FEMALE AND MALE GENETIC EFFECTS ON OFFSPRING PATERNITY: ADDITIVE GENETIC (CO)VARIANCES IN FEMALE EXTRA‐PAIR REPRODUCTION AND MALE PATERNITY SUCCESS IN SONG SPARROWS (MELOSPIZA MELODIA)

Ongoing evolution of polyandry, and consequent extra‐pair reproduction in socially monogamous systems, is hypothesized to be facilitated by indirect selection stemming from cross‐sex genetic covariances with components of male fitness. Specifically, polyandry is hypothesized to create positive genetic covariance with male paternity success due to inevitable assortative reproduction, driving ongoing coevolution. However, it remains unclear whether such covariances could or do emerge within complex polyandrous systems. First, we illustrate that genetic covariances between female extra‐pair reproduction and male within‐pair paternity success might be constrained in socially monogamous systems where female and male additive genetic effects can have opposing impacts on the paternity of jointly reared offspring. Second, we demonstrate nonzero additive genetic variance in female liability for extra‐pair reproduction and male liability for within‐pair paternity success, modeled as direct and associative genetic effects on offspring paternity, respectively, in free‐living song sparrows (Melospiza melodia). The posterior mean additive genetic covariance between these liabilities was slightly positive, but the credible interval was wide and overlapped zero. Therefore, although substantial total additive genetic variance exists, the hypothesis that ongoing evolution of female extra‐pair reproduction is facilitated by genetic covariance with male within‐pair paternity success cannot yet be definitively supported or rejected either conceptually or empirically.     

Rapid and specific identification of nitrile hydratase (NHase)-encoding genes in soil samples by polymerase chain reaction

A polymerase chain reaction (PCR) protocol was developed for the specific detection of genes coding nitrile hydratase (NHase). Primer design was based on the highly conserved sequences found in the coding region of the alpha-subunit gene corresponding to the metal-binding site. Purified genomic DNA from bacterial strains or directly from soil can serve as the target for the PCR, thus affording a simple and rapid method for screening NHase genes. The primer pairs, NHCo1/NHCo2 and NHFe1/NHFe2 yield PCR products corresponding to a partial coding sequence of cobalt and iron NHase genes, respectively. Using the PCR method, both types of iron- and cobalt-NHase-encoding genes were detected in DNA from pure cultures and soil samples. Furthermore consensus primers allowed rapid cloning and expression of novel NHases in Escherichia coli.     

Molecular phylogenetics and generic assessment in the tribe Morindeae (Rubiaceae–Rubioideae): How to circumscribe Morinda L. to be monophyletic?

Most of the species of the family Rubiaceae with flowers arranged in head inflorescences are currently classified in three distantly related tribes, Naucleeae (subfamily Cinchonoideae) and Morindeae and Schradereae (subfamily Rubioideae). Within Morindeae the type genus Morinda is traditionally and currently circumscribed based on its head inflorescences and syncarpous fruits (syncarps). These characters are also present in some members of its allied genera, raising doubts about the monophyly of Morinda. We perform Bayesian phylogenetic analyses using combined nrETS/nrITS/trnT-F data for 67 Morindeae taxa and five outgroups from the closely related tribes Mitchelleae and Gaertnereae to rigorously test the monophyly of Morinda as currently delimited and assess the phylogenetic value of head inflorescences and syncarps in Morinda and Morindeae and to evaluate generic relationships and limits in Morindeae. Our analyses demonstrate that head inflorescences and syncarps in Morinda and Morindeae are evolutionarily labile. Morinda is highly paraphyletic, unless the genera Coelospermum, Gynochthodes, Pogonolobus, and Sarcopygme are also included. Morindeae comprises four well-supported and morphologically distinct major lineages: Appunia clade, Morinda clade (including Sarcopygme and the lectotype M. royoc), Coelospermum clade (containing Pogonolobus and Morinda reticulata), and Gynochthodes–Morinda clade. Four possible alternatives for revising generic boundaries are presented to establish monophyletic units. We favor the recognition of the four major lineages of Morindeae as separate genera, because this classification reflects the occurrence of a considerable morphological diversity in the tribe and the phylogenetic and taxonomic distinctness of its newly delimited genera.     

The WEE1 regulators CPEB1 and miR-15b switch from inhibitor to activators at G2/M

MicroRNAs (miRNAs) in the AGO-containing RISC complex control messenger RNA (mRNA) translation by binding to mRNA 3′ untranslated region (3′UTR). The relationship between miRNAs and other regulatory factors that also bind to mRNA 3′UTR, such as CPEB1 (cytoplasmic polyadenylation element-binding protein), remains elusive. We found that both CPEB1 and miR-15b control the expression of WEE1, a key mammalian cell cycle regulator. Together, they repress WEE1 protein expression during G1 and S-phase. Interestingly, the 2 factors lose their inhibitory activity at the G2/M transition, at the time of the cell cycle when WEE1 expression is maximal, and, moreover, rather activate WEE1 translation in a synergistic manner. Our data show that translational regulation by RISC and CPEB1 is essential in cell cycle control and, most importantly, is coordinated, and can be switched from inhibition to activation during the cell cycle.     

Biomechanical properties of decellularized porcine common carotid arteries

To analyze the effects of decellularization on the biomechanical properties of porcine common carotid arteries, decellularization was performed by a detergent-enzymatic procedure that preserves extracellular matrix scaffold. Internal diameter, external diameter, and wall thickness were measured by optical microscopy on neighboring histological sections before and after decellularization. Rupture tests were conducted. Inner diameter and wall thickness were measured by echo tracking during pressure inflation from 10 to 145 mmHg. Distensibility and incremental elastic modulus were computed. At 10 mmHg, mean diameter of decellularized arteries was 5.38 mm, substantially higher than controls (4.1 mm), whereas decellularized and control arteries reached the same internal diameter (6.7 mm) at 145 mmHg. Wall thickness decreased 16% for decellularized and 32% for normal arteries after pressure was increased from 10 to 145 mmHg. Decellularized arteries withstood pressure >2,200 mmHg before rupture. At 145 mmHg, decellularization reduced compliance by 66% and increased incremental elastic modulus by 54%. Removal of cellular elements from media led to changes in arterial dimensions. Collagen fibers engaged more rapidly during inflation, yielding a stiffer vessel. Distensibility was therefore significantly lower (by a factor of 3) in decellularized than in normal vessels: reduced in the physiological range of pressures. In conclusion, decellularization yields vessels that can withstand high inflation pressures with, however, markedly different geometrical and biomechanical properties. This may mean that the potential use of a decellularized artery as a scaffold for the creation of xenografts may be compromised because of geometrical and compliance mismatch.     

Crystal structure of the receptor-binding protein head domain from Lactococcus lactis phage bIL170

Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry, is subject to lytic phage infections. In the first step of infection, phages recognize the host saccharidic receptor using their receptor binding protein (RBP). Here, we report the 2.30-A-resolution crystal structure of the RBP head domain from phage bIL170. The structure of the head monomer is remarkably close to those of other lactococcal phages, p2 and TP901-1, despite any sequence identity with them. The knowledge of the three-dimensional structures of three RBPs gives a better insight into the module exchanges which have occurred among phages.     

Genetic Connectivity among Populations of an Endangered Snake Species from Southeastern Australia (Hoplocephalus bungaroides, Elapidae)

For endangered species that persist as apparently isolated populations within a previously more extensive range, the degree of genetic exchange between those populations is critical to conservation and management. A lack of gene flow can exacerbate impacts of threatening processes and delay or prevent colonization of sites after local extirpation. The broad-headed snake, Hoplocephalus bungaroides, is a small venomous species restricted to a handful of disjunct reserves near Sydney, Australia. Mark-recapture studies have indicated low vagility for this ambush predator, suggesting that gene flow also may be low. However, our analyses of 11 microsatellite loci from 163 snakes collected in Morton National Park, from six sites within a 10-km diameter, suggest relatively high rates of gene flow among sites. Most populations exchange genes with each other, with one large population serving as a source area and smaller populations apparently acting as sinks. About half of the juvenile snakes, for which we could reliably infer parentage, were collected from populations other than those in which we collected their putative parents. As expected from the snakes' reliance on rocky outcrops during cooler months of the year, most gene flow appears to be along sandstone plateaux rather than across the densely forested valleys that separate plateaux. The unexpectedly high rates of gene flow on a landscape scale are encouraging for future conservation of this endangered taxon. For example, wildlife managers could conserve broad-headed snakes by restoring habitats near extant source populations in areas predicted to be least affected by future climate change.     

Fine-tuning acetyl-CoA carboxylase 1 activity through localization: functional genomics reveals a role for the lysine acetyltransferase NuA4 and sphingolipid metabolism in regulating Acc1 activity and localization

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.     

Modular bioreactor for primary human hepatocyte culture: medium flow stimulates expression and activity of detoxification genes

Down-regulation of detoxification genes, notably cytochrome P450 (CYPs), in primary hepatocyte cultures is a long-standing and major concern. We evaluated the influence of medium flow in this model. Hepatocytes isolated from 12 different liver donors were cultured either in a multichamber modular bioreactor (MCmB, flow rate 250-500 μL/min) or under standard/static conditions, and the expression of 32 genes, enzyme activities and biological parameters were measured 7-21 days later. mRNA expression of genes involved in xenobiotic/drug metabolism and transport, including CYP1A1, 1A2, 2B6, 2C9, 3A4 (and activities for some of them), UDP-glucuronosyltransferase (UGT) 1A1, UGT2B4, UGT2B7, glutathione S-transferase (GSTα), and multidrug resistance protein 1 (MDR1) and MRP2, were specifically up-regulated by medium flow as compared with static controls in all cultures tested. In 2-week-old cultures, expression of detoxification genes reached levels close to or higher than those measured in freshly isolated hepatocytes. In contrast, CYP2D6 and most of other tested genes were not affected by medium flow. We conclude that medium flow specifically interferes with, and up-regulates, the activity of xenosensors and/or the expression of detoxification genes in primary human hepatocytes. Down-regulation of detoxification genes in conventional (static) cultures is therefore partly a consequence of the absence of medium circulation.