Dissecting infectious bronchitis virus-induced host shutoff at the translation level.

Viruses have evolved a range of strategies to utilize or manipulate the host's cellular translational machinery for efficient infection, although the mechanisms by which infectious bronchitis virus (IBV) manipulates the host translation machinery remain unclear. In this study, we firstly demonstrate that IBV infection causes host shutoff, although viral protein synthesis is not affected. We then screened 23 viral proteins, and identified that more than one viral protein is responsible for IBV-induced host shutoff, the inhibitory effects of proteins Nsp15 were particularly pronounced. Ribosome profiling was used to draw the landscape of viral mRNA and cellular genes expression model, and the results showed that IBV mRNAs gradually dominated the cellular mRNA pool, the translation efficiency of the viral mRNAs was lower than the median efficiency (about 1) of cellular mRNAs. In the analysis of viral transcription and translation, higher densities of RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) reads were observed for structural proteins and 5' untranslated regions, which conformed to the typical transcriptional characteristics of nested viruses. Translational halt events and the number of host genes increased significantly after viral infection. The translationally paused genes were enriched in translation, unfolded-protein-related response, and activation of immune response pathways. Immune- and inflammation-related mRNAs were inefficiently translated in infected cells, and IBV infection delayed the production of IFN-ß and IFN-λ. Our results describe the translational landscape of IBV-infected cells and demonstrate new strategies by which IBV induces host gene shutoff to promote its replication. IMPORTANCE: Infectious bronchitis virus (IBV) is a γ-coronavirus that causes huge economic losses to the poultry industry. Understanding how the virus manipulates cellular biological processes to facilitate its replication is critical for controlling viral infections. Here, we used Ribo-seq to determine how IBV infection remodels the host's biological processes and identified multiple viral proteins involved in host gene shutoff. Immune- and inflammation-related mRNAs were inefficiently translated, the translation halt of unfolded proteins and immune activation-related genes increased significantly, benefitting IBV replication. These data provide new insights into how IBV modulates its host's antiviral responses.

Effect of mPEG-PLGA on Drug Crystallinity and Release of Long-Acting Injection Microspheres: In Vitro and In Vivo Perspectives.

PURPOSE: Traditional progesterone (PRG) injections require long-term administration, leading to poor patient compliance. The emergence of long-acting injectable microspheres extends the release period to several days or even months. However, these microspheres often face challenges such as burst release and incomplete drug release. This study aims to regulate drug release by altering the crystallinity of the drug during the release process from the microspheres. METHODS: This research incorporates methoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide) (mPEG-PLGA) into poly(lactide-co-glycolide) (PLGA) microspheres to enhance their hydrophilicity, thus regulating the release rate and drug morphology during release. This modification aims to address the issues of burst and incomplete release in traditional PLGA microspheres. PRG was used as the model drug. PRG/mPEG-PLGA/PLGA microspheres (PmPPMs) were prepared via an emulsification-solvent evaporation method. Scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), and differential scanning calorimetry (DSC) were employed to investigate the presence of PRG in PmPPMs and its physical state changes during release. RESULTS: The addition of mPEG-PLGA altered the crystallinity of the drug within the microspheres at different release stages. The crystallinity correlated positively with the amount of mPEG-PLGA incorporated; the greater the amount, the faster the drug release from the formulation. The bioavailability and muscular irritation of the long-acting injectable were assessed through pharmacokinetic and muscle irritation studies in Sprague-Dawley (SD) rats. The results indicated that PmPPMs containing mPEG-PLGA achieved low burst release and sustained release over 7 days, with minimal irritation and self-healing within this period. PmPPMs with 5% mPEG-PLGA showed a relative bioavailability (Frel) of 146.88%. IN CONCLUSION: In summary, adding an appropriate amount of mPEG to PLGA microspheres can alter the drug release process and enhance bioavailability.

Identification of transcriptionally-active human papillomavirus integrants through nanopore sequencing reveals viable targets for gene therapy against cervical cancer.

Integration of the human papillomavirus (HPV) genome into the cellular genome is a key event that leads to constitutive expression of viral oncoprotein E6/E7 and drives the progression of cervical cancer. However, HPV integration patterns differ on a case-by-case basis among related malignancies. Next-generation sequencing technologies still face challenges for interrogating HPV integration sites. In this study, utilizing Nanopore long-read sequencing, we identified 452 and 108 potential integration sites from the cervical cancer cell lines (CaSki and HeLa) and five tissue samples, respectively. Based on long Nanopore chimeric reads, we were able to analyze the methylation status of the HPV long control region (LCR), which controls oncogene E6/E7 expression, and to identify transcriptionally-active integrants among the numerous integrants. As a proof of concept, we identified an active HPV integrant in between RUNX2 and CLIC5 on chromosome 6 in the CaSki cell line, which was supported by ATAC-seq, H3K27Ac ChIP-seq, and RNA-seq analysis. Knockout of the active HPV integrant, by the CRISPR/Cas9 system, dramatically crippled cell proliferation and induced cell senescence. In conclusion, identifying transcriptionally-active HPV integrants with Nanopore sequencing can provide viable targets for gene therapy against HPV-associated cancers.

Assessment Methods and Intervention Strategies for Cleft-Related Lateral Misarticulation in Chinese-Speaking Children.

The aim of this study was to analyze the characteristics and error speech features of cleft-related lateral misarticulation and provide a basis for clinical evaluation and rational intervention. Participants who were diagnosed with lateral misarticulation after cleft palate repairment were 126 children aged 4, 6 to 16, and 11, and they had complete palatopharyngeal closure, no abnormalities in their speech organs and occlusion, and no hearing or intellectual impairments. The Chinese standard pronunciation clarity word list, the American KAY CSL4500, the Beijing Yangchen YF-16 computer speech analysis workstation, soundproof rooms, Wechsler scales of intelligence-fourth edition, and audiometers were used to evaluate the cleft-related lateral misarticulation. Statistical analysis was performed on the age, gender, error rate, corner of the mouth deviation direction, comorbidity, duration of intervention, period of treatment, and therapeutic effect of concentrated or normal intervention group in different patients. Our results showed that 2 to 3 straight stripes were visible at the onset of consonants /ti:/ /t'i:/, and 3 clear straight lines were visible in /tʂ/, indicating that the lateralized sound had 2 or 3 bursts and lasted for 1 to 2 ms. The onset age of lateralized sound was mostly below 12 years old. Chinese lateralized sound mainly occurred in vowel /i:/, and the occurrence rate of consonants with tongue surface /tɕ]/ /tɕ'/ /ɕ/ was the highest. In addition, the corner of the mouth deviation was also an indicator of lateralization sound, and other types of speech disorders mostly accompanied it. There was a significant difference in the improvement of speech clarity between the concentrated intervention group and the normal group before and after treatment. The 2 groups' average duration and course of treatment were not significantly different. Still, the period of concentrated intervention was shortened considerably, and the speech clarity of both groups of children after treatment exceeded 96%, reaching a normal level.

Identification and Characterization of Colletotrichum Species Associated with Anthracnose Disease of Plum.

Plum (Prunus salicina Lindl.) is commercially cultivated worldwide for the high levels of nutrients in the fruit. In recent years, anthracnose has been severe in some plum planting areas in China, resulting in a large number of necrotic leaves, blight and pre-mature leaf fall. In this study, anthracnose samples of plum leaves were collected from Hezhou, Guilin and Lipu, Guangxi Province, and Meishan city, Abe Tibetan and Qiang autonomous prefecture of Sichuan Province. Characteristics of mycelia on PDA, morphology of appressoria and conidia, and analysis of sequences of several marker regions (internal transcribed spacer [ITS] region, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], histone H3 [HIS3], actin [ACT], ß-tubulin [TUB2], and the intergenic region between apn2 and MAT1-2-1 [ApMat]). The resulting 101 Colletotrichum isolates obtained were identified as eight species: C. fructicola (50.5%), C. siamense (24.8%), C. karsti (8.9%), C. plurivorum (7.9%), C. aeschynomenes (3.9%), C. gloeosporioides (2%), C. celtidis (1%) and C. phyllanthi (1%). Representatives of all eight Colletotrichum species were found to cause disease on wounded leaves of plum seedlings in pathogenicity assays. As far as we are aware, this is the first report of anthracnose of plum caused by C. celtidis and C. phyllanthi in China.

First Report of Anthracnose Caused by Colletotrichum plurivorum on Passion fruit in Guangxi, China.

Passion fruit (Passiflora edulis Sims.) is popular for its rich taste and nutritional value. The planting area of passion fruit in Guangxi has reached 24,300 ha, with an annual output of 380,000 t (Qian 2023). In March 2023, leave spots on more than half of the plants (cv. Qinmi "NO.9"). Moreover, the incidence of disease on the leaves was approximately 20% in Shabu Town, Qinnan District, Qinzhou City, Guangxi, China (N20˚54'-22˚41', E107˚27'-109˚56'). Leaf diseases were orbicular or irregular in shape, white, whitish-grey, yellowish, or gray in color. When leaves were severely affected, larger blotches were formed with yellow halos. For pathogen isolation, three diseased leaf samples were collected from three gardens, respectively, and 5×5 mm tissues were cut from infected margins, surface-disinfected in 75% ethanol for 15 s, followed by 2% sodium hypochlorite for 1 min, rinsed three times with sterile water, and incubated on PDA at 25°C under 12/12 h light/darkness. After 5 days, ninety cultures were isolated, sixty isolates with similar morphology were retained, and three representative isolates BY-1, BY-2, and BY-4 were randomly selected for further study. On PDA, colonies of the three isolates displayed white or grayish-white. Conidia were single-celled, hyaline, and cylindrical, measuring 17.3±1.5 × 6.3±0.7 µm, 17.8±1.7 × 6.0±0.6 µm, and 16.3±1.4 × 6.4±0.6 µm (n=90) for BY-1, BY-2, and BY-4, respectively. Appressoria were single, brown or black, and irregular in shape, measuring 10.2±1.1×6.5±0.5 µm, 10.5±1.3×7.3±0.6, and 10.9±0.8×7.0±0.8 (n=90) for BY-1, BY-2, and BY-4, respectively. These morphological characteristics were similar to Colletotrichum spp. as previously described (Damm et al. 2019). The isolates were further identified by sequencing the internal transcribed spacer (ITS-ITS1/ITS4), glyceraldehyde-3-phosphate dehydrogenase (GAPDH-GDF/GDR), actin (ACT-512F/783R), partial sequences of the chitin synthase 1 (CHS-1-79F/354R), and beta-tubulin 2 (TUB2-T1/Bt2b) (Zhang et al. 2023). All sequences were deposited in GenBank (ITS: OR741759 to OR741761, GAPDH: OR767654 to OR767656, ACT: OR767657 to OR767659, CHS-1: OR767660 to OR767662, TUB2: OR767651 to OR767653). A phylogenetic tree was built with RAxML version 8.2.10 based on concatenated sequences of ITS-GAPDH-ACT-CHS-1-TUB2. The results revealed that the three isolates clustered with C. plurivorum. To confirm the pathogenicity of the three isolates, attached leaves of healthy 5-month-old passion fruit plants were injured in the middle region with sterile toothpicks and inoculated with 20 µL of spore suspension (106 conidia/mL), and the noninoculated control received 0.05% Tween-20 (6 leaves/plant, 3 plants/treatment). The inoculated plants were kept in a greenhouse at 25°C and covered with plastic bags to maintain high humidity. After 9 days, all inoculated leaves were symptomatic, whereas no symptoms were observed in the control. C. plurivorum was reisolated from infected leaves, confirming Koch's postulates. C. plurivorum has been reported to infect Abelmoschus esculentus (Batista et al. 2020) and Carya illinoinensis in China (Zhang et al. 2023). However, this is the first report of anthracnose caused by C. plurivorum on passion fruit in China. The results can provide a robust basis for scientific prevention and control of anthracnose.

Synchronous Computed Tomography-Guided Percutaneous Transthoracic Needle Biopsy and Microwave Ablation for Highly Suspicious Malignant Pulmonary Ground-Glass Nodules.

INTRODUCTION: There is no consensus regarding the most appropriate management of suspected malignant pulmonary ground-glass nodules (GGNs). OBJECTIVE: We aimed to explore the feasibility and safety of synchronous computed tomography-guided percutaneous transthoracic needle biopsy (PTNB) and microwave ablation (MWA) for patients highly suspicious of having malignant GGNs. METHODS: We retrospectively reviewed medical records between July 2020 and April 2023 from our medical center. Eligible patients synchronously underwent PTNB and MWA (either MWA immediately after PTNB [PTNB-first group] or PTNB immediately after MWA [MWA-first group]) at the the physician's discretion. We analyzed the rate of definitive diagnosis and technical success, the length of hospital stay, the postoperative efficacy, and periprocedural complications. RESULTS: Of 65 patients who were enrolled, the rate of definitive diagnosis was 86.2%, which did not differ when stratified by the tumor size, the consolidation-to-tumor ratio, or the sequence of the two procedures (all p > 0.05). The diagnostic rate of malignancy was 83.1%. After the median follow-up duration of 18.5 months, the local control rate was 98.2% and the rate of completed ablation was 48.2%. The rate of perioperative minor and major complications was 44.6% and 6.2%, respectively. The most common adverse events included pain, cough, and mild hemorrhage. Mild hemorrhage took place significantly less frequently in the MWA-first group than in the PTNB-first group (16.7% vs. 45.5%, p < 0.05). CONCLUSION: Synchronous PTNB and MWA are feasible and well tolerated for patients highly suspicious of having malignant GGNs, providing an alternative option for patients who are ineligible for surgical resection.

Fruit Anthracnose on Cavendish bananas Caused by Colletotrichum fructicola in Guangxi, China.

Cavendish banana (Musa spp. AAA group) is one of the main fruit crops worldwide. It is widely planted in Guangdong, Hainan, Guangxi, Fujian and Yunnan provinces in southern China. In November 2020, banana fruits with anthracnose symptoms were collected from Dayu Town (N 23.17°, E 109.80°), Guigang City, and Chengjun Town (N 22.60°, E 110.00°), Yulin City, Guangxi Province, China, where the disease was found on about 70% of the banana plants, and on individual fruit, up to 10% of the surface was covered with symptoms. The symptoms initially began with rust-colored spots on the surface of the immature fruit, which gradually became sunken and cracked as the disease progressed. Small tissues (5×5 mm) from the pericarp at the junction of disease and health were surface-disinfected in 75% ethanol for 10 s, 2% sodium hypochlorite (NaClO) for 1 min, and washed three times in sterile water. Tissue pieces were placed on potato dextrose ager (PDA) and incubated at 25°C. Fifty-nine morphologically similar colonies were obtained after 5 days of incubation, with 100% isolation frequency. Of 59 isolates, GG1-3 isolated from Guigang City and YL4-2 isolated from Yulin City were selected as representative strains for intensive study. Mycelia were off-white for both isolates and conidia obtained from PDA were cylindrical, unicellular, hyaline and obtuse ends, with sizes of 11.5 ± 1.8×3.9 ± 0.8 µm (n=60) and 11.5 ± 1.6×4.1 ± 0.6 µm (n=60) for GG1-3 and YL4-2, respectively (Prihastuti et al. 2009). Genomic DNA was extracted from 7-day-old aerial mycelia using a DNAsecure Plant Kit (Tiangen Biotech, China). The internal transcribed spacer (ITS), the intergenic region of apn2 and MAT1-2-1 (ApMAT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified and sequenced (White et al. 1990; Silva et al.2012; Templeton et al. 1992). Sequences were deposited in GenBank (ITS, OR596961 to OR596962; GAPDH, OR661771 to OR661772; APMAT, OR661773 to OR661774) and showed 100% identities with the corresponding type strains sequences of C. fructicola. Phylogenetic tree was constructed with software raxmlGUI v.2.0.0 based on sequences of multiple loci (ITS, GAPDH and ApMAT) and Maximum Likelihood method. Phylogenetic analysis revealed that the two isolates and C. fructicola were clustered in the same clade, with 94% bootstrap support. According to morphology and phylogenetic analysis, the two isolates GG1-3 and YL4-2 were identified as C. fructicola. For pathogenicity tests, healthy fruits were surface sterilized with 75% ethanol followed by a wash with sterilized water. Five adjacent needle punctures in a 5-mm-diameter circle were made with a sterilized needle on healthy fruits, followed by inoculation with 20 µL of conidial suspension (106 spores/ml), and sterilized water was used as controls. All banana fruit were incubated in a humid chamber at 28°C. After 4 days, all inoculated fruits showed visible symptoms and had rust-colored spots on the margins, while control banana fruits remained symptomless. The fungus was isolated from the inoculated fruit and the isolates were found to match the morphological and molecular characteristics of the original isolates, confirming Koch's hypothesis. To our knowledge, this is the first report of fruit anthracnose on Cavendish bananas caused by C. fructicola in China. This study will provide valuable information for prevention and management of anthracnose on banana fruit.

The cumulative effects of cascade reservoirs control nitrogen and phosphorus flux: Base on biogeochemical processes.

The reservoir serves as a water source, a flood control structure, a navigational aid, and also impacts the downstream ecosystem as well as the reservoir zone. However, debate exists about effectiveness of cascade reservoirs in controlling the transportation of nutrients, particularly in the Yangtze River basin, which has been significantly affected by reservoir development. This research develops a new model X-NPSEM (X with Nitrogen and Phosphorus Steady-state Reservoir Model) based on biogeochemical processes of nitrogen and phosphorus reaction for investigating the dynamic storage capacity of cascade reservoirs at both reservoir- and watershed scales. Then the cumulative effects of cascade reservoirs and the related mechanism were investigated in Fujiang watershed, China. Based on the results, cascade reservoirs retained 16.3 % of nitrogen fluxes and 37.6 % of phosphorus fluxes annually. Downstream reservoirs have higher retention rates of phosphorus (0.48/d) compared to upstream reservoirs (0.10/d), mainly due to inflow sediment. Nitrogen retention rates show seasonal variations: wet season (0.21/d) and dry season (0.17/d). These fluctuations in nitrogen retention are primarily influenced by changes in temperature rather than other factors such as operation period, nitrogen and phosphorus concentration, or the nitrogen/phosphorus ratio. In upstream, the concentration of sediment entering the reservoir plays a decisive role in the transformation of P retention from sink to source. The X-NPSRM coupler model could be used for global reservoir operation and watershed management.

Comparison of binding sites and affinity of flavonol-Cu(II) complexes with the same parent nucleus: Synthesis, DFT prediction, and coordination pattern.

This study explores the coordination dynamics between dietary polyphenols, specifically kaempferol, quercetin, and myricetin, and Cu ions in aqueous environments. A novel synthesis method for flavonol-Cu(II) coordination compounds is introduced, effectively reducing interference from free metal ions. Our results reveal consistent binding patterns of Cu ions with flavonols (2:1 ratio of flavonol to Cu(II)), predominantly at the 4,5 sites. Various analytical techniques are used to validate these coordination ratios and sites. The binding affinity of the flavonols for Cu ions follows a descending sequence: myricetin > quercetin > kaempferol. Notably, coordination with Cu ions enhances the free-radical scavenging activities of these flavonols. These findings hold substantial importance for food chemistry, biology, and medicine, providing crucial insights into the way dietary flavonols form stable structures in environments similar to human body fluids and their interactions with metal ions, opening new possibilities for their application and understanding in diverse scientific domains.

Synergistic interaction of Cu(II) with caffeic acid and chlorogenic acid in α-glucosidase inhibition.

BACKGROUND: Phenolic acids are widespread in foods and are beneficial to human health. However, the role of metal ions in influencing the binding of proteins with phenolic acids that contain the same parent nucleus structure remains unclear. This study investigated the inhibitory effect of caffeic acid (CA) and chlorogenic acid (CHA) on α-glucosidase and the biological effect of copper on this process. RESULTS: It was found that the esterification of CA with quinic acid could increase the fluorescence quenching, conformational change, and inhibitory effect of CHA on α-glucosidase. Copper ions reduced their fluorescence quenching and conformation-changing ability by binding to the neighboring phenolic hydroxyl group but also increased their ability to alter secondary structure and to inhibit α-glucosidase and in vitro anti-glycation. CONCLUSION: Overall, this study shows that the binding of copper ions to the phenolic hydroxyl group adjacent to CA and CHA synergistically inhibited α-glucosidase. The findings will offer a theoretical basis for investigating the properties of metal ions and phenolic acid in food chemistry and their potential applications in the prevention and treatment of diabetes mellitus. © 2023 Society of Chemical Industry.

Negative pressure sealing drainage technology combined with adequate irrigation for oral and maxillofacial space infection can improve serum inflammatory factor levels.

OBJECTIVE: This study aimed to assess the clinical efficacy of combining vacuum sealing drainage with full irrigation in managing oral and maxillofacial space infections and its impact on serum inflammatory factor levels in patients. METHODS: We retrospectively analyzed data from 110 patients with oral and maxillofacial space infections treated at our hospital between February 2018 and March 2022. Among them, 50 patients underwent simple negative pressure closed drainage (control group), while 60 patients received combined full irrigation using 0.9% sodium chloride solution (observation group). We compared clinical treatment outcomes, treatment duration, antibiotic usage duration, quality of life scores, Visual Analogue Scale (VAS) scores, changes in serum IL-6 and TNF-α levels before and after treatment, and the incidence of complications between the two groups. Additionally, we conducted an analysis of risk factors influencing patient prognosis. RESULTS: The observation group exhibited significantly superior treatment efficacy compared to the control group (P < 0.05). Treatment and antibiotic usage durations were shorter in the observation group (P < 0.05). VAS scores after treatment were significantly lower in the observation group (P < 0.05). Serum inflammatory factors improved significantly in both groups after treatment, with a more substantial improvement observed in the observation group (P < 0.05). Post-treatment quality of life was significantly higher, and the incidence of complications was lower in the observation group (P < 0.05). The choice of treatment method independently influenced patient prognosis (P < 0.05). CONCLUSION: Combining vacuum sealing drainage with full irrigation is an effective approach for managing oral and maxillofacial space infections. This treatment leads to improved clinical symptoms, reduced inflammatory responses, decreased pain intensity, and enhanced quality of life while maintaining safety.

Comprehensive investigation on non-volatile and volatile flavor compounds in the Morchella sextelata and Morchella importuna by UPLC-MS/MS and GC × GC-TOF-MS.

Morchella sextelata and Morchella importuna are the main cultivars of morel. However, the key compounds affecting their flavors (taste and odor) are currently unknown. Here, an ultra performance tandem mass spectrometry combined with two-dimensional gas chromatography-time-of-flight mass spectrometry method was used to detect and relatively quantify the metabolites in both morel cultivars. A total of 631 non-volatile compounds and 242 volatile compounds were identified. The odor activity value was calculated to assess the contribution of key odor volatile. The results indicated that M. importuna had a sweeter flavor than M. sextelata. The former posed more prominent mushroom flavor than the latter based on the correlation analysis of the metabolites. The flavor differences of the two morel cultivars are highly relevant with the content of lipids, carbohydrates, amino acids and derivatives, alcohols and ketones. This study provides new insights into the theoretical basis for the flavor differences in both morel cultivars.

Screening and characterization of biocontrol bacteria isolated from Ageratum conyzoides against Collectotrichum fructicola causing Chinese plum (Prunus salicina Lindl.) anthracnose.

Chinese plum (Prunus salicina Lindl.) is a nutritionally and economically important stone fruit widely grown around the world. Anthracnose, caused by Collectotrichum spp., is one of the primary biotic stress factors limiting plum production. Medicinal plants may harbor rhizospheric or endophytic microorganisms that produce bioactive metabolites that can be used as anthracnose biocontrol agents. Here, 27 bacterial isolates from the medicinal plant A. conyzoides with diverse antagonistic activities against C. fructicola were screened. Based on morphological, physiological, biochemical, and molecular characterization, 25 of these isolates belong to different species of genus Bacillus, one to Pseudomonas monsensis, and one more to Microbacterium phyllosphaerae. Eight representative strains showed high biocontrol efficacy against plum anthracnose in a pot experiment. In addition, several Bacillus isolates showed a broad spectrum of inhibitory activity against a variety of fungal phytopathogens. Analysis of the volatile organic compound profile of these eight representative strains revealed a total of 47 compounds, most of which were ketones, while the others included alkanes, alkenes, alcohols, pyrazines, and phenols. Overall, this study confirmed the potential value of eight bacterial isolates for development as anthracnose biocontrol agents.

[Inhibitory Effects and Mechanisms of Three Benzodiazepines on Helicobacter pylori].

Objective To explore the inhibitory effects and mechanisms of benzodiazepines on Helicobacter pylori (Hp).Methods The Hp international standard strain ATCC43504 was treated with benzodiazepines diazepam,midazolam,and remimazolam,respectively.The treatments with amoxicillin and clarithromycin were taken as the positive controls,and that with water for injection as the negative control.The inhibition zone of each drug was measured by the disk diffusion method.The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of each drug against Hp were determined.Hp suspension was configured and treated with diazepam and midazolam,respectively.The bacterial suspension without drug added was used as the control group.The concentration of K+ in each bacterial suspension was measured by an automatic biochemical analyzer before drug intervention(T0)and 1(T1),2(T2),3(T3),4(T4),5(T5),6(T6),and 7 h(T7)after intervention.Hp urease was extracted and treated with 1/2 MIC diazepam,1 MIC diazepam,2 MIC diazepam,1/2 MIC midazolam,1 MIC midazolam,2 MIC midazolam,1 mg/ml acetohydroxamic acid,and water for injection,respectively.The time required for the rise from pH 6.8 to pH 7.7 in each group was determined by the phenol red coloring method.Results The inhibition zones of diazepam,midazolam,remimazolam,amoxicillin,clarithromycin,and water for injection against Hp were 52.3,42.7,6.0,72.3,60.8,and 6.0 mm,respectively.Diazepam and midazolam showed the MIC of 12.5 µg/ml and 25.0 µg/ml and the MBC of 25 µg/ml and 50 µg/ml,respectively,to Hp.The concentrations of K+ in the diazepam,midazolam,and control groups increased during T1-T7 compared with those at T0(all P<0.01).The concentration of K+ in diazepam and midazolam groups during T1-T4 was higher than that in the control group(all P<0.01).The time of inhibiting urease activity in the 1/2 MIC diazepam,1 MIC diazepam,2 MIC diazepam,1/2 MIC midazolam,1 MIC midazolam,and 2 MIC midazolam groups was(39.86±5.11),(36.52±6.65),(38.58±4.83),(39.25±6.19),(36.36±4.61),and(35.81±6.18)min,respectively,which were shorter than that in the acetohydroxamic acid group(all P<0.01)and had no significance differences from that in the water for injection group(all P>0.05).Conclusion Diazepam and midazolam exerted inhibitory effects on Hp,which may be related to the cleavage of Hp cells rather than inhibiting urease.

Influence of lifestyle on suboptimal health: Insights from a national cross-sectional survey in China.

Background: Suboptimal health status (SHS) is a non-clinical or pre-disease state between optimal/ideal health and disease. While its etiology remains unclear, lifestyle is considered one of the most important risk factors. We aimed to examine the effects of lifestyles on SHS through a nationwide survey in China. Methods: We conducted a cross-sectional survey in 148 cities across China between 20 June and 31 August 2022, on 30 505 participants from rural and urban communities gathered through stratified quota sampling. We measured SHS with the Short-Form Suboptimal Health Status Questionnaire (SHSQ-SF). We gathered information on participants' lifestyles (ie, smoking, alcohol consumption, breakfast habits, weekly food delivery frequency, intermittent fasting, sleep duration and physical activities) through face-to-face interview. We determined the relationship between lifestyle and SHS logistic regression analysis by based on odds ratios (ORs) and 95% confidence intervals (CIs). Results: We included 22 897 participants (female: 13 056, male: 9841), 12 108 (52.88%) of whom reported exposure to SHS. After adjusting for demographic characteristics, individuals who currently smoked (OR = 1.165; 95% CI = 1.058-1.283) and those who drank alcohol (OR = 1.483; 95% CI = 1.377.1.596) were at a higher risk of SHS than those who have never done either. In a dose-response way, takeaway food consumption was associated with a higher risk of SHS, while increased frequency of breakfast and mild-intensity exercise conversely reduced said risk. Individuals with shorter sleep duration had a higher risk of SHS when compared to those who slept for more than seven hours per day. Conclusions: We observed a relatively high prevalence of SHS across China, highlighting the importance of lifestyle in health promotion. Specifically, adopting healthy dietary habits, engaging in regular physical activity, and ensuring high-quality sleep are key in preventing SHS. Registration: Chinese Clinical Trial Registry (ChiCTR2200061046).

Online courses for dentist continuing education: A new trend after the COVID-19 pandemic.

Background/purpose: Online courses have been widely used in all levels of education during the COVID-19 pandemic. This study explored the effectiveness of a dentist continuing education (DCE) course through the online devices in Taiwan. Materials and methods: The practicing dentists who participated in the online course of dental radiation technology for DCE offered by the Taiwan Dental Association (TWDA) in October 2022 and in March 2023 were enrolled in this study. The composition of participating dentists was confirmed by the public inquiry system and their learning effectiveness was evaluated by a questionnaire-based survey after the online DCE class. Results: All participating dentists (132 in October 2022 and 117 in March 2023) obtained consistent good learning outcomes in this online DCE course. Of these 249 dentists, there were 170 (68.27%) males and 79 (31.73%) females, 127 (51.00%) dental specialists and 122 (49.00%) general dentists, as well as 50 (20.08%) hospital dentists and 199 (79.92%) clinic dentists. The participation rates for this course of practicing dentists in non-municipalities (4.70%), counties (3.88%), eastern region (8.08%), and outlying islands (3.60%) were much higher than those in municipalities (0.79%), cities (1.16%), and the western region including the northern region (0.88%), central region (1.96%), and southern region (1.94%), respectively. Conclusion: The participating dentists express positive feedback on the online DCE courses, and the online DCE courses can reduce the urban-rural gap in dental education resources. The use of online DCE courses in dental education will be a future trend.

Leaf Spot Caused by Colletotrichum siamense, C. fructicola, and C. aeschynomenes on Ixora chinensis in Guangxi, China.

Ixora chinensis Lam., an important ornamental flower, has become more and more popular in the southwest and southeast regions of China for its bright and abundant flowers (Li et al. 2019). In March 2022, 100% I. chinensis plants showed typical anthracnose symptoms on leaf in Nanning, Guangxi, China (108°22' N, 22°48' E). The central areas of lesions were grayish white with small black particles arranged in a wheel pattern, and the edges of lesions were light red to brown. Three diseased leaf samples were collected from three gardens, respectively. 5×5 mm tissues were cut from infected margins, surface-disinfected in 75% ethanol for 10 s, 2% NaClO for 2 min, rinsed three times in sterilized distilled water, and incubated on PDA at 25°C under 12/12 h light/darkness. Eighty-three morphologically similar colonies were observed on PDA after 5 days, and eight isolates G1-3, G2-1, G3-3, W-1, W-2, LCH2-1, LCH3-3, and LCH4-1 were selected for further study. Genomic DNA of these isolates were extracted from 7-day-old mycelia. Primer pairs ITS1/ITS4, GDF1/GDR1, T1/ßt2b, CHS Ⅰ-79F/CHS Ⅰ-354R, CL1/CL2, ACT-512F/ACT-783R, and MAT1-2-1/apn2 were used to amplify ITS loci and GAPDH, CHS-Ⅰ, CAL, ACT, ApMAT genes, respectively (Yang et al. 2009; Silva et al. 2012; Liu et al. 2015). Sequences have been deposited in GenBank (ITS: OQ771884 to OQ771891, GAPDH: OQ759576 to OQ759583, TUB2: OQ759584 to OQ759591, CHS-1: OQ759568 to OQ759575, CAL: OQ759560 to OQ759567, ACT: OQ759552 to OQ759559, ApMat: OQ759544 to OQ759551). Phylogenetic analysis was performed with raxmlGUI v.2.0.0. based on combined sequences of ITS, GAPDH, TUB2, CHS-1, CAL, ACT, and ApMAT using maximum parsimony analysis. The results revealed that isolates G2-1 and W-2 were clustered with Collectrichum fructicola, G3-3, W-1, G1-3, LCH2-1, and LCH3-3 were clustered with C. siamense, and LCH4-1 was clustered with C. aeschynomenes. Three representative isolates W-2, G3-3, and LCH4-1 were selected for morphology and pathogenicity observation. On PDA, the colonies of three isolates presented white to grey cottony mycelia，from the margin to the center, W-2 was white, grey, and light gray, G3-3 showed light gray, white, and grey, LCH4-1 was white and light gray, respectively. Conidia were all hyaline, one-celled, cylindrical, and straight. The conidial sizes of W-2, G3-3, and LCH4-1 were 11.03 to 17.53 × 4.93 to 8.42 µm (n=100), 10.63 to 19.06 × 3.73 to 6.92 µm (n=100), and 11.61 to 20.39 × 3.65 to 6.67 µm (n=100), respectively. Pathogenicity tests of three isolates were conducted on leaves of 1-year-old I. chinensis plants with and without wounds, three plants for each treatment, and five leaves inoculated for each plant. Conidial suspensions (10 µL, 106 conidia/mL in 0.1% sterile Tween 20) were inoculated on each site. Control group was treated with 0.1% sterile Tween 20. All inoculated sites were covered with wet cotton, and all plants were bagged and placed in the greenhouse to maintain humidity at 25℃. After 10 days, all wounded and inoculated leaves showed leaf spot, whereas unwounded and control leaves remained asymptomatic. Koch's postulates were fulfilled by re-isolating the causal agents from diseased leaves. C. siamense and C. aeschynomenes could cause anthracnose of I. chinensis in China (Liu et al. 2016, Li et al. 2021). However, to our knowledge, this is the first report of C. fructicola infecting I. chinensis in China. This study may provide reference for further epidemiological study and prevention of anthracnose on I. chinensis.

Characterization of a novel hypovirus isolated from the phytopathogenic fungus Colletotrichum camelliae.

Some members of genus Colletotrichum are important plant pathogens. Here, we report a novel positive single-stranded RNA virus, Colletotrichum camelliae hypovirus 1 (CcHV1), from strain GXNN11-2 of Colletotrichum camelliae. The complete genome of CcHV1 is 9907 nucleotides (nt) in length and contains a single large open reading frame (ORF) from nt 352 to 9006. This ORF encodes a polyprotein with four conserved domains, namely UDP-glycosyltransferase, RNA-dependent RNA polymerase (RdRp), peptidase, and DEAD-like helicase. The CcHV1 polyprotein shares the highest similarity with Fusarium concentricum hypovirus 1. Phylogenetic analysis indicated that CcHV1 clustered with members of the genus Betahypovirus within the family Hypoviridae. This is the first report of a hypovirus in a member of the genus Colletotrichum.