Fusarium kuroshium is the primary fungal symbiont of an ambrosia beetle, Euwallacea fornicatus, and can kill mango tree in Japan.

This study identifies fungi associated with Euwallacea fornicatus and determines whether these fungal species play the role of primary symbiont. E. fornicatus adults that emerged from the branches of infested trees in Okinawa main island, Japan, were collected and used to isolate fungi. Fusarium kuroshium and Penicillium citrinum were the most dominant fungal associates of females and males, respectively. F. kuroshium was much more frequently isolated from the head, including mycangia (fungus-carrying organs), of females than any other body parts. We inoculated healthy mango saplings with F. kuroshium or F. decemcellulare, both of which were symbionts of E. fornicatus females infesting mango trees. F. kuroshium decreased leaf stomatal conductance and rate of xylem sap-conduction area and increased length and area of xylem discoloration of the saplings, thereby weakening and killing some. These results suggest that F. kuroshium, a mycangial fungus of E. fornicatus, inhibits water flow in mango trees. This study is the first to report that F. kuroshium causes wilt disease in mango trees and that it is a primary fungal symbiont of E. fornicatus.

The Role of Mycangial Fungi Associated with Ambrosia Beetles (Euwallacea interjectus) in Fig Wilt Disease: Dual Inoculation of Fusarium kuroshium and Ceratocystis ficicola Can Bring Fig Saplings to Early Symptom Development.

The ambrosia beetle, Euwallacea interjectus, is a wood-boring pest and a vector of Ceratocystis ficicola, a pathogenic fungus causing fig (Ficus carica) wilt disease (FWD) in Japan. The ambrosia fungi, Fusarium kuroshium and Neocosmospora metavorans, have been frequently isolated from heads (including mycangia) of wild and reared adult female E. interjectus, respectively. However, the exact mechanisms driving FWD as well as the interactions between F. kuroshium and C. ficicola in fig orchard remain unclear. To verify the role of the mycangial fungi in the FWD progression, fig saplings were subjected to inoculation treatments (T1, F. kuroshium; T2, N. metavorans, reference positive control; T3, C. ficicola; T4, F. kuroshium + C. ficicola, realistic on-site combination). T3 and T4 saplings began wilting approximately 12 days after inoculation, leading to eventual death. Median duration from inoculation to death of the T4 saplings was approximately four days significantly faster than that of the T3 saplings. Xylem sap-conduction test indicated that dysfunction and necrosis area were considerably wider in the T4 saplings than in T3 saplings. These results demonstrate that the synergistic action of F. kuroshium and C. ficicola contributed to accelerated wilting in the saplings. Based on these discoveries, we proposed a model for system changes in the symbiosis between E. interjectus and its associated fungi in FWD in Japan.

Development of an Automated Chemiluminescent Enzyme Immunoassay for Measuring Thrombopoietin in Human Plasma.

Plasma thrombopoietin (TPO) measurements help distinguish between different types of thrombocytopenia but are not feasible in routine clinical practice. We developed a fully automated quantitative chemiluminescent enzyme immunoassay (CLEIA) for measuring TPO (TPO-CLEIA), which is a one-step sandwich-type assay. This assay utilizes a mouse monoclonal capture antibody, which has the neutralizing epitope of the interaction between TPO and the TPO receptor, and a newly generated rabbit monoclonal detector antibody. In analytical performance studies, this assay showed good linearity over the measuring range and high sensitivity. The limit of quantification (LoQ) of this assay was 3.4 pg/mL; low TPO concentration values of almost all healthy individuals exceeded the LoQ value. In clinical validation studies, TPO levels obtained from patients with aplastic anemia (AA) significantly increased, whereas those of patients with immune thrombocytopenia (ITP) were normal or slightly increased. The cutoff value for TPO-CLEIA corresponding to the previously reported values was useful for distinguishing between ITP and AA. These results suggest that TPO-CLEIA can quantify human plasma TPO levels with high accuracy and sensitivity and has the potential to facilitate routine clinical measurement of TPO in patients with various types of thrombocytopenia.

Validation of a new serum granulocyte-macrophage colony-stimulating factor autoantibody testing kit.

Very recently, a modest but significant efficacy of granulocyte-macrophage colony-stimulating factor (GM-CSF) inhalation therapy for the treatment of mild to moderate autoimmune pulmonary alveolar proteinosis (aPAP) has been reported. As the ability to measure the level of GM-CSF autoantibody (GMAb) in the serum is required to decide the indication for this therapy, we developed a high-performance GMAb testing kit for clinical use. As the kit succeeded in reducing nonspecific IgG binding to the ELISA plate, the predictive performance shown in the training study to discriminate aPAP patients from healthy subjects was perfect, providing a cut-off value of 1.65 U·mL-1 in 78 patients with aPAP and 90 healthy subjects in an operator-blinded manner using logistic regression analysis. As in the validation study, serum samples from another 213 patients with aPAP were also blinded and evaluated in an operator-blinded manner against external 207 samples from patients with other types of PAP and patients exhibiting various ground-glass opacities on chest high-resolution computed tomography that require discrimination from PAP. The logistic regression analysis of these validation data sets revealed values of 97.6% and 100% for specificity and sensitivity, respectively. Thus, this new GMAb testing kit is reliable for the diagnosis of aPAP and differential diagnosis of other lung diseases.

Pathogenicity and Distribution of Fusarium solani Isolates Associated with Erythrina Decline in Japan.

Erythrina spp. trees have been declining since the 2000s worldwide, and fungi belonging to Fusarium solani species complex (FSSC) have been suggested to be a causal factor of decline and mortality of Erythrina variegata trees in Okinawa Island, Japan. In addition to the FSSC isolate grouped as "it-1" based on ITS sequence data (previously called strain A), we conducted an inoculation experiment with two isolates grouped as "it-2" (previously strain B), which is genetically close to it-1. Two it-2 isolates originating from two islands showed pathogenicity to E. variegata with the same symptoms as those caused by it-1 isolate. We also found the isolates of it-1 and it-2 were widely distributed, including on Ishigaki Island, ∼400 km south of Okinawa Island across the ocean. All isolates of it-1 and it-2 belong to the ambrosia Fusarium clade of the FSSC, a group of symbionts of ambrosia beetles, including the pathogens of Fusarium dieback in avocados and teas. The detection of ambrosia beetles Euwallacea spp. from our specimens provided information on the vectors of the pathogens. Our present results suggest the fungi of the FSSC could be responsible for the Erythrina decline in other areas with damage.

Prozone-like phenomenon found in chemiluminescent enzyme immunoassay using magnetic particles for measurement of serum anti-single stranded DNA antibody titers: Definition and management.

BACKGROUND: Serum anti-single stranded DNA antibody (anti-ssDNAab) is used as a marker for systemic lupus erythematosus. We found a 'prozone-like phenomenon,' which was different from an original prozone phenomenon, in chemiluminescent enzyme immunoassay using magnetic particles for the measurement of serum anti-ssDNAab titers. We investigated mechanisms of the prozone-like phenomenon and countermeasures to prevent it from being overlooked. METHODS: This study examined 679 samples from patients tested for anti-ssDNAab titer at our hospital. In addition, the BF photometry OD value 2 (OD2), an index of optical density, was monitored simultaneously. RESULTS: The undiluted samples with a prozone-like phenomenon showed extremely lower OD2. Those samples were able to be distinguished from other samples by setting OD2 criteria based on the 95% prediction interval. Significant differences (p < 0.05) were found in the titer ratios (ten-fold diluted against undiluted) between groups with >1.5 and other groups with <1.5 for the ratios of OD2. CONCLUSIONS: We proposed two valuable methods to find a prozone-like phenomenon: one by setting OD2 criteria based on the 95% prediction interval and the other by analyzing the ratios both in titers and OD2 between undiluted and 10-fold diluted samples.

Water retained in tall Cryptomeria japonica leaves as studied by infrared micro-spectroscopy.

Recent studies in the tallest tree species suggest that physiological and anatomical traits of tree-top leaves are adapted to water-limited conditions. In order to examine water retention mechanism of leaves in a tall tree, infrared (IR) micro-spectroscopy was conducted on mature leaf cross-sections of tall Cryptomeria japonica D. Don from four different heights (51, 43, 31 and 19 m). We measured IR transmission spectra and mainly analyzed OH (3700-3000 cm-1) and C-O (1190-845 cm-1) absorption bands, indicating water molecules and sugar groups, respectively. The changes in IR spectra of leaf sections from different heights were compared with bulk-leaf hydraulics. Both average OH band area of the leaf sections and leaf water content were larger in the upper-crown, while osmotic potential at saturation did not vary with height, suggesting higher dissolved sugar contents of upper-crown leaves. As cell-wall is the main cellular structure of leaves, we inferred that larger average C-O band area of upper-crown leaves reflected higher content of structural polysaccharides such as cellulose, hemicellulose and pectin. Infrared micro-spectroscopic imaging showed that the OH and C-O band areas are large in the vascular bundle, transfusion tissue and epidermis. Infrared spectra of individual tissue showed that much more water is retained in vascular bundle and transfusion tissue than mesophyll. These results demonstrate that IR micro-spectroscopy is a powerful tool for visualizing detailed, quantitative information on the spatial distribution of chemical substances within plant tissues, which cannot be done using conventional methods like histochemical staining. The OH band could be well reproduced by four Gaussian OH components around 3530 (free water: long H bond), 3410 (pectin-like OH species), 3310 (cellulose-like OH species) and 3210 (bound water: short H bond) cm-1, and all of these OH components were higher in the upper crown while their relative proportions did not vary with height. Based on the spectral analyses, we inferred that polysaccharides play a key role in biomolecular retention of water in leaves of tall C. japonica.

Physiological and morphological acclimation to height in cupressoid leaves of 100-year-old Chamaecyparis obtusa.

Cupressoid (scale-like) leaves are morphologically and functionally intermediate between stems and leaves. While past studies on height acclimation of cupressoid leaves have focused on acclimation to the vertical light gradient, the relationship between morphology and hydraulic function remains unexplored. Here, we compared physiological and morphological characteristics between treetop and lower-crown leaves of 100-year-old Chamaecyparis obtusa Endl. trees (~27 m tall) to investigate whether height-acclimation compensates for hydraulic constraints. We found that physiological acclimation of leaves was determined by light, which drove the vertical gradient of evaporative demand, while leaf morphology and anatomy were determined by height. Compared with lower-crown leaves, treetop leaves were physiologically acclimated to water stress. Leaf hydraulic conductance was not affected by height, and this contributed to higher photosynthetic rates of treetop leaves. Treetop leaves had higher leaf area density and greater leaf mass per area, which increase light interception but could also decrease hydraulic efficiency. We inferred that transfusion tissue flanking the leaf vein, which was more developed in the treetop leaves, contributes to water-stress acclimation and maintenance of leaf hydraulic conductance by facilitating osmotic adjustment of leaf water potential and efficient water transport from xylem to mesophyll. Our findings may represent anatomical adaptation that compensates for hydraulic constraints on physiological function with increasing height.

Distribution of Streptococcus mutans strains with collagen-binding proteins in the oral cavity of IgA nephropathy patients.

BACKGROUND: IgA nephropathy (IgAN) is the most common primary chronic glomerulonephritis; however, its precise initiating pathogenesis remains unclear. Streptococcus mutans is a major pathogen of human dental caries. S. mutans strains with the cnm gene encoding Cnm, a collagen-binding protein, have been reported to contribute to the development of systemic diseases. However, the contribution of S. mutans with Cnm in the development of IgAN has not been reported. The aim of this study was to investigate the prevalence of cnm-positive S. mutans in IgAN patients and clarify the effects of cnm-positive S. mutans on the histological pathology of IgAN. METHODS: We identified the cnm gene in S. mutans isolated in saliva specimens, which were collected from IgAN patients (n = 53) and control subjects (n = 50). We evaluated the collagen-binding properties of S. mutans in IgAN patients and controls. The clinical parameters and histological scores were also assessed in IgAN patients. RESULTS: The rates of S. mutans isolation in IgAN and control groups were 84.0 and 84.9 %, respectively, not significantly dfferent. cnm-positive strains were significantly more prevalent in the IgAN group than in controls (32.1 vs. 14.0 %, p < 0.05). With regard to collagen-binding assays, the binding rates of cnm-positive strains were significantly higher in the IgAN group than in controls (96.6 vs. 30.0, p < 0.05). In addition, the segmental glomerulosclerosis scores were significantly higher in cnm-positive patients with IgAN than in cnm-negative patients with IgAN (0.94 vs. 0.57, p < 0.05). CONCLUSION: cnm-positive S. mutans strains are potentially associated with the pathogenesis of IgAN.

Preoperative evaluation of deep venous thrombosis in patients with pelvic organ prolapse.

AIM: The incidence of preoperative deep venous thrombosis (DVT) was examined in patients with pelvic organ prolapse (POP). MATERIAL AND METHODS: Preoperative screening for DVT was performed on the basis of D-dimer levels; if D-dimer levels were beyond the cut-off limit (0.5 µg/mL), ultrasound examination of the lower extremities was performed. A total of 75 consecutive patients who were scheduled for POP operation in our department were examined retrospectively. RESULTS: D-dimer levels were elevated in 24 patients (31.6%). Further, DVT was detected in 10 of the 75 patients, resulting in an incidence rate of 13.3% in the present study. D-dimer levels were significantly higher in the patients with DVT (1.25 ± 0.52 µg/mL vs 0.41 ± 0.26 µg/mL). There was no significant difference in age, and although body mass index and the number of diabetes mellitus cases were relatively higher in the patients with DVT than in those without DVT, there were no statistically significant differences. According to a receiver-operator curve, the suggested cut-off D-dimer value was 0.71 µg/mL (sensitivity and specificity was 0.9 and 0.877, respectively). CONCLUSIONS: Although this was a preliminary study, to the best of our knowledge, this is the first report describing the incidence of preoperative DVT in patients with POP. Our study indicates that the incidence of DVT in patients with POP might have been underestimated thus far. Therefore, every surgeon should evaluate patients for DVT prior to any surgical procedure for POP, particularly in those patients with obesity or diabetes mellitus.

Triclocarban-induced change in intracellular Ca²âº level in rat thymocytes: cytometric analysis with Fluo-3 under Zn²âº-free conditions.

Triclocarban (TCC) is an antimicrobial used in personal hygiene products. Recent health concerns arose after TCC was detected in the blood of human subjects who showered with soap containing TCC. In this study, the effect of TCC on intracellular Ca(2+) concentration in rat thymocytes was examined using Fluo-3, an indicator of intracellular Ca(2+). TCC at concentrations ranging from 0.1 µM to 3 µM increased intracellular Ca(2+) concentration biphasically: first by releasing Ca(2+) from intracellular Ca(2+) stores and then inducing Ca(2+) influx through store-operated Ca(2+) channels. The threshold TCC concentration to increase intracellular Ca(2+) concentration in this study was lower than the maximum TCC concentrations reported in human blood samples. Therefore, we anticipate that TCC at concentrations reported in human blood samples might disturb intracellular Ca(2+) signaling in human lymphocytes.

Autoimmune reactivity against precursor form of desmoglein 1 in healthy Tunisians in the area of endemic pemphigus foliaceus.

BACKGROUND: Desmoglein 1 (Dsg1), the pemphigus foliaceus (PF) antigen, is produced as a precursor (preDsg1) and is transported to the cell surface as the mature form (matDsg1). Recent studies show that B cells from North American individuals without pemphigus can potentially produce anti-preDsg1 IgG antibodies, but ELISA screening of large numbers of normal people in North America and Japan hardly ever shows circulating antibodies against preDsg1 or matDsg1. In contrast, in Tunisia, where PF is endemic, anti-Dsg1 IgGs are frequently detected in healthy individuals. OBJECTIVE: To characterize these anti-Dsg1 antibodies from normal individuals in Tunisia. METHODS: Sera from 16 healthy individuals and 9 PF patients in the endemic PF area in Tunisia, and sera from Japanese non-endemic PF patients were analyzed by immunoprecipitation-immunoblotting using recombinant proteins of preDsg1, matDsg1, and domain-swapped Dsg1/Dsg2 molecules. RESULTS: Sera from normal Tunisian individuals reacted to preDsg1 alone (8/16) or more strongly to preDsg1 than to matDsg1 (7/16), while those from all Tunisian PF patients and Japanese non-endemic PF patients reacted similarly to preDsg1 and matDsg1, or preferentially to matDsg1. The epitopes recognized by anti-Dsg1 IgGs from normal Tunisian individuals were more frequently found in the C-terminal extracellular domains (EC3 to EC5), while those in Tunisian endemic PF patients were more widely distributed throughout the extracellular domains, suggesting IgGs against EC1 and EC2 developed during disease progression. CONCLUSIONS: These findings indicate that IgG autoantibodies against Dsg1 are mostly raised against preDsg1 and/or C-terminal domains of Dsg1 in healthy Tunisians in the endemic area of PF.

Prenatal diagnosis of persistent left and absent right superior vena cava.

We report a case of persistent left superior vena cava (LSVC) with absent right superior vena cava (RSVC) diagnosed prenatally. At 27 weeks' gestation, routine fetal ultrasonography showed an abnormal four-chamber view. Fetal echocardiography revealed a markedly enlarged coronary sinus in the four-chamber view. An absent RSVC and a persistent LSVC were demonstrated in the three-vessel view. No additional cardiac malformations were seen before birth. Postnatal imaging confirmed the prenatal diagnosis. The combination of persistent LSVC and absent RSVC without any other cardiac malformations is an extremely rare cardiac anomaly and less commonly detected in utero. Identification of a dilated coronary sinus in the four-chamber view, with atypical features in the three-vessel view, may lead to the antenatal diagnosis of this condition.

Abnormal first-trimester fetal nuchal translucency and amniotic band syndrome.

We report a case of amniotic band syndrome diagnosed prenatally by serial sonographic examinations. Our initial sonographic image showed a large fetal nuchal translucency (NT) at 12 weeks' gestation. Repeated fetal ultrasound images revealed an amniotic band and right upper limb anomaly. Fetal MRI at 19 weeks' gestation revealed right forearm hypoplasia and pseudosyndactyly. The fetus was prenatally diagnosed with amniotic band syndrome and was suspected of having severe functional impairment of the deformed limb. The parents decided to terminate the pregnancy at 21 weeks' gestation. In fetuses with aneuploidy and various structural and genetic abnormalities, the NT thickness is increased in the first trimester. As far as we are aware, this is the first case report of increased NT and limb anomaly associated with amniotic band syndrome. In chromosomally normal fetuses with increased NT, intensive sonographic follow-up should provide grounds for a precise antenatal diagnosis.

Pathogenic epitopes of autoantibodies in pemphigus reside in the amino-terminal adhesive region of desmogleins which are unmasked by proteolytic processing of prosequence.

Pemphigus targets desmogleins (Dsgs), which are thought to be synthesized as inactive precursor proteins with prosequences that are cleaved by substilisin-like proprotein convertases, such as furin, to yield mature adhesive molecules. We hypothesized that some pemphigus pathogenic antibodies (Abs), which presumably interfere with adhesion, only bind the mature form. A pathogenic and three non-pathogenic anti-Dsg1 monoclonal Abs (mAbs) isolated from a pemphigus foliaceus (PF) patient, were used for immunoprecipitation and ELISA of recombinant precursor and mature Dsg1. The pathogenic Ab binds mature Dsg1, whereas non-pathogenic Abs bind either only the precursor or both the precursor and mature Dsg1. Competition ELISA showed that the majority of PF sera target the same or nearby epitopes defined by the pathogenic anti-Dsg1 mAb that blocked >20% binding of 29 out of 40 PF sera. Furthermore, the immunoreactivity of 45 PF sera against the mature Dsg1 was 3.2 fold stronger than that against the precursor Dsg1 by ELISA. Similar results were observed in anti-Dsg3 Abs in 47 pemphigus vulgaris sera, suggesting that most pemphigus sera target epitopes that are unmasked by proteolytic processing. These findings support the idea that at least some pathogenic pemphigus autoantibodies induce the loss of cell adhesion by directly binding the trans-interaction site of Dsgs.