MBL2) leading to dysfunctional protein are related to development of myocardial infarction (MI). The aim of the present study was to study polymorphisms in MBL2 and ficolin genes in relation to the risk of MI.Mannose-binding lectin (MBL) and ficolins activate the complement cascade, which is involved in atherogenesis. Based on a pilot study, we hypothesized that functional polymorphisms in the MBL gene . Functional haplotypes were analyzed and stringent alpha levels of significance were set by permutation testing. Variant MBL2 haplotypes causing MBL deficiency were associated with a two-fold higher risk of MI . Adjustments for conventional cardiovascular risk factors did not substantially influence the association. The ficolins were not associated with MI risk.Using the population-based HUNT Study in Norway, 57133 persons were followed up for a first-time MI from 1995–1997 until the end of 2008. The 370 youngest MI patients were matched by age (range 29–62 years) and gender to 370 controls. A younger population was selected because disease in this group might be less dependent on non-genetic risk factors. The study size was based on power calculation. Polymorphisms in MBL2 variants related to functional MBL deficiency were associated with a doubling of the risk for MI, independent of conventional risk factors. This supports that MBL deficiency may lead to increased atherosclerosis or development of vulnerable plaques.In a young to middle aged and relatively healthy Caucasian population,  Accumulating evidence suggests that atherosclerosis is an inflammatory disease where the innate immune system plays a crucial part in the pathophysiology MBL2) has several polymorphic sites normal, intermediate or deficient serum concentrations of the protein MBL2 and serum concentrations of the protein have been linked to both increased and reduced risk of atherosclerosis and coronary artery disease in different populations The complement system is activated through three possible pathways, denoted as the classic, alternative or lectin pathway. The latter is initiated by mannose-binding lectin (MBL) or by proteins of the ficolin family MBL2 haplotypes corresponding to MBL deficiency in patients with significant coronary artery stenosis compared to patients without significant stenosis, as detailed below. Based on the results from that study, we hypothesised that MBL deficiency is related to the development of myocardial infarction (MI) and that variations in the genes coding for MBL and ficolins may be associated with atherosclerosis.In a pilot study including patients with stable angina pectoris, we found an increased frequency of variant MBL2 and ficolin genotypes with the risk of a first-time MI at young and middle age, i.e. at an age when the genetic influence may be stronger and less dependent on non-genetic risk factors than at older age. The study showed that MBL deficiency was associated with a doubling of the risk for MI, independent of conventional risk factors. This supports a protective role of MBL in atherosclerosis.We therefore performed a case-control study, assessing the association of The study protocols conformed to the Helsinki declaration. Both studies were approved by the Regional Research Ethics Committee in Medicine, Central Norway , and the case-control study was approved by the Data Inspectorate of Norway. The participants in the pilot study gave written informed consent. The HUNT2 participants had signed consent to participate in morbidity and mortality follow-up studies. No minors or children participated in the present study.MBL2 polymorphisms was performed as described in The pilot study was based on data from 236 adults  in a study on circulating plasma markers of inflammation, admitted for first-time elective coronary angiography due to suspected coronary artery disease as detailed previously The case-control study was generated by linkage of population data from the second wave of the Nord-Trøndelag Health Study (HUNT2) to validated information on incident acute MIs.HUNT2 was carried out in 1995–1997 as a population-based study and information was collected through comprehensive questionnaires and a clinical examination. The county of Nord-Trøndelag is fairly representative for Norway as a whole, with only 3% non-Caucasians. All inhabitants 13 years of age and older were invited, and a venous blood sample was drawn from all persons 20 years of age and older. In total, about 75 000 (70%) of those invited attended the study. The inclusion process is described elsewhere There are two primary referral hospitals in the county of Nord-Trøndelag . Data on all acute MI hospitalizations from 1995 (corresponding to the commencement of HUNT2) to the end of 2000 were registered retrospectively, whereas from 2001 registration has been done prospectively. MI was diagnosed according to the European Society of Cardiology/American College of Cardiology consensus guidelines Among participants in HUNT2, the following criteria had to be met to be eligible for the present study: available DNA, and no previous self-reported MI, angina pectoris or stroke. In total, 57 133 individuals met these criteria. We linked these HUNT2 participants to the hospital registrations to ascertain incident cases of MI from baseline at HUNT2 until the end of 2008. During follow-up, 1689 individuals had experienced an MI. Among incident MI patients, the 370 youngest were selected as cases in the study. As controls, we randomly selected 370 participants who were matched to the cases by age (±2 years) and gender. All controls were at risk of MI at the time when the MI occurred in their respective matched case.central obesity,  plus two of the following four criteria 2a) low HDL cholesterol , 2b) hypertension , 2c) fasting plasma glucose ≥5.6 mmol/L or previously diagnosed type 2 diabetes, 2d) fasting triglycerides >1.7 mmol/L.Measurements of blood pressure, height, weight, waist and hip circumference were done as previously described http://www.ncbi.nlm.nih.gov/snp/. The sequences were based on ENSG00000165471 (MBL2), ENSG00000085265 (FCN1), ENSG00000160339 (FCN2), and ENSG00000142748 (FCN3) in Ensemble release 65.DNA was extracted from peripheral blood leukocytes at the HUNT biobank using a commercial kit  or by a robotic method . Genotyping was performed using a combination of polymerase chain reaction (PCR) and pyrosequencing. All of the single nucleotide polymorphisms (SNPs) are found in the online database MBL2 were investigated , C  and D . Wild type is denoted A. The fourth is considered the most important promoter polymorphism: X/Y (rs7096206). These SNPs are inherited in haplotypes. To simplify the interpretation, data are presented by pooling the structural alleles B–D to one allele denoted OY promoter background, thus we used the term YO to denote this defective haplotype. Combining the promoter variant with the A and O alleles results in 6 haplotypes, as shown in normal , intermediate  or deficient , which correspond to serum concentrations of functional MBL Four different SNPs in stigated , Panel AFCN1 −542, rs10120023) was genotyped. In the ficolin-2 gene (FCN2), two amino acid substituting SNPs in exon 8 were included. They are known to cause increased  and reduced  binding capacity of the protein to N-acetylglucosamine, respectively FCN3) is less polymorphic, but a frame-shift variation in position +1637 (rs28357092) of FCN3 is known to cause a 50% reduction of serum ficolin-3 in heterozygotes, and total ficolin-3 deficiency in homozygotes One common polymorphic site in the promoter of the ficolin-1 gene  in Caucasians, a power calculation was performed for the case-control study. To detect an odds ratio (OR) of 2.0, assuming a power of 80% and a 5% significance level, 320 persons were required in each group. In order to account for possible variations in the genotype distribution in small datasets, 370 persons were included in each group. The study population was too small for analyses stratified by gender.Based on an expected frequency of 0.08 of the MBL2 haplotype groups. The Chi-square test was used for comparison of allele frequencies and of frequencies of participants with hypercholesterolemia within the MBL2 haplotype groups. Deviation from the Hardy-Weinberg equilibrium was calculated by using the chi-square test.McNemar's test was used to compare numbers of discordant pairs. Due to non-normal distribution of several variables, the Wilcoxon signed rank test was used to evaluate differences in continuous and ordinal variables between pairs. The Kruskal-Wallis test was used to compare serum total cholesterol, total cholesterol – HDL cholesterol ratio, and triglycerides within the MBL2 haplotype groups and risk of MI. Further models were developed, where traditional risk factors ), the Framingham risk score or the metabolic syndrome were also included.Conditional logistic regression was performed to evaluate associations between the three inferred http://www.r-project.org). All other analyses were performed with Stata/MP for Mac, version 11.2, .All tests were two-sided and the results are presented as means, ORs or HRs ). To avoid false positive conclusions, the alpha level of significance for the comparisons of haplotype frequencies between cases and controls were obtained by permutation testing, using 10,000 permutations. By this method, which is considered the gold standard, a result is significant if the observed p-value is lower than the empirical p-value found under permutation. For other tests, p-values below 0.05 were considered statistically significant. Pilot data was analysed with SPSS for Windows, version 19.0.0 . Permutation testing was performed using the R package, version 2.14.1 . Frequencies of MBL2 and ficolin alleles are available as supporting information . Frequencies of ormation .MBL2 haplotypes causing MBL deficiency were positively associated with MI .Conditional logistic regression showed that variant  with MI . The thr with MI . There wMBL2 haplotypes causing MBL deficiency were associated with a doubling of the risk of MI at middle age (before the age of 62 years). The association was independent of conventional risk factors for MI. Based on the pilot study where associations among variant MBL2 haplotypes, low plasma concentrations of MBL, and increased frequency of significant coronary artery stenosis were demonstrated, increased atherosclerosis is a probable explanation of this finding.In this population-based case-control study we found that variant Previous studies support our finding. However, those studies were performed among patients with severe atherosclerosis MBL2 was more strongly associated with severe atherosclerosis in the youngest patients going through coronary surgery Although the study population was relatively young, conventional cardiovascular risk factors, including hypertension, hypercholesterolemia, smoking and high BMI were also associated with increased risk of MI. Despite incomplete data, there was also a positive association of family history of MI with MI risk. We chose to study people at middle age, anticipating that underlying causes of an early MI would be more likely to be genetic compared to an older age, when non-genetic causes may dominate. Another reason was our previous finding that MBL2 alleles may be correlated with increased carotid plaque area MBL2 haplotypes. Although the link between infections and atherosclerosis is not verified, a combination of MBL deficiency and infection was related both to the development of coronary artery disease Previous studies support a cardio-protective role of MBL MBL2 may be associated with increased risk of cardiovascular disease. A dual effect of MBL has been suggested, as both high and low serum concentrations of MBL were correlated with increased intima-media thickness of the carotid artery in persons with rheumatoid arthritis On the other hand, others have found that high serum MBL-concentrations Analysis of haplotypes with known functional consequences and ensuring stringent alpha levels of significance by permutation testing strengthen the probability of a causal relationship, even if the design of our study did not allow direct causal inference. Our results corroborate that genetically determined MBL deficiency is linked to atherosclerosis. However, we cannot exclude that high MBL concentration and an “eager” complement system may also be harmful in the atherosclerotic process under some circumstances.FCN1 −542 yields some protection, but little is known about the effects of this genetic variation, and more research is needed.None of the ficolin polymorphisms were significantly related to MI. One may speculate that being homozygous for This study was not designed to test improvement of risk prediction. However, the results may generate new hypotheses regarding pathophysiology.MBL2 haplotypes were not assessed in all MI patients from the HUNT2 study; hence it is not known whether the associations are present in older individuals. Serum was not available at the time of the genotyping. However, our pilot study and other previous studies have shown that serum concentrations of functional MBL correspond closely to the genotypes FCN3. All samples with uncertain results were therefore re-sequenced manually. The results were not replicated in a similar cohort, as they were partly confirmatory. Because the included polymorphisms are located closely together and are not covered in previously published genome-wide association studies on early MI, it was not possible to use such data for replication. Although the population in Norway is assumed to be generally representative for the Caucasian population, we cannot exclude the possibility that MBL may be more important in relation to cardiovascular disease in this population compared to others.There are some limitations to our study. MBL2 haplotypes corresponding to functional MBL deficiency were associated with a doubling of the risk for MI in individuals younger than 62 years of age, independent of conventional risk factors. The findings confirm our hypothesis and support that MBL deficiency may lead to increased atherosclerosis or development of vulnerable plaques.The Table S1Primers.(DOCX)Click here for additional data file.Table S2Distribution of MBL2 alleles.(DOCX)Click here for additional data file.Table S3Allele frequencies for FCN1, FCN2 and FCN3.(DOCX)Click here for additional data file.


An oral surgeon needs to know how to better manage such patients, in order to avoid A group of 193 patients (119 men aged between 46 and 82 and 74 women aged between 54 and 76) undergoing OAT for more than 5 years were managed with a standardized management protocol and a 2-months follow-up. The aim of the present study was to apply a protocol, which could provide a safe intra- and postoperative management of patients on OAT.Among the 193 patients, only 2 had postoperative complications.We think that the protocol used in the present study can be used for complete safety in the treatment of this type of patients. The typology of coagulation disorders is extremely heterogeneous. Today, we frequently treat patients taking oral anticoagulant therapy (OAT), a prophylaxis against the occurrence of thromboembolic events ,2.Recent studies on the Italian population indicate an estimated 250.000 patients taking OAT to date. Indications for use of this pharmacological prophylaxis ,4 are coToday, we deal with patients undergoing dicoumarolics therapy, which interfere with the metabolism of vitamin K and therefore with the synthesis of coagulation factors-- II, VII, IX, and X ,8. Theseacenocoumarol: intermediate action, which begins within 24-48 h and ends within 2-4 days.• warfarin: long and delayed action from 3 to 7 days.• Warfarin has a more permanent and long-lasting effect, because of its longer biological half-life (about 36 h).The aim of the present study was to apply a protocol, which could provide a safe intra- and postoperative management of patients on OAT.In the present multicentric study, we focused on a cohort of 193 patients (119 men aged between 46 and 82 years and 74 women aged between 54 and 76 years) undergoing OAT for more than 5 years.preoperative phase, an intraoperative phase, and a postoperative phase including a 2-months follow-up.This study is carried out in compliance with the Helsinki Declaration; it is divided into a 2) can produce thrombocytopenia [Medical history was carried out through specific questionnaires for the diagnosis of hemorrhagic disorders . We askeytopenia .• On the day of the surgery, the dental surgeon has to investigate the patient's hemocoagulative structure through a series of hematoclinical examinations.Counseling between the dental surgeon, patient, and hygienist is necessary to make patients aware of the importance of dental hygiene in preventing dental and periodontal pathologies, which could later require more destructive and invasive therapies .In addition to this, inflammatory or degenerative pathologies of the oral cavity play a crucial role in the genesis of bacterial endocarditis, a non-rare phenomenon among chronic valvulopathy patients and among those with valvular prostheses .Bacterial Endocarditis (BE) is a potentially lethal disease, even if patients are on antibiotics. Mortality can reach up to 30% of cases.British Society Antimicrobial Chemotherapy (BSAC) has revised the cardiac table of patients risking BE. According to these new indications, there are 3 types of patients risking BE:The 1) patients with previous BE; 2) patients with cardiac valve replacement; and 3) patients who underwent surgical shunt creation, especially in the cardiopulmonary area.These new guidelines are no longer ambiguous about performing an antibiotic prophylaxis table  apart frThe aim of antibiotic prophylaxis is to have an effective serum concentration of the active principle during the whole perioperative period.The antibiotic therapy should be given shortly after the beginning of the treatment (1-2 h) and within 6-8 h from surgery; a longer therapy is justified only in case of infected tissues or delayed wound healing.chlorhexidine digluconate 0.12%, 2 times a day for 14 days before surgery.Fifteen days before surgery, periodontal preparation of patients through tartar ablation by means of an ultrasonic equipment. To avoid periodontal hemorrhages, it is necessary to invite patients (before ablation) to do oral rinses for 1 min, with 10 ml of 4.8% solution of tranexamic acid. Moreover, it is important to prescribe to patients oral rinses with The day of surgery, it is necessary to assess the hemocoagulative parameters by taking a blood sample, which will allow to evaluate PT, PTT, and especially the INR measured 24 h before surgery.In the last 3 years, we treated 193 patients on OAT with a standardized management and a 2-months follow-up. Although we evaluated every single clinical case in its uniqueness, we wanted to apply a protocol providing for a safe intra- and postoperative management of patients receiving OAT.There are not any contraindications to the use of local anesthetics: mepivacaine, lidocaine, or articaine are recommended .carpule) and a good anesthesia technique, except for patients who recently had a myocardial infarction or coronary artery bypass surgery (≤ 8 months).The literature does not recommend the use of vasoconstrictors as they could cause tachycardia episodes ; neverthThis will bring 2 advantages:1) The vasoconstrictor will guarantee an optimal zonal ischemia and a longer anesthetic effect, which is very useful when treating hemorrhagic patients [patients ;2) The vasoconstrictor  allows for an effective anesthesia, using a smaller quantity of anesthetic compared to green phials (anesthetic without vasoconstrictor).Among the hemostatic agents that a dental surgeon can use, there are ferric sulfate, oxidized cellulose, gelatin sponges, and anti-fibrinolytics.Surgicel, Tabotamp) consists of a sterile gauze and oxidized cellulose. First, it acts as a mechanical barrier and then as a viscous mass, which works as an artificial clot.Oxidized regenerated cellulose   perform a good local hemostatic action.Antifibrinolytics . As a matter of fact, premedication allows for greater patient compliance during surgery, and the adrenalin plateau caused by intraoperative stress will be avoided.We suggest to our patients oral rinses with chlorhexidine digluconate 0.2% solution .A careful disinfection of perioral and intraoral mucus-dental tissues by using an iodized solution is also necessary. Rinsing the operating field with tranexamic acid solution (4.8%) allows to control hemostasis since initial dieresis.Performing a venous access allows to operate quickly in case of hemorrhagic emergency. In this case, patients could have hypotensive phenomena, which involve difficult identification of the venous access, because vessels could be partially collapsed.In major oral surgery, 2 g of tranexamic acid are administered intravenously (IV) 1 h before surgery (divided into 4 × 0.5 g doses every 15 min) and after preparing the venous access .The cardiac activity and the pressure status have to be steadily monitored using specific equipment .The surgical procedure is performed following a standardized controlled protocol.plexus, intraligamentous, or locoregional anesthesia is performed using a slow infusion of mepivacaine 3%; we have described above what we think about the use of vasoconstrictors, which will bring more benefits than objective risks. Following a periotome syndesmotomy, a luxation of the dental-alveolar gomphosis will be performed. We suggest to reduce to a minimum the use of surgical elevators, because the moments of force of these instruments cause severe trauma to soft tissues along with microlesions to the alveolar ridge. After avulsion, manual reduction of the "greenstick" fracture [A fracture  of the pprimum movens in the residual cyst formation, namely post-extractive alveolites [Accurate alveolar bone cleaning is to perform: by means of an alveolar spoon, we will remove all the possible granulation tissues representing the veolites ,2.A bone rongeur or a bone mill with a turbine motor (continuous irrigation) allows to eliminate the bone roughness, which could damage the soft tissues of the antagonist arch. We compressed the post-extraction alveolus with oxidized regenerated cellulose.absorbable suture  using a "simple running stitch," with a further safety suture using a "simple interrupted stitch"; then, there will be a 30 minute compression of the surgical area, followed by sterile gauze application filled with tranexamic acid. If bleeding stops about 30 min later, it is the clinical demonstration of the technical success of surgery.we suggest an Patients should be discharged at least 60 minutes after surgery (in order to verify the absence of hemorrhagy); we advise a cold and liquid diet during the next 3 days and a tepid and semiliquid diet during the following 3 days.Patients should perform oral rinses for 2 minutes with 10 ml of tranexamic acid, 4 times a day for 7 days . After rDuring the first 30 days after surgery, we suggest patients to rinse their mouth every 12 h with chlorhexidine digluconate 0.12% solution; anti-inflammatory therapy should be prescribed only in case of complex surgeries or if patients experience postoperative pain: 500 mg of paracetamol every 8 hours will be a precaution against hemorrhagic problems caused by the NSAIDs.th day after the surgery.The highest risk of postoperative hemorrhagic diathesis is during the first 12 h after the surgery; our follow-ups allow us to exclude hemorrhagic relapse from the 6In case of postoperative hemorrhage, a buffer imbibed in tranexamic acid should be applied together with compression of the area ; if bleeVicryl 3/0 or 4/0) to avoid trauma caused by suture removal. Non-absorbable sutures are particularly indicated for areas presenting aponeurosis, mimic muscles, frenula, etc. Removal should nonetheless be performed on the 8th day and in the less traumatic way. In case of slight hemorrhages, it is enough to apply a tampon of tranexamic acid and repeat oral rinses for 1 or 2 days.We recommend the use of absorbable sutures . They include intra- and extraoral examinations, a measurement of symptoms of pain by visual analogue scale (VAS), the motivation of patients to oral hygiene, and the possibility of further dental treatments.st one had a slight hematoma in the area of labial commissure, due to the trauma caused by the Farabeuf retractor, but it regressed in a few days. The 2nd one had pains and did not present clot formation in the post-extractive site. Intraoral examination confirmed the diagnosis of post-extractive alveolitis, while the forthcoming pathological medical history indicated that the patient did not follow hygiene rules (smoker) in the immediate postoperative period; the alveolus was cleaned and the symptoms significantly reduced in the follow-up period.Out of 193 treated patients, just 2 of them (only 1.03% of cases) had problems related to surgery: the 1The results of this study show a protocol which allows a safe treatment of patients on OAT.The problems that may principally arise in this type of surgical patients are related to a deficient or excessive clotting, we may also have problems of excessive susceptibility to bacterial infections. With this protocol we have treated 193 patients and we have had none of these problems, except in only 2 cases. These data are significant and show that the management of these patients is possible if we follow a proper protocol such as this one here described.Dental procedures represent a particularly common intervention for patients receiving anticoagulant therapy. The most recent literature indicated that in most cases, no change in the intensity of anticoagulation is needed. As we can highlight in the literature, there are no well-documented cases of severe bleeding in this setting, but there are many documented cases of embolic events in patients whose warfarin therapy was discontinued for dental treatment. If there is a need to control local bleeding, tranexamic acid administration has been used successfully without interrupting anticoagulant therapy ,19.The diathesis of bleeding is significantly related to the intensity of the anticoagulant effect. Therefore, in patients who show a severe bleeding, every effort should be made to maintain the INR at the lower limit of the therapeutic range. For patients with mechanical prosthetic valves (and a persisting risk of increased bleeding), it would be reasonable to aim for an INR range of 2.0 to 2.5. For patients with atrial fibrillation (and a persisting risk of increased bleeding), the anticoagulant intensity can be reduced to an INR range of 1.5 to 2.0 with the expectation that efficacy will be reduced but not abolished. Alternatively, aspirin can be used to replace warfarin in patients with atrial fibrillation, but also with a reduced efficacy in high-risk patients .In the past, the scientific literature often recommended OAT interruption in order to avoid intra- and postoperative hemorrhagic risk: in a research carried out in 1996 in the USA, around 70% of clinicians recommended their patients to interrupt anticoagulant treatment before undergoing dental treatments, with a risk of hemorrhage. In this light, we want to underline how the interruption of an anticoagulant therapy, even for a short time, exposes the patient to thromboembolic risks, even many days after treatment: the analysis of 500 documented cases of patients who suspended an anticoagulant therapy to undergo oral surgery revealed that 5 patients (0.95%) had thromboembolic episodes; 4 of them had fatal thromboembolism, while 1 patient had 2 embolic non-fatal episodes .A randomized study showed how OAT interruption to perform minor oral surgery does not imply any real clinical benefit ,5.Even in those cases in which we are performing surgical treatments particularly invasive, or we are treating a syndromic condition closely related to an augmented bleeding risk, we can emphasize the effectiveness of the association between a conscious administration of tranexamic acid and a well done surgical performance, especially if we follow a well documented protocol ,23.We think that the protocol analyzed in the present study can be used in complete safety for the treatment patients receiving OAT, even though a few factors can alter the patient response to the protocol. The recommendations that urge to maintain the INR at the lower limit of the therapeutic range are still actual, although, the evaluations to do are closely related to each patient, to his general conditions and to the typology of oral surgery to perform.Written informed consent was obtained from the patients for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.FI and FMA participated in the surgical treatment and in the follow-up examinations. MT and SS drafted the manuscript and revised the literature sources. MM and GD participated in the follow-up examinations. FP analyzed the data carried out from the multicentric study and revised the literature sources.ADI revised the literature sources. AMI managed the data collection and contributed to writing the paper. All authors read and approved the final manuscript.


Wistar rats were bilaterally injected with vehicle or CS in the eye anterior chamber, once a week, for 10 weeks. At 3 or 6 weeks of a treatment with vehicle or CS, RON was performed by a single incision in the edge of the neuro-retinal ring at the nasal hemisphere of the optic disk in one eye, while the contralateral eye was submitted to a sham procedure. Electroretinograms (ERGs) were registered under scotopic conditions and visual evoked potentials (VEPs) were registered with skull-implanted electrodes. Retinal and optic nerve morphology was examined by optical microscopy. RON did not affect the ocular hypertension induced by CS. In eyes injected with CS, a significant decrease of retinal (ERG a- and b-wave amplitude) and visual pathway (VEP N2-P2 component amplitude) function was observed, whereas RON reduced these functional alterations in hypertensive eyes. Moreover, a significant loss of cells in the ganglion cell layer, and Thy-1-, NeuN- and Brn3a- positive cells was observed in eyes injected with CS, whereas RON significantly preserved these parameters. In addition, RON preserved the optic nerve structure in eyes with chronic ocular hypertension. These results indicate that RON reduces functional and histological alterations induced by experimental chronic ocular hypertension.Radial optic neurotomy (RON) has been proposed as a surgical treatment to alleviate the neurovascular compression and to improve the venous outflow in patients with central retinal vein occlusion. Glaucoma is characterized by specific visual field defects due to the loss of retinal ganglion cells and damage to the optic nerve head (ONH). One of the clinical hallmarks of glaucomatous neuropathy is the excavation of the ONH. The aim of this work was to analyze the effect of RON in an experimental model of glaucoma in rats induced by intracameral injections of chondroitin sulfate (CS). For this purpose,  Glaucoma is a leading cause of blindness worldwide, characterized by specific visual field defects due to the degeneration of retinal ganglion cells (RGCs) and damage to the optic nerve head (ONH). Elevated intraocular pressure (IOP) is the most important risk factor for the development of glaucoma. However, the underlying mechanisms that link elevated IOP to RGC death are still not fully understood. An experimental model of pressure-induced optic nerve damage would facilitate the understanding of the cellular events leading to RGC death, and how they are influenced by IOP and other risk factors. Recently, we have developed a new model of glaucoma in rats through weekly injections of chondrotin sulfate (CS) in the eye anterior chamber. Acute or chronic intracameral injections of CS significantly increase IOP as compared with vehicle-injected eyes One of the clinical hallmarks of glaucomatous optic neuropathy is the excavation of the ONH, which consists in a progressive posterior displacement of the ONH surface and excavation of the prelaminar tissues beneath the anterior most aspect of the scleral canal, the anterior scleral ring Wistar rat eyes Central retinal vein occlusion (CRVO) is a compartment-like syndrome resulting from increased pressure on the central retinal vein (CRV) within the confined space of the scleral ring which results in tissue ischemia. In 2001, Opremcak and coworkers proposed radial optic neurotomy (RON), in which a microvitreoretinal blade is used to cut the cribriform plate, the scleral ring, and the adjacent sclera in a radial fashion, to alleviate the constriction of the scleral outlet, as a new surgical option for CRVO Since the connective tissues of the anterior scleral canal wall are permanently deformed at early stages of glaucoma In order to assess the effect of RON on functional alterations induced by chronic ocular hypertension, the functional state of retinas from eyes weekly injected with vehicle or CS for 10 weeks with or without RON was analyzed by scotopic electroretinography. The average amplitude of scotopic electroretinogram (ERG) a- and b- waves of rats injected with vehicle or CS for 10 weeks and submitted to RON in one eye and a sham procedure in the contralateral eye at 3 (left panel) or 6 (right panel) weeks of ocular hypertension is depicted in The effect of RON on retinal histological alterations induced by ocular hypertension was examined. A morphometric analysis of retinal sections performed at 10 weeks of treatment with vehicle or CS revealed no differences in the total retina, inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL) thickness (data not shown), whereas a decrease in the number of cells in the ganglion cell layer (GCL) was observed in CS-treated eyes submitted to a sham procedure. RON reduced GCL cell loss, as shown in The optic nerve (ON) from eyes treated with CS for 10 weeks and submitted to sham procedure exhibited an overall loss of staining uniformity and integrity, showing distention and distortion that resulted in a departure from the circular morphology of normal axons. RON performed at 3 or 6 weeks of ocular hypertension significantly preserved ON structure . No fociper se, decreased functional and histological alterations induced by chronic ocular hypertension in the rat eye. Notably, the retinal protection induced by RON was independent from ocular hypertension, as shown by the fact that it did not affect the increase in IOP induced by CS injections.For the first time, the present results indicate that RON, which showed no effect asymptomatic ocular hypertension); ii) 6 weeks of ocular hypertension: decrease in ERG and VEPs, without histological changes (i.e. moderated glaucoma); and iii) 10 weeks of ocular hypertension: further decrease in ERG and VEPs (vs. 6 weeks), and loss of RGCs and optic nerve fibers (i.e. advanced glaucoma) Human primary open angle glaucoma is a progressive optic neuropathy. In the experimental model of glaucoma induced by weekly injections of CS, we have identified different stages, that show the following characteristics: i) 3 weeks of ocular hypertension: no changes in the ERG, VEPs, and retinal morphology  alterations. The present results indicate that the delayed treatment (i.e. at 6 weeks of ocular hypertension) also resulted in a significant protection against experimental glaucomatous damage, both at functional and histological level.Soon after the proposal of RON as a potential treatment modality for CRVO, a debate arose regarding whether the incision of the scleral outlet is a reasonable or dangerous procedure RON performed at 3 or 6 weeks of ocular hypertension prevented and reduced respectively, the decrease in the ERG a- and b-wave and flash VEP N2-P2 amplitude induced by weekly injections of CS, which indicates that RON not only preserved the retinal function, but also the activity of all cells in the pathway from photoreceptors to visual cortex, including RGCs and their axons.In addition to RGCs, the GCL is comprised of a number of displaced amacrine cells. NeuN is a DNA-binding protein that identifies most mature neuronal populations, which has been used as a specific marker for RGCs The precise mechanisms responsible for the retinal protection against glaucomatous damage induced by RON remain to be established. For the past 30 years, discussion has focused on how RGC axons are damaged within the lamina cribrosa, and controversy has centered on whether IOP  or ONH blood supply (the vascular hypothesis) is responsible for ONH axonal damage in this disease. However, consideration of the anatomy of the lamina cribrosa and peripapillary sclera suggests that the classic mechanical and vascular mechanisms of glaucomatous injury are inseparably intertwined While the original rationale of RON was the relief of increased tissue pressure within the optic nerve that results from occlusion of the CRV, the present results suggest that relaxation of the scleral ring of the prelaminar and laminar regions of the ONH may alleviate the IOP-related connective tissue stress, which in turn, could prevent the onset and reduce glaucomatous neuropathy, presumably by counteracting ONH connective tissue damage, maintaining nutritional and oxygen supply, and axoplasmic transport at increased levels of IOP. In addition, since several lines of evidence support that neuroinflammation and oxidative stress can contribute to RGC death A major weakness of the present results relies on the differences between the rat and human eye. Although results from some authors support that the lamina cribrosa in the rat eye is thin and poorly developed Although care must be taken when extrapolating data generated in rodents to humans, our data provide evidence which supports the beneficial effects RON on retinal damage induced by chronic ocular hypertension. Due significance should be given to the fact that the ONH, a very delicate and crucial structure of the eye is being dealt with surgically. However, developments in techniques and technology could increase the margin of safety and efficacy of RON in humans, raising the hope that in the future, benefits of RON against glaucomatous damage could outweigh the risks of this procedure.All animal procedures were in strict accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The ethic committee of the School of Medicine, University of Buenos Aires ) approved this study.Wistar rats  were housed in a standard animal room with food and water ad libitum under controlled conditions of humidity and temperature (21±2°C), under a 12 h light: 12 h dark lighting schedule (lights on at 07.00 h). A total number of 60 animals were used for the experiments, distributed as follows: for IOP, ERG and VEP assessment: 10 animals bilaterally injected with vehicle (control) and 10 animals bilaterally injected with CS submitted to sham procedure or RON at 3 weeks of intracameral treatment, and the same amount of animals submitted to RON or sham procedure at 6 weeks of intracameral treatment . For retina and ON histology: 5 animals bilaterally injected with vehicle (control) and 5 animals bilaterally injected with CS, with RON in one eye and sham procedure in the contralateral eye performed at 3 weeks of intracameral treatment, and the same amount of animals submitted RON in one eye and sham procedure in the contralateral eye at 6 weeks of intracameral treatment Male Rats were anesthetized with ketamine hydrochloride (150 mg/kg) and xylazine hydrochloride (2 mg/kg) administered intraperitoneally. With a Hamilton syringe and a 30-gauge needle, under a surgical microscope with coaxial light, 20 µl of CS  were bilaterally injected into anesthetized rats, and an equal volume of vehicle  was bilaterally injected in control rats. Eyes were focused under a surgical microscope  with coaxial light. The needle moved through the corneoscleral limbus to the anterior chamber with the bevel down. When the tip of the bevel reached the anterior chamber, the liquid progressively increased the chamber's depth, separating the needle from the iris and avoiding needle-lens contact. Applications were made slowly but using a force sufficient to just empty the syringe content (adjusted to 20 µl). Weekly injections were applied at the corneoscleral limbus, beginning from hour 12 and changing the site of the next injection from hour to hour, by rotating the head to achieve better access to the limbus. Injections were performed after application of one drop of 0.5% proparacaine hydrochloride to each eye. Rats showing cataract and animals with phthisis bulbi  were excluded from the experiments. In addition, almost all the animals developed localized corneal edema at the site of the injection that lasted less than 24 h.Rats were anesthetized as already described. After 3 or 6 weeks of treatment with vehicle or CS, animals were subjected to RON in one eye, whereas the contralateral eye was subjected to a sham procedure. RON was performed as follows: eyes were focused under a binocular Colden surgical microscope with coaxial light for fundus visualization and illumination. Using a 30-gauge dental cartridge needle, a scleral puncture was made at 1 mm of the corneoscleral limbus. A single incision at the nasal hemisphere of the optic disk was performed in the edge of the neuro-retinal ring, cutting an equal part of ON and parapapillary retina, avoiding damage to the central retinal vessels. Care was taken to make the stab radial to the optic disk and parallel to the nerve fiber pattern. The sham operated eyes were submitted to a similar procedure , but without any incision.A tonometer  was used to assess IOP in conscious, unsedated rats, as previously described 2 without filter) were amplified, filtered  and averaged . The a-wave was measured as the difference in amplitude between the recording at onset and the trough of the negative deflection and the b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. Runs were repeated 3 times with 5 min-intervals to confirm consistency. Mean values from each eye were averaged, and the resultant mean value was used to compute the group means a- and b-wave amplitude ± SEM. The mean peak latencies and peak-to-peak amplitudes of the responses from each group of rats were compared.Electroretinographic activity was assessed as previously described For VEP recording, two stainless steel electrodes were surgically placed 4 mm lateral to the interhemispheric fissure and 5,6 mm behind bregma (active electrode), as previously described Eyes were enucleated after anesthetic overdose and immersed immediately in a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 1 h. The nictitans membrane was maintained in each eye to facilitate orientation. The cornea and lens were carefully removed, and the posterior portions were fixed for an additional 12 h- period in the same fixative. A cross section of the optic nerve from vehicle and CS-treated eyes was removed 1.5 mm posterior to the globe and postfixed in 1% osmium tetroxide in phosphate buffer. Nerves were processed into epoxy resin, sectioned at 1 µm, and stained with 1% toluidine blue. Eyecups were dehydrated in an alcohol series, and embedded in paraffin. Sections (5 µm thick) were cut along the horizontal meridian through the ONH and stained with H&E.2O2 in phosphate- buffered saline (PBS) for 20 min, and the sections were then incubated overnight at 4°C with a mouse monoclonal anti-Thy-1 antibody . Thy-1 positive signal was developed with the labeled streptavidin-biotin  reagent kit, according to manufacturer's instructions. Some sections were treated without the primary antibodies to confirm specificity. An Olympus BX50 microscope  was used for microscopic observations. Comparative digital images from different samples were grabbed using identical time exposition, brightness, and contrast settings.Antigen retrieval was performed by heating (90°C) slices for 30 min in citrate buffer and then preincubated with 2% normal horse serum, 0.1% bovine serum albumin, and 0.4% Triton X-100 in 0.01 M phosphate-buffered saline for 1 h. The sections were then incubated overnight at 4°C with a mouse monoclonal anti-NeuN antibody , or a goat anti-Brn3a  antibody. An anti-mouse and anti-goat secondary antibody conjugated to Alexa Fluor 568  were used. After immunostaining, the sections were mounted with antifade medium with the fluorescent nuclear stain DAPI . For Thy-1 level assessment, endogenous peroxidase activity was blocked with 0.3% HMicroscopic images were digitally captured with a Nikon Eclipse E400 microscope  attached to a digital camera . The digitalized images were transferred to a Scion Image for Windows analysis system (Scion Corporation Beta 4.0.2).Retinal morphometry was evaluated as previously described ON axon counting was performed as previously described Statistical analysis of results was made by a two-way analysis of variance (ANOVA) followed by Tukey's test, as stated.


Breast density is a well-established risk factor for breast cancer, with assessment of percentage density via a visual analogue scale (VAS) a practical method of measurement strongly associated with risk. We present a method to adjust for inter-observer differences in VAS density estimates and examine the effect of adjustment on the classification of women at high risk of developing breast cancer.A two-stage method is used to make estimates by different observers comparable. Results from all observers are transformed onto the same distribution, then differences in case mix are accounted for. We applied our approach to 13 experienced readers assessing 13,694 screening mammograms from a large clinical study where women are categorised as high risk if they have a 5 to 8% 10-year risk computed by a validated risk model and their breast density is in the top decile of the study population.A total of 1,125 women were assessed as having a 10-year risk of 5 to 8%. Initially 126 of these were also high density, therefore classified as high risk, rising to 147 after density adjustment. After adjustment, 35 women were reclassified from nonhigh to high risk  and 14 women were reclassified from high to nonhigh risk .Adjusting VAS estimates of breast density for inter-observer variation substantially affected which women were classified as high risk of developing breast cancer. If VAS assessment of density is to be used in risk assessment to inform screening strategies, adjustment must be considered.


In Europe, gastric cancer remains diagnosed at advanced stage . Despite curative management combining perioperative systemic chemotherapy and gastrectomy with D1-D2 lymph node dissection, 5-year survival rates of T3 and/or N + patients remain under 30%. More than 50% of recurrences are peritoneal and/or locoregional. The use of adjuvant hyperthermic intraperitoneal chemotherapy that eliminates free cancer cells that can be released into peritoneal cavity during the gastrectomy and prevents peritoneal carcinomatosis recurrences, was extensively evaluated by several randomized trials conducted in Asia. Two meta-analysis reported that adjuvant hyperthermic intraperitoneal chemotherapy significantly reduces the peritoneal recurrences and significantly improves the overall survival. As it was previously done for the evaluation of the extension of lymph node dissection, it seems very important to validate on European or caucasian patients the results observed in trials performed in Asia.GASTRICHIP is a prospective, open, randomized multicenter phase III clinical study with two arms that aims to evaluate the effects of hyperthermic intraperitoneal chemotherapy with oxaliplatin on patients with gastric cancer involving the serosa and/or lymph node involvement and/or with positive cytology at peritoneal washing, treated with perioperative systemic chemotherapy and D1-D2 curative gastrectomy. Peroperatively, at the end of curative surgery, patients will be randomized after preoperatively written consent has been given for participation. Primary endpoint will be overall survival from the date of surgery to the date of death or to the end of follow-up (5 years). Secondary endpoint will be 3- and 5-year recurrence-free survival, site of recurrence, morbidity, and quality of life. An ancillary study will compare the incidence of positive peritoneal cytology pre- and post-gastrectomy in two arms of the study, and assess its impact on 5-year survival. The number of patients to be randomized was calculated to be 306.NCT01882933.EudraCT number: 2012-005748-12, ClinicalTrials.gov identifier: Stomach is the fourth most common digestive cancer, the secSurgery remains the curative treatment of choice for stomach cancer. It consists of a radical subtotal or total gastrectomy with D1 or D2 lymph node Dissection. A logical and reasonable alternative (expert consensus) is therefore to carry out D1 dissection associated with pedicle dissection . This extension corresponds to D2 dissection for antral cancers. When this type of dissection without splenectomy or pancreactectomy is carried out for a cancer of the body or the upper-third of the stomach it is commonly termed D1.5 dissection. Extending the lymph node dissection remains controversial. Two randomized studies and a meta-analysis have shown no benefit from D2 dissection, although a notable benefit was observed in a sub-group of patients with lymph node metastasis-6. More Systemic perioperative chemotherapy is recommended for the curative treatment of stomach cancer in Europe since the publication of the MAGIC and Fédévs 27 months). Two-thirds of the patients include were at stages T3 or T4 and 85% had node positive. The main criticism of this trial is that in 54% of cases lymph node dissection was D0. For many experts, this therefore limits the applicability of adjuvant chemo-radiotherapy following resection. This chemo-radiotherapy can be considered as an alternative for certain patients in good general and nutritional health with lymph node invasion having undergone adequate lymph node Dissection. A retrospective study suggested that replacing FUFA with simplified LV5FU2 (Leucovorin/5-FU) reduces toxicity[Adjuvant chemo-radiotherapy was shown to be effective in a phase III study by MacDonald et al. This testoxicity. Anothertoxicity.vs surgery alone [HR 0.83 (95% CI 0.74-0.94)].Stomach cancer has the highest rate of peritoneal recurrence of all digestive cancers. After curative surgery, the main reason for treatment failure is peritoneal recurrence which, according to the literature, occurs in 40 to 60% of cases, despite extensive surgery including D2 lymph node dissection,16.Several factors favorable to peritoneal recurrence have been identified: invasion of the serosa ,18, deteThe failure rate for curative surgical treatment for patients with stomach cancer is mainly due to peritoneal recurrence. It would therefore seem appropriate to offer preventive treatment to reduce the risk of peritoneal recurrence in at-risk patients and thereby reduce the failure rate. The HIPEC technique is increasingly used in the curative treatment of primary and digestive peritoneal carcinomatosis, in association with cytoreductive surgery-25. It iin vitro. All the collected fluid will be sent to the corresponding lab, where specimens for regular cytological analysis, cell blocks and immunohistochemistry  will be sent to the Centre de Biologie et de Pathologie Est – Hospices Civils de Lyon.Ex vivo separation of lymph node groups in individual containers before the surgical specimen is submitted to the pathologist is recommended. At the end of the gastrectomy procedure the peritoneal washing will be repeated with the same procedure.All the patients will undergo a D1-D2 gastrectomy, carried out according to Japanese guidelines and to the European recommendations for the preservation of spleen and pancreas: lymphad2 + calcium levofolinate 10 mg/m2) as systemic chemotherapy induction for HIPEC 15 min before HIPEC started. At the end of the procedure the patients in the experimental Arm will undergo HIPEC.Following randomization, the patients treated into the experimental Arm receive intravenous 5-FU 400 mg/m2 with 2 Liters of G5%/m2) into the abdomen through the inflow catheter and pulls it out through the drains. A heat exchanger keeps the intraperitoneal fluid at 42°-43°C. After the intraoperative perfusion is complete (30 minutes), the abdomen is closed and the drains are left in place in the postoperative period until the discharge from the peritoneal cavity subsides. With the Closed abdomen technique, surgery is completed with anastomosis and drains positioning as in the Open technique, the abdominal wall sutured. External manual shaking of the abdomen will be carried out for optimization of chemotherapy distribution. The anastomoses will be constructed either before or after HIPEC administration.The HIPEC can be carried out by open or closed abdomen technique. With the Open abdomen technique, after the D2 resection is complete, the inflow catheter is positioned in the gastric resection bed. Drains are placed through separate stab wounds in the abdominal flanks and positioned between the liver and undersurface of the right hemidiaphragm behind the spleen and in the pelvis. Number of drains used is left to the surgeon’s discretion and according to the specifications of the HIPEC circuit used. One of the two temperature probes will be secured near the tip of the inflow catheter and the other in the pelvis. Using a monofilament running suture, the skin edges are secured to the Thompson self retaining retractor, and a plastic sheath is incorporated into these sutures to create an open space beneath. A slit in the plastic cover is made to allow the surgeon hand access to abdomen and pelvis. During the perfusion all the anatomic structures within the abdominal cavity are uniformly exposed to heat and to chemotherapy. A roller pump forces the chemotherapy solution . They will be evaluated with clinical examination daily. Each day for the first week and each third day thereafter, laboratory exams will be performed in order to assess haematological, renal and hepatic function. Locoregional toxicity and systemic toxicity will be evaluated according to the Common Terminology Criteria for Adverse Events (CTC-AE V4.0) from the National Cancer Institute.Once an early postoperative follow-up four weeks after gastrectomy, the visits of follow-up are based on the date of the surgery every 3 months for the first 2 years following surgical procedure and twice a year for the last 3 years. For all patients clinical follow-up with all events and endpoints will be collected and analyzed during 5 years from their inclusion .Patients can be withdrawn from the study under the following circumstances: death, disease progression, initiation of alternate anti-neoplastic therapy, toxicity, intercurrent illness, non compliance (including loss of patient to follow-up), voluntary withdrawal, failure to meet the eligibility criteria.Study participation by individual sites or the entire study may be prematurely terminated, if in the opinion of the sponsor, there is sufficient reasonable cause. Any investigator who wants to discontinue his/her participation to the study must immediately inform the sponsor in writing of this decision. Written notification documenting the reason for study termination will be provided to the investigator by the terminating party.Examples of circumstances that may warrant termination include: failure to enter patients at an acceptable rate, insufficient adherence to protocol requirements, insufficient complete and/or evaluable data, frequency and/or unexpected severity of the toxicity, unacceptable toxicity. Each fatal event occurring within the 60th postoperative days will be immediately reported to the sponsor and to Data and Safety Monitoring Board (DSMB) members. The DSMB will be provided with the description of the fatal events to determine if the death has to be considered as a toxic one.The statistical stopping rules are the following: for each toxic fatal event in the HIPEC arm, an analysis comparing the total number of patients included to the number of patients satisfying maximum toxic fatal event criteria will be performed to determine if the study should be stopped. This analysis will be realized with the Kramar sequential method as impleThis study is designed as a randomized phase III in order to compare the 5-year overall survival in patients with advanced gastric carcinoma randomized to hyperthermic intraperitoneal chemotherapy administration versus that of patients randomized to control arm.The hypotheses are the following: 5-year overall survival in the control group of about 30%,10; 5-yeper protocol analysis). Overall survival will be measured from the date of surgery up to the date of death, regardless of the cause, or to the end of follow-up (5 years). The Kaplan-Meier method will be used to estimate the survival curves. Median survival will also be given and differences between the two groups assessed using a logrank test. This analysis will then be confirmed using a Cox regression analysis taking into account the center effect (frailty Cox model). The conditions for using the Cox regression analysis will be checked.The effectiveness analysis will be carried out on the intention-to-treat population, defined as all patients included. A second analysis will be conducted on the “treated” population, determined according to the treatment actually administered  with their accountability will be described.©) automaton, and with Papanicolaou. For every case, the remaining cell pellet will be submitted to the cell block technique (Shandon Cytoblock Kit®) for Haematoxylin and eosin control and immunohistochemistry. Immunohistochemistry performed on cell blocks using at least CEA  and cytokeratin 20, the Cytokine-like High Mobility Group Box 1 (HMGB1)[A cytological ancillary study will be performed. The aim of this exploratory study is to compare the incidence of positive peritoneal cytology pre- and post-gastrectomy in the two arms of the study (Am A and Arm B), and to assess its impact on 5-year survival. A standardized procedure for peroperative peritoneal cytological sampling was previously approved under a large, French, multi-center prospective study  and will be followed for the pre- and post-gastrectomy sampling. Samples (HMGB1) will als (HMGB1). AdditioThe number of pre- and post-gastrectomy positive cytologies will be calculated. The percentage of pre- and post-gastrectomy positive cytologies will be compared using Mcnemar’s test. A Cox regression model will be used to assess the impact of peritoneal cytology on 5-year overall survival. The conditions for using Mcnemar’s test and the Cox regression analysis will be checked.The study protocol was approved by the Institutional Review Board, the Sud Est IV ethics committee on the 12 of February 2013 and the French National Agency for Medicines and Health (ANSM) on the 26 of April 2013 under the number 2012-005748-12. The study has been registered on the ClinicalTrial.gov website under the identification number NCT01882933. The GASTRICHIP study complies with the Declaration of Helsinki rules, the principles of Good Clincal Pratice guidelines and the Data Protection Act. The trial will also be carried out in keeping with local legal and regulatory requirements.For each patient recruited into the study, written informed consent is essential prior to inclusion into the study after extensive information about the intent of the study, the study regimen, potential associated risks and side effects as well as potential alternative therapies. The investigator will not undertake any diagnostic measures specifically required for the clinical trial until valid consent has been obtained.The benefit of using HIPEC as an adjuvant treatment for advanced gastric cancer has been reported in several randomized studies and two meta-analyses,39. HoweThe chemotherapeutic agents to be used for HIPEC are selected on the basis of their ability to rapidly produce a direct cytotoxic effect (their action should not be dependent on the cell cycle). The concomitant administration of heat increases the cytotoxic effect. The duration of exposure is related to the effectiveness of the intraperitoneal chemotherapy and studies investigating the pharmacokinetics of HIPEC have shown that most of the medication is absorbed during the first hour of perfusion. Furthermore, it has been shown that the rate of postoperative complication is directly linked to the duration of surgical procedures: HIPEC should therefore be administered over the shortest possible period of time in which it is able to produce its cytotoxic effect.Mitomycin C and cisplatin are the two antimitotic agents most commonly used for HIPEC and have therefore been studied the most and shown to meet requirements for this treatment. They have been used in combination by several authors,48-50 fo2 of oxaliplatin for the open-abdomen technique and 350 mg/m2 for the closed-abdomen technique, administered in 2 liters/m2 of 5% glucose with the systemic administration of induction chemotherapy using FU/Leucovorin (400 mg/m2 5-FU and 20 mg/m2 of folic acid) to increase the cytotoxic effect of the oxaliplatin. Glehen et al. recently published a report on the French experience in the curative treatment of gastric peritoneal carcinomatosis[2) in order to avoid an excessive rate of postoperative complications resulting from the experimental treatment.Good results for the treatment of gastric cancer in terms of both response and tolerance have been obtained using oxaliplatin in systemic perioperative chemotherapy protocols. Recentlomatosis. The morThe incidence of signet ring cell adenocarcinoma is steadily increasing in Western countries,53. ReceThe increased incidence of postoperative positive peritoneal cytology has only been suggested in small Asian studies,57. WhilThe authors declare that they have no competing interests.OG and LV have been involved in drafting the manuscript; SB as the methodological advisor; AG, OG, EP, GB, DV and GP have been involved in the study conception and design, assisted in writing the manuscript and have given final approval of the version to be published. All authors read and approved the final manuscript.OG is the study coordinator, obtained the grant and is responsible for the present paper and EP is responsible for the ancillary study.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/183/prepub


Existing approaches typically consider membrane-related information either by using membrane-specific substitution matrices or by assigning distinct penalties for gap creation in transmembrane and non-transmembrane regions. Here, we ask whether favoring matching of predicted transmembrane segments within a standard dynamic programming algorithm can improve the accuracy of pairwise membrane protein sequence alignments. We tested various strategies using a specifically designed program called AlignMe. An updated set of homologous membrane protein structures, called HOMEP2, was used as a reference for optimizing the gap penalties. The best of the membrane-protein optimized approaches were then tested on an independent reference set of membrane protein sequence alignments from the BAliBASE collection. When secondary structure (S) matching was combined with evolutionary information (using a position-specific substitution matrix (P)), in an approach we called AlignMePS, the resultant pairwise alignments were typically among the most accurate over a broad range of sequence similarities when compared to available methods. Matching transmembrane predictions (T), in addition to evolutionary information, and secondary-structure predictions, in an approach called AlignMePST, generally reduces the accuracy of the alignments of closely-related proteins in the BAliBASE set relative to AlignMePS, but may be useful in cases of extremely distantly related proteins for which sequence information is less informative. The open source AlignMe code is available at  This situation has motivated many researchers to turn to remote-template homology modeling, in which the unknown structure of a target sequence is modeled on a known (template) structure of a distantly-related protein, in order to gain insights into membrane protein function. Such studies rely on methods for detecting relationships between two proteins, and subsequently, for accurately aligning their sequences; both of these procedures become increasingly difficult as the similarity between the proteins decreases, particularly below ∼20% identity. The membrane-protein-specific multiple-sequence alignment method PRALINE™ Integral membrane proteins constitute 25–30% of the genes in a given genome Membrane proteins have distinct properties from their water-soluble counterparts, because of their more complex environment. For example, there are only two major classes of structure: α-helical or β-barrel. Also, the membrane-spanning regions of proteins in the α-helical class have a distinctive hydrophobic character. The latter property inspired the early use of hydropathy profiles for locating transmembrane regions in a sequence Aside from the hydropathy-based approach, not many sequence alignment methodologies have been specifically developed for, and trained and tested on, membrane proteins. This reflects in part the limited data available for testing. Only recently have a sufficient number of structures become available that a dataset of known membrane protein structural homologues could be constructed to provide knowledge-based reference alignments, as, for example, in the HOMEP dataset Two multiple sequence alignment (MSA) methods, T-Coffee Other methodologies developed specifically for membrane proteins include the MSA methods STAM Here, we use our membrane protein sequence alignment program, AlignMe, to ask whether favoring matching of transmembrane regions – predicted either using hydrophobicity profiles or more sophisticated transmembrane predictions – increases the accuracy of pairwise alignments relative to the matching of secondary-structure elements alone, in the context of a profile-profile type alignment. We also test different methods for treating substitution rates, using either general or position-specific substitution matrices (PSSMs). We update the dataset of homologous helical membrane proteins for optimization and evaluation of the different strategies. We then compare the best of the different AlignMe strategies to other available methods using the BAliBASE membrane protein reference set 7 Alignment of Membrane proteins) is a protein sequence alignment tool developed in C++, which was designed to allow multiple different (membrane) protein descriptors to be considered simultaneously when defining the similarity between two aligned positions. Thus, structural properties such as transmembrane location or secondary structure represented in the form of profiles, can be combined with sequence and evolutionary information represented in the form of substitution rates (see below for more details). The description of the transmembrane location can be a window-averaged hydropathy plot  at a given alignment position is calculated as a linear combination of values from M input substitution matrices (S), and differences between residue property values (V) from N input profiles:w) for each input is intended to minimize bias towards a specific input. For example, a hydrophobicity scale containing values from –3.0 to 1.0  would be assigned w = 5 when used in combination with a substitution matrix whose values range from –5 to 15 .The similarity Within AlignMe, gap-opening and gap-extension penalties can be assigned different values according to whether the gap is at a terminus  at which an amino acid (A) from one sequence (i) is replaced by the amino acid (B) of the other sequence (j), and of the rate of the reverse substitution:To account for the variability in evolutionary pressure for different positions along the sequence we consider the position-specific substitution rates of the residue types in the two input sequences taken from position-specific substitution matrices (PSSMs). Thus, the similarity is an average of the substitution rate  for MEMSAT-SVM and a version of Uniref90 filtered for transmembrane proteins (from 4th August 2010) for OCTOPUS Three different predictors for α-helical transmembrane segments were tested: TMHMM st August 2010) and Uniref90 (from 28th April 2009), respectively. Each method produces a three-state prediction of the probability of a position being in a coil, α-helix or β-sheet; all three were used as input profiles for AlignMe, with each state contributing one third of the whole. A position was assigned to an α-helix if the predicted probability thereof was >0.5.Two secondary structure predictors were tested: Jufo th March 2010) The original HOMEP dataset contained 36 structures This non-redundant set was then clustered to identify families of related structures. The clustering method  is based on the protein structure distance (PSD) value that is calculated during SKA structural alignments The accuracy of a sequence alignment is often evaluated using a score that counts the fraction of correctly aligned positions with respect to the reference alignment The Alignment Difference (AD) score used here is similar to the mean shift error (MSE) score 6 = 1012 alignments.A Monte Carlo scheme was used to optimize the gap penalty values for each combination of inputs tested. Note that a systematic optimization In each step of the optimization process, AlignMe alignments were created using the current set of gap penalties and evaluated using the AD score. To minimize bias towards large families, all proteins in each HOMEP2 family were aligned with all others in that family and their AD scores were averaged; the overall alignment accuracy score for a given set of gap penalty parameters was the sum over the scores for each family.Starting with a randomly selected set of values (between 0 and 30) for each gap penalty parameter, the search procedure then involved random modifications of one or more gap penalty values from those values, or from the optimal values identified so far. The range of allowed modifications was initially set to be very small  to encourage a detailed examination of the score landscape around the current optimal gap penalty combination. A given combination of gap penalties was accepted if the overall alignment accuracy score was better than the best score found so far, in which case the maximal step size was reset to its initial value. Otherwise, that combination of gap penalties was rejected and the search space was expanded by increasing the maximum step size by 0.06. However, the gap penalty values were limited to the range 0 to 30, with a maximum step size of 30. If no improvements were found after reaching the maximum step size, the search was repeated, starting with the initial maximal step size of 0.06.For each set of input descriptors, this optimization was repeated 20 times with different initial gap penalty values, which was found to be sufficient for reasonable convergence (data not shown). The parameters for which the alignments had the best score were then used for that set of input descriptors. The optimal gap penalties obtained using the JTT membrane substitution matrix were ues e.g. , providiN+M and the parameters did not converge reliably.Optimization of the weights assigned to each input parameter was found to be computationally impractical because the search space increases by the power of Reference 7 set of BAliBASE The so-called ‘core’ regions provided by BAliBASE were not analyzed, as they have been shown to correspond only weakly to conserved secondary structure elements in this set The accuracy of the alignments in the HOMEP2 set was also assessed by building homology models based on each of the alignments, and comparing them to the native structure. For every pair of protein sequences, each protein was modeled using the structure of the other protein as a template. In each case, five models were created using Modeller v9.9 and the one with the best (lowest) DOPE score was evaluated using GDT_TS  and AL4  scores th August 2010, with five iterations, an E-value cut-off of 10−4 and a maximum of 2500 sequences. Sequences in the PSI-BLAST results that were more than twice the length of the query were filtered out. The remaining sequences were clustered using UCLUST Alignments were also calculated with HMAP There are two different possible approaches for generating a MSA from two query sequences and their respective homologues. In the standard approach, all results of both PSI-BLAST searches (including the two query sequences) are combined and aligned as a single large MSA, before extracting out the two query sequences for scoring. The second approach, which we call the “profile-profile” strategy, is to create MSAs for each query and its homologues. The resulting two MSAs or “profiles” are then aligned to one another to create a single MSA, from which the query sequences are then extracted for scoring.HHalign uses a similar strategy to the MSA “profile-to-profile” approach, but each query is described by a hidden Markov model (HMM) based on the results from a PSI-BLAST search , as well as by secondary structure predictions from PSIPRED, generated as described above. Those HMMs were then globally aligned to each other by using the “-mact 0.0″ maximum accuracy flag and by assigning all other parameters their default values.HMAP was also used to calculate profile-to-profile alignments. In this case, one of the sequences was assigned to be the query, and its profile included evolutionary information  combined with predicted secondary-structure from PSIPRED v3.2; the other sequence was assigned to be the template, and its profile was similar except that the secondary structure was assigned from the structure, where available. The two profiles were then globally aligned using HMAP.We also tested TM-Coffee We first describe the selection of input descriptors for AlignMe. To make the comparison between descriptors as fair as possible, gap penalties were optimized for each input tested. To this end, we first constructed an updated set of homologous membrane protein structures , and structure-based sequence alignments of these proteins were used as a reference. Input descriptors were considered to be effective if the AlignMe alignments had both more correctly aligned positions and smaller shift errors, measured as less negative AD scores (see Section 1.4). By also considering the shift error, we expect to help identify methods that are effective for very distantly related proteins. The findings are described for single inputs (Section 2.1) and then for combinations of inputs (Section 2.2). Finally, we compare three of the optimized AlignMe strategies with available alignment programs for the HOMEP2 set (Section 2.3) and the BAliBASE reference set 7 (Section 2.4).Various substitution matrices, hydrophobicity scales, secondary-structure and transmembrane predictions were tested as individual inputs (see Section 1.2).p<10−9). Of the general substitution matrices, the closest agreement with the reference alignments was obtained with the membrane-specific JTT matrix, followed by the general-purpose VTML matrix, although the differences between JTT and the others were not very significant  than those from other approaches (p<0.05). Window-averaging the hydrophobicity values as in a hydropathy plot  did not significantly improve the alignments compared to using PSSMs  is equivalent to using a substitution matrix, except with a focus on one specific physicochemical property. Alignments constructed using hydrophobicity scales were not significantly different from the best of the general substitution matrices  if all nSSMs cf..−4) more accurate than those generated using the best of the hydrophobicity scales . We note that the other differences are not significant (p>0.05) because of a disproportionate contribution of good PSIPRED2.6 alignments in the (large) aquaporin family; this contribution is not reflected in the AD scores in p<0.05) for the HOMEP2 protein set, using DSSP assignments as a reference When representing the sequences as profiles of predicted secondary structure type, the alignments in closest agreement with the reference alignments were obtained using PSIPRED3.2 predictions . Howeverp = 2×10−5), and transmembrane predictions  , followed by secondary structure predictions  . This fiUsing the results for single inputs, we next tested alignments for which the best two or three input descriptors were used in combination, since inclusion of complementary information is expected to progressively improve alignment accuracy . The explanation could be that OCTOPUS predictions of two related proteins match one another better than those of MEMSAT-SVM, or that the OCTOPUS predictions have a simpler form, perhaps providing more orthogonal (complementary) information to the PSSMs than the more detailed profiles obtained from MEMSAT-SVM . Alternatively, the fact that the MEMSAT-SVM values are more evenly distributed over a wider range of values than the OCTOPUS scores  and are thus given a smaller weighting (see Section 1.1) could mean that the MEMSAT-SVM scores can have less influence on the alignments.A potentially useful combination for membrane proteins is evolutionary information plus transmembrane propensity. The latter can be in the form of either a smoothed hydrophobicity value or a transmembrane prediction propensity. Interestingly, in AlignMe, nearly all such combinations resulted in significantly more accurate alignments than those based on the corresponding individual input parameters, but only when gap penalties were allowed to differ between membrane and non-membrane regions  more accurate alignments than when using the transmembrane positions for assigning the thresholds , the latter may be defined using either secondary structure or membrane propensity. We found that using α-helix positions for this distinction  were next tested in combination, which resulted in significantly more accurate alignments than any approach tested so far , with gap penalties of We compared the three AlignMe versions, i.e., with and without secondary structure or membrane matching, to several available multiple-sequence alignment programs, as well as the profile-to-profile alignment program HMAP, and the HMM alignment program HHalign (see Section 1.8). Here, we assess alignments of the training HOMEP2 dataset, first using the structure-based alignments as a reference, and then using the accuracy of homology models built from those alignments as a reference-independent measure.For close homologues  in the HOMEP2 set, the AlignMe alignments are not significantly more accurate than other methods: AlignMeP, AlignMePS, AlignMePST and HHalign alignments all have a high fraction of correctly aligned residues, for example .cf. AlignMePS) does come at the cost of some correctly-aligned positions, especially for sequences with moderate similarity. As mentioned above, for homology modeling of distantly-related pairs of proteins it can be useful to reduce the magnitude of large shift errors since manual adjustment of an alignment can be aided relatively easily by conservation mapping once the helices are approximately aligned. For similar reasons, it is also interesting to know whether the transmembrane helices have been matched to some extent, as many  functional residues lie in these regions. The matching of transmembrane helices in the HOMEP2 set by AlignMe appears to be particularly effective: using AlignMePS and AlignMePST, ≥97% of the known transmembrane helices overlap by at least half of their residues, and ≥62% of the helices (at least 10% more than the next best method) overlap by at least 90% of their residues  is overtraining of the gap penalties and other parameters due to the limited number of membrane protein structures available. We first tested this by using cross-validation: the optimization of AlignMePST was repeated using 20 of the 22 families and the resultant gap penalties used to evaluate the alignment accuracy of the remaining two families. As shown in As a measure of alignment accuracy that is independent of the reference alignments, we also constructed structural (homology) models from the test alignments. The models were compared to the native structures by calculating GDT_TS and AL4 values (see Section 1.7), and also compared to “gold standard” models built using sequence alignments extracted from the SKA structural alignments.cf.Measured by the GDT_TS structural similarity score, several methods have similar accuracy on average . This reThe alignment accuracy of the various methods was also evaluated on an independent data set of membrane protein sequences . This dataset contains manually-curated multiple-sequence alignments, based on PFAM alignments and optimized to improve amino acid and secondary structure matching; no structural information was available at the time to help guide the dataset construction AlignMePS alignments have the most correctly aligned residues in BAliBASE set 7 on average, as well as for 7 of the 8 families , includiTo assess whether the inclusion of transmembrane information is useful for distantly-related proteins, we separated the BAliBASE set into sequences assigned to the same subgroup  or to diWithin the predicted transmembrane regions of the BAliBASE sequences , AlignMeFinally, we also calculated the shift error in the BAliBASE alignments, which is smallest on average for the AlignMePST and AlignMePS alignments, followed by MSAProbs and HHalign, while HMAP has the smallest shift error for the acr and Nat families . MatchinIn this work, we developed a sequence alignment method called AlignMe, which we trained on a dataset of membrane protein structural homologues (HOMEP2). Three different strategies  were assessed using the BAliBASE membrane protein dataset set 7), and compared with other available methods. Overall, the BAliBASE analysis suggests that versions of AlignMe that match secondary structure prediction profiles may be generally useful for aligning membrane proteins  may be discrepancies between the secondary-structure and transmembrane predictions. Quantifying the matching of secondary-structure prediction profiles as described above indicates that the secondary structure profiles match less well in alignments generated with transmembrane predictions (profile difference score for AlignMePST is 0.060) than those without (profile difference score for AlignMePS is 0.055). In other words, transmembrane matching occurs at the expense of secondary-structure matching.A third possible cause of the reduced accuracy for closely-related sequences using AlignMePST is that including a third parameter (the transmembrane prediction) in the score for each position diminishes the contribution of the PSSM in a deleterious way.The above discussion notwithstanding, the BAliBASE results indicate that incorporating transmembrane matching is useful for very distantly-related proteins, particularly for reducing the overall shift error ,9,10. ThOf the other available methods tested, HMAP alignments were most frequently ranked towards the top ,7,8,9,10The profile-to-profile alignments strategy used with MUSCLE and T-Coffee typically resulted in fewer correctly-aligned positions and larger shifts than the other methods tested ,7,8,9,10This study focuses on α-helical membrane protein sequences, so that we obtain gap penalties that are optimal for long helices and are not biased by the inclusion of short β-stranded regions A concern about the current study is the fact that no structural informational was available to aid with the alignments when the BAliBASE reference set 7 was constructed, and therefore it is possible that these alignments contain errors whose effect we cannot yet know As the size of the database of membrane protein structures grows, further assessment of the various methods will be useful. Nevertheless, the results presented here suggest that there is potential for using the specific properties of membrane proteins for training and design in a way that aids the alignment of their sequences.File S1Figure S1 Clustering principle used for generation of the HOMEP2 data set. Figure S2 Correlations between alignment accuracy measures. Figure S3 Comparison of alignment accuracy when using hydrophobicity scales as input descriptors in AlignMe. Figure S4 Profiles of the predicted membrane propensity from the three different transmembrane helix prediction methods tested for AlignMe. Figure S5 Accuracy of HOMEP2 alignments generated by different methods. Figure S6 Accuracy of homology models built from HOMEP2 alignments generated by different methods. Table S1 Proteins in the HOMEP2 data set, listed by family Table S2 Sequence identities between pairs of proteins in the same HOMEP2 family, based on their SKA structural alignments.Contains Figures S1 through S6 and Tables S1 and S2. (DOCX)Click here for additional data file.File S2Dataset of HOMEP2 homologous membrane proteins.(GZ)Click here for additional data file.File S3Manual for AlignMe.(PDF)Click here for additional data file.File S4Source code for AlignMe.(GZ)Click here for additional data file.