It was reported recently that male mice lacking brain serotonin (5-HT) lose their preference for females , suggesting a role for 5-HT signaling in sexual preference. Regulation of sex preference by 5-HT lies outside of the well established roles in this behavior established for the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). Presently, mice with a null mutation in the gene for tryptophan hydroxylase 2 (TPH2), which are depleted of brain 5-HT, were tested for sexual preference. When presented with inanimate  or animate  sexual stimuli, TPH2-/- males show a clear preference for female over male stimuli. When a TPH2-/- male is offered the simultaneous choice between an estrous female and a male mouse, no sexual preference is expressed. However, when confounding behaviors that are seen among 3 mice in the same cage are controlled, TPH2-/- mice, like their TPH2+/+ counterparts, express a clear preference for female mice. Female TPH2-/- mice are preferred by males over TPH2+/+ females but this does not lead to increased pregnancy success. In fact, if one or both partners in a mating pair are TPH2-/- in genotype, pregnancy success rates are significantly decreased. Finally, expression of the VNO-specific cation channel TRPC2 and of CNGA2 in the MOE of TPH2-/- mice is normal, consistent with behavioral findings that sexual preference of TPH2-/- males for females is intact. In conclusion, 5-HT signaling in brain does not determine sexual preference in male mice. The use of pharmacological agents that are non-selective for the 5-HT neuronal system and that have serious adverse effects may have contributed historically to the stance that 5-HT regulates sexual behavior, including sex partner preference. Lmx1b) or tryptophan hydroxylase 2 (TPH2), respectively, suggested for the first time that 5-HT regulates sexual preference [Lmx1b-/- and TPH2-/- mice lost their preference for mice of the opposite sex and this preference could be reinstated by pharmacological replenishment of 5-HT.Serotonin 5-HT) axons innervate virtually all areas of the nervous system from the B1–B9 clusters of neuronal cell bodies located in the mesencenphalon . In its -HT axonseference . In theseference  Lmx1b-/-Interest in TPH2 was reinvigorated when it was discovered that this enzyme existed in two distinct forms . TPH wasIn the course of developing and maintaining our colony of TPH2-/- mice we have noted, apart from occasional bouts of aggression, relatively normal sexual approach and courtship behavior in mating pairs of TPH2-/- mice. The results of studies suggesting that TPH2-/- mice lose their sexual preference ,19 prompad libitum. All mice were socialized , sexually naïve and between 8–14 weeks of age. All behavioral tests were observed and recorded by two independent observers. The Institutional Animal Care and Use Committee of Wayne State University approved the animal care and experimental procedures (Permit Number: A3310–01) and all procedures are in compliance with the NIH Guide for the Care and Use of Laboratory Animals.TPH2−/− mice were generated by deleting exon 1 of the Tph2 gene as described  and wereFilter paper (15.5 × 26.5 cm) was cut to cover the entire bottom of a small mouse cage (16 × 27 × 13 cm). Urine was collected from TPH2+/+ males and TPH2+/+ and TPH2−/− female mice in estrus. Estrus was confirmed by microscopic examination of vaginal smears and is characterized by clusters of cornified squamous epithelial cells . In ordeSexual interactions between 2 mice. This test was performed to determine male interactions with a single, receptive female. Male mice of either genotype were placed individually into a small test cage (16 × 27 × 13 cm) for 30 minutes and immediately thereafter, a TPH2+/+ or TPH2−/− female in estrus was placed into the test cage with the male. The latency to mount and the number of mounts, attacks and intromissions were counted in a 15-min test period.Sexual interactions among 3 mice. This test aimed to determine whether male mice express a sexual preference when presented simultaneously with a choice between a single male and a single receptive female mouse or a choice between two receptive female mice. Male TPH2+/+ or TPH2-/- mice  were placed individually into a small test cage (16 × 27 ×13 cm) for 30 minutes and then two intruder mice were introduced into the cage simultaneously. In one format, the intruder mice were a male TPH2+/+ mouse and a female TPH2+/+ mouse in estrus. In a second format, the intruder mice were a receptive female TPH2+/+ mouse and a receptive female TPH2-/- mouse. The latency and number of mounts, attacks and intromissions were scored for a 15-min period. In order to differentiate between TPH2+/+ and TPH2−/− female intruder mice, tails were coded distinctly with a permanent marker. Because of the complexity of the interactions among 3 mice in the same cage simultaneously , we modified the test to restrict access of the resident mouse with the intruder mice by placing the intruders in wire cups as used in the three chamber social approach test in the study of autism-like behaviors in rodents [ rodents ,44,45. TFemales were housed for 21 days with males in the following genotype combinations, with the female of the pairing listed first: TPH2+/+ x TPH2+/+; TPH2+/- x TPH2+/+, TPH2+/- or TPH2-/-, and TPH2-/- x TPH2+/- or TPH2-/-. Matings that would produce litters that were 100% heterozygous were not carried out. We had no experimental plan for the use of excess numbers of TPH2+/- mice, so these matings were not justifiable from the perspective of animal use guidelines. At the end of this two week period, males were removed and from this time forward, females remained singly housed until giving birth or until the passage of an additional 21 days . A mating was scored as fertile only when a litter was born.Male mice were sacrificed by decapitation and the VNO and MOE were dissected and stored frozen at −80°C until assayed. Frozen tissue was disrupted by sonication in 1% SDS at 95°C and insoluble material was sedimented by centrifugation. Protein was determined by the bicinchoninic acid method and equal amounts of protein (70 μg/lane) were resolved by SDS-polyacrylamide gel electrophoresis and then electroblotted to nitrocellulose. Blots were blocked in Odyssey Infrared Imaging System blocking buffer  for 1 h at room temperature. Primary antibodies against TRPC2 , CNGA2  or GAPDH  were added to blots and allowed to incubate for 16 h at 4°C. Blots were washed 3X in Tris-buffered saline with 0.1% Tween 20 and once with 1X PBS to remove unreacted antibodies and then incubated with fluorescent IRDye anti-IgG secondary antibodies (1:4000) in the dark for 1 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence and the relative densities of TRPC2-, CNGA2- and GAPDH-reactive bands were determined by imaging with an CLx Odyssey Image Station  and quantified using ImageJ software (NIH). Expression levels of TRPC2 and CNGA2 were normalized to GAPDH levels.www.graphpad.com.Data from the urine preference test and sexual preference behavior when using 3 mice simultaneously were analyzed by two-way ANOVA and post hoc comparisons were carried out using Bonferroni’s multiple comparison test. Data from sexual preference behavior in one-on-one pairings were carried out using one-way ANOVA and post hoc comparisons were made using Tukey’s multiple comparison test. Tests of mating success among pairings of genotypes were analyzed using a two-sided Fisher’s Exact test. Student’s t-tests were performed to analyze the normalized levels of TRPC2 and CNGA2 expression. Values of p < 0.05 were deemed statistically significant. All statistical analyses were carried out using GraphPad Prism version 6.02 for Windows, GraphPad Software, San Diego, CA, 2,58 = 53.63, p < 0.0001). An effect of male genotype was not present and the urine scent x genotype interaction was likewise not significant. Post hoc comparisons showed a significant preference by TPH2+/+ and TPH2-/- males for the urine scent of either female genotype over that of TPH2+/+ male urine (p < 0.0001 for each). When males of either genotype were given the choice of urine scents from female TPH2+/+ versus female TPH2-/-, a significant preference  was seen such that both TPH2+/+ males and TPH2-/- males displayed a significant preference for the urine scent of TPH2-/- females over that of TPH2+/+ females (p < 0.05 for both). These data show that male TPH2-/- and TPH+/+ mice have the same preference for females when given a choice of male and female inanimate sexual stimuli. Males of both genotypes also show a significant preference for urine scents of TPH2-/- females over scents from TPH2+/+ females.TPH2+/+ and TPH2-/- male mice were exposed to urine samples from TPH2+/+ males versus urine from either a TPH2+/+ or a TPH2-/- female. The results in 3,35 = 8.60, p < 0.0002) for mounting TPH2-/- females over TPH2+/+ females was shown by both TPH2+/+ (p < 0.05) and TPH2-/- (p < 0.01) males. TPH2+/+ and TPH2-/- males did not differ from each other in the number of mounts when paired with a female of either genotype. The latency to mount did not vary significantly in any of these pairings . Male TPH2-/- mice had significantly more intromissions with TPH2-/- females by comparison to TPH2+/+ females (p < 0.05). While TPH2+/+ males showed a trend toward more intromissions with TPH2-/- females versus TPH2+/+ females, this difference did not reach significance. As seen for mounts, TPH2+/+ males did not differ from TPH2-/- males in the number of intromissions with females of either genotype. We carried out male-on-male pairings but found that sexually-directed behaviors such as mounting and intromissions were completely displaced by overt aggression on the part of TPH2-/- males toward their cage partner , so it was not possible to determine male sexual preference in testes-intact TPH2-/- males. Future studies will carry out male-on-male pairings in castrated TPH2-/- mice in an attempt to reduce aggressive behavior [The results from pairings of individual male and female mice are presented in pairings . The numbehavior ,47.1,36 = 17.30, p = 0.0002) and resident male  were significant for the number of mounts. The intruder genotype x resident genotype interaction was not significant. TPH2+/+ resident males mounted the TPH2-/- female significantly more than the TPH2+/+ female (p < 0.01), and while TPH2-/- males showed a trend to greater numbers of mounts on the TPH2-/- female versus the TPH2+/+ female, this effect did not reach statistical significance. Using the approach of Liu and colleagues [We followed the approach of Liu and colleagues  to deter2,40 = 63.40, p < 0.0001). The interaction between intruder x resident genotype was also highly significant . When placed into a cage containing male and female TPH2+/+ intruders within wire cups, male TPH2+/+ (p < 0.0001) and TPH2-/- mice (p < 0.05) showed a significant preference for investigating female TPH2+/+ mice over TPH2+/+ males. Because of the complexity of the interactions among 3 mice in the same cage simultaneously, we modified the test to restrict access of the intruder mice to the resident mouse by placing the intruders in wire cups ,44,45. TIn light of the data above showing that TPH2-/- females in estrus seemed to be preferred over TPH2+/+ females by males of both genotypes Figs.  and 2, wA null mutation in the gene for TRPC2 ,48 or sulmx1b knockout) or with a null mutation in the gene for TPH2 lose their preference for females [The results presented in this paper show clearly that male mice with a genetic depletion of 5-HT retain their preference for females over males and stand in contrast to a recent study reporting that male mice with significant reductions in the number of 5-HT neurons in brain  or when comparing sexually-directed behaviors in one-on-one pairings of mice  is straightforward. In these cases, we observed that TPH2-/- males express a preference for females over males to the same extent as TPH2+/+ males. However, when examining sex preference by offering a resident male the simultaneous choice of an intruder male and an intruder female, this grouping of 3 mice is extremely complex and involves far more than the resident male solely expressing a sex choice between the two intruder mice. The intruder male and female mice in this paradigm are also faced with several active choices and do not remain passive in the cage while the resident male makes a choice between them. Certainly, the resident male approaches and eventually mounts the female or the male intruder, or both. We frequently observed that the intruder male will also mount the intruder female while the resident male stands by. If the sexual advances of the resident or intruder male are rejected by the intruder female, it often provokes an attack by the rejected male on the other male. Finally, the resident male, especially TPH2-/- males, attack the intruder male and female without obvious provocation as seen in the resident-intruder test of aggressive behavior ,41. For The pharmacological tools used to study the role of 5-HT in determining sexual preference and activity are limited in their specificity and may have contributed to the confusing state of affairs in this area for several reasons. First, early studies showing that reductions in brain 5-HT led to increased sexual activity depended on the use of extremely high doses of the TPH2 inhibitor p-chlorophenylalanine (pCPA). This inhibitor indeed lowers brain 5-HT levels but it also leads to large depletions of dopamine and norepinephrine . pCPA alMice lacking the gene for TPH2 are viable and fertile and, for the most part, have normal morphological and physiological characteristics ,24,26,27In summary, we conclude that a genetic depletion of 5-HT from brain does not have an effect on sexual preference or activity. Previous studies that have implicated 5-HT signaling in these behaviors used pharmacological approaches  that lack specificity for the 5-HT neuronal system and which have numerous, serious adverse effects, both of which make it difficult to attribute observed changes in behavior to 5-HT. The use of inanimate sexual stimuli (urine scents) and pairings between single male and female mice showed clearly that TPH2-/- males prefer females over males. This preference appeared to be lost when TPH2-/- and TPH2+/+ males were given the choice of interacting with a male or female intruder mouse simultaneously. When competing, non-sexually related behaviors that were displayed among 3 interacting mice were controlled by placing the two intruder mice in wire cups, the preference of male TPH2-/- mice for females was affirmed. TPH2-/- females were preferred over TPH2+/+ females in most tests but this effect did not transfer into fruitful matings in TPH2-/- mice. Finally, the finding of normal expression of TRPC2 in the VNO and of CNGA2 in the MOE of TPH2-/- males alone supports the conclusion that the sexual preference of TPH2-/- males for females over males is intact.


Plasmodium falciparum erythrocyte membrane protein 1) encoded by the polymorphic multi-copy var gene family plays an important role in parasite biology and the host-parasite interactions. Sequestration and antigenic variation is an essential component in the survival and pathogenesis of Plasmodium falciparum and contributes to chronic infection . The DBLα domain of PfEMP1 is a potential target for immuno-epidemiological studies and has been visualized as a vaccine candidate against severe malaria. Extensive var gene diversity (DBLα domain of PfEMP1) is an important aspect of pathogenesis in parasite lines. Specific host receptors like heparin, heparan sulphate, blood group A and complement receptor 1 have been reported to bind DBLα domain. Although heparin has been experimentally shown to disrupt the parasite-host interaction and effectively disrupt rosetting, the binding sites for the DBLα domain and mechanism behind heparin-mediated rosette inhibition have not been elucidated.The variant surface antigen PfEMP1 (3D structures and epitopes of DBLα domain in 3D7 and in two parasite isolates have been predicted and compared. Docking studies on DBLα domains with human GAG receptors (heparin and heparan sulphate) to predict the strength of association between the protein–ligand interactions were performed.The DBLα domain structures showed extensive diversity and polymorphism in their binding sites. The linear as well as conformational epitope prediction analysis predicted several B cell epitopes at variable positions in different DBLα domain variants . The docThus such an analysis may lead to the development of novel interference strategies to block red blood cell invasion and provide protection against malaria and provide an insight into the complexities of host parasite interactions.


Recently, we reported that there is significant low field relaxation dispersion in myocardial and fibrotic scar tissue during in vivo T1rho MRI, contributing to contrast enhancement compared to spin echo (T2) MRI. However, we also observed that differences in magnetic field susceptibility between the heart and lung organs and poor RF penetration and field homogeneity in large patients and at 3 T contribute to T1rho quantification errors. Our goal was to determine the influence of static (B0 and B1 field heterogeneity influences T1rho MRI. Tilted rotating frame (TRF) Bloch simulations were performed for a rotary-echo spin lock, varying field heterogeneity and spin lock duration  with random Gaussian noise . Phantom experiments were performed using a homogeneous H2O cylinder (MnCl2 = 0.016%). In vivo experiments were performed in Yorkshire swine (n = 5). Simulated phantom and in vivo images were derived from B0 and B1 maps using the TRF Bloch simulations and were correlated with experimental images at the same spin lock duration.We performed simulations and experiments to explore how B0 field and in the 50 mT/m field gradient in which the B0 field was less than 50 Hz off-resonance. Noticeable variations were observed at 100 Hz off-resonance (100 mT/m gradient). The B0 field in vivo is much less homogeneous than that for the phantom; therefore the T1rho images show much more complex patterns  could result in nutation about an effective field oriented away from the idealized spin lock axis. In vivo results correlated well with simulations and phantoms experiments. Measured T1rho relaxation times in the three identical compartments were constant in the homogeneous Bs Figure . Mean B0Spin lock artifacts were the result of static field heterogeneity predominantly confined to the left ventricular posterolateral wall. Static field heterogeneity should be reduced to less than 10% of the spin lock field amplitude to minimize T1rho quantification errors.The authors gratefully acknowledge support from the National Institutes of Health through awards K99HL108157 and R01HL63904.


The aims of this study were to evaluate the prevalence of Dog Erythrocyte Antigens (DEA) 1, 4, and 7 in Ibizan hounds, to compare the results with the prevalence of DEA in Spanish greyhounds, and to determine the risk of sensitization following the first transfusion of blood not typed for DEA 1 and the probability of an acute hemolytic reaction following a second incompatible transfusion using untyped DEA 1 blood. DEA 1, 4, and 7 status was determined in 92 Ibizan hounds. Results were compared with the previously reported prevalence in Spanish greyhounds. The risks of sensitization and of a hemolytic transfusion reaction were determined amongst Ibizan hounds and between Ibizan hounds and Spanish greyhounds. The prevalence of DEA 1, 4, and 7 was 75%, 98.9%, and 25%, respectively. There was a significantly higher expression of DEA 1 and 7 in Ibizan hounds than in Spanish greyhounds. The probability of sensitization of a recipient dog to DEA 1 with transfusions amongst Ibizan hounds was 18.5% and between Ibizan hounds and Spanish greyhounds was 13.7%. The probability of an acute hemolytic reaction in each group was 3.5% and 1.9%, respectively. There is a higher prevalence of DEA 1 and 7 in Ibizan hounds than in other sighthounds. There is international standardization of seven canine blood groups as categorized by presence of Dog Erythrocyte Antigen (DEA) 1, 3, 4, 5, 6, 7, and 8 , but nonDEA 1 and 7 are the most important blood types with regard to canine blood transfusions. DEA 1 antigen has recently been classified as a unique DEA epitope with variable surface expression   and is pDEA 7 antigens are reported to occur naturally in between 40 and 72% of the canine population , 15, 16.There is also a high prevalence of DEA 4 antigens 98–100%) in the canine population –100% in  and a heAn ideal blood donor does not have blood antigens of types that commonly cause reactions in unmatched recipients. There is no universally agreed definition of a universal canine donor. The most restrictive definition of the universal canine donor would be a dog negative for DEA 1, DEA 3, DEA 5, and DEA 7 and positive for DEA 4. Since 98% of dogs are positive for DEA 4, the rarity of DEA 4 negative dogs means that this antigen is unlikely to influence donor selection. Some transfusion specialists do not exclude DEA 7 positive dogs from the donor pool. In fact the concept of the universal donor in dogs has long been debated since the tests for DEA 3 and DEA 5 antigens are often not available and the quality of these tests is variable; currently typing sera are available only for DEA 1, 4, and 7 antigens. As previously proposed , univershttp://www.fci.be/en/nomenclature/IBIZAN-PODENCO-89.html).Ibizan hounds are medium-size sighthounds originating from the island of Ibiza. They are traditionally used in the Balearic Islands (and less so in Spain and France) to hunt rabbits and other small game. In recent times this breed has been disseminated worldwide through a variety of adoption programs. The Ibizan hound is classified by the Fédération Cynologique Internationale (FCI) in Group 5 (spitz and primitive types) and in section primitive type-hunting dogs. This dog is a typical and robust representative of one of the oldest breeds still in existence (The aims of this study were threefold: (1) to evaluate the prevalence of DEA 1, DEA 4, and DEA 7 blood type in Ibizan hounds; (2) to compare the results with the previously published prevalence of DEAs in other sighthounds ; (3) to determine the risk of recipient sensitization following the first transfusion of blood not typed for DEA 1 and the probability an acute hemolytic reaction following a second incompatible transfusion of blood untyped for DEA 1 both amongst Ibizan hounds and between Ibizan hounds and Spanish greyhounds.In this prospective study EDTA-anticoagulated blood samples were collected from 92 healthy owned Ibizan hounds in January 2015. Dogs were between 1-year and 3-year old, 89 were female (96.7%) and 3 were male (3.3%), and all were living on Ibiza Island, Spain.With the owners' consent cephalic blood samples were collected with a 23 G needle  connected to a 2 mL syringe . They were transferred to a 1 mL EDTA-anticoagulated tube and were stored between 4 and 5°C. All blood samples were collected as part of a study to determine the distribution of blood groups in this breed. This study was conducted according to European legislation (2010/63/EU). In all samples blood typing was performed as described below.DEA 1 status was determined using a commercially available card agglutination technique  according to the manufacturer's instructions and as previously described , 20. TheAnalysis for DEA 4 and 7 antigens was performed by gel column agglutination within microtubes  as previously described  using poAnti-DEA 4 and 7 antibodies were imported and used in this study with the authorization of Italian Health Minister (protocol authorization number 0021278-15/10/2014-DGSAF-DGSAF-P). Blood-typing was performed at the Veterinary Transfusion Unit (REV) of the University of Milan, Milan, Italy.μL of a 0.8% RBC suspension made by suspending 10 μL of the RBC pellet in 1 mL of low ionic strength solution  was mixed with 25 μL of DEA 7 antibodies or with 15 μL of DEA 4 antibodies in the reaction chamber of saline gel columns. For all samples a negative control column with saline solution was included. The gel columns were incubated at 4°C for 30 minutes and were then centrifuged in a special gel column card centrifuge  at 80 × g for 10 minutes. Finally the gel column cards were evaluated for presence and strength of agglutination. Only results validated by negative controls were included in analysis.Briefly 25 The cards were visually interpreted as follows: (0) negative, when all RBCs were at the bottom of the column; 1+, when very few RBC agglutinates were dispersed in the lower part of the gel, with most RBCs at the bottom of the tube; 2+, when all RBCs were agglutinated and dispersed in the gel; 3+, when some RBC agglutinates were dispersed in the upper part of the gel and most of the RBCs formed a red line on the surface of the gel; and 4+, when all RBCs formed a red line on top of the gel. Results were interpreted as negative if no agglutination or 1+ agglutination was present, whereas ≥2+ agglutination reactions were considered positive.The probability of an Ibizan hound becoming sensitized following the first transfusion of blood that was neither cross-matched nor typed for DEA 1 was calculated using the following formula , 10:(1)%The probability of the same dog developing an acute hemolytic reaction with a second incompatible transfusion using untyped blood from any other dog was calculated using the following formula : (2)%  DResults were analysed by absolute prevalence analysis. Using contingency tables or Fisher's exact test the prevalence of DEA 1, 4, and 7 and universal donors in Ibizan hounds calculated in this study was compared with a population of 205 Spanish greyhounds  in which the prevalence of DEA 1, 4, and 7 and universal donors  was 54.6%, 100%, 8%, and 46.7%, respectively .P < 0.05.All statistical analysis was performed using statistical software  with significance set at For DEA 1 blood type, 69 (75%) dogs tested positive and 23 (25%) tested negative. For DEA 4 blood type, 91 (98.9%) dogs tested positive  and 1 (1.1%) dog tested negative. For DEA 7 blood type, 23 (25%) dogs tested positive (16 samples showed 3+ agglutination and 7 samples showed 2+ agglutination) and 69 (75%) were negative (no samples showed agglutination). Of the 92 dogs, 16 (17.4%) were DEA 4 positive only  and 1 (1.1%) dog was negative for all DEA. All control samples in the gel columns tested negative (absence of agglutination).P = 0.005 and P = 0.002, resp.). The prevalence of universal donors (DEA 1 and DEA 7 negative and DEA 4 positive or negative) was lower in Ibizan hounds (P = 0.000008) than in Spanish greyhounds. There was no significant difference in DEA 4 prevalence between the 2 breeds.Ibizan hounds had a significantly higher prevalence of DEA 1 and DEA 7 positivity than Spanish greyhounds (The probability of an Ibizan hound recipient becoming sensitized following the first transfusion of blood from an Ibizan hound donor that was not cross-matched nor typed for DEA 1 was approximately 1 in 5 (18.8%). The probability of the same dog developing an acute hemolytic reaction with a second incompatible transfusion using blood untyped for DEA 1 from any other Ibizan hound was 3.5%.The probability of a recipient Ibizan hound becoming sensitized following the first transfusion of blood from a Spanish greyhound that was not cross-matched nor typed for DEA 1 was approximately 1 in 7 (13.7%). The probability of the same dog developing an acute hemolytic reaction with a second incompatible transfusion using untyped DEA 1 blood from Spanish greyhound was 1.9%.There are breed and geographical differences in the prevalence of different blood group antigens , 15. It In this study the prevalence of DEA 1 expression in Ibizan hounds was 75%, which is higher than previously reported in other pure breeds , 15, 16 All 92 Ibizan hounds were blood typed and only one was DEA 4 negative. This prevalence of DEA 4 expression (98.9%) is in agreement with the prevalence in the general canine population , 21.The prevalence of DEA 7-positive dogs in the study population (25%) was similar to that reported in Golden Retrievers in USA and Brazil (25% and 27% resp.)  but highThe prevalence of universal or “ideal” donors, that is, dogs positive only for DEA 4 or negative for all DEA, in our population of Ibizan hounds, was 18.5%. This is lower than the prevalence of 57.3% found in greyhounds  and in aIn this study the probability of a recipient becoming sensitized and produced antibodies against DEA 1 following the first transfusion of blood that was neither cross-matched nor typed for DEA 1 was 18.8% 1 in 5) amongst Ibizan hounds and 13.7% (1 in 7) between Ibizan hounds and Spanish Greyhounds. These probabilities were lower than previously reported in dogs from Portugal , in Span amongst The gel agglutination technique has been used for decades. This test is sensitive for the detection of DEA 1 and is suited for screening blood donors in a blood bank program , 20. ThiAnother limitation of this study was that the study population of Ibizan hounds was almost exclusively female, mirroring the environment in which Ibizan hounds live. Hunters run these dogs in mostly female packs, as the female is considered the better hunter. In addition we did not know how closely related the dogs used in this study were. It is possible that a closed population of closely related individuals could bias the prevalence of blood types. Finally DEA 3, DEA 5, and Dal blood types were not tested since relevant antisera were unavailable at the time the study was performed.The population of Ibizan hounds studied here showed a different prevalence for DEA 1 and 7 with respect to previous reports of other sighthounds. Although the risk of sensitization and an acute transfusion reaction following incompatible blood transfusion is low, it remains best practice to blood type and cross-match recipients before transfusion.


This study was conducted to analyze the changes of gait parameters between two different footwear size groups due to change of angle between heel and sole of the footwear by keeping the same heel height for both groups.A cross-sectional descriptive study design.The effects of the angle between the sole and the heel of heeled footwear on single and double support time, stride duration and toe off plantar flexion of females.SD = 2.5) and forty nine subjects to the footwear size 40 group with 24.4 (SD = 2.4) years mean age. Both groups were given similar type three centimeters height heeled footwear and there was 6 greater angle locate between heel and sole of size 36 footwear than size 40.Two cameras  were used to capture the walking trials and each individual perform three trials. Captured two dimensional walking trials were analyzed with Motion view 8.0 video analysis software. Independent sample t-test in Statistical Package for Social Sciences Statistics version 17.0 (SPSS Statistics 17.0) was used to analyze the data.Hundred (100) female subjects were participated for the study and fifty one (51) subjects were included to the footwear size 36 group with 23.8years mean age (p<0.05) while the single support time and stride duration was not shown a significance (p>0.05).Mean toe off phase plantar flexion and double support time was shown a significant difference (The angle between heal and sole of footwear significantly deviate gait parameters so rather than heel height footwear type it need to consider about shoe length.


Her2 FISH-amplified cases showed an increasing trend significantly through their corresponding HER2 IHC ordinals by the 2007 and the 2013 criteria, respectively . After excluding equivocal cases, the specificity (100%) and positive predictive value (100%) were unchanged under either the 2007 or the 2013 criteria. The sensitivity (100%), negative predictive value (NPV) (100%), and accuracy (100%) of HER2 IHC were higher under the 2013 criteria than those  under the 2007 criteria. Of the total 49 cases, the number (n = 4) of HER2 IHC equivocal results under the 2013 criteria was 4-fold higher than that (n = 1) under the 2007 criteria (8.16% vs 2.04%). Conclusively, if first tested by IHC, the 2013 criteria caused more equivocal HER2 IHC cases to be referred to Her2 FISH testing than the 2007 criteria. That decreased the false-negative rate of HER2 status and increased the detection rates of HER2 positivity in mucinous EOC.The remarkable success of trastuzumab and other newly developed anti-HER2  therapies in breast, gastric, or gastroesophageal junction cancer patients has supported us to investigate the HER2 status and its possible therapeutic implication in mucinous epithelial ovarian cancer (EOC). However, there is currently no standardization of HER2 scoring criteria in mucinous EOC. In this study, we aimed to compare both the assay performance characteristics of the 2007 and the 2013 American Society for Clinical Oncology and College of American Pathologists scoring methods. Forty-nine tissue microarray samples of mucinous EOC from Asian women were analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) tests using the 2007 and the 2013 criteria, respectively. The overall concordance between IHC and FISH by the 2007 criteria was 97.92 % (kappa = 0.921), and that by the 2013 criteria was 100% (kappa = 1.000). The percentage of  The possible mechanisms of carcinogenesis include activation/amplification of oncogene, inactivation of tumor suppressor genes, inhibition of apoptosis, angiogenesis, and so on. Oncogene amplification causes oncoprotein overexpression and promotes tumor growth. HER2 positivity, in which the HER2 receptor is either overexpressed in the protein stage and/or amplified at the genomic level, has accounted for approximately 20% to 30% of breast cancers and 18% to 35% of mucinous EOCs.9After carefully excluding metastatic mucinous carcinoma and borderline tumors, primary mucinous epithelial ovarian cancer (EOC) makes up approximately 2% to 4% of all ovarian epithelial carcinomas.11 However, there is, so far, no consensus in defining the HER2 positivity in mucinous EOC.12The success experiences of trastuzumab therapy in breast cancer, gastric, or gastroesophageal junction (GEJ) cancer patients and newly developed anti-HER2 drugs encouraged the investigation of anti-HER2 therapy application in other cancers, including mucinous EOC.14 Even though pathology communities adopted the 2007 ASCO/CAP algorithms previously, they will soon be familiar with the new 2013 modified rules all over the world. In this study, we aimed to compare both HER2 assay performance characteristics in mucinous EOC using the 2007 and 2013 ASCO/CAP scoring criteria, respectively.Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are still widely used in assessing the HER2 status of clinical specimens. The American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) proposed original guidelines to test the HER2 status in breast cancers in 2007 and amended those guidelines in 2013 after concerns were raised about false-positive and false-negative HER2 assessments.9 All the experimental samples used in this study were de-linked from direct patient identifiers, and the research was conducted according to International Conference on Harmonization guidelines and complied with all applicable regulations for protection of human subjects of research, including review and approval by the Institutional Review Board, Chung-Shan Medical University Hospital, Taichung, Taiwan.The study materials consisted of 49 cases of mucinous EOC; the characteristics of the TMA derivation were described in our previous report.The HER2 immunostains were performed on the fully automated Ventana Benchmark XT autostainer using pathway antiHER2/neu rabbit monoclonal antibody . HER2 IHC score 3+ breast cancer was used as a positive control. Negative controls were obtained by excluding the primary antibody. The slides were mounted with Permount for microscopic examination, and the images were captured by the NIKON ECLIPSE 50i microscope and NIKON DS-Fi1 Digital Camera System for study comparison.Her2 DNA Probe Kit protocol . The dual-color FISH consisting of 2 labeled DNA probes was performed on sections cut from the same tissue microarray (TMA) blocks. The LSI HER2 probe that spans the entire Her2 gene was labeled in Spectrum Orange, and the CEP17  probe was labeled in SpectrumGreen and hybridized to the alpha satellite DNA located at the centromere of chromosome-17 (17p11.1–q11.1). Counting 2 separate fields of at least 20 cells was essential. We calculated the Her2: CEP17 signal ratio by recording the numbers of Her2 gene (red) and chromosome 17 (green) signals from preselected tumor areas. In most cases, tumor cells from matching sites of IHC analysis were scored for the number of red (Her2) and green (CEP17) signals. Signal photos were taken with the NIKON ECLIPSE 80i fluorescent microscope with a PlanFluor oil objective (100×) using a double band-pass filter that permitted simultaneous green and red colors.The FISH test was performed by the ABBOTT/Vysis PathVysion Her2 FISH tests in this study  assessments, we ran an HER2 control daily in all cases and had 1 pathologist routinely screening the slides.Her2 FISH as the reference standard, the HER2 IHC performance measures were calculated by 2007 and 2013 ASCO/CAP scoring criteria, respectively. Sensitivity was defined as the ratio of HER2 IHC-positive cases among Her2 FISH-amplified patients, specificity was defined as the ratio of HER2 IHC-negative cases among Her2 FISH nonamplified patients, positive predictive value (PPV) was defined as the ratio of Her2 FISH-amplified cases among HER2 IHC-positive patients, negative predictive value (NPV) was defined as the ratio of Her2 FISH nonamplified cases among HER2 IHC-negative patients, as well as accuracy was defined as the ratio of HER2 IHC-positive and Her2 FISH–amplified cases plus HER2 IHC-negative cases and Her2 FISH nonamplified cases among all cases. The overall concordance was defined as the ratio of HER2 IHC-positive and Her2 FISH-amplified cases plus HER2 IHC-negative cases and Her2 FISH nonamplified cases among all nonequivocal IHC cases. Data were analyzed using standard statistical software . All tests were 2-sided and the significance level was 0.05.The consistency between 2007 and 2013 ASCO/CAP IHC results was analyzed by categorized variables using Kappa statistics. The HER2 positivity was defined as having a positive IHC result irrespective of the FISH ratio, plus equivocal or negative IHC result but FISH amplification. Furthermore, we applied the Cochran-Armitage trend test to assess for a trend of positive percentages across the ordinal variables. Regarding Her2 FISH results  by the 2007 and the 2013 ASCO/CAP scoring criteria, they agreed perfectly   by the 2007 criteria but reclassified as having HER2 IHC score 2+  by the 2013 criteria (Table Her2 amplification rates of mucinous EOC (n = 9/49) were the same in both the criteria (18.37% vs 18.37%); the percentage of Her2 FISH amplification increased significantly in a trend through the ordinals of HER2 IHC results  by either the 2007 or the 2013 criteria, respectively (P < 0.001 vs P < 0.001)  cases which were identified based on the 2013 criteria, two cases show FISH amplification and two cases show FISH nonamplification. On the other hand, only one HER2 IHC score 2+ case is identified based on the 2007 criteria and it shows FISH amplification.Under the 2007 and the 2013 ASCO/CAP scoring criteria, we demonstrated that Except for 1 case with a HER2 IHC score 2+  by the 2007 criteria, our data for the relationship between IHC and FISH showed 100% (n = 7/7) in positive concordance, 97.56% n = 40/41) in negative concordance, and 97.92% (n = 47/48) in overall concordance ; whereas except for the 4 cases with HER2 IHC score 2+  by the 2013 criteria, our data for the relationship between IHC and FISH showed 100% (n = 7/7) in positive concordance, 100% (38/38) in negative concordance, and 100% (45/45) in overall concordance   and PPV (100%) by both the 2007 criteria and the 2013 criteria were similar. The sensitivity under the 2007 criteria was lower than that under the 2013 criteria (87.5% vs 100%), the NPV under the 2007 criteria was lower than that under the 2013 criteria (97.6% vs 100%), and the accuracy under the 2007 criteria was lower than that under the 2013 criteria (97.9% vs 100%)  of positive and equivocal results by HER2 IHC and Her2 FISH tests have been down-adjusted, and the 2013 criteria seemed to be less stringent than the 2007 criteria  had Her2 gene amplified by both the 2007 and the 2013 Her2/CEP17 thresholds, respectively (>2.2 vs ≥2.0), but none (n = 0/49) had Her2/CEP17 <2.2/<2.0 with Her2 gene copy numbers ≥6.0 by either the 2007 or the 2013 criteria.The Her2 FISH test in the 2013 criteria was defined as cases showing a Her2/CEP17 <2.0 and an average absolute Her2 signal count per cell of ≥4.0 and <6.0 versus the 2007 criteria, which defined equivocal as cases showing a Her2/CEP17 ratio between 1.8 and 2.2 or Her2 signal count per cell of ≥4.0 and <6.0. Our data revealed that no equivocal Her2 FISH cases (n = 0/49) occurred by both the 2007 and the 2013 criteria.The equivocal The positive HER2 IHC test (score 3+) was defined as circumferential, complete, uniform, and intense staining of >10% of tumor cells in the 2013 criteria versus >30% in the 2007 criteria. Our data revealed that 7 cases (n = 7/49) with HER2 IHC score 3+ existed by both the 2007 and the 2013 criteria. All of them (n = 7) showed homogenous strong, complete membrane staining >30%.The equivocal HER2 IHC result (score 2+) was defined as circumferential membrane staining that is “incomplete” and/or weak/moderate and >10% of tumor cells in the 2013 criteria versus “complete” membrane staining in the 2007 criteria, or complete and circumferential membrane staining that is intense and within ≤10% of tumor cells in the 2013 criteria versus ≤30% in the 2007 criteria. Our data revealed that the 2013 criteria identified more HER2 IHC equivocal cases  than the 2007 criteria  in all 49 cases  was unchanged under 2007 and 2013 criteria. The alterations from HER2 IHC score 0 to 1+ have no clinical relevance. Both the HER2 IHC score 0 and score 1+ have been regarded as “negative” result category. Our data revealed that 25 cases remained HER2 IHC score 0 (negative) and none were upgraded to a score of 1+ by either the 2007 or the 2013 criteria. However, 3 in 16 cases with an IHC score of 1+ (negative) by the 2007 criteria would be reclassified as IHC equivocal (score 2+) result by the 2013 criteria, which therefore required alternative FISH testing  cases would be considered to have negative HER2 status without any further testing on the basis of the 2007 and 2013 ASCO/CAP algorithms. As a result, the rigorous 2007 ASCO/CAP criteria potentially diminished the detection rates of HER2 positivity in mucinous EOC patients in comparison with the undemanding 2013 ASCO/CAP criteria.We identified that 2.04%  or none  of HER2-positive patients would be missed if the 2007 criteria were used. In other words, if the HER2 IHC test was applied first, the 2013 criteria can detect 1 case with However, the lenient 2013 guidelines may permit more patients to go through the second round of testing, so as to avoid the possibilities of missing HER2 positive mucinous patients. Thus, it unfavorably increases the cost of examination and extends the reporting date of HER2 status.Her2 testing in this study on the mucinous EOC. The major reasons were: FISH has been shown to be theoretically easier to interpret due to the stability of the DNA target; the interobserver variation was lower because FISH was an objectively quantitative test; and none with Her2 equivocal FISH existed under both 2007 and 2013 criteria in all 49 cases. So that, the assay performance characteristics of HER2 IHC testing by both 2007 and 2013 criteria were assessed and compared  in mucinous EOC, respectively; both the frequencies of Her2 FISH amplifications by 2007 and 2013 ASCO/CAP scoring criteria were equivalent ; the frequency of HER2 IHC equivocal results by 2007 ASCO/CAP scoring criteria  was less than that by 2013 ASCO/CAP scoring criteria ; and 1 case with Her2 FISH amplification was missing, when first tested by IHC under the 2007 criteria.In summary, we demonstrated that both 2007 and 2013 ASCO/CAP scoring criteria agreed excellently in assessing the HER2 IHC (kappa = 0.903) and When evaluating the HER2 status by IHC first in mucinous EOC, we identified 1 more case with HER2 positivity by the 2013 than that by the 2007 ASCO/CAP guideline scoring criteria. Compared with the 2013 criteria, the more rigorous 2007 criteria resulted in inevitable false-negative IHC, which not only subtracted the opportunity to be referred to FISH testing, but also possibly diminished the detection rates of HER2 positivity in mucinous EOC.


IGF2BP3 (IMP3) is a mRNA binding protein that regulates IGF2 translation and function during embryogenesis. Because IGF2BP3 is undetectable in adult human tissues except the testis, and increased IGF2BP3 expression has been noted in several cancers, it is considered a cancer testis (CT) protein. IGF2BP3 mRNA expression in colorectal cancers (CRC) has not been well studied. This study's aim was to quantitatively assess IGF2BP3 mRNA expression in CRC and, thus, determine if IGF2BP3 has potential as a vaccine target.Data were collected prospectively from CRC patients in an IRB-approved tissue and data bank. Total RNA was isolated and purified from tumor and normal colonic tissue samples and cDNA synthesized. IGF2BP3 expression was analyzed by quantitative PCR (QPCR). Expression levels of IGF2BP3 in tumors and testis were determined and compared. Tumors with levels greater than 0.1% or more of the testis levels were considered positive. Analysis of IGF2BP3 protein expression by immunohistochemistry (IHC) in tumor and normal tissues was also performed.A total of 84 paired tumor and normal tissue specimens were assessed from patients with Stage 2 and 3 CRC; 43% of tumors had IGF2BP3 mRNA expression levels greater than 0.1 % of that of testis and were considered positive. The median tumor expression level was higher in women (p=0.042). No correlation was found between IGF2BP3 mRNA expression and tumor stage or lymph node involvement. IHC was carried out on paired tumor and normal tissue sections from 46 patients; IGF2BP3 staining was noted in 50% of the tumor sections and in 5% of the normal tissue sections.IGF2BP3 mRNA was over expressed in 43% of the tumors whereas the protein was noted in 50% of samples. No correlation between mRNA expression and disease severity was noted. This protein holds promise as a vaccine target, however, a larger study that assesses a more diverse population of patients (Stage 1-4) as well as a study of preoperative serum samples for auto-antibodies to IGF2BP3 are needed to pursue this concept. Surgery remains the mainstay of treatment for patients with colorectal cancer. The last two decades have seen the development of new chemotherapeutic agents and adjuvant and neoadjuvant treatment regimens that have notably improved the long term outcome of these patients. Despite these advances, over 40% of patients who undergo “curative” surgery will progress and develop metastatic disease . TherefoFor many years, numerous investigators around the world have been pursuing the concept of cancer vaccines. The idea of harnessing the patient's immune system to recognize and attack tumor cells is very attractive and likely to be associated with minimal toxicity when compared to conventional chemotherapy agents. Much progress has been made over the past 40 years in regards to our understanding of how the immune system functions and also how the tumor evades immune detection or inhibits the immune response. A very important advance was the approval for clinical use of antibodies that block immune checkpoint proteins ,3, whichCancer/testis (CT) proteins are a category of proteins that hold particular promise as vaccine targets because of their restricted expression . These pIGF2 mRNA binding protein 3 (IGF2BP3) is an mRNA binding protein and cancer testis protein that regulates the translation of IGF2. IGF2BP3 and the other IGF2 mRNA binding proteins (IMPs) regulate IGF2 translation, stability and localization by binding to mRNA ,6. IGF2BAlthough prior investigators have assessed a group of colorectal adenocarcinomas for the presence of IGF2BP3 protein via IHC –17, a quA total of 84 pairs of normal and cancerous colorectal tissue were assessed in this study. There were 39 males and 45 females; average age was 67.5 +/− 14.4 years. The tumor locations were as follows: right, 42 (51%); sigmoid/rectosigmoid, 24 (29%); rectal, 11(13%); and transverse or descending colon, 7(6%). The study was limited to pathologic stage 2 and 3 tumors; there were 48 stage 2 (57%) and 36 stage 3 (43%) tumors.To determine background values for normal tissues, we evaluated the expression of IGF2BP3 in 22 normal adult tissues using semi-quantitative RT-PCR Figure . IGF2BP3All paired samples underwent quantitative PCR as described in the methods section. Of the 84 tumor specimens assessed via RT-PCR, 43 percent of the tumors met the criteria used for scoring positivity of cancer testis proteins, which comprises having expression levels that were greater than observed in normal colonic mucosa and also greater than 0.1 % of the testes expression level (expression levels over 100 in this study) . When thIHC was carried out on 46 paired tumor and normal tissues specimens that collectively covered the full range of RT-PCR protein expression. Positive IGF2BP3 cytoplasmic staining (1+ to 3+ intensity in >10% cells) was noted in 50% of the tumor specimens and 5% of the normal tissue specimens (1+ to 2+) intensity Figure .No significant differences were found in IGF2BP3 mRNA expression levels based on the stage of tumor or presence of lymph node metastases (n=36). There was a non-significant increase in mean expression levels in stage 3 tumors  versus stage 2 tumors  as well as in node-positive group  versus node-negative group . There was however, a significant increase in median IGF2BP3 expression level in women compared to men (p=0.042).The colorectal cancer specimens utilized for this study were taken from IRB approved tissue and data banks organized, managed, and maintained by the Sections of Colon and Rectal Surgery at New York Presbyterian Hospital, Columbia University Health Science Campus and Mount Sinai Roosevelt Hospital in New York City. Samples of tumor tissue and normal adjacent large bowel mucosa were taken from the resected specimens immediately following resection and were rapidly frozen.Demographic data as well as the location of the cancer, the pathology results and the final tumor stage were obtained from the prospective data base. Tumor and paired normal tissue samples from 84 CRC patients ; were studied. Among the study group 6 paired tissue samples were from patients with mucinous tumors.Tumor and normal mucosal samples were obtained from operative specimens after gross evaluation by a pathologist. The tissue samples were placed in separate standard Cryomolds  which were filled with OCT  compound  and placed in liquid nitrogen. The frozen tissue samples were stored in a −80°C freezer.Prior to being included in this study, sections from candidate frozen tumor and normal colonic tissue blocks were taken at −20°C and assessed histologically to confirm the presence of tumor and the quality of each sample. This analysis was done at the Irving Cancer Center of Columbia University in New York City. Poor quality specimens and those with a paucity of tumor cells were not selected for the study.Total RNA was isolated using Qiazol , and purified using the miRNeasy Kit . Briefly, sections of the frozen tissue block (previously embedded in OCT compound) were cut and placed into a 2ml Eppendorf tube, and then homogenized by TissueLyser II  with 1ml Qiazol added. After the aqueous phase was recovered from the chloroform-derived phase separation, another step of acid phenol-chloroform extraction was performed with a phase lock gel tube . RNA precipitation, column binding, on-column DNase digestion, washing and elution were performed according to kit instructions. RNA integrity was confirmed by agarose gel electrophoresis and the concentration was quantified by measuring OD260nm in BioPhotometer .The cDNA first strand reverse transcription was carried out with the ABI High Capacity RNA-to-cDNA Kit . Briefly, 1ug of total RNA and 10ul of 2X reverse transcription buffer and 1ul of 20X enzyme mix were incubated together in a total volume of 20ul at 37°C for 60 minutes followed by 95°C for 5 minutes and held at 4°C. The synthesized cDNA was stored at −20°C until further use.Comparative quantitative PCR was performed with the Mx3005p real-time PCR machine  using the QuantiTect SYBR Green PCR kit . Briefly; PCR was carried out in 20μl total volume containing a final concentration of 1x reaction buffer, 300nM forward and reverse primers  were purchased from Clontech laboratories, Inc.  and Ambion, Inc. .TMREDTaq RedyMix  and 10 pmol of each primer were used. Primers used for amplification were compiled from the literature or were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi)  Primers were designed to have an annealing temperature around 60°C and to encompass introns, thus allowing product discrimination in the case of genomic DNA amplification. Primer specificity was confirmed by aligning with the NCBI sequence database using BLAST http://blast.ncbi.nlm.nih.gov/Blast.cgi. The amplification protocol used was as follows: precycling hold at 95oC for three minutes followed by 35 cycles of denaturation at 95oC for 15 seconds, annealing for 30 seconds  and extension at 72oC for 30 seconds followed by a final extension step at 72oC for 7 minutes. PCR products were separated on 1.5% agarose gels stained with ethidium bromide.RNA samples  were reversed transcribed in a total volume of 20μl using Omniscript RT kit  according to the manufacturer 's protocol using oligo(dT)12-18 primer and RNaseOUT . The cDNA was diluted five times with nuclease free water, and 3 μl of diluted cDNA were used in 25μl PCR reactions. For amplification, JumpStartWith the quantitative PCR results in hand, we selected 46 of our specimens representing a range of RT-PCR protein values from low to high for IHC in order to assess for the presence of IGF2BP3 protein in the tumor specimens and, thus, confirm the RT-PCR expression level results. The sections were cut at 5 mm-thickness and stained with hematoxylin and eosin (H&E). The slides were then reviewed by two pathologists to confirm the presence of tumor in each section. H&E stained slides were screened for optimal tissue and noncancerous tissue adjacent to tumor tissue. Normal colon tissue sections were also assessed after H & E staining for the presence of normal colonic tissue.Immunohistochemistry was performed on fresh frozen slides  using standard IHC protocol. A fresh frozen sample of pancreatic adenocarcinoma was selected as the positive control. IHC staining was performed on 5 mm-thick sections of fresh frozen samples with purified rabbit anti human IGF2BP3 polyclonal antibody . The slides were incubated in H2O2 solution for 10 minutes at room temperature to block the endogenous peroxidase activity. Antigen retrieval was performed by steamer heating in 10mmol/L citrate buffer (pH 6.0). After epitope recovery, slides were incubated with primary antibody IGF2BP3 at 1:100 dilutions overnight at 4°C temperatures. Slides were washed and incubated with secondary anti-rabbit IgG  at 1:200 dilution and tertiary streptavidin-peroxidase conjugate . Slides were treated with chromogen “diaminobenzedine” for antigen detection. Counterstaining was performed with hematoxylin, dehydrated, cleared in xylene, and mounted on coated slides.Immunoreactivity was evaluated independently by two pathologists who assessed the immuno-staining. The percentage of neoplastic cells staining was determined (1 to 100%). IHC staining was considered “positive” when at least 10% or more of neoplastic cells stained positive on a scale of 1 to 3 . IHC staining of 1+ intensity in less than 10% of neoplastic cells was considered negative, and IHC staining of 2–3+ staining in less than10% of cells was considered indeterminate.The demographic and clinical data are expressed as the mean and standard deviation of continuous variables whereas frequencies and percentages were determined for categorical variables. The Fisher exact test (for expected frequency of less than 5) and the Chi-Square test and were utilized for comparison of categorical variables such as the pathologic analysis. The t-test was utilized for comparison of continuous variables. A p value of less than 0.05 was considered statistically significant.The present study is the first to utilize RT-PCR to quantitatively assess IGF2BP3 mRNA expression in a reasonable sized population of colorectal cancers. One earlier study did RT-PCR analyzed a subset of 8 patients out of 203 tumors and demonstrated greater mRNA expression in tumors compared to normal tissues . In the To further assess a prospective cancer testis protein or any other protein as a vaccine target it is useful to assess preoperative plasma or serum specimens for the presence of antibodies to that protein to evaluate the occurrence of spontaneous immune response . The finOf note, in the present study, a non-significant increase in the mean IGF2BP3 mRNA expression levels was noted in the Stage 3 patients when compared to the Stage 2 patients. The same is true regarding the presence of lymph node metastases; a non-significant increase in the mean expression level was noted in the node positive group. Importantly, prior investigators who utilized IHC methods on larger populations (n=165–671) of patients have noted that IGF2BP3 expression was associated with a worse prognosis  –17. The When a subset of tissue sections of the tumors in the present study were assessed via IHC, IGF2BP3 protein was noted in 50 percent of the cases. This result is in the same range as the RT-PCR mRNA result of 43% which is reassuring. Also, of note, the 43% RT-PCR result is closer to the IHC result than the 63% result obtained when the less stringent RT-PCR criteria is applied  which support the use of the stricter criteria. Also, our IHC results are in the same general range as the results of several investigators who utilized IHC methods to assess for IGF2BP3. Li, et al in an IHC study of 203 colon cancers reported that 65% of tumors assessed expressed IGF2BP3 whereas Yuan et al, also using IHC methods, found an expression rate of 56% in a population of 165 colorectal tumors . In contSome shortcomings of this current study include the fact that only stage 2 and 3 patients were considered. Further, as mentioned, no data is provided regarding serum titers of anti-IGF2BP3 antibodies for the study population. Also, this report supplies no oncologic outcome data . Thus, there is a need for larger RT-PCR studies that include patients with Stage 1 and 4 disease as well as antibody data and long term outcome data to shed further light on this topic.In conclusion, this study is the first to perform RT-PCR for IGF2BP3 on a sizable population of colorectal cancer patients. Forty three percent of the tumors examined were found to express IGF2BP3 mRNA when compared to RT-PCR results obtained with each patients normal colonic tissue. Half of the subset of tumors that were assessed via IHC were found to express the IGF2BP3 protein. No significant difference in mRNA expression levels were noted for the Stage 3 vs the Stage 2 patients or node positive vs node negative tumors. Tumor expression of IGF2BP3 may hold promise as a tumor marker and/or vaccine target; however, larger studies are needed as well as determination of auto-antibody titers to IGF2BP3.


Reprogramming of SSC from Tet1 and Tet2 double knockout mice however lacked demethylation of H19 ICR. These results suggest that imprinting erasure during reprogramming depends on the epigenetic landscape of the precursor cell and is mediated by TETs at the H19 locus.Selective methylation of CpG islands at imprinting control regions (ICR) determines the monoparental expression of a subset of genes. Currently, it is unclear whether artificial reprogramming induced by the expression of Yamanaka factors disrupts these marks and whether cell type of origin affects the dynamics of reprogramming. In this study, spermatogonial stem cells (SSC) that harbor paternalized imprinting marks, and fibroblasts were reprogrammed to iPSC (SSCiPSC and fiPSC). The SSCiPSC were able to form teratomas and generated chimeras with a higher skin chimerism than those derived from fiPSC. RNA-seq revealed extensive reprogramming at the transcriptional level with 8124 genes differentially expressed between SSC and SSCiPSC and only 490 between SSCiPSC and fiPSC. Likewise, reprogramming of SSC affected 26 of 41 imprinting gene clusters known in the mouse genome. A closer look at H19 ICR revealed complete erasure in SSCiPSC in contrast to fiPSC. Imprinting erasure in SSCiPSC was maintained even after  Being one of the few exceptions to Mendelian rules, genomic imprinting is an epigenetic mechanism that allows for a subset of genes to be expressed either from the paternally or from the maternally inherited allele2Cell reprogramming, either oocyte-mediatedIn this article we have developed a Systems Biology approach to study the mechanisms involved in the erasure of genomic imprinting. In particular, we show that the reprogramming of spermatogonial stem cells (SSC), a germ-line committed stem cell type that exhibit an androgenetic imprinting pattern in H19 and Igf2rGt(ROSA)26Sortm1(rtTA*M2)JaeCol1a1tm3Jae/J), which allows for a homogeneous and replicable expression of Oct4, Sox2, Klf4 and c-Myc (OSKM) in the presence of doxycyclinePou5f1, a key marker for stemness in SSCId4, another marker of SSC, was expressed at a higher level in Gfra1+ compared to Gfra1- cells, but the differences were not statistically significant (ANOVA p<0.05). A marker of differentiated spermatogonia cKitSSC and fetal fibroblast were obtained from a mouse model harboring Yamanaka factors under the control of a doxycycline inducible promoter . Skin chimerism was detected based on the agouti coat color from injected iPSC, over the white coat color of blastocyst donor CD1 strain. All cell lines were able to contribute to chimeras to a certain degree. In terms of skin chimerism, the 3 SSCiPSC lines outperformed fiPSC lines, resulting in a significantly higher number of pups with a high skin chimerism (>40%) than fiPSC based on Chi-square test .Ins2, Lhcgr, Lhx1, Nanos2, Nr5a1, Serpina 5 and Tcf21 (Tet1 and Tet2) and DNA methylation   . Gene On Dnmt3b) . KEGG pa Dnmt3b)  online.  Dnmt3b) . Out of in vivo differentiation, by analyzing the methylation status of H19 ICR in teratomas. In vivo differentiation of iPSC did not affect the methylation levels at H19 ICR, as teratomas produced either by intramuscular or subcutaneous injections revealed similar methylation patterns as the SSCiPSC line from which they were derived  and in 3 iPSC cell lines derived from each resultant cell type were analyzed . As expe10 derived .Tet1 and Tet2 in Gt(ROSA)26Sortm1(rtTA*M2)JaeCol1a1tm3Jae/J mice by CRISPR/Cas9 systemTet1+Tet2 double knockout (DKO) embryos die in utero likely due to epigenetic defectsTet1 and Tet2 (DKO) and another showing frame-disrupting mutations in Tet2 but an in-frame indel and a non-edited allele in Tet1 (Tet2 KO cells)  . The tese wt SSC A and 4B.in vitro from PGC) resulted in the erasure of the imprinting marks, whereas the fusion with ESC, which also reprogram the B cell genome, does not erase the imprinting marks25Pou5f1 before reprogramming in SSC compared to fibroblastsThe regulation of DNA demethylation at ICR is uncoupled from the global demethylation changes leading to pluripotent states in early embryo. Although imprinting is erased during PGC formation2Pou5f1 locus, and in both cases it seems that there is a conversion of 5 mC to 5 hmC. Tets are the only enzymes known to catalyze 5 mC hydroxylation and both Tet1 and Tet2 are present in EGC and ESC, the two cell types differing in their ability to abolish imprinting marks following cell-fusion based reprogrammingin vitro derivation of pluripotent cells from unipotent GSC  report different patterns of ICR methylation depending on the cell type of origin. Pluripotent stem cells derived from GSC of newborn testis (mGS) lose the imprinting of two paternally methylated ICR H19, Meg3 IG and keep the maternally methylated Rasgrf1, Igf2r and Peg10The notion of a cumulative effect of demethylating agents acting on imprinting erasure rather than a specific mechanism for demethylation at ICR agrees with previous findings in different Systems Biology Approaches. The demethylation of ICR is a late event in cell fusion-based mechanism occurring much more slowly than the demethylation occurring at the in vivo differentiation into teratomas. Genomic imprinting is an epigenetic feature generally overlooked in iPSC and while the imprinting erasure did not impair the differentiation abilities of the SSCiPSC, the functionality of these differentiated cells could be affected. As imprinting has been shown to be affected in other types of iPSC3031A particularly relevant observation is that imprinting erasure was not reversed following Tet2Tet enzymesTet1 and Tet2 were dispensable for SSC reprogramming. A possible explanation for the differences between MEF and SSC reprogramming could be that SSC do not require a mesenchymal-to-epithelial transition, the critical step blocked by Tet deficiencyTet1 and Tet2 are the only Tet members expressed in PGCs or ESCTet1 and Tet2 coincides in time with the conversion of 5 mC to 5 hmC at ICR in PGCs, so they have been suggested to play a role in imprinting erasureTet1 paternal or maternal KO  E9.5–10.5 embryos were observed to dysregulate several imprinted genes by hypermethylation of paternal or maternal ICR, respectivelyTet1 and Tet2 DKO embryos are deprived from 5 hmC. These pups were fertile and some of its progeny shows altered imprinting in H19, Mest, Peg3 and Igf2rTet3, not expressed in PGC but expressed later during gametogenesis and spermatogenesis26Tet1 and Tet2 DKO SSC does not lose their imprinting marks following reprogramming to SSCiPSC, suggesting an essential role of these enzymes in the process. Our findings are in agreement with the critical role for Tet enzymes in the erasure of imprinting mediated by cell fusion between B cells and EGC, where Tet1 depleted EGC were unable to accumulate 5hmC and erase the imprinting marks on the B cell genome following fusionTet enzymes in imprinting erasure in our model, we cannot rule out that passive DNA demethylation may have also played a role.As plausible drivers for imprinting erasure, Tets  are the only known enzymes that convert 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC), an intermediate in the 5 mC demethylation process that undergo passive demethylation as cells divideCollectively, these results highlight that imprinting erasure following Yamanaka factors based reprogramming depends on the epigenetic status of the original cell, with iPSC obtained from highly differentiated cells (fiPSC) being more able to retain the imprinting mark than SSCiPSC. This observation proposes that imprinting marks are specially protected from demethylation and that reprogramming at imprinted loci is a late step in genome reprogramming driven by the cumulative action of demethylating agents, with Tet enzymes playing an essential role. In this regard, Tet mediated imprinting erasure does not seem to require a specific demethylation mechanism, in agreement with the lack of consensus target sequences at ICRsGt(ROSA)26Sortm1(rtTA*M2)JaeCol1a1tm3Jae/J14. Germinal cells were enriched from PND6 testis by differential plating. Briefly, testicular stroma was minced and digested with 1 mg/ml type IA collagenase (Sigma) for 10 min at 37 °C, followed by digestion with 0.05% trypsin-EDTA (Gibco) for 5 min at 37 °C. Cell suspension was purified through a 70 μm filter and plated onto a gelatin coated dish for 2 hours, allowing testicular somatic cells to attach to the plate. Germinal floating cells were subjected to MACS (Miltenyi Biotech) for Gfra1, a SSC membrane markerAll experiments involving vertebrate animals were performed in accordance with the approved guidelines of Beltsville Area Animal Care and Use Committee (BAACUC). All experimental protocols involving vertebrate animals were approved by the Institutional BAACUC committee. Fetal fibroblast and SSC were obtained from the mouse model SSC and fetal fibroblasts were cultured following standard protocols in the presence of 2 μg/ml Doxycycline hyclate (Sigma). For the first passage, individual colonies were picked up and individually trypsinized to obtain clonal lines. Doxycycline concentration was kept at 2 μg/ml for the first 5 passages and then was gradually withdrawn . After passage 8, cells were incubated without doxycycline.3 biopsy of the teratomas was collected to determine DNA methylation. As this procedure does not exclude the possibility of host cell contamination, the percentage of contamination was estimated by a PCR with three primers JaeCol1a1Gt(ROSA)tm3Jae/J iPSC derivatives. Comparison of band intensity between the PCR products amplified from teratomas or from standard mixes of known amounts of wt and transgenic DNA estimated an ~5% host cell contamination in the samples used for DNA methylation  and injected subcutaneously. The same number of cells was injected intramuscularly diluted in culture media. One month after injection, teratoma growth was obvious and the mice were sacrificed and the teratomas collected and fixed in 4% paraformaldehyde. Fixed tissues were dehydrated through ethanol washes, paraffin embedded, sectioned and stained in hematoxylin/eosin (American Histolabs). An inner ~1 mmhylation . This cohylation .SSCiPSC and fiPSC were individualized and injected into CD1 blastocysts (10 iPSC/embryo). The injected blastocysts were transferred by uterotubal embryo transfer to pseudopregnant recipients. Three different lines from each cell type were tested, performing 3 to 5 embryo transfers and obtaining 15 to 30 weaned pups per cell line analyzed . Skin chhttp://david.abcc.ncifcrf.gov/), and differentially expressed genes with p-value ≤ 5*10−5 and fold change ≥ 1.5 were categorized with respect to Molecular Function, Biological Process and Cellular Component. The annotated genes were also mapped into relevant functional groups in a pathway analysis according to the Kyoto Encyclopedia of Genes and Genomes (KEGG).In order to minimize the contamination with the feeder cells, iPSC at passages 13-14 were purified by MACS for SSEA1 following a protocol similar to the one described for SSC enrichment for Gfra1, but using anti-SSEA-1 (CD15) microbeads (Miltenyi Biotech). The purified cells were used for gene expression and DNA methylation analysis. qPCR was performed as previously describedH19-Igf2200 ng of DNA from fibroblasts, FiPS, SSC, SSCiPS and teratomas were treated with bisulfite to convert all unmethylated cytosine to uracil using EZ DNA Methylation-Direct Kit (Zymo Research) according to the manufacturer instructions. A 422 bp fragment of the 2 kb ICR of in vitro transcription  from the plasmid pMJ920 (Addgene 42234) linearized with BstBI and treated with Antarctic phosphatase (NEB). Single guide RNAs (sgRNA) for Tet1 and Tet2, previously described inin vitro transcription  from g-Blocks containing T7 promoter. Both the Cas9 mRNA and the sgRNAs were purified using MEGAclear kit (Life Technologies) and eluted in TE buffer. Cas9 mRNA (100 ng/μl) and sgRNA (20 ng/μl) were injected into two cell embryos obtained from Gt(ROSA)26Sortm1(rtTA*M2)JaeCol1a1tm3Jae/J females. Injected embryos were allowed to develop to the blastocyst stage and transferred to CD1 foster mothers by utero-tubal embryo transfer. Genotyping was performed on DNA extracted from a tail biopsy by using specific primers spanning the target sequence .


We studied the rapid changes in electrical properties of lumbar motoneurons between postnatal days 3 and 9 just before mice weight-bear and walk. The input conductance and rheobase significantly increased up to P8. A negative correlation exists between the input resistance (Rin) and rheobase. Both parameters are significantly correlated with the total dendritic surface area of motoneurons, the largest motoneurons having the lowest Rin and the highest rheobase. We classified the motoneurons into three groups according to their discharge firing patterns during current pulse injection . The delayed onset firing type has the highest rheobase and the fastest action potential (AP) whereas the transient firing group has the lowest rheobase and the less mature AP. We found 32 and 10% of motoneurons with a transient firing at P3–P5 and P8, respectively. About 20% of motoneurons with delayed onset firing were detected at P8. At P9, all motoneurons exhibit a sustained firing. We defined five groups of motoneurons according to their discharge firing patterns in response to ascending and descending current ramps. In addition to the four classical types, we defined a fifth type called transient for the quasi-absence of discharge during the descending phase of the ramp. This transient type represents about 40% between P3–P5 and tends to disappear with age. Types 1 and 2 (linear and clockwise hysteresis) are the most preponderant at P6–P7. Types 3 and 4 (prolonged sustained and counter clockwise hysteresis) emerge at P8–P9. The emergence of types 3 and 4 probably depends on the maturation of L type calcium channels in the dendrites of motoneurons. No correlation was found between groups defined by step or triangular ramp of currents with the exception of transient firing patterns. Our data support the idea that a switch in the electrical properties of lumbar motoneurons might exist in the second postnatal week of life in mice. Electrical properties of developing spinal motoneurons have been studied in several species and at different embryonic and postnatal stages  following current pulse stimulation have been well documented in rat spinal motoneurons  of motoneurons has been found in the neonate rat  to 9 (P9), P0 being the first postnatal day. All surgical and experimental procedures are conformed to the European Communities council directive (86/609/EEC) and approved by our ethics committee . Most of the experimental procedures were described previously  artificial cerebrospinal fluid (ACSF). Then, a laminectomy was performed and the spinal cord and brainstem were removed, taking care to preserve sufficient length of L5 ventral root, placed in a recording chamber and superfused with ACSF containing (in mM): NaCl, 130; KCl, 4; MgClTM three-dimensional hydraulic microdrive. Motoneurons were impaled at a depth of 150–450 μm from the spinal cord surface corresponding to the fifth lumbar segment. Motoneurons were identified by their antidromic action potential (anti AP) evoked following electrical stimulation of the ventral root L5. Intracellular recordings were made either in bridge mode with an output bandwidth of 3.0 kHz or in Discontinuous Current Clamp (DCC) mode, using an Axoclamp 2B amplifier (Axon instruments). Electrode resistance and capacitance were compensated before intracellular recordings. Signals were digitized at 10 kHz by an A/D converter (Digidata 1322 Axon Instruments) and saved on a computer using Clampex 9.2 (Axon instruments).To allow for microelectrode penetration in the spinal cord, the pia was carefully removed medially to L5 ventral root entry, using very fine forceps under binocular control. Fine tip micropipettes for intracellular recordings were made from 1.2 mm filamented glass tubes (Clark Instruments) using a pipette puller . Electrodes were filled with 2 M potassium acetate, and their resistances ranged between 60 and 110 MΩ. The microelectrode was positioned to penetrate the L5 spinal segment with an angle of 30–45° above the horizontal and advanced in the tissue using a NarishigeRin was measured by computing the voltage deflections derived from series of hyperpolarizing and depolarizing constant current pulses  injected into the motoneurons. Measurements were made from the averaged voltage over 50 ms taken from the steady state membrane potential at the end of the pulses. Retained values were the averages of three sets of measurements.Spike potentials were analyzed using the Event Detection/Threshold Search module of Clampfit 9.2. Firing behavior was studied as described previously  if p < 0.01 or (***) if p < 0.005. Results were expressed as means ± SEM or medians with interquartile range when indicated. Graphical representations were obtained using Origin 7.5 (Origin Lab Corporation), Graph pad Prism 6.0 and Corel Draw 12 .Statistical significance was assessed with the non-parametric Permutation with General Score Exact Test for independent or paired samples or the Fisher exact test (StatXact7. Cytel software). The correlation between two sets of data was evaluated by using Pearson’s correlation test or Spearman’s correlation test (Graphpad Prism 6 or StatXact 7). Two groups of data were considered statistically different (*) if All the procedures for morphological studies were described previously .The labeled motoneurons were reconstructed from serial sections (75 μm thick) on Nikon microscope equipped with a computer interfaced motorized stage and TM. Our own database of intracellularly recorded and stained motoneurons with Neurobiotin comprises more than 50 postnatal mouse lumbar motoneurons at different ages between P3 and P10. Among them, 32 motoneurons were fully reconstructed in 3D with NeurolucidaTM. In the present work we used 14 motoneurons at both ages .A single motoneuron was described by up to 18.000 data points, which were stored in a database together with fiducial marks  in ASCII format files. Reconstructed cells were visualized and three-dimensionally analyzed using NeurolucidaThe data base for the electrophysiological study comprises 103 motoneurons from mice aged between P3 and P9. Only neurons displaying a stable membrane potential more negative than −50 mV with overshooting APs during the whole test procedure were kept for analysis. All the motoneurons were identified by recording either the anti AP evoked by the ventral root stimulation Figure .2, n = 3; P8–9: 3212 ± 314 μm2, n = 9; p = 0.04) as well as the mean diameter of primary dendrites . The total dendritic length increases from P3 to P9 by 22%  and the total dendritic surface area by 20%  and P8–P9 (n = 34). The anti AP has comparable amplitude in the two populations  as well as the resting membrane potential  due to ongoing motor axon myelination during this postnatal period.In this series of experiments, a number of parameters of electrical properties was analyzed in two populations of lumbar motoneurons of different ages  compared to the mean Rin in the P3–P5 population . However, the Rin stabilizes between P8 and P9  to 1.76 ± 0.2 nA (n= 31) between P3 and P8 (Table p = 0.009). The mean rheobase is significantly higher at P6/P7  compared to that in P3–P5. Indeed, a significant and negative correlation exists between the rheobase and the Rin  but no significant correlation was found between the gain and the Rin . The mean rheobase was significantly lower at P9  compared to that at P8 . Thus the progression of the rheobase stopped between P8 and P9 in lumbar motoneurons.As expected, the mean Rin was lower in the oldest motoneurons  and between the rheobase and the total dendritic surface area  confirming that the largest motoneurons in the lumbar cord have the lowest Rin and the highest rheobase also during postnatal development. However, we noticed that some motoneurons with similar total dendritic surface area  may have different Rin ranging from 10 to almost 30 MΩ .In twelve motoneurons intracellularly stained and fully reconstructed, significant correlations were found between the Rin and the total dendritic surface area  together with the maximum depolarizing speed which was accelerated from 123 ± 8.69 mV.ms−1 to 149 ± 7.38 mV.ms−1 with age (p < 0.01). Thus younger motoneurons have spikes with lower amplitude and slower time course. No changes with age were seen in AHP amplitude and total AHP duration between P3 and P9. However, AHP half-duration was shorter and half decay time faster in the P8–P9 group (Table As summarized on Table n = 12). The distribution of the different firing patterns is illustrated in pie charts in Figure During the development of spinal motoneurons, different patterns of discharge have been previously described , spike half width , max rise slope  and time to peak  of the AP. The maximum decay slope also shows statistically significant difference between the three groups (not illustrated). All spike parameters  indicate that the delayed firing type do have the fastest AP. The AHP parameters did not show significant differences although there is a tendency in the AHP amplitude to be larger in the delayed group .We then analysed the electrical properties of the subgroups. We focused on the three patterns of discharge firing in motoneurons from P6–P9 animals. We analyzed 14 passive and active electrical properties Figure  suggesti Figures . The gaiFiring pattern was further characterized using increasing and decreasing (triangular) slow current ramps as described previously  whereas some motoneurons exhibit a delayed onset firing pattern up to the second postnatal week. A majority of motoneurons have a sustained firing at all ages between P3 and P9. The sustained firing is present in all motoneurons at P9. The results also show that a counter clockwise hysteresis and/or a prolonged sustained firing in response to current ramp (types 3 and 4) emerge at P8–P9 corresponding to the maturation of L type calcium channels in dendrites of mouse motoneurons. Dendrites of postnatal motoneurons mainly elongated during this short period of time. Although most morphological parameters were not significantly different between P3 and P9 . This proportion is rather low compared to that (65%) observed in lumbar motoneurons recorded in slice . They represent up to 50% of motoneurons at P8–P9 but only 10% at P3–P5 Figure .The non-linear behavior may originate from sodium and/or calcium persistent inward currents (Schwindt and Crill, G85R transgenic mouse model of ALS at 3–4 months of age. Furthermore they found that this population of motoneurons that disappears in SOD1 adult mice was greatly hyperpolarized, which would favour hypoexcitability. We previously showed that lumbar motoneurons from SOD1G85R mice were hypoexcitable very early during the postnatal period having a higher rheobase and lower gain (Bories et al., G93A mice in which an accelerated maturation of lumbar motoneurons might lead to a different time course in the ALS pathology (Quinlan et al., G85R or SOD1G93A mice are needed to elucidate this question.Our results show that the rheobase, input conductance and gain of motoneurons are the highest in the delayed firing group. These results are in agreement with those suggesting that motoneurons with delayed onset firing pattern correspond to the fast motoneurons (Leroy et al., We found rapid changes in the progression of electrical properties of mouse lumbar motoneurons between P3 and P9 whereas the morphology of dendritic arborization evolves slowly. A change of rheobase and Rin progressions occurs, with the disappearance of transient and delayed onset firing types following direct current pulse stimulation, and the emergence of types 3 and 4 discharge patterns following direct ramp current stimulation. We conclude that a switch might exist in the electrical properties of mouse lumbar motoneurons around P8–P9 during the maturation of motor behaviors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HTLV-1 Tax protein is usually undetectable in freshly isolated peripheral blood mononuclear cells (PBMCs). After in vitro culture, the provirus is reactivated and Tax protein is detectable only in a proportion of infected CD4+ T cells: the percentage of Tax+ cells is always lower than the proviral load (PVL). To identify and further analyse the latently infected cells we have measured the expression of tumour suppressor of lung cancer 1 (TSLC1). TSLC1 is a member of the immunoglobulin super family that is expressed on all cells with the exception of PBMCs. It mediates cell-to-cell adhesion by either homophilic or heterophilic interactions with other members of the immunoglobulin family and also signals to the actin cytoskeleton. TSLC1 has been shown to be expressed on primary adult T cell leukaemia (ATL) cells and ATL cell lines. We assayed the expression of TSLC1 in the PBMCs of 13 asymptomatic carriers (AC) and 13 HAM/TSP patients by flow cytometry. We found that the percentage of TSLC1+ CD4+ T cells was positively correlated with the PVL (P < 0.0001). To test whether TSLC1+ cells themselves were infected, we flow sorted TSLC1+ CD4+ T cells of 3 ACs and 3 HAM/TSP patients. A median of 95% of TSLC1+ CD4+ T cells carried the provirus. Whilst only 20% of infected CD4+ T cells expressed Tax, a further 47% were identified on the basis of TSLC1 expression. As the host cytotoxic T cell (CTL) response is an important protective factor, we are now testing the hypothesis that the presence of TSLC1 on the surface of infected cells affects its recognition and subsequent killing by CTLs.


The values of C-reactive protein (CRP) and procalcitonin (PCT) were investigated to determine their effects on postoperative complications in patients with or without systemic inflammatory response syndrome (SIRS).3 and <4.00/mm3. The ability of PCT to predict sepsis and other postoperative complications were determined by performing receiver operative characteristic curve analysis.In 183 patients, in a prospective observational study, serum CRP and PCT values were collected every day starting on postoperative day 1 through day 5. The definition of SIRS includes two or more of the following: temperature >38 or <36°C; heart rate >90 beats/minute; respiratory rate >20/minute; arterial carbon dioxide pressure <32 mmHg; white blood cell count >12,000/mmn = 83) and patients without SIRS . A PCT threshold value of 2.79 ng/ml on postoperative day 1 was able to discriminate postoperative complications in patients with or without SIRS with a sensitivity of 82.5% and a specificity of 70% .All patients were divided post hoc into patients with SIRS  Serum PCT values increased significantly after cardiopulmonary bypass in the SIRS group in comparison with patients without SIRS on postoperative day 1 and remain elevated until postoperative day 5. (2) Serum CRP values also follow a similar pattern, buta CRP threshold value was not obtained to differentiate between postoperative complications in patients with or without SIRS. A PCT threshold value of 2.79 ng/ml on postoperative day 1 is a valuable marker to discriminate between patients with or without SIRS.


The protein kinases ERK1 and ERK2 are the effector components of the prototypical ERK1/2 mitogen-activated protein (MAP) kinase pathway. This signaling pathway regulates cell proliferation, differentiation and survival, and is essential for embryonic development and cellular homeostasis. ERK1 and ERK2 homologs share similar biochemical properties but whether they exert specific physiological functions or act redundantly has been a matter of controversy. However, recent studies now provide compelling evidence in support of functionally redundant roles of ERK1 and ERK2 in embryonic development and physiology. In this review, we present a critical assessment of the evidence for the functional specificity or redundancy of MAP kinase isoforms. We focus on the ERK1/ERK2 pathway but also discuss the case of JNK and p38 isoforms. Phylogenetic analysis of the evolutionary history of MAP kinase genes suggests that vertebrate MAP kinases originated from 3 precursors and have expanded through gene duplication during early vertebrate evolution  kinase pathways are evolutionarily conserved signaling modules that play a key role in transducing extracellular signals into intracellular responses Meloche, . These sErk1 or Erk2 mRNA, suggesting that a single ERK isoform mediates cellular responses in these areas  technology has allowed analysis of the phenotypical consequences of the specific depletion of ERK1 or ERK2 in animals and cells. Erk1−∕− mice develop normally, are viable and fertile, and display no observable phenotype  6.5  also revealed a tight relationship between the extent of development of embryos with different combinations of Erk1 and Erk2 alleles and total ERK1/2 activity in embryonic tissues. As a second approach, we asked whether ERK1 can substitute for ERK2 in mouse embryonic development. We found that ubiquitous expression of an Erk1 transgene fully rescues the placental and embryonic defects observed in ERK2-deficient embryos. ERK1-only mice grow normally, are fertile and do not display any overt phenotype. Expression of transgenic ERK1 also rescued the proliferation defect of ERK2-deficient MEFs and restored normal phosphorylation of a panel of ERK1/2 substrates. Our study provides compelling and definitive evidence for a functionally redundant role of ERK1 and ERK2 kinases during development (Fremin et al., Mek2 at the Mek1 locus rescued the placental phenotype of MEK1-deficient mice (Aoidi et al., We have used complementary genetic approaches to rigorously address the question of ERK1 and ERK2 specificity or redundancy in embryonic development (Fremin et al., Erk1 and Erk2 null mice are attributable to differences in expression levels, with ERK2 being the predominant isoform. The higher expression level of Erk2 in most mammalian tissues can be related to a stronger promoter, although further regulation by post-transcriptional mechanisms cannot be ruled out (Busca et al., Differences in the phenotypes of The above findings underscore the concept that a threshold of global ERK1/2 activity determines developmental progression and phenotypic outcome (Figure Erk1 gene has been lost in all bird lineages and some amphibians, whereas squamates only express ERK1 isoform, despite the presence of both Erk1 and Erk2 genes. The finding that tetrapods can live by expressing only ERK1 or ERK2 provides further demonstration of the functional redundancy of ERK isoforms in animal physiology.The group of Philippe Lenormand recently reported the most detailed analysis of the expression and evolution of ERK1 and ERK2 protein sequences in vertebrates (Busca et al., Jnk1 or Jnk2 gene exhibit distinct phenotypes, suggesting that individual JNK isoforms may serve different signaling functions (Davis, Jnk1−∕− and Jnk2−∕− MEFs proliferate at different rates, a phenotype that has been related to the expression levels of cJun (Tournier et al., The JNK pathway provides another example where different groups have reported contradictory conclusions about the specificity or redundancy of closely related MAP kinase isoforms. JNK1 and JNK2 are ubiquitously expressed in the mouse although their expression levels vary across tissue types. Mice deficient in either s Davis, . In addiJnk2M108G allele was integrated at the endogenous Jnk2 locus by homologous recombination to generate mice expressing analog-sensitive JNK2 kinase. Analysis of MEFs bearing different combination of Jnk1 and Jnk2 alleles revealed that pharmacological inhibition of JNK2 in JNK1 proficient cells caused no change in cJun expression or cell proliferation, contrary to the results obtained in Jnk2−∕− cells. However, both genetic ablation and pharmacological inhibition of JNK2 in Jnk1−∕− cells reduced cJun levels and inhibited cell proliferation. These results demonstrated that JNK1 and JNK2 act redundantly to increase cJun expression and promote cell proliferation. The most likely explanation for the misleading phenotype of Jnk2−∕− cells is that loss of JNK2 leads to increased JNK1 function by a compensatory adaptation mechanism. This adaptation is not observed upon acute inhibition of the kinase and/or in conditions where protein expression is maintained. This study also highlighted the importance of using multiple experimental approaches to interpret the phenotypes of mouse mutants, as discussed above for ERK1 and ERK2.The group of Roger Davis has revisited the proposed negative regulatory role of JNK2 on cJun expression and cell proliferation using a chemical genetic approach (Jaeschke et al., p38α and p38β mouse mutants exhibit distinct phenotypes. Specifically, loss of p38α is embryonic lethal owing to defects in placenta morphogenesis (Adams et al., The p38 MAP kinase pathway regulates numerous cellular processes including adaptation to environmental stress, innate immunity; cell cycle progression and cellular differentiation (Cuenda and Rousseau, p38α and p38β deficient embryos but were absent in single gene knockouts, indicating that that the two isoforms can compensate for each other with respect to these defects (del Barco Barrantes et al., p38β knock-in allele in the p38 knockout background developed to E18.5 and showed rescue of spina bifida and exencephaly defects, but not heart defects. Upon increased dosage from the two additional endogenous p38β alleles, the heart anomalies were rescued and, more importantly, some of the animals survived to adulthood, thereby overcoming the lung defects observed in p38α-deficient animals (del Barco Barrantes et al., The group of Angel Nebreda used a combination of genetic approaches to address the question of the specificity and redundancy of p38α and p38β isoforms (del Barco Barrantes et al., p38α and p38β genes are deficient in sex determination due to reduced expression of the testis-determining gene Sry (Warr et al., Further evidence that p38α and p38β act redundantly comes from work demonstrating that embryos lacking both p38γ and p38δ genes has unveiled key roles of p38γ and p38δ isoforms in tissue regeneration, innate immune responses, inflammation, and tumorigenesis (Escós et al., The two other members of the p38 MAP kinase subfamily also exhibit overlapping functions. p38γ and p38δ share 70% amino acid identity (Cuenda and Rousseau, We have used a combination of genetic approaches together with quantitative analysis of embryonic phenotypes and ERK1/ERK2 activity to demonstrate that ERK1 and ERK2 isoforms are functionally redundant in mouse development and physiology. This conclusion is consistent with the discovery of animal species that express only ERK1 or ERK2 and with studies showing that the two isoforms share similar biochemical properties and substrate specificity. Similarly, by combining multiple experimental approaches, other studies have revealed that JNK and p38 MAP kinase isoforms exert functionally redundant roles. These findings clearly illustrate the importance of using multiple genetic, pharmacological and phylogenetic analyses to define the physiological functions of related signaling proteins.V600E metastatic melanoma and other inhibitors of MAP kinase pathways are undergoing clinical evaluation. It is therefore crucial to determine whether the direct or downstream targets of these inhibitors have specific or redundant functions.The question of MAP kinases redundancy has far-reaching implications. Dysregulation of the ERK1/2, JNK1/2/3, and p38α/β/γ/δ pathways has been causally linked to human congenital syndromes and to a variety of diseases including cancer, arthritis, fibrosis, cardiomyopathies, and neurodegenerative diseases. Small molecule inhibitors of the ERK1/2 pathway have been approved for the treatment of BRAFAll authors listed, have made substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.