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Genome-wide association studies identified single nucleotide polymorphisms on chromosome 7 upstream of KLF14 to be associated with metabolic syndrome traits and increased risk for Type 2 Diabetes (T2D). The associations were more significant in women than in men. The risk allele carriers expressed lower levels of the transcription factor KLF14 in adipose tissues than non-risk allele carriers. To investigate how adipocyte KLF14 regulates metabolic traits in a sex-dependent manner, we characterized high-fat diet fed male and female mice with adipocyte-specific Klf14 deletion or overexpression. Klf14 deletion resulted in increased fat mass in female mice and decreased fat mass in male mice. Female Klf14-deficient mice had overall smaller adipocytes in subcutaneous fat depots but larger adipocytes in parametrial depots, indicating a shift in lipid storage from subcutaneous to visceral fat depots. They had reduced metabolic rates and increased respiratory exchange ratios consistent with increased utilization of carbohydrates as an energy source. Fasting and isoproterenol-induced adipocyte lipolysis was defective in female Klf14-deficient mice and concomitantly adipocyte triglycerides lipase mRNA levels were downregulated. Female Klf14-deficient mice cleared blood triglyceride and NEFA less efficiently than wild type. Finally, adipocyte-specific overexpression of Klf14 resulted in lower total body fat in female but not male mice. Taken together, consistent with human studies, adipocyte KLF14 deficiency in female but not in male mice causes increased adiposity and redistribution of lipid storage from subcutaneous to visceral adipose tissues. Increasing KLF14 abundance in adipocytes of females with obesity and T2D may provide a novel treatment option to alleviate metabolic abnormalities.

Integrin CD103 mediates the adhesion and tissue retention of T cells by binding to E-cadherin which is abundant on epithelial cells. Notably, CD103 is highly expressed on CD8 T cells but conspicuously absent on most CD4 T cells. The mechanism controlling such lineage-specific expression of CD103 remains unclear. Using a series of genetically engineered mouse models, here, we demonstrate that the regulatory mechanism of CD103 expression is distinct between CD4 and CD8 T cells, and that the transcription factor Runx3 plays an important but not an essential role in this process. We further found that the availability of integrin β7 which heterodimerizes with CD103 was necessary but also constrained the surface expression of CD103. Notably, the forced surface expression of CD103 did not significantly alter the thymic development of conventional T cells but severely impaired the generation of MHC-II-restricted TCR transgenic T cells, revealing previously unappreciated aspects of CD103 in the selection and maturation of CD4 T cells. Unlike its effect on CD4 T cell development, however, CD103 overexpression did not significantly affect CD4 T cells in peripheral tissues. Moreover, the frequency and number of CD4 T cells in the small intestine epithelium did not increase even though E-cadherin is highly expressed in this tissue. Collectively, these results suggest that most mature CD4 T cells are refractory to the effects of CD103 expression, and that they presumably utilize CD103-independent pathways to control their tissue retention and residency.

Adult mammalian hematopoiesis is a dynamic cellular process that provides a continuous supply of myeloid, lymphoid, erythroid/megakaryocyte cells for host survival. This process is sustained by regulating hematopoietic stem cells (HSCs) quiescence, proliferation and activation under homeostasis and stress, and regulating the proliferation and differentiation of downstream multipotent progenitor (MPP) and more committed progenitor cells. Inhibitor of DNA binding (ID) proteins are small helix-loop-helix (HLH) proteins that lack a basic (b) DNA binding domain present in other family members, and function as dominant-negative regulators of other bHLH proteins (E proteins) by inhibiting their transcriptional activity. ID proteins are required for normal T cell, B cell, NK and innate lymphoid cells, dendritic cell, and myeloid cell differentiation and development. However, recent evidence suggests that ID proteins are important regulators of normal and leukemic hematopoietic stem and progenitor cells (HSPCs). This chapter will review our current understanding of the function of ID proteins in HSPC development and highlight future areas of scientific investigation.

One of the varied tasks of the Large Animal Rescue Service Switzerland and Liechtenstein (GTRD CH/FL)® is the recovery of animals from manure and cesspools. The aim of the present retrospective study was the evaluation of the rescue protocols of the GTRD CH/FL from such operations and the documentation of a rescue procedure. In the past 25 years, a total of 176 animals have been rescued from manure and cesspools. These included 113 cattle, 51 horses and 12 pigs. All animals could be safely rescued with the animal rescue and transport net (TBTN) or the large animal vertical rescue set (GTVBS). The TBTN is used when the opening of the cesspool is large enough to recover the animal in a horizontal position. The GTVBS is particularly suitable for narrow openings, as the recovery in a -vertical position does not require any constructional modification or the enlargement of the cesspool opening. Both rescue harnesses are characterized by reliable handling and allow gentle recovery.Eine der vielfältigen Aufgaben des Grosstier-Rettungsdienstes Schweiz und Liechtenstein (GTRD CH/FL)® ist die Bergung von Tieren aus Mist- und Jauchegruben. Ziel der vorliegenden retrospektiven Studie war die Auswertung der Bergungsprotokolle des GTRD CH/FL aus solchen Einsätzen und die Dokumentation eines Bergungsablaufs. In den vergangenen 25 Jahren sind insgesamt 176 Tiere aus Mist- und Jauchegruben geborgen worden. Darunter befanden sich 113 Rinder, 51 Pferde und 12 Schweine. Alle Tiere konnten sicher mit dem Tier- Bergungs- und Transportnetz (TBTN) oder dem Grosstier- Vertikalbergungsset (GTVBS) geborgen werden. Das TBTN wird eingesetzt, wenn die Öffnung der Jauchegrube gross genug ist, um das Tier in horizontaler Position zu bergen. Bei engen Öffnungen eignet sich das GTVBS vorzüglich, da durch die Bergung in vertikaler Position die Öffnungen der Gruben nicht mehr vergrössert oder durch andere bauliche Massnahmen verändert werden müssen. Beide Bergungsgeschirre zeichnen sich durch eine zuverlässige Handhabung aus und erlauben eine schonende Bergung.L’une des multiples tâches du Service de sauvetage des grands animaux de Suisse et du Liechtenstein (GTRD CH/FL)® est de sortir des animaux de fosses à fumier ou à lisier. Le but de la présente étude rétrospective était l’évaluation des protocoles de sauvetage du GTRD CH/FL sur la base de ces opérations et la documentation d’un processus de sauvetage. Au cours des 25 dernières années, 176 animaux au total ont été tirés de fosses à fumier ou à lisier. Il s’agissait de 113 bovins, 51 chevaux et 12 porcs. Tous les animaux ont pu être secourus en toute sécurité grâce au filet de sauvetage et de transport des animaux (TBTN) ou à l’équipement de sauvetage vertical pour grands animaux (GTVBS). Le TBTN est utilisé lorsque l’ouverture de la fosse à lisier est suffisamment grande pour récupérer l’animal en position horizontale. Le GTVBS est particulièrement adapté aux ouvertures étroites, car la récupération en position verticale permet de ne pas devoir ou modifier modifiées par d’autres mesures structurelles les ouvertures des fosses. Les deux harnais de sauvetage se caractérisent par une manipulation fiable et permettent une récupération en douceur.Uno degli svariati compiti del Servizio di salvataggio di grandi animali di Svizzera e Liechtenstein (GTRD CH/FL)® è il soccorso di animali intrappolati nelle fosse di letame e liquame. Lo scopo di questo studio retrospettivo era di valutare i protocolli di salvataggio del GTRD CH/FL per tali missioni e di documentarne la procedura di salvataggio. Negli ultimi 25 anni, un totale di 176 animali sono stati recuperati dalle fosse di letame e liquami. Tra questi salvataggi si trovavano 113 bovini, 51 cavalli e 12 suini. Tutti gli animali sono stati salvati con sicurezza utilizzando la rete di salvataggio e trasporto animali (TBTN) o il set di recupero verticale per grandi animali (GTVBS). L’imbracatura TBTN viene utilizzata quando l’apertura della fossa di liquame è abbastanza grande per poter recuperare l’animale in posizione orizzontale. In caso di aperture strette, il GTVBS è molto meglio, perché il salvataggio in posizione verticale significa che le aperture delle fosse non devono essere allargate o modificate con altre misure strutturali. Entrambe le imbracature di recupero sono contraddistinte per la loro maneggevolezza e affidabilità che permette un salvataggio in modo gentile dell’animale.

Supporting the recovery of large carnivores is a popular yet challenging endeavour. Estuarine crocodiles in Australia are a large carnivore conservation success story, with the population having extensively recovered from past heavy exploitation. Here, we explored if dietary changes had accompanied this large population recovery by comparing the isotopes δ 13 C and δ 15 N in bones of crocodiles sampled 40 to 55 years ago (small population) with bones from contemporary individuals (large population). We found that δ 13 C and δ 15 N values were significantly lower in contemporary crocodiles than in the historical cohort, inferring a shift in prey preference away from marine and into terrestrial food webs. We propose that an increase in intraspecific competition within the recovering crocodile population, alongside an increased abundance of feral ungulates occupying the floodplains, may have resulted in the crocodile population shifting to feed predominantly upon terrestrial food sources. The number of feral pigs consumed to sustain and grow crocodile biomass may help suppress pig population growth and increase the flow of terrestrially derived nutrients into aquatic ecosystems. The study highlights the significance of prey availability in contributing to large carnivore population recovery.

Like all taxa, populations of aquatic insects may respond to climate change by evolving new physiologies or behaviors, shifting their range, exhibiting physiological and behavioral plasticity, or going extinct. We evaluated the importance of plasticity by measuring changes in growth, survival and respiratory phenotypes of salmonfly nymphs (the stonefly Pteronarcys californica) in response to experimental combinations of dissolved oxygen and temperature. Overall, smaller individuals grew more rapidly during the 6-week experimental period, and oxygen and temperature interacted to affect growth in complex ways. Survival was lower for the warm treatment, although only four mortalities occurred (91.6% versus 100%). Nymphs acclimated to warmer temperatures did not have higher critical thermal maxima (CTmax), but those acclimated to hypoxia had CTmax values (in normoxia) that were higher by approximately 1°C. These results suggest possible adaptive plasticity of systems for taking up or delivering oxygen. We examined these possibilities by measuring the oxygen sensitivity of metabolic rates and the morphologies of tracheal gill tufts located ventrally on thoracic segments. Mass-specific metabolic rates of individuals acclimated to warmer temperatures were higher in acute hypoxia but lower in normoxia, regardless of their recent history of oxygen exposure during acclimation. The morphology of gill filaments, however, changed in ways that appeared to depress rates of oxygen delivery in functional hypoxia. Our combined results from multiple performance metrics indicate that rising temperatures and hypoxia may interact to magnify the risks to aquatic insects, but that physiological plasticity in respiratory phenotypes may offset some of these risks.

Lipid perturbations contribute to detrimental outcomes in obesity. We previously demonstrated that nervonic acid, a C24:1 ω-9 fatty acid, predominantly acylated to sphingolipids, including ceramides, are selectively reduced in a mouse model of obesity. It is currently unknown if deficiency of nervonic acid-sphingolipid metabolites contribute to complications of obesity. Mice were fed a standard diet, a high fat diet, or these diets supplemented isocalorically with nervonic acid. The primary objective was to determine if dietary nervonic acid content alters the metabolic phenotype in mice fed a high fat diet. Furthermore, we investigated if nervonic acid alters markers of impaired fatty acid oxidation in the liver. We observed that a nervonic acid-enriched isocaloric diet reduced weight gain and adiposity in mice fed a high fat diet. The nervonic acid enrichment led to increased C24:1-ceramides and improved several metabolic parameters including blood glucose levels, and insulin and glucose tolerance. Mechanistically, nervonic acid supplementation increased PPARα and PGC1α expression and improved the acylcarnitine profile in liver. These alterations indicate improved energy metabolism through increased β-oxidation of fatty acids. Taken together, increasing dietary nervonic acid improves metabolic parameters in mice fed a high fat diet. Strategies that prevent deficiency of, or restore, nervonic acid may represent an effective strategy to treat obesity and obesity-related complications.

Significance ICF4 is an inherited disease with early mortality due to immunodeficiency. Although the genetic cause is known to be a mutation in the Lsh (lymphoid-specific helicase) gene, it remains unknown why patients suffer from an impaired immune response. Using conditional Lsh knockout mouse models, we provide evidence that Lsh has a hematopoietic cell-intrinsic role in promoting early B cell development and that deficient B cells fail to conduct the step of DNA end joining that is required to yield a variety of immunoglobulin isotypes during class switch recombination. Our data uncover an essential role of Lsh/HELLS in immunoglobulin production which could lead to improved therapeutic options to treat ICF4 patients., Mutation of HELLS (Helicase, Lymphoid-Specific)/Lsh in human DNA causes a severe immunodeficiency syndrome, but the nature of the defect remains unknown. We assessed here the role of Lsh in hematopoiesis using conditional Lsh knockout mice with expression of Mx1 or Vav Cre-recombinase. Bone marrow transplantation studies revealed that Lsh depletion in hematopoietic stem cells severely reduced B cell numbers and impaired B cell development in a hematopoietic cell-autonomous manner. Lsh-deficient mice without bone marrow transplantation exhibited lower Ig levels in vivo compared to controls despite normal peripheral B cell numbers. Purified B lymphocytes proliferated normally but produced less immunoglobulins in response to in vitro stimulation, indicating a reduced capacity to undergo class switch recombination (CSR). Analysis of germline transcripts, examination of double-stranded breaks using biotin-labeling DNA break assay, and End-seq analysis indicated that the initiation of the recombination process was unscathed. In contrast, digestion–circularization PCR analysis and high-throughput sequencing analyses of CSR junctions and a chromosomal break repair assay indicated an impaired ability of the canonical end-joining pathway in Lsh-deficient B cells. Our data suggest a hematopoietic cell-intrinsic role of Lsh in B cell development and in CSR providing a potential target for immunodeficiency therapy.

Angiotensin II (Ang II) type-2 receptors (AT2R) are expressed in the adult kidney, prominently in renal proximal tubule cells (RPTCs), and play an important role in opposing renal sodium (Na+) retention induced by Ang II stimulation of Ang II type-1 receptor (AT1R). Natriuresis induced by AT1R blockade is due at least in part to AT2R activation and whole body deletion of AT2Rs reduces the natriuretic response to increased blood pressure (BP). The major endogenous AT2R agonist mediating the natriuretic response is Ang III, the Ang II heptapeptide metabolite generated by aminopeptidase A, and the principal nephron site mediating inhibition of Na+ reabsorption by the AT2R is the renal proximal tubule (RPT). AT2Rs induce natriuresis via a bradykinin, nitric oxide and cyclic GMP (cGMP) signaling cascade. Recent studies demonstrated a key role for protein phosphatase 2A (PP2A) in the AT2R-mediated natriuretic response upstream of cGMP. By inducing natriuresis, AT2Rs lower BP in the Ang II-infusion model of hypertension. PP2A activation and the natriuretic response to AT2R stimulation are defective in spontaneously hypertensive rats, a model of primary hypertension in humans. AT2R agonists are candidates for proximal tubule natriuretic agents in Na+ and fluid retention disorders.

BACKGROUND: How signals from activated angiotensin type-2 receptors (AT(2)R) mediate inhibition of sodium ion (Na(+)) reabsorption in renal proximal tubule cells (RPTCs) is currently unknown. Protein phosphatases including protein phosphatase 2A (PP2A) have been implicated in AT(2)R signaling in tissues other than kidney. We investigated whether inhibition of protein phosphatase PP2A reduced AT(2)R-mediated natriuresis and evaluated changes in PP2A activity and localization after renal AT(2)R activation in normal 4- and 10-week-old control Wistar-Kyoto rats (WKY) and 4-week-old pre-hypertensive and 10-week-old hypertensive spontaneously hypertensive rats (SHR). METHODS AND RESULTS: In WKY, direct renal interstitial (RI) administration of selective AT(2)R non-peptide agonist Compound-21 (C-21) increased RI cyclic GMP (cGMP) levels, urine Na(+) excretion (U(Na)V), and simultaneously increased PP2A activity ≅ 2-fold in homogenates of renal cortical tubules. The cGMP and natriuretic responses were abolished by concurrent RI administration of protein phosphatase inhibitor calyculin A (CAL). In RPTCs in response to C-21, PP2A subunits A, B55α and C, but not B56γ, were recruited to apical plasma membranes together with AT(2)Rs. CAL treatment abolished C-21-induced translocation of both AT(2)R and PP2A regulatory subunit B55α to apical plasma membranes. Immunoprecipitation of AT(2)R solubilized from renal cortical homogenates demonstrated physical association of AT(2)R with PP2A A, B55α, and C but not B56γ subunits. In contrast, in SHR, administration of C-21 did not alter U(Na)V or PP2A activity and failed to translocate AT(2)Rs and PP2A subunits to apical plasma membranes. CONCLUSIONS: In RPTCs of WKY, PP2A is activated and PP2A subunits AB55αC are recruited to C-21-activated AT(2)Rs during induction of natriuresis. This response is defective in pre-hypertensive and hypertensive SHR, presenting a potential novel therapeutic target for treating renal Na(+) retention and hypertension.

Foxp3+ Tregs are potent immunosuppressive CD4+ T cells that are critical to maintain immune quiescence and prevent autoimmunity. Both the generation and maintenance of Foxp3+ Tregs depend on the cytokine IL-2. Hence, the expression of the IL-2 receptor α-chain (CD25) is not only considered a specific marker, but also a nonredundant requirement for Tregs. Here, we report that Foxp3+ Tregs in the small intestine (SI) epithelium, a critical barrier tissue, are exempt from such an IL-2 requirement, since they had dramatically downregulated CD25 expression, showed minimal STAT5 phosphorylation ex vivo, and were unable to respond to IL-2 in vitro. Nonetheless, SI epithelial Tregs survived and were present at the same frequency as in other lymphoid organs, and they retained potent suppressor function that was associated with high levels of CTLA-4 expression and the production of copious amounts of IL-10. Moreover, adoptive transfer experiments of Foxp3+ Tregs revealed that such IL-2–independent survival and effector functions were imposed by the SI epithelial tissue, suggesting that tissue adaptation is a mechanism that tailors the effector function and survival requirements of Foxp3+ Tregs specific to the tissue environment.

The interaction between tumor suppressor BRCA2 and DSS1 is essential for RAD51 recruitment and repair of DNA double stand breaks (DSBs) by homologous recombination (HR). We have generated mice with a leucine to proline substitution at position 2431 of BRCA2, which disrupts this interaction. Although a significant number of mutant mice die during embryogenesis, some homozygous and hemizygous mutant mice undergo normal postnatal development. Despite lack of radiation induced RAD51 foci formation and a severe HR defect in somatic cells, mutant mice are fertile and exhibit normal RAD51 recruitment during meiosis. We hypothesize that the presence of homologous chromosomes in close proximity during early prophase I may compensate for the defect in BRCA2-DSS1 interaction. We show the restoration of RAD51 foci in mutant cells when Topoisomerase I inhibitor-induced single strand breaks are converted into DSBs during DNA replication. We also partially rescue the HR defect by tethering the donor DNA to the site of DSBs using streptavidin-fused Cas9. Our findings demonstrate that the BRCA2-DSS1 complex is dispensable for RAD51 loading when the homologous DNA is close to the DSB.

ATP6AP2 expression is increased in the nephron during high-fat diet (HFD) and its knockout (ATP6AP2 KO) reduces body weight (WT) in mice. We evaluated the contribution of ATP6AP2 to urinary glucose (UG) and albumin (Ualb) handling during HFD. We hypothesized that nephron ATP6AP2 KO increases UG and Ualb and minimizes HFD-induced obesity. Eight-week-old male C57BL/6J mice with inducible nephron-specific ATP6AP2 KO and noninduced controls were fed either normal diet (ND, 12% kcal fat) or HFD (45% kcal fat) for 6 months. ATP6AP2 KO mice on ND had 20% (P < 0.01) lower WT compared with controls. HFD-fed mice had 41% (P < 0.05) greater WT than ND-fed control mice. In contrast, ATP6AP2 KO abrogated the increase in WT induced by HFD by 40% (P < 0.05). Mice on HFD had less caloric intake compared with ND controls (P < 0.01). There were no significant differences in metabolic rate between all groups. UG and Ualb was significantly increased in ATP6AP2 KO mice on both ND and HFD. ATP6AP2 KO showed greater levels of proximal tubule apoptosis and histologic evidence of proximal tubule injury. In conclusion, our results demonstrate that nephron-specific ATP6AP2 KO is associated with glucosuria and albuminuria, most likely secondary to renal proximal tubule injury and/or dysfunction. Urinary loss of nutrients may have contributed to the reduced WT of knockout mice on ND and lack of WT gain in response to HFD. Future investigation should elucidate the mechanisms by which loss of renal ATP6AP2 causes proximal tubule injury and dysfunction.

Serine palmitoyltransferase (SPT) long-chain base subunit 1 (SPTLC1) is 1 of the 2 main catalytic subunits of the SPT complex, which catalyzes the first and rate-limiting step of sphingolipid biosynthesis. Here, we show that Sptlc1 deletion in adult bone marrow (BM) cells results in defective myeloid differentiation. In chimeric mice from noncompetitive BM transplant assays, there was an expansion of the Lin(−) c-Kit(+) Sca-1(+) compartment due to increased multipotent progenitor production, but myeloid differentiation was severely compromised. We also show that defective biogenesis of sphingolipids in the endoplasmic reticulum (ER) leads to ER stress that affects myeloid differentiation. Furthermore, we demonstrate that transient accumulation of fatty acid, a substrate for sphingolipid biosynthesis, could be partially responsible for the ER stress. Independently, we find that ER stress in general, such as that induced by the chemical thapsigargin or the fatty acid palmitic acid, compromises myeloid differentiation in culture. These results identify perturbed sphingolipid metabolism as a source of ER stress, which may produce diverse pathological effects related to differential cell-type sensitivity.

An organism’s capacity to cope with stressful experiences is dependent on its ability to appropriately engage central and peripheral systems, such as the hypothalamic-pituitary-adrenal (HPA) axis, to adapt to changing environmental demands. The HPA axis is a primary neuroendocrine mediator of neural and behavioral responses to stress, and dysfunction of this system is linked to increased risk for developing mental health disorders such as depression, anxiety, and post-traumatic stress disorder. However, the mechanisms by which dysregulated HPA function results in abnormal behavioral responses to stress are poorly understood. Here, we tested how corticosterone (CORT)-induced HPA axis disruption affects behavioral responses to stress in male C57BL/6 N mice, and probed correlates of these behaviors in the brain. We show that chronic HPA disruption blunts acute stress-induced grooming and rearing behaviors in the open field test, effects which were accompanied by decreased FOS immunoreactivity in the paraventricular nucleus of the hypothalamus (PVH) and paraventricular nucleus of the thalamus (PVT). Blockade of CORT secretion with metyrapone injection prior to acute stress did not recapitulate the effects of chronic HPA disruption on open field behavior, and acute CORT replacement did not rescue normal behavioral stress responses following chronic HPA disruption. This suggests that under acute conditions, CORT is not necessary for these responses normally, nor sufficient to rescue the deficits of chronic HPA dysregulation. Together, these findings support the hypothesis that chronic HPA dysregulation causes adaptation in stress-related brain circuits and demonstrate that these changes can influence an organism’s behavioral response to stress exposure.

Objective Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of β-adrenergic receptor (βAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis. Methods In an immortalized brown pre-adipocytes cell line, Panx1 currents were measured using patch-clamp electrophysiology. Flow cytometry was used for assessment of dye uptake and luminescence assays for adenosine triphosphate (ATP) release, and cellular temperature measurement was performed using a ratiometric fluorescence thermometer. We used RNA interference and expression plasmids to manipulate expression of wild-type and mutant Panx1. We used previously described adipocyte-specific Panx1 knockout mice (Panx1Adip-/-) and generated brown adipocyte-specific Panx1 knockout mice (Panx1BAT-/-) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of 18F-fluorodeoxyglucose (18F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe. Results In brown adipocytes, Panx1 channel activity was induced either by apoptosis-dependent caspase activation or by β3AR stimulation via a novel mechanism that involves Gβγ subunit binding to Panx1. Inactivation of Panx1 channels in cultured brown adipocytes resulted in inhibition of β3AR-induced lipolysis, UCP-1 expression, and cellular thermogenesis. In mice, adiponectin-Cre-dependent genetic deletion of Panx1 in all adipose tissue depots resulted in defective β3AR agonist- or cold-induced thermogenesis in BAT and suppressed beigeing of white adipose tissue. UCP1-Cre-dependent Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis without affecting beigeing of white adipose tissue and aggravated diet-induced obesity and insulin resistance. Conclusions These data demonstrate that Gβγ-dependent Panx1 channel activation is involved in β3AR-induced thermogenic regulation in brown adipocytes. Identification of Panx1 channels in BAT as novel thermo-regulatory elements downstream of β3AR activation may have therapeutic implications., Highlights • Panx1 channel activation plays a critical role in thermogenic regulation in brown adipocytes. • Panx1 channel activity in brown adipocytes was induced by β3 adrenergic receptor stimulation. • Inactivation of Panx1 channels in cultured brown adipocytes resulted in defective cellular thermogenesis. • In mice, genetic deletion of Panx1 in all adipose tissue depots resulted in defective β3AR agonist- or cold-induced thermogenesis. • Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis and aggravated diet-induced obesity and insulin resistance in mice.

Stress hyperglycemia and insulin resistance are evolutionarily conserved metabolic adaptations to severe injury including major trauma, burns, or hemorrhagic shock (HS). In response to injury, the neuroendocrine system increases secretion of counterregulatory hormones that promote rapid mobilization of nutrient stores, impair insulin action, and ultimately cause hyperglycemia, a condition known to impair recovery from injury in the clinical setting. We investigated the contributions of adipocyte lipolysis to the metabolic response to acute stress. Both surgical injury with HS and counterregulatory hormone (epinephrine) infusion profoundly stimulated adipocyte lipolysis and simultaneously triggered insulin resistance and hyperglycemia. When lipolysis was inhibited, the stress-induced insulin resistance and hyperglycemia were largely abolished demonstrating an essential requirement for adipocyte lipolysis in promoting stress-induced insulin resistance. Interestingly, circulating non-esterified fatty acid levels did not increase with lipolysis or correlate with insulin resistance during acute stress. Instead, we show that impaired insulin sensitivity correlated with circulating levels of the adipokine resistin in a lipolysis-dependent manner. Our findings demonstrate the central importance of adipocyte lipolysis in the metabolic response to injury. This insight suggests new approaches to prevent insulin resistance and stress hyperglycemia in trauma and surgery patients and thereby improve outcomes.

This study aimed to investigate the links between parasites and behavioural traits of juvenile Murray cod (Maccullochella peelii). The Murray cod is an endangered Australian freshwater fish for which restocking programs are in place and there is a growing human consumption market. However, little is known about the parasites of these fish and how these parasites influence their behaviour and survival. Fingerlings and yearling fish were sourced from a hatchery and the wild, and after acclimatisation in the laboratory, variation in behavioural traits was examined using emergence, exploration and predator inspection tests. The fish were then euthanised to determine their age and examined for infection with parasites. Wild fish had more camallanid nematodes and lernaeid copepods than hatchery fish. An information theoretic approach using Akaike's Information Criterion (AIC) indicated that infection with protozoan cysts was an important factor for predicting the latency to emerge and explore a new environment, which was interpreted as reduced "boldness". In contrast, the presence of lernaeid copepods was included in two of the four best models predicting predator inspection, indicating that infected fish were less likely to inspect a predator. Source of fish (wild or hatchery) was found to be a strong influence on behavioural responses in all our tests. All parasites found in the present study are known to result in clinical signs of diseases in their fish hosts, raising the possibility that responses in tests of behavioural traits reflect side effects of infection. Additionally, the effect of host adaptation to not show signs of parasite infection, or more simply that the effects on behaviour are subtle and difficult to reveal with small sample sizes, is discussed. Nonetheless, we propose that it is important that infection with parasites is considered in fish behavioural studies both to assess survival behaviour and to avoid misinterpretation of behavioural tests of animal personality.

Serine palmitoyltransferase complex (SPT) mediates the first and rate-limiting step in the de novo sphingolipid biosynthetic pathway. The larger subunits SPTLC1 and SPTLC2/SPTLC3 together form the catalytic core while a smaller third subunit either SSSPTA or SSSPTB has been shown to increase the catalytic efficiency and provide substrate specificity for the fatty acyl-CoA substrates. The in vivo biological significance of these smaller subunits in mammals is still unknown. Here, using two null mutants, a conditional null for ssSPTa and a null mutant for ssSPTb, we show that SSSPTA is essential for embryogenesis and mediates much of the known functions of the SPT complex in mammalian hematopoiesis. The ssSPTa null mutants are embryonic lethal at E6.5 much like the Sptlc1 and Sptlc2 null alleles. Mx1-Cre induced deletion of ssSPTa leads to lethality and myelopoietic defect. Chimeric and competitive bone marrow transplantation experiments show that the defect in myelopoiesis is accompanied by an expansion of the Lin−Sca1+c-Kit+ stem and progenitor compartment. Progenitor cells that fail to differentiate along the myeloid lineage display evidence of endoplasmic reticulum stress. On the other hand, ssSPTb null mice are homozygous viable, and analyses of the bone marrow cells show no significant difference in the proliferation and differentiation of the adult hematopoietic compartment. SPTLC1 is an obligatory subunit for the SPT function, and because Sptlc1−/− and ssSPTa−/− mice display similar defects during development and hematopoiesis, we conclude that an SPT complex that includes SSSPTA mediates much of its developmental and hematopoietic functions in a mammalian model.

Lymphopenia-induced homeostatic proliferation (LIP) is a critical mechanism for restoring T cell immunity upon lymphodepleting insults or infections. LIP is primarily driven by homeostatic cytokines, such as IL-7 and IL-15, but not all T cells respond with the same efficiency to homeostatic proliferative cues. Although CD8 T cells vigorously proliferate under lymphopenic conditions, naive CD4 T cells are substantially impaired in their response to homeostatic cytokines, and they fail to fully expand. In this study, we show that the availability of IL-2Rβ (CD122), which is a receptor subunit shared by IL-2 and IL-15, affects both the cytokine responsiveness and the LIP of naive CD4 T cells in the mouse. The enumeration of surface IL-2Rβ molecules on murine naive CD4 and naive CD8 T cells revealed a 5-fold difference in IL-2Rβ abundance. Notably, it was the limited availability of IL-2Rβ that impaired CD4 T cell responsiveness to IL-15 and suppressed their LIP. As such, forced IL-2Rβ expression on CD4 T cells by transgenesis bestowed IL-15 responsiveness onto naive CD4 T cells, which thus acquired the ability to undergo robust LIP. Collectively, these results identify IL-2Rβ availability as a new regulatory mechanism to control cytokine responsiveness and the homeostatic proliferation of murine CD4 T cells.

SOCS3 is a cytosolic inhibitor of cytokine signaling that suppresses the activation of cytokine receptor-associated JAK kinases. Mechanistically, SOCS3 is recruited to a site in the cytokine receptors known as the SOCS3-interaction motif, and then binds JAK molecules to inhibit their kinase activity. The SOCS3-interaction motif is found in receptors of the gp130 cytokine family but mostly absent from other cytokine receptors, including γc. Thus, SOCS3 has been considered a selective suppressor of gp130 family cytokines, but not γc cytokines. Considering that γc signaling induces SOCS3 expression in T cells, here we revisited the role of SOCS3 on γc signaling. Using SOCS3 transgenic mice, we found that increased abundance of SOCS3 not only suppressed signaling of the gp130 family cytokine IL-6, but also signaling of the γc family cytokine IL-7. Consequently, SOCS3 transgenic mice were impaired in IL-7-dependent T cell development in the thymus and the homeostasis of mature T cells in peripheral tissues. Moreover, enforced SOCS3 expression interfered with the generation of Foxp3(+) regulatory T cells which requires signaling by the γc family cytokine IL-2. Collectively, we report an underappreciated role for SOCS3 in suppressing γc cytokine signaling, effectively expanding its scope of target cytokines in T cell immunity.

Rabbits are commonly used for sciatic nerve injuries larger than 1.5 cm. This report provides insight into risks and benefits associated with using rabbit models in sciatic nerve injury models and proposes interventions that researchers can use to prevent experimental complications. Fifty-six rabbits from a sciatic nerve injury study that involved a 40 mm sciatic nerve injury were analyzed to examine postoperative complication rates. Autophagy of the phalanges and plantar pressure ulcer development were the most common and serious complications faced. These complications led to 23.2% ( n = 13) of rabbits not being used for data in the original experiment due to euthanasia outside of intended postoperative time points. This increased the cost needed to complete the experiment by $25,038.44. It is our recommendation that alternative models be used instead of rabbits for sciatic nerve injuries. If rabbits must be used, a treatment protocol for preventing autophagy and pressure ulcers is outlined below.

RATIONALE: Previous studies identified a defect in angiotensin III (Ang III)-elicited AT(2) receptor (AT(2)R)-mediated natriuresis in renal proximal tubule cells (RPTCs) of spontaneously hypertensive rats (SHR). OBJECTIVE: This study aimed to delineate in pre-hypertensive SHR kidneys the receptor and/or post-receptor defect causing impaired AT(2)R signaling and renal sodium (Na(+)) retention by utilizing the selective AT(2)R agonist Compound 21 (C-21). METHODS AND RESULTS: Female 4-week-old Wistar-Kyoto (WKY) and SHR rats were studied after 24h systemic AT(1) receptor (AT(1)R) blockade. Left kidneys received 30 min renal interstitial (RI) infusions of vehicle followed by C-21 (20, 40, and 60 ng/kg/min, each dose 30 min). Right kidneys received vehicle infusions. In WKY, C-21 dose-dependently increased urine Na(+) excretion (U(Na)V) from 0.023±0.01 to 0.064±0.02, 0.087±0.01, and 0.089±0.01 μmol/min (P=0.008, P

The actin cytoskeleton plays a central role in establishing cell polarity and shape during embryonic morphogenesis. Daam1, a member of the Formin family of actin cytoskeleton regulators, is a Dvl2-binding protein that functions in the Wnt/Planar Cell Polarity (PCP) pathway. To examine the role of the Daam proteins in mammalian development, we generated Daam-deficient mice by gene targeting and found that Daam1, but not Daam2, is necessary for fetal survival. Embryonic development of Daam1 mutants was delayed most likely due to functional defects in the labyrinthine layer of the placenta. Examination of Daam2 and Daam1/2 double mutants revealed that Daam1 and Daam2 are functionally redundant during placental development. Of note, neural tube closure defects (NTD), which are observed in several mammalian PCP mutants, are not observed in Wnt5a or Daam1 single mutants, but arise in Daam1;Wnt5a double mutants. These findings demonstrate a unique function for Daam genes in placental development and are consistent with a role for Daam1 in the Wnt/PCP pathway in mammals.

SUMMARY Fetal globin genes are transcriptionally silenced during embryogenesis through hemoglobin switching. Strategies to derepress fetal globin expression in the adult could alleviate symptoms in sickle cell disease and β-thalassemia. We identified a zinc-finger protein, pogo transposable element with zinc-finger domain (POGZ), expressed in hematopoietic progenitor cells. Targeted deletion of Pogz in adult hematopoietic cells in vivo results in persistence of embryonic β-like globin expression without affecting erythroid development. POGZ binds to the Bcl11a promoter and erythroid-specific intragenic regulatory regions. Pogz+/− mice show elevated embryonic β-like globin expression, suggesting that partial reduction of Pogz expression results in persistence of embryonic β-like globin expression. Knockdown of POGZ in primary human CD34+ progenitor cell-derived erythroblasts reduces BCL11A expression, a known repressor of embryonic β-like globin expression, and increases fetal hemoglobin expression. These findings are significant, since new therapeutic targets and strategies are needed to treat β-globin disorders., In Brief Gudmundsdottir et al. show that POGZ represses embryonic globin gene expression in mouse and human erythroid cells, in part by regulating Bcl11a expression in vitro and in vivo. The molecular pathways regulated by POGZ may represent potential therapeutic targets to increase fetal globin expression in patients with sickle cell disease and β-thalassemia., Graphical Abstract

Several genes implicated in autism spectrum disorder (ASD) are chromatin regulators, including POGZ. The cellular and molecular mechanisms leading to ASD impaired social and cognitive behavior are unclear. Animal models are crucial for studying the effects of mutations on brain function and behavior as well as unveiling the underlying mechanisms. Here, we generate a brain specific conditional knockout mouse model deficient for Pogz, an ASD risk gene. We demonstrate that Pogz deficient mice show microcephaly, growth impairment, increased sociability, learning and motor deficits, mimicking several of the human symptoms. At the molecular level, luciferase reporter assay indicates that POGZ is a negative regulator of transcription. In accordance, in Pogz deficient mice we find a significant upregulation of gene expression, most notably in the cerebellum. Gene set enrichment analysis revealed that the transcriptional changes encompass genes and pathways disrupted in ASD, including neurogenesis and synaptic processes, underlying the observed behavioral phenotype in mice. Physiologically, Pogz deficiency is associated with a reduction in the firing frequency of simple and complex spikes and an increase in amplitude of the inhibitory synaptic input in cerebellar Purkinje cells. Our findings support a mechanism linking heterochromatin dysregulation to cerebellar circuit dysfunction and behavioral abnormalities in ASD.

Animals show various behavioural, neural and physiological changes in response to losing aggressive encounters. Here, we investigated affective state, which are emotion-like processes influenced by positive or negative experiences, in a territorial fish following aggressive encounters and explore links to bold/shy behavioural traits. Eighteen 15-month old Murray cod (Maccullochella peelii) received three tests in order to determine bold/shy behavioural traits then underwent a typical go/no-go judgement bias (JB) test. The JB apparatus had five adjacent chambers with access provided by a sliding door and fish underwent a training procedure to enter a chamber at one end of the apparatus to receive a food reward but were chased using a net if they entered the chamber at the opposite end. Only one third (N = 6) of fish successfully completed the training procedure (trained fish), and the remaining 12 fish failed to reach the learning criterion (untrained fish). Trained fish housed with a larger aggressive Murray cod for 24 h were significantly less likely to enter intermediate chambers during probe tests compared to control fish, demonstrating a pessimistic response. Trained fish showed "bolder" responses in emergence and conspecific inspection tests than untrained fish, suggesting that shyer individuals were less able to apply a learned behaviour in a novel environment. Our limited sample was biased towards bold individuals but supports the hypothesis that losing an aggressive encounter leads to pessimistic decision-making.

Humanity's reliance on clean water and the ecosystem services provided makes identifying efficient and effective ways to assess the ecological condition of streams ever more important. We used high throughput sequencing of the 16S rRNA region to explore relationships between stream microbial communities, environmental attributes, and assessments of stream ecological condition. Bacteria and archaea in microbial community samples collected from the water column and from stream sediments during spring and summer were used to replicate standard assessments of ecological condition performed with benthic macroinvertebrate collections via the Benthic Index of Biotic Integrity (BIBI). Microbe-based condition assessments were generated at different levels of taxonomic resolution from phylum to OTU (Operational Taxonomic Units) in order to understand appropriate levels of taxonomic aggregation. Stream sediment microbial communities from both spring and summer were much better than the water column at replicating BIBI condition assessment results. Accuracies were as high as 100% on training data used to build the models and up to 80% on validation data used to assess predictions. Assessments using all OTUs usually had the highest accuracy on training data, but were lower on validation data due to overfitting. In contrast, assessments at the order-level had similar performance accuracy for validation data, and a reduced subset of orders also performed well, suggesting the method could be generalized to other watersheds. Subsets of the important orders responded similarly to environmental gradients compared to the entire community, where strong shifts in community structure occurred for known aquatic stressors such as pH, dissolved organic carbon, and nitrate nitrogen. The results suggest the stream microbes may be useful for assessing the ecological condition of streams and especially useful for stream restorations where many eukaryotic taxa have been eliminated due to prior degradation and are unable to recolonize.

Background Previous studies demonstrated that angiotensin (Ang) III, not Ang II, is the predominant endogenous agonist for Ang type‐2 receptor (AT 2R)‐induced natriuresis in normal rats, and that hypertensive 12‐week‐old spontaneously hypertensive rats (SHR) lack natriuretic responses to Ang III. This study tested whether prehypertensive SHR already have defective Ang III‐induced natriuresis and determined possible mechanisms. Methods and Results Female and male normotensive 4‐week‐old SHR and Wistar Kyoto rats were studied after 24‐hour systemic AT 1R blockade. Left kidneys received 30 minute renal interstitial infusions of vehicle followed by Ang III (3.5, 7.0, 14, and 28 nmol/kg per min; each dose for 30 minutes). Right kidneys received vehicle infusions. In 4‐week‐old Wistar Kyoto rats, renal interstitial Ang III increased urine sodium (Na+) excretion but failed to induce natriuresis in 4‐week‐old SHR. Renal Ang III levels were similar between Wistar Kyoto rats and SHR, making increased Ang III degradation as a possible cause for defective natriuresis in SHR unlikely. In Wistar Kyoto rats, renal interstitial Ang III induced translocation of AT 2Rs to apical plasma membranes of renal proximal tubule cells. Simultaneously, Ang III induced retraction of the major Na+ transporter Na+‐H+ exchanger‐3 (NHE‐3) from apical membranes and internalization of Na+/K+ ATPase (NKA) from basolateral membranes of renal proximal tubule cells. Consistent with NHE‐3 and NKA retraction, Ang III increased pSer552‐NHE‐3 and decreased pSer23‐NKA. In contrast, in SHR, intrarenal Ang III failed to induce AT 2R translocation, NHE‐3 or NKA retraction, pSer552‐NHE‐3 phosphorylation, or pSer23‐NKA dephosphorylation. Conclusions These results indicate impaired Ang III/AT 2R signaling as a possible primary defect in prehypertensive SHR., See Editorial Zhuo and Li

Background Sustained pressure overload leads to changes in cardiac metabolism, function, and structure. Both time course and causal relationships between these changes are not fully understood. Therefore, we studied spontaneously hypertensive rats (SHR) during early hypertension development and compared them to control Wistar Kyoto rats. Methods and Results We serially evaluated myocardial glucose uptake rates (Ki) with dynamic 2‐[ 18 F] fluoro‐2‐deoxy‐D‐glucose positron emission tomography, and ejection fraction and left ventricular mass to body weight ratios with cardiac magnetic resonance imaging in vivo, determined glucose uptake and oxidation rates in isolated perfused hearts, and analyzed metabolites, mammalian target of rapamycin activity and endoplasmic reticulum stress in dissected hearts. When compared with Wistar Kyoto rats, SHR demonstrated increased glucose uptake rates (Ki) in vivo, and reduced ejection fraction as early as 2 months of age when hypertension was established. Isolated perfused SHR hearts showed increased glucose uptake and oxidation rates starting at 1 month. Cardiac metabolite analysis at 2 months of age revealed elevated pyruvate, fatty acyl‐ and branched chain amino acid‐derived carnitines, oxidative stress, and inflammation. Mammalian target of rapamycin activity increased in SHR beginning at 2 months. Left ventricular mass to body weight ratios and endoplasmic reticulum stress were elevated in 5 month‐old SHR. Conclusions Thus, in a genetic hypertension model, chronic cardiac pressure overload promptly leads to increased myocardial glucose uptake and oxidation, and to metabolite abnormalities. These coincide with, or precede, cardiac dysfunction while left ventricular hypertrophy develops only later. Myocardial metabolic changes may thus serve as early diagnostic markers for hypertension‐induced left ventricular hypertrophy.

BACKGROUND: Diffusion tensor tractography (DTT) has recently been shown to accurately detect nerve injury and regeneration. This study assesses whether 7-tesla (7T) DTT imaging is a viable modality to observe axonal outgrowth in a 4 cm rabbit sciatic nerve injury model fixed by a reverse autograft surgical technique. METHODS: Transection injury of unilateral sciatic nerve (4 cm long) was performed in 25 rabbits and repaired using a reverse autograft (RA) surgical technique. Analysis of the nerve autograft was performed at 3, 6, and 11 weeks postoperatively and compared to normal contralateral sciatic nerve, used as control group. High-resolution DTT from ex vivo sciatic nerves were obtained using 3D diffusion-weighted spin-echo acquisitions at 7-T. Total axons and motor and sensory axons were counted at defined lengths along the graft. RESULTS: At 11 weeks, histologically, the total axon count of the RA group was equivalent to the contralateral uninjured nerve control group. Similarly, by qualitative DTT visualization, the 11-week RA group showed increased fiber tracts compared to the 3 and 6 weeks counterparts. Upon immunohistochemical evaluation, 11-week motor axon counts did not significantly differ between reverse autograft and control; but significantly decreased sensory axon counts remained. Nerves explanted at 3 weeks and 6 weeks showed decreased motor and sensory axon counts. DISCUSSION: 7-T DTT is an effective imaging modality that may be used qualitatively to visualize axonal outgrowth and regeneration. This has implications for the development of technology that non-invasively monitors peripheral nerve regeneration in a variety of clinical settings.

An unanticipated impact of wind-energy development has been large-scale mortality of insectivorous bats. In eastern North America, where mortality rates are among the highest in the world, the hoary bat (Lasiurus cinereus) and the eastern red bat (L. borealis) comprise the majority of turbine-associated bat mortality. Both species are migratory tree bats with widespread distributions; however, little is known regarding the geographic origins of bats killed at wind-energy facilities or the diversity and population structure of affected species. We addressed these unknowns by measuring stable hydrogen isotope ratios (δ2 H) and conducting population genetic analyses of bats killed at wind-energy facilities in the central Appalachian Mountains (USA) to determine the summering origins, effective size, structure, and temporal stability of populations. Our results indicate that ~1% of hoary bat mortalities and ~57% of red bat mortalities derive from non-local sources, with no relationship between the proportion of non-local bats and sex, location of mortality, or month of mortality. Additionally, our data indicate that hoary bats in our sample consist of an unstructured population with a small effective size (Ne ) and either a stable or declining history. Red bats also showed no evidence of population genetic structure, but in contrast to hoary bats, the diversity contained in our red bat samples is consistent with a much larger Ne that reflects a demographic expansion after a bottleneck. These results suggest that the impacts of mortality associated with intensive wind-energy development may affect bat species dissimilarly, with red bats potentially better able to absorb sustained mortality than hoary bats because of their larger Ne . Our results provide important baseline data and also illustrate the utility of stable isotopes and population genetics for monitoring bat populations affected by wind-energy development.

Carcinogen exposures inscribe mutation patterns on cancer genomes and sometimes bias the acquisition of driver mutations toward preferred oncogenes, potentially dictating sensitivity to targeted agents. Whether and how carcinogen-specific mutation patterns direct activation of preferred oncogenes remains poorly understood. Here, mouse models of breast cancer were exploited to uncover a mechanistic link between strand-biased mutagenesis and oncogene preference. When chemical carcinogens were employed during Wnt1-initiated mammary tumorigenesis, exposure to either 7,12-dimethylbenz(a)anthracene (DMBA) or N-ethyl-N-nitrosourea (ENU) dramatically accelerated tumor onset. Mammary tumors that followed DMBA exposure nearly always activated the Ras pathway via somatic Hras(CAA61CTA) mutations. Surprisingly, mammary tumors that followed ENU exposure typically lacked Hras mutations, and instead activated the Ras pathway downstream via Braf(GTG636GAG) mutations. Hras(CAA61CTA) mutations involve an A-to-T change on the sense strand, whereas Braf(GTG636GAG) mutations involve an inverse T-to-A change, suggesting that strand-biased mutagenesis may determine oncogene preference. To examine this possibility further, we turned to an alternative Wnt-driven tumor model in which carcinogen exposures augment a latent mammary tumor predisposition in Apc(min) mice. DMBA and ENU each accelerated mammary tumor onset in Apc(min) mice by introducing somatic, "second-hit" Apc mutations. Consistent with our strand bias model, DMBA and ENU generated strikingly distinct Apc mutation patterns, including stringently strand-inverse mutation signatures at A:T sites. Crucially, these contrasting signatures precisely match those proposed to confer bias toward Hras(CAA61CTA) versus Braf(GTG636GAG) mutations in the original tumor sets. Our findings highlight a novel mechanism whereby exposure history acts through strand-biased mutagenesis to specify activation of preferred oncogenes.

The breast cancer gene, BRCA2, is essential for viability, yet patients with Fanconi anemia-D1 subtype are born alive with biallelic mutations in this gene. The hypomorphic nature of the mutations is believed to support viability, but this is not always apparent. One such mutation is IVS7+2T>G, which causes premature protein truncation due to skipping of exon 7. We previously identified a transcript lacking exons 4–7, which restores the open-reading frame, encodes a DNA repair proficient protein and is expressed in IVS7+2T>G carriers. However, because the exons 4–7 encoded region contains several residues required for normal cell-cycle regulation and cytokinesis, this transcript's ability to support viability can be argued. To address this, we generated a Brca2 knock-in mouse model lacking exons 4–7 and demonstrated that these exons are dispensable for viability as well as tumor-free survival. This study provides the first in vivo evidence of the functional significance of a minor transcript of BRCA2 that can play a major role in the survival of humans who are homozygous for a clearly pathogenic mutation. Our results highlight the importance of assessing protein function restoration by premature truncating codon bypass by alternative splicing when evaluating the functional significance of variants such as nonsense and frame-shift mutations that are assumed to be clearly pathogenic. Our findings will impact not only the assessment of variants that map to this region, but also influence counseling paradigms and treatment options for such mutation carriers.

The related Rab GTPase-activating proteins (Rab GAPs) AS160 and Tbc1d1 regulate the trafficking of the glucose transporter GLUT4 that controls glucose uptake in muscle and fat cells and glucose homeostasis. AS160- and Tbc1d1-deficient mice exhibit different adipocyte- and skeletal muscle-specific defects in glucose uptake, GLUT4 expression and trafficking, and glucose homeostasis. A recent study analyzed male mice with simultaneous deletion of AS160 and Tbc1d1 (AS160−/−/Tbc1d1−/− mice). Herein, we describe abnormalities in male and female AS160−/−/Tbc1d1−/− mice on another strain background. We confirm the earlier observation that GLUT4 expression and glucose uptake defects of single-knockout mice join in AS160−/−/Tbc1d1−/− mice to affect all skeletal muscle and adipose tissues. In large mixed fiber-type skeletal muscles, changes in relative basal GLUT4 plasma membrane association in AS160−/− and Tbc1d1−/− mice also combine in AS160−/−/Tbc1d1−/− mice. However, we found different glucose uptake abnormalities in isolated skeletal muscles and adipocytes than reported previously, resulting in different interpretations of how AS160 and Tbc1d1 regulate GLUT4 translocation to the cell surface. In support of a larger role for AS160 in glucose homeostasis, in contrast with the previous study, we find similarly impaired glucose and insulin tolerance in AS160−/−/Tbc1d1−/− and AS160−/− mice. However, in vivo glucose uptake abnormalities in AS160−/−/Tbc1d1−/− skeletal muscles differ from those observed previously in AS160−/− mice, indicating additional defects due to Tbc1d1 deletion. Similar to AS160- and Tbc1d1-deficient mice, AS160−/−/Tbc1d1−/− mice show sex-specific abnormalities in glucose and energy homeostasis. In conclusion, our study supports nonredundant functions for AS160 and Tbc1d1.

Summary: Investigating mechanisms that regulate endothelial cell (EC) growth and survival is important for understanding EC homeostasis and how ECs maintain stem cell niches. We report here that targeted loss of Id genes in adult ECs results in dilated, leaky sinusoids and a pro-inflammatory state that increases in severity over time. Disruption in sinusoidal integrity leads to increased hematopoietic stem cell (HSC) proliferation, differentiation, migration, and exhaustion. Mechanistically, sinusoidal ECs (SECs) show increased apoptosis because of reduced Bcl2-family gene expression following Id gene ablation. Furthermore, Id1−/−Id3−/− SECs and upstream type H vessels show increased expression of cyclin-dependent kinase inhibitors p21 and p27 and impaired ability to proliferate, which is rescued by reducing E2-2 expression. Id1−/−Id3−/− mice do not survive sublethal irradiation because of impaired vessel regeneration and hematopoietic failure. Thus, Id genes are required for the survival and regeneration of BM SECs during homeostasis and stress to maintain HSC development. : Gadomski et al. show that Id genes are critical regulators of bone marrow endothelial cell proliferation and survival. Selective loss of Id genes in endothelial cells promotes vessel dilation, apoptosis, and increased permeability, leading to the functional decline of hematopoietic stem cells under steady-state and stress conditions. Keywords: endothelial cells, ID proteins, hematopoietic stem cells, hematopoietic microenvironment, instructive niche, transcription factors, tissue regeneration, progenitor cells, proliferation, bone marrow

Lymphoid tissue inducer (LTi) cells are regarded as a subset of innate lymphoid cells (ILCs). However, these cells are not derived from the ILC common progenitor, which generates other ILC subsets and is defined by the expression of the transcription factor PLZF. Here, we examined transcription factor(s) determining the fate of LTi progenitors versus non-LTi ILC progenitors. Conditional deletion of Gata3 resulted in the loss of PLZF+ non-LTi progenitors but not the LTi progenitors that expressed the transcription factor RORγt. Consistently, PLZF+ non-LTi progenitors expressed high amounts of GATA3, whereas GATA3 expression was low in RORγt+ LTi progenitors. The generation of both progenitors required the transcriptional regulator Id2, which defines the common helper-like innate lymphoid progenitor (ChILP), but not cytokine signaling. Nevertheless, low GATA3 expression was necessary for the generation of functionally mature LTi cells. Thus, differential expression of GATA3 determines the fates and functions of distinct ILC progenitors.

One of the biggest challenges in analyzing high throughput omics data in biological studies is extracting information that is relevant to specific biological mechanisms of interest while simultaneously restricting the number of false positive findings. Due to random chances with numerous candidate targets and mechanisms, computational approaches often yield a large number of false positives that cannot easily be discerned from relevant biological findings without costly, and often infeasible, biological experiments. We here introduce and apply an integrative bioinformatics approach, Biologically Anchored Knowledge Expansion (BAKE), which uses sequential statistical analysis and literature mining to identify highly relevant network genes and effectively removes false positive findings. Applying BAKE to genomic expression data collected from mouse (Mus musculus) adipocytes during insulin resistance progression, we uncovered the transcription factor Krueppel-like Factor 4 (KLF4) as a regulator of early insulin signaling. We experimentally confirmed that KLF4 controls the expression of two key insulin signaling molecules, the Insulin Receptor Substrate 2 (IRS2) and Tuberous Sclerosis Complex 2 (TSC2).

Reprograming of metabolism is one of the central hallmarks of cancer. The majority of cancer cells depend on high rates of glycolysis and glutaminolysis for their growth and survival. A number of oncogenes and tumor suppressors have been connected to the regulation of altered glucose and glutamine metabolism in cancer cells. For example, the oncogene c-Myc plays vital roles in cancer cell metabolic adaptation by directly regulating various genes that participate in aerobic glycolysis and glutaminolysis. Inhibitor of differentiation 1 (Id1) is a helix-loop-helix transcription factor that plays important roles in cell proliferation, differentiation, and cell fate determination. Overexpression of Id1 causes intestinal adenomas and thymic lymphomas in mice, suggesting that Id1 could function as an oncogene. Despite it being an oncogene, whether Id1 plays any prominent role in cancer cell metabolic reprograming is unknown. Here, we demonstrate that Id1 is strongly expressed in human and mouse liver tumors and in hepatocellular carcinoma (HCC) cell lines, whereas its expression is very low or undetectable in normal liver tissues. In HCC cells, Id1 expression is regulated by the MAPK/ERK pathway at the transcriptional level. Knockdown of Id1 suppressed aerobic glycolysis and glutaminolysis, suggesting that Id1 promotes a metabolic shift toward aerobic glycolysis. At the molecular level, Id1 mediates its metabolic effects by regulating the expression levels of c-Myc. Knockdown of Id1 resulted in down-regulation (∼75%) of c-Myc, whereas overexpression of Id1 strongly induced (3-fold) c-Myc levels. Interestingly, knockdown of c-Myc resulted in down-regulation (∼60%) of Id1, suggesting a positive feedback-loop regulatory mechanism between Id1 and c-Myc. Under anaerobic conditions, both Id1 and c-Myc are down-regulated (50–70%), and overexpression of oxygen-insensitive hypoxia-inducible factor 1α (Hif1α) or its downstream target Mxi1 resulted in a significant reduction of c-Myc and Id1 (∼70%), suggesting that Hif1α suppresses Id1 and c-Myc under anaerobic conditions via Mxi1. Together, our findings indicate a prominent novel role for Id1 in liver cancer cell metabolic adaptation.—Sharma, B. K., Kolhe, R., Black, S. M., Keller, J. R., Mivechi, N. F., Satyanarayana, A. Inhibitor of differentiation 1 transcription factor promotes metabolic reprogramming in hepatocellular carcinoma cells.

In natural biological communities, species interact with many other species. Multiple species interactions can lead to indirect ecological effects that have important fitness consequences and can cause nonadditive patterns of natural selection. Given that indirect ecological effects are common in nature, nonadditive selection may also be quite common. As a result, quantifying nonadditive selection resulting from indirect ecological effects may be critical for understanding adaptation in natural communities composed of many interacting species. We describe how to quantify the relative strength of nonadditive selection resulting from indirect ecological effects compared to the strength of pairwise selection. We develop a clear method for testing for nonadditive selection caused by indirect ecological effects and consider how it might affect adaptation in multispecies communities. We use two case studies to illustrate how our method can be applied to empirical data sets. Our results suggest that nonadditive selection caused by indirect ecological effects may be common in nature. Our hope is that trait-based approaches, combined with multifactorial experiments, will result in more estimates of nonadditive selection that reveal the relative importance of indirect ecological effects for evolution in a community context.

Tbc1d1 is a Rab GTPase-activating protein (GAP) implicated in regulating intracellular retention and cell surface localization of the glucose transporter GLUT4 and thus glucose uptake in a phosphorylation-dependent manner. Tbc1d1 is most abundant in skeletal muscle but is expressed at varying levels among different skeletal muscles. Previous studies with male Tbc1d1-deficient (Tbc1d1−/−) mice on standard and high-fat diets established a role for Tbc1d1 in glucose, lipid, and energy homeostasis. Here we describe similar, but also additional abnormalities in male and female Tbc1d1−/− mice. We corroborate that Tbc1d1 loss leads to skeletal muscle-specific and skeletal muscle type-dependent abnormalities in GLUT4 expression and glucose uptake in female and male mice. Using subcellular fractionation, we show that Tbc1d1 controls basal intracellular GLUT4 retention in large skeletal muscles. However, cell surface labeling of extensor digitorum longus muscle indicates that Tbc1d1 does not regulate basal GLUT4 cell surface exposure as previously suggested. Consistent with earlier observations, female and male Tbc1d1−/− mice demonstrate increased energy expenditure and skeletal muscle fatty acid oxidation. Interestingly, we observe sex-dependent differences in in vivo phenotypes. Female, but not male, Tbc1d1−/− mice have decreased body weight and impaired glucose and insulin tolerance, but only male Tbc1d1−/− mice show increased lipid clearance after oil gavage. We surmise that similar changes at the tissue level cause differences in whole-body metabolism between male and female Tbc1d1−/− mice and between male Tbc1d1−/− mice in different studies due to variations in body composition and nutrient handling.

The study of cellular signaling remains a significant challenge for translational and clinical research. In particular, robust and accurate methods for quantitative phosphoproteomics in tissues and tumors represent significant hurdles for such efforts. In the present work, we design, implement and validate a method for single-stage phosphopeptide enrichment and stable isotope chemical tagging, or SPECHT, that enables the use of iTRAQ, TMT and/or reductive dimethyl-labeling strategies to be applied to phosphoproteomics experiments performed on primary tissue. We develop and validate our approach using reductive dimethyl-labeling and HeLa cells in culture, and find these results indistinguishable from data generated from more traditional SILAC-labeled HeLa cells mixed at the cell level. We apply the SPECHT approach to the quantitative analysis of insulin signaling in a murine myotube cell line and muscle tissue, identify known as well as new phosphorylation events, and validate these phosphorylation sites using phospho-specific antibodies. Taken together, our work validates chemical tagging post-single-stage phosphoenrichment as a general strategy for studying cellular signaling in primary tissues.Through the use of a quantitatively reproducible, proteome-wide phosphopeptide enrichment strategy, we demonstrated the feasibility of post-phosphopeptide purification chemical labeling and tagging as an enabling approach for quantitative phosphoproteomics of primary tissues. Using reductive dimethyl labeling as a generalized chemical tagging strategy, we compared the performance of post-phosphopeptide purification chemical tagging to the well established community standard, SILAC, in insulin-stimulated tissue culture cells. We then extended our method to the analysis of low-dose insulin signaling in murine muscle tissue, and report on the analytical and biological significance of our results.

Aplastic anemia (AA) is characterized by hypocellular marrow and peripheral pancytopenia. Because interferon gamma (IFN-γ) can be detected in peripheral blood mononuclear cells of AA patients, it has been hypothesized that autoreactive T lymphocytes may be involved in destroying the hematopoietic stem cells. We have observed AA-like symptoms in our IFN-γ adenylate-uridylate-rich element (ARE)-deleted (del) mice, which constitutively express a low level of IFN-γ under normal physiologic conditions. Because no T-cell autoimmunity was observed, we hypothesized that IFN-γ may be directly involved in the pathophysiology of AA. In these mice, we did not detect infiltration of T cells in bone marrow (BM), and the existing T cells seemed to be hyporesponsive. We observed inhibition in myeloid progenitor differentiation despite an increase in serum levels of cytokines involved in hematopoietic differentiation and maturation. Furthermore, there was a disruption in erythropoiesis and B-cell differentiation. The same phenomena were also observed in wild-type recipients of IFN-γ ARE-del BM. The data suggest that AA occurs when IFN-γ inhibits the generation of myeloid progenitors and prevents lineage differentiation, as opposed to infiltration of activated T cells. These results may be useful in improving treatment as well as maintaining a disease-free status.

// Sandrine Puverel 1, * , Erkan Kiris 1, * , Satyendra Singh 1 , Kimberly D. Klarmann 1, 2 , Vincenzo Coppola 3 , Jonathan R. Keller 1, 2 , Lino Tessarollo 1 1 Mouse Cancer Genetics Program, Center for Cancer Research, NCI, Frederick, MD 21702, USA 2 Basic Science Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, NCI, Frederick, MD 21702, USA 3 The Ohio State University, Department of Cancer, Biology and Genetics, Wexner Medical Center and James Comprehensive Cancer Center, Columbus, OH 43210, USA * These authors have contributed equally to this work Correspondence to: Lino Tessarollo, email: tessarol@mail.nih.gov Keywords: RanBP9, c-Kit signaling, hematopoietic system, spermatogenesis, stem cells Received: April 19, 2016 Accepted: October 26, 2016 Published: November 08, 2016 ABSTRACT c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.

SUMMARY Targeted cancer therapeutics select for drug-resistant rescue subclones (RSCs), which typically carry rescue mutations that restore oncogenic signaling. Whereas mutations underlying antibiotic resistance frequently burden drug-naive microbes with a fitness cost, it remains unknown whether and how rescue mutations underlying cancer relapse encounter negative selection prior to targeted therapy. Here, using mouse models of reversible, Wnt-driven mam-mary cancer, we uncovered stringent counter-selection against Wnt signaling overdose during the clonal evolution of RSCs. Analyzing recurrent tumors emerging during simulated targeted therapy (Wnt withdrawal) by multi-region DNA sequencing revealed polyclonal relapses comprised of multiple RSCs, which bear distinct but functionally equivalent rescue mutations that converge on sub-maximal Wnt pathway activation. When superimposed on native (i.e., undrugged) signaling, these rescue mutations faced negative selection, indicating that they burden RSCs with a fitness cost before Wnt withdrawal unmasks their selective advantage. Exploiting collateral sensitivity to oncogene overdose may help eliminate RSCs and prevent cancer relapse., Graphical Abstract, In Brief Keller and Gunther show that Wnt-driven mammary cancers challenged with simulated targeted therapy (Wnt withdrawal) undergo clonal evolution, which stringently selects for mutations that restore a “just right” level of oncogenic signaling. Therefore, cancer relapses emerge from rare subclones that are encumbered by an untapped vulnerability to oncogene overdose.

Loss of Sirt1 causes increased Hoxa9 expression and expansion of HSPC subsets under hematopoietic stress, resulting in increased DNA damage and exhaustion of long-term progenitors., The (histone) deacetylase Sirt1 is a mediator of genomic and epigenetic maintenance, both of which are critical aspects of stem cell homeostasis and tightly linked to their functional decline in aging and disease. We show that Sirt1 ablation in adult hematopoietic stem and progenitor cells (HSPCs) promotes aberrant HSPC expansion specifically under conditions of hematopoietic stress, which is associated with genomic instability as well as the accumulation of DNA damage and eventually results in a loss of long-term progenitors. We further demonstrate that progenitor cell expansion is mechanistically linked to the selective up-regulation of the HSPC maintenance factor and polycomb target gene Hoxa9. We show that Sirt1 binds to the Hoxa9 gene, counteracts acetylation of its histone target H4 lysine 16, and in turn promotes polycomb-specific repressive histone modification. Together, these findings demonstrate a dual role for Sirt1 in HSPC homeostasis, both via epigenetic regulation of a key developmental gene and by promoting genome stability in adult stem cells.

Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies.

Although most studies of mutualisms focus on a single partner at a time, host species often associate with multiple mutualist partners simultaneously. Because of potential interactions between mutualists, only studying a single type of mutualism could lead to a biased perspective of mutualism benefit and how mutualisms may scale-up to affect communities. The legume Chamaecrista fasciculata engages in a resource mutualism with nitrogen-fixing rhizobia and also forms symbiotic interactions with ants by providing nectar in exchange for defense against herbivores. Although they provide very different benefits to the plant, both mutualists receive carbon resources from the plant. As a result, these two mutualists are likely to interact, potentially competing for carbon resources or mutually benefitting each other via their positive effects on plant hosts. In a full-factorial field experiment, we explored how rhizobia and ants influence one another, C. fasciculata fitness, and the associated arthropod community. Ants reduced plant allocation to rhizobia, but ants also increased rhizobia contamination of uninoculated plants, suggesting that ants may disperse rhizobia. In turn, rhizobia increased ant abundances, with ants preferentially tending plants with rhizobia. Chamaecrista fasciculata received substantial fitness benefits from rhizobia; in contrast, associating with ants reduced fitness. Additionally, the mutualists interacted to influence the abundance of other arthropods found on the plants. Rhizobia increased arthropod abundances, likely because more nitrogen-rich leaf tissue was more attractive to arthropod herbivores, but ants negated these increases. As these results illustrate, multiple mutualists may interact, influencing each other’s abundance and the abundance of other community members.

Widespread flooding in south-eastern Australia in 2010 resulted in a hypoxic (low dissolved oxygen, DO) blackwater (high dissolved carbon) event affecting 1800 kilometres of the Murray–Darling Basin. There was concern that prolonged low DO would result in death of aquatic biota. Australian federal and state governments and local stakeholders collaborated to create refuge areas by releasing water with higher DO from irrigation canals via regulating structures (known as ‘irrigation canal escapes’) into rivers in the Edward–Wakool system. To determine if these environmental flows resulted in good environmental outcomes in rivers affected by hypoxic blackwater, we evaluated (1) water chemistry data collected before, during and after the intervention, from river reaches upstream and downstream of the three irrigation canal escapes used to deliver the environmental flows, (2) fish assemblage surveys undertaken before and after the blackwater event, and (3) reports of fish kills from fisheries officers and local citizens. The environmental flows had positive outcomes; mean DO increased by 1–2 mg L−1 for at least 40 km downstream of two escapes, and there were fewer days when DO was below the sub-lethal threshold of 4 mg L−1 and the lethal threshold of 2 mg L−1 at which fish are known to become stressed or die, respectively. There were no fish deaths in reaches receiving environmental flows, whereas fish deaths were reported elsewhere throughout the system. This study demonstrates that adaptive management of environmental flows can occur through collaboration and the timely provision of monitoring results and local knowledge.