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Food-borne pathogens have been recognized as a major cause of human infections worldwide. Their identification needs to be simpler, cheaper and more reliable than the traditional methods. Here, we constructed a low-cost paper platform for viable pathogenic bacteria detection with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper-based platform to perform a visual test using sandwich hybridization assays. When the RNA products migrated along the platform by capillary action, the gold nanoparticles accumulated at the designated area. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from L. monocytogenes could be detected. It could also be used to specifically detect 20 CFU/mL L. monocytogenes from actual samples. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. This method is suitable for point-of-care applications to detect food-borne pathogens, as it can overcome the false-positive results caused by amplifying nonviable L. monocytogenes. Furthermore, the results can be imaged and transformed into a two-dimensional bar code through an Android-based smart phone for further analysis or in-field food safety tracking.

To the Editor: To provide samples for neonatal mass screening, a few drops of blood are routinely collected from the heel of the newborn infant and allowed to dry on a thick filter paper called the Guthrie card.[1][1] Guthrie cards represent a valuable and comprehensive genetic repository because

Background Accurate sampling of sub-microscopic gametocytes is necessary for epidemiological studies to identify the infectious reservoir of Plasmodium falciparum. Detection of gametocyte mRNA achieves sensitive detection, but requires careful handling of samples. Filter papers can be used for collecting RNA samples, but rigorous testing of their capacity to withstand adverse storage conditions has not been fully explored. Methods Three gametocyte dilutions: 10/μL, 1.0/μL and 0.1/μL were spotted onto Whatman™ 903 Protein Saver Cards, FTA Classic Cards and 3MM filter papers that were stored under frozen, cold chain or tropical conditions for up to 13 weeks . RNA was extracted, then detected by quantitative nucleic acid sequence-based amplification (QT-NASBA) and reverse-transcriptase PCR (RT-PCR). Results Successful gametocyte detection was more frequently observed from the Whatman 903 Protein Saver Card compared to the Whatman FTA Classic Card, by both techniques (p Conclusions This study indicates the Whatman 903 Protein Saver Card is better for Pfs25 mRNA sampling compared to the Whatman FTA Classic Card, and that the Whatman 3MM filter paper may prove to be a satisfactory cheaper option for Pfs25 mRNA sampling. When appropriately dried, filter papers provide a useful approach to Pfs25 mRNA sampling, especially in settings where storage in RNA-protecting buffer is not possible.

We have previously proposed endothelin-2 (EDN2) as a granulosa cell-derived contractile signal that facilitates ovulation. Spatially, Edn2 mRNA expression is restricted to granulosa cells of periovulatory follicles. Temporally, mRNA for this contractile peptide is expressed immediately before follicle rupture. The primary objective of this study was to test the hypothesis that hypoxia mediates EDN2 expression in granulosa cells at ovulation, and if it does, to determine the region within the promoter responsible for this effect. To determine the effect of hypoxia on mRNA expression, immature mice were treated with 5 IU of PMSG followed 48 h later by 5 IU of human chorionic gonadotropin (hCG). Granulosa cells were isolated at 9 h after hCG, cultured under normal or hypoxic conditions, and the expression level of mRNA was compared. mRNA expression was increased when granulosa cells were cultured in a hypoxic environment (p < 0.05). Subsequent promoter analysis found that the 5′ upstream region of the EDN2 promoter (between –1894 bp and –1407 bp) was responsible for hypoxia-mediated changes in EDN2 expression. This promoter region contains multiple sites for potential transcriptional regulation, including that by hypoxia-inducible factor 1 (HIF-1, ACGTG) at –1297 bp. The second objective of this study was to determine whether the progesterone receptor (PR) or cyclooxygenase-2 (COX-2), two key regulators of periovulatory events, controlled EDN2 expression. To accomplish this, gonadotropin-primed mice were treated with RU-486 or indomethacin and expression of mRNA for Edn2 was determined in ovaries collected at 12 h after hCG. Treatment with RU-486 or indomethacin did not affect expression of mRNA for Edn2 (p > 0.05). Taken together, we believe that hypoxia, but not the PR or COX-2, regulate gonadotropin-induced EDN2 expression in the periovulatory follicle.

YopQ in Yersinia enterocolitica (YopK in Yersinia pseudotuberculosis) is a type III secreted protein required for virulence of yersiniae. In this study YopQ expression, secretion and nucleotide sequences of the corresponding yopQ gene from different yersinia strains were analysed. The cytosolic accumulation differed significantly among serotypes of Y. enterocolitica. These differences might be attributable to variations in the nucleotide sequence and their consequences on mRNA secondary structure. An mRNA signal hypothesis has been proposed for YopQ, predicting the coupling of translation and secretion via an mRNA signal. This hypothesis claims a strictly co-translational secretion of YopQ without its intracellular accumulation. The presence of YopQ in the cytosol, even with a closed secretion apparatus, is demonstrated. Moreover, post-translational secretion of YopQ could be demonstrated. These findings do not support the mRNA signal hypothesis for co-translational secretion.

As a first step toward understanding estrogen’s role in neurodevelopment, a PCR cloning strategy was used to isolate complementary DNAs encoding two distinct cytochrome P450 aromatase isoforms in adult zebrafish (Danio rerio) brain and ovary (termed P450aromB and P450aromA, respectively). Sequence and phylogenetic analysis showed that the zebrafish P450arom forms are orthologs of previously identified cyp19b and cyp19a genes in goldfish. On Northern blots, a single 4.4-kb transcript of the P450aromB subtype was identified in brain, and a 2.1-kb transcript of the P450aromA subtype in ovary, but RT-PCR showed a degree of overlapping expression. Both messenger RNA (mRNA) forms were detected in unfertilized eggs and 1.5 hpf (cleavage stage) embryos but declined by 12 hpf, indicating maternal transfer. A secondary rise in mRNAs between 12‐24 hpf indicated the onset of embryonic cyp19b and -a transcription. Both mRNA species accumulated progressively to 120 hpf (early larval stage), but the relative magnitude and pattern of change was isoform specific. Estradiol (E2, 1 mM) advanced and amplified the developmentally programmed accumulation of P450aromB mRNA, and ICI164.384 decreased expressed levels, implying blockade of an endogenous estrogen mediated regulatory component. Conversely, E2 had no effect or decreased P450aromA mRNA. The early embryonic expression of P450aromB and P450aromA isoforms, and differences in developmental programming and estrogen regulation, imply independent regulatory mechanisms and unique functions during major morphogenetic and differentiative events. (Endocrinology 142: 740 ‐750, 2001)

The present study reports the isolation of three complementary DNA (cDNA) clones encoding distinct subtypes of CRF receptors from the diploid catfish (cf) species, Ameiurus nebulosus. The first clone encodes a 446-amino acid protein (cfCRF-R1) that is highly homologous to mouse (m) CRF-R1 (93% identical). The cfCRF-R1 messenger RNA is highly expressed in the brain, and its distribution pattern correlates well with that of mammalian CRF-R1, except for weak expression in the pituitary. When transiently expressed in COS-7 cells, cfCRF-R1 bound CRF, urotensin I, and sauvagine with similar affinities. The second full-length cDNA, which was cloned from catfish heart, encodes a 406-amino acid protein that showed homology to murine CRF-R2 (88%) and when expressed in COS-7 cells preferentially bound sauvagine. The highest level of cfCRF-R2 expression was observed in the heart. The third full-length cDNA clone, which encodes a 428-amino acid protein, is structurally closer to cfCRF-R1 (85%) than to cfCRF-R2 (80%). This novel CRF receptor (cfCRF-R3) bound CRF with a 5-fold higher affinity than urotensin I and sauvagine and was expressed in the pituitary gland, urophysis, and brain. The presence of three different CRF receptors, each with distinct tissue distribution and ligand binding properties, suggests a complex CRF/urotensin I system. (Endocrinology 142: 446 ‐ 454, 2001)

A 3′ RACE technique was used to establish the nucleotide sequence encoding the C-terminal 379 amino acids of rat intestinal Muc3. Unlike the C-terminus of Muc2 and many secretory mucins, Muc3 contains two EGF motifs and a putative transmembrane domain. The mRNA for rat Muc3 is 7.5–8.0 kb.

The genomic RNA of coronaviruses has two unique features. Firstly, it is one of the largest stable RNAs known to exist in nature and is unquestionably the largest viral genomic RNA. The complete sequence of avian infectious bronchitis virus (IBV) RNA shows that its size is 27.6 kilobases (kb) (Boursnell et al., 1987). Although the complete sequences of other Coronavirus genomic RNAs are not yet available, preliminary data from several laboratories indicate that the genomic RNA of mouse hepatitis virus (MHV) is as long as 32 kb (Shieh and Lai, unpublished observations). The large size of these RNAs poses a theoretical quandary for the replication of Coronavirus RNA, considering the high error frequency of RNA-dependent RNA synthesis observed in some systems (Holland et al., 1982; Steinhauer and Holland, 1986). How does Coronavirus RNA replicate faithfully despite the high error frequency of RNA synthesis? It is possible that a proof-reading mechanism operates to correct unavoidable mistakes which are expect d to occur in almost every RNA molecule of this size. Secondly, Coronavirus RNA contains a leader sequence (approximately 72 nucleotides) which is repeated at the 5′-end of every subgenomic mRNA species. This structural organization appears to be similar to that of most of eukaryotic mRNAs which contain leader sequences derived by RNA splicing. Yet the Coronavirus RNA genome does not have consensus splicing signals and the virus replicates exclusively in the cytoplasm (Wilhelmson et al., 1981; Brayton et al., 1981), where there is no conventional RNA splicing machinery. Furthermore, UV transcriptional mapping studies suggest that Coronavirus mRNA species are transcribed independently (Jacobs et al., 1981), instead of being derived by cleavage of a precursor RNA. Thus, a new transcriptional mechanism must operate to transcribe Coronavirus mRNAs.

Poly A containing RNA isolated from embryonic chick calvaria was transferred from 6% formaldehyde 0.75% agarose gels to diazobenzyloxymethyl paper and the paper then hybridized to either nick translated pro alpha 1 collagen cDNA clones, pCg1 or pCg54, or to the nick translated pro alpha 2 collagen cDNA clone, pCg45. From the mobilities of the bands hybridizing most strongly to each, pro alpha 2 collagen mRNA was shown to be slightly larger than pro alpha 1 mRNA; they are 5100 and 4900 nucleotides long respectively. pCg54 also hybridized weakly to two bands of lower mobility, corresponding to RNAs 6.4 and 5.6 kb long. Neither pCg54 nor pCg45 hybridized to type II procollagen mRNA in poly A containing RNA isolated from embryonic chick sterna.

The methylase METTL3 is the writer enzyme of the N6‐methyladenosine (m6A) modification of RNA. Using a structure‐based drug discovery approach, we identified a METTL3 inhibitor with potency in a biochemical assay of 280 nM, while its enantiomer is 100 times less active. We observed a dose‐dependent reduction in the m6A methylation level of mRNA in several cell lines treated with the inhibitor already after 16 h of treatment, which lasted for at least 6 days. Importantly, the prolonged incubation (up to 6 days) with the METTL3 inhibitor did not alter levels of other RNA modifications (i. e., m1A, m6Am, m7G), suggesting selectivity of the developed compound towards other RNA methyltransferases., Writer's block: We developed and characterized a small‐molecule inhibitor of m6A writer METTL3 by protein crystallography, biochemical and cellular assays. It shows high‐nanomolar potency in the biochemical assay, good selectivity against a panel of protein methyltransferases and kinases, and it reduced m6A/A ratio in mRNAs of different cell lines. In addition, we confirmed the selectivity of our compound towards other RNA methyltransferases in living cells.

Abstract: During the last decades, there has been growing interest in using therapeutic messager RNA (mRNA) together with drug delivery systems. Naked, unformulated mRNA is, however, unable to cross the cell membrane and is susceptible to degradation. Here we use graphene quantum dots (GQDs) functionalized with polyethyleneimine (PEI) as a novel mRNA delivery system. Our results show that these modified GQDs can be used to deliver intact and functional mRNA to Huh‐7 hepatocarcinoma cells at low doses and, that the GQDs are not toxic, although cellular toxicity is a problem for these first‐generation modified particles. Functionalized GQDs represent a potentially interesting delivery system that is easy to manufacture, stable and effective., Message delivered! A novel graphene quantum dots based mRNA drug delivery platform was prepared. The results show that these modified GQDs can deliver intact and functional mRNA to Huh‐7 hepatocarcinoma cells at low doses. The transfection efficiency for FGQDs/mRNA complexes was 25 % with a formulation concentration of 4000 ng mRNA/mL, but comparable transfection efficiencies could be achieved at much lower doses if the ratio between carrier and cargo was optimized. This work describes the first steps towards a potentially interesting preparation method for stable and effective mRNA delivery systems.

MicroRNAs constitute a class of endogenous, non‐coding RNAs that influence various processes within the cell. By base‐pairing to partially‐complementary sites located in the 3’ untranslated region of target messenger RNAs, microRNAs participate in post‐transcriptional regulation of the majority of human protein‐coding genes. Their dysregulation has been related to many pathological processes and diseases. Thus, an in‐depth understanding of the microRNA mechanisms of action is crucial. Here, we present a new concept of probe design to achieve an efficient and sequence‐independent miRNA‐mRNA cross‐linking. The new strategy is based on the utilization of a controlled mixture of probes for a chosen miRNA, in which a trioxsalen moiety is introduced at the N 4‐position of a selected cytidine through short oligoethylene glycol‐based linkers. In vitro photo‐cross‐linking experiments with mini‐libraries of probes for microRNAs of interest showed variable cross‐linking efficiencies, demonstrating a general applicability of the presented approach., We describe a new class of miRNA probes with photo‐active trioxsalen attached via oligoethylene glycol‐based linkers. These cross‐link to RNA targets in vitro with efficiencies dependent on sequence and linker‐length. By using controlled mixtures of probes, a general approach to efficient covalent target‐capture independent of miRNAs sequence is proposed.

DNA methylation regulates gene expression by modifying the nucleosome structure of DNA, without altering the gene sequence. It has been reported that DNA methylation reactions are catalyzed by several enzymes. In chickens, thermal conditioning treatment affects the central DNA methylation levels. The purpose of this study was to clarify the changes in DNA methylation and demethylation factors during thermal conditioning in the hypothalamus of 3-day-old chicks. Male chicks (3-days old) were exposed to 40±0.5°C as a thermal conditioning treatment for 1, 2, 6, 9, or 12 h. The control chicks were kept in a thermoneutral zone (30±0.2°C). After thermal conditioning, the mRNA levels of DNA methyltransferase (DNMT)-1, -3a, -3b, and ten-eleven translocation (TET)-1, -2, and -3 in the hypothalamus were measured by q-PCR. The mRNA levels of DNMT-3a and TET-1 were increased by thermal conditioning. Moreover, the expression level of TET-1 increased with the loading time of the thermal conditioning. The gene expressions of DNMT-1, DNMT-3b, TET-2, and TET-3 were not affected by thermal conditioning. Since DNMT-3a is a catalyst for de-novo DNA methylation and TET-1 catalyzes the oxidation of methylated cytosine, it is suggested that the thermal conditioning increased the activation of DNA methylation and demethylation factors, which occur in the hypothalamus of neonatal chicks.

Introduction: Methamphetamine (MA) acts as a powerful oxidant agent, while Rosmarinic Acid (RA) is an effective herbal antioxidant. Oxidative stress-mediated by MA results in apoptosis, and caspase-3 is one of the critical enzymes in the apoptosis process. MA can epigenetically alter gene regulation. In this paper, to investigate the effects of RA on MA-mediated oxidative stress, changes in the level of casp3a mRNA were demonstrated in zebrafish. Methods: The animals were grouped in 3 treatment conditions for the behavioral test: control, MA, MA pretreated by RA, and 6 treatment conditions for the molecular test: control, RA, MA, MA co-treated with RA, MA co-treated with RA/ZnO/chitosan nanoparticle, and ZnO/chitosan nanoparticle. Then molecular and behavioral investigations were carried out, and critical comparisons were made between the groups. MA solution was prepared with a concentration of 25 mg/L, and RA solution was prepared by DPPH test with the antioxidant power of about 97%. Each solution was administered by immersing 20 zebrafish for 20 minutes, once per day for 7 days. The level of casp3a mRNA was quantified by using qRT-PCR. One-sided trapezoidal tank diving test was applied to study behavioral alterations. Results: The qPCR analysis demonstrated the high potential of RA/ZnO/chitosan in counteracting the MA-mediated elevation in casp3a mRNA level. Based on the diving test results of MA-treated fish, MA was found to be anxiolytic compared to the control. While the resulted diving pattern of the MA-treated animals pretreated by RA was novel and different from both the control and MA-treated groups. Conclusion: The potential of RA combined with a suitable nanoparticle against MA-induced oxidative stress was supported. The high efficiency of ZnO/chitosan in increasing RA penetration to the brain cells was evident. MA at a dose of 25 mg/L is anxiolytic for zebrafish. However, the molecular mechanisms involved in these processes should be studied.

B7-H3 isoforms, 4IgB7-H3 (4Ig) and 2IgB7-H3 (2Ig) expression characteristics in acute myeloid leukemia (AML) remain unknown. This study investigated mRNA and membrane protein expression of two isoforms in AML cell lines and de novo patients by using RT-PCR and flow cytometry, and analyzed the B7-H3 promoter methylation state by utilizing RQ-MSP. 4Ig was the dominant B7-H3 isoform. 2Ig mRNA expression rate and abundance were elevated in AML in comparison with controls (P=0.000 and 0.000), while no significant difference in 4Ig (P=0.802, P=0.398). Membrane protein levels of B7-H3 isoforms in AML was higher than controls, detected by total B7-H3 expression rate (P=0.002), total B7-H3 mean fluorescence intensity (MFI) ratio of blast cells and lymphocytes (MFI ratio) (P=0.000), and 4Ig B7-H3 MFI ratio (P=0.005). Compared with 2Iglow group, 2Ighigh patients had older age, lower NPM1 mutation, higher FLT3-ITD mutation, and declining complete remission (CR) rates (P=0.026, 0.012, 0.047, and 0.028). In B7-H3high group, there was a trend towards older age, -M4 and M5, worse karyotype, and lower CR rates, although with no marked difference (P>0.05). The overall survival (OS) of 2Ighigh and B7-H3high groups were shorter than that of 2Iglow and B7-H3low groups in the whole and non-M3 AML cohorts (P=0.006 and 0.046; P=0.003 and 0.032). Besides, an unmethylated B7-H3 promoter was identified. In conclusion, 2Ig mRNA and total B7-H3 isoforms membrane protein tended to have potential diagnostic value for AML. Specifically, high 2Ig mRNA expression and total B7-H3 membrane protein expression indicate worse OS. 4Ig and 2Ig expression are methylation-independent.

Breast cancer, with high morbidity worldwide, is a threat to the life of women. MiR-543 was identified as playing an active part in the development of breast cancer involving multiple molecules. The goal of this study was to explore the molecular mechanisms of the involvement of miR-543 in the development of breast cancer. Quantitative real-time PCR (qRT-PCR) or Western blotting was used to detect mRNA or protein expression. Cell counting kit-8 (CCK-8), and the 5-bromo-2ʹ-deoxyuridine (BrdU), wound healing, and Transwell assays were the main experimental procedures. Furthermore, subcutaneous tumor formation experiments were conducted to detect the function of miR-543 in breast cancer development in vivo. The match of miR-543 and ubiquitin-conjugating enzyme E2T (UBE2T) was detected through a dual-luciferase reporter experiment and RNA pull-down assay. Based on these results, miR-543 exhibited reduced expression in breast cancer tissues and cell lines, whereas UBE2T exhibited high levels. Furthermore, miR-543 directly targeted UBE2T, and a negative correlation between miR-543 and UBE2T was also observed in breast cancer tissues. Moreover, miR-543 overexpression led to inhibition of viability, proliferation, migration, and invasion of breast cancer. Furthermore, miR-543 overexpression undermined the UBE2T promotional effect by inhibiting ERK/MAPK pathway activity in breast cancer cells. Our study revealed that miR-543 impaired breast cancer progression by targeting UBE2T and downregulating UBE2T expression through the ERK/MAPK pathway, which suggested that miR-543 and UBE2T might serve as promising therapeutic gene targets for breast cancer in clinical application.

Acute myeloid leukemia (AML) is a severe hematologic malignancy that threatens human health. Long non-coding RNA (lncRNA) is emerged as a key player in human cancer. Herein, we explored the role of LINC00998 in human AML. LINC00998 was significantly decreased in human AML, which was linked to relapse and poor prognosis. Stable overexpression of LINC00998 inhibited AML cell viability, colony ability, DNA synthesis rate and increased apoptosis. LINC00998 was mainly located in the cytoplasm, in which interacted with ZFP36 ring finger protein (ZFP36), a mRNA destabilizing factor, resulting in increased decay of mammalian target of rapamycin complex 2 (mTORC2), a well-known proto-oncogene in AML. Overexpression of mTORC2 partly blocked the tumor suppressive effects of LINC00998. Importantly, LINC00998 shortened in vivo AML cell survival in xenograft tumor model. Taken together, we found that LINC00998 is a novel tumor-inhibiting lncRNA in human AML. The dysregulation of LINC00998/ZFP36/mTORC2 axis is linked to leukemogenesis and progression.

Acute coronary syndrome (ACS) is a term used to describe major cardiovascular diseases, and treatment of in-stent restenosis in patients with ACS remains a major clinical challenge. Further investigation into molecular markers of ACS may aid early diagnosis, and the treatment of ACS and post-treatment recurrence. In the present study, total RNA was extracted from the peripheral blood samples of 3 patients with ACS, 3 patients with percutaneous coronary intervention (PCI)_non-restenosis, 3 patients with PCI_restenosis and 3 healthy controls. Subsequently, RNA library construction and high-throughput sequencing were performed. DESeq2 package in R was used to screen genes that were differentially expressed between the different samples. Moreover, the intersection of the differentially expressed mRNAs (DEmRNAs) and differentially expressed long noncoding RNAs (DElncRNAs) obtained. GeneCodis4.0 was used to perform function enrichment for DEmRNAs, and lncRNA-mRNA co-expression network was constructed. The GSE60993 dataset was utilized for diagnostic analysis, and the aforementioned investigations were verified using in vitro studies. Results of the present study revealed a large number of DEmRNAs and DElncRNAs in the different groups. We selected genes in the top 10 of differential expression and also involved in the co-expression of lncRNA-mRNA for diagnostic analysis in the GSE60993 dataset. The area under curve (AUC) of PDZK1IP1 (0.747), PROK2 (0.769) and LAMP3 (0.725) were all >0.7. These results indicated that the identified mRNAs and lncRNAs may act as potential clinical biomarkers, and more specifically, PDZK1IP1, PROK2 and LAMP3 may act as potential biomarkers for the diagnosis of ACS.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that causes tremors, gait rigidity, and hypokinesia. We determined the effects of long noncoding RNA small nucleolar RNA host gene 12 (lncRNA SNHG12) on the development of PD. StarBase analysis and dual-luciferase reporter assay verified the interaction between lncRNA SNHG12 and microRNA-138-5p (miR-138-5p). The effects of suppressed lncRNA SNHG12 and increased miR-138-5p levels on mRNA were determined using quantitative real-time-PCR (qRT-PCR) in 1-methyl-4-phenylpyridinium (MPP+) treated SH-SY5Y cells. Increased lactate dehydrogenase (LDH) activity, apoptosis, cleaved-Caspase3/Caspase3 ratio, inflammatory response, reactive oxygen species (ROS) level, decreased cell viability, and superoxide dismutase (SOD) activity were observed in MPP+-stimulated SH-SY5Y cells. Transfection of the lncRNA SNHG12-plasmid reduced neuronal apoptosis, inflammation, and oxidative stress in MPP+-stimulated SH-SY5Y cells that were rescued by adding the miR-138-5p mimic. These results showed that lncRNA SNHG12 could affect neuronal apoptosis, inflammation, and oxidative stress in a PD cell model by regulating miR-138-5p expression. TargetScan and dual-luciferase reporter analysis suggested that miR-138-5p targeted nuclear factor I/B (NFIB). Furthermore, the expression level of NFIB was downregulated after MPP+ stimulation in SH-SY5Y cells. After transfecting with the miR-138-5p inhibitor, NFIB-siRNA, and co-transfecting and detecting NFIB mRNA and protein, we found that miR-138-5p negatively regulated NFIB expression. In conclusion, lncRNA SNHG12 could alleviate neuronal apoptosis, inflammation, and oxidative stress in a PD cell model by regulating the miR-138-5p/NFIB axis, providing new therapeutic targets for patients with PD.

Selenium is an important micronutrient for foetal development. MicroRNAs play an important role in the function of the placenta, in communication between the placenta and maternal systems, and their expression can be altered through environmental and nutritional cues. To investigate the associations between placental selenium concentration and microRNA expression in the placenta, our observational study included 393 mother-child pairs from the New Hampshire Birth Cohort Study (NHBCS) and the Rhode Island Child Health Study (RICHS). Placental selenium concentrations were quantified using inductively coupled plasma mass spectrometry, and microRNA transcripts were measured using RNA-seq. We fit negative binomial additive models for assessing the association between selenium and microRNAs. We used the microRNA Data Integration Portal (mirDIP) to predict the target mRNAs of the differentially expressed microRNAs and verified the relationships between miRNA and mRNA targets in a subset of samples using existing whole transcriptome data (N = 199). We identified a non-monotonic association between selenium concentration and the expression of miR-216a-5p/miR-217-5p cluster (effective degrees of freedom, EDF = 2.44 and 2.08; FDR = 3.08 × 10−5) in placenta. Thirty putative target mRNAs of miR-216a-5p and/or miR-217-5p were identified computationally and empirically and were enriched in selenium metabolic pathways (driven by selenoprotein coding genes, TXNRD2 and SELENON). Our findings suggest that selenium influences placental microRNA expression. Further, miR-216a-5p and its putative target mRNAs could be the potential mechanistic targets of the health effect of selenium.

Although the functional parameters of microRNAs (miRNAs) have been explored to some extent, the roles of these molecules in coronavirus infection and the regulatory mechanism of miRNAs in virus infection are still unclear. Transmissible gastroenteritis virus (TGEV) is an enteropathgenic coronavirus and causes high morbidity and mortality in suckling piglets. Here, we demonstrated that microRNA-27b-3p (miR-27b-3p) suppressed TGEV replication by directly targeting porcine suppressor of cytokine signaling 6 (SOCS6), while TGEV infection downregulated miR-27b-3p expression in swine testicular (ST) cells and in piglets. Mechanistically, the decrease of miR-27b-3p expression during TGEV infection was mediated by the activated inositol-requiring enzyme 1 (IRE1) pathway of the endoplasmic reticulum (ER) stress. Further studies showed that when ER stress was induced by TGEV, IRE1 acted as an RNase activated by autophosphorylation and unconventionally spliced mRNA encoding a potent transcription factor, X-box-binding protein 1 (Xbp1s). Xbp1s inhibited the transcription of miR-27 and ultimately reduced the production of miR-27b-3p. Therefore, our findings indicate that TGEV inhibits the expression of an anti-coronavirus microRNA through the IRE1 pathway and suggest a novel way in which coronavirus regulates the host cell response to infection. Supporting Information The supporting information is available online at 10.1007/s11427-021-1967-x. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

This study is aimed at investigating mechanisms and effects of Krüppel-like factor 16 (KLF16) affects myocardial ischemia-reperfusion. Patients with myocardial ischemia-reperfusion and normal volunteer were collected. C57BL6J male mice were located left anterior descending coronary artery (LAD). H9c2 cell was induced with hydrogen peroxide (H2O2) and Lipopolysaccharide (LPS). Serum KLF16 mRNA expression was increased in myocardial ischemia-reperfusion. Serum mRNA of KLF16 was positive correlation with serum creatine kinase MB (CK-MB) or creatine kinase (CK) levels in patients with myocardial ischemia-reperfusion. The expression of KLF16 mRNA and protein in mice with myocardial ischemia-reperfusion were also increased. The inhibition of KLF16 reduced oxidative stress and inflammation, and presented myocardial ischemia (MI) in vivo model of myocardial ischemia-reperfusion. Mitochondrial transcription factor A (TFAM)/peroxisome proliferator-activated receptor-beta (PPARβ) signal passage is target spot of KLF16 in Myocardial ischemia-reperfusion. TFAM interlink KLF16 in myocardial ischemia-reperfusion. TFAM participate in KLF16 affects myocardial ischemia-reperfusion. PPARβ promoter region KLF16 affects myocardial ischemia-reperfusion. These results firstly demonstrated that knock-out KLF16 reduced oxidative stress and inflammation, and presented MI in vivo model of myocardial ischemia-reperfusion through the induction of PPARβ by TFAM, may provide a novel therapeutic strategy for myocardial ischemia-reperfusion.

Messenger RNA (mRNA) is emerging as an attractive biopolymer for therapy and vaccination. To become suitable for vaccination, mRNA is usually converted to a biomaterial, using cationic peptides, polymers or lipids. An alternative form of converting mRNA into a material is demonstrated that uses branched oligoribonucleotide hybrids with the ability to hybridize with one or more regions of the mRNA sequence. Two such hybrids with hexamer arms and adamantane tetraol as branching element were prepared by solution‐phase synthesis. When a rabies mRNA was treated with the branched hybrids at 1 M NaCl concentration, biomaterials formed that contained both of the nucleic acids. These results show that branched oligoribonucleotides are an alternative to the often toxic reagents commonly used to formulate mRNA for medical applications., Branched oligoribonucleotide hybrids that hybridize to two different loci of mRNA were used to produce a biomaterial that may become useful for vaccination.

Sevoflurane (Sev) is a volatile anesthetic that can inhibit tumor malignancy. Glioma is a main brain problem, but the mechanism of Sev in glioma progression is largely unclear. This study aims to explore a potential regulatory network of long noncoding RNA (lncRNA)/microRNA (miRNA)/mRNA associated with the function of Sev in glioma progression. LncRNA HMMR antisense RNA 1 (HMMR-AS1), miR-7 and cyclin-dependent kinase 4 (CDK4) abundances were examined via quantitative reverse transcription polymerase chain reaction and western blot. Cell viability, invasion, and colony formation ability were analyzed via cell counting kit-8, transwell analysis, and colony formation. The target association was analyzed via dual-luciferase reporter analysis and RNA pull-down. The in vivo function of Sev was investigated by xenograft model. HMMR-AS1 abundance was increased in glioma tissues and cells, and reduced via Sev. Sev constrained cell viability, invasion, and colony formation ability via decreasing HMMR-AS1 in glioma cells. miR-7 expression was decreased in glioma, and was targeted via HMMR-AS1. HMMR-AS1 silence restrained cell viability, invasion, and colony formation ability by up-regulating miR-7 in glioma cells. Sev increases miR-7 abundance via decreasing HMMR-AS1. CDK4 was targeted via miR-7, and highly expressed in glioma. miR-7 overexpression inhibited cell viability, invasion, and colony formation ability via reducing CDK4 in glioma cells. CDK4 expression was reduced by Sev via HMMR-AS1/miR-7 axis. Sev suppressed cell growth in glioma by regulating HMMR-AS1. Sev represses glioma cell progression by regulating HMMR-AS1/miR-7/CDK4 axis.

The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is essential for bone formation, and its imbalance can lead to bone diseases such as osteoporosis. It is reported that PIWI-interacting RNA-36741 (piR-36741) is up-regulated during the osteogenic differentiation, but its role in regulating osteogenic differentiation remains unclear. Here, the primary human BMSCs were used to induce osteogenic differentiation, and the expression of piR-36741 and METTL3 (methyltransferase like 3) was up-regulated during the osteogenic differentiation of BMSCs. Moreover, interference with piR-36741 or METTL3 markedly hindered the osteogenic differentiation of BMSCs, which was manifested by a reduction in osteoblast marker expression (including RUNX2, COL1A1, OPN and OCN), osteogenic phenotype and matrix mineralization. Mechanistically, the piR-36741-PIWIL4 complex directly interacted with METTL3 and prevented METTL3-mediated m6A modification of BMP2 mRNA transcripts, thereby promoting BMP2 expression. And overexpression of BMP2 reversed the inhibitory effect of piR-36741 silence on osteogenic differentiation and the Smad pathway activity. In addition, administration with piR-36741 mimic alleviated ovariectomy-induced osteoporosis in mice. In conclusion, piRNA-36741 overexpression promoted osteogenic differentiation of BMSCs and mitigated ovariectomy-induced osteoporosis through METTL3-mediated m6A methylation of BMP2 transcripts.

Non-membrane-bound compartments such as P-bodies (PBs) and stress granules (SGs) play important roles in the regulation of gene expression following environmental stresses. We have systematically and quantitatively determined the protein and mRNA composition of PBs and SGs formed before and after nutrient stress. We find that high molecular weight (HMW) complexes exist prior to glucose depletion that we propose may act as seeds for further condensation of proteins forming mature PBs and SGs. We identify an enrichment of proteins with low complexity and RNA binding domains, as well as long, structured mRNAs that are poorly translated following nutrient stress. Many proteins and mRNAs are shared between PBs and SGs including several multivalent RNA binding proteins that promote condensate interactions during liquid-liquid phase separation. We uncover numerous common protein and RNA components across PBs and SGs that support a complex interaction profile during the maturation of these biological condensates. These interaction networks represent a tuneable response to stress, highlighting previously unrecognized condensate heterogeneity. These studies therefore provide an integrated and quantitative understanding of the dynamic nature of key biological condensates.

Endocervical adenocarcinoma (EAC) is an aggressive type of endocervical cancer. At present, molecular research on EAC mainly focuses on the genome and mRNA transcriptome, the investigation of small RNAs in EAC has not been fully described. Here, we systematically explored small RNAs in 14 EAC patients with different subtypes using small RNA sequencing. MiRNAs and tRNA-derived RNAs (tDRs) accounted for the majority of mapped reads and the total number of miRNAs and tDRs maintained a relative balance. To explore the correlations between small RNAs expression and EAC with different clinical characteristics, we performed the weighted gene co-expression network analysis (WGCNA) and screened for hub small RNAs. From the key modules, we identified 9 small RNAs that were significantly related to clinical characteristics in EAC patients. Gene ontology and pathway analyses revealed that these molecules were involved in the pathogenesis of EAC. Our work provided new insights into EAC pathogenesis and successfully identified several small RNAs as candidate biomarkers for diagnosis and prognosis of EAC.

The occurrence of distant metastasis is one of the leading causes of death in patients with prostate cancer (PCa). It is confirmed that kinesin protein is associated with a variety of malignancies, and the KIF3 family is related to cancer, but the relationship between KIF3C and prostate cancer is not clear. Our experiments have confirmed that KIF3C is highly expressed in prostate cancer tissues and cell lines. Further, functional tests have proven that KIF3C can promote the growth migration and invasion of PCa. We used Starbase 3.0 to discover that methyltransferase like 3 (METTL3) interacts with KIF3C. Our hypothesis and experiments concluded that METTL3 induced m6A modification on KIF3C, promoting the stabilization of KIF3C-mRNA by IGF2 binding protein 1 (IGF2BP1). The prediction that miR-320d inhibits KIF3C expression by targeting METTL3 using the miRmap website, was later confirmed experimentally. Further, a recovery experiment was used to confirm that miR-320d inhibited the progression of prostate cancer. KIF3C was overexpressed in prostate cancer, promoting its growth migration and invasion was induced by miR-320d/METTL3 in an m6A dependent process.

Cervical cancer (CC) is one of the most common malignancies among women. It has been demonstrated that long coding RNAs (lncRNAs) play a crucial role in CC. The purpose of this study was to investigate the role of the colon cancer associated transcript 2 (CCAT2) lncRNA in CC and elucidate its possible mechanisms of action. The expression of CCAT2, the miR-493-5p microRNA (miRNA), and mRNA was detected using qRT-PCR. Cell viability, proliferation, and migration and invasion were determined using the MTT, colony formation, and transwell assays, respectively. The interactions between miR-493-5p and CCAT2 or cAMP response element-binding protein 1 (CREB1) were verified using the luciferase and RNA pull-down assays. The effects of CCAT2 knockdown on in vivo tumor growth were determined using tumor xenografts and immunohistochemistry assays. The expression of CCAT2 was upregulated in CC cells and tissues. However, the knockdown of CCAT2 inhibited the proliferation and epithelial-mesenchymal transition (EMT) of CC cells in vitro and suppressed tumor growth in vivo. Mechanistically, CCAT2 functions as a competing endogenous RNA (ceRNA) to upregulate the expression of CREB1 by binding to miR-493-5p. The overexpression of CREB1 or downregulation of miR-493-5p antagonized the effect of CCAT2 knockdown on the proliferation and EMT of CC cells. The knockdown of CCAT2 suppressed the aggressiveness of CC via the miR-493-5p/CREB1 axis. Therefore, CCAT2 is likely to be a promising therapeutic target for CC.

Most of Solute carrier family-2 (SLC2) members play a key role of facilitative transporters, and glucose transporter (GLUT) proteins encoded by SLC2s can transport hexoses or polyols. However, the function and mechanism of SLC2s remain unclear in human cancers. Here, we explored the dysregulated expression, prognostic values, epigenetic, genetic alterations, and biomolecular network of SLC2s in human cancers. According to the data from public-omicsrepository, SLC2A4 (GLUT4) was found to be significantly downregulated in most cancers, and higher messenger RNA (mRNA) expression of SLC2A4 significantly associated with better prognosis of breast cancer (BRCA) patients. Moreover, DNA hypermethylation in the promoter of SLC2A4 may affect the regulation of its mRNA expression, and SLC2A4 was strongly correlated with pathways, including the translocation of SLC2A4 to the plasma membrane and PID INSULIN PATHWAY. In conclusion, these results provide insight into SLC2s in human cancers and suggest that SLC2A4 could be an unfavorable prognostic biomarker for the survival of BRCA patients.

Breast-invasive carcinoma (BRCA) is the most frequent and malignant tumor in females. Ceruloplasmin (CP) is a multifunctional molecule involved in iron metabolism, but its expression profile, prognostic potential and relationship with immune cell infiltration in BRCA are unknown. Ceruloplasmin mRNA and protein expression was significantly decreased in BRCA patients according to the Oncomine, UALCAN, GEPIA and TCGA databases. Ceruloplasmin expression was strongly correlated with various clinicopathological features of BRCA patients. BRCA patients with high ceruloplasmin expression exhibited shorter survival times than those with low ceruloplasmin expression based on the Kaplan-Meier plotter and PrognoScan databases. GO and KEGG analyses and GSEA revealed a strong correlation between ceruloplasmin and various immune-related pathways. Ceruloplasmin expression was significantly associated with the infiltration of immune cells into tumor sites by analyzing the TIMER and CIBERSORT. Additionally, ceruloplasmin was positively correlated with immune checkpoints in BRCA. These findings suggest that low ceruloplasmin expression correlates with a favorable prognosis and tumor immune cell infiltration in BRCA patients. Ceruloplasmin may serve as a therapeutic target and predict the efficacy of immunotherapy for BRCA.