Ethanol mediates the interaction between Caenorhabditis elegans and the nematophagous fungus Purpureocillium lavendulum.

Accurately recognizing pathogens by the host is vital for initiating appropriate immune response against infecting microorganisms. Caenorhabditis elegans has no known receptor to recognize pathogen-associated molecular pattern. However, recent studies showed that nematodes have a strong specificity for transcriptomes infected by different pathogens, indicating that they can identify different pathogenic microorganisms. However, the mechanism(s) for such specificity remains largely unknown. In this study, we showed that the nematophagous fungus Purpureocillium lavendulum can infect the intestinal tract of the nematode C. elegans and the infection led to the accumulation of reactive oxygen species (ROS) in the infected intestinal tract, which suppressed fungal growth. Co-transcriptional analysis revealed that fungal genes related to anaerobic respiration and ethanol production were up-regulated during infection. Meanwhile, the ethanol dehydrogenase Sodh-1 in C. elegans was also up-regulated. Together, these results suggested that the infecting fungi encounter hypoxia stress in the nematode gut and that ethanol may play a role in the host-pathogen interaction. Ethanol production in vitro during fungal cultivation in hypoxia conditions was confirmed by gas chromatography-mass spectrometry. Direct treatment of C. elegans with ethanol elevated the sodh-1 expression and ROS accumulation while repressing a series of immunity genes that were also repressed during fungal infection. Mutation of sodh-1 in C. elegans blocked ROS accumulation and increased the nematode's susceptibility to fungal infection. Our study revealed a new recognition and antifungal mechanism in C. elegans. The novel mechanism of ethanol-mediated interaction between the fungus and nematode provides new insights into fungal pathogenesis and for developing alternative biocontrol of pathogenic nematodes by nematophagous fungi. IMPORTANCE Nematodes are among the most abundant animals on our planet. Many of them are parasites in animals and plants and cause human and animal health problems as well as agricultural losses. Studying the interaction of nematodes and their microbial pathogens is of great importance for the biocontrol of animal and plant parasitic nematodes. In this study, we found that the model nematode Caenorhabditis elegans can recognize its fungal pathogen, the nematophagous fungus Purpureocillium lavendulum, through fungal-produced ethanol. Then the nematode elevated the reactive oxygen species production in the gut to inhibit fungal growth in an ethanol dehydrogenase-dependent manner. With this mechanism, novel biocontrol strategies may be developed targeting the ethanol receptor or metabolic pathway of nematodes. Meanwhile, as a volatile organic compound, ethanol should be taken seriously as a vector molecule in the microbial-host interaction in nature.

The significance of systematic lymph node dissection in surgery for early-stage non-small cell lung cancer patients aged ≤40 years.

BACKGROUND: Surgery remains the best option for treating early-stage non-small cell lung cancer (NSCLC), and lymph node dissection (LND) is an important step in this approach. However, the extent of LND in the general age population, especially in young patients, is controversial. This retrospective study aimed to investigate the correlation between systematic lymph node dissection (SLND) and prognosis in young (≤40 years) patients with stage IA NSCLC. METHODS: Clinicopathological data of 191 patients aged ≤40 years who underwent surgical pulmonary resection for stage IA NSCLC between January 2010 and December 2016 were retrospectively collected. Of the patients, 104 received SLND (SLND group), while the other 87 patients underwent sampling or no LND (non-SLND group). The disease-free survival (DFS) and overall survival (OS) curves of the patients from each group were plotted using the Kaplan-Meier method, and the correlations of the patients' clinical factors with prognosis were also analyzed. RESULTS: The median follow-up period was 55 months. During follow-up, 7 patients died, and recurrence or metastasis was detected in 16 patients. Kaplan-Meier analysis revealed no difference in DFS (P=0.132) between the SLND and non-SLND group, but a significant difference was found between the groups in OS (P=0.022). Additionally, there was no statistically pronounced difference in OS or DFS between male and female patients. Multivariate survival analysis showed that the type of SLND, as well as tumor size, is an independent prognostic factor for DFS (HR, 3.530; 95% CI, 1.120-11.119; P=0.031) and OS (HR, 13.076; 95% CI, 1.209-141.443; P=0.034). CONCLUSIONS: For young (age ≤40) stage IA NSCLC patients with pathological invasive adenocarcinoma, intraoperative SLND can improve the DFS and OS. Further studies are needed to verify the most optimal degree of LND in young patients.

Functional Characterization of Core Regulatory Genes Involved in Sporulation of the Nematophagous Fungus Purpureocillium lavendulum.

The nematophagous fungus Purpureocillium lavendulum is a natural enemy of plant-parasitic nematodes, which cause severe economic losses in agriculture worldwide. The production of asexual spores (conidia) in P. lavendulum is crucial for its biocontrol activity against nematodes. In this study, we characterized the core regulatory genes involved in conidiation of P. lavendulum at the molecular level. The central regulatory pathway is composed of three genes, P. lavendulumbrlA (PlbrlA), PlabaA, and PlwetA, which regulate the early, middle, and late stages of asexual development, respectively. The deletion of PlbrlA completely inhibited conidiation, with only conidiophore stalks produced. PlAbaA determines the differentiation of conidia from phialides. The deletion of PlwetA affected many phenotypes related to conidial maturation, including abscission of conidia from conidium strings, thickening of the cell wall layers, vacuole generation inside the cytoplasm, production of trehalose, tolerance to heat shock, etc. Comparative analyses showed that the upstream regulators of the core regulatory pathway of conidiation, especially the "fluffy" genes, were different from those in Aspergillus Besides their roles in conidiation, the central regulators also influence the production of secondary metabolites, such as the leucinostatins, in P. lavendulum Our study revealed a set of essential genes controlling conidiation in P. lavendulum and provided a framework for further molecular genetic studies on fungus-nematode interactions and for the biocontrol of plant-parasitic nematodes.IMPORTANCE Plant-parasitic nematodes cause serious damage to crops throughout the world. Purpureocillium lavendulum is a nematophagous fungus which is a natural enemy of nematodes and a potential biocontrol agent against plant-parasitic nematodes. The conidia play an important role during infection of nematodes. In this study, we identified and characterized genes involved in regulating asexual development of P. lavendulum We found that these genes not only regulate conidiation but also influence secondary-metabolite production. This work provides a basis for future studies of fungus-nematode interactions and nematode biocontrol.

An efficient gene disruption system for the nematophagous fungus Purpureocillium lavendulum.

The fungus Purpureocillium lavendulum (formally Paecilomyces lilacinus) is a natural enemy of insects and plant-parasitic nematodes, and has been used as an important bio-control agent against agricultural pests all over the world. In order to understand the genetic mechanisms governing its biocontrol efficiency and other biological processes, an effective gene disruption system is needed. Here we report the development of an efficient system which integrates selective markers that differ from Purpureocillium lilacinum, a one-step construction method for gene knockout plasmids, and a ku80 knockout strain for efficient homologous recombination. With this system, we effectively disrupted the transcription factors in the central regulation pathway of sporulation and a serine protease which were contributed to nematode infection, demonstrating this system as an efficient gene disrupting system for further characterization of genes involved in the development and pathogenesis of this fungus.

[Association of surfactant protein D gene polymorphisms at rs3088308 and rs721917 with susceptibility to silicosis].

OBJECTIVE: To investigate the relationship between polymorphisms of surfactant protein D (rs3088308 and rs721917) and the susceptibility to silicosis. METHODS: This case-control study included 125 silicosis patients and 125 individuals exposed to industrial dust but without silicosis (control group), who were strictly matched with the case group for age, gender, work type and cumulative length of dust exposure. The rs3088308 and rs721917 polymorphisms of surfactant protein-D were detected in all the participants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The frequencies of T/T, T/A and A/A genotypes of surfactant protein-D rs3088308 locus were 22.2%, 71.2% and 5.6% in the case group, significantly different from the frequencies of 17.6%, 58.4% and 24.0% in the control group, respectively (P<0.05). The frequencies of C/C, C/T and T/T genotypes of rs721917 locus were 17.6%, 56.8% and 25.6% in the case group, similar to the frequencies of 15.2%, 60.0% and 24.8% in the control group, respectively (P>0.05). CONCLUSION: Surfactant protein-D rs3088308 polymorphism is significantly associated with silicosis, and the T allele may be a risk factor for silicosis in individuals exposed to industrial dust.

[Expression of neuropilin 2 in human colorectal carcinoma and its correlation with lymphangiogenesis and lymphatic metastasis of tumor].

OBJECTIVE: To explore the relationship between neuropilin 2 (NRP-2) and lymphangiogenesis and lymphatic metastasis of human colorectal carcinoma (CRC), as well as the expression of NRP-2 in CRC tissues. METHODS: A total of 55 cases of CRC, adjacent and normal tissues of surgical resection were randomly selected at our hospital from March 2010 to January 2012. All pathological findings were confirmed by histopathology. The expression of NRP-2 was detected with reverse transcription (RT)-PCR and immunohistochemistry in parenchymatous and surrounding malignant tissues. Then lymphangiogenesis was marked with D2-40 monoclonal antibody and microlymphatic density (MLD) counted. RESULTS: Significant differences of MLD existed between those of tumor region (39 ± 19) and tumor margin (53 ± 26, P < 0.01). Both the number and shape of lymphangiogenesis were different between the parenchymatous and surrounding tissues. The expression of NRP-2 had a positive correlation with MLD both at the protein level (r = 0.325, P < 0.05) and at the gene level (r = 0.545, P = 0.000). And it was also correlated with the differentiation degree, infiltrative degree, lymphovascular invasion, lymph node metastasis and Dukes tumor staging (all P < 0.05). CONCLUSION: The expression of NRP-2 may regulate lymphangiogenesis and it may play an important role in the incidence and development of CRC.

The elements of human cyclin D1 promoter and regulation involved.

Cyclin D1 is a cell cycle machine, a sensor of extracellular signals and plays an important role in G1-S phase progression. The human cyclin D1 promoter contains multiple transcription factor binding sites such as AP-1, NF-қB, E2F, Oct-1, and so on. The extracellular signals functions through the signal transduction pathways converging at the binding sites to active or inhibit the promoter activity and regulate the cell cycle progression. Different signal transduction pathways regulate the promoter at different time to get the correct cell cycle switch. Disorder regulation or special extracellular stimuli can result in cell cycle out of control through the promoter activity regulation. Epigenetic modifications such as DNA methylation and histone acetylation may involved in cyclin D1 transcriptional regulation.

[Effect of SiO2 on expression of MMP-9/TIMP-1 in human alveolar macrophages in vitro].

OBJECTIVE: To investigate the effect of SiO2 on the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in human silicotic alveolar macrophages (AM). METHODS: Human alveolar macrophages were collected from a silicotic patient by bronchoalveolar lavage and exposed to silicon dioxide for 3 h, 6 h, 12 h, 18 h, 24 h and 36 h. The expression of the MMP-9 in the AM were detected with zymography and immunological method and the expression of the TIMP-1 in the AM with immunological method. RESULTS: The expressions of MMP-9 in the AM increased clearly along with the time, reached peak at 24 h when detected with zymography (average optical density: 3.061+/-0.153 vs 2.851+/-0.164, P<0.05); and reached peak at 18h when detected with immunological method (average optical density: 0.386+/-0.037 vs 0.322+/-0.034, P<0.05). The expression of the TIMP-1 in the AM did not vary when detected with immunological method (P>0.05). CONCLUSION: SiO2 may affect the expression of MMP-9 and MMP-9 activity in the cultured AM.

[Distribution of mast cells and its role in the pathogenesis of Hirschsprung disease].

OBJECTIVE: To investigate the distribution of mast cells (MC) in colon tissue of Hirschsprung disease (HD) and explore the role of mast cells in the pathogenesis of HD. METHODS: Forty-one cases of HD (male 23, female 18), age from 2 months to 15 years, and eight age-matched normal cases were enrolled in this study. The distribution of MC in all layers of colon was examined by immunohistochemistry with mouse antihuman mast cell tryptase monoclonal antibody. RESULTS: The count of MC in all layers of colon aganglionic segments of HD was significantly higher as compared with colon ganglionic segments of HD and normal controls (21.47+/-3.59 vs 3.18+/-0.87, 2.75+/-0.51). The average optical density values(A) of MC in aganglionic and ganglionic segments significantly decreased as compared to normal control (0.38+/-0.10,0.31+/-0.11 vs 0.51+/-0.08). CONCLUSION: Mast cells may play an important role in the pathogenesis of HD.

Comparison of three methods of protein extraction from Dermatophagoides pteronyssinus for two-dimensional electrophoresis.

OBJECTIVE: To explore an effective method of Dermatophagoides pteronyssinus protein extraction suitable for two-dimensional electrophoresis (2-DE) analysis. METHODS: The extracts of Dermatophagoides pteronyssinus were prepared with Coca's solution, lysis buffer of 2-DE, and Trizol reagent, respectively. Bicinchoninic acid (BCA) assay was used to determine the total protein concentration of the samples. The efficiency of different protein extraction methods were evaluated with 2-DE analysis. RESULTS: The concentrations of extracted protein by methods of Coca's solution, lysis buffer, and Trizol reagent were 0.63 g/L, 0.90 g/L, and 0.80 g/L, respectively. The 2-DE analysis results showed that some protein spots in low molecular weight (LMW) range could be detected with the Coca's solution method. With the lysis buffer of 2-DE method, more protein spots in LMW range could be detected, while the medium molecular weight (MMW) protein spots were absent. Several MMW protein spots (174-178 kD and 133 kD) and more LMW protein spots were detected with Trizol reagent method. CONCLUSIONS: Among Coca's solution, lysis buffer of 2-DE, and Trizol reagent, the concentration of extracted protein of Dermatophagoides pteronyssinus by lysis buffer of 2-DE is the highest. However, most protein components of Dermatophagoides pteronyssinus purified mite bodies can be extracted by Trizol reagent, which may generally reflect the whole profile of Dermatophagoides pteronyssinus allergens.

[Clinicopathologic study of distal 2 cm rectal carcinoma intramural spread over dentate line].

OBJECTIVE: To study the extent of distal intramural spread of distal 2 cm rectal carcinoma over the dentate line, and to improve quality of life for these patients through providing pathological evidence of operation mode choice. METHODS: Specimens of thirty patients with rectal carcinoma, operated with ISR(intersphincter resection) or Miles procedure from May 2005 to July 2007, were collected, and their large and histology pathologic slices were examined. Specimens were dissected distally to the dentate line per 2 mm within 1cm and per 5 mm exceed 1cm. The length of distal intramural spread to rectal carcinoma was measured under light microscope. RESULTS: Among the 30 specimens, distal intramural spread over the dentate line was observed only on one case and the invasion length over the dentate line was more than 2 cm, the invasion lengths of the other 29 cases were within the dentate lines. CONCLUSION: Distal 2 cm rectal carcinomas seldom spread over dentate lines. Anus discomfortableness of these patients and other complications could be reduced through reserving more skin over the dentate line, which are important for the improving of quality of life in these patient.