The oligodendrocyte progenitor cells differentiation was skewed with increase in immature oligodendrocytes and astrocytes, which likely cause the observed decrease in white matter, increased neuro-inflammation, together leading to the observed significant cognitive defects.Ionizing radiation causes degeneration of myelin, the insulating sheaths of neuronal axons, leading to neurological impairment. As radiation research on the central nervous system has predominantly focused on neurons, with few studies addressing the role of glial cells, we have focused our present research on identifying the latent effects of single/ fractionated -low dose of low/ high energy radiation on the role of base excision repair protein Apurinic Endonuclease-1, in the rat spinal cords oligodendrocyte progenitor cells’ differentiation. Apurinic endonuclease-1 is predominantly upregulated in response to oxidative stress by low- energy radiation, and previous studies show significant induction of Apurinic Endonuclease-1 in neurons and astrocytes. Our studies show for the first time, that fractionation of protons cause latent damage to spinal cord architecture while fractionation of HZE ( Br. Br31]. As we were expecting that radiation will induce higher neuro-inflammation, it became important to measure radiation induced neuro-inflammation not only in rat spinal cords, but also in the rat brains from these rats.3H] PK11195 . PK11195 is used in positron emission tomography (PET) studies for imaging brain inflammation in vivo as it binds to the peripheral-type benzodiazepine receptor (PBR) expressed by reactive glia and macrophages, as previously described[3[H] PK11195. Non-specific binding is determined on consecutive sections in the presence of excess (20μM) unlabeled PK11195. After completing autoradiography, sections were dried on a slide warmer at 60°C and loaded on KODAK BioMax MR films along with tritium micro scales for three weeks. After three weeks the developed films and digital autoradiograms were obtained by scanning the films on Umax 2100 scanner at 1600 dpi resolution. Quantitative image analysis was performed using ImageJ (NIH) software and manual drawings of regions of interest (ROI) on the accumbens (Acb), anterior Cingulate (Acg), cornu ammonis 1(CA1), cornu ammonis 3 (CA3) and dentate gyrus (dg), frontal cortex (Fr), Occipital cortex (Occ), Substantia Nigra (SN), Striatum (STR), and Ventral Hypothalamus (VHP) regions. ROIs were measured bilaterally, 3–6 measurements/ROI, and a mean value calculated for each animal/side. Nonspecific binding was subtracted from total binding to generate specific binding values translated to nCi/mg via a standard curve generated from the tritium standards. Autoradiography results were analyzed using Statview software. The groups  were compared by two way ANOVA (treatment and region) followed by posthoc comparisons, using Fisher’s PLSD test when indicated. P<0.05 was considered significant.Following exposure and behavioral testing by NORT, rats were decapitated and brains were quickly removed, rinsed with 0.9% saline, and frozen in powdered dry ice. Long-term storage of brains was at −70°C. Frozen brains were sectioned in a cryostat  at −15°C in the coronal plane. Consecutive series of coronal brain sections (20 μm) were collected at 200 μm intervals from frontal cortex (∼3.7 mm from Bregma) to the cerebellum, covering the whole forebrain. On the day of the assay, sections were removed from –70°C and allowed to reach room temperature. TSPO autoradiography was performed with [described, 37. Secet al.[Increased destruction of grey/ white matter, increase in proliferation of ependymal cells at the central canal and altered architecture of the thoracic spinal cord, 3 months post exposure to 1Gy of 250 MeV protons: Our present studies show that at latent time of 3 months post protons exposure, there is destruction of both grey and white matter of T7 thoracic section of middle aged rats’ spinal cord. H & E staining has been routinely used to study changes in spinal cord architecture after spinal cord injury (SCI) , 39. As et al., 41). Noet al.. When thThe effects of single/ fractionated doses of HZE on OPC cell differentiation, and APE1 were measured, revealing altered APE1 induction with most of it appearing outside the nucleus with single dose, which became increasingly nuclear with fractionation, and also showed initial increase in progenitor, immature OL  and astrAnimals were tested in the object recognition task at nine months after protons and X-rays exposure. Proton- irradiated animals showed higher deficit in this task, with lower preference for the novel object of 23.96± 1.68% as compared to X-rays irradiated animals which showed 48.32±13.69% , which wWe were able to study these effects only 9 months post exposure, hence we have data from those rats brains, which indicate a clear increase in the microglial marker translocator protein (TSPO) binding in all brain regions including the hippocampus as shown in Animals were also tested in the object recognition task at six months after protons and HZE exposure. Fe irradiated rats showed most significant deficit with single dose and lower deficit with fractionation where as Protons as well as Si showed some trend towards more deficits with fractionation, although the data with Si NORT was most variable . StatistRadiation research emphasizes understanding the mechanisms of radiation-induced DNA damage as one of the basic regulatory mechanisms governing the outcome. In this paper we have focused on identifying the changes in level of DNA repair protein APE1 induced by oxidative stress, due to HZE radiation in glial cells in comparison to low LET radiation from X-rays and protons. Ionizing radiation causes degeneration of myelin, the insulating sheaths of neuronal axons, leading to neurological impairment. Thus, astronauts exposed to protons and HZE radiation may risk adverse effects during their missions as well as latent health effects . This pa28Si, but with decrease in total APE1 protein  without any significant change in its APE1 endo activity, indicative of single dose inducing APE1, and with HZE dose fraction likely due to more damage, the cells show lowered APE1. As the amount of endonuclease activity detected was so low (0.065 pmoles in control), one cannot conclude if there were changes in its activity as is shown in the APE1 protein content. Further, fractionation resulted in increase in progenitor, immature OL and astrocytes cell types and caused a significant inflammatory response even in those tissues which were not irradiated  and increasingly sparse grey/ white matter architecture with fractionated dose of protons, indicating a possible causal link. There was also a increase in nuclear localization of APE1 in spinal cord OPC, with fractionation of These findings align with previous reports on irradiation induced neuro-inflammation and the ability of neuro-inflammation to spread from the site of injury to relatively remote regions of the central nervous system , 44.Recently, we have also measured these rats heart blood and tissue extracts for changes in inflammatory cytokines, TNF-α and NFκB and found an increase in NFκB and changes in serum TNF-α , indicatin vivo[The increased survival of neonatal and young O-2A progenitor cells observed after fractionation with X-rays or fission neutrons can be attributed to repopulation rather than repair. IR induin vivo. We show56Fe was most detrimental when given as a single dose and deficit declined with dose fractionation. 28Si effects seemed to show trend towards higher deficit with fractionation, although without a level of significance to this trend. We may have seen them achieved if we could have kept these animals for 9 months post exposure, but these rats show more sickness (tumors and weight loss) with time, so they needed to be sacrificed earlier. Age is another limitation as these studies were done in middle aged rats which become old when they are measured for NORT (10–12 month old rats which were measured for NORT 6–9 months post exposure), so one has to consider the effects of aging exacerbating the decrease in cognitive abilities.As previous studies with 250 MeV protons in 2 month old rats  hadn’t eThus, for the first time we have established that fractionation of radiation causes higher degradation of grey/ white matter, inflammation and altered APE1 levels , increased OPC, immature OL, and astrocytes in spinal cord and a very significant neuro-inflammation in brain, which likely leads to the observed significant cognitive defects.S1 FigNFκB was measured from heart tissue by the ChIP assay and TNF-α was measured in rat heart sera using bioactive assay.(TIF)Click here for additional data file.


Focal ischemia may induce pathological alterations in brain areas distant from the primary lesion. In animal models, exofocal neuron death in the ipsilateral midbrain has been described after occlusion of the middle cerebral artery (MCA). Using sequential magnetic resonance imaging (T2- and diffusion-weighted) at 3 Tesla, we investigated acute ischemic stroke patients on days 1, 2, 6, 8, and 10 after stroke onset. Sixteen consecutive patients who had suffered a stroke involving the caudate nucleus and/or putamen of either hemisphere were recruited into the study. Four additional patients with strokes sparing the caudate nucleus and putamen but encompassing at least one-third of the MCA territory served as controls. Ischemic lesions involving striatal structures resulted in hyperintense lesions in ipsilateral midbrain that emerged between days 6 and 10 after stroke and were not present on the initial scans. In contrast, none of the control stroke patients developed secondary midbrain lesions. Hyperintense lesions in the pyramidal tract or the brain stem caused by degeneration of the corticospinal tract could be clearly distinguished from these secondary midbrain gray matter lesions and were detectable from day 2 after ischemia. Co-registration of high-resolution images with a digitized anatomic atlas revealed localization of secondary lesions primarily in the substantia nigra pars compacta. Apparent diffusion coefficient (ADC) values in the secondary lesions showed a delayed sharp decline through day 10. Normalization of ADC values was observed at late measurements. Taken together, our study demonstrates that striatal infarction elicits delayed degenerative changes in ipsilateral substantia nigra pars compacta. Midbrain changes associated with the loss of basal ganglia neurons have been described in experimental models of brain ischemia in rats –6, mice The anecdotal histopathological reports of human autopsy brain tissue published so far indicate nerve cell loss in the ipsilateral substantia nigra during the chronic phase after a large infarction of the basal ganglia , 15. CorNCT00715533). Based on the initial MRI assessment (see below), 16 consecutive patients who had suffered an acute ischemic stroke involving the caudate nucleus and/or putamen of either hemisphere were recruited into the study . Four additional patients who had suffered an acute ischemic stroke sparing caudate nucleus and putamen but typically encompassing at least 1/3 of the middle cerebral artery (MCA) cortical territory served as controls (‘control stroke patients’). Exclusion criteria included severe aphasia or inability to provide informed consent, hemorrhagic stroke, severe brain damage before admission, moderate to severe dementia (MMSE < 18) as well as contraindications to MRI. The protocol had been approved by the local ethics committee and written informed consent was obtained prior to the study from each participant according to the Declaration of Helsinki.Data presented here were collected within the framework of a larger project designated the "1000Plus study”, a prospective, single-center observational investigation , 21. Stu2). On days 6, 8, and 10, the protocol included DWI as described above as well as coronal T2-weighted imaging .All MRI scans were acquired on a 3 T whole-body scanner  dedicated entirely to clinical research. The baseline scan was obtained within 24 hours of stroke onset (day 1). Follow-up scans were performed on days 2, 6, 8, and 10. In some patients, additional later scans were performed as shown in The following definitions apply: (a) ‘Primary ischemic lesion’ means the early T2 hyperintense signal in the MCA territory. (b) The term ‘secondary MRI lesion’ is used for delayed T2/DWI signal changes that emerge in the ipsilateral midbrain after stroke. ROIs of these two lesion types were used independently in further analyses. As described in detail below, lesions were identified separately on T2 and DWI scans. However, because of higher spatial resolution and easier-to-process coronal orientation, volumetry and lesion maps were generated from T2 scans only. In general, DWI lesions matched T2 lesions well. However, because of different scan techniques, we did not calculate the degree of overlap between DWI and T2 lesions.http://www.mricro.com). Lesion segmentation was performed manually by an experienced investigator. To combine left- and right-sided infarcts into a single analysis, all images with right-hemispheric strokes were mirrored right to left.For coronal T2 scans obtained on day 10 (or on the latest available scan before day 10), binary masks distinguishing between the hyperintense-appearing primary and secondary lesions, (coded as “1”), and non-infarcted areas (“0”) were generated for later usage as regions of interests (ROI) and as the basis for subsequent volumetric measurements using the MRIcro software  obtained on day 10 or on the latest available scan between days 6 and 10, respectively, using MRIcro. Imaging data of preceding scans were co-registered with this reference using SPM8 and the mean ADC values of the lesions were quantified for all available time points. For the determination of contralateral ADC values as an intra-subject reference value, ROIs were mirrored to the corresponding contralateral neuroanatomic structures.To visualize the spatial distribution of the primary lesion, DWI data sets on day 10 or on the latest available scan, respectively, and their corresponding ROIs were additionally normalized to the brain template provided by the International Consortium for Brain Mapping (ICBM) using SPM8.Values are presented as means ± SEM. Ipsilateral and contralateral ADC measurements were compared using paired t-tests.Sixteen consecutive striatal stroke patients and four control stroke patients were enrolled into the study. Patients’ baseline characteristics are summarized in Next, we studied the anatomic distributions and relative frequencies of the primary ischemic lesions in relation to the emergence of secondary midbrain changes  and 2A. Finally, we measured ADC values of the ipsilateral secondary exofocal lesions over time. As compared to the corresponding midbrain area of the contralateral hemisphere, ADC values in the ipsilateral lesion showed a delayed sharp decline through day 10 .This study has the following major findings: Ischemic lesions in the striatum lead to secondary changes in ipsilateral midbrain gray matter. Co-registration of high-resolution images at 3 T with a neuroanatomic atlas revealed that in human stroke patients these secondary midbrain changes emerge particularly in the substantia nigra pars compacta. As compared to hyperintense lesions in the pyramidal tract, which are caused by Wallerian degeneration of the corticospinal tract, secondary midbrain hyperintensities were not present on the initial MRI scans but appeared in a delayed fashion between days 6 and 10 after striatal stroke.Cranial MRI provides an excellent anatomic definition of the location of the primary ischemic lesion and can also be used in the subacute and chronic phases after stroke. It is therefore important that clinicians be aware of the delayed MRI changes described here so as not to confuse them with a second stroke. As compared to an earlier study of striatal stroke patients at 0.5 T, which reported the first occurrence of a T2 hyperintensity in the midbrain, on average, at more than 14 days after stroke onset , our stuWhile experimental stroke studies in rats mainly point to involvement of the pars reticulata of the substantia nigra, our study, as well as an earlier MRI study in human patients , found tWhat might be the clinical relevance of delayed exofocal neurodegeneration? In the current study, no apparent clinical deterioration was observed with the onset of secondary midbrain MRI changes. However, using a middle cerebral artery occlusion model in the mouse, we previously demonstrated that antidepressants prevent delayed neurodegeneration in the midbrain and thereby attenuate the depression-like behavioral syndrome that typically evolves in the subacute stages of recovery . We specIn conclusion, exofocal post-ischemic neuronal degeneration in the ipsilateral substantia nigra depends on stroke topography and becomes detectable by MRI between days 6 and 10 after stroke. DWI and T2-weighted imaging at 3 T offer reliable detection of these secondary midbrain changes. The further study of these delayed processes of lesion progression after stroke may aid research in developing new strategies aimed at preventing secondary neuron loss.A potential caveat concerning our data is the relatively small size of the control group. Although the current study focusses primarily on striatal infarctions as related to secondary lesion development, which is only present in this group, the small sample size of the ‘non-striatal’ control group and differences in baseline patient characteristics  of EPND ) in 12 patients ipsilateral to primary lesion (upper panel) compared to mirrored controlateral ROIs (bottom panel). As mentioned in the manuscript, late measurements  were done in single patients and show ADC normalization on day 72 and 144. P-values for the comparison of ipsilateral and contralateral ADC values were calculated using Students paired T-tests.ADC Values (ADC [10(PPTX)Click here for additional data file.


Tropical mountain forests are hotspots of biodiversity hosting a huge but little known diversity of insects that is endangered by habitat destruction and climate change. Therefore, rapid assessment approaches of insect diversity are urgently needed to complement slower traditional taxonomic approaches. We empirically compare different DNA-based species delimitation approaches for a rapid biodiversity assessment of hyperdiverse leaf beetle assemblages along an elevational gradient in southern Ecuador and explore their effect on species richness estimates.Based on a COI barcode data set of 674 leaf beetle specimens (Coleoptera: Chrysomelidae) of 266 morphospecies from three sample sites in the Podocarpus National Park, we employed statistical parsimony analysis, distance-based clustering, GMYC- and PTP-modelling to delimit species-like units and compared them to morphology-based (parataxonomic) species identifications. The four different approaches for DNA-based species delimitation revealed highly similar numbers of molecular operational taxonomic units (MOTUs) (n = 284–289). Estimated total species richness was considerably higher than the sampled amount, 414 for morphospecies (Chao2) and 469–481 for the different MOTU types. Assemblages at different elevational levels (1000 vs. 2000 m) had similar species numbers but a very distinct species composition for all delimitation methods. Most species were found only at one elevation while this turnover pattern was even more pronounced for DNA-based delimitation.Given the high congruence of DNA-based delimitation results, probably due to the sampling structure, our study suggests that when applied to species communities on a regionally limited level with high amount of rare species (i.e. ~50% singletons), the choice of species delimitation method can be of minor relevance for assessing species numbers and turnover in tropical insect communities. Therefore, DNA-based species delimitation is confirmed as a valuable tool for evaluating biodiversity of hyperdiverse insect communities, especially when exact taxonomic identifications are missing. Worldwide, natural habitats are disturbed and destroyed at alarming rates which results in a massive loss of species . EspeciaKnowledge of species numbers, on local as well as on global scale, is important for providing a reference point to estimate biodiversity loss . AlthougWhile debates on how a species should be defined resulted in different competing species concepts  these maBeetles play a crucial role in efforts to estimate the total number of species on Earth ,21 becauExtreme species richness associated with the difficulty of sorting and identifying a poorly known fauna presents a methodological challenge for large-scale biodiversity studies ,46. DNA-In this study, different species delimitation methods were applied to an unexplored leaf beetle fauna in a tropical montane rainforest of southern Ecuador. So far the number of species known from Ecuador is rather low due to the scarce taxonomic information available for this group. The Invertebrate Section of the Museum of Zoology at Pontifical Catholic University of Ecuador in Quito, which holds Ecuador's largest collection of native taxa, harbours ~24,200 Chrysomelidae specimens of which 76% have no identification at all . Especially areas in southern Ecuador are strongly undersampled . The onlBased on species estimates from a set of different delimitation methods we try to infer species diversity and compare it to estimates from parataxonomic morphological species sorting. We test the performance of several delimitation methods and evaluate how much these different yields affect estimates of potential species richness. The study area in the tropical Andes of southern Ecuador is part of a mega-diverse biodiversity hotspot ,74, wherThe Ministerio del Ambiente, Ecuador, permitted us to carry out research and access to study sites was granted by Naturaleza y Cultura Internacional (NCI).Leaf beetle sampling was conducted in November and December 2010 and between May and August 2011 in the mountain forest of Podocarpus National Park (NP) and the adjacent Reserva Biológica San Francisco (RBSF) in southern Ecuador. The area belongs to the Tropical Andes, a biodiversity hotspot ,74, and Sampling was conducted in three different elevational zones within Podocarpus NP and RBSF, namely (1) the 1000 m zone , (2) the 2000 m zone (Estación Científica San Francisco (ECSF) area; 1913–2089 m a.s.l.; lower montane rainforest), and (3) the 3000 m zone   sweep-netting, b) beating of shrubs and smaller trees using a beating-tray and c) hand collection from the vegetation. To complete the overview of species diversity for DNA-based species delimitation, standardized sampling was complemented with additional hand collection and Malaise- as well as light-trapping collections.Beetles were killed and collected in 70% ethanol but transferred into 96% ethanol the same day. For each sampling unit  Chrysomelidae were sorted for preliminary morphospecies and one specimen of each morphospecies of each sample was used for subsequent parataxonomic sorting and molecular analysis.® Blood&Tissue Kit or Qiagen Biosprint 96BS following the manufacturers' protocol. The specimen was subsequently dry mounted and labelled to allow following morphological investigation. The 5'-end of cytochrome c oxidase I  was amplified with the primers LCO1490 and HCO2198, or with LCO and Nancy , and 2.5 μl DNA template, and filled up to 20 μl with sterile H2O. The PCR temperature profile consisted of an initial denaturation at 95° (15 min), followed by 15 cycles at 94° , 55°-40° , 72° , 25 cycles at 50° annealing temperature, and a final extension at 72° (10 min). Products were checked by electrophoresis on a 1.5% agarose gel containing GelRedTM (Biotium Inc.). Successfully amplified DNA fragments were purified using IllustraTM ExoStar  following the manufacturers' protocol.Total genomic DNA was extracted from one to three legs of each specimen, using the Qiagen DNeasyhttp://www.macrogen.com) using the same primers. Sequences are submitted to GenBank (accession numbers KJ677272–KJ677945) and voucher specimens will be deposited in the collections of the Zoological Research Museum Alexander Koenig, Bonn (ZFMK), and the Universidad Técnica Particular de Loja, Ecuador  being subsequently aligned using the implemented MUSCLE algorithm ).Observed and estimated species numbers are higher for molecular species delimitation methods than for morphological species sorting but the differences at a local level are not too great. This might change considerably with a much wider geographical sampling where intraspecific variation is covered more extensively and interspecific divergence decreases as one encounters more closely related, allopatrically distributed species . This miA species accumulation curve that does not reach saturation is frequently found for samples from rainforest communities of insects ,108. In In contrast to expectations from the literature  no signiAdditional Malaise- and light-trapping added more specimens and species with different ecology and habitat preferences  to our sampling. However, the canopy, which is considered to be the most diverse habitat in tropical rainforests ,114, espThe present study provides a rapid biodiversity assessment of the hitherto unstudied leaf beetle fauna of the understorey vegetation of a tropical montane rainforest in Ecuador. It revealed a remarkable diversity of Chrysomelidae and is a good starting point for future, more detailed research on this fauna, also regarding a thorough taxonomic re-examination and the formal description with binary Linnaean names of possibly new species. The large species turnover found between the different elevations suggests the need for further investigation of differences between the communities, particularly along the elevational gradient. This seems even more important since the rates of forest conversion of the Andean mountain forests are high, with Ecuador suffering the highest annual deforestation rate in South-America (-1.9%) ,120.Whereas the integration of different DNA-based approaches for estimating species richness is strongly recommended , the choS1 Fig(EPS)Click here for additional data file.S2 FigColumn 1: Split morphospecies are connected by brackets and share the same colour. Columns 2–7 + 8: MOTUs  and haplotypes splitting a morphospecies are indicated by dark blue fields, those lumping morphospecies by light blue fields, those splitting and lumping morphospecies at the same time by green fields. Red fields indicate differences between the different molecular species delimitation methods.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TablePodocarpus NP = Podocarpus National Park, RBSF = Reserva Biológica San Francisco.(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Epigenetic changes could mediate the association of maternal pre-pregnancy body mass index (BMI) and gestational weight gain (GWG) with adverse offspring outcomes. However, studies in humans are lacking. Here, we examined the association of maternal pre-pregnancy BMI and GWG in different periods of pregnancy with cytosine-guanine (CpG) dinucleotide site methylation differences in newborn cord blood DNA from 88 participants in the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort using the Illumina GoldenGate Panel I. Pyrosequencing was used for validation of the top associated locus and for replication in 170 non-overlapping mother-offspring pairs from the ALSPAC cohort.MMP7, KCNK4, TRPM5 and NFKB1 genes (difference in methylation >5% per 400 g/week greater GWG) . Pre-pregnancy BMI and GWG in mid- or late pregnancy were not associated with differential DNA methylation at any CpG site. Pyrosequencing showed that greater GWG in early pregnancy was associated with increased DNA methylation levels at the top associated CpG site at MMP7, although association did not reach statistical significance (p = 0.302). Greater GWG in mid- (p = 0.167) and late-pregnancy (p = 0.037) were also associated with increased DNA methylation levels at the MMP7 CpG site. In addition, newborns of mothers who exceeded the IoM-recommended GWG had higher DNA methylation levels at the MMP7 CpG site than those of mothers with IoM-recommended GWG (p = 0.080). We failed to replicate findings.After correction for multiple testing greater GWG in early pregnancy (between 0 to 18 weeks of gestation) was associated with increased DNA methylation levels in four CpG sites at MMP7, KCNK4, TRPM5 and NFKB1 genes in offspring cord blood DNA. The specific association of GWG in early pregnancy with the top associated CpG site at MMP7 was not validated using Pyrosequencing and it did not replicate. However, given the potential functional relevancy of the four identified loci, we advocate further exploration of them in larger studies.Greater GWG in early pregnancy was associated with increased methylation at CpG sites at  Greater maternal pre- or early- pregnancy body mass index (BMI) and gestational weight gain (GWG) have been shown to be associated with health outcomes in offspring, including increased birth weight,2, impaiTo our knowledge only one study in humans has examined the association of maternal pregnancy adiposity with offspring DNA methylation patterns. In that study, cord blood white cell DNA methylation was compared between 25 offspring born before their mothers had undergone bypass surgery for severe obesity with 25 matched siblings born after marked weight loss by their mothers following bypass surgery, using the Illumina HumanMethylation450 BeadChip array,16. AlteWe hypothesize that maternal pre-pregnancy BMI and GWG, specifically in early pregnancy, could influence risk of adverse health outcomes in offspring by inducing epigenetic changes. Thus, we aimed to examine whether maternal pre-pregnancy BMI and GWG in different periods of pregnancy were associated with epigenetic marks in newborns, in terms of gene-specific cord blood DNA methylation, and to compare the early pregnancy GWG associations with mid- and late-pregnancy GWG to test whether there was any evidence of specific early pregnancy associations. Our analyses were undertaking in a discovery sample and then in a replication sample, both taken from a large, longitudinal UK cohort of mothers and their offspring.http://www.alspac.bristol.ac.uk)[Data were available from participants in the Avon Longitudinal Study of Parents and Children (ALSPAC), a prospective, population-based mother-child cohort study that recruited over 14 000 pregnant women resident in Avon, UK, between 1991 and 1992 (l.ac.uk),21. A sul.ac.uk), and wer2) was based on the mother’s self-reported weight before pregnancy and maternal report of height at their first questionnaire assessment (~12-18 weeks gestation) [Six trained research midwives abstracted data from obstetric medical records for the entire ALSPAC cohort. Obstetric data abstractions included every measurement of weight entered into the medical records and the corresponding gestational age and date. All pregnancy weight measurements  were used to develop a linear spline multilevel model (with 2 levels: woman and measurement occasion) relating gestational age to weight. Using the entire cohort of women with term pregnancies (≥37 weeks of gestation) fractional polynomial curves were fitted to the data to obtain the average shape of the trajectories of GWG with gestational age . These  models).DNA extracted from cord blood was used for DNA methylation analysis. Site-specific CpG methylation was analysed using bisulphite treated DNA  using the the GoldenGate Cancer Panel I Array  and the GoldenGate Assay Kit with UDG on the Sentrix Universal-96 Array matrix v7A. This panel spans 1,505 CpG sites selected from 807 genes. Arrays were imaged using a BeadArray scanner and image processing and intensity data extracted using Illumina BeadStudio v3.2, methylation module v3.2.5 custom software. Methylation levels  at a given CpG site were estimated as the ratio of signal intensity of the methylated alleles to the sum of methylated and unmethylated intensity signals of the alleles: β = M/(U + M), where M was the fluorescence level of the methylation probe and U was the fluorescence level of the unmethylated probe. The beta values vary from 0 (no methylation) to 1 (100% methylation). Samples were run across four different PCR plates.Based on previous knowledge, the following were considered a priori potential confounding factors because of their possible associations with either maternal pre-pregnancy BMI or GWG and DNA methylation levels: child’s sex and ethnic background, mode of delivery, maternal age at child’s birth, parity, maternal smoking in pregnancy and occupation. Child’s sex, maternal age, parity (0 or 1+) and mode of delivery , and child’s ethnic background (white or non-white) were obtained from the obstetric records. Maternal smoking and occupational (of both the mother and her partner) were reported by the mother in a study questionnaire administered at mean 18 weeks gestation; with data on smoking also reported in subsequent questionnaires administered during pregnancy. Maternal smoking in pregnancy was categorized as no versus yes. Highest parental occupation was used to allocate the children to family social class groups according to the 1991 British Office of Population and Census Statistics classification  following the manufacturer’s protocol. Quantitative bisulphite Pyrosequencing  with Pyro Q-CpG™ Software (version 1.0.6.) was subsequently used to determine the percentage methylation at individual CpG sites. 1ug of DNA was bisulfite modified. Bisulfite treated DNA was added to the first PCR reaction with 12.5 μl Hot Star Taq mastermix (Qiagen) and optimised primer concentrations and annealing temperature. PCR cycling conditions were: 95 degrees C for 15 min, 50 cycles of 95 for 15 secs, 45 degrees C for 30secs, 72 for 15 secs, 72 for 5 min, and 4 for hold/storage. Assays were assessed for amplification bias and reliability as described previously[2 = 0.99). Two independent replicates per sample were processed on separate runs, giving good correlation (R2 between 0.658 and 0.847).A PyroMark MD Pyrosequencing System (Qiagen) was used for the validation of the top associated locus (MMP7_E59). This quantifies DNA methylation in a sequencing-by-synthesis manner providing precise methylation levels of several CpG positions in close proximity. The DNAeviously,27. ZeroThe same Pyrosequencing assay used during the validation study of the top associated locus (MMP7_E59) was used to analyze 192 non-overlapping mother-offspring pairs randomly selected from the 2,183 ALSPAC cohort participants with cord blood DNA available and who were not included in the discovery study. Of these 192 pairs 170 (88%) could be successfully characterized.In the discovery study mixed linear regression models were fitted to estimate the association of pre-pregnancy BMI and GWG in different periods of pregnancy with DNA methylation in offspring cord blood, regressing the methylation status at each CpG site quantified by the Illumina beta value (dependent variable) against maternal pre-pregnancy BMI or GWG (independent variables). Maternal pre-pregnancy BMI and GWG were scaled to be clinically meaningful, examining the variation in CpG site DNA methylation per 1 SD change of maternal pre-pregnancy BMI and per 400 g gain per week of gestation for GWG. Final mIn the validation and replication analyses based on Pyrosequencing, CpG site methylation levels were also analyzed by mixed linear regression models adjusted for child’s sex and maternal age at child’s birth as fixed coefficient and PCR plate as a random batch effect as outlined above.Ethical approval for all aspects of data collection was obtained from the ALSPAC Law and Ethics Committee and the Local Research Ethics Committee in accordance with the guidelines of The Declaration of Helsinki. Written informed consent was obtained for all participants in the study.The discovery study population included 88 mother-offspring pairs. Participants were unrelated newborns born at term and 50% male. Mean (sd) maternal age at birth was 30.2 (3.5) years. In comparison with eligible participants for the present study, mothers included in the discovery study were older, and were less likely overweight or obese before pregnancy, but did not differ in GWG Table .Maternal pre-pregnancy BMI was not found to be associated with differentially methylated DNA at any CpG site in offspring cord blood ; of these, 4 CpG sites remained robust to correction for multiple comparison testing: MMP7_E59 , KNCK4_P171 , TRPM5_E87 , and NFKB1_P496   Table . Six outMAP3K1_E81 ; although it did not remain statistically significant after correction for multiple comparison testing   at one CpG site at MMP7 was validated by reanalyzing CpG site MMP7_E59 in the discovery study using Pyrosequencing. The methylation levels assayed using the GoldenGate platform did not correlate with Pyrosequenced values .The association of early pregnancy GWG with differential methylation at the top associated CpG site at MMP7_E59 and with each other . In agreement with the discovery findings, Pyrosequencing results showed that greater GWG in early pregnancy tended to be associated with higher methylation at the MMP7_E59 CpG site, although the association did not reach statistical significance     maternal age at birth was 28.7 4.4) years. Compared with the discovery study sample those included in the replication study have younger mothers that were more likely overweight or obese before pregnancy, but did not differ in GWG  to be associated with differential methylation at 4 CpG sites at MMP7) and the specific early GWG association with differential methylation at that locus did not validate or replicate.The specific association of early pregnancy GWG with differential methylation at four loci is notable, since early GWG is more likely to reflect maternal fat deposition than later GWG , and so MMP7 gene in relation to greater GWG. Matrix metalloproteinases (MMPs) comprise a large family of structurally related zinc-dependent proteinases that have been classified into subgroups on the basis of their structure, substrate specificity, and cellular localization and include collagenases, gelatinases, stromelysins, and membrane-type (MT-MMPs)[MMP7 is down regulated in obesity[MMP7 would imply lower gene expression, which could translate into a higher risk of adiposity in the offspring in response to an in utero obesogenic environment. However, these associations were not replicated in non-overlapping mother-offspring pairs; thus, present results should be interpreted cautiously and future studies are warranted to confirm the association of greater GWG with differential methylation of MMP7.Our more significant finding in the discovery study was increased methylation of CpG sites at MT-MMPs). MMPs paMT-MMPs). Interes obesity,30. ThesKCNK4, TRPM5 and NFKB1. However, these three identified genes may be worthy of further exploration. KCNK4 encodes for a potassium channel, subfamily K, member 4 known as Trek/TRAAK channels that has been described to be expressed in the central nervous system, modulate neuronal activity, brain metabolism and have a role in neuroprotection[TRPM5 encodes for a thermo-sensitive TRP  channel that is expressed in pancreatic β-cells and could predominantly contribute to pancreatic functions[TRPM5 has been reported to be likely associated with pre-diabetic phenotypes and contribute to the development of type 2 diabetes mellitus[TRPM5 knockout mice exhibit impaired glucose clearance resulting from reductions in insulin secretion[TRPM5 CpG sites in relation to greater maternal GWG; this may in turn result in lower expression of TRPM5 and, thus, perturbed insulin metabolism and increased risk of diabetes in the offspring as it has been previously suggested[NFKB1 encodes for nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, which is responsible for activation of transcription of genes involved in immune responses, inflammation or cell proliferation[NKKB1 gene has been reported to be associated with a range of immune-mediated diseases such as atopy, asthma and related phenotypes[Validation and replication studies using Pyrosequencing were not undertaken for other CpG sites that were identified as being differentially methylated in relation to greater GWG and located in genes otection,32. Knocotection,34. Inteunctions. In humamellitus. Furtherecretion,38. Thisuggested. Finallyferation. Geneticenotypes, which aenotypes,43. TherThe strengths of this study are the prospective design and that potential confounding factors were taken into account in the analyses. Further, we evaluated the association of pre-pregnancy BMI and GWG in distinctive periods of pregnancy with offspring cord blood DNA methylation patterns. To our knowledge no previous human studies have examined these associations, yet these maternal exposures have been shown to be related to later offspring outcomes and it has been suggested these associations may be mediated by epigenetic mechanisms,13,14. TMMP7 CpG sites measured with the GoldenGate array and Pyrosequencing limits the conclusions that can be drawn from the present validation and replication studies. Thirdly, smaller effect sizes in DNA methylation were found and biological significance in terms of changes in gene expression is unknown. However, it may be a general phenomenon in complex diseases and phenotypes, where methylation at any given CpG island or specific CpG sites in affected versus unaffected individuals may vary by less than 10%, indeed they have been reported in many other human epigenetic studies. Moreover, for some genes, evidence exists that a small change in the level of DNA methylation, especially in the lower range, can dramatically alter gene expression levels[MMP7 for validation and replication, which limits the interpretation of results for the other three loci that were identified as being associated with GWG in early pregnancy. Given the likely functional relevance of these and the specificity of association with early GWG we feel that further study of these loci is warranted.However, the study has some limitations. Firstly, DNA methylation changes were assessed in cord blood, which limits to know how identified changes could translate to potential target tissues. However, DNA methylation patterns are largely conserved across tissues suggesting that for population based epidemiological studies methylation markers from easily accessible surrogate tissues could be used as a proxy for methylation in target tissues. Becausen levels. We opteMMP7, KCNK4, TRPM5 and NFKB1 genes in offspring cord blood DNA. These four loci were all potentially functionally relevant but we were only able to take the top associated locus at MMP7 forward for validation using Pyrosequencing and replication in non-overlapping mother-offspring pairs. The specific association with GWG in early pregnancy for that one site was not statistically significantly validated and it did not replicateple. Given this is the first study we are aware of to examine these associations and our findings might reflect limited statistical power, we advocate further exploration of identified loci in larger studies and the study of genome-wide DNA methylation data.We found that greater GWG, specifically in early pregnancy, was associated with increased methylation at 4 CpG sites at The authors declare that they have no competing interests.DAL and CLR: obtained funding for the study and initially designed it. EM and CLR: developed the study aims. EM and AG: conducted the experiments, under the supervision of CLR. EM: performed the statistical analyses and wrote the first draft of the manuscript. EM, DAL and CLR participated in the interpretation of data. All authors help to draft the manuscript, and read and approved the final manuscript.Supplementary material for DNA methylation signatures in cord blood associated with maternal gestational weight gain: results from the ALSPAC cohort.Click here for file


Salmonella species. Mixed infections with tagged Salmonella can be used in combination with probabilistic models to describe the dynamics of the infection process. Here we used mixed oral infections with tagged Salmonella strains to identify bottlenecks in the infection process in naïve and vaccinated mice. We established a next generation sequencing based method to characterize the composition of tagged Salmonella strains which offers a fast and reliable method to characterise the composition of genome-tagged Salmonella strains. We show that initial colonization of Salmonella was distinguished by a non-Darwinian selection of few bacteria setting up the infection independently in gut associated lymphoid tissue and systemic compartments. Colonization of Peyer's patches fuels the sustained spread of bacteria into mesenteric lymph nodes via dendritic cells. In contrast, infection of liver and spleen originated from an independent pool of bacteria. Vaccination only moderately reduced invasion of Peyer's patches but potently uncoupled bacterial populations present in different systemic compartments. Our data indicate that vaccination differentially skews the capacity of Salmonella to colonize systemic and gut immune compartments and provide a framework for the further dissection of infection dynamics.Vaccination represents an important instrument to control typhoid fever in humans and protects mice from lethal infection with mouse pathogenic serovars of  Salmonella infection in non-manipulated and vaccinated mice. We infected mice with mixtures of tagged Salmonella strains and analysed the strain composition in different compartments by high throughput sequencing. This information allowed us to estimate the number of Salmonella invading a given tissue and to describe routes of pathogen dissemination. We show that vaccination only modestly reduces invasion of intestinal lymphoid tissue but had a profound effect on the spread of Salmonella to systemic compartments.Pathogens have evolved strategies to invade, replicate and spread within their hosts. On the contrary, vertebrates have developed sophisticated immune defence mechanisms that limit, and ideally clear, the infection. This dynamic interplay between host and pathogens determines the course of the infection and the development of clinical disease. Knowledge on particularly vulnerable steps in the infection process, i.e. the “Achilles heel” of a pathogen, may guide the development of anti-infective therapies and vaccines. However, for most pathogens we lack detailed information on the dynamics of the infection process. Here we determined bottlenecks, i.e. critical steps during pathogen invasion and spread, after oral  Salmonella enterica serovar Typhi and Paratyphi are estimated to affect some 27 million individuals each year Salmonella can also cause life-threatening invasive disease, in particular in immunocompromised patients and by selected Salmonella lineages in African countries Salmonella typically occurs upon consumption of contaminated water or food and thus safe water supply is the best measure to control typhoid and paratyphoid fever While infections with Salmonella demonstrated protection after oral but not systemic Salmonella infection Salmonella strain yielded high levels of SIgA in the intestine and conferred protection against challenge with fully virulent Salmonella, whereas a non-invasive variant that failed to elicit strong SIgA responses did not provide protective immunity Salmonella strain protected polymeric Ig receptor (pIgR)-deficient mice that fail to transport and secrete Ig into the gut lumen Salmonella by immunization with attenuated SalmonellaAt present two licensed typhoid fever vaccines are available, an injectable capsular polysaccharide vaccine and an orally applied live attenuated bacterial mutant. Parenteral as well as oral vaccines are well tolerated and similarly effective. Still, parenteral and oral vaccines differ with respect to the quality of the induced immune response. Oral vaccines in first instance target gut associated lymphoid tissue (GALT), foremost Peyer's patches (PP), and therein induce a strong gut-directed secretory Ig (SIg) A response Salmonella infection and vaccination , i.e. the number of bacteria recovered under a given condition. In combination with kinetic studies, the CFU adequately describes overall changes in bacterial numbers in a given organ. Still, changes in CFU do not allow disentangling distinct aspects of the infection process such as bacterial growth and death rates, immunological activity, pathogen dissemination and others. However, access to such information could identify critical steps in the infection process and guide the development of vaccines and anti-infective therapies. One way to overcome the limitations of classical infection experiments is to perform co-infections with tagged pathogens and to apply probabilistic models. Such approaches offer unprecedented insights into the dynamics of bacterial populations and may change fundamentally our understanding of bacterial infections Salmonella to identify bottlenecks in the infection process in naïve and vaccinated mice. We exploit stochastic variations in the presence of individual sequence-tagged strains to estimate the number of Salmonella setting up the infection and compare the composition of tags in different compartments to describe their relatedness and deduce routes of pathogen dissemination. We reveal vaccination-sensitive and insensitive steps of the infection process in otherwise non-manipulated wild type mice. Our results suggest that vaccination has only a modest effect on initial colonization but potently restricts pathogen spread in systemic compartments.The difficulty to gauge the contribution of different immune defense mechanisms such as cell-mediated versus humoral immunity to the protection against Salmonella infection process in mice, we performed infections with mixed inocula of genome-tagged strains of Salmonella enterica serovar Typhimurium (S. Typhimurium). For creating such tagged strains, primers carrying a random nucleotide sequence were used to generate genomic integrations, disrupting the endogenous proV gene and introducing an artificial stop codon. A total of 23 strains carrying individual sequence tags was selected for easy-to-discriminate tag sequences and used for further experiments.Vaccination may limit initial invasion, affect subsequent growth of bacteria and/or affect pathogen dissemination. To describe the dynamics of the proV gene is one of three redundant genes under the proU operon, which encodes a glycine-betaine/L-proline binding transport system during high osmolarity situation. Genetic disruption of proV is thought to have no effect on Salmonella virulence S. Typhimurium strain and its tagged variants revealed comparable bacterial loads in the mesenteric lymph nodes (mLN), PP, livers and spleens . This indicated that already 2 days post challenge selection processes profoundly shaped the bacterial population. Moreover, we observed striking differences between compartments, and individual WITS dominating one particular compartment could be less frequent or undetectable in others , mesenteric lymph nodes (mLN), livers and spleens were plated. Individual colonies were picked up, the tagged tectable . Since wn others . In partn others . This fuin vivo and thereby resulting in a disproportionate representation of individual WITS. To distinguish between these alternatives, we performed sequential infection experiments in 129Sv mice. 129Sv mice, which are more resistant than C57BL/6 mice and survive S. Typhimurium infection, were infected with an equal mixture 10 WITS for 60 days. Similar to the findings described in the acute infection, individual WITS were unevenly represented in the mLN 60 days after infection -coded macro, which runs in the Microsoft Excel environment, are available as supplementary material.Mixing the WITS at different proportions allows to shift the range of TSU that can reasonably be determined . IrrespePicking and sequencing of individual clones is poorly suited to determine the frequency of ‘one or more missing WITS’, because a high number of colonies need to be analysed by a low-throughput method. One straightforward method to reliably determine the frequency of ‘missing WITS’ is provided by fluorescence-tagged bacteria. Characterization of nucleotide-tagged bacteria by saturating sequencing techniques, i.e. when all clones are sequenced, represents a second approach (see below).S. Typhimurium strains, here referred to as fluorescent WITS (fWITS). To minimize the impact of the fluorescent marker on Salmonella virulence, all reporter proteins were expressed under control of an inducible promoter that is transcribed at very low rate in vivo but readily inducible in vitro by addition of arabinose to the medium. Two days after i.v. infection with a mixture of three fWITS, all fWITS were represented in individual PP at fairly similar proportions compared to the inoculum and we did not observe ‘one or more missing WITS’ . Reducing the infection dose from 109 to 108 and 107 attenuated Salmonella resulted in a reduction of the TSU per PP -deficient strain (SL1344ΔhilA) or a Salmonella strain lacking a fimbrial subunit (SL1344ΔfimH) showed very low TSU , identifiable by rapid next-generation DNA sequencing, to detail in vivo population dynamics in naïve and vaccinated mice after oral infection.Manipulation of host and pathogen factors can be expected to further alter the outcome of infection dynamics, and to provide fundamental insights into infection pathogenesis. However, the approaches listed above may fall short of describing the fine architecture of pathogen dynamics and relevant information required for the rational design of anti-infective therapies Salmonella dissemination.Following oral infections with a WITS library, we observed a highly uneven representation of WITS in target organs and frequent holes in the WITS library. NGS offered an affordable and fast method to characterize such WITS compositions. In this study typically more than 10-fold more sequences than bacteria present in the sample were analysed – still depending on total CFU and the number of different WITS, far fewer sequences are sufficient to reliably describe the WITS representation in target organs. We propose firstly that holes in the WITS pool can be used to estimate the number of bacteria productively seeding a given compartment; and secondly, that comparison of WITS representation between samples allows describing routes of S. Typhimurium initially seeding target organs. We assume that detectable WITS regularly expand from single bacteria and subsequently grow up to either overrepresented WITS clones or show low net growth and eventually contribute to the overall bacterial load to a much lower degree. Thus, the particular nature of the infected host cell might determine the contribution of the respective bacteria to the overall bacterial population. This observation is consistent with the previously described ‘dormant’ Salmonella, bacteria which are infecting host cells but not undergoing direct replication Both properties of the WITS pool, ‘holes’ and skewed representation of individual WITS, seem interconnected and a consequence of an overall low number of 9Salmonella used for oral infection these values seem surprisingly low. Yet, similar observations were recently reported in the “streptomycin model” of Salmonella infection. In this model despite presence of 109Salmonella in the cecum only about 300 Salmonella reached the draining lymph nodes per day The occurrence of ‘holes’ in the WITS representation can be used to estimate the number of bacteria that initially seeded a compartment. We refer to such values as tissue seeding units – TSU. TSU for PP, mLN, liver and spleen were typically below 50, i.e. less than 50 founder bacteria set up the infection. Considering the number of 10aroA) and wild type S. Typhimurium as well as in susceptible C57BL/6 mice and more resistant 129Sv mice (data not shown). This indicates that metabolic attenuation as well as relevant host factors affecting the killing of incoming Salmonella did not affect the number of bacteria initially breaching the gut barrier. Still the TSU is based on detectable bacteria in a specific organ and we cannot rule out that higher numbers of bacteria initially entered host tissues but are undetectable even in susceptible hosts. Irrespectively, TSU measurement allows uncoupling initial invasion from later steps of the infection process and reveals that entry from the gut lumen into host tissues represents the first bottleneck in oral infections that is overcome by only few bacteria.TSU were similar for attenuated . Experiments were approved by the Lower saxony state office for consumer protection and food safety  under the file numbers TVA 09/1771, TVA 10/0164 and TVA 12/0915.Salmonella strains were derivatives of S. enterica serovar Typhimurium strain SL1344. SL1344ΔaroA and SL1344ΔhilA (SPI-1 deficient) were described previously S. Typhimurium SL1344 was established. Each clone was generated by integrating of a PCR product into the proV gene in the Salmonella chromosomal genome. The PCR product contains a degenerate four nucleotide sequence and a chloramphenicol resistance cassette (amplified from the pKD3-plasmid), flanked by parts of the proV gene. As described previously Salmonella strain. Each clone differed from one another by only 4 bp in the nucleotide tag gene sequence. To select suitable clones for the WITS library, individual colonies were picked, the tagged site amplified by PCR and sequenced. Clones to be included in the library were selected for easy to identify tag sequences with sufficient differences to avoid erroneous assignment of tags. In particular we avoided tag sequences, which by single nucleotide differences/sequencing errors can be interconverted.S. Typhimurium were grown in LB broth, until the culture had reached a density of OD6001.0–1.2. Bacteria were washed with 3% NaHCO3/LB medium, and resuspended to final cell density of 1×109 bacteria  or 2.3×109 bacteria  per 100 µl NaHCO3/LB. Mice were infected by oral gavage with 100 µl bacterial suspension for 2 days (acute infection experiment) or immunized orally with 1×109 live attenuated SL1344 ΔaroA for at least 40 days. Vaccinated mice were given one dose of enrofloxacin  orally at 2.5 mg in 100 µl PBS to clear any remaining Salmonella in the lumen, 2 days before the mice were re-infected orally with 1×109 CFU of SL1344. The actual numbers of inoculated bacteria and of bacteria recovered from tissues were determined by serial dilution plating on selective LB agar plates with appropriate antibiotics (90 µg of streptomycin/ml or/and 100 µg of ampicillin/ml). Organs were dissected and homogenized with Ultra Turrax T18 . Aliquots from these homogenized tissue samples or the whole cell suspensions, were first mixed and vortexed with 3% Triton X-100 (Sigma-Aldrich), then plated on selective LB agar plates and incubated overnight at 37°C.For fluorescence-tagged Salmonella clones colonies were counted under a fluorescence stereomicroscope (Leica MZ16FA).Colonies were washed off in 4 ml PBS on a horizontal shaker. Bacteria suspensions were collected, centrifuged and pellets resuspended. For genomic DNA extraction 4 µl of the cell pellet was transferred to 1 ml of 0.1% SDS, 10 mM Tris HCL, 5 mM EDTA, and incubated at 60°C for 30 minutes to lyse the cells. Genomic DNA was extracted from each sample, by the conventional ‘Phenol-chloroform’ DNA isolation method.ACAGGACGAAGACCGTGAATATGGTTACGTCATTGAGCNNNNTGTGTAGGCTGGAGCTGCTTCGAAGTTC; Forward adapter primer sequence: ACACTCTTTCCCTACACGACGCTCTTCCGATCTACAGGACGAAGACCGTGAATATGG; Reverse adapter primer sequence: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAACTTCGAAGCAGCTCCAG).Ten ng of gDNA from each sample was used as template for a two step PCR reaction creating an amplicon library fully compatible to the multiplexing Illumina TruSeq DNA sequencing protocol .The second step PCR was performed to add the multiplex tag (MID) assigned to each sample, while it extended the final PCR product to 193 base pairs in total length (Forward multiplex adapter primer: QIAquickGel extraction kit (Qiagen) was used to extract and purify the PCR amplicons from the gel according to the manufacturer's instruction. The concentration of the final PCR amplicon library was adjusted and sequenced on Illumina a MiSeq system using MiSeq Reagent Kits (73cycles/323cycles) at the Genome Analytics Group (GMAK) in Brunswick, Germany. The fluorescent images were processed to sequences and transformed to FastQ format using the Real Time Analysis Software RTA 1.17.22 (Illumina). Low quality reads were discarded. Sequences were further processed with in house Microsoft Excel VBA  based macros. Only sequences that comprised intact flanking regions up- and downstream of the nucleotide tag were included in the analysis and the frequency of every individual nucleotide tag was determined. Tag-sequences not represented in the WITS library are likely to be a consequence of sequencing errors and were discarded.Salmonella infection. The efficacy of DT-induced CD11c+ DC depletion in PP, mLN and spleen was confirmed by flow cytometry (data not shown).CD11c-DOG mice Mice were bred at the central animal facility of Hannover Medical School under specified pathogen-free conditions. C57BL/6 mice and 129Sv mice were purchased from the Charles River Laboratory .BiodivR 1.2 software . MHI = 0 indicates that 2 samples do not overlap at all in terms of clonal representation, whereas MHI = 1 implies that both samples share the same set of clones and in similar proportions for each clone. Statistical analysis was performed with GraphPad Prism software. Unpaired nonparametric two-tailed t test (Mann-Whitney test) was used to determine inter-group significant values. Whisker bar lines represent 95% confidence interval range. Statistical differences of the mean values are indicated as follows: *, P value is <0.05; **, P value is <0.01; and ***, P value is <0.001.TSU were determined by a custom made Microsoft Excel VBA  based macro  and provFigure S1Nucleotide-tagging does not affect S. Typhimurium virulence. Mice were infected with a 1∶1 mixture of SL1344 (open boxes) and a WITS library clone (closed circles). Two days after oral infection, CFU were determined for each strain by differential plating of minced mLN and single PP or five days after intra peritoneal infection CFU were determined in liver and spleen (spl). Symbols indicate individual organs and bold lines median CFU from 2 pooled experiments. Numbers indicate organs displaying less than 10 CFU.(TIF)Click here for additional data file.Figure S2WITS did not acquire stable virulence-affecting modifications in vivo. Two 129Sv mice were infected with 10 WITS strains and 60 days after infection the contribution of each strain determined for the mLN. WITS clone 5 has intentionally been excluded because this clone did not grow well in this particular experiment. For each mouse a pair of one over- and one underrepresented WITS was picked (marked by asterisks) and used for a second round of oral infection using both WITS at equal proportions. Similar, WITS compositions were observed in 4 additional mice analysed in 2 independent experiments. After 2 days the mice were sacrificed and the contribution of both strains to the pool of Salmonella in the mLN determined. Numbers indicate the number of colonies analysed.(TIF)Click here for additional data file.Figure S3PCR amplification and NGS results in highly reproducible results. The identical sample was used for independent isolation of gDNA, PCR amplification and sequencing. The plot illustrates the frequency of each WITS in the independent technical replicates. Symbols indicate individual organs pooled from 7 independent experiments.(TIF)Click here for additional data file.Figure S4Qualitative assessment of WITS distribution in different compartments. Scatter plots based on WITS frequencies between compartments in (A) non-vaccinated mice and (B) mice vaccinated with attenuated Salmonella 40–50 days before. Numbers in the lower left corner of the diagram indicate the WITS undetectable in both compartments depicted in the respective diagram.(TIF)Click here for additional data file.Figure S5DC contribute to dissemination of Salmonella to mLN but not systemic compartments. To deplete DC, mice expressing the diphtheria toxin receptor under control of the CD11c promoter p<0.01, ***p<0.001.(TIF)Click here for additional data file.Text S1Provides a brief description of the VBA-coded simulation to estimate TSU.(DOCX)Click here for additional data file.


Escherichia coli (UPEC) are capable of occupying physiologically distinct intracellular and extracellular niches within the urinary tract. This feat requires the timely regulation of gene expression and small RNAs (sRNAs) are known to mediate such rapid adjustments in response to changing environmental cues. This study aimed to uncover sRNA-mediated gene regulation in the UPEC strain UTI89, during infection of bladder epithelial cells. Hfq is an RNA chaperone known to facilitate and stabilize sRNA and target mRNA interactions with bacterial cells. The co-immunoprecipitation and high throughput RNA sequencing of Hfq bound sRNAs performed in this study, revealed distinct sRNA profiles in UPEC in the extracellular and intracellular environments. Our findings emphasize the importance of studying regulatory sRNAs in a biologically relevant niche. This strategy also led to the discovery of a novel virulence-associated trans-acting sRNA—PapR. Deletion of papR was found to enhance adhesion of UTI89 to both bladder and kidney cell lines in a manner independent of type-1 fimbriae. We demonstrate PapR mediated posttranscriptional repression of the P-fimbriae phase regulator gene papI and postulate a role for such regulation in fimbrial cross-talk at the population level in UPEC. Our results further implicate the Leucine responsive protein (LRP) as a transcriptional activator regulating PapR expression. Our study reports, for the first time, a role for sRNAs in regulation of P-fimbriae phase variation and emphasizes the importance of studying pathogenesis-specific sRNAs within a relevant biological niche.Uropathogenic  Escherichia coli (UPEC) are presumably exposed to a deluge of stimuli from epithelial cell contact, urine and host immune factors and we asked if any regulatory sRNAs would play a role in the transition of UPEC from the extracellular niche to the intracellular one. This study employs co-immunoprecipitation using the RNA chaperone Hfq to identify novel virulence-associated sRNAs in intracellular UPEC, followed by high-throughput RNA-seq. We report the identification of a novel sRNA that we designate PapR (P-fimbriae regulator) and elaborate on this discovery by demonstrating a role for PapR in regulation of P-fimbriae—a UPEC surface virulence factor. The results presented in this study offer new insights into the molecular mechanisms of UPEC pathogenesis and a role for sRNA mediated regulation of virulence factors.Recent years have seen an increasing emphasis placed on the role of small RNAs (sRNAs) in the regulation of bacterial gene expression and stress adaptation. The advent of high-throughput sequencing methods has now made it possible to directly monitor the appearance of potentially virulence-associated sRNAs that may contribute to rapid adaptation of the pathogen to a changing environment during infection. Uropathogenic  E.coli (UPEC) are a group of genetically heterogeneous isolates [in vivo[in vitro [Uropathogenic isolates  known toisolates . IBCs suisolates , 4. Whil [in vivo, 5 and iin vitro  studies in vitro .pap, encodes for P-fimbriae that recognize the Gal-α(1–4)β-Gal glycosphingolipid moiety on kidney cells via PapG- the adhesive tip of the fimbria [UPEC possess an extensive repertoire of secreted virulence factors , 9 and s fimbria .pBA), within the regulatory region intergenic to papI and papB. Reversible binding of LRP and PapI to either one of the two GATC sites allows for Dam-mediated methylation of the unbound exposed site, thereby turning pap transcription ON or OFF [papI transcription from the divergent pI promoter [P-fimbrial expression is subject to phase variation involving a reversible epigenetic switch between ON and OFF states which responds to regulator proteins encoded within the operon, PapI and PapB, as well as the global transcription regulators H-NS, cAMP receptor protein (CRP), DNA adenine methylase (Dam) and Leucine responsive protein (LRP) . There aN or OFF . Anotherpromoter .Successful bacterial pathogens efficiently adapt to changing environments inside a host during infection. The past few years have seen considerable efforts dedicated towards understanding the survival strategies that intracellular pathogens adopt and the extent to which small regulatory non-coding RNAs are involved. Small regulatory RNAs (sRNAs), with their short half-life, may constitute an efficient means by which pathogens make snap decisions that favour survival in response to environmental stresses during infection , 19. Rectrans-acting and exert their function via interaction with Hfq—a homohexameric RNA chaperone protein. Several studies have shown Hfq to be important in the scheme of bacterial virulence [The majority of the sRNAs discovered in Gram negative bacteria so far are irulence –25, presirulence , motilitirulence  and epitirulence . Scoutinirulence –31.This study is based on the premise that the altered transcriptional profile of UPEC during infection and intracellular survival should also be reflected in its sRNA repertoire. In order to identify novel pathogenesis-associated sRNAs expressed by intracellular UPEC infecting human bladder epithelial cells in culture, we adopted an established strategy involving an Hfq-RNA co-immunoprecipitation (co-IP) approach followed by RNA-seq. This study demonstrates that the expressed sRNA profile changes substantially in UPEC upon invasion of bladder cells and further reports the identification of PapR, a novel pathogen specific sRNA enriched during infection that plays a role in P-fimbria phase variation.hfq [hfq deletion mutant UTI89Δhfq displayed a reduced ability to adhere to- and invade bladder epithelial cells  version of hfq  on the chfq , to be uhfq . Presencal cells .This observation corresponds with other studies showing that Hfq plays a vital role in regulation of virulence-associated genes during infection . IntraceompA and ompC [Our comparison of the sRNA profiles from culture and infection revealed an increase in the expression of MicA and RybB, known negative regulators of outer membrane (OM) proteins  in the iand ompC , 38. Dsrin silico predictions, two resulted in a stable transcript detectable by Northern blotting and the novel sRNAs were designated PapR and C271  strains, enterohemorrhagic E. coli (EHEC) and Shigella sp. but not in E. coli K-12 . We found that deletion of papR did not affect UTI89 growth rate in LB medium or Epilife cell culture medium  and inner medullary collect ducts of kidney (IMCD3). Bacterial adhesion to both cell lines was tested with untreated and α-D-mannose pre-treated strains in parallel in order to not only estimate the influence of PapR on cell adhesion but also clearly demonstrate type-1 fimbriae independent adhesion. In concurrence with our observations from the agglutination assay, UTI89ΔpapR/pNDM220 demonstrated increased adhesion  to bladder and kidney epithelial cells in a mannose resistant manner , whereas in contrast, the fraction of type-1 expressing cells appeared to be unaffected by papR deletion.The results obtained from agglutination- and adhesion assays indicated that deletion of tibodies . While Uon level ; P-fimbrpap gene cluster is present as a single copy in the UTI89 genome [pap gene cluster is regulated on multiple levels by different but interconnected global regulators along with two regulatory proteins encoded within the pap gene cluster—PapI and PapB. The regulatory genes papI and papB are transcribed from two divergent promoters—pBA and pI. Transcription from the pBA promoter results in a primary transcript which is quickly cleaved by RNase E into separate papB and papA transcripts which differ in their stability as RNA transcripts [in vivo regulatory effect on papI mediated by PapR- a regulatory sRNA located outside the pap-associated pathogenicity island of UTI89, we chose to adopt a well-established approach using pap-lac operon reporter fusions [E. coli K-12 derived strains, DL1504 and DL2121 that lack an endogenous pap gene cluster but are genetically modified to carry a lac operon fusion in the chromosome. Details of DL1504 and DL2121fusion constructs have been illustrated by Braaten et.al [papI and papB genes with their native divergent promoters and the intergenic pap regulatory region between them, with a papB-lacZ operon fusion. In addition, the papB promoter region in the DL2121 strain carries a mutation in the LRP binding site #3 rendering it independent of PapI mediated phase regulation. We found that the DL1504 strain with the pNDM220 empty vector showed the same level of β- galactosidase activity (measured at 17 Miller units) as the parental DL1504 without plasmid and that upon induction of PapR expression with 1mM IPTG the β-galactosidase activity of DL1504/pSK1 was reduced by half. These findings were compounded by the lack of any discernible change in β-galactosidase activity both in DL1504ΔpapI and DL2121  to sites 4, 5, 6 (ON) [The 9 genome  unlike snscripts . In orde fusions , 45, 46.en et.al  and Nou en et.al  respectid DL2121 , indicat, 6 (ON) .In silico predictions revealed a region of near-perfect complementarity spanning 22 nucleotides in PapR and in papI mRNA, including most of the AU rich regions upstream of the PapR terminator  with an inverted terminator stem region, thus abolishing base pairing with the target mRNA while ensuring that the PapR* sRNA would not suffer secondary structural perturbations. As a reporter we constructed a papI::gfp translational fusion as devised previously by Urban and Vogel [papI ORF, the entire length of the gene in addition to a short upstream region at the 5’ end was cloned into a low copy number vector—pXG10, with a constitutively active λPL promoter. PapR and PapR* sRNAs were expressed from pSK1 and pSK1* respectively using 1mM IPTG induction. These assays were performed in an E.coli K-12 background without endogenous pap gene cluster or papR. All transformants were grown aerobically to exponential phase in LB medium and cultured for an additional 30 mins with or without IPTG, after which cells were harvested for total RNA isolation for Northern blotting and whole cell lysates for Western blotting. By detecting RNA levels of papI::gfp and papR in conjunction with protein levels of PapI::GFP, a composite view of PapR-mediated post-transcriptional repression of papI could be visualized. papI::gfp mRNA level (~3 fold) and GFP protein level (~2 fold). The absence of any PapR*-mediated regulation supports the suggested requirement for a direct base pairing interaction between PapR and its papI mRNA target.In order to confirm that down regulation of P-fimbriae was mediated through direct base pairing between PapR and nd Vogel . Owing tE. coli strain UTI89 harvested from within infected bladder epithelial cells was investigated in order to identify novel virulence-associated Hfq-interacting sRNAs. We report the discovery of two sRNAs conserved among related ExPEC strains which were designated PapR and C271.The role played by sRNAs in making rapid cellular decisions and regulating transcription in pathogenic bacteria, although currently underrepresented in the literature, could be a substantial one , 53. In cis- and trans-encoded regulators of gene expression. In Gram negative bacteria, a vast majority of trans-encoded sRNAs are known to depend on Hfq- a global RNA chaperone, for structural stability and regulatory function [Bacterial sRNAs are broadly categorized as function . Bacterifunction , 56.in vitro culture displayed a pronounced tendency towards stress adaptation in the intracellular environment. During infection, UTI89 showed increased expression of sRNAs responding to envelope stress—MicA, RybB and DsrA. It has been known for some time, that cases of acute UPEC-mediated cystitis are associated with the development of tightly clustered intracellular bacterial communities (IBCs) [omp expression when required again during the later IBC stage. DsrA, which was also increased in infection, is a known positive regulator of RpoS [Salmonella over time and in doing so, emphasized the dynamic nature of sRNA expression and the rapid regulation it mediates. At a single time point, our sRNA profile during infection is more likely to be a snapshot view of early adaptations to intracellular survival within bladder epithelial cells.Overall, our survey of UTI89 sRNA profiles during infection and s (IBCs)  and thats (IBCs) . Outer ms (IBCs) . In this of RpoS - an alte of RpoS , 60. Ano of RpoS , GcvB RN of RpoS , the tra of RpoS  and the  of RpoS . Interes of RpoS , a trans of RpoS , 67. Cha of RpoS  demonstrfim operon being highly conserved across UPEC strains, our RNA-seq data did not reveal read counts corresponding to FimR. This could in turn reflect back to the fact that our study involved an Hfq co-IP which might result in a failure to represent asRNAs in the samples since they are less likely to be Hfq dependent [UPEC are known to possess a variety of virulence factors ranging from adhesive fimbriae on the cell surface to secreted toxins that render them potent pathogens. Fimbrial adhesion to the host epithelium is an important first step in early infection and some UPEC strains have been reported to possess as many as ten putative fimbrial operons , and of ependent . We suggpapR in an α-D-mannose resistant manner and a lack of in silico predicted targets from the fim operon, decidedly pointed away from any direct role for PapR in expression of mannose sensitive type-1 fimbriae. Concurrently, by performing parallel adhesion assays where strains were untreated or treated with α-D-mannose prior to infection, it was possible to provide clear evidence of mannose resistant surface fimbriae being responsible for the elevated adhesion observed for UTI89ΔpapR. Infection was carried out in two human cell lines derived from the urinary bladder and inner medullary collecting ducts of the kidney, representing two distinct pathological niches UPEC are known to occupy. Although UTI89ΔpapR demonstrated significantly improved adhesion in both cell lines, this effect was greater in the kidney derived cell line. The kidney cell line also displayed a significant increase in UTI89ΔpapR invasion when compared to UTI89/pNDM220, confirming that the nature of surface fimbriae could influence UPEC tissue tropism. However, this improved adhesion was not followed by a subsequent increase in invasion in the bladder derived cell line; a phenomenon that has been previously reported in relation to P-fimbriae and in vitro infection of bladder epithelial cell lines [hfq in accordance with previous reports [hfq, perhaps indicating that while Hfq does not influence expression of type-1 fimbriae in UTI89, it could have bearing on alternative virulence factors critical for bladder cell adhesion. Since fim and pap operon deletion mutants were not included in the infection assays, improved epithelial cell adhesion of UTI89ΔpapR in the infection assays could not be attributed with certainty to P-fimbriae but rather just to a mannose resistant fimbrial type, While the agglutination and adhesion assays provide hints as to the nature of surface fimbriae expressed at the population level, by fimbriae-specific immunolabelling and flow cytometry we were able to examine single cells to demonstrate a greater fraction of P-fimbriated cells in the PapR null mutant when compared to UTI89wt. Our findings with flow cytometry revealed the presence of both type-1 fimbriated and P-fimbriated bacteria in the population. This could indicate that decisions favouring one type of surface adhesin over another could be made on a population scale, by elevating synthesis of mediators like PapR that would tip the scales in favour of a particular adhesin: type-1 fimbriae [Studies have shown that UPEC infection of the urinary bladder is predominantly associated with the expression of type-1 fimbriae , 71. Howll lines . A reduc reports . It was fimbriae , 73. Thefimbriae  and exprfimbriae . Our obsDespite previous reports suggesting minimal involvement of P-fimbriae in UPEC infections , BucklespapR transcription in an lrp null mutant identifies LRP as a transcriptional activator of PapR synthesis. This finding adds an additional layer of complexity and implies a dual role for LRP in P-fimbriae phase switching under the conditions when bacteria are localized in host cells. A summarizing model illustrating the roles we now have identified for PapR and Lrp in modulation of P-fimbriae phase variation is shown in E.coli. Regulation of PapR could represent an additional indirect means of LRP involvement in type 1and P-fimbrial crosstalk.Pathogens are able to rapidly perceive environmental cues and correspondingly modulate their choice of surface adhesins by a means of phase variation, which is essentially believed to be an immune evasion strategy as well as an adaptive response to boost the odds of bacterial survival in the face of a changing environment . Especiapap operon is regulated by proteins encoded within the gene cluster, PapI and PapB. PapI is known to be involved in ON/OFF phase transition of P-fimbria by interacting with the pap DNA-LRP complex [pBA transcription, to LRP binding sites #4–6, resulting in activation of the pBA promoter. Results ensuing from the β-galactosidase assays using strains with a functional papI (DL1504), papI null strain (DL1504ΔpapI) and papI independent expression system (DL2121), all indicated that PapR targeted papI mRNA and thereby regulated P-fimbrial phase variation. A GFP reporter system was used to not only confirm direct interaction between PapR and its target papI mRNA, but also to identify the region of interaction by employing the pSK1* plasmid construct with an inverted terminal stem loop region. The lack of any reduction in the levels of papI::gfp mRNA as a consequence of PapR* expression from pSK1* plasmid, confirms the in silico predicted region of interaction to hold true.Apart from the global regulators cAMP-CAP, H-NS, Dam methylase and LRP, the  complex , 83. Thiin silico predictions were explored in this study. This however, does not preclude the possibility that PapR might well act on multiple fronts in UPEC.PapR overall expression levels being two fold higher during infection than in the culture grown reference condition, could reflect a need to support type-1 fimbriae expression (preferable during bladder cell invasion) while simultaneously repressing P-fimbriae expression, when infecting bladder epithelial cells. Owing to early experimental evidence suggesting involvement of PapR in P-fimbrial regulation, no alternative mRNA targets from pap regulation, presumably promoting the advantage of a rapid ON/OFF switch favouring the best-suited surface adhesin at the time within at least part of the population, and thereby enhancing the odds of the pathogen’s survival within its specific tissue niche. A few recent studies have reported sRNA-mediated regulation of surface fimbriae in pathogenic bacteria [PapR sRNA represents a new principal addition to the repertoire of macromolecules involved in bacteria , 84 furtE. coli and their expression in the context of infection, should prompt additional efforts to clarify the complete sRNA repertoire that such pathogens possess.Survival of a pathogen and success in infection, hinges on its ability to efficiently sense signals from the host environment and make suitable adaptations  includinE. coli UTI89 with serotype O18:K1:H7 was used for RNA sequencing and strain constructions  culture followed by 3 hours of static growth at 37° C in Epilife cell culture media to replicate infection conditions. Strains for other experiments were grown in LB broth supplemented with 100 μg/ml ampicillin, 40 μg/ml chloramphenicol (Cml) and 40 μg/ml kanamycin (Kan) as required. Expression was induced from the PA1/O4/O3 promoter with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG).A cystitis-derived isolate ructions . E. colihfq, UTI89ΔpapR and UTI89Δlrp were made using pKD3/pKD4 plasmid templates to amplify Cml and Kan resistance cassettes respectively as described by Datsenko and Wanner [et.al.[lrp respectively into the pNDM220 vector as described by Boysen et.al.[papI::gfp translational fusion and confirmed using JMJ732+JMJ733. All relevant primer pairs are listed in The deletion strains UTI89Δd Wanner . Insertir [et.al.. Plasmiden et.al. and consPD07i bladder epithelial cells kindly provided by David Klumpp  were cul2 for the duration of infection to serve as the reference condition. 108 bacterial cells were harvested in IP buffer  and lysed by French press. Cell lysate was incubated with 100 μl of M2 paramagnetic anti-FLAG agarose beads (Sigma) that had been pre-washed twice in IP buffer, for 4 hours at 4°C. Beads were washed twice with cold IP buffer and phenol-chloroform extraction of RNA was performed. Downstream processing of purified co-IP RNA was performed and sequencing was carried out on an Illumina HiSeq2000 platform by Vertis Biotechnology .UTI89Hfq 3xFLAG cultured overnight in LB medium was used to infect PD07i bladder epithelial cells and harvested 3hrs post infection. In parallel, UTI89Hfq 3xFLAG cultured overnight in LB medium was grown statically in Epilife cell culture medium and maintained at 37°C and 5% COThe quality of raw cDNA reads was examined using FastQ and barcodes clipped. Sequence reads (50 nt) were mapped to UTI89 genome (NC_007946.1) and visualized using the Integrated Genome Viewer (Broad Institute). Read counts were calculated using SeqMonk (Babraham Bioinformatics) by normalizing for total reads mapping to the genome in each cDNA library and gene length. Exact duplicate reads were rejected and gene-wise quantitation was performed by counting the number of reads with a minimum of 10 nt overlap to gene annotations. SIPHT  programmCultures were grown statically overnight in LB at 37°C. A 10 mg/ml suspension of bakers’ yeast in PBS was freshly prepared for yeast agglutination and fresh 8% erythrocyte suspension from type A- whole blood was used for hemagglutination. Equal volumes of the overnight bacterial culture was mixed with the yeast/erythrocyte suspensions on a glass slide and observed for visible agglutination with and without addition of freshly prepared 3% α-D-mannose.papR (JMJ834), C271 (JMJ767) and 5S was performed as described previously [et al.[Total RNA was isolated using heavy phase lock gel tubes (5Prime) and Northern blot analysis of eviously . Samplesy [et al. using pr8 bacteria were harvested from appropriate cultures by spinning down cells equivalent to OD600 = 1. Bacterial pellets were then resuspended in 1× SDS loading buffer , boiled for 5 min and separated on a 4–12% Invitrogen NuPage novex Bis-Tris mini gels as detailed by Boysen et.al [~10en et.al . The pro5 events per sample was collected and analyzed using BD FACSDiva software . Strains were distinguished first on the basis of forward scatter area (FSC-A) and side scatter area (SSC-A) and second on the basis of forward scatter area (FSC-A) and FITC fluorescence area (FITC-A). Microscopy was performed using a Leica DMRE fluorescence microscope. Image contrast and addition of scale bars were performed using ImageJ software.Specimens for flow cytometry and immunofluorescence microscopy were fixed with 3.7% paraformaldehyde for 10 min. and washed gently with PBS three times. 2% BSA for 10 min. was used to block nonspecific signals after which samples were washed once with PBS and stored at 4°C overnight. Bacterial strains were stained with 1:500 anti-Fim  and 1:502CO3, 0.27% β-mercaptoethanol) for each replicate in the assay. Samples were incubated with 4 mg/ml ONPG  to allow development of yellow colour and reactions were terminated with addition of 1 M Na2CO3. Optical density was measured at wavelengths of 550 nm and 420 nm and Miller units calculated.Bacteria were resuspended in PBS at appropriate concentrations and 100 μl was used along with 900 μl of Z-buffer  PapR and (B) C271 sRNAs showed a high level of sequence and secondary structure conservation among related Gram-negative strains. Alignments were generated using LocARNA software tool , complem(TIF)Click here for additional data file.S2 Fig(A) Primer extension was performed to map transcription start site of C271 alongside Sanger sequencing reactions to be read from left to right as TACG. (B) Illustration of the genomic context of C271 in UTI89, drawn to scale.(TIF)Click here for additional data file.S3 FigIn silico secondary structure prediction of PapR sRNA using Mfold software. The predicted region of interaction between PapR and papI mRNA is illustrated in red extending over a single stranded region and the terminal stem-loop. The nucleotide bases in the terminal stem that were inverted in the modified PapR* are denoted by an asterisk (*).(TIF)Click here for additional data file.S4 FigpapR/pNDM220 and UTI89ΔpapR/pSK1 were grown at 37°C in aerated LB medium (−) and Epilife cell culture medium (−). Optical density was measured at 600 nm wavelength at 60 min intervals and plotted against time in hours. Mean OD600 from three independent experiments along with their standard deviations was plotted.UTI89/pNDM220, UTI89Δ(TIF)Click here for additional data file.S5 FigpapR/pNDM220, UTI89ΔpapR/pSK1 and UTI89Δhfq for 2 h followed by 1 h of gentamycin treatment to kill extracellular bacteria. Strains were either treated (+) or left untreated (-) with 3% α-D-mannose. Bacterial invasion was assessed by calculating mean CFU counts from three independent experiments and p-value (* <0.05) calculated by t-test.PD07i bladder cells (grey) and IMCD3 kidney medullary collecting duct cells (red) cultured in 24-well plates were infected with UTI89/pNDM220, UTI89Δ(TIF)Click here for additional data file.S6 FigUTI89wt stained with anti-Fim (A) and anti-PapA (B) antibodies were examined by immunofluorescence microscopy to demonstrate staining specificity. The panels on the left represent anti-Fim (Ai) and anti-PapA (Bi) fluorescence images and the panels to the right (Aii and Bii) represent the corresponding phase contrast images. Red arrowheads mark unstained UTI89wt and black arrowheads mark UTI89wt cells that stain positive for the particular fimbriae. Scale bars set at 10 μm. Western blots show single bands corresponding to Fim (Aiii) and PapA (Biii). GroEL was used as the internal loading control.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.