CONSTITUTIVE EXPRESSER OF PATHOGENESIS-RELATED GENES 5 is an RNA-binding protein controlling plant immunity via an RNA processing complex.

Plant innate immunity is capable of combating diverse and ever evolving pathogens. The plasticity of innate immunity could be boosted by RNA processing. Arabidopsis thaliana CONSTITUTIVE EXPRESSER OF PATHOGENESIS-RELATED GENES 5 (CPR5), a key negative immune regulator, is a component of the nuclear pore complex. Here we further identified CPR5 as a component of RNA processing complexes. Through genetic screening, we found that RNA splicing activator NineTeen Complex and RNA polyadenylation factor CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR, coordinately function downstream of CPR5 to activate plant immunity. CPR5 and these two regulators form a complex that is localized in nuclear speckles, an RNA processing organelle. Intriguingly, we found that CPR5 is an RNA-binding protein belonging to the Transformer 2 (Tra2) subfamily of the serine/arginine-rich family. The RNA recognition motif of CPR5 protein binds the Tra2-targeted RNA sequence in vitro and is functionally replaceable by those of Tra2 subfamily proteins. In planta, it binds RNAs of CPR5-regulated alternatively spliced genes (ASGs) identified by RNA-seq. ARGONAUTE 1 (AGO1) is one of the ASGs and, consistent with this, the ago1 mutant suppresses the cpr5 phenotype. These findings reveal that CPR5 is an RNA-binding protein linking RNA processing with plant immunity.

CD25+ B cells produced IL-35 and alleviated local inflammation during experimental periodontitis.

BACKGROUND AND OBJECTIVE: Host immunity is crucial during periodontal inflammations. B cells are considered to have a function of immunoregulation, and TLRs are considered to be crucial in this process. The present study illustrates the potential roles and rules of CD25+ B cells during periodontitis, especially its effect on regulating host IL-35 level and Th1, Th17, and Treg differentiation. MATERIAL AND METHODS: The proportion of local and systemic CD25+ B cell subpopulations from periodontitis models were identified by flow cytometry. To illustrate further mechanism, B cells were cultured with a different type of TLR activators. Expression of IL-10, IL-35, and TGF-ß was detected by ELISA and real-time PCR. We also set adoptive transfer models by using CD25+ B cells. Alveolar bone erosion, proportion of Th1, Th17, and Tregs, and levels of IFN-γ, TNF-α, IL-1ß, and IL-17 were identified. RESULT: Periodontitis induces more CD25+ B cell subpopulations and promotes their IL-10, IL-35, and TGF-ßproduction. TLR activators enhanced Breg proliferation and function. LPS+CpG obviously induced more CD25+ B cell differentiation and production of IL-10, IL-35, and TGF-ß. Adoptive transfer of CD25+ B cells reduces alveolar bone destruction and local Tregs, proportion, especially the local level of IFN-γ and IL-17. In addition, adoptive transfer of CD25+ B cells remedies the pathological change in the proportion of IL-1ß and Th1/Th17 in local lesions. We did not find any significant difference in peripheral blood, regardless of group and detected items. CONCLUSION: Results of the present study clarify that CD25+ B cells enlarged and produced more IL-10, IL-35 and TGF-ß during periodontitis, activation of TLR4 and TLR9 played crucial roles in this process. Also, CD25+ B cells alleviated periodontal inflammation and alveolar bone resorption. Our findings further expanded the potential of B cells during periodontitis.

Astragalus polysaccharide alleviates alveolar bone destruction by regulating local osteoclastogenesis during periodontitis.

Inflammatory imbalance of bone formation/resorption leads to alveolar bone destruction. Astragalus polysaccharide has been confirmed to have anti-inflammatory effects. We sought to disclose the protective effect and its potential mechanisms of astragalus polysaccharide in the periodontitis model. Experimental periodontitis was induced by cotton ligatures for this study. We measured the alveolar bone damage rate, periodontal osteoclasts, proportion of CD4+Foxp3+, CD4+IL-10+, CD4+TGF-ß+ subsets in the gingiva, and RANKL, OPG, TGF-ß+, and IL-10+ level in the gingiva. We also cultured osteoclast precursor cells in the presence of RANKL and astragalus polysaccharide. Osteoclasto-like cells were identified by TRAP staining, mRNA of RANK, TRAP, and TRAF6 were evaluated by real time PCR. We found that astragalus polysaccharide caused significant protection of the alveolar bone via reducing local osteoclasts. It also decreased the proportion of CD4+Foxp3+ cells and upregulated the level of CD4+IL-10+ cells, reduced RANKL, and remedied IL-10 levels. In cell culture experiments, astragalus polysaccharide prohibited the RANKL mediated osteoclast differentiation. The findings of this study disclose the functions and possible mechanisms of astragalus polysaccharide engaged in local osteoclastogenesis, and reveal the considerable effect of astragalus polysaccharide in alveolar bone homeostasis and its likely contribution to host immuno-regulation in periodontitis.

Improved RANKL expression and osteoclastogenesis induction of CD27+CD38- memory B cells: A link between B cells and alveolar bone damage in periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is a bacteria-induced disease that often leads to alveolar bone damage. Its mechanisms were considered to be complicated, involving an imbalance of the formation and resorption of bone. We sought to disclose the antibody-independent function of B cells during periodontitis. MATERIAL AND METHODS: Production of receptor activator for nuclear factor-κB ligand (RANKL) by total lymphocytes or sorted B-cell subsets in gingiva from healthy or experimental periodontitis animals was examined by flow cytometry, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. To define the effects of lymphocytes or B-cell subsets on osteoclastogenesis induction, bone marrow mononuclear cells were culture in culture medium of lymphocytes or cocultured with B-cell subsets. Osteoclasts were enumerated by tartrate-resistant acid phosphatase staining. Constituent ratio of B-cell subsets in healthy or experimental periodontitis was also detected by flow cytometry. RESULT: Gingiva B cells produce more RANKL and support more osteoclastogenesis than T and other lymphocytes, and this potential improved in periodontitis. Memory B cells (CD27+CD38-) decreased their percentage in periodontitis. Memory B cells have the highest propensity for RANKL production. Remarkably, memory B cells from periodontitis animals expressed significantly more RANKL compared to healthy controls. Memory B cells supported osteoclast differentiation in vitro in a RANKL-dependent manner, and the number of osteoclasts was higher in cultures with memory B cells from periodontitis animals than in those derived from healthy ones. Other B-cell subsets have limited impact on osteoclast formation. CONCLUSION: Findings of this study further disclose the roles of B cells engaged in periodontal immunomodulation and reveal the considerable importance of memory B cells in alveolar bone homeostasis and their likely contribution to alveolar bone destruction in periodontitis.

Multiple-step chromosomal integration of divided segments from a large DNA fragment via CRISPR/Cas9 in Escherichia coli.

Although CRISPR/Cas9-mediated gene editing technology has developed vastly in Escherichia coli, the chromosomal integration of large DNA fragment is still challenging compared with gene deletion and small fragment integration. Moreover, to guarantee sufficient Cas9-induced double-strand breaks, it is usually necessary to design several gRNAs to select the appropriate one. Accordingly, we established a practical daily routine in the laboratory work, involving multiple-step chromosomal integration of the divided segments from a large DNA fragment. First, we introduced and optimized the protospacers from Streptococcus pyogenes in E. coli W3110. Next, the appropriate fragment size for each round of integration was optimized to be within 3-4 kb. Taking advantage of the optimized protospacer/gRNA pairs, a DNA fragment with a total size of 15.4 kb, containing several key genes for uridine biosynthesis, was integrated into W3110 chromosome, which produced 5.6 g/L uridine in shake flask fermentation. Using this strategy, DNA fragments of virtually any length can be integrated into a suitable genomic site, and two gRNAs can be alternatively used, avoiding the tedious construction of gRNA-expressing plasmids. This study thus presents a useful strategy for large DNA fragment integration into the E. coli chromosome, which can be easily adapted for use in other bacteria.

N6-Methyladenosine methylase METTL3 contributes to neuropathic pain by epigenetic silencing of mu opioid receptor.

We aimed at exploring the role and mechanism of METTL3-mediated m6A modification in neuropathic pain. Male Sprague-Dawley rats were randomly divided into four groups: Sham operation group (Sham group), chronic constriction injury (CCI) of the sciatic nerve model group (NPP group), intrathecal injection of virus down-regulated METTL3 + CCI model group (M3 + NPP group) and intrathecal injection of negative control virus + CCI model group (Scr + NPP group). The M3 + NPP group and the Scr + NPP group were intrathecally injected with virus nineteen days before operation. The paw withdrawal mechanical thresholds and paw withdrawal latency were respectively recorded one day before operation, three days, five days and seven days after operation. The rats were sacrificed on the seventh day after operation, and their spinal cord tissues were taken. The frozen sections of rats were performed to observe the expression of green fluorescent protein of the virus. The methylation level of RNA, the protein expression of m6A-related enzyme (METTL3) and mu opioid receptor (MOR) in spinal cord tissues of the four groups were measured. Downregulation of METTL3 had no effect on the overall methylation level of the spinal cord, but it could regulate the methylation level of the OPRM1 gene RNA encoding MOR, partially restore the expression of MOR, and relieve pain in rats. In the process of NPP, METTL3 may inhibit the expression of MOR by regulating the methylation level of OPRM1 gene RNA encoding MOR, and ultimately promote the occurrence and development of NPP.

Switched memory B cells promote alveolar bone damage during periodontitis: An adoptive transfer experiment.

Periodontitis is a bacteria-induced disease that often leads to alveolar bone damage. We sought to determine the role and mechanism of switched memory B cells in alveolar bone destruction during periodontitis. Sensitized B cells were sorted and cultured, then their expression of receptor activator for nuclear factor-κB ligand (RANKL), interleukin-6 (IL-6), and interleukin-12 (IL-12) was detected. Using these cells, we prepared adoptive transfer models in which we induced periodontitis. We found that switched memory B cells produced more RANKL in terms of both protein and mRNA levels than other subpopulations. Switched memory B cells expressed more IL-6 and IL-12 mRNA than other subpopulations, but differences in respective protein levels were not significant. Moreover, we found that switched memory B cell transfer resulted in increased alveolar bone loss and periodontal osteoclastogenesis. Moreover, switched memory B cell transfer increased the proportion of Th1 and Th17 cells as well as the expression of RANKL, osteoprotegerin (OPG), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-1ß, IL-6, IL-17A in gingiva, and cervical lymph nodes (CLNs). The outcomes of the present study indicate that switched memory B cells regulate alveolar bone homeostasis via enhancing cytokine expression and increasing proliferation of Th1 and Th17 cells.

[The relationship between periodontitis and arterial sclerosis without genetic confounding].

PURPOSE: The aim of this study was to evaluate periodontitis as a significant risk factor related to arterial sclerosis after eliminating genetic confounding. METHODS: Fifty two monozygotic twin pairs, 104 patients, were divided into 3 groups. group A: 24 pairs, 48 healthy people, as the blank group; group B: 28 patients with periodontitis, as the control group; group C: 28 patients with periodontitis, as the treatment group. The sample in group B and C had the same gene type. The index of CRP, IL-6, TNF-αand IMT were detected before treatment, 6 and 12 week after treatment. The data were analyzed using SPSS 17.0 software package. RESULTS: The index of CRP, IL-6, TNF-α and IMT decreased after treatment in group C, there was no significant change in group A and B at each check point. CONCLUSIONS: Periodontitis can promote the development of sclerosis without the intervening of gene.

Administration of fentanyl via a slow intravenous fluid line compared with rapid bolus alleviates fentanyl-induced cough during general anesthesia induction.

OBJECTIVE: Fentanyl-induced cough (FIC) is a common complication with a reported incidence from 18.0% to 74.4% during general anesthesia induction. FIC increases the intrathoracic pressure and risks of postoperative nausea and vomiting, yet available treatments are limited. This study was designed to investigate whether administering fentanyl via a slow intravenous fluid line can effectively alleviate FIC during induction of total intravenous general anesthesia. METHODS: A total number of 1200 patients, aged 18-64 years, were enrolled, all of whom were American Society of Anesthesiologists (ASA) grade I or II undergoing scheduled surgeries. All patients received total intravenous general anesthesia, which was induced sequentially by midazolam, fentanyl, propofol, and cisatracurium injection. Patients were randomly assigned to receive fentanyl 3.5 µg/kg via direct injection (control group) or via a slow intravenous fluid line. FIC incidence and the severity grades were analyzed with the Mann-Whitney test. Other adverse reactions, such as hypotension, hypertension, bradycardia, tachycardia, hypoxemia, vomiting, and aspiration, during induction were also observed. The online clinical registration number of this study was ChiCTR-IOR-16009025. RESULTS: Compared with the control group, the incidence of FIC was significantly lower in the slow intravenous fluid line group during induction (9.1%, 95% confidence interval (CI): 6.7%-11.4% vs. 55.9%, 95% CI: 51.8%-60.0%, P=0.000), as were the severity grades (P=0.000). There were no statistical differences between the two groups with regard to other adverse reactions (P>0.05). CONCLUSIONS: The administration of fentanyl via a slow intravenous fluid line can alleviate FIC and its severity during induction for total intravenous general anesthesia. This method is simple, safe, and reliable, and deserves clinical expansion.

[Analysis the causes and treatment of postnasal drip syndrome].

OBJECTIVE: To analysis the causes and treatment for postnasal drip syndrome. METHOD: One hundred patients were tested by routine examination of otolaryngology, nasal endoscopy, fiberoptic laryngoscopy and nasal sinuses CT. Choice allergen test and nasopharynx X-ray were taken based on the symptoms and signs. Patients were treated accordingly for different causes, such as flushing the nasal cavity by herbal liquid, using glucocorticoid locally, taking medicine of improving the sticky film cilium function, immunotherapeutic or operation. RESULT: The causes included chronic rhinitis 22 cases (22%), chronic sinusitis or nasal polyp 31 cases (31%), allergic rhinitis 28 cases (28%), adenoid vegetation 16 cases (16%), chronic nasopharyngitis 3 cases (3%). All patients were treated accordingly and total cure rate was 82% after 6 months follow up. CONCLUSION: The causes of postnasal drip syndrome are complicated. It is critical to check the cause and chose adaptable therapy for good result.