Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome.

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspots and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.

Chronic PM2.5 exposure disrupts intestinal barrier integrity via microbial dysbiosis-triggered TLR2/5-MyD88-NLRP3 inflammasome activation.

BACKGROUND: PM2.5, a known public health risk, is increasingly linked to intestinal disorders, however, the mechanisms of its impact are not fully understood. PURPOSE: This study aimed to explore the impact of chronic PM2.5 exposure on intestinal barrier integrity and to uncover the underlying molecular mechanisms. METHODS: C57BL/6 J mice were exposed to either concentrated ambient PM2.5 (CPM) or filtered air (FA) for six months to simulate urban pollution conditions. We evaluated intestinal barrier damage, microbial shifts, and metabolic changes through histopathology, metagenomics, and metabolomics. Analysis of the TLR signaling pathway was also conducted. RESULTS: The mean concentration of PM2.5 in the CPM exposure chamber was consistently measured at 70.9 ± 26.8 µg/m³ throughout the study period. Our findings show that chronic CPM exposure significantly compromises intestinal barrier integrity, as indicated by reduced expression of the key tight junction proteins Occludin and Tjp1/Zo-1. Metagenomic sequencing revealed significant shifts in the microbial landscape, identifying 35 differentially abundant species. Notably, there was an increase in pro-inflammatory nongastric Helicobacter species and a decrease in beneficial bacteria, such as Lactobacillus intestinalis, Lactobacillus sp. ASF360, and Eubacterium rectale. Metabolomic analysis further identified 26 significantly altered metabolites commonly associated with intestinal diseases. A strong correlation between altered bacterial species and metabolites was also observed. For example, 4 Helicobacter species all showed positive correlations with 13 metabolites, including Lactate, Bile acids, Pyruvate and Glutamate. Additionally, increased expression levels of TLR2, TLR5, Myd88, and NLRP3 proteins were noted, and their expression patterns showed a strong correlation, suggesting a possible involvement of the TLR2/5-MyD88-NLRP3 signaling pathway. CONCLUSIONS: Chronic CPM exposure induces intestinal barrier dysfunction, microbial dysbiosis, metabolic imbalance, and activation of the TLR2/5-MyD88-NLRP3 inflammasome. These findings highlight the urgent need for intervention strategies to mitigate the detrimental effects of air pollution on intestinal health and identify potential therapeutic targets.

Frequency and patterns of ribonucleotide incorporation around autonomously replicating sequences in yeast reveal the division of labor of replicative DNA polymerases.

Ribonucleoside triphosphate (rNTP) incorporation in DNA by DNA polymerases is a frequent phenomenon that results in DNA structural change and genome instability. However, it is unclear whether the rNTP incorporation into DNA follows any specific sequence patterns. We analyzed multiple datasets of ribonucleoside monophosphates (rNMPs) embedded in DNA, generated from three rNMP-sequencing techniques. These rNMP libraries were obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late-firing autonomously replicating sequences (ARSs) of the yeast genome, where leading and lagging DNA synthesis starts bidirectionally. We found the preference of rNTP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNTP incorporation changes dramatically within the first 1,000 nucleotides from ARSs, highlighting the Pol δ - Pol ϵ handoff during early leading-strand synthesis. Furthermore, the pattern of rNTP incorporation is markedly distinct between the leading and lagging strands not only in mutant but also in wild-type polymerase cells. Such specific signatures of Pol δ and Pol ϵ provide a new approach to track the labor of these polymerases.

RESCOT: Restriction Enzyme Set and Combination Optimization Tools for rNMP Capture Techniques.

The incorporation of ribonucleoside monophosphates (rNMPs) in genomic DNA is a frequent phenomenon in many species, often associated with genome instability and disease. The ribose-seq technique is one of a few techniques designed to capture and map rNMPs embedded in genomic DNA. The first step of ribose-seq is restriction enzyme (RE) fragmentation, which cuts the genome into smaller fragments for subsequent rNMP capture. The RE selection chosen for genomic DNA fragmentation in the first step of the rNMP-capture techniques determines the genomic regions in which the rNMPs can be captured. Here, we designed a computational method, Restriction Enzyme Set and Combination Optimization Tools (RESCOT), to calculate the genomic coverage of rNMP-captured regions for a given RE set and to optimize the RE set to significantly increase the rNMP-captured-region coverage. Analyses of ribose-seq libraries for which the RESCOT tools were applied reveal that many rNMPs were captured in the expected genomic regions. Since different rNMP-mapping techniques utilize RE fragmentation and purification steps based on size-selection of the DNA fragments in the protocol, we discuss the possible usage of RESCOT for other rNMP-mapping techniques. In summary, RESCOT generates optimized RE sets for the fragmentation step of many rNMP capture techniques to maximize rNMP capture rate and thus enable researchers to better study characteristics of rNMP incorporation.

Strawberry tonoplast transporter, FaVPT1, controls phosphate accumulation and fruit quality.

Phosphorus (P) is essential for plant growth and development, and the vacuole is an important organelle for phosphate storage. However, the tonoplast phosphate transporter in fleshy fruits remains unknown. In this study, based on the strawberry (Fragaria × ananassa) fruit transcriptome data, a tonoplast-localized vacuolar phosphate transporter with SPX and major facilitator superfamily domains, FaVPT1, was identified. FaVPT1 expression was highest in the fruits and could be induced by sucrose. Using transient transgenic systems in strawberry fruit, the downregulation and upregulation of FaVPT1 inhibited and promoted ripening, respectively, and affected phosphate contents, fruit firmness, sugar and anthocyanin contents, and ripening-related gene transcription. FaVPT1 could rescue Pi absorption in both yeast and the Arabidopsis atvpt1 mutant, confirming the similar function of FaVPT1 and AtVPT1, a previously identified tonoplast phosphate transporter in Arabidopsis. The Escherichia coli-expressed SPX domain of FaVPT1 could strongly bind to InsP6 with a Kd of 3.5 µM. The results demonstrate that FaVPT1 is a tonoplast phosphate transporter and regulates strawberry fruit ripening and quality, to a large extent, via sucrose.

Mechanical and Architectural Changes in Animal Bone Following Fast Neutron Irradiation.

ABSTRACT: Damage to healthy bone following exposure to ionizing radiation has been well documented for at least seven decades. Among the reported effects are a transient increase in stiffness and a reduction in breaking strength. These changes have been linked to a decrease in osteoblast proliferation and differentiation, inducing cell cycle arrest, reducing collagen production, and increasing sensitivity to apoptotic agents. In this work, we analyzed some mechanical and structural changes in compact costal bovine bone (Hereford breed, n = 9) subjected to escalating doses of fast neutrons from a 7 Li(p,n) 7 Be reaction. The mean neutron energy was 233 keV with calculated absorbed doses ranging from 0 to 4.05 ± 10% Gy. Samples were subjected to Young's Modulus (YM) and breaking strength testing with a Universal Testing Machine (UTM). We found an increase in Young's Modulus and a decrease in breaking strength as functions of increasing dose equivalent. Optical coherence tomography (OCT) revealed trabecular displacement into compact bone in an irradiated sample (D = 4.05 ± 10% Gy), with breaching of the endosteal wall. OCT further revealed a "crack-like" structure across the irradiated sample, potentially consistent with damage from a proton track resulting from an elastic (n,p) reaction. No previous report has been found on mechanical changes in large mammalian bones following fast neutron doses, nor of the OCT imaging of such samples.

rNMPID: a database for riboNucleoside MonoPhosphates in DNA.

Motivation: Ribonucleoside monophosphates (rNMPs) are the most abundant non-standard nucleotides embedded in genomic DNA. If the presence of rNMP in DNA cannot be controlled, it can lead to genome instability. The actual regulatory functions of rNMPs in DNA remain mainly unknown. Considering the association between rNMP embedment and various diseases and cancer, the phenomenon of rNMP embedment in DNA has become a prominent area of research in recent years. Results: We introduce the rNMPID database, which is the first database revealing rNMP-embedment characteristics, strand bias, and preferred incorporation patterns in the genomic DNA of samples from bacterial to human cells of different genetic backgrounds. The rNMPID database uses datasets generated by different rNMP-mapping techniques. It provides the researchers with a solid foundation to explore the features of rNMP embedded in the genomic DNA of multiple sources, and their association with cellular functions, and, in future, disease. It also significantly benefits researchers in the fields of genetics and genomics who aim to integrate their studies with the rNMP-embedment data. Availability and implementation: rNMPID is freely accessible on the web at https://www.rnmpid.org.

DSBplot: Indels in DNA Double-strand Break Repair Experiments.

Double-strand breaks (DSBs) in DNA are naturally occurring destructive events in all organisms that may lead to genome instability. Cells employ various repair methods known as non-homologous end joining (NHEJ), microhomology mediated end joining (MMEJ), and homology-directed recombination (HDR). These repair processes may lead to DNA sequence variations (e.g., nucleotide insertions, deletions, and substitutions) at the location of the break. Studying DNA DSB repair processes often involves the use of high throughput sequencing assays to precisely quantify the sequence variations near the break with software tools. Often methods of assessing and visualizing these data have not taken into account the full complexity of the sequencing data, such as the frequency, type, and position of the sequence variations in a single comprehensive representation. Here we present a method that allows visualization of the overall variation pattern as well as comparison of these patterns among experimental setups.

RNA-mediated double-strand break repair by end-joining mechanisms.

Double-strand breaks (DSBs) in DNA are challenging to repair. Cells employ at least three DSB-repair mechanisms, with a preference for non-homologous end joining (NHEJ) over homologous recombination (HR) and microhomology-mediated end joining (MMEJ). While most eukaryotic DNA is transcribed into RNA, providing complementary genetic information, much remains unknown about the direct impact of RNA on DSB-repair outcomes and its role in DSB-repair via end joining. Here, we show that both sense and antisense-transcript RNAs impact DSB repair in a sequence-specific manner in wild-type human and yeast cells. Depending on its sequence complementarity with the broken DNA ends, a transcript RNA can promote repair of a DSB or a double-strand gap in its DNA gene via NHEJ or MMEJ, independently from DNA synthesis. The results demonstrate a role of transcript RNA in directing the way DSBs are repaired in DNA, suggesting that RNA may directly modulate genome stability and evolution.

The association between short-term exposure to nitrogen dioxide and hospital admission for schizophrenia: A systematic review and meta-analysis.

BACKGROUND: Ambient air pollution has been identified as a primary risk factor for mental disorders. In recent years, the relationship between exposure to ambient nitrogen dioxide (NO2) and the risk of hospital admissions (HAs) for schizophrenia has garnered increasing scientific interest, but evidence from epidemiological studies has been inconsistent. Therefore, a systematic review and meta-analysis were conducted to comprehensively identify potential correlations. METHODS: A literature search in 3 international databases was conducted before December 31, 2022. Relative risk (RR) and corresponding 95% confidence intervals (CI) were calculated to evaluate the strength of the associations. Summary effect sizes were calculated using a random-effects model due to the expected heterogeneity (I2 over 50%). RESULTS: A total of ten eligible studies were included in the meta-analysis, including 1,412,860 participants. The pooled analysis found that an increased risk of HAs for schizophrenia was associated with exposure to each increase of 10 µg/m3 in NO2 (RR = 1.029, 95% CI = 1.016-1.041, P < .001). However, the heterogeneity was high for the summary estimates, reducing the credibility of the evidence. In 2-pollutant models, results for NO2 increased by 0.3%, 0.2% and 2.3%, respectively, after adjusting for PM2.5, PM10 and SO2. CONCLUSIONS: This study provides evidence that NO2 exposure significantly increases the risk of hospital admission for schizophrenia. Future studies are required to clarify the potential biological mechanism between schizophrenia and NO2 exposure to provide a more definitive result.

Quetiapine Combined with Sodium Valproate in Patients with Alzheimer's Disease with Mental and Behavioral Symptoms Efficacy Observation.

Quetiapine combined with sodium valproate is an effective and more suitable drug treatment for Alzheimer's disease. At present, there are relatively few studies on the combined action mechanism of these two drugs. This study has certain practical value. Alzheimer's disease is a multifaceted, highly genetically heterogeneous neurodegenerative disease. The main clinical manifestations are memory loss, abnormal mental behavior, and loss of various cognitive functions. In order to improve the symptoms of patients with Alzheimer's disease, especially those with mental symptoms, this article combines quetiapine and sodium valproate, two commonly used drugs for the treatment of mental illnesses, and applies them to different levels of Alzheimer's and observes the results of the combination's curative effect. This article introduces Alzheimer's disease and its potential mental behaviors in the method section, and it also introduces the mechanism of action of quetiapine and sodium valproate. For the algorithm, this paper introduces a data mining algorithm to understand the effect of drug efficacy. In the experimental part, firstly, it introduces the experimental objects, the proportion of medicines, and the statistical methods. Secondly, this article covers adverse reactions, inflammatory factors and vascular endothelial indicators, Alzheimer's disease performance, MOAS score, treatment effect evaluation, and satisfaction surveys. It can be seen from the experiment that, in mental behavior, the experimental group decreased from 8.2 before treatment to 0.5, and the control group decreased from 7.1 before treatment to 2.6. It can be seen that the scores of the experimental group changed after receiving the treatment of quetiapine combined with sodium valproate.

Disproportionate presence of adenosine in mitochondrial and chloroplast DNA of Chlamydomonas reinhardtii.

Ribonucleoside monophosphates (rNMPs) represent the most common non-standard nucleotides found in the genome of cells. The distribution of rNMPs in DNA has been studied only in limited genomes. Using the ribose-seq protocol and the Ribose-Map bioinformatics toolkit, we reveal the distribution of rNMPs incorporated into the whole genome of a photosynthetic unicellular green alga, Chlamydomonas reinhardtii. We discovered a disproportionate incorporation of adenosine in the mitochondrial and chloroplast DNA, in contrast to the nuclear DNA, relative to the corresponding nucleotide content of these C. reinhardtii organelle genomes. Our results demonstrate that the rNMP content in the DNA of the algal organelles reflects an elevated ATP level present in the algal cells. We reveal specific biases and patterns in rNMP distributions in the algal mitochondrial, chloroplast, and nuclear DNA. Moreover, we identified the C. reinhardtii orthologous genes for all three subunits of the RNase H2 enzyme using GeneMark-EP + gene finder.

Ribonucleotide incorporation in yeast genomic DNA shows preference for cytosine and guanosine preceded by deoxyadenosine.

Despite the abundance of ribonucleoside monophosphates (rNMPs) in DNA, sites of rNMP incorporation remain poorly characterized. Here, by using ribose-seq and Ribose-Map techniques, we built and analyzed high-throughput sequencing libraries of rNMPs derived from mitochondrial and nuclear DNA of budding and fission yeast. We reveal both common and unique features of rNMP sites among yeast species and strains, and between wild type and different ribonuclease H-mutant genotypes. We demonstrate that the rNMPs are not randomly incorporated in DNA. We highlight signatures and patterns of rNMPs, including sites within trinucleotide-repeat tracts. Our results uncover that the deoxyribonucleotide immediately upstream of the rNMPs has a strong influence on rNMP distribution, suggesting a mechanism of rNMP accommodation by DNA polymerases as a driving force of rNMP incorporation. Consistently, we find deoxyadenosine upstream from the most abundant genomic rCMPs and rGMPs. This study establishes a framework to better understand mechanisms of rNMP incorporation in DNA.

Investigation on the inclusion interaction of 4-sulfonatocalix[n]arenes with 1-(4-nitrophenyl)piperazine.

The inclusion behaviors of 4-Sulfonatocalix[n]arenes (SCXn) (n=4, 6, 8) with 1-(4-nitrophenyl)piperazine (NPP) were investigated by UV spectroscopy and fluorescence spectroscopy at different pH values (pH=3.05, 6.50, 8.40). The UV absorption and fluorescence intensity of NPP remarkably increased in presence of SCXn revealing formation of the inclusion complexes between NPP and SCXn. Moreover, the formation constants (K) of inclusion complexes were also determined by the non-linear fitting method, and the obtained data showed that the formation constants decreasedgradually with the increasing of the pH value. When the pH value was 3.05, the formation constant of NPP with SCX8 reached a maximum of 1.7×10(7) L mol(-1). The stoichiometric ratio was verified to be 1:1 by the continuous variation method. Meanwhile FT-IR and DSC analysis also indicated that NPP could form the inclusion complex with SCXn. In order to explore the inclusion mechanism of NPP with SCXn, 1H NMR and molecular modeling studies were carried out and experimental results showed that the part of benzene ring of NPP penetrated into the hydrophobic cavity of SCXn.