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At least 253 records · Page 14

Comparative Penicillium spp. Transcriptomics: Conserved Pathways and Processes Revealed in Ungerminated Conidia and during Postharvest Apple Fruit Decay

Blue mold, caused by Penicillium spp., is an impactful postharvest disease resulting in significant economic losses due to reduced pome fruit quality and mycotoxin contamination. Using two Penicillium species with different levels of aggressiveness, transcriptomics were implemented in order to identify genes expressed during apple fruit decay and loci expressed in ungerminated conidia. Total RNA was isolated from ungerminated conidia and decayed apple fruit infected with P. expansum R19 or P. polonicum RS1. There were 2442 differentially expressed genes (DEGs) between the R19 and RS1 in apple. Comparisons within species between apple and conidia revealed 4404 DEGs for R19 and 2935 for RS1, respectively. Gene ontology (GO) analysis revealed differential regulation in fungal transport and metabolism genes during decay, suggesting a flux in nutrient acquisition and detoxification strategies. In R19, the oxidoreductase GO category comprised 20% of all DEG groups in apple verses conidia. Ungerminated conidia from both species showed DEGs encoding the glyoxylate shunt and beta-oxidation, specifying the earliest metabolic requirements for germination. This is the first study to identify pre-loaded transcripts in conidia from blue mold fungi, reveal unique genes between species expressed during apple decay, and show the expression dynamics of known fungal virulence factors. These findings will enable development of targeted approaches for blue mold abatement strategies.

59 BASIC BIOLOGICAL SCIENCES↗

Bionutrients-1: Utilizing Genomics and Transcriptomics to Assess the Reliability of Microorganisms for In Situ Nutrient Production on Long Duration Missions

The resupply of current long-duration crewed missions to the ISS relies on ground-launched supplies. As NASA looks toward Mars, ground-based resupply will no longer be an option. Critical nutrients, including vitamin C, vitamin K, folate, and thiamin, degrade during long-term storage, and regular consumption of these nutrients is essential for astronaut health. Another challenge of current food systems is the difficulty of consuming sufficient calories when subsisting on the limited flavors of freeze-dried food, which can lead to weight loss. The inclusion of microorganism-based food systems could alleviate both concerns. For example, the fermentation of rehydrated milk into yogurt with microorganisms genetically incorporating genes to produce critical vitamins would allow for both in situ production of nutrients and a fresh food product with additional flavor profiles. In comparison to plant food production, microorganisms require less flight infrastructure. The BioNutrients-1 mission is demonstrating viability of microbial fermentation food production in microgravity and testing the reliability of this approach for long-duration missions lacking resupply. While the BioNutrients-1 mission includes the collection of multiple phenotypic measurements, this status update will focus on the processing of samples for genomics and transcriptomics analyses as well as the planned analysis pipelines. First, the BioNutrients-1 mission seeks to identify microorganisms capable of surviving long-duration storage at ambient temperatures while maintaining genetic fidelity. To achieve this, nine commonly employed microbial species were stored at ambient temperatures in Stasis Packs on the ISS for five years. The viability and mutation rates will be measured at multiple time points for both flown and ground control samples. From an omics perspective, the changes in the bulk rates of point mutations and genetic rearrangements across the Stasis Pack species during the five years of storage will be determined, providing valuable insights into the potential of these microorganisms for long-duration space missions. Second, the BioNutrients-1 mission is characterizing the impact of microgravity on fermentation. Two strains of the yeast Saccharomyces cerevisiae, each encoding antioxidants (β-carotene or zeaxanthin) were flown to ISS for storage and fermentation within simplified bioreactors (Production Packs). The impact of microgravity on the expression of the antioxidant production genes and general metabolic genes will be determined using RNA sequencing. Ultimately, the transcriptome data will be compared to phenotypic measurements, such as the antioxidant yield, end-state biomass, and the production of EtOH, to determine the impacts of microgravity and long-term storage on microbial fermentation. The findings from this research will be instrumental in understanding the challenges and opportunities of microorganism-based food systems in space missions.

BioNutrients↗

Renin-Angiotensin System (RAS) in Hematopoietic Stem/Progenitor Cells (HS/PC) Predicts Vaso-Reparative Dysfunction and Progression of Diabetic Retinopathy (DR)

Purpose: We tested the hypothesis that loss of angiotensin converting enzyme 2 (ACE2) within diabetic HS/PCs (Hematopoietic Stem/Progenitor Cells) would be detrimental to HS/PC reparative function, and alter their ability to contribute to vascular remodeling in human subjects and rodent models of DR (Diabetic Retinopathy). Methods: Subjects (n52) were recruited as controls (n13) or diabetics (n39) with either no DR, mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of subjects. CD34+ HS/PCs were isolated from peripheral blood. RAS (Renin-Angiotensin System) gene expression and migration was measured. Diabetic ACE2 knockout (KO)C57BL6-Ins2 (Akita) mice at 3, 6 and 9 months of diabetes were compared to age-matched controls. Bone marrow HS/PC populations were analyzed by flow cytometry and migration and proliferation studies performed. Results: ACE2 gene expression in human CD34+ cells from diabetics without DR was increased compared to controls (p0.0437). Mas receptor mRNA was also increased in diabetics without DR, but reduced with the onset of NPDR (p0.0002), suggesting a loss of compensation. DR was associated with CD34+ cell migratory dysfunction. By VESGEN analysis, vessel density measured by several confirming parameters in early NPDR (n3) was greater than in normal retina (n6) in both arteries and veins, which suggests active retinal remodeling. ACE2KO-Akita and Akita cohorts showed reduced retinal thickness by OCT (Optical Coherence Tomography) at 9 months of diabetes. Absence of ACE2 in 9-month Akita mice led to an accelerated increase in acellular capillaries compared to diabetic alone. Electroretinogram (ERG) in ACE2KO-Akita mice resulted in persistent deterioration of the neural retina. Reparative function studies showed that ACE2KO exacerbated diabetes-induced impairment of LK (Low Potassium) cell migration and proliferative functions as early as 3-month of diabetes (p0.0019). Conclusions: Retinopathy and adverse vascular remodeling in subjects with diabetes was associated with a loss of the protective arm of RAS in HS/PCs. Loss of ACE2 exacerbated vascular dysfunction in diabetic mice.

retina↗

Targeted seed EMS mutagenesis reveals a basic helix–loop–helix transcription factor underlying male sterility in sorghum

Abstract Forward genetic screens of mutant populations are fundamental for functional genomics studies. However, isolating independent mutant alleles to molecularly identify causal genes is challenging in species recalcitrant to genetic manipulation. Here, we demonstrate that classic seed ethyl methanesulfonate (EMS) mutagenesis coupled with genome sequencing can overcome this limitation in sorghum. We used this method to generate new mutant alleles of sorghum MALE STERILE 8 (MS8) and identified the causal locus for the ms8 phenotype as Sobic.004G270900, which encodes the sorghum ortholog of maize bhlh122, a basic helix–loop–helix (bHLH) transcription factor required for male fertility in maize. Bulked segregant analysis mapped ms8-1 to a region on chromosome 4 containing Sobic.004G270900. Seeds from heterozygous MS8/ms8-1 plants were mutagenized and screened for chimeric inflorescences containing sectors with white, sterile anthers resembling the ms8-1 homozygous phenotype. DNA sequencing of sterile and fertile sectors from a single chimeric inflorescence revealed two mutations in Sobic.004G270900 within the sterile sector, but not the fertile sector. Isolation of this loss-of-function allele (ms8-2) established Sobic.004G270900 as the causative locus for male sterility in the ms8 mutant. We generated additional alleles of MS8 in a different genetic background using CRISPR/Cas9-based gene editing, where deletions in Sobic.004G270900 also resulted in male sterility. Our work identified a gene underlying male sterility in sorghum and provides a novel and straightforward genetic tool for researchers who lack access to advanced transformation facilities to validate gene candidates. Unlike gene editing, no prior knowledge of candidate genes is required for targeted seed EMS mutagenesis to aid identification of causal loci.

Genetics & Heredity↗

Stacking AsFMT overexpression with BdPMT loss of function enhances monolignol ferulate production in Brachypodium distachyon

To what degree can the lignin subunits in a monocot be derived from monolignol ferulate (ML-FA) conjugates? This simple question comes with a complex set of variables. Three potential requirements for optimizing ML-FA production are as follows: (1) The presence of an active FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme throughout monolignol production; (2) Suppression or elimination of enzymatic pathways competing for monolignols and intermediates during lignin biosynthesis; and (3) Exclusion of alternative phenolic compounds that participate in lignification. A 16-fold increase in lignin-bound ML-FA incorporation was observed by introducing an AsFMT gene into Brachypodium distachyon. On its own, knocking out the native p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (BdPMT) pathway that competes for monolignols and the p-coumaroyl-CoA intermediate did not change ML-FA incorporation, nor did partial loss of CINNAMOYL-CoA REDUCTASE1 (CCR1) function, which reduced metabolic flux to monolignols. However, stacking AsFMT into the Bdpmt-1 mutant resulted in a 32-fold increase in ML-FA incorporation into lignin over the wild-type level.

59 BASIC BIOLOGICAL SCIENCES↗

Elucidating the drought-responsive changes in Poplar cuticular waxes: A GWAS analysis of genes involved in fatty acid biosynthesis

Drought and episodic drought events are major impending impacts of climate change, limiting the productivity of plants and especially trees due to their inherent high transpiration rates. One common mechanism used by plants to cope with drought stress is to change the composition of their leaf cuticular waxes. Cuticular waxes are essential for controlling non-stomatal water loss and are typically composed of a homologous series of very-long-chain fatty acid-derived compounds, as well as flavonoids, tocopherols, triterpenoids, and phytosterols. In this study, we compared the cuticular waxes of 339 natural accessions of Populus trichocarpa (black cottonwood) grown under control and drought conditions in a common garden. A Genome-Wide Association Study (GWAS) was then used to identify candidate genes associated with cuticular wax biosynthesis and/or its regulation. Although no major differences were observed in total wax load when subject to drought conditions, the amounts of the individual wax constituents were indeed responsive to drought. Specifically, changes in alkenes, alcohols, esters, and aldehydes were evident, and suggest that they contribute to the drought response/tolerance in poplar. GWAS uncovered several genes linked to fatty acid biosynthesis, including CER1, CER3, CER4, FATB, FAB1, FAR3, FAR4, KCS, and a homolog of SOH1, as well as other candidate genes that may be involved in coordinating the drought responses in poplar trees. Our findings provide new evidence that genotype-specific shifts in wax composition, rather than total wax accumulation, contribute to drought adaptation in poplar. Additionally, we show that genetic variation in key wax biosynthetic genes drives cuticular wax plasticity in P. trichocarpa under drought, identifying putative molecular targets for improving stress resilience in trees. This study expands our understanding of the adaptative mechanisms of the cuticle and their potential for enhancing drought tolerance in poplar species.

Alkanes↗

Development and In Vivo Evaluation of a MGF110-1L Deletion Mutant in African Swine Fever Strain Georgia

African swine fever (ASF) is currently causing an epizootic, affecting pigs throughout Eurasia, and causing significant economic losses in the swine industry. ASF is caused by African swine fever virus (ASFV) that consists of a large dsDNA genome that encodes for more than 160 genes; few of these genes have been studied in detail. ASFV contains four multi-gene family (MGF) groups of genes that have been implicated in regulating the immune response and host specificity; however, the individual roles of most of these genes have not been well studied. Here, we describe the evaluation of the previously uncharacterized ASFV MGF110-1L open reading frame (ORF) using a deletion mutant of the ASFV currently circulating throughout Eurasia. The recombinant ASFV lacking the MGF110-1L gene (ASFV-G-ΔMGF110-1L) demonstrated in vitro that the MGF110-1L gene is non-essential, since ASFV-G-ΔMGF110-1L had similar replication kinetics in primary swine macrophage cell cultures when compared to parental highly virulent field isolate Georgia2007 (ASFV-G). Experimental infection of domestic pigs with ASFV-G-ΔMGF110-1L produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the MGF110-1L gene from the ASFV genome does not affect viral virulence.

59 BASIC BIOLOGICAL SCIENCES↗

Trade Study for 9 kW Water Membrane Evaporator

Sublimators have been proposed and used in spacecraft for heat rejection. Sublimators are desirable heat rejection devices for short duration use because they can transfer large amounts of heat using little mass and are self-regulating devices. Sublimators reject heat into space by freezing water inside a porous substrate, allowing it to sublimate into vapor, and finally venting it into space. The state of the art thermal control system in orbiting spacecraft is a two loop, two fluid system. The external coolant loop typically uses a toxic single phase fluid that acquires heat from the spacecraft and rejects most of it via a radiator. The sublimator functions as a transient topper for orbiting spacecraft during day pass periods when radiator efficiency decreases. The sublimator interfaces with the internal loop through a built in heat exchanger. The internal loop fluid is non-toxic and is typically a propylene glycol and water solution with inhibitors to prevent corrosion with aluminum fins of the heat exchangers. Feedwater is supplied from a separate line to the sublimator to maintain temperature control of the cabin and vehicle hardware. Water membrane evaporators have been developed for spacecraft and spacesuits. They function similar to a sublimator but require a backpressure valve which could be actuated for this application with a simple fully open or fully closed modes. This technology would be applied to orbital thermal control (lunar or planetary). This paper details a trade study showing that evaporators would greatly reduce the consumable that is used, effectively wasted, by sublimators during start up and shut down during the topping phases of each orbit. State of the art for 9 kW sublimators reject about 870 W per kilogram of mass and 1150 W per liter of volume. If water with corrosion inhibitors is used the evaporators would be about 80% of the mass and volume of the equivalent system. The size and mass increases to about 110% if the internal fluid is 50% propylene glycol/50% water. The true benefit comes from the backpressure valve, that prevents the cyclical shutdown/startup loss of the sublimator and amounts to as much as 0.85 kg per orbit.

Bue, Grant C.↗

Identification of recG genetic interactions in Escherichia coli by transposon sequencing

ABSTRACT Maintaining the integrity of the genome is of utmost importance for cell division and propagation. In Escherichia coli , the RecG protein has been implicated in processing branched recombination intermediates during DNA repair processes, but the primary cellular role(s) of RecG and the repair pathways in which it acts have been difficult to define. To gain additional insight into RecG function, we employed transposon sequencing to identify recG genetic interactions and reveal complementary or redundant functions. The strongest hits from the screen were the dam , uvrD , rnhA , radA , and rep genes. The conditional importance of these five genes in cells lacking recG was confirmed using a plasmid-based assay, revealing synthetic lethal interactions for most double deletion strains. Several of the synthetic lethal gene combinations (with uvrD, rep, and radA , but not rnhA or dam ) were suppressed by deletion of recF or recO , indicating that their genetic relationships involved roles in post-replication gap repair. Additionally, loss of the RecG/SSB interaction phenocopied a recG deletion when combined with dam , uvrD , radA , or rnhA deletions but not with rep . The results reinforce the idea of RecG as a general genome guardian. RecG has at least two functions. It plays an important role in the resolution of joint molecules behind the fork, formed during post-replication gap repair. RecG is also required to suppress genome over-replication caused by unscheduled replication initiation at R-loops, at double-strand breaks caused by dam inactivation, and during replication termination. IMPORTANCE DNA damage and subsequent DNA repair processes are mutagenic in nature and an important driver of evolution in prokaryotes, including antibiotic resistance development. Genetic screening approaches, such as transposon sequencing (Tn-seq), have provided important new insights into gene function and genetic relationships. Here, we employed Tn-seq to gain insight into the function of the recG gene, which renders Escherichia coli cells moderately sensitive to a variety of DNA-damaging agents when they are absent. The reported recG genetic interactions can be used in combination with future screens to aid in a more complete reconstruction of DNA repair pathways in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗

Influence of Space Flight Factors on the Genetic Properties of Streptomyces Lividans 66 (PIJ702)

Gram-positive Streptomyces bacteria display genetic instability in response to external factors. Strain S. lividans 66 harbors the multicopy plasmid pIJ702 with selective and differential marker genes for antibiotic thiostrepton resistance and melanin production. Culture plates of modified ISP agar medium with and without thiostrepton were flown on Foton-M2. Suboptimal flight temperatures, which were simulated for asynchronous ground controls, resulted in slow growth and failure to differentiate and sporulate. Flight samples and asynchronous controls showed a high frequency of failing to express plasmid markers compared to laboratory controls. This was associated with loss of plasmid DNA and likely resulted from suboptimal temperatures for flight cultures and controls. Neither restriction fragment length polymorphism, nor polymerase chain reaction amplification coupled with denaturing gradient gel electrophoresis, revealed differences between pIJ702 DNA from flight vs. control clones. Mutations of the plasmid marker genes resulting from specific spaceflight factors, e.g., microgravity and radiation, were not detected.

Tabakov, V. Yu.↗

Data for Creating Yellow Seed Camelina sativa with Enhanced Oil Accumulation by CRISPR-Mediated Disruption of Transparent Testa 8

Camelina ( Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well-organized seed coat mucilage layer. Transcriptomic analysis of CsTT8-edited seeds revealed significantly increased expression of the lipid-related transcription factors LEC1, LEC2, FUS3, and WRI1 and their downstream fatty acid synthesis-related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced-oil germplasm in camelina. The resulting lines may directly contribute to future net-zero carbon energy production or be combined with other traits to produce desired lipid-derived bioproducts at high yields.

Biofuels↗

Adaptive laboratory evolution and genetic engineering improved terephthalate utilization in Pseudomonas putida KT2440

Poly(ethylene terephthalate) (PET) is one of the most ubiquitous plastics and can be depolymerized through biological and chemo-catalytic routes to its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG). TPA and EG can be re-synthesized into PET for closed-loop recycling or microbially converted into higher-value products for open-loop recycling. Here, in this study, we expand on our previous efforts engineering and applying Pseudomonas putida KT2440 for PET conversion by employing adaptive laboratory evolution (ALE) to improve TPA catabolism. Three P. putida strains with varying degrees of metabolic engineering for EG catabolism underwent an automation-enabled ALE campaign on TPA, a TPA and EG mixture, and glucose as a control. ALE increased the growth rate on TPA and TPA-EG mixtures by 4.1- and 3.5-fold, respectively, in approximately 350 generations. Evolved isolates were collected at the midpoints and endpoints of 39 independent ALE experiments, and growth rates were increased by 0.15 and 0.20 h -1 on TPA and a TPA-EG, respectively, in the best performing isolates. Whole-genome re-sequencing identified multiple converged mutations, including loss-of-function mutations to global regulators gacS, gacA, and turA along with large duplication and intergenic deletion events that impacted the heterologously-expressed tphAB II catabolic genes. Reverse engineering of these targets confirmed causality, and a strain with all three regulators deleted and second copies of tphAB II and tpaK displayed improved TPA utilization compared to the base strain. Taken together, an iterative strain engineering process involving heterologous pathway engineering, ALE, whole genome sequencing, and genome editing identified five genetic interventions that improve P. putida growth on TPA, aimed at developing enhanced whole-cell biocatalysts for PET upcycling.

36 MATERIALS SCIENCE↗

Impact of Selenium and Vitamin E Deficiency on Zika Virus Pathogenesis and Immune Response in Mice

Micronutrient status is recognized to influence host susceptibility to viral infections, yet its impact on Zika virus (ZIKV) pathogenesis remains incompletely understood. We investigated the effects of dietary selenium and combined selenium plus vitamin E deficiency on ZIKV infection outcomes in a type I interferon α/β receptor knockout (Ifnar1 −/− ) murine model. Mice maintained on deficient diets exhibited significantly lower neutralizing antibody titers and reduced levels of key antiviral cytokines (IFN-γ, TNF-α, IFN-α, IFN-β, IL-12p70, CCL5) compared to controls. Correspondingly, higher viral RNA loads were detected in the brains of double-deficient mice, which also experienced greater weight loss and increased mortality. Deep sequencing revealed no major differences in overall viral genome diversity across diet groups; however, specific mutations, including V330L and D67E in the E gene, and V360I in the NS3 gene, were enriched or detected in nutritionally deficient animals. These findings suggest that antioxidant micronutrient deficiency impairs both humoral and cellular immune responses to ZIKV, potentially facilitating enhanced neuroinvasion. While the functional consequences of the identified mutations warrant further investigation, our results underscore the importance of adequate micronutrient intake for optimal antiviral defense. Further studies are needed to clarify the epidemiological significance of these observations.

Biological and medical sciences↗

Diversification of JAZ‐MYC signaling function in immune metabolism

Summary Jasmonate (JA) re‐programs metabolism to confer resistance to diverse environmental threats. Jasmonate stimulates the degradation of JASMONATE ZIM‐DOMAIN (JAZ) proteins that repress the activity of MYC transcription factors. In Arabidopsis thaliana , MYC and JAZ are encoded by 4 and 13 genes, respectively. The extent to which expansion of the MYC and JAZ families has contributed to functional diversification of JA responses is not well understood. Here, we investigated the role of MYC and JAZ paralogs in controlling the production of defense compounds derived from aromatic amino acids (AAAs). Analysis of loss‐of‐function and dominant myc mutations identified MYC3 and MYC4 as the major regulators of JA‐induced tryptophan metabolism. We developed a JAZ family‐based, forward genetics approach to screen randomized jaz polymutants for allelic combinations that enhance tryptophan biosynthetic capacity. We found that mutants defective in all members ( JAZ1/2/5/6 ) of JAZ group I over‐accumulate AAA‐derived defense compounds, constitutively express marker genes for the JA–ethylene branch of immunity and are more resistant to necrotrophic pathogens but not insect herbivores. In defining JAZ and MYC paralogs that regulate the production of amino‐acid‐derived defense compounds, our results provide insight into the specificity of JA signaling in immunity.

jasmonate↗

Magnetic and Electrical Characteristics of Cobalt-Based Amorphous Materials and Comparison to a Permalloy Type Polycrystalline Material

Magnetic component designers are always looking for improved soft magnetic core materials to increase the efficiency, temperature rating and power density of transformers, motors, generators and alternators, and energy density of inductors. In this paper, we report on the experimental investigation of commercially available cobalt-based amorphous alloys which, in their processing, were subjected to two different types of magnetic field anneals: A longitudinal magnetic field anneal or a transverse magnetic field anneal. The longitudinal field annealed material investigated was Metglas 2714A. The electrical and magnetic characteristics of this material were investigated over the frequency range of 1 to 200 kHz and temperature range of 23 to 150 C for both sine and square wave voltage excitation. The specific core loss was lower for the square than the sine wave voltage excitation for the same maximum flux density, frequency and temperature. The transverse magnetic field annealed core materials include Metglas 2714AF and Vacuumschmelze 6025F. These two materials were experimentally characterized over the frequency range of 10 to 200 kHz for sine wave voltage excitation and 23 C only. A comparison of the 2174A to 2714AF found that 2714AF always had lower specific core loss than 2714A for any given magnetic flux density and frequency and the ratio of specific core loss of 2714A to 2714AF was dependent on both magnetic flux density and frequency. A comparison was also made of the 2714A, 2714AF, and 6025F materials to two different tape thicknesses of the polycrystalline Supermalloy material and the results show that 2714AF and 6025F have the lowest specific core loss at 100 kHz over the magnetic flux density range of 0.1 to 0.4 Tesla.

Wieserman, William R.↗

Crystal structure of Escherichia coli thiamine pyrophosphate-sensing riboswitch in the apo state

The thiamine pyrophosphate (TPP)-sensing riboswitch is one of the earliest discovered and most widespread riboswitches. Numerous structural studies have been reported for this riboswitch bound with various ligands. However, the ligand-free (apo) structure remains unknown. Here, we report a 3.1 Å resolution crystal structure of Escherichia coli TPP riboswitch in the apo state, which exhibits an extended, Y-shaped conformation further supported by small-angle X-ray scattering data and driven molecular dynamics simulations. The loss of ligand interactions results in helical uncoiling of P5 and disruption of the key tertiary interaction between the sensory domains. Opening of the aptamer propagates to the gene-regulatory P1 helix and generates the key conformational flexibility needed for the switching behavior. Much of the ligand-binding site at the three-way junction is unaltered, thereby maintaining a partially preformed pocket. Together, these results paint a dynamic picture of the ligand-induced conformational changes in TPP riboswitches that confer conditional gene regulation.

59 BASIC BIOLOGICAL SCIENCES↗

Diploid-dominant life cycles characterize the early evolution of Fungi

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane– and cell-wall–associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

59 BASIC BIOLOGICAL SCIENCES↗

[Dentists on Mars]

The oral health of astronauts, last studied in the mid- 1970s on Skylab, has not been a priority area of study for NASA due to the short length of shuttle stays. But with longer stays on the International Space Station, and planning for a trip to Mars, investigations into how spaceflight affects oral health are needed. The objective of this symposium is to introduce the dental research community to changes occurring in humans who go into space, and how these changes might impact oral health. Dr. Millie Hughes-Fulford, an astronaut-scientist, will review what happens to humans who go into space, and the difficulties of living, and carrying out experiments in space. She will then discuss her research on cell cultures of osteoblasts in space, and in hypergravity Dr. Gerald Sonnenfeld will review immunological changes that occur in spaceflight, and relate the observed decreases in various types of immunological responses to possible effects on oral immunological factors. Dr. Marian Lewis will discuss the effects of spaceflight on gene expression using results from her spaceflight experiments on various cell types. Dr. Jack van Loon, fiom the Dutch Experiment Support Center, will review what is known about bone loss in humans and rats and metatarsal cultures which go into space, and review ground based models (head down bedrest, and tail suspension) that simulate the unloading of spaceflight. Attendees will gain a knowledge of spaceflight research, and information on getting their own experiments in space. The symposium is supported by the NASA Office of Bioastronautics

Duke, P. J.↗