Childhood lead exposure adversely affects neurodevelopment. However, few studies have examined changes in human brain metabolism that may underlie known adverse cognitive and behavioral outcomes.in vivo brain metabolite concentrations as adults, determined by proton magnetic resonance spectroscopy (MRS) in a birth cohort with documented low-to-moderate lead exposure.We examined the association between mean childhood blood lead levels and n = 159; mean age (± SD), 20.8 ± 0.9 years] completed a quantitative, short-echo proton MRS protocol evaluating seven regions to determine brain concentrations of N-acetyl aspartate (NAA), creatine and phosphocreatine (Cr), cholines (Cho), myo-inositol, and a composite of glutamate and glutamine (GLX). Correlation and multiple linear regression analyses were conducted.Adult participants from the Cincinnati Lead Study [Mean childhood blood lead levels were associated with regionally specific brain metabolite concentrations adjusted for age at imaging and Full-Scale intelligence quotient. Adjusted analyses estimated for a unit (micrograms per deciliter) increase in mean childhood blood lead concentrations, a decrease of NAA and Cr concentration levels in the basal ganglia, a decrease of NAA and a decrease of Cho concentration levels in the cerebellar hemisphere, a decrease of GLX concentration levels in vermis, a decrease of Cho and a decrease of GLX concentration levels in parietal white matter, and a decrease of Cho concentration levels in frontal white matter.Gray-matter NAA reductions associated with increasing childhood blood lead levels suggest that sustained childhood lead exposure produces an irreversible pattern of neuronal dysfunction, whereas associated white-matter choline declines indicate a permanent alteration to myelin architecture. Despite public health efforts toward reducing body lead burden, exposure to lead hazards remains an international environmental health problem . Lead haN-methyl-d-aspartate (NMDA) excitatory amino acid receptors  acquired within an MRI examination provides an in vivo measure of brain metabolites such as N-acetyl aspartate (NAA), creatine and phosphocreatine (Cr), glycerolphosphocholine and phosphocholines (Cho), myo-inositol (mI), and a composite of glutamate and glutamine (GLX) at concentration levels on the order of 1–10 millimolar (mM)  blood lead levels tend to have few specific anatomical findings described as characteristic of lead exposure. Neural changes, such as microscopic structural and chemical disruptions, at levels below the detection limits of conventional imaging modalities such as computed tomography or magnetic resonance imaging (MRI) must exist if lead is indeed the source of cognitive deficits and adverse behavioral outcomes. Therefore, a different lar (mM) . These bn = 6) with blood lead concentrations ≥ 27 μg/dL and sex-matched control subjects (n = 6) with blood lead concentrations < 10 μg/dL, all approximately 11 years of age. Lead-exposed children had an average (± SD) blood concentration of 37.7 ± 5.7 μg/dL, whereas controls averaged 5.4 ± 1.5 μg/dL. Bilateral NAA, Cho, and Cr levels in the frontal lobes and hippocampi were reported as reduced in lead-exposed children relative to controls. These studies indicated disruptions in brain metabolism, particularly in the frontal lobe  were followed up quarterly through age 5 years, semiannually from 5 to 6.5 years of age, again at 10 years, and also between the ages of 15 and 17 years. CLS subjects between 19 and 24 years of age (n = 194) were consecutively recruited for this MRS study via mailings, phone calls, and flyers to meet our initial sample size goal of 150 CLS participants. This size was chosen before initiating the imaging aspect of the study, because members of the CLS cohort who were pregnant, incarcerated, or claustrophobic or had metal implanted from surgeries, embedded metal from occupational exposure , or gunshot wounds would not be able have a research MRI examination. Subjects who were known to be diabetic or who had any known neurologic or psychiatric illness were also excluded. The substance use of participants was determined using a clinical urine toxicology screening immunoassay for drugs of abuse, which evaluated amphetamines, barbiturates, benzodiazepine, cocaine, cannabinoids, opiates, and phencyclidine (PCP). (The only positive findings for participants were exclusively cannabinoids.) The alcohol use within the cohort was also assessed via a self-report measure previously described (n = 8), failure to appear for the appointment (n = 7), pregnancy (n = 6), refusal due to claustrophobia (n = 6), nonremovable metal in their body (n = 5), and inability to give informed consent because of cognitive disability (n = 1). Two participants diagnosed with fetal alcohol syndrome as children participated in the imaging and spectroscopy session, but their data are not included in the analyses. Consistent with previous research in this cohort , a longitudinal birth cohort study designed to evaluate the effects of low to moderate environmental lead exposure. The CLS enrolled pregnant women in their first or early second trimester of pregnancy who visited four prenatal clinics within impoverished Cincinnati neighborhoods with a high concentration of older, lead-contaminated housing. Women were excluded if they were known to be addicted to drugs, were diabetic, or had any known neurologic or psychiatric malady. Newborns with birth dates between 1980 and 1985 were subsequently recruited for follow-up studies. Infants were excluded if their birth weight was < 1,500 g or if genetic or other serious medical issues were present at birth. This cohort has been described periodically as cognitive, and behavioral and social assessments were completed , 2001. Nescribed . Thirty-rt e.g., , we founEnrollment of the participants’ mothers in the CLS permitted monitoring of blood lead levels for the participants prenatally, at birth, every 3 months to 60 months of age, and every 6 months from 60 to 78 months of age. The mean of 23 childhood blood lead assessments for each participant from 3 to 78 months of age was employed for comparison analyses. This mean childhood blood lead value has been historically used for comparisons with cognitive and behavioral outcomes , 1993b. 3. We employed a K-means segmentation algorithm using custom software for analysis of the high resolution imaging sequence so as to determine gray matter, white matter, and CSF contributions to each voxel. We then applied a correction factor based on the ratio of volume of CSF to total parenchymal volume and adjusted each metabolite concentration accordingly. LCModel also provides a measure of data quality and reliability, reporting the Cramer–Rao lower-bound bounds  with a quadrature head radiofrequency coil positioned around the head of the participant. An axial three-dimensional, inversion recovery prepped, fast spoiled gradient-echo high-resolution imaging sequence was acquired . Single voxel MRS was acquired with the proton brain examination (PROBE) software  using a point-resolved spectroscopy acquisition mode (PRESS) sequence  and with automatic shimming to achieve water line widths of < 5 Hz. A reference spectrum, acquired within the PROBE acquisition, collected 16 acquisitions of unsuppressed water signal for eddy current and baseline corrections. Data were processed using the LCModel program, a commercially available, user-independent, frequency domain spectral-fitting program that provides measures of metabolite concentrations . Concentd bounds . We requa) neural networks associated with language function are left lateralized; b) most of the study population is right handed and thus likely to be left hemisphere dominant; c) the right hemisphere is prone to technical factors (orientation of slice-selective pulses) that produce signal artifacts  within the spectrum.Seven single voxels, approximately 8 cc in volume, were positioned within the medial frontal gray matter, the left frontal white matter, left parietal white matter, the right temporal lobe at the superior temporal gyrus, the left basal ganglia, left cerebellar hemisphere (gray and white matter), and the cerebellar vermis, respectively . Althougp < 0.05) were considered significant. The metabolites examined included NAA, Cr, Cho, mI, and GLX. Recognizing that other variables could influence the association between metabolite levels and blood lead, the impact of covariates on this relationship were also examined by analyzing the bivariate relationship of the potential covariate with mean childhood blood lead level in each of the sampled brain regions, using a linear regression. Such variables that could potentially contribute included age of participant at time of imaging, Full-Scale intelligence quotient (FSIQ), current drug use (positive only for cannabinoids), sex, birth weight, and gestational age at birth. Covariates that were independently related to the outcome in one or more region at p < 0.10 level were included in the subsequent multiple linear regression analyses. For the linear regression analyses, beta values corresponded to the change in metabolite concentration associated with a 10-μg/dL increase in the mean childhood blood lead level and the 95% confidence intervals (CIs) determined for significance.We investigated the relationship between metabolite levels obtained from MRS analyses in seven brain regions with mean childhood blood lead level initially using Pearson and Spearman correlation coefficients. Correlation values with an alpha (n = 159) who completed the study were predominantly African American (93%), between 19 and 23 years of age, and demonstrated mean childhood blood lead values of 13.3 ± 6.1 μg/dL that ranged from 4.7 to 37.2 μg/dL  increase in mean childhood blood lead concentrations, a decrease of NAA  and Cr  concentration levels in the basal ganglia, a decrease of NAA , and a decrease of Cho  concentration levels in the cerebellar hemisphere, a decrease of GLX  concentration levels in vermis, a decrease of Cho , and a decrease of GLX  concentration levels in parietal white matter, and a decrease of Cho  concentration levels in frontal white matter (Age at time of imaging and FSIQ were the only covariates meeting the criteria established e matter .p = 0.08).We performed a secondary analysis with adjustment for age at the time of imaging only, as FSIQ may be included in the causal pathway linking lead with abnormal metabolite levels. Results of this analysis were generally comparable with those shown, except that the association between lead and NAA concentration in the cerebral hemisphere was no longer statistically significant  and two white-matter regions .Gray-matter sampling within the brain provided information content reflecting neuronal health. Two predominantly gray-matter regions—basal ganglia and cerebellar hemisphere—demonstrated a reduction in NAA with increased mean childhood blood lead levels. This suggests that childhood exposure to lead is associated with some level of neuronal dysfunction occurring within these regions. The diminished Cr levels associated with increased mean childhood blood lead levels further supported impaired neuronal functioning, because Cr levels are thought to reflect the status of cellular energetics. MRS studies reporting results as ratio values with Cr levels serving as an internal reference standard are likely to miss this finding.We anticipated that NAA would inversely correlate with childhood lead exposure in the neuroanatomical regions regulating cognitive and behavioral domains found to be impaired with lead exposure, which include the frontal cortex. From a voxel-based morphometric (VBM) analysis of the volumetric imaging data, we found striking volume loss associated with mean childhood lead exposure within the medial frontal gray matter . The absIncreasing childhood mean blood lead concentrations were also correlated with GLX reductions within the cerebellar vermis as well as the parietal white matter. Although proton MRS sampled under these conditions does not provide information about glutamate specifically, the composite measure sampled with proton MRS implicates abnormal neurotransmitter functioning.We found an inverse association between the mean childhood blood lead levels and the Cho concentration levels within the white matter of the frontal and parietal lobes and the cerebellar hemisphere, respectively. Lead can change white-matter organization and functioning via altered expression of genes essential to myelin formation , delayedThe VBM analyses  for thisThe beta values associated with a 1-μg/dL increase in mean childhood blood lead levels for metabolite concentrations are small, which is physiologically expected. An MRS profile for a brain tumor is typically quite distinct from a region from an acute stroke. In these conditions, metabolite levels dramatically change several orders of magnitude because of the macroscopically observed changes to the brain. For conditions such as low-level lead exposure, where the brain appears relatively normal without lesions, the model of psychiatric diseases such as schizophrenia and bipolar disorder can be considered. These conditions can be clinically diagnosed with high degrees of certainty. However, if a person with a diagnosis of a given psychiatric condition has imaging or spectroscopy performed in a blinded fashion, it is likely to be interpreted within normal limits because of the lack of diagnostic features attributed to the conditions and broad normative values in a clinical setting. In contrast, research studies of such psychiatric conditions compared with appropriately screened and matched healthy control participants can find abnormal metabolite levels associated with a psychiatric condition. Given that the historical blood lead levels of an age- and demographically matched control population would not be known from childhood, nor would the blood lead levels for a young adult be zero, ideal comparison studies with the CLS population are not possible outside of the regression approach applied within this study.The lack of correlative findings within the temporal lobe may arise from technical factors associated with the proton MRS acquisition. Peak broadening due to magnetic susceptibility and field inhomogeneity arises at the interface between the temporal lobe and adjacent structures, including the ear and the temporal bone, resulting in incomplete water suppression, broad line widths of the metabolites, and failure of metabolite quantification. A dedicated MRS examination optimizing the homogeneity of the temporal lobe is necessary to clarify the impact of lead exposure within the temporal lobe. A dedicated study of the hippocampus is also warranted from other reports in the literature providing experimental evidence.The neurochemical changes demonstrated in the adult participants of the CLS reflect the complexities of lead neurotoxicity. Gray-matter reduction of NAA with the basal ganglia and cerebellum is consistent with the concept that sustained childhood lead exposure produces an irreversible pattern of neuronal dysfunction. White-matter choline changes within the frontal and parietal lobes suggest a permanent alteration to myelin architecture.


We describe a death associated with multidrug-resistant tuberculosis and HIV infection outside Africa that can be linked to Tugela Ferry , the town most closely associated with the regional epidemic of drug-resistant tuberculosis. This case underscores the international relevance of this regional epidemic, particularly among health care workers. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) pose an increasing challenge to international health . Treatment was altered from weight-appropriate doses of rifampin, isoniazid, pyrazinamide, and ethambutol to include levofloxacin, amikacin, cycloserine, and protionamide. Because of known sensitivity patterns from isolates at Tugela Ferry and the possibility of XDR TB, para-aminosalicylic acid and linezolid were added to treatment, and intravenous amikacin was changed to capreomycin. Treatment was subsequently changed on the basis of culture results. The patient required 35 days of mechanical ventilation for likely pulmonary immune reconstitution syndrome after initiation of antiretroviral treatment. Although discharged from intensive care after successful treatment of TB, the patient remained hospitalized and died 90 days after first seeking care. Samples cultured from >1 site showed evidence of widespread MDR TB. More than 500 potentially infectious contacts were identified, but no secondary cases of TB have been diagnosed.A presumptive diagnosis of MDR TB was made 7 days after the patient sought care at the hospital. A line probe assay  performed on cervical lymph node aspirates identified M. tuberculosis isolate from the patient to be resistant to rifampin, isoniazid, pyrazinamide, and ethambutol but sensitive to amikacin, capreomycin, moxifloxacin, para-aminosalicylic acid, and linezolid. These resistance and sensitivity characteristics identified the isolate as MDR TB rather than an XDR TB strain. Automated sequencing of rpoB revealed the L533P mutation previously associated with rifampin resistance (–Culture confirmed the ,To explore whether the patient’s isolate was related to the F15/LAM4/KZN strain genotypes associated with the Tugela Ferry outbreak ,. Single-The risk for TB among health care workers in developing countries is well recognized (rpoB mutations suggests that these strains arose independently from the locally circulating F15/LAM4/KZN strain pool, as already suggested for other MDR F15/LAM4/KZN strains (When taken together, the clinical and molecular epidemiologic data in this case support the hypothesis that infection was acquired in South Africa and most likely while the patient was based in Tugela Ferry. Health care workers in South Africa are significantly more likely to be admitted to a hospital with MDR or XDR TB than are population controls (Since this patient left South Africa, multidrug resistance has continued to grow as a challenge to public health, fueled in part by the HIV epidemic (The poor outcome for MDR and XDR TB in HIV-positive patients is well recognized in disease-endemic settings (This report serves as a reminder that XDR or MDR TB and HIV in sub-Saharan Africa represent not just a regional epidemic but also a challenge to international health. Although this challenge has yet to fully emerge, this case highlights the potential risk to health care workers in areas of low and high transmission.


EHP 121(5):607–612; http://dx.doi.org/10.1289/ehp.1205606].Epidemiological studies suggest that noise exposure affects cardiovascular system function. Most studies have focused on links between specific types of chronic loud noises associated with jobsites or roadways and adverse cardiovascular effects including high blood pressure and heart disease. But the biological underpinnings of these relationships have been little explored, particularly with regard to noise encountered during everyday life. A new study finds that lower-intensity everyday noises also can affect the cardiovascular system, although the effects are likely mediated via different pathways than those associated with effects of louder noise [Environmental stimuli influence the cardiovascular system via the autonomic nervous system, which regulates involuntary body functions such as respiration and heartbeat. Noise impacts can be assessed by measuring heart rate and heart-rate variability (HRV). HRV is influenced by sympathetic and parasympathetic activity, which may be considered the accelerator and brakes, respectively, of the autonomic nervous system. These pathways balance each other to ensure optimal heart function, and changes such as reduced HRV have previously been associated with increased risk for adverse cardiovascular effects.The current study took place in Augsburg, Germany, between March 2007 and December 2008, as part of a larger series of investigations by the Rochester Particulate Matter Center. Participants provided their medical history and health information at the start of the study; during its course they completed up to four examinations with individual exposure measurements. For the examinations, which averaged 6 hours, 110 participants wore electrocardiogram equipment that tracked heart-related variables and dosimeters that measured noise and air pollutant (ultrafine particulate matter) exposures as they undertook their normal daily activities.For data analysis, the researchers categorized noise exposures as low-intensity or high-intensity, defined by a cutoff point of 65 dB(A), a weighted measure of perceived loudness. Within these categories, the percent changes in HRV parameters were assessed for each 5-dB(A) increase in noise intensity. Increases within the low-intensity category appeared associated with reduced parasympathetic activity and possibly an increase in sympathetic activity. With increases within the high-intensity category, both sympathetic and parasympathetic activity seemed to increase, but net effects were consistent with the former exceeding the latter. Although these differing responses point to separate underlying mechanisms, both could ultimately reduce HRV and lead to increased cardiovascular risk.The study included a large number of individual-level measurements, control for air pollution effects and physical activity, and separate consideration of low- and high-intensity noise. There may have been residual confounding and data gaps from unmeasured variables, and because the study population was made up of older adults, conclusions may not extend to the general population. Nevertheless, the findings suggest that low-intensity noise activates biological pathways separate from the “fight-or-flight” response triggered by louder noises; the potential health consequences should be explored.


The brainstem nucleus of the solitary tract (NTS) holds the first central neurons in major homeostatic reflex pathways. These homeostatic reflexes regulate and coordinate multiple organ systems from gastrointestinal to cardiopulmonary functions. The core of many of these pathways arise from cranial visceral afferent neurons that enter the brain as the solitary tract (ST) with more than two-thirds arising from the gastrointestinal system. About one quarter of ST afferents have myelinated axons but the majority are classed as unmyelinated C-fibers. All ST afferents release the fast neurotransmitter glutamate with remarkably similar, high-probability release characteristics. Second order NTS neurons receive surprisingly limited primary afferent information with one or two individual inputs converging on single second order NTS neurons. A- and C-fiber afferents never mix at NTS second order neurons. Many transmitters modify the basic glutamatergic excitatory postsynaptic current often by reducing glutamate release or interrupting terminal depolarization. Thus, a distinguishing feature of ST transmission is presynaptic expression of G-protein coupled receptors for peptides common to peripheral or forebrain  neuron sources. Presynaptic receptors for angiotensin (AT1), vasopressin (V1a), oxytocin, opioid (MOR), ghrelin (GHSR1), and cholecystokinin differentially control glutamate release on particular subsets of neurons with most other ST afferents unaffected. Lastly, lipid-like signals are transduced by two key ST presynaptic receptors, the transient receptor potential vanilloid type 1 and the cannabinoid receptor that oppositely control glutamate release. Increasing evidence suggests that peripheral nervous signaling mechanisms are repurposed at central terminals to control excitation and are major sites of signal integration of peripheral and central inputs particularly from the hypothalamus. The nervous system makes a unique contribution to the regulatory mechanisms that help to preserve homeostatic integrity in the face of constantly changing demands. The central nervous system (CNS) serves as a key integrator and coordinator across multiple organ systems and tissues. The CNS and its peripheral neural partners are not only capable of rapidly responding over great distances to coordinate organ function but they also have the capacity to remodel chronically. By orchestrating visceral organ functions, the CNS helps to foster effective system-wide control over the conditions maintaining cellular homeostasis. The homeostatic palate of regulated “states” spans vital energy, temperature, ionic, and metabolic status -based systems that may distinguish particular pathways.One fundamental aspect essential to understanding homeostatic and pathologic complexities is an appreciation of normal regulation and the framework and structure of interactions across organ systems. The goal of this chapter is to focus on the neural signaling interface between incoming information about visceral status and the initiation of CNS regulatory responses. For organ regulation, this afferent-CNS interconnection occurs largely at the caudal third of the nucleus of the solitary tract (NTS). The NTS is a major gateway for cranial visceral afferent information  and unmyelinated subtypes and, for most organs, the slowly conducting C-type afferents greatly outnumber the myelinated class of afferents by 8–10-fold regardless of their cranial or spinal destination , ghrelin, and opioids, a pattern found in other NTS neuron subsets, and these peptides modify the behavior of specific pathways. Thus, peptide neurotransmission appears to be key mechanism of differential control. Glutamate release onto CA-NTS neurons within gastrointestinal pathways is modified by peptides acting presynaptically on ST afferents to modify transmission and manage pathway outcomes  is increasingly recognized as an important signaling partner molecule that is present peripherally as well as in the NTS. GLP-1 is released from both the gastrointestinal tract and from a limited population of preproglucagon (PPG) NTS neurons  approached rates of 30% for C-type afferents while remaining below 5% for A-type afferents , within the NTS. Integration, however, even at second order neurons is highly differentiated through both homo- and heterosynaptic interactions by glutamate and GABA. With multiple “accessory” transmitters present in the NTS , the interaction potential blossoms and assessments require quite elaborate testing to establish cause and effect. Much of this interaction utilizes metabotropic receptors which employ second messenger signaling Conn, . MetabotN-Arachidonoyl-dopamine (NADA) is an endogenous “capsaicin-like” substance that can act at TRPV1 (Huang et al., Fat and membrane lipid metabolites are newly appreciated as CNS signaling molecules (Pingle et al., −/−; Wang and Wang, −/− mice are reported to be resistant to obesity from high fat diets that induce low level inflammation (Motter and Ahern, TRPV1-operated glutamate mechanism offers a new focal point for the integration of multiple signals with substantial clinical importance. Intriguing clinical and basic observations point to multiple risk factors for multi-organ system pathology that relate directly or indirectly to cranial afferents, TRPV1 and NTS signal processing. One extrapolation of these complex correlations concerns inflammation, a well recognized co-factor in multiple disease processes that impact homeostatic systems. In spinal dorsal horn, TRPV1 is part of a chronic pathological transformation of afferent signaling (allodynia/hyperalgesia; Holzer, 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.


One consistent finding among studies using shotgun metagenomics to analyze whole viral communities is that most viral sequences show no significant homology to known sequences. Thus, bioinformatic analyses based on sequence collections such as GenBank nr, which are largely comprised of sequences from known organisms, tend to ignore a majority of sequences within most shotgun viral metagenome libraries. Here we describe a bioinformatic pipeline, the Viral Informatics Resource for Metagenome Exploration (VIROME), that emphasizes the classification of viral metagenome sequences (predicted open-reading frames) based on homology search results against both known and environmental sequences. Functional and taxonomic information is derived from five annotated sequence databases which are linked to the UniRef 100 database. Environmental classifications are obtained from hits against a custom database, MetaGenomes On-Line, which contains 49 million predicted environmental peptides. Each predicted viral metagenomic ORF run through the VIROME pipeline is placed into one of seven ORF classes, thus, every sequence receives a meaningful annotation. Additionally, the pipeline includes quality control measures to remove contaminating and poor quality sequence and assesses the potential amount of cellular DNA contamination in a viral metagenome library by screening for rRNA genes. Access to the VIROME pipeline and analysis results are provided through a web-application interface that is dynamically linked to a relational back-end database. The VIROME web-application interface is designed to allow users flexibility in retrieving sequences  and search results for focused secondary analyses. Genomic Encyclopedia of Bacteria and Archaea [Scientific appreciation of the true extent of microbial diversity and the composition of natural microbial communities now firmly rests on two approaches which utilize environmental DNA sequence data: 1) molecular phylogenetic analysis of single genes which are broadly shared among microbial groups ; and 2) shotgun metagenomic sequencing of environmental DNA. Oftentimes, the principal objective of marker gene studies is to utilize molecular phylogenetic analyses to make inferences about the taxonomic diversity of microorganisms within an environment -5. In an Archaea ,11.In the case of viruses, the lack of a single, universally shared and phylogenetically informative gene has limited the ability of researchers to easily assess the diversity and composition of natural viral assemblages ,12. HoweThe VIROME analysis pipeline relies on three subject protein sequence databases, five annotated databases, the UniVec database, and CD-Hit 454 . The UniThe MetaGenomes On-line (MGOL) peptide database contains nearly 49 million predicted peptide sequences from 137 metagenome libraries and is used to detect similarity to unknown environmental sequences. Within MGOL, nine libraries are described as ‘Eukaryotic’ since they were obtained from cells > 1 µm in size. Thirty-eight are described as ‘Viral’  and 89 are described as ‘Microbial’  are removed from the sequence library and a rRNA-free sequence file is generated. Sequences within this new file are scanned for the presence of tRNAs using tRNAscan-SE [The VIROME bioinformatics pipeline consists of two consecutive steps: 1) sequence quality screening; and 2) sequence analysis ; followeAscan-SE  and openAscan-SE  . Those viral metagenome libraries having the highest frequency of hits to known sequences typically come from marine environments where the hit frequency for longer Sanger reads is around 30% (at a BLAST e-score of ≤0.001) . Sanger These trends indicate that most viral genes are not represented within the major sequence databases. Viral metagenome hit frequencies are even lower when considering smaller, better annotated databases such as SEED and KEGG. As a result, most metagenomic analyses of the genetic and taxonomic composition of viral communities have been based on small sub-populations of sequences within viral shotgun libraries. This subset of sequences has alerted researchers to the ubiquitous presence of metabolic genes within viruses, genes once thought to exist only in cellular life ,35, and To our knowledge, the only other metagenomics analysis pipeline to include analysis against environmental peptides is the Viral Metagenome Annotation Pipeline (VMGAP) . In the Another strength of the VIROME analysis pipeline and web-application interface is the ability to retrieve read sequences, predicted ORFs, predicted peptides and top-hit BLAST results according to a large variety of search criteria. This functionality allows for a broad range of sequence retrieval, from individual sequences to whole libraries. For the researcher, the capability of customized sequence retrieval empowers subsequent sequence-based analyses, especially molecular phylogenetic analyses, which are a cornerstone of molecular ecological studies. In addition to customized searches, the VIROME web-application provides a summary display of BLASTP results organized by criteria such as the taxonomic origin of sequence homologs or functional terms associated with sequence homologs from databases such as KEGG, COG, GO, ACLAME, and SEED. Because VIROME links the sequence information from five annotated databases with UniRef 100 sequences, it is possible to garner a great deal of functional information for those sequences hitting known sequences within UniRef 100. MG-RAST uses a similar strategy with the M5NR non-redundant protein database.In the VIROME web-application interface, views summarizing homology search results according to functional and taxonomic criteria are displayed using fully interactive charts  that are dynamically linked to BLAST data. These summaries provide a ready means for researchers to effectively bin sequences according to a variety of criteria for subsequent analyses such as assembly and clustering. Finally, an important practical concern is that the VIROME pipeline is administered and maintained as a web resource and does not require researchers to have access to advance computing infrastructure . Other metagenomic pipelines such as VMGAP and MetaRep are not available as web-accessible resources and instead require investigators to install and administer the software on local computers, a task requiring significant UNIX systems administration experience ,39. SubmAreas of future development for the VIROME pipeline include the incorporation a sequence assembly component and build search infrastructure to support comparative analyses. Assembly will serve to reduce redundancy in homology search data for a given library, lessen the computational demands of the pipeline, and will be essential for the analysis of Illumina sequence libraries. Currently, no web-accessible metagenomics pipeline includes an assembly step as a routine component of its analysis. With regards to comparative analyses, VIROME output in the form of chart graphics, tab-delimited summary data, and tab-delimited search data can be downloaded and used as input data for qualitative and quantitative comparisons  using third party tools. At present, MetaVir  is the o


We have developed a cell-based model to dissect the mechanisms behind the propagation of tau pathology and assess potential therapeutic strategies.Increasing cognitive impairment and neuronal loss correlate with the spread of tau inclusions through the brain. When present in large quantities, these inclusions contribute to Alzheimer’s disease and other neurodegenerative diseases termed tauopathies. [1]. More recently, experiments both ® 488 label, were added to HEK 293T cells expressing soluble P301S tau. The uptake of exogenous tau and induced fibrillization of endogenous tau was monitored by flow cytometry, immunofluorescence and SDS-PAGE-WB. Specific inhibitors, markers and kinetic experiments were used to examine the mechanisms of tau internalization and interaction within cells. Truncated tau constructs were expressed in cells to assess the structural requirements for induced fibrillization.Filamentous tau from Tg mice expressing P301S tau or filamentous recombinant P301S tau, with or without DyLight® 488, we were able to monitor cellular uptake iand interaction with endogenous tau. This revealed that whilst the majority of cells internalized exogenous tau filaments, only a subset of tau expressing cells retained these and formed inclusions composed of endogenous protein. Studying the kinetics of exogenous tau uptake, along with inhibitors of cell binding and endocytosis, suggested that this process depends on the exogenous tau filaments binding to cell surface proteoglycans and being taken up by a mechanism most consistent with macropinocytosis. We were not able to induce tau fibrillization in cells expressing a N-terminal tau fragment, or tau with fibril forming motif deletions. However, induced tau fibrillization was observed when expressing a C-terminal tau fragment, or solely the repeat region.Hyperphosphorylated filamentous tau from P301S tau mice and non-phosphorylated filamentous recombinant P301S tau both efficiently entered cells and induced the formation of hyperphosphorylated filamentous tau composed of endogenous protein, in a time- and concentration- dependent manner. By labelling filamentous recombinant P301S tau with DyLightWe developed and optimized a cell-based model of pathological tau propagation, whereby the addition of minute quantities of exogenous filamentous tau induces the formation of intracellular hyperphosphorylated filamentous tau by direct interaction with endogenous soluble tau. Using this model we have been able to study mechanisms underlying this process. We have shown that cell surface binding and endocytosis are vital for the uptake of filamentous tau and the subsequent induction of endogenous tau misfolding and propagation to other cells. The expression of truncated tau constructs revealed that the repeat region of tau and the ability to form filaments are necessary for induced fibrillization. This model could be a potential tool to assess mechanism-based therapies.


Islet transplantation is an attractive treatment for selected patients with brittle type 1 diabetes. In the clinical setting, intraportal transplantation predominates. However, due to extensive early islet cell death, the quantity of islets needed to restore glucose homeostasis requires in general a minimum of two donors. Moreover, the deterioration of islet function over time results in few insulin-independent patients after five-year followup. Specific obstacles to the success of islet transplantation include site-specific concerns for the liver such as the instant blood mediated inflammatory reaction, islet lipotoxicity, low oxygen tension, and poor revascularization, impediments that have led to the developing interest for alternative implantation sites over recent years. Within preclinical settings, several alternative sites have now been investigated and proven favorable in various aspects. Muscle is considered a very promising site and has physiologically properties and technical advantages that could make it optimal for islet transplantation. Type 1 diabetes mellitus is a chronic disease with typical onset during childhood or adolescence. Patients suffering from the disease require multiple daily insulin injections coordinated alongside vigilant monitoring of blood glucose levels. For some patients, glucose homeostasis is not optimized despite these efforts, leading to repeated hyperglycemic and hypoglycemic events. Such repeated episodes of severe hypoglycemia, requiring medical assistance, predicate a patient being considered for islet transplantation. Currently, intraportal transplantation is performed in the majority of clinical cases. The liver was selected as the optimal implantation site for islet transplantation in the beginning of the 1970s, the basis of experimental studies by Kemp et al. . Neverth During recent years, interest in alternative implantation sites with the potential to improve long-term results has produced advances in noninvasive methodologies towards the quantification of islet graft mass and function. Positron emission tomography (PET) is one such technique, providing a functional imaging modality that quantifies an in vivo distribution of bioactive compounds labeled with positron-emitting nuclides. Commonly, clinically used PET tracers are labeled with short-lived radionuclides that result in a radiation dose that is both localized and nontoxic, allowing for multiple scans of a single patient.The rapid islet cell death observed in intraportal transplantation is in part caused by an instant blood-mediated inflammatory response (IBMIR) , 5. The In conjunction to the IBMIR, characteristic hypoxic events have also been contributed to early islet cell death. Indeed, during the process of initial islet isolation, the native vascular connections are disrupted, enforcing the islets to depend on oxygen diffusion immediately after transplantation. This process is further complicated when considering that the liver parenchyma has a much lower oxygen tension than can be found in the pancreas . We receIt has been proposed that the high concentrations of immunosuppressive drugs in the portal vein, following intestinal uptake of the drugs, may have a toxic effect on transplanted islet cells in the liver , 12. Mor18F]FDG, a marker for glucose metabolism, imaging both inflammatory lesions and insulitis FDG prior to infusion in murine DTBZ, correlated well to the observable decrease in blood sugar of a preclinical STZ mouse model in a study by Witkowski et al. -labeled exendin-4, a GLP-1R ligand, using SPECT, an imaging modality related to PET.Pattou et al.  showed p18F]FE-DTBZ-d4 (see 18F]L-DOPA (unpublished data) are currently being investigated as biomarkers for transplanted beta cells in two ongoing preclinical studies of intramuscularly transplanted islets in mice.The VMAT2 ligand [Z-d4 see  and the Striated muscle is a promising implantation site and in many regards superior to the liver. However, it is not yet fully characterized regarding islet long-term survival and functionality. The obstacles with early islet cell death observed to date following intramuscular islet transplantation can likely be overcome through remodeling the implantation site. Further studies capable of actualizing effective strategies are clearly needed, within both small and large animal models.


Reductive dehalogenases are the critical enzymes for anaerobic organohalide respiration, a microbial metabolic process that has been harnessed for bioremediation efforts to resolve chlorinated solvent contamination in groundwater and is implicated in the global halogen cycle. Reductive dehalogenase sequence diversity is informative for the dechlorination potential of the site or enrichment culture. A suite of degenerate PCR primers targeting a comprehensive curated set of reductive dehalogenase genes was designed and applied to 12 DNA samples extracted from contaminated and pristine sites, as well as six enrichment cultures capable of reducing chlorinated compounds to non-toxic end-products. The amplified gene products from four environmental sites and two enrichment cultures were sequenced using Illumina HiSeq, and the reductive dehalogenase complement of each sample determined. The results indicate that the diversity of the reductive dehalogenase gene family is much deeper than is currently accounted for: one-third of the translated proteins have less than 70% pairwise amino acid identity to database sequences. Approximately 60% of the sequenced reductive dehalogenase genes were broadly distributed, being identified in four or more samples, and often in previously sequenced genomes as well. In contrast, 17% of the sequenced reductive dehalogenases were unique, present in only a single sample and bearing less than 90% pairwise amino acid identity to any previously identified proteins. Many of the broadly distributed reductive dehalogenases are uncharacterized in terms of their substrate specificity, making these intriguing targets for further biochemical experimentation. Finally, comparison of samples from a contaminated site and an enrichment culture derived from the same site 8 years prior allowed examination of the effect of the enrichment process. Organohalide respiring bacteria utilize chlorinated hydrocarbons as terminal electron acceptors, a respiratory process driven by the activity of a class of enzymes called reductive dehalogenases  have been identified in a wide variety of anaerobic microorganisms, including Sulfurospirillum , including both microarray-based methods and PCR-based protocols  were gathered from in-house datasets, NCBI, and the JGI IMG portal. The genes were aligned using Muscle version 3.8.31 , 0.25 μM dNTPs, 0.5 μM each of forward and reverse primers, and 5 U of Taq DNA polymerase (NEB Inc.) in a total volume of 50 μL. Sample DNA amounts and concentrations are listed in Table Primer performance was tested using pure-strain DNA from A total of 23 samples were tested with the reductive dehalogenase primer suite Table . DNA fro2O instead of elution buffer. PCR products were cloned into the TOPO-TA vector (Sigma) and transformed into TOP-10 chemically competent E. coli (Sigma) under manufacturers directions. Colonies were grown on LB agar plates containing 50 mg/mL kanamycin and 40 mg/mL X-gal. White colonies were screened through PCR with the vector primers T7F and M13R (Sigma). For each reaction, five representative colony PCR reactions with the correct size of insert were sent for Sanger sequencing in both forward and reverse directions with the vector primers . Sequences were assembled into contigs where possible, and identified using BLAST.Select PCR reactions from the KB-1 consortium were cleaned using the Fermentas PCR spin-column clean-up kit with the cleaned product eluted in 35 μL of ddHe-value of 1e−10. The identified rdhA genes were assigned to a primer group based on their best blast-match from the curated rdhA database  under the manufacturer's recommended protocol for a library with 200–300 bp inserts from genomic DNA, excluding optional gel purification steps. The six barcoded sequencing libraries were pooled and sequenced with a single lane of Illumina HiSeq sequencing . The sequences were deconvoluted by barcodes, and pre-processed using tools available on the Galaxy-JGI server. All reads were filtered using Artifact Trimmer to remove low quality, adapter-containing, and low-complexity reads. Passing reads were trimmed to a length of 77 base pairs (4 bases from 5′ and 21 base pairs from 3′ end), criteria based on overall quality statistics. Assemblies were conducted using idba_ud under default parameters , two non-group-specific amplifications of known rdhA genes from the KB-1 culture, one iron-sulfur cluster domain-containing protein from a Dehalococcoides , and a reaction containing phosphatase and methyltransferase sequences from Dehalococcoides. The remaining 13 reactions were visualized as ladders of bands or smears of amplified DNA on an agarose gel. Two of these reactions failed to yield sufficient DNA for cloning. In the remaining eleven reactions, clone sequencing determined that the products were due to non-specific amplification, and included phosphatases, methylases, oxidoreductases, helicases, a metallobetalactamase, and hypothetical proteins, from Dehalococcoides, Geobacter, and other, non-dechlorinating organisms in the KB-1 consortium. From this, the performance of the primer suite was deemed satisfactory: it had detected all of the known rdhA genes within positive controls, and, for the KB-1 consortium, it allowed identification of one previously unknown rdhA gene. The presence of non-specific amplification was expected given the degenerate nature of the primer sequences. The primer suite was expressly designed to be permissive, in order to identify divergent rdhA genes.For one of the positive control DNA samples, the KB-1 mixed microbial consortium, existing clone libraries and metagenome sequencing may not have identified all Dehalobacter sp., a Dehalococcoides mccartyi strain, and a Dehalogenimonas sp. (“WBC-2”) . As expected, putative reductive dehalogenases were present in the samples' assemblies, as were non-specific sequences. The sample assemblies contained 2545–24,981 contigs above a 250 bp length cut-off, and included between 68 and 208 rdhA gene sequences. The average coverage for non-rdhA contigs ranged from 54 to 291x for five of the six samples, in contrast to an average coverage for rdhA contigs of 1868–11,011x. The only sample for which rdhA contigs were not specifically enriched was Lake Faro, the only putatively pristine environment sequenced . This indicates rdhA genes may be at much lower abundance in this sample, and thus both amplification and deep sequencing were required for identification of rdhA from this pristine environment, in the absence of any enrichment for dehalogenating activity.The number of non-specifically-amplified sequences was likely due to two factors: first, all positive PCR reactions were sequenced, even if they presented smeared or laddered bands rather than clear defined bands on an agarose gel, and second, the depth of sequencing conducted on these samples was such that sequences present at very low levels within the cleaned PCR products were expected to be completely sequenced. The depths of coverage for the assembled datasets reveal significant enrichment of rdhA genes were translated and aligned with their closest known homologs and the percent amino acid identity between known and newly-sequenced RdhA proteins calculated. All six of the sequenced samples contain predicted reductive dehalogenases with >90%, and in many cases, >97% sequence identity to characterized reductive dehalogenases whose substrate specificities are known  reductase, while the ISSO samples and the WBC-2 sample contain homologs to both known VC reductases, BvcA and VcrA  or the PceA from rdhA genes either bear significant sequence similarity to uncharacterized rdhA genes with no known substrate specificity, or represent divergent sequences with no closely related database sequence. Approximately one quarter of the rdhA genes identified are perfectly identical on an amino acid level with known RdhA (175 sequences), while one third share 90–99% identity with their closest known homolog (310 sequences) , meaning this is true sequence divergence rather than a long branch artifact from a partial sequence. In all three cases, the low sequence identity precludes a prediction of substrate specificities, but this clade of sequences is specifically active on chlorinated ethenes, suggesting both a putative substrate range and that these genes may be important future targets for classification. Within the primer group 1 phylogeny are sequences homologous to the MbrA TCE-dechlorinating enzyme, as well as a large clade of known and newly determined sequences with no known substrate specificity. The uncharacterized reductive dehalogenase group shares >90% PID within all members, and contains sequences from isolate strains  as well as from all six of the samples from this study. This clade represents broadly distributed reductive dehalogenases, implying the proteins are likely functionally important to the organisms encoding them. The RdhA sequences affiliated with primer group 22, containing the chlorophenol-active CprA from Desulfitobacterium dehalogenans, present another aspect of the identified reductive dehalogenase diversity  Figure . The remy Figure . In contrdhA genes. Each newly published genome sequence has provided novel rdhA sequences, indicating the diversity of the gene family is nowhere near exhaustively covered.The reductive dehalogenase gene family is important within the global chlorine cycle as well as being of industrial significance for remediation efforts  have been identified from the enrichment culture, using degenerate primer-based PCR amplification and sequencing . The last two RdhA are most similar to D. lykanthroporepellens sequences. Notably, none of the Desulfitobacterium-like and Dehalogenimonas-like sequences bear greater than 90% PID to reference sequences, implying these sequences may well be divergent Dehalobacter and Dehalococcoides sequences for which no closely related database sequence exists, which would fit the profile of identified organohalide respiring organisms identified in the culture. Seven of the eight known WL rdhA genes were identified in the WL culture sequences, all but one Dehalococcoides mccartyi gene.The WL culture RdhA bear highest sequence similarity with Dehalococcoidia: 105 Dehalococcoides-like sequences and 42 Dehalogenimonas-like sequences. There are 8 sequences with best matches to Dehalobacter sequences, 5 with Desulfitobacterium, 3 with Ruegeria sp., 1 with Deltaproteobacterium NaphS2, and 1 to the sole Archaeal RdhA, from Ferroglobus placidus DSM 10642. Of the lower abundance organisms, only one Desulfitobacterium-like RdhA has >90% PID to database sequences: all other Desulfitobacterium, Ruegeria, Ferroglobus, and Deltaproteobacterium NaphS2-associated sequences have low PID (<60 PID). Six of the eight previously identified rdhA from the WL culture were present in the WL site groundwater sample, including the one rdhA gene not identified in the current WL culture sample.The predicted community composition of the WL site based on RdhA affiliations is substantially different from the WL enrichment culture. The groundwater RdhA are dominated by homologs to the Dehalobacter sp. dihaloelimination activity on 1,1,2-TCA and Dehalococcoides-mediated VC reduction over the course of 8 years, with a concurrent reduction in community complexity and enrichment over time and through transfers. Despite 8 years of temporal separation and enrichment, the two sequence datasets share 39 RdhA with 99–100% PID, including many of the full-length sequences (Figure The absence of VcrA and the diversity of RdhA host organisms in the WL site sample distinguish it from the WL enrichment culture. These differences are likely the result of selective forces on the enrichment culture for the s Figure . The shaDesign and development of a PCR primer suite targeting the known diversity of reductive dehalogenase homologous genes provided a mechanism for screening environmental and laboratory samples for members of this gene family. Illumina sequencing of six samples' PCR-amplified products resulted in identification of 798 novel reductive dehalogenase sequences. The newly sequenced reductive dehalogenases helped identify broadly distributed gene “types” whose functions may be important within the natural environment, and who will pose interesting targets for further characterization. The sequenced RdhAs also represent new reference sequences spanning a substantial untapped depth of sequence diversity within the gene family. The distribution of the reductive dehalogenases and their taxonomic affiliations within a contaminated site undergoing bioremediation allowed identification of biomarkers for tracking the enrichment culture used for bioaugmentation. A comparison between a contaminated site and an enrichment culture derived from the same site provided insight into the reduced complexity and community composition changes inherent to the enrichment process.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.


Autism spectrum disorders affect significantly more males than females. Understanding sex differences in normal human brain development may provide insight into the mechanism(s) underlying this disparity; however, studies of sex differences in brain development at the genomic level are lacking. Here, we report a re-analysis of sex-specific gene expression from a recent large transcriptomic study of normal human brain development, to determine whether sex-biased genes relate to specific mechanistic processes. We discovered that male-biased genes are enriched for the processes of extracellular matrix formation/glycoproteins, immune response, chromatin, and cell cytoskeleton. We highlight that these pathways have been repeatedly implicated in autism and demonstrate that autism candidate genes are also enriched for these pathways. We propose that the overlap of these male-specific brain transcriptional modules with the same pathways in autism spectrum disorders may partially explain the increased incidence of autism in males. Among the varied genetic, cellular, and clinical phenotypes described in autism spectrum disorders (ASD), the predominance of males versus females affected has stood apart as one of the most replicated findings. The most recent analysis indicates that at least four males are affected for every one female , and theP <0.05 were considered significant.Therefore, we were interested in assessing sex-biased patterns of normal human brain development at the genomic level, in an attempt to gain insight into processes that may underlie sex differences in ASD. We re-analyzed gene expression data from a recent whole-genome transcriptomics study of normal human brain development by Kang and colleagues . This stThe genes with female-biased expression patterns were not significantly enriched for any particular functional categories  . We founde novo DNA mutations (inherited set), such as genome-wide association, copy number variation, linkage, and other association analysis (see Additional file Of unique interest is the finding of an immune system module in normal male-specific brain transcription. While peripheral immune responses have a significant gender dimorphism , the rolnormal male versus female brain development, suggesting that any neurodevelopmental disorder resulting from aberrations in these pathways would be expected to have increased incidence in males. It will be important for future studies to further assess the role of male-specific gene expression programs as they relate to co-expression, dysregulation, and interaction with autism candidate genes in the developing brain, in addition to those of other neurodevelopmental and psychiatric disorders with sex-biased incidence.Overall, these results suggest the hypothesis that sex-specific transcriptional modules may make males more susceptible to neurodevelopmental disorders that result from aberrations – both inherited and environmental – in these pathways. As these four particular pathways are repeatedly implicated in ASD and overlap with pathways implicated by ASD candidate genes, we propose that normal sex differences in the functional genomics underlying human brain development may partly explain the significantly higher incidence of ASD in males. Notably, these four modules are more prominent in ASD: autism spectrum disordersThe authors declare that they have no competing interests.MNZ and OMR conceived of and designed the analysis. MNZ performed the analysis. MNZ and OMR wrote the manuscript. Both authors read and approved the final manuscript.Supplementary data tables. Table S1 presenting genes differentially expressed by sex used for this analysis. Table S2 presenting gene ontology results from DAVID analysis of female genes. Table S3 presenting gene ontology results from DAVID analysis of male genes. Table S4 presenting results from Ingenuity Pathways Analysis of male genes. Table S5 presenting results from Ingenuity Pathways Analysis of female genes. Table S6 presenting results from GeneGo analysis of male genes. Table S7 presenting results from GeneGo analysis of female genes. Table S8 presenting DAVID analysis of all AutKB Genes. Table S9 presenting expression and inherited ASD candidate gene sets. Table S10 presenting expression set DAVID enrichment results. Table S11 presenting inherited set DAVID enrichment results.Click here for file


Mycobacterium tuberculosis comparing whole genome sequencing (WGS) with traditional genotyping, Stefan Niemann and colleagues found that classical genotyping falsely clustered some strains, and WGS better reflected contact tracing.In an outbreak investigation of  Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains.Understanding Hamburg clone”) started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance.During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone , mycobacterial culture (growth of M. tuberculosis from sputum), and chest X-rays. Tuberculosis can be cured by taking several antibiotics daily for at least six months, although the recent emergence of multidrug-resistant M. tuberculosis is making tuberculosis harder to treat.Tuberculosis—a contagious bacterial disease that usually infects the lungs—is a major public health problem, particularly in low- and middle-income countries. In 2011, an estimated 8.7 million people developed tuberculosis globally, and 1.4 million people died from the disease. Tuberculosis is second only to HIV/AIDS in terms of global deaths from a single infectious agent. M. tuberculosis transmission within human populations. Because M. tuberculosis, like all bacteria, accumulates genetic changes over time, there are many different strains (genetic variants) of M. tuberculosis. Genotyping methods have been developed that identify different bacterial strains by examining specific regions of the bacterial genome (blueprint), but because these methods examine only a small part of the genome, they may not distinguish between related transmission chains. That is, traditional strain genotyping methods may not be able to determine accurately where a tuberculosis outbreak started or how it spread through a population. In this longitudinal cohort study, the researchers compare the ability of whole genome sequencing (WGS), which is rapidly becoming widely available, and traditional genotyping to provide information about a recent German tuberculosis outbreak. In a longitudinal cohort study, a population is followed over time to analyze the occurrence of a specific disease.Although efforts to reduce the global burden of tuberculosis are showing some improvements, the annual decline in the number of people developing tuberculosis continues to be slow. To develop optimized control strategies, experts need to be able to accurately track M. tuberculosis isolates of the Haarlem lineage using classical strain typing. The researchers examined each of the 86 isolates from this outbreak using WGS and classical genotyping and asked whether the results of these two approaches correlated with contact tracing data (information is routinely collected about the people a patient with tuberculosis has recently met so that these contacts can be tested for tuberculosis and treated if necessary) and with the spatio-temporal distribution of outbreak cases. WGS of the isolates identified 85 single nucleotide polymorphisms  that subdivided the outbreak into seven clusters of isolates and 36 unique isolates. The WGS results showed that the first isolates of the outbreak were incorrectly clustered by classical genotyping and that one strain—the “Hamburg clone”—started expanding in 1998. Notably, the genome-based clustering patterns were in better accordance with contact tracing data and with the geographical distribution of cases than clustering patterns based on classical genotyping, and they identified eight confirmed human-to-human transmission chains that involved 31 patients and a maximum of three SNPs. Finally, the researchers used their WGS results to estimate that the Hamburg clone emerged between 1993 and 1997, shortly before the discovery of the tuberculosis outbreak through epidemiological surveillance.During long-term (1997–2010) population-based molecular epidemiological surveillance (disease surveillance that uses molecular techniques rather than reports of illness) in Hamburg and Schleswig-Holstein, the researchers identified a large tuberculosis outbreak caused by M. tuberculosis during outbreaks and indicate how WGS data should be interpreted in future genome-based molecular epidemiology studies. WGS has the potential to improve the molecular epidemiological surveillance and control of tuberculosis and of other infectious diseases. Importantly, note the researchers, ongoing reductions in the cost of WGS, the increased availability of “bench top” genome sequencers, and bioinformatics developments should all accelerate the implementation of WGS as a standard method for the identification of transmission chains in infectious disease outbreaks.These findings show that WGS can be used to identify specific strains within large tuberculosis outbreaks more accurately than classical genotyping. They also provide new information about the evolution of http://dx.doi.org/10.1371/journal.pmed.1001387.Please access these websites via the online version of this summary at tuberculosis, including the Global Tuberculosis Report 2012The World Health Organization provides information  on all aspects of Stop TB Partnership is working towards tuberculosis elimination; patient stories about tuberculosis are available (in English and Spanish)The tuberculosis, including information on tuberculosis genotyping (some information in English and Spanish)The US Centers for Disease Control and Prevention has information about tuberculosisThe US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of Tuberculosis Survival Project, which aims to raise awareness of tuberculosis and provide support for people with tuberculosis, provides personal stories about treatment for tuberculosis; the Tuberculosis Vaccine Initiative also provides personal stories about dealing with tuberculosisThe tuberculosis (in English and Spanish)MedlinePlus has links to further information about whole-genome sequencing Wikipedia has a page on  Mycobacterium tuberculosis (Mtb), which is still among the most devastating pathogens Mortality due to infectious diseases remains a major burden, especially in low- and middle-income countries. In an increasingly globalized world, the free movement of humans favors the continuous spread of human pathogens such as 6110 RFLP (restriction fragment length polymorphism) 6110 DNA fingerprinting revealed interesting aspects of actual transmission dynamics, we found that only approximately 50% of the resulting clusters could be confirmed by contact tracing documenting transmission links , number of years of residence in Hamburg (Germany), current address (or whether the patient was homeless), whether the patient was living in a health-care or any public institution, education level and/or professional training (as far as this could be ascertained), the nature of the patient's current employment (if any), details of any previous known exposure to other persons with TB , and the names of the patient's household contacts and/or any close contacts in occupational or crowded settings.To acquire clinical data, the following were also included in the questionnaire: date of first onset of illness , nature of symptoms, date and reason for the diagnostic investigation, latency due to the patient's delay in seeking medical help, the first date of case report to a public health office, associated medical problems , result of tuberculin skin testing (or interferon gamma release assay), chest radiographic findings and results of microbiological analyses, and presence of alcoholism . The sociodemographic and clinical data of each index person acquired from standardized patient questionnaires in the first interview were reassessed immediately after the interview—independently of the cluster study and before the patient's isolate was subjected to RFLP fingerprinting—in order to determine the patient's epidemiologic context, especially his/her possible membership in a high risk group. This profile was intended to help clarify the route of infection and to establish whether the patient's contact information was adequate and whether further Mtb infection could have been transmitted but remained undetected through the omission of probable contact persons. When a patient was recognized to be a cluster member, additional interviews were conducted if possible, i.e., if one or more patients of the same clusters diagnosed earlier were still alive and with known domicile.p-value<0.05 was taken as statistically significant.Categorical data were compared by the Pearson's χ-squared test . The Wilcoxon rank sum test was used to determine whether the distribution of continuous variables differed between two groups; a two-tailed 6110 as a probe, spoligotyping, and 24-locus MIRU-VNTR genotyping were performed by standardized protocols, as described previously 6110 fingerprint patterns. MIRU-VNTR typing revealed only a few differences, consisting of single-locus variations, discriminating the isolates into one large (n = 75) and four smaller (n = 11) groups  groups .Isolated genomic DNA of the Mtb strain 7199/99 was sequenced using 454 pyrosequencing with both standard and paired-end runs. Assembly with GS De Novo Assembler software yielded 124 contigs in five scaffolds. Gap closure was achieved by PCR and Sanger sequencing of the amplicons. The closed genome was corrected for sequencing errors using reads generated by the Illumina platform, and the refined sequence was automatically annotated using the annotation system GenDB For a genome-wide alignment of Mtb strains, we employed the program Mauve Isolated genomic DNA of individual strains was sequenced on the Illumina platform. Resulting reads were mapped to the Mtb H37Rv genome (GenBank ID: NC_000962.2) using the exact alignment program SARUMAN 8 iterations after a burn-in of 106 iterations. Usage of alternative tree priors  in the Bayesian analysis resulted in very similar mutation rates and divergence times. In contrast, when analyses were run on an empty alignment to sample from the prior distribution, divergence times were strongly inflated, suggesting that our results were not an artifact reflecting the priors. Linear regression of root-to-tip distances from a maximum-likelihood tree against dates of isolation was performed by using Path-O-Gen software (available at http://tree.bio.ed.ac.uk/software/pathogen/).Evolutionary rates and divergence times were computed on the basis of an alignment of 3,663,090 sequenced base pairs from each of 86 Mtb isolates using the BEAST software t-test and were significantly different (p<0.05).Relative reproductive fitness of Mtb lineages in the human population was estimated by calculating selection coefficients as the slopes of a least-squares linear regression of the number of cases through time in each lineage. The ratio of two lineages' slopes (αA/αB) provides the relative fitness. The selection coefficient estimates (slopes) were compared using the Student's 6110 DNA fingerprinting and later confirmed by 24-locus MIRU-VNTR typing  were grouped in a single clade, which we termed the “Hamburg clone” , and was continuously isolated until the very end of the study  displayed SNP profiles differing by three SNPs at most , which could be assembled into eight different chains . A high  at most , thus de−7 substitutions per nucleotide site per year , i.e., approximately 0.4 mutations per Mtb genome per year. When analyses were rerun on datasets with sampling dates permutated across isolates, divergence dates were much older and credible intervals much larger  in the patient population . We found that patients infected by the Hamburg clone or by unrelated strains did not differ by age, HIV infection, substance abuse, homelessness, or social milieu  all patients could be assigned to the same social setting in Hamburg.WGS analysis revealed preferential expansion of one specific clone  and esxS (Rv3020c), or consisted of a single-nucleotide deletion, as in mmpl2 (Rv0507). This deletion resulted in the truncation of mmpl2, which is a member of a transmembrane protein family; some of the coding regions are potentially involved in fatty acid transport To fully characterize the genomic background of the preferentially transmitted notation , using snotation . A smallOur study demonstrates that WGS-based typing provides epidemiologically relevant resolution of large, longitudinal Mtb outbreaks much more efficiently than classical genotyping. Genome-based analysis correlated better with contact tracing information and spatio-temporal patterns of the pathogen's spread. In addition, we were able to determine a timescale of evolution of Mtb during clonal expansion over more than a decade, in a population of >80 patients. We estimated a maximum variation of three SNPs in definite human-to-human transmission events as a benchmark for interpreting WGS data for future genome-based molecular epidemiology.6110 patterns differed by as many as 130 SNPs, not supporting direct transmission The efficiency of TB surveillance relies on the capacity to accurately detect outbreaks in the community and to track ongoing transmission chains, which requires the characterization of clinical isolates with high discriminatory power, at strain level 6110 DNA fingerprinting and 24-locus MIRU-VNTR typing, genome sequencing identified a specific clone, named the “Hamburg clone,” as the major causative agent of the studied outbreak in Hamburg and Schleswig-Holstein. In addition, genome-wide polymorphisms revealed several clusters within the outbreak that correlated well with contact tracing data and the known spatio-temporal spread to Schleswig-Holstein in two independent instances, as seen by two distinct clusters , hence a few years before the outbreak was discovered. We note, however, that the actual generation time may be even shorter in the natural population than is indicated here, as our estimate of the evolutionary rate does not include an unknown proportion of mutations possibly removed from the population via selection and drift over 14 y Our data reveal a time-dependent accumulation of genome variation when Mtb spreads in its natural human host population. It shows that Mtb evolves approximately 10-fold more slowly than methicillin-resistant Hamburg clone caused an increased number of cases, including several clusters, compared to the other clones, indicating more effective transmission over a long time period. Given the complex interaction between bacterial, human, and environmental factors influencing TB transmission, several explanations might account for such differential expansion, such as socio-environmental factors, or intensity and duration of exposure to, or characteristics of, source cases Hamburg clone group and patients infected with other isolates , which possibly enhanced the fitness of this particular clone. However, this hypothesis would be difficult to prove, even using available experimental models of infection A final interesting finding revealed by our WGS-based analysis was that the ates see . Both paates see  cannot boups see . TherefoIn conclusion, our study demonstrates the potential of WGS for improved molecular-guided TB surveillance and control. We envision that its progressive effective implementation will be accelerated by the continuously decreasing sequencing costs, broader distribution of so-called bench top genome sequencers Figure S1IS6110 DNA fingerprint, spoligotype, and 24-locus MIRU-VNTR typing patterns of all outbreak strains analyzed. The IS6110 band positions were normalized, so that banding patterns of all strains are mutually comparable. The repeat unit numbers of 24 MIRU loci are shown in yellow shades, with cutoffs ranging from 0 (white) to 35 (red) units. Strains were clustered on the basis of IS6110 fingerprint patterns. Single differences of 24-locus MIRU-VNTR are indicated by asterisks.(TIF)Click here for additional data file.Figure S2Minimum spanning trees of all outbreak isolates specified by the year of isolation. (A) Newly collected isolates were clustered in minimum spanning trees allowing hypothetical nodes. Color codes correspond to (TIF)Click here for additional data file.Figure S3Maximum-likelihood tree displaying sequence variations among outbreak isolates. Each isolate had a specific distance to the root (upper panel). Nomenclature of strains corresponds to the internal laboratory keys. The middle panel illustrates the effect of randomly switching dates across isolates (permutations 1 to 5) on the estimate of divergence time . Root-to-tip distance was plotted against the date of isolation (lower panel).(TIF)Click here for additional data file.Figure S4Circular representation of the Mtb genome 7199/99. The outer rim displays the base-pair scale; the second circle represents all identified coding sequences on either the forward or the reverse strand. The third circle indicates the distribution of large deletions (red), the fourth circle bears all insertions (black), and the fifth shows the positions of IS6110 elements. The sixth circle gives the GC content; the seventh, innermost circle visualizes GC skew.(TIF)Click here for additional data file.Table S1Single nucleotide polymorphisms determined in the isolates investigated.(PDF)Click here for additional data file.Table S2Demographic characteristics of the 86 patients.(PDF)Click here for additional data file.


Visual processing network is one of the functional networks which have been reliably identified to consistently exist in human resting brains. In our work, we focused on this network and investigated the intrinsic properties of low frequency (0.01–0.08 Hz) fluctuations (LFFs) during changes of visual stimuli. There were two main questions to be discussed in this study: intrinsic properties of LFFs regarding (1) interactions between visual stimuli and resting-state; (2) impact of repetition rate of visual stimuli.We analyzed scanning sessions that contained rest and visual stimuli in various repetition rates with a novel method. The method included three numerical approaches involving ICA , fALFF , and Coherence, to respectively investigate the modulations of visual network pattern, low frequency fluctuation power, and interregional functional connectivity during changes of visual stimuli. We discovered when resting-state was replaced by visual stimuli, more areas were involved in visual processing, and both stronger low frequency fluctuations and higher interregional functional connectivity occurred in visual network. With changes of visual repetition rate, the number of areas which were involved in visual processing, low frequency fluctuation power, and interregional functional connectivity in this network were also modulated.To combine the results of prior literatures and our discoveries, intrinsic properties of LFFs in visual network are altered not only by modulations of endogenous factors  and disordered behaviors (early blind), but also exogenous sensory stimuli . It demonstrates that the intrinsic properties of LFFs are valuable to represent physiological states of human brains. Visual network is one of the functional networks which have been reliably identified to consistently exist in human resting brains In our study, we focused on the correlations between exogenous factor modulations and intrinsic properties of LFFs in visual network. We analyzed scanning sessions that contained rest and visual stimuli in various repetition rates with a novel method. The method included three numerical approaches involving Independent Component Analyses (ICA) We performed group ICA-based analyses to obtain visual networks during rest and visual stimulus rate = 2, 4, 8, 16 Hz. In By comparing With higher repetition rate, fALFF in the three areas kept increasing. The largest fALFF occurred in repetition rate = 8 Hz. When repetition rate was even higher, fALFF decreased. This illustrated that low frequency oscillations of visual network fluctuated more dramatically with higher repetition rate. However, the low frequency fluctuations fluctuated the most dramatically in repetition rate = 8 Hz. When repetition rate was even higher, the low frequency oscillations fluctuated less dramatically.With higher visual repetition rate, coherence between both two visual streams and primary visual cortex kept increasing. The largest coherence occurred in repetition rate = 8 Hz. When the repetition rate was even larger, coherence decreased. This showed that higher repetition rate induced stronger interregional functional connectivity in visual network. However, the strongest interregional functional connectivity occurred in repetition rate = 8 Hz. When repetition rate was even higher, weaker interregional functional connectivity occurred.Compared with correlation analyses, coherence analyses are less sensitive to the shape of regional hemodynamic response function (HRF), which varies across regions due to interregional differences in vascular properties Previous literatures demonstrated that inter-individual changes in resting-state, such as aging, gender, and even individual diversities, modified the response to a certain visual stimulus. Aging led to diminished top-down inhibitory control In our study, we used novel methods to focus on the investigation of the overall interactions between visual stimuli and resting-state without discussions on the specific related factors such as aging, gender, or individual diversities. According to the results of our work, when resting-state was replaced by visual stimuli, more areas of visual network participated in visual processing, the low frequency oscillations fluctuated more dramatically, and higher interregional functional connectivity occurred. Briefly, stronger interregional functional connections occurred in visual network during visual stimuli rather than resting-state.In our study, we changed visual repetition rate to alter exogenous sensory stimulus conditions. With increasing visual repetition rate, more areas of visual network participated in visual processing, the low frequency oscillations fluctuated more dramatically, and higher interregional functional connectivity occurred. Briefly, stronger interregional functional connections occurred in visual network when repetition rate was higher. However, the strongest interregional functional connections occurred in repetition rate = 8 Hz. When the repetition rate was even higher, interregional functional connections became weaker. This corresponded to Briicke-Bartley Effects In prior literatures, the relationship between temporal frequency of visual stimuli and biological signals measured by BOLD In our work, we investigated intrinsic properties of LFFs regarding impact of repetition rate using stimulation frequencies between 0–16 Hz. The interregional functional connections were found to peak at 8 Hz. In addition to around 8 Hz peak, previous literatures demonstrated that other peaks were observed for stimulation frequencies at around 20, 40, and 80 Hz Our study focused on the relationship between exogenous factor modulations  and intrinsic properties of LFFs in visual network. According to prior discussions in our work, stronger interregional functional connections occurred in visual network when resting-state was replaced by visual stimuli. With changes of exogenous stimuli , interregional functional connections were also modulated. To combine the results of our work with prior literatures The participant has no history of neurological disorder, medication use, substance abuse, or contraindications to MRI such as claustrophobia.The participant has no metal implants such as a pacemaker or a stent graft.The participant has no make-up or tattoo on the skin.The procedures of this study have been approved by National Taiwan University Hospital, Taipei, Taiwan.The participant understands exactly the procedures of the whole experiment and gives informed written consents to procedures before participation.Fourteen right-handed volunteers , ranging from 20- to 32-years-old, were recruited, trained, and imaged. Handedness was determined by Edinburgh Handedness Inventory The five stimulus conditions in a task period were arranged in randomized order to prevent the results of our study from being affected by a regular pattern of stimuli. The duration of each stimulus condition within a task period was 16 s, which could avoid consistent and gradual reduction in activation caused by long duration of repeated visual stimuli (fMR adaptation) After obtaining scanning sessions that contained rest and visual stimuli in various visual repetition rates, time series of the same stimulus condition in each task period were extracted and combined to become a new time series in a longer duration, as In our study, we focused on three main regions of visual network including primary visual cortex, dorsal stream, and ventral stream and performed regional quantitative analyses to investigate intrinsic properties of LFFs in these regions during visual stimuli at different repetition rates. Particular fMRI experiments Dorsal stream served spatial localization in visual system. There were three stimulus conditions in the task for the localization of primary visual cortex and ventral stream. In the first condition, the participants were asked to see the picture shown in Ventral stream and primary visual cortex respectively served object recognition and receive and transmit visual information in visual system. In the subsequent GLM activation group analysis, BOLD signal time series of the scrambled pictures were compared with those of the object pictures to find the location of ventral stream 2 in-plane voxel resolution; 64×64 image matrix, FOV 256×256 mm2). During the scans for the localization of primary visual cortex, dorsal stream, and ventral stream, the same imaging sequence and parameters were applied. However, 108 whole brain volumes were acquired in each scan for localizing an individual region.Scanning was performed on a Bruker Medspec 3.0 Tesla whole body scanner. During the continuous scan, 480 24-slice whole-brain volumes were acquired using a T2*-weighted gradient-echo, echo-planar-imaging sequence  were acquired in the same slice positions, in order to facilitate the precise determination of the structures corresponding to the functional activation foci.At the end of fMRI data collection, fast spin-echo, 24–slice 2D T1- weighted anatomical images .The five new time series of various stimulus conditions obtained in http://afni.nimh.nih.gov) software http://www.nitrc.org/projects/pestica/) was applied to use temporal ICA-based analyses to determine cardiac and respiratory noises from fMRI data without parallel measurements of physiological signals Further preprocessing was performed using AFNI  were employed to perform a temporally concatenated group ICA on the preprocessed new time series to obtain visual networks during rest and visual stimuli in various repetition rates. We constrained the number of independent components to 20 ICA-based analyses implemented in the GIFT (Group ICA fMRI Toolbox; In our work, we focused on the three main regions of visual network including primary visual cortex, dorsal stream and ventral stream and investigated intrinsic properties of LFFs in these three regions across different visual stimulus conditions. The numerical approaches involving fALFF In fALFF analyses, we calculated fALFF of the three main regions across different repetition rates, as


Inflammatory external root resorption (IERR) after orthodontic treatments is an unusual complication. This case report describes a non-vital maxillary premolar with symptomatic extensive IERR (with a crown/root ratio of 1:1) after receiving orthodontic treatment. The first appointment included drainage, chemo-mechanical preparation of the canal and intra-canal medication with calcium hydroxide (CH) along with prescription of analgesic/antibiotic. The subsequent one-week follow-up revealed the persistence of symptoms and formation of a sinus tract. Finally, extraoral endodontic treatment was planned; the tooth was atraumatically extracted and retrograde root canal filling with calcium enriched mixture (CEM) cement was placed followed by tooth replantation. Clinical signs/symptoms subsided during 7 days postoperatively. The sinus tract also resolved after one week. Six-month and one-year follow-ups revealed complete healing and a fully functional asymptomatic tooth. This case study showed favorable outcomes in a refractory periapical lesion associated with orthodontically induced extensive IERR. The chemical as well as biological properties of CEM cement may be a suitable endodontic biomaterial for these cases. Inflammatory external root resorption (IERR), is one of the complications most frequently associated with inflammation of the periradicular tissues stemming from the microorganism-induced intracanal infection; which mostly leads to progressive damage of root structure and finally tooth loss . Other cExperts are of the opinion that vital teeth with orthodontic induced IERR do not need to undergo endodontic therapy, as this approach does not eliminate the etiologic factor of the condition . HoweverExtraoral endodontic treatment (EET) may be performed with a retrograde approach, which includes cleaning/shaping of the root canal system and filling the prepared root-end with appropriate biomaterial. For this purpose, different materials such as amalgam, gutta-percha, zinc oxide-eugenol cements, glass ionomer cement, gold foil pellets, Cavit, composite resin, mineral trioxide aggregate (MTA) and calcium enriched mixture (CEM) cement have been used , 6; amonThis case report represents the diagnosis and treatment challenges of an EIRR lesion around a symptomatic non-vital maxillary premolar, which had previously been under orthodontic treatment; also extraoral endodontic treatment of this tooth using CEM cement and up to one-year follow-up results are presented.º C. Intraoral examination revealed a diffused swelling in the apical area of the involved tooth, class II tooth mobility and severe tenderness to percussion and palpation; periodontal probing was within the normal range (<3mm). Cold test with Endo-Frost cold spray  elicited no response in involved tooth; the adjacent teeth revealed a positive response to cold test. The periapical x-ray revealed a deep restoration adjacent to the pulp and a large radiolucent periradicular lesion surrounding the open-apex and resorbed root , the involved tooth was isolated and the access cavity was prepared. On entering the pulp chamber, a purulent drainage was observed, and patient’s tenderness immediately subsided. After radiographic working length determination, cleaning and shaping of the root canal was performed; the root canal was irrigated and filled with 5.25% sodium hypochlorite for 15 min along with passive instrumentation. CH powder  and normal saline were mixed to prepare a creamy paste which was then placed within the root canal after canal drying with large paper points . A TID pus tract  and clasAfter administering local anesthesia via infiltration of 2% lidocaine with adrenalin 1:80000, the tooth was atraumatically extracted; meanwhile the apical lesion was curetted and sent for histopathological examination. While holding the tooth in a forceps, the root canal was retrogradely prepared using Peeso reamers #2  and copious saline irrigation; finally the cavity was dried with sterilized paper points. Then CEM cement powder and liquid  was mixed according to the manufacturer’s instructions. CEM was retrogradely inserted and packed within the root canal using a measured plugger. The tooth was replanted into its socket and the accurate repositioning was confirmed radiographically . HistopaThe treated tooth was rechecked after 10 days. The clinical signs/symptoms had resolved and the sinus tract had disappeared. The healing process was uneventful; the periodontal examination showed normal sulcular depth and normal gingival status. Clinical examination at the 6-month and 1-year recalls revealed satisfactory clinical function, the absence of pain/tenderness to percussion/palpation, as well as the absence of tooth mobility and signs/symptoms of inflammation or infection. Periapical radiographs revealed healing of the periapical lesion substituted with newly formed bone; external root resorption of involved tooth ceased without radiographic signs of replacement resorption .Extraoral endodontic treatment (EET) is rewarded by some researchers; and they suggest considering EET as the last option after failure of other endodontic treatments . On the It is stated that EET has several advantages over endodontic surgery since it is less complicated, protracted, invasive, and expensive . In the Management of IERR is based on removing bacteria and their byproducts from the root canal space as well as dentinal tubules to stop the inflammatory processes and allow healing of periodontal tissues . The conSurgical endodontic literature supports a direct cause-and-effect relationship between creations of hermetic apical seal by endodontic filling materials and successful outcomes ; an ideain vivo studies specifically in the field of regenerative endodontics [Sealing ability of CEM cement as root-end filling material is comparable with MTA . An inteodontics  and vitaodontics -32, showodontics , 34. Furodontics  or as a odontics .Extraoral retrograde root canal filling with CEM cement in a refractory apical lesion caused by orthodontically induced extensive IERR, may be a suitable treatment choice in current practice.