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At least 145 records · Page 8

Hemichordates and the Origin of Chordates

At the start of the period of the NASA grant three years ago, we had no information on the organization and development of the body axis of the hemichordate, Saccoglossus kowalevskii. Now we have substantial findings about the anteroposterior axis and dorsoventral axis, and based on this information, we have new insights about the origin of chordates from ancestral deuterostomes. We found ways to obtain and preserve large numbers of embryos and hatched juveniles. We can now collect about 40,000 embryos in the month of September, the time of S. kowalevskii spawning at Woods Hole. Excellent cDNA libraries were prepared from three developmental stages. From these libraries, we directly isolated about 30 gene ortholog sequences by screening and pcr techniques, all of these sequences of interest in the inquiry about the animal's organization and development. We also performed a mid-sized EST project (60,000 randomly picked clones, many of these arrayed). About half of these have been analyzed so far by blastx and are suitable for direct use of clones. We have obtained about 50 interesting sequences from this set. The rest still await analysis. Thus, at this time we have isolated orthologs of 80 genes that are known to be expressed in chordates in conserved domains and known to have interesting roles in chordate organization and development. The orthology of the S. kowalevskii sequences has been verified by neighbor joining and parsimony methods, with bootstrap estimates of validity. The S. kowalevskii sequences cluster with other deuterostome sequences, namely, other hemichordates, echinoderms, ascidians, amphioxus, or vertebrates, depending on what sequences are available in the database for comparison. We have used these sequences to do high quality in situ hybridization on S. kowalevskii embryos, and the results can be divided into three sections-those concerning the anteroposterior axis of S. kowalevskii in comparison to the same axis of chordates, those concerning the dorsoventral axis of S. kowalevskii in comparison to the same axis of chordates, and those concerning the signals and transcription factors found in the endoderm, of S. kowalevskii compared to the signals and transcription factors in the endo-mesodermal cells of Spemann's organizer of chordates.

Gerhart, John↗

Origin, Structure, and Evolution of Grooved Terrain on Ganymede and Comparison to Europa

'Ridge and trough terrain' (RTT) is a generic term for sets of subparallel ridges and troughs observed on several icy outer planet satellites. Ganymede's grooved terrain is the best studied example of ridge and trough terrain. However, the >500 a resolution of Voyager images limited the conclusions that could be drawn regarding its detailed morphology and deformational style, as well as the relationship of bright grooved terrain to dark terrain on Ganymede. The goal of this study was to utilize Galileo high resolution images to examine and categorize the detailed local-scale morphology of ridge and trough terrains on Ganymede, in order to determine the morphological variety and deformational styles of the terrain, and its evolutionary sequence. This included comparison of the ridges and troughs that occur within dark terrain to those within bright terrain. This study addressed implications for the satellite's local-scale stress history, its lithospheric character, and its broader-scale geological history. Conclusions from the Ganymede were applied to gain insight into the origin of ridge and trough terrains on Europa. Attached are the abstracts of the seven papers and list of one popular press article and 13 abstracts which resulted from this study. No subject inventions are associated with this work.

Pappalardo, Robert T.↗

An evolutionarily conserved tryptophan cage promotes folding of the extended RNA recognition motif in the hnRNPR ‐like protein family

Abstract The heterogeneous nuclear ribonucleoprotein (hnRNP) R‐like family is a class of RNA binding proteins in the hnRNP superfamily with diverse functions in RNA processing. Here, we present the 1.90 Å X‐ray crystal structure and solution NMR studies of the first RNA recognition motif (RRM) of human hnRNPR. We find that this domain adopts an extended RRM (eRRM1) featuring a canonical RRM with a structured N‐terminal extension (N ext ) motif that docks against the RRM and extends the β‐sheet surface. The adjoining loop is structured and forms a tryptophan cage motif to position the N ext motif for docking to the RRM. Combining mutagenesis, solution NMR spectroscopy, and thermal denaturation studies, we evaluate the importance of residues in the N ext –RRM interface and adjoining loop on eRRM folding and conformational dynamics. We find that these sites are essential for protein solubility, conformational ordering, and thermal stability. Consistent with their importance, mutations in the N ext –RRM interface and loop are associated with several cancers in a survey of somatic mutations in cancer studies. Sequence and structure comparison of the human hnRNPR eRRM1 to experimentally verified and predicted hnRNPR‐like proteins reveals conserved features in the eRRM.

Biochemistry & Molecular Biology↗

Extent and Origins of Functional Diversity in a Subfamily of Glycoside Hydrolases

Some glycoside hydrolases have broad specificity for hydrolysis of glycosidic bonds, potentially increasing their functional utility and flexibility in physiological and industrial applications. Here, to deepen the understanding of the structural and evolutionary driving forces underlying specificity patterns in glycoside hydrolase family 5, we quantitatively screened the activity of the catalytic core domains from subfamily 4 (GH5_4) and closely related enzymes on four substrates: lichenan, xylan, mannan, and xyloglucan. Phylogenetic analysis revealed that GH5_4 consists of three major clades, and one of these clades, referred to here as clade 3, displayed average specific activities of 4.2 and 1.2 U/mg on lichenan and xylan, approximately 1 order of magnitude larger than the average for active enzymes in clades 1 and 2. Enzymes in clade 3 also more consistently met assay detection thresholds for reaction with all four substrates. We also identified a subfamily-wide positive correlation between lichenase and xylanase activities, as well as a weaker relationship between lichenase and xyloglucanase. To connect these results to structural features, we used the structure of CelE from Hungateiclostridium thermocellum (PDB 4IM4) as an example clade 3 enzyme with activities on all four substrates. Comparison of the sequence and structure of this enzyme with others throughout GH5_4 and neighboring subfamilies reveals at least two residues (H149 and W203) that are linked to strong activity across the substrates. Placing GH5_4 in context with other related subfamilies, we highlight several possibilities for the ongoing evolutionary specialization of GH5_4 enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Molecular mechanism of reflectin’s tunable biophotonic control: Opportunities and limitations for new optoelectronics

Discovery that reflectin proteins fill the dynamically tunable Bragg lamellae in the reflective skin cells of certain squids has prompted efforts to design new reflectin-inspired systems for dynamic photonics. But new insights into the actual role and mechanism of action of the reflectins constrain and better define the opportunities and limitations for rationally designing optical systems with reflectin-based components. We and our colleagues have discovered that the reflectins function as a signal-controlled molecular machine, regulating an osmotic motor that tunes the thickness, spacing, and refractive index of the tunable, membrane-bound Bragg lamellae in the iridocytes of the loliginid squids. The tunable reflectin proteins, characterized by a variable number of highly conserved peptide domains interspersed with positively charged linker segments, are restricted in intra- and inter-chain contacts by Coulombic repulsion. Physiologically, this inhibition is progressively overcome by charge-neutralization resulting from acetylcholine (neurotransmitter)-induced, site-specific phosphorylation, triggering the simultaneous activation and progressive tuning of reflectance from red to blue. Details of this process have been resolved through in vitro analyses of purified recombinant reflectins, controlling charge-neutralization by pH-titration or mutation as surrogates for the in vivo phosphorylation. Results of these analyses have shown that neutralization overcoming the Coulombic inhibition reversibly and cyclably triggers condensation and secondary folding of the reflectins, with the emergence of previously cryptic, phase-segregated hydrophobic domains enabling hierarchical assembly. This tunable, reversible, and cyclable assembly regulates the Gibbs-Donnan mediated osmotic shrinking or swelling of the Bragg lamellae that tunes the brightness and color of reflected light. Our most recent studies have revealed a direct relationship between the extent of charge neutralization and the size of the reflectin assemblies, further explaining the synergistic effects on the intensity and wavelength of reflected light. Mutational analyses show that the “switch” controlling reflectins’ structural transitions is distributed along the protein, while detailed comparisons of the sequences and structures of the recently evolved tunable reflectins to those of their ancestral, non-tunable homologs are helping to identify the specific structural determinants governing tunability.

47 OTHER INSTRUMENTATION↗

Coxsackievirus B4 sewage-isolate induces pancreatitis after oral infection of mice

ABSTRACT Numerous serotypes which belong to the genus Enterovirus (EV) show variability in their virulence and clinical manifestations. They are also known to undergo changes caused by mutations and recombination during their circulation in the environment and the population. Various EV serotypes are prevalent in groundwater, wastewater and surface waters. Our previous studies showed that oral infection induces pancreatitis depending on specific conditions, such as gravidity, in an outbred murine model. Our aim in the present study was to further explore the pancreatic histopathology in an outbred mouse model following oral infection with clinical isolates from a patient who had aseptic meningitis and an isolate from a treated-sewage sample recovered from the residential area of the patient. The isolates were identified as coxsackievirus B4 (CVB4) in tissue culture. The CVB4 sewage-isolate induced pancreatitis after oral infection. In contrast, pancreatitis was absent following infection with the clinical isolates. Comparison of polyprotein sequences showed that the treated-sewage strains differed from the patient's isolates by 9 and 11 amino acids. We conclude that the isolates of clinical and environmental origin differed in their pathogenic properties and showed genetic variation.

Benkoova, Brigita↗

Evolutionary engineering reveals amino acid substitutions in Ato2 and Ato3 that allow improved growth of Saccharomyces cerevisiae on lactic acid

ABSTRACT In Saccharomyces cerevisiae, the complete set of proteins involved in transport of lactic acid across the cell membrane has not been determined. In this study, we aimed to identify transport proteins not previously described to be involved in lactic acid transport via a combination of directed evolution, whole-genome resequencing and reverse engineering. Evolution of a strain lacking all known lactic acid transporters on lactate led to the discovery of mutated Ato2 and Ato3 as two novel lactic acid transport proteins. When compared to previously identified S. cerevisiae genes involved in lactic acid transport, expression of ATO3T284C was able to facilitate the highest growth rate (0.15 ± 0.01 h-1) on this carbon source. A comparison between (evolved) sequences and 3D models of the transport proteins showed that most of the identified mutations resulted in a widening of the narrowest hydrophobic constriction of the anion channel. We hypothesize that this observation, sometimes in combination with an increased binding affinity of lactic acid to the sites adjacent to this constriction, are responsible for the improved lactic acid transport in the evolved proteins.

Baldi, Nicolò↗

Comparative Genomic Analysis of Sulfurospirillum cavolei MES Reconstructed from the Metagenome of an Electrosynthetic Microbiome

Sulfurospirillum spp. play an important role in sulfur and nitrogen cycling, and contain metabolic versatility that enables reduction of a wide range of electron acceptors, including thiosulfate, tetrathionate, polysulfide, nitrate, and nitrite. Here we describe the assembly of a Sulfurospirillum genome obtained from the metagenome of an electrosynthetic microbiome. The ubiquity and persistence of this organism in microbial electrosynthesis systems suggest it plays an important role in reactor stability and performance. Understanding why this organism is present and elucidating its genetic repertoire provide a genomic and ecological foundation for future studies where Sulfurospirillum are found, especially in electrode-associated communities. Metabolic comparisons and in-depth analysis of unique genes revealed potential ecological niche-specific capabilities within the Sulfurospirillum genus. The functional similarities common to all genomes, i.e., core genome, and unique gene clusters found only in a single genome were identified. Based upon 16S rRNA gene phylogenetic analysis and average nucleotide identity, the Sulfurospirillum draft genome was found to be most closely related to Sulfurospirillum cavolei. Characterization of the draft genome described herein provides pathway-specific details of the metabolic significance of the newly described Sulfurospirillum cavolei MES and, importantly, yields insight to the ecology of the genus as a whole. Comparison of eleven sequenced Sulfurospirillum genomes revealed a total of 6246 gene clusters in the pan-genome. Of the total gene clusters, 18.5% were shared among all eleven genomes and 50% were unique to a single genome. While most Sulfurospirillum spp. reduce nitrate to ammonium, five of the eleven Sulfurospirillum strains encode for a nitrous oxide reductase (nos) cluster with an atypical nitrous-oxide reductase, suggesting a utility for this genus in reduction of the nitrous oxide, and as a potential sink for this potent greenhouse gas.

54 ENVIRONMENTAL SCIENCES↗

Phenotypically anchored transcriptomics across diverse agrichemicals reveals conserved pathways and unique gene expression signatures in zebrafish

Agrichemicals such as herbicides, fungicides, insecticides, and biocides are widely used in agriculture, yet some are associated with adverse effects in humans and the environment. While many of these chemicals have been extensively studied in vitro and are included in the EPA’s ToxCast program, comprehensive in vivo comparisons using RNA sequencing across structurally diverse agrichemicals, in a single screening platform, are lacking. In this study, we examined structurally diverse agrichemicals found in the U.S. Environmental Protection Agency’s (EPA) Toxcast Phase I and II library by statically exposing early life stage zebrafish at 6 h post fertilization (hpf) until 120 hpf at concentrations ranging from 0.25 to 100 µM. Morphological outcomes were assessed at 120 hpf across 10 endpoints, including yolk sac edema, craniofacial malformations, and axis abnormalities. Chemicals that produced robust concentration-response relationships were selected for transcriptomic profiling. For transcriptomic analysis, zebrafish were statically exposed to each chemical and sampled at 48 hpf, prior to the onset of morphological effects observed at 120 hpf. Differential expression analysis identified between 0 and 4,538 differentially expressed genes (DEGs) per chemical, with no clear correlation to morphological severity. Both DEG and co-expression network analyses revealed chemical-specific expression patterns that converged on shared biological pathways, including neurodevelopment and cytoskeletal organization. Key regulatory genes such as mylpfa and krt4 were identified within co-expression modules, suggesting their potential role in conserved toxicity mechanisms. Semantic similarity analysis of enriched gene ontology (GO) terms, when compared to existing datasets, highlighted gaps in the annotation of neurodevelopmental processes, indicating that some in vivo effects may not be fully captured by current curated resources. The results provide new insights into the modes of action of diverse agrichemicals and establish a framework for understanding how agrichemical structure relates to biological function in a vertebrate model.

agrichemical↗

Detection of a Large Population of Ultradiffuse Galaxies in Massive Galaxy Clusters: Abell S1063 and Abell 2744

We present the detection of a large population of ultradiffuse galaxies (UDGs) in two massive galaxy clusters, Abell S1063 at z = 0.348 and Abell 2744 at z = 0.308, based on F814W and F105W images in the Hubble Frontier Fields Program. We find 47 and 40 UDGs in Abell S1063 and Abell 2744, respectively. Color–magnitude diagrams of the UDGs show that they are mostly located at the faint end of the red sequence. From the comparison with simple stellar population models, we estimate their stellar mass to range from 10{sup 8} to 10{sup 9} M {sub ⊙}. Radial number density profiles of the UDGs show a turnover or a flattening in the central region at r < 100 kpc. We estimate the total masses of the UDGs using the galaxy scaling relations. A majority of the UDGs have total masses M {sub 200} = 10{sup 10}–10{sup 11} M {sub ⊙}, and only a few of them have total masses M {sub 200} = 10{sup 11}–10{sup 12} M {sub ⊙}. The total number of UDGs within the virial radius is estimated to be N(UDG) = 770 ± 114 for Abell S1063 and N(UDG) = 814 ± 122 for Abell 2744. Combining these results with data in the literature, we fit the relation between the total numbers of UDGs and the masses of their host systems for M {sub 200} > 10{sup 13} M {sub ⊙} with a power law, N(UDG) = M{sub 200}{sup 1.05±0.09}. These results suggest that a majority of the UDGs have a dwarf galaxy origin, while only a small number of the UDGs are massive L* galaxies that failed to form a normal population of stars.

79 ASTRONOMY AND ASTROPHYSICS↗

Three-dimensional Simulations of Massive Stars. II. Age Dependence

We present 3D full star simulations, reaching up to 90% of the total stellar radius, for three 7 M⊙ stars of different ages: zero-age main sequence (ZAMS), mid–main sequence (midMS), and terminal-age main sequence (TAMS). A comparison with several theoretical prescriptions shows that the generation spectra for all three ages are dominated by convective plumes. Two distinct overshooting layers are observed, with most plumes stopped within the layer situated directly above the convective boundary; overshooting to the second, deeper layer becomes progressively more infrequent with increasing stellar age. Internal gravity wave (IGW) propagation is significantly impacted in the midMS and TAMS models as a result of some IGWs getting trapped within their Brunt–Väisälä frequency spikes. A fundamental change in the wave structure across radius is also observed, driven by the effect of density stratification on IGW propagation causing waves to become evanescent within the radiative zone, with older stars being affected more strongly. We find that the steepness of the frequency spectrum at the surface increases from ZAMS to the older models, with older stars also showing more modes in their spectra.

79 ASTRONOMY AND ASTROPHYSICS↗

A mechanistic model and therapeutic interventions for COVID-19 involving a RAS-mediated bradykinin storm

Neither the disease mechanism nor treatments for COVID-19 are currently known. Here, we present a novel molecular mechanism for COVID-19 that provides therapeutic intervention points that can be addressed with existing FDA-approved pharmaceuticals. The entry point for the virus is ACE2, which is a component of the counteracting hypotensive axis of RAS. Bradykinin is a potent part of the vasopressor system that induces hypotension and vasodilation and is degraded by ACE and enhanced by the angiotensin 1-9 produced by ACE2. Here, we perform a new analysis on gene expression data from cells in bronchoalveolar lavage fluid (BALF) from COVID-19 patients that were used to sequence the virus. Comparison with BALF from controls identifies a critical imbalance in RAS represented by decreased expression of ACE in combination with increases in ACE2, renin, angiotensin, key RAS receptors, kinogen and many kallikrein enzymes that activate it, and both bradykinin receptors. This very atypical pattern of the RAS is predicted to elevate bradykinin levels in multiple tissues and systems that will likely cause increases in vascular dilation, vascular permeability and hypotension. These bradykinin-driven outcomes explain many of the symptoms being observed in COVID-19.

60 APPLIED LIFE SCIENCES↗

Vectorcardiographic results from Skylab medical experiment M092: Lower body negative pressure

Vectorcardiograms were recorded via a modified Frank lead system from all crewmen of the three Skylab missions in conjuction with the Lower Body Negative Pressure - M092 Experiment. Data were analyzed by a specially developed computer program (VECTAN). Design of the test sequences allowed direct comparisons of supine resting, Earth based (reference) vectorcardiograms with those taken during lower body negative pressure stress and those obtained at rest in orbit, as well as combinations of these conditions. Results revealed several statistically significant space flight related changes; namely, increased testing and lower body negative pressure stressed heart rates, modestly increased PR interval and corrected QTC interval, and greatly increased P and QPS loop maximal amplitudes. In addition, orientation changes in the QRS maximum vector and the J-vector at rest in space seem quite consistent among crewmen and different from those caused by the application of lower body negative pressure. No clinical abnormalities were observed. Etiology of these findings is conjectured to be, at least in part, related to fluid mass shifts occurring in weightlessness and attendant alterations in cardiovascular dynamics and myocardial autonomic control mechanisms.

Hoffler, G. W.↗

Real Time In Situ Observations of Equiaxed Dendrite Coherency in Al-Cu Alloys Using High-Brilliance, 3rd Generation Synchrotron Sources

In the last decade synchrotron X-ray sources have fast become the tool of choice for performing in-situ high resolution imaging during alloy solidification. This paper presents the results of an experimental campaign carried out at the European Synchrotron Radiation Facility, using a Bridgman furnace, to monitor phenomena during solidification of Al-Cu alloys - specifically the onset of equiaxed dendrite coherency. Conventional experimental methods for determining coherency involve measuring the change in viscosity or measuring the change in thermal conductivity across the solidifying melt Conflicts arise when comparing the results of these experimental techniques to find a relationship between cooling rate and coherency fraction. It has been shown that the ratio of average velocity to the average grain diameter has an inversely proportional relationship to coherency fraction. In-situ observation therefore makes it possible to measure these values directly from acquired images sequences and make comparisons with published results.

Murphy, Andrew G.↗

Transcriptomic Analysis of ISS Crewmembers’ Peripheral Blood Mononuclear Cells Reveals Homeostatic Regulations in Space

The impact of spaceflight on the immune system has been investigated for decades. Studies conducted in cell models, animals and humans suggest that the spaceflight environment affects the innate and acquired immune systems, as the ability to recognize antigens, defend against foreign invaders, and orchestrate repair is significantly hindered. However, the molecular mechanisms behind spaceflight-induced immune dysregulations are still unclear. In this study, blood from eleven (11) International Space Station (ISS) crewmembers was collected before, during and after long duration space missions, as well as from 11 matched ground control subjects. Transcriptomic analysis was performed in isolated peripheral blood mononuclear cells (PBMCs) using the RNA-sequencing technique. In comparison to the blood samples collected from the crewmembers pre-flight, a total of ~1000 genes were found to be upregulated and ~1000 genes downregulated in PBMC collected between 4 and 6 months after they were in space. The most significantly DEGs (differentially expressed genes) include activation of RUBCNL which is an autophagy enhancer and inhibition of GRASP which regulates cell trafficking. Genes involved in cell adhesion, cell cycle progression and other functions were also dysregulated. Pathway analysis of the DEGs indicates mitochondria dysfunction, particularly reduced ATP production in the electron transport chain. Other pathways impacted by spaceflight include glycolysis, autophagy and inflammatory response. Our results suggest that, in space, blood cells may have also experienced energy depletion and reduced metabolism. Consequently, the cells may become autophagic, which is a known homeostatic mechanism for blood cells to become quiescent, but to stay alive. Further analysis of the data shows recovery of the crewmembers after mission and potential differential responses between genders to the space environment. Our data potentially explains some of the physiological changes that have been observed in space such as mitochondria dysfunction, inhibition of T cell activation and telomere lengthening. Comparison of our results with other transcriptomics studies of ISS crewmembers’ blood cells will also be presented.

Maria Moreno Villanueva↗

The Next Generation Virgo Cluster Survey (NGVS). XXIV. The Red Sequence to ∼10{sup 6}L{sub ⊙} and Comparisons with Galaxy Formation Models

We use deep optical photometry from the Next Generation Virgo Cluster Survey (NGVS) to investigate the color–magnitude diagram for the galaxies inhabiting the core of this cluster. The sensitivity of the NGVS imaging allows us to continuously probe galaxy colors over a factor of ∼2 × 10{sup 5} in luminosity, from brightest cluster galaxies to scales overlapping classical satellites of the Milky Way (M{sub g{sup ′}} ∼ −9; M {sub *} ∼ 10{sup 6} M {sub ⊙}), within a single environment. Remarkably, we find the first evidence that the red sequence (RS) flattens in all colors at the faint-magnitude end (starting between −14 ≤ M{sub g{sup ′}} ≤ −13, around M {sub *} ∼ 4 × 10{sup 7} M {sub ⊙}), with the slope decreasing to ∼60% or less of its value at brighter magnitudes. This could indicate that the stellar populations of faint dwarfs in Virgo’s core share similar characteristics (e.g., constant mean age) over ∼3 mag in luminosity, suggesting that these galaxies were quenched coevally, likely via pre-processing in smaller hosts. We also compare our results to galaxy formation models, finding that the RS in model clusters have slopes at intermediate magnitudes that are too shallow, and in the case of semianalytic models, do not reproduce the flattening seen at both extremes (bright/faint) of the Virgo RS. Deficiencies in the chemical evolution of model galaxies likely contribute to the model-data discrepancies at all masses, while overly efficient quenching may also be a factor at dwarf scales. Deep UV and near-IR photometry are required to unambiguously diagnose the cause of the faint-end flattening.

79 ASTRONOMY AND ASTROPHYSICS↗

Shotgun metagenome data of a defined mock community using Oxford Nanopore, PacBio and Illumina technologies

Metagenomic sequence data from defined mock communities is crucial for the assessment of sequencing platform performance and downstream analyses, including assembly, binning and taxonomic assignment. We report a comparison of shotgun metagenome sequencing and assembly metrics of a defined microbial mock community using the Oxford Nanopore Technologies (ONT) MinION, PacBio and Illumina sequencing platforms. Our synthetic microbial community BMock12 consists of 12 bacterial strains with genome sizes spanning 3.2-7.2 Mbp, 40-73% GC content, and 1.5-7.3% repeats. Size selection of both PacBio and ONT sequencing libraries prior to sequencing was essential to yield comparable relative abundances of organisms among all sequencing technologies. While the Illumina-based metagenome assembly yielded good coverage with few misassemblies, contiguity was greatly improved by both, Illumina + ONT and Illumina + PacBio hybrid assemblies but increased misassemblies, most notably in genomes with high sequence similarity to each other. Our resulting datasets allow evaluation and benchmarking of bioinformatics software on Illumina, PacBio and ONT platforms in parallel.

54 ENVIRONMENTAL SCIENCES↗