Erratum: In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli.

This corrects the article 10.3791/65975.

Continuous peripheral electrical nerve stimulation improves cardiac function via autonomic nerve regulation in MI rats.

BACKGROUND: Peripheral electrical nerve stimulation (PENS) reportedly improves cardiac function after myocardial ischemia (MI) by rebalancing the cardiac autonomic nervous system. The dynamic and continuous influence of PENS on autonomic and cardiac function based on cardiac self-repair is not well understood. OBJECTIVES: This study aimed to explore the relationship between autonomic nervous balance and functional cardiac repair after MI and to clarify the optimal acupoint selection and time course for PENS. METHODS: The activities of the superior cervical cardiac sympathetic nerve and vagus nerve were recorded to evaluate the autonomic tone directly. The pressure-volume loop system was used for left ventricular diastolic and systolic function. Noninvasive continuous electrocardiography and echocardiography were performed to analyze heart rate, heart rate variability, and left ventricular function. The effect of continuous PENS (cPENS) or instant PENS (iPENS) on autonomic and cardiac indications was tested. RESULTS: Sympathetic nerve activity and vagus nerve activity increased as compensatory self-regulation on days 7 and 14 post-MI, followed by an imbalance of autonomic tone and cardiac dysfunction on day 28. cPENS at acupoint PC6 maintained autonomic hyperexcitability, improved myocardial systolic and diastolic abilities, and reduced myocardial fibrosis on day 28 post-MI, whereas cPENS at acupoint ST36 had a limited effect. Both iPENS at PC6 and ST36 improved the autonomic and cardiac function of rats in the cPENS groups. CONCLUSION: Rats showed autonomic fluctuations and cardiac dysfunction 28 days post-MI. cPENS produced sympathomimetic action to sustain cardiac self-compensation, but with acupoint specificity. On the basis of cPENS, iPENS evoked autonomic regulation and cardiac benefits without acupoint differentiation.

Synergistic detoxification efficiency and mechanism of triclocarban degradation by a bacterial consortium in the liver-gut-microbiota axis of zebrafish (Danio rerio).

Triclocarban (TCC), an emerging organic contaminant, poses a potential threat to human health with long-term exposure. Here, Rhodococcus rhodochrous BX2 and Pseudomonas sp. LY-1 were utilized to degrade TCC at environmental related concentrations for enhancing TCC biodegradation and investigating whether the toxicity of intermediate metabolites is lower than that of the parent compound. The results demonstrated that the bacterial consortium could degrade TCC by 82.0% within 7 days. The calculated 96 h LC50 for TCC, as well as its main degradation product 3,4-Dichloroaniline (DCA) were 0.134 mg/L and 1.318 mg/L respectively. Biodegradation also alleviated histopathological lesions induced by TCC in zebrafish liver and gut tissues. Liver transcriptome analysis revealed that biodegradation weakened differential expression of genes involved in disrupted immune regulation and lipid metabolism caused by TCC, verified through RT-qPCR analysis and measurement of related enzyme activities and protein contents. 16 S rRNA sequencing indicated that exposure to TCC led to gut microbial dysbiosis, which was efficiently improved through TCC biodegradation, resulting in decreased relative abundances of major pathogens. Overall, this study evaluated potential environmental risks associated with biodegradation of TCC and explored possible biodetoxification mechanisms, providing a theoretical foundation for efficient and harmless bioremediation of environmental pollutants.

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

Promoting high-quality development of acupuncture and moxibustion. / 推进针灸学科的高质量发展.

In the past 20 years, the acupuncture-moxibustion discipline has made a great progress in clinical research, method construction, standard formulation, guideline promotion, basic theory and key scientific issue research. Internationally, the development of acupuncture and moxibustion has gradually begun to pay more attention to the basic issues of the discipline itself from focusing on clinical evidence. The National Institute of Health of USA pays close attention to the construction of acupoint knowledge base and database and to the transformation of peripheral nerve stimulation techniques, which brings forth opportunities and challenges for the development of acupuncture-moxibustion discipline. In the present paper, we analyze the shortcomings of the current development of acupuncture and moxibustion, put forward some strategies for high-quality development in the future, and sort out the basic scientific issues to form an academic consensus. We should employ modern scientific language to express the scientific connotations of the basic theory of acupuncture and moxibustion, and build an open and self-consistent modern theoretical system. In addition, we also should attract more multidisciplinary talents to harmoniously and assiduously work together, insist on continuous innovation to open up a new situation in the transformation of basic scientific research achievements, and establish a new theoretical system of "somato-medicine" represented by acupuncture and moxibustion. In this way, we will guide the acupuncture-moxibustion discipline to make an original contribution to the modern life science and future medicine.

Perspective of Calcium Imaging Technology Applied to Acupuncture Research.

Acupuncture, a therapeutic treatment defined as the insertion of needles into the body at specific points (ie, acupoints), has growing in popularity world-wide to treat various diseases effectively, especially acute and chronic pain. In parallel, interest in the physiological mechanisms underlying acupuncture analgesia, particularly the neural mechanisms have been increasing. Over the past decades, our understanding of how the central nervous system and peripheral nervous system process signals induced by acupuncture has developed rapidly by using electrophysiological methods. However, with the development of neuroscience, electrophysiology is being challenged by calcium imaging in view field, neuron population and visualization in vivo. Owing to the outstanding spatial resolution, the novel imaging approaches provide opportunities to enrich our knowledge about the neurophysiological mechanisms of acupuncture analgesia at subcellular, cellular, and circuit levels in combination with new labeling, genetic and circuit tracing techniques. Therefore, this review will introduce the principle and the method of calcium imaging applied to acupuncture research. We will also review the current findings in pain research using calcium imaging from in vitro to in vivo experiments and discuss the potential methodological considerations in studying acupuncture analgesia.

Caffeine Impaired Acupuncture Analgesia in Inflammatory Pain by Blocking Adenosine A1 Receptor.

Caffeine consumption inhibits acupuncture analgesic effects by blocking adenosine signaling. However, existing evidence remains controversial. Hence, this study aimed to examine the adenosine A1 receptor (A1R) role in moderate-dose caffeine-induced abolishing effect on acupuncture analgesia using A1R knockout mice (A1R-/-). We assessed the role of A1R in physiological sensory perception and its interaction with caffeine by measuring mechanical and thermal pain thresholds and administering A1R and adenosine 2A receptor antagonists in wild-type (WT) and A1R-/- mice. Formalin- and complete Freund's adjuvant (CFA)-induced inflammatory pain models were recruited to explore moderate-dose caffeine effect on pain perception and acupuncture analgesia in WT and A1R-/- mice. Moreover, a C-fiber reflex electromyogram in the biceps femoris was conducted to validate the role of A1R in the caffeine-induced blockade of acupuncture analgesia. We found that A1R was dispensable for physiological sensory perception and formalin- and CFA-induced hypersensitivity. However, genetic deletion of A1R impaired the antinociceptive effect of acupuncture in A1R-/- mice under physiological or inflammatory pain conditions. Acute moderate-dose caffeine administration induced mechanical and thermal hyperalgesia under physiological conditions but not in formalin- and CFA-induced inflammatory pain. Moreover, caffeine significantly inhibited electroacupuncture (EA) analgesia in physiological and inflammatory pain in WT mice, comparable to that of A1R antagonists. Conversely, A1R deletion impaired the EA analgesic effect and decreased the caffeine-induced inhibitory effect on EA analgesia in physiological conditions and inflammatory pain. Moderate-dose caffeine administration diminished the EA-induced antinociceptive effect by blocking A1R. Overall, our study suggested that caffeine consumption should be avoided during acupuncture treatment. PERSPECTIVE: Moderate-dose caffeine injection attenuated EA-induced antinociceptive effect in formalin- and CFA-induced inflammatory pain mice models by blocking A1R. This highlights the importance of monitoring caffeine intake during acupuncture treatment.

Peripheral neural mechanism of visceral pain and acupoint sensitization in 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis model mice. / ç»“è‚ ç‚Žæ¨¡åž‹å°é¼ å† è„ç—›åŠç©´ä½æ•åŒ–çš„å¤–å‘¨ç¥žç»æœºåˆ¶æŽ¢è®¨.

OBJECTIVES: To explore the neural mechanism of visceral pain and related somatic (acupoints) sensitization by using in vivo calcium imaging of dorsal root ganglia (DRG) neurons. METHODS: Eight BALB/c mice were randomly divided into control and model groups, with 4 mice in each group. The colitis model was induced by colorectal perfusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) once daily for 7 days. Mice of the control group received colorectal perfusion of normal saline once daily for 7 days. The location and area of the somatic neurogenic inflammation (cutaneous exudation of Evans blue ï¼»EBï¼½) of the 2 groups of mice were observed after intravenous injection of EB. For pain behavioral tests, sixteen C57BL/6J mice were randomly divided into control and model groups, with 8 mice in each group, and a Von Frey filament was used to stimulate the referred somatic reactive regions in colitis mice, and the number of avoidance and paw withdraw reaction within 10 tests was recorded. For in vivo DRG calcium imaging tests, 24 Pirt-GCaMP6s transgenic mice were randomly and equally divided into control group and colitis model group. The responses of the neurons in L6 or L4 DRG to colorectal distension (CRD), lower back brushing, or mechanical stimulation at the hindpaw were observed using confocal fluorescence microscope. RESULTS: Compared with the control group, the area of EB exudation spot in the hindpaw and lower back regions was increased in the colitis model group (P<0.05), and the avoidance or paw withdraw numbers induced by Von Frey stimulation at the lower back and hindpaw were increased (P<0.01, P<0.05), indicating that colitis induced regional skin (acupoints) sensitization in the lower back and hindpaw regions. Compared with the control group, the percentage of L6 DRG neurons activated by 60 mm Hg CRD in the colitis model mice were apparently increased (P<0.01), the activated neurons mainly involved the medium-sized DRG neurons (P<0.01). In Pirt-GCaMP6s transgenic mice, following brushing the skin of the receptive field (lower back) of L6 DRG neurons, the fluorescence intensity of the brushing-activated DRG neurons and small, medium and large-sized neurons were significantly higher in the colitis model group than those in the control group (P<0.001, P<0.01, P<0.05). After brushing and clamping the skin of the right hindpaw (receptive field of L4 DRG neurons), the percentages of the activated L4 DRG neurons were obviously higher in the colitis model group than those in the control group (P<0.01, P<0.05), while there were no significant changes in the proportion of small, medium and large-sized neurons between the control and colitis model groups. CONCLUSIONS: Colitis may lead to body surface sensitization at the same and adjacent neuro-segments as well as to an increase of the number and activity of the responsive lumbar DRG neurons, among which the L6 DRG neurons at the same neuro-segment as the rectum colon showed an increase in the number of responders and intensity of calcium fluorescence signal while L4 DRG neurons at the level adjacent to the rectum colon showed an increase in the number of responders, suggesting that there may be different mechanisms of peripheral neural sensitization.

Sympathetic-Sensory Coupling as a Potential Mechanism for Acupoints Sensitization.

A series of studies have demonstrated acupoint sensitization, in which acupoints can be activated in combination with sensory hypersensitivity and functional plasticity during visceral disorders. However, the mechanisms of acupoint sensitization remain unclear. Neuroanatomy evidence showed nociceptors innervated in acupoints contribute to the mechanism of acupoint sensitization. Increasing studies suggested sympathetic nerve plays a key role in modulating sensory transmission by sprouting or coupling with sensory neuron/nociceptor in the peripheral, forming the functional structure of the sympathetic-sensory coupling. Notably, the sensory inputs of the disease-induced sensitized acupoint contribute to the homeostatic regulation and also involve in delivering therapeutic information under acupuncture, hence, the role of sprouted sympathetic in acupoint function should be given attention. We herein reviewed the current knowledge of sympathetic and its sprouting in pain modulation, then discussed and highlighted the potential value of sympathetic-sensory coupling in acupoint functional plasticity.

[Relationship between acupoint sensitization and changes in dorsal root ganglion neuron excitability in myocardial ischemia mice].

OBJECTIVE: To investigate the relationship between the sensitization state of acupoints on the surface of the myocardial ischemia (MI) model mice and the changes in the electrophysiological properties of the dorsal root ganglion (DRG) neurons in the corresponding spinal cord segment, and its underlying mechanism. METHODS: Sixty-eight male C57BL/6J mice were randomly divided into control and model groups (34 mice in each group). The model group received an intraperitoneal injection of 160 mg/kg isoproterenol (ISO) to establish the MI model, and the control group received an injection of the same dose of normal saline as the model group. After modeling for about 6 days, MI proportion was measured by HE staining to verify the pathological changes in the heart tissue. Evans blue (EB) dye was injected into the tail vein of mice to reflect the size, location, distribution, and number of exudates on the body surface. Then, whole-cell membrane currents, intrinsic excitability and membrane properties of different types of DRG neurons were evaluated by electrophysiological experiment in vitro. RESULTS: Compared with the control group, the heart size was larger, with pathological outcomes showing enlarged myocardial hypertrophy, destroyed structure of cardiomyocytes, with mononuclear cell infiltration among the cardiomyocytes in the model group. Compared with the control group, the number of EB exudation points was significantly increased (P<0.01), which were mainly concentrated in the epidermis near the T1-T5 segment of the spinal cord, "Feishu" (BL13), "Jueyinshu" (BL14) and "Xinshu" (BL15) in the model group. Compared with the control group, the rheobase and action potential amplitude (APA) of DRG medium-sized neurons were obviously decreased (P<0.01, P<0.05), while the whole-cell membrane currents, the spike numbers, the average instantaneous frequency, and the average discharge frequency were markedly increased (P<0.01). There were no significant alterations in the membrane properties and intrinsic excitability induced by depolarized currents of small-sized neurons between groups. Compared with the control group, the whole-cell membrane currents, spike numbers, and the average instantaneous frequency were significantly increased in the model group(P<0.05, P<0.01) while rheobase was significantly decreased (P<0.05) in DRG medium-sized neurons labeled with biotin and CGRP. CONCLUSION: After the mice were modeled by ISO, the DRG medium-size neurons in the T1-T5 segment of the spinal cord may mediate the sensitization of acupoints on the body surface through their different neuronal membrane properties and intrinsic excitabilities.

Silencing Rac1 and Prex1 Inhibit Epithelial-Mesenchymal Transition in Human Gastric Cancer Cells Induced by Transforming Growth Factor-ß1.

BACKGROUND/AIMS: Transforming growth factor-beta can influence tumor cells, causing epithelial-mesenchymal transition and enhancing their invasion and metastasis ability. Rac1 protein could be used as an independent tumor diagnostic marker and survival predictor. Prex1 is closely related to cell metastasis. In this study, the impact of silencing Rac1 and Prex1 on transforming growth factor-beta 1-induced epithelial-mesenchymal transition and apoptosis of human gastric cancer cells MGC-803 and MKN45 was investigated. MATERIALS AND METHODS: MGC-803 and MKN45 cells received recombinant transforming growth factor-beta 1 (rTGF-ß1) treatments at various concentrations. Cell Counting Kit-8 kit was used to determine cell viability. Rac1 and Prex1 interference vectors were transfected into the rTGF-ß1-treated MGC-803 and MKN45 cells. Cell apoptosis and migration were detected by flow cytometry and scratch test, respectively. Western blot was used to detect the epithelial-mesenchymal transition-related markers E-cadherin, N-cadherin, vimentin, and PDLIM2 expression levels. RESULTS: The rTGF-ß1 (10 ng/mL) could promote MGC-803 and MKN45 cell viability. Silencing Rac1 and Prex1 could increase E-cadherin and PDLIM2 expression, decrease N-cadherin and vimentin expression, inhibit cell viability and migration, and promote apoptosis in rTGF-ß1-treated MGC-803 and MKN45 cells. CONCLUSIONS: Silencing Rac1 and Prex1 could inhibit epithelial-mesenchymal transition, reduce cell viability and migration, and promote apoptosis in human gastric cancer cells.

The Utility of Peripherally Restricted Kappa-Opioid Receptor Agonists for Inhibiting Below-Level Pain After Spinal Cord Injury in Mice.

Pain after spinal cord injury (SCI) can be difficult to treat. Drugs that target the opioid receptor (OR) outside the central nervous system (CNS) have gained increasing interest in pain control owing to their low risk of central side effects. Asimadoline and ICI-204448 are believed to be peripherally restricted KOR agonists withlimited access to the CNS. This study examined whether they can attenuate pain hypersensitivity in mice subjected to a contusive T10 SCI. Subcutaneous (s.c.) injection of asimadoline (5, 20 mg/kg) and ICI-204448 (1, 10 mg/kg) inhibited heat hypersensitivity at both doses, but only attenuated mechanical hypersensitivity at the high dose. However, the high-dose asimadoline adversely affected animals' exploratory performance in SCI mice and caused aversion, suggesting CNS drug penetration. In contrast, high-dose ICI-204448 did not impair exploration and remained effective in reducing both mechanical and heat hypersensitivities after SCI. Accordingly, we chose to examine the potential peripheral neuronal mechanism for ICI-204448-induced pain inhibition by conducting in vivo calcium imaging of dorsal root ganglion (DRG) in Pirt-GCaMP6s+/- mice. High-dose ICI-204448 (10 mg/kg, s.c.) attenuated the increased fluorescence intensity of lumbar DRG neurons activated by a noxious pinch (400 g) stimulation in SCI mice. In conclusion, systemic administration of ICI-204448 achieved SCI pain inhibition at doses that did not induce notable side effects and attenuated DRG neuronal excitability which may partly contribute to its pain inhibition. These findings suggest that peripherally restricted KOR agonists may be useful for treating SCI pain, but the therapeutic window must be carefully examined.

[Involvement of sympathetic nerve activation in acupoint application induced improvement of gastric ulcer and motility in rats with gastric ulcer].

OBJECTIVE: To observe the effect of mustard oil application at "Liangmen" (ST21) on gastric ulcer (GU) and gastric motility and its association with the sympathetic nerve activity in rats with GU, so as to provide experimental basis for improvement of GU by acupoint application. METHODS: Thirty-nine male SD rats were randomly divided into control (n=7), model, acupoint application (AA), medication (guanethidine, an adrenergic sympathetic antagonist) and AA+medication groups (n=8 in each of the latter 4 groups). The GU model was made by applying acetic acid-immersed filter paper onto the gastric antrum. For rats of the AA and AA+medication groups, 50% mustard oil was applied to left ST21 for 10 min, once a day, for 9 consecutive days. Rats of the medication and AA+medication groups received intraperitoneal injection of guanethidine solution (40 mg/kg) beginning from the modeling day on, once a day for 10 consecutive days. The rat's body weight of each group was recorded on the 0th, 1st, 3rd, 7th and 9th day. The intragastric peristaltic wave frequency and the myoelectrical activity (frequency of slow waves, and integration of fast waves) of the gastric smooth muscle were recorded by using PowerLab data acquisition system. The gastric ulcer area was measured after the rats were executed, and histopathological changes of gastric antrum tissues (histopathological score including epithelial cell injury, submucosal edema, hemorrhagic injury, inflammatory cell infiltration score) were observed after H.E. staining. RESULTS: Compared with the control group, the body weight ratio, frequency of gastric peristaltic waves and slow wave frequency of gastric smooth muscle were significantly decreased (P<0.001, P<0.05), while the ulcer area, total histopathological score, epithelial cell injury score, submucosal edema score, hemorrhagic injury score and inflammatory cell infiltration score were significantly increased in the model group (P<0.05, P<0.001). Relevant to the model group, the AA group had a significant increase in the body weight ratio, frequency of gastric peristaltic waves, slow wave frequency, integration of fast waves (P<0.05, P<0.001), and a considerable decrease in the ulcer area, total histopathological score, epithelial cell injury score, submucosal edema score, hemorrhagic injury score and inflammatory cell infiltration score (P<0.05, P<0.001), and the medication group has a significantly decrease in the frequency of gastric peristaltic waves (P<0.05). In comparison with the AA group, the body weight ratio, frequency of gastric peristaltic waves and slow wave frequency of gastric smooth muscle in both medication and AA+medication groups, and the integration of fast waves in the medication group were obviously lower (P<0.05, P<0.001, P<0.01), whereas the levels of ulcer area, total pathological score, submucosal edema score, hemorrhagic injury score and inflammatory cell infiltration score in both medication and AA+medication groups, and the epithelial cell injury score in medication group were significantly higher (P<0.05, P<0.001). CONCLUSION: Application of mustard oil at acupoint ST21 can effectively remit GU caused by acetic acid and regulate gastric rhythmic contraction, which was mediated by sympathetic nerve.

Adrenal sympathetic nerve mediated the anti-inflammatory effect of electroacupuncture at ST25 acupoint in a rat model of sepsis.

Acupuncture plays a vital anti-inflammatory action in sepsis by activating autonomic nerve anti-inflammatory pathways, such as sympathoadrenal medullary pathway, but the mechanism remains unclear. This study aims to explore the optimum parameter of electroacupuncture (EA) stimulation in regulating the sympathoadrenal medullary pathway and evaluate EA's anti-inflammatory effect on sepsis. To determine the optimum parameter of EA at homotopic acupoint on adrenal sympathetic activity, the left adrenal sympathetic nerve firing rate evoked by different intensities of single shock electrical stimulation (ES) at ST25 in healthy male Sprague-Dawley rats were evaluated by in vivo electrophysiological recording, and the levels of norepinephrine (NE) and its metabolites normetanephrine (NMN) were also examined using mass spectrometry. To verify the role of EA at ST25 in sepsis, the rats were given an intraperitoneal injection of lipopolysaccharide (LPS) to induce sepsis model, and survival rate, clinical score, and the level of interleukin (IL)-6, IL-1ß, and IL-10 were evaluated after EA application. We observed that 3 mA is the optimal intensity for activating adrenal sympathetic nerve, which significantly elevated the level of NE in the peripheral blood. For LPS-treated rats, EA at the ST25 apparently increased the survival rate and improved the clinical score compared to the control group. Furthermore, 3 mA EA at ST25 significantly decreased pro-inflammatory cytokines IL-6 and IL-1ß and upregulated anti-inflammatory cytokine IL-10 compared to the LPS-treated group. Overall, our data suggested that 3 mA is the optimal EA intensity at ST25 to activate the sympathoadrenal medullary pathway and exert an anti-inflammatory effect in sepsis.

[Role of animal experiment in acupuncture translational medicine].

The basic biological research of acupuncture originates from clinical practice and serves clinical application. It is the fundamental idea of "acupuncture translational medicine". The animal experiment is indispensable in acupuncture translational medicine, which contributes to simplifying the complex factors affecting the clinical efficacy of acupuncture, observing the effect of the nociceptive stimuli and obtaining the invasive and off-body biomarkers. Focusing on animal experiment, the basic biological research can objectively reveal the structure and function of acupoints, obtain the parameters required by different stimulation methods and clarify the material basis which acupuncture works, so as to provide the references and evidences for acupoint combination and the selection of appropriate stimulation in clinical practice, as well as the development of medical instruments and new drugs.

Evolutionary conservation of nested MIR159 structural microRNA genes and their promoter characterization in Arabidopsis thaliana.

MicroRNAs (miRNAs) are endogenous small RNAs, that are vital for gene expression regulation in eukaryotes. Whenever a pri-miRNA precursor includes another miRNA precursor, and both of these precursors may generate independent, non-overlapping mature miRNAs, we named them nested miRNAs. However, the extent of nested miR159 structural evolutionary conservation and its promoter characterization remains unknown. In this study, the sequence alignment and phylogenetic analysis reveal that the MIR159 family is ancient, and its nested miR159 structures are evolutionary conserved in different plant species. The overexpression of ath-MIR159a, including the 1.2 kb downstream region, has no effect on rescuing the mir159ab phenotype. The promoter truncation results revealed that the 1.0 kb promoter of ath-MIR159a is sufficient for rescuing the mir159ab phenotype. The cis-regulatory elements in the ath-miR159a promoters indicated functions related to different phytohormones, abiotic stresses, and transcriptional activation. While the MybSt1 motif-containing region is not responsible for activating the regulation of the miR159a promoter. The qRT-PCR results showed that overexpression of ath-MIR159a led to high expression levels of miR159a.1-5 and miR159a.1-3 and complemented the growth defect of mir159ab via downregulation of MYB33 and MYB65. Furthermore, continuously higher expression of the miR159a.2 duplex in transgenic lines with the curly leaf phenotype indicates that miR159a.2 is functional in Arabidopsis and suggests that it is possible for a miRNA precursor to encode several regulatory small RNAs in plants. Taken together, our study demonstrates that the nested miR159 structure is evolutionary conserved and miRNA-mediated gene regulation is more complex than previously thought.

Preparation of [18F]Alkenyl Fluorides Using No-Carrier-Added [18F]AgF via Silver-Mediated Direct Radiofluorination of Alkynes.

We have developed a versatile method for the radiosynthesis of [18F]alkenyl fluorides using no-carrier-added [18F]AgF via the silver-mediated direct radiofluorination of alkynes. A variety of electron-deficient internal alkynes were compatible with this strategy and gave the desired product in good regioselectivity and RCY. This method could also be applied to the late-stage radiofluorination and showed potential in nuclear medicine development.

Effects of donor sperm on perinatal and neonatal outcomes resulting from in vitro fertilization-intracytoplasmic sperm injection and embryo transfer cycles: a retrospective cohort study.

Background: The impact of donor sperm on pregnancy outcomes is controversial. The aim of this study was to investigate whether donor sperm in in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment could reduce the rate of live births or increase the incidence of adverse pregnancy outcomes and birth defects in neonates. Methods: This single-centre, retrospective cohort study included 1,559 patients with infertility who received donor sperm at our hospital from 2015 to 2019. All the patients received fresh embryos and underwent first-cycle transfer. After propensity score matching, 4,677 controls who received their partners' sperm were matched at 1:3. Clinical pregnancy, perinatal, and neonatal outcomes were compared between the donor sperm and partner sperm groups. Results: The embryo development was better in the donor sperm group than in the partner sperm group. The high-quality embryo and available embryo rates were significantly higher in the donor sperm group (P<0.05 for both groups). The rate of high-quality embryos transferred from the donor sperm group was higher than that from the partner sperm group (P<0.05). The clinical pregnancy (62.99% vs. 59.65%; P=0.02) and live birth (54.65% vs. 51.59%; P=0.036) rates were higher in the donor sperm group. After adjusting for confounding factors, no significant difference in live birth rates was observed between the two groups (adjusted P=0.057). The low birthweight (18.21% vs. 21.39%; P=0.023) and small for gestational age (SGA) (7.60% vs. 11.97%; P<0.001) rates were lower in the donor sperm group. To exclude the effect of multiple pregnancies, we evaluated neonatal outcomes of singleton pregnancies. No significant differences were noted in preterm and very preterm birth, SGA, mean birthweight, high birthweight, and low birth weight (LBW) and very low birth weight (VLBW) rates (P>0.05 for both groups). Further, no significant between group differences were observed in the ectopic pregnancy rate, early and late spontaneous abortion rates, gestational age, rate of large for gestational age (LGA), and neonatal defects. Conclusions: Compared with partner sperm, donor sperm did not reduce live birth rate and did not increase neonatal LBW or low birth defects.

Caffeine Attenuates Electroacupuncture Effect on Pressure Pain Threshold and Tolerance in Healthy Individuals: A Randomized Controlled Trial.

Introduction: The effect of caffeine on acupuncture analgesia in humans is unclear. This study aimed to investigate whether caffeine-containing beverage intake influences the effect of electroacupuncture (EA) on static quantitative sensory testing (QST) and dynamic QST in healthy subjects. Methods: A total of 40 healthy subjects were enrolled and randomly assigned to receive coffee containing moderate doses of caffeine (coffee group) or non-caffeinated juice (juice group) for 4 weeks. The primary outcome measures were the pressure pain threshold (PPT), pressure pain tolerance (PPTo), and heat pain threshold (HPT) as static QST parameters. Numerical rating scales (NRS) of heat stimulus and nociceptive flexor reflex (RIII reflex), as parameters of dynamic QST, were also examined. EA stimulation with tolerance intensity was performed at ST36 (Zusanli)-GB34 (Yanglingquan) points at weeks 0, 2, and 4. PPT, PPTo, and HPT were detected pre- and post- EA. The NRS scores were examined pre-, during, and post-EA, and 1 min after EA was completed. The RIII reflex was examined pre- and 1-5 min post-EA. Results: At week 0, both groups showed increased PPT and PPTo and decreased NRS scores of heat stimuli and RIII reflex after EA, but HPT was not affected. After 4 weeks, the effects of EA on PPT and PPTo were attenuated in the coffee group compared to the juice group, whereas the effect of EA on the NRS scores and RIII reflex were not influenced. There was no significant difference found at week 2 for these indications. EA also did not affect the HPT in both groups at week 4. Conclusion: Moderate caffeine intake reduced the effects of EA on PPT and PPTo in healthy subjects.

[Comparison of effects of routine electroacupuncture and pre-electroacupuncture in improving lung function in acute lung injury rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) and pre-EA at"Taiyuan"(LU9) on histopathological changes, pulmonary ventilation function, pneodynamics, and contents of TNF-α, IL-1ß and IL-10 in the bronchoalveolar lavage fluid in rats with acute lung injury (ALI), in order to explore their differences of therapeutic effects. METHODS: A total of 92 male SD rats were randomly divided into control, model, routine EA and pre-EA groups, with 23 rats in each group. The ALI rat model was established by instillation of 0.5% lipopolysaccharide solution (5 mg/kg) intratracheally. EA (3 mA, 10 Hz) was applied to bilateral LU9 for 20 min, once 5 h after modeling for rats of the routine EA group, and once on the 1st, 3rd and 5th day respectively before modeling for rats of the pre-EA group. The pulmonary ventilation functions, including dynamic lung compliance (Cdyn), tidal volume (TV)，minute ventilation volume (MV), lung resistance, peak inspiratory flow (PIF), peak expiratory flow (PEF), were measured by using a small animal lung function detection system. The electromyogram (EMG) of the diaphragm muscle was recorded for observing changes of the total integral electromyogram(iEMG), single iEMG and respiration rate. Pathological changes of the lung tissue were scored after H.E. staining. The contents of TNF-α, IL-1ß and IL-10 in the bronchoalveolar lavage fluid were determined by enzyme linked immunosorbent assay. RESULTS: Compared with the control group, the Cdyn, TV, MV and PIF consi-derably decreased (P<0.000 1), and the lung resistance, total iEMG of diaphragm muscle, lung injury score, and contents of TNF-α and IL-1ß were significantly increased (P<0.05, P<0.001, P<0.01) in the model group. In comparison with the model group, the TV, MV and PIF in both routine EA and pre-EA groups,and the Cdyn, total iEMG and single iEMG in the routine EA group, as well as the IL-10 content in the pre-EA group were significantly increased (P<0.01, P<0.001, P<0.05), whereas the lung injury score, and contents of TNF-α and IL-1ß were significantly decreased in the pre-EA group (P<0.05). CONCLUSION: Both pre-EA and routine EA at LU9 can improve the lung function of ALI model rats. Pre-electroacupuncture is related to local immunoregulation, while electroacupuncture is mainly related to the improvement of pulmonary ventilation function and respiratory motility.