Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: Application to a pharmacokinetic study

A rapid, selective and sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher^® 60 RP Select B column (125 mm × 4 mm i.d., 5 μm particle size) using acetonitrile:5 ± 0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0–15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within ±6.3% of nominal values. No matrix effect was observed in this method. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.     

Biochemical and structural consequences of a glycine deletion in the α-8 helix of protoporphyrinogen oxidase

A rare Gly210 deletion in protoporphyrinogen oxidase (PPO) was recently discovered in herbicide-resistant Amaranthus tuberculatus. According to the published X-ray structure of Nicotiana tabacum PPO, Gly210 is adjacent to, not in, the PPO active site, so it is a matter of interest to determine why its deletion imparts resistance to herbicides. In our kinetic experiments, this deletion did not affect the affinity of protoporphyrinogen IX nor the FAD content, but decreased the catalytic efficiency of the enzyme. The suboptimal K_cat was compensated by a significant increase in the K_is for inhibitors and a switch in their interactions from competitive to mixed-type inhibition. In our protein modeling studies on herbicide-susceptible PPO and resistant PPO, we show that Gly210 plays a key role in the αL helix-capping motif at the C-terminus of the α-8 helix which helps to stabilize the helix. In molecular dynamics simulations, the deletion had significant architecture consequences, destabilizing the α-8 helix-capping region and unraveling the last turn of the helix, leading to enlargement of the active site cavity by ∼ 50%. This seemingly innocuous deletion of Gly210 of the mitochondrial PPO imparts herbicide resistance to this dual-targeted protein without severely affecting its normal physiological function, which may explain why this unusual mutation was the favored evolutionary path for achieving resistance to PPO inhibitors.     

Comparison of micrometer‐ and scanning electron microscope‐based measurements of avian eggshell thickness

ABSTRACT The study of avian eggshell structure, including composition, pigmentation, thickness, and strength, has important ecological and economic implications. Previous investigators have used a variety of techniques to derive either direct measures or indirect estimates of eggshell thickness. Assessing the repeatability and method agreement of different techniques is necessary to permit comparison of eggshell thickness values from different studies on various genetic stocks, populations, and species. We recorded and analyzed measurements of eggshell thickness using two methods, micrometers and scanning electron microscopy (SEM), for several Palaeognathae and Neognathae taxa, including nonpasserines and passerines. Applying a tolerance‐interval approach, we found that repeatability of measurements for eggs with thinner shells (<300 μm, all Neognathae taxa) was worse than for eggs with thicker shells (Palaeognathae taxa), but was still statistically and biologically reasonable given that the relative magnitude of intramethod agreements was <11%. Our results support previous predictions that measurements made using a micrometer are comparable to those made using SEM. This finding is particularly important given the relative ease and cost efficiency of the micrometer method. Importantly, these new analyses can be used to validate the use of published data from previous studies of micrometer‐based eggshell thickness for both intra‐ and interspecific comparisons.     

MDA-5 is cleaved in poliovirus-infected cells

Infections with RNA viruses are sensed by the innate immune system through membrane-bound Toll-like receptors or the cytoplasmic RNA helicases RIG-I and MDA-5. It is believed that MDA-5 is crucial for sensing infections by picornaviruses, but there have been no studies on the role of this protein during infection with poliovirus, the prototypic picornavirus. Beginning at 4 h postinfection, MDA-5 protein is degraded in poliovirus-infected cells. Levels of MDA-5 declined beginning at 6 h after infection with rhinovirus type 1a or encephalomyocarditis virus, but the protein was stable in cells infected with rhinovirus type 16 or echovirus type 1. Cleavage of MDA-5 is not carried out by either poliovirus proteinase 2Apro or 3Cpro. Instead, degradation of MDA-5 in poliovirus-infected cells occurs in a proteasome- and caspase-dependent manner. Degradation of MDA-5 during poliovirus infection correlates with cleavage of poly(ADP) ribose polymerase (PARP), a hallmark of apoptosis. Induction of apoptosis by puromycin leads to cleavage of both PARP and MDA-5. The MDA-5 cleavage product observed in cells treated with puromycin is approximately 90 kDa, similar in size to the putative cleavage product observed in poliovirus-infected cells. Poliovirus-induced cleavage of MDA-5 may be a mechanism to antagonize production of type I interferon in response to viral infection.     

Combinatorial treatment of non-small-cell lung cancers with gefitinib and Ad.mda-7 enhances apoptosis-induction and reverses resistance to a single therapy

Activation of the epidermal growth factor receptor (EGFR) contributes to the pathogenesis of non-small-cell lung carcinomas (NSCLC) and gefitinib, a selective reversible EGFR inhibitor, is effective in treating patients with NSCLC. However, clinical resistance to gefitinib is a frequent occurrence highlighting the need for improved therapeutic strategies. Melanoma differentiation associated gene-7 (mda-7)/Interleukin-24 (IL-24) (mda-7/IL-24) displays cancer-selective apoptosis induction when delivered via a replication-incompetent adenovirus (Ad.mda-7). In this study, the effect of Ad.mda-7 infection, either alone or in combination with gefitinib, was analyzed in a panel of NSCLC cell lines carrying wild-type EGFR (H-460 and H-2030) or mutant EGFR (H-1650 and H-1975). While H-2030 and H-1650 cells were sensitive, H-460 and H-1975 cells were resistance to growth inhibition by Ad.mda-7, which was reversed by the combination of Ad.mda-7 and gefitinib. This combination increased MDA-7/IL-24 and downstream effector double-stranded RNA-activated protein kinase (PKR) protein expression, promoting apoptosis induction of NSCLC cells. Inhibition of PKR significantly inhibited apoptosis induction by Ad.mda-7 when administered alone but not when used in combination with gefitinib. The combination treatment also augmented inhibition of EGFR signaling. Our findings indicate that a combinatorial treatment with Ad.mda-7 and gefitinib may provide benefit in the treatment of NSCLC, especially in patients displaying resistance to clinically used EGFR inhibitors.     

Diabetes induces sex-dependent changes in neuronal nitric oxide synthase dimerization and function in the rat gastric antrum

Diabetic gastroparesis is a disorder that predominantly affects women. However, the biological basis of this sex bias remains completely unknown. In this study we tested the hypothesis that a component of this effect may be mediated by the nitrergic inhibitory system of the enteric nervous system. Age-matched male and female Sprague-Dawley rats were studied 8 or 12 wk after streptozotocin (55 mg/kg body wt ip)-induced sustained hyperglycemia and compared with controls. Solid gastric emptying (GE) studies were performed in all the groups. Changes in gastric antrum neuronal nitric oxide synthase (nNOS) mRNA and protein levels were analyzed by real-time PCR and Western immunoblotting, respectively. nNOS dimerization studies were performed using low-temperature SDS-PAGE. In vitro nitrergic relaxation (area under curve/mg tissue wt) was studied after the application of electric field stimulation in an organ bath. Changes in intragastric pressure (mmHg.s) in freely moving rats in the presence or absence of N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) were examined by an ambulatory telemetric method. After diabetes induction, GE is delayed in both male and female rats. However, diabetic females exhibited significant delayed GE than in diabetic males. Compared with male controls, gastric nNOS expression and nitrergic relaxation were substantially elevated in healthy female control rats, accompanied by significantly reduced intragastric pressure. The active dimeric form and dimer-to-monomer ratio of nNOSalpha were also higher in healthy females compared with male rats (P < 0.05). Diabetic females, but not males, showed significant (P < 0.05) impairment in both gastric nNOSalpha dimerization and nitrergic relaxation, accompanied by an increase in intragastric pressure. Our data provide evidence that females may have a greater dependency on the nitrergic mechanisms in health. Furthermore, diabetes seems to affect the nitrergic system to a greater extent in females than in males. Together, these changes may account for the greater vulnerability of females to diabetic gastric dysfunction.     

Synthesis and in vitro antibacterial activity of novel methylamino piperidinyl oxazolidinones

Design and synthesis of a few novel methylamino piperidinyl substituted oxazolidinones are reported. Their antibacterial activities have been evaluated in a MIC assay against broader panel of both susceptible and resistant Gram-positive strains. (S)-N-{3-[3-Fluoro-4-(methyl-{1-[3-(5-nitrofuran-2-yl)-acryloyl]-piperidin-4-yl}-amino)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide 4i has shown comparable antibacterial activity to linezolid and eperezolid in the MIC assay, additionally compound 4i showed good antibacterial activity with an in vitro MIC value of 2-4 microg/mL against linezolid resistant Staphylococcus aureus (linezolid 16 microg/mL).     

CliniProteus: A flexible clinical trials information management system

Clinical trials involve multi-site heterogeneous data generation with complex data input-formats and forms. The data should be captured and queried in an integrated fashion to facilitate further analysis. Electronic case-report forms (eCRF) are gaining popularity since it allows capture of clinical information in a rapid manner. We have designed and developed an XML based flexible clinical trials data management framework in .NET environment that can be used for efficient design and deployment of eCRFs to efficiently collate data and analyze information from multi-site clinical trials. The main components of our system include an XML form designer, a Patient registration eForm, reusable eForms, multiple-visit data capture and consolidated reports. A unique id is used for tracking the trial, site of occurrence, the patient and the year of recruitment.

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Intermittent cyclic process for enhanced biological nutrient removal treating combined chemical laboratory wastewater

The aim of this work was to assess the efficacy for simultaneous enhanced removal of nitrogen and phosphorus including organics treating combined wastewater generated from a chemical laboratory using a bench-scale Intermittent Cyclic Process Bio-reactor (ICPBR). The performance efficacy indicated that the ICPBR system with solid retention time of 15 days achieved optimum efficiency with an overall removal of ammonia nitrogen (NH4-N), phosphorus (PO4-P), and chemical oxygen demand (COD) in the range, 83-92%, 74-93%, and 90-96%, respectively.