Testosterone supplementation increases muscle mass in older men but has not been shown to consistently improve physical function and activity. It has been hypothesized that physical exercise is required to induce the adaptations necessary for translation of testosterone-induced muscle mass gain into functional improvements. However, the effects of testosterone plus low intensity physical exercise training (T/PT) on functional performance and bioenergetics are unknown. In this pilot study, we tested the hypothesis that combined administration of T/PT would improve functional performance and bioenergetics in male mice late in life more than low-intensity physical training alone. 28-month old male mice were randomized to receive T/PT or vehicle plus physical training (V/PT) for 2 months. Compare to V/PT control, administration of T/PT was associated with improvements in muscle mass, grip strength, spontaneous physical movements, and respiratory activity. These changes were correlated with increased mitochondrial DNA copy number and expression of markers for mitochondrial biogenesis. Mice receiving T/PT also displayed increased expression of key elements for mitochondrial quality control, including markers for mitochondrial fission-and-fusion and mitophagy. Concurrently, mice receiving T/PT also displayed increased expression of markers for reduced tissue oxidative damage and improved muscle quality.

Voltage-gated cation channels regulate neuronal excitability through selective ion flux. NALCN, a member of a protein family that is structurally related to the alpha1 subunits of voltage-gated sodium/calcium channels, was recently shown to regulate the resting membrane potentials by mediating sodium leak and the firing of mouse neurons. We identified a role for the Caenorhabditis elegans NALCN homologues NCA-1 and NCA-2 in the propagation of neuronal activity from cell bodies to synapses. Loss of NCA activities leads to reduced synaptic transmission at neuromuscular junctions and frequent halting in locomotion. In vivo calcium imaging experiments further indicate that while calcium influx in the cell bodies of egg-laying motorneurons is unaffected by altered NCA activity, synaptic calcium transients are significantly reduced in nca loss-of-function mutants and increased in nca gain-of-function mutants. NCA-1 localizes along axons and is enriched at nonsynaptic regions. Its localization and function depend on UNC-79, and UNC-80, a novel conserved protein that is also enriched at nonsynaptic regions. We propose that NCA-1 and UNC-80 regulate neuronal activity at least in part by transmitting depolarization signals to synapses in C. elegans neurons.

Receptor type protein tyrosine phosphatase-sigma (PTPσ) is primarily expressed by adult neurons and regulates neural regeneration. We recently discovered that PTPσ is also expressed by hematopoietic stem cells (HSCs). Here, we describe small molecule inhibitors of PTPσ that promote HSC regeneration in vivo. Systemic administration of the PTPσ inhibitor, DJ001, or its analog, to irradiated mice promotes HSC regeneration, accelerates hematologic recovery, and improves survival. Similarly, DJ001 administration accelerates hematologic recovery in mice treated with 5-fluorouracil chemotherapy. DJ001 displays high specificity for PTPσ and antagonizes PTPσ via unique non-competitive, allosteric binding. Mechanistically, DJ001 suppresses radiation-induced HSC apoptosis via activation of the RhoGTPase, RAC1, and induction of BCL-XL. Furthermore, treatment of irradiated human HSCs with DJ001 promotes the regeneration of human HSCs capable of multilineage in vivo repopulation. These studies demonstrate the therapeutic potential of selective, small-molecule PTPσ inhibitors for human hematopoietic regeneration.

Of many lipid transfer proteins identified, all have been implicated in essential cellular processes, but the activity of none has been demonstrated in intact cells. Among these, phosphatidylinositol transfer proteins (PITP) are of particular interest as they can bind to and transfer phosphatidylinositol (PtdIns)--the precursor of important signalling molecules, phosphoinositides--and because they have essential functions in neuronal development (PITPalpha) and cytokinesis (PITPbeta). Structural analysis indicates that, in the cytosol, PITPs are in a 'closed' conformation completely shielding the lipid within them. But during lipid exchange at the membrane, they must transiently 'open'. To study PITP dynamics in intact cells, we chemically targeted their C95 residue that, although non-essential for lipid transfer, is buried within the phospholipid-binding cavity, and so, its chemical modification prevents PtdIns binding because of steric hindrance. This treatment resulted in entrapment of open conformation PITPs at the membrane and inactivation of the cytosolic pool of PITPs within few minutes. PITP isoforms were differentially inactivated with the dynamics of PITPbeta faster than PITPalpha. We identify two tryptophan residues essential for membrane docking of PITPs.

There has been an increased number of studies of nerve transection injuries with the sciatic nerve gap-injury model in the rabbit in the past 2 years. We wanted to define in greater detail what is needed to test artificial nerve guides in a sciatic nerve gap-injury model in the rabbit. We hope that this will help investigators to fully exploit the robust translational potential of the rabbit sciatic nerve gap-injury model in its capacity to test devices whose diameter and length are in the range of those commonly applied in hand and wrist surgery (diameter ranging between 2 and 4 mm; length up to 30 mm). We suggest that the rabbit model should replace the less translational rat model in nerve regeneration research. The rabbit sciatic model, however, requires an effective strategy to prevent and control self-mutilation of the foot in the postoperative period, and to prevent pressure ulcers.

Imprinted genes are differentially expressed by adult stem cells, but their functions in regulating adult stem cell fate are incompletely understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an imprinted gene, regulates hematopoietic stem cell (HSC) self-renewal and regeneration. Deletion of the maternal allele of Grb10 in mice (Grb10m/+ mice) substantially increased HSC long-term repopulating capacity, as compared to that of Grb10+/+ mice. After total body irradiation (TBI), Grb10m/+ mice demonstrated accelerated HSC regeneration and hematopoietic reconstitution, as compared to Grb10+/+ mice. Grb10-deficient HSCs displayed increased proliferation after competitive transplantation or TBI, commensurate with upregulation of CDK4 and Cyclin E. Furthermore, the enhanced HSC regeneration observed in Grb10-deficient mice was dependent on activation of the Akt/mTORC1 pathway. This study reveals a function for the imprinted gene Grb10 in regulating HSC self-renewal and regeneration and suggests that the inhibition of Grb10 can promote hematopoietic regeneration in vivo.

Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in the levels of several plasma membrane lipids including phosphatidylinositol (PtdIns), phosphatidic acid (PtdOH) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. It is widely believed that exchange of lipids between the plasma membrane and endoplasmic reticulum (ER) is required to restore lipid homeostasis during PLC signalling, yet the mechanism remains unresolved. RDGBα (hereafter RDGB) is a multi-domain protein with a PtdIns transfer protein (PITP) domain (RDGB-PITPd). We find that, in vitro, the RDGB-PITPd binds and transfers both PtdOH and PtdIns. In Drosophila photoreceptors, which experience high rates of PLC activity, RDGB function is essential for phototransduction. We show that binding of PtdIns to RDGB-PITPd is essential for normal phototransduction; however, this property is insufficient to explain the in vivo function because another Drosophila PITP (encoded by vib) that also binds PtdIns cannot rescue the phenotypes of RDGB deletion. In RDGB mutants, PtdIns(4,5)P2 resynthesis at the plasma membrane following PLC activation is delayed and PtdOH levels elevate. Thus RDGB couples the turnover of both PtdIns and PtdOH, key lipid intermediates during G-protein-coupled PtdIns(4,5)P2 turnover.

The Sodium Screener© (SS©), as developed by NutritionQuest (Berkeley, CA, USA), was designed to reduce the burden of repeated dietary or urinary sodium measurements, but the accuracy of daily sodium intake estimates has not been reported. Associations were examined between sodium intakes derived from the SS© scores and repeated 24-h recalls (24DR) in two studies with different administration modes. In one study, 102 registered dietitians (RD) completed three Automated Self-Administered 24DRs (ASA24©), version 2014, followed by the SS©; both were self-administered and web-based. In the second sample, (the Study of Household Purchasing Patterns, Eating, and Recreation or SHoPPER), trained dietitians conducted 24DR interviews with 69 community-dwelling adults in their homes; all the community adults then completed a paper-based SS© at the final visit. In the RD study, SS© -predicted sodium intakes were 2604 ± 990 (mean ± Standard deviation (SD)), and ASA24© sodium intakes were 3193 ± 907 mg/day. In the SHoPPER sample, corresponding values were 3338 ± 1310 mg/day and 2939 ± 1231 mg/day, respectively. SS©-predicted and recall sodium estimates were correlated in the RD study (r = 0.381, p = 0.0001) and in the SHoPPER (r = 0.430, p = 0.0002). Agreement between the SS© and 24-h recalls was poor when classifying individuals as meeting the dietary sodium guidelines of 2300 mg/day or not (RD study: kappa = 0.080, p = 0.32; SHoPPER: kappa = 0.207, p = 0.08). Based on repeated 24DR either in person or self-reported online as the criterion for estimating daily sodium intakes, the SS© may require additional modifications.

Tyrosine kinase inhibitors (TKIs) induce molecular remission in the majority of patients with chronic myelogenous leukemia (CML), but the persistence of CML stem cells hinders cure and necessitates indefinite TKI therapy. We report that CML stem cells upregulate the expression of pleiotrophin (PTN) and require cell-autonomous PTN signaling for CML pathogenesis in BCR/ABL+ mice. Constitutive PTN deletion substantially reduced the numbers of CML stem cells capable of initiating CML in vivo. Hematopoietic cell-specific deletion of PTN suppressed CML development in BCR/ABL+ mice, suggesting that cell-autonomous PTN signaling was necessary for CML disease evolution. Mechanistically, PTN promoted CML stem cell survival and TKI resistance via induction of Jun and the unfolded protein response. Human CML cells were also dependent on cell-autonomous PTN signaling, and anti-PTN antibody suppressed human CML colony formation and CML repopulation in vivo. Our results suggest that targeted inhibition of PTN has therapeutic potential to eradicate CML stem cells.

Grains of tetraploid wheat (Triticum turgidum L.) mainly accumulate the non-provitamin A carotenoid lutein-with low natural variation in provitamin A β-carotene in wheat accessions necessitating alternative strategies for provitamin A biofortification. Lycopene ɛ-cyclase (LCYe) and β-carotene hydroxylase (HYD) function in diverting carbons from β-carotene to lutein biosynthesis and catalyzing the turnover of β-carotene to xanthophylls, respectively. However, the contribution of LCYe and HYD gene homoeologs to carotenoid metabolism and how they can be manipulated to increase β-carotene in tetraploid wheat endosperm (flour) is currently unclear. We isolated loss-of-function Targeting Induced Local Lesions in Genomes (TILLING) mutants of LCYe and HYD2 homoeologs and generated higher order mutant combinations of lcye-A, lcye-B, hyd-A2, and hyd-B2. Hyd-A2 hyd-B2, lcye-A hyd-A2 hyd-B2, lcye-B hyd-A2 hyd-B2, and lcye-A lcye-B hyd-A2 hyd-B2 achieved significantly increased β-carotene in endosperm, with lcye-A hyd-A2 hyd-B2 exhibiting comparable photosynthetic performance and light response to control plants. Comparative analysis of carotenoid profiles suggests that eliminating HYD2 homoeologs is sufficient to prevent β-carotene conversion to xanthophylls in the endosperm without compromising xanthophyll production in leaves, and that β-carotene and its derived xanthophylls are likely subject to differential catalysis mechanisms in vegetative tissues and grains. Carotenoid and gene expression analyses also suggest that the very low LCYe-B expression in endosperm is adequate for lutein production in the absence of LCYe-A. These results demonstrate the success of provitamin A biofortification using TILLING mutants while also providing a roadmap for guiding a gene editing-based approach in hexaploid wheat.

Bone marrow (BM) perivascular stromal cells and vascular endothelial cells (ECs) are essential for hematopoietic stem cell (HSC) maintenance, but the roles of distinct niche compartments during HSC regeneration are less understood. Here we show that Leptin receptor-expressing (LepR+) BM stromal cells and ECs dichotomously regulate HSC maintenance and regeneration via secretion of pleiotrophin (PTN). BM stromal cells are the key source of PTN during steady-state hematopoiesis because its deletion from stromal cells, but not hematopoietic cells, osteoblasts, or ECs, depletes the HSC pool. Following myelosuppressive irradiation, PTN expression is increased in bone marrow endothelial cells (BMECs), and PTN+ ECs are more frequent in the niche. Moreover, deleting Ptn from ECs impairs HSC regeneration whereas Ptn deletion from BM stromal cells does not. These findings reveal dichotomous and complementary regulation of HSC maintenance and regeneration by BM stromal cells and ECs.