Allergic conditions in children are a prevalent health concern in Canada. The burden of disease and the societal costs of proper diagnosis and management are considerable, making the primary prevention of allergic conditions a desirable health care objective. This position statement reviews current evidence on dietary exposures and allergy prevention in infants at high risk for developing allergic conditions. It revisits previous dietary recommendations for pregnancy, breastfeeding and formula feeding, and provides an approach for introducing solid foods to high-risk infants. While there is no evidence that delaying the introduction of any specific food beyond six months of age helps to prevent allergy, the protective effect of early introduction of potentially allergenic foods (at four to six months of age) remains under investigation. Recent research appears to suggest that regularly ingesting a new, potentially allergenic food may be as important as when that food is first introduced.
Allergy prevention; Atopic dermatitis; Breastfeeding; Food allergy; Formula feeding; Solid food introduction
Although the roles of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) signaling in KRAS-driven tumorigenesis are well established, KRAS activates additional pathways required for tumor maintenance, inhibition of which are likely to be necessary for effective KRAS-directed therapy. Here we show that the IKK-related kinases TBK1 and IKKε promote KRAS-driven tumorigenesis by regulating autocrine CCL5 and IL-6 and identify CYT387 as a potent JAK/TBK1/IKKε inhibitor. CYT387 treatment ablates RAS-associated cytokine signaling and impairs Kras-driven murine lung cancer growth. Combined CYT387 and MEK inhibitor therapy induces regression of aggressive murine lung adenocarcinomas driven by Kras mutation and p53 loss. These observations reveal that TBK1/IKKε promote tumor survival by activating CCL5 and IL-6 and identify concurrent inhibition of TBK1/IKKε, JAK, and MEK signaling as an effective approach to inhibit the actions of oncogenic KRAS.
KRAS; cytokines; TBK1/IKKε; JAK; lung cancer
Two isoforms of sphingosine kinase, SK1 and SK2, catalyze the formation of the bioactive lipid sphingosine 1-phosphate (S1P) in mammalian cells. We have previously shown that treatment of androgen-sensitive LNCaP prostate cancer cells with a non-selective SK isoform inhibitor, 2-(p-hydroxyanilino)- 4-(p-chlorophenyl)thiazole (SKi), induces the proteasomal degradation of SK1. This is concomitant with a significant increase in C22:0-ceramide and sphingosine levels and a reduction in S1P levels, resulting in the apoptosis of LNCaP cells. In contrast, we show here that a SK2-selective inhibitor, (R)-FTY720 methyl ether (ROME), increases sphingosine and decreases S1P levels but has no effect on ceramide levels and does not induce apoptosis in LNCaP cells. We also show that several glycolytic metabolites and (R)-S-lactoylglutathione are increased upon treatment of LNCaP cells with SKi, which induces the proteasomal degradation of c-Myc. These changes reflect an indirect antagonism of the Warburg effect. LNCaP cells also respond to SKi by diverting glucose 6-phosphate into the pentose phosphate pathway to provide NADPH, which serves as an antioxidant to counter an oxidative stress response. SKi also promotes the formation of a novel pro-apoptotic molecule called diadenosine 5′,5‴-P1,P3-triphosphate (Ap3A), which binds to the tumor suppressor fragile histidine triad protein (FHIT). In contrast, the SK2-selective inhibitor, ROME, induces a reduction in some glycolytic metabolites and does not affect oxidative stress. We conclude that SK1 functions to increase the stability of c-Myc and suppresses Ap3A formation, which might maintain the Warburg effect and cell survival, while SK2 exhibits a non-overlapping function.
Sphingosine kinase; glycolysis; Warburg effect; oxidative stress; diadenosine triphosphate; cancer; proteasome; LNCaP metabolome
Question I have been under the impression that infants should avoid potential allergenic foods such as nuts, cow’s milk, and eggs in order to avoid developing allergic reactions. What advice should I give parents regarding the introduction of food in infancy and the development of food allergy?
Answer There is no evidence that delaying the introduction of any specific food beyond 6 months of age helps to prevent allergy. A recent Canadian Paediatric Society statement recommends no delay in the introduction of food in infancy. Recent research also appears to suggest that early introduction of potentially allergenic foods (at 4 to 6 months of age) might actually provide a form of protection and help prevent allergy, but more research is needed.
Question J’étais sous l’impression qu’on devrait éviter de donner aux nourrissons des aliments potentiellement allergènes comme des noix, du lait de vache et des œufs pour prévenir le développement de réactions allergiques. Quels conseils devrait-on donner aux parents concernant l’introduction des aliments durant la petite enfance et le développement des allergies alimentaires?
Réponse Il n’y a pas de données probantes indiquant que retarder l’introduction d’aliments particuliers après l’âge de 6 mois aide à prévenir les allergies. Une récente déclaration de la Société canadienne de pédiatrie ne recommande aucun délai quant à l’introduction d’aliments durant la petite enfance. De récentes études de recherche semblent aussi faire valoir que l’introduction précoce (entre 4 et 6 mois) d’aliments possiblement allergènes procure une forme de protection et contribue à prévenir les allergies, mais il faudrait plus de recherche à ce sujet.
Upon stimulation by pathogen-associated inflammatory signals, the atypical IκB kinase TBK1 induces type-I interferon expression and modulates NF-κB signaling. Here we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a novel dimeric assembly, created by an extensive network of interactions among the kinase, ubiquitin-like (ULD) and scaffold/dimerization (SDD) domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on Lysine 30 and Lysine 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog IKKε, but not in the canonical IκB kinase IKKβ, which assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IκB kinases in normal and disease physiology and will further development of more specific inhibitors that may be useful as anti-cancer or anti-inflammatory agents.
Macroautophagy, commonly referred to as autophagy, is a protein degradation pathway that occurs constitutively in cells, but can also be induced by stressors such as nutrient starvation or protein aggregation. Autophagy has been implicated in multiple disease mechanisms including neurodegeneration and cancer, with both tumor suppressive and oncogenic roles. Uncoordinated 51-like kinase 1 (ULK1) is a critical autophagy protein near the apex of the hierarchal regulatory pathway that receives signals from the master nutrient sensors MTOR and AMP-activated protein kinase (AMPK). In mammals, ULK1 has a close homolog, ULK2, although their functional distinctions have been unclear. Here, we show that ULK1 and ULK2 both function to support autophagy activation following nutrient starvation. Increased autophagy following amino acid or glucose starvation was disrupted only upon combined loss of ULK1 and ULK2 in mouse embryonic fibroblasts. Generation of PtdIns3P and recruitment of WIPI2 or ZFYVE1/DFCP1 to the phagophore following amino acid starvation was blocked by combined Ulk1/2 double knockout. Autophagy activation following glucose starvation did not involve recruitment of either WIPI1 or WIPI2 to forming autophagosomes. Consistent with a PtdIns3P-independent mechanism, glucose-dependent autophagy was resistant to wortmannin. Our findings support functional redundancy between ULK1 and ULK2 for nutrient-dependent activation of autophagy and furthermore highlight the differential pathways that respond to amino acid and glucose deprivation.
ULK1; ULK2; WIPI1; WIPI2; knockout; MEF; nutrient starvation
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
LC3; autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
The timing of complementary food introduction is controversial. Providing information on the timing of dietary introduction is crucial to the primary prevention of food allergy. The American Academy of Pediatrics offers dietary recommendations that were updated in 2008.
Identify the recommendations that general pediatricians and registered dietitians provide to parents and delineate any differences in counselling.
A 9-item survey was distributed to pediatricians and dietitians online and by mail. Information on practitioner type, gender, length of practice and specific recommendations regarding complementary food introduction and exposure was collected.
181 surveys were returned with a 54% response rate from pediatricians. It was not possible to calculate a meaningful dietitian response rate due to overlapping email databases. 52.5% of all respondents were pediatricians and 45.9% were dietitians. The majority of pediatricians and dietitians advise mothers that peanut abstinence during pregnancy and lactation is unnecessary. Dietitians were more likely to counsel mothers to breastfeed their infants to prevent development of atopic dermatitis than pediatricians. Hydrolyzed formulas for infants at risk of developing allergy were the top choice of formula amongst both practitioners. For food allergy prevention, pediatricians were more likely to recommend delayed introduction of peanut and egg, while most dietitians recommended no delay in allergenic food introduction.
In the prophylaxis of food allergy, pediatricians are less aware than dietitians of the current recommendation that there is no benefit in delaying allergenic food introduction beyond 4 to 6 months. More dietitians than pediatricians believe that breastfeeding decreases the risk of atopic dermatitis. Practitioners may benefit from increased awareness of current guidelines.
Food allergy; Children; Survey; Prevention; Dietary advice
Huntington’s disease (HD) is caused by polyglutamine expansion (exp) in huntingtin (Htt). The type 1 inositol (1,4,5)-triphosphate receptor (InsP3R1) is an intracellular calcium (Ca2+) release channel that plays an important role in neuronal function. In a yeast two-hybrid screen with the InsP3R1 carboxy terminus, we isolated Htt-associated protein-1A (HAP1A). We show that an InsP3R1-HAP1A-Htt ternary complex is formed in vitro and in vivo. In planar lipid bilayer reconstitution experiments, InsP3R1 activation by InsP3 is sensitized by Httexp, but not by normal Htt. Transfection of full-length Httexp or caspase-resistant Httexp, but not normal Htt, into medium spiny striatal neurons faciliates Ca2+ release in response to threshold concentrations of the selective mGluR1/5 agonist 3,5-DHPG. Our findings identify a novel molecular link between Htt and InsP3R1-mediated neuronal Ca2+ signaling and provide an explanation for the derangement of cytosolic Ca2+ signaling in HD patients and mouse models.
The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary strategy for targeting KRAS is to identify gene products that, when inhibited, result in cell death only in the presence of an oncogenic allele1,2. Here we have used systematic RNA interference (RNAi) to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IκB kinase, TBK1, was selectively essential in cells that harbor mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF-κB anti-apoptotic signals involving cREL and BCL-XL that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations identify TBK1 and NF-κB signaling as essential in KRAS mutant tumors and establish a general approach for the rational identification of co-dependent pathways in cancer.
Autophagy, an intracellular degradative pathway, maintains cell homeostasis under normal and stress conditions. Nascent double-membrane autophagosomes sequester and enclose cytosolic components and organelles, and subsequently fuse with the endosomal pathway allowing content degradation. Autophagy requires fusion of autophagosomes with late endosomes, but it is not known if fusion with early endosomes is essential. We show that fusion of AVs with functional early endosomes is required for autophagy. Inhibition of early endosome function by loss of COPI subunits (β′, β, or α) results in accumulation of autophagosomes, but not an increased autophagic flux. COPI is required for ER-Golgi transport and early endosome maturation. Although loss of COPI results in the fragmentation of the Golgi, this does not induce the formation of autophagosomes. Loss of COPI causes defects in early endosome function, as both transferrin recycling and EGF internalization and degradation are impaired, and this loss of function causes an inhibition of autophagy, an accumulation of p62/SQSTM-1, and ubiquitinated proteins in autophagosomes.
The yeast Atg1 serine/threonine protein kinase and its mammalian homologs ULK1 and ULK2 play critical roles during the activation of autophagy. Previous studies have demonstrated that the conserved C-terminal domain (CTD) of ULK1 controls the regulatory function and localization of the protein. Here, we explored the role of kinase activity and intramolecular interactions to further understand ULK function. We demonstrate that the dominant-negative activity of kinase-dead mutants requires a 7-residue motif within the CTD. Our data lead to a model in which the functions of ULK1 and ULK2 are controlled by autophosphorylation and conformational changes involving exposure of the CTD. Additional mapping indicates that the CTD contains other distinct regions that direct membrane association and interaction with the putative human homologue of Atg13, which we have here characterized. Atg13 is required for autophagy and Atg9 trafficking during autophagy. However, Atg13 does not bind the 7-residue dominant-negative motif in the CTD of ULK proteins nor is the inhibitory activity of the CTDs rescued by Atg13 ectopic expression, suggesting that in mammalian cells, the CTD may interact with additional autophagy proteins.
Kocuria, previously classified into the genus of Micrococcus, is commonly found on human skin. Two species, K. rosea and K. kristinae, are etiologically associated with catheter-related bacteremia.
We describe the first case of K. kristinae infection associated with acute cholecystitis. The microorganism was isolated from the bile of a 56-year old Chinese man who underwent laparoscopic cholecystectomy. He developed post-operative fever that resolved readily after levofloxacin treatment.
Our report of K. kristinae infection associated with acute cholecystitis expands the clinical spectrum of infections caused by this group of bacteria. With increasing number of recent reports describing the association between Kocuria spp. and infectious diseases, the significance of their isolation from clinical specimens cannot be underestimated. A complete picture of infections related to Kocuria spp. will have to await the documentation of more clinical cases.
The small GTPase Ras is converted to the active, GTP-bound state during exposure of vertebrate cells to hypothermic stress. This activation occurs more rapidly than can be accounted for by spontaneous nucleotide exchange. Ras–guanyl nucleotide exchange factors and Ras GTPase–activating proteins have significant activity at 0°C in vitro, leading to the hypothesis that normal Ras regulators influence the relative amounts of Ras-GTP and Ras-GDP at low temperatures in vivo. When hypothermic cells are warmed to 37°C, the Raf-Mek-Erk protein kinase cascade is activated. After prolonged hypothermic stress, followed by warming to physiologic temperature, cultured fibroblasts assume a rounded morphology, detach from the substratum, and die. All of these biologic responses are attenuated by pharmacologic inhibition of Mek. Previously, it had been found that low temperature blocks acute growth factor signaling to Erk. In the present study, we found that this block occurs at the level of Raf activation. Temperature regulation of Ras signaling could help animal cells respond appropriately to hypothermic stress, and Ras-Erk signaling can be manipulated to improve the survival of cells in cold storage.