Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses. Received: 8 January 1996 / Revised: 22 March 1996     

Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies

Therapeutic antibodies continue to develop as an emerging drug class, with a need for preclinical tools to better predict in vivo characteristics. Transgenic mice expressing human neonatal Fc receptor (hFcRn) have potential as a preclinical pharmacokinetic (PK) model to project human PK of monoclonal antibodies (mAbs). Using a panel of 27 mAbs with a broad PK range, we sought to characterize and establish utility of this preclinical animal model and provide guidance for its application in drug development of mAbs. This set of mAbs was administered to both hemizygous and homozygous hFcRn transgenic mice (Tg32) at a single intravenous dose, and PK parameters were derived. Higher hFcRn protein tissue expression was confirmed by liquid chromatography-high resolution tandem mass spectrometry in Tg32 homozygous versus hemizygous mice. Clearance (CL) was calculated using non-compartmental analysis and correlations were assessed to historical data in wild-type mouse, non-human primate (NHP), and human. Results show that mAb CL in hFcRn Tg32 homozygous mouse correlate with human (r² = 0.83, r = 0.91, p < 0.01) better than NHP (r² = 0.67, r = 0.82, p < 0.01) for this dataset. Applying simple allometric scaling using an empirically derived best-fit exponent of 0.93 enabled the prediction of human CL from the Tg32 homozygous mouse within 2-fold error for 100% of mAbs tested. Implementing the Tg32 homozygous mouse model in discovery and preclinical drug development to predict human CL may result in an overall decreased usage of monkeys for PK studies, enhancement of the early selection of lead molecules, and ultimately a decrease in the time for a drug candidate to reach the clinic.     

Focus: Microbiome: The Impact of the Gut Microbiota on Drug Metabolism and Clinical Outcome

The significance of the gut microbiota as a determinant of drug pharmacokinetics and accordingly therapeutic response is of increasing importance with the advent of modern medicines characterised by low solubility and/or permeability, or modified-release. These physicochemical properties and release kinetics prolong drug residence times within the gastrointestinal tract, wherein biotransformation by commensal microbes can occur. As the evidence base in support of this supplementary metabolic “organ” expands, novel opportunities to engineer the microbiota for clinical benefit have emerged. This review provides an overview of microbe-mediated alteration of drug pharmacokinetics, with particular emphasis on studies demonstrating proof of concept in vivo. Additionally, recent advances in modulating the microbiota to improve clinical response to therapeutics are explored.     

Detection and Characterization of Clade 1 Reassortant H5N1 Viruses Isolated from Human Cases in Vietnam during 2013

Highly pathogenic avian influenza (HPAI) H5N1 is endemic in Vietnamese poultry and has caused sporadic human infection in Vietnam since 2003. Human infections with HPAI H5N1 are of concern due to a high mortality rate and the potential for the emergence of pandemic viruses with sustained human-to-human transmission. Viruses isolated from humans in southern Vietnam have been classified as clade 1 with a single genome constellation (VN3) since their earliest detection in 2003. This is consistent with detection of this clade/genotype in poultry viruses endemic to the Mekong River Delta and surrounding regions. Comparison of H5N1 viruses detected in humans from southern Vietnamese provinces during 2012 and 2013 revealed the emergence of a 2013 reassortant virus with clade 1.1.2 hemagglutinin (HA) and neuraminidase (NA) surface protein genes but internal genes derived from clade 2.3.2.1a viruses (A/Hubei/1/2010-like; VN12). Closer analysis revealed mutations in multiple genes of this novel genotype (referred to as VN49) previously associated with increased virulence in animal models and other markers of adaptation to mammalian hosts. Despite the changes identified between the 2012 and 2013 genotypes analyzed, their virulence in a ferret model was similar. Antigenically, the 2013 viruses were less cross-reactive with ferret antiserum produced to the clade 1 progenitor virus, A/Vietnam/1203/2004, but reacted with antiserum produced against a new clade 1.1.2 WHO candidate vaccine virus (A/Cambodia/W0526301/2012) with comparable hemagglutination inhibition titers as the homologous antigen. Together, these results indicate changes to both surface and internal protein genes of H5N1 viruses circulating in southern Vietnam compared to 2012 and earlier viruses.     

Genes for tRNAAsp,tRNAPro, tRNATyr and two tRNAsSer in wheat mitochondrial DNA

We have begun a systematic search for potential tRNA genes in wheat mtDNA, and present here the sequences of regions of the wheat mitochondrial genome that encode genes for tRNA^Asp (anticodon GUC), tRNA^Pro (UGG), tRNA^Tyr (GUA), and two tRNAs^Ser (UGA and GCU). These genes are all solitary, not immediately adjacent to other tRNA or known protein coding genes. Each of the encoded tRNAs can assume a secondary structure that conforms to the standard cloverleaf model, and that displays none of the structural aberrations peculiar to some of the corresponding mitochondrial tRNAs from other eukaryotes. The wheat mitochondrial tRNA sequences are, on average, substantially more similar to their eubacterial and chloroplast counterparts than to their homologues in fungal and animal mitochondria. However, an analysis of regions 150 nucleotides upstream and 100 nucleotides downstream of the tRNA coding regions has revealed no obvious conserved sequences that resemble the promoter and terminator motifs that regulate the expression of eubacterial and some chloroplast tRNA genes. When restriction digests of wheat mtDNA are probed with ³²P-labelled wheat mitochondrial tRNAs, <20 hybridizing bands are detected, whether enzymes with 4 bp or 6 bp recognition sites are used. This suggests that the wheat mitochondrial genome, despite its large size, may carry a relatively small number of tRNA genes.     

Effect of Increased Glucose Levels on Na+/K+-Pump Activity in Cultured Neuroblastoma Cells

Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo-inositol metabolism and Na+/K+-pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+-pump activity, myo-inositol metabolism, and myo-inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo-inositol metabolism and partially restored Na+/K+-pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo-inositol metabolism and these abnormalities are associated with a reduction in Na+/K+-pump activity.     

Proline excretion in Escherichia coli: A comparison of an argD + strain and a proline-excreting argD − derivative

In an attempt to deduce the physiological basis of proline excretion in argD ^– strains of Escherichia coli K12, several properties of an argD ⁺ (nonexcreting) and an argD ^– (excreting) derivative were compared. No difference was found in the transport or in the utilization of either proline or its immediate precursor, ¹-pyrroline-5-carboxylate (PCA). Furthermore, no differences were found in the physical or kinetic properties of partially purified preparations of the enzyme mediating the final step in proline biosynthesis, PCA reductase. The specific activity of PCA reductase was, however, consistently higher in crude extracts prepared from the argD ^– mutant.This work was supported by grants from the National Institutes of Allergy and Infectious Diseases (Public Health Service No. AI-10862) and The University of Connecticut Research Foundation (to C. M. B.). J. J. R. was supported by an NDEA Predoctoral Fellowship.     

Laboratory Strains of Bacillus subtilis Do Not Exhibit Swarming Motility

We redemonstrate that SwrA is essential for swarming motility in Bacillus subtilis, and we reassert that laboratory strains of B. subtilis do not swarm. Additionally, we find that a number of other genes, previously reported to be required for swarming in laboratory strains, are dispensable for robust swarming motility in an undomesticated strain. We attribute discrepancies in the literature to a lack of reproducible standard experimental conditions, selection for spontaneous swarming suppressors, inadvertent genetic linkage to swarming mutations, and auxotrophy.Many species of bacteria are capable of flagellum-mediated swimming motility in liquid broth. Of those species, a subset is also capable of a related, but genetically separable, form of flagellum-mediated surface movement called swarming motility (17). Examples of swarming-proficient species include Proteus mirabilis, Vibrio parahaemolyticus, Serratia marcescens, Escherichia coli, Salmonella enterica, and Bacillus subtilis (1, 15, 16, 20, 28). In general, swarming requires a surfactant or wetting agent to reduce surface tension, an increase in flagellar number per cell, and other genetic features that are distinct from swimming (7, 14).There is confusion in the literature concerning the genetic requirements of the swarming phenotype of B. subtilis. It is generally accepted that the ancestral undomesticated strain B. subtilis 3610 exhibits robust swarming motility (18, 20, 33). Swarming motility of strain 3610 requires the production of a secreted surfactant, called surfactin (6, 20), to reduce surface tension and permit surface spreading, and it also requires the protein SwrA to activate flagellar biosynthesis gene expression and increase the number of flagella on the cell surface (5, 20). Some reports claim that domesticated derivatives of 3610, such as the commonly used laboratory strain 168, are also swarming proficient (10, 18, 19, 24). Strain 168, however, is defective in both surfactin production (9, 25) and SwrA (5, 21, 31), and thus, swarming 168 strains challenge the genetic definition of swarming motility. Our lab has never observed swarming in laboratory strains, and here we investigated swarming motility in a reportedly swarming-proficient 168 strain.We obtained a reportedly swarming-proficient 168 strain (13) (generous gift of Simone Séror, Orsay University, Paris-Sud, France) (Table ​(Table1)1) and compared its swarming phenotype to that of 3610 under our standard conditions (20). Swarm plates were prepared one day prior to use with 25 ml of LB medium (10 g Bacto tryptone, 5 g Bacto yeast extract, 5 g NaCl per liter) fortified with 0.7% Bacto agar. To minimize water on the agar surface and thus minimize the potentially confounding influence of swimming motility, plates were dried 20 min prior to inoculation and 10 min postinoculation open-faced in a laminar flow hood. For qualitative swarm assays, plates were centrally inoculated with cells from a freshly grown overnight colony using a sterile stick. For quantitative swarm expansion assays, 1 ml of cells grown to mid-exponential phase (optical density at 600 nm [OD₆₀₀], 0.5) was resuspended in PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄ per liter, pH 7.0) containing 0.5% India ink (Higgins) to an OD₆₀₀ of 10 and centrally spotted (10 μl). Swarm expansion was measured at 0.5-h intervals along a transect on the plate. Plates were incubated at 37°C in 20 to 30% humidity. Whereas strain 3610 was swarming proficient, strain 168 (Orsay) was swarming deficient (Fig. ​(Fig.1A).1A). Thus, strain 168 (Orsay) appeared to behave similarly to all other laboratory strains we have tested previously (20, 21).Open in a separate windowFIG. 1.Swarming motility on LB and B media. In qualitative plate images, colonized agar appears white and uncolonized agar appears black on LB and B media, as indicated. Swarming cells colonize a larger surface area than nonswarming cells. All strains are derivatives of strain 3610 unless otherwise indicated. Bar, 2 cm. (A) Quantitative swarm expansion assays on solid medium and growth in liquid medium of the indicated strains on LB medium (closed symbols) and on B medium (open symbols). To indicate variability in a particular experiment, we have reproduced the quantitative swarm expansion assay of strain 3610 on LB and B media with error bars in Fig. S5 in the supplemental material. (B) Quantitative swarm expansion assays on LB (closed symbols) and B (open symbols) media. The following strains were used: DS3337 (sfp), DS2415 (swrA), DS5106 (168 swrA⁺), DS5758 (168 sfp⁺), and DS5759 (168 swrA⁺ sfp⁺). In all assays, B medium was made according to reference 2 except for strain DS5759, for which B medium was supplemented with 780 μM threonine to compensate for thrC auxotrophy. (C) Swarm plates of the indicated strains on LB medium made with equal parts peptone instead of tryptone. (D) Quantitative swarm expansion assays of the indicated 3610-derived mutant strains on LB medium (closed symbols) and on B medium (open symbols). The following strains were used: DS72 (yvzB), DS2268 (epr), DS3903 (phrC), DS4978 (rapC), DS4979 (oppD), DS2509 (swrB), and DS3649 (degU). All points are averages for three replicates.

TABLE 1.

Strains

Chromatin decondensation in S-phase involves recruitment of Cdk2 by Cdc45 and histone H1 phosphorylation

Cdc45 is required for initiation of DNA replication and fork progression, but its function in these processes remains unknown. We show that targeting Cdc45 to specific chromosomal sites in mammalian cells results in large-scale chromatin decondensation that strongly correlates with histone H1 phosphorylation. Cdk2 is recruited to sites of Cdc45 decondensation, and Cdk2 inhibitors reduce the level of decondensation. Targeting wild-type Cdk2, but not kinase-defective Cdk2, to chromatin is also effective at inducing decondensation involving phospho-H1. Cdc45, Cdk2, Cyclin A, and phospho-H1 associate with chromatin during S-phase, and Cdc45, Cdk2, and an active H1 kinase physically interact. Replicating DNA and phospho-H1 foci colocalize in vivo, and S-phase progression and H1 phosphorylation are directly related and Cdk2 dependent. Because Cdk2 colocalizes with replication foci and H1 regulates higher-order chromatin, we suggest a model in which Cdc45 recruits Cdk2 to replication foci, resulting in H1 phosphorylation, chromatin decondensation, and facilitation of fork progression.     

Contribution of a conserved phenylalanine residue to the activity of Escherichia coli uracil DNA glycosylase

Uracil DNA glycosylase (UDG) excises uracil from DNA to initiate repair of this lesion. This important DNA repair enzyme is conserved in viruses, bacteria, and eukaryotes. One residue that is conserved among all the members of the UDG family is a phenylalanine that stacks with uracil when it is flipped out of the DNA helix into the enzyme active site. To determine what contribution this conserved Phe residue makes to the activity of UDG, Phe-77 in the Escherichia coli enzyme was mutated to three different amino acid residues, alanine (UDG-F77A), asparagine (UDG-F77N), and tyrosine (UDG-F77Y). The effects of these mutations were measured on the steady-state and pre-steady-state kinetics of uracil excision in addition to enzyme.DNA binding kinetics. The overall excision activity of each of the mutants was reduced relative to the wild-type enzyme; however, each mutation gave rise to a different kinetic phenotype with different effects on substrate binding and catalysis. The excision activity of UDG-F77N was the most severely compromised, but this enzyme still bound to uracil-containing DNA at about the same rate as wild-type UDG. In contrast, the decrease in the excision activity of UDG-F77A is likely to reflect a greater reduction in uracil-DNA binding than in the catalytic step. Overall, the effects of the mutations on catalysis are best correlated with the polarity of the substituted residue such that an increase in polarity decreases the efficiency of uracil excision.