Simple Screening Method Research Articles

Hot-Spotting with Thermal Scanning: A Ligand- and Structure-Independent Assessment of Target Ligandability.

Evaluating the ligandability of a protein target is a key component when defining hit-finding strategies or when prioritize among drug targets. Computational as well as biophysical approaches based on nuclear magnetic resonance (NMR) fragment screening are powerful approaches but suffer from specific constraints that limit their usage. Here, we demonstrate the applicability of high-throughput thermal scanning (HTTS) as a simple and generic biophysical fragment screening method to reproduce assessments from NMR-based screening. By applying this method to a large set of proteins we can furthermore show that the assessment is predictive of the success of high-throughput screening (HTS). The few divergences for targets of low ligandability originate from the sensitivity differences of the orthogonal biophysical methods. We thus applied a new strategy making use of modulations in the solvent structure to improve assay sensitivity. This novel approach enables improved ligandability assessments in accordance with NMR-based assessments and more importantly positions the methodology as a valuable option for biophysical fragment screening.

Métodos de cribaje del deterioro cognitivo leve en atención primaria

Diagnosis of mild cognitive impairment (MCI) is always clinical and screening methods only indicate that the patient has a higher risk of this condition. In MCI, there is a slight decline in some cognitive abilities that does not affect activities of daily living and therefore does not produce social or occupational disability. The definitive diagnosis of MCI requires a considerable time investment that is very rarely possible to provide in primary care (PC) consultations. Hence the need for PC physicians to employ rapid and simple screening methods (brief cognitive assessment –BCA-) that allow objective identification of patients likely to have MCI in a few minutes. This article reviews the BCA tools that can truly be applied in less than 10 minutes. The phototest is a brief screening tool that is easy to use and interpret by physicians and is well accepted by patients. Consequently, it is one of the most useful tests in PC for screening of both MCI and dementia. In addition to BCA, instrumental activities of daily living scales should also be applied to differentiate MCI from dementia.

Evaluating the appropriateness of hospital doctors’ requests for pulmonary function tests beyond basic spirometry: results from a prospective observational study

ABSTRACTObjectives: Hospitalists request ‘complete’ pulmonary function tests (PFTs), typically comprising of spirometry, diffusion capacity of the lung for carbon monoxide (DLCO) and absolute lung volumes (ALVs), the results of which assist in the management of patients with respiratory disorders. Recently, concerns have been raised about over-requesting of ‘complete’ PFTs, but there is a paucity of information on the proportion of requests that can be considered clinically inappropriate. This study prospectively evaluated the ‘complete’ PFTs requested in a hospital service and assessed the impact of medical review of the requests.Methods: A six-month prospective study on requests to two teaching hospital PFT laboratories from non-respiratory doctors was undertaken. Requests at one laboratory underwent review by a respiratory doctor (‘intervention laboratory’) while requests at the second laboratory were not reviewed (‘control laboratory’). The appropriateness of requests was measured against pre-specified criteria.Results: PFT requests for 335 subjects were included in the study. In the intervention laboratory, 8 of 110 ALV and 122 of 134 DLCO requests fulfilled pre-specified criteria for appropriate test indications. Fewer ALV (7% vs. 100%, p < 0.001) and DLCO tests (91% vs. 100%, p = 0.031) could have been performed in the intervention laboratory compared to the control laboratory.Conclusion: A considerable proportion of ‘complete’ PFT requests from non-respiratory hospital doctors may be unwarranted. Using a simple screening method, the number of unnecessary PFTs could be reduced, resulting in substantial time and cost savings for hospital PFT laboratories.

Chemical composition and antioxidant, bactericidal, and matrix metalloproteinase inhibition activity of food-related plant

The present study aimed to investigate bio-function, focusing the on antioxidant, bactericidal, and matrix metalloproteinase (MMP) regulatory activity, of food-related plants. Bioassay-coupled chromatography and bioassay guiding, the rapid and simple screening method, was used to identify potential antioxidant and bactericidal components from fifty-five food-related plants. Lonicera japonica exhibited moderate xanthine oxidase (XO) inhibition and antioxidant activity. Rhodiola rosea, provoked strongly inhibition for tyrosinase, lipoxygenase (LOX), XO and MMP-2 activity. The organic fractions of Ligusticum sinense had a significant antimicrobial effect against two Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and two Gram-positive species (Staphylococcus aureus, and Bacillus subtilis). The chemical structures of MMP-2-inhibiting and antimicrobial components was further identified as 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranoside and ferulic acid, respectively, which were determined by 1H and 13C nuclear magnetic resonance (NMR) and mass spectrometry (MS). The results suggest that R. rosea and L. sinense could be a useful natural food preservatives and potential nutraceutical ingredients.

HbA1c screening for new onset diabetes following acute coronary syndrome: is it a worthwhile test in clinical practice?

BackgroundDiabetes and pre-diabetes are prevalent in acute coronary syndrome (ACS) and relate to adverse outcomes. This study used HbA1c to screen for degrees of glucose intolerance amongst patients without known diabetes presenting with ACS.MethodsOver a 1-year period (June 2014–2015) consecutive patients admitted to a single centre cardiology unit with an initial diagnosis of ACS without prior diabetes diagnosis were electronically referred to our diabetes team. Patients were screened for the presence of diabetes by use of an initial HbA1c blood test on day 2 or 3 post admission. If abnormal (≥42 mmol/mol; ≥6.0%), patients were invited for a repeat HbA1c at 2 weeks, and if an intermediate result (42–47 mmol/mol; 6.0–6.4%), for an oral glucose tolerance test (OGTT) at 3 months. Patients were diagnosed with Type 2 diabetes if the repeat HbA1c result was high (≥48 mmol/mol; ≥6.5%) or the OGTT at 3 months confirmed the diagnosis. Other data collected included baseline demographics, risk factors and any history of cardiovascular disease. All patients with ACS were stratified according to the diagnosis and subsequent management.ResultsWe screened 399 patients in total. The mean age was 65 ± 14 years, 268 (67%) were men, 290 (73%) were Caucasian, 95 (24%) were South Asian and 14 (4%) were Afro-Caribbean ethnicity. Of all patients, 57 (14.3%) were diagnosed as pre-diabetes and 43 (10.8%) newly diagnosed diabetes. During the study 28 (7%) patients could not be classified; 6 (1.5%) patients died during the study and 22 (5.5%) patients were missing either initial or repeat HbA1c and were subsequently lost to follow up. Of the baseline variables assessed, there were significantly more patients of South Asian ethnicity in the diabetes group compared to the normal group (42 vs 20%; p = 0.003). There was no difference in detection rates in patients with more severe ACS requiring percutaneous or cardiac surgical intervention.ConclusionsThe use of a simple HbA1c screening method in clinical practice can detect new onset diabetes in approximately 1 in 10 high-risk post ACS patients.

Elemental analysis of tree leaves by total reflection X-ray fluorescence: New approaches for air quality monitoring

This work shows that total reflection X-ray fluorescence (TXRF) is a fast, easy and successful tool to determine the presence of potentially toxic elements in atmospheric aerosols precipitations on tree leaves. Leaves are collected in eleven parks of different geographical areas of the Brescia city, Northern Italy, for environmental monitoring purposes. Two sample preparation procedures are considered: microwave acid digestion and the novel SMART STORE method for direct analysis. The latter consists in sandwiching a portion of the leaf between two organic foils, metals free, to save it from contamination and material loss. Mass composition of macro, micro and trace elements is calculated for digested samples, while relative elemental amount are obtained from direct analysis. Washed and unwashed leaves have a different composition in terms of trace elements. Differentiation occurs according to Fe, Pb and Cu contributions, considered as most representative of air depositions, and probably related to anthropogenic sources. Direct analysis is more representative of the composition of air precipitations. Advantages and drawbacks of the presented methods of sample preparation and TXRF analysis are discussed. Results demonstrate that TXRF allows to perform accurate and precise quantitative analysis of digested samples. In addition, direct analysis of leaves may be used as a fast and simple method for screening in the nanograms range.

Abdominal aortic calcification on a plain X-ray and the relation with significant coronary artery disease in asymptomatic chronic dialysis patients

BackgroundCoronary artery disease (CAD) is common in asymptomatic chronic dialysis patients and plays an important role in their poor survival. Early identification of these high-risk patients could improve treatment and reduce mortality. Abdominal aortic calcification (AAC) has previously been associated with CAD in autopsy studies. Since the AAC can be quantified easily using a lateral lumbar X-ray we hypothesized that the extent of AAC as assessed on a lateral lumbar X-ray might be predictive of the presence of significant CAD in dialysis patients.MethodsAll patients currently enrolled in the ICD2 trial without a history of CABG or a PCI with stent implantation were included in this study. All patients underwent CT-angiography (CTA) and a lateral X-ray of the abdomen. AAC on X-ray was quantified using a previously validated scoring system whereupon the association between AAC and the presence of significant CAD was assessed.ResultsA total of 90 patients were included in this study (71% male, 67 ± 7 years old). Forty-six patients were found to have significant CAD. AAC-score was significantly higher in patients with CAD (10.1 ± 4.9 vs 6.3 ± 4.6 (p < 0.05). Multivariate regression analysis revealed that AAC score is an independent predictor for the presence of CAD with a 1,2 fold higher risk per point increase (p < 0.01). The AAC score has a sensitivity of 85% and a specificity of 57% for the presence of significant CAD.ConclusionThis study shows that abdominal aortic calcification as assessed on a lateral lumbar X-ray is predictive for the presence of significant coronary artery disease in asymptomatic dialysis patients. This simple, non-invasive and cheap screening method could contribute to early identification of patients eligible for further screening of CAD.Trial registrationNTR948, registered 10-4-2007 ; ISRCTN20479861, registered 2-5-2007

Rise in Haemophilus influenzae With Reduced Quinolone Susceptibility and Development of a Simple Screening Method.

β-Lactamase-nonproducing ampicillin-resistant Haemophilus influenzae are prevalent in Japan. Resistance has increased as a consequence of the expanded use of antimicrobial agents, raising concerns about the rise of multidrug (macrolide and fluoroquinolone)-resistant H. influenzae. In this study, we investigated susceptibility to fluoroquinolones in H. influenzae clinical isolates from 2013 to 2014 and identified the amino acid substitutions in quinolone resistance-determining regions of gyrA and parC. All isolates (n = 145) were susceptible to fluoroquinolones; however, some showed reduced susceptibility. The minimum inhibitory concentration of levofloxacin for these strains was 0.063-0.5 µg/mL, and the strains harbored the amino acid substitution S84L in GyrA. Such strains have seen a significant increase. Importantly, all mutants from 2014 were isolated from pediatric patients. In addition, we developed a simple polymerase chain reaction-based screening method for detecting isolates with reduced fluoroquinolone susceptibility. The mutation in GyrA is important as a first step in the development of fluoroquinolone resistance. Hence, detection of reduced susceptible strains may influence the choice of antimicrobial treatment.

Simultaneous determination of dyes of different classes in aquaculture products and spices using HPLC–high-resolution quadrupole time-of-flight mass spectrometry

A simple method of sample preparation, rapid screening, and determination of dyes—veterinary drugs and illegally used dyes (coloring product counterfeiters)—in spices by HPLC–high-resolution quadrupole time-of-flight mass spectrometry is proposed. Sample preparation involves the extraction of analytes from a solid matrix with acetonitrile containing 0.1% of formic acid, twofold dilution of the extract with deionized water, filtration, and chromatographic separation. The limits of detection were 0.01–0.4 ng/g. The procedure of screening and determination of dyes in foods includes the identification of dyes by an accurate monoisotopic mass of the ion (m/z), retention time (t R), and coincidence of the isotopic distribution (mSigma), and, if dyes are detected, their determination by the standard addition method. The relative standard deviation of the results does not exceed 15%. The duration of the analysis is 1–2 h.

Esterase activity inspired selection and characterization of zearalenone degrading bacteria Bacillus pumilus ES-21

Zearalenone (ZEN), mainly produced by Fusarium species, is an estrogenic mycotoxin which causes reproductive disorders in livestock. In this study, we described a simple and rapid method for screening of ZEN-degrading bacteria by esterase activity assay. Soil bacteria strains were first tested for their esterase activities, then active strains were further evaluated for their ZEN-degrading potentials. A bacterial strain named Bacillus pumilus ES-21 was detected to be able to eliminate ZEN in the culture medium. ZEN degradation conditions were optimized through response surface methodology and the result showed that the degradation rate of ZEN by Bacillus pumilus ES-21 was up to 95.7% at the ZEN concentration of 17.9 μg/ml within 24 h. One of the degradation product was proposed to be 1-(3,5-dihydroxyphenyl)-6′-hydroxy-l′-undecen-l0′-one according to LC-TOF-MS/MS analysis. This study provided a strategy for the isolation of ZEN degrading microbes and a promising degrading strain.

A simple screening method for analysis of cell adhesive strength cultured on zein microspheres

Cell adhesion plays an important role in the microcarrier culture. Therefore, it is necessary to find a simple method to evaluate the cell adhesive strength. By the glycerol method, we prepared zein microcarriers. Both microcarrier surface morphology and cells on the microcarriers could be observed clearly when the microcarriers and the cells were stained by acridine orange (AO). Pre-treatment of soaking and sterilizing processes was necessary for cell culture, but it did not alter the morphology of zein microcarriers. Three kinds of cells were cultured on zein microcarriers for 24 h. Vero cells showed the strongest adhesion, while L929 cells were easily detached from the microcarriers after a simple beating operation. The proliferation curve of Vero cells cultured on the microcarriers displayed no difference with that on Cytodex 1.

Simple screening method for copy number variations associated with physical features.

Recent studies of copy number variations (CNVs) associated with physical features, such as body mass index, body height or bone length, have suggested that such CNVs could serve as markers in forensic cases involving unidentified individuals. However, the process of cataloging CNVs has been slow because of the cumbersome nature and low reliability of the procedures involved. Here we describe a simple quantitative real-time PCR (Q-PCR) method for screening of medicolegally useful CNVs, which does not require reference DNA with known copy number. The first step is to prepare a chimeric plasmid vector including one copy each of the single-copy gene-specific sequence as the internal standard, and the target CNV-specific sequence. To assess the validity of this new method, we analyzed CNVs in the LTBP1 and ETV6 gene regions, both of which are candidate CNVs associated with body height. The PCR efficiencies for the single-copy (reference) gene and the target CNV were similar, indicating that quantitation was reliable. Furthermore, simulated analysis of the LTBP1 CNV using mock samples prepared by mixing vectors in varying proportions showed that this analytical method allowed correct determination of the LTBP1 copy number. These results demonstrated that our simple method has considerable potential for screening of trait-related CNVs that would be useful for forensic casework.

Sports and martial arts activities for public health purposes: the musician’s risk profiles and exercise-based health care as a model

The common saying that sports promote good health is in line with main results of sports medicine, but conflicting with the high amount of sports injuries and sports-related diseases. This paper contributes to the discussion of the pros and cons of sports, particularly of the martial arts. It intends to shed light on the benefits of sports in the general public and to explore the enormous and yet unexploited potential of individually tailored sport activities for public health. Comparative analyses and meta-synthesis of empirical studies on the health benefits of sports. Musicians with very heterogeneous work-related risk profiles serve as a model for inductive generalisation. For health promotion and to avoid adverse outcomes, sport activities must take one’s physical status and risk profiles into account and refer to well-adjusted training zones. This encompasses musculoskeletal and biomotor factors, behavioural means to rebalance the nervous system, cardiorespiratory fitness, mental health and psychological benefits, and active pain management and pain relief. Sports organisations provide a wide spectrum of facilities for sports-oriented lifestyles; however, informal experience and relevant publications let us assume that they rather do not explore how to optimise preventative and health-related benefits, except for training with personal coaches. And yet, relatively simple screening methods and individually adjusted intensities, modes, and frequencies of trainings can enhance benefits and greatly contribute to public health, which would, however, require a stronger health awareness of providers and specific education of sports coaches.

A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot

The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting constructs which can also be useful to screen KO and KI mutant mice or cell lines including those generated by CRISPR/Cas9.

Novel HPLC-Based Screening Method to Assess Developability of Antibody-Like Molecules.

The discovery of antibodies that bind to targets with high affinity is now a routine exercise. However, it is still challenging to screen for candidates that, in addition to having excellent biological properties, also have optimal biophysical characteristics. Here, we describe a simple HPLC-based screening method to assess for developability factors earlier in the discovery process.

Considerations for Sample Preparation Using Size-Exclusion Chromatography for Home and Synchrotron Sources.

The success of a SAXS experiment for structural investigations depends on two precise measurements, the sample and the buffer background. Buffer matching between the sample and background can be achieved using dialysis methods but in biological SAXS of monodisperse systems, sample preparation is routinely being performed with size exclusion chromatography (SEC). SEC is the most reliable method for SAXS sample preparation as the method not only purifies the sample for SAXS but also almost guarantees ideal buffer matching. Here, I will highlight the use of SEC for SAXS sample preparation and demonstrate using example proteins that SEC purification does not always provide for ideal samples. Scrutiny of the SEC elution peak using quasi-elastic and multi-angle light scattering techniques can reveal hidden features (heterogeneity) of the sample that should be considered during SAXS data analysis. In some cases, sample heterogeneity can be controlled using a small molecule additive and I outline a simple additive screening method for sample preparation.

Nanostructured Materials Detect Dopamine in Biological Fluids

Dopamine, 2-(3, 4-dihydroxyphenyl)-ethylamine, is an organic base part of the catecholamine and phenethylamine families. Abnormal levels of dopamine generate diseases such as Parkinson's disease, schizophrenia, Attention Deficit Hyperactivity Disorder (ADHD), addiction. Therefore, for early detection of these diseases, fast, simple, reliable and sensitive screening methods are needed. We proposed three reliable stochastic microsensors based on Au-TiO2-graphene and Ag-TiO2-graphene composites modified with basic porphyrine, and azulenes, able to perform both qualitative and quantitative analysis for detection of dopamine in human whole blood and urine samples. The proposed sensors had shown low limits of quantification (0.1 pmol/L) and high sensitivities. The recoveries of dopamine in whole blood and urine samples were higher than 98.00%, with RSD(%) values lower than 1.00%.

Development of a Method to Produce Chromosome Lacking Lines (CLLs) in &amp;lt;i&amp;gt;Nicotiana tabacum&amp;lt;/i&amp;gt; L. “Red Russian”

Monosomic lines of Nicotiana tabacum are helpful to confirm the location of genes on specific chromosomes. In the cross N. nudicaulis and N. tabacum, hybrid seedlings express lethal symptoms, which are controlled by the S subgenome of N. tabacum. To identify the responsible chromosome, we needed to produce chromosome lacking lines (CLLs) of N. tabacum L. “Red Russian” and use them to cross with N. nudicaulis. From a cross of (N. tabacum × N. tomentosiformis) × N. tabacum, 380 BC1 individuals were obtained. Using a Haplo-Q line (a monosomic line lacking the single linkage group 11) and N. tabacum, we found that qPCR is a simple and reliable screening method for CLLs of N. tabacum. The marker PT30342 is located on linkage group 11, and the -Ct value (Ct Actin - Ct PT30342) was 2.0 for a disomic line and was 1.097 for a Haplo-Q line. By the use of flow cytometry, qPCR and chromosome counting together as a screening method, we identified 6 CLLs lacking 2 to 6 chromosomes. Compared with conventional methods, our method is a rapid technique for making and screening CLLs ofthe S or S/T subgenome of N. tabacum. Further, these CLLs will be useful to identify the location of two or more factors on chromosomes controlling a variety of genetic problems affecting breeding. Here, we only made CLLs of the S or S/T subgenome of N. tabacum. We will use the method established in this study to produce CLLs of the T subgenome of N. tabacum, and gather a full set of CLLs of N. tabacum. qPCR could also be applied to the identification of chromosome aberrations in other plants.

Pinning Down the Mechanism of Transport: Probing the Structure and Function of Transporters Using Cysteine Cross-Linking and Site-Specific Labeling.

Transporters are crucial in a number of cellular functions, including nutrient uptake, cell signaling, and toxin removal. As such, transporters are important drug targets and their malfunction is related to several disease states. Treating transporter-related diseases and developing pharmaceuticals targeting transporters require an understanding of their mechanism. Achieving a detailed understanding of transporter mechanism depends on an integrative approach involving structural and computational approaches as well as biochemical and biophysical methodologies. Many of the elements of this toolkit exploit the unique and useful chemistry of the amino acid cysteine. Cysteine offers researchers a specific molecular handle with which to precisely modify the protein, which enables the introduction of biophysical probes to assess ligand binding and the conformational ensemble of the transporter, to topologically map transporters and validate structural models, and to assess essential conformational changes. Here, we summarize several uses for cysteine-based labeling and cross-linking in the pursuit of understanding transporter mechanism, the common cysteine-reactive reagents used to probe transporter mechanism, and strategies that can be used to confirm cysteine cross-link formation. In addition, we provide methodological considerations for each approach and a detailed procedure for the cross-linking of introduced cysteines, and a simple screening method to assess cross-link formation.

Tri-primer-florescence PCR-Sanger sequencing method for screening of full and pre-mutations of FMR1 gene

To screen for CGG repeats in the FMR1 gene among patients with fragile X syndrome and carriers of pre-mutations. Potential full and pre-mutations of the FMR1 gene were detected with a Tri-primer-florescence PCR-Sanger sequencing method. The results were validated with positive and negative controls. All positive and negative controls were confirmed. A male patient was found to have > 200 CGG repeats (full mutation). For a pregnant women who was heterozygous for 35/115 CGG repeats, > 200 CGG repeats were also found with amniotic fluid sample from her fetus who was a male. The result was confirmed by following selective abortion with informed consent. Tri-primer-florescence PCR-Sanger sequencing is a simple, effective and reliable method for routine screening of patients/carriers with full/pre-mutations of the FMR1 gene in the population.