[Transcriptomic analysis of the ΔPaLoc mutant of Clostridioides difficile and verification of its toxicity].

Objective: Comparative analyses of wild-type Clostridioides difficile 630 (Cd630) strain and pathogenicity locus (PaLoc) knockout mutant (ΔPaLoc) by using RNA-seq technology. Analysis of differential expression of Cd630 wild-type strain and ΔPaLoc mutant strain and measurement of its cellular virulence changes. Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioides difficile. Methods: Analysis of Cd630 and ΔPaLoc mutant strains using high-throughput sequencing (RNA-seq). Clustering differentially expressed genes and screening differentially expressed genes by DESeq software. Further analysis of differential genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, cytotoxicity assays of ΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell (Vero) and the human colonic cell (Caco-2) lines. Results: The transcriptome data showed that the ΔPaLoc mutant toxin genes tcdA and tcdB were not transcribed. Compared to the wild-type strain, CD630_36010, CD630_020910,CD630_02080 and cel genes upregulated 17.92,11.40,8.93 and 7.55 fold, respectively. Whereas the hom2 (high serine dehydrogenase), the CD630_15810 (spore-forming protein), CD630_23230 (zinc-binding dehydrogenase) and CD630_23240 (galactitol 1-phosphate 5-dehydrogenase) genes were down-regulated by 0.06, 0.075, 0.133 and 0.183 fold, respectively. The GO and KEGG enrichment analyses showed that the differentially transcribed genes in ΔPaLoc were enriched in the density-sensing system, ABC transport system, two-component system, phosphotransferase (PTS) system, and sugar metabolism pathway, as well as vancomycin resistance-related pathways. Cytotoxicity assays showed that the ΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain. Conclusion: Transcriptional sequencing analysis of the Cd630 and ΔPaLoc mutant strains showed that the toxin genes were not transcribed. Those other differential genes could provide a reference for further studies on the physiological and biochemical properties of the ΔPaLoc mutant strain. Cytotoxicity assays confirmed that the ΔPaLoc mutant lost virulence to Vero and Caco-2 cells, thus laying the foundation for constructing an toxin-attenuated vaccine strain against C. difficile.

[Effect of Modic changes on fusion rate and cage subsidence after transforaminal lumbar interbody fusion].

Objective: To investigate the effects of Modic changes on the fusion rate and cage subsidence after transforaminal lumbar interbody fusion (TLIF). Methods: From January 2015 to January 2018, a total of 186 patients with degenerative lumbar disease who received lumbar instrumentation fusion and monosegmental TLIF with single polyetheretherketone (PEEK) cage in Nanjing Drum Tower Hospital were retrospectively reviewed. Patients with Modic changes at the level where the cage was placed were enrolled as Modic group, and the remaining were assigned into non-Modic group. Disk height, lumbar lordosis and segmental lordosis of the level with TLIF were measured based on the preoperative, postoperative and latest follow-up lateral radiograph. The fusion rate and cage subsidence (more than 2 mm on either endplate) were recorded based on CT scan at the latest follow-up. The Oswestry disability index (ODI) and visual analogue scale (VAS) of pain was used to evaluate the clinical outcome. The data were compared with paired t test between the two groups. Results: In this study, there were 70 males and 116 females with an average age of (55±13) years. There were 99 patients in the Modic group (25 with type 1, 66 with type 2, 8 with type 3), and 87 patients in the non-Modic group. There was no significant difference between Modic group and non-Modic group in demographics and postoperative radiographs. The patients were followed-up for (19±4) months (13 to 48 months). All patients achieved grade 1 or 2 fusion. Cage subsidence was detected in 34 patients (18.3%, 34/186). The incidence of subsidence in Modic group (24.2%, 24/99) was significantly higher than that in non-Modic group (11.5%, 10/87) (χ(2)=5.038, P<0.05), and the incidence of subsidence in type Ⅰ (28.0%, 7/25) and type Ⅱ (24.2%, 16/66) were higher than that in non-Modic group (11.5%, 10/87). There was no significant difference in ODI and VAS between Modic group and non-Modic group before and after the operation and at the latest follow-up (t=0.397-1.568, all P>0.05). Conclusion: Preoperative Modic changes have no impact on fusion rate after transforaminal lumbar interbody fusion, but both type Ⅰ and Ⅱ Modic changes do increase the risk of cage subsidence.

Analysis of changes in joint function and peripheral blood mononuclear cells in patients with systemic lupus erythematosus and intervention effects of different drugs.

OBJECTIVE: To investigate the therapeutic effect of drug therapy with cyclophosphamide and leflunomide on the joint function damage of patients with systemic lupus erythematosus (SLE) and its regulatory effects on expression levels of programmed death receptor 1, Notch signaling pathway genes and interferon-inducible protein 10 in peripheral blood mononuclear cells. PATIENTS AND METHODS: A total of 60 patients with SLE were randomly divided into two groups. They were treated with cyclophosphamide and leflunomide, respectively. The number of painful joints, joint tenderness index, joint swelling index and erythrocyte sedimentation rate of patients before and after treatment were evaluated, and the peripheral blood was collected from patients in the two groups; the peripheral blood mononuclear cells were extracted. RESULTS: We observed that the number of painful joints, joint tenderness index and joint swelling index in cyclophosphamide group were decreased after treatment (p<0.05), and the erythrocyte sedimentation rate was significantly decreased (p<0.05). The number of painful joints, joint tenderness index and joint swelling index in leflunomide group were decreased after treatment (p<0.05), and the erythrocyte sedimentation rate was significantly decreased (p<0.05). The comparisons of changes in joint functions and erythrocyte sedimentation rates between cyclophosphamide group and leflunomide group after drug therapy showed that the curative effect in leflunomide group was superior to that in cyclophosphamide group (p<0.05). The positive expression rate of peripheral blood mononuclear cell Notch1 in leflunomide group after treatment was significantly decreased, and the curative effect was superior to that in cyclophosphamide group (p<0.05). The comparisons of changes in programmed death receptor 1 of lymphocytes and interferon-inducible protein 10 between cyclophosphamide group and leflunomide group after drug therapy showed that the curative effect in leflunomide group was superior to that in cyclophosphamide group (p<0.05). The comparison of positive expression rate of nuclear factor-κB (NF-κB) in peripheral blood mononuclear cells between the two groups after treatment showed that the curative effect in leflunomide group was superior to that in cyclophosphamide group (p<0.05). There were positive correlations of the expression level of programmed death receptor 1 of peripheral blood lymphocytes in SLE patients with double-stranded DNA (ds-DNA) and SLE disease activity index (p<0.05). There were positive correlations of the expression level of peripheral interferon-inducible protein 10 in SLE patients with ds-DNA and SLE disease activity index (p<0.05). CONCLUSIONS: This study proved that both leflunomide and cyclophosphamide have therapeutic effects on the joint functions and immune dysfunction of peripheral blood mononuclear cells of SLE patients; however, and the effect of leflunomide is better. There are positive correlations of SLE disease activity index with the Notch signaling pathway genes, programmed death receptor 1 and interferon-inducible protein 10 in peripheral blood mononuclear cells, suggesting that these factors are related to the immune dysfunction of peripheral blood mononuclear cells.

Galantamine protects against lipopolysaccharide-induced acute lung injury in rats.

Lipopolysaccharide (LPS)-induced endotoxemia triggers the secretion of proinflammatory cytokines and can cause acute lung injury (ALI). The high mobility group box 1 (HMGB1) protein plays an important role as a late mediator of sepsis and ALI. Galantamine (GAL) is a central acetylcholinesterase inhibitor that inhibits the expression of HMGB1. This study evaluated the effects of GAL by measuring levels of inflammatory mediators and observing histopathological features associated with LPS-induced ALI. Sixty 8-10 week old male Sprague-Dawley rats (200-240 g) were randomized into three groups as follows: control group, LPS group (7.5 mg/kg LPS), and LPS+GAL group (5 mg/kg GAL before LPS administration). Histopathological examination of lung specimens obtained 12 h after LPS administration was performed to analyze changes in wet-to-dry (W/D) weight ratio, myeloperoxidase (MPO) activity, and HMGB1 expression level. Additionally, plasma concentrations of tumor necrosis factor-α, interleukin-6, and HMGB1 were measured using an enzyme-linked immunosorbent assay at 0 (baseline), 3, 6, 9, and 12 h after LPS administration. Mortality in the three groups was recorded at 72 h. LPS-induced ALI was characterized by distortion of pulmonary architecture and elevation of MPO activity, W/D weight ratio, and levels of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin-6, and HMGB1. Pretreatment with GAL significantly reduced the LPS-induced lung pathological changes, W/D weight ratio, levels of pro-inflammatory cytokines and MPO activity (ANOVA). Moreover, GAL treatment significantly decreased the mortality rate (ANOVA). In conclusion, we demonstrated that GAL exerted a protective effect on LPS-induced ALI in rats.

[The imaging analysis of the age-related changes on maxillary sinus].

Objective:The aim of this study is to investigate the age-related changes rules of maxillary sinus.Method:The 540 patients (1 080 sides) with normal data of deputy sinus in spiral CT were enrolled,including 270 cases of male and female,age from 7 to 81 years old.They are divided into 9 groups according to the age:Group A at the age of 7-12 years old,Group B at the age of 13-17,Group C at the age of 18-20 years old,Group D at the age of 21-24 years old,Group E at the age of 25-28 years,Group F at the age of 29-35 years old,Group G at the age of 36-40 years old,Group H at the age of 41-65 years old,and Group I is more than 65 years old.By the gender,the patients in each group was divided into male and female groups.There are 30 cases in each group(60 sides).The volumes and the three-dimensional diameters of the maxillary sinus were measured,and the coefficient of gasification of them were calculated.Result:The maxillary sinus volume and 3 D lines have almost the same change trend along with the age between the male and female group;From 7 to 20 ages,they are increased linearly,13 to 17 fastest-growing;18 to 20 years old reached to peak;declined slightly in 21-28 years old,29-35 a second growth peak,and 36 to 40 years old have fallen sharply,to reaching a steady state after 41 years old;The gasification coefficient has no difference among all groups.Conclusion:The volume changes with the age-related on maxillary sinus is in the adolescent stage.It reaches a steady state in the middle and old age stage,and gasification coefficient on maxillary sinus has no age-related changes among all groups.

DNA barcoding of common soft scales (Hemiptera: Coccoidea: Coccidae) in China.

The soft scales (Hemiptera: Coccoidea: Coccidae) are a group of sap-sucking plant parasites, many of which are notorious agricultural pests. The quarantine and economic importance of soft scales necessitates rapid and reliable identification of these taxa. Nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene (barcoding region) and 28S rDNA were generated from 340 individuals of 36 common soft scales in China. Distance-based [(best match, Automated Barcode Gap Discovery (ABGD)], tree-based (neighbor-joining, Bayesian inference), Klee diagrams, and general mixed Yule coalescent (GMYC) models were used to evaluate barcoding success rates in the data set. Best match showed that COI and 28S sequences could provide 100 and 95.52% correct identification, respectively. The average interspecific divergences were 19.81% for COI data and 20.38% for 28S data, and mean intraspecific divergences were 0.56 and 0.07%, respectively. For COI data, multiple methods (ABGD, Klee, and tree-based methods) resulted in general congruence with morphological identifications. However, GMYC analysis tended to provide more molecular operational taxonomic units (MOTUs). Twelve MOTUs derived from five morphospecies (Rhodococcus sariuoni, Pulvinaria vitis, Pulvinaria aurantii, Parasaissetia nigra, and Ceroplastes rubens) were observed using the GMYC approach. In addition, tree-based methods showed that 28S sequences could be used for species-level identification (except for Ceroplastes ceriferus - Ceroplastes pseudoceriferus), even with low genetic variation (<1%). This report demonstrates the robustness of DNA barcoding for species discrimination of soft scales with two molecular markers (COI and 28S) and provides a reliable barcode library and rapid diagnostic tool for common soft scales in China.

Reduced EBP50 expression or mis-localization of the EBP50 protein is associated with the malignant progression of esophageal squamous cell carcinoma.

PURPOSE: The aim of this study was to examine the significance of EBP50 (ezrin-radixin-moesin binding phosphoprotein 50) expression in esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: Real-time PCR (qRT-PCR), western blotting, and immunohistochemical staining were performed to detect EBP50 expression in pairs of ESCCs and matched non-tumor tissues, and the relationships between EBP50 expression and other clinical factors in ESCC were analyzed. An iRNA targeting EBP50 was transfected into EC9706 cells. MTT and plate colony assays were performed to assess the effects of EBP50 down-regulation on cell growth, and flow cytometry was used to evaluate the influence of inhibiting EBP50 on cell cycle progression. RESULTS: The real-time PCR (qRT-PCR), western blotting, and immunohistochemical staining results showed that EBP50 expression was significantly lower in ESCCs compared to matched non-tumor tissues. In addition, decreased EBP50 expression correlated with differentiation, T stage, lymph node (LN) metastasis, and poor prognosis in patients with ESCC. The down-regulation of EBP50 may significantly promote the growth and proliferation of EC9706 cells while accelerating cell cycle progression from the G1to S phase. CONCLUSIONS: EBP50 expression was decreased in ESCC, indicating that EBP50 might play a significant role in the malignant progression of ESCC and be a prognostic marker for patients with ESCC.

Unique histological features of the left atrial posterior wall.

The left atrial posterior wall (LAPW) plays a critical role in atrial fibrillation, but the underlying mechanism remains unclear. In the present study, we sought to characterize the histological features of the LAPW. Different atrial regions were dissected from hearts of normal Sprague-Dawley rats and humans. Haematoxylin/eosin and van Gieson staining were used to analyse atrial cardiomyocyte arrangement and collagen distribution, respectively. Intercellular junctions were evaluated by transmission electron microscopy. In contrast with other atrial regions, the LAPW exhibited more disorganized cardiomyocytes, larger intercellular spaces and variable myocardial fibre arrangement. The proportion of collagen was significantly higher in the LAPW than in other atrial regions. Interestingly, desmosomes were sparse along with intercellular gaps in the LAPW. In summary, distinct disarrangement of cardiomyocytes and an abundance of collagen exist in the LAPW. The sparsity of desmosomes in the LAPW may be related to the heterogeneous distribution and separation of atrial myocytes.

[Clinical and experimental study on wenchangning oral liquor in treating infantile autumn diarrhea].

Gold-Dampness Syndrome of Infantile Autumn diarrhea treated with Wenchangning Oral Liquor was observed clinically. There were 238 cases in the treated group, the cure rate was 81.9%. In comparing with control group (32 cases in Huoxiang Zhengqi Liquid group, 50 cases in gentamycin group), there were highly significant difference between treated and control groups in the cure rate, markedly effective rate, total effective rate, time for stopping diarrhea and for disappearing of clinical symptoms, P < 0.01 and < 0.001. Clinical practice and animal experiments revealed that the drug had no toxic and side-effects. The possible therapeutic mechanisms were; (1) Anti-Rotavirus, anti-diarrhea; (2) Promote the recovery of intestinal digestive and absorptive function; (3) Enhance the immune function of the body; (4) Inhibit the motility of intestinal tract, Wenchangning Oral Liquor is rapid in action, conveniant in administration and safe, conforming to the criteria for the preventing and treating diarrhea of World Health Organization.

Increase in plasma thrombomodulin and decrease in plasma von Willebrand factor after regular radiotherapy of patients with cancer.

In this study, we investigated plasma levels of thrombomodulin (TM) and von Willebrand factor (vWF) in 51 patients suffering from cancer or tumor undergoing 60 cobalt radiotherapy. Plasma TM and vWF antigen were measured by immunoradiometric assay and ELISA, respectively. During radiotherapy, an increase in plasma TM in patients was observed, which was radiation-dose dependent and there was a positive correlation between plasma TM level and radiation doses. However, the level of plasma vWF in the patients was decreased during radiotherapy and there was an inverse correlation between the amount of plasma vWF and radiation doses. Our data indicate that plasma TM is an useful molecular marker for early detection of radiation injury to endothelial cells in patients undergoing radiotherapy.

Effect of new-breviscapine on fibrinolysis and anticoagulation of human vascular endothelial cells.

Cultured confluent human umbilical vein endothelial cells were incubated with new-breviscapine (NB), a flavonoid consisting of 4-OH-scutellarin-7-O-glucuronide (C33H30O18) and FeCl3, MgCl2, and CaCl2, which is first extracted from Erigeron breviscapus (Vant) Hand-Mazz in China, 0, 6.25, 12.5, 25, 50, 100, and 1,000 micrograms.ml-1. The releases of tissue-type plasminogen activator (t-PA), and epoprostenol (Epo) from endothelial cells were stimulated by NB, but no significant effect of plasminogen activator inhibitor (PAI) activity was seen. NB 25-1,000 micrograms.ml-1 induced a production of thrombomodulin (TM) within the cells, an expression of TM on the surface of the cells, and a release of TM from the cells. Our data provide a new evidence that NB is a stimulant to fibrinolysis and anticoagulation of endothelial cells.