Local features determine Ty3 targeting frequency at RNA polymerase III transcription start sites.

Retroelement integration into host genomes affects chromosome structure and function. A goal of a considerable number of investigations is to elucidate features influencing insertion site selection. The Saccharomyces cerevisiae Ty3 retrotransposon inserts proximal to the transcription start sites (TSS) of genes transcribed by RNA polymerase III (RNAP3). In this study, differential patterns of insertion were profiled genome-wide using a random barcode-tagged Ty3. Saturation transposition showed that tRNA genes (tDNAs) are targeted at widely different frequencies even within isoacceptor families. Ectopic expression of Ty3 integrase (IN) showed that it localized to targets independent of other Ty3 proteins and cDNA. IN, RNAP3, and transcription factor Brf1 were enriched at tDNA targets with high frequencies of transposition. To examine potential effects of cis-acting DNA features on transposition, targeting was tested on high-copy plasmids with restricted amounts of 5' flanking sequence plus tDNA. Relative activity of targets was reconstituted in these constructions. Weighting of genomic insertions according to frequency identified an A/T-rich sequence followed by C as the dominant site of strand transfer. This site lies immediately adjacent to the adenines previously implicated in the RNAP3 TSS motif (CAA). In silico DNA structural analysis upstream of this motif showed that targets with elevated DNA curvature coincide with reduced integration. We propose that integration mediated by the Ty3 intasome complex (IN and cDNA) is subject to inputs from a combination of host factor occupancy and insertion site architecture, and that this results in the wide range of Ty3 targeting frequencies.

Cytotoxic Screening and Transcriptomics Reveal Insights into the Molecular Mechanisms of Trihexyl Phosphate-Triggered Hepatotoxicity.

Mounting evidence shows that organophosphate flame retardants (OPFRs), especially aryl- and halogenated-OPFRs, exert various adverse health effects on living organisms. This study evaluated the hepatotoxic effect of trihexyl phosphate (THP) as a long-chain alkyl-OPFR on human hepatocyte cells (LO2) and mouse hepatocyte cells (AML12) by performing screening of cytotoxicity in vitro. In combination with transcriptomic analysis, toxicological mechanisms in vitro were further investigated. Results showed that THP triggered hepatotoxicity in vitro by altering four signaling pathways: endoplasmic reticulum (ER) stress, apoptosis, cell cycle, and the glycolysis signaling pathway. Exposure of LO2 and AML12 liver cells to THP (25 µg/mL) significantly induced ER stress-mediated apoptosis and cell cycle arrest. Meanwhile, downregulation of glycolysis caused the blockage of energy metabolism. Furthermore, the high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) revealed that much of THP was absorbed into the cells and displayed stability in the two liver cell lines. In vivo assays using a mouse model demonstrated that exposure to THP at 400 mg/kg induced the ballooning degeneration of hepatocytes in liver tissue, whereas exposure to THP at 800 mg/kg caused acute liver injury with high alanine aminotransferase levels. This study provides novel insights into the impact of THP on hepatotoxicity in vitro and in vivo and uncovers the underlying toxicological mechanisms, which may serve as a guide for further ecological risk assessment and reasonable application of alkyl-OPFRs.

Investigation on the urinary excretion kinetics of three atractylenolides from crude and processed Atractylodis rhizoma extracts in rats by UPLC-MS/MS.

Atractylodis rhizoma is a frequently-used traditional Chinese medicine in clinical practice, which have the effect of eliminating dampness and tonifying spleen. And after being processed with wheat bran, the dryness of A. rhizoma is reduced, and the function of tonifying spleen is enhanced. Atractylenolides are the major bioactive components of A. rhizoma, including atractylenolide I (AI), atractylenolide Ⅱ (AⅡ) and atractylenolide Ⅲ (AⅢ). The present study aimed to develope a new UPLC-MS/MS method for simultaneous quantification of three atractylenolides in rat urine, and applied to the excretory kinetics in Sprague-Dawley rats after oral administration of crude and processed A. rhizoma extracts. Analytes and internal standard were detected without interference in the multiple reaction monitoring (MRM) mode with positive electrospray ionization. The excretory kinetics parameters were calculated by a urine drug analysis model of drug and statistics (DAS) 3.2.8 software. The t1/2 and Ke of three atractylenolides had no significant difference between crude and processed A. rhizoma, but the recovery accumulative excretion of them in processed A. rhizoma were apparently higher than the crude ones (p<0.05, p<0.01). The results showed that only a small amount of atractylenolides excreted in urine and processing A. rhizoma with wheat bran by stir frying could promote the urinary excretion of them.

NK007 helps in mitigating paclitaxel resistance through p38MAPK activation and HK2 degradation in ovarian cancer.

Ovarian cancer resistance to available medicines is a huge challenge in dire need of a solution, which makes its recurrence and mortality rate further exacerbated. A promising approach to overcome chemoresistance is drug screening from natural products. Here, we report that NK007, a (±)-tylophorine malate isolated from the Asclepiadaceae family, selectively inhibited the proliferation of A2780 and A2780 (Taxol) cells and migration of paclitaxel-sensitive and -resistant ovarian cancer cells. Interestingly, the decline of cell viability, including cell multiplication, clonality, and migration capacity was independent on cell apoptosis. At the molecular level, NK007 considerably induced G1/S arrest and upregulated the expression of phospho-p38 mitogen-activated protein kinase (p-p38MAPK). In addition, hexokinase 2 (HK2) protein degradation was considerably elevated in the presence of NK007, which resulted in the reduction of oxygen consumption rate and extracellular acidification rate. Altogether, our results indicate that NK007, an analog of tylophorine, can overcome paclitaxel (PTX) resistance through p38MAPK activation and HK2 degradation. As an effective, alternative antiresistance agent, NK007 exhibits a promising potential to treat PTX-resistant ovarian cancer.

UPLC-MS/MS of Atractylenolide I, Atractylenolide II, Atractylenolide III, and Atractyloside A in Rat Plasma after Oral Administration of Raw and Wheat Bran-Processed Atractylodis Rhizoma.

Atractylodis Rhizoma is the dried rhizome of Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz and is often processed by stir-frying with wheat bran to reduce its dryness and increase its spleen tonifying activity. However, the mechanism by which the processing has this effect remains unknown. To explain the mechanism based on the pharmacokinetics of the active compounds, a rapid, sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was developed to analyze atractylenolides I, II, and III, and atractyloside A simultaneously in rat plasma after oral administration of raw and processed Atractylodis Rhizoma. Acetaminophen was used as the internal standard and the plasma samples were pretreated with methanol. Positive ionization mode coupled with multiple reaction monitoring mode was used to analyze the four compounds. The method validation revealed that all the calibration curves displayed good linear regression over the concentration ranges of 3.2⁻350, 4⁻500, 4⁻500, and 3.44⁻430 ng/mL for atractylenolides I, II, and III, and atractyloside A, respectively. The relative standard deviations of the intra- and inter-day precisions of the four compounds were less than 6% with accuracies (relative error) below 2.38%, and the extraction recoveries were more than 71.90 ± 4.97%. The main pharmacokinetic parameters of the four compounds were estimated with Drug and Statistics 3.0 and the integral pharmacokinetics were determined based on an area under the curve weighting method. The results showed that the integral maximum plasma concentration and area under the curve increased after oral administration of processed Atractylodis Rhizoma.

Identification of an overprinting gene in Merkel cell polyomavirus provides evolutionary insight into the birth of viral genes.

Many viruses use overprinting (alternate reading frame utilization) as a means to increase protein diversity in genomes severely constrained by size. However, the evolutionary steps that facilitate the de novo generation of a novel protein within an ancestral ORF have remained poorly characterized. Here, we describe the identification of an overprinting gene, expressed from an Alternate frame of the Large T Open reading frame (ALTO) in the early region of Merkel cell polyomavirus (MCPyV), the causative agent of most Merkel cell carcinomas. ALTO is expressed during, but not required for, replication of the MCPyV genome. Phylogenetic analysis reveals that ALTO is evolutionarily related to the middle T antigen of murine polyomavirus despite almost no sequence similarity. ALTO/MT arose de novo by overprinting of the second exon of T antigen in the common ancestor of a large clade of mammalian polyomaviruses. Taking advantage of the low evolutionary divergence and diverse sampling of polyomaviruses, we propose evolutionary transitions that likely gave birth to this protein. We suggest that two highly constrained regions of the large T antigen ORF provided a start codon and C-terminal hydrophobic motif necessary for cellular localization of ALTO. These two key features, together with stochastic erasure of intervening stop codons, resulted in a unique protein-coding capacity that has been preserved ever since its birth. Our study not only reveals a previously undefined protein encoded by several polyomaviruses including MCPyV, but also provides insight into de novo protein evolution.

Retrotransposon profiling of RNA polymerase III initiation sites.

Although retroviruses are relatively promiscuous in choice of integration sites, retrotransposons can display marked integration specificity. In yeast and slime mold, some retrotransposons are associated with tRNA genes (tDNAs). In the Saccharomyces cerevisiae genome, the long terminal repeat retrotransposon Ty3 is found at RNA polymerase III (Pol III) transcription start sites of tDNAs. Ty1, 2, and 4 elements also cluster in the upstream regions of these genes. To determine the extent to which other Pol III-transcribed genes serve as genomic targets for Ty3, a set of 10,000 Ty3 genomic retrotranspositions were mapped using high-throughput DNA sequencing. Integrations occurred at all known tDNAs, two tDNA relics (iYGR033c and ZOD1), and six non-tDNA, Pol III-transcribed types of genes (RDN5, SNR6, SNR52, RPR1, RNA170, and SCR1). Previous work in vitro demonstrated that the Pol III transcription factor (TF) IIIB is important for Ty3 targeting. However, seven loci that bind the TFIIIB loader, TFIIIC, were not targeted, underscoring the unexplained absence of TFIIIB at those sites. Ty3 integrations also occurred in two open reading frames not previously associated with Pol III transcription, suggesting the existence of a small number of additional sites in the yeast genome that interact with Pol III transcription complexes.

In vitro targeting of strand transfer by the Ty3 retroelement integrase.

The Saccharomyces cerevisiae long terminal repeat retrotransposon Ty3 integrates within one or two nucleotides of the transcription initiation sites of genes transcribed by RNA polymerase III. In this study the minimal components required to re-constitute position-specific strand transfer by Ty3 integrase are defined. Ty3 integrase targeted by a synthetic fusion of RNA polymerase III transcription factor IIIB subunits, Brf1 and TBP, mediated position-specific strand transfer of duplex oligonucleotides representing the ends of the Ty3 cDNA. These results further delimit the TFIIIB domains targeted by the Ty3 element and show that IN is the Ty3 component sufficient in vitro to target integration. These results underscore the commonality of protein interactions that mediate transcription and retrotransposon targeting. Surprisingly, in the presence of MnCl(2), strand transfer was TFIIIB-independent and targeted sequences resembling the Ty3 terminal inverted repeat.

Emerging trends and research foci in autophagy of pancreatic cancer: a bibliometric and visualized study.

Objective: The purpose of this study was to analyze the trends by year, country, institution, journal, reference and keyword in publications on the autophagy of pancreatic cancer (PC) and to predict future research hotspots. Methods: The Web of Science Core Collection was used to search for publications. The contributions of various countries/regions, institutes, authors, identified research hotspots, and promising future trends were analyzed using the VOSviewer1.6.16 and CiteSpace6.6.R2 programs. We also summarized autophagy relevant clinical trials of PC. Results: A total of 1293 papers on the autophagy of PC published between 2013 and 2023 were included in the study. The average number of citations per article was 33.76. The China had the most publications, followed by USA, and a total of 50 influential articles were identified through co-citation analysis. Clustering analysis revealed clusters of keywords: metabolic reprogramming and ER stress, mTOR-mediated apoptosis, extracellular trap as the most concerned clusters. The co-occurrence cluster analysis showed pancreatic stellate cell, autophagy-dependent ferroptosis, autophagy-related pathway, metabolic rewiring, on-coding RNA as the highly concerned research topics in recently. Conclusion: The number of publications and research interest have generally increased over the past few years. The China and USA have made prominent contributions to the study of the autophagy of PC. The current research hotspots mainly focus not only on the related modulation, metabolic reprogramming, ferroptosis of tumor cells themselves, but also on tumor microenvironments such as autophagy associated pancreatic stellate cells and new treatments targeting autophagy.

Single-cell RNA-seq and chromatin accessibility profiling decipher the heterogeneity of mouse γδ T cells.

The distinct characteristics of γδ T cells determine their vital roles in the formation of local immune responses and contribute to tissue homeostasis. However, the heterogeneity of γδ T cells across tissues remains unclear. By combining transcriptional and chromatin analyses with a truly unbiased fashion, we constructed a single-cell transcriptome and chromatin accessibility landscape of mouse γδ T cells in the lymph, spleen, and thymus. We also revealed the heterogeneity of γδ T1 and γδ T17 cells across these tissues and inferred their potential regulatory mechanisms. In the thymus, we reconstructed the developmental trajectory and gained further insights into the signature genes from the mature stage, intermediate stage, and immature stage of γδ T cells on the basis of single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin sequencing data. Notably, a novel Gzma+ γδ T cell subset was identified with immature properties and only localized to the thymus. Finally, NR1D1, a circadian transcription factor (TF), was validated as a key and negative regulator of γδ T17 cell differentiation by performing a combined analysis of TF motif enrichment, regulon enrichment, and Nr1d1 knockout mice. In summary, our data represent a comprehensive mapping on the transcriptome and chromatin accessibility dynamics of mouse γδ T cells, providing a valuable resource and reference for future studies on γδ T cells.

Metabolomic study of raw and bran-fried Atractylodis Rhizoma on rats with spleen deficiency.

Atractylodis Rhizoma, a classical Chinese medicine, exhibits unambiguous therapeutic effect on spleen deficiency in China for decades. The aim of the present study was to explore the different effects on the composition and level of endogenous metabolites in rats with spleen deficiency after oral administration of raw and bran-fired Atractylodis Rhizoma, and to explain the mechanism of pharmacodynamic enhancement of the bran-fried Atractylodis Rhizoma from the perspective of metabolomics. With this purpose, spleen deficiency model was established by diet, excessive fatigue and bitter cold diarrhea. Then, Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of vasoactive intestinal peptide (VIP), Somatostatin (SS), substance P (SP) and succinodehydrogenase (SDH) in rats of each group, and to compare the contents of VIP, SS, SP and SDH among groups. UHPLC-Q-TOF-MS based metabolomics was adopted to analyze the plasma from spleen deficiency rats and control rats. Principle component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were utilized to identify differences of metabolic profiles in rats among the control group and the model group；The OPLS-DA were used to analyze the effects of raw and bran-fried Atractylodis Rhizoma on the same metabolites. The results showed that compared with the control group, the contents of VIP, SS, SP and SDH in the plasma of model group decreased, which proved the success of the model group. Compared with model group, the contents of VIP, SS, SP and SDH in the plasma of raw and bran-fried Atractylodis Rhizoma increased, and the effect of bran-fried Atractylodis Rhizoma was better than that of raw Atractylodis Rhizoma. Metabolomics results showed that seventeen different metabolites of spleen deficiency were screened out in the plasma of rats with spleen deficiency compared with the control group. Among them, Nicotinic acid, Dihydrofolic acid, Pantetheine 4'-phosphate and Photophatidylcholine (PC) were the metabolites significantly associated with spleen deficiency, and bran-fried Atractylodis Rhizoma had better intervention and regulation. Through the analysis of metabolic pathways related to these different metabolites of spleen deficiency, and primarily involved in glucosamine metabolism, one carbon pool by folate and so on. This study showed that Atractylodis Rhizoma could provide satisfactory therapeutic effects on spleen deficiency and metabolomics study can be utilized to further understand the molecular mechanisms.

Comparative analysis on bioactivity against three stored insects of Ligusticum pteridophyllum Franch. rhizomes essential oil and supercritical fluid (SFE-CO2) extract.

In order to develop more environmentally benignant insecticides, the Ligusticum pteridophyllum Franch. rhizomes essential oil and supercritical fluid (SFE-CO2) extract were obtained by two published techniques, hydrodistillation and SFE-CO2. The chemical components of this two tested samples were identified by using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detector (GC-FID). Repellent activity and contact toxicity of the obtained samples and myristicin against the adults of Tribolium castaneum (Coleoptera: Tenebrionidae), Lasioderma serricorne (Coleoptera: Anobiidae), and Liposcelis bostrychophila (Psocoptera: Liposcelididae) were compared. Nineteen components were identified in the SFE-CO2 extract. Twelve components were identified in the L. pteridophyllum rhizomes essential oil. SFE-CO2 extract exhibited higher contact toxicity against T. castaneum, L. serricorne, and L. bostrychophila (LD50 = 69.60 µg/adult, 14.58 µg/adult, and 1.69 µg/cm2, respectively) than that of L. pteridophyllum rhizomes essential oil (LD50 = 87.99 µg/adult, 89.82 µg/adult, and 7.87 µg/cm2, respectively). Besides, myristicin (LD50 = 36.46 µg/adult) showed superior contact toxicity against T. castaneum than that of the L. pteridophyllum rhizomes essential oil and SFE-CO2 extract. It possessed potentially practical significance to develop L. pteridophyllum rhizomes into plant pesticide or repellent agent for these stored insect controls. Graphical abstract .

Toxicity and repellent activity of essential oil from Mentha piperita Linn. leaves and its major monoterpenoids against three stored product insects.

The essential oil (EO) from leaves of Mentha piperita was extracted by hydrodistillation. Twenty-one chemical components, accounting for 97.5% of the total oil, were determined by GC-MS and GC-FID. The major chemical components included menthol (41.6%), L-menthone (24.7%), isomenthol (6.3%), and limonene (5.0%). The bioactivity of the obtained EO and its two major components against Tribolium castaneum, Lasioderma serricorne, and Liposcelis bostrychophila adults were evaluated by fumigation, contact, and repellent activity bioassay. The EO showed significant fumigation and contact toxicity against T. castaneum (LC50 = 18.1 mg/L air and LD50 = 2.9 µg/adult, respectively), L. serricorne (LC50 = 68.4 mg/L air and LD50 = 12.6 µg/adult, respectively), and L. bostrychophila (LC50 = 0.6 mg/L air and LD50 = 49.8 µg/adult, respectively) adults. Meanwhile, the repellent effect of the EO on T. castaneum and L. serricorne adults was comparable to that of the positive control at the highest tested concentration. Menthol and L-menthone were two major components in total oil. Among them, L-menthone exhibited significant insecticidal activity on target insects, and menthol showed notable repellent effects. The results indicated that the EO of M. piperita leaves and two tested components have potential to be developed as natural insecticides and repellents for the control of stored product insect pests. Graphical abstract.

Clinical diagnostic value of contrast-enhanced ultrasound and TI-RADS classification for benign and malignant thyroid tumors: One comparative cohort study.

To evaluate the diagnostic efficacy and clinical value of contrast-enhanced ultrasonography (CEUS) plus TI-RADS classification in benign and malignant thyroid tumors compared with either method alone.The informed consent was signed all patients. A total of 370 patients with thyroid tumors of TI-RADS category 3 and 4 were recruited, with 432 thyroid nodules. They respectively received routine ultrasonography and CEUS. The nodules were reclassified according to CEUS scoring, and a combined diagnosis was made. The pathological results were taken as the gold standard. The sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV) and area under the ROC curve were calculated for the 3 diagnostic methods. The diagnostic efficacy was compared by using Student t test, Pearson chi-square (χ) test, McNemar chi-square (χ) test or Z test. Student t test and logistic regression were employed for comparing different imaging features of benign and malignant thyroid tumors on CEUS and risk analysis.Of 432 thyroid nodules, there were 258 malignant nodules (59.72%) and 174 benign ones (40.28%). By logistic regression, 6 suspicious features on CEUS were considered significant for differentiating malignant from benign tumors: slow entry of contrast agents during enhancement stage (OR = 15.610, P = .001), slow time to peak (OR = 7.416, P = .002), non-uniform enhancement (OR = 10.076, P = .023), enhancement pattern (irregular) (OR = 36.233, P = .002), enhancement boundary (unclear) (OR = 25.300, P = .012), and no ring-like enhancement (OR = 25.297, P = .004). CEUS plus TI-RADS classification showed a higher diagnostic efficacy for differentiating between benign and malignant thyroid tumors. The Se was 85.66% (0.806-0.896), Sp 83.33% (0.768-0.884), PPV 88.40% (0.836-0.919), NPV 79.67% (0.729-0.851), and AUC 0.867 ±â€Š0.019 (0.815-0.889). The above indicators were of statistical significance as compared with TI-RADS classification or CEUS alone (P <.05).CEUS can more clearly visualize microvascular distribution of the nodules and offers a new approach to diagnose benign and malignant thyroid tumors. TI-RADS classification plus CEUS is more accurate than TI-RADS classification alone. This combined approach is worthy of clinical popularization.

Directed DNA shuffling of retrovirus and retrotransposon integrase protein domains.

Chimeric proteins are used to study protein domain functions and to recombine protein domains for novel or optimal functions. We used a library of chimeric integrase proteins to study DNA integration specificity. The library was constructed using a directed shuffling method that we adapted from fusion PCR. This method easily and accurately shuffles multiple DNA gene sequences simultaneously at specific base-pair positions, such as protein domain boundaries. It produced all 27 properly-ordered combinations of the amino-terminal, catalytic core, and carboxyl-terminal domains of the integrase gene from human immunodeficiency virus, prototype foamy virus, and Saccharomyces cerevisiae retrotransposon Ty3. Retrotransposons can display dramatic position-specific integration specificity compared to retroviruses. The yeast retrotransposon Ty3 integrase interacts with RNA polymerase III transcription factors to target integration at the transcription initiation site. In vitro assays of the native and chimeric proteins showed that human immunodeficiency virus integrase was active with heterologous substrates, whereas prototype foamy virus and Ty3 integrases were not. This observation was consistent with a lower substrate specificity for human immunodeficiency virus integrase than for other retrovirus integrases. All eight chimeras containing the Ty3 integrase carboxyl-terminal domain, a candidate targeting domain, failed to target strand transfer in the presence of the targeting protein, suggesting that multiple domains of the Ty3 integrase cooperate in this function.