Methyltransferase-like (METTL) homologues participate in Nicotiana benthamiana antiviral responses.

Methyltransferase (MTase) enzymes catalyze the addition of a methyl group to a variety of biological substrates. MTase-like (METTL) proteins are Class I MTases whose enzymatic activities contribute to the epigenetic and epitranscriptomic regulation of multiple cellular processes. N6-adenosine methylation (m6A) is a common chemical modification of eukaryotic and viral RNA whose abundance is jointly regulated by MTases and METTLs, demethylases, and m6A binding proteins. m6A affects various cellular processes including RNA degradation, post-transcriptional processing, and antiviral immunity. Here, we used Nicotiana benthamiana and plum pox virus (PPV), an RNA virus of the Potyviridae family, to investigated the roles of MTases in plant-virus interaction. RNA sequencing analysis identified MTase transcripts that are differentially expressed during PPV infection; among these, accumulation of a METTL gene was significantly downregulated. Two N. benthamiana METTL transcripts (NbMETTL1 and NbMETTL2) were cloned and further characterized. Sequence and structural analyses of the two encoded proteins identified a conserved S-adenosyl methionine (SAM) binding domain, showing they are SAM-dependent MTases phylogenetically related to human METTL16 and Arabidopsis thaliana FIONA1. Overexpression of NbMETTL1 and NbMETTL2 caused a decrease of PPV accumulation. In sum, our results indicate that METTL homologues participate in plant antiviral responses.

Whole-genome analysis revealed the growth-promoting and biological control mechanism of the endophytic bacterial strain Bacillus halotolerans Q2H2, with strong antagonistic activity in potato plants.

Introduction: Endophytes are colonizers of healthy plants and they normally exhibit biocontrol activities, such as reducing the occurrence of plant diseases and promoting plant growth. The endophytic bacterium Bacillus halotolerans Q2H2 (Q2H2) was isolated from the roots of potato plants and was found to have an antagonistic effect on pathogenic fungi. Methods: Q2H2 was identified by morphological observations, physiological and biochemical identification, and 16S rRNA gene sequence analysis. Genes related to the anti-fungal and growth-promoting effects were analyzed using whole-genome sequencing and comparative genomic analysis. Finally, we analyzed the growth-promoting and biocontrol activities of Q2H2 in potato plants using pot experiments. Results: Antagonism and non-volatile substance plate tests showed that Q2H2 had strong antagonism against Fusarium oxysporum, Fusarium commune, Fusarium graminearum, Fusarium brachygibbosum, Rhizoctonia solani and Stemphylium solani. The plate test showed that Q2H2 had the ability to produce proteases, cellulases, ß-1,3-glucanase, dissolved organic phosphate, siderophores, indole-3-acetic acid (IAA), ammonia and fix nitrogen. The suitable growth ranges of Q2H2 under different forms of abiotic stress were pH 5-9, a temperature of 15-30°C, and a salt concentration of 1-5%. Though whole-genome sequencing, we obtained sequencing data of approximately 4.16 MB encompassed 4,102 coding sequences. We predicted 10 secondary metabolite gene clusters related to antagonism and growth promotion, including five known products surfactin, bacillaene, fengycin, bacilysin, bacillibactin, and subtilosin A. Average nucleotide identity and comparative genomic analyses revealed that Q2H2 was Bacillus halotolerans. Through gene function annotation, we analyzed genes related to antagonism and plant growth promotion in the Q2H2 genome. These included genes involved in phosphate metabolism (pstB, pstA, pstC, and pstS), nitrogen fixation (nifS, nifU, salA, and sufU), ammonia production (gudB, rocG, nasD, and nasE), siderophore production (fhuC, fhuG, fhuB, and fhuD), IAA production (trpABFCDE), biofilm formation (tasA, bslA, and bslB), and volatile compound production (alsD, ilvABCDEHKY, metH, and ispE), and genes encoding hydrolases (eglS, amyE, gmuD, ganB, sleL, and ydhD). The potato pot test showed that Q2H2 had an obvious growth-promoting effect on potato roots and better control of Fusarium wilt than carbendazim. Conclusion: These findings suggest that the strain-specific genes identified in bacterial endophytes may reveal important antagonistic and plant growth-promoting mechanisms.

Whole-genome analysis revealed the growth-promoting mechanism of endophytic bacterial strain Q2H1 in potato plants.

Introduction: Endophytes are non-pathogenic inhabitants of healthy plant tissues and have been found to promote plant growth and health. The endophytic bacterial strain Q2H1 was isolated from the roots of the potato and was identified to exhibit growth-promoting effects in potato plants. Methods: Whole-genome sequencing was performed to reveal the mechanism underlying its growth-promoting effect. The obtained sequencing data of approximately 5.65 MB encompassed 5,533 coding sequences. Of note, nine secondary metabolite gene clusters, including siderophore gene clusters, closely associated with plant growth promotion (PGP) were predicted by antiSMASH software. Comparative genomic analysis revealed that Q2H1 belongs to the genus Peribacillus. By gene function annotation, those genes related to plant growth-promoting activities, including indole-3-acetic acid (IAA) synthesis in tryptophan metabolism, siderophore biosynthetic activity, phosphate solubilization, nitrogen fixation, and related genes, were summarized. IAA (14.4 µg/ml) was presumptively produced by Q2H1 using the Salkowski colorimetric method. A total of five genes, namely, phoU, pstB, pstA1, pstC, and pstS, were annotated for phosphate solubilization, which is associated with the ability of the Q2H1 strain to solubilize phosphate under in vitro conditions. Results: It is revealed that genes in the Q2H1 genome associated with nitrogen fixation belonged to three groups, namely, nitrogen fixation (nifU, sufU, salA, and nifS), nitrogen metabolism (nirA, nrtB, and nasA), and glutamate synthesis (glnA, gltB, gltD, and gudB), supported by evidence that Q2H1 grew on medium without nitrogen. We have also identified a siderophore gene cluster located on the chromosome of Q2H1, including seven genes (viz., rbsR, rhbf, rhbE, rhbD, rhbC, rhbA, ddc, and an unknown gene). In the in vitro assay, a prominent brown circle around the colony was produced on the chrome azurol S medium at 48 and 72 h post-inoculation, indicating that the siderophore gene cluster in Q2H1 harbored the ability to produce siderophores. Conclusion: In summary, these findings implied that identifying strain-specific genes for their metabolic pathways in bacterial endophytes may reveal a variety of significant functions of plant growth-promoting mechanisms.

AlkB RNA demethylase homologues and N6 -methyladenosine are involved in Potyvirus infection.

Proteins of the alkylation B (AlkB) superfamily show RNA demethylase activity removing methyl adducts from N6 -methyladenosine (m6 A). m6 A is a reversible epigenetic mark of RNA that regulates human virus replication but has unclear roles in plant virus infection. We focused on Potyvirus-the largest genus of plant RNA viruses-and report here the identification of AlkB domains within P1 of endive necrotic mosaic virus (ENMV) and an additional virus of a putative novel species within Potyvirus. We show that Nicotiana benthamiana m6 A levels are reduced by infection of plum pox virus (PPV) and potato virus Y (PVY). The two potyviruses lack AlkB and the results suggest a general involvement of RNA methylation in potyvirus infection and evolution. Methylated RNA immunoprecipitation sequencing of virus-infected samples showed that m6 A peaks are enriched in plant transcript 3' untranslated regions and in discrete internal and 3' terminal regions of PPV and PVY genomes. Down-regulation of N. benthamiana AlkB homologues of the plant-specific ALKBH9 clade caused a significant decrease in PPV and PVY accumulation. In summary, our study provides evolutionary and experimental evidence that supports the m6 A implication and the proviral roles of AlkB homologues in Potyvirus infection.

[The value of growth differentiation factor-15 and extravascular lung water index in severity grading of acute respiratory distress syndrome patients and their prognosis prediction].

OBJECTIVE: To investigate the value of growth differentiation factor-15 (GDF-15) and extravascular lung water index (EVLWI) in severity grading and prognosis prediction of patients with acute respiratory distress syndrome (ARDS). METHODS: Patients with ARDS aged 18-75 years admitted to the department of respiratory intensive care unit (RICU) of Zhengzhou Central Hospital Affiliated to Zhengzhou University from January 2019 to February 2020 were enrolled. All patients were treated with conventional therapies such as mechanical ventilation, anti-infection, stabilization of water, electrolytes and acid-base environment, blood purification and nutritional support according to their conditions. Besides, the pulse-indicated continuous cardiac output (PiCCO) was performed after admission to the department, and EVLWI before treatment and at 24, 48 and 72 hours of treatment were recorded. Serum GDF-15 level was measured by enzyme linked immunosorbent assay (ELISA) during the same period. Patients were classified as mild, moderate, and severe degree according to the 2012 Berlin Definition of ARDS, and EVLWI and GDF-15 levels in patients with different disease levels before and after treatment were compared. In addition, the length of intensive care unit (ICU) stay, ICU mortality, and 28-day mortality of patients with different GDF-15 or EVLWI levels were analyzed comparatively, with the GDF-15 3 458 ng/L and EVLWI 15 mL/kg as the cut point. RESULTS: A total of 82 patients with ARDS were enrolled, including 22 patients with mild ARDS, 28 patients with moderate ARDS, and 32 patients with severe ARDS. The GDF-15 and EVLWI levels in patients with moderate and severe ARDS at each time point before and after treatment were higher than those in patients with mild ARDS. Both GDF-15 and EVLWI levels in patients with severe ARDS were higher than those in the patients with moderate ARDS. The differences were statistically significant at all the time points except for the difference of GDF-15 levels at 24 hours after treatment (ng/L: 3 900.41±546.43 vs. 3 695.66±604.73, P > 0.05). [GDF-15 (ng/L): 3 786.11±441.45 vs. 3 106.83±605.09 before treatment, 3 895.48±558.96 vs. 3 333.29±559.66 at 48 hours, 3 397.33±539.56 vs. 3 047.53±499.57 at 72 hours; EVLWI (mL/kg): 19.06±1.91 vs. 14.31±1.50 before treatment, 18.56±2.23 vs. 13.26±1.69 at 24 hours, 17.23±1.76 vs. 12.45±1.36 at 48 hours, 15.47±1.81 vs. 11.13±2.19 at 72 hours, all P < 0.05]. According to the cut-off value, there were 23 patients with GDF-15 ≥ 3 458 ng/L and GDF-15 < 3 458 ng/L respectively and there were 23 patients with EVLWI ≥ 15 mL/kg and EVLWI < 15 mL/kg respectively. The length of ICU stay and 28-day mortality in patients with high GDF-15 were significantly higher than those in patients with low GDF-15 [length of ICU stay (days): 21.22±2.69 vs. 15.37±3.14, 28-day mortality: 56.5% vs. 21.7%, both P < 0.05]. The length of ICU stay and 28-day mortality in patients with high EVLWI were also significantly higher than those in patients with low EVLWI [length of ICU stay (days): 18.45±2.61 vs. 14.98±2.75, 28-day mortality: 47.8% vs. 17.4%, both P < 0.05]. CONCLUSIONS: To some extent, GDF-15 and EVLWI levels reflect the severity of patients with ARDS, and high GDF-15 and EVLWI levels are significantly associated with poor prognosis in patients with ARDS.

[Clinical path of mechanical ventilation and atomization inhalation therapy].

In recent years, atomization inhalation therapy has been more and more commonly used in patients with mechanical ventilation. However, the establishment of artificial airway has changed the environment and mode of aerosol delivery. Currently, Expert consensus on atomization inhalation during mechanical ventilation has been established to guide clinical practice. However, many ventilators do not support the treatment of aerosol inhalation due to the tedious procedures and numerous drugs. At the same time, the therapeutic effect of atomization inhalation is affected by many factors, such as ventilator mode selection, parameter setting, heating and humidification, using of artificial nose and filter, etc., which often results in poor clinical effects or even damage to the ventilator. In order to standardize the clinical application of mechanical ventilation atomization inhalation technology and avoid many possible problems in operation, the committee members of Respiratory Therapy Group of Severe Medicine Branch of Henan Medical Association discussed and concluded this clinical path, so as to provide clinical reference for the actual operation and drug administration of mechanical ventilation atomization.

Prolactin rs1341239 T allele may have protective role against the brick tea type skeletal fluorosis.

OBJECTIVE: Prolactin (PRL) has been reported to be associated with increased bone turnover, and increased bone turnover is also a feature of skeletal fluorosis (SF). Autocrine/paracrine production of PRL is regulated by the extrapituitary promoter and a polymorphism in the extrapituitary PRL promoter at -1149 (rs1341239) is associated with disturbances of bone metabolism in other diseases. Here, we have investigated the possibility that the rs1341239 polymorphism is associated with SF, which results from the consumption of brick tea. DESIGN: We conducted a cross-sectional study in Sinkiang, Qinghai, Inner Mongolia in China. Demography survey questionnaires were completed and physical examination and X-ray diagnoses were used to diagnose SF. Brick tea water fluoride intake (IF) and urinary fluoride (UF) were tested by an F-ion selective electrode method. A Sequenom MassARRAY system was used to determine PRL gene polymorphisms. RESULTS: Subjects who were younger than 45 years of age and carried the T allele had a significantly decreased risk of SF [OR = 0.279 (95%CI, 0.094-0.824)] compared to those carrying the homozygous G allele. This phenomenon was only observed in Kazakh subjects [OR = 0.127 (95%CI, 0.025-0.646)]. Kazakh females who carried T alleles has a decreased risk of SF [OR = 0.410 (95%CI, 0.199-0.847)]. For Kazakh subjects which IF is less than 3.5 mg/d, a decreased risk of SF was observed among the participants who carried T alleles [OR = 0.118 (95%CI, 0.029-0.472)]. Overall, subjects with 1.6-3.2 mg/L UF and carried T alleles had a significantly decreased risk of SF [OR = 0.476 (95%CI, 0.237-0.955)] compared to homozygous G allele carriers. This phenomenon was only observed in Kazakh subjects [OR = 0.324 (95%CI, 0.114-0.923)]. CONCLUSIONS: Our results suggested that the PRL rs1341239 T allele decreases the risk of brick tea SF.

Intra-individual variation in urinary iodine concentration: effect of statistical correction on population distribution using seasonal three-consecutive-day spot urine in children.

OBJECTIVE: To determine the effect of statistical correction for intra-individual variation on estimated urinary iodine concentration (UIC) by sampling on 3 consecutive days in four seasons in children. SETTING: School-aged children from urban and rural primary schools in Harbin, Heilongjiang, China. PARTICIPANTS: 748 and 640 children aged 8-11 years were recruited from urban and rural schools, respectively, in Harbin. PRIMARY AND SECONDARY OUTCOME MEASURES: The spot urine samples were collected once a day for 3 consecutive days in each season over 1 year. The UIC of the first day was corrected by two statistical correction methods: the average correction method (average of days 1, 2; average of days 1, 2 and 3) and the variance correction method (UIC of day 1 corrected by two replicates and by three replicates). The variance correction method determined the SD between subjects (Sb) and within subjects (Sw), and calculated the correction coefficient (Fi), Fi=Sb/(Sb+Sw/di), where di was the number of observations. The UIC of day 1 was then corrected using the following equation:[Formula: see text] RESULTS: The variance correction methods showed the overall Fi was 0.742 for 2 days' correction and 0.829 for 3 days' correction; the values for the seasons spring, summer, autumn and winter were 0.730, 0.684, 0.706 and 0.703 for 2 days' correction and 0.809, 0.742, 0.796 and 0.804 for 3 days' correction, respectively. After removal of the individual effect, the correlation coefficient between consecutive days was 0.224, and between non-consecutive days 0.050. CONCLUSIONS: The variance correction method is effective for correcting intra-individual variation in estimated UIC following sampling on 3 consecutive days in four seasons in children. The method varies little between ages, sexes and urban or rural setting, but does vary between seasons.

Modifying Role of GSTP1 Polymorphism on the Association between Tea Fluoride Exposure and the Brick-Tea Type Fluorosis.

BACKGROUND: Brick tea type fluorosis is a public health concern in the north-west area of China. The association between SNPs of genes influencing bone mass and fluorosis has attracted attention, but the association of SNPs with the risk of brick-tea type of fluorosis has not been reported. OBJECTIVE: To investigate the modifying roles of GSTP1 rs1695 polymorphisms on this association. METHODS: A cross-sectional study was conducted. Brick-tea water was tested by the standard of GB1996-2005 (China). Urinary fluoride was tested by the standard of WS/T 89-2006 (China). Skeletal fluorosis was diagnosed by X-ray, the part we scheduled was forearm, shank, and pelvic, then diagnosed the skeletal fluorosis by the standard of WS/192-2008 (China). Gene polymorphism was tested by Sequenom MassARRAY system. RESULT: The prevalence rate in different ethnical participants was different: Tibetan individuals had the highest prevalence rate of skeletal fluorosis. There were significant differences in genotype frequencies of GSTP1 Rs1695 among different ethnical participants (p<0.001): Tibetan, Mongolian and Han subjects with homozygous wild type (GSTP1-AA) genotype were numerically higher than Kazakh and Russian subjects (p<0.001). Compared to Tibetan participants who carried homozygous A allele of GSTP1 Rs1695, Tibetan participants who carried G allele had a significantly decreased risk of skeletal fluorosis (OR = 0.558 [95% CI, 0.326-0.955]). For Kazakh participants, a decreased risk of skeletal fluorosis among carriers of the G allele was limited to non high-loaded fluoride status (OR = 0. 166 [95% CI, 0.035-0.780] vs. OR = 1.478 [95% CI, 0.866-2.552] in participants with high-loaded fluoride status). Neither SNP-IF nor SNP-age for GSTP1 Rs1695 was observed. CONCLUSION: The prevalence rate of the brick tea type fluorosis might have ethnic difference. For Tibetan individuals, who had the highest prevalence rate, G allele of GSTP1 Rs1695 might be a protective factor for brick tea type skeletal fluorosis.