Eukaryotic cells utilize intricate mechanisms for the uptake and intracellular sorting of various macromolecules, such as membrane components and extracellular proteins. Microscopic imaging studies have helped considerably to describe and define important steps of the uptake process. It has been shown that the cytosolic face of the plasma membrane is studded with small, domed assemblages of peripheral membrane proteins. These constitute transitory sorting stations that dynamically remodel the composition of the cell surface in response to both intracellular and extracellular stimuli. Typically, within a minute of forming, these assemblages invaginate to generate “buds” that then detach, generating small (∼60 nm), membrane-bound transport vesicles that deliver their contents (often receptors) to specific intracellular compartments—namely acceptor early endosomes—for further dissemination within the cell . This in2) seems to play a major role in placing coat protomers on the plasma membrane to begin clathrin assembly 22 production in cultured cells leads to an almost immediate dissolution of preexisting clathrin-coated structures at the cell surface Clathrin assembles at discrete patches on the plasma membrane through cooperative interactions involving a large set of endocytic proteins Saccharomyces cerevisiae, the predictable kinetic behavior of coat components has led to a thorough cataloging of temporally resolved protein entry and exit at single-turnover endocytic sites Because transmission electron microscope (EM) images typically reveal isolated, invaginating coated buds all along the cell surface, and clathrin immunolabeling often shows a profusion of small, separated puncta apparently randomly scattered over the surface membrane , it seemYet, in other cell lines PLoS Biology, the Kirchhausen laboratory delves into this issue by utilizing quantitative live-cell imaging of several distinct cell types In this issue of Saffarian et al. provocatively argue that their characterization of general plaque behavior rationalizes a rather discrepant literature, yet the work also raises several fundamental questions. Foremost is whether the plaques they catalog are generally equivalent to the extensive, long-lived, and immobile clathrin patches seen by others. In contrasting ultrastructural studies from other laboratories, the flat patches can be considerably larger 2 for assembly, as consumption of this lipid leads to prompt loss of most clathrin structures and adaptors at the ventral surface Next, we do not understand clearly how, when compared with pits, flat arrays are differentially nucleated and apparently restricted to the adherent surface of the cell. The requirement for a very large excess of hexagonal facets to construct a planar lattice differs obviously from de novo–nucleated buds. Yet, flat lattices must require PtdInsPAnother (possibly related) issue is, what prevents the flat clathrin arrays from forming invaginated buds? Perhaps information can be gleaned from comparison with another membrane compartment where flat clathrin arrays also form with no evidence of rounded bud production: on early endosomes maturing into multivesicular bodies . OriginaS. cerevisiae, the sites of clathrin-mediated endocytosis An alternative model for what prevents the basally located, flat clathrin arrays from forming invaginated buds is that it is due to a unique role for the actin cytoskeleton in plaque formation, coupled with the strength of adhesion of the basal cell surface to the underlying substrate on which cells are growing. Saffarian et al. find that actin nucleation is unnecessary for the budding of spherical coats, although it is worth noting that at the apical surface of polarized epithelial cells, where clathrin buds are incontestably spherical, actin does regulate vesicle internalization Irrespective of mechanism, the current schematic depiction suggests that uptake of a flat clathrin sheet leads to extraneous (noncoated) membrane around the perimeter of the plaque also being internalized . SuperfiBut perhaps the most important lingering question is what the functional significance of the flat clathrin arrays is, if they have no operative relationship to buds. Two different types of clathrin assemblies could be a physical manifestation of compositionally discrete sorting stations operating in parallel to presort cargo at the cell surface. Data to support the idea of cargo-selective clathrin coats are accumulating


Renal cell carcinoma is by far the commonest cancer in this group. Presentation may be with local symptoms. Thus haematuria, usually painless though sometimes with colicky pain secondary to blood clots, is common. Less often, a swelling or aching in the loin may be noticed by the patient. Often and increasingly the tumour is picked up coincidentally during an imaging investigation . Metastatic disease may be evident, in up to a third of patients, as bone, lung or brain secondaries or by systemic features such as weight loss, fever, malaise and anaemia. Such features may also be present with organ-confined disease.α or interleukin-2 does marginally improve progression-free and overall survival, it is at the cost of marked toxicity. However international efforts continue to develop ways of identifying the small minority of patients who will benefit greatly from immunotherapy. More recently knowledge of molecular pathways involved, particularly in clear cell renal carcinoma, has provided a rationale for targeted treatment. Agents have been identified, which target gene products, such as vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF), implicated in tumourigenesis. Such agents have demonstrated significant activity in clinical trials and represent a major advance in treatment  disease where the cancer risk for a variety of tumours including renal carcinoma is transmitted in an autosomal dominant manner. It is now known that the VHL suppressor gene is mutated or silenced in most clear cell renal carcinomas though the initiator(s) of this event are still unknown. Information on risk factors for renal cell carcinoma has come from a large number of international case control and cohort studies , were among the lowest in Europe but reassuring that improvements have particularly been seen during the late 1990s. All patients now have the benefit of having their case discussed by a multidisciplinary team including specialist urology and oncology clinicians, with access to optimal treatments and also information on clinical trials. For example, a new randomised study (SORCE) assessing the function of adjuvant sorafenib (one of the new generation of VEGF inhibitors) in high-risk resected renal cancer has recently been launched. For metastatic disease sunitinib and sorafenib (VEGF inhibitors) have now been licensed in the United Kingdom on the basis of evidence from large randomised controlled clinical trials (


The recent entry of products containing antibacterial agents into healthy households has escalated from a few dozen products in the mid-1990s to more than 700 today. Antibacterial products were developed and have been successfully used to prevent transmission of disease-causing microorganisms among patients, particularly in hospitals. They are now being added to products used in healthy households, even though an added health benefit has not been demonstrated. Scientists are concerned that the antibacterial agents will select bacteria resistant to them and cross-resistant to antibiotics. Moreover, if they alter a person's microflora, they may negatively affect the normal maturation of the T helper cell response of the immune system to commensal flora antigens; this change could lead to a greater chance of allergies in children. As with antibiotics, prudent use of these products is urged. Their designated purpose is to protect vulnerable patients.


While eukaryotes primarily evolve by duplication-divergence expansion (and reduction) of their own gene repertoire with only rare horizontal gene transfers, prokaryotes appear to evolve under both gene duplications and widespread horizontal gene transfers over long evolutionary time scales. But, the evolutionary origin of this striking difference in the importance of horizontal gene transfers remains by and large a mystery.i) their apparent genome size constraint compared to typical eukaryote genomes and ii) their underlying genome expansion dynamics through gene duplication-divergence evolution, as demonstrated by the presence of many tandem and block repeated genes. In principle, this combination of genome size constraint and underlying duplication expansion should lead to a coalescent-like process with extensive turnover of functional genes. This would, however, imply the unlikely, systematic reinvention of functions from discarded genes within independent phylogenetic lineages. Instead, we propose that the long-term evolutionary adaptation of free-living prokaryotes must have resulted in the emergence of efficient non-phylogenetic pathways to circumvent gene loss.We propose that the abundance of horizontal gene transfers in free-living prokaryotes is a simple but necessary consequence of two opposite effects: This need for widespread horizontal gene transfers due to genome size constraint implies, in particular, that prokaryotes must remain under strong selection pressure in order to maintain the long-term evolutionary adaptation of their "mutualized" gene pool, beyond the inevitable turnover of individual prokaryote species. By contrast, the absence of genome size constraint for typical eukaryotes has presumably relaxed their need for widespread horizontal gene transfers and strong selection pressure. Yet, the resulting loss of genetic functions, due to weak selection pressure and inefficient gene recovery mechanisms, must have ultimately favored the emergence of more complex life styles and ecological integration of many eukaryotes.This article was reviewed by Pierre Pontarotti, Eugene V Koonin and Sergei Maslov. With nearly 1,000 fully sequenced genomes, to date, and many more at a draft stage, comparative genomics has already highlighted major differences in the evolution of prokaryote and eukaryote genomes. In particular, a long and to some extent still ongoing debate  has helpα-proteobacteria (with subsequent engulfment of cyanobacteria leading to plastids in plants)  so often because they can", which should also apply, in principle, to unicellular eukaryotes without separation of germline and soma and commonly exposed to foreign DNA, as discussed above.More broadly, one could argue that HGT, or put another way, mixing and matching of genetic elements is the primary mode of life existence that does not call for an explanation  whereasAuthors' responseWe believe that we present a genuine hypothesis which does not concern the existence of any primitive form of life but merely the consequence of the apparent genome size constraint and underlying duplication-divergence evolution of free-living prokaryotes, as we know them. In particular, we would like to stress that the abundance of HGT cannot in itself restrain the size of prokaryote genomes (nor does it imply duplication-divergence evolution of their genomes). So, deriving the abundance of HGT among free-living prokaryotes from their apparent genome size constraint and underlying duplication-divergence evolution is not a circular argument. It is, we believe, a genuine hypothesis, which could in principle be falsified. As pointed out in the paper, this would involve to find a free-living prokaryote lineage having achieved long-term adaptation through systematic gene displacements rather than gene transfers. Although at odd with known evolutionary trends, one cannot exclude a priori the existence of such isolated free-living prokaryotes, which would need to continually reinvent the functions of discarded genes without long-term memory of previous evolutionary successes.Minor comment:p. 3, left: I suggest not lumping viruses and viroids with prokaryotes when considering genome size constraints. I would limit this discussion to cellular life forms in which case a lower limit does seem to exist, although the discovery of tiny endosymbionts like Carsonella pushes this limit surprisingly low.Authors' responseWe have cut this paragraph in two to avoid any confusion.Reviewer 3: Dr Sergei Maslov, Brookhaven National Laboratory, Upton, NY, United States.The manuscript contains a speculative argument that widespread horizontal gene transfer in prokaryotes is an adaptation allowing them to preserve long-term evolutionary memory that would otherwise have been quickly erased by rapid gene turnover.This view goes along with my recent "Home Depot" model of prokaryotic evolution ). In our model prokaryotic genomes are constantly replenished from a common repository with entire metabolic pathways. This process can be compared to constantly buying tools in a hardware store (hence the "Home Depot" metaphor) only to return them once the project is over.Authors' responseWe thank Dr Sergei Maslov for mentioning to us this interesting paper on the "Toolbox model of evolution of prokaryotic metabolic networks and their regulation" [In particular, we would like to point out that, beyond the pivotal role of HGT (which we argue must be widespread under genome size constraint), the premise of the "toolbox model" also appears to rely on an additional finite size constraint of prokaryote evolution, namely, the apparent finite size of their available metabolic universe, Nuniv. Based on current KEGG data, Maslov et al. postulate that there are about Nuniv = 1,800 metabolic compounds that can be metabolized by specific enzymes, which prokaryotes can alternatively acquire or discard through HGT. Then, the toolbox model predicts that the number of new metabolic enzymes that can be acquired and controlled by a single new transcription factor eventually decreases as the size of their metabolic network Nmet approaches Nuniv. Hence, it follows that the interplay between duplication-divergence evolutionary dynamics of prokaryotes under genome size constraints and the finite size of their available metabolic environment might ultimately control not only their requirement for widespread HGT but also the global topology of their metabolic networks.ulation" . In partFrom my standpoint, this manuscript would greatly benefit if authors would accompany their verbal argument with a quantitative model. The model does not have to be realistic it just needs to clearly make the main point of the argument that prokaryotic world would collapse without some sort of gene exchange with a mutualized gene pool. Do authors expect a sharp error-catastrophe-like phase transition *in the absence* of horizontal gene transfer? What numerical constant is best suited to quantify this transition (akin to the number of mutations per genome per generation for error catastrophe)? What is its (approximate) range in prokaryotes? In the section "Testing the hypothesis" authors repeatedly refer to their earlier work (Ref. ) withoutAuthors' responseAfter considering to significantly expand the section on "Testing the hypothesis", we have eventually opted out to greatly simplify the discussion related to refs [Indeed, we felt that the full model developed in [could not be adequately presented and further analyzed within the intended format of an hypothesis paper. to refs ,29. Indeloped in ,29could Similarly, we agree that a simple "error-catastrophe-like model" could be worked out in the case of gene-type independent genomes, but such an homogenous gene-type model could hardly been seen as realistic, as pointed out by S. Maslov. Gene-type dependent HGT should undoubtedly be taken into account as discussed in the section on "Testing the hypothesis".Again, our main intent in this "hypothesis" paper is to put the emphasis on the premises of the argument (i.e. genome size constraints and duplication-divergence evolution) rather than on a specific mathematical model illustrating the resulting need for widespread HGT.


Most of these programs operate on a single contiguous sequence at a time, and the results are generated in a diverse array of readable formats that must be translated to a standardized file format. These translated results must then be concatenated into a single source, and then presented in an integrated form for human curation.High quality annotation of the genes and transposable elements in complex genomes requires a human-curated integration of multiple sources of computational evidence. These evidences include results from a diversity of We have designed, implemented, and assessed a Perl-based workflow named DAWGPAWS for the generation of computational results for human curation of the genes and transposable elements in plant genomes. The use of DAWGPAWS was found to accelerate annotation of 80–200 kb wheat DNA inserts in bacterial artificial chromosome (BAC) vectors by approximately twenty-fold and to also significantly improve the quality of the annotation in terms of completeness and accuracy.Triticum aestivum L.). DAWGPAWS can be applied to annotation efforts in other plant genomes with minor modifications of program-specific configuration files, and the modular design of the workflow facilitates integration into existing pipelines.The DAWGPAWS genome annotation pipeline fills an important need in the annotation of plant genomes by generating computational evidences in a high throughput manner, translating these results to a common file format, and facilitating the human curation of these computational results. We have verified the value of DAWGPAWS by using this pipeline to annotate the genes and transposable elements in 220 BAC insertions from the hexaploid wheat genome ( The relative value of these sequencing efforts is a direct function of the accuracy of the annotation of the resultant sequence assemblies. Genome annotation seeks to delineate the sequence features that occur on the genome, thereby permitting definition of the biological processes responsible for these features [Genomic sequence assemblies are rapidly being published for a great number of species ,2. The sfeatures . In planfeatures ,6.ab initio gene annotation programs as well as sequence similarity searches against databases of previously identified proteins and expressed RNA [ab initio gene finding programs derive full gene models from DNA sequence data based solely on knowledge of the sequence features associated with protein coding domains. Sequence alignments can refine the exon-intron boundaries of these models and provide evidence that computationally predicted genes are actually transcribed in vivo. Existing software can automatically synthesize these data to derive combined evidence gene models [Identification of the genes that have been uncovered in assembled genome sequence data can utilize evidence from both ssed RNA ,7,8. Thee models ,10.ab initio and homology-based approaches have been used to accurately annotate genes in a number of eukaryotic genomes, plant genome annotation efforts cannot focus solely on the annotation of genes due to the risk of conflating genes with transposable elements [ab initio gene annotation programs will often annotate these TE ORFs as genes. Since most TE genes are expressed and represented in cDNA libraries, homology-based searches will indicate that these ORFs are transcribed and they thus may be considered legitimate gene predictions. Simply removing the high-copy-number candidate genes does not alleviate this problem because some true gene families are highly abundant while not all transposable elements are highly repetitive [While this combination of elements . Many TEpetitive . These eab initio results [ab initio transposable element discovery can exploit the fact that many families of TEs occur in high copy number within a host genome [Similar to the prediction of genes, accurate identification of the TEs in genomic sequence data combines homology-based searches and  results -15. Toolt genome -18, or tt genome -21. Homot genome -25 that t genome -29 or let genome -32.The gold standard of genome annotation is the integration and curation of multiple computational results by a knowledgeable biologist . This apHere, we introduce an annotation suite that allows for computational evidences to be generated in an automated fashion, integrates the results from multiple programs and facilitates the human curation of these computational results. This suite was designed to assist a Distributed Annotation Working Group (DAWG) approach for a Pipeline to Annotate Wheat Sequences (PAWS), and we hereafter refer to this effort as DAWGPAWS.The DAWGPAWS workflow Figure  is distrA common thread to each component of the DAWGPAWS package is that computational evidences are translated from the native annotation program output into the standard general feature format (GFF) . The GFFab initio gene annotation programs supported by these scripts include EuGène [ab initio TE annotation programs that can be run in batch mode are Find_LTR [One of the main sets of scripts in the DAWGPAWS package is the batch run program set Table . All of e EuGène , GeneID e EuGène , GeneMare EuGène , and Gene EuGène . The ab Find_LTR , LTR_STRFind_LTR , LTR_FINFind_LTR , LTR_seqFind_LTR , FINDMITFind_LTR , and TanFind_LTR . Batch mFind_LTR , NCBI-BLFind_LTR , RepeatMFind_LTR , and TEnFind_LTR . The fulIn addition to the batch run programs, scripts that convert an individual annotation program output to GFF are also available Table . These pThe DAWGPAWS suite also includes specialized tools for TE annotation. For identification of the highly repetitive regions of a contig, the seq_oligocount.pl program can count the occurrence of oligomers in the query sequence against an index of random shotgun sequences. This program generates all oligomers of length k from the query sequence, and uses the vmatch program  to deterIn addition to the gene and TE annotation-specific scripts included in the DAWGPAWS package, helper applications are also included Table . These CA CLI interface was selected for DAWGPAWS to facilitate the use of our applications in a cluster-computing environment, and to provide stability in program interface across multiple operating systems. While command line interface programs may be daunting to some users, every effort has been made to simplify their use. All of the CLI programs included in the DAWGPAWS suite follow consistent protocols for command line options Table . Help fiThe computational annotation results generated by DAWGPAWS can be directly imported into any genome annotation program that supports GFF. We have used the Apollo program  to visuaThe GBrowse package  can alsoIn addition to visualization and curation of the annotated DNA, it is also possible to transfer the DAWGPAWS results into existing database schema. For example, the CHADO database  can makeThe DAWGPAWS annotation framework has a number of features that make it a good choice to facilitate the workflow in plant genome annotation. The use of configuration files makes it fairly easy to modify the annotation workflow for the species of interest. The configuration files also makes it quite easy to generate results with multiple parameter sets for an individual program. Using multiple parameter sets will be especially useful when working with a genome that has not been annotated before, and for which appropriate annotation parameters have not been identified. Also, while previous annotation pipelines have focused on gene annotation, the DAWGPAWS suite maximizes the quality of TE annotation results. Most plants contain genomes with sizes > 5000 Mb , and areab initio TE prediction programs allowed for the identification of new families of LTR retrotransposons that would have been missed in our previous annotation efforts. Predicted gene models that span these newly discovered families would not have been identified as TEs in the exclusively homology-based searches that were previously used.The current focus of DAWGPAWS in our laboratory is the structural annotation of the genes and TEs in a genome using methods and applications tuned to the Triticeae. In annotation of 220 BACs from hexaploid bread wheat, we found that the DAWGPAWS pipeline increased the rate of individual BAC annotations by twenty-fold. Due to the time required to manually generate annotation results, this previous annotation effort was limited to using the FGENESH annotation program combined with a BLAST search of predicted models against known transposable elements and protein databases . Using tFuture development of DAWGPAWS will incorporate tools for the functional annotation of the predicted genes. Currently, functional annotation can be done within the Apollo program by manually selecting individual gene models and BLASTing these results against appropriate databases. A batch run support for additional local alignment search tools will also be added. The use of NCBI-BLAST is sufficient for most comparisons of sequence contigs against reference databases, but programs such as BLAT  or sim4 ab initio gene annotation programs will also be added to future releases of DAWGPAWS. Augustus [ab initio annotation program that will be useful for gene annotation that seeks to identify all transcripts derived from a single locus. Support for GENEZILLA [de novo and lack species-specific HMM model parameterizations. The addition of the PASA [Support for additional Augustus  is an abENEZILLA  and GlimENEZILLA  gene annENEZILLA  will be the PASA  program the PASA  as well the PASA . Future the PASA , SLAM [7the PASA , and TWIthe PASA  as they The DAWGPAWS annotation workflow provides a suite of command line interface programs that can generate computational evidences for human curation in a high-throughput fashion. We have used the DAWGPAWS pipeline to annotate 220 randomly selected BACs with wheat DNA inserts for both gene and TE content. Our curation efforts on the DAWGPAWS output are implemented in the Apollo program. The tiers file used for visualization of this curation are available as part of the DAWGPAWS package.DAWGPAWS represents an efficient tool for genome annotation in the Triticeae, and can be used in its current form to generate gene and TE computational results for other grass genomes. Minor modifications to the configuration files used by DAWGPAWS can make this program suitable to the generation of computational annotation results for any plant genome. The TE annotation capabilities of DAWGPAWS exceeds any other current genome annotation suite, and makes this package particularly valuable for the great majority of plant genomes, such as wheat or maize, that contain a diverse arrays of TEs that comprise the majority of the nuclear genome.The DAWGPAWS program has been specifically designed to facilitate use of individual component scripts outside of the entire package. Each script can function independently of all other applications in the package, and programs make use of standard input and standard output streams when possible to facilitate integration into existing pipelines. Since this package is being released under the open source GPL (version 3), the suite and its individual components can be used and modified under the terms of the GPL. Template batch run and conversion scripts are provided in a boilerplate format to facilitate extending DAWGPAWS to additional annotation tools. Furthermore, since we have selected the Perl language for the implementation of our package, the addition of new annotation tools can leverage existing modules in the BioPerl toolkit . These mProject Name: DAWGPAWS Plant Genome Annotation PipelineProject Home Page: Operating System: Platform IndependentProgramming Language: PerlOther Requirements: BioPerl 1.4, as well as the annotation programs that scripts are dependent upon.License: GNU General Public License 3Any restrictions to use by non-academics: No restrictionsBAC: Bacterial Artificial Chromosome; cDNA: complementary DNA; CLI: Command Line Interface; EST: Expressed Sequence Tag; flcDNA: full-length complementary DNA; GFF: General Feature Format; GPL: General Public License; HMM: Hidden Markov Model; LTR: Long Terminal Repeat; ORF: Open Reading Frame; pHMM: Profile Hidden Markov Model; TE: Transposable ElementThe authors declare that they have no competing interests.JE developed the pipeline, wrote the software, and drafted the manuscript. JB conceived the study, oversaw pipeline development, and helped draft the manuscript. All authors read and approved the final manuscript.


While the regeneration of a lost tissue is known to mankind for several years, it is only in the recent past that research on regenerative medicine/dentistry has gained momentum and eluded the dramatic yet scientific advancements in the field of molecular biology. The growing understanding of biological concepts in the regeneration of oral/dental tissues coupled with experiments on stem cells is likely to result in a paradigm shift in the therapeutic armamentarium of dental and oral diseases culminating in an intense search for “biological solutions to biological problems.” Stem cells have been successfully isolated from variety of human tissues including orofacial tissues. Initial evidence from pioneering studies has documented the likely breakthrough that stem cells offer for various life-threatening diseases that have so far defeated modern medical care. The evidence gathered so far has propelled many elegant studies exploring the role of stem cells and their manifold dental applications. This review takes you on a sojourn of the origin of stem cells, their properties, characteristics, current research, and their potential applications. It also focuses on the various challenges and barriers that we have to surmount before translating laboratory results to successful clinical applications heralding the dawn of regenerative dentistry. The sojourn of science has unraveled and understood that the secret of life lies in the “DNA,” thanks to Sir James Watson and Crick for their epoch making a historic discovery. In our endeavor to demystify the DNA, we have realized that scientific discoveries in cellular, developmental, and molecular biology have truly revolutionized our collective understanding of the biological processes that could greatly impact and dramatically change our lives in the future.In the new millennium, where biology and biotechnology have replaced chemistry, we are exploring “biological solutions to biological problems.” Owing to the extraordinary advances taking place in the field of cellular and molecular biology, we are on the verge of a paradigm shift, evolving from offering simple mechanical care to consider biological solutions to health promotion, risk assessment, diagnosis, treatment, and even prognosis.Although stem cell technology is just emerging, the regeneration of body parts is hardly a new concept. The regenerative capability of a living creature was recorded as early as 330 BC, when Aristotle observed that a lizard could grow back the lost tip of its tail. Since then, there have been slow but steady attempts at understanding the regenerative capabilities of human being and it is only in the last decade that we have seen an information explosion in the area of stem cell research. Stem cells are likely to revolutionize the entire health care delivery. The time is certainly ripe for all of us to familiarize ourselves with the following: what are stem cells, their characteristics, their potential applications, current research translating to therapy, and possible barriers of its application from the bench to the bedside/chair.They are unspecialized cells with an extraordinary ability to self-renew, capable of differentiating into one or more specialized cell types playing a crucial role in homeostasis and tissue repair.When called into action following an injury, a stem cell self-renews – undergoes cell division and gives rise to one daughter stem cell and one progenitor cell. A progenitor cell is an intermediate cell type formed before it achieves a fully differentiated state. It is regarded as committed to differentiating along a particular cellular developmental pathway of stem cells:Stem cell → Stem cell + Progenitor cell → Differentiated cellBased on their origin, stem cells are categorized either as embryonic stem cells (ESCs) or as postnatal stem cells/somatic stem cells/adult stem cells (ASCs).Totipotency: generate all types of cells including germ cells (ESCs).Pluripotency: generate all types of cells except cells of the embryonic membrane.Multipotency: differentiate into more than one mature cell (MSC).Self-renewal: divide without differentiation and create everlasting supply.Plasticity: MSCs have plasticity and can undergo differentiation. The trigger for plasticity is stress or tissue injury which upregulates the stem cells and releases chemoattractants and growth factors.Among the types of differentiation are:Direct differentiation: a specific type of cell in a special niche developed in a multistep unidirectional pathway .Transdifferentiation: direct conversion of one cell type to another different cell type .Dedifferentiation: a unipotent stem cell becoming a multipotent one.in vitro with HSCs and neuronal cells).Cell fusion: a stem cell fusing with a somatic cell resulting in another lineage  are derived from embryos that are 2–11 days old called blastocysts. They are best grown from supernumerary embryos obtained from Adult stem cells are found in most adult tissues. They are multipotent – capable of differentiating into more than one cell type but not all cell types. The plasDepending on their origin, adult stem cells can be further classified as hemopoetic stem cells (HSCs) and mesenchymal stem cells (MSCs). HSCs are obtained either from cord blood or peripheral blood. MSCs are those that originate from the mesoderm layer of the fetus and in the adult reside in a variety of tissues such as the bone marrow stem cells (BMSCc), limbal stem cells, hepatic stem cells, dermal stem cells, etc.et al. have isolated stem cells from adult human dental pulp (DPSCs) that exhibit a similar immunophenotype to bone marrow stem cells.[Stem cells have also been isolated from orofacial tissues which include adult tooth pulp tissue, pulp tissue of deciduous teeth, periodontal ligament, apical papilla, and buccal mucosa. Gronthos em cells. Stem celem cells. Using a em cells. Recentlyem cells.8Advances in stem cell research have explored many therapeutic avenues and the insight gained by a pilot proof of concept studies promises a plethora of unimaginable benefits:They are useful in functional genomic studies to understand human embryonic gene expression, genomic data mining, and bioinformatics.They serve to study biological processes which help in understanding human developmental disorders like birth defects, cancers, etc.They are new means of creating human disease models for drug discovery and development. It serves as an alternative to animal toxicology thereby hastening the drug to the market.The most crucial and exciting of all stem cell uses is cell-based therapy.Adult stem cells have been used in pilot studies as potential cell-based therapy for various diseases. The following stem cell characteristics make them good candidates for cell-based therapy:Potential to be harvested from patientsHigh capacity of cell proliferation in cultureEase of manipulation to replace existing nonfunctional genes via gene splicing methodsAbility to migrate to host target tissues (homing)Ability to integrate into host tissues and interact with the surrounding tissues.Stem cells derived from either peripheral blood, cord blood, bone marrow, or any adult tissue transported in the right medium to the laboratory is centrifuged, trypsinized, and propagated under ideal conditions and stored in the master cell bank (MCB). The MCB is further passaged to yield colonies of stem cells, given the right inductive signals using appropriate growth factors to allow them to differentiate into required cell types. These are injected or implanted into a patient as cell-based therapy. Homing will ensure that the stem cells reach the site of injury/tissues.Today, the two common methods of cell delivery are intravenous injection (direct delivery of cells) and cell encapsulation systems (indirect delivery of cells using a carrier). The cell encapsulation approach uses a biocompatible, biodegradable material construct that is seeded with cells and implanted into defects in order to regenerate the lost tissue.Stem cells are being explored for a variety of chronic debilitating diseases that have so far escaped remedial measures from traditional allopathic approaches with a hope that cell therapy would repair, repopulate, replace, and rewire tissues and organs regenerating hope and kindling confidence in such therapies.www.osiristx.com). Even in India, we are not far behind since Phase II proof of concept studies has been approved and is underway .Because of the overwhelming success of animal studies, numerous clinical trials are now going on world over.–16 VarioThe regenerative potential of adult stem cells obtained from various sources including dental tissues has been of interest for clinicians over the past years and most research is directed toward achieving the following:Regeneration of damaged coronal dentin and pulpRegeneration of resorbed root, cervical or apical dentin, and repair perforationsPeriodontal regenerationRepair and replacement of bone in craniofacial defectsWhole tooth regeneration.To this date, no restorative material has been able to mimic all physical and mechanical properties of tooth tissue. Furthermore, we have not been successful in providing an ideal solution to certain situations, such as an immature tooth with extensive coronal destruction and reversible pulpitis. If the regeneration of tooth tissue is possible in these situations, it facilitates physiologic dentin deposition that forms an integral part of the tooth thereby restoring structural integrity, minimizing interfacial failure, microleakage, and other consequent complications. Similarly, young permanent teeth that require apexogenesis or apexification are the perfect candidates for the regeneration of pulp as they allow completion of both vertical and lateral root development, improving the long-term prognosis. However, pulp regeneration in fully formed teeth may not be of great benefit, although there is sufficient evidence to say that a restored vital tooth serves longer than a root-canal-treated one.18 Pulp tet al. demonstrated both in vitro and in vivo in animals that dental pulp stem cells (DPSCs) were capable of forming ectopic dentin and associated pulp tissue.[et al. used an in vivo stem cell transplantation system to investigate differential regulation mechanisms of bone marrow stromal stem cells (BMSCs) and DPSCs. DPSCs were found to be able to generate a reparative dentin-like tissue on the surface of human dentin in vivo. This study provided direct evidence to suggest that osteogenesis and dentinogenesis mediated by BMSCs and DPSCs, respectively, may be regulated by distinct mechanisms, leading to the different organization of the mineralized and nonmineralized tissues.[A landmark study conducted by Gronthos p tissue.21 Batoul tissues.et al. demonstrated that the transplantation of ex vivo expanded autologous MSCs can regenerate new cementum, alveolar bone, and periodontal ligament in class III periodontal defects in dogs.[in vitro were successfully reimplanted into periodontal defects in order to promote periodontal regeneration by Hasegawa et al.[Regenerating the periodontium has always been a high priority in craniofacial regenerative biology. Due to the complex structure of the periodontium (consisting of hard and soft tissues), its complete regeneration has always remained a challenge. All the current regenerative techniques such as autologous bone grafts, allografts, or alloplastic materials have limitations and cannot be used in all clinical situations. Therefore, a cell-mediated bone regeneration technique will be a viable therapeutic alternative. Kawaguchi  in dogs. Going a wa et al. A subseqwa et al.et al. used a novel scaffold design with a new fabrication protocol to generate an autologous tissue engineered construct which was used to repair a segmental mandibular defect. The technique promoted osteogenesis and enhanced penetration of bone with blood vessels thereby accelerating tissue regeneration.[et al. showed that a mixture of MSCs and platelet-rich plasma improved bone implant contact and bone density in a mandibular defect.[Craniofacial bone grafting procedures rely on autologous bone grafting, devitalized allogenic bone grafting (using bone from bone bank), and natural/synthetic osteoconductive biomaterials. Autologous bone grafting is limited by donor site morbidity and allogenic bone is often destroyed soon. A long-term outcome using biomaterials relies on their ability to encourage local cells to completely regenerate a defect and results are often not encouraging. If stem cells can be harvested in a scaffold and transplanted into a defect to regenerate the lost tissue, it can alleviate a lot of complications associated with the traditional techniques. Abukawa neration. In a dogr defect. The dever defect. To furthr defect.–31 In suet al. reported the reconstruction of murine teeth using cultured stem cells which when transferred into renal capsules resulted in the development of tooth structures and associated bone.[et al. recently engineered teeth ectopically and transplanted them into an anthrotopic site in a mouse jaw.[et al. used SCAP and PDLSCs and formed a bioroot in mini pigs. SCAP and PDLSCs were seeded in a scaffold and implanted into the sockets of the lower jaw. Postchannels were precreated to leave space for postinsertion and 3 months later the bioroot was exposed and a porcelain crown was inserted. The bioroot developed, and had a natural relationship with the surrounding bone.[A therapeutic option that was unthinkable a few years ago seems an achievable goal today. Even to this day, the replacement of missing teeth has limitations. Although, implants are a significant improvement over dentures and bridges, their fundamental limitation is the lack of natural structural relationship with the alveolar bone . They rely on direct integration of bone on tooth surface which is indeed an unnatural relationship as compared with the natural tooth. Further, they are also associated with a lot of esthetic, functional, and surgical limitations that affect their prognosis. Ohazama ted bone. Nakao etouse jaw. Sonayamaing bone.Despite the significant advances made in medicine over the years, replacing the experienced processes perfected by nature is indeed a difficult proposition. We are yet to get a suitable blood substitute, and still rely on “blood for blood,” and “tissue for tissue.” Although there is little doubt that the best material to replace a tooth or tooth-related tissue is the tooth tissue itself, the question that lies before us is “Can we do it in a way that is predictable and is it practical/clinically feasible?” For any new therapy to be accepted, it should either be more effective than or as effective as the existing therapy with added advantages in terms of technique being simpler, more predictable, long-lasting, time/cost-effective, etc. If the above goal has to be achieved and if laboratory research has to be translated into clinical practice, there are certain barriers that have to be first understood and later overcome.To regenerate tooth/tooth-related tissues, the prerequisites are:Stem cellsSignaling molecules – to induce differentiation into the required tissue type, i.e., into osteoblasts, odontoblasts, cementoblasts, fibroblasts, etc.Scaffold material to support and harvest cellular proliferation.Stem cells obtained from any adult tissue are less, about 1–4%. Further,in vivo studies which support that they is immunologically safe.[The use of pre-existing allogenic cell lines and cell organ culture removes the problem of harvesting cells from the patients themselves and saves considerable time. One of tlly safe.40There are many growth factors that play a role in tissue regeneration. Growth factors are proteins that bind to receptors on the cell and induce cellular proliferation and/or differentiation. Dentin c4446The scaffolds used for regeneration should be biodegradable and the rate of degradation should coincide with the rate of tissue formation. They should be highly porous to facilitate cell seeding and diffusion of nutrients. While cells are fabricating their own natural matrix structure around themselves, the scaffolds should provide structural integrity and then eventually breakdown leaving the newly formed tissue. During this entire process, they should maintain important characteristics of stem cells and yet allow for appropriate differentiation of their progeny.54Several scaffolds have been tried which include both natural and synthetic materials. Natural materials like collagen, alginate, agarose, chitosan5658in vivo. Research is on in making them photopolymerizable to form rigid structures once they are implanted into any tissue site.[To regenerate bone and other body parts, it is preferable to have rigid tissue-engineered scaffold structures as they provide an excellent physical support to the cells. However,61sue site. Another sue site.–69Stem cells derived from all sources hold immense medical promises. Stem cell therapies have virtually unlimited medical and dental applications. While there are several barriers that need to be broken down before this novel therapy can be translated from lab to clinics, it is certain that the future is going to be exciting for all of us. We have moved on from the surgical model of care to the medical model and are likely to move onto the biological model of care. The need of the hour is high-quality research coupled with collaboration between basic scientists and the clinicians. A team effort engaging the expertise of the molecular biologists, immunologists, biomaterial scientists, cell biologists, matrix biologists, and practicing dental surgeons is crucial in attaining the desired goal. Stem cell therapy is no longer science fiction. Recent developments in the technique of stem cell isolation and expansion together with advances in growth factor biology and biodegradable polymer constructs have set a stage for successful tissue engineering of tooth/tooth-related tissues. Stem cell therapy has brought in a lot of optimistic hope amongst researchers, doctors, and not to forget the patients who are the chief beneficiary of this innovation. Stem cells regenerate hope and not all that is happening in research is hype.Remember – “Hope is a prerequisite for any successful scientific innovation.” And one day, very soon, stem cells will be introduced as an alternate/adjuvant in medicine and dentistry.


Structural phylogenomics refers to the combined use of evolutionary and structural information in a bioinformatics analysis. The term phylogenomics refers to two distinct tasks: reconstructing a species phylogeny using multiple genes —allowing a kind of mix-and-match grab-bag of function and structure. The result of these evolutionary innovations is a multiplicity of biological functions and structures that contribute to the diversity of life forms. Consider G protein–coupled receptors (seven-transmembrane receptors found in many eukaryotic species); over 800 have been found in the human genome alone. These genes have diverged from their common ancestor through repeated duplication events, allowing them to recognize hundreds of different ligands and to participate in distinct biological processes. Tracking the evolution of a protein superfamily within and across different species and overlaying the phylogenetic tree with experimental data can allow highly nuanced predictions of function and structure for uncharacterized proteins.Protein superfamilies evolve novel functions and structures through mutations at key positions, gene duplication, internal repeats, and gene fusion and fission events. Many proteins are composed of multiple structural A phylogenomic analysis, as originally defined semi-global  clustering techniques and alignments are also used to construct hidden Markov models  for use in identifying important functional or structural domains in novel sequences. While internal nodes (evolutionary branch points) of protein superfamily phylogenies typically represent speciation and duplication events, in cases where a structural domain-based phylogeny includes sequences with different domain architecture, internal nodes of the tree may also represent gene fusion and fission events; this complicates a phylogenetic analysis, but can yield very powerful insights into the functional roles of individual domains.Structural information can be useful at various points in a phylogenomic inference of function. For instance, most annotation transfer protocols do not differentiate between homologs having only local similarity and those aligning along their entire lengths, and can thus result in errors in annotation. Incorporating domain architecture information , or using a structural phylogenomic clustering method such as FlowerPower A fundamental tenet of phylogenomic inference of function is that the evolutionary tree is correct. Is this assumption reasonable? In our experience estimating phylogenetic trees for protein superfamilies in the PhyloFacts Phylogenomic Encyclopedias positional homology). However, numerous studies have shown that sequence alignment accuracy drops sharply with evolutionary divergence Many factors can affect phylogenetic tree topology accuracy. First, dataset selection (how homologs are selected) can affect phylogenetic accuracy. It is not uncommon to see phylogenetic trees restricted to sequences from whole (fully sequenced) genomes, or from a subset of available species. These restrictions in dataset selection may be convenient, but can cause problems in the estimated phylogeny due to sparse taxon sampling genome trees or gene matrix approaches To mitigate the impact of errors in a multiple sequence alignment, alignment masking is typically performed to remove columns of uncertain homology prior to phylogenetic tree estimation. However, alignment masking protocols appropriate for species phylogeny estimation, where the input is a concatenated alignment of many genes/proteins , or in which different phylogenetic methods may disagree on the branching order. Given these possible problems, how do we evaluate phylogenetic tree methods for use in analysis of protein superfamilies?Phylogenetic tree estimation methods have traditionally been evaluated in two ways: using simulation studies and comparing inferred trees and trusted phylogenies based on fossil data and morphological characteristics. Phylogenetic simulation protocols do not currently model gene duplication events and structural and functional changes, but could presumably be modified to do so. However, while we do not  have the equivalent of fossil evidence for gene families, we do have the rough equivalent of morphological data: abundant 3D protein structures; assays for biochemical function; experimental data indicative of biological process and pathway association, cellular localization, protein–protein interaction, and so on. We propose that these data could be used to evaluate protein superfamily phylogenies estimated from sequence information. Our fundamental assumption in proposing the use of these experimental data to evaluate protein superfamily phylogenies is the following: since evolution is primarily conservative of function and structure, a phylogenetic tree that clusters functionally and structurally similar proteins ought to be more accurate  than one that does not. Unfortunately, this approach has two fundamental limitations. First, functional similarity is not easily quantifiable. Second, evaluating phylogenies based on agreement with annotated function is problematic because of the prevalence of annotation errors and the paucity of experimentally supported annotations.Fortunately, protein structure has several attributes that make it an appealing basis for evaluation of phylogenetic trees. First, structural similarity correlates closely with evolutionary distance, with closely related proteins having higher structural similarity than more distantly related proteins true tree may not be known (or even knowable), but we can assess the predictive power of phylogenetic methods for their actual use in practice.In this article, we are clearly advocating a pragmatic approach to evaluating the effectiveness and utility of phylogenetic tree methods, rather than one that is based on some theoretical or ideological agenda. Simply put, we need a concrete measure of the utility of phylogenetic methods for functional inference. Whether these estimated phylogenies correspond to the If phylogenetic methods have limitations with respect to protein superfamily analysis, how can we improve them? In particular, is it appropriate to use protein structure or other experimental data to improve phylogenetic reconstruction accuracy? For instance, it has been shown that inclusion of phylogenetic information improves the specificity and sensitivity of bioinformatics methods for numerous tasks, including enzyme active site identification In our experience, orthologous groups are typically clustered correctly by most phylogenetic estimation methods. Where methods disagree is at the coarse branching order between paralogous genes.  At these evolutionary branch points, sequence information may be insufficient for phylogenetic resolution, and the use of structural data might prove helpful. If solved structures are available for some of these subtrees, we could use these data to bias the tree topology estimation . This joint analysis of sequence and structural characters is enabled by MrBayes (as shown in complete solution to the problem of protein function prediction, it has been shown to provide significantly higher precision in functional annotation than many other approaches to this task, and provides a unique framework for investigating the changes in protein function and structure explored by evolution.While structural phylogenomics is just one of many methods for predicting protein function (see


Questions have been raised about the stability in vitiligo for the past several years. However, during the same period several large series have been published showing successful melanocyte transplantation outcomes. In spite of repeated expression of concerns about the criteria determining the stability, several thousand patients have undergone transplantation successfully and benefited from these procedures. This by itself should be convincing enough, even to the skeptics and it seems that the debate on whether the ‘stability’ exists is more a theoretical concern than having much practical implications. We have shown that half the patients with recalcitrant bilateral vitiligo vulgaris and nearly 90% of those with segmental vitiligo with one year of stability, as determined by history, show >75% repigmentation at six months. For mostSimple methods such as comparison of pictures taken six months apart and careful history will be largely adequate to determine stability in most of the cases. Careful examination may also give additional clues regarding stability. In progressive vitiligo, often the margins are ill-defined or show different shades of colors. One should refrain from performing surgeries in patients with large surface area involvements as such patients are unlikely to respond regardless of clinical and/or experimental stability.There are several theories of the pathogenesis of vitiligo—biochemical, immunological, genetic and other biological aspects. Vitiligo is now considered as a complex reaction pattern or a syndrome, involving multiple etiologic factors. Thus it We are investigating some of these factors. There are two main biochemical abnormalities associated with vitiligo. These include disturbed antioxidant defense as evidenced by high levels of hydrogen peroxide in the epidermis and elevated serum and urine levels of catacholamines. The latter is thought to be a result of the former. Catecholamines are a better substrate for tyrosinase than tyrosine. This causes production of o-quinones resulting in either heptanation of tyrosinase or generation of reactive oxygen species (ROS) triggering or aggravating melanocyte damage. In our pOxidative stress is proposed as a triggering event in melanocyte degeneration. Imbalance in the oxidant–antioxidant system leads to generation of reactive free radicals. Reactive free radicals bring about lipid peroxidation producing lipid peroxides and lipoxides whose further decomposition yields malondialdehyde, which causes damage to cell membrane or DNA leading to cytotoxicity, mutagenicity and cell death. Antioxidants scavenging them include catalase, glutathione peroxidase, and superoxide dismutase, which are potential markers of stability in vitiligo.Although auto-antibodies to melanocytes have been identified in patients with active vitiligo, at present there is a preponderance of studies in support of cell-mediated auto-immunity in vitiligo. So studySo in summary, while better objective markers are needed to define stability, we should condrutinue to rely on existing recommendations for stability till suc


Autism is a highly heritable complex neurodevelopmental disorder, therefore identifying its genetic basis has been challenging. To date, numerous susceptibility genes and chromosomal abnormalities have been reported in association with autism, but most discoveries either fail to be replicated or account for a small effect. Thus, in most cases the underlying causative genetic mechanisms are not fully understood. In the present work, the Autism Genetic Database (AGD) was developed as a literature-driven, web-based, and easy to access database designed with the aim of creating a comprehensive repository for all the currently reported genes and genomic copy number variations (CNVs) associated with autism in order to further facilitate the assessment of these autism susceptibility genetic factors.AGD is a relational database that organizes data resulting from exhaustive literature searches for reported susceptibility genes and CNVs associated with autism. Furthermore, genomic information about human fragile sites and noncoding RNAs was also downloaded and parsed from miRBase, snoRNA-LBME-db, piRNABank, and the MIT/ICBP siRNA database. A web client genome browser enables viewing of the features while a web client query tool provides access to more specific information for the features. When applicable, links to external databases including GenBank, PubMed, miRBase, snoRNA-LBME-db, piRNABank, and the MIT siRNA database are provided..AGD comprises a comprehensive list of susceptibility genes and copy number variations reported to-date in association with autism, as well as all known human noncoding RNA genes and fragile sites. Such a unique and inclusive autism genetic database will facilitate the evaluation of autism susceptibility factors in relation to known human noncoding RNAs and fragile sites, impacting on human diseases. As a result, this new autism database offers a valuable tool for the research community to evaluate genetic findings for this complex multifactorial disorder in an integrated format. AGD provides a genome browser and a web based query client for conveniently selecting features of interest. Access to AGD is freely available at  Autism is an early onset neurodevelopmental disorder belonging to a group of conditions known as autism spectrum disorders (ASDs) which includes classical autism, pervasive developmental disorder-not otherwise specified (PDD-NOS), and Asperger syndrome . ASDs arSeveral candidate genes have been linked to this highly heritable disorder, but the etiology of most cases remains unknown. Linkage analyses for autism susceptibility loci have suggested the involvement of multiple genes from different chromosomes. Despite the completion of several genome-wide linkage studies for autism, most of the loci identified have not been replicated. Furthermore, association of several candidate genes have been reported and examined in subjects with autism, mainly with no conclusive evidence. As a result, a number of autism susceptibility nucleotide changes have been reported but have not been replicated. These inconsistent results could be in part a reflection of the clinical heterogeneity and varying degrees of severity in ASD.NLGN3 and NLGN4, were reported in individuals with autism spectrum disorders [For example, in 2003 the first evidence of mutations in coding sequences of two X-linked neuroligin genes, isorders . This fiisorders ,6.NLGN3 and NLGN4 genes have been identified in subjects with autism, suggesting that these mutations are not common and occur at a low frequency in the autistic population (less than 1%) [Despite strong supportive evidence for the role of these neuroligin genes in synaptic function, only a few causal mutations in the than 1%) -15. Therde novo than an inherited CNV to be pathogenic, the final causal effect of CNVs might be influenced by other cis- or trans-acting factors in a particular genomic environment, representing in an incomplete penetrance or a variable expressivity for a given CNV. This suggests that due to the complexity of neuropsychiatric disorders, the evaluation of biological relevance of CNVs should be considered in an integrated context [Furthermore, genomic DNA copy number variations (CNVs) including small deletions and duplications of chromosomes, which may affect gene function have been recently reported in association with complex disorders such as autism -19. In a context . For a r context .Recent developments in molecular genetic technologies and knowledge have introduced new avenues to be explored, in particular for complex disorders. A good example is gene regulatory factors such as noncoding RNAs (ncRNAs) which are highly expressed in the nervous system . An estimicroRNAs are small RNA molecules of approximately 22 nt that regulate gene expression by binding to the 3'-untranslated regions (3'UTR) of target mRNA(s), directing translational repression or transcript degradation . It is e5-HT2C mRNA whereby it is subject to posttranscriptional RNA editing and an alternatively spliced exon Vb [5-HT2C mRNA processing than healthy individuals, which may contribute to their clinical symptoms [One of the most studied snoRNAs in humans is HBII-52, located at chromosome 15q11 . In addi exon Vb . Subjectsymptoms . In an asymptoms . AccordipiRNA (Piwi-interacting RNAs) are a newly discovered class of small RNAs, 26-31 nucleotides in length, that are expressed abundantly in the spermatogenic cells . The majSmall interfering RNAs (siRNAs) are about 21 nucleotides in length and derive from double stranded RNA (dsRNA), typically a result of transgenic, viral or other exogenous dsRNA sources . In addiMultiple classes of ncRNAs are highly represented in the nervous system, emphasizing the likelihood that nervous system development and function is heavily dependent on RNA regulatory networks, and alterations of these networks may result in many neurological diseases. It is thought that ncRNAs may provide the key to better understanding the etiology of human diseases, particularly neurological diseases . For exaFragile sites are another important genomic factor in human genetics. Fragile chromosome sites are nonrandom gaps or breaks of variable size that can appear spontaneously or after exposing the cells to chemical agents ,42. BaseThe growing list of autism susceptibility genetic factors and the need to explore the role of gene regulatory elements  warrants the implementation of bioinformatics tools to facilitate a more comprehensive approach evaluating this complex neurodevelopmental disorder. In an effort to make all reported genomic features associated with ASD  and their potential relationship with other genomic features impacting on human disease    relatioAGD stores the lists of autism susceptibility genes and CNVs generated through scientific literature searches. For the susceptibility genes, we included those with at least one suggestive report for their association with autism. Using this broad definition we identified a total of 145 genes after reviewing 1228 articles obtained from PubMed (August 2008) using the search key words "autism" and "gene". The results were carefully evaluated by the PI (Z.T.) and a three-level classification system was used for autism susceptibility genes to reflect their level of association according to the reviewed publications: 3>2>1 whereby category 3 represents the strongest autism candidate genes. This arbitrary classification of genes has been added to provide a quick reference for the importance of each gene in relation to autism while the users may decide not to consider our classification.De novo or familial status of each CNV, as well as the subject's gender was included whenever such information was available in the reference paper. Due to the lack of a standardized formal naming convention for copy number variations that provides a unique identifier for each CNV, an informal identifier has been established for each of the CNVs associated with ASD using the chromosomal band followed by a roman numeral. One such example would be the CNV 17p12_III, which is the third CNV that occurs in the chromosomal band 17p12.For CNVs, those with at least one reported association with ASD were included. The snoRNA data was obtained using the sno/scaRNA coordinate file  from sno-RNA-LBME-db  and the In AGD, data is searchable and displayed in two formats: query based tabular and genome browser. Both tools display the genes, CNVs, ncRNAs, and fragile sites in an easily accessible framework. A web-based tabular query tool enables the user to start the search from any of the above four categories of features. A combination of criteria can then be employed to display other features within a user specified distance of the selected factor's physical chromosomal location Figure . AdditioWhile databases to maintain both autism susceptibility genes (AutDB ) and CNVThis new autism related data resource, AGD, will be routinely updated and upgraded as new information relating to ASD becomes available. Current plans for future developments of AGD are to incorporate additional tools allowing users to correlate currently stored information with linkage analysis studies and noncoding RNA gene targets. Additionally, plans are being formalized to add SNP information into the AGD database expanding search functions and application of this autism resource for the research community.The Autism Genetic Database is a repository resource incorporating all reported autism susceptibility genes and CNVs integrated with the known human noncoding RNAs and fragile sites. The scope of the AGD database distinguishes it from other recently developed data catalogs (i.e., AutDB  and ACRD.The Autism Genetic Database is freely accessible at The authors declare that they have no competing interests.ZT developed and supervised the project. GM and ZT designed the web interface. GM implemented the database and built the data integration pipeline and web interface. ZT extracted and curated autism genetic information from literature. ZT and GM drafted and contributed in witting of the manuscript. All authors read and approved the final version of manuscript.The pre-publication history for this paper can be accessed here:


Therapy-resistant overactivity of detrusor or small capacity and poor compliance, will usually need to be treated by bladder augmentation. Ileal or colonic patches are used frequently for augmenting the bladder, with either intestinal segment appearing to be equally useful. Stomach is rarely used because of the associated complications, but it is the only available intestinal segment for patients with impaired renal function. Concerns regarding long-term effects of associated metabolic acidosis, including abnormalities in linear growth and bone metabolism are misplaced. Ureterocystoplasty offers an attractive urothelium-preserving alternative, avoiding the metabolic complications, mucus production and cancer risk of heterotopic epithelium associated with enterocystoplasty. Though ideal for patients with dilated ureter and nonfunctioning kidney, in patients with functioning kidney it carries added risks associated with transuretero-ureterostomy, mainly obstruction. Ureteral dilatation in meningomyelocele patients is avoidable with proper follow-up and treatment. Therefore they rarely should be candidates for this operation. Alternative urothelium-preserving techniques, such as auto augmentation and seromuscular cystoplasty, have not proven to be as successful as standard augmentation with intestinal segment. Work is in progress on various bioengineering techniques to culture and combine bladder cells in tissue culture for regeneration. Early efforts are exciting, but preliminary. Meningomyelocele (MMC) affects as many as 1 in 1000 births, comprising the most common of pediatric neurogenic bladder and paralysis. Neurogenic bladder in these children presents with various patterns of detrusor-sphincter dysfunction within a wide range of severity. About 15% of neonates with myelodysplasia have no signs of neurological dysfunction at birth. However, there is a high chance of progressive changes in the dynamics of neurological lesions with time. Even babies with normal neuro-urological function at birth have a one in three risk of developing either detrusor sphincter dysynergia or denervation by the time they reach puberty. At birth, the majority of patients have normal upper urinary tract, but nearly 60% of them develop upper tract deterioration due to infection, bladder changes and reflux. MorbiditThe term myelodysplasia includes a group of developmental anomalies that result from defects in neural tube closure. Lesions may include spina bifida occulta, meningocele, meningomyelocele or lipo-meningomyelocele. MMC is by far the most common defect seen and the most detrimental. The term MMC refers to incomplete closure of the vertebral column during embryogenesis, resulting in exposure of the meninges and spinal cord. It is always associated with a constellation of findings - Chiari malformation Type II, which includes hindbrain herniation into the upper cervical canal, dysgenesis or agenesis of corpus callosum, neuronal migration of varying degree and hydrocephalus. AdditionMMC and other neural tube defects represent examples of multifactorial inheritance. Parents of an affected infant have a 1 in 30 chance of producing a second affected offspring. An affected parent, if able to father children has a 3-4% chance of having an affected child. The risk for inheriting this disorder among first degree relative is 1 in 200 and amongst second degree relative is 1 in 100. Maternal folic acid deficiency is an environmental factor strongly associated with neural tube defects (NTDs). SupplemeThe purpose of any classification system is to facilitate the understanding and management of the underlying pathology. In children, the spinal level and extent of congenital lesion are poorly correlated with the clinical outcome. Urodynamic and functional classifications have therefore been more practical for defining the extent of the pathology and planning treatment in children.Bladder is: a) overactive [uninhibited contractions] b) low capacity with poor compliance or c) Underactive/inactive with no effective contractions.The outlet (urethra and sphincter) may be independently: a) overactive causing functional obstruction or b) paralytic with no resistance to urinary flow.Bladder and sphincter work in tandem to make a single functional unit. According to the nature of neurological deficit, the bladder and sphincter may exhibit increased or decreased activity:These conditions may present in different combinations. This is mainly a classification based on urodynamic findings. In meningomyelocele, most patients will present with overactive detrusor and detrusor sphincter dysynergia.Antenatal diagnosis of MMC is made on elevated maternal alpha-protein levels or by sonographic evaluation of NTDs. Biochemical tests include maternal serum alpha fetoprotein (MSAFP), amniotic fluid alpha fetoprotein and amniotic fluid acetyl cholinesterase. The latter two are used as confirmatory tests and should not be regarded as part of routine screening of women at low risk of neural tube defects. Screening by elevated MSAFP  measured at 16-18 weeks of gestation can detect 56-91% of affected fetuses.While technically possible, surgery for MMC is presently not being performed via a fetoscopic approach. Open fet12For families interested in continuing the pregnancy, proper counseling including information about fetal repair, other maternal care issues and prognosis of children should be explained. There is evidence for a better motor level and ambulatory outcome following delivery by elective cesarean section in fetuses who have a bulging lumbosacral MMC and presence of motor function in the affected spinal cord segments in-utero. Fetuses with flat MMC or a large lesion with no motor function below T12, often do not benefit from elective cesarean section.After birth, the infant should be handled with nonlatex gloves as they are increasingly prone to latex allergy/sensitivity. Preterm newborns with MMC are more likely to have congenital heart disease and /or syndromic causes  than term newborns. CompleteAll these infants must be assumed to have a neurogenic bladder because only 5% of MMC can void spontaneously by typical age of toilet training. However,17et al,[In 1981, McGuire et al,Serial radiological imaging for detection of secondary evidence of high detrusor pressures is an effective alternative to urodynamics-based management. In more than 50% patients, observed with periodic radiological imaging, febrile urinary tract infection, hydronephrosis or reflux can be observed by three years of age. However,et al,[Urodynamics may be criticized for lack of standards of performance and interpretation of tests which may lead to unnecessary interventions. McGuire et al, stressedet al, emphasizet al, found thInitial urodynamic testing is performed soon after the state of spinal shock subsequent to neurological closure resolves, usually around six weeks of age. It is reasonable to infuse contrast medium or saline via a pump with a fill rate based on 10% of estimated bladder capacity (approx 5 ml/min). Urodynamic evaluation with electromyographic (EMG) and associated videoscopic evaluation are ideal. Tests performed in an agitated infant are difficult to interpret and several fillings may be needed to verify the findings. A review of the literature reveals that there are few studies that have attempted to characterize urodynamic findings in newborns with MM, 12-32% of patients had normal studies. Detrusor overactivity was noted in 40-76%, the rest revealed underactivity/areflexic detrusor with preserved or diminished compliance. External sphincter had normal EMG activity in approximately 40% and the rest showed evidence of partial or complete denervation.–25et al,[Spindel et al, first reet al, Overall,et al,[et al,[Other researchers have focused attention on detrusor responses. Roach et al, observedl,[et al, performil,[et al, Initial To minimize damage to the upper tracts and bladder.To minimize the social disabilities, from the consequences of neurological deficit and neurogenic bladder dysfunction.From the outset, the goals of urological management are:Management of children with MMC with a neurogenic bladder requires constant observation and adaptation to new problems.et al,[et al,[High-risk infants, that is, those with DLPP greater than 40 cm of water or DSD, are started on anticholinergics (a regime of 0.2 mg/kg dose of oxybutynin twice daily) and intermittent catheterization every three hours throughout the day. In many cases, catheterization at least once during the night (as the infant awakens for feeding) is also recommended. Advocates justify this approach to minimize the deleterious effects of high intravesical pressure, noting that in more than 50% of patients who are observed with periodic radiological imaging, febrile urinary tract infection (UTI), hydronephrosis or reflux develops by three years of age. Eighty pet al, comparedl,[et al, similarll,[et al, Urodynaml,[et al, Infants Widely practiced in the past, it is another option for early management of high intravesical pressures. Indications include intolerance to anticholinergic medications or the failure of pharmacotherapy to improve detrusor compliance sufficiently and maintain low storage pressure despite intermittent catheterization. Rarely, surgery is performed because the parents are unable to perform catheterization owing to their work schedules or other social considerations.Useful in neurogenic bladders which are refractory to anticholinergics and remain in a small-capacity, high-pressure state. Treatment seems to be more effective on bladders with a more active component.32 HoweveUrinary tract infections are common in children with neurogenic bladders. Bacteriuria is found in 60-80% of children with MMC, especially in those undergoing intermittent catheterization and typically does not require treatment.34 SymptoAntibiotic prophylaxis is used in children who have upper tract changes of reflux or hydronephrosis.Therapy-resistant overactivity of detrusor or small capacity and poor compliance, will usually need to be treated by bladder augmentation. Ileal or colonic patches are used frequently for augmenting the bladder, with either intestinal segment appearing to be equally useful. Stomach is rarely used because of the associated complications, but it is the only available intestinal segment for patients with impaired renal function. ConcernsChildren with detrusor overactivity but with underactive sphincters will be severely incontinent. Initial treatment is intermittent catheterization with anticholinergics drugs (as it may reduce the degree of incontinence and offers a much better control over UTIs). At a later stage, the outlet resistance will be increased in order to render them continent. Medical treatment like alpha receptor stimulation has not proven to be effective. SurgicalPatients with MMC and tethered cord syndrome of delayed onset usually present with bladder spasticity and orthopedic foot deformities. It is prudent to consider untethering of cord at the time of onset of symptoms to prevent further deterioration.Newborns with MMC pose a management challenge as few are born with and maintain normal bladder and sphincter function. Interdisciplinary teams of neurosurgeons, urologists, pediatricians orthopedicians, ICU staff, paramedical and nursing staff along with rehabilitation workers are required for optimal management of problems associated with such children. Urodynamics and radiological imaging play an essential role in ascertaining the nature of urological involvement in such patients. Worldwide, management is tailored to these findings. Regardless of whether management is primarily based on urodynamics (with preemptive intervention), radiological imaging (with intervention for development of adverse changes) or a combination of the two, all children with MM require close surveillance for urologic problems, especially during the first few years of life. Goals to protect the bladder and upper tracts and eventually to attain continence, ideally by school age, are discussed with the family from the outset and reinforced during periodic follow-up visits. In the absence of established standardized management protocols, we propose a management algorithm of such patients based on our own institutional experience and literature-based evidence .


The concept of delaying or preventing epithelial transformation remains a viable and attainable goal for the future. Drug-based strategies for chemoprevention of the future may predominantly rely upon targeted therapies with tolerable but defined toxicities for treatment of individuals diagnosed with intraepithelial neoplasias. Foods, diet manipulation strategies, or nutraceuticals may be more appropriate to delay or prevent carcinogenesis progression in healthy populations with genetic or epidemiologic evidence of risk for future transformation. In the case of neoplastic risk, healthy individuals at high risk for future development of a malignancy, for example, individuals with highly penetrant, low-frequency mutations , are usually willing to tolerate surgical procedures such as colectomy, mastectomy, and oophorectomy to reduce cancer risk. Irritating, yet minor, adverse effects  are less acceptable for intervention in healthy populations, who are not ‘diagnosed’ with clear and imminent ‘disease’, but rather are approached to undergo an intervention on the basis of a conceptually abstract ‘epidemiologic risk profile’. Major toxicities such as the recently reported enhanced risk of cardiovascular events associated with the administration of cyclooxygenase-2 inhibitors are unacceptable for any intervention with chemopreventive intent. While these examples represent opposite ends of a continuum of risk–benefit for chemopreventive intervention, risk–benefit for the bulk of individuals at risk for sporadic neoplasms is less clear and poorly defined.Existence of preliminary data to rationalise trials in humans: Epidemiologic data, that is, hypotheses-generating associations, have not accurately predicted prospective cancer preventive outcomes to date with sufficient certainty to justify large-scale, high-cost trials with a cancer end-point ((2) nd-point . Rodent nd-point .Trial design and progression of research in humans: The mixed record of success of the development of novel chemical entities by the pharmaceutical industry as cancer chemopreventive drugs points to the importance of design, implementation, and interpretation of phase I and II research in humans. An optimal cancer preventive dose cannot be extrapolated from doses used for different therapeutic indications. For example, in the chemoprevention of colorectal adenocarcinomas, higher doses of aspirin may be less effective than lower ones, which also protect against cardiovascular disease ((3)  disease .(4) Importance of biomarkers as potential surrogates for preventive efficacy: One might conclude that efforts to develop new chemopreventive interventions should be de-emphasised, and that resources should be redirected to more fundamental research. The experience gained in cancer chemoprevention trials to date provides important lessons that suggest a more rational, scientifically driven mode of development than that which has been often pursued until now. This process should enable a systematic, stepwise, phased exploration in preclinical models and in humans of newly emerging interventions. In the light of these considerations, we discuss below a chemopreventive agent selection strategy of novel drugs and a number of specific agents with putative cancer chemopreventive potential, of which some are currently under active clinical investigation and others originate from novel sources still at the stage of preclinical development.The recent experiences gained in the development of drugs as cancer chemopreventive agents lead to the conclusion that the therapeutic index of proposed interventions should be titrated to relative risk of transformation. Individuals at high risk of developing malignancies with proven pathological evidence of potential transformation , for example individuals with adenomatous polyps, atypical ductal hyperplasia, or oral leukoplakia, might be considered suitable candidates for ‘treatment of their disease’ using interventions consisting of single or combined drug-based approaches. Optimally, such drugs should target specific molecular carcinogenesis profiles obtained via genetic or proteomic profiling of that individual's pathologic lesion. Drugs that target intracellular regulation of proliferation, survival, and angiogenesis may be suitable to be used in such individuals with preneoplasias as compared to those who suffer from advanced full-blown malignancies . Such an approach might start with small, rapidly implemented and brief (60 days or less) translational clinical trials of biomarker modulation by multiple doses of a given intervention, alone or in combination with other mechanism-based agents. Subsequent biomarker-driven trials of drug combinations require duration of a year or more to assess both toxicity and continued biomarker effects prior to consideration of large, population-based cancer end-point trials. Toxicity profiles should include quality of life instruments to reflect the potential adverse nature of such interventions  , when phosphorylated activates signal transduction pathways that enhance cell survival, motility, and proliferation. Inhibitors of EGFR activation via antibodies to the extracellular protein or via tyrosine kinase inhibitory molecules provide promising new approaches to both cancer treatment and prevention. Despite mixed results in cancer treatment, many dysplastic cells also have activated EGFR and are reasonable targets for these agents. Preclinical data P53 targeting with ONYX-015, an adenovirus that replicates only in p53-deficient cells, produced histologic resolution of dysplasis in seven of 19 patients with oral leukoplakia (Peroxisome proliferation activated receptor (PPAR) gamma agonists, such as rosiglitazone, are currently in early phase chemoprevention development. These agents are of particular interest for targeting hormone receptor negative breast epithelial proliferations or dysplasia  concerns over the concept of acceptable therapeutic index for interventions in healthy populations; (2) the necessary preliminary data set required to support the development and implementation of research in humans; (3) the design and progression of research in humans aimed at defining preventive efficacy; and (4) the discovery and validation of biomarkers as potential surrogates for preventive intervention efficacy. Addressing these issues requires reorientation of the selection process of individuals at risk for a future cancer. Individuals at high risk for future transformation are more likely to accept the toxicity risks and financial costs of treatment-related toxicity in exchange for delay of cancer occurrence. Individuals without a known tissue or molecular high-risk factor for cancer will not tolerate toxicity that reduces quality of life.To address these two groups, which represent opposite poles of a risk continuum, development of multiagent pharmacologic interventions for high-risk individuals is warranted. Future combinations of pharmaceutical cancer preventives might consist of agents targeted to specific signal transduction pathways associated with specific epithelial cellular targets. Diet modification, and food or nutritional extracts  with known anticarcinogenesis activity may be more acceptable to healthy populations with known genetic risk based upon gene–environment interactions.


Molecular evolutionary studies in mammals often estimate nucleotide substitution rates within and outside CpG dinucleotides separately. Frequently, in alignments of two sequences, the division of sites into CpG and non-CpG classes is based simply on the presence or absence of a CpG dinucleotide in either sequence, a procedure that we refer to as CpG/non-CpG assignment. Although it likely that this procedure is biased, it is generally assumed that the bias is negligible if species are very closely related.Using simulations of DNA sequence evolution we show that assignment of the ancestral CpG state based on the simple presence/absence of the CpG dinucleotide can seriously bias estimates of the substitution rate, because many true non-CpG changes are misassigned as CpG. Paradoxically, this bias is most severe between closely related species, because a minimum of two substitutions are required to misassign a true ancestral CpG site as non-CpG whereas only a single substitution is required to misassign a true ancestral non-CpG site as CpG in a two branch tree. We also show that CpG misassignment bias differentially affects fourfold degenerate and noncoding sites due to differences in base composition such that fourfold degenerate sites can appear to be evolving more slowly than noncoding sites. We demonstrate that the effects predicted by our simulations occur in a real evolutionary setting by comparing substitution rates estimated from human-chimp coding and intronic sequence using CpG/non-CpG assignment with estimates derived from a method that is largely free from bias.ad hoc methods of ancestral state assignment.Our study demonstrates that a common method of assigning sites into CpG and non CpG classes in pairwise alignments is seriously biased and recommends against the adoption of  In mammals, the methylated form of cytosine (5-methylcytosine or 5mC) is hypermutable . 5mC is As a result of this hypermutability, molecular evolution studies have frequently attempted to estimate CpG and non-CpG substitution rates by separating observed substitutions into those that are inferred to have occurred within and outside CpG dinucleotides. In many previous studies -16, any However, denoting a site as ancestrally CpG based on the presence of a CpG in one of two extant species is clearly biased: this method of ancestral state assignment implies that misclassification of an ancestral non-CpG as CpG in two lineages requires only a single change whereas a minimum of two changes, one in each lineage, must occur to misclassify an ancestral non-CpG as CpG Figure . This isvice versa. However, the key point is that all true non-CpG dinucleotides that are misassigned will, by definition, have experienced at least a single change in one lineage. By incorrectly placing many true non-CpG changes in the CpG class we artificially inflate the estimate of the CpG rate, whilst depressing the estimate of the non-CpG rate. The difference in magnitude of the bias between CpG and non-CpG sites reflects the difference in total numbers of each site type.We first studied the simplest case of DNA sequences that had been evolved to approximate mutational equilibrium. DNA sequences were evolved to approximate mutational equilibrium by generating a random sequence and evolving until each site had experienced on average ten substitutions . Two seqWe next investigated the impact of CpG/non-CpG misassignment in sequence data that were compositionally more realistic than randomly generated sequences at approximate mutational equilibrium. Real mouse coding and intronic sequences were used as ancestral sequences and again, two derived sequences were copied, evolved and the number of CpG and non-CpG changes estimated using CpG/non-CpG assignment. The results of this analysis are presented in Figure The difference in level of estimation bias at fourfold and noncoding sites results from a difference in CpG content. Because of the structure of the genetic code, fourfold degenerate sites are typically enriched for CpG dinucleotides, whereas noncoding sequences are depleted (0.032 vs 0.010 in our murid sequences). With changing CpG frequency, the numbers of misassigned sites will make up different proportions of the total lost or gained from the CpG and non-CpG classes. Thus with decreasing CpG content, the overestimation of the CpG substitution rate becomes greater whereas the underestimation of the non-CpG substitution rate becomes less. Paradoxically, this result implies that whenever CpG/non-CpG assignment is used to estimate substitution rates between closely related species, CpG-rich sequences will appear to be evolving more slowly than CpG-poor sequences.We next investigated whether the effects predicted by our simulations could be observed in a real evolutionary setting. As the CpG/non-CpG assignment bias primarily affects closely related sequences, we estimated substitution rates in human and chimpanzee pairwise alignments of coding and noncoding sequence. As we cannot know the true numbers of CpG and non-CpG substitutions in a real alignment, we sought to compare CpG/non-CpG assignment with a bias-free method. Previous studies -20 have vice versa. Clearly, more reliable removal of the effects of CpG mutation is required prior to any meaningful comparison of rates of nucleotide substitution among different regions of mammalian genomes. Our simulations indicated that exclusion of non-CpG-prone sites (sites preceded by "C" and/or followed by "G"), is one simple and effective method of removing CpG-derived changes. One possible alternative to this is to employ an outgroup species to improve the accuracy of ancestral assignment. However simulations showed that parsimony-based ancestral assignment can introduce additional biases into the estimation of substitution rates [see Additional File Reliable, bias-free estimation of nucleotide substitution rates is a fundamental part of molecular evolutionary studies. Furthermore in order to test hypotheses about natural selection, underlying neutral processes, such as mutational rate variation, must be accounted for and their effects quantified or removed as efficiently as possible. Our study has shown that a commonly used method of effecting such a removal, assigning nucleotide sites to CpG and non-CpG categories, systematically biases the estimation of nucleotide substitution rates. In particular, across small to moderate evolutionary distances, CpG/non-CpG assignment seriously upwardly biases the estimate of the number of CpG changes and downwardly biases the estimate of the number of non-CpG changes. This is due to a simple artefact that causes many more true non-CpG changes to be misassigned as CpG than ad hoc method described here, can be seriously biased and misleading [The bias we describe here also causes the substitution rate within CpG dinucleotides to be substantially overestimated between closely related species. Estimation of the rate of substitution at CpGs has also been attempted using CpG/non-CpG assignment by previous studies ,10. It isleading ,25.The results presented here also have implications for previous comparisons of the substitution rate at fourfold and noncoding sites in mammalian genomes ,14. As aad hoc methods of ancestral state assignment.Our study shows that a commonly used method of assigning sites to CpG or non CpG classes in pairwise alignments is seriously biased. We further note that the effects of CpG and non-CpG misassignment at fourfold and noncoding sites are dependent on differential CpG frequencies, and so these results will apply to any comparison of substitution rates where this is the case. Our results therefore recommend against the adoption of We studied a simple mutation model in which transitions occurred twice as frequently as transversions, and CpG mutations occurred at a different frequency to non-CpG mutations. In all our simulations, two sequences were copied from a single ancestral sequence and evolved. In molecular evolutionary studies fourfold degenerate sites in codons that code for different amino acids in both derived sequences are excluded. To simplify this, in our simulated coding sequences, all nonsynonymous substitutions were considered strongly deleterious and rejected. Qualitatively similar results were obtained when a given proportion of nonsynonymous changes were modelled as neutral (results not shown). In all cases, estimates of the numbers and rates of nucleotide substitution were corrected for multiple hits using the Jukes-Cantor model . This waAncestral sequences in our simulations were derived from two sources: randomly generated sequences that were evolved to reach approximate mutational equilibrium, and real sequences derived from the mouse genome. The former allowed us to quantify the effects of CpG misassignment bias free of the complicating effects of nonequilibrium processes. Real mouse coding and intronic sequences (collected in ref ) were usDJG and PK designed research. DJG performed research and wrote the paper.Supplementary Figures. Figure Click here for file