Development of a rapid quantitative PCR assay for direct detection and quantification of culturable and non-culturable Escherichia coli from agriculture watersheds

A real-time quantitative polymerase chain reaction (Q-PCR) assay was developed for detecting and quantifying Escherichia coli in water samples from agricultural watersheds. The assay included optimization of DNA extraction and purification from water samples, and Q-PCR amplification conditions using newly designed species-specific oligonucleotide primers derived from conserved flanking regions of the 16S rRNA gene, the internal transcribed spacer region (ITS) and the 23S rRNA gene. The assay was optimized using a pure culture of E. coli with known quantities spiked into autoclaved agricultural water samples. The optimized assay was capable of a minimum quantification limit of 10 cells/ml of E. coli in the spiked agricultural water samples. A total of 121 surface water samples from three agricultural watersheds across Canada were analyzed, and results were compared with conventional culture-based enumerations of E. coli. The Q-PCR assay revealed significantly higher numbers of E. coli in water samples than the culture-based assay in each agricultural watershed. The new Q-PCR assay can facilitate the quantification of E. coli in a single water sample in < 3 h, including melt curve analysis, across a range of agricultural water quality conditions.     

Biochar in Combination with Nitrogen Fertilizer is a Technique: To Enhance Physiological and Morphological Traits of Rice (Oryza sativa L.) by Improving Soil Physio-biochemical Properties

The over use of synthetic nitrogen (N) fertilizers is the major anthropogenic cause of low N-use efficiency and environmental damage in wetland rice production. Biochar (B) addition to soil is suggested as a climate change mitigation tool that supports carbon sequestration and reduces N losses and greenhouse gas emissions from the soil. Therefore, this study assessed the effect of four levels of B (0, 10, 20 and 30 t ha^?1) combined with two levels of N (135 and 180 kg ha^?1) on soil health, roots dynamics, physiological attributes, and yield components of rice. The addition of B at 30 t ha^?1 combined with 135 N kg ha^?1 increased chlorophyll content, net photosynthetic rate, biomass, and grain yield by 104%, 64%, 12%, and 30%, respectively, over control. Further, root traits such as total root length (TRL), total root volume (TRV), total root surface area (TRSA), and total average root diameter (TARD) were improved under 30 t ha^?1 combined with 135 N kg ha^?1 by 20%, 13%, 13%, and 25%, respectively, than non-biochar treatment under lower N application. Improvements in these traits resulted from higher N uptake due to improved soil physiochemical properties and soil microbial biomass combined with biochar. Interestingly, enhanced N metabolizing enzyme activities, including nitrate reductase (NR), glutamine synthetase (GS), and glutamine oxoglutarate aminotransferase (GOGAT) in biochar-treated plots, further supported the increases in these traits. Our results revealed that the integration of 30 t B ha^?1 with 135 kg N ha^?1 is a favorable option for enhancing soil health and rice grain yield.

     

Olive Fruit Fly (Bactrocera oleae) Population Dynamics in the Eastern Mediterranean: Influence of Exogenous Uncertainty on a Monophagous Frugivorous Insect

Despite of the economic importance of the olive fly (Bactrocera oleae) and the large amount of biological and ecological studies on the insect, the factors driving its population dynamics (i.e., population persistence and regulation) had not been analytically investigated until the present study. Specifically, our study investigated the autoregressive process of the olive fly populations, and the joint role of intrinsic and extrinsic factors molding the population dynamics of the insect. Accounting for endogenous dynamics and the influences of exogenous factors such as olive grove temperature, the North Atlantic Oscillation and the presence of potential host fruit, we modeled olive fly populations in five locations in the Eastern Mediterranean region. Our models indicate that the rate of population change is mainly shaped by first and higher order non-monotonic, endogenous dynamics (i.e., density-dependent population feedback). The olive grove temperature was the main exogenous driver, while the North Atlantic Oscillation and fruit availability acted as significant exogenous factors in one of the five populations. Seasonal influences were also relevant for three of the populations. In spite of exogenous effects, the rate of population change was fairly stable along time. We propose that a special reproductive mechanism, such as reproductive quiescence, allows populations of monophagous fruit flies such as the olive fly to remain stable. Further, we discuss how weather factors could impinge constraints on the population dynamics at the local level. Particularly, local temperature dynamics could provide forecasting cues for management guidelines. Jointly, our results advocate for establishing monitoring programs and for a major focus of research on the relationship between life history traits and populations dynamics.     

Fuzzy Logic Based Edge Detection in Smooth and Noisy Clinical Images

Edge detection has beneficial applications in the fields such as machine vision, pattern recognition and biomedical imaging etc. Edge detection highlights high frequency components in the image. Edge detection is a challenging task. It becomes more arduous when it comes to noisy images. This study focuses on fuzzy logic based edge detection in smooth and noisy clinical images. The proposed method (in noisy images) employs a 3×3 mask guided by fuzzy rule set. Moreover, in case of smooth clinical images, an extra mask of contrast adjustment is integrated with edge detection mask to intensify the smooth images. The developed method was tested on noise-free, smooth and noisy images. The results were compared with other established edge detection techniques like Sobel, Prewitt, Laplacian of Gaussian (LOG), Roberts and Canny. When the developed edge detection technique was applied to a smooth clinical image of size 270×290 pixels having 24 dB ‘salt and pepper’ noise, it detected very few (22) false edge pixels, compared to Sobel (1931), Prewitt (2741), LOG (3102), Roberts (1451) and Canny (1045) false edge pixels. Therefore it is evident that the developed method offers improved solution to the edge detection problem in smooth and noisy clinical images.     

Thermoregulatory and endocrine responses to light pulses in short-day acclimated social voles (Microtus socialis)

In mammals, nocturnal light pulses (NLP) have been demonstrated to affect physiology and behavior. However, the impact of NLP as a stressor has been less broadly examined. The purpose of this study was to examine the effect of NLP (three 15 min 450 lux light pulses) during each scotophase on both thermoregulation and endocrine stress responses under short-day (SD; 8L:16D) acclimation. Voles were acclimated to either SD (SD voles) or SD+NLP (NLP voles). Resistance to cold was estimated by measurements of body temperature (T_b) during cold exposure (5°C). Daily rhythms of energy expenditure (calculated from oxygen consumption), urine production, and urinary adrenaline and serum cortisol levels were measured. T_b values of SD voles were generally unaffected by the cold stimulus, whereas in NLP voles, resistance to cold was markedly lowered. While SD- and NLP voles showed similar ultradian characteristics in energy expenditure with a period of 3.5 h, mean energy expenditure levels were lowest for voles exposed to NLP-treatment. In SD voles, but not in NLP voles, urine production rates showed clear time variations and were consistently highest for SD voles, with significant differences during the scotophase. Both mean total urinary adrenaline and serum cortisol levels were significantly elevated in NLP-treated voles compared with the control group. Taken together, the results suggest that NLP negatively affects winter acclimatization of thermoregulatory mechanisms of M. socialis, probably by mimicking summer acclimatization, and consequently the thermoregulatory mechanisms respond inappropriately to ambient conditions. One important finding of this study is that NLP may act as a stressor and correspondingly impose a major threat to the physiological homeostasis of M. socialis, such that over-winter survival might be compromised.     

Selective regulation of somatostatin receptor subtype signaling: evidence for constitutive receptor activation

Anterior pituitary hormone secretion is under tonic suppression by hypothalamic somatostatin signaling through somatostatin receptor subtypes (SSTs). Because some hormonal axes are known to be abnormally regulated by ligand-independent constitutively active G protein-coupled receptors, we tested pituitary SSTs for selective constitutive signaling. We therefore differentially silenced endogenous SST2, SST3, and SST5 in somatostatin-sensitive ACTH-secreting mouse AtT-20 pituitary corticotroph cells using small inhibitory RNA (siRNA) and analyzed downstream SSTs-regulated pathways. Transfection with siRNA reduced specific receptor subtype mRNA expression up to 82%. Specificity of receptor silencing was validated against negative controls with different gene-selective siRNAs, concordance of mRNA and cAMP changes, reduced potency of receptor-selective agonists, and phenotype rescue by overexpression of the silenced receptor. Mouse SST3 > SST5 > SST2 knockdown increased basal cAMP accumulation (up to 200%) and ACTH secretion (up to 60%). SST2- and SST5-selective agonist potencies were reduced by SST3- and SST5-silencing, respectively. SST5 > SST2 = SST3 silencing also increased basal levels of ERK1/2 phosphorylation. SST3- and SST5-knockdown increased cAMP was only partially blocked by pertussis toxin. The results show that SST2, SST3, and SST5 exhibit constitutive activity in mouse pituitary corticotroph cells, restraining adenylate cyclase and MAPK activation and ACTH secretion. SST3 mainly inhibits cAMP accumulation and ACTH secretion, whereas SST5 predominantly suppresses MAPK pathway activation. Therefore, SST receptor subtypes control pituitary cell function not only through somatostatin binding to variably expressed cell membrane receptor subtypes, but also by differential ligand-independent receptor-selective constitutive action.     

Characterisation of a cytoplasmic male-sterile hybrid line between Lycopersicon peruvianum Mill. ×Lycopersicon pennellii Corr. and its crosses with cultivated tomato

 The cytoplasmic male-sterile (CMS) line CMS-pennellii (BC₁₀P₂ L. peruvianum×L. pennellii) and its complex hybrids with L. esculentum were studied. The established sterility was classified as the sporogenous type. As a result of the interaction of the genome of L. pennellii and the cytoplasm of L. peruvianum clear changes were established in the profiles of malic enzyme and esterase. Restriction fragment length polymorphism (RFLP) was detected between the mitochondrial (mt) genomes of CMS-pennellii and the cytoplasm donor, L. peruvianum, for two mtDNA probes: atpA and nad3. The established differences in the isozyme pattern and mt genomes are considered as useful markers to distinguish fertile and sterile plants. A breakthrough in the unilateral incompatibility of CMS-pennellii and the incorporation of the genome of L. esculentum on a CMS background is reported. The analysis of the complex hybrids assumes the interaction of two dominant genes – a maintainer gene from L. pennellii and a restorer gene from cultivated tomato. The hybrids produced with L. esculentum provide the basis for the development of a CMS system in cultivated tomato. Received: 25 May 1998 / Accepted: 26 August 1998     

Pulmonary haemorrhage as the earliest sign of severe leptospirosis in hamster model challenged with Leptospira interrogans strain HP358

BackgroundSevere leptospirosis is challenging as it could evolve rapidly and potentially fatal if appropriate management is not performed. An understanding of the progression and pathophysiology of Leptospira infection is important to determine the early changes that could be potentially used to predict the severe occurrence of leptospirosis. This study aimed to understand the kinetics pathogenesis of Leptospira interrogans strain HP358 in the hamster model and identify the early parameters that could be used as biomarkers to predict severe leptospirosis.Methodology/Principal findingsMale Syrian hamsters were infected with Leptospira interrogans strain HP358 and euthanized after 24 hours, 3, 4, 5, 6 and 7 days post-infection. Blood, lungs, liver and kidneys were collected for leptospiral detection, haematology, serum biochemistry and differential expression of pro- and anti-inflammatory markers. Macroscopic and microscopic organ damages were investigated. Leptospira interrogans strain HP358 was highly pathogenic and killed hamsters within 6–7 days post-infection. Pulmonary haemorrhage and blood vessel congestion in organs were noticed as the earliest pathological changes. The damages in organs and changes in biochemistry value were preceded by changes in haematology and immune gene expression.Conclusion/SignificanceThis study deciphered haemorrhage as the earliest manifestation of severe leptospirosis and high levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets serve as a cumulative panel of biomarkers in severe leptospirosis.     

ROS Detoxification and Proinflammatory Cytokines Are Linked by p38 MAPK Signaling in a Model of Mature Astrocyte Activation

Astrocytes are the most abundant glial cell in the retinal nerve fiber layer (NFL) and optic nerve head (ONH), and perform essential roles in maintaining retinal ganglion cell (RGC) detoxification and homeostasis. Mature astrocytes are relatively quiescent, but rapidly undergo a phenotypic switch in response to insult, characterized by upregulation of intermediate filament proteins, loss of glutamate buffering, secretion of pro-inflammatory cytokines, and increased antioxidant production. These changes result in both positive and negative influences on RGCs. However, the mechanism regulating these responses is still unclear, and pharmacologic strategies to modulate select aspects of this switch have not been thoroughly explored. Here we describe a system for rapid culture of mature astrocytes from the adult rat retina that remain relatively quiescent, but respond robustly when challenged with oxidative damage, a key pathogenic stress associated with inner retinal injury. When primary astrocytes were exposed to reactive oxygen species (ROS) we consistently observed characteristic changes in activation markers, along with increased expression of detoxifying genes, and secretion of proinflammatory cytokines. This in vitro model was then used for a pilot chemical screen to target specific aspects of this switch. Increased activity of p38α and β Mitogen Activated Protein Kinases (MAPKs) were identified as a necessary signal regulating expression of MnSOD, and heme oxygenase 1 (HO-1), with consequent changes in ROS-mediated injury. Additionally, multiplex cytokine profiling detected p38 MAPK-dependent secretion of IL-6, MCP-1, and MIP-2α, which are proinflammatory signals recently implicated in damage to the inner retina. These data provide a mechanism to link increased oxidative stress to proinflammatory signaling by astrocytes, and establish this assay as a useful model to further dissect factors regulating the reactive switch.     

Social deprivation and premature mortality: regional comparison across England.

OBJECTIVE--To investigate the pattern and size of the relationship between social deprivation in electoral wards and premature mortality for each health region in England. DESIGN--Ecological study using 1981 census variables and data on mortality for 1981-5. SETTING--14 regional health authorities in England. MAIN OUTCOME MEASURE--Mortality under the age of 65 years from all causes, coronary heart disease, and smoking related diseases in men and women. RESULTS--Increasing deprivation was significantly associated with mortality from all causes, coronary heart disease, and smoking related diseases. The relationship was linear with no apparent threshold. Correlation coefficients were generally greater for deaths from all causes and smoking related diseases and for men compared with women. The slope of the relationship between deprivation and mortality varied among regions. Variations in mortality still existed between regions for equal levels of deprivation. CONCLUSION--Deprivation of an area and premature mortality are strongly linked. The effects of deprivation can be seen throughout the range of affluence and are not limited to the poorest areas. Current targets for reducing coronary heart disease mortality may be achievable if the mortality in poor areas can be reduced to the rates in affluent areas.