Spectrofluorimetric determination of certain biologically active phenothiazines in commercial dosage forms and human plasma

A validated simple and sensitive spectrofluorimetric method was developed for the determination of chlorpromazine hydrochloride, promethazine hydrochloride, trifluperazine hydrochloride, thioridazine hydrochloride, perazine maleate and oxomemazine. The method was based on condensation of malonic acid/acetic anhydride (MAA) under the catalytic effect of the tertiary amine moiety of the studied phenothiazines to provide a deep yellow to brown colour with green florescence. Relative fluorescence intensity of the products was measured at λ_exc 398 nm and λ_em 432 nm. Different variables affecting the reaction were studied and optimized. The method was successfully applied for the determination of the studied drugs in commercial dosage forms. The lower detection limits allowed the application of this method for the determination of the compounds in plasma as an example of a biological fluid. In addition, the method was considered specific for the determination of tertiary amines in the presence of primary and secondary amines; as a result, it was deemed suitable for the determination of the cited drugs in the presence of their degradation products resulting from N‐dealkylation or oxidation of the corresponding sulphoxides or sulphones. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid methods of detecting the target molecule in immunohistology using a bioluminescence probe

We demonstrate a novel rapid direct detection method for immunohistochemistry, using a bioluminescent probe. An anti‐CEA antibody‐fused far‐red bioluminescent protein can monitor the accumulation of this type of probe in tumour tissues. The bimodal spectrum (λ_max = 460 and 675 nm) of this bioluminescent probe is extremely stable under different conditions of pH and ion concentration. The sensitivity of our bioluminescent labelling was at the same level of enzymatic labelling, e.g. peroxidase, as an indirect system. Our novel technique is simple and can shorten the pretreatment time of paraffin sections to around 30 min. The utility of our bioluminescent labelling covers all imaging in vitro, in vivo and ex vivo, suggesting that our antibody‐fused bioluminescent probe has the potential to detect tumour antigens with a high sensitivity in routine immune histological examinations. Copyright © 2012 John Wiley & Sons, Ltd.     

Optimization and validation of spectrofluorimetric method for determination of cefadroxile and cefuroxime sodium in pharmaceutical formulations

A simple, accurate, precise and validated spectrofluorimetric method is proposed for the determination of two cephalosporins, namely, cefadroxile (cefa) and cefuroxime sodium (cefu) in pharmaceutical formulations. The method is based on a reaction between cephalosporins with 1,2‐naphthoquinone‐4‐sulfonate in alkaline medium, to form fluorescent derivatives that are extracted with chloroform and subsequently measured at 610 and 605 nm after excitation at 470 and 460 nm for cefa and cefu respectively. The optimum experimental conditions have been studied. Beer's law is obeyed over the concentrations of 20–70 ng/mL and 15–40 ng/mL for cefa and cefu, respectively. The detection limits were 4.46 ng/mL and 3.02 ng/mL with a linear regression correlation coefficient of 0.9984 and 0.998, and recoveries ranging 97.50–109.96% and 95.73–98.89% for cefa and cefu, respectively. The effects of pH, temperature, reaction time, 1,2‐naphthoquinone‐4‐sulfonic concentration and extraction solvent on the determination of cefa and cefu, have been examined. The proposed method can be applied for the determination of cefa and cefu in pharmaceutical formulations in quality control laboratories. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a dual‐label time‐resolved fluoroimmunoassay for the detection of α‐fetoprotein and hepatitis B virus surface antigen

Time‐resolved fluorometry of lanthanide chelates is one of the most useful non‐isotopic detection techniques and has been used in numerous applications in biomedical science. We developed a time‐resolved fluoroimmunoassay (TRFIA) to quantify α‐fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg) in human serum. Based on a two‐site sandwich protocol, monoclonal antibodies (McAbs) against AFP and HBsAg were co‐coated in 96 microtitration wells and tracer McAbs against HBsAg and AFP were labeled with europium (Eu) and samarium (Sm) chelates, respectively. After application of diluted serum samples, Eu³⁺‐ and Sm³⁺‐McAbs were added and fluorescence signals of Sm³⁺ and Eu³⁺ tracers were collected. Detection limits of AFP and HBsAg were 0.09 mIU/L and 0.01 µg/L, respectively. Measurement ranges of AFP‐TRFIA and HBsAg‐TRFIA were 1–1000 mIU/L and 0.2‐150 µg/L, respectively. Intra‐ and inter‐assay coefficients of variation of AFP‐TRFIA were 3.3‐4.1% and 5.7‐7.2% and for HBsAg‐TRFIA were 2.9‐3.9% and 4.9‐6.8%, respectively. Linear correlation of TRFIA and chemiluminescence immunoassay measurements resulted in a correlation coefficient of 0.9949 for AFP and 0.9940 for HBsAg. For the endurance test, Eu‐labeled McAbs were stable for at least one year at ?20°C and the results of the TRFIA with the same reagents were also reproducible after one year. The availability of a highly sensitive, reliable and convenient AFP/HBsAg TRFIA will allow the quantification of both AFP and HBsAg, thereby providing diagnostic value in various clinical conditions and could be applied for clinical use. Copyright © 2012 John Wiley & Sons, Ltd.     

Spinal cord regeneration: Lessons for mammals from non‐mammalian vertebrates

Unlike mammals, regenerative model organisms such as amphibians and fish are capable of spinal cord regeneration after injury. Certain key differences between regenerative and nonregenerative organisms have been suggested as involved in promoting this process, such as the capacity for neurogenesis and axonal regeneration, which appear to be facilitated by favorable astroglial, inflammatory and immune responses. These traits provide a regenerative‐permissive environment that the mammalian spinal cord appears to be lacking. Evidence for the regenerative nonpermissive environment in mammals is given by the fact that they possess neural stem/progenitor cells, which transplanted into permissive environments are able to give rise to new neurons, whereas in the nonpermissive spinal cord they are unable to do so. We discuss the traits that are favorable for regeneration, comparing what happens in mammals with each regenerative organism, aiming to describe and identify the key differences that allow regeneration. This comparison should lead us toward finding how to promote regeneration in organisms that are unable to do so. genesis 51:529–544. © 2013 Wiley Periodicals, Inc.     

Mx1‐Cre mediated Rgs12 conditional knockout mice exhibit increased bone mass phenotype

Regulators of G‐protein Signaling (Rgs) proteins are the members of a multigene family of GTPase‐accelerating proteins (GAP) for the Galpha subunit of heterotrimeric G‐proteins. Rgs proteins play critical roles in the regulation of G protein couple receptor (GPCR) signaling in normal physiology and human diseases such as cancer, heart diseases, and inflammation. Rgs12 is the largest protein of the Rgs protein family. Some in vitro studies have demonstrated that Rgs12 plays a critical role in regulating cell differentiation and migration; however its function and mechanism in vivo is largely unknown. Here, we generated a floxed Rgs12 allele (Rgs12^flox/flox) in which the exon 2, containing both PDZ and PTB_PID domains of Rgs12, was flanked with two loxp sites. By using the inducible Mx1‐cre and Poly I:C system to specifically delete Rgs12 at postnatal 10 days in interferon‐responsive cells including monocyte and macrophage cells, we found that Rgs12 mutant mice had growth retardation with the phenotype of increased bone mass. We further found that deletion of Rgs12 reduced osteoclast numbers and had no significant effect on osteoblast formation. Thus, Rgs12^flox/flox conditional mice provide a valuable tool for in vivo analysis of Rgs12 function and mechanism through time‐ and cell‐specific deletion of Rgs12. genesis 51:201–209, 2013. © 2013 Wiley Periodicals, Inc.     

DNA methylation profiles provide a viable index for porcine pluripotent stem cells

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor‐intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)‐specific hypomethylated loci (EShypo‐T‐DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso‐4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso‐4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso‐622‐14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum‐free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763–776. © 2013 Wiley Periodicals, Inc.     

Simple and efficient CRISPR/Cas9‐mediated targeted mutagenesis in Xenopus tropicalis

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers. genesis 51:835–843. © 2013 Wiley Periodicals, Inc.     

Evaluation of a novel pneumatic bioreactor system for culture of recombinant Chinese hamster ovary cells

Single use culture systems are a tool in research and biotechnology manufacturing processes and are employed in mammalian cell-based manufacturing processes. Recently, we characterized a novel bioreactor system developed by PBS Biotech. The Pneumatic Bioreactor System? (PBS) employs the Air-wheel?, which is a mixing device similar in structure to a water wheel but is driven by the buoyant force of gas bubbles. In this study, we investigated the physical properties of the PBS system, with which we performed biological tests. In 2 L PBS, the mixing times ranged from 6 (30 rpm, 0.175 vvm) to 15 sec (10 rpm, 0.025 vvm). The k_La value reached upto 7.66/h at 0.5 vvm, even without a microsparger, though this condition is not applicable for cell cultures. Also, when a 10 L PBS equipped with a microsparger was evaluated, a k_La value of upto approximately 20/h was obtained particularly in mild cell culture conditions. We performed cultivation of Chinese hamster ovary (CHO) cells in 2 and 10 L PBS prototypes. Results from the PBS were compared with those from an Erlenmeyer flask and conventional stirred tank type bioreactor (STR). The maximum cell density of 10.6 × 106 cells/mL obtained fromthe 2 L PBSwas about 2 times higher than that from the Erlenmeyer flask (5.6 × 10⁶ cells/mL) andwas similar to the STR (9.7 × 10⁶ cells/mL) when the CHO-S cells were cultured. These results support the general suitability of the PBS system using pneumatic mixing for suspension cell cultivation as a novel single-use bioreactor system.     

Revision of the subfamily Opiinae (Hymenoptera,Braconidae) from Hunan (China), including thirty-six new species and two new genera

The species of the subfamily Opiinae (Hymenoptera: Braconidae) from Hunan (Oriental China) are revised and illustrated. Thirty-six new species are described: Apodesmia bruniclypealis Li & van Achterberg, sp. n., Apodesmia melliclypealis Li & van Achterberg, sp. n., Areotetes albiferus Li & van Achterberg, sp. n., Areotetes carinuliferus Li & van Achterberg, sp. n., Areotetes striatiferus Li & van Achterberg, sp. n., Coleopioides diversinotum Li & van Achterberg, sp. n., Coleopioides postpectalis Li & van Achterberg, sp. n., Fopius dorsopiferus Li, van Achterberg & Tan, sp. n., Indiopius chenae Li & van Achterberg, sp. n., Opiognathus aulaciferus Li & van Achterberg, sp. n., Opiognathus brevibasalis Li & van Achterberg, sp. n., Opius crenuliferus Li & van Achterberg, sp. n., Opius malarator Li, van Achterberg & Tan, sp. n., Opius monilipalpis Li & van Achterberg, sp. n., Opius pachymerus Li & van Achterberg, sp. n., Opius songi Li & van Achterberg, sp. n., Opius youi Li & van Achterberg, sp. n., Opius zengi Li & van Achterberg, sp. n., Phaedrotoma acuticlypeata Li & van Achterberg, sp. n., Phaedrotoma angiclypeata Li & van Achterberg, sp. n., Phaedrotoma antenervalis Li & van Achterberg, sp. n., Phaedrotoma depressiclypealis Li & van Achterberg, sp. n., Phaedrotoma flavisoma Li & van Achterberg, sp. n., Phaedrotoma nigrisoma Li & van Achterberg, sp. n., Phaedrotoma protuberator Li & van Achterberg, sp. n., Phaedrotoma rugulifera Li & van Achterberg, sp. n., Li & van Achterberg,Phaedrotoma striatinota Li & van Achterberg, sp. n., Phaedrotoma vermiculifera Li & van Achterberg, sp. n., Rhogadopsis latipennis Li & van Achterberg, sp. n., Rhogadopsis longicaudifera Li & van Achterberg, sp. n., Rhogadopsis maculosa Li, van Achterberg & Tan, sp. n., Rhogadopsis obliqua Li & van Achterberg, sp. n., Rhogadopsis sculpturator Li & van Achterberg, sp. n., Utetes longicarinatus Li & van Achterberg, sp. n. and Xynobius notauliferus Li & van Achterberg, sp. n. Areotetes van Achterberg & Li, gen. n. (type species: Areotetes carinuliferus sp. n.) and Coleopioides van Achterberg & Li, gen. n. (type species: Coleopioides postpectalis sp. n. are described. All species are illustrated and keyed. In total 30 species of Opiinae are sequenced and the cladograms are presented. Neopius Gahan, 1917, Opiognathus Fischer, 1972, Opiostomus Fischer, 1972, and Rhogadopsis Brèthes, 1913, are treated as a valid genera based on molecular and morphological differences. Opius vittata Chen & Weng, 2005 (not Opius vittatus Ruschka, 1915), Opius ambiguus Weng & Chen, 2005 (not Wesmael, 1835) and Opius mitis Chen & Weng, 2005 (not Fischer, 1963) are primary homonymsandarerenamed into Phaedrotoma depressa Li & van Achterberg, nom. n., Opius cheni Li & van Achterberg, nom. n. andOpius wengi Li & van Achterberg, nom. n., respectively. Phaedrotoma terga (Chen & Weng, 2005) comb. n.,Diachasmimorpha longicaudata (Ashmead, 1905) and Biosteres pavitita Chen & Weng, 2005, are reported new for Hunan, Opiostomus aureliae (Fischer, 1957) comb. n. is new for China and Hunan; Xynobius maculipennis(Enderlein, 1912) comb. n. is new for Hunan and continental China and Rhogadopsis longuria (Chen & Weng, 2005) comb. n. is new for Hunan. The following new combinations are given: Apodesmia puncta (Weng & Chen, 2005) comb. n., Apodesmia tracta (Weng & Chen, 2005) comb. n., Areotetes laevigatus (Weng & Chen, 2005) comb. n., Phaedrotoma dimidia (Chen & Weng, 2005) comb. n., Phaedrotoma improcera (Weng & Chen, 2005) comb. n., Phaedrotoma amputata (Weng & Chen, 2005) comb. n., Phaedrotoma larga (Weng & Chen, 2005) comb. n., Phaedrotoma osculas (Weng & Chen, 2005) comb. n., Phaedrotoma postuma (Chen & Weng, 2005) comb. n., Phaedrotoma rugulosa (Chen & Weng, 2005) comb. n., Phaedrotoma tabularis (Weng & Chen, 2005) comb. n., Rhogadopsis apii (Chen & Weng, 2005) comb. n., Rhogadopsis dimidia (Chen & Weng, 2005) comb. n., Rhogadopsis diutia (Chen & Weng, 2005) comb. n., Rhogadopsis longuria (Chen & Weng, 2005) comb. n., Rhogadopsis pratellae(Weng & Chen, 2005) comb. n., Rhogadopsis pratensis (Weng & Chen, 2005) comb. n., Rhogadopsis sculpta (Chen & Weng, 2005) comb. n., Rhogadopsis sulcifer (Fischer, 1975) comb. n., Rhogadopsis tabidula(Weng & Chen, 2005) comb. n., Xynobius complexus (Weng & Chen, 2005) comb. n., Xynobius indagatrix (Weng & Chen, 2005) comb. n., Xynobius multiarculatus (Chen & Weng, 2005) comb. n.The following (sub)genera are synonymised: Snoflakopius Fischer, 1972, Jucundopius Fischer, 1984, Opiotenes Fischer, 1998, and Oetztalotenes Fischer, 1998, with Opiostomus Fischer, 1971; Xynobiotenes Fischer, 1998, with Xynobius Foerster, 1862; Allotypus Foerster, 1862, Lemnaphilopius Fischer, 1972, Agnopius Fischer, 1982, and Cryptognathopius Fischer, 1984, with Apodesmia Foerster, 1862; Nosopoea Foerster, 1862, Tolbia Cameron, 1907, Brachycentrus Szépligeti, 1907, Baeocentrum Schulz, 1911, Hexaulax Cameron, 1910, Coeloreuteus Roman, 1910, Neodiospilus Szépligeti, 1911, Euopius Fischer, 1967, Gerius Fischer, 1972, Grimnirus Fischer, 1972, Hoenirus Fischer, 1972, Mimirus Fischer, 1972, Gastrosema Fischer, 1972, Merotrachys Fischer, 1972, Phlebosema Fischer, 1972, Neoephedrus Samanta, Tamili, Saha & Raychaudhuri, 1983, Adontopius Fischer, 1984, Kainopaeopius Fischer, 1986, Millenniopius Fischer, 1996, and Neotropopius Fischer, 1999, with Phaedrotoma Foerster, 1862.