DDX21 mediates co-transcriptional RNA m6A modification to promote transcription termination and genome stability.

N6-methyladenosine (m6A) is a crucial RNA modification that regulates diverse biological processes in human cells, but its co-transcriptional deposition and functions remain poorly understood. Here, we identified the RNA helicase DDX21 with a previously unrecognized role in directing m6A modification on nascent RNA for co-transcriptional regulation. DDX21 interacts with METTL3 for co-recruitment to chromatin through its recognition of R-loops, which can be formed co-transcriptionally as nascent transcripts hybridize onto the template DNA strand. Moreover, DDX21's helicase activity is needed for METTL3-mediated m6A deposition onto nascent RNA following recruitment. At transcription termination regions, this nexus of actions promotes XRN2-mediated termination of RNAPII transcription. Disruption of any of these steps, including the loss of DDX21, METTL3, or their enzymatic activities, leads to defective termination that can induce DNA damage. Therefore, we propose that the R-loop-DDX21-METTL3 nexus forges the missing link for co-transcriptional modification of m6A, coordinating transcription termination and genome stability.

Copper-Catalyzed Three-Component Tandem Reaction of Alkynes, α-Diazo Esters, and TMSN3 to Access N-Substituted 1,2,3-Triazoles.

An efficient copper-catalyzed three-component tandem reaction of alkynes, α-diazo esters, and TMSN3 to construct triazoles has been developed. Through this strategy, a number of diverse N-substituted 1,2,3-triazoles were conveniently obtained in moderate to good yields from simple and readily available starting materials using K2CO3 as the base. The mechanism of the tandem Cu-catalyzed azide-alkyne cycloaddition (CuAAC) and Cu-carbenoid-participated C-N coupling reaction has been investigated.

Visible-Light-Initiated Multicomponent Reactions of α-Diazoesters to Access Organophosphorus Compounds.

A simple visible-light-initiated strategy has been established for the construction of organophosphorus compounds via aerobic multicomponent reaction of α-diazoesters, cyclic ethers, and P(O)H compounds under air. A number of phosphonates and phosphinates could be efficiently isolated in moderate to good yields without the use of photosensitizers and metal reagents. This multicomponent reaction has advantages of mild condition, simple operation, eco-friendly energy, good functional-group tolerance, and gram-scale synthesis.

Loss of WTAP Impairs Early Parthenogenetic Embryo Development.

m6A is one of the most common and abundant modifications of RNA molecules present in eukaryotes. The methyltransferase complex, consisting of methyltransferase-like 3 (METTL3), METTL14, and WTAP, is responsible for the m6A modification of RNA. WTAP was identified as an mRNA splicing regulator. Its role as a regulatory subunit of the m6A methyltransferase complex in embryonic development remains largely unknown. To investigate the role of WTAP in porcine early embryonic development, si-WTAP was microinjected into porcine parthenogenetic zygotes. WTAP knockdown significantly reduced the blastocyst rate and global m6A levels, but did not affect the cleavage rate. Betaine was supplemented into the in vitro culture (IVC) to increase the m6A levels. Betaine significantly increased the global m6A levels but did not affect the blastocyst rate. Furthermore, the pluripotency genes, including OCT4, SOX2, and NANOG, were downregulated following WTAP knockdown. The apoptotic genes BAX and CASPASE 3 were upregulated, while the anti-apoptotic gene BCL2 was downregulated in WTAP knockdown blastocysts. TUNEL staining revealed that the number of apoptotic cells was significantly increased following WTAP knockdown. Our study indicated that WTAP has an indispensable role in porcine early embryonic development.

Role of N6-methyl-adenosine modification in mammalian embryonic development.

N6-methyl-adenosine (m6A) methylation is one of the most common and abundant modifications of RNA molecules in eukaryotes. Although various biological roles of m6A methylation have been elucidated, its role in embryonic development is still unclear. In this review, we focused on the function and expression patterns of m6A-related genes in mammalian embryonic development and the role of m6A modification in the embryonic epigenetic reprogramming process. The modification of m6A is regulated by the combined activities of methyltransferases, demethylases, and m6A-binding proteins. m6A-related genes act synergistically to form a dynamic, reversible m6A pattern, which exists in several physiological processes in various stages of embryonic development. The lack of one of these enzymes affects embryonic m6A levels, leading to abnormal embryonic development and even death. Moreover, m6A is a positive regulator of reprogramming to pluripotency and can affect embryo reprogramming by affecting activation of the maternal-to-zygotic transition. In conclusion, m6A is involved in the regulation of gene expression during embryonic development and the metabolic processes of RNA and plays an important role in the epigenetic modification of embryos.

Hydroxyurea affects in vitro porcine oocyte maturation through increased apoptosis and oxidative stress.

Hydroxyurea (HU) is an FDA-approved drug used to treat a variety of diseases, especially malignancies, but is harmful to fertility. We used porcine oocytes as an experimental model to study the effect of HU during oocyte maturation. Exposure of cumulus-oocyte complexes (COCs) to 20 µM (P<0.01) and 50 µM (P<0.001) HU reduced oocyte maturation. Exposure to 20 µM HU induced approximately 1.5- and 2-fold increases in Caspase-3 (P<0.001) and P53 (P<0.01) gene expression levels in cumulus cells, respectively, increased Caspase-3 (P<0.01) and P53 (P<0.001) protein expression levels in metaphase II (MII) oocytes and increased the percentage of apoptotic cumulus cells (P<0.001). In addition, HU decreased the mitochondrial membrane potential (Δφm) (P<0.01 and P<0.001) and glutathione (GSH) levels (P<0.01 and P<0.001) of both cumulus cells and MII oocytes, while increasing their reactive oxygen species (ROS) levels (P<0.001). Following parthenogenetic activation of embryos derived from MII oocytes, exposure to 20 µM HU significantly reduced total blastocyst cell numbers (P<0.001) and increased apoptosis of blastocyst cells (P<0.001). Moreover, HU exposure reduced the rate of development of two-celled, four- to eight-celled, blastocyst, and hatching stages after parthenogenetic activation (P<0.05). Our findings indicate that exposure to 20 µM HU caused significant oxidative stress and apoptosis of MII oocytes during maturation, which affected their developmental ability. These results provide valuable information for safety assessments of HU.

Procyanidin B1 promotes in vitro maturation of pig oocytes by reducing oxidative stress.

Oxidative stress negatively affects the in vitro maturation (IVM) of oocytes. Procyanidin B1 (PB1) is a natural polyphenolic compound that has antioxidant properties. In this study, we investigated the effect of PB1 supplementation during IVM of porcine oocytes. Treatment with 100 µM PB1 significantly increased the MII oocytes rate (p <0.05), the parthenogenetic (PA) blastocyst rate (p <0.01) and the total cell number in the PA blastocyst (p < 0.01) which were cultured in regular in vitro culture (IVC) medium. The PA blastocyst rate of regular MII oocytes activated and cultured in IVC medium supplemented with 100 and 150 µM PB1 significantly increased compared with control (p < 0.01 and p < 0.05). We also evaluated the reactive oxygen species (ROS) levels, mitochondrial membrane potential (Δψm) levels, glutathione (GSH) levels, and apoptotic levels in MII oocytes and cumulus cells following 100 µM PB1 treatment. The results showed that the PB1 supplementation decreased ROS production and apoptotic levels. In addition, PB1 was found to increase Δψm levels and GSH levels. In conclusion, PB1 inhibited apoptosis of oocytes and cumulus cells by reducing oxidative stress. Moreover, PB1 improved the quality of oocytes and promoted PA embryo development. Taken together, our results suggest that PB1 is a promising antioxidant additive for IVM of oocytes.

Targeted point mutations of the m6A modification in miR675 using RNA-guided base editing induce cell apoptosis.

Methylation of the adenine base at the nitrogen 6 position (m6A) is the most common post-transcriptional epigenetic modification of RNA, and it plays a very important role in regulating gene expression. To investigate the role of m6A methylation in the expression of non-coding RNA and miRNA, we used a system of adenine base editors (ABEs). Here, we mutated regions up- and downstream of miRNA 675 m6A modification sites in the H19 locus using HEK293T, L02, MHCC97L, MHCC97H, A549, and SGC-7901 cells. Our results showed that a T-A base transversion had occurred in all cell lines. Moreover, mutation of the regions upstream of the miRNA 675 m6A modification site led to reduced expression of H19 and the induction of cell apoptosis in HEK293T cells. To further confirm our results, L02 and MHCC97L cells were detected using ABEs system. The results indicated increased cell apoptosis and reduced expression of miR675 as well as H19. To confirm the relationship between H19 and miR675 expression, overexpression and knockdown studies were performed. The results showed that reduced HI9 expression induced cell apoptosis through miR675. Taken together, these results indicate that m6A modification can regulate the expression of H19 and miR675 which induce cell apoptosis.

microRNA-670 modulates Igf2bp1 expression to regulate RNA methylation in parthenogenetic mouse embryonic development.

Aberrant epigenetic modification, including N6-methylation of adenosine (m6A), has been frequently reported in embryos derived from parthenogenetic activation (PA). However, the role of Igf2bp1 expression pattern in m6A modification and the mechanism through which Igf2bp1 function is regulated in PA embryos remains elusive. Therefore, in this study, using si-Igf2bp1 and betaine (N,N,N-trimethylglycine, a major methyl donor), we investigated the effect of Igf2bp1 expression in m6A modification on the development of PA embryos. The results indicated that the down-regulation of Igf2bp1 reduced the cleavage and blastula rates of PA embryos. Moreover, m6A expression level was markedly down-regulated following microinjection with si-Igf2bp1. However, the treatment with betaine could significantly restore the m6A level. Further bioinformatics analysis revealed Igf2bp1 as the putative target of microRNA 670 (miR-670). Thus, to confirm this finding, mimics and inhibitor of miR-670 were microinjected into PA embryos. The results demonstrated that miR-670 inhibitor augmented the expression of Igf2bp1 and rescued cleavage and blastula rates. In addition, the miR-670 inhibitor promoted the m6A expression level. TUNEL assay revealed a loss of expression of Igf2bp1 induced cell apoptosis in PA embryos. Taken together, these results demonstrated that miR-670-3p functions as the regulator of Igf2bp1 expression and plays a crucial role in PA development through m6A modification.

ssc-miR-204 regulates porcine preadipocyte differentiation and apoptosis by targeting TGFBR1 and TGFBR2.

MicroRNAs (miRNAs) take part in a variety of biological processes by regulating target genes. Transforming growth factor ß receptor 1 (TGFBR1) and TGFBR2 are crucial members of the TGF-ß family and are serine/threonine kinase receptors. The aim of this study was to explore the functions of ssc-miR-204 in porcine preadipocyte differentiation and apoptosis with regard to the TGFß/Smad pathway. We identified miRNAs predicted to target TGFBR1 and TGFBR2 using a database and selected ssc-miR-204 as a candidate miRNA. ssc-miR-204 overexpression dramatically reduced the levels of TGFBR1 and TGFBR2. However, after transfection with ssc-miR-204 inhibitor, TGFBR1 and TGFBR2 levels were dramatically increased. ssc-miR-204 overexpression dramatically promoted porcine preadipocyte differentiation and apoptosis. After transfection with ssc-miR-204 inhibitor, porcine preadipocyte differentiation and apoptosis were dramatically inhibited. After transfection with ssc-miR-204 mimics, Smad2, Smad3, Smad4, p-Smad2, and p-Smad3 protein levels significantly decreased, and adipogenesis was regulated by inhibiting the TGF-ß/Smad3 signaling pathway. Taken together, these results verified that ssc-miR-204 regulates porcine preadipocyte differentiation and apoptosis by targeting TGFBR1 and TGFBR2.

The perturbed expression of m6A in parthenogenetic mouse embryos.

Parthenogenetically activated oocytes cannot develop to term in mammals owing to abnormal epigenetic modifications. Methylation of the N6 position of adenosine (m6A) is a post-transcriptional epigenetic modification of RNA. To investigate the role of m6A methylation in parthenogenetic (PA) embryonic development, we analyzed METTL3, METTL14, FTO, ALKBH5, YTHDF2, IGF2BP1, and IGF2BP2 expression by quantitative real-time PCR. These genes were found dynamically expressed during the 2-cell, 4-cell, 8-cell, and blastocyst stages of the embryo. Compared to normally fertilized embryos, the expression of these genes was perturbed in PA embryos, especially at the 8-cell stage. Furthermore, immunofluorescence was used to detect m6A expression. The results demonstrated that m6A expression decreased in the 2-cell stage, whereas it increased in the 8-cell stage of PA embryos. Taken together, these results suggest that the expression of RNA methylation-related genes was perturbed, leading to abnormal m6A modification during early development in PA embryos.

The expression of TET3 regulated cell proliferation in HepG2 cells.

Ten-eleven translocation (TET) proteins have been shown to be abnormally expressed in different cancers. To investigate the expression pattern of TET proteins in HepG2 cells, sodium ascorbate was used to treat HepG2 cells. Our results showed that TET1, TET2 and TET3 expression was increased after sodium ascorbate treatment. The TET proteins catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thus, 5mC and 5hmC levels were examined. The results suggested that 5hmC was increased after sodium ascorbate treatment. To further determine the biological function of the TET proteins, si-TET1, si-TET2 and si-TET3 were transfected into HepG2 cells. The results showed that a knock down of TET3 expression stimulated cell proliferation of HepG2 cells. To further understand the effects of TET3 expression on cell proliferation, sodium ascorbate was added to the cells after transfection with si-TET3. The results demonstrated that sodium ascorbate could rescue TET3 expression and inhibit cell proliferation. Taken together, these results indicate that TET3 expression regulated cell proliferation, which is associated with 5hmC in HepG2 cells.

Ascorbic acid improves parthenogenetic embryo development through TET proteins in mice.

The TET (Ten-Eleven Translocation) proteins catalyze the oxidation of 5mC (5-methylcytosine) to 5hmC (5-hydroxymethylcytosine) and play crucial roles in embryonic development. Ascorbic acid (Vc, Vitamin C) stimulates the expression of TET proteins, whereas DMOG (dimethyloxallyl glycine) inhibits TET expression. To investigate the role of TET1, TET2, and TET3 in PA (parthenogenetic) embryonic development, Vc and DMOG treatments were administered during early embryonic development. The results showed that Vc treatment increased the blastocyst rate (20.73 ± 0.46 compared with 26.57 ± 0.53%). By contrast, DMOG reduced the blastocyst rate (20.73 ± 0.46 compared with 11.18 ± 0.13%) in PA embryos. qRT-PCR (quantitative real-time PCR) and IF (immunofluorescence) staining results revealed that TET1, TET2, and TET3 expressions were significantly lower in PA embryos compared with normal fertilized (Con) embryos. Our results revealed that Vc stimulated the expression of TET proteins in PA embryos. However, treatment with DMOG significantly inhibited the expression of TET proteins. In addition, 5hmC was increased following treatment with Vc and suppressed by DMOG in PA embryos. Taken together, these results indicate that the expression of TET proteins plays crucial roles mediated by 5hmC in PA embryonic development.

DNA methylation-mediated silencing of FLT1 in parthenogenetic porcine placentas.

Studies show that perturbed expression of vascular endothelial growth factor (VEGF) family genes is related to abnormal development of parthenogenetic (PA) placenta. In this study, the methylation status of VEGF family genes were compared between PA and normal placentas using bisulfite sequencing PCR (BSP). Results showed no significant difference in the methylation of VEGF-A differentially methylated region (DMR), placental growth factor (PIGF) DMR, and kinase insert domain receptor (KDR) DMR, whereas FMS-like tyrosine kinase 1 (FLT1) DMR was hypermethylated in PA placentas. These results suggested that abnormal methylation of FLT1 DMR might trigger the developmental failure of porcine PA placentas.

[Effects of Acupuncture Stimulation of Bilateral "Hegu" (LI 4) and "Taichong" (LR 3) on Learning-memory Ability, Hippocampal AP 42 Expression and Inflammatory Cytokines in Rats with Alzheimer's Disease].

OBJECTIVE: To observe the effect of acupuncture stimulation of bilateral "Hegu" (LI 4) and "Taichong" (LR 3, the so-called "Four Gate Points") on learning-memory ability, hippocampal interleukin-1 (IL-1) P and IL-2 and amyloid beta (Abeta) 42 levels in Alzheimer's disease (AD) rats,so as to reveal its underlying mechanism in improving AD. METHODS: Male SD rats were randomly divided into sham operation, model, medication and acupuncture groups (n = 12 rats in each group). The AD model was created by microinjection of streptozotocin (10 pL, 3 mg/kg) into the lateral ventricle (repeated the microinjection once two days later). Bilateral LR 3 and LI 4 were punctured with filiform needles and stimulated manually, once a day, 6 days a week for 4 weeks. The rats of the medication group were intragastric perfusion of Donepezil HOI (0.045 mg/kg), once a day for 4 weeks. The learning-memory ability was detected by Morris water maze swimming tests. The immunoactivity of hippocampal Abeta 42 was detected by immunohistochemistry, and the contents of IL-1 P and IL-2 in the hippocampus tissue were determined by ELISA. RESULTS: After modeling, the average escape latency of Morris water navigation task was significantly increased, and the target-platform crossing times of space probe trials were significantly reduced in the model group (P<0.05), suggesting a g-memory ability. After acupuncture intervention, the increased escape latency and the decreased target-platform crossing times were reversed, suggesting an improvement of the learning-memory. The hippocampal Abeta 42 immunoactivity and IL-1 beta content were significantly higher in the model group than in the sham operation group (P<0.05), but the hippocampal IL-2 content was markedly decreased in the model group (P<0. 05). Following the interventions, the increased Abeta 42 expression and IL-1 beta contents, and the decreased IL-2 contents in the hippocampus were also reversed in both the acupuncture and medication groups (P<0.05). CONCLUSION: Acupuncture may improve learning-memory ability in AD rats, which may be associated with its effects in reducing hippocampal Abeta13 42 expression and IL-1beta content and in up-regulating IL-2 level.

[Intervention of electroacupuncture for patients with impaired glucose tolerance].

OBJECTIVE: To explore the regulation on 2-hour postprandial blood glucose (2h PBG) for patients with impaired glucose tolerance (IGT) in the intervention with electroacupuncture. METHODS: Sixty cases of IGT were divided randomly into an electroacupuncture group and a blank control group, 30 cases in each one. In electroacupuncture group, electroacupuncture was applied to Shenshu (BL 23), Pishu (BL 20), Zusanli (ST 36) and Sanyinjiao (SP 6) in the intervention, lasting for 6 sessions. In blank control group, no any intervention was adopted. The levels of fasting blood-glucose (FBG), 2 h PBG with 75 g glucose and hemoglobin Alc (HbAlc) were observed before and after the intervention for the patients in electroacupuncture group, as well as in blank con trol group. RESULTS: The total effective rate was 76.7% (23/30) in electroacupuncture group, which was superior to that of 16.7% (5/30) in blank control group (P < 0.01). 2h PBG [(7.08 +/- 0.74) mmol/L] and HbAlc [(5.74 +/- 0.35)%] were down-regulated significantly after the intervention with electroacupuncture of 6 sessions in electroacupuncture group. The therapeutic effects of it were superior remarkably to those [(8.93 +/- 1.87) mmol/L, (5.97 +/- 0.591)%] in blank control group, indicating statistical significant difference (P < 0.01, P < 0.05). But, there was no any impact for the patients with normal FBG. CONCLUSION: Electroacupuncture may be the means to achieve the clinical effective intervention for the people with IGT and the approach in the prevention and treatment of diabetes at the early stage.

[Effects of different frequencies of electroacupuncture on blood glucose level in impaired glucose tolerance patients].

OBJECTIVE: To Investigate the clinical efficacy of electroacupuncture (EA) at different frequencies for patients with impaired glucose tolerance (IGT). METHODS: A total of 120 IGT outpatients were randomly divided into control, EA-5 Hz, EA-50 Hz, and EA-100 Hz groups (n = 30/group). EA (1 mA) was applied to bilateral Pishu (BL 20), Shenshu (BL23), Zusanli (ST 36) and Sanyinjiao (SP6) for 20 min, once daily for 60 sessions. Body mass index (BMI), fasting blood glucose (FBG) and 2-hour post-prandial blood glucose (2 h PBG) contents were detected by using BAYER Blood Sugar Analyzer and glycosylated haemoglobin (HbA1c) content was detected by enzymatic assay. RESULTS: Following the treatment, both HbA1c and 2 h PBG levels in the EA-5 Hz group were significantly lower than those of the control group and those of pre-treatment in the same one group (P < 0.05, P < 0.01). No significant differences were found between the EA-5 Hz and control groups, between pre-treatment and post-treatment in the EA-5 Hz group in BMI and FBG levels; between the EA-50 Hz and control groups, between the EA-100 Hz and control groups, and between pre-treatment and post-treatment in the EA-50 Hz and EA-100 Hz groups in BMI, FBG,2 h PBG and HbA1c levels (P > 0.05). CONCLUSION: Lower frequency EA of BL 20, BL 23, ST 36 and SP 9 can reduce HbA1c and 2 h PBG levels in IGT patients, suggesting a helpful effect of EA in controlling the development of diabetes.

[Clinical observation on acupuncture for treatment of reflux esophagitis of heat stagnation of liver and stomach type].

OBJECTIVE: To observe the clinical therapeutic effect of acupuncture for treatment of reflux esophagitis of heat stagnation of liver and stomach type. METHODS: Sixty-one cases were randomly divided into an acupuncture group (31 cases) and a medication group (30 cases). The acupuncture group was treated with needles at Zusanli (ST 36), Zhongwan (CV 12), Weishu (BL 21) and Neiguan (PC 6) mainly, once a day; and the medication group was treated with oral administration of 20 mg Omeprazole, once a day. The scores of clincial symptoms, comprehensive therapeutic effect, results of gastroscopy and pathology as well as recurrence rate etc. were observed before and after treatment. RESULTS: After treatment, the scores of symptoms significantly decreased in the two groups (both P < 0.01). The total effective rate of the acupuncture group was 90.3% (28/31), and 90.0% (27/30 )in the medication group, there was no statistical difference between two groups (P > 0.05); results of gastroscopy and esophageal mucosa pathology showed no statistical difference between two groups (both P > 0.05), the recurrence rate 12 weeks after treatment of 9.1% in the acupuncture group was lower than that of 42.9% in the medication grou p (P < 0.05). CONCLUSION: Acupuncture has preferable short and long-term therapeutic effects for treatment of reflux esophagitis of heat stagnation of liver and stomach type.

[Clinical study of the treatment of infant cerebral palsy with warm-reinforcing needling combined with rehabilitation training].

OBJECTIVE: To observe the therapeutic effect of warm-reinforcing needling combined with modern rehabilitation training for infant cerebral palsy (CP). METHODS: Forty cases of cerebral palsy children were randomly divided into rehabilitation (Rehab, n=19) and acupuncture (Acup) + Rehab (n=21) groups. Body acupoints used were Baihui (GV 20), Zusanli (ST 36), Quchi (LI 11), Huantiao (GB 30), Yinlingquan (GB 34), Sanyinjiao (SP 6), etc., and scalp-acupoints Zhisanzhen (the cross-point between the anterior hairline and the median line of the head, 3 cun bilateral to the crossing point), Naosanzhen [Nao-hu (GV 17), 1.5 cun bilateral to GV17], Balance Zone, Motor Zone, etc. . The treatment was conducted once every other day, with 3 months being a therapeutic course, 2 courses altogether. Rehabilitation training included physical training (PT), occupational therapy (OT) training, and speech training (ST), 5 times a week, with 3 months being a therapeutic course, 2 courses altogether. Gross motor function measure (GMFM) and comprehensive function (CF, including cognition, speech, motor, self-care and social-adaptable abilities) were evaluated. RESULTS: After the treatment, of the 19 and 21 cases in Rehab and Acup+ Rehab groups, 12 (63.16%) and 18 (85.71%) experienced marked improvement, 7 (36.84%) and 3 (14.29%) failed, with the effective rates being 63.16% and 85.71% respectively. The therapeutic effect of Acup+ Rehab group was markedly superior to that of Rehab group (P < 0.05). Self-comparison of two groups showed that the scores of CF and GMFM increased significantly in comparison with pre-treatment (P < 0.01), and the scores of F and GMFM of Acup + Rehab group were obviously higher than those of Rehab group (P < 0.01). CONCLUSION: Rehabilitation training combined with acupuncture can apparently improve CP children's motor and comprehensive functions, and the therapeutic effect of AcupWarm-reinf Rehab is evidently superior to that of simple rehabilitation training therapy.