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Joemgu

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Summarize: TECHNICAL FIELD [0001] The invention relates to abrasive particle cleaning products and methods, and, more particularly, to a novel cleaning formulation which environmentally manages interparticle and applicator-to-particle interactions to mechanically position particles for effective cleaning. CROSS REFERENCE TO RELATED APPLICATIONS [0002] (Not applicable) BACKGROUND OF THE INVENTION [0003] For most consumers, cleaning porcelain, for example in a bathroom or other area, is a disagreeable, difficult and, often, ineffectively executed task. More problematic, in geographic areas where water hardness exceeds ideal levels (85% of the United States), cleaning bathroom surfaces, such as sinks, toilets, and tubs is especially challenging due to more serious build-ups of scum, hard water rings, rust, or lime and other mineral deposits. Moreover, cleaning requires that a consumer must usually buy additional items, such as sponges, scrub pads, brushes or applicators before a porcelain cleaner can be used. This creates the possibility that inappropriate implements will be used because they are on hand without purchase, or that the consumer will purchase the wrong applicators. In both cases, the consumer may not even recognize there is a problem. SUMMARY OF THE INVENTION [0004] Perhaps more seriously, cleaning products, in addition to including abrasive constituents, may rely on harsh chemicals designed to attack the constituent elements of built-up stains using a variety of chemical modalities, such as oxidation (for example chlorine-based products, or potentially more aggressive solutions such as muriatic acid, and so forth), surfactant activity and so forth. These attack the hands of the user, for example by burning off one or multiple layers of skin, and adversely affect the environment by leaving poisonous and very long-lived materials in the environment, whether that environment is a river, and ocean or more seriously, a septic field. These concerns may be particularly serious given the widespread use of septic systems and the relatively common occurrence of people having home gardens where, somewhat ironically, they might raise greens, vegetables, fruit and other crops with the objective of having a relatively clean source of food. [0005] Further environmental concerns are raised by the relatively high mobility of chemical agents when they enter the environment. Moreover, many stains may be resistant to chemical-based cleaning compounds. While some products include abrasives, those products that include very fine abrasive particles (typically in a range about approximately 50 microns) in the formulation are most effective, despite the omission of larger potentially more abrasive larger particles. Such very fine abrasive particle formulation products are applied using a sponge, cloth or the like. The combination of sponge and fine particles results in direct mechanical pressing to fix the position of enough abrasive particles to clean a surface with a modest degree of effectiveness, provided that a relatively large patch of sponge is subjected to pressure by the fingers of the user. [0006] The present invention recognizes that larger abrasive particles are most effective when distributed evenly, but tend to accumulate in areas where they are not needed when mechanically scrubbed. They thus tend to scatter away from the point of application of force, using, for example, a sponge. Accordingly, such cleaners are applied in dry powder form evenly over the area to be cleaned, and then scrubbed with a cloth, following which the particles will quickly absorb into the cloth during scrubbing. New particles must then be applied. [0007] Similarly, when using a brush, during scrubbing, larger particles easily slip between bristles to positions where their abrasive effect is substantially compromised. [0008] In contrast, in accordance with the present invention, the viscosity of a cleaning preparation is controllably increased. During cleaning, the top surface of a deposit of cleaning preparation is put in contact with the bottom surface of a sponge, scouring pad or other applicator (such as a cloth) which under the influence of pressure applied by the hands of the individual performing the cleaning, is subjected to movement substantially parallel to the surface being cleaned. [0009] In accordance with the invention, viscosity is controlled in the non-Newtonian fluid in which abrasive particles are suspended to achieve desired abrasive orientation. Relative to the movement of the applicator, the workpiece being cleaned (such as the porcelain surface of a toilet bowl) is stationary, resulting in shear forces which equate to the application of one torque at the work surface associated with the workpiece, and the opposite torque at the work surface associated with the applicator. [0010] This results in rotating particles which are suspended within the relatively high viscosity cleaning preparation. The result is that, particularly in the case of the relatively large particles of abrasive material, suspended abrasives in the inventive cleaning preparation rotate to positions where they tend to lock against further rotation or slippage, thus resulting in more effective use of the forces applied to the cleaning preparation, and, in particular, the abrasive particles entrained therein. Because the invention uses relatively large abrasive particles, they tend to be rotated to a position where relatively pointed abrasive configurations are statically driven against the porcelain surface being cleaned. Thus, movement of the applicator parallel to the workpiece results in dragging these relatively pointed configurations against the workpiece with a minimal area of contact between the workpiece and the abrasives, but with a relatively large amount of force concentrated in and directed against the point of contact of the abrasive with the workpiece. Accordingly, relatively high pressure is concentrated at the point of interface between the abrasive material and the workpiece, thus resulting in substantial scraping forces being applied to accumulations of dirt, stains, mineral accumulations, and so forth deposited on the top surface of the porcelain being cleaned. [0011] In recent years, bathroom cleaning product kits have also been introduced to the market, typically comprising reusable or disposable pads (sometimes incorporating cleaning material) and handles on which they are mounted. However, these kits have shortcomings since they are geared towards cleaning only certain sections of the bathroom. Some bathroom cleaner kits are geared toward cleaning toilet bowls only, whereas others are geared toward cleaning bath tubs only. [0012] While this allows a consumer to purchase a cleaning kit for a specific task, another problem emerges. In order to clean the whole bathroom, which among other things, includes cleaning the bath tub, toilet, and sink, the consumer will have to buy multiple products to get the job done. When the consumer purchases one of these specific cleaner kits, the consumer will also have to acquire additional items, such as sponges, brushes, or scrub pads because accessories that comprise these specialized bathroom cleaner kits may not be designed to be used to clean other bathroom surfaces. [0013] In addition, even the cleaning kits specifically designed to target particular areas of the bathroom have shortcomings in performing the tasks to which they are specifically designed for and dedicated. For example, when purchasing a toilet bowl cleaning kit, a consumer gets the cleaner with just a brush. Moreover, the brush, while it is effective to clean visible portions of the toilet bowl, has a shape which precludes the consumer from most effectively targeting and efficiently cleaning the underside of the toilet bowl periphery where the water ports, which feed the flushing operation, are located. As a result of this, water flow is restricted, the toilet bowl requires multiple flushes and, often, perfectly serviceable toilet bowls are discarded. [0014] Other cleaning kits comprise a cleaner and a sponge. While a sponge will work for large surface areas, one embodiment of the invention provides a cleaner with a separate brush and a sponge, abrasive particles having a range of larger sizes, and, most importantly, a composition with a thickening agent designed to increase the effectiveness of the larger abrasive particles by fixing position and providing for particle rotation to optimal or at least relatively effective positions with the particular applicators used, giving the inventive kit substantial capability over prior art. [0015] In addition, in accordance with the present invention, a brush and/or an applicator with a handle is provided. A preferred embodiment of the invention uses a scouring pad including a sponge and a fibrous top surface. Relatively stiff closely packed bristles or, alternatively, the sponge-based scouring pad are used in the brush of the inventive system because, in combination with the viscous, non-Newtonian nature of the carrier, they effectively trap large particulate particles, such as particles of pumice, and hold them in positions where they tend to be rotationally stabilized and bear against the porcelain surface being cleaned. More particularly, the viscosity of a mixture of the inventive cleaning composition not only maintains the particles in a suspended position which promotes even distribution of abrasive material, but also, working with the viscosity of the suspension maintains the particles proximate to the tips of the bristles, where they can be driven by flexed bristles against the surface being cleaned. In this configuration, the bent brush bristle tip is slanted at, for example, a 60° angle to the surface being cleaned, and the pumice (or other abrasive material) particles are pushed by the flexed bristles against the surface being cleaned and driven toward the forces adjacent the surface being cleaned and the tip of the bristle, where further movement either in the lateral direction (with respect to the brush) or of a rotational nature is prevented as the relatively large abrasive particle is wedged into the applicator surfaces and driven across the surface being cleaned. [0016] The present invention, in this manner, provides a solution to the above discussed problems. At the same time, thickening is provided by a natural agent which is both user-friendly and environment friendly. As compared to prior art harsh chemicals which can damage the skin of the user, whether chlorine bleach, muriatic acid or other infective agents, the inventive composition has the effect of increasing the strength of the cleaner, while without adversely affecting the environment in any substantial way. [0017] The cleaning compound and kit of the present invention provides the consumer a porcelain cleaner kit that may be used to remove hard water rings, rust, lime and mineral deposits on multiple porcelain surfaces, such as toilet bowl inner surfaces, toilet bowl water feeding jets, tubs, and sinks. Thus, the consumer has the flexibility to conform the cleaner kit to the requirements of the job and use it on multiple bathroom surfaces. [0018] For this reason, among others, the inventive ready-to-use porcelain cleaner composition and kit will save the consumer time, and often money, because the consumer will not have a need to purchase additional items to clean a bathroom. Moreover, the inventive system ensures that the combination of applicators and cleaning composition are all properly formulated to work with each other and with a wide variety of porcelain surfaces. [0019] The inventive ready-to-use porcelain cleaner kit has packaged together all components necessary for a wide range of porcelain cleaning jobs and is thus ready for use from the time of purchase. In addition, purchasing a ready-to-use porcelain cleaner kit reduces unnecessary pollution that is generated when the consumer buys these specific bathroom cleaning kits and then must buy additional accessories to clean other bathroom surfaces, and discard unsuitable cleaning compositions, brushes or other applicators. [0020] Furthermore, inventive ready-to-use porcelain cleaning kit has a brush whose shape is designed to specifically target hard to reach areas, such as the underside of a toilet bowl where the water feeding ports are located. [0021] When packaged together, the kit reduces manufacturing costs, assures efficient retail stock placement, and enables the consumer to use the product immediately after purchase because all relevant components are included and matched to the performance characteristics of the cleaning composition. [0022] In accordance with the invention, the pumice particle size distribution used desirably meets the specification of: for 1400 Micron or 14 U.S. Mesh allows 100% passing, 600 Micron or 30 U.S. Mesh allows 99.7-100 passing, 425 Micron or 40 U.S. Mesh allows 47%-67% passing, 300 Micron or 50 U.S. Mesh allows 3% -17% passing, 250 Micron or 60 U.S. Mesh allows 0% to 12% passing. BRIEF DESCRIPTION OF THE DRAWINGS [0023] The operation of the invention will become apparent from the following description taken in conjunction with the drawings, in which: [0024] FIG. 1 is a perspective view of the storage container that houses the cleaning kit components; [0025] FIG. 2 is a perspective view of the porcelain cleaner container; [0026] FIG. 3 is a perspective view of a five-fingered hand protective material; [0027] FIG. 4 is a perspective view of a deformable material incorporating voids; [0028] FIG. 5 is a perspective view of a scrubbing material incorporating voids; [0029] FIG. 6 is a perspective view of an applicator; [0030] FIG. 7 is a perspective view of a specialty brush; [0031] FIG. 8 illustrates cleaning using the inventive formulation; [0032] FIG. 9 is a perspective view of a specialty applicator; [0033] FIG. 10 is a perspective view of a handle portion of the specialty applicator of FIG. 9 ; [0034] FIG. 11 is a perspective view from the top of the sponge of the specialty applicator of FIG. 9, exposing a fibrous cleaning member; and [0035] FIG. 12 is a perspective view from the bottom of the sponge of the specialty applicator of FIG. 9, exposing the attachment member. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0036] Cleaning efficiency of a brush is believed to depend on the access of toothbrush bristles to sheltered areas and their ability to deliver sufficient force to remove accumulations as tufts of bristles travel over surfaces being cleaned. As a first-order approximation, brush stiffness may be said to predominantly depend upon elastic modulus of the bristles, bristle and tuft diameter, the number of tufts of bristles in the brush, the number of bristles per unit area packed into a particular tuft supporting hole, sometimes referred to as the packing factor, and the trim length of bristles. While the above characteristics are believed to be the primary influencers of brush hardness and cleaning efficiency, bristle composition and shape are believed, by some, to have no measurable effect. See, for example, Rawls et al, A Mathematical Model for Predicting Toothbrush Stiffness, available at http://www.ncbi.nlm.nih.gov/pubmed/2079171. [0037] In accordance with the invention, the cooperative relationship between larger particles and a relatively viscous cleaning composition carrier is used to advantage, with the viscosity of the cleaning composition carrier acting to promote interparticle and particle to applicator interactions which result in stabilizing agglomerations of particles and structural abrasive particle interlocking with the applicator to ensure localized and stable accumulations of abrasive materials in configurations designed to apply significant forces to accumulations of foreign matter on porcelain surfaces. [0038] More particularly, in one alcohol-based embodiment of the inventive cleaning composition, the liquid ingredients comprise linear alcohol ethoxylate (a coconut surfactant for cleaning/rinsing/stability), alkyl polyglucoside (sugar cane/coconut surfactant for cleaning/rinsing/stability), xanthan gum (a food grade environmentally friendly thickening agent), essential oils (e.g. lemon, mint, lavender, orange) and calcium carbonate (whitens the color of the mixture). [0039] To make the final formulation, the above liquid ingredients are mixed with pumice, 50% by weight liquid ingredients, and 50% by weight pumice. However, the percentage of pumice can range between 25% and 90% by weight of the final cleaning formulation. However, pumice concentrations between 40% and 65% by weight are preferred, and concentrations between 45% and 60% by weight are particularly preferred. [0040] In an alternative and particularly preferred but water-based embodiment of the invention, the ingredients comprise water—52%, xanthan gum—1.3% (natural thickener & suspending agent for the pumice), glycerin—5% (prevents water from evaporating, derived from coconut oil), laureth—9-1.00% (natural surfactant from coconut oil), peppermint oil—0.20% (natural Indonesian peppermint oil), sodium benzoate—0.50% (fda approved food grade preservative, keeps formula from going rancid), and pumice—40%. [0041] The percentage of pumice can range between 10% and 60% by weight of the final cleaning formulation. However, pumice concentrations between 30% and 60% by weight are preferred, and concentrations between 35% and 50% by weight are particularly preferred. Relative concentrations of the other (liquid) ingredients may, for example, be increased or decreased proportionately. [0042] Suitable thickeners include such gums as guar gum, locust bean gum (also known as carob bean gum), tara gum, xanthan, carrageenan, gellan gum or other vegetable or non-vegetable based thickeners, and may be included in concentrations between 0.3% and 9% by weight of the final composition, although concentrations between 0.5% and 4% by weight are preferred, and concentrations between 0.8% and 2.5% are particularly preferred. Combinations of natural gums, for example guar and xanthan with equal amounts of guar and xanthan have been found to have a synergistic effect. [0043] In accordance with the invention, the preferred abrasive is available from HESS Pumice Products, Inc. of Malad, Id. It comprises an igneous composition, namely pumice. This grade hardness of pumice is sold by Hess as Pumice Grade #2, and comprises a mineral known as pumicite. This is a non-crystalline silica, a mineral which is a member of the rhyolite family. The product used in the invention includes 0.3% impurities none of which are hazardous. The pumice is very white with a GE brightness of 84. Likewise, the product is very hard with a hardness on MOH'S scale of 5.8. Its specific gravity is 2.35 g/cc. Depending on grade the pumice may have a weight of 40-50 pounds per cubic foot. Pumice also has the advantage of being chemically and environmentally inert. [0044] In the inventive formulation, the highly porous nature of the pumice enables it to hold water and air. It is also very close to pH neutral, having a pH at 20° C. range of 7.5-8, and thus from chemical standpoint, not likely to cause any irritation to human skin. [0045] The cleaning composition used in the inventive kit has numerous other advantages. Moreover, tests have shown that is highly effective against a wide range of undesirable porcelain accumulations, while, at the same time, not scratching or otherwise adversely affecting the porcelain being cleaned. It is thus an effective answer to the problem of hard water fixture cleaning, a problem which affects 85% of the country based on information reported by the U.S. Geological Survey. Further, the inventive kit is effective in cleaning porcelain, regardless of age, because, despite significant changes in the manufacturing process, porcelain characteristics upon which the invention depends have not changed. As alluded to above, the product is not harmful to human skin, or even sensitive human skin. Nevertheless, it is very effective in breaking down and cleaning the calcium, magnesium and other mineral buildups, and other accumulations which porcelain suffers from. Thus, effective removal of rust stains, mineral deposits, limescale and oxidation products is achieved. [0046] Moreover, the inventive kit includes scour pads and a contoured applicator handle for ease of cleaning, as appears more fully below. In addition, a special brush is included for the purpose of cleaning rim ports and the secondary feed hole near the drain to allow more efficient water flow to the toilet. At the same time, the inventive system includes no harmful chemicals, which are safe for adults, children and pets due to their chemically benign nature. [0047] Also, as alluded to above, inventive kit is effective against chemically resistant stains that much harsher cleaners including oxidizers such as bleach and acids effectively combat. At the same time, the viscosity of the cleaning preparation of the invention provides for more effective and evenly spread deployment of the pumice abrasive both during application and during use, as well as the time that the inventive composition is sitting on the surface without being worked as the user moves from one area to another in the cleaning operation, while providing for the convenience of deposit on vertical surfaces and, accordingly, easier cleaning. [0048] After use, the container provided with the kit provides for organized storing of the complete system in one place for easy retrieval and a low likelihood of loss of one or more components. [0049] Moreover, in accordance with a preferred embodiment of the invention, packaging materials are selected for suitability for recycling and reuse, thus further increasing the environmentally friendly nature of the product. [0050] In accordance with one embodiment of the invention, scouring pads may optionally be made from biodegradable plastic, with or without incorporated cleaning formulation. In addition, the cleaning brush included in the inventive kit may optionally be made of a wooden handle with natural bristles. Packaging may be made from raw cardboard, without plastic coating, glaze, varnish or other embellishments. For example, the container may be made of raw cardboard printed with a biodegradable ink, such as carbon black suspended in a vegetable oil carrier. [0051] Thus, the entire kit of the present invention may be made biodegradable and of completely sustainable materials. [0052] Referring to FIG. 1, the inventive kit comprises a storage container 10, which is made of a tub 12 and a lid 14 of conventional design. In accordance with a preferred embodiment, both tub 12 and lid 14 are optionally made of raw cardboard. To improve the longevity of storage container 10 (which also serves as the product package), lid 14 may include a plastic rim 16, with a structure substantially similar to that of a conventional ice cream container tub of the type used in retail supermarket sales. [0053] At sale, all components of the kit are contained within storage container 10. These include a small tub 18 of porcelain cleaning composition of the type described above. See FIG. 2. Optionally, tub 18 may be made of plastic or in a more environmentally attractive alternative. In accordance with the present invention tub 18 may be made of cardboard coated with a resinous material such as PVC plastic or a conventional waterproofing coating. A cover 19 is used to close tub 18. [0054] The inventive kit further comprises two pairs of gloves 20, as illustrated in FIG. 3. The gloves may be of conventional design, for example flexible latex rubber of the type typically included in boxes of disposable plastic gloves. Such gloves may be of relatively light design, in so far as the inventive cleaning application is substantially chemically inert. However, the gloves also provide the advantage, largely psychological, of removing the unpleasantness associated with cleaning, especially the cleaning of the porcelain surface of a toilet bowl. While the gloves likely will prevent any germs, bacteria or the like from contacting the hands of the person doing the cleaning, nevertheless, it is recommended, in accordance with the invention that hands be washed after the completion of the cleaning task. [0055] Referring to FIG. 4, the inventive kit further comprises a pair of sponges. Each of the sponges 22 optionally comprises a sponge layer 24 and scouring layer 26. Sponges 22 may be of any one of a number of sizes, for example 4″×2.5″ by 10/16 inches thick. Sponge layer 24 may be 7/16 of an inch thick. Scouring layer 26 may be 3/16 of an inch thick. Scouring layer 26 may be made of fibers of the type conventionally found in scouring sponges. [0056] The kit further comprises a pair of scouring pads 28, as illustrated in FIG. 5. Each scouring pad 28 may be of any one of a number of sizes, for example 4″×2.5″ by 3/16 inches thick. Scouring pad 28 may be of conventional design, comprising fibrous material bonded together. Both scouring pad 28 and scouring layer 26 of sponge 22 may include an abrasive material. Such abrasive material may be pumice of the type described above. However, other abrasives less prone to breaking down and crumbling may optionally be employed. Such other abrasives, but bonded to the fibers, may be designed to maintain their structural integrity under forces of the type and magnitude normally employed by the hand in performing a scrubbing operation. Scouring pad 28 may, optionally, have a more abrasive characteristic than scouring layer 26 of sponge 22. [0057] The inventive kit also comprises a scrubbing brush 30 as illustrated in FIG. 6. Brush 30 has an elongated handle 32 and a brush portion 34. Brush portion 34 comprises six tufts 36 of bristles 38, each, in close proximity to each other. In accordance with a preferred embodiment of the invention, two rows of six tufts each, for a total of 12 tufts may be employed. [0058] A number of typical brushes were evaluated. This evaluation was performed by orienting the bristles at a 45° angle with respect to the weighing surface of a scale, while the body of the brush (within which the bristles were held) was subjected to a force at a 90° angle to the surface of the scale. The force exerted by the bristles in two tufts was measured, allowing stiffness to be determined by noting the displacement distance of the bristle supporting structure of the brush. Results are shown in the chart below: [0000] Brush ID and Tip Description Force Displacement Length #Bristles A. Pink 1.8 oz. 2/16 in. 5/16 in. 26 Toothbrush A. Pink.6 oz. 1/16 in. 5/16 in. 26 Toothbrush B. Black.7 oz. 1/16 in..5 inches 32 Toothbrush C. 1.3 in × 2.3 in. 3 oz. 2/16 in. 11/16 in. 10 Scrubber C. 1.3 in × 2.3 in. 1.5 oz. 1/16 in. 11/16 in. 10 Scrubber D. 1 in. ×.5 in. 2.8 oz. 2/16 in..5 in. 22 Kitchen Brush E. 1 in. ×.5 in. 1.5 oz. 1/16 in..5 in. 22 Kitchen Brush [0059] Brushes C, D and E were found most suitable after tactile inspection of the brushes. Brushes A and B were found less suitable after tactile inspection of the brushes. [0060] The inventive kit also comprises a spatula 40, as illustrated in FIG. 7, which may be made of plastic and includes a rigid handle portion 42 and an applicator portion 44 which may be flexible. Applicator portion 44 has a width of approximately 0.5 inches, although widths between 0.25 inches and 1 inch or even wider will work well. [0061] In accordance with the invention, when it is desired to use the inventive system, the individual doing the cleaning puts on gloves 20. A quantity of cleaning compound 46 contained within tub 18 is scooped up with spatula 40, and applied to the porcelain surface to be cleaned, or, optionally, to brush 30, scouring pad 28 or sponge 22. [0062] In accordance with the invention, it is contemplated that general cleaning will first be done with a sponge 22. Areas requiring more attention are then scoured with scouring pad 28. After that, the most problematic areas may be scoured with brush 30. [0063] Finally, the very effective cleaning may be obtained by applying a quantity of cleaning formulation 46 to a scouring pad 28 and then pressing the rounded end 50 of scrubbing brush 30 into the path and against the porcelain area 52 to be cleaned, as illustrated in FIG. 8. [0064] Turning to FIGS. 9-12, a particularly advantageous embodiment of a scrub brush for applying the cleaning composition of the present invention is illustrated. Generally, brush 110 comprises a handle 112 which has a loop 114 for allowing brush 110 to be hung when not in use. Handle 112 includes a number of finger gripping notches 116. Cleaning is implemented using a fibrous layer 118 which is supported on a sponge 120. Fibrous layer 118 receives the cleaning composition of the present invention and is used to apply it to the workpiece and to scrub the workpiece. Fibrous layer 118 may optionally incorporate abrasive particles. [0065] Handle 112 may be formed of plastic from a single injection molded member together with extension arm 122 and support panel 124. A Velcro attachment member 126 comprising, for example, a fabric with hooks 128 is attached by glue to support panel 124. [0066] Sponge 120 has fibrous layer 118 adhered to it. A mating Velcro® fabric 130 having loops 132 is adhered to the bottom surface of sponge 120. [0067] While an illustrative embodiment of the invention has been described, it is, of course, understood that various modifications of the structures, compositions and methods described herein will be apparent to those of ordinary skill in the art in view of the above disclosure accompanying drawings. Such modifications are within the spirit and scope of the invention, which is limited and defined only by the appended claims.

Summary: A cleaning composition for porcelain comprises abrasive material and a carrier, wherein the carrier is water-based. The abrasive material comprises pumice with a particle size distribution such that at least 35 percent of the particles fall are 250 to 750 microns and wherein the carrier comprises a biodegradable gum in an amount sufficient to act as a thickener and suspending agent for the pumice. The cleaning composition further comprises glycerin and further comprises between 30% and 60% by weight pumice. The cleaning composition further comprises between 0.8% and 2.5% biodegradable gum and a thickener selected from one or more members of the group consisting of guar gum, locust bean gum, tara gum, xanthan, carrageenan, gellan gum and vegetable gum.

big_patent

en

Write a title and summarize: The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca2+ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca2+-regulated proteins that—in mature sperm—are involved in flagellar bending. Cyclic nucleotide-binding proteins play a prominent role in cellular signaling. Their activity is regulated by binding of cAMP and/or cGMP to a highly conserved cyclic nucleotide-binding domain (CNBD), consisting of eight beta sheets that are flanked by three alpha helices [1]. Six different members of the CNBD-containing protein family have been identified: cyclic nucleotide-sensitive ion channels (CNG channels: CNGA1-4, CNGB1,3; HCN channels: HCN1-4) [2], [3], protein kinase A and G (PKA, PKG) [4], [5], a guanine nucleotide-exchange factor (Epac1-2) [6], [7], and a bacterial transcription factor (CAP) [8]. Although the binding of ligand is conveyed to different effector domains like a catalytic kinase domain or a channel gate [9], the CNBD is highly conserved in all those proteins. Here, we report on the identification and functional characterization of an entirely novel member of the CNBD-containing protein family, which we call CRIS (cyclic nucleotide receptor involved in sperm function). We show that CRIS controls sperm function and, thereby, male fertility. A database search identified a cDNA sequence encoding an unknown protein with a CNBD motif. The CNBD is the only functional motif – otherwise, the protein shows no sequence or structural similarity to any other protein. We named the protein CRIS (cyclic nucleotide receptor involved in sperm function). CRIS constitutes a new member within the family of CNBD-containing proteins (Figure 1A). According to the NCBI and Ensembl database, orthologs have been annotated for 52 species (December 2012). We performed a phylogenetic analysis to learn about the evolutionary conservation of CRIS orthologs. CRIS has been identified in all published mammalian genomes (Fig. 1B). Moreover, CRIS exists in some amphibians and reptiles (lizards, softshell turtles, clawed frogs), jawless fish (lampreys), fish-like marine chordata (Branchiostoma), cnidaria (Hydra), tunicates (Ciona), echinodermata (Strongylocentrotus), and insects (Drosophila). CRIS is lacking in birds, fish (e. g. zebrafish), mollusks, nematodes (e. g. C. elegans), and a diverse selection of protostomes. The CNBD of CRIS is characterized by the typical sequence motif of other CNBDs [1]: it is ∼120 residues long and comprises eight beta strands and three alpha helices. The beta strands form a beta barrel and harbor a phosphate binding-cassette (PBC, Figure 2A). To identify commonalities and differences between the CNBD of CRIS and other cyclic nucleotide-regulated proteins, we built a structural model based on the CNBDs of a sea urchin HCN channel (SpIH, 2ptm) [10] and Epac2 (3cf6) [11] (Figure 2B). In this model, we studied the interaction of cAMP with side chain and backbone atoms of the polypeptide. A characteristic Arg residue (R288) in the PBC is in close contact with the negatively charged exocyclic phosphate of cAMP (Figure 2A, red asterisk, Figure 2C). Furthermore, a highly conserved Phe-Gly-Glu (F277/G278/E279) motif in β6 interacts with the 2′-OH ribose moiety of cAMP (Figure 2A, blue asterisks, Figure 2C). Finally, two interactions occur between αC-helix residues and the purine ring of cAMP, one involving F327 (Figure 2C, green asterisk) and another involving N330 (Figure 2C, orange asterisk). High-affinity CNBDs, like those of HCN channels [12] and of a bacterial CNG channel [13], bear an Arg residue at the respective position of N330 [12], [14]. Replacement of this Arg by Ala strongly lowers the ligand affinity of a CNG channel from Mesorhizobium loti (mlCNG) [14], [15], suggesting that this Arg residue sets apart high- from low-affinity CNBDs [1]. In this respect, the CNBD of CRIS probably represents a low-affinity binding domain, like in Epac and mammalian CNG channels, that binds cyclic nucleotides in the micromolar range of concentrations [1], [16], [17]. In summary, the protein-ligand interactions predicted by the CRIS model are similar to those of classical CNBDs, suggesting that CRIS binds cyclic nucleotides. Of note, the KCNH channel family (EAG, ERG, ELK) carry a CNBD that, however, does not bind cyclic nucleotides [18]. The KCNH family members share a conserved sequence motif C-terminal of the αC-helix (LTYNLR in zELK, grey box, Figure 2A); the motif forms a β strand that occupies the binding pocket, suggesting that it serves as an auto-ligand for the channel [18]. However, this motif is absent in all CRIS orthologs, indicating that the CNBD represents a functional CNBD. We experimentally studied binding of cyclic nucleotides to the CNBD using Förster resonance energy-transfer (FRET). The FRET sensor contained the CNBD from mouse CRIS (mCRIS, accession number JN629039) sandwiched between the FRET pair citrine and cerulean (cit-mCNBD-cer, Figure 2D). Similar FRET constructs using CNBDs of other proteins, e. g. Epac, have been successfully employed to detect binding of cyclic nucleotides [19]–[24]. When expressed in HEK293 cells, cit-mCNBD-cer displayed a FRET signal. However, the intracellular distribution was not uniform among cells. In some cells, the FRET sensor was clustered, whereas in other cells, it showed a rather homogenous distribution. In the latter, the FRET signal depended on the intracellular concentration of cyclic nucleotides (Figure 2E). Addition of 8-Br-cAMP, a membrane-permeable cAMP analogue, or NKH477, an activator of adenylyl cyclases, changed the ratio of the cerulean/citrine-FRET: the fluorescence of the acceptor (citrine) was diminished, whereas the fluorescence of the donor (cerulean) was increased (Figure 2E–G). In contrast, 8-Br-cGMP did not change FRET (Figure 2F, G). A mutant construct (cit-mCNBD-R288Q-cer FRET), in which ligand binding was impaired by mutating the conserved arginine in the PBC (R288Q) [25], [26], was rather uniformly distribute throughout the cell, but did not respond to changes in cAMP (Figure 2F, G). These results indicate that CRIS, in fact, is a cyclic nucleotide-binding protein with a preference for cAMP. To unravel the physiological function of CRIS in vivo, we determined the expression pattern of mouse CRIS (mCRIS) by Northern blot, in situ hybridization, Western blot, immunohistochemistry, and mass spectrometry. Northern blot analysis using mRNAs from different tissues revealed that Cris mRNA is only transcribed in testis (Figure 3A). In a similar vein, CRIS protein was detected by different polyclonal and monoclonal antibodies only in immunoblots from lysates of testis. In particular, CRIS was present in precursor cells, but not in cauda sperm from the epididymis (Figure 3B). To verify these results, we performed mass spectrometry. Protein lysates were separated on a 1D gel (SDS-PAGE), lanes were sliced, and analyzed by mass spectrometry. We identified 12 peptides distributed over the entire sequence of CRIS in protein lysates from testis, but not from cauda sperm (Figure 3C). During development, CRIS was detected after day P18 (Figure 3D), i. e. when the first haploid cells – the secondary spermatocytes - emerge. To analyze when CRIS expression starts and ends, we performed in situ hybridization and immunohistochemistry on testis sections: mCris mRNA was expressed in spermatocytes (Figure 3E) and mCRIS protein in late spermatocytes and round spermatids (Figure 3F, G). The distribution of the mCRIS protein within cells is largely uniform, suggesting that CRIS is a cytosolic protein (Figure 3G). The expression of CRIS in certain stages during sperm development and not in mature sperm suggests that CRIS is involved in spermiogenesis, the process that involves the major morphological and function changes during spermatogenesis. To study the function of CRIS in vivo, we generated Cris-deficient mice (CRIS−/−) by homologous recombination in embryonic stem (ES) cells. Exons 5–7, encoding the CNBD, were replaced with a neomycin resistance-cassette (Figure 3H). Disruption of the gene was confirmed by in situ hybridization (Figure 3E), immunohistochemistry (Figure 3F), Southern blotting (Figure 3I), and immunoblotting (Figure 3J). The offspring of heterozygous matings exhibited roughly Mendelian proportions (wild-type (+/+): 33%, heterozygous (+/−): 40%, mutant (−/−): 27%; n = 233), demonstrating that loss of CRIS does not affect embryonic development. CRIS−/− mice are indistinguishable from wild-type and heterozygous littermates regarding appearance, general behavior, and survival rate. Because CRIS is exclusively expressed in testis, we determined testis and epididymis weight of wild-type and mutant males. Whereas epididymis weight was similar, testis weight in mutant males was highly variable compared to wild-type males (Figure 4A): 19% of mutant testes were significantly smaller. Males with reduced testis weight had no cauda sperm and testis morphology was different: the organization of the tubules was altered and no sperm were present in the lumen (Figure 4B). The testis morphology of knockout males with normal testis weight was similar to wild-type testis (Figure 4B). Next, we determined the diameter of the seminiferous tubules from wild-type and knockout testis. Whereas wild-type testis and testis from knockout males with normal testis weight were comparable, the diameter of tubules from knockout males with lower testis weight was reduced by ∼50% (Figure 4C). This correlates well with the lower testis weight observed for those animals. We hypothesized that in knockout males with lower testis weight, spermatogenesis was arrested. Therefore, we analyzed the DNA content of male germ cells using flow cytometry to determine the relative distribution of haploid (1n), diploid (2n), and tetraploid (4n) cells (Figure 4D, E). Mutant testis with lower weight contained only tetraploid and diploid, but no haploid cells (Figure 4D, bottom). The first haploid cells are the secondary spermatocytes. Thus, spermatogenesis in a group of Cris-deficient males stops before the meiotic division and, thereby, avoids the generation of haploid cells, rendering those males infertile. In contrast, mutant males with normal testis weight displayed a distribution of haploid (1n), diploid (2n), and tetraploid (4n) cells similar to wild-type testis (Figure 4E). We analyzed spermatogenesis in more detail using immunohistochemistry. Since spermatogenesis in infertile knockout males stops before the generation of secondary spermatocytes, we investigated whether the transition from primary to secondary spermatocytes proceeds normally in knockout males with normal testis weight. We labeled testis sections with an antibody against MIWI, a piRNA-interacting protein that controls spermatogenesis and is expressed in the later stages of primary spermatocytes, in secondary spermatocytes, and in round spermatids [27]. The staining pattern in wild-type and knockout testis with normal testis weight was similar (Figure 4F), underlining our FACS result that both contained the full set of sperm precursor cells namely spermatogonia, spermatocytes, and spermatids. However, also knockout males with normal testis weight showed a fertility defect: only 60% (13/22, Table 1) of all wild-type females that were mated with mutant males (with normal testis weight) for four weeks became pregnant (compared to 100%, 26/26 in wild-type matings, Table 1). Females that had offspring needed a longer time to become pregnant and their litter size was smaller (Figure 4G, Table 1). The frequency of vaginal plugs was similar between matings with wild-type and mutant males, indicating that mating behavior was normal (Table 1). In vitro fertilization assays were performed to test the ability of mutant sperm to fertilize eggs. Wild-type or mutant sperm were incubated with wild-type oocytes and the number of two-cell stage embryos was counted after 18 h. There was no significant difference between the two genotypes regarding the percentage of two-cell stage embryos (+/+: 48±15%, 312 2-cell stages/655 oocytes; −/−: 61±25%, 481 2-cell stages/793 oocytes, data are presented as mean ± s. d.), indicating that sperm from mutant males are able to penetrate the zona pellucida, the outer layer of the egg. Our results show that CRIS plays an important role during sperm development. However, infertility in Cris-deficient males displays partial penetrance, thereby creating two groups: one with normal testis weight and morphology, which are subfertile, and one with reduced testis weight and aberrant testis morphology, which are infertile. Sperm are propelled by bending of the flagellum [28] and fertility phenotypes often result from defects in sperm motility [29]. Although there was no evidence that CRIS is expressed in mature sperm, we considered the possibility that CRIS controls processes during sperm development that are later required for sperm motility. Therefore, we compared the flagellar beat and motility of wild-type and mutant sperm. In non-capacitated wild-type sperm, the midpiece was straight and the flagellar beat was symmetrical with respect to a line through the midpiece (Figure 5A, dotted line). In contrast, the midpiece of mutant sperm was bent, resulting in a highly asymmetrical flagellar waveform (Figure 5A). Asymmetrical beating is a hallmark of hyperactivation – a swimming behavior that assists sperm to swim through the oviductal mucus, to leave the sperm reservoir at the oviductal isthmus, and to penetrate the egg vestments [30]–[32]. Hyperactivated motility is initiated by a maturation process called capacitation [33], [34]. Mouse sperm are an excellent animal model to study the symmetry of the flagellar beat. Their head is hook-shaped, which allows determining the direction of flagellar bending. In the pro-hook bending state, the flagellum and the hook were pointing towards the same side, whereas in the anti-hook conformation, the flagellum and the hook were facing opposite sides (Figure 5B) [32], [35]. Under capacitating conditions, wild-type mouse sperm displayed a highly asymmetrical flagellar waveform (Figure 5D); flagellar bending in one and the same cell switched between the pro- (bottom row, Figure 5D) and anti-hook conformation (top row, Figure 5D). In contrast, the midpiece of mutant sperm, no matter whether non-capacitated or capacitated, always remained in the anti-hook conformation (Figure 5E). Furthermore, in capacitated mutant sperm the asymmetry of the flagellar waveform was even further enhanced (Figure 5E). We quantified the difference in flagellar bending between wild-type and knockout sperm by calculating the asymmetry index (Figure 5C). An asymmetry index of 0 indicates a symmetric flagellar beat. Bending in the anti-hook conformation is reflected by an increase, bending in the pro-hook conformation by a decrease in the asymmetry index. Under non-capacitating conditions, the asymmetry index of knockout sperm is more positive compared to wild-type sperm. Under capacitating conditions, the asymmetry index of knockout sperm is even further increased, whereas wild-type sperm display both, a positive and a negative asymmetry index, reflecting the switch between the pro- and anti-hook conformation (Figure 5C). The difference in flagellar bending between wild-type and mutant sperm could either result from a defect in the flagellar ultrastructure or in the molecular mechanisms controlling the beat. We compared the ultrastructure of wild-type and mutant sperm using electron microscopy and did not detect any major difference in the 9+2 microtubule structure of the axoneme or any other morphological anomalies (Figure 6A, B, Movie S1). To investigate how the defect in flagellar bending affects sperm motility, we studied the swimming behavior of wild-type and mutant sperm under non-capacitating and capacitating conditions. The swimming behavior can be characterized by the velocity of straight-line path (VSL), which is a measure for progressive swimming: an increase of VSL reflects an increase in progressive motility [36]. The VSL of mutant sperm was lower than that of wild-type sperm under both non-capacitating (Movie S2; Figure 6C, +/+: 67±7 µm/s; −/−: 35±6 µm/s; number of animals: n = 3) and capacitating conditions (Movie S3; Figure 6C, +/+: 56±4 µm/s; −/−: 46±6 µm/s, n = 3). Thus, Cris-deficient sperm swim less progressively than wild-type sperm. We tested whether the asymmetric beat of mutant sperm results from premature capacitation. A hallmark of sperm undergoing capacitation is a characteristic phosphorylation pattern evoked by PKA and tyrosine kinases [37]–[39]. However, there was no difference between the phosphorylation pattern of wild-type and knockout sperm under both non-capacitating and capacitating condition, indicating that the asymmetric beating of knockout sperm does not result from premature capacitation (Figure 6D). Ca2+ plays an important role in sperm motility. It controls chemotactic steering of invertebrate sperm [40]–[42] and Ca2+ entry through CatSper channels promotes the transition to hyperactivated motility [43], [44]. In this respect, the asymmetric flagellar beat of mutant sperm recapitulates the action of an elevated intracellular Ca2+ concentration [Ca2+]i. However, [Ca2+]i between wild-type sperm and sperm from Cris-deficient mice under non-capacitating conditions was not different (+/+: 483±34 nM, −/−: 478±24 nM; number of animals: n = 3). Thus, we hypothesized that the Ca2+ regulation of the flagellar beat rather than [Ca2+]i is altered in CRIS knockout sperm. Therefore, we studied the flagellar beat of wild-type and mutant sperm exposed to a controlled change in [Ca2+]i. Sperm were incubated with a cell-permeant, photoactivatable Ca2+ scavenger (2-Diazo) that rapidly binds free Ca2+ upon flash photolysis [45]. After the UV flash, [Ca2+]i was lower (Figure 6E) and sperm responded with a change in flagellar waveform (Movie S4, S5). We quantified the change in flagellar bending using the asymmetry index (Figure 6F, G). One second before the UV flash, mutant sperm displayed a higher asymmetry index than wild-type sperm (Figure 6G). One second after the flash, the beat of both wild-type and mutant sperm became more symmetrical (Figure 6G). However, the action of lower [Ca2+]i was more dramatic in mutant sperm, which displayed a significantly larger change in asymmetry than wild-type sperm (Figure 6G, H). Thus, the flagellar beat of mutant sperm is not irreversibly locked in the asymmetric state (i. e. anti-hook mode) and is also dependent on [Ca2+]i. In fact, the asymmetry index of mutant sperm at low [Ca2+]i was similar to that of wild-type sperm at resting [Ca2+]i (Figure 6G). This result argues for an altered Ca2+ regulation of the flagellar beat in sperm from Cris-deficient mice. A target for Ca2+ in the sperm flagellum that has been suggested to control sperm motility is the Ca2+/calmodulin-dependent kinase IV (CaMKIV) [46]. To investigate the effect of CaMKIV activity on flagellar bending, we incubated wild-type sperm with 10 µM KN93, an inhibitor of CaMKIV/CaMKII. KN93 did not change the amplitude or frequency of the flagellar beat (amplitude: 31±5 µm (control) vs. 24±10 µm (KN93), frequency: 6±2 Hz (control) vs. 7±3 Hz (KN93), n = 9). However, the asymmetry index of the flagellar beat changed significantly from 0. 26±0. 17 (control) to 0. 03±0. 2 (KN93, p = 0. 02, n = 9), demonstrating that indeed, CaMKIV is involved in the tuning of the flagellar beat asymmetry. However, the drug decreased the asymmetry in wild-type sperm whereas in Cris-deficient mice, the asymmetry is increased. Thus, phosphorylation of proteins by CaMKIV rather augments the asymmetry of flagellar bending, whereas the action of CRIS seems to diminish the asymmetry of flagellar bending. Because we have no evidence that CRIS is expressed in mature sperm, CRIS cannot be directly involved in regulating the Ca2+ sensitivity of flagellar bending. Instead, CRIS expression during spermatogenesis suggests that during sperm development, CRIS might interact with proteins that control flagellar bending in mature sperm. We performed co-immunoprecipitation studies with a CRIS-specific antibody and subsequent mass spectrometry to identify putative binding partners of CRIS in sperm precursor cells. Similar experiments with proteins from sperm precursor-cells of Cris-deficient mice served as a negative control. We identified 27 candidate proteins that fall into three major groups: proteins involved in gene regulation, in flagellar transport, and in cellular signaling (Table 2). During spermatogenesis, gene transcription ceases before mature sperm are formed. A tight control of the temporal and stage-specific gene expression is a prerequisite for the correct differentiation of spermatids into mature sperm [47]. CRIS is expressed in round spermatids. Thus, CRIS might interact with proteins that are involved in regulating gene expression just before transcription stops. Another group comprises proteins involved in flagellar transport; the top candidates being a kinesin-like protein KIF2A and the intraflagellar transport protein IFT172 (Table 2). In Chlamydomonas, proteins involved in intraflagellar transport (IFT) carry cargo between the cytoplasmic basal body and the assembly site at the distal tip of the cilium [48], [49]. In sperm, these mechanisms are poorly understood. ABCF2 is a cytosolic member of the ABC transporter family that has been shown to control cell volume [50]. The putative interaction with IFT172, KIF2A, and ABCF2 in sperm precursor cells suggests that CRIS regulates protein transport into the flagellum during spermiogenesis. We analyzed the expression pattern of IFT172, KIF2A, and ABCF2 in testis and sperm from wild-type and Cris-deficient mice (Figure 7). In testis lysates, we could detect all three proteins. However, there was no obvious difference in expression between wild-type and knockout mice (Figure 7A). Immunohistochemical analysis of testis sections revealed that IFT172 and KIF2A show a punctuated staining in round spermatids, but are predominantly expressed in the flagellum of elongated spermatids and sperm in the testis lumen. However, there was no difference in the staining pattern between wild-type and knockout mice (Figure 7B). The antibody against ABCF2 did not show any specific labeling on testis sections (data not shown). IFT172 and KIF2A were both detected on a Western blot using total sperm lysates, but again, there was no obvious difference between genotypes (Figure 7C). Immunocytochemical analysis of isolated cauda sperm revealed that IFT172 is localized in the midpiece and cytoplasmic droplet, whereas KIF2A is predominantly localized in the principal piece (Figure 7D). However, the staining pattern was similar for wild-type and Cris-deficient mice. Again, the ABCF2 antibody did not show any specific labeling (data not shown). Thus, the overall expression of these putative interaction partners seems to be similar in wild-type and Cris-deficient mice. Also, their localization pattern, analyzed by confocal microscopy, showed no major difference between genotypes. However, it cannot be excluded that these proteins are only slightly mislocalized in the flagellum or a specific interaction with other proteins is missing. The third group encompasses proteins that are involved in cellular signaling. At least two of these proteins - IQCB1 (IQ motif containing B1) and CaMKIV (Ca2+/calmodulin-dependent protein kinase IV) - have been associated with a flagellar function (Table 2). IQCB1 harbors an IQ calmodulin-binding motif; the protein has been linked to retinal and renal ciliopathy [51], [52]. CaMKIV has been proposed to underlie the CaMK-dependent regulation of sperm motility [46]. In Chlamydomonas, Ca2+ controls dynein-driven microtubule sliding by activating calmodulin and CaM kinase [53], [54]. Based on these findings, we propose a model where CRIS regulates protein transport into the axoneme during spermiogenesis through interaction with the transport machinery. In the absence of CRIS, flagellar proteins might be incorrectly assembled or lacking altogether. Fertilization in mammals requires that sperm locate the egg. In the female reproductive tract, sperm are presumably guided by different mechanisms involving chemotaxis, thermotaxis, and rheotaxis [40], [55]–[57]. A precise control of sperm motility is a prerequisite for sperm to reach the site of fertilization and to locate the egg and, thereby, is instrumental for the success of fertilization. Here, we present a new regulator of sperm motility called CRIS. CRIS exists predominantly in species with sperm that are propelled by the beat of the flagellum, but not in nematodes like C. elegans with sperm that do not carry a flagellum and crawl in an amoeboid fashion [58]. This suggests that CRIS is involved in maintaining flagellar function of sperm. Indeed, sperm from Cris-deficient mice display aberrant flagellar bending: their flagellar waveform is highly asymmetrical in the anti-hook conformation. As a result, sperm from mutant males show a substantially reduced swimming velocity along a straight path under both capacitating and non-capacitating conditions. Thus, progressive swimming is strongly reduced. Could this phenotype account for the fertility defect? Successful fertilization requires multiple factors; however, a prerequisite is that sperm reach the site of fertilization. A similar sperm phenotype as in Cris-deficient mice is observed in t haplotype mice [59]. Mouse t haplotypes are naturally occurring variants of chromosome 17 [60]. Sperm from t haplotype mice display a number of defects resulting in infertility. First, progressive swimming is impaired: curvilinear swimming velocity is only 50% and progressive swimming only 20% of that of wild-type sperm. Second, sperm display the “curlicue” phenotype - an augmented effect of Ca2+ on the axoneme, resulting in an anti-hook bending of the midpiece [59], [61]. Third, t haplotype sperm are not able to fuse with the egg, even when artificially transferred to the site of fertilization [62], [63]. Transport studies of these sperm in the female reproductive tract revealed that hardly any sperm are found in the oviduct after mating, indicating that few if any sperm reach the site of fertilization [64]. It was suggested that low progression of these sperm is the primary cause of sterility [64]. Although both the motility phenotypes and the abnormal flagellar bending of sperm from Cris-deficient and t haplotype mice are similar, Cris-deficient sperm can fertilize the egg during IVF. Thus, the t haplotype is more complex and likely involves several genes that contribute to sterility. Cris is located on chromosome 2, ruling out a direct effect of the t haplotype variants on Cris gene-function; however, CRIS might regulate proteins, whose function is also affected in t haplotype mice. Recently, the male contribution to the fertility of the FL1 mouse line was investigated [65]. This mouse line was bred for the phenotype “high fertility” over 158 generations. During this period, the litter size per offspring increased from 10. 4 to 17. 1 [65]. Diallelic breeding demonstrated that about two third of the improved fertility phenotype is contributed by the male. Overall, fewer FL1 sperm were motile and progressive compared to wild-type mice; however, if motile, FL1 sperm displayed improved progression [65]. Taking the results from t haplotype, Cris-deficient, and FL1 mice together, sperm progression through the female genital tract is one of the major determinants of successful fertilization. In sperm from both Cris-deficient and t haplotype mice, the defect in flagellar bending reflects a change in the Ca2+ regulation of flagellar bending rather than altered Ca2+ levels. Ca2+ is an important determinant of flagellar bending in general [66]–[68]. In mammalian sperm, Ca2+ entry via the sperm-specific CatSper channel underlies hyperactivated motility [43], [44]. It has been proposed that distinct Ca2+-signaling pathways control flagellar bending of mouse sperm in either the anti- or pro-hook conformation [35]: Ca2+ entry through CatSper channels predominantly induces pro-hook bending, whereas Ca2+ release from internal stores favors anti-hook bending [35]. Downstream of Ca2+, flagellar bending is governed by Ca2+-binding proteins that indirectly regulate the axonemal motor protein dynein [53], [54]. Ca2+-binding proteins that might control sperm motility are CaM, enkurin, and calaxin [69]–[71]. Calaxin is highly conserved in metazoa and has been first identified in Ciona intestinalis [71]. Calaxin binds to outer dynein arms and directly suppresses microtubule sliding at high Ca2+ concentrations, which is essential for Ca2+-induced asymmetric bending [71]. Enkurin binds to ion channels and to CaM, creating a Ca2+-sensitive signaling platform that has been suggested to control sperm function [70]. Activation of CaMKIV by CaM has been proposed to regulate sperm motility in humans [46]. Interestingly, we identified CaMKIV as a putative interaction partner for CRIS during spermatogenesis (Table 2). We tested the role of CaMKIV in controlling flagellar bending by incubating tethered wild-type sperm with KN93, a CaMKIV blocker. We observed a decrease in the asymmetry index of flagellar bending compared to control sperm. This is opposite to sperm from Cris-deficient mice, which show a higher asymmetry index compared to wild-type sperm. Thus, if CRIS controls CaMKIV function and, thereby, flagellar bending, our results suggest that the effect is rather to repress than to promote CaMKIV function. However, one has to consider that our data do not support expression of CRIS in mature sperm. Therefore, CRIS needs to fulfill its role during sperm development, presumably in a cAMP-dependent manner. The flagellum is formed at the round spermatid stage, which temporally overlaps with CRIS expression. During this stage, proteins are transported into nascent cilia or flagella by the intraflagellar transport machinery [48], [72]. Some of the flagellar proteins, e. g. dynein, are pre-assembled in the cytoplasm before being transported into the flagellum [73]. Our interaction studies provide evidence that CRIS interacts with proteins that control intraflagellar transport (e. g. IFT172, KIF2A). CRIS is a cytosolic protein in spermatids and could, therefore, regulate the pre-assembly of the Ca2+-sensing proteins before they are transported into the sperm flagellum. Recently, the role of two proteins controlling IFT in mouse sperm has been described. RABL2 is a member of the RAS GTPase superfamily and interacts with the IFT machinery to transport a specific set of effector proteins to the sperm flagellum [74]. Rabl2-deficient male mice are infertile and their sperm are immotile. However, RABL2 effector proteins are still transported into the flagellum of Rabl2-deficient mice, but the transport is less effective, showing a 20–30% reduction of protein localization in the sperm flagellum. KIF3A motor protein is responsible for IFT in ciliated cells, but its role in sperm has been ill defined. KIF3A knockout males are infertile due to severe morphological defects of the sperm head and flagellum, indicating that KIF3A is essential for sperm head and tail formation [75]. Interestingly, testis weight of KIF3A knockout males was reduced and KIF3A and its interaction partners show a similar developmental expression pattern as we describe for CRIS. In the same vein, RABL2 also shows a similar developmental expression pattern as described for KIF3A, but also for CRIS [74]. Thus, the few components that have been shown to be involved in sperm IFT are expressed at a similar time point as CRIS during development. The importance of CRIS during sperm development is underlined by the fact that a population of Cris-deficient males is infertile due to a spermatogenic arrest. However, infertility in Cris-deficient males displays partial penetrance, thereby, creating two groups: one with normal testis weight and morphology, which are subfertile, and one with reduced testis weight and aberrant testis morphology, which are infertile. Partial penetrance of infertility has also been reported in other studies. Male mice lacking the α1b-adrenergic receptor can also be grouped according to their fertility defect: 27% of the males are infertile, whereas 73% are subfertile [76]. Partial penetrance of infertility can be attributed to the segregation of genetic modifiers on a hybrid genetic background. Similar results were observed for Hspa4-null males lacking the heat-shock protein 4 [77], for mice lacking the transition nuclear protein 1 (Tnp) [78], or the POU protein sperm-1 [79]. The Cris-deficient mouse line has been backcrossed into the C57Bl/6 background for 8–10 generations. Thus, the segregation of genetic modifiers on a hybrid genetic background, which could affect the penetrance of infertility, is minimized, but cannot be excluded and could account for the partial penetrance of infertility in Cris-deficient male mice. The reproductive phenotype observed in Cris-deficient mice is remarkably similar to that observed in some infertile men suffering from oligoasthenospermia, which is characterized by a lower sperm count and severe motility defects, but normal sperm morphology. Large-scale sequencing approaches need to address whether mutations in the Cris gene underlie fertility defects in human patients. Mice as model system are characterized by a high fecundity. Even if only 59% of Cris-deficient males are fertile, the population can be maintained. In humans, however, a similar defect might result in more severe phenotype and determines the success of a couple to conceive a child. All animal experiments were in accordance with the relevant national and international guidelines and regulations. Animal procedures were approved by the local authorities (LANUV NRW). CRIS orthologs were identified by a protein blast (blastp) in NCBI and an ortholog search in Ensembl (release 65). All sequences were verified using a reciprocal BLAST. CRIS protein sequences were aligned with MAFFT 4. 0. The NJ algorithm was used to construct a phylogenetic tree with ClustalX [80]. Bootstrapping was performed 1,000 times using ClustalX. The tree was visualized with Dendroscope. Bootstrap values are given as percentages. The scale bar shows the amino-acid substitution rate for the NJ (neighbor joining) tree. We used a region encompassing amino-acid residues 202–333 of mCRIS for homology modeling. The structure modeling-server M4T (M4T: a comparative protein structure modeling server (http: //manaslu. aecom. yu. edu/M4T/) [81] was used to investigate the relationship of the CNBD of CRIS with those of other cyclic nucleotide-regulated proteins. A model was built based on the CNBDs of a sea urchin HCN channel (SpIH, 2ptm) [10] and of EPAC2 (3cf6) [11]. Model quality was checked using the ProQ quality prediction server [82]. The LGscore and MaxSub value of the model were 2. 5 and 0. 4, respectively, indicating good to very good model quality. Figures were generated using Maestro (Suite 2011: Maestro, version 9. 2, Schrödinger, LLC). The CNBD protein sequence from the hyperpolarization-activated and cyclic nucleotide-gated ion channel 4 (HCN4) was used to search the database. Regions of a novel sequence (ensemble: 4921517L17Rik) were similar to the CNBD sequence. We used primers derived from this sequence (Table 3) for PCR after reverse transcription of RNA (RT–PCR) from mouse testis to obtain cDNA. For heterologous expression, a hemagglutinin (HA) tag was fused to the C terminus (mCRIS-HA) and cloned into a pcDNA3. 1+ vector (Invitrogen). Sequence alignments were done using ClustalW2. The FRET cer-mCNBD-cit construct was generated by PCR (aa 202–353, Table 3) using mCRIS cDNA as template. Citrine was fused to the N terminus and cerulean tagged with a histidine tag (His10), to the C terminus of the CNBD. GFP variants were amplified with standard primers from pEYFP and pECFP (Clontech). The mutated cer-mCNBD-R288Q-cit construct was generated using a QuikExchange protocol (Table 3, Stratagene). The annotated rat ortholog (NM_001177681) is predicted to encode a ribosomal subunit, however, we could not amplify this sequence from rat testis cDNA. Live-cell imaging was performed using CHO cells on the Olympus CellR system with an IX81 microscope, the MT20 illumination system, and the XM10 CCD camera. CHO cells expressing the FRET sensors were perfused with buffer (140 mM NaCl, 5. 4 mM KCl, 1 mM MgCl2,1. 8 mM CaCl2,5 mM HEPES) with or without 40 µM NKH477,100 µM IBMX (Tocris), and 3 mM 8-Br-cAMP/cGMP (BioLog) 1 min after starting the recording. Cells were excited at 430/25 nm. Fluorescence was detected through 470/24 nm and 535/30 nm bandpass filters. Data was analyzed using the CellR software and Origin (Microcal), expressed as ratio of cerulean to citrine signal, and normalized to the values before perfusion. A peptide comprising amino acids 142VHKKHPDFSFWDKKKQGR159 from mouse CRIS was synthesized and coupled to BSA and OVA (PSL). Rats were immunized subcutaneously and intraperitoneally with a mixture of 50 µg peptide-OVA, 5 nmol CPG oligonucleotide (Tib Molbiol), 500 µl PBS, and 500 µl incomplete Freund' s adjuvant. A boost without adjuvant was given six weeks after the primary injection. Fusion was performed using standard procedures. Supernatants were tested in a differential ELISA with the CRIS peptide coupled to BSA and irrelevant peptides coupled to the same carrier. Monoclonal antibodies that reacted specifically with CRIS were further analyzed in Western blot. Tissue culture supernatant of 4E4 and 4B2, both of rat IgG1 subclass, were used in this study. A polyclonal antibody (1880-1) directed against the same peptide was generated in rabbit and purified using a peptide column. The targeting vector was constructed in a modified pBluescript vector (Stratagene) using a 2. 7 kb genomic fragment as short arm of homology (exon 4+first 95 bp of exon 5) and a 6. 6 kb genomic fragment as long arm of homology (last 134 bp of exon 7+exon 8). In between both arms, a loxP flanked neomycin resistance-gene was inserted, which replaced the 3′ end of exon 5, exon 6 and the 5′ end of exon 7. For negative selection, a diphtheria toxin A (DTA) cassette was inserted at the 3′ end of the long arm. The targeting vector was linearized with SwaI before electroporation into V6. 5 embryonic stem cells [83]. Positive clones were identified by Southern blot analysis using probe 1 and a neo probe, and by PCR (Table 3). Eight-cell embryo injection was performed for two independent clones. Male chimeras were crossed with C57BL/6J females to obtain heterozygous mice. Mice were genotyped by PCR (Table 3). All mice were backcrossed for 10 generations to a C57BL/6J background. Mice used in this study were 2–4 months of age. All animal experiments were in accordance with the relevant guidelines and regulations. Northern blot analyses were performed using a mouse multi-tissue (Clontech) and a mouse reproductive-tissue Northern blot (Zyagen) labeled with a Cris-specific probe (bp 9–325). Northern blots were prehybridized at 68°C for at least 1 h using ExpressHyb solution (Clontech) and PerfectHyb Plus Hybridization buffer (Sigma), respectively. To detect mCRIS mRNA, a 32P labelled DNA probe (bp 9–325; TAKARA Labeling Kit) was used at 1. 5–2×106 cpm/ml in prehybridization solutions at 68°C overnight. After washing (three times in 0. 05% SDS in 2×SSC, once in 0. 1% SDS in 0. 1×SSC, each 20 min at 65°C), signals were detected using Kodak® BioMax™ XAR films (Sigma). Digoxigenin (DIG) -labeled sense and antisense probes were synthesized by in vitro transcription using DIG RNA labeling mixes (Roche). The same cDNA region as for the Northern blot probe was used. Frozen testis sections (18 µm) were fixed for 10 min with 4% paraformaldehyde in PBS. After prehybridization in 50% formamide, 10% dextran sulfate, 100 µg/ml herring sperm DNA in 2×SSC, sections were hybridized with 25–100 ng probe in the same buffer at 42°C overnight. The sections were washed for 30 min at 42°C in 50% formamide/2×SSC and in 50% formamide/0. 2×SSC. For detection, sections were equilibrated in buffer 1 (100 mM Tris/HCl, pH 7. 5,150 mM NaCl), blocked with blocking buffer (0. 5% blocking reagent (Roche) in buffer 1) for 30 min at room temperature, and incubated with 0. 15 U/ml sheep alkaline-phosphatase (AP) -conjugated anti-DIG Fab-fragments (Roche) in blocking buffer for 1 h at room temperature. After washing twice with buffer 1 and equilibration with buffer 3 (100 mM Tris/HCl, pH 9. 5,100 mM NaCl, 150 mM MgCl2), NBT/BCIP (nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate, toluidine salt, Roche) was added to detect bound AP. Testes were either directly embedded in TissueTec (Sakura Finetek), frozen, and sections (18 µm) were fixed for 10 min with 4% paraformaldehyde/PBS or Histochoice (Amresco) or were fixed overnight, cryo-protected in 10 and 30% sucrose, and afterwards embedded in TissueTec. Sperm were immobilized on microscope slides with or without Mitotracker (1 ng/µl; Invitrogen) and fixed for 10 min. To block unspecific binding sites, frozen sections and sperm were incubated for 1 h with blocking buffer (0. 5% Triton-X 100 and 5% ChemiBLOCKER (Millipore) in 0. 1 M phosphate buffer, pH 7. 4). Primary antibodies (Table 4) were diluted in blocking buffer and incubated overnight. Fluorescent secondary antibodies were diluted in blocking buffer containing 0. 5 µg/µl DAPI (Invitrogen) and pictures were taken on a confocal microscope (Olympus FV1000). Periodic acid Schiff' s staining was performed following the manufacturer' s instruction (Sigma). All antibodies are described in Table 4. For heterologous expression, HEK293 or CHO cells were transfected using Lipofectamine 2000 (Invitrogen). Lysates were isolated by homogenizing the tissue and cells in lysis buffer (10 mM Tris/HCl, pH 7. 6,140 mM NaCl, 1 mM EDTA, 1% Triton-X 100,1∶500 mPIC protease inhibitor cocktail) with a tissue homogenizer. After three freeze-thaw cycles, samples were incubated 30 min on ice and then centrifuged at 10,000×g for 5 min at 4°C. Soluble proteins were isolated by homogenizing the tissue and cells in buffer A (20 mM HEPES, 20 mM NaCl, 1 mM EDTA, 0. 1 mM EGTA, pH 7. 4,1∶500 mPIC) with a tissue homogenizer. After three freeze-thaw cycles, samples were incubated 10 min on ice and then centrifuged at 20,000×g for 15 min at 4°C. All samples were heated for 5 min at 95°C prior to separation on SDS-PAGE. For Western blot analysis, proteins were transferred onto PVDF membranes, probed with antibodies and analyzed using a chemiluminescence detection-system. All antibodies are described in Table 4. For reprobing, membranes were incubated in stripping buffer (62. 5 mM Tris/HCl pH 6. 7,100 mM β-mercaptoethanol, 2% SDS) for 30 min at 65°C, washed with PBS, and reprobed. For (co-) immunoprecipitations, 500 µg protein (total lysates) was incubated with 0. 1 ml of the antibody column (monoclonal antibody 4B2 or 4E4 covalently coupled to Protein G) overnight at 4°C, washed four times with lysis buffer, and eluted in 2× SDS-PAGE sample buffer. Before mass spectrometry, proteins were separated on a SDS-PAGE and gel pieces were digested with trypsin. LC-MS/MS was performed on a Micromass CapLC liquid chromatography system and a quadrupole orthogonal acceleration time-of-flight mass spectrometer Q-TOF Ultima (Micromass) equipped with a Z-spray nanoelectrospray source. Peptide separation was performed on a capillary column (PepMap C18,3 µm, 100 Å, 150 mm×75 µm i. d., Dionex). Data was acquired in a data-dependent mode using one MS scan followed by MS/MS scans of the most abundant peak. The MS survey range was m/z 350–1500 and the MS/MS range was m/z 100–2000. The processed MS/MS spectra (MassLynx version 4. 0 software) and the MASCOT server version 1. 9 (Matrix Science Ltd.) were used to search an in-house database, which contained the CRIS sequences. The mass tolerance of precursor and sequence ions was set to 100 ppm and 0. 2 Da, respectively. The search includes variable modifications of cysteins with acrylamide and methionine oxidation. CRIS was accepted as identified if at least two tryptic peptide scores indicate identity or extensive homology. LC-MS/MS analyses of gel-separated proteins were performed on a LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher) equipped with an Eksigent 2D nanoflow LC system (Axel Semrau GmbH). Tryptic peptides were separated on a capillary column (PepMap C18,3 µm, 100 Å, 250 mm×75 µm i. d., Dionex, Idstein) at an eluent flow rate of 250 nl/min using a linear gradient of 2–50% acetonitrile in 0. 1% formic acid (v/v). Mass spectra were acquired in a data-dependent mode with one MS survey scan (with a resolution 60,000) in the Orbitrap and MS/MS scans of the four most intense precursor ions in the LTQ. The MS survey range was m/z 350–1500. The dynamic exclusion time (for precursor ions) was set to 120 s and automatic gain control was set to 106 and 20,000 for Orbitrap-MS and LTQ-MS/MS scans, respectively. The generated peak lists and the MASCOT server (version 2. 2, Matrix Science) were used to search in-house against the SwissProt database (version 2010_10, contains 521,016 sequences, comprising 183,900,292 residues). A maximum of two missed cleavages was allowed and the mass tolerance of precursor and sequence ions was set to 10 ppm and 0. 35 Da, respectively. Acrylamide modification of cysteine and methionine oxidation were considered as possible modifications. Scaffold (version 2. 02, Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications and to generate non-redundant protein lists. Peptide identifications were accepted if they could be established at greater than 70% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99% probability and contained at least two identified peptides. Based on decoy database searches, the false positive rate was estimated to be <1%. Wild-type, heterozygous, or homozygous males (with normal testis weight) were mated with wild-type females (C57Bl/6) for four weeks. Only homozygous males with normal testis weight were used for the analysis of breeding performance. Sperm were isolated by incision of the cauda epididymis in modified TYH medium containing 138 mM NaCl, 4. 8 mM KCl, 2 mM CaCl2,1. 2 mM KH2PO4,1 mM MgSO4,5. 6 mM glucose, 0. 5 mM sodium pyruvate, 10 mM L-lactate, pH 7. 4. For capacitation, the medium contained 3 mg/ml BSA and was supplemented with 25 mM NaHCO3. After 15 min swim out at 37°C and 5% CO2, sperm were counted. All subsequent experiments were performed at room temperature, unless otherwise stated. For isolation of germ cells, testes were decapsulated and incubated in 1 ml Hank' s Balanced Salt Solution (HBSS) containing (20 mM HEPES, 137 mM NaCl, 5. 4 mM KCl, 0. 3 mM Na2HPO4,0. 4 mM KH2PO4,1. 2 mM MgSO4,1. 3 mM CaCl2,6. 6 mM sodium pyruvate, 0. 05% lactate, 5. 6 mM glucose, pH 7. 2) containing 0. 5 mg/ml Collagenase type IA (Sigma) for 30 min at 32°C. The dissociated interstitial cells were removed by two washing steps with HBSS. The seminiferous tubules were then incubated in 1 ml HBSS containing 0. 5 mg/ml Trypsin type XIII (Sigma) and 1 µg/ml DNaseI (Applichem) for 10 min at 32°C. Cell aggregates were sheared gently with a Pasteur pipette. The dispersed seminiferous cells were washed twice by centrifuging at 200×g for 5 min at room temperature. The final cell pellet was resuspended in HBSS and filtered through a Nylon mesh (40 µm mesh). In vitro fertilization (IVF) was performed as described previously [84] and in cooperation with MfD Diagnostics, Wendelsheim, Germany. DNA content analysis using flow cytometry (FACS) was performed as described previously [85]. Sperm motility was studied in shallow observation chambers (depth 150 µm). Cells were either freely swimming or tethered to the glass surface at a lower BSA concentration (0. 3 mg/ml), which resulted in a large fraction of cells that gently adhered to the glass surface. For analysis, we selected those cells attached by the head only and displayed a freely beating flagellum. Sperm motility was recorded under an inverted microscope (IX71; Olympus) equipped with a dark-field condenser and a 10× objective (UPLSAPO; NA 0. 4). The temperature of the microscope was adjusted to 37°C using an incubator (Life Imaging Services). To obtain sharp images of moving sperm, stroboscopic illumination was achieved using a white LED (K2 star; Luxeon) and a custom-made housing and pulse generator (freely swimming: 1 ms, tethered: 2 ms). Images were collected using an EMCCD camera (DU-897D; Andor) for all data shown with the exception of data shown in Figure 5, where a CMOS camera (Dimax; PCO) was used instead. Cells where manually tracked during acquisition using a motorized stage (SCAN IM; Märzhäuser), and the position of the stage was recorded for each frame. The set-up was synchronized using a custom-made acquisition program written in LabVIEW and data acquisition hardware PCI-6040E (National Instruments). Quantification of the flagellar beat was performed using custom-made programs written in MATLAB (Mathworks). The program identified the best threshold for binarization of the image by iteratively reducing the threshold until the expected cell area in the image was achieved. This was followed by a skeleton operation to identify the flagellum. The flagellar beat parameters were averaged during a time window of 1 s around each frame (except for frames at the beginning, the end of the movie, or flanking the UV flash, where the time window was reduced to ∼300 ms). The flagellar asymmetry index was defined as the angle between the line going through the middle of the flagellum and the sperm head and the axis of symmetry of the cell. For alignment of the flagellar-beat envelopes, we used custom-made programs written in LabVIEW. Using defined thresholds, the image was binarized. From a user-defined region-of-interest centered at the cell head, the program determined the location of the head on subsequent frames using a registering procedure. The neck of the cell was identified by applying a mask with the shape of an annulus centered into the sperm head. The annulus had an internal diameter of 16 µm to cover the sperm head, and a 4 µm longer external diameter, enough to resolve the first pixels of the neck. All frames were then rotated and superimposed with a rotation angle equal to the azimuth of the neck region on a reference system centered at the sperm head. Sperm were loaded with 2-Diazo-AM (20 µM; Molecular Probes) for 40 min at 37°C. After incubation, cells were centrifugated (600×g, 8 min), resuspended in TYH buffer containing a lower BSA concentration (0. 3 mg/ml), and allowed to tether onto the chamber wall. Photolysis of 2-Diazo was achieved by 100–200 ms UV flashes from a solid-state light source (Spectra×light engine, Lumencor) and a band-pass filter (H350/50; AHF). Images were collected at 95 frames per second. For Ca2+ recordings, sperm were loaded with Fluo8-AM (5 µM; AAT Bioquest) together with Diazo-2. Excitation of fluorescence was achieved by stroboscopic illumination pulses (1 ms) generated with the cyan light-source of the light-engine Spectra X. Excitation light was filtered through a 475/28 band-pass filter (Bright Line HC, Semrock) and emission was long-pass filtered (510 ALP, Omega optical). Images were collected at 12 frames per second. Sperm were cryo-fixed by high-pressure freezing in 20% dextran (Dextran 40, Mr 40 kDa, Roth) [86], [87]. Frozen samples were freeze-substituted with 0. 2% uranyl acetate in acetone [88]. Finally, samples were infiltrated with Lowicryl (EMS) and polymerized under UV light for several days. Testis sections (50 nm) were generated using an ultra-microtome (UC6, Leica Microsystems), collected on Quanifoil-Cu-grids (EMS), and stained with 2% uranyl acetate and 0. 03% lead citrate. Images were taken with the JEM-2200FS transmission electron-microscope (TEM, JEOL) operating at 200 kV. The tomogram was acquired using the Titan Krios TEM (FEI Company) operating at 300 kV. The tomogram was reconstructed with eTomo [89]. Sperm were loaded with 5 µM Cal-520-AM (ATT Bioquest), 0. 05% pluronic (Invitrogen) in TYH buffer for 45 min at 37°C. After loading, sperm were washed three times with TYH buffer without lactate before starting the measurement. Calibration of [Ca2+]i was performed using a null point method in a rapid-mixing device in the stopped-flow mode (SFM400; Bio-Logic) at 37°C. Sperm were mixed with defined Ca2+ solutions containing ionomycin (1 µM final). The [Ca2+]i equaled the Ca2+ concentration, at which no change in fluorescence was observed. All values are mean ± s. d., unless otherwise stated. Statistical comparisons were carried out with Student' s t test. Statistically significance was accepted at the p<0. 05 level. The 95% confident interval was calculated as 1. 96×s. e. m.

Title: CRIS-A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending Summary: New life begins with the fusion of a sperm and an oocyte. During fertilization, sperm cope with demanding endeavors: they have to locate the egg by directed swimming and penetrate the oocyte' s vestments. Cyclic nucleotides and their targets represent one of the most ancient signaling systems and are important for the control of sperm function. Here, we report the identification and characterization of an entirely novel cyclic nucleotide-binding protein-CRIS-that is crucial for male fertility. CRIS controls sperm development and, thereby, the flagellar beat of sperm. Genetic ablation of CRIS impairs sperm motility hampering steering of sperm through the female genital tract-a deficit that severely reduces male fertility. Thus, we identified an important player in the signaling events that control fertilization, which might open new avenues for the treatment of fertility defects and the design of novel contraceptives.

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Summarize: UVALDE, Texas (AP) — Family members say a Texas couple was killed in a helicopter crash just hours after they were married. Houston TV station KTRK reports that Will Byler and Bailee Ackerman were married Saturday night at a ranch in Uvalde, about 80 miles (125 kilometers) west of San Antonio. Byler's grandfather, William Byler, tells the TV station that the couple died when a helicopter that had departed the wedding reception crashed. The TV station reports that the helicopter's pilot was also killed in the crash. The National Transportation Safety Board says it's investigating the crash and the cause is not yet known. The Houstonian student newspaper reports that the newlyweds were seniors at Sam Houston State University. Two Texas newlyweds were tragically killed when their helicopter crashed after leaving their wedding. Will Byler and Bailee Ackerman, both 23, were killed after they left their wedding reception in the early hours of Sunday morning. Their helicopter reportedly crashed just north of Uvalde. “We celebrated their fairy tale wedding and they were surrounded by their family and friends as they flew off in the family helicopter,” Eric Smith, a family friend, posted on Facebook. “Sadly they crashed into the side of a hill about a mile from the family ranch.” The pilot was also killed. “Please keep everyone associated with this tragic event in your prayers,” Smith wrote. Authorities reportedly discovered the wreckage shortly after sunrise on Sunday, CBS News reported. The Department of Public Safety and Federal Aviation Administration is investigating what caused the crash, according to reports. The newlyweds were both students at Sam Houston University. “It is with the deepest sadness that we announce the tragic passing of two Bearkats Will Byler (Agriculture Engineering senior) and Bailee Ackerman Byler (Agricultural Communication senior) in a helicopter accident departing their wedding," The school’s paper wrote after the deaths. Jessica Stilley, Ackerman’s maid of honor, also took to Facebook to express her grief after the tragedy. “'My sweet Bailee Raye, my heart is broken in a million little pieces as I sit here and think of the rest of my life without my best friend,” Stilley wrote. RELATED STORIES Distraught Father Shares Pictures of Kids Mourning Mom, Unborn Sibling Killed in Crash 9-Year-Old Girl Died Trying to Shield Twin Brothers From Oncoming Car, Uncle Says 12-Year-Old Killed While Trying to Save Her Dog Will Be Buried With Him Gerald Green Lawrence has been identified as the pilot in the helicopter crash that killed 2 newlyweds near Uvalde, Texas, just an hour and a half after the wedding. https://t.co/qV6zRjHDil pic.twitter.com/cI3nXaH4Wi — ABC13 Houston (@abc13houston) November 5, 2018 I spoke w/the step daughter of the pilot who says the flight path was routine for her stepdad and he had flown it many times. The family is in shock over the accident and knew something was wrong when they didn’t hear from anyone hours later. — Charly Edsitty (@CharlyABC13) November 5, 2018 EMBED More News Videos A wedding guest captured a lavish departure by newlyweds before they board a doomed helicopter following their Uvalde, Texas nuptials. EMBED More News Videos Family and friends remember newlyweds killed in helicopter crash A couple who were just married for an hour and a half were killed Saturday night when their helicopter went down in Uvalde, Texas.The groom's grandfather William Byler confirmed to Eyewitness News that the aircraft went down Saturday at their family ranch. His grandson Will Byler, Will's new wife Bailee Ackerman and the aircraft's pilot, Gerald Douglas Lawrence all died in the crash.Eyewitness News spoke to Lawrence's stepdaughter, Amilyn Willard, who said he was a captain in the army and fought in Vietnam.The National Transportation Safety Board is investigating the accident involving a Bell 206B helicopter. The accident happened about 15 miles northwest of Uvalde, according to NTSB's information.The couple's wedding portal on planning website The Knot further confirmed their nuptials taking place on Nov. 3 in Uvalde on Byler's family ranch. Engagement photos also show Byler in his cowboy hat embracing Ackerman.The Sam Houston State University students were surrounded by family and friends as they flew off in the family helicopter.A wedding guest shared video of the couple's lavish departure on board the doomed aircraft:After learning about the newlyweds' fatal crash, the Sam Houston State University Rodeo Team took to Facebook to share their condolences.On Monday afternoon, NTSB officials said the helicopter was in the air for five to 10 minutes when it crashed into a rocky hillside.Craig Hatch, an air safety investigator with the National Transportation Safety Board, said the wreckage is strewn across the hillside that rises above rugged terrain about 80 miles west of San Antonio.The helicopter was owned by the family of William Byler and Lawrence had been a pilot for the family for years.The couple was supposed to be flown to San Antonio where they were to get a flight for their honeymoon destination.Hatch says it's too early to determine what caused the crash but that a preliminary NTSB report will be issued in about two weeks.

Summary: Family members say a Texas couple was killed in a helicopter crash just hours after they were married. Will Byler and Bailee Ackerman were married Saturday at Byler's family ranch in Uvalde, about 80 miles west of San Antonio, reports KTRK. Byler's grandfather, William Byler, tells the TV station that the couple had departed the wedding reception in the family's Bell 206B helicopter, which is a two-bladed model. He says they were married for about an hour and a half when they died. The helicopter's pilot was also killed in the crash. The National Transportation Safety Board says it's investigating and the cause of the crash is not yet known, reports the AP. The Houstonian student newspaper reports that the newlyweds were seniors at Sam Houston State University. They were both 23, per Inside Edition.

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Write a title and summarize: Because species invasions are a principal driver of the human-induced biodiversity crisis, the identification of the major determinants of global invasions is a prerequisite for adopting sound conservation policies. Three major hypotheses, which are not necessarily mutually exclusive, have been proposed to explain the establishment of non-native species: the “human activity” hypothesis, which argues that human activities facilitate the establishment of non-native species by disturbing natural landscapes and by increasing propagule pressure; the “biotic resistance” hypothesis, predicting that species-rich communities will readily impede the establishment of non-native species; and the “biotic acceptance” hypothesis, predicting that environmentally suitable habitats for native species are also suitable for non-native species. We tested these hypotheses and report here a global map of fish invasions (i. e., the number of non-native fish species established per river basin) using an original worldwide dataset of freshwater fish occurrences, environmental variables, and human activity indicators for 1,055 river basins covering more than 80% of Earth' s surface. First, we identified six major invasion hotspots where non-native species represent more than a quarter of the total number of species. According to the World Conservation Union, these areas are also characterised by the highest proportion of threatened fish species. Second, we show that the human activity indicators account for most of the global variation in non-native species richness, which is highly consistent with the “human activity” hypothesis. In contrast, our results do not provide support for either the “biotic acceptance” or the “biotic resistance” hypothesis. We show that the biogeography of fish invasions matches the geography of human impact at the global scale, which means that natural processes are blurred by human activities in driving fish invasions in the world' s river systems. In view of our findings, we fear massive invasions in developing countries with a growing economy as already experienced in developed countries. Anticipating such potential biodiversity threats should therefore be a priority. The deliberate or accidental introduction of species outside their native range is a key component of the human-induced biodiversity crisis, harming native species and disturbing ecosystems processes [1–3]. The greater the introduction of non-natives in a region, the higher the probability that some of them become invasive and will hence cause ecological or economic damage [4,5]. Patterns of non-native species richness are therefore relevant in forecasting the overall impact of invasions on a global scale [5] and should help management authorities to adopt sound, effective conservation policies [5–7]. The process of species invasion consists of three successive stages: initial dispersal, establishment of self-sustaining populations, and spread into the recipient habitat. The last two stages are contingent upon the first one, i. e., if initial dispersal is interrupted, establishment and spread do not occur [8]. Three major hypotheses, which are not necessarily mutually exclusive, have been proposed to explain invasion patterns: the “human activity” [9], “biotic acceptance” [10], and “biotic resistance” [11] hypotheses. The “human activity” hypothesis refers to the three stages of the invasion process (initial dispersal, establishment, and spread), whereas the “biotic resistance” and “biotic acceptance” hypotheses address only the establishment and spread stages [12]. With regards to the establishment stage, the “human activity” hypothesis predicts that, by disturbing natural landscapes and increasing propagule pressure (i. e., the number of individuals released and the frequency of introductions in a given habitat), human activities facilitate the establishment of non-native species [9,13,14]. Everything else being equal, a positive relationship is therefore expected between non-native species richness and quantitative surrogates of propagule pressure and habitat disturbance (e. g., gross domestic product [GDP], percentage of urban area, and human population density [5]). Then, the “biotic acceptance” hypothesis predicts that the establishment of non-native species would be greatest in areas that are rich in native species and with optimal environmental conditions for growth (i. e., “what is good for natives is good for non-natives too” [10]). Everything else being equal, native and non-native species richness should co-vary positively with environmental factors such as energy availability and habitat heterogeneity, which are already recognised as the primary global determinants of native species richness [15,16]. In contrast, the “biotic resistance” hypothesis predicts that species-poor communities will host more non-native species than species-rich communities, the latter being highly competitive and hence readily impede the establishment of non-native species [11,17]. Therefore, a negative relationship is expected between native and non-native species richness. To date, the relative importance of these hypotheses in explaining the variation in non-native species richness had never been tested at the global scale. We tested these hypotheses and report a global map of fish invasions (i. e., the number of non-native fish established per river basin) by using an extensive worldwide dataset of freshwater fish occurrences (i. e., more than 40,000 occurrences of 9,968 fish species) on the river basin scale (1,055 basins covering more than 80% of Earth' s surface). Freshwater fish offer a unique opportunity to identify factors that are responsible for large-scale gradients in non-native species richness for at least two main reasons. First, among vertebrate groups, freshwater fish have been widely introduced over the world [18], which often had subsequent negative consequences on native species and ecosystems integrity [19–23]. Second, as rivers are separated from one another by barriers insurmountable for freshwater fish (land or ocean), they form kind of “biogeographical islands”, whose space is delimited [15]. This implies that the natural and human factors shaping global patterns of non-native species richness can be easily separated. Our results revealed six global invasion hotspots where non-native species represent more than a quarter of the total number of species per basin: the Pacific coast of North and Central America, southern South America, western and southern Europe, Central Eurasia, South Africa and Madagascar, and southern Australia and New Zealand (Figure 1A). According to The World Conservation Union (IUCN) Red List [25], these areas were also characterised by the highest proportion of fish species having a high risk of extinction in the wild (Figure 2). Analysing the absolute number of species, we found that river basins of the Northern Hemisphere host the highest number of non-native fish species (Figure 1B). The human factors considered here to test the “human activity” hypothesis (GDP, population density, percentage of urban area) were found to be positively related to non-native species richness (Table 1), after controlling for the effects of environmental conditions and native species richness. In contrast, the positive correlation between native and non-native richness that was expected by the “biotic acceptance” hypothesis was not significant after controlling for the effects of propagule pressure and habitat disturbance (Table 1). Indeed, the environmental factors displayed either no (net primary productivity) or a weak positive correlation (altitudinal range and basin area) with non-native species richness, after controlling for the effects of propagule pressure and habitat disturbance (Table 1). The negative correlation between native and non-native richness, expected by the “biotic resistance” hypothesis, was not significant after controlling for the effects of environmental conditions, propagule pressure, and habitat disturbance (Table 1). Then, we applied hierarchical partitioning [26–28] that aims to quantify the independent explanatory power of each variable by considering all possible submodels. The deviance explained by the 128 submodels computed in hierarchical partitioning accounted in average for 52% of the total deviance (±7% standard deviation [SD], min = 37%, max = 67%). The human factors had together the greatest independent effect on non-native species richness (70%, Table 2). Among the human factors, the GDP (an economical index of human activities [9]) had the greatest independent explanatory power (43%; Table 2). To a lesser extent, the habitat heterogeneity (i. e., basin area and altitudinal range) and the number of native species also contribute to the variation in non-native species between river basins (Table 2). To test for potential bias in our results due to differences in sampling effort between continents, bootstrap analysis was performed by applying hierarchical partitioning to 1,000 random subsets of 100 basins. For each variable, the independent effect observed did not differ from the 95% bootstrap percentile confidence interval (Table 2), testifying that potential differences in sampling effort between continents hardly affected the results. By using an explanatory modelling approach, we showed that the human activity indicators of the world' s river basins were positively related to the number of established non-native fish species. In addition, they account for most of the global variation in non-native species richness, giving support for the “human activity” hypothesis. More particularly, we highlight that the level of economic activity of a given river basin (expressed by the GDP) strongly determines its invasibility. Three non-exclusive mechanisms may account for this pattern. First, economically rich areas are more prone to habitat disturbances (e. g., dams and reservoirs modifying river flows) that are known to facilitate the establishment of non-native species [7,23,29]. Second, high rates of economic exchanges increase the propagule fluxes of non-native species [6,9] via ornamental trade, sport fishing, and aquaculture [18]. Third, the increased demand for imported products associated with economic development increase the likelihood of unintentional introductions through the import process [6]. The “biotic resistance” hypothesis cannot explain the pattern of fish invasions observed, because no negative relationship between native and non-native species richness was found after controlling for the effects of environmental conditions, propagule pressure, and habitat disturbance. This means that regional species-rich communities are not necessarily a barrier against the establishment of non-native species [17]. Our results are consistent with several studies showing that species-rich fish communities can support higher species richness if the pool of potential colonisers is increased by species introductions [24,30,31]. More generally, our results agree with studies on various taxa that do not report biotic resistance at broad spatial scales [10,11]. Then, we provide no real support for the alternative “biotic acceptance” hypothesis [10] even if native and non-native species richness do respond similarly to some of the environmental gradients tested (i. e., altitudinal range and basin area). Actually, the absence of a significant positive relationship between native and non-native species richness implies that species-rich river basins do not support more non-native species than basins with a low native species richness (i. e., “the rich do not get richer”). This contrasts with numerous continental and regional-scale studies on plants and animals that report a strong matching between native and non-native species richness [10,32–35]. More generally, our results do not agree with the expectation that native and non-native species richness covary positively at macroecological scales [36]. The interpretation of the exact role of human activities (i. e., propagule pressure and habitat disturbance) in driving broad-scale patterns of non-native species richness faced major difficulties in previous continental and regional-scale studies due to covariations between human and natural factors [9,13,34,35]. Indeed, because humans may have preferred to settle in areas providing diverse natural resources, human population was found to be largest in regions with high levels of habitat heterogeneity and energy availability that favour species-rich native fauna and flora [34,37]. This therefore makes it difficult to determine whether the often-reported positive relationship between native and non-native species richness is driven by (i) common responses to habitat heterogeneity and energy availability or (ii) increased propagule pressure and habitat disturbance. Such difficulties were probably related to the spatial extent considered (i. e., a continental or regional extent). Indeed, we found a weak covariation between environmental and human descriptors of the world' s river basins at the global scale (Pearson' s correlation coefficients: r < 0. 35, Table S1). This allowed us to clearly disentangle the relative roles of human activities and environmental conditions in shaping the global pattern of fish invasions. We show that the biogeography of fish invasions at the global scale matches the geography of human impact but not the biogeography of native species. Because increasing the number of non-native species increases the risk of biodiversity loss [4,5], our results have two major implications for future conservation strategies. First, the six global invasion hotspots identified here account for the highest proportion of threatened fish species listed on the IUCN Red List [25]. These areas are also recognised as being biodiversity hotspots (particularly southern Europe, South Africa and Madagascar, southern Australia, and New Zealand [38,39]). Although species classified on the IUCN Red List are threatened by various sources of disturbance (e. g., habitat loss, pollution, species invasion, and overexploitation [25]), non-native species are recognised as a major threat to biodiversity after habitat loss [25,40]. For example, 20% of the 680 species extinctions listed by the IUCN were directly caused by species invasions [2]. Freshwater fish follow the same tendency, as 20% of the species listed by the IUCN are threatened by non-native species [41]. In that context, we recommend that non-native species importations in the six invasion hotspots be prohibited without detailed risk and long term cost-benefits assessments [42]. Special attention should also be given to these areas to design efficient control programs of already-established non-native species. Second, as we provide strong evidence for the “human activity” hypothesis (with a special emphasis on economic activity), we expect that river basins of developing countries will host an increasing number of non-native fish species as a direct result of economic development. This constitutes a serious threat to global biodiversity, because rivers of most developing areas (e. g., southern Asia, western and central Africa) are characterised by high levels of endemism [38]. Anticipating potential biodiversity threats should therefore be a priority, because once they are established, the eradication of a non-native species is extremely difficult and result in high economic costs [43]. Despite the increasing literature on non-native species, this study is, to our knowledge, the first to provide a global map of species invasions for a given taxonomic group and should stimulate others to test the generality of these findings for other taxa at this spatial scale. Such broad-scale analyses would help local researches to focus on non-native species control in the most sensitive areas (e. g., the six invasion hotspots we identified here for freshwater fish). This study should also stimulate researches on freshwater ecosystems by combining the existing global scale databases of physical disturbances [44,45] and the global pattern of fish invasions given here. This would permit to quantify river basins threats by considering simultaneously different sources of disturbance. Such an approach is urgently needed as rivers are among the most threatened ecosystems of the world [46] and as freshwater fish constitute a major source of protein for a large part of the world population [46]. We conducted an extensive literature survey of native and non-native freshwater fish species check lists. Only complete species lists at the river basin scale were considered, and we discarded incomplete check lists such as local inventories of a stream reach or based only on a given family. The resulting database was gathered from more than 400 bibliographic sources including published papers, books, and grey literature databases (references available upon request). Our species database contains species occurrence data for the world' s freshwater fish fauna at the river basin scale (i. e., 80% of all freshwater species described [47] and 1,055 river basins covering more than 80% of Earth' s surface). It constitutes the most comprehensive global database for freshwater fish occurrences at the river basin scale and, to our knowledge, the largest database for a group of invaders. We considered as non-native a species (i) that did not historically occur in a given basin and (ii) that was successfully established, i. e., self-reproducing populations. Estuarine species with no freshwater life stage were not considered in our analyses. The environmental and human databases contain seven variables selected to test (i) the “human activity” hypothesis: human population density (number of people km−2), percentage of urban area and purchase power parity GDP (in US$); (ii) the “biotic acceptance” hypothesis: number of native fish species, basin area (km2), altitudinal range (m), net primary productivity (NPP in kg-carbon m−2 year−1), and (iii) the “biotic resistance hypothesis”: number of native fish species. The area of each river basin was taken from published and unpublished data. The altitudinal range for each river basin was determined from a geographical atlas. We calculated the mean value of NPP, human population density, GDP, and percentage of urban area over the surface area of each basin from 0. 5° × 0. 5° grid data available in the Center for International Earth Science Information Network (CIESIN) and the Atlas of Biosphere [48,49]. The surface area and altitudinal range at the river basin scale are used as quantitative surrogates for habitat heterogeneity [16], which is known to influence native freshwater fish species richness [15,16]. Net primary productivity is used as a quantitative surrogate to river basin energy availability [16] and strongly correlates to native freshwater fish species richness [15,16]. This is verified in our data, as we found that both basin area and NPP are positively correlated to native species richness (partial Pearson' s correlation coefficient: r = 0. 592 and p < 0. 0001 for basin area while controlling for the effect of NPP; r = 0. 514 and p < 0. 0001 for NPP while controlling for the effect of the basin area). Then, the human population density, percentage of urban area, and GDP were used as quantitative surrogates for propagule pressure and habitat disturbance [5,9, 33]. The GDP measures the size of the economy and is defined as the market value of all final goods and services produced within a region in a given period of time. We first mapped the worldwide distribution of (i) the non-native species richness per basin and (ii) the percentage of non-native species per basin (i. e., the ratio of non-native species richness/total species richness). To do that, each basin was delimited by a geographic information system (GIS) using a grid reference of 0. 5° latitude and 0. 5° longitude and then reported on a world map. We used three classes of percentage (Figure 1A) and richness (Figure 1B) of non-native species to draw colour maps. Other maps with more classes were tried and provided similar results. We selected the one that minimised differences in sample size (i. e., number of river basins) between classes. The percentage of non-native species per basin was used to define invasion hotspots where more than a quarter of the species are non-native (i. e., the third class of percentage of non-native species; red areas in Figure 1A). It was preferred to the richness in non-natives due to its independence from native richness and basin area. For each of the three levels of fish invasion ([ 0%–5% ], ]5%–25%], ]25%–95% ]), we determined the percentage of species facing a high to extremely high risk of extinction in the wild, i. e., the vulnerable, endangered, and critically endangered fish species according to the IUCN Red List [25]. The percentage of threatened species should be regarded with caution, because the IUCN Red List for freshwater fish is still incomplete. The percentages of threatened species for the three levels of fish invasion are therefore probably underestimated. Although we recognise the potential biases and limitations of the IUCN listing procedure, the IUCN Red List of threatened species remains the most objective and authoritative system for classifying species in terms of the risk of extinction at the global scale [41,50]. The list of basins for the three levels of invasion is provided in Dataset S1. In this study, to test the three hypotheses (i. e., “human activity”, “biotic acceptance”, and “biotic resistance”), we did not build the best single and parsimonious model by using stepwise selection of a subset of independent variables having a significant effect on the number of non-native species per basin (i. e., predictive approach). Indeed, a single best model is not necessarily the best explanatory model, because minimizing the overall difference between the observed and predicted values does not necessarily equate to determining probable influence in a multivariate setting [26–28,51,52]. In addition, a simple regression model cannot identify situations in which potentially important independent variables are suppressed by other variables due to their high colinearity. When there is colinearity between independent variables, the direct response of the dependent variable to a independent variable may in fact only be an indirect effect owing to high dependence of the considered variable with one or many others [27]. In our dataset, the seven environmental and human variables are not independent (Pearson' s correlation coefficient values ranging from −0. 25 to 0. 79, Table S1). We therefore evaluated the independent explanatory power of each environmental and human variable by using hierarchical partitioning [26–28,51,52], a method based on the theorem of hierarchies in which all possible models in a multiple regression setting are considered jointly to attempt to identify the most likely causal factors (explanatory approach). If we consider k, the number of explanatory variables (X1, Xi, …, Xk), there are 2k possible models (i. e., 128 submodels by considering the seven explanatory variables), including the null model (M0). The Ri is a measure of fit between one independent variable Xi and the dependent variable Y. The fit between each of the seven explanatory variables and the dependent variable Y (number of non-native fish species per basin) was measured by the reduction of deviance generated by introducing a given variable into all of the possible models built with the six other variables within the considered hierarchies. We used a generalised linear model (GLM) with a Poisson error to treat our count data (i. e., the number of non-native fish species per basin). Each explanatory variable was log-transformed to meet the assumptions of normality and homoscedasticity. We consider k! hierarchical orderings of models that always begin with M0 and end with. For any given initial variable Xi, there are (k – 1)! possible hierarchies containing k (k – 1)! models in which Xi appears. For each hierarchy, we evaluate the influence of Xi on each of the k models including Xi (increase in model fit generated by including the variable Xi within each model). The independent influence (Ii) of Xi on Y was obtained by averaging all of the k (k – 1)! increases of fit. This averaging alleviates multicolinearity problems that are ignored by using a simple regression model [26–28]. The joint component Ji (effect caused jointly with the k – 1 other variables) is obtained by subtracting Ii from Ri, with Ri = Ii + Ji. If all explanatory variables were completely independent of one another, there would be no joint contributions [26]. For each variable, the independent and joint contributions are expressed as the percentage of the total explained deviance (R) In our models, the total independent contribution accounts for 75% of the total explained deviance, which means that the joint contribution of each explanatory variable was weak in explaining the global variation in non-native species richness (Figure S1). We therefore quantified the independent effect (IEi) of each variable on the dependent variable Y as the percentage of the total independent contribution, i. e. The significance of the independent effect (IEi) of each variable was determined by a randomization approach (n = 100) which yielded Z-scores [52]. Statistical significance was based on an upper confidence limit of 0. 95. Each variable display a significant independent effect. We applied hierarchical partitioning to a subsample of 597 basins (Afrotropical: 72; Australian: 94; Nearctic: 127; Neotropical: 68; Oriental: 29; Palearctic: 207) for which all seven environmental and human variables used were available. To test potential bias due to differences in sampling effort between continents, hierarchical partitioning was run on 1,000 random subsets of 100 basins among the total of 597 basins. For each variable, we calculated the 95% bootstrap percentile confidence interval of the independent effect (IEi). Hierarchical partitioning was conducted using the ‘hier. part' package [52] version 1. 0–1 implemented on the open source R software [53]. Hierarchical partitioning implemented for linear relationships was relevant to our data, because preliminary analyses did not detected any significant effect of polynomial terms. The hierarchical partitioning results were compared with those obtained with another method (i. e., variation partitioning, [54]). Overall, the results of the two methods were similar, and the variables highlighted as significant by the two approaches were the same. Hierarchical partitioning does not provide information on the form of the relationship (positive or negative) between the number of non-native species and each explanatory variable. To test the “human activity” hypothesis, we analysed the form and the significance of the relationship between each variable related to the “human activity” hypothesis (GDP, percentage of urban area, and population density) and the residuals from a GLM with a Poisson error. This model explains the number of non-native species by using independent variables related to the “biotic resistance” and “biotic acceptance” hypotheses (number of native species, altitudinal range, basin area, and net primary productivity). This allowed us to control for the effects of environmental conditions and native species richness. Then, to test the “biotic acceptance” hypothesis, we analysed the form and the significance of the relationship between each variable related to the “biotic acceptance” hypothesis (i. e., number of native species, altitudinal range, basin area, and net primary productivity) and the residuals from a GLM explaining the number of non-native species by using the human activity–related variables (i. e., GDP, percentage of urban area, and population density). This allowed us to control for the effects of propagule pressure and habitat disturbance. Lastly, to test the “biotic resistance” hypothesis, we analysed the form and the significance of the relationship between the number of native species and the residuals from a GLM explaining the number of non-native species by using independent variables related to the “biotic acceptance” and “human activity” hypotheses (i. e., altitudinal range, basin area, net primary productivity, GDP, percentage of urban area, and population density). This allowed us to control for the effects of environmental conditions, propagule pressure and habitat disturbance. To test the relationship between the model residuals and each explanatory variable, we performed a Spearman rank correlation test, because the model residuals were not normally distributed.

Title: Fish Invasions in the World's River Systems: When Natural Processes Are Blurred by Human Activities Summary: As one of the major threats to biodiversity, the detrimental consequences of biological invasions are widely recognised. Despite this, a global view of invasion patterns and their determinants is still lacking in aquatic ecosystems, reducing our ability to initiate practical actions. Here we report the global patterns of freshwater fish invasion in 1,055 river basins covering more than 80% of Earth' s continental surface. This allows us to identify six major invasion hotspots where non-native species represent more than a quarter of the total number of species. According to the World Conservation Union, these areas are also characterised by the highest proportion of threatened fish species. We also show that the natural factors controlling global biodiversity do not influence the number of non-native species in a given river basin. Instead, human activity-related factors, and particularly economic activity, explain why some river basins host more non-native species. In view of our findings, we fear massive invasions in developing countries with a growing economy as already experienced in developed countries. This constitutes a serious threat to global biodiversity.

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Write a title and summarize: Manifestation contre le déficit de l'Unedic (décembre 2014). JACQUES DEMARTHON / AFP Collectif L’assurance-chômage suscite des inquiétudes légitimes. Ce régime est en déficit sans discontinuer depuis 2009. Sa dette s’élève aujourd’hui à plus de 25 milliards d’euros, avec un déficit annuel de plus de 4 milliards. Rien ne laisse présager un redressement des comptes pour les années à venir si ses règles restent inchangées. La discussion qui s’ouvre le 22 février pour renégocier la convention d’assurance-chômage est une opportunité qu’il faut saisir. Une partie des propositions consiste à réduire la générosité de l’indemnisation chômage, en diminuant son niveau et sa durée, en rendant dégressive cette dernière, ou en intensifiant le contrôle des demandeurs d’emploi. Ces propositions méritent réflexion, mais elles ne sauraient être suffisantes, car elles font peser l’ajustement budgétaire uniquement sur les travailleurs, en laissant entendre qu’ils sont seuls responsables du déficit de l’assurance-chômage. Pourtant, il faut savoir le dire, les entreprises ont aussi leur part de responsabilité. Celles qui embauchent principalement en contrats à durée déterminée (CDD) cotisent peu par rapport aux dépenses qu’elles induisent du fait des indemnités versées à leurs salariés devenus demandeurs d’emploi après la fin du CDD ou de l’intérim. En revanche, les entreprises qui embauchent en contrat à durée indéterminée (CDI) versent généralement des cotisations bien plus importantes que le montant des indemnités de chômage perçues par leurs ex-salariés. Comportement vertueux Or, les pratiques de gestion des ressources humaines des entreprises ont une incidence forte sur les dépenses de l’assurance-chômage : dans bien des cas, plusieurs CDD successifs sur un même poste pourraient être remplacés par un CDI. Tous les contrats courts ne sont pas dus à la nature saisonnière de l’activité, à des imprévus ou au remplacement de salariés absents. En fait, nombre d’entreprises exploitent au mieux les avantages offerts par l’assurance-chômage : aujourd’hui, plus d’une embauche sur deux en CDD est une réembauche dans la même entreprise. Le coût de l’instabilité des emplois est devenu exorbitant : les cotisations chômage pour les seuls salariés en CDD et en intérim sont de 11 milliards inférieures aux allocations qu’ils perçoivent. Et les contrats courts ne sont pas seuls en cause : l’usage de la rupture conventionnelle, notamment pour les seniors, alimente aussi le déficit de l’assurance-chômage. Les entreprises qui embauchent principalement en CDD cotisent peu par rapport aux dépenses qu’elles induisent du fait des indemnités versées à leurs salariés devenus demandeurs d’emploi après la fin de leur contrat Pour résoudre ces problèmes, il faut instituer une modulation de la cotisation chômage employeur en fonction du coût réel mis à la charge de l’assurance-chômage par les entreprises. Dans ce système, le taux de cotisation est modulé en fonction du solde entre les contributions payées par l’entreprise et les allocations versées à ses ex-salariés. L’entreprise qui coûte peu à l’assurance-chômage payerait moins que celle qui l’alimente plus. Les entreprises seraient ainsi incitées à un comportement vertueux de stabilisation de l’emploi. Un tel système existe déjà pour les accidents du travail en France et il constitue on le sait un puissant levier pour inciter les entreprises à réduire accidents et maladies professionnelles. L’assurance-chômage aux Etats-Unis a mis en place un tel système de bonus-malus depuis les années 1930. Dispositif incohérent Un premier pas a déjà été franchi à l’initiative des partenaires sociaux pour infléchir le comportement des entreprises : depuis le 1er juillet 2013, le taux de la contribution patronale d’assurance-chômage est majoré de trois points pour les CDD de moins d’un mois et de 1,5 point pour les CDD de un à trois mois. Mais ce dispositif est loin d’être cohérent : les secteurs qui ont le plus recours à des CDD courts, comme l’intérim ou les emplois saisonniers, sont exonérés. Les CDD d’usage sont majorés à un taux nettement plus faible, de 0,5 point. Au total, cette majoration ne rapporte que 70 millions à l’Unedic ; une goutte d’eau dans les 33 milliards de cotisations. En outre, il n’y a aucun lien entre le coût d’utilisation des contrats courts pour l’assurance-chômage et la modulation de la cotisation. Ainsi, un CDD court est taxé même s’il concerne un salarié qui, ultérieurement, ne consomme pas ses droits à l’indemnisation du chômage. La taxation des contrats courts ne permet pas de cibler les pratiques d’alternance organisée par la même entreprise entre chômage indemnisé et emploi, coûteuses pour l’assurance-chômage. Les cotisations ne sont donc pas systématiquement majorées pour les entreprises dont la gestion de la main-d’œuvre coûte le plus cher à l’assurance-chômage. Pour atteindre cet objectif et stabiliser l’emploi, il faut mettre en place un véritable système de bonus-malus sur les cotisations patronales. Son adoption pourrait améliorer la situation financière de l’assurance-chômage en réduisant la précarité grandissante du travail, véritable fléau que connaît notre pays. Pierre Cahuc (professeur, CREST-ENSAE, Ecole polytechnique) et Jean-Christophe Sciberras (DRH France Solvay, ex-président de l’ANDRH), avec : Stéphane Carcillo (professeur, Sciences Po) ; Marc Ferracci (professeur, université Paris-II) ; François Fontaine (professeur, université Paris-I, Ecole d’économie de Paris) ; Jean-Olivier Hairault (professeur, université Paris-I, Ecole d’économie de Paris ; Francis Kramarz (professeur, CREST-ENSAE, Ecole polytechnique) ; François Langot (professeur, GAINS-TEPP université du Mans) ; Yannick L’Horty (professeur, université Marne-la-Vallée) ; Franck Malherbet (professeur, université de Rouen) ; Radu Vranceanu (professeur, Essec) ; André Zylberberg (directeur de recherche émérite, CNRS, Ecole d’économie de Paris).

Title: Unedic: pour "un bonus-malus sur les cotisations patronales Summary: Les cotisations chômage pour les salariés en CDD et en intérim sont de 11 milliards inférieures aux allocations qu'ils perçoivent. L'entreprise qui coûte peu à l'assurance-chômage doit cotiser moins, et inversement, explique un collectif d'économistes parmi lesquels Pierre Cahuc, Francis Kramarz ou Yannick L'Horty.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Protecting Older Workers Against Discrimination Act''. SEC. 2. FINDINGS AND PURPOSES. (a) Findings.--Congress finds the following: (1) In enacting section 107 of the Civil Rights Act of 1991 (adding section 703(m) of the Civil Rights Act of 1964), Congress reaffirmed its understanding that unlawful discrimination is often difficult to detect and prove because discriminators do not usually admit their discrimination and often try to conceal their true motives. Section 703(m) of the Civil Rights Act of 1964 expressly approved so-called ``mixed motive'' claims, providing that an unlawful employment practice is established when a protected characteristic was a motivating factor for any employment practice, even though other factors also motivated the practice. (2) Congress enacted amendments to other civil rights statutes, including the Age Discrimination in Employment Act of 1967 (referred to in this section as the ``ADEA''), the Americans with Disabilities Act of 1990, and the Rehabilitation Act of 1973, but Congress did not expressly amend those statutes to address mixed motive discrimination. (3) In the case of Gross v. FBL Financial Services, Inc., 557 U.S. 167 (2009), the Supreme Court held that, because Congress did not expressly amend the ADEA to address mixed motive claims, such claims were unavailable under the ADEA, and instead the complainant bears the burden of proving that a protected characteristic or protected activity was the ``but for'' cause of an unlawful employment practice. This decision has significantly narrowed the scope of protections afforded by the statutes that were not expressly amended in 1991 to address mixed motive claims. (b) Purposes.--The purposes of this Act are-- (1) to clarify congressional intent that mixed motive claims shall be available, and that a complaining party need not prove that a protected characteristic or protected activity was the ``but for'' cause of an unlawful employment practice, under the ADEA and similar civil rights provisions; (2) to reject the Supreme Court's reasoning in the Gross decision that Congress' failure to amend any statute other than title VII of the Civil Rights Act of 1964 (with respect to discrimination claims), in enacting section 107 of the Civil Rights Act of 1991, suggests that Congress intended to disallow mixed motive claims under other statutes; and (3) to clarify that complaining parties-- (A) may rely on any type or form of admissible evidence to establish their claims of an unlawful employment practice; (B) are not required to demonstrate that the protected characteristic or activity was the sole cause of the employment practice; and (C) may demonstrate an unlawful employment practice through any available method of proof or analytical framework. SEC. 3. STANDARDS OF PROOF. (a) Age Discrimination in Employment Act of 1967.-- (1) Clarifying prohibition against impermissible consideration of age in employment practices.--Section 4 of the Age Discrimination in Employment Act of 1967 (29 U.S.C. 623) is amended by inserting after subsection (f) the following: ``(g)(1) Except as otherwise provided in this Act, an unlawful practice is established under this Act when the complaining party demonstrates that age or an activity protected by subsection (d) was a motivating factor for any practice, even though other factors also motivated the practice. ``(2) In establishing an unlawful practice under this Act, including under paragraph (1) or by any other method of proof, a complaining party-- ``(A) may rely on any type or form of admissible evidence and need only produce evidence sufficient for a reasonable trier of fact to find that an unlawful practice occurred under this Act; and ``(B) shall not be required to demonstrate that age or an activity protected by subsection (d) was the sole cause of a practice.''. (2) Remedies.--Section 7 of such Act (29 U.S.C. 626) is amended-- (A) in subsection (b)-- (i) in the first sentence, by striking ``The'' and inserting ``(1) The''; (ii) in the third sentence, by striking ``Amounts'' and inserting the following: ``(2) Amounts''; (iii) in the fifth sentence, by striking ``Before'' and inserting the following: ``(4) Before''; and (iv) by inserting before paragraph (4), as designated by clause (iii) of this subparagraph, the following: ``(3) On a claim in which an individual demonstrates that age was a motivating factor for any employment practice, under section 4(g)(1), and a respondent demonstrates that the respondent would have taken the same action in the absence of the impermissible motivating factor, the court-- ``(A) may grant declaratory relief, injunctive relief (except as provided in subparagraph (B)), and attorney's fees and costs demonstrated to be directly attributable only to the pursuit of a claim under section 4(g)(1); and ``(B) shall not award damages or issue an order requiring any admission, reinstatement, hiring, promotion, or payment.''; and (B) in subsection (c)(1), by striking ``Any'' and inserting ``Subject to subsection (b)(3), any''. (3) Definitions.--Section 11 of such Act (29 U.S.C. 630) is amended by adding at the end the following: ``(m) The term `demonstrates' means meets the burdens of production and persuasion.''. (4) Federal employees.--Section 15 of such Act (29 U.S.C. 633a) is amended by adding at the end the following: ``(h) Sections 4(g) and 7(b)(3) shall apply to mixed motive claims (involving practices described in section 4(g)(1)) under this section.''. (b) Title VII of the Civil Rights Act of 1964.-- (1) Clarifying prohibition against impermissible consideration of race, color, religion, sex, or national origin in employment practices.--Section 703 of the Civil Rights Act of 1964 (42 U.S.C. 2000e-2) is amended by striking subsection (m) and inserting the following: ``(m) Except as otherwise provided in this title, an unlawful employment practice is established under this title when the complaining party demonstrates that race, color, religion, sex, or national origin or an activity protected by section 704(a) was a motivating factor for any employment practice, even though other factors also motivated the practice.''. (2) Federal employees.--Section 717 of such Act (42 U.S.C. 2000e-16) is amended by adding at the end the following: ``(g) Sections 703(m) and 706(g)(2)(B) shall apply to mixed motive cases (involving practices described in section 703(m)) under this section.''. (c) Americans With Disabilities Act of 1990.-- (1) Definitions.--Section 101 of the Americans with Disabilities Act of 1990 (42 U.S.C. 12111) is amended by adding at the end the following: ``(11) Demonstrates.--The term `demonstrates' means meets the burdens of production and persuasion.''. (2) Clarifying prohibition against impermissible consideration of disability in employment practices.--Section 102 of such Act (42 U.S.C. 12112) is amended by adding at the end the following: ``(e) Proof.-- ``(1) Establishment.--Except as otherwise provided in this Act, a discriminatory practice is established under this Act when the complaining party demonstrates that disability or an activity protected by subsection (a) or (b) of section 503 was a motivating factor for any employment practice, even though other factors also motivated the practice. ``(2) Demonstration.--In establishing a discriminatory practice under paragraph (1) or by any other method of proof, a complaining party-- ``(A) may rely on any type or form of admissible evidence and need only produce evidence sufficient for a reasonable trier of fact to find that a discriminatory practice occurred under this Act; and ``(B) shall not be required to demonstrate that disability or an activity protected by subsection (a) or (b) of section 503 was the sole cause of an employment practice.''. (3) Certain antiretaliation claims.--Section 503(c) of such Act (42 U.S.C. 12203(c)) is amended-- (A) by striking ``The remedies'' and inserting the following: ``(1) In general.--Except as provided in paragraph (2), the remedies''; and (B) by adding at the end the following: ``(2) Certain antiretaliation claims.--Section 107(c) shall apply to claims under section 102(e)(1) with respect to title I.''. (4) Remedies.--Section 107 of such Act (42 U.S.C. 12117) is amended by adding at the end the following: ``(c) Discriminatory Motivating Factor.--On a claim in which an individual demonstrates that disability was a motivating factor for any employment practice, under section 102(e)(1), and a respondent demonstrates that the respondent would have taken the same action in the absence of the impermissible motivating factor, the court-- ``(1) may grant declaratory relief, injunctive relief (except as provided in paragraph (2)), and attorney's fees and costs demonstrated to be directly attributable only to the pursuit of a claim under section 102(e)(1); and ``(2) shall not award damages or issue an order requiring any admission, reinstatement, hiring, promotion, or payment.''. (d) Rehabilitation Act of 1973.-- (1) In general.--Sections 501(g), 503(d), and 504(d) of the Rehabilitation Act of 1973 (29 U.S.C. 791(g), 793(d), and 794(d)), are each amended by adding after the words ``title I of the Americans with Disabilities Act of 1990 (42 U.S.C. 12111 et seq.)'' the following: ``, including the standards of causation or methods of proof applied under section 102(e) of that Act (42 U.S.C. 12112(e)),''. (2) Federal employees.--The amendment made by paragraph (1) to section 501(g) shall be construed to apply to all employees covered by section 501. SEC. 4. APPLICATION. This Act, and the amendments made by this Act, shall apply to all claims pending on or after the date of enactment of this Act.

Title: Protecting Older Workers Against Discrimination Act Summary: Protecting Older Workers Against Discrimination Act - Amends the Age Discrimination in Employment Act of 1967 to specify that an unlawful employment practice is established when the complaining party demonstrates that age or participation in investigations, proceedings, or litigation under such Act was a motivating factor for any practice, even though other factors also motivated the practice (thereby allowing what are commonly known as "mixed motive" claims). Permits a complaining party to rely on any type or form of admissible evidence, which need only be sufficient for a reasonable trier of fact to find that an unlawful practice occurred. Declares that a complaining party shall not be required to demonstrate that age or retaliation was the sole cause of a practice (thereby rejecting the Supreme Court decision in Gross v. FBL Financial Services, Inc., which requires a complainant to prove that age was the "but-for" cause for the employer's decision). Authorizes the court, on a claim in which an individual demonstrates that age was a motivating factor for any employment practice and in which a respondent demonstrates that the same action would have been taken in the absence of the impermissible motivating factor, to grant declaratory relief, injunctive relief, and attorney's fees and costs directly attributable only to the pursuit of a claim. Prohibits the court in such an instance from awarding damages or issuing an order requiring any admission, reinstatement, hiring, promotion, or payment. Applies the same standard of proof to other employment discrimination and retaliation claims, including claims under the Civil Rights Act of 1964, the Americans With Disabilities Act of 1990, the Rehabilitation Act of 1973, and similar laws concerning federal employees.

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Summarize: City doubles number of walls that spray back To all the late-night revelers who answer nature’s call on city walls: Beware. Again. The Public Works Department is doubling the number of walls painted with pee-repellent paint. In July, DPW installed nine in the Tenderloin, the Mission and South of Market. Eight more will be added this week. The surfaces make urine bounce right back onto the shoes and pants of unsuspecting relief-seekers. And, so far, it seems to be working. “So far so good,” Public Works Director Mohammed Nuru said. “We have had a daily monitoring program, and it seems to be 95 percent successful. There’s only one alley in the Mission that we have had trouble with.” The monitoring system consists of one lucky individual conducting a smell and sight test — anything that smells putrid or looks moist is noted. The trial period for the pee wall program ends in December, and the city will assess its effectiveness then. But it’s already been proved to work in Europe. The paint was first used in Hamburg’s St. Pauli quarter, where beer drinkers often can’t be bothered to find a bathroom. The experiment captured the attention of San Francisco officials. Historically, public urination has been an issue in San Francisco. Legislation banned it in 2002 but that really didn’t work, despite the threat of a $50-to-$500 fine. The Pit Stop program, which provides public restrooms, has contributed to a 17 percent drop in steam cleaning requests since last summer. But city officials wanted to do more. “It went really well in Hamburg,” Nuru said. “Based on that, I think this program is really going to work. It should deter people.” Photo: Scott Strazzante, The Chronicle Image 1 of / 4 Caption Close Image 1 of 4 Buy photo Wayne Howard stands in the corner by a wall that has been coated with pee-repelling paint at the 16th and Mission Street Bart Plaza in San Francisco, Calif., on Thursday, July 30, 2015. Wayne Howard stands in the corner by a wall that has been coated with pee-repelling paint at the 16th and Mission Street Bart Plaza in San Francisco, Calif., on Thursday, July 30, 2015. Photo: Scott Strazzante, The Chronicle Buy this photo Image 2 of 4 Buy photo Two walls have been coated with pee-repelling paint at the 16th and Mission Street Bart Plaza in San Francisco, Calif., on Thursday, July 30, 2015. Two walls have been coated with pee-repelling paint at the 16th and Mission Street Bart Plaza in San Francisco, Calif., on Thursday, July 30, 2015. Photo: Scott Strazzante, The Chronicle Buy this photo Image 3 of 4 Using the wall at 16th Street and Mission as an example, Public Works director Mohammed Nuru demonstrates how the walls deflect liquid onto the clothes of those that urinate on the walls. Using the wall at 16th Street and Mission as an example, Public Works director Mohammed Nuru demonstrates how the walls deflect liquid onto the clothes of those that urinate on the walls. Photo: Cameron Robert, The Chronicle Image 4 of 4 Using the wall at 16th Street and Mission as an example, Public Works director Mohammed Nuru demonstrates how the walls deflect liquid onto the clothes of those that urinate on the walls. Using the wall at 16th Street and Mission as an example, Public Works director Mohammed Nuru demonstrates how the walls deflect liquid onto the clothes of those that urinate on the walls. Photo: Cameron Robert, The Chronicle City doubles number of walls that spray back 1 / 4 Back to Gallery Signs over the walls read, “Hold it! This wall is not a public restroom. Please respect San Francisco and seek relief in an appropriate place.” They don’t explicitly state that the wall will fire back, so some surprises are in store for the unwitting relief-seeker. Paint and installation costs a couple of hundred dollars for each wall. The coating, Ultra-Ever Dry, comes from Ultra-tech, a Florida company in the chemical cleanup and waste management business that also provided the paint for Hamburg. The paint coats an object and creates a surface chemistry and texture with patterns of geometric shapes that have peaks, or high points, that repel most water-based and some oil-based liquids. If it continues to work, even more walls will be painted around the city. Lizzie Johnson is a San Francisco Chronicle staff writer. E-mail: [email protected] Twitter: @lizziejohnsonnn If it’s not winter’s salt and grime, it’s mud and everyday dirt. Keeping your car clean can be a chore – a backbreaking one if you prefer to do it yourself. But Nissan is testing a car that can keep itself clean, thanks to special paints that repel water and oils. Similar technology could eventually allow manufacturers to do away with windshield wipers by effectively fending off water, frost and grime. Meanwhile, new coatings can also help keep a car’s interior cleaner, something especially useful for parents – or those actively involved in sports like camping and trail biking. The Nissan Technical Center in Rolle, Switzerland, is in the midst of testing a specially prepared version of the maker’s little European Note model. The subcompact hatchback has had a layer of a special coating, called Ultra-Ever Dry, applied over its conventional paint finish. Developed by UltraTech International, it’s technically known as a super-hydrophobic and oleophobic paint. In other words, it actually repels both oil and water. That means that most common dirt, grime and oil won’t stick to the vehicle’s sheet metal. The Ultra-Ever Dry finish even works with rain, frost and sleet, Nissan reports, after preliminary testing. "The Nissan Note has been carefully engineered to take the stress out of customer driving,” said Geraldine Ingham, the chief marketing manager for the hatchback. "We are committed to addressing everyday problems our customers face and will always consider testing exciting, cutting edge technology like this incredible coating application." Nissan is testing a version of its Note subcompact, which has a layer of a special coating to make it repel water and oil. Nam Y. Huh / AP For the moment, the maker says it has no plans to offer the coating as a standard feature, but “will continue to consider (it) as a future aftermarket option.” The maker did come up with another interesting paint technology a few years back, a self-healing paint. Dubbed Scratch Shield, it uses an elastic resin mixed into a conventional paint formulation that, when exposed to sunlight, would fill in small scratches within a matter of hours. Unfortunately, few motorists have been willing to pay the premium for the special formulations and Nissan now offers Scratch Shield in only a few markets and not the U.S. The Japanese maker isn’t the only one looking at hydrophobic coatings – which could prove an interesting alternative for keeping windshields clear and clean. Italian designer Leonardo Fioravanti recently rolled out a wiper-less Hidra concept vehicle, which relies on both aerodynamic design to minimize what actually comes close to the windshield – and a special coating designed to keep things from sticking if they do come into contact with the glass. Under a hydrophobic outer coat, a second, nano layer “pushes” dirt off to the sides, while a third layer essentially helps clean off grime. An additional, electrically conductive layer provides the power needed to make it all work. The designers claim the technology behind the Hidra wiper-less windshield could prove reasonably cost-competitive and could wind up in production in a few years, marking the most significant improvement in rain gear since the first hand-operated wipers were introduced more than a century ago. British sports car maker McLaren, meanwhile, has lifted a page from fighter jet construction. It is experimenting with a system that uses high-frequency sound waves to create a sonic barrier to prevent water and dirt from reaching the windshield. Cleanliness is only one reason makers would like to eliminate the windshield wiper. The devices also create plenty of aerodynamic drag, which reduces a vehicle’s fuel economy. Manufacturers also are looking at ways to keep things clean inside their vehicles. Most fabrics are now treated with dirt- and liquid-resistant coatings – or can be ordered with an optional treatment package. Ford says it also tries to design vehicles so it’s easy to wipe or vacuum food or spilled beverages out of cracks and crevices. And to that effect, the new Honda Odyssey minivan this year began offering an optional, built-in vacuum cleaner tucked into its rear cargo compartment. Of course, one challenge is to prevent messes in the first place, says General Motors, which has been conducting research on ways to keep passengers, especially children stuck in back seats known to industry engineers as “the puke zone,” from getting car sick. “We know through other scientific research that even if our eyes are focused on a fixed point – if we can see the outside passing by in the window – our brain is telling us that we are moving,” explained Don Shreves, GM’s Human Factors engineering group manager said. “But if our eyes are at a downward angle and do not see the view outside the vehicle, our bodies become sensitive to motion and increase the chance of sickness.” Among other things, the research has shown GM engineers that passengers are less likely to get sick when DVD screens are mounted overhead.

Summary: San Francisco's experiment with pee-repelling paint is working out so well that the city says it will expand the trial in an attempt to prevent further public urination. Since treating nine of its walls with a liquid-repelling coating that causes urine to bounce back at anyone seeking relief, the city has been sending an unfortunate individual to smell the city's walls and look for moist areas. "So far so good," says Public Works Director Mohammed Nuru. "We have had a daily monitoring program, and it seems to be 95% successful," though he adds there's one particular alley "that we have had trouble with." As a result, officials will apply the Ultra-Ever Dry paint from Florida company Ultra-tech to eight more walls this week, reports the San Francisco Chronicle. Once the program's trial period wraps in December, city officials will review the effectiveness of the paint, which has worked "really well" in Hamburg, Germany, says Nuru. "Based on that, I think this program is really going to work. It should deter people." Signs on the coated walls state: "Hold it! This wall is not a public restroom. Please respect San Francisco and seek relief in an appropriate place." But as they don't explain that the texture produced by the paint creates peaks that repel most water-based and some oil-based liquids, "surprises are in store for the unwitting relief-seeker," notes the Chronicle. Last year, NBC News reported Nissan was testing a vehicle coated with Ultra-Ever Dry paint, meaning soon you may never have to wash your car again.

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Summarize: President Trump reacts to a question about the 9th Circuit at the U.S. Department of the Interior on April 26. (Brendan Smialowski/AFP/Getty Images) In the short time since he took office, President Trump has made no secret of his contempt for the U.S. Court of Appeals for the 9th Circuit, the country’s largest federal appellate court. “A disgraceful decision!” Trump tweeted in February when the California-based court upheld a ruling against his controversial travel ban. The circuit was “in chaos” and “frankly in turmoil,” he said later. Its “terrible decisions,” he said, had been overturned “at a record number.” Tuesday’s ruling against Trump’s sanctuary cities executive order brought another volley of criticisms (even though the decision came not from the appeals court but from a lower court judge in the circuit, which includes courts in nine states, Guam and the Northern Mariana Islands). [Trump blasts ‘ridiculous’ court ruling that blocked his order on sanctuary cities] Reacting to the ruling, Trump told the Washington Examiner that he was “absolutely” considering Republican-backed proposals to split the 9th Circuit into multiple courts. “It’s outrageous,” he said. None of this — the insults, the criticisms, the threats to break up the court — is new. For decades, the 9th Circuit has been a whipping boy for conservatives, who say it suffers from an incorrigible left-wing bias that clouds its decisions. They point to a string of high-profile rulings over the years in favor of liberal causes, and note that most of the court’s jurists are Democratic appointees. They also argue, erroneously, that its decisions get overturned more than any other appeals court. Most of the grumbling happens when the 9th Circuit issues a ruling critics don’t like. The implication is that they couldn’t possibly be wrong on the law in such cases and that their defeats are a product of the court’s ideological tilt. Sometimes things get hyperbolic. Conservative pundits are fond of calling the court the “Ninth Circus” or the “Nutty Ninth.” Newt Gingrich once proposed abolishing it entirely, bashing its opinions as “dictatorial” and “anti-American.” “America can find no more ghoulish poster children for what is fundamentally dishonest about liberal judicial activism than those judges of the 9th U.S. Circuit Court of Appeals,” conservative commentator Mark Q. Rhoads, a former Illinois legislator, once wrote of the court. There was indeed a period when the 9th Circuit was overwhelmingly liberal. In the 1970s, President Jimmy Carter worked with a Democratic Congress to add seats on the court, then stacked it with left-wing jurists. By the end of his term, Carter had installed 15 judges to the court, including the “liberal lion” Judge Stephen Reinhardt. “Carter appointed some of the most liberal judges ever, to any court,” Judge Alex Kozinski, a conservative 9th Circuit judge appointed by President Ronald Reagan, told the New York Times in 2010. Where does the court stand today? That depends on whom you ask. Conservatives say Carter’s legacy is alive and well on the 9th Circuit. As evidence, they cite a number of supposedly liberal-leaning decisions, including a 2002 ruling that found the Pledge of Allegiance unconstitutional because of its “one nation under God” line, as well as the landmark 2012 ruling that threw out California’s same-sex marriage ban. The court’s travel ban ruling appears to have been added to the list of offending decisions. But some legal observers contend that the court, while still generally more liberal than other circuits, has drifted back toward the center. Ben Feuer, chairman of the California Appellate Law Group LLP and a former 9th Circuit clerk, wrote last year that President Barack Obama had appointed less overtly liberal judges to replace retiring Carter appointees. “The court earned its flower-child reputation fairly,” Feuer wrote in the Los Angeles Times. He added: “But whereas the Carter judges reliably took left-leaning positions, the Obama judges are less predictable. That’s a big difference given the 9th Circuit’s mythic liberality.” University of Richmond Law School professor Carl Tobias seemed to agree, calling the 9th Circuit’s liberal reputation “dated.” “The reputation is certainly deserved based on the history of the last 40 years or so,” Hellman told the Associated Press in February. “It’s been more liberal, by which we mean more sympathetic to habeas petitioners, civil rights plaintiffs, anti-trust cases, immigration cases. But it’s less of an outlier now than it was.” Claims that the 9th Circuit is the “most reversed” appeals court are less a matter of debate. Critics of the court like to cite a statistic showing that nearly 80 percent of 9th Circuit rulings that make it to the U.S. Supreme Court get overturned, earning it the “most reversed” moniker. Trump has mentioned the figure on multiple occasions, and did so again in a tweet Wednesday. The claim is misleading at best. The 9th Circuit’s reversal rate was, in fact, 80 percent during the 2015-2016 term, as The Washington Post has reported. But it didn’t have the highest reversal rate that year — nor in any year since 2005. The circuit’s reversal rate was usually higher than the average, but not always. In any case, the Supreme Court only takes up a minuscule fraction of any circuit’s cases in a given year, so a court’s reversal rate, whatever it may be, really doesn’t say much about its jurisprudence. PolitiFact and the Associated Press have reached similar conclusions. Breaking up the 9th Circuit is a different story entirely. Conservative groups and Republicans in Congress have long dreamed of carving a new 12th Circuit, floating legislation year after year to split the court in two. Such a bill was introduced again in February by Sens. Jeff Flake (R-Ariz.) and John McCain (R-Ariz). There’s no shortage of nonpolitical reasons to break up the 9th Circuit. For one, the court takes on thousands more cases than any other circuit, leading some to argue it’s overburdened. It also covers a sprawling area of the country, spanning 15 federal judicial districts. That’s too big, some say. Trump, of course, seems more focused on his ideological battles than the nitty-gritty of the judicial system. As he said in February, “Courts seem to be so political.” More from Morning Mix Portland rose parade canceled after ‘antifascists’ threaten GOP marchers ‘We have to take a stand’: Mormon history scholars file brief against Trump travel ban ‘Mexican heritage’ judge bashed by Trump will oversee deported ‘dreamer’ case WASHINGTON (AP) — President Donald Trump is once again taking aim at a federal appeals court district that covers Western states, saying he is considering breaking up a circuit that is a longtime target of Republicans and is where his first travel ban was halted. Yet it would take congressional action to break up the 9th U.S. Circuit Court of Appeals. Republicans have introduced bills this year to do just that. Asked Wednesday during a White House interview by the Washington Examiner if he'd thought about proposals to break up the court, Trump replied, "Absolutely, I have." "There are many people that want to break up the 9th Circuit. It's outrageous," he told the Examiner. He accused critics of appealing to the 9th district "because they know that's like, semi-automatic." The comments echoed his Twitter criticism of the court Wednesday morning. Trump called U.S. District Judge William Orrick's preliminary injunction against his order stripping money from so-called sanctuary cities "ridiculous" on Twitter. He said he planned to take that case to the Supreme Court. However, an administration appeal of the district court's decision must go first to the 9th Circuit. Republicans have talked for years about splitting the circuit into two appellate courts, but earlier legislative proposals have failed, most recently in 2005. Those battles have often pitted lawmakers from California against members from smaller, more conservative states. Critics say the court has a liberal slant, a high caseload and distances that are too far for judges to travel. The circuit is the largest of the federal appellate courts, representing 20 percent of the U.S. population. It includes California, Alaska, Hawaii, Washington, Oregon, Montana, Idaho, Nevada, Arizona, Guam and the Northern Mariana Islands. The circuit has 29 judges, many more than the 5th, which is the next largest circuit with 17 judges. It was created in 1891 when the American West was much less populated. Democrats have opposed the split. Sen. Dianne Feinstein, D-Calif., was a leading opponent in the 2005 push, which she said was politically motivated. She has suggested adding judges to the court instead. In March, 9th circuit judges appointed by both Democratic and Republican presidents told lawmakers that breaking up the court was a bad idea. The 9th Circuit in February refused to immediately reinstate Trump's ban on travelers from seven predominantly Muslim nations, prompting the administration to release a new, narrower ban. That also has been held up by the courts. President Trump said Wednesday that he has "absolutely" considered proposals that would split up the 9th Circuit Court of Appeals, where judges have blocked two of his executive actions. "Absolutely, I have," Trump said of considering 9th Circuit breakup proposals during a far-ranging interview with the Washington Examiner at the White House. "There are many people that want to break up the 9th Circuit. It's outrageous." "Everybody immediately runs to the 9th Circuit. And we have a big country. We have lots of other locations. But they immediately run to the 9th Circuit. Because they know that's like, semi-automatic," Trump said. His comments came one day after U.S. District Judge William Orrick temporarily blocked Trump's efforts to withhold funds from any municipality that refuses to cooperate with immigration enforcement officers. Orrick, based in San Francisco, argued that Trump had overstepped his authority in January when he directed the Justice Department to put immigration-related conditions on grants for so-called sanctuary cities that may not be directly related to law enforcement. The case, if appealed, would go before the 9th Circuit. Other judges on the court halted two different versions of an executive action aimed at tightening vetting requirements for immigrants from Middle Eastern countries, because both actions called for a temporary suspension of some immigration from several predominantly Muslim countries. "The language could not be any clearer. I mean, the language on the ban, it reads so easy that a reasonably good student in the first grade will fully understand it. And they don't even mention the words in their rejection on the ban," Trump said. "And the same thing with this [sanctuary city decision]. I mean, when you have people that are being enabled to commit crime. And in San Francisco, when you look at Kate Steinle being shot and here is the court, you know, right in that same general area. And when you look at a Kate Steinle, when you look at so many other things." Trump was referring to a young woman in San Francisco, a sanctuary city, who was gunned down by an illegal immigrant in 2015. He has frequently pointed to Steinle's murder as evidence that sanctuary city policies can be harmful to American citizens. "Sanctuary cities have been very, very dangerous, very, very bad. And, you know, we've done a great job on law enforcement, we've done a great job at the border," Trump said. "And all of our most talented people say sanctuary cities are a disaster." Republicans have long criticized the 9th Circuit for its perceived liberal leanings and its enormous geographical reach, which has led to bureaucratic backlogs. GOP lawmakers have repeatedly introduced legislation that would carve out several states under the 9th Circuit's jurisdiction and create a new court designed to lighten the Ninth's caseload. The 9th Circuit hears appeals from courts in nine West Coast states and two U.S. territories. Of its 25 active judges, 18 were appointed by Democratic presidents. Trump said Wednesday that opponents of his policies have engaged in "judge shopping" in their efforts to find a sympathetic judicial platform for their partisan objections. "You see judge shopping, or what's gone on with these people, they immediately run to the 9th Circuit," Trump said. "It's got close to an 80 percent reversal period, and what's going on in the 9th Circuit is a shame."

Summary: Having seen the court block a number of his executive orders during his short time in office, President Trump tells the Washington Examiner he's "absolutely" considering breaking up the 9th Circuit Court of Appeals. "What's going on in the 9th Circuit is a shame," Trump says, adding the 9th Circuit is "outrageous" and his opponents are "judge shopping" by taking cases there. Republicans have been attacking the 9th Circuit for decades, accusing it of being both too liberal and too large, the Washington Post reports. Conservative pundits call it the "Ninth Circus" or the "Nutty Ninth," and Newt Gingrich once proposed getting rid of it entirely for being "anti-American." The first part of Republicans' complaint-that the 9th Circuit is too liberal-isn't as accurate as it used to be. The court's liberal bent declined recently after President Obama placed a handful of centrist judges on it. And the GOP's claim that decisions from the 9th Circuit have the highest rate of being overturned in the Supreme Court is complicated by the fact that the high court takes up a "minuscule fraction" of any courts cases, notes the Post. But Republicans may have a point that the 9th Circuit is too big: The AP reports it has more judges than any other appellate court and covers 20% of the US population. But 9th Circuit judges-appointed by both Democrat and Republican presidents-oppose proposals to break it down into smaller courts. Trump can't break up the court on his own anyway; it would require Congress to act.

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Write a title and summarize: Ectromelia virus (ECTV) is an orthopoxvirus (OPV) that causes mousepox, the murine equivalent of human smallpox. C57BL/6 (B6) mice are naturally resistant to mousepox due to the concerted action of innate and adaptive immune responses. Previous studies have shown that natural killer (NK) cells are a component of innate immunity that is essential for the B6 mice resistance to mousepox. However, the mechanism of NK cell–mediated resistance to OPV disease remains undefined. Here we show that B6 mice resistance to mousepox requires the direct cytolytic function of NK cells, as well as their ability to boost the T cell response. Furthermore, we show that the activating receptor NKG2D is required for optimal NK cell–mediated resistance to disease and lethality. Together, our results have important implication towards the understanding of natural resistance to pathogenic viral infections. Ectromelia virus (ECTV), the causative agent of mousepox, is an orthopoxvirus (OPV) with host specificity for the mouse. It is genetically very similar to vaccinia virus (VACV), the human pathogen variola virus (the agent of smallpox), and monkeypox virus [1], which sporadically infects people in Africa and produced a recent outbreak in the midwestern United States [2,3]. Like smallpox, mousepox is a severe disease with high mortality and infectivity. Thus, mousepox constitutes an excellent model to study smallpox and exanthematous diseases in general [4]. Although all mouse strains can be infected with ECTV, the outcome of the infection following footpad inoculation varies. Some sensitive strains, such as DBA/2, A/J, and BALB/c, develop mousepox and suffer high mortality during the first 2 wk post-infection, whereas other strains, such as C57BL/6 (B6), clear the infection without visible symptoms of systemic disease [5]. The resistance of B6 mice to mousepox is not due to an inherent decreased ability of the virus to replicate in this strain, but is a result of the combined action of the innate and adaptive immune systems [6–8]. Natural killer (NK) cells are innate effector cells serving as a first line of defense against certain viral infections and tumors [9,10]. For example, NK cell–deficient individuals become sick or succumb to normally non-life-threatening infections with the herpes virus human cytomegalovirus (HCMV) or varicella zoster [11–13]. In addition, C57BL/6 mice, which are normally resistant to mouse cytomegalovirus (MCMV), become susceptible when NK cells are depleted [14]. Similarly, NK cells have been shown to be crucial for the resistance of B6 mice to mousepox [15–17]. The activation of NK cells is regulated by a balance of signals transduced by activating and inhibitory receptors [18,19]. It is thought that continuous signaling through inhibitory receptors maintain NK cells in a resting state, and the loss of inhibitory signals (i. e., due to downregulation of MHC class I on target cells) or the expression of ligands for activating receptors on target cells results in NK cell activation [19]. Previous studies have shown that Rmp1, a dominant gene important for the ability of B6 mice to resist mousepox, maps to the “NK complex, ” a region of chromosome 6 that encodes a large number of NK cell–activating and inhibitory receptors [16,18]. Well-defined activating receptors encoded by polymorphic genes within the NK complex in B6 mice include NKR-P1C (NK1. 1, the prototypic marker defining NK cells in B6 mice), the Ly49 family members Ly49H and Ly49D, NKG2D, CD94-NKG2C, and CD94-NKG2E. Cmv1, a gene responsible for the resistance of B6 mice to MCMV infection and also mapped to the NK complex, has been shown to be the activating receptor Ly49H that binds to the MCMV-encoded MHC-like protein m157 expressed on the surface of infected cells [20–23]. On the other hand, the positive identification of any activating NK cell receptor involved in resistance to mousepox is still lacking, even though it has been speculated that Rmp1 might be the prototypic activating receptor NK1. 1 [24]. In this study, we show that NK cells directly contribute to antiviral defenses by curbing virus dissemination to central organs and also indirectly by augmenting the antiviral T cell response. We also demonstrate that the activating receptor NKG2D is involved in the NK cell–mediated resistance to mousepox, whereas NK1. 1 is not. B6 mice are normally resistant to mousepox ([5] and Table 1). To determine whether and when NK cells are required for resistance to mousepox, we depleted NK cells in B6 mice at different times following ECTV infection with either anti-asialo GM1 antiserum or anti-NK1. 1 mAb PK136. Anti-asialo GM1 antiserum depletes NK cells and some activated T cells but not NKT cells, whereas PK136 depletes NK cells and NKT cells but not T cells. Thus, the use of both Abs allowed us to better distinguish the role of NK cells from that of other lymphocyte populations. As shown in Table 1, depletion of NK cells with either Ab on or before day 4 post-infection (PI) with 3,000 pfu ECTV resulted in very high mortality, while mock-depleted mice (normal rabbit sera for anti-asialo GM1 and mouse IgG2a for PK136) were still resistant (data not shown). On the other hand, resistance to mousepox was maintained when mice were depleted of NK cells on day 6 PI, indicating an important role for the physical presence of NK cells at the early, but not later, stages of infection. In additional experiments, we found that five out of five and two out of five PK136-depleted B6 mice succumbed when infected with 100 and 1 pfu ECTV, respectively. Identical results were obtained for BALB/c mice infected with the same virus stock. This shows that the susceptibility of B6 mice depleted of NK cells on the day of infection is similar to that of genetically susceptible BALB/c mice. In agreement with the increased mortality, NK cell depletion resulted in severe necrosis and splenic lymphopenia as compared to non-depleted (intact) B6 mice (Figure 1A). Furthermore, 7 d PI, NK cell–depleted mice had more than 103-fold higher viral titers in both the spleen and liver than intact mice (Figure 1B), indicating a more severe systemic infection in the absence of NK cells. Thus, NK cells are required in the early phase of infection to control viral loads in central organs and to resist lethal mousepox. We next evaluated the NK cell response at different times PI by flow cytometry. NK cell production of IFN-γ and granzyme B (GzB) in the draining lymph nodes (D-LN) was already induced 24 h PI, reaching the peak at 48 h PI when ∼20% of the NK cells produced IFN-γ and 43% produced GzB (Figure 1C and 1D), a time when T cell responses were not yet detected [25]. This was accompanied by a 2- to 4-fold increase in the proportion of NK cells in the D-LN (Figure 1D). NK cell responses in non-draining LN and spleen, however, did not peak until day 5 PI (Figure 1C). In addition, histopathological analysis of the infected footpads on day 2 PI did not show leukocyte infiltration or other signs of inflammation in either intact or NK cell–depleted B6 mice (data not shown). Thus, at very early stages of infection the NK cell response is restricted to the D-LN. To distinguish whether the increase in NK cell number in D-LN resulted from recruitment and/or proliferation, we inoculated mice at different stages of infection with BrdU IP and sacrificed the mice 3 h later to determine BrdU incorporation by flow cytometric analysis. Very few NK cells incorporated BrdU in the D-LN during the first 3 d PI, suggesting increased migration to the D-LN at early times PI. In fact, on day 3 and 5 PI, the BrdU incorporation in NK cells in spleen and liver was higher than in the D-LN (Figure 1E). Thus, the increase of NK cells in the D-LN on day 2 PI is mostly due to recruitment rather than proliferation. A recent report by Hayakawa and Smyth revealed that peripheral NK1. 1+ NK cells can be divided into three subsets according to their expression of CD11b and CD27, which they designated R1 (CD27+, CD11b−), R2 (CD27+, CD11b+), and R3 (CD27−, CD11b+), that represent NK cells at distinct developmental stages [26,27]. R1 cells are immature, whereas R2 and R3 fractionate the mature cells into two populations. While adoptively transferred R2 cells can differentiate into R3 cells, R3 cells appear to be terminally differentiated. We, therefore, investigated whether ECTV infection altered the maturation phenotype of NK cells in the D-LN and found a substantial increase in NK cells with a mature (R3) phenotype on day 2 PI (Figure 1F, upper panels). Of interest, the majority of effectors (IFN-γ+ and/or GzB+) were within this mature NK cell population (Figure 1F, lower panels). Together, these data demonstrate that the increased NK cell number in the D-LN at early time points PI is mainly a result of recruitment. Moreover, the data show that the NK cells responding to ECTV infection are mature or mature very rapidly after infection. We hypothesized that the observed early recruitment to and activation of NK cells in the D-LN may contribute to the prevention of virus dissemination to central organs. Thus, we depleted NK cells in B6 mice with anti-NK1. 1 mAb (PK136) and determined viral titers in spleen and liver on day 3 PI. To rule out a role for T cells at this stage we also depleted both CD4+ and CD8+ T cells by using a combination of anti-CD4 (GK1. 5) and anti-CD8 (2. 43) mAbs. Depletion of NK cells resulted in >103-fold increase in viral loads in the spleen, while a much lower increase in virus titers was observed in mice depleted of T cells. The slight increase in virus titers in the T cell–depleted mice was not statistically significant (p = 0. 09) and was not reproducible. We also observed a significant increase in virus titers on day 3 PI in the livers of NK cell–depleted mice, but not T cell–depleted mice (p = 0. 07) (Figure 1G). Therefore, NK cells, and not T cells, are responsible for limiting the early dissemination of ECTV from the site of infection to central organs. NK cells can control viral infections by producing antiviral cytokines, such as IFN-γ, or by perforin (Pf) -mediated killing of infected cells [28]. Others have shown that B6 mice deficient in either Pf or IFN-γ are susceptible to mousepox [29,30]. However, whether the early control of virus dissemination (day 3 PI) by NK cells requires IFN-γ and/or Pf-mediated killing is unknown. We, therefore, compared virus titers on day 3 PI in the spleens of wild-type (WT), IFN-γ-deficient, and Pf-deficient B6 mice. We found that both IFN-γ- (Figure 1H) and Pf-deficient mice (Figure 1I) had significantly elevated virus titers as compared to B6 mice. Thus, the early control of virus dissemination by NK cells requires both the antiviral effects of IFN-γ and Pf-mediated cytotoxicity. Our previous studies and those of others showed that an optimal CD8+ T cell response is required for resistance to mousepox [8,25,31]. The data above indicate that NK cells have a direct effect in preventing mousepox by curbing virus dissemination during early infection. However, NK depletion at early stages resulted in an increase in spleen and liver viral titers, splenic lymphopenia, liver pathology, and death on day 7 PI (Figure 1 and Table 1), a time when the presence of NK cells was no longer necessary (Table 1) and when the T cell response should have already peaked [25]. This suggested that the absence of NK cells may have an impact on the establishment of the adaptive anti-ECTV T cell response. Thus, we compared in vivo T cell proliferation in intact mice and mice depleted of NK cells on day 0 PI. We found that on day 5 PI (when the T cell response normally peaks in D-LN), only a few CD8+ and CD4+ T cells incorporated BrdU in the D-LN of NK cell–depleted mice, while ∼40% of CD8+ T cells and 20% of CD4+ T cells incorporated BrdU in intact mice (Figure 2A). Mice depleted of NK cells on day 0 PI also had substantially reduced T cell proliferation in spleen and liver on day 7 PI (the time when the T cell response normally peaks in the spleen, data not shown). We also determined the CD8+ T cell response by intracellular staining for the effector molecules, GzB and IFN-γ. On 7 d PI, there was a strong T cell response in spleen with ∼70% of CD8+ T cells producing GzB and more than 10% of CD8+ T cells producing IFN-γ in intact B6 mice (Figure 2B). However, in mice depleted of NK cells on day 0 PI, the CD8+ T cell response was greatly reduced, with only ∼30% of CD8+ T cells producing GzB and ∼5% CD8+ T cells producing IFN-γ. Furthermore, because infected NK cell–depleted mice had severely lymphopenic spleens (Figure 1A), they also had a 16-fold reduction in the absolute number of effector CD8+ T cells as compared with infected intact mice (data not shown). These results indicate that the adaptive T cell response was substantially reduced in the absence of NK cells. To gain insight into the mechanism whereby NK cells become activated during ECTV infection, we explored whether various known NK cell–activating receptors are involved in resistance to mousepox. We first focused on NK1. 1 and on the activating receptors of the Ly49 family. We focused on these receptors because NK1. 1 is not encoded in mousepox-susceptible DBA/2 and BALB/c mice. Furthermore, it has been speculated that NK1. 1 might be Rmp1 [24]. In addition, there is good precedent for the involvement of Ly49H in NK cell–mediated antiviral responses, more specifically to MCMV [20–22]. Ly49D and Ly49H are the only activating receptors of the Ly49 family in B6 mice. Because B6 mice selectively deficient in NK1. 1, Ly49H, or Ly49D are not available, we first used 129/Sve mice, which do not express any of these receptors [22]. Similar to B6 mice, 129/Sve mice were naturally resistant to mousepox but became sensitive when depleted of NK cells with anti-asialo GM1 Ab (PK136 could not be used because 129/Sve mice do not express NK1. 1) (Table 2). This demonstrates that NK cells are also required for 129/Sve mice resistance to mousepox but that NK1. 1, Ly49D, or Ly49H are not essential for resistance because 129/Sve do not possess these receptors. The results above suggested, but did not formally prove that Ly49H, Ly49D, and/or NK1. 1 are not required for the resistance of B6 mice to mousepox, because it remained possible that Ly49H, Ly49D, and/or NK1. 1 play a role in B6 resistance but that other activating receptors substitute their function in 129/Sve mice. Although it is not possible to definitively rule out the participation Ly49H or Ly49D in B6 resistance with currently available reagents, we took advantage of FcɛRIγ-deficient B6 mice [32] to rule out a role for NK1. 1. It is known that NK1. 1 signaling requires association with the ITAM-containing FcɛRIγ adapter [33]. FcɛRIγ-deficient B6 mice, however, were completely resistant to mousepox (Table 2), demonstrating that NK1. 1 is not essential for the resistance of B6 mice to mousepox. In addition, because Abs are required for long-term resistance to mousepox [8,31] and FcɛRIγ is required for Ab-dependent cellular cytotoxicity (ADCC) [32], these data also demonstrate that ADCC is not involved in Ab-dependent resistance to mousepox. We addressed whether the activating receptor NKG2D might be involved in mousepox resistance. NKG2D is expressed by NK cells and some T cells, including γδ-TcR+ T cells and activated CD8+ T cells. Although not polymorphic, this activating receptor is expressed at somewhat lower levels on activated NK cells of mousepox-susceptible NOD and BALB/c mice than in B6 mice ([34] and unpublished data). Because NKG2D-deficient mice are not available, we took advantage of the anti-NKG2D mAb CX5 that, in vivo, blocks binding of NKG2D to its ligands and causes its internalization without depleting NKG2D-bearing cells [35–37]. We inoculated B6 mice with CX5 mAb or control rat IgG 1 d before and 2 d PI. As shown in Table 2, CX5 treatment resulted in 50% mortality suggesting that NKG2D is involved in the NK cell–mediated resistance to mousepox. In mice, NKG2D signals through either DAP10 or DAP12 adapter proteins, whereas Ly49H and D signal through DAP12 [38–40]; therefore, we determined mousepox susceptibility in either DAP12- or DAP10-deficient mice on a B6 mousepox-resistant background. We found that more than 50% of DAP12-deficient mice developed the typical skin rash, but only 10% died following ECTV infection, whereas all DAP10-deficient mice survived without showing ostensible symptoms of mousepox (Table 2). To get further insights into the role of NKG2D and its adapters in resistance to mousepox, we determined cell numbers and virus titers 7 d PI in NKG2D-blocked as well as in DAP12- and DAP10-deficient mice. Results showed that NKG2D blockade and DAP12- or DAP10-deficiency resulted in significantly decreased splenic lymphocytosis as compared with wild type, untreated B6 mice (Figure 3A). More important, the viral loads in NKG2D-blocked mice were almost 103 times higher than in unblocked mice, whereas those of DAP12- and DAP10-deficient mice were also significantly elevated, but to a lesser degree (102 and 10 times higher, respectively) (Figure 3B). Thus, the degree of splenic lymphocytosis and viral loads on day 7 PI was consistent with the lethality and symptoms of mousepox that we observed under the different conditions. Together, these results show that NKG2D is involved in resistance to mousepox. The data also suggest that for this process NKG2D preferentially, but not exclusively, uses DAP12 as the signaling adapter. Most likely, the absence of one adapter is partially compensated by the presence of the other. The data further suggest that Ly49H and Ly49D are not essential for B6 resistance to mousepox because DAP12-deficient mice were only mildly susceptible to mousepox infection. This contrasts with prior studies demonstrating that DAP12-deficient mice on a B6 MCMV-resistant background are exquisitely sensitive to MCMV infection [41]. NKG2D is expressed by NK cells [42] and its expression is further increased during MCMV infection (J. Sun and L. L. Lanier, unpublished data). We, therefore, determined whether ECTV infection also increased NKG2D expression on NK cells. As shown in Figure 4A, NK cells increased NKG2D expression at the cell surface, and the proliferating NK cells after ECTV infection were NKG2Dhigh. The data above showed that NK cells are the main population-controlling early virus dissemination to visceral organs, and that absence of NKG2D signaling results in increased susceptibility to mousepox. However, NKG2D is not only expressed by NK cells but also by some T cells. We hypothesized that if NKG2D has a role in NK cell–mediated resistance to mousepox, NKG2D blockade should result in increased early virus dissemination to organs independent of T cells. To test this hypothesis, we measured early viral titers in the spleens (day 3 PI) of mice treated with the anti-NKG2D mAb CX5. To rule out a role of NKG2D on T cells at this time, we also included groups of mice that were T cell–depleted or NKG2D-blocked and T cell–depleted. As shown in Figure 4B, NKG2D-blocked and T cell–depleted + NKG2D-blocked mice had virus titers that were comparable to those of NK cell–depleted mice and that were significantly higher than those of mice that were only T cell–depleted. Together, these data indicate that NKG2D is involved in the NK cell–mediated resistant to mousepox. We next tested whether NKG2D blockade and DAP10- or DAP12-deficiency affected the recruitment of NK cells to D-LN (Figure 4C, upper panels) or their ability to produce IFN-γ and GzB (Figure 4C, lower panels). However, we found that none of these parameters were decreased. In fact, in both DAP10- and DAP12-deficient mice more NK cells were recruited to the D-LN and a larger proportion produced IFN-γ and GzB as compared with wild-type B6 mice. This indicates that NKG2D is required for effective NK cell–mediated control of ECTV, but is not required for their activation. Because NK cell recruitment and activation in the D-LN was not diminished by NKG2D blockade or DAP12 and DAP10 deficiency (Figure 4C), we hypothesized that NKG2D may be required for optimal NK cell–mediated cytotoxicity. Thus, we examined whether NK cells from NKG2D-blocked mice were defective in their ability to kill various targets. For this purpose, mice were infected with ECTV and treated or not with anti-NKG2D Ab. Five days later, NK cells were purified from their spleens and immediately analyzed by flow cytometry to confirm NKG2D receptor blockade or downregulation in the CX5-treated mice (Figure 4D) and were used as effectors in 4-h 51Cr release assays. Because we were unable to show specific killing of ECTV-infected cells in vitro, we tested the purified NK cells against a variety of uninfected targets that constitutively express NKG2D ligands at their cell surface (MC57G, MEF, and YAC-1) or, as a control, CHO-K1 whose killing is dependent on Ly49D [43,44], but not NKG2D. We found that the NK cells from mice infected with ECTV (whether treated with anti-NKG2D Ab or not) killed all targets much more effectively than those from uninfected mice. This enhanced killing did not require the targets to be infected. However, the NK cells from mice treated with CX5 were less effective killers of NKG2D ligand-bearing target cells as compared to those from untreated mice, except against CHO-K1 cells for which the NK cell killing is dependent on Ly49D and not on NKG2D (Figure 4E). Furthermore, purified NK cells from ECTV-infected intact mice also demonstrated reduced, but not absent cytotoxicity when anti-NKG2D mAb was added to the assay (Figure 4F). Thus, ECTV infection enhances spontaneous NK cell–mediated cytotoxicity, which is partially reduced by NKG2D blockade. Together, these data suggest that NKG2D signaling is not required for the recruitment and activation of NK cells during ECTV infection, but contributes to their optimal ability to kill targets expressing NKG2D ligands. Because anti-NKG2D did not reduce cytotoxicity to the same levels as those in uninfected mice, the data also indicate that activating receptors other than NKG2D are involved in the cytotoxicity induced by ECTV infection. NKG2D-mediated killing requires the recognition of ligands on the surface of target cells. The ligands of NKG2D are host cell–encoded MHC class I–like proteins that are expressed by tumors and stressed cells and also following infection of cells with some viruses. Identified cellular ligands for NKG2D in mice include H60, MULT1, and Rae-1 [45–49]. To determine if ECTV infection induces the upregulation of NKG2D ligands, we infected mouse embryo fibroblasts (MEFs) with 0. 5 pfu/cell ECTV expressing enhanced green fluorescence protein (EGFP), and the expression of NKG2D ligands on infected and uninfected cells was determined by flow cytometry. Consistent with the ability of ECTV-activated NK cells to spontaneously kill them, MEFs constitutively express NKG2D ligands as revealed by staining with mouse NKG2D-Fc fusion protein [50] (Figure 5A). However, infection with ECTV increased NKG2D-Fc staining and resulted in clear upregulation of MULT1 (Figure 5A) but not Rae-1 (data not shown). We also observed increased staining with NKG2D-Fc and anti-pan Rae-1 and anti-MULT1 mAbs in the fibrosarcoma cell line MC57G, and an increase in staining with NKG2D-Fc and anti-pan Rae-1 mAb in peritoneal lymphocytes (data not shown). Moreover, using quantitative RT-PCR, we detected a 2. 8-fold increase of Rae-1 transcripts in the D-LN of ECTV-infected mice at 12 h PI, as compared with uninfected controls (Figure 5C). Thus, these results show that ECTV infection can induce the expression of NKG2D ligands, suggesting that binding of NKG2D with these ligands might result in improved NK cell killing of infected cells in vivo and better control of ECTV dissemination. In this study, we confirmed previous reports demonstrating that NK cells are required for natural resistance to mousepox [15–17]. More importantly, we define several of the mechanisms whereby NK cells afford this resistance. To determine whether and when NK cells are required for resistance to mousepox we depleted mice of NK cells with either anti-asialo GM1 or anti-NK1. 1 Abs at different times PI. Although separately these two approaches have caveats, together they provide conclusive evidence that the presence of NK cells during the first 4 d PI, but not beyond day 5 PI, is essential for resistance to mousepox. First, even though anti-NK1. 1 Ab depletes NKT cells, the loss of mousepox resistance upon Ab treatment cannot be due to the elimination of NKT cells because anti-asialo GM1 Ab does not eliminate this population [51,52]. Furthermore, Parker et al. recently demonstrated that NKT cells are dispensable for resistance to mousepox because ECTV infection of mice deficient in NKT cells (i. e., CD1d- and Vα14Jα281 TCR-deficient mice) did not result in symptoms of mousepox or an increase in virus titers when compared to wild-type B6 mice [17]. Second, although anti-asialo GM1 can eliminate some activated T cells [53], the loss of resistance cannot be attributed to T cell depletion because asialo GM1 is not expressed on virus-specific T cells within the first few days after viral infection. Moreover, we show that anti-NK1. 1 and -asialo GM1 Abs do not affect resistance when given on day 6 PI (the peak of the T cell response). Furthermore, treatment with anti-NK1. 1, but not depletion of T cells one day before infection, resulted in significantly increased virus titers on day 3 PI. In addition, treatment with anti-asialo GM1 Ab accelerated ECTV lethality in RAG-1-deficient mice, which lack adaptive immunity (data not shown). γδ T cell–deficient mice are also resistant to mousepox [17], indicating that the possible depletion of some γδ T cells by anti-asialo GM1 Ab cannot account for the dramatic increase in ECTV lethality after treatment with this Ab. Although work in other laboratories has already shown that depletion of NK cells with either anti-NK1. 1 or asialo-GM1 results in susceptibility to mousepox [6,15,17], depletion at different times PI has not been described previously. Sequential depletion of NK cells allowed us to establish that the presence of NK cells is required only during the very early stages of infection. This is consistent with the concept that NK cells serve as the first line of defense after infection, thereby providing sufficient time to mount a full-fledged T and B cell response. We also established, however, that the contribution of NK cells to mousepox resistance extends beyond the need for their physical presence to kill virus-infected cells early after infection because NK cell depletion before day 4 PI resulted in higher virus titers and death after this time. This is most likely due to the role of NK cells in allowing a potent antiviral T cell response to develop, as discussed below. We also followed the kinetics of NK proliferation and activation in spleen, liver, and D-LN of ECTV-infected mice. Interestingly, although we detected some proliferation, we did not find activated NK cells in liver and spleen on day 3 PI. This was despite the fact that NK cell–mediated control of virus loads in spleen and liver already occurred at this time PI. In fact, the peak of NK cell activation in spleen and liver took place on day 5 PI. On the other hand, the proportion of total NK cells, as well as the proportion of activated NK cells in the D-LN, peaked as early as day 2 PI, and these parameters were still substantially elevated on day 3 PI, notwithstanding that the proliferation of NK cells at these times was not yet detectable in D-LN. In addition, we found that the increase in NK cell numbers in the D-LN on day 2 PI was mostly due to an increase in mature NK cells and that these mature NK cells were preferentially activated. Together, these data suggest that the prompt NK cell response in the D-LN is responsible for the early control of virus loads in central organs and that this response is mostly due to the recruitment and activation of mature NK cells rather than their expansion. To spread to central organs from the footpad, ECTV must pass through the D-LN [5,54–56]. Recently, we have shown that in mousepox-susceptible BALB/c mice, memory CD8+ T cells protect from mousepox, at least in part, by curbing the spread of ECTV from the D-LN to central organs and allowing for the establishment of a full-fledged adaptive response [54]. Our results here suggest that NK cells may use the same strategy, furthering a model where LNs are not only sites where lymphocytes are primed and proliferate but also the place where a major fight against virus spread takes place [54]. In addition to NK cells, strong adaptive T cell responses are essential for resistance to mousepox. Our experiments here show that NK cells have a direct role in controlling ECTV because they reduced virus loads on day 3 PI when the T cell response is still undetectable (data not shown and [25]). However, if the only role of NK cells were direct, one would expect mice depleted of NK cells to become sick, but recover once the adaptive immune system takes control. Yet, while the presence of NK cells on day 6 PI was not required for resistance to mousepox, their presence before day 6 PI was vital for controlling virus titers and preventing pathology and death after this time. This could be explained by the finding that depletion of NK cells on the day of infection resulted in a substantially reduced T cell response. There are three non-excluding possibilities that may explain this effect. First, NK cell production of cytokines such as IFN-γ may directly modulate the adaptive T cell response [28]. A caveat to this hypothesis is that NK cells produce as much or more IFN-γ in ECTV-infected mousepox-susceptible BALB/c and DBA2/J mice as in resistant B6 mice (unpublished data), but these susceptible mice fail to mount an effective T cell response to ECTV. Second, NK cells may indirectly affect the activation of T cells through their interaction with antigen-presenting cells such as macrophages and dendritic cells (DC). This hypothesis has the same caveats as the one above. Third, it is possible that in the absence of NK cells, the uncontrolled virus replication of a highly pathogenic virus overwhelms the T cell response. In support of this possibility, infection of mousepox-susceptible BALB/c mice with a highly attenuated strain of ECTV results in the induction of a strong T cell response (unpublished data), and depletion of NK cells does not affect the T cell response to the poorly pathogenic vaccinia virus (unpublished data) with which ECTV shares most of the dominant CD8+ T cell epitopes [57]. Previous work by Delano and Brownstein showed that Rmp1, a gene important for resistance to mousepox, maps to the NK complex in distal chromosome 6 [16]. Moreover, they proposed that Rmp1 encoded NK1. 1 [24]. However, we found that NK1. 1 is not required for resistance to mousepox. In addition, our work suggests that the activating receptors Ly49D and Ly49H are likely not involved. On the other hand, using in vivo signaling blockade we found that NKG2D is involved in resistance to mousepox and that ECTV infection results in the upregulation of NKG2D ligands in vivo and in vitro. That NKG2D is involved in antiviral responses has precedents. For example, previous studies have shown that cytomegalovirus (CMV) induces the upregulation of NKG2D ligands and that mouse and human CMV encode immune evasion molecules that downregulate NKG2D ligands and are important for their pathogenesis [28]. However, a direct role for NKG2D in the response to poxviruses has not been described previously. Interestingly, Campbell et al. have recently shown that cowpox virus encodes a soluble competitive antagonist of NKG2D [58]. Although this gene has been lost in other OPVs, such as ECTV, VACV, VARV, and MPXV, these data support our finding that NKG2D is involved in resistance to some OPV infections. On the other hand, NKp30, NKp44, and NKp46, but not NKG2D, were found to be responsible for the recognition of VACV-infected cells by human NK cells [59]. Whether this reflects differences between NK cell recognition of different OPVs, differences between human versus mouse NK cells, or both, will require further investigation. Because mouse NKG2D signals through either the adapter protein DAP12 or DAP10, we further tested the susceptibility of mice lacking one or the other adapter. Of interest, when considering virus titers and spleen cellularity, the susceptibility of these two strains of mice was intermediate between intact and NKG2D mAb–blocked B6 mice. This indicates that for resistance to mousepox, the two adapters likely have overlapping, but not completely redundant, effects. Still, DAP12 seems to be the preferred adapter because DAP12-deficient mice were more susceptible to mousepox than DAP10-deficient mice, although it is possible that other DAP12-associated receptors might contribute to resistance to mousepox. Our results predict that DAP10 + DAP12 double-deficient mice would be highly susceptible to mousepox. Unfortunately, these mice are not yet available. NKG2D is expressed by most NK cells but also by activated CD8+ T cells. In fact, we have observed that in ECTV-infected B6 mice a large proportion of virus-specific CD8+ T cells express NKG2D beginning 7 d PI (unpublished data). Thus, the experiments reported here do not rule out the additional contribution of NKG2D in the T cell–mediated resistance to mousepox, a very interesting possibility that we are currently investigating. Nevertheless, our experiments clearly implicate NKG2D in the NK cell–mediated response to ECTV because in vivo NKG2D blockade resulted in enhanced virus loads in central organs before the onset of the T cell response and because in vivo NKG2D blockade reduced the cytotoxicity of ECTV-activated NK cells. Still, several lines of evidence indicate that NKG2D is not the only activating receptor involved in the anti-ECTV NK cell–mediated response and that its most likely role is as a co-stimulator that facilitates cytotoxicity rather than being required for the initial NK cell activation: (1) NK cell depletion was much more effective than NKG2D blockade at rendering B6 mice susceptible to mousepox; (2) NKG2D-blockade did not affect NK cell proliferation (data not shown), IFN-γ and GzB production by NK cells, or recruitment of NK cells into the D-LN of ECTV-infected mice; (3) In vivo and in vitro NKG2D blockade significantly decreased, but did not abrogate, the cytotoxicity of ECTV-activated NK cells. Ongoing studies in our laboratory are aimed at identifying other activating receptor (s) and signaling pathway (s) required for NK cell–mediated resistance to mousepox. In summary, our work demonstrates that NK cells contribute to the natural resistance of B6 mice to mousepox by using direct effector functions (most likely the killing of infected cells) to curb virus dissemination and by supporting a strong adaptive T cell response. Moreover, our data suggest that the activating receptor NKG2D, but not NK1. 1 or Ly49 family members, has a role in this NK cell–mediated resistance to mousepox by promoting optimal NK cell–mediated killing. Thus, our data provide substantial insights into the mechanisms of natural resistance to ECTV and possibly other OPV infections. Together, our work furthers our understanding of host-pathogen interactions and the mechanisms whereby NK cells protect from viral disease. YAC-1 and CHO-K1 cell lines were obtained from Dr. Kerry Campbell (Fox Chase Cancer Center, Philadelphia, Pennsylvania), and A9, MC57G, and BSC-1 cells were obtained from the ATCC. MEF cells were made from day 11 to 13 embryos from B6 mice. As standard tissue culture media, we used RPMI-10 that consisted of RPMI-1640 medium (Invitrogen) supplemented with 10% fetal calf serum (Sigma), 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen), 10 mM Hepes buffer (Invitrogen), and 0. 05 mM 2-mercaptoethanol (Sigma). MEF were grown in DMEM medium containing 15% fetal calf serum. When indicated, RPMI 2. 5 (as above but with 2. 5% FCS) was used. When required, 10 U/ml interleukin 2 (IL2) was added to RPMI 10 (RPMI 10-IL2). All cells were grown at 37 °C and 5% CO2. The production of ECTV stocks for infection of mice and the determination of titers in stocks and organs were done as described previously [8]. To generate ECTV 189898-p7. 5-EGFP, we adapted the method described by Johnston and McFadden [60]. Briefly, a construct containing the ECTV Moscow fragment 189543–189897, the VACV early/late promoter p7. 5, the sequence of EGFP, and the ECTV Moscow fragment 189950–190297, in that order, was made by recombinant PCR and cloned into plasmid Bluescript II SK+ to generate the targeting vector pBS-EVM189898-p7. 5-EGFP. This targeting vector was used to transfect mouse A9 cells using Lipofectamine 2000 as per manufacturer' s instructions (Invitrogen). The transfected cells were infected with wild-type ECTV (Moscow strain, 0. 3 pfu/cell) in 6-well plates. 2 d later, transfected/infected A9 cells were harvested using a rubber policeman, frozen and thawed, and different dilutions of cell lysates were used to infect BSC-1 cells in 6-well plates. 2 h after infection, the cells were overlaid with media containing 0. 5% agarose. 4 d later, green-fluorescent plaques were picked with a pipette tip and used to infect a new set of cells. The purification procedure was iterated five times until all plaques were fluorescent. The resulting virus, ECTV 189898-p7. 5-EGFP, carries EGFP in a non-coding region and is as pathogenic as wild-type ECTV Moscow (not shown). For preparation of ECTV stock for infection of different cell lines, A9 cells were infected with 0. 2 pfu ECTV/cell, and incubated at 37 °C, 5% CO2. After 5 d, the cells were collected, frozen and thawed three times, and then sonicated in a water-bath sonicator. The solid material was pelleted by centrifugation, and the supernatant was stored in aliquots at −80 °C. The DAP10-deficient mice [61,62] (generously provided by Dr. Joe Phillips) and DAP12-deficient mice [63] on the C57BL/6 background were bred at UCSF. All the other mice were bred at the Fox Chase Cancer Center Laboratory Animal Facility in specific pathogen-free rooms or were purchased from Jackson Laboratories. IFN-γ-deficient C57BL/6 mice were generously provided by Dr. Glenn Rall. For infections, sex-matched animals 8–12 wk old were transferred to a biosafety level 3 room. For ECTV infection, anesthetized mice were infected in the left footpad with 25 μl PBS containing 3 × 103 pfu ECTV. Following infections, mice were observed daily for signs of disease (lethargy, ruffled hair, weight loss, skin rash, and eye secretions) and imminent death (unresponsiveness to touch and lack of voluntary movements). Moribund mice were euthanized by halothane inhalation. All of the experimental protocols involving animals were approved by the Fox Chase Cancer Center Institutional Animal Care and Use Committee. For ECTV infection of cells, 3–5 × 105 cells were plated in 6-well plates and cultured overnight to allow cells to adhere. The cells were then infected with 0. 5 pfu ECTV/cell for 18 h, collected, washed, stained, and analyzed for surface expression of various markers. For ECTV infection of peritoneal cells, the mice were euthanized by halothane inhalation and injected i. p. with PBS, the abdomen massaged gently, and the peritoneal cells were collected by aspiration and washed. Depletion of NK cells was performed by i. p. inoculation of 200 μg anti-NK1. 1 mAb PK136 or 20 μl anti-asialo GM1 antisera (Wako), as indicated. Antibody treatment was done 1 d before or on the indicated days after virus infection. For depletion of T cells, mice were injected i. p. with 200 μg anti-CD4 mAb GK1. 5 and 200 μg anti-CD8 mAb 2. 43 1 d before infection. For NKG2D blockade mice were inoculated with purified 200 μg CX5 Ab 1 d before and 2 d PI. Mice were exsanguinated from the orbital cavity to decrease the amount of blood in the liver. The liver was removed and passed through a cell strainer (BD Falcon) to obtain a single cell suspension. The cells were resuspended in 35% Percoll solution (in PBS) containing 100 U/ml heparin and centrifuged at 830 × g for 15 min at room temperature. The upper liquid phase was removed from the tube, the lymphocyte pellet resuspended in 0. 84% NH4Cl solution to lyse the red blood cells, and then washed twice with medium. At the indicated time post-infection (PI), mice were injected with 2 mg BrdU i. p. 3 h later, spleens and lymph nodes (LNs) were removed and made into single cell suspensions. The liver lymphocytes were obtained as described above. The cells were then stained for cell surface molecules, fixed, and permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer' s instructions, incubated with DNase at 37 °C for 1 h, and subsequently stained with FITC-conjugated anti-BrdU mAb (eBiosciences). Determination of cytokine production by intracellular staining was done as described previously [8]. To determinate NK cell responses in LNs, intact organs were incubated at 37 °C for 1 h in media containing 10 μg/ml brefeldin A, made into single cell suspensions, stained, and analyzed as described above. To evaluate NK cell responses in the spleen, the organs were made into single-cell suspensions, RBC were lysed with 0. 84% NH4Cl, and the lymphocytes were washed and incubated at 37 °C for 1 h with brefeldin A, followed by staining and analysis as described. To determine expression of NKG2D ligands, MEFs were infected with 0. 5 pfu ECTV 189898-p7. 5-EGFP for 18 h. Infected cells (∼15%) were identified by EGFP expression. Gated infected and uninfected cells were analyzed for expression of NKG2D ligands by staining with: a PE-labeled rat anti-mouse Rae-1 (IgG2a isotype, R&D Systems) or a PE-labeled rat isotype-matched control IgG2a; with rat anti-mouse MULT1 (IgG2a isotype, R&D Systems) and secondary PE-labeled donkey anti-rat IgG2a or secondary Ab alone as a control; or with mouse NKG2D-human Fc fusion protein (R&D Systems) and an APC-labeled anti-human IgG (BD) secondary Ab or secondary Ab alone as control. NK cells were purified from spleens using anti-CD49b-conjugated microbeads and a LS column (Miltenyi Biotec) according to the manufacturer' s instructions and were stained for flow cytometric analysis with PE-conjugated anti-NKG2D mAb CX5. NK cells were resuspended in RPMI 10, and serially diluted in round-bottom 96-well plates in triplicate in 100 μl/well. The indicated target cells were prepared by incubation with 200 μl 51Cr (0. 1 mCi) in 100 μl of FCS for 2 h. Cells were thoroughly washed, resuspended in RPMI 10, and 50 μl (5×103 targets) were added to the wells containing effector cells. The plates were incubated at 37 °C for 4 h. Where indicated, 20 μg/ml of a neutralizing anti-NKG2D mAb (clone 191004, R&D Systems) was added at the initiation of cytotoxicity assays. 50 μl supernatants were transferred to white 96-well plates containing 75-μl Microscint-40 scintillation fluid (PerkinElmer). Controls included wells with target cells alone for spontaneous release and wells with target cells and 1% Triton-X for maximal release. Radioactivity was measured by using a Packard Topcount instrument (PerkinElmer). Specific lysis was determined by using the formula [ (experimental release − spontaneous release) / (full release − spontaneous release) ]×100. Three animals were used per experimental group. Primers and probes for Rae-1 were purchased from Applied Biosystems. The amplicon was 95 bp and the sequence of the probe was GGAAAAGCCAAGATCAACCTCAAGG. The primers and probe for β-actin were synthesized at the DNA Synthesis Facility in the Fox Chase Cancer Center. The primers and probe used for β-actin were: sense, 5′-CACCGAGGCCCCCCT-3′; anti-sense, 5′-CAGCCTGGATGGCTACGTACA-3′, and the probe was 5′-6-FAM-AACCCTAAGGCCAACCGTGAAAAGATGA-BHQ1–3′. Total RNA extracted from infected cell lines and LNs of infected mice was treated with Dnase I, and the first-strand cDNA was synthesized by using random primers. qRT-PCR was carried out by using the ABI 7500 (Applied Biosystems). The cycling conditions for real-time PCR were: 50 °C for 10 min, followed by 45 cycles of 95 °C for 30 s, and 60 °C for 2 min. Data were analyzed by using the Sequence Detection v1. 2 Analysis Software (Applied Biosystems). We used a two-tailed t test for two samples for means with a confidence level (alpha) of 0. 05 using Excel Analysis Tool Pack (Microsoft). Differences in survival and disease were determined at the FCCC Biostatistics and Bioinformatics Facility using Log Rank Test with the STATE SE/9. 2 software. In all cases, differences were considered significant when p-values were ≤ 0. 05.

Title: A Role for NKG2D in NK Cell-Mediated Resistance to Poxvirus Disease Summary: Ectromelia virus (ECTV) causes mousepox, a murine disease that is the equivalent of human smallpox. ECTV normally penetrates through the periphery but rapidly spreads through the lymphatic system to vital organs. In mousepox-sensitive strains of mice, ECTV infection culminates with either rapid death or overt symptoms of mousepox due to very high loads that the virus reaches in vital organs, particularly the liver. However, some strains of mice such as C57BL/6 (B6) and 129 also become infected with ECTV but naturally resist mousepox by controlling the virus loads in vital organs and clearing the virus without clinical symptoms of disease. Natural killer (NK) cells are cells of the innate immune system previously shown to play an important role in natural resistance to mousepox. However, how NK cells protect from this disease is still unknown. In this paper we show that NK cells directly contribute to antiviral defenses by curbing virus dissemination to vital organs and also indirectly by augmenting the antiviral T cell response. We also demonstrate that optimal protection requires the activating NK cell receptor NKG2D which facilitates killing of ECTV-infected cells. Our work has important implications for the understanding of natural resistance to viral disease.

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Write a title and summarize: The cancer stem cell hypothesis, that a small population of tumour cells are responsible for tumorigenesis and cancer progression, is becoming widely accepted and recent evidence has suggested a prognostic and predictive role for such cells. Intra-tumour heterogeneity, the diversity of the cancer cell population within the tumour of an individual patient, is related to cancer stem cells and is also considered a potential prognostic indicator in oncology. The measurement of cancer stem cell abundance and intra-tumour heterogeneity in a clinically relevant manner however, currently presents a challenge. Here we propose signalling entropy, a measure of signalling pathway promiscuity derived from a sample’s genome-wide gene expression profile, as an estimate of the stemness of a tumour sample. By considering over 500 mixtures of diverse cellular expression profiles, we reveal that signalling entropy also associates with intra-tumour heterogeneity. By analysing 3668 breast cancer and 1692 lung adenocarcinoma samples, we further demonstrate that signalling entropy correlates negatively with survival, outperforming leading clinical gene expression based prognostic tools. Signalling entropy is found to be a general prognostic measure, valid in different breast cancer clinical subgroups, as well as within stage I lung adenocarcinoma. We find that its prognostic power is driven by genes involved in cancer stem cells and treatment resistance. In summary, by approximating both stemness and intra-tumour heterogeneity, signalling entropy provides a powerful prognostic measure across different epithelial cancers. Over recent years considerable evidence has arisen supporting the hypothesis that some cancers are hierarchically organised, akin to the organisation of healthy cells, with a small population of Cancer Stem Cells (CSCs) driving a heterogeneous, hierarchical structure [1,2]. The abundance of CSCs is considered likely to be of prognostic value as well as a source of intra-tumour heterogeneity, a feature that has long been considered of possible prognostic value in oncology [3–6]. Although putative CSCs have been identified by surface marker expression for several malignancies, isolated, and demonstrated to be chemotherapeutic resistant [7–11], it remains a significant challenge to obtain a prognostic measure of their abundance from tumour bulk gene expression profiles across multiple malignancies. Embryonic Stem (ES) cell gene expression signatures are clear candidates for such a measure and indeed have been demonstrated to be prognostic in breast and lung cancer [12–15]. Their overall prognostic significance seems limited, however, and they are unable to discriminate CSCs from the tumour bulk [12,16]. The clinical assessment of intra-tumour heterogeneity also poses a significant challenge, with current experimental approaches requiring multiple biopsies per tumour leaving them severely limited in sample size [17–19]. We posited that an expression based measure of signalling promiscuity may quantify the stemness of a tumour in a manner which is related to intra-tumour heterogeneity, and thus provide us with an improved prognostic model. Here we explore this hypothesis, using an in-silico approach. Specifically, we consider signalling entropy which is computed from the integration of a sample’s genome-wide gene expression profile with an interactome, and provides an overall measure of the signalling promiscuity in the sample [16]. We note that the term signalling entropy was chosen, as opposed to alternatives such as interactome/network entropy, to emphasise the fact that our measure quantifies network traffic (signalling) as opposed to network topology. Importantly, as shown by us previously, signalling entropy correlates with stemness and differentiation potential within distinct cellular developmental lineages [16]. Indeed, we showed that human embryonic stem cells and induced pluripotent stem cells exhibited the highest levels of signalling entropy, with adult stem cells (e. g. hematopoietic stem cells) showing significantly lower values, and terminally differentiated cells exhibiting the lowest entropy values within a lineage [16]. These results were derived mostly from cell-lines, which are characterised by relatively homogeneous cell populations, and were further validated in time-course differentiation experiments [16]. Importantly, we also demonstrated that cancerous tissue displays a higher signalling entropy than its healthy counterpart [16,20], with CSCs showing higher values than the tumour bulk [16]. Thus, signalling entropy provides an approximation of the stemness of a cellular sample. In addition to quantifying stemness of the signalling regime of a homogeneous cell population, signalling entropy, if computed over a heterogeneous cell population, should also quantify the inter-cellular diversity in pathway activation. To investigate this we performed an analytical investigation of signalling entropy, coupled with empirical validation. We derived a sufficient condition on the expression profiles of homogeneous cell populations for signalling entropy to be a measure of intra-sample heterogeneity on average. We subsequently verified that this condition is satisfied by considering 33 distinct adult tissue expression profiles corresponding to 528 pairwise mixtures. Thus, we show that signalling entropy is a good candidate for a correlate of intra-sample heterogeneity. Importantly, because signalling entropy can be computed from a bulk tumour gene expression profile, it allows us to assess the prognostic significance of our measure in large numbers of clinical specimens. We here compute signalling entropy for a total of 5360 tumour samples, focusing on two highly heterogeneous cancers, non-small cell lung cancer (NSCLC) and breast cancer, which constitute the two leading causes of cancer death world-wide [21]. Survival rates for early stage NSCLC are particularly poor [21,22], and identification of prognostic and predictive biomarkers within the stage I stratum is considered a high priority [23]. In breast cancer, the power of gene expression based prognostic indicators, such as OncotypeDX and MammaPrint [24,25], is highly subtype dependent [26,27] and a clinical breast cancer prognostic signature, which is independent of estrogen receptor (ER) status is lacking. Most importantly, current gene expression based prognostic indicators ignore CSC contributions and intra-tumour heterogeneity [17]. Thus, signalling entropy, a measure of both cell anaplasia and intra-tumour heterogeneity, may form the basis of a general and more robust prognostic indicator. By examining gene expression profiles of over 3500 primary breast cancers and 1300 lung adenocarcinomas, we here demonstrate that signalling entropy is prognostic in breast cancer, regardless of ER status, and in lung adenocarcinomas, within the stage I stratum. Signalling entropy is derived from the integration of a sample’s gene expression profile with a human protein interactome, and provides a rough proxy for the overall level of signalling promiscuity in the sample. Briefly, we employ the mass-action principle to derive, for each sample, a stochastic matrix pij, describing the interaction probability of the proteins encoded by genes i and j in the given sample. The signalling entropy is then computed as the normalised entropy rate of the Markov chain described by pij. This entropy rate gives a steady state measure of the disorder (or promiscuity) in signalling information flow over the network in the given sample (Materials and Methods). As shown by us previously, stem cells have a high signalling entropy which decreases during differentiation, a result not forthcoming using other molecular entropy measures [16,28]. Importantly, we also demonstrated that signalling entropy is elevated in CSCs as compared to the tumour bulk [16]. Thus, given a homogeneous cell population, a high signalling entropy suggests that signalling within each cell is very promiscuous and that the cells may therefore have a plastic stem cell like phenotype. However, a heterogeneous sample, consisting of cells with distinct, though not necessarily promiscuous signalling regimes, should also on average display a high signalling entropy, suggesting that signalling entropy may associate with intra-tumour heterogeneity (Fig. 1A). To investigate whether signalling entropy associates with intra-sample heterogeneity, we considered our measure evaluated for three theoretical samples: namely two homogeneous samples consisting only of cell type x or y respectively, and a third heterogeneous sample consisting of a 50: 50 mixture of cell types x and y. It is clear that if cell type x has an expression profile that maximises signalling entropy and cell type y does not, then the signalling entropy of the mixture will be lower than the signalling entropy of x, thus signalling entropy is not a point-wise measure of heterogeneity. However, as most biologically realistic cell types have distinct expression profiles, corresponding to the existence of non-overlapping active pathways between cell type pairs [29], we posited that the signalling entropy of a mixed sample may be higher than that of a homogeneous sample on average. By appealing to detailed balance we examined a closed form expression for signalling entropy. It is a consequence of simple algebra that if signalling entropy is super-additive over the set of biologically admissible expression profiles (i. e., Signalling Entropy (x + y 2) > 1 2 Signalling Entropy (x) + 1 2 Signalling Entropy (y) ) then signalling entropy will on average be elevated in mixed samples as opposed to homogeneous samples (Materials and Methods, S1 Text, S8 Fig, S9 Fig, S10 Fig and S11 Fig). We thus derived a condition for point-wise super-additivity of our measure and then considered a data set of gene expression profiles for 33 distinct adult tissues, representing 528 possible pairwise mixtures [29]. For every possible mixture the derived condition for super-additivity was satisfied (Fig. 1B). Whence the signalling entropies of the mixed samples was significantly higher than that of homogeneous samples on average (Fig. 1C). This provides strong evidence that signalling entropy is a correlate of intra-sample heterogeneity. Thus, signalling entropy associates with tumour stemness in a manner associated with CSC abundance and intra-tumour heterogeneity, making our measure a good candidate for an improved prognostic indicator. In order to assess the prognostic significance of signalling entropy in breast cancer, we first computed its value for each microarray sample of the Molecular Taxonomy of Breast Cancer International Research Consortium dataset (METABRIC) [30], a total of 1980 samples divided into a discovery and validation sets of equal proportion. This data set profiles a large number of clinical variables and thus is a suitable platform to examine the clinical associations of our measure. Using outcome first as a binary phenotype, we observed that patients who died of breast cancer had a higher signalling entropy than patients who were alive at last follow up, a result which was seen in both METABRIC subsets (p < 1e − 7). Using a Cox proportional hazards model, on 5 year censored survival data, we ascertained that high signalling entropy is associated with increased risk of death in breast cancer (c-index = 0. 6, p < 1. 1e − 6). Stratifying patients into 3 groups, representing the 3 tertiles of the signalling entropy distribution, revealed that tumours with a high entropy exhibited a doubling of the hazard rate compared to low entropy tumours. Signalling entropy was found to be associated with tumour grade and ER status, however, its prognostic power was independent of these variables, as well as of stage, p53 status, tumour size and lymph node status (S1 Text, S1 Fig & S2 Fig). In addition, signalling entropy was also found to be independent of a prognostic ES cell signature described by Ben-Porath el al. [12] and the prognostic grade signature described by Sotiriou et al. [31] (S1 Text, S1 Fig & S2 Fig). Signalling entropy was significantly prognostic within each tumour grade strata; notably it was prognostic within the grade 2 stratum in both METABRIC data sets (p < 0. 036), an important result given the difficulty in deciding treatment courses in this intermediate prognosis group [31]. The fact that signalling entropy is prognostic independently of all other measures of cell anaplasia, suggests that our measure may be capturing more than just the stemness of a tumour sample, and that intra-tumour heterogeneity may be contributing to its prognostic power. A recent study by Venet et al. described prognostic associations for a number of random gene expression signatures in breast cancer [32]. To ascertain whether random effects may be driving our findings, we evaluated the prognostic associations of the three random gene expression signatures described by Venet et al.. We found that only one was prognostic in both discovery and validation METABRIC data sets and that its prognostic power was determined by ER status (S3 Fig). To further assess the impact of random effects and the importance of our network, we randomised the gene expression profiles of the METABRIC data sets over the network. Performing 5 randomisations and recomputing signalling entropy for the 1980 samples in both METABRIC data sets, revealed that randomised signalling entropy did not display robust prognostic associations independently of ER status. We are therefore confident that the prognostic power of signalling entropy is not driven by random effects. To further validate the prognostic impact of signalling entropy we considered eight further independent breast cancer data sets. All these datasets described both ER positive and negative tumours with accompanying clinical outcome, profiled on either Affymetrix or Illummina platforms and totalling 1688 samples [33–40], (S1 Table). Meta-analysis revealed that signalling entropy is prognostic across both ER positive and ER negative samples (ER positive: c-index = 0. 63,95% CI = (0. 604,0. 657), p = 8. 5e − 15, ER negative: c-index = 0. 57,95% CI = (0. 538,0. 602), p = 0. 032, Fig. 2A). Five of the additional eight data sets also described histological tumour grade for each sample, allowing us to further confirm that signalling entropy is prognostic within the grade 2 stratum (c-index = 0. 63,95% CI = (0. 581,0. 675), p = 1. 05e − 6, Fig. 2A). These results are in contrast to the performance of MammaPrint, a microarray based breast cancer prognostic signature currently being assessed in the MINDACT trial [41]. In a meta-analysis over the 10 breast cancer validation sets we found that unlike signalling entropy MammaPrint was not significantly prognostic over ER negative samples (Fig. 2B). Another popular breast cancer prognostic assay in clinical trials is OncotypeDX, which uses RT-PCR to quantify the expression of genes associated with survival [25]. Due to differences in the normalisation between RT-PCR and microarrays, a direct comparison between our measure and OncotypeDX is difficult to perform. Moreover, not all the genes required for computing the OncotypeDX recurrence score were present in all the array platforms considered. However, using a microarray version of OncotypeDX, we found that it performed comparably to signalling entropy across both ER positive (signalling entropy vs. OncotypeDX: p = 0. 13) and ER negative samples (signalling entropy vs. OncotypeDX: p = 0. 7, S4 Fig). Thus signalling entropy is prognostic in the two major clinical subtypes of breast cancer and hence is a more robust prognostic indicator than MammaPrint. We next investigated the prognostic power of our measure in lung adenocarcinoma. To evaluate the clinical associations of our measure we first computed signalling entropy for each microarray sample in The Director’s Challenge dataset profiling 398 tumours [42], and for the 455 lung adenocarcinoma RNA-seq tumour samples downloaded from The Cancer Genome Atlas (TCGA) database (http: //cancergenome. nih. gov/). We found that signalling entropy was significantly lower in lung adenocarcinoma patients who were alive at last follow up as opposed to those who had died (p < 0. 03). Fitting Cox proportional hazard models to 3 year censored data revealed that an increased signalling entropy implied a worse prognosis in lung adenocarcinoma (c-index = 0. 6, p < 0. 007). We again separated patients into tertiles of the signalling entropy distribution and found that high signalling entropy conferred almost a doubling of the hazard rate, as assessed over the first 3 years following diagnosis (HR = 1. 9, p < 0. 02). Signalling entropy was found to be associated with tumour stage, grade and smoking status, in both TCGA and Director’s Challenge data sets, yet importantly the prognostic power of signalling entropy was independent of these clinical variables (S1 Text, S5 Fig & S6 Fig). It is of particular note that signalling entropy is significantly prognostic if computed from either microarray or RNA-seq data sets, this result attests to the biological relevance of our measure which is not masked by experimental technique. To validate the prognostic power of signalling entropy in lung adenocarcinoma, we performed a meta-analysis across 4 further independent data sets consisting of a total of 522 lung adenocarcinomas (S2 Table) [43–46]. This revealed that signalling entropy is prognostic across all samples and across stage I samples (all samples: c-index = 0. 58,95% CI = (0. 55,0. 60), p = 1. 9e − 6, stage I: c-index = 0. 56,95% CI = (0. 52,0. 60), p = 0. 037, Fig. 3A). Early stage lung adenocarcinoma suffers from a high relapse rate and it is important to establish more robust prognostic assessments in the stage I subgroup for chemotherapeutic treatment stratification [22]. Sub-staging by size is currently the standard clinical approach to stratify stage I tumours, however, on meta-analysis we found that this stratification, unlike signalling entropy was not significantly prognostic over the stage I stratum (Fig. 3B). Signalling entropy is a clear prognostic indicator in breast cancer, yet its computation requires the expression of many thousands of genes, something which is currently cumbersome and expensive for clinical application. Moreover, our measure associates with tumour grade and ER status in breast cancer and thus the factors driving its prognostic power independently of these variables is unclear. We posited that the prognostic power of our measure, independent of ER status and grade may be captured by the expression of a small number of genes, analogously to the way the prognostic power of tumour grade was captured by the expression of the 97 gene Sotiriou et al. signature [31]. To identify suitable genes representative of signalling entropy’s prognostic power, we first investigated prognostic genes, which were correlated or anti-correlated with signalling entropy independently of grade and ER status, and whose prognostic power was also independent of grade and ER status. We then refined this gene set by fitting a Cox proportional hazards model on 5 year censored data using all the identified genes as covariates and deleting genes which were not significantly prognostic independently of others in the gene set. This resulted in a small set of 81 genes, 10 of which were negatively correlated with signalling entropy and 71 of which were positively correlated S2 Table. A Signalling Entropy prognostic score (SE score) was then defined as the t-statistic evaluating the hypothesis that the 71 positively correlated genes are expressed more highly than the 10 negatively correlated genes (after z-score normalising the data). By using signalling entropy to refine a set of prognostic genes identified by Cox regression, our approach refines the feature selection approach based on correlation with outcome [24]. Consequently, the genes utilised to construct our SE score are both correlated with outcome and with signalling entropy and thus should provide a prognostic indicator representative of signalling promiscuity. Criticism of feature selection for prognostic classifiers based on gene sets ranked by correlation with outcome has stemmed from the considerable discordance of such features between data sets [47,48]. By using signalling entropy to refine the prognostic gene set we found that this gene set instability was reduced. The genes which were both prognostic and correlated with signalling entropy showed more concordance between discovery and validation sets of METABRIC as compared to the genes which were only prognostic. Moreover, this increase in overlap was significantly higher than would be expected by chance (p < 10e − 5, based on re-sampling size matched sets of prognostic genes and assessing overlap). To further confirm this increased rodustness, we derived a set of genes for constructing an SE score from the METABRIC validation set, using an identical procedure to that performed on the discovery set. This gene list was slightly shorter than for the discovery set (55 genes, 34 positively correlated and 13 negatively correlated with signalling entropy) but had an overlap of 4 genes, significantly more than would be expected by chance (p = 0. 012, based on re-sampling size matched sets of prognostic genes and assessing overlap). We provide the lists of prognostic genes both correlated and uncorrelated with signalling entropy as well as the validation set derived SE score genes in S3 Table. Meta-analysis across 9 independent breast cancer data sets revealed that like signalling entropy, the SE score is prognostic across both ER positive and ER negative samples (ER positive: c-index = 0. 63,95% CI = (0. 59,0. 67), p = 4. 6e − 15, ER negative: c-index = 0. 62,95% CI = (0. 58,0. 66), p = 8. 1e − 8, Fig. 4A). Moreover, meta-analysis further demonstrated that the SE score performed comparably to MammaPrint over ER positive samples (SE score vs MammaPrint: p = 0. 18, Fig. 4B), and out-performed MammaPrint over ER negative samples (SE score vs MammaPrint: p = 0. 04, Fig. 4C). Whence the prognostic power of our measure is well captured by the expression of this small set of genes. We next investigated whether a similar SE score could be computed for lung adenocarcinoma. Signalling entropy is correlated with, yet prognostically independent of tumour stage in lung adenocarcinoma, we therefore aimed to derive a score that represented the prognostic power of our measure independently of tumour stage. To achieve this we considered the Director’s Challenge data set of 398 lung adenocarcinomas as a discovery set [42]. We performed an analogous procedure as described above for breast cancer to identify genes associated with signalling entropy’s prognostic power independently of tumour stage in lung cancer, with the only differences being that we adjusted for tumour stage, rather than ER status and grade, and used 3 year censored data rather than 5 year. This resulted in a small set of 29 genes, 8 of which were negatively correlated with signalling entropy and 21 of which were positively correlated (S4 Table). An SE score was then defined again as the t-statistic evaluating the hypothesis that the positively correlated genes are expressed more highly than the negative. Meta-analysis across 5 independent validation data sets revealed that the SE score is prognostic across all samples and across stage I samples (all samples: c-index = 0. 62,95% CI = (0. 59,0. 66), p = 1. 9e − 11, stage I: c-index = 0. 66,95% CI = (0. 60,0. 71), p = 3. 35e − 5, Fig. 5A). We next compared our SE score to a leading gene expression based prognostic indicator for lung adenocarcinoma, the expression of the gene CADM1, which was recently found to be a superior prognostic indicator to many others in the literature [44]. CADM1 expression performed comparably to the SE score in a meta-analysis, however, it was outperformed by pathological tumour stage (CADM1 expression vs stage: p = 0. 03). In contrast the SE score performed comparably to tumour stage (SE score vs stage: p = 0. 13, Fig. 5B). Conventional tumour sub staging by size within the stage I stratum, is established clinical practice, it has thus been suggested that prognostic scores should aim to provide information which complements this staging, rather than seeks to replace it [49]. We therefore evaluated whether prognostic models which combined either the SE score or CADM1 expression with stage Ia/b status within the stage I sub group, outperformed stage Ia/b status alone. We found that the SE score improved over stage Ia/b alone in a meta-analysis across 765 stage I lung adenocarcinomas (SE score+stage vs stage: p = 0. 025), whereas CADM1 expression made no improvement over stage Ia/b (CADM1 expression+stage vs stage: p = 0. 13, Fig. 5C). Whence it may be argued that the SE score provides a stronger candidate prognostic tool than CADM1 expression for clinical application. Another popular prognostic score for lung adenocarcinoma was derived recently by Kratz et al. [22], similarly to OncotypeDX however, this score is based on RT-PCR and thus a direct comparison is difficult. However, a microarray based approximation of the Kratz et al. score was found to perform comparably to signalling entropy both across all samples (SE score vs Kratz et al. score: p = 0. 21) and across stage I samples (SE score vs Kratz et al. score: p = 0. 37, S7 Fig). Given the power of signalling entropy as a prognostic factor in both breast and lung cancer we next investigated which genes and pathways were associated with signalling entropy’s prognostic impact, independently of other clinical variables. To determine which gene sets were enriched among the genes prognostically related to signalling entropy independently of other variables, we considered for breast cancer a list of 320 genes which were prognostic, independent of ER status and grade, and correlated with signalling entropy, again independently of ER status and grade, in both MEATBRIC datasets. For lung adenocarcinoma we considered a list of 158 genes identified as prognostic independently of stage, and correlated with signalling entropy, again independently of stage, in both the Director’s Challenge and TCGA data sets. The two gene lists displayed an overlap of 47 genes (S5 Table displays both gene lists). We performed a gene set enrichment analysis, using a Fisher’s Exact test, comparing each of these gene lists separately against the Molecular Signatures Database [50] (S6 Table shows the top 10 enriched gene sets for both gene lists). The decision to use these gene sets for the enrichment screens, rather than the genes utilised to derive the SE scores was due to them being derived from multiple data sets and thus more robustly representative of signalling entropy’s prognostic associations. We note that gene set enrichment analysis performed on the genes comprising the SE scores gave broadly similar results (S7 Table). The genes found to prognostically associate with signalling entropy in both lung and breast cancer showed considerable concordance in enrichment profiles (even after removal of the 47 genes in the overlap S6 Table). The strongest enrichment was for genes associated with poor survival in lung cancer, histological grade in breast cancer and cell proliferation, supporting the notion that signalling entropy is a prognostic measure of cell anaplasia. In addition, considerable enrichment was found for genes down regulated by the therapeutic agent salirasib and by EGFR inhibitors, as well as for genes as up regulated in cell lines resistant to the chemotherapeutic doxorubicin, supporting the hypothesis that signalling entropy associates with therapeutic resistance. Enrichment was also found for gene sets associated with stem cells and certain CSC pathways. Examples include, genes down-regulated by EZH2, a well known stem cell gene involved in the pathogenesis of several cancers and which plays a documented role in both breast and lung CSCs [51–54]. The set of genes down regulated by CTNNB1 knock-out, a critical component of the Wnt signalling pathway, posited to be important in CSCs and their therapeutic resistance [55] were also enriched. Targets of BMP2 were among the most enriched gene sets in breast but not lung cancer, which is intriguing given the role of this gene specifically in breast CSCs [56]. Enrichment was also found for many gene sets associated with immune system processes. Thus signalling entropy is prognostically related to genes associated with both CSCs and treatment resistance, across multiple malignancies and independently of clinical variables. This result confirms our initial postulate that signalling entropy is a powerful prognostic measure, related both to cell anaplasia and CSCs as well as treatment resistance. The discovery that CSCs show resistance to conventional therapy necessitates an evaluation of their prognostic and predictive value, as well as the development of targeted therapies [8,9]. The notion of tumour cell plasticity raises further challenges [57] with recent discoveries suggesting that CSCs may arise from the tumour bulk by simple changes [58]. This calls into question the notion that CSCs only ever occupy a small proportion of the tumour, and paint a picture of cancer cells as malleable entities capable of generating considerable heterogeneity. Recent observations have also demonstrated the importance of characterising such intra-tumour heterogeneity in the prognostic assessment of epithelial cancers [17]. The measurement of both CSC abundance and intra-tumour heterogeneity in a clinically relevant manner, however, presents a challenge [59]. The majority of currently suggested approaches are limited in sample size, and require the time consuming collection of large new data sets (such as multiple biopsies from single tumours) for validation and proof of concept. Here we have shown that signalling entropy, a measure of pathway promiscuity, which is elevated in CSCs as compared to the tumour bulk is also a potential correlate of intra-tumour heterogeneity. Importantly, our measure is applicable to the plethora of publicly available bulk tumour, genome wide expression data, facilitating swift validation of its prognostic impact on large data sets. By considering 5360 primary tumour samples, we have demonstrated that our measure is a powerful prognostic indicator in both breast and lung cancer. In breast cancer our measure is prognostic within the grade 2 stratum and both ER positive and negative subtypes. In lung adenocarcinoma, our measure is prognostic within the stage I stratum, out-performing tumour size. Signalling entropy is computed from the expression of many thousands of genes and thus is not swiftly translatable. Moreover, it is associated with yet prognostically independent of a number of clinical variables in both breast and lung cancer. We thus used feature selection to derive a small set of genes which capture the prognostic power of signalling entropy independently of other clinical variables, thus representing a more readily applicable quantifier of stemness and intra-tumour heterogeneity. Expression based prognostic indicators for epithelial cancers have been a topic of considerable interest in recent years [22–25,44,60]. Arguably the most successful application has been to breast cancer, where OncotypeDX and MammaPrint are currently in clinical trials for guiding the management of ER positive breast cancer [26,27]. Though powerful, these assays are limited to the ER positive subtype and importantly ignore CSC abundance and intra-tumour heterogeneity. There also exist many more sophisticated prognostic signatures for breast cancer, derived from within the DREAM challenge consortium, and several of which have demonstrated improvement over MammaPrint or OncotypeDX [61–64]. The aim of our work, was first to introduce a prognostic measure of signalling promiscuity, which by approximating CSC abundance and intra-tumour heterogeneity may prove a basis by which to improve the construction of prognostic models for epithelial cancers, and secondly, to compare it to clinically well established or validated signatures such as MammaPrint and OncotypeDX. A direct comparison of signalling entropy to the prognostic indicators from the DREAM challenge, which have not yet entered the clinical setting, is beyond the scope of this work. In comparing signalling entropy to signatures such as MammaPrint it is worth pointing out that a direct comparison is unfair signalling entropy does not involve feature selection. Even so, signalling entropy was found to be more robust than MammaPrint across ER+ and ER- breast cancer. Although signalling entropy was not found to outperform existing prognostic markers in lung adenocarcinoma, by using the SE score, derived by signalling entropy guided feature selection, it was possible to outperform existing state of the art prognostic factors such as CADM1 expression across independent data sets. The nature of signalling entropy as a measure of pathway promiscuity, which correlates with CSCs and associates with intra-tumour heterogeneity [8,9], led us to postulate that it may associate with the phenotypic plasticity of a tumour that enables subversion of therapeutic response. Here we demonstrated that signalling entropy’s prognostic power in epithelial cancers is indeed related to both treatment resistance and CSC pathways. We thus propose signalling entropy as a powerful and readily applicable tool for assessing the prognostic impact of signalling promiscuity across multiple epithelial cancers. In addition to being a strong prognostic factor which outperforms the leading expression based indicators, our measure may also provide insights into intra-tumour heterogeneity, treatment resistance and CSC mechanisms. Signalling entropy was computed in a sample specific manner as described in [16]. Briefly, each sample is first integrated with a Protein Interaction Network (PIN) (see S1 Text) to create a sample specific stochastic matrix, P = (pij). By integrating each sample with the PIN, rather than considering a complete network in which every protein pair can directly interact, we benefit both from a reduction in computational complexity and an improved biological relevance from a focus on direct interactions. Integration with the PIN filters out indirect interactions even if strong correlations are present, making our analysis robust to confounding effects. By using each sample to weigh the PIN we are also reducing the noise present in the network by providing it with a sample-specific biological context. The ith row of P defines a probability distribution describing the rates of reaction of protein i with each of its neighbours in the PIN. These distributions are constructed by appealing to a simplified version of the mass action principle, namely that the rate of a reaction is proportional to the product of the active masses of the reagents involved. We assume that log normalised gene expression is a rough proxy for protein concentration and thus compute P as follows: p i j = E j / ∑ k ∈ N (i) E k, if j ∈ N (i) 0, else (1) where Ej is the log-normalised expression of gene j in the given sample and N (i) denotes the set of direct interaction partners (neighbours) of gene i in the PIN. We note that from this definition ∑j pij = 1 for all j, i. e., P is row stochastic, and the ith row corresponds to the weighted interaction distribution of protein i in the given sample. We note that not all proteins in the PIN have a corresponding probe in the microarray or sequence in the RNA-seq data, consequentially the PIN we consider is the maximally connected component of the original PIN after the removal of missing proteins. For each protein i we then define the local entropy of its interaction distribution, Si, which quantifies the promiscuity of its signalling within the sample: S i = - ∑ j ∈ N (i) p i j log p i j. (2) Signalling entropy is a global measure of signalling promiscuity in a given sample and thus is computed from the entire stochastic matrix pij as the entropy rate, S̃R, of the stochastic process described by pij: S ˜ R = ∑ i π i S i, (3) where πi denotes the stationary distribution of the stochastic matrix, satisfying ∑i πi pij = πj. We note that πi is therefore the non-degenerate eigenvector of P corresponding to the eigenvalue 1 and that by the Perron Frobenius theorem, the existence of πi requires that the matrix P be irreducible; this is guaranteed by the fact that the PIN considered is connected and non-bipartite [65]. The maximum entropy rate of a weighted network, MR, depends solely upon its adjacency matrix, A = (Aij), and can be calculated as the entropy rate of the stochastic matrix pij = Aij νj/λνi, where λ and ν are the dominant eigenvalue and corresponding eigenvector of A, respectively [66]. In order to ensure the results presented in this paper are comparable with those of previous studies on signalling entropy, we will present our findings in terms of normalised signalling entropy: S R = S ˜ R / M R. (4) A closed form expression for signalling entropy is derived and analysed in the S1 Text. R-scripts for the computation of signalling entropy are freely available for download at www. sourceforge. net/projects/signalentropy. We hypothesised that the signalling entropy of a heterogeneous sample generated from a 50: 50 mixture of two homogeneous cell types will be greater, on average, than the signalling entropy of a homogeneous sample. Here we show that if signalling entropy is super-additive then the hypothesis is correct. Let us first define some preliminaries: Let xi ∈ ℝ> 0 be the expression of gene i in cell type X, and denote the vector containing all such variables by x= (xi) i=1n∈Ω⊂ℝ>0, where Ω is some bounded domain. In our analysis x will represent the vector of log normalised gene expression values for a homogeneous sample, we note that as the expression of genes cannot be infinite we bound x within a finite domain Ω, of biologically admissible expression regimes. Our hypothesis on signalling entropy thus amounts to proving the following proposition: Proposition. Let x, y ∈ Ω, then ∫​Ω∫​Ω (SR (x+y2) −SR (x) ) dxdy>0. (5) Let us consider a the following claim: Claim (Super-additivity). Let x, y ∈ Ω then S R x + y 2 > S R (x) 2 + S R (y) 2. (6) It is clear that if the claim is true then the proposition must be true. Notice first that if the claim is true then as it is a strict bound ∃ε > 0 such that S R (x + y 2) > S R (x) 2 + S R (y) 2 + ε. Whence ∫ Ω ∫ Ω S R x + y 2 - S R (x) d x d y > ∫ Ω ∫ Ω S R (y) 2 - S R (x) 2 + ϵ d x d y (7) = | Ω | 2 ϵ (8) > 0, (9) and thus the proposition is true. Thus if signalling entropy is super-additive over homogeneous cell types, this implies that signalling entropy will on average be elevated in heterogeneous mixtures of cell types. These propositions are examined in detail in S1 Text.

Title: Intra-Tumour Signalling Entropy Determines Clinical Outcome in Breast and Lung Cancer Summary: The Cancer Stem Cell (CSC) hypothesis, the idea that a small population of tumour cells have the capacity to seed and grow the tumour, and intra-tumour heterogeneity, the diversity of the cancer cell population within the tumour of an individual patient, have long been considered the basis of potential prognostic indicators in oncology. The identification of CSC based expression signatures and the measurement of intra-tumour heterogeneity, for an assessment of prognostic power in a clinically relevant manner, however, currently presents a challenge. Most proposed methodologies require the collection of new data sets and thus are limited in sample size, making them difficult to validate. Here we consider signalling entropy, a measure of signalling pathway promiscuity, as a means of quantifying the stemness and heterogeneity of any given cancer sample, applicable to publicly available data sets. By considering over 5300 primary tumour samples from both breast and lung cancer patients, we here demonstrate that signalling entropy provides a more robust and general prognostic measure than other leading clinical prognostic indicators.

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Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Library of Congress Administrative Reform Act of 2015''. SEC. 2. AUTHORIZING NATIONAL LIBRARY SERVICE FOR THE BLIND AND PHYSICALLY HANDICAPPED TO PROVIDE PLAYBACK EQUIPMENT IN ALL FORMATS. The first sentence of the Act entitled ``An Act to provide books for the adult blind'', approved March 3, 1931 (2 U.S.C. 135a), is amended by striking ``and for purchase, maintenance, and replacement of reproducers for such sound-reproduction recordings'' and inserting ``and for purchase, maintenance, and replacement of reproducers for any such forms''. SEC. 3. LIBRARY OF CONGRESS NATIONAL COLLECTION STEWARDSHIP FUND. (a) Establishment.--There is hereby established in the Treasury of the United States, as an account for the Librarian of Congress, the ``Library of Congress National Collection Stewardship Fund'' (hereafter in this section referred to as the ``Fund''). (b) Contents of Fund.--The Fund shall consist of the following amounts: (1) Such amounts as may be transferred by the Librarian from amounts appropriated for any fiscal year for the Library of Congress under the heading ``Salaries and Expenses''. (2) Such amounts as may be transferred by the Architect of the Capitol from amounts appropriated for any fiscal year for the Architect of the Capitol under the heading ``Library of Congress Buildings and Grounds''. (3) Such amounts as may be appropriated to the Fund under law. (c) Use of Amounts.--Amounts in the Fund may be used by the Librarian as follows: (1) The Librarian may use amounts directly for the purpose of preparing collection materials of the Library of Congress for long-term storage. (2) The Librarian may transfer amounts to the Architect of the Capitol for the purpose of designing, constructing, altering, upgrading, and equipping collections preservation and storage facilities for the Library of Congress, or for the purpose of acquiring real property by lease for the preservation and storage of Library of Congress collections in accordance with section 1102 of the Legislative Branch Appropriations Act, 2009 (2 U.S.C. 1823a). (d) Continuing Availability of Funds.--Any amounts in the Fund shall remain available until expended. (e) Annual Report.--Not later than 180 days after the end of each fiscal year, the Librarian and the Architect of the Capitol shall submit a joint report on the Fund to the Joint Committee on the Library and the Committees on Appropriations of the House of Representatives and the Senate. (f) Initial 5-Year Plan.--Not later than 6 months after the date of the enactment of this Act, the Librarian shall submit to the Joint Committee on the Library and the Committees on Appropriations of the House of Representatives and the Senate a report providing a plan for expenditures from the Fund for the first 5 fiscal years of the Fund's operation. SEC. 4. EXPANDING USES OF CERTAIN REVOLVING FUNDS. (a) National Audiovisual Conservation Center Fund.--Section 101 of the Library of Congress Fiscal Operations Improvement Act of 2000 (2 U.S.C. 182a) is amended-- (1) in the heading, by striking ``duplication''; and (2) in subsection (a)-- (A) by striking ``duplication and delivery services provided by the Librarian'' and inserting ``the following programs and activities of the Librarian''; (B) by striking the period at the end and inserting a colon; and (C) by adding at the end the following new paragraphs: ``(1) Services related to the duplication and preservation of audiovisual materials and associated collections. ``(2) Storage, inspection, and delivery of audiovisual materials and associated collections.''. (b) Revolving Fund for Gift Shop and Related Services.--Section 102 of such Act (2 U.S.C. 182b) is amended-- (1) in the heading, by striking ``gift shop'' and all that follows and inserting ``sales and other services''; and (2) in subsection (a), by adding at the end the following new paragraphs: ``(5) Traveling exhibitions and exhibition materials. ``(6) Training.''. (c) Inclusion of Tribal Governments in FEDLINK Program.--Section 103(f)(1) of such Act (2 U.S.C. 182c(f)(1)) is amended by inserting after ``Federal Government,'' the following: ``tribal governments (as defined in section 502(c)(3)(B) of title 40, United States Code),''. SEC. 5. AUTHORITY TO ACCEPT GIFTS AND BEQUESTS. (a) Expanding Types of Gifts That May Be Accepted.--The first undesignated paragraph of section 4 of the Act entitled ``An Act to create a Library of Congress Trust Fund Board, and for other purposes'', approved March 3, 1925 (2 U.S.C. 160), is amended-- (1) in the first sentence, by striking ``in the name of the United States'' and all that follows and inserting the following: ``in the name of the United States and in the interest of the Library, its collections, or its service gifts or bequests of personal property, nonpersonal services, voluntary and uncompensated personal services, or money for immediate disbursement.''; (2) in the second sentence, by inserting ``of money'' after ``bequests''; and (3) in the third sentence, by striking ``enter them'' and inserting ``enter the gift, bequest, or proceeds''. (b) Treatment of Gifts of Securities.--The first undesignated paragraph of section 4 of such Act (2 U.S.C. 160) is amended by inserting after the first sentence the following new sentence: ``In the case of a gift of securities, the Librarian shall sell the gift and provide the donor with a receipt from the proceeds of the sale.''. SEC. 6. CONTINUATION OF SERVICE OF RETURNING MEMBERS OF JOINT COMMITTEE ON THE LIBRARY AT BEGINNING OF CONGRESS. (a) Continuation of Service.-- (1) In general.--During the period beginning on the first day of a Congress and ending on the date described in paragraph (2), any Member of Congress who served as a member of the Joint Committee on the Library during the previous Congress shall continue to serve as a member of the Joint Committee. (2) Date described.--The date described in this paragraph is, with respect to a Congress-- (A) in the case of a Member of Congress who is a Member of the House of Representatives, the date on which Members of the House are appointed to serve on the Joint Committee for the Congress; and (B) in the case of a Member of Congress who is a Senator, the date on which Senators are appointed to serve on the Joint Committee for the Congress. (b) Conforming Amendment.--The final undesignated paragraph under the heading ``Senate.'' in section 2 of the Act of March 3, 1883 (chapter 141; 22 Stat. 592) (2 U.S.C. 133), is hereby repealed. (c) Effective Date.--This section and the amendment made by this section shall apply with respect to the One Hundred Fifteenth Congress and each succeeding Congress. SEC. 7. EFFECTIVE DATE. This Act and the amendments made by this Act shall apply with respect to fiscal year 2016 and each succeeding fiscal year.

Title: Library of Congress Administrative Reform Act of 2015 Summary: Library of Congress Administrative Reform Act of 2015 This bill authorizes the Library of Congress (LOC) to purchase, maintain, or replace reproducers for books published either in raised characters, on sound-reproduction recordings, or in any other form (currently limited to reproducers of sound-reproduction recordings) for the use of the blind and for other physically disabled U.S. residents. The bill establishes the Library of Congress National Collection Stewardship Fund, whose amounts may be used directly for preparing collection materials for long-term storage. The use of the LOC revolving fund associated with the national audiovisual conservation center shall include preservation and storage of audiovisual materials and associated collections. Use of the LOC revolving fund currently devoted to the gift shop, decimal classification, photo duplication, and related services shall extend to traveling exhibitions and exhibition materials as well as to training services. The Federal Library and Information Network (FEDLINK) program shall provide specified services on behalf of tribal governments. The types of gifts the LOC may accept shall include bequests of personal property, nonpersonal services, voluntary and uncompensated personal services, or securities. The bill also provides for the continued service on the Joint Committee on the Library in a new Congress of Members of Congress who served on such Committee in a previous Congress.

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Summarize: Lauren Evenden left her baby near empty alcohol bottles and a tattoo kit. She has lost custody of the child after a court heard she was more interested in playing video games than looking after him. A pregnant mother who left her baby near empty alcohol bottles and a tattoo kit has lost custody of the child after a court heard she was more interested in playing video games than looking after him. Lauren Evenden's ground-floor flat in Southampton was filthy and unhygienic, a court heard, and when social workers visited the one-year-old baby, he was wearing a sodden nappy and his cot was full of milk bottles. The 21-year-old, who is pregnant with her second child, failed to take care of her baby, Southampton Crown Court was told. Social services took action after several visits to Evenden's ground-floor flat. On one occasion they found the little boy in a soiled nappy, while on another he was found lying on a urine-stained mattress. Further investigations also revealed the mother, who appeared in court wearing a baseball jacket and floral leggings, had failed to take him for his six-week-old check-up and his inoculations had either been late or missed altogether. Rob Welling, prosecuting, referred to a statement from her previous boyfriend who claimed she was more interested in playing electronic games and watching television than looking after her son. He said feeding was 'chaotic and disorganised' and described how he would have to force her to get up when the baby started to cry at night. And when prosecutors discovered the state of the flat, he added: 'It was apparent the baby had been left in his room longer than he should have been. 'He had been neglected and not looked after.' Social services carried out a further three visits. They found the baby was suffering from a sore because he had been left in a soiled nappy for too long. They arranged for Evenden to have an appointment with the doctor where she was prescribed a cream to treat the sore but she failed to pick it up, the court heard. Mr Welling said 'the final straw' came five days later when social services returned to the premises and got no response. Evenden's ground-floor flat in Southampton was filthy and unhygienic, a court heard, and when social workers visited the one-year-old baby, he was wearing a sodden nappy and his cot was full of milk bottles. It was later discovered that Evenden had left her child while she went out shopping. When she returned social workers found the baby in his room. She claimed that she had asked another resident to look after her baby but Mr Welling told the court this person had 'issues of her own' and the child should not have been left with her. As a result the child has been taken into care and the court was told it was 'unlikely' Evenden would ever get back custody. It was later discovered that Evenden had left her child while she went out shopping. When she returned social workers found the baby in his room. She admitted neglect and received a nine-month suspended prison sentence coupled with 12 months' supervision. She admitted neglect and received a nine-month suspended prison sentence coupled with 12 months' supervision. In mitigation Jamie Gammon, defending, said Evenden had struggled with child issues and had suffered depression. However, the court heard she is now living with her mother, things had improved and her situation, with regard to the unborn child, was being carefully monitored by social services. Recorder Marcus Tregilgas-Davey told Evenden she had failed in her obligations towards his care and welfare over a number of months and that it had not been a single neglect. He said: 'He didn't receive the care he should have done from his mother.'

Summary: Lauren Evenden's Southampton flat was filthy and unhygienic, court heard. When social workers visited the baby, he was wearing a sodden nappy. His cot was full of milk bottles and he had a urine-soaked mattress. It was later discovered that Evenden had left her child while she went out. She claimed to have asked a neighbour to look after the baby boy. She admitted neglect and received nine-month suspended prison sentence. Child has been taken into care. It is unlikely Evenden will get back custody.

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Summarize: SUMMARY OF THE INVENTION The Brush Gopher is a tractor attachment adaptable to most standard farm tractors which have a 3-point hook-up and an engine power of 30 to 150 horse power. The Brush Gopher consists of a steel fabricated unit with fixed opposing curved steel blades applied to trees, brush or roots by vehicular motion working in unison with the tractor&#39;s hydraulic lift. The Brush Gopher also has a lower three sided blade for root digging and grubbing of trees (six inches or less) stumps, brush and bushes. The main elements comprising the unit are the steel main frame; brush shield; tree guide; angle braces supporting gripper and cutting blades which clutch and grip the targeted material; and the grubber blades. The major improvements over the prior art are the abilities of the Brush Gopher to push over; hook and clutch the targeted material; dig and root out the targeted material in continuous operation without operator having to dismount and clean blades of material removed at each cutting. The prior art is structurally weaker and does not have the continuous cleaning capability of this machine. Prior art requires constant up and down dismounting which is a great waste of time and effort. The Brush Gopher is more efficient and safer, due to the Brush Shield component. BRIEF DESCRIPTION OF DRAWINGS The drawing consists of one sheet with three Figures, FIG. 1, FIG. 2 and FIG. 3. FIG. 1 is the Front Elevation of the Brush Gopher. FIG. 2 is the Side Elevation of the Brush Gopher and FIG. 3 is a detail drawing of the Gripper Blades attached to the Main Frame Base. All parts of structural steel. FIG. 1 LIST OF EMBODIMENTS: (FRONT ELEVATION OF BRUSH GOPHER) 1. Structural Steel Main Frame--Top Upright channel section. 2. Structural Steel Main Frame--Bottom Upright angle box section. 3. Structural Steel Main Frame Base angle box section. 4. Brush Shield with equally spaced viewing windows. 5. Steel Rebar Support for the Brush Shield. 6. Viewing window (three equally spaced slots.) 7. Steel Rebar Support for Brush Shield welded to Main Frame Base. 8. Steel Plate welded to Steel Main Frame bottom section with hole drilled. Plate reinforces tractor connection. 9. The 11/8 dia. hole for receipt of bolt to connect tractor arm connection. 10. High strength 11/8&#34; -bolt for connecting tractor arm. 11. One inch plate to form lower each of two lower tractor connections. 12. Half-inch side plate attached to Ref. 11 one inch plate. 13. Steel Gusset plate for support of tractor connection housing. 14-A. Tree Guide Rebar. 14-B. Channel Support for connecting Tree Guide to Main Frame Upright. 15. A 3/4&#34; Rebar Support connecting ends of Tree Guide to Main Frame. 16. Angle Brace for support of Gripper Blade Mount and connected to forward end of gripper mount and also to Main Frame Upright. 17. Gripper Blade Mount for support of Gripper Cutting Blade. 18. Curved Gripper Blade. 19. Grubber Blade. FIG. 2 LIST OF EMBODIMENTS: (SIDE ELEVATION IN TARGET APPROACH MODE.) 1. Structural Steel Main Frame--Top Upright channel section. 2. Structural Steel Main Frame 3. Structural Steel Main Frame Base angle box section. 4. Brush Shield. 7. Steel Rebar Support for Brush Shield welded to Main Frame Base. 8. Steel Plate welded to Steel Main Frame bottom section with hole. 9. The 11/8&#34; dia. hole for receipt of bolt to connect tractor arm. 10. High strength 11/8-&#34; bolt for connecting tractor arm. 12. Half in. plate to form lower connection for tractor connection. 14-A. Tree Guide Rebar. 14-B. Channel Support for connecting Tree Guide to Main Frame Upright. 15. A 3/4&#34; Rebar Support connecting ends of Tree Guide to Main Frame Upright. 16. Angle Brace for support of Gripper Blade Mount and connected to forward ends of gripper mount and also to Main Frame Upright. 17. Gripper Blade Mount for support of Gripper Cutting Blade. 18. Curved Gripper Blade. 19. Grubber Blade. FIG. 3 LIST OF EMBODIMENTS: (DETAIL OF GRIPPER MOUNT AND GRIPPER BLADES) 2. Structural Steel Main Frame Bottom Upright section cut. 3. Structural Steel Main Frame Base (Top View.) 7. Steel Rebar Support (cut section.) 9. The 11/8&#34; Dia. hole location (Top View.) 10. High strength 11/8-&#34; Bolt for connecting tractor arm. (Top View.) 11. One inch plate to form lower tractor connection. (Top View) 12. Half-inch side plate attached to Ref. 11, one inch plate. (Top View.) 17. Gripper Blade Mount for support of Gripper Cutting Blade. (Top View.) 18. Curved Gripper Blade. (Top View.) DETAILED DESCRIPTION General Notes: All parts are fabricated from structural steel to meet American Standards Testing Materials A-36 (ASTM A-36) with the exception of the curved Gripper and Grubber Blades which are to meet American Iron and Steel Institute 4130 (AISI-4130) and the Bolts for connection to tractor which are to meet the ASTM A-325. All connections are to meet American Welding Society (AWS) criteria. BRUSH GOPHER MAIN FRAME--72&#34; tall consisting of top upright section, bottom upright section and base section which is 31&#34; wide. All elements are described below, reference numbers 1 through 19 and as shown on FIG. 1 Front Elevation on attached Drawing 1 of 1. Also FIG. 2 Side Elevation references the same elements with the same reference numbers. FIG. 3 is a detail drawing depicting the top view, looking down, of the Main Frame Base, Gripper Blades as attached to the Gripper Blade Mounts. (Ref. 1, 2 and 3.) 1. Top Upright, 42&#34; long consists of a 6&#34;×2&#34;×1/2&#34; steel channel (ASTM A-36.) This element supports the Brush Shield (Ref. 4) and is attached to the Rebar Support (Ref. 5) at top edge of Main Frame Upright (Ref. 2), all connections welded (AWS.) 2. Bottom Upright (center section) consists of a steel box 24&#34; long welded to bottom edge of Top Upright and made up of two (2) 6&#34;×6&#34;×1/2&#34; steel angles (ASTM A-36.) The bottom edge is welded to the center top of the Main Frame Base (Ref. 3.) The top of Bottom Upright is welded to bottom of Ref. 1. 3. Main Frame Base is 31&#34; wide and consists of steel channel 6&#34;×6&#34;×1/2&#34;, two, (ASTM A-36) which form a box, same as Bottom Upright and is welded to the Bottom Upright at centers (Ref. 2.) BRUSH SHIELD A rectangular element 44&#34;×24&#34; used as a viewing, safety shield welded to Top of Upright of Main Frame. This part is welded to Rebar support at top back and two rebars to support sides at back to Main Frame Base and welded at center of Top Upright center (Ref. 4 with elements 5, 6 and 7.) BRUSH SHIELD 4. A rectangular 44&#34;×24&#34;×1/4&#34; thick steel plate (ASTM A-36). 5. Steel Rebar, 11/2&#34; dia. 44&#34; wide welded to top of Brush Shield at back to act as support and stabilizer for the Brush Shield. 6. Three window, viewing slots for sighting targeted trees, brush and bushes to be removed. Each window is 3&#34; down from top of shield and measure 2&#34;×12&#34;, centered in shield, 12&#34; apart. 7. Two 3/4&#34; thick steel rebars, 65&#34;± long, welded to Ref. 5 Rebar and the Brush Shield (Ref. 4.) The bottom of rebars are welded to Main Frame Base and welded 8&#34; from outer edge on back of Brush Shield for its support. (Ref. 3 and 4.) UPPER TRACTOR CONNECTION--An 1/2&#34; steel plate welded to Main Frame Upright (Ref. 8 to Ref. 2) for support of top tractor connection. (Ref. 8, 9 and 10.) 8. A 6&#34; wide by 4&#34; tall 1/2&#34; thich steel plate welded to top of Main Frame Upright (Ref. 2) drilled through each side section with an 11/8&#34; dia. hole to receive bolt for connection to tractor arm connection. 9. Drilled hole 11/8&#34; dia. in 1/2&#34; plate (Ref. 8.) 10. High Strength Bolt (ASTM A-325), 11/8&#34;± for insertion in hole (Ref. 9) to to hold tractor arm connection. TWO LOWER TRACTOR CONNECTIONS--Connected to ends of Main Frame Base (Ref. 3) containing drilled holes as in Ref. 9, bolts as in Ref. 10 with the addition of three elements of 1&#34; plate, 1/2&#34; side support and 1/2&#34; steel gusset. 11. Steel Plate, 6&#34;×10&#34;×1&#34; acting as support for each tractor connection at ends of Main Frame Base (Ref. 3.) (Ref. 9, 10, 11, 12 and 13.) 12. Side support, 4&#34;×21/2×1/2&#34; thick steel plate welded to 1&#34; steel plate (Ref. 11) that forms the housing for lower tractor connections. 13. Steel Support Gusset, 1/2&#34; triangular plate steel welded to side of 1&#34; Steel Plate (Ref. 11) and bottom of 1/2&#34; steel plate (Ref. 12) for support of housing connection (Ref. 11 and 12.) TREE GUIDE--A 24&#34; wide steel rebar with a channel section welded to its midsection and connected to Bottom Upright of Main Frame (Ref. 2), guide also havtwo rebars as support arms connected to ends of Tree Guide and welded to Bottom Upright of Main Frame. The Tree Guide is formed to jut slightly upward and forward to force or push the targeted tree or brush over, up and out of ground giving mechanical advantage to gripper blades at front of unit (Ref. 18.) Ref. 14-A, 14-B and 15.) TREE GUIDE 14-A. A 11/2×24&#34; steel Rebar welded to front side of Bottom Upright of Main Frame by a steel channel (Ref. 14-B) and to end support rebars (Ref. 15) also welded to Bottom Upright, Main Frame (Ref. 2.) 14-B. A 2&#34;×6&#34; steel channel support connected to center of Tree Guide Rebar (Ref. 14-A) and Bottom Upright of Main Frame (Ref. 2.) A 3/4&#34; Steel Rebar Support connected to each end of Tree Guide (Ref. 14-A) and also connected to Bottom Upright of Main Frame (Ref. 2), all with welded connections (AWS.) ANGLE BRACES to support the Gripper Blade Mounts and Gripper Blades (Ref. 17 and 18) and welded to Bottom Upright of Main Frame (Ref. 2.) 16. Angle brace consists of 3&#34;×2&#34;×5/16&#34; steel angle welded each end to Bottom Upright of Main Frame (Ref. 2) and outer side of Gripper Blade Mount at its forward end (Ref. 17) at 45° angle. GRIPPER BLADE MOUNT is a support for the Gripper Blades (Ref. 18) and is made up of a 6&#34;×6&#34;×1/2&#34; angle 25&#34; long connected at center of Base Main Frame, slotted through box and welded to angle brace at forward end as a support for the Gripper Blade which is welded to bottom side of Gripper Mount. (Ref. 17.) 17. A 6&#34;×6&#34;×1/2&#34; steel angle, 24&#34; long which serves as a mount for the Gripper Blades. This angle is welded on its bottom surface to (Ref. 18) Gripper Cutting Blade and angled through the Main Frame Base (Ref. 2) in fixed position. The Gripper Mount is sharpened on front 6&#34; edge and along 12&#34; of top edge of channel 1/2&#34; surface. This gives added capability to hooking and gripping of targeted material to be removed. GRIPPER CUTTING BLADE is made of steel grader blade curved upward and attached to Gripper Mount (Ref. 17) on its top surface for fixed position and support. The Gripper cutting blades clutch and hook into targeted material (Ref. 18.) 18. A 6&#34;×22&#34;×5/8&#34; curved, fixed cutting blade (made of grader blade) AISI-4130 hardened steel. The curved blade is a 1/2&#34; radius each side of 6&#34; width (front end) and sharpened along the 6&#34; edge and inner edges of 22&#34; length. The blade is welded on its upper surface to Gripper Mount with ends and inner surfaces of Gripping Cutting Blades extending out by 2&#34; from ends and inner sides of Gripper Mounts (Ref. 17.) Also note the curved blade is shaped to resemble a saucer shape with the cutting sharpened 22&#34; length edge curving upward. Front inner edges are 11&#34; apart. The Gripper Cutting Blades&#39; curved edge cut into, scoop, and release timber when blade is withdrawn by tractor&#39;s forward movement, releasing targeted material, allowing continuous cutting without dismounting to clean targeted material off blades. This automatic release of removed material is a great time and labor saving quality over prior art. GRUBBER BLADE is a threepieced blade unit shaped like a &#34; &#34; and welded to the back side and bottom of the Main Frame Base (Ref. 3, FIG. 1 and 2.) This blade unit is made up of Grader Blade (AISI-4130). The Blade uprights are 41/2&#34;×6&#34; solid plates with concave shape, 11&#34; apart at front side and 12&#34; apart at back side of blade unit. The bottom blade is a concave blade with sharpened edge downward. The space between all blades is an open space allowing for dirt, and debris to pass through without clogging, thus giving a continuous operation and release of cleared and grubbed materials. 19. Grubber Blade Unit is a three pieced blade unit with two blades 41/2&#34; ×6&#34;×5/8&#34; concave solid plate blades welded to the bottom 6&#34;×5/8&#34; blade which is 12&#34;± on front side and 13&#34;± on rear or back side, all welded to Main Frame Base (Ref. 3) at center. All blades are concave, with bottom blade curved down, sharpened on front and back edges. The two upright blades are concave with front and back edges sharpened. The Grubber Blade Unit has six sharpened edges, all blades of 5/8&#34; Steel (AISI-4130) or similar to and equal to grader blades. The Grubber Blades root out trees and stumps (6&#34; dia. or less), bushes and brush with downward movements easily releasing the removed material. The following denotes advantages and unique features of the Brush Gopher, attachment for a standard tractor. The Overall Operation of the Brush Gopher is a continuous clearing and grubbing operation with automatic debris release requiring no dismounting and cleaning of blades after each cutting operation. There is no build-up or clogging of removed material as it is cut and removed. The gripper blades clutch, cut and release the targeted material as tractor moves unit forward in continual operation. The grubber blade unit aids this automatic clearing and release by the unique design/invention of the three piece blade unit. The prior art &#34;Tree Puller&#34; is made of square tubular steel units which is a weaker Main Frame Structure and it requires continuous dismounting to clean blades after each cutting operation. The &#34;Brush Gopher&#34;, thus provides a major savings in time and labor cost. Main Frame and Parts are of structural steel members meeting ASTM A-36 making for a stronger and lasting unit. Prior art &#34;Tree Puller&#34; is of square tubular steel material lacking the structural strength of the Brush Gopher. Brush Shield is a steel safety shield with viewing windows, three equally placed in shield for clear sighting of targeted material. The prior art does not have this feature. The Brush Shield deflects hazardous debris from hitting the operator in face or body. Tree Guide gives operator capability of guiding and pushing targeted timber in a selected direction, guiding the fixed Gripper Cutting Blades to grasp or clutch the targeted material. This is a structural steel rebar strongly welded to main frame. Prior art does not have this feature. Gripper Cutting Blades are steel curved blades fixed to gripper mounts connected to Angle Braces. The curved up gripper blades clutch and cut the targeted timber and automatically release the removed material through the vehicular motion and in unison with the tractor&#39;s hydraulic lift as the unit moves forward for continuous operation. There is no stopping the tractor and Brush Gopher to dismount and clean the blades as is necessary in the prior art &#34;Tree Puller.&#34; The forward movement of the Brush Gopher cuts with the Gripper Blades at the same time releasing the blades from targeted material, thus no clogging of blades as in prior art. The Gripper Blades are a unique invention designed by the Inventor for more efficient, clean and cost saving operations. The Gripper Blades to not stick in the wood. Grubber Cutting Blade Unit is a unique invention, improvement over prior art. The 3 piece blade unit is concave in shape and bottom blade is wider in back than in front with side upright blades spaced 11&#34; apart in front and 12&#34; in back, also concave, allowing a clear space for grubbed material to pass through with no clogging of the blades. This feature is an improvement over prior art in that the grubbed or dragged material is automatically released. The debris falls away from the blades and allows for continual operation in unison with the gripper blades. There is no dismounting to manually clear the blades of debris as is the case in the prior art.

Summary: An invention which is an attachment to a standard tractor for use in continuous clearing, uprooting and grubbing of small trees, stumps, roots and brush from the land. The unit is fabricated of structural steel elements specially designed for continuous cutting and release of targeted materials for removal. These elements, specially designed and new to the art include a safety shield with viewing windows which provides protection to the operator from flying, hazardous debris; a steel bar as a guide to the forward cutting blades which also aids in pushing over the targeted timber; a pair of curved blades for gripping and cutting, then releasing the material they cut and lift without clogging the blades. Also the blades do not stick in the wood. This is a unique property as it allows continual operation including clearing the blades of debris saving the operator time and labor dismounting and manually clearing the blades after each cutting operation. The final improvement over prior art is a curved cutting blade unit at the bottom of the invention which grubs roots, stumps and brush after the cutting and uprooting of timbers allowing the debris to pass through without clogging the blades. This feature saves time and effort by not requiring the operator to dismount after each cutting operation and manually clean the blades of debris.

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Summarize: Guns and Gear By Major General Jerry Curry, USA (Ret.) The Social Security Administration (SSA) confirms that it is purchasing 174 thousand rounds of hollow point bullets to be delivered to 41 locations in major cities across the U.S. No one has yet said what the purpose of these purchases is, though we are led to believe that they will be used only in an emergency to counteract and control civil unrest. Those against whom the hollow point bullets are to be used — those causing the civil unrest — must be American citizens; since the SSA has never been used overseas to help foreign countries maintain control of their citizens. What would be the target of these 174, 000 rounds of hollow point bullets? It can’t simply be to control demonstrators or rioters. Hollow point bullets are so lethal that the Geneva Convention does not allow their use on the battle field in time of war. Hollow point bullets don’t just stop or hurt people, they penetrate the body, spread out, fragment and cause maximum damage to the body’s organs. Death often follows. Potentially each hollow nose bullet represents a dead American. If so, why would the U.S. government want the SSA to kill 174,000 of our citizens, even during a time of civil unrest? Or is the purpose to kill 174,000 of the nation’s military and replace them with Department of Homeland Security (DHS) special security forces, forces loyal to the Administration, not to the Constitution? All my life I’ve handled firearms. When a young boy growing up on my father’s farm in Pennsylvania Dad’s first rule of firearms training was, “Never point a gun at someone, in fun or otherwise, unless you intend to shoot them. If you shoot someone, shoot to kill.” I’ve never forgotten his admonition. It stayed with me through my Boy Scout training, when I enlisted in the army as a Private to fight in the Korea War, during my days as a Ranger and Paratrooper and throughout my thirty-four year military career. If this were only a one time order of ammunition, it could easily be dismissed. But there is a pattern here. The National Oceanic and Atmospheric Administration (NOAA) has ordered 46,000 rounds of hollow point ammunition. Notice that all of these purchases are for the lethal hollow nose bullets. These bullets are not being purchased and stored for squirrel or coyote hunting. This is serious ammunition manufactured to be used for serious purposes. In the war in Iraq, our military forces expended approximately 70 million rounds per year. In March DHS ordered 750 million rounds of hollow point ammunition. It then turned around and ordered an additional 750 million rounds of miscellaneous bullets including some that are capable of penetrating walls. This is enough ammunition to empty five rounds into the body of every living American citizen. Is this something we and the Congress should be concerned about? What’s the plan that requires so many dead Americans, even during times of civil unrest? Has Congress and the Administration vetted the plan in public. I fear that Congress won’t take these ammunition purchases seriously until they are all led from Capitol Hill in handcuffs. Why buy all this ammunition unless you plan to use it. Unknown to Congress, Does DHS plan to declare war on some country? Shouldn’t Congress hold hearings on why the Administration is stockpiling this ammunition all across the nation? How will it be used; what are the Administration’s plans? Obama is a deadly serious, persistent man. Once he focuses on an object, he pursues it to the end. What is his focus here? All of these rounds of ammunition can only be used to kill American citizens, though there is enough ammunition being ordered to kill, in addition to every American citizen, also every Iranian, Syrian or Mexican. There is simply too much of it. And this much ammunition can’t be just for training, there aren’t that many weapons and “shooters” in the U.S. to fire it. Perhaps it is to be used to arm illegal immigrants? We have enough military forces to maintain law and order in the U.S. even during times of civil unrest. We have local police, backed up by each state’s National Guard, backed up by the Department of Defense. So in addition to all these forces why does DHS need its own private army? Why do the SSA, NOAA and other government agencies need to create their own civilian security forces armed with hollow nose bullets? Were I the JCS, and if I wasn’t already fully briefed on this matter, I’d stop the purchase of hollow point bullets, ask the secretary of Defense why all this ammunition is being purchased and spread around the country? If I got answers like the ones Congress got during the investigation of Operation Fast and Furious – I’d start tracking all ammunition deliveries nationwide to find out what organizations and units are using them, for what purpose and, if it is not constitutional, prepare to counteract whatever it is that they are doing. This is a deadly serious business. I hope I’m wrong, but something smells rotten. And If the Congress isn’t going to do its duty and investigate this matter fully, the military will have to protect the Constitution, the nation, and our citizens. Jerry Curry is a decorated combat veteran, Army Aviator, Paratrooper, and Ranger, who for nearly forty years has served his country both in the military and as a Presidential political appointee. It didn't take long for the Internet to start buzzing with conspiracy theories after the Social Security Administration posted a notice it was purchasing 174,000 hollow-point bullets. Why is an agency that provides benefits to 56 million retirees, disabled workers, widows and children stockpiling ammunition? Whom are they going to use it on? One website suggested the agency was preparing for civil unrest. And comedian Jay Leno wondered just which senior citizens the agency believes are about to storm its offices. The explanation, it turns out, isn't as tantalizing as an arms buildup to defend against unruly old people. The bullets are for nearly 300 agents who investigate Social Security fraud and made almost 600 arrests last year. Most of the ammunition will be expended on the firing range.

Summary: A routine purchase request from the Social Security Administration has caused a flood of conspiracy theories, reports AP. The agency posted a notice that it's buying 174,000 hollow-point bullets, causing some to believe that it was stockpiling ammo to put down civil unrest. A retired major-general at the Daily Caller speculated that the agency was planning to "arm illegal immigrants," or even kill 174,000 US troops and replace them with security forces "loyal to the Administration, not to the Constitution." The reality is a little tamer. The agency explained that it employs 295 agents with full law-enforcement powers to investigate fraud, the Daily Beast reports. Most of the ammunition will be used on the training range. "Our special agents need to be armed and trained appropriately," the agency says. "They not only investigate allegations of Social Security fraud, but they also are called to respond to threats against Social Security offices, employees, and customers."

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Summarize: 1 of 4. U.S. Vice President Joe Biden (L) and China's Premier Wen Jiabao (R) smile during their meeting at the Zhongnanhai leadership compound in Beijing August 19, 2011. CHENGDU, China (Reuters) - U.S. Vice President Joe Biden on Sunday rejected views that American power is waning and said Washington would never default, wrapping up a China visit that has played down tensions between the world's two biggest economies. "We are still the single best bet in the world, in terms of where to invest," Biden told a university audience in Chengdu, the provincial capital of Sichuan, the southwest province that is the second and last stop of his visit to China. "Please understand that no one cares more about this than we do, since Americans own 87 percent of all our financial assets and 69 percent of all our treasury bonds," Biden said, answering a question about U.S. debt. "So our interest is not just to protect Chinese investment. We have an overarching interest in protecting the investment, while the United States has never defaulted and never will default." "You're safe," he added. Biden also used his speech to renew U.S. calls for Beijing to do more to rein in North Korea and Iran, whose nuclear ambitions have alarmed the West. "The fact is, China and the United States face many of the same threats and share many of the same objectives and responsibilities," he said. But his key theme was, as it has been throughout his five-day visit to China, economic: that the United States can reverse its high debt and low growth, and that China should play a part by buying more American-made goods and services. "I also know that some of you are skeptical about America's future prospects. With that in view, I would like to suggest that I respectfully disagree with that view and will allay your concerns," said Biden. He told the audience to remember that the United States was by far the largest economy in the world, about two and a half times as large as China's. Biden and President Barack Obama, both Democrats, face re-election next year. Biden said the debate with Republicans over how to tackle U.S. fiscal problems would be at the heart of the 2012 presidential election. Chinese Vice President Xi Jinping, who is virtually certain to succeed Hu Jintao as Chinese President in early 2013, has hosted Biden during this visit. Obama administration officials have said they want to build trust with Xi ahead of the transition that begins in late 2012, when Hu gives up his post as general secretary of the ruling Communist Party. Next year would need careful political footwork from both governments, said Biden. "Both our countries are going through a political transition in 2012. It is very important, in my view, that we both are aware of the political sensitivities in each of the countries as they go through that," he said. "NOTHING TO WORRY ABOUT" Sichuan province is a fast-growing example of the inland development that Beijing hopes will power the Chinese economy in coming decades -- and also a slice of the rising consumer power that Washington hopes will buy more U.S. goods and reduce a huge trade deficit with China. With 80 million people, Sichuan enjoyed economic growth of 15.1 percent last year, according to government statistics. Such economic concerns have dominated Biden's visit to China, which began on Wednesday, and has featured a succession of unusually vocal declarations of Beijing's confidence in the U.S. economy, despite Standard & Poor's recent downgrade of the sovereign credit rating of the United States. China has quarreled with the United States on trade, Internet censorship, human rights and U.S. arms sales to Taiwan. While those thorny disputes have not disappeared, they appear to have been overtaken by a shared desire to show confidence and cooperation to a jittery global economy. In Sichuan, Biden raised human rights in general terms. "Liberty unlocks a people's full potential, and in its absence, unrest festers," he told the university audience. Biden told Premier Wen Jiabao on Friday that China had "nothing to worry about" over the safety of its holdings of Treasury debt, and Wen voiced confidence in the resilience of the U.S. economy, troubled by debt worries and sluggish growth. Analysts estimate two thirds of China's $3.2 trillion in foreign exchange reserves, the world's largest, are in dollar holdings, making it the biggest U.S. foreign creditor. Biden will fly to Mongolia on Monday morning for a day before heading onto Japan. (Writing and additional reporting by Chris Buckley and Michael Martina in Beijing; Editing by Yoko Nishikawa) U.S. Vice President Joe Biden's head is framed by the teleprompter as he delivers a speech at Sichuan University in Chengdu in southwestern China's Sichuan province, Sunday, Aug. 21, 2011. Biden says... (Associated Press) U.S. Vice President Joe Biden's head is framed by the teleprompter as he delivers a speech at Sichuan University in Chengdu in southwestern China's Sichuan province, Sunday, Aug. 21, 2011. Biden says... (Associated Press) Vice President Joe Biden said Sunday that the United States and China need to recognize their mutual global concerns and responsibilities and ensure greater fairness in trade and investment conditions. Biden brought a strong message of mutual interdependence on his visit to the southwestern Chinese city of Chengdu on the final day of a five-day visit to the world's second-largest economy and a key U.S. trading partner. "The more we can work together, the more our people can benefit... the more the world can benefit," Biden told students in a speech at Sichuan University. Biden emphasized the frequent exchanges between President Barack Obama and China's Hu Jintao along with government officials in the political and economic field. He said there needed to be more exchanges between their civilian and military leaders over security issues, especially on cybersecurity and maritime issues where the sides view matters from different perspectives. "The fact is, China and the United States face many of the same threats and share many of the same objectives and responsibilities," Biden said. "Our generals should be talking to each other as frequently as our diplomats." Biden said both countries need global stability, which includes preventing Iran and North Korea from obtaining nuclear weapons. He also reasserted that the U.S. will remain a Pacific nation in future, saying that the American presence had benefited regional stability and allowed China to focus on economic development. "Asia and the United States are not separated by this great ocean. We are bound by it," he said. Biden said he recognized frustrations among many Chinese businessmen and officials at the length of time needed to obtain visas to visit the U.S. and said Washington was working on improvements. But he said U.S. companies continue to face major investment barriers in China, a frequent complaint among the business community here. He said U.S. businesses were locked out of entire fields and face "restrictions that no other major economy imposes on us or so broadly." Biden also looked to reassure his audience over the security of China's $1.2 trillion in U.S. Treasury debt following the downgrading of America's credit rating. He said Chinese and U.S. prosperity was key to reviving the global economy. "We're the two biggest engines in the world to be able to do that," he said. Biden was to spend the rest of the day Sunday visiting sites with his Chinese counterpart, Xi Jinping, who is expected to become the country's next leader.

Summary: Vice President Joe Biden wrapped up his five-day visit to China with a strong message of China-US interdependence and firm promises that the United States would never default on its debt, reports Reuters. Speaking in the southwest city of Chengdu earlier today, Biden emphasized the continued strength of the US economy, noting that America's economy is two-and-a-half times larger than China's. He also pointed out that, as the United States owns 87% of its financial assets and 69% its treasury bonds, compared to the 1% of financial assets and 8% of treasury bills owned by China, it was in the US interest to pay its bills, adding "the United States has never defaulted and never will default." Biden called on China to be more forceful in reining in North Korea and Iran, and encouraged Beijing to allow a freer flow of information and greater political dialogue, reports the AP. "Liberty unlocks a people's full potential and in its absence, unrest festers," said Biden. In general, the visit emphasized common ties and building trust between the two countries. "Asia and the United States are not separated by this great ocean," said Biden. "We are bound by it."

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Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. provisional patent application Ser. No. 61/437,749 filed Jan. 31, 2011 which application is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant Nos. CA134128, CA128906 and EB004015 awarded by the National Institutes of Health. The Government has certain rights in the invention. FIELD OF THE INVENTION The invention relates to x-ray-based imaging systems and methods in general and particularly to an imaging system and method that provides x-ray images. BACKGROUND OF THE INVENTION Digital radiography provides a two-dimensional (2-D) image of a three-dimensional (3-D) object resulting in superposition of structures. Stereoscopic imaging is a technique wherein at least two 2-D x-ray projection images, referred to as an image pair, separated by an angle not exceeding 20-degrees (typically, 3 to 10 degrees) are acquired and displayed on a stereo-capable display. Such display may include two monitors each displaying one of the projection images, and the displayed projection images are viewed through cross-polarized mirrors and lenses, resulting in one eye visualizing one image and the other eye the other image. Alternatively, the images can be visualized using “3-D displays” such as those used in consumer electronics. In typical implementation of stereoscopic imaging, the image pair is acquired by physical movement of a single x-ray tube. Digital tomosynthesis is a technique wherein a plurality of 2-D x-ray projections are acquired over a limited angular range not exceeding 180-degrees (typically, 15 to 90 degrees) and mathematically reconstructed to provide a quasi-tomographic or 3-D image of object. This technique has the potential to improve detection of an abnormality in body anatomy and is being actively investigated for breast, chest and abdominal imaging. FDA-approved clinical systems for chest imaging and for breast imaging have been developed by various manufacturers. In typical implementation of digital tomosynthesis, (n+1) projections are acquired over an angular range of −θ to +θ spanning 2θ degrees. Here n is a positive integer. Typically, the peak tube voltage (given in kilovolts peak, or kVp) applied across the anode-cathode of the x-ray tube, and the anode target and x-ray beam filter, referred to target/filter are maintained the same during acquisition of the (n+1) projections. The kVp and target/filter define the x-ray spectral shape, and in combination with the tube current (in millamps), or mAs where mAs is the product of tube current in mA and the x-ray exposure duration in seconds) define the x-ray fluence (photons per unit area). In early studies (see Niklason, L. T., B. T. Christian, L. E. Niklason, D. B. Kopans, D. E. Castleberry, B. H. OpsahlOng, C. E. Landberg, P. J. Slanetz, A. A. Giardino, R. Moore, D. Albagli, M. C. DeJule, P. F. Fitzgerald, D. F. Fobare, B. W. Giambattista, R. F. Kwasnick, J. Q. Liu, S. J. Lubowski, G. E. Possin, J. F. Richotte, C. Y. Wei, and R. F. Wirth, Digital Tomosynthesis in breast imaging. Radiology, 1997. 205(2): p. 399-406; Suryanarayanan, S., A. Karellas, S. Vedantham, S. P. Baker, S. J. Glick, C. J. D&#39;Orsi, and R. L. Webber, Evaluation of linear and nonlinear tomosynthetic reconstruction methods in digital mammography. Acad Radiol, 2001. 8(3): p. 219-24; and Suryanarayanan, S., A. Karellas, S. Vedantham, S. J. Glick, C. J. D&#39;Orsi, S. P. Baker, and R. L. Webber, Comparison of tomosynthesis methods used with digital mammography. Acad Radiol, 2000. 7(12): p. 1085-97), tube current was also maintained the same during acquisition of the (n+1) projections. In current practice, the multiple views required for tomosynthesis require the physical rotation of the x-ray tube for each tomographic view. Although this is technically attainable, the physical movement of the tube is the source of many problems in tomosynthesis. A moving x-ray tube prolongs the exposure time and the duration of physical compression of the breast that in turn increases patient discomfort. Moreover, the resulting longer image acquisition time is more likely to contribute to blurring of the images due to patient motion and physical movement of the x-ray tube. Systems using multiple stationary x-ray sources have been described by others for use in tomosynthesis, particularly for breast imaging (Kautzer et al. US 2005/0226371 A1 and U.S. Pat. No. 7,330,529 B2, Ludwig et al. US 2010/0091940 A1, Zhou et al. U.S. Pat. No. 7,751,528). However, such systems lack a central high power x-ray tube or other type of high-power x-ray source. A tomosynthesis system with a series of stationary sources will have no capability of performing conventional digital mammography because this requires a relatively high power x-ray source to provide sufficient x-ray fluence rate (defined as the number of x-ray photons per unit area per unit time, or x-ray fluence per unit time) that meet mammographic requirements for a reasonably short x-ray exposure, typically between 0.3 to 2.0 second duration. The fixed multi-spot x-ray sources are significantly underpowered and currently they are not capable of delivering the high x-ray fluence needed for mammography in an acceptable time frame to minimize patient motion. Therefore, such systems will be limited to tomosynthesis use only and not mammography. However, a mammographic or radiographic unit that can only operate in the tomosynthesis mode is too limited and it will not be desirable in most medical practices, because most breast imaging centers would prefer a system that can perform both tests. The same reasoning applies to digital radiography and tomosynthesis of other parts of the body such as chest, abdominal and pelvic imaging. Some mammographic and radiographic imaging systems currently manufactured can perform conventional digital mammography and tomosynthesis on demand. In the conventional approach, tomosynthesis can be performed by a mechanical scan of the rotating anode x-ray tube over an arc of about +/−30 degrees from the center and acquiring typically from 15 to 25 images across this scan. The detector may remain stationary or it can rotate and/or move laterally to track the x-ray beam. Each of the 15 to 25 images require a combination of rapid activation of the x-ray tube (termed as “fire”) followed by a mechanical movement to the next position for the next fire or x-ray source activation. This rapid firing and mechanical repositioning creates many problems that lead to a less than optimal tomosynthesis image acquisition. During each firing there is a rise and fall “pulse” of the x-ray tube voltage. The tube current and x-ray output can be hard to control without elaborate and expensive electronic controls. Any irregularities in this pulse can contribute to an increased dose to the patient particularly due to the slow rise and drop of the waveform. Moreover, the x-ray filament is susceptible to the mechanical vibrations of the movement of the tube and this can have a negative effect on the spatial resolution of images. A mechanical “stop-fire-and-go” approach is very problematic because of the mechanical instabilities due to acceleration and deceleration of the mechanical assembly of the x-ray source. Continuous mechanical movement is generally preferred but it also prone to vibrations that can affect the image quality. In addition, during each firing that is of finite duration, the x-ray tube is in continuous motion resulting in blurring that degrades image quality. It typically takes between four to ten seconds for a complete acquisition and this increases the chances for a slight movement of the breast or other part of the body that will degrade the spatial resolution and diagnostic quality of the images. This motion problem can be minimized by applying additional compression on the breast using the pneumatic compression mechanism and plate but this is highly undesirable because of increased pain or discomfort. In chest and abdominal radiography this problem is even more serious because shorter exposures are required, typically in the order of milliseconds in chest imaging. A published international patent application by Ren et al. (WO 2010/060007 A1) discusses some of the issues of mechanical scanning approaches. Other prior art known to the inventors includes the following patents and published applications. U.S. Pat. No. 6,649,914 issued to Moorman et al. on Nov. 18, 2003 is said to describe an x-ray imaging system according to the present invention comprising a stepped scanning-beam x-ray source and a multi-detector array. U.S. Pat. No. 7,099,435 issued to Heumann on Aug. 29, 2006 is said to describe a tomographic reconstruction method and system incorporating Bayesian estimation techniques to inspect and classify regions of imaged objects, especially objects of the type typically found in linear, areal, or 3-dimensional arrays. U.S. Pat. No. 7,545,907 issued to Stewart on Jun. 9, 2009 is said to describe a method of obtaining projection data of an object from a plurality of view angles with respect to the object is provided. The method comprises acts of providing radiation, at each of the plurality of view angles, to an exposure area in which the object is positioned, controlling a radiation energy of the radiation provided at each of the plurality of view angles such that the respective radiation energy is different for at least two of the plurality of view angles, and detecting at least some of the radiation passing through the exposure area at each of the plurality of view angles to obtain the projection data. U.S. Pat. No. 7,551,716 issued to Ruhrnschopf on Jun. 23, 2009 is said to describe scatter correction methods for breast imaging and is relevant to the “scatter compensation in tomosynthesis” aspect of our disclosure. The approach described by Ruhrnschopf uses a pre-computed library of scatter spread functions using Monte Carlo simulations, which is a standard computational tool for scatter estimation. We have published previously Monte Carlo simulations of scatter as a function of tomosynthesis projection angle. See Sechopoulos, I., S. Suryanarayanan, S. Vedantham, C. J. D&#39;Orsi, and A. Karellas, Scatter radiation in digital tomosynthesis of the breast. Med Phys, 2007. 34(2): p. 564-76. U.S. Patent Application Publication No. 20090268865 A1 (Ren et al.) published on Oct. 29, 2009. This patent application is said to describe a method and an apparatus for estimating a geometric thickness of a breast in mammography/tomosynthesis or in other x-ray procedures, by imaging markers that are in the path of x-rays passing through the imaged object. U.S. Pat. No. 7,616,801 issued to Gkanatsios et al. on Nov. 10, 2009 is said to describe a method and system for acquiring, processing, storing, and displaying x-ray mammograms Mp and tomosynthesis images Tr representative of breast slices, and x-ray tomosynthesis projection images Tp taken at different angles to a breast, where the Tr images are reconstructed from Tp images. U.S. Patent Application Publication No. 20100063410 A1 (Avila), published Mar. 11, 2010, describes obtaining a lung cancer risk index based on combining information from multiple sources such as spirometry, chest CT or other x-ray examination including x-ray tomosynthesis with airflow lung function measurements. U.S. Pat. No. 7,680,240 issued to Manjeshwar on Mar. 16, 2010 is said to describe methods for performing image reconstruction that include deriving background projection data for an area outside a targeted field of view of a tomographic image, and reconstructing the tomographic image of the targeted field of view, wherein the background projection data is used in the reconstruction. U.S. Pat. No. 7,697,661 issued to Souchay et al. on Apr. 13, 2010 is said to describe a method wherein the irradiation dose to the breast is distributed in a manner based on the orientation of the x-ray beam followed by filtering of the projections to ensure optimum propagation of the signal-to-noise ratio. U.S. Pat. No. 7,702,142 issued to Ren on Apr. 20, 2010 is said to describe a method and a system for using tomosynthesis projection images of a patient&#39;s breast to reconstruct slice tomosynthesis images such that anatomical structures that appear superimposed in a mammogram are at conforming locations in the reconstructed images. U.S. Pat. No. 7,751,528 issued to Zhou on Jul. 6, 2010 is said to describe using a stationary array of x-ray sources to facilitate tomosynthesis. Other references known to the inventors include the following non-patent literature: Nishikawa, R. M., I. Reiser, P. Seifi, and C. J. Vyborny, A new approach to digital breast tomosynthesis for breast cancer screening, in Medical Imaging 2007: Physics of Medical Imaging, J. Hsieh and M. J. Flynn, Editors. 2007, SPIE. p. 65103C; and Sechopoulos, I., S. Suryanarayanan, S. Vedantham, C. D&#39;Orsi, and A. Karellas, Computation of the glandular radiation dose in digital tomosynthesis of the breast. Med Phys, 2007. 34(1): p. 221-32. A number of problems in attempting to implement a method and system that provides both radiographic and tomographic images have been observed. There is a need for methods and systems that provide radiographic, stereoscopic and tomographic images. SUMMARY OF THE INVENTION According to one aspect, the invention features an x-ray apparatus for making an image. The x-ray apparatus comprises an object holder configured to position an object of interest to allow the making of an image of the object; an x-ray source configured to provide a first x-ray beam having a high x-ray fluence rate to illuminate the object of interest along a first axis; at least one peripheral satellite x-ray source configured to provide at least one secondary x-ray beam having lower x-ray fluence rate than the fluence rate of the first x-ray beam, the at least one secondary x-ray beam configured to illuminate the object of interest along a respective axis that is angularly displaced from the first axis; a detector configured to detect x-ray radiation that has passed through the object of interest from the x-ray source and from the at least one peripheral satellite x-ray source, the detector having an output port configured to provide non-volatile signals representative of the detected x-ray radiation that has passed through the object of interest; a controller configured to command the operation of the x-ray source, configured to command the operation of each of the at least one peripheral satellite x-ray source, and configured to command the operation of the detector to generate the non-volatile signals representative of the detected x-ray radiation that has passed through the object of interest; and a computation unit configured to receive the non-volatile signals representative of the detected x-ray radiation from the detector and configured to manipulate the non-volatile signals representative of the detected x-ray radiation to provide at least one image of the object of interest, the computation unit configured to perform at least one action selected from the group of actions consisting of recording the image of the object of interest, displaying to a user the image of the object of interest, and transmitting the image to a data handling system. In one embodiment, the object of interest is a body part of a living being. In another embodiment, the body part of a living being is a human breast. In yet another embodiment, the x-ray source and at least one peripheral satellite x-ray source are configured to be rotated as a combined unit with reference to the object of interest. In still another embodiment, the x-ray source and at least one peripheral satellite x-ray source are configured to be positioned independently of one another with reference to the object of interest. In a further embodiment, at least one peripheral satellite x-ray source is configured to be operated individually. In yet a further embodiment, the detector is configured to be stationary, or is configured to rotate or move laterally to track an x-ray beam. In an additional embodiment, the controller is configured to control a parameter selected from the group of parameters consisting of an x-ray beam energy, an x-ray beam fluence rate and an x-ray beam duration in response to an orientation of the x-ray beam. In one more embodiment, the apparatus further comprises an anti-scatter grid located in an x-ray beam path. In still a further embodiment, the apparatus further comprises a computational unit configured to apply an x-ray scatter correction method. In one embodiment, at least one image of the object of interest is an image selected from the group of images consisting of a radiographic image, a stereoscopic image, and a tomographic image. In another embodiment, the x-ray source configured to provide a first x-ray beam having a high x-ray fluence rate is a high power source. In a further embodiment, the high power source is selected from the group of sources consisting of a rotating anode source, a high fluence field emission source, and a synchrotron. According to another aspect, the invention relates to a method of making a plurality of images. The method comprises the steps of providing an object of interest for the purpose of making an image of the object; illuminating the object of interest with a first x-ray beam having a high x-ray fluence rate, the first x-ray beam propagating along a first axis; illuminating the object of interest with at least one secondary x-ray beam having lower x-ray fluence rate than the fluence rate of the first x-ray beam, the at least one secondary x-ray beam propagating along a respective axis that is angularly displaced from the first axis; detecting the first x-ray beam and the at least one secondary x-ray beam after they have each passed through the object of interest; generating non-volatile signals representative of the detected x-ray radiation that has passed through the object of interest; manipulating the non-volatile signals representative of the detected x-ray radiation to provide a plurality of images of the object of interest, the plurality of images comprising a stereoscopic image and at least one image selected from the group consisting of a radiographic image and a tomographic image; and performing at least one action of recording the images, transmitting the images to a data handling system, and displaying the images to a user. In one embodiment, the step of illuminating the object of interest with a first x-ray beam, the step of illuminating the object of interest with at least one secondary x-ray beam, the step of detecting the first x-ray beam and the at least one secondary x-ray beam, and the step of generating non-volatile signals representative of the detected x-ray radiation are performed in response to commands from a controller. In another embodiment, the step of illuminating the object of interest with a first x-ray beam and the step of illuminating the object of interest with at least one secondary x-ray beam are performed in any order. In yet another embodiment, the step of illuminating the object of interest with at least one secondary x-ray beam includes illuminating the object of interest with a first of the at least one secondary x-ray beams in a first time interval and illuminating the object of interest with a second of the at least one secondary x-ray beams in a second time interval different from the first time interval. In still another embodiment, the source of a first of the at least one secondary x-ray beams provides x-ray illumination while a source of a second of the at least one secondary x-ray beams is moving. In a further embodiment, at least one of the steps of illuminating the object of interest comprises illuminating the object of interest with an x-ray beam having at least one parameter selected from the group of parameters consisting of x-ray beam energy, x-ray fluence rate and x-ray beam duration, the at least one parameter having a value that is dependent on an orientation of the x-ray beam. In yet a further embodiment, at least one of the steps of illuminating the object of interest with at least one secondary x-ray beam is used to provide one or more of the stereoscopic image, the radiographic image and the tomographic image. In an additional embodiment, the step of illuminating the object of interest with at least one secondary x-ray beam is used for stereotactic localization to obtain samples of the object of interest. In one more embodiment, at least one of the steps of illuminating the object of interest comprises the steps of illuminating the object of interest with an anti-scatter grid in an x-ray beam path; illuminating the object of interest without an anti-scatter grid in the x-ray beam path, and; applying an x-ray scatter correction method comprising the steps of: estimating an x-ray scatter present in an image recorded at a first beam orientation; determining an x-ray scatter present in an image recorded at a second beam orientation different from the first beam orientation by using the estimated x-ray scatter estimated at the first beam orientation; and applying the determined x-ray scatter as a correction for x-ray scatter in an image recorded at the second beam orientation. In still a further embodiment, the step of determining an x-ray scatter present in an image recorded at a second beam orientation is performed using Monte Carlo simulations. In yet another embodiment, the step of determining an x-ray scatter present in an image recorded at a second beam orientation is performed using a library of data that accounts for the range of dimensions and properties of the object. In still another embodiment, the step of applying the determined x-ray scatter as a correction is performed using at least one mathematical procedure selected from the group of mathematical procedures consisting of analytical mathematical operations, iterative mathematical operations, convolution techniques and de-convolution techniques. The foregoing and other objects, aspects, features, and advantages of the invention will become more apparent from the following description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS The objects and features of the invention can be better understood with reference to the drawings described below, and the claims. The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the drawings, like numerals are used to indicate like parts throughout the various views. FIG. 1 is a schematic diagram that shows a system according to principles of the invention that includes a central x-ray source and satellite x-ray source assemblies. FIG. 2 is a schematic diagram that shows a variation of the described approach with the satellite x-ray source assemblies (right and left) spread in an arc away from the central x-ray source, according to principles of the invention. FIG. 3 is a schematic diagram that shows a variation of the described approach that is used when only a conventional mammogram is required. FIG. 4 is another view of the relative positions of a patient, a portion of a patient&#39;s body that is being examined, and the position of the apparatus. FIG. 5 is a schematic diagram that illustrates an embodiment in which the individual sources in the satellite x-ray sources are operated in sequence. FIG. 6 is a schematic diagram that illustrates an embodiment in which the satellite x-ray sources comprise a plurality of discrete individual sources. FIG. 7 is a schematic diagram that illustrates the components of an apparatus according to principles of the invention and the interactions among the components. DETAILED DESCRIPTION Combined Digital Mammography with High Speed Tomosynthesis We describe a method for tomosynthesis imaging that uses a number of x-ray sources positioned on each side (or only on one side if desired) of the conventional central x-ray tube. This preferred embodiment enables fast tomographic image acquisition for tomosynthesis and for stereoscopic x-ray imaging. Image acquisition parameters (kVp, target/filter, mAs), either individually or in combination, can be varied with projection angle during acquisition of the (n+1) projections. We refer to such variation of operating parameters as angle adaptive beam modulation (AABM). In order to overcome the limitations of prior art systems, it is desirable and advantageous to have a series of spatially fixed x-ray sources in an arc path that can activate sequentially or in any desired order to cover the desired angle of exposure in the shortest possible time. We describe a system that is expected to be fully capable of operating as a state-of-the art mammography or radiography system and it also is expected to be capable of operating as a high speed tomosynthesis system by using a combination of a standard high power source and a plurality of peripheral “satellite” x-ray sources. We describe using a combined stationary x-ray source array and a conventional x-ray tube. The x-ray tube can be positioned at the center of the x-ray source array. This allows the use of high x-ray fluence rate projection acquisition using the standard x-ray tube and lower x-ray fluence rate projection acquisitions using the stationary x-ray source array. The combined x-ray tube and stationary x-ray source array can be rotated with reference to an object to be examined in order to facilitate finer angular sampling of the object. Alternatively, each source array can be rotated independently. It is possible in principle to use two or more lower x-ray fluence rate projection acquisitions using the stationary x-ray source array without the high x-ray fluence rate projection to generate some of the images discussed herein below. In various embodiments, the high power source can be a conventional rotating anode source, or a non-conventional higher power x-ray source such as a high fluence field emission source or a synchrotron for the first axis projection rather than the stationary x-ray source. While the first axis can be a central axis (e.g., at zero-degrees in reference to the central ray axis), it is not required that it be a central axis; that is the first axis can be off-center in some embodiments. The preferred embodiments described use one or more banks of stationary sources interposed with a rotating anode x-ray source or a number of lower powered sources interposed with a rotating anode x-ray source. The use of a rotating anode x-ray source is significant so as to provide sufficient x-ray output for digital mammography. Rotating anode x-ray tubes designed for mammography can provide upwards of 5 KW power (Tube voltage: 50 kV, Tube current: 100 mA), whereas stationary sources can only provide tube currents of the order of 3 to 7 mA, as described in a recent article from Zhou&#39;s research group (see Calderon-Colon, X., H. Geng, B. Gao, L. An, G. Cao, and O. Zhou, A carbon nanotube field emission cathode with high current density and long - term stability. Nanotechnology, 2009. 20(32): p. 325707). We describe a dual-use mammographic and tomosynthesis system that is also capable of stereoscopic imaging and stereotactic localization and employs a conventional central x-ray source and detector with an additional set of x-ray sources (satellite sources, 4 ) on each side of a conventional x-ray source ( 1 ) as shown in FIG. 1 and FIG. 2. As is well-known in the x-ray imaging and tomographic imaging arts, once data is acquired using a detector, the data is manipulated in a general purpose programmable computer operating with instructions recorded in non-volatile memory accessible by and readable by the general purpose programmable computer. The images that are extracted from the acquired data can be recorded, can be transmitted to another computational system, and/or can be displayed to a user of the imaging system. FIG. 1 is a schematic diagram that shows several important features of an embodiment of the invention. Illustrated in FIG. 1 are the elements of a digital mammography or tomosynthesis unit with a conventional x-ray tube ( 1 ), which can be a rotating anode x-ray tube or a stationary anode x-ray tube. The x-ray tube has at least one small high heat load focal spot ( 2 ). An x-ray beam collimator ( 3 ) and an x-ray beam filter ( 9 ) are provided to control the high power x-ray beam emanating from the x-ray tube ( 1 ). Also present is an x-ray source assembly ( 4 ) having a plurality of x-ray sources ( 5 ). The x-ray source assembly ( 4 ) also termed “satellite sources”) is constructed of multiple segments which are movable, with a center of rotation ( 12 ) about which each of the x-ray sources ( 5 ) can be rotated. In one embodiment, the x-ray sources ( 5 ) are x-ray sources constructed using field emission x-ray sources. In other embodiments, other types of x-ray sources can be used. Multiple x-ray beam collimators ( 8 ) are provided to collimate the x-ray beam emanating from each of the x-ray sources ( 5 ). The breast ( 6 ) of a patient is illustrated under compression by a breast compression plate ( 11 ). A digital imaging detector ( 7 ) is provided to record the intensity of the x-ray beams ( 10 ) that are used to generate mammographic and tomographic images. The digital imaging detector ( 7 ) can be oriented normal to the axis of the rotating anode x-ray tube ( 1 ) or it can be tilted or shifted to provide angulated views. The digital imaging detector ( 7 ) can be made of amorphous selenium with a thin film transistor (TFT) or optical readout, or an amorphous silicon detector with a scintillator or a photon counting detector. The x-ray sources operate in a range of kVp from about 20 to 150 so that the range that is used for mammography (20 to 50 kVp) and for adult and pediatric radiography (40 to 150 kVp) can be covered. This approach obviates the mechanical scanning of the x-ray tube to perform tomosynthesis. Conventional mammography in the craniocaudal, mediolateral and other mammographic views can be performed by rotating the tube and detector c-arm assembly as in the conventional approach. However, the tomosynthesis acquisition can be acquired by firing the satellite x-ray sources in a predetermined sequence. The satellite x-ray sources are typically of the field emission type (using carbon nanotubes or other components) and they are integrated in groups of two or more as shown in FIG. 1. One advantage of this design is the ability to fire a very rapid sequence of tomosynthesis projections without mechanical movement of the main x-ray source (tube 1 ). This enables a tomographic acquisition in a very short time, typically in the order of approximately one second, that represents at least a tenfold improvement over existing techniques. Moreover, the tomographic acquisition can be performed in the same sequence as the conventional view. For example, the conventional mammographic view can be acquired at a typical exposure time from about one-half second to three seconds followed by or preceded immediately by a tomographic acquisition without repositioning the breast. For chest radiography, the exposure time will be in the millisecond range, typically between 1 millisecond to 1 second. This approach enables for the visualization of conventional and tomographic images that have been acquired in the same position and they can be digitally fused for better correlation between conventional mammographic image and tomographic image acquisition. The use of multiple x-ray sources is expected to require test procedures and calibrations to minimize variation in x-ray beam quality and quantity. FIG. 2 is a schematic diagram that shows a variation of the described approach with the satellite x-ray source assemblies ( 4 right and 4 left) spread in an arc away from the central x-ray source 1. In this embodiment a tomosynthesis acquisition can be made at different angles from the center. Moreover, each satellite source assembly can be positioned in a way that can be rotated independently or in synchrony with the other assembly. FIG. 3 is a schematic diagram that shows a variation of the described approach that is used when only a conventional mammogram is required. In FIG. 3, it is shown how the satellite x-ray source assemblies ( 4 ) can be translated away from the patient (to locations 13 and/or 14 as shown) or can be tilted away from the patient, which motion is not shown. In some embodiments, the satellite x-ray source assemblies ( 4 ) can be moved individually. In some embodiments, the satellite x-ray source assemblies ( 4 ) can be moved as a unit, in which case a mechanical link ( 15 ) between the two satellite x-ray source segments can be provided. In some embodiments, mechanical detents are provided to assure that the satellite x-ray sources ( 4 ) are returned to carefully controlled positions for use in conjunction with the x-ray tube ( 1 ) as previously described. In some embodiments, locating devices such as optical, electrical or magnetic encoders are provided to assure that the satellite x-ray sources ( 4 ) are returned to carefully controlled positions for use in conjunction with the x-ray tube ( 1 ) as previously described. The ability to temporarily displace the satellite x-ray sources ( 4 ) can be helpful in improving patient positioning. Outlines of the location of a patient&#39; head ( 16 ) and body ( 17 ) are indicated in FIG. 3. FIG. 4 is another view of the relative positions of a patient, a portion of a patient&#39;s body that is being examined, and the position of the apparatus. FIG. 4 shows the satellite x-ray sources ( 4 ) in position for providing a tomography image, and in positions (locations 13 and/or 14 as shown) where the satellite x-ray sources are not expected to be used in making a tomographic image. In FIG. 4, the outline of the patient represents a patient closer to the viewer than the apparatus is to the viewer (e.g., one is looking through the patient), and the patient has her back to the viewer. The firing of each x-ray tube can be activated in a number of ways. For example, in one mode of operation, the left source assembly can fire each source sequentially while both assemblies are positioned adjacent to the central x-ray source as shown in FIG. 1. Subsequently, the right assembly fires sequentially while the left assembly moves a predetermined distance counterclockwise (away from the central source). After firing of the selected sources is completed in the right assembly, firing of the sources starts on the left source assembly from its new position farther away from the central x-ray source (tube 1 ). This approach allows the combination of an electronic and mechanical positioning of the focal spot thereby allowing for speed and the ability to acquire projections from various points beyond what is dictated by the number of available satellite x-ray sources. FIG. 1 and FIG. 2 show a symmetric positioning of the satellite x-ray sources on each side of the main x-ray source ( 1 ). In other modes of operation, an asymmetric arrangement may also be desirable. An arrangement using a central x-ray source ( 1 ) in conjunction with just two satellite sources ( 5 ) is also desirable particularly for stereo-mammography. FIG. 5 is a schematic diagram that illustrates an embodiment in which the individual sources in the satellite x-ray sources ( 4 ) are operated in sequence. In FIG. 5 ten individual sources in the satellite x-ray sources ( 4 ) are represented by filled dark circles and are labeled 31 through 40. In the position illustrated, sources 31 through 35 may be fired in a desired sequence, such as in succession or in some other order. While sources 36 to 40 are being fired in the desired sequence, the segment of the satellite x-ray source containing sources 31 - 35 moves in a counterclockwise motion of a pre-defined number of degrees (or pre-defined in units represented by another frame of reference). After the motion is completed, the sources 31 - 35 are located at the positions illustrated by open circles. Sources 31 - 35 can then be fired again in a desired sequence, while the segment of the satellite x-ray source containing sources 36 - 40 moves in a clockwise motion of a pre-defined number of degrees (or pre-defined in units represented by another frame of reference) so that sources 36 - 40 come to be located where the corresponding open circles are illustrated. Sources 36 - 40 can then be fired again in a desired sequence. The movements and firings can be performed as many times as may be desirable or necessary to obtain the images that are sought. FIG. 6 is a schematic diagram that illustrates an embodiment in which the satellite x-ray sources comprise a plurality of discrete individual sources. In FIG. 6, ten individual sources, labeled respectively 21 through 30, are provided instead of the ten sources 31 - 40 illustrated in FIG. 5. While FIG. 5 and FIG. 6 show a total of ten satellite x-ray sources as illustrative embodiments, the number of sources in a satellite x-ray source can be fewer than ten or greater than ten, as individual designs may require, or as may be found to be useful. The number ten is to be understood simply as a useful number for explanation of the apparatus and the method, and is not limiting. Any convenient number of sources can be provided in the apparatus. Scatter Compensation in Tomosynthesis The term “scatter” is commonly used for x-rays that are scattered in tissues and that are detected by the image receptor (the film screen or the digital detector in modern equipment). This x-ray scatter carries incorrect positional information and it degrades the contrast of radiographic images. Scatter is known to be detrimental to image quality. Aggressive measures are taken to suppress its effect in mammography and radiography by using anti-scatter grids. Software based techniques for correcting for the effects of scatter in radiographic imaging have been described. Breast and non-mammographic tomosynthesis would benefit from numerical algorithms and techniques that correct for the effects of scatter. However, such techniques will require accurate estimation of scatter that could vary substantially with each breast or other body part of a patient. However, unlike mammography and radiography, current implementations of digital tomosynthesis do not use an anti-scatter grid and they do not implement numerical scatter correction algorithms to counter the deleterious effect of x-ray scatter. In another preferred embodiment we perform x-ray scatter correction. In this approach we use data from mammography to correct for scatter in tomosynthesis. The current trend is to perform mammography and tomosynthesis with the same system that is capable of performing both tests. Typically, this is implemented by acquiring a tomosynthesis projection sequence either preceding or following a single standard digital radiograph or digital mammogram. During the standard digital radiograph or digital mammogram the anti-scatter grid is in place so as to produce a substantially scatter-reduced image (also referred to as a “reduced x-ray scatter image”). An image produced without using scatter reduction methods will be termed a “full x-ray scatter image.” However, during digital tomosynthesis acquisition the anti-scatter grid is moved out of the x-ray beam to ensure that the anti-scatter grid does not cut-off the x-ray beam during projection acquisition at oblique angles. It is expected that one will be able to utilize the “reduced x-ray scatter image” from the digital radiograph or digital mammogram in combination with the “full x-ray scatter image” from the digital tomosynthesis acquisition that is acquired at a projection angle closest to the digital radiograph or digital mammogram to provide an a priori estimate of scatter for that projection angle. Subsequently, estimates for other projection angles of the tomosynthesis acquisition can be inferred using either analytical approaches that are based on pre-determined variation of scatter with projection angle as described in Sechopoulos, I., S. Suryanarayanan, S. Vedantham, C. J. D&#39;Orsi, and A. Karellas, Scatter radiation in digital tomosynthesis of the breast. Med Phys, 2007. 34(2): p. 564-76, or using accelerated Monte Carlo based approaches that use the a priori information. The position dependent x-ray scatter estimate for each projection angle thus determined can be used for numerical scatter-correction techniques such as those based on convolution approaches, deconvolution approaches and iterative methods. An important aspect in our system and method is the use of the digital mammography image at zero-degree projection angle which has reduced scatter due to the presence of an anti-scatter grid, and we deduce the scatter content for the zero-degree tomosynthesis projection, wherein the anti-scatter grid not present. Once the scatter content at zero-degree tomosynthesis projection is determined, the scatter-content at all other tomosynthesis projection angles can be determined using pre-computed or analytically modeled scatter variation with projection angle. Sparse Stationary X-Ray Source Array During initial development of the stationary x-ray source array technology, it may not be cost efficient to produce a large number of x-ray focal spots that constitute the array or it may not be feasible to position the x-ray focal spots close enough to provide adequate angular sampling. Hence, it is expected that one can use a sparse stationary source array comprising a few focal spot sources that can be rotated with reference to the object for tomosynthesis projection acquisition. In addition, the use of AABM is expected on such a stationary source array which can also be modulated as a function of its rotation about the object. An advantage of a sparse satellite x-ray source array is that its use limits the required range of mechanical movement of the x-ray source, which could improve the use of “step-and-shoot” methods. For example, one could acquire multiple projections corresponding to the location of each source position with the satellite array situated in a first position. One could then rotate the array to the next angular position and the process repeated. This can reduce blurring caused by motion vibrations and angular movement of the x-ray source, because fewer angular transits would be required to obtain a full set of images. Combined X-Ray Tube and Stationary X-Ray Source Array Currently, stationary x-ray source arrays cannot achieve x-ray fluence rates that are as high as that provided by conventional x-ray tubes. Hence, we describe using a conventional x-ray tube for the central (zero-degree) projection and stationary x-ray source for other oblique projections. The conventional x-ray tube will also be used for standard projection imaging such as digital radiography or digital mammography as conventional x-ray tubes have demonstrated the capacity to provide the necessary x-ray fluence rate for such applications. This approach also enables the virtually concurrent acquisition of a conventional mammographic and tomosynthesis image without repositioning the object being imaged. This allows for improved image fusion between the digital radiograph and digital mammogram with the reconstructed tomographic dataset that is free of spatial misregistration due to repositioning of the object being imaged. An advantage of using a conventional x-ray tube for the central (zero-degree) projection is that it allows the use of high fluence imaging needed for standard projection imaging such as digital radiography and digital mammography within an acceptable time frame. Also, the described configuration overcomes blurring due to mechanical movement of the x-ray source. In addition the apparatus and method are also suitable for contrast-enhancement imaging of the object or anatomy, with or without the use of dual-energy technique, wherein images are acquired after injection of contrast media such as intravenously injected iodinated contrast. The described approach also allows for the acquisition of rapid stereoscopic views by using two of the satellite sources to acquire two views that can be viewed in stereo mode. Alternatively, the conventional x-ray tube can provide one image, while one of the satellite sources can provide the second image so that a stereoscopic image pair is obtained and can be viewed in stereo mode. Also, the described approach can be used for tissue sampling such as needle-core biopsy using stereotactic localization, wherein at least one of the satellite sources is used to acquire one of the two views. Alternatively, both views for stereotactic localization can be obtained using satellite sources. Scatter Correction in Digital Tomosynthesis Currently digital tomosynthesis methods do not use any technique for reducing x-ray scatter such as an anti-scatter grid due to grid-cutoff. However, standard projection imaging methods use an anti-scatter grid to reduce x-ray scatter. The combination of digital tomosynthesis with standard digital x-ray imaging appears to be desirable clinically. In such applications, it is expected that the standard digital x-ray imaging acquired with the anti-scatter grid will provide an estimate of “reduced x-ray scatter image” and that the image acquired at a projection angle closest to the standard digital x-ray imaging during digital tomosynthesis acquisition without the anti-scatter grid will provide an estimate of “full x-ray scatter image.” It is expected that these two images at different x-ray scatter conditions can be used to arrive at an estimate of the position-dependent x-ray scatter content for tomosynthesis projection at that projection angle. Position-dependent x-ray scatter content at each tomosynthesis projection angle can then be estimated either analytically using pre-determined numerical methods or using Monte Carlo techniques. Once the position-dependent x-ray scatter content at each projection angle has been estimated, they are used as a priori information for numerical scatter correction techniques that are based on convolution approaches, deconvolution approaches or iterative approaches. An advantage of this feature is that X-ray scatter correction can provide for improved contrast and can reduce artifacts in the image. We are not aware of any prior art that utilizes scatter information under two conditions, i.e., with and without an anti-scatter grid to obtain scatter estimates for scatter correction. Apparatus FIG. 7 is a schematic diagram that illustrates the components of an apparatus according to principles of the invention and the interactions among the components. As illustrated in FIG. 7, an x-ray-based apparatus 710 such as is shown in any of FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, or FIG. 6 is provided to perform the positioning of x-ray sources and an object to be examined A controller 720 is provided that communicates bi-directionally with the apparatus 710. The controller 720 controls the activities of the apparatus 710, and receives data from one or more detectors in the apparatus 710. A computational device 730 communicates with the controller 720, to direct the controller to control the apparatus 710, and to receive from the controller 720 data to be process to generate the one of more stereoscopic, radiographic and tomographic images of the object or interest. The computational device 730 is in one embodiment a general purpose programmable computer provided with instructions recorded on a machine readable medium, and includes a memory upon which the data and/or the generated images can be recorded. The computational device 730 communicates with a display 740, which can display one or more generated images to a user. The display can have one or more display screens, and can operate so as to provide a 3-D stereoscopic image if and when such an image is provided for display. The computational device 730 also includes a user interface that permits a user to initiate operation of the apparatus, and permits a user to request that results be provided as any of a displayed image, a recorded image, recorded data, and data and/or images to be provided to a user at a remote location. Application The invention described herein is directly applicable to digital breast tomosynthesis and digital chest tomosynthesis, which are considered as highly-promising candidates for clinical success. Definitions Recording the results from an operation, data acquisition, or computation, such as for example, recording results at a particular frequency or wavelength, is understood to mean and is defined herein as writing output data in a non-transitory manner to a storage element, to a machine-readable storage medium, or to a storage device. Non-transitory machine-readable storage media that can be used in the invention include electronic, magnetic and/or optical storage media, such as magnetic floppy disks and hard disks; a DVD drive, a CD drive that in some embodiments can employ DVD disks, any of CD-ROM disks (i.e., read-only optical storage disks), CD-R disks (i.e., write-once, read-many optical storage disks), and CD-RW disks (i.e., rewriteable optical storage disks); and electronic storage media, such as RAM, ROM, EPROM, Compact Flash cards, PCMCIA cards, or alternatively SD or SDIO memory; and the electronic components (e.g., floppy disk drive, DVD drive, CD/CD-R/CD-RW drive, or Compact Flash/PCMCIA/SD adapter) that accommodate and read from and/or write to the storage media. Unless otherwise explicitly recited, any reference herein to “record” or “recording” is understood to refer to a non-transitory record or a non-transitory recording. As is known to those of skill in the machine-readable storage media arts, new media and formats for data storage are continually being devised, and any convenient, commercially available storage medium and corresponding read/write device that may become available in the future is likely to be appropriate for use, especially if it provides any of a greater storage capacity, a higher access speed, a smaller size, and a lower cost per bit of stored information. Well known older machine-readable media are also available for use under certain conditions, such as punched paper tape or cards, magnetic recording on tape or wire, optical or magnetic reading of printed characters (e.g., OCR and magnetically encoded symbols) and machine-readable symbols such as one and two dimensional bar codes. Recording image data for later use (e.g., writing an image to memory or to digital memory) can be performed to enable the use of the recorded information as output, as data for display to a user, or as data to be made available for later use. Such digital memory elements or chips can be standalone memory devices, or can be incorporated within a device of interest. “Writing output data” or “writing an image to memory” is defined herein as including writing transformed data to registers within a microcomputer. “Microcomputer” is defined herein as synonymous with microprocessor, microcontroller, and digital signal processor (“DSP”). It is understood that memory used by the microcomputer, including for example instructions for data processing coded as “firmware” can reside in memory physically inside of a microcomputer chip or in memory external to the microcomputer or in a combination of internal and external memory. Similarly, analog signals can be digitized by a standalone analog to digital converter (“ADC”) or one or more ADCs or multiplexed ADC channels can reside within a microcomputer package. It is also understood that field programmable array (“FPGA”) chips or application specific integrated circuits (“ASIC”) chips can perform microcomputer functions, either in hardware logic, software emulation of a microcomputer, or by a combination of the two. Apparatus having any of the inventive features described herein can operate entirely on one microcomputer or can include more than one microcomputer. General purpose programmable computers useful for controlling instrumentation, recording signals and analyzing signals or data according to the present description can be any of a personal computer (PC), a microprocessor based computer, a portable computer, or other type of processing device. The general purpose programmable computer typically comprises a central processing unit, a storage or memory unit that can record and read information and programs using machine-readable storage media, a communication terminal such as a wired communication device or a wireless communication device, an output device such as a display terminal, and an input device such as a keyboard. The display terminal can be a touch screen display, in which case it can function as both a display device and an input device. Different and/or additional input devices can be present such as a pointing device, such as a mouse or a joystick, and different or additional output devices can be present such as an enunciator, for example a speaker, a second display, or a printer. The computer can run any one of a variety of operating systems, such as for example, any one of several versions of Windows, or of MacOS, or of UNIX, or of Linux. Computational results obtained in the operation of the general purpose computer can be stored for later use, and/or can be displayed to a user. At the very least, each microprocessor-based general purpose computer has registers that store the results of each computational step within the microprocessor, which results are then commonly stored in cache memory for later use. Many functions of electrical and electronic apparatus can be implemented in hardware (for example, hard-wired logic), in software (for example, logic encoded in a program operating on a general purpose processor), and in firmware (for example, logic encoded in a non-volatile memory that is invoked for operation on a processor as required). The present invention contemplates the substitution of one implementation of hardware, firmware and software for another implementation of the equivalent functionality using a different one of hardware, firmware and software. To the extent that an implementation can be represented mathematically by a transfer function, that is, a specified response is generated at an output terminal for a specific excitation applied to an input terminal of a “black box” exhibiting the transfer function, any implementation of the transfer function, including any combination of hardware, firmware and software implementations of portions or segments of the transfer function, is contemplated herein, so long as at least some of the implementation is performed in hardware. Theoretical Discussion Although the theoretical description given herein is thought to be correct, the operation of the devices described and claimed herein does not depend upon the accuracy or validity of the theoretical description. That is, later theoretical developments that may explain the observed results on a basis different from the theory presented herein will not detract from the inventions described herein. Any patent, patent application, or publication identified in the specification is hereby incorporated by reference herein in its entirety. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure. While the present invention has been particularly shown and described with reference to the preferred mode as illustrated in the drawing, it will be understood by one skilled in the art that various changes in detail may be affected therein without departing from the spirit and scope of the invention as defined by the claims.

Summary: Systems and methods for providing radiographic, stereoscopic and tomographic images of an object of interest. Examples of objects of interest are body parts of living beings, such as the human breast and the human chest. The apparatus includes a high-fluence rate x-ray source and a plurality of satellite x-ray sources operating at lower fluence rate than the high-fluence rate source. A controller controls the operation and locations of the sources, and the operation of a detector. The method provides procedures in which the operation of the high-fluence source and the satellite sources are individually controlled as to location and orientation relative to the object of interest. In some operations, one satellite source may be operating while another satellite source may be repositioning. By proper control, a reduced x-ray dose and reduced operating time can be attained, thereby improving image quality, patient care, and patient experience.

big_patent

en

Summarize: Suzanne Downer, 61, suffered from high blood pressure that doesn't respond to medication. Around 500,000 people suffer from high blood pressure that doesn't respond to medication. New research has shown that an implant in the leg can help them. Suzanne Downer, 61, a teacher from Welling, Kent, was one of the first in the UK to undergo the procedure, as she tells OONA MASHTA. THE PATIENT. When I was pregnant with my only child, Sophie, 28 years ago, I suffered from pre-eclampsia, a condition which caused me to have very high blood pressure. At its highest it was 220/120 (a healthy reading is 140/90). I had an emergency caesarean at eight months because my blood pressure was so high it could have triggered a stroke. Sophie was healthy, but I was in hospital for three weeks because my blood pressure did not go down. I was put on high doses of medication and warned that a second baby could be dangerous as my pre-eclampsia could worsen next time. Ever since, I've visited my GP every three months to check my blood pressure and medication. But despite different pills and combinations, my blood pressure has remained at around 180/100. I've tried to cut it by staying a healthy weight, avoiding adding salt to food, and exercising frequently. Doctors said it was probably hereditary - my grandfather died of a stroke at 29, and my uncle at 65. Three years ago, things got worse. I spent a week with constant pins and needles in my arms and my blood pressure went as high as 220/110. I was immediately sent to A&E - I had to stay in hospital for observation for 30 hours as doctors feared I was having a heart attack. Then I was referred to a cardiologist. I was on five different blood pressure drugs, with side-effects, including excruciating migraines that lasted up to three days. In 2012 I was referred to Dr Mel Lobo, a hypertension specialist. Tests showed an enlarged heart muscle - a sign it's damaged. My high blood pressure meant my heart had to work harder to pump blood round my body. Dr Lobo gave me a liquid medication called nifedipine, which helped a bit, but when he said there was a mechanical solution I was keen to try it. He said it would involve having a small metal clip implanted in my thigh - it would join a vein and an artery together, so some blood from the artery could leak into the vein, reducing my blood pressure. I would be part of a clinical trial. I had the hour-long procedure in June 2013. First I had local anaesthetic around my groin and they inserted a thin hollow tube into an artery leading to my heart to monitor my blood pressure. Then they inserted the implant in my right thigh. The teacher from Welling, Kent, had an implant fitted in her right thigh. On the operating table my blood pressure dropped to 120/70. It was painful despite the anaesthetic and my right leg was bruised and swollen - I was off work for six weeks. My blood pressure is now around 140/70, slightly raised but a great improvement. I still take some medication, including nifedipine, but it's nothing compared with the cocktail of drugs I used to have. Tests have shown my enlarged heart muscle is starting to heal itself. I no longer live in fear of a heart attack or stroke, or have debilitating side-effects from the drugs. It's a huge relief. THE SPECIALIST. Number of people in the UK believed to have undiagnosed high blood pressure. Dr Mel Lobo is director of the hypertension clinic at Barts Health NHS Trust in London. We need pressure to push blood from the heart to the arteries and the major organs, then back to the heart again. The problem is if your blood pressure is high - which affects more than 15 million Britons - the heart has to work harder to pump blood, which weakens it. Increased pressure can also damage artery walls, and can cause a blockage, leading to a stroke or a heart attack. In a blood pressure reading, the top number (systolic pressure) is the pressure exerted when the heart contracts and pushes blood around the body. The lower number (diastolic pressure) refers to when the heart relaxes between beats. A healthy reading is around 120/80 but high blood pressure is anything above 140/90. She still takes some medication but it's nothing compared with the cocktail of drugs she used to. We try to treat hypertension with lifestyle changes, such as losing weight, reducing alcohol and salt. But high blood pressure can be hereditary, so these may not have an effect. Some patients need medication for the rest of their life. It can include ACE inhibitors, which relax blood vessels, and beta-blockers, which make the heart beat more slowly. Calcium channel blockers widen arteries, and diuretics flush excess water and salt. But all have potential side-effects - ACE inhibitors can trigger a cough, diuretics can lead to erectile dysfunction. And in about 500,000 patients, conventional medical therapy fails to control high blood pressure. There are now alternative, device-based treatments for helping these patients bring down their blood pressure. One is the Coupler device, a paper clip-sized implant inserted in the upper thigh under local anaesthetic. It affects how blood circulates round the body - we effectively transfer blood from a major artery into a major vein, which reduces systolic blood pressure. Once implanted, the Coupler lowers blood pressure by more than 15 per cent and this reduction seems to be long lasting. In a clinical trial of 83 patients, 44 patients were given the device. They experienced significant, lasting reduction in blood pressure, according to a study published last month in the Lancet. The procedure involves making a 2mm incision in the groin and inserting thin tubes into two key blood vessels, the femoral artery and vein. We put a guide wire in to pierce another vein and artery higher in the groin. We then insert the nickel titanium alloy implant; this joins these two vessels together and keeps this passageway open. When blood pressure in the artery becomes dangerously high, small amounts of pressurised blood flows safely from the artery into the vein, reducing blood pressure. The effect is immediate for some, for others it takes a few hours. The procedure is reversible but has not been needed so far. It does have a potential side-effect. Most patients developed leg swelling - the implant can narrow the artery, causing an increase in blood flow in the vein above it, which can lead to swelling. This is often rectifiable with diuretic medication to flush water from the body. However, 29 per cent of patients need a stent - a small tube - inserted in the narrowed arteries to treat the problem. ANY DRAWBACKS? 'The implant is relatively simple to insert, and reversible, the downside is that you undergo a procedure,' says Tom MacDonald, president of the British Hypertension Society and professor of clinical pharmacology at the University of Dundee. 'Some side-effects we know - problems at the insertion site and leg swelling - but some are still unknown, as relatively few people have been treated this way. 'More research could let us help patients with hard-to-control hypertension. But more study is needed before it can be recommended for routine NHS use.' Interested patients can email Barts blood pressure clinic for details. Email: [email protected]

Summary: Suzanne Downer, 61, a teacher from Welling, Kent, has high blood pressure. She's like 500,000 others who don't seem to respond to medication. The mother opted for a new alternative, device to try bring it down. The Coupler is a paper clip-sized implant inserted in the upper thigh. It effectively transfers blood from an artery into a vein, reducing pressure.

cnn_dailymail

en

Summarize: FIELD OF THE INVENTION [0001] The invention relates to a process for the preparation of fish gelatin and fish collagen from fish parts. BACKGROUND OF THE INVENTION [0002] Gelatin is a protein of animal origin which has been known for a very long time and has numerous applications in the food, pharmaceutical, photographic and technical sectors. [0003] It is widespread in animal tissues and organs, such as skin and bones, in the form of its natural precursor, collagen. [0004] Gelatin is obtained industrially from the bones and skins of cattle and pigs, collected from abattoirs. [0005] In both the United States and Europe, beef consumption has been decreasing for several years, gradually reducing the supply of bones and skins originating from this source. This phenomenon has been exacerbated in recent years by the bovine spongiform encephalopathy crisis and the resulting regulations. [0006] As far as gelatins of porcine origin are concerned, their development is being restricted by the discovery of new outlets for the starting material for example pork scratchings and aperitif snacks and by religious considerations forbidding their use in certain countries. [0007] It is therefore particularly important to look for other starting materials which can be used for the manufacture of gelatin. [0008] It is known that fish skin, scales and bones also contain collagen similar to that found in mammals. [0009] Patent GB 235,635 relates to the manufacture of fish glues, gelatins and meals from by-products of marine origin. This principally involves treatment of these by-products with alkali, followed by treatment with sulfurous acid and washing with water to remove the volatile bases responsible for the fishy odor. However, there is no description of the by-products used, the extraction conditions or the characteristics of the gelatins obtained. [0010] For several years, to respond to consumer demand for high-quality fresh products, especially fish fillets, the fishing and canning industry has developed the filleting of fish as soon as they were caught or on land after the frozen fish had been defrosted. This results in the availability of a certain amount of fishing byproducts, particularly fish parts and skins, which can potentially be processed to gelatin. [0011] This has been done especially by NORLAND PRODUCTS, which, since 1985, has been producing aqueous solutions of fish gelatin by the acid extraction of cod skins. However, the corresponding gelatins do not qualify as gelling under the customary conditions for measurement of the gelling strength of gelatins (6.67% gelatin solution kept at 10.degree. C. for 17 h according to Bloom&#39;s method described in “The Science and Technology of Gelatin”, 1977, A. G. WARD and A. COURTS Eds., Academic Press, p. [0012] 507) and remain liquid in solution even when the gelatin concentration is as high as 45%. [0013] Other processes have been developed for improving the quality of the product obtained from fish skins. The process of patent EP 0436266 comprises a step involving treatment with dilute bases, followed by steps involving washing and treatments with different acids. In addition, the gelatin is extracted at neutral pH at a temperature preferably of between 40 and 50 degree. C. [0014] More recently, patent U.S. Pat. No. 5,484,888 reported a process for the preparation of gelatin from kosher fish skins, i.e. skins of fish with scales and fins. [0015] The steps of the process described in said patent take place in a totally alkaline medium, the first step consisting in soaking the skins in an alkaline solution for 3 to 60 days. The extraction is also carried out at an alkaline pH, preferably at a pH equal to 10, and at a temperature of between 45 and 55 degree. C. Finally, in said patent, tuna skins are considered to be unsuitable for the manufacture of gelatin because of the boiling pretreatment to which they are subjected. [0016] U.S. Pat. No. 6,368,656 to Lefebvre relates to a process for the preparation of fish gelatin from fresh or defrosted raw fish skins, which includes the steps of washing of the skins with an aqueous solution, of an oxidizing agent, treatment with acid and hot extraction at an acidic pH. [0017] Most processes to extract fish gelatin from fish wastes (which includes skins, heads and frames) start with raw material which is wet. Furthermore, most processes take a long time to execute the entire processing cycle from the raw material and entering the plant to the finished product which may be either a concentrated liquid gelatin or a dry gelatin. Other processes do not use chemicals to inactivate the natural enzymes that degrade or destroy the gelatin and lower the yield of the gelatin from the wet fish feed material. Once the live fish has been killed, natural enzymes within the fish start to degrade the gelatin. Consequently, it is necessary to process the fish waste as soon as possible or inactivate the enzymes with a chemical that will not degrade the gelatin further. Some types of fish, notably catfish have not produced gelatins of high-quality and have not been sought out as feedstock for gelatin processing. Furthermore, previously described products generate objectionable fish odor in the gelatin. SUMMARY OF THE INVENTION [0018] The present invention therefore relates to an acid process for the preparation of high-quality gelling fish gelatin from fish parts, which comprises steps involving washing of the skins with water, treatment with acid and hot extraction at acid pH. [0019] The starting material consists of fish parts obtained in large quantities when the fish is processed raw. [0020] This is especially the case for tuna in brin, but also for other fish such as sole, tilapia and catfish, in particular the freshwater species Ictalurus punctatus commonly called channel catfish, whose raw fillets are marketed in large quantities. [0021] According to one preferred embodiment, the fish skins originate from fish with scales and fins, for example tuna, tilapia, carp, Nile perch, salmon, sole, especially tropical sole, pollack, hake, mackerel or other similar fish. [0022] According to another embodiment of the invention, the fish skins originate from fish without scales, for example catfish, in particular the freshwater species Ictalurus punctatus commonly called channel catfish, or African sea catfish or North African catfish. [0023] The invention provides a processing method to produce finished gelatin products in a short period of time without suffering a loss in yield. This quick processing method allows for reduced time periods in almost all steps to reach a finished gelatin product. The present invention requires a fewer number of employees, less equipment, less energy, reduced processing per square footage, fewer chemicals and a lower amount of chemicals needed per pound of raw material processed. The amount of waste water is comparatively less. [0024] The present invention reduces the pre-extraction preparation time and the purification steps and additionally has a reduced preparation time period. The extraction step uses an acid based formula to reduce the time required. It is advantageous to use a particular type of acid and concentration level that achieves the separation of the triple helix of the collagen but does not destroy the long molecular chains. Due to the type of acid used, the extraction formula and the temperature held during extraction, fewer chemicals are required. [0025] The present invention allows for the separation of higher gel strength of the gelatin called the Bloom strength. The present invention also removes fish oil from the gelatin without the use of a centrifuge. BRIEF DESCRIPTION OF THE INVENTION [0026] The invention may be understood by reference to the following description taken in conjunction with the accompanying drawings, in which, like reference numerals identify like elements, and in which: [0027] FIGS. 1 a and 1 b illustrates the method steps of the present invention. DETAILED DESCRIPTION [0028] The fish fillets obtained after heading and evisceration are cut up and peeled by hand or machine to leave the fillets on one side and the skins on the other. [0029] The method of the present invention is used to extract gelatin from either wet or dry fish waste, which may include skins, heads and frames, or any other suitable fish wastes. One advantage of the present invention is that the gelatin can be expected in a relatively short amount of time which can be measured from the time the feed material enters the process to the finished product. This short amount of time can be a short as five hours. [0030] In step 102, an enzyme inactivation chemical can be added to the raw fish waste in order to keep the gelatin of the feed material from de-grading. For example, if the transportation trip is longer than approximately 30 minutes, this step is advisable. The enzyme inactivation chemical may include dry calcium hydroxide hydrate in the approximate amounts of 12.5 pounds to 15 pounds per hundred pounds of water in which the fish waste is going to be immersed. Additionally, ice can be added to the above mixture to keep the temperature of the mixture no higher than approximately 5° C. The ice should be added to the mixture before the fish waste is added to the mixture. The mixture of ice and calcium hydroxide hydrate solution tends to draw the blood out of the fish waste and into the solution where the blood can be easily and faster removed at the gelatin processing facility, inactivates the enzymes, and extracts the fats and also dissolves some skin protein that is not gelatin. [0031] In step 104, a rotary screen with optional spray nozzles on the inside and outside of the screen wash the extracted blood and the un-reacted calcium hydroxide hydrate from the fish feed material in approximately 30 minutes after the fish feed material has entered the processing facility. The rotary screen may be supplied with either coldwater or water that can be temperature controlled so as to melt any remaining ice which has not melted with the wet fish feed material. [0032] In step 106, the washed feed fish material is transferred for example by pumping to a preheated, acidified solution which may be water or other suitable material to start the gelatin extraction process. The solution is preheated to approximately 100 Fahrenheit to approximately 170 Fahrenheit in accordance with the type of gelatin that will be extracted and the solution has been pre-acidified to the of range approximately of 1.5 pH to 5.5 pH in accordance with the type the gelatin that will be extracted as shown in step 108. [0033] In order to minimize the gelatin extraction time, the ratio of the hot acidified solution and the fish feed material is approximately 2:1 to 3:1 (solution to fish waste) on a weight basis. In step 110, the fish feed material is placed in the solution in the gelatin extraction tank. The gelatin extraction time preferably does not exceed two hours in duration in order to keep the entire processing time under five hours. [0034] The heated fish feed material and extracted gelatin is pretreated by being filtering so that the untreated or un-reacted fish material is removed before the gelatin clarification process is begun. The filtering in step 112 may be achieved by pumping the fish feed material and extracting the gelatin over a vibrating screen. The hot gelatin solution is transferred to a settling tank in step 114, for example by pumping the hot gelatin solution. At this point, the gelatin extraction tank is available for additional fish material. [0035] The settling tank allows most of the fish oil to rise to the surface where the fish oil is drained off. This process continues until the liquid level approaches the bottom of the tank, and a sight glass aids in this determination. [0036] In step 116, the liquid gelatin solution is filtered to a high clarity for example the liquid gelatin solution may be filtered so that the liquid gelatin solution is approximately 98% transmittance. A plate and frame filter press or a rotary drum vacuum filter may be used to filter the liquid gelatin solution. Additionally, diatomaceous earths may be used in conjunction with the filtering to remove the fine fish material particulates. [0037] In step 118, the remaining traces of fish oil may be removed from the gelatin by incorporating a cellulosic material that absorbs fish oil. This eliminates the need to centrifuge the gelatin to remove the fish oil. The cellulosic material can remove up to approximately 100 percent of the fish oil while a centrifuge can remove approximately 90% of the fish oil. This requires multiple stages of centrifuging. [0038] In step 120, the low molecular weight gelatin is removed from the bulk of the liquid gelatin solution by use of the filter. One filter may be used is an ultrafilter membrane system that has a range of 10,000 to 70,000 molecular weight cut off. This system allows the cut off to be varied and can be changed for different types of gelatin manufactured. The low molecular weight gelatin which has been extracted from the liquid gelatin solution can be used in low high gel strength gelatin products such as liquid protein for beverages and other uses. [0039] In step 122, the concentrated liquid gelatin solution is protected from attack and growth of bacteria, mold or yeast by incorporating a chemical that inhibits this type of growth or it is heat treated to pasteurized the gelatin. [0040] The finished product of concentrated liquid gelatin can be sold as a liquid to customers or dried into a powder or granular form. [0041] While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description herein of specific embodiments is not intended to limit the invention to the particular forms disclosed.

Summary: Process for the preparation of gelatin from fish parts includes the steps of washing the fish parts with an aqueous solution of a calcium hydroxide hydrate and ice to remove blood from the fish parts, treating the washed skins with an acid, and extracting gelatin from the fish skins at a temperature approximately between 100 Fahrenheit to 170 Fahrenheit and at an acidic pH approximately between pH 1.5 to pH 5.5.

big_patent

en

Write a title and summarize: Effectors are microbial-derived secreted proteins with an essential function in modulating host immunity during infections. CfAvr4, an effector protein from the tomato pathogen Cladosporium fulvum and the founding member of a fungal effector family, promotes parasitism through binding fungal chitin and protecting it from chitinases. Binding of Avr4 to chitin is mediated by a carbohydrate-binding module of family 14 (CBM14), an abundant CBM across all domains of life. To date, the structural basis of chitin-binding by Avr4 effector proteins and of recognition by the cognate Cf-4 plant immune receptor are still poorly understood. Using X-ray crystallography, we solved the crystal structure of CfAvr4 in complex with chitohexaose [ (GlcNAc) 6] at 1. 95Å resolution. This is the first co-crystal structure of a CBM14 protein together with its ligand that further reveals the molecular mechanism of (GlcNAc) 6 binding by Avr4 effector proteins and CBM14 family members in general. The structure showed that two molecules of CfAvr4 interact through the ligand and form a three-dimensional molecular sandwich that encapsulates two (GlcNAc) 6 molecules within the dimeric assembly. Contrary to previous assumptions made with other CBM14 members, the chitohexaose-binding domain (ChBD) extends to the entire length of CfAvr4 with the reducing end of (GlcNAc) 6 positioned near the N-terminus and the non-reducing end at the C-terminus. Site-directed mutagenesis of residues interacting with (GlcNAc) 6 enabled the elucidation of the precise topography and amino acid composition of Avr4’s ChBD and further showed that these residues do not individually mediate the recognition of CfAvr4 by the Cf-4 immune receptor. Instead, the studies highlighted the dependency of Cf-4-mediated recognition on CfAvr4’s stability and resistance against proteolysis in the leaf apoplast, and provided the evidence for structurally separating intrinsic function from immune receptor recognition in this effector family. Effectors are intriguing and enigmatic proteins deployed by microbes during host-pathogen interactions [1,2]. Although inhibition of plant immunity during host infection is the main function of these proteins, the manner by which individual effectors perform this task is poorly understood [2]. One of the better understood effectors is CfAvr4, a 135-residue effector protein from the tomato pathogen Cladosporium fulvum, which utilizes a carbohydrate-binding module of family 14 (CBM14) to bind chitin present in fungal cell walls and protect it from hydrolysis by plant-derived chitinases during infection [3–5]. To date, functional orthologues of Avr4 have been identified in a number of fungal species within the Dothideomycete class of fungi and beyond, including the tomato pathogen Pseudocercospora fuligena [6], the banana pathogen Pseudocercospora fijiensis [4], and several others [7]. The majority of Avr4 homologs share a similar cysteine-spacing pattern and contain a distinctive CBM14 domain in their structure, indicating that members of the Avr4 effector family have a conserved role in binding and protecting chitin in fungal cell walls against chitinases [4,6]. Moreover, biochemical analysis between CfAvr4 and its PfAvr4 homolog from P. fuligena has shown that the specificity of these proteins extends further into binding the same length chito-oligosaccharide, i. e. (GlcNAc) 6, suggesting that they share a similar binding-site topography and mechanism of interacting with the ligand [6]. Although protection of chitin seems to be the predominant biological function of the Avr4 family members [4,6], a molecular-level mechanistic understanding of how these effectors bind to their substrate is currently lacking. In this respect, the presence of a CBM14 module in the structure of Avr4 is likely key to its biological function and interaction with chito-oligomers. CBM14s are short modules of approximately 70 residues that bind explicitly to chitin, a long-chain polymer of β (1–4) linked N-acetylglucosamine (GlcNAc) [7]. Currently, only limited information exists as to how CBM14 family members bind to their ligand, but the inclusion of the CBM14 family within the Type C class of CBMs suggests that they lack the extended binding site found in CBM Types A and B [7,8]. However, an experimental validation of this assumption is currently lacking. Despite the lack of information regarding the structural basis for ligand-binding by CBM14 proteins, clues to how Avr4 could possibly interact with its ligand were recently provided by the elucidation of the crystal structure of PfAvr4 from P. fuligena [6]. Like CfAvr4, PfAvr4 utilizes a CBM14 domain to bind specifically to (GlcNAc) 6 oligomers, while binding to higher molecular weight chitin may be facilitated through positive cooperative protein-protein interactions of PfAvr4 molecules [6,9]. Although, a co-crystal of PfAvr4 with chito-oligomers was not attainable, the chitohexaose-binding domain (ChBD) of PfAvr4 was predicted to be located to the C-terminus of the protein [6]. The predicted location of the ChBD in PfAvr4 matches to that of CfAvr4, which used NMR titration experiments to identify residues that may interact with chito-oligomers but was unable to resolve its three-dimensional structure [9]. Next to PfAvr4, the only known structures of CBM14 members are those of tachycitin [10], a small antimicrobial protein from horseshow crab, Der p 23 [11], an allergen from the dust mite Dermatophagoides pteronyssinus, and the ChBD of the human chitotriosidase CHIT1 (ChBDCHIT1) [12,13]. All four structures share a common core containing two β-sheets in a distorted β-sandwich arrangement, a seemingly common fold among CBMs [14]. Next to a conserved biological function, another unexpected commonality shared among several Avr4 family members is their ability to elicit a hypersensitive response (HR) in the presence of the cognate Cf-4 resistance protein from tomato [4,6]. As most Avr4 orthologues share little sequence similarity, it was hypothesized that the indispensability of the CBM14 domain for the function of the protein would make it a prime target for recognition by Cf-4 [4]. However, structure-function analysis with PfAvr4 has shown that point mutations in residues within the predicted ChBD of the protein do not abolish recognition by Cf-4, suggesting that amino acids that directly interact with (GlcNAc) 6 do not form part of CfAvr4’s epitope that is recognized by Cf-4 [6]. However, a shortcoming of the studies on PfAvr4 was that the analysis was based on a predictive model of Avr4’s ChBD instead of structural information regarding the actual carbohydrate-protein interaction. Here, we report the crystal structure of CfAvr4 bound to (GlcNAc) 6, thereby accurately now defining the architecture and amino acid composition of the ChBD and elucidating the underlying molecular mechanism employed by Avr4 family members to bind (GlcNAc) 6 oligosaccharides. The CfAvr4- (GlcNAc) 6 complex showed that, contrary to previous assessments, the ChBD of CfAvr4 extents nearly to the entire length of the protein, which in the presence of the ligand forms a dimeric assembly that laminates two (GlcNAc) 6 oligosaccharides within its structure. Subsequent detailed functional profiling of residues involved in binding (GlcNAc) 6 has further enabled us to quantify their individual contribution to binding affinity, thereby identifying the ones that are most critical to ligand-binding. Further on, by leveraging structural and functional data we reassessed whether the pleiotropic recognition of Avr4 effectors by Cf-4 is based on the perception of residues that directly interact with (GlcNAc) 6 and established that such residues are not targets for recognition by Cf-4, thus provided now strong evidence for structurally separating the ligand-binding function in the Avr4 effector family from recognition by Cf-4. Our previous crystal structure of PfAvr4 proved recalcitrant to structures complexed with chito-oligosaccharides [6]. Consequently, we undertook crystallization of CfAvr4, which has a 10-fold higher binding affinity for (GlcNAc) 6 than PfAvr4, and were successful in obtaining crystals of CfAvr4 in complex with (GlcNAc) 6. CfAvr4 bound in a 1: 1 stoichiometric ratio with the (GlcNAc) 6 ligand and the final structure of the CfAvr4- (GlcNAc) 6 complex was solved at 1. 95Å resolution, with R-factor and R-free values of 16. 7% and 21. 4%, respectively (S1 Table). The X-ray crystal structure of the CfAvr4 monomer spans residues Gln35-Thr113 and is composed of an N-terminal α-helix (H1), a distorted β-sandwich fold formed by a central β-sheet (A) with three anti-parallel β-strands (A1, A2, and A3), a small β-sheet (B) of two anti-parallel β-strands (B4 and B5), and a short C-terminal α-helix (H2) (Fig 1A). Nearly 47% of the structure is organized into α/β secondary structure with the remaining 53% residing in highly ordered loops. The structure clearly shows four disulfide bonds that match the previously determined disulfide pairs of Cys40-Cys70, Cys50-Cys56, Cys64-Cys109, and Cys86-Cys101 [6,15]. A comparison with the PfAvr4 structure shows that the two proteins share a similar fold, with the two structures aligning with a root-mean-squared deviation (RMSD) of 0. 794 Å over 53 α-carbons (Fig 1B and S1A Fig). The only notable discrepancy resides in the proteins’ C-terminus, which in both is comprised of a β-sheet B and an α-helix H2, but CfAvr4 has a two-residue insertion extending the loop connecting the β-strands B1 and B2 (Fig 1C). A search using the DaliLite server [16] revealed that despite the lack of similarity at the amino acid level, CfAvr4 also shares significant structural homology to the CBM14 family members tachycitin (PDB Id: 1DQC), Der p 23 (PDB Id: 4ZCE), and ChBDCHIT1 (PDB Id: 5HBF), aligning to these proteins at an RMSD of 2. 019 Å, 0. 699 Å, and 0. 686 Å, over 36,16, and 38 α-carbons, respectively (Fig 1B and S1B–S1D Fig). However, all three CBM14 proteins lack the N-terminal helix and the large extended loop connecting β-strands A2 and A3 present in CfAvr4 and PfAvr4, plus Der p 23 and ChBDCHIT1 also lack the C-terminal helix. Moreover, three of the four disulfide bonds in CfAvr4 are conserved in tachycitin but only two appear in the Der p 23 and the ChBDCHIT1 structures (S1B–S1D Fig). CfAvr4 was crystallized with (GlcNAc) 6 as an asymmetric unit consisting of two CfAvr4 dimers, with each dimer respectively binding to two (GlcNAc) 6 molecules (S2 Fig and S1 Movie). However, only ~610 Å2 of surface area is buried between the two dimers, suggesting that the tetrameric unit is likely crystallographically induced and that the biologically relevant assembly is a dimer, as previously proposed for PfAvr4 [6,9]. Each dimer consists of two CfAvr4 monomers bound to two parallel (GlcNAc) 6 molecules stacked on top of each other (Fig 2A, S3 Fig and S2 Movie). A single (GlcNAc) 6 chain nearly extends along the entire length of the longitudinal axis of each CfAvr4, with the reducing end located near the N-terminus of the protein and the non-reducing end at the C-terminus. Within each dimer, the stacked (GlcNAc) 6 molecules shift by translation of one sugar ring, with GlcNAc-1 (reducing end) of chain B stacking on top of GlcNAc-2 of chain A and so forth (Fig 2A and S4 Fig). The dimeric assembly creates a 2-fold screw-like rotation of each monomer, such that both sugar and protein are rotated 180° and translated by one sugar unit to create a molecular sandwich that almost entirely encapsulates the parallel-stacked (GlcNAc) 6 molecules within its structure. Surprisingly, the CfAvr4 dimer interface is completely mediated by carbohydrate interactions and no intermolecular protein-protein interactions are observed across the dimer, suggesting that dimerization is a consequence of ligand binding. When considering individual CfAvr4 monomers in the crystallographic asymmetric unit, all four show nearly identical conformations and (GlcNAc) 6 interactions (Fig 2B). Aligned to chain A, chains B, C, and D have an RMSD of 0. 309 Å, 0. 099 Å, and 0. 265 Å, over 67,73, and 68 α-carbons, respectively (Fig 2B). In chains B and D, however, the GlcNAc-6 ring bends toward the protein, deviating from the linearity that is seen in the other two chains. Interestingly, PfAvr4 has been previously crystalized as a dimer as well [6] but a comparison of the CfAvr4- (GlcNAc) 6 dimeric assembly to the ligand-free PfAvr4 dimer showed that two dimer arrangements vary substantially (S5 Fig). For instance, in PfAvr4, the dimeric contacts were mostly water-mediated and only three direct hydrogen-bond interactions between monomer chains were detected [6]. In contrast, CfAvr4 dimerization is mediated exclusively by (GlcNAc) 6, as no direct protein-protein interactions are observed in the CfAvr4- (GlcNAc) 6 dimeric assembly. Instead, several cross linkages are formed where each monomer of CfAvr4 interact with both (GlcNAc) 6 molecules to stabilize the dimeric state of the complex. Further comparison of the dimeric assemblies reveals that upon superposition of the A chain monomers of PfAvr4 and CfAvr4, the B subunit of CfAvr4 is shifted out ~6. 7Å and rotated ~54° relative to the B subunit of PfAvr4 (S5 Fig). This increased separation between the CfAvr4 monomers is caused by enclosing the ligands at the dimerization interface and creating a space large enough for accommodating two (GlcNAc) 6 molecules between the monomers. By modelling the PfAvr4 crystal structure on other chitin-binding proteins, we have previously predicted that PfAvr4’s ChBD resides in the C-terminal domain of the protein consisting of residues on β-strands B4 and B5 and their connecting β-hairpin loop. However, its exact topography and composition could not be precisely defined as even after repeated crystallization attempts a PfAvr4- (GlcNAc) 6 co-crystal was never obtained [6]. Contrary to previous assessments [6,10,15], the chitin hexasaccharide is accommodated in a shallow trench across the longitudinal axis of CfAvr4 with the face of the pyranose rings binding to the protein by means of nonpolar and CH-π interactions, and the ring substituents pointing into the protein core and forming hydrogen bonds with both the main chain and side chains (Fig 3A and S4 Fig). The main facial interaction between CfAvr4 and (GlcNAc) 6 is a CH-π bond between Trp100 and GlcNAc-5. Met51 and Pro53 are also in van der Waals bonding distances of GlcNAc-1 and GlcNAc-3, respectively and contribute to ligand binding. In addition, a number of amino acids in CfAvr4 are also shown to interact with individual sugar ring substituents of the (GlcNAc) 6 substrate (Fig 3B, S4 Fig and S2 Table). For instance, starting from GlcNAc-1, the C6 hydroxyl of GlcNAc-1 forms a hydrogen bond with the main chain carbonyl oxygen of Lys49. The N-acetyl group nitrogen of GlcNAc-2 hydrogen bonds with the main chain carbonyl oxygen of Cys50, while its C3 hydroxyl group hydrogen bonds to the amide oxygen of Gln69. The N-acetyl group nitrogen of GlcNAc-4 forms a water-mediated hydrogen bond interaction with the main chain carbonyl oxygens of both Pro53 and Lys99, an association that is observed in all 4 monomers. The C6 hydroxyl of GlcNAc-5 forms a hydrogen bond with the main chain carbonyl of Cys101, whereas the hydroxyl of Tyr103 is also within hydrogen bonding distance to both the GlcNAc-6 N-acetyl carbonyl and the C3 hydroxyl. In chain B, GlcNAc-6, which does not stack with a carbohydrate subunit from chain A, bends towards the protein so that its N-acetyl group comes within hydrogen bonding distance to the side chain of Asp102 (Fig 3B, S4 Fig and S2 Table). Taken together, the interactions of a CfAvr4 monomer with the (GlcNAc) 6 molecule is mediated by residues Lys49, Cys50, Met51, Pro53, Lys99, Trp100, Cys101, Asp102, and Tyr103, which collectively form at least two water-mediated and nine direct hydrogen bonds with (GlcNAc) 6. Of these, only Trp100 (Trp94 in PfAvr4), Asp102 (Asp96 in PfAvr4), and Trp103 (Tyr97 in PfAvr4) have been previously identified as ChBD residues in PfAvr4 [6]. Interestingly, while no direct protein-protein interactions are observed in the CfAvr4- (GlcNAc) 6 dimeric assembly, there are plenty of sugar-facilitated cross-linkages between the two protein chains (Fig 3B, S4 Fig and S2 Table). For instance, the hydroxyl of Tyr67/A hydrogen bonds to the C6 hydroxyl of the GlcNAc-2/B, whereas the same interaction occurs between Tyr67/B and the GlcNAc-4/A. In a similar way, the amine of Lys84/A hydrogen bonds to the N-acetyl carbonyl of GlcNAc-1/B, whereas Lys84/B interacts with the N-acetyl carbonyl of GlcNAc-3/A. The amide nitrogen of Gln69/A hydrogen bonds with the C3 hydroxyl of GlcNAc-1/B, while Gln69/B hydrogen bonds to C3 hydroxyl of GlcNAc-2/A. Finally, the hydroxyl of Tyr103/A hydrogen bonds to the N-acetyl group of GlcNAc-5/B, but this interaction is not seen between Tyr103/B and the A sugar, as it would have to occur with a GlcNAc-7 unit on the chain A. To assess the functional role that each residue in CfAvr4 involved in binding (GlcNAc) 6 has, we mutated these residues individually and investigated the binding properties via Isothermal Titration Calorimetry (ITC). Specifically, we made the alanine-substitution mutations M51A, P53A, K84A, P87A, W100A, and D102A, and the more conservative mutations Q69N, Y67F and Y103F, to minimize potential protein instability effects. We excluded Lys49 from the mutational analysis since only the main chain of this residue interacts with (GlcNAc) 6, as well as Cys50 and Cys101 because it has been previously shown that mutating these residues compromises the stability of the protein [9,15]. ITC experiments determined that WT-CfAvr4 bound to (GlcNAc) 6 with a dissociation constant (Kd) of 6. 73 ± 1. 49 μM, a binding enthalpy (ΔH) of -38. 37 ± 2. 96 kJ/mol, and a stoichiometric ratio n of 1. 09 ± 0. 12 (S2 Table and S6 Fig). The parameters remained similar irrespectively of whether or not the purification tag was removed from the protein or whether a reverse titration was run, in which concentrated CfAvr4 was titrated into a dilute solution of (GlcNAc) 6, with no evidence of allosteric or dimer dissociation (S2 Table and S6 Fig). The values are also in good agreement with previously reported data except for the stoichiometric ratio [9]. Specifically, our ITC experiment resulted in a stoichiometric ratio of 1: 1, which agrees with the structure, but contrasts the 2: 1 protein: (GlcNAc) 6 stoichiometry proposed previously [9]. Due to this discrepancy, we took great care to verify the concentration of CfAvr4 using a Bradford assay, and quantitated the carbohydrate concentration [17,18]. When assaying the ChBD mutants, mutations M51A, P53A, Y67F, P87A, and Y103F did not affect the Kd substantially and had a small decrease in the ΔH magnitude (S2 Table and S6 Fig). However, mutations W100A and D102A abolished all detectable binding to (GlcNAc) 6, in agreement with the previously reported analogous mutations made in PfAvr4 [6]. Mutations Q69N and K84A both displayed a ~20-fold increase in the Kd raising it to 130. 95 ± 31. 47 μM and 120. 67 ± 18. 77 μM, respectively (S2 Table and S6 Fig). To determine if the reduced affinity for (GlcNAc) 6 of the W100A, D102A, Q69N and K84A mutants was biologically meaningful, we evaluated whether they were able to protect germlings of Trichoderma viride against hydrolysis from chitinases and compared their protective potency to that of the WT-CfAvr4 (S7 Fig). Similar in vitro protection assays were used before to demonstrate and compare the protective properties of CfAvr4, PfAvr4 and other Avr4 family members or mutants thereof [4–6]. As expected, combined addition to pre-germinated germlings of T. viride of BSA (negative control) with chitinases supplemented with basic β-1,3-glucanases inhibited fungal growth, whereas combined application of the enzyme mixture with the WT-CfAvr4 (positive control) enabled fungal growth and survival (S7 Fig). When examining the ChBD mutants, mutants W100A and D102A that do not exhibit any detectable binding to (GlcNAc) 6 (S2 Table and S6 Fig), essentially failed to protect the fungal hyphae against chitinases, thus resulting in poor fungal growth that was comparable to that of the BSA control. Mutants Q69N and K84A that exhibit reduced affinity for (GlcNAc) 6 (S2 Table and S6 Fig), enabled fungal growth to levels comparable to those of the WT-CfAvr4, although at closer inspection of the hyphae many were now seen to bore signs of osmotic injuries such as swollen segments and coagulated cytoplasm (S7 Fig). This indicates that the chitinase treatment had a stronger effect on hyphae treated with the Q69N and K84A mutants as compared to hyphae treated with the WT-CfAvr4 that remained morphologically intact. Collectively, results from the protection assays show that mutations in the ChBD of CfAvr4 that decrease or abolish its affinity for (GlcNAc) 6 also reduce the protein’s ability to protect fungal germlings against chitinases and thus perform its biological function. A previous NMR study of CfAvr4, while unable to determine the structure, showed amide (1HN) backbone chemical shifts upon addition of (GlcNAc) 3 that were assigned to Asn93, Asp94, Asn95, (NDN motif), Asp102, and Tyr103 [9]. The structure of CfAvr4 in complex with (GlcNAc) 6 confirms that Asp102 and Tyr103 directly interact with the ligand. In contrast, the NDN motif is located on the B4-B5 β-hairpin loop and is pointing away from the binding site, where it is at a distance of more than 10Å from the hexasaccharide (S8 Fig). To address this discrepancy between the NMR and crystallographic data, we made mutations N93A, D94A and N95A, and characterized their affinity for (GlcNAc) 6 using ITC (S2 Table). Surprisingly, all three mutations affected the thermodynamic parameters that were indicative of lower affinity for the hexasaccharide, as they increased the Kd by a factor of six (N93A), five (D94A) and three (N95A) (S2 Table). One of the characteristics of CH-π interactions, such as observed between Trp100 and GlcNAc-5, is that they are very sensitive to the electronic structure of the aromatic residues involved in binding [19]. It is thus likely that the nearby NDN motif helps to create a proper electronic environment to facilitate binding, while also providing structural integrity to the C-terminal domain containing Trp100 (see below). The amide side chain of Asn93 is 4. 0 Å from the indole ring nitrogen of Trp100, and further hydrogen bonds to both side chain and main chain of Asn95, thus stabilizing the NDN loop. Therefore, the binding of the hexasaccharide to Trp100 could shift this loop, resulting in the observed backbone NMR chemical shifts [9]. We have previously determined that residues in PfAvr4 that are critical to binding (GlcNAc) 6 do not individually have an effect on PfAvr4’s interaction with Cf-4, as alanine substitution of these residues yields avirulent forms of the protein that elicit a Cf-4-mediated HR. Instead, a strong correlation between receptor activation and Avr4 stability was observed, as ChBD mutants that escape detection by Cf-4 are unstable proteins that are susceptible to proteolytic cleavage in the protease-rich environment of the leaf apoplast [6]. These observations prompt us to suggest that the ligand-binding function of Avr4 is structurally distinct or does not fully overlap with the property of recognition by Cf-4. This is important because it alluded that the molecular basis for the pleiotropic recognition of core effector proteins by single immune receptors is not based on the perception of individual amino acids that define the effectors intrinsic function, as previously hypothesized [4]. The elucidation of the precise structure and amino acid composition of CfAvr4’s ChBD enabled us to address this postulation with higher accuracy and reexamine whether recognition of Avr4 by Cf-4 is mediated through residues directly interacting with (GlcNAc) 6. Therefore, we assessed the ability of Cf-4 to mount an HR upon perception of the WT-CfAvr4 and the ChBD mutants (S2 Table). We also assessed mutants N93A, D94A, N95A, as the NDN motif indirectly affects the protein’s affinity for (GlcNAc) 6. The HR-inducing properties of the effector variants was initially examined by infiltrations of the purified proteins into tomato leaves of cv Purdue 135 (+ Cf-4) and cv Moneymaker (–Cf-4) at concentrations of 5 μg/ml and 10 μg/ml (Fig 4A, S9A Fig and S2 Table). When infiltrated into the leaves of cv Purdue, the WT-CfAvr4 and mutants M51A, P53A, Y67F, K84A, P87A, N95A, W100A and Y103F, all elicited a strong and equal in intensity HR at 5 days post-infiltration (dpi). In contrast, mutants Q69N and D102A elicited an HR at infiltrations with 10 μg/ml but only a weak response at 5 μg/ml, whereas mutants N93A and D94A failed to elicit an HR at both concentrations tested (Fig 4A, S9A Fig and S2 Table). As expected, none of the mutants or the WT-CfAvr4 induced an HR when infiltrated into leaves of cv Moneymaker (S9A Fig). Collectively, these results suggest that residues Gln69, Asn93, Asp94 and Asp102 could be direct targets of recognition by Cf-4 or, alternatively, that they are critical to protein stability and resistance of the protein against proteolytic degradation in the leaf apoplast. To discriminate between these two possibilities, we next transiently co-expressed Cf-4 with WT-CfAvr4 or its individual mutant alleles into leaves of Nicotiana benthamiana, using an Agrobacterium tumefaciens-mediated transformation assay (ATTA) [20]. Cf-4: effector co-infiltrations at cell densities of A6000. 5: A6001. 0 (0. 5: 1 ratio), A6000. 5: A6000. 5 (1: 1 ratio), and A6000. 5: A6000. 25 (2: 1 ratio) all induced an HR in the infiltrated leaf sectors thus resolving that none of our mutants can effectively escape recognition by Cf-4 when present in sufficient amounts in the leaf apoplast (Fig 4B, S9B Fig and S2 Table). We next compared the resilience of the WT-CfAvr4 and of the Q69N, N93A, D94A and D102A mutants against proteolytic degradation by subtilisin. We additionally included in these assays mutants K84A and W100A as, although they trigger a full and equal in intensity HR as the WT-CfAvr4 (S9 Fig), they nonetheless exhibit less affinity for (GlcNAc) 6 (S6 Fig and S2 Table). In all cases, treatment with subtilisin resulted in rapid cleavage reducing the full-length protein to the true mature form of CfAvr4 [3,6] (Fig 4C and S10 Fig). The protease assay showed that with the exception of WT-CfAvr4 and the K84A and W100A mutants (S10 Fig and S2 Table), all other mutants are susceptible to further proteolytic degradation, with mutants N93A and D94A being more so than mutants Q69N and D102A, evidenced by the almost complete disappearance of the band corresponding to the mature CfAvr4 for the N93A and D94A mutants and the decreased intensity of this band for the Q69N and D102A mutants, as compared to the WT-CfAvr4 (Fig 4C). The vulnerability of the mutants to proteolytic degradation is inversely correlated with their ability to elicit a Cf-4 mediated HR (Fig 4B and S2 Table), thus indicating that residues Gln69, Asn93, Asp94 and Asp102 make important contributions to protein stability but they likely do not mediate a direct interaction with Cf-4. Conversely the W100A and K84A mutants, which also show reduced affinity for (GlcNAc) 6 (S6 Fig and S2 Table) but elicited a full HR in tomato and N. benthamiana (S9 Fig), are as resilient to proteolysis as the WT-CfAvr4, evidenced by the equal in intensity band corresponding to the mature CfAvr4 obtained in the subtilisin assay for the three proteins (S10 Fig and S2 Table). Taken together, these results indicate that mutations in residues within the ChBD that decrease or abolish the protein’s affinity for (GlcNAc) 6 do not individually affect recognition by Cf-4 if they do not perturb the stability of the protein. During the past two decades, a great deal of effort has been placed towards deciphering the molecular determinants that define the structural basis of protein–carbohydrate interactions. Such interactions are ubiquitous in nature and at the heart of diverse biological processes of profound importance to human health, plant growth, and microbial disease [8]. Although CBMs may interact with their oligosaccharide in various ways, generally they do not undergo conformational changes when binding to their ligands. Instead the tertiary structure provides a platform for substrate-binding. Binding-site topography is thus key to their binding mode and can be very diverse, ranging from planar surfaces with aromatic residues that stack against the pyranose rings of polysaccharides (Type A CBMs), to grooves or clefts that contain both aromatic and hydrogen-bonding interactions that accommodate long polysaccharide chains (Type B CBMs), and small pockets that bind short oligosaccharide ligands (Type C CBMs) [8]. A recent refinement of these classes further proposed that Type B CBMs bind glycan chains internally (endo-type), whereas Type C modules interact with short mono-, di-, tri-saccharides or the termini of glycans (exo-type) [21]. To date, only limited information exists on how CBM14 family members bind chito-oligomers, although it is assumed that they would exhibit the structural and functional characteristics of Type C lectins [7,8]. The assumption mostly stems from indirect evidence and observations that representative members of this family interact with small oligosaccharide ligands, yet the structural basis of this interaction was so far unknown. The structural characterization of CfAvr4 and of its mode of interaction with (GlcNAc) 6 now provides experimental support that Avr4 may be unique in the CBM14 family in that it has an extended ChBD and can be classified rather as a Type B CBM instead of Type C. This is supported by the fact that Avr4 binds longer polysaccharide chains, whereas its mode of interaction with the substrate is mediated through aromatic residues (i. e. Trp100) and numerous hydrogen bonds with both side chains and main chains. Furthermore, unlike the other structurally-characterized CBM14 family members, CfAvr4 has an additional N-terminal α-helix and an extended loop connecting the α-helix to the first β-strand. The main chain of this loop hydrogen-bonds to (GlcNAc) 6 (Cys50 to GlcNAc-2) and contains the Cys50-Cys56 disulfide bond along with residues Met51 and Pro53 that stack against the pyranose rings of GlcNAc-1 and GlcNAc-3, respectively. Notably, although mutating each side chain individually only slightly reduces the affinity for (GlcNAc) 6, the overall additive effect would be increased. These interactions, along with residues Gln69 and Lys84 that greatly affect binding, are not conserved in tachycitin, Der p 23, and ChBDCHIT1, which are thus likely to have a smaller ChBD that is more similar to the hevein fold and to bind shorter oligosaccharides in par with other Type C CBMs. Next to Avr4, the only information available on the ligand-binding mechanism of a CBM14 family member with a known structure is the ligand-free structure of ChBD of the human chitotriosidase CHIT1 (ChBDCHIT1) [12,13]. CfAvr4 and ChBDCHIT1 share a similar overall fold but the residues that dictate the binding affinity of the two proteins for their ligand are different. For instance, the residues in CfAvr4 essential for binding (GlcNAc) 6, Trp100 and Asp102, are replaced by cysteine (Cys462) and threonine (Thr464) in the ChBDCHIT1 structure (S1D Fig). In ChBDCHIT1, residues Pro451, Leu454, and Trp465 that were identified as crucial for chitin binding [12] are conserved in CfAvr4, aligning to Pro87, Leu90, and Tyr103, respectively. However, although Pro87 and Leu90 sit on the loop connecting the two β-sheets and point toward the ligand binding site suggesting that they will have a role in binding (GlcNAc) 6, the P87A mutation does not have an effect on the binding thermodynamics of CfAvr4, whereas Leu90 is not within binding distance to (GlcNAc) 6, as the closest contact is 3. 3Å between the terminal methyl of the sidechain of chain A and the C6 hydroxyl of GlcNAc-4 of chain B. Interestingly, ChBDCHIT1 binding assays showed that there was no thermodynamic effect on binding when increasing the degree of polymerization beyond the disaccharide of GlcNAc, whereas, Avr4 showed a greatly decreased Kd and more negative ΔH upon an increase to the degree of polymerization of the oligosaccharide. This may indicate a very different binding mechanism between the two ChBDs of the same CBM14 family, which have a conserved fold, or the difference may highlight the vastly different nature of the proteins. CHIT1 is a chitinase with distinct catalytic and ChBD domains and has been implicated in the immune response in humans, whereas Avr4 is a fungal lectin protecting the fungus from plant chitinases. The Avr4 protein most likely needs to bind its ligand with a higher affinity than CHIT1. The structure of the CfAvr4- (GlcNAc) 6 complex further showed that two molecules of CfAvr4 can be joined through the ligand to form a sandwich structure that laminates two (GlcNAc) 6 molecules within the dimeric assembly. This mechanism of ligand-mediated dimerization, to some extent, appears to be unique among carbohydrate-binding proteins, as in most cases dimeric binding involves two molecules of a CBM interacting with the same molecule of the ligand [22–25]. Although it is conceivable that such a dimeric assembly of Avr4 could create a sheltered environment for (GlcNAc) 6, what cannot be determined is whether it is biologically relevant in terms of how the protein interacts with the network of chitin microfibrils present in the fungal cell wall that more accurately represents the biological substrate of Avr4 under in vivo conditions. The cell wall of fungi consists mainly of β-1,3- and β-1,6 glucans cross-linked to randomly oriented microfibrils of chitin, mannans, and glycoproteins that collectively form a three-dimensional layered structure in which glucans and chitin are most frequently positioned closer to the plasma membrane and are overlaid by mannans and glycoproteins [26,27]. Chitin microfibrils in the fungal cell wall are mainly found in the form of randomly oriented short microcrystalline rodlets and to a lesser extent as a network of longer interlaced microfibrils [28,29]. Such an arrangement of a tightly knitted network of chitin would seem to preclude the dimer formation that is seen in the crystal structure. Since, Avr4 binds (GlcNAc) 6 in a 1: 1 stoichiometric ratio, it is plausible that the protein rests as a monomer on the solvent-exposed surface of these microfibrils, thus creating a protective layer against endochitinases. If the case, it suggests that Avr4 may interact differently with free in solution chito-oligosaccharides as compared with chitin fixed in microfibrils. Conditional dimer formation has also been observed in the wheat germ agglutinin (WGA), a chitin binding-lectin with a hevein-like fold which, depending on the solution conditions, forms weak non-obligate and transient homodimers. Specifically, it is shown that although dimerization enables WGA to maximize its ligand binding affinity, the monomers exhibit significant binding affinity as well, thus making the formation of the dimers less mandatory for the function of the protein [30]. Another possibility is that binding of the Avr4 monomer to the chitin aggregate may lift a chitin chain from a microfibril, thus loosening the structure and enabling another monomer to grab hold of the opposite strand, thereby capping the ends of it that would otherwise be accessible to exochitinases. This mode of interaction is somewhat analogous to the model proposed for the enzymatic decrystallization of cellulose by celluloses, in which case the CBM appended to the catalytic domain functions as a wedge that lifts cellulose chains from the cellulose network [31,32]. It should be noted that, depending on the orientation and hydrogen-bonding pattern of its GlcNAc chains, crystalline chitin assembles in nature into mainly three allomorphic forms known as α-, β- or γ-chitin. α-chitin forms antiparallel chains of GlcNAc and is most commonly found in crustaceans, insects, and the cell walls of fungi [33,34], whereas β-chitin forms parallel chains of GlcNAc and is found mainly in diatoms and cephalopods [35–37]. Lastly, γ-chitin forms a mixture of parallel and antiparallel chains of GlcNAc and is found mainly in cocoon fibers of the Ptinus beetle and the stomach of the Loligo squid [38,39]. Despite the differential orientation of the GlcNAc chains in the three crystalline structures of chitin, they all share a similar stacking pattern of these chains, which is similar to the oligosaccharide stacking observed in the CfAvr4- (GlcNAc) 6 complex (S11 Fig). This suggests that Avr4 is likely able to bind all three forms of crystalline chitin, which might explain the broad distribution of CBM14 proteins across nearly all domains of life and their involvement in various biological processes [7]. Our previous work on PfAvr4 led us to hypothesize that the ligand-binding function of Avr4 is structurally distinct or does not fully overlap with the property of recognition by Cf-4 [6]. Instead, a strong correlation between receptor activation and Avr4 stability was observed, as ChBD mutants that evade recognition by Cf-4 embody unstable proteins that are susceptible to proteolytic cleavage in the protease-rich environment of the leaf apoplast [6]. The structural determination of the CfAvr4- (GlcNAc) 6 complex and the elucidation of the precise topography and amino acid composition of CfAvr4’s ChBD enabled us to readdress this postulation now with accuracy and examine whether individual residues that directly interact with (GlcNAc) 6 are targets for recognition by Cf-4. Our results corroborated the previous findings with PfAvr4, as site-directed mutagenesis of residues in CfAvr4’s ChBD yielded effector mutants that are able to trigger a Cf-4 mediated HR when present at sufficient amounts in the leaf apoplast. The studies further highlighted the dependency of Cf-4-mediated HR on CfAvr4’s stability and resistance against proteolysis in the leaf apoplast, as an inverse correlation exists between the intensity of the HR induced by the mutants when infiltrated into tomato leaves of cv. Purdue 135 (+Cf-4) and their susceptibility to subtilisin. These results are also in agreement with early studies showing that race 4 field isolates of C. fulvum produce unstable and protease sensitive isoforms of CfAvr4 as a means of evading recognition by Cf-4 [3,15]. Collectively, these studies emphasize the importance for recognition by Cf-4 of a stable tertiary structure of Avr4 and challenge early postulations that the broad recognition of Avr4 effectors by Cf-4 stems from perceiving residues implicated in binding (GlcNAc) 6. Instead, we hypothesize that immune receptors like Cf-4 could have exploited, during evolution, the need of apoplastic effectors to adopt a well-ordered stable structure in order to withstand proteolytic attack during infections in leaf apoplast, thus perceiving globular fold properties of Avr4. However, it was previously shown that Pro87, a conserved residue among Avr4 effectors, is essential for the Cf-4-mediated HR, as mutating this amino acid to an arginine resulted in a loss of recognition [40]. Our studies show that Pro87 is in close proximity to the ligand and mutating it to alanine results in minimal effect in ligand binding and no effect on Cf-4 recognition, thus corroborating previous finding with PfAvr4 [6]. This suggests that it is likely not the proline that is essential but a small aliphatic residue that is required in this position in order to ensure the structural integrity of the protein. Chitin hexasaccharide was purchased from Megazyme, Inc (Dublin, Ireland). Concentration of WT Avr4 was determined using the theoretical ϵ280 (Avr4_WT) = 17460 M-1 cm-1, which was similar to the calculated ϵ280 determined from a Bradford Assay. Mutants of Avr4 that included aromatic residues had extinction coefficients different from the WT protein and those values, and that of the WT protein, were determined using the ExPASy server [18]. A stock solution of the monomer of N-acetylglucosamine was prepared by dissolving the dry powder in a buffer containing 10 mM Bis-Tris, pH 6. 5,100 mM NaCl to a concentration of 0. 100 g/L using analytic techniques. Standard solution of 0,0. 010,0. 025,0. 050,0. 075 g/L were prepared by serial dilutions. Using glass test tubes and pipets, 100 μL of each standard was mixed with 300μL of concentrated sulfuric acid and vortexed for 10 sec to mix. Solutions were incubated at RT for 10 min and then placed on ice to stop the reaction. Solutions had a maximum absorbance at 322 nm and the concentration curve was determined using the absorbance of each standard at this wavelength. Analytically prepared solution of the di- and tri-saccharide scaled linearly to the calibration curve. To determine the concentration of the chitin hexasaccharide solutions used in ITC, the solutions were diluted into the dynamic range of the calibration curve (S12 Fig) and subjected to the above procedure. The part of the CfAvr4 gene that encodes for the true mature form of the protein (i. e. Lys30-Gln115) [3] was cloned into a modified pCDG-duet-1 vector containing two 6x His tags and a rhinovirus 3C protease cleavage site immediately N-terminal of the MCS1 using the SalI and NotI restriction sites. The vector was transformed into Escherichia coli Rosetta-gami B cells (Novagen) to facilitate formation of disulfide bonds. For expression, 1L cultures were grown at 37°C until OD600 = 0. 5–0. 7 and then cooled to 15°C. Cultures were induced with IPTG at a concentration of 1mM and allowed to express for 18–24 hours. For purification, cells were resuspended in buffer containing 50mM Tris: HCl, pH 8. 0,300mM NaCl, and 5mM imidazole. Cells were lysed using a microfluidizer and the lysate was cleared by centrifugation (39,000xg for 45 min). Cleared lysate was loaded on a 1mL HiFliQ-NiNTA (Anatrace). The column was washed with 10 column volumes (CV) of lysis buffer containing 30mM imidazole followed by a gradient wash, which increased the imidazole concentration to 60mM over 20CV, then washed with an additional 10CV of buffer containing 60mM imidazole. Protein was eluted from the column using lysis buffer containing 300mM imidazole. Collected protein fractions were concentrated to 1/5th original volume, then diluted back to original volume using lysis buffer to reduce imidazole concentration. Protein concentration was checked using A280 and a theoretical ϵ280 = 17460M-1cm-1 The purification tag was removed by adding His-tagged Rhinovirus 3C protease in a 50: 1 Avr4-to-protease ratio and 10μM of BME. The cleavage reaction was conducted at 4°C with stirring for 15 hours. The cleaved tag and protease were removed by flowing cleavage product back over the Ni-NTA column. The collected flow-through was checked for purity on SDS gel. CfAvr4 proved to be unamenable to dialysis. Buffer exchange was conducted via Amicon Ultra-15 Centrifugal Filters (Millipore, 3000 NMWL). The recovered protein was concentrated down to <3mL and diluted to 14mL using the crystallization buffer, 10mM Bis-Tris, pH 6. 5,100mM NaCl. This concentration and dilution was repeated twice more before the final concentration step for full buffer exchange. E. coli-produced CfAvr4 was concentrated to 100mg/mL and mixed with chitin hexasaccharide to reach a final concentration of 80mg/mL and 8mM carbohydrate, a 1: 1 stoichiometric ratio. The protein and ligand were co-crystallized by sitting-drop vapor diffusion in 0. 1M Tris: HCl, pH 8. 5,30% (w/v) PEG-4000,0. 8M LiCl at 4°C. Crystals were harvested and briefly soaked in reservoir buffer supplemented with 30% ethylene glycol prior to flash-cooling in liquid nitrogen. X-ray diffraction data were collected at ALS beamline 8. 3. 1. Crystals belong to space group P21 with unit cell parameters of: a = 39. 83 Å, b = 41. 08 Å, c = 121. 30 Å, β = 97. 871°. A Matthews coefficient [41] was calculated to be 2. 14 Å3/Da (solvent content = 42. 5%) assuming four monomers per asymmetric unit. Phases were determined by molecular replacement using PfAvr4 (PDB: 4Z4A) as a search model. The resulting structure was refined to a resolution of 1. 95Å in the space group P21. The structure was refined to a Rfactor = 16. 70% and an Rfree = 21. 45% using Phenix Refine [42]. Data collection and refinement statistics are shown in S1 Table. The refinement restraints for the hexasaccharide of GlcNAc were generated using Phenix eLBOW [43]. Atomic coordinates along with structure factors have been deposited in the Protein Data Bank, PDB ID: 6BN0. Primers for the site-directed mutagenesis were designed using the Agilent QuikChange Primer Design tool. Primers were purchased from Integrated DNA Technologies (IDT) and mutagenesis reactions were performed using Accuzyme PCR mix. Reaction products were purified and transformed into BL21 (DE3) cells. Colonies were selected and a Miniprep was performed to amplify the DNA. Collected DNA was sequenced to confirm proper, in-frame mutation (QuintaraBio). Some reactions repeatedly resulted in primer duplications and further experiments were performed using the half-reaction procedure to prevent the duplications. Sequence-confirmed mutations were transformed into the Rosetta-gami B cells for expression. All ITC experiments were performed on a TA Instruments low volume NanoITC with a 186uL reaction cell and a reference cell filled with degassed Milli-Q grade water. Reactions were run in a buffer containing 10mM Bis-Tris, pH 6. 5,100 mM NaCl. The reservoir solution was filled with protein solution and continuously stirred while Chitin hexasaccharide solutions titrated in 2. 5μL aliquots for 19 injections, with an initial 1μL injection, for a total injection volume of 48. 5μL. The carbohydrate solutions were made by dissolving the dry powder in buffer to a concentration of 20mM (confirmed using methods described above) and then diluted to the working concentration using the final buffer exchange flow through to match the buffers as closely as possible. The integrated heats, after correction for the heat of dilution were analyzed using the NanoAnalyze software from ITC Technologies. An individual binding site model was used to fit the binding isotherm. All experiments were replicated a minimum of three times. The ability of the WT-CfAvr4 and of ChBD mutants to protect against chitinases was evaluated in in vitro protection assays conducted as described before [6]. Briefly, spores of T. viride (3∙103) were pre-germinated overnight at room temperature in 100 μl of half-strength PDB medium in 96 well plates. The following day, 5 μM of WT-CfAvr4 or ChBD mutants were mixed with 7. 5 units of Zymolyase (Zymo Research cat. no. E1004) and 0. 2 units of bacterial chitinases (Sigma-Aldrich cat. no. C6137) and the mixture was added to the microtitre plate wells containing the pre-germinated spores in a final volume of 150 μl. Plates were incubated at 25°C for 6–8 h before evaluating under the microscope fungal growth and the ability of the proteins to provide protection against chitinases. Zymolase has β-1,3 glucanase and β-1,3-glucan laminaripentaohydrolase activity and is added to the mixture in order to remove the surface glucans from the fungal cell wall and thus expose the underlying chitin layer. Tomato plants of cv Moneymaker (MM), which does not carry Cf-4 or any other functional Cf resistance genes against C. fulvum, and of cv Purdue 135, which expresses a functional Cf-4 resistance gene, were grown in a growth chamber with 16 h of artificial light and 70% humidity at 27°C for 6 weeks. Tomato seeds were obtained from the Tomato Genetic Resource Center (TGRC) at UC Davis. WT-CfAvr4 and mutants thereof with single point mutations at selected amino acids were produced and purified from E. coli Rosetta-gami B cells as description above. Proteins were infiltrated at a concentration of 5 μg/mL and 10 μg/mL into the back side of MM or Purdue 135 tomato leaves using a one mL syringe and the HR response was recorded at 6 days post-infiltrations. The Agrobacterium tumefaciens transient transformation assay (ATTA) was used for the transient co-expression into leaves of Nicotiana benthamiana of Cf-4 with the WT-CfAvr4 and the mutants thereof tested in this study [6,44]. Briefly, N. benthamiana plants were grown in a growth chamber with 16 h of artificial light and 70% humidity, at 25°C for 4 weeks. The true mature form of the WT-CfAvr4 (i. e. between Lys30-Gln115) [3] and of the mutants thereof were singly cloned into the binary expression vector pICH47742 downstream of the PR1A signal sequence of Nicotiana tabacum for targeted secretion into the apoplast, and under the control of CaMV 35S promoter and NOS terminator. The tomato Cf-4 was cloned into pMOG800 as descripted before [20]. Binary vector plasmids were transformed into A. tumefaciens strain GV3101 by electroporation. For the ATTA assay, agrobacteria transformed with pMOG800: Cf-4 was grown in 10mL LB-mannitol medium (LB medium with 10 g/L mannitol) amended with 50 μg/mL kanamycin and 25 μg/mL rifampicin, whereas agrobacteria transformed with pICH47742: WT-CfAvr4 or pICH4774: CfAvr4-mutants were grown in LB-mannitol amended with 100 μg/mL carbenicillin and 25 μg/mL rifampicin. All cultures were incubated at 28°C for 2 days, after which period they were pelleted at 2800g for 15 min, re-suspended in MMAi medium (5 g/L Murashige and Skoog basal salts, 20 g/L sucrose, 10 mM MES, and 200 mM acetosyringone) at an optical cell density (A600) of 2. 0, and further incubated for 2–4 h at room temperature. Agroinfiltrations were performed by mixing agrobacteria containing pMOG800: Cf-4 with agrobacteria containing pICH47742_WT-CfAvr4 or pICH47742: CfAvr4-mutants at three different ratios of optical cell densities, i. e. A6000. 5: A6001. 0, A6000. 5: A6000. 5, and A6000. 5: A6000. 25 of. The mixture of agrobacteria was infiltrated into the leaves of N. benthamiana plants using a 1 mL syringe and the induction of an HR in the infiltrated leaf sectors was evaluated at 5 days post-infiltrations. Control infiltrations were made using only the pMOG800: Cf-4 transformed agrobacteria and the agrobacteria containing pICH47742: CfAvr4 or the mutants thereof without mixing the two cultures, at cell densities of A6000. 25, A6000. 5, and A6001. 0. The WT-CfAvr4 and selected mutants thereof were exposed to the non-specific protease subtilisin in order to determine their resistance against proteolytic degradation. Treatments with subtilisin were made as previously described with minor modifications [6]. Briefly, 40 μM of the E. coli-produced CfAvr4 and the selected CfAvr4 mutants were mixed with 10 mM of calcium chloride and 500 ng/μL of subtilisin (Sigma-Aldrich) in a total volume of 15 μL. Samples were incubated for 30 min at room temperature, after which period they were denatured by mixing with 10 mM PMSF and SDS-PAGE loading buffer, and heating at 98°C for 10 min. Digestion products were visualized on an SDS-PAGE after staining with Coomassie blue. The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www. wwpdb. org (PDB ID code 6BN0).

Title: Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)6 reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein Summary: Microbes mobilize an array of secreted effectors to manipulate their hosts during infections, whereas in response, hosts utilize cognate immune receptors to perceive effectors and mount a defense. To date, the structural basis of effector function and recognition by immune receptors are still poorly understood. Here we present the crystal structure in complex with chitohexaose of CfAvr4, a CBM14 lectin and the founding member of a fungal effector family that binds and protects chitin in fungal cell-walls from chitinases. This is the first structure of a CBM14 protein to be co-crystalized with its ligand that further reveals how Avr4 effectors function. Specifically, by leveraging structural and functional data, we elucidate the molecular basis for ligand-binding by CfAvr4 and show that two effector molecules are brought together through the ligand to form a sandwich structure that laminates two chitohexaose molecules within the dimeric assembly. We further show that recognition of CfAvr4 by the cognate Cf-4 immune receptor is not mediated through residues directly interacting with chitohexaose, thereby structurally uncoupling the ligand-binding function of Avr4 from recognition by Cf-4 and challenging early postulations that the broad recognition of Avr4 effectors by Cf-4 stems from perceiving residues implicated in binding their ligand.

lay_plos

en

Summarize: High-flying tax lawter Cynthia Gibson Beerbower, 65, was found by her housekeeper at her apartment in Fulham, west London, in July this year. A high-flying tax lawyer who worked in the White House was found dead in the bath of her London home after taking an overdose while going through a 'painful' divorce. American-born Cynthia Gibson Beerbower, 65, was found by her housekeeper at her multi-million-pound apartment in Fulham, west London, on July 26 this year. The 'highly intelligent' former Wall Street lawyer had left three notes in her spare bedroom alongside a piece of card which read 'I can't do this anymore', an inquest has heard. The hearing was also told how the note included a request for Mrs Beerbower be buried in Cambridge, where she attended university. Mrs Beerbower had apparently been offered several ambassadorial roles by President Barack Obama in the weeks before her death and was planning a future with her new fiancé. But the hearing at Westminster Coroner's Court heard that Mrs Beerbower was struggling to cope with an ongoing divorce battle and feared her family 'wished her dead'. Her estranged husband also described how Mrs Beerbower had a history of mental illness and had threatened suicide several times. During the hearing, Coroner Fiona Wilcox told the court that a note left by the lawyer said: 'I can't do this any more, I hate you, I am sick'. The court also heard evidence from medical professionals who said Mrs Beerbower had started taking medication that she had not been prescribed. During the hearing, Dr Marios Pierides, a psychiatrist at the Nightingale Hospital in Marylebone, said Mrs Beerbower had visited him before her death. He described how she felt 'lonely' and 'isolated' while going through the process of divorcing her husband John Beerbower. He told the court: 'Cynthia was on various drugs at different time. 'When she came to me she was being treated with various medication, she was on anti-depressants and sleeping pills. 'She explained to me she was going through a very painful period of divorce. Also she was feeling quite lonely and isolated in her residence in Fulham. Dr Pierides added: 'She would say the family wished her dead. But she was, from my point of view, a life affirming person who enjoyed things until the awful circumstances around the divorce happened.' He continued: 'At the same time she was angry and lonely at her circumstances.' The court heard Mrs Beerbower had started taking Phentermine, an appetite suppressor, as well as Diazepam. Neither had been prescribed by the psychiatrist. Dr Pierides told the hearing that the drugs have the capability to 'alter' a person's mental state, adding: 'It could promote psychosis or mood swings. 'It can alter your mental state and you might not know what you're doing.' The American-born Cambridge graduate (left) had been offered several US ambassadorial roles by Barack Obama in the weeks before her death and was planning a future with her new partner, David Shaw (right) Mrs Beerbower's estranged husband, whom she married in 1971, told the court that Mrs Beerbower had threatened suicide in the past. He also said she had previously 'over-medicated' and had mental health problems dating back to the 1970s. Mr Beebower told the inquest: 'I think there were some issues that go all the way back to the middle of the 70s. It was between two and three decades that she had been seeing people.'He said that she became suicidal on occasions, adding: 'She would become very upset or angry and would make these kinds of statements and, for example, lock herself in the bathroom. 'As far as I'm aware she never undertook any action on any of these threats.' The inquest heard Mrs Beerbower was struggling to cope with her divorce from husband John and feared her family 'wished her dead'. Pictured: Mr Beerbower and the couple's daughter Sarah outside Westminster Coroner's Court. Despite her divorce not being finalised, Mrs Beerbower become engaged to David Shaw in May this year, the inquest heard. Mr Shaw described her as a 'highly intelligent women' and said they had started to make plans for the future. He said: 'We were planning for the next 20 years. We were planning about what countries we wanted to go, because she was being offered ambassadorial roles by President Obama.' Mrs Beerbower - one of the few female partners in a Wall Street law firm - had joined the first Clinton administration in 1993. She served as International Tax Counsel under Secretary Lloyd Bentsen before becoming Deputy Assistant Secretary for Tax Policy under Secretary Robert Rubin. She left US Government service in late 1996. Mrs Beerbower retired from the financial business in 2005 and moved to France with her daughter Sarah. Mrs Beerbower was found dead by her housekeeper at her apartment in Fulham, west London (pictured) She and her husband acquired an olive farm in Chateauneuf de Grasse, in the south of France in 2006. From 2012 onward, Cynthia divided her time between homes in Toronto, New York City, the South of France and London, the inquest heard. Home Office pathologist Dr Jon Van Der Walt gave the cause of death as Oxidon, Phetermine and paracetemol toxicity. He said the drugs could have been enough to create a loss of consciousness, or fatal arrhythmia. The coroner said she could not give a verdict of suicide because the drugs Mrs Beerbower was taking could have affected her decision-making. Dr Wilcox said: 'At the time of death she was under the influence of Phentermine - it may well have affected her ability to have clear intentions, or even think clearly in any sensible way.' She instead recorded a narrative verdict, adding: 'She died as a result of poly-drug overdose while under the influence of Phentermine.' For confidential support call the Samaritans in the UK on 08457 90 90 90, visit a local Samaritans branch or click here for details

Summary: Cynthia Beerbower, 65, was found dead at her home in Fulham on July 26. She was offered US ambassadorial roles by Barack Obama before death. But she feared her family 'wished her dead' due to ongoing divorce battle. Husband John Beerbower told hearing she had history of mental illness. Coroner gave narrative verdict, saying suicide could not be proved. Mrs Beerbower joined Clinton administration in 1993 and also worked on Wall Street during high-flying career.

cnn_dailymail

en

Summarize: CROSS REFERENCE TO RELATED APPLICATIONS The present application is a U.S. National Stage under 35 USC 371 filing of International Application Number PCT/US2010/01000, entitled Articulating Laryngoscope filed on Apr. 2, 2010, which is a Nonprovisional Application of U.S. Provisional Application Ser. No. 61/166,037, entitled “FLOWERING LARYNGOSCOPE” filed on Apr. 2, 2009, which are both incorporated herein by reference. FIELD OF THE INVENTION The present invention is related generally to the field of laryngoscopes. BACKGROUND OF THE INVENTION The purpose of the laryngoscope is to aid in intubation. During the intubation process, a laryngoscope is used to open the airways and provide enough light to enable the user to pass an endotracheal tube through the vocal cords, securing the airway so as to provide ventilation to the lungs. Orotracheal intubation by direct laryngoscopy is the method of airway management in critically ill and injured patients, as well as patients undergoing all types of surgery in which general anesthesia is used. Intubation is performed by anesthesiologists, nurse anesthetists, emergency medicine and critical care physicians, dentists and maxillofacial surgeons, veterinarians, and in the out-of-hospital setting by paramedics. Orotracheal intubation is performed many thousands of times daily in the US, and millions of times daily worldwide in operating rooms, emergency departments, intensive care units, and every ambulance in the world. SUMMARY OF THE INVENTION According to the invention, there is provided an articulating laryngoscope, as defined in claims 1 - 37. For a better understanding of the present invention, together with other and further objects thereof, reference is made to the accompanying drawings and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS The present invention is illustratively shown and described in reference to the accompanying drawings, in which: FIGS. 1 and 2 illustrate the side and front views, respectively, of one embodiment of articulating laryngoscope 1 of the present invention in the closed position; FIGS. 3 and 4 illustrate the side and front views, respectively, of one embodiment of articulating laryngoscope 1 of the present invention in the deployed or open position; FIG. 5A is a perspective view of another embodiment of the present invention illustrating extension of the finger members; FIG. 5B is a perspective view of the embodiment of FIG. 5A illustrating relative angular positions of finger members in the extended (open) position; FIG. 6 is a perspective view of the embodiment in FIG. 5A in the closed position; FIG. 7 is a block diagram schematic illustrating exemplary elements of the present invention; and FIGS. 8A-C are illustrations of a protective sleeve; FIGS. 9A-C are illustrations of modular finger holder embodiments; FIG. 10A-B are illustrations of the illumination system of one embodiment of the present invention; FIG. 11 is an illustration of flexion and extension of a finger member; and FIG. 12 is a front view of an individual finger member illustrating 360° rotational capability. DETAILED DESCRIPTION OF THE INVENTION As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about”, even if the term does not expressly appear. Also, any numerical range recited herein is intended to include all sub-ranges subsumed therein. Articulation is defined as a joint on a finger. Actuation is defined as the movement at or about one or more joints. Articulating laryngoscope 1 is designed to be equipped with many functional features including but not limited to, mechanical opening of air passage for ease of intubation, illumination of an air passage during intubation and for examination, delivery of gases and liquids, including medications; suction for removal of fluids including blood and mucus; cauterization to stop bleeding; removal of foreign objects and biopsy specimens; real-time video during intubation to guidance of articulating laryngoscope 1 ; and video recording and camera still images for evaluation and teaching; and surgical instruments including, but not limited to, scalpel, staple and suture. FIGS. 1 and 2 illustrate the side and front views, respectively, of one embodiment of articulating laryngoscope 1 having six (6) fingers or projections or phalanges 2 (terms are interchangeable) arranged in a generally U or C shape configuration 11 in the closed position. The U or C shape configuration 11 provides vision for the user of the articulating laryngoscope 1 to observe the uvula, palatine tonsils, oropharynx, esophagus, larynx, and trachea as articulating laryngoscope 1 is guided into position. Therefore, open side 12 of U or C shape configuration 11 is defined as being facing up in an opposing direction relative to handle 13. Handle 13 can contain PLC 22, trigger or controller 14, AC/DC (battery) power source 29, and interfaces/ports/connections for suction source 27, oxygen source 28, and other fluid delivery input 35 (See FIG. 7 ). Handle 13 operably connects and communicates with fingers 2 at interface 36 by mechanical and electrical means known to one of skilled in the art FIGS. 3, 4, 5 A, 5 B, and 11 illustrate the structure of articulating laryngoscope 1 that provide the function of member actuation to open air passageways. Fingers 2 have joints 3 that couple together a plurality of segments 5 such that at least segments 5 of fingers 2 can articulate, which means movement upward, downward, or in a circular or elliptical path along or about a central axis C. FIG. 5A illustrates segments 5 can have different lengths 6, and thickness or diameters 7. Each segment 5 can be tapered 8 with thickness or diameter decreasing as adjacent segments 5 are attached at joints 3 from proximal end 4 and distal end 9. A plurality of segments 5 form member 19. Joints 3 provide functionality for manipulation of members 19 in many directions when actuated by a trigger 14 or other control mechanism. FIGS. 3 and 4 illustrate side and front views, respectively, of articulating laryngoscope 1 deployed or open position where members 19 extend or flex toward, for example, tissue to open an airway of the oropharynx. FIGS. 5A-B illustrate another actuation of fingers 2 in a “flowering” arrangement where all fingers 2 are extending outwardly away from each other for the maximum opening. Fingers 2 can be manipulated to bring finger ends 15 of members 19 in contact therewith to grab or pinch an object for extraction. With regards to the actuation mechanism, FIG. 6 is an illustration of a closed laryngoscope with one or more channels that can contain embedded wires 21 for controlled finger actuation, fiber optics or light emitting diodes (LED) 22 for illumination or video, and tubes 23 for suction and fluid delivery, including oxygen and medication. Now turning to FIGS. 1 and 7, one embodiment of the actuation mechanism includes a handle 13 with an interface coupler 42 to operably connect handle 13 with fingers 2. Fingers 2 will interlock with handle 13 in such a way as to make physical and electrical communication with AC/DC (battery) power source 28 and PLC 22, both of which are contained in handle 13, and the moveable components in fingers 2. For instance, the actuation may be driven by manual operator energy, for example squeezing trigger 14 mechanical links to finger 2 resulting in the displacement of a physical conducting element such as a metal wire 21. At the interlocking point 36 on handle 13, wire 21 can be coupled to a flexible transducing element 40 in finger 2 or finger tip 15. In one embodiment of flexible transducing element 40 can be spring hinges 41 at joints 3. Displacement of wire 21 in handle 13 will thus result in commensurate displacement of the flexible transducing element 40 in finger 2, resulting in flexion and extension of finger 2 about a central axis C ( FIGS. 5B and 11 ). Each finger 2 has its own central axis C as illustrated in FIGS. 1, 3, 5 B, and 11. Axis C can be linear or non-linear. Flexion is defined is an angular inward movement (interior surface side) φ 1, φ 2, φ 3 (where φ 3 =φ 2 −φ 1 ), etc. of each finger segment 5 from central axis C, about 0° up to about 180°, and any angle therebetween. Extension is defined as an angular outward movement (exterior surface side) θ 1, θ 2, θ 3 (where θ 3 =θ 2 −θ 1 ), etc. of each segment 5 from central axis C, about 0° up to about −180°, and any angle therebetween. Turning now to FIG. 11 for a detailed discussion of actuation. The actuation of the individual fingers 2 can be controlled in such a way as to facilitate flexion (solid image of individual finger 2 ) and extension (dotted line image of individual finger 2 ) in two directions about a central axis C. Though the following disclosure illustrates bi-directional linear motion (up and down), it is within the contemplation of this invention that individual finger 2 can also rotate 360° about axis C ( FIG. 12 ). The path can be circular 52 or elliptical 53 as shown in FIG. 11. This action may be accomplished by integrating two separate conducting element (such as a metal wire 21 ) into finger 2 and handle 13, one which transduces input energy to force for actuation, and the other which places restrictions or enables actuation in one direction about the axis while creating the opposite condition for the opposite direction. For instance, activation of the directional limiter (not shown) may slide two subsurface metal restrictors (not shown) into place and out of place, respectively, on opposite sides of a joint 5, facilitating movement toward the side without a restrictor plate, and inhibiting movement toward the side with a restrictor plate. Conduction of actuation energy will then result in movement in the former direction. As discussed above, members 19 include an embedded actuation mechanism 20. As shown in FIG. 6, one embodiment of actuation mechanism 20 includes a pair of wires 21 extending from proximal end 4 to finger tip 15 at distal end 9. Both wires 21 are connected to trigger 14 to pull one of the wires to control actuation of a particular member 19. Another embodiment of actuation mechanism 20 can be gears (not shown) in to joints 3 linked by rotating rods connected to a motor in handle 13. Yet another embodiment of actuation mechanism 20 can be motors at joints 3 responsive to an electronic stimulus or a signal. Trigger 14 can be connected to actuation mechanism mechanically or electrically, such as by a programmable logic controller (PLC) 22 or controller with logic to determine which wire of the pair of wires to pull to actuate a member 19. One or more strain gauge 23 disposed along the length of members 19 can be used to regulate pressure of members 19 against tissue. The pressure can be monitored by the user on display 30 ( FIG. 7 ) for manual termination of the member actuation when a pressure limit is reached or the termination can be automated with an automatic shutoff when a pressure limit is reached. One embodiment of the present invention can lock finger 2 positions by locking of the trigger at a set displacement. Alternatively, the source of actuation may be electrical energy derived from a DC (battery) power source 29 or AC common line power source, in which case interface coupler 42 at the interface 36 on handle 13 will bring physical contact between conductive electrical wiring (not shown) in the handle and the designated in finger 2, continuing through each finger segment 5 to a successive series of motors, in series or in parallel within each finger 2. Now turning to FIGS. 10A-B illustrating one embodiment of the illumination function of the present invention. Joints 3 can include an opening 10 for light illumination when member 19 is flexed or extended or closed. FIG. 5A illustrates another embodiment for light illumination that includes finger material being made of translucent or clear substrate containing subsurface light sources 16 (fiber optics or LEDs) below the external surface 17 of the material to protect the light source 16 from contamination or prevent malfunctioning of joint 3 due to blockage caused by foreign objects. FIG. 10B illustrates illumination of the oropharynx to view the trachea and esophagus for intubation with the light illumination system of the present invention. Joints 3 can also use opening 10 for suction or other fluid delivery when member 19 is flexed or extended ( FIG. 10A ) or closed. Openings 10 can be disposed along segments 5 in any location, such as midway between joints 3. Finger 2 can include a fluid delivery outlet 25 at finger end 15 capable of delivering forced air or other medical gases (including oxygen), liquids and medicines ( FIG. 2 ) as well as suction. A balloon (not shown) can be fluidly connected to end 15 of finger 2 by fluid delivery line 25 for inflation to open up the passage way or to close off a passage way. Now returning to FIG. 5A illustrate another embodiment of the present invention including webbing 33 between members 19 to function as a barrier to hold back tissues such as the tongue, fluids (such as blood, saliva, mucus), food particles, or other foreign objects that obscure the vision of the user and block the passageway. Webbing 33 can be disposed between one pair of member 19 (as shown in FIG. 5A ) or between all members 19. FIG. 7 illustrates exemplary functions and component connectivity of articulating laryngoscope 1. PLC 22 can control illumination of lights on/off/brightness/direction adjustment; suction on/off/pressure; automatic member actuation shutdown when members 19 exceeds a range of motion limit or when a strain gauge 23 embedded in finger tip 15 exceeds pressure limit; optical focus and field of view; camera on/off/video/still images/run time shutoff/routing of optical signal to display 30 /lens directional adjustment; scalpel actuation measured by depth of cut into tissue and stroke length of cut; oxygen flow rate and mixture; suction flow rate; grasping control logic 32 that accounts for strain gauge 23 readings to adjust actuation of members 19 to assure grasping pressure is not beyond crush limits to avoid destruction or disintegration of object within the patient. Other embodiments of articulating laryngoscope 1 can also include the following features: A. grasping control 32 can include member 19 pinching function to stop, for example, bleeding; B. member 19 can include an endotracheal tube mounted on it; C. member 19 can include a cauterizer mounted on end 15 to stop bleeding; D. member 19 can be constructed in multiple sizes for infants, toddlers, teenager, adults, and animals. Size can also be adapted for dental use, vaginal examinations and procedures, and other cavity examinations and procedures. E. member 19 can include a biopsy needle 51. Fingers 2 can be a monolithic structure formed from a single injection mold. Finger base structure 18 can be rigid at proximal end 4 over segment length 6, where there is no relative movement between members 19. The remaining portion of members 19 can be independently operable and moveable relative to adjacent members 19. Another embodiment of fingers 2 can be a plurality of assembled components wherein a plurality of members 19 are mechanically connected at joints 3 by any conventional means such as ball and socket, hinges, or straps. Materials for fingers 2 and members 19 can include plastic, carbon fiber, polymers, any semi-rigid material, or combination thereof. Material can have antibiotic and healing properties. Members 19 are sufficiently malleable to be self contouring to tissue during actuation that will distribute the force or pressure substantially evenly to prevent point contact for a prolonged period to minimize tissue damage. Materials for handle 13 can include stainless steel, aluminum, plastic, carbon fiber, rubber, polymers, any semi-rigid material, or combination thereof. Now turning to FIGS. 8A-C, disposable sleeve 34 can be slipped over member 19 without webbing to function as a protective covering for reusable fingers 2 to either minimize or eliminate the need for sterilization. One embodiment of sleeve 43 can be used for a single member 19 ( FIG. 8B ). Another embodiment of sleeve 44 can be used for an entire finger 2 assembly ( FIG. 8C ) similar to a glove. Another embodiment of the present invention is modular and includes separate, removable fingers 2 that can be selected for its function (such as suction, fluid delivery, light optics, camera lens, and scalpel) and fitted within a holder for a plurality of fingers 2. Fingers 2 can be made of a low cost, sterile material for disposable purposes or the fingers can be made of materials designed for repeated use and sterilization between uses. Now turning to FIGS. 9A-C with illustrations of embodiments of the present invention with a finger base for modular configurations to hold individual members 2. FIG. 9A illustrates one embodiment of block holder 45 made of malleable material (for example plastic) for individual finger 2. Block holder 45 includes finger attachment devices 46 that can be a hole to receive individual member 2 therein, or a pin to receive individual member 2 thereon. Pin 46 can include a hole therethrough for suction or fluid delivery. Block 45 can directly connect with handle 13. FIGS. 9B-C illustrate holder embodiments 47, 49 being generally U or C shaped and made of malleable material (for example plastic) having hole 48 to receive individual member 2 therein. Holder 47 includes a block 50 that can used to secure holder 47 while inserting and removing individual fingers 2. Individual fingers 2 inserted into holder 47, 49 can directly connect to handle 13. While the disclosure has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the embodiments. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

Summary: Articulating laryngoscope to aid in the intubation of patients by providing illumination of the oral cavity and trachea during the process having, for example, 'fingers' with fiber optic lights at the ends and at joints of the fingers, fingers spread open or 'flower' when the device is deployed, gently retracting and compressing soft tissues in the oral cavity and providing medical professionals with much better illumination of the passageway they are addressing, constructed from a malleable material, including rubber, plastics/polymers, and carbon fiber, instead of hard metal. The fingers may have multiple light sources to ensure a flooding of the patient&#39;s oropharynx with light. Some versions might have fiber-optic cameras connected to one or more fingers for use in teaching and research, and one might have suction capability to facilitate removal of solids and fluids, one embodiment can have at least one finger with a scalpel at its distal end.

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