Soft windowing application to improve analysis of high-throughput phenotyping data.

MOTIVATION: High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors. RESULTS: Here we introduce 'soft windowing', a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources. AVAILABILITY AND IMPLEMENTATION: The method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ANP32E is a histone chaperone that removes H2A.Z from chromatin.

H2A.Z is an essential histone variant implicated in the regulation of key nuclear events. However, the metazoan chaperones responsible for H2A.Z deposition and its removal from chromatin remain unknown. Here we report the identification and characterization of the human protein ANP32E as a specific H2A.Z chaperone. We show that ANP32E is a member of the presumed H2A.Z histone-exchange complex p400/TIP60. ANP32E interacts with a short region of the docking domain of H2A.Z through a new motif termed H2A.Z interacting domain (ZID). The 1.48 Å resolution crystal structure of the complex formed between the ANP32E-ZID and the H2A.Z/H2B dimer and biochemical data support an underlying molecular mechanism for H2A.Z/H2B eviction from the nucleosome and its stabilization by ANP32E through a specific extension of the H2A.Z carboxy-terminal α-helix. Finally, analysis of H2A.Z localization in ANP32E(-/-) cells by chromatin immunoprecipitation followed by sequencing shows genome-wide enrichment, redistribution and accumulation of H2A.Z at specific chromatin control regions, in particular at enhancers and insulators.

Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress.

Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.

The International Mouse Phenotyping Consortium (IMPC): a functional catalogue of the mammalian genome that informs conservation.

The International Mouse Phenotyping Consortium (IMPC) is building a catalogue of mammalian gene function by producing and phenotyping a knockout mouse line for every protein-coding gene. To date, the IMPC has generated and characterised 5186 mutant lines. One-third of the lines have been found to be non-viable and over 300 new mouse models of human disease have been identified thus far. While current bioinformatics efforts are focused on translating results to better understand human disease processes, IMPC data also aids understanding genetic function and processes in other species. Here we show, using gorilla genomic data, how genes essential to development in mice can be used to help assess the potentially deleterious impact of gene variants in other species. This type of analyses could be used to select optimal breeders in endangered species to maintain or increase fitness and avoid variants associated to impaired-health phenotypes or loss-of-function mutations in genes of critical importance. We also show, using selected examples from various mammal species, how IMPC data can aid in the identification of candidate genes for studying a condition of interest, deliver information about the mechanisms involved, or support predictions for the function of genes that may play a role in adaptation. With genotyping costs decreasing and the continued improvements of bioinformatics tools, the analyses we demonstrate can be routinely applied.

Cracking the ANP32 whips: important functions, unequal requirement, and hints at disease implications.

The acidic (leucine-rich) nuclear phosphoprotein 32 kDa (ANP32) family is composed of small, evolutionarily conserved proteins characterized by an N-terminal leucine-rich repeat domain and a C-terminal low-complexity acidic region. The mammalian family members (ANP32A, ANP32B, and ANP32E) are ascribed physiologically diverse functions including chromatin modification and remodelling, apoptotic caspase modulation, protein phosphatase inhibition, as well as regulation of intracellular transport. In addition to reviewing the widespread literature on the topic, we present a concept of the ANP32s as having a whip-like structure. We also present hypotheses that ANP32C and other intronless sequences should not currently be considered bona fide family members, that their disparate necessity in development may be due to compensatory mechanisms, that their contrasting roles in cancer are likely context-dependent, along with an underlying hypothesis that ANP32s represent an important node of physiological regulation by virtue of their diverse biochemical activities.

Acidic nuclear phosphoprotein 32kDa (ANP32)B-deficient mouse reveals a hierarchy of ANP32 importance in mammalian development.

The highly conserved ANP32 proteins are proposed to function in a broad array of physiological activities through molecular mechanisms as diverse as phosphatase inhibition, chromatin regulation, caspase activation, and intracellular transport. On the basis of previous analyses of mice bearing targeted mutations of Anp32a or Anp32e, there has been speculation that all ANP32 proteins play redundant roles and are dispensable for normal development. However, more recent work has suggested that ANP32B may in fact have functions that are not shared by other ANP32 family members. Here we report that ANP32B expression is associated with a poor prognosis in human breast cancer, consistent with the increased levels of Anp32b mRNA present in proliferating wild-type (WT) murine embryonic fibroblasts and stimulated WT B and T lymphocytes. Moreover, we show that, contrary to previous assumptions, Anp32b is very important for murine embryogenesis. In a mixed genetic background, ANP32B-deficient mice displayed a partially penetrant perinatal lethality that became fully penetrant in a pure C57BL/6 background. Surviving ANP32B-deficient mice showed reduced viability due to variable defects in various organ systems. Study of compound mutants lacking ANP32A, ANP32B, and/or ANP32E revealed previously hidden roles for ANP32A in mouse development that became apparent only in the complete absence of ANP32B. Our data demonstrate a hierarchy of importance for the mammalian Anp32 genes, with Anp32b being the most critical for normal development.

The influence of different horseshoes and ground substrates on mid-stance hoof orientation at the walk.

BACKGROUND: Horseshoes with modified contact surfaces combined with deformable ground substrates are used to change hoof orientation during mid-stance, for example, for therapeutic reasons. OBJECTIVES: To measure the effect of horseshoes and ground substrates on sagittal and transverse plane hoof orientation at mid-stance using a dorsal hoof wall mounted triaxial accelerometer. STUDY DESIGN: In vivo experiment, randomised crossover design. METHODS: Differences in sagittal and transverse plane angles between standing and mid-stance of the left front hoof of six horses walking with regular horseshoes, egg bar, toe-wide, medial-wide, lateral-wide and three-degree egg bar shoes on turf, sand and hard ground substrates were assessed with linear mixed models with horseshoe and substrate type as fixed factors (p < 0.05) for each animal. RESULTS: Hoof angles were significantly affected by horseshoe (p < 0.001), surface (p < 0.001) and the combination (p < 0.001). The sagittal plane angle increased in deformable ground substrates at walk-in mid-stance on turf [mean (±standard deviation): 2.6° (±3.8°)] and on sand [2.6° (±4.1°)] across all shoes. The greatest increase was observed with egg bar shoes [turf: 4.37° (±3.82°); sand 4.69° (±3.83°)]. There was a tendency for the hoof to sink laterally into deformable ground substrates among all shoes [turf: 1.11° (±1.49°); sand: 0.93° (±1.93°)]. Medial-wide shoes increased the lateral sinking [turf: 2.00° (±1.63°); sand: 1.79° (±1.58°)]. Lateral-wide shoes reduced the lateral sinking on turf [0.62° (±1.26°)] and induced a marginal medial sinking on sand [-0.007° (±2.03°)]. MAIN LIMITATIONS: The substrate properties were not quantitatively assessed, and observations were limited to front hooves at the walk. A larger sample size would be preferable. CONCLUSIONS: Mid-stance hoof orientation changes with specific combinations of shoes and ground substrates in the walking horse.

CONTEXTO: É especulado que ferraduras com solados diferentes combinadas com superfícies deformáveis podem mudar a orientação do casco durante a fase de apoio, por exemplo, por razões terapêuticas. OBJETIVOS: Mensurar o efeito de diferentes ferraduras e superfícies na orientação do casco nos planos sagital e transversal durante a fase de apoio usando um acelerômetro triaxial acoplado à parte dorsal do casco. DELINEAMENTO DO ESTUDO: Experimento in vivo, delineamento randomizado e cruzado. MÉTODOS: As diferenças entre os ângulos dos planos sagital e transverso nas diferentes fases de apoio do casco do membro anterior esquerdo de seis cavalos ao passo utilizando ferradura normal, oval, oval talonada, de pinça larga e com extensão medial ou lateral na grama, areia ou superfície dura foram avaliadas utilizando modelos mistos lineares com ferradura e tipo de superfície como fatores fixos (P < 0.05) para cada animal. RESULTADOS: Os ângulos do casco foram significativamente afetados pelo tipo de ferradura (P < 0.001), superfície (P < 0.001) e pela combinação de ambos (P < 0.001). O ângulo do plano sagital aumentou em superfícies deformáveis no passo na fase de apoio na grama (média (+/­SD): 2.6 (+/−3.8) graus) e na areia (2.6 (+/−4.1 graus) para todos os tipos de ferradura. O maior aumento foi observado com a ferradura oval (grama: 4.37 (+/− 3.82) graus; areia 4.69 (+/−3.83) graus). Houve uma tendência de o casco rebaixar mais lateralmente em superfícies deformáveis com todas as ferraduras (grama: 1.11 (+/−1.49) graus; areia: 0.93 (+/−1.93) graus). Ferraduras com extensão medial aumentaram o rebaixamento lateral (grama: 2.00 (+/−1.63) graus; areia: 1.79 (+/−1.58) graus). Ferraduras com extensão lateral reduziram o rebaixamento lateral na grama (0.62 (+/−1.26) graus) e induziram o rebaixamento medial na areia (−0.007 (+/−2.03) graus). PRINCIPAIS LIMITAÇÕES: As propriedades das superfícies não foram avaliadas quantitativamente, e as observações foram limitadas aos cascos dos membros anteriores e ao passo. Um número maior de animais no estudo seria desejável. CONCLUSÕES: A orientação do casco na fase de apoio muda de acordo com combinações específicas de ferradura e superfícies no cavalo ao passo.

Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay.

Smg1 is a PI3K-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbor premature termination codons (PTCs) as a result of events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. By using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5. High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from approximately 9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signaling, membrane dynamics, cell death, and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

Computed tomographic guidance for internal fixation of type II distal phalangeal fractures in 51 horses.

BACKGROUND: Type II distal phalanx (P3) fractures are a well-described cause of lameness in horses. Reports on outcome following internal fixation of type II P3 fractures are lacking, and with little emphasis on complications. OBJECTIVE: To describe a technique for internal fixation of type II P3 fractures, and evaluate whether specific variables influenced post-operative complications or a horse's ability to return to work. STUDY DESIGN: Retrospective case series. METHODS: Medical records of 51 horses with CT-guided internal fixation of type II P3 fractures were reviewed. Outcome data were acquired from race records and telephone interviews. Associations between independent variables and outcome were analysed using multivariable logistic regression. RESULTS: Eighty-six per cent (95% CI 74%-94%; n = 44) successfully returned to work. Implant infection (n = 15) and distal interphalangeal joint osteoarthritis (n = 9) were the most common complications, with the latter reducing the likelihood of success (OR = 0.09, 95% CI 0.01-0.7, P = .02). Implant infection increased the time to return to work (HR = 0.5, 95% CI 0.2-0.9, P = .03). The odds of delayed infection decreased by filling the hoof defect with acrylic hoof adhesive rather than poly(methyl methacrylate) and deeply countersinking the screw head (OR = 0.08, 95% CI 0.02-0.38, P = .001); the individual effect of each treatment is unknown. Radiographic healing was not associated with likelihood of success. MAIN LIMITATIONS: Study limitations included variation in follow-up methods, lack of control population for comparison and lack of randomisation of treatment protocols. CONCLUSIONS: Internal fixation of type II P3 fractures is an effective treatment that allows horses to return to athletic use, with similar improved success rates as those reported for conservative management. Infection rates were reduced by deeply countersinking the screw head and filling the hoof defect with an acrylic that mimics hoof wall flexibility and provides a secure seal. Recommencement of training should be based on clinical rather than strictly radiographic findings.

First aid for the laminitic foot: therapeutic and mechanical support.

The goals of mechanical treatment during the acute phase of laminitis are to preserve the lamellar interface by reducing the forces that are compromising its integrity and to make the horse more comfortable. Early decision making is important in managing acute laminitis. This article helps the practitioner to identify some of the commonly used and accepted methods of protecting the laminitic foot. The materials available and the theories behind their use are also described. The laminitic foot needs to be understood before determining methods for its support. Most treatment options involve shifting the weight-bearing forces from compromised areas of the foot (ie, the lamellar interface) to areas more capable of supporting the patient's weight, remembering that the sum of the forces should remain the same. The many treatment options available allow for flexibility and effective management and permit each modality to be combined in infinite ways for hoof support. The goal of therapy is to support the foot and stop the progression of the disease to the chronic phase.

ANP32B Deficiency Protects Mice From Lethal Influenza A Virus Challenge by Dampening the Host Immune Response.

Deciphering complex virus-host interactions is crucial for pandemic preparedness. In this study, we assessed the impact of recently postulated cellular factors ANP32A and ANP32B of influenza A virus (IAV) species specificity on viral pathogenesis in a genetically modified mouse model. Infection of ANP32A-/- and ANP32A+/+ mice with a seasonal H3N2 IAV or a highly pathogenic H5N1 human isolate did not result in any significant differences in virus tropism, innate immune response or disease outcome. However, infection of ANP32B-/- mice with H3N2 or H5N1 IAV revealed significantly reduced virus loads, inflammatory cytokine response and reduced pathogenicity compared to ANP32B+/+ mice. Genome-wide transcriptome analyses in ANP32B+/+ and ANP32B-/- mice further uncovered novel immune-regulatory pathways that correlate with reduced pathogenicity in the absence of ANP32B. These data show that ANP32B but not ANP32A promotes IAV pathogenesis in mice. Moreover, ANP32B might possess a yet unknown immune-modulatory function during IAV infection. Targeting ANP32B or its regulated pathways might therefore pose a new strategy to combat severe influenza.

Inactivation of the retinoblastoma protein family can bypass the HCF-1 defect in tsBN67 cell proliferation and cytokinesis.

Owing to a single missense mutation in the cell proliferation factor HCF-1, the temperature-sensitive tsBN67 hamster cell line arrests proliferation at nonpermissive temperatures, primarily in a G(0)/G(1) state, and displays temperature-sensitive cytokinesis defects. The HCF-1 mutation in tsBN67 cells also causes a temperature-sensitive dissociation of HCF-1 from chromatin prior to cell proliferation arrest, suggesting that HCF-1-chromatin association is important for mammalian-cell proliferation. Here, we report that the simian virus 40 (SV40) early region, in particular, large T antigen (Tag), and the adenovirus oncoprotein E1A can rescue the tsBN67 cell proliferation defect at nonpermissive temperatures. The SV40 early region rescues the tsBN67 cell proliferation defect without restoring the HCF-1-chromatin association, indicating that these oncoproteins bypass a requirement for HCF-1 function. The SV40 early region also rescues the tsBN67 cytokinesis defect, suggesting that the roles of HCF-1 in cell proliferation and proper cytokinesis are intimately linked. The ability of SV40 Tag and adenovirus E1A to inactivate members of the pRb protein family-pRb, p107, and p130-is important for the bypass of HCF-1 function. These results suggest that HCF-1 regulates mammalian-cell proliferation and cytokinesis, at least in part, by either directly or indirectly opposing pRb family member function.

Atp6ap2 ablation in adult mice impairs viability through multiple organ deficiencies.

ATP6AP2 codes for the (pro)renin receptor and is an essential component of vacuolar H+ ATPase. Activating (pro)renin for conversion of Angiotensinogen to Angiotensin makes ATP6AP2 attractive for drug intervention. Tissue-specific ATP6AP2 inactivation in mouse suggested a strong impact on various organs. Consistent with this, we found that embryonic ablation of Atp6ap2 resulted in both male hemizygous lethality and female haploinsufficiency. Next, we examined the phenotype of an induced inactivation in the adult animal, most akin to detect potential effect of functional interference of ATP6AP2 through drug therapy. Induced ablation of Atp6ap2, even without equal efficiency in all tissues (aorta, brain and kidney), resulted in rapid lethality marked by weight loss, changes in nutritional as well as blood parameters, leukocyte depletion, and bone marrow hypoplasia. Upon Atp6ap2 ablation, the colon demonstrated a rapid disruption of crypt morphology, aberrant proliferation, cell-death activation, as well as generation of microadenomas. Consequently, disruption of ATP6AP2 is extremely poorly tolerated in the adult, and severely affects various organ systems demonstrating that ATP6AP2 is an essential gene implicated in basic cellular mechanisms and necessary for multiple organ function. Accordingly, any potential drug targeting of this gene product must be strictly assessed for safety.

BALB/c-congenic ANP32B-deficient mice reveal a modifying locus that determines viability.

We previously found that deletion of the multifunctional factor ANP32B (a.k.a. SSP29, APRIL, PAL31, PHAPI2) resulted in a severe but strain-specific defect resulting in perinatal lethality. The difficulty in generating an adult cohort of ANP32B-deficient animals limited our ability to examine adult phenotypes, particularly cancer-related phenotypes. We bred the Anp32b-null allele into the BALB/c and FVB/N genetic background. The BALB/c, but not the FVB/N, background provided sufficient frequency of adult Anp32b-null (Anp32b(-/-)) animals. From these, we found no apparent oncogenic role for this protein in mammary tumorigenesis contrary to what was predicted based on human data. We also found runtism, pathologies in various organ systems, and an unusual clinical chemistry signature in the adult Anp32b(-/-) mice. Intriguingly, genome-wide single-nucleotide polymorphism analysis suggested that our colony retained an unlinked C57BL/6J locus at high frequency. Breeding this locus to homozygosity demonstrated that it had a strong effect on Anp32b(-/-) viability indicating that this locus contains a modifier gene of Anp32b with respect to development. This suggests a functionally important genetic interaction with one of a limited number of candidate genes, foremost among them being the variant histone gene H2afv. Using congenic breeding strategies, we have generated a viable ANP32B-deficient animal in a mostly pure background. We have used this animal to reliably exclude mouse ANP32B as an important oncogene in mammary tumorigenesis. Our further phenotyping strengthens the evidence that ANP32B is a widespread regulator of gene expression. These studies may also impact the choice of subsequent groups with respect to congenic breeding versus de novo zygote targeting strategies for background analyses in mouse genetics.

CARD9 Promotes Sex-Biased Colon Tumors in the APCmin Mouse Model.

Caspase recuitment domain-containing protein 9 (CARD9) functions in different inflammation pathways to elicit responses to microbial signals and is known to affect intestinal inflammation. Examining the APC(min) mouse model of intestinal tumorigenesis and using stringently controlled, sex- and age-matched pairs of CARD9-competent and CARD9-deficient mice, we have found that CARD9 has a restricted but strong effect on tumorigenesis in the large intestine. We have found that CARD9 reduces viability specifically in males and promotes tumorigenesis specifically in the large intestines of these male mice. To our knowledge, this is the first gene ablation in APC(min) mice that solely affects colon tumors in male subjects and, as such, may have significant clinical implications. Additional data suggest correlative disruption of plasma cytokine expression and immune infiltration of the tumors. We speculate that known sex-specific differences in human colorectal cancer may involve inflammation, particularly CARD9-dependent inflammation.

Isolation and characterization of tetranucleotide microsatellites from Atlantic haddock (Melanogrammus aeglefinus).

Five tetranucleotide microsatellite loci developed from Atlantic haddock (Melanogrammus aeglefinus) are presented. Loci were isolated using a modified magnetic bead-hybridization selection procedure that enriched for tetranucleotide microsatellites. Loci were polymorphic (3-99 alleles per locus; mean, 33.2) and exhibited high levels of observed heterozygosity (0.07-0.99; mean, 0.73) in a sample of 70 haddock collected from the Scotian Shelf in the Northwest Atlantic Ocean. Primer sets for 5 tetranucleotide microsatellites, originally developed from cod (Gmo34), were also tested in Atlantic haddock; one pair yielded readily detectable product and was variable in the population assayed (29 alleles; heterozygosity, 0.96). These loci are suitable for kinship analyses in aquaculture-related applications, and are potentially useful for resolving population structure in the wild.

Targeted ANP32E mutant mice do not demonstrate obvious movement defects.

BACKGROUND: The ANP32 family of proteins have been implicated in neuronal function through biochemical and cellular biology studies in neurons, as well as by recent behavioural studies of a gene-trapped loss-of-function mutation of Anp32e in mice, particularly with respect to fine motor function. A second targeted allele of the Anp32e, however, did not appear to demonstrate neurological phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: Using a stringently controlled cohort of ten-generation backcrossed, co-caged, sex-matched, littermate pairs, we assayed for potential motor defects in the targeted ANP32E-deficient mice. We found no phenotypic difference in any assays. CONCLUSION: Since it is unlikely that the gene-trap is a more complete loss-of-function, our results suggest that ANP32E has no appreciable effect on motor functions and that genetic background differences most likely account for the gene-trap phenomena.

Molecular pathways: tumor cells Co-opt the brain-specific metabolism gene CPT1C to promote survival.

The metabolic adaptations of cancer cells are receiving renewed attention as potential targets for therapeutic exploitation. Recent work has highlighted the importance of fatty acid catabolism through ß-oxidation to cellular energy homeostasis. In this article, we describe recent preclinical studies suggesting that a gene usually expressed only in the brain, carnitine palmitoyltransferase (CPT)1C, promotes cancer cell survival and tumor growth. CTP1C confers rapamycin resistance on breast cancer cells, indicating that this gene may act in a pathway parallel to mTOR-enhanced glycolysis. Because of CPT1C's normally brain-restricted expression and the inability of most drugs to pass the blood-brain barrier, CPT1C may be an ideal candidate for specific small-molecule inhibition. We further speculate that concurrent targeting of CPT1C activity and glycolysis in tumor cells could be a highly effective anticancer approach.

Generation and characterization of the Anp32e-deficient mouse.

BACKGROUND: Accumulated literature suggests that the acidic nuclear phosphoprotein 32 kilodalton (Anp32) proteins control multiple cellular activities through different molecular mechanisms. Like other Anp32 family members, Anp32e (a.k.a. Cpd1, PhapIII) has been conserved throughout vertebrate evolution, suggesting that it has an important function in organismal survival. PRINCIPAL FINDINGS: Here, we demonstrate that the Anp32e gene can be deleted in mice without any apparent effect on their wellbeing. No defects in thymocyte apoptosis in response to various stresses, fibroblast growth, gross behaviour, physical ability, or pathogenesis were defined. Furthermore, combined deletion of Anp32a and Anp32e also resulted in a viable and apparently healthy mouse. SIGNIFICANCE: These results provide evidence that significant functional redundancy exists among Anp32 family members.

Cross-talk between Chk1 and Chk2 in double-mutant thymocytes.

Chk1 is a checkpoint kinase and an important regulator of mammalian cell division. Because null mutation of Chk1 in mice is embryonic lethal, we used the Cre-loxP system and the Lck promoter to generate conditional mutant mice in which Chk1 was deleted only in the T lineage. In the absence of Chk1, the transition of CD4(-)CD8(-) double-negative (DN) thymocytes to CD4(+)CD8(+) double-positive (DP) cells was blocked due to an increase in apoptosis at the DN2 and DN3 stages. Strikingly, loss of Chk1 activated the checkpoint kinase Chk2 as well as the tumor suppressor p53 in these thymocytes. However, the developmental defects caused by Chk1 deletion were not rescued by p53 inactivation. Significantly, even though Chk1 deletion is highly lethal in proliferating tissues, we succeeded in using in vivo methods to generate Chk1/Chk2 double-knockout T cells. Analysis of these T cells revealed an interesting interaction between Chk1 and Chk2 functions that partially rescued the apoptosis of the double-mutant cells. Thus, Chk1 is both critical for the survival of proliferating cells and engages in cross-talk with the Chk2 checkpoint kinase pathway. These factors have implications for the targeting of Chk1 as an anticancer therapy.