Development and validation of a FACS-based lipoprotein localization screen in the Lyme disease spirochete Borrelia burgdorferi

<p>Abstract</p> <p>Background</p> <p>In our previous studies on lipoprotein secretion in the Lyme disease spirochete <it>Borrelia burgdorferi</it>, we used monomeric red fluorescent protein 1 (mRFP1) fused to specifically mutated outer surface protein A (OspA) N-terminal lipopeptides to gather first insights into lipoprotein sorting determinants. OspA:mRFP1 fusions could be detected by epifluorescence microscopy both in the periplasm and on the bacterial surface. To build on these findings and to complement the prior targeted mutagenesis approach, we set out to develop a screen to probe a random mutagenesis expression library for mutants expressing differentially localized lipoproteins.</p> <p>Results</p> <p>A Glu-Asp codon pair in the inner membrane-localized OspA20:mRFP1 fusion was chosen for mutagenesis since the two negative charges were previously shown to define the phenotype. A library of random mutants in the two codons was generated and expressed in <it>B. burgdorferi</it>. <it>In situ </it>surface proteolysis combined with fluorescence activated cell sorting (FACS) was then used to screen for viable spirochetes expressing alternative subsurface OspA:mRFP1 fusions. Analysis of 93 clones randomly picked from a sorted cell population identified a total of 43 distinct mutants. Protein localization assays indicated a significant enrichment in the selected subsurface phenotype. Interestingly, a majority of the subsurface mutant proteins localized to the outer membrane, indicating their impairment in "flipping" through the outer membrane to the spirochetal surface. OspA20:mRFP1 remained the protein most restricted to the inner membrane.</p> <p>Conclusions</p> <p>Together, these results validate this FACS-based screen for lipoprotein localization and suggest a rather specific inner membrane retention mechanism involving membrane anchor-proximal negative charge patches in this model <it>B. burgdorferi </it>lipoprotein system.</p

Public Evidence from Secret Ballots

Elections seem simple---aren't they just counting? But they have a unique, challenging combination of security and privacy requirements. The stakes are high; the context is adversarial; the electorate needs to be convinced that the results are correct; and the secrecy of the ballot must be ensured. And they have practical constraints: time is of the essence, and voting systems need to be affordable and maintainable, and usable by voters, election officials, and pollworkers. It is thus not surprising that voting is a rich research area spanning theory, applied cryptography, practical systems analysis, usable security, and statistics. Election integrity involves two key concepts: convincing evidence that outcomes are correct and privacy, which amounts to convincing assurance that there is no evidence about how any given person voted. These are obviously in tension. We examine how current systems walk this tightrope.Comment: To appear in E-Vote-Id '1

Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase

The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40

A systematic review of methods to assess intake of fruits and vegetables among healthy European adults and children: a DEDIPAC (DEterminants of DIet and Physical Activity) study

Evidence suggests that health benefits are associated with consuming recommended amounts of fruits and vegetables (F&V), yet standardised assessment methods to measure F&V intake are lacking. The current review aims to identify methods to assess F&V intake among children and adults in pan-European studies and inform the development of the DEDIPAC (DEterminants of DIet and Physical Activity) toolbox of methods suitable for use in future European studies. A literature search was conducted using three electronic databases and by hand-searching reference lists. English-language studies of any design which assessed F&V intake were included in the review. Studies involving two or more European countries were included in the review. Healthy, free-living children or adults. The review identified fifty-one pan-European studies which assessed F&V intake. The FFQ was the most commonly used (n 42), followed by 24 h recall (n 11) and diet records/diet history (n 7). Differences existed between the identified methods; for example, the number of F&V items on the FFQ and whether potatoes/legumes were classified as vegetables. In total, eight validated instruments were identified which assessed F&V intake among adults, adolescents or children. The current review indicates that an agreed classification of F&V is needed in order to standardise intake data more effectively between European countries. Validated methods used in pan-European populations encompassing a range of European regions were identified. These methods should be considered for use by future studies focused on evaluating intake of F&V

A process-based model of conifer forest structure and function with special emphasis on leaf lifespan

We describe the University of Sheffield Conifer Model (USCM), a process-based approach for simulating conifer forest carbon, nitrogen, and water fluxes by up-scaling widely applicable relationships between leaf lifespan and function. The USCM is designed to predict and analyze the biogeochemistry and biophysics of conifer forests that dominated the ice-free high-latitude regions under the high pCO2 “greenhouse” world 290–50 Myr ago. It will be of use in future research investigating controls on the contrasting distribution of ancient evergreen and deciduous forests between hemispheres, and their differential feedbacks on polar climate through the exchange of energy and materials with the atmosphere. Emphasis is placed on leaf lifespan because this trait can be determined from the anatomical characteristics of fossil conifer woods and influences a range of ecosystem processes. Extensive testing of simulated net primary production and partitioning, leaf area index, evapotranspiration, nitrogen uptake, and land surface energy partitioning showed close agreement with observations from sites across a wide climatic gradient. This indicates the generic utility of our model, and adequate representation of the key processes involved in forest function using only information on leaf lifespan, climate, and soils

The Tidal Disruption Event AT 2018hyz II: Light Curve Modeling of a Partially Disrupted Star

AT 2018hyz (=ASASSN-18zj) is a tidal disruption event (TDE) located in the nucleus of a quiescent E+A galaxy at a redshift of $z = 0.04573$, first detected by the All-Sky Automated Survey for Supernovae (ASAS-SN). We present optical+UV photometry of the transient, as well as an X-ray spectrum and radio upper limits. The bolometric light curve of AT 2018hyz is comparable to other known TDEs and declines at a rate consistent with a $t^{-5/3}$ at early times, emitting a total radiated energy of $E = 9\times10^{50}$ erg. An excess bump appears in the UV light curve about 50 days after bolometric peak, followed by a flattening beyond 250 days. The light curve shows an excess bump in the UV about 50 days after bolometric peak lasting for at least 100 days, which may be related to an outflow. We detect a constant X-ray source present for at least 86 days. The X-ray spectrum shows a total unabsorbed flux of $\sim 4\times10^{-14}$ erg cm$^{-2}$ s$^{-1}$ and is best fit by a blackbody plus power-law model with a photon index of $\Gamma = 0.8$. A thermal X-ray model is unable to account for photons $> 1$ keV, while the radio non-detection favors inverse-Compton scattering rather than a jet for the non-thermal component. We model the optical and UV light curves using the Modular Open-Source Fitter for Transients (MOSFiT) and find a best fit for a black hole of $5.2\times10^6$ M$_\odot$ partially disrupting a $0.1$ M$_\odot$ star (stripping a mass of $\sim 0.01$ M$_\odot$ for the inferred impact parameter, $\beta=0.6$). The low optical depth implied by the small debris mass may explain how we are able to see hydrogen emission with disk-like line profiles in the spectra of AT 2018hyz (see our companion paper, Short et al.~2020).Comment: 10 pages, 10 figures, published in MNRA

Reconciling carbon-cycle concepts, terminology, and methods

Author Posting. © The Author(s), 2006. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Ecosystems 9 (2006): 1041-1050, doi:10.1007/s10021-005-0105-7.Recent patterns and projections of climatic change have focused increased scientific and public attention on patterns of carbon (C) cycling and its controls, particularly the factors that determine whether an ecosystem is a net source or sink of atmospheric CO2. Net ecosystem production (NEP), a central concept in C-cycling research, has been used to represent two different concepts by C-cycling scientists. We propose that NEP be restricted to just one of its two original definitions—the imbalance between gross primary production (GPP) and ecosystem respiration (ER), and that a new term—net ecosystem carbon balance (NECB)—be applied to the net rate of C accumulation in (or loss from; negative sign) ecosystems. NECB differs from NEP when C fluxes other than C fixation and respiration occur or when inorganic C enters or leaves in dissolved form. These fluxes include leaching loss or lateral transfer of C from the ecosystem; emission of volatile organic C, methane, and carbon monoxide; and soot and CO2 from fire. C fluxes in addition to NEP are particularly important determinants of NECB over long time scales. However, even over short time scales, they are important in ecosystems such as streams, estuaries, wetlands, and cities. Recent technological advances have led to a diversity of approaches to measuring C fluxes at different temporal and spatial scales. These approaches frequently capture different components of NEP or NECB and can therefore be compared across scales only by carefully specifying the fluxes included in the measurements. By explicitly identifying the fluxes that comprise NECB and other components of the C cycle, such as net ecosystem exchange (NEE) and net biome production (NBP), we provide a less ambiguous framework for understanding and communicating recent changes in the global C cycle. Key words: Net ecosystem production, net ecosystem carbon balance, gross primary production, ecosystem respiration, autotrophic respiration, heterotrophic respiration, net ecosystem exchange, net biome production, net primary production

The Early Ultraviolet Light-Curves of Type II Supernovae and the Radii of Their Progenitor Stars

We present a sample of 34 normal SNe II detected with the Zwicky Transient Facility, with multi-band UV light-curves starting at $t \leq 4$ days after explosion, as well as X-ray detections and upper limits. We characterize the early UV-optical colors and provide prescriptions for empirical host-extinction corrections. We show that the $t > 2\,$days UV-optical colors and the blackbody evolution of the sample are consistent with the predictions of spherical phase shock-cooling (SC), independently of the presence of `flash ionization" features. We present a framework for fitting SC models which can reproduce the parameters of a set of multi-group simulations without a significant bias up to 20% in radius and velocity. Observations of about half of the SNe II in the sample are well-fit by models with breakout radii $<10^{14}\,$cm. The other half are typically more luminous, with observations from day 1 onward that are better fit by a model with a large $>10^{14}\,$cm breakout radius. However, these fits predict an early rise during the first day that is too slow. We suggest these large-breakout events are explosions of stars with an inflated envelope or a confined CSM with a steep density profile, at which breakout occurs. Using the X-ray data, we derive constraints on the extended ($\sim10^{15}$ cm) CSM density independent of spectral modeling, and find most SNe II progenitors lose $<10^{-4} M_{\odot}\, \rm yr^{-1}$ a few years before explosion. This provides independent evidence the CSM around many SNe II progenitors is confined. We show that the overall observed breakout radius distribution is skewed to higher radii due to a luminosity bias. We argue that the $66^{+11}_{-22}\%$ of red supergiants (RSG) explode as SNe II with breakout radii consistent with the observed distribution of field RSG, with a tail extending to large radii, likely due to the presence of CSM.Comment: Submitted to ApJ. Comments are welcome at [email protected] or [email protected]

SN 2021zny: an early flux excess combined with late-time oxygen emission suggests a double white dwarf merger event

We present a photometric and spectroscopic analysis of the ultra-luminous and slowly evolving 03fg-like Type Ia SN 2021zny. Our observational campaign starts from $\sim5.3$ hours after explosion (making SN 2021zny one of the earliest observed members of its class), with dense multi-wavelength coverage from a variety of ground- and space-based telescopes, and is concluded with a nebular spectrum $\sim10$ months after peak brightness. SN 2021zny displayed several characteristics of its class, such as the peak brightness ($M_{B}=-19.95$ mag), the slow decline ($\Delta m_{15}(B) = 0.62$ mag), the blue early-time colours, the low ejecta velocities and the presence of significant unburned material above the photosphere. However, a flux excess for the first $\sim1.5$ days after explosion is observed in four photometric bands, making SN 2021zny the third 03fg-like event with this distinct behavior, while its $+313$ d spectrum shows prominent [O I] lines, a very unusual characteristic of thermonuclear SNe. The early flux excess can be explained as the outcome of the interaction of the ejecta with $\sim0.04\:\mathrm{M_{\odot}}$ of H/He-poor circumstellar material at a distance of $\sim10^{12}$ cm, while the low ionization state of the late-time spectrum reveals low abundances of stable iron-peak elements. All our observations are in accordance with a progenitor system of two carbon/oxygen white dwarfs that undergo a merger event, with the disrupted white dwarf ejecting carbon-rich circumstellar material prior to the primary white dwarf detonation.Comment: 19 pages, 16 figures, accepted for publication in MNRA

SN 2022joj: A Peculiar Type Ia Supernova Possibly Driven by an Asymmetric Helium-shell Double Detonation

We present observations of SN 2022joj, a peculiar Type Ia supernova (SN Ia) discovered by the Zwicky Transient Facility (ZTF). SN 2022joj exhibits an unusually red $g_\mathrm{ZTF}-r_\mathrm{ZTF}$ color at early times and a rapid blueward evolution afterwards. Around maximum brightness, SN 2022joj shows a high luminosity ($M_{g_\mathrm{ZTF},\mathrm{max}}\simeq-19.7$ mag), a blue broadband color ($g_\mathrm{ZTF}-r_\mathrm{ZTF}\simeq-0.2$ mag), and shallow Si II absorption lines, consistent with those of overluminous, SN 1991T-like events. The maximum-light spectrum also shows prominent absorption around 4200 \r{A}, which resembles the Ti II features in subluminous, SN 1991bg-like events. Despite the blue optical-band colors, SN 2022joj exhibits extremely red ultraviolet $-$ optical colors at maximum luminosity ($u-v\simeq1.6$ mag and $uvw1 - v\simeq4.0$ mag), suggesting a suppression of flux between $\sim$2500--4000 \r{A}. Strong C II lines are also detected at peak. We show that these unusual spectroscopic properties are broadly consistent with the helium-shell double detonation of a sub-Chandrasekhar mass ($M\simeq1\mathrm{M_\odot}$) carbon/oxygen (C/O) white dwarf (WD) from a relatively massive helium shell ($M_s\simeq0.04$--$0.1\mathrm{M_\odot}$), if observed along a line of sight roughly opposite to where the shell initially detonates. None of the existing models could quantitatively explain all the peculiarities observed in SN 2022joj. The low flux ratio of [Ni II] $\lambda$7378 to [Fe II] $\lambda$7155 emission in the late-time nebular spectra indicates a low yield of stable Ni isotopes, favoring a sub-Chandrasekhar mass progenitor. The significant blueshift measured in the [Fe II] $\lambda$7155 line is also consistent with an asymmetric chemical distribution in the ejecta, as is predicted in double-detonation models.Comment: 24 pages, 11 figures, 6 tables. Submitted to Ap