As totally laparoscopic colorectal surgery is considered challenging and technically demanding with a long steep learning curve, we adopted hand-assisted laparoscopic colorectal surgery as a bridge to totally laparoscopic assisted colorectal surgery. This prospective study aims to highlight the initial experience of a single surgeon with this technique.A prospective analysis of the first 25 cases of hand-assisted laparoscopic colorectal resections which were performed by a single surgeon over a 15-month period. There were 15 males and 10 females with a mean age of 55.5 (range 20-82) years.The indication in majority of cases was cancer (76%). The procedures consisted of 18 (72%) various colectomies and 7 (28%) anterior resections. The operative time ranged between 110-400 (mean 180) min. There was one conversion (4%) and the mean operative blood loss was 80 (range 60-165) ml. The number of lymph nodes retrieved in the cancer cases was 5-31 (mean 15) nodes. The mean length of hospital stay was five (range 3-10) days. The total number of short-term complications was six (24%) and there was one death due to anastomatic leak and multiorgan failure. Long-term complications after a maximum follow up of 30 months were two incisional hernias at the hand port site, but none of the patients developed adhesive small bowel obstruction or late anastomotic stricture. Currently all our colorectal procedures are conducted laparoscopically.Hand-assisted laparoscopic colorectal procedures are easy to learn as a good bridge to master totally laparoscopic colorectal surgery. Since it was first reported in 1991, the numbAll hand-assisted laparoscopic colorectal procedures performed by a single surgeon were prospectively collected. Patients’ demographic data, indications for the procedure, operative time, length of hospital stay, blood loss, short- and long-term complications and mortality were tabulated. In cases of colorectal cancer, the accurate site of lesion was determined preoperatively by colonoscopy and staging included computerized tomography (CT) scan of the chest, abdomen and pelvis. In case of small lesions that were thought to be impalpable at surgery, endoscopic tattooing of the lesions was performed. All patients were admitted the day before surgery and bowel preparation was routinely started as early as possible to avoid gaseous bloating of the colon during the laparoscopic procedure. Prophylactic antibiotics in the form of a single dose of cefazolin 1 g and metronidazole 500 mg, and DVT-prophylaxis (subcutaneous clexane 40 mg) were administered to all colorectal cases.At surgery, the patients were placed in supine position and both legs were kept straight and open to allow passage of the circular staplers in left-sided colectomy. The arm and hand of the opposite side of the planned resection are tugged by the patient’s sides. A beanbag is usually strapped to the table and the patient’s shoulders were supported to avoid slippage during the steep reversed Trendelenburg position. The surgeon stands on the left side of the patient for right hemicolectomy, on the right side for left hemicolectomy and anterior resection and between the patient’s legs for transverse colectomy. All procedures were performed through a midline periumbilical incision (6-7 cm long) for placement of the hand port. Another two trocars (10 mm and 12 mm) were also used for the camera and dissection, electrosurgical and stapling devices . A thirdn = 7) were 8-10 weeks post neoadjuvant chemoradiation. None of the patients needed intraoperative blood transfusion and the mean operative blood loss was 80 (range 60-165) ml. The mean number of lymph nodes retrieved in the cancer cases was 15 (range 5-31) nodes. The mean length of hospital stay was five (range 3-10) days. Since November 2008, all our colorectal procedures have been conducted via totally laparoscopic procedure, while hand-assisted technique is only utilized whenever a difficulty is encountered or conversion is contemplated.Over the 15-month period (July 2006- October 2007), 25 patients  with a mean age of 55.5 (range20-82) years underwent hand-assisted laparoscopic colorectal procedures for various indications; the majority (76%) was for cancer. The procedures consisted of seven sigmoid colectomy (one combined with cholecystectomy), seven left hemicolectomy, four right hemicolectomy, and seven anterior resections . All casShort-term complications: The total number of short-term complications was six (24%); two minor .The ischemic stricture occurred in a patient who underwent HAL left hemicolectomy for proximal descending colon cancer, T3N1M0. He developed a prolonged postoperative ileus, which settled spontaneously. However, he was readmitted with large bowel obstruction two weeks later necessitating emergency resection of the strictured anastomosis, on-table lavage and a primary end- to-end anastomosis. He subsequently received adjuvant chemotherapy and is doing well at 24 months follow up.The ileo-colic anastomatic leak occurred on the fifth postoperative day in an octogenarian (the eldest in the series) with some other comorbidities (ischemic heart disease and hypertension). The leak was recognized and intervention was conducted early, but he developed postoperative renal impairment and failure to wean off the ventilator and died six weeks after his second operation. The third was an iatrogenic injury to the left ureter in a male patient with T3N1M0 upper rectal cancer and diverticular disease who was eight weeks post neoadjuvant chemoradiation. Although, the left ureter was clearly identified in its upper part, it was caught accidently in the stapling device during the pelvic transection of the rectum. The injury was recognized immediately and primary end-to-end repair over a double J-stent was carried out successfully.After a maximum follow up period of 30 months, two patients (8 %) developed incisional hernias at the port site requiring mesh repair, but none developed adhesive small bowel obstruction or anastomotic stricture.interleukin 6 and C-reactive protein than laparoscopic colorectal procedures.[This is the initial experience of a single surgeon with hand-assisted laparoscopic colorectal procedures. It is considered as a “learning curve” experience which was utilized as a bridge towards totally laparoscopic colorectal procedures. Prospective randomized and non-randomized trials have confirmed that hand-assisted laparoscopic colectomy (HALC) offers same minimally invasive benefits as laparoscopic colectomy (LAPC). HALC has shorter operative time, lower conversion rate and has comparable complication rate and length of hospital stay.–12 Howevocedures. Moreoverocedures. There isHALC was introduced to our institution in July 2006 and was performed solely by one surgeon. The aim was to use this hybrid procedure as a bridge towards totally laparoscopic colorectal surgery. Now after the first 25 cases that constitutes this study group, and since November 2008, all colorectal procedures in the author’s unit are performed via totally laparoscopic-assisted technique. Now, the hand-assisted technique is introduced only in difficult laparoscopic cases or to speed up a certain laparoscopic step such as taking down the splenic flexure and difficult rectal dissection in selected cases. It is also used whenever a conversion to open technique is contemplated.As our institution is a tertiary oncology center, the majority of our HALC cases were conducted for malignant cases in high number of cases (76%). There was minimal blood loss and none of the patients needed blood transfusion. This can be explained by the ease with which intra-operative bleeding can be controlled by the inserted hand and also due to the use of versatile blood vessel sealing devices such as the harmonic scalpel and Ligasure. It is also noticeable that there were more left sided and rectal than right-sided procedures. This is attributed to the fact that left-sided colonic cancers are more common than the right-sided ones in our area. Furthermore, most right-sided tumors are dealt with by general surgeons in nearby secondary hospitals, while left-sided and especially rectal cases are usually referred to us for management. All cases in our series were done electively except one case who was admitted as an emergency with large bowel obstruction secondary to a stenosing cancer of the proximal descending colon. He initially underwent endoscopic stenting, and then was optimized to undergo HALC a week later. This highlights the emerging trend of endo-laparoscopy for the management of acute left-sided colonic obstruction.Our initial experience also highlights three interesting short-term complications of laparoscopic colorectal surgery. The first was the ischemic anastomotic stricture, which may be attributed to the stretch on the proximal colon that has occurred during performing the extracorporeal anastomosis. Thrombosis of the terminal vessels feeding the anastomosis was evident during the operation. The second complication was the ileo-colic anastomatic leak in an octogenarian with some other co-morbid conditions. The leak was recognized and intervention was conducted early, but he died of multi-organ failure six weeks after his second operation. The iatrogenic injury to the left ureter was in an elderly man with upper rectal cancer and diverticular disease. The injury was recognized and immediate repair was carried out successfully. It is interesting to note that the ureteric injury occurred in the twenty-fourth hand-assisted case and not early in our experience.The long-term complication of hand-assisted laparoscopic colectomy has been the center of recent debate. It has been postulated that continuous and persistent stretch of the port site may predispose to the development of incisional hernia. Furthermore, placement of hand in the abdomen in HALC increases the risk of postoperative ileus, development of intrabdominal adhesions with future risk of small bowel obstruction. In our sHand-assisted laparoscopic colectomy is advocated as a safe and feasible procedure in malignant as well as benign colorectal conditions. It is easy to learn and combine the advantages of both laparoscopic  and conventional open surgery. It also offers similar short and long-term minimally invasive benefits as laparoscopic colectomy. In our opinion, it is best adopted as an adjunct and a bridge toward adoption of totally laparoscopic colectomy.


MicroRNAs (miRNAs) are key components in post-transcriptional gene regulation in multicellular organisms. As they control cooperatively a large number of their target genes, they affect the complexity of gene regulation. One of the challenges to understand miRNA-mediated regulation is to identify co-regulating miRNAs that simultaneously regulate their target genes in a network perspective.We created miRNA association network by using miRNAs sharing target genes based on sequence complementarity and co-expression patterns of miRNA-target pairs. The degree of association between miRNAs can be assessed by the level of concordance between targets of miRNAs. Cooperatively regulating miRNAs have been identified by network topology-based approach. Cooperativity of miRNAs is evaluated by their shared transcription factors and functional coherence of target genes. Pathway enrichment analysis of target genes in the cooperatively regulating miRNAs revealed the mutually exclusive functional landscape of miRNA cooperativity. In addition, we found that one miRNA in the miRNA association network could be involved in many cooperatively regulating miRNAs in a condition-specific and combinatorial manner. Sequence and structural similarity analysis within miRNA association network showed that pre-miRNA secondary structure may be involved in the expression of mature miRNA's function.On the system level, we identified cooperatively regulating miRNAs in the miRNA association network. We showed that the secondary structures of pre-miRNAs in cooperatively regulating miRNAs are highly similar. This study demonstrates the potential importance of the secondary structures of pre-miRNAs in both cooperativity and specificity of target genes. PCC > 0 represents that x and y are positively correlated with the degree of correlation. On the other hand, PCC < 0 represents that x and y are negatively correlated with a value |PCC|. A positive value of the PCC indicates that miRNAs and genes are co-expressed and a negative value of the PCC indicates that opposite expression pattern exists between them. Considering the conservation of miRNA-target relationship, we retrieved and applied 50,339 and 50,349 human and mouse miRNA-target pairs for the 162 and 7,927 conserved miRNAs and mRNAs by using TargetScan target prediction database (release 4.2) .ij tbetween miRNAs i and jas follows:Target overlap score between a pair of miRNAs is defined as Jaccard similarity coefficient, representing the fraction of shared condition specific targets. We define the target overlap score Targets(i) represents to the set of targets of miRNAs i. We measured the statistical significance of the Jaccard similarity coefficient by using the exact randomization tests [n × n condition specific target overlap matrix T = [ijt] is transformed into an n × n adjacency matrix A = [ija], which encodes the connection strengths between pairs of nodes based on the probabilities related to the Jaccard similarity coefficient. Since the networks considered here are undirected, A is a symmetric matrix with non-negative entries.where on tests . The n ×Figure In the present study, a subnetwork detection algorithm, called MCODE , was apphttp://genome.ucsc.edu/) and tfbsConsFactors. All RefSeq genes genomic locations were taken from hg17. In mouse, regulators of miRNAs were identified in the human genome in the regions orthologous to the mouse. For example, hsa-miR-101 in human and mmu-miR-101 in mouse are orthologous. To assess the statistical significance of the rate of co-occurrence of motif pairs, we used cumulative hypergeometric distribution to calculate the probability of obtaining the rate of co-occurrence, C, equal to or higher than the observed rate of co-occurrence, c', by chance:To investigate the modular nature of global MRAN, we used predicted binding sites for all Position Specific Scoring Matrices (PSSMs) from TRANSFAC version 8.3, as they are defined by the UCSC hg17 genome assembly, in the tfbsConsSites , and edges statistical significance of co-occurrences. Hypergeometric http://www.biocarta.com) pathways to test their functional coherence and meaningfulness. KEGG pathways mainly include cellular processes related to metabolism and biosynthesis. Those on BioCarta cover a wider variety of cellular processes including a large number of signal transduction and immune signalling pathways as well as metabolic and biosynthetic pathways. Pathways with p-value < 0.05 as revealed by the hypergeometric test were considered statistically significant in the present study. To visualize the distinctness of the cooperative modules in terms of functional enrichment, we created heat maps of the significances   and BioCStructural similarity of the members of CMMs was evaluated. T-COFFEE (version 5.05)  was usedThe authors declare that they have no competing interests.YJN designed the study, performed data analysis and wrote the paper. JHK supervised the research.


Many postnatal women are insufficiently physically active in the year after childbirth and could benefit from interventions to increase activity levels. However, there is limited information about the efficacy, feasibility and acceptability of motivational and behavioral interventions promoting postnatal physical activity in the UK.The MAMMiS study is a randomized, controlled trial, conducted within a large National Health Service (NHS) region in Scotland. Up to 76 postnatal women will be recruited to test the impact of two physical activity consultations and a 10-week group pram-walking program on physical activity behavior change. The intervention uses evidence-based motivational and behavioral techniques and will be systematically evaluated using objective measures (accelerometers) at three months, with a maintenance measure taken at a six-month follow-up. Secondary health and well-being measures and psychological mediators of physical activity change are included.The (MAMMiS study will provide a test of a theoretical and evidence-based physical activity behavior change intervention for postnatal women and provide information to inform future intervention development and testing within this population.Current Controlled Trials ISRCTN79011784 During the year following childbirth there are physical and psychological benefits associated with participation in regular physical activity ,2. A gra, setting goals, planning and self-monitoring activity). Three small-scale studies, conducted in the USA, Canada and Australia, recruited healthy women in the year following childbirth and successfully used such approaches to change participation in physical activity ). Participants are advised that regular physical activity means “accumulating at least 30 minutes of moderate-intensity activity at least five times a week” [33] and are eligible for inclusion if they are in contemplation (not regularly active but thinking about starting to be in the next six months) or preparation stages .i) Insufficient Englishii) Pregnancy or those planning to become pregnant in the next six months.iii) Medical contraindications to physical activity, assessed using the Physical Activity Readiness Questionnaire (PAR-Q) [34].iv) Stage of physical activity is assessed as precontemplation (not regularly active and no intention to become active in the next six months), action (regularly active in line with guidelines but only began this in the past six months) or maintenance stage (regularly active in line with guidelines and has been so for longer than six months) according to the Stages of Change questionnaire [32]).No exclusions were made based on characteristics of the baby, that is, women with infants that had been delivered prematurely, had spent time in a special care baby unit and/or had delivered twins or multiple births were eligible for inclusion.t-test to detect change from pre- to post-test between the intervention and control group using a pooled standard deviation of 75 minutes/week. This sample size calculation is based on data from a previous study comparing a 12-week physical activity intervention in insufficiently active breast cancer survivors with usual care [Based on calculations carried out by an independent statistician, 31 participants per group are required in order to detect an effect of 63.83 minutes/week of moderate-vigorous physical activity participation. This assumes power of 90%, 5% significance level with a two-sided unpaired ual care . Comparaual care . This stual care . Assuminual care ,17) up tParticipants are allocated to groups using simple randomization from a computer-generated sequence with block sizes of 4 and 6, generated by an independent person. Following sequence generation, group identifier cards were placed into envelopes alongside a piece of card to block the research team from identifying group allocation. Envelopes were sealed and stacked and participants will be assigned an envelope in the order in which they enroll into the study. This is known as sequentially numbered, opaque, sealed envelopes (SNOSE) .. Exercise  consultations were developed from well-established theoretical models of physical activity change, in particular the Transtheoretical Model (TTM) [.)The intervention consists of a face-to-face physical activity consultation (approximately 45 minutes in length) delivered at the start of a 10-week group pram-walking program. Participants receive a second face-to-face consultation (approximately 25 minutes in length) following the three-month assessments. Consultations are structured and individualized counseling sessions that aim to enhance motivation for physical activity and teach participants self-regulatory strategies associated with adoption and maintenance of an active lifestyle . Consultel (TTM) ). Severael (TTM) -42. Thesel (TTM) -26 and ael (TTM) , alongsiel (TTM)   and planning social support for behavior change . Participants are also invited to attend one group pram-walking session in their local area each week for 10 weeks. A trained walk leader facilitates these and routes have been mapped and risk-assessed as suitable for mothers with prams. Walks are conducted at a moderate-intensity  for 30 to 55 minutes, plus a five-minute warm up and cool down at the start and end of each session. The pram-walking group provides an opportunity to demonstrate a moderate walking pace, provides further social support and prompts for weekly activity. Participants who anticipate difficulties attending are encouraged during the physical activity consultation, to plan alternative physical activity opportunities in line with their personal activity goals. This allows for mothers with difficulties attending walks . Participants who do not attend the pram-walking group in the first two weeks will receive a 10-minute support phone call encouraging efforts towards being more active and offering an opportunity for problem solving barriers to adopting their activity plan. Following the three-month follow-up period a second consultation will provide personalized feedback on changes in physical activity participation and use relapse prevention strategies to encourage participants to continue with an active lifestyle.Further details regarding intervention content and approach to development are available elsewhere . Finally, the intervention and control group will both receive a leaflet after baseline assessments. The “Active living during and after pregnancy” leaflet was developed by NHS Health Scotland and provides information on the physical activity guidelines and suitable activities . The control group will receive no further intervention but will receive standard postnatal management.The CI conducts assessments over two appointments during each measurement period , normally a week apart. These take place either at the university or in participants’ homes, depending on participant preference. The usual order of assessments is demographic details, psychological well-being and fatigue, cardiovascular fitness, height (baseline only), weight and body composition, followed by instructions on wearing the accelerometer (appointment one). Participants return their accelerometer at appointment two where they also complete a questionnaire regarding psychological mediators of physical activity and physical activity participation is assessed using the Seven Day Physical Activity Recall (7-Day PAR) interview.Physical activity change is measured using Actigraph GT3X and GT3X + accelerometers  Accelerometers are worn during waking hours for seven consecutive days (excluding bathing and swimming) on the right hip, with movement assessed using sampling intervals (epochs) of 60 seconds. Accelerometers have been shown to be reliable and valid measures of physical activity participation in community samples, including overweight or obese adults . During . Previous research has found that this is a reliable and valid method for measuring habitual physical activity [, swimming). Participants are also asked whether the activity levels during the previous week were “less than”, “more than” or “about the same” as the last three months and provide reasons for deviations from normal activity. This explanatory data will aid in interpretation of trial data.At the end of the measurement week, the 7-Day PAR interview is administered to participants . This meactivity . The 7-DPsychological well-being is measured using the Adapted General Well-being Index (AGWI) . The oriCardiovascular fitness is measured using the Chester Step Test . The Che2).Height is measured in centimeters (to the nearest cm) using a stadiometer . Height readings are taken twice and averaged at the first baseline appointment. Weight and body composition (% fat mass) are measured using the Tanita portable bioelectrical impedance monitor  according to the measurement protocol specified in the handbook . Weight is measured in kilograms (to the nearest 0.1 kg) and BMI computed as the participant’s weight in kilograms divided by their height in meters squared (kg/mvia a self-completion questionnaire that has been developed for this study from adapted measures used in previous studies [etc.). Mediators measured in this study are specifically those targeted by the intervention methods . Self-efficacy  and outcomes expectancy measures  were sensitive to the most frequent barriers and enablers to activity cited by postnatal women; taken from a survey 667 postnatal women [et al.[Psychological mediators are measured  studies -62. The al women . Intential women . Action al women . Six iteIn line with CONSORT , trial pBlinding of the CI and participants is carried out at baseline since outcomes are taken prior to group allocation. At three- and six-month follow-ups, steps have been taken to minimize potential bias from a lack of blinding of the CI. This includes using objective measures to assess physical activity, fitness weight and body composition according to standardized protocols. Other secondary outcomes are assessed through self-administered questionnaires.Actilife 5 will be used to analyze accelerometer data in the following ways: ‘raw’ accelerometry output  averaged over the measurement period), time (minutes) spent in moderate to vigorous intensity physical activity per week, continuous bouts of at least 10 minutes of moderate-vigorous physical activity (MVPA) and, percentage of time spent in MVPA. The calculation of MVPA is based on accepted cut-points for adult women in free-living conditions -67. Howepost-hoc testing will be used to investigate intervention efficacy (between-groups change to three months) and assess maintenance effects (whether change is sustained to six-month follow-up). Statistical analysis will be conducted on all secondary outcomes, also using this approach.To assess whether change in the main outcome  occurred as a result of change in the psychological mediators, mediation analysis will be conducted using the Sobel test, which involves testing the null hypothesis that there is no difference between the total and direct effects, that is, the assumption that there are no indirect effects of the psychological mediators on physical activity behavior change [Trial data will be analyzed according to intention-to-treat principles with all participants included according to initial group allocation. To minimize the number of statistical tests conducted, Analysis of Covariance (ANCOVA) will be used with a repeated measures design and group allocation as a between groups factor. Covariates, which have been shown to influence physical activity participation, will be included . Population-specific covariates  will also be added to the model. Bonferroni r change . This apr change  approachThis study received favorable ethical approval from NHS Fife and Forth Valley Research Ethics Committee and from the School of Nursing, Midwifery and Health Research Ethics Committee, University of Stirling.Results from this study will be available by the end of 2012. The strengths of this study include the use of a randomized, controlled design, objective measures of physical activity change and a three-month post-intervention follow-up point (six months from baseline). Using this approach, the MAMMiS study will address many of the methodological shortcomings of previous trials. Results from this study may also provide valuable information to inform future physical activity promotion studies with postnatal women. In particular, this trial will provide tentative evidence on the efficacy of a motivational and behavioral intervention including group pram-walking in a UK-setting. As the intervention techniques used in this study have been defined according to previously published taxonomies ,44 (for Other strengths of this study include: conducting mediation analysis to help identify whether psychological constructs targeted through the intervention were in fact responsible for changes in physical activity behavior; including health and well-being measures enabling information to be gathered on the potential impact of physical activity change, which may aid in sample size calculations for future studies interested in these outcomes; and finally, the inclusion of a qualitative arm at the end of the trial will help assess intervention acceptability among postnatal women. These features, alongside outcome and process data, will help advance our understanding of physical activity behavior change in postnatal populations.Physical activity is important for postnatal health and well-being as well as helping to tackle preventable deaths and improve morbidity outcomes and quality of life through preventing or postponing onset of chronic physical and psychological health conditions. Positive physical activity habits adopted during the postnatal period may also go some way to addressing age-related decline in physical activity found among young and middle-aged women . Thus, tThis is an active trial. At the date of submission 65 participants were successfully enrolled.7-Day PAR: Seven day physical activity recall; AGWI: Adapted general well-being index; ANCOVA: Analysis of covariance; CI: Chief Investigator; CHD: Coronary heart disease; CHP: Community health partnership; CONSORT: Consolidated standards of reporting trials; HR: Heart rate; MAMMiS: More active mums in Stirling; MVPA: Moderate-vigorous physical activity; NHS: National health service; PAR-Q: Physical activity readiness questionnaire; PND: Postnatal depression; RCT: Randomized controlled trial; RPE: Rating of perceived exertion; SIMD: Scottish index of multiple deprivation; VAS: Visual analogue scale.The authors declare no competing interests.ASG is a PhD student and CI for the MAMMiS study with responsibility for the development, conduct and reporting of study. ARH and RJMcI are supervisors to ASG. ARH conceived the original idea and secured funding for the original study proposal. All authors read and approved the final version of the manuscript.


At both these occasions, the psychomotor performance (assessed as multiple choice reaction time) and number of correct responses to audio-visual stimuli was assessed at rest and immediately after cessation of maximal intensity exercise. The supplementary low-intensity exercise training increased VO2max and markedly shifted AT toward higher absolute workload. Immediately after completion of the graded exercise to volitional exhaustion, the ballerinas’ psychomotor performance remained at the pre-exercise (resting) level. Neither the resting nor the maximal multiple choice reaction time and accuracy of responses were affected by the supplementary aerobic training. The results of this study indicate that addition of low-intensity aerobic training to regular dance practice increases aerobic capacity of ballerinas with no loss of speed and accuracy of their psychomotor reaction.We investigated whether 6-week low-intensity aerobic training program used as a supplement to regular dance practice might improve both the aerobic capacity and psychomotor performance in female ballet dancers. To assess their maximal oxygen uptake (VO In contrast, 2max and improvement in dance performance following supplementary aerobic training. However, there is no published study concerning this matter in classical ballet dancers. For this reason, we decided to examine whether adding a supplementary low intensity aerobic training program to regular dance practice would improve VO2max and psychomotor performance in classical ballet dancers.Reports on the influence of supplementary training on maximal oxygen uptake in dancers are scarce. corps de ballet (including at least two years immediately preceding the study) they danced on the average about 6 times  per week. They had not been involved in other forms of regular physical activity. After being informed about the purpose of the study, all the subjects signed a written consent to participate in the study. The study protocol was approved by the Ethics Committee of the Academy of Physical Education in Katowice, Poland.Six professional female ballet dancers volunteered for the study. All the subjects started dancing at 9 years of age and were subjected to regular dance training for at least 12 years. During their work as members of the All the volunteers were clinically healthy and in good nutritional status, and their habitual diet was assessed with the use of a questionnaire. The dancers recorded their food intake over a 3-day period just before the commencement of exercise tests, and the daily records were analyzed for energy and macronutrients intake using a computer program Dietus . Basic anthropometric characteristics of the subjects are presented in 2max and anaerobic threshold (AT). All anthropometric measurements, the psychomotor performance test and exercise test were performed both prior to the beginning of aerobic training (pre-T) and following a 6-week supplementary aerobic training (post–T).The experimental protocol consisted of anthropometric measurements, a psychomotor performance test and graded exercise test for the evaluation of VO2) were analyzed continuously (breath-by-breath) for 1 min at rest and at the third minute of each workload using standard technique of open-circuit spirometry (Yeager). Heart rate (HR) was recorded continuously using a PE 3000 Sport Tester .Body composition was assessed using bio-electrical impedance . All subjects cycled on a 828 Monark (Sweden) ergometer with intensity increasing by 30 W every 3 min until volitional exhaustion. Minute ventilation (Ve) and oxygen uptake  using a model Rapid Lab 248 (Bayer Diagnostics) blood gas analyzer.To determine the anaerobic threshold, fingertip capillary blood samples for lactate concentration assessment were taken at rest, at the third minute of each workload, and at the fifth minute of post-exercise recovery. Blood lactate concentration was measured by the standard enzymatic method using commercial kits  and a model UV-1201 UV/VIS Shimadzu spectrophotometer. The sensitivity of the blood lactate assay was 0.1 mmol/l. Additionally, fingertip capillary blood samples were taken both at rest and during maximal exercise for the assessment of acid-base balance parameters (pO−] vs. log exercise intensity) with two segmental linear regressions according to The anaerobic threshold (AT), defined as exercise workload at which the concentration of plasma lactate begins to increase in a nonlinear fashion, was calculated using the log-log method  and 15 “false” (green or yellow light) stimuli. The subjects were asked to press and release, as quickly as possible, a button on the right handlebar of the cycle ergometer in response to a red light, the button on the left handlebar in response to a sound, and not to react to false stimuli. The reaction time was measured with 0.01s accuracy using a reaction time measuring device  and the results were given as a mean of 15 responses to real stimuli.The training program consisted of aerobic cycling exercise  6 days/week, each session lasting 30 min, for successive 6 weeks. The applied workloads were determined based on HR that was kept at 90 beats/min during the first 2 weeks, at 100 beats/min during the next 2 weeks, and at 110 and 120 beats/min in the fifth and sixth week of training, respectively.t test for paired variables. All statistical analyses were carried out using the statistical software package STATISTICA v. 6.0 .All variables are reported as the mean ± S.D. Statistical significance of differences between means was assessed using a 2-way repeated measures ANOVA with training status (pre- or post-) and exercise status  as the repeated measures’ factors, followed by the Bonferroni test when appropriate, or by Student’s Body mass and body composition of the studied subjects before and after supplementary aerobic training are presented in 2), heart rate (HR) and minute ventilation (Ve) (2 (both absolute and relative to body mass and FFM) and maximal Ve were significantly higher after the training (Resting oxygen uptake (VOion (Ve)  did not training .Anaerobic threshold (AT) prior to the supplementary aerobic training appeared at the workload of 73.8 W  and shifEffects of supplementary aerobic training on mean, minimal and maximal choice reaction time at rest and immediately post-exercise are illustrated by the data summarized in There was no significant difference between the pre- and post-exercise reaction time either during the pre-training or post-training trials, and neither the resting, nor the post-exercise reaction time differed between the pre-training and post-training trials.2max tests revealed that the dancers showed low to moderate physical capacity, typical of untrained individuals, which is consistent with earlier reports  has only been measured by non-invasive methods (The relatively low AT level in our study group, which was within the range typical of untrained individuals, was in line with the VO methods , and has methods .These findings demonstrate that the length of professional practice in excess of 4–6 years does not affect aerobic capacity in female classical dancers.2max remaining in the range typical of untrained healthy females. The effect of endurance training on psychomotor performance has not been previously studied in classical ballet dancers. Similar studies performed in athletes have shown a decrease after maximal exercise (In spite of a significant training-related increase in maximal workload there was no significant increase in blood lactate concentration at this workload. This indicates improved aerobic metabolism in the skeletal muscles. In line with this argument was the lack of significant training-related changes in the acid-base balance parameters. Altogether, these data demonstrate considerable benefit from the supplementary training despite VOexercise , whereasexercise ; 1998. Sexercise  in spiteexercise  and to dexercise . Enduranexercise . Despiteexercise ; 2005 anexercise . Importaexercise . Again, The results of this study show that low-intensity endurance training is a suitable supplementary form of physical activity for increasing aerobic capacity in female classical ballet dancers. Notably, this type of training improves endurance without decreasing psychomotor performance during maximal exercise, which seems particularly important in this sport discipline.


MSC in culture typically grow to confluence within a few weeks. They show spindle-shaped fibroblastoid morphology and align to each other in characteristic spatial patterns at high cell density. We present an individual cell-based model (IBM) that is able to quantitatively describe the spatio-temporal organization of MSC in culture. Our model substantially improves on previous models by explicitly representing cell podia and their dynamics. It employs podia-generated forces for cell movement and adjusts cell behavior in response to cell density. At the same time, it is simple enough to simulate thousands of cells with reasonable computational effort. Experimental sheep MSC cultures were monitored under standard conditions. Automated image analysis was used to determine the location and orientation of individual cells. Our simulations quantitatively reproduced the observed growth dynamics and cell-cell alignment assuming cell density-dependent proliferation, migration, and morphology. In addition to cell growth on plain substrates our model captured cell alignment on micro-structured surfaces. We propose a specific surface micro-structure that according to our simulations can substantially enlarge cell culture harvest. The ‘tool box’ of cell migratory behavior newly introduced in this study significantly enhances the bandwidth of IBM. Our approach is capable of accommodating individual cell behavior and collective cell dynamics of a variety of cell types and tissues in computational systems biology.Therapeutic application of mesenchymal stem cells (MSC) requires their extensive  Over the past decade mesenchymal stem cells (MSC) derived from bone marrow, adipose, and many other tissues have intensively been investigated with respect to their tissue regeneration prospects Any therapeutic use of MSC relies on their extensive expansion. One major aspect of cell expansion is the selection of highly potent cells from tissue biopsy. Various protocols have been suggested for effectively isolating MSC with high regenerative potential and homogeneity in vitro.These rapid experimental developments are paralleled by striking progress in the mathematical sciences dedicated to the modeling and simulation of tissue formation dynamics. However, only few approaches address MSC organization on the cellular level During cultivation MSC undergo phenotypic changes that enable their early cell shape-based classification Models of individual migrating cells have reached a very high level of detail and complexity Generalized cell shapes were already introduced by models for Dictyostelium discoideum in vitro. MSC growth dynamics and spatial organization are assessed in terms of the number of cells, mean population radius, and cell-cell alignment. We illustrate the general model behavior by presenting simulation results with essential model parameters being varied. Second, we demonstrate our model's capacity to account for contact guidance by micro-structured substrates and predict a micro-structure that according to our simulations can significantly enhance cell yield in in vitro cell expansion.We first introduce the basics of our new IBM and compare experimental and computational results on MSC expansion Our new IBM builds on previous models of Galle and Drasdo The maximum speed of a podium  domains Cell-cell alignment generally decreases with cell-cell distance. This is demonstrated in  results . This maIn silico reproduction of our experimental results was most sensitive to model parameters that determine the down-regulation of cell proliferation and cell migration as a function of cell density (see Modeling methods section G). Delaying the down-regulation of cell growth to higher cell densities has little effect on the initial phase of cell expansion in which cell-cell alignment generally increases over time - experimentally as well as computationally  temporary changes in the number and position of podia due to probabilistic podium inactivation  and ii) changes in the number of podia phenotype . Down-regulation of random podia activity thus means that the independent podium inactivation rate and the number of podia offset value are simultaneously decreased with cell density. Delaying the down-regulation of random podia activity to higher cell densities generally results in less aligned cells, faster spatial colony expansion, and higher cell numbers . Our resFaster spatial colony expansion is not essentially linked to faster cell population growth as could be presumed based on reduced contact inhibition. This is demonstrated by the variation in spatial colony expansion brought about by changes in cell-substrate friction. These had no effect on the number of cells and resulted in similar cell-cell alignment . As a coPodium length has an effect on cell colonies in that cells with longer podia align more accurately and form larger alignment domains than cells with shorter podia  and 10. In summary, our results indicate that the specific density-dependent down-regulation of cell growth and random podia activity found for the present data maximizes spatial colony expansion and cell-cell alignment at the same time. This strongly suggests that it is finally aimed at maximizing the number of cells per colony given the constraint that each cell seeks to maintain a defined area of contact with the substrate.Next, we demonstrate our model's capability to reproduce experimental data on cell colony growth on micro-structured substrates. The alignment of cell podia to substrate microgrooves is modeled analogous to podia-podia alignment as used in the previous section (MSC on plain substrates). However, other than podia, grooves are never moved.micro) between day 4 and 8 as compared to plain substrates (plain) and measured by colony area increase , has been shown be the basic mechanism of cell directional motion in chemotaxis Another basic question raised by our study is how cells sense properties of substrate micro-structures  on a molecular level and adapt their behavior accordingly. Ensemble physical properties of our model, like cell orientational phase transitions, collective motion, and giant number fluctuations, as found in nematic systems of self-propelled particles in vitro cultivation phase, and thus a reduced probability of cell transformation The ‘tool box’ of cell migratory behavior newly introduced in this study substantially enhances the bandwidth of IBM in computational biology. The high sensitivity of our simulation results on model parameters regulating the density dependence of cell proliferation, migration, and morphology emphasizes the importance of cell-cell interactions in tissue formation. While density dependence of colony formation is a well known characteristic of MSC culture, studies on the impact of cell-cell contacts on MSC expansion are presently still rare. This may be related to the difficulties of non-invasive long-term single cell tracking in MSC culture, which is largely being disabled by cell clustering . In contrast, effects of substrate stiffness and micro/nano-structure on the growth, migration, and differentiation of MSC populations are studied in an increasing number of experimental studies in vitro on plain as well as micro-structured substrates and proposed a microstructure that according to our simulations can significantly enlarge cell harvest in biotechnological applications. We think that our model has high potential for further development and can be evolved to accommodate individual cell behavior and collective dynamics of a variety of cell types and tissues in computational systems biology.In summary, we developed an improved model of proliferating and migrating fibroblast-like cell populations that explicitly represents cell podia and accounts for adjustment of cell behavior in response to cell density. We demonstrated that this model can quantitatively reproduce the spatiotemporal organization of MSC The methods section consists of three mayor parts: Experimental materials and methods, Image analysis methods, and Modeling methods.4 cells/cm2 in tissue culture flasks with Dulbecco's modified Eagle's medium  supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (both Biochrom). Cultures were maintained at 37°C in a humidified atmosphere containing 95% air and 20% O2–5% CO2 . After 48 h, the non-adherent debris were removed by washing twice with phosphate buffered saline  and incubated with fresh medium. Single clones of MSC were randomly chosen and used for daily phase-contrast microscopy  over a period of nine days.Isolation of primary MSCs from sheep and cultivation of the cells: Bone marrow aspirates of 20 ml were obtained from the iliac crests of Merino sheep as previously described in Experimental cell culture micrographs were segmented using fuzzy c-means segmentation The modeling methods section is subdivided into eight parts that explain the interaction between cell bodies as adopted from previous work The newly introduced cell model builds on a previous individual cell-based model of computational tissues as introduced by Galle and Drasdo et al. Isolated cell bodies are represented by elastic spheres of radius l forces . Cell deectively . The defubstrate .The body forces acting on the position of cell center In addition to the above body forces, the model accounts for cell-cell and cell-substrate friction as well as friction associated with cellular reorganization during changes in cell radius according toThe parameters specifying the physical interactions, i.e. the Young modulus The newly introduced cell model expands previous IBMs for computational tissues Random podium generation and activation: Podia of cell ell body . Podia aProtrusion force of the podium tip and traction force between podium tip and cell body: The tip of each podium is assigned a scalar protrusion force dium tip . The celRandom podium inactivation: Podia are inactivated according to the number of podia-related random update rate Podium retraction and final elimination: With its protrusion force switched-off the podium retracts towards the cell body due to its intrinsic contraction force, mimicking actin depolymerization The present model simplifies cells by using a geometric ‘ball and stick’ representation and employing a basically pairwise cell-cell alignment approach. In regions of high cell density this can lead to temporary intersections between cells, which are, however, also frequently observed in cell culture.Podia are represented as cones with spherical tips . Their mreserved . The podreserved . Otherwireserved . If two reserved . The movPodium alignment to microgrooves is handled much like the podium-podium alignment described above with the microgrooves being ‘podia’ that are never moved. Another difference is that the alignment to microgrooves is not executed every time step but rather according to an alignment rate (probability per time). This enables the model cells to cross over microgrooves as is experimentally observed. This behavior is further facilitated by not executing alignment if the podium has already ‘climbed’ the microgroove, i.e. if its tip center is located within the microgroove.Podia dynamics are calculated according to equation (3) and the interaction rules described in the previous paragraph. The dynamics of the cell bodies are calculated according to the balance of forces (equations (1), (2), and (4))for the center and the radius of each cell Here, we present the mathematical formalism for deriving the podia and whole cell movement properties used in the results section. The maximum elongation speed of a newly generated podium is in their  [59]. ThCell proliferation is modeled according to five cell cycle phases. Simulations start with a cell volume The density dependence of cell proliferation, migration, and the number of podia were most important for the present modeling results. Contact inhibition of proliferation and migration of fibroblast-like cells have been described and modeled by others before see also .Figure S1Contact between two cell colonies at day 9 of culture.(TIF)Click here for additional data file.Figure S2Snapshot of a simulation at day 8 of culture with density-dependent contact inhibition of cell growth being disabled.(TIF)Click here for additional data file.Figure S3Cellular growth accelerates spatial colony expansion only if cell migration is low. Mean population radius and number of cells (insets) during simulated cell cultivation. Down-regulation of cell migration as described in the caption of (TIF)Click here for additional data file.Figure S4Podium length accelerates spatial colony expansion and decreases cell number density. Number of cells (top) and mean population radius (bottom) during simulated cell cultivation. The podium length is either short (rameters . Larger (TIF)Click here for additional data file.Figure S5Cell-substrate friction accelerates spatial colony expansion but leaves the number of cells unaffected. Mean population radius and number of cells (inset) for low (red), medium (blue), and high (green) friction corresponding to 0.5, 1.0, and 1.5 times the standard values used for cell body- and podium-substrate friction (friction . The obs(TIF)Click here for additional data file.Figure S6Kinetic exponent for the mean squared displacement (msd) of single non-interacting cells over time for different probabilistic podium inactivation rates. The kinetic exponent (TIF)Click here for additional data file.Figure S7Program flow chart. In each time iteration step, first podia are moved, second bodies, and third radii. Finally, phenotype and proliferation states are updated. The underlying physical, heuristic, and random models are described in the methods section of the main manuscript. Each podium is elongated independently according to the physical model. Overlapping podia are aligned to each other according to the assigned pairwise heuristic. Podia are switched on or off according to random generation and inactivation rates, respectively, which depend on all podia of a given cell. Phenotype  and proliferation  states are regulated according to the local cell density (accounting for cell bodies and podia).(TIF)Click here for additional data file.Video S1Plain substrate - close view.(MPEG)Click here for additional data file.Video S2Plain substrate - far view.(MPEG)Click here for additional data file.Video S3Parallel microgrooved substrate - close view.(MPEG)Click here for additional data file.Video S4Parallel microgrooved substrate - far view.(MPEG)Click here for additional data file.Video S5Starlike microstructured substrate - close view.(MPEG)Click here for additional data file.Video S6Starlike microstructured substrate - far view.(MPEG)Click here for additional data file.


In vivo models that mimic the breast cancer-specific osteolytic bone microenvironment are limited. Previously, we developed a mouse model of tumor-bone interaction in which three mouse breast cancer cell lines were implanted onto the calvaria. Analysis of tumors from this model revealed that they exhibited strong bone resorption, induction of osteoclasts and intracranial penetration at the tumor bone (TB)-interface.Breast cancer is the second leading cause of cancer-related death in women in the United States. During the advanced stages of disease, many breast cancer patients suffer from bone metastasis. These metastases are predominantly osteolytic and develop when tumor cells interact with bone. In this study, we identified and used a TB microenvironment-specific gene expression signature from this model to extend our understanding of the metastatic bone microenvironment in human disease and to predict potential therapeutic targets.in vitro osteoclast model, we demonstrate that our model mimics both the human breast cancer bone microenvironment and osteoclastogenesis. Furthermore, we observed enrichment in various signaling pathways specific to the TB interface; that is, TGF-β and myeloid self-renewal pathways were activated and the Wnt pathway was inactivated. Lastly, we used the TB-signature to predict cyclopenthiazide as a potential inhibitor of the TB interface.We identified a TB signature consisting of 934 genes that were commonly (among our 3 cell lines) and specifically  up- and down-regulated >2-fold at the TB interface in our mouse osteolytic model. By comparing the TB signature with gene expression profiles from human breast metastases and an Our mouse breast cancer model morphologically and genetically resembles the osteoclastic bone microenvironment observed in human disease. Characterization of the gene expression signature specific to the TB interface in our model revealed signaling mechanisms operative in human breast cancer metastases and predicted a therapeutic inhibitor of cancer-mediated osteolysis. Bone is one of the most common sites for metastasis in human breast cancer. Bone metastasis results in cancer-related pain, pathological fracture, hypercalcemia, neurological defects, and immobility; all of which increase the risk of mortality and decrease the quality of life for breast cancer patients -4. WhileWithin the osteolytic lesions of bone metastases, tumor cells interact with osteoclasts (bone resorbing cells) and osteoblasts (bone forming cells), thereby inhibiting normal bone development and ultimately leading to bone destruction -4. As foSeveral animal models have been developed to study the mechanisms governing cancer-mediated osteolysis. However, there is no single animal model that ideally replicates the entire metastatic process from primary breast tumor to bone metastasis. Nevertheless, several models that represent various aspects of bone metastasis have been used successfully to study specific features of the disease. For example, Arguello, et al. developed a model in which melanoma cells injected into the left ventricle of the heart ultimately form bone metastases . This moTo address this question, we reanalyzed gene expression profiles generated from our previous studies using the syngeneic mouse model of breast cancer specific osteolysis that was developed by implanting 3 different cell lines - 4T1 (highly metastatic), Cl66 (moderately metastatic) and Cl66-M2 (low metastatic) - onto the calvariae bone of BALB/C mice -15. The ® Mouse Genome 430A 2.0 Array, as described [Mouse breast cancer cell lines - 4T1, Cl66 (66Cl4) and Cl66M2 - with different metastatic potential -18 were escribed .® were processed and MAS 5.0-normalized using the SimpleAffy [2 MAS 5.0-normalized data was used for subsequent analyses. Fold-change at the TB interface with respect to the TA area for tissues, standard deviation (SD) across TA samples, and median-centered analysis within the TA area were calculated for each of the cell lines  to identify genes up- and down-regulated in the respective samples. The genes were ranked from highest to lowest expression based on the values from fold-change or median-centered analysis.The CEL files for all the samples from Affymetrix GeneChipmpleAffy  program mpleAffy . The logThe following publicly available Affymetrix microarray data were obtained from Gene Expression Omnibus (GEO) : GSE1356The NearestTemplatePrediction algorithm (NTP) ,28 was uThe association of gene signature with known pathways was determined using gene ontology (GO) , pathwayGene symbols were mapped to HG-U133A array probes. They were then used to query the Connectivity Map database .Previously, we transplanted three breast cancer cell lines - 4T1 (highly metastatic), Cl66 (moderately metastatic) and Cl66-M2 (low metastatic) - onto the calvarial bone of BALB/C mice ,14. IrreOur reanalysis illustrates that there is little similarity in gene expression in the TA area samples among the three cell lines Figure . This isThe TA area was grown in a non-canonical tumor microenvironment  and as such can be considered a metastatic tumor. Nevertheless, we still expect that the gene expression profile from the TA area (grown near bone) will resemble previously reported profiles for the cell lines (grown in the breast) used in this study, especially given the fact that the primary tumor and its metastatic tumor have been reported to have similar gene expression profiles . To confIn an effort to translate our findings from our mouse breast tumor model to human disease, we compared the gene expression profile from the TA area of our mouse model to that of primary human breast tumors and cancer cell lines using the NTP algorithm. Specifically, we compared microarray data from 118 primary breast tumor samples  to the gIn order to identify genes that are important for the interaction of breast cancer cells with the tumor microenvironment, we reanalyzed the gene expression at the TB interface and compared that profile to the gene expression profile at the TA area for each of the cell lines. Despite the expected heterogeneity in gene expression from cell line to cell line, we were able to identify 934 genes (TB signature) that were consistently different between the TB interface and the TA area. Among these, 359 were up-regulated and 575 were down-regulated with at least a 2-fold change at the TB interface across all the three cell lines. Figure The gene expression profile of the TB interface was identified relative to the TA area, and, as such, should be enriched for transcriptional processes associated with the TB microenvironment. Indeed, three of the top four genes up-regulated at the TB interface  are well established as mediators of bone metastasis ,41,42. TNext, we compared the specificity of our TB specific gene set against that from the normal bone microenvironment. To this end, we used a public gene expression profile containing data for normal mouse calvarial bone, normal mouse ulnar bone and normal mouse mandibular bone (GSE13563). Our TB signature was compared against this data set using the NTP algorithm. As shown in Figure A primary concern with any animal model is whether it accurately represents human disease. To address this, we applied NTP using the TB signature and publicly available gene expression profiles of human breast metastases  . As showde novo analysis showed that TB interface samples significantly (FDR < 0.2) resemble bone metastases samples but not lung or brain samples. TA area samples also do not resemble any of the metastases   based Sus Figure . Furtherin vitro [The Rankl-mediated differentiation of osteoclast precursors (OCPs) to mature osteoclasts is a key step in breast cancer-specific bone metastasis . Since Rin vitro . The ostin vitro . NTP anaTo assess whether mechanisms that govern bone metastasis in humans are also present in our osteolytic model, we performed Gene Ontology (GO) ; pathwayPathways identified using KEGG analysis that were significantly (FDR p < 0.05) associated with our osteolytic model are shown in Table We also performed enrichment analysis of the TB signature using MSigDB canonical pathway database and GlobalTest package ,38. AmonTo predict a therapeutic agent that specifically targets the TB interface, we queried Connectivity Map database  using thAnimal models that faithfully recapitulate aspects of human breast cancer-specific bone metastasis provide powerful tools to study the complex molecular mechanism(s) by which breast cancer cells metastasize to and interact with the bone microenvironment ,48. PrevThere has been tremendous growth in both the development of high-throughput microarray technology to measure gene expression in tissue and cells and in high-dimensional methods to analyze such data ,50. TogeIn the current study, we applied microarray technology to generate a signature specific to the TB interface of our mouse model. The robustness of our TB-signature is supported by the fact that it was derived from a common set of genes regulated at the TB interface across a heterogeneous set of three mouse breast cancer cell lines. Combining gene expression profiling and molecular pathology, we demonstrated that the TB interface of our model truly represents the tumor microenvironment and not the normal bone microenvironment. Subsequent cross-species comparative transcriptomic analysis demonstrated that many human bone metastases samples are associated with the TB interface in a statistically significant manner. Importantly, there was no association between our breast TB interface and human brain or lung metastases. Together, these data demonstrate that our model specifically mimics human breast (and not lung or brain) cancer bone metastases. Furthermore, analysis of a panel of human breast cancer cell lines predicted 16 that have similar gene expression characteristics to those of the 4T1 tumors. This suggests that our osteolytic model may be adapted to study human breast cancer bone metastasis directly using any of these 16 human cell lines.The TGF-β pathway has a well established role in bone metastasis , and preThe canonical and noncanonical Wnt signaling pathways are involved in the formation, growth and development of normal bone  and bonein vitro differentiated osteoclasts (72 h RANKL treatment) was similar to that of the TB interface. In addition, pathway analysis using the MSigDB showed an enrichment of the TB-signature in a myeloid cell line model [Osteoclasts are derived from hematopoietic precursor cells of the myeloid lineage upon CSF-1 stimulation followed by RANKL-mediated maturation . In our ne model   that are traditionally used to treat hypertension and edema . Althougntiation . These dIn summary, we have demonstrated that the TB microenvironment in our mouse model of osteolytic breast cancer metastasis is highly similar to that of human breast cancer-to-bone metastases. Furthermore, gene expression profile analysis of tumors from this model: (i) identified a TB interface specific gene signature; (ii) revealed signaling pathways that were differentially activated at the TB interface and TA area; (iii) demonstrated a role for osteoclasts in metastatic osteolysis; and (iv) predicted a novel therapeutic agent that specifically targets the TB interface. These data clearly demonstrate that this mouse model can be used to study the cellular and molecular mechanisms driving human breast cancer-to-bone metastasis and osteolysis. Moreover, this model also provides a powerful preclinical setting to test thiazides and other therapeutic agents that specifically target breast cancer osteolysis.WJG is currently an employee of Genomic Health.AS conceived and developed the concept, performed all the analyses and wrote the manuscript. MF performed the animal experiments. CAL contributed to the scientific content. CAL and WJG helped with editing of the manuscript. MF and RKS conceived the idea for the development of the animal model. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/304/prepub


The availability of genome sequences for some of the most devastating eukaryotic plant pathogens has led a revolution in our understanding of how these parasites cause disease, and how their hosts respond to invasion Hyaloperonospora arabidopsidis, Phytophthora infestans, and Phytophthora sojaePhytophthora RXLR effectors contain conserved sequence motifs  in their C-terminal domains that often form tandem repeats One expanded family of effector proteins is defined by the sequence RXLR , which in some cases is followed by an acidic-rich dEER motif (Asp-Glu-Glu-Arg) . The RXLPhytophthora capsici), PexRD2 (from P. infestans), and ATR1 (from H. arabidopsidis) Our laboratories have employed structural biology to investigate the molecular basis of RXLR effector function. A total of four structures have recently been published, those of AVR3a4 and AVR3a11 . Using Hidden Markov Model (HMM)-based sequence searches, these motifs had also been detected in H. arabidopsidis RXLR effector proteins, with 35 out of 134 (26%) containing this fold (HMM score>0), including ATR1 with a low confidence score The studies of Boutemy et al. and Chou et al. independently described the structural homology of AVR3a11 and a domain of ATR1, respectively, to the cyanobacterial four-helix bundle protein KaiA The availability of these four oomycete RXLR effector domain structures, from three different pathogens, allows us to present a detailed analysis of the WY-domain fold. Structural overlays of the conserved WY-domains from each of the effectors are shown in Saprolegnia parasiticaAlbugo labachiiPythium ultimumPhytophthora and Hyaloperonospora proteomes searched previously P. infestans>P. sojae>P. ramorum>H. arabidopsidis) also encode a significantly higher percentage of WY-domains in their secretomes  and Hyaloperonospora (obligate biotrophs), but not P. ultimum, it appears that the WY-domain emerged with biotrophy in this lineage along with the evolution of haustoria and RXLR effectors We believe that the conservation of this “flexible” hydrophobic core fold indicates that a large family of plant pathogenic oomycete effectors may have been derived from a common ancestor. Intriguingly, this effector family has rapidly diverged to gain new and/or adapt existing virulence functions and/or evade detection by plant immune systems. New or modified effector functions may be derived from surface point mutations or indels in the connecting regions between helices (including domain duplication). To test the argument for a common ancestor, we extended previous analyses and searched the proteomes of various organisms using the HMM for the WY-domain, as described in Some of the most notorious and agriculturally important pathogenic oomycetes contain RXLR:WY-domain effectors, suggesting that this structure has been critical for the success of these pathogens. This raises questions such as, why has this fold been preserved and what can it tell us about the function of these proteins? How can we use this knowledge to design novel disease management strategies? Future studies will help define the roles of the WY-domain fold in the virulence mechanisms of these pathogens, in particular how it engages with plant cell targets, and will help to unravel the extent of structural diversity in RXLR effectors. Our initial studies have laid the foundation for new, exciting discoveries addressing the function of oomycete effectors.


The role of bisphosphonates (BP) in early breast cancer (BC) has been considered controversial. We performed a meta-analysis of all randomized controlled trials (RCTs) that appraised the effects of BP on survival in early BC.RCTs were identified by searching the Cochrane Library, MEDLINE databases and conference proceedings. Hazard ratios (HRs) of overall survival (OS), disease-free survival (DFS) and relative risks of adverse events were estimated and pooled.Thirteen trials met the inclusion criteria, evaluating a total of 15,762 patients. Meta-analysis of ten trials which reported OS revealed no statistically significant benefit in OS for BP . Meta-analysis of nine trials which reported the DFS revealed no benefit in DFS (HR 0.95 (0.81–1.12)). Meta-analysis upon menopausal status showed a statistically significant better DFS in the BP-treated patients (HR 0.81(0.69–0.95)). In meta-regression, chemotherapy was negatively associated with HR of survival.Our meta-analysis indicates a positive effect for adjuvant BP on survival only in postmenopausal patients. Meta-regression demonstrated a negative association between chemotherapy use BP effect on survival. Further large scale RCTs are warranted to unravel the specific subgroups that would benefit from the addition of BP in the adjuvant setting. Breast cancer is the most common malignancy among women, accounting for nearly 1 in 3 cancers diagnosed and the second leading cause of cancer death among women in the United States In view of the conflicting data from randomized clinical trials, we performed a systematic review of the literature and a meta-analysis of all randomized trials to evaluate the impact of BP administration on survival in patients with early breast cancer in the adjuvant setting.http://www.clinicaltrials.gov and ,http://www.clinicaltrials.nci.nih.gov. The terms (early OR adjuvant) AND (breast OR mammary) AND  AND (biphosphonates OR bisphosphonates OR clodronate OR pamidronate OR zoledronic acid OR ibandronate) were cross-searched. We scanned references of all included trials and reviews identified for additional studies.We searched the Cochrane Central Register of Controlled Trials, published in The Cochrane Library, PubMed (1966 to 4.2012); EMBASE (1974 to 7.2012); LILACS (1982 to 7.2012); the database of clinical trials in non-metastatic breast cancer; conference proceedings of the American Society of Clinical Oncology (1995–7.2012), San Antonio Breast Cancer Symposium (1995–2012), proceedings of the European Society of Medical Oncology (ESMO); and databases of ongoing and unpublished trials: We included randomized controlled trials that compared BP adjuvant therapy in addition to the standard therapy  with placebo in patients with early breast cancer in the adjuvant setting. We included trials regardless of publication status, date of publication, and language. Two authors (IBA and LV) independently inspected each reference title identified by the search and applied the inclusion criteria.Trials that fulfilled the inclusion criteria were assessed for methodological quality by two authors (IBA and LV). Information about randomization and allocation concealment, blinding, sample size, exclusions after randomization, and the length of follow-up were recorded The primary outcome was overall survival, which was defined as time from randomization to death. Secondary outcomes were disease-free survival (defined as the time from randomization to earliest occurrence of relapse or death from any cause), and toxicity .I2 statistic of inconsistency. Statistically significant heterogeneity was defined as P less than .1 or an I2 statistic greater than 50% I2>50%) we used a random-effects model. Potential sources of heterogeneity were explored through stratifying by type of induction therapy , bisphosphonate's schedule and type of BP, allocation concealment, blinding, and size of studies. All statistical tests were two-sided.Hazard ratios (HRs) and variances for time-to-event outcomes were estimated as described by Parmar et al. We explored potential ground of heterogeneity through meta-regression, assessing the effect of percentage of patients receiving chemotherapy in addition to endocrine therapy in each study on the effect estimates for the primary outcome. Meta-regression was performed on the HR . The regression slope with its SE and significance of the model are reported.The literature search identified 490 trials up until 7.2012, of which 96 were considered potentially relevant. Additional trials were identified by searching conference proceedings and electronic resources of ongoing trials. A total of 15,762 patients were randomly assigned in the thirteen trials included in this meta-analysis. Nevertheless, in 3 studies disease free survival has been the primary endpoint with no report of overall survival data In six trials Allocation concealment was reported as adequate in five trials 2I of heterogeneity −43%, random effect model)  were eligible for the meta-analysis of overall survival t model) . Sensitit model) . The fun2I of heterogeneity −61%, random effect model)  were eligible for the meta-analysis of DFS t model) . The fun2I of heterogeneity −34%, fixed effect model)  . Meta-anIn meta-regression , chemothThe rate of grade 3 or 4 adverse events was reported in thirteen trials The results of our meta-analysis demonstrate a positive effect for BP on survival outcomes when administered in the adjuvant setting, only in post-menopausal patients with breast cancer. Meta-regression appraised the effect of confounders such as chemotherapy on the interaction between BP use and overall survival, and showed a negative association between chemotherapy use and the effect of BP on survival.The primary endpoint of many BP trials in the setting of early breast cancer was a reduction in skeletal related events, while survival had not been an integral endpoint of the study. We attempted to avoid publication bias by searching for and including in our meta-analysis conference proceedings, databases of ongoing trials, and unpublished data, and indeed in the recent San-Antonio Breast Cancer Symposium (2011) two large scale trials in-vitro studies have portrayed the effect of a variety of BP (both nitrogen containing and non-nitrogen bisphosphonates) to hinder tumor cell adhesion, invasion, proliferation and interplay with the bone microenvironment components such as matrix-metalloproteinases (MMPs) Bisphosphonates have been proven to display high affinity for bone and to inhibit bone resorption by osteoclasts Two large cohort studies evaluated breast cancer incidence in healthy postmenopausal women treated with bisphosphonates to prevent bone loss and showed a 29–32% statistically significant reduction in the incidence of invasive breast cancer, lending credence for a potential chemo- preventive effect for BP The biological rationale for the benefit of BP in post-menopausal patients lies in the natural estrogen deficiency at postmenopause which leads to increased osteoclast activity, resulting in decreased bone mineral density (BMD), yet is still considered hypothetical. Bisphosphonates have been shown to be effective in numerous studies in preventing cancer treatment-associated bone loss in postmenopausal women with early breast cancer in vitro studies have indicated there may be a synergistic effect between zoledronic acid and letrozole on breast cancer cell lines Moreover, Meta-analysis of the adverse events as reported in the included studies revealed a significant increase in pyrexia as well as osteonecrosis of the jaw (ONJ). Nevertheless, the incidence of ONJ is extremely low  assay (Oncotype Dx) or a microarray of 70-gene expression profile (Mammaprint) identifies subsets of patients with estrogen receptor positive tumors, who are not treated with chemotherapy; it would therefore be of value to elucidate the role of BP in a randomized trial in this population. This design may define clearly the potential benefit of bisphosphonates while the use of chemotherapy would not hamper their potential anti-tumor effect.Our meta-analysis provides a reflection of the new mounting and highly significant data published in the last 3 years which shed new light on the results of a former published meta-analysis on this matter both regarding efficacy and safety of BP in early breast cancer Checklist S1Checklist items according to PRISMA criteria.(DOC)Click here for additional data file.Figure S1Sensitivity analysis of survival according to median follow up duration. Hazard ratios for each trial are represented by the squares, the size of the square represents the weight of the trial in the meta-analysis, and the horizontal line crossing the square represents the 95% confidence interval (CI). The diamonds represent the estimated overall effect based on the meta-analysis random effect of all trials.(TIFF)Click here for additional data file.Table S1Quality assessment of included studies according to Cochrane criteria. Quality assessment: Low risk-adequate  Unclear – not reported.(PDF)Click here for additional data file.


The pathogen is strictly biotrophic, and thus is completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. Therefore, we have applied a cDNA- amplified fragment length polymorphism (AFLP) approach to analyze gene expression in Mexican lime trees infected by "Ca. Phytoplasma aurantifolia".We carried out cDNA-AFLP analysis on grafted infected Mexican lime trees of the susceptible cultivar at the representative symptoms stage. Selective amplifications with 43 primer combinations allowed the visualisation of 55 transcript-derived fragments that were expressed differentially between infected and non-infected leaves. We sequenced 51 fragments, 36 of which were identified as lime tree transcripts after homology searching. Of the 36 genes, 70.5% were down-regulated during infection and could be classified into various functional groups. We showed that Mexican lime tree genes that were homologous to known resistance genes tended to be repressed in response to infection. These included the genes for modifier of snc1 and autophagy protein 5. Furthermore, down-regulation of genes involved in metabolism, transcription, transport and cytoskeleton was observed, which included the genes for formin, importin β 3, transducin, L-asparaginase, glycerophosphoryl diester phosphodiesterase, and RNA polymerase β. In contrast, genes that encoded a proline-rich protein, ubiquitin-protein ligase, phosphatidyl glycerol specific phospholipase C-like, and serine/threonine-protein kinase were up-regulated during the infection.Candidatus Phytoplasma aurantifolia". These results should help to elucidate the molecular basis of the infection process and to identify genes that could be targeted to increase plant resistance and inhibit the growth and reproduction of the pathogen.The present study identifies a number of candidate genes that might be involved in the interaction of Mexican lime trees with " This is a devastating disease that results in significant economic losses [Ca. Phytoplasma aurantifolia" is transmitted by the leafhopper Hishimonus phycitis (Hemiptera: Cicadellidae) [c losses . Phytoplc losses ,3. It haellidae) .The mechanisms that regulate the distribution of phytoplasmas in the host tissue is still widely unknown. At the tissue level, phytoplasma infection can cause anatomical aberrations such as extensive necrosis the phloem and phloem tissue proliferation, which results in swollen veins . ReducedCatharanthus roseus L.) [Ca. Phytoplasma aurantifolia". Understanding the basis of susceptibility to the pathogen will assist greatly in the development of new control strategies and the identification of pathogen and host factors that are required for disease progression.The molecular details of the infection process are largely unknown. Initial details were obtained from studies of phytoplasma/plant interactions with respect to polyphenol production and the transport of sugar and amino acid and comprehensive differences in gene expression have reported mainly in the experimental host plant periwinkle (seus L.) ,11. Howeseus L.) . In thisU15442), with a pattern similarity coefficient of 0.99. Therefore, the phytoplasma under study was a variant of 16SrII-B and related to "Ca. Phytoplasma aurantifolia".Five months after grafting healthy Mexican lime trees, plants developed the typical symptoms of witches' broom Figure . The resFor each of the 43 primer combinations, 40-100 different transcript derived fragments (TDFs), which ranged from 50 to 800 bp, were visualized as bands Figure .Analysis of the expression profiles of the infected and noninfected samples between replicates revealed 55 differentially expressed TDFs (DE-TDFs) that showed the same pattern in all replicates. Fifty-one of these DE-TDFs were isolated and sequenced. The remaining four DE-TDFs could not be cloned and were excluded from analysis. Out of the 51 sequenced DE-TDFs, 36 showed similarity to known gene sequences in databases  could be assigned to one of the following functional groups: stress response/defense (10 TDFs), cell Metabolism (4 TDFs), protein synthesis/destination (4 TDFs), signal transduction (3 TDFs), transporter (2 TDFs), transcription (1 TDFs), cytoskeleton (1 TDFs) and unknown function (11 TDFs). The molecular function of each individual protein is given in Table To verify the expression patterns that were identified in the cDNA-AFLP study, the expression level of four DE-TDFs was analyzed by real-time RT-PCR. These TDFs were chosen to represent the various functional categories identified Table . Two of Ca. Phytoplasma aurantifolia" by using cDNA-AFLP technique. For this analysis, we used leaf samples from healthy controls and infected plants at the symptomatic stage. The symptomatic stage was chosen because the plant/pathogen interaction is well established but the plant cells are still active and can maintain pathogen survival.In this study, we performed a comparative transcriptomic analysis of healthy Mexican lime trees and those infected by "Ca. Phytoplasma aurantifolia" and Mexican lime trees. We observed transcriptional changes that affected the expression of several genes related to physiological functions that would affect most leaves in infected tissues.As far as we are aware, our study is the first gene expression analysis of the compatible interaction between "The cDNA-AFLP method for global transcriptional analysis is an open architecture technology that is appropriate for gene expression studies in non-model species. This is because prior sequence data are not required for the visual identification of differentially-expressed transcripts, in contrast to other approaches.Sixty-seven percent of the identified DE-TDFs were down-regulated in response to infection, whereas only 33% were up-regulated in response to infection which could reflect the exploitation of cellular resources and the suppression of defence responses by the phytoplasma .Ca. Phytoplasma aurantifolia" were related to defence, cell walls, and response to stress. The expression of autophagy protein 5 was repressed. Autophagy is a survival mechanism that protects cells against unfavourable environmental conditions, such as microbial pathogen infection, oxidative stress, nutrient starvation, and aggregation of damaged proteins [Several genes that were modulated in Mexican lime trees by infection with "proteins . It has proteins  and stimproteins . Biochemproteins . It is l2O2-mediated oxidative cross-linking of preexisting structural cell wall proteins that precedes the activation of transcription-dependent defences. The induction of the hydroxyl proline-rich protein in the present study might reflect a defence mechanism of Mexican lime tree in response to phytoplasma infection.We also identified a cell wall hydroxyl proline-rich protein (GT222039) that was induced in response to the pathogen. Proline-rich proteins are among the major structural proteins of plant cell walls. Environmental stresses can alter the composition of the plant cell wall markedly . It has Another induced protein (GT222056) contained a lysine domain that is found in several enzymes that are involved in degradation of the bacterial cell wall . The rolTwo of repressed genes (GT222036 and GT222036) were identified as a modifier of snc1 (MOS1). Plant resistance (R) genes encode immune receptors that recognise pathogens directly or indirectly and activate defence responses . The expIt has been shown that mos1 mutations reduce the expression of endogenous snc1, which results in the repression of constitutive resistance responses that are mediated by snc1 . It is lCa. Phytoplasma aurantifolia" infections involve the up-regulation of genes that encode components of the lipid metabolism pathway, such as phosphatidyl glycerol specific phospholipase C-like (GT222018). This enzyme regulates the phosphatidylglycerol content via a phospholipase C-type degradation mechanism [Lipid-derived molecules act as signals in plantpathogen interactions, and the roles of jasmonic acid and related oxylipins that are produced from membrane-derived fatty acids through beta-oxidation, are particularly important . During echanism . Anotherechanism .We identified several TDFs that were related to protein metabolism in our study. Among these were genes that encoded ribosomal proteins and enzymes involved in degradation. The expression of two ubiquitin-protein ligases (GT222065 and GT222065) and one 50 S ribosomal protein L15 GT222023) were repressed, whereas another 50 S ribosomal protein L15 (GT222024) was induced. This suggests that the infection results in a general induction of protein turnover, which could reflect an adaptive response in the plants to remove misfolded proteins that have accumulated as a result of stress [ were repThree of the modulated genes had signal transduction and/or gene regulation functions. They corresponded two transducin family protein (GT222030 and GT222029) that were repressed by infection and a serine/threonine protein kinases (GT222061) that was induced during infection. Serine/threonine protein kinases are a group of enzymes that catalyse the phosphorylation of serine or threonine residues in proteins, with ATP or other nucleotides acting as phosphate donors. The phosphorylation of proteins on serine, threonine, or tyrosine residues is an important biochemical mechanism to regulate the activity of enzymes and is used in many cellular processes .The two down-regulated proteins were identified as members of the transducin family and contained WD40 domain. This domain is found in several eukaryotic proteins that with wide variety of functions, which include adaptor/regulatory modules in signal transduction, together with proteins involved in pre-mRNA processing, and cytoskeleton assembly . It is uCa. Phytoplasma aurantifolia". The cDNA-AFLP technique allowed several novel genes to be identified from Mexican lime trees, because a significant proportion of the TDFs are not currently represented in citrus databases. Our data showed that infection resulted in the down-regulation of Mexican lime tress transcripts in all major functional categories. However, certain genes that were required for plant-pathogen interactions were modulated positively during infection at the symptomatic stage. These results will serve as a basis to address new questions and design new experiments to elucidate the biology of plant-phytoplasma interactions and the associated re-programming of the host metabolism. They might also pave the way to identify genes that can be targeted to elevate plant resistance or inhibit the growth and reproduction of the pathogen. However, further research is required to elucidate the roles of these genes in the susceptibility/resistance of Mexican lime tress to "Ca. Phytoplasma aurantifolia", and to determine how strategies might be developed to incorporate these genes into molecular breeding programmes.We believe that this study is the first reported analysis of the expression of genes involved in the interaction of Mexican lime trees with "Ten healthy 1-year-old Mexican lime trees grown in the greenhouse were used in this experiment. Specimens from Mexican lime trees infected with witches' broom were grafted to healthy trees, and specimens from healthy Mexican lime trees were grafted to other healthy trees. The grafted plants were covered for 1 month with plastic bags to increase humidity and were arranged randomly on the greenhouse bench. They were kept under natural light conditions at a temperature of 25-28°C. The branches infected with witches' broom were sampled 20 weeks after inoculation and used for RNA extraction. As a control, RNA was extracted from non-grafted healthy plant leaves that has been grown under similar conditions.et al [et al [Total DNA was extracted from leaf samples (vascular tissues from leaf veins and petioles) using the method described originally by Daire et al  with somet al . Sampleset al  and P7  in accordance with the manufacturer's instructions. cDNA synthesis and cDNA-AFLP analysis were performed for the 10 replicates. First-strand cDNA was synthesised from 2 μg of total RNA using a SuperScript III First Strand Synthesis System  in accordance with the manufacturer's instructions. Second-strand cDNA was sythesised by adding the first-strand cDNA reaction to a reaction mix that contained 15 μl of 10 × cDNAII buffer, 35 U DNA of Polymerase I (Invitrogen), 3 U of RNase H (Invitrogen), and 1 μl dNTPs (25 mM) in a final volume of 150 μl, and incubating for 2 h at 16°C . The resulting double-stranded cDNA was purified in accordance with the method of Powell and Gannon . The conet al. [EcoRI/MseI and PsuI/MseI (Invitrogen). The Sequence of the primers and adapters used for the AFLP reactions are given in Additional File et al. [A 500-ng aliquot of double-stranded cDNA was used for AFLP analysis as described by Bachem et al.  with thee et al. . Selectie et al. . The gelTo select DE-TDFs, the profiles of infected and non-infected samples were compared between replicates. TDFs that differed in abundance between the two types of sample, namely infected and non-infected plants, were selected only when the same pattern was observed in all replicates.The cloning of bands of interest was performed as previously described. BrieflyAfter verification, the recovered products re-amplified using primer pair E-0/M-0 and P-0/M-0 to provide sufficient DNA for cloning. The purified PCR products were cloned into the pGEM-T Easy vector (Promega) and then sequenced.http://www.ncbi.nlm.nih.gov/BLAST/) and The Arabidopsis Information Resource  using the Blastn, Blastx, and tBlastx sequence alignment algorithms [The sequences were compared with those in the non-redundant databases of the National Center for Biotechnology Information  participated in sequence analysis, performed the real-time RT-PCR experiments, and contributed to data interpretation and manuscript writing. MM participated in in the analysis and interpretation of cDNA-AFLP data. SMA participated in plant sample preparation. NHZ, HRZ, and AA participated in sequence analysis, in interpretation of data, in automatic and Gene Ontology assignment. GHS conceived the study, participated in its design and coordination, participated in interpretation of the data, in manual ontology assignments and wrote the manuscript. All authors read and approved the final manuscript.Ca. Phytoplasma aurantifolia" and from healthy plantsAgarose gel electrophoresis of nested PCR product from Mexican lime tree infected by ".Click here for filePrimer sequences used for cDNA AFLP analysis.Click here for filePrimer sequences used for Real-Time PCR analysis.Click here for file