slowly growing natural populations. Various approaches have been adopted in order to improve the sensitivity. These have included the use of multiple probes labelled with a single fluor (Lee et al. 1993); or labelled with multiple fluors (Trebesius et al. 1994) and enzyme-linked probes or detection systems that allow signal amplification (Lebaron et al. 1997, Schonhuber et al. 1999). The latter indirect approach not only has the potential for signal amplification, but may also be used in natural samples showing high levels of autofluorescence. Any thorough identification method has to include positive and negative controls. False-positive results may either be caused by cells emitting autofluorescence upon excitation or by nonspecific binding of the probe to nontarget cells. Samples should therefore be checked for autofluorescence before hybridization and a negative control with a fluorescent oligonucleotide not complementary to rRNA has to be applied to check for sequence-independent nonspecific binding. Such non-specific binding may be due to interaction of the dye compound of the probe with hydrophobic cell components. Failures to detect cells containing target sequences (false-negatives) may originate from cells with either low cellular ribosome content or limited permeability of the cell periphery for the fluorescent probe (Manz et al. 1992). With the rapidly expanding database of 16S rRNA sequences, the problem of probe specificity has become more apparent and the design of probes is becoming increasingly difficult. These problems are also applicable to PCR and other oligonucleotide-dependent techniques. The problem of probe specificity may be overcome by using multiple specific oligonucleotide probes targeting different sites on the rRNA molecule and labelled with different fluorochromes. While a single oligonucleotide target sequence may be found in a number of related taxa, the probability that target sites for three designed oligonucleotides are found in a nontarget organism is, however, much reduced.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1450-1450 ◽  
Author(s):  
Julien Guy ◽  
Iléana Antony-Debré ◽  
Isabelle Arnoux ◽  
Chantal Fossat ◽  
Emmanuel Benayoun ◽  
...  

Abstract Abstract 1450 Background: The WHO 2008 classification emphasizes the role of myeloperoxidase (MPO) detection as the only requirement for assigning myeloid lineage to a blast population, notably for the diagnosis of acute leukemia of ambiguous lineage. WHO highlights flow cytometry (FCM) as the preferred method for MPO detection and EGIL proposed a 10% cut-off for FCM but 3% for cytochemistry. Here we performed a reevaluation of the MPO positivity threshold by FCM comparing as background reference isotype controls or the autofluorescence of residual normal lymphocytes. Methods: A multicenter retrospective trial was set-up which compared retrospectively 128 acute lymphoblastic leukemias (ALL) and 75 acute myeloid leukemias without maturation (AML1), defined as MPO negative or positive by benzidine-based cytochemistry. Blasts were considered MPO positive by flow cytometry when their mean fluorescence intensity exceeded that of blast cells incubated with an isotype control or that of residual lymphocytes in the MPO-stained sample. In order to homogenize the interpretation of MPO staining, five various cut-offs were assessed for both negative controls, respectively 2%, 1%, 0.75%, 0.5% and 0.25%. Besides, as Ratio Fluorescence Intensity (RFI) may be a useful parameter to analyze staining for markers with unimodal distribution, we evaluated the RFI of blast cells MPO fluorescence relative to both controls, comparing mean and median intensities. Results: The harmonized method that was developed to interpret MPO staining by FCM between 4 French centers can be applied regardless of antibodies, permeabilization reagents or instruments used. For both negative controls, the 1% cut-off provided the best discrimination by ROC curve, and was used to assess the percentage of stained blasts in the 203 cases. The EGIL 10% threshold of stained blasts to discriminate between ALL and AML, using the isotype control to assess positivity, provided 100% sensitivity and 85.4% specificity, the optimal threshold being 13% (sensitivity 95.1%, specificity 91.7%). Residual normal lymphocytes proved to be an advantageous alternative reference, a threshold of 28% yielding improved 97.4% sensitivity and 96.1% specificity (Figure). The correlation between both methods was excellent, yet the percentage of positive blasts was higher using lymphocytes as control compared to isotype control. Using the RFI method, the isotype control appeared as the more relevant negative control to discriminate ALL against AML1 with a threshold of 3.42. However, with 90% sensivity and 95,83% specificity, the RFI method was less performing than percentages in this study. Finally, we assessed the relevance of this analysis method and positivity thresholds on 18 AMLs with minimal differentiation (AML0), MPO-negative by definition in cytochemistry, yet liable to be positive in FCM. Interestingly, with these new appropriate thresholds, MPO staining was positive for 10 of 18 AML0 when using lymphocytes as negative controls and only 3 of 17 cases when using an isotype control. Conclusion: i) MPO detection by flow cytometry can be interpreted indifferently of the negative control used if an appropriate threshold is applied; ii) Our analysis method of MPO expression is relevant for ALL and AML discrimination and can be useful for AML0 or mixed phenotype acute leukemia assessment, especially using residual lymphocytes as reference, since the latter bypass the higher non-specific binding of isotype controls on blast cells. Disclosures: No relevant conflicts of interest to declare.


2013 ◽  
Vol 18 (1) ◽  
pp. 86-93
Author(s):  
Gustavo Antônio Martins Brandão ◽  
Rafael Menezes Simas ◽  
Leandro Moreira de Almeida ◽  
Juliana Melo da Silva ◽  
Marcelo de Castro Meneghim ◽  
...  

OBJECTIVE: To evaluate the in vitro ionic degradation and slot base corrosion of metallic brackets subjected to brushing with dentifrices, through analysis of chemical composition by Energy Dispersive Spectroscopy (EDS) and qualitative analysis by Scanning Electron Microscopy (SEM). METHODS: Thirty eight brackets were selected and randomly divided into four experimental groups (n = 7). Two groups (n = 5) worked as positive and negative controls. Simulated orthodontic braces were assembled using 0.019 x 0.025-in stainless steel wires and elastomeric rings. The groups were divided according to surface treatment: G1 (Máxima Proteção Anticáries®); G2 (Total 12®); G3 (Sensitive®); G4 (Branqueador®); Positive control (artificial saliva) and Negative control (no treatment). Twenty eight brushing cycles were performed and evaluations were made before (T0) and after (T1) experiment. RESULTS: The Wilcoxon test showed no difference in ionic concentrations of titanium (Ti), chromium (Cr), iron (Fe) and nickel (Ni) between groups. G2 presented significant reduction (p < 0.05) in the concentration of aluminium ion (Al). Groups G3 and G4 presented significant increase (p < 0.05) in the concentration of aluminium ion. The SEM analysis showed increased characteristics indicative of corrosion on groups G2, G3 and G4. CONCLUSION: The EDS analysis revealed that control groups and G1 did not suffer alterations on the chemical composition. G2 presented degradation in the amount of Al ion. G3 and G4 suffered increase in the concentration of Al. The immersion in artificial saliva and the dentifrice Máxima Proteção Anticáries® did not alter the surface polishing. The dentifrices Total 12®, Sensitive® and Branqueador® altered the surface polishing.


Author(s):  
Nur Hanina Izzati Khairol Mokhtar ◽  
Ainulkhir Hussin ◽  
Aidil Abdul Hamid ◽  
Shahrul Hisham Zainal Ariffin ◽  
Muhammad Ashraf Shahidan

Aims: We aimed to develop a high-throughput lectin assay with minimized background signals to investigate the interactions of lectins and sialic acid glycans, focusing on prostate-specific antigen (PSA). Background: High background signals resulting from nonspecific binding are a significant concern for microtiter plate-based enzyme-linked lectin sorbent assays (ELLSAs), as they can mask specific binding signals and cause false-positive results. Methods: In this study, we constructed an ELLSA based on different washing step parameters, including the number of washing cycles, NaCl and Tween-20 concentrations, and the type of blocking agent and evaluated the effects on both specific and nonspecific binding signals. Furthermore, we performed a PSA binding assay using the optimized ELLSA. Results: The optimal washing parameters based on the highest specific binding signal proposed four cycles of washing steps using a washing buffer containing a high salt concentration (0.5 M NaCl) and mild detergent (0.05% Tween-20). The utilization of the optimized washing parameters in this assay was shown to be sufficient to obtain the optimal binding signals without the use of any blocking agent. Binding assays performed using the optimized ELLSA revealed that the glycan of the PSA sample used in this study mainly consists of terminal α2,6-linked sialic acid, as strongly recognized by Sambucus nigra agglutinin (SNA) with a KD value of 12.38 nM. Conclusion: The ELLSA reported in this study provides a simple yet sensitive assay for sialic acid linkage recognition.


BioTechniques ◽  
2001 ◽  
Vol 30 (6) ◽  
pp. 1268-1272
Author(s):  
C.M. Lisle ◽  
S. Bortolin ◽  
A.S. Benight ◽  
R.A. Janeczko ◽  
R.L. Zastawny

2019 ◽  
Vol 4 (1) ◽  
pp. 16
Author(s):  
Maria Ulfah ◽  
Revika Rachmaniar ◽  
Egi MR. Sudrajat ◽  
Rida W. Fadla ◽  
Hary S. Pinuji

Anredera cordifolia or binahong is one of the Indonesian medicinal plants that is used to treat peptic ulcer. The purpose of this study was to evaluate the effect of the addition of sodium carboxymethylcellulose (CMC) and sorbitol on anti-peptic ulcer activity of A. cordifolia leaves extracts in male Wistar rats. The plants were extracted using decoction method and freeze dried. Three liquid formulas were used i.e., i) a combination of sodium CMC and sorbitol; ii) only sorbitol; iii) extract only. The rats were divided into 6 groups, i.e., positive control (sucralfate 35 mg/kg body weight); negative control (80% ethanol); normal control; and 3 formulas. After the administration of the liquid formula, all groups, except normal control, were given 80% ethanol (l5 ml/kg body weight) to induce peptic ulcer. Antipeptic ulcer activity was evaluated using direct observation on rats gastric mucosa, and histopathology assessment. The result showed that the strongest anti-peptic ulcer  was shown by sorbitol only (96.95% inhibition),  followed by the combination of sodium CMC and sorbitol (92.68% inhibition). The formula which only contained extract showed only  31.70% inhibition.  Statistical analysis showed significant differences between formula 1 and 2 with negative controls. In conclusion, A. cordifolia leaves extract with the addition of sorbitol showed the strongest anti-peptic ulcer activity. Keyword: Anredera cordifolia, peptic ulcer, suspense, Wistar rat.


Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Brandon K Fornwalt ◽  
Takeshi Arita ◽  
Mohit Bhasin ◽  
George Voulgaris ◽  
John D Merlino ◽  
...  

Background- A recent study showed that the most commonly used Tissue Doppler imaging (TDI) parameters to diagnose left ventricular dyssynchrony agree only 50% of the time. Most of these parameters require calculation of the ``time-to-peak” myocardial velocity. This ``time-to-peak” based analysis utilizes only one of >100 data points collected per heart cycle. Methods- We developed and tested a new dyssynchrony parameter, cross-correlation delay (XCD), that utilizes all velocity data points from 3 consecutive beats (~420 points). We hypothesized that XCD would be superior to existing methods at diagnosing dyssynchrony. We tested XCD on 11 members of a positive control group (echocardiographic responders to cardiac resynchronization therapy) and 12 members of a negative control group (normal echocardiogram and 12-lead ECG). We compared XCD to septal-to-lateral delay in time-to-peak (SLD), maximum difference in the basal 2- or 4-chamber times-to-peak (MaxDiff) and standard deviation of the 12 basal and mid-wall times-to-peak (Ts-SD). Results- An XCD threshold of 31ms discriminated between positive and negative controls with 100% sensitivity and specificity (Figure 1 ). SLD, MaxDiff and Ts-SD showed sensitivities of 36, 55 and 100% and specificities of 50, 42 and 50%, respectively. ROC analysis showed XCD and Ts-SD were superior to SLD and MaxDiff in discriminating between positive and negative controls (p<0.01). XCD was the only parameter which decreased after resynchronization in the positive controls (from 160±88ms to 69±61ms, p=0.003). Conclusion- XCD is superior to existing parameters at discriminating patients with LV dyssynchrony from those with normal function. Figure 1. XCD shows the greatest discrimination between positive and negative controls. Dyssynchrony values for each positive control are shown as x’s and values for each negative control are shown as circels. Different dyssynchrony parameters are shown in each subplot (A-D). Threshold values to diagnose dyssynchrony are plotted as horizontal lines in each figure. Note that x’s above the threshold line represent false positives while circles below the threshold line represent false negatives.


2019 ◽  
Vol 79 (2) ◽  
pp. 209-219 ◽  
Author(s):  
Oliver Soutar ◽  
Freya Cohen ◽  
Richard Wall

Abstract Essential oils show promise as natural alternatives to synthetic tick repellents, but few studies have investigated their repellent efficacy in vivo or under field conditions. Here, blanket-drags and standardised walks were employed to evaluate tick acquisition by 1 m2 cotton blankets or cotton trousers, respectively, in woodland edge habitats of known high tick abundance. Blankets and trousers had been treated with one of 5% oregano, rosemary, spearmint or thyme oils, 20% DEET (N,N-diethyl-3-methylbenzamide) (positive control) or ethanol excipient-only (negative control). The number of ticks present on the blankets or trousers differed significantly between treatments: spearmint oil treatments resulted in significantly fewer ticks than the negative controls for both blankets and trousers and significantly fewer ticks were present on the oregano oil treated blankets. For ticks that did attach to the trousers, the rate of drop off within 3 min was significantly higher for trousers treated with spearmint oil or thyme oil than ethanol, oregano oil and rosemary oil. No reduction in repellence was detected over a 24 h period between treatment and testing. The results suggest that 5% oregano and spearmint oils exhibit potential as natural clothing repellents, with an effective equivalence to 20% DEET.


2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Haroon Ashraf ◽  
Kerri Font ◽  
Charles Powell ◽  
Michael Schurr

Objective. The aim of this study was to evaluate wound biomodification by assessing antimicrobial properties present within a human-derived composite amnion-chorion membrane (ACM). Methods. Membranes analyzed were the human-derived ACM BioXclude™ and the porcine-derived collagen membrane Bio-Gide®. Paper discs with and without tetracycline served as positive and negative controls, respectively. The same number of colony-forming units per milliliter for each bacterial species (Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Streptococcus oralis) was inoculated on each of the discs. Discs from each group were removed at 12 and 24 hours and sonicated to remove the bacteria off the membranes. A serial dilution was performed to quantify bacterial growth. Results. The ACM inhibited growth at all time points, with all bacterial strains, identical to the negative control tetracycline discs. The collagen membrane and positive controls did not inhibit growth of any of the bacterial species throughout the 24-hour study period. P<0.05 for microbial growth on ACM or negative control vs. either collagen membrane or positive control. Conclusion. ACM was proven to be as bactericidal as paper discs inoculated with tetracycline at its minimum bactericidal concentration. The ACM bactericidal property may be beneficial in the early wound healing process.


Jurnal Kimia ◽  
2020 ◽  
pp. 24
Author(s):  
R. A. I. K. Maharani ◽  
N. K. Cahyaningsih ◽  
M. D. Abimanyu ◽  
K. W. Astuti

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the treatment options for relieving pain. However, long-term use can trigger gastrointestinal bleeding. Therefore, alternative analgesics which have the same therapeutic effect with lower side effects are needed. Limau (Citrus amblycarpa) is an empirical drug for tingling and cramping. The aim of the study is to determine the analgesic activity of ethanol extract of C. amblycarpa fruit peel. The method used in testing analgesic activity is the Hot Plate method. The study was conducted by dividing 30 mice into 6 groups. The group given CMC-Na 1% was used as a negative control, the group given suspension of sodium diclofenac dose of 6.5 mg/kg of body weight was used as a positive control, and the group given suspension of ethanol extract of C. amblycarpa fruit peel with dose variations 100, 300 and 600 mg/kg of body weight. The test animals were placed on top of the Hot Plate with a temperature of 70°C at 30 minutes after giving suspension test and the response time of mice to heat was observed every 30 minutes for 3 hours with cut off time 15 second. Based on the test results, it can be concluded that the administration of ethanol extract of C. amblycarpa fruit peel with 100, 300 and 600 mg/kg of body weight gave analgesic activity on mice compared to the negative controls (CMC-Na 1%).   Keywords: C. amblycarpa, Fruit Peel, Analgesics, Hot Plate 


2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Brian E Hew ◽  
Ryuei Sato ◽  
Damiano Mauro ◽  
Ilko Stoytchev ◽  
Jesse B Owens

Abstract Safer and more efficient methods for directing therapeutic genes to specific sequences could increase the repertoire of treatable conditions. Many current approaches act passively, first initiating a double-stranded break, then relying on host repair to uptake donor DNA. Alternatively, we delivered an actively integrating transposase to the target sequence to initiate gene insertion. We fused the hyperactive piggyBac transposase to the highly specific, catalytically dead SpCas9-HF1 (dCas9) and designed guide RNAs (gRNAs) to the CCR5 safe harbor sequence. We introduced mutations to the native DNA-binding domain of piggyBac to reduce non-specific binding of the transposase and cause the fusion protein to favor binding by dCas9. This strategy enabled us, for the first time, to direct transposition to the genome using RNA. We showed that increasing the number of gRNAs improved targeting efficiency. Interestingly, over half of the recovered insertions were found at a single TTAA hotspot. We also found that the fusion increased the error rate at the genome-transposon junction. We isolated clonal cell lines containing a single insertion at CCR5 and demonstrated long-term expression from this locus. These vectors expand the utility of the piggyBac system for applications in targeted gene addition for biomedical research and gene therapy.


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