Plasma biochemical values of clinically-normal Australian sea lions (Neophoca cinerea).

Seventeen biochemical constituents were assayed in the blood plasma of clinically-normal Australian sea lions (Neophoca cinerea). The sea lions formed part of a breeding colony which inhabits the southern coast of Kangaroo Island, South Australia. Little variation was found in any of the values obtained from animals of different age and sex. The results were compared with values published for California sea lions (Zalophus californianus), Northern fur seals (Callorhinus ursinus), Harbor seals (Phoca vitulina) and Northern elephant seals (Mirounga angustirostris).     

A pre‐breeding immune challenge delays reproduction in the female dark‐eyed junco Junco hyemalis

Precise timing of life‐history transitions in predictably changing environments is hypothesized to aid in individual survival and reproductive success, by appropriately matching an animal's physiology and behavior with prevailing environmental conditions. Therefore, it is imperative for individuals to time energetically costly life‐history stages (i.e. reproduction) so they overlap with seasonal peaks in food abundance and quality. Female lifetime reproductive fitness is affected by several factors that influence energy balance, including arrival date, timing of egg production, and energetic condition. Therefore, any extra energetic costs during reproduction may negatively affect timing of egg production, and ultimately a female's fitness. For example, mounting an immunological response elicits a high energetic cost, and this transfer of resources towards cell and immune system maintenance could have direct negative effects on reproductive timing. In order to determine whether an immune challenge delays onset of breeding (i.e. egg production), we administered either a humoral immune challenge (keyhole limpet hemocyanin (KLH)) (treatment) or physiological saline (control) to free‐living female dark‐eyed juncos Junco hyemalis in the period immediately prior to egg‐laying (～4 weeks). We found that KLH‐injected females artificially delayed clutch initiation when compared to control females. These data help to refine our understanding of how free‐living birds allocate resources between reproduction and self‐maintenance processes during the critical pre‐laying period of the annual cycle.     

Viruses infecting a warm water picoeukaryote shed light on spatial co-occurrence dynamics of marine viruses and their hosts

The marine picoeukaryote Bathycoccus prasinos has been considered a cosmopolitan alga, although recent studies indicate two ecotypes exist, Clade BI (B. prasinos) and Clade BII. Viruses that infect Bathycoccus Clade BI are known (BpVs), but not that infect BII. We isolated three dsDNA prasinoviruses from the Sargasso Sea against Clade BII isolate RCC716. The BII-Vs do not infect BI, and two (BII-V2 and BII-V3) have larger genomes (~210 kb) than BI-Viruses and BII-V1. BII-Vs share ~90% of their proteins, and between 65% to 83% of their proteins with sequenced BpVs. Phylogenomic reconstructions and PolB analyses establish close-relatedness of BII-V2 and BII-V3, yet BII-V2 has 10-fold higher infectivity and induces greater mortality on host isolate RCC716. BII-V1 is more distant, has a shorter latent period, and infects both available BII isolates, RCC716 and RCC715, while BII-V2 and BII-V3 do not exhibit productive infection of the latter in our experiments. Global metagenome analyses show Clade BI and BII algal relative abundances correlate positively with their respective viruses. The distributions delineate BI/BpVs as occupying lower temperature mesotrophic and coastal systems, whereas BII/BII-Vs occupy warmer temperature, higher salinity ecosystems. Accordingly, with molecular diagnostic support, we name Clade BII Bathycoccus calidus sp. nov. and propose that molecular diversity within this new species likely connects to the differentiated host-virus dynamics observed in our time course experiments. Overall, the tightly linked biogeography of Bathycoccus host and virus clades observed herein supports species-level host specificity, with strain-level variations in infection parameters.Subject terms: Microbial biooceanography, Phylogenetics     

Codon usage bias analysis of genes linked with esophagus cancer

Esophageal cancer involves multiple genetic alternations. A systematic codon usage bias analysis was completed to investigate the bias among the esophageal cancer responsive genes. GC-rich genes were low (average effective number of codon value was 49.28). CAG and GTA are over-represented and under-represented codons, respectively. Correspondence analysis, neutrality plot, and parity rule 2 plot analysis confirmed the dominance over mutation pressure in modulating the codon usage pattern of genes linked with esophageal cancer.     

Cross validation and ruggedness testing of analytical methods used for the quantification of urinary phthalate metabolites

Since the publication of our first analytical method in 2000 to detect and quantify phthalate metabolites in human urine, we have modified the method several times to improve performance, reduce the volume of matrix and solvents used, and to increase the number of analytes in one analytical run. We performed cross method validation and ruggedness testing after each modification to ensure that the analytical method adopted is robust and produces accurate and reproducible data when compared to the previously used method. Here, we present the results from the evaluation of the ruggedness of our analytical approach under variable experimental conditions, using the current analytical method. Minor deviations of the standard experimental conditions, i.e., pH, incubation time, amount of deconjugation enzyme, and incubation temperature, had no effect on final analyte concentrations. Furthermore, we validated the method to ensure accuracy at concentrations beyond the highest calibration standard. The concentrations obtained by using a lower volume of urine agreed well with original levels, suggesting broad linear calibration range as well as complete hydrolysis of the glucuronide conjugates with the standard amount of beta-glucuronidase used for deglucuronidation; also, the time of incubation (90 min) was adequate regardless of the amount of glucuronide present. We also summarize the precision of concentration data acquired by the five different analytical approaches we have used since 2000. The correlation plots of concentration data for each analyte obtained from split sample analysis, using three of these approaches, produced linear curves (R(2)>0.98) with slopes and intercepts that were not statistically different (p>0.05) from 1 and 0, respectively. These results suggest that the data are reproducible and accurate, regardless of the analytical method used. Furthermore, analysis of quality control urine samples made over the years confirmed the stability of the phthalate metabolites in urine at -70 degrees C for several years and the consistency of the analytical measurements obtained by using various methodological approaches over time.     

Interaction of synthetic HA2 influenza fusion peptide analog with model membranes.

The interaction of the synthetic 21 amino acid peptide (AcE4K) with 1-oleoyl-2-[caproyl-7-NBD]-sn-glycero-3-phosphocholine membranes is used as a model system for the pH-sensitive binding of fusion peptides to membranes. The sequence of AcE4K (Ac-GLFEAIAGFIENGWEGMIDGK) is based on the sequence of the hemagglutinin HA2 fusion peptide and has similar partitioning into phosphatidylcholine membranes as the viral peptide. pH-dependent partitioning in the membrane, circular dichroism, tryptophan fluorescence, change of membrane area, and membrane strength, are measured to characterize various key aspects of the peptide-membrane interaction. The experimental results show that the partitioning of AcE4K in the membrane is pH dependent. The bound peptide inserts in the membrane, which increases the overall membrane area in a pH-dependent manner, however the depth of insertion of the peptide in the membrane is independent of pH. This result suggests that the binding of the peptide to the membrane is driven by the protonation of its three glutamatic acids and the aspartic acid, which results in an increase of the number of bound molecules as the pH decreases from pH 7 to 4.5. The transition between the bound state and the free state is characterized by the Gibbs energy for peptide binding. This Gibbs energy for pH 5 is equal to -30.2 kJ/mol (-7.2 kcal/mol). Most of the change of the Gibbs energy during the binding of AcE4K is due to the enthalpy of binding -27.3 kJ/mol (-6.5 kcal/mol), while the entropy change is relatively small and is on the order of 6.4 J/mol.K (2.3 cal/mol.K). The energy barrier separating the bound and the free state, is characterized by the Gibbs energy of the transition state for peptide adsorption. This Gibbs energy is equal to 51.3 kJ/mol (12.3 kcal/mol). The insertion of the peptide into the membrane is coupled with work for creation of a vacancy for the peptide in the membrane. This work is calculated from the measured area occupied by a single peptide molecule (220 A(2)) and the membrane elasticity (190 mN/m), and is equal to 15.5 kJ/mol (3.7 kcal/mol). The comparison of the work for creating a vacancy and the Gibbs energy of the transition state shows that the work for creating a vacancy may have significant effect on the rate of peptide insertion and therefore plays an important role in peptide binding. Because the work for creating a vacancy depends on membrane elasticity and the elasticity of the membrane is dependent on membrane composition, this provides a tool for modulating the pH for membrane instability by changing membrane composition. The insertion of the peptide in the membrane does not affect the membrane permeability for water, which shows that the peptide does not perturb substantially the packing of the hydrocarbon region. However, the ability of the membrane to retain solutes in the presence of peptide is compromised, suggesting that the inserted peptide promotes formation of short living pores. The integrity of the membrane is substantially compromised below pH 4.8 (threshold pH), when large pores are formed and the membrane breaks down. The binding of the peptide in the pore region is reversible, and the pore size varies on the experimental conditions, which suggests that the peptide in the pore region does not form oligomers.     

Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56 U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6 µg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79–84%) transfected muscle fibers with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle.