Identification of lthB, a Gene Encoding a Putative Glycosyltransferase Family 8 Protein Required for Leptothrix Sheath Formation.

The bacterium Leptothrix cholodnii generates cell chains encased in sheaths that are composed of woven nanofibrils. The nanofibrils are mainly composed of glycoconjugate repeats, and several glycosyltransferases (GTs) are required for its biosynthesis. However, only one GT (LthA) has been identified to date. In this study, we screened spontaneous variants of L. cholodnii SP6 to find those that form smooth colonies, which is one of the characteristics of sheathless variants. Genomic DNA sequencing of an isolated variant revealed an insertion in the locus Lcho_0972, which encodes a putative GT family 8 protein. We thus designated this protein LthB and characterized it using deletion mutants and antibodies. LthB localized adjacent to the cell envelope. ΔlthB cell chains were nanofibril free and thus sheathless, indicating that LthB is involved in nanofibril biosynthesis. Unlike the ΔlthA mutant and the wild-type strain, which often generate planktonic cells, most ΔlthB organisms presented as long cell chains under static conditions, resulting in deficient pellicle formation, which requires motile planktonic cells. These results imply that sheaths are not required for elongation of cell chains. Finally, calcium depletion, which induces cell chain breakage due to sheath loss, abrogated the expression of LthA, but not LthB, suggesting that these GTs cooperatively participate in glycoconjugate biosynthesis under different signaling controls. IMPORTANCE In recent years, the regulation of cell chain elongation of filamentous bacteria via extracellular signals has attracted attention as a potential strategy to prevent clogging of water distribution systems and filamentous bulking of activated sludge in industrial settings. However, a fundamental understanding of the ecology of filamentous bacteria remains elusive. Since sheath formation is associated with cell chain elongation in most of these bacteria, the molecular mechanisms underlying nanofibril sheath formation, including the intracellular signaling cascade in response to extracellular stimuli, must be elucidated. Here, we isolated a sheathless variant of L. cholodnii SP6 and thus identified a novel glycosyltransferase, LthB. Although mutants with deletions of lthA, encoding another GT, and lthB were both defective for nanofibril formation, they exhibited different phenotypes of cell chain elongation and pellicle formation. Moreover, LthA expression, but not LthB expression, was influenced by extracellular calcium, which is known to affect nanofibril formation, indicating the functional diversities of LthA and LthB. Such molecular insights are critical for a better understanding of ecology of filamentous bacteria, which, in turn, can be used to improve strategies to control filamentous bacteria in industrial facilities.

Arabidopsis CURLY LEAF functions in leaf immunity against fungal pathogens by concomitantly repressing SEPALLATA3 and activating ORA59.

Arabidopsis non-host resistance against non-adapted fungal pathogens including Colletotrichum fungi consists of pre-invasive and post-invasive immune responses. Here we report that non-host resistance against non-adapted Colletotrichum spp. in Arabidopsis leaves requires CURLY LEAF (CLF), which is critical for leaf development, flowering and growth. Microscopic analysis of pathogen behavior revealed a requirement for CLF in both pre- and post-invasive non-host resistance. The loss of a functional SEPALLATA3 (SEP3) gene, ectopically expressed in clf mutant leaves, suppressed not only the defect of the clf plants in growth and leaf development but also a defect in non-host resistance against the non-adapted Colletotrichum tropicale. However, the ectopic overexpression of SEP3 in Arabidopsis wild-type leaves did not disrupt the non-host resistance. The expression of multiple plant defensin (PDF) genes that are involved in non-host resistance against C. tropicale was repressed in clf leaves. Moreover, the Octadecanoid-responsive Arabidopsis 59 (ORA59) gene, which is required for PDF expression, was also repressed in clf leaves. Notably, when SEP3 was overexpressed in the ora59 mutant background, C. tropicale produced clear lesions in the inoculated leaves, indicating an impairment in non-host resistance. Furthermore, ora59 plants overexpressing SEP3 exhibited a defect in leaf immunity to the adapted Colletotrichum higginsianum. Since the ora59 plants overexpressing SEP3 did not display obvious leaf curling or reduced growth, in contrast to the clf mutants, these results strongly suggest that concomitant SEP3 repression and ORA59 induction via CLF are required for Arabidopsis leaf immunity to Colletotrichum fungi, uncoupled from CLF's function in growth and leaf development.

Porous Pellicle Formation of a Filamentous Bacterium, Leptothrix.

The bacterium Leptothrix cholodnii generates filaments encased in a sheath comprised of woven nanofibrils. In static liquid culture, L. cholodnii moves toward the air-liquid interface, where it forms porous pellicles. Observations of aggregation at the interface reveal that clusters consisting of only a few bacteria primarily grow by netting free cells. These growing clusters hierarchically enlarge through the random docking of other small clusters. We find that the bacteria swim using their polar flagellum toward the interface, where their sheath assists them in intertwining with others and thereby promotes the formation of small clusters. In contrast, sheathless hydrophobic mutant cells get stuck to the interface. We find that the nanofibril sheath is vital for robust pellicle formation as it lowers cell surface hydrophobicity by 60%, thereby reducing their adsorption and enabling cells to move toward and stick together at the air-liquid interface. IMPORTANCE Efficient and sustainable management of water resources is becoming a fundamental issue for supporting growing populations and for developing economic activity. Fundamental to this management is the treatment of wastewater. Microorganisms are the active component of activated sludge that is employed in the biodegradation process of many wastewater treatment facilities. However, uncontrolled growth of filamentous bacteria such as Sphaerotilus often results in filamentous bulking, lowering the efficiency of water treatment systems. To prevent this undesirable condition, strategies based on a fundamental understanding of the ecology of filamentous bacteria are required. Although the filamentous bacterium Leptothrix cholodnii, which is closely related to Sphaerotilus, is a minor inhabitant of activated sludge, its complete genome sequence is known, making gene manipulation relatively easy. Moreover, L. cholodnii generates porous pellicles under static conditions, which may be a characteristic of filamentous bulking. We show that both swimming motility and nanofibril-mediated air-liquid interface attachment are required for porous pellicle formation. These insights are critical for a better understanding of the characteristics of filamentous bulking and might improve strategies to control activated sludge.

Lumi-Map, a Real-Time Luciferase Bioluminescence Screen of Mutants Combined with MutMap, Reveals Arabidopsis Genes Involved in PAMP-Triggered Immunity.

Plants recognize pathogen-associated molecular patterns (PAMPs) to activate PAMP-triggered immunity (PTI). However, our knowledge of PTI signaling remains limited. In this report, we introduce Lumi-Map, a high-throughput platform for identifying causative single-nucleotide polymorphisms (SNPs) for studying PTI signaling components. In Lumi-Map, a transgenic reporter plant line is produced that contains a firefly luciferase (LUC) gene driven by a defense gene promoter, which generates luminescence upon PAMP treatment. The line is mutagenized and the mutants with altered luminescence patterns are screened by a high-throughput real-time bioluminescence monitoring system. Selected mutants are subjected to MutMap analysis, a whole-genome sequencing-based method of rapid mutation identification, to identify the causative SNP responsible for the luminescence pattern change. We generated nine transgenic Arabidopsis reporter lines expressing the LUC gene fused to multiple promoter sequences of defense-related genes. These lines generate luminescence upon activation of FLAGELLIN-SENSING 2 (FLS2) by flg22, a PAMP derived from bacterial flagellin. We selected the WRKY29-promoter reporter line to identify mutants in the signaling pathway downstream of FLS2. After screening 24,000 ethylmethanesulfonate-induced mutants of the reporter line, we isolated 22 mutants with altered WRKY29 expression upon flg22 treatment (abbreviated as awf mutants). Although five flg22-insensitive awf mutants harbored mutations in FLS2 itself, Lumi-Map revealed three genes not previously associated with PTI. Lumi-Map has the potential to identify novel PAMPs and their receptors as well as signaling components downstream of the receptors.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Unveiling the pathophysiology of gestational diabetes: Studies on local and peripheral immune cells.

Gestational diabetes mellitus (GDM) has been associated with impaired maternal immune response. Our aim was to review the available literature linking immune cells profile to GDM, in order to comprehend the role that different subpopulations play in the development of this pathology. We searched in PubMed for studies published in the last decade on circulating levels and placenta expression of immune cells on GDM. We identified 18 studies with several differences regarding the study design, clinical characteristics, number of participants, cell subpopulation and type of sample. Most studies assessed only one subpopulation either in peripheral blood or placenta and did not analyse functional properties of the cells. The most frequently evaluated immune cells were T lymphocytes, especially regulatory T (Tregs), and natural killer (NK) cells in the peripheral blood, and placental macrophages. No studies analysing B cells were identified, and only one study each evaluating γδT cells, dendritic cell (DC) and monocytes was found. Although there are controversies, at least one study reported positive association between GDM and CD4+ (activated), Tregs, Th17 and γδT cells; neutrophil/lymphocyte; NK cell (cytotoxic); macrophages; and monocytes. The number of studies is still small, so caution should be exercised in interpreting the data, and further research is required to validate these findings and establish the role of adaptive and innate immune cells in GDM pathophysiology.

Monocyte profile in peripheral blood of gestational diabetes mellitus patients.

OBJECTIVE: Gestational diabetes Mellitus has been considered an inflammatory disease involving different cells and mediators in its development. The role of innate immune cells in GDM physiopathology remains unclear, therefore this study was conducted to assess monocyte profile in GDM patients. DESIGN: This was a case-control study including 20 glucose-tolerant pregnant women (controls) and 18 GDM patients. METHODS: Flow cytometry was used to assess peripheral blood monocytes subsets (classical, intermediate, non-classical), the expression of TLR4 and CCR2 chemokine receptor (CD192) and cytokines (TNFA, IL6, IL10) secretion by monocytes subsets. In addition, sCD14 serum levels were evaluated by ELISA. RESULTS: We observed increased percentage of CD14+ cells, decreased frequency of intermediate monocytes (CD14+CD16+), and lower percentage of circulating monocytes (classical, intermediate and non-classical) that express TLR4 in the diabetic group compared to controls. Soluble CD14+ serum levels were higher in GDM patients compared to controls. There were no differences in the expression of the CCR2 chemokine receptor and cytokines (TNFA, IL6 and IL10) secretion between the studied groups. CONCLUSIONS: Our results demonstrated that GDM patients present impaired monocyte profile in the peripheral blood, suggesting that these cells are involved in GDM physiopathology.

Cervical cerclage placement decreases local levels of proinflammatory cytokines in patients with cervical insufficiency.

BACKGROUND: Cervical insufficiency is characterized by premature, progressive dilation and shortening of the cervix during pregnancy. If left unattended, this can lead to the prolapse and rupture of the amniotic membrane, which usually results in midtrimester pregnancy loss or preterm birth. Previous studies have shown that proinflammatory cytokines such as interleukin-1ß, interleukin-6, interleukin-8, and tumor necrosis factor alpha are up-regulated in normal parturition but are also associated with preterm birth. Studies evaluating such markers in patients with cervical insufficiency have evaluated only their diagnostic potential. Even fewer studies have studied them within the context of cerclage surgery. OBJECTIVES(S): The objective of the study was to evaluate the impact of local and systemic inflammatory markers on the pathogenesis of cervical insufficiency and the effect of cerclage surgery on the local immune microenvironment of women with cervical insufficiency. STUDY DESIGN: We recruited 28 pregnant women (12-20 weeks' gestation) diagnosed with insufficiency and referred for cerclage surgery and 19 gestational age-matched normal pregnant women as controls. Serum and cervicovaginal fluid samples were collected before and after cerclage surgery and during a routine checkup for normal women and analyzed using a targeted 13-plex proinflammatory cytokine assay. RESULTS: Before surgery, patients with cervical insufficiency had higher levels of interleukin-1ß, interleukin-6, interleukin-12, monocyte chemoattractant protein-1 and tumor necrosis factor alpha in cervicovaginal fluid compared to controls, but after surgery, these differences disappeared. No differences were found in serum of insufficiency versus control women. In patients with insufficiency, the levels of interleukin-1ß, interleukin-6, interleukin-8, monocyte chemoattractant protein-1, and interferon gamma in cervicovaginal fluid declined significantly after cerclage compared with before intervention, but these changes were not detected in serum. CONCLUSION: Compared with normal women, patients with cervical insufficiency have elevated levels of proinflammatory cytokines in cervicovaginal fluid but not in serum, suggesting a dysregulation of the local immune environment. Cerclage intervention led to a significant decline in these proinflammatory cytokines, suggesting that cerclage may help reduce local inflammation in cervical insufficiency.

Growth of children born to renal transplanted women.

Neonates born to transplanted mothers are exposed to immunosuppressive drugs during gestation and have a higher risk of being born prematurely and small for gestational age than the general population. We have prospectively followed up 27 children born to renal transplanted mothers from a single center and 31 healthy children born at term with adequate weight for gestational age. Comparisons of weight and length measurements were made at birth, 1 month (±0.9), 3 months (±1.0), 6 months (±1.0), 9 months (±1.5), and 12 months (±1.49) of age. There were a high rate of prematurity (51.9%) and neonates small for gestational age (40.7%) in the transplant group. At birth, in the transplant group, 28% of neonates had subnormal z-scores for weight and 40%, low z-scores for length. However, at 6 months of age, no significant differences were noticed in mean weight-for-age z-scores between groups (weight -0.43 vs -0.03; length -0.53 vs -0.08). At 12 months of age, comparable mean length-for-age z-scores were observed in both groups (weight 0.01 vs 0.27; length -0.07 vs 0.26). CONCLUSION: Despite high rates of premature births and neonates small for gestational age in the transplant group, there was a good recovery of growth during the first year.. What is Known: • Children born to renal transplanted mothers are exposed to immunosuppressive drugs during gestation [4]. • They have high risk of premature birth and fetal growth restriction, immune alterations at birth, and risk of hospitalization for infection in the first months of life [5]. What is New: • Despite high rates of premature birth and neonates small for gestational age, these infants had good growth recovery by 1 year of age.

Protein expression in human cumulus cells as an indicator of blastocyst formation and pregnancy success.

PURPOSE: The goal for the present study was to implement a technique for protein extraction and identification in human cumulus cells (CCs). METHODS: Forty samples of CCs were collected after ovum pick-up from patients undergoing intracytoplasmic sperm injection (ICSI). Samples were split into the blastocyst group (n = 10), including patients in which all embryos converted into blastocysts, and the non-blastocyst group (n = 10), including patients in which none of the embryos reached the blastocyst stage or the positive-pregnancy (n = 10) and negative-pregnancy group (n = 10). Proteins were extracted and injected into a liquid chromatography system coupled to a mass spectrometer. The spectra were processed and used to search a database. RESULTS: There were 87 different proteins in samples from the blastocyst and non-blastocyst groups, in which 30 were exclusively expressed in the blastocyst group and 17 in the non-blastocyst group. Among the 72 proteins detected in the pregnancy groups, 19 were exclusively expressed in the positive, and 16 were exclusively expressed in the negative-pregnancy group. CONCLUSIONS: CC proteomics may be useful for predicting pregnancy success and the identification of patients that should be included in extended embryo culture programs.

Lymphocyte subsets in human immunodeficiency virus-unexposed Brazilian individuals from birth to adulthood.

Ethnic origin, genetics, gender and environmental factors have been shown to influence some immunologic indices, so that development of reference values for populations of different backgrounds may be necessary. We have determined the distribution of lymphocyte subsets in healthy Brazilian individuals from birth to adulthood. Lymphocyte subsets were determined using four-colour cytometry in a cross-sectional study of 463 human immunodeficiency virus-unexposed children and adults from birth through 49 years of age. Lymphocyte subsets varied according to age, as previously observed in other studies. However, total CD4+ T cell numbers were lower than what was described in the Pediatric AIDS Clinical Trials Group P1009 (PACTG P1009), which assessed an American population of predominantly African and Hispanic backgrounds until the 12-18 year age range, when values were comparable. Naïve percentages and absolute values of CD8+ T cells, as assessed by CD45RA expression, were also lower than the PACTG P1009 data for all analysed age ranges. CD38 expression on both CD4+ and CD8+ T cells was lower than the PACTG P1009 values, with a widening gap between the two studies at older age ranges. Different patterns of cell differentiation seem to occur in different settings and may have characteristic expression within each population.

Measles seroprevalence in adolescents and young adults living with HIV and response to MMR booster in seronegative ones.

OBJECTIVE: The study analyzed people with HIV (PWH) measles seroprevalence and response to MMR booster given to seronegative individuals. DESIGN: A prospective cohort study with four groups: vertically (v-HIV), horizontally infected (h-HIV) individuals, and two control groups. An MMR booster was offered to seronegative individuals. RESULTS: Measles seropositivity and IgG antibodies were significantly lower in v-HIV than in the other groups. All measles seronegative patients responded to booster. CONCLUSION: An MMR booster must be sought during adolescence in vertically PWH.

Development of a Gene Replacement Method for the Filamentous BacteriumLeptothrix cholodniiSP-6.

Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteriaLeptothrix cholodniiSP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation withEscherichia coliS17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes. Graphical overview.

Leptothrix cholodnii Response to Nutrient Limitation.

Microorganisms are widely utilized for the treatment of wastewater in activated sludge systems. However, the uncontrolled growth of filamentous bacteria leads to bulking and adversely affects wastewater treatment efficiency. To clarify the nutrient requirements for filament formation, we track the growth of a filamentous bacterium, Leptothrix cholodnii SP-6 in different nutrient-limited conditions using a high aspect-ratio microfluidic chamber to follow cell-chain elongation and sheath formation. We find that limitations in Na+, K+, and Fe2+ yield no observable changes in the elongation of cell chains and sheath formation, whereas limitations of C, N, P, or vitamins lead to more pronounced changes in filament morphology; here we observe the appearance of partially empty filaments with wide intercellular gaps. We observe more dramatic differences when SP-6 cells are transferred to media lacking Mg2+ and Ca2+. Loss of Mg2+ results in cell autolysis, while removal of Ca2+ results in the catastrophic disintegration of the filaments. By simultaneously limiting both carbon and Ca2+ sources, we are able to stimulate planktonic cell generation. These findings paint a detailed picture of the ecophysiology of Leptothrix, which may lead to improved control over the unchecked growth of deleterious filamentous bacteria in water purification systems.

Tryptophan-derived metabolites and BAK1 separately contribute to Arabidopsis postinvasive immunity against Alternaria brassicicola.

Nonhost resistance of Arabidopsis thaliana against the hemibiotrophic fungus Colletotrichum tropicale requires PEN2-dependent preinvasive resistance and CYP71A12 and CYP71A13-dependent postinvasive resistance, which both rely on tryptophan (Trp) metabolism. We here revealed that CYP71A12, CYP71A13 and PAD3 are critical for Arabidopsis' postinvasive basal resistance toward the necrotrophic Alternaria brassicicola. Consistent with this, gene expression and metabolite analyses suggested that the invasion by A. brassicicola triggered the CYP71A12-dependent production of indole-3-carboxylic acid derivatives and the PAD3 and CYP71A13-dependent production of camalexin. We next addressed the activation of the CYP71A12 and PAD3-dependent postinvasive resistance. We found that bak1-5 mutation significantly reduced postinvasive resistance against A. brassicicola, indicating that pattern recognition contributes to activation of this second defense-layer. However, the bak1-5 mutation had no detectable effects on the Trp-metabolism triggered by the fungal penetration. Together with this, further comparative gene expression analyses suggested that pathogen invasion in Arabidopsis activates (1) CYP71A12 and PAD3-related antifungal metabolism that is not hampered by bak1-5, and (2) a bak1-5 sensitive immune pathway that activates the expression of antimicrobial proteins.

The effect of Gestational Diabetes Mellitus on the fetal compartment.

In indicated preterm births such a Gestational Diabetes Mellitus (GDM), little is known about the role of the amnion membranes. Investigating the role of amnion membrane inflammation in response GDM may suggest novel pathophysiologic mechanisms. We hypothesize that increased GDM inflammatory mediators may weaken the amnion membrane predisposing them to infection. Maternal and fetal serum and amnion membrane biopsies were collected from 20 GDM and 38 normoglycemic subjects (control) who underwent elective cesarean sections. Cytokines and adipokines were evaluated in serum and amnion culture supernatant samples. Amnion membrane biopsies from GDM and control subjects were studied: fresh frozen for RNA analysis for Toll-like receptor expression; cultured with LPS to test membrane permeability, and inflammation LPS + anti-TLR4 for testing mechanism. GDM was associated with higher fetal serum leptin (p = 0.004) and IL-10 (p = 0.04) compared to controls. Amnion membrane explants from GDM had higher levels of IL-6 (p = 0.019), and lower expression of Claudin-4 (p = 0.007) and increased permeability (p = 0.046) compared to controls. GDM membranes treated with LPS showed an increased expression of IL-10 (p = 0.013); IL-6 (p = 0.004) and TNF-α (p = 0.0005) but did not affect membrane permeability. LPS and anti-TLR4 antibody treatment reduced the production of TNF-α in controls (p = 0.03) and GDM (p = 0.007) compared to LPS alone. Fetal inflammatory response seems more balanced in GDM and does not impact membrane permeability function even with an infectious stimulus. Light fetal membrane inflammatory response may explain lack of preterm labor in GDM. Concluding, benign inflammation in the membranes may not be harmful for pregnancy maintenance.

Humoral and cellular immune responses to measles and tetanus: the importance of elapsed time since last exposure and the nature of the antigen.

OBJECTIVE: This study aims to assess the cellular and humoral immune response pre- and post-vaccine rechallenge in healthy adults with previous exposure to measles (virus or vaccine) and different time intervals since last tetanus vaccine. METHODS: Humoral immunity was tested by ELISA, and cellular immunity was tested by intracellular interferon gamma detection after in vitro stimulation with antigens. RESULTS: While cellular immunity was comparable among vaccinated individuals and those who had measles, higher antibody levels were found in those who had the disease in the past. Both antibodies and CD4(+) T cell tetanus immune responses depended on elapsed time since last immunization. Following a vaccine booster, an increase in cellular immunity and antibodies was observed to both tetanus and measles. Measles humoral response was much more intense among individuals previously exposed to a wild virus. CONCLUSIONS: In an era when natural boosters are less frequent, an immune surveillance might be necessary to investigate waning immunity as occurs for tetanus.

Low CD4+ T-cell levels and B-cell apoptosis in vertically HIV-exposed noninfected children and adolescents.

Lymphocyte subsets, activation markers and apoptosis were assessed in 20 HIV-exposed noninfected (ENI) children born to HIV-infected women who were or not exposed to antiretroviral (ARV) drugs during pregnancy and early infancy. ENI children and adolescents were aged 6-18 years and they were compared to 25 age-matched healthy non-HIV-exposed children and adolescents (Control). ENI individuals presented lower CD4(+) T cells/mm(3) than Control group (control: 1120.3 vs. ENI: 876.3; t-test, p = 0.030). ENI individuals had higher B-cell apoptosis than Control group (Control: 36.6%, ARV exposed: 82.3%, ARV nonexposed: 68.5%; Kruskal-Wallis, p < 0.05), but no statistical difference was noticed between those exposed and not exposed to ARV. Immune activation in CD4(+) T, CD8(+) T and in B cells was comparable in ENI and in Control children and adolescents. Subtle long-term immune alterations might persist among ENI individuals, but the clinical consequences if any are unknown, and these children require continued monitoring.

RLP23 is required for Arabidopsis immunity against the grey mould pathogen Botrytis cinerea.

Necrosis- and ethylene-inducing-like proteins (NLPs) are secreted by fungi, oomycetes and bacteria. Conserved nlp peptides derived from NLPs are recognized as pathogen-associated molecular patterns (PAMPs), leading to PAMP-triggered immune responses. RLP23 is the receptor of the nlp peptides in Arabidopsis thaliana; however, its actual contribution to plant immunity is unclear. Here, we report that RLP23 is required for Arabidopsis immunity against the necrotrophic fungal pathogen Botrytis cinerea. Arabidopsis rlp23 mutants exhibited enhanced susceptibility to B. cinerea compared with the wild-type plants. Notably, microscopic observation of the B. cinerea infection behaviour indicated the involvement of RLP23 in pre-invasive resistance to the pathogen. B. cinerea carried two NLP genes, BcNEP1 and BcNEP2; BcNEP1 was expressed preferentially before/during invasion into Arabidopsis, whereas BcNEP2 was expressed at the late phase of infection. Importantly, the nlp peptides derived from both BcNEP1 and BcNEP2 induced the production of reactive oxygen species in an RLP23-dependent manner. In contrast, another necrotrophic fungus Alternaria brassicicola did not express the NLP gene in the early infection phase and exhibited no enhanced virulence in the rlp23 mutants. Collectively, these results strongly suggest that RLP23 contributes to Arabidopsis pre-invasive resistance to B. cinerea via NLP recognition at the early infection phase.

Treatment outcomes of sialendoscopy for submandibular gland sialolithiasis: The minor axis of the sialolith is a regulative factor for the removal of sialoliths in the hilum of the submandibular gland using sialendoscopy alone.

OBJECTIVE: To assess the general guidelines for the removal of sialoliths for submandibular gland sialolithiasis using sialendoscopy alone. METHODS: We analyzed 61 sialoliths treated using sialendoscopy in 42 patients with submandibular gland sialolithiasis. We evaluated the submandibular gland sialoliths and divided each case based upon the location: the Wharton's duct or the hilum. We measured the major and minor axes of the sialoliths using a soft tissue computed-tomography (CT) scan and evaluated the removal rate of the sialoliths using sialendoscopy alone. RESULTS: The removal rate of the sialoliths in the Wharton's duct (52.6%) was significantly higher than that in the hilum of the submandibular gland (26.1%) (P=0.042). The minor axis was significantly correlated to the treatment outcome of sialendoscopy alone for all cases (P=0.030). A significant correlation was observed for cases involving the hilum of the submandibular gland and the measurement of the minor axes of the sialoliths for the treatment outcome of sialendoscopy alone (P=0.009). The major axis showed no correlation with the treatment outcomes of sialendoscopy alone. CONCLUSION: The measurement of the minor axes of the sialoliths with a soft tissue CT scan was correlated with treatment outcome of sialendoscopy alone for all cases, particularly sialoliths in the hilum. The easurement of the major axis showed no correlation with outcomes of sialendoscopy alone.

Diagnostic and treatment effects of sialendoscopy for patients with swelling of the parotid gland when sialoliths are undetected with computed tomography.

Between August 2009 and May 2016, 74 patients underwent sialoendoscopic surgery. 32 patients had parotid gland disease and 9 patients had intermittent swelling of the parotid gland and sialoliths were not detected with CT imaging. 4 patients were diagnosed with idiopathic Stensen's duct stenosis. Sialendoscopy directly confirmed Stensen's duct stenosis in 2 patients. However, the sialendoscope was unable to be inserted in the other 2 patients, who had stenosis of the orifice of the Stensen's duct. Balloon expansion of the duct was performed in these 2 patients and a steroid drug was injected into the duct in one patient. Complete remission was archived in one patient treated with sialendoscopy. Three patients had sialolithiasis. Microsialoliths and/or white floating matter was observed and removed using sialendoscopy. All patients experienced complete remission. In cases of Sjögren syndrome and recurrent parotitis, sialendoscopic surgery was performed, but the symptoms showed no improvement. For patients with microsialoliths, sialendoscopy may be most useful for diagnosis and treatment when the sialoliths are not detected with CT imaging. At present, sialendoscopic surgery have limitation in the treatment of Stensen's duct stenosis and may similarly have limitation in the treatment of Sjögren's syndrome and recurrent parotitis.