Effect of Energy Under-Reporting on Secular Trends of Dietary Patterns in a Mediterranean Population

BackgroundDiet is an important factor in the prevention of chronic diseases. Analysis of secular trends of dietary patterns can be biased by energy under-reporting. Therefore, the objective of the present study was to analyse the impact of energy under-reporting on dietary patterns and secular trends in dietary patterns defined by cluster analysis.ResultsThree clusters, “healthy”, “mixed” and “western”, were identified for both surveys. The “mixed” cluster was the predominant cluster in both surveys. Excluding EUR reduced the proportion of the “mixed” cluster up to 6.40% in the 2000 survey; this caused secular trend increase in the prevalence of the “mixed” pattern. Cross-classification analysis of all participants and PER’ data showed substantial agreement in cluster assignments: 68.7% in 2000 and 84.4% in 2005. Excluding EUR did not cause meaningful (≥15%) changes in the “healthy” pattern. It provoked changes in consumption of some food groups in the “mixed” and “western” patterns: mainly decreases of unhealthy foods within the 2000 and increases of unhealthy foods within the 2005 surveys. Secular trend effects of EUR were similar to those within the 2005 survey. Excluding EUR reversed the direction of secular trends in consumption of several food groups in PER in the “mixed” and “western” patterns.ConclusionsEUR affected distribution of participants between dietary patterns within and between surveys, secular trends in food group consumption and amount of food consumed in all, but not in the “healthy” pattern. Our findings emphasize threats from energy under-reporting in dietary data analysis.     

Properties of spinach yellow mottle, a distinctive strain of tobacco rattle virus

A virus (isolate SYM) obtained from spinach plants in England with a severe yellow mottle disease induced symptoms resembling those of tobacco rattle virus (TRV) in several indicator species but caused systemic necrosis in Chenopodium amaranticolor and C. quinoa. It was transmitted to bait plants grown in soil containing the nematode Trichodorus primitivus. Purified virus preparations contained rod-shaped particles that were predominantly of four modal lengths: 188 nm (L particles), 101 nm (S particles), 57 nm and 48 nm (together called VS particles), containing RNA with mol. wts of 2.4, 1.5, 0.7 and 0.6 million, respectively. L particles (s°₂₀= 300 S) and S particles (230 S) greatly outnumbered VS particles (c. 150 S). All particles contained a single polypeptide species with estimated mol wt of 24 700, slightly larger than those previously reported for tobraviruses. Purified L particles were infective but both L and S particles were needed to induce the production of virus nucleoprotein particles. VS particles were not infective and apparently had no qualitative or quantitative effect on infection by L or by L plus S particles. S particles carried determinants for serological specificity and ability to invade C. amaranticolor systemically. Isolate SYM produced pseudo-recombinants with isolate PRN of TRV. Also, isolates CAM, OR and PRN of TRV, and isolate SYM, were found to be distantly related by three kinds of serological test. No relationship was detected between these isolates and pea early-browning virus in gel-diffusion precipitin tests or electron microscope serological tests, but a distant relationship between isolate SYM and pea early-browning virus was found by micro-precipitin tests. Isolate SYM therefore has closer affinities with TRV than with pea early-browning virus and is considered to be a distinctive strain of TRV.     

Gene-coding assignments of rotavirus double-stranded RNA segments 10 and 11

The gene-coding assignments for genome segments 10 and 11 of a simian virus and two human rotaviruses were determined. For those viruses having a “long” RNA gel pattern (electropherotype), segments 10 and 11 encoded proteins NS₃ and O₄, respectively. The human virus with a “short” electropherotype had the opposite assignments and also differed in (enzyme-linked immunosorbent assay) serotype from the human virus with a long electropherotype.     

Studies on Persistent Infections of Tissue Culture VI. Reversible Changes in Newcastle Disease Virus Populations as a Result of Passage in L Cells or Chick Embryos

Populations of the Victoria strain of Newcastle disease virus (NDV), reisolated from persistently infected L-cell cultures and passed twice in the embryonated hen's egg (NDV_L-E-2), were found to differ strikingly from the original, chick embryo-adapted virus (NDV_o). After exposure of L cells to NDV_o at high multiplicities of infection, all cells became abortively infected; they produced only small aggregates of viral antigen and few, if any, infectious virus particles, but they yielded large amounts of interferon. No cytopathic effects (CPE) were noted, and the cultures survived readily as viral carriers. In contrast, NDV_L-E-2 yielded under similar conditions large quantities of viral antigen and infectious virus particles, but no detectable interferon, and the cultures were rapidly destroyed. This change in “virulence” was at least partially reversible by further serial passages of NDV_L-E-2 in chick embryos, as was evident from a consecutive decrease in CPE with a concomitant increasingly rapid recovery of the L-cell cultures, gradually diminishing yields of infectious viral progeny, and the returning of a capacity to induce interferon synthesis. Thus, NDV_L-E-16 resembled NDV_o in many aspects, except for a less striking reduction in its ability to replicate in L cells. Although a selection of viral variants under the given sets of conditions has not been entirely excluded, the establishment of “avirulence” appears to be largely explained by a gradual accumulation of noninfectious, interferon-inducing components in the course of serial passages in the embryonated hen's egg, and the acquisition of “virulence” by a loss of these components. The evidence is as follows. (i) By a step-wise decrease in the dose of virus and restriction of the analyses to the first infectious cycle, a multiplicity of infection was ultimately reached for all “avirulent” populations at which infected cells produced normal yields of infectious viral progeny; i.e., the interferon-inducing components were diluted to noneffective levels. The lowest multiplicity which resulted in a measurable reduction in infectious virus replication was also the last one to induce detectable interferon synthesis. (ii) All viral clones derived from “avirulent” populations behaved like NDV_L-E-2 rather than like the parent viral suspensions, except that some of them elicited small amounts of interferon in L cells. The interferon-inducing components were reduced or lost in the cloning procedures. The nature of the interferon-inducing components has not been established. These components, which were neutralized by rabbit sera against “virulent” NDV_L-E-2 populations, may represent largely inactive or incomplete virus particles; however, the infectious virus-hemagglutinin ratios of “avirulent” populations were mostly of an order similar to those of “virulent” populations. The interferon-inducing components aborted the infectious process in cells simultaneously invaded by infectious virus particles. The implications of these findings are discussed.     

The formation of “mega‐flocks” depends on vegetation structure in montane coniferous forests of Taiwan

A mixed‐species bird flock is a social assemblage where two or more bird species are moving together while foraging and might benefit from increased foraging efficiency and antipredator vigilance. A “mega‐flock,” which includes flocking species from different vegetation strata, often exhibits high species diversity. Mechanisms for the formation of mega‐flocks have not yet been explored. In this study, we evaluated the influence of vegetation structure and bird species diversity in driving the occurrence of mega‐flocks. We investigated the composition of mixed‐species flocks, local bird communities, and vegetation structure in five vegetation types of two high‐elevation sites in central Taiwan. Mega‐flocks occurred more frequently in pine woodland than later successional stages of coniferous forests. However, species richness/diversity of local bird communities increased along successional stages. Therefore, vegetation variables exhibit more influence on the occurrence of mega‐flocks than local bird communities. Besides foliage height diversity, understory coverage also showed positive effects on flock size of mixed‐species flocks. Our results indicated that pine woodlands with more evenly distributed vegetation layers could facilitate the interactions of canopy and understory flocks and increase the formation of mega‐flocks and thus the complexity of mixed‐species flocks.     

Transcription of Simian Virus 40 II. Hybridization of RNA Extracted from Different Lines of Transformed Cells to the Separated Strands of Simian Virus 40 DNA

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and “revertants” isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and “revertant” cell lines. From 30 to 80% of the “early” strand and from 0 to 20% of the “late” strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.     

Rapid vascular movement of tobraviruses does not require coat protein: evidence from mutated and wild-type viruses

Early studies of the tobravirus Tobacco rattle virus (TRV) described two types of virus isolate with apparently different disease characteristics. M‐type isolates, which contain both viral genomic RNAs and form virus particles, could be passaged by mechanical inoculation and produced rapid but shortlived systemic symptoms. In contrast, NM‐type isolates, which contain only RNA1 and do not form virus particles, were difficult to passage by mechanical inoculation and were very slow to produce systemic symptoms. From the early observations on such isolates made in the 1960s, it has become accepted that M isolates with encapsidated TRV particles move rapidly through the vascular system whereas NM isolates containing only unencapsidated TRV RNA1 move only slowly via plasmodesmata from cell to cell and take many weeks to reach the upper parts of plants. However, we show that NM isolates of TRV and another tobravirus Pea early‐browning virus (PEBV) move into systemic tissue of TV. benthamiana and N. clevelandii by 6 days post inoculation, suggesting that this rapid movement occurs via the vasculature. The systemic movement of TRV and PEBV mutants lacking functional coat protein that have been modified to express the green fluorescent protein were examined by confocal microscopy. This confirmed that the tobraviruses do not require the CP for long distance movement via the phloem, a property that is shared with only a small group of plant viruses.     

High-throughput sequencing reveals the disruption of methylation of imprinted gene in induced pluripotent stem cells

It remains controversial whether the abnormal epigenetic modifications accumulated in the induced pluripotent stem cells (iPSCs) can ultimately affect iPSC pluripotency. To probe this question, iPSC lines with the same genetic background and proviral integration sites were established, and the pluripotency state of each iPSC line was characterized using tetraploid (4N) complementation assay. Subsequently, gene expression and global epigenetic modifications of “4N-ON” and the corresponding “4N-OFF” iPSC lines were compared through deep sequencing analyses of mRNA expression, small RNA profile, histone modifications (H3K27me3, H3K4me3, and H3K4me2), and DNA methylation. We found that methylation of an imprinted gene, Zrsr1, was consistently disrupted in the iPSC lines with reduced pluripotency. Furthermore, the disrupted methylation could not be rescued by improving culture conditions or subcloning of iPSCs. Moreover, the relationship between hypomethylation of Zrsr1 and pluripotency state of iPSCs was further validated in independent iPSC lines derived from other reprogramming systems.     

An X-Ray Scattering Investigation of Wild Cucumber Mosaic Virus and a Related Protein

X-ray scattering data are presented on solutions of wild cucumber mosaic virus and the associated “top component” particles which have little or no RNA. The radii of gyration are 112 Å and 135 Å for bottom and top component, respectively. The radial density distribution within each particle is calculated by Fourier inversion of the scattered amplitudes. The virus particle or bottom component has approximately uniform density with an outer radius of about 140 Å. The transform of the top component shows an almost hollow center extending out to 105 Å with a surrounding shell of high density about 35 Å thick. Thus the RNA would appear to occupy the region inside 105 Å and does not overlap appreciably the region occupied by protein. The virus has associated with it approximately 0.38 gm of water per gm of virus, resulting in an average electron density of 1.25 times that of water.     

Characterization of the Rapidly Labeled Hybridizable RNA Synthesized in L5178Y Mouse Leukemic Cells: Hybridization Lifetime Studies

An attempt is made to characterize the rapidly labeled hybridizable RNA of L5178Y mouse leukemic cells which has been shown to have similar base sequences when synthesized in two different stages of the cell cycle. The size of rapidly labeled RNA molecules was heterogeneous. For labeling times of 20 min or less, the per cent of hybridization was maximal. With longer labeling times, the per cent of hybridization decreased as radioactivity appeared in long-lived species of low hybridization efficiency; the radioactivity profile resembled the optical density profile in sucrose gradients. The lifetime of newly synthesized hybridizable RNA was studied by pulse labeling exponentially growing cells and then “chasing” with nonradioactive uridine. The per cent of hybridization was studied as a function of chase time. Three RNA groups, which comprised different proportions of rapidly labeled hybridizable RNA, were distinguished. The short-lived group had a half-life of 10 min, much less than the values reported in the literature for messenger RNA of mammalian cells. The half-life of 1-1½ hr observed for a medium-lived group more closely corresponds to that of messenger RNA. A long-lived group had a half-life of approximately 20 hr. Specific activity measurements during chase indicate the presence of a “pool” of labeled uridine derivatives. The uridine of this pool appears to be nonexchangeable with but dilutable by exogenous uridine. A nontoxic concentration of actinomycin D was added to the chase media in an attempt to block the “pool effect”. A rapidly degradable RNA was demonstrable both by specific activity and per cent of hybridization measurements.     

Hybridization of Rous Sarcoma Virus Deoxyribonucleic Acid Polymerase Product and Ribonucleic Acids from Chicken and Rat Cells Infected with Rous Sarcoma Virus

Rous sarcoma virus (RSV)-specific ribonucleic acid (RNA) in virus-producing chicken cells and non-virus-producing rat cells infected with RSV was studied by hybridization with the endogenous deoxyribonucleic acid (DNA) product of the RSV virion DNA polymerase system. By hybridizing the total DNA product with excess virion RNA, the product DNA was separated into hybridized (“minus”) and nonhybridized (“plus”) DNA. The “minus” DNA was complementary to at least 20% of the RNA from RSV which remained of high molecular weight after denaturation. A maximum of approximately 65% hybridization was observed between “minus” DNA and RSV RNA or RSV-infected chicken cell RNA. A maximum of about 60% hybridization was observed between “minus” DNA and RSV-infected rat cell RNA. RSV-infected chicken cells contained RSV-specific RNA equivalent to about 6,000 virions per cell. RSV-infected rat cells contained RSV-specific RNA equivalent to approximately 400 virions per cell. Neither cell type contained detectable RNA complementary to virion RNA. The RSV-specific RNA in RSV-infected rat cells did not appear to be qualitatively different from that in RSV-infected chicken cells.     

The Snakelike Chain Character of Unstructured RNA

In the absence of base-pairing and tertiary structure, ribonucleic acid (RNA) assumes a random-walk conformation, modulated by the electrostatic self-repulsion of the charged, flexible backbone. This behavior is often modeled as a Kratky-Porod “wormlike chain” (WLC) with a Barrat-Joanny scale-dependent persistence length. In this study we report measurements of the end-to-end extension of poly(U) RNA under 0.1 to 10 pN applied force and observe two distinct elastic-response regimes: a low-force, power-law regime characteristic of a chain of swollen blobs on long length scales and a high-force, salt-valence-dependent regime consistent with ion-stabilized crumpling on short length scales. This short-scale structure is additionally supported by force- and salt-dependent quantification of the RNA ion atmosphere composition, which shows that ions are liberated under stretching; the number of ions liberated increases with increasing bulk salt concentration. Both this result and the observation of two elastic-response regimes directly contradict the WLC model, which predicts a single elastic regime across all forces and, when accounting for scale-dependent persistence length, the opposite trend in ion release with salt concentration. We conclude that RNA is better described as a “snakelike chain,” characterized by smooth bending on long length scales and ion-stabilized crumpling on short length scales. In monovalent salt, these two regimes are separated by a characteristic length that scales with the Debye screening length, highlighting the determining importance of electrostatics in RNA conformation.     

BeeDoctor,a Versatile MLPA-Based Diagnostic Tool for Screening Bee Viruses

The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called “BeeDoctor”, was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. “BeeDoctor” is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. “BeeDoctor” was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the “BeeDoctor”, virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies.     

Viral Evasion of a Bacterial Suicide System by RNA–Based Molecular Mimicry Enables Infectious Altruism

Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein–RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that “escaped” ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these “pseudo-ToxI” genetic repeats and, in one case, an escape phage had “hijacked” ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA–based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.     

Isolation and identification of lymphocytic and myelogenous leukemia-specific sequences in genomes of gibbon oncornaviruses

Five gibbon ape leukemia virus substrains (two from gibbons with lymphocytic leukemia and three from gibbons with myelogenous leukemia) were examined for unique genomic sequences specific for each form of leukemia. By using sequential adsorption procedures, the genome from each gibbon ape leukemia virus was fractionated into four sets of distinct nucleotide sequences. Based on their hybridization specificities toward DNAs of leukemic tissues, these sequences were designated as follows: (i) “COM,” (ii) “LYM” or “MYE,” (iii) “UNI,” and (iv) “UND.” The COM fraction represented sequences common to all of the viral genomes. The LYM fraction, which was isolated only from gibbon ape leukemia viruses associated with lymphocytic leukemia, represented genomic sequences associated with lymphocytic leukemia since the RNA hybridized at a 4- to 15-fold-higher rate to infected tissue DNA from lymphocytic leukemic gibbons than to infected tissue DNA from myelogenous leukemic gibbons. The MYE fraction, which was isolated only from gibbon ape leukemia viruses associated with myelogenous leukemia, represented genomic sequences associated with myelogenous leukemia since the RNA hybridized at a 5- to 15-fold-higher rate to infected tissue DNA from myelogenous leukemic gibbons than to infected tissue DNA from lymphocytic leukemic gibbons. The UNI fraction contained sequences unique to one virus substrain. The UND fraction contained sequences which varied depending upon the substrains involved in the adsorption procedures. These findings suggest that each gibbon ape leukemia virus examined in this study contains subgenomic sequences that are specifically identifiable only with the form of leukemia from which the virus was isolated.     

The Risks of Sleeping “Too Much”. Survey of a National Representative Sample of 24671 Adults (INPES Health Barometer)

Background

A significant U-shaped association between sleep duration and several morbidity (obesity, diabetes or cardiovascular disease) and mortality risks has been regularly reported. However, although the physiological pathways and risks associated with “too short sleep” (<5 hours/day) have been well demonstrated, little is known about “too much sleeping”.

Purpose

To explore socio-demographic characteristics and comorbidities of “long sleepers” (over 10 hours/day) from a nationally representative sample of adults.

Methods

A cross-sectional nationally representative sample of 24,671 subjects from 15 to 85-year-old. An estimated total sleep time (TST) on non-leisure days was calculated based on a specifically designed sleep log which allows to distinguish “long sleepers” from “short sleepers” (<5 hours/day). Insomnia was assessed according to the International classification of sleep disorders (ICSD-2).

Results

The average TST was 7 hours and 13 minutes (+/− 17 minutes). Six hundred and twelve subjects were “long sleepers” (2.7%) and 1969 “short sleepers” (7.5%). Compared to the whole group, “long sleepers” were more often female, younger (15–25 year-old) or older (above 65 year-old), with no academic degree, mostly clerks and blue collar workers. “Long sleepers” were significantly more likely to have psychiatric diseases and a greater body mass index (BMI). However, long sleep was not significantly associated with the presence of any other chronic medical disease assessed. Conversely, short sleep duration was significantly associated with almost all the other chronic diseases assessed.

Conclusions

In the general population, sleeping too much was associated with psychiatric diseases and higher BMI, but not with other chronic medical diseases.     

Electron Tomography Reveals the Steps in Filovirus Budding

The filoviruses, Marburg and Ebola, are non-segmented negative-strand RNA viruses causing severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. The sequence of events that leads to release of filovirus particles from cells is poorly understood. Two contrasting mechanisms have been proposed, one proceeding via a “submarine-like” budding with the helical nucleocapsid emerging parallel to the plasma membrane, and the other via perpendicular “rocket-like” protrusion. Here we have infected cells with Marburg virus under BSL-4 containment conditions, and reconstructed the sequence of steps in the budding process in three dimensions using electron tomography of plastic-embedded cells. We find that highly infectious filamentous particles are released at early stages in infection. Budding proceeds via lateral association of intracellular nucleocapsid along its whole length with the plasma membrane, followed by rapid envelopment initiated at one end of the nucleocapsid, leading to a protruding intermediate. Scission results in local membrane instability at the rear of the virus. After prolonged infection, increased vesiculation of the plasma membrane correlates with changes in shape and infectivity of released viruses. Our observations demonstrate a cellular determinant of virus shape. They reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of Marburg virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses.     

Development of a challenge-protective vaccine concept by modification of the viral RNA-dependent RNA polymerase of canine distemper virus

We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a “quick response” in vaccine development against well-known and “new” viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.