Mechanism of Microbicidal Action of E-101 Solution, a Myeloperoxidase-Mediated Antimicrobial, and Its Oxidative Products.

E-101 solution is a first-in-class myeloperoxidase-mediated antimicrobial developed for topical application. It is composed of porcine myeloperoxidase (pMPO), glucose oxidase (GO), glucose, sodium chloride, and specific amino acids in an aqueous solution. Once activated, the reactive species hydrogen peroxide (H2O2), hypochlorous acid, and singlet oxygen are generated. We evaluated the treatment effects of E-101 solution and its oxidative products on ultrastructure changes and microbicidal activity against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli Time-kill and transmission electron microscopy studies were also performed using formulations with pMPO or GO omitted. The glutathione membrane protection assay was used to study the neutralization of reactive oxygen species. The potency of E-101 solution was also measured in the presence of serum and whole blood by MIC and minimal bactericidal concentration (MBC) determinations. E-101 solution demonstrated rapid bactericidal activity and ultracellular changes in MRSA and E. coli cells. When pMPO was omitted, high levels of H2O2 generated from GO and glucose demonstrated slow microbicidal activity with minimal cellular damage. When GO was omitted from the formulation, no antimicrobial activity or cellular damage was observed. Protection from exposure to E-101 solution reactive oxygen species in the glutathione protection assay was competitive and temporary. E-101 solution maintained its antimicrobial activity in the presence of inhibitory substances, such as serum and whole blood. E-101 solution is a potent myeloperoxidase enzyme system with multiple oxidative mechanisms of action. Our findings suggest that the primary site where E-101 solution exerts microbicidal action is the cell membrane, by inactivation of essential cell membrane components.

Induction of ß-Lactamase Activity and Decreased ß-Lactam Susceptibility by CO2 in Clinical Bacterial Isolates.

Antimicrobial susceptibility testing of clinical isolates is a crucial step toward appropriate treatment of infectious diseases. The clinical isolate Francisella philomiragia 14IUHPL001, recently isolated from a 63-year-old woman with atypical pneumonia, featured decreased susceptibility to ß-lactam antibiotics when cultivated in 5% CO2. Quantitative ß-lactamase assays demonstrated a significant (P < 0.0001) increase in enzymatic activity between bacteria cultivated in 5% CO2 over those incubated in ambient air. The presence of ß-lactamase genes blaTEM and blaSHV was detected in the clinical isolate F. philomiragia 14IUHPL001 by PCR, and the genes were positively identified by nucleotide sequencing. Expression of blaTEM and blaSHV was detected by reverse transcription-PCR during growth at 5% CO2 but not during growth in ambient air. A statistically significant alkaline shift was observed following cultivation of F. philomiragia 14IUHPL001 in both ambient air and 5% CO2, allowing desegregation of the previously reported effects of acidic pH from the currently reported effect of 5% CO2 on blaTEM and blaSHV ß-lactamases. To ensure that the observed phenomenon was not unique to F. philomiragia, we evaluated a clinical isolate of blaTEM-carrying Haemophilus influenzae and found parallel induction of blaTEM gene expression and ß-lactamase activity at 5% CO2 relative to ambient air. IMPORTANCE ß-Lactamase induction and concurrent ß-lactam resistance in respiratory tract pathogens as a consequence of growth in a physiologically relevant level of CO2 are of clinical significance, particularly given the ubiquity of TEM and SHV ß-lactamase genes in diverse bacterial pathogens. This is the first report of ß-lactamase induction by 5% CO2.

Differential Evolutionary Selection and Natural Evolvability Observed in ALT Proteins of Human Filarial Parasites.

The abundant larval transcript (ALT-2) protein is present in all members of the Filarioidea, and has been reported as a potential candidate antigen for a subunit vaccine against lymphatic filariasis. To assess the potential for vaccine escape or heterologous protection, we examined the evolutionary selection acting on ALT-2. The ratios of nonsynonymous (K(a)) to synonymous (K(s)) mutation frequencies (ω) were calculated for the alt-2 genes of the lymphatic filariasis agents Brugia malayi and Wuchereria bancrofti and the agents of river blindness and African eyeworm disease Onchocerca volvulus and Loa loa. Two distinct Bayesian models of sequence evolution showed that ALT-2 of W. bancrofti and L. loa were under significant (P<0.05; P < 0.001) diversifying selection, while ALT-2 of B. malayi and O. volvulus were under neutral to stabilizing selection. Diversifying selection as measured by ω values was notably strongest on the region of ALT-2 encoding the signal peptide of L. loa and was elevated in the variable acidic domain of L. loa and W. bancrofti. Phylogenetic analysis indicated that the ALT-2 consensus sequences formed three clades: the first consisting of B. malayi, the second consisting of W. bancrofti, and the third containing both O. volvulus and L. loa. ALT-2 selection was therefore not predictable by phylogeny or pathology, as the two species parasitizing the eye were selected differently, as were the two species parasitizing the lymphatic system. The most immunogenic regions of L. loa and W. bancrofti ALT-2 sequence as modeled by antigenicity prediction analysis did not correspond with elevated levels of diversifying selection, and were not selected differently than predicted antigenic epitopes in B. malayi and O. volvulus. Measurements of ALT-2 evolvability made by χ2 analysis between alleles that were stable (O. volvulus and B. malayi) and those that were under diversifying selection (W. bancrofti and L. loa) indicated significant (P<0.01) deviations from a normal distribution for both W. bancrofti and L. loa. The relationship between evolvability and selection in L. loa followed a second order polynomial distribution (R2 = 0.89), indicating that the two factors relate to one another in accordance with an additional unknown factor. Taken together, these findings indicate discrete evolutionary drivers acting on ALT-2 of the four organisms examined, and the described variation has implications for design of novel vaccines and diagnostic reagents. Additionally, this represents the first mathematical description of evolvability in a naturally occurring setting.

Exome sequencing reveals pathogenic mutations in 91 strains of mice with Mendelian disorders.

Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data. Using these new tools, putative pathogenic mutations were identified for 91 (53%) of the strains in our study. Despite the increased power offered by potentially unlimited pedigrees and controlled breeding, about half of our exome cases remained unsolved. Using a combination of manual analyses of exome alignments and whole-genome sequencing, we provide evidence that a large fraction of unsolved exome cases have underlying structural mutations. This result directly informs efforts to investigate the similar proportion of apparently Mendelian human phenotypes that are recalcitrant to exome sequencing.

Kidney adysplasia and variable hydronephrosis, a new mutation affecting the odd-skipped related 1 gene in the mouse, causes variable defects in kidney development and hydronephrosis.

Many genes, including odd-skipped related 1 (Osr1), are involved in regulation of mammalian kidney development. We describe here a new recessive mutation (kidney adysplasia and variable hydronephrosis, kavh) in the mouse that leads to downregulation of Osr1 transcript, causing several kidney defects: agenesis, hypoplasia, and hydronephrosis with variable age of onset. The mutation is closely associated with a reciprocal translocation, T(12;17)4Rk, whose Chromosome 12 breakpoint is upstream from Osr1. The kavh/kavh mutant provides a model to study kidney development and test therapies for hydronephrosis.

A novel allele of Alx4 results in reduced Fgf10 expression and failure of eyelid fusion in mice.

Normal fusion of developing eyelids requires coordination of inductive signals from the eyelid mesenchyme with migration of the periderm cell layer and constriction of the eyelids across the eye. Failure of this process results in an eyelids open at birth (EOB) phenotype in mice. We have identified a novel spontaneous allele of Alx4 that displays EOB, in addition to polydactyly and cranial malformations. Alx4 is expressed in the eyelid mesenchyme prior to and during eyelid fusion in a domain overlapping the expression of genes that also play a role in normal eyelid development. We show that Alx4 mutant mice have reduced expression of Fgf10, a key factor expressed in the mesenchyme that is required for initiation of eyelid fusion by the periderm. This is accompanied by a reduced number of periderm cells expressing phosphorylated c-Jun, consistent with the incomplete ablation of Fgf10 expression. Together, these data demonstrate that eyelid fusion in mice requires the expression of Alx4, accompanied by the loss of normal expression of essential components of the eyelid fusion pathway.