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A promising approach for the treatment of nonalcoholic steatohepatitis (NASH) is the inhibition of enhanced hepatic de novo lipogenesis (DNL), which is the synthesis of fatty acids from nonlipid sources. This study assesses three approaches to DNL suppression in a newly developed dietary NASH mouse model: i) dietary intervention (switch from NASH‐inducing diet to normal diet); ii) inhibition of acetyl‐coenzyme A carboxylase (ACC), the enzyme catalyzing the rate‐limiting step in DNL; and iii) activation of farnesoid X receptor (FXR), a major transcriptional regulator of DNL. C57BL/6J mice on a high‐fat diet combined with ad libitum consumption of a fructose–sucrose solution developed several of the liver histologic features seen in human disease, including steatosis, inflammation, and fibrosis, accompanied by elevated fibrosis biomarkers and liver injury enzymes. Obesity and metabolic impairments were associated with increased intestinal permeability and progression to adenoma and hepatocellular carcinoma. All three approaches led to resolution of established NASH with fibrosis in mice; however, some differences were noted, e.g., with respect to the degree of hepatic steatosis attenuation. While ACC inhibition resulted in elevated blood triglycerides and peripheral obesity, FXR activation prevented peripheral obesity in NASH mice. Comparative transcriptome analysis underlined the translatability of the mouse model to human NASH and revealed novel mechanistic insights into differential regulation of lipid, inflammatory, and extracellular matrix pathways by FXR agonism and ACC inhibition. Conclusion: Novel insights are provided on back translation of clinically observed endpoints of DNL inhibition by targeting ACC or FXR, which are promising therapeutic options for the treatment of NASH, in a newly developed diet‐induced NASH mouse model.

Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.

Dipeptidyl peptidase 9 (DPP9) is a ubiquitously expressed intracellular prolyl peptidase implicated in immunoregulation. However, its physiological relevance in the immune system remains largely unknown. We investigated the role of DPP9 enzyme in immune system by characterizing DPP9 knock-in mice expressing a catalytically inactive S729A mutant of DPP9 enzyme (DPP9ki/ki mice). DPP9ki/ki mice show reduced number of lymphoid and myeloid cells in fetal liver and postnatal blood but their hematopoietic cells are fully functional and able to reconstitute lymphoid and myeloid lineages even in competitive mixed chimeras. These studies demonstrate that inactivation of DPP9 enzymatic activity does not lead to any perturbations in mouse hematopoiesis.

Dipeptidyl peptidase 9 (DPP9) is an intracellular N-terminal post-proline-cleaving enzyme whose physiological function remains largely unknown. We investigated the role of DPP9 enzyme in vivo by characterizing knock-in mice expressing a catalytically inactive mutant form of DPP9 (S729A; DPP9 ki/ki mice). We show that DPP9 ki/ki mice die within 12–18 h after birth. The neonatal lethality can be rescued by manual feeding, indicating that a suckling defect is the primary cause of neonatal lethality. The suckling defect results from microglossia, and is characterized by abnormal formation of intrinsic muscles at the distal tongue. In DPP9 ki/ki mice, the number of occipital somite-derived migratory muscle progenitors, forming distal tongue intrinsic muscles, is reduced due to increased apoptosis. In contrast, intrinsic muscles of the proximal tongue and extrinsic tongue muscles, which derive from head mesoderm, develop normally in DPP9 ki/ki mice. Thus, lack of DPP9 activity in mice leads to impaired tongue development, suckling defect and subsequent neonatal lethality due to impaired survival of a specific subset of migratory tongue muscle progenitors.

Farnesoid X receptor (FXR) agonism is emerging as an important potential therapeutic mechanism of action for multiple chronic liver diseases. The bile acid‐derived FXR agonist obeticholic acid (OCA) has shown promise in a phase 2 study in patients with nonalcoholic steatohepatitis (NASH). Here, we report efficacy of the novel nonbile acid FXR agonist tropifexor (LJN452) in two distinct preclinical models of NASH. The efficacy of tropifexor at

Solar energetic particles (SEPs) constitute an important component of the radiation environment in interplanetary space. Accurate modeling of SEP events is crucial for the mitigation of radiation hazards in spacecraft design. In this study we present two new statistical models of high energy solar proton fluences based on ground level enhancement (GLE) observations during solar cycles 19–24. As the basis of our modeling, we utilize a four parameter double power law function (known as the Band function) fits to integral GLE fluence spectra in rigidity. In the first model, the integral and differential fluences for protons with energies between 10 MeV and 1 GeV are calculated using the fits, and the distributions of the fluences at certain energies are modeled with an exponentially cut-off power law function. In the second model, we use a more advanced methodology: by investigating the distributions and relationships of the spectral fit parameters we find that they can be modeled as two independent and two dependent variables. Therefore, instead of modeling the fluences separately at different energies, we can model the shape of the fluence spectrum. We present examples of modeling results and show that the two methodologies agree well except for a short mission duration (1 year) at low confidence level. We also show that there is a reasonable agreement between our models and three well-known solar proton models (JPL, ESP and SEPEM), despite the differences in both the modeling methodologies and the data used to construct the models.

Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.

Aims. The aim of the study is to investigate the reason for the low productivity of high-energy SEPs in the present solar cycle.Methods. We employ scaling laws derived from diffusive shock acceleration theory and simulation studies including proton-generated upstream Alfven waves to find out how the changes observed in the long-term average properties of the erupting and ambient coronal and/or solar wind plasma would affect the ability of shocks to accelerate particles to the highest energies.Results. Provided that self-generated turbulence dominates particle transport around coronal shocks, it is found that the most crucial factors controlling the diffusive shock acceleration process are the number density of seed particles and the plasma density of the ambient medium. Assuming that suprathermal populations provide a fraction of the particles injected to shock acceleration in the corona, we show that the lack of most energetic particle events as well as the lack of low charge-to-mass ratio ion species in the present cycle can be understood as a result of the reduction of average coronal plasma and suprathermal densities in the present cycle over the previous one.

Analysis is presented for times during a space mission that specified solar proton flux levels are exceeded. This includes both total time and continuous time periods during missions. Results for the solar maximum and solar minimum phases of the solar cycle are presented and compared for a broad range of proton energies and shielding levels. This type of approach is more amenable to reliability analysis for spacecraft systems and instrumentation than standard statistical models.

Modern instrument-simulation techniques offer the possibility of increasing the scientific yield from archival space datasets. In this paper, we report on a simulation of the electron response of the University of Chicago’s Cosmic Ray Nuclei Experiment (CRNE) instrument on the IMP-8 satellite. IMP-8/CRNE returned data from 1973 to 2006. The CRNE particle telescope was designed to measure the isotopic composition of Galactic cosmic-ray (GCR) nuclei and has also been used in many studies of protons and ions above 10 MeV/nucleon from solar energetic particle (SEP) events. But CRNE also functions as a highly-capable detector for solar electrons above 0.5 MeV, an energy range that has not been extensively studied. Utilization of the CRNE electron data has heretofore been limited by the fact that CRNE was never calibrated for electrons. We have therefore used the GEANT4 Monte Carlo simulation package to model the CRNE response to electrons and (separately) protons for multiple energies and incident angles. The results were used to compute the energy- and angle-dependence of the effective area and the energy-dependence of the geometric factor. The response to protons, which was already well understood, was used to verify the mass model, the simulation settings, and the post-processing software. Our simulation of the IMP-8/CRNE electron response now allows analysis of hundreds of relativistic solar electron events observed by CRNE over the years, including studies of evolution of electron energy spectra with high time resolution. We show examples of these results and briefly discuss potential applications to future scientific investigations.

We undertook a quantitative trait locus (QTL) analysis in mice to identify modifier genes that might influence the severity of human iron disorders. We identified a strong QTL on mouse chromosome 9 that differentially affected macrophage iron burden in C57BL/10J and SWR/J mice. A C57BL/10J missense allele of an evolutionarily conserved gene, Mon1a, cosegregated with the QTL in congenic mouse lines. We present evidence that Mon1a is involved in trafficking of ferroportin, the major mammalian iron exporter, to the surface of iron-recycling macrophages. Differences in amounts of surface ferroportin correlate with differences in cellular iron content. Mon1a is also important for trafficking of cell-surface and secreted molecules unrelated to iron metabolism, suggesting that it has a fundamental role in the mammalian secretory apparatus.

The high-energy solar proton data obtained from instruments onboard the IMP-8 spacecraft are used to investigate statistical distributions of event fluences, event durations, and time intervals between adjacent events. The results show that: (1) the event fluences can be approximately fit to a log-normal distribution and (2) the distributions of event durations and time intervals between the events can be represented by Poisson distributions. A virtual data set was generated using these distributions to extend the Solar Probe method [Feynman, J., Tylka, A.J., Reames, D.B., Gabriel, S.B. Near-sun energetic particle environment of Solar Probe-Phase1 final report, JPL Report, 2000] of obtaining the mission-integrated fluences to longer missions. By using the virtual data set generated in this study, we are able to obtain the smooth distribution of mission fluences for missions of any duration, which is not possible when only the available data sets was used.

Alport syndrome is a genetic disease of collagen IV (α3, 4, 5) resulting in renal failure. This study was designed to investigate sex-phenotype correlations and evaluate the contribution of macrophage infiltration to disease progression using Col4a3 knock out (Col4a3KO) mice, an established genetic model of autosomal recessive Alport syndrome. No sex differences in the evolution of body mass loss, renal pathology, biomarkers of tubular damage KIM-1 and NGAL, or deterioration of kidney function were observed during the life span of Col4a3KO mice. These findings confirm that, similar to human autosomal recessive Alport syndrome, female and male Col4a3KO mice develop renal failure at the same age and with similar severity. The specific contribution of macrophage infiltration to Alport disease, one of the prominent features of the disease in human and Col4a3KO mice, remains unknown. This study shows that depletion of kidney macrophages in Col4a3KO male mice by administration of clodronate liposomes, prior to clinical onset of disease and throughout the study period, does not protect the mice from renal failure and interstitial fibrosis, nor delay disease progression. These results suggest that therapy targeting macrophage recruitment to kidney is unlikely to be effective as treatment of Alport syndrome.

The pathogenesis of Bacillus anthracis, the bacterium that causes anthrax, depends on secretion of three factors that combine to form two bipartite toxins. Edema toxin, consisting of protective antigen (PA) and edema factor (EF), causes the edema associated with cutaneous anthrax infections, whereas lethal toxin (LeTx), consisting of PA and lethal factor (LF), is believed to be responsible for causing death in systemic anthrax infections. EF and LF can be transported by PA into the cytosol of many cell types. In mouse macrophages, LF can cause rapid necrosis that may be related to the pathology of systemic infections. Inbred mouse strains display variable sensitivity to LeTx-induced macrophage necrosis. This trait difference has been mapped to a locus on chromosome 11 named Ltxs1 (refs. 7,8). Here we show that an extremely polymorphic gene in this locus, Nalp1b, is the primary mediator of mouse macrophage susceptibility to LeTx. We also show that LeTx-induced macrophage death requires caspase-1, which is activated in susceptible, but not resistant, macrophages after intoxication, suggesting that Nalp1b directly or indirectly activates caspase-1 in response to LeTx.

Chlamydia trachomatis is a bacterial pathogen that is a major cause of blindness and infertility in diverse populations across the world. In an effort to model genetic complexities that are observed in human populations and to identify novel genes involved in susceptibility to C. trachomatis, we have adapted a murine model of systemic infection for use in genetic analysis. In this model, chlamydial colonization and replication is measured in the spleens of mice shortly after intravenous delivery of C. trachomatis L2. Here, we show that C57BL/6J and C3H/HeJ inbred mice are differentially susceptible to this systemic infection. Additionally, fibroblasts cultured from C57BL/6J and C3H/HeJ embryos are differentially permissive for chlamydial replication. We have taken advantage of this natural variation to map quantitative trait loci on Chromosomes 2, 3, and 11 that segregate with the bacterial load in F2 cross progeny during the acute phase of C. trachomatis infection in vivo. To validate our mapping results, we also generated mice that are congenic for a portion of Chromosome 11 from the susceptible parent. This congenic interval confers increased susceptibility to C. trachomatis, both in vivo and in vitro, suggesting that our screen identified at least one gene that is involved in cellular resistance to C. trachomatis replication.

Above a few tens of MeV per nucleon, large, gradual solar energetic particle (SEP) events are highly variable in their spectral characteristics and elemental composition. The origin of this variability has been a matter of intense and ongoing debate. In this paper, we propose that this variability arises from the interplay of two factors—shock geometry and a compound seed population, typically comprising both solar-wind and flare suprathermals. Whereas quasi-parallel shocks generally draw their seeds from solar-wind suprathermals, quasi-perpendicular shocks—by requiring a higher initial speed for effective injection—preferentially accelerate seed particles from flares. Solar-wind and flare seed particles have distinctive compositional characteristics, which are then reflected in the accelerated particles. We first examine our hypothesis in the context of particles locally accelerated near 1 AU by traveling interplanetary shocks. We illustrate the implications of our hypothesis for SEPs with two very large events, 2002 April 21 and 2002 August 24. These two events arise from very similar solar progenitors but nevertheless epitomize extremes in high-energy SEP variability. We then test our hypothesis with correlation studies based on observations of 43 large SEP events in 1997-2003 by the Advanced Composition Explorer, Wind, the Interplanetary Monitoring Platform 8, and GOES. We consider correlations among high-energy Fe/O, event size, spectral characteristics, the presence of GeV protons, and event duration at high energies. The observed correlations are all qualitatively consistent with our hypothesis. Although these correlation studies cannot be construed as proof of our hypothesis, they certainly confirm its viability. We also examine the alternative hypothesis in which a direct flare component—rather than flare particles subsequently processed through a shock—dominates at high energies. This alternative would produce compositional characteristics similar to those of our hypothesis. However, the observed longitude distribution of the enhanced Fe/O events, their spectral characteristics, and recent timing studies all pose serious challenges for a direct flare component. We also comment on measurements of the mean ionic charge state of Fe at high energies. We conclude that shock geometry and seed population potentially provide a framework for understanding the overall high-energy variability in large SEP events. We suggest additional studies for testing this hypothesis.

The B10.Q/J strain of mice was serendipitously discovered to be highly susceptible to infection by the intracellular protozoan parasite,Toxoplasma gondiibut markedly resistant to induction of autoimmune arthritis. We have previously shown that the B10.Q/J phenotype is controlled by a single recessive locus and is associated with lymphocyte hyporesponsiveness to IL-12. Using genetic approaches, we have now localized the B10.Q/J locus to chromosome 9 and established its identity asTyk2, a Janus kinase essential for IL-12 and IFN-α/β cytokine signaling. The B10.Q/JTyk2gene contained a single missense mutation resulting in a nonconservative amino acid substitution (E775K) in an invariant motif of the pseudokinase (Janus kinase homology 2) domain. This mutation appeared to result in the absence of the B10.Q/J-encoded Tyk2 protein, despite presence ofTyk2-specific transcripts. Phenotypically, B10.Q/J cells were indistinguishable fromTyk2-deficient cells, showing impaired signaling and biologic responses to IL-12, IL-23, and type I IFNs. The analogous E782K mutant of human Tyk2 failed to restore IFN-α responsiveness inTyk2null 11,1 cells. Our results indicate a crucial role for Tyk2 in T helper 1-mediated protective and pathogenic immune responses. An additional implication of our findings is that naturally occurring mutations in theTyk2gene may underlie altered susceptibilities to infectious or autoimmune diseases in human and animal populations.

We report heavy-ion composition and spectra for the solar energetic particle (SEP) events of 2001 April 14 and 15, using the combined capabilities of the Advanced Composition Explorer (ACE), Wind, and the Interplanetary Monitoring Platform 8 (IMP-8) to cover the energy range from ∼30 keV nucleon^(-1) to ∼400 MeV nucleon^(-1). These two events are, respectively, the largest impulsive event and the largest ground-level event observed so far in solar cycle 23. These events arose from the same active region and launched into similar interplanetary conditions. Both were associated with large western flares and fast coronal mass ejections (CMEs). However, the two events are distinctly different, thereby providing useful reminders of the fundamental differences between flare- and shock-accelerated SEPs. The detailed observations present challenges for our theoretical understanding of SEP production. Of particular note is the fact that iron has a harder power-law energy spectrum than oxygen above ∼3 MeV nucleon^(-1) in the shock-dominated April 15 event. This spectral difference, which is seen in many other gradual events of various sizes and heliolongitudes, leads to enhanced Fe/O at high energies. Simple shock acceleration models predict the same power-law index for all species. Thus, understanding the origin of this spectral difference will significantly contribute to the resolution of the ongoing debate about the relative roles of CME-driven shocks and flares in producing high-energy solar heavy ions.

Spt3 of Saccharomyces cerevisiae is required for the normal transcription of many genes in vivo. Past studies have shown that Spt3 is required for both mating and sporulation, two events that initiate when cells are at G1/START. We now show that Spt3 is needed for two other events that begin at G1/START, diploid filamentous growth and haploid invasive growth. In addition, Spt3 is required for normal expression of FLO11, a gene required for filamentous growth, although this defect is not the sole cause of the spt3Δ/spt3Δ filamentous growth defect. To extend our studies of Spt3's role in filamentous growth to the pathogenic yeast Candida albicans, we have identified the C. albicans SPT3 gene and have studied its role in C. albicans filamentous growth and virulence. Surprisingly, C. albicans spt3Δ/spt3Δ mutants are hyperfilamentous, the opposite phenotype observed for S. cerevisiae spt3Δ/spt3Δ mutants. Furthermore, C. albicans spt3Δ/spt3Δ mutants are avirulent in mice. These experiments demonstrate that Spt3 plays important but opposite roles in filamentous growth in S. cerevisiae and C. albicans.

Infectious diseases are likely to be a major source of selective pressure on the evolution of the immune system.1 Unfortunately, the functional plasticity and redundancy of the immune system complicates the experimental study of each of its components. Because pathogens exhibit abilities to combat and avoid host immunity, they are excellent tools to dissect the function of each of the components of the immune system.2 In a paper published in this issue, Kamimura et al3 provide an example of how pathogens can be used to reveal specific immune response defects. The authors studied specific alterations introduced into the gp130 gene, which encodes a common subunit of IL-6 family cytokine receptors. Previously, the authors had identified molecular defects in the signals emanating from these mutant receptors. In the current study, the authors evaluated the role of mutant gp130 in resistance to infection with Listeria monocytogenes. The authors chose well in using Listeria as a probe into the functioning of the immune system, since resistance to this pathogen relies on contributions from all the major components of innate and acquired immunity.5 In this article, we will to discuss the general significance of this strategy in the context of understanding the complexities of host immunity. In brief, there are a few mutually complementing genetic approaches that are used to study the host immune response. These approaches can be roughly split into two classes. Many investigators analyze alterations in the host response that are induced by directed genetic modification of an organism. Typical genetic modifications include mouse gene knock-outs, knock-ins, and introduction of transgenes. Because this approach is hypothesis driven, it is widely used, and the results reported by Kamimura et al3 fall in this category. In contrast, genetic mapping allows the identification of genes whose role in host immunity may not yet be suspected. Genetic mapping relies on an unbiased analysis of the immune response of progeny obtained from crosses. While this approach presents several experimental difficulties, it can, unlike the first one, simultaneously reveal both

Genetics can be a powerful approach with which to dissect the biological relationships between pathogens and their hosts. Genetic studies have established that polymorphisms in neuronal apoptosis inhibitory protein 5 (Naip5) appear to explain the entire difference in permissiveness of B6 and A/J macrophages to Legionella replication. The authors examined whether permissiveness of mouse macrophages to Legionella growth was affected by inhibitors of the mitogen activated protein (MAP) kinases, another important class of signaling molecules in the immune system. Importantly, therefore, it was found that macrophages from B6-backcrossed caspase-1 knockout mice were also more permissive for Legionella growth than were wild-type B6 macrophages. Currently, Legionella flagellin is detected by B6 macrophages in a manner dependent on Naip (and possibly Ipaf ), leading to caspase-1 activation, rapid cell death, and nonpermissiveness for Legionella growth. Whether Naip or Ipaf is a direct intracellular receptor for flagellin will likely be difficult to establish convincingly, as even the much more thoroughly characterized Toll and Nod proteins have not been unequivocally demonstrated to bind directly to their putative ligands. It has nevertheless been extremely satisfying to see how the concerted application of mouse and bacterial genetics has led to several insights into the nature of innate macrophage resistance to Legionella.

Background: Inbred mouse strains exhibit striking differences in the susceptibility of their macrophages to the effects of anthrax lethal toxin (LeTx). Previous data has shown that this difference in susceptibility lies downstream of toxin entry into macrophages. A locus controlling this phenotype, called Ltxs1 , has been mapped to chromosome 11, but the responsible gene has not been identified. Results: Here, we report the identification of the Ltxs1 gene as Kif1C , which encodes a kinesin-like motor protein of the UNC104 subfamily. Kif1C is the only gene in the Ltxs1 interval exhibiting polymorphisms between susceptible and resistant strains. Multiple alleles of Kif1C determine the susceptibility or resistance of cultured mouse macrophages to LeTx. Treatment of resistant macrophages with brefeldin-A (which alters the cellular localization of Kif1C) induces susceptibility to LeTx, while ectopic expression of a resistance allele of Kif1C in susceptible macrophages causes a 4-fold increase in the number of cells surviving LeTx treatment. We also show that cleavage of map kinase kinase 3, a target of LeTx proteolysis, occurs in resistant cells. Conclusions: We conclude that mutations in Kif1C are responsible for the differences in the susceptibility of inbred mouse macrophages to LeTx and that proper Kif1C function is required for LeTx resistance. Since the LeTx-mediated proteolysis of map kinase kinase 3 occurs even in resistant cells, Kif1C does not affect cellular entry or processing of LeTx and likely influences events occurring later in the intoxication pathway.

The study of mouse and human genetic variation in infectious disease susceptibility (for review, see Malo and Skamene 1994; Hill 1998) should help to improve our knowledge of disease mechanisms by facilitating the identification of critical host proteins that modulate the infection process. Focusing on differences in disease susceptibility in humans will contribute to making progress in this field, but it is also possible to use mouse genetics to identify genes whose human orthologs are likely to affect the outcome of infections in man. It is impossible to adequately review all of the work that has been done to study the genetics of infectious disease susceptibility and pathogenesis. Therefore, I will discuss studies of two different bacterial infections. Although this is not a comprehensive approach, the examples help to illustrate my view that progress in understanding host susceptibility to infection will be facilitated by genetic studies of mouse models of infectious disease.

Macrophages isolated from C57BL/6J and A/J mice exhibit differences in permissiveness for intracellular replication of L. pneumophila (Yamamoto et al. 1988). This phenotype difference segregates as a single-gene trait in crosses between C57BL/6J and A/J and maps to a locus on distal chromosome 13 (Yamamoto et al. 1991; Yoshida et al. 1991; Dietrich et al. 1995; Beckers et al. 1995). Detailed physical mapping of this locus, called Lgn1, reveals that it contains a series of 50 to 80 kb highly homologous direct repeats and that a cluster of Naip gene paralogs map inside these direct repeats (Scharf et al. 1996; Growney et al. 2000). The region of the human genome that is orthologous to the mouse Lgn1 region also contains a series of highly homologous repeated segments. The human spinal muscular atrophy (SMA) region has what appears to be an inverted duplication of some 500 kb (Lefebvre et al. 1995). This amplified genomic segment contains several transcriptionally active genes, including copies of survival motor neuron (SMN); NAIP; general transcription factor II H, polypeptide 2 (GTF2H2); and small EDRK-rich factor 1 (SERF1) (reviewed by Growney et al. 2000). However, the only gene in common between the amplified segments from the mouse and human Lgn1/SMA intervals is Naip/NAIP (Growney et al. 2000). The fact that the mouse and human Lgn1/SMA regions both have divergently organized sets of closely linked repeats indicates that these amplified segments originated independently in the mouse and human lineages. This observation begs the question of whether the amplification of Naip/NAIP in either mouse or human has any functional significance. Although most of the mouse Naip paralogs are transcriptionally active and encode similar but not identical proteins, it is not known whether these transcripts provide redundant or diverse functions (Huang et al. 1999). These questions about the functionality of the mouse Naip loci are important to the identification of the Lgn1 mutation because the current critical interval for the Lgn1 phenotype contains two different transcriptionally active Naip genes (Naip2 and Naip5) (Growney and Dietrich 2000; Huang et al. 1999). Mapping and sequence analysis of the mouse Lgn1 interval suggests that the Naip genes have arisen through a series of several distinct amplification events emanating from a single ancestral Naip. This model of the origins of the mouse Naip array relies heavily on the sequences (Fig. ​(Fig.1A)1A) of a single exon from the clustered Naip paralogs to build a phylogenetic tree (Growney et al. 2000). A more rigorous basis for determining the relationships of the mouse Naip genes would be to compare their entire genomic sequences. Figure 1 Map of the 129 mouse Naip array and annotation of the genomic sequences. (A) The map of the 129 mouse Naip array that was described previously in Growney et al. (2000) is indicated. The named arrows show the position and orientation of the Naip gene loci. ... In this paper, we report the complete annotated sequence of 26f17, a 220-kb bacterial artificial chromosome (BAC) clone that contains the three Naip genes on the centromere-distal side of the array in the 129 haplotype (Naip1, Naip3, and Naip6) (Fig ​(Fig1A;1A; Growney et al. 2000). In addition, we present three large annotated fragments of genomic sequence from 9045, a 75-kb P1 clone mapping to the central portion of the 129 Naip array (Fig ​(Fig1A;1A; Growney et al. 2000). Our analysis of these genomic sequences has provided additional markers to refine the map of the Lgn1 interval (Growney and Dietrich 2000) and allowed us to refine the previously reported model of the origins of the mouse Naip array.

The orthologous genomic segments on mouse chromosome 13D1-D3 and human chromosome 5q11.2-q13.3 have been extensively studied because of their involvement in two distinct disease phenotypes, spinal muscular atrophy (SMA) in human and susceptibility to Legionella pneumophila (determined by Lgn1) in mice. The overlapping intervals in both species contain genomic amplifications of distinct structure, indicating an independent origin. We have endeavored to construct a comprehensive comparative gene map of the mouse and human Lgn1/SMA intervals in the hopes that the origins and maintenance of the genomic amplifications may become clear. Our comparative gene map demonstrates that the only regional gene in common between the amplified segments in mouse and human is the Lgn1 candidate Naip/NAIP. We have also determined that mice of the 129 haplotype harbor seven intact and three partial Naip transcription units arranged in a closely linked direct repeat on chromosome 13. Several, but not all, of these Naip loci are contained within the Lgn1 critical interval. We present a model for the origins of the mouse and human repetitive arrays from a common ancestral haplotype.

Human chromosome 5q11.2–q13.3 and its ortholog on mouse chromosome 13 contain candidate genes for an inherited human neurodegenerative disorder called spinal muscular atrophy (SMA) and for an inherited mouse susceptibility to infection with Legionella pneumophila (Lgn1). These homologous genomic regions also have unusual repetitive organizations that create practical difficulties in mapping and raise interesting issues about the evolutionary origin of the repeats. In an attempt to analyze this region in detail, and as a way to identify additional candidate genes for these diseases, we have determined the sequence of 179 kb of the mouse Lgn1/SMA interval. We have analyzed this sequence using BLAST searches and various exon prediction programs to identify potential genes. Since these methods can generate false-positive exon declarations, our alignments of the mouse sequence with available human orthologous sequence allowed us to discriminate rapidly among this collection of potential coding regions by indicating which regions were well conserved and were more likely to represent actual coding sequence. As a result of our analysis, we accurately mapped two additional genes in the SMA interval that can be tested for involvement in the pathogenesis of SMA. While no new Lgn1 candidates emerged, we have identified new genetic markers that exclude Smn as an Lgn1 candidate. In addition to providing important resources for studying SMA and Lgn1, our data provide further evidence of the value of sequencing the mouse genome as a means to help with the annotation of the human genomic sequence and vice versa.

In more than 25 years of almost continuous observations, the University of Chicago’s Cosmic Ray Telescope (CRT) on IMP-8 has amassed a unique database on high-energy solar heavy ions of potential relevance to manned spaceflight. In the very largest particle events, IMP-8 /CRT has even observed solar Fe ions above the Galactic cosmic ray background up to ∼800 MeV/nucleon, an energy sufficiently high to penetrate nearly 25 g/cm 2 of shielding. IMP-8 /CRT observations show that high-energy heavy-ion spectra are often surprisingly hard power laws, without the exponential roll-offs suggested by stochastic acceleration fits to lower energy measurements alone. Also, in many solar particle events the Fe/O ratio grows with increasing energy, contrary to the notion that ions with higher mass-to-charge ratios should be less abundant at higher energies. Previous studies of radiation hazards for manned spaceflight have often assumed heavy-ion composition and steeply-falling energy spectra inconsistent with these observations. Conclusions based on such studies should therefore be re-assessed. The significant event-to-event variability observed in the high-energy solar heavy ions also has important implications for strategies in building probabilistic models of solar particle radiation hazards.

Th phenotype development is controlled not only by cytokines but also by other parameters including genetic background. One site of genetic variation between murine strains that has direct impact on Th development is the expression of the IL-12 receptor. T cells from B10.D2 and BALB/c mice show distinct control of IL-12 receptor expression. When activated by Ag, B10.D2 T cells express functional IL-12 receptors and maintain IL-12 responsiveness. In contrast, under the same conditions, BALB/c T cells fail to express IL-12 receptors and become unresponsive to IL-12, precluding any Th1-inducing effects if subsequently exposed to IL-12. Previously, we identified a locus, which we termed T cell phenotype modifier 1 (Tpm1), on murine chromosome 11 that controls this differential maintenance of IL-12 responsiveness. In this study, we have produced a higher resolution map around Tpm1. We produced and analyzed a series of recombinants from a first-generation backcross that significantly narrows the genetic boundaries of Tpm1. This allowed us to exclude from consideration certain previous candidates for Tpm1, including IFN-regulatory factor-1. Also, cellular analysis of F1(B10.D2 × BALB/c) T cells demonstrates that Tpm1 exerts its effect on IL-12 receptor expression in a cell-autonomous manner, rather than through influencing the extracellular milieu. This result strongly implies that despite the proximity of our locus to the IL-13/IL-4 gene cluster, these cytokines are not candidates for Tpm1.

Although disorders of iron metabolism are prevalent, iron transport remains poorly understood. To address this problem, we undertook a positional cloning strategy to identify the causative mutation in mice with microcytic anaemia (mk). Homozygous mk/mk mice have microcytic, hypochromic anaemia due to severe defects in intestinal iron absorption and erythroid iron utilization1–4. We report the identification of a strong candidate gene for mk, and suggest that the phenotype is a consequence of a missense mutation in Nramp2 (ref. 5), a previously identified gene of unknown function. Nramp2 is homologous to Nrampl, a gene active in host defense. If Nramp2 is mk, as the cumulative evidence suggests, our findings have broad implications for the understanding of iron transport and resistance to intracellular pathogens.

As genetic mapping of quantitative trait loci (QTL) becomes routine, the challenge is to identify the underlying genes. This paper develops rigorous genetic tests for evaluation of candidate genes for a QTL, involving determination of allelic status in inbred strains and fine-structure genetic mapping. For the Mom1 modifier of intestinal adenomas caused by ApcMin, these tests are used to evaluate two candidate genes: Pla2g2a, a secretory phospholipase, and Rap1GAP, a GTPase activating protein. Rap1GAP passes the first test but is excluded by a single fine-structure recombinant. Pla2g2a passes both tests and is a strong candidate for Mom1. Significantly, we also find that ApcMin-induced adenomas remain heterozygous for the Mom1 region, consistent with Mom1 acting outside the tumor lineage and encoding a secreted product.

We calculate single event upset (SEU) rates due to protons, alphas, and heavier ions in two satellite systems for the major solar particle events of 1989-92, using a new and complete analysis of GOES proton data and high-energy heavy-ion fluences from the University of Chicago Cosmic Ray Telescope on IMP-8. These measurements cover the entire range of energies relevant to SEU studies and therefore overcome shortcomings of previous studies, which relied upon theoretical or semi-empirical estimates of high-energy heavy-ion spectra. We compare our results to the observed SEU rates in these events. The SEU rates in one device (AMD 93L422s on LEASATs) were overwhelmingly dominated by protons. However, even after taking into account uncertainties in the ground-test cross-section data, we find that at least /spl sim/45% of the SEUs in the other device (Fairchild 93L422s on TDRS-1) must have been caused by heavy ions. Our results demonstrate that both protons and heavy ions must be considered in order to make a reliable assessment of SEU vulnerabilities. Furthermore, the GOES/Chicago database of solar particle events provides a basis for making accurate solar particle SEU calculations and credible worst-case estimates. In particular, measurements of the historic solar particle events of October 1989 are used in "worst week" and "worst day" environment models in CREME96, a revision of NRL's Cosmic Ray Effects on MicroElectronics code.

Spinal muscular atrophy (SMA) is a relatively common, autosomal recessively inherited neurodegenerative disorder that maps to human chromosome 5q13. This region of the human genome has an intricate genomic structure that has complicated the evaluation of SMA candidate genes. We have chosen to study the mouse region syntenic for human SMA in the hope that the homologous mouse interval would contain the same genes as human 5q13 on a simpler genomic background. Here, we report the mapping of such a region to mouse chromosome 13 and to the critical interval for Lgn1, a mouse locus responsible for modulating the intracellular replication and pathogenicity of the bacterium Legionella pneumophila. We have generated a mouse YAC contig across the Lgn1/Sma interval and have mapped the two flanking gene markers for the human SMA locus, MAP1B and CCNB1, onto this contig. In addition, we have localized the two SMA candidate genes, SMN and NAIP, to the Lgn1 critical region, making these two genes candidates for the Lgn1 phenotype. Upon subcloning of the YAC contig into P1s and BACs, we have detected a large, low copy number repeat that contains at least one copy of Naip exon 5. Identification of the Lgn1 gene will either provide a novel function for SMN or NAIP or reveal the existence of another, yet uncharacterized gene in the SMA critical region. Mutations in such a gene might help to explain some of the phenotypic variability among the human SMAs.

Genetic background of the T cell can influence T helper (Th) phenotype development, with some murine strains (e.g., B10.D2) favoring Th1 development and others (e.g., BALB/c) favoring Th2 development. Recently we found that B10.D2 exhibit an intrinsically greater capacity to maintain interleukin 12 (IL-12) responsiveness under neutral conditions in vitro compared with BALB/c T cells, allowing for prolonged capacity to undergo IL-12-induced Th1 development. To begin identification of the loci controlling this genetic effect, we used a T-cell antigen receptor-transgenic system for in vitro analysis of intercrosses between BALB/c and B10.D2 mice and have identified a locus on murine chromosome 11 that controls the maintenance of IL-12 responsiveness, and therefore the subsequent Th1/Th2 response. This chromosomal region is syntenic with a locus on human chromosome 5q31.1 shown to be associated with elevated serum IgE levels, suggesting that genetic control of Th1/Th2 differentiation in mouse, and of atopy development in humans, may be expressed through similar mechanisms.

The intracellular pathogen Legionella pneumophila is unable to replicate in macrophages derived from most inbred mouse strains. Here, we report the mapping of a gene, called Lgn1, that determines whether mouse macrophages are permissive for the intracellular replication of L. pneumophila. Although Lgn1 has been previously reported to map to mouse chromosome 15, we show here that it actually maps to chromosome 13, between D13Mit128 and D13Mit70. In the absence of any regional candidates for Lgn1, this map position will facilitate positional cloning attempts directed at this gene.

Nat. Chem. Biol. 11, 511–517 (2015); published online 1 June 2015; corrected online 15 July 2015 and 11 February 2016 In the version of this article originally published online, the schematic for the construct in Figure 4a was incorrect. A corrected figure has been provided in the HTML and PDF versions of the article.

A genome-wide scan for loss of heterozygosity (LOH) in tumors provides a powerful route to the identification of genes involved in tumorigenesis. This approach has not previously been applied to transgenic mice, despite the considerable advantages they afford for genetic dissection. Here, we report a genome-wide LOH analysis of insulinomas and carcinoid tumors in transgenic mice expressing the simian virus 40 large tumor oncogene. Although the overall genome-wide rate of LOH was quite low, chromosomes 9 and 16 showed high rates of allelic loss. About one-third of tumors showed partial LOH, allowing localization of the likely tumor suppressor genes to intervals of approximately 11 centimorgans. The locus on chromosome 9, named Loh-1, lies in a region with synteny conservation to human chromosomes 3q, 6q12, 15q24, and 3p21, while the locus on chromosome 16, named Loh-2, lies in a region corresponding to human chromosomes 3q and 22q. Of particular note is the synteny conservation with human 3p21, which shows frequent loss in human cancers. These regions do not encode two tumor suppressors, pRB and p53, known to interact with large tumor oncoprotein, suggesting the presence of new genes whose loss of function contributes to multistage tumorigenesis.

We have constructed a genetic map of the mouse genome containing 4,006 simple sequence length polymorphisms (SSLPs). The map provides an average spacing of 0.35 centiMorgans (cM) between markers, corresponding to about 750 kb. Approximately 90% of the genome lies within 1.1 cM of a marker and 99% lies within 2.2 cM. The markers have an average polymorphism rate of 50% in crosses between laboratory strains. The markers are distributed in a relatively uniform fashion across the genome, although some deviations from randomness can be detected. In particular, there is a significant underrepresentation of markers on the X chromosome. This map represents the two-thirds point toward our goal of developing a mouse genetic map containing 6,000 SSLPs.

A model is presented for times during a space mission that specified solar proton flux levels are exceeded. This includes both total time and continuous time periods during missions. Results for the solar maximum and solar minimum phases of the solar cycle are presented and compared for a broad range of proton energies and shielding levels. This type of approach is more amenable to reliability analysis for spacecraft systems and instrumentation than standard statistical models.

Angiogenesis is the process by which new blood vessels are formed from existing vessels. Mammalian populations, including humans and mice, harbor genetic variations that alter angiogenesis. Angiogenesis-regulating gene variants can result in increased susceptibility to multiple angiogenesis-dependent diseases in humans. Our efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have used laboratory animals due to the availability of genome sequence for many species and the ability to perform high volume controlled breeding. Using the murine corneal micropocket assay, we have observed more than ten-fold difference in angiogenic responsiveness among various mouse strains. This degree of difference is observed with either bFGF or VEGF induced corneal neovascularization. Ongoing mapping studies have identified multiple loci that affect angiogenic responsiveness in several mouse models. In this study, we used F2 intercrosses between C57BL/6J and the 129 substrains 129P1/ReJ and 129P3/J, as well as the SJL/J strain, where we have identified new QTLs that affect angiogenic responsiveness. In the case of AngFq5, on chromosome 7, congenic animals were used to confirm the existence of this locus and subcongenic animals, combined with a haplotype-based mapping approach that identified the pink-eyed dilution mutation as a candidate polymorphism to explain AngFq5. The ability of mutations in the pink-eyed dilution gene to affect angiogenic response was demonstrated using the p-J allele at the same locus. Using this allele, we demonstrate that pink-eyed dilution mutations in Oca2 can affect both bFGF and VEGF-induced corneal angiogenesis.

Several methods for analyzing the particle spectra from extremely large solar proton events, called Ground-Level Enhancements (GLEs), have been developed and utilized by the scientific community to describe the solar proton energy spectra and have been further applied to ascertain the radiation exposures to humans and radio-sensitive systems, namely electronics. In this paper 12 GLEs dating back to 1956 are discussed, and the three methods for describing the solar proton energy spectra are reviewed. The three spectral fitting methodologies are EXP [an exponential in proton rigidity (R)], WEIB [Weibull fit: an exponential in proton energy], and the Band function (BAND) [a double power law in proton rigidity]. The EXP and WEIB methods use low energy (MeV) GLE solar proton data and make extrapolations out to approx.1 GeV. On the other hand, the BAND method utilizes low- and medium-energy satellite solar proton data combined with high-energy solar proton data deduced from high-latitude neutron monitoring stations. Thus, the BAND method completely describes the entire proton energy spectrum based on actual solar proton observations out to ~10 GeV. Using the differential spectra produced from each of the 12 selected GLEs for each of the three methods, radiation exposures are presented and discussed in detail. These radiation exposures are then compared with the current 30-day and annual crew exposure limits and the radiation effects to electronics.

Mutations in the human APC gene cause various familial colon cancer syndromes. The Multiple intestinal neoplasia (Min) mouse provides an excellent model for familial colon cancer: it carries a mutant mouse Apc gene and develops many intestinal adenomas. Here, we analyze how this tumor phenotype is dramatically modified by genetic background. We report the genetic mapping of a locus that strongly modifies tumor number in Min /+ animals. This gene, Mom-1 (Modifier of Min-1), maps to distal chromosome 4 and controls about 50% of genetic variation in tumor number in two intraspecific backcrosses. The mapping is supported by a LOD score exceeding 14. Interestingly, Mom-1 lies in a region of synteny conservation with human chromosome 1p35–36, a region of frequent somatic loss of heterozygosity in a variety of human tumors, including colon tumors. These results provide evidence of a major modifier affecting expression of an inherited cancer syndrome.

Technological advances have made possible the development of high-resolution genetic linkage maps for the mouse. These maps in turn offer exciting prospects for understanding mammalian genome evolution through comparative mapping, for developing mouse models of human disease, and for identifying the function of all genes in the organism.

Solar proton events (SPEs) represent the single-most significant source of acute radiation exposure to humans and space systems during lunar and deep-space missions. In this paper several unique solar particle emissions that occurred during solar cycle 23 were investigated. In particular, there were four (4) events that exhibited both a ground level enhancement (GLE) and an energetic solar particle (ESP) feature. These four SPEs occurred on 1) 6 November 1997, 2) 14-18 July 2000, 3) 4-8 November 2001, and 4) 28-29 October 2003. Each event is discussed in detail. Proton integral and differential energy and corresponding rigidity (momentum) spectra were generated based on GOES MEPAD and GOES HEPAD satellite data and ground-based neutron monitor (NM) data. The differential proton spectra are utilized with the NASA Langley Research Center high energy particle transport/dose code, HZETRN 2005, to compute simulated human exposures behind several shielding materials. We have previously shown that a Band function (double power law in particle rigidity) fit more correctly describes the complete event spectra when compared with an exponential rigidity extrapolation that has been in use by the scientific community for several decades, i.e., the exponential extrapolation method consistently under-estimates the human exposure. Comparisons of simulated human radiation exposures are presented for both the Band function fit and the exponential extrapolation methods.

Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.

Solar proton events (SPEs) represent the single-most important source of acute space radiation exposure to humans and radio-sensitive spacecraft systems for lunar and Mars missions. In this paper, a review of the major sola r proton events for the past 5 solar cycles is presented with a focus on those SPEs that produced ground level enhancements (GLEs) as observed/measured by high latitude neutron monitor (NM) stations. Using NM GLE data one can infer the upper energy portion of the SPE e nergy spectrum, which extends up to several GeV (10 3 MeV). Historically, the scientific community has u sed solar proton data in the 10-100 MeV range from early IMP (Interplanetary Monitoring Platform) and, more recently, GOES (Geostationary Operational Environmental Satellite) spacecraft to make radiation exposure estimates. These SPE data were usually fitted using an exponential in rigidity (momentum) assumption (Malitson and Webber, 1963): φ(>P) = No * Exp(-P/Po) where φ is the integral fluence in protons/cm 2 , No is the normalization constant, P is the proton rigidity in million volts (MV), and Po is th e characteristic rigidity in MV. More recently, Xapsos, et al (2000) have determined that a better fit to the SPE data in the 10-100 MeV range is obtained by using a Weibull function. For GLEs, the proton energies extend to several GeV. Recently, Tylka and Dietrich (2008) ha ve utilized NM data and developed methods (Band [1993] function) to extend the solar particle data over the entire particle energy spectrum. SPE radiation assessments can be grossly underestimated if the entire proton energy spectrum is not taken into considerat ion. In this paper, we compare the various fitting methods to estimate the absorbed do se and dose equivalent for six (6) classic GLEs. Finally, detailed radiation exposure analyses are presented using several different radiation-mitigating shielding materials.