Within mouse forebrain, a subset of microRNAs are significantly enriched in synaptoneurosomes (a synaptic fraction containing pinched-off dendritic spines) and a subset are significantly depleted relative to total forebrain homogenate. Here I show that, as a group, the pre-miR hairpin precursors of synaptically enriched microRNAs exhibit significantly different structural features than those that are non-enriched or depleted. Precursors of synaptically enriched microRNAs tend to have a) shorter uninterrupted double-stranded stem segments, and b) more symmetrical bulges containing a single nucleotide on each side. These structural differences may provide a basis for the differential binding of proteins that mediate dendritic transport of pre-miRs, or that prevent pre-miRs from being prematurely processed into mature miRNAs during the transport process.This article was reviewed by I. King Jordan and Jerzy Jurka. The brain expresses a wide variety of miRNAs, some of which show regional and cell type specificity -6. miRNAA recent experimental study of adult mouse forebrain reported the expression of miRNAs in synaptoneurosomes (SYN), a synaptic fraction that is enriched in pinched-off dendritic spines . A signiThese experimental findings suggest that mature miRNAs are formed, at least in part, via processing of pre-miRs locally within dendritic spines ,11. As wAs shown in the present report, the set of SYN-enriched miRNAs do exhibit several structural features that distinguish them from miRNAs that show no enrichment, or that are depleted in synaptic fractions relative to the total forebrain homogenate. This provides independent support for the pre-miR selective transport hypothesis, and suggests a basis for differential interaction of pre-miRs with transport complexes.In our previous study, synaptoneurosomes were prepared, characterized and assayed for miRNA expression as described . MiRNAs Each miRNA (and the predicted folding of its pre-miR) was looked up in miRBase   did not differ significantly in the "top" set (13.9 nt) vs. the "depleted" set (14.1 nt), and multiple sequence alignments (of the entire pre-miR sequence or of loop sequences) failed to reveal any recurring motifs characteristic of the "top" set (not shown). When the "top" set was compared to the "depleted" set, a similar proportion of miRNAs arose from the 5' arm of the pre-miR (8/17 vs. 7/15) and a similar proportion of the miRNAs began with a U (11/17 vs. 8/15). Moreover, the first nucleotide of the mature miRNA was found on a bulge  encoding the mature miRNA. The total length of stem segments in zone 2 was not significantly different across groups (17.94 nt in "top" set vs. 18.53 nt in "depleted" set). However, the longest uninterrupted stem segment present within the pre-miR was 3 nucleotides shorter on average in the "top" set (8.41 ± 0.68) than in the "depleted" set (11.53 ± 1.11), p = 0.02 . The combined SYN-enriched set exhibited 2.1 bulges of this type (1-1) per pre-miR on average, whereas those in the combined non-enriched set contained only 1.1 bulge of this type . FMRP has been implicated in transporting mRNAs to dendrites -16 and cBulges along the hairpin not only serve to interrupt stems but may play a positive role in transport by providing discrete single stranded "handles" for RNA binding proteins. The 1-1 bulge is identified here, for the first time, as a specific feature that may have biological significance, since the increased number of bulges observed in the enriched sets were accounted for almost entirely by 1-1 bulges. Nearly all (34 of 36) SYN-enriched miRNAs arise from pre-miRs that express at least one 1-1 bulge.Selective transport of pre-miRs is likely to be only one of several factors determining the synaptic enrichment of mature miRNAs near synapses, due to the presence of additional biological events that regulate pre-miR processing ,11,17-20miRNA: microRNA; Pre-miR: microRNA hairpin precursor; SYN: synaptoneurosomes.The author declares that they have no competing interests.Smalheiser statistically compared the hairpin precursor structures of miRNAs (pre-miRs) that were previously found to be enriched in synaptoneurosomes to those that are non-enriched or depleted. He finds that pre-miRs enriched in synaptoneurosomes have shorter uninterrupted stem segments and more symmetrical bulges, on average, compared to the non-enriched or depleted set. These structural differences are taken as support for the hypothesis that the synaptically enriched pre-miRs are preferentially transported to dendrites.This manuscript addresses an interesting and open question in miRNA biology. I have two main concerns regarding the data reported here and their interpretation. First of all, the structural differences between pre-miR populations analyzed here appear to be slight, and I am not yet convinced that these structural differences are statistically significant. Second, it is not clear how these structural differences lend support to the preferential transport hypothesis.1. The author uses a parametric statistical test, the Student's t-test, to compare the structural features of pre-miRs. It is not clear whether the underlying distributions of structural features analyzed justify the use of the t-test. Furthermore, the differences revealed between sets appear to be slight and in some cases only marginally significant. It would be more conservative to use analogous non-parametric statistical comparisons, such as the Mann-Whitney U test, of these features. If both parametric and non-parametric methods yield the same results, they will be more convincing. Another more conservative approach would be to simply use simulation by randomly building sets of the same size as the top, depleted etc and measuring differences between the structural features of the simulated sets to get a distribution of differences from which to compute a test statistic. In general, since the sample sizes and differences reported here are small, there is a substantial burden of proof regarding the significance of the structural differences.2. Different pre-miRs show different quantitative levels of enrichment. For the purposes of the statistical analysis conducted here, enrichment bins were used that grouped pre-miRs with similar enrichment or depletion levels. I couldn't help but wonder whether there was any more continuous relationship between pre-miR structural features and enrichment levels. For instance, are the number of stem segments positively correlated with enrichment or are the longest stem segment lengths negatively correlated?e.g. preferential processing)? Is there some reason why the structural features in the enriched set would yield pre-miRs that are preferentially transported? For instance, is there any experimental evidence which suggests that pre-miRs possessing the structural characteristics of the enriched set are preferentially bound by RNA-binding proteins such as FMRP? Is there any reason to expect that slightly shorter contiguous stems and a few more bulges would yield preferential transport?3. The direct support for the preferential transport hypothesis provided by the data in this manuscript is tenuous. The seemingly best evidence for such a hypothesis, namely the correlation between relative synaptic enrichment of pre-miRs and corresponding mature miRNAs, was previously published by this same group. There does not appear to be any known, or even putative, mechanistic connection between the structural differences in enriched versus depleted pre-miRs and preferential transport. In fact, according to the author's own statements, while there is evidence that RNA-binding proteins associate with pre-miRs, there is no evidence that pre-miRs are directly associated with transport complexes. In light of the multiple levels of neuronal miRNA processing regulation, is there any reason that the structural differences uncovered here favor one mechanism (transport) over another  are the ones that get transported. I now acknowledge that the syn enriched mirs are also expressed at lower levels than the syn depleted mirs, which might also reflect the same pre-mir structural differences. But I do not see this as a matter of regulating expression OR regulating transport – I think that the pre-miR structural features are involved in both processes in a unified way. In the revised version, I discuss that there should be a trade off between pre-miR structures that foster rapid dicer binding and cleavage, vs. those that foster incorporation into transport complexes in which cleavage is prevented. Some proteins may actually prefer to bind shorter stems and/or may bind 1-1 bulges.5. I also rewrote the final section where I propose specific predictions.The author uses a statistical approach to demonstrate structural differences between miRNAs enriched in a synaptic fraction of the mouse brain (SYN), relative to the miRNAs in the total forebrain homogenate. He proposes that the distinct structural features  may be involved in transportation of precursors of the SYN-enriched microRNA to dendrites, by a number of hypothetical mechanisms.The hypothesis may be useful for experimental researchers in the field but its presentation is somewhat confusing for a general reader. Specifically, the discussion should be shortened and rephrased to clearly differentiate between predictions based on this study and conclusions based on previously published studies. In the current version there is a disagreement between the abstract and the concluding section.I shortened the Discussion and rewrote the abstract.Scoring scheme for pre-miRs. Contains the raw data used to score pre-miR features.Click here for file


Loss of DNA mismatch repair has been observed in a variety of human cancers. Recent studies have shown that loss of DNA mismatch repair results in resistance to cisplatin but not oxaliplatin, suggesting that the mismatch repair proteins serve as a detector for cisplatin but not oxaliplatin adducts. To identify the signal transduction pathways with which the detector communicates, we investigated the effect of loss of DNA mismatch repair on activation of known damage-responsive pathways, and recently reported that cisplatin differentially activates c-Jun NH2-terminal kinase (JNK) and c-Abl in repair-proficient vs.-deficient cells. In the current study, we directly compared differential activation of these pathways by cisplatin vs. oxaliplatin. The results confirm that cisplatin activates JNK kinase 5.7 ± 1.5 (s.d.)-fold more efficiently in DNA mismatch repair-proficient than repair-deficient cells, and that the c-Abl response to cisplatin is completely absent in DNA mismatch repair-deficient cells. In contrast, there was no detectable activation of the JNK or c-Abl kinases in DNA mismatch repair-proficient or -deficient cells exposed to oxaliplatin. The present study demonstrates that, despite the similarity of the adducts produced by cisplatin and oxaliplatin, they appear to be recognized by different detectors. The DNA mismatch repair system plays an important part in the recognition of cisplatin adducts, and activation of both the JNK and c-Abl kinases in response to cisplatin damage is dependent on the detector function of the DNA mismatch repair proteins. In contrast, this detector does not respond to oxaliplatin adducts. © 1999 Cancer Research Campaign


Nigella sativa with anti cancer acitivity. In the present study we investigated the efficacy of Organic extracts of Nigella sativa seed powder for its clonogenic inhibition and induction of apoptosis in HeLa cancer cell.Cancer remains one of the most dreaded diseases causing an astonishingly high death rate, second only to cardiac arrest. The fact that conventional and newly emerging treatment procedures like chemotherapy, catalytic therapy, photodynamic therapy and radiotherapy have not succeeded in reverting the outcome of the disease to any drastic extent, has made researchers investigate alternative treatment options. The extensive repertoire of traditional medicinal knowledge systems from various parts of the world are being re-investigated for their healing properties. This study progresses in the direction of identifying component(s) from Nigella sativa seedz effectively killed HeLa cells. The IC50 values of methanolic, n-hexane, and chloroform extracts of Nigella sativa were 2.28 μg/ml, 2.20 μg/ml and 0.41 ng/ml, respectively. All three extracts induced apoptosis in HeLa cells. Apoptosis was confirmed by DNA fragmentation, western blot and terminal transferase-mediated dUTP-digoxigenin-end labeling (TUNEL) assay.Methanolic, n-Hexane and chloroform extracts of Nigella sativa seed extracts regulated the expression of pro- and anti- apoptotic genes, indicating its possible development as a potential therapeutic agent for cervical cancer upon further investigation.Western Blot and TUNEL results suggested that  Around one quarter of deaths in the US are accounted for by Cancer. According to the American Cancer Society, on an average, 559,312 people die of the disease each year . In a scUnani, Ayurveda & Siddha and the Chinese system of medicine that have originated from the Arab, India and China respectively. These medicinal systems have identified more than 700 individual herbal extracts as well as several drug preparations which claim to treat and/or prevent several diseases including cancer [There are a number of alternative medicine systems based on traditional theories and philosophy that have originated in specific geographical areas and evolved over the years. The most widely practiced of these include the g cancer .Apoptosis an active physiological process resulting in cellular self-destruction of unwanted cells is absent in cancer cells. Apoptosis is characterised by distinct morphologic changes, including cell shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation, and the formation of apoptotic bodies . Bcl-2 fNigella sativa seed (NS) for testing its in vitro apoptotic activity. It is an annual herb belonging to the family Ranunculaceae and is commonly known as black cumin and is a natural food additive in India and many parts of Asia. The seeds of NS are the source of active ingredient of this plant. In the Unani system, it is regarded as one of the greatest forms of healing medicine available.In present study we chose to investigate the extracts of The bioactivity of the NS seeds has been investigated both in vitro as well as in vivo for a number of therapeutic properties like antioxidant, antihistaminic, anti inflammatory, immune-modulatory, anti viral, anti helimenthic, anti bacterial  and its L and of the pro-apoptotic proteins caspase-9, caspase-8 and caspase-3 and p53. The results led us to hypothesize that certain bioactive compounds in NS seeds might be capable of effectively causing death of cancer cells by up-regulating the expression of certain pro-apoptotic gene and simultaneously down-regulating the expression of anti-apoptotic genes.Cervical cancer forms an important part of cancer sub-types among women and is more common in Hispanic and African-American women than in Caucasians. It is the fifth most common type of cancer among women with 471,000 new cases each year with 11,070 cases in US in 2008 and 3,870 deaths according to the data released by the National Cancer Institute. Considering the significance of this cancer sub-type accounting to a death every 2 minutes, we used HeLa cell lines to analyze the anticancer activity of the NS seed extract and sought to investigate the ability of NS to induce apoptosis and to determine the biochemical mechanisms underlying apoptosis in HeLa cells. To determine the species' antiproliferative activity, we examined the effects of various solvent fractions on cell viability by clonogenic inhibition assay. Induction of apoptosis was assessed by DNA fragmentation, TUNEL assay and western blot for the expression of the anti-apoptotic protein Bcl-2 & bcl-X50 2.28 μg/mL) ,   Fig , n-hexanmL), Fig  and chlo/mL) Fig  signific50 concentration of NS seed extracts as follows: chloroform extract at 0.41 ng/ml, the n-hexane extract at 2.20 μg/ml and the methanolic extract at 2.28 μg/ml to determine whether the bioactive compounds of NS seeds induced HeLa cell death via apoptosis or by necrosis. HeLa cells treated with these extracts showed active apoptosis after 24 hours of treatment; the increase in apoptosis over the untreated population was 21.1% in methanolic extract treated cells, 30% n-hexane extract treated cells and 42% chloroform extract treated cells . HeLa cells were treated with respective ICp53, caspase-3, -8 and -9 was higher in chloroform extract treated cells, followed by n-hexane and methanolic extracts treated cells compared to respective controls. However bcl-2 and bcl-XL were down regulated in n-hexane extract treated cells compared to chloroform and methanolic extract treated and untreated cells , USA, although most of these drugs that claim to cure various ailments are yet to be validated scientifically. According to Parasnis , the fulScreening of medicinal plants for potential anticancer properties has increased greatly over the past few decades. For instance, the US National Cancer Institute has implemented a large-scale project of acquisition and screening of compounds isolated from medicinal plants originating in various parts of the world. These medicinal plants are identified based on ethnopharmacological, chemosystemic and ecological information.Therefore, there is a need for more effective anticancer agents since the most common tumors in adults are resistant to almost all presently available anticancer drugs and the majority of the available drugs have limited anti-solid tumor activity .XL expression was significantly inhibited while p53 and caspase -3,-8 &-9 expression was markedly increased in a concentration-dependent manner. Our results suggest that the NS induced apoptosis by regulating apoptotic genes.Apoptosis is an active physiological process resulting in cellular self-destruction that involves specific morphological and biochemical changes in the nucleus and cytoplasm . Agents N. sativa used apoptosis as their mode of cell death. This was investigated by studying distinct morphological features  and molecular features .The mechanism of action of many anticancer drugs is based on their ability to induce apoptosis ,34. Hencviz. methanol, chloroform and n-hexane. This was to reduce the concentration of the NS seed components in each extract, so that the potent compound(s) can be identified. These extracts regulated the expression of specific target genes. It was found that the chloroform extract led to over expression of pro-apoptotic genes while n-hexane and methanolic extracts led to down regulation of anti-apoptotic genes when compared to respective controls. After sufficient analysis, we could conclude that the NS extracts contain potent component(s) which have the ability to induce apoptosis through caspase and p53 pathway in HeLa cells. However, further studies are required to find the exact compound(s) and their structure(s) as well as to find whether the same extract/compound(s) are also capable of regulating the expression of the same genes in other cancer cell types. In addition, it was observed that the n-haxane extract down- regulated the expression of bcl-2 and bcl-XL and the methanolic and chloroform extract inhibited the expression of bcl-2, though to a smaller extent.The NS seed powder was subjected to three different organic extractions with increasing polarity Efforts towards finding a practical solution in combating this dreadful disease could prove to be of paramount importance. There is no paucity on the availability of umpteen numbers of medicinal plants known to regional communities practicing indigenous healing techniques, which can on scientific evaluation provide an alternative to the presently available options that do not go without severe and painful side-effects. Many herbs have been evaluated in clinical studies and are currently being investigated phytochemically to understand their tumouricidal action against the insidious nature cancer.Ayurvedic therapy has been found to cure these chronic diseases like caner better, which are previously not amenable to treatment by western medical practices . This trMadagascar periwinkle, Vinca rosea), irinotecan (from Camptotheca accuminata) and paclitaxol (from the Pacific yew tree Taxus brevifolia). Future research in this area would help in the identification of safe and effective anticancer drugs.Herbal decoctions consisting of multiple herbs each possessing tremendous potential for a cancer cure are commonly used in Ayurveda. These formulations are reported to work on multiple biochemical pathways and are capable of influencing several organ systems simultaneously. The benefit of a herbal decoction is that it can nourish the body as a whole by supporting various organ systems. More importantly as it has no synthetic part to it, there is very less likelihood of an unexpected adverse effect. Many of the herbs have been scientifically-proven for anti-cancerous properties and are used for the treatment of various cancers ,37. TheyHowever, the potent extracts should be tested on several other cancer cell lines, biochemical and molecular studies carried out using the extracts in animal models to establish their therapeutic efficacy as well as toxicity, and the most potent extracts subjected to HPLC and GC&MS analyses to identify and characterize the efficacious phytotherapeutic and/or bioactive compound(s) in NS.n-hexane, Chloroform, using the Soxhlet apparatus, as represented in the flow chart , Pune, India. Cell lines were maintained and propagated in 90% Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were cultured as adherent monolayer and maintained at 37°C in a humidified atmosphere of 5% COCell viability was assayed where and as required by trypan blue exclusion test  with sliThe Colonogenic inhibition assay was performed as described previously by us . Briefly(Where PE is the plating efficiency).50 value (the concentration of the drug which is capable of bringing about 50% inhibition of colony formation) of the drug used for treatment was determined by plotting a graph with Survival fraction (Y-axis) against the concentrations of the extracts (X-axis) [Fig The ICxis) Fig  &1C.50) of each extract for 24 hours and then harvested. Genomic DNA was extracted by standard salting out method with slight modification [HeLa cells were treated with respective concentration  was used as per the instructions of the manufacturer. The ApopTag kit detects DNA strand breaks in single cells by terminal transferase-mediated dUTP-digoxigenin-end labeling (TUNEL). Briefly, cells were seeded in chamber slides and the next day they were exposed to each organic extracts. After 24 hrs the DNA was tailed with digozigenin-dUTP and cojugated with an anti-digoxigenin fluorescein. The cells were then counterstained with propidium iodide and antifade. The stained cells were observed using Carl-Zeiss epifluorescence microscope using triple band-pass filter. To determine the percentage of cells indicating apoptosis, a total of 1000 cells were counted in each experiment.L antibodies were used at a 1:1000 dilution as the primary antibodies, while horseradish peroxidase-conjugated horse anti-rabbit IgG  was used at a 1:5000 dilution as the secondary antibody. The membrane was then exposed and protein bands were detected using Enhanced Chemiluminescence.After treatment, the cells were collected and washed twice with cold PBS. The cells were then lysed in lysis buffer  and kept on ice for 30 min. The lysates were then centrifuged at 12,000 g at 4°C for 20 min; the supernatants were stored at -70°C until use. The protein concentration was determined by the Bradford method. Aliquots of the lysates (30 μg of protein) were separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane using transfer buffer . After blocking with 5% non-fat dried milk, the membrane was incubated for 2 h with primary antibodies, followed by 30 min with secondary antibodies in milk containing Tris-buffered saline (TBS) and 0.5% Tween. Anti-human p53, caspase-3, caspase-8, -caspase-9, -Bcl-2, and bcl-XThe results of each series of experiments (performed in triplicates) are expressed as the mean values ± standard deviation of the mean (SD).Nigella sativa; NCCS: National Centre for Cell Science; DMSO: Dimethyl Sulfoxide; NCAM: National Council for Alternative Medicine; FBS: Fetal Bovine Serum; DMEM: Dulbecco's Modified Eagle's Medium;TUNEL: terminal transferase-mediated; dUTP: digoxigenin-end labeling; CAM: Complementary and alternative medicine; NS: The authors declare that they have no competing interests.GS is a researcher working in cancer biology and carried the overall study. GS and TNH undertook the Statistical analysis. AM along with JA and DKYL designed the work and interpreted the results. AM, GS and AAA contributed to the writing of the manuscript, which was edited, revised critically and co-ordinated by DKYL. All the authors read and approved the final manuscript.


Prefrontal and cerebellar abnormalities have been associated with higher cognitive deficits in schizophrenia. The current study aimed to show whether or not schizophrenic patients with fronto-cerebellar functional abnormalities show more anhedonia or ambivalence.Regional cerebral metabolic activity was measured using fluoro-D-glucose positron emission tomography and was compared between 24 patients with chronic schizophrenia and 22 healthy normal volunteers. The existence of regional prefrontal hypofunction and regional cerebellar hyperfunction was investigated in each patient. Demographic and clinical variables including the emotional self-report scales were compared between the subgroups of the patients categorized according to the existence and the absence of the regional dysfunctions.Comparisons between each patient and the total normal controls revealed that 14 of the total twenty-four patients had regional hypofrontal functions, whereas 11 patients had regional hypercerebellar functions. Patients with prefrontal hypofunction showed more severe anhedonia than those without prefrontal hypofunction, whereas patients with cerebellar hyperfunction compared to those without cerebellar hyperfunction had more severe ambivalence.It seems that fronto-cerebellar abnormalities may be associated with cardinal emotional features of schizophrenia, such as anhedonia and ambivalence. Schizophrenia is a debilitating disease with heterogeneous phenomenology and diverse symptoms.However, patients with schizophrenia have a deficit in the ability to experience positive emotions related to future activities (anticipatory pleasure) but experience normal levels of positive emotions when directly engaged in pleasant activities (consummatory pleasure).The prefrontal cortex and the cerebellum in the human are two brain regions that are massively larger by approximately one-third when compared to primates that lack capacities for higher cognitive functions.9Abnormalities in higher cognitive functions, hallmarks of schizophrenia, have been consistently connected to disrupted functioning of the prefrontal cortex and the cerebellum.Abnormalities in higher cognitive functions in schizophrenia are associated with prefrontal hypofunction rather than hyperfunction, and cerebellar hypofunction18F-FDG-PET) in each individual patient when compared with total normal controls in order to predict those who would have abnormal functions in the prefrontal cortex and the cerebellum. We then compared the severities of anhedonia and ambivalence between patients with and without prefrontal hypofunction, and between patients with and without cerebellar hyperfunction. We hypothesized that the severities of anhedonia and ambivalence would be significantly different between the patient subgroups.Therefore, we investigated abnormal regional brain functions using 2-deoxy-2-18F-fluoro-glucose-positron emission tomography , and had been in follow-up for more than two years. They fulfilled the DSM-IV criteria for schizophrenia, as diagnosed using the Structured Clinical Interview for DSM-IV (SCID).Each group contained the same number of men and women, and all participants were right-handed. The mean ages of the patient and normal control groups were 30.5 years  and 30.3 years , respectively. The mean years of education were 12.8 (SD=2.0) and 13.0 years (SD=1.6), respectively. Age and educational background were not significantly different between the groups. At the time of positron emission tomography (PET) scanning, the mean duration of illness in the patient group was 8.4 years (SD=3.8). All patients received either one or two neuroleptics, including risperidone, olanzapine, clozapine, aripiprazole and haloperidol, with a mean chlorpromazine- equivalent doseSymptom severity of the patients was assessed using the Positive and Negative Syndrome ScaleImmediately after PET scanning, all subjects reported their own emotional tendencies by filling out four different self-report scales such as the physical and social anhedonia scales,18F-FDG. Afterwards, an 8-min transmission scan was performed using Ge-68 rod sources to correct for attenuation. The emission scan continued in 3-dimensional mode for 15 minutes. Gathered data were then reconstructed in a 128×128×35 matrix with a pixel size of 1.95×1.95×4.25 mm using a 3-dimensional filtered back-projection algorithm employing a transaxial 8.5 mm Hanning Filter and an 8.5 mm axial Ramp Filter.21PET images were acquired using a GE ADVANCE PET scanner  which had an intrinsic resolution of 4.8 mm full width at half maximum (FWHM) and a slice thickness of 4.25 mm for a longitudinal field of view of 15.2 cm. Subjects were asked to lie still with their eyes closed in a quiet, dimly lit room for 40 minutes after receiving an intravenous injection of approximately 185 MBq (5 mCi) of Spatial pre-processing and statistical analyses were performed using Statistical Parametric Mapping . All reconstructed images were spatially normalized into the MNI  standard PET template to remove the inter-subject anatomical variability. An affine transformation was performed to determine the 12 optimal parameters in order to register the brain on the template. Subtle differences between the transformed image and the template were removed by the nonlinear registration method using the weighted sum of the pre-defined smooth basis functions involved in discrete cosine transformation. Spatially normalized images were smoothed by convolution with an isotropic Gaussian kernel with 14 mm FWHM to increase the signal-to-noise ratio and accommodate for variations in subtle anatomical structures.2-tests for categorical variables or independent t-tests for continuous variables. We used the Statistical Package for the Social Sciences (SPSS) , and significance of the differences was accepted when p<0.05 (two-tailed) in the analyses.The effects of global metabolism were removed by normalizing the count of each voxel to the mean count of the brain . In order to demonstrate abnormalities in each case, comparisons were made between each patient and the 22 total normal controls. Significant decreases and increases of the adjusted regional cerebral metabolism were obtained using t-statistics at every voxel. To facilitate interpretation, the t-values were transformed to Z-scores in the standard Gaussian distribution. The clusters consisting of a minimum of 50 contiguous voxels with the threshold of an uncorrected p<0.001 were considered to be significant. According to the a priori hypothesis that there would be prefrontal hypofunction and cerebellar hyperfunction in patients with schizophrenia, the existence or the absence of regional abnormal clusters in the prefrontal cortex and in the cerebellum were categorized into subgroups using the Talaraich coordinate system.As demonstrated in Our unique approach, in which each case and the total normal controls were compared, demonstrated that only 6 patients (25.0%) had both regional hypofrontal and hypercerebellar functions, whereas 5 patients (20.8%) had no abnormalities in these brain regions. It is well known that schizophrenia is a heterogeneous disorder. Previous studies have reported inconsistent brain activities in patients with schizophrenia during the resting state and even during the task-stimulating condition.The patients with prefrontal hypofunction compared to those without prefrontal hypofunction showed more severe physical anhedonia, whereas the patients with cerebellar hyperfunction relative to those without cerebellar hyperfunction exhibited more severe ambivalence . These r25In fact, patients with schizophrenia had a normal internal experience of positive emotion in the moment (consummatory pleasure), but they failed to anticipate or expect that future events would elicit these emotions (anticipatory pleasure).On the other hand, core roles of the cerebellum in the higher cognitive functions include rapid, online error detection, modulation, and coordination, culminating in the production of mental representations in a fine-tuned pattern. The cerebellum is activated in a variety of mental activities including facial recognition, emotional attribution, theory of mind, directed attention and many types of memory.A limitation of our study was that the patients with schizophrenia were all medicated with antipsychotics. There are reports that antipsychotic medications affect cerebellar and even prefrontal functions.In conclusion, this study found that the patients with prefrontal hypofunction tended to have more severe anhedonia than those without prefrontal hypofunction, whereas the patients with cerebellar hyperfunction tended to show more severe ambivalence than those without cerebellar hyperfunction. Therefore, these results support the notion that prefronto-cerebellar abnormalities are associated with cardinal features of schizophrenia, such as anhedonia and ambivalence.


The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.To monitor trafficking of the V-ATPase in the phagocytic pathway of We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway. The enzyme is divided into two sectors, each containing multiple subunits. The peripheral V1 complex is responsible for ATP hydrolysis, and the membrane-spanning V0 complex is responsible for proton translocation. The proton gradient generated by the V-ATPase is used to energize the uptake of small molecules such as neurotransmitters, to regulate extracellular pH, and to acidify the lumen of organelles. Acidification of endosomes and phagosomes by the V-ATPase mediates receptor recycling, activation of lysosomal enzymes, and killing of pathogens. There are several means of regulating V-ATPase activity, including control of V-ATPase subunit expression, intracellular targeting and recycling of V-ATPase-containing vesicles, reversible dissociation of the V0 and V1 domains, and modulation of the coupling ratio between ATP hydrolysis and proton pumping The vacuolar HDictyostelium discoideum have proved to be an excellent system for analyzing the V-ATPase in the endocytic pathway Dictyostelium cells will ingest a wide range of particles including bacteria, yeast, latex beads, glass microspheres, and occasionally other cells. They can serve as hosts for a number of bacterial pathogens that also infect human cells Dictyostelium and mammalian phagocytes, including mechanisms of particle recognition, the role of the cytoskeleton in uptake, the delivery of digestive enzymes, and membrane retrieval Dictyostelium cells as a common marker of the early endosomal compartment.The professional phagocytes of PI(3)P is one of a group of phosphoinositides that specify the identity of compartments along the endocytic pathway by recruiting signaling proteins that carry specific phosphoinositide-binding modules Dictyostelium and subunit a in mammalian cells), we previously demonstrated that shortly after internalization the V-ATPase is delivered to the membrane of new phagosomes by fusion with acidic endosomes that bear the V-ATPase in their membranes Dictyostelium cells rely on phagocytosis for the acquisition of food, so the endocytic pathway is a high-throughput system that ends with the exocytosis of indigestible food residues. Although macrophages do not explicitly mimic this behavior, regulated exocytosis from the endocytic pathway occurs for mammalian cells in several contexts such as healing a torn cell surface Using a fusion of GFP to the large trans-membrane subunit of the V-ATPase , a pH-sensitive fluorophore, to the yeast surface.In the present study, we have employed VatM-GFP to visualize trafficking of the V-ATPase in the endocytic pathway of living Dictyostelium.In live-cell studies of endocytic trafficking, early events are relatively easy to capture because they occur promptly and frequently when a population of phagocytes is presented with appropriate target particles. Thus, we have previously shown that shortly after uptake new bacteria-containing phagosomes are transported along microtubules to the cell center, where they fuse with acidic endosomes (presumptive lysosomes) and become acidified themselves  about five or six minutes after uptake Dictyostelium discoideum strain AX2–214 was transformed by electroporation using vectors for the expression of mRFPmars-LimEΔcoil Dictyostelium was constructed as follows. A SmaI-XhoI 2FYVE restriction fragment was excised from the plasmid pEGFPC1-2FYVE Dictyostelium expression vector pTX-GFP D. discoideum strains were cultivated axenically at 22°C in nutrient medium containing selective agents (G418 and blasticidin) for maintenance of the plasmids. For phagocytosis experiments with living yeast cells, Saccharomyces cerevisiae strain TH2-1B whi5Δ::Rkan) 2HPO4 (pH 9.4). FITC (Sigma F7250) was added to a final concentration of 1 µg/ml, and the suspension was incubated with shaking for 30 minutes at 37°C. The labeled yeast were washed twice with 50 mM Na2HPO4 and twice with 17 mM KH2PO4/Na2HPO4 buffer, pH 6.0 (PB), before being added to Dictyostelium cells. One experiment made use of heat-killed yeast, which had been prepared by boiling for 30 minutes a stock purchased from Sigma (YSC-2); the heat-killed yeast were stored frozen. All yeast cells were washed in PB prior to being added to the D. discoideum cells.r = 66,000) for 3 hours to label all endosomal compartments; yeast were present throughout this incubation. The chamber in which the cells were plated was rinsed to remove the TRITC-dextran immediately before viewing.For certain experiments, cells were incubated in one-third strength HL5 containing 0.75 mg/ml TRITC-dextran  or 2 to 6 hours later (for exocytosis experiments), excess yeast were removed, and the cells were overlaid with a thin layer of agarose For microscopic observation, For some experiments, confocal images were collected using a Zeiss LSM510 laser scanning confocal microscope equipped with a Plan-Apochromat 63x, 1.4 NA DIC objective. Images were acquired at 3.9-second intervals unless otherwise indicated. S65T-GFP was excited with the 488-nm line of an argon laser with a 505–530-nm filter for emission, and mRFP was excited with the 543-nm line of a HeNe laser, with a 560-nm long pass filter for emission. An HFT UV/488/543/633 beam splitter was used.Dictyostelium are illustrated in Early stages of phagosome processing in Dictyostelium cells expressing VatM-GFP as the sole fluorescent protein were incubated with TRITC-dextran to label endocytic compartments. Earlier lower-resolution live cell microscopy studies had shown that fusion generates a compartment that contains both the endosomal marker and the phagocytosed particle To investigate the origin of the vesicles that delivered VatM, Dictyostelium cells expressing moderate levels of GFP-2FYVE, phagocytosis and macropinocytosis proceeded normally, as shown in The delivery of VatM immediately after removal of the actin coat from the phagosome membrane prompted us to define the compartment of the endosomal pathway in which the V-ATPase is acquired. For this purpose we utilized GFP-2FYVE to detect PI(3)P, the phosphoinositide that identifies early endosomes. In E. coli. The proclivity of early endosomes to undergo fusion and fission is evident in the expanded volume and morphological changes of the phagosome in the 252 and 264-second panels. The GFP-2FYVE signal has largely disappeared by six minutes after uptake. For yeast-containing phagosomes, the duration is somewhat longer and more variable.Dictyostelium resembles PI(3)P signaling in mammalian cells Dictyostelium. The interval of GFP-2FYVE binding in Dictyostelium is the same time period during which the V-ATPase is delivered to new phagosomes The timing and morphology of the GFP-2FYVE binding compartment in Dictyostelium cells expressing VatM-GFP with living yeast and examined the cells at intervals. Exocytosis of indigestible yeast carcasses could best be observed at 2 to 6 hours after mixing. Such cells were replete with phagosomes whose membranes were rich in VatM-GFP. Incipient exocytosis was characterized by several minutes of vigorous vesiculation at the surface of a VatM-GFP-labeled phagosome. As shown in Seeking to capture exocytosis events, we mixed A second example of V-ATPase removal prior to exocytosis is shown in We examined the relationship between phagosomal pH and the presence of V-ATPase by feeding cells yeast that had been labeled with FITC, a pH-sensitive fluorophore. FITC fluorescence is quenched at acidic pH, so FITC-yeast in an acidic environment are dim, but brighten when they are neutralized. Removal of the V-ATPase prior to exocytosis of the phagosome appears to be the normal method for retrieval. Although we have recorded only a few examples of the retrieval process using the highly sensitive microscope shown in Exocytosis of a phagosome may occur before the V-ATPase has been fully retrieved, a process we call premature exocytosis. This is observed in situations in which cells containing phagosomes with bulky particles are moving through narrow spaces . To increase the frequency of premature exocytosis, we used the thin layer of agarose that overlay the cells during our experiments, slightly drying the agarose so that it pressed more strongly on the cells. This technique allowed us repeatedly to record this otherwise uncommon event, two examples of which are shown in Constraint in the motion of a bulky phagosome appears to be the trigger for premature phagocytosis. We have recorded 14 cases in which phagosomes with VatM-GFP in their membranes were exocytosed. In every case, the phagosome was immobilized, and in 10 of those cases, the cell was motile and attempting to move away from the position of the phagosome. In the remaining cases, the observation period was too brief to make this judgment, or the cell itself was somewhat flattened by the agarose overlay and was not migrating.Just prior to premature exocytosis, we often observed an abrupt expansion in phagosome volume (11 of the 14 cases described above) followed by separation from the phagosome of one or more large vacuoles whose membranes were rich in VatM-GFP. Such vacuoles were approximately round when they formed and moved away from the phagosome before it was exocytosed. An event of this type is seen in Shortly after separation of the vacuole from the phagosome, the phagosome is exocytosed, transferring the remaining phagosomal V-ATPase to the plasma membrane. Note the association of microtubules with that area of the plasma membrane and the diminishing level of VatM-GFP  revealed that, together with actin, myosin-IB is involved in premature exocytosis. We showed earlier that if a phagosome makes contact with the cortical actin layer, GFP-MyoB becomes transiently enriched at the plasma membrane at the point of contact, and there follows a brief spurt of actin-mediated rocketing that moves the phagosome away from the cortex In premature exocytosis, a sudden expansion of phagosome volume commonly precedes the release of V-ATPase-rich vacuoles from the phagosome. We investigated the source of this volume increase using cells that were maintained in the presence of TRITC-dextran for three hours while they were incubated with yeast, in order to label all endosomal compartments. The cells were rinsed with buffer immediately before they were observed. We were thus able to ask whether the volume increase that preceded vacuole release arose from fusion with TRITC-labeled endosomes or with an unlabeled source. The TRITC-dextran content of the phagosome was diluted by the fusion event, indicating that the source of the fluid influx was at least partly unlabeled . Additi and S3. Another potential source of additional membrane and unlabeled fluid is the contractile vacuole complex, an osmoregulatory compartment that is separate from the endosomal system and does not acquire dextran applied to the external medium bisphosphatidic acid The identity of successive compartments along the endocytic pathway is primarily specified by their phosphoinositide composition We next sought examples of premature exocytosis in cells that had eaten FITC-yeast and were expressing GFP-2FYVE and mRFP-LimEΔ. The present study has visualized trafficking of the V-ATPase in both early and late stages of the endocytic pathway. After the actin filaments that shaped the phagocytic cup and propelled the phagosome away from the cortex had disappeared, V-ATPase-rich vesicles clustered around the new phagosome. Fluid phase content detected within the lumen of several of these vesicles indicated that they were of endosomal origin The novel contribution of the present study is the visualization of multiple routes for retrieval of the V-ATPase from phagosome membranes. In normal retrieval, small vesicles rich in VatM-GFP were carried away from the phagosome membrane over a period of several minutes shortly before exocytosis of the neutralized phagosome. The vigorous movement of both the phagosome and the vesicles during this period was suggestive of microtubule-based transport. We were unable to track the fate of individual vesicles owing to their small size and rapid motion. However, it is appealing to speculate that they may form part of the complement of small vesicles that surround and fuse with a new phagosome. After VatM-GFP had been completely removed, strong actin assembly occurred at several points about the phagosome membrane, apparently positioning the phagosome for exocytosis, which followed a few minutes later. This result is consistent with biochemical analysis of isolated phagosomes Dictyostelium cells prior to exocytosis Dictyostelium cells led to models in which late endosomes filled with fluid phase markers existed for extended periods in a neutralized state prior to exocytosis Our experiments using FITC-yeast showed a correlation between removal of the V-ATPase from the phagosome membrane and a rise in luminal pH. It has been demonstrated in macrophages that inactivation of the V-ATPase by inhibitors results in an elevation in luminal pH owing to a passive proton leak across the phagosome membrane At present there is no information regarding what triggers the removal of the V-ATPase from the phagosome membrane, although increasing acidity within the maturing phagosome may play a role. The V-ATPase itself can act as a pH sensor, interacting with small GTPases in an acidification-dependent manner to modulate vesicular trafficking Dictyostelium amoebae may also encounter narrow passageways as they crawl among soil particles in their natural environment, and this might also occur for mammalian phagocytes migrating within the intercellular spaces of a tissue.An unexpected discovery was that in confined spaces, the phagocytes tended to exocytose a large particle prior to complete removal of the V-ATPase from the phagosome membrane. Premature exocytosis commonly occurred when a cell's mobility in a confined space was limited by a bulky phagosome. In migrating cells, a phagosome is normally kept away from the cell cortex by a brief pulse of actin assembly that is induced each time the phagosome approaches the cortex Dictyostelium cells found that V-ATPase complexes mislocalized to the plasma membrane rapidly became surrounded by clathrin lattices In premature exocytosis, some VatM-GFP from the phagosome membrane was transferred to the plasma membrane. This signal declined rapidly, apparently through the internalization of small vesicles. The rapid rate of removal suggests that a specific and efficient retrieval mechanism is employed. An earlier electron microscopy study of quick-frozen When an immobilized phagosome is pressed against the plasma membrane as a cell attempts to migrate, the phosphoinositide composition of the phagosome membrane changes; it becomes capable of binding PHcrac-GFP, a biosensor for PIP3 and PIP2, phosphoinositides that are normally restricted to nascent and just-sealed endosomes Dictyostelium cells are capable of utilizing three different routes for retrieving the V-ATPase from phagosome membranes. Commonly, V-ATPase retrieval occurs through vesiculation shortly before exocytosis of a neutralized phagosome. When constraint of a bulky phagosome leads to premature exocytosis, the separation of a large vacuole prior to exocytosis allows recovery of a portion of the V-ATPase, and the remainder is rapidly retrieved from the plasma membrane. This versatility presumably accounts for our earlier finding In conclusion, our live cell microscopy studies have revealed that Figure S1Removal of VatM-GFP from the plasma membrane following premature exocytosis. Frames from the time series shown in (2.06 MB TIF)Click here for additional data file.Figure S2Increase in phagosome volume and dilution of fluid phase marker prior to premature exocytosis. (A) The cell was incubated with TRITC-dextran and yeast for three hours, then the medium was replaced with buffer, the cells were covered with a thin layer of agarose that was slightly dried to induce premature exocytosis, and the sample was viewed at once. All endosomal compartments except new macropinosomes were expected to contain TRITC-dextran. (This is the same experiment shown in (3.35 MB TIF)Click here for additional data file.Figure S3Increase in phagosome volume and dilution of fluid phase marker prior to premature exocytosis. (B) This sample was prepared as described in (A) except that the cells were left in buffer for 30 minutes prior to viewing. Thus, late but not early endosomes were expected to contain TRITC-dextran. The results were similar to those in the first experiment, namely, expansion of the phagosome and dilution of the TRITC-dextran (20 seconds), separation of a vacuole (43 seconds), exocytosis of the yeast (79 and 173 seconds), and an increase in TRITC-dextran concentration as the volume of the vacuole is reduced during sorting (173 and 264 seconds). Zeiss LSM510 microscope.(3.11 MB TIF)Click here for additional data file.Figure S4Phagosome expansion and vacuole separation in a cell expressing the contractile vacuole marker dajumin-GFP. The cells were expressing dajumin-GFP and mRFP-LimEΔ; they were mixed with yeast two hours earlier. The cells were covered with a thin layer of agarose that was dried slightly to induce premature exocytosis. A vacuole (arrowhead) separates from the phagosome (39 seconds) and moves away with a tail of actin filaments (53 and 79 seconds). Meanwhile, the phagosome is exocytosed (53 and 79 seconds). No dajumin-GFP label is incorporated into the membrane of the phagosome or the vacuole, arguing that the contractile vacuole system is not the source of the added membrane and fluid. Zeiss LSM510 microscope. Bar, 5 µm.(2.05 MB TIF)Click here for additional data file.Movie S1Delivery of the V-ATPase to a new phagosome. The cell is expressing VatM-GFP and mRFP-LimEΔ. It has just phagocytosed a living yeast cell.(8.16 MB MOV)Click here for additional data file.Movie S2Delivery of the V-ATPase to a new phagosome. The cell is expressing VatM-GFP and has been incubated with TRITC-dextran to label endosomes. The uptake of a heat-killed yeast is shown.(2.03 MB MOV)Click here for additional data file.Movie S3Labeling of a macropinosome with GFP-2FYVE, a biosensor for PI(3)P. The cell is expressing GFP-2FYVE and mRFP-LimEΔ. The nascent macropinosome is initially labeled with the actin marker. Actin disappears and 2FYVE-GFP binding begins after about one minute. It persists for another 2 or 3 minutes as the macropinosome elongates and fragments, behavior consistent with the early or sorting endosome stage.(1.23 MB MOV)Click here for additional data file.Movie S4Labeling by GFP-2FYVE of phagosomes containing bacteria. The cell is expressing GFP-2FYVE and mRFP-LimE and is eating bacteria. New phagosomes are labeled first by the actin marker and then by GFP-2FYVE starting about one minute later and persisting for several minutes. Fusion and fission events characteristic of early phagosomes can be seen during the period of 2FYVE-GFP labeling.(4.84 MB MOV)Click here for additional data file.Movie S5Removal of the V-ATPase from the membrane of a yeast-containing phagosome, followed by actin assembly and exocytosis. The cell is expressing VatM-GFP and mRFP-LimEΔ.(8.65 MB MOV)Click here for additional data file.Movie S6Removal of the V-ATPase from the membrane of a yeast-containing phagosome, followed by actin assembly and exocytosis. Over the same interval, the cell takes up a new yeast. The cell is expressing VatM-GFP and mRFP-LimEΔ.(8.46 MB MOV)Click here for additional data file.Movie S7Exocytosis of a FITC-yeast from a phagosome whose membrane has been depleted of VatM-GFP prior to exocytosis. The cell is expressing VatM-GFP and DdmCherry-LimEΔ. The yeast does not grow brighter upon contact with the extracellular medium.(9.33 MB MOV)Click here for additional data file.Movie S8Premature exocytosis of a yeast-containing phagosome and initial stage of the retrieval of VatM-GFP from the plasma membrane. The cell is expressing VatM-GFP and mRFP-α-tubulin.(7.22 MB MOV)Click here for additional data file.Movie S9Separation of a large V-ATPase-rich vacuole from a yeast-containing phagosome prior to premature exocytosis. The cell is expressing VatM-GFP and mRFP-α-tubulin.(8.50 MB MOV)Click here for additional data file.Movie S10Acidic nature of a prematurely exocytosed phagosome, also showing actin-powered vacuole movement. The cell is expressing VatM-GFP and DdmCherry-LimEΔ. It has eaten a FITC-labeled yeast that is barely visible in the phagosome but brightens upon contact with the higher pH extracellular medium, indicating that the phagosome lumen was acidic.(3.02 MB MOV)Click here for additional data file.Movie S11Premature exocytosis of a phagosome containing a FITC-yeast and dynamics of the vacuole that separates prior to exocytosis. The cell is expressing VatM-GFP and DdmCherry-LimEΔ.(3.37 MB MOV)Click here for additional data file.Movie S12Increase in phagosome volume and dilution of fluid phase marker prior to premature exocytosis. The cell is expressing VatM-GFP and was incubated for 3 hours with TRITC-dextran, then shifted to unlabeled buffer and observed.(9.60 MB MOV)Click here for additional data file.Movie S13Acquisition of GFP-2FYVE label by a vacuole that has separated from a phagosome just prior to premature exocytosis. The cell is expressing GFP-2FYVE and mRFP-LimEΔ; the phagosome contains a FITC-yeast.(3.86 MB MOV)Click here for additional data file.


Among a cohort of 10,106 three-year survivors of childhood cancer, 90 second primary tumours (SPTs) were observed. Within 25 years of 3-year survival about 4% developed a SPT, about 6-fold expected, the relative risk not varying much with increasing follow-up. Following genetic retinoblastoma we observed 30-fold the expected number of SPTs, and over 400-fold the expected number of osteosarcomas. The risk of SPT in the absence of radiotherapy and chemotherapy (inherent risk) following genetic retinoblastoma was 13-fold expected and over 200-fold the expected number of osteosarcomas were observed. There was evidence that both radiotherapy and cyclophosphamide were associated with an increased risk of SPT. After all first primary tumours (FPTs) excluding retinoblastoma we observed almost 5-fold the expected number of SPTs. The inherent risk was 4-fold expected, the relative risks associated with radiotherapy but no chemotherapy, and both radiotherapy and chemotherapy were 6- and 9-fold expected respectively. There were about 20-fold the number of malignant bone tumours expected, most were osteosarcoma; also 7-fold the number of central nervous system tumours expected. There were 8 basal cell carcinomas and it seems likely that radiotherapy was involved in the development of some of these. Radiotherapy appears to have been involved in the development of many of the SPTs observed following all FPTs excluding retinoblastoma, particularly after CNS tumours, Wilms' tumour and Hodgkin's disease. Currently there is insufficient follow-up to examine the risk following chemotherapy. After acute leukaemia there was 20-fold the expected number of central nervous system tumours, though this is based on only 3 cases; whether therapy is directly involved in their development is uncertain. The risks we report are rarely greater than those reported in previous large-scale studies; in most instances they are substantially less. It is very unlikely that many SPTs were missed with our follow-up system so alternative explanations require further investigation; in particular it is possible the lower risks in our data compared to series treated in the United States may be explained, in part, by less combination therapy and lower doses of radiotherapy.


Topiramate might be effective in the treatment of posttraumatic stress disorder (PTSD) because of its antikindling effect and its action in both inhibitory and excitatory neurotransmitters. Open-label studies and few controlled trials have suggested that this anticonvulsant may have therapeutic potential in PTSD. This 12-week randomized, double-blind, placebo-controlled clinical trial will compare the efficacy of topiramate with placebo and study the tolerability of topiramate in the treatment of PTSD.Seventy-two adult outpatients with DSM-IV-diagnosed PTSD will be recruited from the violence program of Federal University of São Paulo Hospital (UNIFESP). After informed consent, screening, and a one week period of wash out, subjects will be randomized to either placebo or topiramate for 12 weeks. The primary efficacy endpoint will be the change in the Clinician-administered PTSD scale (CAPS) total score from baseline to the final visit at 12 weeks.The development of treatments for PTSD is challenging due to the complexity of the symptoms and psychiatric comorbidities. The selective serotonin reuptake inhibitors (SSRIs) are the mainstream treatment for PTSD, but many patients do not have a satisfactory response to antidepressants. Although there are limited clinical studies available to assess the efficacy of topiramate for PTSD, the findings of prior trials suggest this anticonvulsant may be promising in the management of these patients.NCT 00725920 Posttraumatic stress disorder (PTSD) is the only psychiatric disorder that has an etiologic component: exposure to a traumatic event. The disorder is characterized by three symptom clusters: reexperience of the event, avoidance/numbing, and hyperarousal resulting from exposure to a traumatic event. Data frFurthermore, PTSD has a high comorbidity with other psychiatric conditions such as substance abuse/dependence, anxiety disorders, and major depressive disorder causing a worsening prognosis .The development of treatments for PTSD is challenging due to the complexity of the symptoms and psychiatric comorbidities. Psychotherapy and pharmacotherapy are the two main classes of PTSD treatment. In the Cochrane systematic review, Bisson and Andrew  evaluateThe National Institute for Clinical Excellence (NICE)  recommenIn a recently published systematic review, Stein et al  evaluateNevertheless, the bulk of evidence for the efficacy of medication has been with the SSRIs and supports expert consensus guidelines that these medications constitute the first-line medication choice in PTSD.The American Psychiatric Association  practiceThe hypotheses on the etiology of PTSD have suggested that after exposure to traumatic events; the limbic nuclei may become kindled or sensitized, resulting in increased susceptibility to physiologic arousal ,16. The Topiramate is an anticonvulsant with inhibitory activity in animal kindling models and with multiple mechanisms of action: inhibition of carbonic anhydrase, blockade of Na+ channels, inhibition of some high-voltage-activated Ca2+, negative modulating effect on the kainate/AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid) subtype of glutamate receptors, ability to modulate NMDA (Nmethyl-D-aspartate) glutamate receptor and enhancement of GABAergic activity at GABA2 receptors ,18.The blockage or the stabilization of the AMPA (α-amino-3-hydroxy-5 methyl-4 isoxazole propionic acid) receptor may be a possible mechanism responsible for the increase in the threshold for flashbacks and nightmares that may explain the treatment response to topiramate in PTSD . SeveralGlutamatergic neurons play a role in the neuronal plasticity associated with longterm potentiating in adaptation to stress . GlutamaAlthough much of the research associate the glutamatergic systems with learning and memory; the GABAergic (γ-aminobutyric acid) system in the basolateral amygdala, is also involved in the acquisition and extinction of fear memories . ReducedClinical studies with topiramate suggest that it is effective as monotherapy or adjunctive therapy in PTSD. Two open-label studies demonstrated topiramate to be effective and to have a rapid onset of action. Berlant and van Kammen  have repExtensive efforts have been made to establish guidelines based in evidence for PTSD treatment, however there is still no specific drug proved efficacious. Although there are limited clinical studies with topiramate in the treatment of PTSD, the findings of prior topiramate studies suggest that this anticonvulsant may be promising in the management of patients with PTSD.1) To compare the efficacy of topiramate with placebo in the treatment of PTSD. Clinical response will be considered when there are a 20% reduction on CAPS scale scores from baseline and remission when CAPS score is less than 19.2) To study the tolerability of topiramate in the treatment of PTSD, through the dropout rate in each group.The trial will compare the efficacy of topiramate with placebo in the treatment of PTSD, and will study its tolerability through the drop out rate in each group. The primary hypothesis is that topiramate will be superior to placebo in the treatment of PTSD. Secondary research goals includes studying the efficacy of topiramate in social adjustment, functioning and quality of life. Additionally, evaluating treatment adhesion, and studying the efficacy of topiramate in depressive symptoms.All elegible patients admitted at the outpatient clinic from the violence program of Federal University of São Paulo Hospital (UNIFESP) who read and signed a consent inform approved by the UNIFESP Institutional Review Board (IRB) will enter the study. A pre-request to be admitted at this program is having passed through a stressor event which imposed a risk on subjects' physical and/or psychological integrity (DSM-IV criterion A for PTSD diagnostic). Patients looked for an appointment at the program spontaneously, or through health worker recommendation or were called by phone or by mail from a list of subjects which had a PTSD diagnostic after a CIDI submission by trained raters on the Sao Paulo epidemiological catchment area study. This was an epidemiological survey of a random sample of 3000 participants living in the city of Sao Paulo to assess the relationship between exposure to violence and the prevalence of PTSD and common mental disorders.All patients will be administered the Structured Clinical Interviews for DSM-IV Axis I and Axis II  by a trained psychiatrist ,32. PatiThe Structured Clinical Interview for DSM-IV-Axis I and II (SCID-I and II) [•  and II) ,32: is a and II) .The Clinician-Administered PTSD Scale (CAPS) [• e (CAPS) : is a stThe Beck Depression Inventory (BDI) [• ry (BDI) : is a 21MOS 36-Item Short-Form Health Survey (SF-36) [•  (SF-36) : is a scSocial Adjustment Scale (SAS) [• le (SAS) : is a 54Global Assessment of Functioning (GAF) Scale [• F) Scale : is a siThe Clinical Global Impression – Severity scale (CGI-S) [•  (CGI-S) : is a 7-The Clinical Global Impression – Improvement scale (CGI-I) [•  (CGI-I) : is a 7 This is a 12-week randomized, double-blind, placebo-controlled clinical trial that will be conducted at the Federal University of São Paulo Hospital (UNIFESP). The study was approved by UNIFESP Institutional Review Board (IRB), and will follow the Helsinky Declaration, and the local ethical laws. Ethical Committee: 196/96After the SCID-I and II administration, eligible patients will undergo a brief physical examination with height, weight, heart rate and systemic blood pressure measured and a clinical evaluation including electrocardiogram (EKG) and laboratory blood test for complete blood count, fasting glycemia, sodium, potassium, urea, creatinine, T3, T4, and TSH. Women will have a pregnancy test done, to assure they are not pregnant. The psychiatrist who administered the SCID, will also evaluate patients clinical conditions and functionality through CGI-S and GAF. The Clinician-Administered Posttraumatic Stress Scale (CAPS) will be Patients will receive placebo pills and will be re-evaluated by the same psychiatrist one week after. Those having a significant improvement of the symptoms (a CGI-I score less than 3) will be excluded (avoiding premature placebo effects).Those patients without improvement during the placebo trial, and do not show any laboratory and clinical condition exclusion criteria will be randomly assigned to receive either placebo or topiramate, in a 1:1 ratio using computer- generated code. The active and placebo pills will be identical. All research and clinical staff will be blinded to the randomized assignments.Study medication will start at 25 mg/day once daily, at night, and will be increased 25 mg weekly, as tolerated, until complete or nearly complete efficacy is achieved or until 200 mg/day is reached. No other psychotropic medications will be allowed during the study, except for zolpidem (10 mg/day), if needed, for insomnia.Study follow-up visits will be conducted at baseline, and weeks 1, 2, 3, 4, 6, 8, and 12.Symptoms and side effects will be assessed, as well as measurement of systemic blood pressure and pulse rate in each visit by the investigator, who is blind to medication assignment. Compliance will be assessed by counting the amount of pills left in the bottle of medicine in each visit.Treatment will be continued for 12 weeks (active treatment phase); then drug administration will be withdrawn gradually during 2 weeks with reductions of 25/50 mg every three days, according to the final dosage reached.Patients will be assessed until completing 6 months during a follow-up period. If a patient showed a symptomatology worsening during the active treatment phase (12 weeks) when receiving active drug treatment, characterized by a one-point decrease at CGI -I or present serious adverse effects related to medication, he/she will be excluded from the study.The number of subjects calculated for the trial is 72, being 36 on each cell. The sample size was calculated considering a 5% probability of type I error (significance level), and 20% for type II error (power test of 80%), on a bi-caudal test. To the calculation we use an average of 60% for response from PTSD for active drugs, based on published literature, and 30% of placebo response.All data will be analyzed by SPSS (version 13.0). The frequencies of nominal variables from all patients will be calculated. The data analysis will be conducted using a ANOVA comparing each scale during the 2 continuous measures from baseline and 12 weeks with a Between Subject factor was included as independent variable (treatment group). The confidence level that will be used for all comparisons will be 5% (p < .05). All observed Power as a type II error function will be presented with minimum values of significance for each comparison. CAPS scores from baseline and endpoint will be stratified according severity, as nominal data they will be compared using the chi-square test and the Fisher test when there were cells that counted less than five or at least one cell counted zero subjects. For missing data we will use the last observation carried forward strategy for an intention-to-treat analysis.Efficacy analyses will be performed in the intent-to-treat population of patients who received at least one dose of topiramate. The primary efficacy endpoint will be the change in PTSD Scale- CAPS total score from baseline to the final visit at 12 weeks, which will be compared between treatment groups. Missing efficacy data at 12 weeks will be imputed with the last observation carried forward. The secondary outcomes between groups will be comparing the Clinical Global Impressions (CGI), the Beck Depression Inventory (BDI), the Global Assessment of Function (GAF), and the PANAS scales. Other secondary analyses will include an evaluation after 24-weeks (12-week after last patient visit with drug) data with endpoint analyses between the treatment groups covarying for baseline scores. On all secondary outcomes, the Bonferroni correction will be applied for testing multiple variables. Response will be defined as a 20% improvement in the baseline CAPS total or positive symptom scores at 12 weeks and will be compared between treatment groups using a chi-square test. Proportions of patients completing the 12-week double-blind study will be compared between treatment groups using a chi-square test. Adverse events reported by 10% of patients in each treatment group will be compared using a chi-square test. Laboratory values, vital signs and physical examinations are going to be compared from baseline and in reference to normal ranges, and reported as normal or abnormal. Changes in body weight (lb), body mass index  at last visit in the 12-week dataset will be compared as mean changes between treatment groups using t-tests.The authors declare that they have no competing interests.JJM, MFM, developed the design of the randomised clinical trial and participated in writing the article. MSLY is the principal investigator and writer of this manuscript. DDN, MSLY, JPF, NLMV, JBN, NMV and PM will participate as clinical psychiatrists. MCPC will be the study coordinator. RAB and SBA participated on the study design. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Hsp70.1Luciferase (LUC) transgene inserted at a single X chromosome locus of a bull (Bos taurus) was assessed in females after X-chromosome inactivation (XCI). Furthermore, impact of the chromosomal environment on the spontaneous expression of these transgene copies before XCI was studied during early development in embryos obtained after in vitro fertilization (IVF), when the locus was carried by the X chromosome inherited from the bull, and after somatic cell nuclear transfer (SCNT) cloning, when the locus could be carried by the inactive Xi or the active Xa chromosome in a female donor cell, or by the (active) X in a male donor cell.Expression of several copies of the heat-inducible LUC female fibroblasts, i.e. after random XCI, the proportions of late-replicating inactive and early-replicating active XLUC chromosomes were not biased and the proportion of cells displaying an increase in the level of immunostained luciferase protein after heat-shock induction was similar to that in male fibroblasts. Spontaneous transgene expression occurred at the 8-16-cell stage both in transgenic  embryos obtained after IVF and in male and female embryos obtained after SCNT.Transgene copies were mapped to bovine Xp22. In XXLUC chromosome is normally inactivated but at least part of the inactivated X-linked Hsp70.1Luciferase transgene copies remains heat-inducible after random XCI in somatic cells. Before XCI, the profile of the transgenes' spontaneous expression is independent of the epigenetic origin of the XLUC chromosome since it is similar in IVF female, SCNT male and SCNT female embryos.The X HSP70.1 gene promoter linked to firefly luciferase cDNA [P chromosome becomes inactivated by the blastocyst stage [Menck and colleagues have reported a luminescent screening system based on the integration of a transgene composed of scaffold attachment regions flanking the murine ase cDNA ,3. At lease cDNA ,3). Firsst stage ,3 and alst stage . This reSome X-linked genes maintain bi-allelic expression in female cells ,3. On thSimilarly, XCI-related silencing of X-linked transgenes may depend on the insertion site, the transgene's intrinsic properties or other unknown factors. Furthermore, it may vary between cell lineages or during development  induction in both mouse and bovine transgenic embryonic and somatic cells and also spontaneously during embryonic genome activation (EGA) in early mouse transgenic embryos  in December 1999.The remaining of this section is found in [additional file After hybridization with a probe specific for the whole transgene, a unique strong signal was observed on the short arm of the bovine submetacentric X in chromosome preparations from the transgenic bull, precisely in the early-replicating R band Xp22 Figure . The bovLUC chromosome inherited from the bull. Presence of a strong bias would indicate preferential inactivation of one of the X or preferential survival of the cells that inactivate one X. Although normal random XCI is reported for mouse and bovine clones [Since XCI occurs randomly in the somatic bovine lineages ,22, aboue clones ,21,22,26e clones .LUC fibroblast cells cultured in the presence of BrdU during late S phase showed no bias. An equivalent number of BrdU-labeled X chromosomes or inactive Xi (N = 22) and partly BrdU-labeled X chromosomes or active Xa (N = 21) carried copies of the transgene. Furthermore, the transgenes' presence had no visible influence on Xp22 inactivation/activation since the XLUCp22 region replicates late on the Xi , we compared the proportions of luciferase-positive cells after HS induction and immunostaining in this female cell population and the male OV7060 cell population (Table We compared the level of luciferase activity and protein after heat-shock (HS) induction in somatic female cells derived from two fetuses generated by IVF with the sperm of the transgenic original bull and referred to as F616 and BSF731 cells, and in male cells from the original transgenic bull (referred to as OV7060 cells). HS-induced luciferase activity ranged from 0.99 × 10In tissue biopsies of three fetal organs tested from the F616 female fetus, a strong increase in the level of luciferase activity after HS-induction was observed Table . OverallDuring normal development, between the one-cell and the 4-8-cell stages, the level of luciferase activity was null in at least 80% of the IVF and SCNT transgenic bovine embryos and very low in the remaining 20% Figure ; Table 3Before nuclear transfer, the BSF731 female donor cells carried the transgenes either on the Xa or the Xi with a similar probability (see above). However, in female BSF731-derived SCNT embryos, we found no evidence for two sub-populations displaying two different levels and patterns of luciferase activity and the standard error to the mean was similar to that observed in the two other embryo types Table  or was rHsp70.1Luciferase transgene can be similarly active at the 8-16-cell stage in both IVF and cloned embryos, this further suggests that neither the paternal origin of the transgenic XLUC in the case of IVF embryos, nor the origin, male or female, of the somatic cells, in the case of SCNT embryos, prevented spontaneous, oocyte-driven, expression of the X-linked transgenes.Since the LUC, is expected. However, we cannot yet conclude whether the transgenes escaped inactivation after imprinted XCI since the presence of cells in which XCI had not occurred or had occurred randomly is likely.We detected an increased level of luciferase activity after heat-shock induction in IVF female blastocysts [additional File In conclusion, using a species other than mouse and different approaches we have investigated the expression of an X-linked transgene to determine its innocuousness as well as that of the transgene insertion site, and to test its sensitivity to XCI-dependent or -independent silencing. The results indicate that the transgenic X inherited from the cloned bull is normally inactivated/activated in somatic female cells and that at least some of the transgene copies at this locus escape XCI-coupled silencing in these cells. Whether this is due to HS-dependent ,29 or HSThe authors declare that they have no competing interests.DLB and XV generated the bovine bull; XV and JLS generated cell cultures, JML, AB, MC, HH and JLS generated IVF embryos, performed FISH and immuno-detection, and measured luciferase activity and protein concentrations; DLB generated SCNT embryos, JML designed the experiments, performed statistical tests and wrote the draft. HH and XV edited the draft and all authors agreed on the final version of this report.Material and methodsClick here for fileAdditional dataClick here for fileAdditional Table S1Click here for file


Clonal subpopulations of different ploidy values and metastatic capacities, isolated from the RIF-1 mouse sarcoma, have been tested for in vitro X-radiation sensitivity, for in vitro sensitivity to adriamycin and for in vitro and in vivo sensitivity to melphalan and CCNU. Following X-radiation, no consistent differences in the survival curve characteristics (Do and n) of diploid, tetraploid and octoploid cells were observed. In addition no relationship between radiation response and metastatic capacity was observed. For drug response, no marked differences were found in the dose response curves of RIF-1 clones treated in vitro with adriamycin. However, a wide variation in the responses of RIF-1 clones to in vitro melphalan treatment was observed which was independent of both ploidy and metastatic capacity. Although the responses of RIF-1 clones to in vitro CCNU treatment were similarly independent of metastatic capacity, a clear relationship between CCNU sensitivity and ploidy was observed. Thus, all diploid RIF-1 clones were markedly more sensitive to CCNU treatment than either tetraploid or octoploid RIF-1 clones. For both melphalan and CCNU treatment the relative sensitivities in vitro correlated with in vivo sensitivities as assayed by clonogenic cell survival.


Nearly a century has passed since the first report describing antigen-specific immunotherapy (antigen-SIT) was published. Research into the use of antigen-SIT in the treatment of both allergic and autoimmune disease has increased dramatically since, although its mechanism of action is only slowly being unravelled. It is clear though, from recent studies, that success of antigen-SIT depends on the induction of regulatory T (T reg) cell subsets that recognise potentially disease-inducing epitopes. The major challenge remaining for the widespread use of antigen-SIT is to safely administer high doses of immunodominant and potentially pathogenic epitopes in a manner that induces T cell tolerance rather than activation. This review illustrates that intelligent design of treatment agents and strategies can lead to the development of safe and effective antigen-SIT. This, therefore, provides two guiding principles for the design of antigen-specific immunotherapeutic strategies. First, antigen should be directed to tolerogenic rather than immunogenic APC; secondly, administration of antigen should lead to the induction of regulatory T cells.Current treatments for allergic and autoimmune disease treat disease symptoms or depend on non-specific immune suppression. Treatment would be improved greatly by targeting the fundamental cause of the disease, that is the loss of tolerance to an otherwise innocuous antigen in allergy or self-antigen in autoimmune disease (AID). Much has been learned about the mechanisms of peripheral tolerance in recent years. We now appreciate that antigen presenting cells (APC) may be either immunogenic or tolerogenic, depending on their location, environmental cues and activation state . FurtherThe application of allergen-SIT has become increasingly popular since first reported by Leonard Noon in 1911. Subcutaneous (s.c.) or oral/sublingual administration of allergens has been used for the successful treatment of a wide range of allergies including those to bee venom , cow's m+ T cells specific for these epitopes differs. It is now clear that the balance between effector T cell populations on the one hand and IL-10-secreting, suppressive T cells on the other makes the difference between an atopic or healthy immune response. In atopic individuals, the highest proportion of T cells recognising common environmental allergens are IL-4-secreting T helper (Th) 2 cells, whereas IL-10-secreting T cells prevail in healthy individuals [Progress in the development of allergen-SIT has been hindered by a lack of understanding of the underlying immunological mechanisms. In recent years, it has become clear that the ratio of allergen-specific T cells secreting distinct cytokines plays a crucial role in the onset and cessation of allergic diseases. First, it is important to realise that allergic and non-allergic individuals recognise the same T cell epitopes of common allergens  and thatividuals . The impividuals ••. Durinividuals ••,15,16.ividuals .The use of SIT for AID has lagged behind SIT for allergy. This may be because AIDs are more heterogeneous than allergic diseases; the disease-initiating or target antigen may not be known; and/or the immune pathogenesis of AID is associated with epitope spreading  and subsFirst attempts at SIT, in diseases such as multiple sclerosis (MS), were not successful . Weiner + cells in the liver. Expression of antigen in the liver generally enhances the generation of induced FoxP3+ Treg cells and has proven effective in models of uveitis and MS [The administration of self-antigen via plasmid DNA is an attractive approach since co-expression of cytokines and immune modulators can be used to enhance mechanisms of tolerance induction. Early examples of DNA vaccination in AID models produced conflicting results; disease could be either suppressed  or enhans and MS •,34•.Although antigenic protein therapy has been successful in pre-clinical models, the approach has not translated well into the clinic. Nevertheless, there has been success in the use of an alum-based islet-antigen vaccine. GAD-alum treatment led to the preservation of residual insulin secretion in patients with recent-onset T1D •. One ap+ T cells [The alternative to coupling antigen to APC is simply to administer soluble peptide via a tolerogenic route. Much has been learned about the nature of the peptide, dose, route of delivery and timing. The peptide must mimic the naturally processed antigen when bound to major histocompatibility complex (MHC) ; post-tr T cells . Similar T cells . Clinica+ T helper subsets, with Th1, Th2, Th9 and Th17 cells all shown to secrete IL-10 in the face of chronic exposure to antigen . Boiewed in . Importaiewed in •. CombinSome studies suggest that IL-10 secretion is a transitory property of highly stimulated cells, dependent on tissue-specific environmental cues for its maintenance, while others suggest that the IL-10 locus can be genetically modified in terminal differentiation. Recent evidence has shown that sustained high-dose TCR signalling and high levels of IL-12 were required for the induction and maintenance of IL-10 secretion in Th1 cells, through sustained ERK1 and ERK2 MAP kinase phosphorylation ••. HowevThis review has highlighted recent advances in specific immunotherapy for allergic and autoimmune disease. One overriding conclusion is that regulatory mechanisms involving IL-10 are important for effective therapy. Our belief is that basic research should focus on means to target tolerogenic APC; to promote, for example through the use of appropriate adjuvants, secretion of IL-10 from both APC and T cells; and to investigate the mechanisms of dose escalation tolerance. Future clinical trials should focus on patient groups that are most likely to benefit from the treatment, for example major changes should not be expected in advanced stages of disease ; no SIT • of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as: