Effects of silencing hsa_circ_0015326 on proliferation, migration, invasion, and apoptosis of epithelial ovarian cancer cells.

Although the treatment of ovarian cancer has made great progress, there are still many patients who are not timely detected and given targeted therapy due to unknown pathogenesis. Recent studies have found that hsa_circ_0015326 is upregulated in ovarian cancer and is involved in the proliferation, invasion, and migration of ovarian cancer cells. However, whether hsa_circ_0015326 can be used as a new target of ovarian cancer needs further investigation. Therefore, the effect of hsa_circ_0015326 on epithelial ovarian cancer was investigated in this study. At first, si-hsa_circ_0015326 lentivirus was transfected into epithelial ovarian cancer cells. Then real-time fluorescence quantitative PCR (qRT-PCR) was used to detect hsa_circ_0015326 level. The proliferation of ovarian cancer cells was detected by CCK-8 assay. The horizontal and vertical migration abilities of the cells were detected by wound-healing assay and Transwell assay, respectively. Transwell assay was also used to determine the invasion rate. As for the apoptosis rate, it was assessed by flow cytometry. As a result, the expression level of hsa_circ_0015326 in A2780 and SKOV3 was found to be higher than that in IOSE-80. However, after transfecting si-hsa_circ_0015326 and si-NC into the cells, the proliferation, migration, and invasion abilities of A2780 and SKOV3 cells in the si-hsa_circ_0015326 group were significantly reduced in comparison to those in the si-NC and mock groups, while their apoptosis rates were elevated. Collectively, silencing hsa_circ_0015326 bears the capability of inhibiting the proliferation, migration, and invasion of ovarian cancer cells while increasing apoptosis rate. It can be concluded that hsa_circ_0015326 promotes the malignant biological activities of epithelial ovarian cancer cells.

SMaRT-PCR: sampling using masks and RT-PCR, a non-invasive diagnostic tool for paediatric pulmonary TB.

Key challenges in paediatric TB diagnosis are invasive sampling and poor sensitivity of standard methods. This study demonstrates the diagnostic potential of non-invasive sampling of bioaerosols from children using SMaRT-PCR, comprising mask sampling combined with reverse transcriptase-polymerase chain reaction (RT-PCR) for TB.Exhaled bioaerosols were captured on modified N-95 masks in a 10-min talk-cough-breathe process from 51 children (30 with TB confirmed using standard sampling methods and 21 without TB) aged 2-15 years. All mask samples were tested using in-house RT-PCR for 16s and rpoB RNA transcripts. Additional mask samples from children with TB were tested using Xpert® MTB/RIF (n = 3) and Xpert® MTB/RIF Ultra (n = 27).SMaRT-PCR sensitivity for detecting TB among treatment-naïve children was 96% if 16s or rpoB was present, and 75% if both genes were present, comparable to standard methods (71%) in the same cohort. Specificity was better for both genes, at 95%, than 85% for a single gene detection. Mask sampling with Xpert MTB/RIF or Ultra had a sensitivity of only 13%.This is the first study to provide evidence for testing bioaerosols as a promising alternative for detecting paediatric TB. Sampling is non-invasive and simple, with the potential for point-of-care applications. This pilot study also suggests that RNA transcript-based detection may improve TB diagnostic sensitivity in children; however, further investigation is required to establish its adaptability in clinical settings..

Mex-3 RNA binding family member A (MEX3A)/circMPP6 complex promotes colorectal cancer progression by inhibiting autophagy.

RNA-binding proteins (RBPs)-RNA networks have contributed to cancer development. Circular RNAs (circRNAs) are considered as protein recruiters; nevertheless, the patterns of circRNA-protein interactions in colorectal cancer (CRC) are still lacking. Processing bodies (PBs) formed through liquid-liquid phase separation (LLPS) are membrane-less organelles (MLOs) consisting of RBPs and RNA. Previous evidence suggests a connection between PBs dynamics and cancer progression. Despite the increasingly acknowledged crucial role of RBPs and RNA in the accumulation and maintenance of MLOs, there remains a lack of specific research on the interactions between PBs-related RBPs and circRNAs in CRC. Herein, we identify that MEX-3 RNA binding family member A (MEX3A), frequently upregulated in CRC tissues, predicts poorer patient survival. Elevated MEX3A accelerates malignance and inhibits autophagy of CRC cells. Importantly, MEX3A undergoes intrinsically disordered regions (IDRs)-dependent LLPS in the cytoplasm. Specifically, circMPP6 acts as a scaffold to facilitate the interaction between MEX3A and PBs proteins. The MEX3A/circMPP6 complex modulates PBs dynamic and promotes UPF-mediated phosphodiesterase 5A (PDE5A) mRNA degradation, consequently leading to the aggressive properties of CRC cells. Clinically, CRC patients exhibiting high MEX3A expression and low PDE5A expression have the poorest overall survival. Our findings reveal a collaboration between MEX3A and circMPP6 in the regulation of mRNA decay through triggering the PBs aggregation, which provides prognostic markers and/or therapeutic targets for CRC.

Differential gene expression in decidualized human endometrial stromal cells induced by different stimuli.

Decidualization can be induced by culturing human endometrial stromal cells (ESCs) with several decidualization stimuli, such as cAMP, medroxyprogesterone acetate (MPA) or Estradiol (E2). However, it has been unclear how decidualized cells induced by different stimuli are different. We compared transcriptomes and cellular functions of decidualized ESCs induced by different stimuli (MPA, E2 + MPA, cAMP, and cAMP + MPA). We also investigated which decidualization stimulus induces a closer in vivo decidualization. Differentially expressed genes (DEGs) and altered cellular functions by each decidualization stimuli were identified by RNA-sequence and gene-ontology analysis. DEGs was about two times higher for stimuli that use cAMP (cAMP and cAMP + MPA) than for stimuli that did not use cAMP (MPA and E2 + MPA). cAMP-using stimuli altered the cellular functions including angiogenesis, inflammation, immune system, and embryo implantation whereas MPA-using stimuli (MPA, E2 + MPA, and cAMP + MPA) altered the cellular functions associated with insulin signaling. A public single-cell RNA-sequence data of the human endometrium was utilized to analyze in vivo decidualization. The altered cellular functions by in vivo decidualization were close to those observed by cAMP + MPA-induced decidualization. In conclusion, decidualized cells induced by different stimuli have different transcriptome and cellular functions. cAMP + MPA may induce a decidualization most closely to in vivo decidualization.

Predicting condensate formation of protein and RNA under various environmental conditions.

BACKGROUND: Liquid-liquid phase separation (LLPS) by biomolecules plays a central role in various biological phenomena and has garnered significant attention. The behavior of LLPS is strongly influenced by the characteristics of RNAs and environmental factors such as pH and temperature, as well as the properties of proteins. Recently, several databases recording LLPS-related biomolecules have been established, and prediction models of LLPS-related phenomena have been explored using these databases. However, a prediction model that concurrently considers proteins, RNAs, and experimental conditions has not been developed due to the limited information available from individual experiments in public databases. RESULTS: To address this challenge, we have constructed a new dataset, RNAPSEC, which serves each experiment as a data point. This dataset was accomplished by manually collecting data from public literature. Utilizing RNAPSEC, we developed two prediction models that consider a protein, RNA, and experimental conditions. The first model can predict the LLPS behavior of a protein and RNA under given experimental conditions. The second model can predict the required conditions for a given protein and RNA to undergo LLPS. CONCLUSIONS: RNAPSEC and these prediction models are expected to accelerate our understanding of the roles of proteins, RNAs, and environmental factors in LLPS.

Utility of oligonucleotide in upregulating circular RNA production in a cellular model.

Circular RNAs (circRNAs), are a covalently closed, single-stranded RNA without 5'- and 3'-termini, commonly stem from the exons of precursor mRNAs (pre-mRNAs). They have recently garnered interest, with studies uncovering their pivotal roles in regulating various aspects of cell functions and disease progressions. A notable feature of circRNA lies in the mechanism of its biogenesis involving a specialized form of splicing: back-splicing. A splicing process that relies on interactions between introns flanking the circularizing exon to bring the up and downstream splice sites in proximity through the formation of a prerequisite hairpin structure, allowing the spliceosomes to join the two splice sites together to produce a circular RNA molecule. Based on this mechanism, we explored the feasibility of facilitating the formation of such a prerequisite hairpin structure by utilizing a newly designed oligonucleotide, CircuLarIzation Promoting OligoNucleotide (CLIP-ON), to promote the production of circRNA in cells. CLIP-ON was designed to hybridize with and physically bridge two distal sequences in the flanking introns of the circularizing exons. The feasibility of CLIP-ON was confirmed in HeLa cells using a model pre-mRNA, demonstrating the applicability of CLIP-ON as a trans-acting modulator to upregulate the production of circRNAs in a cellular environment.

Genotyping Plasmodium falciparum gametocytes using amplicon deep sequencing.

BACKGROUND: Understanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing. METHODS: A data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach. RESULTS: A 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92-0.99] and 0.92 [95% CI: 0.83-0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28-0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35-3.68]. CONCLUSIONS: A region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.

Role of plasma EBV-DNA load and EBER status on newly diagnosed peripheral T-cell lymphoma.

PURPOSE: To explore the prognostic and therapeutic role of Epstein-Barr Virus (EBV) on peripheral T-cell lymphoma (PTCL). METHODS: Totally 262 newly diagnosed PTCL patients who were hospitalized from January 2014 to December 2022 were retrospectively enrolled. Molecular analysis included 31 eligible patients. EBV-encoded RNA (EBER) presence in tumor tissue and EBV DNA levels in patients at baseline (DNA1) and after 4 cycles of chemotherapy (DNA4) were assessed. RESULTS: Our findings revealed that the EBER-positive cohort exhibited significant differences compared to counterparts in overall survival (OS, P = 0.047) and progression-free survival (PFS, P = 0.009). Both DNA1 and DNA4 were significantly associated with inferior OS. Multivariate analysis demonstrated that DNA4 independently affected PTCL prognosis for OS (hazard ratio = 5.1617; 95% confidence interval 1.1017-24.1831; P = 0.037). Treatment with the cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) plus azacytidine regimen showed a better OS compared to CHOP or CHOP plus etoposide for patients with partially positive EBER and EBER positive statuses (P = 0.192), although the improvement was not statistically significant. This study delineated the genetic paradigm of PTCL, comparing genetic differences by EBV status and found that EBER partially positive plus positive patients were more likely to have DNMT3A (P = 0.002), RHOAG17V (P = 0.023), and TET2 mutations (P = 0.032). CONCLUSION: EBER, DNA1, and DNA4 emerged as sensitive markers for prognosis. CHOP plus azacytidine might present a preferable option for PTCL patients with DNA methylation due to EBV infection.

Salt-Dependent Self-Association of Trinucleotide Repeat RNA Sequences.

Repeat RNA sequences self-associate to form condensates. Simulations of a coarse-grained single-interaction site model for (CAG)n (n = 30 and 31) show that the salt-dependent free energy gap, ΔGS, between the ground (perfect hairpin) and the excited state (slipped hairpin (SH) with one CAG overhang) of the monomer for (n even) is the primary factor that determines the rates and yield of self-assembly. For odd n, the free energy (GS) of the ground state, which is an SH, is used to predict the self-association kinetics. As the monovalent salt concentration, CS, increases, ΔGS and GS increase, which decreases the rates of dimer formation. In contrast, ΔGS for shuffled sequences, with the same length and sequence composition as (CAG)31, is larger, which suppresses their propensities to aggregate. Although demonstrated explicitly for (CAG) polymers, the finding of inverse correlation between the free energy gap and RNA aggregation is general.

Identification of 6-methyladenosine sites using novel feature encoding methods and ensemble models.

N6-methyladenosine (6 mA) is the most common internal modification in eukaryotic mRNA. Mass spectrometry and site-directed mutagenesis, two of the most common conventional approaches, have been shown to be laborious and challenging. In recent years, there has been a rising interest in analyzing RNA sequences to systematically investigate mutated locations. Using novel methods for feature development, the current work aimed to identify 6 mA locations in RNA sequences. Following the generation of these novel features, they were used to train an ensemble of models using methods such as stacking, boosting, and bagging. The trained ensemble models were assessed using an independent test set and k-fold cross validation. When compared to baseline predictors, the suggested model performed better and showed improved ratings across the board for key measures of accuracy.

Liposomes, transfersomes and niosomes: production methods and their applications in the vaccinal field.

One of the most effective strategies to fight viruses and handle health diseases is vaccination. Recent studies and current applications are moving on antigen, DNA and RNA-based vaccines to overcome the limitations related to the conventional vaccination strategies, such as low safety, necessity of multiple injection, and side effects. However, due to the instability of pristine antigen, RNA and DNA molecules, the use of nanocarriers is required. Among the different nanocarriers proposed for vaccinal applications, three types of nanovesicles were selected and analysed in this review: liposomes, transfersomes and niosomes. PubMed, Scopus and Google Scholar databases were used for searching recent papers on the most frequently used conventional and innovative methods of production of these nanovesicles. Weaknesses and limitations of conventional methods (i.e., multiple post-processing, solvent residue, batch-mode processes) can be overcome using innovative methods, in particular, the ones assisted by supercritical carbon dioxide. SuperSomes process emerged as a promising production technique of solvent-free nanovesicles, since it can be easily scaled-up, works in continuous-mode, and does not require further post-processing steps to obtain the desired products. As a result of the literature analysis, supercritical carbon dioxide assisted methods attracted a lot of interest for nanovesicles production in the vaccinal field. However, despite their numerous advantages, supercritical processes require further studies for the production of liposomes, transfersomes and niosomes with the aim of reaching well-defined technologies suitable for industrial applications and mass production of vaccines.

Physiological functions of RIG-I-like receptors.

RIG-I-like receptors (RLRs) are crucial for pathogen detection and triggering immune responses and have immense physiological importance. In this review, we first summarize the interferon system and innate immunity, which constitute primary and secondary responses. Next, the molecular structure of RLRs and the mechanism of sensing non-self RNA are described. Usually, self RNA is refractory to the RLR; however, there are underlying host mechanisms that prevent immune reactions. Studies have revealed that the regulatory mechanisms of RLRs involve covalent molecular modifications, association with regulatory factors, and subcellular localization. Viruses have evolved to acquire antagonistic RLR functions to escape the host immune reactions. Finally, the pathologies caused by the malfunction of RLR signaling are described.

Twelve-month effectiveness and safety of bictegravir/emtricitabine/tenofovir alafenamide in people with HIV from the Canadian cohort of the observational BICSTaR study.

The BICSTaR (BICtegravir Single Tablet Regimen) study is investigating the effectiveness and safety of bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) in people with human immunodeficiency virus (HIV) treated in routine clinical practice. BICSTaR is an ongoing, prospective, observational cohort study across 14 countries. Treatment-naïve (TN) and treatment-experienced (TE) people with HIV (≥18 years of age) are being followed for 24 months. We present an analysis of the primary endpoint (HIV-1 RNA < 50 copies/mL; missing-equals-excluded [M = E]) at month 12 in the BICSTaR Canada cohort, including secondary (CD4 count, CD4/CD8 ratio, safety/tolerability) and exploratory (persistence, treatment satisfaction) endpoints. In total, 201 participants were enrolled in the BICSTaR Canada cohort. The analysis population included 170 participants (TN, n = 10; TE, n = 160), with data collected between November 2018 and September 2020. Of the participants, 88% were male, 72% were White, and 90% had ≥ 1 comorbid condition(s). Median (quartile [Q]1-Q3) age was 50 (39-58) years and baseline CD4 count was 391.5 (109.0-581.0) cells/µL in TN participants and 586.0 (400.0-747.0) cells/µL in TE participants. After 12 months of B/F/TAF treatment, HIV-1 RNA was < 50 copies/mL in 100% (9/9) of TN-active participants and 97% (140/145) of TE-active participants (M = E analysis). Median (Q1-Q3) CD4 cell count increased by +195 (125-307) cells/µL in TN participants and by + 30 (-50 to 123) cells/µL in TE participants. Persistence on B/F/TAF was high through month 12 with 10% (1/10) of TN and 7 % (11/160) of TE participants discontinuing B/F/TAF within 12 months of initiation of treatment. No resistance to B/F/TAF emerged. Study drug-related adverse events occurred in 7% (12/169) of participants, leading to B/F/TAF discontinuation in 4 of 169 participants. Improvements in treatment satisfaction were observed in TE participants. B/F/TAF demonstrated high levels of effectiveness, persistence, and treatment satisfaction, and was well tolerated through month 12 in people with HIV treated in routine clinical practice in Canada.

No evidence for ac4C within human mRNA upon data reassessment.

Cytidine acetylation (ac4C) of RNA is a post-transcriptional modification catalyzed by Nat10. Recently, an approach termed RedaC:T was employed to map ac4C in human mRNA, relying on detection of C>T mutations in WT but not in Nat10-KO cells. RedaC:T suggested widespread ac4C presence. Here, we reanalyze RedaC:T data. We find that mismatch signatures are not reproducible, as C>T mismatches are nearly exclusively present in only one of two biological replicates. Furthermore, all mismatch types-not only C>T-are highly enriched in WT samples, inconsistent with an acetylation signature. We demonstrate that the originally observed enrichment in mutations in one of the WT samples is due to its low complexity, resulting in the technical amplification of all classes of mismatch counts. Removal of duplicate reads abolishes the skewed mismatch patterns. These analyses account for the irreproducible mismatch patterns across samples while failing to find evidence for acetylation of RedaC:T sites.

Clinical mutations in the TERT and TERC genes coding for telomerase components induced oxidative stress, DNA damage at telomeres and cell apoptosis besides decreased telomerase activity.

Telomeres are nucleoprotein structures at the end of chromosomes that maintain their integrity. Mutations in genes coding for proteins involved in telomere protection and elongation produce diseases such as dyskeratosis congenita or idiopathic pulmonary fibrosis known as telomeropathies. These diseases are characterized by premature telomere shortening, increased DNA damage and oxidative stress. Genetic diagnosis of telomeropathy patients has identified mutations in the genes TERT and TERC coding for telomerase components but the functional consequences of many of these mutations still have to be experimentally demonstrated. The activity of twelve TERT and five TERC mutants, five of them identified in Spanish patients, has been analyzed. TERT and TERC mutants were expressed in VA-13 human cells that express low telomerase levels and the activity induced was analyzed. The production of reactive oxygen species, DNA oxidation and TRF2 association at telomeres, DNA damage response and cell apoptosis were determined. Most mutations presented decreased telomerase activity, as compared to wild-type TERT and TERC. In addition, the expression of several TERT and TERC mutants induced oxidative stress, DNA oxidation, DNA damage, decreased recruitment of the shelterin component TRF2 to telomeres and increased apoptosis. These observations might indicate that the increase in DNA damage and oxidative stress observed in cells from telomeropathy patients is dependent on their TERT or TERC mutations. Therefore, analysis of the effect of TERT and TERC mutations of unknown function on DNA damage and oxidative stress could be of great utility to determine the possible pathogenicity of these variants.

Untangling the Molecular Interactions Underlying Intracellular Phase Separation Using Combined Global Sensitivity Analyses.

Liquid-liquid phase separation is an intracellular mechanism by which molecules, usually proteins and RNAs, interact and then rapidly demix from the surrounding matrix to form membrane-less compartments necessary for cellular function. Occurring in both the cytoplasm and the nucleus, properties of the resulting droplets depend on a variety of characteristics specific to the molecules involved, such as valency, density, and diffusion within the crowded environment. Capturing these complexities in a biologically relevant model is difficult. To understand the nuanced dynamics between proteins and RNAs as they interact and form droplets, as well as the impact of these interactions on the resulting droplet properties, we turn to sensitivity analysis. In this work, we examine a previously published mathematical model of two RNA species competing for the same protein-binding partner. We use the combined analyses of Morris Method and Sobol' sensitivity analysis to understand the impact of nine molecular parameters, subjected to three different initial conditions, on two observable LLPS outputs: the time of phase separation and the composition of the droplet field. Morris Method is a screening method capable of highlighting the most important parameters impacting a given output, while the variance-based Sobol' analysis can quantify both the importance of a given parameter, as well as the other model parameters it interacts with, to produce the observed phenomena. Combining these two techniques allows Morris Method to identify the most important dynamics and circumvent the large computational expense associated with Sobol', which then provides more nuanced information about parameter relationships. Together, the results of these combined methodologies highlight the complicated protein-RNA relationships underlying both the time of phase separation and the composition of the droplet field. Sobol' sensitivity analysis reveals that observed spatial and temporal dynamics are due, at least in part, to high-level interactions between multiple (3+) parameters. Ultimately, this work discourages using a single measurement to extrapolate the value of any single rate or parameter value, while simultaneously establishing a framework in which to analyze and assess the impact of these small-scale molecular interactions on large-scale droplet properties.

N6-methyladenosine-modified circSLCO1B3 promotes intrahepatic cholangiocarcinoma progression via regulating HOXC8 and PD-L1.

BACKGROUND: Refractoriness to surgical resection and chemotherapy makes intrahepatic cholangiocarcinoma (ICC) a fatal cancer of the digestive system with high mortality and poor prognosis. Important function invests circRNAs with tremendous potential in biomarkers and therapeutic targets. Nevertheless, it is still unknown how circRNAs contribute to the evolution of ICC. METHODS: CircRNAs in paired ICC and adjacent tissues were screened by circRNAs sequencing. To explore the impact of circRNAs on ICC development, experiments involving gain and loss of function were conducted. Various experimental techniques, including quantitative real-time PCR (qPCR), western blotting, RNA immunoprecipitation (RIP), luciferase reporter assays, RNA pull-down, chromatin immunoprecipitation (ChIP), ubiquitination assays and so on were employed to identify the molecular regulatory role of circRNAs. RESULTS: Herein, we reported a new circRNA, which originates from exon 9 to exon 15 of the SLCO1B3 gene (named circSLCO1B3), orchestrated ICC progression by promoting tumor proliferation, metastasis and immune evasion. We found that the circSLCO1B3 gene was highly overexpressed in ICC tissues and related to lymphatic metastasis, tumor sizes, and tumor differentiation. Mechanically, circSLCO1B3 not only promoted ICC proliferation and metastasis via miR-502-5p/HOXC8/SMAD3 axis, but also eradicated anti-tumor immunity via suppressing ubiquitin-proteasome-dependent degradation of PD-L1 by E3 ubiquitin ligase SPOP. We further found that methyltransferase like 3 (METTL3) mediated the m6A methylation of circSLCO1B3 and stabilizes its expression. Our findings indicate that circSLCO1B3 is a potential prognostic marker and therapeutic target in ICC patients. CONCLUSIONS: Taken together, m6A-modified circSLCO1B3 was correlated with poor prognosis in ICC and promoted ICC progression not only by enhancing proliferation and metastasis via potentiating HOXC8 expression, but also by inducing immune evasion via antagonizing PD-L1 degradation. These results suggest that circSLCO1B3 is a potential prognostic marker and therapeutic target for ICC.

New comparative genomic evidence supporting the proteomic diversification role of A-to-I RNA editing in insects.

Adenosine-to-inosine (A-to-I) RNA editing, resembling A-to-G mutation, confers adaptiveness by increasing proteomic diversity in a temporal-spatial manner. This evolutionary theory named "proteomic diversifying hypothesis" has only partially been tested in very few organisms like Drosophila melanogaster, mainly by observing the positive selection on nonsynonymous editing events. To find additional genome-wide evidences supporting this interesting assumption, we retrieved the genomes of four Drosophila species and collected 20 deep-sequenced transcriptomes of different developmental stages and neuron populations of D. melanogaster. We systematically profiled the RNA editomes in these samples and performed meticulous comparative genomic analyses. Further evidences were found to support the diversifying hypothesis. (1) None of the nonsynonymous editing sites in D. melanogaster had ancestral G-alleles, while the silent editing sites had an unignorable fraction of ancestral G-alleles; (2) Only very few nonsynonymous editing sites in D. melanogaster had corresponding G-alleles derived in the genomes of sibling species, and the fraction of such situation was significantly lower than that of silent editing sites; (3) The few nonsynonymous editing with corresponding G-alleles had significantly more variable editing levels (across samples) than other nonsynonymous editing sites in D. melanogaster. The proteomic diversifying nature of RNA editing in Drosophila excludes the restorative role which favors an ancestral G-allele. The few fixed G-alleles in sibling species might facilitate the adaptation to particular environment and the corresponding nonsynonymous editing in D. melanogaster would introduce stronger advantage of flexible proteomic diversification. With multi-Omics data, our study consolidates the nature of evolutionary significance of A-to-I RNA editing sites in model insects.

RT-qPCR based quantitative analysis of ARO and ADH genes in Saccharomyces cerevisiae and Metschnikowia pulcherrima strains growth white grape juice.

BACKGROUND: Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice. METHODS AND RESULTS: We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method's capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains. CONCLUSIONS: The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.

Curcumin reduces myocardial ischemia-reperfusion injury, by increasing endogenous H2S levels and further modulating m6A.

BACKGROUND: Our previous research shows that Curcumin (CUR) attenuates myocardial ischemia-reperfusion injury (MIRI) by reducing intracellular total RNA m6A levels. However, the mechanism remains unknown. METHODS: For ischemia-reperfusion (IR), H9c2 cells were cultured for 6 h in serum-free low-glycemic (1 g/L) medium and a gas environment without oxygen, and then cultured for 6 h in high-glycemic (4.5 g/L) medium supplemented with 10% FBS and a 21% oxygen environment. The effects of different concentrations of CUR (5, 10, and 20 µM) treatments on signaling molecules in conventionally cultured and IR-treated H9c2 cells were examined. RESULTS: CUR treatment significantly up-regulated the H2S levels, and the mRNA and protein expression of cystathionine γ-lyase (CSE), and down-regulated the mRNAs and proteins levels of thiosulfate sulfurtransferase (TST) and ethylmalonic encephalopathy 1 (ETHE1) in H9c2 cells conventionally cultured and subjected to IR. Exogenous H2S supply (NaHS and GYY4137) significantly reduced intracellular total RNA m6A levels, and the expression of RNA m6A "writers" METTL3 and METTL14, and increased the expression of RNA m6A "eraser" FTO in H9c2 cells conventionally cultured and subjected to IR. CSE knockdown counteracted the inhibitory effect of CUR treatment on ROS production, promotion on cell viability, and inhibition on apoptosis of H9c2 cells subjected to IR. CONCLUSION: CUR attenuates MIRI by regulating the expression of H2S level-regulating enzymes and increasing the endogenous H2S levels. Increased H2S levels could regulate the m6A-related proteins expression and intracellular total RNA m6A levels.