Overexpression of tocopherol biosynthesis genes in guayule (Parthenium argentatum) reduces rubber, resin and argentatins content in stem and leaf tissues.

Natural rubber produced in stems of the guayule plant (Parthenium argentatum) is susceptible to post-harvest degradation from microbial or thermo-oxidative processes, especially once stems are chipped. As a result, the time from harvest to extraction must be minimized to recover high quality rubber, especially in warm summer months. Tocopherols are natural antioxidants produced in plants through the shikimate and methyl-erythtiol-4-phosphate (MEP) pathways. We hypothesized that increased in vivo guayule tocopherol content might protect rubber from post-harvest degradation, and/or allow reduced use of chemical antioxidants during the extraction process. With the objective of enhancing tocopherol content in guayule, we overexpressed four Arabidopsis thaliana tocopherol pathway genes in AZ-2 guayule via Agrobacterium-mediated transformation. Tocopherol content was increased in leaf and stem tissues of most transgenic lines, and some improvement in thermo-oxidative stability was observed. Overexpression of the four tocopherol biosynthesis enzymes, however, altered other isoprenoid pathways resulting in reduced rubber, resin and argentatins content in guayule stems. The latter molecules are mainly synthesized from precursors derived from the mevalonate (MVA) pathway. Our results suggest the existence of crosstalk between the MEP and MVA pathways in guayule and the possibility that carbon metabolism through the MEP pathway impacts rubber biosynthesis.

GLP-1 regulates exercise endurance and skeletal muscle remodeling via GLP-1R/AMPK pathway.

Exercise-induced physical endurance enhancement and skeletal muscle remodeling can prevent and delay the development of multiple diseases, especially metabolic syndrome. Herein, the study explored the association between glucagon-like peptide-1 (GLP-1) secretion and exercise, and its effect on skeletal muscle remodeling to enhance endurance capacity. We found both acute exercise and short-term endurance training significantly increased the secretion of GLP-1 in mice. Recombinant adeno-associated virus (AAV) encoding Gcg (proglucagon) was used to induce the overexpression of GLP-1 in skeletal muscle of mice. Overexpression of GLP-1 in skeletal muscle enhanced endurance capacity. Meanwhile, glycogen synthesis, glucose uptake, type I fibers proportion, and mitochondrial biogenesis were augmented in GLP-1-AAV skeletal muscle. Furthermore, the in vitro experiment showed that exendin-4 (a GLP-1 receptor agonist) treatment remarkably promoted glucose uptake, type I fibers formation, and mitochondrial respiration. Mechanistically, the knockdown of AMPK could reverse the effects imposed by GLP-1R activation in vitro. Taken together, these results verify that GLP-1 regulates skeletal muscle remodeling to enhance exercise endurance possibly via GLP-1R signaling-mediated phosphorylation of AMPK.

Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule.

Production of natural rubber by Parthenium argentaum (guayule) requires increased yield for economic sustainability. An RNAi gene silencing strategy was used to engineer isoprenoid biosynthesis by downregulation of squalene synthase (SQS), such that the pool of farnesyl diphosphate (FPP) substrate might instead be available to initiate natural rubber synthesis. Downregulation of SQS resulted in significantly reduced squalene and slightly increased rubber, but not in the same tissues nor to the same extent, partially due to an apparent negative feedback regulatory mechanism that downregulated mevalonate pathway isoprenoid production, presumably associated with excess geranyl pyrophosphate levels. A detailed metabolomics analysis of isoprenoid production in guayule revealed significant differences in metabolism in different tissues, including in active mevalonate and methylerythritol phosphate pathways in stem tissue, where rubber and squalene accumulate. New insights and strategies for engineering isoprenoid production in guayule were identified.

Radiofrequency catheter ablation of ventricular tachycardia in patients post surgical repair of tetralogy of Fallot / 中华心血管病杂志

Objective: To investigate the clinical and electrophysiological features of ventricular tachycardia (VT) in tetralogy of Fallot (TOF) patients post surgical repair (rTOF) and to analyze the therapeutic effect and prognosis of radiofrequency ablation of rTOF-VT. Methods: This is a retrospective study. Consecutive patients with rTOF-VT, who were treated in Fuwai Hospital from January 2015 to March 2020, were enrolled. All the patients underwent right ventricular voltage mapping following routine cardiac electrophysiological examination, followed by linear or homogenizing radiofrequency ablation based on the low-voltage substrate. The clinical features, 3-dimentional electrophysiological substrate mapping, radiofrequency ablation and long-term prognosis of the enrolled patients were analyzed. Acute ablation success was defined as completion of linear or homogenizing ablation or intraoperative evoked VT as destination of the procedure. Patients were followed up at 3 and 6 months post operation and every year thereafter. The endpoints were sudden cardiac death (SCD) and recurrence of ventricular tachycardia. Results: A total of 20 patients with rTOF-VT were enrolled including 14 males with an age of (35.8±11.8) years. The electrocardiogram identified 23 types of ventricular tachycardia, 19 of which were originated from right ventricular inflow tract outlet. The most common clinical manifestations were heart murmur (19 cases, 95%) and syncope (4 cases, 25%). Electroanatomical substrate mapping was performed in 20 patients and evidenced localized or diffuse scar or low-voltage area of right ventricle. Intraoperative electrophysiological tests provoked ventricular tachycardia in 6 patients (30%), including 5 patients with hemodynamics disturbance. The acute success rate of radiofrequency ablation was 95% (19/20). The follow-up time was (31.1±17.7) months and the recurrence rate of ventricular tachycardia was 30% during follow-up period and 5 cases received repeat radiofrequency ablation and there was no recurrent ventricular tachycardia during follow-up post repeat radiofrequency ablation. Conclusions: The voltage substrate mapping under sinus rhythm is a feasible mapping method for rTOF-VT. Linear or flaky radiofrequency ablation of the slow conduction zone is safe and effective treatment strategy, the recurrence rate after the first radiofrequency ablation is still high, and the effectiveness of repeat radiofrequency ablation is satisfactory in this patient cohort.

Screening of cellulose degradation bacteria from Min pigs and optimization of its cellulase production

BACKGROUND: Cellulose as a potential feed resource hinders its utilization because of its complex structure, and cellulase is the key to its biological effective utilization. Animal endogenous probiotics are more susceptible to colonization in the intestinal tract, and their digestive enzymes are more conducive to the digestion and absorption of feed in young animals. Min pigs are potential sources of cellulase probiotics because of the high proportion of dietary fiber in their feed. In this study, the cellulolytic bacteria in the feces of Min pigs were isolated and screened. The characteristics of enzymes and cellulase production were studied, which provided a theoretical basis for the rational utilization of cellulase and high-fiber food in animal production. RESULTS: In our study, 10 strains of cellulase producing strains were isolated from Min pig manure, among which the M2 strain had the best enzyme producing ability and was identified as Bacillus velezensis. The optimum production conditions of cellulase from strain M2 were: 2% inoculum, the temperature of 35°C, the pH of 5.0, and the liquid loading volume of 50 mL. The optimum temperature, pH and time for the reaction of cellulase produced by strain M2 were 55°C, 4.5 and 5 min, respectively. CONCLUSIONS: Min pigs can be used as a source of cellulase producing strains. The M2 strain isolated from feces was identified as Bacillus velezensis. The cellulase from M2 strain had a good activity and the potential to be used as feed additive for piglets.

Downregulation of a CYP74 Rubber Particle Protein Increases Natural Rubber Production in Parthenium argentatum.

We report functional genomics studies of a CYP74 rubber particle protein from Parthenium argentatum, commonly called guayule. Previously identified as an allene oxide synthase (AOS), this CYP74 constitutes the most abundant protein found in guayule rubber particles. Transgenic guayule lines with AOS gene expression down-regulated by RNAi (AOSi) exhibited strong phenotypes that included agricultural traits conducive to enhancing rubber yield. AOSi lines had higher leaf and stem biomass, thicker stembark tissues, increased stem branching and improved net photosynthetic rate. Importantly, the rubber content was significantly increased in AOSi lines compared to the wild-type (WT), vector control and AOS overexpressing (AOSoe) lines, when grown in controlled environments both in tissue-culture media and in greenhouse/growth chambers. Rubber particles from AOSi plants consistently had less AOS particle-associated protein, and lower activity (for conversion of 13-HPOT to allene oxide). Yet plants with downregulated AOS showed higher rubber transferase enzyme activity. The increase in biomass in AOSi lines was associated with not only increases in the rate of photosynthesis and non-photochemical quenching (NPQ), in the cold, but also in the content of the phytohormone SA, along with a decrease in JA, GAs, and ABA. The increase in biosynthetic activity and rubber content could further result from the negative regulation of AOS expression by high levels of salicylic acid in AOSi lines and when introduced exogenously. It is apparent that AOS in guayule plays a pivotal role in rubber production and plant growth.

Effects of Bruton's tyrosine kinase on the proliferation and differentiation of osteoclasts / 华西口腔医学杂志

OBJECTIVE@#To observe the effect of Bruton's tyrosine kinase (BTK) on the proliferation and differentiation of osteoclasts and to explore the mechanism of BTK on bone destruction in periapical periodontitis.@*METHODS@#After RAW264.7 cells induced with 100 ng·L⁻¹ receptor activator for nuclear factor-κB ligand (RANKL) for 5 days, osteoclast induction was confirmed by light microscopy, tartrate-resistant acid phosphatase (TRAP) staining, and quantitative real-time PCR (RT-qPCR). Then, BTK-small interfering RNA (BTK-siRNA) was transfected into cells induced for 5 days. After 24 h, the expression of TRAP mRNA was measured using RT-qPCR, and the proliferation and differentiation of osteoclasts were detected using CCK-8 and TRAP activity assay. Statistical analysis was performed.@*RESULTS@#After RAW264.7 was induced with RANKL for 5 days, a large number of round, ellipse, irregularly protuberant, and TRAP-positive macrophages were observed under light microscopy. The expression of TRAP mRNA significantly reduced after 24 h of BTK-siRNA transfection (P<0.05). The detection of CCK-8 and TRAP activities showed that the proliferation and differentiation of osteoclasts significantly decreased (P<0.05).@*CONCLUSIONS@#Silencing of BTK can inhibit the proliferation and differentiation of osteoclasts. BTK can be used as a new target for the inhibition of osteoclasts.

Effects of Biodentine on proliferation and osteogenesis of MG-63 cells / 中国组织工程研究

BACKGROUND: As a bioactive substance, Biodentine has good compressive strength, bonding strength and less micro-leakage. It has been successfully applied to a variety of clinical indications. However, much less is known about whether Biodentine can promote osteogenesis. OBJECTIVE: To explore the effects of Biodentine of different concentrations on proliferation and osteogenesis of MG-63 cells. METHODS: The extracts of Biodentine with different concentration gradients (1, 1/2, 1/4, 1/8, 1/16) were prepared. MG-63 cells were cultured in six groups, with the addition of minimum essential medium (control group) and five concentrations of Biodentine extracts. Cell proliferation was detected by cell counting kit-8 assay at 1, 3, 5 and 7 days, and then the best concentration of Biodentine extract was screened. Another MG-63 cells were cultured in minimum essential medium (blank control group) and Biodentine extract of the optimal concentration (experimental group). The expression of osteogenic factor Runx2 mRNA in MG-63 cells was detected by real-time PCR at 1, 3, 5 and 7 days of culture. Then alizarin red staining was used to observe calcified nodules in MG-63 cells at 10 and 14 days of culture. RESULTS AND CONCLUSION: (1) At 1 and 3 days of culture, the number of viable cells in different concentration groups was similar to that in the control group (P ＞ 0.05). At 5 days of culture, compared with the control group, the number of viable cells in MG-63 cells was significantly lowered in the 1 concentration group, showed no significant changes in the 1/2, 1/4, 1/8 concentration groups (P ＞ 0.05), and was significantly increased in the 1/16 concentration group (P ＜ 0.05). Therefore, the 1/16 concentration of Biodentine extract was used for further experiment. (2) Runx2 mRNA expression of the experimental group at 1, 3, 5 and 7 days of culture was 1.14, 5.29, 1.08 and 2.11 times that of the control group, respectively (all P ＜ 0.05). (3) At 10 days of culture, both the experimental group and blank control group showed no mineralized nodules; at 14 days of culture, mineralized nodules were observed in the two groups, and larger and darker nodules were seen in the experiment group. To conclude, Biodentine at certain concentrations can promote the proliferation and osteogenic activity of human osteosarcoma cell line MG-63.

Use of atomic force microscopy to observe the surface and the dynamic forming process of biofilms of Enterococcus faecalis / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To observe the surface of Enterococcus faecalis and the dynamic forming process of those biofilms using atomic force microscopy (AFM) in air condition.</p><p><b>METHODS</b>The surface of Enterococcus faecalis which were dried in air were observed with AFM. We used the cellulose nitrate film to construct the Enterococcus faecalis biofilms model in vitro, and then placed the biofilms under AFM to observe the surface changes of biofilms' development.</p><p><b>RESULTS</b>The cell surfaces of strain Enterococcus faecalis were not regular because of the presence of the amorphous substance on the colony surface, which congregated globular, fibrous structure. Gradually determined that at 6 h the initial biofilm formed and at 24 h the biofilms maintained the steady-state. AFM height images showed topographical changes due to biofilms' development, which were used to characterize several aspects of the bacterial surface, such as the presence of extracellular polymeric substance, and the biofilms' development stage.</p><p><b>CONCLUSION</b>Application of AFM in physiological conditions could be useful in observing Enterococcus faecalis surface ultrastructure and dynamic process of biofilms formation.</p>

Analysis of relationship between the detection of Enterococcus faecalis in post-treatment endodontic disease and clinical symptom or sign / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To detect the Enterococcus faecalis (E. faecalis) in post-treatment endodontic disease, and to analyze the relationship between the occurrence of E. faecalis and clinical symptom.</p><p><b>METHODS</b>108 teeth which need root canal retreatment were collected, and the clinical symptoms and physical signs were recorded. Bacterium samples from root canal were taken, and genome DNA from bacterial samples were extracted. The occurrence of E. faecalis by means of the polymerase chain reaction was investigated.</p><p><b>RESULTS</b>The detection rate of E. faecalis in cases of root canal retreatment was 47.2%, while in cases with symptoms or signs, or cases with both symptoms and signs, the root canal E. faecalis detection rates were 52.6%, 57.9%, 62.5%. The detection rates of E. faecalis between cases with clinical symptom and without clinical symptom demonstrated statistical significance (P < 0.05). The detection rates between cases with both clinical symptom and manifestly aneretic root and cases without clinical symptom and manifestly aneretic root had statistical significance (P < 0.05). In the group of clinical symptom, the detection rate of E. faecalis in cases with biting pain was 66.7%, clearly higher than those without biting pain (P < 0.05).</p><p><b>CONCLUSION</b>The occurrence of E. faecalis in cases of root canal retreatment correlates with clinical symptoms.</p>

Comparisons of carbon storages in Cunninghamia lanceolata and Michelia macclurei plantations during a 22-year period in southern China.

Tree species composition was important for carbon storage within the same climate range. To quantify the dynamics of ecosystem carbon allocation as affected by different tree species, we measured the above- and below-ground biomass accumulation in 22 years, as well as the tissue carbon concentrations of trees in Cunninghamia lanceolata plantation and Michelia macclurei plantation. Results indicated that M. macclurei plantation significantly stored more carbon (174.8 tons/hm2) than C. lanceolata plantation (154.3 tons/hm2). Most of the carbon was found in the soil pool (57.1% in M. macclurei plantation, 55.2% in C. lanceolata plantation). Tree and soil component of M. macclurei plantation possessed significantly higher carbon storage than that of C. lanceolata plantation (p <0.05). No significant difference was found in the carbon storage of understory and forest floor. These results suggest that the broadleaved species (M. macclurei) possesses greater carbon sequestration potential than the coniferous species (C. lanceolata) in southern China.

Secretory expression of K88 (F4) fimbrial adhesin FaeG by recombinant Lactococcus lactis for oral vaccination and its protective immune response in mice.

K88 (F4) fimbrial adhesin, FaeG, was expressed extracellularly in Lactococcus lactis using a nisin-controlled gene expression system. The antibody response and protective efficacy of the recombinant bacteria (L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C(83549) challenge were evaluated in ICR mice. Mice vaccinated with L. lactis [spNZ8048-faeG] had a significantly increased antigen-specific IgG level in the serum and decreased mortality rate (P < 0.05) compared with the control. This indicates that oral immunization of L. lactis [spNZ8048-faeG] can induce an immune-response protection upon challenge with live ETEC in ICR mice.

Effect of different decalcification condition on tooth-clearing technique / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate shrinkage range of cleared teeth caused by nitric acid with different temperature and concentration.</p><p><b>METHODS</b>48 human teeth were root canal-prepared and filled, then randomly and averagely divided into six groups on the basis of temperature and density of nitric acid and the condition of whether or not added the oscillate. Group A was 20 degrees C with 6% nitric acid, group B was 20 degrees C with 6% nitric acid and oscillate, group C was 20 degrees C with 3% nitric acid, group D was 20 degrees C with 3% nitric acid and oscillate, group E was 30 degrees C with 6% nitric acid and oscillate, group F was 30 degrees C with 3% nitric acid and oscillate. After achieving the standard of the decalcification, all the specimens were gradually dehydrated, and then cleared and conserved using methyl salicylate. Time-consumed and shrinkage range of all the specimens were recorded and analyzed.</p><p><b>RESULTS</b>The time of decalcification in group E was the fastest, then was group F, group B. Group C was the last one. The anastole of the specimens was group E > group B > group A, group F > group D > group C, group B > group D, group E > group D, there was significant difference (P < 0.05). Group C had significant difference with other groups (P < 0.05). The anastole rate of the specimens had no significant difference between group A and group B, group C and group D, group B and group F, group D and group F.</p><p><b>CONCLUSION</b>In 20 degrees C, 3% nitric acid with oscillate to carry out the decalcification can use less time and get less anastole. The result of the tooth-clearing technique is the best.</p>

Effect of Enterococcus faecalis on apoptosis rate and the release of matrix metalloproteinase-8 of polymorphonuclear leukocytes / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the release of matrix metalloproteinase-8 (MMP-8) and apoptosis rate of polymorphonuclear leukocytes (PMNs) after PMNs was triggered by Enterococcus faecalis (E. faecalis) in vitro.</p><p><b>METHODS</b>The activated E. faecalis suspension was prepared and added to PMNs suspension as experiment group. As a positive control, phorbol myristate acetate (PMA) was used. As negative control, PMNs suspension was incubated with PBS. The release of MMP-8 was measured at 0, 20, 60, 120 min by ELISA method. E. faecalis lysate acted on PMNs as experiment group, PMNs suspension was incubated with PBS as negative control, samples in two groups were incubated at 37 degrees C for 2, 5, 10, 15 h. The apoptosis rate of PMNs was tested by Flow Cytometry.</p><p><b>RESULTS</b>At 0 min, there was no significant difference of MMP-8 release in the experiment group and positive control (P>0.01); whereas at 60, 120 min, E. faecalis induced a significant lower MMP-8 release compared with the positive control (P<0.01). The apoptosis rate of PMNs in both groups increased along with time, and apoptotic rate in experiment group was higher than that in the control group at 2, 5, 10, 15 h (P<0.01).</p><p><b>CONCLUSION</b>After E. faecalis act on PMNs, no significant release of MMP-8 from PMNs was observed. E. faecalis don't induce PMNs apoptosis delay.</p>

Low light and low ammonium are key factors for guayule leaf tissue shoot organogenesis and transformation.

A new method has been developed for guayule tissue culture and transformation. Guayule leaf explants have a poor survival rate when placed on normal MS medium and under normal culture room light conditions. Low light and low ammonium treatment greatly improved shoot organogenesis and transformation from leaf tissues. Using this method, a 35S promoter driven BAR gene and an ubiquitin-3 promoter driven GUS gene (with intron) have been successfully introduced into guayule. These transgenic guayule plants were resistant to the herbicide ammonium-glufosinate and were positive to GUS staining. Molecular analysis showed the expected band and signal in all GUS positive transformants. The transformation efficiency with glufosinate selection ranged from 3 to 6%. Transformation with a pBIN19-based plasmid containing a NPTII gene and then selection with kanamycin also works well using this method. The ratio of kanamycin-resistant calli to total starting explants reached 50% in some experiments.