CDR identification, epitope mapping and binding affinity determination of novel monoclonal antibodies generated against human apolipoprotein B-100.

In-house generated mAbs to apolipoprotein B-100 (apoB-100) clones hLDL-E8, hLDL-2D8 and hLDL-F5 were extensively studied to determine their complementarity-determining regions (CDRs), binding epitopes and affinity. RT-PCR revealed that all mAbs consisted of kappa light chains and gamma heavy chains. DNA sequencing and bioinformatic analysis showed that the variable gene and protein sequences of their CDRs shared over 50 % identity with the existing databases. The 3D structures of the mAb variable fragments (Fv) with a QSQE score above 0.7 were constructed using the SWISS-MODEL platform. The structural accuracy was confirmed by Ramachandran plots, with 99 % of amino acid residues falling within acceptable regions. Thrombolytic cleavage of apoB-100 and Western blot analysis demonstrated that hLDL-E8 and hLDL-F5 specifically bind to the T3 fragment (aa 1297-3249), whereas hLDL-2D8 binds to the T4 fragment (aa 1-1297). These findings were supported with epitope-binding assays using inhibition ELISA, which indicated that hLDL-E8 binds at different epitopes from hLDL-2D8 and has some overlap with hLDL-F5. Lastly, the binding affinity of the mAbs was examined by indirect ELISA. The average affinity constants (Kaff) for mAbs hLDL-2D8, hLDL-E8 and hLDL-F5 are 1.51 ±â€¯0.69 × 109 Mol-1, 7.25 ±â€¯3.56 × 108 Mol-1 and 4.39 ±â€¯2.63 × 106 Mol-1, respectively. Additionally, the behavior of the antibodies in the dose-response curve revealed that hLDL-F5 may recognize two epitopes of apoB-100 or have very low binding affinity. In contrast, hLDL-2D8 and hLDL-E8 each recognize a single epitope. These findings provide information that will be useful when selecting mAbs for both laboratory and clinical research purposes.

A Skin Testing Strategy for Non-IgE-Mediated Reactions Associated With Vancomycin.

BACKGROUND: Vancomycin infusion reaction (VIR), reportedly mediated through Mas-Related G Protein-Coupled Receptor-X2, is the primary vancomycin-induced immediate drug reaction. Clinically, distinguishing the underlying drug-induced immediate drug reaction mechanisms is crucial for future treatment strategies, including drug restriction, re-administration, and pretreatment considerations. However, the lack of validated diagnostic tests makes this challenging, often leading to unnecessary drug restriction. OBJECTIVE: To determine whether intradermal tests (IDTs) and, separately, the basophil activation test (BAT) differentiate VIR from vancomycin-tolerant subjects. METHODS: This was a cross-sectional study of vancomycin-exposed adults with and without a history of VIR. Data on demographics, allergy-related comorbidities, history of vancomycin exposures, and VIR characteristics were collected. IDT with vancomycin was performed. IDT dose-response EC50, IDT-related local symptoms, and BAT results were compared between groups. RESULTS: A total of 11 VIR and 10 vancomycin-tolerant subjects were enrolled. The most reported VIR symptoms were pruritus (82%), flushing (82%), hives (46%), angioedema (27%), and dyspnea (19%). The IDT dose-response mean EC50 was 328 µg/mL (95% CI, 296-367) in the VIR versus 1166 µg/mL (95% CI, 1029-1379) in the tolerant group (P < .0001). All VIR subjects reported IDT-related local pruritus compared with 60% of tolerant subjects (P = .0185). The %CD63+ basophils were consistently less than 2%, without significant differences between groups (P < .54). CONCLUSIONS: Variations in skin test methodologies could help identify other immediate drug reaction mechanisms beyond IgE. This skin test protocol holds the potential for identifying VIR, particularly in cases where patients have received multiple drugs while BAT is insufficient. Future studies will validate and delineate its predictive value, assessing the risk of VIR.

A novel approach to assess the health risk of aryl hydrocarbon receptor-bound contaminants via inhalation exposure using CYP1A1 expression as a biomarker.

Polycyclic aromatic hydrocarbons (PAHs) and dioxins are potential causes of multiple diseases by activating the aryl hydrocarbon receptor (AhR) pathway. Health risk assessment of chemicals primarily relies on the relative potency factor (RPF), although its accuracy may be limited when solely using EC50 values. The induction of cytochrome P4501A1 (CYP1A1) serves as a biomarker for AhR activation and is an integrator of dioxin-like toxicity. Here, we present a method for evaluating the risks associated with AhR activation using mathematical models of dose-CYP1A1 induction. The dose-effect curves for certain PAHs and dioxins, including Ant, BghiP, 1,2,3,4,7,8-HxCDD, and others, exhibited a non-classical S-shaped form. The toxic equivalent factor (TEF) profiles revealed a broad range of toxic equivalent factor values. The TEFs for PAHs ranged from approximately 0.01 to 6, with higher values being observed when the concentration was less than 10-10 M, with the exceptions of Ace, Phe, and BghiP. Most congeners of dioxins got the lowest TEF value at around 10-10 M, ranging from 0.04 to 1.00. The binding affinity of AhR to ligands did not display a strong correlation with the EC50 of CYP1A1 expression, suggesting that the AhR-mediated effects of PAHs and dioxins are not fixed but instead fluctuate with the dose. Air samples acquired from a parking area were used to compare the proficiency of RPF and our current approach. In the current method, naphthalene and chrysene were the primary contributors of PAHs to AhR-mediated risks in parking lots air samples, respectively. However, the contributions of naphthalene and chrysene could be disregarded in the RPF approach.

A Longitudinal Dose-Response Curve Between Leisure-Time Physical Activity and the Prevalence of Diabetes Based on the Different Levels of Cognitive Function Among Older Adults.

This study investigated a dose-response relationship between Leisure-Time Physical Activity participation (LTPA) and the risk of diabetes and a comparison of the risk across different cognitive function groups among older adults. The Health and Retirement Study data were used from 2012 to 2020 (n = 18 746). This study conducted a Cox Proportional Hazard Regression to investigate the Dose-Response Curve between the prevalence of diabetes and the covariates following a level of LTPA participation. The result presented that the Odds Ratio continuously decreased as the level of LTPA participation increased. Among the three cognitive function groups, the high group (OR = .43, P < .05) and the mid group (OR = .71, P < .05) had a larger negative slope coefficient than the low group. This study found that LTPA participation reduces the risk of diabetes and gives evidence for the importance of cognitive function in reducing the prevalence of diabetes.

Encoding Genetic Circuits with DNA Barcodes Paves the Way for High-Throughput Profiling of Dose-Response Curves of Metabolite Biosensors.

Metabolite biosensors, through which the intracellular metabolite concentrations could be converted to changes in gene expression, are widely used in a variety of applications according to the different output signals. However, it remains challenging to fine-tune the dose-response relationships of biosensors to meet the needs of various scenarios. On the other hand, the short read length of next-generation sequencing (NGS) has greatly limited the design capability of sequence libraries. To address these issues, we describe a DNA trackable assembly method, coupled with fluorescence-activated cell sorting and NGS (Sort-Seq), to achieve the characterization of dose-response curves in a massively parallel manner. As a proof of the concept, we constructed a malonyl-CoA biosensor library containing 5184 combinations with six levels of transcription factor dosage, four different operator positions, and 216 possible upstream enhancer sequence (UAS) designs in Saccharomyces cerevisiae BY4700. By using Sort-Seq and machine learning approach, we obtained comprehensive dose-response relationships of the combinatorial sequence space. Therefore, our pipeline provides a platform for the design, tuning, and profiling of biosensor response curves and shows great potential to facilitate the rational design of genetic circuits.

Radiprodil, a selective GluN2B negative allosteric modulator, rescues audiogenic seizures in mice carrying the GluN2A(N615S) mutation.

BACKGROUND AND PURPOSE: GRIN-related disorders are neurodevelopmental disorders caused by mutations in N-methyl-D-aspartate receptor (NMDAR) subunit genes. A large fraction of these mutations lead to a 'gain of function' (GoF) of the NMDAR. Patients present with a range of symptoms including epilepsy, intellectual disability, behavioural and motor. Controlling seizures is a significant unmet medical need in most patients with GRIN-related disorders. Although several hundred GRIN mutations have been identified in humans, until recently none of the mouse models carrying Grin mutations/deletions showed an epileptic phenotype. The two recent exceptions both carry mutations of GluN2A. The aim of this study was to assess the efficacy of radiprodil, a selective negative allosteric modulator of GluN2B-containing NMDARs, in counteracting audiogenic seizures (AGS) in a murine model carrying the GluN2A(N615S) homozygous mutation (Grin2aS/S mice). EXPERIMENTAL APPROACH: Grin2aS/S mice were acutely treated with radiprodil at different doses before the presentation of a high-frequency acoustic stimulus commonly used for AGS induction. KEY RESULTS: Radiprodil significantly and dose-dependently reduced the onset and severity of AGS in Grin2aS/S mice. Surprisingly, the results revealed a sex-dependent difference in AGS susceptibility and in the dose-dependent protection of radiprodil in the two genders. Specifically, radiprodil was more effective in female versus male mice. CONCLUSION AND IMPLICATIONS: Overall, our data clearly show that radiprodil, a GluN2B selective negative allosteric modulator, may have the potential to control seizures in patients with GRIN2A GoF mutations. Further studies are warranted to better understand the sex-dependent effects observed in this study.

More N fertilizer, more maize, and less alfalfa: maize benefits from its higher N uptake per unit root length.

Root plasticity is fundamental to soil nutrient acquisition and maximizing production. Different soil nitrogen (N) levels affect root development, aboveground dry matter accumulation, and N uptake. This phenotypic plasticity is well documented for single plants and specific monocultures but is much less understood in intercrops in which species compete for the available nutrients. Consequently, the study tested whether the plasticity of plant roots, biomass and N accumulation under different N levels in maize/alfalfa intercropping systems differs quantitatively. Maize and alfalfa were intercropped for two consecutive years in large soil-filled rhizoboxes and fertilized with 6 different levels of N fertilizer (0, 75, 150, 225, 270, and 300 kg ha-1). Root length, root surface area, specific root length, N uptake and yield were all increased in maize with increasing fertilizer level, whereas higher N rates were supraoptimal. Alfalfa had an optimal N rate of 75-150 kg ha-1, likely because the competition from maize became more severe at higher rates. Maize responded more strongly to the fertilizer treatment in the second year when the alfalfa biomass was much larger. N fertilization contributes more to maize than alfalfa growth via root plasticity responses. Our results suggest that farmers can maximize intercropping yield and economic return by optimizing N fertilizer management.

Assessing the impact of neutron relative biological effectiveness on all solid cancer mortality risks in the Japanese atomic bomb survivors.

PURPOSE: Risk analyses, based on relative biological effectiveness (RBE) estimates for neutrons relative to gammas, were performed; and the change in the curvature of the risk to dose response with increasing neutron RBE was analyzed using all solid cancer mortality data from the Radiation Effect Research Foundation (RERF). Results were compared to those based on incidence data. MATERIALS AND METHODS: This analysis is based on RERF mortality data with separate neutron and gamma doses for colon doses, from which organ averaged doses could be calculated. A model for risk ratio variation with RBE was developed. RESULTS: The best estimate of the neutron RBE considering mortality data was 200 (95% confidence interval (CI): 50-1010) for colon dose using the weighted-dose approach and for organ averaged dose 110 (95% CI: 30-350). The ERR risk ratios for all solid cancers combined, for the best fitting neutron RBE estimate and the neutron RBE of 10 result in a ratio of 0.54 (95% CI: 0.17-0.85) for colon dose and 0.55 (95% CI: 0.18-0.87) for organ averaged dose. The risk to dose response curvature became significantly negative (concave down) with increasing RBE, at a neutron RBE of 170 using colon dose and at an RBE of 90 using organ averaged dose for males when fitting a linear-quadratic dose response. For females, the curvature decreased toward linearity with increasing neutron RBE and remained significantly positive until RBE of 80 and 40 using colon and organ averaged dose, respectively. For higher neutron RBEs, no significant conclusion could be drawn about the shape of the dose-response curve. CONCLUSIONS: Application of neutron RBE values higher than 10 results in substantially reduced cancer mortality risk estimates and a significant reduction in curvature of the risk to dose responses for males. Using mortality data, the best fitting neutron RBE is much higher than when incidence data is used. The neutron RBE ranges covered by the overlap in the CIs from both the mortality and incidence analyses are 50-190 using colon dose and in all cases, the best fitting neutron RBE and lower 95% CI are higher than the value of 10 traditionally applied by the RERF. Therefore, it is recommended to consider uncertainties in neutron RBE values when calculating radiation risks and discussing the shape of dose responses using Japanese A-bomb survivors data.

Associations between serum health biomarker concentrations and reproductive performance, accounting for milk yield, in pasture-based Holstein cows in southeastern Australia.

In this single cohort study, we investigated associations between the concentrations of a suite of serum biomarkers measured in the first 30 d of lactation and subsequent reproductive performance measured as mating start date to conception intervals, in pasture-based Holstein cows. A secondary objective was to examine associations between biomarker concentrations and 305-d milk yield to assess whether any positive associations between biomarker concentration and reproductive performance were explained by reduced milk production. The data used had been collected as part of an ongoing project from 2017 to 2020 to compile a data set from a large population of lactating dairy cows. Biomarkers measured were those associated with energy balance (ß-hydroxybutyrate [BHB] and nonesterified fatty acids [NEFA]), protein nutritional status (urea and albumin), immune status (globulin, albumin to globulin ratio and haptoglobin), and macromineral status (calcium and magnesium). Associations between biomarker concentrations and mating start date to conception interval were investigated using Cox proportional hazard models, using between 634 and 1,121 lactations (varying by biomarker) from 632 to 1,103 cows and 11 to 17 mating periods from 10 to 13 herds. Based on hazard ratio (HR) estimates and associated 95% confidence intervals (CI), hazard of conception on any particular day of the herds' mating periods was positively associated with the concentrations of albumin (HR = 1.09; 95% CI: 1.05-1.12), albumin to globulin ratio (HR = 2.82; 95% CI: 1.66-4.79), calcium (HR = 2.01; 95% CI: 1.18-3.43), and magnesium (HR = 2.17; 95% CI: 1.01-4.66), and negatively associated with globulin concentration (HR = 0.98; 95% CI: 0.97 to 1.00). There was also some evidence that NEFA concentration was negatively associated (HR = 0.76; 95% CI: 0.57 to 1.01), and urea concentration positively associated (HR = 1.05; 95% CI: 0.99 to 1.11), with reproductive performance, but no evidence that BHB and haptoglobin concentrations were associated with reproductive performance. Except for NEFA, presence and direction of the associations between the biomarker and milk yield were not discordant with that for reproductive performance. Also, except for NEFA, we found no substantial evidence of nonlinear relationships between biomarker concentration and either reproductive performance or milk yield. Correlations between biomarker concentrations were generally weak, indicating that multibiomarker panels may collectively predict reproductive performance better than any single biomarker. We noted substantial variation in the concentrations of all biomarkers within, and for some biomarkers, between herd-year groups. Collectively, these results indicate that there may be scope to improve biomarker concentrations through nutritional, management, and genetic interventions, and by association, reproductive performance and milk yield may also improve.

You get what you screen for: Standards for experimental design and data fitting in drug discovery.

A common mantra in drug discovery is that "You get what you screen for." This is not a promise that you will always get an effective drug candidate, but rather a warning that inaccuracies in your protocol for screening will more likely produce a compound that fails to be an effective candidate because it matches the properties of your screen, not the desired features of an ideal lead compound. It is with this in mind that we examine the current protocols for evaluating drug candidates and highlight some deficiencies while pointing the way to better methods. Many of the errors in data fitting can be rectified by abandoning the traditional equation-based data fitting methods and adopting the more rigorous mechanism-based fitting afforded by computer simulation based on numerical integration of rate equations. Using these methods bypasses the errors in judgement in choosing the appropriate equation for data fitting and the approximations required to derive those equations. In this chapter we outline the limitations and systematic errors in conventional methods of data fitting and illustrate the advantages of computer simulation and introduce the methods of analysis.

Modeling hormesis using multivariate nonlinear regression in plant biology: A comprehensive approach to understanding dose-response relationships.

For over a century, ecotoxicological studies have reported the occurrence of hormesis as a significant phenomenon in many areas of science. In plant biology, hormesis research focuses on measuring morphological, physiological, biochemical, and productivity changes in plants exposed to low doses of herbicides. These studies involve multiple features that are often correlated. However, the multivariate aspect and interdependencies among components of a plant system are not considered in the adopted modeling framework. Therefore, a multivariate nonlinear modeling approach for hormesis is proposed, where information regarding correlations among response variables is taken into account through a variance-covariance matrix obtained from univariate residuals. The proposed methodology is evaluated through a Monte Carlo simulation study and an application to experimental data from safflower (Carthamus tinctorius L.) cultivation. In the simulation study, the multivariate model outperformed the univariate models, exhibiting higher precision, lower bias, and greater accuracy in parameter estimation. These results were also confirmed in the analysis of the experimental data. Using the delta method, mean doses of interest can be derived along with their associated standard errors. This is the first study to address hormesis in a multivariate context, allowing for a better understanding of the biphasic dose-response relationships by considering the interrelationships among various measured characteristics in the plant system, leading to more precise parameter estimates.

Establishment of ex vivo calibration curve for X-ray induced "dicentric + ring" and micronuclei in human peripheral lymphocytes for biodosimetry during radiological emergencies, and validation with dose blinded samples.

In the modern developing society, application of radiation has increased extensively. With significant improvement in the radiation protection practices, exposure to human could be minimized substantially, but cannot be avoided completely. Assessment of exposure is essential for regulatory decision and medical management as applicable. Until now, cytogenetic changes have served as surrogate marker of radiation exposure and have been extensively employed for biological dose estimation of various planned and unplanned exposures. Dicentric Chromosomal Aberration (DCA) is radiation specific and is considered as gold standard, micronucleus is not very specific to radiation and is considered as an alternative method for biodosimetry. In this study dose response curves were generated for X-ray induced "dicentric + ring" and micronuclei, in lymphocytes of three healthy volunteers [2 females (age 22, 23 years) and 1 male (24 year)]. The blood samples were irradiated with X-ray using LINAC (energy 6 MV, dose rate 6 Gy/min), in the dose range of 0-5Gy. Irradiated blood samples were cultured and processed to harvest metaphases, as per standard procedures recommended by International Atomic Energy Agency. Pooled data obtained from all the three volunteers, were in agreement with Poisson distribution for "dicentric + ring", however over dispersion was observed for micronuclei. Data ("dicentric + ring" and micronuclei) were fitted by linear quadratic model of the expression Y[bond, double bond]C + αD + ßD2 using Dose Estimate software, version 5.2. The data fit has resulted in linear coefficient α = 0.0006 (±0.0068) "dicentric + ring" cell-1 Gy-1 and quadratic coefficient ß = 0.0619 (±0.0043) "dicentric + ring" cell-1 Gy-2 for "dicentric + ring" and linear coefficient α = 0.0459 ± (0.0038) micronuclei cell-1 Gy-1 and quadratic coefficient ß = 0.0185 ± (0.0010) micronuclei cell-1 Gy-2 for micronuclei, respectively. Background frequencies for "dicentric + ring" and micronuclei were 0.0006 ± 0.0004 and 0.0077 ± 0.0012 cell-1, respectively. Established curves were validated, by reconstructing the doses of 8 dose blinded samples (4 by DCA and 4 by CBMN) using coefficients generated here. Estimated doses were within the variation of 0.9-16% for "dicentric + ring" and 21.7-31.2% for micronuclei respectively. These established curves have potential to be employed for biodosimetry of occupational, clinical and accidental exposures, for initial triage and medical management.

An analysis of reproductive outcomes for conceptions of participants of the Air Force Health Study.

Analyses were conducted of reproductive outcomes for conceptions of participants of the Air Force Heath Study. Participants were male Air Force veterans of the Vietnam War. Conceptions were categorized into conceived before and after the start of the participant's Vietnam War service. Analyses accounted for correlation between outcomes for multiple conceptions for each participant. For each of three non-sparsely occurring outcomes, including not live born, miscarriage, and preterm, the probability of occurrence increased substantially when conceived after compared to before the start of Vietnam War service. These results support the conclusion of an adverse effect due to Vietnam War service on these reproductive outcomes. Data for conceptions after the start of Vietnam War service for participants with measured dioxin values were used to estimate dose-response curves for the effect of dioxin exposure on the occurrence of each of the three non-sparsely occurring outcomes. These curves were assumed to be constant up to a threshold and then monotonic after that threshold. For each of the three non-sparsely occurring outcomes, the estimated dose-response curves increased nonlinearly after associated thresholds. These results support the conclusion that the adverse effect due to conception after the start of Vietnam War service is attributable to high enough exposures to dioxin, a toxic contaminant of Agent Orange used for herbicide spraying in the Vietnam War. Sensitivity analyses supported the conclusion that dioxin results were not substantially affected by the assumption of monotonicity, decay due to elapsed time from exposure to measurement, and available covariates.

Different effects of a perioperative single dose of dexamethasone on wound healing in mice with or without sepsis.

Introduction: Sepsis delays wound healing owing to uncontrolled inflammation. A single perioperative dose of dexamethasone is widely used because of its anti-inflammatory effects. However, the effects of dexamethasone on wound healing in sepsis remain unclear. Methods: We discuss the methods to obtain dose curves and explore the safe dosage range for wound healing in mice with or without sepsis. A saline or LPS intraperitoneal injection was applied to C57BL/6 mice. After 24 hours, the mice received a saline or DEX intraperitoneal injection and full-thickness, dorsal wounding operation. Wound healing was observed by image record, immunofluorescence and histological staining. Inflammatory cytokines and M1/M2 macrophages in wounds were determined by ELISA and immunofluorescence, respectively. Results: Dose-response curves reflected the safe dosage range of DEX in mice with or without sepsis, from 0.121 to 2.03 mg/kg and from 0 to 0.633 mg/kg, respectively. we found that a single dose of dexamethasone (1 mg/kg, i.p.) promoted wound healing in septic mice, but delayed wound healing in normal mice. In normal mice, dexamethasone delays inflammation, resulting in an insufficient number of macrophages during the healing process. In septic mice, dexamethasone alleviated excessive inflammation and maintained the balance of M1/M2 macrophages in the early and late healing process. Discussion: In summary, the safe dosage range of dexamethasone in septic mice is wider than that in normal mice. A single dose of dexamethasone (1 mg/kg) increased wound healing in septic mice, but delayed it in normal mice. Our findings provide helpful suggestions for the rational use of dexamethasone.

Modularity and neuronal heterogeneity: Two properties that influence in vitro neuropharmacological experiments.

Introduction: The goal of this work is to prove the relevance of the experimental model (in vitro neuronal networks in this study) when drug-delivery testing is performed. Methods: We used dissociated cortical and hippocampal neurons coupled to Micro-Electrode Arrays (MEAs) arranged in different configurations characterized by modularity (i.e., the presence of interconnected sub-networks) and heterogeneity (i.e., the co-existence of neurons coming from brain districts). We delivered increasing concentrations of bicuculline (BIC), a neuromodulator acting on the GABAergic system, and we extracted the IC50 values (i.e., the effective concentration yielding a reduction in the response by 50%) of the mean firing rate for each configuration. Results: We found significant lower values of the IC50 computed for modular cortical-hippocampal ensembles than isolated cortical or hippocampal ones. Discussion: Although tested with a specific neuromodulator, this work aims at proving the relevance of ad hoc experimental models to perform neuropharmacological experiments to avoid errors of overestimation/underestimation leading to biased information in the characterization of the effects of a drug on neuronal networks.

An analysis of birth defects and developmental disabilities for children of participants of the Air Force Health Study.

Analyses were conducted of the occurrence of eight general categories of birth defects and developmental disabilities for children fathered by participants of the Air Force Heath Study (AFHS). Participants were male Air Force veterans of the Vietnam War. Children were categorized into conceived before and after the start of the participant's Vietnam War service. Analyses accounted for correlation between outcomes for multiple children fathered by each of the participants. For each of the eight general categories of birth defects and developmental disabilities, the probability of its occurrence increased substantially for children conceived after compared to before the start of Vietnam War service. These results support the conclusion of an adverse effect on reproductive outcomes due to Vietnam War service. Data for children conceived after the start of Vietnam War service for participants with measured dioxin values were used to estimate dose-response curves for the effect of dioxin exposure on the occurrence of each of the eight general categories of birth defects and developmental disabilities. These curves were assumed to be constant up to a threshold and then monotonic after that threshold. For seven of the eight general categories of birth defects and developmental disabilities, the estimated dose-response curves increased nonlinearly after associated thresholds. These results support the conclusion that the adverse effect to conception after the start of Vietnam War service may be attributable to high enough exposures to dioxin, a toxic contaminant of Agent Orange used for herbicide spraying in the Vietnam War.

Technical note: Energy dependence of the Gafchromic EBT4 film: Dose-response curves for 70 kV, 6 MV, 6 MV FFF, 10 MV FFF, and 15 MV x-ray beams.

BACKGROUND: EBT4 was newly released for radiotherapy quality assurance to improve the signal-to-noise ratio in radiochromic film dosimetry. It is important to know its dose-response characteristics before its use in the clinic. PURPOSE: This study aims to investigate and compare the dose-response curves of the Gafchromic EBT4 film for megavoltage and kilovoltage x-ray beams with different dose levels, scanning spatial resolutions, and sizes of region of interest (ROI). METHODS: EBT4 film (Lot#07052201) calibration strips (3.5 × 20 cm2 ) were exposed to a 10×10 cm2 open field at doses of 0, 63, 125, 500, 750, 1000 cGy using 6 MV photon beam. EBT4 film strips from the same lot were then exposed to each x-ray beam (6 MV, 6 MV FFF, 10 MV FFF, 15 MV, and 70 kV) at six dose values (50, 100, 300, 600, 800, 1000 cGy). A full sheet (25 × 20 cm2 ) of EBT4 film was irradiated at each energy with 300 cGy for profile comparison with the treatment planning calculation. At two different spatial resolutions of 72 and 300 dpi, each film piece was scanned three consecutive times in the center of an Epson 10000XL flatbed scanner in 48-bit color. The scanned images were analyzed using FilmQA Pro. For each scanned image, an ROI of 2 × 2 cm2 at the field center was selected to obtain the average pixel value with its standard deviation in the ROI. An additional ROI of 1 cm diameter circle was also used to evaluate the impact of ROI shape and size, especially for FFF beams. The dose value, average dose-response value, and associated uncertainty were determined for each energy and relative responses were analyzed. The Student's t-test was performed to evaluate the statistical significance of the dose-response values with different color channels, ROI shapes, and spatial resolutions. RESULTS: The dose-response curves for the five x-ray energies were compared in three color channels. Weak energy dependence was found among the megavoltage beams. No significant differences (average ∼1.1%) were observed for all doses in this study among 6 MV, 6 MV FFF, 10 MV FFF, and 15 MV beams, regardless of spatial resolution and color channel. However, a statistically significant difference in dose-response was observed up to 12% between 70 kV and 6 MV beams. CONCLUSIONS: The dose-response curves for Gafchromic EBT4 films were nearly independent of the energy of the photon beams among 6 MV, 6 MV FFF, 10 MV FFF, and 15 MV. For very low-energy photons (e.g., 70 kV), a separate calibration from the same low-energy x-ray is necessary.

Glyphosate Efficacy in Chloris virgata Sw. in Response to Temperature and Tank Mixing.

Glyphosate alone or a tank mixture of glyphosate and 2,4-D is commonly used for broad-spectrum weed control under fallow conditions in Australia. Air temperature or mixing glyphosate with 2,4-D, may influence the efficacy of glyphosate on feather fingergrass (Chloris virgata Sw.), a problematic summer-season weed of Australia. Dose-response studies were conducted with four populations of feather fingergrass under temperature-controlled glasshouse conditions (35/25 °C and 25/15 °C at 12 h/12 h) to assess the level of glyphosate resistance in relation to temperature regimes. Four parameter log-logistic models were used to develop dose-response curves. Based on plant mortality percentage, LD50 (lethal dose for 50% mortality) values of glyphosate at 25/15 °C for populations Ch, SGM2, SGW2, and CP2 were 137, 60, 650, and 1067 g ae ha-1, respectively. However, at 35/25 °C, the corresponding LD50 values were 209, 557, 2108, and 2554 g ae ha-1, respectively. A similar response was observed for the parameter GR50 (dose for 50% growth reduction) values of glyphosate. These results indicate that populations SGW2 and CP2 are highly glyphosate-resistant and in the summer season, it may be very difficult to control these populations due to poor glyphosate efficacy. These results further suggest that the efficacy of glyphosate for feather fingergrass control can be improved if applied during cooler temperatures (25/15 °C) or the spring season compared with warmer temperatures (35/25 °C) or the summer season. In another study, 2,4-D antagonized glyphosate remarkably in the CP2 (glyphosate-resistant) population but only marginally in the Ch (glyphosate-susceptible) population. Thus, it is not advisable to mix 2,4-D with glyphosate for the control of glyphosate-resistant feather fingergrass populations. The results further suggest that the use of this mixture is useful if the feather fingergrass is not glyphosate-resistant; however, the use of the mixture is to be avoided if the population is glyphosate-resistant in order to not exacerbate the potential resistance problem.

Planar bioluminescent cytotoxicity assay via genetically modified adherent human reporter cell lines, applied to authenticity screening of Saussurea costus root.

The hyphenation of high-performance thin-layer chromatography to effect-directed assays is a very straightforward way to detect individual bioactive zones, and at the same time, to investigate several samples simultaneously. The combination of the separation technique with adherent human cells applied on the same surface was recently shown to be possible. Since on-surface adherent cell assays are in their infancy, a planar bioluminescent cytotoxicity assay was developed to expand the possibilities. Human embryonic kidney (HEK) 293 or HeLa (cervical carcinoma) cells were chosen because of their fast growth rates and high rates of successful transfection, being suitable for the generation of genetically modified reporter cells. For the first time, HeLa cells were visualized on the wettable reversed phase plate surface using digital microscopy. For the generation of bioluminescent reporter cells, vectors for the expression of three luciferase enzymes of various origins were tested. The genetically modified HEK 293T-CMV-ELuc cells were the best suitable for the new planar cytotoxicity assay due to the faster growth rate, robustness, and stronger bioluminescence signal. The stable expression of luciferase under the control of a strong constitutive promoter allowed the cells to be used for the determination of the cytotoxicity of Saussurea costus root samples obtained from the market and to assess the authenticity of these samples. Any cytotoxic zone was detected as a dark zone inhibiting the cell bioluminescence. Five replicates of the dose-response curve confirmed the good assay performance and the cytotoxicity of a zone, which was assigned to costunolide and dehydrocostus lactone. By this, the proof-of-principle of the new planar bioluminescent cytotoxicity assay, which does not require expensive licensing, was successful.

Determining the Dose-Response Curve of Exoelectrogens: A Microscale Microbial Fuel Cell Biosensor for Water Toxicity Monitoring.

Nowadays, the development of real-time water quality monitoring sensors is critical. However, traditional water monitoring technologies, such as enzyme-linked immunosorbent assay (ELISA), liquid chromatography, mass spectroscopy, luminescence screening, surface plasma resonance (SPR), and analysis of living bioindicators, are either time consuming or require expensive equipment and special laboratories. Because of the low cost, self-sustainability, direct current output and real-time response, microbial fuel cells (MFCs) have been implemented as biosensors for water toxicity monitoring. In this paper, we report a microscale MFC biosensor to study the dose-response curve of exoelectrogen to toxic compounds in water. The microscale MFC biosensor has an anode chamber volume of 200 µL, which requires less sample consumption for water toxicity monitoring compared with macroscale or mesoscale MFC biosensors. For the first time, the MFC biosensor is exposed to a large formaldehyde concentration range of more than 3 orders of magnitudes, from a low concentration of 1 × 10-6 g/L to a high concentration of 3 × 10-3 g/L in water, while prior studies investigated limited formaldehyde concentration ranges, such as a small concentration range of 1 × 10-4 g/L to 2 × 10-3 g/L or only one high concentration of 0.1 g/L. As a result, for the first time, a sigmoid dose-response relationship of normalized dose-response versus formaldehyde concentration in water is observed, in agreement with traditional toxicology dose-response curve obtained by other measurement techniques. The biosensor has potential applications in determining dose-response curves for toxic compounds and detecting toxic compounds in water.