Venous abnormalities contribute to the pathophysiology of several neurological conditions. This paper reviews the literature regarding venous abnormalities in multiple sclerosis (MS), leukoaraiosis, and normal-pressure hydrocephalus (NPH). The review is supplemented with hydrodynamic analysis to assess the effects on cerebrospinal fluid (CSF) dynamics and cerebral blood flow (CBF) of venous hypertension in general, and chronic cerebrospinal venous insufficiency (CCSVI) in particular.CCSVI-like venous anomalies seem unlikely to account for reduced CBF in patients with MS, thus other mechanisms must be at work, which increase the hydraulic resistance of the cerebral vascular bed in MS. Similarly, hydrodynamic changes appear to be responsible for reduced CBF in leukoaraiosis. The hydrodynamic properties of the periventricular veins make these vessels particularly vulnerable to ischemia and plaque formation.Venous hypertension in the dural sinuses can alter intracranial compliance. Consequently, venous hypertension may change the CSF dynamics, affecting the intracranial windkessel mechanism. MS and NPH appear to share some similar characteristics, with both conditions exhibiting increased CSF pulsatility in the aqueduct of Sylvius.CCSVI appears to be a real phenomenon associated with MS, which causes venous hypertension in the dural sinuses. However, the role of CCSVI in the pathophysiology of MS remains unclear. The cerebral venous system is often viewed simply as a series of collecting vessels channeling blood back to the heart, yet it also plays an important role in the intracranial hemodynamic/cerebrospinal fluid (CSF) regulatory system (hereafter simply referred to as the hydrodynamic regulatory system), a role that is often overlooked and that appears to influence both perfusion of the brain parenchyma,2 and thDespite having very different pathologies, MS, leukoaraiosis, and NPH all share some common characteristics. In all three conditions, cerebral blood flow (CBF) is reduced-21. Bothet al.[et al.[5, whose course begins in the WM[Since the earliest years of research into MS, there has been suspicion that the venous system might be involved in its etiology, with Dawson, Putnamet al. finding [et al. showed tn the WM, and then the WM-57, leucn the WM, and subn the WM lesions n the WM,59.et al.[P < 0.0001). This finding appears to corroborate the work of Ge et al.[et al., who attributed the reduction in VVV to hypometabolic status in the brain parenchyma of patients with MS, Zivadinov et al. performed a pre-contrast and post-contrast SWI venography experiment, which indicated the reduction in VVV to be due to morphological changes in the cerebral veins of patients with MS. Indeed, such was the clear-cut nature of these venous changes that Beggs et al.[Recently, there has been renewed interest in studying vascular changes associated with MS-62. Thiset al. reportede et al.. Howevers et al. were ablet al.[et al.[et al.[et al.[et al.[et al.[et al.[These findings reinforce a large body of evidence connecting MS with alterations in the cerebral vascular bed. Using tomography, a number of early investigators-71, founet al., identif[et al. studied [et al. found re[et al. reported[et al.. Collect[et al.. Several[et al. found hy[et al. found a [et al., who fouet al.[et al.[et al. did not specifically consider CCSVI, their findings are consistent with those of Zamboni et al., and suggest that venous hypertension may be a feature of MS. Abnormal CSF hydrodynamics have also been implicated in the formation of cortical lesions in MS. Sub-pial lesions, which appear not to be perivenous, cover extensive areas of the cortex, and extend from the surface into the brain[et al.[Venous hypertension in the dural sinuses is known to inhibit absorption of CSF through the arachnoid villi (AV),76. Zambet al. reported[et al. also fouhe brain. They aphe brain,78. Kutz[et al. found suet al.[Leukoaraiosis is a radiological finding, characterized by WM hyperintensities in the periventricular region on T2-weighted MRI scans, which iet al. found a Mirroring the cerebral hemodynamics of MS, several researchers have reported leukoaraiosis to be associated with reduced CBF,83,90,91et al.[Further evidence linking leukoaraiosis with altered venous hemodynamics comes from a series of studies by Chung and co-workers,16,101, et al..In a series of studies, Bateman and co-workers investigated altered venous hemodynamics in a variety of neurological conditions,102,103.et al.[NPH occurs when there is an abnormal accumulation of CSF in the ventricles, causing them to become enlarged, but witet al. independet al.,116. Appet al.. In patiet al.; howeveret al.,102. NPHet al.. It is tet al.[et al.[et al.[CBF has been found to be lower in patients with NPH than in normal controls-123. Thiet al. found th[et al., who rep[et al. attribut[et al., who fou[et al..et al.[A number of researchers have reported marked alterations in CSF dynamics in NPH, with CSF pulsatility in the AoS found to be markedly greater in patients with NPH compared with controls,125-129.et al., who fouet al. or actuaet al.. Althouget al.,130, whiet al.,132. If et al., or alteet al.. Batemanet al. suggesteet al.,134, witet al.. Althouget al..Although there are clear differences in the pathologies of MS, leukoaraiosis and NPH, there are also striking similarities. All three are characterized by: 1) WM changes in the periventricular region; and 2) reduced CBF. The lesions associated with both MS and leukoaraiosis tend to be perivenous in nature, and the changes in CSF dynamics associated with NPH and MS also reveal similarities. This raises intriguing questions as to why these similarities exist. Are there some underlying physical mechanisms that are common to all these conditions?The proximity of immune-cell aggregations to the vasculature is a hallmark of MS. Whereaset al.[Another universal principle found in nature is that of mass transfer. In simple terms, in order for matter to move from one place to another, it must be transported by some mechanism. In biology, the transport of cells and chemicals generally occurs either by: diffusion, by active transport (in the case of ion transport across the cell membrane), or through transport in a bulk fluid such as blood. If diffusion or active transport are the mechanisms at work, then there is a tendency towards higher concentrations of the transported substance near its source and lower concentrations further away. If this simple logic is applied to the formation of perivenous MS lesions, it would suggest that the plaque formation emanates from the blood vessels, rather than the other way round. Indeed, the current thinking appears to support this, suggesting that in MS, plaque formation is precipitated by breaching of the blood–brain barrier (BBB),138,139.et al. that in The mass transport associated with bulk fluids also appears to offer insights into the spatial arrangement of ischemic WM changes, such as those found in leukoaraiosis. Considering oxygen transport in the blood through the cerebral vascular bed, the law of mass transport dictates that as oxygen is supplied to the brain parenchyma, so the oxygen levels in the blood will decrease. Consequently, the oxygen tension in the cerebral arteries will be higher than that in the cerebral veins. Under normal circumstances, this should not cause any problems, but when CBF is greatly impaired, as in both leukoaraiosis,83,90,91et al.[et al.[et al.[et al.[et al.[et al.[There is increasing evidence that hypoxia-like metabolic injury may be a pathogenic component in the formation of MS lesions,86. Wakeet al. found moet al. reported[et al. identifi[et al., who mea[et al. found th[et al. conclude[et al., using a[et al.,147. The[et al., followe[et al.,148,149.et al.[et al.[Several researchers have found similarities between leukoaraiosis and MS,61. Leuket al., investi[et al. to be as[et al., and it [et al.,13,22. T[et al.. Any inc[et al., an actiSo why should some regions of the brain be more vulnerable than others to damage? Perhaps the architecture of the cerebral-venous system provides some clues? While the distal venous regions may be prone to hypoxic stress, the spatial arrangement of the veins may also contribute to their vulnerability. Evidence in support this opinion comes from Schlesinger, who foret al.[The finding that the junction between the medullary and sub-ependymal veins has a high resistance to fluid flow is no surprise. The sub-ependymal veins are collecting vessels, which receive venous blood from a large number of the smaller medullary veins that enter the sub-ependymal veins at approximately 90 degrees. From a fluid-mechanics point of view, this is not a very streamlined configuration, and will result in relatively large pressure drop across this junction. Any stenosis at this junction would therefore greatly increase its resistance, possibly leading to distension of the upstream medullary veins, as Putnam and Adler reported. Consequet al. pointed et al., similaret al.,22, withet al.. Stenosiet al., who meaet al.[Unlike the deep venous system, the superficial system has thin-walled cortical bridging veins that traverse the SAS. Blood flow through these compliant vessels is controlled by sphincters, which regulate discharge into the SSS,159. Thiet al. found GMet al..MS, leukoaraiosis, and NPH all appear, to a greater or lesser extent, to be associated with marked changes in the dynamics of the intracranial CSF system. This suggests that these diseases might be associated with alterations in the intracranial hydrodynamic regulatory system, which controls the volume and pulsatility of the blood in the cerebral vascular bed,166,167.The various pulses associated with the intracranial hydrodynamic system are illustrated in Figure et al.[Close inspection of Figure et al. reportedet al., or alteet al..et al.[et al.[et al.[Although the precise behavior of the intracranial hydrodynamic system under conditions of venous hypertension is unknown, there is evidence that occlusion of the venous-drainage pathways causes blood to accumulate within the cranium. In an experiment involving healthy subjects, Kitano et al. showed t[et al. also per[et al. conclude[et al., who fouet al. published a paper[In 2009, Zamboni  a paper linking  a paper,64 due t a paper. CCSVI h a paper,174, wit a paper,175-179.The results obtained by researchers for CCSVI have been very mixed. For example, some researchers found CCSVI-like venous anomalies to be strongly associated with MS,180-186,et al.[P < 0.001)[et al.[P < 0.0001) with MS.One possible explanation for the discrepancies between studies is the echo color Doppler sonography (ECDS) frequently used to diagnose CCSVI. The floppiness of the vessels involved and the variability of the venous vasculature can lead to erroneous results if ECDS is not undertaken correctly-195. In et al. develope< 0.001). This co[et al., who fouPrevious work,64,196 sFigure Q is the fluid flow rate (ml/min), R is the hydraulic resistance (mmHg.min/ml), and ΔP represents the pressure drop  between the two ends of the vessel. By applying equation 1 to the intracranial system in Figure where et al.[One common feature of CCSVI is stenosis of one or both of the IJVs,197, whiet al..3/beat (assuming 70 beats/min)[20)[3/beat (assuming 70 beats/min), which is close to the mean value of 3.4 mm3/beat reported by Magnano et al.[3/beat reported by Zamboni et al.[From Figure ats/min), is veryats/min). In normats/min). These oats/min), allowinin)[20). Consequo et al. for reduet al.[It is possible to gain an insight into the nature of the hemodynamic changes associated with MS, by undertaking simple hydrodynamic analysis of composite data published by Varga et al.. These dThe data in Table From the data it can be seen that in patients with MS, there is a general reduction in the volume of the vascular bed, which, if approximated to a series of parallel round tubes, equates to a mean reduction in cross-sectional area of the vessels of about 8.4% in patients with MS. According to Poiseuille’s Law:et al.. According to equation 1, hypertension in the dural sinuses would tend to reduce the pressure gradient pushing the blood through the cerebral veins, which in turn would tend to inhibit blood flow. However, when we consider that the CPP is normally in the region of 70 to 90 mmHg, it is unlikely that venous hypertension of less than 5 mmHg, such as that associated with CCSVI, could account for the large reduction in WM CBF reported in patients with MS[where R is the hydraulic resistance of the vessel (mmHg.min/ml) and r is the radius of the vessel (mm), it can be calculated that the 8.4% reduction in average cross-sectional area equates to an approximately 19.3% increase in hydraulic resistance. Given that the blood-flow rate is directly proportional to the hydraulic resistance, this means that the reduction in CBV seen in patients with MS, is more than enough to account for the 15.6% reduction in CBF reported by Varga  with MS-21. Hencet al.[et al.[et al.[Although the above analysis is somewhat simplistic, it does illustrate that cerebral vascular volumetric changes alone appear capable of accounting for the reduction in CBF in the periventricular NAWM in patients with MS. In addition, this finding mirrors those of researchers investigating: 1) reduced CBF,83,90,91et al. of a 10%[et al., who repet al.[et al.[et al.[Further evidence suggesting that occlusion of the cerebral-venous drainage pathways might not be responsible for reduced CBF in patients with MS comes from Moyer et al., who com[et al., who per[et al., who inv[et al.-20, sugg[et al. reportedAlthough much research work has been undertaken into the contribution of venous abnormalities to various neurological conditions, there has generally been a lack of any hydrodynamic analysis to interpret the data collected. Without such analysis, it is possible to misinterpret results and come to potentially erroneous conclusions. In the Venous hypertension in the dural sinuses seems to be associated with marked changes in intracranial compliance. There is sound theoretical reason to believe that this will alter the dynamics of the intracranial CSF system, which in turn may affect the finely tuned intracranial windkessel mechanism. With respect to this, MS and NPH appear to share some similar characteristics. In particular, both conditions seem to be characterized by increased CSF pulsatility in the AoS.Despite conflicting studies, there is increasing evidence that CCSVI is a real physiological phenomenon, and that it is in some way associated with MS. The evidence from CSF-related studies in patients with MS, and the hydrodynamic analysis presented here, suggests that CCSVI causes venous hypertension in the dural sinuses. However, the role that CCSVI might play in the pathophysiology of MS remains unclear, and more work is urgently needed to understand the clinical relevance of this condition.ADC: Apparent diffusion coefficient; AoS: Aqueduct of Sylvius; AV: Arachnoid villi; AVD: Arteriovenous delay; BBB: Blood–brain barrier; CBF: Cerebral blood flow; CBV: Cerebral blood volume; CCSVI: Chronic cerebrospinal venous insufficiency; CNS: Central nervous system; CPP: Cerebral perfusion pressure; CSF: Cerebrospinal fluid; DVA: Developmental venous anomaly; ECDS: Echo color doppler sonography; GM: Grey matter; HIF: Hypoxia-inducible factor; ICP: Intracranial pressure; IJV: Internal jugular veins; JVR: Jugular venous reflux; MRI: Magnetic resonance imaging; MS: Multiple sclerosis; MTT: Mean transit time; NAWM: Normal-appearing white matter; NPH: Normal-pressure hydrocephalus; PVC: Periventricular venous collagenosis; RR: Relapsing–remitting; SAS: Sub-arachnoid space; SSS: Superior sagittal sinus; SWI: Susceptibility-weighted imaging; VVV: Venous vasculature visibility; WM: White matter.The author declares no conflicts of interest.The study was conceived and undertaken by CBB, who also wrote the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/142/prepub


Culture-independent microbiological technologies as well advances in plant genetics and biochemistry provide methodological preconditions for exploring the interactions between plants and their microbiome in the phyllosphere. Improving and combining cultivation and culture-independent techniques can contribute to a better understanding of the phyllosphere ecology. This is essential, for example, to avoid human–pathogenic bacteria in plant food.Most microorganisms of the phyllosphere are nonculturable in commonly used media and culture conditions, as are those in other natural environments. This review queries the reasons for their ‘noncultivability’ and assesses developments in phyllospere microbiology that have been achieved cultivation independently over the last 4 years. Analyses of total microbial communities have revealed a comprehensive microbial diversity. 16S rRNA gene amplicon sequencing and metagenomic sequencing were applied to investigate plant species, location and season as variables affecting the composition of these communities. In continuation to culture-based enzymatic and metabolic studies with individual isolates, metaproteogenomic approaches reveal a great potential to study the physiology of microbial communities  This is a reversible dormancy phase in bacteria when they are unable to undergo a sustained cellular division on or in standard laboratory media Oliver, . This stEscherichia coli O157:H7 on lettuce leaves still produced verotoxins and thus retained their virulent potential.The phenomenon of the VBNC state does indeed occur in the phyllosphere  and developing phyllosphere microbial populations has been addressed in studies by Hunter et al.  and Bali  Cry1Ac protein in transgenic cotton. DGGE profile data and sequences of 16 and 18S rRNA gene fragments suggested that fungi may be more susceptible to Cry1Ac protein than bacteria  used in Gram-negative Proteobacteria as quorum sensing (QS) molecules to regulate density-dependent mechanisms in bacterial communities in the tobacco phyllosphere and determined changes in the composition of this community by DGGE and phospholipid fatty acid analyses. It was suggested that pseudomonads and other AHL-producing Gammaproteobacteria utilize QS-dependent mechanisms to ensure their survival over other epiphytic residents in the nutrient-poor phyllosphere. Hence, AHL QS signals occurring naturally in the phyllosphere could play a role in the interactions between plant-associated bacteria. It is possible that AHL QS signals could be used to suppress pathogens in the phyllosphere of crops.Knowledge of the mechanisms, genes and compounds involved in interactions between microorganisms of the plant microbiome is essential for practical use in biological plant protection. Biological control agents are inherently cultivable microorganisms, and their cultivation is a prerequisite to apply biocontrol strains. Therefore, and for reasons already described in the section ‘Functional structures and metabolic diversity in microbial phyllosphere communities’ above, studies on microbial agents are usually based on cultivation in suitable media. However, a few other studies, which aimed at a more comprehensive assessment of the microbial phyllosphere communities influenced by microorganism–microorganism interactions, were also based on culture-independent approaches Table b. Lv et et al.  identifiE. coli O157:H7, supported the hypothesis that competition for nutrients is the primary mechanism of interactions between phyllospheric microorganisms (Carter et al., E. coli O157:H7 competed for carbon mainly with Actinobacteria, Proteobacteria, Basidiomycota and uncultured fungi and for nitrogen with Proteobacteria, Actinobacteria and uncultured bacteria.Detailed analyses of functional genes in a microbial community of a biofilm established in spinach leaf lysate, which was impaired by co-inoculation with et al. (Xanthomonas campestris pv. vitians, the causative agent of the bacterial leaf spot, and the presence or absence of other phyllosphere bacteria. It is possible that strains of the genus Alkanindiges act as facilitators, and those of Bacillus, Erwinia and Pantoea operate as antagonists of the pathogen.When studying the composition of bacterial communities colonizing the leaves of preharvest field-grown lettuce by pyrosequencing of 16S rRNA gene amplicons, Rastogi et al.  found coThe interaction between microorganisms in the phyllosphere is surely one of the most important issues raising questions that have to be answered before practical applications can be established. However, the research is far from complete. At present, we do not know whether the abundance of plant pathogens is a function of interactions between phyllosphere microorganisms or of the plant genotype. This needs to be clarified through further in-depth studies.et al. (Enterobacteriaceae including many culturable coliforms in the summer samples of field-grown lettuce, reflecting that this is a natural part of the lettuce microbiota instead of being accounted for by faecal contamination.In their large culture-independent survey of leaf surface microbiology, Rastogi et al.  found anE. coli and nontyphoidal Salmonella are enteric human pathogens and do not naturally occur in plants. However, they have been associated with multiple outbreaks of foodborne illness caused by the consumption of fresh-cut leafy vegetables (Teplitski et al., Enterohemorrhagic E. coli and nontyphoidal Salmonella are intrinsically culturable, but they can enter the VBNC state (Dinu & Bach, et al., et al., et al., et al., et al., et al., Enterohemorrhagic et al., et al., et al., et al., et al. (E. coli O157:H7 in utilizing plant nutrients, which is significant to its persistence on plants.Table , et al.  revealedet al., On the other hand, where quantitative evaluation of the persistence of a potential human pathogen following inoculation onto leafy vegetables is the aim of the study, qPCR targeting species-specific genes would be the recommended procedure (Arthurson et al. (et al. (E. coli on diverse fresh-cut leafy greens under preharvest through to postharvest conditions, respectively. Lopez-Velasco et al. (E. coli O157:H7 transformed for GFP expression and kanamycin resistance. The total population of epiphytic bacteria was enumerated on culture plates. Changes in the bacterial community structure during storage were detected by sequencing DGGE bands of 16S rRNA gene amplicons. qPCR was applied for assessing the virulence and stress response genes of E. coli O157:H7 (Table E. coli O157:H7 during storage. The fate of generic E. coli and E. coli O157:H7 during the production, harvest, processing and storage of leafy vegetables was reported in the study by Tomás-Callejas et al. (E. coli isolates and avirulent strains of E. coli O157:H7 were inoculated onto leafy greens in a greenhouse. Their survival was checked by genotyping the generic E. coli strains by REP-PCR and qPCR for the detection of E. coli O157:H7 (Table E. coli as well as E. coli O157:H7 were observed, but individual cells of both populations survived throughout the production and postharvest operations.Cultivation and culture-independent methods were combined in studies by Lopez-Velasco et al.  and Tomá (et al.  to inveso et al.  inoculats et al. . CocktaiFundamental questions regarding the ecology of enteric pathogens, their sources, persistence, distribution and routes of contamination of leafy greens have already been answered using culture and/or culture-independent methods. However, eliminating the risk of plant food contamination and subsequent human disease outbreaks requires clarifying the mechanisms by which enteric pathogens colonize plants and understanding how they can be inhibited or inactivated (Critzer & Doyle, et al. (in situ. Leaf structure and chemistry have been shown to play important roles in differentiating bacterial populations in the phyllosphere and could be prospective breeding targets for managing the phyllosphere microbiota to reduce the growth of phyto- and human pathogens on vegetables and crops. However, the level of research activity into plant–microorganism interactions is merely moderate at present. More attention should be paid to the effects of phyllosphere microorganisms on plants in addition to those of phytopathogens and to the understanding of the crosstalk between microorganisms. Progress in phyllosphere microbiology is not necessarily connected with the application of culture-independent methods as shown in food microbiology to observe enteric human pathogens on fruit and vegetables. However, unlike plate counting, culture-independent techniques have the advantage of also assessing cells at the VBNC stage, which can retain their virulent potential. Consequently, improving and combining cultivation and culture-independent techniques present a challenge to our better understanding mechanisms of the microbial ecology in the phyllosphere.In general, the phyllosphere microbiology has benefited from culture-independent techniques, such as qPCR, microarray assays, 16S rRNA gene amplicon sequencing and whole metagenome shotgun analyses, over the last 4 years. Using metaproteogenomic approaches, Knief et al.  demonstr


According to the literature, the prevalence of supernumerary teeth is 1% to 4% of permanent dentitions; and among these, the presence of fifth mandibular incisor — a supernumerary eumorphic tooth — has rarely been described in literature, and its association with localized aggressive periodontitis is an even more rare entity. This paper reports a very rare case of unusual association of supernumerary eumorphic fifth mandibular incisor with aggressive periodontitis in a Muslim individual, so that these findings generate curiosity and inspire others to carry out further studies and investigations. The condition of supernumerary teeth, or hyperdontia [Online Mendelian Inheritance in Man-187100], is defined as an excess number of teeth compared to the normal dental formula, or existing of teeth additional to the normal series in the dental arches. Their classification is dependent on their position and form. Hyperdontia may occur as a single tooth or multiple teeth, unilateral or bilateral, or in one or both jaws. This classification morphologically can be subcategorized into eumorphic  and dysmorphic (rudimentary) elements. Supernumerary eumorphic teeth have the same morphology as that of the normal teeth, whereas dysmorphic ones are small and conical, tuberculate or odontome in shape.Although there is no consensus on the etiology of supernumerary teeth, one etiologic theory suggests that the supernumerary tooth is created as a result of a dichotomy of the tooth bud. Another 413et al.[143et al.;[et al.[In a survey of 2,000 school-going children, Brook found that supernumerary teeth were present in 2.1% of permanent dentitions. The prevet al. The prevet al. Prevalenet al.13 and itet al.1415 Veryet al.14317 The pret al.143 0.32%, b43et al.; and 0.76.;[et al.The possible association between supernumerary teeth and aggressive periodontitis has been reported in literature.1821 But 18A 25-year-old Indian Muslim man reported to the Department of Periodontics, Teerthanker Mahaveer Dental College and Research Centre, Moradabad, with a chief complaint of bleeding gums and foul smell since 2 to 3 years. He was found to have localized aggressive periodontitis. On routine clinical and radiographic examination, supernumerary eumorphic fifth mandibular incisor was found. We could not differentiate the eumorphic fifth mandibular incisor from the remaining mandibular incisors, clinically or radiographically, neither was there any fusion between crowns and roots in lower incisors. The five mandibular incisors were separate and same in morphology Figures –3. ApartLocalized aggressive periodontitis was diagnosed by ≥5 mm pocket probing depth around all the four first molars with moderate vertical bone loss radiographically. In lower incisors, there was ≥5 mm attachment loss but due to recession, and pocket depth was ≥3 mm.In lower jaw, third molar of right side was impacted, and impaction was mesioangular, whereas the third molar of left side was well erupted and in normal occlusion. Hematological examination consisting of total leukocyte count (TLC), differential leukocyte count (DLC), hemoglobin (Hb), erythrocyte sedimentation rate (ESR), clotting time (CT), bleeding time (BT) revealed no significant findings.Prevalence of supernumerary teeth in mandibular incisor region is 2% of total supernumerary prevalence,13 and it12143In the present case, supernumerary eumorphic mandibular incisor was normal, well individualized with no fusion in roots and crowns. Differentiation of this supplementary tooth from other mandibular incisors was difficult. This fifth incisor mimicked other mandibular incisors in morphology, radiographically and clinically. Hence this type of supernumerary tooth is overlooked most of the time, unless diagnosed by chance by a dentist during clinical and radiographic examination.et al. reported the presence of a supernumerary eumorphic fifth mandibular incisor in a Lebanese consanguineous family where 4 individuals displayed 5 mandibular incisors with the same shape and size, and they hypothesized the possibility of an autosomal recessive inheritance for this nonsyndromic trait.[et al. (2008) reported the case of homozygosity-mapping to identify a homozygous region with different alleles at chromosome 16q 12.2, located at the marker D16S415, which likely harbors the gene underlying this anomaly.[Supernumerary teeth in mandibular incisor region may be seen in some hereditary syndromes .14 They wic trait. Sami et  anomaly. However,et al.[et al.[A possible association between supernumerary teeth and localized aggressive periodontitis has been described in a small number of reported cases.182122 Lo1821et al.; and 0.7l.[et al. So, bothet al. described two identical Black twins with localized juvenile periodontitis, multiple supernumerary teeth and no dental caries. The authors hypothesized that all these three entities were due to genetic influence.[et al. (2004), the association between aggressive periodontitis and supernumerary teeth was suggested to be a random rather than a biologic one.[The first study recognizing the possible connection between supernumerary teeth and periodontitis was a case report by Eley in 1974. In 1981,nfluence. As Odellnfluence. However,ogic one.To conclude, one may think in terms of the correlation between aggressive periodontitis and supernumerary eumorphic mandibular incisor as in this study. This does not mean that both entities have biological connection. However, association between these two entities is definitely a rare one. To prove such biological connection, further studies and genetic investigations are required to be carried out.


This special issue on “New Concepts in Brain Networks” contains articles that review or propose new approaches for investigating brain connectivity. This topic is highly relevant and has been at the forefront of neuroscientific research in recent years. While univariate techniques dominated fMRI data analysis at the onset of fMRI brain mapping, it is now generally accepted that the human brain is a highly multivariate, and dynamic complex system, and more powerful techniques are needed to describe its function adequately. Clearly, such techniques should reflect the complexity and dynamics of brain function.This special topic contains seven articles ranging from reviews of the state of the art to novel clinical applications, and discussions of theoretical background.The paper by Vértes et al.  belongs Hagmann et al.  provide Knösche and Tittgemeyer  review tThe two following papers combine novel algorithmic approaches with clinical applications. Allen et al.  examine A new algorithm for “resting state” fMRI (R-fMRI) is proposed in the paper by Lohmann et al. . The keyA highly sophisticated algorithmic approach is presented by Smith et al. . While tFinally, Kannurpatti et al.  present Overall, this special issue provides an overview of current research on brain networks. This topic is still in its infancy and many new ideas and novel methodologies will be needed in the future.


Most microRNAs have a stronger inhibitory effect in estrogen receptor-negative than in estrogen receptor-positive breast cancers. Copy number variants (CNVs) account for a large proportion of genetic variation in the genome. The initial discoveries of long (> 100 kb) CNVs in normal healthy individuals were made on BAC arrays and low resolution oligonucleotide arrays. Subsequent studies that used higher resolution microarrays and SNP genotyping arrays detected the presence of large numbers of CNVs that are < 100 kb, with median lengths of approximately 10 kb. More recently, whole genome sequencing of individuals has revealed an abundance of shorter CNVs with lengths < 1 kb.We used custom high density oligonucleotide arrays in whole-genome scans at approximately 200-bp resolution, and followed up with a localized CNV typing array at resolutions as close as 10 bp, to confirm regions from the initial genome scans, and to detect the occurrence of sample-level events at shorter CNV regions identified in recent whole-genome sequencing studies. We surveyed 90 Yoruba Nigerians from the HapMap Project, and uncovered approximately 2,700 potentially novel CNVs not previously reported in the literature having a median length of approximately 3 kb. We generated sample-level event calls in the 90 Yoruba at nearly 9,000 regions, including approximately 2,500 regions having a median length of just approximately 200 bp that represent the union of CNVs independently discovered through whole-genome sequencing of two individuals of Western European descent. Event frequencies were noticeably higher at shorter regions < 1 kb compared to longer CNVs (> 1 kb).As new shorter CNVs are discovered through whole-genome sequencing, high resolution microarrays offer a cost-effective means to detect the occurrence of events at these regions in large numbers of individuals in order to gain biological insights beyond the initial discovery. Genetic differences between individuals occur at many levels, starting with single nucleotide polymorphisms (SNPs) , short iet al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [NspI and StyI restriction fragments) at approximately 2-kb resolution, and reported > 1,300 CNVs having a median length of approximately 7.4 kb.Progressively higher resolution microarrays, starting with earlier low resolution bacterial artificial chromosome (BAC) arrays followed by commercially available array comparative genome hybridization (CGH) and SNP genotyping arrays, have steadily driven the discovery of new CNVs and have refined the boundaries of earlier reported CNVs. Specifically, the earliest CNVs described by Sebat et al.  and Iafr [et al. , using B [et al.  used bot [et al.  study, w [et al.  study, w [et al.  study, w [et al.  study ex [et al.  study an [et al.  study exHere in this study, we set out to demonstrate the benefits, as well as limitations, of Affymetrix oligonucleotide arrays with higher resolution than previously available arrays, first in unbiased whole-genome scans to discover CNV regions, and subsequently in localized regions to determine sample-level CNV calls. Our custom arrays were manufactured using standard Affymetrix processes , but witet al. [et al. [et al. [et al. [et al. [et al. [et al. [A fourth custom oligonucleotide array was designed to confirm putative CNV regions identified from the initial genome scans, as well as subsets of CNVs reported in the DGV November 2008), including those reported by Perry et al. , Wang et [et al. , and McC [et al. , and to  [et al.  and Whee [et al.  studies,08, inclu [et al.  studies Our arrays are essentially tiling designs with probe sequences picked from the reference genome (build 36), and are more similar to early BAC and Agilent CGH arrays than to recent genotyping arrays, such as the Affymetrix SNP 6.0 or the Illumina BeadChips, which generate allele-specific signals (with the exception of subsets of non-genotyping copy number probes). To observe copy number events on our arrays, we processed our probe signals with circular binary segmentation (CBS) , a CNV dDNA samples from each of the 90 Yoruba individuals was whole-genome amplified, randomly fragmented, end-labeled with biotin, and then hybridized to the three genome-scan arrays . Probe signals were quantile normalized  across tet al. [et al. [Of the 3,850 putative CNVs having events observed in at least two individuals (defined as high confidence), approximately 67% overlapped at least one record in the DGV (March 2009), while only approximately 44% of the remaining regions having an event in only one individual (singletons) overlapped a DGV record Table . Overlapet al. , and mor [et al. . The putTo experimentally validate a sampling of the putative CNVs, we randomly selected observed events between 400 bp and 10 kb for PCR or quantitative PCR (qPCR). PCR primers were designed to amplify across putative breakpoints, while primers for qPCR were designed within gain regions. Figure These PCR results provided some assurance that the genome scans had relatively low false discovery rates for CNV regions; however, because of the stringent requirements applied to call an event, a noticeable false-negative observation rate was also demonstrated. PCR tests were performed on Yoruba DNAs selected in pairs, whereby an event was observed in one DNA but not the other on the genome-scan arrays. However, the patterns of bands in the PCR gels showed cases of actual losses or gains in 'non-event' DNAs Figure , the delBecause the primary objective of the genome-scans was CNV region discovery, we set stringent requirements for event detection that prioritized low false discovery of regions at the expense of sensitivity to observe sample level calls at those regions. Once CNV regions had been identified in the genome scans, we focused on designing a new array more suited to generating sensitive and reliable sample-level calls, where space on the genome-scan array originally occupied by additional array probes residing outside of CNV regions can now be better used. To optimize array design parameters that would increase sample-level call sensitivity, we designed a small test array with variable probe lengths from 39 to 69 nucleotides, variable probe feature sizes, and 5 replicates of each unique probe, at 150 arbitrarily chosen regions of which 105 were putative CNVs from the genome scan and the remainder were records from the DGV. Filters were not applied to the choice of probe sequences for the test array, which included probes that overlapped any known repetitive regions, including Alu elements. Results from a subset of 12 Yoruba individuals on the small test array suggested the use of 60-nucleotide long probes at 5 micron pitch, with 3 replicates per probe, and the inclusion of probes in repetitive regions, with the exception of Alu elements (data not shown). Probes on the test array corresponding to nearly all Alu elements were not responsive to copy number differences, while probes at other repetitive regions had variable responses that ranged from no change (similar to Alus), reduced response, or full response (similar to non-repetitive regions), with no clear correlation to the class of repeat elements (data not shown). Based on the test array findings, the CNV-typing array was designed to have higher sensitivity for event detection, and includes probes corresponding to repetitive regions (other than Alu elements). Using data from the CNV-typing array, a thorough study of the possible relationships between repeat elements and CNVs is also possible, but is beyond the scope of the current work.et al. [et al. [et al. [There were approximately 98,000 events observed at the putative CNVs across the 90 Yoruba on the CNV-typing array. Nearly 97%  of the putative CNV regions discovered in the genome scans were confirmed to have at least one observed event on the CNV-typing array Table . The higet al.  study, wet al. , includi [et al.  study, p [et al.  study, aet al. [The median length of the confirmed CNVs was 4.4 kb, which was slightly shorter than the median length of the putative CNVs Table . The lenet al.  study, wet al. -21,24-39et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [In order to further compare our results with DGV records at the individual sample level, we selected six recent studies, including the McCarroll et al.  study, w [et al.  and Kidd [et al.  studies  [et al.  study, o [et al.  study ex [et al.  study, 3 [et al.  study, w [et al.  study, wde novo occurrences [de novo events. The approximately 98,000 event calls at 6,368 confirmed CNVs across the 90 Yoruba were grouped by the 30 family trios. Of the total observed events, approximately 10,500 (10.8%) were observed in only the children of trios. The same 30 trios were also part of the McCarroll et al. [et al. [et al. [et al. [et al. referred to as CNVs inferred in offspring but not detected in parents (CNV-NDPs).Since the 90 Yoruba are each members of 30 family trios, we examined the inheritance of events from parents to children. The majority of copy number polymorphisms are inherited , rather urrences . The obsl et al.  study, i [et al.  study ar [et al.  study be [et al.  study beet al. [et al. [et al. [et al. [In order to delineate the observations of false positives in children and false negatives in parents in our work, the trio event calls from the McCarroll et al.  and Wang [et al.  studies  [et al.  and McCa [et al.  studies,et al. [et al. [et al. [et al. [et al. [et al. [et al. [The CNV-typing array has probes corresponding to shorter (< 1 kb) CNVs discovered by sequencing individual genomes ,19, enabet al.  and Whee [et al.  and Whee [et al.  and Whee [et al.  study.et al. [et al. [et al. [et al. [et al. [et al. [et al. [A large majority > 77%) of the shorter CNVs that were discovered by sequencing individuals of Western European descent had at least one observed event in the Yoruba Table . Based o7% of theet al. , Korbel  [et al. , Wang et [et al. , Perry e [et al. , and Kid [et al.  studies, [et al.  and Whee [et al.  studies et al. [et al. [et al. [et al. [That our high resolution genome scans of the 90 Yoruba uncovered as many as 2,690 potentially new CNVs with a median length of approximately 3.0 kb suggests that there are many more CNVs yet to be discovered on the shorter end of the size range. Because of the high resolution of our genome-scan arrays, we were able to delineate neighboring multiple smaller CNVs at regions earlier reported as single larger CNVs, as illustrated in Figure S2 in Additional data file 1. Perry et al.  observed [et al.  study, w [et al.  study, wet al. [et al. [et al. [Even at a resolution of approximately 200 bp, our genome scan detected only a fraction of the CNVs reported in whole-genome sequencing studies  examined in our current work are likely just a fraction of what will eventually be discovered through sequencing more individuals. In the near term, high resolution microarrays offer a cost-effective means to confirm these shorter CNVs, and type large numbers of individuals in order to gain biological insights beyond the initial discovery.Arrays were synthesized following standard Affymetrix GeneChip manufacturing methods utilizing contact lithography and phosphoramidite nucleoside monomers bearing photolabile 5'-protecting groups. Fused-silica wafer substrates were prepared by standard methods with trialkoxy aminosilane as previously described . An imprCandidate 49-mer probe sequences for the three genome-scan array designs were chosen from the non-repetitive regions of the genome, and filtered for extraneous matches to the genome in the central 16 nucleotides, resulting in a total of 32 million unique probes. Rather than placing probes sequentially across the three arrays, probes were dispersed such that every second and third probe against the genome was placed on separate arrays . Because of the inter-digitating of probes across the three designs, the inter-probe interval in any one design between the center positions of neighboring probes is generally 147 bp (the combined length of three probes). However, because probes were filtered out at repetitive regions throughout the genome the overall median interval between neighboring probes on the genome-scan arrays is 196 bp.The CNV-typing array design consists of approximately 800,000 unique probes, with each in triplicate for a total of approximately 2.4 million 60-mer probes. The replicate probes are placed in separated locations on the array to mitigate any regional variations in signals. The approximately 800,000 unique probes are organized into approximately 16,000 partitions, each containing up to 50 unique probes. The probe partitions correspond to putative or reported CNV boundaries. The probes within a partition are evenly spaced along chromosomes, with the exception of regions corresponding to Alu elements and occurrences of high allele frequency SNPs. In order to mitigate any potential compounding effects on signals, probes with a common SNP  in the HapMap repository  within tThe 90 Yoruba individuals are from the HapMap Project ; genomicWhole-genome amplification of genomic DNA samples was performed using the REPLI-g Midi kit  following manufacturer-supplied instructions, starting with 200 ng of input DNA in a 60 μl reaction. Amplified DNA was randomly fragmented by controlled partial digestion with DNase I. The optimal DNA target length for hybridization to the arrays was found to be in the range of 50 to 300 bp, with the majority of fragments at 100 to 200 bp. DNaseI at 2.5 U/μl  was freshly diluted in 10 mM Tris pH 8 to a concentration of 0.3 U/μl; 3 μl of the diluted DNaseI was added to 60 μl of amplified DNA and 7 μl Fragmentation buffer (Affymetrix) at 37°C. To achieve the optimal size range, test fragmentation time courses were first performed using a small amount of the amplified DNA samples, where the incubation varied from 4 to 26 minutes. Following fragmentation, the amplified DNA was ethanol precipitated and resuspended in 33.5 μl water; 1 μl was removed to measure concentration, which was typically approximately 1.5 μg/μl. The fragmented DNA was then end-labeled with biotin using 2.5 μl of 30 mM DNA labeling reagent (Affymetrix) and 5 μl of Terminal Transferase (Affymetrix) in a 50 μl reaction, which included 10 μl of 5× TdT buffer (Affymetrix). Labeling reactions were incubated for 2 hours at 37°C until heat inactivation at 95°C for 10 minutes.The labeled DNAs were hybridized to each array in 200-μl volumes. In addition to 15 μl of approximately 1 μg/μl labeled DNAs, the hybridization solution contained 100 μg denatured Herring sperm DNA , 100 μg Yeast RNA , 20 μg freshly denatured COT-1 DNA , 12% formamide, 0.25 pM gridding oligo (Affymetrix), and 140 μl hybridization buffer, which consists of 4.8 M TMACl, 15 mM Tris pH 8, and 0.015% Triton X-100. Hybridizations were carried out in Affymetrix ovens for 40 hours at 50°C with rotation set at 30 rpm. Following hybridization, arrays were rinsed twice, and then incubated with 0.2× SSPE containing 0.005% Trition X-100 for 30 minutes at 42°C with rotation set at 15 rpm. The arrays were rinsed and filled with Wash buffer A (Affymetrix). Staining with streptavidin, R-phycoerythrin conjugate (Invitrogen) and scanning with the GCS3000 instrument (Affymetrix) were performed as described in the Affymetrix GeneChip SNP 6.0 manual .A sampling of putative CNVs in pairs of Yoruba samples was selected where an event was observed in one DNA but not the other . For standard PCR, putative CNVs having an event segment within a sample in the range 400 bp to 2.5 kb were tested; for quantitative PCR, CNVs having gain segments between 500 bp and 10 kb were tested. Primer sequences for standard PCR were designed from 300-bp candidate regions upstream or downstream of the longest event segments within a sample, and for qPCR, from within the shortest gain segment. Candidate regions having less than 50% RepeatMask (UCSC) were processed in either Primer3  or PrimeSignal intensities were quantile normalized  in sets CBS  was implFor both non-smoothed and smoothed segmentation analyses, gain and loss event thresholds were set to segment mean log2 ratios of > 0.25 and <-0.25, respectively. For each sample, overlapping segments from at least two of three chip designs was required to meet the thresholds in order to call a gain or loss. The boundaries of an individual event were defined by the longest overlap between any two event segments meeting the threshold. A putative CNV was defined as regions having events observed in at least one individual; and the boundaries of a CNV were defined by the longest event among individuals. There were 401 regions where putative CNVs from the non-smoothed segmentation intersected putative regions from the smoothed segmentation. In regions where multiple putative CNVs from the non-smoothed segmentation corresponded to one putative region from the smoothed segmentation, the non-smoothed CNVs were chosen. In regions of one-to-one correspondence, the generally longer putative CNVs from the smoothed segmentation were chosen.et al. [et al. [et al. [et al. [et al. [et al. [et al. [The sample-level event calling thresholds used in the segmentation analysis of the CNV-typing array data were determined by comparing against reference event calls taken primarily from the McCarroll et al.  study an [et al.  study re [et al.  study. T [et al. ,20,30,31 [et al.  study, 2 [et al.  study, t [et al.  study, s [et al.  study haTo assess specificity and sensitivity of event detection in the CNV-typing data, segmentation thresholds were titrated at the 732 McCarroll reference CNVs, and at 6,578 putative CNVs from the genome scan. Any false positives or false negatives in the McCarroll reference event calls will artificially lower the estimates of sensitivity or specificity, respectively, of the CNV-typing array. Figure S6 in Additional data file 1 summarizes the results at seven threshold values that ranged from 0.35 (-0.35) to 0.10 (-10), and shows the trade-off between higher specificity and lower sensitivity. Event thresholds of -0.175 and 0.175 for loss and gain calls, respectively, were chosen; based on further titrations, second-level thresholds of -0.70 and 0.45 were chosen for homozygous deletions and multi-copy gain events, respectively. For each individual Yoruba sample, sets of probes for each CNV were analyzed separately by CBS, and segments with log2 ratios above or below the thresholds were called as events. Probes in the CNV-typing design were grouped into partitions corresponding to known or putative CNVs, where a given CNV may be represented by more than one partition . Although the CNVs vary in the number and density (probes per base-pair) of corresponding probes, the degree of discrimination of log2 ratios above or below the event thresholds were comparable across a range of event lengths and numbers of probes, with only slight loss of discrimination at longer lengths and fewer probes . Microarray raw intensities and chip library files are available at ArrayExpress  under acBAC: bacterial artificial chromosome; CBS: circular binary segmentation; CGH: comparative genome hybridization; CNV: copy number variant; DGV: Database of Genomic Variants; qPCR: quantitative PCR; ROC: receiver operating characteristic; SNP: single-nucleotide polymorphism.All authors are current or former employees of Affymetrix.RR and GF conceived the experiments and designed the genome-scan arrays. HM and GF designed the typing array. PW and GF prepared samples and hybridized the arrays. PW, GF, and HM ran PCRs. HM and JH performed data analysis. HM and GF wrote the manuscript.et al. [et al. [et al. [The following additional data are available with the online version of this paper: Figures S1 to S7 and Tables S3, S5, S6 and S7 , reported CNVs from whole-genome sequencing studies . CNVs with locus_id numbers starting at 100,000 were from the smoothed segmentation analysis. Chromosome locations are on genome build 36. Confirmed CNVs had at least one Yoruba with an event on the CNV-typing array. For confirmed CNVs that overlapped at least one DGV record (March 2009), the closest matching record (variation_id) is listed along with its build 36 coordinates, length, cited reference, and discovery method. Regions were flagged as 'Complex' if both a loss and gain event were observed in the same individual.Click here for fileGel images correspond to 4% agarose (E-gel), gradient polyacrlyamide (PA gel), and 1% agarose (1% gel) electrophoresis gels. DNAs were run in pairs with one having an observed event (Event DNA_ID), and the other without an observed event (non-event DNA_ID). Confirmation calls  were made based on amplicon length differences in each DNA pair, and marked the status of each pair: confirm, maybe (ambiguous), no (no evidence of event), or fail (PCR did not yield expected amplicons). At a subset of regions, amplicon bands were excised and sequenced (seq). The lengths of the putative CNVs are also listed.Click here for file(A) Event calls at confirmed CNVs are compared against consensus references from the Wang et al. [et al. [(B) Calls reported in the McCarroll et al. [et al. [(C) Calls reported in the Wang et al. [et al. [(D) Yoruba trios were arbitrarily assigned trio_ids. The DNA_ids of the 90 Yoruba are listed with the corresponding trio_ids.g et al.  and McCa [et al.  studies.l et al.  study ar [et al.  study.  or gain, the calls were listed as 1 or 3, respectively. For papers with genome positions in build 35, the liftOver utility at UCSC  was usedet al.  study (lClick here for file


Primary melanoma of the adrenal gland is exceptionally rare as demonstrated by the few cases reported in the medical literature, and it has a high fatality rate. We present the case of a patient with two relapses and survival to date.We describe the case of a 58-year-old Caucasian woman who consulted her doctor with symptoms of asthenia, anorexia and weight loss. A mass was palpated in her abdomen at the height of the left hypochondrium. A computed tomographic scan revealed a retroperitoneal mass measuring 10 cm × 15 cm originating in the left adrenal gland. A left nephroadrenalectomy and splenectomy were performed. Histopathologically, the retroperitoneal mass corresponded to a melanoma, and no primary melanoma was found in any other location. The patient was treated with interferon-α-2b. Three years after her diagnosis the patient presented with a retroperitoneal relapse of the mass measuring 7.2 cm, which was removed. Five years after the first relapse a new retroperitoneal relapse mass was diagnosed, which was also removed. Since then the patient has been healthy and free from illness.et al. and Carstens et al., allowed us to diagnose primary melanoma of the adrenal gland.Histological and immunohistochemical studies, together with the criteria described by Ainsworth  Primary melanoma of the adrenal gland is an exceptionally rare occurrence, as demonstrated by the few cases described in the medical literature -11. BothPrimary melanoma of the adrenal gland is usually a voluminous, non-functional tumor showing heterogeneous contrast enhancement on the computed tomographic (CT) scan. The diagnosis is made on the basis of histological and immunohistochemical studies.et al. [et al. [et al. criteria, with the exception of the last one, since our patient is still alive.The adrenal glands can be sites for the metastatic dispersal of cutaneous or visceral melanomas in up to 50% of cases , and hiset al.  establis [et al.  establisPrimary involvement of the adrenal gland is extremely rare. Only 23 patients have been described in the English-language literature, and documentation was complete in only 11 of them [et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [In 1946, Kniseley and Baggentoss [et al.  publishe [et al.  publishe [et al. , Sasidha [et al.  and Cars [et al.  reported [et al.  in 1986, [et al.  in a 42- [et al.  in a 33-et al. [et al. [The mean age of the patients in all of these 12 cases is 50.4 ± 17.2 years, with a median age of 49 years and an age range between 24 and 78 years. Fifty percent of these patients were men, and the initial mean size of the tumors was 10.9 cm ± 3.8 cm with a median size of 10 cm and a range between 6 cm and 17 cm. The most frequent symptom in these cases was pain  and epithelial membrane antigen and the neuroendocrine markers chromogranin A and synaptophysin were negative. A diagnosis of non-pigmented melanoma was established  administered intravenously. Analytical and radiological reviews  were carried out. Thirty-two abdominal CT scans were obtained throughout this period, making it possible to diagnose and treat relapses in a relatively timely manner.Two years after the patient's diagnosis and surgery, during a routine review involving an abdominal CT scan, a solid retroperitoneal mass with areas of necrosis measuring 7 cm × 2 cm × 4.5 cm was found in the left renal fossa, invading the abdominal wall and in direct contact with the intestinal loops. Also, a para-aortic adenopathy with a diameter of 1.8 cm was observed Figure .The patient once again underwent surgery while under general anesthesia. We performed a mid-line suprainfraumbilical incision and located a tumor in the previous surgical area. Its diameter was approximately 10 cm, it seemed to infiltrate the abdominal wall and it was adhered to the splenic angle of the colon, although without infiltration. After the adhesions were removed, we completely removed the tumor, together with a para-aortic adenopathy with a diameter of approximately 3 cm. The post-operative period progressed without any incidents. The pathology of the retroperitoneal lesion and the lymph node showed a tumor entirely similar to the one previously excised.Four years after the initial diagnosis, during a radiological review (chest X-ray and CT scan), pulmonary nodules were detected that were suggestive of pulmonary metastasis. Chemotherapy with dacarbazine and IFNα2b was begun by the oncology service, and one year later the CT scan revealed that the nodules had disappeared.In the CT scan obtained eight years after the initial diagnosis, we observed a nodule measuring 4.5 cm × 2.3 cm in the left renal fossa and a nodule measuring 2.5 cm × 2.5 cm in the mesenteric fat of the descending colon . Melanomas have also been found in the mouth cavity, larynx and bronchi, the esophagus, the rectum, the genitourinary system, the meninges, the ovaries, the uterine cervix, the vagina and the adrenal glands. One of the major obstacles for clinicians is identifying whether the tumor is really a primary tumor or whether it is metastatic .The possibility exists that a tumor lesion of melanocytic cells in the skin or eyes may have gone unnoticed or that a skin lesion removed months or years ago may have been the primary origin of undiagnosed melanoma. Also, some authors have mentioned that primary skin melanoma occasionally reappears spontaneously, and the only clinical symptoms in some of these patients are due to the presence of metastasis in the regional lymphatic ganglia or the internal organs -19. SomeThe adrenal medullary blasts and melanoblasts have a common embryological origin at the neuroectodermal level , similarIn primary melanomas, the four stages of melanogenesis are usually observed . With reBecause at the macroscopic level the melanoma may have a brown or black color, it is necessary to also establish the differential diagnosis with other pigmented adrenal lesions such as pigmented adrenal adenomas . These aAt the macroscopic level, adrenal hematomas may also give rise to confusion; however, under the microscope, hemosiderin-laden macrophages may be seen, and the histological characteristics are completely different. Adrenocortical carcinomas are tumors that require the establishment of a differential diagnosis. It is a larger-sized tumor than the adenoma, and its cells do not stain positively for S-100 or HMB45 .et al. [et al. [The clinical case we present herein meets the criteria of Ainsworth et al.  and Cars [et al. . The tumWritten 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.LGS participated surgical treatment, design and drafted manuscript. SPF participated in the design, literature review and drafted manuscript. MJLP participated in histopathology immunohistochemical study. FAM participated in histopathology and immunohistochemical study. JMS participated in surgical treatment. JARG participated in surgical treatment. All authors read and approved the final manuscript.


The exploitation of DNA for the production of nanoscale architectures presents a young yet paradigm breaking approach, which addresses many of the barriers to the self-assembly of small molecules into highly-ordered nanostructures via construct addressability. There are two major methods to construct DNA nanostructures, and in the current review we will discuss the principles and some examples of applications of both the tile-based and DNA origami methods. The tile-based approach is an older method that provides a good tool to construct small and simple structures, usually with multiply repeated domains. In contrast, the origami method, at this time, would appear to be more appropriate for the construction of bigger, more sophisticated and exactly defined structures. DNA (deoxyribonucleic acid) is the molecule of choice for designed nanostructure fabrication since it has excellent features such as molecular recognition, self-assembly, programmability, predictable nanoscale structure and is easily synthesized . DNA selFabrication of DNA nanostructures was first proposed by Nadrian C. Seeman in the 1980s . Inspireet al. [et al. [et al. [et al. reported the formation of 2D DNA crystals by using only two DNA strands [et al. reported construction of DNA nano-arrays by using three oligos and implementing sequence symmetry [et al. demonstrated formation of three dimensional (3D) DNA crystals using tensegrity triangles as the building blocks [By applying the tile-based motifs and optimizing them, Winfree et al. , LaBean  [et al.  and Feng [et al.  introduc [et al. . Constru [et al. –13. Form [et al. , tensegr [et al.  and six- [et al.  have bee strands , and He symmetry . A reporg blocks .et al. [et al. introduced a way to incorporate some features of both the origami method and the tile based self-assembly to produce weave tile structures [Novel motifs are continuously being introduced, and recently, a half-crossover based structure was reported by Yin et al. , which aructures . They reet al. reported formation of an octahedron by self-folding of a 1.7 kb single-stranded DNA (heavy strand) and a few smaller strands (light strands) [et al. reported construction of a tetrahedron DNA structure. They have also reported conformational changes in the structure due to restriction enzyme digestions [et al. reported the formation of tetrahedron, dodecahedron, buckyball structures [The DNA tile based system has been used to make 3D structures. The first tile based 3D DNA structure was introduced by Seeman . Howeverstrands) . This stgestions  or by stgestions . Formatigestions . Anotherructures  and a DNructures .The addition of dynamic properties to DNA nanostructures is another practice which has been applied over the last decade ,24,27–32et al. [vice versa by multi step hybridization and strand displacement. Later, scientists have programmed the movement of DNA molecules based on toehold strand displacement. Shih and Pierce demonstrated walking of a DNA molecule on top of a DNA tube driven by fuel strands [et al. introduced a new strategy for stepwise walking of a bipedal walker, in which they triggered the forward step by introducing Hg2+ and H+ ions and the backward step by introducing OH− ions and cysteine [Toehold based strand displacement in DNA nanotechnology was initially proposed by Yurke et al. . They de strands , which h strands –36. In a strands . Recentlcysteine .Tile-based DNA nanostructure architecture is a promising method, but there are major drawbacks for the tile-based assembly strategy. First, the design of complex structures using the tile method is a challenge since one needs to design and check the new sequence for each step, which is a time consuming and problematic step. Secondly it is very hard to control production of complex high order nanostructures, and even though some of the structures have finite size and shape, many other structures such as arrays or grids grow as long as sticky strands are available, and therefore there is no control over size. Finally, in order to obtain the predicted structure the strands need to be highly quantitatively controlled.et al. reported formation of the nano arrays by using a long scaffold and some shorter strands [et al. who reported formation of an octahedron by self-folding of a long single-stranded DNA and a few smaller strands [The “DNA origami” method was first proposed and implemented by Paul W. K. Rothemund in 2006 , in whic strands . However strands . Moreove strands .The term origami refers to the Japanese folk art of folding paper into a special shape. The method is called DNA origami since one long strand of DNA is folded to produce the desired structure by the help of smaller staple strands. The origami folding process is described in The origami method is applied to construct 2D and recently 3D DNA nanostructures. In the revolutionary and earliest work , Rothemuet al. reported a tile assembly of the origamies to make higher order self-assembled DNA nanostructures [et al. reported formation of multi-domain DNA origami by using origami four-way junctions [et al. reported construction of the multi-domain DNA origami [Formation of DNA origami from a double stranded DNA (dsDNA) scaffold has been reported  which opructures . Very reunctions , and in  origami . The DNAet al. showed patterning of enhanced green fluorescent protein on top of the origami [et al. demonstrated patterning of the coat of arms of Ukraine by putting streptavidin molecules on top of a rectangular origami [et al. [DNA origamies are interesting since they can be used as platforms for the study of other systems. In the earliest work, patterns (hairpin dumbbells) on top of the origamies were imaged by AFM . DNA ori origami . Kuzyk e origami . Protein [et al. . Additio [et al. .et al. reported gold metallization of branched DNA origami [DNA origami has been used to investigate binding of thrombin molecules to their aptamers . Moreove origami .et al. [et al. positioned carbon nanotubes on top of the DNA origami by aid of interactions between streptavidin molecules and biotinylated DNA strands precisely positioned on the origami and wrapped around the carbon nanotube [et al. reported specific positioning of gold nanoparticles on top of lithographically confined DNA origamies [et al. attached gold nanoparticles to a tube shaped origami and showed formation of plasmonic nanostructures. The exact positioning capability intrinsic to the DNA origami method enabled them to incorporate nanoparticles at very precise positions and thus they could study and prove predicted optical properties of the nanostructures [et al. demonstrated label free detection of RNA molecules by an AFM study of hybridization of the target on top of a rectangular origami [et al. reported a new secondary DNA binding site in the enzyme topoisomerase I [In a very interesting study, Manue et al.  showed pnanotube . In anotnanotube . Furtherrigamies . Positiorigamies . Kuzyk eructures . Ke et a origami . In a ve origami . In very origami . DNA ori origami ,67. DNA  origami  and by umerase I .et al. programmed directed, uniform and continuous translation of a molecular motor along a 100 nm track on flat DNA origami [In a very interesting development, scientists from Hao Yan’s and Erik Winfree’s groups demonstrated a molecular robot which moves along a predefined path on top of a rectangular origami . The mov origami .A number of studies reported the production of 3D DNA origami structures in 2009. William Shih is a pioneer in this field and one might consider the production of an octahedron as the first successful attempt to make 3D DNA origamies . HoweverThere are several strategies for assembling 3D origamies. One method is based on folding flat surfaces against one another through stacking of the helices in separate domains of a flat multi domain origami. The links between the helices could be coaxial, noncoaxial, orthogonal or at any angle. This strategy has also been used to structure complex 2D origami, for example to produce a triangle  and threAnother strategy is to make 3D structures out of multiple layers of DNA origami . Shih anet al. who showed reconfiguration of twisted DNA nanostructures [et al. reported production of a 3D tetrahedral DNA structure which formed from one DNA strand [in vivo, which is a masterful achievement in developing the origami method, since it raises the hope of large scale production of high quality origamies at relatively low cost.Shih’s group reported design and formation of twisted structures, resulting from the insertion or deletion of bases in between the crossovers . A year ructures . They crA strand . This miDNA is a unique molecule for generating nanoscale molecular architectures, with a large number of useful features that facilitate molecular engineering. The tile based method is a promising method when dealing with small and non-complex structures or structures with repeating building blocks. Although tile based structures provided us with a large number of useful and interesting learning steps, the drawbacks of the method necessitated the investigation of another DNA structural method, called DNA origami. The origami method provides a free hand method to design and to construct more sophisticated and highly addressable structures. Currently, using the DNA origami method, construction of large, highly-ordered and complex structures consisting of several hundred thousand atoms  is not only possible, but relatively easily accomplished. The DNA origami method is a very useful tool to generate precisely defined molecular systems for characterization. Some of the demonstrated applications of DNA origami include molecular pattering of surfaces, directed positioning of particles/materials, construction of nanorobots and carriers, drug and material delivery, molecular recognition and sensor applications.et al. [et al. [An example of a high impact application of DNA origami, an enclosed, “smart” molecular nano-carrier, was recently reported by Douglas et al. . They pret al. , to open [et al. , in whicHowever the tile-based and origami methods still suffer from major drawbacks. DNA origamies are not stable in various conditions and require special care. For example, pH has a drastic effect on the structure of DNA nanostructures. In low pH solutions the DNA may be de-purinated and in high pH the hydrogen bonding between the DNA strands will be disrupted. Heating, many chemicals and some organic solvents denature double-stranded DNA. DNase enzymes destroy DNA strands by catalyzing the cleavage of the DNA backbone. Thus handling and storing of samples consisting of DNA structures must be performed carefully. The ions present in solution have a strong impact on DNA structures; at low ionic strength the DNA structures will decompose, and high salt concentrations lead to aggregation of the structures. Molecular tensions and mechanical forces may have negative effects on structures, particularly on extended structures, too.et al. proposed production of origamies from double-stranded DNA, a method that may aid in the use of various scaffold sources [Today, production of DNA origami is based on the use of a very limited number of different scaffolds. To make more sophisticated structures, developing new scaffold production methods will be necessary. A few years ago, Högberg  sources . Moreove sources ,42,82,83 sources ,83,84. E


In their letter, Haighton et al. recommend that we review Bradford Hill guidelines for establishing causality. As noted in our article , we did in vivo (Haighton et al. state that glucuronidated BPA does not appear to be biologically active in mammalian systems; however, glucuronidated BPA can be deconjugated by the placenta or transferred across the placenta, where it can be deconjugated by other fetal tissues . Therefoin vivo .The authors were unclear about how “normal limits” for neurobehavioral assessment are defined. In our study , we admiFinally, Haighton et al. state that other etiologies could be responsible for the abnormal exam at 1 month of age. We stated this exact same point very clearly in our case report . Althoug


The authors wish to acknowledge an error in and and correct an error in their use of a reference. The fourth sentence of the seventh paragraph of the Introduction should read:"A biosensor system, observed by Checa et al. (2011), was capable of detecting Au, quantification of Au was not reported here: However, quantification is crucially important for geochemical exploration [26]."In addition, the authors wish to acknowledge the relevance of an article 'Cerminati S, Soncini FC, Checa SK. Selective detection of gold using genetically engineered bacterial reporters. Biotechnol Bioeng. 2011 Nov; 108(11):2553-60. doi: 10.1002/bit.23213', which relates to the results described in the present article, as follows:A Au biosensor system based on the golTSB regulon and a fluorescent reporter protein was previously developed by Cerminati et al. (2011) and dose-response curves were constructed using KAu(CN)2. While Cerminati et al. (2011) suggested that biosensor systems may be useful for quantifying Au in real soil samples this was not tested in their study. Cerminati et al. (2011) developed a Au biosensor system which under "clean" laboratory conditions was capable of detecting environmentally relevant levels of Au. Our study takes further steps towards the development of a field-ready biosensor system with the development and testing of a selective extraction technique for Au, thermodynamic modelling of Au complexes under experimental conditions and the electrochemical testing of the sensor on complex environmental samples.Similar to the biosensor system presented in Zammit et al. (2013), the Au biosensor developed by Cerminati et al. (2011) was able to quantify Au. However, the gold biosensor developed by Cerminati et al. (2011) itself and the experimental conditions in the Checa et al.  Bacterial sensing of and resistance to gold salts. Mol. Mic. 63: 1307-1318.) and Cerminati et al. (2011) studies varied in a number of important aspects from the ones in Zammit et al. (2013). The Cerminati et al. (2011) biosensor was developed using the golTSB genes from S. typhimurium fused to the gene of a green fluorescent protein (GFP) and expressed in E. coli. In contrast, the biosensor developed for this study used the golTSB genes from S. typhimurium transcriptionally fused to a promoterless lacZ reporter cassette, whose activity was measured electrochemically aimed at future in-field-application. These differences may have contributed to the differences in sensitivity of Au detection reported in both studies. The biosensor developed by Cerminati et al. (2011) was able to quantify Au down to 33.23 nM Au(I). The biosensor developed by Zammit et al. (2013) was able to quantify Au down to a detection limit of 10 nM. However, differences may also be a result of the different Au(I/III)-complexes used in both studies to construct standard curves. While KAu(CN)2 was used by Cerminati et al. (2011), Au(I)-thiosulfate and AuHCl4.3H2O were added to the medium by Zammit et al. (2013). The concentrations of metals tested ranged from 10 *M of Hg(II) to 1 mM of Cu(II) (50 *M of Au was used). These concentrations were not normalized in subsequent analyses. Importantly, in Zammit et al. (2013), cross reactivity of Au(I/III) was tested by mixing the complexes mixed with the other metal ions to show that Au can still be detected by the sensor whereas the Checa et al. (2007) and Cerminati et al. (2011) studies only investigated one metal at a time.


Sir,et al.[Helicobacter pylori infection in patients using NSAIDs” in the previous volume of Saudi Journal of Gastroenterology. They have investigated the accuracy of rapid urease test (RUT) in Helicobacter pylori strains during the gastroscopy process. In response to their article, we present our contradictory evidences about the efficacy of RUT test as the confirmation of H. pylori isolates in biopsy specimen. Detection of H. pylori in clinical samples is an important subject for clinicians for further management of this rogue microorganism. Routinely, diagnosis tests of H. pylori are divided into two major groups:[et al.[H. pylori should be confirmed by using at least any two of thee following methods: RUT, histology, UBT (Urea Breath Test), specific PCR assay, and bacterial culture. Nevertheless, they reported H. pylori-positive strains in their study, while no culture or specific PCR assay were performed in their examination.[H. pylori, while their RUT was primarily reported as being negative. Foroutan et al.[H. pylori strains after culture and specific gene PCR assay.[et al.[H. pylori infection.Foroutan et al. publisher groups:3 invasivs:[et al. It was rs:[et al.3 that thmination. Our datamination. demonstran et al. concludean et al.5 we obseCR assay. In otherCR assay.5 This imy.[et al. study, s