughter stage-defeathering, evisceration, and final chilling-five broiler carcasses were collected from 10 slaughter batches over a year. Additionally, one caecum was obtained from 15 chickens in each slaughter batch to evaluate the flock colonization. In total, 300 samples (150 carcasses and 150 caeca) were collected and enumerated for Campylobacter using standard methods. Campylobacter was pervasive in caecal samples from all slaughter batches, with 86% of carcasses post-defeathering and evisceration stages and 94% post-chilling tested positive for Campylobacter. Campylobacter coli predominates in 55.2% of positive samples, followed by Campylobacter jejuni in 21%, with both species co-existing in 23.8% of the samples. Campylobacter counts in caecal contents ranged from 6.7 to 8.5 log10 CFU/g, decreasing post-defeathering and evisceration to 3.5 log10 CFU/g of neck skin and further to 3.2 log10 CFU/g of neck skin post-evisceration. After chilling, 70% of carcasses exceeded 3 log10 CFU/g of neck skin. Whole-genome sequencing (WGS) of 48 isolates unveiled diverse sequence types and clusters, with isolates sharing the same clusters (less than 20 single nucleotide polymorphisms) between different farms, different flocks within the same farm, as well as in consecutive slaughter batches, indicating cross-contamination. Multiple antimicrobial resistance genes and mutations in gyrA T86I (conferring fluoroquinolone resistance) and an RNA mutation (23S r.2075; conferring macrolide resistance) were widespread, with variations between C. coli and C. jejuni. WGS results revealed that selected virulence genes (pglG, pseD, pseI, flaA, flaB, cdtA, and cdtC) were significantly present in C. jejuni compared to C. coli isolates. This study offers the first insights into Campylobacter dynamics in poultry processing in the UAE. This work provides a base for future research to explore additional contributors to Campylobacter contamination in primary production. In conclusion, effective Campylobacter management demands a comprehensive approach addressing potential contamination sources at every production and processing stage, guided by continued microbiological surveillance and genomic analysis to safeguard public health and food safety.

Unilateral Hearing Loss and Auditory Asymmetry in Mitochondrial Disease: A Scoping Review.

Background/Objectives: Mitochondrial transfer RNA mutations are one of the most important causes of hereditary hearing loss in humans. In most cases, its presentation is bilateral and symmetrical; however, there are numerous cases of single-sided presentation or asymmetrical onset described in the literature that may represent a diagnostic challenge. The aim of this review is to present the evidence of auditory asymmetry in mitochondrial diseases, highlighting the possible presence of cases with atypical presentation. Methods: A review of the English literature to date on hearing loss and mitochondrial diseases was performed using PubMed, Scopus, and Google Scholar databases. The literature review was performed using the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines for scoping review. Results: A total of 10 full-text articles were included in this review, comprising 25 patients with single-sided or asymmetrical hearing loss associated with mitochondrial disease. Conclusions: Sensorineural hearing loss due to mitochondrial disease can represent a complex diagnostic challenge in cases of asymmetric or unilateral presentation. It is critical to recognize this clinical variant and to diagnose it in daily clinical practice.

GTF2H5 Identified as a crucial synthetic lethal target to counteract chemoresistance in colorectal cancer

BackgroundSynthetic lethality (SL) emerges as a novel concept being explored to combat cancer progression and resistance to conventional therapy. Despite the efficacy of chemotherapy in select cases of colorectal cancer (CRC), a substantial proportion of patients encounter challenges, leading to an adverse prognosis of CRC patients. CRC-related SL genes offer a potential avenue for identifying therapeutic targets. MethodsCRC-related SL genes were obtained from the SynLethDB database. The bulk RNA sequencing data, mutation data, and clinical information for treated and untreated CRC patients were enrolled from the UCSC and GEO databases. The Tumor Immunology Single Cell Center database served as the repository for collecting and analyzing single-cell RNA sequencing data. The synergistic killing effect of SL genes and chemotherapeutic drugs on resistant cells was experimentally verified. ResultsIn the present study, pivotal SL genes associated with chemoresistance identified by using WGCNA and CRC patients categorized into two groups based on these genes. Variations between the groups were most pronounced in pathways associated with extracellular matrix remodeling. Further by integrating mutation data, five potential SL genes were discerned, which were highly expressed in the presence of TP53 or KRAS mutations, leading to a severely poor prognosis. Subsequent time series analysis revealed that the expression of GTF2H5 was gradually elevated at different stages of the transition from sensitive to resistant in CRC cells. Finally, it was preliminarily verified by experiments that GTF2H5 may play a key role in driving the drug-resistant transition within CRC cells. ConclusionsThe identification of SL genes that collaboratively interact with chemotherapeutic agents could provide new insights into solving the issue of chemotherapy resistance in CRC patients. And GTF2H5 wields a fundamental influence in inducing chemoresistance in CRC, which provided a potential therapeutic target for CRC.

Molecular Bases and Specificity behind the Activation of the Immune System OAS/RNAse L Pathway by Viral RNA.

The first line of defense against invading pathogens usually relies on innate immune systems. In this context, the recognition of exogenous RNA structures is primordial to fight, notably, against RNA viruses. One of the most efficient immune response pathways is based on the sensing of RNA double helical motifs by the oligoadenylate synthase (OAS) proteins, which in turn triggers the activity of RNase L and, thus, cleaves cellular and viral RNA. In this contribution, by using long-range molecular dynamics simulations, complemented with enhanced sampling techniques, we elucidate the structural features leading to the activation of OAS by interaction with a model double-strand RNA oligomer mimicking a viral RNA. We characterize the allosteric regulation induced by the nucleic acid leading to the population of the active form of the protein. Furthermore, we also identify the free energy profile connected to the active vs. inactive conformational transitions in the presence and absence of RNA. Finally, the role of two RNA mutations, identified as able to downregulate OAS activation, in shaping the protein/nucleic acid interface and the conformational landscape of OAS is also analyzed.

Microbe Profile: Bacteriophage ϕ6: a model for segmented RNA viruses and the evolutionary consequences of viral 'sex'.

Bacteriophage ϕ6 is a segmented dsRNA virus with a lipid envelope, which are unusual traits in bacterial viruses but common in eukaryotic viruses. This uniqueness allowed ϕ6 and its Pseudomonad hosts to serve as a molecular model for RNA genetics, mutation, replication, packaging, and reassortment in both bacterial and eukaryotic viruses. However, an additional uniqueness of ϕ6, created by its high mutation rate, was its use as an experimental system to study key questions such as the evolution of sex (segment reassortment), host-pathogen interactions, mutational load, rates of adaptation, genetic and phenotypic complexity, and game theory.

Physical layer security enhanced scheme based on joint encryption of DNA and RNA data perturbation in CO-OFDM system

—In the work, we propose a new chaotic encryption algorithm for optical physical layer security enhanced in coherent optical orthogonal frequency division multiplexing (CO-OFDM) systems. The proposed algorithm adopts the six-dimensional (6D) hyperchaotic to achieve the generation of key sequences, which can effectively improve the unpredictability and security of the system. The encryption scheme includes two steps, first, the binary sequence is disturbed by DNA and RNA two-stage encoding and RNA mutation, and then the initial encrypted data is injected into the algorithm module with two-dimensional continuous chaotic mapping (2D-SCL) and Brownian motion to scramble successively. Simulations in a 16QAM CO-OFDM system at a symbol rate of 30 Gbaud over 80 km of standard single-mode fiber (SSMF) show that a legitimate user is able to successfully decrypt the received signals, and an illegal eavesdropper obtains a useless signal with a bit error rate (BER) of about 0.5. The scheme can tolerate very small deviations and has a key space of approximately 10294, which can effectively withstand illegal attacks. And the peak-to-average power ratio (PAPR) can be reduced by about 1.01 dB.

Superoxide signal orchestrates tetrathiomolybdate-induced longevity via ARGK-1 in Caenorhabditis elegans

PurposeWhile reactive oxygen species (ROS) have been identified as key redox signaling agents contributing to aging process, which and how specific oxidants trigger healthy longevity remain unclear. This paper aimed to explore the precise role and signaling mechanism of superoxide (O2•−) in health and longevity. MethodsA tool for precise regulation of O2•− levels in vivo was developed based on the inhibition of superoxide dismutase 1 (SOD1) by tetrathiomolybdate (TM) in Caenorhabditis elegans (C. elegans). Then, we examined the effects of TM on lifespan, reproduction, lipofuscin accumulation, mobility, and stress resistance. Finally, the signaling mechanism for longevity induced by TM-O2•− was screened by transcriptome analysis and tested in sod-1 and argk-1 RNAi strains, sod-2, sod-3, and daf-16 mutants. ResultsTM promoted longevity in C. elegans with a concomitant extension of healthy lifespan as indicated by increasing fertility and mobility and reducing lipofuscin accumulation, as well as enhanced resistance to different abiotic stresses. Mechanically, TM could precisely regulate O2•− levels in nematodes via modulating SOD1 activity. An O2•− scavenger Mn(III)TBAP abolished TM-induced lifespan extension, while an O2•− generator paraquat at low concentration mimicked the life prolongation effects. The longevity in TM-treated worms was abolished by sod-1 RNAi but was not affected in sod-2 or sod-3 mutants. Further transcriptome analysis revealed arginine kinase ARGK-1 and its downstream insulin/insulin-like growth factor 1 signaling (IIS) as potential effectors for TM-O2•‾-induced longevity, and argk-1 RNAi or daf-16 mutant nullified the longevity. ConclusionsThese findings indicate that it is feasible to precisely control specific oxidant in vivo and O2•− orchestrates TM-induced health and longevity in C. elegans via ARGK-1-IIS axis.

Revealing Differential RNA Editing Specificity of Human ADAR1 and ADAR2 in Schizosaccharomyces pombe.

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2's preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.

The potentials of nonlinear polarization with hyperspectral imaging of RNA for hepatocellular carcinoma early diagnosis

BackgroundMost cancers acquire numerous genetic changes in proto-oncogenes as well as tumor-suppressor genes. Cancer's early diagnosis remains a challenge. Recently, nonlinear polarization has revealed the potential as a promising tool for early cancer diagnosis. Laser-induced nonlinear polarization can offer a novel fingerprint signature.MethodsIn this study, nonlinear polarization was adopted for the characterization of both DNA and RNA samples from healthy volunteers. Total DNA and RNA were illuminated with a 656-nm LED source, and the resonance frequencies (scattered and re-emitted signals) were captured and recorded using a hyperspectral camera.ResultsChanges in signal frequency as well as phase shift offered a potent means to differentiate DNA (control) from RNA (control). DNA (control) demonstrated characteristic resonance frequencies that differ from total RNA (control) at the 2nd, 3rd, 4th, and 5th harmonics. While DNA demonstrated a phase shift dominating at 0.88 GHz, RNA dominates at 0.106 GHz. The resonance spectral signature of RNA samples from people with hepatocellular carcinoma (HCC) was compared to that of RNA (control). RNA (HCC) demonstrated distinctive frequency signals at 0.014, 0.021, 0.032, and 0.072 GHz. These characteristics feature could facilitate early HCC diagnosis. While RNA (control) dominates at 0.014 and 0.072 MHz, RNA (HCC) dominates at 0.021 and 0.032.ConclusionAs far as we are aware, this is the initial investigation into the use of simple nonlinear polarization to generate spectral fingerprinting signatures of total DNA and RNA. Furthermore, RNA mutations due to HCC were identified via characteristic nonlinear spectral signature.

Unveiling Off-Target Mutations in CRISPR Guide RNAs: Implications for Gene Region Specificity.

The revolutionary CRISPR-Cas9 technology has revolutionized genetic engineering, and it holds immense potential for therapeutic interventions. However, the presence of off-target mutations and mismatch capacity poses significant challenges to its safe and precise implementation. In this study, we explore the implications of off-target effects on critical gene regions, including exons, introns, and intergenic regions. Leveraging a benchmark dataset and using innovative data preprocessing techniques, we have put forth the advantages of categorical encoding over one-hot encoding in training machine learning classifiers. Crucially, we use latent class analysis (LCA) to uncover subclasses within the off-target range, revealing distinct patterns of gene region disruption. Our comprehensive approach not only highlights the critical role of model complexity in CRISPR applications but also offers a transformative off-target scoring procedure based on ML classifiers and LCA. By bridging the gap between traditional target-off scoring and comprehensive model analysis, our study advances the understanding of off-target effects and opens new avenues for precision genome editing in diverse biological contexts. This work represents a crucial step toward ensuring the safety and efficacy of CRISPR-based therapies, underscoring the importance of responsible genetic manipulation for future therapeutic applications.

Simultaneous detection of tumor-derived mutations in DNA and RNA extracted from a single circulating tumor cell based on droplet digital polymerase chain reaction.

e15053 Background: Androgen receptor splice variant 7 (AR-V7) is a constitutively activated isoform of AR and has been associated with resistance to AR-targeted therapies and development of metastatic castration-resistant prostate cancer. Confirmation of AR-V7 expression is important step in prostate cancer diagnosis and determining the appropriate drug, however, the detection of AR-V7 with tissues has limitations, such as the clinical challenge to poorly accessible tumor tissues before and after therapy. In addition, KRAS mutations serve as a prognostic biomarker for the administration of appropriate anti-cancer drugs in patients with various cancers, including non-small cell lung cancer (NSCLC). Here, we developed the process for the simultaneous detection of AR-V7 and KRAS mutation in CTCs using isolation with CytoGen’s Smart Biopsy™ System with droplet digital polymerase chain reaction (ddPCR) with high sensitivity and specificity, overcoming the limitations of tissue biopsy. Methods: ddPCR assays were performed to detect the KRAS G12C mutation using CTC gDNA and to evaluate the absolute levels of AR-V7 using CTC mRNA. Analytical validation was performed using NSCLC NCI-H358 and prostate cancer 22Rv1 cells (10 and 5 cells each), which harbor KRAS G12C and AR-V7, respectively, spiked into 5 mL of healthy blood to mimic CTC. CTCs were isolated using CytoGen's Smart Biopsy™ System. Oligo-dT magnetic beads were used to isolate mRNA from CTCs, and gDNA was isolated from the mRNA-depleted CTC lysates using the AllPrep DNA/mRNA Nano Kit. PCR and droplet reading for each assay were performed on the QX200 AutoDG Droplet Digital PCR System. Results: As a result of CTC KRAS G12C detection using the ddPCR™ KRAS G12/G13 Screening Kit, the mutated KRAS gene was successfully detected in as few as 5 NCI-H358 cells. In the results of CTC AR-V7 detection using the custom TaqMan ddPCR assay, transcripts (AR-V7 and HPRT1) were successfully detected even with only 5 22Rv1 cells. These results suggest that CytoGen's Smart Biopsy™ System could be applied to the simultaneous detection of DNA/RNA mutated genes in clinical NSCLC and prostate cancer patients with detection technology requiring high sensitivity and specificity. Conclusions: Accurately detecting multiple genetic mutations in CTCs is a challenging because CTCs are very rare in the blood. In this study, we developed the process to simultaneously extract and detect DNA mutations and RNA splicing variants in CTCs using CytoGen's Smart Biopsy™ System. This process, along with the results from our ongoing clinical trials, could provide information for the diagnosis and appropriate treatment of patients with various cancers, including NSCLC and prostate cancer.

A novel disulfide death-related genes prognostic signature identifies the role of IPO4 in glioma progression

Background“Disulfide death,” a form of cellular demise, is triggered by the abnormal accumulation of intracellular disulfides under conditions of glucose deprivation. However, its role in the prognosis of glioma remains undetermined. Therefore, the main objective of this study is to establish prognostic signature based on disulfide death-related genes (DDRGs) and to provide new solutions in choosing the effective treatment of glioma.MethodsThe RNA transcriptome, clinical information, and mutation data of glioma samples were sourced from The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA), while normal samples were obtained from the Genotype-Tissue Expression (GTEx). DDRGs were compiled from previous studies and selected through differential analysis and univariate Cox regression analysis. The molecular subtypes were determined through consensus clustering analysis. Further, LASSO analysis was employed to select characteristic genes, and subsequently, a risk model comprising seven DDRGs was constructed based on multivariable Cox analysis. Kaplan-Meier survival curves were employed to assess survival differences between high and low-risk groups. Additionally, functional analyses (GO, KEGG, GSEA) were conducted to explore the potential biological functions and signaling pathways of genes associated with the model. The study also explored immune checkpoint (ICP) genes, immune cell infiltration levels, and immune stromal scores. Finally, the effect of Importin-4(IPO4) on glioma has been further confirmed through RT-qPCR, Western blot, and cell functional experiments.Results7 genes associated with disulfide death were obtained and two subgroups of patients with different prognosis and clinical characteristics were identified. Risk signature was subsequently developed and proved to serve as an prognostic predictor. Notably, the high-risk group exhibited an immunosuppressive microenvironment characterized by a high concentration of M2 macrophages and regulatory T cells (Tregs). In contrast, the low-risk group showed lower half-maximal inhibitory concentration (IC50) values. Therefore, patients in the high-risk group may benefit more from immunotherapy, while patients in the low-risk group may benefit more from chemotherapy. In addition, in vitro experiments have shown that inhibition of the expression of IPO4 leads to a significant reduction in the proliferation, migration, and invasion of glioma cells.ConclusionThis study identified two glioma subtypes and constructed a prognostic signature based on DDRGs. The signature has the potential to optimize the selection of patients for immune- and chemotherapy and provided a potential therapeutic target for glioma.