Genetic Tools Towards A Synthetic Biology Approach For Whole Mitochondrial Genome Engineering

Synthetic biology is an interdisciplinary research field that standardizes and repurposes biological components to better understand life and solve complex problems. As synthetic biology has developed, the goal has become to generate fully controllable biological systems through whole genome engineering (WGE), the cumulation of standardized genome engineering, and DNA delivery methods. In eukaryotes, genetic tools for WGE are limited to the nucleus and present a need to expand to include mitochondria, which maintains its own unique genome. The work presented here begins developing the resources needed to enable whole mitochondrial genome engineering. First, to standardize mitochondrial genome engineering protocols, I cloned the mitochondrial genomes of two diatomaceous algae, Phaeodactylum tricornutum and Thalassiosira pseudonana, as plasmids in bacteria and yeast. Next, a PCR-based engineering method was optimized to generate derivative algal mitochondrial genomes rapidly and inexpensively. After, I sought to adapt an entirely in vivo DNA delivery method, bacterial conjugation, for mitochondrial DNA-delivery. In any scenario modifying bacterial conjugation’s specificity will likely decrease its efficiency of DNA transfer beyond the level of detection. Therefore, I first improved DNA transfer to eukaryotes by generating and screening a deletion plasmid library for the conjugative plasmid, pTA-Mob 2.0. From this data, pSC5 was created that improved DNA delivery to yeast. pSC5 was used to create the pSC5-toxic plasmids that effectively killed yeast and established a novel first-in-the-world conjugation-based antifungal. Together these resources for mitochondrial genome engineering and improved DNA delivery to eukaryotes should improve the feasibility of future endeavors in whole mitochondrial genome engineering

Field measurements of trace gases and aerosols emitted by peat fires in Central Kalimantan, Indonesia, during the 2015 El Nino

Abstract. Peat fires in Southeast Asia have become a major annual source of trace gases and particles to the regional–global atmosphere. The assessment of their influence on atmospheric chemistry, climate, air quality, and health has been uncertain partly due to a lack of field measurements of the smoke characteristics. During the strong 2015 El Niño event we deployed a mobile smoke sampling team in the Indonesian province of Central Kalimantan on the island of Borneo and made the first, or rare, field measurements of trace gases, aerosol optical properties, and aerosol mass emissions for authentic peat fires burning at various depths in different peat types. This paper reports the trace gas and aerosol measurements obtained by Fourier transform infrared spectroscopy, whole air sampling, photoacoustic extinctiometers (405 and 870 nm), and a small subset of the data from analyses of particulate filters. The trace gas measurements provide emission factors (EFs; grams of a compound per kilogram biomass burned) for up to  ∼  90 gases, including CO2, CO, CH4, non-methane hydrocarbons up to C10, 15 oxygenated organic compounds, NH3, HCN, NOx, OCS, HCl, etc. The modified combustion efficiency (MCE) of the smoke sources ranged from 0.693 to 0.835 with an average of 0.772 ± 0.053 (n  =  35), indicating essentially pure smoldering combustion, and the emissions were not initially strongly lofted. The major trace gas emissions by mass (EF as g kg−1) were carbon dioxide (1564 ± 77), carbon monoxide (291 ± 49), methane (9.51 ± 4.74), hydrogen cyanide (5.75 ± 1.60), acetic acid (3.89 ± 1.65), ammonia (2.86 ± 1.00), methanol (2.14 ± 1.22), ethane (1.52 ± 0.66), dihydrogen (1.22 ± 1.01), propylene (1.07 ± 0.53), propane (0.989 ± 0.644), ethylene (0.961 ± 0.528), benzene (0.954 ± 0.394), formaldehyde (0.867 ± 0.479), hydroxyacetone (0.860 ± 0.433), furan (0.772 ± 0.035), acetaldehyde (0.697 ± 0.460), and acetone (0.691 ± 0.356). These field data support significant revision of the EFs for CO2 (−8 %), CH4 (−55 %), NH3 (−86 %), CO (+39 %), and other gases compared with widely used recommendations for tropical peat fires based on a lab study of a single sample published in 2003. BTEX compounds (benzene, toluene, ethylbenzene, xylenes) are important air toxics and aerosol precursors and were emitted in total at 1.5 ± 0.6 g kg−1. Formaldehyde is probably the air toxic gas most likely to cause local exposures that exceed recommended levels. The field results from Kalimantan were in reasonable agreement with recent lab measurements of smoldering Kalimantan peat for “overlap species,” lending importance to the lab finding that burning peat produces large emissions of acetamide, acrolein, methylglyoxal, etc., which were not measurable in the field with the deployed equipment and implying value in continued similar efforts. The aerosol optical data measured include EFs for the scattering and absorption coefficients (EF Bscat and EF Babs, m2 kg−1 fuel burned) and the single scattering albedo (SSA) at 870 and 405 nm, as well as the absorption Ångström exponents (AAE). By coupling the absorption and co-located trace gas and filter data we estimated black carbon (BC) EFs (g kg−1) and the mass absorption coefficient (MAC, m2 g−1) for the bulk organic carbon (OC) due to brown carbon (BrC). Consistent with the minimal flaming, the emissions of BC were negligible (0.0055 ± 0.0016 g kg−1). Aerosol absorption at 405 nm was  ∼  52 times larger than at 870 nm and BrC contributed  ∼  96 % of the absorption at 405 nm. Average AAE was 4.97 ± 0.65 (range, 4.29–6.23). The average SSA at 405 nm (0.974 ± 0.016) was marginally lower than the average SSA at 870 nm (0.998 ± 0.001). These data facilitate modeling climate-relevant aerosol optical properties across much of the UV/visible spectrum and the high AAE and lower SSA at 405 nm demonstrate the dominance of absorption by the organic aerosol. Comparing the Babs at 405 nm to the simultaneously measured OC mass on filters suggests a low MAC ( ∼  0.1) for the bulk OC, as expected for the low BC/OC ratio in the aerosol. The importance of pyrolysis (at lower MCE), as opposed to glowing (at higher MCE), in producing BrC is seen in the increase of AAE with lower MCE (r2 =  0.65)

The demandingness of Nozick’s ‘Lockean’ proviso

Interpreters of Robert Nozick’s political philosophy fall into two broad groups concerning his application of the ‘Lockean proviso’. Some read his argument in an undemanding way: individual instances of ownership which make people worse off than they would have been in a world without any ownership are unjust. Others read the argument in a demanding way: individual instances of ownership which make people worse off than they would have been in a world without that particular ownership are unjust. While I argue that the former reading is correct as an interpretive matter, I suggest that this reading is nonetheless highly demanding. In particular, I argue that it is demanding when it is expanded to include the protection of nonhuman animals; if such beings are right bearers, as more and more academics are beginning to suggest, then there is no nonarbitrary reason to exclude them from the protection of the proviso

A consideration of the challenges involved in supervising international masters students

This paper explores the challenges facing supervisors of international postgraduate students at the dissertation stage of the masters programme. The central problems of time pressure, language difficulties, a lack of critical analysis and a prevalence of personal problems among international students are discussed. This paper makes recommendations for the improvement of language and critical thinking skills, and questions the future policy of language requirements at HE for international Masters students

Field measurements of trace gases and aerosols emitted by peat fires in Central Kalimantan, Indonesia, during the 2015 El Niño

Peat fires in Southeast Asia have become a major annual source of trace gases and particles to the regional-global atmosphere. The assessment of their influence on atmospheric chemistry, climate, air quality, and health has been uncertain partly due to a lack of field measurements of the smoke characteristics. During the strong 2015 El Niño event we deployed a mobile smoke sampling team in the Indonesian province of Central Kalimantan on the island of Borneo and made the first, or rare, field measurements of trace gases, aerosol optical properties, and aerosol mass emissions for authentic peat fires burning at various depths in different peat types. This paper reports the trace gas and aerosol measurements obtained by Fourier transform infrared spectroscopy, whole air sampling, photoacoustic extinctiometers (405 and 870 nm), and a small subset of the data from analyses of particulate filters. The trace gas measurements provide emission factors (EFs; grams of a compound per kilogram biomass burned) for up to ∼ 90 gases, including CO2, CO, CH4, non-methane hydrocarbons up to C10, 15 oxygenated organic compounds, NH3, HCN, NOx, OCS, HCl, etc. The modified combustion efficiency (MCE) of the smoke sources ranged from 0.693 to 0.835 with an average of 0.772 ± 0.053 (n = 35), indicating essentially pure smoldering combustion, and the emissions were not initially strongly lofted. The major trace gas emissions by mass (EF as g kg-1) were carbon dioxide (1564 ± 77), carbon monoxide (291 ± 49), methane (9.51 ± 4.74), hydrogen cyanide (5.75 ± 1.60), acetic acid (3.89 ± 1.65), ammonia (2.86 ± 1.00), methanol (2.14 ± 1.22), ethane (1.52 ± 0.66), dihydrogen (1.22 ± 1.01), propylene (1.07 ± 0.53), propane (0.989 ± 0.644), ethylene (0.961 ± 0.528), benzene (0.954 ± 0.394), formaldehyde (0.867 ± 0.479), hydroxyacetone (0.860 ± 0.433), furan (0.772 ± 0.035), acetaldehyde (0.697 ± 0.460), and acetone (0.691 ± 0.356). These field data support significant revision of the EFs for CO2 (g-8 %), CH4 (g-55 %), NH3 (g-86 %), CO (+39 %), and other gases compared with widely used recommendations for tropical peat fires based on a lab study of a single sample published in 2003. BTEX compounds (benzene, toluene, ethylbenzene, xylenes) are important air toxics and aerosol precursors and were emitted in total at 1.5 ± 0.6 g kg-1. Formaldehyde is probably the air toxic gas most likely to cause local exposures that exceed recommended levels. The field results from Kalimantan were in reasonable agreement with recent lab measurements of smoldering Kalimantan peat for overlap species, lending importance to the lab finding that burning peat produces large emissions of acetamide, acrolein, methylglyoxal, etc., which were not measurable in the field with the deployed equipment and implying value in continued similar efforts. The aerosol optical data measured include EFs for the scattering and absorption coefficients (EF Bscat and EF Babs, m2 kg-1 fuel burned) and the single scattering albedo (SSA) at 870 and 405 nm, as well as the absorption Ångström exponents (AAE). By coupling the absorption and co-located trace gas and filter data we estimated black carbon (BC) EFs (g kg-1) and the mass absorption coefficient (MAC, m2 g-1) for the bulk organic carbon (OC) due to brown carbon (BrC). Consistent with the minimal flaming, the emissions of BC were negligible (0.0055 ± 0.0016 g kg-1). Aerosol absorption at 405 nm was ∼ 52 times larger than at 870 nm and BrC contributed ∼ 96 % of the absorption at 405 nm. Average AAE was 4.97 ± 0.65 (range, 4.29-6.23). The average SSA at 405 nm (0.974 ± 0.016) was marginally lower than the average SSA at 870 nm (0.998 ± 0.001). These data facilitate modeling climate-relevant aerosol optical properties across much of the UV/visible spectrum and the high AAE and lower SSA at 405 nm demonstrate the dominance of absorption by the organic aerosol. Comparing the Babs at 405 nm to the simultaneously measured OC mass on filters suggests a low MAC (∼ 0.1) for the bulk OC, as expected for the low BC/OC ratio in the aerosol. The importance of pyrolysis (at lower MCE), as opposed to glowing (at higher MCE), in producing BrC is seen in the increase of AAE with lower MCE (r2 = 0.65)

Chemical characterization of fine particulate matter emitted by peat fires in Central Kalimantan, Indonesia, during the 2015 El Niño

Fine particulate matter (PM2:5) was collected in situ from peat smoke during the 2015 El Niño peat fire episode in Central Kalimantan, Indonesia. Twenty-one PM samples were collected from 18 peat fire plumes that were primarily smoldering with modified combustion efficiency (MCE) values of 0.725-0.833. PM emissions were determined and chemically characterized for elemental carbon (EC), organic carbon (OC), water-soluble OC, water-soluble ions, metals, and organic species. Fuel-based PM2:5 mass emission factors (EFs) ranged from 6.0 to 29.6 g kg1 with an average of 17:36:0 g kg1. EC was detected only in 15 plumes and comprised 1% of PM mass. Together, OC (72 %), EC (1 %), water-soluble ions (1 %), and metal oxides (0.1 %) comprised 7411% of gravimetrically measured PM mass. Assuming that the remaining mass is due to elements that form organic matter (OM; i.e., elements O, H, N) an OM-to-OC conversion factor of 1.26 was estimated by linear regression. Overall, chemical speciation revealed the following characteristics of peat-burning emissions: high OC mass fractions (72%), primarily water-insoluble OC (8411 %C), low EC mass fractions (1 %), vanillic to syringic acid ratios of 1.9, and relatively high n-alkane contributions to OC (6.2 %C) with a carbon preference index of 1.2-1.6. Comparison to laboratory studies of peat combustion revealed similarities in the relative composition of PM but greater differences in the absolute EF values. The EFs developed herein, combined with estimates of the mass of peat burned, are used to estimate that 3.2-11 Tg of PM2:5 was emitted to atmosphere during the 2015 El Niño peatland fire event in Indonesia. Combined with gas-phase measurements of CO2, CO, CH4, and volatile organic carbon from Stockwell et al. (2016), it is determined that OC and EC accounted for 2.1 and 0.04% of total carbon emissions, respectively. These in situ EFs can be used to improve the accuracy of the representation of Indonesian peat burning in emission inventories and receptor-based models

Immunization with Pre-Erythrocytic Antigen CelTOS from Plasmodium falciparum Elicits Cross-Species Protection against Heterologous Challenge with Plasmodium berghei

BACKGROUND: The Plasmodium protein Cell-traversal protein for ookinetes and sporozoites (CelTOS) plays an important role in cell traversal of host cells in both, mosquito and vertebrates, and is required for successful malaria infections. CelTOS is highly conserved among the Plasmodium species, suggesting an important functional role across all species. Therefore, targeting the immune response to this highly conserved protein and thus potentially interfering with its biological function may result in protection against infection even by heterologous species of Plasmodium. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, we developed a recombinant codon-harmonized P. falciparum CelTOS protein that can be produced to high yields in the E. coli expression system. Inbred Balb/c and outbred CD-1 mice were immunized with various doses of the recombinant protein adjuvanted with Montanide ISA 720 and characterized using in vitro and in vivo analyses. CONCLUSIONS/SIGNIFICANCE: Immunization with PfCelTOS resulted in potent humoral and cellular immune responses and most importantly induced sterile protection against a heterologous challenge with P. berghei sporozoites in a proportion of both inbred and outbred mice. The biological activity of CelTOS-specific antibodies against the malaria parasite is likely linked to the impairment of sporozoite motility and hepatocyte infectivity. The results underscore the potential of this antigen as a pre-erythrocytic vaccine candidate and demonstrate for the first time a malaria vaccine that is cross-protective between species

All-sky search for periodic gravitational waves in LIGO S4 data

We report on an all-sky search with the LIGO detectors for periodic gravitational waves in the frequency range 50-1000 Hz and with the frequency's time derivative in the range -1.0E-8 Hz/s to zero. Data from the fourth LIGO science run (S4) have been used in this search. Three different semi-coherent methods of transforming and summing strain power from Short Fourier Transforms (SFTs) of the calibrated data have been used. The first, known as "StackSlide", averages normalized power from each SFT. A "weighted Hough" scheme is also developed and used, and which also allows for a multi-interferometer search. The third method, known as "PowerFlux", is a variant of the StackSlide method in which the power is weighted before summing. In both the weighted Hough and PowerFlux methods, the weights are chosen according to the noise and detector antenna-pattern to maximize the signal-to-noise ratio. The respective advantages and disadvantages of these methods are discussed. Observing no evidence of periodic gravitational radiation, we report upper limits; we interpret these as limits on this radiation from isolated rotating neutron stars. The best population-based upper limit with 95% confidence on the gravitational-wave strain amplitude, found for simulated sources distributed isotropically across the sky and with isotropically distributed spin-axes, is 4.28E-24 (near 140 Hz). Strict upper limits are also obtained for small patches on the sky for best-case and worst-case inclinations of the spin axes.Comment: 39 pages, 41 figures An error was found in the computation of the C parameter defined in equation 44 which led to its overestimate by 2^(1/4). The correct values for the multi-interferometer, H1 and L1 analyses are 9.2, 9.7, and 9.3, respectively. Figure 32 has been updated accordingly. None of the upper limits presented in the paper were affecte

Search for gravitational waves from binary inspirals in S3 and S4 LIGO data

We report on a search for gravitational waves from the coalescence of compact binaries during the third and fourth LIGO science runs. The search focused on gravitational waves generated during the inspiral phase of the binary evolution. In our analysis, we considered three categories of compact binary systems, ordered by mass: (i) primordial black hole binaries with masses in the range 0.35 M(sun) < m1, m2 < 1.0 M(sun), (ii) binary neutron stars with masses in the range 1.0 M(sun) < m1, m2 < 3.0 M(sun), and (iii) binary black holes with masses in the range 3.0 M(sun)< m1, m2 < m_(max) with the additional constraint m1+ m2 < m_(max), where m_(max) was set to 40.0 M(sun) and 80.0 M(sun) in the third and fourth science runs, respectively. Although the detectors could probe to distances as far as tens of Mpc, no gravitational-wave signals were identified in the 1364 hours of data we analyzed. Assuming a binary population with a Gaussian distribution around 0.75-0.75 M(sun), 1.4-1.4 M(sun), and 5.0-5.0 M(sun), we derived 90%-confidence upper limit rates of 4.9 yr^(-1) L10^(-1) for primordial black hole binaries, 1.2 yr^(-1) L10^(-1) for binary neutron stars, and 0.5 yr^(-1) L10^(-1) for stellar mass binary black holes, where L10 is 10^(10) times the blue light luminosity of the Sun.Comment: 12 pages, 11 figure

Search for Gravitational Waves Associated with 39 Gamma-Ray Bursts Using Data from the Second, Third, and Fourth LIGO Runs

We present the results of a search for short-duration gravitational-wave bursts associated with 39 gamma-ray bursts (GRBs) detected by gamma-ray satellite experiments during LIGO's S2, S3, and S4 science runs. The search involves calculating the crosscorrelation between two interferometer data streams surrounding the GRB trigger time. We search for associated gravitational radiation from single GRBs, and also apply statistical tests to search for a gravitational-wave signature associated with the whole sample. For the sample examined, we find no evidence for the association of gravitational radiation with GRBs, either on a single-GRB basis or on a statistical basis. Simulating gravitational-wave bursts with sine-gaussian waveforms, we set upper limits on the root-sum-square of the gravitational-wave strain amplitude of such waveforms at the times of the GRB triggers. We also demonstrate how a sample of several GRBs can be used collectively to set constraints on population models. The small number of GRBs and the significant change in sensitivity of the detectors over the three runs, however, limits the usefulness of a population study for the S2, S3, and S4 runs. Finally, we discuss prospects for the search sensitivity for the ongoing S5 run, and beyond for the next generation of detectors.Comment: 24 pages, 10 figures, 14 tables; minor changes to text and Fig. 2; accepted by Phys. Rev.