Characterization of Aspergillus section Nigri species populations in vineyard soil using droplet digital PCR.

Identification of populations of Aspergillus section Nigri species in environmental samples using traditional methods is laborious and impractical for large numbers of samples. We developed species-specific primers and probes for quantitative droplet digital PCR (ddPCR) to improve sample throughput and simultaneously detect multiple species in each sample. The ddPCR method was used to distinguish Aspergillus niger, Aspergillus welwitschiae, Aspergillus tubingensis and Aspergillus carbonarius in mixed samples of total DNA. Relative abundance of each species measured by ddPCR agreed with input ratios of template DNAs. Soil samples were collected at six time points over two growing seasons from two raisin vineyards in Fresno County, California. Aspergillus section Nigri strains were detected in these soils in the range of 102 -105  CFU g-1 . Relative abundance of each species varied widely among samples, but in 52 of 60 samples, A. niger was the most abundant species, ranging from 38 to 88% of the total population. In combination with total plate counts, this ddPCR method provides a high-throughput method for describing population dynamics of important potential mycotoxin-producing species in environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate the utility of ddPCR as a means to quantify species of Aspergillus section Nigri in soil. This method eliminates the need for isolation and sequence identification of individual fungal isolates, and allows for greater throughput in measuring relative population sizes of important (i.e. mycotoxigenic) Aspergillus species within a population of morphologically indistinguishable species.

Detection and discrimination of four Aspergillus section Nigri species by PCR.

Species of Aspergillus section Nigri are not easily distinguished by traditional morphological techniques, and typically are identified by DNA sequencing methods. We developed four PCR primers to distinguish between Aspergillus niger, Aspergillus welwitschiae, Aspergillus carbonarius and Aspergillus tubingensis, based on species-conserved differences in the calmodulin gene sequence. PCR amplification from total DNA using these primers was species specific; no amplification occurred from nontarget species DNA for each primer pair. Species-specific PCR could distinguish between species in mixed DNA templates, indicating a utility in determining culture uniformity of isolated Aspergillus strains. In addition, with these primer sets, each species could be detected in soil following mixed-species inoculation with Aspergillus spores. This indicates that PCR with these species-specific primers may be useful in determining the distribution of Aspergillus species in environmental samples without the need for species identification from isolated strains, as well as detecting species that may be infrequently isolated by culture-based methods.

Assessing transmission of crop diseases by insect vectors in a landscape context.

Theory indicates that landscape composition affects transmission of vector-borne crop diseases, but few empirical studies have investigated how landscape composition affects plant disease epidemiology. Since 2006, Bemisia tabaci (Gennadius) has vectored the cucurbit yellow stunting disorder virus (CYSDV) to cantaloupe and honeydew melons (Cucumis melo L.) in the southwestern United States and northern Mexico, causing significant reductions in yield of fall melons and increased use of insecticides. Here, we show that a landscape-based approach allowing simultaneous assessment of impacts of local (i.e., planting date) and regional (i.e., landscape composition) factors provides valuable insights on how to reduce crop disease risks. Specifically, we found that planting fall melon fields early in the growing season, eliminating plants germinating from seeds produced by spring melons after harvest, and planting fall melon fields away from cotton and spring melon fields may significantly reduce the incidence of CYSDV infection in fall melons. Because the largest scale of significance of the positive association between abundance of cotton and spring melon fields and CYSDV incidence was 1,750 and 3,000 m, respectively, reducing areas of cotton and spring melon fields within these distances from fall melon fields may decrease CYSDV incidence. Our results indicate that landscape-based studies will be fruitful to alleviate limitations imposed on crop production by vector-borne diseases.

Bacterial population dynamics during the ensiling of Medicago sativa (alfalfa) and subsequent exposure to air.

AIMS: To describe, at high resolution, the bacterial population dynamics and chemical transformations during the ensiling of alfalfa and subsequent exposure to air. METHODS AND RESULTS: Samples of alfalfa, ensiled alfalfa and silage exposed to air were collected and their bacterial population structures compared using 16S rRNA gene libraries containing approximately 1900 sequences each. Cultural and chemical analyses were also performed to complement the 16S gene sequence data. Sequence analysis revealed significant differences (P < 0·05) in the bacterial populations at each time point. The alfalfa-derived library contained mostly sequences associated with the Gammaproteobacteria (including the genera: Enterobacter, Erwinia and Pantoea); the ensiled material contained mostly sequences associated with the lactic acid bacteria (LAB) (including the genera: Lactobacillus, Pediococcus and Lactococcus). Exposure to air resulted in even greater percentages of LAB, especially among the genus Lactobacillus, and a significant drop in bacterial diversity. CONCLUSIONS: In-depth 16S rRNA gene sequence analysis revealed significant bacterial population structure changes during ensiling and again during exposure to air. SIGNIFICANCE AND IMPACT OF THE STUDY: This in-depth description of the bacterial population dynamics that occurred during ensiling and simulated feed out expands our knowledge of these processes.

Isolation and identification of ochratoxin A-producing Aspergillus section Nigri strains from California raisins.

AIMS: To determine incidence and levels of ochratoxin A (OTA) in California raisins and to isolate and characterize OTA-producing fungi from California raisin vineyard populations. METHODS AND RESULTS: Forty raisin clusters sampled from four California vineyards in the San Joaquin Valley were analysed for OTA content using immunoaffinity and HPLC methods. OTA was detected in 93% of the samples, at levels from 0·06 to 11·4 ng g⁻¹. From these raisin samples, a total of 400 strains of Aspergillus were isolated and analysed for OTA production. Twelve isolates (3%), from five raisin samples, produced OTA. These isolates were identified as Aspergillus carbonarius, based on morphological characteristics and multilocus sequence analysis. Levels of OTA produced by these isolates on raisin agar ranged from 0·9 to 15 µg g⁻¹. CONCLUSIONS: OTA is a common contaminant of raisin vineyards, but average levels are much lower than EU regulatory limits for dried fruit. The primary species responsible for OTA contamination in California raisins is A. carbonarius. SIGNIFICANCE AND IMPACT OF THE STUDY: This study illustrates that low-level OTA contamination of raisins occurs in California and that ecological studies of A. carbonarius within the Aspergillus section Nigri population on raisins are warranted to monitor ochratoxigenic potential of the crop.

Effluent carbonaceous biochemical oxygen demand (CBOD) characterization for modern pulp and paper facilities.

As receiving water quality models are being used to address dissolved oxygen issues requiring an increased degree of resolution, a more refined characterization of effluent CBOD can become an important aspect of the analysis. The selection and use of kinetic models to identify effluent specific parameters can have a significant impact on this characterization. This study modeled effluents from six pulp and paper facilities in order to reassess the kinetic models, the data, and experimental design used for a typical effluent characterization. The dual first order model fit these effluents with significantly less error than the traditional first order model suggesting a significant fraction of the CBOD is slowly degradable. Because the dual first order model produces a more refined characterization of CBOD kinetics than the first order model, it places an increased demand upon the data used to inform the parameter estimates. Therefore, analysis of the precision of the parameter estimates and methods for improving estimation precision via experimental design are also discussed.

Evaluation of Trap Cropping for Control of Diamondback Moth (Lepidoptera: Plutellidae) in a Broccoli Production System.

Trap cropping, in which a trap crop is planted near a cash crop, has been used successfully for reducing pest damage in some agricultural systems. We used a meta-analysis of extensive data on two trap cropping systems, diamondback moth, Plutella xylostella (Linnaeus), exploiting cabbage and Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) exploiting maize, to show that oviposition preference for, and high larval mortality on trap crops are important indicators of effectiveness of trap cropping systems. We then evaluated Indian mustard (Brassica juncea var. juncea L. Czern.) (Capparidales: Brassicaceae) and yellow rocket (Barbarea vulgaris W. T. Aiton) (Brassicales: Brassicaceae) as trap crops for protecting broccoli (Brassica oleracea var. italica Plenck) (Capparidales: Brassicaceae) against diamondback moth in Yuma, AZ, using planting configurations compatible with current practices for commercial production and without use of insecticides. In oviposition choice tests, both yellow rocket and Indian mustard were highly preferred over broccoli in the field. Furthermore, the number of larvae and pupae was significantly lower on yellow rocket and Indian mustard compared to broccoli, indicating relatively high mortality on these trap crops. Nevertheless, during the fall and spring growing seasons, no significant differences in the number of individuals on broccoli or proportion of broccoli crowns infested at harvest occurred between plots with trap crops relative to plots exclusively planted to broccoli. Thus, with the plant density and planting patterns used and without use of insecticides, there was no evidence that trap cropping was effective for reducing diamondback moth infestation of broccoli.

Protease-activated receptor-1 impedes prostate and intestinal tumor progression in mice.

Essentials Protease activated receptor-1 (PAR-1) has been proposed to drive cancer progression. Surprisingly, PAR-1 deletion accelerated tumor progression in two distinct experimental settings. PAR-1 deletion was shown to limit the apoptosis of transformed epithelial cells. Thrombin- and activated protein C-mediated PAR-1 activation have unique effects on tumor cell biology. SUMMARY: Background Multiple studies have implicated protease-activated receptor-1 (PAR-1), a G-protein-coupled receptor activated by proteolytic cleavage of its N-terminus, as one target coupling thrombin-mediated proteolysis to tumor progression. Objective To analyze the role of PAR-1 in the setting of two distinct spontaneously developing tumor models in mice. Methods We interbred PAR-1-deficient mice with Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, which spontaneously develop prostate tumors, and adenomatous polyposis coli Min (APCMin/+ ) mice, which spontaneously develop intestinal adenomas. Results Analyses of TRAMP mice with advanced disease (30 weeks) revealed that PAR-1 deficiency resulted in significantly larger and more aggressive prostate tumors. Prostates collected at an earlier time point (12 weeks of age) revealed that PAR-1 promotes apoptosis in transformed epithelia. In vitro analyses of TRAMP-derived cells revealed that activated protein C-mediated PAR-1 cleavage can induce tumor cell apoptosis, suggesting that tumor cell-intrinsic PAR-1 functions can limit tumor progression. Paralleling results in TRAMP mice, PAR-1-deficient APCMin/+ mice developed three-fold more adenomas than PAR-1-expressing mice, and the adenomas that formed were significantly larger. Moreover, loss of PAR-1 expression was shown to limit apoptosis in transformed intestinal epithelial cells. Conclusions Together, these results demonstrate a previously unrecognized role for PAR-1 in impeding tumor progression in vivo. These results also offer a cautionary note suggesting that long-term PAR-1 inhibition could increase malignancy risk in some contexts.

Field evaluation of Cucurbit yellow stunting disorder virus transmission by Bemisia tabaci.

Cucurbit yellow stunting disorder virus (CYSDV) is a whitefly-transmitted Crinivirus (Closteroviridae) that impacts melon production in many parts of the world including the USA. It has been responsible for melon crop loss in the southwestern U.S. since 2006 when it was first identified. Control strategies have revolved mainly around chemical control, but research to identify suitable products and approaches to implementing them have lagged. The current study investigated the performance of four systemic insecticides in the field while concurrently tracking CYSDV disease progression after controlled and natural whitefly inoculation of young melon plants. Assessments of virus incidence were made using two different visual observation methods in concert with ELISA analyses of leaf disks samples collected biweekly. Infection rates were consistently lowest in plots treated with the butenolide insecticide flupyradifurone while dinotefuran was second in efficacy measures. Flupyradifurone also held whitefly densities to their lowest numbers relative to the other treatments. Two other insecticides, imidacloprid and cyantraniliprole, exacerbated virus incidence in multiple trials. Further investigation into the anomalous finding of increased virus incidence due to insecticide application is ongoing.

Protease-activated receptor-1 inhibits proliferation but enhances leukemia stem cell activity in acute myeloid leukemia.

Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1- cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.

Specificity of serotonin reuptake inhibitors in the treatment of obsessive-compulsive disorder. Comparison of fluvoxamine and desipramine.

To evaluate whether serotonin reuptake inhibition is critical to the treatment of obsessive-compulsive disorder, 40 outpatients with a principal diagnosis of obsessive-compulsive disorder were randomized in a double-blind fashion to 8 weeks of treatment with either the serotonin reuptake inhibitor fluvoxamine maleate (n = 21) or the norepinephrine reuptake inhibitor desipramine hydrochloride (n = 19). Fluvoxamine was significantly better than desipramine in reducing the severity of obsessive-compulsive symptoms, as measured by the Yale-Brown Obsessive Compulsive Scale and by the global response rate ("responder" equaling "much improved"). Eleven of 21 patients were responders with fluvoxamine compared with 2 of 19 patients with desipramine. Fluvoxamine, but not desipramine, was also effective in reducing the severity of "secondary" depression. Fluvoxamine-induced improvement in symptoms of obsessive-compulsive disorder was not correlated with the severity of baseline depressive symptoms. This study provides additional evidence that the acute serotonin reuptake properties of a drug are predictive of its anti-obsessive-compulsive efficacy. It is hypothesized that the mechanism of action of serotonin reuptake inhibitors in obsessive-compulsive disorder may be related to chronic treatment-induced adaptive changes in presynaptic serotonin receptor function (eg, autoreceptor desensitization) and/or indirect influences on dopaminergic function (eg, in the basal ganglia).

Clinical phenomenology and neurobiology of cocaine abstinence: a prospective inpatient study.

OBJECTIVE: This study was undertaken to document symptoms and changes in dopaminergic function emerging after abrupt cessation of cocaine use. METHOD: After admission, 22 patients with DSM-III-R cocaine dependence were observed drug free for 3 weeks. The patient-rated Ribicoff Abstinence Rating Scale, Symptom Rating Scale, Physical Symptom Scale, Patient Rated Anxiety Scale, Beck Depression Inventory, and visual analogue scales for 16 subjective states were completed daily, and nurses rated 13 patients with the global anxiety and depression items of the Short Clinical Rating Scale. Serial blood samples were obtained three times weekly, and the patients' levels of prolactin, growth hormone (GH), and homovanillic acid (HVA) were measured. Their prolactin and GH values were compared with those of matched normal subjects. RESULTS: A total of 62 subjective symptom variables were evaluated. At baseline, the symptom ratings were mildly elevated. At 3 weeks there were significant decreases from baseline in 28 variables and nearly significant decreases in six additional variables. Nurse-rated anxiety and depression also changed, but in a more variable pattern. There was a small but significant increase from baseline over time in plasma prolactin, but there were no significant changes in GH or HVA. The patients' prolactin and GH values did not differ from those of the normal subjects. CONCLUSIONS: These findings suggest that symptoms after inpatient cessation of uncomplicated cocaine addiction are relatively mild and decrease linearly over the first month. Evidence of dysregulated central dopamine function was limited. The findings do not support routine use of pharmacological agents in the inpatient management of such patients.

Role of fibrinogen- and platelet-mediated hemostasis in mouse embryogenesis and reproduction.

Studies of mice with genetic deficits in specific coagulation factors have shown that many, but not all, components of the hemostatic system serve an essential role in mouse embryogenesis and pregnancy. Although the developmental failures observed in these mice are typically associated with severe hemorrhage, it is uncertain whether the role of coagulation factors in embryogenesis and reproduction is specifically tied to their function in thrombus formation and prevention of blood loss (i.e. hemostasis). Here, we show (i) that a complete loss of fibrinogen- and platelet-dependent hemostatic capacity does not reproduce the developmental defects occurring in mice with either deficits in thrombin generation or unfettered thrombin consumption; (ii) that the essential role of fibrinogen in the maintenance of pregnancy does not involve interaction with platelets; and (iii) that the previously described in utero growth retardation of gene-targeted mice lacking NF-E2, a transcription factor critical for megakaryopoieis, is not caused by a loss of platelet-dependent hemostatic function. In addition, we demonstrate (iv) that fibrinogen can confer physiologically relevant hemostatic function in the absence of platelets, but that a complete loss of hemostatic capacity results if a combined absence of these components is genetically imposed. These findings support the notion that the function of coagulation factors for in utero development and pregnancy is mechanistically distinct from their ability to mediate the formation of hemostatic platelet-fibrin(ogen) aggregates.

Genetic manipulation of fibrinogen and fibrinolysis in mice.

Vascular integrity is maintained by a sophisticated system of circulating and cell associated hemostatic factors that control local platelet deposition, the conversion of soluble fibrinogen to an insoluble fibrin polymer, and the dissolution of fibrin matrices. However, hemostatic factors are likely to be biologically more important than merely maintaining vascular patency and controlling blood loss. Specific hemostatic factors have been associated with a wide spectrum of physiological processes, including development, reproduction, tissue remodeling, wound repair, angiogenesis, and the inflammatory response. Similarly, it has been proposed that hemostatic factors are important determinants of a variety of pathological processes, including vessel wall disease, tumor dissemination, infectious disease, and inflammatory diseases of the joint, lung, and kidney. The development of gene targeted mice either lacking or expressing modified forms of selected hemostatic factors has provided a valuable opportunity to test prevailing hypotheses regarding the biological roles of key coagulation and fibrinolytic system components in vivo. Genetic analyses of fibrin(ogen) and its interacting factors in transgenic mice have proven to be particularly illuminating, often challenging long standing concepts. This review summarizes the key findings made in recent studies of gene targeted mice with single and combined deficits in fibrinogen and fibrinolytic factors. Studies illustrating the role and interplay of these factors in disease progression are highlighted.

Gold nanoparticles containing redox-active supramolecular dendrons that recognize H2PO4-.

Gold nanoparticles have been functionalized with thiol dendrons containing three redox active amidoferrocenyl or silylferrocenyl units; using cyclic voltammetry, these dendronized gold nanoparticles recognize H2PO4-.

Regional cerebral blood flow during acute and chronic abstinence from combined cocaine-alcohol abuse.

Regional cerebral blood flow (rCBF) was assessed using SPECT and HMPAO in ten cocaine abusers within 72 h of last cocaine use and then after 21 days of abstinence. In comparison to normals the cocaine abusers had significantly reduced rCBF in 11 of 14 brain regions with the largest reductions in the frontal and parietal cortex and greater rCBF in the brain stem. These perfusion defects appeared to be primarily due to combined alcohol and cocaine abuse and frontal but not parietal defects appeared to resolve partially during 21 days of abstinence.

Soft tissue sarcomas of infancy.

Malignancy occurring during the neonatal period (defined as the first 28 days of life) is over 3 times the incidence of other pediatric age groups. Of all neoplasia occurring in infants, benign and malignant, 25% are soft tissue tumors. Differentiating the benign lesions from the 15% that are malignant can be difficult. This article discusses the epidemiology, differential diagnosis, evaluation, and treatment of infants with soft tissue sarcomas. Fibrosarcoma and rhabdomyosarcoma are also discussed at length. The authors review other rare tumors as well. The impact on diagnosis of molecular techniques is included when appropriate. A multidisciplinary team approach for treatment of these infants is recommended.

Transient tachycardia- and bradycardia-dependent left anterior and left posterior hemiblocks. Effects of isoproterenol.

Tachycardia- and bradycardia-dependent, left anterior and left posterior hemiblocks as transient phenomena were registered in two patients spontaneously, and especially as a consequence of isoproterenol infusion. A chronic trifascicular type of A-V block was present in the first case, whereas in the second case a bradycardia-dependent left posterior hemiblock was registered during an acute myocardial infarction. In the first patient the isoproterenol effects were: (1) a shortening of the refractoriness and an increase of the conduction velocity in the injured fascicle, (2) an increase in the slope of phase-4 depolarization on the left posterior fascicle, and (3) a presumably shifting toward zero of threshold potential on the left anterior fascicle. Isoproterenol effects disappeared from 30 to 40 min after it was discontinued. In the second case the bradycardia-dependent left posterior hemiblock was registered during very fast heart rates (150 beats min). This finding supports the view that enhanced phase-4 depolarization is the main factor in the development of bradycardia-dependent intraventricular blocks in the course of acute myocardial ischemia.

Dopaminergic responsivity during cocaine abstinence: a pilot study.

This preliminary study investigated dopamine (DA) function in six hospitalized cocaine-dependent subjects (DSM-III-R) who received 1.5 mg/kg of active cocaine by mouth, t.i.d., for 3 days followed by 9 days of placebo cocaine. During early and late abstinence from cocaine, plasma growth hormone (GH), homovanillic acid (HVA), prolactin, and 3-methoxy-4-hydroxyphenethyleneglycol responses to the placebo-controlled administration of oral L-dopa 250 mg/carbidopa 25 mg (Sinemet) were measured. Sinemet caused significantly greater placebo-corrected increases in GH and HVA during early as compared with late abstinence. Acute abstinence from cocaine may be associated with increased DA responsivity, which normalizes over time.