The lipocalin apolipoprotein D (Apo D) is upregulated in peripheral nerves following injury and in regions of the central nervous system, such as the cerebral cortex, hippocampus, and cerebellum, during aging and progression of certain neurological diseases. In contrast, few studies have examined Apo D expression in the brainstem, a region necessary for survival and generally less prone to age-related degeneration. We measured Apo D expression in whole human brainstem lysates by slot-blot and at higher spatial resolution by quantitative immunohistochemistry in eleven brainstem nuclei . In contrast to cortex, hippocampus, and cerebellum, apolipoprotein D was highly expressed in brainstem tissue from subjects  with no history of neurological disease, and expression showed little variation with age. Expression was significantly stronger in somatomotor nuclei  than visceromotor or sensory nuclei. Both neurons and glia expressed Apo D, particularly neurons with larger somata and glia in the periphery of these brainstem centers. Immunostaining was strongest in the neuronal perinuclear region and absent in the nucleus. We propose that strong brainstem expression of Apo D throughout adult life contributes to resistance against neurodegenerative disease and age-related degeneration, possibly by preventing oxidative stress and ensuing lipid peroxidation.  Apolipoprotein D is a glycoprotein first isolated from the plasma HDL fraction and also found in smaller amounts in VLDL and LDL. The amino acid sequence shows a high homology with the lipocalin family but not with other apolipoproteins. The main role of Apo D is still uncertain, but by analogy with other lipocalins it may serve as a transporter of small hydrophobic molecules between the circulation and tissue . The genThe Apo D protein has since been detected in many organs, tissues, and body fluids of both humans and other species , and is In the human peripheral nervous system (PNS), Apo D is synthesized and secreted by fibroblasts and Schwann cells under basal conditions and upregulated during regenerative processes that follow injury. Following sciatic nerve injury, Apo D mRNA and protein expression levels increase to levels much higher than all other apolipoproteins ,6. Like The human central nervous system (CNS) expresses a unique Apo D isoform with lower molecular weight (29 kD) than that founded in CSF or plasma (32 kD). In humans and other mammals, immunohistochemical and hybridohistochemical techniques have demonstrated Apo D on the surfaces of glial cells , pial cells, perivascular cells, and neurons ,8–13. Apolipoprotein D expression is more intense in white matter than grey matter due to particularly strong expression in oligodendrocytes, where it may be involved in myelination . On the Increased expression of Apo D in the CNS has been observed in neurodegenerative and neuropsychiatric disorders  samples were gathered retrospectively from pathology archives of necropsies performed for diagnostic purposes; 2) patient identities were anonymized and completely delinked from unique identifiers; and 3) there was no risk to the participants.All human brain tissue samples were provided by the Department of Pathologic Anatomy of the Central Hospital of Asturias and obtained from necropsies within 6 h of death. These samples were the same as those used in previous studies by our group -10,21. TApo D levels were quantified using a slot blot technique . SamplesThe blots of Apo D were dried and bands quantified as relative optical densities (arbitrary units) using a digital scanner  and ImageJ 1.37c  .After fixation, tissue blocks were washed in distilled water, dehydrated, cleared in butyl acetate, and embedded in paraffin. Sections were cut at 10 μm thickness and mounted on "SuperFrost Plus" (Mentzel-Glasse) slides, dried at 36 °C for 24 h, deparaffined in xylene, and partially rehydrated by incubation in a graded alcohol series (2 min per concentration). In each case, every 80th or 160th section was stained by the Nissl/Myelin technique  and exam2O2  to quench endogenous peroxidase activity, washed in distilled water, and treated with PBS. After blocking non-specific binding by incubation in BSA (30 min), sections were incubated with the antibody against human Apo D  overnight at 4 °C. Immunoreactivity was detected using the Extravidin biotin peroxidase staining kit (Sigma Extra 3), and peroxidase activity visualized by incubation with Sigma Fast DAB (Sigma D4168) at room temperature for 30 min. Finally, the sections were counterstained using a modified formaldehyde thionin method [For immunohistochemistry, fixed sections were permeablized with triton X , washed in distilled water, treated with Hn method , dehydraFor quantification of Apo D expression, the chromogenic signal was selected using Adobe Photoshop CS 8.0.1  and density measured by ImageJ 1.37c  according to a procedure developed by our group . BrieflyAll statistical calculations were conducted using SPSS 15.0 for Windows. Data sets were first tested for normality using the Kolmogorov-Smirnov test with Lilliefors correction. Expression levels of Apo D were compared among nuclei by the Mann-Whitney U test due to non-homogeneity of variance. A P value < 0.05 was considered statistically significant. The levels of Apo D expression were measured in 26 human brainstem samples from patients ranging in age from 32 to 96 years. Densitometric analysis of slot blots showed robust and relatively stable expression throughout adult life, particularly between the forth and eighth decades . RegressExpression of Apo D was also measured at the cellular level in 11 pontine and medullar nuclei of both sensorial and motor functional systems . All speAll somatomotor and branchimotor system nuclei studied showed robust Apo D immunoreactivity throughout life. The hypoglossal nucleus (XII), located in the middle and lower third of the medulla oblongata, is part of the somatic motor column of the brainstem. This somatomotor nucleus  exhibiteabducens nucleus (III) is also part of the somatic motor column of the brainstem and shares many characteristics with XII , a nucleus with well described topographic organization, contained many large Apo D-positive neurons and showed the highest Apo D expression of all nuclei studied . The denThe immunoreactivity of Roller’s nucleus  was alsoThe DX  and NST The general and special somatosensory nuclei of the brainstem are located in the pons and medulla oblongata. We also studied IO  due to iThe VNC consists of inferior (IVN), lateral (LVN), medial (MVN), and superior (SVN) components. The IVN contains neurons of a broad range of sizes, but medium-size neurons are predominant . The IVNUnlike forebrain regions and cerebellum, we found intense Apo D expression in many neurons and glial cells in young human brainstem nuclei. Immunostaining for Apo D was usually restricted to the neuronal cytoplasm, with highest intensity in the perinuclear region. When the signal was strong, it tended to fill the entire cell, even the neurites, but has been not observed in the cell nucleus. In general, larger neurons showed more intense staining for Apo D than smaller neurons. The number of Apo D-positive glial cells appeared highest at the periphery compared to the core of these nuclei . FinallyIn the present study, we found that expression of Apo D in medulla oblongata and pons is already high in young subjects, contrary to other areas of the brain previously studied ,9, and wIn human frontal cortex, expression is higher in white matter than grey matter due to higher synthesis by oligodendrocytes ,9,13,34.Increased expression of Apo D in the CNS has been associated with aging, neurodegenerative diseases, and neuropsychiatric disorders. However, the majority of studies on Apo D have focused on cerebral and cerebellar cortices and the hippocampus, areas particularly prone to pathological changes with age and during the progression of neurodegenerative diseases. In all these areas, Apo D expression in young subjects was lower in grey matter than white matter, and increased in both tissues with age and following injury. Apo D expression has also been linked to cellular senescence and growth arrest in cell culture ,35, and The underlying molecular mechanisms of CNS aging include instability of nuclear and mitochondrial genomes, neuroendocrine dysfunction, oxidative stress, altered calcium metabolism, and inflammation-mediated neuronal damage . A growiWe observed strong Apo D expression in all studied nuclei of the medulla oblongata and pons from both young and old subjects, and no statistically significant increase in Apo D expression with age. Thus, Apo D is not a marker of aging in the brainstem. Due to these high levels of Apo D, we speculate that human brainstem is not exposed to damaging OS during aging, that Apo D expression in this brain region is uniquely regulated, or that the predominant role for Apo D in this brain region is distinct from that in other regions. It has long been known that Apo D is critical for remyelination of rat sciatic nerve, as evidenced by the > 40-fold increase in expression in regenerating nerve compared to non-injured control nerve . ElevateFinally, Apo D may also regulate neuritogenesis and synaptogenesis. Co-cultures of adipocytes with peripheral neurons showed enhanced neurite and synapse formation. It has reported that apolipoproteins D and E3 exert neurotrophic and synaptogenic effects in dorsal root ganglion cell cultures . ConsideThe effects of aging on the CNS have been studied for more than a century, but only a few works have focused on effects to the brainstem. We previously reported that aging is not associated with neuronal loss in brainstem nuclei , only a Although the full spectrum of Apo D functions is unclear, the current study strongly suggests that this molecule is critical for resistance to age-related neural damage. Neurons that do not synthesize or import Apo D from surrounding glial cells are more vulnerable to age- and disease-related death. Diseases or injuries of the brainstem region are normally incompatible with life, requiring stable basal expression of Apo D (and possibly other neuroprotective factors). The results of this study support the hypothesis that Apo D is a critical neuroprotective molecule against neurodegenerative disease and the ravages of age.


Particularly, since the first published double-blind randomized placebo-controlled trial in a paradigm using allogeneic cord blood and erythropoietin to treat cerebral palsy under immunosuppression showed beneficial therapeutic effects in infantile cerebral palsy, long-held doubts about the efficacy of this new cell therapy are dispelled and a revision of therapeutic views upon an ailment, for which there is no cure at present, is warranted. Hence, this review will summarize the available information on autologous cord blood therapy for cerebral palsy and that on the relevant experimental work as far as potential mechanisms and modes of action are concerned.About 17 million people worldwide live with cerebral palsy, the most common disability in childhood, with hypoxic-ischemic encephalopathy, preterm birth, and low birth weight being the most important risk factors. This review will focus on recent developments in cell therapy for infantile cerebral palsy by transplantation of autologous umbilical cord blood. There are only 4 publications available at present; however, the observations made along with experimental data  About 17 million people worldwide live with cerebral palsy, the most common disability in childhood, with hypoxic-ischemic encephalopathy, preterm birth, and low birth weight being the most important risk factors . Among 1in vivo and in vitro may be of utmost importance clinically, so that a review at an early developmental stage of this new therapeutic concept seems justified. Particularly, since the first published double-blind randomized placebo-controlled trial in a paradigm using allogeneic cord blood and erythropoietin to treat cerebral palsy under imunosuppression showed beneficial therapeutic effects in infantile cerebral palsy [This review will focus on recent developments in cell therapy for infantile cerebral palsy by transplantation of autologous umbilical cord blood because therapeutic strategies using cord blood in other human neurologic entities, including those for lysosomal storage disorders , have real palsy . Thus, lHowever, transplantation of allogeneic cord blood for cerebral palsy is limited by immunologic responses of the host (host-versus-graft reaction) and therefore requires immunosuppression, which in turn causes both inflammatory and neurotoxic side effects as well as reduced therapeutic efficacy depending on the degree of HLA mismatch , 12. Henin vitro and in vivo, using a different animal model (“Levine”) of perinatal cerebral hypoxic-ischemia in newborn rats to increase effectiveness [2 inhalation for 80 minutes on postnatal day (PN) 7. The neurodevelopment of the rat at PN 7 is equivalent to that of the human brain at birth [First mention of the concept of amelioration of perinatal brain damage by neuroregeneration using cord blood dates back to 2002, when our preliminary data in chronically prepared fetal sheep showed a highly specific invasion of human umbilical cord blood mononuclear cells (huMNC) into the damaged brain region, that is, “homing,” in a model of cerebral ischemia . This wativeness , 15. Theat birth . These sat birth . Unlike at birth , thus prat birth  , thus reducing glial scar formation  so that But not only the somatosensory motor behavior, also gross and fine motor function as well as muscle strength were improved upon transplantation of huMNC after hypoxic-ischemic brain damage in newborn rats both medium and long term . These bhttp://www.genecards.org/cgi-bin/carddisp.pl?gene=CD68). Interestingly, the CD68 (ED1) immunosignals in brain regions representing cortical motor areas for fore and hind limbs were significantly smaller in transplanted animals, an effect that was long-lasting (6 weeks) and irrespective of transplantation site of huMNC [Glial cells play a crucial role in neuroregeneration, specifically, activation of astrocytes as part of the inflammatory response may be involved in the formation of glial scars that limit neuronal plasticity after brain damage in newborn rats . This reof huMNC , 29, sugof huMNC .The distribution of gap junction protein CX43 reflected that of GFAP, while a zone of upregulation was observed in the inner glial wall of astrocytes and microglia, which separated the lesion core from the surrounding parenchyma. Again there was a reduced CX43 expression after huMNC transplantation, suggestive of less glial scar formation. The observed changes were quantified by Western blot analysis and quantitative real-time PCR of GFAP and CX43 on proteins and mRNA at 1 day (P9), 2 weeks (P21), and 6 weeks (P51) after transplantation, revealing already after 1 day a downregulation of astrocytic protein and mRNA  Figure . These rIn summary, transplantation of huMNC reduces glial scar formation and improves motor deficits medium and long term after hypoxic-ischemia in the newborn rat. This is paralleled by reduced infiltration of activated microglia/macrophages and decreased expression of GFAP, CX43, and SHP-1 mRNA and protein, all of which are known to increase after hypoxic-ischemia –34. ImpoThe mechanisms by which transplanted huMNC exert functional recovery in the brain are largely unknown. Hence, studies on the cellular and molecular level are required. Recently, using our model of hypoxic-ischemia in newborn rats , the effFurthermore, expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) revealed already two days after the insult that both growth factors, known to inhibit apoptosis and inflammation (BDNF) and to promote vasodilatation, angiogenesis, and neuroprotection (VEGF), declined in untreated but showed no change (BDNF) or an increase (VEGF) in transplanted animals . Thus, bIn our first set of experiments on transplantation of huMNC after hypoxic-ischemic brain damage in newborn rats, we hypothesized that—unlike in control animals that received a transplantation without brain lesion—the blood brain barrier must have been at least transiently destroyed  to explaClinical data on autologous cord blood transplantation for treatment of cerebral palsy are scarce –45 and hPapadopoulos and coworkers first reported on 2 toddlers with diagnosed cerebral palsy that received a combination of autologous cord blood, low dose subcutaneous granulocyte colony stimulating factor (G-CSF) injection before and/or after cord blood transfusion, and hyperbaric oxygen therapy . Though The largest cohort of autologous cord blood transplantation for cerebral palsy has been published by Lee and coworkers , who repFrom these 5 children, MRI FA values were evaluated to assess white matter integrity in 26 regions of interest (ROI). In 3 regions  significant changes after treatment were noted when compared with pretreatment values. The authors conclude that intravenous cord blood infusion seems to be practical and safe and has yielded potential benefits in children with cerebral palsy .n = 20), it is noteworthy that within the subgroup of hypoxic-ischemic encephalopathy , the non-responders were significantly older  than the responders who showed improvements after treatment , reports on only 3  patients showing adverse effects . These pVital signs and pulse-oximetry were monitored every 5 minutes during infusion and every 30 minutes for 2 to 4 hours after infusion . Thus, oIn order to avoid giving false hopes to patients and their families, it will be of utmost importance for ongoing and future clinical trials  to definFrom an experimental point of view, treatment for acute hypoxic-ischemic brain damage using mononuclear cells from human umbilical cord blood was shown to be effective, at least in the model that we and others employed. Also, insights in some important aspects regarding potential mechanisms and modes of action involved have been gathered. However, since there is virtually no information neither on the optimum amount of cells needed nor on the ideal timing of transplantation after the insult to be effective, future work should also focus on these clinically important questions.From a clinical point of view, intravenous autologous cord blood therapy for infantile cerebral palsy is safe, however, based on the preliminary uncontrolled clinical data available, it appears only to be effective in certain cases. It is important to notice that in the majority of uncontrolled cases it was not effective. Future work and ongoing clinical trials primarily must define both the groups of brain disorders responding and the therapeutic window, in which autologous cord blood therapy may be effective, in order to avoid giving false hopes to the patients and their families. Time matters—in several respects.


Recent discoveries have revealed the central role of PRDM9 in mammalian recombination. The precise function of this protein, however, remains poorly understood, as do the causes for its rapid evolution and its role in reproductive isolation. During mammalian meiosis, double-strand breaks are deliberately made throughout the genome and then repaired, leading to the exchange of genetic material between copies of chromosomes. How the locations of breaks are specified was largely unknown until a fortuitous confluence of statistical genetics and molecular biology uncovered the role of PRDM9, a DNA binding protein. Many properties of this protein remain mysterious, however, including how it binds to DNA, how it contributes to male infertility—both in humans, and in hybrid mice—and why, in spite of its fundamental function in meiosis, its binding domain varies extensively among humans and across mammals. We present a brief summary of what has recently been learned about PRDM9 in different fields, focusing on the puzzles yet to be resolved. Homologous recombination refers to the process by which DNA is broken and exchanged between copies of chromosomes. It is essential to the proper alignment and segregation of chromosomes during meiosis, with double-strand breaks serving to initiate the homology search and crossovers (one of the possible resolutions of recombination) tethering homologs together in order to ensure proper disjunction How hotspot locations and intensities are specified remained obscure until recently, when an epigenetic modification  was shown to be an important mark for the initiation of recombination in yeast and mice Prdm9 to a difference in recombination activity between mouse strains Prdm9-null mice show arrest of gametes in meiotic prophase I and impaired double-strand break repair CCCCaGTGAGCGTtgCc” enriched in hotspots that tend to be used in African populations but rarely in Europeans In 2009, two groups independently associated a region containing PRDM9 emerged in a completely distinct context: as the first (and to date only) locus shown to underlie hybrid sterility in mammals In spite of this rapid progress, however, a number of pieces do not fit into the puzzle, notably the tenuous relationship observed in sperm-typing experiments between PRDM9 variants, their predicted motifs, and the resulting recombination activity PRDM9 genotype, even when the hotspots do not contain an exact match to the 13-bp or the 17-bp motif  PRDM9 zinc fingers are highly diverse among humans, with over 20 variants already described In order to better understand the dominance relationships, we reanalyzed hotspot activity from previous sperm-typing studies, focusing on A-type and C-type variants see . As showEven so, the large variation in activity seen among A-type and C-type variants for the same set of hotspots remains a puzzle cis  can also affect binding affinity of PRDM9 and could contribute to the variability seen among individuals  and Hst1f (for fertility) variants of the zinc fingers of PRDM9 from M. mus domesticus and the Hstws and Hstwf alleles  in M. mus musculusM. mus musculus together with Hst1s and Hstws at Prdm9; all other combinations of Prdm9 alleles are fertile, as are female F1  that had never segregated together in the same population e.g., . F1 offsimer cf. . Thus, sPRDM9 per se compromises fertility within humans  were significantly enriched among fertile men We note that, within a single subspecies, mice carrying the sterility allele are fertile The residues of PRDM9 zinc fingers in contact with DNA show an unusually high rate of change in both rodents and primates Alternatively, the zinc finger could be evolving rapidly unrelated to its role in recombination per se: for example, PRMD9 could have a role in suppressing selfish elements in the genome Some of the incongruous observations might be explained if PRDM9 is responsible for the specification of all or almost all hotspots; if PRDM9 variants interact with one another and are dosage sensitive, and if the first half of the zinc fingers also affects binding. What is now required is a diverse set of experiments contributed from many fields, ranging from structural and molecular biology to speciation and evolutionary biology. Further knowledge about the structure of PRDM9, its binding properties and its possible cofactors, as well as its characterization in other species, will then allow us to address questions raised by recent findings, notably: Given the hundreds of thousands of motif instances in the genome to which PRDM9 could bind, how are recombination hotspots specified? How does the zinc finger evolve to find new motifs without deleterious effects on alignment and segregation, and what are the constraints on the state space of possible motifs? Is its rapid change due specifically to its role in recombination or is the change in hotspot activity a pleiotropic consequence of some other function Table S1The recombination activity of different variants atPRDM9.(DOC)Click here for additional data file.


Recent evidence suggests that many complex diseases are caused by genetic variations that play regulatory roles in controlling gene expression. Most genetic studies focus on nonsynonymous variations that can alter the amino acid composition of a protein and are therefore believed to have the highest impact on phenotype. Synonymous variations, however, can also play important roles in disease pathogenesis by regulating pre-mRNA processing and translational control. In this study, we systematically survey the effects of single-nucleotide variations (SNVs) on binding affinity of RNA-binding proteins (RBPs). Among the 10,113 synonymous SNVs identified in 697 individuals in the 1,000 Genomes Project and distributed by Genetic Analysis Workshop 17 (GAW17), we identified 182 variations located in alternatively spliced exons that can significantly change the binding affinity of nine RBPs whose binding preferences on 7-mer RNA sequences were previously reported. We found that the minor allele frequencies of these variations are similar to those of nonsynonymous SNVs, suggesting that they are in fact functional. We propose a workflow to identify phenotype-associated regulatory SNVs that might affect alternative splicing from exome-sequencing-derived genetic variations. Based on the affecting SNVs on the quantitative traits simulated in GAW17, we further identified two and four functional SNVs that are predicted to be involved in alternative splicing regulation in traits Q1 and Q2, respectively. With the advent of microarray and next-generation sequencing technology, the RNA-binding consensus sequences of several RBPs have recently been identified [Alternative splicing is an important level of gene regulation and greatly contributes to proteome diversity . In humaentified -8.Nonsynonymous single-nucleotide polymorphisms (SNPs) alter the amino acid composition of a protein; their effects on protein functions can be predicted by many bioinformatics tools, including SIFT , PolyPheIn this study, we present a computational strategy to systematically characterize the potential capability of single-nucleotide variations (SNVs) to regulate alternative splicing. Focusing on the exonic SNVs identified in each of the seven populations in the 1000 Genomes Project, we found that the minor allele frequencies (MAFs) for the synonymous SNVs residing in alternatively spliced exons and potentially disrupting the binding of RBPs are more comparable to nonsynonymous SNVs than to other SNVs, suggesting that they are under similar selection pressure. This result suggests the importance of regulatory SNVs that are associated with certain diseases or phenotypes and the importance of integrating biological annotation into genetic association studies. Furthermore, from the SNVs that are simulated to be associated with the phenotypes distributed by Genetic Analysis Workshop 17 (GAW17) , we idenGenotypes of SNVs for 697 individuals from seven populations  were derived from the sequence alignment files created in the 1000 Genomes Project and distributed by GAW17. We considered 24,487 exonic SNVs (both synonymous and nonsynonymous) within 3,205 autosomal genes. For phenotypes, the GAW17 committee carried out 200 replicate simulations on traits Q1 and Q2 and disease liability for 697 individuals; the simulations were based on an answer sheet of associated SNVs for each trait .As a proof of concept, we focus our analysis on nine RBPs whose binding affinity has been characterized using an in vitro assay called RNAcompete . Using aTo evaluate the potential of a SNV to alter the binding affinity of an RBP, for each RBP we first characterized the distribution of the preference scores for the binding and nonbinding events; a higher preference score indicates higher binding potential. We used the mean plus 3 times the standard deviation as the cutoff to distinguish binding and nonbinding events. As indicated in Figure PSmin and PSmaj are the RBP’s preference score for the minor allele and the major allele, respectively; b and n denote score distributions for the binding and nonbinding events, respectively; and PSmin) under the distribution indicating binding events (b). A positive or negative AC value indicates that the minor allele will cause a gain or loss of binding affinity, respectively.where We used the AltEvent track in the UCSC  Genome Browser to identify the SNVs residing in the exons that can be alternatively spliced . This trWe calculated the capability of changing the binding affinity of each RBP for all 24,487 exonic SNVs, based on the AC score (Eq. (1)), which is defined as the logarithmic ratio of the binding likelihood of the minor and major alleles. A more extreme positive or negative AC value indicates a higher possibility of altering an RBP’s binding affinity (gain or loss of the binding). For each RBP, the AC values for all the exonic SNVs follow a normal-like distribution; one example, for SFRS1 (or SF2), is shown in Figure To illustrate the regulatory role of SNVs, we focused our analysis on 10,113 synonymous SNVs, of which 1,851 candidates potentially affect the binding of at least one of the nine RBPs. Interestingly, some of the variations potentially affect the binding of more than one RBP: 173 variations affect bindings of 2 RBPs and 10 variations affect 3 RBPs. We clustered these 183 synonymous SNVs based on their AC values. Clearly, some of the SNVs can potentially cause the gain or loss of the binding of two RBPs. For instance, Vts1 and RBM4 share 32 SNVs in the heat map Figure . Similart test was used to quantitatively measure the significance of MAF differences between different categories; p-values are labeled in Figure To characterize the disease relevance of predicted synonymous SNVs that potentially affect RBP binding, we further divided all the synonymous SNVs into four (nonoverlapping) categories: (1) synonymous SNVs affecting RBP binding but not located within alternatively spliced exons (SS+RBP−Alt), (2) synonymous SNVs located within alternatively spliced exons but not affecting RBP binding (SS+Alt−RBP), 3) synonymous SNVs not only affecting RBP binding but also located within alternatively spliced exons (SS+RBP+Alt), and (4) synonymous SNVs located outside alternatively spliced exons and not affecting any RBP binding (SS−RBP−Alt). We examined the MAFs of the SNVs in the different categories based on all 697 individuals distributed by GAW17. We found that the average MAFs for the category SS+RBP+Alt are similar to the ones for the nonsynonymous SNVs in all the populations and that the average MAFs for the other three categories are markedly higher (Figure  synonymoBased on our observations, we propose a workflow to prioritize variations identified in the exome sequencing Figure . First, Distributed by GAW17, each of traits Q1, Q2, and disease liability are simulated to be affected by a particular set of nonsynonymous SNVs . We furtWe present a strategy to prioritize synonymous SNVs based on their likely capacity to change the binding affinity of an RBP and thereby affect pre-RNA splicing. Synonymous variations within alternatively spliced exons that affect RBP binding appear to be under similar selection pressure as nonsynonymous SNVs and therefore are candidates for functional SNVs affecting the phenotype. Synonymous SNVs that are outside the AltEvent or that do not affect RBP binding are under less selection pressure and therefore are considered less likely to be functional. We also show that some RBPs may have antagonistic relations when binding to synonymous variations, whereas others share the common consequence of gain or loss of binding. Trait-specific regulatory SNVs indicate that some nonsynonymous SNVs not only result in amino acid substitution but also regulate alternative splicing.Our proposed workflow provides an applicable way to identify phenotype-associated variations involved in alternative splicing. It should be noted that both nonsynonymous and synonymous variations that affect alternative splicing can be identified by using this workflow. The results of this analysis will lead to novel hypotheses for investigating the mechanisms of disease-causing mutations.The nine RBPs analyzed in this study are properly assessed for their binding preferences on RNA 7-mers . WithoutThe authors declare that there are no competing interests.MT, YW and YL contributed to the design of the study. MT, YW and YL designed and performed the computational modelling and drafted the manuscript. GW, JJ, HJE, JRS, and YW participated in coordination, discussions related to result interpretation and revision of the manuscript. All authors read and approved the final manuscript.


This study constitutes our next step toward a full investigation of how intrinsic membrane and synaptic parameters affect the electrical activity of a half-center oscillator (HCO) model and how different parameter regimes influence stability and modulatability of the HCO model’s output. In our previous study , we consspiking, silent, asymmetric activity, plateau, irregular spikes, asymmetric bursting, one burst, irregular period, unbalanced, and functional ; the isolated neuron instances were split into seven groups: spiking, silent, bistable, plateau, irregular spikes, irregular period, and regular bursting [We systematically  exploredregularly bursting (isolated) neuron instances compose only a small minority of functional HCOs (424 instances); the vast majority was composed of spiking neurons . Also, our data suggest that regular bursting isolated neurons form functional HCOs that are more robust to variations in synaptic parameters than those formed by spiking isolated neurons. Approximately 8.2%  of the functional HCOs have bursting characteristics that are similar to those of the living animal [By querying the database we started analyzing the relationships between the neuronal parameters of the model instances within the same group and across groups. Model instances whose neurons are synaptically isolated are called isolated neuron instances. We found that g animal .We used dimensional stacking (the NDVis tool) to visualize the distribution of each group of model instances versus the neuronal parameters. While each group has its own distribution within the parameter space, the groups of bistable and regular bursting isolated neuron instances separate large zones of spiking and silent model instances.st component has the greatest impact (variance > 60%). This component is mostly influenced by the maximal conductances of NaP, K2 and Leak, and the ELeak (linear combination of the parameters). Then, we applied PCA to our group of functional HCOs showing realistic leech bursting characteristics and we found that the first 6 (out of 8) are the most important principal components for this group (sum of variances > 96%). However, unlike in the regular bursting group, none of these principal components is dominant. This suggests that this group has complex non-linear relationships between its parameters and to explore them we need to use more sophisticate computational methods that the (linear) PCA.To explore in depth the interaction of parameters that lead to the different activity groups we have identified, we used the Principal Component Analysis (PCA) method. When we applied the PCA to the group of regular bursting isolated neuron instances, we found that only 4 (out of 6) components are important for this group (sum of variances > 95%) and that the 1


During 2011, 5 persons in the area of Lazio, Italy were infected with a monophyletic strain of hepatitis E virus that showed high sequence homology with isolates from swine in China. Detection of this genotype in Italy parallels findings in other countries in Europe, signaling the possible spread of strains new to Western countries. Recent evidence indicates that the family Hepeviridae may contain several genera and that viruses from some genera can be transmitted from animals to humans and vice versa (>24 subgenotypes (Hepatitis E virus (HEV) represents the major etiologic agent of enterically transmitted, non-A, non-B hepatitis. One third of the world population is estimated to have been infected with HEV, although the global extent of infection is unknown ; Other causes of acute liver injury, including drug toxicity, autoimmune hepatitis, and infection by hepatitis A, B, and C viruses, as well as by cytomegalovirus and Epstein-Barr virus, were excluded . All patThe 5 case-patients lived in the same area (maximum distance apart: 27 km) . The casSerum samples were tested for RNA by reverse transcription-nested PCR by using primers designed within open reading frame (ORF) 1 and ORF2 (The similarity of the strain from this outbreak to strains from recent autochthonous HEV genotype 4 infections in Europe was relatively low: 73.5% in the overlapping 37–172-nt ORF1 region of the isolate from Germany (GenBank accession no. EU879120) .An outbreak of HEV infection caused by genotype 4 in Italy involved 5 case-patients who lived in the same area and did not travel to disease-endemic areas. The high genetic similarity in ORF1 and ORF2 among the 5 HEV isolates supports a point-source outbreak and not sustained local circulation of this strain. Epidemiologic information did not identify the transmission route; available data ruled out direct transmission among patients, and parenteral transmission is unlikely because none of the patients had received blood transfusions, tattoos, or drug injections.Three patients had IgG against hepatitis A virus (HAV); no information on HAV vaccination status was available. However, the presence of these antibodies does not necessarily imply higher than average levels of exposure to enterically transmitted viruses; a HAV seroprevalence >60% has been reported in persons in the birth cohorts of the 5 case-patients in central Italy  is considered the most likely source of infection with HEV genotype 3 in Europe , which swww.comuni-italiani.it/statistiche/stranieri.html). Available data do not support correlations between immigration from China and spread of this HEV genotype in Lazio.Possible sources of infection with this HEV strain that cannot be ruled out include contaminated food from abroad and direct introduction through infected immigrants from China or other countries in Asia. However, the proportion of immigrants from Asia in this area of Italy, 0.77%, is lower than the national average  was observed between HEV isolates from human cases in northern and southern France and the strain isolated from swine in Belgium (In summary, this outbreak of HEV genotype 4 infection in Italy was not linked to infection by imported foods or persons traveling from endemic areas, which suggests the possibility that newly imported strains might spread this virus to new areas. Molecular characterization of HEV outbreaks in Europe is needed to implement epidemiologic mapping of infection with introduced strains of HEV and subsequent circulation.Methods used to generate partial open reading frame 2 sequences, including the 537-nt region of sequences from southern France described by Colson et al. (


Pumice is an extremely effective rafting agent that can dramatically increase the dispersal range of a variety of marine organisms and connect isolated shallow marine and coastal ecosystems. Here we report on a significant recent pumice rafting and long-distance dispersal event that occurred across the southwest Pacific following the 2006 explosive eruption of Home Reef Volcano in Tonga. We have constrained the trajectory, and rate, biomass and biodiversity of transfer, discovering more than 80 species and a substantial biomass underwent a >5000 km journey in 7–8 months. Differing microenvironmental conditions on the pumice, caused by relative stability of clasts at the sea surface, promoted diversity in biotic recruitment. Our findings emphasise pumice rafting as an important process facilitating the distribution of marine life, which have implications for colonisation processes and success, the management of sensitive marine environments, and invasive pest species. Pumice rafting is an important, but poorly understood and little known natural phenomenon that reflects a dynamic interplay between volcanism, the atmosphere and oceans, and marine biology. Such geological rafts have been suggested as a long-distance dispersal mechanism that can overcome physiological limitations on dispersal ranges for many marine species; they provide intermittent contact between shallow marine and coastal ecosystems that otherwise remain isolated by vast stretches of deep ocean 22 emissions (∼25 kilotons) were measured during the subplinian phase 2 degassing and hydrothermal venting until at least December 2008, 28 months after the eruption.After 22 years of dormancy, a category 2–3 (Volcanic Explosive Index) dacitic eruption at Home Reef from 7–16 August 2006 produced a new but temporary volcanic island (pumice cone) and a large floating mass of pumice initially extending over >440 km2Lepas sp.) attached to the pumice was made The large pumice raft produced in the eruption on August 7 2006, moved northeast towards the Vava’u Islands of northern Tonga and subsequently headed northwest and westwards reaching Fiji by mid-September 2006 3 magma in the Home Reef eruption to be >2.5×1012 . Importantly, each clast is a potential raft opportunity for an organism, emphasising the sheer abundance of rafting vehicles available immediately following a volcanic eruption.Pumice is an extremely effective rafting agent that can dramatically increase the dispersal range of a variety of marine organisms For rafting to be successful, distances travelled and frequency of dispersal events are of principal importance, whereas rate, transport direction and duration of rafts can significantly impact on rafted taxa abundance and diversity −1 . This drift rate, by utilising surface currents, is significant when compared to the potential drift rates and distances of pelagic larvae Drift trajectories of Tonga-derived pumice have been mapped using observations and sightings of stranded pumice 11 clasts. Most significantly, each one of these pumice clasts represents a rafting opportunity for organism(s).Successful dispersal not only depends on transport direction and velocity of floating items, but also on their total abundance in a particular region Positive relationships exist between raft size and number of travellers Lepas anserifera) were prolific in the early infestation of pumice, with some pumice lapilli collected from ‘live’ rafts in Tonga carrying >220 individuals. These numbers (12) and surviving transport to eastern Australia (∼8×1011), translate into the long-distance rafting of >10 billion individuals or colonies for some taxa. In some cases, the numbers of individuals rafted will have increased along the raft trajectory because several species will have reached sexual maturity during the rafting event . These data thus indicate a large biomass is ferried during pumice rafting events, especially in tropical waters resulting in high propagule pressure for many taxa. The high population numbers indicated here , nudibranchs and hydroids/scyphozoa formed a proximal or early colonising assemblage . Continued biotic recruitment of corals, bivalves, serpulids, anemones , cyanobacterial colonies of Order Oscillatoriales, gastropods (dominantly Recluzia sp.) and oysters (Crassostrea spp), during the pumice raft voyage from Tonga to Australia (attachment between 2–7 months), added to the proximally and early recruited assemblage, to form the middle colonising assemblage. The late assemblage continued this diversification trend, recruiting organisms from tropical and subtropical waters in eastern Australia from March to December 2007 with new recruits including macroalgae , calcareous algae, and occasionally macroalgae. Epibiont paucity on the dorsal sides of pumice clasts largely reflects the exposed surface environment due to the pumice floating with some freeboard, resulting in persistent solar radiation and to a lesser extent, air exposure. Solar radiation and desiccation have been reported to negatively affect colonization of littoral benthic communities Biotic succession involving the disappearance of early attached biota is not an obvious feature of the 2006–2007 rafting event. All previously attached biota continued to grow and survive along the pumice trajectory , with thAttention to transport and connectivity issues of marine communities has increased dramatically in the past decade, driven by concerns over the spread of invasive species, marine reserve design for improved conservation, fisheries resources, and climate-change effects 2 quadrats over pumice strandlines on beaches – this was to provide a representative and achievable sampling of the stranded pumice material given the volume of pumice deposited and length of coastline (>2500 km) along which stranding occurred. At other locations, samples of stranded pumice were collected but over a larger area of the beach and these are referred to as “representative” in Pumice raft material produced by the 2006 Home Reef eruption and examined in this study was collected from two main locations: the Vava’u Group of islands, Tonga, and from eastern Australia . All nec−2. More than 4900 clasts have been individually examined, measured and described – this includes material preserved in alcohol (N = 505), or dried (N = 4479). Epibionts were divided into two basic groupings: colonial  for which percent coverage of individual pumice clasts was visually estimated, or solitary, where individuals could be counted per pumice clast . For each clast, the following data were collected following examination using a binocular microscope: 1) maximum and minimum clast lengths; 2) pumice textural type; 3) evidence for recent clast breakage; 4) biological keel development and location of attachment/occurrence of organisms to either the dorsal or ventral sides of the pumice; 5) total number of plant and invertebrate species; 6) % epibiont coverage of pumice clast; 7) for cyanobacteria, fleshy/macroalgae, calcareous algae, cheilostome Bryozoa - occurrence, % coverage, number of species, types; 8) for gastropods, goose barnacles, bivalves - occurrence, number of species, number of individuals, types and shell lengths; 9) for corals, acorn barnacles and anemones - occurrence, number of species, number of individuals, diameter; 10) for serpulids - occurrence, number of individuals; 11) for forams, arthropods, nudibranchs, sponges, isopods/amphipods and egg casings - occurrence, number of species, number of individuals, types; and 12) for hydroids/scyphozoans - occurrence, number of species, types, and % coverage.The number of clasts was counted for each sample site listed in Virtually all pumice clasts were highly abraded and rounded, and most clast abrasion predated epibiont recruitment as the epibionts have grown over abraded surfaces. Later abrasion affected pumice samples washed across reefs, and strand samples collected from reef atolls . In other cases, species level identification could not be made due to the very juvenile forms present on clasts and this has been a particular issue for the attached corals – the rapid transit and stranding of the pumice limited the growth time available, and virtually all coral spats observed on pumice collected in April-May 2007 were <2 mm in diameter. In addition, little taxonomic work and species documentation are presently available for many of the attached biota for the SW Pacific and Eastern Australia with which to compare.3 is based on the measured areal extent 2 and an estimated raft thickness of 10 cm. Spheres were initially inserted at random locations within a prismatic volume surrounded by fixed bounding walls. The distribution of sphere diameters matched the measured maximum linear dimensions of 4,875 clasts obtained from locations given in −3. Using this number density, a pumice raft of 0.16 km3 would contain a minimum of 2.5×1012 clasts. Since the diameters of the spheres were given by the maximum linear dimensions of measured clasts, this value is considered a lower bound on the number of clasts comprising the pumice raft.Numbers of pumice clasts produced by the eruption have been estimated in the following way. Discrete Element The trajectories of the pumice rafts were calculated as a combination of the surface currents and the direct action of winds and waves on the rafts. The surface currents are derived using the methodology of Bonjean & Lagerloef We conducted an analysis of similarity (ANOSIM) using a Bray-Curtis similarity metric and 9999 permutations using Primer 6 . Details on the calculation of the pumice trajectories are given in the Materials and Methods section. Grey areas without bathymetric information represent continental shelves of <1000 m depth, where geostrophic ocean currents were not calculated.(GIF)Click here for additional data file.Appendix S1Data sources to historical (<200 a) pumice raft-producing eruptions shown in.(DOCX)Click here for additional data file.


Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator that represents a promising target for the treatment of several metabolic diseases. Administration of recombinant wild type FGF21 to diabetic animals leads to a dramatic improvement in glycaemia and ameliorates other systemic measures of metabolic health. Here we report the pharmacologic outcomes observed in non-human primates upon administration of a recently described FGF21 analogue, LY2405319 (LY). Diabetic rhesus monkeys were treated subcutaneously with LY once daily for a period of seven weeks. The doses of LY used were 3, 9 and 50 mg/kg each delivered in an escalating fashion with washout measurements taken at 2, 4, 6 and 8 weeks following the final LY dose. LY therapy led to a dramatic and rapid lowering of several important metabolic parameters including glucose, body weight, insulin, cholesterol and triglyceride levels at all doses tested. In addition, we observed favorable changes in circulating profiles of adipokines, with increased adiponectin and reduced leptin indicative of direct FGF21 action on adipose tissue. Importantly, and for the first time we show that FGF21 based therapy has metabolic efficacy in an animal with late stage diabetes. While the glycemic efficacy of LY in this animal was partially attenuated its lipid lowering effect was fully preserved suggesting that FGF21 may be a viable treatment option even in patients with advanced disease progression. These findings support continued exploration of the FGF21 pathway for the treatment of metabolic disease. The conventional approach to discover novel drugs for diabetes mellitus is traditionally centered on agents able to lower blood glucose with little to no attention paid to other measures of metabolic health. However, long term clinical outcomes with the molecules developed via this methodology have been mixed at best. This has led to a steady though guarded rise in attention towards novel so-called “glucose plus” targets that act via multifaceted mechanisms thus offering additional metabolic benefits beyond improvements in glycemic control Pichia pastoris.We have previously demonstrated a diverse spectrum of pharmacologic actions in diabetic monkeys following treatment with human recombinant FGF21 Macaca mulatta).Following the initial studies in rodents In this report we show that LY administration led to dramatic and rapid improvements in metabolic parameters including lowering of circulating glucose/insulin, ameliorated plasma lipid profiles and reduced body weight. These effects were observed in the presence of treatment-emergent LY antibodies. Importantly, in an animal with advanced disease progression, the effects of LY on circulating lipids and body weight were readily present even though its glycemic efficacy was significantly attenuated. In conclusion, our new data with LY, an engineered FGF21 variant, support further exploration of FGF21-based therapies for the treatment of metabolic disorders, including studies in patients at the advanced stages of disease development.Pichia pastoris as described LY was expressed and purified from All animal protocols in this study were approved by the Eli Lilly and Co. Animal Use and Care Committee. All monkeys were single housed in stainless steel cages for the duration of the protocol to allow for measurement of food intake as previously described , water was provided ad libitum. Monkeys were housed in a temperature controlled facility (21°C ±3°C) with a 12 h light/dark cycle. Animals were provided with environmental enrichment and were routinely monitored for signs of illness or distress by the veterinary care staff throughout the duration of the study. However, at the time of cessation of dosing none of the animals had shown any physical signs of distress or discomfort. None of the animals were sacrificed at any point during the study.To establish a metabolic baseline, vehicle was administered daily by subcutaneous injection for 6 to 15 consecutive days to a total of 6 diabetic rhesus monkeys of either sex that had no prior exposure to any FGF21 based molecule. Subsequently, daily subcutaneous injections of LY were administered in a dose-escalating fashion  for a total of 7 weeks  followed by a 60-day follow-up period (no injections). A 1- to 2-day washout period was included after each of the three LY treatment periods to allow for washout of the drug prior to drawing samples for assessment of immunogenicity. Volumes administered ranged from 0.06 mL/kg to 1 mL/kg. Sites of subcutaneous administration were rotated among multiple sites on the thorax/abdomen  and sites were recorded for each dose.During each period, the following parameters were monitored as functional biomarkers of LY pharmacodynamic activity: daily fed blood glucose and food intake, weekly body weight, bi-weekly fasted insulin, leptin, and adiponectin, and plasma lipids at the end of each treatment period.Prior to the lead in period we determined that one of the 6 animals selected for the study displayed significantly more advanced disease progression than the other members of the cohort, as evidenced by extremely low insulin levels in the context of overt hyperglycemia and low body weight as compared to the rest of the animals in the treatment cohort that were hyperinsulinemic and obese. However, to provide insight into FGF21 biology in the context of severe diabetic pathology we included this animal in the study and present some of the data obtained in this monkey separate to that of the remaining members of the cohort, both to highlight FGF21 effects in late stage disease and also to prevent distortion of the values reported for the other animals. All pharmacokinetic and immunogenicity data in Blood glucose, fructosamine, and triglycerides (TGs) were determined by Antech Diagnostics . Fasting plasma insulin and glucagon were measured using enzyme-linked immunosorbent assay (ELISA) kits from Linco Diagnostic Services, Inc. . Total cholesterol, low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) were measured using a Hitachi 912 clinical chemistry analyzer. Cytokine analysis was done by Rules-Based Medicine, Inc.  using human antigen Multi-Analyte Profiles (MAP) kits. All clinical chemistry, lipid composition, cytokine, and other analyses presented in this report were performed on the blood samples collected 24 h after the last dose unless stated otherwise.Serum samples were analyzed for concentrations of immunoreactive LY protein using anti-LY antibodies by the sandwich ELISA method. Samples were analyzed in duplicate and, when appropriate, at multiple dilutions.LY immunogenicity was assessed using a qualitative ELISA for the detection of LY-reactive antibodies in rhesus monkey serum samples with a detection limit of 1,150 relative ng/mL and LY tolerance of ≤1.0 µg/mL.Data are presented as mean ±SEM. Data were analyzed using repeated-measures analysis applied for multi-dose designs utilizing post hoc analysis (Tukey). Differences were considered significant when p = <0.05.Exposures to LY following its chronic subcutaneous administration to diabetic rhesus monkeys were evaluated by sandwich ELISA. LY levels were measured in fasting plasma samples collected weekly during the lead in and treatment phase. The mean plasma concentration of LY from each dosing phase increased in a dose dependent manner from 3 to 50 mg/kg . There wAll 6 animals demonstrated positive anti-LY2405319 antibody titers during the study. LY-reactive antibodies were measured in serum samples collected during the vehicle lead-in and 1–2 days following the final dose for each treatment period. Additional samples were collected during week 2 and 3 of the follow-up period, and during weeks 4, 6, and 8 of the follow-up period. Treatment-emergent anti-LY immunoglobulins were detected in all 6 monkeys with titers ranging from 10 to >31,250 . The repSimilar to the effects observed with administration of wild type FGF21 in rodents Food consumption was assessed daily and converted to weekly caloric intake. Following two weeks of LY treatment food intake was dramatically suppressed when compared to basal values similar to the results of the previous studies with FGF21 in this species Prior to LY administration, mean baseline plasma glucose was above 200 mg/dL , approxiMean fasting plasma triglyceride concentrations were significantly reduced during LY administration, with near-maximal efficacy already observed after only 1 week of treatment at the lowest dose, and only slight additional reductions with higher doses during the remainder of the treatment period . Thus, ain vivo action Recently our group and others have reported FGF21-induced regulation of the circulating levels and improved total body sensitivity to a multitude of other metabolic factors such as insulin, leptin and adiponectin highlighting one of the major mechanisms of FGF21 To date, it has not been determined whether FGF21 based therapy, often deemed to correct hyperglycemia via an insulin sensitization mechanism Interestingly, and in spite of the fact that J-15 had lower body weight at the beginning of the study, LY still remained potently efficacious to induce body weight loss . This boin vivo efficacy in rodent models of obesity and diabetes with no significant differences in the mechanism of action when compared to wild type FGF21 Non-human primates are thought to develop type 2 diabetes in a very similar manner to that observed in humans and thus offer an ideal test system for testing of anti-diabetic therapeutics Importantly, we show here that LY displays therapeutic characteristics similar to wild type FGF21 in the rhesus monkey. As with FGF21 administration, LY was able to rapidly and robustly improve and maintain tight glycemic control within the normal range over the course of the treatment while also delivering dramatic reductions in both circulating lipids and body weight. Together, these findings confirm that our engineered variant of the native FGF21 protein, LY, demonstrates therapeutic characteristics similar to FGF21 in non-human primates. The maximal efficacy of LY and wild type FGF21 are indeed similar which is likely due to the fact that neither FGF21 nor LY are able to lower glucose beyond the normal range in these species . Neverthin vivo adiponectin secretagogue. Given that FGF21 is unable to lower glucose in adiponectin null mice Recently we and others have reported that the metabolic effect of FGF21 is heavily reliant on engagement of the FGFR1/KLB complex in white adipose tissue in vivo, able to induce a multitude of the metabolic effects in a parallel manner via an array of diverse adipose centric mechanisms While caution must be exercised due to the single case nature of the data, the fact that LY remained partially efficacious in an animal at the late stage of disease development opens the possibility that FGF21 based therapy may have therapeutic utility in patients with well advanced type 2 diabetes. Intriguingly, and even though the glycemic efficacy of LY did appear to be partially blunted, adiponectin levels in J-15 were increased robustly and to a similar degree as in the other 5 obese monkeys , as withOur data show that the FGF21 based clinical candidate, LY, is robustly efficacious in diabetic rhesus monkeys where it is able to induce the full gamut of FGF21 biology. Chronic administration of the compound led to dramatic improvements in both serum glucose and lipids in addition to robust effects on food intake and body weight. Furthermore, in an animal with late stage diabetes, FGF21 therapy retained the majority of its efficacy with attenuation of only glycemic aspects of FGF21-mediated pharmacology. In conclusion, this work provides further evidence to support the investigation of FGF21 as a potential multifaceted “glucose plus” agent and supports further discovery and development of FGF21 based therapies.


Manganese (Mn), an essential trace element, is important for plant health. In plants, Mn serves as a cofactor in essential processes such as photosynthesis, lipid biosynthesis and oxidative stress. Mn deficient plants exhibit decreased growth and yield and are more susceptible to pathogens and damage at freezing temperatures. Mn deficiency is most prominent on alkaline soils with approximately one third of the world's soils being too alkaline for optimal crop production. Despite the importance of Mn in plant development, relatively little is known about how it traffics between plant tissues and into and out of organelles. Several gene transporter families have been implicated in Mn transport in plants. These transporter families include NRAMP , YSL (yellow stripe-like), ZIP (zinc regulated transporter/iron-regulated transporter [ZRT/IRT1]-related protein), CAX (cation exchanger), CCX , CDF/MTP , P-type ATPases and VIT (vacuolar iron transporter). A combination of techniques including mutant analysis and Synchrotron X-ray Fluorescence Spectroscopy can assist in identifying essential transporters of Mn. Such knowledge would vastly improve our understanding of plant Mn homeostasis. With an increasing human population, there is a growing demand for improvements in crop production. Mineral nutrients such as iron (Fe), copper (Cu), and zinc (Zn) are essential for both plants and animals, but maintaining optimal micronutrient levels presents challenges in both the plant and animal kingdoms. To combat human nutrient deficiency, efforts are being made to increase the bioavailability of nutrients in the edible portions of plants , on which the majority of characterization work has been carried out. The transporter families will be organized based on their putative function of transporting Mn into or out of the cytoplasm. Table A. thaliana and rice) and tissue localization  of the transporters is shown in Figures This review will focus on the micronutrient Mn. Despite its importance in cellular processes, such as photosynthesis and protecting cells against reactive oxygen species (ROS), little is known about transporters essential for Mn uptake and storage in the cell. We describe common and emerging techniques for assaying Mn localization and accumulation within the plant. In addition, we detail the protein families implicated in Mn transport. Particular emphasis will be placed on the model plant species Mn is a micronutrient element required for normal plant growth and development. It is essential for most photosynthetic organisms as a component of the oxygen-evolving complex in photosystem II (PSII), which catalyzes the water-splitting reaction to produce oxygen and provides electrons for the photosynthetic electron transport chain , the most commonly found forms in biological systems are Mn2+, Mn3+ and Mn4+with Mn4+ being the least stable  , thereby reducing cytoplasmic Mn , where there is a predominance of Mnsmf1, fet3fet4, ctr1, and zrt1zrt2, which are unable to transport Mn, Fe, Cu, and Zn, respectively, across the plasma membrane  pumps both Ca2+ and Mn2+ in the Golgi for detoxification purposes or for use as a cofactor for Golgi-localized proteins , phytosiderophores (PS), phytate and organic acids  /energy dispersive X-ray analysis (EDAX) has been used to provide detailed electron micrographs of tissue from hyperaccumulator plants along with energy dispersive X-ray spectra from regions of interest  , which can transport amino acid-containing compounds and their derivatives . Therefore, YSLs in non-grasses most likely use NA for intercellular and intracellular metal transport  are found in the apical PM of Arabidopsis root hairs  superfamily  between transmembrane domain 4 and transmembrane domain 5  can mediate vacuolar sequestration of Mn and confer resistance to high Mn stress . Expression of AtCCX3 in yeast rescues mutants defective in either PM or vacuolar import of Mn2+  which make up the CaCA superfamily  in transmembrane domains 2 and 4, which is not found in the Zn- or Fe/Zn-CDFs  and subgroup 9 . To date, AtMTP11 is the only functionally characterized Arabidopsis Mn-CDF. Yeast transformed with AtMTP11are more tolerant to Mn2+  identified two genes, BmMTP10 and BmMTP11, which were named after their orthologs in A. thaliana. Much like AtMTP11, these proteins are associated with the Golgi and are hypothesized to efflux excess Mn via the secretory pathway. The conservation of Mn-CDF function among multiple plant species supports the importance of these transporters in Mn detoxification and cellular homeostasis.Two MTP11 orthologs (PtMTP11.1 and PtMTP11.2) in poplar are believed to have a similar function as AtMTP11 due to their localization to Golgi-like compartments and ability to complement Arabidopsis 2+ pumps that remove Mn2+ from the cytosol and deliver it into their respective endomembrane compartment.The endomembrane system is essential for coordinating ion homeostasis in the cell. Several transporters that localize to the vacuolar membrane are described to facilitate metal uptake and release. However, there are also transporters in other endomembrane compartments that are equally important. Currently, there are two characterized proteins in Arabidopsis, AtECA1  and AtECA3, which are localized to the ER and Golgi Apparatus, respectively  and three in rice (OsECA1-3)  found in mammals. SERCAs, PMCAs (plasma membrane Ca2+-ATPases) and SPCAs (secretory pathway Ca2+-ATPases) comprise three distinct subfamilies of P2-type Ca-ATPases in animal cells , shows high amino acid sequence similarity to ECA2 (77%) in comparison to ECA1 and ECA3 , which transports Fe and Mn into the vacuole in yeast  are localized to the vacuole. They transport Mn, Fe and Zn in yeast, however physiological studies of mutants suggest that they are only Fe and Zn transporters in planta (Zhang et al., CCC1 (CaMn, although essential for plant survival, can be toxic to plants. Over the past few decades, several transporter families have been identified that play a role in Mn homeostasis. Interestingly, many of the transporters that translocate Mn have broad specificity for other ions, particularly divalent cations. For example, transporters such as IRT1, originally described as an Fe transporter in the root, can also transport Mn into the plant. In addition, CAX family members along with ECA proteins were originally thought to transport Ca; it was only subsequent research that discovered their roles in Mn transport as well. Despite the advancements in identifying Mn transporters, there is still a lot unknown regarding the molecular mechanisms controlling Mn homeostasis in plants. Many Mn transporters are not differentially regulated at the transcriptional level in response to Mn stress. Further research can explore the possibility that many of the transporters are under post-transcriptional or post-translational control. The degradation of OsNRAMP3 in response to Mn toxicity in rice may be a common mechanism used by plants to tolerate stress conditions (Yamaji et al., A. thaliana in crop species is a necessary step toward agronomic advancement. Research involving identification of genes involved in Mn homeostasis can be exploited to generate plants that are able to thrive in suboptimal soil conditions which would increase crop production and guarantee food security. For example, if we breed plants with high Mn uptake efficiency in the root, we can grow plants in highly alkaline soils where plants usually succumb to Mn deficiency. In conjunction, developing plants with increased Mn storage capability can assist in either soil Mn detoxification or improved growth of plants in acidic soils.Further characterization of orthologs of Mn transporters found in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The advent of high-throughput technology challenges the traditional histopathological classification of cancer, and proposes new taxonomies derived from global transcriptional patterns. Although most of these molecular re-classifications did not endure the test of time, they provided bulk of new information that can reframe our understanding of human cancer biology. Here, we focus on an immunologic interpretation of cancer that segregates oncogenic processes independent from their tissue derivation into at least two categories of which one bears the footprints of immune activation. Several observations describe a cancer phenotype where the expression of interferon stimulated genes and immune effector mechanisms reflect patterns commonly observed during the inflammatory response against pathogens, which leads to elimination of infected cells. As these signatures are observed in growing cancers, they are not sufficient to entirely clear the organism of neoplastic cells but they sustain, as in chronic infections, a self-perpetuating inflammatory process. Yet, several studies determined an association between this inflammatory status and a favorable natural history of the disease or a better responsiveness to cancer immune therapy. Moreover, these signatures overlap with those observed during immune-mediated cancer rejection and, more broadly, immune-mediated tissue-specific destruction in other immune pathologies. Thus, a discussion concerning this cancer phenotype is warranted as it remains unknown why it occurs in immune competent hosts. It also remains uncertain whether a genetically determined response of the host to its own cancer, the genetic makeup of the neoplastic process or a combination of both drives the inflammatory process. Here we reflect on commonalities and discrepancies among studies and on the genetic or somatic conditions that may cause this schism in cancer behavior. It was about a decade ago when the first studies attempting to re-classify cancer according to global transcript analysis were published -3. A couUnder certain conditions, inflammation seems to promote carcinogenesis, whereas in other situations it seems to have anti-tumor effects. The intensity and nature of the inflammation could explain this apparent contradiction ,5. In moLong ago, it was suggested that spontaneous regression or involution of malignant melanoma could be explained in terms of cellular immunity . Cochranin vitro and in reconstituted xenografts. Interestingly, together with the effector component of the immune response, the inflamed tumor phenotype also displayed the presence of immune inhibitory mechanisms including the expression of indoleamine-2,3-dioexygenase, PD-L1 and T regulatory cells. These findings suggest that tumors of the inflamed phenotype do not discriminate among various components of the immune response as they can sustain immune effector and immune regulatory functions simultaneously; it is probably the overall balance between the two vectors that determines the ultimate fate of these cancers individual cancers. More recently, the same group identified a similar pattern predictive of favorable outcome in patients vaccinated with dendritic cell loaded with multiple tumor antigen-derived peptides [701 (pSTAT1) and higher pSTAT1/pSTAT3tyr705 in cancer cells of patients with tumors displaying this type of infiltrate and this was associated with longer overall survival [Prospective molecular profiling of melanoma metastases undergoing immunotherapy with the systemic administration of high dose interleukin (IL)-2 suggested that lesions likely to undergo complete regression were immunologically activated before therapy . Recentlpeptides . Similarpeptides ,30. An ipeptides ,31 or anpeptides . Moschospeptides  observedsurvival . Treatmesurvival -37. As asurvival . Similarsurvival  who alsoInterestingly, others did not observe such a relationship between presence of tumor infiltrating lymphocytes and improved survival in patients with metastatic melanoma although the absence of tumor infiltrating lymphocytes predicted the development of lymph node metastases .The dichotomy between an immunologically active or a quiescent cancer phenotype is not exclusive of melanoma. Several studies described a neoplastic phenotype enriched in immunologic signatures or immune infiltrates that could be observed in cancer of ovarian -44, coloT cells represent the dominant immune infiltrate in ovarian cancer and the presence of CD8-expressing T cells within the intra-epithelial compartment is a strong predictor of improved survival ,43,70. AIntra-epithelial T cell infiltrates in ovarian cancer have been associated with enhanced expression of IFN-α, IL-2 and HLA class I molecules ,83 suggeIn 1998, Naito et al reported a correlation between infiltration of colon cancers by CD8 expressing T cells and improved survival . It was In addition, the evidence that regulatory T cells can lose FoxP3 , effectoA similar portrait was recently described for breast cancer . MoreoveFurthermore, Denkert et al  observedBesides the preclinical evidence suggesting that anticancer immune responses contribute to the success of chemotherapy, Ladoire S. et al  observedInterestingly, an older study observed an opposite pattern whereby lymphocytic infiltrate was associated with worse 5 year disease-free survival and with reduced estrogen receptor expression . HoweverWallace et al.  reportedTogether with gene expression profiling, immunohistochemical (IHC) validation of gene expression data and tissue microarrays (TMAs) are useful tools to clarify the prognostic relevance of gene expression in discrete tumor tissues ,101. Howin vitro. It is interesting to note that the constitutive activation of cancer cells observed in vitro through the detection of the phosphorylation of STAT-1 is generally modest; yet it corresponds to a transcriptional profile fully associated with the coordinate expression of ISGs activated downstream of the STAT-1/IRF-1 axis. Moreover, it appears that this constitutive activation of pSTAT-1, though mild, predisposes cell lines to higher sensitivity to further stimulation with IFN-γ [Although gene expression analysis has proven accurate in the characterization of the tissue microenvironments supporting the relevance of ongoing immune responses, it cannot provide information about the source cell expressing immune effectors gene. Based on transcriptional evidence, it is generally assumed that signatures of immune activation identified by testing whole cancer tissue reflect activation of genes expressed by infiltrating immune cells. However, we recently compared immunologic signatures of primary pancreatic cancer tissues pancreatic cancer cell lines and observed activation of innate immunity including activation of ISGs in both . Moreoveth IFN-γ ,104.in vivo perpetuate a positive feedback loop whereby the cancer cells not only invite immune cells to the tumor microenvironment but they are also more sensitive to pre-inflammatory factors secreted by immune cells. A recent commentary outlines different characteristics of the tumor cells that may explain paradoxical observations on tumor infiltrating cells of the immune system [Currently, no correlative study has been reported to test whether the signatures observed in immune activated tumors are also present in cell lines derived from the same tumors. It is important to note, however, that expression of various transcription factors associated with IFN signaling by cancer cells has been broadly described in various tumors  and it he system .kB [Constitutive production of immune stimulatory molecules and activation of immune mechanisms in cancer cells is likely to drive the homing of immune cell within the tumor microenvironment as recently demonstrated in experimental models ,109. It kB -112. MorkB . Thus, tkB  or immunkB  that canConstitutive expression and activation of STAT-3, a common oncogenic signaling pathway, has been clearly associated with cancer progression and poor prognosis . MoreoveIt could be argued that infiltration of the immune cells into the tumor site is a characteristic dictated by the genetic background of the host . Indeed It is also possible that the genetics of the tumor may drive infiltration of different cells of the immune system to the tumor site ,78,81,82Several studies emphasized on IRF-1 as a transcriptional activator that facilitates a Th1 immune phenotype and leads to tumor infiltration of CD8+ lymphocytes associated with a favorable prognosis in cancer patients ,102,118.et al. [The importance of a Th1 immune environment has also been observed by Dieu-Nosjean et al.  who obsekB [What are the mechanisms leading to constitutive activation of these transcriptional factors in cancer? While strides have been made in the understanding of the activation of the JAK/STAT pathway in leukemia  very litkB -123. SinkB ,124-126,It has been clearly shown that patients with early stage (stage II) or more advanced cancers suffer impaired immune function. The most striking example is decreased activation of ISGs and decreased ability to phosphorylate STAT-1 by circulating immune cells -129. TheIt is becoming clear that tumors can be segregated into at least two categories independently of their histology. Of them one bears a signature consistent with a Th1 type of immune activation which in turn is associated with, lymphocytic infiltrate, better prognosis and enhanced likelihood to respond to therapy. It remains unknown, why this dichotomy occurs, whether it depends on the genetic makeup of individuals bearing the disease or it is due to somatic mutations within cancer cells. It is also unknown, whether these signatures of strong clinical relevance are reflected by changes in the peripheral circulation. We believe that future studies comparing tumor tissue characteristics with the peripheral circulation, along with clinical data will be key to better classify cancers.The authors declare that they have no competing interests.FMM conceived of the manuscript, and participated in its design, coordination, analysis and interpretation of literature data.MLA participated in the acquisition, analysis and interpretation of literature data,All the authors made intellectual contributions and have been involved in drafting the manuscript, and approved the final manuscript.


Human parvovirus 4 infections are primarily associated with parenteral exposure in western countries. By ELISA, we demonstrate frequent seropositivity for antibody to parvovirus 4 viral protein 2 among adult populations throughout sub-Saharan Africa , which implies existence of alternative transmission routes. Parvoviridae, although viruses showing 61%–63% sequence similarity to PARV4 have recently been described in pigs and cows (––Human parvovirus 4 (PARV4) was originally detected in plasma from a person at risk for infection with HIV through injection drug use  in the United Kingdom, Italy, and Germany  of PARV4 genotype 1 to expand investigations of epidemiology and transmission of the virus , populations in sub-Saharan Africa, particularly in Central Africa, show high rates of exposure that cannot plausibly be accounted for by parenteral exposure. For example, the highest rate of seropositivity was observed among blood donors in Burkina Faso and Democratic Republic of the Congo who were uniformly negative for HCV antibodies, as well as for HIV-1 antibodies, by third-generation screening. In this setting, HCV infections are a frequent correlate of multiple blood transfusions and use of unsterilized needles in medical treatment or vaccination, as well as injection drug use. The high rate of seropositivity among HCV screen-negative samples from all 4 countries in Africa provides strong evidence for an alternative route of PARV4 transmission that is largely or entirely absent in Western countries.These findings are consistent with PCR-based evidence for PARV4 viremia, presumably associated with acute infection, among young children in rural Ghana . This association with HIV appears initially consistent with higher frequencies of PARV4 seropositivity reported among HIV (and HCV) co-infected injection drug users in Western countries. However, in the latter group, the association with HIV-1 was thought to reflect a greater frequency of illicit injection with shared needles (,Although this study leaves many questions on the transmission of PARV4 unanswered, the striking differences in seroprevalence and risk-group associations between sub-Saharan Africa and Western countries provides the basis for future more detailed investigations of its transmission routes, epidemiology, and potential clinical outcomes of infections. The previously noted sequence homogeneity of PARV4 nucleotide sequences between variants detected in Western countries (


RNA polymerase (pol) III transcription is responsible for the transcription of small, untranslated RNAs involved in fundamental metabolic processes such mRNA processing (U6 snRNA) and translation (tRNAs). RNA pol III transcription contributes to the regulation of the biosynthetic capacity of a cell and a direct link exists between cancer cell proliferation and deregulation of RNA pol III transcription. Accurate transcription by RNA pol III requires TFIIIB, a known target of regulation by oncogenes and tumor suppressors. There have been significant advances in our understanding of how TFIIIB-mediated transcription is deregulated in a variety of cancers. Recently, BRF2, a component of TFIIIB required for gene external RNA pol III transcription, was identified as an oncogene in squamous cell carcinomas of the lung through integrative genomic analysis. In this review, we focus on recent advances demonstrating how BRF2-TFIIIB mediated transcription is regulated by tumor suppressors and oncogenes. Additionally, we present novel data further confirming the role of BRF2 as an oncogene, extracted from the Oncomine database, a cancer microarray database containing datasets derived from patient samples, providing evidence that BRF2 has the potential to be used as a biomarker for patients at risk for metastasis. This data further supports the idea that BRF2 may serve as a potential therapeutic target in a variety of cancers. Cancer is a major health problem afflicting millions of Americans annually and despite tremendous research and treatment advances, is still the leading cause of death amongst men and women younger than age 85 years . A dominLike all eukaryotic polymerases, RNA pol III cannot recognize its target promoters directly and accurate initiation requires TFIIIB -4. In hiB-related factor 2) shares structural features with TFIIB and BRF1  is a ubiquitous and conserved protein kinase with growth-promoting and oncogenic properties. CK2 is abnormally active in a variety of human cancers . COPA was previously described and utilized to identify oncogenic chromosomal aberrations such as the TMPRSS2:ETS fusion gene in prostate cancer by Tomlins et al . Based oLastly, we investigated if there was a correlation between BRF2 overexpression and clinical outcome. Using breast carcinoma studies, we performed a meta-analysis across 10 different breast carcinoma datasets studying recurrence, metastasis and death, Figure Recently, RNA pol III transcription has been the focus of a Phase I and pharmacokinetic study. Hammond-Thelin et al. studied the effects of a novel nucleoside analog inhibitor (TAS-106) of RNA pol I, II and III, in patients with advanced solid malignancies . PreviouBy elucidating the mechanism(s) by which RNA pol III transcription is both regulated and deregulated, it will be possible to further understand the mechanism(s) by which aberrant activity of the general transcription machinery contributes to cancer development. Deregulation of RNA pol III transcription in cancers coupled with the observation that TFIIIB, specifically BRF2-TFIIIB, is commonly a target of deregulation in a variety of cancers demonstrates that RNA pol III transcription is indeed a key player in tumorigenesis and could serve as a novel target in the development of pharmacological agents.The authors declare that they have no competing interests.SC reviewed the literature, wrote and drafted the manuscript; LS corrected and finalized the manuscript. All authors read and approved the final version.


Major advances have been made in recent years in our knowledge and understanding of the microbial communities involved in ammonia oxidation in a wide range of habitats, including Chinese agricultural soils. In this mini-review, we focus our attention on the distribution and community diversity of ammonia-oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) in Chinese soils with variable soil properties and soil management practices. The niche differentiation of AOB and AOA in contrasting soils have been functionally demonstrated using DNA-SIP (stable isotope probing) methods, which have shown that AOA dominate nitrification processes in acidic soils, while AOB dominated in neutral, alkaline and N-rich soils. Finally, we discuss the composition and activity of ammonia oxidizers in paddy soils, as well as the mitigation of the greenhouse gas nitrous oxide (N2O) emissions and nitrate leaching via inhibition of nitrification by both AOB and AOA.Ammonia (NH Nitrification is a major process in the nitrogen (N) cycling, including a two-step process, the oxidation of ammonia to nitrite and subsequently nitrite to nitrate Prosser, . AmmoniaChina represents an enormous agricultural landscape with diverse soil types and pH. Research on nitrification has received much attention since the 1980's due to large increases in the use of ammonia-based N fertilizers and their associated environmental consequences. The discovery of archaeal ammonia oxidation also stimulated major research interest on ammonia oxidizers in Chinese soils. In this mini-review, we will summarize recent research findings on community composition and distribution of ammonia oxidizers in Chinese soils, environmental factors that affect the abundance and activity of AOB and AOA, and discuss the relative importance of AOB and AOA in ammonia oxidation in different soil systems.amoA gene, variants of which are commonly found in both bacteria and archaea. With the availability of culture-independent molecular ecology techniques such as denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), cloning, sequencing, and quantitative PCR in China, new insights into the diversity and distribution of AOB and AOA targeting the amoA genes in various Chinese soils have been rapidly achieved during the last five years. The abundance and composition of AOB and AOA in acidic red soils (pH 3.7–6.0), for example, were first investigated at a long-term field experiment station located at Qiyang , which had received continuous fertilization practice for 16 years  converts ammonia to hydroxylamine. The alpha (A) subunit of the AMO enzyme is encoded by the Nitrosospira amoA cluster 10, 11 and 12 in acidic red soils, while Nitrosospira amoA clusters 3a.1, 3a.2 and Nitrosomonas amoA cluster 6 and 7 are mainly distributed in alkaline and neutral soils  whereas that of group 1.1b was positively correlated with pH . Meta-analysis and high-throughput sequencing analysis by Gubry-Rangin et al.  was positively correlated with both AOB and AOA abundance, a pronounced shift of community composition was only observed in AOA but not in AOB in response to the long-term fertilization treatments  with different amounts of urea-N had a clear effect on the structure of AOB community, while the AOA structure remained stable during the period of investigation  gas. The development of nitrification inhibitors has therefore played an instrumental role in controlling ammonia oxidation rates, and thus reducing N losses through nitrate leaching and N2O emissions  on AOB and AOA community composition, abundance and function. DCD is a non-volatile commercially-available compound which inhibits nitrification by covalently binding to the active site of, and thereby inactivating the AMO enzyme , which has been widely used as a suicide inhibitor of AMO (De Boer et al., 2H2 inhibition of nitrification in an alkaline Chinese soil, Xia et al. (2H2, although the inhibition of AOB growth was greater. Interestingly, various studies using C2H2 as an inhibitor of nitrification have yielded different results with reference to the microbial groups that dominate ammonia oxidation. For example, ammonia oxidation in microcosms containing a Scottish agricultural soil was dominated by AOA whose growth was suppressible by C2H2-treatment (Offre et al., 2H2 treatment of German agricultural soils that AOB ammonia oxidation were more functionally important than AOA in the same soils.Another important nitrification inhibitor is acetylene (Ca et al.  revealed2O emissions under aerobic conditions (Wrage et al., 2O emissions. A recent report analyzing stable isotopic signatures of N2O emissions showed that AOA may be largely responsible for N2O emissions from marine environments (Santoro et al., Nitrifier denitrification is considered a significant source of soil N2O, a potent greenhouse gas. Integrated investigations of AOA and AOB communities using complementary methodologies are expected to facilitate identification of nitrification and denitrification activities of archaeal and bacterial ammonia oxidizers under different soil conditions. In order to reduce greenhouse gas emissions and nutrient loss from agricultural soils, it is imperative to gain a better understanding of the microbial communities involved and their specific contributions to nitrification and soil N cycling in general. This will be critical for increasing our ability to develop better strategies for N cycle management and to improve the N use efficiency, while at the same time minimizing adverse environmental impacts.Studies on the ecological distribution and community dynamics of AOB and AOA across a wide range of soil habitats and management regimes in China have demonstrated broad physiological diversity and distinct ecophysiology under contrasting soil and climatic conditions. However, it remains to be ascertained whether different functions, or possible functional redundancy, of phylogenetically distinct AOB and archaeal groups exist under different environmental conditions. In order to better understand soil N dynamics, research on the spatial and temporal variations of AOB and AOA activity and their unique contributions to nitrification is urgently needed, particularly in water-logged rice paddy soils where biogeochemical activity is high due to frequent oxic/anoxic shifts. Increased magnitude and frequency of N fertilizer application to agricultural soils stimulates the emissions of NThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


LRRK2) gene, which is responsible for both autosomal dominant and sporadic Parkinson’s disease (PD), in Caucasians (CEU), Japanese (JPT) and Chinese (CHB) from HapMap and Chinese (CHS), Malays (MAS) and Indians (INS) from the Singapore Genome Variation Project (SGVP) utilizing the traditional heatmaps and targeted analysis of LRRK2 gene via Monte Carlo simulation through varLD scores of these ethnic groups. Both heatmaps and targeted analysis showed that LD pattern of JPT was different from that of INS (P=0.0001); while LD pattern of CEU was different from that in Asian except for INS . Our study suggests that there is a higher chance to detect associations between PD and those trait-associated SNPs of LRRK2 gene found in Caucasian studies in INS, while those found in Japanese studies are likely to be better replicated among CHB.Genome Wide Association Studies (GWASs) have identified trait-associated polymorphisms via a hypothesis-free approach. However, it is challenging when attempting to reproduce GWAS findings in different populations as it fundamentally relies on the similar patterns of linkage disequilibrium (LD) between the unknown causal variants and the genotyped single nucleotide polymorphisms (SNPs). To address this potential limitation, we examined the regional LD pattern of leucine-rich repeat kinase 2 ( Parkinson’s disease (PD), a chronic common neurodegenerative disorder, has an estimated 1%–2% of individuals in those over the age of 65 years, and more than 4% in those more than 85 years . As an illustration, we determined the linkage disequilibrium between rs2046932, which was associated with PD at genome wide significance level in Japanese , and nei and INS .Within some of these potential constraints, it is challenging when attempting to reproduce primary GWAS findings from one population in other populations. Replicating an association across diverse populations fundamentally relies on the similar patterns of LD between the unknown causal variants and the genotyped SNPs, and greater inconsistencies in replication are expected in genomic regions where patterns of LD are different in different populations . With thAs LD variation can confound replication candidate gene studies with large sample size due to the not strong LD with causal variant in different populations , knowledLRRK2 among CEU and INS, and among JPT, CHS and CHB, suggesting that there is a higher likelihood to detect the association between PD and those trait-associated SNPs of LRRK2 gene found in Caucasian studies in INS, those LRRK2 SNPs found in Japanese studies are likely to be better replicated among CHB when allelic frequencies are similar in Caucasians and INS, or in JPT and CHB, respectively. Our findings will be useful for many of the future genetic association and fine mapping studies, and also meta-analyses evaluating LRRK2 tagging and functional variants across different ethnic populations. However, considering the minimal role of LRRK2 as a candidate and susceptibility gene in PD pathogenesis among Indian by other authors [In conclusion, our study highlighted evidence of similar regional linkage disequilibrium of  authors ,24,25, c