ΦX174 Attenuation by Whole-Genome Codon Deoptimization.

Natural selection acting on synonymous mutations in protein-coding genes influences genome composition and evolution. In viruses, introducing synonymous mutations in genes encoding structural proteins can drastically reduce viral growth, providing a means to generate potent, live-attenuated vaccine candidates. However, an improved understanding of what compositional features are under selection and how combinations of synonymous mutations affect viral growth is needed to predictably attenuate viruses and make them resistant to reversion. We systematically recoded all nonoverlapping genes of the bacteriophage ΦX174 with codons rarely used in its Escherichia coli host. The fitness of recombinant viruses decreases as additional deoptimizing mutations are made to the genome, although not always linearly, and not consistently across genes. Combining deoptimizing mutations may reduce viral fitness more or less than expected from the effect size of the constituent mutations and we point out difficulties in untangling correlated compositional features. We test our model by optimizing the same genes and find that the relationship between codon usage and fitness does not hold for optimization, suggesting that wild-type ΦX174 is at a fitness optimum. This work highlights the need to better understand how selection acts on patterns of synonymous codon usage across the genome and provides a convenient system to investigate the genetic determinants of virulence.

The path from geology to indoor radon.

It is generally accepted that radon emission is strongly influenced by the geological characteristics of the bedrock. However, transport in-soil and entry paths indoors are defined by other factors such as permeability, building and architectural features, ventilation, occupation patterns, etc. The purpose of this paper is to analyze the contribution of each parameter, from natural to man-made, on the radon accumulation indoors and to assess potential patterns, based on 100 case studies in Romania. The study pointed out that the geological foundation can provide a reasonable explanation for the majority of the values recorded in both soil and indoor air. Results also showed that older houses, built with earth-based materials, are highly permeable to soil radon. Energy-efficient houses, on the other hand, have a tendency to disregard the radon potential of the geological foundation, causing a higher predisposition to radon accumulation indoors and decreasing the general indoor air quality.

New data on Amynodontidae (Mammalia, Perissodactyla) from Eastern Europe: Phylogenetic and palaeobiogeographic implications around the Eocene-Oligocene transition.

Amynodontidae is a family of Rhinocerotoidea (Mammalia, Perissodactyla) known from the late Early Eocene to the latest Oligocene, in North America and Eurasia. European Amynodontidae are very rare, and all remains belong almost exclusively to a single post-Grande Coupure genus from the Oligocene, Cadurcotherium. The "Grande Coupure" defines an extinctions and dispersal-generated originations event in Europe that is nearly contemporaneous with the Eocene-Oligocene transition. Perissodactyls are one of the major groups affected by this event: Palaeotheriidae went almost extinct during this crisis, whereas Rhinocerotidae appeared for the first time in Europe. Study of fossiliferous Eastern-European localities from this age is crucial for the understanding of this crisis. We report here three new localities of Amynodontidae in Eastern Europe. Two of them are dated from the Eocene (Morlaca, Romania; Dorog, Hungary), whereas the other is either Late Eocene or Early Oligocene (Dobârca, Romania). The skull from this latter locality belongs unexpectedly to the same individual as a previously described mandible attributed to "Cadurcodon" zimborensis. As a result, this specimen can be allocated to its proper locality, Dobârca, and is assigned to a new genus, Sellamynodon gen. nov. It is characterised by an extraordinary growth of the nuchal crest, a unique character among amynodontids. Along with this remarkable material from Dobârca, two specimens from another Romanian locality, Morlaca, have been recently discovered and are dated from the Late Eocene. They belong, as well as new material from Dorog (Middle Eocene, Hungary), to the genus Amynodontopsis, also found in North America. The new Hungarian material represents the earliest occurrence of Amynodontidae in Europe. New phylogenetic hypotheses of Rhinocerotoidea are proposed, including the new material presented here, and show that Amynodontidae may be closer to the polyphyletic family 'Hyracodontidae' than to Rhinocerotidae. Amynodontidae, with their deep preorbital fossa and extremely reduced premolars, display in fact a very derived condition, compared to rhinocerotids.

A dinosaurian facial deformity and the first occurrence of ameloblastoma in the fossil record.

Despite documentation of various types of neoplastic pathologies encountered in the vertebrate fossil record, no ameloblastic tumours have been recognised so far. Ameloblastoma is a benign neoplasic tumour with a strong preponderance for the mandible. Here, we report for the first time the presence of an ameloblastoma neoplasm in the lower jaw of a specimen referred to the derived non-hadrosaurid hadrosauroid dinosaur Telmatosaurus transsylvanicus from the uppermost Cretaceous of the Haeg Basin in Romania. The location, external appearance and internal structure of the pathological outgrowth provide clear evidence for the diagnosis of ameloblastoma in Telmatosaurus. This report extends the range of pathologies encountered in hadrosauroid dinosaurs. In addition, recognition of an ameloblastoma neoplasm in a taxon lying close to the origin of 'duck-billed' hadrosaurid dinosaurs confirms the predisposition of this clade towards neoplasia pathologies already in its basal members.

Reconstruction of a Large Anterior Ear Defect after Mohs Micrographic Surgery with a Cartilage Graft and Postauricular Revolving Door Flap.

A novel postauricular revolving door island flap and cartilage graft combination was employed to correct a large defect on the anterior ear of an 84-year-old man who underwent Mohs micrographic surgery for an antihelical squamous cell carcinoma. The defect measured 4.6 × 2.4 cm and spanned the antihelix, scapha, a small portion of the helix, and a large segment of underlying cartilage, with loss of structural integrity and anterior folding of the ear. The repair involved harvesting 1.5 cm(2) of exposed cartilage from the scaphoid fossa and then sculpting and suturing it to the remnant of the antihelical cartilage in order to recreate the antihelical crura. The skin of the posterior auricle was then incised just below the helical rim and folded anteriorly to cover the cartilage graft. The flap remained attached by a central subcutaneous pedicle, and an island designed using the full-thickness defect as a stencil template was pulled through the cartilage window anteriorly to resurface the anterior ear. This case demonstrates the use of the revolving door flap for coverage of large central ear defects with loss of cartilaginous support and illustrates how cartilage grafts may be used in combination with the flap to improve ear contour after resection.

Red Iron-Pigmented Tooth Enamel in a Multituberculate Mammal from the Late Cretaceous Transylvanian "Hateg Island".

Mammals that inhabit islands are characterized by peculiar morphologies in comparison to their mainland relatives. Here we report the discovery of a partial skull associated with the lower jaws of a Late Cretaceous (≈70 Ma) multituberculate mammal from the Carpathian "Hateg Island" of Transylvania, Romania. The mammal belongs to the Kogaionidae, one of the rare families that survived the Cretaceous-Paleogene mass extinction in Europe. The excellent preservation of this specimen allows for the first time description of the complete dentition of a kogaionid and demonstration that the enigmatic Barbatodon transylvanicus presents a mosaic of primitive and derived characters, and that it is phylogenetically basal among the Cimolodonta. Another peculiarity is the presence of red pigmentation in its tooth enamel. The red coloration is present on the anterior side of the incisors and on the cusps of most of the teeth. Energy-dispersive X-ray spectrometry (EDS) analysis reveals that the pigmented enamel contains iron, as in living placentals. Such a red pigmentation is known in living soricine shrews and many families of rodents, where it is thought to increase the resistance of the enamel to the abrasion that occurs during "grinding" mastication. The extended pattern of red pigment distribution in Barbatodon is more similar to that in eulipotyplan insectivores than to that in rodents and suggests a very hard diet and, importantly, demonstrates that its grasping incisors were not ever-growing. As inferred for other endemic Transylvanian vertebrates such as dwarf herbivorous dinosaurs and unusual theropod dinosaurs, insularity was probably the main factor of survival of such a primitive mammalian lineage relative to other mainland contemporaries of the Northern hemisphere.

Exploring naphthyl-carbohydrazides as inhibitors of influenza A viruses.

A library of hydrazide derivatives was synthesized to target non-structural protein 1 of influenza A virus (NS1) as a means to develop anti-influenza drug leads. The lead compound 3-hydroxy-N-[(Z)-1-(5,6,7,8-tetrahydronaphthalen-2-yl)ethylideneamino]naphthalene-2-carboxamide, which we denoted as "HENC", was identified by its ability to increase the melting temperature of the effector domain (ED) of the NS1 protein, as assayed using differential scanning fluorimetry. A library of HENC analogs was tested for inhibitory effect against influenza A virus replication in MDCK cells. A systematic diversification of HENC revealed the identity of the R group attached to the imine carbon atom significantly influenced the antiviral activity. A phenyl or cyclohexyl at this position yielded the most potent antiviral activity. The phenyl containing compound had antiviral activity similar to that of the active form of oseltamivir (Tamiflu), and had no detectable effect on cell viability.

G-quadruplex-generating polymerase chain reaction for visual colorimetric detection of amplicons.

We have developed a self-reporting polymerase chain reaction (PCR) system for visual colorimetric gene detection and distinction of single nucleotide polymorphisms (SNPs). Amplification is performed using target-specific primers modified with a 5'-end tail that is complementary to a G-quadruplex deoxyribozyme-forming sequence. At end-point, G-quadruplexes are forced to fold from PCR-generated duplex DNA and then are used to colorimetrically report the successful occurrence of PCR by assaying their peroxidase activity using a chromogenic substrate. Furthermore, primer design considerations for the G-quadruplex-generating PCR system have allowed us to visually distinguish SNPs associated with Mycobacterium tuberculosis drug resistance alleles.

Structure-based design of supercharged, highly thermoresistant antibodies.

Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that "supercharges" proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated ≈30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives.

First evidence of reproductive adaptation to "island effect" of a dwarf Cretaceous Romanian titanosaur, with embryonic integument in ovo.

BACKGROUND: The Cretaceous vertebrate assemblages of Romania are famous for geographically endemic dwarfed dinosaur taxa. We report the first complete egg clutches of a dwarf lithostrotian titanosaur, from Totesti, Romania, and its reproductive adaptation to the "island effect". METHODOLOGY/FINDINGS: The egg clutches were discovered in sequential sedimentary layers of the Maastrichtian Sânpetru Formation, Totesti. The occurrence of 11 homogenous clutches in successive strata suggests philopatry by the same dinosaur species, which laid clutches averaging four ∼12 cm diameters eggs. The eggs and eggshells display numerous characters shared with the positively identified material from egg-bearing level 4 of the Auca Mahuevo (Patagonia, Argentina) nemegtosaurid lithostrotian nesting site. Microscopic embryonic integument with bacterial evidences was recovered in one egg. The millimeter-size embryonic integument displays micron size dermal papillae implying an early embryological stage at the time of death, likely corresponding to early organogenesis before the skeleton formation. CONCLUSIONS/SIGNIFICANCE: The shared oological characters between the Hateg specimens and their mainland relatives suggest a highly conservative reproductive template, while the nest decrease in egg numbers per clutch may reflect an adaptive trait to a smaller body size due to the "island effect". The combined presence of the lithostrotian egg and its embryo in the Early Cretaceous Gobi coupled with the oological similarities between the Hateg and Auca Mahuevo oological material evidence that several titanosaur species migrated from Gondwana through the Hateg Island before or during the Aptian/Albian. It also suggests that this island might have had episodic land bridges with the rest of the European archipelago and Asia deep into the Cretaceous.

In vitro selection of RNA aptamers to a small molecule target.

This unit describes the selection of aptamers from a single-stranded RNA pool that bind to small molecule targets. Aptamers generated by this type of selection experiment can potentially function as receptors for small molecules in numerous applications, including medical diagnostics, therapeutics, and environmental monitoring. This unit describes two modes of selection, one by column filtration and one by batch selection.

Motifs from the deep.

Because of the increasing recognition of the importance of non-coding RNAs in gene regulation, there is considerable interest in identifying RNA motifs in genomic data. In a recent report in BMC Genomics, Breaker and colleagues describe a new algorithm for identifying functional noncoding RNAs in metagenomic sequences of marine organisms, a strategy that may be particularly effective for discovering new and unique riboswitches.

A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families.

Most basophilic serine/threonine kinases preferentially phosphorylate substrates with Arg at P-3 but vary greatly in additional strong preference for Arg at P-2 or P-5. The structural basis for P-2 or P-5 preference is known for two AGC kinases (family of protein kinases A, G, and C) in which it is mediated by a single pair of acidic residues (PEN+1 and YEM+1). We sought a general understanding of P-2 and P-5 Arg preference. The strength of Arg preference at each position was assessed in 15 kinases using a new degenerate peptide library approach. Strong P-2 or P-5 Arg preference occurred not only in AGC kinases (7 of 8 studied) but also in calmodulin-dependent protein kinase (CAMK, 1 of 3) and Ste20 (STE) kinases (2 of 4). Analysis of sequence conservation demonstrated almost perfect correlation between (a) strong P-2 or P-5 Arg preference and (b) acidic residues at both PEN+1 and YEM+1. Mutation of two kinases (PKC-theta and p21-activated kinase 1 (PAK1)) confirmed critical roles of both PEN+1 and YEM+1 residues in determining strong R-2 Arg preference. PAK kinases were unique in having exceptionally strong Arg preference at P-2 but lacking strong Arg preference at P-3. Preference for Arg at P-2 was so critical to PAK recognition that PAK1 activity was virtually eliminated by mutating the PEN+1 or YEM+1 residues. The fact that this specific pair of acidic residues has been repeatedly and exclusively used by evolution for conferring strong Arg preference at two different substrate positions in three different kinase families implies it is uniquely well suited to mediate sufficiently good substrate binding without unduly restricting product release.