ALDH2 genotype on the exposure to locally formed acetaldehyde via the saliva without ethanol ingestion.Acetaldehyde associated with alcoholic beverages was recently classified as carcinogenic (Group 1) to humans based on uniform epidemiological and biochemical evidence. ALDH2  deficient alcohol consumers are exposed to high concentrations of salivary acetaldehyde and have an increased risk of upper digestive tract cancer. However, this interaction is not seen among ALDH2 deficient non-drinkers or rare drinkers, regardless of their smoking status or consumption of edibles containing ethanol or acetaldehyde. Therefore, the aim of this study was to examine the effect of the ALDH2 genotypes of 17 subjects were determined by PCR-RFLP. The subjects rinsed out their mouths with 5 ml of 40 vol% alcohol for 5 seconds. Salivary ethanol and acetaldehyde levels were measured by gas chromatography.The ALDH2 genotypes.Acetaldehyde reached mutagenic levels rapidly and the exposure continued for up to 20 minutes. The mean salivary acetaldehyde concentrations did not differ between For ALDH2 deficient subjects, an elevated exposure to endogenously formed acetaldehyde requires the presence of ethanol in the systemic circulation.Our findings provide a logical explanation for how there is an increased incidence of upper digestive tract cancers among ALDH2 deficient alcohol drinkers, but not among those ALDH2 deficient subjects who are locally exposed to acetaldehyde without bloodborne ethanol being delivered to the saliva. Thus, ALDH2 deficient alcohol drinkers provide a human model for increased local exposure to acetaldehyde derived from the salivary glands. ALDH2) gene is 10-fold that for alcoholics without the mutation [Cancers of the upper digestive tract are often found at an advanced stage, remain difficult to treat and have a high mortality rate. The risk for upper digestive tract cancer for alcoholics who have an impaired ability to eliminate acetaldehyde due to a single point mutation in the mitochondrial aldehyde dehydrogenase 2  of the ALDH2 gene, which contains a single nucleotide polymorphism (SNP) and results in the synthesis of an inactive ALDH2 enzyme [ALDH2*2 genotype, alcohol consumption and upper digestive tract cancer risk found in epidemiological data and the elevated local acetaldehyde exposure of ALDH2*2 carriers who consume alcohol provide convincing evidence for the specific carcinogenic potential of acetaldehyde in the pathogenesis of upper digestive tract cancers. Based on these findings, the International Agency for Research on Cancer (IARC) recently classified acetaldehyde associated with alcoholic beverages i.e. present in alcoholic beverages and/or formed endogenously from ethanol to be a Group 1 carcinogen in humans [ALDH2 is a low 2 enzyme . In addi2 enzyme -14. Unifn humans .In addition to alcohol consumption, smoking is also a generally accepted major etiological factor for upper digestive tract cancers . The mulALDH2 genotype significantly potentiates the risk of upper digestive tract cancer, there seems to be no apparent increased ESCC risk with ALDH2 deficient non-drinkers [ALDH2 genotype and raises the question about whether the ALDH2 genotype only has an effect when systemic ethanol is available.Unlike the case for alcohol ingestion, in which the drinkers ,19. Nevedrinkers . This suALDH2 genotype on the exposure of the upper digestive tract to salivary acetaldehyde when ethanol is only rinsed in the mouth, but not ingested.Our present study follows up on our earlier paper in which 7 ALDH2 deficient individuals and 13 individuals with the functional ALDH2 enzyme ingested 0.5 g/kg of ethanol and their salivary acetaldehyde levels were followed thereafter at 20 minute intervals for a period of 240 minutes . At eachALDH2*2 genotype [20 healthy Eastern Asian volunteers  were recruited into the study. The mean age of the participants was 25.7 years range: 21-38 years) and the mean body mass index (BMI) 20.9 (range: 18.0-27.8). All the participants were of Chinese origin and moderate alcohol drinkers i.e. they consumed <20 drinks/week (men) or <14 drinks/week (women). Half of the volunteers reported having a history of alcohol-related flushing symptoms, which are known to correlate significantly with the -38 yearsThe study was approved by the co-ordinating Ethics Committee, Hospital District of Helsinki and Uusimaa (Finland). Signed informed consent to participate in the study was obtained from each study participant.The participants were instructed to abstain from drinking alcohol for 24 hours before the study visit day and to fast for two hours before giving samples. Smoking was prohibited on the day of the study. Salivary samples which were used for genotyping were collected prior to the ethanol rinsing experiment. For the rinsing procedure the volunteers gave a baseline salivary sample and then rinsed their mouths with 5 ml of 40 vol% alcohol for 5 seconds, after which the oral contents were discharged and salivary samples were collected at 30 s, 2 min, 5 min, 10 min, 15 min and 20 min after discharging. The participants also answered a questionnaire regarding their oral health, alcohol use, smoking, diet and medication.ALDH2 gene (rs671). The PCR protocol included one cycle of 95°C for 5 min, 40 cycles of 98°C for 10 s, 60°C for 30 s, and 74°C for 45 s and a final cycle of 74°C for 2 min. A 430-bp DNA fragment that contained the polymorphic site of ALDH2 was amplified by PCR using the forward primer 5′-TCAAATTACAGGGTCAACTGCT-3′ and the reverse primer 5′-GGCTGGGTCTTTACCCTCTC-3′ (Sigma-Aldrich). The PCR reaction required 7.5 µl of distilled water, 12.5 µl of 2X Xtreme Buffer, 2.5 µl of 2 mM deoxyribonucleotide triphosphates, 10 pmol each for the two ALDH2 primers, and 0.5 U of KOD Xtreme DNA polymerase  in a total volume of 25 µl. PCR products were digested using AcuI according to the manufacturer’s instructions (New England Biolabs Inc.). The 430 bp ALDH2*1 fragment was cut into two fragments of 296 and 134 bp. The ALDH2*2 allele (2*/2*) was not cut. Fragments were analyzed by using gel electrophoresis on a 2% agarose gel. Samples of five randomly selected participants were analyzed twice to assess the reliability of the genotyping protocol.The genotyping protocol used was modified from Hayashida et al. . Whole s50 µl of 6 M perchloric acid was added to 450 µl of saliva to stop organic reactions, after which the samples were immediately sealed in 20 ml vials and stored at -20°C until analysis. Dual or triple parallel samples were collected at each time point whenever possible to confirm analytical reliability. Acetaldehyde and ethanol concentrations were measured by headspace gas chromatography as previously described . 100 µM Target sample size (6) was calculated for an effect size of 1.5 SD with an alpha level of 0.05 and a power of 0.80 using data from our previous study with ALDH2 deficient subjects . InteracALDH2*2 genotype, four (67%) were smokers. Of the participants without the ALDH2*2 genotype, four (36%) were smokers. The genotyping of one smoker was unsuccessful. No chronic illnesses were reported. Aside from one participant using oral medication for birth control, no regular medications were reported. Antibiotics had not been used for at least 30 days. Two participants (10%) reported following a non-lactose diet. One participant (5%) followed a vegetarian diet. Only one participant (5%) reported consuming products that contain lactic acid bacteria.All of the participants reported brushing their teeth at least twice per day, three participants (15%) also used mouthwashes. Nine participants (45%) were smokers and all reported consuming <20 alcohol drinks/week (men) or <14 alcohol drinks/week (women). Of participants with the ALDH2*1/*1 genotype, five the ALDH2*1/*2 genotype and one participant the ALDH2*2/*2 genotype  and those with the deficient ALDH2*2 allele  found that there were no statistically significant differences in the mean in vivo acetaldehyde levels of the saliva samples for any of the time points . Also, analysis of the area under the curve didn’t show a statistically significant difference between the genotype groups . There were no statistically significant differences in levels of salivary acetaldehyde between male and female subjects or between ALDH2 genotype variants. Also, the interaction between smoking and salivary acetaldehyde concentration was not statistically significant .After the 5 s of oral exposure to 40 vol% alcohol, salivary acetaldehyde concentrations rose quickly and peaked at 2 min , Table 1e points . The intALDH2*1 allele  and those with the deficient ALDH2*2 allele  found that there were no statistically significant differences in the mean in vivo ethanol levels of the saliva samples for any of the time points . There were no statistically significant differences in levels of salivary ethanol between male and female subjects or between ALDH2 genotype variants. Also, the interaction between smoking and salivary ethanol concentration was not statistically significant .Measured ethanol levels peaked at 30 s and thereafter declined rapidly . Detectae points . The intWe have earlier demonstrated that after an oral ingestion of a moderate dose (0.5 g/kg) of ethanol, salivary acetaldehyde levels measured at 20 minute intervals are 2-3 times higher among ALDH2 deficient individuals (n = 7) than in those with functional ALDH2 enzyme n = 13) . In that . In thaOur present results show for the first time that after a brief oral exposure to non-ingested ethanol, the concentration of salivary acetaldehyde of ALDH2 deficient subjects is not significantly higher than that of subjects with normal ALDH2 activity. It should be noted, however, that at the 2 min time point, means of salivary acetaldehyde between the groups showed a near-significant difference (p=0.06) which may become significant should the number of study subjects be considerably higher. At high concentrations of salivary ethanol (mean ranging from 248 to 899 mM) that are seen at the 2 and 5 min timepoints, enzymatic activity of the small salivary glands located in the oral mucosa may also contribute to our findings. At the 10 and 15 min time points salivary ethanol concentrations had decreased to 5-30 mM, a level that is comparable to those found after oral ingestion of alcohol .Km ADH enzymes in the gingiva and the lingual mucosa [No measurable levels of acetaldehyde or ethanol were found from the baseline salivary samples taken before subjects rinsed their mouths with 40 vol% alcohol. This is in accordance with earlier findings showing that without the presence of ethanol or tobacco smoke, normal saliva does not contain measurable levels of acetaldehyde ,24. The l mucosa  and alsol mucosa . In concl mucosa .ALDH2 genotyping protocol in order to decrease the risk of sample contamination. Our study did not include the analysis of other genetic factors involved in ethanol metabolism, such as the ADH1B genotype. This warrants further studies that should focus on the possible role of ADH polymorphisms in the exposure of the oral cavity to ethanol without its ingestion.We used direct PCR in our Acetaldehyde is widely present in the environment and has been found to be mutagenic and carcinogenic in vitro and in animal experiments -29. A reKm ALDH enzymes and the presence of high Km ADH enzymes in the gingiva and the lingual mucosa may further increase the local exposure to carcinogenic concentrations of salivary acetaldehyde [Acetaldehyde in concentrations of 100 µM and above has been shown to result in an exponential increase in mutagenic DNA lesions . Acetaldaldehyde .H. pylori status combined with high consumption of pickled food has recently been shown to result in a 27-fold risk of noncardia gastric cancer [The official criterion for alcoholic beverages is that they contain 2.8 vol% or more of alcohol and their consumption is systematically followed and used in cancer epidemiology. However, many non-alcoholic beverages and edibles produced by fermentation processes may contain low but significant levels of ethanol in addition to mutagenic concentrations of acetaldehyde ,35,36. Tc cancer . Thus feWhen combined, these results imply that the presence of ethanol in the systemic circulation is a key factor for the increased exposure of the upper digestive tract mucosa to endogenously formed acetaldehyde encountered with ALDH2 deficient consumers of alcohol. Likewise, these findings provide a logical explanation for the epidemiological findings that show that ALDH2 deficiency increases the risk of upper digestive tract cancer for alcohol drinkers, but not for non-drinkers who are exposed to acetaldehyde that is derived from sources that do not associate with the presence of ethanol in the systemic blood circulation ,19. SuchALDH2*2 carriers, but not for ALDH2 deficient non-drinkers and rare drinkers, regardless of their smoking status and possible consumption of edibles that contain ethanol or acetaldehyde. Thus, ALDH2 deficient alcohol drinkers provide a human model for increased local exposure to acetaldehyde derived from the salivary glands every time when they are drinking alcoholic beverages.In conclusion, our present study supports earlier findings that show that the elevated levels of carcinogenic acetaldehyde found in the saliva of ALDH2 deficient individuals appear to be derived from the parotid glands ,13, whic


Dear EditorBeta Thalassemia major is a genetic disease with an autosomal recessive pattern and is differentiated by severe microcytic hypochromic hemolytic anemia with hepatosplenomegaly, ineffective erythropoiesis and bone marrow expansion. The β-glIn the last decade, numerous studies have been published about distribution and frequency of mutations within the HBB gene in some regions of the Iran.5] These These5] We firstly attempted also to fully analyze the beta globin gene in 130 blood transfusion dependent individuals in the two closely located towns, namely Eizeh and Baq-malek, After investigation a cohort of 200 beta thalassemia major patients and more than 1000 carriers in the Khuzestan Province in a previous study,8] We[8] We7][ We[8] We


Quantifying chromosomal instability (CIN) has both prognostic and predictive clinical utility in breast cancer. In order to establish a robust and clinically applicable gene expression-based measure of CIN, we assessed the ability of four qPCR quantified genes selected from the 70-gene Chromosomal Instability (CIN70) expression signature to stratify outcome in patients with grade 2 breast cancer.in silico. We assessed the ability of CIN4 to stratify outcome in an independent cohort of patients diagnosed between 1999 and 2002. 185 formalin-fixed, paraffin-embedded (FFPE) samples were included in the qPCR measurement of CIN4 expression. In parallel, ploidy status of tumors was assessed by flow cytometry. We investigated whether the categorical CIN4 score derived from the CIN4 signature was correlated with recurrence-free survival (RFS) and ploidy status in this cohort.AURKA, FOXM1, TOP2A and TPX2 (CIN4), were selected from the CIN70 signature due to their high level of correlation with histological grade and mean CIN70 signature expression We observed a significant association of tumor proliferation, defined by Ki67 and mitotic index (MI), with both CIN4 expression and aneuploidy. The CIN4 score stratified grade 2 carcinomas into good and poor prognostic cohorts  and its predictive power was confirmed by multivariate analysis outperforming MI and Ki67 expression.The first clinically applicable qPCR derived measure of tumor aneuploidy from FFPE tissue, stratifies grade 2 tumors into good and poor prognosis groups. Chromosomal instability (CIN) is a key determinant of biological behavior of breast cancer The potential utility of a gene expression based measure of CIN is further emphasized by its complex relationship with histological grade All microarray data sets used in this analysis were normalized by robust multi-array average (RMA) Focusing on histological grade, we evaluated 185 invasive breast carcinomas consisting of 63 grade 1, 62 grade 2 and 60 grade 3 FFPE tissue samples regardless of other pathological features from the Buda MÁV Hospital (1999–2002), diagnosed and graded by a single pathologist (J.K.). Recurrence-free survival (RFS) time was defined either by loco-regional relapse or the appearance of a distant metastasis, and whichever shorter if both applicable. The study was a retrospective analysis. General written consent was obtained from all patients at time of surgery. The samples were anonymised for the study. The study was approved by the Ethical Committee of the Semmelweis University (IKEB #7/2008 and #7-1/2008).In line with bioinformatics, the clinicopathological properties of the selected 185 breast cancer patients were analyzed. The mean age of patients was 58.8±12.8 years . Among the histological types, invasive ductal carcinoma was the most common overall (65.9%), but less frequent types of cancer were also included in the analysis (8.1%): 1 papillary, 1 tubular, 1 micropapillary and 3 mucinous carcinomas in the grade 1 group; 1 micropapillary and 1 mucionous in grade 2; and 4 with medullary features, 2 metaplastic and 1 micropapillary carcinomas in grade 3 cancers. Tumor size, frequency of vascular invasion, presence of necrosis, Nottingham Prognostic Index (NPI) and number of relapses showed an increase, while ER, PgR expression and RFS decreased with higher grade . When chFive to ten 5 µm thick sections were used from each case for RNA purification after assessment of cellularity on HE stained slides (minimum of 70% tumor cell content was required). RNA was extracted with Qiagen FFPE RNeasy kit according to the manufacturer's protocol . High Capacity RNA-to-cDNA kit was used to reverse transcribe 1000 ng of RNA . The Eppendorf epMotion 5070 robotic system was used to transfer samples and reagents to 384-well full-skirted white plates . The qPCR was performed in duplicates with Taqman® Assays  in Gene Flow cytometry was performed for the analysis of ploidy. Briefly, a 50 µm section was cut from all the FFPE blocks. A scroll of tissue was placed in a microcentrifuge tube and xylene was added to remove the paraffin wax. The tissue was then serially re-hydrated through 100%, 95%, 70% and 50% ethanol for 5 minutes respectively at room temperature. The tissue was washed twice with distilled water. A suspension of nuclei was made by incubating the tissue in a 0.5% pepsin solution  prepared in 0.9% saline at pH 1.5. Incubation was carried out at 37°C for 30 minutes. The nuclei were washed once with PBS, stained with propidium-iodine and analyzed using the Calibur 1 FACS mashine and CellQuest software . DNA index was assigned as follows: diploids were ‘1.0’, a tumor with a DNA index greater than 1.10 was classified as aneuploid The assignment of each patient into two cohorts using the CIN4 expression signature was performed in the R statistical environment  using the package Prediction Analysis for Microarrays  as described previously In order to select a more limited set of genes that optimally reflect the CIN70 signature we retrieved expression profiles for the CIN70 signature genes from 10 publicly available breast cancer datasets The CIN70 genes were then ranked by Pearson's correlation coefficients to the CIN score (mean CIN70 expression) within these breast cancer microarray cohorts [supplementary references]. In order to derive a clinically applicable qPCR expression signature for use in FFPE tissue, containing fewer genes with equivalent information reflecting mean CIN70 expression For 5 of the 10 breast cancer microarray cohorts histological grade was also available. The above listed 4 genes were also highly correlated with histological grade (Pearson correlation coefficient above 0.7).Next, we assessed the association individually on data sets between the mean-expression level of CIN4 and clinical outcome across the same 10 cohorts containing expression data from 1928 breast cancers. We observed significant discriminative power  by CIN4 for the stratification of good from poor clinical outcome in all of the breast cancer cohorts [supplementary references]. Therefore, the expression of CIN4 appears almost as efficient at predicting cancer outcome as the extended CIN25 and CIN70 signatures. We were able to compare the performance in silico of the CIN4 signature to a number of previously published predictors of outcome such as CIN25, CIN70 B4GALT3, SLC9A3R2 and PUM1 were previously chosen based on their low variability in gene expression datasets described previously Expression of CIN4 was assessed in a retrospective cohort of 185 patients for which we had FFPE primary breast cancer samples available from Buda MÁV hospital, treated between 1999 and 2002 . Kaplan-In order to define a threshold for CIN4 expression for distinct outcome groups, we trained the PAM algorithm using the continuous CIN4 gene expression signature to discriminate clinical outcome of 63 patients with grade 1 breast cancer compared to the poorer outcome associated with 60 grade 3 breast cancers from within this cohort of 185 patients. Using this CIN4 expression threshold that best distinguished grade 1 compared to grade 3 breast cancers, the PAM defined CIN4 score was established, and we assessed the ability of this CIN4 score to stratify cancer outcome in the remaining 62 patients with grade 2 breast cancer from this 185 patient cohort.Using this threshold, the CIN4 score was able to stratify patients with grade 2 breast cancers into good (44 patients) and poor (18 patients) prognostic groups  .For the identified genes that have been previously described to be of prognostic value, we have evaluated the prognostic power of AURKA, FOXM1, TOP2A, and TPX2 separately. Although, in public breast cancer datasets  all the genes showed strong predictive potential Click here for additional data file.Figure S2In silico comparison of the performance of CIN4 vs. CIN25, CIN70, Ki67, genetic grade, HOXB13:IL17BR index, Genomic Grade Index, the 21-gene recurrence score and NKI70 in the GIS, JBI and JBI1 datasets  (A), and assessing the additive power of CIN4 (B).(PDF)Click here for additional data file.Figure S3Kaplan-Meier curves of Ki67 and mitotic index performances in the tissue samples of grade 2 breast tumors.(TIF)Click here for additional data file.Figure S4Individual prognostic performance of AURKA, FOXM1, TOP2A and TPX2 in breast cancer datasets as compared to CIN4 signature (groups split at median expression).(PDF)Click here for additional data file.Figure S5Correlation of CIN4 and markers used in the determination of immunophenotype. CIN4 and A) ER, B) PR, and C) HER2 expression, and D) HER2/chromosome 17 score determined by FISH.(TIF)Click here for additional data file.Figure S6CIN4 expression in ER-negative and ER-positive tumors.(TIFF)Click here for additional data file.Table S1The public datasets analysed in the study and their supplementary references.(XLS)Click here for additional data file.Table S2The probes used for the qPCR evaluation in the study.(XLS)Click here for additional data file.Table S3Expression of control genes in the breast cancer tissue samples according to grade.(XLS)Click here for additional data file.Table S4Performance of the individual components of CIN4 in tissue samples of grade 2 breast tumors.(XLS)Click here for additional data file.Table S5Correlation of the clinicopathological/prognostic variables with each other in grade 2 breast tumours only. Pearson correlation coefficients displayed, significant correlation: gray highlight.(XLS)Click here for additional data file.


The precise timing of events in the brain has consequences for intracellular processes, synaptic plasticity, integration and network behaviour. Pyramidal neurons, the most widespread excitatory neuron of the neocortex have multiple spike initiation zones, which interact via dendritic and somatic spikes actively propagating in all directions within the dendritic tree. For these neurons, therefore, both the location and timing of synaptic inputs are critical. The time window for which the backpropagating action potential can influence dendritic spike generation has been extensively studied in layer 5 neocortical pyramidal neurons of rat somatosensory cortex. Here, we re-examine this coincidence detection window for pyramidal cell types across the rat somatosensory cortex in layers 2/3, 5 and 6. We find that the time-window for optimal interaction is widest and shifted in layer 5 pyramidal neurons relative to cells in layers 6 and 2/3. Inputs arriving at the same time and locations will therefore differentially affect spike-timing dependent processes in the different classes of pyramidal neurons. Timing is a central concept in cortical function. At the network level, information is encoded in the spiking of neurons and there is much debate about the level of precision that is important +, K+ and Ca2+ channels Recently it has become clear that the input/output function of pyramidal neurons is also profoundly influenced by the computational properties of the dendritic tree itself + channels followed by a slower Ca2+-dependent component nd component is particularly pronounced and typically drives the soma to fire a burst of APs nd component contributes to further somatic depolarization but does not necessarily trigger axonal firing. The dendritic and axonal spike initiation zones are coupled by the influence of the backpropagating action potential (bAP) that lowers the threshold for the initiation of the dendritic spike. This phenomenon, known as “backpropagation activated calcium spike firing” (BAC firing) Neocortical pyramidal neurons have a spike initiation zone in the apical dendrite In this paper, we investigated the time window of coincidence detection in L2/3, L5 and L6 pyramidal neurons of the somatosensory cortex in rats using simultaneous dual patch-clamp recordings from the cell body and apical dendrite and we show that all three types of pyramidal neurons have a specific time window for somato-dendritic spike interaction.The study was approved by the Veterinary office of the Canton Bern, Switzerland, permission number 90/08.2PO4, 25 NaHCO3, 1 MgCl2, 2 CaCl2, and 25 glucose; pH 7.4. Parasagittal slices, 300 µm thick, were cut from the tissue block with a vibratome (Microm) and kept at 37°C for 30 min and then at room temperature until use.Experiments were performed in somatosensory neocortical slices from postnatal day 28–49 Wistar rats (n = 26) using procedures described previously All experiments were performed at 32.0±0.5°C. Single pyramidal neurons were identified using infrared Dodt gradient contrast or oblique illumination and a CCD camera . Slices were perfused with the same extracellular solution mentioned above. Recording pipettes were filled with intracellular solution containing the following: 130 mM K-gluconate, 5 mM KCl, 30 mM HEPES, 10 mM Phospho-kreatine, 4 mM MgATP, and 0.3 mM GTP; pH 7.3. The somatic pipette contained in addition 10–50 µM Alexa 594 (Invitrogen), 100 µM Oregon Green BAPTA-1 , and 0.2% Biocytin (Sigma). Dual whole-cell voltage recordings were performed from the soma and dendrites (6–10 and 20–40 MΩ pipette resistances respectively) using Axoclamp 2A (Axon Instruments) and Dagan BVC-700A amplifiers (Dagan Corporation). Data were acquired with an ITC-18 board (Instrutech) and custom software written for the Igor environment (Wavemetrics). After recordings, slices were fixed and stained as described previously The dendritic recording was made at least 20 min after establishing the somatic recording to allow intracellular spread of the dyes from the soma. Dendrites were targeted with infrared-scanning gradient contrast (IR-SGC) All statistics were calculated using commercial software . If not otherwise indicated values represent means ± s.e.m. All data were tested for normality and equal variance. Statistical comparisons of spike thresholds were performed using 2-way repeated measurement ANOVA to test for effects of time versus baseline  or for tinj) was injected into the dendrite  & 2/3 in the somatosensory neocortex of rats , no significant effect of layers  but a significant effect of the interaction between layers and time . Post hoc test showed that the threshold reduction was significantly different for L5 pyramidal neurons compared to L6 and L2/3 for many time points, whereas L6 and L2/3 pyramidal neurons were only different from each other at one time point. The presence of an AP had the greatest effect on L5 pyramidal neurons reducing the threshold by 41±7%. Furthermore, the coincidence detection time window for L5 was extended relative to L6 and L2/3 pyramidal neurons (To compare the time windows for somato-dendritic coupling between the different pyramidal cell classes we normalized the values at the different Δt's to the threshold for generating a dendritic spike without an axonal AP . 2-way r neurons .In summary, we found that the coincidence timing curve for the initiation of dendritic spikes in L5 pyramidal neurons was wider than for L6 and for L2/3 pyramidal neurons. L6 and L2/3 pyramidal neurons exhibited similar coincidence detection windows to each other but were narrower than in L5 cells implying these cells require more precise synaptic inputs for this effect. The bAP had the greatest relative effect on dendritic spike generation in L5 neurons however the baseline threshold in L5 neurons was much larger than in L2/3 and L6 neurons . Thus, tWhat are the implications of timing differences between pyramidal cell classes? We predict that processes in the dendritic tree which are influenced by the coupling of bAPs with local dendritic membrane potential such as STDP The active and passive properties of L6, L2/3 and L5 pyramidal tuft dendrites are similar but not identical The functional consequence of coincidence detection in pyramidal neurons depends also on the particular inputs that are associated. The cortical layer of the cell bodies and basal dendrites of pyramidal neurons determines the proximal input In conclusion, we have shown that all pyramidal neurons of the rat somatosensory cortex can associate inputs arriving at their distal and proximal dendritic trees in a limited time window that varies between cell classes. This suggests that pyramidal neurons operate in a similar way on the input which reaches the different cortical layers they are covering.


Changes to the molecular structure of Cav1.2 channels could affect sensitivity of the channels to blockade by CCBs. Recently, extensive alternative splicing was found in Cav1.2 channels that generated wide phenotypic variations. Cardiac and smooth muscles express slightly different, but functionally important Cav1.2 splice variants. Alternative splicing could also modulate the gating properties of the channels and giving rise to different responses to inhibition by CCBs. Importantly, alternative splicing of Cav1.2 channels may play an important role to influence the outcome of many cardiovascular disorders. Therefore, the understanding of how alternative splicing impacts Cav1.2 channels pharmacology in various diseases and different organs may provide the possibility for individualized therapy with minimal side effects.Calcium channel blockers (CCBs) are widely used to treat cardiovascular diseases such as hypertension, angina pectoris, hypertrophic cardiomyopathy, and supraventricular tachycardia. CCBs selectively inhibit the inward flow of calcium ions through voltage-gated calcium channels, particularly Ca The T type Cav3.1 and L type Cav1.3 channels are expressed in the sinus node cells and modulate pacemaker activityCalcium ions play a critical role in muscle function. Voltage-gated calcium channels (VGCCs) govern the depolarization induced Ca1 subunit is the basic structure of the channel, while the β, α2δ and/or γ subunits interact with the α1 subunit and play a modulatory role. Calcium channel blockers (CCBs) are widely used in clinical practice to treat cardiovascular disorders from hypertension to angina pectoris, arrhythmia, Raynaud syndrome, and cerebral vasospasm, etc. The basic effect of CCBs is to inhibit VGCCs by binding to the pore forming α1 subunit and the Cav1.2 channel is the major target of CCBs.VGCCs are composed of multiple subunits. The pore forming α1 subunit of Cav1.2 channelThree classes of small molecule CCBs are currently in clinical use: 1,4-dihydropyridines (DHPs), phenylalkylamines (PAAs), and benzothiazepines (BTZs). They all bind to the αv1.2 channel were identified to link with antihypertensive outcomeGenetic factors determine drug response taking into consideration many other factors such as age, sex, body weight, and heath status. Pharmacogenomics provides information on the linkage of genetic factors to drug responses and may also provide the basis for the use of safer and more efficient medications to patients. In hypertension, genetic associations with antihypertensive response have been established for diuretics, beta-blockers, ACE inhibitors and angiotensin1 receptor blockers. However, most of the information is lacking in calcium channel blockers. Recently, three single nucleotide polymorphisms (SNPs) of Ca1 subunit of Cav1.2 channelv1.2 channels under physiological and pathophysiological conditions and the influence of such changes on pharmacology. The proteomic structure of Cav1.2 channels could change under pathological conditions due to alternative splicing. The way we view individualized medicine in treating cardiovascular diseases may need to be expanded beyond pharmacogenomics.Alternative splicing is a post-transcriptional modification process. Multiple functional variants could be generated from a single gene. Recently, a large number of alternatively spliced exons have been identified within the pore-forming αCACNA1C, codes for the α1 subunit and contains 55 exons. At least 19 exons are subjected to alternative splicingThe human Cav1.2 gene, The binding site for CCBs is mainly composed of the transmembrane segments 5 and 6 (S5 and S6) of domains I to IV. By using photoaffinity labeling, antibody mapping, and chimeric study, DHPs were found to bind IIIS5, IIIS6 and IVS6 segments, while IIIS6 and IVS6 are the binding sites for PAAs and DTZs2+ influx through Ca2+ channels in cardiac and vascular smooth muscles. However, there exist variable responses to blockade of Cav1.2 channels by CCBs within the two tissues. For example, vascular smooth muscles are more sensitive to DHPs than cardiac muscles. One obvious reason is that calcium channels in smooth muscle possess a higher binding affinity than in cardiac musclev1.2 channels are locked in an inactivated state which favors the DHP blockThe pharmacological effect of CCBs depends on their inhibition of Cav1.2 channel is generally divided into a cardiac isoform (Cav1.2a) and a smooth muscle isoform (Cav1.2b). Cav1.2a channel is the predominant channel in heart while Cav1.2b channel in smooth muscles. Cav1.2a channel contains the combination of exons 1a/8a/-9*/32/33v1.2b) contained exons 1b/8/9*/32/33Cav1.2b channel is more sensitive to DHP block than Cav1.2a channel which is similar to the observations in native heart and blood vesselsThe Cav1.2 channel activity is also regulated by phosphorylationCav1.2 channels in heart and blood vessels, numerous splice variants are found to be expressed in cardiovascular systemv1.2 channels in arterial smooth muscles relates with the left shifted window currents recorded in native smooth musclesAlthough there exist predominant Cav1.2 channels are crucial for cardiovascular functions as deletion of the gene in mouse leads to embryonic lethalityv1.2 channels was linked to many diseasesv1.2 gene was reported in Timothy syndrome, a disorder characterized by dysfunction in multiple organ systems, including heart, skin, eyes, teeth, immune system and brain2+ ions will result in the lengthening of action potential, leading to cardiac arrhythmia and sudden death. The levels of expression of exon 8 and 8a is different in various organs and tissues and thus the location of the mutations in exon 8 or 8a would determine the severity of the symptoms and the involvement of other organs. CCBs are ideal to treat the patients by reducing the Ca2+ influx from mutant channels.Cav1.2 channels has identified to be altered in cardiovascular disorders. Mutually exclusive exons 31 and 32 are developmentally regulatedet alet alAlternative splicing of Cav1.2 channels highlights a novel way towards individualized medication. Besides SNPs, post transcriptional modification produces Cav1.2 channels with huge variability both in structure and function. Each person could express slightly different splice variants in different tissues. But the functional impact could be enormous. Furthermore, under pathological conditions, the splice patterns can be altered. Such alteration could be variable at different stages of the disease. Thus, each patient could express a signature pattern of Cav1.2 channels generated by alternative splicing. This provides possible targets for individualized medication. However, many questions need to be addressed first and chief of which is how the splicing profile from different organs of a patients can be achieved. The nature of alternative splicing makes it impossible to get such information simply from blood. Also the length of the gene and multiple splicing sites makes it difficult to determine combinatorial profiles for the expression of the many alternatively spliced exons in the full length Cav1.2 channel transcripts. The next obstacle is to select suitable splice variants as targets for drug discovery and development. Most of the current CCBs in use are not designed against one splice variant without affecting others. The understanding of alternative splicing of Cav1.2 channels is far from complete. One example is the hemichannels generated by misspliced exonsv1.2 channels remains mostly unclear.The progress in the study of alternative splicing of Cav1.2 channels to cardiovascular pharmacology and pathophysiology. However, the knowledge in other organs and systems are mostly lacking. For example, the splicing pattern in nervous system is not well studied. Considering the higher expression of Cav1.2 channels in neurons, CCBs in treating nervous system disorders could attract more attention if neuronal specific CCB is discovered one day in the future. In conclusion, we presented another consideration for the development or discovery of drugs against Cav1.2 channels that may be efficacious in the management of cardiovascular disorder.In this review, we discussed the progress in relating alternative splicing of Ca


PLA2G6 gene have variable phenotypic outcome including infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, idiopathic neurodegeneration with brain iron accumulation and Karak syndrome. The cause of this phenotypic variation is so far unknown which impairs both genetic diagnosis and appropriate family counseling. We report detailed clinical, electrophysiological, neuroimaging, histologic, biochemical and genetic characterization of 11 patients, from 6 consanguineous families, who were followed for a period of up to 17 years. Cerebellar atrophy was constant and the earliest feature of the disease preceding brain iron accumulation, leading to the provisional diagnosis of a recessive progressive ataxia in these patients. Ultrastructural characterization of patients’ muscle biopsies revealed focal accumulation of granular and membranous material possibly resulting from defective membrane homeostasis caused by disrupted PLA2G6 function. Enzyme studies in one of these muscle biopsies provided evidence for a relatively low mitochondrial content, which is compatible with the structural mitochondrial alterations seen by electron microscopy. Genetic characterization of 11 patients led to the identification of six underlying PLA2G6 gene mutations, five of which are novel. Importantly, by combining clinical and genetic data we have observed that while the phenotype of neurodegeneration associated with PLA2G6 mutations is variable in this cohort of patients belonging to the same ethnic background, it is partially influenced by the genotype, considering the age at onset and the functional disability criteria. Molecular testing for PLA2G6 mutations is, therefore, indicated in childhood-onset ataxia syndromes, if neuroimaging shows cerebellar atrophy with or without evidence of iron accumulation.Mutations in  PLA2G6 mutations (PLAN) constitutes a heterogeneous group of clinical entities which encompasses infantile neuroaxonal dystrophy , atypical neuroaxonal dystrophy (NAD), idiopathic neurodegeneration with brain iron accumulation including Karak syndrome  ), following a conventional protocol  using suOphthalmologic examinations were occasionally limited because many of the patients were young and because of the cognitive complications of the neurodegenerative disorder. The assessment of optic disk pallor was done using indirect ophthalmoscopy. All patients had visual evoked responses (VEPs) using flashing lights and P100 was the most important wave to identify. All patients had brain computer tomography (CT) and/or magnetic resonance imaging (MRI) [Siemens 1.5 Tesla].vastus lateralis) was undertaken in 4 patients and examined by standard histological techniques. Two glutaraldehyde-fixed muscle specimens were available for electron microscopy (EM) examination and processed as described by Weis et al. ), and malnutrition (requiring feeding through gastrostomy tube) and bed sores in another girl aged 9.8 years (F1 [P1]). Two patients died at ages of 12.8 years (F3 [P1]) and 28 years (F6 [P1]). The causes of death were, respectively, respiratory infection due to H1N1 swine flu and septicaemia following bed sores.Physical examination  revealedAbsent or delayed evoked potentials (VEP) were seen in 5/11 (45%) patients . In the Distal axonal-type sensorimotor neuropathy was evident in 9 (90%) patients who had nerve conduction studies . The compound muscle action potentials (CMAPs) morphology was diphasic and the peak amplitudes were recorded. With the exception of one (F5 [P1]), all patients had either unobtainable or low amplitude CMAPs for the peroneal nerve, recorded from the extensor digitorum brevis (EDB) muscle; while in the upper limbs 3/9 patients (33%) showed normal CMAP amplitudes. The median sensory nerve action potentials (SNAPs) recorded in 9 patients were normal in 7 (77%) and reduced in one (F6 [P2]), while sural SNAPs were within the normal range in 4/10 (40%) patients.Brain imaging  was undeglobus pallidus, seen as reduced signal on T2, FLAIR and/or diffusion weighted image (DWI) sequence in the initial scans , 3.1 years (F1 [P1]) and 3.3 years (F2 [P1]), respectively . His affected brother (F6 [P3]) showed clear features of brain iron deposition on MRI done at a younger age  whereas cerebellar atrophy was shown in all patients.Eight (80%) of the 10 children who had MRI scans showed evidence of increased iron deposition in the al scans  and 4. Oal scans  did not ectively . Repeateectively , showed substantia nigra was revealed by the initial MRI in 6 of 10 (60%) patients at a median age of 10.3 years . Three of the patients with initial normal substantia nigra signal had their MRI at 1.3 year (F2 [P2]), 2.8 years (F4 [P1]), and 3.3 years (F2 [P1]) but no subsequent scans. The fourth patient (F5 [P1]), had his initial MRI at the age of 4.2 years, but showed substantia nigra iron deposition when imaging was repeated 3 years 4 months later (Increased iron in the hs later . With imhs later  and 4.c oxidoreductase (complex II + coenzyme Q + complex III) of 4.0 mU/mg (control range = 8.2-44), and decylubiquinol: cyt c oxidoreductase (complex III) of 41 mU/mg (control range = 54-434). Citrate synthase activity was at the lowest reference value . By contrast, the ratios between enzyme activity of the respiratory chain and citrate synthase were not remarkable. The relatively low activity of all enzymes on protein base, combined with the normal enzyme activity ratio’s, lead us to conclude that there is a relatively low mitochondrial content in the muscle of patient [F6 (P1)], rather than a deficiency of one or more respiratory chain enzymes. To confirm these results we determined the density of the intermyofibrillar mitochondria in the patient’s [F6 (P1)] biopsy and compared it to an age and gender matched healthy individual’s muscle specimen taken from the same location  using electron microscopy. We found that the patient’s muscle showed a significant decrease in the mitochondrial density (0.255 +/- 0.05/µm2) in comparison to the control (0.379 +/- 0.06/µm2) (p=0.0001).Histological examination of muscle biopsies from 4 patients revealed non-specific neurogenic features. In contrast, electron microscopy  revealedElectron microscopic analysis of the muscle biopsy (taken at the age of 3 years) of patient F1 (P1)  with INAPANK2 gene for 5 families while all the affected members of these were homozygous for the microsatellite markers flanking the PLA2G6 locus . All affected individuals were homozygous for the identified mutation while parents, for whom the DNA was available, were heterozygotes for the mutations and unaffected siblings were either heterozygotes or homozygous for the wild type allele  the age at onset of the disease manifesting as ataxia, and (ii) the evolution of the disease at the functional level. We expressed the results as a graph with exponential trend curves. A curve of the evolution of the age at onset of ataxia and a second one showing evolution of the age at which patients reach functional Stage 7 (wheelchair bound) were formulated. The disease duration was also considered in the graph . We coulPLA2G6 mutations.The present cohort represents one of the largest collections of patients from one country and the same ethnic background (Arabs) with neurodegeneration associated with In the present study, 6 children   had claslocus caeruleus in the disease, which has been documented pathologically in patients with PLA2G6 mutations ) developed complex partial seizure at 21 years of age, which was confirmed by EEG. Another 2 siblings with INAD had brief tonic seizures with normal EEG in one and non-specific changes in another. Fast rhythms on EEG were observed in 2 patients with classic INAD without clinical seizures . These fOptic atrophy occurs in the majority of children with infantile and atypical NAD whereas nystagmus and strabismus are also common -6,28,29.Neurophysiologic studies revealed features of distal axonal neuropathy in all 4 patients with Karak syndrome phenotype. Also 3 of 5 (60%) children with classic INAD had features of axonal neuropathy on NCS. This is similar to the findings in other cohort of patients with INAD [PLA2G6 in mice (iPLA2b-/-) was documented to lead to the development of cerebellar atrophy by the age of 13 months [corpus callosum thinning) was evident in 2 patients (F1 [P1] and F5 [P1]) and borderline changes were detected initially in one of them (F5 [P1]). Similarly, one of 5 reported patients ) and at 4.2 years for another (F1 [P1]). The latter child had an earlier MRI at 3.1 years which revealed globus pallidus hypointensity on DWI only  showed a relatively low mitochondrial content in the muscle. This is compatible with the observed ultrastructural abnormalities seen by electron microscopy, and is also in agreement with the proposed role of PLA2G6 in protecting the mitochondrial membrane from peroxidation  with classic INAD and F6 [P1] with Karak syndrome phenotype). This membranous material is probably derived from the sarcoplasmic reticulum/myotubular system and could have resulted from the disrupted membrane homeostasis following disruption of iPLAodelling . To the xidation .This is the first time that such a genotype phenotype correlation was highlighted in a cohort of PLAN patients, even in studies which included a group of patients belonging to the same ethnic background. Indeed, in the reported series , there wPLA2G6 gene mutations, five of which are novel. We have shown that the phenotype of neurodegeneration associated with PLA2G6 mutations is variable in this cohort of patients who belong to the same ethnic background but, in terms of functional disability, was influenced by the genotype. Nevertheless, cerebellar atrophy is the constant and earliest feature of the disease, and precedes brain iron accumulation, leading to the provisional diagnosis of a recessive progressive ataxia in these patients. It is noteworthy that variable combinations of sites of degeneration and associated symptoms are often observed in childhood-onset recessive ataxias including epilepsy and abnormal cognition [PLA2G6 mutations is, therefore, indicated in childhood-onset ataxia syndromes, if neuroimaging shows cerebellar atrophy with or without evidence of brain iron accumulation. Considering the age at onset and the functional disability, there is an evidence of genotype-phenotype correlation. However, the wide intra- and inter-familial variability of the disease in physiological, psychiatric and other clinical aspects, cannot be explained only by single homozygous mutations in the PLA2G6 gene. At least, other environmental and/or genetic factors  might probably modulate the disease presentation.In conclusion, we describe the phenotypic and genetic spectrum of 11 patients followed for a maximum period of 17 years and report six underlying ognition -36. ElecVideo S1Patient F2 (P2) with INAD, aged 2.4 years, showing tetraparesis and brisk reflexes (including adductor reflex) despite absence of leg stiffness (note the frog position of the lower limbs).(WMA)Click here for additional data file.


TRPM7, a cation channel of the transient receptor potential channel family, has been identified as a ubiquitous magnesium transporter. We here show that TRPM7 is expressed in endothelial cells isolated from the umbilical vein (HUVEC), widely used as a model of macrovascular endothelium. Quiescence and senescence do not modulate TRPM7 amounts, whereas oxidative stress generated by the addition of hydrogen peroxide increases TRPM7 levels. Moreover, high extracellular magnesium decreases the levels of TRPM7 by activating calpains, while low extracellular magnesium, known to promote endothelial dysfunction, stimulates TRPM7 accumulation partly through the action of free radicals. Indeed, the antioxidant trolox prevents TRPM7 increase by low magnesium. We also demonstrate the unique behaviour of HUVEC in responding to pharmacological and genetic inhibition of TRPM7 with an increase of cell growth and migration. Our results indicate that TRPM7 modulates endothelial behavior and that any condition leading to TRPM7 upregulation might impair endothelial function. TRPM6 and -7 the first molecularly defined components of the mammalian Mg transport machinery. TRPM6 and -7 show the unique functional duality of being an ion channel and a kinase. TRPM7, which is ubiquitously expressed, was initially thought to play a prominent role in intracellular Mg homeostasis, whereas TRPM6 controls systemic Mg homeostasis by regulating Mg transport in the kidney and in the gut Magnesium (Mg), the second most abundant intracellular cation, plays a major role in regulating endothelial function TRPM7 mimics the effects of Mg deficiency in these cells The presence of functional TRPM7 channels in human endothelial cells has been demonstrated TRPM7 stimulate cell proliferation TRPM7 induces cell cycle arrest. In addition to the significant elevation of TRPM7 in the vasculature of MgL mice TRPM7 transcript in HUVEC exposed to shear stress has been described In endothelial cells derived from the umbilical vein (HUVEC), two independent reports have shown that siRNAs transiently silencing To this purpose, it is noteworthy that different types of endothelial cells including HUVEC have very low levels of TRPM7 current which shows no significant increase in response to fluid flow Although scarce, the data reported until now point to a potential regulatory role for TRPM7 in the maintenance of vascular integrity Because increasing evidence suggests that TRPM7 might contribute to the pathophysiology of the vasculature in general and of the endothelium in particular, we explored the modulation of the expression of TRPM7 in human endothelial cells and the effects of its inhibition on some aspects of endothelial function.4in vitro, HUVEC were used at low population doublings . PD were calculated as log2 (number of cells at time of subculture/number of cells plated). We define senescent cells as the culture that do not increase the cell number and remain subconfluent for 2 weeks. We confirmed the senescent phenotype with senescence-associated-beta galactosidase activity assay as described Primary HUVEC isolated from the umbilical vein (American Type Culture Collection) were cultured in M199 containing 10% fetal bovine serum (FBS), 1 mM glutamine, 1 mM penicillin and streptomycin, Endothelial Cell Growth Factor (150 µg/ml), 1 mM sodium pyruvate and heparin (5 units/ml) on 2% gelatin-coated dishes 2O2 (100 µM) for 30 min and then part of the samples immediately lyzed while the remaining was maintained in culture for 24 additional h before lysis. In other experiments HUVEC were treated with trolox (10 µM) In some experiments HUVEC were treated with HTRPM7, we utilized the pGIPZ shRNAmir (0.2 µg/cm2) (Open Biosystems). The construct was transfected into 3×105 HUVEC using Arrest-in Transfection Reagent (Open Biosystems). Transfection with Non-Silencing (scrambled) pGIPZ shRNAmir was used as control.To silence For proliferation assays, the cells were seeded at low density in growth medium. At the end of the experiments, the cells were trypsinized, stained with a trypan blue solution (0.4%) and the viable cells were counted using a Burker chamber. In some experiments the cells were treated with 2-aminoethoxydiphenyl borate  or Co(III)-hexaammine (250 µM) (Sigma Aldrich).in vitro model of wound repair as previously described Migration was determined using an TRPM7 amplification cycle consisted of 1 min at 95°C, 45 sec at 54°C and 1 min at 72°C using 400 nmol/l of each primer in a final volume of 25 µl. The reaction was stopped after 35 cycles. One fifth of the reaction mix was separated on a 2% agarose gel. The sequences of the TRPM7 primers are the following: sense 5′-CTTATGAAGAGGCAGGTCATGG-3′ and antisense 5′-CATCTTGTCTGAAGGACTG-3′ (size of the amplified fragment: 213 bp). RT-PCR with specific primers for actin was performed to normalize .Cells were lyzed in 1 ml Trizol and RNA was purified. RNA was quantified using Nanodrop ND-1000 spectrophotometer and electrophorezed on a 1% agarose gel containing 2.2 M formaldehyde before reverse transcription. Using the Transcriptor first-strand cDNA synthesis kit (Roche Diagnostics), cDNA was synthesized from 2 µg of total RNA using oligo-dT and 5 units of reverse transcriptase at 50°C for 60 min, followed by heating to 85°C for 5 min. PCR amplification was carried out using 1/50 of the final RT reaction. Each HUVEC were lyzed in lysis buffer . Protein concentration was determined using the Bradford protein assay (Bio-Rad). Cell extracts (100 µg/lane) were resolved by 8% SDS-PAGE, transferred to nitrocellulose sheets at 100 mA for 16 h, and probed with anti-TRPM7 (Chemicon) and anti-actin (Sigma Aldrich) antibodies. Secondary antibodies were labelled with horseradish peroxidase . The SuperSignal chemiluminescence kit (Pierce) was used to detect immunoreactive proteins. All the results shown were reproduced at least three times and a representative Western blot is shown. Densitometry was performed by the ImageJ software on 3–5 different blots.2 solutions. The experiments were performed in triplicate and repeated 5 times with similar results. Data are shown as the mean ± standard deviation.NOS activity was measured in the conditioned media by the Griess method To evaluate whether the expression of TRPM7 is related to cell growth, we compared proliferating vs quiescent HUVEC for their amounts of TRPM7. On culture plates, HUVEC grow until they form a perfect monolayer. At this stage, cells stop growing and become quiescent. This pattern can be shown by the arrest of thymidine incorporation 3H-Thymidine incorporation assay 10 h after seeding the cells, half of the samples were growth arrested by exposure to a starvation medium. After 48 h, the cells were lyzed and Western blot performed. No significant modulation of the total amounts of TRPM7 was observed in cells rendered quiescent by starvation vs proliferating cells . Similarin vitro passage when compared to early passage, young cells , an inhibitor of the proteasome, bafilomycin (100 nM), which inhibits lysosomal activity, or calpeptin (5 and 10 µg/ml), a specific m- and μ-calpain inhibitor TRPM7 expression is increased by oxidative stress in monocytes and PC12 cells 2O2 (100 µM), from which hydroxyl radicals are produced by the Fenton reaction, rapidly and stably increased TRPM7 levels after 30 min and up to 24 h. In addition, the antioxidant trolox (10 µM) prevented TRPM7 accumulation in HUVEC cultured in 0.1 mM Mg containing medium (We then tried to understand the mechanisms underlying the increase of TRPM7 levels in HUVEC cultured in low extracellular Mg. Since i) low Mg promotes oxidative stress in endothelial cells g medium .To determine whether TRPM7 contributes to the proliferation of endothelial cells, we transiently transfected HUVEC with a specific shRNA or with a non-silencing shRNA sequence as a control. TRPM7 levels were dramatically reduced 24 and 48 h after transfection and increased thereafter . We deteTRPM7 as well as treatment with 2-APB (50 µM) and Co(III)hexaammine (250 µM) induced HUVEC migration  is a vasoprotective factor with anti-atherogenic properties ilencing .TRPM7 is an important regulator of Mg homeostasis and adequate concentrations of Mg contribute to maintain normal endothelial functions TRPM7 expression is enhanced by oxidative stress in monocytes In general, the expression of ion channels is rather stable, being conductivity regulated by gating mechanisms linked to signalling cascades. Indeed, in vascular smooth muscle cells exposed to fluid flow, TRPM7 expression does not change and the increase in TRPM7 current is due to its translocation to the plasma membrane In HUVEC cultured in high Mg, the levels of TRPM7 decrease. We hypothesize that this downregulation might protect the cells against Mg overload which would negatively influence TRPM7 channel activity. Indeed, as intracellular Mg increases, TRPM7 activity declines. In addition, too much intracellular Mg would generate imbalances of ion concentrations, in particular interfering with calcium homeostasis Last issue to consider is the role of TRPM7 in regulating HUVEC function.TRPM7 and inhibiting its activity with 2-APB or Co(III)hexaammine. The fact that both pharmacological and genetic inhibition of TRPM7 have no effect on NO production is relevant, since our results with shRNA against TRPM7 are in disagreement with a previous report showing that silencing TRPM7 stimulates NO production in an ERK pathway dependent fashion An adequate production of NO is a marker of endothelial function to the point that in clinical practise the examination of vasodilatation in response to stimuli that release NO is routinely employed to assess endothelial function TRPM7 silencing. In particular, it is worth to note that human microvascular endothelial cells are arrested in the G1 phase of the cell cycle upon silencing TRPM7TRPM7 silencing significantly impairs HMEC motility Both genetic and pharmacological inhibition of TRPM7 induce HUVEC proliferation. To our knowledge, HUVEC are unique in responding with an increase of cell growth to The evidence that TRPM7 inhibition impacts on endothelial cell migration and proliferation might have pathophysiologic significance. Indeed, low levels of TRPM7 might facilitate the re-endothelization of vascular injuries, thus preventing excessive subintimal proliferation of smooth muscle cells and reducing the risk of vascular complications. If this theory proves true, maintaining low levels of TRPM7 might contribute to vascular integrity and, in case of damage, to vascular repair.


Overall, our data demonstrate that while subtle differences exist in vitro the metabolic effects in vivo of FGF19/FGF21 are indistinguishable, supporting a shared mechanism of action for these two hormones in the regulation of energy balance.Fibroblast growth factors 19 (FGF19) and 21 (FGF21) have emerged as key regulators of energy metabolism. Several studies have been conducted to understand the mechanism of FGF19 and FGF21 action, however, the data presented has often been inconsistent and at times contradictory. Here in a single study we compare the mechanisms mediating FGF19/FGF21 actions, and how similarities/differences in actions at the cellular level between these two factors translate to common/divergent physiological outputs. Firstly, we show that in cell culture FGF19/FGF21 are very similar, however, key differences are still observed differentiating the two. In vitro we found that both FGF's activate FGFRs in the context of βKlotho (KLB) expression. Furthermore, both factors alter ERK phosphorylation and glucose uptake with comparable potency. Combination treatment of cells with both factors did not have additive effects and treatment with a competitive inhibitor, the FGF21 delta N17 mutant, also blocked FGF19's effects, suggestive of a shared receptor activation mechanism. The key differences between FGF21/FGF19 were noted at the receptor interaction level, specifically the unique ability of FGF19 to bind/signal directly via FGFR4. To determine if differential effects on energy homeostasis and hepatic mitogenicity exist we treated DIO and ob/ob mice with FGF19/FGF21. We find comparable efficacy of the two proteins to correct body weight and serum glucose in both DIO and ob/ob mice. Nevertheless, FGF21 and FGF19 had distinctly different effects on proliferation in the liver. Interestingly,  In mammals the fibroblast growth factors (FGFs) play diverse roles in the regulation of many cellular processes ranging from development to survival Previous publications have demonstrated that FGF15/19 and FGF21 bind to the βKlotho (KLB) isoform of the Klotho family while FGF23 has distinct affinity for the αKlotho (KL) subtype FGF23 plays a well described role in phosphate metabolism and has not previously been shown to affect energy balance We demonstrate here that on both a molecular and whole organism level there are many similarities in the action of FGF19 and FGF21. While FGF21 showed no direct FGFR binding, FGF19 was able to bind FGFR4 independent of KLB. In functional studies we show in 3T3-L1 fibroblasts expressing KLB, both FGF19 and FGF21 were able to stimulate glucose uptake with similar pharmacodynamic properties. When 3T3-L1 adipocytes were treated with a combination of both FGF19 and FGF21 we saw no additive or synergistic effect. Furthermore, treatment with an inhibitory truncated form of FGF21 (termed ΔN17) For both in vitro and in vivo studies FGF19, FGF21 and ΔN17 were generated as previously described All animals were individually housed in a temperature-controlled (24°C) facility with 12 h/12 h light/dark cycle. Animal protocols in this study were approved by the Eli Lilly and Co. Animal Use and Care Committee (Protocol No. 09012).Male C57Bl/6J mice (n = 6 per group) (Taconic Farms) were maintained on a calorie-rich diet consisting of 40% fat, 39% carbohydrate, and 21% protein caloric content  and had free access to food and water before randomization by weight. Mice were administered either FGF19 or FGF21 for a period of 7 days via continuous infusion using osmotic minipumps  at the doses specified. Following sacrifice glucose levels were determined using Precision G Blood Glucose Testing System .Male ob/ob mice (n = 6 per group) (Taconic Farms) were maintained on a standard chow diet  and had free access to food and water before randomization by weight. Mice were administered either FGF19 (1 mg/kg/day) or FGF21 (1 mg/kg/day) for a period of 7 days via continuous infusion using osmotic minipumps . Following sacrifice glucose levels were determined using Precision G Blood Glucose Testing System .Male ob/ob mice (n = 6 per group)  were maintained on a standard chow diet  and had free access to food and water before randomization by weight. Mice were administered with either FGF21, ΔN17 or a combination of both via daily injection at doses indicated for a period of 3 days after which serum was collected for analysis. Prior to sacrifice and blood collection on day 3 the fasted cohorts were deprived of food overnight. Following sacrifice glucose levels were determined using Precision G Blood Glucose Testing System .On day 1 of the study, an osmotic minipump  containing 5-bromo-2-deoxyuridine  was implanted subcutaneously into each 9-week-old male C57bl/6J mouse . Each mouse was given daily subcutaneous injections of either phosphate-buffered saline , FGF19 (2 mg/kg/day) or FGF21 (2 mg/kg/day) for 7 consecutive days. At the end of the 7-day study samples of liver were collected from each mouse, placed in 10% neutral-buffered formalin, processed routinely, and embedded in paraffin. Multiple tissue sections were produced from each paraffin block, stained with Hematoxylin & Eosin (H&E), or immunolabeled for BRDU by routine immunohistochemical methods as outlined below. The H&E tissue sections were evaluated routinely for microscopic changes Cellular incorporation of BrdU was detected by digesting deparaffinized tissue sections with 0.1% protease (Sigma Aldrich) and treating the sections with 2N hydrochloric acid. Sections were blocked with CAS BLOCK , incubated with a rat antibody to BrdU , and bound rat antibody was detected with biotinylated rabbit antibody to rat IgG . Tissue sections were quenched with Peroxidase Blocking Solution  and retained biotin was detected with Vectastain Elite ABC kit (Vector Laboratories). Reaction sites were visualized with DAB Substrate-Chromagen System  followed by DAB enhancer . Sections were counterstained with hematoxylin.BiaCore studies were performed on a BiaCore 2000 instrument . Proteins were covalently immobilized on censor chip CM4 using amine coupling according to the manufacturer's protocol. Typically, 100–50 response units (RU) were immobilized on individual flow cells of the sensor chip. BSA  was immobilized on flow cell 1 as a negative control. Proteins suspended in HBS-P  were then injected for 30 min at a flow rate of 30 ml/min using the kinject command. KD kinetic constants were calculated by BiaEvaluation 4.1 software using a 1∶1 Langmuir model.Cells were treated as indicated for 3 h and glucose uptake was assayed as previously described Cells were treated as indicated for 5 min and subsequently lysed. Total ERK phosphorylation was assessed using an AlphaScreen SureFire Phospho-ERK1/2 Assay Kit (Perkin Elmer) according to the manufacturer's instructions and an EnVision Multilabel Microplate Reader Model 2103 (Perkin Elmer) with the AlphaScreen HTS Turbo option was used for signal detection.RNA was isolated from tissues using TRIzol reagent  or by homogenization of frozen samples in Lysing Matrix D shaker tubes  and was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit . Reactions were performed in triplicate on an ABI Prism 7900HT (PE Applied Biosystems) and were normalized to either 36B4 mRNA or 18S rRNA. ssays-on-Demand Gene Expression Products (PE Applied Biosystems) were as follows: hEGR1, Hs00152928_m1; hFGFR1, Hs00915142_m1; hFGFR2, Hs01552926_m1; hFGFR3, Hs00179829_m1; hFGFR4, Hs01106908_m1; hKL, Hs00183100_m1; hKLB, Hs00545621_m1; mFGFR1, Mm00438930_m1; mFGFR2, Mm01269930_m1; mFGFR3, Mm00433294_m1; mFGFR4, Mm01341852_m1; mKL, Mm00473122_m1; mKLB, Mm00502002_m1; rFGFR1, Rn00577234_m1, rFGFR2, Rn01269940_m1; rFGFR3, Rn00584799_m1; rFGFR4, Rn01441815_m1; rKL, Rn00580123_m1.Data are presented as mean ±SEM. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test where appropriate. Differences were considered significant when P = <0.05.Prior to testing FGF19 and FGF21 for activity in cell based assays we measured expression of FGF receptors and Klotho subtypes in the cell lines we used via RT-qPCR. We found that the expression of FGFR isoforms and the Klotho co-factors differed greatly between the lines. In 3T3-L1 fibroblasts we saw high levels of FGFR1 in addition to lower expression of FGFR2 and only traces of FGFR3 with no detectable FGFR4, KL or KLB . In Hep3In order to assess the specificity and functional significance of the interaction between FGF19, FGF21, FGF23 and the Klotho family we conducted studies in which we expressed either KL or KLB in 3T3-L1 fibroblasts. We chose 3T3-L1 fibroblasts as neither KL or KLB is natively present in these cells To determine if differentiation from fibroblast to adipocyte alters the response of 3T3-L1 cells to the hormone-like FGFs we treated 3T3-L1 adipocytes with either FGF19 or FGF21 and assessed their effects on ERK phosphorylation and glucose uptake. The expression of FGFRs in these cells is very similar to that which we showed for 3T3-L1 fibroblasts As both 3T3-L1 and Hep3B cells express appreciable amounts of FGFRs we turned to L6 cells to confirm our findings as these cells have been reported previously to have vanishingly low expression of both FGFRs and KLs in vitro as a competitive antagonist and leads to inhibition of FGF21 mediated signaling by binding to KLB and blocking FGF21 mediated receptor activation We have previously demonstrated that N terminally truncated FGF21 (ΔN17) acts To determine if the inhibition of FGF19/21 signaling we see in vitro translates to effects on metabolic parameters in vivo we examined fed and fasted glucose levels in ob/ob mice treated with FGF21, ΔN17 or a combination of both. In fasted animals FGF21 reduced glucose, an effect blocked by combination with ΔN17. Interestingly, we found that in fasted animals, treatment with ΔN17 partially blocked the reduction in serum glucose one normally observes in fasted animals, suggesting ΔN17 may interfere endogenous glucose homeostasis in the fasted state . FurtherFGF19 has previously been reported to induce mitogenicity in animals Our group and others have previously reported on the efficacy of FGF21 in the treatment of obesity in animal models In an effort to compare efficacy of the factors in another model of obesity we examined the effects of chronic FGF19 and FGF21 infusion in ob/ob mice. Interestingly ob/ob mice seem to display a differential response to FGF treatment when compared to WT mice. In both the FGF19 and FGF21 treatment groups there was a significant attenuation of body mass accrual over the 7 day treatment period, furthermore, the magnitude of the effect was greater in FGF21 treated animals when compared to FGF19 treatment . FGF19 tThe therapeutic potential of both FGF19 and FGF21 in the treatment of metabolic disorders has been discussed extensively in the literature Our group and others have previously reported that the metabolic activity of FGF21 or lack of thereof is determined by the presence or absence of the cofactor KLB In 3T3-L1/KLB fibroblasts we saw FGF19 and FGF21 mediated signaling and glucose uptake with FGF21 more potent than FGF19. We did not see any effect of FGF23 in the 3T3-L1/KLB cells consistent with previous data showing specificity for KL alone In cells which predominantly express FGFR4 but do not express KLB, FGF19 was active but FGF21 was not. In the presence of KLB, both FGF19 and FGF21 can signal via FGFR4 but FGF19 appears to be significantly more potent than FGF21. This was evident in 3T3-L1 fibroblasts stably expressing FGFR4 in which FGF19 was able to act directly in the absence of KLB and induce glucose uptake, while FGF21 did not have any activity. This finding is important as while previous reports have shown binding of FGF19 to FGFR4 On a side note, while L6 cells have previously been reported to be free of background signaling due to a paucity of FGFR expression We found no synergistic or additive effect on glucose uptake when cells were treated with both FGF19 and FGF21 simultaneously. This indicates that these two factors share a common signaling pathway via which they regulate glucose transport in cell culture.in vivo ΔN17 is also able to block the glucose lowering action of exogenous FGF21 in both fed and fasted mice. In both fed and fasted ob/ob mice treated with FGF21 we see the usual glucose lowering effect we have reported previously This commonality of the two factors extended to our studies of inhibition using the competitive antagonist ΔN17 which we have previously shown is effective in inhibiting FGF21 at the receptor activation level in vitro. In the present study we observed that not only does ΔN17 inhibit downstream FGF21 signaling but also shows a similar efficacy in blocking FGF19 mediated effects. These data support the hypothesis that in cell culture models FGF21 and FGF19 operate by activation of a similar signaling cascade. Furthermore, we go on to demonstrate that in vivo administration of dN17 alone affected plasma glucose but only in the fasted state. Given the KLB antagonistic nature of ΔN17s mode of action, and the absence of effects on glucose homeostasis in a fed mice treated with the protein, we hypothesize that even though a substantial amount of FGF21 is detected in plasma of fed ob/ob mice, it is likely present in a non-functional form which is unable to interact with endogenous KLB in the manner described previously It is also important to note that As several previous studies have noted mitogenic effects in animal models following treatment with FGF19 and absence of thereof with FGF21, we examined both FGF19 and FGF21 in an in vivo setting. In our hands FGF19 dosing led to a very significant increase in proliferation in the liver while FGF21 had no effect. Our data support earlier work suggesting FGFR4 binding by FGF19 may mediate its mitogenic effects Supporting this hypothesis is the finding that treatment with FGF19 improves glucose tolerance in DIO FGFR4KO mice suggesting activation of FGFR4 is not required in the mediation of at least some of the metabolic effects of this factor To date direct comparisons of FGF19 and FGF21 treatment in animal models have not been conducted. Here we show in DIO mice both FGF19 and FGF21 have beneficial effects in the treatment of metabolic dysregulation. It has been previously demonstrated that in DIO models ranging from rodents to primates that FGF21 treatment is able to correct the abnormal metabolic parameters evoked by prolonged high fat diet feeding Here we show that the metabolic effects of treatment with either FGF19 or FGF21 are almost identical. The main difference noted was increased potency of FGF21 when compared to FGF19 in terms of its effect on weight loss. Other effects such as the glucose lowering component of their action were indistinguishable, supporting the hypothesis of a shared mechanism of action.In conclusion, our study demonstrates that the effects of FGF19 and FGF21 both in vitro and in vivo show a high degree of similarity. This interchangeability between the factors likely results from the ability of both to bind KLB and FGFRs. In mice, treatment with FGF19 and FGF21 both led to amelioration of the obese phenotype with significant improvements in all parameters tested. Our data demonstrate that both in vitro and in vivo FGF19 and FGF21 are able to potently activate the KLB/FGFR complex and that this activation likely mediates the positive metabolic outcomes we observe. Our data lend further support for further investigation of both FGF21 and FGF19 as potential therapies for obese/diabetic humans.


LIM domain-binding factor 2 (LDB2) gene in this region had the strongest association with body weight for weeks 7–12 and with average daily gain for weeks 6–12. This GGA4 region was previously reported to contain body weight QTL. GGA1 and GGA18 had three SNP effects on body weight with genome-wide significance. Some of the SNP effects with the significance of “suggestive linkage” overlapped with previously reported results.Chicken body weight is an economically important trait and great genetic progress has been accomplished in genetic selective for body weight. To identify genes and chromosome regions associated with body weight, we performed a genome-wide association study using the chicken 60 k SNP panel in a chicken F2 resource population derived from the cross between Silky Fowl and White Plymouth Rock. A total of 26 SNP effects involving 9 different SNP markers reached 5% Bonferroni genome-wide significance. A chicken chromosome 4 (GGA4) region approximately 8.6 Mb in length (71.6–80.2 Mb) had a large number of significant SNP effects for late growth during weeks 7–12. The  Body weight is an economically important trait for broiler chickens. The identification of DNA polymorphisms and causative genes affecting body weight provides necessary molecular information for marker assisted selection and gene based selection to improve quantitative traits Blood samples of chickens were collected from the brachial vein by standard venipuncture procedure #XK622, approved by the Animal Welfare Committee of China Agricultural University.The study population was the China Agricultural University chicken F2 resource population that was produced from reciprocal crosses of Silky Fowl and White Plymouth Rock which consisted of four half-sibling pedigrees. In this study, 278 individuals of three generations were included. Body weights of the 229 F2 animals were measured weekly from birth to 12 weeks of age, average daily weight gains (ADG) were calculated from birth to 6 weeks of age (ADG6) and from 6 weeks to 12 weeks of age (ADG12). Basic statistics of phenotype data are displayed in Genomic DNA extraction from blood was performed with phenol/chloroform method, and DNA concentration was diluted to 50 ng/ul. The quality and concentration of genomic DNA fulfilled the requirements for the Illumina Infinium SNP genotyping platform. Genotyping using the Illumina 60 K Chicken SNP Beadchip was carried out at the Illumina-certified service provider, DNA LandMarks Inc., Canada. Quality control was assessed in GenomeStudio v2008.1 Pairwise linkage disequilibrium (LD) measured by r2 values for the F2 population and the parental breeds  were calculated for each chromosome using PLINK (v1.07) We assessed the F2 population structure using MDS analysis available from the PLINK software. All autosomal SNPs were pruned using the indep-pairwise option, with a window size of 25 SNPs, a step of 5 SNPs, and r2 threshold of 0.2 Genome-wide association analyses were carried out in PLINK. Linear regression analyses for body weights were performed with the first MDS component, sex, batch, and birth weight as covariates. While the statistical model for ADGs included the first MDS component, sex, and batch as covariates. Measures of SNP effects were calculated by the EPISNP2 package (v3.4) 2 values exceeding 0.40. Using this approach, the estimated number of independent SNP markers and LD blocks was 25,941, so that the threshold P-value of the 5% Bonferroni genome-wide significance was 1.92×10−6 (0.05/25941). The threshold P-value for the significance of “suggestive linkage” that allows one false positive effect in a genome-wide test −5 (1/25941). Empirical genome-wide P-values were obtained from 25,000 permutations for each SNP using the maxT function in PLINK.The threshold P-value of the 5% Bonferroni genome-wide significance was calculated based on the estimated number of independent markers and LD blocks for autosome markers 2<0.2 using the first two MDS components  showed that a chicken  chromosome 4 (GGA4) region was strongly associated with body weight for weeks 7–12 and with average daily gain for weeks 6–12. A total of 26 SNP effects involving 9 different SNP markers reached 5% Bonferroni genome-wide significance under the LD conditions , and 19 of these 26 SNP effects reached 5% empirical genome-wide significance from permutation tests  gene had the strongest association with late growth (body weights from 7 to 12 weeks of age and ADG12 from 6 to 12 weeks of age). LDB2 is capable of binding to a variety of transcription factors, and is of vital importance during brain development and blood vessel formation TBC1D1 gene was highly significant for body weight at 12 weeks of age. TBC1D1 was reported to be a candidate gene for obesity in humans TBC1D1 haplotype has been under selection during domestication in broiler chickens LOC769270 gene had strong association with late growth (body weights from 11 to 12 weeks of age and ADG12). LOC769270 is a hypothetical protein coding which was bioinformatically predicted in chicken only.The oculocutaneous albinism II (OCA2) gene had highly significant effects on body weight in weeks 11–12. The association between OCA2 and body weight in chicken was the first report in this study but the SNP effect in OCA2 overlapped with a reported body weight QTL region detected in intercrossed lines involving White Plymouth Rock background OCA2 gene is associated with body weight and body size in mouse OCA2 gene could be relevant to growth traits.One SNP on GGA1 in the For early growth traits, only one SNP  on GGA18 had significant association with body weight at 2 weeks of age. The lack of SNP effects on early growth traits could be due to epistatic interaction that may explain more of the genetic variance of early growth than single gene effects −5)  . These eglypican 6 (GPC6) gene, glypican 5 (GPC5) gene, and gga-mir-17-92 cluster, and is located within the QTL for bodyweight identified in previous studies using the same F2 population as in this study Two SNPs located at 151 and 152.3 Mb on GGA1 had effects on body weight in weeks 11–12 and ADG12. This region harbors Popeye domain-containing protein 1 (POPD1) gene on GGA3 had effects on body weight in weeks 10–12 and ADG12, and a polymorphism (Gga_rs15178951) 31 kb downstream of BMP7 gene on GGA20 had effects on body weight in weeks 11–12. They overlapped with QTL regions reported by two studies A SNP (Gga_rs14373757) within the A previous study DYNC1I1 located at 23.9 Mb on GGA2 was associated with body weight in week 6 and ADG6, and two SNPs both located within the Opioid-binding protein/cell adhesion molecule-like (OPCML) gene on GGA24 were found to be associated with body weight in week 12 and ADG12. A SNP (Gga_rs14269721) within the gene Cbfa2t2 on GGA20 was in association with body weight in week 12 and ADG12. This is a new QTL identified in this study only.Other previous studies In summary, our GWAS detected 26 SNPs with genome-wise significance and 128 SNPs with the significance of suggestive linkage. Most of these SNPs were reported for the first time. Many of the SNP effects overlapped with previously reported QTL regions, providing evidence towards confirmation of QTL effects. The results are also helpful for identifying the exact QTL locations because of the much improved map resolution of the 60 k SNP panel over the map resolution of microsatellite markers used by most of previous reports on chicken QTL effects.Figure S1Manhattan plot of genome-wide association analysis for body weight traits. The dashed line indicates genome-wise significance of suggestive association , and the solid line declares genome-wise 5% significance with a p-value threshold of 1.92×10−6.(PDF)Click here for additional data file.Table S1Associated SNP with genome-wise significance of suggestive association for body weight traits.(XLS)Click here for additional data file.


There is an error in Figure 4. The correct version of Figure 4 can be seen here: [^] Also, in reference to this correction, in the tenth paragraph of the Results and Discussion section, the sentence "The absence of any interaction with the P3 side-chain explains the diversity of residues (Q|G|K|N|E) observed at this position in MNV cleavage junctions [14], a feature that is also shared by picornavirus 3Cpro cleavage junctions [29], [30]" should correctly say "The absence of any interaction with the P3 side-chain explains the diversity of residues (Q|G|H|N|E) observed at this position in MNV cleavage junctions [14], a feature that is also shared by picornavirus 3Cpro cleavage junctions [29], [30]."


To the Editor: After the emergence of pandemic (H1N1) 2009 virus, measures for its control were taken quickly  when a clinical suspicion of pandemic influenza was established  2009 virus had been made in the respondent’s household.>20×/d. Three workers were vaccinated against seasonal and pandemic influenza, while only 1 was vaccinated against pandemic (H1N1) 2009 alone. None took oseltamivir. Five positive samples were identified (13.8% of the study population) being obtained from four doctors and one nurse, all women. The 4 doctors had signs and symptoms for 24–48 hours consisting of fever, general indisposition, and coughing; none of the 4 required hospitalization. The nurse was a woman 26 years of age with no influenza symptoms and with a positive PCR result on week 5. None of these 5 workers had received any influenza vaccination.At the outset, 60 members of the hospital staff volunteered to participate. Those who missed >4 sample tests, or >2 consecutive ones, were considered to have abandoned the study. Of the 36 staff members who completed the study, 27 were women (75%). The participants’ average age was 37 years (CI 95%: 34.8–39.4). Sixteen were doctors, 16 were nurses, 2 were nursing auxiliary staff, and 2 were hospital orderlies. During the monitoring period, 5 (13%) subjects exhibited coughing, 7 (20%) had runny noses, 3 (8%) experienced painful swallowing, 6 (16%) had headaches, and 1 (2%) felt generally unwell. Nearly 75% stated they washed their hands with antiseptic lotion Three workers reported that a diagnosis of pandemic (H1N1) 2009 influenza had been made with respect to a member of their household, but none of the workers had a positive PCR result. The distribution of positive PCR results in our hospital during the study is shown in the It had previously been hypothesized that the incidence of asymptomatic cases would be higher than the incidence of symptomatic cases  2009 during the study period.Our study began during the week in September 2009 in which the overall rate of incidence of pandemic (H1N1) 2009 in Spain reached 77.8 cases per 100,000 inhabitants  had been vaccinated against the novel form of the influenza A virus, and none of them had positive PCR results for pandemic (H1N1) 2009 virus. On the other hand, 5 (15%) of workers not vaccinated had a positive PCR result. This finding suggests that, despite the climate of uncertainty concerning the evolution of the influenza outbreak, hospital workers had a greater fear of possible side effects of the vaccine than of the disease itself.