A dielectric structure including solid dielectric regions incorporating a plurality of regions of vacuum or gas is provided. The dielectric constant of the regions of solid dielectrics can have a dielectric constant greater than 4. Each of the plurality of regions of vacuum or gas or the regions of solid dielectrics may be anisotropic with an aspect ratio of at least four. The smallest average dimension of a plurality of regions of vacuum or gas and/or solid dielectrics can have a length of less than 1 micron. The dielectric structure may have a higher electrical energy density in the regions of vacuum or gas than in the solid matrix. One or more electrodes of the capacitive structure can be coated with a solid insulating layer without an interface between a region of vacuum or gas and electrode.

Dielectrics provide electrical insulation to prevent undesired current flow in electrical equipment, including capacitors, transmission lines, circuit substrates, semiconductor devices, mechanical supports, and other applications. Dielectrics are limited to operation at electric fields at or below their dielectric strength, the electric field level at which the dielectric fails to prevent current flow below an acceptable level. The maximum electric field at which a dielectric can reliably be operated is dependent on the applied voltage, the thickness of the dielectric, and the spatial relationship of conductors and dielectrics in the system, among other factors. Since the dielectric strength is limited by the material properties, manufacturing quality, and operating conditions, the thickness of the dielectric often must be increased to enable operation at higher voltage. As the thickness of the dielectric increases to support higher voltage operation, the size of the devices, such as capacitors and transmission lines, increases. In the case of capacitors, increasing the dielectric thickness reduces the capacitance density, defined as the capacitance per unit area, and thus requires a larger capacitor area to obtain an equivalent total capacitance as a capacitor incorporating a thinner dielectric. The additional material required to increase both the thickness and area of the dielectric causes the volume and weight of the dielectric and overall device to be much larger. Since many applications for capacitors and other electrical devices incorporating dielectrics would benefit from smaller and/or lighter components, materials capable of operating at higher electric field are needed to reduce the size and weight of the components incorporating the dielectrics. The electrical energy density, defined as the energy stored in a dielectric per unit volume or per unit mass, is commonly used as a metric to evaluate the size and/or weight of dielectrics based on their material properties. By increasing a dielectric's electrical energy density, components incorporating that dielectric can often be made smaller and lighter.
The electrical energy density, We [J/m3], can be calculated for a medium with a relative permittivity, which is commonly referred to as the dielectric constant, εr, in an electric field, E [V/m], by the following equation. The permittivity of free space, ε0 [F/m], is approximately 8.85×10−12 F/m.
      W    e    =            1      2                  ɛ      0                  ɛ      r                  E      2
The electrical energy density scales with the square of the electric field and linearly with the dielectric constant. Therefore, increasing the operating electric field, which is limited by the dielectric strength of the medium, and/or the dielectric constant, which is dependent on the dielectric medium and is often a function of the operating temperature, frequency, and electric field, will increase the electrical energy density. While many efforts to increase the electrical energy density have sought to increase both the dielectric constant and the dielectric strength, and thus operating electric field, increasing the dielectric strength of a dielectric medium to increase the operating electric field can provide much more significant gains in electrical energy density due to the squared term.
Electrostatic capacitors store energy in an electric field. The term electrostatic capacitor used here is intended to differentiate from electric double layer capacitors and should be understood to include devices used with an alternating current (AC) or transient signal as well as direct current (DC). While capacitors have conventionally used dielectrics to separate the positive and negative charges of a capacitor, electric double layer capacitors, which are often called supercapacitors or ultracapacitors, do not typically have a dielectric separating the charges. Rather, conducting electrolyte and electrodes are in direct contact, and charges are not transferred across the interface unless the applied voltage is above a threshold voltage of a few volts. Thus, while the capacitance density and energy density of electric double layer capacitors is typically greater than those of electrostatic capacitors, the voltage of electric double layer capacitors is limited. While electric double layer capacitors can be arranged in series to create assemblies with higher operating voltage, either the capacitance of each electric double layer capacitor in the series stack must be approximately equal to evenly distribute the voltage across each element of the assembly or additional voltage control hardware must be used to prevent any individual capacitor from operating at too high of voltage. Thus, electric double layer capacitors are not suitable for providing high energy density in many high voltage applications.
Capacitors capable of operating at higher voltages than electric double layer capacitors (i.e., electrostatic capacitors) typically have a much lower capacitance density and energy density than electric double layer capacitors. There is a need for improvements in the field of electrostatic capacitors to increase energy density and power density while maintaining or improving capacitance density, and thus improvements to the dielectrics in electrostatic capacitors are needed. There are several other important performance factors related to dielectrics for electrostatic capacitors and other applications that can also be improved with new dielectric materials. First, many dielectric applications require both the capability to withstand high and low temperature extremes, temperature cycling, and to have a small variation in dielectric properties with respect to temperature. Second, it is also often critical for there to be minimal variation of the dielectric properties with respect to the applied voltage/operating electric field in the dielectric or other operational or environmental factors. For example, to remain within specification for certain applications, capacitor dielectrics must not change more than a predetermined percentage from their standard value over the range of voltages, frequencies, and temperatures of use. Solid and liquid dielectrics can have large variation due to effects of voltage/electric field, frequency, and temperature due to the dependence of the dielectric constant on these parameters. Conventional dielectrics with high dielectric constants, such as ferroelectrics, often have large variance with environmental and operating variables. In the case of capacitors, stability of the capacitance and voltage rating with respect to temperature and applied voltage would greatly expand the applicability of capacitors with high energy density storage capabilities. Third, many AC and radio frequency (RF) applications require low dielectric losses to operate efficiently at high frequencies. Some capacitors and cables operate with low-loss dielectrics based on foams produced with low dielectric constant polymers (e.g. polyethylene or polypropylene with εr<4), or simply gas or vacuum as the electrical insulator. However, these low-loss dielectrics have poor dielectric strength due to their implementation, limiting their use in devices for high electric field operation for high energy and power density. Fourth, many applications require improved reliability, which typically requires derating of the operating conditions (e.g., operating electric field in the dielectric of a capacitor) from the maximum capability of the dielectric to enable a long operating lifetime. Dielectrics with a higher dielectric strength can be operated with a larger derating factor, thus increasing lifetime and reliability, while operating at the same electric fields of other dielectrics with lower derating factors. Fifth, to achieve operation at very high electric fields, film capacitors, which are commonly made with polymer film dielectrics, have been developed with the ability to be operational after one or more failure events within the dielectric. This capability, commonly referred to as self-clearing or self-healing, enables operation beyond the limitations of the weakest points of the dielectric, where a material defect or contamination would otherwise cause premature failure of the entire device. This self-clearing or self-healing property is enabled both by using very thin electrodes and the appropriate dielectric. The dielectric material is a critical factor in determining the self-clearing or self-healing capability, and some dielectrics have been found to be incompatible with the methods used for self-clearing. Due to the known shortcomings of dielectrics known in the art, a novel approach to dielectric materials is required to satisfy the many requirements on dielectrics over the range of operating conditions.
Materials for High Energy Density Capacitors—Conventional high energy density capacitors are based on solid dielectrics of polymers, ceramics, or composites of polymers and ceramics. In some cases, liquid dielectrics, such as insulating oils, are used in addition to a solid dielectric. Significant efforts have been made in recent decades to mature thin-film polymer manufacturing for high energy density capacitors. Biaxially oriented polypropylene (BOPP) is commercially available in films with thicknesses on the order of microns that can be operated at field strengths on the order of 300 MV/m. These thin-film polymers are routinely used in practice, and the energy density is particularly high when made with self-healing electrodes capable of isolating points of failure in the dielectric through the vaporization of the electrode around the failure point with the surge in current at the fault. These polymer-based thin films are limited in the range of operating temperature due to the properties of the polymer. In particular, the polymer will soften or otherwise degrade at high temperature such that the dielectric strength and energy density are reduced. The polymer properties are also dependent on the glass transition temperature, which can affect low temperature performance. Other acrylic-based polymers with higher operating temperature have been commercialized at thicknesses of less than 1 um. While the small thickness and different polymer chemistry are improvements from other polymer capacitor technologies, the technology has reached its limits with operation demonstrated at near 1,000 MV/m for sub-micron polymer thicknesses.
Other approaches to achieve a high capacitive energy density use high dielectric constant materials. Some polymers, such as poly vinylidene fluoride (PVDF) and other polymers with polar groups, can have dielectric constants greater than 10. However, the dielectric strength of these polar polymers is typically much lower than the previously discussed BOPP and acrylic-based thin-film polymers. The polar groups also cause significant losses under AC signals, which make them unsuitable for many applications. Ceramic materials with dielectric constants ranging up to several thousand have been used in capacitors. The ceramics with the highest dielectric constants are ferroelectrics such as barium titanate and strontium titanate. While these ceramics have many applications, their use in high energy density capacitors is limited due to ferroelectric saturation, which causes a large decrease in the dielectric constant with increased voltage. These perovskite ceramics also exhibit high dielectric losses at RF frequencies and can have significant variation of properties with respect to temperature. Other ceramics are applied in high energy density capacitors. Aluminum electrolytic capacitors operate the aluminum oxide dielectric layer at fields up to 800 MV/m. With a dielectric constant of approximately 10, a field strength of 800 MV/m corresponds to 28.3 J/cm3, though the operating field and energy density is typically lower. Tantalum capacitors, with a dielectric constant of 25, are also used in high energy density applications but typically at lower field strengths than aluminum oxide. While these ceramics are at the state of the art of commercially available capacitors, the opportunities for increases in energy density are limited as the ceramics are already operated near their theoretical breakdown limit (1.38 GV/m for trigonal aluminum oxide and 370 MV/m for cubic tantalum oxide). Another area of active work for high energy density capacitors is polymer-ceramic composites. These composites of ceramic particles in a polymer matrix typically exhibit a higher dielectric constant than the unloaded polymer and a higher dielectric strength than the ceramic of the same thickness of the composite. However, the dielectric strength is often determined by the dielectric strength of the polymer, so the polymer-ceramic composite approach has little or no advantage to polymer multi-layer capacitors in which the polymer layer is on the sub-micron scale as is done in acrylic-based polymer multi-layer capacitors.

Dielectric structures for electrical insulation with vacuum or gas


A contact pin - contact bushing unit comprises a contact pin (3), having two spring-mounted, parallel legs (6) bordering an elongated hole formed by punching. The legs are employed for insertion into the opening of a circuit board. The legs (6) terminate in a shoulder (7) which serves to demarcate the insertion path. The contact bushing is formed by a contact spring bushing (2) with several contact springs (8). The contact springs extend roughly from the area of a clamping shoulder (8), which determines the fixture point of one of their ends, at a distance from the bushing axis to the area of the front pin insertion hole (13). In an intermediate area of the contact bushing, the contact springs display the shortest distance from the bushing axis. The shoulder bordering the contact pin insertion path is formed by the indicated clamping shoulder (7) of the contact spring bushing (2).

The present invention relates to a contact pin contact bushing structural unit, in which the contact pin includes two approximately parallel legs for insertion into the hole of a circuit board. The legs border an elongated hale created by stamping, are spring-mounted, and end in a shoulder which serves to mark the boundary of the insertion path.
A contact pin--contact bushing structural unit of this type is known as disclosed in U.S. Pat No. 29,513. In this unit, both the contact pin and the contact bushing are formed from punched-out contact sheet metal. The contact bushing, which adjoins the shoulder terminating the insertion path of the contact pin, has the form of a tongue which displays a punched-out opening and which is bent backwards onto itself, along an axis that extends on a diagonal to the pin axis and that runs substantially through the area of the punched-out contact opening. The tongue has a certain elasticity which determines the contact force with which the tongue rests against a counter-contact pin introduced into the punched-out opening. When there are inconsistencies in the dimensions, which is ultimately unavoidable, there are relatively high contact resistances for small insertion forces, and vice versa. High insertion forces, which for their own part contribute to desirably small contact resistances, are impossible to achieve, however, whenever there are very many contact pin - contact bushing structural units of the described type fixed to a shared circuit board for the purpose of coupling the units with a counter-plug device comprising a number of counter-pins of the appropriate dimensions.

Process for making contact pin - contact bushing structural unit


Systems and methods to predict and present availability and value of retail spaces in shopping malls. Robots are dispatched to temporary retail spaces to collect at least a portion of operation data stored in a database with leasing data of temporary retail spaces in a mall, tenant coordination data, and subscriber data. A web-based application predicts availability of the temporary retail spaces in the mall based on the leasing data, the tenant coordination data, and the operation data. The web-based application includes an interactive plan of the mall as a user interface for subscribers to access data related to the temporary retail spaces, and process applications for leasing the temporary retail spaces.

U.S. Pat. App. Pub. No. 2009/0149992 discloses a robot capable of traveling within a predetermined pathway area.
U.S. Pat. App. Pub. No. 2010/0094463 discloses a robot having a position recognition section that recognizes a current position of the robot within a guide zone. The robot is controlled to move to each of guide locations in the guide zone. At each of the guide locations, the robot transmits contents information corresponding to the guide location to a mobile terminal held by a person to be guided near the robot.
U.S. Pat. No. 7,860,647 discloses a traffic condition prediction table used to predict vacancy of a parking lot as an accommodating facility for presentation on a navigation device.
U.S. Pat. App. Pub. No. 20140365251 discloses a vacancy rate calculation apparatus for tables in a restaurant.
The disclosures of the above discussed patent documents are hereby incorporated herein by reference.

This patent is subject to a terminal disclaimer.

Robotic systems and methods in prediction and presentation of resource availability


A system for publishing digital content is described which, in one embodiment, includes a beacon device associated with a first user configured to transmit identification information and information indicating its current position. A browsing device includes a display configured and adapted to display to a second user a representation of a local geographical area, and to selectively display to the second user content information associated with the first user, based at least in part on whether the location of the beacon device is within the local geographical area.

The last few years have seen an avalanche of content published via electronic media. Blogs make it very simple to voice one's opinion on virtually any subject. Publishers such as YouTube have amassed millions of clips made by amateurs, from teenagers singing a popular song, to serious pieces about politics and current events. Many people simply want to express themselves, and have their message reach out to the public. Accordingly, systems and methods which can enable users to locate people, goods, and services in their immediate surroundings are desirable.
The internet allows a user to connect with merchants and friends all around the world. It makes no difference where a person is located geographically. While this makes world wide advertising of goods and services easy, it makes it difficult to browse the immediate surroundings, or a predefined geographic area, for interesting information. The internet can help you find a specialized part dealer on the other side of the globe, but it is not very useful when you want to browse around for a doctor in a crowd of people. Similarly, cell phone technology allows users to make and receive calls all over the world, but does very little to aid users in locating people and information in a user's immediate surroundings. Mobile navigation systems are useful in providing directions, static information about particular locations (e.g. shops and service stations near your current location), and even providing dynamic information such as traffic status on major roadways. However, as of yet there has not yet been a system for individual users to publish or advertise their own content to a localized audience.
Currently, content published on the web is accessible to anyone across the world. There is little or no possibility to publish to a given local geographic area. To some degree, the Internet has become so large that it is now difficult to browse for things that are in your local area.


A coordinate measuring machine includes a probe having a measurement unit, a movement mechanism for moving the probe and a host computer for controlling the movement mechanism. The host computer includes: a compensation-amount calculating unit for calculating a compensation amount for compensating an error in the position of the measurement unit caused by the movement of the probe; and a compensator for compensating the error in the position of the measurement unit based on the compensation amount calculated by the compensation-amount calculating unit. The compensation-amount calculating unit calculates a translation-compensation amount for compensating a translation error of the probe and a rotation-compensation amount for compensating a rotation error of the probe based on a rotation frequency of the movement of the probe.

1. Field of the Invention
The present invention relates to a profile measuring instrument.
2. Description of Related Art
A known profile measuring instrument includes: a probe having a measurement unit for measuring an object; a movement mechanism for moving the probe; and a controller for controlling the movement mechanism, the measurement unit being moved along a surface of the object to measure a profile of the object (see, for instance, Patent Literature 1: JP-A-55-154408 and Patent Literature 2: JP-A-07-324928).
A coordinate measuring machine according to the Patent Literature 1 uses acceleration of respective driven components or acceleration obtained by a drive motor in order to compensate measurement errors caused by a deflection of a movement mechanism on account of inertial force during the scanning movement of the probe, i.e. dynamic deflection (dynamic Abbe's error) caused by a deformation of the movement mechanism.
The CMM (Coordinate Measuring Machine) disclosed in the Patent Literature 2 calculates a compensation value representing deflection characteristics of the movement mechanism as a function of the probe position and acceleration in order to compensate the aforementioned dynamic Abbe's error.
FIG. 12 is an illustration showing a model of Abbe's error to be compensated by a conventional profile measuring instrument.
Specifically, the CMM disclosed in the Patent Literature 2 models the Abbe's error as a spring SP connecting a probe position Ps (xs, ys, zs) and a compensated probe position Pc (xc, yc, zc) as shown in FIG. 12. A spring constant K of the spring SP represents the deflection characteristics of the movement mechanism, which are dependent on the probe position Ps.
Accordingly, for instance, a relationship between the probe position xs and the compensated probe position xc in X-axis direction can be expressed by the following formula (1) based on the spring constant K and a force F applied on the spring SRxc=xs−K·F  (1)
Further, since the force F is caused on account of the inertial force during the scanning movement of the probe, the force F is proportional to acceleration a of the probe. In other words, the relationship between the probe position xs and the compensated probe position xc can be expressed by the following formula (2), where Ka represents a proportion constant between the force F and the acceleration a of the probe.xc=xs−K·Ka·a  (2)
As described above, the CMM disclosed in the Patent Literature 2 can compensate the Abbe's error based on a function of the probe position Ps and the acceleration a.
However, since the CMM disclosed in the Patent Literature 2 compensates the Abbe's error based on the function of the probe position Ps and the acceleration a, the Abbe's error cannot be appropriately compensated when the model of the Abbe's error has to be expressed by a high-order transfer function.
Specifically, since the CMM disclosed in the Patent Literature 2 compensates the Abbe's error based on the function of the acceleration a, if one tries to transform the model of the Abbe's error into a function of, for instance, frequency (e.g. a function of rotation frequency of the scanning movement of the probe according to the present invention, which will be described below), only a second-order term of the function can be expressed. Further, the model of the Patent Literature 2 cannot be directly transformed into a transfer function. In the Patent Literature 2, instead of a given mass M, the force F is obtained by acceleration (movement condition) of the mass M to be used for compensation.


A belt detachment tool 10 includes a detacher 11 and a pulley engagement portion 12. The detacher 11 includes a belt-climbing surface 11a′, a belt-sliding surface 11b′, and a belt-transferring surface 11c′ disposed therebetween. At least a belt contact portion of the belt-transferring surface 11c′ has a radius of curvature of 5-11 mm in plan view.

The present invention relates to belt detachment tools used to detach a power transmission belt wound over pulleys.
Many automobiles and general industrial machinery employ a belt transmission apparatus that includes a power transmission belt wound over pulleys and serves as a power transmitter for transmitting power from a source of motion.
In assembling such a belt transmission apparatus, a power transmission belt that is wound over and attached on pulleys is sometimes temporarily detached from the pulleys, e.g., to adjust the tension. After the adjustment, the power transmission belt is attached to the pulleys again.
Japanese Unexamined Patent Publication (Japanese Translation of PCT Application) No. 2004-533579 describes a belt detachment tool for the above-described case. This belt detachment tool has a curved body including a pulley engagement surface and a belt engagement surface that constitute an edge of the belt detachment tool.


The invention provides a method of inhibiting a bacterial RNA polymerases. The invention has applications in control of bacterial RNA polymerase activity, control of bacterial gene expression, control of bacterial growth, antibacterial chemistry, and antibacterial therapy.

RNA Polymerase
Bacterial infections remain among the most common and deadly causes of human disease. Infectious diseases are the third leading cause of death in the United States and the leading cause of death worldwide (Binder et al. (1999) Science 284, 1311-1313). Multi-drug-resistant bacteria now cause infections that pose a grave and growing threat to public health. It has been shown that bacterial pathogens can acquire resistance to first-line and even second-line antibiotics (Stuart B. Levy, The Challenge of Antibiotic Resistance, in Scientific American, 46-53 (March, 1998); Walsh, C. (2000) Nature 406, 775-781; Schluger, N. (2000) Int. J. Tuberculosis Lung Disease 4, S71-S75; Raviglione et al., (2001) Ann. NY Acad. Sci. 953, 88-97). New approaches to drug development are necessary to combat the ever-increasing number of antibiotic-resistant pathogens.
RNA polymerase (RNAP) is the molecular machine responsible for transcription and is the target, directly or indirectly, of most regulation of gene expression (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Cramer, P. (2002) Curr. Opin. Structl. Biol. 12, 89-97; Murakami & Darst (2003) Curr. Opin. Structl. Biol. 13, 31-39; Borukhov & Nudler (2003) Curr. Opin. Microbiol. 6, 93-100; Landick, R. (2001) Cell 105, 567-570; Korzheva & Mustaev (2001) Curr. Opin. Microbiol. 4, 119-125; Armache, et al. (2005) Curr. Opin. Structl. Biol. 15, 197-203; Woychik & Hampsey (2002); Cell 108, 453-463; Asturias, F. (2004) Curr. Opin. Genet Dev. 14, 121-129; Cramer, P. (2004) Curr. Opin. Genet. Dev. 14, 218-226; Geiduschek & Kassayetis (2001) J. Mol. Biol. 310, 1-26). Bacterial RNAP core enzyme has a molecular mass of ˜380,000 Da and consists of one β′ subunit, one β subunit, two α subunits, and one ω subunit; bacterial RNAP holoenzyme has a molecular mass of ˜450,000 Da and consists of bacterial RNAP core enzyme in complex with the transcription initiation factor σ (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Cramer, P. (2002) Curr. Opin. Structl. Biol. 12, 89-97; Murakami & Darst (2003) Curr. Opin. Structl. Biol. 13, 31-39; Borukhov & Nudler (2003) Curr. Opin. Microbiol. 6, 93-100). Bacterial RNAP core subunit sequences from Gram-negative bacterial species, Gram-postive bacterial species, and Thermococcus-Deinococcus-clade bacterial species exhibit moderate amino acid sequence conservation (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Iyer, et al. (2004) Gene 335, 73-88). Eukaryotic RNAP I, RNAP II, and RNAP III contain counterparts of all bacterial RNAP core subunits, but eukaryotic-subunit sequences and bacterial-subunit sequences exhibit only limited amino acid sequence conservation (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Cramer, P. (2002) Curr. Opin. Structl. Biol. 12, 89-97).
Crystallographic structures have been determined for bacterial RNAP and eukaryotic RNAP II (Zhang et al., (1999) Cell 98, 811-824; Cramer et al., (2000) Science 288, 640-649; Naryshkin et al., (2000) Cell 101, 601-611; Kim et al., (2000) Science 288, 1418-1421; Korzheva et al., (2000) Science 289, 619-625; Ebright, R. (2000) J. Mol. Biol. 304, 687-689; Cramer et al., (2001) Science 292, 1863-1876; Gnatt et al., (2001) Science 292, 1876-1882; Mekler et al., (2002) Cell 108, 599-614; Murakami et al., (2002) Science 296, 1280-1284; Murakami et al., (2002) Science 296, 1285-1290; Vassylyev et al., (2002) Nature 417, 712-719; Bushnell et al., (2004) Science 303, 983-988; Westover et al., (2004) Science 303, 1014-1016; Armache, et al., (2003) Proc. Natl. Acad. Sci. USA 100, 6964-6968). Moreover, cryo-EM structures have been determined for bacterial RNAP and eukaryotic RNAP I (Opalka, et al. (2000) Proc. Natl. Acad. ScL USA 97, 617-622; Darst, et al. (2002) Proc. Natl. Acad. Sci. USA 99, 4296-4301; DeCarlo, et al. (2003) J. Mol. Biol. 329, 891-902).
Structures also have been determined for RNAP complexes with nucleic acids, nucleotides and inhibitors (Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363; Campbell, et al. (2005) EMBO J. 24, 674-682; Artsimovitch, et al. (2004) Cell 117, 299-310; Tuske, et al. (2005) Cell 122, 541-522; Temiaov, et al. (2005) Mol. Cell 19, 655-666; Vassulyev, et al. (2005) Nature Structl. Biol. 12, 1086-1093; Gnatt, et al. (2001) Science 292, 1876-1882; Westover, et al. (2004a) Science 303, 1014-1016; Westover, et al. (2004b) Cell 119, 481-489; Ketenberger, et al. (2004) Mol. Cell 16, 955-965; Bushnell, et al. (2002) Proc. Natl. Acad. Sci. USA. 99, 1218-1222; Kettenberger, et al. (2005) Natl. Structl. Mol. Biol. 13, 44-48).
The structures reveal that RNAP-bacterial or eukaryotic—has a shape reminiscent of a crab claw. The two "pincers" of the "claw" define the active-center cleft that can accommodate a double-stranded nucleic acid-and which has the active-center Mg2+ at its base. The largest subunit (β′ in bacterial RNAP) makes up one pincer, termed the "clamp," and part of the base of the active-center cleft. The second-largest subunit (β in bacterial RNAP) makes up the other pincer and part of the base of the active-center cleft.
Based on crystallographic structures for bacterial RNAP and eukaryotic RNAP biophysical results, and biochemical results, models have been proposed for the structures of transcription initiation and elongation complexes (Gnatt et al., (2001) Science 292, 1876-1882; Ebright, R. (2000) J. Mol. Biol. 304, 687-689; Naryshkin et al., (2000 Cell 101, 601-611; Kim et al., (2000) Science 288, 1418-1421; Korzheva et al., (2000) Science 289, 619-625; and Mekler et al., (2002) Cell 108:599-614).
Bacterial RNAP is a proven target for antibacterial therapy (Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) Trends Biochem. Sci. 29, 159-162). The suitability of bacterial RNAP as a target for antibacterial therapy follows from the fact that bacterial RNAP is an essential enzyme (permitting efficacy), the fact that bacterial RNAP subunit sequences are conserved (providing a basis for broad-spectrum activity), and the fact that bacterial RNAP subunit sequences are only weakly conserved in eukaryotic RNAP I, RNAP II, and RNAP III (providing a basis for therapeutic selectivity).
The rifamycin antibacterial agents—notably rifampicin, rifapentine, and rifabutin—function by binding to and inhibiting bacterial RNAP (Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105, 621-632; Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363). The rifamycins bind to a site on bacterial RNAP adjacent to the RNAP active center and sterically and/or allosterically prevent extension of RNA chains beyond a length of 2-3 nt (Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105, 621-632; Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363).
The rifamycins are in current clinical use in treatment of Gram-negative and 1.0 Gram-positive bacterial infections (Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105, 621-632; Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363). The rifamycins are of particular importance in treatment of tuberculosis; the rifamycins are first-line anti-tuberculosis agents and are the only anti-tuberculosis agents able rapidly to clear infection and prevent relapse (Mitchison, D. (2000) Int. J. Tuberc. Lung Dis. 4, 796-806). The rifamycins also are of importance in treatment of bacterial infections relevant to biowarfare or bioterrorism; combination therapy with ciprofloxacin, clindamycin, and rifampicin was successful in treatment of inhalational anthrax following the 2001 anthrax attacks (Mayer, et al. (2001) JAMA 286, 2549-2553), and combination therapy with ciprofloxacin and rifampicin, or doxycycline with rifampicin, is recommended for treatment of future cases of inhalational anthrax (Centers for Disease Control and Prevention (2001) JAMA 286, 2226-2232).
The clinical utility of the rifamycin antibacterial agents is threatened by the existence of bacterial strains resistant to known rifamycins (Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105, 621-632; Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363). Resistance to rifamycins typically involves substitution of residues in or immediately adjacent to the rifamycin binding site on bacterial RNAP—i.e., substitutions that directly decrease binding or function of rifamycins. A significant and increasing percentage of cases of tuberculosis are resistant to rifampicin (1.4% of new cases, 8.7% of previously treated cases, and 100% of cases designated multidrug-resistant, in 1999-2002; Schluger, N. (2000) Int. J. Tuberc. Lung Dis. 4, S71-S75; Raviglione, et al. (2001) Ann. N.Y. Acad. Sci. 953, 88-97; Zumia, et al. (2001) Lancet Infect. Dis. 1, 199-202; Dye, et al. (2002) J. Infect. Dis. 185, 1197-1202; WHO/IUATLD (2003) Anti-tuberculosis drug resistance in the world: third global report (WHO, Geneva)). Strains of bacterial bioweapons agents resistant to rifampicin can be, and have been, constructed (Lebedeva, et al. (1991) Antibiot. Khimioter. 36, 19-22; Pomerantsev, et al. (1993) Antibiot. Khimioter. 38, 34-38; Volger, et al. (2002) Antimicrob. Agents Chemother. 46, 511-513; Marianelli, et al. (204) J. Clin. Microbiol. 42, 5439-5443).
In view of the public-health threat posed by rifamycin-resistant bacterial infections, there is an urgent need for new antibacterial agents that (i) target bacterial RNAP (and thus have the same biochemical effects as rifamycins), but that (ii) target sites within bacterial RNAP distinct from the rifamycin binding site (and thus do not show cross-resistance with rifamycins). (See Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S (2004) Trends Biochem. Sci. 29, 159-162.)
RNA Polymerase Secondary Channel
Crystallographic structures have been determined for bacterial RNAP and eukaryotic RNAP II (Zhang et al., (1999) Cell 98, 811-824; Cramer et al., (2000) Science 288, 640-649; Cramer et al., (2001) Science 292, 1863-1876; Gnatt et al., (2001 Science 292, 1876-1882; and Ebright, R. (2000) J. Mol. Biol. 304, 687-689), and, based on the crystallographic structures, biophysical results, and biochemical results, models have been proposed for the structures of transcription initiation and elongation complexes (Gnatt et al., (2001 Science 292, 1876-1882; Ebright, R. (2000) J. Mol. Biol. 304, 687-689; Naryshkin et al., (2000 Cell 101, 601-611; Kim et al., (2000) Science 288, 1418-1421; Korzheva et al., (2000) Science 289, 619-625; and Mekler et al., (2002) Cell 108:599-614).
The models for the transcription elongation complex imply that nucleic acids completely fill the active-center cleft of RNAP, and thus the only route by which incoming nucleoside-triphosphate substrates ("NTPs") can access the active center is through a ˜25 Å long, ˜10 Å wide tunnel—the "secondary channel," "pore," "nucleotide-uptake channel," or "nucleotide-entrance channel,"—that bores through the floor of the active-center cleft of RNAP and extends to the exterior surface of RNAP opposite the active-center cleft (Gnatt et al., (2001) Science 292, 1876-1882; Ebright, R. (2000) J. Mol. Biol. 304, 687-689; and Korzheva et al., (2000) Science 289, 619-625).
The models for the transcription elongation complex imply that the RNAP secondary channel mediates multiple biochemical activities important for function of RNAP, including: uptake of NTPs, release of pyrophosphate product, release of abortive-RNA and edited-RNA products, interaction with RNA product during transcriptional pausing, interaction with RNA product during transcriptional arrest, interaction with RNA product during editing, and interaction with the elongation factors GreA and GreB.
Physically blocking the RNAP secondary channel with a small molecule can inhibit one or more of these activities and thereby can inhibit function of RNAP (WO 2004/023093; Mukhopadhyay et al. (2004) Mol. Cell 14, 739-751; Adelman et al. (2004) Mol. Cell 14, 753-762). For example, physically blocking the RNAP secondary channel with a small molecule can prevent uptake of NTPs by RNAP and thus can prevent transcription (WO 2004/023093; Mukhopadhyay et al. (2004) Mol. Cell 14, 739-751; Adelman et al. (2004) Mol. Cell 14, 753-762).
A region within the RNAP secondary channel—a region that comprises amino acids 736-747 and 779-781 of the RNAP β′ subunit in RNAP from Escherichia coli—is a useful target for compounds that inhibit transcription, including, by way of example, the lariat peptide microcin J25 (MccJ25) and derivatives thereof (WO 2004/023093; FIG. 1). This region includes residues that are conserved in RNAP from bacterial species, but that are not conserved, and indeed are radically different, in RNAP from eukaryotic species (FIG. 1). This region forms a ˜5 Å shallow pocket within the wall of the RNAP secondary channel (FIG. 2). A compound that binds to this region of a bacterial RNAP can physically block the secondary channel of a bacterial RNAP, can prevent uptake of NTPs by a bacterial RNAP, can inhibit bacterial transcription, can inhibit bacterial gene expression, and can inhibit bacterial growth.
The secondary-channel region referred to above in RNAP from Escherichia coli is similar in amino acid sequence in RNAP from other species of bacteria and is called herein the "bacterial RNAP homologous secondary channel amino acid sequence" (FIG. 1). (For example, amino acid residues 736-747 and 779-781 of the β′ subunit of RNAP from Escherichia coli are similar to amino acid residues 740-751 and 783-785 of the β′ subunit of RNAP from Bacillus subtilis (FIG. 1). Thus, a molecule that binds to this region and inhibits an activity associated with the secondary channel in RNAP from Escherichia coli also may bind to this region and inhibit an activity associated with the secondary channel in RNAP from other species of bacteria. Therefore, molecules found to have antibacterial activity (through binding to this region and inhibiting an activity associated with the secondary channel) against Escherichia coli also may be found to have antibacterial activity against other species of bacteria.
In contrast, the secondary-channel region referred to above differs radically in amino acid sequence between bacterial RNAP and eukaryotic RNAP, including human RNAP I, human RNAP II, and human RNAP III (FIG. 1). This allows for identification of molecules that bind, in a secondary-channel-region-dependent fashion, to a bacterial RNAP, but that do not bind, or that bind substantially less well, to a eukaryotic RNAP. This also allows for identification of molecules that inhibit, in a secondary-channel region-dependent fashion, an activity of a bacterial RNAP, but that do not inhibit, or that inhibit substantially less well, an activity of a eukaryotic RNAP. This differentiation is important, because it permits the identification of bacterial-RNAP-selective binding molecules and bacteria-selective inhibitors.
In the three-dimensional structure of a bacterial RNAP, the secondary-channel region referred to above is located near to, but does not overlap, the rifamycin binding site (FIG. 3). The distance between the center of the secondary-channel region referred to above and the center of the rifamycin binding pocket is ˜25 Å.
The invention provides, by way of example only, a bacterial RNAP homologous secondary channel amino acid sequence corresponding to, and alignable with, residues 736-747 and 779-781 of the β′ subunit of RNAP from Escherichia coli, as well as with corresponding residues of the β′ subunit of Bacillus subtilis, Haemophilus influenzae, Vibrio cholerae, Pseudomonas aeruginosa, Treponema pallidum, Borrelia burgdorferi, Xyella fastidiosa, Campylobacter jejuni, Neisseria meningitidis, Rickettsia prowazekii, Thermotoga maritima, Chlamydia trachomatis, Mycoplasma pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis, Synechocystis sp., Aquifex aeolicus, Deinococcus radiodurans, Thermus thermophilus, and Thermus aquaticus.
Ligands that bind to the secondary channel of, and inhibit an activity of, a bacterial RNAP are known in the art. Such ligands include, for example, the lariat-peptide microcin J25 (MccJ25)(WO 2004/023093; Mukhopadhyay et al. (2004) Mol. Cell 14, 739-751; Adelman, et al. (2004) Mol. Cell 14, 753-762; Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483), substituted derivatives of the lariat-peptide MccJ25 (WO 2004/023093; U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006; Mukhopadhyay, et al. (2004) Mol. Cell 14, 739-751), and minimized derivatives of lariat-peptide MccJ25 (WO2005/024040; Semenova, et al. (2005) J. Bacteria 187:3859-3863). The references cited above are incorporated herein in their entirety.
Lariat-Peptide MccJ25
The antibacterial peptide microcin J25 (MccJ25) inhibits bacterial RNAP by binding within, and obstructing, the secondary channel of bacterial RNAP (WO 2004/023093; Mukhopadhyay et al. (2004) Mol. Cell 14, 739-751; Adelman, et al. (2004) Mol. Cell 14, 753-762). MccJ25 has the sequence (Sequence ID NO. 1): Gly1-Gly2-Ala3-Gly4-His5-Pro6-Val7-Glu8-Tyr9-Phe10-Val11-Gly12-Ile13-Gly14-Thr15-Pro16-Ile17-Ser18-Phe19-Tyr20-Gly21 cyclic(8→1) peptide (Bayro, et at. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483; FIG. 4, line 1).
MccJ25 has an unusual "lariat-peptide" covalent structure (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483; FIG. 4, line 1). MccJ25 is 21 amino acids in length and consists of an 8-residue cyclic segment—with a backbone-sidechain amide linkage between the backbone nitrogen atom of residue Gly1 and the side-chain carboxyl group of Glu8—followed by a 13-residue linear segment.
MccJ25 further has an unusual "lariat-protoknot" three-dimensional structure (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483; FIG. 5). In the three-dimensional structure of MccJ25, the linear segment of the lariat loops back and penetrates and threads through the cycle of the lariat, essentially as a thread through the eye of a needle. The linear segment is irreversibly locked in place and trapped within the cycle by steric constraints imposed by the aromatic sidechains of Phe19 and Tyr20, which bracket the cycle, with the aromatic sidechain of Phe19 being on located on one face of the cycle and the aromatic sidechain of Tyr20 being located on the other face of the cycle.
The lariat-peptide/lariat-protoknot structure of MccJ25, with irreversible trapping of the linear segment of the lariat within the cycle of the lariat, results in exceptional resistance to denaturation (complete resistance to tested thermal and chemical denaturants; Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483) and exceptional resistance to proteolysis (complete resistance to tested mesophilic endo- and exopeptidease; Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483).
The lariat-peptide/lariat-protoknot structure of MccJ25 is generated by a MccJ25-specific biosynthetic system. MccJ25 is produced by bacterial strains harboring a plasmid-borne lariat-peptide/lariat-protoknot biosynthetic cassette, consisting of a gene for MccJ25 precursor, two genes for factors that process McJ25 precursor into mature MccJ25, and one gene for a factor that exports MccJ25 from the cell (Solbiati, et al. (1999) J. Bacteriol. 181, 2659-2662). The gene names and functions are as follows:
mcjA; encodes the MccJ25 precursor (McjA)
mcjB; encodes a MccJ25 processing factor (McjB)
mcjB; encodes a MccJ25 processing factor (McjC)
mcjD; encodes a MccJ25 export factor (McjD)
The MccJ25 precursor is a 58-residue peptide consisting of a 37-residue N-terminal pro-sequence (residues numbered as −37 to −1) and a 21-residue C-terminal segment having the same amino acid sequence as mature MccJ25 (residues numbered as 1 to 21) (Solbiati, et al. (1999) J. Bacteriol. 181, 2659-2662).
Processing of the MccJ25 precursor to yield mature MccJ25 entails two reactions: (1) cleavage of the backbone-backbone amide linkage between residue −1 and residue 1, resulting in removal of the 37-residue N-terminal pro-sequence; and (2) formation of a backbone-sidechain amide linkage between the backbone nitrogen atom of residue 1 and the sidechain carboxyl of residue 8, resulting in cyclization of residues 1-8 and entrapment of residues 9-21 (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483). The MccJ25 processing factor McjC exhibits amino acid sequence similarity to amidotransferases of the Asn-synthase/Gln-hydrolase class, which catalyze transfer of ammonia or an amine from an amide donor to a carboxyl acceptor (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383). It has been proposed that McjC participates in both reactions in processing of the MccJ25 precursor to yield mature MccJ25, acting on pre-folded MccJ25 precursor to catalyze transfer of the backbone nitrogen atom, also known as the α-amino group, of residue 1 from the backbone amide linkage between residue −1 and residue 1 to the sidechain carboxyl of residue 8 (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383).
It has not been possible to date to re-create the lariat-peptide/lariat-protoknot structure of MccJ25 without use of the above-described MccJ25-specific biosynthetic system. Attempted chemical synthesis of MccJ25 yields a product having a lariat-peptide covalent structure but not having a lariat-protoknot three-dimensional structure (i.e., not having the linear segment of the lariat looped back and penetrating and threading through the cyclic segment of the lariat; the resulting material exhibits no detectable biological activity (Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483). Chemical synthesis of a linear analog of MccJ25 yields a product having neither a lariat-peptide covalent structure nor a lariat-protoknot structure; the resulting material exhibits no detectable biological activity (Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483). Production of a recombinant linear analog of MccJ25, by use of a nucleic acid sequence encoding the linear analog of MccJ25 likewise yields a product having neither a lariat-peptide covalent structure nor a lariat-protoknot structure and exhibiting no detectable biological activity.
The MccJ25 lariat-peptide/lariat-protoknot biosynthetic cassette is organized as a gene cluster, with gene order mcjA, mcjB, mcjC, mcjD (Solbiati, et al. (1996) J. Bacteriol. 178, 3661-3663; Solbiati, et al. (1999) J. Bacteriol. 181, 2659-2662). The size of the MccJ25 lariat-peptide/lariat-protoknot biosynthetic cassette is ˜4,500 bp.
The MccJ25 lariat-peptide/lariat-protoknot biosynthetic cassette can be expressed in an original, naturally occurring, Mcc25-producer strain, resulting in MccJ25 production (Solbiati, et al. (1996) J. Bacteriol. 178, 3661-3663; Solbiati, et al. (1999) J. Bacteriol. 181, 2659-2662).
The MccJ25 lariat-peptide/lariat-protoknot biosynthetic cassette also can be introduced into, and expressed in, a surrogate host strain, resulting in surrogate-host MccJ25 production (Solbiati, et al. (1999) J. Bacteriol. 181, 2659-2662; Blond, et al. (1999) Eur. J. Biochem. 259, 747-755).
Because the nucleotide sequence of the mcjA gene determines the amino acid sequence of the MccJ25 precursor, and because, in MccJ25 biosynthesis, the MccJ25 precursor is processed to yield mature MccJ25, a change in the nucleotide sequence of the mcjA gene ("mutation") can result in a corresponding change in the amino acid sequence of MccJ25 ("substitution"). The relationship between a mutation in the mcjA gene and a corresponding substitution in MccJ25 is determined by, and is predictable from, the universal genetic code.
Introduction of a mutation into the mcjA gene can be accomplished in straightforward fashion by use of molecular-biology and directed-evolution procedures known in the art, including, for example, random mutagenesis, site-directed mutagenesis, and gene synthesis (WO 2004/023093; U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006; Mukhopadhyay, et al. (2004) Mol. Cell 14, 739-751; see also Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
Accordingly, mcjA derivatives containing mutations, and corresponding MccJ25 derivatives containing substitutions, can be, and have been, prepared (WO 2004/023093; U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006; Mukhopadhyay, et al. (2004) Mol. Cell 14, 739-751). This approach is limited to production of MccJ25 derivatives containing substitutions at a subset of residue positions, since other residue positions cannot be substituted without disruption of processing or export (U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006). Nevertheless, this approach provides a source of MccJ25 derivatives having different useful properties, including, for example, high affinity for a target of interest, high potency for inhibition of a reaction of interest, or suitability for site-specific incorporation of a detectable group such as a fluorochrome (WO 2004/023093; U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006; Mukhopadhyay, et al. (2004) Mol. Cell 14, 739-751).
In the same manner, "libraries" of mcjA derivatives containing single mutations or small numbers of mutations, and corresponding "libraries" of MccJ25 derivatives containing single substitutions or small numbers of substitutions, can be, and have been, prepared (U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006). This approach is limited to production of libraries of MccJ25 derivatives containing substitutions at a subset of residue positions, since other residue positions cannot be substituted without disruption of processing or export (U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006). Nevertheless, this approach, optionally combined with screening of "libraries" by use of procedures known in the art, provides a further source of MccJ25 derivatives with different useful properties, including, for example, high potency for inhibition of a reaction of interest, or suitability for site-specific incorporation of a detectable group such as a fluorochrome (U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006).
MccJ25 inhibits Gram-negative bacterial RNAP by binding within, and obstructing, the secondary channel of Gram-negative bacterial RNAP (WO 2004/023093; Mukhopadhyay et al. (2004) Mol. Cell 14, 739-751; Adelman, et al. (2004) Mol. Cell 14, 753-762). Through inhibition of Gram-negative bacterial RNAP, MccJ25 exhibits antibacterial activity against certain Gram-negative bacterial species, including the Gram-negative enterics Escherchia coli and Salmonella sp. (Delgado, et al. (2001) 183, 4543-4550; Yuzenkova, et al. (2002) 277, 50867-50875). MccJ25 does not inhibit Gram-positive bacterial RNAP or Thermococcus-Deinococcus bacterial RNAP, and, accordingly, MccJ25 not inhibit Gram-positive bacterial RNAP or Thermococcus-Deinococcus bacterial growth (Yuzenkova, et al. (2002) 277, 50867-50875).
The binding site for MccJ25 within bacterial RNAP is remote from the binding site for rifamycins and from the sites of substitutions that confer resistance to rifamycins (WO 2004/023093; Mukhopadhyay et al. (2004) Mol. Cell 14, 739-751; Adelman, et al. (2004) Mol. Cell 14, 753-762). Accordingly, MccJ25 exhibits no cross-resistance with rifamycins and retains full ability to inhibit RNAP derivatives resistant to rifamycins.
MccJ25, as a direct consequence of its lariat-peptide/lariat-protoknot structure, exhibits two features useful for drug design and drug discovery:                (1) MccJ25 is genetically encoded (through genetic encoding of a precursor and processing factors; Solbiati, et al. (1996) J. Bacteriol. 178, 3661-3663; Solbiati, et al. (1999) J. Bacteriol. 181, 2659-2662), permitting efficient production by fermentation (Blond, et al. (1999) Eur. J. Biochem. 259, 747-755) and permitting efficient construction of derivatives by molecular-biology or directed-evolution procedures (WO 2004/023093; U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006; Mukhopadhyay, et al. (2004) Mol. Cell 14, 739-751).        (2) MccJ25 is resistant to denaturation and proteolysis (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483).
This combination of features is not only useful, but also unusual. Most compounds do not exhibit this combination of features. (Most peptides and proteins exhibit only the first feature. Most non-peptide, non-protein compounds exhibit only the second feature.)
Non-MccJ25-Related Lariat Peptides
We refer herein to compounds according to general structural formula (I) as "non-MccJ25-related lariat peptides."
wherein:                (i) X is an amino acid residue containing a backbone nitrogen atom;        (ii) Y is an amino acid residue containing a side-chain carboxyl group;        (iii) α is a peptide segment of from about 5 to about 8 amino acid residues;        (iv) β is a peptide segment of from about 6 to about 15 amino acid residues;        (v) there is an amide bond between the backbone nitrogen atom of X and the side-chain carboxyl of Y; and        (vi) wherein the compound has less than 25% amino acid sequence identity with microcin J25 (MccJ25).
Non-MccJ25-related lariat peptides are known in the art and include the siamycins [siamycin I (also known as MS-271, NP-06, and FR901724; Yano, et al. (1996) Bioorg. Med. Chem 4, 115-120; Katahira, et al. (1996) Bioorg. Med. Chem 4, 121-129; Chokekijchai, et al. (1995) Antimicrob. Agents Chemother. 39, 2345-2347; Nakashima, et al. (1996) Biol. Pharm. Bull. 19, 405-412; Detlefsen, et al. (1995) J. Antibiot. 48, 1515-1517), siamycin II (Detlefsen, et al. (1995) J. Antibiot. 48, 1515-1517; Constantine, et al. (1995) J. Biomol. NMR 5, 271-286), and siamycin III (also known as RP 71955 and aborycin; Helynck, et al. (1993) J. Antibiot. 46, 1756-1757; Frechet, et al. (1994) Biochem. 33, 42-50; Potterat, et al. (1994) Liebigs Annalen der Chemie 7, 741-743)], RES-701-n [RES-701-1 (Yamasaki, et al. (1994) J. Antibiot. 47, 276-280; Katahira, et al. (1995) Bioorg. Med. Chem. 3, 1273-1280), RES-701-2 (Yano, K. et al. (1995) J. Antibiot. 48, 1368-1370; Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220), RES-701-3 (Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220), and RES-701-4 (Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220)], propeptin (Kimura, et al. (1997) J. Antibiot. 50, 373-378), anantin (Wyss, et al. (1991) J. Antibiot. 44, 172-180), and lariatins [lariatin A and lariatin B (Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491)] (FIG. 4).
Known non-MccJ25-related lariat peptides have lariat-peptide covalent structures similar to the lariat-peptide covalent structure of MccJ25 (Yano, et al. (1996) Bioorg. Med. Chem 4, 115-120; Katahira, et al. (1996) Bioorg. Med. Chem 4, 121-129; Chokekijchai, et al. (1995) Antimicrob. Agents Chemother. 39, 2345-2347; Nakashima, et al. (1996) Biol. Pharm. Bull. 19, 405-412; Detlefsen, et al. (1995) J. Antibiot. 48, 1515-1517; Constantine, et al. (1995) J. Biomol. NMR 5, 271-286; Helynck, et al. (1993) J. Antibiot. 46, 1756-1757; Frechet, et al. (1994) Biochem. 33, 42-50; Potterat, et al. (1994) Liebigs Annalen der Chemie 7, 741-743; Yamasaki, et al. (1994) J. Antibiot. 47, 276-280; Katahira, et al. (1995) Bioorg. Med. Chem. 3, 1273-1280; Yano, K. et al. (1995) J. Antibiot. 48, 1368-1370; Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220; Kimura, et al. (1997) J. Antibiot. 50, 373-378; Wyss, et al. (1991) J. Antibiot. 44, 172-1801; Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491; FIG. 4). The known non-MccJ25-related lariat peptides have lengths of 16-21 residues (FIG. 4). The known non-MccJ25-related lariat peptides contain either: (1) an 8-residue cyclic segment—with a backbone-sidechain amide bond between the backbone nitrogen atom of Xaa1 and the sidechain carboxyl of Glu8 or Asp8-followed by a 9- to 12-residue linear segment; or (2) a 9-residue cyclic segment—with a backbone-sidechain amide bond between the backbone nitrogen atom of Xaa1 and the sidechain carboxyl of Asp9-followed by a 7- to 12-residue linear segment (FIG. 4).
Known non-MccJ25-related lariat peptides have lariat-protoknot three-dimensional structures similar to the lariat-protoknot three-dimensional structure of MccJ25. Three-dimensional structures have been determined for several known non-MccJ25-related lariat peptides, including siamycin I (Katahira, et al. (1996) Bioorg. Med. Chem 4, 121-129), siamycin II (Constantine, et al. (1995) J. Biomol. NMR 5, 271-286), siamycin III (Frechet, et al. (1994) Biochem. 33, 42-50)], RES-701-1 (Katahira, et al. (1995) Bioorg. Med. Chem. 3, 1273-1280), and lariatin A (Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491). In each case, the compound has been found to have a lariat-protoknot structure, in which the linear segment of the lariat loops back, and penetrates and threads though the cyclic segment of the lariat (Katahira, et al. (1996) Bioorg. Med. Chem 4, 121-129; (Constantine, et al. (1995) J. Biomol. NMR 5, 271-286; Frechet, et al. (1994) Biochem. 33, 42-50); Katahira, et al. (1995) Bioorg. Med. Chem. 3, 1273-1280; Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491).
The known non-MccJ25-related lariat peptides exhibit no significant amino acid similarity to MccJ25 (less than 25% sequence identity; FIG. 4).
The known non-MccJ25-related lariat peptides are produced by bacterial producer strains: Streptomyces sp. strains for siamycins, RES-701-n, and anantin (Yano, et al. (1996) Bioorg. Med. Chem 4, 115-120; Katahira, et al. (1996) Bioorg. Med. Chem 4, 121-129; Chokekijchai, et al. (1995) Antimicrob. Agents Chemother. 39, 2345-2347; Nakashima, et al. (1996) Biol. Pharm. Bull. 19, 405-412; Detlefsen, et al. (1995) J. Antibiot. 48, 1515-1517; Constantine, et al. (1995) J. Biomol. NMR 5, 271-286; Helynck, et al. (1993) J. Antibiot. 46, 1756-1757; Frechet, et al. (1994) Biochem. 33, 42-50; Potterat, et al. (1994) Liebigs Annalen der Chemie 7, 741-743; Yamasaki, et al. (1994) J. Antibiot. 47, 276-280; Katahira, et al. (1995) Bioorg. Med. Chem. 3, 1273-1280; Yano, K. et al. (1995) J. Antibiot. 48, 1368-1370; Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220; Wyss, et al. (1991) J. Antibiot. 44, 172-1801), a Microbispora sp. strain for propeptin (Kimura, et al. (1997) J. Antibiot. 50, 373-378), and a Rhodococcus sp. strain for lariatins (Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491).
It has not been possible to date to re-create the lariat-peptide/lariat-protoknot structure of a non-MccJ25-related lariat peptide without use of the Streptomyces sp., Microbispora sp. and Rhodococcus sp. producer strains. Attempted chemical synthesis of RES-701-1 yields a product having a correct lariat-peptide covalent structure but not having a correct lariat-protoknot three-dimensional structure (i.e., not having the linear segment of the lariat looped back and penetrating and threading through the cyclic segment of the lariat); the resulting material exhibits only 1/700 the biological activity of authentic RES-701-1 (Katahira, et al. (1995) Bioorg. Med. Chem. Lett. 5, 1595-1600; He, et al. (1995) Bioorg. Med. Chem. Lett. 5, 621-626). Chemical synthesis of a linear analog of RES-701-1 yields a product having neither a lariat-peptide covalent structure nor a lariat-protoknot structure; the resulting material exhibits no detectable biological activity (He, et al. (1995) Bioorg. Med. Chem. Lett 5, 621-626). These negative results are reminiscent of negative results obtained for attempted chemical synthesis of MccJ25 and for chemical synthesis and recombinant production of a linear analog of MccJ25 (Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483)
No lariat-peptide/lariat-protoknot biosynthetic cassette for non-MccJ25-related lariat peptides have been described in prior art.
As a consequence, surrogate-host expression of non-MccJ25-related lariat peptides has not been accomplished in prior art.
As a further consequence, production of substituted derivatives of non-MccJ25-related lariat peptides has not been accomplished in prior art.
As a further consequence, production of libraries of substituted derivatives of non-MccJ25-related lariat peptides has not been accomplished in prior art.
The known non-MccJ25-related lariat peptides have been reported to have useful properties, including antibacterial activity for siamycins (Yano, et al. (1996) Bioorg. Med. Chem. 4, 115-120; Potterat, et al. (1994) Liebigs Annalen der Chemie 7, 741-743), propeptin (Kimura, et al. (1997) J. Antibiot. 50, 373-378), and lariatins (Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491); antiviral activity for siamycins (Chokekijchai, et al. (1995) Antimicrob. Agents Chemother. 39, 2345-2347; Nakashima, et al. (1996) Biol. Pharm. Bull, 19, 405-412; Detlefsen, et al. (1995) J. Antibiot. 48, 1515-1517; Lin, et al. (1996) Antimicrob. Agents Chemother. 40, 133-138), endothelin type B receptor antagonist activity for RES-701-n (Morishita, et al. (1994) J. Antibiot. 47, 269-275; Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220), prolyl endopeptidase inhibition activity for propeptin (Kimura, et al. (1997) J. Antibiot. 50, 373-378), and atrial natriuretic factor receptor antagonist activity for anantin (Wyss, et al. (1991) J. Antibiot. 44, 172-1801).
The known non-MccJ25-related lariat peptides have not been reported in prior art to inhibit bacterial RNAP.

Non-MCCJ25-related lariat-peptide inhibitors of bacterial RNA polymerase

Rutgers, The State University of New Jersey


An apparatus for entering alphabetic data into a computer or other electronic or mechanical devices with a keyboard, wherein: the alphabet letters are placed in an ordered arrangement, the vowels are grouped together, the vowels are all placed on the left side of the keyboard, each vowel has the same color code, the consonants are arranged in groups, the consonant groups are arranged from left to right in alphabetic group order, each consonant group has a specific color code, two groups of consonants have the same color, and letters in each consonant group are arranged vertically across the three rows of the keyboard, with the exception of the JK consonant group which is arranged horizontally on a single row of the keys.

The success of the Remington Standard No. 2 typewriter in 1878, which used the QWERTY keyboard, led to the universal adoption of the QWERTY layout. It was an open design and is hailed as one of the successful examples of open standards for an industry. This layout was designed to slow down the rate of typing of the operator. The early mechanical system could not keep up with the rate the keys were being struck. The operator could press keys in sequence faster than the machine could return the previous key to its starting position. There was no consideration given to the ease of learning the keyboard.
The QWERTY keyboard was arranged to scatter the most struck keys to slow down the speed of the operator. For instance, a frequently pressed vowel "a" is pressed by the small finger of the left hand, which is a weak finger and the mechanical typewriters of the time required a forceful stroke to bring the key up to strike the ribbon and make an impression. The QWERTY keyboard was laid out in three rows with 10 keys in the top row, 9 in the second row, and 7 in the third row, with the keys arranged in diagonal columns to allow the keys to depress a lever.
However, the QWERTY keyboard did have one significant design feature: efficiency. The design of the keyboard with three rows with 10 letters in the first row, 9 letters in the second row and 7 letters in the third row. This created a middle home row for the fingers to rest and then every key is then either in the home row, or one row up or one row down.
The QWERTY keyboard to a beginning typist is completely random and each letter has to be painfully memorized by repetition. It takes weeks if not months to train a typist to a high level of skill. Despite this significant limitation, the ubiquitous presence of the QWERTY keyboard on all typewriters made changing to an easier to learn system commercially unfeasible.
Typists were regarded as high skill employees and typing speed and accuracy was considered a desirable feature of employees. Subsequently, there was a search for a faster keyboard. August Dvorak was granted U.S. Pat. No. 2,040,248 (1936) for a new keyboard. The Dvorak keyboard was designed for maximum speed by carefully measuring the distance the operator had to go to type each letter. Ease of learning the keyboard was not considered. Again, to a beginning typist the keyboard was a random arrangement of letters and the finger to type each letter had to be learned by rote and extensive practice. The Dvorak keyboard layout comprised, from top to bottom, 7, 10 and 9 keys in each of three rows. This differentiated it from the QWERTY design of three rows of 10, 9, and 7 keys. The Dvorak keyboard never made significant headway for typewriters because the marginal difference in speed was more than negated by the long period of training necessary to change from one random arrangement of letters to another random arrangement of letters.
Starting in the 1970's with the introduction of the personal computer, the typing keyboard was separated from the physical computer. The QWERTY keyboard arrangement was adopted by manufacturers for the computer keyboards. Once again, the slight advantage in speed provided by the Dvorak keyboard did not make up for the long learning time. The Dvorak keyboard remains available for computers but only occupies an insignificant share of the market.
Thus, the main keyboard layouts had as their central concern the speed of the typist and developed their systems in response to this concern, but they had opposite goals. The intention of the QWERTY system was to slow down typing speed while the Dvorak system was to speed up typing. The Dvorak and QWERTY designers never paid any consideration to the difficulty of learning the keyboard layout. To a new learner of the keyboard layout, both the QWERTY and Dvorak systems appear as random arrangements that must be painstakingly learned through repetition.
In a study of computer users, the average typing rate was thirty-three words per minute to transcribe a document and the rate for composition was nineteen words per minute. This indicates that the overwhelming majority of computer users are not typing at any great rate of speed. Therefore, improving anyone's rate of typing from sixty-five to seventy words per minute for a skilled typist by having the typist learn a completely new keyboard arrangement is not a worthwhile endeavor that offsets the disability in quickly learning the keyboard. In contrast, providing a keyboard that is easier to learn and easier to use would provide a significant savings in learning times for beginning typists and aid slow typists.
Over the decades typing has changed from a skilled trade of secretarial work to a mass skill such as driving a car. Today, children in grade school learn keyboarding skills in the seventh grade, if not earlier. The mass use of cell phones and computers has made using a keyboard almost universal for all professions. Thus, the QWERTY keyboard system's random arrangement of letters now presents an even greater barrier and considerable waste of time, effort and money learning a keyboard layout meant for a mechanical keyboard of the 1900's.
Based on the above, there exists a need in the art for a keyboard arrangement of letters that enable the beginning learner and any user to quickly and efficiently identify the position of each of the letters on the keyboard. In order to accomplish this, the letters must be arranged in a manner that takes into account the learned knowledge of the user, that is, the learned order of the alphabetic sequence of letters and the distinction between the vowels and the consonants.


A cushion, which is a solid structure formed by a cardboard, includes a first baseboard, a second baseboard, a third baseboard, a fourth baseboard, a fifth baseboard and at least one supporting board that are joined to each other. Those junctions are foldable. The fourth baseboard has a first baffle flap and a plurality of the first cut lines. The fifth baseboard has a second baffle flap and a plurality of the second cut lines. When the first baffle flap and the second baffle flap are side matched to form the solid structure, the portion of the fourth baseboard between the first cut lines and the portion of the fifth baseboard between the second cut lines are folded toward each other to form a concave. The supporting board is joined to a lateral side of the opening of the solid structure and folded to be a supporting structure to cover the opening.

1. Field of Invention
The invention relates to a cushion and, in particular, to a cushion designed as an integrated structure.
2. Related Art
Nowadays, the electronic products such as computers, scanners, and computer peripherals become the indispensable equipments in the modern life. In order to protect the electronic products from being damaged on the shipping way, the corners and edges of the electronic products must be supported and protected with buffer materials before the electronic apparatus are put into the carton.
Regular buffer materials for aforesaid purpose are commonly molded from foamed plastics. However, with raising the environmental consciousness, the cardboards have been intensively used for packing the electronic products so that the environmental pollutions can be reduced and the protection purpose is attained concurrently.
With reference to FIG. 1, the conventional cushion 1 made of the cardboard comprises a bearing base 11, and a plurality of fastened elements 12. The bearing base 11 is formed by folding a cardboard. The bearing base 11 has a bottom wall 111 and two lateral walls 112. The lateral walls 112 are respectively disposed at the opposite sides of the bottom wall 111 and perpendicular to the bottom wall 111. Two slots 113 are disposed near two opposite ends of the bottom wall 111. The fastened elements 12 are also respectively formed by folding a cardboard. Each of the fastened elements 12 has a plurality of the slots 121 used to be engaged with the slots 113 of the bearing base 111 so that the lateral walls 112 can be fixed on the fastened elements 12. Therefore, referring to the FIG. 2, two cushions 1 can be respectively disposed at two opposite ends of a product 2 to protect the product 2 from impact.
However, only the fastened elements 12 are used to fix the two ends of the bearing base 11, which is a simple structure, so that the conventional cushion 1 is easy to be damaged during the long-time shipment. In addition, because the cushion 1 is assembled by a plurality of components, the assembling procedures are complex, resulting in more manufacturing time and higher cost.
Therefore, according to the above-mentioned problems, it is a subjective to provide a cushion with a rigid structure, which is easily assembled and has lower manufacturing cost.


A collection pipette that collects a microscopic object includes a first tube part, a second tube part connected to an end of the first tube part, and a third tube part connected to the other end of the first tube part. The longitudinal direction of the third tube part intersects with the longitudinal direction of the first tube part, and is parallel to the longitudinal direction of the second tube part. For example, the length in the longitudinal direction of the third tube part is shorter than the length in the longitudinal direction of the first tube part.

In the field of biotechnology, a micromanipulation system for finely manipulating microscopic objects used when DNA solution or a cell is injected into a cell or an egg under microscopic observations has been known. Patent Literature 1 discloses a cell collection apparatus including a manipulator for sucking cells. Moreover, Patent Literature 2 discloses a cell culture dish the surface of which is provided with a plurality of microcontainers with different sizes.

Tubular instrument and manipulation system