Impact of volume and expression time in an AAV-delivered channelrhodopsin.

Optogenetics has revolutionised neuroscience research, but at the same time has brought a plethora of new variables to consider when designing an experiment with AAV-based targeted gene delivery. Some concerns have been raised regarding the impact of AAV injection volume and expression time in relation to longitudinal experimental designs. In this study, we investigated the efficiency of optically evoked post-synaptic responses in connection to two variables: the volume of the injected virus and the expression time of the virus. For this purpose, we expressed the blue-shifted ChR2, oChIEF, employing a widely used AAV vector delivery strategy. We found that the volume of the injected virus has a minimal impact on the efficiency of optically-evoked postsynaptic population responses. The expression time, on the other hand, has a pronounced effect, with a gradual reduction in the population responses beyond 4 weeks of expression. We strongly advise to monitor time-dependent expression profiles when planning or conducting long-term experiments that depend on successful and stable channelrhodopsin expression.

Analysis of the effect of air temperature on ammonia emission from band application of slurry.

Field application of liquid animal manure (slurry) is a significant source of ammonia (NH3) emission to the atmosphere. It is well supported by theory and previous studies that air temperature effects NH3 flux from field applied slurry. The objectives of this study was to statistically model the response of temperature at the time of application on cumulative NH3 emission. Data from 19 experiments measured with the same system of dynamic chambers and online measurements were included. A generalized additive model allowing to represent non-linear functional dependences of the emission on the temperature revealed that a positive response of the cumulative NH3 emission on the temperature at the time of application up to a temperature of approximately 14 °C. Above that, the temperature effect is insignificant. Average temperature over the measuring period was not found to carry any additional information on the cumulative NH3 emission. The lack of emission response on temperature above a certain point is assumed to be caused by drying out of the slurry and possible crust formation. This effect is hypothesized to create a physical barrier that reduce diffusion of NH3 to the soil surface, thereby lowering the emission rate. Furthermore, the effect of the interaction between soil type and application technique and the effect of dry matter content of the slurry was derived from the model, and found to be significant on cumulative NH3 emission predictions.

Total Phosphorus Determination in Soils Using Laser-Induced Breakdown Spectroscopy: Evaluating Different Sources of Matrix Effects.

Laser-induced breakdown spectroscopy (LIBS) is a potential alternative to wet chemical methods for total soil phosphorus determination, but matrix effects related to physical and chemical sample properties need to be further understood. The aim of this study was to explore matrix effects linked to particle size distribution and chemical form of phosphorus on LIBS response and the ability of LIBS to predict total phosphorus in a range of different soil types. Univariate calibration curves were developed by spiking the soils with increasing doses of phosphorus, and limits of detection for LIBS determined phosphorous (P) (LIBS-P) were calculated. Different particle size distributions in otherwise identical soils were obtained by four milling treatments and effects of chemical form of phosphorus were examined by spiking soils with identical amounts of phosphorus in different chemical compounds. The LIBS-P response showed a high correlation (R2 > 0.99) with total phosphorus for all soils. Yet, the sensitivity of LIBS differed significantly among soils, as the slope of the calibration curves increased with increasing sand content, resulting in estimated limits of detection of 10 mg kg-1 for the sandiest and 122 mg · kg-1 for the most clayey soils. These limits indicate that quantitative evaluation of total phosphorus in sandy and loamy sandy soils by LIBS is feasible, since they are lower than typical total phosphorus concentrations in soil. A given milling treatment created different particle size distributions depending on soil type, and consequently different LIBS-P results. Thus, procedures that specify the required degree of homogenization of soil samples prior to analysis are needed. Sieving after milling could be an option, but that should be tested. The soils spiked with Fe(III) phosphate, potassium phosphate and phytic acid had similar LIBS-P, except for soils with hydroxyapatite, which resulted in markedly lower response. These results suggested that matrix effects related to the chemical nature of phosphorus would be minor for non-calcareous soils in humid regions, where apatites comprise only a small fraction of total phosphorus. Strategies to overcome matrix effects related to particle size and content of apatite-phosphorus by combining multivariate models and soil type groupings should be further investigated.

Growth and Phenology of Vulpia Myuros in Comparison with Apera Spica-Venti, Alopecurus Myosuroides and Lolium Multiflorum in Monoculture and in Winter Wheat.

Vulpia myuros has become an increasing weed problem in winter cereals in Northern Europe. However, the information about V. myuros and its behavior as an arable weed is limited. Field and greenhouse experiments were conducted in 2017/18 and 2018/19, at the Department of Agroecology in Flakkebjerg, Denmark to investigate the emergence, phenological development and growth characteristics of V. myuros in monoculture and in mixture with winter wheat, in comparison to Apera spica-venti, Alopecurus myosuroides and Lolium multiflorum. V. myuros emerged earlier than A. myosuroides and A. spica-venti but later than L. multiflorum. Significant differences in phenological development were recorded among the species. Overall phenology of V. myuros was more similar to that of L. multiflorum than to A. myosuroides and A. spica-venti. V. myuros started seed shedding earlier than A. spica-venti and L. multiflorum but later than A. myosuroides. V. myuros was more sensitive to winter wheat competition in terms of biomass production and fecundity than the other species. Using a target-neighborhood design, responses of V. myuros and A. spica-venti to the increasing density of winter wheat were quantified. At early growth stages "BBCH 26-29", V. myuros was suppressed less than A. spica-venti by winter wheat, while opposite responses were seen at later growth stages "BBCH 39-47" and "BBCH 81-90". No significant differences in fecundity characteristics were observed between the two species in response to increasing winter wheat density. The information on the behavior of V. myuros gathered by the current study can support the development of effective integrated weed management strategies for V. myuros.

Nitrate leaching from suction cup data: Influence of method of drainage calculation and concentration interpolation.

The use of suctions cups is a common practice for estimating nitrate (NO3 -N) leaching under agricultural systems despite the various uncertainties associated with the approach. One major uncertainty is water flux, which is required for calculating NO3 -N leaching loads from measured concentrations. Another problem is the interpolation of NO3 -N concentrations between measurement days. We investigated how differences in water flux, obtained from two different models (EVACROP and APSIM), affect NO3 -N leaching loads. The effect of interpolation of NO3 -N concentrations based on days or drainage was also addressed. The models were set up according to a 2-yr field experiment with spring barley (Hordeum vulgare L. Quinch) with different levels of N fertilization rates on a loamy soil at Flakkebjerg, Denmark. Due to small differences in measured NO3 -N concentrations between sequential samplings, the method of interpolation did not significantly affect NO3 -N leaching in the two periods investigated. Although there is no standard against which leaching losses from different approaches can be tested, results highlight that the modeling of water uptake as affected by N supply influences the amount of drainage and thus calculated NO3 -N leaching. Therefore, for experiments with varying N fertilization levels, the APSIM model, which accounts for N nutrition on crop water use, is likely more accurate. For common fertilization rates, the simpler EVACROP seems appropriate. Thus, when using suction cup data for testing models or for evaluating mitigation options for nitrate leaching, the use of an appropriate model for estimating water fluxes is important.

Methane fluxes from a rewetted agricultural fen during two initial years of paludiculture.

Rewetting agricultural peatland abates carbon dioxide (CO2) emission, but the resulting waterlogged anaerobic soil condition may create hotspots of methane (CH4) emissions. In this study, we measured CH4 emissions from side-by-side replicated plots in an agricultural fen cultivated with reed canary grass under a control and two experimental rewetting (i.e., paludiculture) conditions as either continuously flooded to soil surface or semi-flooded where water from the flooded plots intruded from sub-surface. Fluxes were measured for two successive years at 1-2 week intervals (total 59 measurement dates) using static chambers. Annual emissions were estimated by trapezoidal linear interpolation of the measured fluxes between the measurement dates. Two-year time-weighted average ground water tables (GWT) in the flooded, semi-flooded and control plots were 1, 3 and 9 cm below soil surface, respectively. The annual average emissions from flooded plots were 82 and 116 g CH4 m-2 yr-1 in Year 1 and 2, respectively, which were significantly higher than the emissions from semi-flooded plots (35 and 69 g CH4 m-2 yr-1 in Year 1 and 2, respectively) and from control plots (3 and 9 g CH4 m-2 yr-1 in Year 1 and 2, respectively). Overall, the results showed that the GWT in paludiculture should be maintained few cm below soil surface during high temperature periods to prevent risks of high CH4 emissions.

Seed size and burial depth influence Zostera marina L. (eelgrass) seed survival, seedling emergence and initial seedling biomass development.

Seed burial in the sediment is critical for successful seedling establishment in seagrasses because it protects from predation and dispersal into unsuitable sites, and it may enhance germination by exposing the seeds to suitable germination stimuli. However, relatively little is known about the fate of buried seeds and their ability to emerge from greater depths. The goal of this study was to determine seed survival in the sediment, seedling emergence success and initial seedling biomass of Zostera marina in relation to burial depth and to evaluate if large seeds, having larger energy reserves, are more tolerant to burial than small seeds. Seeds from a perennial Z. marina population were buried at 7 different sediment depths (0.1-8 cm), and seeds sorted by size (large and small) were buried at depths of 2, 4 and 6 cm in outdoor mesocosms. Total seedling emergence after 2 months was significantly affected by seed burial depth, with maximum values in the top 2 cm of the sediment (48.1-56.7% of planted seeds), and a marked decline below 4 cm depth to only 5% seedling emergence at the deepest burial depth of 8 cm. Moreover, seeds had shorter time to emergence from shallow compared to deep burial depths. At all burial depths, a small fraction of seeds (<10%) died after germination but before emerging, and 15-30% remained viable after 6 months. Seed mortality was the major limitation to seedling recruitment from the deeper burial depths. The effect of seed size on seedling emergence success and time was not clear, but heavier seeds displayed greater longevity and gave rise to seedlings of significantly higher biomass, indicating that the mobilization of metabolic reserves may be important during initial seedling development.

Vitellogenin expression in the oilseed rape pest Meligethes aeneus.

When investigating insecticide resistance of pest insects, for example, the pollen beetle Meligethes aeneus, it is relevant to differentiate toxicological and molecular genetic data between male and female specimens. A molecular sex determination method would allow resistance testing to be run without prior sorting of the samples. A one-step quantitative RT-PCR method for quantification of the yolk protein vitellogenin expression in the pollen beetle was established. The expression level of vitellogenin relative to tubulin was determined. Pollen beetles were tested at different time points during their development to determine if vitellogenin is a reliable molecular marker for detection of sexually mature females. The differentiation between females and males by relative expression of vitellogenin to tubulin is conditional regarding the life cycle. Sexually mature females and males could easily be distinguished, whereas immature specimens could not be seperated. Vitellogenin expression is a successful marker for identification of sexually mature pollen beetles. Females from the spring populations showed vitellogenin expression when the temperature was above 10.2°C. Further, detailed observations of vitellogenin throughout the spring indicated a strong relationship between daily temperatures and vitellogenin expression, which is an indicator of oviposition ability.

Effect of poplar trees on nitrogen and water balance in outdoor pig production - A case study in Denmark.

Nitrate leaching from outdoor pig production is a long-standing environmental problem for surface and groundwater pollution. In this study, the effects of inclusion of poplar trees in paddocks for lactating sows on nitrogen (N) balances were studied for an organic pig farm in Denmark. Vegetation conditions, soil water and nitrate dynamics were measured in poplar and grass zones of paddocks belonging to main treatments: access to trees (AT), no access to trees (NAT) and a control without trees (NT), during the hydrological year April 2015 to April 2016. Soil water drainage for each zone, simulated by two simulation models (CoupModel and Daisy), was used to estimate nitrate leaching from the zones in each paddock. N balances (input minus output) for the treatments were computed and compared. The results showed that, in terms of annual water balance and regardless of treatment, simulated evapotranspiration of poplar was 560-569 and 489-498 mm for CoupModel and Daisy, respectively, and corresponding evapotranspiration of grass-clover was 250 and 400 mm, against precipitation of 1076 mm. Simulated drainage below the root zone varied as 620-723 mm for Daisy and 568-958 mm for CoupModel, the higher end of the latter being probably overestimated. Annual nitrate leaching ranged from 32 kg N ha-1 in the poplar zone of NAT up to 289 kg N ha-1 in the control grass zone of NT. The poplar zone showed significantly lower nitrate leaching, by 75-80%, compared to the grass zone. For the control NT treatment, nitrate leaching was approximately 50% higher in the grass zone closest to the hut compared to the grass zone further away. NT treatment also had the largest surface N balance of 468 kg N ha-1 compared to 436 and 397 kg N ha-1 for AT and NAT, respectively. When N losses by leaching and volatilisation were included, soil N balances were 118, 157 and 113 kg N ha-1 for AT, NAT and NT, respectively. Overall, the two simulation models were found useful tools for analyses of water balance for complex agroforestry systems. The findings collectively suggest that it is possible to decrease nitrate leaching from outdoor pig production on sandy soils by inclusion of poplar trees. Additional measures are nevertheless needed to reduce N losses on a mean area basis in paddocks with 20% tree cover.

A new DNA sensor system for specific and quantitative detection of mycobacteria.

In the current study, we describe a novel DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. The presented work is the first to describe how the catalytic activities of a member of the type IA family of topoisomerases can be exploited for specific detection of bacteria. The principle for detection relies on a solid support anchored DNA substrate with dual functions namely: (1) the ability to isolate mycobacterial TOP1A from crude samples and (2) the ability to be converted into a closed DNA circle upon reaction with the isolated enzyme. The DNA circle can act as a template for rolling circle amplification generating a tandem repeat product that can be visualized at the single molecule level by fluorescent labelling. This reaction scheme ensures specific, sensitive, and quantitative detection of the mycobacteria TOP1A biomarker as demonstrated by the use of purified mycobacterial TOP1A and extracts from an array of non-mycobacteria and mycobacteria species. When combined with mycobacteriophage induced lysis as a novel way of effective yet gentle extraction of the cellular content from the model Mycobacterium smegmatis, the DNA sensor system allowed detection of mycobacteria in small volumes of cell suspensions. Moreover, it was possible to detect M. smegmatis added to human saliva. Depending on the composition of the sample, we were able to detect 0.6 or 0.9 million colony forming units (CFU) per mL of mycobacteria, which is within the range of clinically relevant infection numbers. We, therefore, believe that the presented assay, which relies on techniques that can be adapted to limited resource settings, may be the first step towards the development of a new point-of-care diagnostic test for tuberculosis.

Catch Crop Residues Stimulate N2O Emissions During Spring, Without Affecting the Genetic Potential for Nitrite and N2O Reduction.

Agricultural soils are a significant source of anthropogenic nitrous oxide (N2O) emissions, because of fertilizer application and decomposition of crop residues. We studied interactions between nitrogen (N) amendments and soil conditions in a 2-year field experiment with or without catch crop incorporation before seeding of spring barley, and with or without application of N in the form of digested liquid manure or mineral N fertilizer. Weather conditions, soil inorganic N dynamics, and N2O emissions were monitored during spring, and soil samples were analyzed for abundances of nitrite reduction (nirK and nirS) and N2O reduction genes (nosZ clade I and II), and structure of nitrite- and N2O-reducing communities. Fertilization significantly enhanced soil mineral N accumulation compared to treatments with catch crop residues as the only N source. Nitrous oxide emissions, in contrast, were stimulated in rotations with catch crop residue incorporation, probably as a result of concurrent net N mineralization, and O2 depletion associated with residue degradation in organic hotspots. Emissions of N2O from digested manure were low in both years, while emissions from mineral N fertilizer were nearly absent in the first year, but comparable to emissions from catch crop residues in the second year with higher precipitation and delayed plant N uptake. Higher gene abundances, as well as shifts in community structure, were also observed in the second year, which were significantly correlated to NO 3 - availability. Both the size and structure of the nitrite- and N2O-reducing communities correlated to the difference in N2O emissions between years, while there were no consistent effects of management as represented by catch crops or fertilization. It is concluded that N2O emissions were constrained by environmental, rather than the genetic potential for nitrite and N2O reduction.

Detection of the Malaria causing Plasmodium Parasite in Saliva from Infected Patients using Topoisomerase I Activity as a Biomarker.

Malaria is among the major threats to global health with the main burden of disease being in rural areas of developing countries where accurate diagnosis based on non-invasive samples is in high demand. We here present a novel molecular assay for detection of malaria parasites based on technology that may be adapted for low-resource settings. Moreover, we demonstrate the exploitation of this assay for detection of malaria in saliva. The setup relies on pump-free microfluidics enabled extraction combined with a DNA sensor substrate that is converted to a single-stranded DNA circle specifically by topoisomerase I expressed by the malaria causing Plasmodium parasite. Subsequent rolling circle amplification of the generated DNA circle in the presence of biotin conjugated deoxynucleotides resulted in long tandem repeat products that was visualized colorimetrically upon binding of horse radish peroxidase (HRP) and addition of 3,3',5,5'-Tetramethylbenzidine that was converted to a blue colored product by HRP. The assay was directly quantitative, specific for Plasmodium parasites, and allowed detection of Plasmodium infection in a single drop of saliva from 35 out of 35 infected individuals tested. The results could be determined directly by the naked eye and documented by quantifying the color intensity using a standard paper scanner.

Genetic diversity within and among aecia of the wheat rust fungus Puccinia striiformis on the alternate host Berberis vulgaris.

An isolate of the fungus Puccinia striiformis, causing yellow (stripe) rust on cereals and grasses, was selfed on the alternate (sexual) host, Berberis vulgaris. This enabled us to investigate genetic variability of progeny isolates within and among aecia. Nine aecial clusters each consisting of an aecium (single aecial cup) and nine clusters containing multiple aecial cups were selected from 18 B. vulgaris leaves. Aeciospores from each cluster were inoculated on susceptible wheat seedlings and 64 progeny isolates were recovered. Molecular genotyping using 37 simple sequence repeat markers confirmed the parental origin of all progeny isolates. Thirteen molecular markers, which were heterozygous in the parental isolate, were used to analyse genetic diversity within and among aecial cups. The 64 progeny isolates resulted in 22 unique recombinant multilocus genotypes and none of them were resampled in different aecial clusters. Isolates derived from a single cup were always of the same genotype whereas isolates originating from clusters containing up to nine aecial cups revealed one to three genotypes per cluster. These results implied that each aecium was the result of a successful fertilization in a corresponding pycnium and that an aecium consisted of genetically identical aeciospores probably multiplied via repetitive mitotic divisions. Furthermore, the results suggested that aecia within a cluster were the result of independent fertilization events often involving genetically different pycniospores. The application of molecular markers represented a major advance in comparison to previous studies depending on phenotypic responses on host plants. The study allowed significant conclusions about fundamental aspects of the biology and genetics of an important cereal rust fungus.

Temporal and Spatial Variability of Fungal Structures and Host Responses in an Incompatible Rust-Wheat Interaction.

Information about temporal and spatial variability of fungal structures and host responses is scarce in comparison to the vast amount of genetic, biochemical, and physiological studies of host-pathogen interactions. In this study, we used avirulent wild type and virulent mutant isolates of Puccinia striiformis to characterize the interactions in wheat carrying yellow rust Yr2 resistance. Both conventional and advanced microscopic techniques were used for a detailed study of morphology and growth of fungal colonies and associated host cell responses. The growth of the wild type isolates was highly restricted due to hypersensitive response (HR, plant cell death) indicated by autofluorescence and change in the shape of the affected plant cells. The host response appeared post-haustorial, but large variation in the time and stage of arrest was observed for individual fungal colonies, probably due to a delay between detection and response. Some colonies were stopped right after the formation of the primary infection hyphae whereas others formed highly branched mycelia. HR was first observed in host cells in direct contact with fungal structures, after which the defense responses spread to adjacent host cells, and eventually led to encasement of the fungal colony. Several cells with HR contained haustoria, which were small and underdeveloped, but some cells contained normal sized haustoria without signs of hypersensitivity. The growth of the virulent mutants in the resistant plants was similar to the growth in plants without Yr2 resistance, which is a strong indication that the incompatible phenotype was associated with Yr2. The interaction between P. striiformis and wheat with Yr2 resistance was highly variable in time and space, which demonstrate that histological studies are important for a deeper understanding of host-pathogen interactions and plant defense mechanisms in general.

Microbial N Transformations and N2O Emission after Simulated Grassland Cultivation: Effects of the Nitrification Inhibitor 3,4-Dimethylpyrazole Phosphate (DMPP).

Grassland cultivation can mobilize large pools of N in the soil, with the potential for N leaching and N2O emissions. Spraying with the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) before cultivation was simulated by use of soil columns in which the residue distribution corresponded to plowing or rotovation to study the effects of soil-residue contact on N transformations. DMPP was sprayed on aboveground parts of ryegrass and white clover plants before incorporation. During a 42-day incubation, soil mineral N dynamics, potential ammonia oxidation (PAO), denitrifying enzyme activity (DEA), nitrifier and denitrifier populations, and N2O emissions were investigated. The soil NO3- pool was enriched with 15N to trace sources of N2O. Ammonium was rapidly released from decomposing residues, and PAO was stimulated in soil near residues. DMPP effectively reduced NH4+ transformation irrespective of residue distribution. Ammonia-oxidizing archaea (AOA) and bacteria (AOB) were both present, but only the AOB amoA transcript abundance correlated with PAO. DMPP inhibited the transcription of AOB amoA genes. Denitrifier genes and transcripts (nirK, nirS, and clades I and II of nosZ) were recovered, and a correlation was found between nirS mRNA and DEA. DMPP showed no adverse effects on the abundance or activity of denitrifiers. The 15N enrichment of N2O showed that denitrification was responsible for 80 to 90% of emissions. With support from a control experiment without NO3- amendment, it was concluded that DMPP will generally reduce the potential for leaching of residue-derived N, whereas the effect of DMPP on N2O emissions will be significant only when soil NO3- availability is limiting. IMPORTANCE: Residue incorporation following grassland cultivation can lead to mobilization of large pools of N and potentially to significant N losses via leaching and N2O emissions. This study proposed a mitigation strategy of applying 3,4-dimethylpyrazole phosphate (DMPP) prior to grassland cultivation and investigated its efficacy in a laboratory incubation study. DMPP inhibited the growth and activity of ammonia-oxidizing bacteria but had no adverse effects on ammonia-oxidizing archaea and denitrifiers. DMPP can effectively reduce the potential for leaching of NO3- derived from residue decomposition, while the effect on reducing N2O emissions will be significant only when soil NO3- availability is limiting. Our findings provide insight into how DMPP affects soil nitrifier and denitrifier populations and have direct implications for improving N use efficiency and reducing environmental impacts during grassland cultivation.

The parental origin correlates with the karyotype of human embryos developing from tripronuclear zygotes.

OBJECTIVE: It has previously been suggested that embryos developing from intracytoplasmic sperm-injected (ICSI) zygotes with three pronuclei (3PN) are endowed with a mechanism for self-correction of triploidy to diploidy. 3PN are also observed in zygotes after conventional in vitro fertilization (IVF). The parental origin, however, differs between the two fertilization methods. Whereas the vast majority of 3PN IVF zygotes are of dispermic origin and thus more likely to have two centrioles, the 3PN ICSI zygotes are digynic in origin and therefore, more likely to have one centriole. In the present study, we examine whether the parental origin of 3PN embryos correlates with the karyotype. METHODS: The karyotype of each nucleus was estimated using four sequential fluorescence in situ hybridizations-each with two probes-resulting in quantitative information of 8 different chromosomes. The karyotypes were then compared and correlated to the parental origin. RESULTS: 3PN ICSI embryos displayed a significantly larger and more coordinated reduction from the assumed initial 3 sets of chromosomes than 3PN IVF embryos. CONCLUSION: The differences in the parental origin-and hence the number of centrioles-between the 3PN IVF and the 3PN ICSI zygotes are likely to be the cause of the differences in karyotypes.

Practical use of a uterine score system for predicting effects on interval from calving to first insemination and non-return rate 56 in Danish dairy herds.

A detailed study of 398,237 lactations of Danish Holstein dairy cows was undertaken. The objective was to investigate the information gained by evaluating vaginal discharge in cows from 5 to 19 days post-partum (p.p.) using an ordinal scale from 0 to 9. The study focused on the interval from calving to first insemination (CFI) and the non-return rate 56 days after first insemination (NR56), adjusted for the confounders milk production and body condition score (BCS). For the analyses, BCS was evaluated on the same day that the uterine score was made. Milk production was defined as test-day milk yield in the first month p.p. The study showed that the evaluation of vaginal discharge according to this score system permitted ranking of cows according to CFI and NR56, i.e. an increasing uterine score was associated with a significantly longer time from calving to first insemination and significantly reduced the probability of success of the first insemination. Reproductive success was already affected if the uterine score had reached 4 (i.e. before the discharge smelled abnormally). The negative effect on CFI and NR56 increased as the uterine score increased, which suggested that the uterine scoring system was a useful guide to dairy producers.

The proto-oncogene TWIST1 is regulated by microRNAs.

Upregulation of the proto-oncogene Twist1 is highly correlated with acquired drug resistance and poor prognosis in human cancers. Altered expression of this multifunctional transcription factor is also associated with inherited skeletal malformations. The mammalian Twist1 3'UTRs are highly conserved and contain a number of potential regulatory elements including miRNA target sites. We analyzed the translational regulation of TWIST1 using luciferase reporter assays in a variety of cell lines. Among several miRNAs tested, miR-145a-5p, miR-151-5p and a combination of miR-145a-5p + miR-151-5p and miR-151-5p + miR-337-3p were able to significantly repress Twist1 translation. This phenomena was confirmed with both exogenous and endogenous miRNAs and was dependent on the presence of the predicted target sites in the 3'UTR. Furthermore, the repression was sensitive to LNA-modified miRNA antagonists and resulted in decreased migratory potential of murine embryonic fibroblast cells. Understanding the in vivo mechanisms of this oncogene's regulation might open up a possibility for therapeutic interference by gene specific cancer therapies.

Droplet microfluidics platform for highly sensitive and quantitative detection of malaria-causing Plasmodium parasites based on enzyme activity measurement.

We present an attractive new system for the specific and sensitive detection of the malaria-causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer-sized products detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/µL. Moreover, the setup allowed for detection of Plasmodium parasites in noninvasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide, and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate detection of even a few parasites is becoming increasingly important for the continued combat against the disease. We believe that the presented droplet microfluidics platform, which has a high potential for adaptation to point-of-care setups suitable for low-resource settings, may contribute significantly to meet this demand. Moreover, potential future adaptation of the presented setup for the detection of other microorganisms may form the basis for the development of a more generic platform for diagnosis, fresh water or food quality control, or other purposes within applied or basic science.

Variation of polyphenols and betaines in aerial parts of young, field-grown Amaranthus genotypes.

Amaranthus hybridus and Amaranthus mantegazzianus are commonly cultivated and the entire young fresh plants consumed as vegetables in regions of Africa and Asia. A. hybridus and A. mantegazzianus were cultivated at four sites in three climate regions of the world: Santa Rosa, Argentina; Lleida, Spain; and Prague and Olomouc, both in the Czech Republic. The contents of flavonoids (isoquercitrin, rutin, nicotiflorin), hydroxybenzoic acids (protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid), hydroxycinnamic acids (caffeic acid, p-coumaric acid, ferulic acid), hydroxycinnamyl amides (N-trans-feruloyltyramine, N-trans-feruloyl-4-O-methyldopamine), and betaines (glycinebetaine, trigonelline) were determined. The variation in phytochemical content due to species and cultivation site was analyzed utilizing the multivariate statistical methods of principal component analysis (PCA) and graphical model (GM). The Argentinean samples differed from the three other locations due to higher contents of most compounds. The samples from Spain and the Czech Republic differed from each other in the content of the negatively correlated metabolites trigonelline and the flavonoids. The two amaranth species were separated primarily by a higher content of trigonelline and the two hydroxycinnamyl amides in A. mantegazzianus. The GM showed that the quantities of the different analytes within each compound group were intercorrelated except in the case of the betaines. The betaines carried no information on each other that was not given through correlations with other compounds. The hydroxycinnamic acids were a key group of compounds in this analysis as they separated the other groups from each other (i.e., carried information on all of the other groups). This study showed the contents of polyphenols and betaines in the aerial parts of vegetable amaranth to be very dependent on growth conditions, but also revealed that some of the compounds (trigonelline and the two hydroxycinnamyl amides) may be useful as features of a taxonomic classification.