Oxidative Pathways of Deoxyribose and Deoxyribonate Catabolism.

Using genome-wide mutant fitness assays in diverse bacteria, we identified novel oxidative pathways for the catabolism of 2-deoxy-d-ribose and 2-deoxy-d-ribonate. We propose that deoxyribose is oxidized to deoxyribonate, oxidized to ketodeoxyribonate, and cleaved to acetyl coenzyme A (acetyl-CoA) and glyceryl-CoA. We have genetic evidence for this pathway in three genera of bacteria, and we confirmed the oxidation of deoxyribose to ketodeoxyribonate in vitro. In Pseudomonas simiae, the expression of enzymes in the pathway is induced by deoxyribose or deoxyribonate, while in Paraburkholderia bryophila and in Burkholderia phytofirmans, the pathway proceeds in parallel with the known deoxyribose 5-phosphate aldolase pathway. We identified another oxidative pathway for the catabolism of deoxyribonate, with acyl-CoA intermediates, in Klebsiella michiganensis. Of these four bacteria, only P. simiae relies entirely on an oxidative pathway to consume deoxyribose. The deoxyribose dehydrogenase of P. simiae is either nonspecific or evolved recently, as this enzyme is very similar to a novel vanillin dehydrogenase from Pseudomonas putida that we identified. So, we propose that these oxidative pathways evolved primarily to consume deoxyribonate, which is a waste product of metabolism. IMPORTANCE Deoxyribose is one of the building blocks of DNA and is released when cells die and their DNA degrades. We identified a bacterium that can grow with deoxyribose as its sole source of carbon even though its genome does not contain any of the known genes for breaking down deoxyribose. By growing many mutants of this bacterium together on deoxyribose and using DNA sequencing to measure the change in the mutants' abundance, we identified multiple protein-coding genes that are required for growth on deoxyribose. Based on the similarity of these proteins to enzymes of known function, we propose a 6-step pathway in which deoxyribose is oxidized and then cleaved. Diverse bacteria use a portion of this pathway to break down a related compound, deoxyribonate, which is a waste product of metabolism. Our study illustrates the utility of large-scale bacterial genetics to identify previously unknown metabolic pathways.

Hidden diversity of soil giant viruses.

Known giant virus diversity is currently skewed towards viruses isolated from aquatic environments and cultivated in the laboratory. Here, we employ cultivation-independent metagenomics and mini-metagenomics on soils from the Harvard Forest, leading to the discovery of 16 novel giant viruses, chiefly recovered by mini-metagenomics. The candidate viruses greatly expand phylogenetic diversity of known giant viruses and either represented novel lineages or are affiliated with klosneuviruses, Cafeteria roenbergensis virus or tupanviruses. One assembled genome with a size of 2.4 Mb represents the largest currently known viral genome in the Mimiviridae, and others encode up to 80% orphan genes. In addition, we find more than 240 major capsid proteins encoded on unbinned metagenome fragments, further indicating that giant viruses are underexplored in soil ecosystems. The fact that most of these novel viruses evaded detection in bulk metagenomes suggests that mini-metagenomics could be a valuable approach to unearth viral giants.

Genome-wide identification of bacterial plant colonization genes.

Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system. Among the genes we identified were some with obvious colonization-related roles in motility and carbon metabolism, as well as 44 other genes that had no or vague functional predictions. Independent validation assays of individual genes confirmed colonization functions for 20 of 22 (91%) cases tested. To further characterize genes identified by our screen, we compared the functional contributions of P. simiae genes to growth in 90 distinct in vitro conditions by RB-TnSeq, highlighting specific metabolic functions associated with root colonization genes. Our analysis of bacterial genes by sequence-driven saturation mutagenesis revealed a genome-wide map of the genetic determinants of plant root colonization and offers a starting point for targeted improvement of the colonization capabilities of plant-beneficial microbes.

Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus.

The 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics. VIDEO ABSTRACT.

A virtual outpatient department provides a satisfactory patient experience following endoscopy.

PURPOSE: The purpose of this study was to investigate telephone follow-up of post-endoscopy patients as an alternative to attendance at the outpatient department. METHODS: Access to outpatient appointments is often a target for improvement in healthcare systems. Increased outpatient clinic capacity is not feasible without investment and extra manpower in an already constrained service. Outpatient attendance was audited at a busy colorectal surgical service. A subset of patients appropriate for follow-up in a "virtual outpatient department" (VOPD) were identified. A pilot study was designed and involved telephone follow-up of low-risk endoscopic procedures. Patient satisfaction was assessed using the Medical Interview Satisfaction Scale (MISS), which is a standardised survey of patient satisfaction with healthcare experiences. This was conducted via anonymous questionnaire at the end of the study. RESULTS: Of a total of 166 patients undergoing endoscopy in the time period, 79 were prospectively recruited to VOPD follow-up based on eligibility criteria. Overall, 67 (84.8 %) were successfully followed up by telephone consultation; nine patients (11.4 %) were contacted by mail. The remaining three patients (3.8 %) were brought back to the OPD. Patients recruited were more likely to be younger (55.82 ± 14.96 versus 60.78 ± 13.97 years, P = 0.029) and to have had normal examinations (49.4 versus 31.0 %, χ (2) = 5.070, P = 0.025). Nearly three quarters of patients responded to the questionnaire. The mean scores for all four aspects of the MISS were satisfactory, and overall patients were satisfied with the VOPD experience. CONCLUSION: VOPD is a target for improved healthcare provision, with improved efficiency and a high patient satisfaction rate.

Multicenter Evaluation of Rectal cancer ReImaging pOst Neoadjuvant (MERRION) Therapy.

OBJECTIVE: The aim of this study was to evaluate the utility of reimaging rectal cancer post-CRT (chemoradiotherapy) with magnetic resonance (MR) imaging of the pelvis for local staging and computed tomography of thorax, abdomen, and pelvis (CT TAP) to identify distant metastases. BACKGROUND: The success of neoadjuvant CRT for locally advanced rectal cancer has changed an already complex management algorithm. There is no consensus whether patients should be restaged before surgery. METHODS: Data from 5 institutions with prospectively maintained databases including patients who received neoadjuvant CRT for locally advanced rectal cancer were acquired. Only patients who had been staged pre- and post-CRT with MR imaging and CT TAP were included. MR findings were correlated with histopathological stage using weighted κ (kappa) statistics to test agreement, where a κ value of less than 0.5 was deemed unacceptable. RESULTS: A total of 285 patients fulfilled the criteria for the study; 84% had American Joint Committee for Cancer stage 3 disease pre-CRT, and the remainder had stage 2 disease. Fourteen patients did not proceed to surgery post-CRT-2 were observed as "complete responders," and the remainder either had unresectable disease or were unfit for surgery. MR imaging could not predict T stage (κ = 0.212) or nodal involvement (κ = 0.336). Most pertinently, MR imaging was unable to detect a complete pathological response (κ = 0.021), nor could it discriminate T4 disease (κ = 0.445). CT TAP restaging altered management in 6.7% of patients, who had metastatic disease. CONCLUSIONS: MR reimaging using standard protocols is of limited value in determining surgical approaches; a better modality of local restaging is required.

De novo assembly of highly diverse viral populations.

BACKGROUND: Extensive genetic diversity in viral populations within infected hosts and the divergence of variants from existing reference genomes impede the analysis of deep viral sequencing data. A de novo population consensus assembly is valuable both as a single linear representation of the population and as a backbone on which intra-host variants can be accurately mapped. The availability of consensus assemblies and robustly mapped variants are crucial to the genetic study of viral disease progression, transmission dynamics, and viral evolution. Existing de novo assembly techniques fail to robustly assemble ultra-deep sequence data from genetically heterogeneous populations such as viruses into full-length genomes due to the presence of extensive genetic variability, contaminants, and variable sequence coverage. RESULTS: We present VICUNA, a de novo assembly algorithm suitable for generating consensus assemblies from genetically heterogeneous populations. We demonstrate its effectiveness on Dengue, Human Immunodeficiency and West Nile viral populations, representing a range of intra-host diversity. Compared to state-of-the-art assemblers designed for haploid or diploid systems, VICUNA recovers full-length consensus and captures insertion/deletion polymorphisms in diverse samples. Final assemblies maintain a high base calling accuracy. VICUNA program is publicly available at: http://www.broadinstitute.org/scientific-community/science/projects/viral-genomics/ viral-genomics-analysis-software. CONCLUSIONS: We developed VICUNA, a publicly available software tool, that enables consensus assembly of ultra-deep sequence derived from diverse viral populations. While VICUNA was developed for the analysis of viral populations, its application to other heterogeneous sequence data sets such as metagenomic or tumor cell population samples may prove beneficial in these fields of research.

Genome-wide patterns of intrahuman dengue virus diversity reveal associations with viral phylogenetic clade and interhost diversity.

Analogous to observations in RNA viruses such as human immunodeficiency virus, genetic variation associated with intrahost dengue virus (DENV) populations has been postulated to influence viral fitness and disease pathogenesis. Previous attempts to investigate intrahost genetic variation in DENV characterized only a few viral genes or a limited number of full-length genomes. We developed a whole-genome amplification approach coupled with deep sequencing to capture intrahost diversity across the entire coding region of DENV-2. Using this approach, we sequenced DENV-2 genomes from the serum of 22 Nicaraguan individuals with secondary DENV infection and captured ∼75% of the DENV genome in each sample (range, 40 to 98%). We identified and quantified variants using a highly sensitive and specific method and determined that the extent of diversity was considerably lower than previous estimates. Significant differences in intrahost diversity were detected between genes and also between antigenically distinct domains of the Envelope gene. Interestingly, a strong association was discerned between the extent of intrahost diversity in a few genes and viral clade identity. Additionally, the abundance of viral variants within a host, as well as the impact of viral mutations on amino acid encoding and predicted protein function, determined whether intrahost variants were observed at the interhost level in circulating Nicaraguan DENV-2 populations, strongly suggestive of purifying selection across transmission events. Our data illustrate the value of high-coverage genome-wide analysis of intrahost diversity for high-resolution mapping of the relationship between intrahost diversity and clinical, epidemiological, and virological parameters of viral infection.

Synergistic phage-antibiotic combinations for the control of Escherichia coli biofilms in vitro.

The potential application of phage therapy for the control of bacterial biofilms has received increasing attention as resistance to conventional antibiotic agents continues to increase. The present study identifies antimicrobial synergy between bacteriophage T4 and a conventional antibiotic, cefotaxime, via standard plaque assay and, importantly, in the in vitro eradication of biofilms of the T4 host strain Escherichia coli 11303. Phage-antibiotic synergy (PAS) is defined as the phenomenon whereby sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacteria's production of virulent phage. Increasing sub-lethal concentrations of cefotaxime resulted in an observed increase in T4 plaque size and T4 concentration. The application of PAS to the T4 one-step growth curve also resulted in an increased burst size and reduced latent period. Combinations of T4 bacteriophage and cefotaxime significantly enhanced the eradication of bacterial biofilms when compared to treatment with cefotaxime alone. The addition of medium (10(4) PFU mL(-1)) and high (10(7) PFU mL(-1)) phage titres reduced the minimum biofilm eradication concentration value of cefotaxime against E. coli ATCC 11303 biofilms from 256 to 128 and 32 µg mL(-1), respectively. Although further investigation is needed to confirm PAS, this study demonstrates, for the first time, that synergy between bacteriophage and conventional antibiotics can significantly improve biofilm control in vitro.

Whole genome deep sequencing of HIV-1 reveals the impact of early minor variants upon immune recognition during acute infection.

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.

Highly sensitive and specific detection of rare variants in mixed viral populations from massively parallel sequence data.

Viruses diversify over time within hosts, often undercutting the effectiveness of host defenses and therapeutic interventions. To design successful vaccines and therapeutics, it is critical to better understand viral diversification, including comprehensively characterizing the genetic variants in viral intra-host populations and modeling changes from transmission through the course of infection. Massively parallel sequencing technologies can overcome the cost constraints of older sequencing methods and obtain the high sequence coverage needed to detect rare genetic variants (< 1%) within an infected host, and to assay variants without prior knowledge. Critical to interpreting deep sequence data sets is the ability to distinguish biological variants from process errors with high sensitivity and specificity. To address this challenge, we describe V-Phaser, an algorithm able to recognize rare biological variants in mixed populations. V-Phaser uses covariation (i.e. phasing) between observed variants to increase sensitivity and an expectation maximization algorithm that iteratively recalibrates base quality scores to increase specificity. Overall, V-Phaser achieved > 97% sensitivity and > 97% specificity on control read sets. On data derived from a patient after four years of HIV-1 infection, V-Phaser detected 2,015 variants across the -10 kb genome, including 603 rare variants (< 1% frequency) detected only using phase information. V-Phaser identified variants at frequencies down to 0.2%, comparable to the detection threshold of allele-specific PCR, a method that requires prior knowledge of the variants. The high sensitivity and specificity of V-Phaser enables identifying and tracking changes in low frequency variants in mixed populations such as RNA viruses.

Internally deleted WNV genomes isolated from exotic birds in New Mexico: function in cells, mosquitoes, and mice.

Most RNA viruses exist in their hosts as a heterogeneous population of related variants. Due to error prone replication, mutants are constantly generated which may differ in individual fitness from the population as a whole. Here we characterize three WNV isolates that contain, along with full-length genomes, mutants with large internal deletions to structural and nonstructural protein-coding regions. The isolates were all obtained from lorikeets that died from WNV at the Rio Grande Zoo in Albuquerque, NM between 2005 and 2007. The deletions are approximately 2kb, in frame, and result in the elimination of the complete envelope, and portions of the prM and NS-1 proteins. In Vero cell culture, these internally deleted WNV genomes function as defective interfering particles, reducing the production of full-length virus when introduced at high multiplicities of infection. In mosquitoes, the shortened WNV genomes reduced infection and dissemination rates, and virus titers overall, and were not detected in legs or salivary secretions at 14 or 21 days post-infection. In mice, inoculation with internally deleted genomes did not attenuate pathogenesis relative to full-length or infectious clone derived virus, and shortened genomes were not detected in mice at the time of death. These observations provide evidence that large deletions may occur within flavivirus populations more frequently than has generally been appreciated and suggest that they impact population phenotype minimally. Additionally, our findings suggest that highly similar mutants may frequently occur in particular vertebrate hosts.

Recent advances in bacteriophage therapy: how delivery routes, formulation, concentration and timing influence the success of phage therapy.

OBJECTIVES: Bacteriophages are bacteria-specific viruses that infect and, in the case of obligately lytic phages, destroy their host bacteria. Phage therapy has been used therapeutically to combat bacterial infections since their discovery. This paper reviewed recent in-vivo phage therapy studies, with a distinct focus on the effect of delivery routes, phage concentration and timing of administration on the success of the therapy. KEY FINDINGS: It was found that the most successful route of administration for the treatment of systemic infections was via the parenteral route. Oral delivery is mainly used to treat gastrointestinal infections. However, in some cases phages can also reach the systemic circulation. Local delivery (skin, ears, teeth) has proved extremely successful in the treatment of topical infections, as has the inhalation of phages for the treatment of lung infections. The ability of phages to prevent biofilm formation on medical devices has received much attention, mainly in the area of catheter coatings. This review also highlights areas in which phage therapy needs substantial development. Many papers were lacking in formulation details, with crude phage stocks being used in most cases. No phage stability data were included in any of the papers. SUMMARY: The review concluded that although phage therapy is an excellent alternative for the treatment of bacterial infections, optimisation of formulations and long-term stability data is required before it can be widely used within a clinical setting.

PriSM: a primer selection and matching tool for amplification and sequencing of viral genomes.

SUMMARY: PriSM is a set of algorithms designed to select and match degenerate primer pairs for the amplification of viral genomes. The design of panels of hundreds of primer pairs takes just hours using this program, compared with days using a manual approach. PriSM allows for rapid in silico optimization of primers for downstream applications such as sequencing. As a validation, PriSM was used to create an amplification primer panel for human immunodeficiency virus (HIV) Clade B. AVAILABILITY: The program is freely available for use at: www.broadinstitute.org/perl/seq/specialprojects/primerDesign.cgi.

Evidence for negative selection on the gene encoding rhoptry-associated protein 1 (RAP-1) in Plasmodium spp.

Assessing how natural selection, negative or positive, operates on genes with low polymorphism is challenging. We investigated the genetic diversity of orthologous genes encoding the rhoptry-associated protein 1 (RAP-1), a low polymorphic protein of malarial parasites that is involved in erythrocyte invasion. We applied evolutionary genetic methods to study the polymorphism in RAP-1 from Plasmodium falciparum (n=32) and Plasmodium vivax (n=6), the two parasites responsible for most human malaria morbidity and mortality, as well as RAP-1 orthologous in closely related malarial species found in non-human primates (NHPs). Overall, genes encoding RAP-1 are highly conserved in all Plasmodium spp. included in this investigation. We found no evidence for natural selection, positive or negative, acting on the gene encoding RAP-1 in P. falciparum or P. vivax. However, we found evidence that the orthologous genes in non-human primate parasites (Plasmodium cynomolgi, Plasmodium inui, and Plasmodium knowlesi) are under purifying (negative) selection. We discuss the importance of considering negative selection while studying genes encoding proteins with low polymorphism and how selective pressures may differ among orthologous genes in closely related malarial parasites species.

Outcomes of planned hospital birth attended by midwives compared with physicians in British Columbia.

BACKGROUND: The impact of midwifery versus physician care on perinatal outcomes in a population of women planning birth in hospital has not yet been explored. We compared maternal and newborn outcomes between women planning hospital birth attended by a midwife versus a physician in British Columbia, Canada. METHODS: All women planning a hospital birth attended by a midwife during the 2-year study period who were of sufficiently low-risk status to meet eligibility requirements for home birth as defined by the British Columbia College of Midwives were included in the study group (n=488). The comparison group included women meeting the same eligibility requirements but planning a physician-attended birth in hospitals where midwives also practiced (n=572). Outcomes were ascertained from the British Columbia Reproductive Care Program Perinatal Registry to which all hospitals in the province submit data. RESULTS: Adjusted odds ratios for women planning hospital birth attended by a midwife versus a physician were significantly reduced for exposure to cesarean section (OR 0.58, 95% CI 0.39-0.86), narcotic analgesia (OR 0.26, 95% CI 0.18-0.37), electronic fetal monitoring (OR 0.22, 95% CI 0.16-0.30), amniotomy (OR 0.74, 95% CI 0.56-0.98), and episiotomy (OR 0.62, 95% CI 0.42-0.93). The odds of adverse neonatal outcomes were not different between groups, with the exception of reduced use of drugs for resuscitation at birth (OR 0.19, 95% CI 0.04-0.83) in the midwifery group. CONCLUSIONS: A shift toward greater proportions of midwife-attended births in hospitals could result in reduced rates of obstetric interventions, with similar rates of neonatal morbidity.

Outcomes of planned home births versus planned hospital births after regulation of midwifery in British Columbia.

BACKGROUND: The choice to give birth at home with a regulated midwife in attendance became available to expectant women in British Columbia in 1998. The purpose of this study was to evaluate the safety of home birth by comparing perinatal outcomes for planned home births attended by regulated midwives with those for planned hospital births. METHODS: We compared the outcomes of 862 planned home births attended by midwives with those of planned hospital births attended by either midwives (n = 571) or physicians (n = 743). Comparison subjects who were similar in their obstetric risk status were selected from hospitals in which the midwives who were conducting the home births had hospital privileges. Our study population included all home births that occurred between Jan. 1, 1998, and Dec. 31, 1999. RESULTS: Women who gave birth at home attended by a midwife had fewer procedures during labour compared with women who gave birth in hospital attended by a physician. After adjustment for maternal age, lone parent status, income quintile, use of any versus no substances and parity, women in the home birth group were less likely to have epidural analgesia (odds ratio 0.20, 95% confidence interval [CI] 0.14-0.27), be induced, have their labours augmented with oxytocin or prostaglandins, or have an episiotomy. Comparison of home births with hospital births attended by a midwife showed very similar and equally significant differences. The adjusted odds ratio for cesarean section in the home birth group compared with physician-attended hospital births was 0.3 (95% CI 0.22-0.43). Rates of perinatal mortality, 5-minute Apgar scores, meconium aspiration syndrome or need for transfer to a different hospital for specialized newborn care were very similar for the home birth group and for births in hospital attended by a physician. The adjusted odds ratio for Apgar scores lower than 7 at 5 minutes in the home birth group compared with physician-attended hospital births was 0.84 (95% CI 0.32-2.19). INTERPRETATION: There was no increased maternal or neonatal risk associated with planned home birth under the care of a regulated midwife. The rates of some adverse outcomes were too low for us to draw statistical comparisons, and ongoing evaluation of home birth is warranted.