CRISPR Dependency Screens in Primary Hematopoietic Stem Cells Identify KDM3B as a Genotype Specific Vulnerability in IDH2- and TET2-Mutant Cells.

Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Mutation order in acute myeloid leukemia identifies uncommon patterns of evolution and illuminates phenotypic heterogeneity.

Acute myeloid leukemia (AML) has a poor prognosis and a heterogeneous mutation landscape. Although common mutations are well-studied, little research has characterized how the sequence of mutations relates to clinical features. Using published, single-cell DNA sequencing data from three institutions, we compared clonal evolution patterns in AML to patient characteristics, disease phenotype, and outcomes. Mutation trees, which represent the order of select mutations, were created for 207 patients from targeted panel sequencing data using 1 639 162 cells, 823 mutations, and 275 samples. In 224 distinct orderings of mutated genes, mutations related to DNA methylation typically preceded those related to cell signaling, but signaling-first cases did occur, and had higher peripheral cell counts, increased signaling mutation homozygosity, and younger patient age. Serial sample analysis suggested that NPM1 and DNA methylation mutations provide an advantage to signaling mutations in AML. Interestingly, WT1 mutation evolution shared features with signaling mutations, such as WT1-early being proliferative and occurring in younger individuals, trends that remained in multivariable regression. Some mutation orderings had a worse prognosis, but this was mediated by unfavorable mutations, not mutation order. These findings add a dimension to the mutation landscape of AML, identifying uncommon patterns of leukemogenesis and shedding light on heterogeneous phenotypes.

Jak2V617F Reversible Activation Shows Its Essential Requirement in Myeloproliferative Neoplasms.

Gain-of-function mutations activating JAK/STAT signaling are seen in the majority of patients with myeloproliferative neoplasms (MPN), most commonly JAK2V617F. Although clinically approved JAK inhibitors improve symptoms and outcomes in MPNs, remissions are rare, and mutant allele burden does not substantively change with chronic therapy. We hypothesized this is due to limitations of current JAK inhibitors to potently and specifically abrogate mutant JAK2 signaling. We therefore developed a conditionally inducible mouse model allowing for sequential activation, and then inactivation, of Jak2V617F from its endogenous locus using a combined Dre-rox/Cre-lox dual-recombinase system. Jak2V617F deletion abrogates MPN features, induces depletion of mutant-specific hematopoietic stem/progenitor cells, and extends overall survival to an extent not observed with pharmacologic JAK inhibition, including when cooccurring with somatic Tet2 loss. Our data suggest JAK2V617F represents the best therapeutic target in MPNs and demonstrate the therapeutic relevance of a dual-recombinase system to assess mutant-specific oncogenic dependencies in vivo. SIGNIFICANCE: Current JAK inhibitors to treat myeloproliferative neoplasms are ineffective at eradicating mutant cells. We developed an endogenously expressed Jak2V617F dual-recombinase knock-in/knock-out model to investigate Jak2V617F oncogenic reversion in vivo. Jak2V617F deletion abrogates MPN features and depletes disease-sustaining MPN stem cells, suggesting improved Jak2V617F targeting offers the potential for greater therapeutic efficacy. See related commentary by Celik and Challen, p. 701. This article is featured in Selected Articles from This Issue, p. 695.

FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Deep generative model deciphers derailed trajectories in acute myeloid leukemia.

Single-cell genomics has the potential to map cell states and their dynamics in an unbiased way in response to perturbations like disease. However, elucidating the cell-state transitions from healthy to disease requires analyzing data from perturbed samples jointly with unperturbed reference samples. Existing methods for integrating and jointly visualizing single-cell datasets from distinct contexts tend to remove key biological differences or do not correctly harmonize shared mechanisms. We present Decipher, a model that combines variational autoencoders with deep exponential families to reconstruct derailed trajectories (https://github.com/azizilab/decipher). Decipher jointly represents normal and perturbed single-cell RNA-seq datasets, revealing shared and disrupted dynamics. It further introduces a novel approach to visualize data, without the need for methods such as UMAP or TSNE. We demonstrate Decipher on data from acute myeloid leukemia patient bone marrow specimens, showing that it successfully characterizes the divergence from normal hematopoiesis and identifies transcriptional programs that become disrupted in each patient when they acquire NPM1 driver mutations.

Mutation order in acute myeloid leukemia identifies uncommon patterns of evolution and illuminates phenotypic heterogeneity.

Acute myeloid leukemia (AML) has a poor prognosis and a heterogeneous mutation landscape. Although common mutations are well-studied, little research has characterized how the sequence of mutations relates to clinical features. Using published, single-cell DNA sequencing data from three institutions, we compared clonal evolution patterns in AML to patient characteristics, disease phenotype, and outcomes. Mutation trees, which represent the order of select mutations, were created for 207 patients from targeted panel sequencing data using 1 639 162 cells, 823 mutations, and 275 samples. In 224 distinct orderings of mutated genes, mutations related to DNA methylation typically preceded those related to cell signaling, but signaling-first cases did occur, and had higher peripheral cell counts, increased signaling mutation homozygosity, and younger patient age. Serial sample analysis suggested that NPM1 and DNA methylation mutations provide an advantage to signaling mutations in AML. Interestingly, WT1 mutation evolution shared features with signaling mutations, such as WT1-early being proliferative and occurring in younger individuals, trends that remained in multivariable regression. Some mutation orderings had a worse prognosis, but this was mediated by unfavorable mutations, not mutation order. These findings add a dimension to the mutation landscape of AML, identifying uncommon patterns of leukemogenesis and shedding light on heterogenous phenotypes.

Single-cell genotypic and phenotypic analysis of measurable residual disease in acute myeloid leukemia.

Measurable residual disease (MRD), defined as the population of cancer cells that persist following therapy, serves as the critical reservoir for disease relapse in acute myeloid leukemia and other malignancies. Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of more effective curative treatments. Discriminating between residual leukemic clones, preleukemic clones, and normal precursors remains a challenge with current MRD tools. Here, we developed a single-cell MRD (scMRD) assay by combining flow cytometric enrichment of the targeted precursor/blast population with integrated single-cell DNA sequencing and immunophenotyping. Our scMRD assay shows high sensitivity of approximately 0.01%, deconvolutes clonal architecture, and provides clone-specific immunophenotypic data. In summary, our scMRD assay enhances MRD detection and simultaneously illuminates the clonal architecture of clonal hematopoiesis/preleukemic and leukemic cells surviving acute myeloid leukemia therapy.

Hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer.

Clonal hematopoiesis (CH) is defined as clonal expansion of mutant hematopoietic stem cells absent diagnosis of a hematologic malignancy. Presence of CH in solid tumor patients, including colon cancer, correlates with shorter survival. We hypothesized that bone marrow-derived cells with heterozygous loss-of-function mutations of DNMT3A, the most common genetic alteration in CH, contribute to the pathogenesis of colon cancer. In a mouse model that combines colitis-associated colon cancer (CAC) with experimental CH driven by Dnmt3a+/Δ, we found higher tumor penetrance and increased tumor burden compared with controls. Histopathological analysis revealed accentuated colonic epithelium injury, dysplasia, and adenocarcinoma formation. Transcriptome profiling of colon tumors identified enrichment of gene signatures associated with carcinogenesis, including angiogenesis. Treatment with the angiogenesis inhibitor axitinib eliminated the colon tumor-promoting effect of experimental CH driven by Dnmt3a haploinsufficiency and rebalanced hematopoiesis. This study provides conceptually novel insights into non-tumor-cell-autonomous effects of hematopoietic alterations on colon carcinogenesis and identifies potential therapeutic strategies.

CXCL8/CXCR2 signaling mediates bone marrow fibrosis and is a therapeutic target in myelofibrosis.

Proinflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution are yet to be fully elucidated. Previously, we identified a critical role for combined constitutive JAK/STAT and aberrant NF-κB proinflammatory signaling in MF development. Using single-cell transcriptional and cytokine-secretion studies of primary cells from patients with MF and the human MPLW515L (hMPLW515L) murine model of MF, we extend our previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. Hematopoietic stem/progenitor cells from patients with MF are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to an exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L-adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway among patients with MF.

Molecular predictors of immunophenotypic measurable residual disease clearance in acute myeloid leukemia.

Measurable residual disease (MRD) is a powerful prognostic factor in acute myeloid leukemia (AML). However, pre-treatment molecular predictors of immunophenotypic MRD clearance remain unclear. We analyzed a dataset of 211 patients with pre-treatment next-generation sequencing who received induction chemotherapy and had MRD assessed by serial immunophenotypic monitoring after induction, subsequent therapy, and allogeneic stem cell transplant (allo-SCT). Induction chemotherapy led to MRD- remission, MRD+ remission, and persistent disease in 35%, 27%, and 38% of patients, respectively. With subsequent therapy, 34% of patients with MRD+ and 26% of patients with persistent disease converted to MRD-. Mutations in CEBPA, NRAS, KRAS, and NPM1 predicted high rates of MRD- remission, while mutations in TP53, SF3B1, ASXL1, and RUNX1 and karyotypic abnormalities including inv (3), monosomy 5 or 7 predicted low rates of MRD- remission. Patients with fewer individual clones were more likely to achieve MRD- remission. Among 132 patients who underwent allo-SCT, outcomes were favorable whether patients achieved early MRD- after induction or later MRD- after subsequent therapy prior to allo-SCT. As MRD conversion with chemotherapy prior to allo-SCT is rarely achieved in patients with specific baseline mutational patterns and high clone numbers, upfront inclusion of these patients into clinical trials should be considered.

Synthesis of Plasmonically Active Titanium Nitride Using a Metallic Alloy Buffer Layer Strategy.

Titanium nitride (TiN) has emerged as a highly promising alternative to traditional plasmonic materials. This study focuses on the inclusion of a Cr90Ru10 buffer layer between the substrate and thin TiN film, which enables the use of cost-effective, amorphous technical substrates while preserving high film quality. We report best-in-class TiN thin films fabricated on fused silica wafers, achieving a maximum plasmonic figure of merit, -ϵ'/ϵ″, of approximately 2.8, even at a modest wafer temperature of around 300 °C. Furthermore, we delve into the characterization of TiN thin film quality and fabricated TiN triangular nanostructures, employing attenuated total reflectance and cathodoluminescence techniques to highlight their potential applications in surface plasmonics.

Digital Interventions to Enhance Readiness for Psychological Therapy: Scoping Review.

BACKGROUND: Psychological therapy is an effective treatment method for mental illness; however, many people with mental illness do not seek treatment or drop out of treatment early. Increasing client uptake and engagement in therapy is key to addressing the escalating global problem of mental illness. Attitudinal barriers, such as a lack of motivation, are a leading cause of low engagement in therapy. Digital interventions to increase motivation and readiness for change hold promise as accessible and scalable solutions; however, little is known about the range of interventions being used and their feasibility as a means to increase engagement with therapy. OBJECTIVE: This review aimed to define the emerging field of digital interventions to enhance readiness for psychological therapy and detect gaps in the literature. METHODS: A literature search was conducted in PubMed, PsycINFO, PsycARTICLES, Scopus, Embase, ACM Guide to Computing Literature, and IEEE Xplore Digital Library from January 1, 2006, to November 30, 2021. The PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews) methodology was applied. Publications were included when they concerned a digitally delivered intervention, a specific target of which was enhancing engagement with further psychological treatment, and when this intervention occurred before the target psychological treatment. RESULTS: A total of 45 publications met the inclusion criteria. The conditions included depression, unspecified general mental health, comorbid anxiety and depression, smoking, eating disorders, suicide, social anxiety, substance use, gambling, and psychosis. Almost half of the interventions (22/48, 46%) were web-based programs; the other formats included screening tools, videos, apps, and websites. The components of the interventions included psychoeducation, symptom assessment and feedback, information on treatment options and referrals, client testimonials, expectation management, and pro-con lists. Regarding feasibility, of the 16 controlled studies, 7 (44%) measuring actual behavior or action showed evidence of intervention effectiveness compared with controls, 7 (44%) found no differences, and 2 (12%) indicated worse behavioral outcomes. In general, the outcomes were mixed and inconclusive owing to variations in trial designs, control types, and outcome measures. CONCLUSIONS: Digital interventions to enhance readiness for psychological therapy are broad and varied. Although these easily accessible digital approaches show potential as a means of preparing people for therapy, they are not without risks. The complex nature of stigma, motivation, and individual emotional responses toward engaging in treatment for mental health difficulties suggests that a careful approach is needed when developing and evaluating digital readiness interventions. Further qualitative, naturalistic, and longitudinal research is needed to deepen our knowledge in this area.

Geographic distribution of family physicians in Japan and the USA: a cross-sectional comparative study.

INTRODUCTION: Solutions for geographic maldistribution of physicians is challenging around the world, but primary care specialists are expected to resolve this issue. This study compares the geographic distribution of family physicians in Japan and the USA, both of which are developed countries without a major system for physician allocation by the public sector; however, the two countries differ greatly in the maturity of family medicine (ie length of its history as part of the healthcare system and the population of qualified family medicine experts). METHODS: This cross-sectional comparative study used publicly available online databases for Japan in 2018 and 2017 in the USA. The municipalities in Japan and counties in the USA were divided into quintile groups according to population density. The number of family physicians per unit population in each group of areas was calculated, and was evaluated with a residual analysis. The geographic distribution of all physicians in Japan was simulated assuming that the proportion of family physicians among all physicians in Japan (0.16%) was increased to match that in the USA (11.8%). RESULTS: Of 320 084 physicians in Japan and 899 244 in the USA, 519 (77.2%) family physicians in Japan and 105 999 (100%) in the USA were included. The distribution of family physicians in Japan was noticeably shifted to areas with the lowest population density. In contrast, family physicians in the USA were distributed equally across areas. The distribution of physicians of other specialties (general internists, pediatricians, surgeons and obstetricians/gynecologists) was shifted heavily to areas with the highest population densities in both countries. The simulation analysis showed the geographic maldistribution of the total number of physicians improved substantially if the proportion of family physicians in Japan is increased to match that in the USA. CONCLUSION: The distribution of family physicians is more equitable than that of other medical specialists; however, an immature family medicine system can lead to an aggregation of family physicians in rural areas. This aggregation supports equity due to the broader scope of practice required by family physicians in rural areas. In countries where family medicine has not yet matured as a specialty, provided that the equitable aggregation of family physicians in rural areas can be maintained, increasing the number of family physicians as a proportion of the total number of physicians may improve the geographic maldistribution of the total number of physicians.

Temporal and Spatial Assessment of the Corneal Response to UV Cross-Linking Using 3-Dimensional In Vivo Confocal Microscopy.

ABSTRACT: The goal of this study was to evaluate the temporal and spatial pattern of wound healing following UV corneal cross-linking (CXL) using 3-dimensional (3-D) confocal imaging in vivo. Using a modified Heidelberg Retinal Tomograph with Rostock Corneal Module confocal microscope, we performed 3-D scans on two patients at multiple time points after CXL. Patient 1 showed a normal post-CXL wound healing response, with initial subbasal nerve loss and keratocyte apoptosis in the anterior stroma, followed by partial restoration of both the nerve plexus and stromal keratocytes by 6 months. In patient 2, in addition to anterior corneal damage, pyknotic nuclei were observed in the posterior stroma 7 days after CXL. Acellular areas were present in the posterior stroma at 3 months, with only partial keratocyte repopulation at 6 months. Regeneration of the subbasal nerve plexus was also delayed. Three-dimensional confocal imaging allowed these unusual wound healing responses to be identified in the absence of any corresponding clinical observations.

Transition Metal Synthetic Ferrimagnets: Tunable Media for All-Optical Switching Driven by Nanoscale Spin Current.

All-optical switching of magnetization has great potential for use in future ultrafast and energy efficient nanoscale magnetic storage devices. So far, research has been almost exclusively focused on rare-earth based materials, which limits device tunability and scalability. Here, we show that a perpendicularly magnetized synthetic ferrimagnet composed of two distinct transition metal ferromagnetic layers, Ni3Pt and Co, can exhibit helicity independent magnetization switching. Switching occurs between two equivalent remanent states with antiparallel alignment of the Ni3Pt and Co magnetic moments and is observable over a broad temperature range. Time-resolved measurements indicate that the switching is driven by a spin-polarized current passing through the subnanometer Ir interlayer. The magnetic properties of this model system may be tuned continuously via subnanoscale changes in the constituent layer thicknesses as well as growth conditions, allowing the underlying mechanisms to be elucidated and paving the way to a new class of data storage devices.

An integrated pipeline for comprehensive analysis of immune cells in human brain tumor clinical samples.

Human tissue samples represent an invaluable source of information for the analysis of disease-specific cellular alterations and their variation between different pathologies. In cancer research, advancing a comprehensive understanding of the unique characteristics of individual tumor types and their microenvironment is of considerable importance for clinical translation. However, investigating human brain tumor tissue is challenging due to the often-limited availability of surgical specimens. Here we describe a multimodule integrated pipeline for the processing of freshly resected human brain tumor tissue and matched blood that enables analysis of the tumor microenvironment, with a particular focus on the tumor immune microenvironment (TIME). The protocol maximizes the information yield from limited tissue and includes both the preservation of bulk tissue, which can be performed within 1 h following surgical resection, as well as tissue dissociation for an in-depth characterization of individual TIME cell populations, which typically takes several hours depending on tissue quantity and further downstream processing. We also describe integrated modules for immunofluorescent staining of sectioned tissue, bulk tissue genomic analysis and fluorescence- or magnetic-activated cell sorting of digested tissue for subsequent culture or transcriptomic analysis by RNA sequencing. Applying this pipeline, we have previously described the overall TIME landscape across different human brain malignancies, and were able to delineate disease-specific alterations of tissue-resident versus recruited macrophage populations. This protocol will enable researchers to use this pipeline to address further research questions regarding the tumor microenvironment.

UVB mutagenesis differs in Nras- and Braf-mutant mouse models of melanoma.

BRAF-mutant melanomas are more likely than NRAS-mutant melanomas to arise in anatomical locations protected from chronic sun damage. We hypothesized that this discrepancy in tumor location is a consequence of the differential sensitivity of BRAF and NRAS-mutant melanocytes to ultraviolet light (UV)-mediated carcinogenesis. We tested this hypothesis by comparing the mutagenic consequences of a single neonatal, ultraviolet-AI (UVA; 340-400 nm) or ultraviolet-B (UVB; 280-390 nm) exposure in mouse models heterozygous for mutant Braf or homozygous for mutant Nras Tumor onset was accelerated by UVB, but not UVA, and the resulting melanomas contained recurrent mutations affecting the RING domain of MAP3K1 and Actin-binding domain of Filamin A. Melanomas from UVB-irradiated, Braf-mutant mice averaged twice as many single-nucleotide variants and five times as many dipyrimidine variants than tumors from similarly irradiated Nras-mutant mice. A mutational signature discovered in UVB-accelerated tumors mirrored COSMIC signatures associated with human skin cancer and was more prominent in Braf- than Nras-mutant murine melanomas. These data show that a single UVB exposure yields a greater burden of mutations in murine tumors driven by oncogenic Braf.

A high throughput dynamic method for characterizing thermodynamic properties of catalyzed magnesium hydrides by thermogravimetric analysis.

The use of the conventional pressure-composition-temperature (PCT) method to determine the thermodynamics of metal hydrides is a time-consuming process. This work presents an efficient method based on thermogravimetric analysis (TGA), to characterize the thermodynamic parameters. Through cycling catalyzed magnesium hydride in a TGA apparatus under a flowing gas with a constant hydrogen partial pressure, TGA curves can be used to determine absorption/desorption equilibrium temperatures. Based on the van't Hoff analysis, the reaction enthalpies and entropies can be derived from the equilibrium temperatures as a function of hydrogen pressure. Using the results of this work we calculated the hydrogenation and dehydrogenation enthalpies, which are 79.8 kJ per mol per H2 and 76.5 kJ per mol per H2, respectively. These values are in good agreement with those reported values using the PCT method. These results demonstrate that the TGA can be an efficient and effective method for measuring thermodynamic parameters of metal hydrides.

Synthetic biological circuit tested in spaceflight.

Synthetic biology has potential spaceflight applications yet few if any studies have attempted to translate Earth-based synthetic biology tools into spaceflight. An exogenously inducible biological circuit for protein production in Arabidopsis thaliana, pX7-AtPDSi (Guo et al. 2003), was flown to ISS and functionally investigated. Seedlings were grown in a custom built 1.25 U plant greenhouse. Images recorded during the experiment show that leaves of pX7-AtPDSi seedlings photobleached as designed while wild type Col-0 leaves did not, which reveals that the synthetic circuit led to protein production during spaceflight. Polymerase chain reaction analysis post-flight also confirms that the Cre/LoxP (recombination system) portions of the circuit were functional in spaceflight. The subcomponents of the biological circuit, estrogen-responsive transcription factor XVE, Cre/LoxP DNA recombination system, and RNAi post-transcriptional gene silencing system now have flight heritage and can be incorporated in future designs for space applications. To facilitate future plant studies in space, the full payload design and manufacturing files are made available.