Gene/Protein Disease Symptom Drug Enzyme Compound
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Stir bar soptive extraction (SBSE) coupled to thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) and membrane-assisted solvent extraction (MASE) coupled to large volume injection-programmed temperature vaporisation-GC-MS (LVI-PTV-GC-MS) were optimised for the simultaneous determination of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalate esters (PEs), nonylphenols (NPs), polybrominated biphenyls (PBBs) and polybrominated diphenyl ethers (PBDEs) in water samples. In the case of SBSE-TD, variables affecting the extraction (extraction time, addition of sodium chloride or methanol and sample volume) and desorption (cryofocusing temperature, desorption time and temperature, vent pressure and desorption flow) were fitted for the simultaneous determination. The extraction solvent nature (n-hexane, cyclohexane, n-heptane, ethyl acetate, toluene, dichloromethane or cyclohexane:ethyl acetate mixtures), as well as the addition of methanol (0-30%) and sodium chloride (0-20%), the extraction temperature (30-60 degrees C), shaking speed (250-750 rpm) and extraction time (5-150 min) were studied for the simultaneous membrane-assisted preconcentration. Finally, PTV-LVI variables such as injection volume (100-600 microL), injection speed (10-40 microL s(-1)), vent pressure (0-12.7 psi), vent time (0.05-0.8 min), vent flow (30-80 mL min(-1)), cryofocusing temperature (20-70 degrees C), split flow (20-100 mL min(-1)) and split time (1-5 min) were optimised. The optimisation was carried out by means of experimental design approaches in most of the cases. Precision (approximately 3-19% for both SBSE-TD and MASE-LVI-PTV), accuracy (approximately 80-120% for both SBSE-TD and MASE-LVI-PTV), limits of detection (LoDs) (0.1-222 ng L(-1) for MASE-LVI-PTV and 0.03-20.4 ng L(-1) for SBSE-TD in dependence of substance) and linearity (from 25 ng L(-1) up to at least 500 ng L(-1) for both procedures) were established for both procedures. Finally, the developed methods were applied to the determination of the free concentrations of PAHs, PCBs, PEs, NPs, PBBs and PBDEs in natural water samples (estuarine water and sea water) from the Bilbao estuary (Northern Spain) and comparable results were obtained with both procedures.
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PMID:Simultaneous preconcentration of a wide variety of organic pollutants in water samples. Comparison of stir bar sorptive extraction and membrane-assisted solvent extraction. 1899 Mar 98

In this paper, effectiveness of six different commonly applied extraction techniques for the determination of robenidine in poultry feed has been compared. The sample preparation techniques included shaking, Soxhlet, Soxtec, ultrasonically assisted extraction, microwave - assisted extraction and accelerated solvent extraction. Comparison of these techniques was done with respect to the recovery extraction, temperature and time, reproducibility and solvent consumption. Every single extract was subjected to clean - up using aluminium oxide column (Pasteur pipette filled with 1g of aluminium oxide), from which robenidine was eluted with 10ml of methanol. The eluate from the clean-up column was collected in a volumetric flask, and finally it was analysed by HPLC-DAD-MS. In general, all extraction techniques were capable of isolating of robenidine from poultry feed, but the recovery obtained using modern extraction techniques was higher than that obtained using conventional techniques. In particular, accelerated solvent extraction was more superior to other techniques, which highlights the advantages of this sample preparation technique. However, in routine analysis, shaking and ultrasonically assisted extraction is still the preferred method for the solution of robenidine and other coccidiostatics.
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PMID:Comparison of extraction techniques of robenidine from poultry feed samples. 1907 6

A high-density cell culture method was successfully established in P. pastoris with the alcohol oxidase I (AOXI) promoter in order to produce large quantities of recombinant human angiostatin (AS) which has been reported to have antiangiogenic activity. A preliminary study on fermentation conditions in shaking flasks indicated that adequacy of biomass is beneficial to obtain more products. The fermentation was carried out in a 10 l bioreactor with 5 l modified growth medium recommended by Invitrogen at 30 degrees C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density reached A(600) = 125, methanol-PTM4 trace salts was added to induce the expression of AS. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by the addition of 7 M NH(4)OH and the biomass was maintained at about A(600) = 200. After 60 h of induction, the secreted AS was 153 mg/l. The recombinant AS inhibited the angiogenesis on CAM and suppressed the growth of B16 melanoma in C57BL/6J mice (P \0.01).
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PMID:Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture. 1912 68

Theeffects of four extracting solvents [absolute ethanol, absolute methanol, aqueous ethanol (ethanol: water, 80:20 v/v) and aqueous methanol (methanol: water, 80:20 v/v)] and two extraction techniques (shaking and reflux) on the antioxidant activity of extracts of barks of Azadirachta indica, Acacia nilotica, Eugenia jambolana, Terminalia arjuna, leaves and roots of Moringa oleifera, fruit of Ficus religiosa,and leaves of Aloe barbadensis were investigated. The tested plant materials contained appreciable amounts of total phenolic contents (0.31-16.5 g GAE /100g DW), total flavonoid (2.63-8.66 g CE/100g DW); reducing power at 10 mg/mL extract concentration (1.36-2.91), DPPH(.) scavenging capacity (37.2-86.6%), and percent inhibition of linoleic acid (66.0-90.6%). Generally higher extract yields, phenolic contents and plant material antioxidant activity were obtained using aqueous organic solvents, as compared to the respective absolute organic solvents. Although higher extract yields were obtained by the refluxing extraction technique, in general higher amounts of total phenolic contents and better antioxidant activity were found in the extracts prepared using a shaker.
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PMID:Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. 1955 90

Efficient porcine interferon-alpha (pIFN-alpha) expression in high density recombinant Pichia pastoris cultivation was achieved in a 5 l bench-scaled bioreactor. The results indicated that a high and stable oxygen uptake rate (OUR) during induction phase was closely related with pIFN-alpha production efficiency. The multi-variables clustering and analysis results showed that the achievement of a high and stable OUR relied on a higher glycerol consumption rate during fed-batch culture phase and a moderate methanol level (around 10 g/l) during induction phase. In the high and stable OUR environments (200-300 mmol/l/h), the highest pIFN-alpha antiviral activity could reach a level of 6.7 x 10(6) IU/ml, which was more than 10-300-folds higher than those obtained at lower OUR (80-200 mmol/l/h) using the same bioreactor and those obtained in shaking flasks. Clustering and analysis of the specific growth and glycerol consumption rates data during culture phase could detect the ill fermentation state at early stage, potentially provided a simple and effective fault alarming/diagnosis method for the achievement of stable pIFN-alpha production.
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PMID:Effective and stable porcine interferon-alpha production by Pichia pastoris fed-batch cultivation with multi-variables clustering and analysis. 1964 59

We produced high pathogenic avian influenza H5N1 haemagglutinin protein HA1 in recombinant Pichia pastoris in a 10 L fermentor, to establish a high-density cell fermentation method. We studied the effects of different factors such as culture temperature, induced temperature, methanol feeding methods, trace elements on the growth of Pichia pastoris, the yield and the biologic activity of recombinant HA1 protein. The culture temperature in pre-induced and induced stage were optimized at 25 degrees C to adapt cell growth and recombinant protein expression, and induced temperature at 25 degrees C also resulted in higher biologic activity of rHA1 than at 30 degrees C. The binding activity of rHA1 against a wide-spectrum neutralizing antibody was susceptible to the presence of any trace elements, although trace elements would essentially benefit for the cell fermentation. As a conclusion, the expression level of rHA1 produced with optimized fermentation process reached 120 mg/L, which was 10.5 times higher than the one produced in regular shaking flask. The resultant high-density cell fermentation can likely produce rHA1 of H5N1 in large scale.
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PMID:[Expression of H5N1 avian influenza virus haemagglutinin protein in pichia pastoris by high-density cell fermentation]. 1967 Jun 49

The sensitivity and specificity of a novel method of screening for cocaine in hair, based on matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS), have been evaluated. The method entails a rapid extraction procedure consisting of shaking 2.5 mg pulverised hair at high frequency in the presence of an acidic solution (160 microL of water, 20 microL of acetonitrile and 20 microL of 1 M trifluoroacetic acid) and a stainless-steel bullet. Following centrifugation, the supernatant is dried under a nitrogen stream, and the residue is reconstituted in 10 microL of methanol/trifluoroacetic acid (7:3; v/v). One microlitre of the extract is deposed on a MALDI sample holder previously scrubbed with graphite; an alpha-cyano-4-hydroxycinnamic acid (matrix) solution is electrosprayed over the dried sample surface to achieve a uniform distribution of matrix crystals. The identification of cocaine is obtained by post-source decay experiments performed on its MH(+) ion (m/z 304), with a limit of detection of 0.1 ng/mg of cocaine. A total of 304 hair samples were analysed in parallel by MALDI-MS and a reference gas chromatography-MS method. The obtained results demonstrate specificity and sensitivity of 100% for MALDI-MS. Evidence of cocaine presence was easily obtained even when hair samples exhibiting particularly low cocaine levels (<0.5 ng/mg) were analysed.
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PMID:Validation of a fast screening method for the detection of cocaine in hair by MALDI-MS. 2006 51

To prepare recombinant fox growth hormone (fGH), we amplified its cDNA from silver fox pituitary tissue by RT-PCR and cloned into yeast shuttle vector pPIC9K down stream of a-factor signal peptide sequence by SnaB I and Not I restriction sites. The recombinant secretion vector pPIC9K/fGH, linearized by Sal I, was transformed into histidine-deficient Pichia pastoris strain GS115 by electroporation. We selected His+ -transformed methylotropic (His+, Mut+) yeast using histidine-absent medium containing dextrose (MD) or methanol (MM) as the only carbon source, and then screened the recombinant GS115 with multi-copy fGH genes by G418. The secretive expression of fGH was performed under the induction of methanol in shaking flask culture. The results showed that the fGH cDNA sequence amplified in this paper was basically in consistence with the published in GenBank. We achieved the secretive expression of recombinant fGH identified by SDS-PAGE and Western blotting. The fGH expression level was 119 mg/L, accounted for 34% of total proteins in fermentation medium.
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PMID:[Cloning and expression of fox growth hormone gene in Pichia pastoris]. 2011 90

The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha. We used assembly PCR to synthesize the native gene HPV16L1-N and the codon optimized gene HPV16L1. The synthesized genes were cloned into pMOXZa-A vector to generate plasmids pMOXZ-HPV16N and pMOXZ-HPV16. The expression cassettes MOXp-HPV16L1(N)-AOXTT were cloned into YEp352 vector and transferred into H. polymorpha. After methanol inducement, the expression of L1 protein in H. polymorpha was detected from the codon optimized gene HPV16L1 rather than the native gene HPVI6L1-N. The parameters for induced cultivation for strain HP-U-16L with HPV16L1 were investigated in shaking flask cultures. After induced cultivation in YPM (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 12 h at 37 degrees C for 72 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by H. polymorpha.
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PMID:[Optimized expression of the L1 protein of human papillomavirus in Hansenula polymorpha]. 2011 96

The risk assessment of hydrophobic organic compounds (HOCs) in aquatic toxicity or bioconcentration tests is a challenge due to their low aqueous solubilities, sorption and losses leading to poorly defined exposure and reduced test sensitivity. Passive dosing overcomes these problems via the continual partitioning of HOCs from a dominating reservoir loaded in a biocompatible polymer such as silicone, providing defined and constant freely dissolved concentrations and eliminating spiking with co-solvents. This study characterised the performance of a passive dosing format for aquatic tests with small organism such as invertebrates and algae, consisting of PDMS silicone cast into the base of the glass test vessel. The PDMS silicone was loaded by partitioning from a methanol solution containing PAHs (logK(OW) 3.56-6.63) as model compounds, followed by removal of the methanol with water. This resulted in highly reproducible PDMS silicone HOC concentrations. When shaking, release of PAHs into aqueous solution was rapid and reproducible, and equilibrium partitioning was reached within 5h for all compounds. The buffering capacity was sufficient to maintain stable concentrations over more than 10 weeks. This format was applied in a 48h Daphnia magna immobilisation assay to test the toxicity of a range of PAHs at their aqueous solubility. D. magna immobilisation did not show a trend with aqueous solubility or hydophobicity (K(OW)) of the PAHs. However, the immobilisation data for all compounds could be fitted with one maximum chemical activity response curve. Those PAHs with the lowest maximum chemical activities resulted in no immobilisation. Naphthalene and phenanthrene showed full toxicity at aqueous solubility, and passive dosing was also used for the concentration-response testing of these compounds. The freely dissolved aqueous concentrations causing 50% immobilisation (EC-50) were 1.96 mg L(-1) for naphthalene and 0.48 mg L(-1) for phenanthrene. Therefore, passive dosing is a practical and economical means of improving the exposure of HOCs in aquatic toxicity or bioconcentration tests.
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PMID:Controlling and maintaining exposure of hydrophobic organic compounds in aquatic toxicity tests by passive dosing. 2017 Sep 70


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