Rationally designed families of orthogonal RNA regulators of translation

Our ability to routinely engineer genetic networks for applications is limited by the scarcity of highly specific and non-cross-reacting (orthogonal) gene regulators with predictable behavior. Though antisense RNAs are attractive contenders for this purpose, quantitative understanding of their specificity and sequence-function relationship sufficient for their design has been limited. Here, we use rationally designed variants of the RNA-IN-RNA-OUT antisense RNA-mediated translation system from the insertion sequence IS10 to quantify >500 RNA-RNA interactions in Escherichia coli and integrate the data set with sequence-activity modeling to identify the thermodynamic stability of the duplex and the seed region as the key determinants of specificity. Applying this model, we predict the performance of an additional ~2,600 antisense-regulator pairs, forecast the possibility of large families of orthogonal mutants, and forward engineer and experimentally validate two RNA pairs orthogonal to an existing group of five from the training data set. We discuss the potential use of these regulators in next-generation synthetic biology applications.     

Rickettsia Phylogenomics: Unwinding the Intricacies of Obligate Intracellular Life

Background

Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular α-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs) of open reading frames (ORFs) will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs) from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs).

Methodology/Principal Findings

We present 1823 representative (no gene duplications) and 259 non-representative (at least one gene duplication) rickettsial OGs. While the highly reductive (∼1.2 MB) Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group), TG (typhus group), TRG (transitional group), and SFG (spotted fever group) rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions.

Conclusion/Significance

Collectively, we determined the relative conservation and distribution of 14354 predicted ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets.     

Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms

Carotenoids are produced by all photosynthetic organisms, where they play essential roles in light harvesting and photoprotection. The carotenoid biosynthetic pathway of diatoms is largely unstudied, but is of particular interest because these organisms have a very different evolutionary history with respect to the Plantae and are thought to be derived from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes. Furthermore, diatoms have an additional xanthophyll-based cycle for dissipating excess light energy with respect to green algae and higher plants. To explore the origins and functions of the carotenoid pathway in diatoms we searched for genes encoding pathway components in the recently completed genome sequences of two marine diatoms. Consistent with the supplemental xanthophyll cycle in diatoms, we found more copies of the genes encoding violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP) enzymes compared with other photosynthetic eukaryotes. However, the similarity of these enzymes with those of higher plants indicates that they had very probably diversified before the secondary endosymbiosis had occurred, implying that VDE and ZEP represent early eukaryotic innovations in the Plantae. Consequently, the diatom chromist lineage likely obtained all paralogues of ZEP and VDE genes during the process of secondary endosymbiosis by gene transfer from the nucleus of the algal endosymbiont to the host nucleus. Furthermore, the presence of a ZEP gene in Tetrahymena thermophila provides the first evidence for a secondary plastid gene encoded in a heterotrophic ciliate, providing support for the chromalveolate hypothesis. Protein domain structures and expression analyses in the pennate diatom Phaeodactylum tricornutum indicate diverse roles for the different ZEP and VDE isoforms and demonstrate that they are differentially regulated by light. These studies therefore reveal the ancient origins of several components of the carotenoid biosynthesis pathway in photosynthetic eukaryotes and provide information about how they have diversified and acquired new functions in the diatoms.     

Pollinator Specificity and Convergence in Fly-pollinated Pleurothallis(Orchidaceae) Species: A Multiple Population Approach

We studied the floral biology of 12 populations of five rupicolousPleurothallis(Orchidaceae) species occurring in campo rupestre vegetationat nine localities in Brazil. All of these species are pollinatedby flies belonging to the families Chloropidae and Phoridae.In the five Pleurothallis species studied, all conspecific populationsattracted the same pollinator species. All pollinators werefemales; they laid eggs in flowers of the two nectarless species,but never in the flowers of nectar-presenting species. The twopairs ofPleurothallis species with similar flower morphologiesand odours attracted the same pollinators: P. johannensis -P. fabiobarrosii, pollinated by Tricimba sp. (Chloropidae) andP. teres - P. ochreata pollinated by Megaselia spp. (Phoridae).There was no overlap in the distribution of thePleurothallisspecies that shared pollinators. Despite similarities in floralmorphology and odour, genetic data show that these species pairsare not each other's closest relatives. We hypothesize thatthese similarities are due to convergence in allopatric speciesthat evolved similar pollination mechanisms. Conversely, thereare reasons to believe that adaptation to different pollinationmechanisms occurred in the closely related species P. johannensisand P. teres.Copyright 2001 Annals of Botany Company Pleurothallis, Orchidaceae, floral biology, fly-pollination, Chloropidae, Phoridae, pollinator specificity, campo rupestre     

Social interaction in synthetic and natural microbial communities

Social interaction among cells is essential for multicellular complexity. But how do molecular networks within individual cells confer the ability to interact? And how do those same networks evolve from the evolutionary conflict between individual‐ and population‐level interests? Recent studies have dissected social interaction at the molecular level by analyzing both synthetic and natural microbial populations. These studies shed new light on the role of population structure for the evolution of cooperative interactions and revealed novel molecular mechanisms that stabilize cooperation among cells. New understanding of populations is changing our view of microbial processes, such as pathogenesis and antibiotic resistance, and suggests new ways to fight infection by exploiting social interaction. The study of social interaction is also challenging established paradigms in cancer evolution and immune system dynamics. Finding similar patterns in such diverse systems suggests that the same ‘social interaction motifs’ may be general to many cell populations.     

Helicobacter pylori antimicrobial resistance in a pediatric population

Background

The increasing prevalence of Helicobacter pylori (H. pylori) antimicrobial resistance, primarily for clarithromycin decreases the success of treatment. The aim of this study is to determine the local pattern of first‐line antimicrobials resistance and the eradication rate.

Material and Methods

Prospective cohort study of H. pylori infected patients (positive histological or cultural exams) treated at Centro Materno‐Infantil do Norte from January of 2013 to October of 2017. Susceptibility to 4 antibiotics: amoxicilin, metronidazole, clarithromycin, and levofloxacin were analyzed by E‐test (phenotypic resistance). The E‐test was chosen because it is simple and cost‐effective for routine susceptibility testing. Point mutations that confer clarithromycin resistance were surveyed (genotypic resistance). Eradication of H. pylori infection was defined by a negative urea breath test or fecal antigen 6‐8 weeks after the end of treatment.

Results

Of a total of 74 H. pylori infected patients, 16 were excluded because they had previous H. pylori treatment or severe systemic disease. Median age of infection cases was 15 years (3‐17 years). Eradication regimen used in all patients combined the use of 3 antibiotics (amoxicillin and metronidazole or clarithromycin) and proton pump inibhitor for 14 days and was tailored according antimicrobial susceptibility. 79.5% of the patients completed the treatment. The resistance rate for metronidazole and clarithromycin was 3.3% and 23.3%, respectively. There was no resistance for amoxicilin and levofloxacin. The rate of genotypic resistance to clarithromycin was 37.2%. The eradication rate was 97.8%.

Conclusions

The authors found a high resistance rate of H. pylori for clarithromycin in this northern portuguese pediatric center. This factor should determine a change in local current treatment, contraindicating the use of clarithromycin as a first‐line treatment for H. pylori infection in children. The high eradication rate maybe explained for the eradication treatment tailored according antimicrobial susceptibility.     

Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection

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Histomorphological and histochemical characteristics of the intestine of the Senegal sole, Solea senegalensis.

The Senegal sole, Solea senegalensis has been exploited extensively in aquaculture from different countries; at present an intensive production of larvae and adults is being achieved with some nutritional problems. Since this species displays very different life styles and feeding habits at different stages of its life history (larvae, metamorphosis, adults), and because digestive mucins are implicated in different physiological processes including: increase of digestive efficiency, promotion of macromolecules-absorption, buffering of intestinal fluid, prevention of proteolytic damage to the epithelium and defence against bacteria, etc., we studied the histomorphological aspects, as well as the histochemical distribution of carbohydrates, (PAS, Alcian Blue), proteins (Bromophenol Blue), lipids (Oil Red O, Black Sudan B) and glycoproteins (Horseradish peroxidase-conjugated lectins) in the intestinal epithelium of adult Solea senegalensis specimens. Our data are compared with those obtained in larvae and adults of this and other fish species. Primary and secondary folds, microvilli of the intestinal enterocytes, as well as mucous or goblet cells were observed with a scanning electron microscope. Enterocytes and mucocytes of the intestine of adult Solea senegalensis were characterized by a rich supply of sugar and oligosaccharides. Carbohydrates (glycogen and mucins), proteins and lipids were present in cytoplasm and microvilli--brush border--of the enterocytes, which contain GalNAc, GlcNAc, Man, Glc and sialic acid-N-acetyl-D-galactosamine glycoconjugates. Intestinal mucous cells were strongly or weakly stained with Alcian Blue (pH 2.5 and 1). PAS reactivity was intense in numerous goblet cells, but some cells were PAS unreactive or weakly stained. Some goblet cells were positive for Bromophenol Blue but numerous cells were unstained; thus many proteins and possibly lipids may be conjugated with sugars. A similar reactivity to WGA and to neuraminidase-WGA was identified in some intestinal goblet, which were Con A unreactive, indicating the absence of Man and/or Glc and NANA glycoconjugates. GalNAc residues were only scarcely present in glycoproteins of some goblet cells.     

Transferrin trojan horses as a rational approach for the biological delivery of therapeutic peptide domains.

One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein, such as human serum transferrin. To examine whether this is feasible, a peptide sequence cleavable by the human immunodeficiency virus type 1 protease (VSQNYPIVL) was inserted into various regions of human serum transferrin, and the resultant proteins were tested for function. Experimentally, molecular modeling was used to identify five candidate insertion sites in surface exposed loops of human serum transferrin that were distant from biologically active domains. These insertions were cloned using polymerase chain reaction mutagenesis, and the proteins were expressed using a baculovirus expression vector system. Analysis of the mutant proteins provided a number of important findings: (a) they retained native human serum transferrin function, (b) the inserted peptide sequence was surface exposed, and most importantly, (c) two of these mutants could be cleaved by human immunodeficiency virus-1 protease. In conclusion, this investigation has validated the use of human serum transferrin as a carrier protein for functional peptide domains introduced into its structure using protein engineering. These findings will be useful for developing a novel class of therapeutic agents for a broad spectrum of diseases.