Angiogenesis has developed into a major area of cancer research. Recently, several newly identified signalling pathways have been shown to play a role in both normal and pathological (including tumour) angiogenesis. Several of the molecules involved in these pathways have potential as novel anti-cancer therapeutic targets including members of the ephrin/Eph receptor, Notch/delta, sprouty, hedgehog and roundabout/slit families. These developments are reviewed. Extensive research into the molecular mechanisms involved in vessel formation has identified proangiogenic factors such as vascular endothelial growth factor (VEGF) and the angiopoietins, together with antiangiogenic factors such as the thrombospondins and transcription factors leading to their expression like Id1  anchor, whereas the ephrin B ligands are inserted into the plasma membrane via a transmembrane region followed by a conserved cytoplasmic domain. The ephrins bind to two families of transmembrane EphA and EphB tyrosine kinase receptors. While the A-type ephrins preferentially bind to the EphA receptors and the B-type ephrins to the EphB receptors, within each subclass, considerable promiscuity of receptor binding has been demonstrated.α 1, -3 and -4. The activation of Notch upon ligand binding is accompanied by proteolytic processing that releases an intracellular domain of Notch (NICD) from the membrane. The intracellular domain then translocates to the nucleus where it associates with the CSL family of DNA-binding proteins to form a transcription factor. This complex initiates the transcription of a set of target genes, including the Enhancer of Split group E(spl) and others; in this way Notch functions as a membrane-bound transcription factor. That Notch is active during human vascular development is illustrated by the cerebral disorder, cerebral autosomal-dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL), which arises from a mutation in Notch3. CADASIL is characterised by stroke, vascular dementia and arteriopathy of cerebral arterioles.Jagged1 was inactivated in the mouse embryo  promoter in mice during embryonic development, the expression of this activated Notch results in growth and developmental delay and embryonic lethality at embryonic day 10 , desert hedgehog (Dhh) and Indian hedgehog (Ihh). Of these, Shh is the most widely expressed during development and lack of Shh is embryonically lethal with multiple defects in early to mid-gestation. Ihh is less widely expressed and mice deficient in Ihh are able to survive until late gestation but die due to skeletal and gut defects. Dhh-deficient mice are viable but display peripheral nerve and male fertility defects.Hedgehog proteins have been shown to act as morphogens in numerous different tissues during embryonic development. They are 19 kDa proteins that interact with heparin on the cell surface through an N-terminal basic domain and are tethered to the surface through cholesterol and fatty acyl modification. Hedgehog signalling is crucial for the formation of limb, lung, gut, hair follicles and bone. There are three human homologues of the Signalling by all three Hedgehog proteins occurs through interaction with the Patched1 receptor, which then activates the transcription factors Gli1, Gli2 and Gli3. The downstream targets of the Gli gene products include both patched and Gli themselves, thus patched and Gli are both components and targets of the Hh signalling pathway.in vivo. Thus, when Shh is administered to aged mice it induced new vessel growth in ischaemic hind limbs. The Shh-induced vessels showed a characteristically large diameter. Despite this, Shh had no effect in vitro on endothelial-cell migration or proliferation but did induce the expression of proangiogenic VEGF and angiopoietins-1 and -2 from interstitial mesenchymal cells. The indirect nature of hedgehog signalling has been confirmed in zebrafish  signalling during tracheal development. It is an intracellular protein, localised to the inner leaflet of the plasma membrane by a cysteine-rich domain. In Drosophila, Spry is expressed at the tips of growing primary branches of the tracheal system, in the eye imaginal disc, the embryonic chordotonal organ precursors and in the midline glia. There are four isoforms of Spry in mammals, each having a highly conserved C terminus but variable N terminus. They are all expressed in restricted patterns in the embryo in early development, showing dose correlation with sites of FGF signalling suggesting that Spry proteins may function as negative regulators of FGF signalling during vertebrate development as well as in Drosophila. In fact, a decrease in Spry2 expression in the mouse results in increased lung branching morphogenesis. As with tracheal development, the process of angiogenesis requires receptor tyrosine kinase signals. In addition, FGF, VEGF, platelet-derived growth factor, ephrin B and Tie-2 are all components of both tracheal and blood vessel development. In view of these many similarities between Drosophila trachea development and mammalian angiogenesis in terms of gene function, therefore, it was anticipated that Spry would also have a role in angiogenesis.Sprouty (Spry) was first identified in in vitro were transfected with mSpry4 there was a decrease in cell migration and cell cycle arrest at the G1/S phase with no apoptosis. The action of Spry4 appears to be via receptor tyrosine kinase pathways since there was a reduction in both basal and bFGF or VEGF-induced MAPK phosphorylation in Spry4-expressing cells. MAPK signalling is involved in the regulation of proliferation, migration and differentiation during angiogenesis and the inhibition of tyrosine kinase-stimulated MAP kinase activation probably accounts for the observed cell cycle arrest. To date it is not known whether the other sprouty proteins have similar antiangiogenic properties since the mode of action of sprouty in angiogenesis is very much in its infancy.Direct evidence for a role for Spry in angiogenesis comes from a study in which the mouse Spry4 was overexpressed in the developing endothelium of a mouse embryo using an adenoviral vector , a novel roundabout receptor restricted to endothelial cells was unexpected (In situ analysis has shown magic roundabout to be absent from adult tissues, but strongly expressed on the vasculature of tumours including those of the brain, bladder and colon metastasised to the liver (Roundabouts and slits comprise a family of signalling molecules thought to be restricted to cells of neuronal lineage. Roundabout was so named because of the neuronal phenotype arising from its deletion in expected . In situIt is arguably true to say that few researchers in the field of angiogenesis expected the growth of new blood vessels to be a simple event, nevertheless the molecular complexity of the process and the number of pathways involved has been a surprise. A significant outcome of this complexity has been the pleasing number of potential new targets available for therapeutic intervention. Studies of antiangiogenesis and vascular targeting are clearly at an exciting stage.


To characterize the cornea of individuals with Thygeson’s superficial punctate keratitis (TSPK) at the cellular level by laser confocal biomicroscopy.Both corneas of three patients with TSPK referred to Yamaguchi University Hospital were imaged with a laser confocal biomicroscope. Morphological changes were evaluated for each layer of the cornea.The number of Langerhans cells was greatly increased in the basal cell layer of the focal corneal epithelium and in Bowman’s layer in the four eyes affected by TSPK. Aggregates of these cells were associated with the subepithelial nerve plexus. Langerhans cells were also evident in the unaffected eyes of the two patients with unilateral TSPK, although their numbers were much smaller than those in the affected eyes. Topical treatment with betamethasone phosphate resulted in the virtual disappearance of Langerhans cells from the affected eyes.The prominent association of Langerhans cells with TSPK suggests that the activation of these cells by inflammatory conditions might contribute to the pathogenesis of this disorder. Thygeson’s superficial punctate keratitis (TSPK) is characterized clinically by ocular irritation, tearing, photophobia, and visual disturbance but has minimal clinical manifestations in the cornea -3. It usThe Heidelberg Retinal Tomograph II, Rostock Cornea Module  has recently been introduced into clinical practice in ophthalmology -9. GivenWe described the characterization of three patients with TSPK by in vivo imaging with the HRTII-RCM in this present report. Our findings suggest that immunological reactions mediated by LCs may contribute to the pathogenesis of TSPK.Three unrelated patients with TSPK referred to the cornea service at Yamaguchi University Hospital were enrolled in the study. They reported ocular irritation, tearing, photophobia, or foreign-body sensation in one (two patients) or both (one patient) eyes. Slitlamp microscopic examination revealed that characteristic elevated and coarse granular opacities that stained with fluorescein in the affected eyes . CorrectWith written informed consent, we examined both corneas of each patient by HRTII-RCM imaging on their first visit to our clinic. The study was also approved by the Institutional Review Board of Yamaguchi University Hospital.A drop of local anesthetic  was instilled into the lower fornix of each eye, and a drop of polymer gel  was applied to the microscope probe to allow for optical coupling of the microscope objective lens to the cornea. HRTII-RCM imaging was performed at the central and focal cornea including the coarse granular opacities. Images of all layers of the cornea were collected and saved. All images were analyzed with a personal computer (12 inch monitor with a display resolution of 1024 by 768 pixels) and under the same lighting conditions so as to avoid variation in image contrast on the monitor. Three observers viewed the digitally stored images frame by frame in a masked manner. The most representative images from each corneal layer were selected for evaluation and ranked according to the severity of change.Most of the abnormalities in the eyes affected by TSPK were confined to the basal cell layer of the corneal epithelium, the subepithelial nerve plexus, Bowman's membrane, and the anterior stroma, with the middle and deep regions of the stroma and endothelial cells being largely normal. The changes observed were similar in the four affected eyes of the three patients but they varied in severity.2 and 15.0±2.21 cell/mm2 in the basal cell layer, respectively. The number of LC in TSPK affected eyes significantly increased.Superficial cells of the corneal epithelium showed no substantial changes in cell size, shape, or brightness (data not shown) in the local region including the coarse granular opacities of the affected eyes. In wing cell layer and basal cell layer, we could not detect any abnormalities in cell shape and cell number. In contrast, an increase in the number of dendritic cells was apparent in the basal cell layer of the corneal epithelium in all affected eyes . The num2 and 21.9±4.42 cell/mm2 in the nerve plexus, respectively. Variability in the morphology of dendritic cells was not obvious in the unaffected eyes of these two patients . These abnormalities were most pronounced in the region closest to Bowman's membrane. Images of the posterior stroma and corneal endothelium appeared normal in the affected eyes.We also obtained post-therapeutic HRTII-RCM images for patient #3 . The numWith the use of HRTII-RCM, we have obtained in vivo images of the central cornea in eyes affected by TSPK. Although dendritic cells were observed in the unaffected healthy corneas of the two patients with unilateral TSPK, these cells were greatly increased in number and formed aggregates in both the basal cell layer of the corneal epithelium and in Bowman’s layer of the affected eyes. The dendritic cells in Bowman’s layer appeared associated with the nerve fibers that form the nerve plexus of the corneal epithelium. Medical intervention reduced the numbers of these dendritic cells to normal values.With use of HRTII-RCM, we were able to observe abnormalities of cell shape and cell density in the eyes affected by TSPK. In the affected eyes, accumulation of dendritic cells was apparent in the focal lesions. Tandem scanning confocal microscopy of eyes affected by TSPK previously revealed multiple highly reflective filamentous structures in Bowman’s layer of the corneal epithelium, but these structures were not conclusively identified as dendritic cells . In addiElectron microscopy has previously revealed dendritic cells in close contact with nerve fiber bundles in the cornea of individuals with ocular viral infection . In the In conclusion, we have demonstrated the accumulation and aggregation of LCs in the basal cell layer of the corneal epithelium and in association with the subepithelial nerve plexus in the affected eyes of patients with TSPK. These observations suggest that TSPK might result, at least in part, from immune responses to an unknown inciting agent.


DNA-binding domains (DBDs) are essential components of sequence-specific transcription factors (TFs). We have investigated the distribution of all known DBDs in more than 500 completely sequenced genomes from the three major superkingdoms  and documented conserved and specific DBD occurrence in diverse taxonomic lineages. By combining DBD occurrence in different species with taxonomic information, we have developed an automatic method for inferring the origins of DBD families and their specific combinations with other protein families in TFs. We found only three out of 131 (2%) DBD families shared by the three superkingdoms. All sequence-specific transcription factors (TFs) contain DNA-binding domains (DBDs), evolutionary units that mediate the specificity of the TF–DNA interaction. Domain-based analysis of TFs is thus effective functionally as well as phylogenetically. TFs and their binding targets have been under intensive study, and previous key publications on TFs and DBDs tended to focus on specific phylogenetic groups Z-score of a number of TFs containing a particular DBD family of interest was calculated for each family to highlight the organisms in which the family is expanded relative to other species. Orange indicates positive Z-scores and thus a relative expansion of the DBD in that particular lineage and blue indicates negative Z-scores or a contraction. The distinct expansion pattern of different groups of DBDs in prokaryotes and eukaryotes implies that the DBD families are highly specific to two types of cells: nucleate and anucleate.Earlier, we have introduced a heatmap representation to aid visualisation of the expansion and contraction of DBD families in order to investigate the distribution of DBDs in different lineages In addition to the heatmap, we have developed a new simple method for inferring the origin of protein domains. By combining DBD family occurrence with taxonomic information from the NCBI taxonomy tree, we demonstrate that the method is able to estimate when each DBD family emerged. We term this the taxonomic limit. The same method is used to estimate when the combinations of DBDs and other protein families in TFs emerged. We provide the taxonomic conservation density, which is the fraction of species containing the DBD out of the total number of species within taxonomic clades see .Using our method, we found that 19 out of 131 (15%) DBDs have cellular organisms as their taxonomic limits (shared by more than one superkingdom). Eleven of these DBDs are shared by Archaea and Bacteria but not Eukaryota, and only three (2%) are shared by all three superkingdoms b. When wFocusing on the prokaryotic genomes, helix-turn-helix is by far the commonest DBD structure The heatmap shown in Examples of prokaryotic phylum-specific DBDs include WhiB, a DBD specific to Actinobacteria that regulates mycelium formation. The FlhC and FlhD TFs, with Proteobacteria (Gram-negative) as their taxonomic limit, have been shown to be global regulators involved in many cellular processes, including flagella transcriptional activators In contrast to the uniform DBD occurrence in Bacteria, The most notable difference in the Metazoa is between vertebrates and invertebrates. Although the majority of DBDs found in animals are present in both groups, the expansion tends to be less pronounced in the invertebrates. The DBDs with particularly extensive expansion in vertebrates include STAT , T-box (body plan and organogenesis) and p53 (cell cycle arrest and apoptosis). DBDs such as IRF (interferon regulator factor) and Churchill  are absent from invertebrates, which might reflect the more elaborate immune and nervous systems in vertebrates. In contrast, the Runt and GCM families regulate fundamental developmental processes in both vertebrates and invertebrates, and are equally expanded in both groups.Metazoa and Fungi are phylogenetically closer and share more DBD families with Viridiplantae . In accoMonosiga brevicollis, a marine choanoflagellate that is thought to be the closest sequenced unicellular relative of animals M. brevicollis, and many DBDs specific to animals  and their partner domains (square nodes) in bacterial TFs are mostly (31 out of 44) shared by more than one bacterial phylum . For insSpecific DBD–partner domain combinations are observed in animals, fungi and plants. The eukaryotic-specific Tub family, for instance, occurs in a single-domain TF in more than 25% of eukaryotic TFs (green border node). It occurs also downstream of the SOCS_box domain only in animals, and co-occurs exclusively with F-box in plants b. This fAnother distinctive feature of eukaryotic TF architectures not found in prokaryotes is the repetition of the same DBD family within a single TF chain (self-looping arrows in The partner domains in eukaryotic TFs have more diverse functions than those in Bacteria, and the commonest function is to mediate protein–protein interaction and dimerisation. This is thought to be important to the formation of composite protein modules, a crucial step towards combinatorial regulation. Examples of these families include BTB, Bromodomain, SAM, ANK and hATC.DBDs are essential to all sequence-specific TFs because they regulate the specificity of TF–DNA binding, which in turn governs differential expression and determines physiological diversity in different species across the tree of life. With this analysis of conserved and lineage-specific DBDs, and TF architectures using our new method for inferring taxonomic limits, we contribute new insights into the global picture of the TF repertoire and its evolution. Our findings can facilitate the experimental design of high-throughput studies on transcriptional regulators, e.g. Refs We demonstrate a limited conservation of DBD families between prokaryotes and eukaryotes. Only 15% of known DBDs have cellular organisms as their taxonomic limit, as opposed to 33% of all Pfam domains. Lineage-specific DBD repertoires can be seen at the eukaryotic kingdom level: only 29% of eukaryotic families are shared by more than two superkingdoms. Prokaryotic DBDs are less specific to the major bacterial phyla, with 49% of families being shared. In addition to DBD, the variety in DBD and partner domain combination adds another level of complexity to TF structures. The specific DBD families and TF architectures in different lineages can be used as signatures for the genetic regulatory circuits in diverse phylogenetic groups. Knowledge of the phylogenetic distribution of DBD families and their domain combinations can improve methods for remote homology detection


Drosophila, the beneficial effects of rapamycin in diseases related to HD are autophagy-dependent. We will also discuss the importance of autophagy in early stages of development and its possible contribution as a secondary disease mechanism in forms of fronto-temporal dementias, motor neuron disease, and lysosomal storage disorders.Efficient protein turnover is essential for the maintenance of cellular health. Here we review how autophagy has fundamental functions in cellular homeostasis and possible uses as a therapeutic strategy for neurodegenerative diseases associated with intracytosolic aggregate formation, like Huntington's disease (HD). Drugs like rapamycin, that induce autophagy, increase the clearance of mutant huntingtin fragments and ameliorate the pathology in cell and animal models of HD and related conditions. In  Both of these systems are responsible for the efficient degradation and turnover of proteins within the cell. Failure of either the UPS or autophagy has been associated with disease while the upregulation of these processes has been shown to ameliorate certain disease pathologies.The proteasome is responsible for the selective degradation or recycling of short-lived cytosolic and nuclear proteins, but also regulates the turnover of some long-lived proteins. Attachment of a chain of at least four covalently bonded ubiquitin molecules to a protein targets it for degradation by the proteasome. The proteasome, a 2000 kDa, multisubunit protein complex has a hollow barrel that contains its proteolytic components. Because of the small size of the proteasome's proteolytic core, client proteins generally require unfolding prior to degradation. Due to the size of the narrow barrel of the proteasome and the specificity of the process, many proteins are unable to be degraded by the UPS, including oligomers or proteins in organelles Macroautophagy, more commonly referred to as autophagy, is responsible for the bulk degradation of long-lived cytosolic proteins and organelles. Unlike proteasomal degradation, autophagic degradation is thought to be largely non-specific. Degradation of macromolecules or organelles by autophagy requires the formation of double-membrane vesicles called autophagosomes and subsequent fusion of autophagosomes with lysosomes. This fusion produces an autophagolysosome within which the cargo is degraded by acidic lysosomal hydrolases.2Despite an advanced understanding of the UPS, many aspects of autophagy remain largely unknown. The discovery of pathways that induce autophagy has contributed to the progression of our understanding of autophagy as a degradative pathway and its implications in human health.Autophagy is induced under starvation conditions, while it is inhibited under nutrient-rich conditions These early screens provided a foundation for the understanding of autophagy in yeast and higher eukaryotes by identifying key proteins and interactions. From the assessment of protein interactions and autophagosome morphology, autophagy degradation has been found to consist of six main steps: induction, cargo selection and packaging, autophagosome formation and completion, recycling of autophagy regulators, fusion of the autophagosome with the lysosome, and cargo degradation. These steps rely on the function of key autophagy genes (Atg genes) 3In light of the importance of the degradative pathways in mammalian disease, a branch of autophagy research has studied the role of autophagy in normal development and maintenance of health. It has been shown that autophagy is important during critical developmental stages in which nutrients are restricted. Termination of the direct fetal nutrient supply from the mother presents a stressful situation for the newborn infant. During this transition period, autophagy provides the necessary nutrients to the infant through increased turnover of proteins. This has been shown experimentally in a transgenic mouse model expressing GFP fused LC3 to visualise autophagosomes in vivo −/− mice and a significant weight reduction in the Atg7−/− mice. Atg5 +/− or wildtype littermates (21–24 h). These experiments support the role of autophagy in normal developmental processes that occur in the absence or diminished levels of nutrient supply, lending further importance to the need for the functional turnover of amino acids.To assess the specific role of autophagy, Mizushima's and Tanaka's groups studied the effect of starvation during this critical period in Atg5 and Atg7 knockout mice, i.e. autophagy-deficient mice. These mice develop normally with only a slightly lower birth weight for the Atg5As a systemic deficiency of autophagy genes in these mice causes neonatal death within a day of birth, conditional autophagy-deficient mice were generated to assess the role of autophagy in homeostasis of various tissues later in development. In the absence of a predisposition to genetic disease, both Atg5 and Atg7 conditional knockout mice and ubiquitous knockout mice develop ubiquitinated protein aggregates and varying degrees of tissue deterioration. Conditional Atg5 and Atg7 knockout mice suffer not only from nutrient deprivation during starvation periods but also develop dysfunctional hepatic tissue and progressive neurodegeneration Drosophila p62 orthologue in autophagy-deficient flies A recent study analysed the role of p62/A170/SQSTM1 in relation to inclusion formation in autophagy-deficient mice. Loss of p62 in autophagy-deficient mice decreased levels of polyubiquitinated proteins and inclusions. The authors suggested that this was because p62 was a key mediator of inclusion formation Drosophila melanogaster mutants. Although Atg7 mutant flies are morphologically normal, they are sensitive to stressful conditions and show a decreased level of cell death-induced autophagy during metamorphosis, a necessity during normal development Additional characterisation of autophagy deficiency has been performed in Atg7 Drosophila have also contributed to an understanding of the roles autophagy may play in ageing. Atg8a mutant flies have reduced lifespan, while transgenic overexpression of Atg8a extends the fly lifespan by 56% and increases its resistance to oxidative stress and reduces the accumulation of ubiquitinated proteins Studies in 4Autophagosomes need to ultimately fuse with lysosomes. The endocytic pathway is necessary for maturation of autophagosomes via the fusion of autophagosomes with endosomes, prior to autophagosome–lysosome fusion 5The relay of signals within and between cells is dependent upon efficient internal transport. Neuronal cells are particularly dependent on the accurate and timely conversion of external stimuli to an intercellular response via the movement of internal cargo. Directed movements over long distances, such as that seen in motor neurons, are dependent upon microtubules via dynein and kinesin motors. Dyneins move cargo centripetally towards the minus-end of microtubules, i.e. towards the microtubule organising centre near the nucleus. Plus-end-directed kinesins move cargo in the opposite direction, centrifugally, outwards into the cytoplasm and the plus-end of microtubules  in mouse models 6The turnover and recycling of integral membrane proteins from the plasma membrane is carried out largely by different functions of the endocytic pathway. Simple sorting by endosomes recycles proteins, returning them to the plasma membrane. A more intricate degradative system utilises the multivesicular body (MVB), a specialised vesicular structure generated by invagination of the endosomal membrane to form lumenal vesicles. The fusion of MVBs with lysosomes releases the vesicles into the acidic lumen of the lysosomes within which hydrolases degrade the vesicles and their cargo. The sorting of integral membrane proteins into the MVB pathway is dependent upon monoubiquitination of the protein, a signal that destines the protein for incorporation into the lumenal vesicles of MVBs In addition to known roles in HIV budding Expression of a deletion mutant of CHMP2B, synonymous to the splice mutant, increases neuronal and dendritic loss through an apoptosis-independent pathway The effect of ESCRT dysfunction on autophagy has been studied by the expression of mutants or the knockdown of genes in all three complexes and associated proteins:  are a group of at least 40 neurodegenerative or myopathic diseases defined by the accumulation of undegraded lysosomal substrates within the lysosomal lumen The current understanding of autophagy and its dependence upon lysosomes has led researchers to study the effects of LSDs on both basal autophagy and the induction of autophagy. Fusion of autophagosomes with lysosomes is necessary for the degradation of cargo by the lysosomal acidic hydrolases. Without this fusion event autophagosomes and their cargo accumulate, thus increasing toxic species within cells. A recent study These examples suggest that autophagy deficiency may be an important secondary disease mechanism in a range of conditions.8Intracellular protein misfolding/aggregation are features of many late-onset neurodegenerative diseases, called proteinopathies. These include Alzheimer's disease, Parkinson's disease (PD), tauopathies, polyglutamine expansion diseases – such as Huntington's disease (HD) – and various spinocerebellar ataxias (SCAs), like SCA3. Currently, there are no effective strategies to slow or prevent the neurodegeneration or muscular dystrophy resulting from these diseases in humans. Due to the size of the narrow barrel of the proteasome and specificity of the process, most large aggregate-prone proteins are precluded from entering the structure Drosophila HD model and transgenic HD mice The discovery of target of rapamycin (TOR) as a pivotal regulator of autophagy induction in eukaryotes These findings are replicable in other proteinopathy models. For instance, rapamycin reduces toxicity and enhances clearance of aggregate-prone proteins, like a polyalanine expansion protein and tau . These results provide proof-of-principle in the ability of autophagy to clear aggregate-prone proteins Unfortunately, long-term use of rapamycin can cause adverse side effects such as poor wound-healing and general immunosupression. Mammalian target of rapamycin (mTOR) regulates a range of pathways , and its major side-effects can be attributed to autophagy independent pathways. Also, how mTOR regulates mammalian autophagy is still not understood. Further insight into how this occurs will hopefully provide safer, more specific druggable targets suitable for long-term treatment.Drosophila and zebrafish models of HD.Currently, there exist very few pharmacological treatment options for neurodegenerative proteinopathies. The disaccharide trehalose has also been shown to reduce aggregation and induce autophagy in an mTOR-independent manner, but this process and the pathway through which it acts remain unknown A recent study has shown tantalising effects of lithium in the treatment of amyotrophic lateral sclerosis (ALS) in humans and mouse models of this disease. Lithium carbonate (when given in addition to the benchmark drug riluzole), significantly delayed the onset of disability and death in human ALS patients, compared to those administered riluzole alone. Treatment of G93A ALS mice with lithium carbonate delayed death and onset of disease progression. Tissue collected from these mice showed an increase autophagic activity and a general decrease in disease phenotypes such as ubiquitin and alpha synuclein accumulation 9Today 31 genes have been identified that play a specific role in the normal function of autophagy The progression of research in the field of autophagy has taken the understanding of autophagy from a means of survival during starvation to a possible means of treating neurodegeneration pharmacologically. Progress in the understanding of autophagy has emphasised the importance of autophagy in cellular homeostasis, adaptation to starvation, prevention of neurodegeneration in animal models, and the significance of its deficiency in the development of neurodegenerative disorders. The overall relevance of autophagy in disease far exceeds the breadth of this review. With its implicated roles ranging from viral infection and cancer to liver and cardiac diseases (reviewed in


Dentin dysplasia is a rare hereditary disturbance of dentin formation characterized by defective dentin development with clinically normal appearing crowns, severe hypermobility of teeth and spontaneous dental abscesses or cysts. Radiographic analysis shows obliteration of all pulp chambers, short, blunted and malformed or absent roots and peri-apical radiolucencies of non carious teeth.We present a case of dentin dysplasia type I in a 12-year-old Iranian boy, and the clinical, radiographic and histopathologic findings of this condition and treatment are described.There are still many inconclusive issues in the diagnosis and management of patients with dentin dysplasia. The diagnostic features of this rare disturbance will remain incompletely defined until additional cases have been described. Early diagnosis of the condition and initiation of effective regular dental treatments may help these patients to prevent or delay loss of dentition. Dentin dysplasia (DD) is an autosomal dominant hereditary disturbance in dentin formation, which may present with either mobile teeth or pain associated with spontaneous dental abscesses or cysts. It is a rare anomaly of unknown etiology that affects approximately one patient in every 100,000  but it wThis article describes an uncommon case of DD type I, subtype 1a, in a 12-year-old Iranian boy, highlighting the clinical and radiographic variations of the defect as confirmed by histopathologic examination.A 12-year-old Iranian boy was referred to the Pedodontics Department of Shaheed Beheshti University Medical Sciences, Dental School due to excessive painful swelling of his mandibular left cheek region. Clinical examination showed that the following teeth were present in his mouth:7654321 12 C45677654321 12 C4567The crowns of his teeth had normal morphologic characteristics, but the color of his teeth was slightly more yellow than expected for a patient of his age Figure . Oral hyOn the basis of the clinical and radiographic appearance, a diagnosis of DD type I, subtype 1a, was suspected. Clinical and radiographical examination of the patient's parents and siblings revealed no cases of DD type I, and there were no previous cases of this disturbance in the familial history. The following treatment plan was formulated: dietary and oral hygiene instructions, fluoride supplements, surgical enucleating of the cystic lesion at the left first permanent molar region, restoration of the carious teeth, and extraction of the left primary canine and primary first molar, which were carious and mobile.The cystic lesion was enucleated and sent for histopathological examination. Based on the pedodontist's and surgeon's view, it was decided to extract the remaining root of the lower left first permanent molar during surgery because of extensive caries. The histopathological features were consistent with the clinical diagnosis of a radicular cyst. The cystic cavity was lined with a variable thickness of non keratinized stratified squamous epithelium with arch-shaped appearance and exocytosis in the underlying connective tissue was severely infiltrated by chronic inflammatory cells. Extravasated red blood cells and hemosiderin pigments were also seen Figure . The extet al. [et al. [The pathogenesis of DD is still unknown in the dental literature. Logan et al.  proposed [et al.  proposed [et al. , but in  [et al. . Some as [et al.  but our There were no variations in the morphology of the affected teeth in our patient but there are reports that have suggested possible variations in the morphology of teeth affected by this type of dysplasia ,12. HistManagement of patients with dentinal dysplasia has presented dentists with many problems. Extraction has been suggested as a treatment alternative for teeth with pulp necrosis and peri-apical abscess. Follow-up and routine conservative treatment is another choice of treatment plan in DD . AnotherDentin dysplasia type I is a rare inherited abnormality of the dentin that leads to premature exfoliation of the primary and permanent teeth. Early diagnosis of the condition is important for initiation of effective preventive treatment. In this regard, the pediatric dentist has an important role in the early diagnosis of this disorder and in guiding patients in the selection of measures to prolong the retention of affected teeth.DSPP: dentin sialophosphoprotein.Written informed consent was obtained from the patient's parents 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.LT wrote and supervised the manuscript. FM carried out the pathologic issues and took the ground section. MM and LM carried out all dental treatments and completed the literature review.


In silico analyses of the properties shared among the promoters of co-expressed genes facilitates the identification of transcription factors (TFs) involved in the co-regulation of those genes. Our previous experience with microarray data led to the development of a database suitable for the examination of regulatory motifs in the promoters of co-expressed genes.A growing number of gene expression-profiling datasets provides a reliable source of information about gene co-expression. cis-Regulatory Elements in the Mammalian Genome) system designed for in silico studies of the promoter properties of co-regulated mammalian genes. The cREMaG system offers an analysis of data obtained from human, mouse, rat, bovine and canine gene expression-profiling studies. More than eight analysis parameters can be utilized in user-defined combinations. The selection of alternative transcription start sites and information about CpG islands are also available.We introduce the cREMaG (in silico studies of mammalian transcriptional gene regulation. The resource is freely available at http://www.cremag.org.Using the cREMaG system, we successfully identified TFs mediating transcriptional responses in reference gene sets. The cREMaG system facilitates  It is now estimated that more than 5% of the mammalian genome encodes functional information, including regions involved in the regulation of gene expression, whereas only 1.5% of the mammalian genome contains protein-coding information Methods for mining gene sequences for transcriptionally relevant information have become possible with the growing body of knowledge about mammalian genomes, gene expression and regulation of gene expression  database is presented, which may help to resolve the above problems and allow for the discovery of TFs responsible for the regulation of co-expressed genes. Moreover, its successful application is demonstrated.Here, the new cREMaG  and phastCons scores for multiple alignments of 30 vertebrate genomes were retrieved from UCSC.For every TSS, sequences 10 kbp upstream and 5 kbp downstream of the TSS  content (%GC) was >50% and the ratio of observed to expected CpG dinucleotide frequencies (CpGobs/CpGexp) was >0.6 Promoter sequences were scanned with TFBS matrices obtained from the JASPAR database and the public release of the TRANSFAC database using the TFBS BioPerl module Three types of background sequences were prepared: random, core promoter and conserved promoter. To develop a random promoter background, 20 Mbp of random sequence was generated with equal numbers of all four nucleotides. To develop a core promoter background dataset, sequences containing the region 200 bp upstream of the gene start position for every gene in a genome were retrieved and concatenated. To develop a conserved promoter background, sequences with conservation scores higher than 75% for every gene were obtained and concatenated. The resulting random, core and conserved promoter sequences were scanned for all JASPAR and public TRANSFAC TFBSs at every integer matrix score threshold from 60% up to 100%. The frequencies at all thresholds were stored in a set of TFBS frequency tables.Random TFBS occurrence and Background sequence, are combined with the TFBS frequency tables to define a matrix score threshold for each matrix. The threshold with the most similar (least-distant) background frequency (random occurrence) to the user-defined frequency in the user-defined type of sequence is set as optimal.Two user-defined parameters, TFBSs from all alternative promoters for a particular gene meeting the user-defined criteria are combined into one pool. This procedure is repeated for all genes from the query set. The total length of queried sequences filtered for user-defined parameters is computed. The fold-difference in TFBS frequency is computed by dividing the observed TFBS frequency by the background frequency. The probable number of genes with particular TFBS hit obtained by chance is computed. For each particular TFBS matrix, the fold-change in the frequency of genes containing a particular TFBS compared with a predicted background frequency is computed. Z-scores are computed based on the fold distribution for all TFBS matrices. The fold distribution is Gaussian. The p-value was defined as the probability of obtaining a specific range of z-scores using the standard normal distribution. The p-values for frequency fold and gene fold are computed separately. Moreover, a proportion p-value is computed, defined as the proportion of TFBS hits that can be explained by chance.promoter properties section of the database is updated daily. Adding a new batch of position weight matrices for TFBS requires computation of all genes in the database and thus requires about one month of computation. Therefore, the repository of TFBS matrices will be updated at least once a year.The cREMaG system was designed for easy and continuous updates. The Ensembl database, which is the sequence resource for the cREMaG database, is updated several times a year. A Perl script continuously retrieves and analyzes new sequences and orthologs and stores them in the MySQL database used by cREMaG. The Mus musculus, Rattus norvegicus, Homo sapiens, Bos taurus and Canis familiaris. Five types of IDs are supported: Ensembl IDs, Entrez Gene IDs, gene symbols and Illumina and Affymetrix microarray IDs. It is recommended to use Ensembl Gene IDs because the engine of cREMaG is based on Ensembl IDs. Next, the IDs should be passed into the text field. It is recommended that users use between five and one hundred genes. Queries of fewer than five genes may yield unreliable results, and queries of more than 300 will slow down the analysis. Finally, the user may select whether to use all alternative TSSs, only the most distal TSSs, only the most highly expressed TSSs  or TSSs with or without CpG islands.In step I, the user is asked to fill in the five-field form . First, The queried IDs are converted to Ensembl Gene IDs. Every identified Ensembl Gene ID is translated into a gene symbol. Next, cREMaG searches for all TSSs belonging to genes identified previously by the Ensembl Gene ID. If a gene symbol corresponding to more than one Ensembl Gene ID will be queried, all of the corresponding genes will be included in the analysis.In step II, the user is asked to confirm the identified genes and alternative promoters . The resConservation threshold parameter is responsible for the elimination of TFBSs with conservation scores lower than the selected threshold. It is possible to select values from 60% (low conservation) to 90% (very high conservation). The Top percent of conserved regions parameter is responsible for the elimination of the TFBSs with the lowest conservation scores. It is possible to select values between 1% (only the most conserved TFBSs) and 100% . The Maximum number of most conserved TFBSs parameter was designed to analyze an equal number of TFBSs for every promoter independent of its conservation level. The Coding/non-coding sequence parameter allows users to search TFBSs in coding elements, non-coding elements or both. The Length of upstream segment parameter allows users to choose how many base pairs upstream of the TSS are used for the analysis . The Length of downstream segment parameter allows the user to choose how many base pairs downstream of the TSS are taken for analysis (0 bp to 5000 bp). The Random TFBS occurrence (optimized matrix score threshold) parameter determines how restrictive the matrix score threshold should be set . The Random TFBS occurrence parameter is combined with the Select background parameter to determine the background sequence type , and together they determine the random TFBS occurrence rate.In step III, the user is asked to select the analysis parameters . The ConGenes column of the table contains the symbols of genes containing particular TFBS in their promoters. TFBSs number shows the total number of TFBSs found in all included promoters. TFBSs Expected number is the number of expected occurrences of a TFBS matrix and is computed based on the length of sequences used for the analysis and the random false-positive rate selected by the user. TFBSs Fold is the result of dividing the TFBS number by the TFBSs Expected number. The TFBSs Fold p is the p-value showing how the TFBS fold stands out from the fold distribution. Genes Number is the number of genes containing a particular TFBS. Genes Expected Number is the expected number of genes containing a particular TFBS. Genes Fold is the result of dividing the Genes Number by the Genes Expected number. The Genes Fold p is the p-value showing how Genes Fold stands out from the fold distribution. The Proportion p-value is the proportion of TFBS hits that can be explained by chance. The fourth table shows the fold-differences of the most overrepresented binding sites from the TRANSFAC database and contains the same information as the JASPAR table. The fifth table shows the average lengths of the CpG islands within the query set and within the whole genome and the p-value for this comparison.In the last step, the user is provided with five tables . The firCamkcre4Creb1; Crem–/– animals. The genes analyzed are widely recognized in the literature as immediate early genes (IEG) -4, 1st rank). Moreover, SRF was also found  as a true-positive regulator of IEG -4). The table with the query results is provided in the To demonstrate the functionalities of cREMaG, we utilized the results from gene expression profiling in the striatum of cocaine-treated mice lacking CREB in the forebrain, accompanied by the deletion of CREMHomer1 gene has multiple transcript forms with distinct alternative TSSs. Homer1a is a CREB-inducible isoform of Homer1 widely described in the literature, and its transcription start site is the most proximal from the beginning of the gene Homer1 was submitted as a mouse gene symbol into the cREMaG system. In step II of the analysis, there was the possibility of selecting from four TSSs. The three cAMP response elements (CRE) were found only at the core promoter of the Homer1a isoform. This example shows the advantages of using the promoter of only the inducible transcriptional isoform when searching for overrepresented binding sites. While including all transcription start sites provides noise in the final results, the selection of particular promoters for the analysis may give a higher chance for true-positive results.The cREMaG system was also designed to analyze the promoters of particular transcriptional variants. The To demonstrate accuracy of cREMaG, we compared it with other available on-line tools: oPOSSUM, CORE_TF, TFM-Explorer and Pscan The cREMaG database was designed for the analysis of gene expression patterns obtained from microarray expression data. cREMaG allows for the identification of overrepresented TFBSs in the promoter regions of genes from co-expressed gene sets. Besides cREMaG, there are other systems suitable for the analysis of overrepresented transcription factor binding sites in mammalian genes, including oPOSSUM, PAP, CORE_TF, TFM-Explorer, Pscan and Genomatix Stable and well-established data repositories were selected to build cREMaG. cREMaG uses matrices from two publicly available TFBS matrices repositories, JASPAR and the public release of TRANSFAC Promoters stored in cREMaG were curated for their evolutionary conservation, based on the assumption that functional elements evolve more slowly than nonfunctional elements Optimization of the matrix score threshold was resolved previously in the PATSER, MATCH and Genomatix tools Our previous experience with microarray studies http://www.cremag.org address. However, if you find the cREMaG database useful for your work, please cite this paper.The database is freely available to academic and non-academic users at the Table S1List of Affy IDs of CREB-dependent genes from gene expression profiling in the striatum of cocaine-treated mice lacking CREB in the forebrain .(0.02 MB XLS)Click here for additional data file.Table S2Query results of CREB-regulated gene-set.(0.02 MB XLS)Click here for additional data file.Table S3The gene sets, with the original IDs, RefSeq numbers, promoter sequences and results of cREMaG comparison with other tools.(0.60 MB XLS)Click here for additional data file.


There is little research on the relationship between key socioeconomic variables and alcohol related harms in Australia. The aim of this research was to examine the relationship between income inequality and the rates of alcohol-attributable hospitalisation and death at a local-area level in Australia.We conducted a cross sectional ecological analysis at a Local Government Area (LGA) level of associations between data on alcohol caused harms and income inequality data after adjusting for socioeconomic disadvantage and remoteness of LGAs.The main outcome measures used were matched rate ratios for four measures of alcohol caused harm; acute (primarily related to the short term consequences of drinking) and chronic (primarily related to the long term consequences of drinking) alcohol-attributable hospitalisation and acute and chronic alcohol-attributable death. Matching was undertaken using control conditions  at an LGA level.A total of 885 alcohol-attributable deaths and 19467 alcohol-attributable hospitalisations across all LGAs were available for analysis. After weighting by the total number of cases in each LGA, the matched rate ratios of acute and chronic alcohol-attributable hospitalisation and chronic alcohol-attributable death were associated with the squared centred Gini coefficients of LGAs. This relationship was evident after adjusting for socioeconomic disadvantage and remoteness of LGAs. For both measures of hospitalisation the relationship was curvilinear; increases in income inequality were initially associated with declining rates of hospitalisation followed by large increases as the Gini coefficient increased beyond 0.15. The pattern for chronic alcohol-attributable death was similar, but without the initial decrease. There was no association between income inequality and acute alcohol-attributable death, probably due to the relatively small number of these types of death.We found a curvilinear relationship between income inequality and the rates of some types of alcohol-attributable hospitalisation and death at a local area level in Australia. While alcohol-attributable harms generally increased with increasing income inequality, alcohol-attributable hospitalisations actually showed the reverse relationship at low levels of income inequality. The curvilinear patterns we observed are inconsistent with monotonic trends found in previous research making our findings incompatible with previous explanations of the relationship between income inequality and health related harms. The harms caused by drinking are mediated by a variety of social and contextual factors operating at both individual and community levels. For example, social class  has been shown to independently account for the occurrence of alcohol caused harms . Other sA considerable amount of work in social epidemiology has focused upon income inequality or disparity. Income disparities arise when income is unequally distributed across a given population, irrespective of the absolute income levels of the population. Income inequality has been shown to be associated with a variety of health and social outcomes including rates of all-cause mortality , violentIncome inequality can be measured in a number of ways, but is most often measured through the Gini coefficient in the field of health research . The GinVarious mechanisms have been postulated through which income inequality may manifest an effect on health outcomes ,14. Typiet al . For th10threvision, Australian Modification (ICD-10-AM). Hospitalisation data analysed in this report cover the 1999/2000 fiscal year while the mortality data were obtained for the 2000 calendar year.Two types of alcohol related harm were examined; alcohol-attributable hospitalisation and alcohol-attributable mortality. Data on hospitalisations was obtained from the AIHW's National Hospital Morbidity Database (NHMD) which is a compilation of clinical information on hospital separations across all public and almost all private hospitals in Australia. Mortality data were sourced from the ABS Mortality Datafile, which is a compilation of details of all Australian deaths obtained from state and territory Death Registries. Both data sources contain information on age, sex, principal diagnosis, external cause and LGA of residence for all cases. Unfortunately the NHMD does not include detailed data on place of residence for Queensland or South Australian hospitalisations, meaning that these two states were not included in the analysis of alcohol-attributable hospitalisations. Principle diagnosis and any applicable external causes are coded on both datasets according to International Classification of Diseases et al .There was a total of 630 Australian LGAs included in our dataset. The number of events varied across LGAs with some LGAs having few or zero cases or controls. LGAs with zero cases or controls for a given outcome measure were not included in the analysis of that outcome. As hospitalisation data from Queensland and South Australia were unavailable, LGAs from these two states were not included in the analysis of alcohol-attributable hospitalisations. The number of LGAs included in each analysis is specified in relation to each of the models below.There were 885 alcohol-attributable deaths and 19467 alcohol-attributable hospitalisations across all LGAs. Table There was a relatively large number of alcohol-attributable hospitalisations and 373 LGAs with corresponding Gini coefficients available for analysis of the acute alcohol-attributable hospitalisations and 349 LGAs with corresponding Gini coefficients available for analysis of the chronic alcohol-attributable hospitalisations. Table Figures Table Figures This study is the first to provide evidence of a relationship between income inequality and alcohol-attributable harm in Australia. The nature of the relationship was consistent across acute and chronic alcohol-attributable hospitalisations and was similar for chronic alcohol-attributable deaths. In general the results showed that increasing LGA-level income inequality was associated with increasing rates of alcohol-attributable harm, after adjusting for general socio-economic disadvantage and remoteness of LGAs. While these relationships appeared strong and robust, there was no evidence of a relationship between income inequality and acute alcohol-attributable deaths, possibly due to the relatively small number reported.et al [et al's matched analysis (where they included other injury death as controls) showed a clear monotonic trend with no statistically significant differences in odds between different percentiles of their Gini coefficient. In this way even our findings in relation to the association between the Gini coefficient and mortality differ from those found in previous research.The relationship between income inequality and health outcomes such as all-cause mortality remains controversial ,14. Howeet al  in relatAs indicated, the relationship between income inequality and poor health has been potulated to result from a variety of direct or indirect causal paths ,14. ProcThe findings in relation to remoteness showed that inner and outer-regional areas of the country were less likely to experience alcohol-attributable harm than major cities and remote areas of Australia. These geographic variations contrast with previous Australian research that has generally shown metropolitan areas to have lower rates of alcohol-attributable harm than non-metropolitan areas . This coThis study has several limitations. First, the study was ecological meaning that there is the potential for ecological bias. However, the Gini coefficient, our exposure variable, is not subject to a classical ecological fallacy because it is a characteristic the LGA that has been measured. Second, the study was cross-sectional in nature. Ready interpretation of cross-sectional ecological studies requires the exposure  and outcome  to occur within a similar timeframe. In this framework it is reasonable to infer that hazardous/high-risk drinking produced the acute alcohol-attributable outcome for which a relationship to income inequality was observed . The same is not necessarily true of the chronic alcohol-attributable conditions that result from sustained patterns of risky/high-risk drinking over time. In this case we need to assume relative stability in persons' residence over time, and relatively static levels of inequality. The validity of these assumptions is unknown.In this study we found a curvilinear relationship between income inequality and the rates of some types of alcohol-attributable hospitalisation and death at a local area level in Australia. While alcohol-attributable harms generally increased with increasing income inequality, alcohol-attributable hospitalisations actually showed the reverse relationship at low levels of income inequality. These curvilinear patterns we observed are inconsistent with monotonic trends found in previous research making our findings incompatible with previous explanations of the relationship between income inequality and health related harms. This has significant implications for public policy initiatives directed towards reducing income inequalities within LGAs, as it suggests that any effects on alcohol-attributable harms may not be uniform across different levels of income inequality.The authors declare that they have no competing interests.All authors contributed to the design of the study. PD led the writing of the manuscript and DJ and PD led the statistical analyses. TC and PC extracted the data for analysis. All authors contributed to the drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The ovary undergoes several changes after the                        menopause. The main characteristics of the postmenopausal ovary are a loss of                        follicles and several changes as a consequence of apoptotic processes. Signs of                        atrophy and fibrosis are evident. Primordial follicles are usually absent in                        postmenopause, whereas corpora atretica, hemorrhagica and albicantia, scar                        tissue, and simple follicular cysts are common.                                in vitro and in vivo["germinal                                    epithelium." During the                            adult, reproductive period of life, OSE is mainly involved in the physiological                            process of ovulation. Ovulation induces cyclic rupture and regenerative repair                            of the ovarian coelomic epithelium. This process of repeated disruption and                            repair accompanied by the complex remodeling reflects a somatic stem/progenitor                            cell-mediated process in the mammalian ovaries; a label-retaining cell                            population in the coelomic epithelium of the adult mouse ovary has already been identified as possible somatic stem/progenitor cells [Ovarian surface epithelium (OSE) is an                            important structure of the human ovary and is involved in both reproductive                            function and ovarian tumor formation. Primordial germ cells (PGCs) in embryonic                            ovaries are of extraovarian origin, but those developing during the fetal                            period are derived from the OSE. PGCs in the fetal ovary express most, but not                            all of the markers associated with pluripotent cells  and can d in vivo. Becauseor cells .       The                            OSE layer gradually flattens, but is always present, even in menopausal females                            of advanced age . ApoptotIt seems that human OSE stem                            cells retain the characteristics of embryonic stem cells. Based on previous                            experience , Virant-in vitro fertilization program,                            these cells developed into round oocyte-like cells  and Deleted in azoospermia-like (Dazl) are highly                            expressed in aged mouse ovaries with complete oocyte depletion [Stra 8 gene                            and failed to develop further. These cells retained the capacity to develop into                            oocytes when transplanted and exposed to a young host environment. Premeiotic                            germ cells apparently persist in aged atrophic mouse ovaries but are blocked in                            their ability to undergo meiosis and transition into oocytes.                                        Recently, the Tilly's group                            performed important experiments on the adult mouse ovary. They found that the                            germline-specific meiosis-commitment genes epletion . In the de novo folliculogenesis and oogenesis in the adult ovary                            cannot be ruled out. It is difficult to prove de novo                            oogenesis/folliculogenesis in humans, because there are strict limitations on                            the methodologies allowed in in vivo studies. Tilly's group was the                            first to confirm this in the mouse model. They demonstrated cells expressing                            the meiotic protein SCP3 in juvenile and adult mouse ovaries after the previous                            elimination of the primordial follicle reserve with the cell toxin busulfan [In spite of the                            persistent dogma that the number of follicles and oocytes in the mammalian                            ovary is set at birth and depleted over the course of life, a role of ovarian                            stem cells in busulfan . They fobusulfan . Furtherbusulfan .       busulfan . Zou andbusulfan  infectedde novo oogenesis and folliculogenesis in the                            adult human ovary: firstly, the presence of stem cells in the human adult OSE [Two main scientific                            facts support the idea of dult OSE ,7 and indult OSE  as well dult OSE , and secdult OSE .       Most ovarian cancers arise from the OSE -                            mesothelial surface lining of the ovaries or from invaginations of this lining                            into the superficial ovarian cortex which form cortical inclusion cysts. These                            cysts are thought to be precursor lesions of ovarian carcinomas.                            Epithelial-mesenchymal transition, a transcriptional program inducing                            maintenance of the mesenchymal phenotype, plays a role in tumor progression and                            metastasis . Aggressde novo oogenesis/folliculogenesis) due to                            different natural blockades in the ovary, but they can be involved in the                            formation of ovarian cancer. Because they are accumulated in the OSE layer,                            they can quickly develop into ovarian tumors. This might be the reason for the                            clinical experience of a higher incidence of epithelial ovarian cancer in                            older, postmenopausal women [Stem cells                            present in the OSE layer of postmenopausal women can no longer be involved in                            reproductive function (al women  and for al women .


Background and purpose Two-stage revision remains the gold standard in the treatment of infected knee arthroplasty. Lately, good long-term results of direct exchange arthroplasty have been reported. The purpose of this literature review is to compare the clinical outcome achieved with one-stage revision and two-stage revision with different types of spacers.Methods A thorough systematic review of literature was undertaken to idenepsy reports on the treatment alternatives. Papers written in English or including an English abstract, published from 1980 through 2005, and reporting either the success rate in eradication of infection or the clinical status achieved were reviewed. 31 original articles describing the results of 154 one-stage exchange arthoplasties and of 926 two-stage exchange arthoplasties were included. The depth of detail in the description of materials and methods varied markedly, making it impossible to perform a meta-analysis. Instead, a descriptive review of the results is presented.Results With a follow-up of 12–122 months, the overall success rate in eradication of infection was 73–100% after one-stage revisions and 82–100% after two-stage revisions. Reinfection rates were the lowest in series where articulating cement spacers were used, though the follow-up was relatively short. Studies using articulating spacers reported the highest average postoperative ranges of motion. Otherwise, no correlations were observed between the clinical outcome and the length of follow-up, the type of revision, or the type of spacer. The clinical outcome (knee scores and range of motion) of the one-stage revisions was no different from that of the two-stage revisions.Interpretation Two-stage exchange is an effective treatment. Mobile spacers may further improve the range of motion. More experience in one-stage revision is required in order to define its role in the management of infected knee arthroplasties. Two-stage exchange arthroplasty has remained the gold standard in the treatment of infected total knee arthroplasties (TKAs) for over two decades . The oriVery little has been published on one-stage revision in the literature , but proIn January 2006, we searched several international databases using highly sensitive though somewhat unspecific search strategies, which are described in detail in Appendix 1. The literature search strategies were created in co-operation with a librarian. The research plan was reviewed and approved by the Finnish Office for Health Technology Assessment (see http://finohta.stakes.fi/).Two of the present authors (EJ and IS) reviewed the search results independently and classified the references found in terms of whether they should be included on basis of the title of the paper , whetherFinally, 25 original studies published in English  and 6 stData about materials, methods, and results of each original article included was extracted into a specific form independently by EJ and IS. Each study was assessed using a checklist (Appendix 2). The checklist, based on previously published criteria for evaluation of the internal validity  and geneThe materials extracted by EJ and IS were compared to each other and conflicting data were re-checked from the original papers and corrected after discussion.The principal outcomes were (1) the rates of new and recurrent infections and (2) the clinical outcome—measured as postoperative clinical knee score and range of motion—following the revision arthroplasty. Due to heterogeneity and to the low calculated quality scores in the reports, the study materials were not pooled using meta-analytical techniques. Instead, several graphs were prepared for the principal outcome variables in order to visualize any possible trend after sorting the materials by a potentially explanatory variable. A qualitative and descriptive summary of the results is also presented.The studies reported the results of 926 two-stage and 152 one-stage arthroplasties . The numNone of the studies included used a randomized or controlled trial setting. In 2 studies of two-stage revisions, historical comparison groups were used to analyze the effect of spacer type on treatment outcome . 5 studiIn all studies, patients were given intravenous antibiotics after the first-stage operation, usually for 4–6 weeks. The details of antibiotic treatment  were often unavailable, however, and only half of the studies reported the exact length of the interim period between the two stages . After reimplantation arthroplasty, a common protocol seems to have been to continue intravenous antibiotics until the results of operative bacterial cultures were ready. There appeared to be a correlation between publication year (rather than spacer type) and the duration of the interim period, with the most recent studies reporting shortest interim periods (data not shown). After one-stage revisions, antibiotics were given intravenously for periods ranging from 1–2 weeks  to 4–6 wThere were no statistically significant differences in patient demographics , types oSuccess in infection management was analyzed in three ways. Firstly, the total number of infections appearing after treatment was recorded, supplemented by the number of recurrent infections and of new infections . After the index revision, deep knee infection was detected in 0–31% of cases. The treated infections recurred in 0–18% of the cases. The new infection rate varied from 0 to 31%.The length of follow-up did not appear to affect the rate of recurrent infections, but the reports with follow-up of < 4 years had few new infections .The lowest rates of recurrent infection were reported in studies where no spacer was used (0–5%) . Also, rKnee Society knee score and Hospital for Special Surgery knee score were used to measure the clinical outcome in most studies. Even more frequently, postoperative range of motion or maximal flexion was reported . PreoperThe highest average postoperative ranges of motion or maximal flexion exceeding 100 degrees (104–112 degrees) were achieved with articulating spacers , but theData that can allow calculation of the change from preoperative to latest postoperative range of motion or clinical score were available in 9 studies. Except for 1 report , conside2 studies comparing static and mobile spacers have been published . ClinicaDespite the relatively high number of reports on the treatment of infected TKAs with exchange arthroplasty, there is a lack of prospective comparative trials. Most of the studies reviewed were retrospective case series and failed to describe materials, surgical technique, or both in enough detail—thus making it difficult to compare the studies to each other. The studies published after the literature searches of our study  do not aBecause of the heterogeneity of the studies and their materials, we could not use meta-analytical techniques. Instead, the studies were reviewed descriptively, which limits the objectivity of conclusions and leaves room for interpretative disagreement. To minimize the confounding effect caused by heterogeneous materials, only papers reporting a pure series of either one-stage or two-stage revision for deep knee infection were included. Consequently, some potentially relevant papers  were excThere was—and there still is—no question about the role of the two-stage approach being the gold standard in the management of infected knee replacements. Thus, our aim was to detect any factors that might have a relationship with the outcome variables under study. A trend suggesting such a relationship was found between the type of spacer and the rate of recurrence of infections, and also between the type of spacer and postoperative range of motion . However, as it appears that recurrence rate has declined over the past 2 decades  may haveSpacers to be used during the interim period between resection and reimplantation operations were introduced to maintain joint cavity and to prevent contractures of periarticular soft tissues and, to thereby facilitate the reimplantation. Mobile spacers are thought to ease patients’ ambulation and prevent soft tissue contractures and muscle atrophy. In our study, no factors that could be reliably related to improved postoperative outcome were detected. A slight trend suggesting some advantage of mobile spacers over other types was seen, but only concerning range of motion .Though advantages of spacers of any type could not be proven, the results of our review and of the most recent reports  stronglyA deep infection is an expensive complication  and the Although reinfection rates of only 9% and 11% after one-stage revision have been reported , the higOf the different modifications of the conventional two-stage approach, the recently reported protocol with shorter (2–3-week) antibiotic treatment has resulted in reasonable eradication rates , but at One of the major problems with the comparisons between the different treatment approaches used in this study is the possibility of selection bias. It is possible that the patients selected for one-stage revision or two-stage revision with mobile spacer prosthesis were healthier than those patients for whom two-stage revision was performed, for example, without any spacers. In such cases, the comparisons may be biased in favor of treatment alternatives, to optimize the changes to eliminate the infection. Such selection by surgeon’s preference would bias comparisons in favor of treatment alternatives other than the conventional two-stage exchange arthroplasty. Inclusion and exclusion criteria, and also the primary diagnoses and patient comorbidity indices were seldom reported. For these reasons, the confounding effects of host status and type of infection—and also of possible selection bias—could not be analyzed.The studies reviewed report rather similar success rates of 82–100% in eradication of infection. The rates are comparable to those of the more recently published series and to those of series published in languages other than English  and 4. TThe factors that affect the outcome of exchange arthroplasty remain largely unknown. Clinical experience and studies on infected hip and knee arthroplasty suggest that host status (comorbidity), type of infection (acute vs. chronic), and the condition of the environment of the joint involved contribute to the outcome of infected knee replacement , 2002. TMost reports on exchange arthroplasty performed for infected knee arthroplasty are of poor methodological quality, and no unbiased comparative studies exist. The previously reported series included in this systematic analysis yielded similar success rates, but the factors that could predict the outcome are mostly unknown. In the light of our findings, two-stage revision with delayed reimplantation remains the gold standard. It seems reasonable to employ mobile spacers where possible, as they do not compromise the attempts to eradicate infection but may improve postoperative outcome. Despite some promising preliminary reports, the value of and indications for direct exchange arthroplasty remain unclear. Future studies focusing on these treatments should preferably use a prospective randomized setting and compare the new approach to the gold standard of two-stage exchange arthroplasty.


Arabidopsis thaliana genome, the Arabidopsis community and the annotator centers have been working on the improvement of gene annotation at the structural and functional levels. In this context, we have used the large CATMA resource on the Arabidopsis transcriptome to search for genes missed by different annotation processes. Probes on the CATMA microarrays are specific gene sequence tags (GSTs) based on the CDS models predicted by the Eugene software. Among the 24 576 CATMA v2 GSTs, 677 are in regions considered as intergenic by the TAIR annotation. We analyzed the cognate transcriptome data in the CATMA resource and carried out data-mining to characterize novel genes and improve gene models.Since the finishing of the sequencing of the The statistical analysis of the results of more than 500 hybridized samples distributed among 12 organs provides an experimental validation for 465 novel genes. The hybridization evidence was confirmed by RT-PCR approaches for 88% of the 465 novel genes. Comparisons with the current annotation show that these novel genes often encode small proteins, with an average size of 137 aa. Our approach has also led to the improvement of pre-existing gene models through both the extension of 16 CDS and the identification of 13 gene models erroneously constituted of two merged CDS.This work is a noticeable step forward in the improvement of the Arabidopsis genome annotation. We increased the number of Arabidopsis validated genes by 465 novel transcribed genes to which we associated several functional annotations such as expression profiles, sequence conservation in plants, cognate transcripts and protein motifs. Arabidopsis thaliana and its first annotation by the international Arabidopsis community [Since the finishing of the whole genome sequencing of the model plant ommunity , gene prommunity . Indeed,ommunity . The latommunity . In addiommunity ,6, compaommunity ,8, or coommunity .In the framework of the European CATMA project ,11, a miAvailable public transcriptome data produced with the CATMA micro-arrays were used to investigate these questions . The dat++ database [++ also contains TAIR gene annotations, available transcript sequences and the latest version of the Eugene predictions (v1.59) for the Arabidopsis genome. The gene extremities were extended using overlapping cognate transcript sequences (EST and cDNA). This improved definition of UTRs allowed us to discard GSTs which are outside annotated CDS but which overlap extended transcriptional units. Similarly, GSTs mapped less than 300 bp away from the extremity of a predicted CDS without cognate transcripts were not selected since they could correspond to the unknown UTR region of the corresponding mRNA. The 677 GSTs mapped outside TAIR annotated genes, pseudogenes or known RNA genes  were selected as novel candidate genes. Among these 677 candidates, 28 occur in Eugene models, which extend TAIR CDS models. The corresponding expression data provide an improvement of CDS annotation  is always confirmed by the coherent extension of similarities with homologous proteins . For instance, we found 63 novel genes expressed in one leaf sample only, which is significantly more than the 38.6 expected  based on the 136 leaf samples.The comparison of the transcription data obtained from 522 hybridized samples for the 465 novel genes and all the 21 260 Arabidopsis genes tagged by a CATMA v2 GST shows interesting features at the functional level. Most newly identified genes are detected in a limited number of experimental conditions  according to the TAIR annotation but two different gene models were predicted by Eugene. For 13 loci, the transcriptome results show that two GSTs associated with the same gene provide opposite ratios in the same experiment, thus suggesting that they actually match two different genes . Furthermore, the analysis of the transcriptome data from 522 hybridized samples brings an additional functional dimension with numerous expression conditions of these novel and corrected genes. The biological and biochemical roles of the large majority of the novel genes remain unknown since only 15% of them share similarities with proteins of known function  between the two samples co-hybridized on the same array, is given for each spot. A normalized logarithm intensity for each sample is also calculated. This is done according to the within-array correction proposed by Yang and Thorne [For each CATMA array, the raw data are the logarithm of median feature pixel intensity at 635 nm (red) and 532 nm (green) wavelengths; no background is subtracted. A normalization per array is performed to remove systematic biases. First, spots that are considered badly formed features are excluded. Then, a global intensity-dependent normalization is performed using the lowess procedure  to corred Thorne .Since each comparison of two samples is performed in dye-swap, the log-ratio between the two co-hybridized samples is defined as the average of the normalized log-ratios of the two arrays of a dye-swap, and the intensity signal of a sample is defined as the average of the normalized logarithm intensities of the two arrays of a dye-swap.a posteriori (MAP) rule does not classify it on the component with the highest mean and such that one of the calculated posterior probabilities is greater than 10-4. Once the threshold is defined, an intensity signal is declared as significant when it is greater than the hybridization threshold and the associated GST is declared hybridized. Otherwise, the intensity signal is not significant and we consider that transcription of the corresponding gene is not detected.We have developed a new statistical procedure to determine the set of probes whose intensity signal is considered significant, since existing procedures are either an arbitrary threshold based on an estimation of a local background or require the knowledge of a population of non-hybridized probes . Our proi.e. when for one of the two arrays the spot corresponding to the gene was a badly formed feature.We focus on distinct GSTs supposed to match the same gene, declared differentially expressed and which have log-ratio of opposite sign. To do that, a differential analysis is performed per dye-swap with a paired t-test on the normalized log-ratios. The number of observations per spot is inadequate for calculating a gene-specific variance. For this reason, it is assumed that the variance of the log-ratios is the same for all genes, and spots displaying extreme specific variances  are excluded. The raw P values are adjusted by the Bonferroni method, which controls the Family Wise Error Rate (FWER) . When th++ database [Searches of cognate transcripts, RACE-PCR products, previous lost annotation, and of homologous proteins in Arabidopsis or in other species have been carried out by sequence comparisons (BLASTn or BLASTp) with GenBank Release 159. Additional information such as PFAM motifs, MPSS tags, GST position and Eugene v1.59 CDS models have been retrieved from the FLAGdbdatabase ,18.Primers for RT-PCR were designed using Primer3  with theSA designed the study, performed data-mining and drafted the manuscript. MLMM performed the statistical analyses. VB selected candidate GSTs and participated in exploitation of transcriptome data. LT carried out RT-PCR, participated in production of transcriptome data. FB and SB participated in production of transcriptome data. PEJ and MI provided RNA pollen samples. VT designed the GSTs and the primers for RT-PCR. TS developed the Eugene software and improved the manuscript. AL participated in design study and helped to draft the manuscript. JPR conceived of the study and generated transcriptome data. All authors have read and approved the final manuscript.Information about identification and function of the 465 novel genes. 1: ID of GSTs selected outside TAIR models and exhibiting a transcription signal  and exhibiting a transcriptional signal (with a web link to the CATdb database  for addiClick here for fileInformation about detected erroneous gene merging. 1 and 2: ID of the two GSTs used to detect the gene merging (with a web link to the CATdb database  for addiClick here for fileIdentification in GEO  or ArrayClick here for file


Cystic adventitial disease of the popliteal artery is a rare cause of non-atheromatous claudication. It usually requires surgery to improve the distance walked by patients.We report the case of a 44-year-old Caucasian man with unilateral symptomatic popliteal cysts extending to his genicular branches and associated with multilevel stenosis of his anterior tibial artery. A surgical evacuation of the cysts successfully restored his arterial patency and led to an objective haemodynamic improvement but was associated with early recurrence of symptoms.We suggest that the involvement of the genicular branches in cystic adventitial disease of the popliteal artery is a possible indicator of extensive adventitial degeneration and unfavourable clinical prognosis. Cystic adventitial disease (CAD) of the popliteal artery (PA) is a rare but well-recognized non-atheromatous cause of claudication. Since it was first described in 1954 [Histopathological features of the disease are cystic collections of mucinous material containing varying combinations of mucopolysaccharides and mucoproteins within the adventitial layer of the artery. The cysts exert extrinsic pressure on the arterial lumen, which accounts for the clinical manifestations of chronic lower limb ischemia, mainly intermittent claudication, and limb pain with absent distal pulses. Its aetiology is uncertain, with theories arguing about the possible degenerative, embryonic or ganglionic nature of the disease .Diagnosis is usually achieved with duplex ultrasound, computed tomography (CT), or magnetic resonance imaging (MRI). Various approaches of treatment have been described, including percutaneous cyst aspiration, open incision and cyst enucleation, endovascular stenting, excision of the cyst with autologous vein graft reconstruction, and bypass surgery .We report the case of a patient with unilateral claudication secondary to multiple adventitial cysts of the popliteal artery with additional involvement of the genicular arteries.A 44-year-old Caucasian man who works as a personal trainer presented with a four week history of unilateral (right) leg claudication occurring at a distance of 150 meters and exacerbated by running. He had no significant personal medical history, smoked five cigarettes per day, and engaged in extreme sports and vigorous exercise. His body mass index (BMI) was normal, although he had been morbidly obese ten years prior to presentation.On examination, all of our patient's lower limb pulses were palpable beside the dorsalis pedis on both his feet. His Doppler ankle-brachial pressure index (ABPI) was 1.09 on the affected side, with a 40 mmHg post-exercise pressure drop. Duplex ultrasound revealed three adventitial popliteal cysts, the largest measuring 3.4 cm (length) by 0.8 cm (diameter). A magnetic resonance angiogram confirmed a high-grade stenosis in his symptomatic proximal popliteal artery, as well as multiple stenoses in both his anterior tibial arteries but with a three-vessel bilateral run-off. An MRI scan of the affected popliteal fossa, performed to accurately assess the relations of the cysts to the surrounding structures and to exclude any other pathology, additionally showed involvement of his genicular arteries  limb occurring at a distance of more than half a mile and after exercise. A repeat Duplex scan demonstrated that his popliteal artery was widely patent with no evidence of recurrent stenoses. His ABPI was 1.36 with no pressure fall after exercise. In the absence of radiological evidence of popliteal artery stenosis, our patient was advised to avoid strenuous exercise, with a view to proceed to further imaging if symptoms recurred.et al. [Although CAD of the popliteal artery was first described more than five decades ago, there is a growing published interest in the diagnosis and management of this rare condition -7. Our cet al.  that focMultiple treatment options have been employed in the management of the disease. Despite reports of spontaneous resolution of symptoms , the majBecause of the rarity of the disease and the lack of large studies involving long follow-up examinations, the recurrence rate of previously treated CAD of the popliteal artery, or any associated risk factors, are not precisely known; it is, however, presumed to vary between six percent and ten percent, irrespective of the treatment method, with onset of recurrent symptoms between one and 21 months -15.The proposed mechanism for an adventitial cyst to become symptomatic involves a sufficient increase of the pressure within the fluid-filled cyst during muscle exertion, thus resulting in haemodynamically significant endoluminal stenosis . CommuniCAD of the popliteal artery, although uncommon, should be considered in the differential diagnosis in young patients presenting with claudication, particularly if there are no risk factors for peripheral vascular disease. Our report raises the possibility that the extension of CAD to the genicular arteries could be a predictor of higher risk of recurrence, either as an indicator of cysts communicating with the knee synovium or as a marker of the involvement of smaller vessels elsewhere. Vascular surgeons should thus be encouraged to report similar cases in order to better identify risk factors of unsuccessful outcome based on larger series. Also, patients should be warned that they may not experience complete resolution of their symptoms despite objective evidence of surgical patency.The authors declare that they have no competing interests.EY performed the literature search and compiled data presented in this report. PT undertook the management of our patient from the time of his initial presentation to his surgery and follow-up examination. He also revised the manuscript draft. Both authors read and approved the final manuscript.Written informed consent was obtained from our 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.