Tick-borne encephalitis virus (TBEV) is transmitted to vertebrates by taiga or forest ticks through bites, inducing disease of variable severity. The reasons underlying these differences in the severity of the disease are unknown. In order to identify genetic factors affecting the pathogenicity of virus strains, we have sequenced and compared the complete genomes of 34 Far-Eastern subtype (FE) TBEV strains isolated from patients with different disease severity . We analyzed the complete genomes of 11 human pathogenic strains isolated from the brains of dead patients with the encephalitic form of the disease (Efd), 4 strains from the blood of patients with the febrile form of TBE (Ffd), and 19 strains from patients with the subclinical form of TBE (Sfd). On the phylogenetic tree, pathogenic Efd strains formed two clusters containing the prototype strains, Senzhang and Sofjin, respectively. Sfd strains formed a third separate cluster, including the Oshima strain. The strains that caused the febrile form of the disease did not form a separate cluster. In the viral proteins, we found 198 positions with at least one amino acid residue substitution, of which only 17 amino acid residue substitutions were correlated with the variable pathogenicity of these strains in humans and they authentically differed between the groups. We considered the role of each amino acid substitution and assumed that the deletion of 111 amino acids in the capsid protein in combination with the amino acid substitutions R16K and S45F in the NS3 protease may affect the budding process of viral particles. These changes may be the major reason for the diminished pathogenicity of TBEV strains. We recommend Sfd strains for testing as attenuation vaccine candidates. Its circulation areas are in the forested zones of many European countries and northern Japan, China, and Mongolia. In Russia, the natural foci of the infection are spread across Kaliningrad Oblast in the west to Sakhalin Island in the east TBEV is a member of the virus genus Flavivirus, of the family Flaviviridae. This genus includes approximately 80 species, many of which are pathogenic in humans, such as West Nile virus, dengue, yellow fever, Japanese encephalitis, etc. The clinical course of TBE in the Far East is recognized to be more severe than in other Eurasian areas The exact genetic basis of these differences in the form and severity of the disease is unclear. Previously, the variable severity was associated with the virus subtype As a rule, in order to determine the virulence and pathogenicity of viral strains, their neuroinvasiveness and neurovirulence in mice or changes in the growth characteristics of cell culture have been studied. However, there is no clear correlation between the virulence of strains in mice and strain pathogenicity in humans In our previous studies, we compared the genomes of several strains isolated from patients with subclinical forms of the disease and indicated specific mutations in strains with different pathogenicity The length of the nucleotide sequences of the genomes ranged from 10,405 to 11,103 nucleotides ). Moreover, 8,131 positions were constant in all of the analyzed TBEV strains. Mutations were most often located in the third codon position and were found at 2,245 positions of the genome (21.6%). The aligned sequences of the genomes, 3′ UTR, and polyproteins are not shown, but can be provided upon request. We used maximum likelihood estimation for discrete data analysis to perform phylogenetic analysis of the complete sequenced genomes compared to the previously reported genome sequences of the FE subtype and prototype SIB (Vasilchenko) and WE (Neudoerfl) subtypes of TBEV . The anaThe pathogenic Efd strains are divided into two branches, which may be due to the geographical isolation of the speciation areas of the ancestral forms of the virus. Indeed, the strains of cluster II include those related to the Sofjin strain isolated in Russia, Primorye, and the strains of cluster III include those related to the Senzhang strain isolated in northern China. The Sfd strains form a separate cluster (I), but they have a common ancestor with the strains of cluster II. We did not find the Sfd strains to have a common ancestor with those of cluster III. TBEV strains (Fdf) that induced febrile disease did not form a specific separate cluster, except for the Shkotovo-94 strain, which formed a separate branch. Three other strains, i.e. Kiparis-94, Primorye–89, and Primorye–52, induced febrile disease, and are located in clusters II and I. The abnormal location of these strains will be discussed later in the “Exceptions” section.The genomic 5′ non-translated region is conserved and has 131 nucleotides. None of the positions correlated with changes in strain pathogenicity, and single mutations were mainly found in pathogenic strains in the left part of the fragment . In otheThe genomic 3′ UTR is heterogeneous in length, ranging from 31 to 728 nucleotides in different strains, depending on the length of deletions . AlthougThe first variant strains described with a longest 3′ UTR deletion of 697 nucleotides covering the region from 9 to 705 bp  are the Most strains, except for those with extensive deletions, and two strains, Spassk-72 and Primorye-437, lacking the terminal portion but with a relatively extended 3′ UTR, have conserved terminal portions of 327 nucleotides in length. This conserved region contains a number of mutations that do not correlate with the pathogenicity of strains and mutations -C245T and -A302G, which differ between groups of strains with different pathogenicity.The predicted 2D structure of 3′ UTR consists of several loops. The extreme loop within the range from −1 to −85 nucleotides (CR1) is conserved in all strains. The 5′ UTR and 3′ UTR cyclization disclose the SLB in the 5′ NTR  and partUnlike mosquito-borne flaviviruses, the start codon is located in the SLC, which does not participate in the formation of the 5′ UAR and remains unchanged. The 5′ UTR positions 109–128 were completely conserved in all of the analyzed strains, with no nucleotide substitutions . The 3′ Nevertheless, the Spassk-72 strain retained pathogenic properties despite the absence of cyclization motifs.Amino acid sequences of polyproteins are more conserved than nucleotide genome sequences. Thus, the observed strain group had only 198 positions (<6.4%) with a substitution of at least one amino acid residue. The polyproteins of pathogenic TBEV (Efd) strains had a length 3414 aa, but Sfd strains had single amino acid deletions in the 111 position of the capsid protein.Since most amino acid residues in polyproteins are constant, for convenience, we removed the conserved amino acid residues from the analysis and saved only those positions in which we observed at least one amino acid substitution . It is oWe have sequenced and analyzed the nucleotide sequences of the complete genomes of 34 FE TBEV strains isolated from patients with variable disease severity. The FE TBEV subtype was chosen because its strains normally induce severe disease in humans, which allowed us to collect a sufficient number of strains with diametrically opposite pathogenicity in humans The severity of TBE may depend on both characteristics of the human host and the state of their immune system, and the specifics of the TBEV genome. In this report, we did not consider the state of the immune system of patients, the location of the tick bite, the exposure time and other factors, which may affect the severity of the disease, but instead focused on the specifics of the genome of TBEV strains isolated from patients with variable disease severity. We previously suggested that specific mutations of the TBEV genome leading to amino acid substitutions could determine differences in disease severity There are several reasons for the analysis of complete genomes in a large number of closely related TBEV strains. Spontaneous random single mutations can incorporated into viral RNA during replication by RNA-dependent RNA polymerases and cannot be corrected as the RdRp lack 3′ exonuclease activity. The number of such mutations should increase with an increasing number of passages, if we exclude the possibility of elimination of the viral RNA mutant variants in subsequent passages in another organism of the same species. There is fear that continuous passages of TBEV in suckling mice may have resulted in viral adaptation to mice and induced some of the mutations that were not present in viruses isolated from human cells Secondly, analysis of strains of only one, and not a few subtypes, eliminates mutations acquired by the strains of different subtypes during a prolonged period of evolution, which can greatly complicate the analysis. For instance, the Sofjin strain of the FE TBEV subtype differs from the Vasilchenko strain of the SIB subtype by 1,486 mutations in the protein-coding domain, resulting in 174 amino acid substitutions. The number of available SIB strains is significantly lower than the number of substitutions; therefore, it is not possible to identify those substitutions that affect the pathogenicity of the virus.The neuropathogenic potential of TBEV includes two different properties of the virus, i.e. neuroinvasiveness and neurovirulence dS) and non-synonymous (dN) substitution rates calculated for the complete coding region of the genome by codeml in the PAML package dN/dS (max)  = 0.0615 and dN/dS (mean)  = 0.0416 indicate no adaptive selection in the analyzed sample group of strains, which corresponds to previously reported data on other flaviviruses The length of virus RNA of the analyzed strains ranged from 10,404 kb (Primorje-208) to 11,103 kb (Svetlogorie), depending on the size of the deletion in the 3 ′NTR. There are three clearly visible clusters in the phylogenetic tree . ClusterThe specifics of the 5′ and 3′ UTR sequences may change TBEV pathogenicity Thus, mutations detected in the 5′ UTR and 3′ UTR sequences of Efd and Sfd strains do not correlate with pathogenicity and are likely to be random. Furthermore, mutations in the 3′ UTR, including those that differ between Efd and Sfd strains, do not affect the pathogenicity and are random. However, it is possible that unidentified specific nucleotide sequences in the 3′ UTR can affect the position and size of deletions that occur when the virus is transmitted from tick cells to mammalian cells.One feature of the 3′ UTR in the analyzed strains is the presence of extended deletions that vary with respect to position and size across strains. The mechanism of the occurrence of these deletions, their role and their importance to the evolution of the viral population are uncertain; hence, this problem requires a detailed discussion. Previous studies have shown a different degree of influence from the artificial introduction of extended deletions of different lengths, starting from the stop codon to the core element, on the survival and pathogenicity of viral strains Analysis of our data also indicates that extended deletions have no significant effect on the pathogenicity of the strain and the efficiency of virus replication in mammalian cells, if they do not affect the conservative end portion of the 3′ UTR of approximately 325 nucleotides. This region of the 3′ UTR includes regulatory elements and sequences involved in the cyclization of viral RNA . If thisTBEV is known to circulate in nature by cyclic transmission from ticks to small mammals, followed by return to the ixodid tick vector, where the virus propagates and persists for a long period. The stage of virus survival in mammalian cells is necessary, but insignificant in terms of time The studies by Labuda et al. confirm this suggestion. They showed that TBEV is most effectively transmitted from infected to naïve ticks during co-feeding on an animal A terminal core element with highly conserved nucleotide sequences was previously found in all analyzed TBEV strains. This conserved site has been indicated in many publications We identified similar strains with the extended deletion in the group of Sfd strains  and in the group of Efd strains (Primorye-52 and Primorye-739). The latter two strains induced a subclinical form of the disease; hence, an extensive deletion in the 3′ UTR dramatically reduces the pathogenicity of the strains in humans. We suggest that the extensive deletion in the 3′ UTR in the Primorye-739 and Primorye-52 strains probably occurred during the initial stages of infection in patients, which resulted in a reduction in the pathogenicity of the virus and a subclinical course of the disease.The Spassk-72 strain, which is located in a cluster of pathogenic strains and has an unusual extended deletion in the 3′ UTR, including the core element, was isolated from a patient with fatal encephalitis. This may be due to the content in the human body of a considerable amount of virus variants with a full-length 3′ UTR, which was not observed by us.Unfortunately, our method of genome sequencing is not suitable for the quantitative determination of the ratio of full-length and deleted variants of the virus; therefore, it is necessary to develop a novel approach to the analysis of 3′ UTR variability during the transmission of TBEV from ticks to different mammalian cells. It is possible that the recently reported approach based on using a circular polymerase extension cloning (CPEC) reaction can be adapted The viral replication cycle includes several stages, i.e. attachment of the virus to the surface of the target cell, RNA replication, and synthesis and processing of the viral protein and assembly of viral particles Amino acid substitutions in various parts of the polyprotein can influence different stages of viral replication and strain pathogenicity, but the exact mechanisms behind this influence are unknown. The role of each amino acid is also unclear. Most amino acid substitutions found in the viral population may be incidental and (or) determined by the adaptation of the virus to various environments, meaning that they cannot affect the pathogenicity of strains. We found 198 positions with substitutions of the amino acids in the polyprotein with a length of 3414 aa . The subBelow, the function of each protein and the possible role of key amino acid substitutions are provided in more detail.Viral particles of all flaviviruses have a nucleocapsid surrounded by a lipid bilayer membrane, inside of which there are two fixed structural proteins, the surface protein, E (approximately 54 kDa), and precursor membrane protein, prM (approximately 27 kDa) Cleavage of the capsid protein from the polyprotein occurs in two stages:int).- In the first stage, the cleavage of C-prM, by host signalases, forms a membrane-bound protein C with a signal peptide  in the capsid protein of Sfd strains that can affect the packing density of the capsid protein , but theThe other structural proteins prM and E both contained specific substitutions , which are located in the transmembrane domains and differ between Efd and Sfd strains . These aThus, only one protein from the group of structural proteins had amino acid substitutions that correlated with the Efd and Sfd groups and may influence the pathogenicity of strains. Substitutions in the transmembrane domain of the capsid protein may affect strain pathogenicity at the budding stage through the formation of capsidless subviral particles. The structural proteins prM and E did not have substitutions that could affect the pathogenicity of the analyzed strains; therefore, the attachment of viral particles to the cells or viral penetration into cells will probably not significantly influenced the pathogenicity of viral strains.The non-structural protein NS1 is a highly conserved glycoprotein with 12 invariant cysteine residues. Unlike other non-structural proteins, NS1 is secreted and accumulates in the serum of infected subjects The non-structural protein NS2A is a small membrane-bound protein that participates in RNA replication by binding to the 3′ UTR with the proteins of the replicative complex The non-structural protein NS2B is a small membrane-bound protein that forms a complex with viral protein NS3, which has protease catalytic activity. Substitutions in the hydrophilic part of the protein affect the catalytic activity of the complex The non-structural protein NS3 is a 70-kDa soluble protein with protease, helicase, NTPase and 5′-terminal RNA triphosphatase activities The non-structural protein NS4A is a membrane protein with four transmembrane domains. Moreover, the C-terminal site, denoted as a 2K fragment, serves as a signal sequence for the translocation of the protein NS4B into the lumen of the endoplasmic reticulum  and the most conserved among the flaviviruses. It has RNA-dependent RNA polymerase and methyltransferase activities 2) The NS5 protein of the Sfd strains contained the key substitutions S634T, R677K, I692V and A724S, which may affect the pathogenicity of TBEV. These substitutions are located in the RNA-dependent RNA polymerase domain (RdRp) of NS5 . The subAmino acid substitutions in the NS5 protein can affect the neurovirulence of strains not only through changes in the efficiency of replication of viral RNA or inhibiting of interferon production, but also via interaction with certain cellular proteins which may affect pathogenic properties of the TBEV. Thus, recently published paper indicated All TBEV strains analyzed contained KFK-D motifs in the protein NS5  and caused neurological disease in white mice, but had different neurovirulence for humans. These facts confirm our previous data on the absence of unequivocal correlation between neurovirulence of strains in mice and humans We have shown that the strain position on the phylogenetic tree and the presence of certain key amino acid substitutions correlates well with the pathogenicity of TBEV strains. As a rule, the strains of cluster I were isolated from persons with subclinical course of the disease, whereas the strains of clusters II and III caused the severe fatal form of encephalitis. However, there were several exceptions to this rule, some of which can be explained by a more detailed study.The most rigorous exceptions to this rule are the strains Primorje-86 and Primorje-90, which are Sfd strains based on their molecular characteristics, but were isolated from individuals who had a posthumous diagnosis of “encephalitis” in 1986 and 1990. We have no way to explain this discrepancy, beyond a speculation on the inaccuracy of the diagnosis or the presence of severe immunodeficiency or other comorbidities that could cause an adverse outcome on the background of infection by Sfd strains of TBEV.The Primorye-52 and Primorye-739 strains caused a subclinical form of the disease, despite the fact that they are located in clusters II and III on the phylogenetic tree and have key amino acids specific to pathogenic Efd strains. The reason for this discrepancy may be the presence of the longest deletion in the 3′ UTR, including the entire core element, which hinders the effective propagation of the virus in human cells. Thus, this exception confirms our suggestion that removal of the longest fragment in the 3′ UTR may significantly reduce the pathogenicity of TBEV strains.Other exceptions are the strains that cause the febrile form of the disease, which results in the recovery of patients and which can considered as a weakened form of encephalitis. The Shkotovo strain forms a separate branch of the phylogenetic tree between the Efd and Sfd strains and is different from the Efd strains by the presence of the key amino acids 16K and 45F in the viral protease NS3, which are characteristic of the Sfd strains. This indicates that the substitutions 16K and 45F in the viral protease NS3 may reduce the pathogenicity of these strains.The Ffd strain Primorye-94 had a V179A substitution in NS4B, which is specific to the Sfd strains, but its role in reducing the pathogenicity of strains is unknown. Additionally, this strain has many other unique amino acid substitutions: V141I in protein E, K47R in protein NS1, L69F, M161I and R228K in NS2B, A68V and L143I in NS3, and A92V in NS4A. It is likely that a combination of this set of substitutions reduces the pathogenicity of the strain, but there is no direct evidence of the impact of these changes on pathogenicity.The strains Primorye-82 and Kiparis-94 represent Sfd strains, but with increased virulence, as they caused the febrile form of the disease. Each strain has several single amino acid substitutions at different sites of the polyprotein . At the Thus, deletions in the structural capsid protein and amino acid substitutions in the non-structural proteins NS3 and NS5 may reduce the virulence of TBEV via effects on viral RNA replication, polyprotein processing and the assembly of viral particles. Amino acid substitutions found in the E protein did not correlate with the degree of pathogenicity of TBEV strains and probably do not affect virus attachment to cells. Substitutions in the 3′ UTR do not play a significant role in changing the pathogenicity of strains, but more research is needed to understand the mechanism of these deletions, their extent and position.Identifying the role and importance of each key amino acid substitution in the pathogenicity of TBEV strains requires additional studies using site-directed mutagenesis techniques with a full-length infectious cDNA clone. However, this type of work requires a biosafety level 4 laboratory and non-human primates to test the neurovirulence potential of new virus variants.At the same time, Sfd strains can be recommended for testing as candidates of the live attenuated vaccine without additional modifications. These strains can be divided into 3 groups according to the presence of specific substitutions in the E protein and four groups according to the presence and the length of the deletions in the 3′ UTR.Mouse experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals according to the USSR Ministry of Public Health Regulation No. 1189 of 10.10.1983. Experiments were performed according to the USSR Ministry of Public Health Regulations No. 755 of 12.09.1977 and No. 701 of 27.07.1978 for the humane treatment of animals. All experimental procedures were approved and performed according to the Research Institute of Epidemiology and Microbiology of the Siberian Branch of RAMS (Vladivostok) Animal Care and Use Committee (protocol # 1 on 30.01.2008).The isolation of TBEV strains was performed as previously described 2, 200 U of MMLV RT  and Milli-Q water  was incubated at 42°C for 30 min. Samples of cDNA for transportation were mixed with equal volumes of isopropanol and stored as a suspension.Total RNA was extracted from 100 µL of a 10% brain or plasma suspension in PBS using Trizol LS  according to the manufacturer's recommendations. Then, RNA was precipitated, washed twice using 1 mL of 80% ethanol and air-dried for 5 min. The RNA precipitate was resuspended in 50 µL of fresh Milli-Q water and used as a template in a reverse transcriptase reaction as soon as possible. To prepare cDNA by reverse transcription, a mixture of 5 µL of RNA, 50 pM of hexanucleotide primers, 200 mM of each NTP, 5 mM MgClPairs of oligonucleotide primers for amplification were designed based on previously published sequences for the Sofjin, Senzhang, 205, Glubinnoe and Aina strains and optimized to generate 38 overlapping fragments for sense and antisense strains with an average length of about 1000 base pairs . To amplFor each sequencing reaction, approximately 50 to 100 ng of purified dsDNA were mixed with 3.2 pmol of one of the primers and with a reaction cocktail containing the four dye-labeled dideoxynucleotide terminators . The cycle sequencing parameters used were as described in the manufacturer's protocol . Reaction products were precipitated by ethanol-acetate as described in the manufacturer's recommendations. The pellet was resuspended in 16 µL of template suppression reagent, heated for 2 min at 95°C, and kept on ice until it was loaded into the Applied Biosystems Prism 3100 sequencer.BioEdit v.7.2.0 sequence alignment editor) (http://www.mbio.ncsu.edu/bioedit/bioedit.html). The nucleotide sequence was translated into the amino acid sequence online at the Bioinformatics Resource Portal (http://web.expasy.org/translate/). The nucleotide and translated amino acid sequences of the strains were deposited in the international database GenBank (http://www.ncbi.nlm.nih.gov/) under the accession numbers shown in Overlapping nucleic acid sequences were combined for analysis and edited with a software package Phylogenetic analysis of nucleotide and amino acid sequences of the complete genomes of the analyzed virus strains and the previously reported genome sequences of the TBEV strains of Far East subtypes , the Vasilchenko strain as the prototype strain of the Siberian subtype, and Neudoerfl as the prototype strain of the Western subtype was performed using the Maximum Likelihood (ML) method. The phylogenetic tree was estimated using the ML method TreeFinder Synonymous (dS) and non-synonymous (dN) substitution rates were calculated by CodeML in the PAML package http://mfold.rna.albany.eduRNA secondary structure predictions of the UTRs of viral RNA were performed using the mfold program, which is available from http://www.cbs.dtu.dk/services/SignalP/) Prediction of the location and the possibility of signal sequence cleavage were carried out online at the SignalP 4.1 Server  was taken as a template for homology modeling as this model has a high resolution (2.45 Å) and the sequence identity between the target and the template is greater than 25% The crystal structure of the capsid protein C from West Nile Virus, subtype Kunjin (PDB code 1sfk) was taken as a template for homology modeling The crystal structure of the dengue virus RNA-dependent RNA polymerase (RdRP) catalytic domain, at a resolution of 1.85 Å (PDB code 2j7u), was chosen as a template 2 in order to allow adjustment of the solvent molecules. After this, free molecular dynamics simulations were performed using the NPT  ensemble by applying a time step of 2 fs. The temperature was kept constant at 300 K. The Berendsen pressure control scheme was used to keep the pressure in the vicinity of 1 bar during the simulation with a relaxation time of 2 ps and a default compressibility parameter for water equal to 44.6×10−6 bar −1. The system was considered equilibrated after 1 ns. The MD simulation without restraints was carried out for 20 ns for both complexes of NS2b/NS3, for 5 ns for both complexes of capsid protein C, and for 5 ns for both complexes of RdRP.The molecular dynamics (MD) simulations were performed using Amber software version 11. The Amber 99 force field was applied for the complexes Figure S1The scheme of the 3′ UTR and the position of deletions.(TIF)Click here for additional data file.Figure S2The similarity matrix of genomes and polyproteins. Pathogenic strains are shown in bold font and strains with the febrile form of TBEV are shown in red.(TIF)Click here for additional data file.Figure S3Predicted secondary structure of Sofjin strain 5′ UTR.(TIF)Click here for additional data file.Figure S4Predicted secondary structures 3′ UTR of Sfd strain Primorye – 320 and Efd strain Svetlogorie. Circles indicate random nucleotide substitutions, and squares indicate characteristic substitutions that differ from groups of Efd and Sfd strains.(TIF)Click here for additional data file.Figure S5Predicted secondary structures of the interaction between at the 5′ and 3′ ends of the RNA of the TBE virus pathogenic strain Svetlogorie. 5′ and 3′ flanking sequences connected by a RNA circle insert; the AUG initiation codon is boxed. The constant loop SLA and SLC and the new UAR (Upstream AUG) region are shown.(TIF)Click here for additional data file.Figure S6Amino acid residue substitutions and positions in viral proteins. The key amino acid residues in Sfd strains differ from the amino acid residues of pathogenic strains, indicated in bold and in gray blocks. The strains that cause disease of varying severity are color-coded according to the legend in (TIF)Click here for additional data file.Figure S7A map of Primorye (Far East Russia) showing the location of sites in which patients were bitten by ticks. Isolated Efd strains are shown in black, Sfd strains in green and strains with the febrile form of TBEV are shown in red.(TIF)Click here for additional data file.Figure S8Prediction of the most probable C-prM signalase cleavage sites. (A) Efd strain Svetlogorie, highlighted red square amino acid Leu that deleted in Sfd strains. (B) Sfd strain Primorye-320.(TIF)Click here for additional data file.Figure S9Predicted tertiary structure of the capsid protein. (A) The tertiary structure of the capsid protein dimer for the pathogenic strain Dalnegorsk. (B) The tertiary structure of the capsid protein dimer for the Sfd strain Primorye – 270.(TIF)Click here for additional data file.Figure S10Transmembrane domains predictions in Efd strain Svetlogorie and Sfd strain Primorye – 320. (A) Capsid proteins. (B) prM proteins. (C) Envelope proteins. (D) NS2B proteins. (E) NS4B proteins. Amino acid positions that differ in the Efd and Sfd strains are identified by colored lines.(TIF)Click here for additional data file.Figure S11Aligned amino acid sequences of the flavivirus RNA polymerase domain. Designations: TBEV – tick-borne encephalitis virus, strain Dalnegorsk, accession: FJ402886; OHFV - Omsk hemorrhagic fever virus, strain Guriev, accession: BAH78736; KFDV - Kyasanur forest disease virus, accession: AAQ91607; POWV - Powassan virus, strain Nadezdinsk-1991, accession: ACD88752; YFV - Yellow fever virus, strain Uganda 2010, accession: AEQ35299; DV2 - Dengue virus 3, isolate DENV-3/US/BID-V1473/2002, accession: ACD13417; DV2 - Dengue virus 2, strain D2/TO/UH39/1974, accession: ADM26233; JEV - Japanese encephalitis virus, isolate SC0415, accession: AEO72405; WNV - West Nile virus, accession: ADZ96248.(TIF)Click here for additional data file.Table S1Results of prediction signal peptide cleavage.(DOCX)Click here for additional data file.Table S2List of primers used for amplification and sequencing of complete genomes.(DOCX)Click here for additional data file.


A multidisciplinary team is essential for a high quality ECMO service. However, few health professionals have any knowledge on ECMO and, in addition, there are discrepancies between the level of expertise among these professionals. Thus, we evaluated the impact of a basic theoretical ECMO course on the knowledge gain of different healthcare professionals.A basic six-hour theoretical course on ECMO was taught to a heterogeneous audience formed by 12 medical students, 10 nurses, 4 perfusionists and 4 physicians. Content included the following topics: 1) concept of ECMO; 2) usage scenarios; 3) aspects of the circuit; 4) differences between pulmonary and cardiac ECMO; 5) management of patients on ECMO; 6) aspects of cannulation; 7) complications; 8) concept of E-CPR; and 9) importance of the multidisciplinary team. Questionnaires for self-evaluation, graduating from 1 to 5 the level of expertise on the referred topics, were conducted immediately before and after the course.Twenty-six  questionnaires were completed. The three most clear topics before the course were the importance of the multidisciplinary team , the concept of ECMO  and usage scenarios , while the three less clear were the concept of E-CPR , management of patients on ECMO  and aspects of cannulation . An important, although not significant, discrepancy between the level of expertise among the professional categories was present before the course . After the course, a significant knowledge gain was observed in all the topics (p<0.001). Perfusionists presented lower gain than others participants (p=0.05). At the end, there was an homogenization between the level of expertise among the audience .A basic theoretical ECMO course is important, not only to raise the expertise on ECMO, but also to level different professionals for future advanced training and specialization.


Several aspects of the epidemiology of 2009 (H1N1) pandemic influenza have not been accurately determined. We sought to study whether the age distribution of cases differs in comparison with seasonal influenza.We searched for official, publicly available data through the internet from different countries worldwide on the age distribution of cases of influenza during the 2009 (H1N1) pandemic influenza period and most recent seasonal influenza periods. Data had to be recorded through the same surveillance system for both compared periods.For 2009 pandemic influenza versus recent influenza seasons, in USA, visits for influenza-like illness to sentinel providers were more likely to involve the age groups of 5–24, 25–64 and 0–4 years compared with the reference group of >64 years . Pediatric deaths were less likely in the age groups of 2–4 and <2 years than the reference group of 5–17 years . In Australia, notifications for laboratory-confirmed influenza were more likely in the age groups of 10–19, 5–9, 20–44, 45–64 and 0–4 years than the reference group of >65 years . In New Zealand, consultations for influenza-like illness by sentinel providers were more likely in the age groups of <1, 1–4, 35–49, 5–19, 20–34 and 50–64 years than the reference group of >65 years .The greatest increase in influenza cases during 2009 (H1N1) pandemic influenza period, in comparison with most recent seasonal influenza periods, was seen for school-aged children, adolescents, and younger adults. Since the spring of 2009, the globe has witnessed an influenza pandemic due to a variant H1N1 influenza virus In this regard, we sought to explore the differences between the 2009 (H1N1) pandemic influenza period and recent seasonal influenza periods regarding the age distribution of influenza cases.We searched, up to March 2010, in the official websites of major health organizations or institutions worldwide for publicly available surveillance data on the age distribution of influenza cases. Specifically, we searched in the websites of the World Health Organization, the European Centre for Disease Prevention and Control, the United Kingdom Health Protection Agency and Office of National Statistics, the French Institute for Public Health Surveillance, the Swedish Institute for Communicable Disease Control, the United States Centers for Disease Control and Prevention (CDC) and the Department of Defense Global Emerging Infections Surveillance and Response System, the Public Health Agency of Canada, the Australian Government Department of Health and Ageing and the National Notifiable Diseases Surveillance System, the New Zealand Ministry of Health and the Institute of Environmental Science and Research, the South African National Institute for Communicable Diseases, the Pan American Health Organization, the Argentinean Ministry of Health, the Mexican Department of Health, the Chilean Ministry of Health, and the Brazilian Ministry of Health. We also searched for additional relevant influenza surveillance data, through web links from the above websites. Finally, we retrieved official data on the type and antigenic characterization of circulating influenza viruses during each influenza season and country analyzed.We included in our analysis epidemiological data referring to the 2009 (H1N1) pandemic influenza period (from mid April 2009 until the end of 2009) and one or more of the most recent influenza seasons, provided that they were recorded with the same surveillance system and similar methodology. We included data on influenza cases that had been diagnosed either clinically or with appropriate relevant laboratory methods, regardless of the level of health-care they received. We synthesized the retrieved data into meaningful and comparable age groups. We calculated the percentage of cases in the different age groups for both the pandemic and the seasonal influenza periods. We constructed graphical representations of the percentile distribution of cases into age groups between the pandemic and, cumulatively, the seasonal influenza periods. We also calculated the percentage difference in the recorded number or rate of cases, between the pandemic and the seasonal influenza periods, for each age group.We compared the distribution of cases into age groups between the pandemic and, cumulatively, the seasonal influenza periods, using the chi-square test. A p value <0.05 was considered statistically significant. We also calculated the odds ratio (OR) and 95% confidence interval (CI) for a case of influenza to belong to each specific age group compared with a reference age group, between the pandemic and, cumulatively, the seasonal influenza periods. We used the older age group in each dataset as the reference group. For the above analyses, we used the SPSS Statistics v.17.0 software .We identified comparable data on the age distribution of influenza cases between the 2009 (H1N1) pandemic period and most recent seasonal influenza periods that were provided by the CDC, the Australian Government Department of Health and Ageing, and the New Zealand Institute of Environmental Science and Research. In P<0.001; In Australia, the National Notifiable Diseases Surveillance System provided data regarding the number of cases of laboratory-confirmed influenza . In 2009In New Zealand, the Institute of Environmental Science and Research, a governmental research institute, provided data regarding the average weekly consultation rate for influenza-like illness in sentinel practices per 100000 patient population reported by these practices . In 2009We used the above presented data on the average weekly sentinel consultation rate for influenza-like illness in New Zealand to approximately calculate the absolute number of cases of influenza-like illness reported by the sentinel practices during the pandemic and seasonal influenza periods that we evaluated . SpecifiP<0.001). For the pandemic influenza period versus the seasonal influenza period, a case of influenza was more likely to belong into the age group of less than 1 year , 1–4 years , 35–49 years , 5–19 years , 20–34 years , and 50–64 years , compared with the reference group of those more than 65 years.The distribution of influenza-like illness cases into age groups differed between the 2009 pandemic influenza period and the 2008 seasonal influenza period , the CDC provided data regarding the number of visits to sentinel providers for influenza-like illness . During In the USA, the CDC provided data regarding the laboratory-confirmed influenza-associated hospitalization rate for the general population . RelevanWe compared the age distribution of laboratory-confirmed influenza-associated hospitalization rates between the pandemic period of 1 Sep 2009 – 2 Jan 2010 and the whole 2008–2009 influenza season, because corresponding age-specific data were provided only for these periods. During the above-mentioned 2009 (H1N1) pandemic influenza period, compared with the 2008–2009 seasonal influenza period, the cumulative laboratory-confirmed influenza-associated hospitalization rate increased primarily for the age groups of 18–49 years and 50–64 years ; the increase in the hospitalization rate was relatively lower for the age group of 5–17 years (380%), followed by the groups of those more than 65 years and those up to 4 years of age . As we could not reliably retrieve accurate data regarding the size and age distribution of the reference population for the hospitalization rates analyzed above, we did not proceed to calculate the respective raw hospitalization data.P = 0.003, In USA, the CDC provided data regarding the number of laboratory-confirmed influenza-associated pediatric deaths . For theA common finding in the surveillance data from major national health organizations worldwide that we identified for the purposes of this study is that the age group of elderly individuals (more than 65 years of age) was the least one affected during the 2009 (H1N1) pandemic influenza period, in comparison with the most recent seasonal influenza periods. This particularly refers to laboratory-confirmed disease incidence and presentation with influenza-like illness to sentinel providers, according to data recorded in different countries. Regarding the age groups mostly affected by 2009 (H1N1) pandemic influenza in comparison with the most recent seasonal influenza periods, we noted certain differences according to the level of influenza diagnosis , the severity of cases , and country. For 2009 (H1N1) pandemic influenza, in comparison with seasonal influenza, laboratory-confirmed influenza cases and laboratory-confirmed influenza-associated hospitalizations mostly increased for school-age children, adolescents, and younger adults, according to data from Australia and USA, respectively. Pediatric deaths associated with laboratory-confirmed influenza increased mostly for school-age children and adolescents, according to US data. The greatest increase in influenza-like illness presentations to sentinel providers was noted for school-age children, adolescents and young adults in USA, while, in New Zealand, this was noted for infants and preschool children.Differences in the age distribution of cases of 2009 (H1N1) pandemic influenza in comparison with seasonal influenza can be attributed to differences in the degree of preexisting specific immunity in different age groups. Specifically, elderly individuals appear to have had protective immunity more frequently than other age groups against the 2009 (H1N1) pandemic influenza virus due to prior immunologic encounters with antigenically similar viruses many years before. Increasing age has been positively associated with the presence of cross-reactive antibodies against the 2009 (H1N1) pandemic influenza virus, which can prevent infection Although 2009 (H1N1) pandemic influenza, compared with seasonal influenza, appears to have affected mainly school-age children, adolescents, and younger adults, regarding outpatient presentations for influenza-like illness in USA or incidence of laboratory-confirmed disease in Australia, there appears to have been a shift towards young and middle-aged adults for hospitalizations in USA. This could be explained by the more frequent co-existence of known risk factors for influenza in the latter age groups, compared with younger patients Similar observations as the ones made in our analysis regarding a greater impact of the 2009 (H1N1) pandemic influenza on younger patients, compared with seasonal influenza, have also been made for other influenza pandemics Similar findings regarding the impact of 2009 (H1N1) influenza pandemic by age in terms of mortality were noted in another study that modeled excess pneumonia & influenza mortality in the general population in the USA during 2009–2010 and the past 47 influenza seasons Seroprevalence studies evaluating the temporal changes in the prevalence of antibodies against the 2009 (H1N1) pandemic influenza virus in the community have helped in the clarification of the evolution of the disease amongst different age groups. A study that applied statistical modeling to evaluate sequential seroprevalence data from England noted that during the second wave of 2009 (H1N1) pandemic influenza (September 2009 to February 2010) the cumulative incidence of infection was higher in the age group of 5–14 years, followed by the age group of 1–4 years, and those of 15–24 and 25–44 years The findings of our analysis should be interpreted with caution for several reasons. First, the recorded cases of influenza reflect only a small subset of the actual clinical symptomatic cases, the majority of which is not expected to seek medical care The comparative data we identified on the age distribution of cases of 2009 (H1N1) pandemic influenza and seasonal influenza, recorded by the same surveillance systems, derived from a relatively small number of sources. The data we included referred to developed countries , which have similar socioeconomic conditions, health care systems, and age distribution of the total population. They cannot be highly representative of countries under development, in many of which the life expectancy of the population is lower and the birth rate is greater, and, thus, the percentage of younger individuals in the population is higher. Differences between countries could also exist regarding the epidemiology of influenza, the surveillance and diagnostic methods used, including the definitions for syndromic surveillance, and the attitude of the public, healthcare professionals and administrative organizations towards influenza Apart from biological differences in the disease characteristics An important relevant consideration refers to potential differences in vaccination coverage of different age groups of the general population between the pandemic and seasonal influenza periods. This is plausible given the differences in relevant public health recommendations and vaccination policies. Still, for most of the 2009 (H1N1) pandemic influenza period examined in the context of this study, the specific influenza vaccine was not available, and the vaccination coverage of the general population was considerably low in most countries An additional parameter, however, that can affect the age distribution of seasonal influenza cases is the predominant influenza strain in a certain region. H1 influenza A strains tend to have greater predilection for younger individuals compared with H3 strains, while influenza B is typically associated with milder illness than influenza A In conclusion, the comparative analysis of publicly available data from 3 different countries, recorded by the same surveillance systems, shows that during 2009 (H1N1) influenza pandemic period the age distribution of cases shifted towards children, adolescents, and younger adults, in comparison with recent seasonal influenza periods. The comparative burden of the pandemic influenza might have been greater for young and middle-aged adults when hospitalizations are concerned. These data could aid public health authorities to better organize appropriate response strategies for future pandemics.


According to the National Heart, Lung, and Blood Institute of the National Institute of Health, hypotension refers to an abnormally low blood pressure (BP). In childhood, hypotension can be determined according to two different definitions: BP below the 5th percentile or below two standard deviations (SDs) of the mean for age and gender . A graphWe used a computerized calculation method to develop high-resolution graphics containing curves with 5,841 points each, to depict the main percentiles associated with low BP for boys from 1 to 17 years old in the 50th percentile of height. Each point represents the calculation of the polynomial equation that includes the statistical processing of the last Report on Blood Pressure . We alsoFive monotonic curves of systolic BP for boys representing the 50th, 25th, 10th, 5th, and 2.275th (-2SD) percentiles were built (Figure The adjustment of systolic BP for height is of little significance, and the updated graphic can be used to diagnose low systolic BP for boys. Clinical studies are necessary to determine the systolic BP percentile that better represents clinically significant hypotension.


The obvious inference is that, in respect to defense response, the ratio  increases under different stresses and, by destroying cellular components, may cause cell death. In addition, ROS are part of the complex network of transduction signals that induce defense reactions against stress or, alternatively, trigger programmed cell death, and key questions are the levels of each ROS that, respectively determine defense and death responses of the cell. The answer to those questions is difficult because there are several patterns of cell death that frequently appear mixed and are hardly distinguishable. Moreover, although considerable progresses have been achieved in the determination of the levels of specific ROS, critical questions remain on the ROS level in specific cell compartments. By considering chloroplasts as the main source of ROS in photosynthetic tissues at light, a comparison of the levels in stress and senescence of the chloroplastic activities involved in the generation and scavenging of ROS suggests plausible differences in the levels of specific ROS between stress defense and death. In effect, the three activities of the chlororespiratory chain increase similarly in stress defense response. However, in senescence, superoxide dismutase (SOD), that converts superoxide anion radical ( Reactive oxygen species (ROS) were formerly known for their damage effects on cellular components. Later, evidences, mainly accumulated along the last decade of twentieth century, showed at the turn of the millennium  additionThe involvement of ROS in the senescence of photosynthetic tissues provides a good system to size the magnitude of the challenges and to follow progresses to understand the integration of ROS signals within the networks controlling cell cycle and responses to stress. To distinguish between the ROS mediated effects in stress and in cell death, we address two fundamental aspects related to the roles of ROS in the programmed senescence of photosynthetic tissues under field conditions at light: (1) sources and sinks of ROS, and (2) signals that immediately follow ROS in the transduction networks. Programed senescence and death of photosynthetic leaves probably represents the highest amount of biomass and number of cells in the Earth suffering programmed cell death (PCD). Senescence of photosynthetic fruit tissues is a case of programmed senescence, not immediately followed of death, which is part of the maturation of most fleshy fruits and, as such, of high economic relevance. Similitude and differences between the senescence of leaf and the ripening of fruits were recognized since long time and we will refer the last exclusively for recent advances related to the involvement of components of the ROS generation machinery in fruit maturation. In a lesser extension, we will refer to a few processes where ROS signals are also involved and provide insights to understand leaf senescence under field conditions. Among them, the senescence and death of animal cell provides a lot of research advances that may be relevant. Senescence of non-photosynthetic plant cell, cell death associated to hypersensible response (HR), leaf senescence associated to diverse abiotic stresses  and stress responses in general are fields where recent investigations provide insight relevant to the involvement of ROS as node signals in networks controlling cell processes.There are different types of cell senescence and death, which frequently make difficult the comparison of the research results in different organisms, in different tissues of an organism and of different types of senescence and death of a specific tissue. For a comparison with the best characterized types of cell senescence and death in animals , and in Figure 1 summarized the best-known ROS, their transformation and deleterious effects on cell components. In addition to the main ROS found in non-photosynthetic cells: superoxide anion radical (2O2), and hydroxyl radical (HO∙); another ROS, the singlet oxygen (1O2), is produced in chloroplast by transfer of excitation from triplet excited chlorophyll (3Chl*) to O2. Hydroperoxyl radical  consuming H2O2.2O2 and HO∙ , are also generated in other cell compartments, especially in mitochondria and peroxisomes where H2O2 is destroyed by catalase. To prevent the generation of excess 1O2, heat dissipation of 3Chl* excitation is enhanced through zeaxanthin formed by the xanthophyll cycle . Then, transporters of the PET become over-reduced, the production of ROS increases and, depending on the environmental severity and the rapidity of the correction responses, it can destroy cell components producing the syndrome of photo-oxidative stress and eventually causing cell death  are mainly unspecific and could hardly be envisaged as related to their mechanism of interaction with other signal molecules of the regulatory network of the cell. However, effects other than damage are conceivable for H2O2. On the other hand, being 1O2, ∙ short-lived, the more stable (and permeable through membranes) H2O2 seems the plausible ROS signal candidate that interacts with other components of the regulatory cellular network and generates genetic and other responses of the cell to stress and PCD. Moreover, as H2O2 is substrate of catalase and diverse PXs, it could conceivably interact  with proteins downstream in a signal cascade of a cellular network. H2O2 is also the product of the sequence of transformations of 1O2 and Figure 1), which, in a first approximation, suggests that the increase of these ROS was finally senses by an increase of H2O2 and that, in the simplest imaginable mechanism, H2O2 would be the unique ROS signal of the cellular networks. From a broad perspective, the transformations 1O2 to 2O2 should be no other than stages of signal transduction until the last ROS before non-ROS nodes. As we will see, facts are not so simple. Certainly, there many evidences that H2O2 is a signal, at least in the transduction cascades of the responses to stress, but there are strong evidences suggesting that 1O2 could also be signals that, independently of their transformation to H2O2, originate cascade signals involved in leaf senescence.Damage effects of ROS  to  and, thus, it decreases the efficiency of photosynthesis. At the photo-physical stage, the heath dissipation of excited chlorophyll to reduce the formation of 1O2 also impairs the use of light energy in photosynthesis. Therefore, plants have evolved mechanisms to regulate the processes of generation and scavenging of ROS by adjusting them to different environmental conditions in order to minimize ROS damage and maximize photosynthesis yield. The adjusting is got through appropriate level of zeaxanthin, SOD, and PX .In effect, the dynamic of ROS generation and scavenging in the healthy and mild-stressed leaves seems to change in senescence. As pointed above, in health photosynthesizing leaf,  OH-) to . HO∙ is uced PET . Among cuced PET , in addi, and PX  for any 2O2 would be a less harmful alternative than the formation of 1O2 under transitory high light. The photo-inhibition of photosystem II  and the drain of electrons  ensure the fine-tuning of the redox level of the transporters of electrons in the cyclic PET. The complete sequence of reactions is:and results in a global respiratory process named chlororespiration :Figure 2.that is schematized in In the field, rapid increases and fluctuations of the intensity of light, high and low temperatures, hydric stress, and aggressive chemicals rapidly change the rates of electron supply (from PSII) and consumption . In this scenario, to maintain functional the cyclic PET and minimize damages by ROS, the activities of the chlororespiratory chain  must be high in comparison with more stable or mild environments. Accordingly, the levels of the Ndh complex increase under different stresses  and Ndh 2O2 formed by over-expressed SOD. Chloroplast lacks catalase but contains several PXs (among them plastoquinol PX) whose increase in senescence must efficiently consume the low amount of H2O2 produced by chloroplast lacking SOD in senescing leaves. In this line, in adult-senescent leaves, over-expression of the Ndh complex and chloroplastic PX and under-expression of chloroplastic SOD must increase the level of 2O2 in respect to the response of young leaves. As the Ndh complex provides electrons, per se, it must increase the redox level of the transporters and, consequently, the formation of 1O2 and 1O2 and 1O2 and When adult-senescent leaves are subjected to different stresses, Ndh complex level and activity as well as those of plastid PX increase in a similar way than in young fully photosynthesis active leaves. In contrast, the induction of SOD in response to stress becomes progressively impaired when leaves enter senescence . The resndhF gene and Ndh complex  fail to respond. Then, fine-tuning of the redox level of the electron transporters of cyclic PET is broken early in leaf senescence and gives way to their over-reduction and to a growing spiral of ROS production and death.Summarizing the evidences discussed above, the decrease of SOD and the increase of the Ndh complex play crucial roles in senescence by determining an increase of 2O2 increases the expression of the plastid ndh genes, the levels of the Ndh complex and the NADH dehydrogenase activity of the Ndh complex by phosphorylation of the NDH-F subunit  mutant  mutant . As poin1O2, ∙), H2O2 is sufficiently stable and presumably permeable through chloroplast membranes to connect with the cytosolic network of signals that control specific gene expression in the nucleus. Immediate targets of H2O2 are not yet known and plausible candidates in cytosol, or also into the chloroplast, are 2-cysteine peroxiredoxins (PRDX). These could also act as redox signals  kinases that are involved in the H2O2 production and in the regulation of death, mainly in the response to biotic and abiotic stresses  2O2 and its derivative HO∙, are the chloroplast ROS signals involved in programmed leaf senescence, the obvious question refers to its (their) immediate target. As mentioned above, 1O2 or Figure 1) and, essentially, the same amino acids are modified by 2O2. Therefore, the oxidation of cysteine residues of proteins by 2O2 in chloroplast and cytosol. However, no precise or specific protein target is yet known that initiates a signal after oxidation by 2O2 for draining the excess of electrons, should impair the rate of cyclic PET collapsing the thylakoid membrane potential and, thus, shooting the production of more 1O2 that would become the key initial signal that compels the cell to death at light. As pointed above, to maintain the leaf functional and healthy, the chloroplast forms other ROS than 1O2. Therefore, the low level of 1O2 must be an objective for enduring cell. For senescence and death, 1O2 probably initiates a signal chain linked to the damage of polyunsaturated fatty acids.If Figure 1 shows, 1O2 attacks polyunsaturated fatty acids. The first products of the attack of polyunsaturated fatty acids of membrane lipids by 1O2 are 13-hydroperoxy derivate fatty acids. Among them, 13-hydroperoxy linoleic acid is transformed in chloroplasts to the oxylipin -12-oxo-phytodienoic acid, which is transformed into jasmonic acid (JA) in peroxisomes and further, in cytosol, to related compounds as methyl jasmonate . In this regard, it is significant that treatments with JA increase the production of 1O2  in Arabidopsis. In this way, the induction of the SAG for MYB must obstruct the expression of the chloroplastic SOD, hence contributing, in addition to the mechanisms described above, to the growing spiral of the 1O2 and It would be concluded that the initiation of the program for leaf death is the fail to properly stimulate the expression chloroplastic SODs under mild stress, but the meaning of starting point is fading in an autocatalytic spiral of events as that proposed. Hence, among SAGs, MYB transcription factors have been described  that binnescence .ArabidopsisCsd2 gene also includes two W-boxes (TGAC(T)) recognized by WRKY transcription factors  at the different steps of complex transduction networks that control stress response and PCD by WRKY53. To add more complexity, several proteins control the expression of the WRKY53 gene and the binding of the WRKY53 protein to DNA upstream of genes expressed in senescence. Therefore, through variable responses to JA and H2O2, the complex network of WRKY proteins could be crucial determining the transition of the defense response to PCD as a consequence of increased 1O2 plus 2O2 ratio.The regulatory upstream region of the  factors , some of factors . The genand H2O2  should dand H2O2  may be i2O2 in cytosol or non-chloroplast organelles could be relevant. In addition, hormonal factors related to developmental processes, as seed filling and shadowing by upper leaves, and nutritional factors can produce a mixture senescence mechanisms, one of which could be dominant in some plants and environments.The model described for leaf senescence requires, at least in its first stages, light acting on the photosynthesis machinery. But the syndrome of leaf senescence would be more complex in field where alternating light and dark periods during the day could overlap light dependent senescence with dark dependent senescence. Under this condition, the involvement of a high production of HIn contrast to animals, the development of plants is strongly affected by environmental factors and, not surprisingly, ROS are involved as signals in developmental processes leading to cell death and in the defense response against environmental stress. Difficulties to determine the levels of some specific ROS in the different cell compartments open key questions as the levels and mechanisms through which ROS control different issues such as death and defense. In addition, and similarly to other signals of the networks controlling developmental processes, the level of ROS are subjected to multiple cross, feed-forward, and feed-back effects and poorly known factors, as iRNA and epigenetic modifications, that make difficult to identify a precise cause to effect chain of events explaining the final response of the plant.1O2, 2O2, and HO∙ ) are well known. In addition, the levels of the enzymes involved in the generation and scavenging of most ROS can be accurately determined in different cell compartments and in different stress and senescence conditions. From enzyme data, qualitative level differences between stress and senescence for each ROS can reasonably be proposed for different cell compartments which progresses in ROS determination (1O2 + 2O2 for the initiation of senescence at light, could be extended or modified after comparison of enzymatic activities in stress responses with those in other types of senescence, including the senescence of non-photosynthetic tissues. The results must facilitate the identification of proteins that directly interact with specific ROS in the regulatory cellular networks. In a first approach, the complex of WRKY proteins deserves full attention. In addition, the results would provide a wide perspective to investigate more precisely the control of genes for generation and scavenging of ROS in stress and PCD.However, in contrast to most other signals, the successive steps for enzymatic, and non-enzymatic generation and scavenging of the main ROS (mination  could teThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A comprehensive self-administered diet history questionnaire (DHQ: 150-item semi-quantitative questionnaire) and a brief self-administered DHQ (BDHQ: 58-item fixed-portion–type questionnaire) were developed for assessing Japanese diets. We compared the relative validity of nutrient intake derived from DHQ with that from the BDHQ, using semi-weighed 16-day dietary records (DRs) as reference.Ninety-two Japanese women aged 31 to 69 years and 92 Japanese men aged 32 to 76 years completed a 4-nonconsecutive-day DR, a DHQ, and a BDHQ 4 times each (once per season) in 3 areas of Japan .No significant differences were seen in estimates of energy-adjusted intakes of 42 selected nutrients  between the 16-day DRs and the first DHQ (DHQ1) or between the DR and the first BDHQ (BDHQ1) for 18 (43%) and 14 (33%) nutrients, respectively, among women and for 4 (10%) and 21 (50%) nutrients among men. The median (interquartile range) Pearson correlation coefficients with the DR for energy-adjusted intakes of the 42 nutrients were 0.57 (0.50 to 0.64) for the DHQ1 and 0.54 (0.45 to 0.61) for the BDHQ1 in women; in men, the respective values were 0.50 (0.42 to 0.59) and 0.56 (0.41 to 0.63). Similar results were observed for the means of the 4 DHQs and BDHQs.The DHQ and BDHQ had satisfactory ranking ability for the energy-adjusted intakes of many nutrients among the present Japanese population, although these instruments were satisfactory in estimating mean values for only a small number of nutrients. We previously examined the validity of the DHQ and BDHQ in terms of food group intake and found that they had similar and satisfactory validity for a wide range of food groups among adult Japanese women and men in terms of their ability to both estimate median values and rank individuals in a population.4 However, the validity of these questionnaires with regard to nutrient intake has not been compared. Because a finding of satisfactory validity for food groups does not reflect satisfactory validity for nutrients, we must examine the validity of nutrient intakes for the DHQ and BDHQ despite the existence of a validation study for food group intakes.Because dietary questionnaires do not necessarily estimate true food intake, their validity should be evaluated. Several US studies have compared the validity of short and long versions of questionnaires,Here, we compared the relative validity of energy and nutrient intakes derived from the DHQ and BDHQ among healthy women and men in Japan against 16-day semi-weighed dietary records (DRs).4,8 Briefly, the study was conducted in 3 areas of Japan, namely, Osaka (urban), Nagano , and Tottori . In each area, we recruited apparently healthy women aged 30 to 69 years who were willing to participate with a cohabitating husband, such that each of the 10-year age class strata had 8 women, regardless of the age of the husbands. Thus, a total of 96 women and 96 men were invited. Before the study, the study purpose and protocol were explained at group orientations. Written informed consent was obtained from each participant. The study did not undergo ethical approval because it was conducted before the advent of ethical guidelines for epidemiology research in Japan. However, the use of the data of this study was approved by the Ethics Committee at the University of Tokyo Faculty of Medicine (No. 3421). Ultimately, 92 women aged 31 to 69 years and 92 men aged 32 to 76 years who completed the protocol were included in the present analysis.Details of the study design, participant characteristics, and dietary assessment methods have been reported elsewhere.Between November 2002 and September 2003, the participants completed four 4-nonconsecutive-day semi-weighed DRs, 1 in each season, at intervals of approximately 3 months, ie, November and December 2002 (autumn), February 2003 (winter), May 2003 (spring), and August and September 2003 (summer) . Each ofThe participants also answered the DHQ and BDHQ 4 times, once in each season, at intervals of approximately 3 months from November 2002 to September 2003. In each season, the DHQ and BDHQ were answered approximately 2 days before the start of the dietary recording period, in the order of BDHQ before DHQ . Respons3,4,8–11 The DHQ consists of 7 sections: (1) general dietary behavior, (2) usual cooking methods, (3) consumption frequency and amount of alcoholic beverages, (4) consumption frequency and semi-quantitative portion size of selected food and nonalcoholic beverage items, (5) dietary supplements, (6) consumption frequency and semi-quantitative portion size of staple foods, soup for noodles, and miso (fermented soybean paste) soup, and (7) open-ended items for foods consumed more than once a week but not appearing in the DHQ.The DHQ is a 16-page semi-quantitative questionnaire that asks about the consumption frequency and portion size of selected foods to estimate the dietary intake of 150 food and beverage items during the preceding month.4 The BDHQ consists of 5 sections: (1) intake frequency of food and nonalcoholic beverage items, (2) daily intake of rice and miso soup, (3) frequency of drinking and amount per drink for alcoholic beverages, (4) usual cooking methods, and (5) general dietary behavior.The BDHQ is a 4-page fixed-portion questionnaire that asks about the consumption frequency of selected foods, but not about portion size, to estimate the dietary intake of 58 food and beverage items during the preceding month.12 while standard portion sizes and the sizes of bowls for rice and cups for miso soup were derived from several recipe books for Japanese dishes.3,4 Because the use of dietary supplements is uncommon in Japan ,12 we did not considered information on dietary supplements that was derived from the DHQ.Food and beverage items contained in the DHQ and BDHQ were selected from foods commonly consumed in Japan, mainly from a food list used in the National Health and Nutrition Survey of Japan,Standard Tables of Food Composition in Japan.13 Because intakes of most nutrients were positively correlated with energy intake, energy-adjusted values were also calculated by the residual method using a regression model, while the density method was used to compute the amount of each nutrient consumed daily, as a percentage from daily energy intake for energy-containing nutrients or per 10 MJ of daily energy intake for non–energy-containing nutrients.14 All statistical analyses were performed on log-transformed values to account for non-normality. Relative validity of the DHQ and BDHQ were assessed in terms of their ability to estimate representative values by comparing mean values and ranking ability, using Pearson correlation coefficients. We compared nutrient intakes derived from the first DHQ (DHQ1) and the first BDHQ (BDHQ1) with those from the four 4-day DRs. Although the reference period differed between DHQ1 or BDHQ1 and the four 4-day DRs (1 conducted in each season), our purpose here was to examine whether a single DHQ or BDHQ for dietary habits during the previous month could represent habitual dietary intake over a longer period. Additionally, we also examined the mean of the 4 DHQs and BDHQs (mDHQ and mBDHQ) using the same method to compare annual intake from the questionnaires with that of the DR.All statistical analyses were done using SAS statistical software, version 9.2  and were performed separately for women and men. Crude values for the intake of energy and 42 selected nutrients were estimated based on the intake of food items obtained with the DR or respective questionnaire and the corresponding food composition list in the t-test using 1-sided values. A P value less than 0.05 was considered to indicate a significant difference. Pearson correlation coefficients between the DR and DHQ1, BDHQ1, mDHQ, and mBDHQ were then calculated. Additionally, because a 16-day DR may be insufficient to account for intra-individual variation, we also calculated deattenuated correlation coefficients using intraindividual and interindividual variances.1 Correlation coefficients obtained from the DHQ and BDHQ were compared using the Meng-Rosenthal-Rubin method to compare overlapping correlation coefficients,15 with tested correlation coefficient pairs considered statistically different when z was greater than 1.96, with a significance level of 5%.Energy and nutrient intakes in the crude and energy-adjusted models for DR, DHQ1, BDHQ1, mDHQ, and mBDHQ are presented as means. Statistically significant differences between the DR and each of the questionnaires were determined with the paired Selected characteristics of the participants are shown in Table Pearson correlation coefficients for estimates of crude and energy-adjusted nutrient intakes and energy intake on the DR versus the DHQ and BDHQ are shown in Table We examined the relative validity of energy and nutrient intakes estimated by the DHQ and BDHQ, using a 16-day DR as reference. For energy and many  energy-adjusted nutrients, mean intakes estimated by DHQ1 and BDHQ1 were significantly different from those estimated by the DR. The results for mDHQ and mBDHQ were similar. These results suggest that the DHQ and BDHQ are satisfactory for estimating mean values for only a limited number of nutrients.However, for many  correlations with energy-adjustment and many  deattenuated correlations with energy-adjustment, values greater than 0.4 were observed between the DR and DHQ1 and between the DR and BDHQ1. Although the correlation with the DR was slightly higher for mDHQ and mBDHQ than for DHQ1 and BDHQ1, the latter questionnaires had reasonable correlations for many nutrient intakes. Furthermore, correlation coefficients between the DR and DHQ1 and between the DR and BDHQ1 did not significantly differ for almost all nutrients, which was also the case for mDHQ and mBDHQ. Thus, the DHQ and BDHQ had satisfactory ranking ability for many nutrients in this population.5–7 all have shown reasonable correlations between the 2 versions. Our results in a Japanese population are consistent with those findings. Wakai reviewed the validity of dietary questionnaires developed and validated in Japan16 and showed that medians of coefficients between DRs and questionnaires on energy and nutrients ranged from 0.31 to 0.56 in the studies investigated. Our present DHQ and BDHQ thus performed similarly to these other Japanese dietary questionnaires. Wakai then considered long (97 or more food items) versus short questionnaires (<70 items) and reported that the median correlation coefficient for nutrients in an individual questionnaire ranged from 0.42 to 0.52 for the long form and from 0.31 to 0.45 for the short form. On the basis of those results, he concluded that long questionnaires had slightly higher validity in the estimation of nutrient intake. In the present study, the corresponding values for women were 0.57 for the DHQ and 0.54 for the BDHQ, with respective values for men of 0.50 and 0.56. Our study indicated that, unlike the results of the review, the BDHQ did not necessarily have a worse correlation as compared with the DHQ.Of the several US studies that have compared short and long versions of the same questionnaire with regard to energy and nutrient intake,17 Although it is not clear why the median correlation coefficient of the DHQ was higher than that of the BDHQ in women while the opposite was true in men, the degree of the effects of questionnaire length and misestimation of portion size may differ by sex. Because of its lesser burden, the BDHQ might be preferred in the investigation of many of the nutrients examined here. However, it should be mentioned that correlations of some nutrients  were higher for the DHQ than for the BDHQ. Further, the DHQ was better than the BDHQ in estimating mean intakes of vitamin C and some other nutrients. These results indicate that the DHQ is more suitable than the BDHQ in estimating intakes of some nutrients.There are several explanations for why both the 150-item DHQ and the 58-item BDHQ had relatively high correlations. First, the food and beverage items in the BDHQ may accurately reflect the foods commonly consumed in Japan. Second, the DHQ requires more time to complete than the BDHQ, which may reduce the accuracy of responses for the former questionnaire. Third, the BDHQ differs from the DHQ in that it does not ask about portion size. Given that misestimation of portion size is a source of error in answering these questionnaires, some participants might have inaccurately answered at least some of the DHQ questions on portion size.18–24 However, the correlations of these nutrients from mDHQ and mBDHQ were slightly higher than those from DHQ1 and BDHQ1. If we were to examine the intakes of these nutrients in an epidemiologic study using the DHQ or BDHQ, multiple DHQs and BDHQs would be needed.Although the median correlations for both the DHQ and BDHQ were relatively good, in men, correlation coefficients for retinol in the DHQ1 and BDHQ1, α-carotene in the DHQ1, and n-3 polyunsaturated fatty acid in the BDHQ1 were much lower than those of other nutrients in the present study and much lower than those of any nutrient in other studies.1 Biomarkers are a better standard for some nutrients, and any errors in such markers are independent of errors in the questionnaire. Validity testing of the DHQ for Japanese adults by using 24-hour urine, serum, or doubly labeled water methods has revealed satisfactory validity for some nutrients.9–11 Validation studies with quantitative biomarkers are also necessary for the BDHQ. Second, because the algorithm used to calculate food intake from the BDHQ was written using previous information19 and other unpublished observations, it had insufficient reliability. Finally, the generalizability of the present results is limited because the participants were not representative of the general Japanese population and might have been highly health conscious. Additionally, while all the women answered the questionnaires themselves, a considerable number of men (32%) answered with the help of their wives. The validity of the 2 questionnaires in men might have been lower had they answered the questionnaires themselves.Several limitations of this study should be mentioned. First, although we assumed that energy and nutrient intakes derived from the DR were the standard, DRs are susceptible to measurement error due to erroneous recording and potential changes in eating behavior. Nevertheless, as compared with 24-hour dietary recall or other instruments that rely on memory, errors in DRs are thought to have a lower correlation with errors in the DHQ and BDHQ.In conclusion, this study showed that the DHQ and BDHQ had satisfactory ranking ability for energy-adjusted intakes of many nutrients, as compared with DRs, in a population of Japanese men and women, although ability of these instruments to estimate mean values was satisfactory for only a limited number of nutrients. Additionally, although the correlations obtained from mDHQ and mBDHQ were better than the respective values from DHQ1 and BDHQ1, even a single DHQ and BDHQ had reasonable correlations for many nutrient intakes. The validity of the BDHQ was similar to that of the DHQ. These findings support the idea that, for many energy-adjusted nutrients, both the DHQ and BDHQ can be used in large-scale epidemiologic studies in Japan.


The crystal structure of the ternary complex of NAD+-dependent formate dehydrogenase from the methylotrophic bacterium Moraxella sp. C-1 with the cofactor (NAD+) and the inhibitor (azide ion) was established at 1.1 A resolution. The complex mimics the structure of the transition state of the enzymatic reaction. The structure was refined with anisotropic displacitalicents parameters for non-hydrogen atoms to a R factor of 13.4%. Most of the nitrogen, oxygen, and carbon atoms were distinguished based on the analysis of the titalicperature factors and electron density peaks, with the result that side-chain rotamers of histidine residues and most of asparagine and glutamine residues were unambiguously determined. A comparative analysis of the structure of the ternary complex determined at the atomic resolution and the structure of this complex at 1.95 A resolution was performed. In the atomic resolution structure, the covalent bonds in the nicotinamide group are somewhat changed in agreitalicent with the results of quantum mechanical calculations, providing evidence that the cofactor acquires a bipolar form in the transition state of the enzymatic reaction. The structurally simplest formate dehydrogenases are NAD FDH, EC .2.1.2, w+ and formate. FDHs from different organisms  function as dimers consisting of two identical subunits with a molecular weight from 35 to 50 kDa. The molecular mechanism of FDH is characterized by the direct transfer of a hydride ion from the substrate to the C4 atom of the nicotinamide ring of NAD+ without additional proton transfer steps, which usually occurs in reactions catalyzed by related NAD+-dependent dehydrogenases. Hence, the FDH-catalyzed reaction is a convenient model for studying the mechanism of hydride ion transfer in the active site of NAD+-dependent hydrogenases by methods of quantum mechanics and molecular dynamics [Formate dehydrogenases belong to a large superfamily of D-isomer specific 2-hydroxyacid dehydrogenases [The knowledge of the three-dimensional structure of the enzyme under study with accurate atomic coordinates is of great importance for the investigation of the molecular mechanisms of the enzyme. Based on X-ray diffraction data at atomic resolution, the protein structure can be refined with anisotropic displacitalicent parameters for individual non-hydrogen atoms, which substantially increases the reliability of structural information and allows the determination of atomic coordinates with 0.02 A accuracy . At this+-dependent dehydrogenases, such as horse liver alcohol dehydrogenase (five structures) [Lactobacillus brevis (three structures) [Plasmodium falciparum (one structure) [Moraxella sp. C-1 (MorFDH) with NAD+ and an inhibitor (azide ion), which mimics the transition state of the enzymatic reaction, was established at atomic resolution (1.1 A).Currently, 937 protein structures with atomic resolution are available in the Protein Data Bank . This is approximately 1.6 % of the total number of structures in the PDB. Atomic resolution structures were solved only for three NADuctures) , and lacructure) . The avaructure)  and 1.55ructure) . In thisCrystals of the MorFDH-NAD+-azide ternary complex were grown by the hanging drop vapor diffusion method. Recombinant full-size MorFDH was expressed and purified according to the procedure described in . The puro.X-ray diffraction data were collected at a wavelength of 0.8166 A using a MAR165 CCD detector at the EMBL X11 beamline at the DORIS storage ring of the DESY synchrotron (Hamburg). To decrease radiation damage in the protein crystal and increase the resolution due to a decrease in the thermal displacements of the atoms, the crystal was cooled with nitrogen (100 K) using a Cryojet cryocooler during data collection. Before freezing in a stream of nitrogen, the crystal was soaked in a cryosolution  glycerol) for 30 s. Two X-ray diffraction data sets were collected from the same crystal. The first data set was collected in the resolution range 20-1.5 A with a short exposure time; the second data set, in the resolution range 20-1.1 A with a longer exposure time. This made it possible to avoid overexposure of low-resolution reflections, which is often the case when the exposure time is high, and to measure weak high-resolution reflections with high accuracy. The diffraction data sets were processed with DENZO and SCALEPACK program packages . The cry+-azide ternary complex at 1.95 A resolution [Since these crystals are isomorphous with the crystals that were used for the structure determination of the MorFDH-NADsolution , the latsolution . The consolution . The corsolution . The errsolution . The dif+-azide structure lowered the R factor by 5.3 % and the Rfree, by 5.2 %. The mean weighted error of the atomic coordinates characterized by the diffraction precision index (DPI) also decreased from 0.036 to 0.028 A. For the previously published structure of the MorFDH-NAD+-azide complex at 1.95 A resolution, this value was 0.141 A [The great advantage of atomic resolution is the high ratio of observations to parameters, due to which the temperature factors of non-hydrogen atoms can be refined anisotropically. In the isotropic approximation, three positional parameters and the isotropic temperature factor of each non-hydrogen atom are refined, whereas six anisotropic thermal parameters are used instead of one isotropic temperature factor for each atom  in the anisotropic refinement. This approach allows a much more accurate interpretation of the diffraction data. The inclusion of the anisotropic parameters into the crystallographic refinement of the MorFDH-NAD+-azide complex is depicted in + molecule, four azide ions, and six glycerol molecules were located. The NAD + molecule and one azide ion are bound in the active site of the enzyme, whereas three azide ions and all glycerol molecules are located on the surface of the protein globule. Like in the structure solved at 1.95 A resolution, the C-terminal residues 392-399 were located in the atomic resolution structure. These eight residues were not visible in electron density maps calculated for the previously solved structure of the holo-form of FDH from the bacterium Pseudomonas sp. 101 [The polypeptide chain fold of the solved structure of the ternary MorFDH-NAD sp. 101 . Neverth+-azide structures solved at 1.1 A and 1.95 A resolution is 0.30 A, which indicates that they are identical. The maximum deviation is 1.1 A (for the residue Ser18). The atomic coordinates for 18 amino acid side chains located on the surface of the protein globule differ by more than 1 A, with the maximum deviation being 9 A (for the residue Arg26). These differences may be associated with the properties of the crystals grown in slightly different crystallization conditions or by the erroneous location of the side chains in the structure at lower resolution.The rms deviation of the coordinates of all C≤ atoms of the MorFDH-NADA considerable difference of the structure at atomic resolution is that the number of water molecules located in this structure is 2.4 times larger than that in the structure solved at 1.95 A resolution . It should be noted that 169 water molecules have virtually identical positions in both structures. In fact, only conserved highly ordered water molecules, which are located in the cavities of the protein globule and in the first solvent shell, were located in the structure at 1.95 A resolution. The atomic resolution structure allowed the much more accurate and reliable determination of the solvent structure in the crystal structure of the MorFDH-NAD+-azide complex. +-azide complex at 1.95 A resolution, only five residues were found to adopt two different conformations. A considerable improvement of the quality of the electron density maps for the atomic resolution structure allowed the identification of ten such residues. The side chains of five lysine residues  and one glutamine residue (Glu397) were located only partially, which is evidence that these residues exist in several conformations. All residues adopting alternative conformations and with missing side chain atoms in the model are on the surface of the protein globule, which accounts for their high conformational flexibility.Due to the high accuracy of diffraction data, the atomic resolution allows the more precise determination of the alternative conformations of residues. In the structure of the MorFDH-NAD0 with respect to the actual conformation in the protein crystal, the B factor of the nitrogen atom will be considerably lower, whereas the B factor of the oxygen atom will be considerably higher than the average temperature factors of covalently bonded atoms. A similar situation (higher B factors of the nitrogen atoms N≤ and N≤ and lower B factors of the carbon atoms C≤ and C≤) is observed also for incorrectly rotated histidine side chains. In addition, the diffraction data at atomic resolution allows one to differentiate between carbon, oxygen, and nitrogen atoms based on electron density maps, because atoms with more electrons have higher electron density peaks +-azide complex at 1.1 A resolution were confirmed, and the side chains of the Gln66 and Asn135 residues were found to be rotated compared to their conformation in the 1.95 A resolution structure. It was not always possible to unambiguously choose the isomer based on the analysis of the temperature factors, which is an indication that both rotational isomers are present in the structure.A considerable advantage of atomic resolution diffraction data is that carbon, oxygen, and nitrogen atoms can be reliably differentiated, which allows the identification of rotational isomers of asparagine, glutamine, and histidine side chains. The rotational isomers of these residues are usually determined by analyzing the hydrogen bonds with adjacent atoms, and in some case definite conclusions cannot be drawn. The X-ray diffraction data at atomic resolution allows one to determine the isomers of asparagine and glutamine residues by analyzing the temperature factors of the side chain nitrogen and oxygen atoms. If the carboxamide group of asparagine or glutamine in the model is rotated by 180+ molecule and azide ion is depicted in +-azide complex is considered as a stable analog of the transition state of the enzymatic reaction [The structure of the active site of MorFDH containing the bound NADreaction , 22.+-azide ternary complex may reflect important details of the structural organization of the transition state in the FDH-catalyzed reaction.The X-ray diffraction data at atomic resolution confirm that the carboxamide group of the cofactor in the active site of MorFDH is fixed in the trans conformation (the 07 atom is directed to the C4 atom) by hydrogen bonds with active site residues. According to quantum-mechanical calculations in the gas phase, the trans conformation is 2 kcal/mol less favorable than the cis conformation . The anaPreviously, it has been shown by calculation methods, using combined molecular dynamics and quantum mechanics studies, that the cofactor molecule adopting the energetically activated trans conformation in the transition state of the FDH-catalyzed reaction may take on the properties of the so-called bipolar form . A similDue to the high accuracy of the atomic resolution diffraction data, the influence of standard stereochemistry on the atomic coordinates of the nicotinamide group of the cofactor can be weakened in the course of the crystallographic structure refinement. After a separate refinement cycle using the REFMAC program with weaker bond length constraints for the nicotinamide group (by increasing the rms bond length deviation from 0.02 A to 0.20 A), the C7-07 bond length was 1.29 A, which is 0.06 A smaller than the standard value, and the C3-C7 bond length was 1.43 A, which is 0.07 A longer than the standard value. These differences are only 1.3 times larger than the error in the bond lengths estimated as the sum of two mean-weighted errors in the atomic coordinates . Neverth+-dependent dehydrogenases.To sum up, the atomic resolution structure of FDH was determined for the first time, which provides a deeper insight into the details of the molecular mechanism of this enzyme and the hydride ion transfer in the active sites of NADThis work was supported by the Russian Federal Agency for Science and Innovation  and the Russian Foundation for Basic Research (grant № 08-04-00830-a). The authors thank the italicBL Hamburg Outstation for the allotted time on the synchrotron radiation source and Alexander Popov personally for assistance with diffraction data collection.