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Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier function and increases susceptibility to infections. Herein, we hypothesized that somatic mutations together with cigarette smoke create a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from controls (n=33) or cancer cases (n=143), we conducted 16S rRNA gene sequencing (MiSeq), with RNA-seq data from lung cancer cases in The Cancer Genome Atlas (n=1112) serving as the validation cohort. We demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue, indicating a shift in the overall microbial community in lung cancer patients as compared to those without cancer. Lung cancer cases were classified by the relative abundance of two taxa, Variovorax and Streptococcus, with an increase in Variovorax abundance in tumors as compared to non-tumor adjacent paired lung tissue (FDR corrected P=0.013). The species of Variovorax was identified histologically, and also by two additional 16S rRNA strategies (Resphera Insight analysis and PacBio sequencing). A group of taxa were associated with squamous cell carcinoma (SCC), of which Acidovorax were enriched in smokers (P =0.0013). Further, these taxa, including Acidovorax, exhibited higher abundance among the subset of SCC cases with TP53 mutations, an association not seen in adenocarcinomas (AD). Therefore, we observed a microbiome-gene and a microbiome-exposure interaction in SCC lung cancer tissue. Together, these results open the door for future biomarker research that could be used to improve screening and direct mechanistic studies of lung cancer therapy. Citation Format: Leigh Greathouse, James White, Valery Bliskovsky, Ashley Vargas, Eric Polley, Elise Bowman, Mohammed Khan, Ana Robles, Brid Ryan, Amiran Dzutsev, Giorgio Trinchieri, Marbin Pineda, Sven Bilke, Paul Meltzer, Marina Walther-Antonio, Garth Ehrlich, Joshua Mell, Joshua Earl, Sergey Balashov, Archana Bhat, Alex Valm, Clayton Deming, Sean Conlan, Julia Oh, Julie Segre, Curtis Harris. Microbiome-TP53 gene interaction in human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4925. doi:10.1158/1538-7445.AM2017-4925

Tumor cells contain nonsynonymous somatic mutations that alter the amino acid sequences of the proteins encoded by the affected genes. Those alterations are foreign to the immune system and may therefore represent tumor-specific neoantigens capable of inducing antitumor immune responses. Somatic mutational and neoantigen density has recently been shown to correlate with long-term benefit from immune checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma, suggesting that a high density of neoepitopes stemming from somatic mutations may enhance clinical benefit from blockade of immune checkpoints that unleash endogenous responses to these mutation-associated neoantigens (MANAs). Expression of the programmed cell death ligand 1 (PD-L1) in tumors or tumor-infiltrating immune cells has been associated with responses to PD-1 blockade; however, PD-L1 expression or other immune biomarkers have not been sufficient to fully explain therapeutic outcomes. Among the patients that initially respond to PD-1 blockade, some become resistant to the therapy. Up-regulation of alternate immune checkpoints, loss of HLA haplotypes, or somatic mutations in HLA or JAK1/JAK2 genes have been proposed as mechanisms of evasion to immune recognition in some patients, but the mechanisms underlying response and acquired resistance to immune checkpoint blockade have remained elusive. To examine mechanisms of resistance to immunotherapy, we performed genome-wide sequence analysis of protein coding genes and T-cell receptor (TCR) clonotype analysis, followed by functional assays of autologous T-cell activation of patients who demonstrated initial response to immune checkpoint blockade but ultimately developed progressive disease.Of a cohort of 42 NSCLC patients treated with single-agent PD-1 or combined PD-1 and CTLA4 blockade, we identified all consecutive cases that at the time of the analysis developed acquired resistance (two subjects treated with nivolumab and two with ipilimumab and nivolumab) and where paired tumor specimens were available both before and after therapy. To examine the landscape of genomic alterations and associated neoantigens, we performed whole exome sequencing of tumors from these patients. Pretreatment and postprogression specimens were obtained from the same anatomic location or from sites in close anatomic proximity. We examined multiple immune-related parameters of peptides stemming from somatic alterations using a computational multidimensional neoantigen prediction platform. This approach allowed for identification of peptides within mutated genes that were predicted to be processed and presented by MHC class I proteins and therefore had the potential to elicit an immune response. The algorithm evaluated the binding of mutant peptides (8-11mers) to patient-specific HLA class I alleles and ranked the neoantigens according to MHC binding affinity, antigen processing, and self-similarity. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. While algorithm-based predictions of antigenicity are valuable in narrowing down the large number of peptides capable of being generated by a mutation to a set potential antigenic peptides presented by self-MHC alleles, functional T cell recognition is critical to evaluate immune responsiveness. To this end, we developed a sensitive approach for assessing T-cell response to candidate MANAs (cMANAs) that utilized next-generation sequencing of TCR-Vb CDR3 regions as a measure of T-cell clonality. To evaluate T-cell recognition of eliminated neoantigens, purified peripheral blood T cells from the patients described above were stimulated with autologous peripheral blood mononuclear cells (PBMCs) loaded with cMANA peptides in a ten-day culture system. We subsequently used TCR next-generation sequencing to assess the differential abundance of neoantigen-specific T cell clonotypes in these expanded T cell populations. In order to further investigate the importance of eliminated MANAs, we generated peptides from retained and gained MANAs and assessed their potential to elicit a MANA-specific T-cell expansion. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. These findings indicate that patient-derived T cells recognized the eliminated neoantigens and suggest that these neoantigens were relevant targets for the achievement of initial therapeutic response to checkpoint blockade. Conceptually, neoantigen loss occurs through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations. To evaluate the contribution of these mechanisms to the loss of neoantigens, we analyzed the tumors both before and after therapy using the SCHISM pipeline and incorporating mutation frequency, tumor purity, and copy number variation to infer the fraction of cells containing a specific mutation (mutation cellularity). Consistent with our hypothesis, we observed both mechanisms of neoantigen elimination: loss of truncal changes through genetic events involving chromosomal deletions and loss of heterozygosity (LOH) and loss of subclonal neoantigens either by LOH or through elimination of tumor subclones. Both truncal and subclonal changes were among the eliminated neoantigens that were functionally validated. To evaluate the impact of changes in neoantigen landscape on cytotoxic T-cell receptor repertoire, we analyzed serially collected PBMCs, prior to immunotherapy initiation, at clinical response, and at resistance. We hypothesized that loss of neoantigens would lead to a decrease in clonality of cytotoxic TCR clonotypes, thus reflecting tumor immune evasion at the time of emergence of resistance. We observed peripheral T-cell expansion of a subset of the top 100 most frequent intratumoral clones, with the most frequent clones reaching up to a 44-fold increase in abundance in the blood at the time of response, followed by a decrease to pretreatment levels at the time of resistance. As a comparison, such decreases in TCR frequencies were not observed in a NSCLC patient with durable response to PD-1 blockade and no change in intratumoral TCR frequencies was seen in a NSCLC patient with primary resistance to PD-1 blockade. Taken together, these observations suggest that TCR expansion may be both a useful measure of response to checkpoint blockade and an indicator of acquired therapeutic resistance through neoantigen loss. As immune checkpoint therapy has become standard of care for many cancer types, the development of acquired resistance is being recognized more commonly. Through our comprehensive genomic analyses, we have identified changes in the genomic landscape of tumors during immune checkpoint blockade. These analyses show that emergence of acquired resistance is associated with loss of mutations encoding for putative tumor-specific neoantigens, through both elimination of tumor subclones and chromosomal loss of truncal alterations. Using a new approach to assess neoantigen reactivity by T cells, we found that some of these eliminated mutations indeed encoded peptides recognized by T cells in the peripheral circulation of the respective patients. Given that the antitumor efficacy of checkpoint blockade likely involves release of endogenous T-cell responses to tumor antigens generated by coding mutations, our findings are consistent with a mechanism of acquired resistance to immune checkpoint blockade that involves therapy-induced immune editing of MANAs. Acquisition of somatic resistance mutations is a common mechanism of therapeutic resistance to targeted therapies. However, elimination of genomic alterations and more specifically loss of somatic mutations through subsequent genetic events is uncommon in the context of natural tumor evolution or therapeutic resistance. Elimination of mutation associated antigens by a T-cell-dependent immunoselection process has been proposed as a mechanism of cancer immunoediting in melanoma after adoptive T cell transfer; however, the evolution of neoantigen loss as an escape mechanism under the selective pressure of immune checkpoint blockade in lung cancer has not been previously studied. In addition, we examined a variety of other mechanisms that have been proposed in the development of resistance to immunotherapies. We did not observe any differences in PD-L1 expression in tumor cells between responsive and resistant tumor samples. Likewise, we evaluated known potential genomic mechanisms of immunotherapy resistance; however, there were no new genomic alterations in the CD274 gene encoding for PD-L1, PDCD1 encoding for PD-1, CTLA4, JAK1 or JAK2 genes, in HLA genes, beta 2 microglobulin, or other antigen presentation-associated genes. This work demonstrates for the first time that acquired resistance to anti-PD-1 or anti-PD1/anti-CTLA4 therapy in lung cancer can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of tumor neoantigen reactivity may mitigate the development of acquired resistance. Citation Format: Valsamo Anagnostou, Kellie N. Smith, Patrick M. Forde, Noushin Niknafs, Rohit Bhattacharya, James White, Vilmos Adleff, Jillian Phallen, Neha Wali, Carolyn Hruban, Violeta B. Guthrie, Kristen Rodgers, Jarushka Naidoo, Hyunseok Kang, William Sharfman, Christos Georgiades, Franco Verde, Peter Illei, Qing Ka Li, Edward Gabrielson, Malcolm Brock, Cynthia Zahnow, Stephen B. Baylin, Rob Scharpf, Julie R. Brahmer, Rachel Karchin, Drew M. Pardoll, Victor E. Velculescu. Evolution of neoantigen landscape during immune checkpoint blockade in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr NG01. doi:10.1158/1538-7445.AM2017-NG01

When I was in boot camp, our drill instructor told us that assume makes an ass out of u and me. It was true then, and it is true today. In this instance, assumptions came into play several times, both by the worker and by the companies involved. The good news is that it did not result in a fatality, but that does not relieve the pain and suffering that the employee had to endure. This same type of scenario is likely repeated at many job sites throughout the United States. Multiple contractors, dozens--maybe hundreds--of workers, power system equipment and devices; all of these have to be taken into consideration when performing maintenance activities. It can become a blur. People are people, and people make mistakes. That is why we have OSHA regulations, NFPA 70E, company procedures, policies, etc. Most if not all of us have either been involved in accidents or know people who have been. It's not like it's a secret that people make mistakes, but talk to some and they seem to think only others have that failing. Safety is not about just any one procedure or rule. It's about slowing down, making a plan, and executing that plan. There are plenty of tools available to help us: policies, procedures, codes, standards, federal regulations, and state and local laws. I am not about to say that the worker involved in this incident was not taking safety seriously, but he failed to follow some fundamental safety rules like test-before-touch. If he had taken just that one step, there would be nothing to write about.

Temporary protective grounding serves a critical safety function when working on electrical power systems. Although many field service personnel are well familiar with the requirements of using temporary protective grounds, many others have only a partial understanding of important aspects of it. This paper reviews the more important aspects of the application of temporary protective grounds, including sizing, order of placement and removal, results of misapplication, and common rules for working with temporary protective grounds.

OBJECTIVE Neurosarcoidosis is a rare granulomatous disease that can result in cranial neuropathy, chronic meningitis, and intracranial granuloma formation. Meningeal involvement may cause focal nodular enhancement that can simulate common cranial base tumors. The objective of the current study is to further define the clinical features of neurosarcoidosis in a large cohort of patients, focusing on characteristics relevant to the skull base surgeon. STUDY DESIGN Retrospective series. SETTING Two tertiary academic referral centers. PATIENTS Consecutive patients diagnosed with neurosarcoidosis. INTERVENTION(S) Review of clinical presentation, physical examination, radiologic findings, biopsy results, and laboratory testing. MAIN OUTCOME MEASURES Prevalence and distribution of cranial neuropathy, radiologic features of meningeal enhancement, and patterns of simulated tumors. RESULTS A total of 305 patients met study criteria. The mean age at diagnosis was 47 years and 53% were female. The optic nerve was the most commonly involved cranial nerve, followed by the trigeminal and the facial nerve. Meningeal enhancement was present in 67% of cases with 17% demonstrating focal or multicentric nodular enhancement simulating tumor. The most common locations of inflammatory tumor development included the cavernous sinus, petrous temporal bone, and sphenoid wing; six patients had bilateral internal auditory canal lesions, several mimicking neurofibromatosis type II. CONCLUSION Establishing the diagnosis of neurosarcoidosis remains challenging. Meningeal involvement and cranial neuropathy often mimic other more common conditions. Careful review of patient history and clinical imaging can reveal important clues toward the diagnosis of neurosarcoidosis. The clinician must maintain a high index of suspicion in patients with atypical presentation to avoid misdiagnosis and facilitate early medical treatment.

Electrical power systems maintenance is recognized by NFPA 70E and NFPA 70B as critical to being able to work safely on or near electrical systems and equipment. Even though many upper-level managers think of maintenance as strictly an overhead function, the costs involved with a lack of maintenance can far exceed the outlay required to maintain power systems properly.

Given the lack of urology specific directives for the periprocedural management of anticoagulant and antiplatelet medications, the AUA (American Urological Association) and ICUD (International Consultation on Urological Disease) named an international multidisciplinary panel to develop consensus based recommendations.A systematic literature review was queried by a methodologist for 3 questions. 1) When and in whom can anticoagulant/antiplatelet prophylaxis be stopped in preparation for surgery? 2) What procedures can be safely performed without discontinuing anticoagulant/antiplatelet prophylaxis? 3) What periprocedural strategies can adequately balance the risk of major surgical bleeding vs the risk of major thrombotic event? Hematology and cardiology guidelines, and 79 articles were selected for full review.Multidisciplinary management of anticoagulant/antiplatelet medications for patients with recent thromboembolic events, mechanical cardiac valves, atrial fibrillation and cardiac stents would reduce the high morbidity and mortality of inexpertly discontinuing or modifying these lifesaving therapies. No elective procedures requiring interruption of dual antiplatelet therapies should be performed with a recent bare metal or drug eluting stent. The risk of significant bleeding complications is low for patients who require continuation of aspirin for ureteroscopy, transrectal prostate biopsies, laser prostate outlet procedures and percutaneous renal biopsy. Open extirpative prostate and renal procedures can be performed with a low risk of significant hemorrhage for patients on aspirin and those requiring heparin based bridging strategies. The current literature does not give direction on the timing of the resumption of anticoagulant/antiplatelet prophylaxis other than that it be resumed as soon as the risk of bleeding has decreased.A total of 2,674 nonredundant article abstracts were obtained and assessed for relevance to key questions outlined by the panel. Overall 106 articles were selected for full text review and accepted or rejected based on the relation to the topic, quality of information and key questions. A total of 79 articles were accepted. Reasons for rejection (27 articles) included abstract only (12), insufficient information or unrelated to topic (13) and redundancy (2). We extracted study design, patient population, followup period and results from accepted articles, which serve as the evidence base.

Lung cancer is the most dreaded of all cancers because of the higher mortality rates associated with it worldwide. The various subtypes of lung cancer respond differently to a particular treatment regime, which makes the therapeutic interventions all the more complicated. The concept of cancer stem cells (CSCs) is based primarily on the clinical and experimental observations that indicate the existence of a subpopulation of cells with the capacity to self-renew and differentiate as well as show increased resistance to radiation and chemotherapy. They are considered as the factors responsible for the cases of tumor relapse. The CSCs may have significant role in the development of lung tumorigenesis based on the identification of the CSCs which respond during injury. The properties of multi-potency and self-renewal are shared in common by the lung CSCs with the normal pluripotent stem cells which can be isolated using the similar markers. This review deals with the origin and characteristics of the lung cancer stem cells. The role of different markers used to isolate lung CSCs like CD44, ALDH (aldehyde dehydrogenase), CD133 and ABCG2 (ATP binding cassette sub family G member 2) have been discussed in detail. Analysis of the developmental signaling pathways such as Wnt/β-catenin, Notch, hedgehog in the regulation and maintenance of the lung CSCs have been done. Finally, before targeting the lung CSC biomarkers for potential therapeutics, challenges faced in lung cancer stem cell research need to be taken into account. With the accepted notion that the CSCs are to blame for cancer relapse and drug resistance, targeting them can be an important aspect of lung cancer therapy in the future.

STUDY OBJECTIVE To determine the outcome in patients with sleep complaints and normal polysomnograms (PSG). BACKGROUND PSG is used in the evaluation of persons with sleep complaints. There is a small amount of literature available on patients with sleep complaints and normal PSGs. METHODS PSGs interpreted as normal at the sleep laboratory at Wake Forest University Baptist Medical Center over a 3-year period were considered for inclusion. All PSG requests were screened for appropriateness. Follow-up data were obtained by structured telephone interview (within a period of 5 months of PSG) and chart review. This interview requested: (1) main sleep complaint, (2) diagnosis given by the ordering physician, (3) treatment for the sleep complaint, (4) further studies, and (5) overall satisfaction. RESULTS Forty-six patients were identified with a normal PSG, and 27 (58%) were successfully contacted for the telephone interview. Of these 27 patients, 8 were men and 19 were women. Fifty-two percent of patients were not given a diagnosis by the referring physician after their normal PSG to explain their sleep symptoms. Eight patients (29%) were offered treatment for their sleep complaints despite their normal PSGs. Twenty patients (74%) reported no change in their sleep quality since the PSG. Most patients (66%) did not find the PSG helpful. CONCLUSIONS The majority of patients with sleep complaints and normal PSG were not given a clear diagnosis by the referring physician, and sleep complaints persist after a normal PSG. Further evaluation by a sleep specialist may be appropriate.

Este trabalho analisa alguns aspectos da fotodegradação heterogênea do polipropileno (PP) e compósitos de PP com talco. Amostras moldadas por compressão e por injeção foram expostas à radiação ultravioleta em laboratório por períodos de até 48 semanas. Procedimentos sistemáticos de exposição e coleta de material para análise foram adotados visando-se minimizar as variabilidades inerentes deste estudo. As amostras foram caracterizadas por cromatografia de permeação em gel, espectroscopia de infravermelho, calorimetria diferencial de varredura, difração de raios-X, comportamento mecânico e microscopia ótica e eletrônica. As heterogeneidades na fotodegradação do PP podem estar relacionadas com três principais aspectos: (i) natureza semi-cristalina do polímero; (ii) perfil de degradação; (iii) variações estruturais em diferentes posições nos corpos de prova. As consequências da fotodegradação heterogênea foram discutidas, enfatizando-se os efeitos nas propriedades físicas.In this paper some characteristics of the heterogeneous photodegradation of polypropylene (PP) and PP containing talc were analyzed. Samples produced by injection and compression moulding were exposed to ultraviolet radiation in the laboratory for periods of up to 48 weeks. Systematic procedures for exposure and sampling were adopted in an attempt to minimize the inherent variabilities of this type of study. The specimens were characterized by gel permeation chromatography, infrared spectroscopy, differential scanning calorimetry, X-ray diffraction, mechanical properties and light and electron microscopy. The heterogeneities in PP photodegradation can be related to three main aspects: (i) the semi-crystalline nature of PP; (ii) the depth-profile of degradation; (iii) the structural variations in different positions along the test bar. The consequences of these heterogeneities were discussed with emphasis on the effects on physical properties.

The aim of this study was to investigate the effect of gallic acid (GA) on liver fibrosis induced by carbon tetrachloride (CCl4). Male BALB/c mice were randomly divided into four groups: normal control group (group A), CCl4-induced liver injury control group (group B), and CCl4 induction with GA of low dose (5 mg/kg) and high dose (15 mg/kg) treatment group (group C and group D). GA was intra-gastric given for mice once a day after 2 weeks of CCl4 induction. Animals were killed at the eighth week. Degrees of fibrosis and collagen percentage were measured. Hyaluronic acid (HA), type IV collagen (cIV), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γ-GT) were determined. Expression of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) mRNA levels were examined by RT-PCR. Western blotting was carried out to evaluate the changes of MMP-2 protein. HE and VG stainings showed GA in a dose-dependent manner improved significantly the fibrosis condition in CCl4-injured mice (P < 0.05 or P < 0.01). Also, the concentrations of HA, cIV, and MDA, as well as the serum levels of ALT, AST, and γ-GT were markedly reduced by GA (P < 0.05 or P < 0.01), and decreases in MMP-2, TIMP-1 mRNA, and MMP-2 protein were observed as well (P < 0.05 or P < 0.01). GA could exert protective effect on liver injury and reduce liver fibrosis induced by CCl4 in mice, which might be through the inhibition of hepatic stellate cell activity.

Background Lymphatic filariasis is a neglected tropical disease afflicting more than 120 million people, while another 1.3 billion people are at risk of infection. The nematode worm Brugia malayi is one of the causative agents of the disease and exists in a mutualistic symbiosis with Wolbachia bacteria. Since extensive lateral gene transfer occurs frequently between Wolbachia and its hosts, we sought to measure the extent of such LGT in B. malayi by whole genome sequencing of Wolbachia-depleted worms. Results A considerable fraction (at least 115.4-kbp, or 10.6%) of the 1.08-Mbp Wolbachia w Bm genome has been transferred to its nematode host and retains high levels of similarity, including 227 w Bm genes and gene fragments. Complete open reading frames were transferred for 32 of these genes, meaning they have the potential to produce functional proteins. Moreover, four transfers have evidence of life stage-specific regulation of transcription at levels similar to other nematode transcripts, strengthening the possibility that they are functional. Conclusions There is extensive and ongoing transfer of Wolbachia DNA to the worm genome and some transfers are transcribed in a stage-specific manner at biologically relevant levels.

Background: guidelines have changed in relation to the indication of antiplatelet therapy for the primary and secondary prevention of stroke. Of interest is how the proportion of patients who had or had not taken antiplatelet agents prior to an incident stroke has changed over time, whether the type of antiplatelet agents used has altered and whether prior antiplatelet use is associated with a particular ischaemic stroke subtype. Methods: a stroke register was retrospectively examined. All ischaemic stroke patients admitted between January 2004 and March 2011 to a single University Hospital with a catchment population of 750,000 were included. We excluded those who were on anticoagulants prior to the ischaemic stroke. Results: a total of 4,307 ischaemic stroke patients [male 47.5%, mean age 77.6 (SD 11.7) years] were included. Of them, 54.7% (SD 2.2%) were not on any antiplatelet therapy prior to their incident stroke. The type and pattern of antiplatelet use prior to stroke did not change significantly during the 7-year study period, and there were no statistically significant differences between different ischaemic stroke subtypes with regards to prior antiplatelet use. Conclusions: our findings highlight the requirement to improve currently available risk prediction scores as well as the potential clinical impact of antiplatelet resistance within the at risk population who are already on antiplatelets. These findings also indicate that targeting of multiple risk factors may be very important in stroke prevention.

Background: Research to understand and control microbiological risks associated with the consumption of fresh fruits and vegetables has examined many environments in the farm to fork continuum. An important data gap however, that remains poorly studied is the baseline description of microflora that may be associated with plant anatomy either endemically or in response to environmental pressures. Specific anatomical niches of plants may contribute to persistence of human pathogens in agricultural environments in ways we have yet to describe. Tomatoes have been implicated in outbreaks of Salmonella at least 17 times during the years spanning 1990 to 2010. Our research seeks to provide a baseline description of the tomato microbiome and possibly identify whether or not there is something distinctive about tomatoes or their growing ecology that contributes to persistence of Salmonella in this important food crop. Results: DNA was recovered from washes of epiphytic surfaces of tomato anatomical organs; leaves, stems, roots, flowers and fruits of Solanum lycopersicum (BHN602), grown at a site in close proximity to commercial farms previously implicated in tomato-Salmonella outbreaks. DNA was amplified for targeted 16S and 18S rRNA genes and sheared for shotgun metagenomic sequencing. Amplicons and metagenomes were used to describe "native" bacterial microflora for diverse anatomical parts of Virginia-grown tomatoes. Conclusions: Distinct groupings of microbial communities were associated with different tomato plant organs and a gradient of compositional similarity could be correlated to the distance of a given plant part from the soil. Unique bacterial phylotypes (at 95% identity) were associated with fruits and flowers of tomato plants. These include Microvirga, Pseudomonas, Sphingomonas, Brachybacterium, Rhizobiales, Paracocccus, Chryseomonas and Microbacterium. The most frequently observed bacterial taxa across aerial plant regions were Pseudomonas and Xanthomonas. Dominant fungal taxa that could be identified to genus with 18S amplicons included Hypocrea, Aureobasidium and Cryptococcus. No definitive presence of Salmonella could be confirmed in any of the plant samples, although 16S sequences suggested that closely related genera were present on leaves, fruits and roots. © 2013 Ottesen et al.; licensee BioMed Central Ltd.

This paper reviews some of the more common electrical safety myths and misconceptions field electrical workers often have. Many of these misconceptions continue in spite of training and education. Some are handed down from worker to worker, similar to tribal knowledge. The real danger from these misconceptions is that they impede the truth and cause electrical workers to misjudge hazards and risks.

Summary Purpose Although topiramate is widely prescribed for epilepsy and migraine, there is no intravenous product. We have developed an injectable topiramate formulation in which the drug is solubilized in a cyclodextrin matrix, Captisol® (Ligand Pharmaceuticals, Inc., La Jolla, CA). Our long-term goal is to evaluate intravenous topiramate for the treatment of neonatal seizures. Prior to studies in newborns, we carried out an investigation of injectable topiramate's safety and pharmacokinetics in adult patients. Methods Twenty adult volunteers with epilepsy or migraine on stable, on maintenance topiramate therapy were given 25 mg of a stable-labeled intravenous topiramate over 10 min, followed by their usual oral doses. Vital signs were taken, electrocardiography studies (ECGs) were recorded, and the infusion sites were periodically examined prior to and up to 24 h after dosing. Blood samples were collected prior to administration and serially for 96 h thereafter. Plasma concentrations of both stable-labeled and regular topiramate were measured using liquid chromatography-mass spectrometry (LC-MS). Concentration-time data were analyzed using a noncompartmental approach with WinNonlin 5.2 (Pharsight Corporation, Mountain View, CA, U.S.A.). Key Findings Seven patients experienced one or more of the following minor adverse events including nausea and vomiting (1), tingling around the lips (1), paresthesia in the arms and legs (1), and a mild vasovagal response with intravenous catheter placement (1). Included in the adverse events were four patients with epilepsy who had seizures consistent with their histories. There were no changes in heart rate, blood pressure, or ECG results, and there were no infusion site reactions. Pharmacokinetic parameters (mean ± standard deviation [SD]) determined following the intravenous dose included absolute bioavailability: 110 ± 16%, distribution volume: 0.79 ± 0.22 L/kg, clearance: 2.03 ± 1.07 L/h, and elimination half-life: 27.6 ± 9.7 h. Distribution volume, half-life, and clearance were significantly altered by enzyme-inducing drugs. Significance A single 25-mg dose of intravenous topiramate caused minimal infusion site or systemic adverse effects in patients taking oral topiramate. Pharmacokinetic results show that oral topiramate is completely absorbed and that its steady-state elimination half-life is longer than previously assumed, which permits once or twice daily dosing even in the presence of enzyme-inducing drugs. The information from this study can inform the design of subsequent studies in adults, older children, and newborns, including controlled clinical trials intended to determine the efficacy and safety of intravenous topiramate for neonatal seizures.

There are 10× more bacterial cells in our bodies from the microbiome than human cells. Viral DNA is known to integrate in the human genome, but the integration of bacterial DNA has not been described. Using publicly available sequence data from the human genome project, the 1000 Genomes Project, and The Cancer Genome Atlas (TCGA), we examined bacterial DNA integration into the human somatic genome. Here we present evidence that bacterial DNA integrates into the human somatic genome through an RNA intermediate, and that such integrations are detected more frequently in (a) tumors than normal samples, (b) RNA than DNA samples, and (c) the mitochondrial genome than the nuclear genome. Hundreds of thousands of paired reads support random integration of Acinetobacter-like DNA in the human mitochondrial genome in acute myeloid leukemia samples. Numerous read pairs across multiple stomach adenocarcinoma samples support specific integration of Pseudomonas-like DNA in the 5′-UTR and 3′-UTR of four proto-oncogenes that are up-regulated in their transcription, consistent with conversion to an oncogene. These data support our hypothesis that bacterial integrations occur in the human somatic genome and may play a role in carcinogenesis. We anticipate that the application of our approach to additional cancer genome projects will lead to the more frequent detection of bacterial DNA integrations in tumors that are in close proximity to the human microbiome., Author Summary There are 10× more bacterial cells in the human body than there are human cells that are part of the human microbiome. Many of those bacteria are in constant, intimate contact with human cells. We sought to establish if bacterial cells insert their own DNA into the human genome. Such random mutations could cause disease in the same manner that mutagens like UV rays from the sun or chemicals in cigarettes induce mutations. We detected the integration of bacterial DNA in the human genome more readily in tumors than normal samples. In particular, extensive amounts of DNA with similarity to Acinetobacter DNA were fused to human mitochondrial DNA in acute myeloid leukemia samples. We also identified specific integrations of DNA with similarity to Pseudomonas DNA near the untranslated regulatory regions of four proto-oncogenes. This supports our hypothesis that bacterial integrations occur in the human somatic genome that may potentially play a role in carcinogenesis. Further study in this area may provide new avenues for cancer prevention.

Somatic, nonsynonymous genetic alterations present in cancer can lead to the formation of novel protein sequences and thus production of immunogenic “non-self” neoantigens. Those neoantigens with sufficient expression will be processed and presented on MHC molecules, subsequently inducing a tumor-specific T cell response. Emerging data from clinical studies suggest that neoantigen load and composition is associated with the efficacy of some immunotherapies. As neoantigens play central a role in the cancer-immunity cycle, it is critical to identify the most potent immunogenic neoantigens effectively and accurately. Here we have leveraged highly accurate cancer whole exome (WES) analyses from FFPE tumor tissue with a state-of-the-art analysis protocol to identify and prioritize candidate neoantigens most likely to promote an immune response. ImmunoSelect-R utilizes somatic variants from WES to ensure detection of true somatic peptides and minimize false positives, and provides accurate HLA typing from whole exome sequencing data with >99.9% sensitivity and specificity. When applied to a set of experimentally validated neoantigens, ImmunoSelect correctly classified 18 out of 19 as strong neoantigen candidates, suggesting a sensitivity of greater than 90%. Moreover, in a small set of 10 patients, ImmunoSelect consistently ranked experimentally validated neoantigens within top 20% of all neoantigen candidates derived from whole exome sequencing. In summary, ImmunoSelect is able to identify and prioritize candidate neoantigens from cancer exome sequencing results effectively and accurately, enabling personalized cancer vaccine development, adoptive T-cell transfer, and prediction of response to checkpoint inhibitors Citation Format: James White, John Simmons, Sam Angiuoli, Mark Sausen, Sian Jones, Lisa Kann, Manish Shukla, Maria Sevdali, Victor Velculescu, Luis Diaz, Theresa Zhang. Accurate identification and prioritization of candidate neoantigens from cancer exome sequencing [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A039.

Colorectal cancer (CRC) continues to be the second leading cause of cancer-related deaths globally. While multiple environmental factors are associated with incidence of CRC, multiple studies have reported that CRC is frequently linked with an altered colonic microbiota both in terms of taxonomic composition as well as structural organization. However, differences in study design and analysis methodologies among CRC microbiome profiling studies have generated inconsistent findings. To identify bacterial taxa consistently associated with CRC, we applied a uniform microbiome analysis methodology to sequence datasets followed by meta-analysis. Raw 16S rRNA amplicon sequence datasets from 12 studies including two new Malaysian cohorts were collected and analyzed using the Resphera Insight high-resolution taxonomic assignment protocol. Taxonomic abundances within each dataset were then assessed for significant enrichment or depletion in colorectal carcinoma samples versus controls. Alpha diversity and differences in predicted functional capacity were assessed utilizing QIIME and PICRUSt, respectively. Several bacterial species were consistently enriched in colorectal carcinoma samples relative to healthy controls including Fusobacterium nucleatum, Fusobacterium necrophorum, Leptotrichia trevisanii, Bacteroides fragilis, Parvimonas micra, Peptostreptococcus stomatis, and Gemella morbillorum. In contrast to other studies that have generally described Fusobacteria as enriched in CRC relative to controls, we identified multiple fusobacterial members that were often not strongly linked with CRC including Fusobacterium varium and Cetobacterium somerae. Evaluating functional predictions inferred from taxonomic composition, we observed consistent enrichment of specific KEGG categories in CRC including glycosyltransferases and carbohydrate digestion and absorption. Applying a uniform high-resolution analysis protocol for microbiome profiling enabled detection of specific species that were strongly associated with CRC across multiple studies. These findings demonstrate that higher level analyses (e.g. those at the genus or family level) may be insufficient for detection of indicator taxa in some populations. Citation Format: James R. White, Julia Drewes, Cynthia L. Sears. High-resolution microbiome profiling and meta-analysis yields insight into microbial consortia associated with colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 844.

Somatic, nonsynonymous genetic alterations present in cancer can lead to the formation of novel protein sequences and thus production of immunogenic “non-self” neoantigens. Some of those neoantigens will be processed, presented on MHC molecules, and induce tumor-specific T cell responses. Because neoantigens play central roles in the cancer-immunity cycle, it is critical to identify the most potent immunogenic neoantigens effectively and accurately. Combining PGDx's highly accurate cancer exome analyses (CancerXome™) with in silico neoantigen prediction, we have launched ImmunoSelect-R™ that identifies and prioritizes the most relevant mutation-derived neoantigens. To ensure detection of true somatic mutations and prevent false positive mutations from confounding neoantigen identification, ImmunoSelect utilizes CancerXome that delivers unparalleled cancer whole exome sequencing accuracy, achieving 95% sensitivity and 97% positive predictive value at 10% mutant allele frequency with 150x coverage. ImmunoSelect also provides accurate HLA typing from whole exome sequencing with >99.9% sensitivity and specificity. Once exome-based mutations and novel open-reading-frames are identified and HLA genotypes defined, ImmunoSelect utilizes state of art bioinformatics pipelines for prediction and prioritization of the most relevant neoantigens. When applied to a set of experimentally validated neoantigens, ImmunoSelect identified 18 out of 19 of them as being strong neoantigen candidates, suggesting a sensitivity of greater than 90%. Moreover, ImmunoSelect consistently ranked experimentally validated neoantigens within top 20% of all neoantigen candidates derived from whole exome sequencing. In summary, ImmunoSelect is able to identify and prioritize candidate neoantigens from cancer exome sequencing results effectively and accurately, enabling personalized cancer vaccine development, adoptive T-cell transfer, and prediction of response to checkpoint inhibitors Citation Format: James White, Sam Angiuoli, Mark Sausen, Sian Jones, Lisa Kann, Manish Shukla, Maria Sevdali, Victor Velculescu, Luis Diaz, Theresa Zhang. Identify and prioritize candidate neoantigens from cancer exome sequencing with unmatched accuracy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 528.

Background Enriching environmental samples to increase the probability of detection has been standard practice throughout the history of microbiology. However, by its very nature, the process of enrichment creates a biased sample that may have unintended consequences for surveillance or resolving a pathogenic outbreak. With the advent of next-generation sequencing and metagenomic approaches, the possibility now exists to quantify enrichment bias at an unprecedented taxonomic breadth. Findings We investigated differences in taxonomic profiles of three enriched and unenriched tomato phyllosphere samples taken from three different tomato fields (n = 18). 16S rRNA gene meteganomes were created for each of the 18 samples using 454/Roche’s pyrosequencing platform, resulting in a total of 165,259 sequences. Significantly different taxonomic profiles and abundances at a number of taxonomic levels were observed between the two treatments. Although as many as 28 putative Salmonella sequences were detected in enriched samples, there was no significant difference in the abundance of Salmonella between enriched and unenriched treatments. Conclusions Our results illustrate that the process of enriching greatly alters the taxonomic profile of an environmental sample beyond that of the target organism. We also found evidence suggesting that enrichment may not increase the probability of detecting a target. In conclusion, our results further emphasize the need to develop metagenomics as a validated culture independent method for pathogen detection.

This paper looks at the most common issues causing circuit breaker malfunctions. Power circuit breakers protect electrical power systems from damage and people from injury or death if a fault were to occur during maintenance or operation of the system. If the circuit breakers are not maintained they cannot be depended upon to function in accordance with the manufacturer's specifications. This paper reviews findings of Shermco's Circuit Breaker Shop while working on customer's circuit breakers.

Celiac disease (CD) is a unique autoimmune disorder in which the genetic factors (DQ2/DQ8) and the environmental trigger (gluten) are known and necessary but not sufficient for its development. Other environmental components contributing to CD are poorly understood. Studies suggest that aspects of gluten intake might influence the risk of CD occurrence and timing of its onset, i.e., the amount and quality of ingested gluten, together with the pattern of infant feeding and the age at which gluten is introduced in the diet. In this study, we hypothesize that the intestinal microbiota as a whole rather than specific infections dictates the switch from tolerance to immune response in genetically susceptible individuals. Using a sample of infants genetically at risk of CD, we characterized the longitudinal changes in the microbial communities that colonize infants from birth to 24 months and the impact of two patterns of gluten introduction (early vs. late) on the gut microbiota and metabolome, and the switch from gluten tolerance to immune response, including onset of CD autoimmunity. We show that infants genetically susceptible to CD who are exposed to gluten early mount an immune response against gluten and develop CD autoimmunity more frequently than at-risk infants in which gluten exposure is delayed until 12 months of age. The data, while derived from a relatively small number of subjects, suggest differences between the developing microbiota of infants with genetic predisposition for CD and the microbiota from infants with a non-selected genetic background, with an overall lack of bacteria of the phylum Bacteriodetes along with a high abundance of Firmicutes and microbiota that do not resemble that of adults even at 2 years of age. Furthermore, metabolomics analysis reveals potential biomarkers for the prediction of CD. This study constitutes a definite proof-of-principle that these combined genomic and metabolomic approaches will be key to deciphering the role of the gut microbiota on CD onset.

Carbamazepine is a widely prescribed antiepileptic drug. Owing to the lack of an intravenous formulation, its absolute bioavailability, absolute clearance, and half-life in patients at steady state have not been determined. We developed an intravenous, stable-labeled (SL) formulation in order to characterize carbamazepine pharmacokinetics in patients. Ninety-two patients received a 100-mg infusion of SL-carbamazepine as part of their morning dose. Blood samples were collected up to 96 hours after drug administration. Plasma drug concentrations were measured with liquid chromatography-mass spectrometry, and concentration-time data were analyzed using a noncompartmental approach. Absolute clearance (l/hr/kg) was significantly lower in men (0.039 ± 0.017) than in women (0.049 ± 0.018; P = 0.007) and in African Americans (0.039 ± 0.017) when compared with Caucasians (0.048 ± 0.018; P = 0.019). Half-life was significantly longer in men than in women as well as in African Americans as compared with Caucasians. The absolute bioavailability was 0.78. Sex and racial differences in clearance may contribute to variable dosing requirements and clinical response.

In 2010 OSHA made changes to its regulations concerning cranes and derricks. These changes will have a significant impact on the training of crane and derrick operators and on how these are operated. As with any major change to an OSHA regulation, there will probably be costs associated with compliance to these changes

Author Manuscript date: 2013 February 05., Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin and vagina. Much of this diversity remains unexplained, although diet, environment, host genetics and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human-associated microbial communities, the Human Microbiome Project has analysed the largest cohort and set of distinct, clinically relevant body habitats so far. We found the diversity and abundance of each habitat’s signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and among individuals. The project encountered an estimated 81–99% of the genera, enzyme families and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology and translational applications of the human microbiome.

The CloVR-16S pipeline employs several well-known phylogenetic tools and protocols for the analysis of 16S rRNA sequence datasets: A) Mothur – a C++-based software package used for clustering 16S rRNA sequences into operational taxonomic units (OTUs). Mothur creates OTUs using a matrix that describes pairwise distances between representative aligned sequences and subsequently estimates within-sample diversity (alpha diversity); B) The Ribosomal Database (RDP) naïve Bayesian classifier assigns each 16S sequence to a reference taxonomy with associated empirical probabilities based on oligonucleotide frequencies; C) Qiime – a python-based workflow package, allowing for sequence processing and phylogenetic analysis using different methods including phylogenetic distance (UniFrac) for within-(alpha diversity) and between-(beta diversity) sample analysis; D) Metastats and custom R scripts used to generate additional statistical and graphical evaluations. Though some of the different protocols used in CloVR-16S overlap in purpose (e.g. OTU clustering), the end-user benefits from their overall complementary nature as they focus on different aspects of the phylogenetic analysis. CloVR-16S accepts as input raw multiplex 454-pyrosequencer output, i.e. pooled pyro-tagged sequences from multiple samples, or alternatively, pre-processed sequences from multiple samples in separate files. This protocol is available in CloVR beta versions 0.5 and 0.6.

The CloVR-Metagenomics pipeline employs several well-known tools and protocols for the analysis of metagenomic whole-genome shotgun (WGS) sequence datasets: A) UCLUST – a C++-based software package for clustering redundant DNA sequences and removing artificial 454 replicates; B) BLASTX and BLASTN for functional and taxonomic assignment of sequences, respectively; C) Metastats and custom R scripts to generate additional statistical and graphical evaluation. The CloVR-Metagenomics pipeline accepts as input multiple fasta files (1 sample per file) and a corresponding tab-delimited metadata file that specifies features associated with the samples, which are used for comparative analysis. This protocol is available in CloVR beta version 0.5 and 0.6.

The CloVR-Microbe pipeline performs the basic processing and analysis steps required for standard microbial single-genome sequencing projects: A) Whole-genome shotgun sequence assembly; B) Identification of protein and RNA-coding genes; and C) Functional gene annotation. B) and C) are based on the IGS Annotation Engine (http://ae.igs.umaryland.edu/), which is described elsewhere (K Galens et al. submitted). The assembly component of CloVR- Microbe can be executed independently from the gene identification and annotation components. Alternatively, pre-assembled sequence contigs can be used to perform gene identifications and annotations. The pipeline input may consist of unassembled raw sequence reads from the Sanger, Roche/454 GS FLX or Illumina GAII or HiSeq sequencing platforms or of combinations of Sanger and Roche/454 sequence data. The pipeline output consists of results and summary files generated during the different pipeline steps. Annotated sequence files are generated that are compatible with common genome browser tools and can be submitted to the GenBank repository at NCBI. This protocol is available in CloVR beta versions 0.5 and 0.6.

This paper is an overview of significant changes that were acted upon during the October NFPA 70E ROC meeting conducted in Savannah, GA. This paper does not attempt to cover all changes that were made to the 70E.

The CloVR-16S pipeline employs several well-known phylogenetic tools and protocols for the analysis of 16S rRNA sequence datasets:A) Mothur [1] – a C++ - based software package used for clustering 16SrRNA sequences into operational taxonomic units (OTUs). Mothur creates OTUs using a matrix that describes pairwise distances between representative aligned sequences and subsequently estimates within-sample diversity (alpha diversity);B)The Ribosomal Database (RDP) naive Bayesian classifier [2] assigns each 16S sequence to a reference taxonomy with associated empirical probabilities based on oligonucleotide frequencies;C) Qiime [3] – a python-based workflow package, allowing for sequence processing and phylogenetic analysis using different methods including phylogenetic distance (UniFrac [4]) for within- (alpha diversity) and between- (beta diversity) sample analysis;D) Metastats [5] and custom R scripts used to generate additional statistical and graphical evaluations.Though some of the different protocols used in CloVR-16S overlap in purpose (e.g. OTU clustering), the end-user benefits from their overall complementary nature as they focus on different aspects of the phylogenetic analysis. CloVR-16S accepts as input raw multiplex 454-pyrosequencer output, i.e. pooled pyrotagged sequences from multiple samples, or alternatively, pre-processed sequences from multiple samples in separate files. This protocol is available in CloVR beta versions 0.5 and 0.6.

The CloVR-16S pipeline employs several well-known phylogenetic tools and protocols for the analysis of 16S rRNA sequence datasets: A) Mothur [1] – a C++ - based software package used for clustering 16SrRNA sequences into operational taxonomic units (OTUs). Mothur creates OTUs using a matrix that describes pairwise distances between representative aligned sequences and subsequently estimates within-sample diversity (alpha diversity); B)The Ribosomal Database (RDP) naive Bayesian classifier [2] assigns each 16S sequence to a reference taxonomy with associated empirical probabilities based on oligonucleotide frequencies; C) Qiime [3] – a python-based workflow package, allowing for sequence processing and phylogenetic analysis using different methods including phylogenetic distance (UniFrac [4]) for within- (alpha diversity) and between- (beta diversity) sample analysis; D) Metastats [5] and custom R scripts used to generate additional statistical and graphical evaluations. Though some of the different protocols used in CloVR-16S overlap in purpose (e.g. OTU clustering), the end-user benefits from their overall complementary nature as they focus on different aspects of the phylogenetic analysis. CloVR-16S accepts as input raw multiplex 454-pyrosequencer output, i.e. pooled pyrotagged sequences from multiple samples, or alternatively, pre-processed sequences from multiple samples in separate files. This protocol is available in CloVR beta versions 0.5 and 0.6.

Background Tomato (Solanum lycopersicum) consumption has been one of the most common causes of produce-associated salmonellosis in the United States. Contamination may originate from animal waste, insects, soil or water. Current guidelines for fresh tomato production recommend the use of potable water for applications coming in direct contact with the fruit, but due to high demand, water from other sources is frequently used. We sought to describe the overall bacterial diversity on the surface of tomato fruit and the effect of two different water sources (ground and surface water) when used for direct crop applications by generating a 454-pyrosequencing 16S rRNA dataset of these different environments. This study represents the first in depth characterization of bacterial communities in the tomato fruit surface and the water sources commonly used in commercial vegetable production. Results The two water sources tested had a significantly different bacterial composition. Proteobacteria was predominant in groundwater samples, whereas in the significantly more diverse surface water, abundant phyla also included Firmicutes, Actinobacteria and Verrucomicrobia. The fruit surface bacterial communities on tomatoes sprayed with both water sources could not be differentiated using various statistical methods. Both fruit surface environments had a high representation of Gammaproteobacteria, and within this class the genera Pantoea and Enterobacter were the most abundant. Conclusions Despite the major differences observed in the bacterial composition of ground and surface water, the season long use of these very different water sources did not have a significant impact on the bacterial composition of the tomato fruit surface. This study has provided the first next-generation sequencing database describing the bacterial communities living in the fruit surface of a tomato crop under two different spray water regimes, and therefore represents an important step forward towards the development of science-based metrics for Good Agricultural Practices.

The Brazos-Trinity Basin on the slope of the Gulf of Mexico passive margin was drilled during Integrated Ocean Drilling Progam Expedition 308. The buried anaerobic sediments of this basin are largely organic-poor and have few microbial inhabitants compared with the organic-rich sediments with high cell counts from the Peru Margin that were drilled during Ocean Drilling Program Leg 201. Nucleic acids were extracted from Brazos-Trinity Basin sediments and were subjected to whole-genome amplification and pyrosequencing. A comparison of the Brazos-Trinity Basin metagenome, consisting of 105 Mbp, and the existing Peru Margin metagenome revealed trends linking gene content, phylogenetic content, geological location and geochemical regime. The major microbial groups (Proteobacteria, Firmicutes, Euryarchaeota and Chloroflexi) occur consistently throughout all samples, yet their shifting abundances allow for discrimination between samples. The cluster of orthologous groups category abundances for some classes of genes are correlated with geochemical factors, such as the level of ammonia. Here we describe the sediment metagenome from the oligotrophic Brazos-Trinity Basin (Site 1320) and show similarities and differences with the dataset from the Pacific Peru Margin (Site 1229) and other pyrosequenced datasets. The microbial community found at Integrated Ocean Drilling Program Site 1320 likely represents the subsurface microbial inhabitants of turbiditic slopes that lack substantial upwelling.

The relationship between tunicates and the uncultivated cyanobacterium Prochloron didemni has long provided a model symbiosis. P. didemni is required for survival of animals such as Lissoclinum patella and also makes secondary metabolites of pharmaceutical interest. Here, we present the metagenomes, chemistry, and microbiomes of four related L. patella tunicate samples from a wide geographical range of the tropical Pacific. The remarkably similar P. didemni genomes are the most complex so far assembled from uncultivated organisms. Although P. didemni has not been stably cultivated and comprises a single strain in each sample, a complete set of metabolic genes indicates that the bacteria are likely capable of reproducing outside the host. The sequences reveal notable peculiarities of the photosynthetic apparatus and explain the basis of nutrient exchange underlying the symbiosis. P. didemni likely profoundly influences the lipid composition of the animals by synthesizing sterols and an unusual lipid with biofuel potential. In addition, L. patella also harbors a great variety of other bacterial groups that contribute nutritional and secondary metabolic products to the symbiosis. These bacteria possess an enormous genetic potential to synthesize new secondary metabolites. For example, an antitumor candidate molecule, patellazole, is not encoded in the genome of Prochloron and was linked to other bacteria from the microbiome. This study unveils the complex L. patella microbiome and its impact on primary and secondary metabolism, revealing a remarkable versatility in creating and exchanging small molecules.

BackgroundThe widespread popularity of genomic applications is threatened by the "bioinformatics bottleneck" resulting from uncertainty about the cost and infrastructure needed to meet increasing demands for next-generation sequence analysis. Cloud computing services have been discussed as potential new bioinformatics support systems but have not been evaluated thoroughly.ResultsWe present benchmark costs and runtimes for common microbial genomics applications, including 16S rRNA analysis, microbial whole-genome shotgun (WGS) sequence assembly and annotation, WGS metagenomics and large-scale BLAST. Sequence dataset types and sizes were selected to correspond to outputs typically generated by small- to midsize facilities equipped with 454 and Illumina platforms, except for WGS metagenomics where sampling of Illumina data was used. Automated analysis pipelines, as implemented in the CloVR virtual machine, were used in order to guarantee transparency, reproducibility and portability across different operating systems, including the commercial Amazon Elastic Compute Cloud (EC2), which was used to attach real dollar costs to each analysis type. We found considerable differences in computational requirements, runtimes and costs associated with different microbial genomics applications. While all 16S analyses completed on a single-CPU desktop in under three hours, microbial genome and metagenome analyses utilized multi-CPU support of up to 120 CPUs on Amazon EC2, where each analysis completed in under 24 hours for less than $60. Representative datasets were used to estimate maximum data throughput on different cluster sizes and to compare costs between EC2 and comparable local grid servers.ConclusionsAlthough bioinformatics requirements for microbial genomics depend on dataset characteristics and the analysis protocols applied, our results suggests that smaller sequencing facilities (up to three Roche/454 or one Illumina GAIIx sequencer) invested in 16S rRNA amplicon sequencing, microbial single-genome and metagenomics WGS projects can achieve cost-efficient bioinformatics support using CloVR in combination with Amazon EC2 as an alternative to local computing centers.

AC impedance methods have been utilized to explore surface conduction mechanisms on printed circuit boards (PCBs) containing various types of solder flux contaminants. Residues from water-soluble, rosin-based, and no-clean fluxes were analyzed and evaluated for their potential impact on reliability. Impedance data for intentionally contaminated PCBs having several circuit line geometries were obtained at different relative humidities. An equivalent circuit model is presented that fits the data obtained. It is used to evaluate and distinguish among ohmic, kinetic, and diffusion effects and to predict the environmental conditions that may be detrimental for various line geometries.

The stable benzyl azide (I) containing a boronate ester allows regioselective 1,3-dipolar cycloaddition with terminal olefins followed by a Cu-catalyzed Suzuki coupling of the resulting triazoles such as (IIIa).

Background The field of neural prosthetics aims to develop prosthetic limbs with a brain-computer interface (BCI) through which neural activity is decoded into movements. A natural extension of current research is the incorporation of neural activity from multiple modalities to more accurately estimate the user's intent. The challenge remains how to appropriately combine this information in real-time for a neural prosthetic device. Methodology/Principal Findings Here we propose a framework based on decision fusion, i.e., fusing predictions from several single-modality decoders to produce a more accurate device state estimate. We examine two algorithms for continuous variable decision fusion: the Kalman filter and artificial neural networks (ANNs). Using simulated cortical neural spike signals, we implemented several successful individual neural decoding algorithms, and tested the capabilities of each fusion method in the context of decoding 2-dimensional endpoint trajectories of a neural prosthetic arm. Extensively testing these methods on random trajectories, we find that on average both the Kalman filter and ANNs successfully fuse the individual decoder estimates to produce more accurate predictions. Conclusions Our results reveal that a fusion-based approach has the potential to improve prediction accuracy over individual decoders of varying quality, and we hope that this work will encourage multimodal neural prosthetics experiments in the future.

The human body plays host to thousands of bacterial species in a variety of ecosystems. Until recently, microbial communities have been impossible to investigate thoroughly, as the vast majority of bacteria cannot be cultured through laboratory techniques. New technologies (e.g. high-throughput sequencing, 16S rRNA surveys) allow us to deeply sample the genetic content of a microbial environment in order to estimate its overall composition and functional capacity. Recent studies in this context have revealed that human obesity has a microbial component: obese gut microbiomes are distinct from the lean population. This result indicates potential therapeutic approaches to treating obesity by manipulating gut microflora. However, our limited knowledge of the microbial interactions in the gut hinders our ability to design future experiments or effective treatments. Using 16S rRNA time-series sequence data from obese individuals on a one-year diet, we employ mathematical modeling to study microbial population dynamics in the human gut. The model indicates several interspecific interactions in this microbial community, and we investigate the impact of prebiotic and probiotic therapies for obesity through simulation. To our knowledge, this is the first model to quantitatively describe the in vivo microbial dynamics of the human gut using sequence data, and we hope this application illustrates the insight mathematical modeling can bring to the field of metagenomics.

Background: Glucagon-like peptide 1 (GLP-1) and pancreatic polypeptide (PP) are intestinal hormones that are involved in the post-prandial satiety response. We sought to assess meal-related changes in these hormones in young children and determine whether differences exist between normal weight (NW) and overweight (OW) children. Methods: Seven to 11 year-old healthy NW (n = 20) and OW (n = 12) volunteers were given a standardized breakfast and lunch following an overnight fast and had measurements of GLP-1 and PP over 9 hours. We characterized whether GLP-1 and PP changed from the pre-prandial to the post-prandial state and whether the serum levels corresponded to reported appetite. Results: GLP-1 did not increase after eating, did not decline prior to the next meal, and did not correspond to satiety ratings in either group. PP increased post-prandially in OW children after both breakfast and lunch, but in the NW group PP only increased after breakfast. PP levels did not decline in either group as the next meal approached. Conclusions: In our study of school-age children, feeding had little effect on GLP-1 secretion and a variable effect on serum PP levels. Observed differences in the GLP-1 and PP responses between the NW and OW groups do not suggest there is an intrinsic abnormality in their secretion that causes weight gain.

Numerous studies are currently underway to characterize the microbial communities inhabiting our world. These studies aim to dramatically expand our understanding of the microbial biosphere and, more importantly, hope to reveal the secrets of the complex symbiotic relationship between us and our commensal bacterial microflora. An important prerequisite for such discoveries are computational tools that are able to rapidly and accurately compare large datasets generated from complex bacterial communities to identify features that distinguish them. We present a statistical method for comparing clinical metagenomic samples from two treatment populations on the basis of count data (e.g. as obtained through sequencing) to detect differentially abundant features. Our method, Metastats, employs the false discovery rate to improve specificity in high-complexity environments, and separately handles sparsely-sampled features using Fisher's exact test. Under a variety of simulations, we show that Metastats performs well compared to previously used methods, and significantly outperforms other methods for features with sparse counts. We demonstrate the utility of our method on several datasets including a 16S rRNA survey of obese and lean human gut microbiomes, COG functional profiles of infant and mature gut microbiomes, and bacterial and viral metabolic subsystem data inferred from random sequencing of 85 metagenomes. The application of our method to the obesity dataset reveals differences between obese and lean subjects not reported in the original study. For the COG and subsystem datasets, we provide the first statistically rigorous assessment of the differences between these populations. The methods described in this paper are the first to address clinical metagenomic datasets comprising samples from multiple subjects. Our methods are robust across datasets of varied complexity and sampling level. While designed for metagenomic applications, our software can also be applied to digital gene expression studies (e.g. SAGE). A web server implementation of our methods and freely available source code can be found at http://metastats.cbcb.umd.edu/., Author Summary The emerging field of metagenomics aims to understand the structure and function of microbial communities solely through DNA analysis. Current metagenomics studies comparing communities resemble large-scale clinical trials with multiple subjects from two general populations (e.g. sick and healthy). To improve analyses of this type of experimental data, we developed a statistical methodology for detecting differentially abundant features between microbial communities, that is, features that are enriched or depleted in one population versus another. We show our methods are applicable to various metagenomic data ranging from taxonomic information to functional annotations. We also provide an assessment of taxonomic differences in gut microbiota between lean and obese humans, as well as differences between the functional capacities of mature and infant gut microbiomes, and those of microbial and viral metagenomes. Our methods are the first to statistically address differential abundance in comparative metagenomics studies with multiple subjects, and we hope will give researchers a more complete picture of how exactly two environments differ.

This paper analyzes different computational methods for real-time decoding of neural signals in primary motor cortex (M1). Specifically, we compare different classifiers as well as different Principal Component Analysis (PCA)-based pre-classification strategies to identify how to proceed in terms of the necessary trade-off between computational complexity and accuracy. Our methods are applied to neural data in monkey, recorded while performing dexterous hand and finger movement tasks. We show that differences due to selection of a classifier using the same feature set are statistically significant for reduced sets of neurons, and specifically that neural networks are to be preferred to a linear classifier. Furthermore, we show that using PCA-based methods prior to neural network-based classification yields statistically equal real-time decoding accuracies using less than 20% of principal components. We therefore conclude that performing PCA prior to classification with a smaller feature space statistically provides the same or better decoding accuracies as those obtained using a larger feature space and a linear or non-linear classifier.

Summary An intravenous formulation of carbamazepine (CBZ) was administered to 113 (60 male; 53 female) persons with epilepsy aged 19–87 years. Subjects received 100 mg of study drug as replacement for 100 mg of their usual morning dose of CBZ. There were no significant changes in blood pressure or heart rate suggesting that this formulation can be developed as replacement therapy for persons unable to take oral CBZ.

Hierarchical clustering is a popular method for grouping together similar elements based on a distance measure between them. In many cases, annotations for some elements are known beforehand, which can aid the clustering process. We present a novel approach for decomposing a hierarchical clustering into the clusters that optimally match a set of known annotations, as measured by the variation of information metric. Our approach is general and does not require the user to enter the number of clusters desired. We apply it to two biological domains: finding protein complexes within protein interaction networks and identifying species within metagenomic DNA samples. For these two applications, we test the quality of our clusters by using them to predict complex and species membership, respectively. We find that our approach generally outperforms the commonly used heuristic methods.