Acidosis is a major cause of death in patients with malaria, although what causes acidosis is still unclear. One possibility is that hypovolemia contributes to the problem, and that rehydration therapy could be of benefit. Now, Sanjeev Krishna and colleagues have shown that in children with severe malaria dehydration is not severe and is not correlated with other measures of disease severity. “The optimum resuscitation approach in severe childhood malaria remains to be defined,” says Nick White , the academic editor of the paper. “The relative advantages of blood, colloids, and crystalloids need to be characterized.”Every year around 200 million people worldwide contract malaria, of whom over a million die. The vast majority of those who die are children under five years, mostly in Africa, since young children have had little chance to acquire any immunity. Fluid resuscitation is generally considered to be a cornerstone of treatment—but how much fluid should be given? Some researchers believe that surrogate signs of fluid depletion—such as tachycardia, reduced capillary refill time, and reduced urine excretion—suggest that there is substantial volume depletion. The reason that the amount of fluid given matters so much is that giving too much, especially of hypotonic solutions, can lead to electrolyte imbalance, especially hyponatremia and hypokalemia.Research efforts have been hampered by not having an easy way to assess in patients the fluid depletion in different compartments of the body, i.e., total body water and extracellular and intracellular water volume. Krishna and colleagues used heavy-water distribution to calculate the total body water and bromide distribution to determine the extracellular volume in 19 children with moderately severe malaria and 16 with severe malaria in Gabon. By subtracting extracellular volume from total body water, they were able to calculate intracellular volume for each child. They also used a less invasive and more rapid method of determining water volumes based on using bioelectrical impedance to calculate the volume.None of the children were severely dehydrated (defined as more than 100 ml/kg depletion), and only three of the children with severe anemia had fluid depletion, which was moderate (60–90 ml/kg depletion). “This challenges the view that dehydration is a major contributor to the pathology of this frequently lethal disease,” says White.So based on these data, obtained from a carefully studied, albeit small group of children, what should people who treat children with malaria do? The authors' first recommendation is that clinicians should think again about how vigorously they rehydrate children, and if they have access to ways of assessing fluid volume more precisely, they should do so . And certainly the methods used by Krishna and colleagues should undergo wider testing in larger groups of children to confirm their usefulness. Until the worldwide efforts to prevent malaria come to fruition, refining the management of infected children will remain a cornerstone of the efforts against this devastating disease.


What defines a sex? Although we tend to think there are only two - males and females - there are many different ways to mix and match the attributes of sexes From a human perspective, sexes seem a relatively simple thing to get one's head around—there are females, and there are males. But our perspective seems biased and narrow when applied to life as a whole, says evolutionary biologist Laurence Hurst of the University of Bath, United Kingdom.“If you were a single-celled alga sitting in a pond, you wouldn't see the world as splitting into males and females.”In fact, different species have evolved a bewildering number of ways to mix and match the attributes of sexes. Some do not have males and females, but have adaptations that mean each individual performs a specific role during sex. There are other species of which every member is sexually equivalent, but individuals nevertheless divide into groups for the purposes of mating. And in some species, individuals make both eggs and sperm . This biPogonomyrmex, queens must mate with two types of males to produce both reproductive individuals and workers . Species with mating types, rather than males and females, aren't limited to two interbreeding groups: the ciliate protozoan Yet most isogamous species have only two mating types. This seems perverse—it excludes half the population as potential mates without gaining the benefits of specialization in sexual biology. With William Hamilton, Hurst came up an explanation for this apparent inefficiency.Two-group mating systems, they proposed, evolved as a way for genes in the nucleus to police the DNA in organelles. Cellular structures with their own genomes, such as mitochondria and chloroplasts, can divide more rapidly than the cells that house them. If the inheritance of organelles was biparental, selfish mutations in their DNA could spread rapidly, Hurst and Hamilton showed. A nuclear gene that enforces uniparental inheritance of organelles, along with a label that allows such cells to recognize each other so that their nuclear genes can share the benefits of cytoplasmic policing, should be favored.C. rheinhardtii, for example, comes in two mating types called plus and minus. When the two fuse, the plastid of the minus cell is detroyed. Most isogamous species that fuse cells have a similar mechanism. Male-killer parasites such as Wolbachia, a parasite of arthropods, show the selection pressure that intracellular passengers can exert . And cellfusion experiments hint that biparental inheritance of organelles does indeed cause problems, says Hurst. “Hybrids are often rubbish, but they can be better if a drug is administered that inhibits the mitochondria of one cell line.”The mating biology of isogamous species offers considerable support for this idea. The aforementioned The species that have lots of mating types, such as ciliate protozoa, exchange nuclear DNA, but not cytoplasm, and hence not intracellular organelles. Since individuals are freed from the need to police their organelles or keep out parasites, selection favors the widest assortment of possible mates, and thus the evolution of a large number of mating types so that one's own type—which one can't mate with—is a small subset of the population. It is possible to imagine species with cytoplasmic policing likewise having many mating types, but such a situation would be much more prone to break down and be invaded by selfish agents than one with two clearly defined types, which is what we usually see in nature. Some have argued that cytoplasmic policing might also be a selective force for different-sized gametes. Sperm could be small so that they do not import mitochondria into the egg.More than a decade after he devised it, Hurst's is still the leading hypothesis explaining the number of mating types in a species. But experimental evidence remains frustratingly elusive. “I wouldn't say I was entirely satisfied,” says Hurst. “We've got all these ideas, and they turn out to be quite hard to test—there's no simple thing one can do on a single species.” There are species where the uniparental inheritance of organelles is not so strictly enforced, says Hoekstra, such as yeasts and plants. “It's not easy to see if selection [on organelles] is strong enough,” he says.S. commune, with its thousands of mating types, each sexual encounter involves only two cells. Nor are we likely to find a species that defies this pattern. The technical difficulties of combining more than two sets of genetic information into one individual, and of parceling out that information during meiosis, must be vast, says Brian Charlesworth of the University of Edinburgh. “We've reached the point of two cells fusing, and stuck with that; two cells are probably just as good as three,” he says.Yet even in a species such as Pogonomyrmex rugosus and P. barbatus. The hybrids have not yet been classed as a new species, but they are well established across the southwestern United States, and there is no evidence of contemporary gene flow between hybrids and their parent species.The ant colonies that Parker suggests have three parents are a hybrid of the species Each ant has one parent if it is male, because male ants are produced from unfertilized eggs, or two if it is female. But each sex also comes in two genetic strains. If a queen mates with a male of her own strain, her offspring will be queens, and if she mates with a male from the other strain, the sperm will give rise to workers. So, for a colony to function fully it—and the queens it produces, because workers raise queens—must have two fathers and one mother. And if any one group were to disappear, the population as a whole would go extinct—unlike fungal mating types, where it's easy to imagine that the species would carry on if a few disappeared. “If you lose any one, the whole thing collapses,” says Parker. “It's really different from any other system.”Pogonomyrmex has four sexes: the males and females of each strain. The idea is particularly potent if one views a social insect colony as a “superorganism,” with the workers equivalent to the cells of a body. It's as if a female mates with one male to produce her offspring's somatic cells, and another to produce its germ cells. The ants form chaotic mating swarms, so most queens have no problem mating multiply and getting sperm from males of both strains, although one would expect that males would strongly favor mating with females of their own strain.So, Parker argues, It's not known how the system originated. Separating the worker and reproductive castes by genetics—other social insects do this by environment, that is, by rearing workers and reproductives differently—may allow selection to operate more efficiently on each lineage, and the workers may benefit from hybrid vigor: field researchers report them as being highly aggressive. In an echo of Hurst's hypothesis, the system also mixes mitochondrial and nuclear genes differently in queens and workers.Pogonomyrmex's mating types sexes, arguing that to define sexes in yet another way only confuses the picture further. “[The ants] are an interesting system, but I wasn't persuaded by Parker's interpretation,” Charlesworth says. “I'm not a fan of the idea that it's useful to use the word ‘sex’ to describe compatibility between mating types—it muddies the waters.” Others are more positive towards Parker's interpretation: “It deserves to be taken seriously,” says evolutionary biologist Eörs Szathmáry of the Collegium Budapest in Hungary. “He's thrown a stone in the water—now we need to see what kinds of ripples it makes. You can't falsify a definition in the way you can a hypothesis; what determines their fate is whether people find them useful or not.”Some evolutionary biologists, such as Charlesworth, do not consider Species in which some individuals give up their reproductive opportunities to form part of a breeding group, such as slime molds, might have a system similar to that of the ants, Parker believes. “There may be hidden mating incompatibilities,” he says. “Now [that] people know to look, we're going to start seeing more of these systems.”One option for dividing up the sexes is “both”—hermaphroditism. This might seem like an ideal solution—everyone becomes a potential partner, and everyone can bear offspring. In practice, however, hermaphroditism is uncommon among multicellular animals. The reasons are similar to those explaining why evolution favors unequal-sized gametes—once sexes have evolved, it's better to commit all one's resources to one role or the other, rather than try and be a jack-of-all-trades. After all, there are many good uses for mating resources other than simply producing eggs or sperm. An animal could defend a territory or provide parental care, for example.Hermaphroditism, however, is useful if one's sexual options are severely limited. In particular, it can be favored when encounters with potential mates are extremely rare. It makes no sense for an animal to invest heavily in the biological equipment of maleness, say, if it will have almost no opportunities to use it: better to hedge your bets. Animals with low or unpredictable population densities and those that are immobile, have poor senses, or lack long-distance signalling are often hermaphroditic. These include sponges, worms—whether flat, nematode, or annelid—and many molluscs . Most hermaphrodites still need to find at least one mate in their lifetimes: the cost of inbreeding prevents self fertilization from becoming common.Helix aspersa snails shoot calcareous love darts into one another. And when the marine flatworm Pseudobiceros bedfordi mates, each worm has two penises, which they fence with in a battle to smear one another with sperm without being fertilized themselves in the process (Hermaphroditic animals have some weird sexual adaptations.  process  and 2.Such oddities result when the mating opportunities of a hermaphroditic species increase, and specialization starts to become more favorable, says evolutionary biologist Nico Michiels, of the University of Muenster in Germany. In a species with two separate sexes, males and females often have different ideas about whether a mating is a good idea—males tend to be keener, females tend to be choosier. The result can be an evolutionary arms race, with each sex evolving adaptations that help them get their way. By exercising mate choice, each sex has some influence on the types of adaptations that evolve—anything too outlandish is unlikely to be favored. This counterbalance is not present in hermaphrodites, however. Rather than having one half of a species resist a particular mating strategy, the whole species is just as likely to adopt it. “Hermaphrodites run into awkward and bizarre mating conflicts,” says Michiels.Michiels believes that hermaphroditism was the ancestral state for animals, and thinks that we might be able to find the relics of this past in contemporary species with separate sexes. To test these ideas, he is searching for groups containing closely related hermaphroditic and bisexual species. Such taxa are very rare, however.


Mycobacterium ulcerans (MU), as the third most prevalent mycobacterial disease. In Ghana, there have been more than 2000 reported cases in the last ten years; outbreaks have occurred in at least 90 of its 110 administrative districts. In one of the worst affected districts, Amansie West, there are arsenic-enriched surface environments resulting from the oxidation of arsenic-bearing minerals, occurring naturally in mineral deposits.In 1998, the World Health Organization recognized Buruli ulcer (BU), a human skin disease caused by Proximity analysis, carried out to determine spatial relationships between BU-affected areas and arsenic-enriched farmlands and arsenic-enriched drainage channels in the Amansie West District, showed that mean BU prevalence in settlements along arsenic-enriched drainages and within arsenic-enriched farmlands is greater than elsewhere. Furthermore, mean BU prevalence is greater along arsenic-enriched drainages than within arsenic-enriched farmlands.The results suggest that arsenic in the environment may play a contributory role in MU infection. Buruli ulcer (BU) is a skin disease, which usually begins as a painless nodule or papule and may progress to massive skin ulceration. If untreated BU may lead to extensive soft tissue destruction, with inflammation extending to deep fascia. The parts of the body most affected are the extremities. Subsequent complications may include contractural deformities. The main form of treatment is wide excisional surgery, including amputation of limbs, which requires prolonged hospitalization and is thus a significant burden on hospital resources and budgets.In recent years, there has been increased incidence of BU in West Africa , Mexico, French Guyana, Papua New Guinea and Australia. The disease seems to affect mostly impoverished inhabitants in remote and rural areas; children are the most vulnerable, accounting for about 70% of the cases . The WorMycobacterium ulcerans (MU), which was first described in Bainsdale, Australia, in 1948 [The causative agent of BU is  in 1948 . From th in 1948 ,6. Porta in 1948  have sug in 1948 .Epidemiological data suggest that environmental factors such as climate, soil, geology, geochemistry, etc. may indirectly influence or contribute to MU infection . In addiSince MU is known to be present in nature although its reservoir is not known and since the epidemiology of BU is still unclear, there is a need to have a better understanding of environmental, ecological, and behavioural factors that predispose to infection. Spatial analysis potentially contributes important information leading to the understanding of the epidemiology and etiology of BU. The main objective of this paper is to explore relationships between some spatial environmental factors and the prevalence of BU.P values range from 0.09 to 0.46. The buffers with the highest P values  are 100 m for drainage channels and 400 m for farmlands. With these buffers 24 of the 61 settlements  fall within 100 m of arsenic-enriched drainage channels  levels of arsenic. Arsenic interacts with and inhibits several enzymes in the body  leading The results of this study reveal spatial dependency of BU prevalence upon proximity to drainage channels and farmlands containing > 15 ppm arsenic. Proximity implies chronic exposure to and/or ingestion of elevated concentrations of arsenic, which influences susceptibility to infection.It has been consistently theorized that BU is acquired when MU enters the body through a skin rupture ,27. HoweSeveral dermatological diseases  are related to arsenic ingestion and exposure . BioaccuIn a case study, Amofah et al.  reportedSpatial analysis of data provides opportunities for epidemiologists to study associations between environmental factors and spatial distribution of diseases . A geogrIn this study, topographic map data, stream sediment geochemical data for arsenic, ASTER satellite imagery and locations of settlements with BU cases were the basic data inputs into the GIS. Spatial data processing was carried out (a) to delineate arsenic-enriched catchment basins based on arsenic concentrations in stream sediment samples, (b) to delineate farmlands from ASTER satellite imagery and determine arsenic-enriched farmlands based on catchment basin data and (c) to extract drainage channels from the topographic map and determine arsenic-enriched drainage channels based on arsenic-enriched catchment basins. Proximity analysis was undertaken to determine spatial relationships between BU-affected areas and the arsenic-enriched areas determined from the data inputs.The study area is in Ghana, where the first case of BU was reported in 1971 and, between 1991 and 1997, more than 2000 cases have been reported . The dis2. The district capital, Manso Nkwanta, is about 40 km south of Kumasi. The district is drained by the Offin and Oda rivers. Vegetation in the district is composed mainly of secondary forests, thicket, forb regrowth  and swamp vegetation. Vegetation thrives in ferric fluvisols, which are the major soil types in the district. These soils have been developed through yearly rainfall ranging from 125 to 200 cm with temperatures of 22 to 30°C. The landscape of the district varies from gentle to broken.The Amansie West District lies between latitudes 6°N and 6°45'N and longitudes 1°30'W and 2°15'W. It covers an area of about 1,136 kmThe district is underlain by Lower Proterozoic Birimian and, to a lesser extent, Tarkwaian rocks. Throughout Ghana, Birimian rocks of West Africa are mainly volcanic greenstones with intervening sedimentary rocks and granitoid intrusions, in places containing deposits composed of pyrite, arsenopyrite, minor chalcopyrite, sphalerite, galena, native gold and secondary hematite .The district has about 310 settlements  with a population in 2000 of 108,726. There are approximately equal percentages of males and females , of whom 70% are farmers and 22% are engaged in legal and 'galamsey'  mining.2, comprising 61 settlements and including the Bilpraw goldmine (formerly a treasure mine of the Ashanti Kings). The BU cases per settlement range from 1–29.The study area is the east-central part of the Amansie West District , with an area of about 623 kmThe following are the sources of spatial data input to the GIS.• Incidence of BU per settlement in 1999, obtained from Korle-BU Teaching Hospital, Accra, Ghana.• Settlement population estimates for 2000, projected by the Ministry of local government and rural development.• Topographic map , a single sheet covering the study area, obtained from the Survey Department, Accra, Ghana.• Location map  of stream sediment samples collected in part of the Amansie West District in 1992 and list of arsenic concentrations determined in these samples, obtained from the Geological Survey Department, Accra, Ghana.• Boundary map  of the district, obtained from the Amansie West District Administration.• ASTER imagery (level 1B) acquired on 15/01/2002, obtained from the US Geological Survey.• Landuse/landcover map of Ghana (traced on Landsat TM data of 1998 and published in the same year), obtained from the Remote Sensing Application Unit (RSAU), University of Ghana, Legon.The GIS operations were carried out in three principal steps: (1) spatial data capture; (2) generation of spatial factor maps; and (c) spatial data analysis. The GIS operations were carried out using ILWIS (Integrated Land and Water Information Systems), a GIS software package developed by the International Institute for Geo-information Science and Earth Observation (ITC) in the Netherlands.The different analog maps were scanned then georeferenced (by defining the x and y coordinates of the corner points of the maps) into a UTM coordinate system. From the scanned map, spatial data were captured by screen digitizing. From the topographic map, rivers, streams and gullies were digitised as line segments as were elevation contours. The boundaries of the district were digitised as line segments and then polygonized. The locations of centres of 61 settlements (identifiable on the topographic map) were digitised as points and the BU incidence in 1999 was recorded as spatial attribute of each settlement.From the stream sediment sample location map, the locations of the samples were digitised as points and the arsenic concentrations (in ppm) were recorded as a spatial attribute of each sample. The ASTER imagery was also georeferenced to the same coordinate system using eight reference points (tie points), which were selected in the image and which could be identified in the topographic map. Using an affine transformation, a root mean square error (RMSE) of 0.58 pixel was obtained in georeferencing the ASTER imagery.For each of the settlements with incidence of BU the percentage prevalence of BU was calculated. Prevalence expresses cases of a disease in terms of the proportion of the population afflicted at a specified time . It is eThe spatial factor maps generated from the stream sediment geochemistry data for use in the spatial analysis were: (a) map of arsenic-enriched catchment basins; (b) map of arsenic-enriched farmlands; (c) map of arsenic-enriched drainage channels.The stream sediment geochemical data for arsenic were initially analysed statistically to determine a threshold value that divides the data into background  classes and anomalous  classes of arsenic concentrations. The data are lognormally distributed and, after removing obvious outliers in the data, a geometric mean of 8.9 ppm As and standard deviation of 2.8 ppm As were obtained. The threshold value was therefore set at 15 ppm As . The spatial distribution of arsenic was then mapped through the generation of a catchment basin anomaly map in which a sample catchment basin is assigned the geochemical attribute of the corresponding sample ,46. Gene• creation of a raster digital elevation model (DEM) through interpolation of elevation contours;• generation of raster map of drainage lines; and• calculation of sample catchment basin boundaries via an iterative calculation procedure involving the DEM and the raster map of drainage lines.The catchment basin map of arsenic concentrations was then classified into a binary map showing arsenic-normal areas (with ≤ 15 ppm As) and arsenic-enriched areas (with > 15 ppm As) as shown in Figure A supervised classification of ASTER imagery was carried out to distinguish between the major landcover/landuse classes known in the area. These landcover classes are (a) forest areas, (b) residential areas or settlements (bare of vegetation), and (c) farmlands. Using the available landuse/landcover map and topographic map as references, training pixels of known landuse/landcover classes were selected using a colour composite of ASTER bands 2, 3 and 4. These three bands gave the highest optimal index factor (OIF), which indicates the combination of three spectral bands that provide optimum information about landcover . The boxTo determine arsenic-enriched farmlands, a Boolean AND operation was performed by crossing the catchment basin anomaly map and the classified landcover/landuse image. About 21% of the total area of farmlands in the classified image is arsenic-enriched.A Boolean AND operation was performed by crossing the catchment basin anomaly map and the raster map of drainage lines. About 22% of the total length of drainage lines is indicated to be arsenic-enriched.The inhabitants of a settlement earn their livelihoods by exploiting the resources of the surrounding land. This land influences their exposure to infections and to environmental factors that dispose to infections. Proximity analysis was therefore used to determine spatial relationships between BU prevalence per settlement and (i) arsenic-enriched farmlands and (ii) arsenic-enriched portions of the drainage system. The proximity analysis was carried in two principal steps. First, maps of distances from arsenic-enriched farmlands and arsenic-enriched portions of the drainage system were generated. Second, the point map of BU prevalence per settlement was overlaid on (or crossed with) each of these maps.A buffer is a zone of specified distance around a selected map feature. A GIS creates buffer zones around selected map features such as arsenic-enriched farmlands and arsenic-enriched portions of drainage systems. Around each of these, buffers were set at intervals of 100 m up to 1000 m. Each buffer zone map was crossed with BU prevalence data of settlement to determine how many of these fall within and outside of the buffer.At each increasing interval of 100 m, a test of the significance of the difference of the mean BU prevalence within the buffer and outside of the buffer is made using the t-statistic:,  are the sample means,  is the pooled sample variance, i nand j nare the sample sizes from population i and j. Using ij tand degrees of freedom given by i n+ jn-2, a t distribution look-up table provides the probability, P that the means are significantly different.where AAD carried out the research and drafted the manuscript. EJMC guided parts of the research and both EJMC and MH reviewed the manuscript.


Genes use a simple language—written in the molecules of DNA—to build thousands of proteins in a dizzying variety of sizes and shapes. With only four different nucleotide building blocks, DNA codes for the 20 different amino acids (each with their own structures and properties) that provide the foundation for the enormous diversity of protein form and function. This diversity makes the systematic study of all the proteins of a given organism , a challenging enterprise.Interactions between proteins underlie nearly every fundamental process within the cell. They can form higher-order multiprotein complexes like those involved in transcription and replication, help transport proteins to their proper location in the cell, and participate in signaling pathways. Because of their importance, disruption of these interactions can have disastrous consequences. For example, the loss of the ability of a normal cellular protein called Src to bind to certain other proteins can be associated with cancer progression. The determinants of these interactions are poorly understood, but in many cases these interactions are mediated by small pieces of the proteins, which are called peptides. Peptides serve as the starting point for the novel strategy reported in this issue.Gianni Cesareni and colleagues have added to the repertoire of proteomic analysis by devising a global strategy to investigate protein–protein interactions on an organismal level using yeast as a model organism. The authors select a protein of interest from yeast, which can be thought of as the “bait” for which they wish to identify protein-binding partners. They start by looking at a number of different previously identified peptides that bind the bait protein. Commonalities between the sequences of these peptides form the “consensus” binding sequence, a base framework of protein sequence from which many possible variations can be derived. Since the protein sequences of all proteins (the proteome) in yeast can be deduced from the sequenced genome, the authors can scan the proteome for proteins that contain the consensus, or a closely related, sequence. These proteins could potentially bind the bait protein. Peptide sequences from these identified proteins are synthesized chemically and arrayed on a membrane, which is bathed in a solution containing the bait protein. After washing off the excess bait protein, they can figure out where it remains on the membrane and therefore tell which peptides the bait protein has bound. The proteins corresponding to these peptides are candidate binding proteins that are validated by further experimentation.The protein–protein interactions identified by this approach can be used to extend the network of known interactions in the proteome. This will enable researchers to draw functional linkages between proteins, whether they are involved in a basic biological process or in human disease. By examining whole families of proteins, it may also aid in elucidating the underlying determinants of binding specificity, which would provide clues to the biomechanisms underlying cell processes. These insights could lead to methods for manipulating these interactions, perhaps even in cases of human disease, as in the case of Src and cancer. This approach can readily be applied to the proteomes of more complex organisms like humans and adds to the growing number of experimental strategies available to researchers in proteomics.


Cell reports the involvement of an RNA helicase in the pathway by which HIV exports partially spliced and unspliced RNA out of the nucleus. This suggests the ubiquity of RNA helicases in RNA export from the nucleus, and has novel mechanistic implications.Nucleo-cytoplasmic transport of RNA is one of many cellular pathways whose illumination has progressed hand in hand with understanding of retroviral mechanisms. A recent paper in  HIV must export to the cytoplasm not only full length genomic and structural gene-encoding RNA species, but also partially and fully spliced RNAs which code for a variety of overlapping structural and accessory genes. Whereas the fully spliced HIV RNA species readily exit the nucleus and undergo translation, the partially spliced and unspliced species require the interaction of the HIV-1 Rev protein with the cis-acting, viral RRE (Rev responsive element) for expression. Simpler retroviruses, such as the type-D Mason-Pfizer monkey virus (MPMV) solve a similar problem by encoding a cis-acting, constitutively active CTE (constitutive transporter element) in the 3'UTR of the viral genome, which uses a distinct pathway . Of lessThe Rev/RRE pathway involves the Ran-CRM-1, shuttling system: In the nucleus, Ran-GEF converts Ran-GDP to Ran-GTP. Ran-GTP bound CRM-1 binds the NES  domain of Rev, which is in turn bound to the RRE, and enables CRM-1 to transport the resulting RNA/protein complex as cargo into the cytoplasm, presumably through CRM-1 interactions with nucleoporins. In the cytoplasm, Ran-GAP converts Ran-GTP to Ran-GDP, releasing the REV/RNA cargo. The asymmetric distribution of Ran-GEF and Ran-GAP between nucleus and cytoplasm ensures a constant RAN-GTP/GDP gradient to facilitate CRM-1 recycling and continued Rev/RRE export. CTE-mediated RNA export is based on binding to the Tap-NXT complex, and export through an unrelated transport pathway, which, ironically, is responsible for the bulk of mRNA transport .Cell, report the involvement of the RNA helicase, DDX3, in the Rev/RRE pathway [Enter the laboratories of Kuan-Teh Jeang and Larry Kleiman, who, in a comprehensive study in the October 29th issue of  pathway . Interesin vitro (in a manner independent of its NES). Although no data are presented concerning direct in vitro binding of DDX3 to Rev, for such an interaction to exist and to be significant, DDX3 would have to bind one of the three identified functional regions in Rev. These include the NES, the coincident NLS/RBD  and the Rev multimerization domain, which flanks the RBD [A number of findings not only provided evidence consistent with a role for DDX3 in the Rev export pathway, but also provided mechanistic insights. DDX3, though constitutively cytoplasmic, shuttles between nucleus and cytoplasm in a manner dependent on CRM-1, as evidenced by inhibition by leptomycin B (LMB), which inhibits CRM-1 export, but not Tap export. DDX3 also localizes to nuclear outer membranes in a speckle formation and co-immunoprecipitates with nucleoporins, in addition to having a distribution in the cytoplasm. In cell lysates, DDX3 co-immunoprecipitates with either Rev protein or CRM-1. Although the studies on cell lysates fail to distinguish between pairwise binding and ternary complex formation, ternary complex formation is a distinct possibility. DDX3 directly binds CRM-1  the RBD . No regiIn the tradition of good research, the results of Yedavalli et al.  pose as On a highly speculative note, the intimate involvement of a DEAD box helicase in the Rev pathway, and the observation that DDX3 has a cytoplasmic localization, in addition to its nuclear membrane localization, recalls some old data from two independent laboratories, indicating that in some systems Rev may not act on export, but on post export events, to promote Rev/RRE dependent expression ,18. The Finally there is the possibility that specific helicases will be valid therapeutic targets for antiretroviral chemotherapy. Although targeting a cellular gene involved in a viral pathway risks inhibiting a necessary cellular function, it avoids the overwhelming problem posed by rapid viral mutation to resistance.RRE: Rev Responsive ElementMPMV: Mason-Pfizer monkey virusCTE: Constitutive Transport ElementNES: Nuclear Export SignalLMB: Leptomycin BNLS: Nuclear Localization SignalRBD: RNA Binding DomainNone. The opinions expressed are those of the author and do not necessarily express the opinion of the FDA.


Anti-ischemic therapy, in particular beta-blockers, is the most commonly employed drug for the control of myocardial ischemia in patients with stable coronary artery disease. Its widespread use also in patients with suspected coronary artery disease has important practical, clinical diagnostic and prognostic implications because diagnostic tests are heavily influenced by its effects. In the present review, the pathophysiological mechanisms of ischemia protection by antianginal therapy are described. Not all stressors are created equal in front of the different classes of antianginal drugs and on their turn the different classes of drugs exert different levels of protection on inducible ischemia. Several clinical implications can be drawn: From the diagnostic viewpoint antianginal therapy decreases test sensitivity, offsetting the real ischemic burden for a too high percentage of false negative tests. From the prognostic viewpoint test positivity in medical therapy identifies a group of subjects at higher risk of experiencing cardiac death and positivity on medical therapy can be considered a parameter of ischemia severity. Nonetheless in patients with known coronary artery disease the ability of antianginal therapy to modify the ischemic threshold at stress testing represent a powerful means to assess therapy efficacy. From a practical viewpoint, the use of antianginal therapy at time of testing has advantages and disadvantages which are largely dependent on the purpose a test is performed: if the purpose of testing is to diagnose ischemia, it should be performed in the absence of antianginal medications. If the purpose of testing is to assess the protective effects of antianginal therapy, the test should be performed on medications. Anti-ischemic therapy, in particular beta-blockers, is the most commonly employed drug for the control of myocardial ischemia in patients with stable coronary artery disease. Its widespread use also in patients with suspected coronary artery disease has important practical, clinical diagnostic and prognostic implications because diagnostic tests are heavily influenced by its effects. The diagnostic and prognostic impact of anti-ischemic therapy on stress testing is largely ignored but not negligible. The issue raises several questions: How to evaluate patients at time of testing for myocardial ischemia? How to interpret a stress test performed on anti-ischemic therapy? Are the stressors employed for the detection of myocardial ischemia created equal in relation to the different classes of drugs used in clinical practice? Is stress testing able to assess the efficacy of medical therapy in patients with known coronary artery disease? Has the protection of anti-ischemic therapy on inducible myocardial ischemia any impact on long-term survival?The answer to all these issues relies on the mechanism through which myocardial ischemia is induced by the different stressors (exercise or pharmacologic such as dipyridamole and dobutamine) employed during stress testing. Test exploring organic coronary artery stenosis can induce ischemia by two basic mechanisms: 1. an increase in oxygen demand, exceeding the fixed supply and 2. flow maldistribution due to inappropriate coronary arteriolar triggered by a metabolic/pharmacologic stimulus . During In the presence of coronary atherosclerosis, appropriate arteriolar dilation can paradoxically exert detrimental effects on regional myocardial perfusion, causing overperfusion of myocardial layers or regions already well perfused in resting conditions at the expense of regions or layers with a precarious flow balance in resting conditions . Anti-isHowever, experimental studies on the model of the exercising dog have shown that beta-blockers protect myocardium from stress-induced myocardial blood flow-function relation: for a given transmural flow, there is a rise of subendocardial and a fall of subepicardial flow, with an improved regional performance . This saOn the basis of these premises and taking into consideration that the markers of inducible myocardial ischemia  are very different therefore expressing a different sensitivity to the action of anti-ischemic therapy at time of testing, it is clear that medical therapy affects test results  A negative test on medical therapy might represent a false negative result, therefore it is advisable to repeat the test off therapy in order to assess the real ischemic burden through the conventional stress echocardiographic parameters ,37. – i.Patients may be undergoing various forms of antianginal therapy at the time of testing, both an advantage and a disadvantage for stress echocardiography testing. The disadvantage is that antianginal therapy reduces sensitivity, since stress-induced wall motion abnormalities are caused by the development of obligatory myocardial ischemia. The advantage is that the effect of therapy can be assessed using an objective, primary ischemic end-point such as changes in stress-induced wall motion abnormalities. The presence of ischemia can be titrated on the basis of the ischemic-free stress time and the extent and severity of the induced dyssynergy. The various forms of stress are differently affected by various forms of therapy. In patients with known or suspected coronary artery disease, ongoing anti-ischemic therapy at the time of testing heavily modulates the prognostic value of pharmacological stress echo. In presence of concomitant anti-ischemic therapy, a positive test is more prognostically malignant and a negative test less prognostically benign. However, the decision to remove a patient from beta-blocker therapy for stress testing should be made on an individual basis and should be done carefully to avoid a potential hemodynamic rebound effect, which can lead to accelerated angina or hypertension . No majo


In vivo studies were conducted to quantify the effectiveness of low-level direct electric current for different amounts of electrical charge and the survival rate in fibrosarcoma Sa-37 and Ehrlich tumors, also the effect of direct electric in Ehrlich tumor was evaluate through the measurements of tumor volume and the peritumoral and tumoral findings.6 and 1 × 105 viable tumor cells, respectively. For each type of tumor four groups (one control group and three treated groups) consisting of 10 mice randomly divided were formed. When the tumors reached approximately 0.5 cm3, four platinum electrodes were inserted into their bases. The electric charge delivered to the tumors was varied in the range of 5.5 to 110 C/cm3 for a constant time of 45 minutes. An additional experiment was performed in BALB/c male mice bearing Ehrlich tumor to examine from a histolological point of view the effects of direct electric current. A control group and a treated group with 77 C/cm3 (27.0 C in 0.35 cm3) and 10 mA for 45 min were formed. In this experiment when the tumor volumes reached 0.35 cm3, two anodes and two cathodes were inserted into the base perpendicular to the tumor long axis.BALB/c male mice, 7–8 week old and 20–22 g weight were used. Ehrlich and fibrosarcoma Sa-37 cell lines, growing in BALB/c mice. Solid and subcutaneous Ehrlich and fibrosarcoma Sa-37 tumors, located dorsolaterally in animals, were initiated by the inoculation of 5 × 103, respectively.Significant tumor growth delay and survival rate were achieved after electrotherapy and both were dependent on direct electric current intensity, being more marked in fibrosarcoma Sa-37 tumor. Complete regressions for fibrosarcoma Sa-37 and Ehrlich tumors were observed for electrical charges of 80 and 92 C/cmHistopathological and peritumoral findings in Ehrlich tumor revealed in the treated group marked tumor necrosis, vascular congestion, peritumoral neutrophil infiltration, an acute inflammatory response, and a moderate peritumoral monocyte infiltration. The morphologic pattern of necrotic cell mass after direct electric current treatment is the coagulative necrosis. These findings were not observed in any of the untreated tumors.The data presented indicate that electrotherapy with low-level DEC is feasible and effective in the treatment of the Ehrlich and fibrosarcoma Sa-37 tumors. Our results demonstrate that the sensitivity of these tumors to direct electric current and survival rates of the mice depended on both the amount of electrical charge and the type of tumor. Also the complete regression of each type of tumor is obtained for a threshold amount of electrical charge. Several investigators have reported encouraging results from experimental low-level direct current therapy (DEC) in different types of tumor [The use of electric current in the treatment of malignant tumors has been known since the beginning of the 19of tumor . These sThe dose-response relationships obtained in these studies indicate that the DEC effectiveness depends on both the type of tumor and therapeutic scheme . Lack of guidance has become an obstacle to introduce the electrochemical treatment (EChT) in the clinic oncology. This is due to the lack of standardization of the EChT method regarding DEC doses and electrode array. Ren et al.  studied In spite of these results, the efficacy of DEC treatment has been controversial since an optimum electrode array and a threshold amount of electrical charge for each type of tumor have not been established. We believe that the procedure to determine the amount of electrical charge for each type of tumor is completely destroyed is more feasible to implement than that for the optimum electrode array, which involves several variables, such as polarity, number, and orientation of the electrodes. The knowledge of the optimum values of these parameters may lead to maximize the antitumor effectiveness of DEC and minimize their adverse effects in the organism. This allows the establishment of a therapeutic procedure for the tumor treatment in animals and in clinical oncology.The aim of this study is to test the hypothesis that the responses of the tumors treated with DEC is dependent on dose. Ehrlich and fibrosarcoma Sa-37 tumors were used. The survival rates of the mice bearing of these two types of tumor were determined. The antitumor effects of DEC were also evaluated through the peritumoral and tumoral findings in Ehrlich tumor.The experiment was run in accordance with Good Laboratory Practice rules and animals protection laws. The experiment was approved by the ethical committee of Oriente University, which follows the guideline from the Cuban Animal Ethical Committee. BALB/c male mice, 7–8 week old and 20–22 g weight were used. They were supplied from the National Center for Production of Laboratory Animals (CENPALAB), Havana City, Cuba, and were kept in standard laboratory conditions with water and food ad libitum. Animals were healthy  and were maintained in plastic cages inside a room at a constant temperature of 23 ± 2°C and relative humidity of 65 %, and a natural day-night cycle. During therapy the animals were firmly fixed on wooden boards, so all treatments were performed in the absence of anesthesia. All treated animals showed uneasy and quick breathing during fixation.Ehrlich and fibrosarcoma Sa-37 cell lines, growing in BALB/c mice, were received from the Center for Molecular Immunology, Havana City, Cuba. Both cell lines are being maintained in the Cell Culture Collection of the Department of Pathologic Anatomy, Hospital "Conrado Benítez", Santiago de Cuba, Cuba.6 viable tumor cells in 0.2 ml of 0.9 % NaCl, while fibrosarcoma Sa-37 solid and subcutaneous tumors located dorsolaterally in animals, were initiated by the inoculation of 1 × 105 viable tumor cells in 0.2 ml of 0.9 % NaCl. For both tumors, the viability of the cells was determined by Trypan blue dye exclusion test and it was over 95 %. Cell count was made using an hematocytometer.The Ehrlich and fibrosarcoma Sa-37 ascitic tumor cell suspensions, transplanted to the BALB/c mouse, were prepared from the ascitic forms of the tumors. Ehrlich solid and subcutaneous tumors, located dorsolaterally in animals, were initiated by the inoculation of 5 × 10. The mean tumor volume with the corresponding standard deviation of three determinations was calculated in each experimental group. Mice with non-palpable tumor at day 60 after the treatment were designated as cured.Tumor growth was followed by measuring three perpendicular tumor diameters  with a vernier caliper. The tumor volume was estimated using the equation days) was determined for each individual tumor as the time needed to double the initial tumor volume. For each experimental group the mean DT and its standard deviation were calculated.Tumor doubling time , slight (+), moderate (++) and severe (+++).To supply electrochemical treatment, a high stability and low noise DEC source was built at the National Center for Applied Electromagnetism (CNEA). The electrode configuration consisted of a multi-electrode array formed by two anodes and two cathodes inserted into the base perpendicular to the tumor long axis keeping about 3 mm distance between them. Cathode and anode were connected in alternate sequence. This multi-electrodes array was proposed taking into account the results reported by Chou et al. . All ele3 were used. From this range of electrical charge three values were chosen to show the DEC effectiveness in both types of tumors. When the tumors reached approximately 0.5 cm3 in BALB/c mice, a single shot electrotherapy was supplied (zero day). For each type of tumor four groups consisting of 10 mice each randomly divided were formed. For Ehrlich tumor the groups formed were: control group (CG1), treated group with electrical charge of 36 C/cm3 (18.0 C in 0.5 cm3) and 6.7 mA for 45 min (TG1-1), treated group with 63 C/cm3 (31.5 C in 0.5 cm3) and 11.7 mA for 45 min (TG1-2), and treated group with 92 C/cm3 (46.0 C in 0.5 cm3) and 17 mA for 45 min (TG1-3). For fibrosarcoma Sa-37 tumor the groups formed were: control group (CG2), treated group with 36 C/cm3 (18 C in 0.5 cm3) and 6.7 mA for 45 min (TG2-1), treated group with 63 C/cm3 (31.5 C in 0.5 cm3) and 11.7 mA for 45 min (TG2-2), and treated group with 80 C/cm3 (40.0 C in 0.5 cm3) and 14.8 mA for 45 min (TG2-3).In order to find the thresholds of the electrical charge for which Ehrlich and fibrosarcoma Sa-37 tumors are completely destroyed, different amounts of electrical charge in the range of 5.5 to 110 C/cm3 (52.5 C in 0.5 cm3) and 19.4 mA for 45 min was supplied to 10 mice (5 mice bearing Ehrlich tumor and 5 mice bearing fibrosarcoma Sa-37 tumor). Also the dose of 110 C/cm3 (55 C in 0.5 cm3) and 20.3 mA for 45 min was supplied to 10 mice (5 mice bearing Ehrlich tumor and 5 mice bearing fibrosarcoma Sa-37 tumor). These doses were used to evaluate the therapeutic and adverse effects of the DEC above 100 C/cm3. For each type of tumor was formed a control group of 10 mice.The dose of 105 C/cm3 (27.0 C in 0.35 cm3) and 10 mA for 45 min (TG-A). This treated group was divided in three subgroups TG1-A, TG2-A and TG3-A to show the tumor and peritumoral findings at 1, 2 and 4 days after DEC treatment. Each experimental group was formed by 6 mice. When the Ehrlich tumor volumes reached 0.35 cm3, two anodes and two cathodes were inserted into the base perpendicular to the tumor long axis and a single shot electrotherapy was supplied (zero day).In order to examine from the histolological point of view the effects of direct electric current in Ehrlich tumor two experimental groups were formed: a control group (CG-A) and a treated group with 77 C/cmIn all experiments, before treatment the DEC was increased gradually step by step for two minutes until the desired intensity. During treatment it was constant and continually monitored. The voltage was also continually monitored. It varied, in accordance with the change of tissue resistance during the current application, between 5 and 25 V. The total electrical charge was calculated in real time. After a single application of the intended dose, the treatment was stopped. In this case, the current was decreased step by step for two minutes until its intensity was 0 mA. During electrotherapy, mice were firmly restrained, without obvious discomfort; therefore no anesthesia was necessary.In the control groups, four electrodes were placed into the base perpendicular to the tumor long axis without applying any direct current (0 mA). The animals of this group were firmly fixed but without DEC and showed uneasy and quick breathing during their fixation.Survival rates of the mice bearing both types of the tumors were determined for each experimental group. The survival rate (in %) was defined as the ratio between the number of live animals and the total number of animals, multiplied by 100 %. Survival checks mortality were made daily.The histologic cuts from each tumor were made according to the largest diameter. They were fixed in a 10 % formal solution and processed by the paraffin method.Hematoxylin and eosin staining was used. Each cut was divided into four microscopic fields in order to calculate the necrosis percentage through panoramic lens. This percentage was calculated as the ratio between the necrosis area and the tumor total area, multiplied by 100 %.The nonparametric statistical criterion of one-tailed Wilcoxon-Mann-Whitney rank sum was used to compare volumes between the treated groups with DEC and their respective control groups. Survival curves for the three different mice treatment groups for each tumor type were estimated by using the Kaplan-Meier product limit estimator .McNemar's statistical criterion was used for comparing the main histopathological findings in peritumoral zones in animals from CG-A and TG-A. P values of less than 0.05 were considered significant. The mean value and its mean standard error were reported for each experimental group.As it is shown in Table 3 that the tumors partially regressed for 2 and 4 days, respectively. For these same doses the fibrosarcoma Sa-37 tumor reached their respective partial regressions for 4 and 5 days. Both eventually outgrew again.The results shown in this study revealed that the sensitivity of the Ehrlich and fibrosarcoma Sa-37 tumors was dose dependent. The sensitivity to DEC of both types of tumors increased with the increase of the amount of electrical charge Table  and 2. T3 ; however, for the other doses there were significant differences (P < 0.05).The overall survival curves of the mice bearing Ehrlich and fibrosarcoma Sa-37 tumors are shown in figures The cured mice were sacrificed at 100 days post-treatment. Before sacrifice, the animals were active and in good physical condition with adequate body weight. They had good posture and coats of hair. After sacrifice, the histopathological findings in each of these mice showed complete disappearance of the tumor and evidence of healing. In the treated mice a very little necrotic tissue remained within a fibrous scar. Serology and histological finding of the organs did reveal neither abnormalities nor metastases (results not shown).3, during the first 24 hours after DEC treatment. The histological findings showed both severe alterations in liver and kidney and an increase in the weight of these organs. Metastases were not observed in any of these mice.The death of a mouse 1-day after DEC treatment was observed in TG1-3. The histological findings revealed damages in the lungs due to hemorrhage and a small circular necrosis. Metastases were not observed in this mouse. It was also observed the death of a mouse 25 days post-treatment in TG1-3 and 50 days in TG2-3 due to the cannibalism shown by the mice, probably because of the blood present in the tumors after DEC treatment. All the mice died for amounts of electrical charge above 100 C/cmThe histopathological findings revealed that in the Ehrlich untreated tumors (CG-A) the necrotic area was mainly central and it constituted approximately from 20 % of the tumor total area Fig. . HoweverThere was a lack of well defined necrosis zones surrounding the electrodes. The morphologic pattern of the necrotic cell mass observed is the coagulative necrosis. The dead tissue becomes both swollen and firm in consistency. Preservation of the basic profile of the coagulated cancerous cell and nuclear karyolysis were also observed. The lysed erythrocytes was also observed. This type of necrosis was accompanied by accumulation of neutrophil polymorphonuclear leucocytes.Lymphocytes (L) and plasmatic cells, named CP, were observed in all the tumors in CG-A and TG-A but there were no significant differences Table , Fig. 8.In this experiment no mouse died from intercurrent disease during or after the treatment. Before sacrifice, the animals were active and in good physical condition with adequate body weight. They had good posture and coats of hair.The results of this study demonstrated that DEC has a marked antitumor effect because a single-shot electrotherapy delivered via four platinum electrodes inserted into the base of the fibrosarcoma Sa-37 and Ehrlich murine tumors significantly retarded their growths when compared with their respective control groups. The fact that tumor regression increases with the increase of the amount of electrical charge may be explained because the induced necrosis by DEC into the tumor depends directly on its intensity, a matter that is in agreement with the results of Robertson et al. [We believe that the necrosis is the predominant mechanism of cell death, by the cellular tumefaction (or cellular swelling), cell rupture, breakdown of organelles and acute inflammatory response observed during the first 4 days post-treatment in all treated tumors, result that agrees with that previously reported by Dodd et al.  and HolaThe necrosis may be due to the ischemia observed in all tumors treated with DEC, which could lead to an irreversible cell injury of the tumor cells and therefore to cellular death. This fact could be related with other experimental findings found after DEC treatment, such as: degradation of phospholipids, lost of high energy phosphate and increase of the intracellular calcium , membranThe prolonged acute inflammation observed during 4 days after DEC treatment may be explained by the persistent leukocyte infiltrate also observed in the peritumoral findings. This persistent leukocyte infiltrate  becomes a harmful agent because during the chemotaxis they amplify the effects of the initial inflammatory stimulus through the liberation of potent mediators  that lead to both endothelial and tissue damages. This leukocyte infiltrate may also activate the immune system . In all 3, respectively, may suggest that each tumor model has its threshold of electric charge from which it is completely destroyed. This threshold depends on the electric nature of the tumor and their physiological characteristics . This fact explains the cure of the mice and why the tumors do not duplicate their initial volumes during the observation time  declare that they have no competing interestsHCC conceived the study, and participated in its design and coordination. Also, he carried out the inoculation of the tumor cells in the mice, the measure of the tumor volumes and the survival rate of mice as well as elaborated the manuscript. MCSQ participated in the design of the study and participated in the measure of the histological findings of the organs and tumor and peritumoral findings and as well as elaborated the manuscript. LEBC carried out the inoculation of the tumor cells in the mice, conceived and participated in the design of the study and performed the statistical analysis as well as elaborated the manuscript. RNPB and DSL participated in the design of the study and contributed to elaboration of this manuscript. MFS participated in the design of the study and carried out the serology. All authors read and approved the final manuscript. OGP and TRG participated in the design of the study and contributed to elaboration of this manuscript. All authors read and approved the final manuscript. JLMF participated in the design of the study and performed the statistical analysis.The pre-publication history for this paper can be accessed here:


Functional information is lacking for many of the hypothetical proteins encoded within sequenced genomes. Richard Roberts proposes that a community-based approach might offer an effecient way to fill the function gap During the last few years, we have seen enormous strides in our abilities to sequence genomes, and the information that has poured out of these sequences is quite astonishing. With more than 150 complete genome sequences now available and many laboratories rushing into microarray analysis, proteomic initiatives, and even systems biology, it seems an appropriate time to consider not just the opportunities those sequences present, but also their shortcomings. By far the most serious problem is the quality and degree of completeness of the annotation of those genomes. Most troublesome are the large numbers of open reading frames that have been identified by computer programs, but remain labeled as a “conserved hypothetical protein” when they occur in more than one genome or simply a “hypothetical protein” when they appear unique to the genome in question. Between them, these two categories of annotated open reading frames often represent more than half of the potential protein-coding regions of a genome.These annotations highlight just one portion of our ignorance about the information content of genomes and our lack of fundamental knowledge about the function of so many of the building blocks of cells. Unless we rectify this situation, it is likely to undermine many of the other “-omic” efforts currently underway. Here I advocate a rather straightforward approach to address this problem—focused initially on the bacterial genomes. In contrast to the numerous proposals for big science initiatives to understand the fundamental workings of biological organisms, I propose a small science, relatively low-tech approach that could have a dramatic pay off. A relatively small investment could yield a massive amount of information that would greatly enhance our current efforts to use genomic approaches to study life.The initial proposal is directed at deciphering the role of the “hypothetical proteins” encoded in the microbial genomes and would involve a community-wide approach to determine the function of these hypotheticals based on solid, old-fashioned biochemistry. The essence of the idea is to undertake an interdisciplinary effort that couples our current bioinformatics capabilities to predict protein function with a directed exploration by experimental laboratories to test those predictions. I would encourage a consortium of bioinformaticians to produce a list of all of the conserved hypothetical proteins that are found in multiple genomes, to carry out the best possible bioinformatics analysis, and then to offer those proteins to the biochemical community as potential targets for research into their function. To energize laboratories with appropriate expertise to participate in this community-wide effort, I suggest that a special program be set up by one or more of the funding agencies so that laboratories undertaking the investigation of any particular protein receive a small grant upfront as a supplement to an existing grant. Upon completion of the project and the identification of the function, they would receive a further supplement to that grant as a reward. In this way, one might hope to rally some of the best biochemical talent and apply it to this problem of determining function for a wide range of new proteins. The cost of such an operation could be quite minimal, and the bureaucracy and review process could be equally simple. Here is a case where a modest infusion of funds could greatly enhance our ability to annotate both existing and new genome sequences and ensure that our current investments in genomic sequences yield the richest biological harvest possible. There are two key steps in the proposed plan.The first step is to encourage some bioinformaticians with appropriate expertise in the functional annotation of genomes to form a consortium and undertake the assembly of a list of prime targets for which an experimental demonstration of function would be most valuable. Three general classes of such genes come to mind: (1) The conserved hypothetical genes. These belong to the set of genes that have orthologs in many other genomes, but for which no function has been experimentally determined in any case. A recent success among such genes is illustrated in These sets of targets would be combined and arranged into a prioritized list in which each was accompanied by the best assessment of potential function. The priorities would be based on which genes were most likely to prove broadly informative. For instance, a conserved hypothetical gene that occurred in most genomes would be of higher priority than one that had only two orthologs. The list would be on a public Web site where these targets and the predicted functions could be examined and modified by alternative or additional predictions from other groups to guide future experimentation. As function was derived, that information could be presented and the target removed from the main list.The second step would be to invite experimentalists to peruse the list and find those potential genes whose protein products might lie within their realm of expertise so that they could use their experimental knowledge and reagents to quickly test for function. Initially, I would advocate allowing laboratory teams to pick and choose among the list and sign up to study just one of these open reading frames. I would recommend allowing one laboratory per open reading frame in the initial stages. A laboratory wishing to sign up would generate a short document highlighting why its expertise might be suitable for a particular protein. A one-page proposal should suffice, with no experimental plan demanded. At this point, a small panel could choose among competing efforts and the laboratory chosen would be given a small grant and up to six months to carry out its analysis. If it was successful in delineating the function of their target protein, a paper would be written and submitted for peer review. If the paper was accepted for publication, then an additional sum would be allocated as a supplement to the laboratory's existing grant. If, after six months, a laboratory had not managed to delineate the function, it would submit a short report describing the approaches that have been tried, with the results of its analyses. This would be posted on the public Web site and that target would then become open for analysis by other laboratories, under the same conditions as before.Escherichia coli, I would not demand that the biochemical experiments be done on the E. coli gene. Any of the orthologs would do, so long as the similarity was sufficiently strong to give high expectations that function would be conserved. In fact, for a laboratory that happened to be already working on one of the homologs, this program might provide an added bonus and greatly speed its work. I would also encourage both biochemical and genetic approaches, since one can never be certain when one method might be better than another. The list would, of course, also include conserved genes not found in E. coli, but commonly distributed in other genomes. In particular, I would make a pitch for including all genes in Mycoplasma genitalium, which, as the free-living organism with the fewest genes, might be the most suitable as a model system for in-depth understanding of its biology.While the initial list of target genes should probably be based on a well-studied and experimentally tractable organism such as Saccharomyces cerevisiae genome (This proposal for experimental attack on hypothetical genes is really a very traditional approach that becomes large-scale simply because of the parallel nature of the implementation. It resembles the successful approach used by the Europeans to achieve the complete sequence of the e genome . The resE. coli, one gene, hemK, was found that had no immediately known protein product (hemK gene might encode this enzyme. Subsequent hemK homologs were annotated as putative protoporphyrinogen oxidases and soon the “putative” was dropped. Then one group noticed that the gene product contained protein sequence motifs typical of DNA adenine methyltransferases. From then on, the annotations in GenBank alternated between these two assignments.During studies of the genetics of heme biosynthesis in  product . Since ohemK gene is of ubiquitous occurrence from humans to Chlamydia, and yet until 2002 its true biochemical function was unknown. At that point, two groups (E. coli the HemK gene product was an N5 glutamine methyltransferase that transferred a methyl group from S-adenosylmethionine to the amide nitrogen of a specific glutamine residue in the protein chain release factors prfA and prfB. Particularly noteworthy was the observation that the hemK gene is positioned immediately adjacent to the prfA gene in many microbial genomes! Here is a case where bioinformatics suggested strongly that the hemK gene encoded a methyltransferase, but an experiment was needed to identify the substrate. Since the adjacent gene in the genome encoded the substrate, it might have been possible to make that prediction too. There are additional paralogs of hemK in several genomes, but their biochemical activity and substrates remain to be identified.This o groups  demonstr


Benign fibrous histiocytomas (BFH) usually presents as a small benign lesion that predominantly occurs in the skin. Only few cases of BFH arising from bone have been reported, its occurrence in pelvic bones is even rarer.os ilium which was larger than earlier reported BFH of the bone. Surgical resection was performed successfully including resection of the inner pelvic ring and reconstruction of the linea terminalis using a non-vascularized fibular autograft. At 18 months after tumor resection and reconstruction of the pelvic ring, with interposition of a free vascularized fibula graft patient has an excellent clinical oncological and functional outcome.A 34-year-old female presented with BFH at a rare anatomical location on both sides of the Non vascularized fibular autograft is a useful reconstructive procedure in select patients. Benign fibrous histiocytoma (BFH) is a tumor that occurs predominantly in the skin  and most commonly in younger individuals. The tumor typically presents as a painless nodule varying in size from a few millimeters to several centimeters.BFH of the bone has been a subject of increasing interest within the past few years. The term was initially introduced by Dominok in 1980 to describe a cystic lesion in the femur of a 66-year-old man -6.Histologically, BFH arising from soft tissues cannot be distinguished from those arising from bone . Pain isOn radiography the lesion is characterized by osteolytic lesions with a well defined sclerotic margin . MagnetiA local recurrence rate of 5 to 25 % is reported in literature. The recurrence rate is typically related to the size of the tumor . Consequilium with a large extra- and intrapelvic soft-tissue component . We are of the opinion that stabilization of the inner pelvic ring is necessary to prevent stress fracture of the remaining bone bridge.After mobilization of the patient an increasing diastasis of the symphysis was noted Figure . This miOncologically the marginal tumor resection has proved to be an adequate treatment. Despite high recurrence rates, especially in the case of large BFH , no locaThe author(s) declare that they have no competing interests.PN prepared the manuscript and participated in the operations.KL carried out the radiographies and MR imaging and contributed the section on imagingMW performed histological analysis and wrote pathology part of manuscriptLB and DP critically reviewed the manuscript for its scientific content and performed surgery.


An increasing number of studies have profiled tumor specimens using distinct microarray platforms and analysis techniques. With the accumulating amount of microarray data, one of the most intriguing yet challenging tasks is to develop robust statistical models to integrate the findings.n = 305 samples) on a common probability scale, we developed a 90-gene meta-signature, which strongly associated with survival in breast cancer patients. Given the set of independent studies using different microarray platforms which included spotted cDNAs, Affymetrix GeneChip, and inkjet oligonucleotides, the individually identified classifiers yielded gene sets predictive of survival in each study cohort. The study-specific gene signatures, however, had minimal overlap with each other, and performed poorly in pairwise cross-validation. The meta-signature, on the other hand, accommodated such heterogeneity and achieved comparable or better prognostic performance when compared with the individual signatures. Further by comparing to a global standardization method, the mixture model based data transformation demonstrated superior properties for data integration and provided solid basis for building classifiers at the second stage. Functional annotation revealed that genes involved in cell cycle and signal transduction activities were over-represented in the meta-signature.By applying a two-stage Bayesian mixture modeling strategy, we were able to assimilate and analyze four independent microarray studies to derive an inter-study validated "meta-signature" associated with breast cancer prognosis. Combining multiple studies (The mixture modeling approach unifies disparate gene expression data on a common probability scale allowing for robust, inter-study validated prognostic signatures to be obtained. With the emerging utility of microarrays for cancer prognosis, it will be important to establish paradigms to meta-analyze disparate gene expression data for prognostic signatures of potential clinical use. DNA microarray analysis has been shown to be a powerful tool in various aspects of cancer research . With thτ2. The effect size was defined to be the difference between the tumor and normal sample means divided by pooled standard deviation. From a Bayesian perspective, Wang et al. [More systematic approaches have been proposed for integration of findings from multiple studies using different array technologies. Rhodes et al.  have prog et al.  used datg et al. . The larpoe  was introduced as a new scale and used in the context of molecular classification [poe in a framework to meta-analyze microarray data. Several desirable features of using poe as a new expression scale include the following: 1. poe provides a scaleless measure and thereby facilitates data integration across microarray platforms; 2. poe is a model-based transformation with direct biological implications in the context of gene expression data, as it is estimated based on a method that adopts an underlying mixture distribution that accommodates over-, under-, and unchanged expression categories; 3. poe unmasks differential expression patterns in microarray data by offsetting the influence of extreme expression values [poe allows merging of samples on the unified scale rather than using gene-specific summaries.A recent study proposed a Bayesian mixture model based transformation of DNA mi-croarray data with potential features applicable to meta-analysis of microarray studies . The basfication . The plan values ; 4. DataIn recent publications of breast cancer microarray studies, several groups have explored the hypothesis that the capacity to metastasize is intrinsic to the tumor and therefore can be revealed by gene expression pattern. Four independent studies have correlated gene expression profiles generated from distinct DNA microarray platforms to breast cancer prognosis -13. Amonpoe. We applied our method to the aforementioned breast cancer DNA microarray data sets. With the strength of the poe transformation and data integration, our goal was to develop an inter-study validated meta-signature that predicts relapse-free survival in breast cancer patients with improved statistical power and reliability.In this proof-of-concept study, we propose a two-stage meta-analysis of microarrays based on poe. The Bayesian hierarchical model employed for estimation borrows strength across genes by assuming further distributions for the gene-specific parameters (see Methods). For data integration purposes, we focused on a common set of 2,555 genes that were profiled in each of the four studies. Although the cost for compiling common genes is a loss of potential predictive features, it is not unreasonable to assume, given the analogous hypothesis explored in each study, that the common set represents the most relevant genes of interest for breast cancer prognosis. The resulting values of poe represent signed probability of differential expression for gene j in sample i, and thus provide a unified measure across studies. Further, the transformation improves contrast in each data set by removing the influence of extreme expression values. In stage 2, the expression profile of tumor samples from multiple studies were combined on the poe scale to generate a meta-cohort. The benefit of data integration using poe is twofold. First, it improves power of statistical analysis by increasing the sample size. Such integration of independent data sets renders sensitivity to those small yet consistent expression changes for certain genes. Second, it reduces the chance of false positive features due to artifacts from a single study, and allows reliable findings across studies. In this paper, we integrated four breast cancer microarray data sets of distinct platforms , not all studies have the time to recurrence information available and instead provide data on recurrence within a certain time interval . To deal with this issue, we utilized a dichotomization where a bad outcome is recurrence during followup and a good outcome is remaining recurrence-free for at least three years. The additional constraint for the good outcome group is to reduce potential bias introduced by short censoring due to insufficient length of follow-up. This is particularly relevant in cross-study analysis, given the heterogeneity in patient recruitment criteria and study designs. Accordingly, of the combined meta-cohort (n = 305) of breast cancer patients, 48.9% were in the poor outcome group, whereas 51.1% in the good outcome group. The sample sizes for each study are shown in Table P ≤ 0.001) in all cross-validation steps, representing a cohort of essential genes strongly associated with breast cancer recurrence. By random chance, there would be on average 2.5 genes to be found significant at P ≤ 0.001 in a set of 2,555. By finding 23 genes with a P ≤ 0.001, it is clear that there are much more predictive features than would be expected by random chance.Each gene was then associated with the recurrence status by a logistic regression within a leave-one-out cross validation scheme, and rank-ordered by the significance level of the coefficient. As a result, 23 genes held up as significant predictor of recurrence  as a linear combination of the P < 0.0001). In spite of the heterogeneity of the combined patient population, the meta-signature predicted the odds of recurrence for a patient showing a high risk signature as four times of the odds of recurrence for a patient showing a low risk signature. Several studies have implicated that the lymph node status is one of the principal clinical factors to classify patients in relation to the risk of relapse of breast cancer [By minimizing the misclassification error, we obtained a 90 gene meta-signature that reliably predicts outcome in the meta-cohort. This meta-signature classified 122 patients into a high risk group, where 84 (69%) of them had a recurrence. On the other hand, the signature classified 183 patients into a low risk group, where 118 (64%) of them did not recur by the end of the followup. By cross-tabulating the risk groups predicted by the meta-signature and the actual recurrence status, we obtained an estimated odds ratio of 4.0 . Similarly for nodal positive patients, a cohort thought to be at high risk of recurrence, the meta-signature identified 79 (51.6%) of the LN+ patients to have, in fact, lower recurrence risk over time . A multivariate logistic regression model suggested that the meta-signature is an independent predictor of the recurrent status with respect to nodal status in the meta-cohort (OR = 3.7(2.3–6.1), P < 0.0001).Kaplan-Meier analysis provides further evidence that the meta-signature was a significant prognostic index of breast cancer recurrence in the meta-cohort Figure . The estTo comprehend the potential gains of such two-stage meta analysis over individual analysis in each single study cohort, we constructed study-wise gene expression signatures using the same method. By minimizing the misclassification errors, we obtained a signature consisting 10, 60, 100, and 130 genes for Sorlie, van't Veer, Sotiriou, and Huang study cohort respectively . The resMeta-analysis accounts for such heterogeneity of the individual signatures in two ways. First its overlap with the study-specific signatures ranged from 3–40%. The excluded genes are likely to be cohort-specific findings that can not be replicated. Second, the meta-signature recruited 41 genes not previously picked by any of the single cohort signature, likely representing predictive features with small but consistent effects previously masked in single studies. When examining the performances of the gene signatures, the meta-signature showed a comparable or better performance compared with the individually optimized signatures both in the odds ratio estimates  terms and then grouped by functional classes. Based on the hypergeometric distribution, we calculated the significance of over-representation of a particular process in the signature. Figure poe scale. In particular, poe provides a unified platform-free scale, and simultaneously enhances the intrinsic contrast in the expression data. Furthermore, combining sample pools on the poe scale mitigates the influence of potential artifacts from a single study. The benefit of such data integration is reflected on two counts. One, integrated sample cohorts improve the reliability of the findings by guarding against false positive results from a single study. Two, it increases the statistical power to detect small consistent effects that can be otherwise masked by inadequacy of the sample size of an individual data set. By implementing this modeling approach, we were able to combine information from four microarray studies to build an inter-study validated meta-signature for predicting survival in breast cancer patients.Several important issues to consider when integrating microarray studies include use of different gene expression measurement scales, varying analytical power and reliability of the results for individual studies. To account for these issues, we proposed a two-stage mixture modeling strategy, the strength of which was built on the mixture model based transformation and the subsequent data integration on the As described earlier, a common set of 2555 genes was used in this meta-analysis, as it is important to provide the same context for data-driven estimation of the posterior probabilities. Although we assume the common set comprises the most biologically relevant genes, the loss of potential predictive genes, however, may offset the statistical power of the analysis. For example, one of our recent studies has established the polycomb protein EZH2 to be an independent predictor of breast cancer survival outcome. This geFunctional annotation of the meta-signature revealed genes such as Cyclin E and BCL2, which were previously shown to be correlated with survival outcome in breast cancer ,24. A stIn conclusion, a distinction of the analysis presented here relative to those by other authors ,6 is thaThe breast cancer microarray data sets were obtained at the author's websites from four recently published studies -13. EachX with elements xij, where i = 1, ..., mk, j = 1, ..., n . The expression measurement xij can be the ratio of the two fluorescent dye hybridization intensities for the spotted cDNA arrays[E be a latent class variable, and eij indicates over-, under- or normal expression for each entry of the R matrices. We have:Each of the four raw data sets was treated as an expression matrix NA arrays,12 and tNA arrays, or averNA arrays. Let E beij are latent and not directly observed from the data. We were interested in estimating the probabilities of eij being 1 or -1 given the observed raw expression xij, which were denoted as  and . Estimates of these latent quantities were obtained under a Bayesian mixture model setting. In particular, we assume the raw expression xij falls into one of the three expression categories. For each gene j, the expression then arises from a mixture of three distributions:The values of xij|eij = 1) ~ fj1,(·), (xij|eij = 0) ~ fj0,(·), and (xij|eij = -1) ~ fj-1,(·). and  = Peij = -1) are the multinomial probabilities for eij. With the specifications of models, we can calculate the latent quantities by Bayes' rule:respectively. Here, U refers to a uniform distribution and N refers to a normal distribution. By noting that the supports for the two uniform distributions are disjoint, the probabilities of differential expression are mutually exclusive with the forms:poe = p+ - p-. As a result, poe ranges from -1 to 1, and can be interpreted as the signed conditional probability of differential expression.A one dimension measure can thus be constructed as To borrow strength across genes, the estimation of the gene-specific parameters was formulated under a Bayesian hierarchical model setting. Given the large amount of parameters, prior distributions were specified to model the variation of the gene-specific parameter estimates, in particular, were obtained for each of the five data sets , we defined outcome groups as recurred during followup and remained relapse-free for at least 3 years. In particular, Let t* can be specified with clinical knowledge. We chose t* = 3 years in this study. We then consider constructing classifiers using y; note that y = 1 corresponds to the poor outcome group and y = 0 to the good outcome group. The sample sizes for each study are shown in Table where j, we fit the following univariate logistic regression model using data from all studies:Logistic regression was used to build a classifier for prognosis. For each gene x* is the rescaled value that allows data integration across multiple studies. The esti-mated values of βj, , are then used to form a risk score using a variation of the compound covariate predictor method [x1, ..., xD, the risk index is given as .where r method ,29; for , where , and  is the effect estimate for gene j in the combined meta-cohort without the ith sample. The risk index for sample i is a weighted linear combination of the expression profiles of the top D genes, where the ranking of the genes is based on their corresponding significance in the univariate logistic model fit. Classification of sample i to the risk groups is then based on the ith leave-one-out risk index, i.e.,  = I{RIi >c} with c being the empirical quantiles (40th - 70th) of the RI's. The number of genes D in a classifier is treated as a parameter and optimized to minimize the prediction error rates.If we want to assess the performance of the classifier, we must deal with the issue of training and testing the model using the same data. An "honest" estimate of the prediction error rate is obtained using leave-one-out cross-validation. Define a risk index The list of the top cumulative genes in the meta-signature was obtained by ranking all 2,555 genes by the number of times in the leave-one-out cross-validation steps that each one had a P-value from the univariate logistic regression less than 0.001.poe pro-files of the meta-signature. Yellow represents high probability of over-expression and blue represents high probability of under-expression. For heat maps of raw data matrices, we preprocessed the data by mean centering and then dividing by the standard deviation for each row. The means and the standard deviations used in the normalization were the relapse-free (RF) sample means and variances for each study data. The values for the recurrence (R) samples after standardizing then represented the number of standard deviations over or under the mean RF sample expression.We used the treeview software  to generRS, DG, and AC designed the study. RS carried out the statistical analysis and prepared the manuscript. DG and AC supervised the analysis and participated in the manuscript preparation. All authors read and approved the final manuscript.Plots of misclassification rates. The PDF file lists plots of misclassification error rates for classifiers identified in each individual study cohort and the meta-cohort.Click here for filePlots of Kaplan-Meier curves. The PDF file lists Kaplan-Meier plots for study-wise cross-validation of the individually identified signatures. A gene signature was trained in one study cohort and used to validate in each of the other study cohorts as testing sets.Click here for fileMeta-signature gene list. The excel file contains a list of Unigene ID, gene symbol, and full name of the 90 genes in the meta-signature.Click here for fileet al. study data .POE imputation of the Sorlie data. The excel file contains a table of imputed signed probability matrix transformed from the Sorlie Click here for fileet al. study data .POE imputation of the van't Veer data. The excel file contains a table of imputed signed probability matrix transformed from the van't Veer Click here for fileet al. study data .POE imputation of the Sotiriou data. The excel file contains a table of imputed signed probability matrix transformed from the Sotiriou Click here for fileet al. study data .POE imputation of the Huang data. The excel file contains a table of imputed signed probability matrix transformed from the Huang Click here for file