Continuous positive airway pressure (CPAP) treatment for acute cardiogenic pulmonary edema can have important benefits in acute cardiac care. However, coronary care units are usually not equipped and their personnel not adequately trained for applying CPAP with mechanical ventilators. Therefore we investigated in the coronary care unit setting the feasibility and outcome of the simple Boussignac mask-CPAP (BCPAP) system that does not need a mechanical ventilator.BCPAP was introduced in a coronary care unit where staff had no CPAP experience. All consecutive patients transported to our hospital with acute cardiogenic pulmonary edema, a respiratory rate > 25 breaths/min and a peripheral arterial oxygen saturation of < 95% while receiving oxygen, were included in a prospective BCPAP group that was compared with a historical control group that received conventional treatment with oxygen alone.During the 2-year prospective BCPAP study period 108 patients were admitted with acute cardiogenic pulmonary edema. Eighty-four of these patients (78%) were treated at the coronary care unit of which 66 (61%) were treated with BCPAP. During the control period 66 patients were admitted over a 1-year period of whom 31 (47%) needed respiratory support in the intensive care unit. BCPAP treatment was associated with a reduced hospital length of stay and fewer transfers to the intensive care unit for intubation and mechanical ventilation. Overall estimated savings of approximately € 3,800 per patient were achieved with the BCPAP strategy compared to conventional treatment.At the coronary care unit, BCPAP was feasible, medically effective, and cost-effective in the treatment of acute cardiogenic pulmonary edema. Endpoints included mortality, coronary care unit and hospital length of stay, need of ventilatory support, and cost (savings). Many cases of acute cardiogenic pulmonary edema (ACPE) are treated with an oxygen mask and pharmacologic treatment including diuretics and vasodilators ,2. When A simple CPAP device which does not require a ventilator might therefore be useful to treat patients with ACPE in the CCU and reduce the need for more invasive treatment in the ICU. The Boussignac CPAP (BCPAP) system is a small lightweight plastic cylinder that is directly connected to a face mask which has been shown to be effective for ACPE in the emergency department and in prehospital care ,10. Alth2). BCPAP can generate a fraction of FiO2 that approaches 100% [2O at a flow of 8 L/min, 5 cm H2O at a flow of 15 L/min and 10 cm H2O at a flow of 23 L/min. This was measured during CPAP treatment with a handheld electronic pressure analyzer .The BCPAP face mask system  which is CE-approved and approved by the Food and Drug Administration is a simple and lightweight (10 g) disposable cylindrical plastic device without sensors, mechanical valves and without heavy tubing. A jet flow of air and or oxygen generated in this plastic tube creates a flow dependent pressure . We usedhes 100% . The gen2) < 95% while receiving oxygen [This study was conducted in a regional university hospital with a 12 bed CCU and 4 ICU's with 47 beds. ACPE was defined as acute dyspnea associated with a past of ischemic heart disease or cardiomyopathy and physical signs consistent with pulmonary edema including a respiratory rate > 25 breaths/min and a peripheral arterial oxygen saturation . The first ten patients treated with BCPAP on the CCU were observed by the medical and nursing staff of the ICU to ensure a safe and sufficient CPAP treatment.The institutional review board of the hospital approved the study design that compared a historical control group with a prospective BCPAP group. Verbal consent was obtained and recorded from patients to use their medical data. (METc 2004.195)2O and an FiO2 of 80–100%. BCPAP pressure and FiO2 were decreased when there was a clinical improvement in SpO2 and heart rate. Patients were preferably kept in a sitting position during BCPAP. In order to facilitate successful implementation of BCPAP both medical and nursing staff was involved in the implementation process. During a training session of 45 minutes we introduced the BCPAP system as well as a protocol , ventimask (FiO2 40–60%) or nasal catheter  and on ICU admission with endotracheal intubation. For this purpose we evaluated all records from patients admitted to the CCU and the ICU's for ACPE.Data from the prospective BCPAP cohort of patients were compared with the data from a historical control group. The prospective part of the study was conducted over a period of 24 months. Patients in the control group were admitted in the same CCU and hospital in a previous 12 month period and treated with oxygen through non-rebreathing mask  or means with standard deviation (SD) for continuous variables, or as group percentages for categorical variables. Statistical analysis was performed using Student's t, Mann Whitney, Wilcoxon signed rank test, Fisher exact or Chi-square test. Overall comparisons were based on intention-to-treat: all patients treated at the CCU or ICU for ACPE were compared between the control and the BCPAP period, irrespective of whether they actually received BCPAP. All statistical analyses were performed using commercially available software .Sixty-six patients were included in the control group and 108 patients in the BCPAP group Figure . Patient2 91%. IQR 88–93) showed adequate spontaneous improvement. The median duration of BCPAP treatment was 105 minutes (IQR 60–172 minutes). Pressure measurements showed a predicted CPAP level in 76% of the BCPAP patients. In only two patients a flow of more than 23 L/min had to be given to achieve the desired BCPAP level. Sixty-four of 66 patients maintained stable gas exchange after BCPAP was stopped. Two of the 66 patients required follow-up respiratory support in the ICU after BCPAP treatment in the CCU. One patient with a large myocardial infarction and support with an intra-aortic balloon pump had to be intubated and the other received ventilator mask-CPAP. No other complications of the BCPAP treatment were reported and 88% of the staff assessed the implementation of BCPAP as simple and uncomplicated.No problems in the practical use of the BCPAP system were identified. Eighteen patients who were admitted to the CCU were eligible for BCPAP but did not receive this treatment and received oxygen alone. The main reason was because there were insufficient signs of serious respiratory distress and staff believed that the patients . Both groups showed a significant increase in SpO2 after 2 hours  with a similar SpO2 after 2 hours. Heart rate in the BCPAP group was significantly decreased after 2 hours of treatment (median 104/min to 86/min), in contrast to the unchanged heart rate in the control group (median 102). In the 2 to 6 hours after discontinuation of BCPAP, the saturation or heart rate did not deteriorate again.Baseline median SpOAfter implementing BCPAP on the CCU, the number of intubations showed an absolute decrease of 14% (p < 0.001) compared to the control period with conventional treatment. Likewise the number of ICU admissions showed an absolute decrease of 25% (p < 0.001) compared to the control period.The median CCU-stay in de BCPAP group was 1 day with a median hospital length of stay of 6 days. In the control group the median CCU-stay was 2 days and the median hospital length of stay was 14 days. There was no significant difference between the mortality rates in both groups (BCPAP 22% versus control 24%).As a shift was found in route of admittance, resulting in more patients in the BCPAP group admitted to the CCU compared to the ICU, costs changed accordingly. In the control group, mean total costs for ACPE patients were € 13,754 per patient.In the BCPAP group  mean total costs were € 9,968. This resulted in a saving of € 3,787 per candidate for the new treatment strategy compared to the traditional strategy.We demonstrated that for patients with acute cardiogenic pulmonary edema it is possible to use a simple form of mask-CPAP. The introduction of BCPAP at our CCU decreased endotracheal intubations and the related ICU admissions. Our study also underscores that CPAP, as delivered by mask, is superior to conventional oxygen treatment in improving oxygenation and decreasing heart rate. Moreover, no complications were observed with the use of the BCPAP system on our CCU. The decreasing number of endotracheal intubations with related ICU admissions after implementation of BCPAP on our CCU led to notable cost reductions. Savings were approximately € 3,800 per candidate for the strategy that included BCPAP compared to the conventional strategy.The treatment of ACPE with non-invasive CPAP was already described in de first half of the previous century by both Poulton  and Bara2/FiO2 ratio, subjective dyspnea score, and respiratory and heart rates with CPAP [ACPE patients show significant improvement in the PaOith CPAP ,20. At tith CPAP . More coith CPAP ,22. TherA tight fitted facemask can cause discomfort but it is fundamental for achieving adequate positive pressure in the airways during CPAP. The patients at our CCU were systematically queried about their experiences with BCPAP treatment and they found that the reduction of dyspnea outweighed any discomfort of the facemask. These findings were exemplified by a patient who was successfully treated during many episodes of ACPE with BCPAP: although initially wary about a facemask he subsequently easily accepted it for many times . AlthougThe effectiveness of BCPAP has also been demonstrated in prehospital settings . Future There are some limitations of the present study. First, the data from the control group is retrospective and ACPE is not always easy to diagnose. Based on the evaluation of all patient records great care was taken to include all patients who were admitted to our hospital with ACPE according to established criteria . Second,The results of this study underscore the utility of the Boussignac CPAP system as a simple, effective and cost saving modality on a CCU for patients with ACPE.The author(s) declare that they have no competing interests.WD, HR, and MN executed the study, collected data and drafted the manuscript. WD, TJ, IH, WN, KV, FZ and MN participated in conception, design, analysis and interpretation. TJ, IH, WN, LA, FZ and MN assisted in writing the manuscript. TJ, LA, FZ, and MN revised the manuscript critically for important intellectual content, and all gave final approval of the manuscript.The pre-publication history for this paper can be accessed here:


Dystocia, difficult labour, is a common but also complex problem during childbirth. It can be attributed to either weak contractions of the uterus, a large infant, reduced capacity of the pelvis or combinations of these. Previous studies have indicated that there is a genetic component in the susceptibility of experiencing dystocia. The purpose of this study was to identify susceptibility genes in dystocia.OXT) on chromosome 20 and oxytocin-receptor (OXTR) on chromosome 3.A total of 104 women in 47 families were included where at least two sisters had undergone caesarean section at a gestational length of 286 days or more at their first delivery. Study of medical records and a telephone interview was performed to identify subjects with dystocia. Whole-genome scanning using Affymetrix genotyping-arrays and non-parametric linkage (NPL) analysis was made in 39 women exhibiting the phenotype of dystocia from 19 families. In 68 women re-sequencing was performed of candidate genes showing suggestive linkage: oxytocin (We found a trend towards linkage with suggestive NPL-score (3.15) on chromosome 12p12. Suggestive linkage peaks were observed on chromosomes 3, 4, 6, 10, 20. Re-sequencing of OXT and OXTR did not reveal any causal variants.Dystocia is likely to have a genetic component with variations in multiple genes affecting the patient outcome. We found 6 loci that could be re-evaluated in larger patient cohorts. Dystocia, defined as prolonged and difficult labour, is a common obstetric problem affecting 6-8% of all deliveries . It is aParturition is regulated by many factors and the mechanisms regulating labour and the expulsion of the child are incompletely understood . Animal We used the Swedish Medical Birth Registry  that covFollowing the telephone interviews and the study of the medical records we concluded that the phenotype is heterogeneous since not all of the women had dystocia as an indication for their caesarean section. Given that the primary focus of the study was uterine dysfunction rather than other reasons for dystocia and to enable us to pick the affected sib pairs with the most uniform phenotype for genetic analysis we divided the diagnosis of dystocia into four subgroups: certain dystocia, likely dystocia, unlikely dystocia and no dystocia. These subgroups are described below:1. Certain dystocia: Definite diagnosis , all subsequent deliveries by caesarean section or instrumental delivery and no other indications.3. Unlikely dystocia: No induction of labour, short delivery time (< 20 h), other indication for caesarean i.e. relative disproportion, child ≥ 5000 g, subsequent deliveries without caesarean section or instrumental delivery.4. No dystocia: Absolute disproportion, breech presentation, caesarean section before start of labour, delivery misclassified as caesarean but was actually vaginal.Additional sisters and cousins were invited to participate in families where several women were identified with delivery-related problems. Of the initial 150 women, 56 were excluded and 10 new cases added making the study population a total of 104. Of these 104 women, 83 provided a blood sample for the genetic analysis. Only families with women in group 1 and 2 were used in the genetic analysis with non-parametric linkage (NPL). See Table ® Mapping 10 K 2.0 array containing approximately 10,000 SNP markers following the manufacturer's instructions .Single nucleotide polymorphism (SNP) genotyping was performed for 18 affected sib-pairs and one family with three affected siblings using the Affymetrix GeneChipMERLIN  was usedOXT) and oxytocin receptor (OXTR) genes. Cases were not matched with the reference group, but were used to establish an estimate of the population frequency of potential mutations. Exons including 100 bp flanking sequence on both sides and 1 kb upstream of the first exon were amplified using polymerase chain reaction (PCR). Purified PCR products were sequenced using DYEnamic ET dye terminator kit following manufacturer's instructions  and electrophoresed using a MegaBACE 1000 instrument . Sequence analysis was performed using the MegaBACE Sequence Analyser 3.0 software (Amersham Biosciences) and Staden package computer programs [Genomic DNA from 68 affected individuals with dystocia and a historic reference material consisting of 107 healthy women without adverse obstetric history who had given a written consent to participate in studies on complications of pregnancy were used to sequence oxytocin  exhibiting a phenotype of certain or likely dystocia. Since many of the subjects were post-term, 53 (51.0%) of them had undergone induction of labour. In 16 of the 47 families (34.0%), the mother had had dystocia or some other obstetric problem such as instrumental or breech delivery. Thirty-eight (36.5%) women said that the delivery had been a bad or very bad experience and 22 (21.2%) said that their first delivery had negatively influenced the number of children they had born. Clinical characteristics have been collected in Table .1% exhibAccording to simulations performed with Merlin, significant P-value (identified by an NPL-score reached in 5% of simulations) would have corresponded to an NPL-score of 3.64, while suggestive P-value (identified by an NPL-score reached at least once per simulation) would have corresponded to and NPL-score of 1.98. Our best linkage peak was located on chromosome 12p12 (NPL score 3.15), and other peaks with suggestive linkage were found on chromosomes 3, 4, 6 and 20. Linkage results are summarized in Table OXT and OXTR exons including splice sites and putative promoter regions in five individuals with dystocia and one control to detect common variations. One known polymorphism was detected within the OXT locus, but did not differ in allele frequency from that expected from dbSNP data. We were unable to sequence exon 2 due to high (> 70%) GC content. Sixteen variations were identified within the OXTR locus, of which four were novel and the remaining 12 were included in dbSNP with rs numbers. Eleven OXTR polymorphisms were selected for sequencing in 68 dystocia cases  and oxytocin receptor (OXTR), both of which are obvious candidate genes, did not allow us to identify any potential causal mutations. However, it is possible that we have overseen regulatory variants affecting gene expression, mRNA stability or localization of protein product. It is also possible that we have overlooked true candidate genes on regions showing suggestive linkage on chromosomes 4, 6, 10 and 12. We chose comparison with a healthy historic reference material for the re-sequencing of OXT and OXTR. Evidently this group differs from our cases regarding age, BMI, gestational length and birth weight but we did not manage to find any mutations. If we actually had found any mutations a comparison with matched controls would of course have been preferable.According to our knowledge, this is the first paper assessing the genetic origin of dystocia through non-parametric linkage analysis. There is strong suggestive evidence of linkage at chromosome 12p12 and we found several possible genes in the areas of interest but none that struck as being solely responsible for this condition. The re-sequencing of oxytocin  declined to participate directly at the telephone interview and no case of offending a woman by this direct approach was encountered. On the contrary, the majority of women were willing to donate blood samples without any compensation. Thus, the direct approach appears feasible and has great potential to increase the scientific value of medical registers.This study indicates that dystocia is a complex disease that is probably not caused by a single locus disease allele. Knowledge of the genetic architecture of complex diseases is still incomplete and it is possible that the risk for any common disease is dependent on a large number of loci, each with a number of low frequency disease-predisposing alleles . The stuDuring evolution there is balance between mutation and selection and since dystocia-causing mutations would not be brought on to the next generation in the absence of the possibility of caesarean section, there is most likely genetic heterogeneity responsible for the phenotype. The disease-causing alleles can of course also be propagated by male offspring, but we have so far not studied any possible male phenotype.Since the occurrence of dystocia is likely to involve allelic variations in several different loci we think that it might be very cumbersome and costly to collect a large enough sample set to reach significance in any single locus with genome-wide linkage analysis. An alternative and complimentary approach would be to focus on extensive re-sequencing of candidate genes in affected individuals and controls to assess genetic variability within candidate loci and identify possible causal variants. We have focused on the maternal genotypes but during pregnancy it is probably of interest to take the genetics of the child into account as well. For example, our study does not assess whether dystocia risk is affected by the properties of the fetus, but this might be worthwhile to pursue further.In the overall assessment of a woman giving birth the obstetrician might find the knowledge that epidemiological studies have shown an increased risk of dystocia in a primiparous woman if her mother or sister has experienced the same problem to be useful. Our study indicates that there actually might be a genetic background for dystocia through a strong suggestive evidence of linkage at chromosome 12p12 and also at 5 other loci that might be of interest. We believe that larger studies including patients with a well defined phenotype of dystocia might lead to new insights into the aetiology and physiology of this important condition.The authors declare that they have no competing interests.MA participated in planning the study, collected the material, participated in the genetic and statistical analysis and wrote the main part of the manuscript. HP and KK participated in designing the study, and made the main part of the NPL-analysis and re-sequencing as well as the statistical analysis. JK and MW originated the study, participated in its design and helped in writing the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/11/105/prepub


A surgical resection is currently the preferred treatment for esophageal cancer if the tumor is considered to be resectable without evidence of distant metastases (cT1-3 N0-1 M0). A high percentage of irradical resections is reported in studies using neoadjuvant chemotherapy followed by surgery versus surgery alone and in trials in which patients are treated with surgery alone. Improvement of locoregional control by using neoadjuvant chemoradiotherapy might therefore improve the prognosis in these patients. We previously reported that after neoadjuvant chemoradiotherapy with weekly administrations of Carboplatin and Paclitaxel combined with concurrent radiotherapy nearly always a complete R0-resection could be performed. The concept that this neoadjuvant chemoradiotherapy regimen improves overall survival has, however, to be proven in a randomized phase III trial.The CROSS trial is a multicenter, randomized phase III, clinical trial. The study compares neoadjuvant chemoradiotherapy followed by surgery with surgery alone in patients with potentially curable esophageal cancer, with inclusion of 175 patients per arm.The objectives of the CROSS trial are to compare median survival rates and quality of life , pathological responses, progression free survival, the number of R0 resections, treatment toxicity and costs between patients treated with neoadjuvant chemoradiotherapy followed by surgery with surgery alone for surgically resectable esophageal adenocarcinoma or squamous cell carcinoma. Over a 5 week period concurrent chemoradiotherapy will be applied on an outpatient basis. Paclitaxel (50 mg/m2) and Carboplatin (Area-Under-Curve = 2) are administered by i.v. infusion on days 1, 8, 15, 22, and 29. External beam radiation with a total dose of 41.4 Gy is given in 23 fractions of 1.8 Gy, 5 fractions a week. After completion of the protocol, patients will be followed up every 3 months for the first year, every 6 months for the second year, and then at the end of each year until 5 years after treatment. Quality of life questionnaires will be filled out during the first year of follow-up.This study will contribute to the evidence on any benefits of neoadjuvant treatment in esophageal cancer patients using a promising chemoradiotherapy regimen.ISRCTN80832026 Esophageal cancer is a highly lethal disease, as reflected by an overall 5-year survival rate of 10%. The totaSurgical resection is currently the preferred treatment for esophageal cancer if a patient is fit enough to undergo major surgery and the tumor is considered to be resectable without evidence of distant metastases (cT1-3 N0-1 M0). However, approximately 30% of operated patients, clinically considered to have resectable disease, have microscopically irradical resections performed on. -6The goals of neoadjuvant chemotherapy are a reduction of recurrence from occult lymphatic and/or distant metastases with improvement of survival and possible tumor shrinkage with an increased radical resectability rate. In many of the performed phase II studies the patients who had objective response to chemotherapy had a significantly better survival compared to non-responding patients.,8The number of randomized phase III studies comparing neoadjuvant chemotherapy followed by surgery versus surgery alone is limited. -13 The rChemotherapy and radiotherapy can interact in several ways. Both treatment modalities may be active against different tumor cell populations (additive effect), the chemotherapy may be effective against micrometastases while radiation is active locoregionally . Chemotherapy may synchronize cells in a vulnerable phase for radiotherapy, decrease repopulation after radiotherapy and, by shrinking a tumor, enhance reoxygenation, which is advantageous for radiotherapy.,172 given 3 or 4 days before the start of radiotherapy and 4 courses afterwards. × (Glomerular Filtration Rate + 25).It is possible that some patients will experience asymptomatic bradycardia during the Paclitaxel infusion. In addition, hypersensitivity reactions are possible and generally occur within the first few minutes of initiating the infusion. For these reasons, it is recommended that there is constant supervision and that the vital signs are monitored every fifteen minutes during Paclitaxel administration. Thereafter, patients may be observed and heart rate and blood pressure checked if necessary, according to clinical symptoms.A total dose of 41.4 Gy will be given in 23 fractions of 1.8 Gy, 5 fractions per week, starting the first day of the first cycle of chemotherapy. All patients will be radiated by external beam radiation, using 3-D conformal radiation technique. The patient will be positioned in supine position. Reproducibility will be assessed by orthogonal laser beams.The Gross Tumor Volume (GTV) is defined by the primary tumor and any enlarged regional lymph nodes., and wilThe Planning Target Volume (PTV) will provide a proximal and distal margin of 4 cm, in case of tumor extension into the stomach, a distal margin of 3 cm will be chosen. A 2 cm radial margin around the GTV will be provided to include the area of subclinical involvement around the GTV and to compensate for tumor motion and set-up variations.Both lungs will be contoured. The heart will be contoured on all slices; its cranial border will include the infundibulum of the right ventricle and the apex of both atria., and wilRadiation therapy will be delivered using a multiple field technique. Treatment can be given with the combination of anterior/posterior, oblique or lateral field. Customized blocks or a multi-leaf collimator will be used to shape the treatment fields. All patients will undergo a 3D planning. Beams-eye-view (BEV) displays will be used to ensure optimal target volume coverage and optimal normal tissue sparing. The most appropriate technical solutions  will be chosen as long as they comply with the International Commission on Radiation Units and Measurements (ICRU) 50/62 safety margins and homogeneity requirements.Dose-Volume-Histograms (DVHs) of both lungs, the heart and spinal cord will be obtained for all patients. DVHs will mainly be used to document the normal tissue damage. DVHs may also help to select the most appropriate treatment plan. The volume of lung tissue that receives 20 Gy or more will not exceed 30% of the total lung volume (V20 Gy lung < 30%). The volume of the heart that receives 40 Gy will not exceed 30% of the heart volume (V40 Gy heart < 30%) and the volume of the liver that receives 30 Gy will not exceed 60% of the total liver volume (V30Gy liver < 60%). There is no limit for the maximal length of involved esophagus as long as the normal tissue criteria are not exceeded.The risks for severe pneumonitis for patients treated under this protocol will be minimized as the volume of both lungs will be limited by the use of BEV planning and field-shaping . The spinal cord tolerance (50 Gy) will not be exceeded with this technique.Radiation therapy will be delivered with megavoltage equipment with photon energies of equal to or greater than 6 MV. A multileaf collimator or individually shaped blocks will be used to shape the irradiation portal according to the planning target volume.The prescription dose will be specified at the ICRU 50/62 reference point, which will be the isocenter for most patients. The daily prescription dose will be 1.8 Gy at the ICRU reference point and the 95% isodose must encompass the entire planning target volume (PTV). The maximum to the PTV must not exceed the prescription dose by > 7% (ICRU 50/62 guidelines). Tissue density inhomogeneity correction will be used. Portal images will be obtained during the first fraction of all fields. On indication portal images will be repeated.9/L and/or platelets < 50 × 109/L, chemotherapy will be delayed by 1 week until recovery above these values. In case of febrile neutropenia (granulo's < 0.5 × 109/L and fever > 38.5°C) or in case of severe bleeding or requiring ≥ 2 units of platelet transfusions further chemotherapy will be withheld.If on day 8, 15, 22, 29, and 36 the WBC are < 1.0 × 10Hypersensitivity reactions will be classified as mild, moderate or severe. Definitions and management guidelines are outlined in table Radiotherapy, especially concurrent with chemotherapy can lead to acute esophagitis. In some cases medical support and/or a feeding tube will be necessary. In the event of grade 4 radiation induced esophagitis both chemotherapy and radiotherapy will be withheld until the esophagitis has recovered to grade 3. Other acute complications of the radiation therapy are erythema, cough, nausea, fatigue and weight loss. In the first weeks to six months after the irradiation radiation pneumonitis or fistula formation can occur.Patients randomized for surgery alone will be treated as soon as possible after randomization. In the chemoradiation arm, surgery will be performed preferably within 6 weeks after the completion of the chemoradiation. For carcinomas proximal to the tracheal bifurcation a transthoracic esophageal resection with a two field lymph node dissection is preferred. For carcinomas distal of the tracheal bifurcation but proximal to the gastro-esophageal junction, a transthoracic approach with a two field lymph node dissection or a transhiatal approach can be performed, depending on both patient characteristics and local expertise. For distA wide local excision including the N1 lymph nodes is carried out in both techniques including a standard excision of the lymph nodes around the celiac axis (separately collected with left gastric artery marked by a suture). The continuity of the digestive tract will be restored by a gastric tube reconstruction or a colonic interposition procedure with an anastomosis in the neck.The resection specimen will be evaluated essentially using the standard protocol . The most recent UICC protocol is used for TNM-classification and stage grouping.,33 In thThe pathology report should contain the following: site of the tumor/lesion, type and grade of the tumor, extension into the esphageal wall, resection margins, therapy effects (Mandard score), lymph node status including the site and the number of nodes with therapy effects.After completion of the protocol, patients will be followed up every 3 months for the first year, every 6 months for the second year, and then at the end of each year until 5 years after treatment, to document late toxic effects and, if applicable, disease relapse or progression, and death. Patients in the chemoradiation arm will be asked to fill out quality of life questionnaires before and after therapy and every three months during the first year of follow up. Patients randomized for surgery alone will fill out the questionnaires before surgery and every three months during the first year of follow-up.Data will be analyzed according to the 'Intention to treat' principle. We believe that an estimated difference in median survival of 16 months  versus 22 months  would justify applying this regimen in the future. We calculated that for this purpose 350 patients, 175 patients per arm, have to be enrolled. We assumed a two-sided significance level of 0.05 and a power of 0.80.[The responsible physician will inform the patient about the background and present knowledge on the drugs under study with special reference to known activity and toxicity. It must be emphasized that the patient is allowed to refuse the treatment either before or at any time during the study. Before the patient is entered in the study the patient's written consent will be obtained. The principal investigator (AvdG) will ensure that this study will be carried out in agreement with the "Declaration of Helsinki, Tokyo, Venice" or the laws and regulation of the country, whichever provides greater protection of the individual. The study has been approved by the institutional ethical review committee.Any unexpected clinical adverse event or abnormal laboratory test value that is serious, including death or overdose, occurring during the course of the study, irrespective of the treatment received by the patient, must be reported to the study coordinator within one working day of occurrence.An adverse event is serious if it is fatal or life threatening, permanently disabling, requiring hospitalization other than for planned treatment or if it is an overdose. A death occurring during treatment within 4 weeks after stopping treatment, whether treatment-related or not must be reported to the study coordinator.A number of phase III trials have been performed comparing chemoradiotherapy followed by surgery versus surgery alone.,36-44 Onet al. reported a 13% absolute difference in survival at 2 years in favor of those patients receiving neoadjuvant chemoradiotherapy.[Several meta-analyses have been performed over the years, with varying conclusions.,45-49 Thotherapy. However,Currently most patients in Europe and the US diagnosed with esophageal cancer present with adenocarcinomas located in the distal esophagus. Many of these patients are nowadays routinely treated with neoadjuvant chemoradiotherapy. Unfortunately, this policy is based on data of less than 200 patients with an adenocarcinoma of the esophagus treated with concurrent neoadjuvant chemoradiotherapy, as shown by the latest meta-analysis. Therefore, we believe surgery alone is still an acceptable treatment for patients with resectable esophageal cancer.Based on the favorable results of our phase II trial with a neoadjuvant chemoradiotherapy regimen consisting of weekly administrations of Carboplatin and Paclitaxel combined with concurrent radiotherapy we initiated a phase III trial comparing this regimen with surgery alone in a sufficiently large group of patients. The accrual of this trial, a total of 350 randomized patients, is expected to be completed in the last quarter of 2008. This study will contribute to the evidence on any benefits of neoadjuvant treatment in esophageal cancer patients using a promising chemoradiotherapy regimen.The authors declare that they have no competing interests.MvH drafted the manuscript. AvdG and LBK co-authored the writing of the manuscript. All other authors participated in the design of the study during several meetings and are local investigators at participating centers. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Tetraodon nigroviridis and Takifugu rubripes. These correspond to Homo sapiens chromosomes 4, 5, 8 and 10.One of the many gene families that expanded in early vertebrate evolution is the neuropeptide (NPY) receptor family of G-protein coupled receptors. Earlier work by our lab suggested that several of the NPY receptor genes found in extant vertebrates resulted from two genome duplications before the origin of jawed vertebrates (gnathostomes) and one additional genome duplication in the actinopterygian lineage, based on their location on chromosomes sharing several gene families. In this study we have investigated, in five vertebrate genomes, 45 gene families with members close to the NPY receptor genes in the compact genomes of the teleost fishes H. sapiens, M. musculus, D. rerio, T. rubripes and T. nigroviridis. 26 gene families, including the NPY receptor genes, (plus 3 described recently by other labs) showed a tree topology consistent with duplications in early vertebrate evolution and in the actinopterygian lineage, thereby supporting expansion through block duplications. Eight gene families had complications that precluded analysis (such as short sequence length or variable number of repeated domains) and another eight families did not support block duplications . RT-PCR carried out with several tissues in T. rubripes revealed that all five NPY receptors were expressed in the brain and subtypes Y2, Y4 and Y8 were also expressed in peripheral organs.Chromosome regions with conserved synteny were identified and confirmed by phylogenetic analyses in We conclude that the phylogenetic analyses and chromosomal locations of these gene families support duplications of large blocks of genes or even entire chromosomes. Thus, these results are consistent with two early vertebrate tetraploidizations forming a paralogon comprising human chromosomes 4, 5, 8 and 10 and one teleost tetraploidization. The combination of positional and phylogenetic data further strengthens the identification of orthologs and paralogs in the NPY receptor family. The evolutionary relationships of the NPY-receptor family receptors in vertebrates have been difficult to resolve due to uneven evolutionary rates and because some subtypes are missing in some classes of vertebrates. By using information on chromosomal location, initially in pig and human ,2, we suGene duplication by tetraploidization in the chordate lineage was proposed by Susumu Ohno in 1970 , based uIn addition to the Hox-chromosome similarities, Lundin also reported resemblance within three other groups of human chromosomes. One group consisted of Hsa4 and Hsa5 , later fThe sizes of the quadrupled paralogous gene regions have been difficult to determine because of numerous chromosomal rearrangements during the approximately 500 Myr since the tetraploidizations. Several vertebrate genome projects have recently been reported or are in progress, but due to incomplete assembly of the sequences into contigs or scaffolds, let alone chromosomes, these cannot always be used to analyze conserved synteny or paralogous gene regions. Another complicating factor has been the uneven divergence rates in some of the daughter genes after the duplications ,33-35 thOur laboratory has previously reported that the genes encoding NPY (neuropeptide Y)-family receptors, which belong to the superfamily of rhodopsin-like G-protein-coupled receptors (GPCRs), are located in the paralogon comprised of the human chromosomes Hsa4, 5 and 10 , althougTetraodon. nigroviridis and Takifugu. rubripes, and performed phylogenetic analyses of these gene families. This approach has been named "transitive homology" .The sequenced RT-PCR products revealed that the Y4 sequence contained an extended second extracellular loop in pes Fig. . This exp 3 Fig. . Comparisee Fig. . The T. T. nigroviridis NPY receptor genes. In addition to these, another 9 families were included due to linkage to the NPY receptors in T. rubripes. Phylogenetic analysis of these 45 families (including the NPY receptor family) confirmed 26 to be compatible with an expansion in vertebrate evolution before the origin of gnathostomes. Neighbor-joining trees are shown in Fig. T. nigroviridis was 556 (Y4), 310 (Y8a), 375 (Y8b), 487 (Y2) and 370 (Y7) according to the 34.1d version of the Ensembl database. This number of genes (2098) represents approximately 7.5% of the total gene number in the T. nigroviridis genome . The corresponding human orthologs situated on chromosomes 4, 5, 8/2/7 and 10 are dispersed over a large portion of these chromosomes. The synteny group associated with T. nigroviridis Y8a/Y8b seems to have been broken up in the human genome because some families are located on chromosome 8 and a few are on chromosomes 2 and 7. In addition to the 35 gene families with at least three members on the T. nigroviridis chromosomes, 127 gene families were identified that are represented on two of the chromosomes [see Additional file A total of 35 gene families were found with members in three or four of the regions harboring the 4), 310 Ya, 375  and human . These results are in agreement with previous results investigating other genes present on Hsa 4, 5, 8 and 10 [The statistical test of the investigated paralogous regions based on a binomial test as used previously by Vienne et al.  shows th8 and 10 .T. nigroviridis and families known to be linked to the NPY receptor genes in the T. rubripes genome. Three families that also support block or chromosome duplications were recently analyzed by other groups and are therefore not described here, namely two subfamilies of adrenergic receptors and the ADAM family of metzincins [Descriptions of the 25 subfamilies linked to the NPY receptor genes investigated in detail in this study are given below, i.e., the families that have members on 3 or 4 of the paralogous chromosomal regions in tzincins ,45.T. nigroviridis and families studied due to members being present on the NPY receptor scaffolds in T. rubripes) see Additional file Danio rerio genome) as well as comparison with additional genomes not available at the onset of this study.Eight families with members on these chromosomes showed a tree topology seemingly inconsistent with expansion in early vertebrate evolution i.e. with several outgroup sequences dispersed among the vertebrate sequences, suggesting earlier origin of these paralogs. In addition to these families, eight families had to be left out of the analysis because of short sequence length, high conservation and therefore uninformative alignments or difficulties to generate reasonable alignments due to varying numbers of repeated domains. For a complete list of all families in the regions investigated  includes proteins with a metalloprotease domain, disintegrin and spacer domains and a number of trombospondin repeats. Members of this protein family have been implicated in several diseases . The humThe ankyrin gene family is one of the families earlier found to be part of a paralogon ,14 with 2+ ions [A core domain consisting of four repeated units, each about 70 amino acids long, characterizes the annexin family. Each repeat unit contains 5 alpha helices that usually contain a "type 2" motif for binding of Ca2+ ions . The phyThis family contains two members in the human genome (AP3M1 and AP3M2) on Hsa8 and Hsa10 consistent with early vertebrate block duplication. They are members of a larger family of proteins called adaptins that are important for vesicular transport common to all eukaryotes ,52. The The CNNM protein family  contains four members in both the human and the mouse genomes ,54. ThesThe dual specificity phosphatases (or MAP kinase phosphatases) are evolutionarily conserved enzymes that are important in the regulation of apoptosis, cell proliferation and cell differentiation. They exert their effects by dephosphorylating and thereby inactivating MAP kinases . The recT. nigroviridis is a subfamily of the fibroblast growth factors (FGFs) suggesting that both ligands and receptors were duplicated in the same time window. Hurst and Lercher observed that many ligand genes are linked to their receptor genes  and that this could be due to block duplication that would keep the ratio of gene products at the same level as before duplication [The fibroblast growth factor receptors comprise one of the families already described as being a part of this paralogon ,14. The lication . Our analication -59.The HNRNP family is a family of proteins that contain two RBD or RRM domains. These proteins are known to interact with telomeric repeats d(TTAGGG) and to 3'splice sites r(UUAG/G) and have also been proposed to have a role in the regulation of mRNA stability. One of the human members, HNRPD has previously been mapped to Hsa 4q21 and been shown to have a highly conserved ortholog in the mouse genome . Our preLGI1 (leucine-rich gene – glioma inactivated) was discovered by positional cloning in 1998 and found to be mainly expressed in neural tissues, particularly in the brain. The gene is localized to human chromosome 10q24, a region found to be rearranged or deleted in several types of malignant brain tumors. Because of this LGI1 was proposed to be a tumor suppressor gene [LGI1 were found in the human genome and it was shown that these four genes constitute a subfamily of leucine rich repeat (LRR) genes [The gene sor gene . Three aR) genes . The topA subfamily of the large superfamily of mitogen-activated protein kinases (MAPK) was identified as being part of this paralogon. This subfamily contains the human members MAPK8, MAPK9 and MAPK10 . These proteins are involved in a wide variety of cellular processes like cell growth, proliferation, differentiation, immunity and development . For a rThe MXD protein family has four members in the human genome. These proteins are characterized by a common basic-helix-loop-helix leucine zipper domain necessary for the formation of heterodimers with other proteins, such as Myc and Max . The funAll intermediate filaments have a similar structural organization with a central alpha-helical rod domain that begins and ends with highly conserved aa-motifs necessary for correct assembly. Intermediate filament proteins have earlier been viewed as scaffolding structural proteins but more and more data suggest much more dynamic roles for these proteins . The phyThe tachykinin receptors are GPCRs that bind the amidated neuropeptides substance P, neurokinin A and neurokinin B as well as some other related peptides . In vertOxoglutarate dehydrogenase  makes up a part of the enzyme complex responsible for conversion of α-ketoglutarate to succinyl coenzyme A in the Krebs cycle . The genThe PDZ and LIM domain-containing family is a multi domain protein family characterized by the presence of one PDZ and one or several LIM domains. Both the ABLIM family described above and the PDLIM family described here belong to the same superfamily . Our anaSaccaromyces cerevisiae). This family contains three closely related members of duplicated genes in the human lineage clearly duplicated after 2R but the two fish genes support the paralogon described here. The function of these proteins is unknown but they are thought to be involved in intra-mitochondrial protein sorting. One of the human genes is present on Hsa5 and earlier observed to be linked to one member of the MXD family (see above) [This family contains a conserved domain named MSF (after the protein MSF1 found in the yeast e above) .The SAMD8 family (here named after one of its members) contains three different genes in the human genome: SAMD8 , TMEM23 or Mob  and SMS2 (Sphingomyelin synthase 2). Proteins in this family all contain a SAM domain and 4–6 transmembrane domains and constitute a subset of a larger family of spingomyelin synthases . Our anaDanio rerio is indicative of any extra duplication.The secreted frizzled related proteins  have five members in the human genome, three of which are located in the regions analyzed here. The SFRP family is characterized by a frizzled domain and a C-terminal netrin domain. Proteins of the SFRP family have been implicated in regulating Wnt-frizzled signalling either by interacting with Wnts or the frizzled receptors ,76. Our The SORB or vinexin family has three members in the human genome namely vinexin, CAP (c-Cbl associated protein)/ponsin and ArgBP2 (Arg-binding protein 2). Common for all these proteins is that they contain a sorbin homology domain (pfamID PF02208) and three SH3 domains . MembersThe two RNA binding proteins TIA1 (RNA-binding protein TIA-1)  and TIARTetraspanins are a large family of membrane proteins with four transmembrane domains (hence the name). These proteins are present in a wide variety of organisms and it has been proposed that the tetraspanin-like proteins present in plants share a common origin with tetraspanins in animals . TetraspC. elegans and implicated in regulation of netrin signalling since mutation of unc-5 causes neural migration defects while ectopic expression of unc-5 causes netrin-dependent redirection of axon growth in some neurons. Four different paralogs of UNC-5-like proteins have been found in vertebrates. These proteins contain two extracellular immunoglobulin-like domains and two extracellular trombospondin type 1 (TSP_1) domains [The transmembrane protein UNC-5 was first characterized in  domains  and thre domains . The neu domains . The topThe ZIMP family of proteins contains a conserved SP-Ring/Miz domain, which they share with other PIAS proteins (protein inhibitors of activated STATS). In human, this family contains two members on Hsa7 and Hsa10 . Both thT. nigroviridis genome has two additional members resulting from the teleost fish tertaploidization on Tni2 and Tni Unr.The ZINK family contains two proteins (zink finger protein 703 and zink finger protein 503) in the human genome. Not much is known about the functions of these proteins. They both contain a classical C2H2 zinc finger domain (pfam ID PF00096). The positional and phylogenetic information for these two genes are in agreement with block duplication before the actinopterygian-sarcopterygian split. The T. nigroviridis and T. rubripes genomes. We also used the information from the analyses of neighboring gene families to assign orthology and paralogy of the NPY receptor genes. The present study identified the receptors Y2, Y4, Y7, Y8a and Y8b in the two pufferfish genomes  and Y7 is still present in chicken [In the tetrapod lineage, Y7 and Y8 are present in the frog  chicken  while bo chicken . Finally chicken , but theT. rubripes genes revealed that both Y4 and Y8a have received insertions in the coding region after divergence from the lineage leading to zebrafish, as seen for the melanocortin receptor genes MC2R and MC5R in pufferfish [T. rubripes. The largest of the Y8a introns is 2.5 kb and carries a small exon that extends extracellular loop 2 with 21 amino acids. In the Y8b gene one cryptic intron is spliced and this shortened splice variant is present in a minor proportion of the mRNAs and presumably leads to a nonfunctional partial receptor protein. This cryptic intron splice site is also present in the medaka gene found in the genome database. We suggest that the Y4 insertion was also probably a reinserted intron that subsequently lost its splice signals, but thanks to its small size and the maintained reading frame it could be tolerated as a protein expansion in extracellular loop 2. Functional expression of the receptors will be necessary to see if the extensions of loop 2 in Y4 and Y8a affect the ligand-binding properties of the receptors. Studies of the anatomical distribution of the mRNAs for the five NPY receptors in T. rubripes, as detected by RT-PCR, show that all five are present in the brain and eye. Interestingly, Y8a and Y8b differ greatly in their distribution in that Y8b is expressed in all organs investigated whereas Y8a shows the narrowest distribution of all receptors, although Y8a and Y8b are most closely related to each other having originated in 3R. Functional information is still missing for many fish NPY-family receptors. The pufferfish NPY system is more complicated than in most other vertebrates because there are four peptide ligands due to 3R duplicates of both NPY and its relative PYY [Our characterization of the five fferfish , despitefferfish . In the tive PYY .Several authors have discussed what criteria one should have for identifying paralogous regions and to safely infer that block duplications or chromosome duplications have occurred -96. We aIt has been suggested that the observed patterns of paralogy within genomes, often interpreted to be the remnants of large-scale duplications, could be the result of convergent evolution . We beliAmong the gene neighbors of the NPY receptor genes, we analyzed 44 gene families with members on the chromosomes bearing NPY-receptor genes. In fact, chromosomes orthologous to three of the four human chromosomes were all found to be present in duplicate in the fish genomes, consistent with the 3R teleost tetraploidization see Fig. , 8 and 9Eight gene families had to be excluded from analysis because they had too many family members to achieve a reasonably clear picture of their evolutionary relationships or problems in analyzing them due to too high sequence conservation to be informative or varying number of protein domains making it hard to obtain unequivocal alignments. More thorough analysis using diagnostic positions and comparisons of intron-exon structure could be used to clarify the relation of the different paralogs of these families. Thus, our study provides a minimal estimate of the number of gene families that expanded simultaneously with the NPY receptor genes.Out of the 44 gene families a total of 26+3 support block/chromosome duplications whereas eight gene families are unsupportive of the block duplication scheme with invertebrate sequences located between the vertebrate paralogs. The reasons for this may be earlier expansions of these families, uneven evolutionary rates among the daughter genes, gene conversion between family members or simply translocation of distant family members to the chromosomes in this study.We uncovered evidence for extra fish duplicates, in agreement with 3R, for 18 of these 26 gene families. However, because we were forced to exclude several gene family members as they lacked one or a few protein domains, this is certainly an underestimate of the true number (see above). It has been pointed out earlier that the number of genes retained after 3R is rather low as compared to after 1R and 2R . One gooT. nigroviridis chromosomes may further corroborate the conserved synteny between the chromosomal regions in this study, although they need to be analyzed phylogenetically as well. Among these 127 pairs of paralogs, all ten possible combinations of the five T. nigroviridis chromosomes are represented. The lack of sequence data from several important species is still a limiting factor in investigation of the early vertebrate chromosome duplications. In particular, sequences from the cephalochordate Branchiostoma floridae (first draft version of assembly recently released but with limited data on gene linkage) and representatives of the jawless and cartilaginous fishes will help to date the block/chromosome duplication events when more extensive gene contigs have been generated.The fact that there are 127 gene families with members on two of the investigated Saccharomyces cerevisiae) duplicated by tetraploidization around 100 million years ago [Arabidopsis thaliana produced by the most recent polyploidizations in this species that took place 20 to 60 million years ago [One interesting observation is that members of some of the gene families studied here have been reported to interact with each other in signaling networks or possess domains that commonly interact with each other. This opens for the possibility that blocks of genes that were duplicated simultaneously can co-evolve and therefore one might detect subfunctionalization of entire gene networks and not only of different paralogs within one family. Examples of such families according to the BioGRID database of protein interactions ,102 are ears ago  as well ears ago ,106. Theears ago -110 statears ago ,112.In earlier analyses of these paralogous regions using several other gene families, the order of duplication events has been inferred based on the phylogenetic trees giving highest support for the ,  topology . In our T. rubripes and T. nigroviridis and compared the chromosomal regions where the receptor genes are located in one additional fish and two mammalian species. The conserved synteny shows that many of the gene families were located together in the same chromosome regions of the common ancestor of gnathostomes more than 400 Myr ago [In summary, we have characterized the NPY receptor repertoire in the two pufferfishes  Myr ago   according to manufacturer's protocol. Purified total RNA was reverse transcribed and single-strand 5'RACE-ready cDNA was prepared using SMART RACE cDNA Amplification Kit . Primers used for the receptors as well as an internal actin control are listed in Table Total RNA was isolated from eleven T. nigroviridis genome, all Ensembl Gene IDs for genes positioned four megabases on each side of the receptor genes were downloaded and saved in an Excel file. In addition to Ensembl Gene IDs the file contained information on Ensembl Family ID and family description as well as information about chromosomal position and human orthologs. This list was sorted based on Ensembl Family ID and all multiple entries for the same gene due to multiple transcripts were removed. The gene families containing members close to three or more of the T. nigroviridis NPY receptor genes were used for phylogenetic analysis. In addition to gene families found in this way, several families with members on the NPY receptor gene harboring scaffolds in T. rubripes were included [see Additional file T. nigroviridis, T. rubripes, Danio rerio, Homo sapiens and Mus musculus. Invertebrate sequences representing at least one outgroup species were included in order to root the trees. For the main part of the trees both Drosophila melanogaster and Ciona intestinalis was used as outgroups. In cases where no clear orthologs could be found in these two species we used sequences from other invertebrate genomes available in the Ensembl database [Starting from the confirmed receptor genes in the database  . In cases where a large number of sequences were included in the Ensembl family, the initial phylogenetic tree was used to find relevant sub families represented by multiple vertebrate sequences and at least one outgroup sequence. The identified sub families were realigned and subjected to neighbor-joining analysis as described above. In addition to gene families represented on three or more of the T. nigroviridis genomes using the binomial test as described earlier by Vienne et al. [The position of paralogs were statistically tested for random distribution in both the human and e et al. . Genes be et al. .T. rubripes and wrote part of the manuscript. DL conceived and initiated the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.TAL performed the phylogenetic and chromosome analyses and drafted and coordinated the manuscript, FO performed many of the initial analyses, GS participated in the phylogenetic and chromosome analyses and contributed to the manuscript. L–GL spawned the concept of duplication of these chromosomal regions and contributed conceptually. SB participated in the design of experiments and contributed conceptually. BV performed the experimental work in Neighbor-joining trees for the 26 gene families analyzed in detail.Click here for fileQuartet puzzling trees for the 26 gene families analyzed in detail.Click here for fileTetraodon nigroviridis.List of gene families linked to the NPY receptor genes in Click here for fileCompressed archive of alignment files for all analyzed families in fasta format.Click here for fileSummary of statistical testing of paralogous regions.Click here for file


Anticipation of resource availability is a vital skill yet it is poorly understood in terms of neuronal circuitry. Rodents display robust anticipatory activity in the several hours preceding timed daily access to food when access is limited to a short temporal duration. We tested whether this anticipatory behavior could be generalized to timed daily social interaction by examining if singly housed male mice could anticipate either a daily novel female or a familiar female. We observed that anticipatory activity was moderate under both conditions, although both a novel female partner and sexual experience are moderate contributing factors to increasing anticipatory activity. In contrast, restricted access to running wheels did not produce any anticipatory activity, suggesting that an increase in activity during the scheduled access time was not sufficient to induce anticipation. To tease apart social versus sexual interaction, we tested the effect of exposing singly housed female mice to a familiar companion female mouse daily. The female mice did not show anticipatory activity for restricted female access, despite a large amount of social interaction, suggesting that daily timed social interaction between mice of the same gender is insufficient to induce anticipatory activity. Our study demonstrates that male mice will show anticipatory activity, albeit inconsistently, for a daily timed sexual encounter. The ability to anticipate events is crucial for survival. In particular, timing activity to take advantage of resource availability helps optimize energy usage and avoid predation. The most well-studied model of this phenomenon is food anticipatory activity (FAA), in which temporally restricting the availability of food results in an increase in activity prior to the time of feeding Since daily restricted water access To test for the ability of male mice to anticipate novel social interaction, a group of C57BL/6J male mice (n = 12) were divided into two groups: the experimental group (n = 6) received restricted access to a novel female for an hour each day during the light cycle from Zeitgeber Time (ZT) 9 to ZT 10  while the control group (n = 6) was disturbed slightly at both ZT 9 and ZT 10 to control for handling artifacts. To monitor activity levels, these mice were video recorded in their home cages for 23 h, starting at ZT10 (only while single housed) at days 0, 7, 14, 21, and 28 of this treatment. The videos were analyzed by an automated behavior recognition system, HomeCageScan 3.0 We examined the temporal aspects of home-cage activity to determine whether male mice show an increased activity  in the hours prior to daily female access . We noteWe next asked whether the one hour of daily female exposure influenced the amount of total activity . There were no significant differences in the overall activity between mice with restricted female access and control mice (Mann-Whitney Test) . BecauseWe repeated the timed female access experiment but changed two parameters to test how anticipation of social interaction is influenced by 1) mating experience and 2) familiar partners. First, both the experimental (n = 8) and control (n = 8) groups of male mice were cohabitated with a female for seven days (from day -7 until day 0). Second, starting on day 0, each mouse in the experimental group was paired daily with the same female for one hour for an entire week. At the end of the week of exposure to the same female for one hour the mice were video recorded to measure their activity. The male was assigned to a new, virgin female at the end of the weekly recording, and this procedure was repeated for five weeks total (with recordings starting at day -7 in this experiment). Control mice were handled in parallel with experimental mice but not exposed to females with the exception of the initial cohabitation period during day -7 to day 0 of the experiment.We examined the temporal aspects of home-cage activity to determine whether male mice show an increased activity in the hours preceding to daily female access . We noteIn order to determine the amount of social and sexual interaction during the restricted female access, we video recorded the hour of female access on days 15, 28, and 29. Days 15 and 29 were with novel female partners, while day 28 was interaction with a female with which the mice had been partnered repeatedly with for seven days in a row. This enabled us to assess if males interact with novel females differently than they interact with females they have grown accustomed to. Interaction between the male and female mice was quantified by manual annotation of video recordings in which the behaviors were categorized as sniff body, approach, chase, walk away, mount and attempt mount, thrust and/or ejaculation, or non-interacting. The male mice were awake during the entire one hour period (data not shown). Most of the interaction between the males and females was exhibited in the form of sniffing: the males sniffed the female a median of 62 times on day 15, 42.5 times on day 28, and 50 times on day 29 . The difRestricted feeding is the most well-documented example of a behavioral manipulation that induces AA, and self-imposed changes in the timing of food intake have been shown to contribute to the AA observed in rats limited to 1 h of water access each day, with free access to food In experiment 1, mice with restricted female access spent a similar amount of time as control mice with their noses in the food bin from ZT 5-9 except at day 28, where female-access males had a significant increase in food bin entry  . There wIn Experiment 2, there was no statistically significant difference in the time mice with restricted female access spent with their noses in the food bin relative to control mice during the four hours prior to female access (ZT 5-9)  . Mice wiOne possible explanation for the AA observed in mice in experiments 1 and 2 would be that these mice were disturbed and kept awake for an hour daily. To test this hypothesis in a non-social context, a new cohort (n = 17) of C57BL/6J male mice was divided into three groups: one group received running wheels  for two Mice that received running wheels did not exhibit any increase in activity during the four hours prior to wheel-running access relative to the mice in the control group or mice that received domes  . The totTo quantify the amount of activity observed in mice receiving domes, running wheels, or the controls, videos recorded during at least an hour and a half of the two hours of running wheel or dome access and were manually annotated using the Caltech Behavior Annotator software. Individual frames were labeled as running on the wheel, interacting with the wheel or dome (but not running), awake but not interacting with the wheel or dome, sleeping inside the dome, or sleeping outside the dome. On days 7 and 14, mice that received access to running wheels were awake during the entire recording period, in contrast to the mice that received shelter domes or the bedding disturbance controls . The datFrom our analysis above, we concluded that the moderate AA observed in social interaction experiments 1 and 2 was neither due to self-imposed restriction of feeding behavior nor increased daily timed physical activity but it was not clear if the AA was caused by social stimulation or anticipation of a sexual interaction. To disambiguate between these possibilities, we performed a restricted social interaction experiment using female-female pairs. Female C57BL/6J mice were single housed for one week, after which they were given one hour access to a female C57BL/6 Tyr partner  from ZT 9 to 10 in their home cage for 28 days consecutively. The same female-female partners were maintained for all 28 days of the experiment.We examined the temporal aspects of home-cage activity to determine whether female mice show an increased activity hours prior to daily female access . There wTo more rigorously quantify the level of interaction between male-female and female-female pairs, we made use of a dual mouse automatic tracking system. Screenshots of the tracker locating the center of masses of both mice while at different positions in the cage is shown in In restricted social interaction experiment 1, in which virgin males were given a novel female each day, a modest AA when we normalized for total activity was evident from two weeks of restricted female access until the experiment was terminated at four weeks . In sociA similar study of restricted socio-sexual interactions in rats reported that scheduled mating at ZT6 significantly increased food intake during the following 6 h, and that the number of rats showing AA to daily mate access significantly decreased when post-coital feeding was prevented . Thus, we examined our data set to determine if eating behavior was related to restricted female access. In social experiment 1, there was a correlation between anticipatory food bin entry and AA  but therWhen given restricted access to running wheels, mice are awake during the entire time they have running wheel access . PreviouOne of the more interesting results of this experiment was the fact that robust AA was not consistent within animals, as it suggests that AA need not be expressed daily in order to persist  and 2K. Although our results suggest that non-sexual social stimulus alone is not enough to induce AA in females, this result can be tested in male mice with restricted access to females in estrus in comparison to restricted access to ovariectomized females or to those not in estrus. In addition, future experiments can be improved by allowing males to be cohabitated with females for a week prior to restricted access, thereby allowing them to both gain sexual experience and recover from their previous subordinate status. To account for the loss of interest in mating post-ejaculation and other factors that influence AA caused restricted female access, it will be important in future studies to both record activity and social interaction with a higher sampling rate, ideally every day. The time of day for sexual and/or social interaction is another variable that could be modified, as rats show a much higher interest in copulation in the early part of the dark cycle et al.Taken together, these results and possible future iterations of social/sexual anticipatory conditions suggest that limited mate access causes mild AA, albeit this is far less robust than that observed with restricted food access. Importantly, these results suggest that food is not a wholly unique zeitgeber for the anticipatory arousal system, a question recently phrased by Webb ad libitum access to LabDiet Laboratory Rodent Diet 5001 and water and were on a 13 hours of light 11 hours of dark cycle.All experiments were approved by the Caltech Animal Care and Use Committee. Male and female C57BL/6 mice ranging from 10–12 weeks of age were ordered from Jackson Labs and single housed for forty-eight to seventy-two hours prior to initiating each experiment. All mice were allowed The videos of singly housed mice in the home cage were analyzed by an automated behavior recognition system, HomeCageScan 3.0 Twelve C57BL/6J male mice were divided into two groups: one group of six mice received restricted access to a C57BL/6J female, the other group of six mice was used to control for handling conditions. At the start of each week, one virgin female was deposited into the cage of each experimental male mouse at ZT 9 and removed at ZT 10. Each of the six females were given to a novel male partner for the next six days. On the seventh day of each week, the male mice were recorded for twenty-three hours prior to receiving the same female from the first day of the week. The female mice were returned to group housing  at the end of each hour, and sacrificed after seven days of interaction. To control for handling conditions, control mice were moved to the hood and had their food rack lifted at ZT 9 and ZT 10. All male mice were video recorded in their home cages at days 0, 7, 14, 21, and 28 of this treatment.Sixteen C57BL/6J male mice were cohabitated with a female partner for seven days prior to the experiment. Starting on day 0, the male mice were divided into two groups, one group of eight experimental mice that received restricted access to a female and another group of eight mice that was used to control for handling condition. A novel virgin female was placed in the cage of each experimental mouse at ZT 9 and removed and returned to group housing  at ZT 10:00. Each experimental male received access to the same partner from ZT 9 to ZT 10 each day for seven days. The mice were video-recorded from ZT 10 until ZT 9 the following day (for 23 hours) every seven days. At the end of each recording, the female was sacrificed and the male received a new virgin female partner in its cage for one hour for seven days. To control for handling conditions, control mice were moved to the hood and had their food wire lifted at ZT 9 and ZT 10:00. All male mice were recorded for twenty-three hours on day -7 , 0, 7, 14, 21, 28, and 35. The one hour interaction between mice were recorded on day 15, 28, and 29 for quantifying social interaction. Interaction behaviors were defined from the perspective of the male mouse and labeled as either sniff body, chase, thrust, or mount.Sixteen C57BL/6 female mice, 10 weeks old, were singly housed for six to eight days prior to their first restricted social interaction with a C57BL/6J- Tyr(c-2J)/J female (17-21 weeks old). Starting on day 0, the female mice were divided into two groups, one group of eight experimental mice that received restricted access to a stable female partner for one hour daily from ZT 9 to ZT 10 for the entire duration of the experiment and another group of eight mice that was used as a control and handled twice daily at the same times as the experimental mice. Female C57BL/6- Tyr(c-2J)/J (which have a white coat color caused by a single gene mutation in an otherwise C57BL/6 genetic background) were single housed at all times except when interacting with C57BL/6J females. The C57BL/6J female mice were video-recorded from ZT 10 until ZT 9 (for 23 hours) weekly . The one hour of social interaction (ZT 9-10) was also recorded weekly for quantifying of social interaction. Interaction behaviors were defined from the perspective of the C57BL/6 (black mice), and labeled as one of the following: groom (black mouse grooming white mouse), receive groom (white mouse grooming black mouse), sniff body (black mouse with its nose in contact with the white mouse), receive sniff (white mouse with its nose in contact with the black mouse), chase (any instance where the black mouse approached the white mouse at a high velocity), repeated pawing (any time when the white mouse was moving and the black mouse touched its paws with it for more than three times), of wrestling (any instance with a large amount of interaction and movement and the dominant mouse was unidentifiable).Nonparametric tests were chosen for analysis of behavioral data as activity patterns did not follow a normal distribution. Statistical significance tests and multiple regressions were generated using GraphPad Instat. Sixteen male mice received access to a low-profile running wheel for twenty-four hours. The next day, the mice were divided into three groups: one group of six mice received access to a low-profile running wheel from ZT 8 to ZT 10, one group of five mice received access to a dome from ZT 8 to ZT 10 (to control for the presence of a large novel object), and one group of five mice had a corner of their bedding slightly disturbed at ZT 8 and ZT 10. The mice were recorded on day 0, 7, 14 from ZT 10 until ZT 8 the following day and on day 21 from ZT 10 until ZT 10 the next day. The mice did not receive dome or wheel access on day 21 of the experiment.On days 0, 7, and 14, the mice were video-recorded during at least one and a half hours of the two hours of wheel access and dome access. Controls were recorded on day 7 and day 14 during this time. Caltech Behavior Annotator software was used to manually the behavior as either running on the wheel, interacting with the dome or the wheel, awake but not interacting, sleeping inside the wheel or dome, or sleeping outside the wheel or dome. Running was defined as having all four feet on the wheel and moving. Interaction was defined as any contact with the wheel or dome or any time the mouse and the dome or wheel appeared to overlap from the camera's point of view. Any time the mouse was awake but not either running or interacting with the wheel was considered awake but not interacting. The amount of interaction for one mouse in the running wheel group was excluded from day 7, as his wheel was not functional on that day.