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Determination of the In Vivo Pharmacodynamic Profile of Cefepime against Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli at Various Inocula. Dana Maglio, 2004.Cefepime was evaluated in vivo against two inoculum sizes of four strains of Escherichia coli that produced extended-spectrum beta-lactamases (ESBLs) in a murine neutropenic thigh infection model to characterize the pharmacodynamic activity of cefepime in the presence of ESBL-producing bacteria and to evaluate if differences in lengths of cefepime exposure are required with various inocula . Three strains possessed a single enzyme each: TEM-10, TEM-12, and TEM-26 . The fourth strain possessed two TEM-derived ESBLs and a third uncharacterized enzyme . Two non-ESBL-producing E . coli strains were included for comparison . Mice received various doses of cefepime to achieve a spectrum of percentages of time the drug was above the MIC (%T>MICs) for each isolate at both inocula . No significant difference in cefepime exposure was required to achieve similar bactericidal effects for ESBL- and non-ESBL-producing isolates when the starting inoculum was 105 CFU of E . coli per thigh . The increased MICs observed in vitro for the ESBL-producing strains at 107 CFU/ml did not predict the amount of exposure required to achieve a comparable level of bactericidal activity in vivo at the corresponding starting inoculum of 107 CFU/thigh . Compared to the cefepime exposure in tests with the lower inoculum (105 CFU/thigh), less exposure was required when the starting inoculum was 107 CFU/thigh (%T>MIC, 6% versus 26%), such that similar doses (in milligrams per kilogram of body weight) produced similar bactericidal effects with both inocula of ESBL-producing isolates . Equivalent exposures of cefepime produced similar effects against the microorganisms regardless of the presence of ESBL production . Pharmacodynamic profiling undertaken with conventional cefepime MIC determinations predicted in vivo microbial outcomes at both inoculum sizes for the ESBL-producing isolates evaluated in this study . These data support the use of conventional MIC determinations in the pharmacodynamic assessment of cefepime . In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris. Wouter Vervecken, 2004.The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells . In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues . This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues . Here, we describe engineering of the P . pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans . This was accomplished by inactivation of OCH1 and overexpression of an  -1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and ß-1,4-galactosyltransferase retained in the Golgi apparatus . The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins . The procedures which we developed allow glycan engineering of any P . pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides .
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