Direct Screening Method Research Articles

Performance assessment of an equine metabolomics model for screening a range of anabolic agents.

Despite their ban, Anabolic Androgenic Steroids (AAS) are considered as the most important threat for equine doping purposes. In the context of controlling such practices in horse racing, metabolomics has emerged as a promising alternative strategy to study the effect of a substance on metabolism and to discover new relevant biomarkers of effect. Based on the monitoring of 4 metabolomics derived candidate biomarkers in urine, a prediction model to screen for testosterone esters abuse was previously developed. The present work focuses on assessing the robustness of the associated method and define its scope of application. Several hundred urine samples were selected from 14 different horses of ethically approved administration studies involving various doping agents' (AAS, SARMS, β-agonists, SAID, NSAID) (328 urine samples). In addition, 553 urine samples from untreated horses of doping control population were included in the study. Samples were characterized with the previously described LC-HRMS/MS method, with the objective of assessing both its biological and analytical robustness. The study concluded that the measurement of the 4 biomarkers involved in the model was fit for purpose. Further, the classification model confirmed its effectiveness in screening for testosterone esters use; and it demonstrated its ability to screen for the misuse of other anabolic agents, allowing the development of a global screening tool dedicated to this class of substances. Finally, the results were compared to a direct screening method targeting anabolic agents demonstrating complementary performances of traditional and omics approaches in the screening of anabolic agents in horses.

Discovery of diphenyl ether–degrading Streptomyces strains by direct screening based on ether bond–cleaving activity

Diphenyl ethers (DEs), which are widely used in the agricultural and chemical industries, have become hazardous contaminants in the environment. Although several DE-degrading bacteria have been reported, discovering new types of such microorganisms could enhance understanding of the degradation mechanism in the environment. In this study, we used a direct screening method based on detection of ether bond-cleaving activity to screen for microorganisms that degrade 4,4'-dihydroxydiphenyl ether (DHDE) as a model DE. Microorganisms isolated from soil samples were incubated with DHDE, and strains producing hydroquinone via ether bond cleavage were selected using hydroquinone-sensitive Rhodanine reagent. This screening procedure resulted in the isolation of 3 bacteria and 2 fungi that transform DHDE. Interestingly, all of the isolated bacteria belonged to one genus, Streptomyces. To our knowledge, these are the first microorganisms of the genus Streptomyces shown to degrade a DE. Streptomyces sp. TUS-ST3 exhibited high and stable DHDE-degrading activity. HPLC, LC-MS, and GC-MS analyses revealed that strain TUS-ST3 converts DHDE to its hydroxylated analogue and generates hydroquinone as an ether bond-cleavage product. Strain TUS-ST3 also transformed DEs other than DHDE. In addition, glucose-grown TUS-ST3 cells began to transform DHDE after incubation with this compound for 12h, and produced 75μM hydroquinone in 72h. These activities of streptomycetes may play an important role in DE degradation in the environment. We also report the whole genome sequence of strain TUS-ST3.

Online SFE-SFC-MS/MS colony screening: A high-throughput approach for optimizing (-)-limonene production

Conventional analysis of microbial bioproducers requires the extraction of metabolites from liquid cultures, where the culturing steps are time consuming and greatly limit throughput. To break through this barrier, the current study aims to directly evaluate microbial bioproduction colonies by way of supercritical fluid extraction-supercritical fluid chromatography-triple quadrupole mass spectrometry (SFE-SFC-MS/MS). The online SFE-SFC-MS/MS system offers great potential for high-throughput analysis due to automated metabolite extraction without any need for pretreatment. This is the first report of SFE-SFC-MS/MS as a method for direct colony screening, as demonstrated in the high-throughput screening of (-)-limonene bioproducers. Compared with conventional analysis, the SFE-SFC-MS/MS system enables faster and more convenient screening of highly productive strains.

Potential Biocontrol Activities of Populus Endophytes against Several Plant Pathogens Using Different Inhibitory Mechanisms

The plant microbiome can be used to bolster plant defense against abiotic and biotic stresses. Some strains of endophytes, the microorganisms within plants, can directly inhibit the growth of plant fungal pathogens. A previously isolated endophyte from wild Populus (poplar), WPB of the species Burkholderia vietnamiensis, had robust in vitro antifungal activity against pathogen strains that are highly virulent and of concern to Pacific Northwest agriculture: Rhizoctonia solani AG-8, Fusarium culmorum 70110023, and Gaemannomyces graminis var. tritici (Ggt) ARS-A1, as well as activity against the oomycete, Pythium ultimum 217. A direct screening method was developed for isolation of additional anti-fungal endophytes from wild poplar extracts. By challenging pathogens directly with dilute extracts, eleven isolates were found to be inhibitory to at least two plant pathogen strains and were therefore chosen for further characterization. Genomic analysis was conducted to determine if these endophyte strains harbored genes known to be involved in antimicrobial activities. The newly isolated Bacillus strains had gene clusters for production of bacillomycin, fengicyn, and bacillibactin, while the gene cluster for the synthesis of sessilin, viscosin and tolaasin were found in the Pseudomonas strains. The biosynthesis gene cluster for occidiofungin (ocf) was present in the Burkholderia vietnamiensis WPB genome, and an ocf deletion mutant lost inhibitory activity against 3 of the 4 pathogens. The new isolates lacked the gene cluster for occidiofungin implying they employ different modes of action. Other symbiotic traits including nitrogen fixation, phosphate solubilization, and the production of auxins and siderophores were investigated. Although it will be necessary to conduct in vivo tests of the candidates with pathogen-infected agricultural crops, the wild poplar tree microbiome may be a rich source of beneficial endophyte strains with potential for biocontrol applications against a variety of pathogens and utilizing varying modes of action.

Investigating Antimicrobial Characteristics/Advantages of Australian Wood Species for Use in Food Packaging—A Feasibility Study

The antimicrobial characteristics of Australian commercial timber species were studied using a direct screening method and two different microorganisms. The effectiveness of timber samples was compared with plastic and paper samples during an incubation period of 24 h at 37 °C. The initial data with E. coli and S. aureus showed no difference in performance between the plastic and paper samples and the softwood samples tested. Hardwood samples, however, showed an inhibition zone when tested with S. Aureus. The data showed similar trends of inhibition zones developed for sterilised and non-sterilised samples of spotted gum and shining gum species. The observed data showed promising antimicrobial characteristics for both veneer and solid samples of hardwood species. Further studies investigating the type of extractives, their role in antimicrobial characteristics and differences in the type of surface exposed to the microbial contamination from the point of view of timber’s anatomical properties are proposed.

Multi-Objective Design Optimization of a Tubular Permanent Magnet Linear Generator With 120° Phase Belt Toroidal Windings

The utilization of 120° phase belt toroidal windings (120°-TWs) can bring power density improvement in a tubular permanent magnet linear generator (TPMLG). Unfortunately, the TPMLG with 120°-TWs (120°-TPMLG) usually involve multiple structural parameters, and the interaction or even conflict among those structural parameters would have strong impact on generator performances in the design optimization process. To alleviate this problem, a multi-objective design optimization method is proposed to conduct a multi-objectives optimization, which aims to obtain the high-power density and low-detent force. First, the comprehensive sensitivity analysis method, namely global sensitivity analysis, is adopted to evaluate the sensitivities of the structural parameters on the performances of the 120°-TPMLG. According to the evaluation results, the structural parameters are divided into three levels: non-sensitive level, mild-sensitive level, and strong-sensitive level. Then, to improve the whole design optimization efficiency, the direct-screening method, the surface method, and the genetic algorithm is implemented in the three levels, respectively. And the optimal structural parameters of the 120°-TPMLG are finally determined. To verify the feasibility of the 120°-TPMLG and the effectiveness of the proposed design optimization method, the performances of the optimal 120°-TPMLG are analyzed and compared with the initial one and the same dimensions TPMLG with traditional toroidal windings (T-TPMLG) in detail. It shows that the performances of the optimal 120°-TPMLG meet the design requirements. Moreover, the detent force is largely decreased and the power density is notably improved. The results prove that the proposed multi-objective design optimization method is available for the 120°-TPMLG.

Major Constituents of Cannabis Vape Oil Liquid, Vapor and Aerosol in California Vape Oil Cartridge Samples.

During the E-cigarette or Vaping product use Associated Lung Injury (EVALI) outbreak of August 2019 to February 2020, the California Department of Public Health, Food and Drug Laboratory Branch received numerous cannabis vape oil cartridge investigation samples from throughout the state. Many of these products were directly linked to patients; others were collected as part of investigations. We determined the major ingredients and additives in twelve unused cannabis vape oil cartridge samples obtained before (n = 2) and during the EVALI outbreak (n = 10) in California from September 2018 to December 2019. We tested for major constituents in vape oil liquid, vape oil vapor, and vape oil aerosol phases. A nontargeted Gas Chromatography Mass Spectrometry direct injection screening method was developed for vape oils, a headspace heating module used for vape oil vapors and a solid-phase microextraction (SPME) vaping rig for aerosols generated by vaping. We have identified more than 100 terpenes and natural extracts, 19 cannabinoids, and other potential toxic additives such as Vitamin E Acetate, Polyethylene Glycols, and Medium Chain Triglycerides. We determined more terpenes and minor cannabinoids can be produced via vaporizing and aerosolizing the vape oil. Delta9-THC and potential toxic additives were found at lower levels in the vapor and aerosol than in the vape liquid.

Native Mass Spectrometry for the Study of PROTAC GNE-987-Containing Ternary Complexes.

PROteolysis TArgeting Chimeras (PROTACs) promote the degradation, rather than inhibition, of a drug target as a mechanism for therapeutic treatment. Bifunctional PROTAC molecules allow simultaneous binding of both the target protein and an E3‐Ubiquitin ligase, bringing the two proteins into close spatial proximity to allow ubiquitinylation and degradation of the target protein via the cell's endogenous protein degradation pathway. We utilized native mass spectrometry (MS) to study the ternary complexes promoted by the previously reported PROTAC GNE‐987 between Brd4 bromodomains 1 and 2, and Von Hippel Lindeau E3‐Ubiquitin Ligase. Native MS at high resolution allowed us to measure ternary complex formation as a function of PROTAC concentration to provide a measure of complex affinity and stability, whilst simultaneously measuring other intermediate protein species. Native MS provides a high‐throughput, low sample consumption, direct screening method to measure ternary complexes for PROTAC development.

Screening Methods for Isolation of Biocontrol Epiphytic Yeasts against Penicillium digitatum in Lemons.

Worldwide, the green rot caused by Penicillium digitatum is one of the most aggressive postharvest diseases of lemons. Searching for sustainable alternatives to chemical fungicides, epiphytic yeasts as potential biocontrol agents were isolated from citrus fruits using a tailor-made selective medium. For disclosing their antagonistic potential against P. digitatum, obtained isolates were subjected to direct screening methods, both in vitro and in vivo. In the course of the primary in vitro screening that comprised dual culture assays, 43 yeast strains displaying antagonistic activities against the pathogen were selected. Subsequently, such strains were subjected to an in vivo screening that consisted of a microscale test, allowing the selection of six yeast strains for further analysis. In the final screening using macroscale in vivo tests, three strains (AcL2, AgL21, and AgL2) displaying the highest efficiencies to control P. digitatum were identified. The protection efficiencies in lemons were 80 (AcL2), 76.7 (AgL21), and 75% (AgL2). Based on sequence analysis of the PCR amplified D1/D2 domains of the 26S rRNA genes, they were identified as representatives of the species Clavispora lusitaniae. Interestingly, the strains exhibited a broad action spectrum among citrus fruits as they were also able to combat the green mold disease in grapefruit and two orange varieties. The direct screening methods applied in this study favored the recovery of efficient candidates for application as biological control agents to combat fungal infestations of citrus fruits.

Direct screening of a target-specific covalent binder: stringent regulation of warhead reactivity in a matchmaking environment.

A peptide-type covalent binder for a target protein was obtained by direct and stringent screening of a warhead-modified peptide library on the robust T7 phage. The aryl fluorosulfate (fosylate) warhead was activated only in a matchmaking microenvironment created between the target protein and an appropriate peptide during the reactivity/affinity-based co-selection process of extended phage display.

DISCOVER: A facile structure-based screening method for vinyl compound producing microbes

Here we report a novel structure-based microbial screening method for vinyl compound discovery, DISCOVER (direct screening method based on coupling reactions for vinyl compound producers). Through a two-step screening procedure based on selective coupling reactions of terminal alkenes, the thiol-ene reaction (1st step of screening) and Mizoroki-Heck reaction, followed by iodine test (2nd step of screening), microbes producing vinyl compounds like itaconic acid (IA) can be isolated from soil samples. In the 1st step of screening, soil sources are plated on agar medium supplemented with an antimicrobial agent, α-thioglycerol (TG), and a radical initiator, VA-044 (VA). In the 2nd step of screening, vinyl compounds produced in the cultures are labelled with iodobenzene via the Mizoroki-Heck reaction, followed by an iodine test, leading to the detection and characterisation of labelled products. We evaluated the validity of DISCOVER using IA and its producer Aspergillus terreus. Experimental data supported our hypothesis that IA reacts with TG in the medium via the thiol-ene reaction and consequently, A. terreus rapidly forms colonies on the agar medium because of the loss of the antimicrobial activity of TG. Using DISCOVER, high throughput and selective isolation of A. terreus strains producing IA was possible from soils.

Impedance microbiology to speed up the screening of lactic acid bacteria exopolysaccharide production

Bacterial production of exopolysaccharides (EPS) is of increasing interest near food manufacturers, biotechnology industries and nutritionists because of their different roles. Several analytical methods are available for recovery, quantification and characterization of EPS from lactic acid bacteria (LAB) in food. However, direct screening method for production of EPS is still based on the visual observation of filamentous texture of the colonies developed on supplemented solid growth media. To overcome weaknesses of many currently used screening methods, we propose adopting impedance microbiology to evaluate the EPS production from LAB in milk. In this work we have proven that the peculiar shape of capacitance curve of Lactobacillus delbrueckii subsp. bulgaricus 2214, measured in milk by means of a BacTrac 4300® system, is due to production of EPS. Besides the pH measurement, the amounts of EPS evaluated after 0, 8, 13 and 55 h of incubation in milk, were in agreement with the evaluation of gene expression and confirmed by the observations by confocal laser scanning microscopy and by transmission electron microscopy.With the aim to verify the applicability of the proposed method, the drop entity of the capacitance curve (ΔE%) of 22 EPS-producing (EPS+) LAB strains and one negative (EPS-) control was evaluated both in broth medium and in milk. The positive ΔE% value found for all of the strains cultivated in the clear broth medium allowed to confirm the EPS production, simply observing a strain-dependent amount of EPS on surface of the measurement electrodes of the device. When the same EPS+ strains were cultivated in milk, the obtained ΔE% values showed that only a few of them were able to produce EPS in this environment, supporting their diversified ability to utilize lactose for this purpose.Results obtained by this multidisciplinary study demonstrate that impedance microbiology represents a suitable method to overcome the limits of the most commonly used methods to screen LAB for EPS production in milk. Moreover, these results also open a door to the application to other food and beverages, in which the EPS produced in situ could be of great interest for food industry.

Ask them all: Self-report universal prenatal substance use screening in the United States

ABSTRACTBackground: Increasing rates of opioid use in the United States have created a national public health crisis. Substance use in pregnancy increases risk for poor birth outcomes. National advisory groups recommend screening all women for prenatal substance use. However, there is no single approach or tool recommended for universal prenatal substance use screening.Objective: This study seeks to evaluate the effectiveness of universal screening using a direct approach self-report of substance use in pregnancy.Method: A retrospective review of de-identified data to evaluate women’s willingness to self-report substance use through direct screening. Data obtained from 24 months of maternal self-report were analyzed to assess the overall effectiveness of a direct screening method during the prenatal period.Results: Findings revealed maternal substance use disclosure rates of 9.7%, higher than the 5.4% United States national average.Conclusion: Increasing awareness of maternal prenatal substance use offers opportunity for education and support early in pregnancy. Findings suggest the direct method may support maternal self-report of substance use in universal prenatal screening. Additional study with diverse populations and clinical settings is needed.

The Assessment and Impact of Careless Responding in Routine Outcome Monitoring within Mental Health Care

Careless responding by mental health patients on self-report assessments is rarely investigated in routine care despite the potential for serious consequences such as faulty clinical decisions. We investigated validity indices most appropriate for detecting careless responding in routine outcome monitoring (ROM) in mental health-care. First, we reviewed indices proposed in previous research for their suitability in ROM. Next, we evaluated six selected indices using data of the Brief Symptom Inventory and the Mood and Anxiety Symptom Questionnaire from 3,483 outpatients. Simulations showed that for typical ROM scales the index, Mahalanobis distance, and inter-item standard deviation may be too strongly confounded with the latent trait value to compare careless responding across patients with different symptom severity. Application of two different classification methods to the validity indices did not converge in similar prevalence estimates of careless responding. Finally, results suggest that careless responding does not have a substantial biasing effect on scale-score statistics. We recommend the person-fit index to screen for random careless responding in large ROM data sets. However, additional research should further investigate methods for detecting repetitive responding in typical ROM data and assess whether there are specific circumstances in which simpler validity statistics or direct screening methods perform similarly as the index.

Direct screening method to assess antimicrobial behavior of untreated wood

The objective of the present study was to develop a simple and direct screening method to assess antimicrobial activities of wood material. Wood samples consisted of sawdust and wood discs cut in transversal (RT) and tangential (LT) direction, from four different wood species which were tested against four important bacteria in hospital hygiene. Area of inhibition was observed around sawdust embedded wells and wooden discs placed on agar. This study showed that wood disc diffusion can be used to easily and rapidly screen antimicrobial properties of untreated wood, while sawdust diffusion needs further work to do this.

Enzyme immobilized on polyamidoamine-coated magnetic microspheres for α-glucosidase inhibitors screening from Radix Paeoniae Rubra extracts accompanied with molecular modeling

In this study, a method for direct screening and identification of α-glucosidase inhibitors (AGIs) from extracts of natural products was established based on polyamidoamine (PAMAM) coated magnetic microspheres. A facile route to synthesize the magnetic PAMAM was employed and α-glucosidase was successfully covalently attached to its surface through cross linking of glutaraldehyde. Using the enzyme-loaded magnetic microspheres, potential inhibitors were fished out from crude extracts directly, followed by structure confirmation. The inhibitory activities of the screened components were further investigated by the enzyme-loaded magnetic microspheres. The Fe3O4 @PAMAM@α-Glu microspheres displayed favorable dispersibility, fast magnetic separation, large enzyme binding amount (42.9 μg•mg−1) and high enzyme activity. Moreover, the α-glucosidase on the surface of PAMAM coating maintained high storage stability and remarkable reusability. Taking advantage of specific interaction of the α-glucosidase with AGIs, the materials could selectively capture a known AGI (+)-catechin under the interference of an inactive compound salicylic acid, with a binding capacity as high as 15.4%. Additionally, using the Fe3O4 @PAMAM@α-Glu microspheres in the inhibition assay, the enzymatic reaction could be stopped by magnetic separation instead of the traditional addition of Na2CO3 solution, which not only eliminated the disturbance of termination reagent to the results, but also reused the immobilized α-glucosidase. The screening and inhibitory activity verification of potential ligands in Radix Paeoniae Rubra (“Chi-shao” in Chinese) extracts were achieved by using Fe3O4 @PAMAM@α-Glu microspheres, demonstrating practical applicability of our method. Therefore, the magnetic PAMAM-based screening approach could be a feasible and alternative strategy for discovering enzyme inhibitors from natural product extracts.

Multimode Optimization Research on a Multiport Magnetic Planetary Gear Permanent Magnet Machine for Hybrid Electric Vehicles

In this paper, by incorporating the new concept of the noncontact magnetic planetary gear into a permanent magnet brushless machine, a new multiport magnetic-planetary-gear permanent magnet machine is proposed. To achieve flexible combination and split of the power and torque among different electrical or mechanical ports in the machine, a multiobjective optimization design method considering multioperational modes is proposed and investigated. To obtain high optimization design efficiency, the whole optimization is divided into three steps based on various driving modes. In each step, the multobjective genetic algorithm, response surface method, and direct screening method are utilized, respectively. Moreover, the basic no-load characteristics and torque performances of the proposed machine are also studied. Finally, a prototype machine based on the optimized structure dimensions is manufactured and tested. Both the theoretical and experimental analysis verifies the effectiveness of the investigated machine and the proposed method.

Rapid Colorimetric Assay for Detection of Listeria monocytogenes in Food Samples Using LAMP Formation of DNA Concatemers and Gold Nanoparticle-DNA Probe Complex.

Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL−1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity, and accuracy were 100, 90.20, and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

A Novel Method for Anti-HLA Antibody Detection Using Personalized Peptide Arrays.

BackgroundHLA mismatches are the primary cause of alloantibody-mediated rejection (AMR) in organ transplantation. To delineate antigenic and immunogenic potentials among individual HLA mismatches, information regarding antibody specificity at the epitope level, instead of the allelic level, is needed.MethodsThis study explores a direct screening method for HLA linear epitopes in kidney transplant patients. We custom synthesized a large panel of 15-residue HLA peptides in an array format and measured alloantibody reactivity to these peptides from the sera of post and/or pretransplant patients. Two design concepts for the arrays were followed: a standard array of a fixed panel of peptides or personalized arrays. The standard array contains 420 peptides derived from a predetermined set of HLA-DQ allelic antigens based on templates also used in the single-antigen beads assay.ResultsThe array detected distinct antiserum patterns among transplant subjects and revealed epitope levels of specificity largely in accordance with the single-antigen results. Two personalized arrays that each included donor-derived peptides of HLA-A, -B, -C, -DQ, and -DR sequences were separately designed for 2 transplant subjects. The personalized arrays detected de novo antibodies following transplantation. The new method also showed superior sensitivity to a single-antigen assay in one of the cases whose pathological diagnosis of AMR occurred before single-antigen assay could detect antibodies.ConclusionsThis pilot study proved the feasibility of using personalized peptide arrays to achieve detection of alloantibodies for linear HLA epitopes associated with distinct donor-recipient mismatches. Single or multiple reactive epitopes may occur on an individual HLA molecule, and donor-specific HLA-DQ-reactivity among 5 kidney transplant subjects revealed patterns of shared epitopes.

The Potential of Raman Spectroscopy for the Classification of Fish Fillets

Since fishery products represent an important and globally growing food resource, there is also an increased incidence rate of intentional mislabelling of fish products and restaurant frauds globally. In the present study, Raman spectroscopy, as a fast and non-invasive technique, was applied using a laser at a wavelength of 532 nm for the classification of deep frozen fish fillets. Without any preparation, muscle tissue of 12 fish types was analysed with a Raman device, and according to a hierarchical cluster analysis of their spectra, three groups were identified: (1) the carotenoid group, fish from salmonid family; (2) the freshwater group, fish that was reared in fresh or brackish water; and (3) the saltwater group, fish reared in saltwater. Thus, it is demonstrated that Raman spectroscopy can be used as a direct, non-expensive and fast screening method before proceeding with standard methods for the identification of fish fillets.