Opsins: Evolution in waiting

Complete vertebrate genome sequencing has revealed a remarkable stability and uniformity in the protein-coding gene set, which at first glance might suggest that gene duplication events are relatively rare. This may be a red herring, or at least a red cichlid, as the Lake Malawi cichlid fishes show rapid and extensive duplication and diversification of their retinal cone photoreceptor opsin genes.

Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States

In Late summer 1999, an outbreak of human encephalitis occurred in the northeastern United States that was concurrent with extensive mortality in crows (Corvus species) as well as the deaths of several exotic birds at a zoological park in the same area. Complete genome sequencing of a flavivirus isolated from the brain of a dead Chilean flamingo (Phoenicopterus chilensis), together with partial sequence analysis of envelope glycoprotein (E-glycoprotein) genes amplified from several other species including mosquitoes and two fatal human cases, revealed that West Nile (WN) virus circulated in natural transmission cycles and was responsible for the human disease. Antigenic mapping with E-glycoprotein-specific monoclonal antibodies and E-glycoprotein phylogenetic analysis confirmed these viruses as WN. This North American WN virus was most closely related to a WN virus isolated from a dead goose in Israel in 1998.

Evaluation and comparison of mammalian subcellular localization prediction methods

Background: Determination of the subcellular location of a protein is essential to understanding its biochemical function. This information can provide insight into the function of hypothetical or novel proteins. These data are difficult to obtain experimentally but have become especially important since many whole genome sequencing projects have been finished and many resulting protein sequences are still lacking detailed functional information. In order to address this paucity of data, many computational prediction methods have been developed. However, these methods have varying levels of accuracy and perform differently based on the sequences that are presented to the underlying algorithm. It is therefore useful to compare these methods and monitor their performance. Results: In order to perform a comprehensive survey of prediction methods, we selected only methods that accepted large batches of protein sequences, were publicly available, and were able to predict localization to at least nine of the major subcellular locations (nucleus, cytosol, mitochondrion, extracellular region, plasma membrane, Golgi apparatus, endoplasmic reticulum (ER), peroxisome, and lysosome). The selected methods were CELLO, MultiLoc, Proteome Analyst, pTarget and WoLF PSORT. These methods were evaluated using 3763 mouse proteins from SwissProt that represent the source of the training sets used in development of the individual methods. In addition, an independent evaluation set of 2145 mouse proteins from LOCATE with a bias towards the subcellular localization underrepresented in SwissProt was used. The sensitivity and specificity were calculated for each method and compared to a theoretical value based on what might be observed by random chance. Conclusion: No individual method had a sufficient level of sensitivity across both evaluation sets that would enable reliable application to hypothetical proteins. All methods showed lower performance on the LOCATE dataset and variable performance on individual subcellular localizations was observed. Proteins localized to the secretory pathway were the most difficult to predict, while nuclear and extracellular proteins were predicted with the highest sensitivity.

Propionibacterium acnes:infection beyond the skin

Propionibacterium acnes is a Gram-positive bacterium that forms part of the normal flora of the skin, oral cavity, large intestine, the conjunctiva and the external ear canal. Although primarily recognized for its role in acne, P. acnes is an opportunistic pathogen, causing a range of postoperative and device-related infections. These include infections of the bones and joints, mouth, eye and brain. Device-related infections include those of joint prostheses, shunts and prosthetic heart valves. P. acnes may play a role in other conditions, including inflammation of the prostate leading to cancer, SAPHO (synovitis, acne, pustulosis, hyperostosis, osteitis) syndrome, sarcoidosis and sciatica. If an active role in these conditions is established there are major implications for diagnosis, treatment and protection. Genome sequencing of the organism has provided an insight into the pathogenic potential and virulence of P. acnes. © 2011 Expert Reviews Ltd.

Health and population effects of rare gene knockouts in adult humans with related parents

Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3222 British Pakistani-heritage adults with high parental relatedness, discovering 1111 rare-variant homozygous genotypes with predicted loss of gene function (knockouts) in 781 genes. We observed 13.7% fewer than expected homozygous knockout genotypes, implying an average load of 1.6 recessive-lethal-equivalent LOF variants per adult. Linking genetic data to lifelong health records, knockouts were not associated with clinical consultation or prescription rate. In this dataset we identified a healthy PRDM9 knockout mother, and performed phased genome sequencing on her, her child and controls, which showed meiotic recombination sites localized away from PRDM9-dependent hotspots. Thus, natural LOF variants inform upon essential genetic loci, and demonstrate PRDM9 redundancy in humans.

Variabilidade genética de Leishmania spp. circulantes entre seres humanos e cães e infecção de flebotomíneos em áreas endêmicas para as leishmanioses no Rio Grande do Norte

Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector’s species and the parasite’s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.

Variabilidade genética de Leishmania spp. circulantes entre seres humanos e cães e infecção de flebotomíneos em áreas endêmicas para as leishmanioses no Rio Grande do Norte

Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector’s species and the parasite’s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.

2012 09 February Jean Weissenbach interview

Jean Weissenbach, telephone interview by Kathryn Maxson and Robert Cook-Deegan, conducted from Durham, NC 09 February 2012. Jean Weissenbach, a leader in French genetic mapping, directed the French national sequencing center, Généthon, during the HGP and was instrumental in helping to build agreement to the Bermuda Principles in France.

2011 18 August Mark Guyer-Jane Peterson interview

Mark Guyer and Jane Peterson, in-person interview with Kathryn Maxson and Robert Cook-Deegan, conducted in Rockville, MD (NIH campus), 18 August 2011. Mark Guyer and Jane Peterson were grants program officers at the NIH during the HGP, and were some of the longest-standing employees in the HGP administrative structure. Both witnessed the transformation of the Office of Genome Research into the National Center for Human Genome Research and, finally, the National Human Genome Research Institute. They were close participants in the history of the Bermuda Principles within the NIH.

Intraclonal heterogeneity is a critical early event in the development of myeloma and precedes the development of clinical symptoms.

The mechanisms involved in the progression from monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) to malignant multiple myeloma (MM) and plasma cell leukemia (PCL) are poorly understood but believed to involve the sequential acquisition of genetic hits. We performed exome and whole-genome sequencing on a series of MGUS (n=4), high-risk (HR)SMM (n=4), MM (n=26) and PCL (n=2) samples, including four cases who transformed from HR-SMM to MM, to determine the genetic factors that drive progression of disease. The pattern and number of non-synonymous mutations show that the MGUS disease stage is less genetically complex than MM, and HR-SMM is similar to presenting MM. Intraclonal heterogeneity is present at all stages and using cases of HR-SMM, which transformed to MM, we show that intraclonal heterogeneity is a typical feature of the disease. At the HR-SMM stage of disease, the majority of the genetic changes necessary to give rise to MM are already present. These data suggest that clonal progression is the key feature of transformation of HR-SMM to MM and as such the invasive clinically predominant clone typical of MM is already present at the SMM stage and would be amenable to therapeutic intervention at that stage.

Personalized cancer medicine: molecular diagnostics, predictive biomarkers, and drug resistance.

The progressive elucidation of the molecular pathogenesis of cancer has fueled the rational development of targeted drugs for patient populations stratified by genetic characteristics. Here we discuss general challenges relating to molecular diagnostics and describe predictive biomarkers for personalized cancer medicine. We also highlight resistance mechanisms for epidermal growth factor receptor (EGFR) kinase inhibitors in lung cancer. We envisage a future requiring the use of longitudinal genome sequencing and other omics technologies alongside combinatorial treatment to overcome cellular and molecular heterogeneity and prevent resistance caused by clonal evolution.

Improving photofermentative hydrogen production through metabolic engineering and DOE (Design of Experiments)

A l’heure actuelle, les biocarburants renouvelables et qui ne nuit pas à l'environnement sont à l'étude intensive en raison de l'augmentation des problèmes de santé et de la diminution des combustibles fossiles. H2 est l'un des candidats les plus prometteurs en raison de ses caractéristiques uniques, telles que la densité d'énergie élevée et la génération faible ou inexistante de polluants. Une façon attrayante pour produire la H2 est par les bactéries photosynthétiques qui peuvent capter l'énergie lumineuse pour actionner la production H2 avec leur système de nitrogénase. L'objectif principal de cette étude était d'améliorer le rendement de H2 des bactéries photosynthétiques pourpres non sulfureuses utilisant une combinaison de génie métabolique et le plan des expériences. Une hypothèse est que le rendement en H2 pourrait être améliorée par la redirection de flux de cycle du Calvin-Benson-Bassham envers du système de nitrogénase qui catalyse la réduction des protons en H2. Ainsi, un PRK, phosphoribulose kinase, mutant « knock-out » de Rhodobacter capsulatus JP91 a été créé. L’analyse de la croissance sur des différentes sources de carbone a montré que ce mutant ne peut croître qu’avec l’acétate, sans toutefois produire d' H2. Un mutant spontané, YL1, a été récupéré qui a retenu l'cbbP (codant pour PRK) mutation d'origine, mais qui avait acquis la capacité de se développer sur le glucose et produire H2. Une étude de la production H2 sous différents niveaux d'éclairage a montré que le rendement d’YL1 était de 20-40% supérieure à la souche type sauvage JP91. Cependant, il n'y avait pas d'amélioration notable du taux de production de H2. Une étude cinétique a montré que la croissance et la production d'hydrogène sont fortement liées avec des électrons à partir du glucose principalement dirigés vers la production de H2 et la formation de la biomasse. Sous des intensités lumineuses faibles à intermédiaires, la production d'acides organiques est importante, ce qui suggère une nouvelle amélioration additionnel du rendement H2 pourrait être possible grâce à l'optimisation des processus. Dans une série d'expériences associées, un autre mutant spontané, YL2, qui a un phénotype similaire à YL1, a été testé pour la croissance dans un milieu contenant de l'ammonium. Les résultats ont montré que YL2 ne peut croître que avec de l'acétate comme source de carbone, encore une fois, sans produire de H2. Une incubation prolongée dans les milieux qui ne supportent pas la croissance de YL2 a permis l'isolement de deux mutants spontanés secondaires intéressants, YL3 et YL4. L'analyse par empreint du pied Western a montré que les deux souches ont, dans une gamme de concentrations d'ammonium, l'expression constitutive de la nitrogénase. Les génomes d’YL2, YL3 et YL4 ont été séquencés afin de trouver les mutations responsables de ce phénomène. Fait intéressant, les mutations de nifA1 et nifA2 ont été trouvés dans les deux YL3 et YL4. Il est probable qu'un changement conformationnel de NifA modifie l'interaction protéine-protéine entre NifA et PII protéines (telles que GlnB ou GlnK), lui permettant d'échapper à la régulation par l'ammonium, et donc d'être capable d'activer la transcription de la nitrogénase en présence d'ammonium. On ignore comment le nitrogénase synthétisé est capable de maintenir son activité parce qu’en théorie, il devrait également être soumis à une régulation post-traductionnelle par ammonium. Une autre preuve pourrait être obtenue par l'étude du transcriptome d’YL3 et YL4. Une première étude sur la production d’ H2 par YL3 et YL4 ont montré qu'ils sont capables d’une beaucoup plus grande production d'hydrogène que JP91 en milieu d'ammonium, qui ouvre la porte pour les études futures avec ces souches en utilisant des déchets contenant de l'ammonium en tant que substrats. Enfin, le reformage biologique de l'éthanol à H2 avec la bactérie photosynthétique, Rhodopseudomonas palustris CGA009 a été examiné. La production d'éthanol avec fermentation utilisant des ressources renouvelables microbiennes a été traitée comme une technique mature. Cependant, la plupart des études du reformage de l'éthanol à H2 se sont concentrés sur le reformage chimique à la vapeur, ce qui nécessite généralement une haute charge énergetique et résultats dans les émissions de gaz toxiques. Ainsi le reformage biologique de l'éthanol à H2 avec des bactéries photosynthétiques, qui peuvent capturer la lumière pour répondre aux besoins énergétiques de cette réaction, semble d’être plus prometteuse. Une étude précédente a démontré la production d'hydrogène à partir d'éthanol, toutefois, le rendement ou la durée de cette réaction n'a pas été examiné. Une analyse RSM (méthode de surface de réponse) a été réalisée dans laquelle les concentrations de trois facteurs principaux, l'intensité lumineuse, de l'éthanol et du glutamate ont été variés. Nos résultats ont montré que près de 2 moles de H2 peuvent être obtenus à partir d'une mole d'éthanol, 33% de ce qui est théoriquement possible.

Ruthenibacterium lactatiformans gen. nov., sp.nov., an anaerobic, lactate-producing member of the family Ruminococcaceae isolated from human faeces

Two novel strains of Gram-stain-negative, rod-shaped, obligately anaerobic, non-spore-forming, non-motile bacteria were isolated from the faeces of healthy human subjects. The strains, designated as 585-1T and 668, were characterized by mesophilic fermentative metabolism, production of d-lactic acid, succinic acid and acetic acid as end products of d-glucose fermentation, prevalence of C18 : 1 ω9, C18 : 1 ω9 aldehyde, C16 : 0 and C16 : 1 ω7c fatty acids, presence of glycine, glutamic acid, lysine, alanine and aspartic acid in the petidoglycan peptide moiety and lack of respiratory quinones. Whole genome sequencing revealed the DNA G+C content was 56.4–56.6 mol%. The complete 16S rRNA gene sequences of the two strains shared 91.7/91.6 % similarity with Anaerofilum pentosovorans FaeT, 91.3/91.2 % with Gemmiger formicilis ATCC 27749T and 88.9/88.8 % with Faecalibacterium prausnitzii ATCC 27768T. On the basis of chemotaxonomic and genomic properties it was concluded that the strains represent a novel species in a new genus within the family Ruminococcaceae , for which the name Ruthenibacterium lactatiformans gen. nov., sp. nov. is proposed. The type strain of Ruthenibacterium lactatiformans is 585-1T (=DSM 100348T=VKM B-2901T).

Étude de la plasticité génomique des algues vertes de l’ordre Chlamydomonadales

Les récents progrès en génomique ont conforté la complexité de l’origine des algues; d’un point de vue de la phylogénie des hôtes de l’endosymbiose, les algues forment un groupe évolutif polyphylétique. Les algues vertes forment deux embranchements majeurs : les Streptophyta et les Chlorophyta. Les chlorophytes comprennent la majorité des algues vertes connues et se regroupent en quatre classes. La première, les Prasinophyceae, occupe la position la plus basale, tandis que l’ordre d’embranchement des trois autres classes (Ulvophyceae, Trebouxiophyceae et Chlorophyceae) demeure encore incertain. Pour clarifier les relations évolutives chez les Clorophyceae, huit génomes chloroplastiques appartenant à la lignée des Chlamydomonadales, lignée majeure des Chlorophyceae, ont été séquencés et analysés. Des études phylogénétiques ont confirmé les classifications préétablies et de nouveaux clades se sont vus formés. Les génomes de ces algues chlorophycéennes ont révélé une architecture conservée avec un certain nombre de caractères spécifiques à la classe des Chlamydomonadales. L’analyse de leurs caractères moléculaires a révélé des génomes marqués par la réduction ou le réarrangement de leur répertoire génomique comparativement aux génomes chloroplastiques des algues vertes plus ancestrales.

Macrophage adaptation leads to parallel evolution of genetically diverseEscherichia colismall-colony variants with increased fitness in vivo and antibiotic collateral sensitivity

Small-colony variants (SCVs) are commonly observed in evolution experiments and clinical isolates, being associated with antibiotic resistance and persistent infections. We recently observed the repeated emergence of Escherichia coli SCVs during adaptation to the interaction with macrophages. To identify the genetic targets underlying the emergence of this clinically relevant morphotype, we performed whole-genome sequencing of independently evolved SCV clones. We uncovered novel mutational targets, not previously associated with SCVs (e.g. cydA, pepP) and observed widespread functional parallelism. All SCV clones had mutations in genes related to the electron-transport chain. As SCVs emerged during adaptation to macrophages, and often show increased antibiotic resistance, we measured SCV fitness inside macrophages and measured their antibiotic resistance profiles. SCVs had a fitness advantage inside macrophages and showed increased aminoglycoside resistance in vitro, but had collateral sensitivity to other antibiotics (e.g. tetracycline). Importantly, we observed similar results in vivo. SCVs had a fitness advantage upon colonization of the mouse gut, which could be tuned by antibiotic treatment: kanamycin (aminoglycoside) increased SCV fitness, but tetracycline strongly reduced it. Our results highlight the power of using experimental evolution as the basis for identifying the causes and consequences of adaptation during host-microbe interactions.