Dependence of Laser-induced Breakdown Spectroscopy Results on Pulse Energies and Timing Parameters Using Soil Simulants

The dependence of some LIBS detection capabilities on lower pulse energies (<100 mJ) and timing parameters were examined using synthetic silicate samples. These samples were used as simulants for soil and contained minor and trace elements commonly found in soil at a wide range of concentrations. For this study, over 100 calibration curves were prepared using different pulse energies and timing parameters; detection limits and sensitivities were determined from the calibration curves. Plasma temperatures were also measured using Boltzmann plots for the various energies and the timing parameters tested. The electron density of the plasma was calculated using the full-width half maximum (FWHM) of the hydrogen line at 656.5 nm over the energies tested. Overall, the results indicate that the use of lower pulse energies and non-gated detection do not seriously compromise the analytical results. These results are very relevant to the design of field- and person-portable LIBS instruments.     

Usefulness of a new online patient-specific quality assurance system for respiratory-gated radiotherapy

PurposeThe accuracy of gated irradiation may decrease when treatment is performed with short “beam-on” times. Also, the dose is subject to variation between treatment sessions if the respiratory rate is irregular. We therefore evaluated the impact of the differences between gated and non-gated treatment on doses using a new online quality assurance (QA) system for respiratory-gated radiotherapy.MethodsWe generated dose estimation models to associate dose and pulse information using a 0.6 cc Farmer chamber and our QA system. During gated irradiation with each of seven regular and irregular respiratory patterns, with the Farmer chamber readings as references, we evaluated our QA system’s accuracy. We then used the QA system to assess the impact of respiratory patterns on dose distribution for three lung and three liver radiotherapy plans. Gated and non-gated plans were generated and compared.ResultsThere was agreement within 1.7% between the ionization chamber and our system for several regular and irregular motion patterns. For dose distributions with measured errors, there were larger differences between gated and non-gated treatment for high-dose regions within the planned treatment volume (PTV). Compared with a non-gated plan, PTV D_95% for a gated plan decreased by −1.5% to −2.6%. Doses to organs at risk were similar with both plans.ConclusionsOur simple system estimated the radiation dose to the patient using only pulse information from the linac, even during irregular respiration. The quality of gated irradiation for each patient can be verified fraction by fraction.     

Background correction of two-colour cDNA microarray data using spatial smoothing methods

The analysis of two-colour cDNA microarray data usually involves subtracting background values from foreground values prior to normalization and further analysis. This approach has the advantage of reducing bias and the disadvantage of blowing up the variance of lower abundant spots. Whenever background subtraction is considered, it implicitly assumes locally constant background values. In practice, this assumption is often not met, which casts doubts on the usefulness of simple background subtraction. In order to improve background correction, we propose local background smoothing within the pre-processing pipeline of cDNA microarray data prior to background correction. For this purpose, we employ a geostatistical framework with ordinary kriging using both isotropic and anisotropic models of spatial correlation and 2-D locally weighted regression. We show that application of local background smoothing prior to background correction is beneficial in comparison to using raw background estimates. This is done using data of a self-versus-self experiment in Arabidopsis where subsets of differentially expressed genes were simulated. Using locally smoothed background values in conjunction with existing background correction methods increases the power, increases the accuracy and decreases the number of false positive results.     

Tuning neocortical pyramidal neurons between integrators and coincidence detectors

Do cortical neurons operate as integrators or as coincidence detectors? Despite the importance of this question, no definite answer has been given yet, because each of these two views can find its own experimental support. Here we investigated this question using models of morphologically-reconstructed neocortical pyramidal neurons under in vivo like conditions. In agreement with experiments we find that the cell is capable of operating in a continuum between coincidence detection and temporal integration, depending on the characteristics of the synaptic inputs. Moreover, the presence of synaptic background activity at a level comparable to intracellular measurements in vivo can modulate the operating mode of the cell, and act as a switch between temporal integration and coincidence detection. These results suggest that background activity can be viewed as an important determinant of the integrative mode of pyramidal neurons. Thus, background activity not only sharpens cortical responses but it can also be used to tune an entire network between integration and coincidence detection modes.     

The effects of physical parameters on laser-induced breakdown spectroscopy analysis of intact tablets

With the advent of the Food and Drug Administration initiatives to investigate and encourage the use of process analytical technologies, laser-induced breakdown spectroscopy (LIBS) is considered an excellent analytical tool to understand the processability of solid dosage form. In this article, the feasibility of the LIBS system for quantitation of active drug within a solid dosage form, as well as the effects of various physical parameters on its signal, is investigated. A model drug containing chlorine and sulfur was used. The examination of the specificity and reproducibility of the measurements led to the use of chlorine and carbon as the internal standard. An overall relative SD of 1.1% for the signal was found. For quantitation purposes, calibration curves using compound-X in formulated tablets were generated. It was found that curves generated from roller-compaction tablets generally gave higher LIBS signal than those generated using direct-compressed (DC) process. To investigate these differences, effect of LIBS signals from several physical properties of the tablets were examined. It was found that unmilled compound-X used in the manufacture of the tablets gave a LIBS signal 30% lower than when milled compound-X was used. However, by using multiple crushing-recompression DC process of the milled compound-X, the LIBS results were comparable with those found from both processed tablets using milled compound-X. Other physical parameters, such as wide ranges of granule size and tablet hardness found in the typical manufacturing process, had limited effect on the LIBS signal. From these results, it was noted that for accurate quantitation, it is necessary to use the same physical properties of compound-X and the same manufacturing process in the calibration standards as the actual samples.     

The use of metal-catalyzed oxidation to suppress background staining caused by marker amplification reagents and the effects of this oxidation on the stability of antibody-antigen complexes in immunohistochemistry

Marker amplification is a powerful technique for visualizing immunohistochemically deposited markers that otherwise would be invisible. Amplification usually is achieved with physical developers, which are solutions that contain a source of silver(I) plus a reducing agent. When the marker is present in extremely small quantities, prolonged incubation in the developer is required and unwanted background staining in the form of type III argyrophilia becomes problematic. Suppression of type III argyrophilia can be achieved by metal-catalyzed oxidation using the copper/H₂O₂ system, which normally is applied immediately prior to amplification. Because there is no reason, in principle, why metal-catalyzed oxidation should not be employed at earlier stages in the immunohistochemical staining procedure, we investigated whether earlier oxidation might confer any advantages over the traditional methodology. Immunocolloidal gold combined with two light insensitive physical developers was chosen as the model system, because visualization by light microscopy requires extended periods in the developers. Moreover, the system does not suffer from problems concerning endogenous enzyme- or non-enzyme-catalyzed marker deposition. Applying metal-catalyzed oxidation at each stage of the immunohistochemical procedure revealed that the technique could be employed successfully prior to staining, but not following the primary or secondary antibodies. In the latter cases, specific immunolocalization was lost entirely and only generalized nonspecific staining was seen. A limited investigation into the mechanism of metal-catalyzed oxidation of aldehyde fixed tissue sections suggested that it involved the formation of aldehyde groups. We suggest that the application of metal-catalyzed oxidation prior to immunohistochemical staining would have the advantages of both suppressing type III argyrophilia and inhibiting unwanted endogenous peroxidase activity. We also suggest that metal-catalyzed oxidation might reduce the affinity of tissue for other transition metals, such as copper, whose potential for improving marker amplification techniques has been demonstrated previously in dot-blot model systems.     

Safely Coupling Livestock and Crop Production Systems: How Rapidly Do Antibiotic Resistance Genes Dissipate in Soil following a Commercial Application of Swine or Dairy Manure?

Animal manures recycled onto crop production land carry antibiotic-resistant bacteria. The present study evaluated the fate in soil of selected genes associated with antibiotic resistance or genetic mobility in field plots cropped to vegetables and managed according to normal farming practice. Referenced to unmanured soil, fertilization with swine or dairy manure increased the relative abundance of the gene targets sul1, erm(B), str(B), int1, and IncW repA. Following manure application in the spring of 2012, gene copy number decayed exponentially, reaching background levels by the fall of 2012. In contrast, gene copy number following manure application in the fall of 2012 or spring of 2013 increased significantly in the weeks following application and then declined. In both cases, the relative abundance of gene copy numbers had not returned to background levels by the fall of 2013. Overall, these results suggest that under conditions characteristic of agriculture in a humid continental climate, a 1-year period following a commercial application of raw manure is sufficient to ensure that an additional soil burden of antibiotic resistance genes approaches background. The relative abundance of several gene targets exceeded background during the growing season following a spring application or an application done the previous fall. Results from the present study reinforce the advisability of treating manure prior to use in crop production systems.     

Stress-induced thermotolerance of ventilatory motor pattern generation in the locust, Locusta migratoria

Ventilation is a crucial motor activity that provides organisms with an adequate circulation of respiratory gases. For animals that exist in harsh environments, an important goal is to protect ventilation under extreme conditions. Heat shock, anoxia, and cold shock are environmental stresses that have previously been shown to trigger protective responses. We used the locust to examine stress-induced thermotolerance by monitoring the ability of the central nervous system to generate ventilatory motor patterns during a subsequent heat exposure. Preparations from pre-stressed animals had an increased incidence of motor pattern recovery following heat-induced failure, however, prior stress did not alter the characteristics of the ventilatory motor pattern. During constant heat exposure at sub-lethal temperatures, we observed a protective effect of heat shock pre-treatment. Serotonin application had similar effects on motor patterns when compared to prior heat shock. These studies are consistent with previous studies that indicate prior exposure to extreme temperatures and hypoxia can protect neural operation against high temperature stress. They further suggest that the protective mechanism is a time-dependent process best revealed during prolonged exposure to extreme temperatures and is mediated by a neuromodulator such as serotonin.     

High-Yield Deblocking of Amino Termini of Recombinant Immunoglobulins with Pyroglutamate Aminopeptidase

For larger proteins, efficient deblocking prior to Edman sequencing is especially important to obtain quality, extended sequencing data which is limited by the stepwise accumulation of background from the random acid hydrolysis of the protein. Therefore, any portion that remains blocked contributes to the undesirable background. We report an optimized procedure for the removal of pyroglutamate (pGlu) by pyroglutamate aminopeptidase (PGAP) and demonstrate its use for the quantitative deblocking of several humanized recombinant antibodies (rIgGs). The rIgGs with blocked heavy chain provided an advantageous system in which removal of pGlu from the heavy chain was determined as a ratio of the deblocked heavy chain to the light chain in the first cycle of sequencing; i.e., the light chain was used as an internal standard. The reaction temperature, reaction time, enzyme-to-substrate ratio, denaturation, and reduction/carboxymethylation prior to digestion, and different commercial enzymes were evaluated. The optimized procedure involves reduction/carboxymethylation in guanidine buffer, buffer exchange by gel-permeation chromatography, and overnight PGAP digestion at 37°C. Five different rIgGs, including one with blocked heavy and light chains, were deblocked in nearly quantitative yields using this procedure.     

The relationship between hsp 70 localization and heat resistance

Using indirect immunofluorescence we have investigated the kinetics of nuclear accumulation and removal of hsp 70 in HA-1 Chinese hamster fibroblasts exposed to elevated temperatures. The kinetics of accumulation of hsp 70 in the nuclei were found to be time/temperature dependent at all temperatures tested (42-45 degrees C). At a given temperature, the fraction of cells manifesting nuclear localization of hsp 70 increased with exposure time. For a given duration of heating, the fraction of cells manifesting nuclear localization of hsp 70 increased with the temperature. The kinetics of the nuclear accumulation of hsp 70 were similar for normal HA-1 cells, their heat-resistant variants, and transiently thermotolerant cells (triggered by prior exposure to a brief heat shock or to sodium arsenite). Upon return to 37 degrees C after heat shock, the kinetics of removal of the hsp 70 associated with the nucleus was dependent on the severity of the initial heat challenge. However, for a given heat dose, the decay of nuclear localization of hsp 70 was more rapid in thermotolerant and heat-resistant cells than in their normal counterparts. These results suggest that the increased levels of hsp 70 associated with the transient or permanently heat-resistant state may play a direct role in restoring and/or repairing heat-induced nuclear and nucleolar alterations associated with heat-induced cell killing. Furthermore, they also suggest that the heat-resistant state may involve ameliorated repair of heat-induced cellular alterations.     

Notch signaling coordinates the patterning of striatal compartments

Numerous lines of evidence suggest that Notch signaling plays a pivotal role in controlling the production of neurons from progenitor cells. However, most experiments have relied on gain-of-function approaches because perturbation of Notch signaling results in death prior to the onset of neurogenesis. Here, we examine the requirement for Notch signaling in the development of the striatum through the analysis of different single and compound Notch1 conditional and Notch3 null mutants. We find that normal development of the striatum depends on the presence of appropriate Notch signals in progenitors during a critical window of embryonic development. Early removal of Notch1 prior to neurogenesis alters early-born patch neurons but not late-born matrix neurons in the striatum. We further show that the late-born striatal neurons in these mutants are spared as a result of functional compensation by Notch3. Notably, however, the removal of Notch signaling subsequent to cells leaving the germinal zone has no obvious effect on striatal organization and patterning. These results indicate that Notch signaling is required in neural progenitor cells to control cell fate in the striatum, but is dispensable during subsequent phases of neuronal migration and differentiation.     

消落带生态系统氮素截留转化的主要机制及影响因素

消落带是陆地与水体(河流、湖泊、水库、湿地以及其他特殊水体)之间的生态过渡带,具有独特的生态水文学和生物地球化学过程,是截留和转化NH₄⁺、NO₃^-等非点源氮素进入水体的最后一道生态屏障.整合已有相关研究成果发现: 1)植物固持作用改变氮素在土壤-植被-土壤-大气中相对存在位置;2)微生物反硝化作用将氮素从系统内永久性地去除,是消落带生态系统氮素截留转化的主要机制,但其相对贡献率仍有很大的不确定性.在不同流域背景条件下,影响消落带生态系统氮素生物地球化学循环的主要生态因子变化较大,很难确定地下水位高低、植被状况、微生物属性和土壤基质等哪一个生态因子是驱动消落带生态系统氮素循环的关键因子.研究方法的局限性、大的时空尺度数据的缺乏及对植被宽度认识的模糊性,是导致消落带生态系统氮素截留转化结果变异性大的主要原因.因此,应在消落带生态系统具体研究区位环境因子基础上,利用数学模型、GIS、RS等分析方法及同位素示踪和气体联用测定等定量分析技术,从不同时空尺度研究消落带生态系统氮素的循环与转化规律,以实现消落带生态系统氮素截留转化最优化,为消落带生态系统的科学管理提供理论基础.     

Capturing the Dynamics of a Hybrid Multiscale Cancer Model with a Continuum Model

Cancer is a complex disease involving processes at spatial scales from subcellular, like cell signalling, to tissue scale, such as vascular network formation. A number of multiscale models have been developed to study the dynamics that emerge from the coupling between the intracellular, cellular and tissue scales. Here, we develop a continuum partial differential equation model to capture the dynamics of a particular multiscale model (a hybrid cellular automaton with discrete cells, diffusible factors and an explicit vascular network). The purpose is to test under which circumstances such a continuum model gives equivalent predictions to the original multiscale model, in the knowledge that the system details are known, and differences in model results can be explained in terms of model features (rather than unknown experimental confounding factors). The continuum model qualitatively replicates the dynamics from the multiscale model, with certain discrepancies observed owing to the differences in the modelling of certain processes. The continuum model admits travelling wave solutions for normal tissue growth and tumour invasion, with similar behaviour observed in the multiscale model. However, the continuum model enables us to analyse the spatially homogeneous steady states of the system, and hence to analyse these waves in more detail. We show that the tumour microenvironmental effects from the multiscale model mean that tumour invasion exhibits a so-called pushed wave when the carrying capacity for tumour cell proliferation is less than the total cell density at the tumour wave front. These pushed waves of tumour invasion propagate by triggering apoptosis of normal cells at the wave front. Otherwise, numerical evidence suggests that the wave speed can be predicted from linear analysis about the normal tissue steady state.     

Integrin-like allosteric properties of the catch bond-forming FimH adhesin of Escherichia coli

FimH is the adhesive subunit of type 1 fimbriae of the Escherichia coli that is composed of a mannose-binding lectin domain and a fimbria-incorporating pilin domain. FimH is able to interact with mannosylated surface via a shear-enhanced catch bond mechanism. We show that the FimH lectin domain possesses a ligand-induced binding site (LIBS), a type of allosterically regulated epitopes characterized in integrins. Analogous to integrins, in FimH the LIBS epitope becomes exposed in the presence of the ligand (or "activating" mutations) and is located far from the ligand-binding site, close to the interdomain interface. Also, the antibody binding to the LIBS shifts adhesin from the low to high affinity state. Binding of streptavidin to the biotinylated residue within the LIBS also locks FimH in the high affinity state, suggesting that the allosteric perturbations in FimH are sustained by the interdomain wedging. In the presence of antibodies, the strength of bacterial adhesion to mannose is increased similar to the increase observed under shear force, suggesting the same allosteric mechanism, a shift in the interdomain configuration. Thus, an integrin-like allosteric link between the binding pocket and the interdomain conformation can serve as the basis for the catch bond property of FimH and, possibly, other adhesive proteins.     

Influence of organic shock loads in an ASBBR treating synthetic wastewater with different concentration levels

Safe application of the anaerobic sequencing biofilm batch reactor (ASBBR) still depends on deeper insight into its behavior when faced with common operational problems in wastewater treatments such as tolerance to abrupt variations in influent concentration, so called shock loads. To this end the current work shows the effect of organic shock loads on the performance of an ASBBR, with a useful volume of 5L, containing 0.5-cm polyurethane cubes and operating at 30 degrees C with mechanical stirring of 500 rpm. In the assays 2L of two types of synthetic wastewater were treated in 8-h cycles. Synthetic wastewater I was based on sucrose-amide-cellulose with concentration of 500 mg COD/L and synthetic wastewater II was based on volatile acids with concentration ranging from 500 to 2000 mg COD/L. Organic shock loads of 2-4 times the operation concentration were applied during one and two cycles. System efficiency was monitored before and after application of the perturbation. When operating with concentrations from 500 to 1000 mg COD/L and shock loads of 2-4 times the influent concentration during one or two cycles the system was able to regain stability after one cycle and the values of organic matter, total and intermediate volatile acids, bicarbonate alkalinity and pH were similar to those prior to the perturbations. At a concentration of 2000 mg COD/L the reactor appeared to be robust, regaining removal efficiencies similar to those prior to perturbation at shock loads twice the operation concentration lasting one cycle and stability was recovered after two cycles. However, for shock loads twice the operation concentration during two cycles and shock loads four times the operation concentration during one or two cycles filtered sample removal efficiency decreased to levels different from those prior to perturbation, on an average of 90-80%, approximately, yet the system managed to attain stability within two cycles after shock application. Therefore, this investigation envisions the potential of full scale application of this type of bioreactor which showed robustness to organic shock loads, despite discontinuous operation and the short times available for treating total wastewater volume.     

NEUROENDOCRINE REGULATION OF PROTEIN ACCUMULATION BY THE TRANSPARENT ACCESSORY REPRODUCTIVE GLAND OF MALE RHODNIUS PROLIXUS

Surgical removal of the corpus allatum or neurosecretory cells of the brain results in a marked reduction in protein accumulation in the transparent accessory reproductive gland (TARG) of the male. Topical application of C₁₈ juvenile hormone stimulates a dose-dependent accumulation of protein in allatectomized males. These results suggest that a direct or indirect stimulation by the neuroendocrine system is normally required for TARG protein synthesis.     

Experimental and computational studies of the G[UUCG]C RNA tetraloop

In prokaryotic ribosomal RNAs, most UUCG tetraloops are closed by a C-G base-pair. However, this preference is greatly reduced in eukaryotic rRNA species where many UUCG tetraloops are closed by G-C base-pairs. Here, biophysical properties of the C[UUCG]G and G[UUCG]C tetraloops are compared, using experimental and computational methods. Thermal denaturation experiments are used to derive thermodynamic parameters for the wild-type G[UUCG]C tetraloop and variants containing single deoxy substitutions in the loop. A comparison with analogous experiments on the C[UUCG]G motif shows that the two RNA species exhibit similar patterns in response to the substitutions, suggesting that their loop structures are similar. This conclusion is supported by NMR data that suggest that the essential UUCG loop structure is maintained in both tetraloops. However, NMR results show that the G[UUCG]C loop structure is disrupted prior to melting of the stem; this behavior is in contrast to the two-state behavior of the C[UUCG]G molecule. Stochastic dynamics simulations using the GB/SA continuum solvation model, run as a function of temperature, show rare conformational transitions in several G[UUCG]C simulations. These results lead to the conclusion that substitution of a G-C for a C-G closing base-pair increases the intrinsic flexibility of the UUCG loop.     

The lectin receptor kinase LecRK-I.9 is a novel Phytophthora resistance component and a potential host target for a RXLR effector

In plants, an active defense against biotrophic pathogens is dependent on a functional continuum between the cell wall (CW) and the plasma membrane (PM). It is thus anticipated that proteins maintaining this continuum also function in defense. The legume-like lectin receptor kinase LecRK-I.9 is a putative mediator of CW-PM adhesions in Arabidopsis and is known to bind in vitro to the Phytophthora infestans RXLR-dEER effector IPI-O via a RGD cell attachment motif present in IPI-O. Here we show that LecRK-I.9 is associated with the plasma membrane, and that two T-DNA insertions lines deficient in LecRK-I.9 (lecrk-I.9) have a 'gain-of-susceptibility' phenotype specifically towards the oomycete Phytophthora brassicae. Accordingly, overexpression of LecRK-I.9 leads to enhanced resistance to P. brassicae. A similar 'gain-of-susceptibility' phenotype was observed in transgenic Arabidopsis lines expressing ipiO (35S-ipiO1). This phenocopy behavior was also observed with respect to other defense-related functions; lecrk-I.9 and 35S-ipiO1 were both disturbed in pathogen- and MAMP-triggered callose deposition. By site-directed mutagenesis, we demonstrated that the RGD cell attachment motif in IPI-O is not only essential for disrupting the CW-PM adhesions, but also for disease suppression. These results suggest that destabilizing the CW-PM continuum is one of the tactics used by Phytophthora to promote infection. As countermeasure the host may want to strengthen CW-PM adhesions and the novel Phytophthora resistance component LecRK-I.9 seems to function in this process.     

Equivalence of background and bleaching desensitization in isolated rod photoreceptors of the larval tiger salamander

Psychophysical experiments have shown an equivalence between sensitivity reduction by background light and by bleaches for the human scotopic system. We have compared the effects of backgrounds and bleaches on the light-sensitive membrane-current responses of isolated rod photoreceptors from the salamander Ambystoma tigrinum. The quantum catch loss was factored out from the desensitization due to bleaching to give the fraction of "extra" desensitization due to adaptation. For backgrounds, desensitization is well described by the Weber/Fechner equation. The extra desensitization after bleaches can also be described by the Weber/Fechner equation, if an "equivalent" background produced by bleaching is made linearly proportional to the fraction of pigment bleached. A background which produces an extra desensitization of a factor of two is equivalent to a fractional bleach of approximately 6%. Equivalent background and bleaching desensitizations were associated with similar reductions in circulating current. There is a linear relation between log flash sensitivity and decrease in circulating current. Equivalent background and bleaching desensitizations were associated with similar increases in cGMP phosphodiesterase and guanylate cyclase activity. These were inferred from membrane current changes after steps into lithium or IBMX solutions. There were also similar reductions in the integration times of dim flash responses for equivalent desensitizations produced by backgrounds and bleaches. These results suggest that the equivalence between background and bleaching found psychophysically may arise at the very earliest stages of visual processing and that these two processes of desensitization have similar underlying mechanisms.