Although glucose deprivation mainly induces autophagy via AMPK activation and the subsequent inhibition of mTORC1, we will also comment other signalling pathways, as well as evidences indicating that, under certain conditions, autophagy can be activated by glucose. A better understanding on how glucose regulates autophagy not only will expand our basic knowledge of this important cell process, but it will be also relevant to understand common human disorders, such as cancer and diabetes, in which glucose levels play an important role.Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focus on the signalling pathways by which environmental glucose directly,  Three different forms of autophagy coexist in most cells : MicroauMicroautophagy, a still poorly characterized type of autophagy in mammalian cells, consists in the direct engulfment of cytoplasmic components in intralysosomal tubules and vesicles, which are produced by various modifications of the lysosomal membranes. Chaperone-mediated autophagy is a selective lysosomal pathway for the degradation of cytosolic proteins with KFERQ-like sequences, in which both cytosolic and lysosomal chaperones facilitate the transport of these proteins for degradation into the lysosomal lumen through a multimer of the lysosomal protein LAMP2A spanning the lysosomal membrane. Macroautophagy is the most important and better known form of autophagy. Initially, cytoplasmic components are surrounded by a double membrane, with still an unclear origin, forming a vacuole, which is called an autophagosome. Autophagosomes fuse with endosomes and lysosomes to form autolysosomes with an acidic pH and where the degradation of the sequestered material by lysosomal hydrolases occurs. While autophagy has been usually considered a nonselective and an in bulk degradation process, there are also selective autophagic pathways. For example, the selective degradation of mitochondria, peroxisomes or ribosomes by autophagy is called, respectively, mitophagy, pexophagy or ribophagy. In this paper, unless otherwise stated, the term “autophagy” will only refer to macroautophagy, and we will not review here the two other forms of autophagy.e.g. [Autophagy is regulated by a high number of extracellular stimuli, which are transduced through a large variety of signalling pathways with the kinase mTORC1 occupying a central position in most of them ,3. In mae.g. ). In conet al. [Autophagy, like protein degradation , in spitet al. . ATP (adATG (autophagy-related) genes provided insights into the molecular basis of autophagy. Many of the codified proteins in yeast are conserved in mammals, but there are also mammalian specific ATG proteins. Now, the role of some, but not all, of the more than 30 identified Atg proteins is known and has been discussed elsewhere [Most of our current knowledge on the molecular machinery of autophagy derives from studies in yeast that, initially, were mainly carried out in the laboratories of Yoshinori Ohsumi in Japan, Michael Thumm in Germany and Dan Klionsky in USA. In the late 90s, two different forms of autophagy were recognized in yeast: micro- and macroautophagy . The firlsewhere ,11,12. MSaccharomyces cerevisiae in a medium without glucose induces the synthesis of gluconeogenic enzymes  and also of the galactose and maltose transporters. When these cells are subsequently incubated in a medium containing glucose, the transcription of these genes is suppressed and the induced proteins are degraded [In yeast, autophagy is mainly affected by nitrogen starvation, but glucose starvation plays also an important role, as we will see below in a few examples. The growth of degraded  in the gdegraded .Pichia pastoris and Hansenula polymorpha when they are incubated in a medium containing methanol [H. polymorpha [P. pastoris [Something similar occurs with the methylotrophic yeasts methanol ,16. Thismethanol . In the pastoris . Pexophapastoris ,18,19. Opastoris , and othpastoris .S. cerevisiae via inhibition by glucose of the expression of Snf1p, the closest yeast homologue of AMPK (AMP-activated protein kinase). Both Snf1p and the cyclin-dependent kinase Pho85p have been described to control autophagy in opposite directions, activating and inhibiting it, respectively, via the autophagic proteins Atg1 and Atg13 [S. cerevisiae maintained in a medium with plenty of nutrients. Under these conditions, autophagy is inhibited via PKA (cAMP-dependent protein kinase A) and also, and independently, via TorC1 [The data mentioned above indicate that some types of specific autophagy are induced by glucose. However, other experiments indicate that glucose could also produce in yeast the opposite effect, namely an inhibition of autophagy, at least in the case of the nonselective autophagy. For example, this can occur in nd Atg13 . Anotheria TorC1 ,24. Bothia TorC1 . In thisia TorC1 , becauseia TorC1 , althougIn summary, while it is known that depletion of nitrogen mainly inactivates TorC1 and induces autophagy, the signalling pathways by which glucose-deprivation produces the same effect are less known. It is possible that in yeast TorC1 mainly responds to nitrogen levels while the response to carbon levels occurs mainly via the Ras/PKA pathway  and also1, responsible for ATP hydrolysis, and Vo, responsible for proton translocation. In yeast, glucose is important to modify the activity of the V-ATPase independently of protein synthesis [1 domain remain attached to V0, while with glucose this figure rises to 50–70% [1 and V0 portions produces the loss of their respective functions. To explain how glucose increases the V1–Vo coupling, an activation of the proton pump Pma1 at the plasma membrane that produces an increase in cytosolic pH [In addition to the sequestration step, glucose in yeast can also affect positively autophagy at later steps, for example regulating the assembly of the V-ATPase, which, as mentioned before, consumes ATP. The V-ATPase is a supramolecular complex responsible for the acidification of organelles in eukaryotic cells  and has ynthesis . Thus, io 50–70% . This resolic pH  and a resolic pH  have beeIn summary, glucose in yeast can both induce and inhibit autophagy. For example, glucose induces a selective autophagy during an adaptive phase to specifically remove enzymes or organelles previously overproduced, and increases also the assembly and the activity of the V-ATPase and, thus, of the vacuolar acid hydrolases. However, glucose in a nutrient-rich medium can also suppress nonspecific autophagy to prevent an unnecessary autodigestion of the cell, and this mainly occurs via Ras/cAMP/PKA but also via TorC1 and Sch9 and by inactivation of Snf1p.In mammalian cells, as in yeast, autophagy is a process highly regulated by nutrients. Over the last years, the effects of amino acids on autophagy have been extensively studied. However, the role of glucose is less known, despite its implication in diseases such as diabetes or different types of cancer. In mammals low and high blood glucose levels induce the production and release of, respectively, glucagon by alpha cells and insulin by beta cells of pancreas. It is also well known that, for example in liver, glucagon activates autophagy while insulin inhibits it. However, the amount of glucose in the environment may also produce an autophagic response unrelated to these hormones. This response is mainly due to the role of glucose as an important determinant of the cellular energy status, but also to other glucose effects, such as oxidative stress and ROS accumulation. In the following sections we will not review the glucose effects on autophagy mediated by hormones, because they have been extensively described elsewhere, and we will deal exclusively with those other glucose effects.via PI3K (phosphatidylinositol 3-kinase), of the V-ATPase [et al. [As in yeast, in mammalian cells the availability of glucose promotes the assembly, V-ATPase  favourinV-ATPase . Since l [et al.  showed i [et al. ,36.In contrast to these data, most investigators consider that in mammalian cells glucose inhibits autophagy ,38,39,40KIP1, induce autophagy when ATP decreases in cells incubated without glucose. Others, such as p38, ERK  or JNK , respond to ROS generated by an excessively low or high availability of glucose, activating autophagy. Finally, we will also mention the possible role of IKK (IκB kinase) and FOXO (forkhead class O) proteins in the induction of autophagy by glucose, which occurs without an apparent relation with ATP or ROS. All this reflects the existence in mammalian cells of a complicated network that interconnects different signalling pathways in the regulation of autophagy by glucose.In the following sections, we will discuss different signalling pathways that regulate autophagy by glucose. In spite of the apparent importance of PKA in the signalling pathways by which glucose regulates autophagy in yeast, its relevance in mammalian cells remains elusive and, therefore, will not be mentioned here. Some of the implicated proteins, such as AMPK, p53 or p27KIP1 [Glucose is an important and immediate source of energy in the form of ATP. Therefore, it is logical to consider that the regulation of autophagy by glucose may be explained by changes in energetic levels. As mentioned above, AMPK is an important stimulator of autophagy that is activated by a drop in ATP levels produced, for example, by glucose withdrawal . InductiKIP1  . For exP levels . More reP levels ,62. UndeP levels . Furtherapamycin . Finallyapamycin ,64.Since AMPK is an energy sensor, its activity is important in many human diseases, in most cases in relationship with glucose levels and autophagy. This is highlighted for example in carcinomas in which the glycoprotein MUC1 is overexpressed. These cells incubated under low glucose conditions (1 mM) show a higher AMPK activity than normal cells  and, sin-/- AMPKMEFs incubated in glucose free medium [e.g., SESTRIN 2 and DAPK-1 (death-associated protein kinase 1). SESTRIN 2 activates AMPK and induces TSC2 phosphorylation to inhibit mTORC1 [AMPKB1 [DRAM (damage-regulated autophagy modulator) and TSC2 [Glucose deprivation induces the phosphorylation of p53 by AMPK in Ser15 (Ser18 in mice) ,67,68. Te medium , suggeste medium . In low e medium , where it mTORC1 . DAPK-1 t mTORC1 . Other gand TSC2 ,74. The The cytoplasmic-nuclear shuttling of p53 has a major role in autophagy regulation . WhereasKIP1 through LKB1/AMPK, allowing its stabilization [KIP1, even upon glucose starvation. Reconstitution of these cells with LKB1 produces an increase in both total and P-T198 p27KIP1 levels in glucose free medium, as well as in ACC phosphorylation [KIP1 is activated by LKB1/AMPK during glucose deprivation. Moreover, glucose deprivation induces the expression of p27KIP1, while moderate increments in D-(+)-glucose reduces it [KIP1 is ubiquitinated by SIAH/SIP and degraded by proteasomes [KIP1 is associated with a decrease in cell motility, but the effect on autophagy was not studied.Glucose deprivation increases the phosphorylation  of the cyclin-dependent kinase inhibitor p27lization ,77. HeLarylation . All theduces it . Conversteasomes . This deKIP1 by AMPK under glucose starvation determines, in addition to its stabilization, its cellular localization. Thus, in NIH3T3 cells expressing a constitutively active form of AMPK, only 10% of p27 KIP1 presents a nuclear localization, whereas in cells that express a dominant negative AMPK this percentage increases up to 70% [KIP1 is sequestered in the cytosol by 14–3–3 proteins, which prevent its binding to the IMPORTINα, responsible of its transport to the nucleus [Phosphorylation of p27p to 70% . This ca nucleus .KIP1 has been shown to induce autophagy under glucose deprivation conditions, since a prolonged incubation without glucose produces an increment in the number of autophagic vacuoles in +/+p27 MEFs, while autophagy is repressed in -/-p27 MEFs. In addition, ectopic expression of the phosphomimic p27 T198D mutant is sufficient to induce autophagy [p27utophagy .KIP1 activates autophagy is not known, it could be mediated by its effect on the RHOA-GTPase. Cytosolic p27KIP1 prevents the activation of RHOA-GTPase [Although the precise mechanism by which p27A-GTPase ,82 and iA-GTPase . et al. [et al. [Apart from variations in the energy levels, there are other changes that take place in the cells depending on the availability of glucose. Both glucose deprivation ,85 and het al.  establis [et al.  showed tThis increase in ROS levels is subsequently sensed by proteins such as AMPK , p38MAPKp38MAPK is activated upon oxidative stress caused both by glucose deprivation and hyperglycemia ,90. TherStarting with the positive effect on autophagy, it is known that once the p38MAPK is activated by different stimuli it can phosphorylate BCL-2 , allowinAs for an inhibitory role, it has been described, for example, that phosphorylation of p38MAPK increases its affinity with the p38 Interacting Protein (p38IP), competing with ATG9 for its binding. As a result, there is a decrease in the formation of the p38IP/ATG9 complex, needed for autophagosome formation . However2O2 levels, which ultimately will lead to the activation of the ASK1/SEK1/JNK pathway [Another effect of oxidative stress due to both a high glucose concentration or glucose deprivation is the increased expression and activation of JNK ,103. For pathway .A high glucose concentration leads to an interaction of JNK with other regulatory pathways such as ERK/p53  or NF-κBet al. [ERK1/2 is also considered an activator of autophagy, because its inhibition lessens the induction of autophagy produced by different stimuli ,107. In et al.  have alset al. [Since it is known that glucose induces both ROS and AMPK, which activate ERK1/2 ,111, it et al.  that shoIn addition to the signalling pathways related with energy changes and ROS induction that appear to be implicated in the regulation of autophagy in response to glucose levels, there are others implicated in this regulation. Here we will describe the role of NF-κB, SIRT1 and FOXO see .Several studies have established an effect of a high glucose concentration in the activation of IKK , which cet al. [et al. [Independently of the above-mentioned reports, it has been shown a positive correlation in the activities of NF-κB and autophagy. Thus, Copetti et al.  describe [et al.  recently [et al. . Finally [et al. . IKK can [et al. .Although the direct relationship among glucose availability, induction of NF-κB and a subsequent effect on autophagy has not been described yet, these data suggest a possible role of NF-κB on glucose-induced autophagy. Therefore, more studies are needed to verify this possibility.+-dependent deacetylase that has been reported to deacetylate, upon nutrient deprivation (HBBS medium), ATG5, ATG7 and ATG8 proteins to induce autophagy [RAB7 (a GTPase protein implicated in the fusion of autophagosomes with endosomes and lysosomes) [GABARAPL1, LC3 and ATG12 (which participates in the elongation of the autophagic vesicles) genes [via SIRT1/FOXO1/3.SIRT1 is a NADutophagy . Under sutophagy ,123. As sosomes) , GABARAPIn addition to its obvious importance for a better understanding of the physiological roles of autophagy, the issue of the regulation of autophagy by the available glucose levels has also important pathological implications. We will present here only two examples in relationship with two important pathologies: cancer and diabetes.The induction of autophagy under conditions of glucose deprivation is very important for the maintenance of cell homeostasis. When the availability of glucose is a limiting factor and thus depletion of energy occurs, as is the case in most cancers, an increased autophagy allows to obtain energy from the superfluous cell constituents. Cancer cells survive and even grow in poor nutrient microenvironments thanks to an induction of autophagy via AMPK, p53 or JNK . In the We have also discussed above that glucose deprivation, as well as hyperglycemia, induces autophagy by signalling pathways mediated by ROS production. This is of great importance in the pathology of diabetes, where the production of ROS by hyperglycemia has been shown to be an important factor influencing its clinical complications . The hypin vivo during ischemia/reperfusion [Diabetic patients are at significantly higher risk of ischemia/reperfusion injury . During erfusion ,138. Treerfusion . In the erfusion . This imerfusion .e.g.,p53 and p27KIP1, also activated by ATP depletion. In addition, ROS also influence autophagy in response to glucose deprivation or hyperglycemia, by signalling pathways that implicate other kinases such as p38MAPK, JNK or ERK1/2. Finally, glucose-induced autophagy can be also regulated by IKK/NF-κB or FOXO, independently of ROS or the ATP levels and AMPK function.Although some of the signalling pathways that orchestrate in yeast the regulation of autophagy by glucose have been conserved in mammalian cells, a more complex signalling network appears to be involved in these cells. Here, AMPK, which is activated by a deficit in energy, is the most important kinase of these signalling pathways. This kinase exerts its effect mainly, but not exclusively, through the mTOR kinase, the main nutrient sensor in the cells. However, there are other proteins that are also implicated in the regulation of autophagy downstream of AMPK in the response to glucose deprivation, The importance of the regulation of autophagy by glucose is highlighted by human diseases in which glucose levels are affected. For example, autophagy serves to preserve cell integrity in cancer cells growing in glucose-limiting environments and in pancreatic β-cells under diabetes. Therefore, understanding the mechanisms of autophagy regulation by glucose is of major interest in order to better understand these and other pathologies.


Diet is a modifiable factor that may affect sleep, but the associations of macronutrient intakes with insomnia are inconsistent. We investigated the associations of protein, fat, and carbohydrate intakes with insomnia symptoms.In this cross-sectional analysis of 4435 non-shift workers, macronutrient intakes were assessed by the brief-type self-administered diet history questionnaire, which requires the recall of usual intakes of 58 foods during the preceding month. Presence of insomnia symptoms, including difficulty initiating sleep (DIS), difficulty maintaining sleep (DMS), and poor quality of sleep (PQS) were self-reported. Logistic regression analysis was used to estimate odds ratios (ORs) and 95% CIs adjusted for demographic, psychological, and behavioral factors, as well as medical histories.Low protein intake  was associated with DIS  and PQS , while high protein intake  was associated with DMS . Low carbohydrate intake  was associated with DMS .Protein and carbohydrate intakes in the daily diet were associated with insomnia symptoms. The causality of these associations remains to be explained. Diet is a behavioral factor that may influence sleep.13 A cross-sectional study reported an association between high fat intake and short sleep duration.14 An interventional study reported that consumption of a supplement high in carbohydrate  45 minutes before bedtime significantly increased rapid eye movement sleep as compared with a low carbohydrate supplement .15 Another interventional study reported that tryptophan from dietary protein reduced sleep latency in those with long sleep latency and increased deep sleep as recorded by sleep electroencephalography.16 However, studies of the associations of usual intakes of protein, fat, and carbohydrate with insomnia symptoms are scarce and inconclusive.Demographic factors ,To better understand the factors related to better sleep hygiene, we investigated the associations of protein, fat, and carbohydrate in the daily diet with 3 insomnia symptoms in a large population of middle-aged Japanese workers.In this cross-sectional study, we used data from the Aichi Workers’ Cohort of cardiovascular disease. The cohort included 6651 civil servants (5179 men and 1472 women) in Aichi Prefecture—an urban and suburban area in central Japan. The cohort consisted of civil servants aged 35 to 66 years at recruitment in 2002 (participation rate: 62%). Most workers in the cohort were engaged in clerical work; police officers, firefighters, and public school teachers were not included in the cohort. The baseline survey in 2002 included a self-administered questionnaire on the absence or presence of insomnia symptoms, dietary habits, other lifestyle factors, psychological factors, and medical histories.n = 1008), who were mostly employed by public hospitals, were excluded from the present analysis because the behavior changes and circadian rhythm disruption associated with shift work may confound observed associations. We also excluded participants with missing values in variables for shift work (n = 127), nutrient intake (n = 78), and the covariates described below . We also excluded those with extremely low or high energy intakes , which left 4835 for the analysis.Shift workers , waking up often in the middle of the night (difficulty maintaining sleep [DMS]), and not feeling refreshed when waking up in the morning . The response choices were dichotomous (yes or no).17–19 Regarding the energy-adjusted nutrient intakes determined by the BDHQ used in this study, a validation study that utilized 16-day weighed dietary records is regarded as the gold standard and reported the following correlation coefficients  between the 2 methods: 0.62/0.49 for protein, 0.70/0.65 for fat, and 0.78/0.67 for carbohydrate.19The participants’ dietary habits during the preceding month were assessed with the brief-type self-administered diet history questionnaire (BDHQ), which is a simple version of the self-administered diet history questionnaire (DHQ). Both have been validated and used for nutritional studies in Japan.20 These dietary reference values were established for primary prevention of hypertension, hyperlipidemia, stroke, and myocardial infarction and were based on epidemiologic studies and nutritional experiment studies.20 Percentage of carbohydrate energy intake was classified into 3 categories , as was fat intake . The middle intake category of carbohydrate and fat is the recommended level.20 As for protein, we used quartiles of % energy because there is no established standard.Intakes of carbohydrate and fat were categorized according to values that corresponded to the national dietary goals for preventing lifestyle-related diseases, as recommended in Dietary Reference Intakes for Japanese, 2010.Body mass index (BMI) was calculated by using measured height and weight. Mental well-being was assessed by a question asking whether participants considered their life meaningful and worth living. Responses were classified into 4 categories . Perceived stress was assessed by asking, “Do you have much stress in your daily life?”. Responses were classified into 4 categories . Smoking status was classified into 3 categories ; regular exercise into 2 categories ; alcohol intake estimated by BDHQ (g/day) into 5 categories ; and coffee drinking habit was classified into 2 categories . Medical histories of hypertension, hyperlipidemia, diabetes, angina, myocardial infarction, and stroke were self-reported. A positive history of hypertension, hyperlipidemia, and diabetes was defined as regular doctor's appointments, or prescribed medication, for the respective condition.P-values for the interaction of sex and alcohol intake with intakes of protein and carbohydrate. Two-sided P-values less than 0.05 were regarded as statistically significant. All computations were performed using SPSS version 18.0 for Windows.Logistic regression analysis was used to estimate odds ratios (ORs) and 95% CIs for the 3 insomnia symptoms associated with intakes (% energy) of protein, fat, and carbohydrate adjusted for age, sex, BMI, total energy intake, perceived stress, mental well-being, smoking status, regular exercise, alcohol intake, coffee drinking habit, and medical histories. Age, BMI, and total energy intake were used as continuous variables in the analysis. Testing for linear trend was performed by applying the median intakes of protein, fat, and carbohydrate to each category and analyzing them as continuous variables. For additional analyses of thresholds of insomnia symptoms, participants with a protein intake of 16% or more of total energy were integrated as a reference group in the analysis of DIS and PQS. Those with a protein intake less than 19% were integrated as a reference group in the analysis of DMS. Participants with a carbohydrate intake of 50% or more were integrated as the reference group in the analysis of DMS. Stratified analyses according to sex and alcohol intake were performed to examine confounding and effect modification. For the latter, we calculated P < 0.01) for DIS and DMS, 0.022 (P = 0.12) for DIS and PQS, and −0.079 (P < 0.01) for DMS and PQS. Subjects excluded due to missing values were older (50.7 vs 48.3 years), more likely to be female (22.0% vs 18.6%), and had a higher DIS prevalence (17.3% vs 12.8%).The mean age of participants was 48.3 years. Overall, 54.4% of the study subjects reported having at least 1 of the 3 insomnia symptoms. Overall, 6.7% reported having 2 symptoms, and 2.2% had all 3 symptoms. The prevalences of DIS, DMS, and PQS were 12.8%, 20.3%, and 32.4%, respectively  was inversely associated with DIS and PQS but positively associated with DMS. Carbohydrate intake (% energy) was inversely associated with DMS Table . In multP for interaction for DIS, 0.06; DMS, 0.36; PQS, 0.14), by protein intake and alcohol intake , by carbohydrate intake and sex , or by carbohydrate intake and alcohol intake .In analyses stratified by sex, a protein intake of less than 16% was marginally significantly associated with DIS in men OR 1.30) but not in women (OR 1.11). A protein intake of less than 16% was significantly associated with PQS in men (OR 1.26) but not in women (OR 1.13). A protein intake of 19% or more was significantly associated with DMS in men (OR 1.41) but not in women (OR 1.40). A carbohydrate intake of less than 50% was marginally significantly associated with DMS in men (OR 1.21) but not in women (OR 1.02). A positive association of protein intake with DMS, and marginal inverse associations with DIS and PQS, were also observed in nondrinking subjects . The present study included a large sample size of a general working population that was relatively homogeneous in educational level, income, and work environment and evaluated a large number of covariates to control for potential confounding. In addition, 3 questions were used to assess insomnia symptoms, and a validated dietary questionnaire was used to determine macronutrient intakes.We found that low protein intake was associated with DIS and PQS, while high protein intake was associated with DMS. Low carbohydrate intake was also associated with DMS. Previous findings on the associations of protein and carbohydrate intakes with insomnia symptoms have been inconsistent. A cross-sectional study found an association between low protein and carbohydrate intakes and self-reported symptoms of insomnia .16 Low protein intake could lead to low availability of tryptophan and cause insomnia symptoms. Paradoxically, high protein intake could lower brain tryptophan levels since other competitive large neutral amino acids (LNAAs) contained in protein prevent tryptophan from crossing the blood brain barrier,23,24 which might also cause insomnia symptoms. Another possibility is that high protein intake facilitates catecholamine syntheses by supplying the amino acid tyrosine,25 which could increase alertness.26 Therefore, both too little and too much protein could cause insomnia symptoms. We carried out an additional analysis of the association of protein intake with having at least 1 of the 3 insomnia symptoms and found that the association was U-shaped , which could support our hypothesis. Because the BDHQ did not allow estimation of tryptophan intake, further studies are needed to elucidate the associations between amino acid intakes, such as tryptophan and tyrosine, and insomnia symptoms in this and other populations.Our findings of associations between protein intake and insomnia symptoms might seem inconsistent, since protein intake was inversely associated with DIS and PQS but positively associated with DMS. However, it is possible that some amino acids from dietary protein are associated with insomnia symptoms. For example, tryptophan is a dietary amino acid that serves as a precursor for brain serotonin, a sleep-inducing agent.14,21,22,27 In addition, although alcohol intake was associated with both fat intake and presence of insomnia symptoms, fat intake was not related to presence of insomnia symptom even after statistical adjustment for alcohol intake. In the future, prospective cohort studies must confirm whether such associations are present in different samples.Although presence of insomnia symptoms and fat intake were both inversely associated with sleep duration, no association between fat intake and insomnia symptoms was observed, which was consistent with the findings of previous studies.27 Previous experimental studies suggest that increases in blood glucose level after carbohydrate intake are associated with early sleep onset and deep sleep,15,28 possibly due to the specific effect of carbohydrate on tryptophan transport. By triggering secretion of insulin, carbohydrate lowers the levels of all serum amino acids except tryptophan. This results in higher serum tryptophan levels (relative to LNAAs), which allows tryptophan to cross the blood brain–barrier much more easily than other LNAAs.23,29A previous study found that low carbohydrate intake was associated with shorter sleep duration.30 This may simply be due to the different definitions of insomnia used in the present and previous studies. Both the PSQI and the questionnaire used in the present study are self-reported. However, the PQSI also assesses sleep quantity and quality by inquiring about 18 items and generates a score to define insomnia.31 The question items used in the present study only assess sleep quality by inquiring about the 3 separate insomnia symptoms. Therefore, caution is warranted in interpreting the differences in prevalence across studies. Another study of a Japanese working population, which used a definition similar to that in the present study , found a similar prevalence (24.0% in men and 28.6% in women vs 29.3% in men and 29.4% in women in the present study).32 Fourth, we relied solely on self-reports for the definition of insomnia. However, studies have indicated that self-reported insomnia shows at least moderate agreement with polysomnographic findings.33,34 Fifth, the validity and reliability of the questionnaire items on symptoms of insomnia were not evaluated. Although we included 3 domains of insomnia , which is consistent with the definition of insomnia in the International Classification of Diseases-10 Research Diagnostic Criteria (ICD-10 RDC), the questionnaire did not include frequency and duration of symptoms, which should be at least 3 times per week for at least 1 month according to the ICD-10 RDC. Therefore, presence of a symptom depended on participant perception, and there is thus a possibility of over- or under-reporting. However, such over- or under-reporting would be unrelated to nutrient intakes, and we believe that it would have weakened the association. In addition, participants who had at least 1 insomnia symptom were more likely to have high perceived stress , failure to complete a task in time , which is consistent with ICD-10 RDC criteria that require daytime dysfunction for a diagnosis of insomnia. Moreover, previous studies that used similar items to assess insomnia symptoms have shown similar prevalences of insomnia symptoms.32,35–37 Sixth, depression was not assessed in this study. Although severely depressed workers would not have participated, individuals with mild to moderate depression could have been included, which might have confounded the association, since depression would alter both diet and sleep.38,39 Future studies should include a validated assessment to screen for depression. Last, in women, the association of dietary habit with sleep was insignificant and attenuated, possibly due to the smaller sample size, higher prevalence of insomnia symptoms, and smaller interindividual differences in nutrients intakes as compared with men. However, the directionality of the results was similar in men and women, and no significant effect modification was observed. Thus, we believe that our decision to combine men and women in the analysis was appropriate.There are some limitations to our study. First, causality cannot be inferred from a cross-sectional study. It is possible that workers with symptoms of insomnia may have altered their nutritional intakes. However, the relation between diet and insomnia symptoms is not well-known, and it is thus unlikely that lay individuals would change their diet because of their awareness of such symptoms. Therefore, we speculate that diet influenced insomnia symptoms. Second, the percentage of subjects excluded due to missing values was relatively large (14%). Although the excluded subjects were more likely to have DIS, there were no statistical differences in macronutrient intakes between participants and nonparticipants with DIS. Therefore, we believe that our finding was not due to systematic selection of samples due to missing values. Third, the proportion of individuals with at least 1 of the 3 insomnia symptoms (54.4%) was higher than the prevalence of insomnia in previous reports (30% to 45%), as assessed by the Pittsburgh Sleep Quality Index (PSQI), in the Japanese working population.In conclusion, we found associations between a protein intake of less than 16% of total energy and both DIS and PQS, a protein intake of 19% or more and DMS, and a carbohydrate intake of less than 50% and DMS. Further prospective studies are needed to determine the causal relationships.Abstract in Japanese.


In view of the limited data available on the conservative treatment of patients with congenital scoliosis (CS), early surgery is suggested in mild cases with formation failures. Patients with segmentation failures will not benefit from conservative treatment. The purpose of this review is to identify the mid- or long-term results of spinal fusion surgery in patients with congenital scoliosis.Retrospective and prospective studies were included, reporting on the outcome of surgery in patients with congenital scoliosis. Studies concerning a small numbers of cases treated conservatively were included too. We analyzed mid-term (5 to 7 years) and long-term results (7 years or more), both as regards the maintenance of the correction of scoliosis and the safety of instrumentation, the early and late complications of surgery and their effect on quality of life.A small number of studies of surgically treated patients were found, contained follow-up periods of 4-6 years that in the most cases, skeletal maturity was not yet reached, and few with follow-up of 36-44 years. The results of bracing in children with congenital scoliosis, mainly in cases with failure of formation, were also studied.Spinal surgery in patients with congenital scoliosis is regarded in short as a safe procedure and should be performed. On the other hand, early and late complications are also described, concerning not only intraoperative and immediate postoperative problems, but also the safety and efficacy of the spinal instrumentation and the possibility of developing neurological disorders and the long-term effect these may have on both lung function and the quality of life of children.Few cases indicate the long-term results of surgical techniques, in the natural progression of scoliosis. Similarly, few cases have been reported on the influence of conservative treatment.In conclusion, patients with segmentation failures should be treated surgically early, according to the rate of deformity formation and certainly before the pubertal growth spurt to try to avoid cor- pulmonale, even though there is lack of evidence for that in the long-term. Furthermore, in patients with formation failures, further investigation is needed to document where a conservative approach would be necessary. Noted in about 1 in 1000 births, congenital scoliosis is the most common congenital spinal disorder, followed by congenital kyphosis and lordosis [1 - 2]. The vertebral disorders that cause Congenital Scoliosis may be due to either failure of formation or failure of segmentation or a combination of these, leading to a mixed deformity [1 - 4]. Complete failure of formation leads to hemivertebrae with the absence of one pedicle and a region of the vertebral body, while incomplete failure of formation leads to a wedged vertebra [1 - 4]. Both types of malformations may be lateral, causing scoliosis; posterolateral, causing lordoscoliosis; dorsal, causing lordosis; anterolateral, causing kyphoscoliosis or ventral, causing kyphosis . Patients with proximal thoracic deformity who require fusion of more than four segments, especially those with rib anomalies, are at the highest risk of developing restrictive pulmonary disease . FurtherApart from clinical functionally indicators, quality of life is also affected. Compared with healthy peers, congenital scoliosis patients treated with early spinal fusion present differences in the PFT and Quality of Life (QOL) scores at 6.9 years follow-up. Patients with thoracic fusions had shorter spines, worse pulmonary function, and more pain than the non-thoracic fused. In addition, they presented lower physical functioning and physical summary values, compared with healthy children . A contrCrankshafting was observed in 15% of the patients, more often with larger curves and earlier fusions .Thompson et al Table  41], re, re41], Marks et al Table 42], exa, exa42],). Low back pain began 22 years after surgery (at 23 years) and cervical pain 24 years after surgery (at 25 years) [Winter and Smiths study a case with a 44-year follow-up after surgery at the age of 1 year for congenital scoliosis (Table 15 years) . Anterio). The levels of the hemivertebrae were from T8 to L5. Spine-related anomalies were noted in five cases, including one case of skin dimple, one case of cervical rib and three cases of spinal bifida. Other congenital anomalies were noted in 3 cases with congenital heart diseases, 2 cases with genitourinary anomalies, 2 cases with gastrointestinal anomalies, one case with craniofacial deformity and 2 cases with mental retardation. Preoperatively, the 19 patients who underwent posterior instrumentation surgery, showed curves between 24° and 65°, which improved postoperatively at a range between 17° and 52°. No neurological complications were observed after surgery. The complications that occurred were one case with superficial wound inflammation, 1 case of loss of repair and one case with broken screws. Nevertheless, during the follow-up, there was a progression from 20° to 53°. Three skeletally mature patients experienced severe back pain, which improved after fusion in situ [Te Chen and Wang studied single level hemivertebrae in 22 patients, 8 male and 14 female, over an average of 19.3 years (Table 1 in situ . Interes in situ .). Of these, four were skeletally mature, with Risser signs of 4 and 5, while the rest immature with Risser signs of 0 to 3. The pre-operative curve was 32.9°, the operation it was 11.2° and, in the last follow-up 9.4°. Immediate complications included mild radiculopathy, followed by a complete recovery and superficial wound infections. In one patient, the spinal implants were removed due to rod fracture, while one patient presented hook displacement, which necessitated hook reinsertion. In addition, a patient presented pseudarthrosis that required bone grafting and revision of posterior instrumentation to treat progressive kyphosis.Another study with long-term follow-up of thoracolumbar hemivertebrae resection by double approach, by Bollini et al.  involved1), with a mean follow-up of 51 years involved seven case reports on congenital scoliosis [The last study, by Winter and Lonstein Table , with a coliosis , some trAnother retrospective study, by Ruf, Jensen et al, relating to hemivertebra resection, examined the impact of intervention in 41 patients who underwent 51 consecutive hemivertebrae resections  has been reduced with the Chêneau braces applied from initially 17° to 9° at the age of 16 years, during the last SIR . His VC  was 1.640 ml, 3% of the predicted value (Figure Another two conservatively treated cases of congenital scoliosis caused by wedged vertebrae were reported by Cheneau, Grivas et al . The firDue to the limited number of references in the literature, further research is deemed necessary to document whether health-related signs and symptoms improve in the long-term, when spinal fusion is performed in patients with congenital scoliosis. It is also necessary to identify their role in the natural history of congenital scoliosis and their impact on a growing spine.The same would be desirable for cases where conservative treatment was applied.Although studies were reported on case series of patients with formation failures which followed conservative approach, its indications are not yet sufficiently documented.On the contrary, patients with segmentation failures should be treated surgically as early as possible, according to the rate of deformity formation and certainly before pubertal growth spurt to try to avoid cor pulmonale, even though there is lack of evidence for that in the long-term.The authors declare that they have no competing interests.All the authors read and approved the final manuscripts. ΑΚ: Participated in the literature search on PubMed and in drafting the paper. TBG: Participated in the literature search on PubMed and in drafting the paper. HRW: Had the idea for the study, participated in the manuscript drafting, the PubMed search, and provided the figures. DT: Participated in the manuscript drafting, copyediting and research of databases.


It has been suggested that soy isoflavones have protective effects against breast cancer. However, data from epidemiological studies are not conclusive. A recent meta-analysis showed that soy intake was inversely associated with breast cancer risk in Asian but not Western populations, which indicates that protection against breast cancer may require that women consume levels of soy typical in Asian diets. In addition to the amount of soy isoflavones consumed, the form and food source of isoflavones, timing of isoflavone exposure, estrogen receptor status of tumors, and equol-producer status and hormonal profile of individuals may modify the association between soy isoflavone intake and the risk of breast cancer. These factors might explain the heterogeneity of results from studies. This present report contrasts background data from Japanese and Western women to identify the potential modifying of these factors. Isoflavones also inhibit the activity of key enzymes that convert androgens to estrogens.2 In addition, isoflavones have been shown to be anti-proliferative,3 proapoptic,4 anti-angiogenic,5 anti-oxidative,6 and anti-inflammatory.7 Taken together, these findings indicate that soy isoflavone may be a potent agent for preventing breast cancer. However, some data suggest that isoflavones promote breast cancer. Indeed, it has been shown that genistein stimulates the growth of estrogen-sensitive breast cancer cells in ovariectomized mice.8,9 Because of the apparent complexity of the relationships between isoflavones and breast cancer in laboratory studies, data from human studies are particularly important. However, current data from epidemiological studies are not conclusive. There is some epidemiological evidence of an inverse association between isoflavone and breast cancer, but it is inconsistent.10 Thus, no consensus has emerged regarding the preventive aspects of isoflavones.There has been much interest in the potentially protective role of soy in breast cancer development. Soybeans are the main source of isoflavones, which are classified as phytoestrogens. Laboratory data show that isoflavones have a wide range of biological actions. First, they have an affinity for estrogen receptors in vitro.11 A meta-analysis of 8 studies showed that Asian women consuming the highest amount (≥20 mg/day) of dietary isoflavones had a 29% reduction in breast cancer risk, as compared with those with low consumption (<5 mg/day) of isoflavones. An approximately 50% reduction in risk was reported in a prospective study of Japanese women.12 In contrast, a meta-analysis of 11 studies of women eating Western diets found no association between isoflavone intake and breast cancer risk. In that analysis, the median for the highest intake was 0.8 mg/day. Therefore, women may need to consume the amount of soy in Asian diets to gain protection against breast cancer. Alternatively, the protective effects may only be present when exposure to soy occurs early in life. Differences in (1) the form and source of isoflavones, (2) the biological response to isoflavones among ethnic groups, and (3) the interactions with individuals’ hormonal profiles may also explain the heterogeneity of the findings. This present report attempts to address these issues by contrasting background data from Japanese and Western women.Recently, Wu et al conducted 2 separate meta-analyses of studies carried out in Asian and Western populations.13,14 suggests that lifestyle changes, including a change in dietary isoflavone intake, may play a role. According to the Cancer Incidence in Five Continents Vol IX,15 the incidence rate for breast cancer in 1998–2002 was 32.0 per 100 000 among Japanese women in Osaka, Japan; 105.6 per 100 000 among white women in Hawaii; and 107.5 per 100 000 among Japanese women in Hawaii.The incidence rate of breast cancer is historically much lower in Japan than in Western countries. One possible explanation for this is that Japanese consume much more soy foods containing isoflavones. The fact that the incidence rate of breast cancer among Japanese immigrants rises as the length of time in the host country increases16 for more than 50 years. About 20 000 individuals from 6000 households in a randomly selected 300 districts are invited to participate in this survey every year. Foods and beverages consumed by a family are weighted and recorded for 3 consecutive days (1-day records have been used since 1995). According to the NNS reports, the mean intake of soy products was 67.2 g/day in 1975 and 57.7 g/day in 2005. Recently, the Food Safety Commission in Japan estimated isoflavone intake (expressed as aglycone) among Japanese, using the 2002 NNS data (response rate unknown). The median and the 95th percentile isoflavone intakes were 18 and 70 mg/day, respectively. Mean estimates of daily consumption reported in other studies in Japan ranged from 26 to 54 mg.17 A database of the isoflavone content of Japanese foods, based on a validated direct measurement method, was first reported by Kimira et al.18 In a report from the Takayama study—a prospective study of residents in Takayama city, Gifu, Japan19—the mean estimate, based on a validated food frequency questionnaire, was 19.6 mg per 1000 kcal among 15 724 female residents aged 35 years or older .20 Daily intake of isoflavones is estimated to be between 0.5 and 3 mg in the United States21,22 and less than 2 mg in European countries.23 In a recent study of 8809 adults from the National Health and Nutrition Examination Survey (NHANES), the mean isoflavone intake, based on one 24-hr diet recall, was 1.2 mg.24 Changing dietary habits have been reported in migrant populations. For example, soy intake among Japanese-Americans appears to be higher than that of US whites and lower than that of Japanese living in Japan. Takata et al25 reported that mean isoflavone intake, estimated from a food frequency questionnaire, was 11.6, 7.2, and 3.5 mg per 1000 kcal in Japanese women in Gifu, and Japanese and white women living in Hawaii, respectively. According to the 2002 NNS data in Japan, isoflavone intake was associated with age. Even among adults, younger age groups tended to consume less isoflavones: the reported mean daily intake of isoflavones was 19.4, 21.5, 23.5, 27.4, 33.6, and 29.6 mg per day for adults aged 20–29, 30–39, 40–49, 50–59, 60–69, and 70+ years, respectively. Soy isoflavone intake will likely decrease among Japanese in the future.The nutritional status of Japanese has been evaluated annually by the National Nutritional Survey (NNS)26 collected 24-hour urine and serum samples from Japanese women enrolled in the Japan Public Health Center-based (JPHC) prospective study. The mean levels for daidzein and genistein were 17.0  and 14.2 (14.1) µmol/day, respectively, in urine. The corresponding values in plasma were 119.9 (135.8) and 475.3 (510.4) nmol/L, respectively. These values were reasonably correlated with isoflavone intake estimated from food-frequency questionnaires or diet records (r = 0.22–0.48). Similar values in the urine, plasma, and serum of Japanese women have been reported in other studies.27–29 Even in spot urine samples, isoflavone levels were well correlated with intakes estimated from diet records (r = 0.30 for daidzein and 0.27 for genistein).30 In a study of 59 women aged 20 to 45 years, the median values were 4.0 and 3.2 nmol/mg creatinine for daidzein and for genistein, respectively, in first morning urine specimens.31 Another study of 419 women reported that the mean daidzein level was 12.8 nmol/mg creatinine in spot urine samples collected at approximately 2:00 PM.32 Among Western populations, reported urinary isoflavone levels have been much lower. The geometric mean levels were 0.27 and 0.08 nmol/mg creatinine for daidzein and genistein, respectively, in urine specimens of participants in the NHANES between 1999 and 2000.33 In a study of UK women, the geometric means for daidzein and genistein were 0.55 and 0.28 nmol/mg creatinine, respectively, in spot urine, and 7.9 and 15.2 µmol/L, respectively, in serum.34Biomarkers are also useful in estimating exposure to soy isoflavones. Yamamoto et al22 Such uses are increasing.35 Recently, new isoflavone datasets for non-soy based foods have been developed to estimate isoflavone intake more accurately. Horn-Ross et al22 assessed the isoflavone intake in 447 non-Asian women in the San Francisco Bay area, using a new database. Mean intakes of genistein and daidzein were estimated to be 1.5 and 1.3 mg/day, respectively. Major sources were tofu, doughnuts, soymilk, white bread, and canned tuna. Interestingly, doughnuts accounted for about 20% of the average of daily intake of genistein and 15% of daidzein intake. It is likely that Japanese women are also consuming isoflavones from such “hidden” sources of soy. Using several databases of non-soy foods, such as cereals, eggs, dairy foods, meats, fish, nuts, and vegetables,21,36–39 and assuming that processed meats and fish contain 2% soy protein by weight, isoflavone intake from these non-soy foods was estimated to be approximately 0.68 mg per 1000 kcal among the women in the Takayama study (unpublished data). This value is substantially lower than the isoflavone estimate from soy foods .Sources of isoflavones also differ between Japanese and Western populations. The intake of traditional soy-based foods is high in Japan. Based on data from the Takayama study, the most common soy foods are tofu, miso (soybean paste), soybeans, fried tofu, natto (fermented soybeans), and soymilk. The fermented soy foods (miso and natto) accounted for about 40% of total isoflavone intake. In Western populations, the consumption of traditional soy foods is substantially lower, and a portion of total isoflavone consumption is derived from soy protein and soy flour added to a variety of foods.39 Knowing the bioavailability of the different isoflavone forms is thus fundamental. Watanabe et al conducted a kinetic study of Japanese men after ingestion of 60 grams baked soybean powder (kinako).40 Differences in the form of isoflavone (aglycone vs glucoside) are believed to alter isoflavone pharmacokinetics and, hence, the association between soy intake and breast cancer risk. However, there is no consensus among studies on the bioavailability of aglycone vs glucoside.41 In addition to isoflavone form, the matrix in which it is delivered is likely to have an effect on pharmacokinetics. It is also possible that an individual’s background diet may modify the association of isoflavone intake with breast cancer risk by affecting the pharmacokinetics of metabolism of isoflavones. In addition, frequency of ingestion may affect bioavailability of isoflavones.41 An optimum steady-state serum isoflavone level would be expected from consuming relatively small doses of soy throughout the day, as opposed to a single dose at one time. Indeed, Gardner et al42 observed that plasma isoflavone concentrations remained constant when soy foods were consumed at breakfast, lunch, and dinner.In fermented soy products, like natto or tempeh, aglycones are the principal form of isoflavone, whereas in unfermented soy products, like soy milk or soy supplements, the glucoside form is predominant.43 Equol, a metabolite of daidzein, has been identified in the urine and blood samples of some but not all humans. Intestinal bacteria play an essential role in daidzein metabolism. Several candidate bacteria for daidzein metabolism, including one reported by Ueno et al,44 have been suggested, but the human intestinal bacteria responsible for daidzein metabolism are yet to be identified.45 Equol binds with greater affinity to estrogen receptors than its parent compound daidzein,46 and it is superior to all other isoflavones in its antioxidant activity,47 which suggests that its overall potency is greater. The prevalence of equol producers appears to be higher in Japanese than in whites,27,48,49 which might explain the additional benefits conferred on Japanese women in terms of reduced breast cancer risk.Metabolism of isoflavone can vary greatly between individuals, even when they consume an identical amount of isoflavone-containing food.50 possibly because of alterations in gene expression and morphological changes in the mammary glands. The details have been described elsewhere.51 To this author’s knowledge, 3 case-control studies have examined the association between soy intake during childhood or adolescence and the risk of breast cancer among Asian women.52–54 All reported a significant inverse association. Another case-control study of Canadian women also observed a significant inverse association between adolescent isoflavone intake and breast cancer risk.55 A recent prospective study of Chinese women confirmed that a high intake of soy foods during adolescence was associated with a reduced risk of breast cancer.56 Unfortunately, there has been no such study among Japanese women.In vivo studies have consistently shown that prepubertal or pubertal exposure to genistein reduces the incidence and/or multiplicity of chemically induced mammary tumors in experimental animals,57 Some epidemiological studies observed associations between pre- and perinatal characteristics, such as birth order and birth weight, and subsequent risk of breast cancer.58 There has been no study assessing the association between maternal soy intake during pregnancy and the risk of breast cancer in offspring; however, by altering the estrogen environment, soy exposure in utero may affect the subsequent risk of breast cancer.Perinatal factors are also thought to influence subsequent breast cancer development. Trichopoulous hypothesized that the developing breast is influenced by the fetal environment, particularly variations in hormone concentrations, which could mediate subsequent breast cancer development.59 reported that in 7 Japanese women at delivery the mean levels of genistein and daidzein were 83.9 and 45.5 nmol/L, respectively, in maternal plasma, and 165 and 58.8 nmol/L, respectively, in umbilical cord plasma. They observed high correlations between levels in maternal and umbilical plasma . A study by the present author, which included 194 pregnant women, supported the hypothesis that isoflavone can be transferred from mother to fetus.30 In that study, we also included an assessment of maternal dietary intake of soy during pregnancy and measurements of estrogen levels in maternal and umbilical cord blood; the mean soy isoflavone intake was 21.7 mg per day. The geometric mean levels for serum genistein and daidzein were 116.5 and 50.2 nmol/L, respectively, in maternal blood at delivery, and 126.9 and 38.6 nmol/L, respectively, in umbilical cord blood. Estradiol level in umbilical cord blood was unrelated to isoflavone levels in both maternal and umbilical cord blood.In utero soy exposure occurs among Japanese. Adlercreutz et al60 reported that soy-based formulas contained approximately 32 to 47 µg/ml of isoflavone, indicating that a 4-month old infant fed soy formula would consume 28 to 47 mg of isoflavones per day. In their study, the mean plasma concentrations of genistein and daidzein were 2530 and 1160 nmol/L, respectively, in 7 infants fed soy-based formulas. An isoflavone intake of 3 mg for Japanese infants is not low, if we take into account the body size of babies; however, it would be much lower than that available from a soy-based formula. Badger et al61 reported substantial differences between exposure to soy between Japanese and Americans. If these differences are real, then Japanese fetuses would be exposed to the same high isoflavone levels that were present in maternal circulation. Serum isoflavone levels among Japanese infants would then be expected to decline at birth and remain low, until soy foods were introduced as baby food. Thereafter, the level would increase to the usual adult levels as a result of habitual intake. American infants fed soy formula would be expected to have high serum levels starting immediately after birth and persisting until they were weaned, at which time levels would drop and likely remain low thereafter.Infants in Japan are weaned on to soy products between 6 and 12 months of age, after which they continue to receive isoflavone-containing foods indefinitely; tofu and miso soup are common baby foods. Based on intake frequencies of tofu, miso soup, natto, soybean flour, and soymilk reported by 288 mothers, 6-month-old Japanese infants consumed about 3.1 mg of soy isoflavone per day (unpublished data). Setchell et al62,63 Because isoflavones have an affinity for estrogen receptors, the protective effect of soy intake may be pronounced for estrogen receptor-positive tumors. To date, 6 studies have assessed the association of soy or isoflavone with breast cancer risk, with regard to the estrogen receptor status of tumors.34,64–68 Some of these studies, including 1 conducted in Japan, observed an inverse association of soy intake with the risk of estrogen-positive tumors,64,65,67 but not with the risk of other tumor types.34,66,68 The incidence rate of estrogen receptor-positive tumors is increasing in the United States.69 In addition, it has been reported that the prevalence of estrogen receptor-positive expression in normal breast tissue from Japanese women was lower than that in white women.70 This may simply be a result of the lower incidence of breast cancer among Japanese women. The determinants of estrogen receptor status in normal breast tissue and tumors are not known.Epidemiological studies have found that several hormone-related lifestyle factors, such as nulliparity, earlier age at menarche, higher body mass index (BMI), and use of postmenopausal hormones were related to elevated risk of estrogen receptor-positive, but not estrogen receptor-negative, cancers.71 Evidence from case-control studies has supported a protective role for soy in premenopausal women versus postmenopausal women.10 The effects of soy and isoflavone on the level of circulating estrogen have been examined in many intervention studies. A recent meta-analysis of intervention studies revealed that although soy and isoflavone consumption did not affect estradiol or estrone, it did reduce FSH and LH in premenopausal women.72 Moreover, in postmenopausal women, soy and isoflavone were associated with a small but nonsignificant increase in total estradiol. These observations lend tentative support to the above hypothesis. However, in a cohort study that observed a significant inverse association between soy isoflavone intake and breast cancer risk among Japanese women, the association was somewhat stronger in postmenopausal women than in premenopausal women.12 A similar tendency was noted in another recent cohort study of Asian women.68 In addition, some studies have observed lower endogenous estradiol levels in low-risk populations than in high-risk populations.73,74 Although postmenopausal estrogen levels should not be compared when measured by different laboratories, estrogen levels appear to be low among postmenopausal Japanese women .75–79 Such a low-estrogen environment, which likely reflects low body fat mass, may favor the protective effects of soy on breast cancer in Japanese women. Some studies of soy and breast cancer risk included analyses stratified by BMI but not by estrogen level. The results were not consistent: one observed a somewhat stronger association in women with high BMI,55 but others noted no significant differences.68,80It has been suggested that isoflavones have stimulatory effects in low-estrogen environments, and that in high-estrogen environments, they block the effects of estrogen.The causes of breast cancer remain unclear. Established risk factors include age, race/ethnicity, reproductive factors, and obesity. Unfortunately, there are few modifiable factors. Dietary factors have been implicated in the etiology of breast cancer and soy/isoflavone has been a candidate for dietary intervention. However, existing evidence from epidemiological studies is not conclusive and there have been few prospective studies of the issue. Estimation of soy and isoflavone intake, like other dietary components, is subject to measurement error. In addition, the existence of hidden sources of soy makes it difficult to estimate total isoflavone intake accurately, especially among “nonconsumers” of soy-based foods.There is a need for more, methodologically sound, prospective studies—with extensive exposure measurement. To interpret the data, we need to consider the factors described above, namely, isoflavone dose, forms and sources of isoflavone, timing of isoflavone exposure, and the equol-producer status, estrogen-receptor status, and hormonal profile of individuals, as these are all factors that potentially modulate the association between soy intake and breast cancer risk. Other dietary, environmental, and genetic factors may also modify the association. Future studies need to address these questions by including samples large enough to detect the factors that are capable of modifying the associations between soy and breast cancer risk.


We identified a rare p.A152T variant in MAPT exon 7 in two (of eight) patients with clinical presentation of parkinsonism and postmortem finding of neurofibrillary tangle pathology. Two siblings of one patient also carried the p.A152T variant, and both have progressive cognitive impairment. Further screening identified the variant in two other cases: one with pathologically confirmed corticobasal degeneration and another with the diagnosis of Parkinson's disease with dementia. The balance of evidence suggests this variant is associated with disease, but the very varied phenotype of the cases with the mutation is not consistent with it being a fully penetrant pathogenic mutation. Interestingly, this variation results in the creation of a new phosphorylation site that could cause reduced microtubule binding. We suggest that the A152T variant is a risk factor associated with the development of atypical neurodegenerative conditions with abnormal tau accumulation.Microtubule-associated protein tau  Here, we report two patients with atypical parkinsonian disorder and abnormal tau accumulation at postmortem, both of whom were identified to carry the A152T variation. We then screened a larger series of tauopathy cases for this mutation (PSP and CBD) and some cases with idiopathic Parkinson's disease to determine the nature of the phenotype that seemed to be associated with this variant and found an additional case with CBD as well as a case of Parkinson's disease in which there was prominent tangle pathology.Tau belongs to the family of the microtubule-binding proteins (MT)  and is e (17q21) . MAPT muia (FTD)  but alsoia (FTD) . Recentlauopathy . With th2All subjects in this report had provided written consent to perform neuropathological and genetic studies. The access to clinical records and pathological material at the Queen Square Brain Bank (QSBB) has generic ethical approval from a London Multi-Centre Research Ethics Committee under a license from the Human Tissue Authority.2.1After postmortem the brains were divided midsagittally. One half brain was immediately frozen and stored at −80 °C, whereas the other half was immersed and fixed in 10% neutral formalin for 3 weeks. Tissue blocks were processed using standard protocols. We performed hematoxylin and eosin, Luxol fast blue/cresyl violet, Congo red staining on 7-μm-thick sections and also used the modified Bielschowsky and Gallyas silver impregnation methods. Immunohistochemistry with antibodies to phospho-tau (AT8 clone recognizing Ser202/Thr205), 3-repeat (3R) and 4-repeat (4R) tau isoforms , ubiquit2.2MAPT gene open reading frame in eight cases that had received a clinical diagnosis of postencephalitic parkinsonism (PEP) or clinical parkinsonism with unclassifiable tauopathy at postmortem. PEP is a rare clinical entity characterized by the development of parkinsonism after the development of encephalitis . Initial postmortem studies indicated PEP in case 1, described later in the article, and was previously reported as such . The original neuropathological investigation did not reach a conclusive diagnosis in case 2, which had been categorized as “parkinsonism associated with unclassifiable neurofibrillary tangle pathology”.In our initial DNA-sequencing study, we sequenced the entire 2.3PARK2 and LRRK2 genes had also been previously fully sequenced without finding any changes. Three of these eight cases have been previously reported   . After t2.4research.microsoft.com/enus/um/redmond/projects/mscompbio/FisherExactTest/. A p-value less than 0.01 was considered statistically significant.Fisher exact tests were conducted using an online tool 2.5MAPT exon 7 in DNA derived from the brains of the QSBB archival collection, including 114 cases with PSP   Environmental Genome Project, Seattle, WA [URL: evs.gs.washington.edu/niehsExome/] [accessed on 03/2012]) revealed similarly low frequencies of the A152T variant  reported earlier were identified to carry a heterozygote nonsynonymous variant in exon 7  (accession numbers ctively) A and 1B. variant   . At age Neuropathological review of this case has confirmed significant degree of neuronal loss in the substantia nigra, which was most severe in the ventrolateral tier. The tau pathology in the substantia nigra included neurofibrillary tangles (NFTs) and numerous, either fine or coarse, neuropil threads (NTs). Sparse NFTs and NTs were also seen in the midbrain tegmentum and, in addition, amyloid plaque-associated abnormal neurites were observed in the midbrain tectum. There were scattered NFTs and tau-positive threads in the striatum; occasional NFTs and threads were also seen in the globus pallidus and subthalamic nucleus. There was a single NFT with sparse NTs in the cerebellar dentate nucleus. There were amyloid-β (Aβ)-positive diffuse and mature plaques in the frontal, parietal, and temporal cortices. The tau pathology, which included classical NFTs, NTs, and plaque-associated neurites, was severe in the CA1 hippocampal subregion, entorhinal cortex, and fusiform gyrus, and it corresponded to Braak and Braak stage IV . The tauPARK2 and LRRK2 genes would indicate that an, as yet, unidentified neurodegenerative process was responsible for the underlying nigral cell loss. An alternative hypothesis is that the mild subcortical tau pathology with deposition of both 3R- and 4R-tau is related to the MAPT p.A152T mutation and an atypical tauopathy is responsible for the nigral cell loss.After re-visiting the neuropathology of this case, our findings, as noted earlier, were not compatible with a pathological diagnosis of PEP. One might argue that the tau pathology, including the abundant NFTs and NTs in the substantia nigra, and the milder tau pathology in the subthalamic nucleus and globus pallidus can be entirely “age-related” . In this3.1.33.1.3.1When in her late 50s, this English woman began to develop a symmetrical akinetic rigid syndrome. Two years later, examination revealed hypomimia, drooling, predominant axial rigidity with mild symmetrical limb rigidity, brisk deep tendon reflexes, and flexor plantar response. A diagnosis of Parkinson's disease was made; however, there was no response to 700 mg/day of levodopa therapy. Her symptoms gradually deteriorated, and 7 years later, she developed dysarthria and dysphagia. In her mid 60s, she started to experience daily episodic dystonic spasms with an opisthotonus posture and oculogyric crisis. Examination in between these episodes revealed normal pursuit and saccadic eye movements, fixed retrocollis, slow tongue movement, anarthria, severe axial and limb rigidity, and dystonic posturing of the feet. Her cognitive function remained intact. The presence of oculogyric crisis prompted the neurologist to revise the diagnosis to PEP in the last year of her life. She died after disease duration of 10 years. However, in view of her lack of levodopa response, short disease duration, and the lack of encephalitis history, the clinical diagnosis of PEP would be very unlikely.She has three younger sisters . The secReview of this case with immunohistochemistry using the AT8, 3R-tau, and 4R-tau antibodies indicated that the overall neuropathological findings fulfilled the diagnostic criteria for PSP , as ther3.2MAPT gene in 114 PSP, 8 CBD, and 48 idiopathic Parkinson's disease cases from the QSBB archival collection. The mutation was identified in a case of CBD. More surprisingly, it was also found in one of the idiopathic Parkinson′s disease cases.After obtaining the findings outlined earlier in the article, we screened exon 7 of the 3.2.13.2.1.1The CBD case developed symptoms in the mid-sixth decade, with early word finding difficulty and right-sided rigidity and gait difficulty evolving through nonfluent aphasia to mutism, increasing dependency, and death 11 years after symptom onset. There was no documented family history of dementing illness. The pathology included achromatic swollen neurons in the neocortex and limbic areas, numerous pretangles and NFTs, dense meshwork of tau-positive threads, astrocytic plaques in the frontal, temporal, and parietal cortices, and tau-positive threads and some coiled bodies in subcortical white matter. Additionally there were numerous TDP-43-positive neuronal cytoplasmic inclusions (NCIs) and neuronal intranuclear inclusions in globus pallidus, caudate, and putamen and severe cell loss in substantia nigra.3.2.23.2.2.1123I]FP-CIT DAT images, a diagnosis of Parkinson's disease with dementia was made when he was in his early 50s. He was commenced on levodopa therapy with moderate response. His cognitive function deteriorated markedly in the following year with fluctuating consciousness, disorientation, urinary incontinence, severe dementia, visual hallucination, myoclonus, and left-sided predominant akinetic rigidity. He died 7 years after the onset of his first symptoms.In his late 40s, this man developed an anxiety disorder and poor concentration, which resulted in him getting dismissed from his job. In the following year, he started to stutter and was diagnosed with depression. Two years later, he had memory loss, executive impairment, and slowness in movement. After the subsequent findings of markedly reduced tracer reuptake in the basal ganglia on [Neuropathological findings included moderate to severe cell loss in the substantia nigra in association with α-synuclein-positive Lewy bodies and Lewy neurites. Frequent cortical Lewy bodies were observed in the frontal, temporal, parietal, cingulate gyrus, and transentorhinal cortices, corresponding to Braak stage 6 , diffuse4MAPT gene that contributed to those cases with complex tauopathies, previously often diagnosed as PEP. Our finding of the p.A152T MAPT mutation in two of eight of these cases, together with the previous report from Our intention in this study was to try and understand whether there were genetic factors in the MAPT mutations, the tau pathology was very varied, both in morphology and distribution: being 4 repeat in cases 2 and 3 and a classic mixture of 3 and 4 repeat in cases 1 and 4.However, the identification of the variant in a case of idiopathic PD was a surprise, even though this case is unusual for such a young case in having a considerable burden of tau pathology. Additionally, the disease picture in all the cases we report varies both clinically and pathologically, and although we show some evidence for segregation in the single family in which this analysis was possible, in most cases family history was not reported and, unlike in classic cases with MAPT splice and P301L mutations do. The mutated residue is next to Thr153 (T153), a phosphorylation site which is a target for proline-directed kinases such as mitogen-activated protein kinase (MAPK), cell division protein kinase 5 (cdk5), and glycogen synthase kinase-3 alpha (GSK3α). Using an antibody against the phosphorylated T153 (pT153), The position of the variant is interesting in that it creates an additional phosphorylation site in the tau protein, and tau hyperphosphorylation has been repeatedly suggested to contribute to tangle formation and cell death sites . One plaLRRK2 mutations offer a precedent for this complex relationship between mutations and disease. Some mutations  cause disease in an almost fully penetrant autosomal dominant fashion  and H1N1 influenza A virus pandemics in Europe from the late 1910s until the mid-1920s . It has 5We conclude that the rare p.A152T variant is likely to increase the susceptibility to the development of neurodegenerative conditions with abnormal tau accumulation. Further functional studies are necessary to fully dissect the functional consequences and precise pathogenic mechanisms associated with this mutation. The evidence for the pathogenicity of this variant is not strong enough for these data to be used in genetic counseling.None of the authors have potential or actual conflicts of interest, and all the authors have seen the manuscript before submission.MRC/Wellcome Trust through a PD Centre Grant, and by the Wellcome Trust. The funding source had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The work was funded by the Reta Lila Weston Foundation, the PSP Brain Bank, USA Parkinson's Disease Foundation and the There is no animal work in the manuscript, and the human work on blood and pathology materials has been carried out in compliance with UK regulations.


The patterns of emergence and diversification of the families of ubiquitin ligases provide insights about the evolution of the eukaryotic ubiquitination system. U-box ubiquitin ligases (UULs) are proteins characterized by containing a peculiar protein domain known as U box. In this study, the origin of the animal UUL genes is described.UBE4A, UBE4B, UIP5, PRP19, CHIP and CYC4) were already present in the ancestor of all current metazoans and the seventh (WDSUB1) is found in placozoans, cnidarians and bilaterians. The fact that only 4 - 5 genes orthologous to the human ones are present in the choanoflagellate Monosiga brevicollis suggests that several animal-specific cooptions of the U box to generate new genes occurred. Significantly, Monosiga contains five additional UUL genes that are not present in animals. One of them is also present in distantly-related protozoans. Along animal evolution, losses of UUL-encoding genes are rare, except in nematodes, which lack three of them. These general patterns are highly congruent with those found for other two families  of ubiquitin ligases.Phylogenetic and structural data indicate that six of the seven main UUL-encoding genes found in humans  are parallel indicates that there are underlying, linage-specific evolutionary forces shaping the complexity of the animal ubiquitin system. In eukaryotes, protein ubiquitination is a key biochemical mechanism involved in multiple cellular processes, which range from its main role in the control of protein quality and protein levels, as part of the ubiquitin-proteasome system, to the regulation of gene expression -3. UnderIn some cases, the origin of these E3s can be traced back to early eukaryotic evolution. For example, three of the twelve subfamilies of the RBR family  ,8 or theUBE4A and UBE4B genes , CHIP , UIP5 (a. k. a. UBOX5), PRP19 , CYC4  and WDSUB1 [ACT1 (a. k. a. TRAF3IP2) has been described as encoding a ubiquitin ligase that may contain a very divergent U box [To further determine the generality of this pattern, I decided to undertake the study of another important type of E3 proteins, the U-box ubiquitin ligases (UULs). UULs were discovered more than ten years ago . They ard WDSUB1 ,12,14. Rnt U box .parkin gene are a well-known cause of familial Parkinson disease, and therefore it is of significant interest that Chip is acting also on the products of several other familial Parkinson disease genes, such as SNCA  and ; [LRRK2 ,24. The ; [LRRK2 -28), Mal; [LRRK2 ) and, fi; [LRRK2 ,31) indiy levels .PRP19 encodes a splicing factor required for the activation of the spliceosome in both Saccharomyces cerevisiae [Drosophila and mouse Prp19 null mutants are lethal [UBE4A and UBE4B, are also related to a yeast gene, Ufd2. This non-essential gene was first characterized as encoding a protein able to elongate polyubiquitin chains (E4 protein) [Caenorhabditis elegans, the products of the orthologs of UBE4B and CHIP, called respectively Ufd-2 and Chn-1, form a complex with the chaperone Cdc-48  and ubiquitinate another chaperone, called Unc-45, which is required for correct muscle formation [Ube4b mutations in homozygosis die before birth, while heterozygotes show multiple nervous system anomalies that worsen with age [UBE4B also leads to neurodegeneration in parallel to the generation of ubiquitin inclusions [CYC4, originally called Cyp-60, was first characterized as encoding a divergent cyclophilin, which was only much later shown to be also a UUL [C. elegans, the ortholog of CYC4, called mog-6, is involved in the switch from spermatogenesis to oogenesis in hermaphrodite worms [WDSUB1 are totally unknown.Much less is known of the functions of the rest of UULs. revisiae  and mammrevisiae . It is previsiae ,35 Althorevisiae , the pree lethal -38. Two protein) . Evidencprotein) . As descprotein) ,39. The protein) ,41). In ormation ,43. It hormation . Presencormation . Whetherwith age . Overexpclusions . CYC4, oso a UUL ,48. Someso a UUL . The bioso a UUL ,50. In Cte worms . The laste worms . The rolMonosiga brevicollis. I show that these genes follow the same evolutionary pattern as other E3s: diversification in early animal history followed by simplification in particular lineages. In most groups, these UUL-encoding genes are very conservative, but, as described for other ubiquitin ligase families (see above), nematode species turn out once again to be exceptional, given that they have lost three UUL-encoding genes. The functional implications of these results are discussed.Research in UUL proteins and UUL-encoding genes in animals would benefit by having them included in an evolutionary framework, which provides three different types of data. First, the patterns of presence/absence of the genes in different species, useful not only to understand the evolution of the genes, but also to determine the best animal models and eventually to better understand the phenotypes found in mutants . Second, the speed of the evolution of a same gene in different species, which may suggest either differences in their evolutionary constraints or new functional roles. Finally, the patterns of evolution of the structures of the proteins, which, if changes are present, may lead also to the generation of novel functional hypothesis, testable in the laboratory. Here, I describe the evolutionary history of all UUL-encoding genes in animals, using when needed external data, most especially from the genome of the closest animal relative sequenced so far, the choanoflagellate Monosiga brevicollis present in the databases (circa April 2010) were selected. Monosiga can be used as a convenient outgroup to establish the genes that were already present when animals emerged, given that choanoflagellates are considered the closest living relatives of animals [To generate comprehensive databases of UULs, TblastN searches were performed using the sequences of the human U-box proteins as queries against the nr, htgs, gss, est and wgs databases of the National Center for Biotechnology Information (NCBI) . The seq animals . From th animals ,56 to co animals .Amphimedon queenslandica was reported [A. queenslandica that were incorporated to generate final alignments.While this paper was being revised, the final draft genome sequence of the sponge reported . Given tFrom those alignments, phylograms were obtained using three different methods of phylogenetic reconstruction. First, Neighbor-joining (NJ) trees were characterized using MEGA 4 . Second,Figure Chip: Chip proteins are characterized by containing 3 N-terminal tetratricopeptide repeats (TPRs), involved in protein-protein interactions, and a C-terminal U-box  and annelids . Exceptionally, the gene was not found in the choanoflagellate M. brevicollis. In the alignments, it was particularly noticeable the high level of sequence divergence in the genes of Caenorhabditis nematode species. This is obvious in Figure Brugia malayi, while Caenorhabditis sequences are separated from the rest of the tree and have much longer branches. Given that Drosophila and Caenorhabditis are both ecdysozoans, their divergence from the lineage that gave rise to humans occurred at the same time. Therefore, one would expect to find similar degrees of divergence when the human proteins are compared to either the fly or nematode proteins. However, for Chip that expectation is not fulfilled. Along the conserved region of 279 amino acids analyzed, the resemblance between human and Drosophila Chip proteins  is much higher than the one found in the comparison human Chip vs. nematode Chip .1) species  and annelids , were also detected. As described above for Chip, nematode Prp19 protein sequences  appear as very divergent in the tree  was substantially higher  than the similitude between human and Caenorhabditis elegans .2) scovered  and planscovered ,66. The scovered  and 7 C-e Figure . Again, UBE4A and UBE4B: all the proteins related to Saccharomyces cerevisiae Ufd2p, among them UBE4A and UBE4B, contain a large stretch of similarity , which lack UBE4A. Fragments of a single Ufd2-like gene were detected in Monosiga brevicollis, being thus likely that the UBE4A/UBE4B duplication occurred after the choanoflagellate/metazoan split. In addition of those shown in Figures UBE4A and UBE4B orthologs were found in molluscs , annelids (both found in Capitella and Hirudo) and platyhelminthes . For UBE4B, high divergence of nematode  and also Ciona and Schmidtea sequences is again suggested by the long branches observed in Figure Drosophila melanogaster UBE4B  than to the Caenorhabditis elegans orthologous protein .3, 4) database ), N-termdatabase ). The du Figures . The excCYC4: CYC4 proteins are characterized by having an N-terminal U box and a cyclophilin (a. k. a. Pro_isomerase) domain near to their C terminus , annelids  and platyhelminthes (Schmidtea). Notably, once again nematode sequences appear in the tree as highly divergent .5) s Figure . This tyt Figure . This tiUIP5: The structure of the protein encoded by this gene  and well conserved within animals. Therefore, Figure UIP5 exists. Most animals, including sponges, contain a UIP5 gene, but, interestingly, nematodes such as Caenorhabditis and Brugia have lost it. In lophotrochozoans, UIP5 genes have been detected in molluscs , but not in annelids or platyhelminthes. A potential duplicate is observed in the tetraploid amphibian Xenopus laevis  e Figure  is peculs Figure .WDSUB1: the protein encoded by this gene is characterized by containing 7 WD40 repeats and a SAM_2 domain in addition of the U box  but not in structure, given that it can be deduced that it encodes a protein with 2 SAM domains but without WD40 repeats . Finally, a few sequences having just the SAM and U box domains, but lacking the WD40 domain, have been also detected . Thus, WDSUB1-related genes appeared before the emergence of animals, but bona fide WDSUB1 genes, encoding proteins with the characteristic WD40 - SAM - U box signature, seem to have originated more recently, after the ancestors of sponges diverged from the ancestor of the rest of animals. Notably, WDSUB1 genes are again absent in nematodes  and also in the fruit flies of the Drosophila genus, although they are present in some other insect species  s Figure . They haAct1, has been recently described [Act1 with the rest of U boxes is very weak (data not shown), the evolutionary conservation of this gene was also analyzed. It was found to be restricted to mammals. In addition of determining the orthologs of the known human UUL genes, it was also significant to establish whether additional genes existed that were present in other species but not in humans. Along these searches, I detected sequences that correspond to an additional UUL-encoding gene in the cnidarian Nematostella vectensis  and surprisingly, five extra genes in the choanoflagellate Monosiga brevicollis . None of these sequences contained the conserved regions characteristic of the seven canonical UUL-encoding genes. The structures of their predicted proteins are detailed in Figure Nv_UBOXa, Mb_UBOXd and Mb_UBOXe) encode proteins lacking any similarity to other proteins outside the U box domain  domain, a protein-protein interaction domain present in a large number of intra- and extracellular proteins [Mb_UBOXc is predicted to encode a protein with a glycosyl hydrolase domain. Specific searches were performed to detect genes related to these six exceptional ones in other species. In just one case, Mb_UBOXa, clearly related sequences were detected in a few protozoans of distantly related groups, such as the heterolobosean Naegleria gruberi (Accession number XM_002676557.1), the unclassified anaerobic flagellate Trimastix pyriformis (Acc. No. EC839610.1) and the diplomonad Spironucleus vortens (Acc. No. GH177899.1) among others. All these sequences potentially encode proteins with the same structure that the one generated by Mb_UBOXa. Therefore, although five of the six novel genes discovered in cnidarians or choanoflagellates seem to be lineage-specific, Mb_UBOXa is an ancient gene which is not present in animals.As it was indicated in the Introduction, a potential eighth UUL-encoding human gene, escribed . AlthougAct1) originated long time ago. Proteins structurally identical to the seven corresponding human UULs were already present in early animal evolution, given that they are found in placozoans, cnidarians and bilaterians. Since then, losses have been scarce, although WDSUB1 has been lost independently several times . TblastN searches against the databases at Nembase4, which compiles all the available information for nematode ESTs, confirmed that orthologs of those three genes are not detected in any species. It is also interesting that only a few lineage-specific new genes have been detected. Among all the organisms examined, it turns out that it is the simplest, the unicellular choanoflagellate Monosiga brevicollis, the one with the most extensive set of U box ubiquitin ligases. The data so far available suggest that this is largely due to the emergence of additional, novel genes in choanoflagellates.The most parsimonious hypothesis to explain the evolutionary origin of the UUL-encoding genes described in this study is depicted in Figure s Figure . A notabChip, PRP19, CYC4, which are present in many other eukaryotic groups) or in early animal evolution . The final result concerns the unexpectedly large number of UUL-encoding genes found in choanoflagellates but not in animals, a total of five . However, all UUL-encoding genes are present in Ciona .C. elegans. This may facilitate a fast determination in the nematode of the functions of these proteins and perhaps of the ubiquitination system as a whole.In conclusion, the analysis of the U-box ubiquitin ligases in animals confirms and extends our previous findings about the general patterns of diversification that explain the current diversity of E3 proteins in animals. An additional result that may have experimental interest is the detection of a general simplification of the ubiquitin ligase set of proteins in a model species such as CHIP sequences. Text file containing the aligned sequences in fasta format. Used to generate the phylogram shown in Figure Click here for filePRP19 sequences. Text file containing the aligned sequences in fasta format. Used to generate Figure Click here for fileUBE4A sequences. Text file containing the aligned sequences in fasta format. Used to generate Figure Click here for fileUBE4B sequences. Text file containing the aligned sequences in fasta format. Used to generate Figure Click here for fileCYC4 sequences. Text file containing the aligned sequences in fasta format. Used to generate Figure Click here for fileUIP5 sequences. Text file containing the aligned sequences in fasta format. Used to generate Figure Click here for fileWDSUB1 sequences. Text file containing the aligned sequences in fasta format. Used to generate Figure Click here for file