Attenuation of phytofungal pathogenicity of Ascomycota by autophagy modulators.

Autophagy in eukaryotes functions to maintain homeostasis by degradation and recycling of long-lived and unwanted cellular materials. Autophagy plays important roles in pathogenicity of various fungal pathogens, suggesting that autophagy is a novel target for development of antifungal compounds. Here, we describe bioluminescence resonance energy transfer (BRET)-based high-throughput screening (HTS) strategy to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 that is critical for autophagosome formation. We identified ebselen (EB) and its analogs ebselen oxide (EO) and 2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PT) as inhibitors of fungal pathogens Botrytis cinerea and Magnaporthe oryzae ATG4-mediated ATG8 processing. The EB and its analogs inhibit spore germination, hyphal development, and appressorium formation in Ascomycota pathogens, B. cinerea, M. oryzae, Sclerotinia sclerotiorum and Monilinia fructicola. Treatment with EB and its analogs significantly reduced fungal pathogenicity. Our findings provide molecular insights to develop the next generation of antifungal compounds by targeting autophagy in important fungal pathogens.

Nuclear effectors of plant pathogens: Distinct strategies to be one step ahead.

Nuclear effector proteins released by bacteria, oomycete, nematode, and fungi burden the global environment and crop yield. Microbial effectors are key weapons in the evolutionary arms race between plants and pathogens, vital in determining the success of pathogenic colonization. Secreted effectors undermine a multitude of host cellular processes depending on their target destination. Effectors are classified by their localization as either extracellular (apoplastic) or intracellular. Intracellular effectors can be further subclassified by their compartment such as the nucleus, cytoplasm or chloroplast. Nuclear effectors bring into question the role of the plant nucleus' intrinsic defence strategies and their vulnerability to effector-based manipulation. Nuclear effectors interfere with multiple nuclear processes including the epigenetic regulation of the host chromatin, the impairment of the trans-kingdom antifungal RNAi machinery, and diverse classes of immunity-associated host proteins. These effector-targeted pathways are widely conserved among different hosts and regulate a broad array of plant cellular processes. Thus, these nuclear sites constitute meaningful targets for effectors to subvert the plant defence system and acquire resources for pathogenic propagation. This review provides an extensive and comparative compilation of diverse plant microbe nuclear effector libraries, thereby highlighting the distinct and conserved mechanisms these effectors employ to modulate plant cellular processes for the pathogen's profit.

The nuclear effector MoHTR3 of Magnaporthe oryzae modulates host defence signalling in the biotrophic stage of rice infection.

Fungal effectors play a pivotal role in suppressing the host defence system, and their evolution is highly dynamic. By comparative sequence analysis of plant-pathogenic fungi and Magnaporthe oryzae, we identified the small secreted C2 H2 zinc finger protein MoHTR3. MoHTR3 exhibited high conservation in M. oryzae strains but low conservation among other plant-pathogenic fungi, suggesting an emerging evolutionary selection process. MoHTR3 is exclusively expressed in the biotrophic stage of fungal invasion, and the encoded protein localizes to the biotrophic interfacial complex (BIC) and the host cell nucleus. The signal peptide crucial for MoHTR3' secretion to the BIC and the protein section required for its translocation to the nucleus were both identified by a functional protein domain study. The host-nuclear localization of MoHTR3 suggests a function as a transcriptional modulator of host defence gene induction. After ΔMohtr3 infection, the expression of jasmonic acid- and ethylene-associated genes was diminished in rice, in contrast to when the MoHTR3-overexpressing strain (MoHTR3ox) was applied. The transcript levels of salicylic acid- and defence-related genes were also affected after ΔMohtr3 and MoHTR3ox application. In pathogenicity assays, ΔMohtr3 was indistinguishable from the wild type. However, MoHTR3ox-infected plants showed diminished lesion formation and hydrogen peroxide accumulation, accompanied by a decrease in susceptibility, suggesting that the MoHTR3-induced manipulation of host cells affects host-pathogen interaction. MoHTR3 emphasizes the role of the host nucleus as a critical target for the pathogen-driven manipulation of host defence mechanisms and underscores the ongoing evolution of rice blast's arms race.

Alternative splicing diversifies the transcriptome and proteome of the rice blast fungus during host infection.

Alternative splicing (AS) contributes to diversifying and regulating cellular responses to environmental conditions and developmental cues by differentially producing multiple mRNA and protein isoforms from a single gene. Previous studies on AS in pathogenic fungi focused on profiling AS isoforms under a limited number of conditions. We analysed AS profiles in the rice blast fungus Magnaporthe oryzae, a global threat to rice production, using high-quality transcriptome data representing its vegetative growth (mycelia) and multiple host infection stages. We identified 4,270 AS isoforms derived from 2,413 genes, including 499 genes presumably regulated by infection-specific AS. AS appears to increase during infection, with 32.7% of the AS isoforms being produced during infection but absent in mycelia. Analysis of the isoforms observed at each infection stage showed that 636 AS isoforms were more abundant than corresponding annotated mRNAs, especially after initial hyphal penetration into host cell. Many such dominant isoforms were predicted to encode regulatory proteins such as transcription factors and phospho-transferases. We also identified the genes encoding distinct proteins via AS and confirmed the translation of some isoforms via a proteomic analysis, suggesting potential AS-mediated neo-functionalization of some genes during infection. Comprehensive profiling of the pattern of genome-wide AS during multiple stages of rice-M. oryzae interaction established a foundational resource that will help investigate the role and regulation of AS during rice infection.

A rice gene encoding glycosyl hydrolase plays contrasting roles in immunity depending on the type of pathogens.

Because pathogens use diverse infection strategies, plants cannot use one-size-fits-all defence and modulate defence responses based on the nature of pathogens and pathogenicity mechanism. Here, we report that a rice glycoside hydrolase (GH) plays contrasting roles in defence depending on whether a pathogen is hemibiotrophic or necrotrophic. The Arabidopsis thaliana MORE1 (Magnaporthe oryzae resistance 1) gene, encoding a member of the GH10 family, is needed for resistance against M. oryzae and Alternaria brassicicola, a fungal pathogen infecting A. thaliana as a necrotroph. Among 13 rice genes homologous to MORE1, 11 genes were induced during the biotrophic or necrotrophic stage of infection by M. oryzae. CRISPR/Cas9-assisted disruption of one of them (OsMORE1a) enhanced resistance against hemibiotrophic pathogens M. oryzae and Xanthomonas oryzae pv. oryzae but increased susceptibility to Cochliobolus miyabeanus, a necrotrophic fungus, suggesting that OsMORE1a acts as a double-edged sword depending on the mode of infection (hemibiotrophic vs. necrotrophic). We characterized molecular and cellular changes caused by the loss of MORE1 and OsMORE1a to understand how these genes participate in modulating defence responses. Although the underlying mechanism of action remains unknown, both genes appear to affect the expression of many defence-related genes. Expression patterns of the GH10 family genes in A. thaliana and rice suggest that other members also participate in pathogen defence.

Novel Dual-Targeting Antibody Fragment IDB0062 Overcomes Anti-Vascular Endothelial Growth Factor Drug Limitations in Age-Related Macular Degeneration.

Purpose: Repeated administration of anti-vascular endothelial growth factor drugs to treat age-related macular degeneration leads to resistance. To overcome this drawback, we developed the novel recombinant dual-targeting antibody fragment IDB0062, which is comprised of the anti-vascular endothelial growth factor A Fab and neuropilin 1-targeting peptide, and we assessed its properties. Methods: We compared the in vitro activity of IDB0062 and conventional drugs using cell proliferation, wound healing, and Transwell assays. The in vivo efficacy of IDB0062 was determined using mouse choroidal neovascularization and oxygen-induced retinopathy models. To evaluate the ocular distribution of IDB0062, we intravitreally administered IDB0062 and ranibizumab to cynomolgus monkeys and measured the retinal drug levels. Results: IDB0062 effectively inhibited not only vascular endothelial growth factor A in vitro but also placenta growth factor 2, vascular endothelial growth factor B, and platelet-derived growth factor BB, which induce vascular endothelial growth factor A-independent angiogenesis. In addition, IDB0062 showed non-inferior efficacy compared with aflibercept in vivo despite the low selectivity for mouse vascular endothelial growth factor A. In the monkey intravitreal pharmacokinetic study, IDB0062 improved drug distribution in the retina compared with ranibizumab, confirming the accelerated onset of pharmacological action when IDB0062 is injected in the vitreous humor. Conclusions: Through neuropilin 1 binding, IDB0062 can improve the efficacy and accelerate the onset of pharmacological action in the posterior segment, which is targeted for macular degeneration, thereby improving drug responsiveness in drug-resistant patients. Translational Relevance: Considering its novel mechanism of action, IDB0062 may help in controlling resistance to conventional anti-vascular endothelial growth factor drugs in clinical settings.

The Membrane-Bound Protein, MoAfo1, Is Involved in Sensing Diverse Signals from Different Surfaces in the Rice Blast Fungus.

To establish an infection, fungal pathogens must recognize diverse signals from host surfaces. The rice blast fungus, Magnaporthe oryzae, is one of the best models studying host-pathogen interactions. This fungus recognizes physical or chemical signals from the host surfaces and initiates the development of an infection structure called appressorium. Here, we found that protein MoAfo1(appressorium formation, MGG_10422) was involved in sensing signal molecules such as cutin monomers and long chain primary alcohols required for appressorium formation. The knockout mutant (ΔMoafo1) formed a few abnormal appressoria on the onion and rice sheath surfaces. However, it produced normal appressoria on the surface of rice leaves. MoAfo1 localized to the membranes of the cytoplasm and vacuole-like organelles in conidia and appressoria. Additionally, the ΔMoafo1 mutant showed defects in appressorium morphology, appressorium penetration, invasive growth, and pathogenicity. These multiple defects might be partially due to failure to respond properly to oxidative stress. These findings broaden our understanding of the fungal mechanisms at play in the recognition of the host surface during rice blast infection.

Ultrafast carbothermal reduction of silica to silicon using a CO2 laser beam.

We report the extraction of silicon via a carbothermal reduction process using a CO2 laser beam as a heat source. The surface of a mixture of silica and carbon black powder became brown after laser beam irradiation for a few tens of seconds, and clear peaks of crystalline silicon were observed by Raman shift measurements, confirming the successful carbothermal reduction of silica. The influence of process parameters, including the laser beam intensity, radiation time, nitrogen gas flow in a reaction chamber, and the molar ratios of silica/carbon black of the mixture, on the carbothermal reduction process is explained in detail.

Two nuclear effectors of the rice blast fungus modulate host immunity via transcriptional reprogramming.

Pathogens utilize multiple types of effectors to modulate plant immunity. Although many apoplastic and cytoplasmic effectors have been reported, nuclear effectors have not been well characterized in fungal pathogens. Here, we characterize two nuclear effectors of the rice blast pathogen Magnaporthe oryzae. Both nuclear effectors are secreted via the biotrophic interfacial complex, translocated into the nuclei of initially penetrated and surrounding cells, and reprogram the expression of immunity-associated genes by binding on effector binding elements in rice. Their expression in transgenic rice causes ambivalent immunity: increased susceptibility to M. oryzae and Xanthomonas oryzae pv. oryzae, hemibiotrophic pathogens, but enhanced resistance to Cochliobolus miyabeanus, a necrotrophic pathogen. Our findings help remedy a significant knowledge deficiency in the mechanism of M. oryzae-rice interactions and underscore how effector-mediated manipulation of plant immunity by one pathogen may also affect the disease severity by other pathogens.

Transcriptome Profiling of the Rice Blast Fungus Magnaporthe oryzae and Its Host Oryza sativa During Infection.

The rice blast (fungal pathogen: Magnaporthe oryzae and host: Oryza sativa) is one of the most important model pathosystems for understanding plant-microbe interactions. Although both genome sequences were published as the first cases of pathogen and host, only a few in planta transcriptome data during infection are available. Due to technical difficulties, previously reported fungal transcriptome data are not highly qualified to comprehensively profile the expression of fungal genes during infection. Here, we report the high-quality transcriptomes of M. oryzae and rice during infection using a sheath infection-based RNA sequencing approach. This comprehensive expression profiling of the fungal pathogen and its host will provide a better platform for understanding the plant-microbe interactions at the genomic level and serve as a valuable resource for the research community.

Preparation of Transparent Conductive Electrode via Layer-By-Layer Deposition of Silver Nanowires and Its Application in Organic Photovoltaic Device.

Solution processed transparent conductive electrodes (TCEs) were fabricated via layer-by-layer (LBL) deposition of silver nanowires (AgNWs). First, the AgNWs were coated on (3-Mercaptopropyl)trimethoxysilane modified glass substrates. Then, multilayer AgNW films were obtained by using 1,3-propanedithiol as a linker via LBL deposition, which made it possible to control the optical transmittance and sheet resistance of multilayer thin films. Next, thermal annealing of AgNW films was performed in order to agent their electrical conductivity. AgNW monolayer films were characterized by UV-Vis spectrometer, field emission scanning electron microscopy, optical microscopy, atomic force microscopy and sheet resistance measurement by four-point probe method. The high performances were achieved with multilayer films, which provided sheet resistances of 9 Ω/sq, 11 Ω/sq with optical transmittances of 71%, 70% at 550 nm, which are comparable to commercial indium tin oxide (ITO) electrodes. Finally, an organic photovoltaic device was fabricated on the AgNW multilayer electrodes for demonstration purpose, which exhibited power conversion efficiency of 1.1%.

A novel approach to investigate hypoxic microenvironment during rice colonization by Magnaporthe oryzae.

Because molecular oxygen functions as the final acceptor of electrons during aerobic respiration and a substrate for diverse enzymatic reactions, eukaryotes employ various mechanisms to maintain cellular homeostasis under varying oxygen concentration. Human fungal pathogens change the expression of genes involved in virulence and oxygen-required metabolisms such as ergosterol (ERG) synthesis when they encounter oxygen limitation (hypoxia) during infection. The oxygen level in plant tissues also fluctuates, potentially creating hypoxic stress to pathogens during infection. However, little is known about how in planta oxygen dynamics impact pathogenesis. In this study, we investigated oxygen dynamics in rice during infection by Magnaporthe oryzae via two approaches. First, rice leaves infected by M. oryzae were noninvasively probed using a microscopic oxygen sensor. Second, an immunofluorescence assay based on a chemical probe, pimonidazole, was used. Both methods showed that oxygen concentration in rice decreased after fungal penetration. We also functionally characterized five hypoxia-responsive genes participating in ERG biosynthesis for their role in pathogenesis. Resulting insights and tools will help study the nature of in planta oxygen dynamics in other pathosystems.

Production of germanium nanoparticles via laser pyrolysis for anode materials of lithium-ion batteries and sodium-ion batteries.

Germanium nanoparticles were synthesized and subjected to study as anode materials for lithium ion batteries and sodium ion batteries. Laser pyrolysis of GeH4 was used to produce germanium nanoparticles and the average diameter of these nanoparticles was easily controlled by regulating sensitizer gas flow rates during the process. 60 and 10 nm diameter nanoparticles were synthesized and micron-size powder was purchased and these three pure germanium powder samples were tested as the anode materials of lithium ion batteries and sodium ion batteries in terms of cycle retention, long term cycles and the kinetics of reactions. Experimental results showed that the smallest powder sample which is synthesized, average 10 nm, exhibited excellent performances in both kinds of batteries. According to the results, the characteristics of batteries improved as the size of germanium powder decreased consistently. Pure germanium was thoroughly investigated as an anode of metal-ion batteries with regard to its powder size. The experimental data and synthesis approach of germanium nanoparticles suggested in this research would be a good example for the utilization of elemental germanium in high performance batteries.

Identification of a Spotted Leaf Sheath Gene Involved in Early Senescence and Defense Response in Rice.

Lesion mimic mutants (LMMs) commonly exhibit spontaneous cell death similar to the hypersensitive defense response that occurs in plants in response to pathogen infection. Several lesion mimic mutants have been isolated and characterized, but their molecular mechanisms remain largely unknown. Here, a spotted leaf sheath (sles) mutant derived from japonica cultivar Koshihikari is described. The sles phenotype differed from that of other LMMs in that lesion mimic spots were observed on the leaf sheath rather than on leaves. The sles mutant displayed early senescence, as shown, by color loss in the mesophyll cells, a decrease in chlorophyll content, and upregulation of chlorophyll degradation-related and senescence-associated genes. ROS content was also elevated, corresponding to increased expression of genes encoding ROS-generating enzymes. Pathogenesis-related genes were also activated and showed improved resistance to pathogen infection on the leaf sheath. Genetic analysis revealed that the mutant phenotype was controlled by a single recessive nuclear gene. Genetic mapping and sequence analysis showed that a single nucleotide substitution in the sixth exon of LOC_Os07g25680 was responsible for the sles mutant phenotype and this was confirmed by T-DNA insertion line. Taken together, our results revealed that SLES was associated with the formation of lesion mimic spots on the leaf sheath resulting early senescence and defense responses. Further examination of SLES will facilitate a better understanding of the molecular mechanisms involved in ROS homeostasis and may also provide opportunities to improve pathogen resistance in rice.

Silicon Nanocanyon: One-Step Bottom-Up Fabrication of Black Silicon via in-Lasing Hydrophobic Self-Clustering of Silicon Nanocrystals for Sustainable Optoelectronics.

We report a novel one-step bottom-up fabrication method for multiscale-structured black Si, which is characterized by randomly distributed microscale Si layers covered with sub-100 nm protrusions with submicron boundary grooves. The unique multiscale structure, suggested as a "nanocanyon," effectively minimizes light reflection over a broad spectrum by diversifying the scattering routes from the nanotextured surface to the wide distributed boundary micronanoscale grooves. This structure was achieved by hydrophobic clustering and local aggregation of instantaneously melted Si nanocrystals on a glass substrate under laser irradiation. This method can replace the complicated conventional silicon processes, such as patterning for selective Si formation, texturing for improved absorption, and doping for modifying the electrical properties, because the proposed method obviates the need for photolithography, chemical etching, vacuum processes, and expensive wafers. Finally, black Si photosensor arrays were successfully demonstrated by a low-cost solution process and a laser growth sintering technique for microchannel fabrication. The results show the great potential of the proposed fabrication method for low-cost and sustainable production of highly sensitive optoelectronics and as an alternative to conventional wafer-based photosensor manufacturing techniques.

Role of the Histone Acetyltransferase Rtt109 in Development and Pathogenicity of the Rice Blast Fungus.

Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 plays important roles in maintaining genome integrity and surviving DNA damage. Here, we investigated the implications of Rtt109-mediated response to DNA damage on development and pathogenesis of the rice blast fungus Magnaporthe oryzae (anamorph: Pyricularia oryzae). The ortholog of Rtt109 in M. oryzae (MoRtt109) was found via sequence homology and its functionality was confirmed by phenotypic complementation of the Saccharomyces cerevisiae Rtt109 deletion strain. Targeted deletion of MoRtt109 resulted in a significant reduction in acetylation of H3K56 and rendered the fungus defective in hyphal growth and asexual reproduction. Furthermore, the deletion mutant displayed hypersensitivity to genotoxic agents, confirming the conserved importance of Rtt109 in genome integrity maintenance and genotoxic stress tolerance. Elevated expression of DNA repair genes and the results of the comet assay were consistent with constitutive endogenous DNA damage. Although the conidia produced from the mutant were not impaired in germination and appressorium morphogenesis, the mutant was significantly less pathogenic on rice leaves. Transcriptomic analysis provided insight into the factors underlying phenotypic defects that are associated with deficiency of H3K56 acetylation. Overall, our results indicate that MoRtt109 is a conserved histone acetyltransferase that affects proliferation and asexual fecundity of M. oryzae through maintenance of genome integrity and response to DNA damage.

MoJMJ1, Encoding a Histone Demethylase Containing JmjC Domain, Is Required for Pathogenic Development of the Rice Blast Fungus, Magnaporthe oryzae.

Histone methylation plays important roles in regulating chromatin dynamics and transcription in eukaryotes. Implication of histone modifications in fungal pathogenesis is, however, beginning to emerge. Here, we report identification and functional analysis of a putative JmjC-domain-containing histone demethylase in Magnaporthe oryzae. Through bioinformatics analysis, we identified seven genes, which encode putative histone demethylases containing JmjC domain. Deletion of one gene, MoJMJ1, belonging to JARID group, resulted in defects in vegetative growth, asexual reproduction, appressorium formation as well as invasive growth in the fungus. Western blot analysis showed that global H3K4me3 level increased in the deletion mutant, compared to wild-type strain, indicating histone demethylase activity of MoJMJ1. Introduction of MoJMJ1 gene into ΔMojmj1 restored defects in pre-penetration developments including appressorium formation, indicating the importance of histone demethylation through MoJMJ1 during infection-specific morphogenesis. However, defects in penetration and invasive growth were not complemented. We discuss such incomplete complementation in detail here. Our work on MoJMJ1 provides insights into H3K4me3-mediated regulation of infection-specific development in the plant pathogenic fungus.

ZnO decorated germanium nanoparticles as anode materials in Li-ion batteries.

Germanium exhibits high charge capacity and high lithium diffusivity, both are the key requirements for electrode materials in high performance lithium ion batteries (LIBs). However, high volume expansion and segregation from the electrode during charge-discharge cycling have limited use of germanium in LIBs. Here, we demonstrate that ZnO decorated Ge nanoparticles (Ge@ZnO NPs) can overcome these limitations of Ge as an LIB anode material. We produced Ge NPs at high rates by laser pyrolysis of GeH4, then coated them with solution phase synthesized ZnO NPs. Half-cell tests revealed dramatically enhanced cycling stability and higher rate capability of Ge@ZnO NPs compared to Ge NPs. Enhancements arise from the core-shell structure of Ge@ZnO NPs as well as production of metallic Zn from the ZnO layer. These findings not only demonstrate a new surface treatment for Ge NPs, but also provide a new opportunity for development of high-rate LIBs.

Prospective randomized clinical trial of hydrophilic tapered implant placement at maxillary posterior area: 6 weeks and 12 weeks loading.

PURPOSE: Early loading of implant can be determined by excellent primary stability and characteristic of implant surface. The implant system with recently improved surface can have load application 4-6 weeks after installing in maxilla and mandible. This study evaluated the effect of healing period to the stability of hydrophilic tapered-type implant at maxillary posterior area. MATERIALS AND METHODS: This study included 30 patients treated by hydrophilic tapered-type implants (total 41 implants at maxilla) and classified by two groups depending on healing period. Group 1 (11 patients, 15 implants) was a control group and the healing period was 12 weeks, and Group 2 (19 patients, 26 implants) was test group and the healing period was 6 weeks. Immediately after implant placement, at the first impression taking, implant stability was measured using Osstell Mentor. The patients also took periapical radiographs after restoration delivery, 12 months after restoration and final followup period. The marginal bone loss around the implants was measured using the periapical radiographs. RESULTS: All implants were survived and success rate was 97.56%. The marginal bone loss was less than 1mm after 1 year postoperatively except the one implant. The stabilities of the implants were not correlated with age, healing period until loading, insertion torque (IT), the diameter of fixture and the location of implant. Only the quality of bone in group 2 (6 week) was correlated with the stability of implant. CONCLUSION: Healing period of 6 weeks can make the similar clinical prognosis of implants to that of healing period of 12 weeks if bone quality is carefully considered in case of early loading.

Clinical evaluation of sinus bone graft in patients with mucous retention cyst.

BACKGROUND: Mucous retention cyst refers to a cyst made by expansion due to the blockage of the salivary gland near the maxillary sinus, and it is surrounded by epithelial cells. Most of them are small; therefore, they cannot be found well and are frequently with antral polyp. The aim of this study was to evaluate the clinical prognosis of sinus bone graft in patients with mucous retention cyst. METHODS: This study was performed retrospectively on 23 patients who had sinus bone graft. Group 1 was 8 patients (10 sinuses) who had a mucous retention cyst, and group 2 was 15 patients (17 sinuses) who had no pathologic history about the maxillary sinus. For these patients, sinus bone graft was performed using the lateral approach technique. The total 51 implants were placed 6.22 weeks on the average after sinus bone graft. Sinus membrane perforation during operation, postoperative complications, marginal bone loss after restorative function, implant success rate, and survival rate were analyzed. RESULTS: There was no complication in group 1, and there were three complications in group 2. In group 2, two cases of implants failed. The types of postoperative complications consisted of two minor infections and one wound dehiscence. Two implants of total 51 implants were removed, and the survival rate of implants was 96.08 % (group 1 100 %, group 2 93.5 %). The total success rate of implants was 92.2 % (group 1 95 %, group 2 90.3 %). CONCLUSIONS: The clinical prognosis was not affected by the presence of mucous retention cyst.