Tsc1 knockout mice despite significantly elevated mTORC1-S6K1 activity. Neither up- nor down-regulation of the mTORC1-S6K1 axis in vivo or in vitro had any effect on phospho-FMRP S499 levels. In addition, FMRP S499 phosphorylation was unaltered in S6K1-knockout mice. Collectively, these data strongly suggest that FMRP S499 phosphorylation is independent of mTORC1-S6K1 activity and is not altered in TSC.Hyperactive mammalian target of rapamycin (mTOR) is associated with cognitive deficits in several neurological disorders including tuberous sclerosis complex (TSC). The phosphorylation of the mRNA-binding protein FMRP reportedly depends on mTOR complex 1 (mTORC1) activity via p70 S6 kinase 1 (S6K1). Because this phosphorylation is thought to regulate the translation of messages important for synaptic plasticity, we explored whether FMRP phosphorylation of the S6K1-dependent residue (S499) is altered in TSC and states of dysregulated TSC-mTORC1 signaling. Surprisingly, we found that FMRP S499 phosphorylation was unchanged in heterozygous and conditional  TSC1 or TSC2, which are upstream regulators of mTOR kinase activity in mTOR complex 1 (mTORC1). Most patients are born heterozygous for either TSC gene and experience additional inactivating mutations during development leading to loss of heterozygosity Tsc1 or Tsc2 heterozygosity is sufficient to impair neuroplasticity and learning and memory despite the absence of brain malformations and clinical seizures Tsc2+/− mice are rescued by rapamycin treatment indicating that these deficits are reversible and mTORC1-dependent. In adult Tsc2+/− mice, an ERK inhibitor, but not rapamycin, rescued plasticity-dependent deficits, which is consistent with increased ERK activity in these mice Tsc1+/− mice, however, do not display increased ERK activity Altered mTOR signaling is a shared feature of many neurodevelopmental disorders that display high rates of mental retardation with comorbid autistic features such as TSC and fragile X syndrome (FXS) FMR1 (the X-linked gene encoding FMRP) results in FXS, the leading cause of inherited intellectual disabilities and autism FMRP is an mRNA-binding protein that regulates the translation of ∼4–6% of brain mRNAs, many of which are involved in neuroplasticity Tsc1 knockout mice despite significantly elevated mTORC1-S6K1 activity. Subsequent experiments revealed that neither up- nor down-regulating mTORC1-S6K1 signaling activity in vivo or in vitro has any effect on pFMRP levels, indicating that the mTORC1-S6K1 pathway plays no role in regulating S499 FMRP phosphorylation.Because FMRP is absent in FXS and would be predicted to be hyperfunctional in TSC, it has been hypothesized that S6K1-dependent FMRP S499 hyperphosphorylation in TSC might explain some of the opposite phenotypes observed in these two models of autism y/+Fmr1 mice that were absent in y/−Fmr1 mice  and pFMRP S499 (referred to as pFMRP antibody). FMRP belongs to a small family of proteins that includes the fragile X-related proteins 1 and 2 (FXR1 and FXR2) and shares ∼70–80% homology with FXR1/2 in the N-terminal region but essentially no homology in the C-terminal region invitro as previously reported We next verified that S6K1 can phosphorylate FMRP S499 Tsc1+/− mice, we first examined the phosphorylation levels of the mTORC1-dependent site on S6K1 T389 (pS6K1), which functions as an activating phosphorylation. We secondarily characterized phosphorylation of the S6K1-dependent sites on ribosomal protein S6 S240/244. We used the hippocampus of 2 month-old male Tsc1+/− mice to avoid potential variations in tFMRP levels between gender e.g., pFMRP:tFMRP). Despite significantly decreased levels of TSC1 and TSC2 in Tsc1+/− compared to Tsc1+/+ mice  mice were bred against wt/−;Emx1-Tsc1Cre mice to yield mixed litters of forebrain-specific Tsc1 wildtype, heterozygous, and knockout mice. In Emx1-Cre mice, Cre recombinase is expressed in forebrain glial and glutamatergic progenitors beginning around E9.5 Tsc1 , dominant negative TSC2 (TSC2DN), constitutively active Rheb (RhebCA), and hyperactive mTOR (mTORhyper) . Rapamycin injections were performed daily for 5 days and PF-4708671 mice were injected once and sacrificed two hours post-injection. Both rapamycin and PF-4708671 significantly decreased mTORC1-S6K1 activity as demonstrated by reduced pS6 S240/244 levels, however, pFMRP levels were unchanged , this was found to be due to enhanced ERK-RSK signaling rather than mTORC1 activity Tsc1+/− telencephalon In order to study the mTORC1-S6K1-FMRP connection, we first examined whether mTORC1 pathway activity is increased in +/−Tsc1 mice and in the forebrain of conditional Tsc1 knockout mice, we could not detect elevated FMRP S499 phosphorylation. Because we found that S6K1 can phosphorylate recombinant human FMRP in vitro, we tested whether our inability to detect increased FMRP S499 phosphorylation may be because the mTORC1-S6K1 axis modulates FMRP S499 phosphorylation independent of the TSC pathway or because basal FMRP S499 phosphorylation is saturated. We therefore tested whether either overexpression of other inputs to mTORC1 or inhibition of mTORC1-S6K1 activity would alter FMRP S499 phosphorylation.Importantly, despite evidence of elevated mTORC1-S6K1 activity in P2 fractions of in vitro or in vivo, affected the degree of FMRP S499 phosphorylation. Finally, FMRP S499 phosphorylation is identical between S6k1+/+ and S6k1−/− mice, indicating that S6K1 is dispensable for normal FMRP S499 phosphorylation. This finding is in contrast with data in a previous study None of the manipulations that altered mTORC1-S6K1 activity, either in vitroCollectively, these data demonstrate that S6K1 is not required for phosphorylation of endogenous FMRP at S499. Furthermore, these data indicate that the mTORC1-S6K1 pathway does not regulate the phosphorylation of FMRP S499 in any way. This negative finding is consistent with the fact that the sequence surrounding FMRP S499, EASNApS, contains none of the features that would render it a good S6K1 candidate. The preferred phosphorylation motif for S6 kinases is relatively well preserved, RXRXXpS (where X = any amino acid and p denotes the phosphorylated residue). With rare exception S6 kinase substrates deviate from this sequence dFmr1 drosophila highlights the biological significance of this phosphorylation site Our findings open clear questions. In particular, it is important to identify the kinase(s) responsible for FMRP S499 phosphorylation. The recent finding that phospho-mimetic FMRP but not phospho-dead FMRP can fully rescue Our initial interest was to investigate the contribution of dysregulated FMRP S499 phosphorylation to TSC. To our surprise we were unable to find a link between mTORC1-S6K1 and S499 phosphorylation under any condition. Although our findings strongly suggest that another kinase is responsible for the phosphorylation of FMRP S499, the mTORC1-S6K1 pathway may yet regulate the phosphorylation of other FMRP residues.All animal research protocols were approved by the Institutional Animal Care and use Committee, Yale University.Tsc1−/− mice (see description below). +/−Tsc1 mice (+ for wildtype (wt) and - for mutant (mut) alleles, NCI), also noted Tsc1Het in figure legends, were generated by David J. Kwiatkowski  and were of mixed background: B6;129S4, C57BL/6J, BALB/cJ and 129SvJae. y/−Fmr1 and y/+Fmr1 mice, which are knockout (KO) and WT mice, respectively, were a gift from Dr. Leonard Kaczmarek, Yale University, New Haven, CT. +/+S6k1 and −/−S6k1 samples  were a gift from Dr. Kat Takeda, National Jewish Health, Denver, CO. To generate forebrain-specific conditional −/−Tsc1 (Tsc1KO) mice, we bred flox/+;Emx1-Cre−/−; R26R-Tsc1tdTomato+/+ (where +/+ connotes the presence of the tdTomato gene) to +/−;Emx1-Cre+/+;R26R-Tsc1tdTomato−/− mice. Emx1-Cre mice were kindly provided by Dr. Cardin . R26R-tdTomato mice were obtained from the Jackson Labs. fl/+Tsc1 mice (Jackson Labs) were also generated by David J. Kwiatkowski. With the exception of Tsc1:Emx1-Cre transgenic mice, all mice used in this study were 2 months old. Because all flox/−Tsc1 mice die by P20 due to seizure, only P7 mice were used in this study.We used male wild-type and transgenic mice except for conditional in house or using Genetyper services (www.genetyper.com). For in-house and Genetyper-assisted genotyping we used a three-primer protocol that allows for the simultaneous detection of wild type, mutant and floxed Tsc1 alleles: TSC1F4536∶5′ – AGG AGG CCT CTT CTG CTA CC; TSC1R4830∶5′ – CAG CTC CGA TGA AGT G; TSC1R6548∶5′ – TGG GTC CTG ACC TAT CTC CTA.Genotyping was performed either Mice were anesthetized with isoflurane followed by decapitation. Brains were acutely dissected in ice-cold Hank’s Balanced Salt Solution , snap frozen in liquid nitrogen and stored at −80°C. For whole cell lysates, samples were homogenized in RIPA buffer supplemented with DNase I (8 U/10 ml), 1x HALT protease/phosphatase inhibitor cocktail (Pierce #78443), and okadaic acid (100 nM). Protein concentrations were quantified using a standard BCA protein assay (Pierce #23225). For a given experiment, samples were diluted to the same concentration with lysis buffer and then boiled in an equal volume of 2x Laemmli buffer at 99°C for 5 minutes.2O supplemented with HALT. Cytoplasmic and P2 fraction protein concentrations were quantified and the samples were boiled in an equal volume of 2x Laemelli Sample Buffer. We validated that P2 fractions were enriched for synaptic proteins PSD95 and SAPAP3 and relatively depleted of nonsynaptic proteins such as α–tubulin and the glial protein GFAP Tissues were homogenized in P2 lysis buffer  with micro-pestles (RPI #199222); 20 strokes/sample. Homogenates were centrifuged at 1000 g for 10 minutes at 4°C and the pellet discarded. Supernatants were recentrifuged at 10,000 g x 15 minutes at 4°C and the supernatant set aside as the cytoplasmic fraction. The pellet (P2 fraction) was resuspended in P2 buffer and centrifuged again at 10,000 g for 15 minutes at 4°C. The supernatant was discarded and the P2 fraction was resuspended in 50 mM Tris-HA single CD1 adult mouse hippocampus was homogenized in 300 µl phosphatase assay lysis buffer . The sample was centrifuged at 13,000 rcf for 10 minutes at 4°C. 800U lambda phosphatase (NEB #P0753S) was added to 100 µl supernatant and the sample incubated at 37°C for 30 minutes. The reaction was terminated by addition of an equal volume of 2x Laemmli buffer.Antibodies and usage parameters are listed in Transfections were formed using PolyJet transfection reagent (SignaGen) according to the manufacturer’s protocol. Vectors and their sources are listed in Rapamycin , PF-470867 (Symansis), Bisindolylmaleimide V (B5) , and okadaic acid  were used as indicated in the text. Okadaic acid was dissolved in ethanol (vehicle 2) for N2a cell culture experiments and DMSO when used to supplement lysis buffers. (S)-DHPG was purchased from Tocris and diluted in water.Kinase assays were performed by Kinexus (Canada). Recombinant FMRP S500 and S500D were generated as previously described 2. When cells reached approximately 70% confluence in six-well plates, the medium was replaced with pre-warmed complete media and treatment was begun one hour later. For in vitro experiments, individual treatments and transfections were performed between two and six times prior to performing the combined experiments represented in in vitro experiments). Cell lysis was performed on ice. Cells were rinsed twice with ice-cold 1X phosphate-buffered saline Laemmli and lysed in N2a lysis buffer . Cells were then scraped from the wells and lysates centrifuged at 16,000 RCF for 20 minutes at 4°C. The supernatant was added to 6X Laemmli sample buffer to a final concentration of 1X sample buffer and boiled for 5 minutes at 99°C.In general, N2a cells were cultured in complete media (Dulbecco’s Modified Eagle Medium (DMEM) (Gibco 11965-092), 5% fetal bovine serum (FBS) (Gibco 16140-071), and 1% Penicillin-Streptomycin (Gibco 15140-122) in a 37°C incubator at 5% COAll western blots were performed using 10% tris-gylcine gels and protein transferred to PVDF membranes according to a standard wet transfer protocol. In cases where cross-blot normalization was required a standard sample was loaded on each gel to account for inter-gel variability. In general, the optimal linear range for each antibody was determined using the appropriate sample type  prior to experimental immunoblot assays. In some cases, limited linear ranges were run on the same gel (that is 80% and 120% of a control sample were loaded in end lanes) to ensure detectability of minor changes in protein signals. Densitometry was performed using Image J without background correction or rolling ball adjustments. All phospho-protein signals were normalized to total protein signals from the same blot. For phospho-proteins, adequate removal of phospho-antibody was verified by probing with secondary alone after stripping the membrane. Raw ratios of phospho-protein normalized to total protein or total protein normalized to loading control  were calculated in Microsoft Excel.in vitro experiments, the data is represented as the % change from the control lane on the same membrane; as such, control lanes are without error bars. Statistical significance was determined using Mann-Whitney U, one-way ANOVA or two-way ANOVA using Dunnett’s and Turkey’s post-hoc tests where indicated. P<0.05 was considered significant. Data are shown as mean ± standard of the mean (SEM) unless otherwise specified.Statistical analysis was performed on raw densitometric ratios using GraphPad 6. For data presentation, values were normalized to control data such that control groups were always = 1. For


Due to its high energy consuming characteristics, brown adipose tissue (BAT) has been suggested as a key player in energy metabolism. Cold exposure is a physiological activator of BAT. Intermittent cold exposure (ICE), unlike persistent exposure, is clinically feasible. The main objective of this study was to investigate whether ICE reduces adiposity in C57BL/6 mice. Surprisingly, we found that ICE actually increased adiposity despite enhancing Ucp1 expression in BAT and inducing beige adipocytes in subcutaneous white adipose tissue. ICE did not alter basal systemic insulin sensitivity, but it increased liver triglyceride content and secretion rate as well as blood triglyceride levels. Gene profiling further demonstrated that ICE, despite suppressing lipogenic gene expression in white adipose tissue and liver during cold exposure, enhanced lipogenesis between the exposure periods. Together, our results indicate that despite enhancing BAT recruitment, ICE in mice increases fat accumulation by stimulating de novo lipogenesis. Obesity is a metabolic disease characterized by overexpansion of white adipose tissue (WAT). Although genetic predisposition is important in the development of obesity, chronic positive energy balance has been considered the main cause of obesity in the general population. Therefore, correcting energy imbalance is an ideal therapy for obesity. Unfortunately, commonly used therapeutic approaches such as dieting and exercise are not efficient at containing the obesity epidemic.WAT and BAT are the two main types of fat in mammals. WAT is the primary energy depot that stores energy as triglyceride-enriched lipid droplets. By contrast, BAT is considered as an energy dispenser that consumes significant amounts of chemical energy toward thermogenesis Cold exposure activates BAT thermogenesis. However, prolonged exposure to cold in humans has been limited by cardiovascular and respiratory complications Here, by using C57BL/6 mice, we have investigated whether and how ICE alters adiposity. Similar to human subjects and rats, ICE induced BAT recruitment in mice. Unexpectedly, ICE induced fat accumulation, an effect that cannot be attributed to hyperphagia or stress. Remarkably, ICE induced lipogenic gene expression in both WAT and liver during the non-exposure period. Therefore, our results demonstrate that in spite of inducing BAT recruitment, ICE increases de novo lipogenesis in WAT and liver then enhances fat accumulation in mice.C57BL/6 mice at 12–16 weeks of age housed under standard conditions with ad libitum access to food were used. They were singly caged for all cold exposure studies to avoid huddling. All cold exposure started at 8:30–9:00 am. For acute cold exposure (ACE), single caged mice were transferred to a 4°C room or a room (23–25°C) next to the cold room to ensure the mice have a similar environment, such as noise and light. The cold room is 20 meters away from the EchoMRI system. After each scanning, which took 1.5 min, the mouse was immediately returned to cold room or the place where control mice located. Therefore, the interruption of cold exposure was reduced to minimal 4–5 min. For ICE, the exposure time to cold air was gradually increased: 3 hours on the first day and an additional hour for each subsequent day up to 6 hours per day. This was followed by a 6-hr cold exposure daily for 8–10 days. For feeding restriction during ICE, each mouse was provided a daily quota equal to its own baseline food intake measured one week prior to the ICE. Tissue samples were collected immediately after cold exposure (ACE) or at 9:00 PM in the ad libitum state (ICE). All animal protocols were approved by the University of California San Diego Institutional Animal Care and Use Committees.Body composition was determined by MRI scanning of conscious mice . Each scan took 1.5–2 minutes. During ACE, body composition was scanned hourly. For ICE, mice were scanned before and after each cold exposure. Interscapular BAT and inguinal and epididymal WAT were weighed at the end of the ICE study.2) was normalized to lean body mass measured just prior to the clorimetry.Energy expenditures were measured using the Oxymax Lab Animal Monitoring System . Mice were individually housed in the chamber at room temperature with ad libitum access to food and water and under regular light/dark cycles. Oxygen consumption  were homogenized in 2∶1 chloroform∶methanol to extract triglycerides. After adding NaCl to 0.88%, samples were centrifuged and the aqueous phase decanted. The organic phase was evaporated and then dissolved in isopropranol. Triglyceride levels were measured using a commercial kit . Hepatic TG secretion rates were measured after injection (i.v.) of Poloxamer-407 to block LPL as previously described Sections of fixed fat tissue stained with H&E were imaged at 20× magnification. To size adipocytes using ImageJ software , the area of measurement was randomly chosen. Adipocytes were outlined to measure the cross-section areas, excluding those that were too small or too large  to avoid errors in the outlining process.Previously described procedures were used Data are expressed as mean ± standard error of the mean (SEM). Student t-tests and ANOVA were used to determine p values. P<0.05 was considered statistically significant.We investigated the effects of both acute and intermittent cold exposures (ACE and ICE) on adiposity. We found that ACE for 5 hours induced progressive loss of body weight, fat mass, and even lean mass . The redSimilar to ACE, ICE also induced a significant loss of body fat during each cycle of cold exposure . This loSimilar to results from cold acclimation studies BAT activation has been suggested to increase energy expenditure and improve glucose and lipid metabolism, especially in the obese state. However, our study showed that ICE did not significantly alter glycemic levels either in the fasting state  or afterHaving shown that ICE increased BAT recruitment and body fat, we then investigated its effect on energy expenditure using indirect calorimetry to compare each mouse before and after ICE treatment. To our surprise, ICE caused no significant alteration in oxygen consumption in the basal state . HoweverCold exposure often increases food intake in rodents Cold exposure is a physical stress, and stress has been implicated in the development of obesity Both epididymal and inguinal fat pads expanded significantly in ICE mice . InteresTo investigate the mechanisms underlying ICE-enhanced fat accumulation, we examined the expression and activation status of key regulators of adipogenesis and lipogenesis in WAT and liver. During cold exposure, there were significant increases in the phosphorylation of hormone sensitive lipase (HSL) in both epididymal  and inguIn contrast, 9 hours after recovery from ACE, the expression of FAS and SREBP1c in epididymal fat  and liveWe also compared the expression of lipogenic genes between ICE and control mice under regular housing conditions. We found robustly increased mRNA levels of de novo lipogenic genes, such as FAS, acetyl-CoA carboxylase (ACC1), glycerol-3-phosphate acyltransferase (GPAT), LXRα and SREBP1c, in WAT  and liveThe enlarged epididymal fat mass of ICE mice and their smaller adipocyte size imply that ICE enhances adipogenesis. PPARγ and C/EBPα, master transcription factors for adipocyte differentiation and adipogenesis, were both significantly upregulated in epididymal fat of ICE mice , consistBAT-mediated thermogenesis is a calorie-consuming process that might be utilized to correct the energy surplus that underlies obesity in humans. Consistent with previous studies, our study indeed showed that ICE increases BAT recruitment. In addition, we found a significant reduction of body fat within hours of cold exposure in both our ACE and ICE protocols. Surprisingly, between successive rounds of cold exposure in the ICE protocol, we observed re-expansion of adiposity to a level beyond the basal level of the preceding cycle. This finding of a net increase in fat mass in our ICE mice is in line with a previous mouse study Another important finding of this study is that cold exposure profoundly impacts not only BAT but also liver and WAT, which might play a critical role in expanding adiposity between cold exposure periods. Under physiological conditions, cold exposure induces a series of responses from various organs to maintain body temperature, including BAT activation and TG mobilization. Consistent with previous reports Adipose tissue can expand through hypertrophy (increase in cell volume) and/or hyperplasia (increases in cell number). Our study showed that ICE increased the mass of both inguinal and epididymal fat. Consistent with previous reports Energy balance plays a critical role in controlling fat accumulation. Cold exposure induces food intake in rodents. Our study indeed showed that ICE transiently increased food intake. However, our dietary intervention study showed that food restriction did not prevent ICE from causing expansion of adiposity, arguing against hyperphagia as the major cause of ICE-induced fat accumulation. On the other hand, previous studies have demonstrated that cold exposure increases energy expenditure in mice WAT and BAT are distinct adipose tissues that are essential to energy storage and dissipation respectively. Despite playing opposite roles in energy expenditure, they act in concert to maintain energy homeostasis under physiological conditions. Our current study found that despite the induction of BAT recruitment and the reduction of WAT mass during cold exposure, the net effect of repetitive cold exposure is enhanced basal de novo lipogenesis and lipid accumulation in mice. Because fatty acids are the main fuel for non-shivering thermogenesis, increased lipid accumulation may help the mice cope with subsequent cold exposure. Therefore, we conclude that ICE increases body fat accumulation in mice by stimulating de novo lipogenesis during non-cold-exposure period.Fig. S1Cold exposure reduces body fat, but more body fat accumulated next day before cold exposure. Body composition was scanned before and after each round of cold exposure.(TIF)Click here for additional data file.Fig. S2Effect of ICE on liver histology. After 12 days of ICE treatment, liver tissues were collected and stained with hematoxylin and eosin.(TIF)Click here for additional data file.


Although local factors, including human population growth, land use change, and water management influence Suwa and Torne, the general patterns of ice seasonality are similar for both systems, suggesting that global processes including climate change and variability are driving the long-term changes in ice seasonality.Lake and river ice seasonality (dates of ice freeze and breakup) responds sensitively to climatic change and variability. We analyzed climate-related changes using direct human observations of ice freeze dates (1443–2014) for Lake Suwa, Japan, and of ice breakup dates (1693–2013) for Torne River, Finland. We found a rich array of changes in ice seasonality of two inland waters from geographically distant regions: namely a shift towards later ice formation for Suwa and earlier spring melt for Torne, increasing frequencies of years with warm extremes, changing inter-annual variability, waning of dominant inter-decadal quasi-periodic dynamics, and stronger correlations of ice seasonality with atmospheric CO Quantitative, direct annual observations by humans of climatic variables starting before 1840s and the Industrial Revolution are rare. Most studies of long-term climate have used time series of human observations that begin near the start of the Industrial Revolution, and rely on paleo-chronologies to assess climate conditions prior to the start of the Industrial Revolution12Lake and river ice seasonality (dates of ice freeze and breakup) responds sensitively to climatic change and variability4564568910114omiwatari in Japanese. This important event was followed by a purification process by the priests at the shrine and a celebration that was recorded by at least 15 generations of Shinto priests since 1443. The starting point and direction of the ridge was used to forecast the harvest, temperature, and precipitation for that year1415The Suwa and Torne ice records were collected primarily for religious or economic purposes. The long-term ice freeze record for Lake Suwa, defined as the complete freezing of the lake surface, was collected by Shinto priests observing a legend whereby the male god Takeminakata would cross the lake to visit the female god Yasakatome at her shrine on the other side of the lake. The crossing was evidenced by the god’s footsteps on the ice that left a sinusoidal ice ridge known as the The record for the date of ice breakup for Torne River, defined as when the ice was generally moving, was started by a merchant named Olof Ahlbom in 1693. The timing of ice breakup continued even after Olof Ahlbom fled the Russian troops and then returned to Tornio, Finland following the end of the Russian occupation from 1715–1721. The ice breakup time series has been preserved because of Torne’s important role in trade, transportation, food, and recreation beginning in the 17th and 18th centuries. Logistical factors include: i) proximity to two towns, Tornio, Finland and Haparanda, Sweden with their own newspaper and monitoring organizations, ii) weather journals conserved by Anders Hellant, and iii) ice breakup guessing competitions for which competitors guess the hour and minute of the annual spectacular ice breakup helped preserve the record17182. The Tenryu River flows through Suwa and the lake’s water volume turnover is every 39 days. Suwa has recently developed hot springs surrounded by four resort towns with a population of 91,000 inhabitants141919The two systems differ greatly in geography and local conditions. Suwa is a shallow temperate lake  with a mean depth of 4.7 m and area of 13.3 kmThe extent and annual grain of the Suwa and Torne ice records allowed us to address how climate and variability differed before and after the start of the Industrial Revolution. More specifically, after the start of the Industrial Revolution: i) Do ice dates reveal more rapid warming? ii) Do warm extremes increase in frequency? iii) Does inter-annual variability increase? iv) Do frequencies of quasi-periodic dynamics  change? and v) Do drivers  of ice seasonality change?Both Suwa and Torne exhibited more rapid rates of change consistent with warming with later ice freeze and earlier ice breakup following the start of the Industrial Revolution . For SuwThe prevalence of extreme warm years has been increasing over time for both Suwa and Torne . Suwa diFor Suwa and Torne, the timing of ice-freeze and breakup indicate that interannual variability differs among some 30-year non-overlapping windows . For SuwBefore the Industrial Revolution, the ice records of Suwa and Torne both exhibited inter-annual and multi-decadal quasi-cycles with significant periods between 8 and 64 years . Torne a2) concentrations and local seasonal air temperatures (March for Suwa and January-April for Torne). In addition, NAO was significant for Torne after the start of the Industrial Revolution, while for Suwa ENSO was not a significant predictor in either period , ice freeze in Suwa is 4.6 days per decade later or approximately 24 times more rapid than the early period 1443–1683), whereas ice breakup in Torne is 0.66 days per decade earlier or twice as fast than the early period. For Suwa, additional evidence of increased rates of warming starting in the 1810’s is provided by diaries of temperature and precipitation, ice fre83, where2319Although these two inland waters have uniquely long time series including years before the Industrial Revolution, there are hundreds of inland waters exhibiting patterns of later ice freeze and earlier ice breakup after the start of the Industrial Revolution456711132623et al.5The frequency of extreme warm years has been increasing over time both for Suwa and Torne . For exaet al. concluded that the increase in warm extremes resulted primarily from change in the meanCharacterizing patterns of interannual variability are complex for Suwa and Torne. Periods of increasing and decreasing variability are apparent. For Suwa, in the early period, there was no trend in interannual variability. The recent period for Suwa shows the highest variability, with the most recent 30 years being the most variable observed across the entire period of record. On the other hand for Torne, the interannual variability in the timing of ice breakup is consistently decreasing. A 90-year record of air temperature352 concentrations may have stabilized the positive phase of the NAO. The stabilization of the NAO in the positive phase associated with increasing CO2 concentrations appears to be contributing to changing ice phenology in the direction of a warming climate37Contrary to our expectations, the same quasi-periodic dynamics did not persist throughout the time series for either Suwa or Torne. Rather, quasi-periodic dynamics tended to weaken and we observed a loss of interdecadal periodicities after the start of the Industrial Revolution. Climate studies have suggested changing periodicities in the North Atlantic Oscillation (NAO) and El Niño Southern Oscillation (ENSO) over time37383940412 concentrations  were important explanatory factors for later ice freeze in Suwa and earlier ice breakup in Torne. Similarly, Tanaka and Yoshino documented a correlation of 0.77 between winter air temperatures (December-January) and freeze date in Suwa between 1945–1978 indicating that warmer winters are associated with later ice freeze171920454656910Following the start of the Industrial Revolution, increasing air temperatures and atmospheric CO3 sec−1 with a maximum capacity of 600 m3 sec−1 3 day−1 in 1926 to 11,000 m3 day−1 in 1959 to 15,000 m3 day−1 in 20153 day−1 to approximately 13,000 households, at an average temperature of 65 degrees celsius. Evidently, the waters from the hot spring is now almost all being used for human consumption. Although Suwa is influenced by a variety of local factors, the urban heat island, hot springs, and geyser do not provide substantial heat input to the lake to explain the recent prevalence of no-freeze years and later freeze dates alone. Even with these local changes, the ice thickness and ice duration have been strongly related to air temperatures presenting a strong signal of climate change and variability4315Suwa is influenced by several anthropogenic factors including: land use, human population, flood control gate, and hot springs. The Suwa watershed is 40 times larger than the lake and includes 50% forest and wilderness, 11% rice paddies and dry fields, 6% residential, and the remaining land use includes forest preserves, golf courses, roads and rail corridors, and an amusement parkth century, 162 log-driving dams were constructed on Torne, but later demolished in the 1970s that may have sped up the runoff1720Torne is one of the largest unregulated rivers in Scandinavia, but is influenced by several local anthropogenic factors including urban development, bridge construction, hydropower development on a tributary of Torne, and demolition of constructed dams19202 emissions and air temperatures continue to rise, the male god may soon never cross the lake again to visit the female god as he has in Shinto legend for centuries.These analyses based on direct human observations of ice for Suwa and Torne over 570 and 320 years are consistent with climate change based on later ice formation and earlier spring melt, without relying on instruments, modelling results, or inferences from paleo records. We advance knowledge of climate change and variability using two rare datasets that are well within the direct human perception of the world. As Suwa no longer freezes every year, the male god, Takeminakata, is no longer able to walk across the lake to see the female god, Yasakatome, every yearWe obtained ice freeze dates for a 572-year period from 1443–2014 for Lake Suwa and a 321-year record of river ice breakup dates for Torne River from 1693–2013 from the National Snow and Ice Data Center54st, therefore we converted dates to day of year, using a zero to represent the calendar day January 1st.Ice-freeze dates for Suwa were first recorded in 1443. Ice freeze date, defined as the first date of complete cover, was decided by observers from the shoreline. The name of the family observing ice freeze is provided, although the unique name of the observer is not given but would have included at least 15 generations of observers14141643141643141643Ice breakup dates  from Torne have been recorded since 1693 and were also converted to day of year. Data were collected from observation journals, newspapers, weather monitoring services in Tornio, Finland, and Haparanda, Sweden, the Finnish Environment Institute, and local guessing competitions1718192 programWe obtained data from paleo- and historical records that may be important to ice freeze date on Lake Suwa and ice breakup date on Torne River . We acquet al. compare, calibrate, and evaluate over 15 sources of data summarizing sailing records in the Stockholm harbour in an effort to validate the reconstruction of January-April air temperaturesWe acquired air temperatures for both locations. For Suwa, we obtained reconstructed March air temperatures from Kyoto, Japan that were derived from cherry blossom phenology records obtained from diaries of emperors, aristocrats, and monks from 854–1995We used segmented regression to test for abrupt changes in the trend of ice dates in Torne. Specifically, we wanted to test when a shift in the temporal trend of ice date may have occurred. To estimate the timing and magnitude of a change in the slope of ice dates, we used continuous segmented regression (CSR) models. In CSR, trend lines on either side of the estimated breakpoint intersect (hence making them “continuous”), but are allowed to have different slopes. In general, a CSR takes the formi are the years of the time series, β0 is the intercept of the regression (ice date on year 0), β1 is the trend in ice date prior to any breakpoint (ice date per year), the ak are the breakpoints , the βk+1 are the changes in the temporal trend at each of the k breakpoints compared to the trend prior to the breakpoint, and the εi are the errors. Note that the βk+1 parameters indicate the effect on ice date of years elapsed since the previous breakpoint once the breakpoint has passed.The Torne time series began in 1693 and ended in 2013, thus x = 1, 2, … 321. Ice breakup dates for Torne ranged from day 117 to day 160, and the ice melted each year of the time series. For Torne, we fit CSR parameters using ordinary least squares using the lm function in the statistical programming language R.st of the designated “year”); however, there were 37 years when the lake did not freeze. Treating no-freeze years as missing data or as a constant date would result in biased results if we employed the regression techniques used for Torne. Thus, calculating trends for Suwa ice dates required a statistical approach distinct from that used in Torne. We can consider the lake an instrument that measures a value which we call ice date. This instrument indicates the favorability of conditions for ice formation, and if we understand the lake instrument to censor measurements at 62, then the no-freeze years can be encoded as ice dates of 62. We consider Suwa as an instrument with output of ice date that is censored at an upper limit, L = 62. As such, the observed yi are related to L and the latent variable The Suwa time series was split into an early period (1443–1683) and a late period (1923–2014) . A break2 (the variance of ε in Eq. 1), can be calculated as:To address this censoring of Suwa ice dates while fitting the parameters, we used a Tobit regression model. For a Tobit regression model with an upper limit (right censoring) of the response variable, the log likelihood of observing data given the parameters β (as in Eq. 1) and σi) during freeze years, as well as the probabilities that ice date was censored (unobserved) during no-freeze years. Thus, the β in the Tobit regression model indicate the effect of unit change in X on the latent variable, where φ(.) and Φ(.) are the probability and cumulative density functions of the normal distribution, respectively. The first term is the standard normal likelihood, and applies to observations for which an ice date was observed. The second term reflects the probability of the observation being censored, and applies to no-freeze years. Given parameter values, Eq. 3 reflects the probabilities of observing the ice dates  was selected. The model with the lowest AIC value was chosen as the most parsimonious modelWe compared AIC values from CSR models containing one or two breakpoints to multiple regression models containing only year or only year and yearLike the breakpoint models, the polynomial model indicates that trends in ice date are becoming steeper over time. The four models were of similar AIC for the Torne data, with the AIC decreasing as model complexity increased from 2155.8 to 2152.8 . Comparinormfit function). All possible 30-year non-overlapping windows were examined to ensure the pattern in variability was not sensitive to the specific start-date used. The variability is presented in units of days and differs in baseline magnitude between the two lakes.For gross climate variability, we analyzed the standard deviation of non-overlapping 30-year windows of ice dates. Although all permutations of window sizes were evaluated, the general pattern was not influenced by window size. Therefore, we chose 30-year windows to assess long-term variability in climate by accounting for intra-annual, inter-annual and most multi-decadal variation attributed to weather, Quasi-biennial oscillation, El Nino Southern Oscillation, solar sunspots, and shorter multi-decadal variation in the range of 20–30 years. Before the standard deviation was calculated, any linear trend in the data within the 30-year window was removed to remove potential bias due to trend. For Suwa, the missing (unobserved) ice dates were removed, reducing the number of observations in the window and widened the standard deviation confidence interval. No-ice years were treated as censored values and included in the maximum likelihood fit of standard deviation . For the Suwa data wavelet analysis, the omitted middle period contained the majority of missing values. The missing values in the early and late period  were replaced with the average of the time series, a conservative approach to prevent spurious oscillation detection that can occur with interpolated datasets. Tornio had no missing or no-ice observations. The Morlet basis function was chosen for its frequency identifying characteristics. For the continuous wavelet transform , signifi2, El Niño Southern Oscillation index (Suwa only), North Atlantic Oscillation index (Torne only), and Sunspots (Torne only). In each system, the relationships between ice date and each of the climate drivers were explored using data before and after the breakpoint. In Suwa, the duration of the period on both sides of the breakpoint was 75 years (1581–1655 and 1923–1997), and the time periods were chosen to maximize duration on either side of the breakpoint while minimizing the inclusion of years for which ice data were missing. In Torne, we used the full time series on either side, giving 174 years in the first portion (1693–1866) and 146 years in the second portion (1867–2013).We explored linear relationships between ice date and the following climate drivers : air temperature (Air °C), atmospheric COscaled = (x−μ)/σ, where μ is the mean of x, and σ is its standard deviation) the time series. These transformations were applied separately to early and late periods , and for the driver variables, we scaled . As described for the analysis of temporal trends, for Torne Eq. 4 was fit with ordinary least squares, and with the Tobit regression for Suwa. For each regression, we assumed there was a possibility that the residuals of the regression would be autocorrelated; to estimate coefficient standard errors in the presence of autocorrelation, we used a bootstrapping procedure where the randomized residuals retained the autocorrelation structure of the regression residuals.where y is ice date, βp AR parameters and q MA parameters. An ARMA model has the general formTo characterize the autocorrelation structure of the residuals in Eq. 4, we fit an autoregressive moving average ) model with t are the residuals ε from Eq. 4, and μ is the mean of the residuals, which is 0. The βi are the AR parameters, the αj are the MA parameters, and the εt-j are the residuals. We applied Eq. 5 to the ε of Eq. 4. We selected among ARMA models using AIC, and allowed model complexity to vary from ARMA to ARMA . These models were fit and selected using the stepwise procedure implemented in the auto.arima function in the forecast R package. We then simulated an ARMA process using the fitted ARMA parameters; the variance of the innovations in the simulated ARMA process was the maximum likelihood estimate acquired in fitting Eq. 5 to the residuals.The yFit Eq. 4Separate residuals from Step 1 into before and after periodFit and select an ARMA time series model (Eq. 5) to each set of residualsUse the model from Step 3 to simulate new sets of residuals from fitted time series modelAdd the residuals simulated in Step 4 to fitted values  and after (subscript 2) the breakpoint in the following manner:1) and after (β2) the breakpoints, and s.e. are the standard errors of those coefficients. We also corrected these p-values to control for multiple tests and to maintain constant family wise error rates. We performed the Holm-Bonferroni correction, and no conclusions about significance among pairs changed.where Z is the z-score to be compared under the standard normal curve, the β are the regression coefficients before .


In situ fixation (ISF) is standard treatment for slipped capital femoral epiphysis (SCFE) to stabilize the epiphysis and to prevent further slip. The aim of this study was to clarify the incidence of slip progression after ISF and its prognostic factors.We retrospectively reviewed 53 hips in 49 consecutive SCFE patients who underwent single screw ISF and were followed until physeal closure. Clinical and radiographic findings were viewed to assess progression of the posterior tilting angle (PTA).2 = 0.148, p = 0.005.Mean PTA was 33.4 degrees  at ISF and 35.9 degrees  at physeal closure with progression of PTA of 2.5 degrees . Slip progression occurred in 28 of 53 hips (53%), and more than five degrees of progression occurred in 14 hips (26%). Multiple regression analysis revealed that point of screw insertion  was a significant prognostic factor for progression of the slip by the following formula: (progression of PTA) = -1.523 + 2.701 × (point of screw insertion), RThe current study showed that a screw inserted from the lateral side to the intertrochanteric line prevented postoperative slip progression.The online version of this article (doi:10.1186/1471-2474-15-317) contains supplementary material, which is available to authorized users. Slipped capital femoral epiphysis (SCFE) is the most frequent hip problem in adolescents –4. MechaSometimes osteotomy is needed to realign the proximal femur . In situThe study protocol was approved by the institutional review board  and all the patients gave written informed consent. This retrospective observational cohort study included only SCFE patients who underwent single screw ISF as the initial treatment from 1996 to 2010 and were followed until physeal closure. Patients with avascular necrosis, endocrine disorders, multiple screw fixation, and preventive fixation for asymptomatic contralateral hip were excluded. Joint- based analysis was applied in this study. The patients were admitted to the hospital and kept in bed with one kg of positional traction until the operation.The Lauenstein method was applied for the lateral image as follows: care was always taken to keep the patient’s trunk in a semilateral decubitus position, 45 degrees leaning to the affected side and keeping the patient’s thigh on the platform with 90 degrees hip flexion. posterior tilting angle (PTA) was measured by neck-shaft angle using the Southwick procedure . ISF wasRadiological evaluation of PTA included the screw position , 17, theWe registered 112 consecutive hips in 98 SCFE patients (75 boys and 23 girls) from 1996 to 2010. Initially, ISF was performed on 77 hips in 64 SCFE patients. Subsequently, we excluded four hips in four patients with avascular necrosis, six hips in three patients with endocrine disorders, six hips in six patients with double screw fixation, and six hips with preventive fixation for asymptomatic contralateral hip. Of the remaining 55 hips, 53 hips  in 49 patients (40 boys and 9 girls) were observed until physeal closure with a 96% follow-up rate. The mean time to physeal closure, i.e. follow-up period, was 12.9 months .Mean age at ISF was 11.8 years  and mean BMI was 24.7 . Mean duration from first visit to our hospital to ISF was 9.5 days . Onset of symptoms was acute in 12 hips, acute on chronic in 12, and chronic in 29. Functionally, 48 hips were stable and 5 were unstable.Cohen’s quadratic weighted kappa for inter-observer reliability was 0.700 for the screw position, 0.886 for the point of screw insertion, and 0.798 for the distribution of threads. ICC was 0.977 in PTA. Cohen’s quadratic weighted kappa for intra-observer reliability was 0.938 for the screw position, 0.876 for the point of screw insertion, and 0.588 for the distribution of threads. ICC was 0.989 in PTA.Mean PTA at ISF was 33.4 degrees . Mean PTA at physeal closure was 35.9 degrees  with progression of PTA of 2.5 degrees . We performed an additional osteotomy after physeal closure in four hips in which PTA was more than 45 degrees and range of motion was restricted with the Drehmann sign . There wp = 0.001). Rate of lateral insertion was significantly higher in the slip-retention group than in the slip-progression group (p = 0.011). Acute on chronic type of SCFE was more in the slip-retention group than in the slip-progression group with a statistical difference (p = 0.043). Furthermore, Table p = 0.004). PTA at physeal closure was also significantly larger in the medial group than in the lateral group . Consequently, progression of PTA was 3.9 degrees for the medial group and 1.2 degrees for the lateral group with a statistical difference , and more than five degrees of progression occurred in 14 hips (26%). Table 2 = 0.148, p = 0.005.Multiple regression analysis revealed that point of screw insertion  was an independent prognostic factors for slip progression, and could be predicted by the following formula: (progression of PTA) = (progression of PTA) = -1.523 + 2.701 × (point of screw insertion), RA lateral point of screw insertion was an independent prognostic factor for slip progression after ISF in SCFE patients. The current study showed that a single screw inserted from lateral to intertrochanteric line prevented postoperative slip progression without the risk of screw penetration into the joint. One of the reasons may be because the thickness of the cortical bone at the proximal femoral metaphysis increases from the medial to the lateral aspect and the screw can go through a longer distance in the metaphysis, resulting in secure stability of the screw Figure . GoodwinHowever, best practices for ISF have been controversial –25. SeveWard et al. reported that mean time to physeal closure was 13 months with single screw fixation and longer with eccentric placement of the screw . In geneSome biomechanical and clinical studies suggest that double screws rather than a single screw is better, due to increased rotational stability under torsional loading conditions , 30, 31.Upasani et al. reported that maximum stability was gained when 40%-60% of the threads engaged the epiphysis using a 16 mm-thread single screw . Carney Patient characteristics such as the growth spurt, endocrinologic disorders, unstable slip, or acute-on-chronic slip were previously reported as risk factors, although they were not found to be significant factors in this study.There were several limitations to this study. First, because of its retrospective nature, patient characteristics varied, and each operation was performed according to the surgeon’s preference and best judgment. A prospective comparative trial is desirable to validate our results. Second, all the measurements were based on simple radiographs, and may have included some measurement error. However, inter-observer and intra-observer measurements were shown statistically to be closely related. Thus, we conclude that radiographic evaluation is still the gold standard for pediatric patients. Further study is needed using computed tomography or magnetic resonance imaging to determine whether these are more reliable or more accurate.The incidence of slip progression after ISF in SCFE was 53% (28 of 53 hips). More than five degrees of progression occurred in 14 hips (26%). Lateral screw insertion was a favorable prognostic factor in ISF.


Moringa oleifera respectively, and recognized for their chemopreventive and medicinal properties. In contrast to the well-studied SFN, little is known about the molecular pathways targeted by GMG-ITC. We investigated the ability of GMG-ITC to inhibit essential signaling pathways that are frequently upregulated in cancer and immune disorders, such as JAK/STAT and NF-κB. We report for the first time that, similarly to SFN, GMG-ITC in the nanomolar range suppresses IL-3-induced expression of STAT5 target genes. GMG-ITC, like SFN, does not inhibit STAT5 phosphorylation, suggesting a downstream inhibitory event. Interestingly, treatment with GMG-ITC or SFN had a limited inhibitory effect on IFNα-induced STAT1 and STAT2 activity, indicating that both isothiocyanates differentially target JAK/STAT signaling pathways. Furthermore, we showed that GMG-ITC in the micromolar range is a more potent inhibitor of TNF-induced NF-κB activity than SFN. Finally, using a cellular system mimicking constitutive active STAT5-induced cell transformation, we demonstrated that SFN can reverse the survival and growth advantage mediated by oncogenic STAT5 and triggers cell death, therefore providing experimental evidence of a cancer chemopreventive activity of SFN. This work thus identified STAT5, and to a lesser extent STAT1/STAT2, as novel targets of moringin. It also contributes to a better understanding of the biological activities of the dietary isothiocyanates GMG-ITC and SFN and further supports their apparent beneficial role in the prevention of chronic illnesses such as cancer, inflammatory diseases and immune disorders.Sulforaphane (SFN) and moringin (GMG-ITC) are edible isothiocyanates present as glucosinolate precursors in cruciferous vegetables and in the plant  Chronic pathological conditions such as cancer, inflammatory diseases and immune disorders figure among the leading causes of mortality worldwide ,2. Dietac-Myc, Pim-1, Bcl-x, Osm or Cis [We also reported recently that SFN inhibits STAT5-mediated transcription . STAT5 -benzyl ITC, also called moringin (thereafter referred to as GMG-ITC), recently attracted the attention of scientists for its chemopreventive activity. GMG-ITC is produced by myrosinase-catalyzed hydrolysis of glucomoringin (GMG) [Moringa oleifera is renowned for its medicinal virtues and nutritional value. All parts of the plant  are consumed or used in preparations for the treatment of various ailments, including infections, inflammation, hypertension and diabetes [in vitro and in vivo, notably through the inhibition of the NF-κB pathway [Beside SFN, another edible ITC from the plant in (GMG) –59. Moridiabetes . GMG-ITCdiabetes  and was  pathway ,61–63. T pathway ,64, GMG- pathway . On the  pathway . Beside In this study, we investigated the potential inhibitory activity of GMG-ITC on a number of signaling pathways often deregulated in chronic disorders , using the well-characterized SFN as a reference compound. We show—to our knowledge for the first time—that GMG-ITC, similarly to SFN, strongly inhibits STAT5-mediated transcription without altering STAT5 activation by phosphorylation. Whereas both ITCs exerted a limited inhibitory activity toward STAT1/STAT2-mediated transcription, we confirmed that they are potent inhibitors of NF-κB signaling, GMG-ITC being more efficient than SFN. Altogether, our data identified STAT5, NF-κB and, to a lesser extent, STAT1/STAT2 as molecular targets of GMG-ITC and SFN, therefore further supporting the proposed role of these natural ITCs as valuable dietary chemopreventive agents.Moringa oleifera Lam. (fam. Moringaceae) seeds and purified by HPLC (>99% purity), as previously described [Dimethyl sulfoxide (DMSO) and curcumin were purchased from SIGMA (D-2650 and 08511 respectively). R,S-Sulforaphane (SFN) was from LKT Laboratories (S8044). Moringin (GMG-ITC) was produced by myrosinase-catalyzed hydrolysis of glucomoringin isolated from escribed ,58. Comp2 in a humidified incubator. HeLa cells  were grGermany; ) was groGermany; . Non-indFor drug treatments in RT-qPCR and Western blot assays, HeLa and Ba/F3 cells were incubated with 0.4–10 μM SFN or GMG-ITC, 10–50 μM curcumin, or with 0.01–0.05% DMSO (vehicle), as indicated, for 30 minutes prior to cytokine stimulation. DMSO was adjusted to either 0.01% or 0.05% final, as specified, in all conditions. IFNα stimulation of HeLa cells was performed by incubating cells with 3 ng/mL rhIFNα2a (ImmunoTools) for either 30 minutes  or 4 hours . TNF stimulation of HeLa cells was carried out for 1 hour in the presence of 30 ng/mL TNF . For IL-3 stimulation of Ba/F3 cells, cells were washed twice in RPMI 1640 and rested in RPMI 1640 supplemented with 10% heat-inactivated FCS and penicillin/streptomycin for 6 hours before addition of 5 ng/mL IL-3 for either 5 minutes  or 30 minutes .5 and 1 x 105 cells/mL respectively, and 1 μL was used for cDNA synthesis using iScript cDNA Synthesis kit  following the manufacturer's instructions. Quantitative PCR was performed on a RotorGene Q (Qiagen) as previously described [Following cell harvest, Ba/F3 and HeLa cells were lysed in iScript RT-qPCR sample preparation reagent  at a concentration of 4 x 10escribed , using 0escribed ,54,68,69escribed ,54,69. D5 IL-3-growing Ba/F3 cells and 0.6 x 104 HeLa cells pre-treated 30 minutes with 0.1–100 μM SFN, 0.02–20 μM GMG-ITC, or with 0.1% DMSO (vehicle). DMSO concentration was adjusted to 0.1% in all conditions. Duration of incubation with the WST-1 reagent was 2 hours for Ba/F3 cells and 4 hours for HeLa cells, to be comparable to the duration of drug treatment in the gene expression analysis experiments. Data are expressed as a percentage of cytotoxicity relative to vehicle. Each WST-1 assay was performed in triplicate and repeated at least three times for each cell line. Results of one representative experiment are shown. Raw data  are available in the WST-1 assays  were performed to measure changes in mitochondrial dehydrogenase activity as previously described ,46, on 0The number of living and dead cells was evaluated by Trypan Blue exclusion following SFN or GMG-ITC treatment, as previously described ,46. ViabWhole-cell Brij protein lysis and Western-blot analyses were performed as described ,54. AntiCis, Osm and c-Myc was inhibited by SFN in a dose-dependent manner, while expression of the housekeeping gene 36b4 remained unaffected  and G1P3 being more strongly inhibited (75% and 100% inhibition by 10 μM SFN and GMG-ITC respectively). Interestingly, basal expression of c-Myc remained unaffected upon treatment with up to 10 μM SFN or GMG-ITC  cytotoxicity in the presence of 10 μM SFN or 2 μM GMG-ITC and increased (60%) cytotoxicity at 100 μM SFN or 20 μM GMG-ITC . TherefoWe next investigated whether treatment with SFN or GMG-ITC had an effect on STAT1 and/or STAT2 phosphorylation, using non-toxic ITC concentrations. HeLa cells were pre-treated for 30 minutes with 10 μM SFN or 2 μM GMG-ITC and stimulated with IFNα2a for 30 minutes. STAT1 and STAT2 phosphorylation was evaluated by Western blot using phospho-specific antibodies. Treatment of IFNα-stimulated HeLa cells with SFN or GMG-ITC had no major effect on STAT1 and STAT2 phosphorylation and did not affect their respective protein levels . Given tin vitro and in vivo [IL-8, IL-6) and of the housekeeping gene S9 was assessed by RT-qPCR . Complete inhibition of NF-κB target gene expression was observed at 10 μM SFN or GMG-ITC, while inhibition only reached 8–24% (depending on the target gene) upon treatment with the same concentration of curcumin. Expression of the control gene S9 remained mostly unchanged in all conditions. Together, these experiments reveal that both SFN and GMG-ITC inhibit NF-κB signaling with a greater potency than that mediated by the well-characterized NF-κB inhibitor curcumin. Beside, GMG-ITC appears to be more potent at 2 μM than SFN in inhibiting TNF-induced gene expression.A number of independent reports indicate that both SFN and GMG-ITC inhibit NF-κB signaling  in vivo ,61,62,74 in vivo . However in vivo . HeLa ce RT-qPCR . TNF-indin vivo [The ability of natural compounds such as the ITCs SFN and GMG-ITC to inhibit multiple signaling pathways controlling cell proliferation, cell survival, immune modulation and inflammation, make them attractive agents in particular for cancer prevention. To assess the chemopreventive activity of natural compounds, we developed a cellular model of STAT5-induced oncogenic transformation, so-called Ba/F3-tet-on-1*6 . Ba/F3-tin vivo ,68,76,77Doxycycline-dependent expression and phosphorylation of FLAG-tagged STAT5A-1*6 was verified by Western blot using FLAG-, STAT5- and phospho-STAT5-specific antibodies . STAT5A-This study aimed to analyse and compare the inhibitory activities of two natural ITCs, sulforaphane (SFN) and moringin (GMG-ITC). We focused on a number of signaling pathways essential for the control of cell proliferation, apoptosis, immune response and inflammation and known to be dysregulated in various chronic diseases. Gene expression analyses revealed that SFN and GMG-ITC differentially affected the investigated signaling pathways. We found that both SFN and GMG-ITC inhibit IL-3-induced STAT5 signaling at concentrations as low as 0.4 μM. This is to our knowledge the first demonstration of an inhibitory effect of GMG-ITC on STAT5-mediated transcription. We also showed for the first time that both ITCs inhibit to some extent IFNα-induced STAT1 and STAT2 activity, although the effective concentration was 25-times higher (10 μM) than that required for inhibition of STAT5 activity. In both cases, inhibition of JAK/STAT signaling did not take place at the level of STAT protein phosphorylation, suggesting a downstream inhibitory event. Interestingly, SFN and GMG-ITC specifically impaired STAT5-dependent (Ba/F3) but not basal (HeLa) c-Myc expression, highlighting the specificity of action of these ITCs. Furthermore, SFN and GMG-ITC inhibit TNF-induced NF-κB signaling, thus supporting previous studies performed in other experimental systems ,61–63. IThe observation that STAT5 target gene expression is similarly inhibited by SFN and GMG-ITC treatment may suggest a similar inhibitory mechanism. This possibility is supported by the observation that neither SFN nor GMG-ITC treatment interfere with STAT5 phosphorylation. We showed before that SFN treatment does not affect STAT5 activation (phosphorylation), nor its binding to DNA, and only marginally interferes with STAT5-mediated recruitment of RNA polymerase II . FurtherG1P3, ISG15, STAT1 and IRF9) was differentially affected upon SFN and GMG-ITC treatment, in favor of a promoter-specific regulation. Further investigations will be necessary to address the level of inhibition by these natural ITCs. It should be noted that, in contrast to STAT1/2/5 proteins, constitutive and IL-6-induced STAT3 phosphorylation was shown to be reduced upon treatment by SFN in a number of cancer cell lines [The finding that SFN and GMG-ITC treatment does not alter STAT1 and STAT2 phosphorylation similarly suggest that inhibition of IFN-stimulated gene expression takes place at the transcriptional level. This proposition is further supported by the fact that expression of the ISGs investigated  and as density of living cells (number of living cells/mL). To better visualize SFN-induced cell growth inhibition in non-induced cells, the Y-axis scale of the corresponding graph was expanded (lower left graph).(EPS)Click here for additional data file.S1 FileRT-qPCR CT values; WST-1 OD values; uncropped Western blots.(PDF)Click here for additional data file.