Molecular evolution of VP3, VP1, 3Cpro and 3Dpol coding regions in coxsackievirus group A type 24 variant isolates from acute hemorrhagic conjunctivitis in 2011 in Okinawa,Japan

A large acute hemorrhagic conjunctivitis (AHC) outbreak occurred in 2011 in Okinawa Prefecture in Japan. Ten strains of coxsackievirus group A type 24 variant (CA24v) were isolated from patients with AHC and full sequence analysis of the VP3, VP1, 3C^pro and 3D^pol coding regions performed. To assess time‐scale evolution, phylogenetic analysis was performed using the Bayesian Markov chain Monte Carlo method. In addition, similarity plots were constructed and pairwise distance (p‐distance) and positive pressure analyses performed. A phylogenetic tree based on the VP1 coding region showed that the present strains belong to genotype 4 (G4). In addition, the present strains could have divided in about 2010 from the same lineages detected in other countries such as China, India and Australia. The mean rates of molecular evolution of four coding regions were estimated at about 6.15 to 7.86 × 10^?3 substitutions/site/year. Similarity plot analyses suggested that nucleotide similarities between the present strains and a prototype strain (EH24/70 strain) were 0.77–0.94. The p‐distance of the present strains was relatively short (<0.01). Only one positive selected site (L25H) was identified in the VP1 protein. These findings suggest that the present CA24v strains causing AHC are genetically related to other AHC strains with rapid evolution and emerged in around 2010.

     

Sequencing and Analysis of Globally Obtained Human Respiratory Syncytial Virus A and B Genomes

Background

Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in children globally, with nearly all children experiencing at least one infection by the age of two. Partial sequencing of the attachment glycoprotein gene is conducted routinely for genotyping, but relatively few whole genome sequences are available for RSV. The goal of our study was to sequence the genomes of RSV strains collected from multiple countries to further understand the global diversity of RSV at a whole-genome level.

Methods

We collected RSV samples and isolates from Mexico, Argentina, Belgium, Italy, Germany, Australia, South Africa, and the USA from the years 1998-2010. Both Sanger and next-generation sequencing with the Illumina and 454 platforms were used to sequence the whole genomes of RSV A and B. Phylogenetic analyses were performed using the Bayesian and maximum likelihood methods of phylogenetic inference.

Results

We sequenced the genomes of 34 RSVA and 23 RSVB viruses. Phylogenetic analysis showed that the RSVA genome evolves at an estimated rate of 6.72 × 10^-4 substitutions/site/year (95% HPD 5.61 × 10^-4 to 7.6 × 10^-4) and for RSVB the evolutionary rate was 7.69 × 10^-4 substitutions/site/year (95% HPD 6.81 × 10^-4 to 8.62 × 10^-4). We found multiple clades co-circulating globally for both RSV A and B. The predominant clades were GA2 and GA5 for RSVA and BA for RSVB.

Conclusions

Our analyses showed that RSV circulates on a global scale with the same predominant clades of viruses being found in countries around the world. However, the distribution of clades can change rapidly as new strains emerge. We did not observe a strong spatial structure in our trees, with the same three main clades of RSV co-circulating globally, suggesting that the evolution of RSV is not strongly regionalized.     

A testis‐specific gene within a widely expressed gene: Contrasting evolutionary patterns of two differentially expressed mammalian proteins encoded by a single gene,CAMK4

Understanding the patterns of genetic variations within fertility‐related genes and the evolutionary forces that shape such variations is crucial in predicting the fitness landscapes of subsequent generations. This study reports distinct evolutionary features of two differentially expressed mammalian proteins [CaMKIV (Ca²⁺/calmodulin‐dependent protein kinase IV) and CaS (calspermin)] that are encoded by a single gene, CAMK4. The multifunctional CaMKIV, which is expressed in multiple tissues including testis and ovary, is evolving at a relatively low rate (0.46–0.64 × 10^?9 nucleotide substitutions/site/year), whereas the testis‐specific CaS gene, which is predominantly expressed in post‐meiotic cells, evolves at least three to four times faster (1.48–1.98 × 10^?9 substitutions/site/year). Concomitantly, maximum‐likelihood‐based selection analyses revealed that the ubiquitously expressed CaMKIV is constrained by intense purifying selection and, therefore, remained functionally highly conserved throughout the mammalian evolution, whereas the testis‐specific CaS gene is under strong positive selection. The substitution rates of different mammalian lineages within both genes are positively correlated with GC content, indicating the possible influence of GC‐biased gene conversion on the estimated substitution rates. The observation of such unusually high GC content of the CaS gene (≈74%), particularly in the lineage that comprises the bovine species, suggests the possible role of GC‐biased gene conversion in the evolution of CaS that mimics positive selection.     

Evaluation of consistency in quantification of gene copy number by real‐time reverse transcription quantitative polymerase chain reaction and virus titer by plaque‐forming assay for human respiratory syncytial virus

The plaque‐forming assay is the standard technique for determining viral titer, and a critical measurement for investigating viral replication. However, this assay is highly dependent on experimental technique and conditions. In the case of human respiratory syncytial virus (RSV) in particular, it can be difficult to objectively confirm the accuracy of plaque‐forming assay because the plaques made by RSV are often small and unclear. In recent studies, RT‐qPCR methods have emerged as a supportive procedure for assessment of viral titer, yielding highly sensitive and reproducible results. In this report, we compare the viral replication, as determined by plaque‐forming assay, and the copy numbers of RSV genes NS1, NS2, N, and F, as determined by RT‐qPCR. Two real‐time PCR systems, SYBR Green and TaqMan probe, gave highly similar results for measurement of copy numbers of RSV N genes of virus subgroups A. We determined the RSV gene copy numbers in the culture cell supernatant and cell lysate measured at various multiplicities of infection. We found that copy number of the RSV N gene in the culture supernatant and cell lysate was highly correlated with plaque‐forming units. In conclusion, RT‐qPCR measurement of RSV gene copy number was highly dependent on viral titer, and the detailed comparison between each gene copy number and virus titer should be useful and supportive in confirming RSV plaque‐forming assay and virus dynamics. The technique may also be used to estimate the amount of RSV present in clinical specimens.

     

Two sites of photoinhibition of the electron transfer in oxygen evolving and Tris-treated PS II membrane fragments from spinach

Photoinhibition was analyzed in O₂-evolving and in Tris-treated PS II membrane fragments by measuring flash-induced absorption changes at 830 nm reflecting the transient P680⁺ formation and oxygen evolution. Irradiation by visible light affects the PS II electron transfer at two different sites: a) photoinhibition of site I eliminates the capability to perform a stable charge separation between P680⁺ and Q_A ^- within the reaction center (RC) and b) photoinhibition of site II blocks the electron transfer from Y_Z to P680⁺. The quantum yield of site I photoinhibition (2–3×10^-7 inhibited RC/quantum) is independent of the functional integrity of the water oxidizing system. In contrast, the quantum yield of photoinhibition at site II depends strongly on the oxygen evolution capacity. In O₂-evolving samples, the quantum yield of site II photoinhibition is about 10^-7 inhibited RC/quantum. After selective elimination of the O₂-evolving capacity by Tris-treatment, the quantum yield of photoinhibition at site II depends on the light intensity. At low intensity (<3 W/m²), the quantum yield is 10^-4 inhibited RC/quantum (about 1000 times higher than in oxygen evolving samples). Based on these results it is inferred that the dominating deleterious effect of photoinhibition cannot be ascribed to an unique target site or a single mechanism because it depends on different experimental conditions (e.g., light intensity) and the functional status of the PS II complex.Abbreviations A₈₃₀ absorption change at 830 nm - P680 primary electron donor of PS II - PS II photosystem II - Mes 2(N-morpholino)ethansulfonic acid - Q_A, Q_B primary and secondary acceptors of PS II - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbohydrazide - FWHM fullwidth at half maximum - Ph-p-BQ phenyl-p-benzoquinone - PFR photon fluence rate - Pheo pheophytin - RC reaction center     

Molecular Characterization of Human Respiratory Syncytial Virus in the Philippines, 2012-2013

Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infections in infants and children worldwide. We performed molecular analysis of HRSV among infants and children with clinical diagnosis of severe pneumonia in four study sites in the Philippines, including Biliran, Leyte, Palawan, and Metro Manila from June 2012 to July 2013. Nasopharyngeal swabs were collected and screened for HRSV using real-time polymerase chain reaction (PCR). Positive samples were tested by conventional PCR and sequenced for the second hypervariable region (2^nd HVR) of the G gene. Among a total of 1,505 samples, 423 samples were positive for HRSV (28.1%), of which 305 (72.1%) and 118 (27.9%) were identified as HRSV-A and HRSV-B, respectively. Two genotypes of HRSV-A, NA1 and ON1, were identified during the study period. The novel ON1 genotype with a 72-nucleotide duplication in 2^nd HVR of the G gene increased rapidly and finally became the predominant genotype in 2013 with an evolutionary rate higher than the NA1 genotype. Moreover, in the ON1 genotype, we found positive selection at amino acid position 274 (p<0.05) and massive O- and N-glycosylation in the 2^nd HVR of the G gene. Among HRSV-B, BA9 was the predominant genotype circulating in the Philippines. However, two sporadic cases of GB2 genotype were found, which might share a common ancestor with other Asian strains. These findings suggest that HRSV is an important cause of severe acute respiratory infection among children in the Philippines and revealed the emergence and subsequent predominance of the ON1 genotype and the sporadic detection of the GB2 genotype. Both genotypes were detected for the first time in the Philippines.     

Variable evolutionary rates in the molecular evolution of mammalian growth hormones

In mammals pituitary growth hormone (GH) shows a slow basal rate of evolution (0.22 ± 0.03 × 10^–9 substitutions/amino acid site/year) which appears to have increased by at least 25–50-fold on two occasions, during the evolution of primates (to at least 10.8 ± 1.3 X 10^–9 substitutions/amino acid site/year) and artiodactyl ruminants (to at least 5.6 ± 1.3 X 10^–9 substitutions/amino acid site/year). That these rate increases are real, and not due to inadvertent comparison of nonorthologous genes, was established by showing that features of the GH gene sequences that are not expressed as mature hormone do not show corresponding changes in evolutionary rate. Thus, analysis of nonsynonymous substitutions in the coding sequence for the mature protein confirmed the rate increases seen in the primate and ruminant GHs, but analysis of nonsynonymous substitutions in the signal peptide sequence, synonymous substitutions in the coding sequence for signal peptide or mature protein, and 5 and 3 untranslated sequences showed no statistically significant changes in evolutionary rate. Evidence that the increases in evolutionary rate are probably due to positive selection is provided by the observation that in the cases of both ruminant and primate GHs the periods of rapid evolution were followed by a return to a slow rate similar to the basal rate seen in other mammalian GHs. Differences between the biological properties of GHs have been identified which may relate to these periods of rapid adaptive molecular evolution. On the basis of sequence data currently available (but excluding rodent GHs which show an intermediate rate, the basis of which is not clear) for most (90%) of evolutionary time mammalian GHs have been in the slow phase of evolution, with possibly most of the few amino acid substitutions that have occurred being neutral in nature. But most (80%) of the amino acid substitutions that have been introduced into GH during the course of mammalian evolution have been accepted during the rapid phases and were adaptive in nature.     

Molecular clock of silent substitution: At least six-fold preponderance of silent changes in mitochondrial genes over those in nuclear genes

Summary For each of eleven different types of nuclear genes, comparisons of the protein coding sequences were made between human, mouse and rat pairwisely, and the evolutionary rate of silent substitution, v _S ^nucl. , was estimated. It is shown that the v _S ^nucl. is not only very high (=5.37×10^–9/site/yr), but also approximately uniform for different genes regardless of the types, which confirms our previous results (Miyata et al. 1980b). This is in sharp contrast to the rate of protein evolution which differes greatly from protein to protein. Furthermore the v _S ^nucl. is shown to be approximately constant with respect to different divergence times, at least within a short time period (75 Myr). Based on these observations, we propose a new molecular clock which has several advantages over a protein clock. Using this clock, we show that the rate of amino acid replacement in the immunoglobulin Ck gene of b4 rabbit is unexpectedly high, almost comparable to the rate of silent changes. This rate may be the highest one for protein evolution that we know so far. We further examine the rate of silent substitutions in mitochondrial genes comparing mouse and rat. Surprisingly the rate is extremely high (35×10^–9/site/yr), at least 6-times as high as the corresponding rate of nuclear genes. Based on the estimate, we discuss a possible origin of the rapid rate found in mitochondrial DNA.     

Application of a stability statistic to international maize yield trials

Summary Genotype x environment (GE) interaction encountered in experiments complicates genotype selection and varietal recommendation. The integration of yield and stability of genotypes into a single parameter may make selection and recommendation easier. Kang developed a rank-sum method that allows selection for both yield and the stability variance statistics ( _i ² or s _i ² ) of Shukla. The objective of this research was to compare the rank-sum selection method to selection based on yield alone in five international maize (Zea mays L.) yield trials. Ranks were assigned for yield (the highest mean yield received a rank of 1) and for _i ² and s _i ² (the lowest value received a rank of 1). The yield and _i ² ranks and/or the yield and s _i ² ranks for each genotype were summed. Each trial contained two reference entries (REs). Yield rank or rank-sum of each genotype was compared to yield rank or rank-sum of the best RE (BRE). GE interaction was significant for all trials. Heterogeneity in the GE interaction due to the linear effect of a covariate (differences in fertility and/or cultural practices) was significant in Trials 1, 2, and 5. Overall, in all trials, 29 genotypes were selected on the basis of yield alone. On the basis of _i ² and yield rank-sum, 32 genotypes were identified, with 11 being lower yielding than the 29 yield-based selections. On the basis of s _i ² and yield rank-sum, 31 genotypes were selected, with 11 being lower yielding than the yield-bases selections. Obviously, yield is sacrificed when the rank-sum method is used in the selection process. However, selection based on yield alone may not be adequate when GE interaction is significant because of testing in diverse environments.     

Stone age diseases and modern AIDS

Background

Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of ~10^-4 substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases with fidelities approximately 10 000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA virus evolution rates were first suggested in evolution experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminivirus mutation rates remain unanswered.

Results

We determined the short-term evolution rate of MSV using full genome analysis of virus populations initiated from cloned genomes. Three wild type viruses and three defective artificial chimaeric viruses were maintained in planta for up to five years and displayed evolution rates of between 7.4 × 10^-4 and 7.9 × 10^-4 subs/site/year.

Conclusion

These MSV evolution rates are within the ranges observed for other ssDNA viruses and RNA viruses. Although no obvious evidence of positive selection was detected, the uneven distribution of mutations within the defective virus genomes suggests that some of the changes may have been adaptive. We also observed inter-strand nucleotide substitution imbalances that are consistent with a recent proposal that high mutation rates in geminiviruses (and possibly ssDNA viruses in general) may be due to mutagenic processes acting specifically on ssDNA molecules.     

Adaptive evolution of chloroplast genomes in ancestral grasses

The grass family, Poaceae, is one of the most successful families among angiosperms. Although it has long been suggested that the chloroplast genomes of the Poaceae have undergone an elevated evolutionary rate compared to other angiosperms, little was known about the details of this phenomenon. By using chloroplast genome data from 31 seed plants species, we recently showed that episodic rate acceleration occurred in the common ancestral branch of the core Poaceae (a clade formed by rice Oryza sativa, wheat Triticum aestivum, maize Zea mays and their allies) accompanied by elevated non-synonymous/synonymous rate ratio, while the rate and the non-synonymous/synonymous rate ratio reverted to the low level typical of most monocot species in the terminal branches. It was further shown that positive selection or adaptive evolution operated in several chloroplast proteins during the evolution of ancestral grasses, and the amino acid sites which putatively experienced positive selection have been identified. These findings illustrate the importance of future works of structural biological research of chloroplasts to understand the background of the evolution of the successful group, Poaceae.Key words: rate acceleration, positive selection, non-synonymous/synonymous rate ratio, Poaceae, structural biologyThe grass family, Poaceae, is one of the largest plant families, comprising about 10,000 species including the most important agricultural plants, rice, wheat and maize, as well as grass-dominated ecosystems which comprise about one-third of Earth''s vegetative cover and support a vast number of animals.¹ The chloroplast genes of the grass family Poaceae are known to have undergone accelerations in their evolutionary rates,^2,3 yet little was known about the details of this acceleration. It has become increasingly feasible to estimate the phylogenetic tree of angiosperms and to clarify the tempo and model of molecular evolution by using chloroplast genome sequences.^4–6 By using chloroplast genome data from 13 monocot species and 18 species from dicots and gymnosperms (31 species in total), we recently examined the details of this phenomenon from several aspects.Figure 1 shows the Poales + Musa part of the chloroplast ML tree of the 31 species, and the elongated branches of Poaceae show the rate acceleration in that particular group. Moreover, longer distances of the Poaceae species from Musa than the Typha/Musa distance by more than two times both in terms of non-synonymous and synonymous substitutions (Fig. 1) indicate that both types of substitutions have undergone rate acceleration along the line leading to Poaceae. To explore the pattern of rate change during the course of grass evolution more in detail, we estimated the time-scale of Angiosperm phylogeny with a relaxed clock based on the Bayesian method implemented in MCMCTREE program of PAML.⁷ As is apparent from Figure 1, the molecular evolutionary rate (substitution rate) differs among different lineages, and therefore we used the relaxed clock method which takes account of the evolution of the evolutionary rate in estimating the divergence times and the pattern of rate change. Based on fossil evidence, we assumed the followings in calibrating the relaxed clock; (1) the Gymnosperm/Angiosperm divergence occurred at 280–310 Ma (million years ago),^5,6 (2) the divergence of Poales from other monocots occurred before 115 Ma,^8,9 and (3) the most basal divergence in eudicots occurred before 125 Ma.⁵Open in a separate windowFigure 1Poales + Musa part of the chloroplast genome tree from 31 seed plant species. The branch lengths are proportional to the estimated lengths by the ML with the codon-substitution model (CODEML in PAML⁷). Non-synonymous (d_N) and synonymous (d_S) distances of Poales from Musa and ω = d_N/d_S (along branches) were estimated by the same program.We further gave a constraint of >65 Ma for the Zea/Oryza divergence based on the recent finding of 65 Ma grass phytoliths in dinosaur coprolites which places the diversification of the grasses to the Cretaceous period.^10,11 As a result, it was found that the rate acceleration was limited to the common ancestral branch of Poaceae after they diverged from Musa and that the rate reverted to the slow rate typical of most monocot species in the terminal branches. Even when the constraint was removed, almost the same pattern of rate change was obtained, suggesting that our conclusion regarding accelerated rate in the ancestral grasses followed by the reverted slow rate in contemporary Poaceae is robust.Non-synonymous/synonymous rate ratio (ω = d_N/d_S) is widely used as an indicator of positive selection or adaptive evolution.¹² Figure 1 also indicates a pronounced increase of ω ratio in the common ancestral branch of Poaceae after their divergence from Typha, followed by reversion in the terminal branches to the lower level typical of basal lineages. The elevation of the ω ratio can be due either by adaptive evolution or by relaxation of selective constraints. An ω value higher than 1 is usually regarded as an evidence of adaptive evolution, but since the ω values shown in the figure averages over the entire protein-encoding genes, we would not obtain such a high value even if positive selection operated in some regions of some proteins. To identify positively selected sites, among 75 protein-encoding genes, we at first selected 14 genes, for which the model with higher ω in the ancestral grass branch than others is significantly better than the model with homogeneous ω, and by using the branch-site model,^13,14 we identified 5 genes (atpE, cemA, clpP, rpoB and rps11) which have p value of the branch-site likelihood ratio test less than 0.05 and contain positively selected sites. The amino acid sites and substitutions identified to have experienced positive selection are as follows; atpE (2T→K, 17S→C, 41A→N, 64M→W, 132V→W), cemA (55N→R, 76Y→K, 161W→F, 190I→F, 204I→C), clpP (26R→V, 48V→T, 86F→T, 112I→P, 134E→R, 182T→D), rpoB (90R→F, 338G→K, 1026G→N), rps11 (54V→P, 62A→S, 82A→R, 105L→S, 115R→A, 120L→R) where the numberings of amino acid sites are those of Zea mays.¹⁵ We anticipate that these amino acid substitutions might have relevance to the successful evolution of grasses. To clarify the implication of these findings, structural biological studies of chloroplast proteins on how the amino acid changes affect their functions are needed.Rates of molecular evolution can be potentially linked to life history of organisms. By comparing evolutionary rates of chloroplast, nuclear and mitochondrial genes across five groups of angiosperms, Smith and Donoghue¹⁶ found that the rates are generally low in trees/shrubs compared to related herbs. Our finding, however, suggests that the pattern of rate change during evolution is more complicated than has previously been anticipated, and highlights the need for distinguishing rates of internal branches and those of terminal branches rather than averaging along a lineage in addressing this complicated problem.As Theodosius Dobzhansky¹⁷ wrote, nothing in biology makes sense except in the light of evolution, and the functional background of the molecular machinery in chloroplasts should be interpreted in the light of evolution. We hope our molecular evolutionary analysis of the chloroplast genomes is the first step towards this goal, and hope collaboration of molecular evolutionists with structural biologists becomes fruitful in the future.     

The role of reactive oxygen species and subsequent DNA-damage response in the emergence of resistance towards resveratrol in colon cancer models

In spite of the novel strategies to treat colon cancer, mortality rates associated with this disease remain consistently high. Tumour recurrence has been linked to the induction of resistance towards chemotherapy that involves cellular events that enable cancer cells to escape cell death. Treatment of colon cancer mainly implicates direct or indirect DNA-damaging agents and increased repair or tolerances towards subsequent lesions contribute to generate resistant populations. Resveratrol (RSV), a potent chemosensitising polyphenol, might share common properties with chemotherapeutic drugs through its indirect DNA-damaging effects reported in vitro. In this study, we investigated how RSV exerts its anticancer effects in models of colon cancer with a particular emphasis on the DNA-damage response (DDR; PIKKs-Chks-p53 signalling cascade) and its cellular consequences. We showed in vitro and in vivo that colon cancer models could progressively escape the repeated pharmacological treatments with RSV. We observed for the first time that this response was correlated with transient activation of the DDR, of apoptosis and senescence. In vitro, a single treatment with RSV induced a DDR correlated with S-phase delay and apoptosis, but prolonged treatments led to transient micronucleations and senescence phenotypes associated with polyploidisation. Ultimately, stable resistant populations towards RSV displaying higher degrees of ploidy and macronucleation as compared to parental cells emerged. We linked these transient effects and resistance emergence to the abilities of these cells to progressively escape RSV-induced DNA damage. Finally, we demonstrated that this DNA damage was triggered by an overproduction of reactive oxygen species (ROS) against which cancer cells could adapt under prolonged exposure to RSV. This study provides a pre-clinical analysis of the long-term effects of RSV and highlights ROS as main agents in RSV''s indirect DNA-damaging properties and consequences in terms of anticancer response and potent resistance emergence.In spite of chemotherapy and systematic screening for people at risk, the mortality rate of colorectal cancer (CCR) remains high and stable, with 6 00 000 deaths per year. The 5-year survival ranges from 90% in patients with stage I to 10% in patients with stage IV disease.¹ This low success rate in the treatment of CCR results from many failures associated with high resistance and the risk of metastasis. Resistance has been related to the adaptations that inhibit the ability of tumour cells to die by apoptosis. These adaptations are well known to implicate other mechanisms that control the pro-survival/pro-apoptotic balance including autophagy and senescence.^{2, 3} Senescence constitutes a powerful resistance mechanism towards CCR chemotherapy, which typically involves DNA-damaging agents nowadays.⁴ Indeed, cancer cells respond to DNA damage by initiating the DNA-damage response (DDR), which induces cell cycle delay, more prolonged growth arrests (senescence) and apoptosis of the lethally damaged cells. The DDR is initiated by the phosphatidylinositol 3-kinase-related kinases (PIKKs) ATM, ATR and DNA-PKcs that elicit DNA repair, concomitant cell cycle progression and apoptosis regulations through the Chk1/2-p53-p21 signalling cascade.⁵One promising approach to counteract chemoresistance could be the use of adjuvants. Among them, resveratrol (trans-3,4′,5-trihydroxystilbene; RSV) has been shown to prevent or delay the different steps of carcinogenesis^{6, 7} through its capacity to inhibit cell cycle progression, to induce apoptosis,^{8, 9} autophagic-related cell death¹⁰ and senescence via DNA damage.^{11, 12} Because of its low toxicity in animal models⁶ and in humans^{13, 14, 15}, RSV has been proposed as a potent adjuvant to sensitise cancer cells to various anticancer drugs,^{16, 17, 18} cytokines (e.g. TRAIL),¹⁹ and ionising radiation.²⁰ Recently, phase I/II clinical trials have shown that administration of RSV was correlated with a 5% reduction of tumour growth in patients with confirmed CCR, despite its low bioavailability in its unmetabolised form.²¹ Accordingly, we have proved in vitro that RSV metabolites were able to induce the DDR, subsequent S-phase delay and apoptosis and improved the efficacy of anticancer drugs.²²Considering that RSV behave as an apparent DNA-damaging agent, we investigated its long-term effects towards CCR models with a particular emphasis on its DNA-damaging properties and the related consequences in vitro and in vivo in terms of resistance. We demonstrated that RSV-induced DNA damage was triggered by an overproduction of reactive oxygen species (ROS) against which cancer cells could adapt under prolonged exposure to RSV. These effects have to be considered pre-clinically to further point out RSV as a potent chemosensitising agent.     

MPK‐1/ERK is required for the full activity of resveratrol in extended lifespan and reproduction

Resveratrol (RSV) extends the lifespan of various organisms through activation of sirtuin. However, whether RSV‐mediated longevity is entirely dependent upon sirtuin is still controversial. Thus, understanding additional mechanisms concerning the genetic requirements for the biological activity of RSV needs to be clarified to utilize the beneficial effects of RSV. In this study using Caenorhabditis elegans as a model system, we found that MPK‐1 (an ERK homolog) signaling is necessarily required for RSV‐mediated longevity of sir‐2.1/sirtuin mutants as well as for wild‐type worms. We demonstrated that MPK‐1 contributes to RSV‐mediated longevity through nuclear accumulation of SKN‐1 in a SIR‐2.1/DAF‐16 pathway‐independent manner. The positive effect of RSV in regulating lifespan was completely abolished by RNA interference against mpk‐1 in the sir‐2.1 and daf‐16 mutants, strongly indicating that the MPK‐1/SKN‐1 pathway is involved in RSV‐mediated longevity, independently of SIR‐2.1/DAF‐16. We additionally found that RSV protected worms from oxidative stress via MPK‐1. In addition to organismal aging, RSV prevented the age‐associated loss of mitotic germ cells, brood size, and reproductive span through MPK‐1 in C. elegans germline. Therefore, our findings not only provide new mechanistic insight into the controversial effects of RSV on organismal longevity, but additionally have important implications in utilizing RSV to improve the outcome of aging‐related diseases.     

Local interleukin-10 production during respiratory syncytial virus bronchiolitis is associated with post-bronchiolitis wheeze

Background

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants. Following RSV bronchiolitis, 50% of children develop post-bronchiolitis wheeze (PBW). Animal studies have suggested that interleukin (IL)-10 plays a critical role in the pathogenesis of RSV bronchiolitis and subsequent airway hyperresponsiveness. Previously, we showed that ex vivo monocyte IL-10 production is a predictor of PBW. Additionally, heterozygosity of the single-nucleotide polymorphism (SNP) rs1800872 in the IL10 promoter region was associated with protection against RSV bronchiolitis.

Methods

This study aimed to determine the in vivo role of IL-10 in RSV pathogenesis and recurrent wheeze in a new cohort of 235 infants hospitalized for RSV bronchiolitis. IL-10 levels in nasopharyngeal aspirates (NPAs) were measured at the time of hospitalization and the IL10 SNP rs1800872 genotype was determined. Follow-up data were available for 185 children (79%).

Results

Local IL-10 levels during RSV infection turned out to be higher in infants that later developed physician diagnosed PBW as compared to infants without PBW in the first year after RSV infection (958 vs 692 pg/ml, p = 0.02). The IL10 promoter SNP rs1800872 was not associated with IL-10 concentration in NPAs.

Conclusion

The relationship between high local IL-10 levels during the initial RSV infection and physician diagnosed PBW provides further evidence of the importance of the IL-10 response during RSV bronchiolitis.     

Molecular characterization of Streptococcus pneumoniae,particularly serotype19A/ST320, which emerged in Krasnoyarsk,Russia

Streptococcus pneumoniae , a common human pathogen, colonizes the nasopharynx and causes diseases including acute otitis media (AOM). Herein, pneumococcal serotype distributions in children before and after PCV7 vaccination and in patients with pneumococcal disease in Siberian Russia (Krasnoyarsk) are reported. Analyses included antimicrobial susceptibility testing, sequence typing (ST), pulsed field gel electrophoresis, virulence‐related surface protein gene (VSG) typing with novel primers and structural analysis by scanning electron microscopy. In healthy children (HC) prior to administration of PCV7, drug‐susceptible serotype23F/ST1500 was a major pneumococcal genotype. In the PCV7 trial, multidrug‐resistant serotype19A/ST320 emerged in vaccinees after PCV7, exhibiting a PCV7‐induced serotype replacement. Multidrug‐resistant serotype19A/ST320 was evident in patients with AOM. Community‐acquired pneumonia (CAP) isolates showed genetic similarities to the AOM (ST320) genotype, constituting a common non‐invasive AOM–CAP group. In contrast, meningitis isolates were more divergent. Overall, 25 ST types were identified; five (20%) of which were Krasnoyarsk‐native. Regarding VSGs, PI‐1 (rlrA /rrgB ), PI‐2 (pitA /B ), psrP and cbpA were present at 54.3%, 38.6%, 48.6%, and 95.7%, respectively, with two major VSG content types, PI‐1^?/PI‐2^?/psrP ⁺/cbpA ⁺ and PI‐1⁺/PI‐2⁺/psrP ^‐/cbpA ⁺, being found for HC and non‐invasive diseases, respectively. A major clone of serotype19A/ST320 (PI‐1⁺/PI‐2⁺) produced the longest pneumococcal wire (pilus) structures in colonies. ST1016 (PI‐1^?/PI‐2^?) in HC had HEp‐2 cell‐adherent pili. These results suggest that serotype19A/ST320 and related genotypes, with the VSG content type PI‐1⁺/PI‐2⁺/psrP ^?/cbpA ⁺, emerged in vaccinees after PCV7 in Siberia, accompanying diseases in non‐vaccinated children, and that some genotypes (serotypes19A/ST320 and 18/ST1016) produced novel pneumococcal structures, predicting their roles in colony formation and adherence.

     

Massive parallel MHC genotyping: titanium that shines

The power of population genetic analyses is often limited by sample size resulting from constraints in financial resources and time to genotype large numbers of individuals. This particularly applies to nonmodel species where detailed genomic knowledge is lacking. Next‐generation sequencing technology using primers ‘tagged’ with an individual barcode of a few nucleotides offers the opportunity to genotype hundreds of individuals at several loci in parallel ( Binladen et al. 2007 ; Meyer et al. 2008 ). The large number of sequence reads can also be used to identify artefacts by frequency distribution thresholds intrinsically determined for each run and data set. In Babik et al. (2009 ), next‐generation deep sequencing was used to genotype several major histocompatibility complex (MHC) class IIB loci of the European bank vole ( Fig. 1 ). Their approach can be useful for many researchers working with complex multiallelic templates and large sample sizes.

Figure 1 Open in figure viewer PowerPoint Hypothetical example of parallel genotyping of two individuals using individually bar‐coded primers. Polymerase chain reactions (PCRs) are performed separately for each individual using a forward primer with a unique Tag‐sequence of four nucleotides. After sequencing of pooled PCR products, sequences can be sorted by their forward primer Tag (Tag‐sorting error rate was estimated < 0.1%). Rare sequences most likely represent artefacts and due to the large amount of sequences obtained (up to 10⁶) the artefact threshold can be determined intrinsically for each data set and was estimated to be around 3% in the case of bank vole MHC class IIB genes ( Babik et al. 2009 ). Photos by Gabriela Bydlon.     

Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes

Plant pathogenic microbes deliver effector proteins inside host cells to modulate plant defense circuitry and enable parasitic colonization. As genome sequences from plant pathogens become available, genome-wide evolutionary analyses will shed light on how pathogen effector genes evolved and adapted to the cellular environment of their host plants. In the August 2007 issue of Plant Cell, we described adaptive evolution (positive selection) in the cytoplasmic RXLR effectors of three recently sequenced oomycete plant pathogens. Here, we summarize our findings and describe additional data that further validate our approach.Key words: plant-microbe interactions, effectors, gene families, positive selectionA diverse number of plant pathogens, including bacteria, oomycetes, fungi and nematodes, deliver effector proteins inside host cells to modulate plant defense circuitry and enable parasitic colonization.^1–8 Because these so-called cytoplasmic effectors function inside plant cells and produce phenotypes that extend to plant cells and tissues, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host.^9,10 In a study published in the August 2007 issue of Plant Cell, we and our collaborators examined the extent to which positive selection (adaptive evolution) has shaped the evolution of the cytoplasmic effectors of three recently sequenced oomycete plant pathogens Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica (Genome Sequencing Center at Washington University).¹¹     

Molecular characterization of the porcine testis-specificphosphoglycerate kinase 2 (<Emphasis Type="Italic">PGK2</Emphasis>) gene and its association with male fertility

We have isolated and characterized the porcine testis-specific phosphoglycerate kinase 2 (PGK2) gene, and 1665 bp of full-length PGK2 cDNA were also compiled using modified rapid amplification 5-RACE and 3-RACE information. The results of genomic and cDNA sequences of the porcine PGK2 gene demonstrated that it is a single-exon intronless gene with a complete open reading frame of 1251 bp encoding a PGK protein of 417 amino acids. Real-time quantitative PCR results showed that PGK2 mRNA was solely expressed in the testis. There was a lower amount of PGK2 expression in the testis of a 10-month-old herniated boar and a very small amount of PGK2 expression in the testis of an 8–week-old cryptorchid piglet compared to an adult boar. Two SNPs in the PGK2 gene (SNP-A: T427C; SNP-B: C914A) resulting in amino acid substitutions (SNP-A: Ser¹⁰²–Pro¹⁰²; SNP-B: Thr²⁶⁴–Lys²⁶⁴) were detected and genotyped among six pig breeds. The nucleotide C at SNP-A responsible for the amino acid exchange to proline could lead to the loss of a casein kinase II (CK2) phosphorylation site in the PGK2 peptide. Association analyses between PGK2 genotypes and several traits of sperm quantity and quality were performed. The results showed that SNP-B has a positive significant effect on semen volume in the breed Pietrain (p=0.08), i.e., boars carrying genotype CC revealed an increased volume of 49 ml compared with boars having the genotype AA.The nucleotide sequence data reported in this article have been submitted to GenBank and have been assigned the accession numbers AY500132 and AY486962.