Genome-Wide Identification and Expression Pattern Profiling of the Aquaporin Gene Family in Papaya (Carica papaya L.).

Aquaporins (AQPs) are mainly responsible for the transportation of water and other small molecules such as CO2 and H2O2, and they perform diverse functions in plant growth, in development, and under stress conditions. They are also active participants in cell signal transduction in plants. However, little is known about AQP diversity, biological functions, and protein characteristics in papaya. To better understand the structure and function of CpAQPs in papaya, a total of 29 CpAQPs were identified and classified into five subfamilies. Analysis of gene structure and conserved motifs revealed that CpAQPs exhibited a degree of conservation, with some differentiation among subfamilies. The predicted interaction network showed that the PIP subfamily had the strongest protein interactions within the subfamily, while the SIP subfamily showed extensive interaction with members of the PIP, TIP, NIP, and XIP subfamilies. Furthermore, the analysis of CpAQPs' promoters revealed a large number of cis-elements participating in light, hormone, and stress responses. CpAQPs exhibited different expression patterns in various tissues and under different stress conditions. Collectively, these results provided a foundation for further functional investigations of CpAQPs in ripening, as well as leaf, flower, fruit, and seed development. They also shed light on the potential roles of CpAQP genes in response to environmental factors, offering valuable insights into their biological functions in papaya.

Differential gene expression during floral transition in pineapple.

Pineapple (Ananas comosus var. comosus) and ornamental bromeliads are commercially induced to flower by treatment with ethylene or its analogs. The apex is transformed from a vegetative to a floral meristem and shows morphological changes in 8 to 10 days, with flowers developing 8 to 10 weeks later. During eight sampling stages ranging from 6 h to 8 days after treatment, 7961 genes were found to exhibit differential expression (DE) after the application of ethylene. In the first 3 days after treatment, there was little change in ethylene synthesis or in the early stages of the ethylene response. Subsequently, three ethylene response transcription factors (ERTF) were up-regulated and the potential gene targets were predicted to be the positive flowering regulator CONSTANS-like 3 (CO), a WUSCHEL gene, two APETALA1/FRUITFULL (AP1/FUL) genes, an epidermal patterning gene, and a jasmonic acid synthesis gene. We confirm that pineapple has lost the flowering repressor FLOWERING LOCUS C. At the initial stages, the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was not significantly involved in this transition. Another WUSCHEL gene and a PHD homeobox transcription factor, though not apparent direct targets of ERTF, were up-regulated within a day of treatment, their predicted targets being the up-regulated CO, auxin response factors, SQUAMOSA, and histone H3 genes with suppression of abscisic acid response genes. The FLOWERING LOCUS T (FT), TERMINAL FLOWER (TFL), AGAMOUS-like APETELAR (AP2), and SEPETALA (SEP) increased rapidly within 2 to 3 days after ethylene treatment. Two FT genes were up-regulated at the apex and not at the leaf bases after treatment, suggesting that transport did not occur. These results indicated that the ethylene response in pineapple and possibly most bromeliads act directly to promote the vegetative to flower transition via APETALA1/FRUITFULL (AP1/FUL) and its interaction with SPL, FT, TFL, SEP, and AP2. A model based on AP2/ERTF DE and predicted DE target genes was developed to give focus to future research. The identified candidate genes are potential targets for genetic manipulation to determine their molecular role in flower transition.

The origin and evolution of sex chromosomes, revealed by sequencing of the Silene latifolia female genome.

White campion (Silene latifolia, Caryophyllaceae) was the first vascular plant where sex chromosomes were discovered. This species is a classic model for studies on plant sex chromosomes due to presence of large, clearly distinguishable X and Y chromosomes that originated de novo about 11 million years ago (mya), but lack of genomic resources for this relatively large genome (∼2.8 Gb) remains a significant hurdle. Here we report S. latifolia female genome assembly integrated with sex-specific genetic maps of this species, focusing on sex chromosomes and their evolution. The analysis reveals a highly heterogeneous recombination landscape with strong reduction in recombination rate in the central parts of all chromosomes. Recombination on the X chromosome in female meiosis primarily occurs at the very ends, and over 85% of the X chromosome length is located in a massive (∼330 Mb) gene-poor, rarely recombining pericentromeric region (Xpr). The results indicate that the non-recombining region on the Y chromosome (NRY) initially evolved in a relatively small (∼15 Mb), actively recombining region at the end of the q-arm, possibly as a result of inversion on the nascent X chromosome. The NRY expanded about 6 mya via linkage between the Xpr and the sex-determining region, which may have been caused by expanding pericentromeric recombination suppression on the X chromosome. These findings shed light on the origin of sex chromosomes in S. latifolia and yield genomic resources to assist ongoing and future investigations into sex chromosome evolution.

TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability.

For comprehensive studies of the brain structure and function, fluorescence imaging of the whole brain is essential. It requires large-scale volumetric imaging in cellular or molecular resolution, which could be quite challenging. Recent advances in tissue clearing technology (e.g. CLARITY, PACT) provide new solutions by homogenizing the refractive index of the samples to create transparency. However, it has been difficult to acquire high quality results through immunofluorescence (IF) staining on the cleared samples. To address this issue, we developed TSA-PACT, a method combining tyramide signal amplification (TSA) and PACT, to transform samples into hydrogel polymerization frameworks with covalent fluorescent biomarkers assembled. We show that TSA-PACT is able to reduce the opacity of the zebrafish brain by more than 90% with well-preserved structure. Compared to traditional method, TSA-PACT achieves approximately tenfold signal amplification and twofold improvement in signal-to-noise ratio (SNR). Moreover, both the structure and the fluorescent signal persist for at least 16 months with excellent signal retention ratio. Overall, this method improves immunofluorescence signal sensitivity, specificity and stability in the whole brain of juvenile and adult zebrafish, which is applicable for fine structural analysis, neural circuit mapping and three-dimensional cell counting.

[Cloning, expression and purification of fructose-2, 6-bisphosphatase gene CpF2KP in papaya].

Papaya, which is mainly cultivated in the southeastern region of China, is one of the four famous fruits in Lingnan. It is favored by people because of its edible and medicinal value. Fructose-6-phosphate, 2-kinase/fructose-2, 6-bisphosphatase (F2KP) is a unique bifunctional enzyme with a kinase domain and an esterase domain that catalyzes the synthesis and degradation of fructose-2, 6-bisphosphate (Fru-2, 6-P2), an important regulator of glucose metabolism in organisms. In order to study the function of the gene CpF2KP encoding the enzyme in papaya, it is particularly important to obtain the target protein. In this study, the coding sequence (CDS) of CpF2KP, with a full-length of 2 274 bp, was got from the papaya genome. The amplified sequence of full-length CDS was cloned into the vector PGEX-4T-1 which was double digested with EcoR I and BamH I. The amplified sequence was constructed into a prokaryotic expression vector by genetic recombination. After exploring the induction conditions, the results of SDS-PAGE showed that the size of the recombinant GST-CpF2KP protein was about 110 kDa. The optimum IPTG concentration and temperature for CpF2KP induction were 0.5 mmol/L and 28 ℃, respectively. The purified sin[A1] gle target protein was obtained after purifying the induced CpF2KP protein. In addition, the expression level of this gene was detected in different tissues, and showed that the gene was expressed at the highest level in seeds and the lowest in pulp. This study provides an important basis for further revealing the function of CpF2KP protein and studying the involved biological processes of this gene in papaya.

Transcriptome analysis of sugarcane reveals rapid defense response of SES208 to Xanthomonas albilineans in early infection.

BACKGROUND: Diseases are the major factor affecting the quality and yield of sugarcane during its growth and development. However, our knowledge about the factors regulating disease responses remain limited. The present study focuses on identifying genes regulating transcriptional mechanisms responsible for resistance to leaf scald caused by Xanthomonas albilineans in S. spontaneum and S. officinarum. RESULTS: After inoculation of the two sugarcane varieties SES208 (S. spontaneum) and LA Purple (S. officinarum) with Xanthomonas albilineans, SES208 exhibited significantly greater resistance to leaf scald caused by X. albilineans than did LA Purple. Using transcriptome analysis, we identified a total of 4323 and 1755 differentially expressed genes (DEGs) in inoculated samples of SES208 and LA Purple, respectively. Significantly, 262 DEGs were specifically identified in SES208 that were enriched for KEGG pathway terms such as plant-pathogen interaction, MAPK signaling pathway, and plant hormone signal transduction. Furthermore, we built a transcriptional regulatory co-expression network that specifically identified 16 and 25 hub genes in SES208 that were enriched for putative functions in plant-pathogen interactions, MAPK signaling, and plant hormone signal transduction. All of these essential genes might be significantly involved in resistance-regulating responses in SES208 after X. albilineans inoculation. In addition, we found allele-specific expression in SES208 that was associated with the resistance phenotype of SES208 when infected by X. albilineans. After infection with X. albilineans, a great number of DEGs associated with the KEGG pathways 'phenylpropanoid biosynthesis' and 'flavonoid biosynthesis' exhibited significant expression changes in SES208 compared to LA Purple that might contribute to superior leaf scald resistance in SES208. CONCLUSIONS: We provided the first systematical transcriptome map that the higher resistance of SES208 is associated with and elicited by the rapid activation of multiple clusters of defense response genes after infection by X. albilineans and not merely due to changes in the expression of genes generically associated with stress resistance. These results will serve as the foundation for further understanding of the molecular mechanisms of resistance against X. albilineans in S. spontaneum.

Preparation and characterization of astaxanthin-loaded microcapsules and its application in effervescent tablets.

BACKGROUND: Astaxanthin is a type of keto-carotene with potential health benefits. However, astaxanthin has poor solubility and stability, resulting in its low oral bio-availability. Microcapsules can be used to improve the water solubility, stability and oral bio-availability of lipophilic bioactive compounds. Effervescent tablets can further improve the stability, smell and taste of microcapsules, and are more easily accepted by consumers. RESULTS: Astaxanthin-loaded microcapsules were prepared by layer-by-layer assembly and freeze-drying technologies. Sodium caseinate and κ-carrageenan were applied as wall materials. The prepared microcapsules had good flow properties and encapsulation efficiencies (> 85%). Fourier transform infrared spectroscopy demonstrated that the mechanisms of layer-by-layer self-assembly between sodium caseinate and κ-carrageenan might be electrostatic adsorption and hydrogen bonding. The preparation process and excipients did not affect the antioxidant effect of astaxanthin. The in vitro simulated digestion study showed that microcapsules were mainly dissolved and digested in the simulated intestinal solution. Compared with its raw material, microencapsulation could improve the bio-accessibility of astaxanthin greatly. Then, astaxanthin-loaded microcapsules were incorporated into effervescent tablets by wet granulation and tablet-pressing methods. The dissolution of astaxanthin from effervescent tablets was over 90% in 2 h, which indicated a good dissolution effect. A cytotoxicity study revealed that astaxanthin loaded effervescent tablets had a good biocompatibility. Encapsulating astaxanthin-loaded microcapsules in effervescent tablets can improve its chemical stability. CONCLUSION: Effervescent tablets containing microcapsules could be used to improve the solubility, stability and bio-accessibility of lipophilic bioactive compounds. © 2022 Society of Chemical Industry.

Electroacupuncture Alleviates Neuropathic Pain through Regulating miR-206-3p Targeting BDNF after CCI.

Background: Electroacupuncture (EA) has benefits for neuropathic pain. However, the underlying mechanisms are still unknown. The current study explores the underlying mechanisms of EA in neuropathic pain of chronic constriction injury (CCI) rats. Material/Methods. Overall, 126 Sprague-Dawley (200-250 g) rats were divided into nine groups randomly: the sham-operated, CCI, CCI+EA, CCI+sham EA, CCI+NS, CCI+AAV-NC, CCI+AAV-miR-206-3p, CCI+EA+NS, and CCI+EA+AAV-miR-206-3p groups. The animals were sacrificed 14 days postsurgery. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests were used to determine differences in neurobehavioral manifestations. qPCR, western blotting, and immunofluorescence (IF) were carried out to detect the expression levels of miR-206-3p, BDNF, BAX/Bcl-2, TNF-α, and IL-6. Nissl staining was measured to observe morphological changes in neurons. Transmission electron microscopy (TEM) was employed to evaluate microscopic changes in dorsal horn synapses. Results: Hyperalgesia was reduced markedly by EA in the CCI model. The expression level of miR-206-3p was elevated, whereas the expression levels of BDNF, BAX/Bcl-2, TNF-α, and IL-6 were decreased in EA-treated CCI rats. However, a miR-206-3p inhibitor partially abrogated the analgesic effect of EA and resulted in poor behavioral performance and the BDNF, BAX/Bcl-2, TNF-α, and IL-6 expression was elevated as well. Conclusions: EA can relieve neuropathic pain by regulating the miR-206-3p/BDNF pathway, thus exerting anti-inflammatory and antiapoptotic effect.

SunUp and Sunset genomes revealed impact of particle bombardment mediated transformation and domestication history in papaya.

Transgenic papaya is widely publicized for controlling papaya ringspot virus. However, the impact of particle bombardment on the genome remains unknown. The transgenic SunUp and its progenitor Sunset genomes were assembled into 351.5 and 350.3 Mb in nine chromosomes, respectively. We identified a 1.64 Mb insertion containing three transgenic insertions in SunUp chromosome 5, consisting of 52 nuclear-plastid, 21 nuclear-mitochondrial and 1 nuclear genomic fragments. A 591.9 kb fragment in chromosome 5 was translocated into the 1.64 Mb insertion. We assembled a gapless 9.8 Mb hermaphrodite-specific region of the Yh chromosome and its 6.0 Mb X counterpart. Resequencing 86 genomes revealed three distinct groups, validating their geographic origin and breeding history. We identified 147 selective sweeps and defined the essential role of zeta-carotene desaturase in carotenoid accumulation during domestication. Our findings elucidated the impact of particle bombardment and improved our understanding of sex chromosomes and domestication to expedite papaya improvement.

Water Treadmill Training Ameliorates Neurite Outgrowth Inhibition Associated with NGR/RhoA/ROCK by Inhibiting Astrocyte Activation following Spinal Cord Injury.

Spinal cord injury (SCI) often results in damage to or degeneration of axons. Crosstalk between astrocytes and neurons plays a pivotal role in neurite outgrowth following SCI. Rehabilitative training is a recognized method for the treatment of SCI, but the specific mechanism underlying its effect on axonal outgrowth in the central nervous system (CNS) has not yet been determined. A total of 190 adult male SD rats weighing 200-250 g were randomly divided into eight groups for use as animal models of SCI. Rats were subjected to water treadmill training (TT) for 7 or 14 d. The Basso-Beattie-Bresnahan (BBB) motor function scale, hematoxylin-eosin (HE) staining, Nissl staining, Western blotting, and immunofluorescence were used to measure tissue morphology and the degree of neurological deficit and to determine quantitative expression and accurate localization of the corresponding proteins. We found that TT decreased tissue structure damage and improved functional recovery. TT also promoted the regeneration of neurons and reduced SCI-induced apoptosis SCI around the lesion, as well as significantly increasing the expression of GAP43 and NF200 after SCI. In addition, TT significantly inhibited the injury-induced increase in the expression of proinflammatory factors. Moreover, TT reduced the activation of astrocytes and microglia, accompanied by the reduced expression of C3d and increased expression of S100A10. Finally, TT effectively reduced the level of chondroitin sulfate proteoglycan (CSPG) surrounding the lesion and inhibited the NGR/RhoA/ROCK signaling pathway in neurons after SCI. Overall, we found that TT played a novel role in recovery from SCI by promoting axonal outgrowth associated with NGR/RhoA/ROCK signaling by inhibiting astrocyte activation after SCI.

Electroacupuncture suppresses spinal nerve ligation-induced neuropathic pain via regulation of synaptic plasticity through upregulation of basic fibroblast growth factor expression.

BACKGROUND: Improving synaptic plasticity is a good way to alleviate neuropathic pain. Electroacupuncture (EA) is currently used worldwide to treat this disease, but its specific mechanisms of action need further investigation. Evidence has suggested that basic fibroblast growth factor (bFGF) plays an important role in promoting nerve regeneration and can promote the expression of vascular endothelial growth factor (VEGF). OBJECTIVE: In this study, we examined the effects of EA on synaptic plasticity and its underlying mechanism. METHODS: A spinal nerve ligation (SNL) rat model was established. NSC37204 (a specific inhibitor of bFGF) was used to determine the relationship between bFGF and putative EA-mediated improvements in synaptic plasticity. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were assessed to evaluate hyperalgesia in rats with SNL. Tissue morphology was detected by hematoxylin-eosin (HE) and Nissl staining, while neural plasticity and its molecular mechanisms were examined by Western blotting, quantitative real-time polymerase chain reaction (qPCR), dual-label immunohistochemistry and transmission electron microscopy. RESULTS: We found that EA improved synaptic plasticity, consistent with higher levels of expression of bFGF and VEGF. Contrary to the beneficial effects of EA, NSC37204 promoted synaptic reconstruction. Furthermore, EA-induced improvements in the neurobehavioral state and improved synaptic plasticity were blocked by NSC37204, consistent with lower expression levels of bFGF and VEGF. CONCLUSION: These findings indicate that EA suppresses SNL-induced neuropathic pain by improving synaptic plasticity via upregulation of bFGF expression.

Methylation related genes affect sex differentiation in dioecious and gynodioecious papaya.

Morphological, genic and epigenetic differences often exist in separate sexes of dioecious and trioecious plants. However, the connections and relationships among them in different breeding systems are still unclear. Papaya has three sex types, which is genetically determined and epigenetically regulated, and was chosen as a model to study sex differentiation. Bisulfite sequencing of genomic DNA extracted from early-stage flowers revealed sex-specific genomic methylation landscapes and seasonally methylome reprogramming processes in dioecious and gynodioecious papaya grown in spring and summer. Extensive methylation of sex-determining region (SDR) was the distinguishing epigenetic characteristics of nascent XY sex chromosomes in papaya. Seasonal methylome reprogramming of early-stage flowers in both dioecy and gynodioecy systems were detected, resulting from transcriptional expression pattern alterations of methylation-modification-related and chromatin-remodeling-related genes, particularly from those genes involved in active demethylation. Genes involved in phytohormone signal transduction pathway in male flowers have played an important role in the formation of male-specific characteristics. These findings enhanced the understanding of the genetic and epigenetic contributions to sex differentiation and the complexity of sex chromosome evolution in trioecious plants.

Advances in NK cell production.

Immunotherapy based on natural killer (NK) cells is a promising approach for treating a variety of cancers. Unlike T cells, NK cells recognize target cells via a major histocompatibility complex (MHC)-independent mechanism and, without being sensitized, kill the cells directly. Several strategies for obtaining large quantities of NK cells with high purity and high cytotoxicity have been developed. These strategies include the use of cytokine-antibody fusions, feeder cells or membrane particles to stimulate the proliferation of NK cells and enhance their cytotoxicity. Various materials, including peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB), induced pluripotent stem cells (iPSCs) and NK cell lines, have been used as sources to generate NK cells for immunotherapy. Moreover, genetic modification technologies to improve the proliferation of NK cells have also been developed to enhance the functions of NK cells. Here, we summarize the recent advances in expansion strategies with or without genetic manipulation of NK cells derived from various cellular sources. We also discuss the closed, automated and GMP-controlled large-scale expansion systems used for NK cells and possible future NK cell-based immunotherapy products.

Anti-Tumor Activity of Expanded PBMC-Derived NK Cells by Feeder-Free Protocol in Ovarian Cancer.

Natural killer (NK) cells have shown great therapeutic potential against a wide range of cancers due to their pan-specific target recognition. Numerous reports indicate that NK cell immunotherapy is an effective therapeutic approach for treating hematological malignancies, but shows limited effects against solid tumors. In this study, several models of ovarian cancer (OC) were used to test the anti-cancer effects of NK cells derived from human peripheral blood mononuclear cells and expanded using a feeder cell-free expansion system (eNKs). The results show that eNKs exhibit potent inhibitory activity on tumor growth in different ovarian cancer xenograft mice (i.e., solid tumors, abdominal metastatic tumors, and ascites), importantly, in a dose-dependent manner. Moreover, adoptive transfer of eNKs resulted in significant reduction in ascites formation in OC peritoneal tumor models, and especially in reducing intraperitoneal ascites. We found that eNKs could migrate to the tumor site, retain their activity, and proliferate to maintain high cell counts in cutaneous xenograft mice. In addition, when increased the infusion with a high dose of 12 × 107 cells/mouse, Graft-versus-host disease could be induced by eNK. These data show that eNK cell immunotherapy could be a promising treatment strategy for ovarian cancers, including solid tumors and ascites.

Expression profiling of the Dof gene family under abiotic stresses in spinach.

DNA-binding with one finger (Dof) are plant-specific transcription factors involved in numerous pathways of plant development, such as abiotic stresses responses. Although genome-wide analysis of Dof genes has been performed in many species, but these genes in spinach have not been analyzed yet. We performed a genome-wide analysis and characterization of Dof gene family in spinach (Spinacia oleracea L.). Twenty-two Dof genes were identified and classified into four groups with nine subgroups, which was further corroborated by gene structure and motif analyses. Ka/Ks analysis revealed that SoDofs were subjected to purifying selection. Using cis-acting elements analysis, SoDofs were involved in plant growth and development, plant hormones, and stress responses. Expression profiling demonstrated that SoDofs expressed in leaf and inflorescence, and responded to cold, heat, and drought stresses. SoDof22 expressed the highest level in male flowers and under cold stress. These results provided a genome-wide analysis of SoDof genes, their gender- and tissue-specific expression, and response to abiotic stresses. The knowledge and resources gained from these analyses will benefit spinach improvement.

Evolutionary analysis and functional characterization of SiBRI1 as a Brassinosteroid receptor gene in foxtail millet.

Brassinosteroids (BRs) play important roles in plant growth and development. Although BR receptors have been intensively studied in Arabidopsis, those in foxtail millet remain largely unknown. Here, we show that the BR signaling function of BRASSINOSTEROID INSENSITIVE 1 (BRI1) is conserved between Arabidopsis and foxtail millet, a new model species for C4 and Panicoideae grasses. We identified four putative BR receptor genes in the foxtail millet genome: SiBRI1, SiBRI1-LIKE RECEPTOR KINASE 1 (SiBRL1), SiBRL2 and SiBRL3. Phylogenetic analysis was used to classify the BR receptors in dicots and monocots into three branches. Analysis of their expression patterns by quantitative real-time PCR (qRT-PCR) showed that these receptors were ubiquitously expressed in leaves, stems, dark-grown seedlings, roots and non-flowering spikelets. GFP fusion experiments verified that SiBRI1 localized to the cell membrane. We also explored the SiBRI1 function in Arabidopsis through complementation experiments. Ectopic overexpression of SiBRI1 in an Arabidopsis BR receptor loss-of-function mutant, bri1-116, mostly reversed the developmental defects of the mutant. When SiBRI1 was overexpressed in foxtail millet, the plants showed a drooping leaf phenotype and root development inhibition, lateral root initiation inhibition, and the expression of BR synthesis genes was inhibited. We further identified BRI1-interacting proteins by immunoprecipitation (IP)-mass spectrometry (MS). Our results not only demonstrate that SiBRI1 plays a conserved role in BR signaling in foxtail millet but also provide insight into the molecular mechanism of SiBRI1.

[Effect of electroacupuncture on angiopoietin-1 in spinal cord of rats with neuropathic pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the behavior, histomorphology and the expression of angiopoietin-1 (Angpt-1) in rats with spinal nerve injury, so as to explore its mechanism on neuropathic pain. METHODS: Forty-five male SD rats were randomly divided into sham, model and EA groups (n=15 rats in each group). Spinal nerve ligation (SNL) of the L5 lumbar vertebra was performed to establish a rat model of neuropathic pain. The rats in the EA group were given EA at "Zusanli" (ST36) and "Kunlun" (BL60) of the operation side with continuous wave at a frequency of 2 Hz and an intensity of 1.5 mA once a day, 30 minutes each time for 7 days. The sham group only exposed L5 spinal nerves without ligation. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were observed and recorded before modeling and on days 3,5,7,10,12 and 14 after modeling. L4-L6 segments of spinal cord were taken and the morphological changes of spinal dorsal horn were observed by HE staining. The changes of spinal dorsal horn nerve fiber structure were observed by silver plating staining. Angpt-1 expression was detected by Western blot and immunohistochemistry. RESULTS: Compared with the sham group, the model group had significant reductions in MWT and TWL at each time point (P<0.01); compared with the model group, the EA group had significant increases in MWT and TWL on days 10,12 and 14 after intervention (P<0.05, P<0.01). HE staining showed that in the model group, the spinal dorsal horn showed degeneration and necrosis of neurons, nuclear fixation and shrinkage, and loose surrounding tissues. The degree of tissue damage of the EA group was milder than that of the model group. The silver staining results showed the model group had obvious neuronal fibrillary tangles, while there were fewer neuronal fibrillary tangles in the EA group. Compared with the sham group, the Angpt-1 expression in the model group was significantly decreased (P<0.01), and compared with the model group, the EA group had a significant increase in the expression of Angpt-1 (P<0.01). CONCLUSION: EA can promote the recovery of nerve function in SNL rats by up-regulating Angpt-1 expression.

Auxin and cytokinin mediated regulation involved in vitro organogenesis of papaya.

In vitro organogenesis is a multistep process which is largely controlled by the balance between auxin and cytokinin. Previous studies revealed a complex network regulating in vitro organogenesis in Arabidopsis thaliana; however, our knowledge of the molecular mechanisms underlying de novo shoot formation in papaya (Carica papaya) remains limited. Here, we optimized multiple factors to achieve an efficient and reproducible protocol for the induction of papaya callus formation and shoot regeneration. Subsequently, we analyzed the dynamic transcriptome profiles of samples undergoing this process, identified 5381, 642, 4047, and 2386 differentially expressed genes (DEGs), including 447, 66, 350, and 263 encoding transcription factors (TFs), in four stage comparisons. The DEGs were mainly involved in phytohormone modulation and transduction processes, particularly for auxin and cytokinin. Of these, 21 and 7 candidate genes involved in the auxin and cytokinin pathways, respectively, had distinct expression patterns throughout in vitro organogenesis. Furthermore, we found two genes encoding key TFs, CpLBD19 and CpESR1, were sharply induced on callus induction medium and shoot induction medium, indicating these two TFs may serve as proxies for callus induction and shoot formation in papaya. We therefore report a regulatory network of auxin and cytokinin signaling in papaya according to the one previously modeled for Arabidopsis. Our comprehensive analyses provide insight into the early molecular regulation of callus initiation and shoot formation in papaya, and are useful for the further identification of the regulators governing in vitro organogenesis.

Electroacupuncture improves neuronal plasticity through the A2AR/cAMP/PKA signaling pathway in SNL rats.

Improvements in neuronal plasticity are considered to be conducive to recovery from neuropathic pain. Electroacupuncture (EA) is regarded as an effective rehabilitation method for neuropathic pain. However, the effects and potential mechanism associated with EA-induced repair of hyperesthesia are not fully understood. Evidence has suggested that the adenosine A2A receptor (A2AR) and the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway play an important role in improving neuropathic pain. Here, we examined the function of EA in promoting neuronal plasticity in spinal nerve ligation (SNL) rats. The A2AR antagonist SCH58261, A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-50-N-ethylcarboxamido adenosine HCl (CGS21680) and A2AR siRNA were used to confirm the relationship between A2AR and the cAMP/PKA pathway as well as the effects of A2AR on EA-induced improvements in neurobehavioral state and neuronal plasticity. Mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), HE staining, Western blotting, RT-PCR, immunofluorescence, enzyme-linked immunosorbent assay, Nissl staining, silver staining, Golgi-Cox staining and transmission electron microscopy were used to evaluate the changes in neurobehavioral performance, protein expression, neuronal structure and dendrites/synapses. The results showed that EA and CGS21680 improved the behavioral performance, neuronal structure and dendritic/synaptic morphology of SNL rats, consistent with higher expression levels of A2AR, cAMP and PKA. In contrast to the positive effects of EA, SCH58261 inhibited dendritic growth and promoted dendritic spine/synaptic remodeling. In addition, the EA-induced improvement in neuronal plasticity was inhibited by SCH58261 and A2AR siRNA, consistent with lower expression levels of A2AR, cAMP and PKA, and worse behavioral performance. These results indicate that EA suppresses SNL-induced neuropathic pain by improving neuronal plasticity via upregulating the A2AR/cAMP/PKA signaling pathway.

Electroacupuncture may alleviate neuropathic pain via suppressing P2X7R expression.

Neuropathic pain is a severe problem that is difficult to treat clinically. Reducing abnormal remodeling of dendritic spines/synapses and increasing the anti-inflammatory effects in the spinal cord dorsal horn are potential methods to treat this disease. Previous studies have reported that electroacupuncture (EA) could increase the pain threshold after peripheral nerve injury. However, the underlying mechanism is unclear. P2X7 receptors (P2X7R) mediate the activation of microglia and participate in the occurrence and development of neuropathic pain. We hypothesized that the effects of EA on relieving pain may be related to the downregulation of the P2X7R. Spinal nerve ligation (SNL) rats were used as a model in this experiment, and 2'(3')-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a P2X7R agonist. We found that EA treatment decreased dendritic spine density, inhibited synaptic reconstruction and reduced inflammatory response, which is consistent with the decrease in P2X7R expression as well as the improved neurobehavioral performance. In contrast to the beneficial effects of EA, BzATP enhanced abnormal remodeling of dendritic spines/synapses and inflammation. Furthermore, the EA-mediated positive effects were reversed by BzATP, which is consistent with the increased P2X7R expression. These findings indicated that EA improves neuropathic pain by reducing abnormal dendritic spine/synaptic reconstruction and inflammation via suppressing P2X7R expression.