T6SS and T4SS Redundantly Secrete Effectors to Govern the Virulence and Bacterial Competition in Pectobacterium PccS1.

Previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and virulence in many gram-negative bacteria. However, the role of T6SS in virulence in Pectobacterium atrosepticum remains controversial. We examined a closely related strain, PccS1, and discovered that its T6SS comprises a single-copy cluster of 17 core genes with a higher identity to homologs from P. atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS1, we found that three of the five VgrGs could be classified into group I VgrGs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and were dependent on either the T6SS or type IV secretion system (T4SS). This interaction directly governed virulence and competition. Meanwhile, supernatant proteomic analyses with strains defective in the T6SS and/or T4SS confirmed that effectors, such as FhaB, were secreted redundantly to control the virulence and suppress host callose deposition in the course of infection. Notably, this redundant secretion mechanism between the T6SS and T4SS is believed to be the first of its kind in bacteria.

Multiple genes deletion based on Cre-loxP marker-less gene deletion system for the strains from the genus of Pectobacterium.

OBJECTIVE: To introduce the Cre-loxP system for constructing marker-less multiple-gene deletion mutants in Pectobacterium, overcoming limitations of antibiotic markers and enhancing the understanding of pathogenic mechanisms. RESULTS: Firstly, a plasmid named pEX18-Cre, containing a sacB sucrose suicide gene, was constructed to express Cre recombinase in Pectobacterium. Secondly, a mutant in which the loxP-Km fragment replaced the target gene was obtained through homologous recombination double-crossover with the chromosome. Finally, pEX18-Cre was introduced into the mutant to excise the DNA between the loxP sites, thereby removing the markers and achieving multiple gene deletions. By utilizing the Cre-loxP system, we successfully constructed multiple marker-less gene deletion mutants in Pectobacterium strains. CONCLUSIONS: The Cre-loxP system efficiently creates marker-less multiple-gene deletion mutants, enhancing the study of Pectobacterium pathogenic mechanisms by overcoming antibiotic marker limitations.

Exploring the feasibility of preoperative tumor-bed boost, oncoplastic surgery, and adjuvant radiotherapy schedule in early-stage breast cancer: A phase II clinical trial.

BACKGROUND: Oncoplastic breast-conserving surgery (OBCS) improves satisfaction in patients who would fare otherwise sub-optimal cosmetic outcome, while brings challenge in tumor-bed identification during adjuvant radiotherapy. The ultra-hypofractionated breast radiotherapy further shortens treatment sessions from moderately hypofractionated regimens. To circumscribe the difficulty in tumor-bed contouring and the additional toxicity from larger boost volumes, we propose to move forward the boost session preoperatively from the adjuvant radiation part. Thus, the present study aims to evaluate the feasibility of a new treatment paradigm of preoperative primary-tumor boost before breast-conserving surgery (BCS) or OBCS followed by adjuvant ultra-hypofractionated whole-breast irradiation (u-WBRT) for patients with early-stage breast cancer. METHODS: There was a phase II study. Patients younger than 55 years old, with a biopsy confirmed mono-centric breast cancer, without lymph node involvement were enrolled. Preoperative primary-tumor boost was given by a single 10 Gy in 1 fraction, and BCS or OBCS was conducted within two weeks afterwards. Adjuvant u-WBRT (26 Gy/5.2 Gy/5 f) was given in 6 weeks postoperatively without any boost, after the full recovery from surgery. Surgical complications and patient-reported outcomes, as assessed via Breast-Q questionnaires, were documented. A propensity score matching approach was employed to identify a control group at a 1:1 ratio for BREAST-Q outcomes comparison. RESULTS: From May 2022 to September 2023, 36 patients were prospectively enrolled. Surgical complications were observed in 7 cases (19.4%), including 3 cases with Clavien-Dindo (CD) grade 1-2 and 4 cases with CD grade 3 complications. All but four patients (11.1%) started the planned u-WBRT within one week after the pre-defined due dates postoperatively (≤49 d). Four patients (11.1%) developed grade 2 radiodermatitis after chemotherapy initiation. Compared to the study group, the control patients reported higher scores in chest physical well-being ( P =0.045) and in their attitudes towards arm swelling ( P =0.01). No significant difference was detected in the other of domains (Satisfaction with Breasts, Sexual and Psychosocial Well-Being, and Adverse Effects of Radiation). With a median follow-up period of 9.8 months (2.4-18.9 mo), none had any sign of relapse. CONCLUSION: This Phase II clinical trial confirmed the technical and safety feasibility of novel radiation schedule in patients undergoing BCS or OBCS. According to the BREAST-Q questionnaire, patients who underwent novel radiation schedules reported lower satisfaction in chest physical well-being. A randomized controlled trial is necessary to further investigate these findings. Additionally, long-term follow-up is required to assess oncological outcomes.

A genome-wide association study reveals that epistasis underlies the pathogenicity of Pectobacterium.

Pectobacterium spp. are important bacterial pathogens that cause soft rot symptoms in various crops. However, their mechanism of pathogenicity requires clarity to help control their infections. Here, genome-wide association studies (GWAS) were conducted by integrating genomic data and measurements of two phenotypes (virulence and cellulase activity) for 120 various Pectobacterium strains in order to identify the genetic basis of their pathogenicity. An artificial intelligence-based software program was developed to automatically measure lesion areas on Chinese cabbage, thereby facilitating accurate and rapid data collection for virulence phenotypes for use in GWAS analysis. The analysis discovered 428 and 158 loci significantly associated with Pectobacterium virulence (lesion area) and cellulase activity, respectively. In addition, 1,229 and 586 epistasis loci pairs were identified for the virulence and cellulase activity phenotypes, respectively. Among them, the AraC transcriptional regulator exerted epistasis effects with another three nutrient transport-related genes in pairs contributing to the virulence phenotype, and their epistatic effects were experimentally confirmed for one pair with knockout mutants of each single gene and double gene. This study consequently provides valuable insights into the genetic mechanism underlying Pectobacterium spp. pathogenicity. IMPORTANCE Plant diseases and pests are responsible for the loss of up to 40% of food crops, and annual economic losses caused by plant diseases reach more than $220 billion. Fighting against plant diseases requires an understanding of the pathogenic mechanisms of pathogens. This study adopted an advanced approach using population genomics integrated with virulence-related phenotype data to investigate the genetic basis of Pectobacterium spp., which causes serious crop losses worldwide. An automated software program based on artificial intelligence was developed to measure the virulence phenotype (lesion area), which greatly facilitated this research. The analysis predicted key genomic loci that were highly associated with virulence phenotypes, exhibited epistasis effects, and were further confirmed as critical for virulence with mutant gene deletion experiments. The present study provides new insights into the genetic determinants associated with Pectobacterium pathogenicity and provides a valuable new software resource that can be adapted to improve plant infection measurements.

High-level production of a cold-active B-mannanase from Bacillus subtilis BS5 and its molecular cloning and expression.

Mannanases can be useful in the food, feed, pulp and paper industries. In this research a Bacillus subtilis strain (named Bs5) which produced high-level beta-mannanase was isolated. Maximum level of beta-mannanase (1231.41 U/ml) was reached when Bacillus subtilis Bs5 was grown on konjac powder as the carbon source for nine hours at 32 degrees C. The beta-mannanase was a typical cold-active enzyme and its optimal temperature of 35 degrees C was the lowest among those of the known mannanases from bacteria. In addition, the optimal pH was 5.0 and much wide pH range from 3.0-8.0 was also observed in the beta-mannanase. These properties make the beta-mannanase more attractive for biotechnological applications. The DNA sequence coding the beta-mannanase was cloned and the open reading frame consisted of 1089 bp encoding 362 amino acids. A phylogenetic tree of the beta-mannanase based on the similarity of amino acid sequences revealed that the beta-mannanase formed a cluster with the beta-mannanases of Bacillus subtilis, which was separated from the mannanases of fungi and other bacteria. The beta-mannanase gene could be expressed in Escherichia coli and the recombinant beta-mannanase was characterized by Western blot. This study provided a new source of carbohydrate hydrolysis enzyme with novel characteristics from Bacillus subtilis.

Efficacy and safety of Zicuiyin decoction on diabetic kidney disease: A multicenter, randomized controlled trial.

BACKGROUD: Zicuiyin (ZCY) decoction created by Xichun Zhang in the Qing dynasty has been used on diabetes mellitus and complications for more than two centuries in China. Huangkui capsule (HKC) is a listed Chinese patent medicine to treat diabetic kidney disease (DKD). To determine whether ZCY is non-inferior to HKC in the treatment of DKD, a multicenter, parallel-control, open-label, randomized clinical trial was conducted. METHODS: In this clinical trial, 88 DKD patients were recruited at three centers in Tianjin from January 2018 to December 2019. They were randomized to receive HKC (2.5 g, TID) or ZCY (crude drug amount 75 g, 150 ml, BID) for eight weeks based on routine treatment. The primary outcome was the change of estimated glomerular filtration rate (eGFR). The secondary outcomes included change of serum creatinine (SCr), urinary albumin excretion rate, 24 h urinary protein, urinary albumin-creatinine ratio, glycosylated hemoglobin A1c, symptom scores, and microbiota compositions profiles. RESULTS: The change of eGFR in HKC and ZCY groups were -7.08 ± 24.65 and 2.57 ± 18.49 ml/min/1.73 m2, respectively (p < 0.05). The 95% lower confidence limit for the difference between the estimated means was 1.93 ml/min/1.73 m2, establishing the superiority of ZCY. Compared to HKC, ZCY could significantly decrease SCr and symptom scores (p < 0.05). There were no significant differences in other outcomes between the two groups (p > 0.05). ZCY ameliorated gut microbiota dysbiosis, including increased Prevotellaceae and Lactobacillaceae and decreased Enterobacteriales, Clostridiaceae and Micrococcaceae. No severe adverse events were reported in any group. CONCLUSIONS: ZCY had better efficacy in improving and protecting kidney function. It would be an alternative option to treat DKD, especially those who decline eGFR and gut microbiota dysbiosis. TRIAL REGISTRATION: Chinese Clinical Trial Registry: ChiCTR-OON-17012076. Registered July 21, 2017.

Transcriptome of Pectobacterium carotovorum subsp. carotovorum PccS1 infected in calla plants in vivo highlights a spatiotemporal expression pattern of genes related to virulence, adaptation, and host response.

Bacterial pathogens from the genus Pectobacterium cause soft rot in various plants, and result in important economic losses worldwide. We understand much about how these pathogens digest their hosts and protect themselves against plant defences, as well as some regulatory networks in these processes. However, the spatiotemporal expression of genome-wide infection of Pectobacterium remains unclear, although researchers analysed this in some phytopathogens. In the present work, comparing the transcriptome profiles from cellular infection with growth in minimal and rich media, RNA-Seq analyses revealed that the differentially expressed genes (log2 -fold ratio ≥ 1.0) in the cells of Pectobacterium carotovorum subsp. carotovorum PccS1 recovered at a series of time points after inoculation in the host in vivo covered approximately 50% of genes in the genome. Based on the dynamic expression changes in infection, the significantly differentially expressed genes (log2 -fold ratio ≥ 2.0) were classified into five types, and the main expression pattern of the genes for carbohydrate metabolism underlying the processes of infection was identified. The results are helpful to our understanding of the inducement of host plant and environmental adaption of Pectobacterium. In addition, our results demonstrate that maceration caused by PccS1 is due to the depression of callose deposition in the plant for resistance by the pathogenesis-related genes and the superlytic ability of pectinolytic enzymes produced in PccS1, rather than the promotion of plant cell death elicited by the T3SS of bacteria as described in previous work.

[Effect of okam on inflammation and remodeling of airway of mice with ovalbumin induced asthma].

OBJECTIVE: To investigate the effect of okam on inflammation and remodeling of airway in mice with ovalbumin (OVA) induced asthma. METHODS: Thirty-two mice of Kunming strain were divided into four groups randomly: model group, glucocorticoid inhalation group, okam group and control group, with 8 mice in each group. The asthmatic mice model was reproduced by combined injection and aerosol inhalation of OVA. The mice in model group received normal saline (0.3 ml) gavage daily. The mice in glucocorticoid inhalation group received budesonide (0.4 ml, 200 mug) and normal saline (3.6 ml) inhalation. The mice in okam group were gavaged with okam daily (50 mg/kg). The controls were given normal saline instead of OVA sensitization. All mice were sacrificed 42 days later, followed by lavage of tracheo-bronchial tree of the right lung, and the right lung was saved for pathological examination. The total cell number and differentiation in bronchoalveolar lavage fluid (BALF) were counted under microscope. The expression of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) in BALF were assessed by enzyme linked immunosorbent assay (ELISA). The histological changes in the bronchi and alveoli were evaluated after hematoxylin and eosin (HE) staining. The expression of matrix metalloproteinase-9 (MMP-9) as well as the tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by immunohistochemistry. RESULTS: Compared with the model group, the total cell count and IL-4 level in BALF, and the score of pathological changes in the broncho-alveolar tissue in okam group or glucocorticoid inhalation group were lower significantly, and the IFN-gamma level elevated markedly (all P<0.01). The MMP-9, TIMP-1 expression in glucocorticoid inhalation group and the TIMP-1 expression in okam group were decreased greatly (P<0.05 or P<0.01). All of above indexes showed marked differences between control group and okam group (P<0.05 or P<0.01). There were significant changes in the total cell count, IFN-gamma, pathological changes, MMP-9 and TIMP-1 between the glucocorticoid inhalation group and the okam group (P<0.05 or P<0.01). CONCLUSION: Okam may alleviate inflammation of the bronchial and degrade the development of airway remodeling to some degree.

[Values of impulse oscillometry for pulmonary function and adhesion molecules in childhood asthma].

OBJECTIVE: Impulse oscillometry (IOS) is a novel technique for the evaluation of pulmonary function. Soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) are definitive indicators for the severity of asthma. This study aimed to explore the relationship of IOS pulmonary function with sICAM-1 and sVCAM-1, and their values in childhood asthma. METHODS: IOS via Master Screen System for pulmonary function was performed in 40 children with acute asthma and 25 healthy children. Twenty-three of 40 children with acute asthma were re-tested for IOS pulmonary function at remission. sICAM-1 and sVCAM-1 levels were measured in 23 children with acute asthma, 20 asthmatic children at remission and 16 healthy children. RESULTS: The parameters of IOS pulmonary function, R5, R20, R5-R20, X5, Fres and Zrs in children with acute asthma were significantly higher than in asthmatic children at remission and in normal controls (q= 2.91-15.61, P < 0.01 or 0.05). There were significant differences in R5, R5-R20, Fres and Zrs between the asthmatic children at remission and normal controls (q= 3.08- 9.19, P < 0.01 or 0.05). sICAM-1 and sVCAM-1 levels in children with acute asthma were significantly higher than in asthmatic children at remission and in normal controls (q= 6.23-26.15, P < 0.01). The asthmatic children at remission had higher levels of sICAM-1 and sVCAM-1 than the normal controls (q=16.86, 12.46, P < 0.01). R5-R20 positively correlated with sICAM-1 and sVCAM-1 in children with acute asthma (r=0.45, 0.57, P <0.05). CONCLUSIONS: IOS for pulmonary function and sICAM-1 and sVCAM-1 may be used to evaluate the severity and therapeutic effects of childhood asthma. A correlation exists between IOS pulmonary function and sICAM-1 and sVCAM-1.

Comparison of sonographic characteristics of primary thyroid lymphoma and anaplastic thyroid carcinoma.

BACKGROUND: Although primary thyroid lymphoma (PTL) and anaplastic thyroid carcinoma (ATC) both account for a rare portion of the morbidity of all thyroid malignancies, the therapeutic methods and prognosis for these two diseases are different. The purpose of this study was to investigate the sonographic characteristics of PTL and ATC and to compare the sonographic findings of PTL and ATC. METHODS: The study included 42 patients with histopathologically proven PTL (n=27) and ATC (n=15). The Clinical characteristics and sonographic findings were retrospectively reviewed and compared between the two groups. RESULTS: The mean age of patients with ATC was not significantly different from that in patients with PTL (P=0.601). The female-to-male ratio of patients with ATC was significantly lower than that of patients with PTL (P=0.029). Both PTL and ATC commonly present as a relatively large, solid mass on sonography with compressive symptoms, in which hoarseness was seen more frequently in ATC group (66.7%) than in PTL group (14.8%) (P=0.001). There is no significant difference in thyroid size, nodular size, margin, shape, echo texture, echogenicity, cystic change, vascularity and local invasion on sonography between ATC and PTL groups. Echogenic strands, markedly hypoechoic and enhanced posterior echo were seen more frequently in PTL group (92.6%, 92.6%, and 85.2%, respectively) than those in ATC group (6.7%, 60.0%, and 33.3%, respectively) (P<0.05), and calcification was seen more frequently in ATC group (80.0%) than in PTL group (0%) (P<0.001). Three ultrasound patterns were observed for PTL including diffuse type (25.9%), nodular type (48.2%) and mixed type (25.9%), while all ATC cases presented with nodular type (100.0%). Associated Hashimoto's thyroiditis occurred more frequently in PTL group (59.3%) than in ATC group (20.0%) (P=0.023). CONCLUSIONS: Certain sonographic features as a markedly hypoechogenicity, the presence of an enhanced posterior echo and linear echogenic strands, lack of calcification and associated Hashimoto's thyroiditis were valuable for distinguishing PTL from ATC. In contrast, heterogeneous echogenicity, uncircumscribed margin, irregular shape, and vascular pattern were not specific features for differential diagnosis.

The decreased expression of miR-625 predicts poor prognosis of esophageal squamous cell carcinoma.

BACKGROUND: Previous study has detected the expression of miR-625 in esophageal squamous cell carcinoma (ESCC) and found that miR-625 was related to tumor depth, stage, and metastasis of ESCC. However, the prognostic value of miR-625 in ESCC has not yet been reported. METHODS: Real-time quantitative PCR was employed to measure the expression level of miR-625 in clinical ESCC tissues. Survival curves were made using the Kaplan-Meier method, and the log rank test was used to analyze the differences between clinicopathological characteristics and survival in ESCC patients. RESULTS: The expression level of miR-625 in ESCC tissues was significantly lower than that in adjacent non-tumor tissues (1.00 ± 0.38 vs. 3.25 ± 1.83, P < 0.0001). Low miR-625 expression was observed to be closely correlated with lymph node metastasis (P = 0.01), distant metastasis (P = 0.007), tumor differentiation (P = 0.04), and advanced TNM stage (P = 0.005). The 5-year overall survival rate in the low expression group was 38.1%, compared with 68.8% in the high expression group (log-rank test, P = 0.001). Multivariate Cox regression analysis showed that miR-625 expression level (HR = 3.72, 95% CI: 1.36-8.78, P = 0.005) was an independent factor in predicting the overall survival of ESCC patients. CONCLUSION: Our findings provide the convincing evidence for the first time that the down-regulation of miR-625 may serve as a novel molecular marker to predict the aggressive tumor progression and unfavorable prognosis of ESCC patients.