CHD7), which is an ATP-dependent chromatin remodeller, have been identified as the cause of CHARGE syndrome. Here, we review recent work aimed at understanding the mechanism of CHD7 function in normal and pathological states, highlighting results from biochemical and in vivo studies. The emerging picture from this work suggests that the mechanisms by which CHD7 fine-tunes gene expression are context specific, consistent with the pleiotropic nature of CHARGE syndrome.CHARGE syndrome is a rare genetic syndrome characterised by a unique combination of multiple organ anomalies. Dominant loss-of-function mutations in the gene encoding chromodomain helicase DNA binding protein 7 ( CHD7 genotype.The expressivity and penetrance of CHARGE phenotypes show little correlation with the The hypothesis that mutations or polymorphisms in other genes modify disease phenotypes in humans remains unproven.CHD7 mutations can affect its nucleosome remodelling activity in vitro.CHARGE-associated in vivo and the effects of this activity on gene expression require further study.The role of CHD7 in regulating nucleosome positioning CHD7 pleiotropy may result from its interactions with cell type-specific transcription factors.CHD7 recruitment to cell type-specific enhancers may underlie context-specific roles. Advances in genomics over the past few years have led to impressive achievements in the identification of mutations responsible for rare genetic diseases, especially those with simple, monogenetic aetiologies CHD7 gene (MIM 608892) were identified in patients with CHARGE syndrome CHD7 encodes a chromatin-remodelling factor (CRF), which implied an epigenetic aetiology for CHARGE syndrome. CRFs have evolved to deal with the additional complexity and hindrance that the tight association of eukaryotic DNA with histone proteins imposes on replication and transcription ; cranial nerve dysfunction ; semicircular canal anomalies (94%); coloboma  intronic DNA to capture intron–exon boundaries, of seven patients with clinically typical CHARGE syndrome, for whom standard Sanger sequencing failed to identify a CHD7 mutation, identified two intronic (c.5051-15T>A and c.5405-17G>A) mutations in the CHD7 gene in two patients .In the remaining 5–10% of patients with clinically typical CHARGE syndrome, the apparent lack of CHD7, it is still possible that other genes are involved in CHARGE syndrome. In 2004, the gene encoding sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3E (SEMA3E) was shown to be interrupted by a de novo balanced translocation [t] in a patient with CHARGE syndrome SEMA3E has a major role: only one de novo missense mutation in SEMA3E was found in a cohort of 24 patients with CHARGE syndrome, and Sema3e mutant mice do not phenocopy any of the cardinal features of CHARGE syndrome Despite most patients carrying mutations in TBX1) Other genetic alterations that can phenocopy CHARGE syndrome, resulting in a clinical diagnosis of CHARGE syndrome, include chromosomal aberrations  modify phenotypic penetrance or expressivity in CHARGE syndrome?A recent study found that heterozygous expression of a stabilised, transcriptionally inactive variant of p53 during mouse development resulted in a constellation of phenotypes typical of CHARGE syndrome Chd7 heterozygous mouse model. If this proves to be possible, pharmacological inhibition of p53 in utero might represent an experimental approach for preventing certain CHARGE syndrome phenotypes, akin to observations with Treacher Collins syndrome Chd7 heterozygous embryos will significantly advance our understanding of the pathological mechanisms underlying CHARGE syndrome.It is not yet known whether reducing p53 activity will be able to rescue specific CHARGE syndrome phenotypes in an appropriate In summary, studies in mouse models have suggested that CHD7 functions in a context-dependent manner by interacting with, and affecting the expression or activation of, tissue-specific transcriptional regulators and signalling molecules. The application of next-generation sequencing approaches to screen for putative disease-modifying mutations or polymorphisms in these CHD7-interacting pathways is an important next step in understanding the pathophysiology of CHARGE syndrome (see Outstanding Questions Box).CHD7 mutations in patients with CHARGE syndrome just over 10 years ago, clinical genetic, animal, and biochemical studies have led to important insights into the causes of CHARGE syndrome phenotypes. These studies have also led to the identification of novel roles for CHD7 in development and disease. If we want to understand exactly how CHD7 functions in vivo, new experimental approaches will need to be used. For instance, genome-wide analysis of H3K27me3 or H3K36me3 histone modifications might reveal altered patterns in CHD7-deficient mammalian cells. DNA accessibility assays should be used to identify functionally relevant changes in nucleosome organisation at key regulatory regions in CHD7-deficient cells. Such experiments will not only be relevant to understanding CHARGE syndrome, but will also provide critical insights into how CRFs of the CHD family fine-tune gene expression in vivo. Loss-of-function mutations in several CHD genes are associated with intellectual disability and neurodevelopmental disorders CHD genes disrupts neural development by similar or distinct mechanisms. Furthermore, all CHD proteins (including CHD7) have been implicated in cancer and much work is needed to decipher the underlying mechanisms, which are likely to involve mechanisms of deregulated developmental signalling and genomic instability  noncoding regions that control CHD7 expression; (ii) genes that encode proteins that interact with CHD7 and/or regulate CHD7 activity; or (iii) different genes that result in a similar constellation of developmental phenotypes?Can ‘modifier’ genes that alter expressivity or penetrance of specific phenotypes be identified in patients?Are different organ defects in CHARGE syndrome caused by shared mechanisms? Is the hypothesis that CHD7 controls central developmental pathways or processes and that deregulation of one common pathway is responsible for several major developmental phenotypes correct? Perhaps a model whereby CHD7 has a unique, context-specific function in each cell type and developmental stage where it acts, through facilitating or repressing the activity of enhancer elements, is more accurate (How is CHD7 recruited to chromatin? Are interactions with histone modifications or TFs by themselves sufficient or does efficient, stable CHD7 recruitment and chromatin association require cooperative interactions?What is the primary mechanism of CHD7 action on the chromatin template? Once recruited, does CHD7 alter DNA accessibility by destabilising DNA–histone interactions leading to ‘indiscriminate’ nucleosome remodelling so that the final effect of nucleosome remodelling is determined by local DNA context (What is the contribution, if any, of CHD7-interacting proteins on CHD7 action? For example, to what extent is the interaction of CHD7 with chromatin-modifying enzymes or other CRF such as PBAF essential for some of its functions?Since the first identification of


TGFB3) and TNF-α (TNF) gene polymorphisms and male infertility. A total of 846 subjects, 423 diagnosed with male infertility and 423 fertile men were enrolled. TGFB3  and TNF (rs1800629:-308G > A) gene polymorphisms were genotyped. No association between TNF genotype and infertility was observed. As for TGFB3, the genotypes distribution was similar in infertile and fertile men. However, rs2284792 minor allele frequency was significantly higher among infertile subjects. Heterozygous rs2284792 AG genotype was associated with increased odds for infertility  and similar results were observed for G allele carrier status . Heterozygosity in TGFB3 rs3917158 was also associated with the infertility . The TGFB3 variant genotypes were associated with lower spermatozoa motility parameters in fertile men. The results indicate that variants in TGFB3 gene may be associated with male infertility. However, the findings require further replication and validation.Factors affecting the blood-testis barrier function may be involved in testicular damage and male infertility. Two cytokines play an important role in the barrier regulation, namely transforming growth factor beta 3 (TGF-β3) and tumor necrosis factor (TNF-α). The aim of this study was to investigate the potential association between TGF-β3 ( Infertility is a common problem affecting one in six couples, and in 50% it is attributed to the male factor resulting from acquired and/or congenital abnormalities, in which 60–75% is found to be idiopathic1Most of male infertility genetic association studies explored factors involved in spermatogenesis. However, it seems reasonably to explore also an impact of genetic determinants of the blood-testis barrier integrity. Factors affecting the barrier function may be directly involved in the development of testicular damage and problems with fertility. The blood-testis barrier is a very restrictive blood-tissue barrier, and creates a specialized microenvironment for development and maturation of germ cells. It consists of three components: anatomical (tight junctions), physiological (transporters) and immunological. The barrier components restrict passage of molecules and cells  and regulate movement of substances in or out of the lumen  as well as limit access by the immune system cells and sequester the majority of autoantigenic germ cells . The blood-testis barrier is not a static structure, as it undergoes extensive restructuring during spermatogenesis to meet the needs of germ cells and Sertoli cells. The anatomical barrier tight junctions undergo remodeling  to allow adaptation to Sertoli cell changes (but remain static for their function as integrity of the junctions remains intact) and transporter expression also adapts to various demands during spermatogenesis. Disruption of the barrier function and integrity  lead to testicular injury and infertility4Two crucial cytokines are involved in the regulation of the blood-testis barrier, namely transforming growth factor beta 3 (TGF-β3) and tumor necrosis factor (TNF-α). These cytokines determine the homeostasis of tight junctions and basal ectoplasmic specialization-structural proteins, proteases, protease inhibitors, and other extracellular proteins (e.g. collagen) in the seminiferous epithelium. Some of those molecules are known regulators of focal contacts between the extracellular matrix and other actively migrating cells, such as macrophages, fibroblasts or malignant cells. Experimental studies revealed that administration of IL-1β or TNF-α impair integrity of the blood-testis barrierin vitro, the amount of TNF-α produced by Sertoli cells declines significantly, indicating that it can downregulate the Sertoli cell tight junction–barrier function. Co-incubation of Sertoli cells with recombinant TNF-α results in disruption of the integrity of tight junction barrier, as shown in experiments quantifying the transepithelial electrical resistance across the Sertoli cell monolayerTNF gene (coding for TNF-α) with male infertility. It was documented that TNF-α activity depends on the polymorphic alleles of TNF. A single nucleotide polymorphism (SNP) in TNF gene promoter is a result of guanine for adenine transition (−308G > A). In vitro transfection studies revealed higher expression of −308A minor allele in comparison to −308GIn the testis, TNF-α, a proinflammatory cytokine, is secreted by germ cells, predominantly pachytene spermatocytes and round spermatides as well as Sertoli cells, whereas its receptors are restricted to Sertoli cellsin vitroIn vitro studies documented that recombinant TGF-β3 led to disruption of Sertoli cell monolayerin vivo using the CdCl2-treated rat model of infertility, i.e. an increase of TGF-β3 was paralleled by decreased content of occludin and ZO-1TGFB3) in the pathology of connective tissue. A significant association was found between TGF-β3 gene (TGFB3) polymorphism and fibrosis in patients with sarcoidosis (SNP 4875A > G and 17369T > C)Another cytokine which plays an important role in the regulation of the blood-testis barrier function is transforming growth factor-β3 (TGF-β3), which is the most abundant form of TGF-β in the testis. It is produced by Sertoli cells, and its level declines when Sertoli cell tight junction barrier is being assembled Standard sperm parameters of the study subjects are presented in TNF genotype and male infertility  was higher among infertile subjects in case of all the studied SNPs, reaching statistical significance level for TGFB3 rs2284792 (p = 0.025). Heterozygous rs2284792 AG genotype was associated with increased odds for infertility  and similar results were observed for G allele carrier status . As for TGFB3 rs3917158, only in the case of heterozygous subjects, an association with infertility was significant . For all other SNPs, only marginally significant associations were observed when dominant model was applied . No association was observed between ertility . As for p < 0.1, . As all ilibrium , they shTGFB3 SNPs, haplotype frequencies were assessed, in both infertile and control groups. It was revealed, that 5 SNPs form two most frequent haplotypes T_C_A_T_C (estimated frequency of 0.750 among infertile and 0.786 among fertile man), and C_T_G_C_T . However, frequencies of the haplotypes did not differ significantly between infertile cases and controls , higher percentage of non-progressive motility , as well as decreased total motility and higher percentage of immotile spermatozoa among variant carriers (rs2268626 and rs2284792). TNF rs1800629 variant was associated with lower percentage of spermatozoa with progressive motility. In infertile subjects the genetic associations and sperm parameters were mostly not observed.When the studied tile men . HoweverTNF and TGFB3, and male infertility.The normal function of the blood–testis barrier protects developing germ cells against harmful agents and immunological influences. Protection is necessary because many agents or immune system cells can disturb the delicate process of meiotic cell division, thus leading to infertility. The barrier include structural component build of tight junctions as well as functional elements being mainly membrane transporters, e.g. glycoprotein P. TNF-α and TGF-β3 are two crucial cytokines, that are involved in the regulation of the blood-testis barrier, affecting both its structure as well as activity of functional componentsTNF rs1800629 polymorphism and male infertility in a Polish population. This observation is contrary to the report of Tronchon et al. from French population, who revealed significantly increased frequency of the TNFα -308 A allele in infertile male patients with testicular failure or with altered sperm motility compared with patients with normal sperm parameterset al. are in keeping with French observations. i.e. substitution level from G to A in the TNF-α gene was significantly higher in the infertile subjects as compared to fertile controlset al. documented significant overrepresentation of TNF -308AA carriers among patients diagnosed with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermiaOur study did not reveal significant association between TGFB3 polymorphism. The results of the study demonstrated similar  distribution of the studied TGFB3 genotypes in infertile and fertile men. However, higher frequency of the minor alleles  in infertile men, being significant for TGFB3 rs2284792. It was also found that heterozygous rs2284792 AG genotype was associated with significantly increased odds for infertility and similar results were observed for G allele carrier status. In the case of TGFB3 rs3917158 a significant association with infertility in heterozygous subjects was found, with odd ratio of 1.37. Analysis in the subgroup of infertile men with semen abnormalities according to the WHO criteria (65.1% of infertile subjects) revealed a significant association of TGFB3 rs2284792 variant with infertility; differences were significant for overall comparison, dominant model and allele frequency. Significant association for TGFB3 rs3917158 SNP could also be observed for that subgroup analysis   . Those dTGFB3 polymorphism with male infertility can be also explained by other findings of the present study. It was found that the TGFB3 polymorphism may affect sperm parameters. The study revealed that TGFB3 polymorphism was associated with sperm motility parameters, i.e. TGFB3 variant genotypes were associated with lower percentage of spermatozoa with progressive motility (reaching significance level for rs2268626), higher percentage of non-progressive motility , as well as decreased total motility and higher percentage of immotile spermatozoa among fertile men variant allele carriers (rs2268626 and rs2284792). No associations were found in the whole infertile men group, possibly due to more pronounced dysfunction of spermatozoa. However, separate analysis of sperm parameters in normozoospermic infertile men and infertile men with semen abnormalities revealed no significant genotype-related differences, but lower percentage of motile spermatozoa was observed in TGFB3 rs2268626 and rs2284792 minor homozygotes .In conclusion, the results of the present study may suggest an association between Chlamydia trachomatis infection, without history of pelvic inflammatory disease or abdominal operations. Male factor exclusion criteria included: clinical picture suggestive of obturatory azoospermia; history of testicular, epididymis or accessory gland infection; testicular torsio, maldescence or injury; varicocele; co-existing systemic disease; history of mumps.The study was carried out on 423 consecutive, otherwise healthy male patients, aged 32.9 ± 4.5 years (range: 19–52 years) without any chromosomal abnormalities, undergoing semen analysis due to infertility workup. The inclusion criteria were as following: age 18 to 56 years; no children from current or previous relations with at least a year history of at least a year of regular (2–3 weekly), unprotected sexual activity without conception; female partners aged up to 35 years with regular menstrual bleedings and/or progesterone levels in the luteal phase of the cycle >10 ng/ml, normal transvaginal ultrasound examination, negative testing for The control group consisted of 423 healthy males, aged 34.3 ± 8.3 years, (range: 21–62) recruited among consecutive men accompanying their female partners at term labor at the Department of Reproductive Medicine and Gynecology, Siedlecka str., Police, Poland. Paternity was confirmed by women; however the possible paternal discrepancy was additionally checked based on blood group verification.Both the men undergoing infertility workup as well as the fertile controls were of Polish origin, recruited within the same geographical region.Semen samples were collected by masturbation after two to seven days of abstinence from sexual activity. Among infertile men, full seminograms, allowing for detailed data analysis were available for 344 patients. As for the fertile controls, that group was recruited among men accompanying their female partners at term labor, and only 124 of men from this group agreed to provide sperm sample for analysis (29.3%). However, that subgroup is representative for the whole study control group (no significant differences for age nor ethnicity) and provides necessary data on sperm parameters in healthy Polish males.Sperm parameters were evaluated manually within one hour after the sample collection. Concentration, motility and morphology of spermatozoa were assessed according to the 2010 World Health Organization guidelinesThe study was approved by the Ethics Committee of Pomeranian Medical University (KB-0012/116/10) and conducted according to the Declaration of Helsinki. All participants provided written informed consent prior to participating in the study.TNF, previously associated with higher gene expression . Five tag SNPs with frequencies >0.1, mapping the TGFB3 gene  were selected with tag SNP picker using data for Caucasian population from the International HapMap Project (http://www.hapmap.org). MAFs are given for CEU population (Utah residents with Northern and Western European ancestry) from HapMap database. All the SNPs are located in gene introns. Genomic DNA was extracted from 200 mL of whole blood samples using Gene-MATRIX Quick Blood DNA Purification Kit . Commercially available, pre-validated allelic discrimination TaqMan real-time PCR assays  were used for genotyping. Fluorescence data was captured using ViiA 7 Real-Time PCR System  after 40 cycles of PCR.All the subjects were genotyped for rs1800629  SNP in the promoter region of the 2 test. Continuous variables were compared between groups using Mann-Whitney U-test (comparison between two groups) or Kruskal-Wallis test (more than 2 groups). Associations between categorical variables were assessed by the Chi2 test, with Yate’s corrections for n < 5 . The EH program  was used to estimate haplotype frequencies. Linkage disequilibrium (LD) was measured: the D’ was calculated using 2LD software and squared correlation coefficient (r2) was evaluated. A p level of less than 0.05 was considered statistically significant.Genotype distribution was tested for Hardy-Weinberg equilibrium using ChiHow to cite this article: Droździk, M. et al. TGFβ3 (TGFB3) polymorphism is associated with male infertility. Sci. Rep.5, 17151; doi: 10.1038/srep17151 (2015).


Hydrocephalus is an under-recognized presentation of progressive supranuclear palsy (PSP) and dementia with Lewy bodies (DLB).We describe four normal pressure hydrocephalus (NPH)-like presentations of pathology-proven PSP (n = 3) and DLB (n = 1) and review the literature on the hydrocephalic presentation of these atypical parkinsonisms.Despite the presence of ventriculomegaly disproportionate to the extent of parenchymal atrophy, all patients demonstrated early postural impairment and/or oculomotor abnormalities that encouraged a diagnostic revision. Hallucinations were the only early atypical manifestation of the hydrocephalic DLB presentation.and PSP) actually represent the hydrocephalic presentation of selected neurodegenerative disorders.Early postural impairment, falls, oculomotor impairment, and/or hallucinations are inconsistent with the diagnosis of NPH and suggest PSP or DLB as the underlying NPH mimic. We postulate that previously reported cases of “dual” pathology  contains supplementary material, which is available to authorized users. Of the 12 patients who underwent VPS placement, definite gait improvement was present in only 6 by one year and just 4 after 3 years, one third of the original VPS-treated cohort. Remarkably, pathology available in 5 patients in the VPS-treated cohort revealed the presence of neurodegenerative disorders, progressive supranuclear palsy (PSP) and dementia with Lewy bodies (DLB), each accounting for a case.Hydrocephalus as an imaging finding is commonly interpreted as potentially representing normal pressure hydrocephalus (NPH) largely because of the favorable therapeutic implications . Howeverlacement . Betweensustained response to VPS placement is paramount to justifying this invasive procedure. We examined four patients referred to us for suspected NPH but whose pathology demonstrated PSP and DLB. The preliminary diagnosis of NPH in these patients was based on the interpretation of imaging features, namely the presence of hydrocephalus judged disproportionate to the extent of surrounding parenchymal atrophy. While a careful neurological examination suggested further NPH work up was unwarranted in three patients, cerebrospinal fluid diversion in one (case 4) supported this diagnosis by yielding subjective benefits in gait and urinary function. In addition to early impairment postural reflexes, other clinical red flags against NPH emerged between 12 and 24 months. We suggest that PSP and DLB, as previously reported for Alzheimer disease (AD), is capable of generating a neuroimaging profile suggestive of communicating hydrocephalus, with ventriculomegaly disproportionate to the extent of parenchymal atrophy, prompting a diagnostic consideration for NPH and potentially misdirecting treatment toward cerebrospinal fluid diversion.Because of the large volume of NPH referrals to tertiary care centers, predicting a Four consecutive patients referred to our center for suspected NPH for whom brain autopsy was obtained were identified in our center from the years 2007–2012. After evaluation in our center, VPS placement was discouraged for the first three patients but pursued in the fourth to no avail. Written informed consent was obtained from all patients for the publication of patient-related data and videotapes.This 68-year-old woman developed balance impairment followed by falls, mostly forward, urinary incontinence, and intermittently slurred speech over a 2-year period. Head CT demonstrated mild to moderate hydrocephalus Figure A, promptThis 79-year-old man developed difficulty with “complex” leg movements, such as those required when walking across a railroad or stepping on a train wagon. Brain MRI obtained within one year was interpreted as mild hydrocephalus Figure B, promptThis 73-year-old man initially exhibited falls, visual hallucinations, irritability and combative behavior. Within two months he was unable to ambulate independently and had noticeable memory loss. Brain MRI taken at 3 months from symptom onset demonstrated moderate ventriculomegaly without overt atrophy in the apical cuts, suggesting the diagnosis of NPH Figure C. HoweveThis 72-year-old man with progressive gait and balance impairment underwent a brain MRI, which upon presence of hydrocephalus, hyperdynamic fluid signal in the third ventricle, and entrapped sulci in several cortical areas Figure D prompteThere are two major shortcomings in the appraisal of NPH as an entity, namely the absence of clinical and pathological biomarkers and the reliance on short-term response to drainage for its definition , which oBesides early impairment of postural reflexes, our patients had early cognitive impairment, either predating or occurring concurrently with gait impairment. Cognitive impairment is a regrettable component of the “classic” NPH triad. Of the 8 cognitively impaired VPS-treated patients in the Mayo Clinic series, only 1 continued to benefit at 3 years . SimilarA major pitfall of the interpretation of these data is to assume that these patients had “co-occurrence” of AD and NPH , 6, rathPSP appears to be the most common etiology of suspected NPH with a parkinsonian phenotype Table . It was Our cases and the review of the literature highlight several key clinically relevant messages: 1) early cognitive impairment is the most important “cognitive red flag” of the NPH triad and predicts an alternative diagnosis; 2) early impairment of postural reflexes, with or without falls, is the most common “motor red flag” of the NPH triad, and should suggest PSP or DLB; 3) visual hallucinations are a strong clinical biomarker of synucleinopathies  and theiIn sum, hydrocephalus in the setting of postural impairment and/or oculomotor abnormalities most probably represents PSP or DLB. Hallucinations may be the only early manifestation of the hydrocephalic presentation of DLB. An initial promising response to VPS placement does not exclude the possibility of an underlying neurodegenerative disease. Furthermore, a fully formed “classic” triad of gait impairment, urinary incontinence, and cognitive impairment appears to be more common in conditions other than NPH. The accumulating evidence suggests that early postural impairment, falls, oculomotor impairment, or hallucinations are inconsistent with the diagnosis of NPH regardless of ventricular size. Recognition of the hydrocephalic presentation of PSP and DLB can avoid unnecessary and potentially harmful shunting procedures.Additional file 1: Video S1: Patient 1 shows growling dysarthria, restriction of upgaze with supranuclear vertical gaze palsy, appendicular dystonia with dysmetria, wide-based, short-stride gait with erect posture, mild retrocollis, and impaired postural reflexes. (MP4 11 MB)Additional file 2: Video S2: Patient 2 demonstrates upgaze restriction with nystagmus but no square-wave jerks or slow saccades. There was marked gait impairment with wide base support and retropulsion on pull test. (MP4 17 MB)Additional file 3: Video S3: Patient 3 shows marked retrocollis, severe dysarthria and ophthalmoplegia. Patient is videotaped over the course of a “bad spell”, in hindsight interpreted as part of his DLB-associated cognitive fluctuations. (MP4 13 MB)Additional file 4: Video S4: Patient 4 shows a wide based, unsteady gait. Disappearance of corrective saccades to the optokinetic strip was the early clue as to his subtle saccadic impairment, and predated a later development of supranuclear vertical gaze palsy. (MP4 13 MB)Authors’ original file for figure 1Authors’ original file for figure 2Below are the links to the authors’ original submitted files for images.


Osteoporosis has become a major public health problem, it is characterised by loss of bone mass and architecture due to disturbance in bone remodelling. Most current treatments retard bone loss but have no stimulating effect on bone formation . OsteocyFour different compositions were seeded with MLO-Y4 cells and accommodated in two different cultures (3D scaffolds versus 2D films). After 5 days of culture, Sost gene expression was analysed using Real-time PCR in all groups and the data was normalised to a housekeeping gene (18s).There was a robust statistically significant increase in MLO-Y4 gene expression for Sost when cultured on a Collagen-Hydroxyapatite (HA) substrate compared to a Collagen-only substrate. Furthermore, the 3D dimensionality enhanced gene expression across all different compositions.This study has demonstrated that scaffold composition and dimensionality has a significant influence upon regulation of MLO-Y4 gene expression. This also indicates that Sost gene is regulated by both composition and dimensionality. The ability to stimulate MLO-Y4 cell line to express Sost sufficiently will offer a precious tool for researchers to further study sclerostin secretion, identify novel regulators of Sost gene expression and investigate them to develop new therapeutic agents that may offer advantage over the currently available treatments for osteoporosis.


Spatial variations in composition of marine microbial communities and its causes have largely been disclosed in studies comprising rather large environmental and spatial differences. In the present study, we explored if a moderate but temporally permanent climatic division within a contiguous arctic shelf seafloor was traceable in the diversity patterns of its bacterial and archaeal communities. Soft bottom sediment samples were collected at 10 geographical locations, spanning spatial distances of up to 640 km, transecting the oceanic polar front in the Barents Sea. The northern sampling sites were generally colder, less saline, shallower, and showed higher concentrations of freshly sedimented phytopigments compared to the southern study locations. Sampling sites depicted low variation in relative abundances of taxa at class level, with persistent numerical dominance by lineages of Gamma- and Deltaproteobacteria . The Archaea, which constituted 0.7–1.8% of 16S rRNA gene copy numbers in the sediment, were overwhelmingly (85.8%) affiliated with the Thaumarchaeota. Beta-diversity analyses showed the environmental variations throughout the sampling range to have a stronger impact on the structuring of both the bacterial and archaeal communities than spatial effects. While bacterial communities were significantly influenced by the combined effect of several weakly selective environmental differences, including temperature, archaeal communities appeared to be more uniquely structured by the level of freshly sedimented phytopigments. Microbial community similarities tend to show a distance decay relationship, implying that the phylogenetic composition of communities becomes increasingly dissimilar with increasing geographical distance. It is now generally accepted that both contemporary environmental parameters and historical contingencies, maintained by dispersal limitation, may contribute to this beta-diversity. Hence, the classical Baas Becking statement “Everything is everywhere, but the environment selects” is questioned as a universal model for explaining the observed variation in microbial community composition  is part of the circumpolar Arctic Continental Shelf. It extends northwards from the northern coasts of Norway and Russia to the Arctic Ocean, and is delimited by the Novaya Zemlya and the Norwegian Sea along the east-west axis. With an average depth of 230 m, it is the deepest of the Arctic shelf seas. It is also characterized by less coastal erosion and river water inflow than other Arctic shelf seas  and phaeophytin were determined by a Turner 7000 fluorometer  from readings at 665 nm in ethanol extracts before and after treatment with 1 M acetic acid  and sand/gravel (>63 μm). Total organic carbon (TOC) content was analyzed by a LECO CS-200 Analyzer . Sediment chlorophyll Total DNA was extracted from duplicate 0.5 g samples of each site using the PowerSoil™ DNA Isolation kit  according to the manufacturer's instructions. The concentration and quality of extracted DNA were determined by a NanoDrop ND-1000 spectrophotometer .−1 to 10−3). Standard curves for threshold cycle (Ct) vs. logarithm of the start concentration of 16S rRNA gene copies, from 106 to 101, were established with Escherichia coli K10 for Bacteria and Methanoplanus petrolearius DSM11571 for Archaea. This corresponded to E. coli genomic DNA being serially diluted from 0.76 to 0.76 × 10−5 ng and M. petrolearius diluted from 1.56 to 1.56 × 10−5 ng. Genomic standards were included in each qPCR run to ensure linearity and expected slope values of the Ct/log[gDNA] curves.Quantification of 16S ribosomal RNA genes was used for the estimation of prokaryotic cell densities. Quantitative real-time PCR (qPCR) was performed on an ABI 7500Fast real-time PCR system  using primers 27F/338R for Bacteria and A571F/915R for Archaea (see Supplementary Table Taq polymerase (Invitrogen), and 10 ng of genomic DNA template. The thermocycler (Applied Biosystems) conditions were: initial denaturation step at 94°C for 5 min; 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; a final extension at 72°C for 5 min. To minimize potential random PCR biases, each sample was amplified in sextuplicate (triplicates of each DNA isolation). Correctly sized amplification products were extracted from the gel by use of the QIAquick Gel Extraction kit , and replicate samples were pooled and purified one more time with Agencourt AMPure XP beads . Equal amounts of amplicons from each PCR run were pooled and subjected to multiplex pyrosequencing using a 454/Roche GS-FLX Titanium instrument  installed at the Norwegian High Throughput Sequencing Centre . The bacterial (BM) and archaeal (AM) amplicons were sequenced separately, as was a second bacterial preparation from sampling station 6 (D6). The latter was subjected to a deeper sequencing effort than used in the multiplex analysis. The raw sequence data have been submitted to the EMBL database under the accession numbers ERP003605 (BM dataset), ERP003606 (AM dataset), and ERP003607 (D6 dataset).Tagged PCR primers for each sampling station were constructed by adding unique oligonucleotides to the universal forward primers 27F for Bacteria and 571F for Archaea (Supplementary Table http://greengenes.lbl.gov) using the PyNAST tool, as integrated in the QIIME package  pipeline  based on the Hellinger transformed Bray-Curtis distance metric was used  were subsequently constructed in R from the standardized environmental variables to quantify their relative importance and test the significance of the individual environmental factors by using the glm function. To partition the possible community structuring effects of geography and environmental factors, partial Mantel tests were used  with 104 Monte Carlo permutations. The same procedure was used for testing the relationships between geographical and environmental distances. Tests for correlations between bacterial and archaeal abundances and environmental variables were also based on Spearman rank correlation coefficients.The combinations of environmental variables that best explained community variation among the sampling stations were obtained as the ones generating maximum rank correlations between the environmental and community distance matrices , as did the additional environmental factors water depth , salinity , and Chl a/phaeophytin ratio , the latter being used as indicator of freshly sedimented phytopigment material. On the other hand, the grain size distribution and organic content of the sediment showed a more random variation between the sampling stations. Principal component ordination, based on the environmental data, separated the sampling stations in accordance with the south-north dichotomy along PC1  to the northern ones (6–10), while station 5 was in a transitional temperature zone . Furthermore, the bacterial gene abundance showed significant relationships with temperature , and phytopigment ratio , while no correlations were found between the abundance and environmental data for the Archaea.Bacterial 16S rRNA gene copy numbers varied in the range of 3.1 × 10es Table , the quaThe sequence datasets comprised 65 904, 139 590, and 164 880 qualified reads (excluding reads representing singletons) from the sequencing of the bacterial (BM) and archaeal (AM) multiplex amplicons of the transect and a deeper bacterial sequencing of station 6 (D6). A high bacterial diversity was confirmed in this Barents Sea sediment sample as the numbers of unique OTUs obtained both from the multiplex BM and the single station D6 material exceeded 5500 at ≥97% sequence identity Table . The 21-Nitrosopumilus. Besides the Thaumarchaeota, phylotypes representing the Miscellaneous Crenarchaeotal Group . The five bacterial taxa that generated most explained difference between the two regions were the proteobacterial orders Nitrosomonadales (p = 0.006), Rhodospirillales (p = 0.009), Marinicellales (p = 0.009), Desulfuromonadales (p = 0.010) and the uncultured proteobacterial group Sva0853 (p = 0.010). For the Archaea, just the variation in the Parvarchaeota (p = 0.001) and Thaumarchaeota (p = 0.021) tag abundances gave significant contributions to explained regional difference . The zero-distance bacterial Bray-Curtis index value of 0.65  and by spatial autocorrelations of the same environmental factors (p = 0.001). Hence, relationships were optimized between combinations of environmental parameters and community variation by the bioenv procedure, and significant relationships were found between these combinations of environmental factors and community distances  in which Schauer et al.  have fouThe temperature difference between the southern and northern sampling stations constitutes a stable oceanographic feature of the western Barents Sea (Ingvaldsen and Loeng, a/phaeophytin ratio and the concentration of Chl a have been used as estimators of freshness of sedimented phytoplanktonic material in e.g., the western Barents Sea. Positive relationships between the content of sediment phytopigments and bacterial growth and production has been demonstrated (Jørgensen and Boetius, a (Robidart et al., At the time of our sampling effort in late May, winter sea ice had retracted from around sampling stations 5–6 to about station 8 and was partly disintegrated even further north. The spring bloom, which is particularly intensive in the 20–50 km marginal zone south of the ice edge (Sakshaug et al., No significant community structuring effects of water depth or salinity were observed. Previous studies documenting effects of water depth are founded on substantially wider depth ranges than the less than 300 m in the present study. In two studies based on pyrosequence data comprising sampling sites from surface level to the deep ocean floor, up to 3.0% of the sediment bacterial community variation was found to be explained by water depth (Zinger et al., There is the possibility that unmeasured environmental variables contributed significantly to community variation through the sampling range. Possible unaccounted variables include the levels of inorganic nutrients (Wu et al., Several studies have confirmed microbial community distance decay relationships in marine habitat types like pelagic water (Monier et al., In conclusion, our data consolidated previous findings regarding the bacterial alpha-diversities of marine shelf seafloor sediments but reinforced the significance of the Thaumarchaeota as the principal archaeal group in this type of environment. Furthermore, the study confirmed that biogeographical structures are detectable in marine sediment prokaryotic communities by deep 16S rRNA gene sequencing, even where high dispersal rates combined with weak environmental filtration counteract the build-up of beta-diversity patterns. This may have implications for the practicality of employing such approaches to monitoring microbial effects of e.g., the predicted rise in air and water temperatures in the polar regions, including the Barents Sea, in the years to come. This climate change is expected to be manifested in the microbial communities (Kirchman et al., 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.


These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs.NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM) but not high (50 μM) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α NMDA receptors (NMDARs) play an essential role in the regulation of synaptic strength in the brain  is a member of the serine protease family most well known for its role in vascular thrombolysis where it activates the zymogen plasminogen to form plasmin, which degrades fibrin and remove blood clots  receptor antagonist and potassium channel blocker, 4-aminopyridine  was a generous gift from Boehringer Ingelheim  or purchased from Biopur . For some experiments the excipients in Actilyse® tPA were removed by dialysis against HEPES . Receptor-associated protein (RAP) was produced as a glutathione S-transferase fusion protein and purified by glutathione-affinity chromatography. The glutathione S-transferase tag was removed by thrombin cleavage prior to use. The active site mutant human tPA (S478A) was purchased from Molecular Innovations .Recombinant human tPA , with half medium changes 24 h following plating and then at 7 day intervals.The use of animals in this research was approved by the University of Auckland Animal Ethics Committee. Primary hippocampal cultures were prepared from embryonic (E18) Wistar rats as described previously  according to the manufacturer’s instructions. Calcium responses were recorded on an Envision plate reader  using the following settings: excitation filter, FITC 485 nm; emission filter, 520 nm. Each well was recorded individually, with 15 s of baseline recording, followed by injection of agonist and recording of the response for a further 45 s. Antagonists were added manually 5 min (tPA) or 15 min , prior to recording. Raw fluorescence data were converted to ΔF/F0; where F0 is the average fluorescence over the first 15 s of recording prior to addition of agonist (baseline) and ΔF is Fmax−F0.All experiments were performed on cultures maintained for 14–17 days ijky = μ + iα + jb + ij(αb) + ijke where ijky is the transformed AUC measurement for the kth technical replicate for treatment i in plate j, μ + iα is the mean transformed AUC measurement for treatment i, jb is the random effect associated with plate j, ij(αb) is the plate by treatment random effect  and ijke is the random error associated with technical replicate k for treatment i in plate j. The R command lmer from the package lme4  + (1|plate_id:treatment). All statistical tests were planned comparisons between a treatment and the appropriate control and were calculated as contrasts from the fitted models with p-values obtained using degrees of freedom determined by the Kenward-Roger method using packages lsmeans  from 0 to 45 s was determined and used as the dependent variable for all statistical analyses. These data were first transformed using a square root transform to ensure that there was homogeneity of variance and an approximately normal distribution of residuals in the fitted statistical models. The data for all NMDA treatments were analyzed as a complete set. This data set contained 24 individual plates of cells, 121 treatment by plate biological replicates and 351 individual data points. The data for the bicuculline and 4-AP treatments were analyzed separately, consisting of 4 plates of cells, 19 treatment by plate replicates and 53 individual data points. Statistical analysis was conducted in R 3.0.2 , using a Fluo-4-based calcium assay and a high speed fluorometric plate reader. Treatment with the NMDAR agonist NMDA alone (5–100 μM) resulted in a rapid and concentration-dependent increase in intracellular calcium levels  contains a number of excipients, including L-arginine, which has been suggested to be toxic to neurons  and a second commercial recombinant tPA, Biopur tPA, which does not contain excipients. Both produced similar inhibition of the 5 μM NMDA-mediated calcium response  and the potassium channel blocker 4-AP  and the use-dependent NMDAR open channel blocker MK-801 (10 μM) both significantly inhibited the calcium response of neurons treated with 50 μM NMDA while the L-type voltage-gated calcium channel blocker nimodipine (10 μM) had no significant effect on calcium levels Figures . The cal478 residue mutated to alanine. In contrast to tPA, the enzymatically-inactive tPAS478A had no effect on 5 μM NMDA-mediated changes in calcium levels . This result suggested an off-target effect of tPA-STOP and so it was not used in any further experiments. In a second approach we tested the effect of an enzymatically-inactive tPA mutant, with the active site Ser Figures .2-antiplasmin. α2-antiplasmin (140 nM) alone did not affect 5 μM NMDA-induced calcium flux and did not block the effect on tPA on 5 μM NMDA-induced calcium flux (Figures To investigate if tPA’s effects on intracellular calcium levels involved tPA-mediated activation of plasmin, cultures were preincubated with α Figures . This su Figures  experime Figures . RAP als Figures  suggestiIn this study we have explored the effect of the proteolytic enzyme tPA on NMDA receptor-induced calcium flux in primary cultures of rat hippocampal neurons. Our results reveal that tPA’s effects on NMDA-mediated changes in intracellular calcium levels vary with the concentration of NMDA. They support an inhibitory effect of tPA on calcium flux activated by low concentrations of NMDA or synaptic action potentials, through a mechanism that requires the enzymatic activity of tPA.A receptor antagonist bicuculline to relieve this inhibition, leading to bursts of action potentials and activity-dependent calcium transients mediated largely by calcium influx through synaptic NMDA receptor (Hardingham et al., We found that pre-treatment of cultured hippocampal neurons with tPA had no effect on intracellular free calcium levels following stimulation with 50 μM NMDA. This relatively high concentration of NMDA has been reported to directly activate post-synaptic NMDARs (Bading et al., To the best of our knowledge this is the first study to report an inhibitory effect of tPA on NMDA receptor-mediated calcium flux in neurons. Several other groups have investigated the effect of tPA on intracellular calcium levels (Nicole et al., 2-antiplasmin. While, no such change was observed (Figures Our data support tPA inhibition of NMDA-mediated changes in calcium levels through a proteolytic mechanism. The changes could involve direct effects of tPA on the NMDA receptor to inhibit calcium entry but we cannot exclude changes in intracellular calcium also involving differential release of calcium from intracellular stores. Treatment of cultures with a catalytically inactive tPA mutant did not inhibit NMDA-mediated calcium flux. tPA has been proposed to cleave the GluN1 subunit of the NMDA receptor and potentiate NMDA-induced calcium influx (Nicole et al.,  Figures , these etPA has also been shown to interact with the NMDA receptor via the low-density LRP1. Several studies have implicated LRP1 as the major tPA receptor in the brain facilitation subsequent downstream signaling (Zhuo et al., Another potential target of tPA is GluN2D-containing NMDARs. tPA has been proposed to potentiate GluN2D-containing NMDA receptor-dependent activation in cortical neurons activated with 50 μM NMDA with receptor activation monitored by quantitation of ERK signaling or increased neuronal death (Baron et al., A receptor function. These effects require tPA to be proteolytically active and appear not to involve plasminogen as a substrate or LRP as part of a receptor-mediated mechanism. Our data provide additional evidence for the involvement of tPA in modulating NMDA receptor function and suggest a further level of complexity to the way that tPA may influence neuronal physiology and pathology. Further research is needed to determine which NMDA receptor subtypes are affected and how the effects relate to hippocampal synaptic plasticity in vivo (Liu et al., In conclusion, our study has found that tPA can inhibit NMDA receptor-mediated changes in intracellular calcium levels in cultured primary rat embryonic hippocampal neurons when NMDARs are activated with either low concentrations of NMDA or through activation of synaptic NMDARs by blocking GABAThe 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.


Bric- a-Brac/Tramtrack/Broad/ Pox virus and Zinc finger) domain class that is expressed in many elements of the developing larval nervous system. It has roles in innate behaviors such as larval locomotion and adult courtship. In performing tissue-specific knockdown with the Gal4-UAS system we identified a new behavioral phenotype for lov: larvae failed to burrow into their food during their growth phase and then failed to tunnel into an agarose substratum during their wandering phase. We determined that these phenotypes originate in a previously unrecognized role for lov in the tracheae. By using tracheal-specific Gal4 lines, Lov immunolocalization and a lov enhancer trap line, we established that lov is normally expressed in the tracheae from late in embryogenesis through larval life. Using an assay that monitors food burrowing, substrate tunneling and death we showed that lov tracheal knockdown results in tracheal fluid-filling, producing hypoxia that activates the aberrant behaviors and inhibits development. We investigated the role of lov in the tracheae that initiates this sequence of events. We discovered that when lov levels are reduced, the tracheal cells are smaller, more numerous and show lower levels of endopolyploidization. Together our findings indicate that Lov is necessary for tracheal endoreplicative growth and that its loss in this tissue causes loss of tracheal integrity resulting in chronic hypoxia and abnormal burrowing and tunneling behavior.The Drosophila protein Jim Lovell (Lov) is a putative transcription factor of the BTB/POZ ( As they transition from feeding to pupation, the larvae undertake a wandering phase in which they move away from food and seek out a site to settle for the prepupal and pupal molts. This wandering phase is a vulnerable period in the life cycle when exposure to environmental risks such as desiccation or predators can occur , 2. Prevlov in other larval and adult behaviors [lov might affect these responses. lov encodes a putative transcription factor of the BTB/POZ domain family [lov acts to specify the terminal differentiation of particular classes of neurons and that its roles in behavior reflect these functions.Our prior studies have already identified roles for ehaviors , 6 and sn family  and immulov in different subsets of neurons we undertook neuronal cell-type specific knockdown of lov, using the Gal4 system [lov RNAi. A series of neuron-specific Gal4 lines were screened for their effects on development and behavior when driving lov RNAi. Given that lov is expressed in almost all of the external sense organ (eso) neurons of the embryonic PNS we particularly sought Gal4 drivers that would express lov RNAi uniquely in eso lineages. The transcription factor Cut is essential for the maintenance of eso lineages [cut locus that drive expression in the embryonic esos have been identified [cut-Gal4 driver that expresses specifically in these esos has been generated. However, a cut-Gal4 driver that directs transcription in the esos of the adult wing margin is available [cut(ue)-Gal4 to denote the cutupstream enhancer it contains, was therefore included in our screen.In order to dissect out the individual roles of 4 system  in combilineages  in both lineages . Regionsentified , but no vailable , 12 -Gal4 produced partial lethality in the larval phase that proved to be associated with an absence of burrowing and tunneling behaviors. In investigating these behaviors we uncovered a new role for lov in the growth of the larval tracheae that appears to be the origin of these behavioral defects. The damage produced by tracheal lov knockdown leads to hypoxia, which has been shown previously to cause larvae to exit their food burrows [lov.In contrast to the other Gal4 lines tested with  burrows . We showlov RNAi lines were available from the Drosophila RNAi Consortia  for these studies. We used Semi-Q RT-PCR to assess the effectiveness of these constructs at depleting lov RNA in the late embryonic nervous system when driven by the elav-Gal4 driver (lov RNAi construct (HMSO01126) from the TRiP facility proved most effective and was used as the lov RNAi construct for all the studies described here.Three 4 driver . The Vallov RNAi driven by multiple neuronal drivers, and all but one produced behavioral anomalies  without affecting overall viability. The unique lethality seen for cut(ue)-Gal4 > lov RNAi animals was therefore investigated further, beginning with a characterization of the early cut(ue)-Gal4 expression pattern. Using UAS-mCherry as a reporter [cut(ue)-Gal4 is highly regional within the tracheal dorsal trunks (DTs). It is largely limited to the three posterior sections, with weak expression in metamere Tr9 and very strong expression in i) metamere Tr10, ii) the connection between the DTs posterior to Tr10 (termed here the “bridge”), and iii) the final segment of the DTs adjacent to the spiracles  that gives Gal4 expression in the tracheae and other cell types is known and used within the tracheal community [cut(ue)-Gal4 line, which lies more than 80 kb upstream of the cut coding sequence, was not previously known to regulate expression in the larval airways.We initially screened for effects of reporter , we detereporter . Furtherpiracles . Cut is ommunity . Howevercut(ue)-Gal4 produces larval lethality when driving lov RNAi clearly indicates that lov is also expressed in the embryonic/larval tracheal system. Our prior analysis of embryonic Lov expression did not detect Lov protein in the developing tracheae [lov PGawB enhancer trap [91Ylov-Gal4. Previously, we investigated the Gal4 pattern from this insertion in the adult brain, in connection with its effects on adult gravitaxis behavior [91Ylov-Gal4 in early embryogenesis, but later in development, mCherry expression faithfully mirrored the expression pattern of Lov in the nervous system that we have characterized previously [That tracheae , but forcer trap  mutationbehavior . Using aeviously . However91Ylov-Gal4 expression in the tracheae is a faithful representation of lov gene activity by staining third instar larval tracheae with Lov antibody. Nuclear Lov staining was detectable in all the epithelial cells and fusion cells of the DTs . We conclude that 91Ylov-Gal4 faithfully reports the lov expression pattern from late embryogenesis into larval life and reveals both neural and tracheal lov expression at these stages. The Lov tracheal expression pattern includes the regions of the tracheae in which cut(ue)-Gal4 is active. Thus the lethality of cut(ue)-Gal4 > lov RNAi larvae reflects previously unknown activity in the tracheae for both lov and the cut(ue)-Gal4 construct.We confirmed that this  the DTs . Our pricut(ue)-Gal4 > lov RNAi larvae indicated two behavioral abnormalities: they stayed away from their food and did not tunnel into an agarose substratum during wandering. To further characterize and quantitate these behaviors and to confirm their origin in lov knockdown in the tracheae, we generated and used an assay that assesses larval food burrowing, substratum tunneling, and overall development -Gal4, which is expressed throughout the tracheal system from early in its inception in embryogenesis [cut(ue)-Gal4 and 91Ylov-Gal4. The PG142 Gal4 line was not used because this driver also gave Gal4 expression in the larval musculature. The expression patterns of the three chosen Gal4 lines in first instar larvae are compared in 91Ylov-Gal4 > UAS mCherry larvae do not express mCherry in these spiracles (data not shown).Initial observation of elopment  and examogenesis , cut(ue)lov tracheal knockdown with the three Gal4 lines produced effects of differing severity on larval growth and behavior. cut(ue)-Gal4 > lov RNAi, about half of the larvae were found away from their food in the first 2–3 days after hatching -Gal4, no larvae tunneled in the agarose substratum (cut(ue)-Gal4, no larvae survived long enough to attempt pupation. For 91Ylov-Gal4 > lov RNAi, essentially no defects in the parameters measured were detected: larvae grew and developed to adults like controls, burrowing in their food and tunneling during wandering in a wild type manner -Gal4 expression is very weak in most of the tracheal network, but is strong in the most posterior sections of the tracheal DTs, close to the spiracles. btl-Gal4 and 91Ylov-Gal4 are expressed throughout the tracheal epithelial cells but 91Ylov-Gal4 expression does not begin until the tracheal system is fully formed and is much weaker than btl-Gal4 expression -Gal4 > lov RNAi phenotype was also enhanced in lov hemizygotes, resulting in growth, behavior and viability defects more like those seen with btl-Gal4  that showed poor growth, failed burrowing and tunneling, and high pre-adult death showed strong penetrance of the tracheal fluid-filling phenotype. For btl-Gal4 > lov RNAi and btl-Gal4/lov def > lov RNAi larvae, which show the most severe developmental effects, the entire tracheal system becomes fluid-filled as early as day 2 after hatching in 100% of the larvae (cut(ue)-Gal4 > lov RNAi larvae, although the fluid-filling phenotype is 100% penetrant, the expressivity is more limited than in btl-Gal4 > lov RNAi larvae -Gal4 driver -Gal4 > lov RNAi larvae will leave the interior of a yeast pile like hypoxic larvae, we placed them under mounds of yeast paste and monitored their behavior as compared to controls. As shown in cut(ue)-Gal4 /+) larvae stayed in the food, cut(ue)-Gal4 > lov RNAi larvae came out of the yeast pile in a manner comparable to that of wild type larvae subjected to 1% oxygen -Gal4 > lov RNAi larvae as purely neuronal responses to hypoxia is complicated by the recognition that long- lasting hypoxia depresses metabolism and produces both growth and behavioral defects [cut(ue)-Gal4 > lov RNAi larvae that survive to wandering stage and fail to tunnel are noticeably smaller than controls -Gal4>lov RNAi is that these defects are indirect responses to prolonged hypoxia and reflect the fact that the larvae are too physiologically challenged to burrow and tunnel.In wild type larvae, these hypoxia-induced behaviors are rapidly initiated by sensory neurons some of which contain atypical soluble guanylyl cyclases that act as oxygen sensors (reviewed in ). Howeve defects , 26. cutcontrols  and are e growth  and at d91Ylov-Gal4 /lov def larvae with that of cut-Gal4 > lov RNAi larvae provides insight on this point -Gal4 > lov RNAi -Gal4 > lov RNAi larvae, the mutant phenotypes of these larvae probably result from neural defects caused by the 91Ylov insertion -Gal4 > lov RNAi larvae: although forward locomotion rates are comparable and low for the two genotypes, 91Ylov-Gal4 hemizygous larvae are also hampered by spontaneous backward motion -Gal4 > lov RNAi larvae do not burrow (cut(ue)-Gal4 > lov RNAi are not due to indirect effects on growth and locomotion but rather are a direct neural response to hypoxia.A comparison of the locomotor activity of is point . In the f larvae  so they tion see  rather td motion . Nevertheir food  and showeir food , quantiteir food , whereast burrow  and theit burrow . This colov tracheal knockdown larvae we examined the expression of genes known to be induced by low oxygen in larval life. Responses to hypoxia show strong developmental regulation in Drosophila. In particular, late embryos and early larvae show very limited changes in gene expression in hypoxia, with no increase in transcripts for the canonical indicator of hypoxia—lactate dehydrogenase (LDH) [aerobic glycolysis (lactate production in the presence of oxygen), with concomitant upregulation of LDH [btl-Gal4 > lov RNAi larvae die before this stage, we thus focused on determining whether wandering cut(ue)-Gal4 > lov RNAi larvae/early prepupae show hypoxia-induced transcriptional changes.To identify direct evidence of hypoxia in se (LDH) . This prn of LDH . In contn of LDH . Given tCG10160, the gene for LDH ; CG11652, the gene for dDPH1, a component of diphthamide synthesis; CG4608, the gene for the FGF receptor, Branchless, and CG31543, the gene for Fatiga, the prolyl hydroxylase that regulates levels of the key hypoxia-induced transcription factor HIF-α (Sima in Drosophila) [fatiga and dDPH1 was detected, with branchless transcripts showing a minor but consistent upregulation relative to control (data not shown). In contrast, transcripts for LDH were consistently upregulated about two fold (2.3 +/- 0.5) in cut(ue)-Gal4 > lov RNAi larvae as compared to controls (cut(ue)-Gal4 > lov RNAi larvae are hypoxic.We used Semi-Q RT-PCR to probe for transcripts from the four genes shown previously to undergo the greatest hypoxia-induced upregulation at this developmental stage ; these asophila) . No conscontrols . Given t91Ylov-Gal4 hemizygous larvae are phenotypically distinct from larvae with tracheal knockdown of lov, both in terms of behavior and morphology. As discussed above, behaviorally they differ in that they display spontaneous backward locomotion and some tunneling activity and morphologically they have air-filled tracheae. In addition, although, like tracheal lov RNAi larvae, they are smaller than controls . Further, the tracheae in btl-Gal4 > lov RNAi larvae inflate normally just before hatching. Thus the tracheal leakiness detected here appears to reflect a role for lov during tracheal growth and development in larval life.We found only minor structural defects in the late embryonic/early larval tracheal system of cut(ue)-Gal4 > lov RNAi larvae (data not shown), even though there are fluid-filled tracheal regions very close to the spiracles in these larvae.One cause of larval tracheal fluid-filling is loss of the lipid water barrier within the posterior spiracles. A simple assay in which a dye is added to semi-liquid food makes spiracle water entry easily detectable . Using tcut(ue)-Gal4 driver. Under this driver, adjacent sections of the tracheal trunks showing weak (Tr9) or strong (Tr10) lov RNAi knockdown can be compared. We therefore developed a protocol to dissect out the dorsal trunks from late stage larvae to examine these tracheal regions in cut(ue)-Gal4 > lov RNAi and control animals. Given that the septate junctions  provide the barrier to fluid passage through the tracheal epithelial cell layer [cut(ue)-Gal4 > lov RNAi and control larvae. At the light microscope level, no differences in localization of these proteins between control and experimental tissues was detected -Gal4 driver produces a very specific pattern of Gal4 expression in the DTs, with low levels in Tr9, high levels in Tr10, and even higher levels in the bridge and the extreme terminal sections of the DTs adjacent to the spiracles. We found that, in cut(ue)-Gal4 > lov RNAi, the size of the DT epithelial cells decreases dramatically in the region of high Gal4 expression. Tr9 cells appear somewhat smaller than in control larvae and Tr10 cells are considerably reduced compared to Tr9 cells (cut(ue)-Gal4 > lov RNAi larvae these cells were so small and disorganized that they were usually no longer attached to the spiracles. A break in the continuity of the DTs that would allow hemolymph entry was therefore present at this position.However, in performing these immunolocalization studies, an unexpected finding emerged. As shown in r9 cells . In wildlov knockdown, in most dissected DTs the Tr10 metamere was sufficiently intact to allow further analysis. DAPI nuclear staining revealed that the nuclei in Tr10 cells were noticeably smaller than those in Tr9 and there appeared to be more cells in this region than in the equivalent control region (cut(ue)-Gal4 > lov RNAi and cut(ue)-Gal4 /+ larvae established that, whereas in controls, the nuclear DNA content of cells in Tr9 and Tr10 is similar, in lov knockdown larvae the DNA content of Tr10 cells is about half that of Tr9 cells (cut(ue)-Gal4 > lov RNAi tracheae as an indication of the number of cells in each segment. Whereas the cell numbers for Tr9 and Tr10 are comparable in controls, in the lov knockdown tracheae, we quantitated a 50% increase in cell number for Tr10 over Tr9 -Gal4, does not express in any neural elements. Rather, we hypothesize that the guanylyl cyclase-expressing neurons identified by the Morton laboratory as oxygen-sensing neurons [lov that is disrupted to initiate these behaviors involves growth and polyploidization of the tracheal epithelial cells.The failed food burrowing and substratum tunneling identified by this work led us to discover a new role for  neurons  are the lov knockdown under the cut(ue)-Gal4 driver support this hypothesis. The DTs were frequently broken in the region posterior to Tr10 where the lov knockdown levels were highest. This region, and the adjacent Tr10, were always fluid-filled in the lov knockdown larvae.How might this decreased growth lead to loss of the barrier properties of the tracheal cells? As part of the 200-fold increase in weight during larval life , the larlov knockdown precedes the tracheal damage and fluid-filling also produced by lov RNAi. As an alternative it could be argued that an unidentified initial defect causes tracheal leakage and then the presence of fluid in the tracheae inhibits tracheal growth. For example, despite our findings from light microscopy, we could propose that the septate junctions are not functional in lov knockdown, leading to fluid entry and subsequent growth inhibition. Preliminary experiments (data not shown), in which weaker lov knockdown in the tracheae was achieved by countering cut(ue)-induced Gal4 activity with leaky Gal80 expression, make this possibility unlikely. In these cut(ue)-Gal4/Gal80 > lov RNAi larvae, Tr10 growth is inhibited but there is no fluid accumulation in the tracheae.This explanation for the origin of the tracheal damage assumes that loss of growth and endopolyploidy induced by cut(ue)-Gal4>lov RNAi larvae is low when compared to that detected in comparable stage larvae exposed to 4% oxygen for six hours [dDPH, 11x increase, fatiga, 8x increase, branchless, 11x increase) we found small, in some cases inconsistent, changes in our cut(ue)-Gal4 > lov RNAi larvae.Our data indicate that the amount of tissue hypoxia in ix hours . Althougcut(ue) driver, we interpret this finding to mean that tissue hypoxia is mostly limited to regions surrounding the affected tracheal sections, that is, Tr9, Tr10 and the bridge region. The Morton laboratory has shown that the terminal sensory cones located on abdominal segments A8 and A9 contain neurons expressing oxygen-sensing guanylyl cyclases and are major oxygen sensing organs for the larva [cut(ue)-Gal4 > lov RNAi larvae and we propose they undergo sufficient oxygen deprivation in these mutant animals to activate the behavioral responses characterized here.Given the highly regional fluid-filling of the tracheae produced with the he larva , 31, 34.The morphogenesis of the Drosophila larval tracheal system during embryogenesis requires no growth by mitosis and cell division (for reviews see –37). InsSubsequently, as for most of the larval tissues, the large increase in size of the tracheal system in larval life is accomplished by endoreplicative growth, that is, multiple rounds of DNA replication without nuclear or cytoplasmic division. This growth produces large cells each containing a single large polyploid nucleus. The precise point at which this endoreplicative growth is initiated is not known, but previous work indicates that most of the larval tissues have undergone at least one round of endoreplication prior to larval hatching .lov knockdown since late in embryogenesis reveals that these cells are smaller, more numerous and reduced in DNA content. Comparisons to nuclei/cells in the imaginal discs, which remain diploid in larval life, make it clear however that the lov knockdown DT cells have undergone some endoreplicative growth. These findings raise the possibility that lov has a role in the initial commitment to endoreplication at the very end of embryogenesis, when lov expression in the DTs is initiated, and that when lov is depleted, this transition is delayed and instead some growth by mitotic divisions occurs. As discussed below, the cells in Tr2 of the DTs only grow by regular cell divisions, demonstrating that mitotic growth can occur in the larval DTs.Our examination of DT cells that have experienced lov knockdown, it is likely that it underestimates the effects of a complete loss of lov function on these parameters. One factor causing an under-estimation of cell number increase (conservative definition of the posterior Tr10 boundary) is described above. Other factors that probably lessen the effects detected include the activity of the cut(ue) enhancer in Tr9 as well as in Tr10  enhancer is strikingly limited, immediately suggesting a role for cut in regional specialization of individual DT metameres. It is noteworthy that the region of highest activity of this enhancer is the region closest to the spiracles, posterior to the “bridge” structure, where polyploidization of the tracheal epithelial cells is detectably lower than in metameres Tr9 and Tr10 (see above). Regional transcriptional specialization in the tracheal system has already been demonstrated [cut expression across each tracheoblast cell cluster [cut regulation of regional specialization within the tracheal system therefore exits. However, other enhancers associated with the cut gene are known to regulate cut expression in the spiracles and tracheae [cut in the DTs may differ considerably from that produced by the cut(ue) enhancer alone. Quantitation of Cut tracheal protein expression is needed to resolve this issue.The Gal4 expression generated by the nstrated . Furthernstrated . Studies cluster , 43. A ptracheae , 44 and lov tracheal knockdown, 91Ylov-Gal4 hemizygous larvae show no indications of tracheal permeability but display backward movement, failure of systemic growth and poor survival to adulthood. These phenotypes are similar to those of 47lov, a deletion mutation generated from the 91Ylov chromosome in which 1.4 kb of DNA upstream of the 91Ylov PGawB transposon insertion site are missing [47lov mutant embryos, Lov expression in many elements of the developing larval nervous system is lost, suggesting that the phenotypes generated are neural in origin [91Ylov-Gal4 hemizygous larval phenotypes, like those of 47lov, are also neural in origin.Unlike larvae with  missing . In lov4n origin . It seem91Ylov PGawB insertion site is ~ 500 bp upstream of the start site for transcripts B and D of the lov gene [47lov indicated that the region around this site contains a regulatory element(s) controlling the expression of transcript D in neural tissues. The Gal4 expression pattern for the 91Ylov PGawB insertion described here reveals the presence of additional regulatory sequences in this region that control lov expression in the larval tracheae. The tracheal phenotypes identified here demonstrate the effects of specific knockdown of this component of lov expression in larval life. However, the 47lovand 91Ylov -Gal4 phenotypes probably derive from direct disruption of neural regulatory elements within this same DNA region and thus affect neural roles for the gene in larvae.The lov gene . Our prelov is expressed strongly and almost exclusively in the developing nervous system [lov are probably neural in origin. In contrast, these current studies define an additional route by which lov may activate behavioral responses and in general, they underscore the fact that genes can influence behavior in multiple, often indirect, ways.The immediate stimulus for a behavioral response is typically neural. From the middle stages of embryogenesis until hatching, s system  and all BL27327 -Gal4); BL8807 ; BL4961(Df (2R) K10 b pr Bl c  /SM1); the CyO-GFP balancers from stocks BL5702  and BL6662  were used to prepare lov deficiency chromosome/CyO-GFP stocks. lov RNAi stocks used were as follows; #10739, from the Vienna Drosophila Resource Center; #JF02205  and #HMS01126  from the TRiP Project at Harvard Medical School. Canton-S was used as the wild type (+) stock for these studies. 91Ylov was identified in a previous screen [The following stocks were obtained from the Bloomington Drosophila Stock Center (BDSC); Petri plates (10 cm) with a layer (3–4 mm) of 2% agarose were generated and 10 newly hatched larvae of a given genotype were placed in a central well containing a standard amount (0.8 gms) of yeast paste . At least 50 larvae for each genotype were examined. Growth, burrowing and tunneling behavior, and survival were observed daily through to adulthood. Once adults had emerged, plates were washed extensively with water to remove debris and dried for 1–2 days. Images of the tunneling patterns on the plates were then taken with a Biorad GelDoc Universal Hood II system, using a black background. Under these conditions the tunnels appeared as white strips against the dark background. Tunneling was then quantitated from negatives of these images by calculating the total number of dark pixels in tunnels per plate using NIH image J. An initial round of studies used agarose plates without mold inhibitor. After noting that mold growth in the tunnels increased the pixel count on Image J, 0.25% methyl p-hydroxybenzoate (Nipagen) was included in all plates. Comparisons of tunneling activity were only performed between sets of plates that were either all + Nipagen or—Nipagen.btl-Gal4, UAS-Actin-GFP/CyO, GFP larvae were selected using a Leica MX FluoIII fluorescence microscope. For imaging, larvae were immobilized either with ether vapor before mounting in 70% glycerol or by heating for a few seconds on a 70oC hot plate [Embryos from the appropriate crosses were collected on grape plates for four hours and after hatching, larvae were raised on mounds of yeast paste on grape plates at 22oC. For crosses involving ot plate  after molov def /CyO twi-2x GFP stock was used for crosses to generate hemizygous larvae and non-GFP larvae were selected at 72 hours AEL then reared separately to 90–92 hours AEL. For assay, each larva was transferred to a clean 2% agarose plate, given one minute to acclimatize and then forward strides, backward strides and head turns were scored over a one minute period under a dissecting microscope. Assays were performed at 18oC, >50 larvae of each genotype were analyzed. Student’s t-test was used to compare all other genotypes to the cut(ue)-Gal4/+ control.Embryos from appropriate crosses were collected on grape plates for four hours. The midpoint of this four hours was used as the Average Egg Lay time (AEL). Larvae were fed on mounds of yeast paste on grape plates at 22oC and assayed 90–92 hours after AEL. A Actin57B, which is abundantly expressed in larvae, and ribosomal protein gene rp49 were used as control genes for standardizing expression. Primers used were:RNA was isolated from embryos or larvae using Trizol lysis as previously . RNA wasactin forward: 5’ TTCCAAGCCGTACACACCGTAACT 3’actin reverse: 5’ TCATCACCGACGTACGAGTCCTTCT 3’rp49 forward: 5' TACAGGCCCAAGATCGTGAA 3'rp49 reverse: 5' CACGTTGTGCACCAGGAACT 3'lactate dehydrogenase set 1 forward: 5’ CATCCTTGTCAATGCCATGTTC 3’lactate dehydrogenase set 1 reverse: 5’ TGCTTATGGTGTCCAATCCC 3’      set 2 forward: 5' CTGAAGAACCCCCAGATCAC 3'      set 2 reverse: 5' GCAAAATGGTATCGGGACTG 3'branchless forward: 5' TTGCCTGTATCTCTGCATGG 3'branchless reverse: 5' TCGTGTAGGTGCTCAGCTTG 3'fatiga forward: 5' GACAAGATCCGAGGCGATAA 3'fatiga reverse: 5' CGCTCCCTGATGTGGTAGTT 3'dDPH1 forward: 5' TGCCAGAGACAACGAAGATG 3'dDPH1 reverse: 5' GCCTTGTCTTCCGAGTGTTC 3’Larvae were dissected in phosphate buffered saline (PBS) with 1mM EDTA, following the protocol described by Ramachandran and Budnik . The intFixed dissected larval tracheae were blocked in BBXN . Primary antibodies used were i) guinea pig anti-Lov, prepared in two guinea pigs  against the Lov protein fragment used previously to generate rabbit and guinea pig Lov antisera , 47, andDissected tracheae were treated sequentially with 0.1% Triton in PBS for 10 min, 0.1M ammonium chloride in PBS for 10 min, 50 mg/μl RNAse A (Thermo scientific) in PBS for 1 hour and finally DAPI  for 5 min. Tracheae were then detached from the cuticle for mounting. To quantitate DAPI intensity, image series were taken via a Zeiss LSM 710 confocal microscope with a 40x oil immersion objective. Nikon NIS-Element C software was then used to build 3D images of nuclei and quantitate DAPI intensity for each selected nucleus. An average of six nuclei were analyzed in each tracheal segment, and tracheae from eight individual larvae were analyzed per genotype. To quantitate cell numbers in Tr9 and Tr10 of the tracheal system, a Zeiss Axioplan2 was used, and 10 individual tracheal pairs were analyzed for each genotype.S1 Figlov RNAi lines were driven by elav-Gal4. Embryos (12–16 hrs after egg laying) were collected for RNA extraction. Semi-Q RT-PCR was used for transcript quantification. lov and actin PCR products for each RNA sample were run in parallel in separate agarose gel lanes. RNA preparations from two sets of embryos gave identical results. See All the (TIF)Click here for additional data file.S2 Figcut(ue)-Gal4 > lov RNAi and btl-Gal4 > lov RNAi larvae, failure to burrow (larvae outside the food) is seen but this is associated with greater larval death (as opposed to transition to pupation) for btl-Gal4 > lov RNAi larvae. (b) In the lov hemizygous condition, cut(ue)-Gal4/lov def > lov RNAi larvae outside the food die in greater numbers rather than pupating. Larvae were assayed in batches of 10. At least five batches per genotype were examined. Error bars = +/- SEM.Larvae were placed in tunneling assays one day after hatching as described in Material and Methods. (a) for both (TIF)Click here for additional data file.


The current Ebola virus (EBOV) outbreak in West Africa is unprecedented in terms of both its size and duration, and there has been speculation and concern regarding the potential for EBOV to increase in virulence as a result of its prolonged circulation in humans. Here we investigate the relative potency of the interferon (IFN) inhibitors encoded by EBOVs from West Africa, since an important EBOV virulence factor is inhibition of the antiviral IFN response. Based on this work we show that, in terms of IFN antagonism, the West African viruses display no discernible differences from the prototype Mayinga isolate, which corroborates epidemiological data suggesting these viruses show no increased virulence compared with those from previous outbreaks. This finding has important implications for public health decisions, since it does not provide experimental support for theoretical claims that EBOV might gain increased virulence due to the extensive human-to-human transmission in the on-going outbreak. Concerns have been raised regarding potential increase in the virulence of the Ebola virus during the current West African outbreak. Here the authors show that the ability to inhibit interferon response, one of the aspects of Ebola virus virulence, does not differ between the prototype Mayinga and isolates from the current outbreak. Of particular concern is the unprecedented number of human hosts encountered by the virus, which has given rise to fears that it will exhibit changes in virulence or transmissibility through adaptation, although such changes have so far not been reported. Genetic variation is observed between the West African Makona variant viruses and those associated with previous outbreaks, although only a very limited number of mutations have been observed among sequenced samples taken from EHF patients during the current outbreak23Ebola virus (EBOV) is a negative-sense, single-stranded RNA virus that causes a severe haemorrhagic fever in humans with high case fatality rates, ranging from 47 to 91%  response58VP24 likewise fills multiple roles during the viral life cycle. It regulates both transcription and replication131416171920In this study, we examine several variants of VP35 and VP24 corresponding to the different genotypes of Makona variant EBOVs circulating in West Africa for their ability to inhibit the IFN response. Importantly, we find no apparent differences in the expression of these proteins in mammalian cells, their function in viral genome transcription and replication or their ability to inhibit the IFN response, which does not support a potential for increased virulence of EBOV Makona via this mechanism.Based on multiple sequence alignments, we cloned all unique (on the amino acid level) VP35 and VP24 sequences found among the EBOV Makona variant genotypes published at the time of submission  and increasing amounts of the different Makona VP24 variants. After 24 h, cells were stimulated with 1,000 IU human IFN-β to initiate IFN signalling and subsequent induction of the ISRE promoter. The two distinct Makona VP24 sequences examined here were as effective as Mayinga VP24 in inhibiting the IFN-β signalling activity  with no 1921343536Our experimental studies have found no evidence for differences in the function of VP35 and VP24 from any of the currently published sequence variants of the Makona variant of EBOV, either among each other or in comparison to the prototype EBOV Mayinga. While EBOV virulence is almost certainly multifactorial in natureAlso conceptually, hypervirulence, while a common feature of zoonotic pathogens due to a lack of co-evolution with their dead-end hosts, is usually considered maladaptive. Indeed, after switching hosts it is expected that a virus rather attenuates to a lower level of virulence resulting in a longer time window for its transmission, which is limited during debilitating acute infectionset al.Finally, it is important to note that despite extensive and on-going human-to-human transmission and the fact that our sequences came from samples spanning 9 months of the outbreak, we observed only three different protein sequences for VP35 and two for VP24. Since submission of this manuscript, additional sequences have continued to be published, including 175 near-complete sequences reported recently by Tong The coding regions of the VP35 genes from EBOV Yambuku isolate Mayinga and EBOV Makona isolates C05 and C15 , as well as the coding regions of the VP24 genes from Mayinga and C05, were inserted into pCAGGS ref.  using st−1) (Life technologies) in 5% CO2 in a humidified incubator at 37 °C.Human embryonic kidney 293 cells (ATCC CRL-1573) were cultured in Dulbecco's minimal essential medium (Sigma-Aldrich) supplemented with 10% (v/v) foetal bovine serum (heat inactivated at 56 °C for 30 min) (Life technologies), 1% L-glutamine (2 mM) (Life technologies) and 1% penicillin/streptomycin  antibody 293 cells were seeded in 96-well plates 1 day before transfection for 30–50% confluence at the time of transfection. Initial transfection was performed using TransIT-LT1 (Mirus) according to the manufacturer's instructions. Each experimental well-received the following plasmids, which have been previously describedRenilla luciferase  for normalization and pCAGGS-VP35 . Wells receiving <500 ng of the VP35 construct received empty pCAGGS to compensate for differences in total plasmid mass. Twenty-four hours later, a second transfection was performed, again using TransIT-LT1, with 200 ng of a pEF construct encoding amino acids 1–200 of the human RIG-I CARD domain, which had been constructed from pEF-BOS RIG-I kindly provided by Kate Fitzgerald (Addgene plasmid # 27236)IFN production assays were performed as previously described293 cells were seeded in 12-well plates 1 day before transfection for 30–50% confluency at the time of transfection. Transfection was performed using TransIT-LT1 according to the manufacturer's instructions. Plasmids transfected included 250 ng firefly luciferase in a pISRE reporter plasmid (Agilent)Differences in reporter activity in the presence of Mayinga or the various Makona variant proteins at each transfection amount were calculated and analysed by one-way analysis of variance followed by Tukey's multiple comparison test using Prism 6.04.How to cite this article: Dunham, E. C. et al. Assessing the contribution of interferon antagonism to the virulence of West African Ebola viruses. Nat. Commun. 6:8000 doi: 10.1,038/ncomms9000 (2015).Supplementary Figure 1 and Supplementary Methods