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Proper embryonic and postnatal skeletal development require coordination of myriad complex molecular mechanisms. Disruption of these processes, through genetic mutation, contributes to variation in skeletal development. We developed a high-throughput N-ethyl-N-nitrosourea (ENU)-induced saturation mutagenesis skeletal screening approach in mice to identify genes required for proper skeletal development. Here, we report initial results from live-animal X-ray and dual-energy X-ray absorptiometry (DXA) imaging of 27,607 G3 mice from 806 pedigrees, testing the effects of 32,198 coding/splicing mutations in 13,020 genes. A total of 39.7% of all autosomal genes were severely damaged or destroyed by mutations tested twice or more in the homozygous state. Results from our study demonstrate the feasibility of in vivo mutagenesis to identify mouse models of skeletal disease. Furthermore, our study demonstrates how ENU mutagenesis provides opportunities to create and characterize putative hypomorphic mutations in developmentally essential genes. Finally, we present a viable mouse model and case report of recessive skeletal disease caused by mutations in FAM20B. Results from this study, including engineered mouse models, are made publicly available via the online Mutagenetix database. © 2021 American Society for Bone and Mineral Research (ASBMR).

Background Atopy, the overall tendency to become sensitized to an allergen, is heritable but seldom ascribed to mutations within specific genes. Atopic individuals develop abnormally elevated IgE responses to immunization with potential allergens. To gain insight into the genetic causes of atopy, we carried out a forward genetic screen for atopy in mice. Methods We screened mice carrying homozygous and heterozygous N-ethyl-N-nitrosourea (ENU)-induced germline mutations for aberrant antigen-specific IgE and IgG1 production in response to immunization with the model allergen papain. Candidate genes were validated by independent gene mutation. Results Of 31 candidate genes selected for investigation, the effects of mutations in 23 genes on papain-specific IgE or IgG1 were verified. Among the 20 verified genes influencing the IgE response, eight were necessary for the response, while 12 repressed IgE. Nine genes were not previously implicated in the IgE response. Fifteen genes encoded proteins contributing to IgE class switch recombination or B-cell receptor signaling. The precise roles of the five remaining genes (Flcn, Map1lc3b, Me2, Prkd2, and Scarb2) remain to be determined. Loss-of-function mutations in nine of the 12 genes limiting the IgE response were dominant or semi-dominant for the IgE phenotype but did not cause immunodeficiency in the heterozygous state. Using damaging allele frequencies for the corresponding human genes and in silico simulations (Monte Carlo) of undiscovered atopy mutations, we estimated the percentage of humans with heterozygous atopy risk mutations. Conclusions Up to 37% of individuals may be heterozygous carriers for at least one dominant atopy risk mutation.

Adenosine monophosphate deaminase 3 (Ampd3) encodes the erythrocyte isoform of the adenosine monophosphate (AMP) deaminase gene family. Mutations in this gene have been reported in humans, leading to autosomal-recessive erythrocyte AMP deaminase deficiency. However, the mutation is considered clinically asymptomatic. Using N-ethyl-N-nitrosourea mutagenesis to find mutations that affect peripheral lymphocyte populations, we identified 5 Ampd3 mutations (Ampd3guangdong, Ampd3carson, Ampd3penasco, Ampd3taos, and Ampd3commanche) that strongly correlated with a reduction in naive CD4+ T and naive CD8+ T-cell populations. Causation was confirmed by targeted ablation of Ampd3. Knockout mice had reduced frequencies of CD62LhiCD44lo CD4+ naive and CD8+ naive T cells. Interestingly, these phenotypes were restricted to T cells circulating in peripheral blood and were not seen in T cells from secondary lymphoid organs (lymph nodes and spleen). We found that reduction of naive T cells in the peripheral blood of Ampd3−/− mice was caused by T-cell–extrinsic factor(s), which we hypothesize to be elevated levels of adenosine triphosphate released by Ampd3-deficient erythrocytes. These findings provide an example in which disruption of an erythrocyte-specific protein can affect the physiological status of lymphocytes in peripheral blood.

Using random germline mutagenesis in mice, we identified a viable hypomorphic allele (boh) of the transcription-factor-encoding gene Ovol2 that resulted in obesity, which initially developed with normal food intake and physical activity but decreased energy expenditure. Fat weight was dramatically increased, while lean weight was reduced in 12-week-old boh homozygous mice, culminating by 24 weeks in massive obesity, hepatosteatosis, insulin resistance, and diabetes. The Ovol2

Forward genetic studies use meiotic mapping to adduce evidence that a particular mutation, normally induced by a germline mutagen, is causative of a particular phenotype. Particularly in small pedigrees, cosegregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the identification of mutations causing immune phenotypes in mice by creating Candidate Explorer (CE), a machine-learning software program that integrates 67 features of genetic mapping data into a single numeric score, mathematically convertible to the probability of verification of any putative mutation–phenotype association. At this time, CE has evaluated putative mutation–phenotype associations arising from screening damaging mutations in ∼55% of mouse genes for effects on flow cytometry measurements of immune cells in the blood. CE has therefore identified more than half of genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus. The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.

Embryonic formation and patterning of the vertebrate spinal column requires coordination of many molecular cues. After birth, the integrity of the spine is impacted by developmental abnormalities of the skeletal, muscular and nervous systems, which may result in deformities, such as kyphosis and scoliosis. We sought to identify novel genetic mouse models of severe spine deformity by implementing in vivo skeletal radiography as part of a high-throughput saturation mutagenesis screen. We report selected examples of genetic mouse models following radiographic screening of 54,497 mice from 1275 pedigrees. An estimated 30.44% of autosomal genes harbored predicted damaging alleles examined twice or more in the homozygous state. Of the 1275 pedigrees screened, 7.4% presented with severe spine deformity developing in multiple mice, and of these, meiotic mapping implicated N-ethyl-N-nitrosourea alleles in 21% of pedigrees. Our study provides proof of concept that saturation mutagenesis is capable of discovering novel mouse models of human disease, including conditions with skeletal, neural and neuromuscular pathologies. Furthermore, we report a mouse model of skeletal disease, including severe spine deformity, caused by recessive mutation in Scube3. By integrating results with a human clinical exome database, we identified a patient with undiagnosed skeletal disease who harbored recessive mutations in SCUBE3, and we demonstrated that disease-associated mutations are associated with reduced transactivation of Smad signaling in vitro. All radiographic results and mouse models are made publicly available through the Mutagenetix online database with the goal of advancing understanding of spine development and discovering novel mouse models of human disease., Summary: We report selected mouse models of spine deformity following mutagenesis across 30% of autosomal genes, results of which are made publicly available to advance understanding of spine development and disease.

Endoplasmic reticulum (ER) calcium (Ca(2+)) stores are critical to proteostasis, intracellular signaling, and cellular bioenergetics. Through forward genetic screening in mice, we identified two members of a new complex, Pacs1 and Wdr37, which are required for normal ER Ca(2+) handling in lymphocytes. Deletion of Pacs1 or Wdr37 caused peripheral lymphopenia that was linked to blunted Ca(2+) release from the ER after antigen receptor stimulation. Pacs1‐deficient cells showed diminished inositol triphosphate receptor expression together with increased ER and oxidative stress. Mature Pacs1 (−/−) B cells proliferated and died in vivo under lymphocyte replete conditions, indicating spontaneous loss of cellular quiescence. Disruption of Pacs1‐Wdr37 did not diminish adaptive immune responses, but potently suppressed lymphoproliferative disease models by forcing loss of quiescence. Thus, Pacs1‐Wdr37 plays a critical role in stabilizing lymphocyte populations through ER Ca(2+) handling and presents a new target for lymphoproliferative disease therapy.

Autism spectrum disorder (ASD) is a constellation of neurodevelopmental disorders with high phenotypic and genetic heterogeneity, complicating the discovery of causative genes. Through a forward genetics approach selecting for defective vocalization in mice, we identified Kdm5a as a candidate ASD gene. To validate our discovery, we generated a Kdm5a knockout mouse model (Kdm5a-/-) and confirmed that inactivating Kdm5a disrupts vocalization. In addition, Kdm5a-/- mice displayed repetitive behaviors, sociability deficits, cognitive dysfunction, and abnormal dendritic morphogenesis. Loss of KDM5A also resulted in dysregulation of the hippocampal transcriptome. To determine if KDM5A mutations cause ASD in humans, we screened whole exome sequencing and microarray data from a clinical cohort. We identified pathogenic KDM5A variants in nine patients with ASD and lack of speech. Our findings illustrate the power and efficacy of forward genetics in identifying ASD genes and highlight the importance of KDM5A in normal brain development and function.

Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Forward genetics provides an excellent tool to find, in an unbiased manner, genes that are essential to these processes. Using N -ethyl- N -nitrosourea mutagenesis in mice in combination with a screening protocol using optical coherence tomography (OCT) and automated meiotic mapping, we identified 11 mutations presumably causative of retinal phenotypes in genes previously known to be essential for retinal integrity. In addition, we found multiple statistically significant gene-phenotype associations that have not been reported previously and decided to target one of these genes, Sfxn3 (encoding sideroflexin-3), using CRISPR/Cas9 technology. We demonstrate, using OCT, light microscopy, and electroretinography, that two Sfxn3 −/− mouse lines developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium and disruption of the external limiting membrane. Using single-cell RNA sequencing of retinal cells isolated from C57BL/6J mice, we demonstrate that Sfxn3 is expressed in several bipolar cell subtypes, retinal ganglion cells, and some amacrine cell subtypes but not significantly in Müller cells or photoreceptors. In situ hybridization confirmed these findings. Furthermore, pathway analysis suggests that Sfxn3 may be associated with synaptic homeostasis. Importantly, electron microscopy analysis showed disruption of synapses and synaptic ribbons in the outer plexiform layer of Sfxn3 −/− mice. Our work describes a previously unknown requirement for Sfxn3 in retinal function.

Buildings are complex systems whose energy consumption depends on the interplay of multiple physical and human factors. This paper explored the performance of single-family detached dwellin...

Choi et al. identify mice with a metabolic disorder and severe lymphoid and myeloid hypoplasia resulting from mutation of PDIA6. The ER-resident oxidoreductase PDIA6 is necessary for folding of stroma-derived Wnt3a, BAFF, and IL-7 proteins, which are necessary for hematopoiesis., In a forward genetic screen of N-ethyl-N-nitrosourea (ENU)–induced mutant mice for aberrant immune function, we identified mice with a syndromic disorder marked by growth retardation, diabetes, premature death, and severe lymphoid and myeloid hypoplasia together with diminished T cell–independent (TI) antibody responses. The causative mutation was in Pdia6, an essential gene encoding protein disulfide isomerase A6 (PDIA6), an oxidoreductase that functions in nascent protein folding in the endoplasmic reticulum. The immune deficiency caused by the Pdia6 mutation was, with the exception of a residual T cell developmental defect, completely rescued in irradiated wild-type recipients of PDIA6-deficient bone marrow cells, both in the absence or presence of competition. The viable hypomorphic allele uncovered in these studies reveals an essential role for PDIA6 in hematopoiesis, but one extrinsic to cells of the hematopoietic lineage. We show evidence that this role is in the proper folding of Wnt3a, BAFF, IL-7, and perhaps other factors produced by the extra-hematopoietic compartment that contribute to the development and lineage commitment of hematopoietic cells.

SARS-CoV-2 infection causes diverse outcomes ranging from asymptomatic infection to respiratory distress and death. A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoV) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Therefore, we analyzed samples from the same individuals before and after SARS-CoV-2 infection or vaccination. We found hCCCoV antibody levels increase after SARS-CoV-2 exposure, demonstrating cross-reactivity. However, a case-control study indicates baseline hCCCoV antibody levels are not associated with protection against SARS-CoV-2 infection. Rather, higher magnitudes of pre-existing betacoronavirus antibodies correlate with more SARS-CoV-2 antibodies following infection, an indicator of greater disease severity. Additionally, immunization with hCCCoV spike proteins before SARS-CoV-2 immunization impedes generation of SARS-CoV-2 neutralizing antibodies in mice. Together, these data suggest pre-existing hCCCoV antibodies hinder SARS-CoV-2 antibody-based immunity following infection and provide insight on how pre-existing coronavirus immunity impacts SARS-CoV-2 infection, which is critical considering emerging variants., Graphical Abstract, A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoV) impacts susceptibility to SARS-CoV-2 infection. Lin et al. analyze hCCCoV antibodies in the same individuals before and after SARS-CoV-2 infection, finding pre-existing betacoronavirus antibodies may hinder SARS-CoV-2 effective immunity following infection.

Objectives To estimate birth prevalence of congenital cytomegalovirus (cCMV) in HIV-exposed uninfected children born in the current era of combination antiretroviral therapy and describe cCMV-related neurodevelopmental and hearing outcomes. Study design The Surveillance Monitoring for ART Toxicities cohort study follows HIV-exposed uninfected children at 22 sites in the US and Puerto Rico. Birth cCMV prevalence was estimated in a subset of participants who had blood pellets collected within three weeks of birth and underwent ≥1 of 6 assessments evaluating cognitive and language development including an audiologic examination between 1 and 5 years of age. Detection of CMV DNA by polymerase chain reaction testing of peripheral blood mononuclear cells was used to diagnose cCMV. Proportions of suboptimal assessment scores were compared by cCMV status using Fisher exact test. Results Mothers of 895 eligible HIV-exposed uninfected children delivered between 2007 and 2015. Most (90%) were on combination antiretroviral therapy, 88% had an HIV viral load of ≤400 copies/mL, and 93% had CD4 cell counts of ≥200 cells/μL. Eight infants were diagnosed with cCMV, yielding an estimated prevalence of 0.89% (95% CI, 0.39%-1.75%). After adjusting for a sensitivity of 70%-75% for the testing method, projected prevalence was 1.2%-1.3%. No differences were observed in cognitive, language and hearing assessments by cCMV status. Conclusions Although birth cCMV prevalence in HIV-exposed uninfected children born to women with well-controlled HIV is trending down compared with earlier combination antiretroviral therapy-era estimates, it is above the 0.4% reported for the general US population. HIV-exposed uninfected children remain at increased risk for cCMV.

The primary goals of modern genetics are to identify disease-causing mutations and to define the functions of genes in biological processes. Two complementary approaches, reverse and forward genetics, can be utilized to achieve this goal. Reverse genetics is a gene-driven approach that comprises specific gene targeting followed by phenotypic assessment. Conversely, forward genetics is a phenotype-driven approach that involves the phenotypic screening of organisms with randomly induced mutations followed by subsequent identification of the causative mutations (i.e. those responsible for phenotype). In this article, we focus on how forward genetics in mice can be utilized to explore dermatologic disease. We outline mouse mutagenesis with the chemical N-ethyl-N-nitrosourea (ENU) and the strategy utilized to instantaneously identify mutations that are causative of specific phenotypes. We also summarize the types of phenotypic screens that can be performed to explore various aspects of dermatologic disease.

T follicular helper cells (TFH) participate in germinal center (GC) development and are necessary for B cell production of high-affinity, isotype-switched antibodies. In a forward genetic screen, we identified a missense mutation in Prkd2, encoding the serine/threonine kinase protein kinase D2, which caused elevated titers of immunoglobulin E (IgE) in the serum. Subsequent analysis of serum antibodies in mice with a targeted null mutation of Prkd2 demonstrated polyclonal hypergammaglobulinemia of IgE, IgG1, and IgA isotypes, which was exacerbated by the T cell-dependent humoral response to immunization. GC formation and GC B cells were increased in Prkd2-/- spleens. These effects were the result of excessive cell-autonomous TFH development caused by unrestricted Bcl6 nuclear translocation in Prkd2-/- CD4+ T cells. Prkd2 directly binds to Bcl6, and Prkd2-dependent phosphorylation of Bcl6 is necessary to constrain Bcl6 to the cytoplasm, thereby limiting TFH development. In response to immunization, Bcl6 repressed Prkd2 expression in CD4+ T cells, thereby committing them to TFH development. Thus, Prkd2 and Bcl6 form a mutually inhibitory positive feedback loop that controls the stable transition from naive CD4+ T cells to TFH during the adaptive immune response.

Peak demand energy costs are vastly greater than non-peak energy costs, which create unique opportunities for utility-owned, localized renewable energy development. A small, mountain utility’s energy supply contract terms have considerable impact on peak demand-related expenses, with annual savings exceeding 40% expected in an upcoming contract transition. Furthermore, utility-scale electrical peak shaving through grid-tied PV systems represent a valuable source of financial savings without necessitating costly energy storage mechanisms. Savings reaped from avoided cost premiums can, in turn, be invested in continued renewable energy development.

LPS-responsive beige-like anchor (LRBA) protein deficiency in humans causes immune dysregulation resulting in autoimmunity, inflammatory bowel disease (IBD), hypogammaglobulinemia, regulatory T (T reg ) cell defects, and B cell functional defects, but the cellular and molecular mechanisms responsible are incompletely understood. In an ongoing forward genetic screen for N -ethyl- N -nitrosourea (ENU)-induced mutations that increase susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice, we identified two nonsense mutations in Lrba . Although T reg cells have been a main focus in LRBA research to date, we found that dendritic cells (DCs) contribute significantly to DSS-induced intestinal inflammation in LRBA-deficient mice. Lrba −/− DCs exhibited excessive IRF3/7- and PI3K/mTORC1-dependent signaling and type I IFN production in response to the stimulation of the Toll-like receptors (TLRs) 3, TLR7, and TLR9. Substantial reductions in cytokine expression and sensitivity to DSS in LRBA-deficient mice were caused by knockout of Unc93b1 , a chaperone necessary for trafficking of TLR3, TLR7, and TLR9 to endosomes. Our data support a function for LRBA in limiting endosomal TLR signaling and consequent intestinal inflammation.

The SMCR8-WDR41-C9ORF72 complex is a regulator of autophagy and lysosomal function. Autoimmunity and inflammatory disease have been ascribed to loss-of-function mutations of Smcr8 or C9orf72 in mice. In humans, autoimmunity has been reported to precede amyotrophic lateral sclerosis caused by mutations of C9ORF72. However, the cellular and molecular mechanisms underlying autoimmunity and inflammation caused by C9ORF72 or SMCR8 deficiencies remain unknown. Here, we show that splenomegaly, lymphadenopathy, and activated circulating T cells observed in Smcr8(−/−) mice were rescued by triple knockout of the endosomal Toll-like receptors (TLRs) TLR3, TLR7, and TLR9. Myeloid cells from Smcr8(−/−) mice produced excessive inflammatory cytokines in response to endocytosed TLR3, TLR7, or TLR9 ligands administered in the growth medium and in response to TLR2 or TLR4 ligands internalized by phagocytosis. These defects likely stem from prolonged TLR signaling caused by accumulation of LysoTracker-positive vesicles and by delayed phagosome maturation, both of which were observed in Smcr8(−/−) macrophages. Smcr8(−/−) mice also showed elevated susceptibility to dextran sodium sulfate-induced colitis, which was not associated with increased TLR3, TLR7, or TLR9 signaling. Deficiency of WDR41 phenocopied loss of SMCR8. Our findings provide evidence that excessive endosomal TLR signaling resulting from prolonged ligand–receptor contact causes inflammatory disease in SMCR8-deficient mice.

BackgroundPersons who are infected with human immunodeficiency virus (HIV) are at high risk of human papillomavirus (HPV)-associated cancers. The objectives are to compare antibody titers to HPV 6, 11, 16, and 18 and rate of abnormal cytology between perinatally HIV-infected (PHIV) and perinatally HIV-exposed, uninfected (PHEU) youth.MethodsThis is a prospective observational cohort study of HPV4 vaccinated youth performed as part of the multicenter Pediatric HIV/AIDS Cohort Study Adolescent Master Protocol. Seroconversion and geometric mean titer (GMT) against HPV types 6, 11, 16, and 18 were calculated. Vaccine effectiveness included rates of abnormal cervical cytology and genital warts.ResultsSeroconversion to HPV 6, 11, 16, and 18 occurred in 83%, 84%, 90%, and 62% of 310 vaccinated PHIV youth compared to 94%, 96%, 99%, and 87% of 148 vaccinated PHEU youth, respectively (P < .05 for all comparisons). GMTs were lower in the PHIV vs PHEU within each category of HPV4 doses received. Higher GMTs were associated with younger age, lower HIV type 1 RNA viral load, and higher CD4% at first HPV4 vaccination, as well as shorter duration between last vaccine dose and antibody specimen. Abnormal cytology occurred in 33 of 56 PHIV and 1 of 7 PHEU sexually active vaccinated females, yielding incidence rates per 100 person-years of 15.0 (10.9 to 20.6) and 2.9 (0.4 to 22.3), respectively.ConclusionAntibody titers to HPV4 were lower for all serotypes in PHIV compared to PHEU youth. Protection against abnormal cytology was also diminished in sexually active PHIV females.

Myosin ID (MYO1D) is a member of the class I myosin family. We screened 48,649 third generation (G3) germline mutant mice derived from N-ethyl-N-nitrosourea-mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). We found and validated mutations in Myo1d as a cause of increased susceptibility to DSS-induced colitis. MYO1D is produced in the intestinal epithelium, and the colitis phenotype is dependent on the nonhematopoietic compartment of the mouse. Moreover, MYO1D appears to couple cytoskeletal elements to lipid in an ATP-dependent manner. These findings demonstrate that MYO1D is needed to maintain epithelial integrity and protect against DSS-induced colitis., Summary: Using random germline mutagenesis and screening of mice, we determined that loss of MYO1D function in nonhematopoietic tissues renders mice susceptible to colitis induced by dextran sodium sulfate challenge.

Innate behaviors are genetically encoded, but their underlying molecular mechanisms remain largely unknown. Predator odor 2,4,5-trimethyl-3-thiazoline (TMT) and its potent analog 2-methyl-2-thiazoline (2MT) are believed to activate specific odorant receptors to elicit innate fear/defensive behaviors in naive mice. Here, we conduct a large-scale recessive genetics screen of ethylnitrosourea (ENU)-mutagenized mice. We find that loss of Trpa1, a pungency/irritancy receptor, diminishes TMT/2MT and snake skin-evoked innate fear/defensive responses. Accordingly, Trpa1−/− mice fail to effectively activate known fear/stress brain centers upon 2MT exposure, despite their apparent ability to smell and learn to fear 2MT. Moreover, Trpa1 acts as a chemosensor for 2MT/TMT and Trpa1-expressing trigeminal ganglion neurons contribute critically to 2MT-evoked freezing. Our results indicate that Trpa1-mediated nociception plays a crucial role in predator odor-evoked innate fear/defensive behaviors. The work establishes the first forward genetics screen to uncover the molecular mechanism of innate fear, a basic emotion and evolutionarily conserved survival mechanism., TMT is a chemical that evokes innate defensive behaviors yet the molecular mechanisms are not well understood. Here the authors perform a large-scale forward genetics screen in mice and identify Trpa1, a pungency/irritancy receptor, as a chemosensor for predator odor-evoked innate fear and defensive behaviors.

Wnt signaling out on a limb The Wnt/β-catenin signaling pathway is central to hematopoietic stem cell (HSC) survival, renewal, and differentiation. Both excessive and insufficient β-catenin can have adverse consequences, so “destruction complexes” ensure its precise regulation. Using a mouse forward genetic screen, Choi et al. identified a mutation in the gene limb region 1–like ( Lmbr1l ) that severely impaired lymphocyte development and function. LMBR1L interacted with multiple members of the Wnt/β-catenin signaling pathway. The authors found that LMBR1L works with an endoplasmic reticulum–localized ubiquitin ligase to serve as a second brake on this system in lymphocytes. Deleting β-catenin decreased apoptosis and restored proliferative capacity in an Lmbr1l -deficient mouse T cell line. Science , this issue p. eaau0812

The NLRP3 inflammasome responds to microbes and danger signals by processing and activating proinflammatory cytokines, including interleukin 1β (IL-1β) and IL-18. We found here that activation of the NLRP3 inflammasome was restricted to interphase of the cell cycle by NEK7, a serine-threonine kinase previously linked to mitosis. Activation of the NLRP3 inflammasome required NEK7, which bound to the leucine-rich repeat domain of NLRP3 in a kinase-independent manner downstream of the induction of mitochondrial reactive oxygen species (ROS). This interaction was necessary for the formation of a complex containing NLRP3 and the adaptor ASC, oligomerization of ASC and activation of caspase-1. NEK7 promoted the NLRP3-dependent cellular inflammatory response to intraperitoneal challenge with monosodium urate and the development of experimental autoimmune encephalitis in mice. Our findings suggest that NEK7 serves as a cellular switch that enforces mutual exclusivity of the inflammasome response and cell division.

Socio-indexical cues and paralinguistic information are often beneficial to speech processing as this information assists listeners in parsing the speech stream. Associations that particular populations speak in a certain speech style can, however, make it such that socio-indexical cues have a cost. In this study, native speakers of Canadian English who identify as Chinese Canadian and White Canadian read sentences that were presented to listeners in noise. Half of the sentences were presented with a visual-prime in the form of a photo of the speaker and half were presented in control trials with fixation crosses. Sentences produced by Chinese Canadians showed an intelligibility cost in the face-prime condition, whereas sentences produced by White Canadians did not. In an accentedness rating task, listeners rated White Canadians as less accented in the face-prime trials, but Chinese Canadians showed no such change in perceived accentedness. These results suggest a misalignment between an expected and an observed speech signal for the face-prime trials, which indicates that social information about a speaker can trigger linguistic associations that come with processing benefits and costs.

Computational inference of mutation effects is necessary for genetic studies in which many mutations must be considered as etiologic candidates. Programs such as PolyPhen-2 predict the relative severity of damage caused by missense mutations, but not the actual probability that a mutation will reduce/eliminate protein function. Based on genotype and phenotype data for 116,330 ENU-induced mutations in the Mutagenetix database, we calculate that putative null mutations, and PolyPhen-2-classified “probably damaging”, “possibly damaging”, or “probably benign” mutations have, respectively, 61%, 17%, 9.8%, and 4.5% probabilities of causing phenotypically detectable damage in the homozygous state. We use these probabilities in the estimation of genome saturation and the probability that individual proteins have been adequately tested for function in specific genetic screens. We estimate the proportion of essential autosomal genes in Mus musculus (C57BL/6J) and show that viable mutations in essential genes are more likely to induce phenotype than mutations in non-essential genes., Programs such as PolyPhen-2 predict the relative severity of damage by missense mutations. Here, Wang et al estimate probabilities that putative null or missense alleles would reduce protein function to cause detectable phenotype by analyzing data from ENU-induced mouse mutations.

Objective. To compare the mean national enrollment rates of underrepresented minority (URM) students in a pharmacy school with mean rates in California pharmacy schools, and identify barriers faced by URM students during the application process. Methods. The American Association of Colleges of Pharmacy (AACP) enrollment data from 2005 to 2014 were used to compare the demographics of California pharmacy schools with the average enrollment of URM students in pharmacy schools nationally. A survey was administered to students in the 2017 and 2018 classes at Touro University California College of Pharmacy to identify common barriers that students faced in pursuing pharmacy education. Results. The average enrollment of URM in pharmacy programs nationally was 12.3% in 2005, compared to 12.4% in 2014. The average enrollment of URM in California pharmacy schools was 9.4% in 2005 compared to 8.5% in 2014. The top barriers to pursuing pharmacy education that students reported included the cost of tuition (43.4%), prerequisite requirements (36.9%), and obtaining letters of recommendation (32.3%). Conclusion. The average enrollment of URM students in pharmacy schools nationally has remained higher than that in California pharmacy schools across the years studied. California pharmacy programs should develop strategies to alleviate the barriers identified and further diversify pharmacy education.

Acidosis, a critical aspect of central nervous system (CNS) pathophysiology and a metabolic corollary of the hypoxic stem cell niche, could be an expedient trigger for hippocampal neurogenesis and brain repair. We recently tracked the function of our isoxazole stem cell-modulator small molecules (Isx) through a chemical biology-target discovery strategy to GPR68, a proton (pH) sensing G protein-coupled receptor with no known function in brain. Isx and GPR68 coregulated neuronal target genes such as Bex1 (brain-enriched X-linked protein-1) in hippocampal neural progenitors (HCN cells), which further amplified GPR68 signaling by producing metabolic acid in response to Isx. To evaluate this proneurogenic small molecule/proton signaling circuit in vivo, we explored GPR68 and BEX1 expression in brain and probed brain function with Isx. We localized proton-sensing GPR68 to radial processes of hippocampal type 1 neural stem cells (NSCs) and, conversely, localized BEX1 to neurons. At the transcriptome level, Isx demonstrated unrivaled proneurogenic activity in primary hippocampal NSC cultures. In vivo, Isx pharmacologically targeted type 1 NSCs, promoting neurogenesis in young mice, depleting the progenitor pool without adversely affecting hippocampal learning and memory function. After traumatic brain injury, cerebral cortical astrocytes abundantly expressed GPR68, suggesting an additional role for proton-GPCR signaling in reactive astrogliosis. Thus, probing a novel proneurogenic synthetic small molecule's mechanism-of-action, candidate target, and pharmacological activity, we identified a new GPR68 regulatory pathway for integrating neural stem and astroglial cell functions with brain pH.

Rationale: Transgenic Notch reporter mice express enhanced green fluorescent protein in cells with C-promoter binding factor-1 response element transcriptional activity (CBF1-RE x4 -EGFP), providing a unique and powerful tool for identifying and isolating “Notch-activated” progenitors. Objective: We asked whether, as in other tissues of this mouse, EGFP localized and functionally tagged adult cardiac tissue progenitors, and, if so, whether this cell-based signal could serve as a quantitative and qualitative biosensor of the injury repair response of the heart. Methods and Results: In addition to scattered endothelial and interstitial cells, Notch-activated (EGFP + ) cells unexpectedly richly populated the adult epicardium. We used fluorescence-activated cell sorting to isolate EGFP + cells and excluded hematopoietic (CD45 + ) and endothelial (CD31 + ) subsets. We analyzed EGFP + /CD45 − /CD31 − cells, a small ( Conclusions: A dynamic Notch injury response activates adult epicardium, producing a multipotent cell population that contributes to fibrosis repair.

With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.

The E2F1 transcription factor, which is deregulated in most human cancers by mutations in the p16-cyclin D-Rb pathway, has both oncogenic and tumor-suppressive properties. This is dramatically illustrated by the phenotype of an E2F1 transgenic mouse model that spontaneously develops tumors in the skin and other epithelial tissues but is resistant to papilloma formation when subjected to a two-stage carcinogenesis protocol. Here, this E2F1 transgenic model was used to further explore the tumor-suppressive property of E2F1. Transgenic expression of E2F1 was found to inhibit ras-driven skin carcinogenesis at the promotion stage independent of the type of promoting agent used. E2F1 transgenic epidermis displayed increased expression of p19(ARF), p53, and p21(Cip1). Inactivation of either p53 or Arf in E2F1 transgenic mice restored sensitivity to two-stage skin carcinogenesis. While Arf inactivation impaired tumor suppression and p21 induction by E2F1, it did not reduce the level of apoptosis observed in E2F1 transgenic mice. Based on these findings, we propose that E2F1 suppresses ras-driven skin carcinogenesis through a nonapoptotic mechanism involving ARF and p53.

The ARF tumor suppressor participates in a p53-dependent apoptotic pathway that is stimulated in response to some oncogenic stimuli. The E2F1 transcription factor is a critical downstream target of the Rb tumor suppressor and, when active, can promote proliferation as well as apoptosis. The finding that E2F1 transcriptionally regulates the ARF gene has led to the suggestion that ARF contributes to E2F1-induced apoptosis. Counter to this hypothesis, this study demonstrates not only that ARF is unnecessary for E2F1 to induce apoptosis but also that inactivation of ARF actually enhances the ability of E2F1 to promote apoptosis. Inactivation of ARF also cooperates with E2F1 activity to promote entry into the S phase of the cell cycle. This relationship between ARF and E2F1 is demonstrated in transgenic epidermis in vivo and in mouse embryo fibroblast cultures in vitro. In contrast, the ability of Myc to induce apoptosis is diminished in the absence of ARF. E2F1 induces the accumulation of p53 in the absence of ARF, and this is associated with the phosphorylation of p53 on several residues. These findings demonstrate that ARF is a negative regulator of E2F1 activity and is not required for E2F1-induced apoptosis.