CHH methylation of genes associated with fatty acid and jasmonate biosynthesis contributes to cold tolerance in autotetraploids of Poncirus trifoliata.

Polyploids have elevated stress tolerance, but the underlying mechanisms remain largely elusive. In this study, we showed that naturally occurring tetraploid plants of trifoliate orange (Poncirus trifoliata (L.) Raf.) exhibited enhanced cold tolerance relative to their diploid progenitors. Transcriptome analysis revealed that whole-genome duplication was associated with higher expression levels of a range of well-characterized cold stress-responsive genes. Global DNA methylation profiling demonstrated that the tetraploids underwent more extensive DNA demethylation in comparison with the diploids under cold stress. CHH methylation in the promoters was associated with up-regulation of related genes, whereas CG, CHG, and CHH methylation in the 3'-regions was relevant to gene down-regulation. Of note, genes involved in unsaturated fatty acids (UFAs) and jasmonate (JA) biosynthesis in the tetraploids displayed different CHH methylation in the gene flanking regions and were prominently up-regulated, consistent with greater accumulation of UFAs and JA when exposed to the cold stress. Collectively, our findings explored the difference in cold stress response between diploids and tetraploids at both transcriptional and epigenetic levels, and gained new insight into the molecular mechanisms underlying enhanced cold tolerance of the tetraploid. These results contribute to uncovering a novel regulatory role of DNA methylation in better cold tolerance of polyploids.

Diagnostic Errors in Initial Misdiagnosis of Foreign Body Aspiration in Children: A Retrospective Observational Study in a Tertiary Care Hospital in China.

Background: Foreign body aspiration (FBA) in children is a common emergency that can easily be missed, leading to delays in treatment. Few large cohort studies have focused on errors in diagnostic assessment. The main purpose of this study was to analyze factors contributing to the initial misdiagnosis of FBA in children. Methods: We retrospectively reviewed the charts of 226 children diagnosed with FBA at the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University from January 2018 to November 2020. Cases were divided into two groups according to whether or not patients were initially misdiagnosed. The clinical characteristics of the two groups were then compared. The Diagnosis Error Evaluation and Research (DEER) taxonomy tool was applied to cases with initial misdiagnosis. Results: Of the 226 included children with a final diagnosis of FBA, 153 (67.7%) were boys. Ninety percent of patients were under 3 years old. More than half (61.9%) of the children were referred from primary institutions, and 38.1% visited tertiary hospitals directly. A total of 80 (35.4%) patients were initially misdiagnosed. More than half of misdiagnosed children received an alternative diagnosis of bronchiolitis (51.3%), the most common alternative diagnosis. Test failures (i.e., errors in test ordering, test performance, and clinician processing) were primarily responsible for the majority of initial diagnostic errors (76.3%), followed by failure or delay in eliciting critical case history information (20.0%). Characteristics significantly associated with initial misdiagnosis were: presentation over 24 h (OR 9.2, 95% CI 4.8-17.5), being referred from primary institutions (OR 8.8, 4.1-19.0), no witnessed aspiration crisis (OR 7.8, 3.0-20.3), (4) atypical signs or symptoms (OR 3.2, 1.8-5.7), foreign body not visible on CT (OR 36.2, 2.1-636.8), foreign body located in secondary bronchi (OR 4.8, 1.3-17.2), organic foreign body (OR 6.2, 1.4-27.2), and history of recurrent respiratory infections (OR 2.7, 1.4-5.3). Children with misdiagnosis tended to have a longer time from symptom onset to the definitive diagnosis of FBA (P < 0.001). Conclusions: More than one-third of children with FBA were missed at first presentation. Errors in diagnostic testing and history taking were the main reasons leading to initial misdiagnosis.

Enhanced ROS scavenging and sugar accumulation contribute to drought tolerance of naturally occurring autotetraploids in Poncirus trifoliata.

Tetraploids have been reported to exhibit increased stress tolerance, but the underlying molecular and physiological mechanisms remain poorly understood. In this study, autotetraploid plants were identified by screening natural seedlings of trifoliate orange (Poncirus trifoliata). The tetraploids exhibited different morphology and displayed significantly enhanced drought and dehydration tolerance in comparison with the diploid progenitor. Transcriptome analysis indicated that a number of stress-responsive genes and pathways were differentially influenced and enriched in the tetraploids, in particular those coding for enzymes related to antioxidant process and sugar metabolism. Transcript levels and activities of antioxidant enzymes (peroxidase and superoxide dismutase) and sucrose-hydrolysing enzyme (vacuolar invertase) were increased in the tetraploids upon exposure to the drought, concomitant with greater levels of glucose but lower level of reactive oxygen species (ROS). These data indicate that the tetraploids might undergo extensive transcriptome reprogramming of genes involved in ROS scavenging and sugar metabolism, which contributes, synergistically or independently, to the enhanced stress tolerance of the tetraploid. Our results reveal that the tetraploids take priority over the diploid for stress tolerance by maintaining a more robust system of ROS detoxification and osmotic adjustment via elevating antioxidant capacity and sugar accumulation in comparison with the diploid counterpart.

Development of a Tandem Repeat-Based Polymerase Chain Displacement Reaction Method for Highly Sensitive Detection of 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB) is one of the most destructive diseases in citrus production worldwide. Early detection of HLB pathogens can facilitate timely removal of infected citrus trees in the field. However, low titer and uneven distribution of HLB pathogens in host plants make reliable detection challenging. Therefore, the development of effective detection methods with high sensitivity is imperative. This study reports the development of a novel method, tandem repeat-based polymerase chain displacement reaction (TR-PCDR), for the detection of 'Candidatus Liberibacter asiaticus', a widely distributed HLB-associated bacterium. A uniquely designed primer set (TR2-PCDR-F/TR2-PCDR-1R) and a thermostable Taq DNA polymerase mutant with strand displacement activity were used for TR-PCDR amplification. Performed in a regular thermal cycler, TR-PCDR could produce more than two amplicons after each amplification cycle. Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment. The sensitive level was proven to be 100× higher than conventional PCR and similar to real-time PCR. Data from the detection of 'Ca. L. asiaticus' with filed samples using the above three methods also showed similar results. No false-positive TR-PCDR amplification was observed from healthy citrus samples and water controls. These results thereby illustrated that the developed TR-PCDR method can be applied to the reliable, highly sensitive, and cost-effective detection of 'Ca. L. asiaticus'.

Comparative analysis of cluster versus conventional immunotherapy in patients with allergic rhinitis.

The present study aimed to compare the efficacy and safety of subcutaneous immunotherapy with Dermatophagoides pteronyssinus standardized extract administered in conventional and cluster immunotherapy regiments for patients with persistent allergic rhinitis. A total of 60 patients with moderate to severe allergic rhinitis caused by dust mites were treated for 1 year with either conventional immunotherapy (n=30) or cluster immunotherapy (n=30). Nasal conjunctival symptoms and signs were assessed to evaluate the clinical efficacy of the two regimens, and the incidence of local and systemic adverse reactions were also evaluated. The findings demonstrated that the cluster regimen reduced the duration between the initial and maintenance dose by >60%, and resulted in a significant improvement, as compared with the conventional regimen, after 6 weeks of observation (P<0.05). However, the incidence of local and systemic adverse reactions in the cluster regimen during the dose accumulation phase and the dose maintenance phase was not significantly different, as compared with the conventional immunotherapy regimen. These results suggest that cluster immunotherapy is efficacious and safe to treat patients who are clinically sensitive to dust mites.

GSTM1 null genotype is a risk factor for laryngeal cancer.

It remains unclear whether the Glutathione S-transferase M1 (GSTM1) null genotype influence laryngeal cancer development. This study aimed to investigate the interactions among GSTM1 genotype with regard to laryngeal cancer development. We searched online electronic databases (PubMed, EMBASE and CNKI). The strength of association between the GSTM1 genotype and laryngeal cancer risk was assessed by calculating OR with 95% CI. Finally, a total of 25 case-control studies with 2999 cases and 4942 controls on the association between GSTM1 genotype and laryngeal cancer risk were included in this meta-analysis. The overall result showed that the GSTM1 null genotype was related to an increased risk of laryngeal cancer (OR = 1.34; 95% CI, 1.09-1.63). Subgroup analysis was performed according to ethnicity. The results showed that Asians had an increased risk of laryngeal cancer (OR = 1.90; 95% CI, 1.40-2.57), while no significant increased risk was observed in Caucasians (OR = 1.15; 95% CI, 0.97-1.36). In conclusion, this meta-analysis suggested that GSTM1 null genotype was significantly associated with increased laryngeal cancer risk.

[Missed diagnosis in 1 case of esophageal foreign body of button battery in infant: a case report and review of the literature].

To be on the alert on infants with esophageal foreign body, and to pay more attention to the button battery esophageal foreign body, the clinical data of a 12-month-old infant with button battery esophageal foreign body, which was missed diagnosis for up to 4 months, is analyzed. And the related literature is reviewed. An esophagoscopy was carried out to remove the foreign body. A favorable outcome was achieved. When the infants have unexplained gastrointestinal symptoms, we should consider the possibility of an esophageal foreign body. We should pay attention to the button battery due to its highly corrosive to the esophagus. Timely diagnosis, reasonable operation are the keys to cure.

Nitric oxide is involved in dehydration/drought tolerance in Poncirus trifoliata seedlings through regulation of antioxidant systems and stomatal response.

Nitric oxide (NO) is a component of the repertoire of signals implicated in plant responses to environmental stimuli. In the present study, we investigated the effects of exogenous application of NO-releasing donor sodium nitroprusside (SNP) and nitric oxide synthase inhibitor N(G)-nitro-L-arginine-methyl ester (L-NAME) on dehydration and drought tolerance of Poncirus trifoliata. The endogenous NO level was enhanced by SNP pretreatment, but decreased by L-NAME, in the hydroponic or potted plants with or without stresses. Under dehydration, leaves from the SNP-treated hydroponic seedlings displayed less water loss, lower electrolyte leakage and reactive oxygen species accumulation, higher antioxidant enzyme activities and smaller stomatal apertures as compared with the control (treated with water). In addition, pretreatment of the potted plants with SNP resulted in lower electrolyte leakage, higher chlorophyll content, smaller stomatal conductance and larger photosynthetic rate relative to the control. By contrast, the inhibitor treatment changed these physiological attributes or phenotypes in an opposite way. These results indicate that NO in the form of SNP enhanced dehydration and drought tolerance, whereas the inhibitor makes the leaves or plants more sensitive to the stresses. The stress tolerance by NO might be ascribed to a combinatory effect of modulation of stomatal response and activation of the antioxidant enzymes. Taken together, NO is involved in dehydration and drought tolerance of P. trifoliata, implying that manipulation of this signal molecule may provide a practical approach to combat the environmental stresses.

Cloning and molecular characterization of a mitogen-activated protein kinase gene from Poncirus trifoliata whose ectopic expression confers dehydration/drought tolerance in transgenic tobacco.

The mitogen-activated protein kinase (MAPK) cascade plays pivotal roles in diverse signalling pathways related to plant development and stress responses. In this study, the cloning and functional characterization of a group-I MAPK gene, PtrMAPK, in Poncirus trifoliata (L.) Raf are reported. PtrMAPK contains 11 highly conserved kinase domains and a phosphorylation motif (TEY), and is localized in the nucleus of transformed onion epidermal cells. The PtrMAPK transcript level was increased by dehydration and cold, but was unaffected by salt. Transgenic overexpression of PtrMAPK in tobacco confers dehydration and drought tolerance. The transgenic plants exhibited better water status, less reactive oxygen species (ROS) generation, and higher levels of antioxidant enzyme activity and metabolites than the wild type. Interestingly, the stress tolerance capacity of the transgenic plants was compromised by inhibitors of antioxidant enzymes. In addition, overexpression of PtrMAPK enhanced the expression of ROS-related and stress-responsive genes under normal or drought conditions. Taken together, these data demonstrate that PtrMAPK acts as a positive regulator in dehydration/drought stress responses by either regulating ROS homeostasis through activation of the cellular antioxidant systems or modulating transcriptional levels of a variety of stress-associated genes.

Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response.

Polyamines, small aliphatic polycations, have been suggested to play key roles in a number of biological processes. In this paper, attempts were made to investigate the possibility of improving dehydration tolerance of citrus in vitro plants by exogenous application of spermine (Spm). 'Red Tangerine' (Citrus reticulata Blanco) in vitro plants pretreated with 1 mM Spm exhibited less wilted phenotype and lower water loss and electrolyte leakage than the control under dehydration. Spm-pretreated plants contained higher endogenous polyamine content during the course of the experiment relative to the control, particularly at the end of dehydration, coupled with higher expression levels of ADC and SPMS. Histochemical staining showed that the Spm-pretreated leaves were stained to a lower extent than those without Spm pretreatment, implying generation of less reactive oxygen species (ROS). On the contrary, activities of peroxidase (POD) and superoxide dismutase (SOD) in the Spm-pretreated samples were higher than the control at a given time point or during the whole experiment, suggesting that Spm exerted a positive effect on antioxidant systems. In addition, significantly smaller stomatal aperture size was observed in Spm-pretreated epidermal peels, which showed that stomatal closure was promoted by polyamines. All of these data suggest that Spm pretreatment causes accumulation of higher endogenous polyamines and accordingly leads to more effective ROS scavenging (less tissue damage) and stimulated stomatal closure (lower water loss) upon dehydration, which may function collectively to enhance dehydration tolerance.

[Differentially expressed gene profiles of cochlea in rats induced by acute and chronic sodium salicylate injection].

OBJECTIVE: To study the mechanism of electrophysiologic changes caused by different type of sodium salicylate injection. METHODS: Decapitated three group rats ( acute injected, chronic injected and normal rats ) separately, dissected the temporal bones to collect cochlea, and the otic capsules were removed. Then the cochlear materials from each groups were pooled and homogenized respectively, extracted the total RNA, obtained cDNA from purified total RNA by reversed transcription, cDNA were transcripted to cRNA probes in vitro. Hybridized the cRNA probes with tester chip to evaluate the quality of probes, if good, hybridized the probes with real chip. Obtained three gene expression profiles of different groups of cochlea Analyzed the differentially expressed genes among three groups by SOM. Analogized the SOM result to electrophysiologic changes. Then analyzed the genes in clusters of analog results by Gene Ontology. Then the genes in clusters of analog results were analyzed by Gene Ontology. Hsp27 was chosen to validate the result of gene chip using real time quantitative reverse transcription PCR ( RTQ RT-PCR). RESULTS: The probes was good, and the chip hybridization results was credible. We obtained 6 clusters genes by SOM analysis, in which we choose cluster 3 and cluster 4 as candidate cluster. There were 46 genes in cluster 3 and 30 genes in cluster 4 employing GO analysis, which involved in cell communication, cell motility, metabolism, immune response and nerve ensheathment, et al. The result of RTQ RT-PCR showed high concordance with that of gene chip. CONCLUSION: It's a new method to study the mechanism of electrophysiologic changes caused by sodium salicylate by gene chip and SOM analysis.