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2.
Methods Mol Biol ; 2633: 195-211, 2023.
Article in English | MEDLINE | ID: mdl-36853466

ABSTRACT

Polymerase Chain Reaction (PCR) products have been traditionally characterized by cloning and cycle sequencing. However, when quick sequencing data are required, the cloning step may be omitted and PCR products can be sequenced directly. We describe here a sequencing protocol that involves the gold standard Big Dye chemistry in a low throughput format using one of the latest sequencing platforms the ABI Seqstudio. Our cycle sequencing protocol follows the following steps: (1) purification of the PCR product with a spin column-based kit; (2) quality & quantity assessment of the PCR product with the use of spectrophotometry & gel electrophoresis; (3) setup and amplification of the cycle sequencing reaction; (4) Capillary Electrophoresis; (5) Sequence Data Analysis.


Subject(s)
Data Analysis , Electrophoresis, Capillary , Polymerase Chain Reaction , Spectrophotometry
3.
Toxicol Rep ; 9: 699-712, 2022.
Article in English | MEDLINE | ID: mdl-35433275

ABSTRACT

Crinum jagus (C. jagus; J. Thomps.) Dandy (Liliaceae) is a pantropical plant known for its medicinal values and pharmacological properties. The study assessed the protective effects and changes in oxidative stress indices due to C. jagus leaf extracts on the toluene-induced liver and kidney injuries in rats. The study was conducted on 8-week-old male Wistar rats (n = 80), weighing 243.3 ± 1.42 g. Group I, 1 ml/kg distilled water for 7 days; Group II, 4.5 ml/kg toluene once, 1 ml/kg distilled water for 7 days; Group III, 4.5 ml/kg toluene once, 500 mg/kg methanolic extract for 7 days; Group IV, 4.5 ml/kg toluene once, 500 mg/kg aqueous extract for 7 days; Group V, 500 mg/kg methanolic extract for 7 days; Group VI, 500 mg/kg aqueous extract for 7 days; Group VII, 500 mg/kg of vitamin C for 7 days; Group, VIII, 4.5 ml/kg toluene once, 500 mg/kg vitamin C for 7 days, all administrations were given by oral gavage. The phytochemical contents, absolute and relative organ weights of liver and kidneys, liver and kidney function tests, antioxidant status, as well as histological tests were analyzed using standard protocols. The tannins, flavonoids, and polyphenols were in highest concentration in both extracts, content in methanol extract (57.04 ± 1.51 mgg-1, 35.43 ± 1.03 mgg-1, 28.2 ± 0.34 mgg-1 respectively) > aqueous extract (18.74 ± 1.01 mgg-1, 13.43 ± 0.47 mgg-1, 19.65 ± 0.21 mgg-1 respectively). In the negative control group (II), bodyweights significantly (P < 0.05) reduced by 22%, liver weight and kidney weight significantly (P < 0.05) increased by 42% and 83% respectively, liver-to-bodyweight and kidney-to-bodyweight ratios increased significantly (P < 0.05); serum liver function tests (LFTs) i.e., bilirubin, alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (GGT), and serum kidney function tests (creatinine and urea) were significantly (P < 0.05) elevated; oxidant status (tissue malondialdehyde; MDA) was significantly (P < 0.05) elevated, antioxidant status i.e., tissue superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) levels was significantly (P < 0.05) reduced; with markedly visible renal and hepatic histopathological findings, compared to the normal control group. In C. jagus extract test groups (III and IV), the parameters were significantly (P < 0.05) alleviated and reversed to normal/near normal compared to the negative control. The LFTs, kidney function tests, and antioxidant status were significantly (P < 0.05) more improved with the methanol extract test and standard control groups compared to the aqueous extract test group; Also, the methanol extract test group showed better histological features than the aqueous extract test and standard control groups. The methanolic extract shows better antioxidant potential due to the availability of more nonenzymatic antioxidants (tannins, flavonoids, and polyphenols). The findings showed that toluene is a very aggressive xenobiotic due to the promotion of oxidative stress and peroxidation of cellular lipids, but C. jagus leaves provide significant protection through the reducing power of nonenzymatic antioxidants and their ability to induce endogenous antioxidant enzymes (SOD, CAT, and glutathione reductase or GR) causing reduced cellular lipid peroxidation and tissue damages, quickened tissue repair, and improved cell biology of liver and kidneys during toluene toxicity. The methanol leaf extract provides better protection and should be advanced for more experimental and clinical studies to confirm its efficacy in alleviating oxidative stress tissue injuries, specifically due to toluene.

4.
Sci Rep ; 12(1): 2094, 2022 02 08.
Article in English | MEDLINE | ID: mdl-35136108

ABSTRACT

The aim of this study was the isolation and molecular characterization of fungi from untreated refinery effluent by using multiple conserved genes. The Fungi isolated were characterized based on PCR amplification and genomic sequencing of the internal transcribed spacer region (ITS), partial ß-tubulin (BenA), calmodulin (CaM), and RNA polymerase second large subunit (RPB2) genes, along with morphological characterization. The obtained sequences were subjected to BLAST analysis and the corresponding fungal isolates were assigned species names after comparison with representative sequences available in GenBank. Fifteen (15) Fungi species belonging to four genera of Aspergillus, Penicillium, Fusarium, and Trichoderma with Aspergillus as the predominant genus were identified. Therefore these genes should be used as molecular markers for species level identification of fungi (especially Aspergillus and Penicillium as proven in this study.


Subject(s)
Fungi/genetics , Oil and Gas Industry , Phylogeny , Wastewater/microbiology , Calmodulin/genetics , DNA, Intergenic , Fungi/isolation & purification , Genes, Fungal , RNA Polymerase II/genetics , Tubulin/genetics
5.
Adv Exp Med Biol ; 820: 3-47, 2015.
Article in English | MEDLINE | ID: mdl-25417014

ABSTRACT

In this chapter, we study the effects of the acceleration gravity on the sedimentation deposition probability, as well as the aerosol deposition rate on the surface of the Earth and Mars, but also aboard a spacecraft in orbit around Earth and Mars as well for particles with density ρ p = 1,300 kg/m³, diameters d p = 1, 3, 5 µm, and residence times t = 0.0272, 0.2 , respectively. For particles of diameter 1 µm we find that, on the surface of Earth and Mars the deposition probabilities are higher at the poles when compared to the ones at the equator. Similarly, on the surface of the Earth we find that the deposition probabilities exhibit 0.5 and 0.4 % higher percentage difference at the poles when compared to that of the equator, for the corresponding residence times. Moreover in orbit equatorial orbits result to higher deposition probabilities when compared to polar ones. For both residence times particles with the diameters considered above in circular and elliptical orbits around Mars, the deposition probabilities appear to be the same for all orbital inclinations. Sedimentation probability increases drastically with particle diameter and orbital eccentricity of the orbiting spacecraft. Finally, as an alternative framework for the study of interaction and the effect of gravity in biology, and in particular gravity and the respiratory system we introduce is the term information in a way Shannon has introduced it, considering the sedimentation probability as a random variable. This can be thought as a way in which gravity enters the cognitive processes of the system (processing of information) in the cybernetic sense.


Subject(s)
Aerosols/pharmacokinetics , Gravitation , Respiratory System/metabolism , Static Electricity , Aerosols/chemistry , Aerosols/metabolism , Algorithms , Earth, Planet , Extraterrestrial Environment , Humans , Inhalation Exposure , Mars , Models, Biological , Particle Size , Probability , Spacecraft
6.
Forensic Sci Int ; 245: 51-7, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25447174

ABSTRACT

The Tudor warship the Mary Rose sank in the Solent waters between Portsmouth and the Isle of Wight on the 19th of July 1545, whilst engaging a French invasion fleet. The ship was rediscovered in 1971 and between 1979 and 1982 the entire contents of the ship were excavated resulting in the recovery of over 25,000 objects, including the skeleton of a small to medium sized dog referred to as the Mary Rose Dog (MRD). Here we report the extraction and analysis of both mitochondrial and genomic DNA from a tooth of this animal. Our results show that the MRD was a young male of a terrier type most closely related to modern Jack Russell Terriers with a light to dark brown coat colour. Interestingly, given the antiquity of the sample, the dog was heterozygotic for the SLC2A9 gene variant that leads to hyperuricosuria when found in modern homozygotic animals. These findings help shed light on a notable historical artefact from an important period in the development of modern dog breeds.


Subject(s)
Body Remains , DNA Fingerprinting/veterinary , Dogs/genetics , Ships , Accidents , Animals , DNA/isolation & purification , Glucose Transport Proteins, Facilitative/genetics , History, Medieval , Homozygote , Male , Microsatellite Repeats , Polymorphism, Single Nucleotide
7.
Nanoscale ; 3(3): 1218-24, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21258715

ABSTRACT

We have previously demonstrated that chitosan derivative N-octyl-O-sulfate chitosan (NOSC), which presents important pharmacological properties, can suspend single walled carbon nanotubes (SWNTs) up to 20 times more effectively than other chitosan derivatives in an aqueous environment. In an attempt to further investigate the impact of different molecular weights of chitosan to the solubilization and anticoagulant properties of these hybrids an array of NOSC derivatives varying their molecular weight (low, medium and high respectively) was synthesised and characterised by means of FT-IR spectroscopy, NMR spectroscopy and thermal gravimetric analysis (TGA). Microwave and nitric acid purified SWNTs, characterised by FT-IR spectroscopy, transmission electron microscopy (TEM) and Raman spectroscopy, were colloidally stabilised by these polymers and their anticoagulant activity was assessed. The results revealed that the low molecular weight NOSC coated SWNTs exhibit the highest activity when 0.5 mg mL(-1) NOSC solutions are used, activity which is similar to that of the free polymer. Preliminary studies by exposure of these hybrids to Brine Shrimp (Artemia) cysts revealed no effect on the viability of sub-adult Artemia. Our findings suggest the possibility of tailoring these nanomaterials to bear the required properties for application as biocompatible building blocks for nanodevices including biosensors and biomaterials.


Subject(s)
Blood Coagulation/drug effects , Chitosan/chemistry , Chitosan/pharmacology , Nanotubes, Carbon/chemistry , Nanotubes, Carbon/ultrastructure , Anticoagulants/chemical synthesis , Anticoagulants/pharmacology , Excipients/chemistry , Humans , Materials Testing , Molecular Weight , Particle Size
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