We developed a method to make a various high quality random peptide libraries for evolutionary protein engineering based on a combinatorial DNA synthesis.16 diversity, and the construction of a library with random sequence coding 120 amino acids containing few stop codons.A split synthesis in codon units was performed with mixtures of bases optimally designed by using a Genetic Algorithm program. It required only standard DNA synthetic reagents and standard DNA synthesizers in three lines. This multi-line split DNA synthesis (MLSDS) is simply realized by adding a mix-and-split process to normal DNA synthesis protocol. Superiority of MLSDS method over other methods was shown. We demonstrated the synthesis of oligonucleotide libraries with 10Owing to the flexibility of the MLSDS method, it will be able to design various "rational" libraries by using bioinformatics databases. In Table F = 1.00)  Table . So far,In order to make long ORFs, we assembled 8 units of the oligomers. Stem sequences of them did not have any stop codons. A DNA library encoding 120 amino acids plus nine 5'- and 3'-flanking semi-random di-peptides  was constructed  and purity (data of PAGE) of synthesized DNA. With an in vivo selection, there is a diversity limit by the transformation step. But with an in vitro selection, there is no such limitation. Thus exploration of huge sequence space by in vitro virus : [T]: [G]: [C] = x1 : x2 : x3 : x4 and x5 : x6 : x7 : x8, respectively. And for example, when L = 3, the expected alanine composition p1 is given by:Computer calculations to determine the optimum molar mixing ratio of four bases in the codon synthesis step were performed by using Mathematica (Wolfram Research). We made a GA program for this purpose. Firstly, the target amino acid composition, for the full-length sequence without deletion.L-dimensional optimization problem) having the solution xi as integer . The reason for 6-digits "integer" was to simplify the DNA synthesizer handling and also to simplify the calculation. As the fitness F of x in the GA, we took a correlation coefficient between the expected (or designed) amino acid composition and the target amino acid composition:We solved an integer-programming problem  considering the single nucleotide deletion.In the additional WORD file , the detail of calculation method is described for the the expected amino acids composition Click here for file


Governments around the world have turned their attention to the issue of taxpayer access to primary research literature stemming from publicly funded investigations The global debate over access to primary research literature heated up this summer, fueled by a slew of congressional and parliamentary recommendations, claims of political victory by critics and proponents of open access, and redoubled lobbying efforts on every side of the issue. After months of often dizzying rhetoric from virtually all camps, one concrete development has indisputably emerged from the fray: governments around the world have begun to take an interest in the question of who can and can't read the results of the scientific research they fund. “We are convinced,” concluded a recent report from the Science and Technology Committee of the United Kingdom's House of Commons, “that the amount of public money invested in scientific research and its outputs is sufficient to merit Government involvement in the publishing process” . United PLoS Biology and PLoS Medicine, that currently make their content immediately free online in centralized repositories. A US House of Representatives Committee on Appropriations, for example, recently passed language that would allow many, though not all, publishers six months between the date of publication of NIH-funded research articles and the date of their deposition in a free-to-use archive. While such pronouncements may sow fear in the hearts of some scientists and publishers, concerns that governments are poised to tell researchers where or how to publish seem largely unfounded. Both the UK report and rumblings from the US government suggest that any legislative dictates on access to scientific literature are likely to be structured to minimize potentially deleterious implications for established, subscription-based journals, for-profit and not-for-profit alike. Mandates for open access to articles summarizing the results of publicly funded research would not be mandates for scientists to submit work only to the handful of journals, like included in publicly funded electronic archives like PubMed Central, as the US Congress might, would be less a paradigm shift or a radically interventionist mandate than a sensible extension of existing policy for most governments and their funding agencies.In any case, it is a perfectly reasonable premise that governments should attach conditions to grants mandating public access to resulting peer-reviewed, published articles. Making funding for research contingent on the results of the work being disseminated as widely as possible is hardly a revolutionary proposition. All funders expect, of course, that scientists won't simply stash their findings in a desk drawer. Most, like NIH, include in their mission statements clauses about “fostering the communication of medical and health sciences information” . The US Increasingly, it seems, this is the view being adopted by policy makers—that it is the status quo, rather than prospective policy revision, that is anomalous or hard to justify. “We would be very surprised,” the Science and Technology Committee notes, “if Government did not itself feel the need to account for its investment [in research] in the publishing process. We… hope that this report will be a catalyst for change” .Washington Post last year  report was the product of a seven-month investigation, featuring some 127 submissions of written evidence and four days of oral testimony from the likes of Nature Publishing Group, Reed Elsevier, and indeed, the Public Library of Science. NIH has promised a period of public comment on its plan for implementing the Appropriations Committee's requirement before moving forward, in addition to the information-gathering meeting of publishers in July and subsequent meetings hosted by Dr. Zerhouni. All told, the current spate of government attention to the issue of public access to research results seems methodical, inclusive, and likely to prove productive for scientific communities and the public.As a matter of sheer principle, it strikes many people as odd that “anyone can download medical nonsense from the Web for free, but citizens must pay to see the results of carefully conducted biomedical research that was financed by their taxes,” as Rick Weiss noted on the front page of the ast year . While n


As key regulators of mitotic chromosome segregation, the Aurora family of serine/threonine kinases play an important role in cell division. Abnormalities in Aurora kinases have been strongly linked with cancer, which has lead to the recent development of new classes of anti-cancer drugs that specifically target the ATP-binding domain of these kinases. From an evolutionary perspective, the species distribution of the Aurora kinase family is complex. Mammals uniquely have three Aurora kinases, Aurora-A, Aurora-B, and Aurora-C, while for other metazoans, including the frog, fruitfly and nematode, only Aurora-A and Aurora-B kinases are known. The fungi have a single Aurora-like homolog. Based on the tacit assumption of orthology to human counterparts, model organism studies have been central to the functional characterization of Aurora kinases. However, the ortholog and paralog relationships of these kinases across various species have not been rigorously examined. Here, we present comprehensive evolutionary analyses of the Aurora kinase family.Phylogenetic trees suggest that all three vertebrate Auroras evolved from a single urochordate ancestor. Specifically, Aurora-A is an orthologous lineage in cold-blooded vertebrates and mammals, while structurally similar Aurora-B and Aurora-C evolved more recently in mammals from a duplication of an ancestral Aurora-B/C gene found in cold-blooded vertebrates. All so-called Aurora-A and Aurora-B kinases of non-chordates are ancestral to the clade of chordate Auroras and, therefore, are not strictly orthologous to vertebrate counterparts. Comparisons of human Aurora-B and Aurora-C sequences to the resolved 3D structure of human Aurora-A lends further support to the evolutionary scenario that vertebrate Aurora-B and Aurora-C are closely related paralogs. Of the 26 residues lining the ATP-binding active site, only three were variant and all were specific to Aurora-A.In this study, we found that invertebrate Aurora-A and Aurora-B kinases are highly divergent protein families from their chordate counterparts. Furthermore, while the Aurora-A family is ubiquitous among all vertebrates, the Aurora-B and Aurora-C families in humans arose from a gene duplication event in mammals. These findings show the importance of understanding evolutionary relationships in the interpretation and transference of knowledge from studies of model organism systems to human cellular biology. In addition, given the important role of Aurora kinases in cancer, evolutionary analysis and comparisons of ATP-binding domains suggest a rationale for designing dual action anti-tumor drugs that inhibit both Aurora-B and Aurora-C kinases. The Auroras are a conserved family of serine/threonine kinases which have essential functions in cell division ,2. In miDrosophila melanogaster (fruitfly) that caused defective spindle-pole formation [Drosophila was found to have a second Aurora homolog [Caenorhabditis elegans, similarly has two Aurora-like genes [Saccharomyces cerevisiae and Schizosaccharomyces pombe, have a single Aurora, known as increase-in-ploidy 1 (Ipl1) [Xenopus laevis, which has two kinases; Aurora-A and Aurora-B [Model organism studies have played a pivotal role in functional characterization of Aurora kinases. Aurora kinases were first identified as mutant alleles in ormation . Subsequ homolog , and theke genes ,6. The f1 (Ipl1)  and Auro1 (Ipl1) , respectAurora-B ,10. MoreAurora-B .The Aurora kinases are mitotic kinases that generally associate with chromosomes, often in complexes with other proteins, and interact with cytoskeletal components in cell division. The three mammalian Aurora kinases appear at specific locations during mitosis. Aurora-A, the "polar kinase", primarily associates with the separating centrosomes while Aurora-B, the "equatorial kinase", is a chromosomal passenger protein . The leaRecent studies indicate that all three Aurora kinases have strong associations with cancer. Aurora-A has been mapped to a region in the human chromosome 20q13.2-13.3) that is amplified in cancer cell lines and primary tumors .2-13.3 t. TransfeDespite the importance of model organisms in understanding Aurora kinase function, the evolutionary relationships among these variants are unclear. Two previous phylogenetic analyses of Aurora kinases were incomplete because the contemporary complement of Aurora kinases was unavailable  or certaTakifugu rubripes [Danio rerio [Ciona intestinalis [In order to construct a comprehensive phylogenetic tree, GenBank was searched for all possible Aurora kinases. In addition to previously published Aurora kinase sequences, further chordate and urochordate Aurora homologs were found by using mammalian Aurora-A, Aurora-B and Aurora-C protein sequences as queries in BLASTP or TBLASTN  searchesrubripes , the zebio rerio , and thestinalis .Multiple sequence alignments show that the Aurora kinase family is highly conserved among species Fig. . PairwisC. intestinalis. The use of alternative kinase families, other than PLK4, to root the tree did not alter the internal topology of the Aurora clade. Although its genome sequence is incomplete, C. intestinalis likely has only a single Aurora homolog since other probable kinase open reading frames associated with the next top five BLASTP [Phylogenetic trees constructed using four methodologies, all rooted using polo-like kinases type 4 (PLK4), show that all vertebrate Auroras form a clade distinct from those of invertebrates Fig. . The phye BLASTP  hits didXenopus laevis, rodents and humans as well as a new putative ortholog in T. rubripes.Among true vertebrates, our phylogenetic tree shows that the Aurora kinases underwent two major gene duplication events. The first split in cold-blooded vertebrates lead to the formation of two Aurora subfamilies. One branch encompasses all known vertebrate Aurora-A sequences in a single orthologous lineage that includes fishes, amphibians and mammals. This family includes previously identified Aurora-A kinases in X. laevis) and fish (D. rerio and T. rubripes). Searches of T. rubripes and D. rerio protein and DNA sequence databases detected several other putative serine/threonine kinase homologs but none were Auroras according to phylogenetic analyses. Thus, cold-blooded vertebrates appear to have only a single Aurora-A ortholog, a single Aurora-B-like homolog, and lack an Aurora-C ortholog. Conversely, Aurora-A, Aurora-B and Aurora-C appear to be ubiquitous to mammals  where they are encoded by separate chromosomal loci. It would appear that mammalian Aurora-B and Aurora-C evolved from a duplication event involving the ancestral Aurora-B found in cold-blooded vertebrates. This depiction of the evolutionary relationships of vertebrate Auroras was consistently determined by four different phylogenetic methods with high bootstrap or Bayesian posterior probability values  and S. pombe (Ark1). Other basal branches are the amitochondrial fungi, Encephalitozoon cuniculi, and the kinetoplast protist, Leishmania major [The Aurora kinases of plants and invertebrates are all outgroup lineages to chordates / urochordates Fig. . Althougia major .C. elegans and D. melanogaster, occupy separate early branches and are not, as their current names suggest, orthologs to either Aurora-A or Aurora-B of vertebrates. An unrooted phylogenetic tree with only model organism species shows the same topology of vertebrate Auroras as the more species-rich tree rooted by PLK4 kinases  and ZM4Evolutionary analysis shows that cell division Aurora kinases, while consistent in theme throughout eukaryotes, have undergone lineage-specific expansions and specialization in metazoans. Aurora-C is the least known of the Aurora kinases. Yet as an evolutionary innovation in mammals, further studies are very much warranted from the perspectives of better understanding its potential roles in both cell replication and tumor progression. A better functional understanding of Aurora-C would help clarify the evolutionary relationships of Aurora-B and Aurora-C in mammals relative to the ancestral Aurora-BC in cold-blooded vertebrates. Additionally, the close evolutionary and structural relationships between mammalian Aurora-B and Aurora-C offers the tantalizing opportunity to design dual kinase inhibitors that might circumvent potential tumor cell resistance to mono-target chemotherapeutics.Takifugu rubripes, Danio rerios and Ciona intestinalis homologs were obtained by BLASTP and TBLASTN [T. rubripes and D. rerios had orthologs to X. laevis Aurora-A and Aurora-B but not mammalian Aurora-C. C. intestinalis had a single Aurora-like kinase.All Aurora kinase orthologs and paralogs were initially collected from GenBank nonredundant protein database by performing separate searches using BLASTP  with hum TBLASTN  of speciPLK4 kinases were selected as the outgroup for phylogenetic analyses because they were the most similar non-Aurora kinases to either human Aurora-A, Aurora-B or Aurora-C in multiple BLASTP  searches6 generations, burn-in values were set for 104 generations, and trees sampled every 100 generations. All trees were visualized using the program TREEVIEW v1.6.6 [We constructed phylogenetic trees using distance neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood quartet puzzling (QP), and Bayesian posterior probabilities (BP). NJ trees were based on pair wise distances between amino acid sequences using the programs NEIGHBOR and PROTDIST (Dayhoff option) of the PHYLIP 3.6 package . The proW v1.6.6 .For the Aurora kinase phylogeny rooted with PLK4 kinases shown in Fig. For the unrooted phylogeny of Aurora kinases of model organisms shown in Fig. The SwissPDBviewer program  was usedJRB conceived the study, performed the phylogenetic analysis and drafted the manuscript. KKK performed the 3D structural analyses and contributed to the draft manuscript. MLB participated in the preliminary phylogenetic analysis and contributed to the draft manuscript. PS and DRP both contributed to the draft of the manuscript and added key references.Multiple sequence alignment of edited Aurora and Plk4 kinases used to produce the phylogeny shown in Fig. Click here for fileAccession numbers (gi), Locus link Ids (LID) or genome predicted protein Ids for Aurora and PLK kinases as labelled in the Supplementary Data multiple sequence alignment file S1.Click here for file


Manmade nanoparticles range from the well-established multi-ton production of carbon black and fumed silica for applications in plastic fillers and car tyres to microgram quantities of fluorescent quantum dots used as markers in biological imaging. As nano-sciences are experiencing massive investment worldwide, there will be a further rise in consumer products relying on nanotechnology. While benefits of nanotechnology are widely publicised, the discussion of the potential effects of their widespread use in the consumer and industrial products are just beginning to emerge. This review provides comprehensive analysis of data available on health effects of nanomaterials. Scientists world-wide are continuing to discover unique properties of everyday materials at the sub micrometer scale ,2. This While benefits of nanotechnology are widely publicised, the discussion of the potential effects of their widespread use in the consumer and industrial products are just beginning to emerge ,8. Both In this review we summarise the known facts about nanomaterial hazards, discuss the potential entry points of nanoparticles into the human body, explore their likely pathways inside the body and analyse published experimental results on the bioactivity of nanomaterials.Human skin, intestinal tract and lungs are always in direct contact with the environment. Whereas skin acts as a barrier, lungs and intestinal tract also allow transport (passive and/or active) of various substances like water, nutrients or oxygen. Because of that fact they are likely to be a first port of entry for nanomaterials journey into the human body. Our knowledge in this field mainly comes from drug delivery  and toxicology (xenobiotics) studies. Human skin functions as a strict barrier and no essential elements are taken up through the skin (except radiation necessary to build up vitamin D). The lungs exchange oxygen and carbon dioxide with the environment, and some water escapes with warm exhaled air. The intestinal tract is in close contact with all the materials taken up orally; there all nutrients (except gasses) are exchanged between the body and the environment.2 in area, and is at most places covered with a relatively thick first barrier (10 micron) which is build of strongly keratinised dead cells  and alveoli (gas exchange areas). Human lungs contain about 2300 km of airways and 300 million alveoli (gas exchange areas)  is predominantly by macrophage phagocytosis. The mucociliary escalator is an efficient transport system pushing the mucus, which covers the airways, together with the trapped solid materials towards the mouth. The phagocytosis of particles and fibres results in activation of macrophages and induces the release of chemokines, cytokines, reactive oxygen species, and other mediators; this can result in sustained inflammation and eventually fibrotic changes. The phagocytosis efficiency can be affected by the  characteristics of the solid material (see below); moreover, fibres too long to be phagocytized  will only be cleared very slowly.Laboratory exposure studies have shown that if the inhaled concentrations are low, such that the deposition rate of the inhaled particles is less than the mechanical alveolar macrophage-mediated clearance rate in the lung, then the retention half time is about 70 days (steady-state lung burden during continuous exposure). If the deposition rate of the inhaled particles exceeds this clearance rate, the retention half time is significantly increased, reflecting an impaired or prolonged alveolar macrophage-mediated clearance function with continued accumulation of lung burden (overload). Inhaled fibres, which are persistent in the alveoli, can interact with the pulmonary epithelial cells or even penetrate the alveolar wall and enter the lung tissue. These fibres are often described as being in the "interstitial" where they may lie between or within the cells making up the alveolar walls. Bio-persistent solid materials, certainly those particles containing mutagenic substances or asbestos fibres or silica, which remain for years in the lungs, increase the risk of developing cancer.et al [et al [It has been reported recently that nanotubes show a sign of toxicity , confirmet al  and Lam l [et al , which dl [et al  concludel [et al  their rel [et al .2/g), it was found that the biological effects  were dependent on specific surface area and not particle mass. Similar findings were reported in earlier studies on tumorigenic effects of inhaled particles. It was shown that tumour incidence correlated better with specific surface area than with particle mass [Clearance from the lung depends not only on the total mass of particles inhaled but also on the particle size and, by implication, on particle surface, as shown in the following studies. A sub-chronic 3 months inhalation exposure of rats to ultrafine (~20 nm) and fine (~200 nm) titanium dioxide (TiO2) particles demonstrated that the ultrafine particles cleared significantly slower, showed more translocation to interstitial sites and to regional lymph nodes when compared to the fine TiO2 particles . By compcle mass ,28.3) study [3) study [Comparing the health effects of chronically inhaled TiO2 particles with distinctly different sizes, it is remarkable that the low exposure (10 mg/m3) study  resulted3) study . The inhIn summary, most nano-sized spherical solid materials will easily enter the lungs and reach the alveoli. These particles can be cleared from the lungs, as long as the clearance mechanisms are not affected by the particles themselves or any other cause. Nano-sized particles are more likely to hamper the clearance resulting in a higher burden, possibly amplifying any possible chronic effects caused by these particles. It is also important to note that specific particle surface area is probably a better indication for maximum tolerated exposure level than total mass. Inhaled nano-fibres  also can enter the alveoli and their clearing would, in addition, depend on the length of the specific fibre. Recent publications on the pulmonary effects of carbon nanotubes confirm the intuitive fear that nano-sized fibre can induce a rather general non-specific pulmonary response.Reports on the surface properties of nanoparticles, both physical and chemical, stress that nanoparticles differ from bulk materials. Their properties depend heavily on the particle size. Therefore, nanoparticles are not merely small crystals but an intermediate state of matter placed between bulk and molecular material. Independently of the particle size, two parameters play dominant role. The charges carried by the particle in contact with the cell membranes and the chemical reactivity of the particle .Polycationic macromolecules show a strong interaction with cell membranes in vitro. A good example can be found in the Acramin F textile paint system. Three poly-cationic paint components exhibited considerable cytotoxicity  in diverse cell cultures, such as primary cultures of rat and human type II pneumocytes, and alveolar macrophages and human erythrocytes. The authors argued that the multiple positive charges play an important role in the toxic mechanism ,33. Biocet al [Dekie et al  concludeet al  suggesteet al . Therefoet al [Geiser et al  studied et al ,43. On tet al . Specifiet al .Although the type of particle does not seem to play an important role in whether it is embedded in the surfactant lining of the alveoli, the embedding process itself is crucial. Particle-cell interaction is possible only after the immersion of the particulates in the lining fluid and research is needed to study this phenomenon in detail in relation to inhaled nanoparticles. Logically, as described in the report for silica , the reaThe impact of inhaled particles on other organs has only recently been recognised. Most research has concentrated on the possible consequences of particle related malfunction of the cardio-vascular system, such as arrhythmia, coagulation  etc. Howet al [99mTc) labelled carbon particles to the blood. These particles, which are very similar to the ultrafine fraction of actual pollutant particles, diffused rapidly – within 5 minutes – into the systemic circulation  but, unfortunately, no radioactivity measurements with blood were reported [et al [The literature on the translocation of very small particles from the lungs into the blood circulation is limited and often conflicting. A recent study has reported deposition and clearance over 2 h of an ultrafine (60 nm) reported . In agrereported , Kawakamreported  have repreported  that aerreported  and othereported ,16,57 hareported . A recenreported  has provd [et al . In inhad [et al .Passage of solid material from the pulmonary epithelium to the circulation seems to be restricted to nanoparticles. The issue of particle translocation still need to be clarified: both the trans-epithelial transport in the alveoli and the transport via nerve cells. Thus, the role of factors governing particle translocation such as the way of exposure, dose, size, surface chemistry and time course should be investigated. For instance, it would be also very important to know how and to what extent lung inflammation modulates the extra-pulmonary translocation of particles.Long non-phagocytizable fibres (in humans longer than 20 micron) will not be effectively cleared from the respiratory tract. The main determinants of fibre bio-persistence are species specific physiological clearance and fibre specific bio-durability . In the alveoli the rate at which fibres are physically cleared depends on the ability of alveolar macrophages to phagocytose them. Macrophages containing fibres longer than their own diameter may not be mobile and be unable to clear the fibres from the lung. The bio-durability of a fibre depends on dissolution and leaching as well as mechanical breaking and splitting. Long fibres in the lung can disintegrate, leading to shorter fibres that can be removed by the macrophages. Bio-persistent types of asbestos, where breakage occurs longitudinally, result in more fibres of the same length but smaller diameter. Amorphous fibres break perpendicular to their long axis ,61, resuIt is self-evident that the slower the fibres are cleared (high bio-persistence), the higher is the tissue burden and the longer the fibres reside in a tissue the higher is the probability of an adverse response. A milestone was set by Stanton et al ,63 who uThe bio-durability of fibres with a diameter < 100 nm will probably not differ from larger inhalable fibres. Therefore, great caution must be taken in case of the contact with nano-fibres, Bio-durability tests must be performed before releasing any products containing them. Carbon nanotubes, which are of high technical interest, are one of the materials which need to be tested in depth concerning bio-persistence and cancer risk. The first toxicological studies indicated that carbon nanotubes can be a risk for human health -24, whilAlready in 1926 it was recognised by Kumagai  that parThe kinetics of particle translocation in the intestine depends on diffusion and accessibility through mucus, initial contact with enterocyte or M-cell, cellular trafficking, and post-translocation events. Charged particles, such as carboxylated polystyrene nanoparticles  or thoseParticulates, once in the sub-mucosal tissue, are able to enter both lymphatic and capillaries. Particles entering the lymphatic are probably important in the induction of secretory immune responses while those which enter the capillaries become systemic and can reach different organs. In one study , the bodCrohn's disease is characterised by transmural inflammation of the gastrointestinal tract. It is of unknown aetiology, but it is suggested that a combination of genetic predisposition and environmental factors play a role. Particles (0.1–1.0 micron) are associated with the disease and indicated as potent adjuvants in model antigen-mediated immune responses. In a double-blind randomised study, it has been shown that a particle low diet  alleviates the symptoms of Crohn's disease . AlthougDiseases other than of gut origin also have marked effects on the ability of GIT to translocate particles. The absorption of 2-micron polystyrene particles from the PP of rats with experimentally induced diabetes is increased up to 100-fold (10% of the administered dose) compared to normal rats . HoweverFrom the literature cited above it is clear that engineered nanoparticles can be taken up via the intestinal tract. In general the intestinal uptake of particles is better understood and studied in more detail than pulmonary and skin uptake. Because of this advantage it is maybe possible to predict the behaviour of some particles in the intestines but precaution should be taken. For those nanoparticles designed to stabilise food or to deliver drug via intestinal uptake other, more demanding, rules exist and should be followed before marketing these compounds.Skin is an important barrier, protecting against insult from the environment. The skin is structured in three layers: the epidermis, the dermis and the subcutaneous layer. The outer layer of the epidermis, the stratum corneum (SC), covers the entire outside of the body and only contains dead cells, which are strongly keratinized. For most chemicals the SC is the rate-limiting barrier to percutaneous absorption (penetration). The skin of most mammalian species is, on most parts of the body, covered with hair. At the sites, where hair follicles grow, the barrier capacity of the skin differs slightly from the "normal" stratified squamous epidermis. Most studies concerning penetration of materials into the skin have focussed on whether or not drugs penetrate through the skin using different formulations containing chemicals and/or particulate materials as a vehicle. The main types of particulate materials commonly used are: liposomes; solid poorly soluble materials such as TiO2 and polymer particulates and submicron emulsion particles such as solid lipid nanoparticles. The penetration of these particulate carriers has not been studied in detail.et al in [TiO2 particles are often used in sunscreens to absorb UV light and therefore to protect skin against sunburn or genetic damage. It has been reported by Lademann et al in  that micet al in . A diffeet al in , who arget al in  demonstret al in  into theet al in ,87,82 buPenetration of non-metallic solid materials such as biodegradable poly microparticles, 1 to 10 micron with a mean diameter of 4.61 ± 0.8 micron was studied after application on to porcine skin. The number of microparticles in the skin decreased with the depth (measured from the airside towards the subcutaneous layer). At 120 micron depth (where viable dermis present) a relatively high number of particles was found, at 400 micron (dermis) some micro-particles were still seen. At a depth of 500 micron no microparticles were found . In the Emzaloid™ particles, a type of submicron emulsion particle such as liposomes and nonionic surfactant vesicles (niosomes), with a diameter of 50 nm to 1 micron, were detected in the epidermis in association with the cell membranes after application to human skin . The autA recent review by Hostynek  stated tet al [Only limited literature on nanoparticles penetrating the skin is available, but some conclusions can already be drawn. Firstly, penetration of the skin barrier is size dependent, nano-sized particles are more likely to enter more deeply into the skin than larger ones. Secondly, different types of particles are found in the deeper layers of the skin and at present it is impossible to predict the behaviour of a particle in the skin. And finally, materials, which can dissolve or leach from a particle , or break into smaller parts , can possibly penetrate the skin. We did not find any direct indication that particles, that had penetrated the skin, also entered the systemic circulation. The observation that particles in the skin can be phagositized by macrophages, Langerhans cells or other cells is a possible road towards skin sensitisation. Tinkle et al  have shoGlass fibres and Rockwool fibres are widely distributed man-made mineral fibres because of their multiple applications, mainly as insulation materials, which have become important for replacing asbestos fibres. In contact with the skin, these fibres can induce dermatitis through the mechanical irritation. Why these fibres are such strong irritant has not been examined in detail. In occlusion irritant patch tests in humans it was found that Rockwool fibres with a diameter of 4.20 ± 1.96 micron were more irritating than those with a mean diameter of 3.20 ± 1.50 micron. The fact that "small" fibres can cause strong skin irritation has been known for a long time, e.g. itching powder. It is also commonly accepted that some types of man made fibres can easily induce non-allergic dermatitis. Although this is common knowledge, it is not clear what makes these fibres irritants. In search for reports on skin irritation caused by fibres with a diameter of < 100 nm no information could be found, indicating that more research is needed.The body distribution of particles is strongly dependent on their surface characteristics. For example, coating poly(methyl methacrylate) nanoparticles with different types and concentrations of surfactants significantly changes their body distribution . CoatingOral uptake (gavage) of polystyrene spheres of different sizes (50 nm to 3 micron) in female Sprague Dawley rats (for 10 days at a dose of 1.25 mg/kg/day) resulted in systemic distribution of the nanoparticles. About 7% (50 nm) and 4% (100 nm), was found in the liver, spleen, blood and bone marrow. Particles larger than 100 nm did not reach the bone marrow and those larger than 300 nm were absent from blood. No particles were detected in heart or lung tissue .Irrespective of the uptake route, the body distribution of particles, is most dependent on the surface characteristics and the size of the particles. It is an important issue in drug-design in order to help to deliver medication to the right target. In unintentional uptake of nanoparticles these characteristics can strongly influence the accumulation of a specific type of particle in the particular body site.et al [Epidemiological studies have reported a close association between particulate air pollution and cardiovascular adverse effects such as myocardial infarction . The latet al  studied et al . It is iPollutant particles such as diesel exhaust particles (DEP), may cause a marked pulmonary inflammation within an hour after their deposition in the lungs. Moreover, intratracheal instillation of DEP promotes femoral venous and arterial thrombosis in a dose-dependent manner, already starting at a dose of 5 μg per hamster (appr. 50 μg/kg). Subsequent experiments showed that prothrombotic effects persisted at 6 h and 24 h after instillation  and confirmed that peripheral thrombosis and pulmonary inflammation are not always associated . Solid iEpidemiologic studies have provided valuable information on the adverse health effects of particulate air pollution in the community, indicating that nanoparticles act as an important environmental risk factor for cardiopulmonary mortality. Particle-induced pulmonary and systemic inflammation, accelerated atherosclerosis, and altered cardiac autonomic function may be part of the patho-physiological pathways, linking particulate air pollution with cardiovascular mortality. Also, it has been shown that particles deposited in the alveoli lead to activation of cytokine production by alveolar macrophages and epithelial cells and to recruitment of inflammatory cells. An increase in plasma viscosity, fibrinogen and C-reactive protein has been observed in samples of randomly selected healthy adults in association with particulate air pollution ,98,99.A number of reports on cellular uptake of micro- and nano- sized particles has been published. Reports on particle uptake by endothelial cells ,101, pulOne of the promising alleys of nanotechnology is organ- or cell- specific drug delivery mediated by nanoparticles -114. It Also, other processes such as tight junction modulation or P-glycoprotein (Pgp) inhibition also may occur . OberdörIt has been shown that nanoparticles, that enter the liver, can induce oxidative stress locally. A single  and repeated (14 days) intravenous administration of poly-isobutyl cyanoacrylate  or polystyrene  nanoparticles induced a depletion of reduced glutathione (GSH) and oxidised glutathione (GSSG) levels in the liver, as well as inhibition of superoxide dismutase (SOD) activity and a slight increase in catalase activity. The nanoparticles did not distribute in the hepatocytes, implicating that the oxidative species most probably were produced by activated hepatic macrophages, after nanoparticle phagocytosis.Uptake of polymeric nanoparticles by Kupffer cells in the liver induces modifications in hepatocyte antioxidant systems, probably due to the production of radical oxygen species . We havein vitro particles are less cytotoxic when dosed in a medium with high protein content. In the lungs, mucus or surfactant is present, in which antioxidants are present, but these can be easily neutralised when a high number of oxidative compounds is inhaled.Although the contact with nanomaterials in the lungs and intestinal tract shows many similarities important differences between inhalation and ingestion of nanomaterials exist from the toxicological point of view. In the intestinal tract a complex mix of compounds – such as secreted enzymes, ingested food, bacteria of the gut flora, etc – is present, which can interact with the ingested nanomaterial. Non-specific interaction often reduces the toxicity of the ingested material. It has been described that The transit through the intestinal tract is a relatively fast process, the continuous decay and renewal of the epithelium makes sure that nanomaterials will not remain long in the intestinal tract. The presence of solid material in the lumen of the intestines will not automatically induce an inflammatory response. Inhaled materials < 10 micron and > 5 micron will not enter the alveolar spaces of the lungs, and therefore these will be cleared easily in healthy persons via the muco-ciliary escalator. Particles that are smaller than 5 micron will deposit in the alveolar space via Brownian movement. In the alveoli, water insoluble materials can only be removed via phagocytosis by macrophages or other cells, or via transportation through the epithelium to the interstitium or systemic circulation. These processes are often accompanied by the onset of (persistent) inflammation. The particles themselves can – depending on the physical-chemical characteristics of the material – remain for a long period in the alveoli.In the intestinal tract, the ingested materials are stressed from acidic (stomach) to basic conditions. The shift in pH markedly changes the solubility and the ionic state of the material via changing the surface characteristics. In the lungs, the milieu of the lumen is more constant.Particles in the nano-size range can certainly enter the human body via the lungs and the intestines; penetration via the skin is less evident. It is possible that some particles can penetrate deep into the dermis. The chances of penetration depend on the size and surface properties of the particles and also on the point of contact in the lung, intestines or skin. After the penetration, the distribution of the particles in the body is a strong function of the surface characteristics of the particles. A critical size might exist beyond which the movement of the nanoparticles in parts of the body is restricted. The pharmaco-kinetic behaviour of different types of nanoparticles requires detailed investigation and a database of health risks associated with different nanoparticles  should be created. The presence of the contaminates, such as metal catalysts present in nanotubes, and their role in the observed health effects should be considered along with the health effect of the nanomaterials.The increased risk of cardiopulmonary diseases requires specific measures to be taken for every newly produced nanoparticle. There is no universal "nanoparticle" to fit all the cases, each nanomaterial should be treated individually when health risks are expected. The tests currently used to test the safety of materials should be applicable to identify hazardous nanoparticles. Proven otherwise, it would be a challenge for industry, legislators and risk assessors to construct a set of high throughput and low cost tests for nanoparticles without reducing the efficiency and reliability of the risk assessment. Nanoparticles designed for drug delivery or as food components need special attention.


Noroviruses  being a leading cause of acute gastroenteritis outbreaks, the impact of NV infection is at present unknown and little information is available about strains circulating in Italy. In April 2002 an outbreak of gastroenteritis occurred in the province of Bari , involving several households.Despite A retrospective cohort study was performed in order to assess risk factors associated with illness. All households where a case occurred were included in the study. Faecal specimens were collected from ill individuals. NV-specific RT-PCR was performed. Eleven samples of mussels were collected from fish-markets involved in the outbreak. A nested PCR was used for mussel samples.One hundred and three cases, detected by means of active surveillance, met the case definition. Raw shellfish eating was the principal risk factor for the disease, as indicated by the analytic issues . NVs were found by means of RT-PCR of all the stool specimens from the 24 patients tested. Eleven samples of shellfish from local markets were tested for the presence or NVs; six were positive by nested PCR and genotypes were related to that found in patients' stools.This is the first community outbreak caused by NVs related to sea-food consumption described in Italy. The study confirms that the present standards for human faecal contamination do not seem to be a reliable indicator of viral contaminants in mussels. Norovirus , one of four genera in the Caliciviridae family, includes a group of morphologically similar but genetically different single-stranded RNA viruses. NVs represent the most important cause of non-bacterial gastroenteritis worldwide. In industrialised countries NVs may be responsible for up to 80% of all outbreaks of gastroenteritis [nteritis . OutbreaContaminated food or water commonly represents the main source of infection. Epidemics spread by the faecal-oral route, even if transmission may also occur through direct person-to-person contact or aerosolised viral particles.The incubation period of NV gastroenteritis is 24–48 hours and symptoms include vomiting, diarrhoea, abdominal pain, low-grade fever, headache and myalgia.Italy has no specific surveillance system for viral gastroenteritis and laboratory diagnosis is only carried out in a few cases. Therefore the impact of NV infection is currently unknown and little information is available about circulating strains. Outbreak investigations are usually performed by local health units. Gastroenteritis notifications are often too delayed to identify the etiologic agent and source of infection correctly.Puglia, a region in the South-East of Italy, has about four million inhabitants. Bari  is the capital. Raw mussels are largely consumed in Bari and, in general, in Puglia, especially at Christmas and Easter. In 1994 raw seafood consumption was the source of a small cholera outbreak. Moreover, hepatitis A is endemic in this region.This report describes a large outbreak of NV gastroenteritis that involved several households in Bari province.st and April 7th 2002, in the province of Bari . In Italy, at Easter many people eat in restaurants and the day after Easter most people go picnicking. In the province of Bari seafood  are often consumed on these occasions. The Regional Epidemiological Office, alerted by the Local Health Unit, performed a field study, collecting data from all General Practitioners and all Emergency Units in the province. Active surveillance was conducted until April 15th 2002 (one week after the last case onset). The aim of the investigation was to describe the outbreak and to identify the etiologic agent, the source of infection and the means of transmission.The outbreak occurred during the Easter holidays, between March 31st – April 7th, who had diarrhoea (three or more loose stools in any 24 hour period) or vomiting (at least one episode). Fever greater than or equal to 38°C, abdominal pain or nausea were considered additional symptoms. A "probable" secondary case was defined as illness in a household with onset of symptoms more than 24 hours after the primary case.A case was defined as illness in a resident in the area during the period March 31A retrospective cohort study was performed in order to assess risk factors associated with illness.All households where a case occurred were included in the study.Information, collected by means of a standardised questionnaire, included: 1) demographic individual characteristics such as age, gender, occupation; 2) type of food consumed during the last 72 hours before onset of symptoms; 3) if ill, type, date and time of onset of symptoms.To assess the association between food consumption and disease, relative risks (RR) and 95% confidence intervals (95% CI) were calculated. Age and gender were compared between ill and unaffected individuals by the chi squared and Mann-Whitney tests. A p-value less than 0.05 was considered significant. Variables associated to the illness in the univariate analysis were included in the stratified analysis; summary RR and 95% CI were calculated from the formulas of Greenland and Robins . CollectFaecal specimens were collected from ill individuals. Part of the specimens was refrigerated and processed within 12 hours to detect ova and parasites by direct microscopy and common bacteria by standard methods. The rest was stored at -20°C until examination by NV-specific reverse transcription-polymerase chain reaction (RT-PCR).Viral nucleic acid extraction and purification from stool specimens was performed as previously reported . RT-PCR Eleven samples of mussels were collected from two fish-markets from which the cases had bought the shellfish they consumed. Mussel samples were also processed within 12 hours to detect common bacteria by standard methods.Then a nested PCR was used. The procedure for mussel processing as well as viral RNA extraction and purification has been previously described in full .The primers used for first round PCR were JV12 and SM31. Nested PCR was carried out with the use of the primers SR33 for negative strand DNA synthesis and SR48 and SR46 for positive strand synthesis of genogroup I (GI) and genogroup II (GII) sequences, respectively [The 333-bp or 123-bp amplification products from cases and from mussels were subjected to sequencing with PCR primers. When required, cloning was carried out on PCR products. Sequences obtained were aligned with those available in the GenBank.One hundred and three subjects met the case definition; 22 of them were defined as "probable" secondary cases. Fifty eight (56.3%) of the 103 cases were female; the mean age was 42.6 years , nausea (58.3%), diarrhoea (53.4%), abdominal pain (47.6%), fever (16.5%). No difference was observed in the clinical pattern by gender. Age was significantly higher in ill individuals with severe diarrhoea (lasting more than 24 hours or undergoing specific treatment) . No difference in age was found for the other symptoms.st at 5,00 pm and lasted eight days. The epidemic curve shows a single peak on April 2nd (35 cases between 1:00 and 12:00 AM) and a right tail probably due to secondary cases  in the days before the onset of symptoms of relatives. All 36 healthy subjects were interviewed. Nineteen (52.8%) of them were female, the mean age was 42.5 years. Gender and age were not significantly different between healthy and ill individuals (table An attack rate of 74.1% (103/139) was observed. In the univariate analysis, the association between raw mussel consumption and illness was significant . The attack rate according to raw mussel consumption was 86.3% (69/80).Even cooked mussel consumption was associated with illness in the univariate analysis . No other food was associated with illness . Such association was not significant in the "ate raw mussels" stratum . The relative risk according to cooked mussel consumption was 1.38 (95% CI: 1.00 – 1.91) after correction for raw mussels consumption .Both stool and mussel samples were negative for parasites and bacterial enteropathogens. All stool samples from 24 cases were positive for NV by RT-PCR and 5 of 11 mussel samples by nested PCR. The sequences of strains revealed great heterogeneity. In fact, the simultaneous occurrence of GI and GII viruses within the same outbreak was observed. One sample of mussels showed the presence of a mixed genogroup. Sequence analysis showed that strains from 19 cases and 3 mussel samples had identical sequences and belonged to GII, clustering narrowly to the strain NLV/Tarrag/238/2001/Sp, thus strengthening the epidemiologic link of the mussels to the cases. The nucleotide sequences from three cases and 2 mussel samples formed two other distinct clusters showing the best fit with the strains Saitama U25 and Khs1-1997-JP. Sequences from two further cases and one mussel sample were assigned to genogroup I and showed a high degree of identity with the strain NLV/Steinbach/EG/2001/CA.Three familial groups consisting of 7 cases and three members of a further family group from the outbreak showed identical sequences and were classified as NV GII (the main cluster of 19 cases). A fourth member of the latter family group was positive for one of the two GI strains. The other strains investigated by sequence analysis were unrelated to those from these familial groups.In Italy a national NV outbreaks database does not exist, although NVs are the leading cause of gastroenteritis outbreaks in Europe as in the rest of the western countries . MoreoveThis investigation showed the causative role of mussel consumption in the outbreak, confirmed by laboratory tests on both stools and food samples.The heterogeneity of viral strains associated with the consumption of contaminated mussels is no surprise due to the peculiar features of shellfish. In fact, bivalve shellfish are filter feeders and tend to accumulate whatever pollutants are in the water which can result in viral contamination from a multitude of possible sources affecting many individuals. In any case, further studies should be carried out to clarify this issue . The lowIn the 1990s, NVs were identified as the primary pathogen associated with shellfish-borne gastroenteritis in the United States . Since tRaw shellfish consumption is very frequent among the people of Puglia. Mussels sold in Puglia come from a large number of suppliers: from countries within the European Union (EU) and from other Mediterranean countries. The frequent shellfish consumption had already been blamed for the cases of cholera that occurred in Puglia in 1994, during the Christmas period, when a major epidemic was reported in Albania, on the opposite coast of the Adriatic sea ; moreoveMoreover, our data showed cooked mussels also played a significant role. In fact cooking might not completely inactivate NVs and because of the low infectious dose, even a limited residual contamination can result in illness .The epidemic curve shows a usual pattern consistent with a common point source. However, person to person transmission may have played a role in the last phase of the outbreak. On the other hand, such a means of transmission of NV is well documented.This investigation confirms the importance of field study in gastroenteritis outbreaks, as well as obtaining stool specimens from patients and food samples for laboratory analysis in a short time .The present standards for human faecal contamination do not seem to be a reliable indicator of viral contaminants in mussels . SeafoodThis episode confirms that large NV outbreaks occur in Italy but only an accurate investigation can recognise this pathogen and that current regulations and commercial practices need to be revised to assure the safety of shellfish consumption and to improve control of future outbreaks.None declared.MC and GB carried out microbiological assays and drafted the manuscript. CG participated in the design of the study and performed the statistical analysis. MQ conceived the study as well as participating in its design and coordination. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


It has been generally accepted that E-NTPase/E-NTPDase plays regulatory role in purinergic signalling, but other functions may yet be discovered.E-NTPase/E-NTPDase is activated by millimolar concentrations of Ca2+and Mg2+ in vivo.In this article it is proposed on the basis of published data that E-NTPase/E-NTPDase could play a role in the influx and efflux of CaAttenuation of extracellular Ca2+ influx by rat cardiac sarcoplasmic anti-E-NTPase antibodies and oligomerization studies on mammalian CD39 conclusively point towards the existence of a new channel in the membrane. Further studies on these properties of the E-NTPase/E-NTPDase may provide detailed mechanisms and identify the potential patho-physiological significance. Excitable cells exhibit an action potential, a substantial general depolarization of the plasma membrane, in response to depolarizing stimuli; influx of Ca2+ occurs via plasma membrane Ca2+ channels and/or release from sarco (endo) plasmic reticulum via ryanodine-receptor Ca2+ channels which regulate the excitation – contraction coupling i results from influx of Ca2+ across the plasma membrane and Ca2+ release from the endoplasmic reticulum. Ca2+ release from the SER depends on the binding of inositol 1,4,5-triphosphate (InsP3) to its receptor Ca2+channels, and also on Ca2+ binding to ryanodine receptor – Ca2+channels.In non-excitable cells, the increase in i play a role in ROCC opening [2+]I, increase in [cAMP]i i  and acti [cAMP]i .2+ channels must be a highly regulated event involving physical movement of channel components inclusive of the alteration in channel protein conformation; Also, an extracellular source of free energy (ΔG) could be of critical importance. This might be supplied by E-NTPase/E-NTPDase mediated hydrolysis of NTP/NDP. Co-ordination of this process might play a role in the opening of Ca2+ channels, independently of membrane depolarization or other factors.Opening of Cai. Extracellular metabolism  o/ [NDP] o hydrolysis might be utilized for opening the channel formed by the homo-oligomeric ENTPase/E-NTPDase.2+/Mg2+ transport processes, and the experimental evidence obtained in favor of the whole proposal.outlines the intracellular and extracellular factors that would influence the termination of the Ca2+/Mg2+ channel. Fig. 2+/Mg2+ channel gate. Fig. Membrane depolarization could locally alter protein conformation. This in turn could potentially induce post-translational modification in the (intracellular) monomer subunits of the E-NTPase/E-NTPDase, followed by translocation to the membrane (depending on the tissue type(s) and functional requirement(s)) Fig. . Fig. 2 NPPB (5-nitro-2-(3-phenyl propylamine benzoic acid), a potent inhibitor of CFTR (cystic fibrosis transmembrane receptor) and p-glycoprotein mediated ATP release o concentration is <1 mM and restores [Mg2+]i when the heart is perfused with Ringer buffer containing 5 × 10-7 M Mg2+. Failure of either of these two transport mechanisms may result in a rise in [Mg2+]i, impairing the contractile machinery of the myocardium i = Extracellular Nucleotide triphosphate; [NDP]0 = Extracellular Nucleotide diphosphate; ROCC = Receptor Operated Ca2+ channel; SER = Sarco (Endo) plasmic reticulum; i [cAMP]= Cytoplasmic or intracellular cAMP; PMCA = Plasma membrane Ca2+pump ATPase.VOCC = voltage-operated Ca2+ channel; LGCCS = Ligand gated non-specific cation channels. RACC = Stretch/Receptor activated non-specific Ca2+channels. SUR = Sulfonylurea Receptor Proteins. CD36 = Thrombospondin receptor on platelets. CD39 = Ecto Ca2+/Mg2+apyrase.


The extended model, presented in this paper, also includes the following physiologically important phenomena: desensitization of calcium channels; internalization of the dimerized receptors and recycling of some of the internalized receptors; an increase in Gq concentration near the plasma membrane in response to receptor dimerization; and basal rates of synthesis and degradation of the receptors. With suitable choices of the parameters, good agreement with a variety of experimental data of the LH release pattern in response to pulses of various durations, repetition rates, and concentrations of GnRH were obtained. The mathematical model allows us to assess the effects of internalization and desensitization on the shapes and time courses of LH response curves.In a previous study, a model was developed to investigate the release of luteinizing hormone (LH) from pituitary cells in response to a short pulse of gonadotropin-releasing hormone (GnRH). The model included: binding of GnRH to its receptor (R), dimerization and internalization of the hormone receptor complex, interaction with a G protein, production of inositol 1,4,5-trisphosphate (IP The purpose of our work is to use mathematics and machine computation to understand the dynamics of this important and interesting physiological system.Gonadotropin-releasing hormone (GnRH) is released by the hypothalamus in a pulsatile fashion and stimulates luteinizing hormone (LH) and follicle stimulating hormone (FSH) release by pituitary cells by a complex series of signaling processes. Although there is substantial information about various individual steps in the signaling system, there is less understanding of how these components interact to give rise to the overall behavior of the system. The frequency of pulses varies throughout the menstrual cycle increasing markedly just prior to ovulation. And, it has been observed in IP3), release of calcium from the endoplasmic reticulum (ER), entrance of calcium into the cytosol via voltage gated membrane channels, pumping of calcium out of the cytosol via membrane and ER pumps, and the release of luteinizing hormone (LH). Cytosolic calcium dynamics were simplified and it was assumed that there is only one pool of releasable LH. Despite these and other simplifications, the model results matched experimental curves and enabled us to understand the reasons for the qualitative features of the LH release curves in response to GnRH pulses of short durations and different concentrations both in the presence and absence of external calcium. We note that Heinze et al, (t) and the rate of return of receptors to the membrane is k11n[HR](t - 40)χ(t - 40). To simplify notation, we write [HR]40 = [HR](t - 40)χ(t - 40).We make the following simple assumption about the recyling of receptors  receptor concentration is R0 = 10-2 nM, and the magnitude of P0 is chosen so that approximately  of the resting amount of receptor is produced per hour, thus ensuring a slow recovery to the steady state receptor concentration in the absence of GnRH.Since only 60% percent of the internalized receptors are returned to the membrane after exposure to GnRH, there would not be a full recovery of receptors in the membrane. In the model we therefore include a low basal rate of receptor synthesis, k33 is the parameter that determines the rate of increased concentration of GQ at the membrane in response to the formation of HRRH. We are assuming a finite pool of GQ that can be transported from the cytoplasm to the immediate vicinity of the plasma membrane. This pool is assumed to be regulated by the amount of HRRH for only the first 20 minutes, and after this time the rate of increase is negligible  - k666[CAC]2     (8)ERR = k-6 = 5 min-1.The change in cytosolic calcium concentration, CAER, is then determined by the rate constant ERR, which is the rate of extrusion, multiplied by the fraction of open channels, CHO, and the difference in concentration between the calcium concentration in the cytoplasm and the endoplasmic reticulum. As in Blum et al. , calciumk8 , we haveAs in the previous model, the volume of the ER is assumed to be 1/20 of the volume of the cytosol. CAC is determined by the rate of calcium efflux through ion channels in the ER membrane minus the rate at which calcium is being pumped back into the ER, plus the rate of calcium entry from the plasma membrane. The function VSR denotes the rate of calcium influx from extracellular calcium into the cytosol and depends on E with rate constant k8  and on C to GnRH . Since t to GnRH . We furtata; see  for morewheret) = (k8E(t) + k88[CAC](t) - k888([CAC])(t))2) × VSRO(t)     (11)VSR(and VSRO satisfies the following.t) ≤ 1     (13)0 ≤ VSRO – (14). These non-linear equations cannot be solved analytically but solutions can be obtained by machine computation. To do this, we used the solver ODE45 in Matlab.The values of the rate constants are given in Table In Figure Figures r0 = 0.6) of the internalized receptors are returned to the membrane. Thus, in the presence of continuous exposure to GnRH, the total number of receptors in the membrane continues to decline as shown in Figure Figures Figures The previous model  was intAs shown in Figure [HR]. However, the decline in the effector, E, which leads to the formation of IP3 (see equation (6)) is only 25% (data not shown) because of the substantial, rapid rise in GQ . For a pulse period of 1 hour, the same amount of LH is released in response to each pulse for each GnRH concentration (Panel C). In vivo, one would not expect desensitization, so this result is consistent with experimental observations that LH pulses of the same magnitude occur approximately once an hour except just prior to ovulation . Note aExperiments have been performed to examine LH release in response to different concentrations of GnRH. King et. al.  performeKing et al.  also perk11 = 0) or calcium channel densensitization to zero (v0 = 0 = v1) and compared the results to the full model for continuous and pulsatile exposures. In the full model, in response to continuous exposure there is initial rapid increase in LH release followed by a decrease to basal levels at about 40 minutes . If, in addition, the rate of internalization of receptors is decreased from k11 = 0.08/n nM-1·min-1 to k11 = 0.04/n nM-1·min-1, the response to the second pulse of GnRH was very similar to that observed experimentally, as shown in Figure Stojilkovic et al.  exposed k11 = 0.08/n to k11 = 0.8/n. Although this decreased the amount LH released appreciably on the second pulse, the amount of LH released was not reduced to a comparably low level as observed experimentally. We therefore also decreased the amount of return of internalized ET receptors to the membrane, r0, from 60% to 10%. As shown in Fig. In the experiments of Stojilkovic et al. , the fir-1 to 0.4 min-1. This resulted in approximately 70% decrease in the magnitude of response to the second pulse of ET, but further increase in v1 did not cause any further reduction in magnitude. Since there is evidence suggesting that the calcium channels in the ER desensitize in response to GnRH , we intq/11 proteins closely associated with the plasma membrane. With these additions we are now able to examine the behavior of the model system over medium term (up to three hours) exposures to GnRH and to a variety of pulsatile exposures. We have compared the model behavior to many such different experiments and found that it shows the essential response properties of the gonadotrophs. Furthermore, since the model includes many of the intracellualar physiological processes, we have used the model to investigate and understand the mechanisms that give rise to the various experimental results.We have extended our previous model to include receptor internalization and partial degradation, outer membrane calcium channel desensitization, basal levels of receptor synthesis and destruction, and an increase in the number of Gv1  from 0.02/min to 0.005/min we obtain a good match to the rat data (closed circles in Figure v1 from 0.02/min to 0.05/min we obtain a good match to the hamster data (stars in Figure We note that the response of gonadotrophs to GnRH depends on the method of cell preparation, the stage of the estrous cycle, and the particular animals and species used. Thus, the real physiological parameters will vary in these different situations. Therefore, one would not expect that our model with the fixed set of "standard" parameters (used for the simulations in Figures We used parameter variation to investigate whether receptor internalization or outer membrane calcium channel desensitization plays the major role in LH release desensitization and concluded that outer membrane calcium channel desensitization is more important, at least in the experiments of Janovick and Conn . We alsoIt is important to note that the model ignores a number of processes that play a role in the long-term response to GnRH. In gonadotrophs, depending on the frequency and duration of exposure to pulses of GnRH, there may be an increase or decrease in the number of receptors in the cell membrane due to changes in gene expression and/or mRNA translation ,9,8,30. Washington and Reed contributed mostly to the mathematical development, Conn contributed to the physiological analysis, and Blum contributed to both.The authors declare that they have no competing interests.


Early lifetime exposure to dietary or supplementary vitamin D has been predicted to be a risk factor for later allergy. Twin studies suggest that response to vitamin D exposure might be influenced by genetic factors. As these effects are primarily mediated through the vitamin D receptor (VDR), single base variants in this gene may be risk factors for asthma or allergy.951 individuals from 224 pedigrees with at least 2 asthmatic children were analyzed for 13 SNPs in the VDR. There was no preferential transmission to children with asthma. In their unaffected sibs, however, one allele in the 5' region was 0.5-fold undertransmitted (p = 0.049), while two other alleles in the 3' terminal region were 2-fold over-transmitted (p = 0.013 and 0.018). An association was also seen with bronchial hyperreactivity against methacholine and with specific immunoglobulin E serum levels.The transmission disequilibrium in unaffected sibs of otherwise multiple-affected families seem to be a powerful statistical test. A preferential transmission of vitamin D receptor variants to children with asthma could not be confirmed but raises the possibility of a protective effect for unaffected children. Supportin vitro  and in v studies , also ep studies ,5 report studies .Vitamin D has been used for many years in various doses and preparations to prevent rickets, a disease usually induced by poor dietary calcium intake and sun deprivation. It seems that widespread "historical" rickets in industrial countries was also a genetic disease. A formal twin analysis yielded a 91% concordance rate in monozygotic twins compared to 23% in dizygotic twins . Also a 2D3 binds to nuclear vitamin D receptor (VDR), which exists from under 500 to over 25,000 copies / cell in many human tissues including thymus, bone marrow, B and T cells and lung alveolar cells [Fok1 restriction site  in asthma families did not find an association [The active vitamin D metabolite 1,25(OH)ar cells . The genar cells . The VDRar cells ,10. Whilociation , positivociation  as well ociation .FokI SNP. All three LD blocks have now been covered by additional SNPs in our asthma family sample.So far, dbSNP catalogued 117 SNPs in the VDR when a resequencing approach of the VDR published in June 2004 found 245 SNPs . In thisThe sample analyzed here consisted of 951 individuals from 224 pedigrees contributing 11,383 genotypes.Mean pedigree size was 4.4; the number of phase-known individuals ranged from 221 to 305 (with the exception of rs2853563) in affected, and from 24 to 41 in the unaffected, children. Except for rs2853563, the minor allele frequency always exceeded 19%. In two families both parents had asthma, in 82 only one parent had asthma and in 140 families both parents were disease-free.Of the markers tested, only marker rs2239186 showed a slightly reduced transmission ratio in asthmatic children . In unaffected children three markers showed significantly altered ratios: 0.5 for hCV2880804, and 2.0 and 1.9 in rs1989969 and rs2853564, respectively. Excess transmission was generally more pronounced in unaffected children , the age distribution of affected and unaffected children was also compared , measured as quantitative trait locus (QTL) by using the slope of the dose-response curve in a standardized methacholine challenge protocol, also indicated an association with the same three SNPs identified in unaffected sibs. Further dichotomizing BHR by comparing the upper versus all other quartiles also yielded significant associations . The sum score of all specific IgE serum levels (RAST) was associated with markers rs1989969 (P = 0.021) and rs2853564 (P = 0.018), while total IgE was not found to be associated.Table DS. Again, no significant transmission distortion was found in affected children, while one haplotype showed a 5.1-fold over-transmission in unaffected children (P = 0.009).Finally, a 4-locus haplotype was constructed of those SNPs associated in the TDTThis study addresses a previously described association of VDR SNPs with asthma and related phenotypic traits. Although a preferential transmission of vitamin D receptor variants to children with asthma could not be confirmed, it raises the possibility of a protective effect in unaffected children. Although the effect is rather moderate, several SNPs support this association. If any, the association is probably more related to RNA turnover than to a structural modification of the receptor as there was no association with LD block "B" that codes for the translated exons. Gene expression can be varied over a 100-fold range by subtle modifications of the 3'-terminal sequence , which wThis notion is partially in contrast with a previous study of 7 VDR SNPs in the CAMP (Childhood Asthma Management Program) study of 582 nuclear families where SNP rs7975232 (akin ApaI) in intron 8 showed a highly significant effect . A confiunpublished own observation). CYP24A1 is particular interesting as it is the major enzyme of the degradation pathway that showed a 97-fold increase after vitamin D treatment of rats [It is unlikely that increased or decreased vitamin D sensitivity is simply mediated by a genetic variation in the VDR. Vitamin D requires several enzymatic steps to be activated, transported and degraded; receptor signalling requires several co-factors and all of these may contribute additive or multiplicative effects on vitamin D sensitivity. The co-activator retinoic acid receptor RXR may itself affect Th1 and Th2 development . Other t of rats  or 12-fo of rats .The number of genes regulated by vitamin D has been recently extended beyond those genes with known VDRE promoter motifs  to another 150 up- and down-regulated regulated genes -23. The th and 70th percentiles) did not provide much information for linkage analysis [In addition to its biological context, this study also has some implications on the statistical analysis as the rather low number of unaffected sibs seemed to contribute to a few positive associations. Despite enormous efforts to map complex genetic diseases, SNP association studies are often lacking power . Linkageanalysis . Althouganalysis ).s of 3.2) has been estimated to be approx. 250 [Transmission disequilibrium testing of unaffected child – parent trios originating from families with another two affected offsprings, may be a powerful alternative. As there is a strong ascertainment bias of these families toward a genetic risk, as well as a disease causing environmental factor, being unaffected is an extreme phenotype ("being sane in an insane world"). The transmission to unaffected children can be seen as an independent cross-match to the transmission to affected children. The high power of testing unaffected sibs has already been predicted on theoretical grounds . The numrox. 250 . A samplrox. 250 . In thisrox. 250  is unlikThe transmission disequilibrium in unaffected sibs in otherwise multiple-affected families seems to be a powerful test. A preferential transmission of vitamin D receptor variants to children with asthma could not be confirmed but raises the possibility of a protective effect in unaffected children.rd birthday a history of at least three years of recurrent wheezing and should not have any other airway disease diagnosed. Unaffected siblings were also sampled if they were at least 6 years old, eligible for pulmonary function testing and did not have doctor-diagnosed asthma.The German asthma sib pair families were collected in 26 paediatric centres in Germany and Sweden for an initial genome-wide linkage scan. In these families at least two children were required with confirmed doctor-diagnosed asthma, while prematurity or low birth weight of the children were excluded, along with any other severe pulmonary disease. All affected children should have after their 3On the first home visit a complete pedigree of the family was drawn and information collected in a questionnaire. Participants were examined for several associated phenotypes. Pulmonary function tests were performed by forced flow volume tests and bronchial challenge was done by methacholine.Briefly, pulmonary function tests were performed by forced expiration in a sitting position using a nose-clip. Forced flow volume tests were performed until three reproducible loops were achieved. Of these the trial with the maximum sum of FVC and FEV1.0 was used for the analysis. Bronchial challenge with methacholine was done with increasing doses of 0, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, 25 mg/ml during 5 consecutive breaths with 14 mg delivered from a de Vilbiss 646 nebulizer chamber by using a breath-triggered pump ZAN 200 . The provocation was stopped either with the occurrence of symptoms or a fall of 20% from the baseline FEV1.0 and the slope of the dose-response curve calculated.Total IgE was determined with an ELISA . The allergens tested were birch (betula verruscose) ALK SQ108, hazel (corylus avellana) ALK SQ113, the herbs ribworth (plantago lanceolata) ALK N342 and mugwort ALK SQ312, mixed grass ALK SQ299, dust mite dermatophagoides farinae ALK SQ 504, dermatophagoides pteronyssimus ALK SQ 503, cat dander ALK SQ555 and dog dander ALK SQ 553, and fungi , which were bought in one batch and stored at +5°C until analysis.The original family collection  has beenDNA was isolated from peripheral white blood cells using Qiamp  or Puregene isolation kits . From the VDR we selected 16 SNPs where 13 could finally be analyzed. They had to be polymorphic, complete, received a high calling score, passed paternity checks and were in Hardy-Weinberg equilibrium. The following three markers have been excluded from the analysis: rs2239179 (contaminant in mass spectrum peak), rs2853559  and rs797523 (typing error in primer sequence).Genotyping was performed using MALDI-TOF mass spectrometry of allele-specific primer extension products Table  generate2-test as well as the transmission to their unaffected sibs. An extension of the classical TDT [DS). The standardized linkage disequilibrium coefficient D' and the correlation coefficient R2 were calculated for each pair of SNPs in parents by using the R package "genetics". All analyses were cross-checked by using SIBPAIR software. Haplotypes were estimated using TDTPHASED in the UNPHASED package [Clinical data and genotypes were transferred to a SQL 2000 database by using Cold Fusion 4.0 scripts. Statistical analyses were performed using R 1.8.1  by accesical TDT  was also package  and tran package  for the VDR vitamin D receptorVDRE vitamin D responsive elementSNP single nucleotide polymorphismMALDI-TOF matrix assisted laser desorption ionisation – time of flightDSP discordant sib pairsTDT transmission disequilibriumLD linkage disequilibriumQTL quantitative trait locusBHR bronchial hyperreactivityFEV1 forced volume during the 1st secondThe author developed the idea presented in this paper, initiated the study, applied for funding, developed protocols, participated in the clinical survey, planned the laboratory analysis, did the statistical analysis, and drafted the report. This manuscript contains no patient identifiable information.


Recombinant adenoviral vectors are highly efficient for in vitro and in vivo gene delivery. They can easily be produced in large numbers, transduce a wide variety of cell types and generate high levels of transgene expression.The AdEasy system is a widely used system for generating recombinant adenoviral vectors, which are created with a minimum of enzymatic manipulations and by employing homologous recombination in E. coli.In this paper we describe an alternative simplified method for screening recombinant DNA within the AdEasy system. This Duplex-PCR-method is independent of the transgene or insert and can be used for the complete AdEasy system. It is characterized by a simple standard protocol and the results can be obtained within a few hours. The PCR is run with two different primer sets. The primers KanaFor and KanaRev hybridizise with the Kanamycin resistence gene and AdFor and AdRev detect the adenoviral backbone. In case of recombinant clones, two diagnostic fragments with a size of 384 bp and 768 bp are generated.The practicability of this method was verified with three different transgenes: Cytosin Deaminase (AdCD), p53 (Adp53) and Granulocyte Macrophage Colony Stimulating Factor (AdGM-CSF). Recombinant clones are indicated by two diagnostic fragments and are then suitable for further processing.In summary, the presented protocol allows fast detection of recombinants with an easy technique by minimizing the amount of necessary steps for generating a recombinant adenovirus. This method is time sparing and cost-effective. Recombinant adenoviral vectors are highly efficient for in vitro and in vivo gene delivery. They can easily be produced in large numbers, transduce a wide variety of cell types and generate high levels of transgene expression. The AdEasy system is a widely used, simplified system for generating recombinant adenoviral vectors, which are created with a minimum of enzymatic manipulations and by employing homologous recombination in E. coli . The sysThe presented Duplex-PCR is performed as follows: After Co-transformation of the Pme I-digested shuttle vector with the adenoviral backbone plasmid to E. coli (BJ 5183) and plating on agar (selection on kanamycin), half of the overnight grown colonies are picked and used directly as template for the colony-PCR. The other half of the colony is used for inoculation with LB-kanamycin and then incubation at 37°C. Only the positive, recombinant clones which have been detected by PCR are grown overnight, a minipreparation of DNA is then performed the next morning.The PCR is run using two different primer sets. The primers KanaFor (5' CAA GAT GGA TTG CAC GCA GG 3') and KanaRev (5'AAG GCG ATA GAA GGC GAT GC 3') hybridize to the Kanamycin resistence gene and AdFor (5'GGC TGC TCT GCT CGG AAG AC 3') and AdRev (5'GGC ATA CGC GCT ACC CGT AG 3') detect the adenoviral backbone.2, 100 mM dNTPs and 0,2 Units Taq polymerase. The bacteria are denatured at 95°C for 10 minutes. The PCR-products are amplified by 40 cycles of annealing at 58°C (30 sec), extension at 72°C (30 sec) and denaturation at 94°C (30 sec). In our experience, this procedure produced the best results without generating false positive clones  is size fractionated with 80 V for 1 h in 1% agarose in the presence of ethidium bromide and the resulting bands visualized with ultraviolet illumination.The DNA obtained by small scale alkali lysis from the recombinants is then extracted twice with a phenol-chloroform protocol, precipitated and carefully resuspended in 20 μl RNAse-free water. The construct is linearized with PacI and directly transfected into 911 cells, which are monitored for cytopathic effects, i.e. production of recombinant adenoviruses. The cytopathic effect is usually seen within 5 to 10 days. The expression of the transgene is confirmed by Western blot analysis. The practicability of our procedure was verified with three different transgenes: Cytosin Deaminase (AdCD), p53 (Adp53) and Granulocyte Macrophage Colony Stimulating Factor (AdGM-CSF).The conventional way of screening for recombinants after Co-transformation of the linearized shuttle vector with the adenoviral backbone vector in E. coli is by plating on LB/kanamycin, growing the bacteria overnight, then picking the colonies and growing them again for 10–15 hours. Minipreps are then performed and the size is evaluated on agarose gels. A restriction digest with three different restriction enzymes is then done and finally again another agarose gel is run. This is a relatively time-consuming and laborious procedure, which takes about 2 days.In contrast, the presented alternative protocol allows fast detection of recombinants with a simplified technique by minimizing the amount of necessary steps for generating a recombinant adenovirus. The method is time sparing and cost-effective. In our experience, the above described protocol showed no problems with false negative clones. After optimisation of the PCR protocol, we were able to run the conventional screening method (e.g. by restriction digest) for recombinant clones at the same time as the presented simplified PCR. We found no differences in regard to the final results for the two methods, but it has to be kept in mind that only a limited number of recombinant adenoviruses were actually generated with the new technique. Furthermore, we exclusively then continued our work with the AdCD-virus. Therefore it cannot be ruled out that under other conditions the presented technique may produce other results than the conventional technique. The positive clones were processed and finally transfected into 911 cells. After harvesting recombinant adenovirus, we infected cells with AdCD and checked the expression of the Cytosin deaminase protein by Western blot. The functionality of the gene was proven by FACS analysis. We confirmed expression of the protein as well as its functionality.The presented protocol allows fast detection of recombinant clones within the AdEasy system with an easy, cost-effective technique. Therefore, this procedure is a potential alternative for screening recombinants within the AdEasy system.DA designed the study and was responsible for manuscript preparation, MK, IB optimised the PCR protocol, PK was responsible for manuscript preparation, MB contributed to manuscript preparation and JW was responsible for study design and manuscript preparation. All authors read and approved the final manuscript.


Public health strategies are needed to curb antimalarial drug resistance. Theoretical argument points to an association between malaria transmission and drug resistance although field evidence remains limited. Field observations, made in Zimbabwe, on the relationship between transmission and multigenic drug resistance, typified by chloroquine, are reported here.in vivo chloroquine failure events for five sites in relation to natural endemicity and spraying history.Periodic assessments of the therapeutic response of uncomplicated falciparum malaria to chloroquine in two selectively sprayed or unsprayed health centre catchments, from 1995 – 2003. Cross-sectional analysis of During selective house spraying, the chloroquine failure rate for the sprayed catchment decreased, such that, after four years, the odds of chloroquine failure were 4× lower than before start of spraying in the area . Chloroquine failure odds for the sprayed area became 4× lower than contemporaneous failure odds for the unsprayed area , although the likelihood of failure was not significantly different for the two catchments before selective spraying started . When spraying ended, in 1999, the drug failure odds for the former sprayed area increased back 4 fold by 2003 . High altitude areas with naturally lower transmission exhibited a 6× lower likelihood of drug failure than low-lying areas . Compared to sites under ongoing annual spraying, areas that were last sprayed 3–7 years ago experienced a 4-fold higher probability of chloroquine failure .Reduced transmission is associated with suppressed levels of resistance to chloroquine and presumably other regimens with multigenic drug resistance. It seems the adoption of transmission control alongside combination chemotherapy is a potent strategy for the future containment of drug-resistant malaria. The escalation of parasite drug resistance has persisted as a major obstacle to malaria control for decades -3. OwingPlasmodium falciparum has repeatedly demonstrated the ability to develop resistance to practically any drug upon wider introduction, as illustrated by multi-drug resistance, especially in South East Asia [in vitro resistance to proguanil/atovaquone [However, ast Asia -15. Thusast Asia ,16,17 whovaquone -22. Addiin vivo multigenic drug resistance, typified by chloroquine.Mathematical models have been proposed suggesting a relationship between malaria transmission and the evolution of drug resistance, though some workers suggest a positive association ,24 whileZimbabwe, on the southern fringes of malaria in Africa, experiences seasonal and potentially epidemic transmission characterized by a non-immune population with high probability of drug treatment -30. The The study was based at five health centres located in the low-lying (<600 m above sea level) hyperendemic transmission zone as well as those in the higher altitude 600 – 1200 m asl) mesoendemic transmission zone bordering the malaria-free central watershed . Transverse assays for Pfmdr1 and Pfcrt mutations associated with chloroquine resistance were carried out in these two sites during the 1998–99 transmission season. Further assessments of in vivo chloroquine therapeutic response were carried out cross-sectionally in another three sites where treatment change to chloroquine (CQ) + sulfadoxine/pyrimethamine (SP) was not yet being implemented due to temporary unavailability of SP. Malarial incidence was determined retrospectively for all sites using available health centre records.The study was a prospective assessment of the therapeutic response of in vivo therapeutic efficacy of chloroquine was assessed using the standard WHO (1996) protocol [The protocol . Since tInclusion of all age groups was on the rationale that there is no premunition in the population. Recruited patients were thus a representative sample of the symptomatic population which presents for treatment with chloroquine in the primary health care system. The radical asexual parasite elimination criterion was adopted because persistent asexual parasitaemia poses a risk of complications in non-immunes.P. falciparum Pfmdr1 gene and at codon 76 of the P. falciparum chloroquine resistance transporter gene (Pfcrt), which are associated with chloroquine resistance [Pfcrt codon, additional restriction sites were included in the target PCR product to serve as internal controls for complete digestion.Amino acid polymorphisms at codons 86 and 1246 of the sistance ,33, weresistance ,35. ApprThe study was approved by respective provincial medical health authorities and by the Medical Research Council of Zimbabwe. Patient participation was by the informed consent of the patients themselves or guardians, in the case of children.On the grounds of low malarial incidence, the catchments of Sahumani clinic, in Mutasa district and Burma Valley clinic, in Mutare district Fig , were reThere were no malaria statistics for the two health centres prior to 1998, (1997 for Sahumani). However, during the selective annual spraying, the risk of contracting malaria in the sprayed Burma Valley catchment was at least 2.6 fold lower than for Sahumani : 4.18 (1.485 – 11.782), p = 0.004, n = 146) Chloroquine efficacy assessments for 2003 were not conducted in Sahumani as the treatment was changed that year to chloroquine plus sulfadoxine/pyrimethamine.Pfmdr1 and Pfcrt codons associated with chloroquine resistance were examined in pre-treatment patient samples from 1998 and 1999 in Burma Valley and Sahumani . Resistance-associated mutations at amino acid codons 86 and 1246 of Pfmdr1, and codon 76 of Pfcrt, were more prevalent in the Sahumani area (Table Amino acid polymorphisms on A scatterplot of chloroquine therapeutic failure rate with malarial incidence suggested a positive association Fig . In steaP. falciparum malaria calls for public health strategies to maximize the useful life of antimalarials. According to the findings of the present study, reduced transmission, due to vector control or high altitude, was associated with suppressed levels of in vivo therapeutic failure and genotypic resistance to chloroquine. Assuming that chloroquine resistance has a multigenic mechanism, as is the general consensus [The build up of drug-resistant onsensus ,36,37, tPfcrt and Pfmdr1, despite the lower transmission. This paradoxical result suggests that the sprayed area probably favoured more genetic out-crossing, resulting in recombination break down of drug-resistant haplotypes. The genetic out-crossing may partly explain the association of low drug resistance with the house spraying. Further studies are needed to verify this relationship in more areas.From the Burma Valley and Sahumani cross-sectional assays, there was, in the sprayed catchment, a higher likelihood of infections carrying mixed mutated and wild type codons, for both In Burma Valley, despite drug pressure, the proportion of resistant parasites decreased during spraying, and subsequently resurged after the spraying stopped. This is reminiscent of the decrease in proportion of chloroquine-resistant parasites reported in China , and morin vivo fitness burden that is thought to ensue following cessation of drug use. In the present results, drug selection advantage for the resistant parasites appeared to be directly counteracted by independent survival limiting factors, such as vector control and high altitude. This has important implications for control as it means that drug-resistant P. falciparum can be contained during drug use. Furthermore, costly acquisition of immunity in the resident population is, presumably, not the only prerequisite for curbing drug resistance.What is distinct about the current results is that the fitness cost for resistance appeared to occur in the sporogonic phase, as distinguished from an de facto stronghold for malaria, eradication so far remains only an academic prospect, as the malaria burden continues to increase [The present results afford field evidence supporting the continuation of sustainable malaria vector control programmes. Similar findings were reported for Uganda , althougincrease . It seemReduced transmission due to house spraying or high altitude is associated with suppressed levels of phenotypic and genotypic resistance to chloroquine and presumably other multigenically encoded drug regimens. Transmission control implemented with combination chemotherapy seems a potent approach for the future containment of drug-resistant malaria.SM was the principal investigator responsible for the study design, data collection, analysis and drafting of the manuscript. SLM carried out essential co-ordination of project activities. RM and TC afforded technical input on the manuscript and facilitated field data collection. SKC made vital contributions in original proposal development, seeking of funding, and edited the manuscript. KPD provided crucial inputs in the study design, genetic analysis, general direction and co-ordination of the study and the writing up of the manuscript.