A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry-based confirmatory method was redeveloped and validated for the simultaneous determination of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscles. The analytes were extracted from minced chicken muscle with acetonitrile and ammoniated water mixture. A second extraction with ethyl acetate was followed by evaporation and dissolution of the residue in ammoniated methanol before defatting with n-hexane. Finally, the extract was further cleaned up by dispersive solid phase extraction using C-18 end-capped dispersive material. The validation protocol was adapted from the European Commission Decision 2002/657/EC and all the performance characteristics were successfully satisfied. The recoveries of all the analytes were found to be in the range of 86.4-108.1% and the precision values, within day and between days, ranged from 2.7% to 11% and 4.4% to 16.3%, respectively. The method was tested in various incurred samples and applied to analyse a wide range of random poultry market samples (n = 120) collected from three cities of the Punjab, Pakistan. Chloramphenicol and florfenicol residues were detected at low levels in less than 11% of the samples. Chloramphenicol was detected only in 4 samples with the concentration range of 0.17-0.477 µg kg, whereas the levels of florfenicol/florfenicol amine residues detected in 9 samples ranged from 8.7 to 32.8 µg kg. Moreover, most of the florfenicol residues were identified as tissue bound, extractable only after strong acid hydrolysis.
LC/MS-based variant profiling of lipid A component of endotoxic lipopolysaccharides of Pasteurella multocida type B:2, a causative agent of haemorrhagic septicaemia in water buffalo and cattle.
BACKGROUND
Hepatitis C virus genotype 3a (HCV G3a) is highly prevalent in Pakistan. Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV, medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease. Furthermore, from natural products, active compounds against vital HCV proteins like non-structural protein 3 (NS3) protease could be identified to prevent viral proliferation in the host.
AIM
To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.
METHODS
Full-length NS3 without co-factor non-structural protein 4A (NS4A) and codon optimized NS3 protease in fusion with NS4A were expressed in
Escherichia coli
. The expressed protein was purified by metal ion affinity chromatography and gel filtration. Citrus fruit extracts were screened using fluorescence resonance energy transfer (FRET) assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry (MS)/MS technique. Among different polyphenols, highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.
RESULTS
NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein. Furthermore, in enzyme kinetic studies, NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3. So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease. FRET assay was developed and validated by the half maximal inhibitory concentration (IC
50
) values of commercially available inhibitors. Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91% of protease activity. Among the compounds identified by LCMS analysis, hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of -10.98.
CONCLUSION
Fused NS4A-NS3 protease is functionally more active, which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC
50
value of 23.32 µmol/L.
The direct discharge of azo dyes and/or their metabolites into the environment may exert toxic, mutagenic, and carcinogenic effects on exposed fauna and flora. In this study, we analyzed the metabolites produced during the degradation of an azo dye namely Reactive Black 5 (RB5) in the bacterial augmented-floating treatment wetlands (FTWs), followed by the investigation of their underlying toxicity. To this end, a FTWs system was developed by using a common wetland plant Phragmites australis in the presence of three dye-degrading bacteria (Acinetobacter junii strain NT-15, Pseudomonas indoloxydans strain NT-38, and Rhodococcus sp. strain NT-39). We found that the FTW system effectively degraded RB5 into at least 20 different metabolites with the successful removal of color (95.5%) from the water. The fish toxicity assay revealed the non-toxic characteristics of the metabolites produced after dye degradation. Our study suggests that bacterially aided FTWs could be a suitable option for the successful degradation of azo dyes, and the results presented in this study may help improve the overall textile effluent clean-up processes.
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