Streptococcus pneumoniae is a common bacterium that is present in the nasopharynx of many children and some adults, where it causes no harm to its carrier but can be transmitted to others. If it moves beyond the nasopharynx, however, it can cause ear infections or invasive disease, such as pneumonia or meningitis. Invasive disease from this organism occurs especially in children, the elderly, and individuals with weakened immune systems.The protective effect of antibodies against bacterial pneumonia has been appreciated since the 1930s, when it was shown that serum therapy—the transfer of serum from an immunized animal to a patient with acute disease caused by the same bacterial strain—could reduce mortality from pneumococcal pneumonia by half. Subsequent development of vaccines based on the bacterium's polysaccharide capsule, which could protect against infection, confirmed that an endogenous antibody response can provide protection against invasive disease.One challenge for vaccine development has been the existence of many different serotypes (the same species of bacteria but with different composition of the polysaccharide capsule). As protection usually doesn't extend to different serotypes, vaccination with capsule components from different serotypes is necessary to ensure broad protection. Such vaccines have been shown to be efficient and safe. They are now recommended in many countries for infants and toddlers, and for people over 65—the two age groups in which invasive disease is most common—and for others who are at increased risk of pneumococcal disease .Even without vaccination, however, most exposed individuals will never get invasive disease. Instead, they develop natural immunity against the different serotypes, though this immunity gradually declines with old age. Marc Lipsitch and colleagues wanted to understand the immunological basis of this natural immunity, and specifically whether it was due to anticapsular antibodies.If protection from invasive disease is due to acquiring anticapsular antibodies against each of the pneumococcal serotypes, they argued, this would lead to two predictions about the age distribution of disease caused by the different serotypes in the non-vaccinated population. First, for serotypes that are more common and therefore encountered earlier in life, children should develop immunity more quickly, causing disease from these types to drop off earlier in life than disease from the less common types. Second, protection against invasive disease from a particular serotype should coincide with the acquisition of antibodies against that serotype, on both the individual and population level.Neither prediction was borne out by the actual data the researchers analyzed, suggesting that there is more to natural immunity against pneumococcal disease than just anticapsular antibodies. The study doesn't demonstrate what the additional components are, but additional research might not just teach us about our immune system but also provide clues for further vaccine development. As the authors say, “A better understanding of the mechanisms that underlie natural immunity to pneumococcus could pave the way for the development of more effective, species-specific pneumococcal vaccines.”


Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.The atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures.This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis. After the development of serological tests for hepatitis A and B viruses in the 1970s it became clear that an additional agent accounted for approximately 90% of transfusion-associated hepatitis  [Flavivirideae family. Diagnostic tests for anti-HCV antibodies developed thereafter proved that HCV was indeed the predominant cause of NANBH [The novel agent, hence termed hepatitis C virus (HCV), currently infects approximately 3% of the world's population and it was classified within the of NANBH . In viewRational drug design has not been the primary way for discovering major therapeutics. However, recent successes in the area give reason to expect that drug discovery projects will increasingly be structure based. One of the possible targets for drug development against HCV is the NS3 protease variants. HCV RNA is translated into a polyprotein that during maturation is cleaved into functional components. One component, nonstructural protein 3 (NS3), is a 631-residue bifunctional enzyme with protease and helicase activities.The N-terminal portion of the NS3 protein was predicted to contain a serine protease domain as judged from conserved sequence patterns and by homology to Flavi- and Pestiviruses -6. The NGenome sequencing efforts are providing us with complete genetic blueprints for hundreds of organisms, including humans. We are now faced with assigning, understanding and modifying the functions of proteins encoded by these genomes. This task is generally facilitated by 3D structures , which aComparative modeling uses experimentally determined protein structures to predict conformation of other proteins with similar amino acid sequences. For modeling of proteins was used restrained-based modeling implemented in the program MODELLER . The modWe modeled the structure of variants of NS3 protease variants available in the National Center for Biotchnology Information (Genbank), using structural bioinformatics tools. Knowledge of the three-dimensional structure variants will undoubtedly aid the design of useful inhibitors that may be used as a drug against hepatitis C virus. In order to speed up the modeling process all NS3 models were generated in a Beowulf cluster . The potential of the structural bioinformatics for development of new antiviral drugs is discussed.The identity between the sequences of a bifunctional protease structure  ,38  was used to generate homology models for docking studies. Binding of an inhibitor to the active site of an enzyme is typically connected with local and possibly also global structural rearrangement of the enzyme (induced-fit mechanism). Therefore structure-based drug design preferentially relies on the crystal structures of enzyme-inhibitor complexes containing bound inhibitors of similar chemical structures to the compounds being designed. Such complexes offer more detailed and accurate picture of the inhibitor-enzyme interactions and structural complementarity between the inhibitor and the active site. The homology models of the variants of NS3 protease which used the NS3 complexed with an inhibitor are more adequate to docking simulations. The atomic coordinates of all water molecules were removed from the templates.The analysis of the Ramachandran diagram φ-ψ plots of the 1CU1 structure (template) were used to compare the overall stereochemical quality of the NS3 protease variants structures against template solved by biocrystallography  was developed to archive protein structure identified in infectious agents such as NS3 protease variants from hepatitis C virus. All supporting data related to the 3D structures modeling, such as protein codes, atomic coordinates in PDB format from modeled proteins, fasta sequence, links to others databases and various information about the protein were arranged in the MySQL  databaseLarge scale protein homology modeling, in which whole sequence databases or whole genomes are used as input into automated modeling algorithms, have been reported by several groups . By utilInhibition studies have shown that NS3 is only modestly inactivated by classic serine protease inhibitors such as chloromethylketones or phenylmethyl sylfonylfluoride . The strMolecular modeling is usually the method of choice when there is a clear relationship of homology between the sequence of a target protein and at least one known structure. This computational technique is based on the assumption that tertiary structures of two proteins will be similar if their sequences are related, and it is the approach most likely to give accurate results . There aNext, the spatial restraints and CHARMM energy terms enforcing proper stereochemistry  were com1-χ2, χ-1, χ-3, χ-4 and ω values for each of the 20 amino acid types) and covalent geometry  these average values were calculated using PROCHECK [The overall stereochemical quality of the final models for each NS3 protease variants from hepatitis C virus was acessed by the program PROCHECK . The rooPROCHECK . The VerPROCHECK -30.NJFS carried out the molecular modeling and participated in the building and design of the database tables, HAA and CEB carried of analysis of the models and created database interface, FPS selected the primary sequences of NS3 protease variants of the hepatitis C virus, IMVGCM participated of the study, PR conceived the study and suggestions in this manuscript, JRRP participated in the study and suggestions in this manuscript, WFA conceived the study, and participated in its analysis and coordination. All authors read and approved the final manuscript.


Several tumors can exist as multiple lesions within a tissue. The lesions may either arise independently, or they may be monoclonal. The importance of multiple lesions for tumor staging, progression, and treatment is subject to debate. Here we use mathematical models to analyze the emergence of multiple, clonally related lesions within a single tissue. We refer to them as multi-focal cancers. We find that multifocal cancers can arise through a dynamical interplay between tumor promoting and inhibiting factors. This requires that tumor promoters act locally, while tumor inhibitors act over a longer range. An example of such factors may be angiogenesis promoters and inhibitors. The model further suggests that multifocal cancers represent an intermediate stage in cancer progression as the tumor evolves away from inhibition and towards promotion. Different patterns of progression can be distinguished: (i) If tumor inhibition is strong, the initial growth occurs as a unifocal and self contained lesion; progression occurs through bifurcation of the lesion and this gives rise to multiple lesions. As the tumor continues to evolve and pushes the balance between inhibition and promotion further towards promotion, the multiple lesions eventually give rise to a single large mass which can invade the entire tissue. (ii) If tumor inhibition is weaker upon initiation, growth can occur as a single lesion without the occurrence of multiple lesions, until the entire tissue is invaded. The model suggests that the sum of the tumor sizes across all lesions is the best characteristic which correlates with the stage and metastatic potential of the tumor. The occurrence of multiple lesions is observed in a variety of cancers. That is, not one, but several lesions are observed within a given tissue. Multiple lesions can occur by two basic mechanisms -5. EitheIn this paper we focus on multiple lesions with a monoclonal origin. We will refer to them as "multifocal" cancers. The mechanism by which such multifocal cancers are generated, and their relation to the stage and metastatic potential of the cancer, are not fully understood . Yet, thWe start with a simple model which describes tumor growth in relation to the production of promoters and inhibitors. We then extend this model to describe the local spread of cancer cells across space (tissue), and examine somatic evolution of cells away from tumor inhibition and towards promotion.C; promoters, P; and inhibitors, I. It is assumed that both promoters and inhibitors can be produced by cancer cells. In addition, inhibitors may be produced by healthy tissue. The model is given by the following set of differential equations which describe cancer growth as a function of time,We consider a basic mathematical model which describes the growth of a cancer cell population, assuming that the amount of blood supply influences the rate of cell division. The model includes three variables: the population of cancer cells, r. Growth is assumed to be density dependent and saturates if the population of cancer cells becomes large (expressed in the parameter ε). In addition, the growth rate of the cancer cells depends on the balance between promoters and inhibitors, expressed as P/(I + 1). The higher the level of promoters relative to inhibitors, the faster the growth rate of the cancer cell population. If the level of promoters is zero, or the balance between promoters and inhibitors in heavily in favor of inhibitors, the cancer cells cannot grow and remain dormant [δ. Promoters are produced by cancer cells at a rate ap and decay at a rate bp. Inhibitors are produced by cancer cells at a rate aI and decay at a rate bI. In addition, the model allows for production of inhibitors by normal tissue at a rate ξ.The equations are based on a previous study . The pop dormant -26. Canci) The cancer cells cannot grow and consequently go extinct.That is, C = 0, P = 0 and I = 0. The cancer goes extinct in the model because we only consider cells which require the presence of promoters for division. If the level of promoters is not sufficient, the rate of cell death is larger than the rate of cell division. In reality, however, it is possible that a small population of non-angiogenic tumor cells survives. Here, we omit this for simplicity. (ii) The population of cancer cells grows to significant levels, that is, C = .The analysis of the model above is presented in detail in the Materials and Methods section. It suggests the following patterns. There are two outcomes. . The exact condition is given by (9). In this case, the outcome is either failure of cancer growth, or successful growth to large numbers. Which outcome is achieved depends on the initial conditions. Successful growth is only observed if the initial number of cancer cells lies above a threshold. Then, enough promoters are initially produced to overcome inhibition. This provides an important barrier to the successful growth of cancers. It could explain why it is difficult for cancers to escape angiogenesis inhibition, and why autopsies often reveal the existence of multiple small, non-pathogenic tumors which have failed to progress [How do the parameter values influence the outcome of cancer growth? The cancer extinction outcome is always stable. The reason is as follows. The cancer cells require promoters to grow. The promoters, however, are produced by the cancer cells themselves. If we start with a relatively low initial number of cancer cells, this small population cannot produce enough promoters to overcome the presence of inhibitors. Consequently, the cancer fails to grow and goes extinct. This outcome is always a possibility, regardless of the parameter values. Significant cancer growth can be observed if the intrinsic growth rate, progress .In this section, we introduce space into the above described model. We consider a one-dimensional space along which tumor cells can migrate. The model is formulated as a set of partial differential equations and is written as follows,Dc. Inhibitors can also diffuse across space, and this is described by the diffusion coefficient DI. It is generally thought that inhibitors act over a longer range, while promoters act locally [ξ. As before, numerical simulations indicate that results are not changed qualitatively by this simplification. As mentioned above, the model considers tumor spread across space. It is important to point out that we do not consider long-range metastatic spread. Instead, we consider local spread of a tumor within a tissue, such as the breast, liver, brain, or esophagus.The model assumes that tumor cells can migrate, and this is described by the diffusion coefficient  locally ,24. TherHere we investigate the process of tumor growth and progression in relation to the degree of inhibition and promotion. A mathematical analysis is presented in the Materials and Methods section. Here we present biological insights and results of numerical simulations.aI/bI >>ap/bp). This corresponds to the early stages when the tumor is generated. We then investigate how tumor growth changes as the degree of inhibition is reduced relative to the level of promotion . We consider the following parameter regions . Only a small number of cells which do not require promotion for survival would remain.uni-focal). Note that this does not mean that it is in principle impossible to generate more lesions. It means that the space between lesions is bigger than the space provided for cancer growth within the tissue.2. If the degree of inhibition is weaker, the cancer cells can grow. The spread across space is, however, self-limited Figure . The can3. As the production of inhibitors is further reduced, we enter another parameter region. Now fewer inhibitors diffuse across space. We observe that multiple lesions or foci are formed Figure . They ar4. If the degree of inhibition is further reduced and lies below a threshold, spread of inhibitors is sufficiently diminished such that the tumor cells can invade the entire space and tissue Figure . In biolIn summary, as the relative degree of inhibition is reduced, the patterns of tumor growth change from absence of significant growth, to a single self-limited tumor, to the occurrence of multiple foci, and to the maximal invasion of the tissue by tumor cells. Multi-focal cancers may arise through the dynamical interplay between long range inhibition and local promotion. The following section will examine this in the light of somatic evolution.We have shown how the pattern of cancer growth can depend on the relative balance of promoters and inhibitors. Here we consider these results in the context of somatic evolution, and suggest some clinical implications.At early stages of cancer progression, the balance between inhibitors and promoters is in favor of inhibition. Inhibitors are likely to be produced by healthy cells (e.g. in the context of angiogenesis), and they are more abundant than an initiating population of transformed cells. In the context of angiogenesis, specific mutations have been shown to result in the enhanced production of promoters or reduced production of inhibitors in cancer cells. Our model has shown that such mutants have to be produced at a relatively high frequency, so that a sufficient number of promoting cells are present in order to ensure that enough promoters are produced to overcome the effect of inhibition.Once the promoting cells have succeeded to expand, cancer progression can occur in a variety of ways according to the model. How the cancer progresses depends on how much the balance between promotion and inhibition has been shifted in favor of promotion. We distinguish between three possibilities Figures , 3 &4.(i) The balance between inhibition and promotion has been shifted only slightly in favor of promotion, such that self-limited growth of the cancer is observed Figure . That is(ii) The first mutation shifts the balance between promoters and inhibitors to a lager extent which is sufficient to result in the generation of multiple lesions Figure . The mul(iii) Finally, assume that the initial mutation shifts the balance so much in favor of promotion that maximal invasion of the tissue is possible Figure . Now we In summary, the model predicts different modes of cancer progression in relation to the evolution away from tumor inhibition and towards promotion. A single cancer lesion may spread across the tissue without the occurrence of multiple lesions. Alternatively, the cancer can first grow as a single, self-contained lesion. This can then bifurcate to give rise to multiple foci, either as a result of additional mutations, or as a result of the natural pathway by which multiple foci are generated, depending on the degree of tumor promotion conferred by the initial mutation. Further evolutionary events can then induce the multiple foci to become a single, maximally invasive mass. The occurrence of multiple foci therefore represents an intermediate stage in tumor progression towards malignancy.The models discussed here show that multiple foci with a monoclonal origin can develop through a dynamical interplay between tumor promoters and inhibitors. The cancer can only grow to high loads as a single mass if it has largely escaped all inhibitory effects. Otherwise, the cancer is likely to grow via the generation of a relatively small and self limited tumor which then bifurcates into multiple foci until it finally invades the entire tissue. The occurrence of multiple foci is therefore an intermediate stage in cancer progression. The higher the number of foci, the further advanced the stage of cancer progression.A clinically important step in carcinogenesis is the process of metastasis. That is, the spread of tumor cells to the lymph node, entry into the blood supply, and the spread to other tissues. Various studies have investigated the metastatic potential of multi-focal compared to uni-focal cancers ,28,29. IFurther, it is important to note that studies which aim to assess the correlation between multi-focality and metastatic potential should not only concentrate on the number of foci, but also on the size of the foci. As we have shown with the model, cancer progression might start with a small single lesion which can be considered uni-focal. It can then bifurcate to give rise to multiple foci, and finally spread through the entire tissue. When such spread occurs, the multiple foci turn into a big and single mass, and this would again be considered uni-focal. Hence, the cumulative size or volume of the tumor is likely to be the best predictor of malignant progression.In conclusion, we suggest that the balance between tumor promoting and inhibiting factors might be an important driving force which determines the pattern on cancer progression, and can account for the occurrence of multi-focal cancers. The best worked out example of such promoter-inhibitor dynamics is angiogenesis. In this context, inhibitors are produced both by healthy tissue cells and by tumor cells. During the course of progression, tumor cells can mutate and evolve to produce less inhibitors and more promoters. The initial establishment of an angiogenic cell line is the most difficult step. Since the promoting factors are produced by angiogenic cells themselves, their initial abundance has to be sufficiently high, such that the balance can be shifted away from inhibition. This enables the population of cancer cells to expand beyond a very small size. This growth can then give rise to a self-limited uni-focal cancer which can bifurcate to give rise to multi-focal cancers. Further evolutionary events can finally lead to maximal tissue invasion. If the initial mutation allows the cells to sufficiently escape from inhibition, cancer progression can occur as a single expanding mass without the occurrence of multi-focality. These arguments not only apply to angiogenesis, but to any tumor promoting and inhibiting factors where inhibitors act over a long range while promoters act locally. Therefore, the therapeutic use of inhibitors should be further explored. This is an active area of research in the context of angiogenesis , and theHere we present mathematical methodology used to analyze the equations described in the text.P = CaP/bP). It is convenient to denoteHere we discuss a linear stability analysis of system (1–3). Let us first simplify the problem by using a quasistationary approach, that is, we will assume that the level of promoters adjusts instantaneously to its steady-state value 2 - 4εγ > 0, then there are exactly three positive equilibria in the system. If either of these conditions is violated, the  solution is the only  stable point.It is obvious that if Stability analysis can be performed by the usual methods. For the  equilibrium, the Jacobian isC±, I±), we get the following Jacobian,that is, this equilibrium is always stable. For the points  we have eigenvaluesand for the solution (Y ± ≡ 2bIW + δ(ε - γ - W ± Γ). We can see that solution  is always unstable and we will not consider it any longer. Solution , which we call for simplicity  from now on, is stable as long aswhere Y+ > 0     (9)P with C defined by , we can rewrite equation (4) asHere we present a linear analysis of system (4–6). As before, we are going to assume that promoters adjust instantaneously to their equilibrium level. By replacing This equation together with equation (6) gives a Turing model.) is a stable equilibrium. Of course, this solution also satisfies the system of PDEs, . Let us consider a wave-like deviation from this spatially uniform solution:Let us go back to the system of ODEs, (7–8), and assume that solution  can be written asλ2 + λ(bI - α + (DC + DI)ω2) + aIβ - (bI + DIω2)(α - DCω2) = 0.     (12)λ, with the spatial frequency of the perturbation, ω. The stability conditions now are given byThis is the dispersion relation which connects the growth-rate, bI - α + (DC + DI)ω2 > 0,     (13)aIβ - (bI + DIω2)(α - DCω2) > 0.     (14)) of the system of ODEs, (7–8), are obtained automatically from the conditions above by setting ω = 0:Note that the stability conditions for solution aIβ - bI α > 0.     (16)Inequality (13) is always satisfied because of inequality (15). Let us derive conditions under which the spatially uniform solution is unstable. This requires that condition (14) is reversed. This can be expressed as follows:F(ω) ≡ DIDCω4 - ω2γ1 + γ2 < 0.     (17)where we denoted for simplicity,γ1 = αDI - bIDC, γ2 = αIβ - αbI > 0.ω = 0, with a positive leading term. The points, ±|ω|, satisfyingThis is a fourth order polynomial, symmetrical with respect to the line ω, ±ωc. The condition F(ωc) < 0 defines that the uniform solution  is unstable.correspond to the two minima of the left hand side of inequality (17). Let us call these values of F(ω) for different values of aI, see Figure aI, F(ω) is strictly positive, and the spatially uniform solution is stable. As aI increases, the function F(ω) crosses the line F = 0. The critical value of aI, aI,c, for which F(ωc) = 0, is determined fromLet us plot the function αDI - bIDC)2 = 4DIDC(aIβ - αbI), has only very narrow regions of ω corresponding to negative values. More precisely, Δω ~ L-1, where L is the spatial dimension of the system. In terms of parameter aI, we require that it is sufficiently close to aI,c. Then, we can calculate the "most unstable" wavenumber, that is, ωc defined by equation (18), with aI,c. This value will determine the spatial period of the solution,The applicability of the above analysis depends on the parameters of the system. First of all, we need conditions (15–16) to be satisfied. They mean that without diffusion, a positive, spatially uniform solution is stable. Next, we need to be in a In numerical simulations described in this paper, we used the following (Neumann) boundary conditions:aI is below the critical, aI <aI,c. The system exhibits bistability. If we start in the vicinity of a  solution, then cancer will not grow and decay to zero. If we start from a point  in the domain of attraction of the solution , then the system will develop towards this positive spatially homogeneous stationary solution.The simulation results are presented in the main body of the paper. Here we discuss the behavior of the system in the light of the analysis presented above. Let us assume that the value aI >aI,c, but make sure that it is sufficiently close to aI,c . Again, if the initial conditions are close to the zero solution, then the zero state will be the state that the system will attain. However, if we start in the vicinity of the  state, we will observe interesting behavior. Solution  is now unstable, and we will see "ripples" developing on top of this solution. This is Turing instability. The spatial period of the ripple was calculated in the previous section. Long-time evolution of this state is of course not in the realm of linear stability analysis, but we can predict that the spatial scale of the resulting solution will be given by (19).Next, let us suppose we have aI is much higher than critical. Now, solution  is unstable even in the system of ODEs. However, a periodic solution will develop, unless the initial condition is in the domain of attraction of the zero solution. The spatial scale of the periodic solution is determined intrinsically by the parameters of the system, and it grows with aI. Intuitively this is easy to understand, because higher values of aI correspond to higher levels of inhibition, so the distance between regions of large C will become larger. Note that the exact period of the periodic solution is adjusted to fit the boundary conditions of the system. For instance, with the Neumann boundary conditions, the boundary points are forced to be troughs of the wave-like pattern. In other words, the period of the solution must be an integer fraction of L.Finally, let us assume that


New York Times bestseller list and winning accolades from the likes of Jimmy Carter. A wide range of inherited disorders falls under the rubric of muscular dystrophy, but all involve some form of progressive muscle wasting. Stepanek's condition impaired nearly all of his body's functions, but other more common forms, including Emery-Dreifuss muscular dystrophy (EDMD), selectively target skeletal muscle and induce cardiac abnormalities.In June, muscular dystrophy patients lost one of their most passionate advocates to a rare form of this degenerative neuromuscular disorder—thirteen-year-old Mattie Stepanek. In his short life, Stepanek wrote five volumes of inspirational poetry, topping the EDMD is caused by mutations in either of two genes: one encodes lamin A, a structural protein associated with the nucleus, and the other encodes a nuclear membrane protein called emerin. Lamins, a major component of the structural network that supports the nuclear envelope, help the nuclear envelope maintain structural integrity and absorb mechanical stress without rupturing.  Emerin binds to proteins that regulate gene transcription. Emerin and lamins are found in most cell types, yet EDMD attacks only skeletal muscles, major tendons, and the cells that regulate cardiac muscle contraction. So where does this tissue specificity come from?One theory suggests that emerin selectively targets proteins that specifically regulate gene expression in EDMD-affected tissues. Another theory proposes that emerin provides structural support to the nuclear envelope and that emerin mutations are most destructive in tissues subjected to mechanical stress—like skeletal muscle and tendons. Current evidence supports both models. Recent studies suggest that emerin forms complexes with actin—the mother of all structural proteins. Actin proteins can join together (polymerize) to form a variety of filaments. However, given longstanding doubts that actin exists in the nucleus, let alone functions there, researchers were unsure what the findings might indicate. Now James Holaska, Amy Kowalski, and Katherine Wilson propose that emerin not only functions as a structural protein in the nucleus but that it does so by interacting with actin.Evidence that emerin and lamin A can form multiprotein complexes comes primarily from experiments in test tubes. To get a sense of the physiological significance of these findings, Wilson and colleagues purified emerin-binding proteins from the nuclei of living cells. They found that emerin binds to polymerized actin and, in fact, appears to stimulate polymerization. By binding and “capping” a specific end of the actin filament, emerin prevents filament de-polymerization (disassembly), effectively increasing the rate of actin polymerization by four- to twelve-fold. The authors propose that emerin “promotes the formation of a nuclear actin cortical network,” which could serve to anchor membrane proteins and lamin filaments to the inner nuclear membrane and thus enhance the structural integrity of the nuclear envelope. Whether emerin also interconnects the lamin and actin filament networks at the nuclear envelope—which could significantly reinforce its mechanical strength—will have to await further study.Muscle contraction places enormous stress on cell membranes. These results suggest that actin-based networks, in addition to lamin networks, support the structural integrity of the nuclear envelope. Defects in proteins involved in either network could compromise nuclear structure, which could in turn disrupt the cell's gene expression program, for example, or rupture the cell membrane, killing the cell. Subtle defects in proteins important for muscle cell integrity can cause several forms of muscular dystrophy. Now it appears that emerin defects could cause EDMD in part by compromising the mechanical integrity of nuclei in muscle cells and tendons.


In the mouse zygote the paternal genome undergoes dramatic structural and epigenetic changes. Chromosomes are decondensed, protamines replaced by histones and DNA is rapidly and actively demethylated. The epigenetic asymmetry between parental genomes remains at least until the 2-cell stage suggesting functional differences between paternal and maternal genomes during early cleavage stages.Here we analyzed the timing of histone deposition on the paternal pronucleus and the dynamics of histone H3 methylation  immediately after fertilization. Whereas maternal chromatin maintains all types of histone H3 methylation throughout the zygotic development, paternal chromosomes acquire new and unmodified histones shortly after fertilization. In the following hours we observe a gradual increase in H3/K4mono-methylation whereas H3/K4tri-methylation is not present before latest pronuclear stages. Histone H3/K9di-methylation is completely absent from the paternal pronucleus, including metaphase chromosomes of the first mitotic stage.Parallel to the epigenetic asymmetry in DNA methylation, chromatin modifications are also different between both parental genomes in the very first hours post fertilization. Whereas methylation at H3/K4 gradually becomes similar between both genomes, H3/K9 methylation remains asymmetric. It is now generally accepted that the properties of a particular DNA sequence in cells are not solely defined by the nucleotide sequence itself, but by "epigenetic" modifications as well. Epigenetic modifications imply the methylation of cytosine residues in CpG dinucleotides and covalent modifications of core histones. These modifications allow for flexible, but heritable at the same time, reprogramming of the genome.In histone H3 five lysine residues can be methylated  . MethylaThe fertilization of mouse egg causes dramatic changes in organization of both paternal and maternal genomes. Initially arrested in metaphase II oocyte completes the meiosis, forming haploid maternal pronucleus and extruding the second polar body. The densely packed with protamines sperm DNA decondences, protamines get exchanged by histones and DNA undergoes active demethylation. The demethylation in the early mouse zygote occurs asymmetrically on paternal DNA and affects different classes of repetitive and single copy sequences, but not the control regions of imprinted genes ,8. PreviHere we examine time dependent changes of chromatin structure in the mouse zygote, focusing on the dynamics of the acquisition of histones in the paternal pronucleus and methylation status of histone H3 at positions K4 and K9.in vitro fertilization of mature mouse oocytes. Histones and methylated H3/K4 and H3/K9 were detected by using indirect immunofluorescence. In our experiments we used antibodies, which specifically recognize mono- or tri-methylated forms of H3/K4, and di-methylated H3/K9. The zygotes were analyzed after 3, 5, 8, 10, 12 and 18 hours incubation of mature oocytes with capacitated sperm from donor males. After 18 hours most of embryos were found to be at metaphase and some already at telophase stage of the first mitotic division. Even using in vitro fertilization, the obtained zygotes are not completely synchronous in their development and it is more appropriate to use PN stages classification, which is based on the morphological changes of both pronuclei [In order to obtain mouse zygotes at different stages of development and to provide the precise timing for fertilization we used 0/early PN1 stages (approx. 3–5 hours p.f.), exactly when the global demethylation starts [We performed the immunostaining against core histones (anti-PanHistone antibodies) in all the stages tested in combination with antibodies, recognizing the specific methylated forms of histone H3. This served as a positive control for the immunostaining procedure and allowed us to follow the dynamics of histone acquisition in the paternal pronucleus. Histones were first detected shortly after the penetration of sperm into the oocyte and the beginning of the decondensation of sperm chromatin. According to PN stages classification we could clearly detect histones on paternal pronucleus at late PNn starts  Fig. 1)0/early P1 (approx. 5 hours p.f.) stage  and the difference in antibodies staining intensity between paternal and maternal pronuclei becomes indistinguishable in the last pronuclear stage PN5 (approx 12 hours p.f.)  Fig.  and in mf.) Fig. . The facet al. suggest that H3/K9 methylation does not depend on DNA methylation [et al. [1 stage, i.e. at a time when we first observe the appearance of H3/K4mono-methylation  procedures were performed as described in [2 incubator for 3, 5, or 8 hours. For longer incubation time the oocytes were incubated with sperm in HTF medium for 8 hours and then transferred into the drop of pre-gasses and pre-warmed M16 medium and incubated further for 2, 4 or 10 hours.As sperm and oocytes donors we used (C57BL/6 X CBA)Focedures . Sperm iribed in . BrieflyAfter the removal of zona pellucida by treatment with Acidic Tyrode's solution fertilized oocytes were fixed for 20 min in 3.7% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. The fixed zygotes were blocked overnight at 4°C in 1% BSA, 0.1% Triton X-100 in PBS. After blocking the embryos were incubated in the same solution with either anti PanHistones , anti mono-methyl H3/K4 , anti tri-methyl H3/K4  or anti di-methyl H3/K9 , and anti-rabbit secondary antibodies coupled with Rhodamine Red-X (Jackson ImmunoResearch Laboratories Inc.). After final washes the zygotes were placed on slides and mounted with a small drop of Vectashield (VectorLab) mounting medium containing 0.5 μg 4,6-diamino-2-phenylindole (DAPI). At least 20 zygotes have been analyzed for each stage of zygotic development.The slides were analyzed on Zeiss Axiovert 200 M inverted microscope equipped with the fluorescence module and B/W digital camera for imaging. The images were captured, pseudocoloured and merged using AxioVision software (Zeiss).KL conducted the experimental part of the work and co-wrote the manuscript. JW coordinated the study and co-wrote the manuscript. All authors read and approved the final manuscript.


On the Origin of Species—the remains were initially described as either those of a “brutish” race or of someone disfigured by disease. As Darwinian evolution caught on, so did the realization that these fossils were evidence of an earlier human species. Scientists have been debating Neanderthal's place in human evolution ever since.After miners unearthed a skull and bones in a Neander Valley cave in Germany in 1856—three years before the publication of An ongoing question concerns the possibility that Neanderthals and early humans mated, since they likely crossed paths during thousands of years of European cohabitation. In a new study, Mathias Currat and Laurent Excoffier present a simulation model based on what we know about the population density and distribution of Neanderthals and Cro-Magnons. Their results complement recent genetic and morphological evidence indicating that early human and Neanderthal interbreeding was unlikely.The notion that modern Europeans directly descended from Neanderthals has mostly yielded to two competing models: One postulates that modern humans arose in Africa about 130,000 years ago and completely replaced coexisting archaic forms with no interbreeding, while the other proposes a gradual transition with interbreeding.Though mounting genetic evidence  suggests Neanderthals and early humans did not breed, the evidence has been inconclusive. It's possible, for example, that any Neanderthal gene “leakage” could have been lost through genetic drift if the mating populations were small. And because so few fossils are available to analyze, previous studies could rule out only Neanderthal contributions over 25%.Currat and Excoffier's model refines various parameters—such as geographic boundaries, local population variations, range expansion, and competition for resources—based on archeological and demographic data for both populations. Evidence suggests modern humans replaced Neanderthals over 12,500 years, for example, which constrains the speed at which modern humans could expand.The authors started with a scenario based on a set of “plausible” parameter values—their basic scenario—and then varied the local interbreeding rate and, for example, the population size and location of Cro-Magnons, to produce eight alternate scenarios describing how Cro-Magnon colonization of Europe might have proceeded. They estimated the likely proportion of Neanderthal gene contributions to the modern gene pool using “coalescent simulations,” which generate the genealogies and diversity of genes in local populations based on simulations of their population densities and migration histories. The simulations show that if Neanderthals bred with Cro-Magnons without constraints over thousands of years, Neanderthal contributions to the modern gene pool “would be immense.” Surprisingly, the simulations also show that even a very small mixing should lead to high levels of Neanderthal DNA in modern humans.What could account for this counterintuitive result? Given a low population density with small local breeding populations, any introduction of Neanderthal genes would decrease the frequency of Cro-Magnon genes of that population; if these Neanderthal integrations take place as the Cro-Magnon population is expanding, newly acquired Neanderthal genes would also be amplifiedSince no Neanderthal mitochondrial DNA has been found in modern-day Europeans, the authors modeled the maximum number of interbreeding events that would support this observation. The estimated maximum number of events, it turns out, falls between 34 and 120—extremely low values, Currat and Excoffier conclude, “given the fact that the two populations must have coexisted for more than 12,000 years.”While the authors acknowledge their simulations suggest rather than reflect reality, their model does incorporate real historical data such as Cro-Magnon expansion over time and local population growth. At a value of only 0.1%, their new estimate of the rate of interbreeding is about 400 times lower than previous estimates and provides strong support that Neanderthals and Cro-Magnon didn't interbreed and may even have been different species.


There are already 40 cities in Africa with over 1 million inhabitants and the United Nations Environmental Programme estimates that by 2025 over 800 million people will live in urban areas. Recognizing that malaria control can improve the health of the vulnerable and remove a major obstacle to their economic development, the Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the Systemwide Initiative on Malaria and Agriculture convened a multi-sectoral technical consultation on urban malaria in Pretoria, South Africa from 2nd to 4th December, 2004. The aim of the meeting was to identify strategies for the assessment and control of urban malaria. This commentary reflects the discussions held during the meeting and aims to inform researchers and policy makers of the potential for containing and reversing the emerging problem of urban malaria. Anopheles mosquito breeding decreases with increasing proximity to the centre of urban areas [Africa's population will almost triple by the year 2050. This expansion will occur primarily in urban areas and by 2025, 800 million people will live in urban communities. Especially affected will be West Africa, where the urban population annual growth rate of 6.3% is more than twice the rate of the total population growth. Today in the humid forest zone, more people live in cities than in rural areas and in twenty years time, two out of three West Africans will live in urban centres. While many of Africa's health problems are common to both urban and rural environments, recognizing and meeting the public health challenges in these growing cities is becoming increasingly urgent. Malaria has been considered a predominantly rural disease in Africa, primarily because suitable vector breeding sites are scarce in highly populated areas. Yet, although studies have shown that an areas -3, transan areas ,5.nd-4th December 2004 to develop an evidence-based approach for evaluating and controlling urban malaria. Participants were drawn from seven sub-Saharan countries, Europe, North America and South Asia . Recognizing the need for extensive cross-sectoral involvement and collaboration in dealing with the challenge of urban malaria, representatives from the research/ academic, NGO, development, policy-making and donor communities co-operated in the process to identify key knowledge gaps and opportunities for control. Included in the group were sociologists, clinical epidemiologists, entomologists and control specialists.The Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the International Water Management Institute/ Systemwide Initiative on Malaria and Agriculture convened a meeting in Pretoria 2Urbanization is a recent phenomenon in Africa: in 1960 there were no African cities with one million inhabitants, today there are forty. Has malaria become a serious problem within these huge cities and their peri-urban environs? Data presented from studies in a number of sub-Saharan African cities  showed clearly that malaria is a considerable urban health problem in Africa. The studies demonstrated great heterogeneities in malariometric indices both between and within cities. It was recognized that not only the major cities of Africa, but also many medium sized regional towns, home to a large proportion of the Africa population, have considerable levels of malaria . With maMisdiagnosis of malaria is a serious problem everywhere, but in areas of low malaria endemicity presumptive treatment of all fevers as malaria can result in over 75% of cases being misdiagnosed as malaria [Effective provision of appropriate treatment also remains a serious challenge in urban settings. The Abuja Declaration stated that by 2005 "At least 60% of those suffering from malaria have prompt access to and are able to use correct, affordable and appropriate treatment within 24 hours of onset of symptoms." Despite the fact that access to quality health care is better on average in urban compared to rural zones, the formal public health facilities are often the last source of treatment used along the pathway to cure. Often malaria care initially involves leftover medicines from the home , the purchase of cheaper herbal medicines or unprescribed conventional medicines. The problems of obtaining treatment from a health facility may be exacerbated by the need to obtain permission from an authority figure, absence from work and loss of income, the need to raise money to fund both the treatment and associated costs such as travel . With HoThe highly focal nature of urban malaria requires targeting of interventions to specific urban districts, and therefore, requires detailed information on each area in advance. However, relationships between administrative boundaries, environment and population distribution are complex in urban areas, which makes them difficult to sample and characterize in a representative way. Strategies for population-representative sampling must incorporate a range of environments and populations to identify accurately environmental and other risk factors. This may be further complicated as urban populations can be highly mobile and in peri-urban areas there may be a high rate of turnover in groups of lower socio-economic status. Presentations from South, East and West Africa clearly demonstrated that Geographic Information Systems (GIS)-based approaches are valuable tools for assessing heterogeneities in risk factors for urban malaria, and for subsequent implementation and monitoring of interventions.Experienced researchers believe that the urban environment has advantages for the effective delivery of appropriate interventions. A number of studies have demonstrated that higher rates of coverage with insecticide-treated bednets can be achieved in urban areas ,11, althLarval control, achieved either by source reduction or larviciding, can be community directed and may be feasible in certain settings as part of a comprehensive, integrated vector management strategy. There is optimism in some communities about its efficacy and the results of further research into the costs and benefits of such interventions are awaited with interest. Environmental modifications may also be feasible if partners from the community and outside the health sector are engaged. Work from Sri Lanka has demonstrated how a very effective scheme to control malaria by modification of irrigation structures was accepted by the agricultural community because of the financial and water savings that the scheme introduced . HoweverThe conclusions of the meeting have been summarised in the Pretoria Statement on Urban Malaria Figure . While iAll authors participated as session chairs in the technical workshop and were instrumental in producing the summary conclusions. All authors read and approved the final manuscript.List of attendees and affiliationsClick here for file


The same molecular qualities that endowed DNA with its capacity to carry hereditary information make it a powerful tool to explore the self-assembly of complex nanostructures How did life emerge from a soup of chemicals? How do patterns such as schools of fish form from individuals? How do voting patterns emerge? Such a diverse array of problems seems completely unrelated. However, they all involve “emergence of complexity.” When individuals come together, they form patterns, structures, and organizations that cannot be discerned from the individuals alone. The study of the emergence of complexity is one of the most active and important areas of research. It is important not only for understanding nature, but also for technological applications, including the fabrication of large-scale integrated nanocircuits using a bottom-up approach, and the preparation of multifunctional “smart” nanomaterials.DNA evolved to be the primary carrier of genetic information because of its extraordinary chemical properties. These same properties also make DNA an excellent system for the study of self-assembly and self-organization. Two complementary molecules of single-stranded DNA have paired bases that bond with each other and form the well-known double helix structure. Two molecules of double-stranded DNA (duplexes) can further associate together if they have complementary single-stranded overhangs (sticky ends). Intermolecular interactions can be precisely predicted by Watson–Crick basepairing (adenine to thymidine and guanine to cytosine). And, these interactions are structurally understood at the atomic level. Given the diversity of the DNA sequences, we can easily engineer a large number of pairs of DNA duplexes that associate with each other with sequence specificity and in a well-defined fashion. This property is not common among other molecular systems. Small organic and inorganic molecular pairs can interact with each other with specificity and in well-defined structures, but the number of such pairs is limited and their chemistry varies greatly. Protein molecules, such as antibody–antigen pairs, have great diversity and high specificity. However, it is extremely difficult, if not impossible, to predict how proteins interact with each other. In contrast, DNA as a molecular system fulfills all the aforementioned criteria.In nature, DNA occurs predominantly as a linear molecule, and if its conformations were limited to linearity, it would not be very useful for studying self-assembly. Fortunately, branched DNA structures can be engineered. Holliday junctions, for example, are intermediates that occur during genetic recombination. To model Holliday junctions, a stable four-arm junction has been constructed in which, by design, no two strands are fully complementary to each other  Figure A. For exExtensive studies have shown that the four-arm junction adopts an X-shape structure B under pOne simple example of self-assembly is the formation of two-dimensional (2D) periodic arrays or 2D crystals. This is also one of the greatest successes in the field of DNA self-assembly . The firi molecule takes the input from the Xi and Yi−1 molecules. In other words, the values of the Xi and Yi−1 molecules determine what Y molecule will be incorporated. There are four different types of Y molecules, whose inputs are , , , and . These four, and only four, Y molecules are enough to satisfy any input combination. Two C molecules connect the input and output molecules, which is necessary for the characterization but not essential for the self-assembly process. Sticky ends between the X molecules and the C molecules are longer than those between Y molecules and between Y and X or C molecules. Thus, the C and X molecules assemble first to form the inputs because the association between longer sticky ends is more stable than those between the shorter ones. Then the output Y molecules assemble to the assembled C and X molecules. In that study (Inspired by early theoretical suggestions , experimat study , two difPLoS Biology have extended the algorithmic self-assembly strategy from 1D to 2D (Winfree and co-workers in this issue of 1D to 2D . This ac1D to 2D . ExternaIn principle, a wide range of 2D patterns could be generated with the same set of molecules and the same strategy, changing only the first row of the assembly, which specifies the external inputs. Realization of this goal will critically rely on the elimination of assembly errors, or the introduction of error corrections .The current work represents a neat approach to understanding the emergence of complexity. It integrates both simulation and wet chemistry. It also provides a plausible approach to nanofabrications. Over the last decade, a variety of methods have been developed, which use biomacromolecules as templates to fabricate nanostructures . Limited


Severe acute respiratory syndrome (SARS) is a new infectious disease caused by a novel coronavirus that leads to deleterious pulmonary pathological features. Due to its high morbidity and mortality and widespread occurrence, SARS has evolved as an important respiratory disease which may be encountered everywhere in the world. The virus was identified as the causative agent of SARS due to the efforts of a WHO-led laboratory network. The potential mutability of the SARS-CoV genome may lead to new SARS outbreaks and several regions of the viral genomes open reading frames have been identified which may contribute to the severe virulence of the virus. With regard to the pathogenesis of SARS, several mechanisms involving both direct effects on target cells and indirect effects via the immune system may exist. Vaccination would offer the most attractive approach to prevent new epidemics of SARS, but the development of vaccines is difficult due to missing data on the role of immune system-virus interactions and the potential mutability of the virus. Even in a situation of no new infections, SARS remains a major health hazard, as new epidemics may arise. Therefore, further experimental and clinical research is required to control the disease. Severe acute respiratory syndrome (SARS) is the first new infectious disease of this millennium. SARS has originated from Southern China at the end of 2002 and has a high mortality and morbidity. Within a period of six months beginning at the end of 2002, the disease has affected more than 8,000 people and killed nearly 800 . The disSARS is caused by a novel, SARS-associated coronavirus (SARS-CoV) -6 which Shortly after the initial global alert, the WHO initiated a collaborative multi-center research project on SARS diagnosis, led by eleven principal laboratories in nine countries . Using mParallel to the progress made in the epidemiology and clinical diagnosis which has recently been demonstrated by numerous case reports, clinical studies and definitions , scientiWhile other reviews have focused on the epidemiology, clinical presentation and potential treatment of SARS, the present overview aims to analyze and present the currently available data on molecular mechanisms of SARS. In this respect, it is important to underline that in the present state of no specific drug or vaccine being available, research on molecular mechanism is crucial to identify potential treatment targets.Prior to the development of therapeutic regimes based on molecular mechanisms of the disease, the causative agent had to be isolated and analysed. Soon after the fast establishment of the international WHO laboratory network, rapid progress was made in the identification process of the causative agent, and it was reported that SARS is most probably caused by a novel strain of the family of coronaviruses -6. TheseSoon after the identification of a new coronavirus as the causative agent of SARS and of a southern Chinese province as the first area of occurrence, animal species of this area have been speculated to be the origin of the SARS-CoV. As analysis of the SARS-CoV genetic sequence revealed large differences to any other currently known coronaviruses in humans or domestic animals ,17, it wUntil the identification of the new SARS-CoV, the coronaviruses have been divided into three subgroups, which differ with respect to their genome . The firDespite the fact that this new virus most likely jumped to humans from wild animal species, it has remarkably well adapted to the human organism as shown by its high person-to-person transmissibility.The structure of the SARS viral RNA is organized in 13–15 open reading frames (ORF) and contains a total of approximately 30,000 nucleotides ,16,17.Recently, 61 SARS-CoV sequences derived from the early, middle, and late phases of the SARS epidemic together with two viral sequences from palm civets were analyzed . GenotypOnly ORFs exceeding fifty amino acids in translational capacity are considered relevant as they contain the sequences for the structural and functional properties of the virus and are therefore of potential interest for the development for future therapeutic strategies. The comparison of the different SARS-CoV ORFs with those of other coronaviruses reveals a familiar pattern of structural gene arrangement with replicase and protease genes (gene 1a-1b) and the spike (S), envelope (E), membrane (M) and nucleocapsid (N) genes in a typical 5'- to 3' order of appearance . The propro, one of the two papain-like proteinases operating on cleavage sites at the N-terminus of the polyproteins , also called 3C-like protease (3CLpro), is responsible for the cleavage of all the remaining proteins encoded by gene 1a-1b [Two-thirds of the SARS RNA is organized in the gene 1a-1b. The sequence of this gene is highly conserved among all coronaviruses . ORFs 1as Figure . The maine 1a-1b ,30.Apart from gene 1, coronavirus genes are known to be usually expressed from subgenomic mRNAs. They share a common leader sequence at the 5'-end and initiate at different places in the genome extending toward the 3'-end of the virus genome . Some ORpro and Mpro) were addressed as they are involved in replication, transcription or post-translational polyprotein processing [So far, five to eight subgenomic mRNAs were found in SARS-CoV-infected cells ,27. Thieocessing .In conclusion, research in the area of coronavirus gene expression is important to delineate components which directly affect SARS-CoV virulence.The structural proteins of the new SARS-CoV are potential targets for new treatment options. The new SARS-CoV only contains the three envelope proteins, spike (S), envelope (E), and membrane (M) but not the hemagglutinin-esterase (HE) protein, which is present in some coronaviruses of the second group.The spike glycoprotein is responsible for the characteristic spikes of the SARS-CoV Figure . Intra- The SARS-CoV has also been reported to cause the formation of syncytia in vivo, but so far only under the condition of cultured Vero cells . The SARAn interesting feature of the SARS-CoV and other coronaviruses is the resistance against the gastrointestinal fluids despite the lipid composition of their envelope. It has been reported that the SARS-CoV can survive in diarrheal stool for four days and also, patients with SARS often suffer from gastrointestinal symptoms with the virus to be detected in the stool . As the -4 for RNA-dependent RNA polymerases [It is unclear when and how novel pathogens such as the SARS-CoV cross the barriers between their natural reservoirs and human populations, leading to the epidemic spread of novel infectious diseases . As withymerases , RNA virymerases . This obymerases .Next to mutations, a further threat of the SARS-CoV is based on the ability of coronaviruses to undergo RNA recombination at a high frequency . For a vAlthough human coronaviruses are characteristically causing self-limiting short diseases, the question of potential chronic SARS infections is of major importance for a future disease control. If the SARS-CoV is able to cause a chronic persistent infection, chronic carriers may serve as sources for new SARS outbreaks. However, the detection of SARS-CoV in stool of patients for longer periods than 6 weeks after hospital discharge has not been reported so far. Therefore, the danger of chronic carriers may not be relevant. In contrast to common human coronavirus infections with short durations, most animal coronaviruses cause persistent infections. As an example, the feline coronavirus FIPV infects animals which then continue to shed virus for periods reaching up to seven months after infection without carrying disease symptoms . Also, TThe mean incubation period of SARS was estimated to be 6.4 days . The mean reported time from the onset of clinical symptoms to the hospital admission varied between three and five days .Main clinical features of the disease are in the initial period common symptoms such as persistent fever, myalgia, chills, dry cough, dizziness, and headache. Further, although less common symptoms are sore throat, sputum production, coryza, vomiting or nausea, and diarrhea ,51. SpecThe clinical course of the disease seems to follow a bi- or triphasic pattern. In the first phase viral replication and an increasing viral load, fever, myalgia, and other systemic symptoms can be found. These symptoms generally improve after a few days. In the second phase representing an immunopathologic imbalance, major clinical findings are oxygen desaturation, a recurrence of fever, and clinical and radiological progression of acute pneumonia. This second phase is concomitant with a fall in the viral load. The majority of patients is known to respond in the second phase to treatment. However, about 20% of patients may progress to the third and critical phase. This phase is characterized by the development of an acute respiratory distress syndrome (ARDS) commonly necessitating mechanical ventilation.Rapid progress has been made in understanding the clinical presentation of SARS in adults and children -56. In cThe course of the disease in teenagers more likely resembles adults in concerning clinical presentation and disease progression . SARS maCoronaviruses are known to exert their effects by cytocidal and immune-mediated mechanisms. In vitro studies using cell culture assays have shown that coronavirus infection commonly results in cytopathic effects such as cellular lysis or apoptosis . Also, tNext to cytocidal effects, also immune-mediated mechanisms of both the innate and adaptive immune system seem to contribute to the pathogenesis of SARS-CoV infections. In this respect, it has been shown that in MHV infection, T cells and cytokines play an important role in development of the disease . Also, hFor SARS-CoV infections, it has been reported that there seems to be an inflammatory cell influx consisting in particular of macrophages in the airways, and a massive release of cytokines during the peak of the infection ,68. It iThe pharmacotherapy for SARS with anti-inflammatory steroids is controversial and largely anecdotal . It was The most obvious gene which is likely to be a key modifier of SARS pathomechanisms is the spike (S) protein gene. As known for other coronaviruses, it does not only affect viral pathogenesis by determining the target cell specificity but also by other mechanisms. In this respect, a single mutation in the S gene of MHV has significant effects on the viral virulence and tissue tropism . Also, mThe primary target cells of SARS-CoV infection are respiratory epithelial cells. As the virus can also be detected in stool specimen and patients with SARS often also have gastrointestinal symptoms, epithelial cells of the gastrointestinal tract also seem to be major target cells. Next to these epithelial cells, the SARS-CoV has also been found in macrophages and many other cells as it has been detected in not only in the respiratory tract and stool specimen but also in the blood, liver, kidney and urine . In thisThe SARS-CoV target cell specificity is determined by the spike protein affinity to cellular receptors. In contrast to the all group III coronaviruses and the SARS-CoV for which the receptors have not been finally analyzed, it is known that group I coronaviruses bind to aminopeptidase N (CD13) as receptors , while gRecently, it was shown that a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), efficiently binds the S1 domain of the SARS-CoV S protein. SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Also, anti-ACE2 but not anti-ACE1 antibodies blocked viral replication on Vero E6 cells, indicating that ACE2 is a functional receptor for SARS-CoV  which waRecently, the C-type lectin CD209L  was discovered to be a further human cellular glycoprotein that can serve as an alternative receptor for SARS-CoV . The intAfter binding to the receptor, the next molecular step of potential use for the development of anti-SARS drugs is the virus entry into the cells. While most coronaviruses enter their target cells via plasma membrane fusion, a further entry mechanism may be acidic pH-dependent endocytosis . FocusinAfter the binding to a host cell receptor and entry into the cells, the molecular steps of transcription, translation and protein processing display further potential targets for new therapeutic strategies. In this respect, the RNA-dependent RNA polymerases (SARS-CoV RdRp) may be a potential target for a future anti-SARS therapy. A recent study located its conserved motifs and built a three-dimensional model of the catalytic domain . The autpro) or the 3C-like proteinase (3CLpro), and two auxiliary, papain-like proteinases (PL1pro and PL2pro). The latter two are responsible for the cleavage of the viral polyproteins, pp1a and pp1ab, at three sites near the amino-terminus, while the Mpro processes these proteins at as many as 11 additional sites. Interestingly, SARS-CoV lacks the PL1pro [pro [pro on PL1pro cleavage sites has been shown in IBV and HCoV [pro gene in other coronavirus genomes, SARS-CoV displays a domain within ORF1a that lacks any detectable sequence homology and has therefore been named the SARS-unique domain (SUD) [Also, protease activity is crucial for SARS-CoV RNA replication and protein processing ,85, and e PL1pro ,17, but pro [pro . This isand HCoV . Roughlyin (SUD) . It is npros of TGEV [pro in the crystalline state. The X-ray structure of the complex revealed binding of the P1 glutamine, P2 leucine, and P4 threonine side chains of this compound to the respective subsites in the substrate-binding cleft, in agreement with the pronounced specificity for cleavage by the Mpro after the substrate sequence -Xaa-Leu-Gln. The structure also showed the expected covalent attachment of the methyl ketone group at P1 of the inhibitor to the catalytic cysteine of the enzyme.Crystal structures have been determined for the M of TGEV , HCoV 22 of TGEV , and, mo of TGEV . They al of TGEV ,87,88. T of TGEV  have synpros of HCoV 229E and TGEV, the crystal structure of the SARS-CoV Mpro revealed some surprises [pro from the viral polyprotein, which probably involves a pH-dependent step.In spite of 40% and 44% sequence identity, respectively, to the Murprises . Within pro was employed by Yang et al. [The same hexapeptidyl chloromethylketone inhibitor used by Anand et al.  in theirg et al.  to charapro resembled that of AG7088 in complex with its target, the 3C proteinase of human rhinovirus [On the basis of their crystallographic work, Anand et al.  found thinovirus , even thinovirus  thereforinovirus .pro and screened 29 approved and experimental drugs against a model of the SARS CoV proteinase as well as the experimental structure of the transmissible gastroenteritis virus (TGEV) proteinase [Other studies used molecular dynamics simulations of the Moteinase . It was oteinase . HoweverFurther potential targets are the E and M proteins Figure  as they With regard to the multitude of potential epithelial target cells, specific endogenous drug delivery systems may also be of relevance. In this respect, the family of peptide transporters consisting of PEPT1 and PEPT2 which are differentially expressed in potentially infected cells of the respiratory tract ,95, smalAs most patients develop an immunity against the SARS-CoV and survive the infection, the possibility of creating an effective and safe vaccine seems to exist . There aLive-attenuated coronavirus vaccines can be generated by deletions in "group-specific genes". The deletions of these genes do not change replication properties but attenuate the virus . ExampleWhole killed vaccines are generally safe and easy to generate. In fact, this technique has been applied in veterinary medicine to generate vaccines for BoCV and IBV . Also, aUsing molecular biology techniques to generate large quantities of recombinant viral proteins, recombinant subunit vaccines, e.g. against the spike protein, are expected to be created relatively easy as shown by two recent studies ,117. EigAn approach using recombinant vectored vaccines with DNA or a viral vector could be a promising target. The DNA prime and adenovirus or MVA boost approach which is currently analysed for a potential use in the development of HIV vaccines, may also offer a strategy to prevent SARS infections. In this respect, a multi-valent approach which induces both humoral and T cell-mediated host responses seems to be the most attractive strategy.From the field of veterinary medicine, data on this approach are already available: A recombinant fowlpox with the S1 gene of IBV was demonstrated to be relatively protective against IBV . Also, aA further strategy is based on the use of epitopes which can be delivered using viral or DNA vectors. Such an epitope-based strategy for coronavirus vaccination has already been reported  and the In summary, the important development of SARS vaccines can be approached using several techniques which should ideally encompass the induction of both humoral and cell-mediated mechanisms. As coronavirus vaccines in animals have partly been reported to cause an enhancement of viral infections , a cautiIn summary, the onset of the SARS epidemic in different continents has led to the formation of a successful laboratory network to identify the molecular mechanisms underlying the SARS infection. Next to the development of early diagnostic tests and effective treatment strategies, it is most important to orchestrate research activities which lead to the development of vaccines and antiviral agents, as there is no established therapy to date. Even now in a situation of only a handful of new cases, SARS remains a major global health hazard which may reappear.


Tula hantavirus carrying recombinant S RNA segment (recTULV) grew in a cell culture to the same titers as the original cell adapted variant but presented no real match to the parental virus. Our data showed that the lower competitiveness of recTULV could not be increased by pre-passaging in the cell culture. Nevertheless, the recombinant virus was able to survive in the presence of the parental virus during five consecutive passages. The observed survival time seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature. Recombination in RNA viruses serves two main purposes: (i) it generates and spreads advantageous genetic combinations; and (ii) it counters the deleterious effect of mutations that, due to the low fidelity of viral RNA polymerases and lack of proofreading, occur with high frequency . The purHantavirus, family Bunyaviridae) have a tripartite genome comprising the L segment encoding the RNA-polymerase, the M segment encoding two external glycoproteins, and the S segment encoding the nucleocapsid (N) protein [et al [Hantaviruses . These data confirmed our earlier observation [in situ formed recombinant S RNA disappeared from the mixture after four passages [First, we designed RT-PCR primers able to discriminate between non-recombinant (V-type) and recombinant (REC-type) types of TULV S RNA. The resullts presented in Fig. ervation  that thepassages , one shoin vivo situation, from one rodent to another. If transmission is performed in a sampling-like fashion – and this seems to be the case for hantaviruses [Although relatively short, the observed survival time of the recTULV in the presence of the original variant TUL02 seems to be sufficient for transmission of a recombinant virus, in a hypothetical aviruses  – the reaviruses  supportsaviruses ,21.The genetic swarm of S RNA molecules from the recTULV is represented almost exclusively by the variant with a single break point located between nt332 and nt368. The proportion of the dominant variant is larger in the passaged recTULV  than in the freshly formed mixture of recS RNAs  . Thus, rThe data presented in this paper show that the recTULV presents no real match to the original cell adapted variant and that the lower fitness of the recombinant virus can not be increased by pre-passaging in cell culture. The observed survival time of the recTULV in the presence of the parental virus seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature.3 – 104 FFU/ml.RecTULV (clone 5) was purified from the mixture it formed with the original variant, TULV02, using two consequent passages under terminal dilutions . After t6 cells) were infected with the 1:1 mixture of recTULV and TULV02, approximately 104 FFU altogether. After 7–12 days the supernatant (~20 ml) was collected and RNA was extracted from the cells with TriPure™ isolation reagent, Boehringer Mannheim. Aliquots (2 ml) of the supernatant were used to infect fresh cells; the rest was kept at -70°C. The following nine passages were performed in the same way.Vero E6 cells (5 × 10RT was performed with MuLV reverse transcriptase (New England Biolabs); for PCR, AmpliTaq DNA polymerase  was used. To monitor the presence of TULV S RNA on passages, RT-PCR was performed with primers VF738  and VR855 . To monitor the presence of recTULV S RNA, RT-PCR was performed with primers RECF738  and RECR855 . All four primers were perfect matches to the homologous sequences; to the heterologous sequences, the forward primers have five mismatches while the reverse primers have six. Alternatively, complete S segment sequences of both variants of TULV were amplified using a single universal primer  and thenThe author(s) declare that they have no competing interests.AngP participated in the design of the study, carried out the experiments and helped to draft the manuscript. AlexP participated in the design of the study and drafted the manuscript. Both authors read and approved the final manuscript.


Do you want to know about evolution? Brian and Deborah Charlesworth provide an excellent and concise account of the core issues for a broad range of readers Evolution: A Very Short Introduction, is no mean feat.Their book is one of a series of short introductions, published by Oxford University Press, covering an eclectic array of subjects that aim to provide an accessible yet stimulating read for anyone wanting a thorough introduction to a topic. In this small volume, the Charlesworths have succeeded on both fronts and provide an excellent account of the core issues for a broad range of readers. One of the reasons for the book's appeal is that the authors draw on a range of carefully chosen human traits to illustrate their points. By contrast, most evolutionary textbooks (other than those purely on human evolution) tend to focus on nonhuman organisms. As with traits in every other organism, many human and human-related characteristics have evolved via genetic drift and natural selection, and they provide an effective means of convincing readers of the reality and relevance of evolution. For example, to explain how mutation can cause the loss of a function, the authors discuss the relatively poor sense of smell in humans, as compared with many other mammals, using an example of a vestigial ‘pseudogene’ of a human olfactory receptor gene. They also discuss tooth decay, enzyme aesthetics, heritable differences, cancer and other diseases, and the ability to taste and so on.Although the topics the Charlesworths choose to focus on are certainly appropriate, they provide only a brief mention of one important process—development. Evolutionary developmental biology is a burgeoning field that can provide interesting and important insights into our understanding of the mechanisms of evolution. For example, the absence of eyes in cavefish, rather than being the result of a degenerative process, might be the result of selection on genes that govern feeding morphology, a selection process that has included suppression of eye development . Such deIn Chapter 7, the authors discuss five topics that have traditionally been hard to understand from an evolutionary point of view. These ‘difficult problems’ are ageing, altruism, human consciousness, complex adaptations, and the origin of living cells. Difficult problems can be interpreted in two ways: those that, although hard to solve, have either been explained or will eventually be explained by modern evolutionary theories, and those problems that cannot be fully resolved with our current understanding but leave room for learning about additional mechanisms or factors. The Charlesworths generally consider only those problems of the former type—the explained ones. However, I think that some of the more intractable problems should be described in more detail. For instance, complex adaptation might be fully explained by mutations and natural selection, but additional unknown mechanisms might be essential for the evolution of the complex traits. I realize that opponents of modern evolutionary theory, such as creationists, have often cited these traditional problems to support their conclusion that modern evolutionary theory is wrong; but progress always depends on the consideration of new ideas, and there might be important mechanisms still to be discovered that play a key role in evolution. Describing potentially intractable problems might also spur on young readers who are thinking of studying evolutionary biology with the hope that there are still some theoretical battles to be conquered.The Blind Watchmaker by Richard Who is the target audience of this book? For many books, the topics chosen and the writing style can perhaps provide clues to the nature of the readers. For instance, In Japan, there seem to be few people who deny the facts of evolution, although there are many ideologically motivated books opposing natural selection and Darwinism. To convince creationists of evolution is usually extremely difficult, if not impossible, because they will never doubt their assumption that God created humankind. Education of young and curious people, however, can make a difference. This is where I think the book will be most successful, but this book should not just be limited to young people—I can recommend it to anyone who wants to know about evolution. Moreover, I can recommend it to Japanese students not only as an introduction to evolution, but also as an exercise in reading a well-written and engaging English text.


Immunotherapy in patients with Alzheimer's disease (AD) is rapidly becoming a hot topic of modern geriatric and clinical gerontology. Current views see immunization with Aβ peptide, the amyloidogenic protein found in senile plaque of AD patient's brains, or the infusion of preformed antibody specific for human Aβ, as possible therapeutic approaches to improve the cognitive status in the disease. Animal models of the disease have provided positive results from both approaches. Thus, an initial clinical trial using immunization with human Aβ in AD patients was started, but then shortly halted because of an unusually high incidence (6%) of meningoencephalitis. A long and currently ongoing debate in the scientific community about the pro or contra of vaccination or passive immunization with Aβ in AD is thereafter started. Here, the authors would like to stress few points of concern regarding these approaches in clinical practice. Immunotherapy in patients with Alzheimer's disease (AD) is rapidly becoming a hot topic of modern geriatric and clinical gerontology. M.E. Weksler  in the aCurrent views see immunization with Aβ peptide, the amyloidogenic protein found in senile plaque of AD patient's brains, or the infusion of preformed antibody specific for human Aβ, as possible therapeutic approaches to improve the cognitive status in the disease.Animal models of the disease have provided positive results from both approaches, since either vaccination with human Aβ or infusion of preformed antibodies specific for Aβ, have resulted in a decrease of amyloid plaques density in the brain of amyloid precursor protein (APP) transgenic mice ,3. ImproAn initial clinical trial using immunization with human Aβ in AD patients was started and then shortly halted because of an unusually high incidence (6%) of meningoencephalitis (see the Weksler article ). A longHere, we would like to stress few points of concern regarding these approaches in clinical practice.1) The claimed animal model for AD is unfortunately incomplete even if useful model for the human disease.2) Vaccination with human Aβ in mice induces an immune response against a foreign protein, i.e. human Aβ, and the mouse Aβ homolog does not appear to be involved. On the contrary, Aβ vaccination in man may potentially induce an autoimmune like disease in the brain and other peripheral tissues of susceptible patients. At the moment we do not have the ability to predict which patients will suffer destructive immune responses.3) The effects of both vaccination or passive immune therapy in AD brains might be non-specific, as already suggested by a recent report  and by t4) Vaccine therapy is not always effective in the elderly, since immune defects of variable degree are often present in old persons . In old 5) Historically vaccination has been successful in the prevention of the diseases much and much less in the therapy of ongoing diseases. A significant gain would be achieved by Aβ vaccination or passive immune therapy, if these manipulations will work in the very early stages of the disease. At the moment, no clinical data on this topic are available and those from animal models have not extensively addressed this topic.6) A general consideration on AD is also mandatory. In fact, clinical signs of dementia show up after extensive synapse and neuron loss have already occurred in the brain. How can an immune response restore an already compromised nervous circuit or revive dead neurons? The very modest decrement of cognitive deterioration rate claimed in a proportion of AD patients receiving the vaccine is a marginal therapeutic goal. In fact, the disease has not been cured, and the modest clinical slow down in the AD progression takes big prices, i.e. patients and care givers will continue to suffer the catastrophic effects of the disease for a longer time.We feel that Aβ vaccination and in a less extent passive immune therapy are now exciting experimental protocols for animal research and experimental neurology investigations, probably premature for clinical applications.


The gefitinib compassionate-use programme has enabled >39,000 patients worldwide to receive gefitinib  treatment. This paper reports the outcome of gefitinib treatment in Chinese patients who enrolled into the 'Iressa' Expanded Access Programme (EAP) at the Peking Union Medical College Hospital.Thirty-one patients with advanced or metastatic non-small-cell lung cancer (NSCLC) that had progressed after prior systemic chemotherapy were eligible to receive oral gefitinib 250 mg/day as part of the EAP. Treatment was continued until disease progression or unacceptable toxicity occurred. The impact of treatment on disease-related symptoms and quality of life (QoL) was evaluated with the Chinese versions of European Organization for Research and Treatment of Cancer Quality of Life Questionnaires (EORTC QLQ-C30 and QLQ-LC13).Gefitinib was well tolerated. Adverse events (AEs) were generally mild (grade1 and 2) and reversible. The most frequent AEs were acneform rash and diarrhoea. Only one patient withdrew from the study due to a drug-related AE. The objective tumour response rate was 35.5% ; median progression-free survival was 5.5 months ; median overall survival was 11.5 months . The QoL response rates for five functioning scales and global QoL varied from 56–88%. The main symptom response rates varied from 44–84%. QoL and symptom response were correlated with objective tumour response.Gefitinib demonstrated safety and efficacy as monotherapy in this series of Chinese patients with advanced NSCLC and was also associated with remarkable symptom relief and improvement in QoL. Although clinical trials are needed to confirm these positive findings, the data suggest that treatment with gefitinib may be beneficial for some Chinese patients who do not respond to chemotherapy and have poor prognosis. Platinum-based combination chemotherapy is the standard first-line treatment for patients with advanced non-small-cell lung cancer (NSCLC). Although meta-analysis of clinical trials proved that combination chemotherapy for advanced NSCLC is superior to best supportive care  and new Many solid tumours express or highly express the epidermal growth factor receptor (EGFR). In lung cancer, deregulation of EGFR is seen mainly in NSCLC: EGFR is highly expressed in NSCLC at levels varying from 32–80% -6. AbnorGefitinib is an orally active small molecule EGFR tyrosine kinase inhibitor (EGFR-TKI). The tolerability and efficacy of gefitinib have been explored extensively in the West and Japan. In a Phase I trial, Nakagawa et al compared the safety profile, pharmacokinetic parameters and antitumour activity of gefitinib in patients with solid malignant tumours in Japan, the USA and Europe, and no significant difference was found for ethnicity . HoweverPatients with advanced NSCLC who had no alternative therapeutic options have been able to receive gefitinib treatment in a worldwide compassionate-use programme. Here we report the outcome of treatment with oral gefitinib 250 mg/day in Chinese patients with advanced NSCLC participating in the compassionate-use programme at Peking Union Medical College Hospital.9/L, absolute neutrophil count >1.5 × 109 cells/L, platelet count >100 × 109/L); liver function ; renal function ; PaO2 >60 mmHg.Patients aged ≥18 years with histologically or cytologically confirmed advanced or metastatic NSCLC were eligible for enrolment into the compassionate-use programme after providing written, informed consent. They were required to have failed prior chemotherapy, and had no other treatment options available. Patients who had received chemotherapy were included if the treatment ended >28 days prior to the study. Other eligibility criteria included: adequate bone marrow function , pregnancy and lactation.One oral gefitinib tablet (250 mg) was taken at about the same time each day. Gefitinib was administered every day without interruption unless disease progression or unacceptable toxicity occurred.2 during therapy. Patients were evaluated after the first and third cycles of therapy, then every three cycles. Tumour response was evaluated according to Response Evaluation Criteria In Solid Tumors [In this study, 28 days of treatment was defined as one treatment cycle. Baseline evaluation included medical history and physical examination, electrocardiogram, chest X-ray, thorax computed-tomography scan and ultrasonography of the upper abdomen. Laboratory investigations included complete blood counts, urinalysis, and renal and liver function tests. Performance status (PS) was evaluated according to Eastern Cooperative Oncology Group (ECOG) criteria. Brain magnetic resonance imaging and radionuclide bone scans were only performed if metastatic disease was suspected according to the clinical manifestations of each patient. Regarding interstitial lung disease, we paid attention to clinical respiratory symptoms  and radiographic findings of patients, and monitored PaOd Tumors . In lineQoL was assessed using the Chinese version of the European Organization for Research and Treatment of Cancer (EORTC) core questionnaire, the Quality of Life Questionnaire (QLQ)-C30 (version 3.0) and the supplemental lung-cancer-specific module QLQ-LC13 . FollowiLogistical regression test models were used to identify baseline factors  that might independently predict tumour response. Median progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan-Meier method and a log-rank test was used to detect differences OS between strata.2 test or Fisher's exact test.Changes in symptoms and QoL were assessed in two different ways. Firstly, the mean scores of QoL and disease-related symptoms at baseline were compared with those at the end of the second cycle of treatment using a paired-sample t test. Secondly, the response rates of symptoms and QoL were calculated. For the assessment of symptoms and QoL, the classification system suggested by Stephens et al was used . The resThirty-one eligible patients were enrolled into the EAP at Peking Union Medical College Hospital between October 2002 and October 2003. Patient characteristics are listed in Table All patients were assessed for toxicity. Twenty-three (74%) patients had at least one AE. Treatment-related toxicities are listed in Table The assessment of patient response is listed in Table vs. 4.4 months).Median PFS was 5.5 months ; median OS was 11.5 months . The tumor response was associated with improved OS. Median OS of patients with response was significantly higher than that of those with no response (log-rank test p = 0.0058)  patients returned QoL questionnaires. All 25 patients returned the questionnaires at the first cycle of therapy, 23  at the second cycle of therapy, and 18  at the third cycle. The most frequently reported general symptoms were fatigue (100%) and appetite loss (68%), while dyspnoea (100%) and coughing (84%) were the most frequently reported respiratory symptoms. The changes in the mean scores for symptom scales, functioning and global QoL scales are presented in Table Patient responses for functioning, global QoL and main symptoms are shown in figure Haemoptysis had the highest response rate (84%), the rate of improvement (63%) being just lower than that of appetite loss among the five symptoms. The response rates for dyspnoea and coughing were 56% and 68%, respectively. For fatigue and appetite, the response rates were 44% and 80%, respectively.Differences in response rates for symptoms and QoL between patients with or without objective tumour response are shown in Table A daily oral 250 mg dose of gefitinib was well tolerated in this series of Chinese patients with advanced NSCLC, who received gefitinib as part of a compassionate-use programme. AEs were mild and reversible and different to those associated with conventional chemotherapy, such as neutropenia, thrombocytopenia and neuropathy. No haematological or neural toxicity was observed in the study. As with prior clinical trials evaluating the toxicity of gefitinib, the most frequently reported AEs were acneform rash and diarrhoea (grade 1 or 2). Four patients developed grade 1 or 2 stomatitis during treatment, which has previously been associated with gefitinib treatment in 7.8% of patients in IDEAL 1 but was not reported in IDEAL 2 .Severe acute interstitial pneumonia is the most serious AE that has been linked with gefitinib therapy. Inoue et al recently reported that 4 of 18 patients receiving gefitinib in their clinic developed severe acute interstitial pneumonia . The worThe response rate (35.5%) was higher than that of recently presented Phase II trials . The DCRs were 54.4% and 42% for the 250 mg/day dose in IDEAL 1 and 2, respectively, in contrast to 58.1% in our study. Unlike IDEAL 2, which was performed in the USA, IDEAL 1 was a global trial that recruited patients from 43 centres across Europe, Australia, South Africa and Japan and included a total of 102 Japanese patients with advanced NSCLC. The response rate for Japanese patients was higher than that of non-Japanese patients . However, the difference in response rate for ethnicity was not verified using multivariate logistical regression analysis. The difference in response rate between Japanese and non-Japanese patients was attributed to bias of baseline predictive factors of patients  ,10.QoL is an important endpoint for assessment of gefitinib treatment and has been included in some phase I and phase II trials. In these trials, the Functional Assessment of Cancer Therapy – Lung questionnaire was used to assess QoL and symptoms. Its validation and sensitivity were verified. Significant improvements in symptoms and QoL were observed in IDEAL 1 and 2. In IDEAL 1, symptom and QoL improvement with a dose of 250 mg/day were 40.3% and 23.9%, respectively, and in IDEAL 2 they were 43.1% and 34.3%, respectively. In both trials, improvements in symptoms and QoL correlated well with tumour response .In our patient series, the EORTC QLQ-C30 and its supplement module QLQ-LC13 were used for assessment of QoL and symptoms. Together, they are thought to be one of the best developed instruments for lung cancer assessment . The queIn conclusion, the data from this patient series show that an oral dose of gefitinib 250 mg/day is well tolerated and has significant antitumour activity in Chinese patients with advanced NSCLC who had failed previous chemotherapy and for whom no other treatment options were available. Although clinical trials are needed to confirm these positive findings, the data suggest that treatment with gefitinib may be beneficial for some Chinese patients who do not respond to chemotherapy and have poor prognosis.'Iressa' is a trademark of the AstraZeneca group of companiesNone declared.XLM and LYL designed the experiments and wrote the manuscript. XTZ, SLW and MZW participated in patient's follow-up and tumour response evaluation.The pre-publication history for this paper can be accessed here: