Characterization of regulatory genes Plhffp and Plpif1 involved in conidiation regulation in Purpureocillium lavendulum.

Purpureocillium lavendulum is an important biocontrol agent against plant-parasitic nematodes, primarily infecting them with conidia. However, research on the regulatory genes and pathways involved in its conidiation is still limited. In this study, we employed Agrobacterium tumefaciens-mediated genetic transformation to generate 4,870 random T-DNA insertion mutants of P. lavendulum. Among these mutants, 131 strains exhibited abnormal conidiation, and further in-depth investigations were conducted on two strains (designated as #5-197 and #5-119) that showed significantly reduced conidiation. Through whole-genome re-sequencing and genome walking, we identified the T-DNA insertion sites in these strains and determined the corresponding genes affected by the insertions, namely Plhffp and Plpif1. Both genes were knocked out through homologous recombination, and phenotypic analysis revealed a significant difference in conidiation between the knockout strains and the wild-type strain (ku80). Upon complementation of the ΔPlpif1 strain with the corresponding wildtype allele, conidiation was restored to a level comparable to ku80, providing further evidence of the involvement of this gene in conidiation regulation in P. lavendulum. The knockout of Plhffp or Plpif1 reduced the antioxidant capacity of P. lavendulum, and the absence of Plhffp also resulted in decreased resistance to SDS, suggesting that this gene may be involved in the integrity of the cell wall. RT-qPCR showed that knockout of Plhffp or Plpif1 altered expression levels of several known genes associated with conidiation. Additionally, the analysis of nematode infection assays with Caenorhabditis elegans indicated that the knockout of Plhffp and Plpif1 indirectly reduced the pathogenicity of P. lavendulum towards the nematodes. The results demonstrate that Agrobacterium tumefaciens - mediated T-DNA insertion mutagenesis, gene knockout, and complementation can be highly effective for identifying functionally important genes in P. lavendulum.

Clinical evaluation of resorbable polylactic acid (PLA) intracanal posts for primary incisor restoration. Randomized controlled clinical trial.

This randomized, controlled clinical trial compares the clinical performance of glass-fibre and resorbable polylactic acid (PLA) intracanal posts used to restore carious primary incisors in young patients. The study sample includes 180 primary upper central incisors of 90 children aged 3 to 4 years. All patients were randomly divided into two equal groups of 45 children who received PLA and glass-fibre (GFP) intracanal posts. The clinical assessment of incisor restorations was carried out immediately upon completion and at months 3, 6 and 12 according to the following criteria: anatomical form, marginal adaptation, surface roughness, marginal pigmentation, colour match, secondary caries and contact point. The Gingival Index (GI), the Bleeding Index (Cowell modification; mBI), and bite force (BF) were measured. At the 3-month follow-up, the occlusal BF of patients who received PLA posts was higher than the baseline; the GI and mBI scores were lower, by contrast (p < 0.05). This tendency was even more pronounced 6 and 12 months after the restoration. The incidence of side effects or symptoms (apical inflammation, cervical fracture, loosening of the crown) after the PLA posts was significantly lower than after the GFP (p < 0.05). No statistically significant differences were present between the two groups with respect to colour matching, anatomical form, marginal adaptation, marginal pigmentation, surface roughness, occlusal contact and secondary caries. Based on the results, applying PLA intracanal posts and cyanoacrylate to residual anterior crowns in young children can improve their gingival health, reduce side effects, and increase the likelihood of successful restoration.

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 Growth and Conidiation of Purpureocillium lavendulum Are Co-Regulated by Nitrogen Sources and Histone H3K14 Acetylation.

Plant-parasitic nematodes cause severe economic losses to agriculture. As important biocontrol agents, nematophagous fungi evolved the ability to obtain nitrogen sources from nematodes. However, the impact of nitrogen sources on the growth and development of these fungi is largely unknown. In this study, we aimed to better understand how nitrogen sources could influence vegetative growth and conidiation through epigenetic regulation in the nematophagous fungus, Purpureocillium lavendulum. Through nutrition screening, we found a phenomenon of the fungus, limited colony extension with a large amount of conidia production when cultured on PDA media, can be altered by adding ammonia nitrate. Characterized by site-directed mutagenesis, the histone H3K14 acetylation was found to be involved in the alternation. Furthermore, the acetyltransferase PlGCN5 was responsible for H3K14 acetylation. Knockout of Plgcn5 severely diminished conidiation in P. lavendulum. Chip-seq showed that H3K14ac distributed in conidiation regulating genes, and genes in the MAPK pathway which may be the downstream targets in the regulation. These findings suggest that histone modification and nitrogen sources coordinated lifestyle regulation in P. lavendulum, providing new insight into the mechanism of growth regulation by nutritional signals for the carnivorous fungus.

Comparison of curative effect between different retrograde filling materials in young permanent molar root canal therapy.

The first mandible and maxilla permanent molars are the first permanent teeth that grow next to the deciduous teeth and may decay due to carelessness. Their caries can spread to the pulpal tentacles and cause pulpal and periapical diseases. In the current study, we tried to compare the curative effect of different retrograde filling materials, i.e. white MTA, gray MTA, Portland cement, and IRM, in young permanent molar root canal therapy. Because IL-1ß stimulates bone degradation by osteoclasts, IL-1ß gene expression was also measured for further evaluation. For this purpose, 400 students (240 boys and 160 girls) aged 8 to 11 years referred to the Pediatric Dental Center for first permanent molar root canal therapy were selected during two years. After recording the demographic characteristics of each patient, the first permanent molar teeth were examined by a general dentist with Abslang and decayed teeth were considered to have both discolorations in their grooves and apparent opacity. The patients, who need root canal therapy, were divided into four groups. The first group was treated with gray MTA. The second group was treated with white MTA. The third group received Portland cement for root canal therapy. The fourth group was treated with IRM. Also, IL-1ß gene expression was evaluated by the real-time PCR technique. Relative changes in gene expression in PBMC cells were performed using One Way ANOVA. SPSS 18 software was used to determine the correlation of gene expression in PBMCs. The results showed that there was no significant difference between the groups in terms of age (p = 0.12) and gender (p = 0.24). Also, the need for endodontic treatment in the mandible (n = 278) was higher than the maxilla (n = 85) and both jaws (n = 37). But there was no significant difference between the groups in terms of the need for endodontic treatment (p = 0.32). The results of Pearson correlation coefficients between studied groups in terms of IL-1ß gene expression showed that gray MTA and white MTA were not statistically different, but MTAs were generally different from Portland cement and IRM, with higher IL-1ß gene expression. In general, the results showed that the teeth in the vicinity of gray MTA and white MTA showed a more appropriate response than Portland cement and IRM, so the use of MTA and its preference over other materials is recommended. In the case of Portland cement, more studies are needed to reach a conclusion comparing this material with MTA.

Evaluation of P53 gene expression by immunohistochemistry to diagnosis oral precancerous lesions.

Oral Precancerous lesions include leukoplakia, erythroplakia, and mucosa palate changes due to reverse smoking. Assessing the prevalence of these lesions in a cross-sectional study can be effective in the timely prevention and treatment of lesions, in any community. Hence, in the present study, evaluation of P53 gene expression was done by immunohistochemistry method to diagnosis oral precancerous lesions. For this purpose, 111 Chinese patients (54 women and 57 men) were selected for examination. The age range of these patients was 22 to 69 years, and their average age was 32.6 years. All patients were examined by one physician. Oral mucosa was used for immunohistochemical evaluations. All samples taken from patients' mucosa were evaluated by one pathologist under a light microscope. 80 cases of the 111 patients were smokers and 27 were non-smokers. Among the 80 smokers, 56.25% had leukoplakia, 3.75% had erythroplakia, and 40% had mucosa palate changes. Regarding non-smokers, 74.07% had leukoplakia and 25.93% had erythroplakia. None of the non-smokers had mucosa palate changes. In terms of the lesion location, in patients with leukoplakia 89.23%, and patients with erythroplakia 90% of the lesion was located in the cheek mucosa and buccal vestibule. Also, in patients with leukoplakia 9.23%, and patients with erythroplakia 10% of the lesion was located in the lips vestibular mucosa. Only 1.54% of leukoplakia had a lesion in the vermilion border, and none of the erythroplakia patients had a lesion on the vermilion border. 76 patients (68.46%) showed positive expression of the P53 gene. The expression level of the P53 gene did not show a significant relationship with age, and the genders did not have a statistically significant difference in terms of gene expression. The expression level of the P53 gene was 59.8% in leukoplakia, 70% in erythroplakia, and 40% in Mucosa palate changes. The present study showed that the evaluation of P53 gene expression was well able to detect oral precancerous lesions and their severity by increasing their expression rate.