The standard of care for colorectal peritoneal carcinomatosis is evolving from chemotherapy to cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) for patients with disease limited to the peritoneum. Peritoneal carcinomatosis from colorectal cancer treated with chemotherapy alone results in median survival of 5 to 13 months, whereas CRS with HIPEC for early peritoneal carcinomatosis from colorectal cancer resulted in median survival of 48-63 months and 5 year survival of 51%.Completeness of cytoreduction and limited disease are associated with longer survival, yet early peritoneal carcinomatosis is undetectable by conventional imaging. Exploratory laparotomy can successfully identify early disease, but this approach can only be justified in patients with high risk of peritoneal carcinomatosis. Historical data indicates that patients presenting with synchronous peritoneal carcinomatosis, ovarian metastases, perforated primary tumor, and emergency presentation with bleeding or obstructing lesions are at high risk of peritoneal carcinomatosis. Approximately 55% of these patient populations will develop peritoneal carcinomatosis. We hypothesize that performing a mandatory second look laparotomy with CRS and HIPEC for patients who are at high risk for developing peritoneal carcinomatosis from colorectal cancer will lead to improved survival as compared to patients who receive standard of care with routine surveillance.This study is a prospective randomized trial designed to answer the question whether mandatory second look surgery with CRS and HIPEC will prolong overall survival compared to the standard of care in patients who are at high risk for developing peritoneal carcinomatosis from colorectal cancer (CRC). Patients with CRC at high risk for developing peritoneal carcinomatosis who underwent curative surgery and subsequently received standard of care adjuvant chemotherapy will be evaluated. The patients who remain without evidence of disease by imaging, physical examination, and tumor markers for 12 months after the primary operation will be randomized to mandatory second look surgery or standard-of-care surveillance. At laparotomy, CRS and HIPEC will be performed with intraperitoneal oxaliplatin with concurrent systemic 5-fluorouracil and leucovorin. Up to 100 patients will be enrolled to allow for 35 evaluable patients in each arm; accrual is expected to last 5 years.ClinicalTrials.gov ID: NCT01095523 In the United States, approximately 108,070 patients are diagnosed with colon cancer and 40,740 patients with rectal cancer per year  . 214  and hyperthermic intraperitoneal chemotherapy (HIPEC) -13. The Verwaal et al  performeUnfortunately, PC is usually detected late in the course of disease secondary to late presentation of symptoms and difficulty with detection based upon imaging techniques and tumor markers. Once symptomatic disease develops, complete resection is more difficult to achieve. The only reliable means of detection is repeat laparotomy, but this operation cannot be justified in all patients with CRC. High risk group of patients who may benefit -28 have Given the impact on survival with CRS and HIPEC, Elias et al.  performeChemotherapeutic drugs used for intra-operative management are based on the most effective systemic regimens which also demonstrate good results regarding intraperitoneal delivery. Systemic oxaliplatin has demonstrated significant activity against metastatic CRC with an objective response rate of 24% as a single agent and 55% in combination with 5-FU and leucovorin (FOLFOX) ,29,30. FIn summary, for approximately 50% of patients who develop PC from CRC, a complete resection may be potentially curative. Patients with high-risk primary CRC have an incidence of PC greater than 50%, yet modern imaging and tumor or serum markers fail to detect recurrence at an early stage. We hypothesize that MSLS with CRS and HIPEC in patients who are at high risk for recurrent PC will lead to improved survival compared to standard surveillance. Therefore, a prospective randomized trial has been designed to evaluate MSLS with CRS and HIPEC versus treatment of PC once disease is detected, during routine surveillance, by imaging or symptoms.The Mandatory Second Look Surgery (MSLS) for high Risk Colorectal Cancer trial was approved by the Institutional Review Board (IRB) of the National Cancer Institute, National Institutes of Health, Bethesda, Maryland.This study is a randomized controlled trial. The trial schema is illustrated in Figure Registration of patients onto this study will take place within 24 hours of the patient signing the consent by faxing a completed eligibility checklist to the Central Registration Office. This trial will consist of three stages: Lead-in Stage includes the time period from 3 months to 14 months following resection. Patients who present for evaluation prior to month 11 post resection will be enrolled in this stage prior to randomization. Stage 1 includes MSLS with HIPEC and CRS and the recovery period. Patients who are randomized to receive surgical intervention will be entered on stage 1. Stage 2 is the follow up period. Patients who are randomized to surveillance will be directly entered on stage 2; patients who are randomized to the MSLS arm will be entered on stage 2 upon recovery from the operative procedure. After 11-14 months following primary resection, the patients will be stratified and randomized by the Central Registration Office to undergo MSLS with HIPEC and CRS or standard-of-care surveillance (SC) without surgery. The Central Registration Office will notify the study coordinator of the results of randomization. Patients must be randomized within 2 weeks of the baseline imaging studies. Patients who are randomized to the MSLS arm must be treated within 2 weeks of randomization.Patients will be stratified according to two factors: 1: Presenting with PC, which was completely resected, and/or ovarian metastases (incidence of recurrent PC > 60%) vs. presenting with tumor perforation, T4 lesions that required adjacent organ resection, and/or emergency presentation with bleeding or obstructing lesions (incidence of recurrent PC < 40%), and 2: Receiving adjuvant chemotherapy for less than 6 months vs. > 6 months. This will generate 4 strata.The primary objective of the trial is to determine if there is a difference in overall survival among patients who are at high risk for CRC PC and are randomized to receive either SC alone or MSLS with HIPEC and CRS when indicated.Based upon results in the literature, patients who would be eligible to be randomized on this trial and who receive SC alone would be expected to have an estimated 25% 5-year survival from the date of randomization. The goal of this study will be to determine if the use of a MSLS with HIPEC and CRS will result in patients having survival that is associated with an increase to 50% 5-year survival. Following registration, patients who are eligible for randomization will be randomized between SC and MSLS with HIPEC and CRS and followed for survival. Patients will be stratified for synchronous peritoneal carcinomatosis and ovarian metastasis vs. perforated primary and emergency with bleeding or obstruction, and prior chemotherapy ≤ 6 months vs. >6 months. Kaplan-Meier curves and a two-tailed log-rank test will be the primary analysis methods. Assuming exponential survival curves, the hazard rate for the systemic therapy is 0.0231, or approximately a 2.3% probability of dying each month when the 5-year survival probability is 25%. If we assume that the MSLS arm will have an associated survival of 50% at 5 years, this survival corresponds to a hazard rate of 0.0116, and the resulting hazard ratio for the comparison of the two overall survival curves would be 1.991. Following the principles of a phase 2.5 design, to compare these curves and detect a difference with a 0.10 one-tailed log-rank test, a total of 35 evaluable subjects per arm  will need to be randomized over a five year period, followed for an additional two years from the date of entry of the last patient, and observation of 38 total deaths in order to have 80% power to compare the curves.It is expected that 80% of registered patients will be eligible for the randomization. Thus, in order to enroll 70 randomized patients, a total of up to 100 evaluable patients may be enrolled. The patients who present prior to 11 months will be enrolled in the lead-in stage in which some patients may develop symptomatic or image-detected disease. These patients will not be eligible for randomization, but the number of randomized patients relative to enrolled patients will be noted in order to determine the denominator of patients who develop peritoneal recurrence within the first year of treatment. Registration will be permitted to continue until 70 patients have been randomized, and thus it is possible that up to 5-10 additional patients beyond 70 may be randomized. This will permit a very small increase in power. Patients will be analyzed as randomized; only patients found to be grossly inevaluable for any reason will not be included in the final analysis once randomized.Progression-free survival will also be evaluated using Kaplan-Meier curves and a two-tailed log rank test, as a secondary endpoint. In addition, a prognostic factor evaluation using Cox proportional hazards modeling will take place after the study has concluded in order to identify if there are factors which can be identified that are associated with overall or progression-free survival in patients randomized to treatments on this trial; this analysis will be interpreted as a secondary endpoint.It is expected that 20 patients per year can be accrued onto this trial, and thus accrual will be completed in approximately 5 years. Allowing for a very small number of inevaluable patients, the accrual ceiling will be set at 100 patients in order to permit 95 registrations and 70-75 randomized patients.Careful evaluation to ascertain the toxicity and clinical response will be performed. The principal investigator will monitor the data and toxicities in order to identify trends quarterly. The principal investigator will be responsible for revising the protocol as needed to maintain safety. The NCI IRB will review submitted adverse events monthly to evaluate trends and will require a follow-up plan from the principal investigator whenever a trend is identified.The study will be monitored by the NCI/CCR Data Safety and Monitoring Board (DSMB) on an annual basis to evaluate the safety of the two arms. The serious adverse events  will be reported according to type of toxicity, maximal grade noted per patient, and for toxicities with at least a possible attribution to the therapy provided on that arm. Comparisons will be made between the two arms using Cochran-Armitage tests for trend, or other appropriate methods, to determine if there is increased toxicity associated with either arm.Beginning at the first annual DSMB meeting after 35 patients have been randomized, annual interim evaluations will be performed to determine if there is sufficient evidence to terminate accrual because of a better than expected improvement in survival.• Histologically confirmed colorectal adenocarcinoma.• Curative resection (NED at closure) for CRC which was (1) perforated into the peritoneal cavity, (2) associated with minimal PC which was completely excised at the time of initial operation, (3) T4 lesion that required en bloc resection of additional organs, (4) associated with ovarian metastases or (5) emergency presentation with lesions associated with obstruction and/or bleeding.Note: Patient who presented at the time of diagnosis with limited extra abdominal metastases may be eligible if the lesions were completely resected and the patient remains NED.◦ • Received at least 3 months of standard of care adjuvant therapy and is disease free by conventional imaging at time of registration and randomization.Note: The imaging will be reviewed by an experienced radiologist and a surgical attending prior to enrollment.◦ • Greater than or equal to 18 years of age.• Clinical performance status of ECOG ≤ 2.• Stable serum CEA levels not indicating recurrence.• No history of prior/other malignancies within the 2 years prior to enrollment with the exception of basal cell carcinoma.• Active systemic infections, coagulation disorders, or other major medical illnesses precluding major surgery.• Prior experimental therapy with novel agents.• Prior HIPEC.• History of brain metastases.• Childs B or C cirrhosis or with evidence of severe portal hypertension by history, endoscopy, or radiologic studies or with evidence of moderate to severe ascites.• Weight less than 40 kg.• History of congestive heart failure and/or an LVEF < 40%.• Significant COPD or other chronic pulmonary restrictive disease with PFT's indicating an FEV1 less than 50% or a DLCO less than 40% predicted for age.All patients randomized to the MSLS arm will undergo exploratory laparotomy at the Surgery Branch, NCI. The overall goal for surgical therapy is to determine whether clinically occult metastases exist and to render the patient NED with negative margins if metastases are detected. All previous dissection planes should be open as feasibly and safely as possible which requires an exploratory laparotomy because most recurrences detected at sites of prior operative therapy that often have adhesions preventing laparoscopic evaluation of these areas. Determination of NED status will be based upon visual inspection, frozen biopsy of any suspicious lesions, and ultrasound of liver with biopsy as indicated. Peritoneal washings will be performed in all patients. Sixteen16 peritoneal biopsies will be done - 4 from each quadrant. Data from these investigations will be used at the final analysis of the risks and benefits of this approach. Pre- and post-operative peritoneal disease will be recorded using the PCI and the Gilly's methodology ,36. If a2). Stable perfusion parameters are obtained and the peritoneal cavity is warmed to a minimum of 41°C prior to starting the clock for perfusion time and a maximum of 43°C. The perfusion will be continued for 30-35 minutes. During the perfusion, constant physical manipulation of the abdomen (shaking) will be maintained for the entire perfusion period to assure even distribution of the perfusate. The heater coil will be maintained at 46-48°C. Peritoneal temperature will be measured continuously by four probes placed immediately beneath the peritoneal surface on either side of the abdomen and in the pelvis. The patient's core temperature will be measured with an esophageal probe (which correlates well with pulmonary artery temperatures) and maintained at less than 41°C using a cooling blanket and ice packs around the legs and head. At the end of the perfusion, the abdomen will be re-opened and the perfusate irrigated from the abdominal cavity. All intraperitoneal drug dosages will be calculated on ideal body weight. 5-FU (400 mg/m2) and leucovorin (20 mg/m2) will be administered intravenously 15 minutes prior to HIPEC to potentiate the activity of oxaliplatin (460 mg/m2 diluted in 2.0 L/m2 of D5W via the perfusion circuit).For the HIPEC procedure, two large bore catheters (thoracostomy tubes) will be inserted through the abdominal wall incision, one over the right lobe of the liver and one in the pelvis. The abdominal skin will be closed and the catheters connected to a perfusion circuit. The perfusate passes from a reservoir through a roller pump, heat exchanger, and then into the abdominal cavity. Efflux from a second catheter is then recirculated through the reservoir and pump. The perfusion flow rate will be maintained at 2.0 L/min and a perfusate volume will be maintained which moderately distends the abdominal cavity correlating with intra abdominal pressures of 5 to 15 mm Hg  vs. similar patients who receive standard of care surveillance.• To determine recurrence-free survival from the time of randomization in both arms.• To investigate selection criteria for patients who might benefit from MSLS with HIPEC and CRC.• To determine the percentage of patients who are diagnosed with recurrent peritoneal disease (gross vs. microscopic) at exploration.• To determine the percentage of patients with recurrent disease at MSLS who are successfully debulked to R0.• To determine the rate of clinical detection of PC during the pre-randomization period and thereby determining the denominator for this cohort of patients.rd most common malignancy in the United States accounting for over 100,000 new cases per year [Colorectal cancer is the 3per year . Despiteper year . CRS andper year ,13,23. TThis trial tests the strategy of mandatory second-look laparotomy for patients at high risk of recurrent peritoneal carcinomatosis versus the current standard-of-care of routine surveillance. The high risk patients previously have been defined as having limited and resected peritoneal disease at initial operation, ovarian metastases, tumor perforation, T4 lesions with en bloc adjacent organ resection, and emergency presentation with bleeding and obstruction. Given that this patient population has high risk of peritoneal recurrence that translates into very poor survival, a strategy as invasive as an mandatory exploratory second-look surgery is justified to determine its impact on survival, yet this proposal should be subjected to rigorous scientific scrutiny prior to implementation outside of a clinical trial. If the primary objective of increasing overall survival is met, this trial may change the current management of patients at high risk for developing recurrent peritoneal carcinomatosis to one of aggressive, early, surgical intervention with strong evidence to show that patients will benefit from this strategy. Hopefully, this treatment will significantly increase long-term survival among patients with peritoneal carcinomatosis.The authors declare that they have no competing interests.IA is the principal investigator for the study described in the manuscript. RTR and IA are responsible for the concept and design. RTR, JLD, CDK, SMS, MAT, and IA made significant contributions to protocol validity, design, drafting, and revising of the manuscript. SMS developed the statistical considerations for the trial. RTR, JLD, CDK, SMS, MAT, and IA contributed to the scientific accuracy of the manuscript. IA gave the final approval for the final version to be published.


A major problem in the chemotherapy of colorectal cancers is their resistance to most cytotoxic drugs which may be due to insufficient cellular transport. Drugs conjugated to monoclonal antibodies recognising tumour antigens may overcome these difficulties by providing access of active agents to the tumour cells. The anti-tumour monoclonal antibody shown to localise in patients with colorectal cancer, 791T/36, has been investigated as a potential targeting antibody. Eight cell lines were established from surgically resected material and were shown to bind 791T/36 antibody. They were screened for their sensitivity to methotrexate, 5-fluorouracil and daunomycin. Although 5-fluorouracil is the drug of choice for chemotherapy of colorectal cancer it was the most cytotoxic drug in only 2 of the 8 cell lines. Only the 4 cell lines which were resistant to methotrexate showed less cytotoxicity with methotrexate than 5-fluorouracil. The cell lines which were resistant to methotrexate were more sensitive to 791T/36-methotrexate conjugates. Daunomycin was the most cytotoxic drug in 4 of the 8 cell lines. However, a similar cytotoxicity was observed for free drug and 791T/36 daunomycin in the two lines tested. Selective monoclonal antibody drug conjugates may offer a solution to treatment of tumours which are resistant to classical chemotherapeutic agents. This is the first report to show that newly established cell lines that are resistant to classical chemotherapeutic agents are rendered sensitive when the drug enters the cell as a drug monoclonal antibody carrier.


Bmal1 and that these rhythms can be rescued by virally-mediated restoration of Bmal1 expression in the dorsomedial nucleus of the hypothalamus (DMH) but not in the suprachiasmatic nucleus (site of the master light-entrainable circadian pacemaker). These results, taken together with controversial DMH lesion results published by the same laboratory, appear to establish the DMH as the site of a Bmal1-dependent circadian mechanism necessary and sufficient for food anticipatory rhythms. However, careful examination of the manuscript reveals numerous weaknesses in the evidence as presented. These problems are grouped as follows and elaborated in detail: 1. data management issues , 2. failure of evidence to support the major conclusions, and 3. missing data and methodological details. The Fuller et al results are therefore considered inconclusive, and fail to clarify the role of either the DMH or Bmal1 in the expression of food-entrainable circadian rhythms in rodents.Daily feeding schedules generate food anticipatory rhythms of behavior and physiology that exhibit canonical properties of circadian clock control. The molecular mechanisms and location of food-entrainable circadian oscillators hypothesized to control food anticipatory rhythms are unknown. In 2008, Fuller et al reported that food-entrainable circadian rhythms are absent in mice bearing a null mutation of the circadian clock gene  Circadian rhythms in mammals are regulated by a master circadian pacemaker located in the suprachiasmatic nucleus (SCN) ,2. This st century were limited to a few laboratories, and yielded primarily negative findings. With the advent of powerful new molecular biological techniques, and recognition of the importance of food as a time-cue for circadian oscillators outside of the SCN [Bmal1, and are rescued by virally-mediated restoration of Bmal1 expression selectively in the DMH (but not in the SCN). The two studies appear to establish that the DMH contains Bmal1-dependent circadian oscillators that are both necessary and sufficient for the expression of food-entrainable behavioral and temperature rhythms in rodents.Efforts to localize food-entrainable circadian oscillators for behavior began over 30 years ago, but until the turn of the 21 the SCN ,10, more the SCN  reported the SCN  exploiteThese studies potentially constitute a seminal demonstration of localization of function in the mammalian brain. However, despite considerable effort, other laboratories, using rats or mice, have so far been unable to confirm either the lesion or the gene knockout results -18. ConsA critical task for peer reviewers is to evaluate whether the evidence presented in a new study supports the authors' substantive conclusions. Before undertaking this task, the reviewers must have confidence that the evidence has been presented both fully and accurately. Accuracy is generally assumed, unless there are clear indications  that errors may have occurred. Inspection of the figures provided in Fuller et al  suggestsBmal1-/- mouse by adeno-associated viral (AAV)-BMAL1 injection into the DMH. Figure S3B in the original supplementary materials was another double-plot of body temperature intended to illustrate failure of recovery of food anticipation following injection of AAV-BMAL1 into the SCN. However, the two double-plots . Fig. S3D, in which the trace is derived from the data shown in fig. S3B, was also incorrect." The unidentified 'error' presumably refers to the mismatch between the two figures. It is not clear how the duplicate plots could appear to be identical except for one critical segment immediately preceding mealtime. This could be a peculiarity of the algorithms used to generate the 'actogram' style plots, but neither the original paper nor the supplementary materials provide information on the plotting conventions of this software. Regardless, the fact that a significant misalignment occurred raises concerns about the accuracy of other figures in the paper.1a. The Fuller et al  paper wa1b. Careful examination of the other 'actograms' and average waveforms in Fuller et al suggest that other alignment errors may well have occurred. One striking indicator of possible errors is the unusual direction and rapidity of change of body temperature in several of the waveforms. According to the figure legends, Figure two and corrected Figure S3 depict waveforms of body temperature averaged over days 10–14 of 4-h/day restricted feeding. Figures two A and C Fig.  here and1c. Data misalignment in Fuller et al is also indicated by mismatches between waveforms and actogram-style plots. In Figure S3 (the Corrected version on-line) the waveforms depicted in panels three C and D Fig.  here are'circadian rise in body temperature during the presumptive dark cycle, CT12-24', in reference to Figure two C in the main text). However, the published literature on food-restricted mice in DD  indi indi'cirThe preceding analysis raises legitimate concerns about data management in Fuller et al . In the Bmal1-/- mice selectively restored Bmal1 expression in these structures, and selectively rescued light-entrainable and food-entrainable circadian rhythms, respectively (a double dissociation). To demonstrate that restoration of Bmal1 expression was indeed spatially restricted to the target structure, it is necessary to provide autoradiographs of Bmal1 expression in one or more whole coronal sections of the brain, showing expression in the target structure and no expression outside of this structure in brain regions that normally express Bmal1 in wildtype mice at that phase of the DMH rhythm. Figure S4 in Fuller et al shows Per1 expression in whole brain coronal sections, but the autoradiographs provided to illustrate Bmal1 expression in AAV-BMAL1 mice were cropped to include only the target area , and very different from comparable autoradiographs in the original Bunger et al [Bmal1 knockout study. The autoradiographs therefore appear at odds with the claim in the text that Bmal1 expression in this mouse was limited to the DMH.Given the authors' claim that  S4 Fig.  here arehe brain . HoweverS4G Fig.  here, ider et al Bmal1 knoBmal1-/- mice were rescued by intra-DMH injection of AAV-BMAL1. However, the 'actogram' style double-plot of body temperature that is provided as evidence of functional rescue  the temperature scale ranges from 34–38°C degrees, while in Figure two B (null mutant) the temperature scale ranges from 30–38°C. Similarly, in Fuller et al's Figure S3A (heterozygote) the temperature scale ranges from 35–38°C, while in Figure S3D (null mutant) it ranges from 31–37°C. This has the effect of compressing the waveform in the Bmal1-/- example, reducing the apparent slope of a regression line drawn through the temperature waveform prior to mealtime. If the scales are made equivalent, the waveforms look more similar. The body temperature waveforms in these Bmal1-/- examples may have more ultradian variation, but body temperature clearly is rising over the 1–4 hours preceding mealtime. Moreover, as discussed in Point 1c above, there are strong indications that the waveform in Fig. S3D  requiring us to arouse them by gentle handling after presentation of the food to avoid their starvation and death during restricted feeding". Hungry mice (even if asleep) will arouse and orient immediately when the door of their isolation chamber is opened at mealtime to place food in the cage. A mouse that has to be physically handled to be aroused is very likely either sick or profoundly hypothermic (or both). In the original text, the authors do not report body weight or food intake data. In their Reply [Bmal1-/- mice did not lose body weight, then why did they exhibit bouts of hypothermia severe enough to prevent them from arousing spontaneously when food was placed in the cage?These concerns appear to be warranted. Fuller et al report that ir Reply  to the Tir Reply , the autBmal1-/- mice are somehow protected from this effect, two laboratories have independently collected food intake and body weight data from wildtype and Bmal1-/- mice fed according to the procedure of Fuller et al . In one lab , the null mutants lost significant weight when the food was placed on the cage tops during two days of ad-lib food access . Evidently, even without food restriction, Bmal1-/- mice have physical limitations that may impair their ability to reach food available in standard cage top food hoppers. Food was therefore placed on the floor for 5 additional baseline days and 4 days of food restriction (4 h/day). A 25% weight loss 'endpoint' criterion was established for termination of the experiment, to prevent death. Three of 5 Bmal1-/- mice reached the 25% weight loss criterion on day 3 of restricted feeding and one reached it on day 4. Weight loss in the remaining Bmal1-/- mouse was 18% on day 4. Weight loss in 6 wildtype mice averaged 7.5% after one day of restricted feeding, and 9% after 4 days.There are numerous studies in the literature showing that wildtype mice and rats typically lose weight when food restricted, particularly when the duration of food access is 4 h or less. To evaluate whether Bmal1-/- and 5 wildtype mice, of various ages, with body weights in the 22–28 gm range. These mice were maintained in breeder cages with corn cob bedding and nesting material, in an ambient temperature of 22.5°–25.5°C. Food was placed on the cage tops within 4.5 cm of the floor. When food was abruptly restricted to 4 h/day for 4 days, wildtype mice remained within ± 2% of starting weight, while the two Bmal1-/- mice lost 8% and 9% body weight, respectively. The second set consisted of 7 Bmal1-/- mice and 6 age-matched (5–8 weeks) wildtype mice. These mice were housed in standard recording cages with locked running wheels, in DD and 22 – 23°C, with food placed on the cage tops as in Fuller et al. Under these conditions, when food was abruptly limited to 4 h/day for 10 days, the Bmal1-/- mice lost weight dramatically, all but one reaching the 25% endpoint criterion within 3–9 days , two sets of wildtype and null mutant mice were tested. The first set consisted of 2 Bmal1-/- mice abruptly restricted to 3 h food/day [Bmal1-/- mice died under this feeding protocol, but mortality rates dropped to zero when a gradual food restriction protocol was adopted. Thus, the impact of food restriction schedules on body weight in mice is affected by environmental conditions  and feeding protocol . Given the methodological details provided by Fuller et al [Bmal1-/- mice did not lose body weight is puzzling.Note that in both laboratories, body weights were measured after the daily mealtime, which underestimates weight loss sustained by the mice at meal onset, 20 h after their last meal. The less severe weight loss evident in the first set tested by Pendergast and Yamazaki may be a result of warmer housing conditions  that would have reduced energy expenditure. Notably, Fuller et al  state thfood/day . In thater et al ,30, the In this section, we identify data or methodological details that are missing from the manuscript, but that are required to support the major claims, to assess the reliability of the reported effects, or to replicate the experiments.3a. The total number of mice used, and the numbers per group, are not reported .Bmal1-/- mice develop progressive arthropathy and become less mobile, which could attenuate the expression of food anticipatory activity and impair their ability to retrieve food and eat a sufficient amount in 4 h to remain healthy.3b. The age of the mice at the time of behavioral testing is not reported. Age is a non-trivial methodological detail; as noted, by the age of 14 weeks Bmal1 rescue and circadian rhythm rescue are not reported. It is technically non-trivial to limit spread of microinjected viral vector to a relatively small target structure . The degree of difficulty is increased when bilateral injections are made (both have to limit expression to the target). To guide replication studies, it would be simple and appropriate to report the total number of mice receiving injections, the number in which Bmal1 expression was restored unilaterally, bilaterally or outside of the target area, and the number in each category that exhibited rescue of rhythms.3c. The success rates for injection placement, Bmal1+/-, Bmal-/- and AAV-BMAL1 mice. This graph shows body temperature rising in anticipation of mealtime in the heterozygous mice and in DMH-rescued AAV-BMAL1 mice but not in the Bmal1-/- mice. As noted, the authors state that Bmal1-/- mice "often slept or were in torpor through the window of restricted feeding, requiring us to arouse them by gentle handling after presentation of the food to avoid their starvation and death during restricted feeding". If some animals were severely hypothermic (< 31°C) at mealtime, it should be made clear in the figure legend as to whether these mice were included in the group averages. If these mice, or certain days, were not included, this should be indicated and the exclusion criteria clearly specified.3d. Figure two D in Fuller et al  is a ploBmal1-/- mice tested in LD 12:12 "may go into torpor during the night and forage more consistently during the day". However, no rationale or evidence is provided to support this statement. A reasonable assumption is that the incidence of torpor would increase with time since the last meal. Consequently, torpor should be far more likely to occur during the light period just before mealtime, particularly in Bmal1-/- mice that are supposed to have no circadian rhythms in LD , Fuller et al state that food restricted cool laboratory (22°C)torpor is a normal defense mechanism used by mice when faced with a 20-h fast in a '. In the 'corrected' supplementary materials , cage temperature is reported as 22 ± .1°C. However, in the previous version of the supplementary materials , cage temperature was reported as 24 ± .1°C. It is not clear which version is correct, because the change was made without being noted in the list of Corrections published in the Oct. 31 issue.3f. In the Reply  to the TBmal1-/- mice exhibit robust food anticipatory behavioral rhythms if they are gradually adapted to restricted feeding and if food pellets are placed in the cage rather than on cage tops, where it may be difficult for physically frail Bmal1-/- mice to reach. In the Reply [The Technical Comment  on the Fhe Reply  to the Cmeasure food-seeking behaviors rather than circadian rhythms'. According to Fuller et al, locomotor activity measured by overhead motion sensors is a 'food-seeking behavior' that reflects homeostatic factors , whereas activity and temperature measured by telemetry are 'unrelated circadian-driven physiological responses'. Food anticipatory activity rhythms exhibited by Bmal1-/- mice in other laboratories [Bmal1 knockout, and Gooley et al by DMH-ablation [measuring different things'.4a. In their studies of food anticipatory rhythms, Gooley et al  and Fullratories ,17,18 arablation . Accordimeasuring different things'.However, these arguments are neither intuitive nor consistent with available evidence. It is not at all clear how a properly functioning transponder in the intraperitoneal cavity, such as used by Fuller et al, could fail to register activity that is sufficient to trigger an overhead motion sensor, such as used by other labs. Thus, it seems unlikely from the outset that telemetry and overhead motion sensors could be differentially sensitive to homeostatic and circadian factors. In fact, three labs have conducted direct comparisons of the two measures in rats  and miceunrelated circadian-driven physiological response' is similarly unsubstantiated. Fuller et al provide no theoretical rationale for how body temperature could be immune to homeostatic factors  and thermogenic effects of eating and ongoing locomotor activity; in fact, their own data indicate that temperature is drastically affected by food restriction (their mice were severely hypothermic). Unsubstantiated claims about the special value of temperature as a circadian endpoint for studies of food anticipation are doubly disconcerting, as these may encourage the unnecessary use of invasive and expensive procedures .Fuller et al's claim that body temperature is also an ''the assertions made by Mistlberger et al. concerning the role of the DMH in food-entrainable circadian rhythms are not accurate. We and Mieda et al. did not find that the DMH is one of "a number of" brain regions whose clock gene expression is "synchronized by scheduled feeding." What was found is that the DMH is the only region of the brain that has self-sustained cycles of clock gene expression induced de novo by restricted feeding'. These comments are remarkable in misrepresenting both the Technical Comment and the results of Mieda et al [mPer1 and mPer2 gene expression that persists during total food deprivation. These data do not establish that DMH clock gene rhythms are 'self sustained', i.e., generated intrinsically. DMH clock gene rhythms could be driven by inputs from elsewhere in the brain or the periphery, related to sensory, motor, metabolic or other correlates of food anticipatory rhythms in behavior or physiology. There certainly are many other brain regions in which clock gene rhythms are reset by restricted feeding and persist during food deprivation [Per gene expression in the DMH are induced 'de novo' by daytime feeding is itself not accurate. While this seems to be generally overlooked, Figures 4 and 5 in Mieda et al [Per1 gene expression in the DMH of ad-lib fed mice, with an approximate 3–4 fold increase in expression at ZT13 compared to ZT7. The amplitude of the rhythm only appears low in those figures due to the scaling of the y-axis. During restricted feeding, the phase of the rhythm was shifted and its amplitude increased at least 2-fold. Thus, DMH rhythms are reset and amplified, not induced de novo, by daytime restricted feeding schedules. A rhythm of DMH Per1 expression in ad-lib fed mice has also been reported by Moriya et al [Per2 expression have been observed in the DMH, arcuate nucleus and adjacent anterior hypothalamic areas in mediobasal hypothalamic explants from ad-lib fed PER2:luc mice [4b. Fuller et al , in theida et al . Mieda erivation -39. Moreda et al  clearly ya et al , one or luc mice . Thus, cBmal1-dependent circadian mechanism in the DMH is sufficient to drive food-entrainable rhythms of activity and body temperature in mice. While numerous, these flaws are by no means minor. Our overriding objective here is to promote methodological rigor, to enable us to arrive at a clear view of how the brain synchronizes behavior and physiology to daily feeding opportunities. When new, potentially seminal findings are presented, we are obligated to scrutinize the evidence carefully to ensure that it meets standards sufficient to become part of the foundation for future work. The evidence as presented in Fuller et al [In this review of Fuller et al ,30 we haer et al  clearly The authors declare that they have no competing interests.


Ceratitis Capitata (DIPTERA: Tephritidae) is a major agricultural pest in Argentina. One main cause for the success of non-contaminant control programs based on genetic strategies is compatibility between natural and laboratory germplasms.The Mediterranean fruit fly A comprehensive characterization of the fruit fly based on genetic studies and compatibility analysis was undertaken on two founder populations from the provinces of Buenos Aires and Mendoza, used in pioneering sterile male technique control programmes in our country. The locations are 1,000 km apart from each other.We compared the genetic composition of both populations based on cytological, physiological and morphological characterization. Compatibility studies were performed in order to determine the presence of isolation barriers. Results indicate that the Buenos Aires germplasm described previously is partially different from that of the Mendoza population. Both laboratory colonies are a reservoir of mutational and cytological polymorphisms. Some sexual chromosome variants such as the XL and the YL resulting from attachment of a B-chromosome to the X-chromosome or Y-chromosome behave as a lethal sex-linked factor. Our results also show incompatibility between both germplasms and pre-zygotic isolation barriers between them. Our evidence is consistent with the fact that polymorphisms are responsible for the lack of compatibility.The genetic control mechanism should be directly produced in the germplasm of the target population in order to favour mating conditions. This is an additional requirement for the biological as well as economic success of control programs based on genetic strategies such as the sterile insect technique. The analysis of representative samples also revealed natural auto-control mechanisms which could be used in modifying pest population dynamics. Ceratitis capitata (Wiedemann), is the most economically important agricultural pest insect in the world. It belongs to the Tephritidae family, the “true fruit flies”, which is the target of large-scale eradication and suppression programs based on genetic strategies like the sterile insect technique (SIT)The Mediterranean fruit fly (medfly), At present a particular transgenic strain of the medfly Reared medflies must display morphological, physiological and behavioural features that are as close as possible to those of their wild counterparts. Any departure from the “wild” characteristics could cause the failure of an SIT program Considerable genetic variation in natural populations of the medfly from different geographic regions has been previously reported by different researchers A comprehensive characterization of the fruit fly based on genetic studies and compatibility analysis was undertaken on two founder populations from the provinces of Buenos Aires and Mendoza. These materials were used in pioneering sterile male technique control programs in our country.The success of non-contaminant control methods based on genetic strategies depends on compatibility between natural and laboratory germplasms.The utility of insect colonies depends on the laboratory conditions in which they are established and the precision with which they are managed. In fruit fly colonies, large and genetically variable founder populations are collected and carefully maintained in laboratory environments that provide -according to the investigator's criteria- optimal quantities and qualities of diet and space so as to promote the highest possible levels of survival for all developmental stages. However, some genotypes are lost, and this is not always a consequence of rearing.C. capitata (Wied.).At least three unmanageable events contribute to genetic drift in laboratory colonies of C. capitata is a “polyphage” and “multivoltine” species, so, generally, founder karyotypes do not really represent the whole genetic pool of the natural population. The collection of samples is mainly performed in the most economically important host-fruits during the same period every year. Limited knowledge of the biology and oviposition strategies of the fruit fly in nature, and within economically unimportant host-fruits, is further obstacle to improving the collection of samples. For this reason, we are probably losing a great deal of the natural genetic variability, since there may be different genetic associations between this fruit fly and other host fruits that are economically unimportant.Sampling itself, especially if sample size is small, could favour genetic drift. The medfly Differential adaptation of the founder genotypes: laboratory conditions fit some genotypes but not others.Evolutionary processes caused either by accidental changes in laboratory conditions or by mutation events, which are beyond control. These changes are a consequence of rearing and can be studied because: a) there is a large amount of individuals per generation; b) the life cycle of fruit flies is shortened and the number of generations per year increases.Cochlyomia hominivorax (Coquerel). A program to eradicate this pest in the United States, based on the release of sterile blowflies, failed because a state of reproductive incompatibility developed between wild-type and laboratory-reared individuals. Later on, a chromosomal polymorphism affecting the genital morphology of wild type females was associated with isolation barriers The importance of analyzing genetic variability was demonstrated in the screw-worm ARG 17 have been studied through the years. Next, a picture of this variability is summarised. Variation in the number of internal orbital bristles or spatulated hairs was observed in males. It was determined that in females, a gene is responsible for the increase in rostrum orbital hairs. It was demonstrated that these genes have a pleiotropic effect and variable expression Est-1 gene, a pupal esterase with two codominant alleles – was reported by some authors Previous work performed in our laboratory demonstrated the existence of different chromosomal polymorphisms within geographic populations from the provinces of Buenos Aires, Tucumán, Mendoza and Río Negro A and YB and affecting the long arm of the Y-chromosome, were reported, but they alter neither the sexual determining factor nor the fertility of carrier individuals B and YA chromosomes were 0.6 and 0.4 Polymorphisms, named YARG 17L- is derived from the attachment of a B-chromosome to the XS chromosome, and its inheritance was also reported by these authors. Homozygous female XLXL were never found A polymorphism affecting the length of the X-chromosome is present within a stock originated in the same ecological niche as 1 offspring, of an allele previously described as a recesive one; 3) individuals from the Mendoza population showed a high frequency of chromosomal polymorphism; 4) 16% of the chromosomes tested by backcrossing showed distorted segregation.One of our studies showed isolation barriers between individuals from this laboratory (origin: Province of Buenos Aires) and those from a laboratory colony in the Province of Mendoza C. Capitata in our country. One of them is from North Central Buenos Aires province – in the plains region, with a temperate, rainy climate. The other colony comes from the province of Mendoza, which is in an artificial oasis in a pre-Andean desert area. The locations are 1,000 km apart from each other. The purpose of our present study is to analyze the structure of both Argentinian C. capitata founder populations from different geographic origins. This is to determine whether they are compatible, and establish if their cytological and/or morphological complexities can account for the lack of compatibility. The analysis is based on the description of physiological behaviors and chromosomal polymorphisms as well as their possible associations. In keeping with this design, the karyotype of the samples and its possible incidence on viability was studied.Colonies from Buenos Aires and Mendoza have been periodically analyzed and used in pioneering sterile male technique programs to control ARG 17 genetic variability Analyzing ARG 17 presented morphological mutants. In addition, polymorphisms affecting rostrum pigmentation and thorax vertex basal pattern were observed. Despite efforts to elucidate their genetic control, it was not possible to find a simple explanation for them and it has been suggested that multiple gene effects might have been involved (unpublished).Karyotypical analyses of the colony were carried out throughout the generations. Cytological studies demonstrated the existence of sexual chromosome polymorphisms , sexual A  in the United States, based on the release of sterile blowflies, failed because a state of reproductive incompatibility developed between wild-type and laboratory-reared individuals. A chromosomal polymorphism affecting the genital morphology of wild type females was associated with isolation barriers Ceratitis capitata) and the failure to control it A program to eradicate the screw-worm  the number of females in the population and b) damage to fruits. Another improvement on SIT-based control programs was the construction of transgenic strains of the medfly harbouring a tetracycline-repressible transactivator (tTA) that causes lethality in the heterozygous progeny Ceratitis capitata populations. Additionally, we discovered natural auto-control mechanisms, such as the sex-linked lethals causing distorted sexual ratios.Present data provide consistent evidence that, in order to avoid pre-zygotic isolation barriers between target and laboratory populations, the genetic control mechanism should be produced directly in the treated population's germplasm. This is an additional condition for the success of the control programs based on genetic strategies such as the Sterile Insect Technique for controlling The periodic study of colonies reveals precious information on naturally occurring control mechanisms such as those detected within the colonies, which could be used by geneticists in order to modify pest population dynamics.Materials were maintained following the technique described by Terán ARG 17, which has been maintained as a closed population, and to date (35 years later), no inbreeding problems have been observed. It is at present the reference base material of the laboratory. Some data about this strain have already been reported and, in this paper, they have been summarised for easier understanding.It was originated in 1965 at the Institute of Plant Pathology (Eng. Turica) with samples from San Pedro  and samples from the area around Castelar , both of which localities are in the Province of Buenos Aires. This material was used in the SIT control programmes in the original area. It received recurrent introductions of wild material from the same areas . A sample of 2,428 pupae (mean weight = 9.614 mg/pupae) was carried to the Insect Laboratory (I.G.E.A.F.) in 1973. A bottle-neck was observed during the following generation: of over 55,000 laid eggs, only 9,830 imagines, or 20%, were recovered. Of these, over 30% died during the first three days. The remaining imagines gave rise to Mendoza 1, and the other one used in 1994 as Mendoza 2. These colonies were also employed in pioneering studies for control programmes (SIT) in that province.Mendoza founder populations represent two colonies founded with specimens taken from different host-fruits and localities of Mendoza  which received recurrent introductions throughout successive generations. The colony used in our experiments during 1986 will be referred to as Mendoza 1 was a sample of approximately 6,000 pupae taken from the Mendoza laboratory population. Mendoza 2 was a sample of approximately 28,000 pupae received from the Mendoza laboratory population (G0).We assembled 73 F1 families from the Mendoza 2 colony. A “family,” as termed by Lerner 1.T5038 Y+/X nig strain A sexual chromosome. Autosome 2 carries the cuticular marker nig (niger), a recesive black pupa and imago mutation ARG 17 germplasm 100 generations ago. All the females are homozygous for the marker nig  and all the males are wild-type , because they are heterozygous for the niger gene. In the present work, this auto-sexing strain was used to measure compatibility with Mendoza germplasm. The advantage of having an auto-sexing strain is that this material makes it possible to recognize and separate males from females in immature stages such as the pupal stage and avoid releasing sterile females.The T15879 Y+/X nig Strain (with Mendoza germplasm) was used to reconstitute the auto-sexing mechanism of the T5038 strain made up in the Mendoza germplasm.The We assembled 94 families in the following way:T5038×1 female T5038×1 female Mendoza I50 families: 1 male T5038×1 female Mendoza I16 families: 1 maleT15879×1 female T15879×1 female Mendoza I28 families: 1 male ARG 17 was performed. The chromosomal constitution of flies was periodically determined from 1973 up to now. Morphological studies were performed on pupae and imagines, analyzing colour segregation, rostrum pigmentation and thorax vertex basal pattern in both sexes, number of spatulated hairs in males, and number of orbital rostrum hairs in females.A morphological and cytological analysis of the reference laboratory strain T5038 and T15879 strains with Mendoza 1 germplasm was performed in order to use an autosexing strain to control Mendoza wild population. The number of matings was studied in order to determine the male preference. The offspring colour segregation in the pupa and adult stages was analyzed in order to determine its maternal origin. Using this method, the offspring from crossings with female T5038 should be black and the offspring from crossings with Mendoza females should be wild type. For this purpose, 94 families were assembled in the following way: 50 families with one male T5038 (+) and two females, T5038 (nig) and Mendoza 1 (+); 16 families with one male T5038 (+) and one female Mendoza 1 (+); 28 families with one male T15879 (+) and two females, T15879 (nig) and Mendoza 1 (+).A test of compatibility of the translocated Mendoza 2 colony was conducted on samples of the G1 and on the F1 and F2 progenies of 73 assembled families, studying karyotypes and physiological and morphological characters.The analysis of genetic variability within the 1 larvae sample (N = 13) of Mendoza 2 and on 36 derived laboratory strains (N = 81). We obtained cytological data from 1 to 4 individuals out of 5 chromosome spreads belonging to 5 specimens per strain.We conducted the karyotypical study on a GThe chromosomal constitution of the flies was determined through the cytological analysis of mitotic metaphases in the cerebral ganglion cells from third instar larvae. Ganglion cells were stained with 2% lacto-propionic orcein for 5 hours at 25°C, as described by The sexual chromosome variant size ratios were calculated measuring the length of a pair: the shorter chromosome variant against the other one. The relative chromosome length was the mean value obtained from repeated measurements of at least 10 different metaphase plates within each chromosome spread of a larvae sample.1 egg-hatching were tested. Those couples which did not lay eggs the first time were given a consecutive second opportunity to be tested.The first egg-laying opportunity and fertility measured through F2 from Mendoza 2 was analyzed through the study of their pupal viability, sex ratio and spontaneous segregation of mutants.The FFor the statistical analysis of the sexual index, the G-test with a null hypothesis for a sex ratio of 50∶50 was used Cytological and physiological data were compared.1 and F2 segregations were analyzed in terms of pupa colour, imago colour, or eye colour mutants.This section should provide enough detail to allow full replication of the study by suitably skilled investigators. Protocols for new methods should be included, but well-established protocols may simply be referenced. We encourage authors to submit, as separate supporting information files, detailed protocols for newer or less well-established methods. These are published online only, but are linked to the article and are fully searchable.Pupae and imago from G


Traumatic anterior dislocation of the shoulder is a common occurrence increasingly being treated arthroscopically. This study aims to determine the outcome of arthroscopic anterior stabilization using bioknotless anchors and analyze the motion in a subset of these patients.The outcome of 20 patients who underwent arthroscopic anterior stabilization using the bioknotless system was studied (average follow-up 26 months). Four of these patients underwent motion analysis of their shoulder pre- and post-operatively.15% were dissatisfied following surgery and the recurrence of instability was also 15%. Those who were dissatisfied or suffered recurrent symptoms had statistically significant lower constant scores at the final follow up. Pre-operative motion analysis showed a disordered rhythm of shoulder rotation which was corrected following surgery with minimal loss of range of motion.Our success rate was comparable to similar arthroscopic techniques and results published in the literature. Patient satisfaction depended more on return to usual activities than recurrence of symptoms. There was very little reduction in range of movement following surgery and the rhythm of shoulder motion, particularly external rotation in abduction was improved.Four retrospective series. Traumatic anterior dislocation of the shoulder is a very common condition, with an approximate incidence of 10-20 per 100,000 per year. with appBetween October 2004 and April 2006, 20 shoulders in 20 patients underwent arthroscopic anterior stabilization of the shoulder using bioknotless anchors. All patients were assessed pre-operatively and operated on by the two senior authors (VK and RW). There were 16 males and 4 females with an average age of 28 (range 17 to 53). Patients had an average of four dislocations (minimum two) prior to surgery and were deemed to be suitable candidates for arthroscopic stabilization. All patients presented initially with traumatic anterior dislocation of the shoulder. Sixteen patients participated in recreational contact sports and three at a regional or national level. Ten had occupations involving heavy manual labor.The fastrack system is a spatial tracking system that uses an electromagnetic field to determine the 3D position and orientation of markers in space. A biotechnologist (AR) placed the makers on the bony landmarks of the sternum , scapula , humerus and elbow  and wrist (the ulnar head); a complete picture of shoulder range and rate of motion can be built. It has a fast sampling rate (30 Hz) and has been shown to be an effective and valid method for the assessment of upper limb motion. Six patiPatients are placed in the beach chair position under general anesthetic and interscalene brachial plexus block. Examination under anesthesia is carried out followed by a standard diagnostic arthroscopy. All patients were found to have increased anterior translation of the humeral head and one had increased posterior translation in addition. All patients were found to have a Bankart lesion, seven of whom had an additional Hill-Sachs lesion and three also had SLAP lesions. Repair of the Bankart and/or SLAP lesion was performed using the bioknotless anchors as described elsewhere.10 For smAll patients were placed in a sling in internal rotation post-operatively. Once pain allowed, they were taught pendulum exercises and encouraged to perform these daily.After 1 week they were reviewed by a physiotherapist who commenced passive range of movement exercises. Assisted active and active mobilization was introduced over the following 6 to 12 weeks. External rotation beyond neutral was avoided for 6 weeks and combined external rotation and abduction for 12 weeks. All patients were advised to avoid contact sports and overhead weight-bearing activities for 6 months.Follow-up assessment was carried out by the first author and consisted of constant score evaluation in both affected and unaffected shoulders, patient satisfaction, and recurrence of instability or dislocation. Post-operative radiographs were not routinely obtained and were only done if clinically indicated. Fastrack analysis was carried out on those patients that had undergone pre-operative analysis. The average length of follow-up was 26 months (range 18 to 35).P<0.05, Student’s t-test).Average constant scores returned to 85%±7.7% of that measured in the contralateral arm . FifteenOf the 16 sportsmen and women, 10 returned to their pre-injury level of participation and 7 of 10 patients returned to heavy physical occupations. Overall, 13 patients returned to their previous activities and 7 did not (35%). We did not encounter any other surgical complications. Fastrack analysis revealed a very slight reduction in range of motion post-operatively. External rotation in abduction measured in the four patients was reduced by 4° ± 4°. The range of motion correlated with that measured clinically. Dynamically, it was noted that the velocity of motion, represented by the gradient of the curve, was much more constant following surgery . The reaThe open capsulolabral repair remains the gold standard against which other methods are measured. There is considerable evidence showing low recurrence in the order of 5-10%, with high patient satisfaction many years post-operatively. There arThe knotless anchor, developed by Thal, enables Our rates of re-dislocation and instability, 10% and 5%, respectively, are in broad agreement with other studies using the bioknotless anchor.10 ShoulderThe range of motion following open surgery is usually reduced. The greater soft tissue dissection (when compared with arthroscopy) and division/repair of subscapularis lead to loss of external rotation (ER), particularly when combined with abduction. However, it is precisely this movement that causes the greatest amount of apprehension when examining the patient with anterior shoulder instability. It has been suggested that by reducing the amount of external rotation, the patient no longer enters their “apprehension zone” and therefore no longer suffers with symptomatic instability. One study comparing transglenoid sutures with open repair found that 45% of patients in the open group lost some ER (10-40°) but there were no re-dislocations. The arthroscopic group had no loss of motion but a 60% recurrence of instability, suggesting that reducing the range of motion reduces recurrence.Both imbrication of redundant capsule and closure of the rotator interval have been recommended in addition to repair of the Bankart lesion and intuitively are both likely to reduce the overall range of motion. Even usiQualitatively, the graphs showing range of motion over time show a much smoother velocity of shoulder movement post-stabilization . Pre-opeOur results are broadly in line with others reporting their experience with the bioknotless anchor. The fastrack analysis demonstrates that patients who have a successful outcome have on average, a slight decrease of external rotation, but this is not as significant as that seen in open stabilization. The velocity of the external rotation arc is smoother post-operatively and the interruption or “catch” seen during preoperative analysis is abolished. Our study suggests that arthroscopic stabilization is an effective procedure which restores stability without significantly decreasing range of motion, contrary to the results seen with open stabilization.


Three dimensional echocardiography (3DE) is a new, rapidly evolving modality for cardiac imaging. Important technological advances have heralded an era where practical 3DE scanning is becoming a mainstream modality. We review the modes of 3DE that can be used. The literature has been reviewed for articles that examine the applicability of 3DE to congenital heart defects to visualize anatomy in a spectrum of defects ranging from atrioventricular septal defects to mitral valve abnormalities and Ebstein's anomaly. The use of 3DE color flow to obtain echocardiographic angiograms is illustrated. The state of the science in quantitating right and left ventricular volumetrics is reviewed. Examples of novel applications including 3DE transesophageal echocardiography and image-guided interventions are provided. We also list the limitations of the technique, and discuss potential future developments in the field. Complex intra-cardiac anatomy and spatial relationships are inherent to congenital heart defects (CHD). Beginning over thirty years ago and until recently, the clinician's ability to image the heart by echocardiography has been limited to two-dimensional techniques. In the iRecognition of these limitations of 2DE led to burgeoning research and clinical interest in the modality of 3DE. Early reconstructive approaches were based on 2DE image acquisitions that were subsequently stacked and aligned based on phases of the cardiac cycle, in order to recreate a 3DE dataset. While thTechnologic advances that have facilitated the maturation of 3DE techniques include the following:Matrix transducersBeam forming and steering in three spatial dimensionsDisplay of three-dimensional informationSoftware for quantificationTwo important advances in transducer technology, namely the organization of elements and the use of novel piezoelectric materials, have been the structural basis for improvements in 3DE matrix transducers.Contemporary matrix-array transducers comprise as many elements in the elevational dimension as they do in the azimuthal dimension, with over 60 elements in each of these dimensions. While the elements are arranged in a two-dimensional grid, this array generates 3DE images. In order to be able to steer in the elevational plane, each element must be electrically independent from all other elements, and each element must be electrically active. The technology and electrical circuitry to electrically insulate and connect each element became commercially available in 2002. As a result, the contemporary matrix array transducer consists of thousands of electrically active elements that independently steer a scan line left and right, as well as up and down.The piezoelectric material in an ultrasound transducer is a fundamental determinant of system image quality. Piezoelectric transducer elements are responsible for delivery of ultrasound energy into the scanned tissue and for converting returning ultrasound echoes into electric signals. Their coupling efficiency in converting electrical energy to mechanical energy or vice versa is a key determinant of image quality, Doppler sensitivity and penetration. To create an overall piezoelectric effect, these elements must be subject to the application of an external electric field to align dipoles within polycrystalline materials. For almost forty years, a ceramic polycrystalline material, PZT (lead-zirconate-titanate) or PZT composites, has been the standard piezoelectric material used in medical imaging. This material is a uniform powder that is mixed with an organic binder; the resulting compound is baked into a dense polycrystalline structure. At its best, it achieves ~ 70% alignment of dipoles due to imperfect alignment of the individual dipoles. This leads to a corresponding constraint in the electromechanical coupling efficiency of the material.One example of new piezoelectric material involves growing crystals from a molten ceramic material, resulting in a homogenous crystal with fewer defects, lower losses and no grain boundaries.–10 When 11Beam forming constitutes the steering and focusing of transmitted and received scan lines. For 3DE, this means that beam former must be steered both in the azimuthal and elevational planes. This is achieved both in the ultrasound system and within the transducer itself, using highly specialized integrated circuits to create a 3D trapezoid of acoustic information that is processed. These 3DE data are summed, processed and finally placed into rectangular space using a 3D scan converter.Two-dimensional computer displays consist of rows and blocks of picture elements, termed pixels, that comprise a 2D image. In contrast, a 3DE data set consists of bricks of pixels, termed volume elements or voxels. However, even for a 3DE data set, the two-dimensional nature of the display imposes restrictions on the ability to appreciate depth. As shown initially by the ancient Greeks and subsequently rediscovered during the Renaissance, perspective is used to simulate the appearance of 3D depth, providing objects the appearance of being close to or deeper / further away from the screen. The process of adding perspective is done by casting a light beam through the collection of voxels. The light beam either hits enough tissue so as to render it opaque, or it keeps shining through transparent voxels so as to render it transparent. More recent algorithms apply different hues to the front of the data set (nearest to the screen) as opposed to voxels that are far from the screen . The useQuantification requires segmentation of structures of interest from the acquired data. Since myocardial motion occurs in three spatial dimensions, 2DE planes are inherently incapable of capturing the entire motion. Two-dimensional techniques for quantitation are based on geometric formulas that rely on assumptions regarding the shapes of cardiac structures. However, these assumptions are frequently incorrect. In contrast, 3DE acquisitions include the entire extent of the structure, thus minimizing the possibility of foreshortening of the apex or any geometric assumptions regarding shape. Three-dimensional quantitative software tools have the potential to quantify cardiac structures accurately regardless of their shape. Advances in the software tools for processing 3DE datasets have mirrored the rapid advances in transducer technology that have occurred over the past five years.3DE volumetric techniques rely on definition of chamber cavities, that is, the blood-endocardium interface. The software constructs this interface by using a process known as surface rendering, and represents it as a mesh of points and lines. This software-generated mesh is calculated for every frame of acquisition, thus providing a moving cast of the cavity of the ventricle during the cardiac cycle. Since this is digital data, it provides for ease of computation of global and regional volumes, synchrony as well as parametric displays of endocardial excursion and timing of contraction .3DE quantification tools for the left ventricle (LV) are more technologically advanced than for other cardiac structures. Until recently, 3DE LV quantification tools employed the method of disk summation. With improvements in computing speeds and programming, newer tools have been developed to provide instantaneous tracking of the blood pool - endocardium interface at each frame of acquisition. This provides a surface-rendered model that is displayed as a mesh of lines and points. However, these approaches are still based on some basic 3D geometric assumptions regarding left ventricular shape, and therefore their application cannot be extended to the right ventricle or to univentricular hearts. Given the complex shape and architecture of the right ventricle, it is not surprising that tools for quantifying right ventricular (RV) volume have been slower to mature. Until very recently, these tools utilized the method of disks for volumetrics. Novel soQuantitative software for the mitral valve provides the ability to perform sophisticated analyses of the nonplanar shape of the mitral annulus and to measure 3D structures including annular diameters, commissural lengths, leaflet surface areas.16 QuantiElectronically-steered 3DE systems have two major modes of scanning: live and EKG-gated. The live mode is the only one where the system scans in 3D real-time. A defining characteristic of this mode is: if the transducer comes off the chest, the image disappears. The live 3D mode can also be operated within a three-dimensionally shaped zoom box. Live 3DE modes provide narrow (20 to 30 degrees in the elevation plane) datasets that have high voxel density. Live 3DE can be obtained on patients with arrhythmias or with an active precordium with no potential for motion or stitch artifacts.EKG-gated modes are required to provide wider volumes while maintaining adequate frame rates . Gating Contemporary 3DE systems provide the user with tools to vary frame rate, 3D volume size and image resolution. Increasing the requirement in one of these causes a drop in the other two, all things being equal.Given the potential for motion and stitch artifacts with gated modes and the need for high spatial and temporal resolution, it has been our practice to use live 3DE to delineate anatomy. We reserve the use of gated modes for the following:Complicated cardiac anatomy that requires extensive offline croppingTargets that do not fit within a live 3DE windowQuantitation of chamber volumes3DE color flow demonstrations of regurgitant jets or shunt flows.Live 3DE imaging has been commercially available for trans-thoracic and fetal applications since 2002. Live 3DE transesophageal echocardiographic imaging became commercially available in 2007. This has yielded images never before seen on the beating heart . We anti3DE imaging has three broad areas of clinical application among patients with congenital heart disease: visualization of morphology, volumetric quantitation of chamber sizes and flows, and image-guided interventions.Dating from an early stage in the development of 3DE technology, the structural complexity that is inherent to congenital heart disease has been identified as fertile substrate for exploration using 3DE.–19et al, described the role of 3DE in delineating congenital abnormalities of the mitral valve.[et al, demonstrated that 3DE allowed comprehensive assessment of the anatomy of double orifice mitral valve.[et al, demonstrated the additive value of 3DE and improved image quality using intraoperative epicardial 3DE to delineate the anatomy of atrioventricular (AV) valves.[et al, studied 41 patients with AV valve abnormalities and found that 3DE imaging was helpful in delineating the morphology of the valve leaflets and their chordal attachments, the subchordal apparatus, the mechanism and origin of regurgitation, and the geometry of the regurgitant volume.[et al, examined the role of 3DE in patients with Ebstein's anomaly of the tricuspid valve. They demonstrated that 3DE provided clear visualisation of the morphology of the valve leaflets, including the extent of their formation, the level of their attachment, and their degree of coaptation. They were also able to visualize the mechanism of regurgitation or stenosis.[3DE is valuable in delineating the morphology of the atrioventricular valves. Espinola-Zavaleta al valve. Lu et alal valve. Rawlins ) valves. Seliem et volume. Vettukatstenosis.et al, studied 52 datasets on 51 patients with atrioventricular septal defects (AVSD) and showed that gated 3DE views could be cropped to obtain en face views of the atrial and ventricular septa.[Hlavacek ar septa. These viar septa.,8. Theseet al, showed that 3DE reconstructions provided unique en face views of atrial and ventricular septal defects.[et al, studied 38 patients with atrial and / or ventricular septal defects using 3DE, and compared their results to 2DE and surgical findings. They demonstrated novel 3DE views of both atrial and ventricular septal defects and improved accuracy of quantification of the size of the defect by 3DE compared to 2DE (r = 0.92 vs. r = 0.69).[Tantengco  defects. Cheng et = 0.69). This app = 0.69). We have 3DE color flow Doppler has been used to provide echocardiographic ‘angiograms’ of flow patterns in the aortic arch (coarctation of the aorta), the branch pulmonary arteries (the Lecompte maneuver) and across Blalock-Taussig shunts. These auet al, examined the role of 3DE in 8 children who subsequently underwent surgery for congenital aortic valvar stenosis. They showed that 3DE was accurate in providing measurements of aortic valve annulus, number of valve leaflets, in identifying sites of fusion of the leaflets as well as nodules and excrescences that characterized dysplastic valves.[et al, studied 16 patients with subaortic stenosis. Using a form of 3DE reconstruction known as multiplanar reconstruction, which provides access to an unlimited number of 2DE planes, they demonstrated abnormalities of mitral valve leaflet or chordal apparatus attachments (14 patients), abnormal ventricular muscle band (11 patients) and abnormal increased aorto-mitral separation (2 patients).[Sadagopan c valves. Bharuchaatients).et al, evaluated 4 patients with left ventricular non-compaction using 3DE.[Baker sing 3DE. They fouet al, compared 3DE measurements of LV volumetrics to those obtained using MRI.[et al, evaluated the feasibility of 3DE LV volumetrics, as well as the resource utilization, learning curve, inter- and intra-observer reproducibility of this technique. The study design involved 15 datasets and four observers who had varying degrees of (self-rated) experience with 3DE quantitation.[3DE quantitation provides both proven and potential value for pediatric echocardiography. Bu sing MRI. Their sttitation. They fouet al recently demonstrated excellent correlations between LV mass measured by 3DE and MRI.[Studies in adults have validated 3DE as an accurate method for measuring LV mass.–37 While and MRI.et al, examined the association between LV dysfunction and intra-LV dyssynchrony in children using 3DE.[The 3DE approach to measuring intra-LV dysynchrony utilizes the American Society of Echocardiography's 16-segment model of the LV. It measusing 3DE. They stuet al, reported a large series of adults who underwent RV volumetric measurements using MRI and 3DE using the disk summation method.[et al, studied RV volumes by 3DE using a new and robust protocol that utilizes multiplanar reconstruction and tracing with semi-automated border detection in 16 children with congenital heart disease.[http://www.pediatricheartnetwork.org/svrforhealthcareproviders.asp).Until recently, the accuracy of 3DE measurement of RV volume and ejection fraction had not been evaluated. This is unsurprising given the complex architecture of the right ventricle, and the technical difficulty of imaging it adequately. Recently, Gopal n method. Niemann  disease. They fouMultiplanar reconstruction tools provide the ability to not only visualize but also to trace and measure the area of valvar regurgitant orifices at the level of the vena contracta. While this technique is new and has not been validated, it has been shown to be feasible, and has already yielded new insights into the shape of regurgitant jets, and holds promise as a tool to enhance the echocardiographer's ability to serially quantify valve regurgitation.–43et al, developed a technique for quantifying non-aliased 3DE color flow jets in vitro.[2 = 0.93). 3DE quantification of color flow has recently been validated in adults by comparison to cardiac outputs obtained by thermodilution.[Pemberton in vitro. They valin vitro. They comdilution. Applicatdilution.et al, demonstrated the use of live 3DE to guide the performance of endomyocardial biopsy in children.[et al, have extended the concept of image-guided intervention to the very novel approach of epicardial live 3DE-guided, open-chest, closed-heart, off-bypass cardiac surgery.[in vitro validation of the ability of live 3DE to guide the performance of common surgical tasks. More recently, they have undertaken closure of small atrial septal defects in the porcine model using live 3DE guidance. With the recent introduction of live 3D TEE, we have been able to obtain spectacular images of cardiac chambers, septal structures and valves. Live 3D TEE is being used to guide catheter manipulations, trans-septal procedures and closure of atrial septal defects[The high temporal and spatial resolution of trans-thoracic and particularly trans-esophageal echocardiography has ignited interest in the potential uses of live 3DE to guide interventions. Scheurer children. In their surgery.–52 They l defects [Figuresl defects,11.The learning curve with 3DE is steep but negotiable. Our experience would suggest that the success of 3DE in a program requires advocacy and an investment of time by both echocardiographers and sonographers. The acceptance of 3DE is improving on a global level, albeit at an early stage of the technology cycle. We have developed and implemented an interactive teaching course that utilizes simulations using 3DE datasets with rehearsal and direct mentoring; this has been shown to be useful in overcoming the steep part of the learning curve.Many of the limitations of 2DE imaging extend to 3D echocardiography. Patients who have poor windows for 2DE examinations are unlikely to have good 3DE windows. Structures that are difficult to visualize by 2DE because they are thin and parallel to the imaging plane, such as the atrial septum in a teenager as viewed from an apical view, will probably remain difficult to visualize from that imaging window by 3DE.Given the early stage of development of this technology at the current time, it is unsurprising that governing bodies such as the American Society of Echocardiography have not yet developed formal protocols.Over the next decade, advances in the 3DE arena will involve technical enhancements such as improved image resolution, holographic displays, a wide range of validated software tools for quantification, and enhancements to work flow. New, multi-modality applications will increasingly bring 3DE into the mainstream. Refinements in transducer technology will make high-resolution 3DE available across the spectrum of patient sizes. We anticipate a 3DE TEE probe miniaturized for pediatric usage. With the growing interest in multi-modality imaging, 3DE volumetric data will eventually be integrated with the pressure data that is available during cardiac catheterization, yielding pressure-volume loops that can be obtained as a matter of routine clinical practice.


Rho GTPases are a distinct subfamily of the superfamily of Ras GTPases. The best-characterised members are RhoA, Rac and Cdc42 that regulate many diverse actions such as actin cytoskeleton reorganisation, adhesion, motility as well as cell proliferation, differentiation and gene transcription. Among the 20 members of that family, only Rac2 and RhoH show an expression restricted to the haematopoietic lineage.RhoH was first discovered in 1995 as a fusion transcript with the transcriptional repressor LAZ3/BCL6. It was therefore initially named translation three four (TTF) but later on renamed RhoH due to its close relationship to the Ras/Rho family of GTPases. Since then, RhoH has been implicated in human cancer as the gene is subject to somatic hypermutation and by the detection of RHOH as a translocation partner for LAZ3/BCL6 or other genes in human lymphomas. Underexpression of RhoH is found in hairy cell leukaemia and acute myeloid leukaemia.Some of the amino acids that are crucial for GTPase activity are mutated in RhoH so that the protein is a GTPase-deficient, so-called atypical Rho GTPase. Therefore other mechanisms of regulating RhoH activity have been described. These include regulation at the mRNA level and tyrosine phosphorylation of the protein's unique ITAM-like motif. The C-terminal CaaX box of RhoH is mainly a target for farnesyl-transferase but can also be modified by geranylgeranyl-transferase. Isoprenylation of RhoH and changes in subcellular localisation may be an additional factor to fine-tune signalling.Little is currently known about its signalling, regulation or interaction partners. Recent studies have shown that RhoH negatively influences the proliferation and homing of murine haematopoietic progenitor cells, presumably by acting as an antagonist for Rac1. In leukocytes, RhoH is needed to keep the cells in a resting, non-adhesive state, but the exact mechanism has yet to be elucidated. RhoH has also been implicated as a regulatory molecule in the NFκB, PI3 kinase and Map kinase pathways. The recent generation of RhoH knockout mice showed a defect in thymocyte selection and TCR signalling of thymic and peripheral T-cells. However, RhoH-deficient mice did not develop lymphomas or showed obvious defects in haematopoiesis. The superfamily of mammalian Ras like proteins comprises over 150 members that can be subdivided into five main families, namely Ras, Rho, Rab, Arf and Ran and all control rather specifically one particular facet of cell metabolism. While Ras is an important regulator of proliferation ,3. Rho GAs is the case for most of the other Ras-like GTPases, Rho proteins are monomeric 20–30 kDa GTP binding proteins that act as molecular switches. They are turned on when bound to GTP and switched off when bound to GDP . Three mStructurally, all Rho GTPases contain a Rho family specific, exposed insert of 13 amino acids that has a function in binding to effectors and regulators  , althouRhoH is thought to mainly act as a negative regulator for such diverse processes as proliferation, survival, migration and engraftment of haematopoietic progenitor cells  and was Thirteen years ago, Dallery and co-workers set out to identify new potential oncogenes by cloning chromosomal translocation breakpoints that were found in human lymphomas . They haThe initial characterisation showed that RhoH was a 191 amino acid containing protein and a novel homologue of the Ras family. While it had 27% identity to H-RAS it showed up to 45% identity with members of the Rho family. It also contained the Rho typical insert motif, although shortened to 7 amino acids, which confirmed that classification. However, the absence of residues crucial for the catalytic activity of the proteins q27;p13) translocation with LAZ3/BCL6 in a non-Hodgkin lymphoma cell line  transloc-34, most;4q27;p13Reciprocal chromosomal translocations with oncogenes are found in a number of non-Hodgkin lymphomas. The occurring translocations are rather specific for the type of lymphoma and they are often the primary event causing the disease. Follicular lymphoma (FL) for example, is associated with a translocation involving the BCL2 gene. However, rearrangements involving LAZ3/BCL6, eventually interfering with LAZ3/BCL6 protein expression, are also found . Bcl6 isThe importance of LAZ3/BCL6 translocations was investigated by Akasaka et al. . The autA different RHOH translocation was found in a case of multiple myeloma (or plasma cell myeloma). Chromosomal abnormalities and translocations seem to increase in pathogenesis in this bone marrow based plasma cell neoplasm . In thisRecently, a novel class of genetic aberration called aberrant somatic hypermutation (ASHM) has been described . SomaticIn mature B-cell neoplasms, the occurrence of ASHM and the mutation frequency of RHOH vary. While some cancers have low rates of ASHM in general , others Although Hodgkin lymphomas (HL) are targeted frequently by ASHM (55–80%), the RHOH gene is not mutated at high frequency . Both suThe third group of lymphoid tumours, where abnormalities of RHOH are reported, are immunodeficiency-associated disorders such as the B-cell post-transplant lymphoproliferative disorder (PTLD). PTLD is a lymphoid proliferation or lymphoma that may result from immunosuppression after receiving solid organ or bone marrow allografts. While the polymorphic form of the disorder rarely undergoes ASHM (12.5%), the monomorphic form, representing a progression of the disease, had ASHM in the majority of cases investigated (66.7%) . This isTwo recent publications highlight the fact that RhoH is underexpressed in hairy cell leukaemia (HCL) and acute myeloid leukaemia (AML), respectively ,56.In vitro reconstitution of RhoH expression was sufficient to repress CD11c surface expression.HCL is a rare disease, comprising only 2% of lymphoid leukaemia cases. The origin of the disease is not known and effective treatment is not available . The disin vivo xenograft model, RhoH reconstitution leads to the inhibition of malignant progression. The tumour burden of the mice was reduced by 70% suggesting that elevated levels of RhoH protected the animals against mortality [CD11c is the integrin alpha X subunit that together with the beta2 chain acts as a leukocyte specific integrin. So the authors asked whether RhoH plays a role in intercellular adhesion. Indeed, cells reconstituted with RhoH showed decreased cell proliferation and homotypic and heterotypic adhesion, respectively. In a transwell assay, the authors could demonstrate that RhoH reconstitution reduced transendothelial migration, a central process in the pathogenesis of the disease. In an ortality .The expression level of RhoH was also investigated in bone marrow samples from AML patients and underexpression of RHOH on the mRNA level was found to be a prognostic marker for bad prognosis, overall survival as well as disease-free survival . This isIt is to be expected that RhoH expression levels can modulate the activity of other proteins involved in cell survival and proliferation as well. It will therefore be of crucial importance to gain a better understanding of RhoH as a modulator of signalling cascades in order to better understand its biological implications in cancer patients.In comparison to the number of studies on the putative role of RhoH in cancer, only a very limited number of publications deal with the function of RhoH as a signalling molecule. This can be attributed in part to the fact that all mutations of the RHOH gene were found to occur in the non-coding part of the gene. This suggests that in human haematological malignancies the absence of RhoH is problematic rather than activating mutations or overexpression of the protein. The recent finding that overexpression of RhoH in hairy cell leukaemia reduces the transformation potential  might stSeven years after the initial discovery of RhoH, the first study that described several aspects of the signalling of RhoH in various haematopoietic cell types was published . Due to As Rho GTPases characteristically play a function in cell morphology and the organisation of the actin cytoskeleton, the effects of RhoH expression on actin polymerisation were studied in non-haematopoietic NIH 3T3 and MDCK cells. The authors describe RhoH to be diffusely distributed in the cytoplasm and they saw no difference in the morphology of PDGF stimulated cells in the presence or absence of RhoH overexpression . HoweverThe antagonistic function of RhoH had already been described in the initial study of Li et al., where RhoH was shown to reduce the RhoA, Rac1 or Cdc42-induced activation of the transcription factor NFκB. Jurkat cells stimulated with TNFα show decreased NFκB activity in the presence of RhoH . InitialSince RhoH is GTPase-deficient, other mechanisms must be available to regulate the activity or function of the protein, one being the possibility of transcriptional regulation. Li et al. tested a couple of stimuli on Jurkat cells and reported that PMA reduces endogenous RhoH transcripts after 60–80 min by 80% of the baseline level, while TNFα treatment did not act as a regulator of RhoH mRNA levels . So far In a study aimed at identifying regulators of lymphocyte adhesion factor 1 (LFA-1), RhoH was found to be of importance . LFA-1 iin vitro for their ability to respond to a cytokine stimulus [in vivo [Supporting the theory that RhoH acts as a negative regulator of Rac signalling and other signalling cascades, it was found that RhoH negatively influences proliferation, migration and engraftment of haematopoietic bone marrow derived progenitor cells . To assestimulus . In resp[in vivo . However[in vivo ,28. Neve[in vivo ,57.Because RhoH is expressed strongest in T-cells, it was tested early on whether differentiation of T-cells into Th1 and Th2 subsets leads to varying RhoH expression patterns . Indeed,Using RhoH-deficient mice, the analysis of the function of RhoH in T-cells was investigated in more detail. Two groups independently showed that RhoH acts as a positive regulatory factor for thymocyte selection and T-cell receptor signalling ,28. RhoH- CD25+ DN3 cells. During thymocyte positive selection, TCR mediated signalling results in upregulation of markers for positive selection, such as CD5 and CD69. In RhoH-deficient mice the expression of these markers was reduced, suggesting that RhoH plays a role in thymocyte positive selection. Although peripheral thymocytes expressed the TCR complex at normal levels, anti-CD3ε stimulation triggered only a reduced proliferative response suggesting that the down-regulation occurred downstream of the TCR complex [RhoH-deficient mice were found to have abnormally low levels of T-lymphocytes and the cellularity of their thymus was app. 60% lower than in control animals, due to reduced proliferative cell expansion. In mutant mice, the number of double negative thymocytes was reduced by 60%, indicating that the development between double negative (DN) to double positive (DP) stages was disturbed; this was also reflected in an elevated number of CD44 complex . Injecti6–8YXXL/I. A closer look at the RhoH sequence revealed that RhoH contains a sequence that the authors dubbed an ITAM-like motif, where the (iso)leucines at position +3 are replaced by an alanine. Interestingly, this motif is absent in any of the other Rho GTPases. Further mutational analysis confirmed the theory that the two tyrosines Y73 and Y83 in RhoH are essential for recruitment of Zap70. Deletion of the two SH2 domains in Zap70 known to bind to the TCR CD3ζ chain also abolished interaction with RhoH.In order to identify the potential interaction partner of RhoH downstream of the TCR, a pulldown assay was performed using GST-coupled RhoH and lysates from Jurkat cells. A specifically interacting protein was analysed by mass spectrometry and found to be Zap70. Zap70 is a tyrosine kinase of the Syk family that is expressed in T-cells and NK-cells which interacts with the CD3ζ chain of the TCR complex . The phyLike the RhoH-deficient mice, Zap70 null mice have a developmental block of the maturation at the double negative stage and peripheral thymocytes that are unresponsive to TCR mediated stimuli . This suThe haematopoietic GTPase RhoH is an atypical family member of the Rho GTPases as it is constitutively active and not regulated through the classical cycling between GTP- and GDP-bound state. The protein was originally cloned as a fusion transcript with LAZ3/BCL6 in a non-Hodgkin lymphoma cell line  and has Even though to date no other obvious defects of the RhoH-deficient mice have been reported, it is to be expected that RhoH will be an important regulatory molecule in a number of other signalling cascades. In an early study it was shown that stimulation of neutrophils with GM-CSF leads to an upregulation of RhoH mRNA, suggesting that RhoH may play a role in diseases characterised by neutrophilic inflammation such as chronic obstructive pulmonary disease (COPD) . It willGTP: guanosine 5'-triphosphate; GDP: guanosine 5'-diphosphate; GM-CSF: granulocyte-macrophage colony-stimulating factor; CNS: central nervous system.The authors declare that they have no competing interests.KFK wrote the manuscript and FF performed experiments and revised the manuscript. Both authors read and approved the final manuscript.