The LysR-Type Transcriptional Regulator CrgA Negatively Regulates the Flagellar Master Regulator flhDC in Ralstonia solanacearum GMI1000.

The invasion and colonization of host plants by the destructive pathogen Ralstonia solanacearum rely on its cell motility, which is controlled by multiple factors. Here, we report that the LysR-type transcriptional regulator CrgA (RS_RS16695) represses cell motility in R. solanacearum GMI1000. CrgA possesses common features of a LysR-type transcriptional regulator and contains an N-terminal helix-turn-helix motif as well as a C-terminal LysR substrate-binding domain. Deletion of crgA results in an enhanced swim ring and increased transcription of flhDC In addition, the ΔcrgA mutant possesses more polar flagella than wild-type GMI1000 and exhibits higher expression of the flagellin gene fliC Despite these alterations, the ΔcrgA mutant did not have a detectable growth defect in culture. Yeast one-hybrid and electrophoretic mobility shift assays revealed that CrgA interacts directly with the flhDC promoter. Expressing the ß-glucuronidase (GUS) reporter under the control of the crgA promoter showed that crgA transcription is dependent on cell density. Soil-soaking inoculation with the crgA mutant caused wilt symptoms on tomato (Solanum lycopersicum L. cv. Hong yangli) plants earlier than inoculation with the wild-type GMI1000 but resulted in lower disease severity. We conclude that the R. solanacearum regulator CrgA represses flhDC expression and consequently affects the expression of fliC to modulate cell motility, thereby conditioning disease development in host plants.IMPORTANCERalstonia solanacearum is a widely distributed soilborne plant pathogen that causes bacterial wilt disease on diverse plant species. Motility is a critical virulence attribute of R. solanacearum because it allows this pathogen to efficiently invade and colonize host plants. In R. solanacearum, motility-defective strains are markedly affected in pathogenicity, which is coregulated with multiple virulence factors. In this study, we identified a new LysR-type transcriptional regulator (LTTR), CrgA, that negatively regulates motility. The mutation of the corresponding gene leads to the precocious appearance of wilt symptoms on tomato plants when the pathogen is introduced using soil-soaking inoculation. This study indicates that the regulation of R. solanacearum motility is more complex than previously thought and enhances our understanding of flagellum regulation in R. solanacearum.

Ralstonia solanacearum elicitor RipX Induces Defense Reaction by Suppressing the Mitochondrial atpA Gene in Host Plant.

RipX of Ralstonia solanacearum is translocated into host cells by a type III secretion system and acts as a harpin-like protein to induce a hypersensitive response in tobacco plants. The molecular events in association with RipX-induced signaling transduction have not been fully elucidated. This work reports that transient expression of RipX induced a yellowing phenotype in Nicotiana benthamiana, coupled with activation of the defense reaction. Using yeast two-hybrid and split-luciferase complementation assays, mitochondrial ATP synthase F1 subunit α (ATPA) was identified as an interaction partner of RipX from N. benthamiana. Although a certain proportion was found in mitochondria, the YFP-ATPA fusion was able to localize to the cell membrane, cytoplasm, and nucleus. RFP-RipX fusion was found from the cell membrane and cytoplasm. Moreover, ATPA interacted with RipX at both the cell membrane and cytoplasm in vivo. Silencing of the atpA gene had no effect on the appearance of yellowing phenotype induced by RipX. However, the silenced plants improved the resistance to R. solanacearum. Moreover, qRT-PCR and promoter GUS fusion experiments revealed that the transcript levels of atpA were evidently reduced in response to expression of RipX. These data demonstrated that RipX exerts a suppressive effect on the transcription of atpA gene, to induce defense reaction in N. benthamiana.

Ectopic expression of the TAL effector AvrXa7 in Xanthomonas citri subsp. citri hinders citrus canker symptom formation by modulating transcriptional profile of citrus genes.

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a serious bacterial disease that affects citrus trees worldwide. The ectopic expression of TAL effector AvrXa7 in Xcc suppressed canker development. The Xcc strain expressing avrXa7 induced a yellow symptom around the inoculation site. Transcriptome analysis revealed 315 differentially expressed genes, which were categorized into several functional groups. The more interesting genes were those involved in the biosynthesis of terpene and ethylene. In particular, the linoleate 13 S-lipoxygenase gene CsLOX2-1 was found to possess the AvrXa7 binding sequence in the promoter region. The recognition of AvrXa7 to the CsLOX2-1 promoter was subsequently confirmed by yeast one-hybrid and electrophoretic mobility shift experiments. This demonstrated that the TALE effector AvrXa7 promotes CsLOX2-1 expression by directly binding to the promoter sequence. Our findings contribute a valuable clue to identifying the potential genes that can be used to prevent citrus canker.

Molecular study on the carAB operon reveals that carB gene is required for swimming and biofilm formation in Xanthomonas citri subsp. citri.

BACKGROUND: The carA and carB genes code the small and large subunits of carbamoyl-phosphate synthase (CPS) that responsible for arginine and pyrimidine production. The purpose of this work was to study the gene organization and expression pattern of carAB operon, and the biological functions of carA and carB genes in Xanthomonas citri subsp. citri. METHODS: RT-PCR method was employed to identify the full length of carAB operon transcript in X. citri subsp. citri. The promoter of carAB operon was predicted and analyzed its activity by fusing a GUS reporter gene. The swimming motility was tested on 0.25% agar NY plates with 1% glucose. Biofilm was measured by cell adhesion to polyvinyl chloride 96-well plate. RESULTS: The results indicated that carAB operon was composed of five gene members carA-orf-carB-greA-rpfE. A single promoter was predicted from the nucleotide sequence upstream of carAB operon, and its sensitivity to glutamic acid, uracil and arginine was confirmed by fusing a GUS reporter gene. Deletion mutagenesis of carB gene resulted in reduced abilities in swimming on soft solid media and in forming biofilm on polystyrene microtiter plates. CONCLUSIONS: From these results, we concluded that carAB operon was involved in multiple biological processes in X. citri subsp. citri.

Xanthomonas citri subsp. citri type III effector PthA4 directs the dynamical expression of a putative citrus carbohydrate-binding protein gene for canker formation.

Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker, elicits canker symptoms in citrus plants because of the transcriptional activator-like (TAL) effector PthA4, which activates the expression of the citrus susceptibility gene CsLOB1. This study reports the regulation of the putative carbohydrate-binding protein gene Cs9g12620 by PthA4-mediated induction of CsLOB1 during Xcc infection. We found that the transcription of Cs9g12620 was induced by infection with Xcc in a PthA4-dependent manner. Even though it specifically bound to a putative TAL effector-binding element in the Cs9g12620 promoter, PthA4 exerted a suppressive effect on the promoter activity. In contrast, CsLOB1 bound to the Cs9g12620 promoter to activate its expression. The silencing of CsLOB1 significantly reduced the level of expression of Cs9g12620, which demonstrated that Cs9g12620 was directly regulated by CsLOB1. Intriguingly, PhtA4 interacted with CsLOB1 and exerted feedback control that suppressed the induction of expression of Cs9g12620 by CsLOB1. Transient overexpression and gene silencing revealed that Cs9g12620 was required for the optimal development of canker symptoms. These results support the hypothesis that the expression of Cs9g12620 is dynamically directed by PthA4 for canker formation through the PthA4-mediated induction of CsLOB1.

Developing an enhanced chimeric permuted intron-exon system for circular RNA therapeutics.

Rationale: Circular RNA (circRNA) therapeutics hold great promise as an iteration strategy in messenger RNA (mRNA) therapeutics due to their inherent stability and durable protein translation capability. Nevertheless, the efficiency of RNA circularization remains a significant constraint, particularly in establishing large-scale manufacturing processes for producing highly purified circRNAs. Hence, it is imperative to develop a universal and more efficient RNA circularization system when considering synthetic circRNAs as therapeutic agents with prospective clinical applications. Methods: We initially developed a chimeric RNA circularization system based on the original permuted intron-exon (PIE) and subsequently established a high-performance liquid chromatography (HPLC) method to obtain highly purified circRNAs. We then evaluated their translational ability and immunogenicity. The circRNAs expressing human papillomavirus (HPV) E7 peptide (43-62aa) and dimerized receptor binding domain (dRBD) from SARS-CoV-2 were encapsulated within lipid nanoparticles (LNPs) as vaccines, followed by an assessment of the in vivo efficacy through determination of antigen-specific T and B cell responses, respectively. Results: We have successfully developed a universal chimeric permuted intron-exon system (CPIE) through engineering of group I self-splicing introns derived from Anabaena pre-tRNALeu or T4 phage thymidylate (Td) synthase gene. Within CPIE, we have effectively enhanced RNA circularization efficiency. By utilizing size exclusion chromatography, circRNAs were effectively separated, which exhibit low immunogenicity and sustained potent protein expression property. In vivo data demonstrate that the constructed circRNA vaccines can elicit robust immune activation (B cell and/or T cell responses) against tumor or SARS-CoV-2 and its variants in mouse models. Conclusions: Overall, we provide an efficient and universal system to synthesize circRNA in vitro, which has extensive application prospect for circRNA therapeutics.

Factors affecting perception of laypeople and dental professionals toward different smile esthetics.

Abstract Background: /purpose: Several factors such as identity, income, and age potentially associated with smile perceptions. This study aimed to identify the factors affecting the smile esthetic perception in different identities (layperson, general dentist and orthodontist) and to detect the extent of their association with smile perception. Materials and methods: Extraoral photographs in frontal, lateral, and three-quarter views were shot and adjusted on Adobe Photoshop into 95 smile photographs with different smile patterns. Based on these photographs, the investigators were asked to fill the online questionnaire. Pearson chi-square test and logistic regression were used for statistical analyses. Results: Identity, gender, age, and treatment experience were noted to affect smile esthetic perception. In addition, the perception of smile esthetics was significantly different among frontal, lateral, and three-quarters views regarding the arc ratio, most posterior teeth exposure, upper teeth exposure, and lower teeth exposure. Conclusion: Identity, gender, age, and treatment experience influence the smile esthetics perception, with a significant difference in the results of the esthetic perception based on the 3 smile views. Of all demographic factors, identity had a strong relation to the perception of smile attractiveness. Nevertheless, additional studies are needed to realize how the demographic factors influence people's perception of smile esthetics, particularly in the three-quarter and lateral views.

Bridging Connectivity Issues in Digital Access and Literacy: Reflections on Empowering Vulnerable Older Adults in Singapore.

This article describes a ground-up initiative for a volunteer-run digital literacy program in Singapore targeting vulnerable older adults, focusing on the barriers faced in running this program and training these beneficiaries. It further offers possible solutions to overcome these hurdles, providing insight for individuals or organizations seeking to start similar ground-up initiatives.

Prevalence and risk factors for hypercalcemia among non-dialysis patients with chronic kidney disease-mineral and bone disorder.

PURPOSE: To examine the prevalence and risk factors for hypercalcemia among non-dialysis chronic kidney disease (CKD) patients with mineral and bone disorder (MBD). METHODS: A retrospective cohort study was conducted in Singapore General Hospital, involving all CKD stage 4 and 5 pre-dialysis patients who were on treatment for MBD in June 2016. Each patient was followed up for 1 year and screened for hypercalcemia episodes. Mild, moderate and severe hypercalcemia were defined as corrected calcium of 2.47-3.00, 3.01-3.50 and ≥ 3.51 mmol/l respectively. Patients who were on dialysis, post-renal transplant, post-parathyroidectomy or had no calcium levels taken during the study period were excluded. Details related to patients' clinical information and hypercalcemia episodes were collected. Multivariate logistic regression analysis was performed to evaluate risk factors for hypercalcemia. RESULTS: Of 557 patients, 75 (13.4%) patients developed hypercalcemia. There were 120 (97.6%) mild and 3 (2.4%) moderate hypercalcemia episodes. The daily elemental calcium intake from phosphate binders and usage of vitamin D analogues did not differ between patients with and without hypercalcemia (p > 0.05). After adjusting for covariates, lower baseline iPTH level [odds ratio (OR) 0.96, 95% CI 0.93-0.99], history of hypercalcemia in past 1 year (OR 11.11, 95% CI 3.36-36.75) and immobility (OR 3.34, 95% CI 1.34-8.40) were associated with increased hypercalcemia risk. CONCLUSION: Hypercalcemia affects a significant proportion of pre-dialysis patients with MBD. More studies should be undertaken to evaluate other risk factors associated with hypercalcemia.

Antihistamines prescribed off-label among paediatric patients at a tertiary care hospital setting in Malaysia.

Background Antihistamines are widely prescribed to children but should be used with caution in young children. Objective To determine the paediatric prescribing pattern of antihistamines with a focus on the off-label prescribing and factors that influence such prescribing. Setting Paediatric wards of a tertiary care hospital setting in Malaysia. Methods The pharmacy-based computer system and medical records were used to collect the required data. Labelling status of each antihistamine was determined based on the information provided in the product leaflets. Main outcome measure Antihistamines prescribed off-label and factors associated with such prescribing. Results Of the 176 hospitalised children aged <18 years prescribed with an antihistamine in the year 2012, 60.8 % received it in an off-label manner. Of 292 antihistamine prescription items, 55.5 % were prescribed off-label. Loratadine (35.3 %) was the most frequently prescribed antihistamine and chlorpheniramine maleate (34.0 %) was the most common antihistamine prescribed off-label. The main reason for the off-label prescribing of antihistamines was prescribing at higher than the recommended dose (30.2 %). Binary logistic regression showed that children aged <2 years (OR 12.65; 95 % CI 2.87-55.67) and the number of medications received (OR 1.14; 95 % CI 1.00-1.29) were significant predictors for the off-label prescribing of antihistamines. Conclusion Prescribing antihistamines for children in an off-label manner was prevalent at the studied locations and warrants further investigation on the consequences of such prescribing.

[Apolipoprotein B is associated with metabolic syndrome in Chinese pedigrees with familial hyperlipidemia].

OBJECTIVE: To investigate the prevalence of metabolic syndrome (MS) as well as the potential predictors in families with familial combined hyperlipidemia (FCHL), familial hypertriglyceridemia (FHTG), familial hypercholesterolemia (FH) and normolipidemic families in China. METHODS: The prevalence of MS was identified among 70 different families with 560 individuals aged > or = 20, including 43 FCHL families with 379 individuals, 3 FHTG families with 30 individuals, 16 FH families with 102 individuals and 8 normolipidemic families with 49 individuals. Diagnosis of MS was based on the modified criteria of National Cholesterol Education Program, US, substituting body mass index for waist circumference. Multivariate logistic regression was used to analyze the association between MS and different pedigrees. RESULTS: MS was identified in 60.7% of the FCHL patients and 71.4% of the FHTG patients. The prevalence of MS in the family members was 36.7% for the FCHL families, 33.3% for the FHTG families, 17.6% for the FH families, and 16.3% for the normolipidemic families, with an odds ratio (OR) of 2.97 (95% CI 1.29 to 7.07) in the FCHL families compared with in the normolipidemic families. Multivariate logistic regression showed an association between apolipoprotein (apo) B and MS with an OR of 1.05 (1.03 to 1.07) in the FCHL families, an OR of 1.26 (1.03 to 1.55) in the FHTG families, and an OR of 1.07 (1.01 to 1.12) in the FH families, independent of variables such as age, gender, apoA1, and LDL cholesterol, but showed no association in the normolipidemic families (P >0.05). Similarly, apo A1 provided an OR of 0.95 (0.94 to 0.97) in the FCHL families and an OR of 0.94 (0.90 to 0.99) in the FH families, but neither in the FHTG families nor in the normolipidemic families (both P >0.05). CONCLUSION: Apo B may be regarded as a relevant factor in the assessment of MS in FCHL, FHTG and FH families in Chinese. However, this finding needs to be verified by prospective studies in diverse ethnicities and warrants additional studies to elucidate the possible mechanisms linking apoB to MS.

Location of the mandibular canal and thickness of the occlusal cortical bone at dental implant sites in the lower second premolar and first molar.

The objective of this study was to evaluate the location of the mandibular canal and the thickness of the occlusal cortical bone at dental implant sites in the lower second premolar and lower first molar by using dental cone-beam computed tomography (CBCT). Seventy-nine sites (47 second premolar and 32 first molar sites) were identified in the dental CBCT examinations of 47 patients. In this study, 4 parameters were measured: (1) MC-the distance from the mandibular canal to the upper border of the mandible; (2) CD-the distance from the mandibular canal to the buccal border of the mandible; (3) MD-the distance from the mandibular canal to the lingual border of the mandible; (4) TC-the thickness of the cortical bone at the occlusal side. A statistical analysis was employed to compare the size and differences between these 4 parameters at the lower second premolar and lower first molar. Regarding the MC and MD, the experimental results showed no statistical difference between the first molar and second premolar. However, the TC for the second premolar was greater than that of the first molar. Thus, careful consideration is necessary in choosing the size of and operation type for dental implants.