Treatment of postsurgical trigeminal neuralgia with Fu's subcutaneous needling therapy resulted in prompt complete relief: Two case reports.

RATIONALE: Trigeminal neuralgia is a recurrent unilateral transient electroshock-like pain. Fu's subcutaneous needling (FSN), used to treat the musculoskeletal problems, has not been reported in this field. PATIENT CONCERNS: The pain extent of case 1 had no reduction after the previous microvascular decompression, the pain of case 2 relapsed 4 years after the microvascular decompression. DIAGNOSES: Postsurgical trigeminal neuralgia. INTERVENTIONS: FSN therapy was applied on the muscles around the neck and face area, which the myofascial trigger points were palpated in these muscles. The FSN needle was inserted into the subcutaneous layer and the needle tip was pointed toward the myofascial trigger point. OUTCOMES: The following outcome measurements were observed before and after treatment, including numerical rating scale, Barrow Neurology Institute Pain Scale scores, Constant Face Pain Questionnaire scores, Brief Pain Inventory-Facial scores, Patient Global Impression of Change scores, and medication dosage. The follow-up surveys were made after 2 and 4 months respectively. The pain of Case 1 was significantly reduced after 7 times FSN treatments and the pain of Case 2 was even disappeared after 6 times FSN treatments. LESSONS: This case report suggested that FSN can relieve postsurgical trigeminal neuralgia safely and effectively. Clinical randomized controlled studies are needed to be further conducted.

[Effect of electroacupuncture on endometrial receptivity and IVF-ET pregnancy outcomes in patients with diminished ovarian reserve].

OBJECTIVE: To explore the effect of electroacupuncture on endometrial receptivity and the pregnancy outcomes of in vitro fertilization and embryo transfer (IVF-ET) in the patients with diminished ovarian reserve (DOR). METHODS: Sixty-eight patients of DOR undertaken IVF-ET were randomized into an observation group (34 cases, 2 cases dropped off) and a control group (34 cases, 1 case dropped off). In the control group, endometrial preparation was performed according to the routine protocol. In the observation group, on the base of the treatment as the control group, acupuncture was applied to Geshu (BL 17), Shenshu (BL 23), Mingmen (GV 4), Shiqizhui (EX-B 8), Ciliao (BL 32), Zhongliao (BL 33), Tianshu (ST 25), Qihai (CV 6) and Guanyuan (CV 4), etc. Electric stimulation was given at Ciliao (BL 32)-Zhongliao(BL 33), Tianshu (ST 25)-Zigong (EX-CA 1), with disperse-dense wave, 2 Hz/15 Hz in frequency and tolerable current in intensity. Electroacupuncture was given once every two days, 3 times weekly, lasing 3 menstrual cycles till 1 day before embryo transfer. The endometrial thickness and morphology were observed on the day of human chorionic gonadotropin (HCG) of egg retrieval cycle, the day of endometrial transformation in frozen-thawed embryo transfer (FET) cycle and the day of embryo transfer in both groups successively; as well as HCG positive rate, clinical pregnancy rate, embryo implantation rate and live birth rate. RESULTS: In the observation group, the proportion of type A endometrium on the embryo transfer day was higher than those on HCG day of the egg retrieval cycle and the endometrial transformation day of FET cycle (P<0.05), and also higher than that of the control group (P<0.01). In the observation group, HCG positive rate, clinical pregnancy rate, embryo implantation rate and live birth rate were 75.0% (24/32), 71.9% (23/32), 47.4% (27/57) and 56.3% (18/32) respectively, and all higher than 36.4% (12/33), 30.3% (10/33), 18.0% (11/61) and 15.2% (5/33) in the control group separately (P<0.01). CONCLUSION: Electroacupuncture improves the endometrial receptivity and IVF-ET pregnancy outcomes in the patients of diminished ovarian reserve.

Viral RNA N6-methyladenosine modification modulates both innate and adaptive immune responses of human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in humans. A well-known challenge in the development of a live attenuated RSV vaccine is that interferon (IFN)-mediated antiviral responses are strongly suppressed by RSV nonstructural proteins which, in turn, dampens the subsequent adaptive immune responses. Here, we discovered a novel strategy to enhance innate and adaptive immunity to RSV infection. Specifically, we found that recombinant RSVs deficient in viral RNA N6-methyladenosine (m6A) and RSV grown in m6A methyltransferase (METTL3)-knockdown cells induce higher expression of RIG-I, bind more efficiently to RIG-I, and enhance RIG-I ubiquitination and IRF3 phosphorylation compared to wild-type virion RNA, leading to enhanced type I IFN production. Importantly, these m6A-deficient RSV mutants also induce a stronger IFN response in vivo, are significantly attenuated, induce higher neutralizing antibody and T cell immune responses in mice and provide complete protection against RSV challenge in cotton rats. Collectively, our results demonstrate that inhibition of RSV RNA m6A methylation enhances innate immune responses which in turn promote adaptive immunity.

Nonsegmented Negative-Sense RNA Viruses Utilize N6-Methyladenosine (m6A) as a Common Strategy To Evade Host Innate Immunity.

N6-Methyladenosine (m6A) is the most abundant internal RNA modification catalyzed by host RNA methyltransferases. As obligate intracellular parasites, many viruses acquire m6A methylation in their RNAs. However, the biological functions of viral m6A methylation are poorly understood. Here, we found that viral m6A methylation serves as a molecular marker for host innate immunity to discriminate self from nonself RNA and that this novel biological function of viral m6A methylation is universally conserved in several families in nonsegmented negative-sense (NNS) RNA viruses. Using m6A methyltransferase (METTL3) knockout cells, we produced m6A-deficient virion RNAs from the representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae and found that these m6A-deficient viral RNAs triggered significantly higher levels of type I interferon compared to the m6A-sufficient viral RNAs, in a RIG-I-dependent manner. Reconstitution of the RIG-I pathway revealed that m6A-deficient virion RNA induced higher expression of RIG-I, bound to RIG-I more efficiently, enhanced RIG-I ubiquitination, and facilitated RIG-I conformational rearrangement and oligomerization. Furthermore, the m6A binding protein YTHDF2 is essential for suppression of the type I interferon signaling pathway, including by virion RNA. Collectively, our results suggest that several families in NNS RNA viruses acquire m6A in viral RNA as a common strategy to evade host innate immunity.IMPORTANCE The nonsegmented negative-sense (NNS) RNA viruses share many common replication and gene expression strategies. There are no vaccines or antiviral drugs for many of these viruses. We found that representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae among the NNS RNA viruses acquire m6A methylation in their genome and antigenome as a means to escape recognition by host innate immunity via a RIG-I-dependent signaling pathway. Viral RNA lacking m6A methylation induces a significantly higher type I interferon response than m6A-sufficient viral RNA. In addition to uncovering m6A methylation as a common mechanism for many NNS RNA viruses to evade host innate immunity, this study discovered a novel strategy to enhance type I interferon responses, which may have important applications in vaccine development, as robust innate immunity will likely promote the subsequent adaptive immunity.

Porcine Epidemic Diarrhea Virus Deficient in RNA Cap Guanine-N-7 Methylation Is Attenuated and Induces Higher Type I and III Interferon Responses.

The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.

Integrated miRNA-seq analysis reveals the molecular mechanism underlying the effect of acupuncture on endometrial receptivity in patients undergoing fertilization: embryo transplantation.

Endometrial receptivity (ER) is the main factor affecting in vitro fertilization (IVF) and embryo transplantation. Previous studies have shown that acupuncture might be useful for increasing ER. However, the underlying microRNA (miRNA) molecular mechanisms deserve to be further elucidated. In this study, we performed small RNA sequencing of endometrial samples from infertile women who either underwent acupuncture therapy or did not. Differentially expressed microRNAs (DEmiRNAs) were identified and used to predict target genes. Then, the functional and pathway were analyzed for the target genes. Moreover, quantitative reverse transcription PCR (qRT-PCR) was performed to validate the RNA-seq results. Finally, the miRNA-gene network was conducted by Cytoscape. A total of 39 DEmiRNAs were identified between the acupuncture group and the control group. The functional enrichment analysis suggested that the target genes of the DEmiRNAs were significantly enriched in GO biological process (BP) terms associated with transcription, such as regulation of DNA-templated transcription and positive regulation of DNA-templated transcription. The pathway analysis showed that DEmiRNAs might be involved in acupuncture therapy via Endocytosis, Axon guidance, Oxytocin signaling, the Hippo pathway, and Estrogen signaling pathways. Significant downregulation of hsa-miR-449a and hsa-miR-449b-3p, and significant upregulation of hsa-miR-3135b and hsa-miR-345-3p in the RNA-seq results were validated by qRT-PCR. Besides, these four DEmiRNAs and their 34 target genes conducted a miRNA-gene network. Our results predict that hsa-miR-449a, hsa-miR-3135b and hsa-miR-345-3p may underly mechanisms by which acupuncture therapy help increase ER and promote endometrium receptivity in preparation for in vitro fertilization and embryo transplantation.

[Research progress on the mechanism of acupuncture and moxibustion in treatment of endometriosis].

The clinical and experimental researches of acupuncture and moxibustion on endometriosis during past 15 years were collected. The research progress underling the mechanism of acupuncture and moxibustion on endometriosis was summarized. By taken Chinese characters, i.e. "acupuncture-moxibustion" and "endometriosis" as well as the English words, i.e. "acupuncture therapy" and "endometriosis" as the searching words, the relevant articles were retrieved from Pubmed, Embase, Chinese China National Knowledge Infrastructure, Wanfang Database, Chongqing VIP and the China Biology Medicine Disc etc. The retrieval time was from January 2003 to October 2018. As a result, 41 articles of clinical studies and 18 articles of animal experiments were screened. Of them, 20 articles (including 18 articles of animal experiments and 2 articles of clinical trial) are related to the mechanism underling acupuncture and moxibustion on endometriosis. In term of the impacts on growth factors, enzymes and enzyme inhibitors, the mechanism of acupuncture and moxibustion was explored on the analgesia for endometriosis, the inhibition of ectopic tissue angiogenesis and invasive adhesion, as well as the regulation of immune function and endocrine. Moreover, the limitation and prospect on the current researches were proposed so as to provide the more reliable evidence for clinical treatment of endometriosis with acupuncture and moxibustion.

Whole Transcriptome Sequencing Reveals How Acupuncture and Moxibustion Increase Pregnancy Rate in Patients Undergoing In Vitro Fertilization-Embryo Transplantation.

BACKGROUND: In vitro fertilization and embryo transfer (IVF-ET) technology has been widely used in the therapy of refractory infertility. Previous studies showed that acupuncture can effectively increase the clinical pregnancy rate of IVF-ET. However, the molecular mechanism is unknown. MATERIALS AND METHODS: In this study, we performed whole transcriptome sequencing for endometrial samples from infertile women who underwent acupuncture and moxibustion therapy or not. Differentially expressed noncoding RNAs (ncRNAs) and mRNAs were identified and their functions were predicted. Besides, a competitive endogenous RNA network was constructed to further interpret the molecular mechanism of acupuncture and moxibustion therapy on infecund patients. In addition, real-time PCR was applied to validate the RNA-seq results. RESULTS: We identified 317 differentially expressed mRNAs and 82 ncRNAs in acupuncture and moxibustion therapy group compared with control group. Functional enrichment analysis suggested that these genes were significantly enriched in GO-BP terms associated with cellular transport, such as ATP hydrolysis coupled proton transport, vacuolar acidification, transferrin transport, and proton transport and metabolic process, including small molecule metabolic process and metabolic process. Pathway enrichment analysis enriched 11 terms, including oxidative phosphorylation, synaptic vesicle cycle, mineral absorption, and metabolic pathways. Four of five selected differentially expressed genes were validated by real-time PCR. CONCLUSION: Our results suggested that acupuncture and moxibustion therapy might increase the pregnancy rate of patients undergoing IVF-ET by the regulation of ncRNAs.

Circular RNA sequencing reveals the molecular mechanism of the effects of acupuncture and moxibustion on endometrial receptivity in patients undergoing infertility treatment.

The present study aimed to determine the profile of differentially expressed circular RNAs (circRNAs) in infertile patients treated with acupuncture and moxibustion and verify the role of acupuncture and moxibustion in altering endometrial receptivity (ER). High­throughput RNA sequencing and bioinformatics analysis of samples from six pairs of patients treated with or without acupuncture and moxibustion were conducted. The reliability of high­throughput RNA sequencing was validated using reverse transcription­quantitative PCR. The most significant circRNA functions and pathways were selected by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A circRNA­miR­mRNA interaction network was constructed to determine the connection between circRNAs, microRNAs (miRs), and mRNAs. High­throughput RNA sequencing identified 2,653 circRNAs. A total of 86 circRNAs was differentially expressed, of which 57 were upregulated and 29 were downregulated, between the acupuncture and moxibustion group and the control group. In the GO analysis, the identified BP terms were chromatin modification, positive regulation of transcription from RNA polymerase II promoter involved in unfolded protein response, oxidative DNA demethylation, regulation of transcription from RNA polymerase II promoter in response to hypoxia, and regulation of smooth muscle cell differentiation. The identified CC terms were nucleoplasm, nucleolus, nucleus, histone acetyltransferase complex, and annulate lamellae. The identified MF terms were methylcytosine dioxygenase activity, chromatin binding, zinc ion binding, histone binding, and protein binding. In the KEGG pathway analysis, the identified pathways were protein processing in endoplasmic reticulum, degradation of aromatic compounds, shigellosis, mTOR signaling pathway, bacterial invasion of epithelial cells, and prostate cancer. Circ­SFMBT2, circ­BACH1, and circ­LPAR1 were significantly upregulated (P<0.05) and associated with numerous miRs and mRNAs. Acupuncture and moxibustion could impact ER by regulating the expression of circRNAs.

De-novo characterization of the soft-shelled turtle Pelodiscus sinensis transcriptome using Illumina RNA-Seq technology.

The soft-shelled turtle Pelodiscus sinensis is a high-profile turtle species because of its nutritional and medicinal value in Asian countries. However, little is known about the genes that are involved in formation of their nutritional quality traits, especially the molecular mechanisms responsible for unsaturated fatty acid and collagen biosynthesis. In the present study, the transcriptomes from six tissues from Pelodiscus sinensis were sequenced using an Illumina paired-end sequencing platform. We obtained more than 47 million sequencing reads and 73954 unigenes with an average size of 754 bp by de-novo assembly. In total, 55.19% of the unigenes (40814) had significant similarity with proteins in the National Center of Biotechnology Information (NCBI) non-redundant protein database and Swiss-Prot database (E-value <10(-5)). Of these annotated unigenes, 9156 and 11947 unigenes were assigned to 52 gene ontology categories (GO) and 25 clusters of orthologous groups (COG), respectively. In total, 26496 (35.83%) unigenes were assigned to 242 pathways using the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). In addition, we found a number of highly expressed genes involved in the regulation of P. sinensis unsaturated fatty acid biosynthesis and collagen formation, including desaturases, growth factors, transcription factors, and extracellular matrix components. Our data represent the most comprehensive sequence resource available for the Chinese soft-shelled turtle and could provide a basis for new research on this turtle as well as the molecular genetics and functional genomics of other terrapins. To our knowledge, we report for the first time, the large-scale RNA sequencing (RNA-Seq) of terrapin animals and would enrich the knowledge of turtles for future research.