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Fluorescence quenching reaction of chlorophyll a by tris(acetylacetonate)iron(iii) in various solvents

Leesakul N, Masen D, Grampp G

Sains Malaysiana, 2017;46:1369-1377.

hlorophyll a is known as the prevailing light absorbing pigment giving a strong absorption and fluorescence emission in visible region. Quenching reactions of the chlorophyll a fluorescence by Fe(acac)3 were precisely investigated in various organic solvents which are benzene toluene, ethanol, methanol, dmf, dmso and acetonitrile. Electron transfer performance of chlorophyll a by Fe(acac)3 was investigated from oxidative quenching reaction. Herein, the simplified Rehm-Weller relationship was used to calculate the free energy change of the photo-induced electron transfer reaction. Emission intensity decreased when the concentration of Fe(acac)3 quencher was increased. Non-linear Stern-Volmer plots are found to be affected by inner filter effect more than the ground state complex formation. Rate of quenching reactions (kq) were determined from the Stern-Volmer equation with corrected inner filter effect. The rates of quenching reactions occurred faster in high viscous solvents.

Matched MeSH terms: Fluorescence
Biomolecular interaction of a platelet aggregation inhibitor, 3,4-methylenedioxy-β-nitrostyrene with human serum albumin: multi-spectral and computational characterization

Kabir MZ, Roslan AA, Ridzwan NFW, Mohamad SB, Tayyab S

J Biomol Struct Dyn, 2020 Jun;38(9):2693-2703.
PMID: 31271347 DOI: 10.1080/07391102.2019.1640133

Molecular interaction of the 3,4-methylenedioxy-β-nitrostyrene (MNS), an inhibitor of platelet aggregation with the main transport protein, albumin from human serum (HSA) was explored using absorption, fluorescence and circular dichroism (CD) spectroscopy in combination with in silico analyses. The MNS-HSA complexation was corroborated from the fluorescence and absorption spectral results. Implication of static quenching mechanism for MNS-HSA system was predicted from the Stern-Volmer constant, KSV-temperature relationship as well as the bimolecular quenching rate constant, kq values. Stabilization of the complex was affirmed by the value of the binding constant (Ka = 0.56-1.48 × 104 M-1). Thermodynamic data revealed that the MNS-HSA association was spontaneously driven mainly through hydrophobic interactions along with van der Waal's interaction and H-bonds. These results were well supported by in silico interpretations. Far-UV and near-UV CD spectral results manifested small variations in the protein's secondary and tertiary structures, respectively, while three-dimensional fluorescence spectra displayed microenvironmental fluctuations around protein's fluorophores, upon MNS binding. Significant improvement in the protein's thermostability was evident from the temperature-stability results of MNS-bound HSA. Binding locus of MNS, as identified by competitive drug displacement findings as well as in silico analysis, was found to be located in subdomain IIA (Sudlow's site I) of the protein.Communicated by Ramaswamy H. Sarma.

Matched MeSH terms: Fluorescence
Fulltext Centromeres of Cucumis melo L. comprise Cmcent and two novel repeats, CmSat162 and CmSat189

Setiawan AB, Teo CH, Kikuchi S, Sassa H, Kato K, Koba T

PLoS One, 2020;15(1):e0227578.
PMID: 31945109 DOI: 10.1371/journal.pone.0227578

Centromeres are prerequisite for accurate segregation and are landmarks of primary constrictions of metaphase chromosomes in eukaryotes. In melon, high-copy-number satellite DNAs (SatDNAs) were found at various chromosomal locations such as centromeric, pericentromeric, and subtelomeric regions. In the present study, utilizing the published draft genome sequence of melon, two new SatDNAs (CmSat162 and CmSat189) of melon were identified and their chromosomal distributions were confirmed using fluorescence in situ hybridization. DNA probes prepared from these SatDNAs were successfully hybridized to melon somatic and meiotic chromosomes. CmSat162 was located on 12 pairs of melon chromosomes and co-localized with the centromeric repeat, Cmcent, at the centromeric regions. In contrast, CmSat189 was found to be located not only on centromeric regions but also on specific regions of the chromosomes, allowing the characterization of individual chromosomes of melon. It was also shown that these SatDNAs were transcribed in melon. These results suggest that CmSat162 and CmSat189 might have some functions at the centromeric regions.

Matched MeSH terms: In Situ Hybridization, Fluorescence
Determination of four lignans in Phyllanthus niruri L. by a simple high-performance liquid chromatography method with fluorescence detection

Murugaiyah V, Chan KL

J Chromatogr A, 2007 Jun 22;1154(1-2):198-204.
PMID: 17418855 DOI: 10.1016/j.chroma.2007.03.079

A new and simple analytical method using HPLC with fluorescence detection was developed for the simultaneous determination of four lignans (phyllanthin, hypophyllanthin, phyltetralin and niranthin) in Phyllanthus niruri L. plant samples. Optimal separation was achieved with an isocratic mobile phase consisting of acetonitrile-water (55:45 v/v). The method recorded limits of detection (S/N=5) for phyllanthin at 0.61 ng/mL, hypophyllanthin at 6.02 ng/mL, phyltetralin at 0.61 ng/mL and niranthin at 1.22 ng/mL, being 80, 8, 80 and 40 times, respectively, lower when compared with those derived using HPLC-UV detection. The limits of quantification (S/N=12) were 4.88 ng/mL for phyllanthin and phyltetralin, 9.76 ng/mL for niranthin and 24.4 ng/mL for hypophyllanthin showing 40, 8 and 20 times, respectively, lower than those from the UV detection method. The within-day and between-day accuracy for the four lignans were between 98.1% and 102.9% while their precision values were below 2.2%. The mean recovery was between 92.5% and 110.1%. The method was then successfully applied for the quantification of lignans in P. niruri plant samples. The highest amount of lignans was found in the leaves followed by fruits, branches and stem, whilst the roots have the least amount of lignans.

Matched MeSH terms: Fluorescence
Use of computational and wet lab techniques to examine the molecular association between a potent hepatitis C virus inhibitor, PSI-6206 and human serum albumin

Abubakar M, Mohamed SB, Abd Halim AA, Tayyab S

Spectrochim Acta A Mol Biomol Spectrosc, 2023 Jun 05;294:122543.
PMID: 36868020 DOI: 10.1016/j.saa.2023.122543

This study explores the plausible molecular interaction between a potent hepatitis C virus inhibitor, PSI-6206 (PSI), and human serum albumin (HSA), a primary transporter in blood plasma. Results obtained from both computational viz. molecular docking and molecular dynamics (MD) simulation and wet lab techniques such as UV absorption, fluorescence, circular dichroism (CD), and atomic force microscopy (AFM) complemented each other. While docking results identified PSI binding to subdomain IIA (Site I) of HSA by forming six hydrogen bonds, MD simulations signified the complex stability throughout the 50,000 ps. A consistent cutback in the Stern-Volmer quenching constant (Ksv) along with rising temperatures supported the static mode of fluorescence quenching in response to PSI addition and implied the development of the PSI-HSA complex. This discovery was backed by the alteration of the HSA UV absorption spectrum, a larger value (>1010 M-1.s-1) of the bimolecular quenching rate constant (kq) and the AFM-guided swelling of the HSA molecule, in the presence of PSI. Moreover, the fluorescence titration results revealed a modest binding affinity (4.27-6.25×103 M-1) in the PSI-HSA system, involving hydrogen bonds, van der Waals and hydrophobic interactions, as inferred from ΔS = + 22.77 J mol-1 K-1 and ΔH = - 11.02 KJ mol-1values. CD and 3D fluorescence spectra reminded significant adjustment in the 2° and 3° structures and modification in the Tyr/Trp microenvironment of the protein in the PSI-bound state. The results obtained from drug competing experiments also advocated the binding location of PSI in HSA as Site I.

Matched MeSH terms: Spectrometry, Fluorescence
In vitro interactions of two pesticides, propazine and quinoxyfen with bovine serum albumin: Spectrofluorometric and molecular docking investigations

Duman B, Erkmen C, Zahirul Kabir M, Ching Yi L, Mohamad SB, Uslu B

Spectrochim Acta A Mol Biomol Spectrosc, 2023 Nov 05;300:122907.
PMID: 37257323 DOI: 10.1016/j.saa.2023.122907

Binding mechanisms of two selected pesticides, propazine (PRO) and quinoxyfen (QUI) with bovine serum albumin (BSA) was examined using fluorescence, absorption and molecular docking methods. Intrinsic fluorescence of BSA was quenched in the presence of both PRO and QUI. The quenching was ascertained to be conversely linked to temperature, which suggested the contribution of static quenching process in the PRO-BSA and QUI-BSA complex formations. This results were validated by the enhancement in absorption spectrum of BSA upon binding with PRO and QUI. Binding constant values (Kf = 9.55-0.60 × 10-3 M-1 for PRO-BSA system; Kf = 7.08-5.01 × 102 M-1 for QUI-BSA system) and number of binding site (n) values for the PRO-BSA and QUI-BSA systems at different temperatures affirmed a weak binding strength with a set of equivalent binding sites on BSA. Thermodynamic data obtained for both the PRO-BSA and QUI-BSA interactions predicted that the association process was spontaneous and non-covalent contacts such as hydrophobic interactions, van der Waals forces and hydrogen bonds participated in the binding reactions. This result was further supported by the molecular docking assessments. Three-dimensional spectral results revealed the microenvironmental alterations near tryptophan (Trp) and tyrosine (Tyr) residues in BSA by the addition of PRO and QUI. The docking analysis demonstrated the binding pattern for the PRO-BSA and QUI-BSA systems and disclosed the preferred binding site of both PRO and QUI as site I (subdomain IIA) of BSA.

Matched MeSH terms: Spectrometry, Fluorescence
Fulltext Genome Assembly and Analysis of the Flavonoid and Phenylpropanoid Biosynthetic Pathways in Fingerroot Ginger (Boesenbergia rotunda)

Taheri S, Teo CH, Heslop-Harrison JS, Schwarzacher T, Tan YS, Wee WY, et al.

Int J Mol Sci, 2022 Jun 30;23(13).
PMID: 35806276 DOI: 10.3390/ijms23137269

Boesenbergia rotunda (Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have a high flavanone and chalcone content. Here we report the genome analysis of B. rotunda together with a complete genome sequence as a hybrid assembly. B. rotunda has an estimated genome size of 2.4 Gb which is assembled as 27,491 contigs with an N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has a 72% repeat content, with class I TEs occupying ~67% of the assembled genome. Fluorescence in situ hybridization of the 18 chromosome pairs at the metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. An SSR analysis identified 238,441 gSSRs and 4604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in the leaf to 53% CpG, 18% CHG and 25% CHH in the embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in the watery callus. B rotunda has a relatively large genome with a high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics of B. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.

Matched MeSH terms: In Situ Hybridization, Fluorescence
Fulltext A coumarin-based fluorescent probe as a central nervous system disease biomarker

Yap AC, Mahamad UA, Lim SY, Kim HJ, Choo YM

Sensors (Basel), 2014 Nov 10;14(11):21140-50.
PMID: 25390405 DOI: 10.3390/s141121140

Homocysteine and methylmalonic acid are important biomarkers for diseases associated with an impaired central nervous system (CNS). A new chemoassay utilizing coumarin-based fluorescent probe 1 to detect the levels of homocysteine is successfully implemented using Parkinson's disease (PD) patients' blood serum. In addition, a rapid identification of homocysteine and methylmalonic acid levels in blood serum of PD patients was also performed using the liquid chromatography-mass spectrometry (LC-MS). The results obtained from both analyses were in agreement. The new chemoassay utilizing coumarin-based fluorescent probe 1 offers a cost- and time-effective method to identify the biomarkers in CNS patients.

Matched MeSH terms: Spectrometry, Fluorescence/methods*
A simple and sensitive fluorescence based biosensor for the determination of uric acid using H2O2-sensitive quantum dots/dual enzymes

Azmi NE, Ramli NI, Abdullah J, Abdul Hamid MA, Sidek H, Abd Rahman S, et al.

Biosens Bioelectron, 2015 May 15;67:129-33.
PMID: 25113659 DOI: 10.1016/j.bios.2014.07.056

A novel optical detection system consisting of combination of uricase/HRP-CdS quantum dots (QDs) for the determination of uric acid in urine sample is described. The QDs was used as an indicator to reveal fluorescence property of the system resulting from enzymatic reaction of uricase and HRP (horseradish peroxidase), which is involved in oxidizing uric acid to allaintoin and hydrogen peroxide. The hydrogen peroxide produced was able to quench the QDs fluorescence, which was proportional to uric acid concentration. The system demonstrated sufficient activity of uricase and HRP at a ratio of 5U:5U and pH 7.0. The linearity of the system toward uric acid was in the concentration range of 125-1000 µM with detection limit of 125 µM.

Matched MeSH terms: Fluorescence; Fluorescence Resonance Energy Transfer
Capillary electrophoresis for the analysis of paralytic shellfish poisoning toxins in shellfish: comparison of detection methods

Keyon AS, Guijt RM, Gaspar A, Kazarian AA, Nesterenko PN, Bolch CJ, et al.

Electrophoresis, 2014 May;35(10):1496-503.
PMID: 24591173 DOI: 10.1002/elps.201300353

Paralytic shellfish toxins (PSTs) are produced by marine and freshwater microalgae and accumulate in shellfish including mussels, oysters, and scallops, causing possible fatalities when inadvertently consumed. Monitoring of PST content of shellfish is therefore important for food safety, with currently approved methods based on HPLC, using pre- or postcolumn oxidation for fluorescence detection (HPLC-FLD). CE is an attractive alternative for screening and detection of PSTs as it is compatible with miniaturization and could be implemented in portable instrumentation for on-site monitoring. In this study, CE methods were developed for C(4) D, FLD, UV absorption detection, and MS-making this first report of C(4) D and FLD for PSTs detection. Because most oxidized toxins are neutral, MEKC was used in combination with FLD. The developed CZE-UV and CZE-C(4) D methods provide better resolution, selectivity, and separation efficiency compared to CZE-MS and MEKC-FLD. The sensitivity of the CZE-C(4) D and MEKC-FLD methods was superior to UV and MS, with LOD values ranging from 140 to 715 ng/mL for CZE-C(4) D and 60.9 to 104 ng/mL for MEKC-FLD. With the regulatory limit for shellfish samples of 800 ng/mL, the CZE-C(4) D and MEKC-FLD methods were evaluated for the screening and detection of PSTs in shellfish samples. While the CZE-C(4) D method suffered from significant interferences from the shellfish matrix, MEKC-FLD was successfully used for PST screening of a periodate-oxidized mussel sample, with results confirmed by HPLC-FLD. This confirms the potential of MEKC-FLD for screening of PSTs in shellfish samples.

Matched MeSH terms: Spectrometry, Fluorescence/methods*
Fluorescence-in-situ-hybridization in the surveillance of urothelial cancers: can use of cystoscopy or ureteroscopy be deferred?

Ho CC, Tan WP, Pathmanathan R, Tan WK, Tan HM

Asian Pac J Cancer Prev, 2013;14(7):4057-9.
PMID: 23991952

BACKGROUND: Fluorescence in situ hybridization (FISH) testing may be useful to screen for bladder carcinoma or dysplasia by detecting aneuploidy chromosomes 3, 7, 17 and deletion of the chromosome 9p21 locus in urine specimens. This study aimed to assess the sensitivity, specificity, positive and negative predictive value of FISH in a multi-ethnic population in Asia.
MATERIALS AND METHODS: Patients with haematuria and/or past history of urothelial cancer on follow-up had their voided urine tested with FISH. Patients then underwent cystoscopy/ ureteroscopy and any lesions seen were biopsied. The histopathological reports of the bladder or ureteroscopic mucosal biopsies were then compared with the FISH test results.
RESULTS: Two hundred sixty patients were recruited. The sensitivity and specificity of the FISH test was 89.2% and 83.4% respectively. The positive (PPV) and negative predictive values (NPV) were 47.1% and 97.9%. By excluding patients who had positive deletion of chromosome 9, the overall results of the screening test improved: sensitivity 84.6%; specificity 96.4%; PPV 75.9% and NPV 97.9%.
CONCLUSIONS: UroVysion FISH has a high specificity of detecting urothelial cancer or dysplasia when deletion of chromosome 9 is excluded. Negative UroVysion FISH-tests may allow us to conserve health resources and minimize trauma by deferring cystoscopic or ureteroscopic examination.

Matched MeSH terms: In Situ Hybridization, Fluorescence/methods*
Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing

Moo EK, Abusara Z, Abu Osman NA, Pingguan-Murphy B, Herzog W

J Biomech, 2013 Aug 9;46(12):2024-31.
PMID: 23849134 DOI: 10.1016/j.jbiomech.2013.06.007

Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in cell responses to mechanical stimuli that depend on the microscopic approach and the thresholding methods used and may result in contradictory interpretations.

Matched MeSH terms: Microscopy, Fluorescence, Multiphoton*
Surface enhanced Raman scattering and plasmon enhanced fluorescence in zinc-tellurite glass

Amjad RJ, Sahar MR, Dousti MR, Ghoshal SK, Jamaludin MN

Opt Express, 2013 Jun 17;21(12):14282-90.
PMID: 23787617 DOI: 10.1364/OE.21.014282

We report significant enhancements in Er(3+) luminescence as well as in Raman intensity in silver nanoparticles embedded zinc-tellurite glass. Surface enhanced Raman scattering effect is highlighted for the first time in tellurite glass containing silver NPs resulting in an enhanced Raman signal (~10 times). SAED manifest the growth of Ag(0) nanoparticles along the (111) and (200) crystallographic planes having average diameter in the range 14-36 nm. Surface plasmon resonance bands are observed in the range 484-551 nm. Furthermore, four prominent photoluminescence bands undergo significant enhancements up to 3 times. The enhancement is majorly attributed to the local field effect of silver NPs.

Matched MeSH terms: Spectrometry, Fluorescence/instrumentation*
Characterization of erythrosine B binding to bovine serum albumin and bilirubin displacement

Mathavan VM, Boh BK, Tayyab S

Indian J. Biochem. Biophys., 2009 Aug;46(4):325-31.
PMID: 19788065

The interaction of crythrosine B (ErB), a commonly used dye for coloring foods and drinks, with bovine scrum albumin (BSA) was investigated both in the absence and presence of bilirubin (BR) using absorption and absorption difference spectroscopy. ErB binding to BSA was reflected from a significant red shift of 11 nm in the absorption maximum of ErB (527 nm) with the change in absorbance at lamdamax. Analysis of absorption difference spectroscopic titration results of BSA with increasing concentrations of ErB3 using Benesi-Hildebrand equation gave the association constant, K as 6.9 x 10(4) M(-1). BR displacing action of ErB was revealed by a significant blue shift in the absorption maximum, accompanied by a decrease in absorbance difference at lamdamax in the difference spectrum of BR-BSA complex upon addition of increasing concentrations of ErB. This was further substantiated by fluorescence spectroscopy, as addition of increasing concentrations of ErB to BR-BSA complex caused a significant decrease in fluoresccnce at 510 nm. The results suggest that ErB binds to a site in the vicinity of BR binding site on BSA. Therefore, intake of ErB may increase the risk of hyperbilirubinemia in the healthy subjects.

Matched MeSH terms: Spectrometry, Fluorescence/methods
A unique "turn-on" fluorescence signalling strategy for highly specific detection of ascorbic acid using carbon dots as sensing probe

Fong JFY, Chin SF, Ng SM

Biosens Bioelectron, 2016 Nov 15;85:844-852.
PMID: 27290666 DOI: 10.1016/j.bios.2016.05.087

Carbon dots (CDs) that showed strong blue fluorescence were successfully synthesised from sodium alginate via furnace pyrolysis. The single step pyrolytic synthesis was simple to perform while yielded CDs with high photostability, good water solubility and minimum by-products. In order to design the probe with "turn-on" sensing capability, the CDs were screened against a series of metal cations to first "turn-off" the fluorescence. It was found that ferric ions (Fe(3+)) were most responsive and effective in quenching the fluorescence of CDs. Based on this observation, the conditioning of the probe was performed to ensure the fluorescence was completely quenched, while not overloading the system with Fe(3+). At the optimised condition, the CDs-Fe(3+) mixture served as a highly specific detection probe for ascorbic acid (AA). The analytical potential of the probe was evaluated and showed a good linear range of response for AA concentration of 24-40μg/mL. The selectivity study against other possible co-existing species was carried out and proved that our unique "turn-on" fluorescence signalling strategy was highly effective and selective towards AA as the target analyte. The probe was demonstrated for quantification of AA in real samples, which was the commercially available vitamin C supplement. The result showed good accuracy with minimum deviation from standard method adopted for validation purpose.

Matched MeSH terms: Spectrometry, Fluorescence/methods
Cd(2+) Triggered the FRET "ON": A New Molecular Switch for the Ratiometric Detection of Cd(2+) with Live-Cell Imaging and Bound X-ray Structure

Aich K, Goswami S, Das S, Mukhopadhyay CD, Quah CK, Fun HK

Inorg Chem, 2015 Aug 3;54(15):7309-15.
PMID: 26192906 DOI: 10.1021/acs.inorgchem.5b00784

On the basis of the Förster resonance energy transfer mechanism between rhodamine and quinoline-benzothiazole conjugated dyad, a new colorimetric as well as fluorescence ratiometric probe was synthesized for the selective detection of Cd(2+). The complex formation of the probe with Cd(2+) was confirmed through Cd(2+)-bound single-crystal structure. Capability of the probe as imaging agent to detect the cellular uptake of Cd(2+) was demonstrated here using living RAW cells.

Matched MeSH terms: Fluorescence Resonance Energy Transfer/methods*
Fulltext Epidermal growth factor receptor (EGFR) gene alteration and protein overexpression in Malaysian triple-negative breast cancer (TNBC) cohort

Zakaria Z, Zulkifle MF, Wan Hasan WAN, Azhari AK, Abdul Raub SH, Eswaran J, et al.

Onco Targets Ther, 2019;12:7749-7756.
PMID: 31571924 DOI: 10.2147/OTT.S214611

Background: Epidermal growth factor receptor (EGFR) is a member of the ErbB family of tyrosine kinase receptor proteins that plays important roles in tumour cell survival and proliferation. EGFR has been reported to be overexpressed in up to 78% of triple-negative breast cancer (TNBC) cases suggesting it as a potential therapeutic target. The clinical trials of anti-EGFR agents in breast cancer showed low response rates. However, a subgroup of patients demonstrated response to EGFR inhibitors highlighting the necessity to stratify patients, who might benefit from effective combination therapy that could include anti EGFR-agents. Population variability in EGFR expression warrants systematic evaluation in specific populations.
Purpose: To study EGFR alterations and expressions in a multi ethnic Malaysian TNBC patient cohort to determine the possibility of using anti-EGFR combinatorial therapy for this population.
Patients and methods: In this study, we evaluated 58 cases of Malaysian TNBC patient samples for EGFR gene copy number alteration and EGFR protein overexpression using fluorescence in-situ hybridization (FISH) and immunohistochemistry (IHC) methods, respectively.
Results: EGFR protein overexpression was observed in about 30% while 15.5% displayed high EGFR copy number including 5.17% gene amplification and over 10% high polysomy. There is a positive correlation between EGFR protein overexpression and gene copy number and over expression of EGFR is observed in ten out of the 48 low copy number cases (20.9%) without gene amplification.
Conclusion: This study provides the first glimpse of EGFR alterations and expressions in a multi ethnic Malaysian TNBC patient cohort emphasising the need for the nationwide large scale EGFR expression evaluation in Malaysia.

Matched MeSH terms: Fluorescence; In Situ Hybridization, Fluorescence
Latanoprost Quantification in Ocular Implants and Tissues: HPLC-Fluorescence vs HPLC-UV

Soliman K, Jirjees F, Sonawane R, Sheshala R, Wang Y, Jones D, et al.

J Chromatogr Sci, 2021 Jan 01;59(1):64-70.
PMID: 33047781 DOI: 10.1093/chromsci/bmaa078

Anti-glaucoma latanoprost-loaded ocular implants provide prolonged delivery and enhanced bioavailability relative to the conventional eye drops. This study aims at the development and validation of a reversed-phase high-performance liquid chromatography method for quantitative analysis of nanogram levels of latanoprost in the eye, and for the first time, compares the use of fluorescence vs ultraviolet (UV) detectors in latanoprost quantification. The mobile phase was composed of acetonitrile:0.1% v/v formic acid (60:40, v/v) with a flow rate of 1 mL/min and separation was done using a C18 column at temperature 40°C. The fluorescence excitation and emission wavelengths were set at 265 and 285 nm, respectively, while the UV absorption was measured at 200 nm. The latanoprost concentration-peak area relationship maintained its linearity (R2 = 0.9999) over concentration ranges of 0.063-10 μg/mL and 0.212-10 μg/mL for the fluorescence and UV detectors, respectively. The UV detector showed better precision, while the fluorescence detector exhibited higher robustness and greater sensitivity, with a detection limit of 0.021 μg/mL. The fluorescence detector was selected for quantification of latanoprost released from ocular implants in vitro and in porcine ocular tissues. The developed method is a robust, rapid and cost-effective alternative to liquid chromatography-mass spectrometry for routine analysis of latanoprost released from ocular implants.

Matched MeSH terms: Spectrometry, Fluorescence/methods*
Conformational analysis of champedak galactose-binding lectin under different urea concentrations

Kameel NI, Wong YH, Shuib AS, Tayyab S

Plant Physiol Biochem, 2016 Jan;98:57-63.
PMID: 26642433 DOI: 10.1016/j.plaphy.2015.11.007

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.

Matched MeSH terms: Fluorescence; Spectrometry, Fluorescence
Fulltext A comparative analysis on the binding characteristics of various mammalian albumins towards a multitherapeutic agent, pinostrobin

Feroz SR, Sumi RA, Malek SN, Tayyab S

Exp Anim, 2015;64(2):101-8.
PMID: 25519455 DOI: 10.1538/expanim.14-0053

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.

Matched MeSH terms: Spectrometry, Fluorescence/methods

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