Monoallelically expressed genes that exert their phenotypic effect in a parent-of-origin specific manner are considered to be subject to genomic imprinting, the most well understood form of epigenetic regulation of gene expression in mammals. The observed differences in allele specific gene expression for imprinted genes are not attributable to differences in DNA sequence information, but to specific chemical modifications of DNA and chromatin proteins. Since the discovery of genomic imprinting some three decades ago, over 100 imprinted mammalian genes have been identified and considerable advances have been made in uncovering the molecular mechanisms regulating imprinted gene expression. While most genomic imprinting studies have focused on mouse models and human biomedical disorders, recent work has highlighted the contributions of imprinted genes to complex trait variation in domestic livestock species. Consequently, greater understanding of genomic imprinting and its effect on agriculturally important traits is predicted to have major implications for the future of animal breeding and husbandry. In this review, we discuss genomic imprinting in mammals with particular emphasis on domestic livestock species and consider how this information can be used in animal breeding research and genetic improvement programs. Mammals are diploid organisms characterized by the presence of complete sets of paternally- and maternally inherited chromosomes in each somatic cell. Normal mammalian development requires that the paternal and maternal copy of each gene  is expressed correctly, with each copy having the potential to be expressed equally  in each cell. However, a subset of mammalian autosomal genes has been identified where expression is restricted to one of the two parentally inherited chromosomes in a parent-of-origin specific manner; such genes are said to be imprinted. Imprinted genes on autosomal chromosomes can affect both male and female offspring, and such imprinting effects do not arise as a consequence of sex chromosome inheritance. Rather, ‘classically defined’ autosomal imprinting is a consequence of the parental origin of each allele such that, in general, paternally expressed/maternally imprinted genes are transcriptionally silenced on the maternally inherited chromosome only, while maternally expressed/paternally imprinted genes are silenced solely on the paternally inherited chromosome . Not allXIST), which initiates the chromosome-wide gene silencing of the X-chromosome from which the XIST transcript is derived. Interestingly, preferential inactivation of the paternally derived X-chromosome involving XIST transcripts has been reported in the placental tissue of XX female mammals, a process known as ‘imprinted X inactivation’  have been documented; however, only 25, 21, and 14 experimentally validated imprinted genes/loci have been reported for cattle, pigs and sheep, respectively . Initialdatabase  only 40 database . Furtherdatabase .PEG3 domain was recently shown to result in the loss of paternal MIMT1 expression in the brain and cotyledon of all carrier fetuses. This mutation is thought to be responsible for late fetal mortality and stillbirth in 85% of the offspring inheriting the causative mutation from the founding sire; it has been postulated that the remaining 15% of progeny inheriting the mutation survive due to incomplete silencing of the maternally inherited MIMT1 allele  at these sites result in dysregulated expression of reciprocally imprinted genes leading to developmental disorders . In catt1 allele , 2012.conflict theory’ of genomic imprinting, which states paternally expressed imprinted genes  have evolved to actively promote fetal growth and development, thereby maximizing maternal resources to offspring bearing a particular paternal genome during gestation. In contrast, maternally expressed imprinted genes  have evolved to suppress fetal growth, thereby causing a more uniform distribution of maternal resources to all offspring carrying a particular maternal genome, despite possessing different paternal genomes  and/or chromatin-associated proteins . Such epigenetic “imprints” can serve to mediate the local expression of genes, either through transcriptional activation, transcriptional attenuation or complete transcriptional silencing. In mammals, parent-of-origin-specific expression due to genomic imprinting is reliant on the existence of epigenetic differences between the two parental alleles resulting in their differential expression in the same nucleus .vice versa) in the subsequent generation. These parent-of-origin imprints can be then inherited by all daughter cells through mitosis following fertilization, potentially resulting in imprinted gene expression patterns throughout the lifespan of the animal ; and fourthly, the imprint must be erased and reset in the germ line such that appropriate parent-of-origin identity is established in the gametes for the subsequent generation .3) by DNA methyltransferase enzymes to the 5′ carbon of cytosine residues that exist primarily in CpG dinucleotides  , the most extensively studied epigenetic marks associated with genomic imprinting is DNA methylation . In mamm strand] . Cytosin strand] ].Figure 1). Thus, silenced or repressed gene expression is generally observed from the hypermethylated DMR . In eukaryotic cell nuclei, DNA is tightly packed into chromatin such that the DNA double-helix is wrapped around the histone octameric core to form the basic chromatin unit, the nucleosome. The N-terminal regions of histone proteins that protrude from the nucleosome can undergo various post-translational modifications  that can regulate gene expression -mediated repression of gene expression, macro ncRNAs, (which are often 100s of kb in length) can elicit cis-regulatory effects on gene expression, and thereby can generate allele-specific imprinting effects on gene expression  is well established for X-chromosome inactivation in female mammals . Long ncpression .In general, studies of imprinted gene regulation and imprinted gene clusters are revealing a complex interplay between DNA methylation, histone modifications, higher-order chromatin structure, RNA-mediated epigenetic effects and transcription, which are all involved in the establishment of the primary genomic “imprint” .de novo methylation has also been shown to coincide with global changes in histone tail modifications, which are not observed in female germ cells during this period of development  including somatic cell nuclear transfer (SCNT) cloning studies. ART involves the isolation, handling, manipulation and culture of gametes, and early embryos, usually after hormonal stimulation. As discussed above, major epigenetic reprogramming events occur during gametogenesis and early embryonic development and it has been proposed that ART exposes the epigenome to external factors that may interfere with the correct establishment and maintenance of genome imprints. For example, superovulation, embryo culturing and cryopreservation can affect methylation profiles and gene expression at imprinted loci . EpigeneAnother example of an epigenetic-associated developmental disorder is LOS. LOS is an overgrowth disorder in domesticated ruminants bearing phenotypic similarities to Beckwith–Wiedemann syndrome , and is characterized by excessive birth weight, enlarged tongue, umbilical hernia, enlarged internal organs and hypoglycemia . Both BWH19 gene and the paternally expressed/maternally imprinted IGF2 gene, which encodes a fetal mitogen. Studies have shown that both genes are under the control of a single ICR that is unmethylated on the maternal allele and methylated on the paternal allele. In mice, binding of the CCCTC-binding factor (zinc finger protein), CTCF, to the non-methylated ICR inhibits maternal expression of Igf2 by preventing interaction of its promoter with downstream enhancers; however, the H19 promoter has access to the downstream promoters resulting in its maternal expression , which is concomitant with biallelic expression of KCNQ1OT1 and downregulation of CDKN1C, a negative regulator of cell proliferation  at two ICRs, that independently regulate the expression of two clusters of reciprocally imprinted genes on human chromosome 11p15, are associated with BWS . One imppression . The secnal copy . Paternal allele . In humaferation , 2010.H19-IGF2 and the KCNQ1OT1-CDKN1C loci and ART-generated fetuses, especially in offspring displaying LOS or which had died shortly after birth —a mode of non-Mendelian inheritance termed ‘polar overdominance’  gene within the imprinted DLK1-DIO3 gene cluster on ovine chromosome 18  genes .The complex interplay between different epigenetic and genetic mechanisms in regulating mammalian imprinted gene expression is aptly illustrated by the callipyge phenotype in sheep, which is responsible for a ~30% increase in skeletal muscle (most notably at the hindquarters), a corresponding ~8% reduction in fat content and improved feed efficiency . This phminance’ . The calosome 18 . This cl+/patC) display overexpression of the paternally expressed DLK1 and PEG11 protein-coding transcripts in skeletal muscle tissue relative to non-callipyge animals . In contrast, individuals that inherit the callipyge mutation on the maternal chromosome  display upregulation of maternal long ncRNAs and miRNAs in cis relative to wild-type  and callipyge animals  gene—the encoded product of which removes acetyl groups from histone proteins resulting in increased chromatin condensation and repressed transcription—has also been observed in callipyge animals relative to non-callipyge animals suggesting a role for histone modification in regulation of expression at the DLK1-DIO3 imprinted cluster -mediated cleavage of PEG11 transcripts .A recently refined model of the polar overdominance observed for the callipyge phenotype suggests that stically , 2014. Ihenotype . Furthernscripts ; however of DLK1 . It has DLK1-DIO3 imprinting domain) in regulating complex phenotypes. Indeed, the callipyge phenotype demonstrates that the mammalian ‘hard-wired’ genome is not the single repository of regulatory information that has phenotypic effects, but that the ‘soft-wired’ epigenome—the collective term for epigenetic mechanisms that regulate gene expression—also has an important role in determining phenotype  and epigenetic mechanisms  transcriptional repressor. Animals inheriting a sire-derived ‘A’ nucleotide display a three-fold increase in IGF2 expression in post-natal muscle relative to those animals inheriting a sire-derived ‘G’ nucleotide, which results in increased muscle mass and a corresponding reduction in body fat  are subject to imprinting, several of these have been shown to have major effects on complex mammalian phenotypes. In mice, for example, studies have demonstrated the contribution of imprinted genes to variation in adiposity and body weight, muscle traits, metabolism, and disease susceptibility and resistance to infectious disease . Genetic content . This SNbody fat .Collectively, these studies highlight the important role played by epigenetically regulated loci in contributing to heritable phenotypic variation, making them attractive targets for candidate gene association studies and also inclusion in genome-wide scans that incorporate imprinting/parent-of-origin effects in domestic livestock species.a priori based on their biological function  has resulted in remarkable rates of genetic improvement and has led to the development of several elite high-performance livestock populations, most notably the Holstein-Friesian dairy breed. Initially, systematic science-based improvement of domestic livestock used quantitative genetic evaluation of phenotypic data generated from managed populations or pedigrees, such that individual animals displaying increased performance  for desired traits were selected as candidate parents for subsequent generations. In the last two decades, however, there has been a paradigm shift in animal genetic improvement research involving data generated from molecular genetic markers, which has been concomitant with advances in genome sequencing and genotyping technologies, bioinformatics and biostatistics. SNPs and simple tandem repeat (STR) loci represent two of the most abundant DNA sequence polymorphisms within the mammalian genome and are the predominant genetic markers used in genotype-phenotype association studies. The methods that form the basis of these programs involve testing for associations between measured traits  and genetic marker genotypes. The genetic markers used can be distributed across the whole genome  or be situated within or proximal to genes selected for analysis 2 progeny of experimental cross-bred pig populations . Collectively, these results suggest that the GNAS and PEG3 loci play an important role in bovine growth and development, fertility and health  distributed across 15 bovine autosomes influencing growth and carcass traits; two of these QTL encompassed the bovine imprinted G3 genes . Subsequd health .IGF2 and IGF2R genes and meat quality, milk production and growth traits in beef and dairy cattle populations  with neighboring variants in the proximal INS (insulin) gene, which contributes to the regulation of lactation . However, for many imprinted loci, transcriptional silencing is only partial .The documented biological roles of imprinted genes in regulating mammalian growth and development together with the accumulating genotype–phenotype association data in domestic livestock species/populations, suggests that loci subject to genomic imprinting represent an important reservoir of genetic variation that may be exploited in selective breeding programs . However partial , which cFigure 3).Furthermore, different forms of parent-of-origin effects can also generate phenotypic differences between genotype classes at the same locus. For example, polar overdominance, as exemplified by the callipyge phenotype, can result in phenotypic differences between reciprocal heterozygotes; in addition, under a model of polar overdominance, one of the heterozygous states will display a phenotypic value greater than all three other genotypes, which themselves show no differences in phenotypic values. Conversely, a model of polar underdominance, whereby one of the two reciprocal heterozygotes has a phenotypic value less than all three other phenotypically equivalent genotypes, has been reported in mice . FinallyFor many industrialized countries, the original MAS concept for livestock breeding has been supplanted by ‘genome-wide selection’ or ‘genomic selection’ using 1000s of genetic markers distributed across the genome. Genomic selection in livestock was originally proposed by Genomic selection strategies are largely unconcerned with knowledge of the genes and causal variants that directly affect phenotypes . HoweverThe phenomena outlined above, demonstrate that imprinting parent-of-origin effects may complicate traditional quantitative genetic models used in phenotypic association studies. This review illustrates that imprinted gene expression can have a major effect on phenotypic traits in domestic livestock populations. Furthermore, imprinting is an important factor to consider in the models used for future the genetic improvement of domestic livestock for those genomic regions where imprinted gene expression is known to occur and to affect economically important traits included in the selection index.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.) and from Science Foundation Ireland Investigator Grants awarded to DEM (Nos: SFI/01/F.1/B028 and SFI/08/IN.1/B2038) and CS (Nos: 02/IN.1/B49 and 08/IN.1/B1931).We gratefully acknowledge the funding support from the University of Connecticut, the Irish Department of Agriculture, Food and the Marine Research Stimulus Program (project numbers RSF-06-406, RSF-06-0353, and RSF-06-0409


Epigenetic mechanisms play a role in human disease but their involvement in pathologies from the central nervous system has been hampered by the complexity of the brain together with its unique cellular architecture and diversity. Until recently, disease targeted neural types were only available as postmortem materials after many years of disease evolution. Current in vitro systems of induced pluripotent stem cells (iPSCs) generated by cell reprogramming of somatic cells from patients have provided valuable disease models recapitulating key pathological molecular events. Yet whether cell reprogramming on itself implies a truly epigenetic reprogramming, the epigenetic mechanisms governing this process are only partially understood. Moreover, elucidating epigenetic regulation using patient-specific iPSC-derived neural models is expected to have a great impact to unravel the pathophysiology of neurodegenerative diseases and to hopefully expand future therapeutic possibilities. Here we will critically review current knowledge of epigenetic involvement in neurodegenerative disorders focusing on the potential of iPSCs as a promising tool for epigenetic research of these diseases. A major challenge to model neurodegenerative disorders has been the inaccessible nature of the specific neural cell types targeted by disease which are usually available only in postmortem state. Recent somatic cell reprogramming protocols have contributed to overcome such a difficulty. Reprogramming of somatic cells to pluripotency can be currently achieved by different methods including somatic cell nuclear transfer (SCNT), fusion of somatic and pluripotent cells, included ectopic expression of defined sets of pluripotency transcription factors (TF) in adult somatic cells to generate induced pluripotent stem cells (iPSCs), and direct reprogramming of adult somatic cells to induced neurons (iN) by empirically determined cocktails of neurogenic factors –5. In neParkinson disease (PD) is a neurodegenerative disorder associated with the progressive loss of dopaminergic neurons (DAn) in the substantia nigra pars compacta (SNpc) resulting in the cardinal motor symptoms of bradykinesia, rigidity, tremor, and postural instability , 9. Due Whereas only 5–10% of cases with neurodegenerative disorders such as AD, PD, or ALS are Mendelian disorders caused by pathogenic mutations in disease-associated genes, the vast majority of cases are considered sporadic resulting from the complex interplay of genetic risk factors and largely unknown environmental conditions , 20. HowHere we will review the potentiality and current challenges of iPSC-based models to investigate epigenetic regulation of neurodegenerative disorders in the context of other existing patient specimens and disease models.There are three major categories of epigenetic modifications including DNA methylation, histone posttranslational modifications, and noncoding RNAs (ncRNAs) which encompass microRNAs (miRNA), small noncoding RNAs (sncRNA), and long noncoding RNAs (lncRNA) . The fir“Epigenetics” can be defined as mitotically and/or meiotically heritable changes in gene expression which occur without changes in the DNA sequence. These epigenetic modifications mediate the execution of cell-type specific genomic programs activated in response to interoceptive as well as environmental stimuli. Conceptually, epigenetic mechanisms include long-term silencing, transcription, posttranscriptional RNA processing, translation, X chromosome inactivation, genomic imprinting, DNA replication and repair, and the maintenance of genomic integrity . These e3) to the 5′ position resulting in 5-methylcytosine (5mC). This reaction is catalyzed by specific enzymes termed de novo DNA methyltransferase (DNMT) and it occurs at expense of ATP and S-adenosylmethionine as methyl donor.DNA methylation is an important regulatory mechanism of gene expression in eukaryotes. In mammals and humans DNA methylation mostly affects the cytosine (C) base when it is followed by a guanine (G). These CpG sites can be clustered in the so-called “CpG islands” (GCI) but DNA methylation can also be present in non-CG contexts . Both CpG and non-CG methylation are detected in the mouse and human brain , 60 but DNMT is expressed during neural development and in adult brain in a tissue- and cell-specific manner including areas of active neurogenesis  and adulOther enzymes such as the oxidoreductases of the Ten-Eleven Translocation (TET) family are responsible for the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) . MembersIn addition to DNA methylation, the conformation of the chromatin is also regulated by histone posttranslational modifications. In eukaryotic chromatin, the genomic DNA is packed around histone proteins forming the so-called nucleosome, which consists of 147 base pairs of DNA wrapped around a histone octamer containing 2 copies each of the core histones H2A, H2B, H3, and H4 . The nucThere are more than 100 different histone posttranslational modifications which can affect different histone amino acid residues including lysine (K), arginine (R), serine (S), threonine (T), and glutamate (E) . Of thesε-amino groups of lysine residues of histones neutralizes their positive charge thereby relaxing chromatin structure [For instance, methylation of H3K4 can inhibit the binding of HDAC therefore favoring acetylation whereas acetylation of H3K18 facilitates the engagement of HAT . Methylatructure  commonlytructure . Per contructure . Histonetructure  . The spWith the exception of HD which is a largely monogenic disease, most of the patients with other neurodegenerative disorders such as AD, PD, or ALS are considered sporadic or idiopathic. In these cases, the disease is expectably driven by the cumulative and/or synergistic effect of genetic risk variants together with largely unknown environmental conditions –153 whosEnvironmental conditions include the exposition of an individual to drugs, toxins, metabolites, or other external stimuli. However, the environment can also be considered as the single cell microenvironment encompassing external cellular stimuli, inflammatory responses, or signaling from nearby cells. Yet both of these macro- and microenvironmental conditions have been shown to contribute to the modification of the epigenome by ultimately inducing interoceptive cell signaling cascades . These eIn principle, iPSC models are ideally suited for drug development due to their limitless self-renewal capacity allowing the production of large quantities of cells and to their high differentiation properties into disease-specific cell types. However, iPSC models have not yet been extensively used in large-scale drug screenings in neurodegenerative disorders due to the clonal variation associated with stochastic gene mutation  and alsoEpigenetic drugs currently explored in human disease models include most prominently histone deacetylation inhibitors, DNA methylation inhibitors, and histone acetylation activators . ConceptSpecifically in PD, other studies illustrate that epigenetic drugs can be useful to modulate disease aspects related to epigenetic deregulation. Thus, a recent study in a human DAn model and mouse organotypic brain slice cultures has shown that the treatment with the HDAC inhibitor sodium butyrate (NaBu) upregulates the expression of oxidative stress-sensitive protein kinases (PKCs) and augments DAn apoptotic cell death . Since tIn summary, let alone that epigenetic research of neurodegenerative disorders is an emerging field, the identification of epigenetic therapeutic targets for AD, PD, or ALS is at its infancy. In this scenario, iPSC-based models may be useful not only to detect epigenetic changes associated with these diseases but also to explore the ability of candidate epigenetic drugs to correct epigenetic alterations and to design novel therapeutic strategies. To date, this goal seems to be technically feasible only for small or medium scale drug studies or for very specific drugs. Yet future patient-specific iPSC-based systems using improved cell reprogramming protocols are expected to pave the way out for epigenetic research ultimately intending the delivery of personalized epigenetic therapies.Although recent works have provided evidence about the involvement of epigenetic changes in neurodegenerative diseases and supported the use of iPSC-derived neural models to explore epigenetic alterations in these disorders, several questions remain to be answered.A first question is whether iPSC-derived neurons really mimic the epigenetic and expression features occurring in the affected brain areas of the patients. Epigenetic changes relevant to neurodegenerative diseases are expected to reflect the complex interaction of genetic background, environmental factors, and gene expression in the context of the brain. Accordingly, it would be necessary to assess whether iPSC-derived neurons obtained from patient fibroblasts do faithfully recapitulate the molecular events occurring in the complex cell microenvironment of the brain thus representing good models of disease. A direct approximation would encompass the comparison and determination of the level of coincidence of epigenetic marks in iPSC-derived neurons and in their patient brain cell counterparts as investigated in a recent study . HoweverSecond, assuming that iPSC systems represent good disease models for neurodegenerative diseases, a new venue would be open to explore epigenetic changes in specific genetic loci by using high resolution whole-genome methodologies, including commercially available methylation arrays, whole-genome bisulphite sequencing, or whole-genome histone marks analyses in patients and controls. In order to implement our comprehension of disease mechanisms, these data could be analyzed by integrative biology methods interrogating the epigenome, the transcriptome, and the known risk genetic loci detected in genomewide association studies (GWAS) –197. In Third, assuming that these neurons recapitulate causative epigenetic alterations, a new opportunity will be offered to explore the capacity of different epigenetic-modifying drugs to modulate pathologic epigenetic patterns and then observe their respective functional effects and establish cause-effect mechanisms. In this way, iPSC-derived neurons could represent a valid tool to test potential therapeutic drugs reversing the pathological epigenetic changes and to monitor disease-relevant processes such as alpha-synuclein or amyloid deposition, autophagy alterations, or mitochondrial dysfunction. In summary, iPSC-derived neurons models have promising perspectives and open new avenues for biological mechanistic studies, drug discovery and testing, and clinical therapy of neurological disorders related with epigenetic changes.


Gasterosteus aculeatus) is a well-known model organism. Through identification of genetic variations under parallel selection, i.e., variations shared among related but independent lineages, evidence of positive selection is obtained. In this study, we investigated whole-genome resequencing data from the marine and freshwater groups of three-spined sticklebacks from diverse areas along the Pacific and Atlantic Ocean coastlines, and searched for GCNVs under parallel selection.Understanding the genetic basis of adaptive evolution is one of the major goals in evolutionary biology. Recently, it has been revealed that gene copy number variations (GCNVs) constitute significant proportions of genomic diversities within natural populations. However, it has been unclear whether GCNVs are under positive selection and contribute to adaptive evolution. Parallel evolution refers to adaptive evolution of the same trait in related but independent lineages, and three-spined stickleback  contains supplementary material, which is available to authorized users. Analyss e.g., , 9)..cis-elemThese approaches focused on positive selection on variations due to nucleotide substitutions. However, it has recently been revealed that copy number variations (CNVs), or gains or losses of DNA segments, constitute a significant proportion of genomic diversity –15. BecaGasterosteus aculeatus) are an excellent system to investigate parallel evolution [parallel selection on the globally shared, standing SNVs in the same genes in different freshwater populations, providing strong evidence that positive selection on these SNVs contributed to the adaptive evolution toward the freshwater environments [Parallel evolution, which is the adaptive evolution of the same trait in related but independent lineages, can provide evidence of positive selection, because genetic drift is unlikely to produce concerted changes in independent lineages . The marvolution . This spvolution , 23. Preronments –26. Receronments  reportedWe downloaded whole-genome resequencing data of 10 marine and 10 freshwater individuals of three-spined sticklebacks  from NCp < 0.05) for each mapping option. Collectively, these results suggested that the 24 GCNVs were likely due to parallel selection. Note that the 2.3× coverage of the resequencing data [Twenty-four genes showed significant differences in the numbers of mapped reads between both groups Figure . Among ting data  would haneurexophilin and PC-esterase domain family member 3 (NXPE3) overlapped with a region that was reported as a CNV in a marine group of three-spined sticklebacks [sulfotransferase (SULT), NOD-like receptor (NLR), apolipoprotein L (APOL), kinesin family (KIF), and myosin heavy chain (MyHC). The finding that the identified GCNVs included genes in multigenic families was consistent with the idea that GCNVs of multigenic family genes are more likely to occur than those of single-copy genes. This is because, fatal effects due to copy-number changes of multigenic family genes tend to be less than those of single-copy genes [APOL[KIF[SULT[MyHC in fish [Among the identified GCNVs, klebacks . In addipy genes . It woules [APOL, KIF[31IF[SULT in prima in fish .An important characteristic of the 24 GCNVs likely under parallel selection was that they frequently appeared at close locations on the genomes  and two genes that were homologous to MyHC . A previous study showed parallel divergences between littoral and pelagic phenotype pairs of three-spined stickleback MHC genes, which are key genes in the immune system and would be associated with parasite communities in each habitat [Among the 24 GCNVs likely under parallel selection, larger gene copy numbers were more frequently associated with the freshwater group   was also supported by at least one within-read-length SNV position pair in three individuals of the marine group . This gene was reported to be overexpressed in the brooding tissue of pregnant specimens of a species in genus Syngnathus[NXPE3 within marine populations [NXPH3 is a neuropeptide-like molecule that functions in brain [NXPE3 copy numbers in the freshwater group might have been associated with their anadromous behavior [The primates . Anotherresponse . The dupngnathus, which bulations . NXPH3 iin brain , and neubehavior .APOL2 and ENSGACG00000003408 were higher in the freshwater group than those in the marine group highly significantly (p < 0.005 after Bonferroni correction) under the short photoperiod condition. The short photoperiod condition resembled winter, thus these genes might have contributed to the fitness though the overwinter survival [If the two strongly supported freshwater-increased GCNVs actually contributed to the parallel evolution of the three-spined sticklebacks, the amount of transcription products of these genes should be important for the adaptation. Thus, we analyzed microarray data of gills of three-spined sticklebacks in marine and freshwater groups under the short and long photoperiod conditions , and evasurvival .In this study, we showed the possibility that GCNVs underwent positive selection in the parallel evolution of the three-spined sticklebacks and had a role in the adaptation to the freshwater environment. It would be notable that many CNVs were found in a marine population of three-spined sticklebacks , which sThe three-spined stickleback genome sequence (BROADS1.56) and the annotated gene models were taken from the Ensembl database . The geet al.[A resequencing dataset of 10 marine and 10 freshwater individuals was previously generated using an Illumina Genome Analyzer II , which yielded approximately sixty million reads (approximately 2.3×) per individual . The acThe sequenced reads from each individual were mapped to the stickleback genome using the Bowtie 0.12.8 software   were counted using the ‘intersectBed’ command in bedtools . Becausep value based on a permutation test. We randomly reallocated the 10 freshwater and 10 marine individuals into two groups 10,000 times, performed the same analyses, and obtained the null distribution of numbers of GCNVs.To confirm that the number of identified GCNVs under parallel selection was significantly larger than that expected by chance , we calculated an empirical If the identified GCNVs involved gene duplications or multiplications, three or more different allelic sequences should be observed within the gene in each individual of each group, because three or more different allelic sequences cannot originate from a diploid genome. Thus, we examined whether three or more different allelic sequences were observed in the identified GCNVs Figure C.within-read-length SNV position pairs). The numbers of different nucleotide pairs for each of the within-read-length SNV position pairs were counted, where each nucleotide pair was supported by multiple reads. Finally, we selected GCNVs that showed three or more different nucleotide pairs in at least three individuals of either group.For each of the identified GCNVs, SNVs were called by applying the SAMtools/BCFtools pipeline  to the rFor each GCNV likely under parallel selection, we obtained functional annotations of the gene from the Ensembl database. If the functional annotations were unavailable, BLASTX searches  against http://cibex.nig.ac.jp) with the accession number CBX139. Two marine and freshwater datasets were treated as biological replicates. If multiple probes were mapped to one transcript, the median signal intensity of these probes was used. After removing intra-gene probes, genes with significant expression-value differences between the marine and freshwater groups were identified using the eBayes method in the limma package [Microarray data of gills of two families of pure marine and pure freshwater crosses under short and long photoperiods  were dow package .Additional file 1: Table S1: Summary of the resequencing datasets of 10 marine and 10 freshwater sticklebacks. (PDF 49 KB)Additional file 2: Table S2: Numbers of SNV pairs in which three or more allelic sequences were observed for each GCNVs. (PDF 48 KB)


In the last decade, it has become apparent that toll-like receptor (TLR) signaling can play an important role in ovarian cancer (OC) progression. Interestingly, TLR activation in immune cells can help activate an anti-tumor response, while TLR signaling in tumor cells themselves is often associated with cancer-promoting inflammation. For example, it has been shown that TLR activation in dendritic cells can result in more effective antigen presentation to T cells, thereby favoring tumor eradication. However, aberrant TLR expression in OC cells is associated with more aggressive disease  and has also been implicated in resistance to mainstream chemotherapy. The delicate balance of TLR activation in the tumor microenvironment in different cell types altogether help shape the inflammatory profile and outcome of tumor growth or regression. With further studies, specific activation or repression of TLRs may be harnessed to offer novel immunotherapies or adjuvants to traditional chemotherapy for some OC patients. Herewith, we review recent literature on basic and translational research concerning therapeutic targeting of TLR pathways for the treatment of OC. Ovarian cancer (OC) has the most devastating death rate of gynecological cancers with only 44% of women surviving 5 years after diagnosis –4. The lAs aggressive OC often correlates with an immunosuppressive leukocyte population in the tumor environment, efforts to modulate these cells to potentiate an anti-tumor immune response are ongoing –12. Of pToll-like receptors expressed in leukocytes  serve a crucial function at the start of the immune response, activating numerous pro-inflammatory pathways resulting in cytokine secretion, and further activation of immune cells, including the adaptive immune response –23. It iIn 2005, Adams et al. first described the rationale for TLR3 agonist therapy for advanced OC . In 2009Earlier this year, Bellora and colleagues demonstrated that TLR activation in tumor-associated macrophages (TAMs) obtained from OC patients resulted in a shift from an M2 to an M1-polarization phenotype . This isRecently, a TLR8-specific agonist, VTX-2337 , entered Phase II clinical trials for OC patients with chemotherapy-resistant and recurring disease . The PhaTLR9 ligands have similarly received interest as potential treatments for OC, specifically in combination with other immunomodulatory agents . In 2009In 2009, Zhou and colleagues reported on the expression of TLRs in human ovarian tissue samples, including both normal and neoplastic  tissue . It was The effects of TLR signaling in cancer cells have been extensively investigated for TLR4, perhaps the best-studied PRR. In 2005, Huang and colleagues reported on its expression and activation in numerous mouse cancer cell lines . They dein vitro scratch essays revealed the increased migratory capabilities of tumor cells expressing higher TLR9 levels (in both ovarian and breast tumor cells). It was also reported that higher TLR9 expression was more common in poorly differentiated tumors (hormone-receptor-negative tumor cells were found to have more TLR9); thus, these tumors have fewer targeted therapeutic options. In addition, it was found that OC patients with metastatic disease had elevated levels of hypo-methylated DNA (TLR9 ligand) in their serum. Further, the authors offered even more evidence of the detrimental effects of TLR9 signaling in OC cells, showing the co-localization of TLR9 and its ligand, as well as NF-κB activation, which was proportional to the levels of TLR9 expression. Significantly, NF-κB appears to be constitutively activated in numerous cancer types, whereby it is associated with highly aggressive disease and poor disease outcome, highlighting the potential of TLR targeting to inhibit this important inflammatory switch in tumor cells  has been associated with disease aggressiveness and poor clinical outcome . Berger or cells , 49.In addition to the PAMPs that can activate TLRs , endogenous ligands for these molecules have also been identified . For insThe last decade of research on TLR activity and its implications in OC progression indicate that inhibition of certain TLRs in cancer cells and/or TLR stimulation in immune cells may be of therapeutic benefit in some patients. While immune activation by means of TLR stimulation can generate an anti-cancer effect, the cytokine profile following TLR activation in tumor cells typically favors an immunosuppression that can potentiate immune-tolerance and promote angiogenesis, furthering tumor growth. Figure 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.


Testicular (germ cell) cancer is a model of a chemocurable malignancy and tends to have a favourable prognosis even in advanced stages due to exquisite sensitivity to platinum-based chemotherapy. However, both acute and longer-term toxicities of multiagent chemotherapy remain significant as causes of morbidity, very occasionally mortality, and impaired quality-of-life. Here, we report a case of acute cerebral venous sinus thrombosis occurring within 10 days of chemotherapy initiation in a young patient without comorbidities, whose only predisposing factors were malignancy, chemotherapy, and perhaps mild dehydration. The clinical presentation was also unusual with headache of moderate severity only without focal or global neurologic deficits. We suspect that cisplatin may have had direct vasculotoxic effects. The patient recovered fully after short-duration anticoagulation but oncologists must remain aware of unusual and unpredictable complications of cytotoxic treatment.  Testicular cancer is the commonest solid malignancy in young men and since the development and application of cisplatin-based cytotoxic chemotherapy  in the 19/L) and neutropaenia (0.0 × 109/L), and moderate thrombocytopaenia (53 × 109/L) in keeping with recent chemotherapy administration. On the second day of admission he had a brief and self-terminating episode of loss-of-consciousness. Unenhanced CT scan of the head showed hyperdensity in the distal aspect of the superior sagittal sinus close to the torcula  nonseminomatous germ cell tumour was undergoing multiagent cytotoxic chemotherapy with curative intent. He had no significant medical comorbidity but was a cigarette smoker and moderately overweight. He had undergone right inguinal orchidectomy which revealed predominant (99%) embryonal carcinoma  with a minor (1%) malignant teratoma intermediate component. Staging CT scan showed lymphadenopathy in the retroperitoneum, pelvis , superior mediastinum, and left axilla. Four days after the first dose of intramuscular bleomycin as part of the bleomycin, cisplatin, and etoposide (BEP) chemotherapy regimen, he complained of recent onset headache over a 7–10-day period. He described it as a generalised, moderately severe constant “pressure” headache that was worse on waking in the morning and exacerbated by coughing and straining. There were no other neurologic symptoms. He had also vomited on two occasions in the first 5 days following cisplatin and had several episodes of loose stools. On examination, there was no focal neurologic deficit and he was alert and orientated. Laboratory investigations showed mild hyponatraemia (133 mM), raised serum urea (7.8 mM), and profound leucopaenia  although this should be considered prior to anticoagulation in unprovoked CVST. He had no family history of recurrent venous thromboembolism or miscarriage in female relatives. CVST has been previously described as occurring in various malignancies including cervical cancer [Cerebral venous sinus thrombosis (CVST) is an uncommon condition that typically affects young females with obvious risk factors such as pregnancy or oral contraceptive pill use or patients with coexistent local inflammatory pathology or inherited thrombophilias. It is a rare cause of cerebrovascular disease and is probably underrecognised , 8. In ol cancer , colorecl cancer , and Ewil cancer . In keepl cancer , perhapsAlthough venous thromboembolism (VTE) is common in patients with malignant disease, particularly those undergoing chemotherapy, it typically affects the lower-limb deep veins and/or pulmonary arterial tree leading to well-recognised and appreciated clinical syndromes. It remains important for the practising oncologist to be vigilant for unusual sites of venous thrombosis and to investigate and treat these in an expedient manner. We note that primary prophylaxis against VTE in cancer patients, whilst a reasonable consideration, has not been proven to improve overall survival in the setting of newly diagnosed lung cancer with an increase in bleeding events .In terms of investigation, in this case we proceeded directly to cerebral imaging on the basis of the symptoms and clinical scenario, although measurement of plasma D-dimer/fibrin degradation product levels may have a role particularly in excluding CVST in low-moderate risk patients . This caOur patient had a favourable outcome with the minimal duration of 3 months of anticoagulation with radiological evidence of thrombus resolution and disappearance of symptoms. However, there remains uncertainty as to the optimal duration of anticoagulant treatment in this rare disease; international guidelines suggest 3–12 months of therapy individualised for each patient . The rolIn conclusion, although the outlook for patients with advanced testicular cancer is highly favourable, chemotherapy remains potentially hazardous as illustrated in this case and a high index of suspicion is required to assess for and manage complications to allow safe delivery of treatment.


Olfactory ensheathing cell (OEC) transplantation is a candidate cellular treatment approach for human spinal cord injury (SCI) due to their unique regenerative potential and autologous origin. The objective of this study was, through a meta-epidemiologic approach, (i) to assess the efficacy of OEC transplantation on locomotor recovery after traumatic experimental SCI and (ii) to estimate the likelihood of reporting bias and/or missing data. A study protocol was finalized before data collection. Embedded into a systematic review and meta-analysis, we conducted a literature research of databases including PubMed, EMBASE, and ISI Web of Science from 1949/01 to 2014/10 with no language restrictions, screened by two independent investigators. Studies were included if they assessed neurobehavioral improvement after traumatic experimental SCI, administrated no combined interventions, and reported the number of animals in the treatment and control group. Individual effect sizes were pooled using a random effects model. Details regarding the study design were extracted and impact of these on locomotor outcome was assessed by meta-regression. Missing data (reporting bias) was determined by Egger regression and Funnel-plotting. The primary study outcome assessed was improvement in locomotor function at the final time point of measurement. We included 49 studies  in the final analysis. The overall improvement in locomotor function after OEC transplantation, measured using the Basso, Beattie, and Bresnahan (BBB) score, was 20.3% (95% CI 17.8–29.5). One missing study was imputed by trim and fill analysis, suggesting only slight publication bias and reducing the overall effect to a 19.2% improvement of locomotor activity. Dose-response ratio supports neurobiological plausibility. Studies were assessed using a 9-point item quality score, resulting in a median score of 5 (interquartile range [IQR] 3–5). In conclusion, OEC transplantation exerts considerable beneficial effects on neurobehavioral recovery after traumatic experimental SCI. Publication bias was minimal and affirms the translational potential of efficacy, but safety cannot be adequately assessed. The data justify OECs as a cellular substrate to develop and optimize minimally invasive and safe cellular transplantation paradigms for the lesioned spinal cord embedded into state-of-the-art Phase I/II clinical trial design studies for human SCI. This meta-analysis study examines the effects of transplanting olfactory ensheathing cells in rodents with experimental spinal cord injury, finding evidence for significant recovery and identifying aspects of the procedure that influence the effect size. Spinal cord injury converts into a debilitating disease affecting millions of chronic patients worldwide. Despite increased molecular knowledge over the last decades, no causal pharmacological or cellular therapy has proven effective so far. Due to their unique regenerative capabilities and their autologous origin, olfactory ensheathing cells (OECs) constitute an appealing candidate for topical cell transplantation. In contrast to few and heterogeneous experimental reports of OEC transplantation after spinal cord injury in humans, a considerable number of preclinical studies have been conducted applying OEC transplantation in rodent models. We set out to conduct a systematic review and meta-analysis to assess preclinical efficacy of OEC transplantation. We detected a significant overall increase of functional neurological recovery in animals after OEC transplantation compared to the control group. This effect was not distorted by publication bias. We identified several specific hallmarks of the cell transplantation procedure that determine the effect size of the transplantation. Our findings delineate conditions for optimized OEC transplantation into lesioned spinal cords and its relevance for effective translation to human trials. Cellular transplantation strategies applying different cellular sources have been tested in experimental spinal cord injury (SCI) models as a possible treatment to propagate functional recovery –3. RestoThe viability as autologous graft and genetic stability of OECs supports their feasibility for clinical translation. OECs comprise a unique and highly specialized cell type, physiologically located at the border between the central nervous system (CNS) and peripheral nervous system (PNS), since the olfactory system is part of both the PNS and CNS .After olfactory nerve injury, olfactory receptor neurons located in the olfactory epithelium (PNS) can extend axons that enter the olfactory bulb (CNS), thus representing a highly specialized aspect of physiological axonal regeneration in the mammalian CNS –9. This p-value of about 0.05  score  for eval2 = 96.4%, 62 experiments, 1,164 animals) when including various test paradigms . Meta-regression showed several aspects of study design that were significantly associated with different observed efficacies:Overall, OEC transplantation after traumatic SCI improved locomotor recovery by 23.6%  was associated with greater efficacy than subacute (1–2 wk) or chronic time windows (4–8 wk) after SCI : Microsurgical resection of fibrotic scar tissue from the lesion site was associated with a reduced degree of neurological recovery. In particular, in the chronic phase, surgical resection after a scar has been formed was detrimental, whereas manipulations during the acute phase appear less detrimental , only very low effect sizes were observed , the efficacy was very low , relating to a maximum score value of 9. All studies were published in peer-reviewed journals, in which 28 studies reported blinded assessment of outcome and 25 did not. Only 17 studies (32%) out of 53 reported random allocation to treatment or control group. None of the studies made use of sample size calculation, and only one used blinded induction of injury. None of the single study quality items accounted for a significant amount of heterogeneity.The combined 9-point item quality score revealed a trend for the relationship between effect size and study quality, but showed no statistical significance. Studies with lower-quality score items correspond with higher effect sizes, whereas higher quality studies associate with lower effect size .We assessed the dataset for missing data by applying funnel plotting and Egger regression analysis . PublicaTrim and fill analysis was used to identify theoretically missing experiments based on the funnel plot’s symmetry. Only one missing experiment was imputed, suggesting that present publication bias for this cohort was only minor, adjusting the effect size only marginally from 20.3% to 19.2% . The finAlthough there is reason to believe that studies at risk of bias might give overstatements of efficacy, it is also plausible that such studies may give less precise estimates. Therefore, in a post hoc analysis, we investigated whether studies with a higher quality score (i) were closer to the overall calculated effect size or (ii) had higher precision (1/SE) . The modThis systematic review and meta-analysis of preclinical animal literature identified 49 studies of olfactory ensheathing cell transplantation reporting 23.6% improvement of functional outcome. Data from 1,164 animals comprising 62 experiments were stratified for neurobiological and study characteristics, which accounted for significant proportion of in-between study heterogeneity. Motor-score sub-analysis was confined to studies including assessments of neurobehavioral recovery applying the most commonly used BBB-score characterized by smaller effect sizes (20.3%) compared to other measures of motor recovery . Statistical techniques to detect and correct for publication bias revealed a slight overestimation of effect sizes due to theoretically missing experiments resulting in a corrected estimated efficacy of 19.2%. Besides minimizing variability to enable the most accurate reproduction , an altBulb- and mucosa-derived OECs differ substantially, for example, with regards to their proliferative ability over time ,29. OECsDespite migratory properties and the ability to intermingle with host scar-forming cells, the localization of transplantation matters. Injection to rostral and caudal parenchyma juxtaposed to the lesion site is significantly more effective than to the lesion core. This is in line with the proposed underlying neurobiological mechanisms such as neuroprotection and the propagation of neuroplasticity and remyelination, which do not apply to the pan-necrotic lesion core. Multiple injections with smaller volumes are associated with elevated locomotor effects compared to a reduced number of injections, which in turn require larger cell numbers and injection volumes. Considering technical aspects of transplantation, it appears that neurotoxicity generated by injection into the spinal cord itself is comparatively low (likely as long as no vessel is affected) and depends largely on the amount of volume being injected. The results favour multiple injections over large cell deposits through singular injection (declining benefit-risk ratio with increasing injection volume). The data also suggest that the toxicity of invasive transplantation is largely determined by the injected volume and the attributable hydrodynamic dissection pressure . In brien = 2 out of 566). Reported adverse effects included fever, mild anemia, syringomyelia, cerebrospinal fluid leakage, aseptic meningitis, and sensory and motor deterioration. The safety being claimed in some reviews summarizing experimental OEC mode of action was challenged by a recent clinical case report identifying an intramedullar tumor formation 8 y after spinal mucosal OEC transplantation presenting with progressive pain [A recent meta-analysis summarized 10 treatment series reporting on 1,193 chronic SCI patients and demonstrated an improvement rate of 39% on the American Spinal Injury Association (ASIA) impairment scale after chronic SCI  and OEC ive pain . The tumThe following limitations apply to this study. Our analysis depends on the validity of locomotor recovery scales used, which has been questioned . HoweverDue to the unique properties of fostering a plethora of neurobiological repair mechanisms including axon sprouting, myelination, and neuroprotection, autologous OECs are considered a candidate cellular source for transplantation into the lesioned spinal cord. This systematic meta-analysis reports a substantial overall effect of OEC transplantation after experimental SCI. The data suggests more effective OEC transplantation paradigms, (i) when being derived from the olfactory bulb compared with mucosa-derived OECs, (ii) when injected to rostral-caudal parenchyma compared with the injury epicenter, and (iii) when being fractionated, allowing for smaller injection volumes. Invasive surgical resection of the fibrotic scar at the lesion site should be avoided. Publication bias was minimal and affirms the translational potential in terms of preclinical efficacy. Safety issues cannot be addressed here sufficiently, given that rodent models rarely study long-term follow-up. Therefore, late complications in humans cannot be addressed sufficiently. At present, the ideal cell for transplant-mediated CNS repair has not been identified. Based on validated efficacy, OECs qualify as autologous cell source to elucidate and optimize cell invasive transplantation paradigms after human SCI as a component for escalated treatment concepts, e.g., by applying phase II trials with informative secondary endpoints aiming to characterize treatment responders and patients prone to develop transplantation-associated complications.http://www.dcn.ed.ac.uk/camarades/files/PROSPERO%20Protocoll%20OEG.pdf). The methodology and statistical approach is described in greater detail elsewhere [The study protocol was finalized in advance of any data collection and is accessible online : (olfactory OR olfactory bulb OR olfactory lamina propria OR olfactory ensheathing cells) AND . Search results were limited to animals. The animal filter was modified for the search in PubMed . Two invStudies were included if they reported the effects of OEC transplantation in animal models after spinal cord injuries, including contusion, compression, hemisection, transection, and photochemical injury. We included SCI experiments comparing functional outcome in a group of animals receiving OEC treatment with a control group receiving no treatment or vehicle treatment. Non-traumatic models of SCI were excluded, as well as studies reporting only combined treatments. For studies to be included they had to report the number of animals for each group, the mean effect size, and its standard deviation or standard error of the mean.We extracted details of individual study characteristics from each publication, and when a single publication reported more than one experiment, these data were extracted and treated as independent experiments. Study characteristics were extracted, including the gender and breed of the animals, time route and dose of transplantation, anaesthetic, and method of injury, as well as adjuvant treatment. Functional outcome was assessed for each experiment. Where data were expressed in text, numerical values were extracted. Where the outcome was expressed graphically only, Universal Desktop Ruler  was used to visually extract the data points. Only the final time point of the assessment of functional recovery was included.As an assessment for “risk of bias,” we assessed the methodological quality of each study using a modified 9-point item quality checklist, adapted from the CAMARADES  quality checklist : (i) repp < 0.05. In the regression model, the variance in the dependent variable that is accounted for by covariates is used to calculate an adjusted R2, a measure of how much residual heterogeneity is explained by the model [A normalized effect size (ES) for each comparison was calculated, defined as the improvement of outcome in the treatment group compared with that in the control group with reference to the outcome of an untreated, unlesioned, “sham” animal. The attributed size of the control group was adjusted if a single control group was compared to more than one treatment group. We used DerSimonian and Laird random effects model meta-regression to calculate an overall estimate of effect size. The analysis was stratified according to the method of injury, type of treatment, time of application, number of transplanted cells, quality assessment score, adjuvant treatment, time of assessment, medium used for cell culture, transplant origin, assessment of OEC purity, details of surgical procedure, and type of anaesthetic. Meta-regression was used to determine how much heterogeneity can be explained by study design characteristics. Random effects meta-regression was conducted by taking into account both within-study and between-study variance using STATA13 with a significance level of he model . FiguresS1 Checklist(DOC)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Granulomatosis with polyangiitis is a rare multisystemic autoimmune disorder predominantly affecting the upper and lower respiratory tracts and the kidneys, and rarely affecting other organ systems. Tuberculosis can mimic the presentation of granulomatosis with polyangiitis, and both can occur simultaneously in the same patient. Here we report what we believe to be the first case of concurrent granulomatous breast lesions and hemorrhagic retinal angiopathy in a Sri Lankan woman with refractory granulomatosis with polyangiitis complicated by probable tuberculosis.A 48-year-old Sri Lankan Moorish woman presented with a 6-month history of ulcerating bilateral breast lumps, a 3-month history of non-healing painful ulcers on the palate, and sudden bilateral painless loss of vision. Retinoscopy confirmed left-sided retinal hemorrhages and bilateral panuveitis. An examination of her respiratory system showed bilateral coarse crepitations. Histologic examination of the palatal and breast lesions showed chronic granulomatous inflammation. Her levels of immune markers were elevated but her renal function was normal. Chest radiography showed bilateral mid-zone and lower-zone infiltrates with cavitation and small pleural effusions. Her serum proteinase 3 anti-neutrophil cytoplasmic antibody titer and the level of adenosine deaminase in her pleural fluid were significantly elevated. She was diagnosed with generalized granulomatosis with polyangiitis complicated with probable pulmonary tuberculosis, and was started on methylprednisolone and cyclophosphamide pulse therapy with anti-tuberculous treatment. She later developed cerebral vasculitis, indicating refractory disease, and was treated with second-line rituximab with excellent response.Proteinase 3 anti-neutrophil cytoplasmic antibody may be a valuable diagnostic marker in patients with atypical symptoms of granulomatosis with polyangiitis or in the presence of probable tuberculosis. Retinal vascular angiopathy needs to be diagnosed and treated early to prevent the development of complete blindness. Concomitant cytotoxic and anti-tuberculous treatments may be safe and effective in patients with simultaneous refractory disease with probable tuberculosis. Granulomatosis with polyangiitis (GPA), also known as Wegener’s granulomatosis, is a rare multisystem autoimmune disorder predominantly affecting the upper and lower respiratory tracts and the kidneys . It has Here, we report what we believe to be the first case of concurrent granulomatous breast lesions and hemorrhagic retinal angiopathy, which occurred in a Sri Lankan woman with refractory GPA complicated with probable pulmonary TB.A 48-year-old Sri Lankan Moorish woman from Colombo presented to our emergency treatment unit with bilateral sudden-onset painless loss of vision. There was no associated tearing, irritation, or red eyes. Six months previously she had noted bilateral, slowly growing breast lumps for which she has not taken medical advice, on cultural grounds. The lumps subsequently became ulcerated, with intense pain and discomfort Fig. . She alsA general examination revealed that our patient was of average build with no lymphadenopathy. She did not consent to a genital examination. An examination of her respiratory system showed bilateral diffuse coarse crepitations. There was no dullness over her lung fields and no bronchial breathing was present. Her skin and nails appeared normal with no stigmata of chronic disease. Results from cardiovascular, rheumatological, and neurological examinations were normal.Our patient’s light perception was absent bilaterally and she had no direct or indirect light reflexes, suggesting a bilateral visual pathway lesion. Results from magnetic resonance imaging of her brain and orbits were normal, excluding any gross abnormality of the posterior visual pathway. Indirect retinoscopy showed retinal hemorrhage due to retinal vascular angiopathy in her left retina with bilateral panuveitis Fig. . Visual Our patient’s immune markers were elevated, with an erythrocyte sedimentation rate of 135 mm/h and C-reactive protein level of 96 mg/dL. Chest radiography showed bilateral mid-zone and lower-zone infiltrates with cavitation and small pleural effusions Fig. . ContrasAn ultrasound scan of her breast showed multiple hypoechoic ill-defined areas suggestive of abscesses. A biopsy of the abscess wall showed well-formed granulomas with collections of histiocytes and multinucleated giant cells. There was no caseation to indicate TB or cytological abnormalities suggestive of malignancy. Histologic examination of the palatal ulcers indicated a similar pattern of granulomatous inflammation with some necrotic areas. Screening for human immunodeficiency virus was negative and her normal levels of angiotensin-converting enzyme excluded sarcoidosis. Her renal function and results of a full urine report were normal. However, her serum PR3-ANCA titer was significantly elevated at 190.74 IU/mL  although her myeloperoxidase-neutrophil cytoplasmic antibody, anti-nuclear antibody, and rheumatoid factor titers were all normal. Her pleural fluid adenosine deaminase (ADA) level was significantly elevated at 101 IU/L .The pulmonary and oral manifestations together with strong positivity for PR3-ANCA confirmed the diagnosis of generalized GPA in our patient . This wa2 by intravenous infusion once weekly for 4 weeks, with intravenous methylprednisolone 1,000 mg/day for 3 days followed by prednisone 1 mg/kg per day [Our patient was started on intravenous methylprednisolone  and intravenous cyclophosphamide at a dose of 15 mg/kg every 2 weeks for the first three pulses, followed by infusions every 3 weeks for the next 3–6 pulses, as per European League Against Rheumatism recommendations . She was per day , 12. AspWe believe this is the first reported case of concurrent granulomatous breast lesions and vasculitis-like hemorrhagic retinal angiopathy in a patient with GPA. The presence of ADA-positive cavitating lung lesions suggested probable TB, even though this was not confirmed by microbiological tests. Given that the radiological pattern of the pulmonary lesions was indicative of GPA, in the absence of a tissue diagnosis our patient was considered to have coexisting pulmonary TB on the basis of pleural fluid ADA. The prevalence of TB in urban Sri Lanka 13.9 %) is higher than in rural areas (2.2 %) [.9 % is hThe presence of blindness and organ-threatening GPA created a management challenge in terms of the timing of cytotoxic treatment in our patient. We opted to treat both conditions simultaneously, with close monitoring of organ function. The appearance of a new symptom complex of multiple infarctions on magnetic resonance imaging raised the possibility of treatment for refractory disease. Our patient showed an excellent clinical response to second-line rituximab in the absence of renal involvement. The recent RITUXVAS trial, which was conducted among patients with newly diagnosed GPA or microscopic polyangiitis with renal involvement, found no difference between the rituximab and control groups, while B-cell return was associated with relapse .Although TB can be associated with elevated serum PR3-ANCA levels, the histology in the current case was more indicative of GPA than TB . The intAlthough GPA affecting the breast is rare, it should nonetheless be considered in the evaluation of any breast mass. Retinal vascular angiopathy needs to be diagnosed and treated early to prevent the development of complete blindness. A full systemic evaluation is thus essential in patients diagnosed with GPA. Sarcoidosis, immune deficiency, and disseminated malignancy should be excluded in a patient presenting with breast lumps; pulmonary and ocular lesions, the nature of the lesions, histological results, and biomarkers are important for making a differential diagnosis in such cases. PR3-ANCA is a valuable diagnostic marker in patients with atypical symptoms of GPA or concurrent similar illness such as TB. Simultaneous cytotoxic treatment, including rituximab, and ATT can be safe and effective in patients with refractory GPA and probable TB.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


To investigate whether compliance of patients to antibiotic treatment is better when antibiotics are administered once than multiple times daily.We performed a systematic search in PubMed and Scopus databases. Only randomized controlled trials were considered eligible for inclusion. Compliance to antibiotic treatment was the outcome of the meta-analysis.Twenty-six studies including 8246 patients with upper respiratory tract infections in the vast majority met the inclusion criteria. In total, higher compliance was found among patients treated with once-daily treatment than those receiving treatment twice, thrice or four times daily . Likewise, children that received an antibiotic twice-daily were more compliant than those receiving the same antibiotic thrice-daily . Higher compliance was also found among patients receiving an antibiotic once compared to those receiving an antibiotic of different class thrice or four times daily . The finding of better compliance with lower frequency daily was consistent regardless of the study design, and treatment duration.This meta-analysis showed that compliance to antibiotic treatment might be associated with higher when an antibiotic is administered once than multiple times daily for the treatment of specific infections and for specific classes of antibiotics.  The frequency of the daily dosing is among others a factor affecting compliance to treatment... No heterogeneity was detected in this analysis ]. Also, hichildren.,30 No siI2 = 97%). One study reporting on adults showed higher compliance with once versus thrice-daily regimens . The compliance was not different nor between children receiving twice and those receiving thrice-daily regimens . No heterogeneity was detected in this analysis (I2 = 0%).No significant difference in compliance was found between children receiving once-daily regimen and those receiving thrice-daily regimen . and  respectively. Considerable heterogeneity was detected in both analyses .Definition of compliance: received all versus high amount of the medication. The analysis regarding the definition of compliance showed that when as compliant patients were defined those who received all the medication, there was higher compliance in patients receiving the drug once-daily than those receiving the drug multiple times daily . Considerable heterogeneity was detected in both analyses (I2 = 96%).Duration of treatment: ≤ 7 versus > 7 days. When analysis was performed according to the duration of the antibiotic treatment, the compliance was higher in the once-daily group compared to the multiple times daily group, both in studies with short duration of treatment (≤ 7 days)  and in those with longer duration (> 7 days) [1867 patients RR = 1.34 ]. No heterogeneity was detected in the former analysis (I2 = 19%), while considerable in the latter analysis (I2 = 99%).This meta-analysis revealed that patients who received antibiotic treatment once-daily had higher compliance than those who received antibiotic treatment multiple times daily. Of interest, this finding was observed both in open-label and in single-blind RCTs.Previous studies have suggested that the clinical effectiveness with once-daily regimens may be non-inferior to multiple daily dosing regimens.–52 RegarExpectedly, the more times daily patients took a medication, the less compliant were. In fact, the lowest percentages of compliance were observed among patients treated with thrice-daily regimens or four times daily regimen in one study. The mostHigh compliance may lead to clinical success but low compliance may result in treatment failure, emergence of resistant strains, and increased healthcare costs through relapses of infection and hospitalizations.–57 ApartTo our knowledge this is the first meta-analysis study focusing on the compliance to antibiotic treatment according to the number of doses per day. The finding of the meta-analysis is consistent with a previous review that studied the association between dose regimens and medication compliance. In that Our findings should be interpreted in view of important limitations. First, considerable statistical heterogeneity was detected in most analyses. In addition, it should be emphasized that compliance was not included among the primary outcomes in none but one included study. FurthermIn conclusion, considering the limitations surrounding this meta-analysis once-daily antibiotic treatment might be associated with higher compliance than treatment administered multiple times daily in specific populations, for specific sites of infections and specific classes of antibiotics. Since higher compliance to treatment may imply higher clinical effectiveness, the frequency of the dosing schedule of an antibiotic is an additional parameter that could be considered before prescription.S1 PRISMA Checklist(DOC)Click here for additional data file.


A 63-year-old woman who had hypopituitarism was re-admitted to our hospital because of fever, diarrhea and disturbance of consciousness with life-threatening arrhythmia due to prolongation of the QT interval. She has been treated with hydrocortisone consequently, and has shown few ventricular arrhythmias with normalization of the QT interval. There have been several reports showing the case of prolonged QT interval with adrenal insufficiency, but there are few reports of isolated adrenocorticotropic hormone deficiency without any electrolytes imbalance that showed polymorphic ventricular tachycardia associated with QT prolongation. We discuss some possible mechanisms of how adrenal insufficiency causes life-threatening arrhythmia. Since lack of glucocorticoid hormone might induce prolongation of the QT interval, patients with adrenal insufficiency should be paid attention as candidates of lethal arrhythmias particularly when exposed to excessive stresses. Adrenal crisis is a life-threatening emergency, however, the relation between adrenal crisis and life-threatening arrhythmia is not known. We describe a case with adrenal crisis and polymorphic ventricular tachycardia (VT).3 μl, respectively. The rapid test of influenza type A was positive. Ultrasound echocardiography (UCG) showed normal cardiac size and function of the left ventricle. We intravenously administered hydrocortisone with large volume of physiological saline and started rapid ventricular pacing at the rate of 120 bpm. In the second day of the treatments, arrhythmias were not observed and the QT interval was normalized (QTc 394 ms) , and IKr  by inducing expressions of the serum- and glucocorticoid-inducible kinase (SGK1) [K channels induce outward potassium currents, lack of glucocorticoid may extend duration of action potentials by reducing expressions of SGK1 and these ion channels [It is well known that hypothyroidism causes QT prolongation , but here (SGK1) . Since tchannels . In factchannels .Long-QT syndrome causes life-threatening ventricular arrhythmias such as Torsades de Pointes. Since lack of glucocorticoid hormone might induce prolongation of the QT interval, patients with adrenal insufficiency should be paid attention as candidates of lethal arrhythmias particularly when exposed to excessive stresses.This study has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. We have obtained the informed consent from the patient.


Xestospongia muta, from the Caribbean. In combination with a gene-specific approach, our goals were to (1) characterize genetic evidence for nitrogen cycling in X. muta, an important limiting nutrient on coral reefs (2) identify which prokaryotic symbiont lineages are metabolically active and, (3) characterize the metabolic potential of the prokaryotic community. Xestospongia muta expresses genes from multiple nitrogen transformation pathways that when combined with the abundance of this sponge, and previous data on dissolved inorganic nitrogen fluxes, shows that this sponge is an important contributor to nitrogen cycling biogeochemistry on coral reefs. Additionally, we observed significant differences in gene expression of the archaeal amoA gene, which is involved in ammonia oxidation, between coral reef locations consistent with differences in the fluxes of dissolved inorganic nitrogen previously reported. In regards to symbiont metabolic potential, the genes in the biosynthetic pathways of several amino acids were present in the prokaryotic metatranscriptome dataset but in the host-derived transcripts only the catabolic reactions for these amino acids were present. A similar pattern was observed for the B vitamins . These results expand our understanding of biogeochemical cycling in sponges, and the metabolic interchange highlighted here advances the field of symbiont physiology by elucidating specific metabolic pathways where there is high potential for host-prokaryote interactions.Compared to our understanding of the taxonomic composition of the symbiotic microbes in marine sponges, the functional diversity of these symbionts is largely unknown. Furthermore, the application of genomic, transcriptomic, and proteomic techniques to functional questions on sponge host-symbiont interactions is in its infancy. In this study, we generated a transcriptome for the host and a metatranscriptome of its microbial symbionts for the giant barrel sponge,  Marine sponges are widespread and ecologically important members of many marine benthic habitats  of processes driven by sponge-associated prokaryotes and the environmental effects on those processes that influence both biogeochemical cycling and host-microbe interactions is essential to fully understand the multiple roles of sponges in benthic marine ecosystems. Recent studies aimed at understanding the taxonomic diversity and functional roles of sponge symbionts have utilized high throughput sequencing methods. These studies have led to the discovery of eukaryotic-like domains in symbiont-derived proteins  were sampled at 15 m depth at ~09:00 from each of three locations: Rock Bottom Reef, Little Cayman, Cayman Islands (LC) , North Perry Reef, Lee Stocking Island (LSI) , Bahamas, and Conch Reef, Key Largo, FL (FL) . Sponge pieces were cut from the top rim of the sponge (“pie slice” including both pinacoderm and mesohyl) and placed in a plastic bag with seawater in a cooler until reaching the laboratory 15–20 min later. Each sponge sample was then placed in RNAlater  or DNA buffer  while one sponge sample from each location was used to generate the metatranscriptome. From the metatranscriptome dataset, the three sponges samples were pooled for assembly but were analyzed individually for EMIRGE analysis as described below.As part of the study described in Fiore et al.  replicat−1) and samples were sent to the University of Illinois (Urbana-Champaign) for library construction and sequencing. There the eukaryotic rRNA was removed from 1 μg of total RNA with Ribominus Eukaryote kit (Invitrogen) and library construction was performed using a TruSeq RNA Sample Prep kit  per the manufacturer's instructions. The libraries from the three sponge samples plus six coral libraries treated exactly the same  and Prinseq .The ribosomal sequences from the EMIRGE analysis were classified using the Ribosomal Database Project (RDP) classifier v. 2.5  and as prokaryotic (n = 3723) were extracted in MEGAN and used to parse the original assembly file of contigs into separate files. These files were then individually used in BLASTx searches as described above and visualized in MEGAN. Contig sequences in all KEGG pathways that were examined were extracted and manually checked for quality by a BLASTx against NCBI database. A contig was discarded if it did not match to the expected gene based on the KEGG annotation. Phylogenetic information for each contig in a KEGG pathway was not available in MEGAN, therefore, prokaryotic contigs were classified by phyla if the top two matches were the same phyla and the query sequences was at least 50% homologous to the expected and top sequence. If this standard was not met, or if the top BLAST match was unclassified , the contig was binned as unclassified. Poriferan contigs were confirmed if they matched to the sponge Amphimedon queenslandica using BLASTx, even if the homology was less than 50%. If poriferan contigs did not match to A. queenslandica they were discarded. In MEGAN, contigs were normalized to 100,000 per dataset for comparison between the putative sponge and prokaryotic mRNA datasets. Quality trimmed reads and associated metadata were deposited in the CAMERA database under accession number CAM_P_0001214 and are now available through the iMicrobe database under the same accession number (http://data.imicrobe.us/project/view/128).Putative mRNA (poriferan and prokaryotic) contiguous sequences (contigs) from the CLC Workbench assembly were used in a BLASTx search against the NCBI RefSeq protein database , nitrification , denitrification , and anaerobic ammonium oxidation  were selected as target amplification products. Genomic DNA was used as the template in these gene-specific reactions. However, for expression studies  of archaeal amoA genes, extracted RNA was used to synthesize cDNA.Because of the role that sponges play in the cycling of DIN, the signature genes involved in nitrogen fixation  and stored in DNA buffer were used in CTAB (hexadecyltrimethylammonium bromide) genomic DNA (gDNA) extractions along with water samples collected near the sponges at the same time. Seawater (~1 L) was collected adjacent to the same sponges described above and filtered through a 0.2 μm membrane filter . Filters were cut in half  and also minced into smaller pieces to increase surface area for DNA extraction. Samples for both sponges and filters were processed as described in Fiore et al.  and OR2 (5′-ATGGTGTTGGCGGCRTAZAKYGCCATCAT-3′)   and qnorB7R (GGNGGRTTDATCADG)  and nirK5R (GCCTCGATCAGRTTRTGG)  and Arch-amoAR (5′-GCGGCCATCCATCTGTATGT- 3′) , 2.5 μl of 10× Titanium Taq buffer, 0.2 mM dNTPs , 0.4 μM of each primer, and 25 ng of genomic DNA template. However, for the second step of the Escherichia coli using the pGEM-T easy vector system  and JM109 chemically competent cells according to manufacturers instructions (Promega). Transformed cells were sent to Functional Biosciences Inc.  for sequencing on an ABI 3730xi DNA sequencer.The triplicate reactions were pooled and electrophoresed on a 1% agarose gel and bands of appropriate size were extracted using Qiaquick gel extraction kit . Gel extracted products were cloned in nifH and amoA (see below) gene sequences were deposited in GenBank .Chromatograms resulting from Sanger sequencing were manually checked for quality, and vector sequence was trimmed in the program Geneious v4.8.4 (Biomatters Ltd.). Trimmed sequences were then queried by BLAST against the NCBI nr database using Geneious and matching sequences were compiled. Pairwise alignments in Geneious were also performed for each sequence and its closest BLAST match, which were then translated and manually checked for errors. These sequences and the closest matches from BLAST queries were aligned  and the alignment was used to create a neighbor-joining tree in Geneious. amoA between the different locations. The archaeal amoA gene was chosen for quantitative expression analysis as this gene has previously been sequenced from X. muta  was confirmed using PCR methods described above.Total RNA was also extracted as described for the metatranscriptome analysis and converted to cDNA using RETROscript (Ambion) and random decamers following the manufacturer's protocol. The synthesized cDNA was used to quantify differential expression of archaeal Δ, ΔCt values were used to determine the difference in the relative expression between groups using the single sample with the highest Δ, ΔCt values (FL) as a calibrator in determining relative expression between the three locations. The calculated values were log + 1 transformed and tested for normality  and JMP v 10.0.2.Quantitative real time PCR (QRT-PCR) was performed using genomic DNA for normalization of sponge samples from each location . The green, non-sulfur, Chloroflexi, and Acidobacteria were also prominent OTUs in all three samples, although these varied in abundance among the three, with the highest abundance in the FL sponge compared to the LC and LSI sponges. The classes within these phyla also varied between the sponges, with Anaerolineae (Chloroflexi) more abundant in the LSI sponge than the FL and LC sponges. Other notable groups in terms of known functional activity include archaeal OTUs (Euryarchaeota and Thaumarchaeota), Nitrospirae and Planctomycetes, which were present in all sponge samples.In all three sponge samples the most abundant OTUs (OTUs with the highest NormPrior), were assigned to the phylum Proteobacteria, and within this phylum, most were assigned to the class Gammaproteobacteria Figure . The secde novo assembly was selected for use in the analyses described below. Of the CLC assembled contigs, 3,727 contigs were assigned to Bacteria or Archaea Domains, while 7,976 were assigned to the phylum Porifera based on the LCA algorithm results in MEGAN. Of the prokaryotic contigs, most were bacterial in origin (93%), while 7% were archaeal in origin.The CLC Workbench assembly of non-ribosomal RNA reads yielded 35,219 contigs, or transcriptional features, with N50 = 1010, an average contig length of 979 bp, and GC content of 43%  were classified by KEGG as metabolism (~19%), followed by genetic information processing (~10%), and environmental information processing (~7%) Figure . Cellulanar genes that encode a nitrate oxidoreductase , nirK genes encoding for nitrite reductases , and an nrfA gene encoding for an ammonia-forming cytochrome c nitrite reductase . Other transcripts involved in nitrogen cycling were part of assimilatory pathways, with prokaryotic transcripts corresponding to reactions that incorporate ammonia into amino acids , cystathione beta-lyase , and sulfite reductase . Sulfur assimilation related features from the host were mainly related to adenylylsulfate (APS) metabolism.Sulfur assimilation via different pathways in the host sponge and in the prokaryotic community was also observed Figure . In the 1) compound metabolism, specifically formaldehyde, formate, carbon monoxide, and potentially methane , a 5,10-methenyltetrahydromethanopterin cyclohydrogenase , and a phosphosulfolactate synthase . Transcript matches to genes involved in formaldehye metabolism were recovered such as frmA, a glutathione-independent formaldehyde dehydrogenase, formate dehydrogenase (fdh), alcohol dehydrogenase enzymes, and many carbon monoxide dehydrogenase transcripts (cdhA)   were successfully amplified from LC and LSI Xestospongia muta samples and from the water column. Sequences from FL were recovered, but were all low matches to the database and did not align with the other nifH sequences so were not analyzed further. The nifH-deduced amino acid sequences from X. muta that could be analyzed fell into cyanobacterial and proteobacterial groups  , Myxosarcina sp. (87–89%) and the cyanobacterium UCYN-A (83–85%). Proteobacterial nifH-deduced amino acid sequences from the current study were most similar to either Alphaproteobacteria such as Bradyrhizobium japonicum (80–87%), or to Gammaproteobacteria such as Vibrio spp. (96–97%), and Azotobacter chroococcum (95–96%). Seawater-derived nifH-deduced amino acid sequences generally clustered together within each cluster, with the exception of sequence SW1.7 classified as Gammaproteobacteria, and sequence SW3.7, classified as Cyanobacteria.Gene sequences encoding the nitrogen fixing enzyme nitrogenase (qnorB) sequences (n = 22) were successfully recovered from Xestospongia muta at all locations. Gene-deduced amino acid sequences (qnorB) recovered from X. muta were more similar to each other than to other known qnorB-deduced amino acid sequences from other bacteria and were ~36% similar to Alcaligenes faecalis (AM284323). One high quality sequence of the nitrate reductase gene, nirK, was recovered. Clone XmFL3m_3nirK (KJ020980) matched to several uncultured bacterial nirK genes as well as to Pseudomonas aeruginosa  and Sinorhizobium sp. .Nitric oxide reductase gene  to those documented previously by Lopez-Legentil et al.  = 11.67, p = 0.0086], with LSI and LC having significantly higher expression relative to FL  but no difference between each other   assigned terms were different between the metatranscriptome (prokaryotic transcripts) and the sponge transcriptome . While the most abundant GO biological process for both sets of transcripts was oxidation-reduction for the prokaryotic dataset electron transport, purine base metabolism, ribosome biogenesis and translation transcripts were the next most abundant. In the sponge dataset, signal transduction, protein phosphorylation, serine family metabolism and proteolysis were the next most abundant GO biological processes.Amphimedon queenslandica as annotated using the KEGG databases . Additionally, prokaryotic and sponge-derived transcripts involved in tryptophan metabolism were characterized . Within the tryptophan metabolic pathway, transcripts were recovered that are involved in indole metabolism including, indole 3-pyruvate decarboxylase , aldehyde dehydrogenase , and nitrile hydratase  . The biosynthetic pathway of lysine was entirely represented by prokaryotic transcripts and included at least eight different phyla, notably, Poribacteria and Thaumarchaeota  biosynthetic pathway was represented by poribacterial and bacteroidetes transcripts for 7,8-diaminonanoate transaminase  and biotin synthase  biosynthetic pathway, but with more prokaryotic phyla involved . A key enzyme in riboflavin synthesis, riboflavin synthase, was represented by proteobacterial transcripts . Both prokaryotic and sponge transcripts were involved in the conversions of FAD to/from riboflavin. Transcripts for the thiamin (vitamin B1) biosynthetic pathway included genes involved many early precursor compounds but also important genes such as thiG . No transcripts involved in the final steps leading to thiamin phosphate and thiamin were recovered, but sponge-derived transcripts did dominate the catabolism of thiamin. Prokaryotic transcripts corresponding to cobalamin (vitamin B12) biosynthesis were also recovered , and pantothenate. The biotin vitamin B biosynthed Table .A. queenslandica predicted genes were recovered . Eukaryotic-like domain containing proteins were detected in the metatranscriptome (prokayrotic) dataset, including two transcriptional features encoding leucine-rich repeat containing proteins, as well as tetratricopeptide repeat domains (TPR), and ankryin repeat domains (AR) . Host transcripts involved in the cellular responses  proteins, heat-shock proteins) of the host to stress and/or bacteria or viruses that were homologous to R) Table .Xestospongia muta, is abundant on Caribbean coral reefs and has been shown to be an important mediator of inorganic nitrogen cycling on these coral reefs  and protein-coding (mRNA) genes within a metatranscriptome. As previously discussed there are limits to the interpretation of studies comparing rRNA and rDNA genes of prokaryotes to address simultaneously the question of taxonomy and metabolic activity  and nitrate + nitrite (NO−x) from Xestospongia muta led to the hypothesis that anaerobic nitrogen transformations occur in this sponge in addition to aerobic nitrification  were amplified from X. muta sponges. Proteobacerial and cyanobacterial nifH gene sequences were recovered, similar to what has been observed previously in sponges . The DNRA gene, nrfA, was also recovered in the metatranscriptome from a deep-sea sponge , corresponding to high DOM in the Florida Keys from allochthonous input  or particulate organic nitrogen (PON), or both, whereas the prokaryotic community obtains most of its nitrogen from NH3 via glutamine synthetase and glutamate synthase (GS-GOGAT) pathways. If there were transfer of nitrogen from symbionts to the sponge this would suggest that it may be in the form of amino acids or other nitrogenous compounds rather than as NH3. This notion is supported by the expression of genes encoding for the enzymes histidine ammonia lyase and aminomethyltransferase, which are involved in amino acid metabolism and NH3 formation, in the sponge dataset.Ammonia is also assimilated as a source of nitrogen for the prokaryotic community of 1 compounds, such as methanol, formaldehyde, or carbon monoxide, as sources of carbon and energy has been characterized in diverse prokaryotic groups  were overrepresented relative to the bacterioplankton dataset in a metagenome of the sponge Cymbastela concentrica  are similar to what has been observed in the analysis of other sponge transcriptomes  biosynthetic pathway was particularly striking, with transcripts corresponding to the key enzymes leading to biotin detected that were homologous to Poribacteria and Bacteroidetes genes. Genes involved in biosynthesis of B vitamins were documented in a poribacterial genome previously . A similar pattern was observed for the riboflavin (vitamin B2) biosynthesis pathway, with prokaryotic transcripts corresponding to the production of riboflavin and sponge transcripts corresponding to the conversion between riboflavin and the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).The notion that vitamins produced by sponge microbes could be beneficial to the host has been proposed previously  (Doxey et al., Xestospongia muta, are heterotrophic filter feeders, they may be able to obtain these metabolites from their food but hosting prokaryotes that provide a continuous source of these important metabolites might be more advantageous and predictable in time and space in a relatively nutrient poor environment.A recent study that examined the microbial communities of phylogenetically divergent sponges suggested that there might be selective pressure for vitamin producing prokaryotes (Fan et al., Xestospongia muta-associated prokaryotes and potential points of interaction between the sponge and its symbiotic prokaryotes. Complementary methods of sequencing approaches along with physiological studies including metabolite studies are needed to effectively advance our understanding of host-microbe relationships in sponges. The metabolic pathways described here provide specific target areas for future experimental studies focused on sponge-associated microbes.The approach described here has provided novel insights into the metabolic potential of 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.