Characterization of salt interferences in second-harmonic generation detection of protein crystals.

Studies were undertaken to assess the merits and limitations of second-harmonic generation (SHG) for the selective detection of protein and polypeptide crystal formation, focusing on the potential for false positives from SHG-active salts present in crystallization media. The SHG activities of salts commonly used in protein crystallization were measured and quantitatively compared with reference samples. Out of 19 salts investigated, six produced significant background SHG and 15 of the 96 wells of a sparse-matrix screen produced SHG upon solvent evaporation. SHG-active salts include phosphates, hydrated sulfates, formates and tartrates, while chlorides, acetates and anhydrous sulfates resulted in no detectable SHG activity. The identified SHG-active salts produced a range of signal intensities spanning nearly three orders of magnitude. However, even the weakest SHG-active salt produced signals that were several orders of magnitude greater than those produced by typical protein crystals. In general, SHG-active salts were identifiable through characteristically strong SHG and negligible two-photon-excited ultraviolet fluorescence (TPE-UVF). Exceptions included trials containing either potassium dihydrogen phosphate or ammonium formate, which produced particularly strong SHG, but with residual weak TPE-UVF signals that could potentially complicate discrimination in crystallization experiments using these precipitants.

Fourier magnitude of the field incident on a random scattering medium from spatial speckle intensity correlations.

Spatial speckle intensity correlations are used to determine the spatial Fourier magnitude of a field incident on a random scattering medium. The patterned beam is scanned across the scattering medium, and the speckle pattern on the opposite side is imaged at each beam position. A theory based on a Green's function representation is used to reconstruct the spatial Fourier magnitude of the patterned incident field.

Verification of biomechanical methods employed in a comprehensive study of mild traumatic brain injury and the effectiveness of American football helmets.

Concussion, or mild traumatic brain injury, occurs in many activities, mostly as a result of the head being accelerated. A comprehensive study has been conducted to understand better the mechanics of the impacts associated with concussion in American football. This study involves a sequence of techniques to analyse and reconstruct many different head impact scenarios. It is important to understand the validity and accuracy of these techniques in order to be able to use the results of the study to improve helmets and helmet standards. Two major categories of potential errors have been investigated. The first category concerns error sources specific to the use of crash test dummy instrumentation (accelerometers) and associated data processing techniques. These are relied upon to establish both linear and angular head acceleration responses. The second category concerns the use of broadcast video data and crash test dummy head-neck-torso systems. These are used to replicate the complex head impact scenarios of whole body collisions that occur on the football field between two living human beings. All acceleration measurement and processing techniques were based on well-established practices and standards. These proved to be reliable and reproducible. Potential errors in the linear accelerations due to electrical or mechanical noise did not exceed 2% for the three different noise sources investigated. Potential errors in the angular accelerations due to noise could be as high as 6.7%, due to error accumulation of multiple linear acceleration measurements. The potential error in the relative impact velocity between colliding heads could be as high as 11%, and was found to be the largest error source in the sequence of techniques to reconstruct the game impacts. Full-scale experiments with complete crash test dummies in staged head impacts showed maximum errors of 17% for resultant linear accelerations and 25% for resultant angular accelerations.

How predictable are aphid population responses to elevated CO2?

Experiments investigating the population responses of aphids to CO2 enrichment have yielded results suggesting that aphid populations will be both larger under elevated CO2 and that they will be smaller under elevated CO2. Most studies have failed to reject the null hypothesis of no difference in population sizes due to atmospheric CO2 concentration. This diversity of results has led some investigators to conclude that aphid responses are not general, and that every aphid-plant interaction may be unique and unpredictable a priori. We use a single, general, mathematical model to consider the population responses of cereal aphids to grass grown under different CO2 concentrations. The model shows that it is possible to explain any of the three observed results: larger populations, smaller populations, or no difference, and that which of these three outcomes arises may depend critically on the interaction between aphid nitrogen requirements and the nitrogen fertility of the soil. The model also shows that the qualitative results will depend on how sensitive the aphid species is to increases in its own density. Past studies have shown that aphids increase their production of winged offspring in response to increasing aphid density. The model predicts that, in general, aphid species that have lower nitrogen requirements and that are less sensitive to their own density will be more likely to have larger populations in elevated CO2 compared to ambient CO2. Differences between aphid species (and clones) in their nitrogen requirements and the strength of their density-dependent response have not been widely reported in the literature. Also, the nitrogen fertility of the soil has rarely been manipulated in experiments on aphid responses to rising CO2 levels. The model suggests that the diversity of population responses of aphids may be both understandable and predictable in the context of such an interaction.

A proposed new biomechanical head injury assessment function - the maximum power index.

Recently, several cases of mild traumatic brain injury to American professional football players have been reconstructed using instrumented Hybrid III anthropomorphic test dummies ATDs. The translational and rotational acceleration responses of injured and uninjured players' heads have been documented. The acceleration data have been processed according to all current head injury assessment functions including the GSI, HIC and GAMBIT among others. A new hypothesis is propounded that the threshold for head injury will be exceeded if the rate of change of kinetic energy of the head exceeds some limiting value. A functional relation is proposed, which includes all six degrees of motion and directional sensitivity characteristics, relating the rate of change of kinetic energy to the probability of head injury. The maximum value that the function achieves during impact is the maximum power input to the head and serves as an index by which the probability of head injury can be assessed.

A distance to the galaxy NGC4258 from observations of Cepheid variable stars.

Cepheid variable stars pulsate in a way that is correlated with their intrinsic luminosity, making them useful as 'standard candles' for determining distances to galaxies; the potential systematic uncertainties in the resulting distances have been estimated to be only 8-10%. They have played a crucial role in establishing the extragalactic distance scale and hence the value of the Hubble constant. Here we report observations of Cepheids in the nearby galaxy NGC4258; the distance calculated from the Cepheids is 8.1 +/- 0.4 Mpc, where the uncertainty does not include possible systematic errors. There is an independently determined geometric distance to this galaxy of 7.2 +/- 0.5 Mpc, based on the observed proper motions of water masers orbiting the central black hole; the distances differ by 1.3sigma. If the maser-based distance is adopted and the Cepheid distance scale revised accordingly, the derived value of the Hubble constant would increase by 12 +/- 9%, while the expansion age of the Universe would decrease by the same amount.

Lymphoproliferative responses of specific-pathogen-free chickens to Mycoplasma gallisepticum strain PG31.

Mycoplasma gallisepticum (MG) is one of the aetiologic agents of chronic respiratory disease in chickens and infectious sinusitis in turkeys. We investigated humoral and cellular immune mechanisms following experimental infection with four different strains of MG. Peripheral blood leukocytes (PBL) obtained from chickens were examined for proliferation using antigen preparations of whole cell MG as stimuli in vitro. A consistent lymphoproliferative response was observed against the homologous whole cell antigens in the group of chickens infected with strain PG31. Significant lymphoproliferation was detected as early as 1 week post-infection. We further characterized antigen-specific proliferation by measuring the production of interferon and nitric oxide by the PBL of infected chickens. Consistent with lymphoproliferation, we also detected the presence of interferon and nitric oxide in vitro in antigen-stimulated cultures. These results indicate a possible role of cell-mediated immune responses in the development of immunity following MG infection in chickens.

Identification of Mycoplasma iowae by colony immunoblotting utilizing monoclonal antibodies.

The utility of monoclonal antibody Mab-2C in identification of Mycoplasma iowae (MI) by colony immunoblotting technique was explored. Colony immunoblots of reference MI strains, field isolates, and mycoplasmas recovered from experimentally inoculated turkey embryos were probed with Mab-2C. The monoclonal antibody identified colonies of all the MI isolates tested and did not cross-react with colonies of M. gallisepticum, M. synoviae, or M. meleagridis. In western immunoblots of 22 MI field isolates, Mab-2C showed immunoreactivity with an antigen of approximately 45 kD molecular weight. No phenotypic variation of the epitope recognized by Mab-2C was observed in colony immunoblots of MI colonies. The monoclonal antibody reported here can be used for identification of MI colonies by a simple and rapid colony immunoblot method.

Monoclonal antibodies specific to Mycoplasma iowae.

Monoclonal antibodies (MAbs) against Mycoplasma iowae (MI) were produced to identify common immunogenic determinants shared between antigenically heterogenous MI. Twenty-four MAbs were produced against MI. With western immunoblotting, all 24 MAbs recognized a 45,000-MW protein (p45) of MI strain I-695. One of the MAbs characterized, MAb 2C, identified p45 antigen in western immunoblots with six laboratory strains and 24 field isolates of MI. MAb 2C did not cross-react with Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis, or nonpathogenic avian mycoplasmas. Triton X-114 phase separation of MI proteins showed that p45 is an integral membrane protein. In immunofluorescent staining and immunoelectron microscopy of MI, MAb 2C reacted with surface antigen(s). These MAbs specific to MI may be used in detection and diagnosis of MI infections.

Toward a comprehensive biomechanical injury cost model.

The proposed biomechanical injury cost model utilizes surrogate-based injury assessment functions to predict the probability of occurrence and the probable cost of specific injuries to the head, thorax, abdomen, and lower extremities. The resulting probability cost is a function of the number, location, and severity of injuries. As more precise injury assessment functions and more accurate cost estimates become available, the model will become an effective tool for comparing and classifying different injury patterns.

Effect of temperature on the stability of avian influenza virus antigens under different storage conditions.

The combined effect of time and temperature on the stability of two avian influenza virus (AIV) isolates concentrated with polyethylene glycol (PEG), stored at different temperatures, and used in the preparation of avian influenza vaccine was evaluated in turkeys at 24 hr and at 12, 24, 30, 36, and 42 months of storage. The differences detected between antibodies raised in turkeys by vaccines made from isolates under different storage conditions, times, and temperatures were not significant (P > 0.05), especially with vaccines prepared from one isolate. Virus recovery rates following challenge studies of vaccinated birds were similar. However, birds that were vaccinated twice had lower rates of virus recovery from the trachea, lungs, pancreas, and fecal samples following challenge infection. The results suggest that if stable isolates of AIV can be identified, such isolates can be rapidly concentrated with PEG and stored at -20 C or -196 C for at least 42 months without any loss of potency in the vaccine prepared from these isolates. This would reduce the costs associated with vaccine storage and subsequent expiration dates.

Description of osteomyelitis lesions associated with Actinomyces pyogenes infection in the proximal tibia of adult male turkeys.

Actinomyces pyogenes was isolated from osteomyelitis lesions from the proximal tibia of mature tom turkeys. Gram-stained impression smears of the lesions resulted in bacteria that appeared as club-shaped, gram-positive pleomorphic rods. The bacteria grew better in a reduced-oxygen environment. The lesions were well demarcated and cavernous, ranging from purulent to caseous in consistency.

Virulence factors of Escherichia coli associated with colisepticemia in chickens and turkeys.

Four hundred twenty turkey and 80 chicken Escherichia coli isolates from colisepticemic birds were examined for the following properties: heat-labile toxin (LT), heat-stable enterotoxin, verotoxin, colicinogenicity, hemolysin, and hydroxamate/aerobactin production. Twenty-four (5.7%) of the 420 turkey isolates and six (7.5%) of the 80 chicken isolates produced an LT that was cytotoxic for both Vero and Y-1 cells. In contrast, 48 (11.4%) of the turkey isolates and 18 (22.5%) of the chicken isolates produced a distinct LT that was cytotoxic only for Vero cells. In addition, 64 (80.0%) of the chicken isolates and 309 (74.0%) of the turkey isolates produced aerobactin. Colicinogenicity occurred in 51 (64.0%) of the chicken isolates, with 41 (51.0%) producing colicin V. By contrast, 254 (61.0%) of the turkey isolates produced a colicin, of which 176 (42.0%) produced colicin V. None of the chicken and turkey isolates produced hemolysin or heat-stable enterotoxin.

Efficacy of avridine and liposomes as adjuvants for avian influenza virus antigens in turkeys.

Four-week-old turkeys were vaccinated with H4N8, H5N2 and H7N3 inactivated avian influenza viruses (AIV) in one of the following adjuvants: avridine, positively charged liposomal avridine, liposomal avridine with covalent attachment to the AIV antigens and oil-emulsion; and without any adjuvant. Two vaccinations were given at a 4-week interval. Antibody concentrations in sera and in respiratory lavages were measured over a period of 8 weeks. The turkeys responded with high cumulative geometric mean HI titres to the H4N8, H5N2 and H7N3 components of the vaccines, while the cumulative geometric mean HI titres for the antigens without adjuvant were significantly (P < 0.05) lower. The most efficacious adjuvant, positively charged liposomal avridine, gave the highest local immune response as measured by total antibody specific to AIV in respiratory lavages. The difference in weight gains between vaccinated and non-vaccinated turkeys was significant (P<0.05). Virus isolations 2 weeks post-challenge decreased in vaccinated turkeys.

Enhancement of antibody response of turkeys to trivalent avian influenza vaccine by positively charged liposomal avridine adjuvant.

Trivalent avian influenza (AIV) antigens (H4N8, H5N2 and H7N3), mixed with positively charged, negatively charged and neutral avridine-containing liposomes, and oil-emulsion were subcutaneously administered to 6-week-old turkeys. Charged liposomal avridine adjuvant, either positive or negative, produced a better antibody response than uncharged liposomal avridine or oil-emulsion adjuvants when used in a trivalent avian influenza vaccine. The antibody response to the different antigens was generally greater to the positively charged adjuvanted vaccine compared with the negatively or neutral charged or oil-emulsion adjuvanted vaccines and these differences were significant (P less than 0.05) with the three antigens. The results suggest that the positively charged liposomal avridine plays a significant role as adjuvant to the AIV antigens.

A quantitative measurement of the effect of avian influenza virus on the ability of turkeys to eliminate Pasteurella multocida from the respiratory tract.

The effect of avian influenza virus (AIV) infection on the ability of turkeys to eliminate Pasteurella multocida from the respiratory tract was evaluated. Four-week-old turkeys were experimentally infected with an apathogenic AIV subtype (H5N2) by the oculonasal route and subsequently superinfected with P multocida (Urbach strain) by the intranasal route three days after infection with AIV. Quantitative clearance of P multocida from the trachea and lung was determined using a pour plate technique on samples collected at intervals after infection. Samples from turkeys which had been infected with AIV were found to yield more P multocida than those from turkeys which had not been infected with AIV. The numbers of P multocida increased in infected birds to a greater extent than in birds which had not been infected with the virus. The present study suggests that AIV infection may contribute to the increased numbers and a decreased clearance of P multocida in turkeys.

Endotoxin lipopolysaccharide from Escherichia coli and its effects on the phagocytic function of systemic and pulmonary macrophages in turkeys.

The effect of Escherichia coli lipopolysaccharide (LPS) on the competence of pulmonary macrophages and phagocytic cells from the systemic circulation of turkeys was examined using luminol-enhanced zymosan-stimulated chemiluminescence. The results showed a rapid and accelerated oxidative burst in both systemic and pulmonary macrophages in LPS-treated turkeys that was significantly greater than in untreated controls. However, this increased oxidative metabolism induced by LPS was not associated with enhanced intracellular bacterial killing by pulmonary macrophages. Turkeys treated with LPS showed a highly significant decrease in pulmonary bactericidal activity against Staphylococcus aureus challenge, indicating a defect in pulmonary macrophage function induced by LPS.

Serological response in broiler chicks to different commercial Newcastle disease and infectious bronchitis vaccines.

Broiler chicks were administered vaccines against Newcastle disease and infectious bronchitis (both Arkansas and Massachusetts strains) at 2 weeks of age as either primary or secondary vaccinations. The vaccine was administered as a spray at 2 weeks of age to chicks that had received Newcastle disease vaccine alone, bronchitis vaccine alone, both vaccines in combination, or no vaccine at day 1 in the hatchery. The Newcastle disease hemagglutination-inhibition response was significantly lower in chicks receiving Newcastle disease vaccine as a secondary vaccine at 2 weeks than in those receiving the vaccine as a primary vaccination at that age. In contrast, the bronchitis hemagglutination-inhibition response was significantly higher in chicks receiving bronchitis vaccine as a secondary vaccination at 2 weeks than in those receiving the vaccine as a primary vaccination at that age.