A comparative analysis of two primate and two rodent genomes suggests that telomerase was utilized, in some instances, for the repair of DNA double-strand breaks during mammalian evolution. In vertebrates, tandem arrays of TTAGGG hexamers are present at both telomeres and intrachromosomal sites ). We previously showed that, in primates, ITSs were inserted during the repair of DNA double-strand breaks and proposed that they could arise from either the capture of telomeric fragments or the action of telomerase.Homo sapiens and Pan troglodytes) and two rodent (Mus musculus and Rattus norvegicus) genomes allowed us to describe organization and insertion mechanisms of all the informative ITSs present in the four species. Two novel observations support the hypothesis of telomerase involvement in ITS insertion: in a highly significant fraction of informative loci, the ITSs were introduced at break sites where a few nucleotides homologous to the telomeric hexamer were exposed; in the rodent genomes, complex ITS loci are present in which a retrotranscribed fragment of the telomerase RNA, far away from the canonical template, was inserted together with the telomeric repeats. Moreover, mutational analysis of the TTAGGG arrays in the different species suggests that they were inserted as exact telomeric hexamers, further supporting the participation of telomerase in ITS formation.An extensive comparative analysis of two primate (These results strongly suggest that telomerase was utilized, in some instances, for the repair of DNA double-strand breaks occurring in the genomes of rodents and primates during evolution. The presence, in the rodent genomes, of sequences retrotranscribed from the telomerase RNA strengthens the hypothesis of the origin of telomerase from an ancient retrotransposon. The vertebrate telomeres consist of extended arrays of the TTAGGG hexamer. The specialized function of the telomerase enzyme, together with a multitude of telomere-binding proteins, is required to maintain sufficiently long telomeres, assuring stability to the linear eukaryotic chromosomes. Telomerase is an atypical reverse transcriptase that adds telomeric repeats to chromosome ends, overcoming the limitations of the replicative apparatus that would cause shortening of the termini at each replication round. Telomerase is composed of two moieties: a protein endowed with reverse transcriptase activity (telomerase reverse transcriptase (TERT)), and an RNA molecule (telomerase RNA component (TERC)) -3. TelomRepetitions of the telomeric hexamer at intrachromosomal sites, the so called interstitial telomeric sequences (ITSs), have been described in many species, including primates and rodents -16. In pA direct involvement of telomerase in ITS insertion is conceivable in view of the mounting evidence for the sharing of factors between the machineries for DNA double-strand break repair and telomere maintenance -27. In pHomo sapiens, Pan troglodytes, Mus musculus and Rattus norvegicus to analyze all the ITSs present in them. We were thus able to demonstrate that the same mechanisms for ITS insertion, previously identified in primates, are also operating in rodents. Furthermore, we obtained evidence that, in rodents, portions of TERC other than the canonical hexameric template can be retrotranscribed during the process; this observation, together with the results obtained by a comparative analysis of all ITS loci, suggests that telomerase can contribute to DNA double-strand break repair.In order to investigate the possible role of telomerase in ITS insertion, we took advantage of the availability of the nearly complete sequence of the genomes of 4 sequence as query, we performed a BLAT search [M. musculus or mouse and R. norvegicus or rat) and two species of the Primates order, hominidae family (H. sapiens or human and P. troglodytes or chimpanzee). We found 306 and 326 ITS loci in the mouse and rat genomes, respectively, and 100 and 110 ITS loci in the human and chimpanzee genomes, respectively, containing four or more TTAGGG repeated units. Subtelomeric type loci consisting of tandemly oriented exact and degenerate TTAGGG repeats were preliminarily removed since they are probably the product of recombination events involving telomeres [Using the (TTAGGG)T search ,35 for aelomeres . This opFor each mouse ITS locus, we searched the orthologous rat locus by using up to 20 kb of the sequence comprising the ITS as query for a BLAT search against the rat genome database. Similarly, the mouse loci orthologous to rat ITS loci were searched in the mouse genome database. For 128 mouse and 120 rat loci the orthologous loci in the other species were either not identifiable or grossly rearranged . In 58 loci the telomeric repeats were conserved in both species , hence they were inserted in the genome of a common ancestor of mouse and rat (more than 12-14 million years ago (MYA)) . FinallyThe same type of comparative analysis was carried out for the 83 human and the 79 chimpanzee ITSs. The majority (75 loci) of the primate ITSs  were present in both species , hence they originated before the human/chimpanzee split, that is, more than 6 MYA . Only foBy comparing the flanking sequence of each ITS-containing locus with the sequence of the corresponding empty locus in the two Rodentia and the two Primates species, we could define the mechanism of insertion at each informative locus . We found that the ITSs were inserted with the same mechanisms previously described in primates , which tSurprisingly, at some rodent loci, the ITS was added together with a sequence homologous to a portion of a TERC distant from the telomeric template. These loci and the proposed mechanism of insertion are discussed below.The analysis of the length of all the interstitial telomeric arrays  has shown that the length of the ITSs is similar in mice as compared to rats and in humans as compared to chimpanzees  were observed , we wondered whether their frequency was correlated with the age of the insertion event. Considering that the species-specific ITSs were inserted in the genome more recently than the conserved ones, we compared the frequency of mismatches in species-specific and in conserved ITSs. In all four species, the number of mismatches per telomeric unit is significantly lower in the 'young' (species-specific) compared to the 'old' (conserved) ITSs Table ; therefoIf telomerase was directly involved in the insertion of ITSs at break sites, we would expect, in the ancestral sequence, a non-random presence of nucleotides in register with the inserted telomeric repeats. In fact, the presence of 1-5 nt microhomology to the telomeric hexamer at the 3' end of a break site is known to favor so called 'chromosome healing', that is, the creation of a new telomere at a break site by telomerase ,41. We tThe results of this analysis showed a strikingly high frequency of nucleotides in register with the inserted telomeric repeats . An exhaustive BLAT search of loci containing TERC-like sequences was performed in the genome of the four species using the TERC genes as query. In the primate genomes no homologies were scored besides the TERC gene itself. On the contrary, in the mouse, 14 loci containing portions of the TERC sequence different from the repeat template were found adjacent to telomeric repeats Table . Three lFigure In our previous work  we descrIt has been suggested that the presence of telomeric-like repeats at interstitial sites may cause chromosomal instability -47; in lAlthough the four basic mechanisms of ITS insertion are shared between primates and rodents, the presence, at 14 mouse ITS loci, of sequences homologous to the 3' domain of TERC revealed that, in rodents, an additional mechanism, involving TERC retrotranscription, was active. This pathway is present only in the rodents and is discussed below.Another difference between the two orders is the length of the ITSs Figure : about 4The proportion of primate ITSs conserved in both species, and therefore inserted before the human-chimpanzee split, is very high (more than 90%), and significantly higher than in rodents 24%)  compared to the 'old' (conserved) ITSs Table : the 'olIn our previous work, we proposed that the ITSs could be inserted at DNA double-strand break sites either by telomerase or by the capture of telomeric fragments . The resTwo independent sets of data presented in this work point to a direct role of telomerase in ITS formation. In the first place, in a highly significant number of species-specific loci, the break site, which occurred in the ancestral sequence, exposed from one to eight nucleotides in register with the inserted telomeric hexamers. Even more significant in this regard is the observation that, at 14 mouse ITS loci, sequences homologous to the 3' domain of the RNA component of telomerase, far away from the hexamer template, which is located near the 5' end of the RNA, were inserted together with the telomeric repeats  proteins may play a role in different steps of this process, as well as in the simple insertion of telomeric repeats. In particular, besides Ku, which is known to bind the telomerase RNA component, the DNA-PK catalytic subunit may be involved in the activation of factors responsible for the final end-joining. In addition, the observation that sequences at the break site are modified during ITS insertion . In addThe data presented here corroborate our hypothesis that the insertion of interstitial telomeric repeats is a consequence of a peculiar pathway of DNA double-strand break repair and extend this conclusion from primates to rodents; we might, therefore, infer that this pathway is more general and probably operates also in other eukaryotes. We also showed that, although rarely, portions of the telomeric RNA other than the canonical template for the telomeric repeats can be retrotranscribed during the process, strongly suggesting the participation of telomerase. These telomerase driven repair processes occurring during evolution constitute a previously undescribed mechanism of genome plasticity and support the hypothesis, based on the structural similarity between telomerase and retrotransposon reverse transcriptases, that an ancient retrotransposon may have provided a DNA-end maintaining activity to the eukaryotic chromosome -67.4 sequence was used as query for a BLAT search [M. musculus: University of California Santa Cruz (UCSC) Genome Browser database, March 2005), rat , human  and chimpanzee  [The (TTAGGG)T search  in the ger 2003) ,69.M. musculus; NR_001567, R. norvegicus; NR_001566, H. sapiens; gnl|ti|236061930, P. troglodytes). Sequences were aligned using the multiple sequence alignment software, MultAlin [A BLAT search of loci containing TERC-like sequences was performed in the genome of the four species using the TERC genes as query   human-specific ITS loci; (b) chimpanzee-specific ITS loci; (c) ITS loci inserted before the human-chimpanzee split for which the insertion mechanism was described previously; (d) ITS loci conserved in human and chimpanzee and inserted within repetitive elements. Additional data file The following additional data are available with the online version of this paper. Additional data file The four mechanisms of ITS insertion previously described .Click here for fileThe 128 mouse loci for which the orthologous rat loci were either not found or grossly rearranged and the 120 rat loci not found or rearranged in the mouse genome database.Click here for fileThe 58 ITS loci conserved in the two rodent species.Click here for fileMouse-specific and rat-specific ITSs together with the mechanism of their insertion and the number of nucleotides in register with the inserted telomeric repeats.Click here for fileThe 75 loci conserved in the two primate species and the three human loci for which the orthologous chimpanzee loci were not found or were grossly rearranged.Click here for file(a) Human-specific ITS loci; (b) chimpanzee-specific ITS loci; (c) ITS loci inserted before the human-chimpanzee split for which the insertion mechanism was described previously; (d) ITS loci conserved in human and chimpanzee and inserted within repetitive elements.Click here for fileThis figure shows how the mechanism of ITS insertion at DNA double strand break sites was deduced.Click here for fileThe sequence of all the TERC-ITS loci found in the rodent genomes and a description of their organization.Click here for file


Tumors of the cerebellum and brainstem account for half of all brain tumors in children. The realization that cerebellar lesions produce clinically relevant intellectual disability makes it important to determine whether neuropsychological abnormalities occur in long-term survivors of pediatric cerebellar tumors. Little is known about the neurobehavioral sequale resulting specifically from the resection of these tumors in this population. We therefore reviewed neuropsychological findings associated with postoperative cerebellar mutism syndrome and discuss the further implications for cerebellar cognitive function. Postoperative cerebellar mutism syndrome (CMS) is characterized by severely diminished or absent speech output as well as other neurological, cognitive, and behavioral impairments. The incidence of CMS in children who underwent surgery of the cerebellum is estimated to be between 8 and 31%.1 CMS has 36et al. studied the effect of isolated cerebellar hemispheric tumors post-operatively and compared them with the effect of temporal tumors. –50 47–50In conclusion, these findings show that the cerebellum is important in cognitive and affective functioning. Besides the similarity of frontal impairment of executive functions, there are also additional challenges, especially in visual-spatial domains, attention and short-term memory. The pattern of these dysfunctions, involving many different cognitive functions, supports the idea that cerebellar cognitive and affective symptoms cannot be explained by one uniform cerebellar dysfunction, but are rather a consequence of the many pathways connecting the cerebellum and the cerebrum in both directions. It is important to point to the psychopathological problems of these children. Both early and long-term neuropsychological follow-up and specific intervention until integration into professional life seem mandatory for children after posterior fossa tumors. Follow-up of all patients treated for a posterior fossa tumor in childhood should include extensive neuropsychological testing at regular intervals. This may be of benefit for school planning and later work planning.In children with CMS, executive, lingual and affective dysfunctions are seem to be related to tumor type, localization, time frame of the diagnosis, and receiving radio-/ chemotherapy after the operation. As briefly, in patients with medulloblastoma, at postoperative radio-/ chemotherapy period, and in the early age of diagnose, the incidence of CMS with executive, lingual, and affective dysfunctions are common. In addition, in patients with vermian lesions affective dysfunction and in patients with hemispherical lesions lingual and executive dysfunctions are usually affected. The improving survivals of these patients with the advances in treatment modalities may decline their life quality if they were evaluated only by their neurological deficits. Prior detection of problems due to executive, language and affective dysfunction, and multidisiplinary management strategies should be considered in the follow-up therapies. Patients have improved quality of life if they receive pharmacological, speech threapy, and indivudual educational support with physiciatric examination. Furthermore, a detailed understanding of cerebellary functions may give further insight into ADHD, autism, and schizophrenia-like physiciatric disorders.


In vivo observations of the mouse microcirculation canhardly be performed due to technical difficulties, limitingthe knowledge that could be obtained from gene manipulatedmice models. The aim of the present study was tocheck the applicability of a novel optical system, the orthogonalpolarization spectral technology, to study the mousemicrocirculation. In anaesthetized mice, the spinotrapeziusmuscle microcirculation was observed in situ. The diameterof precapillary arterioles was measured before and aftera pharmacological or hormonal stimulation. High-contrastimages of the muscle microcirculation were obtained andsignificant vasodilatation of arterioles was observed aftertopical applications of acetylcholine, sodium nitroprusside,and insulin. As compared to conventional techniques, orthogonalpolarization spectral imaging makes it possible toassess and study microvascular beds in mice, which were inaccessibleuntil now, allowing the use of gene manipulatedmice to investigate, for example, the mechanisms involvedin the development of diabetic microangiopathy.


DNA methylation by the Dnmt family occurs in vertebrates and invertebrates, including ascidians, and is thought to play important roles in gene regulation and genome stability, especially in vertebrates. However, the global methylation patterns of vertebrates and invertebrates are distinctive. Whereas almost all CpG sites are methylated in vertebrates, with the exception of those in CpG islands, the ascidian genome contains approximately equal amounts of methylated and unmethylated regions. Curiously, methylation status can be reliably estimated from the local frequency of CpG dinucleotides in the ascidian genome. Methylated and unmethylated regions tend to have few and many CpG sites, respectively, consistent with our knowledge of the methylation status of CpG islands and other regions in mammals. However, DNA methylation patterns and levels in vertebrates and invertebrates have not been analyzed in the same way.in silico along the phylogenetic tree. We observed a gradual transition from fractional to global patterns of methylation in deuterostomes, rather than a clear demarcation between vertebrates and invertebrates. When we applied this methodology to six piscine genomes, some of which showed features similar to those of invertebrates.Using a new computational methodology based on the decomposition of the bimodal distributions of methylated and unmethylated regions, we estimated the extent of the global methylation patterns in a wide range of animals. We then examined the epigenetic changes The mammalian global DNA methylation pattern was probably not acquired at an early stage of vertebrate evolution, but gradually expanded from that of a more ancient organism. In contrast, the DNA methylation status has been investigated widely, from mammals to invertebrate deuterostomes, such as the sea urchin, revealing that large parts of these genomes are subject to stable methylation, even in invertebrates [In mammals, DNA methylation is achieved by the collaboration of several DNA methyltransferases; 3 family . Howevertebrates .It is well documented that the global patterns and levels of DNA methylation are distinct between vertebrates and invertebrate deuterostomes . In the Curiously, especially to computational biologists, methylated and unmethylated regions can be reliably predicted from the local frequency of CpG dinucleotides . Separate.g., fishes and invertebrate chordates.So far, however, DNA methylation patterns and methylation levels have not been analyzed in the same way in vertebrates and invertebrates. The prevalent view that the DNA methylation of the two groups differs seems to have hindered us from examining their common features, despite the use of orthologous enzymes in both systems. It is unlikely that the change in the methylation pattern happened suddenly at a specific evolutionary stage. An abrupt change in the pattern of methylation could be deleterious to organisms. If a gradual transition from the mosaic to the global methylation pattern occurred, traces of the transition might be apparent in extant organisms located evolutionarily near the transition zone; in silico.To test this possibility of a gradual transition, we developed a new computational methodology to estimate the distributions of methylated and unmethylated regions in various animal genomes. This methodology does not confidently predict the methylation status or level in a specific region, but predicts the proportion of methylated and unmethylated regions from local frequencies of CpG dinucleotides. It is well known that in genomic imprinting and X-inactivation, one member of a pair of alleles with identical sequences sometimes exhibits a different level of DNA methylation, and these variations in methylation levels are often dependent on the developmental stages, tissues, or cell types . The levTo identify typical patterns of mosaic and global DNA methylation, we first arbitrarily chose four 2-Mb contiguous regions from four distinctive species and drew the changes in the ratios of observed over expected CpG numbers, hereafter called the "CpG score" (as in the UCSC database), along the genomic coordinates Figure . A mosaiStrongylocentrotus purpuratus (purple sea urchin), Branchiostoma floridae (Florida lancelet), Ciona intestinalis (ascidian), Danio rerio (zebrafish), Xenopus tropicalis (western clawed frog), Anolis carolinensis (green anole), Gallus gallus (chicken), Ornithorhynchus anatinus (platypus), Monodelphis domestica (gray short-tailed opossum), Canis familiaris (dog), Mus musculus (mouse), and Homo sapiens (human) to represent various clades, namely, echinoderms, cephalochordates, urochordates, fishes, amphibians, reptiles, avians, monotremes, marsupials, carnivores, rodents, and primates  to be 0.299, 0.124, and 0.063, respectively, which are very similar to those of the chicken , Oryzias latipes (Japanese medaka), Gasterosteus aculeatus (three-spined stickleback), Takifugu rubripes (torafugu), and Tetraodon nigroviridis (spotted green pufferfish). Intriguingly, the two ratios show a wide range of values in these non-tetrapod vertebrates  and mm8 , respectively.We downloaded the CpG island annotation data from We used the DNA methylation data of Human Epigenome Project Release 26th June 2006 ,20. We uvice versa. Consecutive statuses were clustered into a methylated or unmethylated region and the whole set of the lengths was used for the final statistical analysis to estimate the extent of global methylation pattern.We used the barycenter of the two decomposed normal distributions as the boundary of the methylated and unmethylated regions. In this way, every sliding window of 2 kb, moved in steps of 100 bp, was assigned to one of the two regions, producing binary data for each 100 bp covering the whole genomes. To smooth the binary data, a 2-kb sliding window analysis was performed again and the methylation status was decided based on whether methylation was dominant over unmethylation or The authors declare that they have no competing interests.KO conceived of the study, designed the study, and drafted the manuscript. KAM designed the study and analyzed the data. KN participated in the coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.Table S1. The genomic coordinates and their methylation status for the ascidian DNA methylation analysis. The ci2 chromosome name, start position, end position, methylation status, and CpG score are tabulated. Methylated and unmethylated statuses are represented by 1 and 0, respectively.Click here for file


The blood flow of tumours growing in rat mammary glands previously exposed to 1500 R X-rays was 52% that of same-sized tumours in unirradiated host tissue. About 2 months after irradiation, the blood flow of the mammary gland was raised and that of the skin was unchanged, compared with the corresponding unirradiated tissue. Tumours growing in preirradiated and unirradiated mammary glands responded similarly to bolus injections of noradrenaline, angiotensin II, and isoprenaline. The responses of the irradiated tissues to these drugs were, however, not always the same as those of the corresponding unirradiated tissues. Exposure of the lungs to approximately 1500 R X-rays made the animals uniquely sensitive to bolus injections of noradrenaline and angiotensin II.


Histone acetylation is one of the key regulatory mechanisms controlling transcriptional activity in eukaryotic cells. In higher eukaryotes, a number of nuclear histone acetyltransferase (HAT) enzymes have been identified, most of which are part of a large multisubunit complex. This diversity, combined with the large number of potentially acetylable lysines on histones, suggested the existence of a specific regulatory mechanism based on the substrate specificity of HATs. Over the past decade, intensive characterisations of the HAT complexes have been carried out. However, the precise mode of action of HATs, and particularly the functional differences amongst these complexes, remains elusive. Here we review current insights into the functional role of HATs, focusing on the specificity of their action. Studies based on biochemical as well as genetic approaches suggested that HATs exert a high degree of specificity in their acetylation spectra and in the cellular processes they regulate. However, a different view emerged recently from genomic approaches that provided genome-wide maps of HAT recruitments. The careful analysis of genomic data suggests that all HAT complexes would be simultaneously recruited to a similar set of loci in the genome, arguing for a low specificity in their function. In this review, we discuss the significance of these apparent contradictions and suggest a new model that integrates biochemical, genetic and genome-wide data to better describe the functional specificity of HAT complexes. Histone post-translational modifications have shown to be key regulators among transcription regulation mechanisms ,2. Histoin vitro acetylation assays were carried out to investigate the substrate specificity of distinct HATs. These analyses showed that HATs exert a certain degree of specificity for particular lysine residues on different histone tails. Second, in vivo gene inactivation studies allowed testing the HAT specificity by observing phenotypical effects caused by ablation of a particular HAT. Interestingly, most of these studies argued for a high degree of specificity in the developmental or gene expression phenotypes. More recently, availability of new high-throughput technologies such as chromatin immunoprecipitation sequencing (ChIP-seq) allowed the investigation of the recruitment of HATs and the deposition of acetylation marks at a genome-wide scale , whereas for most of the nuclear HATs only a single gene is present in nonvertebrate species. This observation raised the question of the functional importance of closely related paralogs in vertebrates. Answers were partially obtained by crossing mice carrying individual homozygous or heterozygous KOs of the given paralogs and comparing double KOs to that of the single KOs. For example, while the single CBP or p300 heterozygous mutants exert a milder phenotype than the homozygous mutants, the mice heterozygous for both HATs show phenotypes similar to the homozygous depletion of one or the other paralog . This reTaken together, these studies suggest that, amongst HATs, a high degree of functional specificity exists, with the exception of closely related paralogs that rather reflect the importance of gene expression dosage than functional specificity.In the past few years, our understanding of genome regulation has tremendously progressed due to the introduction of ChIP combined with microarray (ChIP-chip) or with high-throughput sequencing (ChIP-Seq). These experiments have allowed us to analyse the presence of a particular genomic feature at the genome-wide scale. Recently, several systematic ChIP-seq studies have been published, providing genome-wide mapping data for HATs and acetylation marks in resting human T cells ,13.This resource has given us the opportunity to analyse the specificity of genome-wide HAT recruitments. We have first extracted genome-wide distributions of five HATs [namely, p300, CBP, MOF , PCAF, and Tip60) and 18 acetylation marks and then systematically analysed the presence of each of the 18 acetylation marks at the binding sites of the five distinct HATs. Interestingly, this analysis revealed that, when comparing the genome-wide binding sites of a given HAT with that of the 18 different histone acetylation marks, no specific or unique acetylation pattern for any of the five HATs could be identified Figures  and 4b. per se would be requested for proper activation.The detected co-occurrence of HATs and the consequent broad acetylation patterns observed can suggest two different scenarios. First, since in ChIP cell populations rather than a single cell are analysed, we cannot exclude that the recruited HATs could occur in a stochastic manner. This would imply that in a given cell, one HAT is recruited to a particular locus, while in another cell, a distinct HAT is recruited instead to the same locus. In this scenario, the observed lack of histone acetylation specificity would come from the lack of specificity in the mechanisms that recruit HATs to particular loci. In this case, the acetylation specificity would not be an important feature for regulation, but only the acetylation et al. [In the second scenario, HATs would work collaboratively. In all cells, all the studied HATs would be permanently recruited and released at every bound loci in a dynamic fashion. This scenario implies also that many transcriptional coactivators can be dynamically recruited all the time to a set of regulated loci to modify (acetylate) the given chromatin environment. Such a mechanism for coactivator action has already been suggested by Hager et al. . Thus, gin vivo action of HAT complexes  that are not challenged, one can assume that they exert only "routine" gene expression programs compared to the major transcriptional changes happening during differentiation of a tissue or an organism. We hypothesize that the genome-wide data obtained in the resting somatic cells reflect a "maintenance stage" in the gene activation process  that may not be the case in resting differentiated somatic cells. During cell differentiation, the high complexity level of transcriptional regulatory networks is a prerequisite for the orchestration of the time-controlled initiation of the proper transcriptional programs. However, in resting somatic tissues, where the number of newly initiated transcription processes is likely to be lower than during cell commitment, the large panel of activators would redundantly maintain activation states. This could explain why high redundancy in the recruitment of HATs is observed in resting cells, while their functional specificity is needed to achieve proper embryo development.Drosophila [The above-presented data  is focusosophila ,25. The osophila , these ein vitro stem cell differentiation systems [The very recent improvement of the genome-wide mapping technologies and the development of novel ChIP-grade antibodies against HATs and complex-specific subunits will allow researchers to verify these genome-wide observations and provide new insights into the functional roles of HATs. For example, the time course analysis of different HAT recruitments upon dynamic activation of a particular gene networks, for example,  systems , should HAT-containing complexes are key components of chromatin-mediated transcriptional regulatory networks. Proper understanding and modelling of their mode of action and function within these networks is a prerequisite for accurate prediction of the behaviour of transcriptional systems. Recent technology breakthrough in the postgenomic area allowed new insights into the function of HATs, but were often interpreted with the liability of ignoring previously available biochemical and genetic data, leading to oversimplified models. Here we propose novel considerations that could reconcile the different lines of evidence in a unified model that describes the mode of action of HATs more precisely. Our current model proposes that HATs are differently required, depending on the stage of gene activation, with a high functional specificity in the early gene activation stage and with a less-specific functionality in the later maintenance stage.The authors declare that they have no competing interests.ARK, KA and LT contributed to the study design. KA and ARK performed the bioinformatics analyses. KA, ARK, DD, JT, OP and LT wrote the manuscript. All authors read and approved the final manuscript.


Leishmania amastigotes within macrophages from skin and popliteal lymph node biopsies. At necropsy, lesions were found in different organs, but it was particularly striking to observe large areas of pallor in the myocardium. Histological examination revealed an intense chronic inflammatory reaction in many organs, and numerous macrophages were found to contain amastigote forms of Leishmania. The inflammatory reaction was especially severe in the heart, where large areas of the myocardium appeared infiltrated with huge numbers of mononuclear immune cells, causing cardiac muscle atrophy and degeneration. Despite the severe inflammation, the number of parasitized macrophages was low in the myocardium, as revealed by immunohistochemical staining of Leishmania amastigotes. Because cardiac involvement is not usually described in this condition, this dog represents a very rare case of canine visceral leishmaniasis with affection of the myocardium.A dog presented with cutaneous nodules, enlarged lymph nodes and oedema in limbs, face and abdomen. The diagnosis of visceral leishmaniasis was established by identification of  Leishmania that is transmitted to the dog by the bite of blood-sucking sand flies (Phlebotomus species). The parasite reproduces itself within the host's macrophages and, as a consequence, causes an intense mononuclear inflammatory reaction consisting of mononuclear immune cells. In the Mediterranean countries, L. infantum is the species usually implicated in canine visceral leishmaniasis, a severe systemic form of the disease characterized by progressive wasting due to the involvement of multiple organs such as spleen, liver, lymph nodes, bone marrow, kidneys and skin [Canine leishmaniasis is a zoonotic parasitic disease caused by an intracellular protozoan of the genus and skin ,2. The cand skin . In this9/l), thrombocytopenia (54 × 109/l) and hypoalbuminemia (2.10 g/dl). Blood urea nitrogen (BUN) and creatinine values were under normal ranges, but urine specific gravity was 1.025, with protein level of 2+. An electrocardiogram (ECG) showed low amplitude of the QRS complex with normal cardiac rate and rhythm. Routine histological evaluation of biopsies obtained from the skin and the popliteal lymph node revealed a chronic non-suppurative inflammation consisting mainly of macrophages and some plasma cells and lymphocytes. Small vacuoles containing basophilic bodies morphologically consistent with Leishmania amastigotes were also seen in the cytoplasm of a few macrophages and therefore, visceral leishmaniasis was diagnosed.An 11-year-old female Basset Hound was presented to the Hospital Clinico Veterinario Rof Codina with a history of swelling of the head and limbs. On initial evaluation, abnormal physical examination findings included non-ulcerated cutaneous nodules, a generalized mild lymphadenomegaly of the superficial lymph nodes, a stiff gait and oedema in limbs, face and abdomen. Haematological abnormalities included normochromic anaemia , lymphopenia/monocytopenia  and Giemsa stains, and evaluated by light microscopic examination. The presence of antibody .The most important histopathological finding was the presence of an intense chronic inflammatory reaction composed of mononuclear cells  in most organs. In the heart, the myocardium showed a dense accumulation of macrophages between the muscle fibers Fig  and alsoLeishmania amastigotes. In lungs, kidneys and skin, amastigote forms of Leishmania were seen inside macrophages, but with restricted distribution in the inflammatory infiltrate. Notably, the parasite load was extremely low in the heart, since only a few macrophages were parasitized, requiring careful search to identify them in a background of severe inflammation.The parasite load was high in spleen, lymph nodes and liver, organs where most of the macrophages had large cytoplasmic parasitophorous vacuoles with round basophilic nucleus and distinct bar-shaped paranuclear kinetoplast, morphological features compatible with Leishmania antigens, resulting in a strong labelling of amastigotes inside macrophages (Fig The identification of parasitized macrophages was greatly facilitated in all organs, but especially in the heart, by the immunohistochemical detection of ages Fig .Leishmania amastigotes has been undoubtedly demonstrated [Cardiac involvement in canine visceral leishmaniasis appears to be anecdotal, since only a very few cases of myocardiopathy during the course of the disease have been reported in the literature to date -7. Howevnstrated .Leishmania. Therefore, affection of the myocardium and, as a consequence, impairment of the cardiac function must be added to the plethora of lesions and clinical signs that may be present in dogs with the visceral form of this chronic parasitic disease.In the case we report here, the occurrence of subcutaneous oedemas was attributed to hypoalbuminemia as a result of nephropathy with protein loss, with changes in the ECG that correlated with this initial diagnosis. After the definitive diagnosis of visceral leishmaniasis, renal dysfunction can be explained by glomerulonephritis due to deposition of immune complexes, while changes in ECG may be also explained by the occurrence of myocarditis caused by The authors declare that they have no competing interests.MLP and JMN were responsible for the necropsy and the histological examination. NA and DF were responsible for the immunohistochemistry. FM, AG and MLS were responsible for the clinical examination of the dog. All authors were involved in drafting the manuscript and gave final approval of the manuscript.


The etiology of seizure disorders in lung cancer patients is broad and includes some rather rare causes of seizures which can sometimes be overlooked by physicians. Paraneoplastic limbic encephalitis is a rather rare cause of seizures in lung cancer patients and should be considered in the differential diagnosis of seizure disorders in this population.This case report describes the new onset of seizures in a 64-year-old male patient receiving chemotherapy for a diagnosed stage IV non-small cell lung carcinoma. After three cycles of therapy, he was re-evaluated with a chest computed tomography which showed a 50% reduction in the tumor mass and in the size of the hilar and mediastinal lymphadenopathy. Twenty days after the fourth cycle of chemotherapy, the patient was admitted to a neurological clinic because of the onset of self-limiting complex partial seizures, with motionless stare and facial twitching, but with no signs of secondary generalization. The patient had also recently developed neurological symptoms of short-term memory loss and temporary confusion, and behavioral changes. Laboratory evaluation included brain magnetic resonance imaging, magnetic resonance spectroscopy of the brain, serum examination for 'anti-Hu' antibodies and stereotactic brain biopsy. Based on the clinical picture, the patient's history of lung cancer, the brain magnetic resonance imaging findings and the results of the brain biopsy, we concluded that our patient had a 'definite' diagnosis of paraneoplastic limbic encephalitis and he was subsequently treated with a combination of chemotherapy and oral steroids, resulting in stabilization of his neurological status. Despite the neurological stabilization, a chest computed tomography which was performed after the 6th cycle showed relapse of the disease in the chest.Paraneoplastic limbic encephalitis is a rather rare cause of new onset of seizures in patients with non-small cell lung carcinoma. Incidence, clinical presentation, laboratory evaluation, differential diagnosis, prognosis and treatment of this entity are discussed. The etiology of seizure disorders in patients with cancer is broad. Intracranial metastasis, adverse drug reactions, drug withdrawal or intoxication, metabolic disturbances and infections are the most common causes, but the differential diagnosis also includes rarer causes which can sometimes be overlooked by physicians treating such patients. We report a case of paraneoplastic limbic encephalitis (PLE) which is a rather rare cause of seizures in patients with non-small cell lung carcinoma.4N2M0) undifferentiated large cell lung carcinoma was diagnosed in a 64-year-old Greek man. He was a smoker with a smoking history of 60 pack-years. Twenty-two years earlier, he had been diagnosed with a seminoma of the left testicle, for which he had been treated with surgical resection and adjuvant regional radiotherapy.Stage IV  scan revealed a mass in the left upper lobe, lymphadenopathy in the left hilum and the mediastinum, and two small nodules in the right lower lobe.A brain CT scan showed an edematous area with no contrast enhancement in the left temporal lobe, but the patient, who had no neurological symptoms and had a normal neurological clinical examination, refused further investigation using magnetic resonance imaging (MRI). An abdominal CT scan and a bone scan were negative for metastases.The patient was started on intravenous chemotherapy with a combination of carboplatin, etoposide and epirubicin every 28 days, and after three cycles of therapy he was re-evaluated using CT. The chest CT showed a 50% reduction in the mass in the left upper lobe and in the size of the hilar and mediastinal lymphadenopathy. There was no change in the nodules in the right lower lobe, or in the appearance of the abdominal or brain CT scans.Twenty days after the fourth cycle of chemotherapy, the patient was admitted to a neurological clinic because of the onset of self-limiting complex partial seizures, including motionless stare and facial twitching, with no signs of secondary generalization. His relatives stated that, during the previous 2 weeks, the patient had developed neurological symptoms of short-term memory loss and temporary confusion, and behavioral changes including anxiety and depression. He was started on anticonvulsants  and soon after underwent a brain MRI, which showed findings of cerebral gliomatosis  is associated with the greatest frequency and diversity of paraneoplastic syndromes, including Cushing's syndrome, syndrome of inappropriate antidiuretic hormone secretion and rare paraneoplastic neurological syndromes [The most common paraneoplastic neurological syndromes are Lambert-Eaton myasthenic syndrome and paraneoplastic encephalomyelitis (PEM). Paraneoplastic neurological syndromes are caused by autoimmune processes triggered by cancers and directed against antigens common to both the cancer and the nervous system, designated as onconeural antigens. Autoantibodies against onconeural antigens, strongly associated with cancer and detected by several laboratories in a reasonable number of patients with well defined paraneoplastic neurological syndromes, have been described as 'well characterized' paraneoplastic antibodies .PEM is characterized pathologically by neuronal loss and inflammatory infiltrates in particular areas of the nervous system. The location and severity of the neuronal loss, which may be confined to one area of the nervous system or involve multiple areas over time, predict the clinical symptoms of the patient .The predominant neurological syndrome of PEM at diagnosis is sensory neuropathy. Other common neurological syndromes associated with PEM are cerebellar ataxia, limbic encephalitis (LE), brainstem encephalitis, intestinal pseudo-obstruction and parietal encephalitis.PLE is clinically characterized by subacute cognitive dysfunction with severe memory impairment, seizures and psychiatric features, including depression, anxiety and hallucinations. Hypothalamic dysfunction may occur, with somnolence, hyperthermia and endocrine abnormalities. PLE may present as an isolated neurological syndrome or as a part of PEM. It may occasionally be associated with thymoma, or testicular, bladder, colon or kidney cancer, or non small cell lung cancer (NSCLC), but SCLC is by far the most frequent underlying tumor. Because LE is associated relatively often with cancer, it is characterized as a 'classical' paraneoplastic neurological syndrome .Clinical diagnosis of PLE associated with lung cancer is difficult. Generally, the following criteria must be fulfilled :a) clinical picture of seizures, memory loss and/or confusion or psychiatric symptoms suggesting involvement of the temporal lobes or limbic system.b) temporal relationship  between the onset of neurological symptoms and diagnosis of lung cancer. PLE usually precedes the diagnosis of cancer by a median time of 8 months, but may also appear after tumor diagnosis, either in the first 6 months  or when the tumor is in remission .c) absence of metastatic, metabolic , infectious , vascular (ischemia or hemorrhage), drug-related  or chemotherapy  treatment-related causes that could account for the neurological dysfunction.d) abnormal MRI of the head characterized by a high-intensity signal on T2-weighted images and atrophy (and sometimes enhancement with contrast injection) on T1-weighted images in one or both medial temporal lobes. In selected difficult cases, co-registration of 18F-fluorodeoxyglucose positron emission tomography may further improve the sensitivity of imaging. CT scans may show no contrast-enhancing lesion in the temporal lobe, but are not sensitive or specific for the diagnosis of PLE.Other analyses which can help in the diagnosis of PLE are :a) serum or cerebrospinal fluid (CSF) examination positive for 'anti-Hu' antibodies (autoantibodies generated against the Hu antigen found in the nucleus of neurons). Anti-Hu antibodies have been consistently reported in PLE associated with lung cancer (about 50–60% of patients with SCLC and PLE have anti-Hu antibodies), although their absence does not rule out the syndrome . PLE in b) CSF analysis assists in making the diagnosis of PLE by detecting inflammatory abnormalities  supporting the diagnosis of an inflammatory or immune-mediated neurological disorder and by confirming the absence of malignant cells, excluding  the presence of leptomeningeal metastases.c) electroencephalography is useful in assessing whether changes in the level of consciousness or behavior are related to temporal lobe seizures.d) biopsy from MRI enhancing areas usually reveals perivascular cuffing, interstitial infiltrates of lymphocytes, microglial proliferation, gliosis and neuronal degeneration, and confirms the absence of malignant cells.In 2004, an international panel of neurologists established diagnostic criteria that divide patients with a suspected paraneoplastic neurological syndrome into 'definite' and 'probable' categories, based on the presence or absence of a typical clinical picture, cancer and specific autoantibodies [The prognosis of PEM remains poor, in terms of survival and neurological impairment. The median survival time is 11.8 months after onset of the PEM, with a 3-year actuarial survival of 20% . In patiPatients with PLE and lung cancer who are negative for anti-Hu antibodies seem to improve more often after treatment of their cancer than those who have anti-Hu antibodies. The Anti-Hu(+) patients usually die from complications of their neurological status, whereas in the Anti-Hu(-) group, death is due to progression of the lung cancer. The limited stage of the disease, irrespective of anti-neuronal antibody status, is associated with neurological improvement after tumor treatment.The treatment of LE is generally unsatisfactory. There are two aspects of the treatment :a) removing the antigen source by treating the underlying malignancy.b) suppressing the immune reaction with the use of immunosuppressive drugs or therapies.The treatment of the underlying malignancy appears to be more effective on neurological outcome than the use of immune modulation. In a study of 200 patients with anti-Hu-associated PEM , there wAlthough immunotherapy alone is probably not effective in the majority of the patients, a trial with it should be considered when anti-neoplastic treatment is not possible because the tumor cannot be found or when PEM appears during or after tumor treatment. There are no established protocols for immunotherapy. Immunotherapies include methylprednisolone, azathioprine, tacrolimus, intravenous immunoglobulins +/- cyclophosphamide or methylprednisolone, plasma exchange and removal of plasma IgG by immunoadsorption with a protein A column.Although there is no generally effective treatment for all such patients, early diagnosis and treatment of the tumor seem to give the best chance to stabilize the disease, especially in patients negative for anti-Hu antibodies . For thiParaneoplastic limbic encephalitis is a possible cause of seizures in patients with lung cancer. New onset of paraneoplastic limbic encephalitis in patients with already diagnosed lung cancer is usually associated with progression or relapse of the disease. Patients with paraneoplastic limbic encephalitis and lung cancer who are negative for anti-Hu antibodies are more likely to improve after treatment of the tumor and have lower chances of developing paraneoplastic encephalomyelitis than those who have anti-Hu antibodies. Early diagnosis and treatment of the tumor offer the best chances for improvement in patients with paraneoplastic limbic encephalitis.The authors declare that they have no competing interests.VV and EM drafted the final version of this manuscript. CG helped to draft the manuscript. AK participated in data collection and treated the patient. CG conducted the brain biopsy. SP collected the histological photos and rendered an interpretation. EV evaluated the radiological findings. PC conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Previous studies have shown that a particular site in the periaqueductal gray (PAG), the rostrolateral PAG, influences the motivation drive to forage or hunt. To have a deeper understanding on the putative paths involved in the decision-making process between foraging, hunting, and other behavioral responses, in the present investigation, we carried out a systematic analysis of the neural inputs to the rostrolateral PAG (rlPAG), using Fluorogold as a retrograde tracer. According to the present findings, the rlPAG appears to be importantly driven by medial prefrontal cortical areas involved in controlling attention-related and decision-making processes. Moreover, the rlPAG also receives a wealth of information from different amygdalar, hypothalamic, and brainstem sites related to feeding, drinking, or hunting behavioral responses. Therefore, this unique combination of afferent connections puts the rlPAG in a privileged position to influence the motivation drive to choose whether hunting and foraging would be the most appropriate adaptive responses. Previous studies from our laboratory, examining the neural basis ofmorphine-induced inhibition of maternal behavior, brought up the suggestion ofa rather unsuspected and integrative role of the periaqueductal gray (PAG) ininfluencing the selection of adaptive behavioral responses , 2.Examining the neural basis underlying maternalbehavior inhibition by low doses of morphine in morphine-experienced dams, wefound that morphine treatment induces a behavioral switch from maternal topredatory behavior. Hence, morphine-challenged dams, tested in an environmentcontaining both pups and roaches (which served as prey), clearly preferredhunting instead of nursing . We haveThe results of behavioral, neuronal immediate early gene activation, andlesion experiments indicate that a particular site in the PAG, at the level ofthe oculomotor nucleus, located in the outer half of the lateral column, andreferred to as the rostrolateral PAG (rlPAG), should be responsible for thisswitching from maternal behavior to prey hunting in morphine-treated dams . First,To start unraveling this circuit, in thepresent study, we performed a comprehensive investigation on the rlPAG afferentconnections, using the Fluorogold as retrograde tracer. A number of retrogradetract-tracing studies have investigated the afferent sources of inputs to thePAG, but they used a much less sensitive retrograde tracer  and were based on largeinjection sites encompassing different PAG functional domains . TheretOverall, the present results support the ideathat the rlPAG combines a unique set of inputs rendering this regionparticularly suitable for influencing the decision-making process betweenhunting, foraging, and other behavioral responses.n = 18) weighing 190–220 g andapproximately 90 days of age at the beginning of the experiments. Food and waterwere available ad libitum tothe animals in light-controlled (06:00 AM to 06:00 PM) andtemperature-controlled (23–25°C)rooms. Conditions of animal housing and all experimental procedures wereconducted under institutional guidelines of the Committee on Animals of the  andthe Committee on the Care and Use of Laboratory Animal Resources, National Research Council.Subjectswere adult female Wistar rats (μm) by applying a +3 μA current, pulsed at 7-second intervals, with aconstant-current source . After a survival time of 7–12 days, theanimals were deeply anesthetized with sodium pentobarbital  andperfused transcardially with a solution of 4.0% paraformaldehyde in 0.1 Mphosphate buffer, pH 7.4; the brains were removed and left overnight in asolution of 20% sucrose in 0.1 M phosphate buffer at 4°C. The brainswere then frozen, and five series of 30-μm-thick sections were cut on a slidingmicrotome in the transverse  plane and collected from the caudalmedulla through the rostral tip of the prefrontal cortex. One complete serieswas processed for immunohistochemistry with an antiserum directed againstFluorogold  at a dilution of 1:5000. The antigen-antibodycomplex was localized with a variation of the avidin-biotin complex system (ABC)[Animals were anesthetized with amixture of ketamine  andxylazine , andunilateral iontophoretic deposits of a 2% solution of Fluorogold were placed stereotaxically into the rlPAG . Deposits were made over 5 minutes through a glass micropipette . The appearance of arepresentative Fluorogold injection site in the rlPAG is illustrated in Retrogradely labeled neurons were found in theisocortex, amygdala and in the septal region. No retrogradely labeled cellswere found in the hippoccampal formation.In the isocortex, a large number of retrogradely labeled neurons was observed inthe prelimbic, infralimbic, anterior cingulate, and secondary motor areasFigures , in addiIn the lateral septal nucleus, a relatively sparse number of marked cells were observedin the rostral part of the nucleus, distributed mainly through the dorsalregion of the ventrolateral zone Figures . In the At the thalamus, a substantial retrograde labeling was found in theventral part of the zona incerta Figures . No markIn thepreoptic region, a substantial retrograde labeling was found in the median preopticnucleus Figures , in addiAt the anterior hypothalamic levels, the anterior part of the anteriorhypothalamic nucleus presented a dense cluster of retrogradely labeled cells,which were distributed in the region that seems to overlap, at least partially,with a territory known to contain a large number of neurons expressingenkephalin  Figures. MoreoveAt tuberal levels, a dense number ofretrogradely labeled cells was found in the anterior part of the ventromedialnucleus, in addition to a moderate number of marked cells in theretrochiasmatic area, the ventrolateral and central parts of the ventromedialnucleus, the tuberal nucleus, and rostral parts of the posterior hypothalamicnucleus Figures . FurtherAt the mammilary levels, a large number of labeled neurons were found in the dorsal premammilary nucleus, mostlydistributed in the dorsal part of the nucleus . FinallyAt mesodiencephalic levels, a large number ofmarked cells were found in the precommissural nucleus . In the At rostral pontine levels, a fewlabeled cells were found in the laterodorsal tegmental nucleus, as well as inthe central lateral, dorsal lateral, and ventral lateral parts of theparabrachial nucleus .It should be noted that, in theexperiments with Fluorogold deposits centered in the rlPAG, retrograde labelingwas mostly ipsilateral. However, some of the main sources of projections tothis area, including the prefrontal cortex, retrochiasmatic area, anterior hypothalamicnucleus, ventromedial hypothalamic nucleus, lateral hypothalamic area, zonaincerta, precommissural nucleus, and the PAG, also displayed conspicuousretrograde labeling contralateral to the injection site.The results of the present retrograde axonaltract-tracing study suggest that the rlPAG receive inputs from several widelydistributed areas in the forebrain and, to a lesser extent, from the brainstem,as well. Prefrontal cortical areas represent the major telencephalic source ofinputs to the rlPAG. In addition, clear telencephalic inputs appear to arisefrom the gustatory, visceral, and perirhinal cortical areas, as well as fromthe medial part of the central amygdalar nucleus and the interfascicularnucleus of the bed nuclei of the stria terminalis. In the diencephalon, massiveinputs to the rlPAG arise from several hypothalamic sites, including the medianpreoptic nucleus, anterior hypothalamic nucleus, retrochiasmatic area, anterior,and ventrolateral parts of the ventromedial nucleus, dorsal premammillarynucleus (PMd), and several districts of the lateral hypothalamic area. Incontrast, the rlPAG appears to receive inputs from considerably fewer brainstemsites, where significant retrograde labeling was found in other parts of thePAG  and in the intermediatelayers of the lateral part of the superior colliculus.In agreement with previous anterogradetract-tracing studies, medial prefrontal cortical areas, including the infralimbic,prelimbic, anterior cingulated, and secondary motor areas, appear to representone of the most important afferent sources of projections to the rlPAG –20. In lThe present findings also revealed that the rlPAG istargeted by several hypothalamic districts. At preopotic levels, the rlPAGseems to receive substantial inputs from the median preoptic nucleus, inaddition to somewhat sparse inputs from the anteroventral preoptic nucleus. Both of these projections have been confirmed by previous PHAL studies . AtanteThe present results also revealedthat a small number of brainstem sites appear to innervate the rlPAG. Evidentretrograde labeling was found in the precommissural nucleus, the dorsomedialand ventrolateral parts of the PAG, and the lateral part of the intermediatelayer of the superior colliculus. All of these projections have been confirmedthrough PHAL anterograde tract-tracing method [As mentioned above, previous retrogradetract-tracing studies have investigated the afferent sources of inputs to thePAG, but they were based on large injection sites, providing just an overallpicture of the afferent inputs to the PAG without differentiating among theparticular PAG domains . By deteThe evidence just discussed supports the idea that different functional PAG domainsare likely to have particular sets of afferent inputs, and next, we willprovide a discussion on how the diverse inputs to the rlPAG may be related toits function on hunting and foraging behavior.As commented in the Introduction, the rlPAG is an opioid sensitive site, which iscritical for influencing the motivation drive to hunt and forage, and is likelyto be part of a neural circuit involved in the decision-making process between hunting, foraging, andother behavioral responses. The present findings help to reveal this complexnetwork, and reinforce the rlPAG's nodal role in integrating a wealth ofdifferent kinds of information likely to influence foraging or huntingactivity.One of the main findings of the present investigation is that the rlPAG appears to beparticularly driven by inputs from medial prefrontal cortical areas. In thepresent context, it is noteworthy that, in the prefrontal cortex, the anterior cingulate, the prelimbic, and theinfralimbic cortices have been associated with diverse emotional, cognitive, andmnemonic functions that underlie attentional and decision-making processes , 40. In As commented in theIntroduction, the rlPAG upregulated Fos expression in animals performinginsect hunting, and in the present study, we have shown that the rlPAGintegrates inputs from a number of neural sites related to the circuitryunderlying predatory hunting. Thus, we have presently found that the medialpart of the central amygdalar nucleus provides a significant projection to the rlPAG. The medial part of the central amygdalar nucleus is part of a distinctamygdalar circuit comprising; the anteriorpart of the cortical nucleus, the anterior part of the basomedial nucleus and theposterior part of the basolateral nucleus, which has been shown to be mobilizedduring insect predation and seems to be particularly involved in processingvisceral, gustatory and olfactory information –44. The The rlPAG also seems to be innervated by lateral hypothalamic regionsactivated during predatory hunting, namely, the parasubthalamic nucleus and theregion containing cells expressing MCH andhypocretin/orexin . The parWe have presently shown that the rlPAG is also targeted by the lateralpart of the intermediate layer of the superior colliculus, which we have alsopreviously shown to be mobilized during insect hunting . In factThe rlPAG is also significantly targeted by hypothalamic districts thatdo not seem to be directly involved in controlling feeding or hunting behavior,namely, the median preoptic nucleus, the retinoceptive region of the lateralhypothalamic, area and the dorsal premammillary nucleus. The median preopticnucleus is classically known to respond to plasma osmolarity and influencedrinking behavior , and thiHere, it is important toconsider that functional studies using electrical or chemical stimulation havesuggested a role for the lateral PAG in defensive responses. In the rat,chemical stimulation with kainic acid (KA) in the region of the rlPAG producedstrong backward defense , a kind In addition, the concept of a distinct functional column for the entire lateralPAG should also be revised. In fact, the role here assigned to the rlPAG doesnot seem to apply to the caudal half of the lateral PAG, since lesionsencroaching upon this latter PAG region do not seem to affect the motivationaldrive to hunt , 33.Overall, the present findingssupport the idea that the rlPAG has a central role in a complex networkcontrolling the decision-making processbetween hunting, foraging, and other behavioral responses. On one hand, therlPAG appears to be importantly driven by medial prefrontal cortical areasinvolved in controlling attentional and decision-making processes. Onthe other hand, the rlPAG receives a wealth of information from differentneural sites related to feeding, drinking, or hunting behavioralresponses. Therefore, this uniquecombination of afferent connections puts the rlPAG in a privileged position toinfluence the motivation drive to choose whether hunting and foraging are the most appropriate adaptiveresponses.The supplementary material contains a list of abbreviations mentioned in the article.Click here for additional data file.


The urethra is a usual site of introduction of foreign bodies for autoerotic stimulation. We present an unusual case of bladder perforation caused by foreign body that was self-inserted in the urethra and consequently slipped inside the bladder in a 29-year-old female patient with psychiatric disease. The patient was referred to our department for macroscopic hematuria and abdominal pain. Imaging studies revealed the presence of a foreign body in the pelvic area which had perforated the left lateral wall of the bladder. The foreign body was removed via open cystotomy. In psychiatric patients hematuria and pelvic pain may result from insertion of a foreign body in the bladder usually during masturbation. Foreign Bodies (FBs) usually enter the bladder via migration from the urethra either during clitoris or penis masturbation. Other rare causes are iatrogenic or migration from surrounding structures, vagina, cervix, uterus, or rectum. The aim of our study is to discuss the diagnostic and therapeutic implications in a challenging case of bladder perforation caused by the insertion of a foreign body in the bladder during masturbation.We present the case of a 29-year-old female suffering from dementia which was eventually found to have inserted a pencil into the urethra for the purpose of masturbation. The pencil accidentally slipped into the urinary bladder and the patient was referred to the emergency room a few days after the incident due to macroscopic hematuria, dysuria, and lower abdominal pain. On initial questioning the patient did not reveal the insertion of FB in her urethra. Therefore, imaging studies were undertaken as in the routine evaluation of macroscopic hematuria. Bladder ultrasonography (US) , and intA wide range of foreign bodies that were self-introduced in the urethra and bladder has been reported in both sexes. The insertion of objects such as eyebrow pencil, wrist watch, Blu-Tack, cable, rubber tube, electrical wire, cocaine, hair, ballpoint pen, or even cucumber has been reported in the literature –9. The most common reason for self-insertion of a foreign body into the urethra is of erotic or sexual nature, usually for masturbation –13. The In the majority of these cases clinical presentation varies from asymptomatic to swelling of the external genitalia, dysuria, poor urinary stream or retention, bloody or purulent urethral discharge, and ascending urinary tract infection , 11. In With regard to management and depending on the type of foreign body and its location, various methods of removal have been described, including meatotomy, cystoscopy, internal or external urethrotomy, suprapubic cystostomy, Fogarty catheterization, and injection of solvents . EndoscoIn cases where endoscopic techniques are unsuccessful, then one has to resolve to open surgery as in our case. Endoscopic retrieval of a foreign body from the male urethra and bladder may be more challenging due to the longer urethral length and the occasional presence of obstructive BPH in older men. On the contrary, endoscopic retrieval is facilitated by the shorter length and wider diameter of the female urethra. For objects stuck in the penile urethra, external urethrotomy is recommended , while fThe case presents a potentially serious complication of an accidentally introduced foreign body into the urinary bladder during masturbation. Macroscopic hematuria and abdominal pain should raise the suspicion of a foreign body insertion in the urinary bladder, especially in psychiatric patients.


From being a mere (though important) diagnostic tool, radiology has evolved to become an integral part of therapy in portal hypertension today. Various procedures are currently available, the choice depending on the etiology and location of disease, the pathoanatomy, and the symptomatology. The main aim of any procedure is to reduce the portal pressure by either direct or indirect methods. This can be achieved with transjugular intrahepatic portosystemic shunt (TIPS), recanalization of the hepatic vein outflow, recanalization of the portal vein and its tributaries, recanalization of dysfunctional portosystemic shunts, partial splenic embolization, and embolization of arterioportal shunts. When any of these procedures cannot be performed due to anatomical or physiological reasons, the symptoms can often be controlled effectively with embolization of varices or balloon-occluded retrograde transvenous obliteration of varices (BRTO). This article briefly describes the procedures, their results, and their current status in the treatment of portal hypertension. The role of the radiologist in the management of portal hypertension (PHT) has undergone a significant metamorphosis over the last few decades. Initially, it was limited to determining the presence and cause of PHT, using angiographic techniques such as percutaneous splenoportography, transhepatic portography, and arterioportography. For many years these procedures were conducted frequently for planning surgical treatment; later, they were replaced by safer and equally reliable modalities such as USG, CT scan and CT angiography (CTA) and MRI and MRI angiography (MRA).Progressively, the focus changed from diagnostic to therapeutic procedures. Interventions involving the portal venous system were introduced in the 1970s, beginning with transhepatic embolization for control of gastric and esophageal variceal bleeding. The subsequent decade saw an expansion in the variety of therapeutic interventions, with procedures such as transjugular intrahepatic portosystemic shunt, portal vein recanalization, balloon-occluded retrograde transvenous obliteration of varices, hepatic venous outflow angioplasty, and revision of surgical shunts being rapidly introduced one after the other. Since then radiological interventions have become established methods in the treatment of PHT.Transjugular intrahepatic portosystemic shunts (TIPS)Recanalization of hepatic venous outflowRecanalization of the occluded portal vein and its tributariesEmbolization of arterioportal fistulaPartial splenic embolizationRevision of occluded surgical or radiological portosystemic shuntsInterventions that reduce portal blood pressure:Percutaneous transhepatic variceal embolizationBalloon retrograde obliteration of gastric varices (BRTO)Percutaneous peritoneovenous shuntInterventions to palliate symptoms related to portal hypertension :The primary goal in treating portal hypertension is reduction in the portal venous pressure itself. This should mitigate the complications of portal hypertension such as bleeding from varices and congestive gastroenteropathy, accumulation of ascites and hydrothorax, or the hepatorenal syndrome, etc. When it is not possible to achieve this primary goal, various procedures can be offered to palliate or control the symptoms related to portal hypertension. The various portal vein interventions can be broadly categorized as:TIPS is a portosystemic shunt created within the liver parenchyma between the hepatic vein and the portal vein. The procedure involves many steps: (1) puncture of the jugular vein, (2) cannulation of the hepatic vein, (3) passage of a long needle from the hepatic vein, through the liver parenchyma and into the portal vein, (4) dilatation, with an angioplasty balloon, of the parenchymal tract created by the needle, and (5) stent deployment to ensure patency of the tract. Usually an 8-10 mm diameter stent is chosen to adequately decompress the hypertensive portal circulation to achieve a final portosystemic gradient of less than 12 mm Hg .TIPS was conceived as early as 1969, when Rösch reported this method in a series of dog experiments using Teflon tubes as stents..2]2]3) and severe coagulopathy (INR >5).313031]Arterioportal fistulae (APF) are a rare cause of PHT. They may be congenital or secondary to trauma, surgery, percutaneous biopsy or other liver procedures, and liver tumors. Although silent most of the times, some patients with large APFs can present with features of PHT such as bleeding, ascites, and splenomegaly. The preferred treatment is transarterial embolization of the feeding artery, using coils, detachable balloons, or glue. The procedure is usually successful, provided the fistula is not too large and is accessible.PSE is performed to diminish inflow of blood into the portal vein, with secondary reduction of the portal venous pressure. The procedure involves superselective catheterization and embolization of the intrasplenic arterial branches, usually with polyvinyl alcohol particles. This achieves reduction of portal vein pressure, reduction in splenic size, and improvement in hypersplenism-induced thrombocytopenia. The resuPTE was the earliest intervention performed for portal hypertension and was first described by Lunderquist and Vang in 1974 to treat intractable variceal bleeding. In this technique, the portal vein is catheterized by a percutaneous transhepatic approach and the gastric vein feeding the varix is embolized with ethanol, steel coils, or cyanoacrylate glue . When fiBRTO is a technique that is popular in Japan for control of gastric varices through a natural gastrorenal shunt. The technique involves advancing a balloon catheter from the femoral vein into the outlet of the gastrorenal shunt. Following balloon occlusion of the shunt, sclerosant (5% ethanolamine oleate) is injected retrogradely to fill the gastric varices. After adequate contact of the sclerosant with the variceal wall, the sclerosant is aspirated and the balloon catheter withdrawn. Considered by many to be as effective as TIPS in controlling gastric variceal bleeding, it has an added advantage in that it augments portal blood flow by occluding the natural shunt that takes blood away from the portal vein. This improves liver function in cirrhotic patients and also prevents encephalopathy, a problem commonly associated with TIPS. However,Surgical peritoneovenous shunts have been replaced by TIPS in most centers but are still offered to patients who cannot tolerate a TIPS. However, surgery in this high-risk group is associated with the morbidity of general anesthesia. These shunts can now be inserted by radiologists with less risk as the procedure is done under local anesthesia. In addition, venous entry puncture can be more precise, and access is easier, with USG guidance.There are various interventional procedures that can be offered to patients with PHT. The choice of the procedure is based on the etiology of PHT, the symptoms, the clinical status, and the results of imaging studies. Most procedures now offer high success rates, good mid- and long-term results, and significantly less morbidity than the corresponding surgical procedures, and this has led to the emergence of interventional radiology as the procedure of choice in controlling PHT and its complications.


The history of allergic disease goes back to 1819, when Bostock described his own ‘periodical affection of the eyes and chest’, which he called ‘summer catarrh’. Since they thought it was produced by the effluvium of new hay, this condition was also called hay fever. Later, in 1873, Blackley established that pollen played an important role in the causation of hay fever. Nowadays, the definition of allergy is ‘An untoward physiologic event mediated by a variety of different immunologic reactions’. In this review, the term allergy will be restricted to the IgE-dependent reactions. The most important clinical manifestations of IgE-dependent reactions are allergic conjunctivitis, allergic rhinitis, allergic asthma and atopic dermatitis. However, this review will be restricted to allergic rhinitis. The histopathological features of allergic inflammation involve an increase in blood flow and vascular permeability, leading to plasma exudation and the formation of oedema. In addition, a cascade of events occurs which involves a variety of inflammatory cells. These inflammatory cells migrate under the influence of chemotactic agents to the site of injury and induce the process of repair. Several types of inflammatory cells have been implicated in the pathogenesis of allergic rhinitis. After specific or nonspecific stimuli, inflammatory mediators are generated from cells normally found in the nose, such as mast cells, antigen-presenting cells and epithelial cells (primary effector cells) and from cells recruited into the nose, such as basophils, eosinophils, lymphocytes, platelets and neutrophils (secondary effector cells). This review describes the identification of each of the inflammatory cells and their mediators which play a role in the perennial allergic processes in the nose of rhinitis patients.


Nitrogen-fixing symbioses between legumes and bacteria of the family Rhizobiaceaeinvolve differentiation of both plant and bacterial cells. Differentiation of plant root cells isrequired to build an organ, the nodule, which can feed and accommodate a large populationof bacteria under conditions conducive to nitrogen fixation. An efficient vascular system isbuilt to connect the nodule to the root, which delivers sugars and other nutrients to thenodule and removes the products of nitrogen fixation for use in the rest of the plant. Cellsin the outer cortex differentiate to form a barrier to oxygen diffusion into nodules, whichhelps to produce the micro-aerobic environment necessary for bacterial nitrogenaseactivity. Cells of the central, infected zone of nodules undergo multiple rounds ofendoreduplication, which may be necessary for colonisation by rhizobia and may enableenlargement and greater metabolic activity of these cells. Infected cells of the nodulecontain rhizobia within a unique plant membrane called the peribacteroid or symbiosomemembrane, which separates the bacteria from the host cell cytoplasm and mediates nutrientand signal exchanges between the partners. Rhizobia also undergo differentiation duringnodule development. Not surprisingly, perhaps, differentiation of each partner is dependentupon interactions with the other. High-throughput methods to assay gene transcripts,proteins, and metabolites are now being used to explore further the different aspects ofplant and bacterial differentiation. In this review, we highlight recent advances in ourunderstanding of plant cell differentiation during nodulation that have been made, at leastin part, using high-throughput methods.


Many cases of coeliac disease, a gastrointestinal autoimmune disorder caused by sensitivity to gluten, can remain in a subclinical stage or undiagnosed. In a significant proportion of cases (10–15%) gluten intolerance can be associated with central or peripheral nervous system and psychiatric disorders.A 38-year-old man was admitted as to our department an inpatient for worsening anxiety symptoms and behavioural alterations. After the addition of second generation antipsychotic to the therapeutic regimen, the patient presented neuromotor impairment with high fever, sopor, leukocytosis, raised rhabdomyolysis-related indicators. Neuroleptic malignant syndrome was strongly suspected. After worsening of his neuropsychiatric conditions, with the onset of a frontal cognitive deficit, bradykinesia and difficulty walking, dysphagia, anorexia and hypoferraemic anaemia, SPET revealed a reduction of cerebral perfusion and ENeG results were compatible with a mainly motor polyneuropathy. Extensive laboratory investigations gave positive results for anti-gliadin antibodies, and an appropriate diet led to a progressive remission of the encephalopathy. Coeliac disease is an inflammatory disease of the upper small intestine resulting from gluten ingestion . The diaThe literature describes cases of cerebral perfusion abnormalities in untreated coeliac patients ,10. TherIn May 2001, a 38-year-old man with anxious-depressive symptoms was referred to us for psychiatric assessment. These symptoms, occurring sporadically for around two years, had worsened following a protracted absence from work (due to a disabling right wrist fracture). The patient had a history of surgical operations to correct kyphoscoliosis.He was diagnosed with reactive depressive disorder in the context of personality disorder NOS (not otherwise specified) and put on paroxetine 10 mg with benzodiazepines. Following the appearance of bizarre behaviours and heteroaggressiveness towards family members, anti-psychotic therapy  was added. In May 2002, worsening anxiety symptoms and behavioural alterations that could not be managed at home culminated in the patient's hospitalisation in our department for re-assessment and review of therapy. Two days after the addition of risperidone 2 mg to the existing therapeutic regimen  the patient presented muscle rigidity, cramp-like muscle pain and increased osteo-tendinous reflexes leading to bradykinesia and difficulty walking. Withdrawal of the anti-psychotic drug did not improve the picture significantly. Laboratory investigations revealed raised CK (536 U/l) and a brain CT-scan showed an area of hypodensity of possible ischaemic origin in the posterior fossa, as well as moderate deepening of the cortical sulci in the frontal-temporal region bilaterally. EEG showed mild, non-specific, non-focal abnormalities.3), and raised rhabdomyolysis-related indicators (CK 1216 U/l and LDH 718 U/l). The patient was transferred to the infectious diseases department. Since neuroleptic malignant syndrome was strongly suspected, the anti-psychotic was withdrawn and dantrolene 50 mg/day and cardiorespiratory support were started, substantially resolving the acute symptomatology. The patient developed an Enterococcus faecalis infection of the urinary tract and deep-vein thrombosis (DVT), which were treated with antibiotic therapy and subcutaneous heparin.The severe anxiety symptoms and behavioural alterations persisted and a week later anti-psychotic treatment was re-introduced. The clinical picture, already characterised by neuromotor impairment, worsened abruptly and unexpectedly, with the onset of high fever (39°C), sopor, acute respiratory insufficiency with peripheral cyanosis, leukocytosis  continued to lack a plausible explanation.In mid-June, a further worsening of his neuropsychiatric conditions prompted his readmission to our department. During this second stay, he displayed the progressive onset of a frontal cognitive deficit together with affective lability, behavioural and affective regression, and verbal and motor stereotypes. Furthermore, the appearance of dysphagia and anorexia led to significant weight loss (20 kg in two months) which necessitated parenteral nutrition.The patient was then sent to the Neuropathology Unit at the "C. Besta" Neurological Institute in Milan, where single photon emission computed tomography (SPECT) revealed a reduction of perfusion and thus of neuronal activity and density in the right superior and middle frontal gyri, the left superior frontal gyrus, and the left medial temporal and occipital gyri; electroneurography (ENeG) results were compatible with a mainly motor polyneuropathy. Subsequently, mild hypoferraemic anaemia was found. Gluten sensitivity tests (part of further and more extensive laboratory investigations) gave positive results for anti-gliadin (IgG 32 UI/ml), anti-endomysial and anti-transglutaminase antibodies). A appropriate diet was instituted and led to a progressive remission of the encephalopathy and an improvement in the psychiatric symptoms and the lesions detected on SPECT and ENeG, which, at follow up, were no longer present. After a period of rehabilitation, this patient is still followed by our psychiatric service for mild anxiety symptoms. He takes olanzapine 2.5 mg and derives benefit from the treatment.The diagnostic process in this patient proved particularly complicated. This is, in fact, a case of clinical onset of coeliac disease in adulthood, without signs of malabsorption and with exclusively psychiatric involvement . In our view, this clinical picture could be attributed to the SPET-documented frontal hypoperfusion. This would indeed explain the lack of benefit of the initial psychopharmacological treatment and the progressive worsening of the symptoms that, together with the onset of behavioural disinhibition, necessitated the patient's hospitalisation. During hospitalisation, the administration of anti-psychotic drugs triggered the onset of neuroleptic malignant syndrome, which, initially atypical (without fever and leukocytosis) and then full blown, slowed down the diagnostic process and delayed the recognition of the true nature (organic) of the aetiology. We cannot rule out the possibility that this coeliac patient presented a particular susceptibility to this rare complication associated with the use of second-generation antipsychotic drugs , given tAfter the resolution of the acute picture, there remained a progressively worsening psycho-organic syndrome, secondary to the above-mentioned cerebral involvement, and neuromotor deficits due to the polyneuropathy detected on ENeG. The case we describe recalls literature reports of an adult-onset progressive frontal, subcortical-type cognitive deficit, characterised by confusion, personality disturbances and associated neurological  pictures, coinciding with the exacerbation of a malabsorption syndrome .The appearance of the first signs pointing to malabsorption, i.e., the weight loss and hypoferraemic anaemia, finally prompted us to investigate a possible autoimmune aetiology, and to test for anti-gliadin antibodies, for which the patient was positive.The diagnostic hypothesis of gluten encephalopathy was confirmed by the remission of the symptoms and of the lesions observed on SPECT following the institution of an appropriate diet.In addition to the objective diagnostic difficulties presented by this case, we wish to add a further, ideological consideration, relating to an unwitting tendency of colleagues from other specialist disciplines to stigmatise patients classified as "psychiatric". Indeed, both the possible organic aetiology of the clinical picture and the concomitant medical disorders presented by "psychiatric" patients are often underestimated, slowing down the diagnostic process and the taking of the necessary therapeutic measures.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.NP has made substantial contributions to the acquisition of clinical data; VS has given the final approval of the version to be published; BE has made substantial contributions to the acquisition of clinical data; PB participated in the analysis and interpretation of clinical data; CC was involved in drafting the manuscript; CC was involved in revising the manuscript critically.All authors read and approved the final manuscript.