The positive and negative predictive values of specific IgE to penicillins are not well established for penicillin hypersensitivity. One reason may be that serum IgE levels to penicillin diminish over time. The objective in this study was to investigate variations in serum half-life (T½) for specific IgE to penicillins (s-IgE) and to evaluate the outcome of penicillin challenges in patients with previous but not present specific IgE to penicillins.Two subgroups were investigated. All included patients had a history of penicillin allergy with reported symptoms such as urticaria/angioedema or unclassified cutaneous rash. T½ of specific IgE to penicillins was calculated based on sera from 29 patients with repeated measurements of s-IgE. Twenty-two patients with a previous positive s-IgE was followed and challenged with penicillin when IgE had become negative.The T½ for s-IgE varied between the 26 patients with decreasing s-IgE from 1.6 months to 76.4 months and 52% had a T½ of less than a year. The three patients with stable and increasing IgE-values showed T½ approaching infinity A total of 29 challenges with β-lactams were performed. Four different patterns were seen when evaluating the clinical reaction to challenge (positive/negative) and post-challenge boost of s-IgE (yes/no). Eight (36.4%) had negative challenge and negative post-challenge s-IgE, eight (36.4%) negative challenge, but positive post-challenge s-IgE levels. 3 (13.6%) had positive challenge and positive post-challenge s-IgE whereas 3 (13.6%) were challenge positive, but had negative post-challenge s-IgE.Specific IgE to penicillins declines over time stressing the importance of a close time relation between diagnostic work-up and clinical reaction. Reversal of previously positive s-IgE may still be associated with positive penicillin challenges and/or re-boostering of s-IgE to positivity. Correct diagnosis of allergy to penicillin is of great importance, but in contrast to what is found in a patient with a history of grass pollen allergy, discrepancy between skin test, measurement of specific IgE and outcome of challenge is often seen in patients with suspected penicillin allergy. Recommendations to patients based on false-negative results can lead to severe adverse reactions whereas false-positive results can lead to administration of less effective and more expensive drugs. We have previously demonstrated , 2 that In the period September 2003 to October 2013, our serum bank contains samples from 2791 adult patients with suspected penicillin allergy, who had performed measurements of s-IgE to penicillins. Hereof a total of 287 adult patients had a positive s-IgE to one or more penicillins (benzylpenicillin (pen G), phenoxymethylpenicillin (pen V), ampicillin (AMP), amoxicillin (AX)).A total of 29 patients (10 men and 19 women) had 3 or more measurements of s-IgE over a period of up to 59 months. These were included for determination of T½ of s-IgE , Figure The serum bank contained sera from forty-two patients followed prospectively with repeated measurements of s-IgE that had reverted to negative test results. Of these twenty-two (12 men and 10 women) agreed to participate in the present study. All patients had negative skin prick test (SPT) and intracutaneous test (ICT) to penicillins prior to penicillin challenge, Figure The β-lactam eliciting the initial reaction (culprit drug) was pen V in 15 cases (62.5%), AX in 5 (20.8%) and DX in 2 (8.3%).According to the history reactions commencing within 1 h after drug intake were classified as immediate reactions (IR) and occurred in 7 patients. Reactions commencing 1 h or longer after drug intake were classified as nonimmediate reactions (NIR) and seen in 15 patients. Twelve of the 22 patients reported urticaria and/or angioedema  and 10 patients with unclassified cutaneous rash . Sixteen of the 22 patients had experienced their initial clinical reaction within one year prior to measurement of the positive s-IgE (thirteen even within 6 months), two within 1–5 years and 4 more than five years prior to blood sampling.All patients showed undetectable levels of s-IgE at the time of penicillin challenge. The challenge tests were performed according to procedures in our two previous studies , 2, withThe Regional Scientific Ethical Committees for Southern Denmark has approved the study . All patients gave written informed consent for the study as well as consenting to storage of their blood in the serum bank.S-IgE levels to pen G, pen V, AMP, AX and MDM (minor determinant mix) were measured using ImmunoCAP . A positive result was defined as a value ≥ 0.35 kU/L. Total IgE levels were measured in 20 of the 22 patients included in the penicillin challenge in patients with previous IgE sensitization to penicillins .k, where k is the positive value of the terminal slope of the IgE concentration-time curve. The majority of the patients (n = 20) had experienced their initial clinical reaction within one year prior to measurement of the positive s-IgE, three within 5 years and 6 more than five years prior to blood sampling.Determination of the elimination half-life (T½) of s-IgE was calculated using WinNonlin 6.3 . T½ was calculated by the following equation: T½ = ln /k, whereData were analyzed for significance using the Fischer’s exact test. Probability values of < 0.05 were considered statistically significant. The statistical analyses were performed with STATA 13, StataCorp, College Station, Texas, USA.The study included 51 cases  were included in both the penicillin challenge in patients with previous IgE sensitization to penicillin and in the estimation of T½ of s-IgE.For estimation of T½ of s-IgE, 29 (19 women + 10 men) patients with three or more consecutively serum measurements of specific IgE to one or more penicillins were included. Median level (25–75 percentiles) for s-IgE at inclusion was 4.3 kU/l (1.4-18.5). Figure Twenty-two patients had a previous positive s-IgE; all were skin test negative to the four penicillins (data not shown). The demographic data, results of specific IgE determination and outcome of their challenge(s) are presented in Table We describe for the first time the outcome of penicillin challenge in patients with a history of penicillin allergy and with previous but not present measurable levels of specific IgE to penicillins. Further, we describe for the first time variations in serum half-life of specific IgE in patients, sensitized to a penicillin.The present study confirms that levels of s-IgE decline over time with different rates in the single patient, neither related to initial level nor to positivity of reaction. Possible underlying mechanisms behind this variable decay is unknown, but in concordance with previous studies having shown that specific IgE antibody levels tend to decrease over time in the absence of new contact with the hapten –12. A deT½ for s-IgE varied from 1.6 to 76.4 months. In approximately half of the patients T½ was less than a year. Our results thus imply, that a considerable number of penicillin allergic patients will present with a negative s-IgE depending on the time elapsed since their clinical reaction had taken place.A total of 29 challenges with β-lactam antibiotics were performed in 22 patients with previous positive specific IgE to penicillin and negative skin tests. Four different patterns were seen when evaluating the clinical reaction to challenge (positive/negative) and post-challenge boost of s-IgE (yes/no). Eight patients had negative clinical reaction to challenge and negative post-challenge s-IgE, 8 patients had negative challenge, but positive post-challenge specific IgE levels. Three patients had positive clinical reaction to challenge and positive post-challenge s-IgE whereas 3 patients were challenge positive, but had negative post-challenge s-IgE. The six cases with positive challenge should be classified as allergic to penicillin and the 8 cases with negative challenge and negative post-challenge s-IgE should be classified as not being allergic. It is difficult to decide how the 8 patients with negative challenge and subsequent positive post-challenge s-IgE should be classified and they should probably be considered as still being allergic. The appearance of s-IgE after the challenge may be interpreted as a booster reaction. A conclusion can, however, not be drawn without actually challenging the patients once again. Patients with negative s-IgE at the time of investigation and a case history of reaction several years prior to investigation may have been s-IgE positive at the time of reaction. The 22 patients in the present study would have been classified as s-IgE negative if investigated in average 2.7 years after the clinical reaction took place (range 0–8 years). The frequency of positive outcomes of penicillin challenge in the present study  was similar to the findings in our previous study [Several studies have evaluated , 15–20 tMeasurement of s-IgE is recommended in patients with a history of an immediate reaction (IR) . Due to In a study from 2013 Johansson et al.  indicateRegarding the challenges with penicillin in patients with a previous but not present positive s-IgE, a negative sIgE was not associated with tolerance in 63.6% of the tested patients and even a tolerated readministration of penicillins  may lead to boostering of sIgE with unknown significance, but possibly reoccurrence of penicillin allergy. Further the more indistinct outcomes in the patients with negative challenge but positive post-challenge s-IgE, no firm conclusion can be drawn without challenging the patients once again. Our results thus suggest that a negative outcome of the 7-day oral challenge should be followed by measurement of specific IgE 4 weeks later, and only if this proved negative, the patient should be classified as tolerant to penicillin.An IgE response to penicillin is often a transient phenomenon. In this study approximately half of the patients had a serum half-life of specific IgE to penicillin of less than a year, suggesting that the majority of patients with IgE sensitization at the time of reaction have converted to IgE negativity if measured more than a year after the incident took place. This is supported by the finding that we found the same frequency of positive outcomes during penicillin challenge in the present study compared to our previous study, where all patients  challenged never had had demonstrable IgE to penicillin  in contr


However, technical issues limited the sensitivity of analysis of pre-syncytial blastoderm embryos and precluded studies of oocytes after stage 13. We developed methods to analyze stage 14 oocytes and pre-syncytial blastoderm embryos, and found that stage 14 oocytes make Bcd protein, that bcd RNA and Bcd protein distribute in matching concentration gradients in the interior of nuclear cycle 2–6 embryos, and that Bcd regulation of target gene expression is apparent at nuclear cycle 7, two cycles prior to syncytial blastoderm. We discuss the implications for the generation and function of the Bcd gradient.Bicoid (Bcd) protein distributes in a concentration gradient that organizes the anterior/posterior axis of the Drosophila embryo. It has been understood that DOI:http://dx.doi.org/10.7554/eLife.13222.001 As an embryo develops, a single cell transforms into a collection of different types of cells. One protein that is crucial for this process in fruit fly embryos is Bicoid. Thirty years ago, scientists discovered that Bicoid protein is concentrated at the head end of the embryo and gradually decreases in amount towards the rear end. This concentration gradient of Bicoid protein organizes the embryo body and regulates the expression of many genes, thus directing the cells to develop different identities.Several assumptions had been made about how this gradient is established. It was thought that in the unfertilized egg, the mRNA molecules that will be translated to produce Bicoid proteins are stored in an inactive state in the region of the egg that later develops into the embryo’s head. In the embryo, the mRNA molecules were believed to remain in the head region while being translated, with the newly formed proteins then gradually spreading from this site to create the Bicoid gradient. It was also thought that no Bicoid proteins are stored in the unfertilized egg. However, no known methods were sensitive enough to investigate these assumptions.Now, using newer and more sensitive methods, Ali-Murthy and Kornberg show that Bicoid protein is present in the unfertilized fruit fly egg in the same region as the mRNA molecules that make Bicoid. Furthermore, the Bicoid gradient forms when the embryo has fewer than 32 nuclei, much earlier in development than previously thought. The Bicoid protein also does not appear to spread passively towards the rear of the embryo, but is transported in a more orchestrated manner.Overall, Ali-Murthy and Kornberg’s results suggest that the early fruit fly embryo is more organized and actively regulated than had been previously understood. This paves the way for further studies that use sensitive techniques to investigate this early stage of development.DOI:http://dx.doi.org/10.7554/eLife.13222.002 Whetherg laying , or is p dispute .After fertilization, nuclei divide rapidly and synchronously eight times in the interior of the embryo, moving outward in a choreographed sequence that places them simultaneously at the surface at nuclear cycle 9 (nc9). The five division cycles that follow delineate the syncytial blastoderm stages nc10-nc14. Nuclear divisions cease at nc14, whereupon the nuclei begin to individuate into single cells and gastrulation ensues. Various measures, including in situ hybridization , RT-PCR Oogenesis provides the Drosophila egg with a rich dowry of mRNA that is essential to the development of the early, pre-cellular embryo, and for a number of reasons, the period that precedes the maternal-to-zygotic transition has been considered to depend only on maternal stores and to be independent of the zygotic genome. One, the early nuclear divisions are so rapid (9.6 min) that productive gene expression has been deemed impossible. Two, molecular analyses of transcriptional activity have almost universally failed to detect RNA synthesis at pre-syncytial blastoderm stages, even as the sensitivity of the detection methods has increased. Three, comprehensive genetic screens for mutants defective in early development identified many genes that are required maternally, but found no evidence for genes that must be active in the zygote prior to cellularization at nc14 ; 1986.Although these observations have substantiated the idea that the gene products supplied by the mother during oogenesis are sufficient for first thirteen cleavage cycles, this conclusion is based on negative findings, and because it depends on the sensitivity of the analysis, it leaves open the possibility that more sensitive methods might detect zygotic transcripts expressed from a small number of active genes or might recognize phenotypes in mutant embryos that were not revealed by then available histological techniques. Drosophila embryos are heavily populated with yolk and glycogen granules that impede histological studies, and have few obvious morphological features that can be evaluated for dependence on genotype. In addition, the idea that rapidly dividing nuclei are incapable of expression has no experimental basis because the capacity for transcription and translation at early nuclear cycles has not been analyzed. It is possible therefore that the normal transcriptional processes are sufficient for transcription units that are small  gene  gene . En protr to nc7 . These fThe work described here investigates the development of the early embryo further, focusing on the formation and function of the Bcd gradient. To do so, we needed to develop methods that overcome the technical impediments that have limited analysis of the early stages. For instance, because Drosophila fertilization cannot be made synchronous and because females hold their embryos for variable periods prior to laying, an embryo of any particular stage must be identified histologically and individually, and methods must be used that are sensitive enough to detect expression from a small number of active genes in the small number of nuclei. Although molecular detection methods have improved, antibody staining and in situ hybridization have been ineffective due to the large number of yolk and glycogen granules that reduce signal to noise and sensitivity. By using sensitive methods that we modified for studies of the early embryo, we found that the processes that generate the Bcd protein gradient are more complex and operate earlier than had been appreciated, and that the function of the Bcd gradient begins prior to formation of the syncytial blastoderm.Krüppel (Kr) is a 'gap'” gene that is expressed by pre-cellular, syncytial blastoderm embryos in a central band that spans approximately 20% embryo length. Although the earliest reported expression detected by in situ hybridization is nc10  if mothers were wild type (had two bcd copies) and 54–75% if mothers had six. This is indicative of the dependence of Kr expression on maternal Bcd , this result suggests that nuclei are polarized prior to their arrival at the cortex and it is consistent with the conclusion that the nuclear dots mark sites of Kr transcription.The number of nuclei with fluorescent dots at nc7 and nc8 was small, and although the position of these marked nuclei is in the central region of the embryo where the Kr band will be robust at later cycles, it is important to know whether the dots in these nuclei mark nuclear transcripts. Chromosomes in nuclei at the cortex of syncytial blastoderm embryos are organized and polarized such that the centromeric regions congregate apically and the telomeres congregate basally . Chromosexuperentia (exu) or staufen (stau). Function of the exu and stau genes during oogenesis is essential to localize bcd mRNA at the anterior pole and for the Bcd gradient in syncytial blastoderm embryos , follicle cells completely envelope the oocyte , the chostage 12  and 13 , and because of the spatial relationship between these distributions, it posits that the Bcd protein gradient is a direct consequence of the location of bcd mRNA  from the anterior pole is continuous during both the pre-syncytial blastoderm and syncytial blastoderm stages PC4/CyO; cn1 exu4 bw1/CyO, l(2)DTS1001; w*; Df(2R)exu2/CyO; bcdE1; Diap11 st1 kniri-1 bcd6 rnroe-1 pp/TM3, Sb1; Dp(bcd:GFP); bcdE1/TM3; +/CyO, Dp(bcd); bcdE1/TMDGRP-208; stau3; 10, b1 pr1 Bl1 c1/SM1; Dp(bcd:GFP)Df(2R)Kr (4x bcd); p (6x bcd)(WT) Primary antibodies: rabbit α-Bcd (M. Biggin); rabbit α-GFP ; sheep α-DIG ; mouse α-Lamin . Secondary antibodies (Life Technologies): goat α-rabbit, Alexa 488; goat α-rabbit, Alexa 555; donkey α-rabbit, Alexa 555; donkey α-sheep, Alexa 488; donkey α-sheep, Alexa 555; goat α-mouse, Alexa 488; goat α-mouse, Alexa 555.Embryos were de-chorionated in 50% bleach, washed with 0.1% Triton X-100 and transferred to a 20 ml vial containing fixative , 50 mM EGTA (pH 8.0), 10.24% formaldehyde (Ultrapure), and shaken for 1.5 hr. The lower phase was removed and embryos were de-vitellinized by vigorous shaking in 4 ml cold methanol (MeOH) for 1 min, transferred to a 1.5 ml (Eppendorf) tube, rinsed 3x with MeOH and stored at -20°C in MeOH.Embryos (0.05 ml) were rocked in 1:1 MeOH:EtOH in a 1.5 ml (Eppendorf) tube for 5 min, and rinsed with EtOH (2 x 5 min). 0.9 ml EtOH was removed, 0.9 ml xylene was added, and after rocking for 1 hr, liquid was removed and embryos were rinsed with 1 ml EtOH (2 x 5 min).Embryos were rinsed by rocking in 1 ml MeOH (2 x 5 min), rinsed in 1:1 MeOH:0.5% formaldehyde in PBT  for 5 min, rocked in 5% formaldehyde in PBT (1 hr), rinsed by rocking in 1 ml PBT (2x) followed by rocking in 1 ml PBT (4 x 10 min).Embryos were incubated with rocking for: (1) 10 min at room temperature in 1 ml 1:1 hybridization solution ; (2) 10 min in 1 ml HybS at 55°C; (3) 45 min in 1 ml fresh HybS at 55°C; and (4) overnight in fresh HybS at 55°C.5 μl probe (prepared according to Embryos were rinsed at 55°C in 1 ml HybS (3x) that was pre-warmed to 55°C for 15 min, washed in 1 ml HybS (6 x 30 min), washed in 1:1 HybS:PBT at room temperature for 10 min and washed in PBT at room temperature for 30 min. Embryos were rocked in 1 ml blocking solution (1:5 Roche blocking reagent:PBT) for 1 hr, in 0.5 ml fresh blocking solution with 0.12 mg pre-adsorbed α-Digoxigenin antibody overnight at 4°C, washed in 1 ml PBT at room temperature (10 x 30 min) and in 1 ml blocking solution at room temperature for 1 hr. Embryos were incubated for 1.5 hr in 0.5 ml secondary antibody in blocking solution, rinsed in 1 ml PBT (2x), stained with DAPI (0.1 μg/ml) in 1 ml PBT (5 min), washed in 1 ml PBT (3-4x) at 4°C (2–14 hr), mounted in Vectashield on a slide and sealed under a coverslip with nail polish.Embryos were de-chorionated in 50% bleach, washed with 1% Triton X-100 and transferred to a 20 ml vial containing fixative , 1 ml 16% formaldehyde (Ultrapure)) and shaken for 25 min The lower phase was removed and embryos were de-vitellinized by vigorous shaking in 4 ml cold MeOH for 1 min, transferred to a 1.5 ml (Eppendorf) tube, rinsed 3x with MeOH and stored in MeOH at -20°C.Embryos were rinsed 2x with PBT , washed by rocking for 4 x 15 min in 1 ml PBT, rocked for 1 hr in 1 ml PBTi , rocked for 2 hr in 1 ml hybridization buffer, rinsed in 1 ml PBT (2x) and rocked in 1 ml PBT for 15 min Embryos were incubated for 1 hr in 1 ml blocking solution followed by incubation for 14+ hr at 4°C in 0.5 ml blocking solution + primary antibody, rinsed by rocking at room temperature in 1 ml PBT for 30 min (10x), and blocked for 1 hr in 1 ml blocking solution. For secondary antibody, embryos were incubated with secondary antibody in 0.5 ml blocking solution for 1.5 hr at room temperature, rinsed in 1 ml PBT (2x), stained with DAPI in 1 ml PBT (5 min), washed in 1 ml PBT (3-4x) at 4°C for 2–14 hr, mounted in Vectashield on a slide and sealed under a coverslip with nail polish.Stage 14 oocytes were dissected from 5 pairs of ovaries  that were obtained from 4–5 day old, well-fed females. Stage 14a and 14b accounted for 96–97% of stage 14 oocytes. Approximately 3% were stage 14c and 14d. To obtain larger numbers of stage 14c and 14d oocytes, five pairs of ovaries were isolated and incubated in PBS for 20 hr at room temperature; the relative proportion of stage 14 oocytes in these preparations was 14a, 0%; 14b, 2–3%; 14c, 75–80%; 14d, 15–20%. Stage 14a and 14b oocytes were not extruded from the distal tip of the ovary; 14c and 14d oocytes were wholly or partially in a lateral branch of the oviduct. The stage 14c and 14d oocytes were sensitive to hypochlorite treatment and had not completed meiosis I, and were therefore designated as not activated . OocytesOvaries were dissected in PBS from 4–5 day old females. Stage 14 oocytes were separated from the ovaries and transferred to a 20 ml vial containing fixative , 50 mM EGTA (pH 8.0), 10.24% formaldehyde (Ultrapure), and shaken for 1 hr. The lower phase was removed, 5 ml cold MeOH was added and after 1 min vigorous shaking, the upper phase was removed and oocytes were transferred to a 1.5 ml (Eppendorf) tube, rinsed with MeOH (3x) and stored at -20°C in MeOH.Fixed oocytes in MeOH were transferred to a glass cavity slide and viewed under a dissecting microscope. Fine forceps were used to hold the dorsal appendages and the dorso-lateral side of the chorion and vitelline membrane were sliced with a fine needle. Oocytes freed of chorion and vitelline membrane were stored at -20°C in MeOH.Oocytes were rinsed 2x with 1 ml PBT  and with rocking in 1 ml PBT  for 1 hr, in 1 ml HybS for 2 hr, and rinsed with 1 ml PBT (2x) and with rocking in 1 ml PBT for 15 min.Oocytes were rocked in 1 ml blocking solution (1:5 Roche blocking reagent:PBT) for 1 hr, and in 0.5 ml pre-adsorbed primary antibody in blocking solution at 4°C overnight, washed with rocking in 1 ml PBT at room temperature  at 4°C for 2–14 hr, mounted in Vectashield on a slide and sealed under a coverslip with nail polish.Same as for oocyte antibody staining (above).Same as for embryo in situ hybridization (above).Embryos were mounted with slight pressure under coverslips and orientation was determined by analysis of serial optical sections. Images were obtained with a Leica SPE confocal microscope and processed with ImageJ.Total RNA was prepared from five embryos with Zymo Research RNA MicroPrep kits (Cat. #R1060) and quantified by absorbance with a nanodrop spectrophotometer. cDNA was prepared using Applied Biosystem High Capacity RNA-to-cDNA kits (Cat. #4387406) starting with approximately 200 ng RNA. The Q-PCR reactions were carried out with a BioRad C1000 Touch Thermal Cycler and SsoAdvanced SYBR Green according to manufacturers’ instructions. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Drosophila pre-syncytial blastoderm" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Allan Spradling as the Reviewing Editor and James Manley as the Senior Editor.Thank you for submitting your work entitled "Bicoid gradient formation and function in the The following individuals involved in review of your submission have agreed to reveal their identity: Michael Eisen and Markus Noll (peer reviewers).The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Drosophila egg and early embryo provide much of our current insight into how maternal factors contribute to early developmental patterning. The formation of the bicoid protein gradient by the regulated translation of maternally localized bcd mRNA represents a key aspect of this process, one that has become the textbook example of a "morphogen gradient." However, the mechanisms which shape the gradient remain imperfectly understood and competing models have been put forward. The consensus opinion among the reviewers is that the work presented here adds to our understanding of Drosophila embryo patterning and Bcd gradient formation.The Kr is expressed as nascent transcripts by nuclear cycle 7 and 8 (nc7 and 8) at the proper location (2) in nuclei that are already polarized , i.e., prior to their arrival at the cortex. (3) Bcd protein is visible in a concentration gradient already at nc2 to nc6, which depends also at early presyncytial blastoderm stages on the presence of maternal Stau and Exu proteins. (4) At these early presyncytial blastoderm stages, the Bcd protein gradient extends posteriorly in what the authors call a medial plume rather than at the cortex as at later stages. This proves that the process of gradient formation must be more complex than previously thought. (5) The distribution of bcd RNA is very similar to that of Bcd protein also at these early cleavage stages (nc2 – nc6), with the medial plume reaching its maximum intensity at nc4. (6) The authors detected Bcd protein already at nc1 and (7), most astonishingly, in stage 14c and 14d oocytes, again coincident with the anterior location of bcd RNA in oocytes. (8) In contrast to bcd RNA, which was evident in oocytes at earlier stages, Bcd protein was not detected before stage 14c. This demonstrates that the translational block of bcd mRNA is relieved prior to fertilization and that Bcd protein is present precisely where bcd mRNA is sequestered.Several important findings are reported. (1) The gap gene bcd RNA and protein, and by contradicting some aspects of the leading model for how the gradient is produced.The authors describe a different shape and demonstrate that zygotic gene expression starts earlier than the pre-cellular blastoderm stage by forcing a revision in the previously accepted timing and spatial distribution of bcd mRNA that, by translation, produces the Bcd protein gradient although the authors are more conservative. The most important conclusion is that mechanisms of morphogen gradient formation in general are not dependent on passive diffusion, overthrowing a tenet maintained over many decades, but "appear to involve dispersion along cytoskeletal cables."In the Discussion, the authors propose a two-step model of Bcd gradient formation, which differs from an earlier model mainly by the initial gradient formation of the medial plume during nc2-6. This gradient is followed by a cortical gradient, consistent with a model previously proposed . This latter model and the model proposed by the authors are consistent with the gradient being formed by the A small number of relatively minor revisions are now requested.Drosophila embryos. While the authors document expression earlier than previously reported, an achievement that likely required improved sensitivity of DIG in situ hybridization as well as of antibody staining of early embryos to achieve a much better signal-to-noise ratio. However, the manuscript does not include experiments directly comparing different in situ hybridization methods, including in particular smFISH, which is generally accepted to have greater sensitivity in many applications. It remains unclear why the low level expression described here was not detected in previous studies. Consequently, statements about the relative sensitivity of various methods should be removed from the manuscript unless data from such tests are added.1) The major weakness of the paper relates to the authors' claim to have developed new and more sensitive methods for detecting RNA and protein in preblastoderm bcd RNA translational block is relieved in late stage 14 oocytes is interesting but because follicles were incubated in vitro for an extended period, the possibility of artifact remains. A recent study shows that stage 14 oocytes about to be ovulated can be recognized by the loss of posterior follicle cells . The authors may wish to investigate whether the onset of bcd translation takes place before or after ovulation, in vivo.2) The finding that the 3) The authors should more adequately reference earlier literature, which shows that nuclear foci of in situ hybridization are often sites of nascent transcription. A small number of relatively minor revisions are now requested. 1) The major weakness of the paper relates to the authors' claim to have developed new and more sensitive methods for detecting RNA and protein in preblastoderm Drosophila embryos. While the authors document expression earlier than previously reported, an achievement that likely required improved sensitivity of DIG in situ hybridization as well as of antibody staining of early embryos to achieve a much better signal-to-noise ratio. However, the manuscript does not include experiments directly comparing different in situ hybridization methods, including in particular smFISH, which is generally accepted to have greater sensitivity in many applications. It remains unclear why the low level expression described here was not detected in previous studies. Consequently, statements about the relative sensitivity of various methods should be removed from the manuscript unless data from such tests are added.bcd and Kr in situ hybridization with FISH, Quantigene and DIG probes. The text in the Results now reads:A supplemental figure to Kr transcription prior to nc10, we evaluated various different techniques for in situ hybridization, and determined that a procedure we developed that uses DIG probes was the most sensitive for our studies of Kr and bcd transcripts in pre-cellular embryos  The finding that the bcd RNA translational block is relieved in late stage 14 oocytes is interesting but because follicles were incubated in vitro for an extended period, the possibility of artifact remains. A recent study shows that stage 14 oocytes about to be ovulated can be recognized by the loss of posterior follicle cells . The authors may wish to investigate whether the onset of bcd translation takes place before or after ovulation, in vivo.The following sentence has been added to the Methods:“Results of in situ hybridization and antibody staining with oocytes obtained from freshly dissected ovaries and from ovaries incubated ex vivo were indistinguishable.”3) The authors should more adequately reference earlier literature, which shows that nuclear foci of in situ hybridization are often sites of nascent transcription.The earlier literature showing foci of in situ hybridization as sites of nascent transcript has been cited as follows:Kr band”.”“In situ hybridization can detect sites of nascent transcript production as points of staining or fluorescence , and the nc10-13 embryos we analyzed had bright dots in most or all nuclei in the “


The TRIM5α restriction factor interferes with retroviral infections by inhibiting an early step of viral replication. TRIM5α activity was recently proposed to be regulated by the SUMO machinery and one SUMO consensus conjugation site as well as three putative SUMO interacting motifs (SIMs) were identified within TRIM5α sequence. Whereas mutation of the SIM sequences was found to abolish TRIM5α antiviral activity, mutation of the consensus SUMO conjugation site did not affect its restriction capacity, although this putative site has never been shown to be actually a SUMO substrate.in vitro and in cellulo and that Lysine 10 is the main SUMOylation site. Mutation of the consensus SUMO conjugation motif in position 10 abrogated SUMOylation at this position, but did not disrupt TRIM5α antiviral activity.Here we further demonstrate that TRIM5α relies on the SUMO machinery to promote restriction, since SUMO1 overexpression enhances TRIM5α-mediated retroviral inhibition whereas knockdown of SUMO1 or E2 SUMO conjugating enzyme Ubc9 prevents restriction. Furthermore, we show for the first time that TRIM5α is SUMOylated both Altogether, our results confirm that the SUMO machinery is involved in TRIM5α-mediated retroviral restriction, and demonstrate that TRIM5α is a SUMO 1 and SUMO 2 substrate. The inability to abrogate TRIM5α antiviral activity by mutating its main SUMO conjugation motif supports the notion that non-covalent interaction with SUMO or SUMOylated proteins rather than TRIM5α direct SUMOylation is required. TRIM5α is a restriction factor that interferes with an early step of retroviral replication in a species-dependent manner. Rhesus macaque TRIM5α (rhTRIM5α) is a potent inhibitor of HIV-1 infection, whereas its human counterpart (hTRIM5α) only weakly restricts HIV-1 . TRIM5α Most TRIM proteins, including TRIM5α, self-associate into corpuscules, either cytoplasmic or nuclear . One of via an ATP-dependent enzymatic pathway involving 4 enzymes, a SUMO protease, an E1-activating enzyme, an E2-conjugating enzyme (Ubc9) and an E3 ligase, via a mechanism essentially analogous to that of ubiquitin [per se, proteins can also interact non-covalently with SUMO or SUMO-conjugated proteins via a SUMO-interacting motif (SIM) [SUMOs are small ubiquitin-like proteins that are posttranslationally conjugated to other proteins, thereby regulating a wide range of biological processes, including transcription, DNA repair, chromosome dynamics and subcellular localization of target proteins . The biquitin ,8. Besidif (SIM) . Three Snzyme Ubc and an Eif (SIM) . It is sWe first evaluated the implication of SUMO machinery in TRIM5α-mediated antiviral activity. For this purpose, HeLa cells were transfected or not with small amounts of rhTRIM5α-expressing plasmid, in order to obtain a moderate block of viral infection that can be modulated. We tested the effect of siRNA-mediated knockdown of endogenous SUMO1, Ubc9 (the E2 SUMO conjugating enzyme) or PIAS1 (an E3 SUMO ligase) expression on rhTRIM5α anti-HIV activity. As shown in Figure In order to confirm these results, we tested whether SUMO1 was capable of modulating rhTRIM5α-mediated HIV-1 restriction by transfecting SUMO1 in HeLa cells expressing or not rhTRIM5α. The expression level of rhTRIMα was reduced compared to panel 1A, in order to limit the amplitude of HIV-1 restriction. As shown in Figure in situ proximity ligation assay (PLA). Cells were probed with anti-TRIM5α and anti-SUMO1 primary antibodies, followed by species-specific PLA probes. Our results show close proximity of TRIM5α with SUMO1, with an average of 38.6 spots per cell +/- 8.9 (SD), compared with 0.41 spots per cell +/- 0.39 for the PLA probes alone control. These results confirm that endogenous TRIM5α is conjugated by SUMO or interacts with SUMOylated proteins. Similar results were obtained following infection with HIV-1 (data not shown).To confirm interaction of TRIM5α with SUMO or SUMOylated proteins, we performed Duolink in vitro SUMOylation assay using purified recombinant E1 (SAE1 and SAE2 subunits) and E2 (Ubc9) enzymes, recombinant SUMO1 or SUMO2, and increasing doses of GST-tagged PIAS1 E3-ligase or GST alone, as control. As a substrate, we used in vitro translated 35S-labelled human or rhesus macaque TRIM5α. As shown in Figure in vitro. Interestingly, we observed an enhancement of SUMO1 and SUMO2 conjugation to both human and rhesus macaque TRIM5α when the reaction was performed with increasing concentrations of GST-PIAS1.In order to determine whether TRIM5α itself can be a substrate for SUMO modification, we employed an in cellulo. Since no high molecular weight form of TRIM5α could be detected in wt HeLa cells (not shown), we co-transfected human or rhesus macaque HA-tagged TRIM5α with Ubc9 in order to boost the SUMOylation machinery. Two days post-transfection, cell extracts were analyzed by western-blot, revealing two bands, a major band at 55 kDa, which corresponds to unmodified TRIM5α, and a faint band at approximately 75 kDa, which corresponds to the expected size for TRIM5α conjugated to one SUMO moiety  and LK263KP (S2) in hTRIM5α, and VK10EE (S1) and LK265KP (S2) in rhTRIM5α. The first potential SUMO-conjugation site, referred to as S1, is located at the N-terminal extremity of the protein, before the RING domain, whereas the second putative site, S2, is located between the coiled-coil and the B30.2 domain  ,8. TRIM5in vitro SUMOylation assay. As shown in Figure To determine whether either of these lysines are acceptor sites for SUMO1 conjugation, two single K to R point mutants were generated in human and rhesus macaque TRIM5α at K10 (S1m) and K263 or K265 (S2m), as well as double mutants (S1S2m). These constructs were tested in the in vitro, rhTRIM5α S1m showed the same anti-HIV-1 activity than the wild-type protein, thus demonstrating that TRIM5α SUMOylation is not required for retroviral restriction, at least in HeLa cells (Figure Finally, we looked at the phenotype of S1 mutants by testing their capacity to restrict retroviral infections. Despite dramatic SUMOylation defects s Figure C.in cellulo nor in vitro. This report is the first to demonstrate that TRIM5α is a SUMO substrate and to experimentally confirm that K10 is the main SUMOylation site. Previous groups have attempted to detect TRIM5α SUMOylation but without success [in vitro nickel pulldown assay required optimization of SUMO and Ubc9 expression and of binding conditions, which together may point to the fact that SUMOylation is transitory and affects only a small proportion of a given protein. Since rhTRIM5α K10R mutant is still able to restrict HIV-1, our results support the fact that the capacity of TRIM5α to interact with SUMOylated proteins, via its SIMs, rather than its direct SUMOylation, is required for its antiretroviral activity [Although TRIM5α was suspected to be SUMOylated and that K10 was identified as a SUMOylation consensus site -11, TRIM success -11. Indeactivity . We are


Competitive beverages are drinks sold outside of the federally reimbursable school meals program and include beverages sold in vending machines, a la carte lines, school stores, and snack bars. Competitive beverages include sugar-sweetened beverages, which are associated with overweight and obesity. We described competitive beverage availability 9 years after the introduction in 2004 of district-wide nutrition standards for competitive beverages sold in Boston Public Schools.In 2013, we documented types of competitive beverages sold in 115 schools. We collected nutrient data to determine compliance with the standards. We evaluated the extent to which schools met the competitive-beverage standards and calculated the percentage of students who had access to beverages that met or did not meet the standards.Of 115 schools, 89.6% met the competitive beverage nutrition standards; 88.5% of elementary schools and 61.5% of middle schools did not sell competitive beverages. Nutrition standards were met in 79.2% of high schools; 37.5% did not sell any competitive beverages, and 41.7% sold only beverages meeting the standards. Overall, 85.5% of students attended schools meeting the standards. Only 4.0% of students had access to sugar-sweetened beverages.A comprehensive, district-wide competitive beverage policy with implementation support can translate into a sustained healthful environment in public schools. Competitive beverages are drinks sold outside of the federally reimbursable school meals program and include beverages sold in vending machines, a la carte lines, school stores, and snack bars. More than 90% of middle and high school students and 55% of elementary students in the United States have access to competitive beverages ,2. CompeHealth and government organizations have identified competitive beverage standards and policies as important strategies for improving children’s health –12. As pResearch on the effectiveness of district-level competitive beverage policy on availability has produced mixed findings. In 2 studies, SSBs were less likely to be available when prohibited by district policy ,14, but In 2004, the Boston Public School (BPS) Committee approved a mandatory district-level policy for competitive beverage nutrition standards, including a ban on all SSBs. The standards have been in place for nearly a decade, but the extent to which the policy has been implemented and sustained is unknown. The objective of this study was to examine the level of compliance with the competitive beverage policy 9 years after its implementation. Our evaluation may help inform implementation of the HHFK Smart Snacks in Schools regulation as schools nationwide work toward achieving the nutrition standards. We hypothesized that most schools would meet the competitive beverage nutrition standards and that most BPS students would attend schools that meet the standards 9 years after implementation of the district-wide policy.We used a cross-sectional post-test study design to conduct an onsite audit of the availability of competitive beverages in all 122 BPS buildings from March through June 2013.The BPS district was the first in Massachusetts to develop district-level nutrition policy and standards for competitive foods and beverages . The comIn 2010 and 2011, BPS health and wellness staff consulted with more than 400 individuals, including principals, teachers, school staff, parents and guardians, students, out-of-school coordinators, community partners, and national experts to inform the development of an implementation toolkit. The toolkit was a guide to encourage school staff, students, and families to create and support a cultural shift in the school food and beverage environment . It inclIn 2013, when we conducted our evaluation, the competitive beverage nutrition standards did not allow the sale of SSBs, beverages containing artificial sweeteners, or beverages containing caffeine . In elementary schools, bottled water was the only competitive beverage allowed; 100% juices and milks were not permitted. Total sugars in flavored milk, allowed only in middle schools and high schools, were limited to 22 grams or fewer per 8-ounce serving, and only 8-ounce servings or less were allowed. Serving sizes of 100% fruit or vegetable juices were limited to 4 ounces in middle schools and 8 ounces in high schools .Trained research assistants used digital photographs and a standardized protocol to document beverages for sale at each access point in 122 BPS buildings. The standardized protocol was a paper-and-pencil survey developed by Harvard researchers. Data collectors documented the type and location of each access point, the type and size of each beverage, and the total number of slots for each beverage at each access point. Of the 122 BPS buildings surveyed, 7 school buildings were excluded because beverage standards did not apply to the populations served: 4 early child care and education centers, 1 adult technical academy, 1 counseling and intervention center, and 1 administrative building.We scheduled visits to schools one week ahead of time. We defined an access point as any vending machine, a la carte cafeteria sale, school store, or cafe where competitive beverages were sold. We recorded the type, brand, flavor, portion size, percentage milk fat, and percentage juice for each unique beverage at each access point.We collected nutrient and ingredient information for each beverage from manufacturer websites, including information on calories , total fat (g), sugar (g), and artificial sweetener or caffeine (yes/no). Beverages were classified into mutually exclusive categories: water, 100% juice (fruit or vegetable), unflavored milk, flavored milk, SSBs, diet drinks , or other . We did not include the availability of competitive beverages in teacher’s lounges because researchers were unable to consistently access the lounges during school hours and because they were generally not accessible to students.We used publicly available student enrollment data for each school for the 2012–2013 academic year to analyze demographics and to calculate the percentage of students with access to competitive beverages by grade level .We assessed the number and type of unique competitive beverages available at each access point and determined which met competitive beverage nutrition standards. We classified a school as having met the standards if every sold beverage met the standards or if none were sold. We classified schools that did not meet the nutrition standards into 2 categories: those that sold SSBs and those that did not sell SSBs. We made this distinction because the negative health effects of SSBs are more significant than the negative health effects of other beverages that do not meet standards . For buildings that accommodated mixed grade levels , we applied the standards applicable to the lowest grade level. To determine the proportion of schools meeting the competitive beverage standards, we summed the number of schools that had no competitive beverages and the number of schools that fully met the standards and divided this sum by the total number of schools, stratified by grade level. We used logistic regression to determine whether beverages were more likely or less likely to meet the nutrition standards according to access point type.We determined the percentage of students without access to any competitive beverages, the percentage of students with access only to competitive beverages meeting the standards, and the percentage of students with access to noncompliant beverages (further classifying noncompliant beverages as SSBs or non-SSBs). We then summed the number of students attending each school classified into each of these 4 categories and divided this number by the total number of students enrolled in BPS, stratified by grade level. We used SAS version 9.3 (SAS Institute Inc) to conduct all analyses. This study was determined to be exempt by the Harvard T.H. Chan School of Public Health Committee on Human Subjects.Of the 115 school buildings included in analysis, 78 were elementary schools, 13 were middle schools, and 24 were high schools. Of the total population of 56,259 students, 40.8% were Hispanic and 36.7% were non-Hispanic black; 72.2% were eligible to receive free or reduced-price meals .Of the 115 schools, 103 (89.6%) of schools met the competitive beverage nutrition standards; 86 (74.8%) sold no competitive beverages, and 17 (14.8%) sold only competitive beverage that met the standards. Elementary schools most frequently met the standards (93.6%), followed by middle schools (84.6%) and high schools (79.2%). Among elementary and middle schools, most met the standards because no competitive beverages were sold (88.5% of elementary schools and 61.5% of middle schools). Competitive beverages were sold at 47 access points in 29 schools. Most access points were vending machines (28 of 47 [59.6%]) or a la carte cafeteria sales (17 of 47 [36.2%]) . We founOf the 12 schools in which competitive beverages were sold that did not meet the standards, 9 schools sold 100% juices, low-fat unflavored milk, or non-calorically sweetened water; 4 of these schools were elementary schools that sold 100% juice or low-fat milk (instead of nonfat milk). SSBs were sold in vending machines in 1 elementary school and 1 high school and as a la carte items in another high school . Low-fat flavored milks were sold at 1 middle school and artificially sweetened water was sold at another middle school. Diet beverages were sold at 1 high school and tea, coffee, or fruit smoothies at another high school.By percentage of students, most (88.4%) elementary school students and almost half of middle school students (44.9%) did not have any access to competitive beverages. Although 81.2% of high school students had access, most (56.6%) had access only to beverages that met the standards. Overall, 61.4% of students did not have access to any competitive beverages, and 24.1% had access only to competitive beverages that met standards, comprising 85.5% of the students district-wide . Only 4.Our study is a unique evaluation of a district-level competitive beverage policy 9 years after implementation. We provided evidence for sustainability and effectiveness in ensuring access to healthy competitive beverages. The overwhelming majority of Boston Public Schools (89.6%) were compliant with the competitive beverage nutrition standards. Most schools (74.8%) met the standards because no competitive beverages were sold, and 14.8% sold only competitive beverages that met the standards. District-wide, 85.5% of BPS students attended schools meeting the competitive beverage nutrition standards. Our study demonstrates that a comprehensive district-wide policy, in coordination with ongoing professional education, community-identified tools, and technical assistance training, can translate into a sustained healthier environment.Nationwide, approximately 40% of district wellness policies ban soda as competitive beverages in schools, and fewer than 20% of elementary and 10% of middle schools or high schools ban other types of SSBs . Other nThe BPS district succeeded in nearly eliminating the sale of SSBs, the beverages most strongly associated with negative health effects –9. Only Sustained adherence to the competitive beverage standards in the BPS district may have contributed to decreased consumption of SSBs among students. In 2006, two years after implementation of the ban on SSBs, the daily consumption of SSBs declined significantly among high school students (−0.30 servings per day), while no such decline was found nationally during that time . Nine yeThis study has several strengths. First, we conducted an onsite audit with objective measures to document competitive beverages sold rather than relying on school staff reports, which are subject to recall and social desirability biases. We surveyed every school in the BPS district, providing a complete assessment of competitive beverage availability. We also provided detailed information on district-level competitive beverage policy implementation, a unique contribution to the scientific literature because details on implementation strategies are scarce. Finally, we conducted the assessment 9 years after the policy was implemented.This study also has several limitations. One, we did not have baseline data on competitive beverage availability before policy was implemented. Thus, we do not know the extent to which competitive beverages were available before implementation, and we were unable to measure the magnitude of any changes in availability after implementation. However, the BPS school committee identified the need for a policy restricting the availability of competitive beverages, which suggests that competitive beverages were overly available and that standards were warranted. Two, we lacked a control group to exclude the possibility that adherence to the competitive beverage policy was part of a larger trend. However, national estimates indicate that SSBs are still widely offered as competitive beverages, and the availability of SSBs in the BPS district is well below national averages . Three, Nine years after implementation of a district-wide competitive beverage policy, 89.6% of schools met the standards, 85.5% of students attended schools in which the environment supported these standards, and only 4.0% of students had access to any SSBs. These findings and policy implementation strategies may be particularly encouraging to school districts nationally as they work to comply with HHFK Smart Snacks in Schools requirements . Our stu


The acid corrosion inhibition process of mild steel in 1 M HCl by 4-[methoxy]coumarin (ATC), has been investigated using weight loss technique and scanning electron microscopy (SEM). ATC was synthesized, and its chemical structure was elucidated and confirmed using spectroscopic techniques (infrared and nuclear magnetic resonance spectroscopy).ads) were calculated. Quantum chemical parameters such as highest occupied molecular orbital energy, lowest unoccupied molecular orbital energy  and dipole moment (μ) were calculated and discussed. The results showed that the corrosion inhibition efficiency increased with an increase in both the EHOMO and μ values but with a decrease in the ELUMO value.The results indicated that inhibition efficiencies were enhanced with an increase in concentration of inhibitor and decreased with a rise in temperature. The adsorption equilibrium constant (K) and standard free energy of adsorption  atom, N (nitrogen) atom and O (oxygen) atom. It is very important to use corrosion inhibitors to prevent metal dissolution and minimize acid consumption –4. The mThe chemicals utilized were supplied by Sigma-Aldrich and the purity checked by TLC (thin layer chromatography). Infrared spectra were obtained on a Thermo Scientific, NICOLET 6700 FTIR spectrometer. Nuclear magnetic resonance spectra were obtained on a JEOL JNM-ECP 400. Elemental microanalysis, was carried out using a model 5500-Carlo Erba C.H.N elemental analyzer.−1 , 291.2 ; 3079.1 (C–H aromatic),1752.3 cm−1 , 1591.1 cm−1, 1635.3 cm−1 (C=C aromatic); Anal. Calcd. for C12H9N3O3S: C 52.36 %, H 3.30 %, N 15.26 %. Experimentally: C 51.64 % H 2.92 % and N 14.94 %s.This compound was synthesized in good yield according to the previously described procedures , 29. Phocleaning procedures described in ASTM G1-03 test method , 3231, 3HOMO , E LUMO  and μ (dipole moment) were calculated and discussed.The molecular optimization was carried out using the density function theory (DFT)/B3LYP with basis set 6-31G. Quantum chemical calculations such as E Corrosion rate inhibition efficiencies were calculated for various concentrations of ATC for the duration 1, 2, 3, 4, 5, 10 and 24 h, at 303 K are shown in Figs. A differentiation of the inhibition efficiencies of ATC on mild steel in acidic medium with and without of different concentrations of ATC at various temperatures  indicates that corrosion efficiency rise with increasing of concentration and reduced with temperature rise . This technique has been found to be successful in providing insights into the chemical reactivity and selectivity in terms of global parameters such as electro-negativity (v), hardness (g) and softness (S), and local softness (sđ~r Þ) , 45. TheThe Mulliken charge distribution of (ATC) is presented in Table Our research demonstrate that the synthesized macromolecule represents an excellent inhibitor for materials in acidic solutions. The efficiency of this macromolecule had maximum inhibition efficiency up to 96 % at 0.5 mM and diminish with a higher temperature degree, which is revealing of chemical adsorption. Inhibitor molecules were absorbed by metal surface and follow Langmuir isotherms low and establishes an efficient macromolecule inhibitor hading excellent inhibitive properties due to entity of S (sulfur) atom, N (nitrogen) atom and O (oxygen) atom. SEM (Scanning electron microscope) measurements were confirming the figuration of a protective metal surface. Inhibition study of synthesized macromolecules obviously expose their function in the protection of MS in 1 M HCl.


Ursus maritimus). Population structure and space use of polar bears have been challenging to quantify because of their circumpolar distribution and tendency to range over large areas. Knowledge of movement patterns, home range, and habitat is needed for conservation and management. This is the first study to examine the spatial ecology of polar bears in the Foxe Basin management unit of Nunavut, Canada. Foxe Basin is in the mid-Arctic, part of the seasonal sea ice ecoregion and it is being negatively affected by climate change. Our objectives were to examine intrapopulation spatial structure, to determine movement patterns, and to consider how polar bear movements may respond to changing sea ice habitat conditions. Hierarchical and fuzzy cluster analyses were used to assess intrapopulation spatial structure of geographic position system satellite-collared female polar bears. Seasonal and annual movement metrics  and home-range fidelity (static and dynamic overlap) were compared to examine the influence of regional sea ice on movements. The polar bears were distributed in three spatial clusters, and there were differences in the movement metrics between clusters that may reflect sea ice habitat conditions. Within the clusters, bears moved independently of each other. Annual and seasonal home-range fidelity was observed, and the bears used two movement patterns: on-ice range residency and annual migration. We predict that home-range fidelity may decline as the spatial and temporal predictability of sea ice changes. These new findings also provide baseline information for managing and monitoring this polar bear population.Climate change has been identified as a major driver of habitat change, particularly for sea ice-dependent species such as the polar bear ( William Henry Burt was an early proponent of an integrated approach to wildlife management conservation, when he identified the need to understand a species’ behavior and spatial patterns Burt . Today, Ursus maritimus) are one such vulnerable species, due to their high trophic level, specialized diet, and reliance on the distribution of sea ice habitat for foraging  based on-ice composition, circulation, duration, and how bears respond to sea ice dynamics , with Hudson Strait  and Foxe Basin  being similar in size. All three areas are shallow (predominantly <200 m deep), productive seas that undergo an annual sea ice cycle from ice free to almost total ice cover  (the main prey species), bearded seals (Erignathus barbatus), harbour seals (Phoca vitulina), harp seals (Pagophilus groenlandicus), walrus (Odobenus rosmarus), bowhead , narwhal (Monodon monoceros), and beluga (Delphinapterus leucas)  by remote injection using a dart delivered from a helicopter. All bears were caught on land, during the ice-free season, following standard capture and handling methods  linked to Argos satellites  were deployed on 45 adult female polar bears with cubs-of-the-year, yearlings, and 2-year olds, as well as, two females without offspring in August–October, 2007–2009.Location data were collected at 3- or 4-h intervals. We used GPS quality location data (accuracy <10 m) with the exception of two bears whose collars provided only Doppler shift quality locations for part of the year, of which we used classes 1, 2, and 3 (accurate to <1 km). Daily locations at 13:00 GMT or nearest recorded values were used for the cluster analysis and overlap analyses, and all locations were used to calculate movement rates and home-range size.We defined five seasons based on sea ice dynamics, ice concentration, and ringed seal life history, similar to those used by Ferguson et al.  and ParkIf a bear entered a maternity or temporary den, the locations were excluded in the movement rate calculations but were included in the home range and cluster analyses. Denning was identified when a bear stopped moving for several weeks or months, and renewed movement was not attributed to sea ice movement. All suspected dens were on land.t-test (Rabe-Hesketh and Everett m = 1.5) and the diagonal metric given the dimensions of the study area. FUZME has been applied to determine caribou (Rangifer tarandus) herd membership  convex polygon (MCP) home ranges. Annual and seasonal MCPs were calculated using Hawth’s Tools Beyer  in ArcMaMovement rates and Euclidean distances between locations were calculated using Hawth’s Tools in ArcMap v9.3.1 . All available locations were used to calculate monthly movement rates and for each month a bear had to have ≥15 days of location data to be included. The total on-ice time (days) was calculated for each bear as the date on ice at freeze-up to the date on land the following year.α = 0.05 and were performed using SPSS. Means are presented with ± 1 standard error (SE).To compare differences between the movement metrics, we used a one-way analyses of variance (ANOVA) and Tukey’s HSD post hoc test for pair-wise comparisons. ANOVA was used to test for significant trends in monthly and seasonal movement metrics. All statistical tests and comparisons used Home range fidelity was measured by calculating individual interannual seasonal and annual home-range overlap. Nine females from Foxe Basin and Hudson Strait had sufficient data over two consecutive years to be included in the overlap calculations. We used both static and dynamic overlap Powell , which wt-squared test , showing that each bear was spatially independent , Hudson Strait (HS) and Hudson Bay (HB) Fig.A,B. The test Fig.. The fuztest Fig. split thdent see .2 and varied from 19,633 km2 to 401,351 km2. The mean annual home range sizes differed between clusters within the FB population . There were also differences in the seasonal home range sizes. The freeze-up home ranges differed among clusters , with those in FB and HS smaller than in HB . Winter home ranges also differed among clusters , with FB being smaller than HS and HB .The mean annual MCP home range area was 115,918 ± 15,382 kmF2,26 = 3.57, P = 0.04) when HB (0.8 km/h) bears moved slower than HS (1.3 km/h) and FB (1.2 km/h) bears. Mean monthly on-ice movement rates from December to July declined in HB , increased in HS , and there was no trend in FB  . FB bears were on the ice 31 days longer  than bears in HS and 56 days longer  than bears in HB . Polar bears moved onto the sea ice in FB the earliest , and HS the latest . The mean date that bears left the sea ice for land also differed among regions  and FB bears left the ice for land latest  and HB bears earliest .The mean number of days on the sea ice differed among the regions (n = 9). The mean annual static overlap was 72 ± 6% (range 24–92%). The mean seasonal static home range overlap ranged from 36 ± 10% during the ice-free season to 60 ± 7% during freeze-up  overlap was positive with mean Jacobs’ index of 0.3 ± 0.06 (range 0.2–0.6) Fig.. A positWithin the Foxe Basin population, female polar bear locations on the seasonal sea ice were distributed in three clusters and each cluster generally corresponded with the main marine regions. There were differences in the movement metrics between clusters. At the individual level, the bears showed annual and seasonal fidelity to their home ranges and moved independently of each other on the sea ice.Intrapopulation spatial structure has been observed in other polar bear populations. Cluster analyses in the Barents Sea and Kara Sea populations found that the spatial clusters were related to sea ice habitat use: One cluster used seasonal fast ice in the near shore, and the other used multiyear drift ice off shore , the clusters were likely unrelated to differential use of sea ice habitats. Further, there were no apparent barriers  to polar bear movement, and both prey and denning habitats were widely available over the region. Polar bears were capable of moving across the entire study area but they did not, instead they exhibited regional clusters that adhered to marine regions of Foxe Basin, Hudson Strait, and Hudson Bay. The clusters arose from individual home-range fidelity and may be a product of learned behavior that develops in a predictable and resource-rich environment  have been related to foraging strategies and ultimately with the structure and quality of sea ice habitat. In the Canadian Arctic and Barents Sea, female polar bears using pelagic or active pack ice habitat had larger annual ranges than the near-shore or landfast ice bears  that results in emergent population level patterns and strategies  (Mueller and Fagan Within the context of climate change, understanding polar bear movement patterns are important as a means to anticipate their potential behavioral plasticity for responding to changes in sea ice habitat phenology, distribution, and loss. Research (Ferguson et al. Polar bear populations can be affected by the dual stresses of habitat change and hunting Lentfer . Both fa


Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism.The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species  Caenorhabditis elegans, and Drosophila123The insulin signaling pathway, one of the most widely distributed pathways among invertebrate species, is conserved among eukaryotes, including mammals, The insulin signal is transmitted via the insulin receptor (IR). Vertebrates possess more than one homologous IR family member, including the IR, the type-I insulin-like growth factor receptor (IGF-IR), and the insulin receptor-related receptor (IRR)8911Drosophila melanogaster and C. elegans1617Numerous insulin signaling pathways in invertebrates and vertebrates have been found to be relatively well conserved, including the ligand, its receptors, and the final effectors1415Lymnaea stagnalis (seven ILPs)1920Aplysia californica (one ILP)Anodonta cygnea (six ILPs)Lottia gigantea (four ILPs)Crassostrea gigas (one ILP)626A. cygnea for recombinant piscine IGF-I and porcine insulin has been studied. The results indicate that this receptor shares similar binding properties with vertebrates IRRsMytilus trossolusA. californicaC. gigas, CgRas, CgPten, and CgP70S6K were found based on their sequence conservationIn mollusks, insulin-like peptides (ILPs) have been found in Rhipicephalus (Boophilus) microplusPenaeus vannameiPanulirus argusInsulin can stimulate the PI3K and MAPK pathways to increase glucose transport and glycogen and lipid synthesis, diminish gluconeogenesis, inhibit glycogenolysis and lipolysis, and regulate the expression of specific genesPinctada fucata, a marine bivalve mollusk that is mainly cultivated in China and Japan, has very high economic value in terms of pearl production, and it is also an important research topic regarding biomineralization, growth, genetic variation, and resistance to disease. In our previous study, we characterized the insulin-related peptide receptor of P. fucata (pfirr) gene, which is involved in regulating developmental processes in P. fucataP. fucata. Moreover, the MAPK and PI3K signaling pathways have not been studied in P. fucata. Additionally, it is unknown whether pfIRR can regulate these signaling pathways, and whether these two pathways interact with each other. Furthermore, it is not known whether the insulin/IGF-I responsive machinery in P. fucata is correlated with carbohydrate/glycogen metabolism via these signaling pathways.The pearl oyster, P. fucata, we first tested the interaction between human recombinant IGF-I (hrIGF-I) and pfIRR, and the effect of hrIGF-I-mediated pfIRR stimulation in activating the MAPK and PI3K signaling cascades in P. fucata oocytes. Second, we examined the effect of the pfIRR inhibitor PQ401 on hrIGF-I-mediated Akt/protein kinase B (PKB) and MAPK phosphorylation. Third, we measured the effects of the MEK inhibitor PD98059 and the PI3K inhibitor wortmannin on the activation of the hrIGF-I-induced PI3K and MAPK signaling pathways downstream of pfIRR in P. fucata oocytes. Fourth, we analyzed the effects of hrIGF-I on glycogen content and glucose levels, the phosphorylation of Akt/PKB at amino acid residues Thr308 (T308) and Ser473 (S473), as well as p44/42 MAPK, and the expression of genes following the intramuscular (i.m.) injection of hrIGF-I.To investigate the insulin signaling pathway in Escherichia coli and purified  with 1:1,250, 2,500, 5,000, 10,000, 20,000, 40,000, and 80,000 dilutions of the antisera . The conin vitro co-immunoprecipitation (Co-IP) analysis using P. fucata oocytes was employed. The results showed that pfIRR was co-immunoprecipitated by hrIGF-I (−11–10−6 M) when using anti-hrIGF-I antibodies for the Co-IP  for the Co-IP  for various periods of time and with different concentrations of hrIGF-I for same time. The phosphorylation of p44/42 MAPK was examined by determining the ratio of phospho- to total p44/42 MAPK. As shown in −11 to 10−7 M , and a stronger reduction was achieved by treatment with 50 μM PD98059. Pretreatment with PD98059 at 50, 60, and 80 μM concentrations for 1 h completely inhibited the basal and hrIGF-I-stimulated phosphorylation of T308 and S473 of Akt/PKB .To detect the duration of the PD98059-meidated inhibition of PI3K activity, we analyzed the phosphorylation of T308 and S473 after treatment with 60 μM PD98059 for 0.5, 1.5, and 2 h. Basal MAPK phosphorylation was completely prevented by treatment with PD98059 for 0.5 and 1.5 h, but not for 2 h. hrIGF-I-stimulated MAPK phosphorylation exhibited the greatest decrease after 1.5 h of PD98059 treatment. Pretreatment with 60 μM PD98059 for 0.5 to 2 h completely inhibited the basal and hrIGF-I-stimulated phosphorylation of T308 and S473 of Akt/PKB .To determine whether PI3K is involved in hrIGF-I-mediated ERK pathway activation, we assessed the effect of the PI3K inhibitor wortmannin on hrIGF-I-induced MAPK phosphorylation. Basal and hrIGF-I-stimulated phosphorylation of T308 and S473 were completely prevented by treatment with 20, 40, and 60 μM wortmannin for 1 h. Pretreatment with wortmannin lowered basal and hrIGF-I-stimulated MAPK phosphorylation, and a slight dose-dependent inhibition was observed .To detect the duration of the wortmannin-mediated inhibition of ERK activity, we analyzed MAPK phosphorylation after treatment with 40 μM wortmannin for 0.5, 1, and 2 h. Basal and hrIGF-I-stimulated phosphorylation of T308 and S473 were completely prevented by treatment with wortmannin for 1.5 and 2 h. Pretreatment with 40 μM wortmannin for 1.5 h, but not for 0.5 h, resulted in the greatest decreases in basal and hrIGF-I-stimulated MAPK phosphorylation. HrIGF-I-stimulated, but not basal, MAPK activity was lowered by wortmannin treatment for 2 h .in vivo  control, the glycogen content in the digestive glands (DGs) and adductor muscles (AMs) significantly increased . The gluin vivo .We analyzed four downstream genes of the hrIGF-I signaling pathway: the pfirr, glucokinase (GK), glycogen synthase kinase-3β (GSK-3β), and PP1 genes. The relative expression levels of the pfIRR, GK, and PP1 genes significantly increased in DGs after hrIGF-I injection, whereas there were no differences in expression levels for hrIGF-I-treated or non-treated AMs . HoweverThe protein expression of pfIRR, GK, PP1, phospho-GS, GS, phospho-GSK-3β, and GSK-3β was measured by Western blotting. The expression of pfIRR and PP1 increased in DGs after hrIGF-I injection, whereas no difference was observed for hrIGF-I-treated or non-treated AMs , which wE. coli. High avidity antisera were obtained, which were functional in ELISA and western blotting assays. The crude antisera that were purified by a protein A affinity column showed that the antibody is highly specific for the TK domain of pfIRR.In this study, we report the generation and evaluation of a polyclonal antibody against pfIRR by expressing a fragment encoding the TK domain of pfIRR in −11–10−6 M) when using the anti-hrIGF-I antibody for Co-IP . The effect of hrIGF-I on Akt activation was also sustained for at least 6 h. The maximal activation of T308 phosphorylation was obtained at 10−9 M hrIGF-I, while the maximal activation of S473 phosphorylation occurred at 10−8 M hrIGF-I. Although the maximal activation level of phospho-T308-Akt was achieved at 180 min, the signal was very strong compared with that in the other lanes; thus, it may be an outlier.Our experiments indicated that the MAPK and PI3K pathways are activated following hrIGF-I stimulation in Full Akt activation requires the dual phosphorylation of S473 by mammalian target of rapamycin companion 2 (mTORC2)–rapamycin-insensitive companion of mTOR complex (RICTOR), integrin-linked kinase 1 (ILK)-1, or DNA-dependent protein kinase (DNAPK) as well as the phosphorylation of T308 by PDK142P. fucata embryosIn previous studies, a diaryl urea compound, PQ401, was found to antagonize IGF-IR autophosphorylation in cultured human MCF-7 cells, with a half-maximal inhibitory concentration of 12 μM, as well as the growth of cultured breast cancer cells in serum at 10 μMP. fucata, the results of hrIGF-I signaling show that PI3K activity is decreased following MAPK inhibition, as also occurs during HGF signaling.We used the MEK inhibitor PD98059 and the PI3K inhibitor wortmannin to examine the role of MAPK and PI3K in regulating the hrIGF-I signaling pathway. Treatments with PD98059 inhibited basal and hrIGF-I-mediated MAPK and Akt activities, indicating that hrIGF-I activates the MAPK signaling pathway, and that MEK is involved in hrIGF-I-mediated PI3K pathway activation. In mammals, active ERK can influence the PI3K/Akt pathway via phosphorylation of GAB1 and IRS on several serine residues that are adjacent to p85 PI3K-binding sites (three YXXM motifs)4649−9 M).PD98059 inhibited MAPK activation at 50–80 μM concentrations. T The reduced but not inhibited MAPK activation by hrIGF-I stimulated demonstrated that MAPK activation is not regulated by only one signaling pathway, and it indicates that Ras/MAPK → PI3K/Akt cross-talk that can amplify MAPK signaling occurs upstream of MEK. The maximal inhibition time for PD98059 in our study was 2 h. The positive influence of the Ras/MAPK pathway on the PI3K/Akt pathway was not affected by the inhibitor concentration or the stimulation time, which may be because the signal strength (hrIGF-I dose) and the stimulation time (0.5 h) in these inhibition experiments were the same. The basal activities of MAPK and Akt were inhibited by different doses of PD98059 and different treatment times, whereas after treatment, hrIGF-I stimulated the phosphorylation of MAPK, but not Akt, at either T308 or S473. This may be because MAPK is more sensitive than Akt to the dose of hrIGF-I used , whereas PI3K inhibition dramatically decreases ERK phosphorylation at low physiological EGF dosesP. fucata in regard to carbohydrate/glycogen metabolism induction by hrIGF-I demonstrated that the mechanism of the hrIGF-I responsive machinery is conserved in P. fucata. The glycogen content in various tissues  was examined  were obtained from the Marine Biology Research Station at Daya Bay of the Chinese Academy of Sciences . The oysters were cultivated in floating net cages in the sea under natural conditions. All animal experiments were conducted in accordance with the guidelines and approval of the respective Animal Research and Ethics Committees of the Chinese Academy of Sciences.Oocytes were obtained directly from the gonad with a Pasteur pipet, filtered through 120-μm nylon mesh to remove debris, and washed three times with filtered (0.45-μm filter) natural seawater at room temperature (26 °C).Reagents were obtained from the following sources: hrIGF-I from Shanghai Prime Gene Bio-Tech. Co., Ltd. ; dimethyl sulfoxide from Sigma–Aldrich ; hrIGF-I monoclonal antibody from Abcam ; rabbit IgG from Beyotime , phospho-T308 and phospho-S473Akt/PKB, Akt/PKB, phospho-p44/p42 MAPK/Erk 1/2, p44/p42 MAPK/Erk 1/2 antibodies, wortmannin, and PD98059 from Cell Signaling Technology ; GK antibody, GS antibody and PP1  antibody from Sangon Biotech ; phospho-GS (Ser641) antibody from Sino Biological ; alpha-tubulin polyclonal antibody from Proteintech ; horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody from Abbkine .P. fucata IRR was made by Shanghai Biomodel Organism Science & Technology Development Co., Ltd . An 837-bp cDNA fragment encoding the TK domain of IRR (GenBank accession number: JX121113) was amplified and cloned into the BamHI/XhoI sites of the pET28a expression vector , and histidine-tagged TK was expressed in E. coli and purified with a Ni2+-NTA column.A polyclonal antibody against Polyclonal antibody was raised against the purified TK domain in two rabbits. After immunization, the antisera were collected and purified by a protein A affinity column. The titers of the antisera were determined by ELISA. Western blot analyses were conducted to characterize the polyclonal antibody.−6, 10−7, 10−8, 10−9, 10−10, and 10−11 M hrIGF-I. The oocytes were lysed and immunoprecipitated using the Pierce Classic IP Kit . The lysates were co-immunoprecipitated using anti-hrIGF-I (10 μg) antibodies, subjected to SDS-PAGE (10%), and detected by western blotting with an anti-pfIRR antibody (10 μg). Rabbit IgG served as a negative control. The Co-IP lysates obtained using anti-pfIRR antibodies (15 μg) were detected by western blotting with anti-hrIGF-I (10 μg). IgG served as a negative control.Oocytes were treated with different concentrations of hrIGF-I for 4 h at 4 °C. hrIGF-I was dissolved in PBS (0.2 M) at a concentration of 0.02 mM, and then the oocytes were separately treated with 10Cell lysates were separated by SDS-PAGE. After transfer to polyvinylidene fluoride membranes , the membranes were blocked in 5% bovine serum albumen (BSA) (Thermo Fisher Scientific) in Tris-buffered saline-Tween 20 (TBST). The blots were incubated with 1:1,000 to 1:5,000 dilutions of the indicated antibodies in blocking buffer overnight at 4 °C. Then, blots were washed with TBST and incubated with a 1:5,000 dilution of an HRP-conjugated anti-rabbit secondary antibody in blocking buffer for 1 h at room temperature, followed by further washing. The SuperSignal® West Pico chemiluminescent substrate was used to detect HRP according to the manufacturer’s instructions (Pierce). Positive signals from the target proteins were visualized using a TANON5200 image analyzer .−7 M) for various periods of time , and then analyzed by western blotting to detect the phosphorylation of Akt/PKB at T308 and S473, as well as the amounts of Akt/PKB, phosphorylated p44/42 MAPK, p44/42 MAPK, and α-tubulin. Oocytes were incubated with hrIGF-I at various concentrations  for 30 min. Lysates was subjected to western blotting with appropriate antibodies. The densitometry ratio of phospho-p44/42/total p44/42, and phospho-Akt/total Akt were measured by Tanon Gel Imaging System software.Oocytes were incubated with hrIGF-I (10−9 M) for 30 min. Lysates were subjected to western blotting to detect the phosphorylation of Akt/PKB at T308 and S473, as well as the amounts of Akt/PKB, phosphorylated p44/42 MAPK, and p44/42 MAPK.Oocytes were incubated without or with increasing concentrations of PQ401 for 1 h, or with 40 μM PQ401 for 0.5, 1.5, and 2 h, and then treated with or without hrIGF-I (10−9 M) for 30 min. Lysates were subjected to western blotting to detect the phosphorylation of Akt/PKB at T308 and S473, as well as the amounts of Akt/PKB, phosphorylated p44/42 MAPK, and p44/42 MAPK.Oocytes were incubated without or with increasing concentrations of PD98059 or wortmannin for 1 h, or with 60 μM PD98059 or 40 μM wortmannin for 0.5, 1.5, and 2 h, and then treated with or without hrIGF-I (10Oysters were injected i.m. with hrIGF-I (0.06 μg/g wet body weight) or an equal volume of PBS as a control for 3 h. The oysters were removed for hemolymph sampling and then sacrificed. Hemolymph samples were processed immediately. Glucose levels were determined according to the instructions of the manufacturer of the Glucose Assay Kit . The DGs and AMs were quickly dissected and divided into three parts. One part was processed immediately for glycogen content according to the instructions of the manufacturer of the Liver/Muscle Glycogen Assay Kit (Nanjing Jiancheng Bioengineering Institute), while another was frozen in liquid nitrogen and stored at −80 °C for subsequent RNA extraction. Real-time polymerase chain reaction (PCR) was performed as described previouslyHow to cite this article: Shi, Y. and He, M.-x. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata. Sci. Rep.6, 22063; doi: 10.1038/srep22063 (2016).


Monitoring drift ice in the Arctic and Antarctic regions directly and by remote sensing is important for the study of climate, but a unified modeling framework is lacking. Hence, interpretation of the data, as well as the decision of what to measure, represent a challenge for different fields of science. To address this point, we analyzed, using statistical physics tools, satellite images of sea ice from four different locations in both the northern and southern hemispheres, and measured the size and the elongation of ice floes (floating pieces of ice). We find that (i) floe size follows a distribution that can be characterized with good approximation by a single length scale  Sea ice is an important constituent of our planet’s surface, covering almost The measurement of morphological properties of ice floes could inform us on key properties regarding the rheology of sea ice and help the reconstruction of the number of floes of a given scale starting from incomplete measurements or measurements at different scales . The rich spectrum of behaviors of sea ice and the available data have already attracted statistical mechanics investigators34781011drift ice, focusing on the size and shape of the individual floes that drift ice is composed of. Remote sensing makes the access to these data simple, by the exploitation of popular services such as Google Maps. Since floe diameters vary across several orders of magnitude, depending on season, location, and weather, we ask whether common patterns emerge, and how they may be related to the physics of ice fragmentation.Here, we take an empirical approach to the question of defining useful observables regarding sea-ice morphological properties, and analyze data obtained from satellite images of sea ice detached from the shoreline, called finite-size scaling form, which only depends on a single length scale. Scaling is a significant concept, commonly found in critical phenomena and self-organized criticality, but also in diverse fields including ecology17Regarding ice-floe size, while some of the previous studies found evidence for scale-free distributions5These results lead to the conclusion that floe size alone cannot be a fully informative measurement of sea-ice properties. For this reason, we widen the analysis of floe contours by considering also their shapes. Surprisingly, while particle size is widely considered in the literature—e.g., for classifying new fragmentation physicscharacteristic length and a shape disorder parameter—that we advocate as useful for comparing ice-floe images taken in different regions and conditions.In brief, our results can be summarized as follows. (Ia) Ice-floe sizes follow a simple scaling form, hence the size distribution is characterized by a single length scale. (Ib) This feature is predicted by diverse models; therefore, size alone is only moderately informative about the physical processes at play. (II) Ice-floe shape asymmetries are reproduced by a novel geometric model, which should set a more stringent constraint on the possible relevant physical processes. (III) Points (Ia) and (II) suggest two independent scalar quantities—a Montagu Island area ; ice floes 2 m to 100 m wide; image taken in October 2003 by the QuickBird-2 satellite; resolution ~2.5 m/pixel; retrieved from Google MapsHopen Island area ; ice floes 2 m to 150 m wide; image taken in June 2009 by the GeoEye-1 satellite; resolution ~1.7 m/pixel; retrieved from Google Earth.Svalbard area ; ice floes 60 m to 5 km wide; image taken in June 2001 by the Landsat 7 satellite; resolution 30 m/pixel; retrieved from the U.S. Geological SurveyKara Sea (north hemisphere); ice floes 150 m to 5 km wide; image taken in March 2000 by the Landsat 7 satellite; resolution 30 m/pixel; retrieved from the U.S. Geological SurveyWe employed four data sets, composed of visible-light imagery taken by two different satellites in four different locations in the north and south hemispheres see :Montagu All images were segmented by the Potrace algorithm, and contour information for each detectable floe was gathered. More details about the satellite imagery and the data analysis can be found in the Methods section. We obtained approximately First, we focus on the linear size of the floes. The segmentation procedure yields each contour The measurement of two axes . This suTo rationalize the observed scaling behavior, we consider statistical models of fragmentation. We briefly review two classes of existing models; the first is a stochastic dynamics for the number of floes of a given size, the second is a deterministic geometric description of crack propagation in brittle fracture26l at time In the first class of models, the physical aspects of the fragmentation process are summarized by a rate the first term is due to floes of size When no source is present, The second class of available models is that of brittle fracture. Fragment size distributions are obtained by considering the propagation of cracks in the material. Older theories32stationary states of processes happening at time scales much faster than the seasonal variations. The advantage of this description is that it provides a natural setting for the observation that the same scaling form agrees with empirical data taken in different periods of the year .The steady state Additionally, we propose here a complementary formulation belonging to the first kind of models, which assumes that the empirical distributions could be the In order to proceed, one has to specify It should be stressed that all models are necessarily simplified descriptions and neglect some of the phenomena caused by the very complex interaction between sea ice and the environmentSumming up, the three models described above are compatible with the following scaling form for the fragment size distribution,The previous section has shown that the characteristic length shape factor,aspect ratio, i.e., the ratio between the lengths of the longer and the shorter axis.When considering contour data, the gyration radius used in the definition of We have measured shown in  are peakVoronoi tessellation generated by the seedsTo interpret and characterize these data, we introduce a simple stochastic geometric model, whose only parameter is linked to the distribution of seed locations. Specifically, the seeds are placed randomly following what is known as a “simple point process” in probability; the actual implementation is described below. To fix the ideas, this model may be interpreted as describing the refreezing of floes during winter as the coalescence of radially growing ice floes started from randomly-located seeds, but its formulation is purely geometric and one cannot exclude that it may be derived from alternative physical interpretations. Once the seeds are fixed, the shapes of the resulting floes are obtained as the cells of the i=1, …, n2, be the points of a regular triangular lattice embedded in The simplest point process is arguably the Poisson point process, whose realizations are sets of random independent points. We employ the following generalization, aimed at introducing repulsive correlation between points in an easily controllable way. Refer to Simulating the model for several values of pressive , suggestThe solidity of such a measure is supported by the following observations. (i) Results are not sensitive to the number of seeds , location  and ice age and type .The sea ice system is complex, and several processes are at play, influencing each other. Further data analysis and modeling may attempt to assess, for instance, the relevance of melt pond formation on the thinning surface, which enhances fracturing, or of the development of pressure ridges. Our data are relatively limited, and leave the physical interpretations open, but the regularities observed in the floe shapes express important constraints that any realistic theory should satisfy. The main result of this work is the introduction of two novel independent scalar observables, or “order parameters” for ice floes, summarizing the properties of the distribution of sizes and of shape anisotropies respectively. Regarding floe size, data and different models of fracture suggest a scenario where the spectrum of the different forces at play is reducible, to a good approximation, to a single dominant scale, Considering shape elongation, we find that, as in the case of floe size, a single parameter fully characterizes the distribution in the whole available range of variability. However, differently from size, we found no universal scaling function accounting for the shape anisotropy data. On the other hand, we showed how a simple geometric model of ice accretion and coalescence (related to random Voronoi tessellations) realizes a one-parameter family of distributions fully capturing the shape fluctuations of ice floes, condensing once again the statistics of the elongations to a single dimensionless parameter, the shape disorder 2 ice layer on MarsIt would be interesting to study the shape of ice fragments in other systems, both geological  was taken by the GeoEye 1 satellite on June 13, 2009; catalog ID: 1050410001E370002 in the north-east sector of the image, available in high resolution through the Google Earth application. Spectral bands and exact resolutions for images 1 and 2 could not be determined; if one assumes, as is likely, that Google services use multispectral (red+green+blue) images then the pixel resolutions are 2. Only the quadrants with less than We give here more details about the satellite imagery used. Each data set is composed of a single satellite image. Image 1 (Montagu Island) was taken by the QuickBird 2 satellite on October 16, 2003; catalog ID: 1010010002631B00All images were first made monochromatic by application of a threshold on pixel intensity. This threshold is gauged by sight, but we tested that its precise value does not have a relevant impact on the results, as long as it lies between the average ice value and the average background value. Then the contours of single ice floes were extracted  using th2 snapshot has a While pixel size imposes a cutoff on the minimum floe-size detectable, image size has an influence on the statistics of large objects. Underestimation of the number of large floes can be quantifiedHow to cite this article: Gherardi, M. and Lagomarsino, M. C. Characterizing the size and shape of sea ice floes. Sci. Rep. 5, 10226; doi: 10.1038/srep10226 (2015).


Internal fixation with intramedullary devices has gained popularity for the treatment of intertrochanteric femoral fractures, which are common injuries in the elderly. The most common complications are lag screw cut out from the femoral head and femoral fracture at the distal tip of the nail. We report here a rare complication of postoperative lag screw migration into the pelvis with no trauma. The patient was subsequently treated with lag screw removal and revision surgery with total hip arthroplasty. This case demonstrated that optimal fracture reduction and positioning of the lag screw are the most important surgical steps for decreasing the risk of medial migration of the lag screw. Furthermore, to prevent complications, careful attention should be paid to subsequent steps such as precise insertion of the set screw. Intertrochanteric femoral fractures are common injuries in the elderly. An estimated 1.66 million hip fractures occurred worldwide in 1990, and this number is expected to increase to 6.26 million by 2050 . FurtherHere we present a rare complication of postoperative lag screw migration into the pelvis with no trauma. The patient was subsequently treated with lag screw removal and revision surgery with total hip arthroplasty.A 63-year-old female was injured after falling off a bicycle and transported to a nearby emergency hospital by ambulance. On initial physical examination, tenderness and swelling were noted over the trochanteric region. She could not move her left leg actively because of severe left hip pain. She had no distal neurovascular deficits or other clinically apparent orthopedic or systemic injuries. Radiography revealed a trochanteric fracture in the left femur, AO type 31-A1.2 . She undThe patient tolerated the procedure well and the postoperative period was uneventful. At the most recent followup 2 years after the revision surgery, she had fully recovered activities of daily living and did not require a walking aid.Postoperative medial lag screw migration into the pelvis is a rare complication. Although it has been termed “the Z effect,” a description more appropriate for 2-screw devices, the same phenomenon has also been noted in implants with a single femoral head fixation element, including the gamma nail , 7. To oIn our case, a short gamma 3 nail was placed, with the lag screw locked rotationally by the set screw, which controls not only rotation but also the excessive medial and lateral movement of the lag screw. The gamma nail is biomechanically constructed so that the lag screw slides in the lateral direction. One major advantage of this mechanism is controlled compression of the fracture with weight bearing, which helps in fracture healing , 12. In In summary, we report a rare case of postoperative lag screw migration into the pelvis treated with total hip arthroplasty. This case shows that optimal fracture reduction and positioning of the lag screw are the most important surgical steps in decreasing the risk of medial migration of the lag screw. Moreover, careful attention must be paid to subsequent steps such as set screw insertion to prevent complications.