Identification of a damage-associated molecular pattern (DAMP) receptor and its cognate peptide ligand in sweet potato (Ipomoea batatas).

Sweet potato (Ipomoea batatas) is an important tuber crop, but also target of numerous insect pests. Intriguingly, the abundant storage protein in tubers, sporamin, has intrinsic trypsin protease inhibitory activity. In leaves, sporamin is induced by wounding or a volatile homoterpene and enhances insect resistance. While the signalling pathway leading to sporamin synthesis is partially established, the initial event, perception of a stress-related signal is still unknown. Here, we identified an IbLRR-RK1 that is induced upon wounding and herbivory, and related to peptide-elicitor receptors (PEPRs) from tomato and Arabidopsis. We also identified a gene encoding a precursor protein comprising a peptide ligand (IbPep1) for IbLRR-RK1. IbPep1 represents a distinct signal in sweet potato, which might work in a complementary and/or parallel pathway to the previously described hydroxyproline-rich systemin (HypSys) peptides to strengthen insect resistance. Notably, an interfamily compatibility in the Pep/PEPR system from Convolvulaceae and Solanaceae was identified.

DNA Methylation in the Fields of Prenatal Diagnosis and Early Detection of Cancers.

The central objective of the metamorphosis of discovery science into biomedical applications is to serve the purpose of patients and curtail the global disease burden. The journey from the discovery of DNA methylation (DNAm) as a biological process to its emergence as a diagnostic tool is one of the finest examples of such metamorphosis and has taken nearly a century. Particularly in the last decade, the application of DNA methylation studies in the clinic has been standardized more than ever before, with great potential to diagnose a multitude of diseases that are associated with a burgeoning number of genes with this epigenetic alteration. Fetal DNAm detection is becoming useful for noninvasive prenatal testing, whereas, in very preterm infants, DNAm is also shown to be a potential biological indicator of prenatal risk factors. In the context of cancer, liquid biopsy-based DNA-methylation profiling is offering valuable epigenetic biomarkers for noninvasive early-stage diagnosis. In this review, we focus on the applications of DNA methylation in prenatal diagnosis for delivering timely therapy before or after birth and in detecting early-stage cancers for better clinical outcomes. Furthermore, we also provide an up-to-date commercial landscape of DNAm biomarkers for cancer detection and screening of cancers of unknown origin.

Tetranorsesquiterpenoids as Attractants of Yucca Moths to Yucca Flowers.

The obligate pollination mutualism between Yucca and yucca moths is a classical example of coevolution. Oviposition and active pollination by female yucca moths occur at night when Yucca flowers are open and strongly scented. Thus, floral volatiles have been suggested as key sensory signals attracting yucca moths to their host plants, but no bioactive compounds have yet been identified. In this study, we showed that both sexes of the pollinator moth Tegeticula yuccasella are attracted to the floral scent of the host Yucca filamentosa. Chemical analysis of the floral headspace from six Yucca species in sections Chaenocarpa and Sarcocarpa revealed a set of novel tetranorsesquiterpenoids putatively derived from (E)-4,8-dimethyl-1,3,7-nonatriene. Their structure elucidation was accomplished by NMR analysis of the crude floral scent sample of Yucca treculeana along with GC/MS analysis and confirmed by total synthesis. Since all these volatiles are included in the floral scent of Y. filamentosa, which has been an important model species for understanding the pollination mutualism, we name these compounds filamentolide, filamentol, filamental, and filamentone. Several of these compounds elicited antennal responses in pollinating (Tegeticula) and non-pollinating (Prodoxus) moth species upon stimulation in electrophysiological recordings. In addition, synthetic (Z)-filamentolide attracted significant numbers of both sexes of two associated Prodoxus species in a field trapping experiment. Highly specialized insect-plant interactions, such as obligate pollination mutualisms, are predicted to be maintained through "private channels" dictated by specific compounds. The identification of novel bioactive tetranorsesquiterpenoids is a first step in testing such a hypothesis in the Yucca-yucca moth interaction.

Species-Specific Induction of Plant Volatiles by Two Aphid Species in Apple: Real Time Measurement of Plant Emission and Attraction of Lacewings in the Wind Tunnel.

Upon damage by herbivores, plants release herbivory-induced plant volatiles (HIPVs). To find their prey, the pest's natural enemies need to be fine-tuned to the composition of these volatiles. Whereas standard methods can be used in the identification and quantitation of HIPVs, more recently introduced techniques such as PTR-ToF-MS provide temporal patterns of the volatile release and detect additional compounds. In this study, we compared the volatile profile of apple trees infested with two aphid species, the green apple aphid Aphis pomi, and the rosy apple aphid Dysaphis plantaginea, by CLSA-GC-MS complemented by PTR-ToF-MS. Compounds commonly released in conjunction with both species include nonanal, decanal, methyl salicylate, geranyl acetone, (Z)-3-hexenyl acetate, (Z)-3-hexenyl butanoate, (Z)-3-hexenyl 2-methyl-butanoate, (E)-ß-caryophyllene, ß-bourbonene and (Z)-3-hexenyl benzoate. In addition, benzaldehyde and (E)-ß-farnesene were exclusively associated with A. pomi, whereas linalool, (E)-4,8-dimethyl-1,3,7-nonatriene were exclusively associated with D. plantaginea. PTR-ToF-MS additionally detected acetic acid (AA) and 2-phenylethanol (PET) in the blends of both trees attacked by aphid species. In the wind tunnel, the aphid predator, Chrysoperla carnea (Stephens), responded strongly to a blend of AA and PET, much stronger than to AA or PET alone. The addition of common and species-specific HIPVs did not increase the response to the binary blend of AA and PET. In our setup, two host-associated volatiles AA + PET appeared sufficient in the attraction of C. carnea. Our results also show the importance of combining complementary methods to decipher the odor profile associated with plants under pest attack and identify behaviourally active components for predators.

Remodeling the epigenome and (epi)cytoskeleton: a new paradigm for co-regulation by methylation.

The epigenome determines heritable patterns of gene expression in the absence of changes in DNA sequence. The result is programming of different cellular-, tissue- and organ-specific phenotypes from a single organismic genome. Epigenetic marks that comprise the epigenome (e.g. methylation) are placed upon or removed from chromatin (histones and DNA) to direct the activity of effectors that regulate gene expression and chromatin structure. Recently, the cytoskeleton has been identified as a second target for the cell's epigenetic machinery. Several epigenetic 'readers, writers and erasers' that remodel chromatin have been discovered to also remodel the cytoskeleton, regulating structure and function of microtubules and actin filaments. This points to an emerging paradigm for dual-function remodelers with 'chromatocytoskeletal' activity that can integrate cytoplasmic and nuclear functions. For example, the SET domain-containing 2 methyltransferase (SETD2) has chromatocytoskeletal activity, methylating both histones and microtubules. The SETD2 methyl mark on chromatin is required for efficient DNA repair, and its microtubule methyl mark is required for proper chromosome segregation during mitosis. This unexpected convergence of SETD2 activity on histones and microtubules to maintain genomic stability suggests the intriguing possibility of an expanded role in the cell for chromatocytoskeletal proteins that read, write and erase methyl marks on the cytoskeleton as well as chromatin. Coordinated use of methyl marks to remodel both the epigenome and the (epi)cytoskeleton opens the possibility for integrated regulation (which we refer to as 'epiregulation') of other higher-level functions, such as muscle contraction or learning and memory, and could even have evolutionary implications.

Synergistic Effects of Volatiles from Host-Infested Plants on Host-Searching Behavior in the Parasitoid Wasp Lytopylus rufipes (Hymenoptera: Braconidae).

Herbivore-induced plant volatiles (HIPVs) are important cues for natural enemies to find their hosts. HIPVs are usually present as blends and the effects of combinations of individual components are less studied. Here, we investigated plant volatiles in a tritrophic system, comprising the parasitoid wasp Lytopylus rufipes Nees (Hymenoptera: Braconidae), the Oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae), and Japanese pear, Pyrus pyrifolia 'Kosui', so as to elucidate the effects of single components and blends on wasp behaviors. Bioassays in a four-arm olfactometer, using either shoots or their isolated volatiles collected on adsorbent, revealed that female wasps preferred volatiles from host-infested shoots over those from intact shoots. Analyses identified (Z)-3-hexenyl acetate (H), linalool (L), (E)-ß-ocimene (O), (E)-3,8-dimethyl-1,4,7-nonatriene (D), and (E,E)-α-farnesene (F). Among them, only F was induced by infestation with G. molesta. When tested singly, only O and D elicited positive responses by L. rufipes. Binary blends of HO and DF elicited a positive response, but that of HD elicited a negative one, even though D alone elicited a positive response. Remarkably, wasps did not prefer either the ODF or HL blends, but showed a highest positive response to a quinary blend (HLODF). These results show that synergism among volatiles released from host-infested plants is necessary for eliciting high behavioral responses in L. rufipes, enabling L. rufipes to find its host efficiently.

A herbivore-induced homoterpene volatile is emitted from Basella alba leaves.

An irregular C11 homoterpene, (E)-4,8-dimethylnona-1,3,7-triene (DMNT) was identified as a major component of the volatile compounds emitted from Basella alba leaves induced by herbivore. The terpenes including DMNT were not detected from the leaves infected by Botrytis cinerea. These results suggested that volatile emission from B. alba leaves was induced by herbivory but not by a fungal infection.

Identification and functional analysis of two P450 enzymes of Gossypium hirsutum involved in DMNT and TMTT biosynthesis.

The homoterpenes (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) are major herbivore-induced plant volatiles that can attract predatory or parasitic arthropods to protect injured plants from herbivore attack. In this study, DMNT and TMTT were confirmed to be emitted from cotton (Gossypium hirsutum) plants infested with chewing caterpillars or sucking bugs. Two CYP genes (GhCYP82L1 and GhCYP82L2) involved in homoterpene biosynthesis in G. hirsutum were newly identified and characterized. Yeast recombinant expression and enzyme assays indicated that the two GhCYP82Ls are both responsible for the conversion of (E)-nerolidol to DMNT and (E,E)-geranyllinalool to TMTT. The two heterologously expressed proteins without cytochrome P450 reductase fail to convert the substrates to homoterpenes. Quantitative real-time PCR (qPCR) analysis suggested that the two GhCYP82L genes were significantly up-regulated in leaves and stems of G. hirsutum after herbivore attack. Subsequently, electroantennogram recordings showed that electroantennal responses of Microplitis mediator and Peristenus spretus to DMNT and TMTT were both dose dependent. Laboratory behavioural bioassays showed that females of both wasp species responded positively to DMNT and males and females of M. mediator could be attracted by TMTT. The results provide a better understanding of homoterpene biosynthesis in G. hirsutum and of the potential influence of homoterpenes on the behaviour of natural enemies, which lay a foundation to study genetically modified homoterpene biosynthesis and its possible application in agricultural pest control.

Expression of lima bean terpene synthases in rice enhances recruitment of a beneficial enemy of a major rice pest.

Volatile terpenoids play a key role in plant defence against herbivory by attracting parasitic wasps. We identified seven terpene synthase genes from lima bean, Phaseolus lunatus L. following treatment with either the elicitor alamethicin or spider mites, Tetranychus cinnabarinus. Four of the genes (Pltps2, Pltps3, Pltps4 and Pltps5) were up-regulated with their derived proteins phylogenetically clustered in the TPS-g subfamily and PlTPS3 positioned at the base of this cluster. Recombinant PlTPS3 was able to convert geranyl diphosphate and farnesyl diphosphate to linalool and (E)-nerolidol, the latter being precursor of the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Recombinant PlTPS4 showed a different substrate specificity and produced linalool and (E)-nerolidol, as well as (E,E)-geranyllinalool from geranylgeranyl diphosphate. Transgenic rice expressing Pltps3 emitted significantly more (S)-linalool and DMNT than wild-type plants, whereas transgenic rice expressing Pltps4 produced (S)-linalool, DMNT and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT). In laboratory bioassays, female Cotesia chilonis, the natural enemy of the striped rice stemborer, Chilo suppressalis, were significantly attracted to the transgenic plants and their volatiles. We further confirmed this with synthetic blends mimicking natural rice volatile composition. Our study demonstrates that the transformation of rice to produce volatile terpenoids has the potential to enhance plant indirect defence through natural enemy recruitment.

Attractiveness of Host Plant Volatile Extracts to the Asian Citrus Psyllid, Diaphorina citri, is Reduced by Terpenoids from the Non-Host Cashew.

Diaphorina citri is a vector of the bacterial causative agent of Huanglongbing (HLB = Citrus greening), a severe disease affecting citrus crops. As there is no known control for HLB, manipulating insect behaviour through deployment of semiochemicals offers a promising opportunity for protecting citrus crops. The behavioural responses of D. citri to plant volatiles, and the identity of these plant volatiles were investigated. Volatiles were collected from host plants Murraya paniculata, Citrus sinensis, C. reshni, C. limettioides, Poncirus trifoliata, and from non-host plants Psidium guajava, Mangifera indica, Anacardium occidentale. In behavioural assays, female D. citri spent more time in the arms containing volatiles from either M. paniculata or C. sinensis compared to the control arms. When D. citri was exposed to volatiles collected from A. occidentale, they preferred the control arm. Volatiles emitted from the other studied plants did not influence the foraging behaviour of D. citri. Chemical analyses of volatile extracts from C. sinensis, M. paniculata, and A. occidentale revealed the presence of the terpenoids (E)-4,8-dimethylnona-1,3,7-triene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) in higher amounts in A. occidentale. In further behavioural bioassays, female D. citri spent less time in arms containing a synthetic blend of DMNT and TMTT compared to the control arms. Female D. citri also spent less time in arms containing the synthetic blend in combination with volatile extracts from either M. paniculata or C. sinensis compared to the control arms. Results suggest that higher release of the two terpenoids by A. occidentale make this species unattractive to D. citri, and that the terpenoids could be used in reducing colonisation of citrus plants and therefore HLB infection.

Engineering DMNT emission in cotton enhances direct and indirect defense against mirid bugs.

INTRODUCTION: As an important herbivore-induced plant volatile, (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) is known for its defensive role against multiple insect pests, including attracting natural enemies. A terpene synthase (GhTPS14) and two cytochrome P450 (GhCYP82L1, GhCYP82L2) enzymes are involved in the de novo synthesis of DMNT in cotton. We conducted a study to test the potential of manipulating DMNT-synthesizing enzymes to enhance plant resistance to insects. OBJECTIVES: To manipulate DMNT emissions in cotton and generate cotton lines with increased resistance to mirid bug Apolygus lucorum. METHODS: Biosynthesis and emission of DMNT by cotton plants were altered using CRISPR/Cas9 and overexpression approaches. Dynamic headspace sampling and GC-MS analysis were used to collect, identify and quantify volatiles. The attractiveness and suitability of cotton lines against mirid bug and its parasitoid Peristenus spretus were evaluated through various assays. RESULTS: No DMNT emission was detected in knockout CAS-L1L2 line, where both GhCYP82L1 and GhCYP82L2 were knocked out. In contrast, gene-overexpressed lines released higher amounts of DMNT when infested by A. lucorum. At the flowering stage, L114 (co-overexpressing GhCYP82L1 and GhTPS14) emitted 10-15-fold higher amounts than controls. DMNT emission in overexpressed transgenic lines could be triggered by methyl jasmonate (MeJA) treatment. Apolygus lucorum and its parasitoid were far less attracted to the double edited CAS-L1L2 plants, however, co-overexpressed line L114 significantly attracted bugs and female wasps. A high dose of DMNT, comparable to the emission of L114, significantly inhibited the growth of A. lucorum, and further resulted in higher mortalities. CONCLUSION: Turning down DMNT emission attenuated the behavioral preferences of A. lucorum to cotton. Genetically modified cotton plants with elevated DMNT emission not only recruited parasitoids to enhance indirect defense, but also formed an ecological trap to kill the bugs. Therefore, manipulation of DMNT biosynthesis and emission in plants presents a promising strategy for controlling mirid bugs.

Structural modification of (3E)-4,8-dimethyl-1,3,7-nontriene enhances its ability to kill Plutella xylostella insect pests.

BACKGROUND: Plant secondary metabolites and their modified derivatives play an important role in the discovery and development of novel insecticides. The natural plant product (3E)-4,8-dimethyl-1,3,7-nontriene (DMNT) has been proven to be able to effectively repel and kill the lepidopteran insect pest Plutella xylostella. RESULTS: In this study, four oxygenated derivatives of DMNT were synthesized by allylic hydroxylation and subsequent etherification or esterification. Bioassays on P. xylostella larvae showed that the compounds DMNT-OCH3 (2), DMNT-OCy (3) and DMNT-OAc (4) were more toxic to the larvae than DMNT alone. The most pronounced effect was observed for compound 2, which showed a 22.23% increase in lethality at a concentration of 0.25 µm. Moreover, the peritrophic matrix (PM) barrier in the insect midgut was more severely damaged by compounds 2, 3 and 4 than by DMNT. The median lethal concentration (LC50 , 48 h) of compounds 2, 3 and 4 on P. xylostella was determined to be 0.98, 1.13 and 1.11 mg mL-1 , respectively, which is much lower than the commercial insecticides eucalyptol (2.89 mg mL-1 ) and thymol (2.45 mg mL-1 ). CONCLUSION: These results suggested that oxygenated DMNT derivatives offer a significantly improved killing effect over DMNT on P. xylostella. This work has provided a basis for further design, structural modification and development of DMNT as botanical insecticides. © 2023 Society of Chemical Industry.

Methylation is maintained specifically at imprinting control regions but not other DMRs associated with imprinted genes in mice bearing a mutation in the Dnmt1 intrinsically disordered domain.

Differential methylation of imprinting control regions in mammals is essential for distinguishing the parental alleles from each other and regulating their expression accordingly. To ensure parent of origin-specific expression of imprinted genes and thereby normal developmental progression, the differentially methylated states that are inherited at fertilization must be stably maintained by DNA methyltransferase 1 throughout subsequent somatic cell division. Further epigenetic modifications, such as the acquisition of secondary regions of differential methylation, are dependent on the methylation status of imprinting control regions and are important for achieving the monoallelic expression of imprinted genes, but little is known about how imprinting control regions direct the acquisition and maintenance of methylation at these secondary sites. Recent analysis has identified mutations that reduce DNA methyltransferase 1 fidelity at some genomic sequences but not at others, suggesting that it may function differently at different loci. We examined the impact of the mutant DNA methyltransferase 1 P allele on methylation at imprinting control regions as well as at secondary differentially methylated regions and non-imprinted sequences. We found that while the P allele results in a major reduction in DNA methylation levels across the mouse genome, methylation is specifically maintained at imprinting control regions but not at their corresponding secondary DMRs. This result suggests that DNA methyltransferase 1 may work differently at imprinting control regions or that there is an alternate mechanism for maintaining methylation at these critical regulatory regions and that maintenance of methylation at secondary DMRs is not solely dependent on the methylation status of the ICR.

Preference Test of Plutella xylostella Larvae upon DMNT Treatment.

We describe a method to test the preference of insects in response to (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). We use a device that includes a horizontal glass tube, two grooves (with activated carbon), air flow, rubber stoppers/tubes, transparent glass containers (optional), and a holder for the glass tube (optional). Equal amounts of activated carbon in the groove (removable) are placed at both ends to avoid air contamination. The air flow is generated by an air pump. In the closed device, different samples are placed at each end of the glass tube. The air pump at the top of the glass tube forms an air flow that converges to the middle site of the glass tube. In each test, insect larvae are located in the middle of the glass test tube. If the test samples release DMNT that can be sensed by insects, the insects will selectively move to one specific end of the glass tube. The number of insects that move to each end will be recorded for further studies. This method can also be used to test the preference of insects in response to other volatile compounds.

Volatile DMNT directly protects plants against Plutella xylostella by disrupting the peritrophic matrix barrier in insect midgut.

Insect pests negatively affect crop quality and yield; identifying new methods to protect crops against insects therefore has important agricultural applications. Our analysis of transgenic Arabidopsis thaliana plants showed that overexpression of pentacyclic triterpene synthase 1, encoding the key biosynthetic enzyme for the natural plant product (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), led to a significant resistance against a major insect pest, Plutella xylostella. DMNT treatment severely damaged the peritrophic matrix (PM), a physical barrier isolating food and pathogens from the midgut wall cells. DMNT repressed the expression of PxMucin in midgut cells, and knocking down PxMucin resulted in PM rupture and P. xylostella death. A 16S RNA survey revealed that DMNT significantly disrupted midgut microbiota populations and that midgut microbes were essential for DMNT-induced killing. Therefore, we propose that the midgut microbiota assists DMNT in killing P. xylostella. These findings may provide a novel approach for plant protection against P. xylostella.

Engineering Nicotiana tabacum for the de novo biosynthesis of DMNT to regulate orientation behavior of the parasitoid wasps Microplitis mediator.

BACKGROUND: (E)-4,8-dimethylnona-1,3,7-triene (DMNT), one of the homoterpenes, is thought to contribute to plant indirect defense against insect herbivores. DMNT-enriched plants have great application potential to regulate insect behavior in the 'push & pull' strategy of pest management. However, de novo biosynthesis of DMNT in plants without a homoterpene metabolic pathway in their wild type is still not achieved, and the role of DMNT played in these plants and their interacted insects remains unclear. RESULTS: Cytochrome P450s and terpene synthases involved in homoterpenes biosynthesis in cotton plants were employed to generate DMNT-releasing tobacco plants. Single GhTPS14 transgenic Nicotiana tabacum only emitted (E)-nerolidol, the precursor of DMNT. Transgenic tobaccos expressing single GhCYP82Ls were unable to produce DMNT or TMTT, while DMNT was detected when exogenous (E)-nerolidol was added. Compared to wild-type plants, only co-expression of GhCYP82Ls and GhTPS14 in transgenic tobaccos triggered the constitutive release of single-component DMNT. Furthermore, DMNT-emitting transgenic tobacco plants, whether infested with Helicoverpa armigera larvae or not, significantly incited orientation behavior of parasitoid wasps Microplitis mediator. CONCLUSION: Wild type N. tabacum plants have no DMNT metabolic pathway. DMNT could be de novo biosynthesized via co-expression of GhCYP82Ls and GhTPS14. What is more, the parasitoid wasp M. mediator could be recruited by DMNT-releasing transgenic tobaccos, especially by H. armigera-infested transgenic tobaccos, suggesting the potential roles of engineered N. tabacum in regulating the behavioral preference of M. mediator.

Epigenetic Reprogramming of Lineage-Committed Human Mammary Epithelial Cells Requires DNMT3A and Loss of DOT1L.

Organogenesis and tissue development occur through sequential stepwise processes leading to increased lineage restriction and loss of pluripotency. An exception to this appears in the adult human breast, where rare variant epithelial cells exhibit pluripotency and multilineage differentiation potential when removed from the signals of their native microenvironment. This phenomenon provides a unique opportunity to study mechanisms that lead to cellular reprogramming and lineage plasticity in real time. Here, we show that primary human mammary epithelial cells (HMECs) lose expression of differentiated mammary epithelial markers in a manner dependent on paracrine factors and epigenetic regulation. Furthermore, we demonstrate that HMEC reprogramming is dependent on gene silencing by the DNA methyltransferase DNMT3A and loss of histone transcriptional marks following downregulation of the methyltransferase DOT1L. These results demonstrate that lineage commitment in adult tissues is context dependent and highlight the plasticity of somatic cells when removed from their native tissue microenvironment.

[Regulation and mechanism of miR-148a on cardiomyocyte differentiation induced by 5-aza in mesenchymal stem cells].

OBJECTIVE: To investigate the expression of miR-148a in the process of myocardial differentiation of human mesenchymal stem cells (hMSCs) induced by 5-azacytidine (5-aza) and study the effects of miR-148a on myocardial differentiation of hMSCs. METHODS: The immunofluorescence analysis was used to detect the expressions of the associated mark genes of cardiac specific protein (α-MHC) in the process of myocardial differentiation of hMSCs induced by 5-aza. qRT-PCR and Western blot were used to analysis the expressions of miR-148a and DNA methyltransferase 1 (DNMT1) after myocardial differentiation of hMSCs, respectively. The expression of α-MHC after transfection with synthetic miR-148 mimics and miR-148a inhibitor was examined by Western blot. We used bioinformatics analysis to predict the potential target of miR-148a, and the dual luciferase report gene system was used to verify the predication. After co-transfected with DNMT1 shRNA and miR-148a inhibitors, hMSCs were used to explore the regulatory role and mechnism of miR-148a in the process of myocardial differentiation of hMSCs. RESULTS: α-MHC was increased significantly after induced by 5-azacytidine. miR-148a was increased significantly in cardiomyocyte differentiation of hMSCs, while the gene and protein expression levels of DNMT1 were decreased significantly in this progress (P<0.01). The expression of α-MHC was up-regulated significantly in hMSCs when miR-148a was induced into cardiomyocyte differentiation and overexpressed. Instead, downregulation of miR-148a suppressed α-MHC expression (P<0.01). Knockdown of DNMT1 blocked the role of miR-148a in differentiation of hMSCs. CONCLUSIONS: miR-148a was upregulated in cardiomyocyte differentiation of hMSCs, and miR-148a promoted myocardial differentiation of hMSCs via targeting DNMT1.

Prenatal stress decreases Bdnf expression and increases methylation of Bdnf exon IV in rats.

There is ample evidence that exposure to stress during gestation increases the risk of the offspring to develop mood disorders. Brain-derived neurotrophic factor (Bdnf) plays a critical role during neuronal development and is therefore a prime candidate to modulate neuronal signaling in adult offspring of rat dams that were stressed during gestation. In the current study, we tested the hypothesis that alterations in Bdnf expression in prenatally stressed (PNS) offspring are mediated by changes in DNA methylation in exons IV and VI of the Bdnf gene. We observed decreased Bdnf expression in the amygdala and hippocampus of prenatally stressed rats both at weaning and in adulthood. This decrease in Bdnf expression was accompanied by increased DNA methylation in Bdnf exon IV in the amygdala and hippocampus, suggesting that PNS-induced reduction in Bdnf expression may, at least in part, be mediated by increased DNA methylation of Bdnf exon IV. Expression of DNA methyltransferases (Dnmt) 1 and 3a was increased in PNS rats in the amygdala and hippocampus. Our data suggest that PNS induces decreases in Bdnf expression that may at least in part be mediated by increased DNA methylation of Bdnf exon IV.

Hypoxia: a master regulator of microRNA biogenesis and activity.

Hypoxia, or low oxygen tension, is a unique environmental stress that induces global changes in a complex regulatory network of transcription factors and signaling proteins to coordinate cellular adaptations in metabolism, proliferation, DNA repair, and apoptosis. Several lines of evidence now establish microRNAs (miRNAs), which are short noncoding RNAs that regulate gene expression through posttranscriptional mechanisms, as key elements in this response to hypoxia. Oxygen deprivation induces a distinct shift in the expression of a specific group of miRNAs, termed hypoxamirs, and emerging evidence indicates that hypoxia regulates several facets of hypoxamir transcription, maturation, and function. Transcription factors such as hypoxia-inducible factor are upregulated under conditions of low oxygen availability and directly activate the transcription of a subset of hypoxamirs. Conversely, hypoxia selectively represses other hypoxamirs through less well characterized mechanisms. In addition, oxygen deprivation has been directly implicated in epigenetic modifications such as DNA demethylation that control specific miRNA transcription. Finally, hypoxia also modulates the activity of key proteins that control posttranscriptional events in the maturation and activity of miRNAs. Collectively, these findings establish hypoxia as an important proximal regulator of miRNA biogenesis and function. It will be important for future studies to address the relative contributions of transcriptional and posttranscriptional events in the regulation of specific hypoxamirs and how such miRNAs are coordinated in order to integrate into the complex hierarchical regulatory network induced by hypoxia.