Drosophila and other metazoan genomes predicts widespread use of post-transcriptional regulation in cellular and developmental processes. Previous studies identified roles for several RNA-binding proteins in dendrite branching morphogenesis of Drosophila larval sensory neurons. To determine the larger contribution of post-transcriptional gene regulation to neuronal morphogenesis, we conducted an RNA interference screen to identify additional Drosophila proteins annotated as either RNA-binding proteins or translation factors that function in producing the complex dendritic trees of larval class IV dendritic arborization neurons. We identified 88 genes encoding such proteins whose knockdown resulted in aberrant dendritic morphology, including alterations in dendritic branch number, branch length, field size, and patterning of the dendritic tree. In particular, splicing and translation initiation factors were associated with distinct and characteristic phenotypes, suggesting that different morphogenetic events are best controlled at specific steps in post-transcriptional messenger RNA metabolism. Many of the factors identified in the screen have been implicated in controlling the subcellular distributions and translation of maternal messenger RNAs; thus, common post-transcriptional regulatory strategies may be used in neurogenesis and in the generation of asymmetry in the female germline and embryo.The large number of RNA-binding proteins and translation factors encoded in the  Dendrites are neuronal structures that receive sensory and synaptic input, allowing a neuron to perceive and respond to its surrounding environment. Dendrites display diverse and often complex arborization patterns that are integral to neuron function, as they determine the type of sensory or synaptic input a neuron is capable of processing. Although a myriad of dendritic arbor morphologies has been reported, the molecular mechanisms that regulate dendrite morphogenesis and the establishment of specific arborization patterns are only partly understood. Recent studies have begun to identify the molecular components that are involved in generating cell type specific arborization patterns. These studies have largely focused on the roles of transcription factors that regulate the overall arborization architecture of specific neural subtypes , Pumilio (Pum), and the Fragile X mental retardation protein (FMRP) direct some aspects of dendrite development and branching , approximately 400 branch points were routinely detected within the field of view.The number of branch points and the total branch length were quantified in Z series projections of ddaC neurons imaged on a Leica SP5 confocal microscope. Images shown in Drosophila genome encodes at least 400 proteins annotated as mRNA binding proteins, proteins with known RNA binding domains, and/or translation factors excluding ribosomal proteins , pumilio (pum), smaug (smg), and glorund (glo) . At the time the screen was performed, independent RNAi lines were only available for a small subset of the positive candidates. We were able to confirm dendrite defects for eight of them (brain tumor (brat) and 4EHP, with mutants and have shown that they function together with nos and pum to regulate dendrite morphogenesis (oskar (osk) and rumpelstiltskin (rump), also confirmed roles in class IV da neurons , U2 small nuclear riboprotein auxiliary factor 38 (U2af38), and x16 resulted in an increase in the number of branch points due to an excess of higher order branches (U2af38) or decreased , however, indicating that branches are shorter than in wild-type control neurons. The decrease in total dendrite length for mbl and x16 reflects shortening of primary as well as higher order branches, such that the dendritic tree no longer covers the entire field. In contrast to the evenly spaced dendritic branches of control neurons, knockdown of all three genes also caused defects in the spacing of dendrites, with clustering of terminal branches and resulting gaps in coverage within the arbor.Knockdown of branches . Total dembryonic lethal abnormal vision (elav) and sans fille (snf), had no affect on the number of branch points but did reduce total dendrite length, resulting in an elevated ratio of branch points to length like that of mbl, U2af38, and x16 RNAi neurons. Similarly, the shortening and clustering of branches in elav and snf RNAi neurons produced coverage defects , CG11334, Gemin3 (Gem3), Dead-box-1 (Ddx1), arrest (aret), mind bomb 2 (mib2), squid (sqd), spoonbill  resulted in both branch loss and a concomitant decrease in total dendritic length , loquacious (loqs), CG6418, CG5705, and CG5800 RNAi, the number of branch points was reduced disproportionately to dendrite length , which produced characteristic trees with truncated main branches and few, short higher order branches. Less severe but consistent phenotypes were observed for an additional eIF3 subunit gene, eIF3-p40, as well as for subunits of eIF2B , eIF4a, and eIF4E-4  generally produced milder phenotypes  RNA binding domain, resulted in a phenotype resembling that of translation initiation factors. A severe branching deficit and even greater effect on dendrite length was produced by RpS3 RNAi, leaving truncated and denuded branches  the incomplete knockdown associated with RNAi. Indeed, while mutational analysis has shown roles for pum and dfmr1 in class IV da neuron dendrite morphogenesis (pum nor dfmr1 RNAi had an effect. Similarly RNAi knockdown of staufen (stau), which encodes a double-stranded RNA binding protein, did not result in any overt dendritic defects whereas dendritic branching was reduced in stau mutant larvae (data not shown). Nonetheless, the screen was ultimately successful in identifying many RBPs and translation factors not previously known to function in dendrite morphogenesis. Moreover, we found that certain classes of RBPs or translation factors were associated with particular phenotypes, suggesting that specific points of mRNA regulation may influence distinct dendrite morphogenetic processes.Our RNAi screen inevitably underestimates the extent of RBP involvement due to (1) the existence of a potentially large number of RBPs without annotated RBDs  splicing factor families involved in alternative splicing (Drosophila Down syndrome cell adhesion molecule (Dscam) mRNA in a cell culture assay , suggesting that they may be transported to dendrites. Consistent with this idea, initiation factors including eIF2-alpha and eIF2-beta, both of which were positive in our screen, have been identified as components of neuronal transport ribonucleoprotein granules , suggesting that the RBPs they encode may regulate effectors of these pathways in da neurons. During pupariation, Drosophila class IV da neuron dendrites are eliminated through a pruning process in preparation for the formation of new projections necessary for the adult nervous system. This dendritic pruning involves dendrite severing in response to hormonal stimulation, local caspase activation in dendrites, and engulfment by phagocytes . In addition, 76% of RBPs with da neuron functions are expressed in the larval central nervous system, including 82% of those with ovarian expression. Both oocytes and neurons may share an elevated demand for post-transcriptional gene regulation compared to other cell types. Early developmental events in Drosophila rely on maternally expressed genes, which is thought to allow for rapid embryonic development  as well as dfmr1, glo, nos, pum, stau, and smg have been shown to play roles in establishing asymmetries in the oocyte and early embryo by regulating the localization or local translation of target mRNAs (References in FlyBase gene reports: http://flybase.org). Moreover, both nos and osk mRNAs are transported into da neuron dendrites, consistent with a local regulatory function for Nos and Osk proteins (The majority of elopment . Thus, p


Large-scale tumor sequencing projects are now underway to identify genetic mutations that drive tumor initiation and development. Most studies take a gene-based approach to identifying driver mutations, highlighting genes mutated in a large percentage of tumor samples as those likely to contain driver mutations. However, this gene-based approach usually does not consider the position of the mutation within the gene or the functional context the position of the mutation provides. Here we introduce a novel method for mapping mutations to distinct protein domains, not just individual genes, in which they occur, thus providing the functional context for how the mutation contributes to disease. Furthermore, aggregating mutations from all genes containing a specific protein domain enables the identification of mutations that are rare at the gene level, but that occur frequently within the specified domain. These highly mutated domains potentially reveal disruptions of protein function necessary for cancer development.APC, TP53 and KRAS. In addition, we found significant mutation frequency within specific domains located in these genes, as well as within other domains contained in genes having low mutation frequencies. These domain “peaks” were enriched with functions important to cancer development including kinase activity, DNA binding and repair, and signal transduction.We mapped somatic mutations from the protein coding regions of 100 colon adenocarcinoma tumor samples to the genes and protein domains in which they occurred, and constructed topographical maps to depict the “mutational landscapes” of gene and domain mutation frequencies. We found significant mutation frequency in a number of genes previously known to be somatically mutated in colon cancer patients including Using our method to create the domain landscapes of mutations in colon cancer, we were able to identify somatic mutations with high potential to drive cancer development. Interestingly, the majority of the genes involved have a low mutation frequency. Therefore, themethod shows good potential for identifying rare driver mutations in current, large-scale tumor sequencing projects. In addition, mapping mutations to specific domains provides the necessary functional context for understanding how the mutations contribute to the disease, and may reveal novel or more refined gene and domain target regions for drug development. APC gene have been shown to be highly prevalent in colorectal tumors [http://provocativequestions.nci.nih.gov).The advent of high-throughput, whole-genome DNA sequencing has enabled the evaluation of normal and tumor tissue samples from hundreds of patients in a single study, revealing both germline and somatic mutations with potential involvement in cancer susceptibility, initiation and development. However, distinguishing the handful of somatic mutations expected to initiate and maintain tumor growth, so-called driver mutations, from mutations that play no role in cancer development, passenger mutations, is still a major hurdle to fully understanding the mechanisms of the disease and to the design of more effective treatments. Most current, state-of-the-art studies take a gene-centric approach to the problem -6, identl tumors ,2,7. UnfFurthermore, gene-centric approaches to classifying driver and passenger mutations make no distinction between mutations in different sites on the gene, disregarding important information about the functional context of the site of the mutation. A recent study by Vidal’s team demonstrated the potential of gene-centric approaches to mischaracterize mutations . The autet al. in 2006, significant enrichments of proteins containing the MH1 and MH2 domains were found [et al., whereas 70% of the mutations in MH2-containing proteins fell within the MH2 domain, the enrichment of the MH1 domain turned out to be misleading as further analysis revealed that none of mutations in the MH1-containing proteins occurred inside of the MH1 domain itself [In this study, we introduce a new approach for the analysis of cancer somatic mutations based on the study of these mutations at the protein domain level. We argue that since protein domains define the structural and functional units of the proteins, mapping mutations not only to the genes in which they occur, but also to individual protein domains, adds functional information critical to the accurate assessment of the impact of the mutations. Analysis of the positions of individual oncogenic mutations discovered from several independent studies has revealed significant clustering of these mutations at specific positions within the catalytic domain of several protein kinases ,11,12. On itself . Similarn itself . Domain Our approach for identifying mutations relevant to cancer development specifically maps somatic mutations to the individual domains in which they occur, resulting in a more accurate measure of enrichment of mutations at the domain level. By mapping mutations to individual domains, our method avoids potentially misleading conclusions from gene-based domain enrichment analyses and provides an inherent functional explanation for how the mutations contribute to disease. We performed an exome-wide study of somatic mutations, including single nucleotide variants (SNVs) and short insertions and deletions (indels), from 100 colon adenocarcinoma patients obtained from The Cancer Genome Atlas (TCGA) project . To implet al. later followed up the study using a larger set of transcripts and revised statistics for identifying significantly mutated genes [ APC, KRAS, TP53 and others for colorectal cancer, but is dominated by a larger number of infrequently mutated gene “hills”. Here, we compare the gene-based mutational landscape of a much larger set of colon tumor samples to the landscape revealed by Wood et al., and show significant clustering of mutations in many of the previously identified CAN genes.We also introduce new terminology to describe the distribution of somatic mutations at the protein domain level: the “domain mutational landscape” consisting of a topographic representation of mutation frequencies within individual protein domains from whole-genome, cancer sequencing studies, and “domain peaks” defined as protein domains mutated at high-frequency in tumor genomes of the same or different tumor types. Sjöblom and collaborators performed the first large-scale analysis of breast and colorectal cancer mutations . Using aed genes . The autIn addition, we also describe the domain mutational landscape of colon cancer, and demonstrate how this landscape reveals major properties that cannot be revealed by gene-based landscapes. We show how highly mutated domain peaks can be missed by gene-centric methods when the individual genes containing the domains are not mutated at high frequencies. We also show how focusing only on significantly mutated genes can miss instances where mutations occurring within a shared domain are actually the more relevant functional contributors to the cancer. Therefore, due to its ability to identify and functionally characterize somatic mutations with high potential to drive cancer development, we expect our novel domain-centric method to become an integral tool for the analysis of data from future large-scale cancer sequencing studies.In this study, we compared the gene and domain mutational landscapes of somatic mutations present in tumor samples from 100 colon adenocarcinoma patients participating in the TCGA project. Mutations in protein coding genes such as single nucleotide variants causing amino acid changes (nonsynonymous SNVs or nsSNVs), short insertions or deletions causing a shift in the reading frame (frame shift mutations) and mutations causing the gain or loss of a stop codon (stop-gain or stop-loss mutations) are generally expected to be the most likely candidates for driving disease development. Therefore, we identified all somatic mutations of these types in addition to nonframeshift insertions and deletion mutations occurring within the protein coding regions of the tumor samples . In total, 21,572 mutations were identified in the 100 samples, yielding an average of 215 mutations per patient. Approximately 80% of the mutations were nsSNVs, 12% were frame shift mutations, 7% were stop-gain mutations, 0.15% were stop-loss mutations, and 1% were nonframeshift insertion mutations  annotations for all genes and domains containing somatic mutations in our colon and breast cancer sets. A subsequent enrichment analysis of GO terms annotated to gene peaks in our colon cancer landscape revealed an overrepresentation of genes annotated with GO terms related to signal transduction, kinase activity, DNA damage response and the regulation of apoptosis. A list of the top overrepresented GO terms from the enrichment analysis of gene peaks for colon and breast cancer can be found in Additional file et al. and Wood et al. identified significant clustering of mutations in the “genomic landscapes” of human breast and colorectal cancers. Despite the need of larger samples to reach more accurate conclusions [Previous studies by Sjöblom clusions ,19,20, tBy mapping mutations not only to the genes, but also to the individual domains they occurred in, we were able to construct the mutational landscapes for both genes and domains for 100 colon cancer patients Figure  and 1B. et al. revealed similar topographies: a few highly mutated gene mountains along with a much larger number of still significantly mutated gene hills. There was a relatively small overlap between the 154 genes identified by our study and the 140 CAN genes; only six genes were found to be significant in both studies. As noted, the two studies also used different tumor samples and different statistical models to determine significant mutation frequencies. Yet, despite these differences, four of the top five colorectal CAN genes  ranked in the top twenty genes with the highest normalized mutation frequency, and the fifth top CAN gene (PIK3CA) was identified to have a significantly mutated domain. We also identified seven genes with significant mutation frequency from the Cancer Gene Census [NRAS and BRAF. A GO term enrichment analysis of all 154 significantly mutated genes in our study identified enrichment in many biological processes and molecular functions known to be disrupted in cancer development including signal transduction, regulation of apoptosis, regulation of cell proliferation and DNA damage response.Comparison of our gene-based landscape for colon cancer to the landscape constructed by Wood TIGD7 and JRKL genes. Although TIGD7 and JRKL are both homologs of the Jrk “jerky” gene associated with epilepsy in mice [TP53 and APC, respectively. However, mapping mutations to the individual domains illustrates the value of our domain-centric method to provide the essential functional context to explain the role the mutations in cancer development. The GO term enrichment analysis for significantly mutated domains confirmed enrichment of significantly mutated domains with functions important to cancer development including kinase activity, DNA binding and repair, and signal transduction.Our analysis of the gene landscape resulted in the re-identification of genes with known cancer association and confirmation on enrichment of genes involved in processes critical to cancer development, which validates that our method can identify significantly mutated genes relevant to cancer, and also provides evidence that the method can be applied to other specified regions within the genome, including domain regions. The main focus of this study, however, was the construction of the domain mutational landscape for colon cancer and its comparison with the gene-based mutational landscape. In total, we identified 45 domains with significant mutation frequency in the colon tumor samples. Again, the landscape was characterized by mountains and hills, similar to that of the gene-based landscape, with the highest peaks in the P53, APC_crr and CENP-B_N domains. The CENP-B_N domain, a known DNA-binding domain , receive in mice , they doTP53 mutations [APC gene contains seven repeats of the APC_crr domain that bind to the Arm domains of the beta-catenin protein in addition to thirteen other distinct domains [APC’s ability to bind and down-regulate beta-catenin, critically impairing its function as a tumor suppressor gene in the Wnt signalling pathway [Our study of the domain landscape of cancer mutations also highlights the relevance of considering the modularity of the proteins when studying somatic mutations. Is the whole protein responsible for the disruption that promotes tumor growth, or are only some of the functional units of the proteins relevant? For instance, the P53 domain, also known as the P53 DNA-binding domain, contains over 90% of the known utations , even thutations ). The AP domains . Truncat pathway ,27.PIK3CA gene ranked in the top five highest, normalized mutation frequencies in the breast cancer set , CTTNBP2 (2 mutations), FILIP1 (5 mutations), and FILIP1L (1 mutation). Interestingly, FILIP1L is a highly conserved protein known to inhibit proliferation and migration and increase apoptosis in endothelial cells [FILIP1L gene was not significant in our study, CortBP2 ranked in the top 75 domains with the highest mutation frequency, suggesting a novel role in colon cancer development for FILIP1L and the other genes containing mutations in the CortBP2 domain. As with any in silico analysis, however, the identification of domains and genes with suspected roles in cancer development can only generate new hypotheses that must ultimately be experimentally validated.The examples discussed above, in which significant domain peaks correspond to at least one significant gene peak only constitute 14 of the 45 significantly mutated domains from the colon tumor set. The other 31 domain peaks correspond to genes without significant mutation frequencies which are undetected in the gene landscape. Because these domains do not occur in significantly mutated genes, they would likely not be found by traditional, gene-centric studies, but may reveal the disruption of potentially critical functional mechanisms within the cancer tissues. One of these peaks corresponds to the cortactin-binding protein-2 domain, CortBP2, that was mutated in four genes, al cells . This anal cells ,39. WhilNew methods are critically needed to distinguish mutations that drive tumor initiation and development from the millions of variants being identified in current large-scale tumor sequencing projects. Our novel, domain mutational landscape approach for identifying potential driver mutations in significantly mutated domains reveals many properties that traditional gene landscapes cannot reveal while also adding the functional context necessary for understanding how individual mutations contribute to cancer development. We also compared the mutational landscapes for breast and colon cancer, demonstrating the potential for the domain landscape to identify functional similarities among different cancer types. Determining which mutations are most important for tumorigenesis will shed new light on the selective pressures experienced during the process and will ultimately provide a new set of gene and domain targets for drug development.http://tcga-data.nci.nih.gov/tcga/) using the mutation files from the ucsc.edu_COAD.IlluminaGA_DNASeq.Level_2.1.0.0 and ucsc.edu_COAD.SOLiD_DNASeq.Level_2.1.0.0 directories downloaded on September 13th, 2011 and the genome.wustl.edu_BRCA.IlluminaGA_DNASeq.Level_2.3.0.0 directory downloaded on September 21st, 2011 respectively. The individual patient records listed both somatic and germline SNVs and short insertions and deletions (indels) in addition to the tumor and matched normal tissue genotypes for each mutation. The patient records were filtered to remove germline mutations and mutations that did not pass the quality control filter. A union set of somatic SNVs and indels was created for each cancer type from the individual patient records by identifying all somatic mutations present in at least one patient. The genotypes of all patients were then examined for each mutation in the union set to count the number of times the mutation occurred somatically in the patient population. The ANNOVAR program was used to filter out mutations present in dbSNP (release 130) in order to remove mutations known to be polymorphic, therefore unlikely to be cancer driver mutations [Controlled access, whole-exome mutation data for 100 colon adenocarcinoma patients and 522 breast invasive carcinoma patients were downloaded from the TCGA Data Portal   for all  et al. and Wood et al. studies [CAN genes: candidate cancer genes identified by the Sjöblom studies ,2; GO: GNLN and TAP participated in the preparation and analysis of the data and drafting the manuscript. DP developed the algorithm for computing the mutation frequency significance thresholds. MGK conceived of the study, participated in the analysis, and helped to draft the manuscript. All authors read and approved the final manuscript.The authors declare that they have no competing interests.Contains supplementary tables S1 to S7 Table S1 – Mutation counts for breast cancer Table S2 – Shared significantly mutated genes in colon and breast cancer Table S3 – Shared significantly mutated domains in colon and breast cancer Table S4 – GO terms enriched in domains significantly mutated in colon cancer Table S5 - GO terms enriched in domains significantly mutated in breast cancer Table S6 – GO terms enriched in genes significantly mutated in colon cancer Table S7 - GO terms enriched in genes significantly mutated in breast cancerClick here for fileTop genes highly mutated in colon cancer tumor genomes Complete list of significantly mutated genes from the colon cancer set. Gene name, representative protein length, and number of mutations are listed.Click here for fileTop genes highly mutated in breast cancer tumor genomes Complete list of significantly mutated genes from the breast cancer set. Gene name, representative protein length, and number of mutations are listed.Click here for fileTop domains highly mutated in colon cancer tumor genomes Complete list of significantly mutated domains from the colon cancer set. Gene name, Pfam domain accession, number of mutations, cumulative domain length, and genes containing mutations in the domain are listed.Click here for fileTop domains highly mutated in breast cancer tumor genomes Complete list of significantly mutated domains from the breast cancer set. Gene name, Pfam domain accession, number of mutations, cumulative domain length, and genes containing mutations in the domain are listed.Click here for file


The decline noticed in several fertility traits of dairy cattle over the past few decades is of major concern. Understanding of the genomic factors underlying fertility, which could have potential applications to improve fertility, is very limited. Here, we aimed to identify and study those genes that associated with a key fertility trait namely estrous behavior, among genes expressed in four bovine brain areas , either at the start of estrous cycle, or at mid cycle, or regardless of the phase of cycle.OXT, AVP, POMC, MCHR1), but also genes whose association with estrous behavior is novel and warrants further investigation.An average heat score was calculated for each of 28 primiparous cows in which estrous behavior was recorded for at least two consecutive estrous cycles starting from 30 days post-partum. Gene expression was then measured in brain tissue samples collected from these cows, 14 of which were sacrificed at the start of estrus and 14 around mid cycle. For each brain area, gene expression was modeled as a function of the orthogonally transformed average heat score values using a Bayesian hierarchical mixed model. Genes whose expression patterns showed significant linear or quadratic relationships with heat scores were identified. These included genes expected to be related to estrous behavior as they influence states like socio-sexual behavior, anxiety, stress and feeding motivation (OXT and AVP play major roles in regulating estrous behavior in dairy cows. Genes related to neurotransmission and neuronal plasticity are also involved in estrous regulation, with several genes and processes expressed in mid-cycle probably contributing to proper expression of estrous behavior in the next estrus. Studying these genes and the processes they control improves our understanding of the genomic regulation of estrous behavior expression.Several genes were identified whose expression levels in the bovine brain associated with the level of expression of estrous behavior. The genes  Maintaining good fertility and thereby optimum reproductive performance in dairy cows is of great economic importance for the dairy industry. Knowledge on factors influencing fertility is already being applied to improve or regulate fertility. For example, the importance of limiting negative energy balance in early lactation cows for proper reproductive performance is well recognized ,2. InsigAmong the fertility traits, the expression of estrous behavior (heat), a key fertility trait that marks the fertile period in cows, has decreased both in duration and intensity over generations of cows selected for high milk yield . Short hIn an effort to understand the genomic regulation of estrous behavior expression in dairy cows, a microarray experiment was set up to study gene expression levels in 4 different brain areas and the anterior pituitary of cows sacrificed at either the start of estrus (day0 of estrous cycle) or at mid-cycle (day12). Differential gene expression analysis between day0 and day12 cows for each of these tissues revealed a limited number of significant genes in the anterior pituitary alone and none in the brain areas (detailed results not reported here). When the trait of interest is quantitative, as in this case (estrous behavior quantified as heat score), the grouping of individuals into qualitative classes dilutes the available information. Therefore, the association between gene expression and phenotypic trait may be better analyzed using the individual quantitative measurements. Using this approach in an earlier study, we identified a set of a few hundred probes out of approximately 24,000 probes on a bovine microarray corresponding to genes whose level of expression in the anterior pituitary of experimental cows associated with the degree to which the individual cows expressed estrous behavior . Among tThe objective here is to identify and study those genes that associated with estrous behavior, among genes expressed in four bovine brain areas , either at the start of estrous cycle, or at mid cycle, or regardless of the phase of cycle.The degree of estrous behavior expression was quantified as a cow's average heat score using heat scores recorded from at least two consecutive cycles Table . The datAVP and MCHR1, a linear trend was observed in HC and AM respectively whereas for OXT, a quadratic trend was observed in DH.Additional file OXT, POMC, AVP, CCK, CGA) or neurotransmitter receptors  and metabolizing enzymes . We could also identify 5 more sets of genes, each consisting of between 5 to 10% of the total genes. These included genes associated with: "transport and localization" , "transporter activity" , "metabolism" (e.g. glucose and amino acid metabolism or steroidogenesis), "cell cycle" (here including processes linked to DNA folding and repair) and "multi-organism processes". In the last category we included immune system related genes like CTLA4, IL1RL1, MARCO, FCRLA and IL33.Of the total 640 unique probes found associated with heat score in all analyses and brain areas, 372 had gene annotations. Sets of genes associated with functional categories which we group as: "transcription and regulation of gene expression", "detection of and responses to stimuli" and "signaling pathways" each made up about 15% of the genes that were identified. In the category "detection of and responses to stimuli" we included the genes involved in neurotransmission through encoding for neurotransmitters/hormones  found in the different brain areas and analyses is provided in Additional file Variation in the behavioral trait to express estrous behavior was found to be associated with variation in the expression of genes in the cow's brain areas: DH, VH, HC and AM and also the AP, an endocrine gland pivotal in synchronizing estrous behavior with hormonal and ovarian events preceding ovulation. The choice of these tissues for this study was based on their reported involvement in regulating female sexual behavior. Although genes differ in their influence on specific traits, it is tempting to consider the AM to be of relatively higher importance for regulating estrous behavior as the largest number of associated genes was found here at day0 and because AM is known for its central role in regulating emotions. Key genes and biological processes as identified from the lists of heat score associated genes are discussed next and linked to estrous behavioral expression, though the links are not always as expected.CGA gene, which encodes for the alpha subunit of glycoprotein hormones , with associations being time and brain area specific. It seems that cows with clear expression of estrous behavior have relatively high expression of this gene in the hypothalamus around mid-cycle and low expression around start of estrus.An association with heat scores was detected for the OXT gene was found associated in the HC and DH whereas the AVP gene was found associated in the HC and AM. The known functional properties of these genes contributed to several enriched GO terms in the DAVID analysis, especially those related to female mating behavior. Oxytocin is released within the brain where it acts on specific oxytocin receptors to elicit effects like female sexual receptivity, grooming behavior and partner bonding [AVP is associated with sexual behavior and bonding [OXT gene deletion [OXT and AVP in several brain areas is in line with the above mentioned findings and suggests a major influence of these genes on estrous behavior expression in dairy cows.The  bonding . In the  bonding ,13. Simi bonding  and its  bonding ,16. In mdeletion . Studiesdeletion  demonstrCCK, POMC, MCHR1, GABRA6, HTR2A and DRD2, which associated with heat score in at least one brain area, are known to modulate emotional states like anxiety and satiety [DRD2) signaling in the mediobasal hypothalamus affects female sexual motivation and receptivity [HTR2A and DRD2) and gamma-aminobutyric acid (GABA) (linked to GABRA6) in the mediobasal hypothalamus being triggered by such cues only when ewes are in estrus [HTR2A) associated with heat score at day12 in VH. Studies in female rats and hamsters have shown the inhibitory and facilitatory effects of serotonin receptor agonists and antagonists on the hypothalamic regulation of sexual receptivity [The genes  satiety -21 or eveptivity . Interaceptivity . The pern estrus . We founeptivity ,26, and eptivity .PTGDS, PTGIS, PTGFR) that regulate prostaglandin functioning. In the central nervous system, prostaglandins are involved in functions like thermoregulation and influencing neuronal morphology. Prostaglandins are also known to be under the influence of estradiol [Noteworthy, are the heat score associated genes  may be explained by the effect of the neurotransmitter, acetylcholine on arousal, plasticity and reward. The products of the muscarinic cholinergic receptor genes, CHRM1 and CHRM3, are Gq-protein coupled receptors whose activation releases intracellular Ca2+ via the phospholipase C - inositol 1,4,5-trisphosphate signaling pathway [PLCB2, ITPKA) and several protein kinases were also found associated to heat score. These findings may be explained based on the hypothesis put forward by Kow and Pfaff [2+.The present association between heat scores and the expression of  pathway . The gennd Pfaff  that theTAC3 gene encodes the protein tachykinin (or neurokinin B), which in humans has been considered a critical regulator of gonadotropins  via regulating GnRH secretion [PEBP1, MOBP, LTA4H and KCNN2. PEBP1 has been suggested to be involved in chronic stress-induced memory impairment [MOBP has been linked to mood disorders [LTA4H to depression in women [KCNN2 to anxiety and stress responses [The finding of expected processes related to estrus as described in the previous section supports assumed neurophysiological mechanisms underlying female sexual behavior in dairy cows. Here, examples are given of more novel candidate genes and mechanisms. The ecretion . The prepairment , MOBP haisorders , LTA4H tin women , and KCNesponses .LIPN1 in mice seems to establish a cross-talk between reproduction and metabolic events [POU1F1 gene, which encodes transcription factors involved in activation of growth hormone and prolactin, was found associated with heat score in the AP on day0. This gene may contribute in part to the generally observed reduction in estrous behavior in high producing cows. The heat score associated gene GARNL1 is noted here because it has been linked to egg productivity in laying hens [The gene c events  and was ing hens  and, thuCTLA4, IL1RL1, MARCO, FCRLA, IL33, CCL26 and CXCL10, indicating the importance of cell-cell interactions. It has been shown that immunoglobulin superfamily proteins may play important roles in brain developmental processes and the functioning of neuronal networks in adults because they provide the ideal structure for protein-protein interactions and, thus, cell-cell interactions [A number of heat score associated genes have been linked to the immune system, e.g. ractions . Remodelractions , may alsATP1A3), which was found to be associated to heat score at estrus in AP in our earlier study was also found in VH. ATP1A3 has been implicated in rapid-onset dystonia parkinsonism (RDP), characterized by sudden onset of neurological symptoms over hours to a few days [The gene product Sodium/potassium-transporting ATPase subunit alpha-3  gene has been found associated in DH. However, care needs to be taken to interpret the results of the combined analysis where an associated gene was also found in one or both of the separate analyses. There could be cases where the day0+day12 combined analysis found an associated gene because of the effect of a strong association found in one of the separate analyses. An example of this case is HTR2A which did not associate with heat scores on day0, but did on day12 (R2 = 0.27) and also weakly (R2 = 0.1) in the day0+day12 combined analysis, probably as a carryover effect.Including data from both day0 and day12 cows in a combined analysis not only revealed genes that were associated with estrous behavior regardless of phase of cycle but also resulted in greater statistical power and helped reveal some associated genes which could not be found in the separate analysis. For example, SLC17A7 found in VH at day0 and in the combined analysis and also in the AP, is known to mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells and may also mediate the transport of inorganic phosphate [NKD1, found in several brain areas across all analyses, was seen to contribute to several GO terms related to binding and signal transduction. It has been reported to have a role as an antagonist of Wnt signaling pathway which may influence the development of neurons in dorsal midbrain [ANO8, also found associated in several brain areas and analyses, is a member of the anoctamin family which has been implicated in calcium ion-activated chloride channels that perform several important functions including neuronal excitability.Interesting observations were also made by investigating associated genes that appeared in several brain areas and analyses. For example, the gene hosphate . The genmidbrain , suggestOXT and AVP play major roles in regulating estrous behavior along with genes affecting neurotransmission and neuronal plasticity. Genes, whose expression in mid-cycle associated with estrous behavior, may contribute to preparing the cow for the next estrus. Figure To summarize, several genes were found here whose expression levels in the brain areas were associated with the degree to which cows express estrous behavior. For some of these genes, there is a known function relating them to processes regulating estrous behavior, while for others, the current association suggests such a relation. We propose that the genes OXT, AVP, POMC, MCHR1), but also genes whose association with estrous behavior is novel and warrants further investigation. Studying these genes and the processes they control improves our understanding of the genomic regulation of estrous behavior expression, ultimately leading to better management strategies or tools to improve or monitor reproductive performance.The study predicted estrous behavior associated genes in the 4 brain areas at two time points of the estrous cycle. These included genes expected to be related to estrous behavior as they influence states like socio-sexual behavior, anxiety, stress and feeding motivation  onwards till their time of sacrifice which varied between 77 and 139 DIM. The estrous behavior recorded in these cows were quantified as heat scores and the scores from multiple consecutive cycles per cow were averaged to obtain the average heat score per cow . Of the http://www.bovineoligo.org/. This array was amongst the few whole genome bovine spotted microarrays available at the time this experiment was performed in the year 2007-08. The choice of microarray technology over RNA sequencing, which was not a well established technology then, was appropriate for fulfilling the objectives of this study in a precise and cost effective manner. The procedures followed for RNA preparation and microarray hybridization were as described in our earlier study [RNA extracted from brain tissue samples were hybridized on Bovine 24K oligonucleotide (70-mer) microarrays designed and produced by the Bovine Oligonucleotide Microarray Consortium (BOMC), USA er study . The purhttp://www.ebi.ac.uk/arrayexpress).The array experiment data is publicly accessible on ArrayExpress  package  within Ber study . Gene ex [et al.  that emp [et al.  coded inn the AP , here wen the AP . Howevern the AP . We perfhttp://www.ensembl.org release 56 (October 2009) provided on the EADGENE website by the authors of the oligo-set re-annotation pipeline, sigReannot [The original annotation of the bovine 24 K oligonucleotide microarray provided by BOMC dates back to June 2007. For our analysis, we used the bovine oligonucleotide array probe re-annotation (Version 5) based on Ensembl gReannot . For theFor gaining insight into biological processes underlying estrous behavior, we performed functional analyses of the sets of estrous behavior associated genes (study sets) identified in the 3 analyses for the 4 brain areas and the anterior pituitary. To increase the accuracy of functional analysis, we used the re-annotated probes information. The probe re-annotation also provides the Ensembl gene ID of the orthologous human genes for 14,585 probes, which we used in the functional analysis instead of bovine genes so as to benefit from the greater gene annotation information available for human species.http://david.abcc.ncifcrf.gov/ that integrates biological data from several sources including GO [Functional analysis was done using DAVID bioinformatics resources ,53, a fruding GO  and bioluding GO  thresholuding GO  pathway AM: Amygdala; AP: Anterior Pituitary; BOMC: Bovine Oligonucleotide Microarray Consortium; DAVID: Database for Annotation, Visualization and Integrated Discovery; DH: Dorsal Hypothalamus, EADGENE: European Animal Disease Genomics Network of Excellence for Animal Health and Food Safety; FDR: False Discovery Rate; FSH: Follicle stimulating hormone; GnRH: Gonadotropin-releasing hormone; GO: Gene Ontology; HC: Hippocampus; KEGG: Kyoto Encyclopedia of Genes and Genomes; LH: Luteinizing hormone; QTL: Quantitative trait loci; SD: Standard Deviation; TSH: Thyroid stimulating hormone; VH: Ventral Hypothalamus;AACW performed the microarray experiments. AK performed the data analysis and drafted the manuscript. HAM and RFV helped with the statistical analysis. BB, HW, MFWP and MAS helped with the biological interpretation of the results. All authors read and helped in improving the manuscript.Association patterns between gene expression and heat score of estrous behavior associated genes identified in the four brain areas and anterior pituitary. See legend at end of table.Click here for fileVenn diagrams showing the number of overlapping probes between the different brain areas per analysis. The figures in brackets represent the total number of estrous behavior associated probes found in each brain area per analysis. Here, AM - Amygdala; HC - Hippocampus; DH - Dorsal Hypothalamus; VH - Ventral Hypothalamus; AP - Anterior Pituitary.Click here for fileSignificantly enriched Gene Ontology and KEGG pathway terms in the estrous behavior associated gene lists. Table shows all terms with p < 0.10 and includes gene ontology terms in the 3 categories: Biological Process, Molecular function and Cellular component.Click here for fileCollection of brain samples and pituitary from the experimental cows. Procedure followed for collection of anterior pituitary and brain samples: amygdala, hippocampus, dorsal hypothalamus and ventral hypothalamus (with pictures).Click here for file


The production of ethanol has been considered as an alternative to replace part of the petroleum derivate. Brazil and the US are the leading producers, but more environmentally friendly alternatives are needed. Lignocellulose has an enormous potential but technology has to be still improve in order to economically produce ethanol. The present paper reviews the potential and problems of this technology and proposes the study of a group of microorganisms with the largest genetic pool, marine microorganism. In 2010, the human population was estimated to be 6.8 billion and projected to reach 7.6 billion in 2020   , totally replaced lead additives as octane enhancer. Since 2000, the almost uncontrolled growth of supply and demand of ethanol, replacing MTBE (methyl tertiary- butyl ether), also alleviated some of the dependence to oil. In 2005, the renewable fuel standard (RFS) program was created under the Energy Policy Act (EPAct of 2005), and established the first renewable fuel volume mandate in the United States, this program required 7.5 billion gallons of renewable fuel to be blended into gasoline by 2012  of 2007 to include diesel, in addition to gasoline to increase the volume of renewable fuel required to be blended into transportation fuel from 9 billion gallons in 2008 to 36 billion gallons (bgy) by 2022. This constitutes a four-fold increase over current biofuel production and 25% of current motor fuel use. The mandate envisions eventual substitution of advanced cellulosic biofuels for currently produced corn based ethanol. But while achieving this goal, it doubles the use of corn based ethanol despite widespread concerns about increased food prices  , nitrate reducers or denitrifiers (reduces NO3- to N2), Mn (IV) reducers (reduce MnO2 to Mn (II)), Fe (III) reducers (reduces Fe2O3 or FeO (OH) to Fe (II)), sulfate reducers (reduces SO22- or S- to HS), or methanogens (reduce CO2 to CH4). They also found that strong iron ion chelators and polyethylene glycols could mitigate the inhibition.Cellulases can also be susceptible to interference from various noncellulosic substances. Tejirian and Xu  showed cBoth anaerobic bacteria, as well as anaerobic fungi, are known to produce multienzyme complexes termed cellulosomes; in contrast, aerobic microorganisms produce their cellulase components as single enzymes. It has been suggested that the cellulosome provide anaerobic microorganisms with an advantage to degrade cellulose more efficiently, since cellulosomal cellulases degrade cellulose in a simultaneous manner rather than in a sequential manner , which involves four discrete process steps. Simultaneous saccharification and fermentation (SSF) consolidates hydrolysis and fermentation of cellulose hydrolysis products into one process step, with cellulase production and fermentation of hemicellulose hydrolysis products occurring in two additional separate process steps. Simultaneous saccharification and co fermentation (SSCF) involves two process steps: cellulase production and a second step in which cellulose hydrolysis and fermentation of both cellulose and hemicellulose hydrolysis products occurs Lee .This technology has several problems; including the growth inhibition by harmful products formed during biomass hydrolysis, resulting in low alcohol concentration. These problems contribute to the high cost of lignocellulosic ethanol by increasing capital expenditure, reducing product yields, and increasing water volumes that must be handled as part of relatively dilute product streams. Similar to the traditional fermentation, the major process steps for cellulosic fermentation plant are as follows:Pretreatment: Among of the chemical treatments used dilute acid at higher temperature has been widely used as it is highly active, inexpensive and easy to execute. In this step, biomass feedstock undergoes a process to break down the hemicellulose fraction of the feedstock into a mixture of soluble five-carbon sugars, such as xylose and arabinose, and soluble six-carbon sugars, such as mannose, galactose, and glucose, it also reduce cellulose crystallinity and increase the porosity of the materials , reciprocally control biofilm formation  , the use of blockers or inducers (QS) as a enhancing tool could also result in the isolation of right strains needed by the industry. In addition, phages are known to carry a variety of host genes, not only in a negative effect but as it has been shown it can also augment the metabolism, immunity and distribution of the host in many unexpected ways (Rohwer and Vega Thurber, The author declare that he have no competing interests.


Orexin, cardiovascular function, and hypertension. In normal rats, central administration of orexin can induce significant increases in arterial blood pressure (ABP) and sympathetic nerve activity (SNA), which can be blocked by orexin receptor antagonists. In spontaneously hypertensive rats (SHRs), antagonizing orexin receptors can significantly lower blood pressure under anesthetized or conscious conditions. (2) Orexin, respiratory function, and central chemoreception. The prepro-orexin knockout mouse has a significantly attenuated ventilatory CO2 chemoreflex, and in normal rats, central application of orexin stimulates breathing while blocking orexin receptors decreases the ventilatory CO2 chemoreflex. Interestingly, SHRs have a significantly increased ventilatory CO2 chemoreflex relative to normotensive WKY rats and blocking both orexin receptors can normalize this exaggerated response. (3) Orexin, central chemoreception, and hypertension. SHRs have higher ABP and SNA along with an enhanced ventilatory CO2 chemoreflex. Treating SHRs by blocking both orexin receptors with oral administration of an antagonist, almorexant (Almxt), can normalize the CO2 chemoreflex and significantly lower ABP and SNA. We interpret these results to suggest that the orexin system participates in the pathogenesis and maintenance of high blood pressure in SHRs, and the central chemoreflex may be a causal link to the increased SNA and ABP in SHRs. Modulation of the orexin system could be a potential target in treating some forms of hypertension.In this review we focus on the role of orexin in cardio-respiratory functions and its potential link to hypertension. (1)  Mounting evidence suggests that orexin is not only important to sleep-wake cycle and feeding regulation but also to respiratory and cardiovascular regulation. Furthermore, orexin may play role in some types of hypertension.Orexins, also known as hypocretins (Hcrt), are two excitatory neuropeptides, orexin A and orexin B (OX-A and OX-B) or hypocretin 1 and hypocretin 2 (Hcrt1 and Hcrt 2), produced by neurons primarily located in the lateral hypothalamic area (LHA)  in hypothalamic slices prepared from orexin neuron/enhanced green fluorescent protein (EGFP) transgenic mice , the cell bodies of orexin neurons are strictly located in the perifornical area and lateral and dorsal hypothalamic areas , and PVN in Wistar rats  and activates neurons of the lateral paragigantocellular area , e.g., neurons in the hypothalamic PVN and the RVLM  significantly reduced the number of neurons activated by OX-A, while co-application of OX1R and OX2R antagonists completely eliminated orexin A-induced depolarization  plays a crucial role in the regulation of circulation and blood pressure Guyenet, , and mann Figure . Furthern Figure .in vivo have demonstrated that orexin participates in blood pressure regulation. Transgenic orexin deficient animals, both prepro-orexin knockout (pp-OX KO) mice  with RNAi or antagonizing OX1Rs can block blood pressure and HR responses to acute panic stress  or the RVLM in anesthetized SHRs significantly decreased blood pressure  orexin participates in the regulation of cardiovascular homeostasis, (b) exogenous orexins can increase SNA and blood pressure in normal animals, and (c) transgenic orexin deficient animals have lower resting blood pressure, have led two independent groups to test the hypothesis that orexin may participate in the development of neurogenic hypertension in spontaneously hypertensive rats (SHRs)  gene is down-regulated in the adrenal gland in SHR and stroke-prone SHR (SHRSP) relative to normotensive WKY rats. This raises an interesting question of the role of peripheral orexin on cardiovascular function. As mentioned above, orexin neurons are exclusive located in the lateral hypothalamus and send projections to many brain locations. However orexins are found in many peripheral tissues, and the peripheral orexin distributions and functions have been discussed by recent reviews  , or a selective OX2R antagonist (TCS OX2 29) into RVLM can partially block the OX-A induced increased SBP and HR in SIHR  Transgenic orexin deficient animals have a lower resting blood pressure. (2) Blocking OXRs significantly lowers blood pressure and SNA in adult SHRs. (3) PP-OX mRNA and gene expression are upregulated in the CNS in hypertensive rats and mice. Based on these findings we suggest that an overactive orexin system in the CNS may participate in the pathogenesis and maintenance of high blood pressure in certain forms of hypertension. Further, modulation of the orexin system could be a potential target in treating some forms of hypertension.In normal animals, activation of OXRs by orexin in the CNS or in areas critically involved in SNA regulation, e.g., the RVLM and spinal cord, induces sympathetically mediated hypertension and tachycardia, which can be attenuated by OXR antagonists. Transgenic orexin deficient animals have a lower resting blood pressure. Spontaneously hypertensive rats and mice may have an overactive orexin system as (1) blocking OXRs produces significant anti-hypertensive effects in SHRs, (2) the expression of OX-A mRNA in increased in RVLM in SHRs and (3) the pp-OX/Hcrt gene is upregulated in hypothalamus in hypertensive BPH/2J mice. Orexin is important in the regulation of SNA and blood pressure and an overactive orexin system may be pathologically linked to the development of neurogenic hypertension.1R and/or OX2R mRNA is also found abundantly in many respiratory nuclei in the brainstem with a pattern matching that of OX-containing axon terminals  in the RTN significantly reduced the respiratory response to hypercapnia (7%CO2) with a substantial effect in wakefulness ; the CO2 chemoreflex was not significantly changed in sleep in the dark period and wakefulness and sleep in the light period of the diurnal cycles  Orexin may be a link between breathing and sleep-wake status.In summary: (1) Orexin neurons are intrinsically chemosensitive and send projections to other central chemoreceptor sites, e.g., the RTN and medullary raphe. (2) Orexin may regulate breathing and central chemoreception directly and/or indirectly by recruiting other brainstem chemoreceptor sites, e.g., the RTN and medullary raphe. (3) Orexin modulation of the COAnatomically, many cardiovascular and respiratory related nuclei are closely intertwined within similar regions of the brain, or are even synaptically connected. Many of these nuclei are part of the respiratory-sympathetic network that is critical to the regulation of respiratory and sympathetic activity and blood pressure  induced greater increases in ventilation (E), blood pressure and SNA than did hypoxia (10%O2/93%N2)  Many putative central chemoreceptors sites, e.g., the RTN, NTS and medullary raphe, are involved in the regulation of SNA and blood pressure. (2) Activation of central chemoreceptors by hypercapnia significantly increases blood pressure, SNA and respiration with and without peripheral chemoreceptors. These studies suggest that the hypercapnic induced increase in ABP is due to sympatho-excitation via activation of the central chemoreceptors. The central chemoreceptors directly or indirectly regulate sympathetic vasomotor tone and blood pressure, and can activate the sympathetic outflow in a tonic manner independent of the effects on the central respiratory pattern generator  channels in young pre-hypertensive SHRs , or hyperoxic hypercapnia (7%CO2/93%O2) when peripheral chemoreceptors are suppressed, is significantly higher in both wakefulness and sleep than that of the normotensive WKY control rats , ABP remains significantly above the normal even at 21 days post CSD  Can antagonism of OXRs be a target to treat such an augmented CO2 response and the associated hypertension in SHR? At present, we have found that antagonism of both OXRs using an orally administered dual orexin receptor antagonist, Almxt: (1) significantly reduced the exaggerated CO2 chemoreflex in SHR to the same level as measured in the control normotensive WKY rats  The orexin knockout mouse has a decreased respiratory hypercapnic chemoreflex as well as a lower resting ABP  while SHRs have severe hypertension, a hyperactive central hypercapnic reflex, and possibly an overactive orexin system. Blocking both orexin receptors in SHR can normalize the exaggerated hypercapnic chemoreflex and significantly lower blood pressure, which suggests the overactive central chemoreceptors may be an important link in neurogenic hypertension with orexin as mediator in SHRs.Orexin knockout mice have both a severely attenuated hypercapnic chemoreflex and lower resting blood pressure, while SHRs have severely increased blood pressure and enhanced peripheral and central chemoreflexes with a possibly upregulated central orexin system. Antagonism of both orexin receptors can significantly lower SNA and blood pressure and normalize that central hypercapnic chemoreflex in SHRs. We suggest that overactive central chemoreceptors may be an important link to the development and maintenance of high blood pressure in SHRs with orexin as a key mediator.2 chemoreceptors is associated with sympatho-excitation, hypertension and tachycardia. In SHRs, the overactive central and peripheral chemoreflex may play an important role in the development of neurogenic hypertension. Orexin links the respiratory and sympathetic nervous systems and an overactive orexin system may be the cause of hyperactive central CO2 chemoreflex in SHRs and thus the associated hypertension. Antagonism of OXRs can normalize the overactive CO2 central chemoreflex and significantly lower ABP and SNA in SHRs. Based on the data obtained from SHRs we hypothesize that modulation of the orexin system could be a potential target in treating some forms of hypertension.Activation of central COThe 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.


Dengue has emerged as the most important life-threatening illness in the world, especially in Asian countries around Hong Kong, where the incidence of dengue hemorrhagic fever (DHF) is much greater than other continents. However, little is known about molecular and cellular processes sustaining egress of dengue virus in the host cell.To better understand the viral and cellular determinants of dengue virus egress, we have established a dengue VLP producing stable cell line (HeLa-prME) and demonstrated that dengue VLP was able to mimic the budding and egress process of dengue viruses so that it constitutes a safe and convenient tool for the study of egress of dengue virus. Under the support of RFCID grant, HeLa-prME cells were used to screen a siRNA library that included 122 cellular membrane trafficking genes.Our screen results revealed that knockdown of ADP-ribosylation factor (ARF) 1 and ARF6 had significant effects on VLP production by HeLa-prME cell. Experiments with other ARF proteins, which were not included in the siRNA library, showed that the ARF4/ARF5 double knockdown could inhibit VLP production but had no effect on secretion of other proteins such as soluble dengue E protein, suggesting the specificity of their involvement in VLP production. Further experiments using real virus showed that the depletion of ARF4/5 by siRNA could significantly reduced the replication of dengue 1 virus, dengue 4 virus and yellow fever virus, confirming the important role of ARF4 and ARF5 for not only dengue viruses but also other flaviviruses.Our study uncovered the importance of class II ARFs in the egress of dengue virus. Results from this project provided information on mechanism of dengue virus assembly and its dependence on cellular machineries.


There is concern that antibiotic resistance can potentially be transferred from animals to humans through the food chain. The relationship between specific antibiotic resistant bacteria and the genes they carry remains to be described. Few details are known about the ecology of antibiotic resistant genes and bacteria in food production systems, or how antibiotic resistance genes in food animals compare to antibiotic resistance genes in other ecosystems. Here we report the distribution of antibiotic resistant genes in publicly available agricultural and non-agricultural metagenomic samples and identify which bacteria are likely to be carrying those genes. Antibiotic resistance, as coded for in the genes used in this study, is a process that was associated with all natural, agricultural, and human-impacted ecosystems examined, with between 0.7 to 4.4% of all classified genes in each habitat coding for resistance to antibiotic and toxic compounds (RATC). Agricultural, human, and coastal-marine metagenomes have characteristic distributions of antibiotic resistance genes, and different bacteria that carry the genes. There is a larger percentage of the total genome associated with antibiotic resistance in gastrointestinal-associated and agricultural metagenomes compared to marine and Antarctic samples. Since antibiotic resistance genes are a natural part of both human-impacted and pristine habitats, presence of these resistance genes in any specific habitat is therefore not sufficient to indicate or determine impact of anthropogenic antibiotic use. We recommend that baseline studies and control samples be taken in order to determine natural background levels of antibiotic resistant bacteria and/or antibiotic resistance genes when investigating the impacts of veterinary use of antibiotics on human health. We raise questions regarding whether the underlying biology of each type of bacteria contributes to the likelihood of transfer via the food chain. American consumers and producers are increasingly aware of concerns over antibiotic resistance in food. In animal agriculture, the specific concern is that the use of antibiotics in food animals promotes the growth of antibiotic resistant bacteria that can then be transferred to humans via food processing and distribution systems While a broad conceptual framework has been worked out describing how use of antibiotics in food animals has the potential to adversely impact human health In microbial community metagenomic studies, total DNA is isolated from a complex sample, and then subjected to high-throughput sequencing. Unlike 16S rRNA sequencing which relies on a PCR step to target the 16S rRNA gene specifically, in metagenomic studies there is no PCR step, and the entire genomic DNA of a sample is sheared and sequenced directly. This direct sequencing allows for the collection of quantitative data that is not possible with 16S rRNA-based studies. Metagenomic sequencing theoretically provides information on all of the genes present in a sample, and data analysis tools such as MG-RAST Antibiotic resistance is a broad term, and is commonly used to denote the presence and/or expression of at least one antibiotic resistance element. There are multiple classes of antibiotics, multiple mechanisms of resistance within classes, and multiple genes within each functional group. In this study, we used the MG-RAST classification of resistance to antibiotic and toxic compounds (RATC) to categorize functional genes associated with antibiotic resistance, and quantified based on absolute numbers or percent within a metagenome or RATC class.Table S1). Previous work evaluating the distribution of functional genes in metagenomes from nine biomes reported the mean percentage of genes assigned virulence functions was 9.79% In order to determine the distribution of genes associated with resistance to antibiotic and toxic compounds (RATC) from agricultural, environmental, and human-associated samples, we analyzed publicly available metagenomic datasets . In other words, microbial communities from marine environments have a smaller percentage of their total genetic resources allocated to RATC genes, suggesting that the bacteria in these habitats encounter fewer antibiotic and toxic compounds, compared to bacteria from the terrestrial and gastrointestinal samples. Based upon prevalence, 22 of the 26 metagenomes could be grouped into one of four classes–fecal (4.04 to 3.75% RATC), gastrointestinal tract (3.03 to 2.66% RATC), aquatic terrestrial ecosystems (2.24 to 2.15% RATC), and aquatic marine ecosystems (2.00 to 0.70%RATC). The percentage RATC in ungrouped metagenomes including kimchi, whale fall, soy phyllosphere, and farm soil ranged from 3.09 to 3.62%. An obvious relationship between increased incidence of RATC and cell density is evident from these samples. Survival in microbially-dense, very active environments  compared to lower density environments (aquatic ecosystems) may involve coping with toxic metabolites and antibiotics. What is the role for anthropogenic antibiotic use and prevalence of RATC? Certainly samples within fecal and gastrointestinal tract groups were likely exposed to anthropogenic antibiotics at some point in time, but the high rate of RATC in kimchi, whale fall, and termite gut, which probably were never treated with antibiotics, indicate that the effect of antibiotic exposure on percent RATC is more complicated.The RATC group of genes represents a subset of virulence genes that ranged from 0.70% to 4.04% of classified metagenome gene sequences Figure 2The current metagenomic analysis affords a first glimpse of antibiotic gene distribution across metagenomes. For example, although the four North American costal samples display the same general distribution of RATC genes, slight differences can be observed for specific RATC classes and locations. The Chesapeake Bay metagenome has a higher proportion of assignments in the tripartite Gram negative multi-drug resistance (MDR) (2.6%) and tetracycline resistance (2.6%) categories compared to the Gulf of Main, Key West, and the Gulf of Mexico , and a lower proportion of the acriflavin resistance cluster and beta-lactamase resistance . Difference in RATC distribution can also be observed in the cattle rumen and fecal samples, with both fluoroquinolone resistance and tetracycline resistance genes being lower in feces  than in rumen samples . The differences between the fecal and rumen samples likely reflect the underlying biological functions of the two gastrointestinal locations, and serve as a reminder of the dynamic nature of the ecology of antibiotic resistance genes in food production settings. Two other metagenomes of interest are the fish gut and the fermented kimchi. The fish gut contains a much higher proportion of mercury resistance, mercuric reductase, and cobalt/zinc/cadmium genes than any other metagenome in the analysis . The day 29 kimchi fermentation contains proportionately more of the two-protein Gram positive MDR genes (12.3%) compared to any other metagenome in the analysis . Both the fish and the kimchi results make sense in light of what we already know about the biology of the two systems: fish are known to concentrate mercury, and vegetable fermentations specifically select for Gram-positive organisms. The data set assembled for the current analysis contains three human fecal and three cattle rumen metagenomes. Within each “replicated” metagenome type, the RATC values are fairly similar. While all of the metagenome results await confirmation via replication over multiple samples and over time, the limited data available at this point suggests that there are characteristic RATC profiles for specific metagenomes. Previous work examining metagenomic profiles revealed biome-associated metabolic profiles, including gene assignments to the functional category of “virulence”. The current study extends the conclusions of Disndale et al. Figure S1). Of these RATC categories, beta-lactamase resistance, MDR efflux pumps, fluoroquinolone resistance, cobalt/zinc/cadmium resistance, and acriflavin resistance genes were present in all 26 metagenomes. This broad distribution across agricultural, environmental, and human-associated samples indicates that these mechanisms of antibiotic resistance are functionally important in many habitats, however the current analysis is unable to determine if these genes have been transferred between habitats.All metagenomes examined contained antibiotic resistance genes. Thirty-three different RATC categories were represented in the 26 metagenomes examined , suggesting that there are differences in the numbers and kinds of antibiotic resistance in these general categories of metagenomes. Specifically, agricultural and human-associated samples each form distinct clusters within quadrant III, and aquatic samples, including a coastal marine cluster, were distributed within quadrant IV and nearby in quadrant I . The majority of beta-lactamase resistance genes in this particular set were associated with the soy phyllosphere (23%), followed by Galapagos (19%), canine (17%), cattle fecal (10%), and Key West (9%). Across the 16 metagenomes, Beta-lactamase genes were most frequently associated with Alpha- and Gammaproteobacteria, which together account for 36% of all Beta-lactamase genes in the 16 metagenomes studied. It is not clear why the leaf of a soybean plant contained the largest proportion of beta-lactamase genes from the 16 metagenomes examined; however extended-spectrum beta-lactamase gene sequences have been identified on both conventional and organic retail spinach Beta-lactam antibiotics Figure 4MDR efflux pumps Figure 5Fluoroquinolone resistance Figure 6Overall, 90% of Tetracycline resistance genes from the 16 metagenomes sampled were associated with canine and animal agriculture metagenomes Figure 7The canine metagenome accounted for 34% of all vancomycin resistance genes found in the 16 metagenomes examined Figure 8Escherichia coliAs with all gene-based surveys, the data indicate only the presence or absence of the target gene, but provide no information on whether the gene came from a living or dead bacterium, or if the gene is expressed in any particular habitat. The concept of HGT, where living bacteria can pick up genes released by dead bacteria, is a key element in the model describing how antibiotic resistance genes from animals can cause clinical health problems for humans. Knowing which bacteria are likely carrying specific antibiotic resistance genes raises the question of whether some bacteria are more relevant than others in the discussion of antibiotic resistance in food animals. For example, it has long been known that the major source of bacterial contamination on beef during processing comes from the animal hide, not the feces Our results support those of Wright et al. Antibiotic resistance genes are common in both agricultural and non-agricultural habitats, although differences exist in the diversity and taxonomic distribution of the bacteria associated with these genes in agricultural and non-agricultural metagenomes. Since antibiotic resistance genes are a natural part of both human-impacted and pristine habitats, presence of these resistance genes in any specific habitat therefore is not sufficient to indicate or determine impact of anthropogenic antibiotic use.Figure S1RATC assignments for each of 26 metagenomes. Percent of RATC genes in each metagenome that are assigned to each RATC class. The highest values are denoted in red, the lowest values in green.(PDF)Click here for additional data file.Table S1Metagenome Statistics.(DOC)Click here for additional data file.Table S2Resistance classes and genes used in this study.(DOC)Click here for additional data file.Table S3Distribution of five categories of resistance genes across 16 metagenomes, listed as percent of total genes in that category for all 16 metagenomes.(DOC)Click here for additional data file.


But the real scientific data show that:(a) Dental amalgam is by far the main source of human total mercury body burden. This is proven by autopsy studies which found 2-12 times more mercury in body tissues of individuals with dental amalgam. Autopsy studies are the most valuable and most important studies for examining the amalgam-caused mercury body burden.(b) These autopsy studies have shown consistently that many individuals with amalgam have toxic levels of mercury in their brains or kidneys.(c) There is no correlation between mercury levels in blood or urine, and the levels in body tissues or the severity of clinical symptoms. SCENIHR only relied on levels in urine or blood.(d) The half-life of mercury in the brain can last from several years to decades, thus mercury accumulates over time of amalgam exposure in body tissues to toxic levels. However, SCENIHR state that the half-life of mercury in the body is only "20-90 days".(e) Mercury vapor is about ten times more toxic than lead on human neurons and with synergistic toxicity to other metals.(f) Most studies cited by SCENIHR which conclude that amalgam fillings are safe have severe methodical flaws. SCENIHR  from the European Commission claim : "ExposuSCENIHR did not cite any autopsy studies, which are the most reliable studies for assessing mercury levels in tissues.An approx. 2-5-fold increase of mercury levels in blood and urine in living individuals with dental amalgam as well as a 2-12 fold increase in several body tissues was observed in deceased individuals with dental amalgam . AdditioAccording to these studies, dental amalgam is responsible for at least 60-95% of mercury deposits in human tissues. This was not acknowledged by SCENIHR.SCENIHR  state thIn contrast to this claim studies have shown that mercury (Hg) from dental amalgam is transformed into organic mercury compounds by microorganisms in the human gastrointestinal tract -31. LeisInorganic mercury levels of 0.02 ng Hg/g (2 μl of 0.1 μMolar Hg in 2 ml substrate) led to the total destruction of intracellular mircrotubuli and to the degeneration of axons . In otheMercury vapor inhalation in doses which also occur in humans with many amalgam fillings and chewing led to pathological changes in the brains of animals after 14 days ,36.In a recent autopsy study, it was found that individuals with more than 12 amalgam fillings have more than 10-times higher mercury levels in several tissues including the brain, compared to individuals with only 0-3 amalgam fillings .The average mercury level in the brain of EU citizens with more than 12 amalgam fillings was 300 ng Hg/g brain tissue , which iIn another autopsy, individuals with more than 10 amalgams have 504 ng Hg/g in their kidney tissues  and 83.3 ng Hg/g in the liver   is very low".A simple literature research reveals quite the opposite: Dentists working with amalgam have an increased mercury exposure ,181,182.Even 30 years after cessation of mercury exposure, dental nurses showed significant adverse health effects . In spitSCENIHR stated that "There is no evidence of any association between amalgam restorations and either male or female fertility or obstetric parameters". As a proof of this statement, SCENIHR cited just one study, which examines only semen parameters in men. However, other studies point to the opposite direction, especially when examining women:Female dental assistants exposed to amalgam showed a higher rate of infertility . Women wA 7,5-fold increased level of mercury was found in the cerebrospinal fluid (CSF) of MS patients . It woulMS patients who had their amalgam fillings removed showed fewer depressions, less aggression and less psychotic and compulsory behaviors when compared to a group of MS patients with amalgam fillings . They alSCENIHR state that "there is no evidence for a relationship between Amyotrophic Lateral Sclerosis (ALS) and mercury"In contrast to the statement of SCENIHR, there are many studies which suggest that mercury may play a pathogenetic role in ALS:Mercury vapor is absorbed by motor neurons  where itAmong the most frequently reported symptoms due to amalgam fillings are: Chronic fatigue, headache, migraine, increased susceptibility to infections, muscle pain, lack of concentration, digestion disorders, sleeping disorders, low memory capacity, joint pain, depression, heart sensations, vegetative disregulation, mood disorders and many more ,230-234.Until recently it was not possible to differentiate between „amalgam-sensitive" and „amalgam-resistant" persons by their mercury levels in blood or urine or an epicutaneous test (patch test) ,21. HoweSCENIHR did not mentioned any susceptibility parameters which make a significant proportion of the population more susceptible to mercury from dental amalgam:It is known that mercury exposure leads to aberrant urine porphyrine profiles in dentists  and autiA genetic polymorphism of coproporphyrinoxidase (CPOX4) ,201 leadThe critical question here is the effect of mercury vapor exposure on brain porphyrine profiles since an aberrancy in brain heme has been associated with the inability to remove beta-amyloid protein from brain cells, which in turn may lead to Alzheimer's disease .It should be noted that porphyrins lead to heme, and heme is critical for several biochemical mechanisms: (i) heme is the oxygen carrying cofactor for haemoglobin, (ii) heme is a critical cofactor for the P450 class of enzymes that are responsible for detoxifying xenobiotics from the body, (iii) heme is a necessary cofactor for one of the complexes in the electron transport system of mitochondria and therefore ATP-synthesis.Therefore, mercury inhibition of heme production could have a multitude of secondary effects causing human complaints and illnesses.In spite of the fact that 85% of the dentists and dental technicians tested showed mercury related toxicities in both behavior and physiological parameters, and 15% showed an increase of mercury induced neurological deficits with polymorphism of the CPOX4 gene, organized dentistry and SCENIHR still maintain that amalgams do not cause any significant medical problems because the urine and blood levels are below safety limits.Another genetic polymorphism of the brain derived neurotrophic factor (BNDF) increases also the susceptibility to very low level mercury exposure ,187.It could be shown that amalgam sensitive persons are significantly more likely to be carriers of the apolipoprotein E4-allel (APO-E4) than symptom free controls and that they are less likely to carry the APO-E2 ,234. APOReduced glutathione (GSH) is the main natural chelator for heavy metals in the body due to its sulfhydryl-containing cysteine. Only mercury, which is bound to glutathione (or selenium), is capable of leaving the body via urine or biliary excretion. Thus, a high level of glutathione is crucial for mercury metabolism. It has been described that polymorphisms in genes leading to impaired GSH production cause higher retention of inorganic and organic mercury in the body. Other factors which may increase susceptibility to low dose mercury exposure, e.g. low levels of selenium, abnormal reaction of neutrophil granulocytes, activity of super oxide dismutase, D4-receptor positive methionine synthetase and impaired methionine transulfuration- and methylation pathways (about 15% of the population), led to decreased mercury protecting agents, like S-adenyl-methionine, cysteine, GSH and metallothionine ,245-247.Significant improvement of health and above mentioned diseases (including Multiple Sclerosis and other autoimmune diseases) have been reported after amalgam removal (in most studies with elaborate protective measures to minimize mercury exposure) ,248-251.SCENIHR stated that "There is no evidence of a causal relationship between dental amalgam and autism" and "... that no link has been yet established between vaccines, thimerosal and autism".Nonetheless other authors come to opposite conclusions:"...mercury exposure altered cell number and cell division; these impacts have been postulated as modes of action for the observed adverse effects in neuronal development. The potential implications of such observations are evident when evaluated in context with research showing that altered cell proliferation and focal neuropathologic effects have been linked with specific neurobehavioral deficits ." Cheuk and Wong (2006) in patients diagnosed with attention-deficit hyperactivity disorder and Desoto and Hitlan (2007) in patients diagnosed with autistic disorders found significant elevations in blood mercury levels in comparison with controls ,254. AdaRecent brain pathology studies have revealed elevations in mercury levels and mercury-associated oxidative stress markers in patients diagnosed with autistic disorders. The level of mercury in the urine of autistic children shows an increase of 3-5 times after appropriate treatment with the mercury chelator DMSA compared to healthy children . AutistiZahir et al. (2005) described that the access of mercury"...to man through multiple pathways air, water, food, cosmetic products and even vaccines increase the exposure. Fetuses and infants are more susceptible to mercury toxicity. Mothers consuming diet containing mercury pass the toxicant to fetuses and to infants through breast milk. Decreased performance in the areas of motor function and memory has been reported among children exposed to presumably safe mercury levels [...] Mercury has been found to be a causative agent of various sorts of disorders, including neurological, nephrological, immunological, cardiac, motor, reproductive and even genetic. Recently heavy metal mediated toxicity has been linked to diseases like Alzheimer's, Parkinson's, Autism, Lupus, Amyotrophic lateral sclerosis, etc.".Some studies which found no associations between mercury exposure and autism have severe methodical flaws .In order to study toxic effects it is necessary to compare at least two samples: one that was exposed to the substance in question and one that was not. One of the main problems in most of the amalgam studies is that the vast majority did not incorporate a true control group that had never been exposed to dental amalgam. Even when comparing samples with and without dental fillings, the sample without dental fillings had been exposed to dental amalgam earlier in life. The studies cited frequently not only by SCENIHR as a proof of the putative harmlessness of amalgam do not use "proper" non-amalgam control groups. There is a prominent example to describe:The Swedish twin study  actuallySCENIHR also cited Zimmer et al. (2002) as a proof of the safety of amalgam. But this study compared two groups exposed to amalgam  in terms of mercury levels in body fluids and psychometric tests. The mean number of amalgam fillings was identical in both groups. They found equal mercury levels in both amalgam groups. Zimmer et al. p. 210) conclude: "Thus, mercury released from amalgam fillings was not a likely cause of complaints reported by the amalgam sensitive subjects" [0 concludSCENIHR based their statement about the safety of dental amalgam also on two children amalgam trials. These studies show severe methodical flaws:In two randomised trials on children it was evaluated whether mercury containing dental amalgam had adverse neuropsychological or renal effects ,278. HeaPower calculation  indicates that psychological illness, having prevalence of 6.7% in the composite-treated children, would have to have had a prevalence of at least 14.5% in the amalgam group to have an 80% chance of being proven statistically (observed was 9.0%). Similarly for neurological illness, observed prevalences in the composite group  would have needed at least 4.5% prevalence in the amalgam group to be significant. From the authors it was concluded that "there is no reason to discontinue use of mercury amalgam"  and thatThe first conclusion is a classic error: Due to its lack of power, the study provides false reassurance that mercury is 'safe'. To effectively evaluate the effect sizes seen, the trial should have been much larger (1500-2500/group).2/day mercury was released from a "non-mercury releasing amalgam" and this remained constant over the study period of 2 years [Urine porphyrin profiles and markers of oxidative stress, which are elevated in individuals with dental amalgam ,119 were 2 years .Mean mercury urine levels were significantly higher in the amalgam groups ,278, altOne of the included criteria for the two studies was "no interfering health conditions" including neurodevelopmental disorders. The Centers for Disease Control and Prevention (CDC) in Atlanta (USA) reports that 1 in 6 American children have a neurodevelopmental disorder. However, above mentined papers conclude that amalgams should remain a viable clinical option in dental restorative treatment  and theyThere has been an alarmingly increase of mercury in our environment  and humand largest user group in the EU [In the European Union (EU) the usage of amalgam amounts to 120 tons yearly. Dentists are the 2n the EU ,284.Recent calculations done by Hylander ,285 showThis mercury from dental amalgam  enter into the environment. When including environmental costs into the economic calculation , amalgam is the most costly dental material as was shown by Hylander and Godsite .The SCENIHR amalgam expert group consisted of one engineer (chairman), four dentists, a toxicologist and two veterinarians. The chairman has tight contacts to the industry. No experts for medicine or environmental medicine were included. One must wonder why it were the dentists who represented the strongest party in SCENIHR.Due to their education and clinical experience, dentist are not able to judge medical systemic adverse side effects caused by dental amalgam, like multiple sclerosis, autism, autoimmunity, Alzheimer's disease, psychiatric diseases etc. Usage of dental amalgam may increase worldwide (increasing caries epidemic in undeveloped countries which constitute the highest percentage of the world`s population ). Today, dental organisations are the only trade group of health professionals who endorse the use of a product that is primarily made of mercury. Every amalgam patent has been produced according to dental organisations specifications ,288. ThiThe author declare that he have no competing interests.


A 71-year-old man with history of asthma, non-insulin-dependent diabetes mellitus, and chronic obstructive pulmonary disease was treated for squamous cell carcinoma of the oral cavity. He underwent a partial mandibular resection with plate reconstruction followed by subsequent radiation therapy. A neck dissection was performed during the initial operative procedure. Eight months later, the patient was noted to have significant pain and development of intraoral fistulas. In addition, there was jaw swelling, difficulty opening the mouth fully (trismus), and bone destruction, which resulted in exposure of the mandibular reconstruction plates with evidence of underlying bony necrosis.What is the most likely diagnosis for this patient?What management options are available for treatment of this condition?How does prior biphosphate therapy influence this condition?How does hyperbaric oxygen (HBO) therapy affect the success rate of surgery?Osteonecrosis is a nonfatal bone condition in which damage and/or necrosis occurs as a result of poor local blood supply. Osteonecrosis occurs in 1 in 100,000 people (0.001%), affecting 20,000 new patients a year typically between the ages of 20 and 50 years.2-Prior bisphosphonate therapy has also been implicated as a cause of bone necrosis (bisphosphonate-associated osteonecrosis of the jaw) particularly at the lingual aspect of the posterior mandible.3,6,7Osteoradionecrosis is caused by damage done by radiation to small arteries, depriving the bone of oxygen. It can be caused spontaneously when bone decays at a faster rate than it is created. It can also result when irradiated bone is subjected to trauma, most commonly tooth extraction (84%) and oncological surgery 12%). Symptoms include pain, swelling, inability to properly open the jaw (trismus), malocclusion, and exposed bone and pathologic fractures.%. Sympto,Specifically, ORN of the mandible is characterized by 3 grades of severity. Grade I ORN is the most commonly observed, where the alveolar bone is exposed. This can be treated with HBO therapy, a process in which the patient is given 100% oxygen at hyperbaric pressures. Oxygen can diffuse into the bloodstream at a faster rate than at normal atmospheric pressure to restore vitality in the affected bone.2Grade II ORN is defined when the condition can no longer be treated effectively with HBO therapy. This usually requires the removal of dead bone (sequestrectomy) and/or the excavation of tissue to drain the infected bone (saucerization).2,Grade III ORN is characterized by the presence of a pathologic fracture. This is often accompanied by frank bony necrosis.2


B cells are efficient antigen-presenting cells (APCs), relying on antigen uptake through the B cell receptor (BCR). The mechanism of antigen recognition remains a topic of debate; while the prevalent view holds that antigens need to be multivalent for BCR activation, monovalent antigens can also initiate B cell responses. In this review, we describe the steps required for antigen uptake, processing, and loading of peptides onto MHC Class II compartments in B cells for efficient presentation to CD4 T cells, with a special focus in the initial steps of BCR recognition of antigen. This, in turn, influences the activation state of the B cells and their ability to engage CD4 T cell help. The nature of the antigen and strength of interaction (affinity) for BCR are thus essential features that determine whether processed antigen can be efficiently presented to CD4 T cells. Here, we review the sequential steps required for BCR recognition of antigen, internalization, processing, and loading onto MHC II, with special emphasis on the early events of BCR–antigen interaction required for efficient B cell activation.Professional antigen-presenting cells (APCs) take up antigen through phagocytosis, fluid-phase pinocytosis, or receptor-mediated endocytosis for processing, loading of the digestion products onto MHC Class II (MHC II) and presentation to CD4 T cells. The ability of B cells to present antigen to CD4 T cells was recognized over three decades ago, but they were deemed less efficient than dendritic cells or macrophages due to their comparatively lower capacity to take up antigen non-specifically. In contrast, antigen recognition through the BCR is a far better  means to present antigen to CD4 T cells compared to non-specific antigen uptake , which receive continuous inputs by means of lymphatic fluid originating in peripheral tissues and delivered to lymph nodes through the afferent vessels (lymph node) or via blood, which carries lymphocytes and antigen through the trabecular artery (spleen) . Small, The BCR discriminates tonic signals delivered to B cells in their resting state, required for survival, from activating signals that lead to differentiation and antibody production. It is generally accepted that antigen binding leads to BCR clustering and internalization of antigen; however, the steps required to achieve such clustering, the antigen valency, and even whether clustering is required at all for activation remains controversial. Total internal reflection fluorescence microscopy suggests that most BCRs are monomeric on the cell surface and aggregate upon ligand binding  in a manDiffusion of the BCR in resting cells is constrained by the actin cytoskeleton: areas rich in actin and the ezrin–radixin–moesin (ERM) family of proteins, which are proteins that link plasma membrane components to the actin cytoskeleton, are associated with slow-diffusing BCRs. Alteration of the actin cytoskeleton by exposure of B cells to depolymerizing agents such as Latrunculin A or Cytochalasin D leads to BCR signaling in the absence of antigen, and increase the rate of diffusion of the BCR . The tet2+ flux, but did so inefficiently, while monomers failed to induce any response. Surprisingly, dimers could activate equally well, regardless of whether NIP-molecules were placed on adjacent lysines or whether separated by 24 amino acids, a finding difficult to reconcile with the expected molecular distances between variable regions in the same BCR or achievable upon clustering  in the non-covalently associated Igα and Igβ heterodimers within seconds. This leads to Syk kinase and PLCγ2 recruitment and phosphorylation, Ca in Ref. , 29]. Si in Ref. . As anti in Ref.  presumabIn the ER, newly synthesized MHC II αβ dimers associate with the invariant chain (Ii), a chaperone that delivers MHC II to the endocytic pathway. There, proteases such as Cathepsin S and L stepwise cleave Ii to finally yield the class-II-associated invariant chain peptide, CLIP, which remains associated with the peptide-binding groove to prevent premature loading with peptides. MHC II has been shown to localize to unique MIIC compartments, distinct from endosomes or lysosomes , 33. In The actin cytoskeleton plays a role in antigen presentation by B cells; its regulation is shared with components of the BCR pathway such as Syk, Btk, and Vav . The actB cells are programed to present antigen to T cells primarily after BCR-mediated internalization of the antigen. The process of antigen recognition through the BCR not only triggers drastic changes in B cell gene expression profiling, but also affects endocytic trafficking and surface molecule expression. These steps require antigens that are sufficiently strong to exceed the threshold required for activation. When this criterion is met, BCR clusters, B cells expand in size, with concomitant enhanced diffusion rates of the BCR to capture more antigens. B cells then contract, internalize antigen, proteolyze it and present the resulting peptides on Class II MHC products. How exactly these processes are coordinated remains to be uncovered: is there a minimum BCR signaling level required to internalize antigen, or regardless of the signal strength will any antigen bearing the epitope be internalized? Is BCR crosslinking required for triggering gene expression, internalization, and antigen presentation, or just for a few of these processes? The valency of antigen and affinity are essential determinants to fully activate these processes, but which aspects of signaling and antigen presentation are directly affected remain to be established. B cell survival requires tonic signaling via the BCR, and activation triggers must therefore be finely tuned to prevent activation by non-optimal BCR ligands and autoantigens. A better understanding of how BCR recognition of antigen is tuned to control downstream processes and outcomes of B cell activation will require additional BCR animal models with homogeneous B cell populations; these models will enhance our knowledge for the rational design of therapies aimed to boost B cell responses or control B cell malignancies and autoimmunity.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.