Conformational analysis of champedak galactose-binding (CGB) lectin under different urea concentrations was studied in phosphate-buffered saline (pH 7.2) using far-ultraviolet circular dichroism (far-UV CD), tryptophan (Trp) fluorescence and ANS fluorescence. In all cases, CGB lectin displayed a two-step, three-state transition. The first transition (from the native state to the intermediate state) started at ∼2.0 M urea and ended at ∼4.5 M urea, while the second transition (from the intermediate state to the completely denatured state) was characterized by the start- and end-points at ∼5.75 M and ∼7.5 M urea, respectively, when analyzed by the emission maximum of Trp fluorescence. A marked increase in the Trp fluorescence, ANS fluorescence and -CD values at 218 nm (-CD218 nm) represented the first transition, whereas a decrease in these parameters defined the second transition. On the other hand, emission maximum of the Trp fluorescence showed a continuous increase throughout the urea concentration range. Transformation of tetramer into monomer represented the first transition, whereas the second transition reflected the unfolding of monomer. Far-UV CD, Trp fluorescence and ANS fluorescence spectra were used to characterize the native, the intermediate and the completely denatured states of CGB lectin, obtained at 0.0 M, 5.0 M and 9.0 M urea, respectively. The intermediate state was characterized by the presence of higher secondary structures, increased ANS binding as well as increased Trp fluorescence intensity. A gradual decrease in the hemagglutination activity of CGB lectin was observed with increasing urea concentrations, showing complete loss at 4.0 M urea.
The interaction of pinostrobin (PS), a multitherapeutic agent with serum albumins of various mammalian species namely, goat, bovine, human, porcine, rabbit, sheep and dog was investigated using fluorescence quench titration and competitive drug displacement experiments. Analysis of the intrinsic fluorescence quenching data revealed values of the association constant, K(a) in the range of 1.49 - 6.12 × 10(4) M(-1), with 1:1 binding stoichiometry. Based on the PS-albumin binding characteristics, these albumins were grouped into two classes. Ligand displacement studies using warfarin as the site I marker ligand correlated well with the binding data. Albumins from goat and bovine were found to be closely similar to human albumin on the basis of PS binding characteristics.
We present barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel insitu analyses (BOLORAMIS), a reverse transcription-free method for spatially-resolved, targeted, in situ RNA identification of single or multiple targets. BOLORAMIS was demonstrated on a range of cell types and human cerebral organoids. Singleplex experiments to detect coding and non-coding RNAs in human iPSCs showed a stem-cell signature pattern. Specificity of BOLORAMIS was found to be 92% as illustrated by a clear distinction between human and mouse housekeeping genes in a co-culture system, as well as by recapitulation of subcellular localization of lncRNA MALAT1. Sensitivity of BOLORAMIS was quantified by comparing with single molecule FISH experiments and found to be 11%, 12% and 35% for GAPDH, TFRC and POLR2A, respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglial cells. We used fluorescence in situ sequencing to detect error-robust 8-base barcodes associated with each of these genes. We then used this data to uncover the spatial relationship among cells and transcripts by performing single-cell clustering and gene-gene proximity analyses. We anticipate the BOLORAMIS technology for in situ RNA detection to find applications in basic and translational research.
Matched MeSH terms: In Situ Hybridization, Fluorescence/methods*
Together with isocitrate dehydrogenase (IDH) mutation, co-deletion of 1p19q (1p19q codel) is a prerequisite for diagnosis of oligodendroglioma, making it imperative that histopathology laboratories introduce testing for 1p19q codel. To date there is still no consensus reference range and cut-offs that confirm deletion of 1p or 19q. We embarked on determining our reference range in 11 formalinfixed, paraffin-embedded non-neoplastic brain tissue using fluorescence in situ hybridisation (FISH) with the Vysis 1p36/1q25 and 19q13/19p13 FISH Probe Kit (Abbott Molecular Inc., USA). At same time we attempted to validate our methodology in 13 histologically-confirmed IDH-mutant oligodendrogliomas. For 1p, percentage cells with deletion (range=8-23%; mean±SD = 15.73±5.50%) and target: control (1p36:1q25) ratio (range = 0.89-0.96; mean±SD = 0.92±0.03) in non-neoplastic brain, differed significantly (p<0.000) from oligodendroglioma (percentage cells with deletion: range = 49-100%; mean±SD = 82.46±15.21%; target:control ratio range:0.50-0.76; mean±SD = 0.59±0.08). For 19q, percentage cells with deletion (range = 7-20%; mean±SD = 12.00±3.49%) and target:control (19q13/19p13) ratio (range:0.90-0.97; mean±SD = 0.94±0.02) in non-neoplastic brain also differed significantly from oligodendroglioma (percentage cells with deletion: range = 45-100%; mean±SD = 82.62±18.13%; target:control ratio range:0.50-0.78; mean±SD = 0.59±0.09). Using recommended calculation method, for diagnosis of 1p deletion, percentage of cells showing deletion should be >32-33% and/or target:control ratio <0.83. For 19q, percentage of cells showing deletion should be >22% and target:control ratio <0.88. Using these cut-offs all 13 oligodendroglioma demonstrated 1p19q codel.
Matched MeSH terms: In Situ Hybridization, Fluorescence*
In this report we demonstrate the role of fluorescence in situ hybridisation (FISH) and conventional cytogenetic methods in clinically and cytogenetically confirmed cases of microdeletion syndromes. A total of nine cases were referred to the Cytopathology and Cytogenetic Unit, Hospital Universiti Kebangsaan Malaysia (HUKM) from 2002 to 2004. They include three Prader-Willi syndrome, three DiGeorge syndrome, one Williams syndrome, one Miller-Dieker syndrome and one Kallmann syndrome. Blood samples from the patients were cultured and harvested following standard procedures. Twenty metaphases were analysed for each of the cases. FISH analysis was carried out for all the cases using commercial probes (Vysis, USA): SNRPN and D15S10 for Prader-Willi syndrome, LIS1 for Miller Dieker syndrome, ELN for Williams syndrome, KAL for Kallmann syndrome, TUPLE 1 and D22S75 for DiGeorge syndrome. Conventional cytogenetic analysis revealed normal karyotypes in all but one case with structural abnormality involving chromosomes 9 and 22. FISH analysis showed microdeletions in all of the nine cases studied. This study has accomplished two important findings ie. while the FISH method is mandatory in ruling out microdeletion syndromes, conventional cytogenetics acts as a screening tool in revealing other chromosomal abnormalities that may be involved with the disease.
Matched MeSH terms: Fluorescence; In Situ Hybridization, Fluorescence
In October 2011, the National International Health Regulations (IHR) 2005 Focal Point for Malaysia received notification from the United States’ Centers for Disease Control and Prevention (CDC) of a probable Sarcocystis outbreak amongst 23 travellers from six countries who had vacationed on Tioman Island between June and August 2011. The Ministry of Health, Malaysia (MOH) in collaboration with the Department of Veterinary Services, Malaysia (DVS) conducted a cross sectional study in November 2011 to determine the presence of Sarcocystosis among humans, animals and in the environment in Tioman Island. Epidemiological investigations conducted involved a community health survey of 44 residents in Kampung Salang, Tioman and review of outpatient attendance cards for suspected or confirmed cases of Sarcocystosis. Twenty-eight fresh stool samples were collected and sent to the National Public Health Laboratory (NPHL) for detection of Sarcocystis oocysts using fluorescence microscopy. Water samples taken from 27 water sampling points around the island were processed and analysed under the fluorescence microscope using ultraviolet (UV) light at the Institute for Medical Research (IMR) to detect the presence of Sarcocystis sporocyst. DVS collected 84 faecal samples from four types of domesticated animals and then analysed them at the Veterinary Services Centre in Tioman Island for Sarcocystis oocysts and other parasitic ova and cysts using qualitative Floatation Technique. The results showed that Sarcocystis was not present in humans, animals and in the environment in Tioman Island during the study period. Further surveillance among humans, wildlife and the environment is needed to determine Sarcocystis endemicity in Tioman Island.
It is known from our previous studies that kiwifruits, which are used in common human diet, have preventive properties of coronary artery disease. This study describes a combination of (1)H NMR spectroscopy, multivariate data analyses and fluorescence measurements in differentiating of some kiwifruit varieties, their quenching and antioxidant properties. A total of 41 metabolites were identified by comparing with literature data Chenomx database and 2D NMR. The binding properties of the extracted polyphenols against HSA showed higher reactivity of studied two cultivars in comparison with the common Hayward. The results showed that the fluorescence of HSA was quenched by Bidan as much as twice than by other fruits. The correlation between the binding properties of polyphenols in the investigated fruits, their relative quantification and suggested metabolic pathway was established. These results can provide possible application of fruit extracts in pharmaceutical industry.
A magnetic nanographene oxide sorbent as a selective sorbent for the magnetic solid-phase extraction combined with high-performance liquid chromatography and fluorescence detection was developed and proved to be a robust method for zearalenone determination in corn samples. Optimum extraction of zearalenone (20 mg magnetic nanographene oxide sorbent, extraction for 15 min, desorption time of 15 min using 1 mL of 0.5% formic acid in methanol) resulted in low limits of detection (05 mg/L) and quantitation (0.13 mg/L) and good linearity range of 0.13-1.25 mg/L with the correlation coefficient of 0.9957. Acceptable recoveries (79.3-80.6%) with relative standard deviations below 4% and satisfactory intra- and interday precisions (2-7.4%) were achieved. Additionally, the proposed method has been proved to be good in several aspects: easily prepared sorbent with high affinity to zearalenone, convenient and fast procedure, and high extraction efficiency.
The ability of human serum albumin (HSA) to bind medium-sized hydrophobic molecules is important for the distribution, metabolism, and efficacy of many drugs. Herein, the interaction between pyrene, a hydrophobic fluorescent probe, and HSA was thoroughly investigated using steady-state and time-resolved fluorescence techniques, ligand docking, and molecular dynamics (MD) simulations. A slight quenching of the fluorescence signal from Trp214 (the sole tryptophan residue in the protein) in the presence of pyrene was used to determine the ligand binding site in the protein, using Förster's resonance energy transfer (FRET) theory. The estimated FRET apparent distance between pyrene and Trp214 was 27Å, which was closely reproduced by the docking analysis (29Å) and MD simulation (32Å). The highest affinity site for pyrene was found to be in subdomain IB from the docking results. The calculated equilibrium structure of the complex using MD simulation shows that the ligand is largely stabilized by hydrophobic interaction with Phe165, Phe127, and the nonpolar moieties of Tyr138 and Tyr161. The fluorescence vibronic peak ratio I1/I3 of bound pyrene inside HSA indicates the presence of polar effect in the local environment of pyrene which is less than that of free pyrene in buffer. This was clarified by the MD simulation results in which an average of 5.7 water molecules were found within 0.5nm of pyrene in the binding site. Comparing the fluorescence signals and lifetimes of pyrene inside HSA to that free in buffer, the high tendency of pyrene to form dimer was almost completely suppressed inside HSA, indicating a high selectivity of the binding pocket toward pyrene monomer. The current results emphasize the ability of HSA, as a major carrier of several drugs and ligands in blood, to bind hydrophobic molecules in cavities other than subdomain IIA which is known to bind most hydrophobic drugs. This ability stems from the nature of the amino acids forming the binding sites of the protein that can easily adapt their shape to accommodate a variety of molecular structures.
Matched MeSH terms: Fluorescence Resonance Energy Transfer/methods*
A simple analytical method using HPLC with fluorescence detection was developed for the simultaneous determination of four lignans, phyllanthin (1), hypophyllanthin (2), phyltetralin (3) and niranthin (4) from Phyllanthus niruri L. in plasma. The method recorded limits of detection for 1, 2, 3 and 4 as 1.22, 6.02, 0.61 and 1.22 ng/ml, respectively, at a signal-to-noise ratio of 5:1 whereas their limits of quantification were 4.88, 24.41, 4.88 and 9.76 ng/ml, respectively, at a signal-to-noise ratio of 12:1. These values were comparable to those of other sensitive methods such as gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-MS (HPLC-MS) and HPLC-electrochemical detection (HPLC-ECD) for the analysis of plasma lignans. A further advantage over known methods was its simple protocol for sample preparation. The within-day and between-day accuracies for the analysis of the four lignans were between 87.69 and 110.07% with precision values below 10.51%. Their mean recoveries from extraction were between 91.39 and 114.67%. The method was successfully applied in the pharmacokinetic study of lignans in rats. Following intravenous administration, the lignans were eliminated slowly from the body with a mean clearance of 0.04, 0.01, 0.03 and 0.02 l/kg h and a mean half-life of 3.56, 3.87, 3.35 and 4.40 h for 1, 2, 3 and 4, respectively. Their peak plasma concentration upon oral administration was 0.18, 0.56, 0.12 and 0.62 microg/ml, respectively, after 1h. However, their absorption was incomplete with a calculated absolute oral bioavailability of 0.62, 1.52, 4.01 and 2.66% for 1, 2, 3 and 4, respectively.
Interaction between a local anesthetic drug, articaine (ART) and human serum albumin (HSA) was investigated in the absence and presence of paracetamol (PAR) and caffeine (CAF) using spectroscopic, voltammetric, and computational techniques for the first time. The results demonstrated that increasing concentrations of ART in HSA solution led to a decrease in HSA fluorescence signal, indicating the ART-HSA complex formation via the static quenching mechanism. The binding strength of the complex was moderate (binding constant, Ka = 5.87 × 103 M-1 in fluorescence and 6.31 × 103 M-1 in voltammetric at 298 K). Thermodynamic analysis (ΔS = +28.32 J mol-1 K-1; ΔH = -30.17 kJ/mol) of the binding reaction suggested involvement of hydrophobic interactions, van der Waal's forces and hydrogen bonding in stabilizing the ART-HSA complex. Significant microenvironmental alterations near the Trp and Tyr residues of HSA consequent to the ART-HSA complex formation. ART predominantly binds to Sudlow's site I of HSA with more negative binding energy and stronger hydrophobic interactions compared to Site II. The stability of the ART-HSA complex at Site I over a 100 ns timeframe, supported by stable hydrogen bonding and compact HSA structure throughout the molecular dynamics simulations. The effect of PAR and CAF on the binding strength between ART and HSA was also examined, and presence of PAR and CAF in the reaction mixture produced significant reduction in the binding affinity of ART to HSA. These findings underscore the competitive binding between ART, PAR, and CAF, which impacts their pharmacokinetics and efficacy. This study provides valuable insights into the complex interactions between anesthetic drugs and common pharmaceuticals, potentially guiding clinical practices and drug development.
Fluorescence in situ hybridization (FISH) is increasingly gaining importance in clinical diagnostics settings. Due to the ability of the technique to detect chromosomal abnormalities in samples with low cellularity or containing a mixed population of cells even at a single-cell level, it has become more popular in cancer research and diagnosis. Here, we describe the FISH technique for detection of PAX8-PPARγ translocation in follicular thyroid neoplasms, and the optimal protocol for the detection of this fusion gene using in archival formalin-fixed paraffin-embedded (FFPE) thyroid tissue sections.
Matched MeSH terms: Microscopy, Fluorescence; In Situ Hybridization, Fluorescence/methods*
The effect of room temperature ionic liquid (RTIL) on the formation of the fluorescence ternary complex oxalate-sodium morin-5-sulfonate (NaMSA)-Aluminium(III) has been studied. In weakly acidic medium and in the presence of RTIL, 1-Butyl-3-methylimidazolium hexafluorophosphate (BMIM-PF6), total complex formation is achieved as compared with the formation of the binary complex of NaMSA-Aluminium(III). The fluorescence characteristics of the system allowed the establishment of a very sensitive method for the spectrofluorimetric determination of oxalate ion. The ternary complex formed its highest fluorescence signal at 513 nm and excitation at 420 nm. In these conditions, the method produces a detection limit of 0.57 ng mL(-1). The procedure has been satisfactorily applied to the determination of oxalate ion in a vegetal tissue (spinach leaves).
Aptamers are single stranded DNA or RNA oligonucleotides that have high affinity and specificity towards a wide range of target molecules. Aptamers have low molecular weight, amenable to chemical modifications and exhibit stability undeterred by repetitive denaturation and renaturation. Owing to these indispensable advantages, aptamers have been implemented as molecular recognition element as alternative to antibodies in various assays for diagnostics. By amalgamating with a number of methods that can provide information on the aptamer-target complex formation, aptamers have become the elemental tool for numerous biosensor developments. In this review, administration of aptamers in applications involving assays of fluorescence, electrochemistry, nano-label and nano-constructs are discussed. Although detection strategies are different for various aptamer-based assays, the core of the design strategies is similar towards reporting the presence of specific target binding to the corresponding aptamers. It is prognosticated that aptamers will find even broader applications with the development of new methods of transducing aptamer target binding.
Overexpression of the epidermal growth factor receptor-related gene ErbB2 occurs in 18% to 25% of patients with breast cancer in Western countries and is associated with a poor prognosis. The prevalence of ErbB2-positive tumors in Asia is unclear, partly because data are limited. The objective of this review was to summarize the reported prevalence of ErbB2-positive tumors from a large sample of Asian patients and to examine ErbB2 assessment methods in Asia. From searches of MEDLINE, local language journals, and local and international conference proceedings as well as locoregional breast cancer experts' recommendations, the authors selected up to 5 studies each from India, Korea, Malaysia, the Philippines, Singapore, Taiwan, and Thailand that reported ErbB2 results based on assessment with immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). The reported prevalence of ErbB2-positive tumors in 22 studies on 24,671 patients, of whom 14,398 patients were assessed for ErbB2 status, varied widely (range, 6%-65%) as did the assessment methods used. Most studies (n=21) used IHC to assess ErbB2 status, but definitions for positivity varied. When robust assessment methods were used, the median prevalence was 19% based on strong IHC staining (IHC3+; n=9812 patients) and 25% based on FISH (n=681 patients). Data on the prevalence of ErbB2-positive breast cancer in Asia are limited. The current survey indicated that the prevalence in Asia may be similar to that in Western countries; thus, up to 1 in 4 Asian patients with breast cancer potentially could benefit from ErbB2-targeted treatment. A standard, reliable ErbB2 assessment method available to patients across Asia is urgently required.
Matched MeSH terms: In Situ Hybridization, Fluorescence
A total of 126 local and imported samples of commercial white and black pepper in Malaysia were analysed for aflatoxins B1, B2, G1 and G2 (AFB1, AFB2, AFG1, AFG2) content using high-performance liquid chromatography (HPLC) with a fluorescence detector (FD). An acetonitrile-methanol-water (17 : 29 : 54; v/v) mixture was used as a mobile phase and clean-up was using an immunoaffinity column (IAC). Seventy out of 126 (55.5%) samples were contaminated with total aflatoxins, although only low levels of aflatoxins were found ranging from 0.1 to 4.9 ng g(-1). Aflatoxin B1 showed the highest incidence of contamination and was found in all contaminated samples. There was a significant difference between type of samples and different brands (p < 0.05). The results showed black peppers were more contaminated than white peppers.
A study to quantify the free glutamic acid content of six processed foods, 44 dishes and 26 condiments available in Malaysia was performed using high-performance liquid chromatography with a fluorescence detector (HPLC-FRD). Recovery tests were carried out with spiked samples at levels from 6 to 31 mg g(-1). High recovery in different matrices was achieved ranging from 88% +/- 13% to 102% +/- 5.12%, with an average of 97% +/- 8.92%. Results from the study revealed that the average free glutamic acid content ranged from 0.34 +/- 0.20 to 4.63 +/- 0.41 mg g(-1) in processed foods, while in prepared dishes it was as low as 0.24 +/- 0.15 mg g(-1) in roti canai (puffed bread served with curry or dhal) to 8.16 +/- 1.99 mg g(-1) in dim sum (a small casing of dough, usually filled with minced meat, seafood, and vegetables, either steamed or fried). Relatively, the content of free glutamic acid was found to be higher in condiments at 0.28 +/- 0 mg g(-1) in mayonnaise to 170.90 +/- 6.40 mg g(-1) in chicken stock powder.
This article reviews progress in the application of electrophoretic techniques for the separation of nanoparticles. Numerous types of nanoparticles have recently been synthesised and integrated into different products and procedures. Consequently, analytical methods for the efficient characterisation of nanoparticles are now required. Several studies have revealed that gel electrophoresis can readily be used for separating nanoparticles according to their size or shape. However, many other studies focused on separation of nanoparticles by CE. In some cases nanoparticles could be separated by CZE, simply using pure buffer as the BGE. In other studies, buffer additives (most often SDS) were used, enabling fast separations of metallic nanoparticles by size. Other CE methods also allowed for separation of nanoparticle conjugates with biomolecules. Dielectrophoresis is yet another electrophoretic technique useful in separation and characterisation of nanoparticles; particularly nanotubes. Detection methods often used after electrophoretic separation include UV/Vis absorption and fluorescence spectroscopy. Examples of recent and relevant older reports are presented here. The authors conclude that electrophoretic methods for nanoanalysis can provide inexpensive and efficient tools for quality assurance and safety control; and as a consequence, they can augment transfer of nanotechnologies from research to industry.
Green fluorescent protein (GFP) is a versatile reporter protein and has been widely used in biological research. However, its quantitation requires expensive equipment such as a spectrofluorometer. In the current study, a gel documentation imaging system using a native polyacrylamide gel for the quantitation of GFP was developed. The assay was evaluated for its precision, linearity, reproducibility, and sensitivity in the presence of Escherichia coli cells and was compared with the spectrofluorometric method. Using this newly established, gel-based imaging technique; the amount of GFP can be quantified accurately.
The appearance of dental porcelains is comparable to natural teeth. This study discusses the effect of Y2O3 addition to the fluorescent property of dental porcelains. The composition of dental porcelains contained Y2O3 as the fluorescent agent and base frit. The combinations of Y2O3 added consist of a series with 0.5, 1.0, 1.5, 2.0 and 2.5 wt% respectively, based on the total composition. In the extreme condition, fluorescent agents are added from 5.0 up to 10.0 wt%. In order to enhance the fluorescent property of dental porcelains, an opacifiying agent, cerium oxide (CeO2) was also added to dental porcelains composition. The fluorescent property was determined using Spectroline EF-1400C/F that emits 240 nm wavelength ultraviolet light. The microstructure was examined by Scanning Electron Microscope (SEM). The result shows that, the fluorescent properties displayed are similar to natural teeth when subjected to ultraviolet light. SEM micrograph was able to show the fluorescent agent dispersed in glass phase. Increasing additions of Y2O3 gave the fluorescent properties near to natural teeth.