Linking phylogenetic identity and biogeochemical function of uncultivated marine microbes with novel mass spectrometry techniques

The combined activities of diverse heterotrophic marine microorganisms significantly shape global biogeochemical cycles, but models of these activities are currently limited to aggregate microbial community processes, and it remains unclear how community structure and the functional roles of specific microbial taxa should be integrated into these models. Therefore, understanding the contributions of specific microbial populations toward net community processes remains a critical step for determining the appropriate taxonomic resolution that should be employed in morec omplex models of ecosystem processes. The application of ‘omics’ methods, such as metagenomics and metaproteomics, has revealed the phylogenetic diversity of natural microbial communities and the functional potential of discrete populations. From the integration of these observations, an understanding of microbial community dynamics has emerged in which niche processes influence general patterns of community structure at broad taxonomic scales across spatial and temporal resource gradients. Within these dynamic microbial systems, the partitioning of specific resources is governed by resource preferences and competitive interactions that cannot be ascertained with ‘omics’ approaches alone. In order to link phylogenetic identity with ecological function, and characterize resource partitioning in coastal marine microbial communities, we applied novel mass spectrometry techniques to stable isotope probing (SIP) experiments conducted on microbes sampled from coastal North Pacific surface waters.Chapter 2 presents the first application of proteomic stable isotope probing (proteomic SIP) to track 13C-labeled substrates into the proteomes of planktonic marine microbial communities. We developed two metrics for describing observations of label incorporation into peptides. Label frequency is a measure of protein synthesis activity that is calculated as the ratio of labeled to total detected peptide mass spectra for a defined set of proteins. Average enrichment is a measure of substrate specialization, and is calculated from the average percent of stable isotope content measured for a set of labeled peptides. Using these metrics, we compared the assimilation of 13C-labeled amino acids by abundant microbial taxa over two time-points, 15 and 32 hours. The communities sampled from Newport, OR and Monterey Bay, CA exhibited similar behaviors. Alteromonadales and Rhodobacterales proteomes had significantly high label frequency at the first time-point but had diverging trajectories in the second time-point indicating that Rhodobacterales held a competitive advantage as the amended substrate became depleted.In Chapter 3, we examined the assimilation of six 13C-labeld substrates by microbial taxa sampled from Monterey Bay, CA. Comparisons between relative abundance shifts and substrate assimilation were inconsistent, emphasizing the need for caution when interpreting relative abundances shifts in microbial community experiments. Specialization patterns were significantly conserved among abundant populations within class level divisions, suggesting that resource preferences have deep evolutionary origins, but variation in the activities of individual species or strains within these lineages may be driven by other environmental factors, such as resource concentrations or temperature, or pressure from grazing and viral lysis. Although activity measures were also conserved among these classes, measures of activity divided the seclades into higher or lower activity, suggesting different strategies for responding to increased nutrient availability.Finally, Chapter 4 explores physiological bases for observations of diverging levels of activity among microbial taxonomic lineages. We found that substrate additions resulted in reproducible taxonomic and functional changes in the whole community metaproteome, and that relative abundances of specific protein functional assignments differentiated abundant taxa. Comparisons of the protein functional profiles (i.e., the relative abundances of mass spectra assigned to functional categories) for specific taxa,between time-points and also between treatments, generally revealed minimal changes int he expressed proteins, which suggests some inherent overall stability in the functions of these taxa, despite environmental changes. However, there were significantly different amounts of variation observed in the proteomes of abundant taxa; higher levels of which correlated with higher observed label frequency, greater numbers of detected ribosomes,and larger nitrogen requirements encoded in their genomes. Taken together, these observations suggest that the capacity of organisms to respond rapidly to increased nutrient availability relies on the ability to transition into states of increased proteinsynthesis, and that this strategy does not select for reductions in nitrogen requirements.However, these adaptations for exploiting abundant resources were not correlated with other physiological features, such as the abundance of transporters, motility proteins, and gene regulatory mechanisms.The outcome of these experiments, enabled by the concurrent use of ‘omics’ and novel mass spectrometry methods, was a deeper understanding of how resource preferences of individual microbial taxa impact carbon cycling processes within complex marine microbial communities. Although SIP approaches can only access assimilatory processes, they complement established ‘omics’ techniques by revealing interactions such as competition for and partitioning of resources that can only be examined by simultaneous measuring of whole community dynamics and the relative contributions of individual populations.Keywords: Proteomic SIP, marine microbial ecology, metagenomics, metaproteomics, stable isotope prob

Tissue-selective expression of a conditionally-active ROCK2-estrogen receptor fusion protein

The serine/threonine kinases ROCK1 and ROCK2 are central mediators of actomyosin contractile force generation that act downstream of the RhoA small GTP-binding protein. As a result, they have key roles in regulating cell morphology and proliferation, and have been implicated in numerous pathological conditions and diseases including hypertension and cancer. Here we describe the generation of a gene-targeted mouse line that enables CRE-inducible expression of a conditionally-active fusion between the ROCK2 kinase domain and the hormone-binding domain of a mutated estrogen receptor (ROCK2:ER). This two-stage system of regulation allows for tissue-selective expression of the ROCK2:ER fusion protein, which then requires administration of estrogen analogues such as tamoxifen or 4-hydroxytamoxifen to elicit kinase activity. This conditional gain-of-function system was validated in multiple tissues by crossing with mice expressing CRE recombinase under the transcriptional control of cytokeratin14 (K14), murine mammary tumor virus (MMTV) or cytochrome P450 Cyp1A1 (Ah) promoters, driving appropriate expression in the epidermis, mammary or intestinal epithelia respectively. Given the interest in ROCK signaling in normal physiology and disease, this mouse line will facilitate research into the consequences of ROCK activation that could be used to complement conditional knockout models

Prosthetic Knee

Amputations, specifically lower limb amputations, are common in Sub Saharan Africa and across the broader global community largely due to infection and disease. Our project, The Prosthetic Knee Team, partners with the orthopedic workshop at the CURE International Hospital in Kijabe, Kenya to create a prosthetic knee design for a specific type of amputation known as a Knee Disarticulation (also called through-knee). Currently, the orthopedic workshop is only able to provide one very expensive prosthetic knee option for these patients, and they often elect to undergo a second surgery, a trans-femoral amputation, because the cost of the second surgery and trans-femoral prosthesis combined is less than the currently available through-knee prosthetic. The goal of our project is to provide the orthopedic workshop with a manufacturable prosthetic knee design that provides through-knee amputees with a cheaper prosthetic option and removes the need to have a second amputation above the knee. Throughout the past two semesters, our focus was on organizing collected data, researching knee-disarticulations, and communicating with our client to more fully understand the scope of our project. After determining that moving forward our project will be manufacturing the prosthetic knees at Messiah College’s machine shop and shipping them to CURE Kenya to be fit on patients, we began to brainstorm potential design ideas. We are presently working on modifying and improving our chosen design to best meet all of the specifications laid out by our partner. Those specifications include minimized thigh-lengthening, low weight, maximized stability and durability, and aesthetically pleasing.https://mosaic.messiah.edu/engr2020/1017/thumbnail.jp

Another Shipment of Six Short-Period Giant Planets from TESS

We present the discovery and characterization of six short-period, transiting giant planets from NASA\u27s Transiting Exoplanet Survey Satellite (TESS) -- TOI-1811 (TIC 376524552), TOI-2025 (TIC 394050135), TOI-2145 (TIC 88992642), TOI-2152 (TIC 395393265), TOI-2154 (TIC 428787891), & TOI-2497 (TIC 97568467). All six planets orbit bright host stars (8.

Modeling Kepler transit light curves as false positives: Rejection of blend scenarios for Kepler-9, and validation of Kepler-9d, a super-Earth-size planet in a multiple system

Light curves from the Kepler Mission contain valuable information on the nature of the phenomena producing the transit-like signals. To assist in exploring the possibility that they are due to an astrophysical false positive, we describe a procedure (BLENDER) to model the photometry in terms of a "blend" rather than a planet orbiting a star. A blend may consist of a background or foreground eclipsing binary (or star-planet pair) whose eclipses are attenuated by the light of the candidate and possibly other stars within the photometric aperture. We apply BLENDER to the case of Kepler-9, a target harboring two previously confirmed Saturn-size planets (Kepler-9b and Kepler-9c) showing transit timing variations, and an additional shallower signal with a 1.59-day period suggesting the presence of a super-Earth-size planet. Using BLENDER together with constraints from other follow-up observations we are able to rule out all blends for the two deeper signals, and provide independent validation of their planetary nature. For the shallower signal we rule out a large fraction of the false positives that might mimic the transits. The false alarm rate for remaining blends depends in part (and inversely) on the unknown frequency of small-size planets. Based on several realistic estimates of this frequency we conclude with very high confidence that this small signal is due to a super-Earth-size planet (Kepler-9d) in a multiple system, rather than a false positive. The radius is determined to be 1.64 (+0.19/-0.14) R(Earth), and current spectroscopic observations are as yet insufficient to establish its mass.Comment: 20 pages in emulateapj format, including 8 tables and 16 figures. To appear in ApJ, 1 January 2010. Accepted versio

A Habitable-zone Earth-sized Planet Rescued from False Positive Status

We report the discovery of an Earth-sized planet in the habitable zone of a low-mass star called Kepler-1649. The planet, Kepler-1649 c, is 1.06$^{+0.15}_{-0.10}$ times the size of Earth and transits its 0.1977 +/- 0.0051 Msun mid M-dwarf host star every 19.5 days. It receives 74 +/- 3 % the incident flux of Earth, giving it an equilibrium temperature of 234 +/- 20K and placing it firmly inside the circumstellar habitable zone. Kepler-1649 also hosts a previously-known inner planet that orbits every 8.7 days and is roughly equivalent to Venus in size and incident flux. Kepler-1649 c was originally classified as a false positive by the Kepler pipeline, but was rescued as part of a systematic visual inspection of all automatically dispositioned Kepler false positives. This discovery highlights the value of human inspection of planet candidates even as automated techniques improve, and hints that terrestrial planets around mid to late M-dwarfs may be more common than those around more massive stars.Comment: 11 pages, 3 figures, 1 table. Accepted for publication in ApJ

KOI-54: The Kepler Discovery of Tidally Excited Pulsations and Brightenings in a Highly Eccentric Binary

Kepler observations of the star HD 187091 (KIC 8112039, hereafter KOI-54) revealed a remarkable light curve exhibiting sharp periodic brightening events every 41.8 days with a superimposed set of oscillations forming a beating pattern in phase with the brightenings. Spectroscopic observations revealed that this is a binary star with a highly eccentric orbit, e = 0.83. We are able to match the Kepler light curve and radial velocities with a nearly face-on (i = 5 degrees.5) binary star model in which the brightening events are caused by tidal distortion and irradiation of nearly identical A stars during their close periastron passage. The two dominant oscillations in the light curve, responsible for the beating pattern, have frequencies that are the 91st and 90th harmonic of the orbital frequency. The power spectrum of the light curve, after removing the binary star brightening component, reveals a large number of pulsations, 30 of which have a signal-to-noise ratio greater than or similar to 7. Nearly all of these pulsations have frequencies that are either integer multiples of the orbital frequency or are tidally split multiples of the orbital frequency. This pattern of frequencies unambiguously establishes the pulsations as resonances between the dynamic tides at periastron and the free oscillation modes of one or both of the stars. KOI-54 is only the fourth star to show such a phenomenon and is by far the richest in terms of excited modes.NASA, Science Mission DirectorateNASA NNX08AR14GEuropean Research Council under the European Community 227224W.M. Keck FoundationMcDonald Observator

Two Earth-sized planets orbiting Kepler-20

Since the discovery of the first extrasolar giant planets around Sun-like stars, evolving observational capabilities have brought us closer to the detection of true Earth analogues. The size of an exoplanet can be determined when it periodically passes in front of (transits) its parent star, causing a decrease in starlight proportional to its radius. The smallest exoplanet hitherto discovered has a radius 1.42 times that of the Earth's radius (R Earth), and hence has 2.9 times its volume. Here we report the discovery of two planets, one Earth-sized (1.03R Earth) and the other smaller than the Earth (0.87R Earth), orbiting the star Kepler-20, which is already known to host three other, larger, transiting planets. The gravitational pull of the new planets on the parent star is too small to measure with current instrumentation. We apply a statistical method to show that the likelihood of the planetary interpretation of the transit signals is more than three orders of magnitude larger than that of the alternative hypothesis that the signals result from an eclipsing binary star. Theoretical considerations imply that these planets are rocky, with a composition of iron and silicate. The outer planet could have developed a thick water vapour atmosphere.Comment: Letter to Nature; Received 8 November; accepted 13 December 2011; Published online 20 December 201

A First Comparison of Kepler Planet Candidates in Single and Multiple Systems

In this letter we present an overview of the rich population of systems with multiple candidate transiting planets found in the first four months of Kepler data. The census of multiples includes 115 targets that show 2 candidate planets, 45 with 3, 8 with 4, and 1 each with 5 and 6, for a total of 170 systems with 408 candidates. When compared to the 827 systems with only one candidate, the multiples account for 17 percent of the total number of systems, and a third of all the planet candidates. We compare the characteristics of candidates found in multiples with those found in singles. False positives due to eclipsing binaries are much less common for the multiples, as expected. Singles and multiples are both dominated by planets smaller than Neptune; 69 +2/-3 percent for singles and 86 +2/-5 percent for multiples. This result, that systems with multiple transiting planets are less likely to include a transiting giant planet, suggests that close-in giant planets tend to disrupt the orbital inclinations of small planets in flat systems, or maybe even to prevent the formation of such systems in the first place.Comment: 13 pages, 13 figures, submitted to ApJ Letter