Flight IV technical report for experiment 74-37 contained polycrystalline solidification in low-G

Experiments were performed to study the effect of a low-gravity environment on the columnar-to-equiaxed transition (CET) during polycrystalline solidification. Solutions of H2O-30 wt% NH4Cl and H2O-37 wt% NH4Cl were solidified in semicylindrical molds with radial heat extraction. Both solutions were quenched from the same soak temperature (90 C); the respective superheat temperatures were, therefore, approximately 57 and 23 C. The lower superheat resulted in a completely columnar structure, and the higher superheat resulted in a 1/3 columnar - 2/3 equiaxed microstructure; these results were independent of the relationship between heat flow direction and gravity. Grain multiplication mechanisms observed were showering, thermal inversion driven convection cells, and compositionally induced density inversion driven convection cells

Voltage-Dependent Structural Interactions in the Shaker K+ Channel

Using a strategy related to intragenic suppression, we previously obtained evidence for structural interactions in the voltage sensor of Shaker K+ channels between residues E283 in S2 and R368 and R371 in S4 (Tiwari-Woodruff, S.K., C.T. Schulteis, A.F. Mock, and D.M. Papazian. 1997. Biophys. J. 72:1489–1500). Because R368 and R371 are involved in the conformational changes that accompany voltage-dependent activation, we tested the hypothesis that these S4 residues interact with E283 in S2 in a subset of the conformational states that make up the activation pathway in Shaker channels. First, the location of residue 283 at hyperpolarized and depolarized potentials was inferred by substituting a cysteine at that position and determining its reactivity with hydrophilic, sulfhydryl-specific probes. The results indicate that position 283 reacts with extracellularly applied sulfhydryl reagents with similar rates at both hyperpolarized and depolarized potentials. We conclude that E283 is located near the extracellular surface of the protein in both resting and activated conformations. Second, we studied the functional phenotypes of double charge reversal mutations between positions 283 and 368 and between 283 and 371 to gain insight into the conformations in which these positions approach each other most closely. We found that combining charge reversal mutations at positions 283 and 371 stabilized an activated conformation of the channel, and dramatically slowed transitions into and out of this state. In contrast, charge reversal mutations at positions 283 and 368 stabilized a closed conformation, which by virtue of the inferred position of 368 corresponds to a partially activated (intermediate) closed conformation. From these results, we propose a preliminary model for the rearrangement of structural interactions of the voltage sensor during activation of Shaker K+ channels

Central Metabolic Responses to Ozone and Herbivory Affect Photosynthesis and Stomatal Closure.

Plants have evolved adaptive mechanisms that allow them to tolerate a continuous range of abiotic and biotic stressors. Tropospheric ozone (O3), a global anthropogenic pollutant, directly affects living organisms and ecosystems, including plant-herbivore interactions. In this study, we investigate the stress responses of Brassica nigra (wild black mustard) exposed consecutively to O3 and the specialist herbivore Pieris brassicae Transcriptomics and metabolomics data were evaluated using multivariate, correlation, and network analyses for the O3 and herbivory responses. O3 stress symptoms resembled those of senescence and phosphate starvation, while a sequential shift from O3 to herbivory induced characteristic plant defense responses, including a decrease in central metabolism, induction of the jasmonic acid/ethylene pathways, and emission of volatiles. Omics network and pathway analyses predicted a link between glycerol and central energy metabolism that influences the osmotic stress response and stomatal closure. Further physiological measurements confirmed that while O3 stress inhibited photosynthesis and carbon assimilation, sequential herbivory counteracted the initial responses induced by O3, resulting in a phenotype similar to that observed after herbivory alone. This study clarifies the consequences of multiple stress interactions on a plant metabolic system and also illustrates how omics data can be integrated to generate new hypotheses in ecology and plant physiology

Yap1-Driven Intestinal Repair Is Controlled by Group 3 Innate Lymphoid Cells

Intestinal repair is driven by epithelial stem cells, but how these stem cells are instructed to initiate repair was unknown. Here, Romera-Hernández et al. report that epithelial proliferation after damage is independent of the stem cell-protective signal IL-22 but requires ILC3-dependent amplification of regenerative YAP1 signaling in stem cells.Tissue repair requires temporal control of progenitor cell proliferation and differentiation to replenish damaged cells. In response to acute insult, group 3 innate lymphoid cells (ILC3s) regulate intestinal stem cell maintenance and subsequent tissue repair. ILC3-derived IL-22 is important for stem cell protection, but the mechanisms of ILC3-driven tissue regeneration remain incompletely defined. Here we report that ILC3-driven epithelial proliferation and tissue regeneration are independent of IL-22. In contrast, ILC3s amplify the magnitude of Hippo-Yap1 signaling in intestinal crypt cells, ensuring adequate initiation of tissue repair and preventing excessive pathology. Mechanistically, ILC3-driven tissue repair is Stat3 indepe

ReCombine: A Suite of Programs for Detection and Analysis of Meiotic Recombination in Whole-Genome Datasets

In meiosis, the exchange of DNA between chromosomes by homologous recombination is a critical step that ensures proper chromosome segregation and increases genetic diversity. Products of recombination include reciprocal exchanges, known as crossovers, and non-reciprocal gene conversions or non-crossovers. The mechanisms underlying meiotic recombination remain elusive, largely because of the difficulty of analyzing large numbers of recombination events by traditional genetic methods. These traditional methods are increasingly being superseded by high-throughput techniques capable of surveying meiotic recombination on a genome-wide basis. Next-generation sequencing or microarray hybridization is used to genotype thousands of polymorphic markers in the progeny of hybrid yeast strains. New computational tools are needed to perform this genotyping and to find and analyze recombination events. We have developed a suite of programs, ReCombine, for using short sequence reads from next-generation sequencing experiments to genotype yeast meiotic progeny. Upon genotyping, the program CrossOver, a component of ReCombine, then detects recombination products and classifies them into categories based on the features found at each location and their distribution among the various chromatids. CrossOver is also capable of analyzing segregation data from microarray experiments or other sources. This package of programs is designed to allow even researchers without computational expertise to use high-throughput, whole-genome methods to study the molecular mechanisms of meiotic recombination

Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators

Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on differential single-cell gene expression analysis of mesenchymal osteolineage cells close to and further removed from hematopoietic stem/progenitor cells to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. Amongst the genes which were preferentially expressed in proximal cells, we functionally examined three secreted or cell surface molecules not previously connected to HSPC biology: the secreted RNase Angiogenin, the cytokine IL18 and the adhesion molecule Embigin and discovered that all of these factors are HSPC quiescence regulators. Our proximity-based differential single cell approach therefore reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance understanding of microenvironmental regulation of stem cell function

Expression of Plet1 controls interstitial migration of murine small intestinal dendritic cells.

Under homeostatic conditions, dendritic cells (DCs) continuously patrol the intestinal lamina propria. Upon antigen encounter, DCs initiate C-C motif chemokine receptor 7 (CCR7) expression and migrate into lymph nodes to direct T cell activation and differentiation. The mechanistic underpinnings of DC migration from the tissues to lymph nodes have been largely elucidated, contributing greatly to our understanding of DC functionality and intestinal immunity. In contrast, the molecular mechanisms allowing DCs to efficiently migrate through the complex extracellular matrix of the intestinal lamina propria prior to antigen encounter are still incompletely understood. Here we show that small intestinal murine CD11b <sup>+</sup> CD103 <sup>+</sup> DCs express Placenta-expressed transcript 1 (Plet1), a glycophoshatidylinositol (GPI)-anchored surface protein involved in migration of keratinocytes during wound healing. In the absence of Plet1, CD11b <sup>+</sup> CD103 <sup>+</sup> DCs display aberrant migratory behavior, and accumulate in the small intestine, independent of CCR7 responsiveness. RNA-sequencing indicated involvement of Plet1 in extracellular matrix-interactiveness, and subsequent in-vitro migration assays revealed that Plet1 augments the ability of DCs to migrate through extracellular matrix containing environments. In conclusion, our findings reveal that expression of Plet1 facilitates homeostatic interstitial migration of small intestinal DCs

Functional differences between human NKp44- and NKp44+ RORC+ innate lymphoid cells

Human RORC+ lymphoid tissue inducer cells are part of a rapidly expanding family of innate lymphoid cells (ILC) that participate in innate and adaptive immune responses as well as in lymphoid tissue (re) modeling. The assessment of a potential role for innate lymphocyte-derived cytokines in human homeostasis and disease is hampered by a poor characterization of RORC+ innate cell subsets and a lack of knowledge on the distribution of these cells in adults. Here we show that functionally distinct subsets of human RORC+ innate lymphoid cells are enriched for secretion of IL-17a or IL-22. Both subsets have an activated phenotype and can be distinguished based on the presence or absence of the natural cytotoxicity receptor NKp44. NKp44+ IL-22 producing cells are present in tonsils while NKp44- IL-17a producing cells are present in fetal developing lymph nodes. Development of human intestinal NKp44+ ILC is a programmed event that is independent of bacterial colonization and these cells colonize the fetal intestine during the first trimester. In the adult intestine, NKp44+ ILC are the main ILC subset producing IL-22. NKp44- ILC remain present throughout adulthood in peripheral non-inflamed lymph nodes as resting, non-cytokine producing cells. However, upon stimulation lymph node ILC can swiftly initiate cytokine transcription suggesting that secondary human lymphoid organs may function as a reservoir for innate lymphoid cells capable of participating in inflammatory responses

Repertoire of Intensive Care Unit Pneumonia Microbiota

Despite the considerable number of studies reported to date, the causative agents of pneumonia are not completely identified. We comprehensively applied modern and traditional laboratory diagnostic techniques to identify microbiota in patients who were admitted to or developed pneumonia in intensive care units (ICUs). During a three-year period, we tested the bronchoalveolar lavage (BAL) of patients with ventilator-associated pneumonia, community-acquired pneumonia, non-ventilator ICU pneumonia and aspiration pneumonia, and compared the results with those from patients without pneumonia (controls). Samples were tested by amplification of 16S rDNA, 18S rDNA genes followed by cloning and sequencing and by PCR to target specific pathogens. We also included culture, amoeba co-culture, detection of antibodies to selected agents and urinary antigen tests. Based on molecular testing, we identified a wide repertoire of 160 bacterial species of which 73 have not been previously reported in pneumonia. Moreover, we found 37 putative new bacterial phylotypes with a 16S rDNA gene divergence ≥98% from known phylotypes. We also identified 24 fungal species of which 6 have not been previously reported in pneumonia and 7 viruses. Patients can present up to 16 different microorganisms in a single BAL (mean ± SD; 3.77±2.93). Some pathogens considered to be typical for ICU pneumonia such as Pseudomonas aeruginosa and Streptococcus species can be detected as commonly in controls as in pneumonia patients which strikingly highlights the existence of a core pulmonary microbiota. Differences in the microbiota of different forms of pneumonia were documented

Low Levels of DNA Polymerase Alpha Induce Mitotic and Meiotic Instability in the Ribosomal DNA Gene Cluster of Saccharomyces cerevisiae

The ribosomal DNA (rDNA) genes of Saccharomyces cerevisiae are located in a tandem array of about 150 repeats. Using a diploid with markers flanking and within the rDNA array, we showed that low levels of DNA polymerase alpha elevate recombination between both homologues and sister chromatids, about five-fold in mitotic cells and 30-fold in meiotic cells. This stimulation is independent of Fob1p, a protein required for the programmed replication fork block (RFB) in the rDNA. We observed that the fob1 mutation alone significantly increased meiotic, but not mitotic, rDNA recombination, suggesting a meiosis-specific role for this protein. We found that meiotic cells with low polymerase alpha had decreased Sir2p binding and increased Spo11p-catalyzed double-strand DNA breaks in the rDNA. Furthermore, meiotic crossover interference in the rDNA is absent. These results suggest that the hyper-Rec phenotypes resulting from low levels of DNA polymerase alpha in mitosis and meiosis reflect two fundamentally different mechanisms: the increased mitotic recombination is likely due to increased double-strand DNA breaks (DSBs) resulting from Fob1p-independent stalled replication forks, whereas the hyper-Rec meiotic phenotype results from increased levels of Spo11-catalyzed DSBs in the rDNA