Fluopyram Suppresses Population Densities of Heterodera glycines in Field and Greenhouse Studies in Michigan.

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, causes significant damage to soybean production annually. Fluopyram is a fungicide commonly used in soybean seed treatments intended to control soilborne fungal pathogens; however, recent studies have also suggested inhibitory effects on SCN. We examined the effects of a fluopyram seed treatment, ILeVO, on SCN reproduction, sudden death syndrome (SDS) development, and yield in a 3-year field study. Overall, fluopyram had a significant effect on yield (P = 0.046) and end-of-season SCN eggs and second-stage juveniles (Pf, P = 0.033) but no significant effect on SCN reproduction (Rf) or SDS disease index (P > 0.05). Post hoc tests indicated that fluopyram increased yield and suppressed SCN quantities. However, Rf was consistently greater than 1 whether or not the seed was treated with fluopyram, indicating that SCN populations were still increasing in the presence of fluopyram. A follow-up greenhouse study indicated that fluopyram reduced SCN relative to nontreated controls, as observed in the field, but only reduced SCN DNA within roots of a susceptible cultivar. These results indicate that fluopyram can suppress SCN quantities relative to nontreated seed but may not successfully reduce nematode populations without the use of additional management strategies.

Identification of Fungal Communities Within the Tar Spot Complex of Corn in Michigan via Next-Generation Sequencing.

Tar spot is a fungal disease complex of corn that has been destructive and yield limiting in Central and South America for nearly 50 years. Phyllachora maydis, the causal agent of tar spot, is an emerging corn pathogen in the United States, first reported in 2015 from major corn producing regions of the country. The tar spot disease complex putatively includes Monographella maydis (syn. Microdochium maydis), which increases disease damage through the development of necrotic halos surrounding tar spot lesions. These necrotic halos, termed "fish-eye" symptoms, have been identified in the United States, though Monographella maydis has not yet been confirmed. A recent surge in disease severity and loss of yield attributed to tar spot in the United States has led to increased attention and expanded efforts to understand the disease complex and how to manage it. In this study, next-generation sequencing of the internal transcribed spacer-1 (ITS1) ribosomal DNA was used to identify fungal taxa that distinguish tar spot infections with or without fish-eye symptoms. Fungal communities within tar spot only lesions were significantly different from communities having fish-eye symptoms. Two low abundance OTUs were identified as Microdochium sp., however, neither were associated with fish-eye symptom development. Interestingly, a single OTU was found to be significantly more abundant in fish-eye lesions compared to tar spot lesions and had a 91% ITS1 identity to Neottiosporina paspali. In addition, the occurrence of this OTU was positively associated with Phyllachora maydis fish-eye symptom networks, but not in tar spot symptom networks. Neottiosporina paspali has been reported to cause necrotic lesions on various monocot grasses. Whether the related fungus we detected is part of the tar-spot complex of corn and responsible for fish-eye lesions remains to be tested. Alternatively, many OTUs identified as Phyllachora maydis, suggesting that different isolate genotypes may be capable of causing both tar spot and fish-eye symptoms, independent of other fungi. We conclude that Monographella maydis is not required for fish-eye symptoms in tar spot of corn.

Increased micronucleus frequency in exfoliated cells of the buccal mucosa in hairdressers.

Hairdressers are exposed daily to chemical substances, such as dyes, chemical straighteners and curling chemicals, which can be absorbed, inhaled or possibly ingested. We analyzed the frequency of micronuclei (MNC) in exfoliated cells of the buccal mucosa of 50 hairdressers and 50 controls in Pelotas, RS, Brazil. An assessment was carried out on the incidence of MNC, binucleated cells (BNC), broken egg cells (BEC), budding cells (BC), and the sum of anomalies (SA), in 2000 cells per individual. The data were analyzed with SPSS, using the Mann-Whitney U-test, α = 0.05. The mean number of anomalies in hairdressers was 2.02 ± 3.60 MNC; 8.50 ± 5.07 BNC; 9.06 ± 3.83 BEC; 0.32 ± 0.62 BC, and 19.90 ± 9.61 SA; in controls it was 0.36 ± 1.06 MNC; 5.20 ± 4.73 BNC; 5.92 ± 2.67 BEC; 0.10 ± 0.36 BC, and 11.58 ± 6.67 SA; the differences for all parameters were significant. The non-occupational factors did not significantly influence the alterations. A significant increase of BEC (P = 0.003) was observed in the hairdressers and SA (P = 0.033) in females. The lowest income level influenced MNC (P = 0.044), and the habit of not smoking influenced SA (P = 0.020). We concluded that exposure to substances used by hairdressers is genotoxic for men.

Buccal micronucleus frequency is associated with age in Down syndrome.

Down syndrome has been linked to premature aging and genomic instability. We examined the frequency of micronucleus (MN) and binucleated cells in the oral mucosa of Down syndrome patients and healthy controls matched by age and gender, addressing the effect of age and family income. Down syndrome individuals had an increased number of MN (14.30 +/- 9.35 vs 4.03 +/- 1.71; P<0.001) and binucleated cells (0.97 +/- 1.3 vs 0.33 +/- 0.66; P<0.05) per 2000 cells. Micronucleus frequency of Down syndrome individuals correlated positively with age (r = 0.437; P = 0.009), and the older (> or =21) Down syndrome age group (30.8 +/- 8.4 years old) had about 2-fold more micronuclei (P < or = 0.05) than did the younger group (<21). Average family income did not correlate with MN frequency in controls (r = -0.948; P = 0.183), but a borderline negative correlation was seen in DS subjects (r = -0.9484; P = 0.0516). Individuals whose average income was ten times minimum wages had about 2-fold less MN than those receiving around minimum wage. We conclude that the buccal MN assay is a useful and minimally invasive method for monitoring genetic damage in humans and could be used as a tool to evaluate age-associated genomic instability in Down syndrome.

Mutagenic potential of water from Pelotas Creek in Rio Grande do Sul, Brazil.

Water resource degradation is one of mankind's greatest worries, as it causes direct and indirect damage to the associated biota. We initiated a water monitoring study in Pelotas Creek in 2003 in order to assess the mutagenic effect of the creek's waters. Allium cepa cells exposed to water samples and a chronically exposed macrophyte were analyzed, through evaluation of the mitotic index, mitotic anomalies, interphase anomalies, and total anomalies. Five points were chosen along the lower course of Pelotas Creek, from which water samples and floating pennywort (Hydrocotyle ranunculoides, Apiaceae) were collected in 2006 and 2007. The enteric bacterium Escherichia coli was found at all sampling points; in the physical-chemical analysis, a few variables exceeded permitted limits, pH (from 6 to 9), chloride (250 mg/L), hardness (from 10 to 200 mg CaCO(3)/L), and conductivity (100 microOmega/cm). There was an increased number of cytogenetic anomalies in exposed A. cepa cells and in the pennywort in 2006 relative to 2007, which may be explained by the increased rainfall, which was three times greater in 2007 at some stations than in 2006.Omega/cm). There was an increased number of cytogenetic anomalies in exposed A. cepa cells and in the pennywort in 2006 relative to 2007, which may be explained by the increased rainfall, which was three times greater in 2007 at some stations than in 2006.

Increasing mutagenicity of São Gonçalo Channel waters based on the Allium cepa test.

The São Gonçalo Channel is of great importance to the conservation of local biodiversity; it also is a water supply source of the city of Pelotas, Brazil, and the surrounding region. We examined the mutagenic activity of its waters. The following items were seasonally investigated in Allium cepa root radicular meristem cells: mitotic index, mitotic anomalies, interphase anomalies, and total anomalies. Water samples were collected from four different stations, Lock Dam, Santa Bárbara Channel, Pelotas Creek, and Barra do Laranjal. A drinking water negative control was used. For each sampling station, 8000 cells were counted, 2000 of which by repetition. The data were computed on a database (SPSS), and then analyzed by the chi-square test and the Mann-Whitney U-test. In 2005, the channel water provoked a significantly greater number of anomalies than the control water. The number of anomalies increased in 2007. This suggests that there was an increase in toxic substances in the channel over the years.

Genotoxicity evaluation in chronic renal patients undergoing hemodialysis and peritoneal dialysis, using the micronucleus test.

Patients with chronic renal disease have an increased incidence of cancer. It is well known that long periods of hemodialysis treatment are linked to DNA damage due to oxidative stress. This genotoxic effect may cause the loss of chromosome fragments, or even entire chromosomes, which form micronuclei after cell division, and can be detected by the micronucleus test. In the present case-control study, we evaluated the genotoxic effect of hemodialysis treatment in 20 patients undergoing hemodialysis, and 20 subjected to peritoneal dialysis, matched for gender and age with 40 controls. Genetic damage was assessed by examining the frequency of micronuclei in 2000 exfoliated buccal cells per individual. Our results revealed that patients undergoing hemodialysis treatment have a significantly higher frequency of micronucleated cells (MNC; 5.60 +/- 5.31) compared to control subjects (1.50 +/- 2.01, p < 0.01). Interestingly, the same was not observed for the peritoneal dialysis patients who showed no significant differences in MNC (2.85 +/- 2.96) frequency compared to control individuals (3.25 +/- 3.85). In addition, we evaluated the possible association between creatine levels, smoking, alcohol intake, age, duration of treatment, and incomes of the individuals (separately analyzed according to their gender) and the frequency of micronuclei. The results reported here indicate that the duration of treatment is the only factor associated with increased MNC frequency among hemodialysis patients (Spearman coefficient of 0.414, p = 0.01). The number of MNC found in individuals with six years or less of treatment was significantly lower (2.91 +/- 2.74) compared to patients with seven or more years of treatment (8.89 +/- 5.96, p < 0.05). Overall, peritoneal dialysis may be a safer choice of treatment, but further studies need to be performed to investigate the risks and benefits of both treatments.

Genotoxicity evaluation in chronic renal patients undergoing hemodialysis and peritoneal dialysis, using the micronucleus test

Patients with chronic renal disease have an increased incidence of cancer. It is well known that long periods of hemodialysis treatment are linked to DNA damage due to oxidative stress. This genotoxic effect may cause the loss of chromosome fragments, or even entire chromosomes, which form micronuclei after cell division, and can be detected by the micronucleus test. In the present case-control study, we evaluated the genotoxic effect of hemodialysis treatment in 20 patients undergoing hemodialysis, and 20 subjected to peritoneal dialysis, matched for gender and age with 40 controls. Genetic damage was assessed by examining the frequency of micronuclei in 2000 exfoliated buccal cells per individual. Our results revealed that patients undergoing hemodialysis treatment have a significantly higher frequency of micronucleated cells (MNC; 5.60 ± 5.31) compared to control subjects (1.50 ± 2.01, p < 0.01). Interestingly, the same was not observed for the peritoneal dialysis patients who showed no significant differences in MNC (2.85 ± 2.96) frequency compared to control individuals (3.25 ± 3.85). In addition, we evaluated the possible association between creatine levels, smoking, alcohol intake, age, duration of treatment, and incomes of the individuals (separately analyzed according to their gender) and the frequency of micronuclei. The results reported here indicate that the duration of treatment is the only factor associated with increased MNC frequency among hemodialysis patients (Spearman coefficient of 0.414, p = 0.01). The number of MNC found in individuals with six years or less of treatment was significantly lower (2.91 ± 2.74) compared to patients with seven or more years of treatment (8.89 ± 5.96, p < 0.05). Overall, peritoneal dialysis may be a safer choice of treatment, but further studies need to be performed to investigate the risks and benefits of both treatments.

Cytogenetics of genetic counseling patients in Pelotas, Rio Grande do Sul, Brazil.

From 1986 to 2002, we examined the chromosomal composition of 916 patients attended by two genetic counseling services in the city of Pelotas, in the Brazilian State of Rio Grande do Sul, to determine the genetic causes of their disturbances. Patterns of G-banding using trypsin and Giemsa (GTG) and C-banding using barium and Giemsa (CBG) were studied using phytohemagglutinin M-stimulated lymphocytes cultured from peripheral blood. Among the patients, 110 had Down's syndrome, 7 had Edward's syndrome, 4 had Patau's syndrome, 29 had Turner's syndrome, 5 had Klinefelter's syndrome, and 3 had "cri-du-chat" syndrome. Abnormal chromosomes were observed in 29.3% of the patients. Most of these (56.3%) were numerical abnormalities, with the remaining being structural variants.

Patau syndrome with a long survival. A case report.

Trisomy 13 is a clinically severe entity; 85% of the patients do not survive beyond one year, and most children die before completing six months of age. We report a female child, 28 months old, white, the fourth child of a non-consanguineous couple, who presented trisomy 13. The child was born at term, from a vaginal delivery, weighing 2600 g. At birth, she was cyanotic, icteric, spastic, and cried weakly. The initial clinical examination detected polydactyly in the left hand, congenital clubfoot and convex soles, ocular hypertelorism, a low nasal bridge, numerous hemangiomas distributed throughout the body, cardiomegaly, and perimembranous inter-ventricular communication. There was no cleft lip or palate. On physical examination at 18 months old, the child weighed 6,900 g, had a cephalic perimeter of 41 cm, a thoracic perimeter of 43 cm and was 76 cm tall. At 28 months, she weighed 10,760 g and was 88.5 cm tall. Neuropsychomotor development retardation was evident from birth and, according to the psychologist and the social assistant of APAE (Handicapped Parents and Friends Association) in Canguçu, Rio Grande do Sul, there was a noticeable improvement after physiotherapy and recreational sessions.

Patau syndrome with a long survival: a case report

Trisomy 13 is a clinically severe entity; 85% of the patients do not survive beyond one year, and most children die before completing six months of age. We report a female child, 28 months old, white, the fourth child of a non-consanguineous couple, who presented trisomy 13. The child was born at term, from a vaginal delivery, weighing 2600 g. At birth, she was cyanotic, icteric, spastic, and cried weakly. The initial clinical examination detected polydactyly in the left hand, congenital clubfoot and convex soles, ocular hypertelorism, a low nasal bridge, numerous hemangiomas distributed throughout the body, cardiomegaly, and perimembranous inter-ventricular communication. There was no cleft lip or palate. On physical examination at 18 months old, the child weighed 6,900 g, had a cephalic perimeter of 41 cm, a thoracic perimeter of 43 cm and was 76 cm tall. At 28 months, she weighed 10,760 g and was 88.5 cm tall. Neuropsychomotor development retardation was evident from birth and, according to the psychologist and the social assistant of APAE (Handicapped Parents and Friends Association) in Cangucu, Rio Grande do Sul, there was a noticeable improvement after physiotherapy and recreational sessions.

Cytogenetics of genetic counseling patients in Pelotas, Rio Grande do Sul, Brazil

From 1986 to 2002, we examined the chromosomal composition of 916 patients attended by two genetic counseling services in the city of Pelotas, in the Brazilian State of Rio Grande do Sul, to determine the genetic causes of their disturbances. Patterns of G-banding using trypsin and Giemsa (GTG) and C-banding using barium and Giemsa (CBG) were studied using phytohemagglutinin M-stimulated lymphocytes cultured from peripheral blood. Among the patients, 110 had Down's syndrome, 7 had Edward's syndrome, 4 had Patau's syndrome, 29 had Turner's syndrome, 5 had Klinefelter's syndrome, and 3 had [quot ]cri-du-chat[quot ] syndrome. Abnormal chromosomes were observed in 29.3% of the patients. Most of these (56.3%) were numerical abnormalities, with the remaining being structural variants.

Occupational genotoxicity risk evaluation through the comet assay and the micronucleus test

The micronucleus (MN) test and the alkaline single cell gel or comet assay were applied to exfoliated cells of the buccal mucous in order to evaluate the genotoxic risk associated with occupational exposure of 10 storage battery renovation workers, and 10 car painters, with age matched controls, in Pelotas, RS, in southern Brazil. In the MN test, 2000 exfoliated buccal cells were analyzed for each individual, while 100 cells were examined in the comet assay. In the comet test, both comet tail length and a damage index were calculated. Highly significant effects of occupational exposure were found with both the MN test and the comet assay (P<0.001). The comet assay was found to be rapid, of simple visualization, and it is a sensitive technique for measuring and analyzing DNA damage in human cells

Occupational genotoxicity risk evaluation through the comet assay and the micronucleus test.

The micronucleus (MN) test and the alkaline single cell gel or comet assay were applied to exfoliated cells of the buccal mucous in order to evaluate the genotoxic risk associated with occupational exposure of 10 storage battery renovation workers, and 10 car painters, with age matched controls, in Pelotas, RS, in southern Brazil. In the MN test, 2000 exfoliated buccal cells were analyzed for each individual, while 100 cells were examined in the comet assay. In the comet test, both comet tail length and a damage index were calculated. Highly significant effects of occupational exposure were found with both the MN test and the comet assay (P<0.001). The comet assay was found to be rapid, of simple visualization, and it is a sensitive technique for measuring and analyzing DNA damage in human cells.

Evaluation of genotoxicity through micronuclei test in workers of car and battery repair garages

In this study, the micronuclei test (MNT) was applied in exfoliated cells of buccal mucosa, in order to evaluate the genotoxic risk associated with occupational exposure of mechanics, storage battery renovation workers, and car painters. For each individual, 3000 exfoliated buccal cells were analyzed. There was a significantly higher frequency of micronucleated cells (MNC) in the exposed workers than in controls. Smoking and drinking habits, age, and working time did not represent significant factors in terms of increasing the production of micronuclei (MN), when the control and the exposed groups were compared. These results allowed to conclude that the studied individuals belong to a risk group and should periodically undergo biological monitoring and proper care

Immunolocalisation of phospholipase D1 on tubular vesicular membranes of endocytic and secretory origin.

We have examined the localisation of overexpressed phospholipase D1 (PLD1) using antibodies against its amino- and carboxyl-terminal domains. PLD1 overexpressed in COS-7 cells showed variable distribution by immunofluorescence but was mainly in punctate structures in the perinuclear region and at the plasma membrane. Downregulation by an anti-sense plasmid resulted in almost exclusively perinuclear distribution in punctate structures that contained immunoreactivity for the endogenous KDEL receptor and the early endosomal antigen EEA1 protein. Influenza haemagglutinin (HA) and HA-derived mutants designed to locate primarily to secretory or endocytic membranes were present in PLD1-positive membranes. Immunofluorescence analysis in permanent CHO cell lines that express PLD1 inducibly confirmed the presence of PLD1 on both endocytic and secretory membranes. Analysis of PLD1 distribution by immunocytochemistry and electron microscopy of intact CHO cells and of isolated membranes revealed that PLD1 was present in tubulovesicular elements and multivesicular bodies. Some of these were close to the Golgi region whereas others stained positive for endocytic cargo proteins. Morphometric analysis assigned the majority of PLD1 immunoreactivity on endosomal membranes and a smaller amount on membranes of secretory origin. PLD1, via signals that are currently not understood, is capable of localising in tubulovesicular membranes of both endocytic and secretory origin.

Internalization-competent influenza hemagglutinin mutants form complexes with clathrin-deficient multivalent AP-2 oligomers in live cells.

Most membrane proteins are endocytosed through clathrin-coated pits via AP-2 adaptor complexes. However, little is known about the interaction of internalization signals with AP-2 in live cells in the absence of clathrin lattices. To investigate this issue, we employed cells cotransfected with pairs of antigenically distinct influenza hemagglutinin (HA) mutants containing different internalization signals of the YXXZ family. To enable studies on the possible association of the naturally trimeric HAs into higher order complexes via binding to AP-2, we exploited the inability of HAs from different influenza strains to form mutual trimers. Thus, we coexpressed HA pairs from different strains (Japan and X:31) bearing similar cytoplasmic tails mutated to include internalization signals. Using antibody-mediated immunofluorescence co-patching on live cells, we demonstrate that internalization-competent HA mutants form higher order complexes and that this clustering depends on the strength of the internalization signal. The clustering persisted in cells treated with hypertonic medium to disperse the clathrin lattices, as validated by co-immunoprecipitation experiments. The clustering of HAs bearing strong internalization signals appears to be mediated via binding to AP-2, as indicated by (i) the coprecipitation of alpha-adaptin with these HAs, even in hypertonically treated cells; (ii) the co-localization (after hypertonic treatment) of AP-2 with antibody-mediated patches of these mutants; and (iii) the dispersal of the higher order HA complexes following chlorpromazine treatment, which removes AP-2 from the plasma membrane. These results suggest that even in the absence of clathrin lattices, AP-2 exists in multivalent complexes capable of simultaneously binding several internalization signals from the same family.

A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion.

A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23 degrees C rather than 37 degrees C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37 degrees C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4 degrees C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.

Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3.

BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. RESULTS: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. CONCLUSIONS: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.