Apparatus for preparing mimics of suspended particles in the troposphere and their controlled deposition onto individual lung cells in culture with measurement of downstream biological response

Inhalation exposure to particles <10 microm in size that are suspended in the troposphere (PM10) is a factor in respiratory and cardiovascular diseases. The extent of the injury, local to systemic inflammation, is dependent on the number, size, and composition of the particles to which an individual is exposed. The physical properties of and compounds on PM10 that are responsible for these adverse effects on human health are the subject of intense investigation. Here, we report a laboratory method that involved the creation of 1-120 particles per trial that were of known size and composition, followed by deposition of them directly onto individual human lung cells within a cell culture, and after an incubation period, a downstream biological response was measured. To illustrate this methodology, particles that each contained 50 pg of lipopolysaccharide were created and deposited onto individual cells over a region <0.36 mm2 within a genetically modified A549 cell culture. The biological readout was the relative expression of intercellular cell adhesion molecule 1 after 24 h of incubation using an immunocytochemistry assay. The apparatus and methodology introduced here enables studies at the interface between the relevant but diverse areas of atmospheric particle chemistry and lung cell biology to identify the chemical and physical factors of PM10 that cause/exacerbate respiratory and cardiovascular diseases by triggering various biological pathways.     

Engineering thermal stability in RNA phage capsids via disulphide bonds

The RNA bacteriophages, a group that includes phages Qbeta and MS2, have a number of potential bionanotechnological applications, including cell specific drug delivery and as substrates for the formation of novel materials. Despite extensive sequence identity between their coat protein subunits, and an almost identical three-dimensional fold, Qbeta and MS2 capsids have dramatically different thermal stabilities. The increased stability of Qbeta has been correlated with the inter-subunit disulphide bonds present in that capsid and not present in MS2. We have tested this hypothesis directly using mass spectrometry. Analysis of the dissociated coat protein subunits suggests that inter-molecular disulphides are formed at the capsid five-fold but may not be at the three-fold axes. This conclusion has been tested by engineering disulphide cross-links into either the five-fold or three-fold positions of the recombinant MS2 capsid. Five-fold cross-linking results in a mutant with stability properties similar to those of Qbeta. Three-fold cross-linking results in a mutant unable to assemble T = 3 shells, implying that five-fold structures are on pathway to capsid assembly in these phages. The results demonstrate how it is possible to redesign the physical properties of phage shells and may be of general relevance to future applications of viruses and virus-like particles.     

Genetic basis and risk factors for infectious and noninfectious diseases in US Holsteins. I. Estimation of genetic parameters for single diseases and general health

Health data collected from 1996 to 1999 from 177 herds in Minnesota and Wisconsin were analyzed to establish genetic basis for infectious and noninfectious diseases. Three types of health traits were targeted. First, available infectious conditions were used to identify animals that are superior in their general immunity (including innate immunity) for infectious diseases. Generalized immunity may be thought of as a combination of immune responses to a variety of immune system challenges. Second, single infectious and noninfectious diseases were analyzed separately. Third, infectious reproductive diseases as one category of related conditions, and cystic ovary disease as one category of 3 related noninfectious ovary disorders were studied.Data were analyzed using a threshold model that included herd, calving year, season of calving, and parity as cross-classified fixed factors; and sire and cow within sires as random effects. Days at risk and days in milk at the beginning of a record were included by fitting the days as continuous covariates in the model. A heritability value of 0.202 +/- 0.083 was estimated for generalized immunity. Heritability values of 0.141 and 0.161 were estimated for uterine infection and mastitis, respectively. Heritability of single noninfectious disorders ranged from 0.087 to 0.349. The amount of additive genetic variance recovered in the underlying scale of noninfectious disorders tended to zero when combining multiple conditions. The study supports combining infectious diseases into categories of interest but we do not recommend the same approach for noninfectious disorders.     

Geographic Variation in Attraction to Human Odor Compounds by Aedes aegypti Mosquitoes (Diptera: Culicidae): A Laboratory Study

Previous investigations of Aedes aegypti response to human odor components have revealed a number of compounds that attract host-seeking females. However, such studies have utilized only a small number of long-term laboratory Ae. aegypti colonies. Using laboratory y-olfactometers, we studied the attraction of four different Ae. aegypti populations (North Queensland, Australia; Florida, USA; Singapore; and Minas Gerais, Brazil) to a key attractant compound from human skin, lactic acid. Combinations of lactic acid with ammonia and a fatty acid (caproic acid) were also investigated. The aims were to determine the extent of variation in lactic acid dose response among populations and to see whether all four populations responded equally to combinations of human odor components. Although all Ae. aegypti populations were attracted to lactic acid, there were differences in the threshold dose: Florida 0.03 μg/min, Singapore 0.17 μg/min, North Queensland 1.92 μg/min, and Brazil 10.27 μg/min. Attraction to lactic acid alone (maximum <40%) was significantly lower than for human odor (>87% for all populations). Significant increases in attraction were observed when lactic acid was combined with ammonia or caproic acid, although not for all populations. In addition, the highest doses of caproic acid tested decreased attraction when combined with lactic acid. The divergent responses to host kairomones seen here may be evidence of adaptation to locally available hosts in different parts of the geographic range of Ae. aegypti.