Transcriptome analysis of the synergistic mechanisms between two strains of potato virus Y in Solanum tuberosum L.

Many viruses employ a process known as superinfection exclusion (SIE) to block subsequent entry or replication of the same or closely related viruses in the cells they occupy. SIE is also referred to as Cross-protection refers to the situation where a host plant infected by a mild strain of a virus or viroid gains immunity against a more severe strain closely related to the initial infectant. The mechanisms underlying cross-protection are not fully understood. In this study, we performed a comparative transcriptomic analysis of potato (Solanum tuberosum L.) leaves. The strains PVYN-Wi-HLJ-BDH-2 and PVYNTN-NW-INM-W-369-12 are henceforth designated as BDH and 369, respectively. In total, 806 differentially expressed genes (DEGs) were detected between the Control and JZ (preinfected with BDH and challenge with 369) treatment. Gene Ontology (GO) analysis showed that the response to external biological stimulation, signal transduction, kinase, immunity, redox pathways were significantly enriched. Among these pathways, we identified numerous differentially expressed metabolites related to virus infection. Moreover, our data also identified a small set of genes that likely play important roles in the establishment of cross-protection. Specifically, we observed significant differential expression of the A1-II gamma-like gene, elongation factor 1-alpha-like gene, and subtilisin-like protease StSBT1.7 gene, with StSBT1.7 being the most significant in our transcriptome data. These genes can stimulate the expression of defense plant genes, induce plant chemical defense, and participate in the induction of trauma and pathogenic bacteria. Our findings provided insights into the mechanisms underlying the ability of mild viruses to protect host plants against subsequent closely related virus infection in Solanum tuberosum L.

Acupuncture and Moxibustion Inhibited Intestinal Epithelial-Mesenchymal Transition in Patients with Crohn's Disease Induced by TGF- ß 1/Smad3/Snail Pathway: A Clinical Trial Study.

OBJECTIVE: To explore whether acupuncture combined with moxibustion could inhibit epithelialmesenchymal transition in Crohn's disease by affecting the transforming growth factor ß 1 (TGF- ß 1)/Smad3/Snail pathway. METHODS: Sixty-three patients with Crohn's disease were randomly divided into an observation group (31 cases) receiving moxibustion at 43 °C combined with acupuncture, and a control group (32 cases) receiving moxibustion at 37 °C combined with sham acupuncture using a random number table. Patients were treated for 12 weeks. Crohn's Disease Activity Index (CDAI) was used to evaluate disease activity. Hematoxylin-eosin staining and transmission electron microscopy were utilized to observe the morphological and ultrastructural changes. Immunohistochemistry was used to detect the expression of transforming growth factor ß 1 (TGF-ß 1), T ß R1, T ß R2, Smad3, Snail, E-cadherin and fibronectin in intestinal mucosal tissues. RESULTS: The decrease of the CDAI score, morphological and ultrastructural changes were more significant in observation group. The expression levels of TGF- ß 1, Tß R2, Smad3, and Snail in the observation group were significantly lower than those before the treatment (P<0.05 or P<0.01). After treatment, the expression levels of TGF-ß 1, TßR2, and Snail in the observation group were significantly lower than those in the control group (all P<0.05); compared with the control group, the expression of fibronectin in the observation group was significantly decreased, and the expression of E-cadherin was significantly increased (all P<0.05). CONCLUSIONS: Moxibustion at 43 °C combined with acupuncture may suppress TGF-ß 1/Smad3/Snail pathway-mediated epithelial-mesenchymal transition of intestinal epithelial cells in Crohn's disease patients by inhibiting the expression levels of TGF-ß 1, Tß R2, Smad3, and Snail. (Registration No. ChiCTR-IIR-16007751).

Potato Virus Y Strain N-Wi Offers Cross-Protection in Potato Against Strain NTN-NW by Superior Competition.

Potato virus Y (PVY) is one of the most economically important pathogens of potato. PVY exhibits different phenotypes in dissimilar potato cultivars. Previously, we observed that two recombinant isolates, PVYN-Wi-HLJ-BDH-2 (BDH) and PVYNTN-NW(SYR-II)-INM-W-369-12 (369), exhibited different virulence levels in potato cultivar Kexin 13 despite high genome sequence identity. Indeed, 369 induced severe necrosis and plant death in severe cases in Kexin 13 and severe mosaic in cultivar Yanshu 8, whereas BDH caused mainly mosaic symptoms on the plants of both cultivars. We hypothesized that preinfection of plants with BDH could cross-protect them from 369 infection, and not vice versa. Challenge inoculation, either by mechanical wounding or through grafting, with 369 on plants that were preinfected with BDH did not augment the symptom expression in both cultivars. Reverse transcription quantitative PCR analysis showed that, after challenge inoculation with 369, the titer of the isolate on BDH-preinfected plants remained at a low level (about 3 × 104 copy/µl) during the tested time course (0 h to 30 days). In contrast, in plants that were preinoculated with buffer (mock) and challenge inoculated with 369, the titer of 369 increased continuously until reaching its highest level of about 2 × 107 (Yanshu 8) and about 4 × 108 (Kexin 13) during the time course. Surprisingly, in plants that were preinfected with 369 and challenge inoculated with BDH, the accumulation of BDH reached nearly the same level as that in plants that were preinoculated with buffer and challenge inoculated with BDH. Taken together, these results suggest that PVYN-Wi mediated cross-protection against PVYNTN-NW(SYR-II) by superior competition and better fitness.

Effectiveness of herb-partitioned moxibustion combined with electroacupuncture on polycystic ovary syndrome in patients with symptom pattern of kidney deficiency and phlegm-dampne.

OBJECTIVE: To observe the effect of herb-partitioned moxibustion combined with electroacupuncture on polycystic ovary syndrome (PCOS) in patients with symptom pattern of kidney deficiency and phlegm-dampness. METHODS: Totally 62 PCOS patients who met the inclusion criteria were randomly divided into intervention group and control group. In the intervention group, acupoints were subjected to (43 ± 1) ℃ herb-partitioned moxibustion and electroacupuncture treatment. In the control group, acupoints were subjected to (37 ± 1) ℃ herb-partitioned moxibustion and sham electroacupuncture treatment. Treatment was provided 3 times per week for 12 weeks, and the treatment was stopped during menstruation. After the treatment, the clinical efficacy, the Traditional Chinese Medicine (TCM) symptom pattern score, basal body temperature (BBT) levels, menstrual cycle, and related sex hormone levels of the two groups were evaluated. RESULTS: Sixty patients were eventually included in the study (30 in the intervention group and 30 in the control group). There were not obvious between-group differences in the baseline efficacy parameters (all P > 0.05). (a) After treatment, the two groups have improved in terms of clinical efficacy, TCM symptom pattern score, menstrual cycle and, BBT (P < 0.01, < 0.05), and the intervention group was better than the control group (P < 0.01, < 0.05). (b) In the intervention group, the estradiol (E2) and P450 aromatase (P450arom) levels were increased (all P < 0.05), the testosterone (T) level was decreased (P < 0.05), and the luteotropic hormone (LH) and anti-Müllerian hormone (AMH) levels were significantly decreased (P < 0.01). In the control group, E2 level was increased (P < 0.05), and LH level was decreased (P < 0.05). After treatment, the T, LH, and AMH levels of the intervention group were decreased compared with those of the control group (all P < 0.05), and the P450arom level was increased (P < 0.05). CONCLUSIONS: Herb-partitioned moxibustion combined with electroacupuncture can effectively improve the related clinical symptoms of PCOS patients with kidney deficiency and phlegm-dampness. Herb-partitioned moxibustion combined with electroacupuncture may inhibit the overexpression of AMH to increase the expression level of P450arom in ovarian granulosa cells, thereby reconstructing the dependence of follicular development on FSH, and finally improving abnormal follicular development and hyperandrogenism in PCOS patients with kidney deficiency and phlegmdampness. TRIAL REGISTRATION NUMBER: ChiCTR1900024182.

[Professor SHI Yin's experience in treatment of obese polycystic ovary syndrome based on spleen and kidney functions].

Professor SHI Yin's clinical experience in treatment of obese polycystic ovary syndrome (PCOS) accompained with infertility with acupuncture and moxibustion in view of spleen and kidney functions is summarized. Professor SHI believes PCOS is commonly seen in obese women of childbearing age, accompained with infertility, with kidney and spleen involved in terms of the disorder location. In pathogenesis, spleen and kidney deficiency is the primary and obstruction of phlegm (damp) in collateral is the secondary. According to clinical differentiation, the main acupoints are selected from the thoroughfare and conception vessels, the kidney meridian and the spleen/stomach meridian and the corresponding acupoint prescriptions and techniques are used individually, aiming at tonifying kidney, strengthening spleen, benefiting qi, resolving phlegm, eliminating damp, resolving stasis, activating blood circulating and regulating menstruation for successful pregnancy. Professor SHI proposes the theory as "taking syndrome/pattern differentiation as the root, equal consideration of the property of acupoints and that of herbal medicine, similarity of acupoint selection with herbal prescription, as well as determination of reinforcing and reducing techniques of acupuncture and moxibustion". It fully embodies the idea of TCM as "syndrome/pattern differentiation and treatment determination" and displays the curative effect of acupoints and the techniques of acupuncture and moxibustion, being worthy of clinical reference.

Effectiveness of Electroacupuncture for Simple Obesity: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

OBJECTIVE: To evaluate the effectiveness of electroacupuncture in the treatment of simple obesity. METHODS: Randomized clinical trials concerning electroacupuncture as a treatment of simple obesity published prior to October 31, 2019, were searched in the following Chinese and English databases: Chinese National Knowledge Infrastructure (CNKI), WanFang Database, China Science and Technology Journal Database (VIP), Chinese Biomedical Literature Database (CBM), PubMed, Cochrane Library, Web of Science, and Scopus. After data collection and quality evaluation, meta-analysis was performed using RevMan 5.3 software and Stata 15.0 software. RESULTS: A total of 13 studies involving 937 patients with simple obesity were included in the meta-analysis. Results revealed that the total effective rate (RR = 1.29, 95% CI [1.13, 1.48]; P=0.0002), BMI (MD = -1.82, 95% CI [-2.21, -1.43]; P < 0.000), waist circumference (MD = -2.39, 95% CI [-3.95, -0.84]; P=0.003), hip circumference (MD = 0.31, 95% CI [-2.37, 2.99]; P=0.82), waist-hip ratio (MD = -0.05, 95% CI [-0.07, -0.03]; P < 0.00), and body fat rate (MD = -1.56, 95% CI [-2.35, -0.78]; P=0.0001) in the electroacupuncture group were superior to those in the control group. Analysis of acupoint clustering and correlation using SPSS 24.0 and Clementine 12.0 revealed the highest statistical support for acupoint groups CV12-CV4 and CV12-ST25-CV4, while ST36-CV12-ST25, SP6, and ST40-ST24-SP15-ST37-CV4 were found to be validly clustered acupoints. CONCLUSION: For treating simple obesity, electroacupuncture is superior to other interventions such as acupuncture, acupoint catgut embedding therapy, and simple lifestyle modification for improvement in body fat rate, waist circumference, and waist-hip ratio, although not hip circumference. Acupoint analysis revealed that ST25, CV12, CV4, SP6, and ST36 can form the basis for electroacupuncture therapy for the treatment of simple obesity.

[Herbal cake-partitioned moxibustion improves intestinal epithelial barrier dysfunction by suppressing TNF-α-mediated apoptosis pathway of intestinal epithelium in rats with Crohn's disease].

OBJECTIVE: To observe the effect of herbal cake-partitioned moxibustion (Moxi) on tumor necrosis factor (TNF)-α/ TNF receptor 1 (TNFR1)-associated death domain (TRADD) / Fas-associated death domain (FADD) pathway-mediated apoptosis of intestinal epithelial cells in Crohn's disease (CD) rats, so as to explore its underlying mechanisms in the treatment of CD. METHODS: Forty-eight SD male rats were randomly divided into normal, model, Moxi and medication groups (n＝12 rats in each). The CD model was established by intra-annual perfusion of 2，4，6-trinitrobenzene sulfonic acid (TNBS) solution (TNBS∶50% alcohol＝2∶1, 3 mL/kg), once every 7 days, 4 times altogether. For rats of the Moxi group, moxibustion was given to "Tianshu" (ST25) and "Qihai" (CV6), two moxa-cones every time, once daily for 10 days. For rats of the medication group, intragastric perfusion of mesalazine solution was given twice daily for 10 days. After the treatment, the colonic epithelium tissue was sampled. The epithelial cells were purified and cultured to establish an in vitro intestinal epithelial barrier, and added with TNF-α (a pro-inflammatory factor, 100 ng/mL) in the culture medium for 24 h for making an increased epithelial permeability model. The permeability of intestinal epithelial cell barrier was evaluated by detecting the fluorescence yellow transmittance of the TNF-α-incubated cell medium. Western blot was used to detect the expression levels of TNFR1, TRADD, receptor-interacting protein 1 (RIP1), FADD and zinc finger protein A20 (A20, a ubiquitination enzyme for inhibiting activation of TRADD and RIP1) of the cultured intestinal epithelium cells. The apoptosis of the TNF-α-incubated intestinal epithelial cells was detected by flow cytometry. RESULTS: After modeling and compared with the normal group, the fluorescence yellow transmittance of intestinal epithelia cells, apoptosis rate, and expression levels of TNFR1, TRADD, and RIP1 proteins were significantly increased (P<0.001, P<0.01), and the expression of A20 was significantly decreased (P<0.01) in the model group. In comparison with the model group, the fluorescence yellow transmittance of intestinal epithelial cells, the apoptosis rate and expression levels of TRADD, RIP1 and FADD were remarkably down-regulated (P<0.001, P<0.01), and the expression of A20 was significantly up-regulated (P<0.01) in both the Moxi and medication groups. CONCLUSION: Herbal cake-partitioned moxibustion may down-regulate the permeability of intestinal epithelial barrier and the apoptosis of intestinal epithelial cells by way of suppressing TNF-α-mediated cellular apoptosis pathway of intestinal epithelium in CD rats.

Genetic Diversity of Potato virus Y in Potato Production Areas in Northeast China.

In 2011-2014, ELISA or nucleic acid spot hybridization (NASH) testing for common potato viruses or Potato spindle tuber viroid (PSTVd) was performed on 500 leaf samples collected in potato fields in the northeast provinces Heilongjiang and Inner Mongolia, China. The results revealed that 38.4% (Heilongjiang) and 27.7% (Inner Mongolia) were positive for Potato virus Y (PVY). To unveil the strain composition and population structure of PVY in the region, the multiplex RT-PCR described by Chikh-Ali et al. was performed on all of the ELISA-PVY-positive samples. Of the 158 samples whose PVY strain scenarios could be determined, PVYNTN-NW-SYR-II and PVYN-Wi were the most abundant strains, occurring in 58.9 and 47.5% samples, followed by PVYNTN-NW-SYR-I (31.0%), PVYN:O (19.6%), Eu-PVYNTN (7.6%), NA-PVYN (1.3%), and PVYO (0.6%). In the 84 single-strain-infected samples, PVYN-Wi accounted for 41.7%, PVYNTN-NW-SYR-II for 40.5%, PVYNTN-NW-SYR-I for 14.3%, and PVYN:O and Eu-PVYNTN for 3.6% each. Seven isolates representing PVYNTN-NW-SYR-I (HLJ-6-1 and HLJ-9-4), PVYNTN-NW-SYR-II (INM-W-369-12 and SC-1-1-2), PVYN:O (HLJ-30-2), and PVYN-Wi (HLJ-BDH-2 and HLJ-C-429) were sequenced and analyzed molecularly. Whereas the sequence identities for isolates belonging to the same strain group were >98.5%, they fell for isolates belonging to different strain groups to 92.7-98.1% at the genome level and 96.1-98.4% at the polyprotein level. Interestingly, the exact location of the recombination events varied among isolates within a strain group. Phylogenetic analysis of all 42 full length PVY sequences from China indicated that most clustered to various recombinant groups, despite the fact that the PVY isolates were isolated from at least five host species. Pathological analysis of four isolates representing PVYN:O, PVYN-Wi, PVYNTN-NW-SYR-I, and PVYNTN-NW-SYR-II revealed that the PVYNTN-NW-SYR-II isolate incited the most severe symptoms on potato cultivar Kexin 13, followed by PVYNTN-NW-SYR-I, PVYN:O and PVYN-Wi. The PVYNTN-NW-SYR-I and PVYNTN-NW-SYR-II isolates also caused necrotic ringspots on the tubers of Kexin 13, indicating their ability to induce the potato tuber necrotic ringspot disease in potato.

[Characteristics of P1 gene of potato virus Y in Heilongjiang].

Objective: Based on different potato virus Y isolates gene sequencing, we studied the diversity of potato virus Y strains, to provide information for molecular detection, prevention and control of the virus. Methods: P1 gene of 15 samples of potato virus Y of Heilongjiang Province was cloned and then the sequences of genes were analyzed by using phylogenetic tree. Results: Samples were divided into two groups. According to a comparative analysis, 10 samples have highly conservative and homologous genes. They are the dominant population in the research area and have certain genetic distance to other domestic samples and foreign samples. In another group, 5 samples differ significantly with local dominant population in term of P1 gene. These 5 samples also have some differences and their P1 genes are close to those of other domestic samples and foreign samples. By comparing PVY strain data provided by uploaded sequences in GenBank, it found that P1 gene of test samples is similar with PVYNTN-NW strains. These 15 samples as well as other domestic samples are evolved from PVYN strains. Conclusions: The P1 gene analysis demonstrated that PVY is influenced by environment significantly and PVY of 10 samples in Heilongjiang develops local characteristics in the long-term evolution. The later 5 samples reflect that most PVY in China may be introduced by foreign cultivars. At the same time, PVY spreads through regional resource exchange and tuber transportation in China.

Evidence of timing effects on acupuncture: A functional magnetic resonance imaging study.

According to Traditional Chinese Medicine there is an optimum time to administer acupuncture at a particular acupoint. The present study used functional magnetic resonance imaging to investigate the timing effects of acupuncture at the Zusanli (ST36) acupoint. A total of 10 healthy volunteers and 10 post-stroke patients were recruited. The subjects received acupuncture stimulation at ST36 during two time periods: between 7:00 a.m. and 9:00 a.m. (the AM condition) and between 3:00 p.m. and 5:00 p.m. (the PM condition), seven days later. Blood oxygenation level-dependent signals were captured while the patient was receiving the acupuncture stimulation. The results showed a stronger activation in the AM condition than in the PM condition in both healthy and stroke subjects. The significant regions in the healthy subjects included the prefrontal cortex, cingulum, thalamus and cerebellum; for the stroke patients, the significant regions were the cuneus, supplementary motor area and inferior parietal gyrus. Timing can therefore modulate brain activation patterns during acupuncture in healthy subjects and stroke patients; however, the modulation effect appears to differ between the two subject groups. Further studies are required to explore the timing effects of acupuncture at different acupoints in different populations.

Effect of integrated cognitive therapy on hippocampal functional connectivity patterns in stroke patients with cognitive dysfunction: a resting-state FMRI study.

Objective. This study aimed to identify abnormal hippocampal functional connectivity (FC) following ischemic stroke using resting-state fMRI. We also explored whether abnormal hippocampal FC could be modulated by integrated cognitive therapy and tested whether these alterations were associated with cognitive performance. Methods. 18 right-handed cognitively impaired ischemic stroke patients and 18 healty control (HC) subjects were included in this study. Stroke subjects were scanned at baseline and after integrated cognitive therapy, while HCs were only scanned at baseline, to identify regions that show significant correlations with the seed region. Behavioral and cognitive assessments were obtained before each scan. Results. During the resting state, we found abnormal hippocampal FC associated with temporal regions, insular cortex, cerebellum, and prefrontal cortex in stroke patients compared to HCs. After integrated cognitive therapy, however, the stroke group showed increased hippocampal FC mainly located in the prefrontal gyrus and the default mode network (DMN). Altered hippocampal FC was associated with cognitive improvement. Conclusion. Resting-state fMRI may provide novel insight into the study of functional networks in the brain after stroke. Furthermore, altered hippocampal FC may be a compensatory mechanism for cognitive recovery after ischemic stroke.

Electroacupuncture promotes neural cell proliferation in vivo through activation of the ERK1/2 signaling pathway.

The aim of this study was to investigate the effect of electroacupuncture (EA) on cell proliferation and its molecular mechanisms. Sixty rats were randomly divided into 5 groups: sham operation control (SC), ischemia control (IC), EA, EA and DMSO injection (ED), EA and U0126 injection (EU). All the groups, with the exception of SC, underwent middle cerebral artery occlusion (MCAO), and DMSO or U0126 was injected into the rat in the ED or EU group 30 min prior to MCAO. Cell proliferation was evaluated by proliferating cell nuclear antigen (PCNA) immunostaining. The changes of cell cycle proteins (cyclin D1, CDK4, cyclin E, CDK2, p21 and p27) and the ERK1/2 pathway activation were examined by RT-PCR and western blot analysis. The results showed that the positive cell numbers of PCNA immunostaining in the EA and ED groups were more than those in the IC group (P<0.05). The mRNA and protein levels of p21 or p27 were obviously increased, however, the mRNA and protein levels of cyclin D1, CDK4, cyclin E and CDK2 were reduced in the IC and EU groups. The findings suggested that EA activates the ERK1/2 signaling pathway to protect brain injury during cerebral ischemia. However, this positive effect of EA can be blocked by U0126.

Electroacupuncture enhances hippocampal NSCs proliferation in cerebral ischemia-reperfusion injured rats via activation of notch signaling pathway.

The purpose of this study was to investigate the mechanisms by which electroacupuncture (EA) enhances hippocampal neural stem cells (NSCs) proliferation in cerebral ischemia-reperfusion (I/R) injured rats. A total of 72 male adult Sprague-Dawley rats were randomly divided into the sham operation control group (SC), the ischemia control group (IC) and the EA group. Middle cerebral artery occlusion (MCAO) was performed to establish the focal cerebral I/R injury model. Proliferation of hippocampal NSCs in cerebral I/R injured rats was determined by the Nestin immunohistochemical staining. Activation of the notch signaling pathway was detected by Western blotting and reverse transcription polymerase chain reaction analysis. The serum level of neurotrophic factors, e.g., the brain-derived neurotrophic factor (BDNF) and the Glial cell line-derived neurotrophic factor (GDNF), was measured using enzyme-linked immunosorbent assay (ELISA). The results showed that EA at Quchi (LI11) and Zusanli (ST36) acupoints significantly alleviated neurological deficits, reduced infarct volumes and promoted the proliferation of hippocampal NSCs in cerebral I/R injured rats. The crucial signaling molecules in the notch signaling pathway were activated and the secretion of BDNF and GDNF was increased upon EA. The protein and mRNA levels of Cyclin D1, Cdk4 and p-Rb were increased, while p21 and p27 transcripts were suppressed by notch signaling. These results suggest that the up-regulatory effect of EA on the notch signaling pathway and neurotrophic factor secretion may result in the promotion of NSCs proliferation and consequently a therapeutic effect on cerebral ischemia.