Immunization by a bacterial aerosol

By manufacturing a single-particle system in two particulate forms (i.e., micrometer size and nanometer size), we have designed a bacterial vaccine form that exhibits improved efficacy of immunization. Microstructural properties are adapted to alter dispersive and aerosol properties independently. Dried "nanomicroparticle" vaccines possess two axes of nanoscale dimensions and a third axis of micrometer dimension; the last one permits effective micrometer-like physical dispersion, and the former provides alignment of the principal nanodimension particle axes with the direction of airflow. Particles formed with this combination of nano- and micrometer-scale dimensions possess a greater ability to aerosolize than particles of standard spherical isotropic shape and of similar geometric diameter. Here, we demonstrate effective application of this biomaterial by using the live attenuated tuberculosis vaccine bacille Calmette-Guérin (BCG). Prepared as a spray-dried nanomicroparticle aerosol, BCG vaccine exhibited high-efficiency delivery and peripheral lung targeting capacity from a low-cost and technically simple delivery system. Aerosol delivery of the BCG nanomicroparticle to normal guinea pigs subsequently challenged with virulent Mycobacterium tuberculosis significantly reduced bacterial burden and lung pathology both relative to untreated animals and to control animals immunized with the standard parenteral BCG.     

Unsteady penetration of a target by a liquid jet

It is widely acknowledged that ceramic armor experiences an unsteady penetration response: an impacting projectile may erode on the surface of a ceramic target without substantial penetration for a significant amount of time and then suddenly start to penetrate the target. Although known for more than four decades, this phenomenon, commonly referred to as dwell, remains largely unexplained. Here, we use scaled analog experiments with a low-speed water jet and a soft, translucent target material to investigate dwell. The transient target response, in terms of depth of penetration and impact force, is captured using a high-speed camera in combination with a piezoelectric force sensor. We observe the phenomenon of dwell using a soft (noncracking) target material. The results show that the penetration rate increases when the flow of the impacting water jet is reversed due to the deformation of the jet–target interface––this reversal is also associated with an increase in the force exerted by the jet on the target. Creep penetration experiments with a constant indentation force did not show an increase in the penetration rate, confirming that flow reversal is the cause of the unsteady penetration rate. Our results suggest that dwell can occur in a ductile noncracking target due to flow reversal. This phenomenon of flow reversal is rather widespread and present in a wide range of impact situations, including water-jet cutting, needleless injection, and deposit removal via a fluid jet.Ceramic materials, although having a reputation for being inherently brittle, have been used for different armor systems for almost a century. The application of ceramic-based armor ranges from protection of aircraft and personnel against small-caliber threats, to vehicle armor designed to defeat long-rod penetrators and shaped charges.Here, we consider thick, well-confined ceramic armor systems designed to withstand the high-velocity impact of heavy-metal long-rod penetrators. Such systems are extensively used to study the dynamic penetration properties of ceramics over relatively long time frames: the confinement prevents/reduces the macroscopic cracking of the ceramic target and thus provides a more controlled experimental setting where statistical effects of cracking are minimized.The time-resolved penetration behavior of ceramic armor has been extensively studied over the past 45 y using flash radiography, a technique used for impact experiments involving optically opaque targets (1–4). As sketched in Fig. 1, the typical penetration–time behavior of a ceramic target impacted by a metallic long rod is characterized by three cases depending on the impact velocity . Case I: Below a critical impact velocity, the metallic long rod erodes completely on the target surface without significant penetration (Fig. 1A). This phenomenon is commonly referred to as interface defeat and the critical transition velocity is labeled “.”Open in a separate windowFig. 1.Sketches illustrating the three regimes of penetration of ceramic targets by long-rod penetrators as a function of projectile impact velocity . (A) For , the long-rod projectile is eroded on the target surface without significant penetration. This regime is called “interface defeat.” (B) For slightly larger than , the projectile dwells on the target surface for at least 29 μs before penetration commencing at a high rate. (C) For , penetration into the target occurs after a much shorter dwell period. (D) Sketch of typical penetration rate versus time curves for the three cases I–III. (A and B adapted from ref. 10; C adapted from ref. 12.)Case II: For velocities slightly higher than the transition velocity, a phase of projectile defeat followed by penetration can be observed (Fig. 1B). The phase without penetration has been termed dwell because the penetrator “dwells” on the target surface for a duration called dwell time. Case III: As the impact velocity increases well beyond the transition velocity, dwell times decrease until they become too short to be measured (Fig. 1C) (5).The most widely used explanation for the phenomenon of dwell is cracking-induced loss of penetration resistance of the target material. This explanation is mainly based on postmortem examination of recovered targets (6, 7). A region of intense microcracking and cone cracks immediately under the impact site was observed in cross-sections of ceramic targets subjected to impacts just below the transition velocity. Consequently, it is hypothesized that dwell occurs during the time when the ceramic transitions from an intact to a damaged state (5, 8).However, three critical failings of this hypothesis can be identified. First, whereas microcracking (comminution) was observed in silicon carbide (SiC), no comminution occurred in tungsten carbide (WC) and boron carbide (B₄C) targets, even though they display the phenomenon of dwell. Second, the process of cracking takes place on a timescale of the order of 1 μs (9), whereas dwell times can be up to two orders of magnitude higher (10, 11). Finally, there is experimental evidence that the penetration rate during dwell is not zero, indicating that the target material is pushed away or removed already during dwell (12). To conclude, the cracking-induced loss of penetration resistance of the target does not provide a satisfactory explanation for the origins of dwell.In search for an alternative explanation, we note that Renström et al. (13) demonstrated that the loading from the impacting long rod on the ceramic targets is predominantly inertial (or hydrodynamic) in nature. This raises the question of whether fluid–structure interaction (FSI) between the fluid-like projectile and the deformable target might offer another possible explanation for the dwell phenomenon. FSI effects have been studied in the context of sand sprays impacting deformable structures (14, 15). Similar to the impacting long rod, the loading by the sand is also primarily inertial in nature. In such cases there is a substantial increase in the momentum transmitted by the sand to a deformable structure compared with sand impact on a rigid structure.In the ceramic target experiments with which we are concerned, the eroding projectile seems to exhibit a flow alteration due to the deformation of the target surface (Fig. 1). This flow alteration is also observed in numerical simulations of related experiments (16). The situation closely resembles a water jet hitting a dimpled rigid surface as shown in Fig. 2, a textbook example from which we know that the transmitted momentum and the impact force increase with increasing curvature of the surface (17). Considering the rate of momentum of the fluid striking the target surface and the fluid leaving the target surface, the force exerted on the target by the jet (in the direction of the incoming jet) is given bywhere is the angle between incoming and exiting fluid, is the material density of the jet, and the cross-sectional area of the jet. This simple expression can be used to gauge what phenomenon might occur when a jet impacts a deforming target (for cases when the jet velocity is much larger than the penetration rate). With increasing deformation of the surface of the target, decreases, resulting in an increase in the force ––it is this FSI effect that we hypothesize results in the unsteady penetration rate of the target. Numerical simulations, such as those in ref. 16, suggest that the impact pressure is affected by the penetration–deformation of the target surface, but they show no clear correlation between the impact pressure variations and the associated penetration rates.Open in a separate windowFig. 2.Schematic view of the proposed FSI mechanism resulting in an unsteady penetration rate due to the impact of a fluid jet with velocity . (A) During the initial stages of the jet impact, the target surface is flat and the fluid spreads horizontally. (B) As the jet deforms the target and penetrates at depth δ, it creates a dimple at the impact site; the flow pattern changes, resulting in backflow of the fluid with a velocity and a consequent increase in the impact force , which increases as the jet exit angle decreases.This study aims to explore the FSI during projectile impact by using scaled analog experiments. These are designed to circumvent the two major challenges associated with the previously described ceramic target experiments, namely: (i) to obtain direct impact force measurements, and (ii) to achieve a sufficient spatial and temporal resolution of the 3D problem with a very limited number of 2D images. We therefore perform experiments wherein a low-velocity water jet impacts on a translucent gel.We show that our scaled analog experiments reproduce the penetration versus time responses observed for ceramic targets impacted by long-rod penetrators, albeit at much lower impact velocities and consequent penetration rates. Results presented here include three impact velocities: slightly lower, slightly higher, and significantly higher than the transition velocity . Similar to the ceramic target experiments, the loading times in our scaled experiments are much longer than the time for elastic wave propagation through the target. This implies that on the timescale of the loading, the target is in static equilibrium insomuch as the force exerted by the jet on the target is equal to the reaction force between the target and the support structure. Thus, to quantify the FSI effect we perform direct impact force measurements by placing the translucent gel target on a high-sensitivity piezoelectric force sensor. The force measured by the transducer is equal to the impact force. We present these measurements in terms of a nominal impact pressure defined aswhere is the cross-sectional area of the incoming jet.We also perform a series of control creep penetration experiments wherein the penetration force is kept constant by applying a fixed load on a steel-rod penetrator with cross-sectional area , i.e., equal to that of the water jet. The control experiments are designed such that the penetration pressure is equal to that exerted by the water jet on a rigid flat target:The contrast between the water-jet penetration versus time responses and those in the control creep experiments will serve to demonstrate that it is the FSI effect that results in the observed unsteady penetration response.     

Transplantation of a myelodysplastic syndrome by a long-term repopulating hematopoietic cell

The myelodysplastic syndromes (MDS) comprise a group of premalignant hematologic disorders characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). Although it is well established that many malignancies can be transplanted, there is little evidence to demonstrate that a premalignant disease entity, such as MDS or colonic polyps, can be transplanted and subsequently undergo malignant transformation in vivo. Using mice that express a NUP98-HOXD13 (NHD13) transgene in hematopoietic tissues, we show that a MDS can be transplanted to WT recipients. Recipients of the MDS bone marrow displayed all of the critical features of MDS, including peripheral blood cytopenias, dysplasia, and transformation to AML. Even when transplanted with a 10-fold excess of WT cells, the NHD13 cells outcompeted the WT cells over a 38-week period. Limiting-dilution experiments demonstrated that the frequency of the cell that could transmit the disease was ≈1/6,000–1/16,000 and that the MDS was also transferable to secondary recipients as a premalignant condition. Transformation to AML in primary transplant recipients was generally delayed (46–49 weeks after transplant); however, 6 of 10 secondary transplant recipients developed AML. These findings demonstrate that MDS originates in a transplantable, premalignant, long-term repopulating, MDS-initiating cell.     

Sympatric speciation by allochrony in a seabird

The importance of sympatric speciation (the evolution of reproductive isolation between codistributed populations) in generating biodiversity is highly controversial. Whereas potential examples of sympatric speciation exist for plants, insects, and fishes, most theoretical models suggest that it requires conditions that are probably not common in nature, and only two possible cases have been described for tetrapods. One mechanism by which it could occur is through allochronic isolation-separation of populations by breeding time. Oceanodroma castro (the Madeiran or band-rumped storm-petrel) is a small seabird that nests on tropical and subtropical islands throughout the Atlantic and Pacific Oceans. In at least five archipelagos, different individuals breed on the same islands in different seasons. We compared variation in five microsatellite loci and the mitochondrial control region among 562 O. castro from throughout the species' range. We found that sympatric seasonal populations differ genetically within all five archipelagos and have ceased to exchange genes in two. Population and gene trees all indicate that seasonal populations within four of the archipelagos are more closely related to each other than to populations from the same season from other archipelagos; divergence of the fifth sympatric pair is too ancient for reliable inference. Thus, seasonal populations appear to have arisen sympatrically at least four times. This is the first evidence for sympatric speciation by allochrony in a tetrapod, and adds to growing indications that population differentiation and speciation can occur without geographic barriers to gene flow.     

IgG glycan hydrolysis by a bacterial enzyme as a therapy against autoimmune conditions

EndoS from Streptococcus pyogenes efficiently hydrolyzes the functionally important and conserved N-linked glycan of IgG in human blood. Repeated i.v. administration of EndoS in rabbits completely hydrolyzes the glycans of the whole IgG pool, despite the generation of anti-EndoS antibodies. EndoS administration had no apparent effects on the health of the animals. EndoS hydrolysis of the IgG glycan has profound effects on IgG effector functions, such as complement activation and Fc receptor binding, suggesting that the enzyme could be used as an immunomodulatory therapeutic agent against IgG-mediated diseases. We demonstrate here that EndoS indeed has a protective effect in a mouse model of lethal IgG-driven immune (or idiopathic) thrombocytopenic purpura. EndoS pretreatment of pathogenic antibodies inhibits the development of disease, and the enzyme also rescues mice from already established disease when severe thrombocytopenia and s.c. bleeding have developed. These results identify EndoS as a potential therapeutic agent against diseases where pathogenic IgG antibodies are important and further emphasize antibody glycans as possible targets in future therapies against antibody-mediated autoimmune conditions.     

Rapid protection in a monkeypox model by a single injection of a replication-deficient vaccinia virus

The success of the World Health Organization smallpox eradication program three decades ago resulted in termination of routine vaccination and consequent decline in population immunity. Despite concerns regarding the reintroduction of smallpox, there is little enthusiasm for large-scale redeployment of licensed live vaccinia virus vaccines because of medical contraindications and anticipated serious side effects. Therefore, highly attenuated strains such as modified vaccinia virus Ankara (MVA) are under evaluation in humans and animal models. Previous studies showed that priming and boosting with MVA provided protection for >2 years in a monkeypox virus challenge model. If variola virus were used as a biological weapon, however, the ability of a vaccine to quickly induce immunity would be essential. Here, we demonstrate more rapid immune responses after a single vaccination with MVA compared to the licensed Dryvax vaccine. To determine the kinetics of protection of the two vaccines, macaques were challenged intravenously with monkeypox virus at 4, 6, 10, and 30 days after immunization. At 6 or more days after vaccination with MVA or Dryvax, the monkeys were clinically protected (except for 1 of 16 animals vaccinated with MVA), although viral loads and number of skin lesions were generally higher in the MVA vaccinated group. With only 4 days between immunization and intravenous challenge, however, MVA still protected whereas Dryvax failed. Protection correlated with the more rapid immune response to MVA compared to Dryvax, which may be related to the higher dose of MVA that can be tolerated safely.     

Recognition of trimethyllysine by a chromodomain is not driven by the hydrophobic effect

Posttranslational modifications of histone proteins regulate gene expression via complex protein-protein and protein-DNA interactions with chromatin. One such modification, the methylation of lysine, has been shown to induce binding to chromodomains in an aromatic cage [Nielsen PR, et al. (2002) Nature 416:103-107]. The binding generally is attributed to the presence of cation-pi interactions between the methylated lysine and the aromatic pocket. However, whether the cationic component of the interaction is necessary for binding in the aromatic cage has not been addressed. In this article, the interaction of trimethyllysine with tryptophan is compared with that of its neutral analog, tert-butylnorleucine (2-amino-7,7-dimethyloctanoic acid), within the context of a beta-hairpin peptide model system. These two side chains have near-identical size, shape, and polarizabilities but differ in their charges. Comparison of the two peptides reveals that the neutral side chain has no preference for interacting with tryptophan, unlike trimethyllysine, which interacts strongly in a defined geometry. In vitro binding studies of the histone 3A peptide containing trimethyllysine or tert-butylnorleucine to HP1 chromodomain indicate that the cationic moiety is critical for binding in the aromatic cage. This difference in binding affinities demonstrates the necessity of the cation-pi interaction to binding with the chromodomain and its role in providing specificity. This article presents an excellent example of synergy between model systems and in vitro studies that allows for the investigation of the key forces that control biomolecular recognition.     

Lattice defects induced by microtubule-stabilizing agents exert a long-range effect on microtubule growth by promoting catastrophes

Microtubules are dynamic cytoskeletal polymers that spontaneously switch between phases of growth and shrinkage. The probability of transitioning from growth to shrinkage, termed catastrophe, increases with microtubule age, but the underlying mechanisms are poorly understood. Here, we set out to test whether microtubule lattice defects formed during polymerization can affect growth at the plus end. To generate microtubules with lattice defects, we used microtubule-stabilizing agents that promote formation of polymers with different protofilament numbers. By employing different agents during nucleation of stable microtubule seeds and the subsequent polymerization phase, we could reproducibly induce switches in protofilament number and induce stable lattice defects. Such drug-induced defects led to frequent catastrophes, which were not observed when microtubules were grown in the same conditions but without a protofilament number mismatch. Microtubule severing at the site of the defect was sufficient to suppress catastrophes. We conclude that structural defects within the microtubule lattice can exert effects that can propagate over long distances and affect the dynamic state of the microtubule end.

Microtubules are cytoskeletal polymers that rapidly switch between phases of growth and shortening, and this behavior, termed dynamic instability, plays a crucial role in the formation, maintenance, and reorganization of microtubule arrays during cell division, migration, and differentiation (1, 2). The transition from growth to shrinkage, an event called catastrophe, is known to occur when the protective cap of guanosine triphosphate (GTP)–bound tubulin subunits is reduced or lost, but the underlying mechanisms are still the subject of investigation (3, 4). One interesting property of microtubules is that the frequency of catastrophes depends on microtubule age: Microtubules that are growing for a longer time have a higher chance to switch to depolymerization (5, 6). Changes occurring at the microtubule end, such as loss of individual protofilaments or end tapering, have been shown to promote catastrophe (7–9). In principle, it is also possible that the catastrophe frequency at the plus end is affected by structural features in the microtubule lattice farther away from the tip, but this possibility has so far remained untested.Structural studies have established that tubulin can form tubes with different protofilament numbers (10), dependent on the species, nucleation template, presence of different microtubule-associated proteins, and other properties of the polymerization reaction (e.g., ref. 11; reviewed in ref. 12). An important consequence of the structural plasticity of the microtubule lattice (13) is the formation of lattice defects, such as sites where a microtubule gains or loses one or more protofilaments (11, 14–17). A recent cryoelectron tomography analysis showed that in some cell types, such as Drosophila neurons, variations and transitions in protofilament number are readily detectable (18) and are thus likely to be physiologically relevant. Switches in protofilament number can be introduced during microtubule growth, and their presence may affect microtubule dynamics in different ways. For example, defects can be repaired through tubulin incorporation, and the resulting islands of GTP-tubulin can trigger microtubule rescue (19–22). On the other hand, the presence of defects could potentially also induce catastrophes (as proposed in ref. 14), since conformational properties of the microtubule lattice might propagate over some distances (23).To study the relationship between lattice defects and microtubule catastrophes, one should be able to directly correlate the presence of defects with the dynamics of microtubule ends. We recently found that fluorescent analogs of microtubule-stabilizing agents (MSAs) can be used to induce microtubule lattice defects that can be visualized by fluorescence microscopy. When present at low concentrations, MSAs preferentially bind to microtubule plus ends that enter a “precatastrophe” state (24), which is manifested by the gradual loss of the GTP cap and reduced recruitment of end-binding (EB) proteins that detect GTP-bound microtubule lattice (25–27). Strong accumulation of MSAs at precatastrophe microtubule ends leads to the formation of stabilized patches of microtubule lattice, where the tube is incomplete and keeps incorporating GTP-tubulin but is not fully repaired (24). When microtubules switch to depolymerization, such persistent lattice defects, which coincide with the hotspots of MSA binding, can induce repeated rescues and, therefore, they were termed “stable rescue sites” (24).Here, we used MSA-induced lattice defects to address two questions. First, what prevents complete repair of an MSA-induced persistent lattice defect? And second, does the presence of such a persistent defect affect the dynamics of the microtubule plus end? Since different MSAs are known to affect the number of protofilaments (15, 28–33), we hypothesized that persistent lattice defects could be associated with the changes in protofilament number and thus could not be fully repaired for geometrical reasons. We tested this idea by generating stable microtubule seeds with one MSA and then elongating them in the presence of another MSA, with the same or different preference for protofilament number. Use of fluorescent MSAs allowed us to directly follow drug binding. We found that precatastrophe microtubule ends accumulated MSAs in all conditions; however, the outcome of drug binding was different. When there was no mismatch in protofilament number between the seeds and the elongation conditions, drug accumulations were short in duration and length, and microtubule growth beyond such sites was processive. In contrast, when, based on the MSA properties, a mismatch in protofilament number could be expected, large and persistent drug accumulations were formed. The existence of such mismatches was confirmed by cryoelectron microscopy (cryo-EM) and by measuring microtubule growth rate, which became higher with increasing protofilament number. When microtubule ends extended beyond a mismatch-containing lattice defect, they displayed elevated catastrophe frequency. Laser-mediated severing of a microtubule at the site of the persistent defect reduced catastrophe frequency at the plus end. Our data demonstrate that local perturbations in microtubule structure can affect the state of the dynamic end at a distance of several micrometers.     

Reduction of myocardial infarct size by a fluorocarbon-oxygenated reperfusate

This investigation assesses whether the size of an acutely revascularized myocardial infarct (MI) could be reduced by altering the composition of the initial reperfusate. Nineteen open-chest dogs underwent 4-hour occlusion of the left anterior descending coronary artery and were then assigned to a treatment group: 12 dogs to selective intracoronary infusion of the modified reperfusate over 30 minutes before resumption of blood flow for 60 minutes and 7 to a control group (90 minutes of unmodified blood reperfusion). The modified reperfusate consisted of 500 ml of a fluorocarbon-oxygenated crystalloid solution (PO2 650 mm Hg; total O2 content 5.5 vol%) whose composition was adjusted by decreasing Ca++ (0.25 mM), increasing pH (7.60) and adding glucose (1.8 g/liter). Four hours after occlusion, technetium-99m-labeled microspheres were injected into the left atrium. After 90 minutes of reperfusion, the heart was removed and sliced transversely. Areas not perfused by microspheres (areas at risk) were traced, planimetered and compared with the areas of necrosis after incubation in triphenyltetrazolium chloride. Areas were then converted into weights. In control dogs, the weight of necrotic myocardium was not significantly different from the weight at risk (5.0 +/- 0.7 vs 7.0 +/- 0.8 g, respectively [mean +/- standard error of the mean]), whereas it was markedly reduced in treated dogs (5.9 +/- 0.5 vs 9.4 +/- 0.7 g, respectively, p less than 0.001). The weight of salvaged myocardium was 3.4 +/- 0.5 g in treated dogs vs 1.9 +/- 0.4 g in the control group (p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylsalicylic Acid-Induced Gastrointestinal Bleeding Determined by a 51Cr Method on a Day-to-Day Basis

The daily gastrointestinal blood loss caused by plain and microencapsulated acetylsalicylic acid (ASA) tablets was compared. Fourteen healthy, male volunteers participated in a double-blind, cross-over study, lasting 38 days. Before drug administration a median gastrointestinal bleeding of 0.9 ml/24 h was observed. During oral intake of 1.5 g ASA twice a day for 5 days, an increased faecal blood loss was seen in all volunteers. The increase was significant for both plain and microencapsulated ASA (p < 0.01). Plain ASA tablets, however, caused a greater faecal blood loss than the microencapsulated tablets (p = 0.05), maximum median levels being 6.2 ml/24 h and 3.9 ml/24 h, respectively. An optimal design of radiochromium studies for determination of drug-induced gastrointestinal blood loss is discussed.     

Characterization of an Exopolymeric Flocculant Produced by a Brachybacterium sp.

We evaluated the bioflocculant production potential of an Actinobacteria, which was isolated from a freshwater environment in the Eastern Cape province of South Africa. 16S rDNA nucleotide sequencing analyses revealed that the actinobacteria belongs to the Brachybacterium genus, and the sequences were deposited in the GenBank as Brachybacterium sp. UFH, with accession number {"type":"entrez-nucleotide","attrs":{"text":"HQ537131","term_id":"312064211","term_text":"HQ537131"}}HQ537131. Optimum fermentation conditions for bioflocculant production by the bacteria include an initial medium pH of 7.2, incubation temperature of 30 °C, agitation speed of 160 rpm and an inoculum size of 2% (vol/vol) of cell density 3.0 × 10⁸ CFU/mL. The carbon, nitrogen and cation sources for optimum bioflocculant production were maltose (83% flocculating activity), urea (91.17% flocculating activity) and MgCl₂ (91.16% flocculating activity). Optimum bioflocculant production coincided with the logarithmic growth phase of the bacteria, and chemical analyses of the bioflocculant showed 39.4% carbohydrate and 43.7% protein (wt/wt). The mass ratio of neutral sugar, amino sugar and uronic acids was 1.3:0.7:2.2. Fourier transform infrared spectroscopy (FTIR) indicated the presence of carboxyl, hydroxyl and amino groups, amongst others, typical for heteropolysaccharide and glycosaminoglycan polysaccharides. Bioflocculant pyrolysis showed thermal stability at over 600 °C, while scanning electron microscope (SEM) imaging revealed a maze-like structure of interlaced flakes. Its high flocculation activity suggests its suitability for industrial applicability.     

Regulation of flagellar dynein activity by a central pair kinesin

The motility of cilia and flagella is powered by dynein ATPases associated with outer doublet microtubules. However, a flagellar kinesin-like protein that may function as a motor associates with the central pair complex. We determined that Chlamydomonas reinhardtii central pair kinesin Klp1 is a phosphoprotein and, like conventional kinesins, binds to microtubules in vitro in the presence of adenosine 5'-[beta,gamma-imido]triphosphate, but not ATP. To characterize the function of Klp1, we generated RNA interference expression constructs that reduce in vivo flagellar Klp1 levels. Klp1 knockdown cells have flagella that either beat very slowly or are paralyzed. EM image averages show disruption of two structures associated with the C2 central pair microtubule, C2b and C2c. Greatest density is lost from part of projection C2c, which is in a position to interact with doublet-associated radial spokes. Klp1 therefore retains properties of a motor protein and is essential for normal flagellar motility. We hypothesize that Klp1 acts as a conformational switch to signal spoke-dependent control of dynein activity.     

Chemoprevention of colorectal cancer by aspirin: a cost-effectiveness analysis.

BACKGROUND & AIMS: The aim of the study is to compare the cost-effectiveness of aspirin and colonoscopy in the prevention of colorectal cancer. METHODS: A Markov process is used to follow a hypothetical cohort of 100,000 subjects aged 50 years until death. Four strategies are compared: (1) no intervention, (2) colonoscopy once per 10 years and every 3 years in subjects with polyps, (3) chemoprevention with 325 mg of daily aspirin, and (4) combination of the second and third strategies. The various strategies are compared calculating incremental cost-effectiveness ratios (ICERs). RESULTS: The expected number of colorectal cancers is 5904 per 100,000 subjects. Colonoscopy prevents 4428 colorectal cancers and saves 7951 life-years at an ICER of $10,983 per life-year saved compared with no intervention. Aspirin prevents 2952 colorectal cancers and saves 5301 life-years at an ICER of $47,249 per life-year saved compared with no intervention. The cost of aspirin therapy plus management of aspirin-related complications was reported to be $172 per year per patient. Varying the annual aspirin-related costs between $50 and $200 results in ICER changes between $4617 and $57,080, with the 2 strategies breaking even at $70. Applying aspirin chemoprevention plus colonoscopy screening concomitantly yields an ICER of $227,607 per life-year saved compared with screening colonoscopy alone. CONCLUSION: As compared with colonoscopy once per 10 years, the use of aspirin to prevent colorectal cancer saves fewer lives at higher costs. The high complication cost and the lower efficacy of aspirin render screening colonoscopy a more cost-effective strategy to prevent colorectal cancer.     

Suppression of hepatitis C virus replication by cyclosporin a is mediated by blockade of cyclophilins

BACKGROUND & AIMS: Cyclosporin A specifically suppresses hepatitis C virus (HCV) replication in vitro at clinically achievable concentrations. In this study, we investigated the mechanisms of action of cyclosporin A against HCV replication. METHODS: The in vitro effects of cyclosporin A on HCV replication were analyzed using an HCV replicon system that expresses chimeric luciferase reporter protein. RESULTS: The significant effects of cyclosporin A on expression of an HCV replicon and the absence of such effects of FK506, which shares mechanisms of action with cyclosporin A, suggested the involvement of intracellular ligands of cyclosporin A, the cyclophilins. Transient and stable knockdown of the expression of cytoplasmic cyclophilins A, B, and C by short hairpin RNA-expressing vectors suppressed HCV replication significantly. A cyclosporin analogue, cyclosporin D, which lacks immunosuppressive activity but exhibits cyclophilin binding, induced a similar suppression of HCV replication. Furthermore, cyclosporin A treatment of Huh7 cells induced an unfolded protein response exemplified by expression of cellular BiP/GRP78. Treatment of cells with thapsigargin and mercaptoethanol, which induce the unfolded protein responses, suppressed HCV replication, suggesting that the cyclosporin-induced unfolded protein responses might contribute to the suppression of HCV protein processing and replication. CONCLUSIONS: The anti-HCV activity of cyclosporin A is mediated through a specific blockade of cyclophilins, and these molecules may constitute novel targets for anti-HCV therapeutics.     

Homologous ligands accommodated by discrete conformations of a buried cavity

Conformational change in protein–ligand complexes is widely modeled, but the protein accommodation expected on binding a congeneric series of ligands has received less attention. Given their use in medicinal chemistry, there are surprisingly few substantial series of congeneric ligand complexes in the Protein Data Bank (PDB). Here we determine the structures of eight alkyl benzenes, in single-methylene increases from benzene to n-hexylbenzene, bound to an enclosed cavity in T4 lysozyme. The volume of the apo cavity suffices to accommodate benzene but, even with toluene, larger cavity conformations become observable in the electron density, and over the series two other major conformations are observed. These involve discrete changes in main-chain conformation, expanding the site; few continuous changes in the site are observed. In most structures, two discrete protein conformations are observed simultaneously, and energetic considerations suggest that these conformations are low in energy relative to the ground state. An analysis of 121 lysozyme cavity structures in the PDB finds that these three conformations dominate the previously determined structures, largely modeled in a single conformation. An investigation of the few congeneric series in the PDB suggests that discrete changes are common adaptations to a series of growing ligands. The discrete, but relatively few, conformational states observed here, and their energetic accessibility, may have implications for anticipating protein conformational change in ligand design.The importance of conformational flexibility in protein–ligand interactions is widely acknowledged. Structural studies of model systems such as dihydrofolate reductase (1, 2), cyclophilin A (3), adenylate kinase (4), and others (5, 6) have suggested that conformational changes in the protein are coupled to progress along the catalytic reaction coordinate, and that local fluctuations can affect coupling between binding and global transitions (7). For signal transduction, the importance of such conformational changes has long been recognized (8), and has been emphasized by recent experimental (9) and computational studies (10). Accordingly, molecular dynamics simulations of protein–ligand complexes are now widely considered for ligand design (11–16).Despite the attention lavished on protein conformational change overall, the incremental protein accommodations that might be expected over a series of ligand perturbations have received less consideration. Indeed, in the teeth of the “methyl, ethyl, propyl, butyl…futile” aphorism and the many medicinal chemistry programs that explore such incremental perturbations, surprisingly few crystal structures of congeneric ligands bound to a single protein are publicly available. Of the few there are, none resolve decisively how a protein might accommodate a congeneric series of ligands. If we define a congeneric series as one with at least six ligands related through an incremental change in functionality, then only 13 of these are known in the Protein Data Bank (PDB), and all but 2 (bold and underlined in SI Appendix, Table S4) of these undergo little conformational change upon ligand binding—a point to which we return. Conversely, ligand binding leads to substantial conformational changes in therapeutic targets such as aldose reductase (17), dihydrofolate reductase (2), and tRNA-guanine transglycosylase (18), but here the perturbations among the ligands have often not been systematic enough to disentangle changes in ligand size and polarity, making it harder to isolate the receptor conformational changes involved and their origins. In addition, similar ligands can adopt dissimilar binding modes in the same protein (19).Ideally, one would like series of ligands where size and physical properties are increased incrementally without introducing other perturbations that could change binding determinants. Correspondingly, one would like a site where the growing ligand forces receptor accommodations. One system that recommends itself is the cavity site in T4 lysozyme created by the substitution Leu99→Ala (L99A cavity). Formed in the hydrophobic core of the protein, the resulting 150-ų cavity is sequestered from solvent and is almost entirely apolar. Seminal studies by Matthews and colleagues demonstrated that this cavity can bind aryl hydrocarbons (20, 21), and since then the cavity and related mutants have become model systems for ligand recognition (22–29). Contributing to this status has been the commercial availability of thousands of likely ligands, many closely related to one another. This is something that is untrue of larger, more complicated binding sites, where fewer likely ligands are readily available, and fewer still in congeneric series.Here we determine the structures of eight alkyl benzenes in complex with the L99A cavity, including complexes with benzene, toluene, ethylbenzene, n-propylbenzene, sec-butylbenzene, n-butylbenzene, n-pentylbenzene, and n-hexylbenzene, as well as the apo cavity, at resolutions ranging from 1.39 to 1.80 Å. Because only benzene may be readily accommodated by the apo cavity but most other members of the series bound with greater affinity, this series seemed well-suited to exploring ligand-provoked conformational changes. We asked whether the cavity continuously adapted its conformation to these incremental enlargements in ligand size or whether instead the cavity jumped to discrete conformational states. Using multiconformational crystallographic refinement, we investigated whether the new conformations of the cavity were accessible to the ground state or were more distant in energy. Expecting continuous changes among conformations, we were surprised by the structures that emerged. Comparison with other series in the PDB suggests that these types of protein changes may be common, with implications for anticipating protein accommodation in ligand design.