Genome Editing Method for the Anaerobic Magnetotactic Bacterium Desulfovibrio magneticus RS-1.

Magnetosomes are complex bacterial organelles that serve as model systems for studying bacterial cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related Alphaproteobacteria of the genus Magnetospirillum that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have been found in physiologically and phylogenetically diverse bacteria. Due to a lack of molecular genetic tools, the mechanistic diversity of magnetosome formation remains poorly understood. Desulfovibrio magneticus RS-1 is an anaerobic sulfate-reducing deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified 10 genes in the conserved magnetosome gene island of D. magneticus that are essential for its magnetic phenotype. However, this screen likely missed mutants with defects in crystal size, shape, and arrangement. Reverse genetics to target the remaining putative magnetosome genes using standard genetic methods of suicide vector integration have not been feasible due to the low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis in D. magneticus using a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the upp gene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacing kupM, a gene identified in our previous screen as a magnetosome formation factor, with a streptomycin resistance cassette. Overall, our results show that targeted mutagenesis using a replicative plasmid is effective in D. magneticus and may also be applied to other genetically recalcitrant bacteria.IMPORTANCE Magnetotactic bacteria (MTB) are a group of organisms that form intracellular nanometer-scale magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membranes, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization and are potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from that of the cubooctahedral-shaped magnetite crystals of the genetically tractable MTB within the Alphaproteobacteria Here, we present a method for genome editing in Desulfovibrio magneticus RS-1, a cultured representative of the deeply branching MTB of the class Deltaproteobacteria This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB

Oral Health Beliefs of Alaska Native Dental Patients

This study investigated the oral health beliefs of Alaska Native dental patients via a questionnaire with 4-point Likert scales. Respondents (n = 136) reported high perceived importance of keeping natural teeth. Females viewed oral health as more important than males. Respondents generally did not view dentists as readily available (M = 2.86, SD = 0.66). Perceived availability and efficacy of dentists were positively correlated (r = 0.219, p = 0.021). Beliefs regarding seriousness of dental problems and importance of oral health were strongly correlated (r = 0.547, p \u3c 0.001). Age predicted perceived seriousness of oral health problems, dentist availability, and prevention benefits. Access to dental care is perceived as a barrier among Alaska Natives generally and elders in particular. Oral health education and prevention messages should utilize the existing belief that keeping natural teeth is important. Public information about available services and transportation logistics may decrease perceived availability barriers

Maternal residential pesticide use and risk of childhood leukemia in Costa Rica.

Evidence suggests that early-life exposure to pesticides inside the home may be associated with childhood leukemia, however data from Latin American countries are limited. We examined whether self-reported maternal residential pesticide use and nearby pesticide applications-before and after child's birth-were associated with acute lymphoblastic leukemia (ALL) in the Costa Rican Childhood Leukemia Study (CRCLS), a population-based case-control study (2001-2003). Cases (n = 251 ALL) were diagnosed between 1995 and 2000 (age <15 years at diagnosis) and were identified through the Costa Rican Cancer Registry and National Children's Hospital. Population controls (n = 577) were drawn from the National Birth Registry. We fitted unconditional logistic regression models adjusted for child sex, birth year, and socioeconomic status to estimate the exposure-outcome associations and also stratified by child sex. We observed that self-reported maternal insecticide use inside the home in the year before pregnancy, during pregnancy, and while breastfeeding was associated with increased odds of ALL among boys [adjusted Odds Ratio (aOR) = 1.63 (95% confidence interval [95% CI]: 1.05-2.53), 1.75 (1.13-2.73), and 1.75 (1.12-2.73), respectively. We also found evidence of exposure-response relationships between more frequent maternal insecticide use inside the home and increased odds of ALL among boys and girls combined. Maternal report of pesticide applications on farms or companies near the home during pregnancy and at any time period were also associated with ALL. Our study in Costa Rica highlights the need for education to minimize pesticide exposures inside and around the home, particularly during pregnancy and breastfeeding

Development of an In Vivo Exsheathment Assay of Infective L3 Haemonchus contortus Larvae in Fistualated Sheep

Purpose: Gastrointestinal nematodes (GIN) hinder the sustainable production of small ruminants on pasture and parasite resistance to chemical dewormers is becoming a growing concern. Condensed tannin containing legume forages are being tested to evaluate their anti-parasitic properties and potential contribution to an overall parasite control program for small ruminants such as sheep and goats. One of the most pathogenic GIN of small ruminants is Haemonchus contortus. The final step to full infectivity of H. contortus third stage larvae (L3) is exsheathment in the rumen. The objective of this study was to establish an in-vivo exsheathment assay in fistulated sheep as a tool to evaluate the ability of several varieties of birdsfoot trefoil, a condensed tannin containing forage, to inhibit the exsheathment of H. contortus L3. Methods: The larvae used in the experiment was harvested from the feces of infected donor lambs using a modified Baermann’s technique. In addition, two in-vitro exsheathment assays using either dilute bleach solution or overnight incubation after exposure to CO2 were used for comparison against in-vivo exsheathment. Three different methods of containment of the infective L3 in a porous PVC tube within the rumen were evaluated. 1) Histology cassettes fitted with nylon mesh with a pore size of 8 microns. 3000 larvae were pipetted into the cassettes and the cassettes sealed. To prevent the escape of L3 various methods of sealing the cassettes were tried including super glue, clamps, rubber bands, and clay. 2) Dialysis tubes. 2000 larvae were pipetted into dialysis tubing with varying molecular weight cut offs (50 to 1,000 kD). 3) Small (5 ml) polypropylene sample jars with 8 micron mesh cover. 2000 L3 pipetted into polypropylene sample jars covered with an 8 micron mesh. Each method was tested by suspending the larvae in the various containment systems within a porous PVC tube and then removing a container after 60 and 120 minutes of incubation within the rumen. The percentage of viable and exsheathed infective L3 larvae were determined by microscope. Criteria used to evaluate the efficacy of containment were the percent of recovered L3 from the containment system and the percent of total L3 exsheathed. Results: Results from the histology cassette technique show that, although some L3 were exsheathed, the histology cassettes were not able to be sealed to the degree necessary to prevent L3 from escaping, thus potentially infecting the sheep. Results are still pending on the other two methods as we continue to collect data. Conclusion: Development of an in-vivo exsheathment assay is critical in determining the anthelmintic efficacy of condensed tannin containing legume forages. In order to see if the forages affect exsheathment it is necessary to have an in-vivo assay that is able to provide reliable control-exsheathment from which you can compare exsheathment statistics after being fed the forages

The effects of minimal tillage, contour cultivation and in-field vegetative barriers on soil erosion and phosphorus loss.

Runoff, sediment, total phosphorus and total dissolved phosphorus losses in overland flow were measured for two years on unbounded plots cropped with wheat and oats. Half of the field was cultivated with minimum tillage (shallow tillage with a tine cultivator) and half was conventionally ploughed. Within each cultivation treatment there were different treatment areas (TA). In the first year of the experiment, one TA was cultivated up and down the slope, one TA was cultivated on the contour, with a beetle bank acting as a vegetative barrier partway up the slope, and one had a mixed direction cultivation treatment, with cultivation and drilling conducted up and down the slope and all subsequent operations conducted on the contour. In the second year, this mixed treatment was replaced with contour cultivation. Results showed no significant reduction in runoff, sediment losses or total phosphorus losses from minimum tillage when compared to the conventional plough treatment, but there were increased losses of total dissolved phosphorus with minimum tillage. The mixed direction cultivation treatment increased surface runoff and losses of sediment and phosphorus. Increasing surface roughness with contour cultivation reduced surface runoff compared to up and down slope cultivation in both the plough and minimum tillage treatment areas, but this trend was not significant. Sediment and phosphorus losses in the contour cultivation treatment followed a very similar pattern to runoff. Combining contour cultivation with a vegetative barrier in the form of a beetle bank to reduce slope length resulted in a non-significant reduction in surface runoff, sediment and total phosphorus when compared to up and down-slope cultivation, but there was a clear trend towards reduced losses. However, the addition of a beetle bank did not provide a significant reduction in runoff, sediment losses or total phosphorus losses when compared to contour cultivation, suggesting only a marginal additional benefit. The economic implications for farmers of the different treatment options are investigated in order to assess their suitability for implementation at a field scale

Conservation in the dark? the information used to support management decisions

The management requirements for protected areas are frequently complex and urgent; as a result, managers often need to act quickly and make decisions with limited supporting evidence at their disposal. Despite demands for high-quality information, it is unclear how much of this evidence conservation practitioners use to assist with their decision making. We investigated the information used to manage protected areas, based on the evidence reported by practitioners when evaluating their management performance. We examined the management of over 1000 protected areas run by two Australian conservation agencies - Parks Victoria and the New South Wales Department of Environment and Climate Change - an unprecedented scope for this type of study. We found that very few conservation practitioners use evidence-based knowledge to support their management. The evidence used varies with the management issue, reserve type, and reserve size. Around 60% of conservation management decisions rely on experience-based information, and many practitioners report having insufficient evidence to assess their management decisions. While experience plays an important role in conservation management, the apparent lack of evidence-based information to support decision making in the reserves has the potential to compromise outcomes and jeopardize the investment made in protected areas for conservation

Intraspecific Differences in Molecular Stress Responses and Coral Pathobiome Contribute to Mortality Under Bacterial Challenge in \u3ci\u3eAcropora millepora\u3c/i\u3e

Disease causes significant coral mortality worldwide; however, factors responsible for intraspecific variation in disease resistance remain unclear. We exposed fragments of eight Acropora millepora colonies (genotypes) to putatively pathogenic bacteria (Vibrio spp.). Genotypes varied from zero to \u3e90% mortality, with bacterial challenge increasing average mortality rates 4-6 fold and shifting the microbiome in favor of stress-associated taxa. Constitutive immunity and subsequent immune and transcriptomic responses to the challenge were more prominent in high-mortality individuals, whereas low-mortality corals remained largely unaffected and maintained expression signatures of a healthier condition (i.e., did not launch a large stress response). Our results suggest that lesions appeared due to changes in the coral pathobiome (multiple bacterial species associated with disease) and general health deterioration after the biotic disturbance, rather than the direct activity of any specific pathogen. If diseases in nature arise because of weaknesses in holobiont physiology, instead of the virulence of any single etiological agent, environmental stressors compromising coral condition might play a larger role in disease outbreaks than is currently thought. To facilitate the diagnosis of compromised individuals, we developed and independently cross-validated a biomarker assay to predict mortality based on genes whose expression in asymptomatic individuals coincides with mortality rates

User-Centered Evaluations with Older Adults: Testing the Usability of a Mobile Health System for Heart Failure Self-Management

Many older adults living with heart failure struggle to follow recommended self-management routines. To help older adults with heart failure more effectively and efficiently self-manage their disease, we developed Engage, a mobile health application promoting the performance, logging, and sharing of routine self-management behaviors. This paper reports on the usability evaluation of the Engage system with 15 older adults with heart failure and informal caregivers. In two phases, participants used Engage during a task-based usability test (n=5) and a scenario-based usability test (n=10). Usability and performance data were assessed through video-recorded observation and the administration of the system usability scale (SUS) and NASA Task Load Index (TLX). We found that task-based testing was useful in quickly identifying problems within our application, but scenario-based testing elicited more valuable feedback from older adults. A comparison of the different evaluation methods used and the discussion of the challenges encountered provide multiple implications for the practice of usability testing of mobile health products with older adults

Settlement of larvae from four families of corals in response to a crustose coralline alga and its biochemical morphogens

Healthy benthic substrates that induce coral larvae to settle are necessary for coral recovery. Yet, the biochemical cues required to induce coral settlement have not been identified for many taxa. Here we tested the ability of the crustose coralline alga (CCA) Porolithon onkodes to induce attachment and metamorphosis, collectively termed settlement, of larvae from 15 ecologically important coral species from the families Acroporidae, Merulinidae, Poritidae, and Diploastreidae. Live CCA fragments, ethanol extracts, and hot aqueous extracts of P. onkodes induced settlement (>10%) for 11, 7, and 6 coral species, respectively. Live CCA fragments were the most effective inducer, achieving over 50% settlement for nine species. The strongest settlement responses were observed in Acropora spp.; the only non-acroporid species that settled over 50% were Diploastrea heliopora, Goniastrea retiformis, and Dipsastraea pallida. Larval settlement was reduced in treatments with chemical extracts compared with live CCA, although high settlement (>50%) was reported for six acroporid species in response to ethanol extracts of CCA. All experimental treatments failed (< 10%) to induce settlement in Montipora aequituberculata, Mycedium elephantotus, and Porites cylindrica. Individual species responded heterogeneously to all treatments, suggesting that none of the cues represent a universal settlement inducer. These results challenge the commonly-held notion that CCA ubiquitously induces coral settlement, and emphasize the critical need to assess additional cues to identify natural settlement inducers for a broad range of coral taxa

CGH and SNP array using DNA extracted from fixed cytogenetic preparations and long-term refrigerated bone marrow specimens

<p>Abstract</p> <p>Background</p> <p>The analysis of nucleic acids is limited by the availability of archival specimens and the quality and amount of the extracted material. Archived cytogenetic preparations are stored in many laboratories and are a potential source of total genomic DNA for array karyotyping and other applications. Array CGH using DNA from fixed cytogenetic preparations has been described, but it is not known whether it can be used for SNP arrays. Diagnostic bone marrow specimens taken during the assessment of hematological malignancies are also a potential source of DNA, but it is generally assumed that DNA must be extracted, or the specimen frozen, within a day or two of collection, to obtain DNA suitable for further analysis. We have assessed DNA extracted from these materials for both SNP array and array CGH.</p> <p>Results</p> <p>We show that both SNP array and array CGH can be performed on genomic DNA extracted from cytogenetic specimens stored in Carnoy's fixative, and from bone marrow which has been stored unfrozen, at 4°C, for at least 36 days. We describe a procedure for extracting a usable concentration of total genomic DNA from cytogenetic suspensions of low cellularity.</p> <p>Conclusions</p> <p>The ability to use these archival specimens for DNA-based analysis increases the potential for retrospective genetic analysis of clinical specimens. Fixed cytogenetic preparations and long-term refrigerated bone marrow both provide DNA suitable for array karyotyping, and may be suitable for a wider range of analytical procedures.</p