The application provides products, which may trigger pro-apoptotic or pro-death effects on neoplastic cells and/or tissues of the human nervous system, more particularly on neoplastic cells and/or tissues of the human CNS. The products of the application can be used as anti-proliferative or tumoricid agents in the treatment and/or palliation and/or prevention of neoplasms of the human nervous system, more particularly of glioblastoma or brain stem glioma. Therefore, the application also relates to compositions, such as pharmaceutical compositions or drugs, which comprise at least one of said products, as well as to the biotechnological and medical applications of said products and compositions.

Tumors develop when, in a healthy organ, cells display uncontrolled growth and division beyond the normal limits and escape cell death mechanisms.
Viruses are masters for manipulating the proliferation or death of the host cell they infect, mainly by interfering with crucial endogenous interactions. Rabies virus (RABV) is a neurotropic virus, which causes fatal encephalitis in mammals. RABV has developed a particular strategy to ensure its propagation in the nervous system. Its virulence correlates with the ability of infected neurons to survive.
Virulent RABV strains have been described to preserve the integrity of the neuronal network, thereby favoring the spreading of the virulent strain within the Central Nervous System (CNS), although they have also been described to have no positive or a negative impact on neurite outgrowth, for example no positive or a negative impact on axon outgrowth of rat motoneurons in culture (Guigoni and Coulon, 2002).
Attenuation of laboratory strains in the search for candidate live anti-RABV vaccines led to the observation that RABV attenuation is linked to their ability to trigger cell death. WO 03/048198 relates to the G protein of RABV and to fragments thereof of at least 100 amino acids, which induce the disruption of the neuronal cell integrity and the formation of apoptotic bodies. WO 03/048198 describes that the effect of these apoptotic bodies is to stimulate a humoral immune response, preferably a B-dependent humoral immune response.
Préhaud et al., 2010 have described that the various partners of attenuated rabies virus strains include PTPN4, MAST2, MAST1, DLG2, MDPZ. A cell—penetrating peptide (Cyto13-att; SEQ ID NO: 11) encoding the PDZ binding site (PDZ-BS) of the attenuated ERA rabies virus strain induces cell death in the neuroblastoma cell line SK-N-SH, whereas peptides from the virulent VIR strain PDZ-BS (Cyto13-vir; SEQ ID NO: 14) or peptides lacking the last four amino acids (Cyto9-Δ; SEQ ID NO: 17) do not (Préhaud et al., 2010).

Products useful for the treatment of malignant neoplasms of the human nervous system

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE


A drag balance for measuring drag forces on a model is described which mos within a hollow region of a streamlined strut. The strut is attached to a carriage for towing the model through a medium. The drag balance has a ground frame attached inside the strut and a balance frame attached to the flat plate at the base of the strut. The ground frame and balance frame are connected by flexures which allow only a small amount of movement of the balance frame and model relative to the ground frame and strut in the towing direction. Strain gages are attached to one or more of the flexures to determine the amount of bending in the flexures and thus obtain a measure of the drag forces exerted on the model. Mounting the drag balance within the streamlined strut minimizes turbulence and allows the drag balance to be connected directly to the model at the base of the strut such that drag forces on the model are accurately determined.

(1) Field of the Invention
The present invention relates generally to the measurement of drag forces on a surface, and more particularly to a strut-mounted drag balance for measuring drag force on a flat plate moving through a medium.
(2) Description of the Prior Art
The performance of a vehicle moving through a medium is limited by the drag force of the medium on the vehicle. To improve efficiency and reduce the power required to move the vehicle through the medium, numerous drag reduction methods have been tried. It is customary to test drag reduction methods by model testing in towing basins or wind and water tunnels where a scaled model of the vehicle is towed through the medium or the medium is flowed past the model. To measure drag forces on the model, a towing post, or strut is attached to the model through a load cell, or drag balance. Typical prior art drag balances, as exemplified by U.S. Pat. No. 5,343,742 to Cusanelli et al., consist essentially of a hollow metal cube, generally four inches on a side, with portions of the sides removed, leaving planar top and bottom surfaces connected by four legs. The strut is attached to the top surface and the model is attached to the lower surface. Forces exerted on the model cause bending in the legs which is measured using strain gages attached to the legs. When the model is submerged in the medium such that the towing post extends into the medium, the strut is streamlined to minimize extraneous drag forces exerted on the strut, and the drag balance is placed within the scaled model. Testing of drag reduction methods which do not rely on vehicle shape to reduce drag, e.g., polymer injection and magneto hydrodynamic turbulence control devices, is complicated by the difficulty of predicting drag on a model shaped as a scaled vehicle. It is preferable to test such drag reduction methods using a flat plate model since drag calculations are much simpler for a flat plate model than for a hydrodynamically shaped model, thus actual test results can be easily compared to theoretical predictions. Since the flat plate has no interior volume, the drag balance cannot be placed within the plate. The cubic configuration of current drag balances would cause considerable turbulence if placed between the strut and the flat plate. If the drag balance were placed above the strut, the drag forces exerted on the strut itself would corrupt the drag reduction measurements. Additionally, The size of current drag balances also prevents their placement within an efficiently streamlined strut.

The United States of America as represented by the Secretary of the Navy


An apparatus, system and method for telepresence communications in an environment of a virtual location between two or more participants at multiple locations. First perspective data descriptive of the perspective of the virtual location environment experienced by a first participant at a first location and feature data extracted and/or otherwise captured from a second participant at a second location are processed to generate a first virtual representation of the second participant in the virtual environment from the perspective of the first participant. Likewise, second perspective data descriptive of the perspective of the virtual location environment experienced by the second participant and feature data extracted and/or otherwise captured from features of the first participant are processed to generate a second virtual representation of the first participant in the virtual environment from the perspective of the second participant. The first and second virtual representations are rendered and then displayed to the first and second participants, respectively. The first and second virtual representations are updated and redisplayed to the participants upon a change in one or more of the perspective data and feature data from which they are generated. The apparatus, system and method are scalable to two or more participants.

There are many situations in which one or more individuals would like to observe and possibly interact with objects or other individuals. When two or more individuals need to meet and discuss issues of mutual interest, a common approach is a physical (face-to-face) meeting. This type of meeting has the advantage of direct personal contact and gives the individuals the ability to communicate most effectively, since eye contact may be maintained, and physical gestures such as facial expressions, hand movements, and body posture are readily evident. For most meetings, this is the preferred medium of exchange since large amounts of information may be exchanged transparently if the information is at the location of the meeting.
In certain situations, such as communication over long distances, arranging such face-to-face meetings can be time-consuming or prohibitively expensive. In these situations, the most common way to exchange information is over the telephone, via e-mail or by teleconferencing. Each of these approaches has serious drawbacks. Telephone conversations provide none of the visual cues that may be important when making a business decision. Telephone conversations are also difficult to manage when more than two individuals need to be involved in the meeting. E-mail or regular postal services are much slower than an in-person meeting and provide none of the visual or even audio cues that are present in in-person meetings. The use of video teleconferencing equipment allows individuals at remote locations to meet and exchange information through the use of audio/visual communication.
There is, however, a substantial difference between an in-person meeting between two or more people and a meeting using a video teleconferencing system. The latter does not provide the same experience as the former. In an in-person meeting, we see the other person in three dimensions, in color and at the right size, and each participant at their appropriate physical position. More importantly, we have the ability to make and maintain eye contact. This visual information contributes to a sense of presence of the individual. The current state of the art in video teleconferencing provides none of these benefits. Video teleconferencing also does not provide the nuances of facial and body movement available from a personal meeting, since the entire image is transmitted at the same scale. Therefore, the in-person impact of a frown or smile is likely to be greater than when using a video teleconferencing system since the correct aspect and detail of the area around the mouth is not transmitted in a video teleconference. Moreover, exchange of non-personal information, such as reports, documents, etc., resident at a particular location to others participating in a teleconference may be limited. It is therefore difficult to transmit personal and non-personal information of a desirable quality and quantity using existing teleconferencing technology.

This patent is subject to a terminal disclaimer.

Method system and apparatus for telepresence communications utilizing video avatars


A wind turbine blade is provided. The wind turbine blade has multiple vortex generators, each projecting from a surface of the blade and having a predetermined length. The vortex generators are arranged on a strip. The width of the strip is several times larger than the length of a vortex generator.

Wind turbines are provided with a rotor shaft which is part of an electrical generator producing electricity during movement of the rotor relative to a stator of the generator. The stator comprises a number of coils, the rotor comprises a number of permanent magnets so that an electric voltage is induced when the rotor is turned. Typically the rotor comprises three wind turbine blades.
Conventionally vortex generators are attached on wind turbine blades in order to reduce a tendency of separation of the air flow on the surface of wind turbine rotor blades. Another effect is that vortex generators counteract stall.
In WO 00/15961 A1 a wind turbine blade comprising multiple vortex generators is disclosed. Each vortex generator is wedge-shaped and extending perpendicularly out from the blade surface. There is a high risk of the vortex generators being damaged or torn off during handling of the blade, e.g. when the blade is lifted in lifting straps, due to the fragile design of the vortex generators. Vortex generators may be formed integrally with the blade, whereby each vortex generator is produced as a separate member having a bottom face which is secured to the surface of the blade by adhesion.
In WO 2008/113350 A2 a wind turbine blade with multiple vortex generators is proposed which are arranged in two or more parallel rows.
Another wind turbine blade with vortex generators is known from EP 1 314 885 A1. The vortex generators are arranged on a strip which is fixed on a surface of the rotor blade.
Experience has shown that high centrifugal forces and aerodynamic forces acting on the attached strips of vortex generators pose a risk for them to rip off over time Vortex generators installed at the tip end part of the rotor blade are worst affected by this problem. The efficiency of the vortex generators is reduced, furthermore it is both time consuming and expensive to re-fit them again.


Example implementations may relate to methods and systems for detecting an event in a physical region within a physical space. Accordingly, a computing system may receive from a subscriber device an indication of a virtual region within a virtual representation of the physical space such that the virtual region corresponds to the physical region. The system may also receive from the subscriber a trigger condition associated with the virtual region, where the trigger condition corresponds to a particular physical change in the physical region. The system may also receive sensor data from sensors in the physical space and a portion of the sensor data may be associated with the physical region. Based on the sensor data, the system may detect an event in the physical region that satisfies the trigger condition and may responsively provide to the subscriber a notification that indicates that the trigger condition has been satisfied.

Physical spaces may be used for retail, manufacturing, assembly, distribution, and office spaces, among others. Over time, the manner in which these physical spaces are designed and operated is becoming more intelligent, more efficient, and more intuitive. As technology becomes increasingly prevalent in numerous aspects of modern life, the use of technology to enhance these physical spaces becomes apparent. Therefore, a demand for such systems has helped open up a field of innovation in sensing techniques, data processing, as well as software and user interface design.


A system for measuring the awareness of a subject including an imaging device adapted to acquire data representative of an image of the subject, and an awareness processor connected to the imaging device, wherein the awareness processor is adapted to classify the awareness of the subject according to the position of the subject.

A primary task when operating a vehicle, such as, driving an automobile, flying a plane, conducting a train or the like, is to monitor vehicular movement to ensure safe passage of the vehicle and its contents. Often times, however, a vehicle operator will become distracted. Some common distractions include fatigue, talking on or dialing a phone, interacting with passengers, reading road signs, or the like. Such distractions tend to direct the attention of the operator away from this primary task, and contribute to many, possibly avoidable, accidents. Human factors research, moreover, shows that a distracted driver reacts slower to unpredictable and potentially dangerous events, each of which might be avoided provided increased operator awareness.
To address these and additional issues previous systems have been proposed wherein devices periodically or randomly require an operator to manually respond to an awareness indicator by pressing a button, or the like. If a response is not received, the device generates an alarm alerting the operator of potential danger. Other proposed devices attempt to monitor driver awareness based on heart metrics. For example, the device may measure fluctuations in heart rate, blood pressure, or irregular heart beat patterns. While these attempts, allegedly, increase driver awareness during times of fatigue, they are crude measures that are susceptible to false signals.

System and method to determine awareness


A current detecting unit detects three phase current of a three-phase inverter from current flowing between the inverter and a DC power supply. The current detecting unit includes a specified voltage vector generating portion that generates a specified voltage vector indicating a voltage vector that three-phase voltage of the inverter should follow, and a specified voltage vector correcting portion that corrects the generated specified voltage vector. The inverter is controlled in accordance with the specified voltage vector after the correction.

1. Field of the Invention
The present invention relates to a motor control device that drives and controls a motor. In addition, the present invention relates to a current detecting unit, an inverter controlling device and a system interconnecting device.
2. Description of Related Art
In order to perform a vector control of a motor by supplying three-phase AC power to the motor, it is necessary to detect current values of two phases (e.g., U-phase current and V-phase current) among three phases including U-phase, V-phase and W-phase. Although two current sensors (current transformers or the like) are usually used for detecting current values of two phases, the use of two current sensors causes an increase of cost of the entire system equipped with the motor.
For this reason, there is provided a conventional method in which bus current (DC current) between an inverter and a DC power supply is sensed by a single current sensor, and current values of two phases are detected from the sensed bus current. This method is also called a single shunt current detecting method.
FIG. 37 shows a general block diagram of a conventional motor driving system in which the single shunt current detecting method is adopted. An inverter (PWM inverter) 202 is equipped with half bridge circuits for three phases, each of which includes an upper arm and a lower arm, and it converts a DC voltage from a DC power supply 204 into a three-phase AC voltage by switching the individual arms in accordance with a specified three-phase voltage value given by a controller 203. The three-phase AC voltage is supplied to a three-phase permanent-magnet synchronous motor 201, so that the motor 201 is driven and controlled.
A line connecting the individual lower arms in the inverter 202 with the DC power supply 204 is called a bus line 213. A current sensor 205 transmits a signal indicating bus current that flows in the bus line 213 to the controller 203. The controller 203 does sampling of an output signal of the current sensor 205 at appropriate timing so as to detect phase current of a phase in which a voltage level becomes a maximum value (maximum phase) and phase current of a phase in which a voltage level becomes a minimum value (minimum phase), i.e., current values of two phases.
If voltage levels of phases are separated from each other sufficiently, current values of two phases can be detected by the process described above. However, if the maximum phase of voltage and an intermediate phase become close to each other, or if the minimum phase of voltage and the intermediate phase become close to each other, it is difficult to detect current values of two phases. Note that description of the single shunt current detecting method including description of a reason why it becomes difficult to detect current values of two phases will appear later with reference to FIGS. 3, 4 and 5A-5D.
Considering this, in a certain conventional method, if current values of two phases cannot be detected by the single shunt current detecting method in a certain period, current values of three phases are estimated from past current information in the period. More specifically, d-axis current and q-axis current obtained by converting past current values of three phases are inversely converted into current values of three phases so as to estimate them.
In another conventional method, if current values of two phases cannot be detected by the single shunt current detecting method in a certain period, a pulse width of a PWM signal for each arm in the inverter is corrected based on gate signals of three phases in the period.
A usual correction example of a specified voltage value (pulse width) that corresponds to the above-mentioned correction is shown in FIG. 38. In FIG. 38, the horizontal axis indicates time, while 220u, 220v and 220w show voltage levels of the U-phase, the V-phase and the W-phase, respectively. Since a voltage level of each phase follows the specified voltage value (pulse widths) for each phase, they are considered to be equivalent. As shown in FIG. 38, the specified voltage value (pulse width) of each phase is corrected so that "maximum phase and intermediate phase" as well as "minimum phase and intermediate phase" of the voltage do not approach each other closer than a predetermined distance. Thus, voltages of individual phases do not become close to each other to the extent that current values of two phases cannot be detected, and current values of two phases can be detected stably.
In this way, correction with respect to voltage is beneficial. On the contrary, it is desired to provide a technique that can realize a desired correction for maintaining the state where motor current can be detected by a simple process. In addition, without limiting to motor current, it is important to develop a correction technique that can be realized by a simple process.

Motor control device and current detecting unit


A castor apparatus having a body, a first wheel, a second wheel and an impact dissipation device. The castor apparatus can be positioned at the end of an arm of a chair base. The castor apparatus includes an impact dissipation device that redirects force transmitted through the arm and the castor onto the ground. A method is also disclosed.

1. Field of the Disclosure
The invention relates in general to a chair assembly, and more particularly, to a castor apparatus which is usable with a chair base, as well as a chair base with a castor apparatus. The disclosure is particularly useful in an office chair environment, however, it is not limited thereto.
2. Background Art
The use of office chairs is ubiquitous. Generally, office chairs are configured to last for years. Many office chairs are coupled to a central column that is coupled to a chair base. The chair base includes a plurality of outwardly extending arms that terminate with a castor. The castors are positioned so that they can be rotated relative to the arm so that they can change rolling direction. This is particularly useful for quickly directing the chair in the direction desired. This is achieved by having the axle of the wheels of the castor being offset from the axle defining the rotation of the castor itself relative to the arm of the chair base.
While this ability of the castor to rotate is quite helpful when translating the chair across the ground, there are problems. Due to the offset, when a load is applied to the chair, the castor is directed outwardly as the axis of rotation of the wheels is offset from the axis of rotation of the castor vis-à-vis the arm. The outward force can be quite large when the load increases, and, so much so that the castor can break off from the arm, or the arm can break itself.

Castor apparatus for a chair base and a chair base with a castor apparatus


Apparatus and method for joining composite structures of aircrafts are disclosed. An example apparatus includes an inboard composite wing panel having a first end portion configured to be coupled to upper and lower inboard fittings composed of a metallic material and an outboard composite wing panel having a first end portion configured to be coupled to upper and lower outboard fittings composed of a metallic material. A side of body rib has a plate shaped first end portion composed of a metallic material. A fuselage frame is configured to be joined at one end of the upper inboard fitting, and the upper and lower inboard fittings and the upper and lower outboard fittings are coupled to the side of body rib at the plate shaped first end portion.

An aircraft wing of a commercial aircraft or jetliner is typically assembled prior to being attached to a fuselage of the aircraft. For example, an aircraft wing assembly typically includes a front spar and a rear spar each of which extends in the span direction of the wing, and structural members such as, for example, stringers and ribs that connect to the front and rear spar to form a skeletal structure or framework of the aircraft wing. A wing panel or skin, which may be formed with stringers, is coupled to the skeletal structure or framework to form an aircraft wing.
To couple or attach the aircraft wing to the fuselage, commercial aircrafts employ wing-fuselage attachment joints. For example, a wing-fuselage joint couples an aircraft wing to a central wing box supported by the fuselage of the aircraft. The aircraft wing is often attached to the central wing box via an upper wing panel joint and a lower wing panel joint. Typically, an upper wing panel joint employs a double plus chord composed of aluminum to couple an upper wing panel of the aircraft wing to the central wing box and a lower wing panel joint employs a T-chord to couple a lower wing panel of the aircraft wing to the central wing box.
Further, because of their relatively high strength-to-weight ratios, composite materials such as carbon-fiber reinforced plastics are often used in aircraft wings to reduce weight and increase performance. For example, the upper and lower wing panels of an aircraft wing or central wing box may be composed of a carbon composite or carbon fiber reinforced polymer, while wing spars, ribs and other frame structures are manufactured from metallic materials such as aluminum, steel, etc.
However, directly attaching carbon composites such as carbon-fiber reinforced plastics to aluminum structures can induce corrosion (e.g., galvanic corrosion) due to chemical and electro-chemical reactions with the surrounding environment. For example, composite materials such as carbon fibers are good electrical conductors and they produce a large galvanic potential with the aluminum alloys of an airplane structure. Thus, corrosion can occur when moisture forms on a composite/aluminum interface due to significantly different electrochemical potentials of these materials. Therefore, in some instances, coupling a composite wing panel directly to a plus chord composed of aluminum may cause an undesirable corrosive effect at that joint. Plus chords composed of titanium protect against such undesirable corrosive effect, but significantly increase the costs of the aircraft.

Apparatus and methods for joining aircraft composite structures


Devices, softwares and methods limit the total Modem-On-Hold (“MOH”) timeout time of a single log-in session. When a logged in user requests a Modem-On-Hold session, a server counts the cumulative time spent during individual MOH sessions. Once a limit is exceeded, then subsequent MOH requests are denied.

1. Field of the Invention
The present invention is related to the field of modems and network servers, and more specifically to modems and servers that can request a Modem-On-Hold and can deny such a request under certain conditions.
2. Description of the Related Art
Networks, such as the internet, are increasingly used for communications. Typically a user subscribes with an Internet Service Provider ("ISP") that is connected to the internet. The user has a host device, such as a personal computer. The host device can communicate over a telephone line with a network device of the ISP, such as a server. Then the user and the ISP exchange data over the modem connection, typically in binary form.
The host device includes a modem, which communicates over the telephone line. The server has individual ports, one for each user that calls in with a modem to log in.
Recently modems that are made according to the International Telecommunications Union (ITU) V.92 standard provide a new feature, which is called the Modem-On-Hold ("MOH"). The modem sends a MOH request to temporarily discontinue ("place on hold") the modem connection. The server receives the MOH request, and grants it by temporarily discontinuing the modem connection.
The MOH feature is useful to the user, because the telephone line is temporarily released. Then it can be used to receive an incoming call, or to place an outgoing call.
The MOH feature, however, is detrimental to the internet service provider. While the modem connection is temporarily discontinued, the port assigned to it by the server remains committed to the user, but unused. As such, it may not be assigned to another user. A number of users could abuse the privilege, employing the MOH feature many times in a single log-in session.

Devices, softwares and methods for selectively limiting the total modem-on-hold timeout time of a single log-in session


The sizing of virtual machines is optimized based on projected performance metrics. All virtual machine configuration resources are normalized by a processing device. The normalized resources for the virtual machine configurations are then stored in a catalogue. An application is then profiled to obtain resource demand estimates for each virtual machine configuration and a base performance is calculated for the application. The base performance is used to predict performance estimates on all virtual machine configurations in the catalogue. Accordingly, a virtual machine configuration having a lowest response time is selected.

The present invention relates to virtual machine optimization, and more specifically, to optimizing the use of available resources through sizing and consolidating virtual machines based on projected performance metrics.
A contemporary virtual machine (VM) is a software implementation of a machine (i.e., a computer) that executes programs like a physical machine. The VM typically emulates a physical computing environment, but requests for central processing unit (CPU), memory, hard disk, network and other hardware resources are managed by a virtualization layer which translates these requests to the underlying physical hardware. VMs are created within a virtualization layer, such as a hypervisor or a virtualization platform that runs on top of a client or server operating system. The virtualization layer is typically used to create many individual, isolated VMs within a single, physical machine. Multiple VMs are typically used in server consolidation, where different services that were previously run on individual machines are instead run in isolated VMs on the same physical machine. Contemporary VMs may be moved, copied, and reassigned between host servers to improve hardware resource utilization.
Contemporary cloud computing providers allow a VM to be managed over the Internet across a number of hardware devices while only allocating as much space as needed at any one time. Typically, cloud computing providers offer a tiered pricing subscription plan for users to rent VMs on which to run their own computer applications. Users typically choose from a number of different VM configurations to meet their computing needs. For example, at each increasing price tier, the CPU cores, memory, disk space, and network bandwidth of the VM configuration are generally increased to minor the increased resources provided. This allows a user to select and pay for a VM configuration that meets the user's target resource demands.

Optimization of virtual machine sizing and consolidation

INTERNATIONAL BUSINESS MACHINES CORPORATION


A method of obtaining luminance and chromaticity of white in the RGBW display using RGB display, including; obtaining spectral values of a q grayscale of red sub-pixels, green sub-pixels and blue sub-pixels of the RGB display; obtaining spectral values of red, green and blue sub-pixels of a color filter in the RGB display; calculating a spectral value of the q greyscale of white in the RGB display as a spectral value of the q grayscale of the white sub-pixels of the RGBW display; calculating tristimulus values WXq, WYq and WZq of the q greyscale of the white sub-pixels of the RGBW display; using the stimulus value WYq as a luminance of the q greyscale of the white sub-pixels of the RGBW display, and using the formulae

Field of the Invention
The present invention belongs to display technical field, more specifically, the present invention relates to a method of obtaining luminance and chromaticity of white in RGBW display device using RGB display device.
Description of Prior Art
The present display devices, such as liquid crystal display device and organic light emitting diode (OLED) display device, etc., all use red (R) sub-pixels, green (G) sub-pixels and blue (B) sub-pixels to display a picture. However, in order to increase penetration rate, luminance and energy efficiency of the display device so as to ensure energy saving and environment protection, a display device displaying the picture using red (R) sub-pixels, green (G) sub-pixels, blue (B) sub-pixels and white (W) sub-pixels has already been developed. In such case, if luminance and chromaticity of the white (W) sub-pixels in the display device can be obtained, then the design on white balance and driving of the display device can be further improved.

Method of obtaining luminance and chromaticity of white in RGBW display device using RGB display device

Abstract: x    =                            WX          q                                      WX            q                    +                      WY            q                    +                      WZ            q                                                              and            y    =                  WY        q                              WX          q                +                  WY          q                +                  WZ          q                    to calculate a chromaticity (x, y) of the white sub-pixel of the RGBW display.

SHENZHEN CHINA STAR OPTOELECTRONICS TECHNOLOGY CO., LTD.


This invention provides a locking wedge assembly capable of exerting a predetermined pressure in at least one direction. Such assembly removably secures a male connection member to a female connection member of an articulated coupling arrangement. Such articulated coupling arrangement used to secure one end of a first railway car to an adjacent end of said railway car in a semi-permanent fashion. The assembly includes a wedge-shaped member having a surface engageable with a surface located in an opening in a side wall portion of a cavity at an outer end of the female member and another surface engageable with a surface on a shaft member of a spherical bearing carried by such male member. An aperture is formed through such wedge-shaped member along a longitudinal axis thereof. A bolt member is disposed through this aperture and an aperture formed through a bottom wall on the outer end of such female member adjacent the side wall opening. At least one force exerting device is positioned intermediate either the top surface of the wedge member and the bottom surface of the head of such bolt on the bottom surface of the bottom wall and the top surface of the nut carried by the bolt.

Prior to the development of the present invention, it is well known in today's modern railroad industry that couplers are used to connect adjacently disposed ends of a pair of railway cars together. Further, on any of these railway cars which may possibly be used in interchange type service, these standard couplers must have received approval by the Association of American Railroads (AAR) prior to their being installed on such adjacently disposed ends of the railway cars.
It is generally well recognized, in the railroad industry, that such couplers will normally be required to serve a number of functions in this application. For example, standard couplers are used to facilitate the connection and the unconnecting of individual railway cars to and from, respectively, a train consist. Another function served by these standard couplers is that they enable such individual railway cars to more easily negotiate the curved portion of the track which will be encountered during operation of the train on the track structure. Additionally, such standard couplers perform the function of allowing such railway cars to be more easily and readily combined, thereby making up a train consist. These standard couplers further permit such adjacent ends of the railway cars to be easily and readily separated into individual cars as necessary for either loading or unloading cargo thereto or therefrom, respectively.
In more recent times, however, in the railroad industry it has come to be generally recognized that a significant number of relative important advantages can be achieved by the interconnection of a number of railway cars to form a substantially semi-permanent unit. This has been particularly the situation, for example, where such railway cars are specifically designed for use in what is commonly referred to in the railroad industry as "piggyback" or intermodal service. One of the primary reasons for this is that the cargo which is to be either loaded or unloaded is either brought to or removed from, respectively, predetermined central locations. Generally, such central locations are usually owned and operated by the railroads. This cargo is normally either over-the-road trailers or large containers which usually are used to ship cargo by oceangoing vessels.
The individual railway cars which have been connected together in this substantially semi-permanent manner are commonly known in the railroad industry as either a "5-pack" or as a "10-pack". Except at the extreme outermost ends of each 5 or 10-pack unit, the use of such standard couplers discussed above is not required on these 5 or 10-pack units. Because of their dedicated service, these 5 or 10-pack units will generally only be broken apart on a periodical basis. This is the primary reason why such standard couplers are not required in this particular application. In most cases, for example, the breaking of these 5 or 10-pack units will normally only occur only when some maintenance must be carried out on either an individual coupler component or on certain other critical components positioned on the railway car which will require an individual car to be removed from such 10-pack unit on a temporary basis. In the railroad industry, it has become quite obvious that with the use of some type of semi-permanent coupling arrangement it is possible for them to achieve a rather significant reduction in their operating and maintenance costs. These cost reductions can be attributed to a variety of reasons. At a minimum, these reasons include a significant reduction in the weight of the railway equipment. Such weight reduction results in a rather significant reduction in the amount of energy required to move a train consist over the track structure. Because in this semi-permanent coupling arrangement fewer railway trucks are required, there is not only a reduction in equipment costs achieved but this also results in a reduction of the maintenance requirements.
Now, however, with the relatively extensive use of such substantially semi-permanent coupling arrangement, the railroad industry in conjunction with the railway equipment suppliers have determined that it is of critical importance for a close-buttoned relationship to be maintained between the couplers draft components present in a particular coupling arrangement. Further contributing to the importance of this close-buttoned relationship requirement is the ever increasing loads which must be carried by modern railway cars and train consists in order for the railroads to be competitive. This close-buttoned relationship has been found necessary, for example, so that the detrimental effects of the impact forces which are normally encountered during in-track train operation can be reduced to an acceptable level. In this manner, the possible damage that could be incurred by the cargo and/or the railway equipment can be held to an absolute minimum. Such impact forces are generally encountered during normal buff operation of the train consist.
With the above discussion in mind, attention is now directed to a particular prior art type articulated coupling arrangement used for the purpose of connecting adjacent ends of a pair of railway cars together in such semi-permanent fashion. This prior art articulated coupling arrangement is taught in U.S. Pat. No. 4,258,628. As has been illustrated therein, this particular articulated coupling apparatus includes a male connection member and a female connection member. The male connection member is secured at one end thereof to one end of a first railway car body member and the female connection member is secured at one end thereof to an adjacent end of second railway car body member.
The female connection member, in this prior art coupling arrangement, is rotatably engaged in a center plate bowl portion of the bolster member positioned intermediate the side frames of the railway track member. Such rotatable-type engagement is accomplished in a manner that is generally well known in the railway art. The outer end portion of the male connection member is disposed for relative movement, at the outer end portion of such female connection member.
A pin member is utilized in this coupling arrangement to connect the outer end portion of the male connection member within the cavity of such female connection member together thereby forming such substantially semi-permanent coupling. This pin member is positioned in a vertical plane. Additionally, this pin member is positioned in aligned apertures which are formed in each of such male connection member and such female connection member. It is of significance to note that, as is taught in this prior art reference, the aperture formed in such male connection member for receiving such pin member therein must be formed somewhat larger than such pin member itself. In this coupling arrangement, this is necessary to permit certain movements that are required of the coupling apparatus during service to be achieved.
It should additionally be noted that a rear surface portion of such apertures formed in such male connection member and which will receive such pin member therein includes a horizontally disposed concave configuration and a vertically disposed convex configuration. This particular configuration is desirable in this coupling arrangement in order for both the male connection member and the female connection member to be able to move in each of a horizontal direction and a vertical direction with respect to one another. Another substantial area of surface contact between the rear surface of such pin aperture and the pin member itself is provided by this configuration at the same time.
Such male connection member, adjacent the outer end surface of the outer end portion thereof, includes a convex configuration. This convex configuration on the outer end surface of such male connection member abuttingly engages a complementary concave surface which is formed on a front face portion of a follower member.
As illustrated, in this coupling arrangement, this follower member is disposed within the rear portion of such cavity located in the outer end portion of such female connection member. This follower member, on the rear face portion thereof, includes a pair of vertically disposed slot-like cavities formed therein. A first portion of a resilient member is positioned within each of these vertically disposed slot-like cavities. Each such resilient member includes a second portion which extends outwardly from the rear face portion of such follower member.
In this manner, a vertically disposed wedge-like element can be engaged with the exposed outermost surface area of each such resilient element. Such wedge-like element being a necessary component so that during service of this coupling arrangement such follower member and the male connection member can be urged forwardly. Consequently, the rear surface portion of the aperture formed in such outer end portion of the male connection member will be maintained substantially in mating engagement with such pin member at all times.
In this coupling arrangement, because the majority of the articulated connecting members used are normally manufactured as cast components, such mating engagement being maintained between such pin members and the rear surface portion of this aperture disposed in the male connection member is essential. Furthermore, in attempting to maintain the manufacturing costs of this coupling arrangement as low as possible, such cast connecting components will normally receive very little, if any, finish machining necessary to provide either the required or the desirable dimensional control. In other words, these cast connecting members will generally be used as cast. As a result of this manufacturing cost-saving practice, it is quite often difficult to provide an articulated coupling apparatus that will be self-adjusting under the various wear conditions which such coupling apparatus will normally be subjected to during operation. It is important, nevertheless, to minimize as much as possible the slack encountered in the various coupling connections during such in track service.
Other prior art type coupling devices are taught U.S. Pat. No. 3,716,148 and Canadian Patent Number 1,231,078.

Locking wedge assembly to removably secure a male connection member in an articulated-type coupling arrangement


A disposable absorbent garment includes a topsheet, a backsheet, and an absorbent core disposed between the topsheet and the backsheet. The topsheet, backsheet and absorbent core are assembled together to form a layered assembly including a front region, a back region, and a crotch region disposed between the front region and the back region. The layered assembly has a longitudinal plane that extends substantially centrally through the front, back and crotch regions. Each of the front and back regions further includes a pair of ear portions extending in substantially opposite lateral directions with respect to the longitudinal plane. Further, each of the ear portions includes a layered section of a material layer and a stretchable member extendible between a contracted configuration and an extended configuration. The material layer is secured to the stretchable member when the stretchable member is disposed in an extended configuration and the outside surface of the material layer is disposed in a substantially planar configuration. In this way, the material layer is, thereafter, extendible with the stretchable member from a contracted configuration to an extended configuration wherein its outer surface is returned to its substantially planar configuration.

The present invention relates generally to disposable absorbent garments and, more particularly, to a disposable absorbent garment having a stretchable ear portion and a method for making such a disposable absorbent garment.
Disposable absorbent garments contemplated by the invention include disposable diapers, disposable pull-on garments, and the like. These garments are worn about the lower torso or waist of the user so as to receive and contain urine and other bodily wastes. Disposable diapers are particularly intended for use on infants who depend on a caretaker to fit the diaper. Typically, the infant is laid down while the caretaker places the garment around the waist region of the infant and then secures two ends on each side of the diaper together.
Disposable pull-on garments include training pants, pull-on diapers, disposable underwear, and adult incontinence garments. It is generally expected that the user of any one of these garments will be able to put on and take off the garment on his\her own. As for training pants, these garments are intended for use on a young child just before or about the time the child is ready to graduate from diapers to regular underpants (i.e., during toilet training). Training pants (and other disposable pull-on pants) have closed sides such that the user or care giver raises the garment about the user's legs to put it on and slips the garment downward about the user's legs to take it off. Thus, training pants (and other pull-on pants) are designed to be put on and taken off in the same manner by which regular underpants are put on and taken off. This feature is particularly advantageous in toilet training because it provides an early introduction to underpants and, more specifically, trains the child to wear regular underpants. Similarly, it is advantageous that the training pants fit and look very much like regular underpants so that the child is comfortable in making the transition from training pants to regular underpants.
The principal elements that typically makeup the disposable absorbent garments described above are a liquid-permeable inner layer, a liquid-impermeable outer layer and an absorbent core sandwiched between the inner and outer layers. Elastic members may also be incorporated into different parts of the garments. For example, elastic members may be positioned longitudinally along the diaper, generally outboard of the absorbent core to effect a seal around the buttocks, legs or both of the user. In addition, several elastic members (e.g., in the form of elongated elastic threads) may be positioned laterally throughout the waist region (including the side waist regions) of a disposable absorbent garment to allow the garment to stretch when it is put on and then during wear. In this way, the garment can stretch to accommodate variations in waist size and leg size of the user, while fitting snugly about the waist and legs and without sagging. One drawback, however, is that the elastic strands are visible on the outer surfaces of the garment and can interfere to some extent with decorative or instructional printing displayed on the outer surfaces of the garment.
It is also known to provide disposable garments with separate side waist regions in the form of elastic side panels such as those disclosed in U.S. Pat. No. 4,938,753. The elastic side panels described therein have side edges which are attached to the outer edges of a central absorbent core assembly. The elastic side panels may be made from various elastomeric materials or combinations thereof and provide a more uniform fit generally more snug than side waist regions having elongated elastic strands. Moreover, the elastic side panels provide outer surfaces which are more uniform than the outer surfaces on side waist regions having elongated elastic strands. However, the elastomeric material for the side panel does not typically provide for a smooth, even outer surface when the side panels are in either the relaxed state or the stretched state. Accordingly, the side panels are not particularly conducive to having decorative or instructional printing displayed thereon.
Alternatively, it is known to provide a unitary pair of training pants with elasticized ear flaps such as those disclosed in U.S. Pat. No. 5,246,433. The elasticized ear flaps are formed by sandwiching a relaxed elastomeric element between two material layers of the ear flaps, and adhesively attaching the material layers to the relaxed elastomeric element to form an elastic laminate. The design also calls for mechanically stretching the elastic laminate such that the material layers permanently deform. Although the side waist region according to this design generally provides a more uniform fit than a side waist region having elongated elastic strands, the outside surfaces of the side waist region are not conducive to having decorative or instructional print displayed thereon because the mechanically stretched material layers are uneven and cannot fully return to their original undistorted configuration.

Disposable absorbent garment having stretchable side waist regions


The present invention relates to a composition comprising a plurality of polynucleotide probes. The composition can be used as array elements in a microarray. The present invention also relates to a method for selecting polynucleotide probes of the composition.

The present invention relates to a composition comprising a plurality of polynucleotide probes for use in research and diagnostic applications.
DNA-based arrays can provide a simple way to explore the expression of a single polymorphic gene or a large number of genes. When the expression of a single gene is explored, DNA-based arrays are employed to detect the expression of specific gene variants. For example, a p53 tumor suppressor gene array is used to determine whether individuals are carrying mutations that predispose them to cancer. The array has over 50,000 DNA probes to analyze more than 400 distinct mutations of p53. A cytochrome p450 gene array is useful to determine whether individuals have one of 18 known polymorphisms of two human cytochrome p450 genes. These polymorphisms can cause increased drug metabolism, drug resistance or drug toxicity.
DNA-based array technology is especially relevant to the rapid screening of expression of a large number of genes. There is a growing awareness that gene expression is affected in a global fashion. A genetic predisposition, disease or therapeutic treatment may affect, directly or indirectly, the expression of a large number of genes. In some cases the interactions may be expected, such as where the genes are part of the same signaling pathway. In other cases, such as when the genes participate in separate signaling pathways, the interactions may be totally unexpected. Therefore, DNA-based arrays can be used to investigate how genetic predisposition, disease, or therapeutic treatment affects the expression of a large number of genes.
It would be advantageous to prepare DNA-based arrays that can be used for monitoring the expression of a large number of genes coding for signaling pathway polypeptides, including different types of receptor, transducer and effector-like polypeptides. The present invention provides for a composition that can be employed in an array-format for detecting changes in expression of a large number of genes coding for different signaling pathway polypeptides.
In one aspect, the present invention provides a composition comprising a plurality of polynucleotide probes, wherein each of said polynucleotide probes comprises at least a portion of a gene coding for a signaling pathway polypeptide. The plurality of polynucleotide probes can comprise I) first polynucleotide probes, wherein each of said first polynucleotide probes comprises at least a portion of a gene coding for a receptor-like polypeptide; II) second polynucleotide probes, wherein each of said second polynucleotide probes comprises at least a portion of a gene coding for a transducing polypeptide; III) third polynucleotide probes, wherein each of said third polynucleotide probes comprises at least a portion of a gene coding for an effector-like polypeptide; or combinations thereof.
More particularly, in one preferred embodiment the composition comprises a plurality of polynucleotide probes wherein each gene coding for a signaling pathway polypeptide is at least a portion of a sequence selected from the group consisting of SEQ ID Nos: 1-1490. In a second preferred embodiment, the composition comprises a plurality of polynucleotide probes comprising at least a portion of at least 1000 of the sequences of SEQ ID Nos: 1-1490. In a third preferred embodiment, the composition comprises a plurality of polynucleotide probes wherein said polynucleotide probes comprise at least a portion of substantially all the sequences of SEQ ID Nos: 1-1490. The polynucleotide probes can be complementary DNAs, clone DNAs and the like.
The composition is particularly useful as hybridizable array elements in a microarray for monitoring the expression of a plurality of target polynucleotides. The microarray comprises a substrate and hybridizable array elements. The microarray of this invention is particularly useful in the diagnosis and treatment of cancer, an immunopathology, a neuropathology and the like.
In another aspect, the present invention encompasses an expression profile that can reflect the levels of a plurality of target polynucleotides in a sample. The expression profile comprises the microarray and a plurality of detectable complexes. Each detectable complex is formed by having at least one of the target polynucleotides hybridizing to at least one of the hybridizable array elements and further comprises a labeling moiety for detection. The expression profile of this invention is particularly useful in the diagnosis and the treatment of cancer, an immunopathology, a neuropathology and the like.
In yet another aspect, the invention provides a method for selecting a plurality of polynucleotide probes, said method comprising (I) obtaining a plurality of query sequences; (II) screening said query sequences against one or more databases comprising annotated sequences to identify sequence hits; and (III) selecting said sequence hits with the highest homology (top hits) to said annotated sequences. The query sequences can be expression sequence tags (ESTs) or full length gene coding sequences, which are electronically screened using preferably the Basic Local Alignment Search Tool (BLAST) algorithm. In one embodiment, the highest homology is identified as a BLAST score equal to or above 100 at a P-value equal to or below 10xe2x88x9210 against the GenPept database. In a second embodiment, the highest homology is identified as a percent sequence identity equal to or above 80% and a BLAST score equal to or above 250 against the GenBank Primate database. In a third embodiment, the highest homology is identified as a percent identity equal to or above 75% and a BLAST score equal to or above 250 against the GenBank Rodent database. In a fourth embodiment, the highest homology is identified as the match with the lowest P-value when searches are performed against GenPept, GenBank Primate or GenBank Rodent databases.
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
The Sequence Listing is a compilation of nucleotide sequences obtained by sequencing clone inserts (isolates) of different cDNA libraries. Each sequence is identified by a sequence identification number (SEQ ID No:), by the clone number from which it was obtained and by the cDNA library from which the sequence was obtained.
Table 1 is a list of the sequences according to their SEQ ID Nos:. For SEQ ID Nos: 1-1049 (homologous to GenBank sequences) the first column contains Incyte clone numbers. The second column contains a relevant GenBank identification number match, if any. The last column contains an annotation associated with the referenced GenBank identification number along with the genus species or source name. For SEQ ID Nos: 1050-1490 (exact matches to GenBank) the first column contains the GenBank identification number. The second column contains an annotation associated with the referenced GenBank identification number along with the genus species or source name.
Table 2 is a list of the cDNA libraries and a description of the preparation of the cDNA libraries.
Definitions
The term xe2x80x9cmicroarrayxe2x80x9d refers to an ordered arrangement of hybridizable array elements. The array elements are arranged so that there are preferably at least about 10 different array elements, more preferably at least 100 array elements, and most preferably at least 1,000 array elements, on a 1 cm2 substrate surface. The maximum number of array elements is unlimited, but is at least 100,000 array elements. Furthermore, the hybridization signal from each of the array elements is individually distinguishable. In a preferred embodiment, the array elements comprise polynucleotide probes.
A xe2x80x9cpolynucleotidexe2x80x9d refers to a chain of nucleotides. Preferably, the chain has from 100 to 10,000 nucleotides, more preferably from 150 to 3,500 nucleotides. The term xe2x80x9cprobexe2x80x9d refers to the ability of the polynucleotide to hybridize with a target polynucleotide to form a polynucleotide probe/target complex. A xe2x80x9ctarget polynucleotidexe2x80x9d refers to a chain of nucleotides to which a polynucleotide probe can hybridize by base pairing. In some instances, the sequences will be complementary (no mismatches). In other instances, there may be a 5% mismatch.
A xe2x80x9cpluralityxe2x80x9d refers preferably to a group of at least 10, more preferably to a group of at least 100, and even more preferably to a group of at least 1,000, members. The maximum number of members is unlimited, but is at least 100,000 members.
A xe2x80x9cportionxe2x80x9d means a stretch of at least 100 consecutive nucleotides. A xe2x80x9cportionxe2x80x9d can also mean a stretch of at least 100 consecutive nucleotides that contains one or more deletions, insertions or substitutions. A xe2x80x9cportionxe2x80x9d can also mean the whole coding sequence of a gene. Preferred portions are those that lack secondary structure as identified by using computer software programs such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth, Minn. LASERGENE (DNASTAR, Madison, Wis.) macDNAsis (Hitachi Software Engineering, South San Fransisco, Calif., and the like.
The term xe2x80x9cgenexe2x80x9d or xe2x80x9cgenesxe2x80x9d (Hitachi Software Engineering, South San Francisco, Calif. refers to the partial or complete coding sequence of a gene. The phrase xe2x80x9cgenes coding for signaling pathway polypeptidesxe2x80x9d refers to genes that code for polypeptides that likely participate in signaling pathways and include those listed in Table 1.
The phrase xe2x80x9cquery sequencesxe2x80x9d refers to sequences whose identity or homology is being investigated. A xe2x80x9cdatabasexe2x80x9d is a repository of information which is preferably accessible by electronic means. xe2x80x9cAnnotated sequencesxe2x80x9d are sequences whose identity has already been determined and preferably exist in a database. The phrase xe2x80x9cpercent sequence identityxe2x80x9d refers to the percentage of identical match found in a comparison of two or more amino acid or nucleic acid sequences.
The Invention
The present invention provides a composition comprising a plurality of polynucleotide probes, wherein each polynucleotide probe comprises at least a portion of a gene coding for a signaling pathway polypeptide (SPP). Preferably, the sequences of the polynucleotide probes are selected from those sequences presented in the Sequence Listing. In one preferred embodiment the composition comprises a plurality of polynucleotide probes wherein each gene coding for a signaling pathway polypeptide is at least a portion of a sequence selected from the group consisting of SEQ ID Nos: 1-1490. In a second preferred embodiment, the composition comprises a plurality of polynucleotide probes comprising at least a portion of at least 1000 of the sequences of SEQ ID Nos: 1-1490. In a third preferred embodiment, the composition comprises a plurality of polynucleotide probes wherein said polynucleotide probes comprise at least a portion of substantially all the sequences of SEQ ID Nos: 1-1490.
The composition is particularly useful when it is used as hybridizable array elements in a microarray. The microarray can be used for large scale genetic or gene expression analysis of a large number of target polynucleotides. The microarrays can be used in the diagnosis of diseases and in the monitoring of treatments where altered expression of SPPs cause disease, such as in cancer, an immunopathology, a neuropathology, and the like. The microarrays can also be used to investigate an individual""s predisposition to a disease, such as cancer, an immunopathology, a neuropathology, and the like.
When the composition of the invention is employed as hybridizable array elements in a microarray, the array elements are organized in an ordered fashion so that each element is present at a specified location on the substrate. Because the array elements are at specified locations on the substrate, the hybridization patterns and intensities (which together create a unique expression profile) can be interpreted in terms of expression levels of particular genes and can be correlated with a particular disease or condition or treatment.
The composition comprising a plurality of polynucleotide probes can also be used to purify a subpopulation of mRNAs, cDNAs, genomic fragments and the like, in a sample. Typically, samples will include the target polynucleotides of interest and other nucleic acids which may enhance the hybridization background in the sample. Therefore it may be advantageous to remove these nucleic acids. One method for removing the additional nucleic acids is by hybridizing the sample containing target polynucleotides with immobilized polynucleotide probes under hybridizing conditions. Those nucleic acids that do not hybridize to the polynucleotide probes are washed away. At a later point, the immobilized target polynucleotide probes can be released in the form of purified target polynucleotides.
Polynucleotide Probes
This section describes the selection of probe sequences for the plurality of polynucleotide probes. The probe sequences are derived from genes that code for signaling pathway polypeptides (SPPs) and can include gene sequences that fit in one of three different functional sequence groups (I through III). As a result, the composition of polynucleotide probes comprises sequences derived from genes of one of these functional sequence groups, the combination of any two of these functional sequence groups or from the combination of all three functional sequence groups. In a preferred embodiment, the composition comprises polynucleotide probes comprising sequences derived from all three functional sequence groups.
The functional sequence groups are divided as follows. Functional sequence group I comprises sequences for genes coding for receptor-like polypeptides. These polypeptides are able to sense the external environment of a cell and initiate a cascade of events. Included in this functional sequence group are binding proteins, receptor tyrosine kinases, G protein receptors, seven transmembrane domain receptors, tyrosine kinase receptors and the like. Functional sequence group II comprises sequences for genes coding for transducing polypeptides. These polypeptides transmit and amplify signals received from the receptor-like polypeptides. Included in this functional group are G proteins, growth and differentiation proteins, serine/threonine phosphatases, tyrosine phosphatases, phosphodiestereases, phospholipases, ras-related proteins, serine/threonine kinases, MAP kinases, adenylyl cyclases and the like. Functional sequence group III comprises sequences for genes coding for effector-like polypeptides. The effector-like polypeptides may perform a cellular function as a result of having sensed the signals from the transducing polypeptides. Included in this functional sequence group are cell matrix adhesion proteins, cell-cell adhesion proteins, ion channels, chemokines, cyclooxygenases, cytokines, hormones, nitric oxide synthases, proteases, protease inhibitors, transcription factors, transporter proteins and the like.
Genes for the functional sequence groups are selected by screening a large number of cDNA libraries, such as those described in Table 2, to discover clone inserts with sequences (listed in the Lifeseq databases) which are matches to genes coding for SPPs. The matches can be exact matches (100% identity) or homologous. As used herein, xe2x80x9chomologousxe2x80x9d refers to sequence similarity between a reference sequence and at least a portion of a newly sequenced clone insert, and can refer to either a nucleic acid or amino acid sequence. Preferably, regions of homology are identified using BLAST (Basic Local Alignment Search Tool). (See Altschul, S. F. (1993) J. Mol. Evol 36: 290-300; and Altschul et al. (1990) J. Mol. Biol. 215: 403-410). BLAST involves first finding similar segments between the query sequence and a database sequence, then evaluating the statistical significance of any matches that are found and finally reporting only those matches that satisfy a user-selectable threshold of significance. Alternatively, other search algorithms can be employed such as FASTA, a rapid sequencing algorithm described by Lippman and Pearson (1988; PNAS 85:2444-2448); ClustalW, a multiple sequence alignment program for DNA or proteins (Thompson et. al. (1994) Nucl. Acid Res. 22: 4673-4680); and the like.
In one preferred embodiment, full length gene coding sequences derived from the clone inserts are used as query sequences against sequences in public databases, such as the GenPept and GenBank databases (human, primate, and rodent databases). These databases contain previously identified and annotated sequences. In another embodiment, expression sequence tags (ESTs) are used as query sequences.
Top hit annotation is then performed. When an alignment between the query sequence and a sequence in any of the databases has a statistically significant score, the query sequence is annotated with the annotation of that sequence (resulting match). Sequences with the same annotation are placed in the same protein function tree, i.e., the tyrosine kinase tree, the serine/threonine kinase tree, the G protein tree and the like. A database employing protein functions to analyze sequence data is disclosed in copending patent application entitled xe2x80x9cDatabase System Employing Protein Function Hierarchies for Viewing Biomolecular Sequence Dataxe2x80x9d, Ser. No. 08/812,290, herein incorporated by reference. Several protein function trees are then combined to form functional sequence groups.
After identifying those sequences that have been annotated to the different protein function groups, polynucleotide probes are generated from these sequences. These sequences are provided in SEQ ID Nos: 1-1490 in the Sequence Listing. Table 1 provides the annotation for the referenced identification number for SEQ ID Nos: 1-1490.
The resulting composition can comprise polynucleotide probes that are not redundant, i.e., there is no more than one polynucleotide probe to represent a particular gene. Alternatively, the composition can comprise polynucleotide probes that are redundant, i.e., a gene is represented by more than one polynucleotide probe.
The selected polynucleotide probes may be manipulated further to optimize the performance of the polynucleotide probes as hybridization probes. Some probes may not hybridize effectively under hybridization conditions due to secondary structure. To optimize probe hybridization, the probe sequences are examined using a computer algorithm to identify portions of genes without potential secondary structure. Such computer algorithms are well known in the art, such as OLIGO 4.06 Primer Analysis software (National Biosciences), LASERGENE (DNASTAR) or MAcDNASIS (Hitachi). These programs can search nucleotide sequences to identify stem loop structures and tandem repeats and analyze the G+C content of the sequence (those sequences with a G+C content greater than 60% are excluded). Alternatively, the probes can be optimized by trial and error. Experiments can be performed to determine whether probes and target polynucleotides hybridize optimally under experimental conditions.
Where the number of different polynucleotide probes is desired to be greatest, the probe sequences are extended to assure that different polynucleotide probes are not derived from the same gene, i.e., the polynucleotide probes are not redundant. The probe sequences may be extended utilizing the partial nucleotide sequences derived from EST sequencing by employing various methods known in the art. For example, one method which may be employed, xe2x80x9crestriction-sitexe2x80x9d PCR, uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2: 318-322).
Polynucleotide Probes
This section describes the polynucleotide probes. The polynucleotide probes can be DNA or RNA, or any RNA-like or DNA-like material. The polynucleotide probes can be sense or antisense polynucleotide probes. Where target polynucleotides are double stranded, the probes may be either sense or antisense strands. Where the target polynucleotides are single stranded, the nucleotide probes are complementary single strands.
In one embodiment, the polynucleotide probes are complementary DNAs (cDNAs). The size of the DNA sequence of interest may vary, and is preferably from 100 to 10,000 nucleotides, more preferably from 150 to 3,500 nucleotides.
In a second embodiment, the polynucleotide probes are clone DNAs. In this case the size of the DNA sequence of interest, i.e., the insert sequence excluding the vector DNA, may vary from 100 to 10,000 nucleotides, more preferably from 150 to 3,500 nucleotides.
The polynucleotide probes can be prepared by a variety of synthetic or enzymatic schemes which are well known in the art. The probes can be synthesized, in whole or in part, using chemical methods well known in the art. (Caruthers et al. (1980) Nucleic. Acids Res. Symp. Ser. (2). Alternatively, the probes can be generated, in whole or in part, enzymatically.
Nucleotide analogues can be incorporated into the polynucleotide probes by methods well known in the art. The only requirement is that most of the incorporated nucleotide analogues must serve to base pair with target polynucleotide sequences. For example, certain guanine nucleotides can be substituted with hypoxanthine which base pairs with cytosine residues. However, these base pairs are less stable than those between guanine and cytosine. Alternatively, adenine nucleotides can be substituted with 2, 6-diaminopurine which can form stronger base pairs than those between adenine and thymidine.
Additionally, the polynucleotide probes can include nucleotides that have been derivatized chemically or enzymatically. Typical chemical modifications include derivatization with acyl, alkyl, aryl or amino groups.
The polynucleotide probes can be immobilized on a substrate. Preferred substrates are any suitable rigid or semirigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which the polynucleotide probes are bound. Preferably, the substrates are optically transparent.
Probes can be synthesized, in whole or in part, on the surface of a substrate by using a chemical coupling procedure and an ink jet application apparatus, such as that described in PCT publication WO95/251116 (Baldeschweiler et al.). Alternatively, the probe can be synthesized using a self-addressable electronic device that controls when reagents are added (Heller et al. U.S. Pat. No. 5,605,662) or by photolysis using imaging fibers for light delivery (Healey et al. (1995) Science 269: 1078-80).
Complementary DNA (cDNA) can be arranged and then immobilized on a substrate. The probes can be immobilized by covalent means such as by chemical bonding procedures or UV. In one such method, a cDNA is bound to a glass surface which has been modified to contain epoxide or aldehyde groups. In another case, a cDNA probe is placed on a polylysine coated surface and then UV cross-linked (Shalon et al. PCT publication WO95/35505, herein incorporated by reference). In yet another method, a DNA is actively transported from a solution to a given position on a substrate by electrical means (Heller et al. U.S. Pat. No. 5,605,662). Alternatively, individual DNA clones can be gridded on a filter. Cells are lysed, proteins and cellular components degraded and the DNA coupled to the filter by UV cross-linking.
Furthermore, the probes do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group. The linker groups are typically about 6 to 50 atoms long to provide exposure to the attached polynucleotide probe. Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with one of the terminal portions of the linker to bind the linker to the substrate. The other terminal portion of the linker is then functionalized for binding the polynucleotide probe.
The polynucleotide probes can be attached to a substrate by dispensing reagents for probe synthesis on the substrate surface or by dispensing preformed DNA fragments or clones on the substrate surface. Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions simultaneously.
Sample Preparation
In order to conduct sample analysis, a sample containing target polynucleotides is provided. The samples can be any sample containing target polynucleotides and obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.
The target polynucleotides can be DNA or RNA. The DNA or RNA can be isolated from the sample according to any of a number of methods well known to those of skill in the art. For example, methods of purification of nucleic acids are described in Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes. Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed. Elsevier Science, New York, N.Y. (1993). In a preferred embodiment, total RNA is isolated using the TRIZOL total RNA isolation reagent (Life Technologies Gaithersburg, Md.) and mRNA is isolated using oligo d(T) column chromatography or glass beads.
Alternatively, the target polynucleotides may be derived from DNA or RNA. When target polynucleotides are derived from an mRNA, the target polynucleotides can be a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from that cDNA, an RNA transcribed from the amplified DNA, and the like. When the target polynucleotide is derived from DNA, the target polynucleotide can be DNA amplified from DNA or RNA reverse transcribed from DNA. In yet another alternative, the targets are target polynucleotides prepared by more than one method.
When target polynucleotides are amplified it is desirable to amplify the nucleic acid sample and maintain the relative abundances of the original sample, including low abundance transcripts. Total mRNA can be amplified by reverse transcription using a reverse transcriptase and a primer consisting of oligo d(T) and a sequence encoding the phage T7 promoter to provide a single stranded DNA template. The second cDNA strand is polymerized using a DNA polymerase and a RNAse which assists in breaking up the DNA/RNA hybrid. After synthesis of the double stranded cDNA, T7 RNA polymerase can be added and RNA transcribed from the second cDNA strand template (Van Gelder et al. U.S. Pat. No. 5,545,522). RNA can be amplified in vitro, in situ or in vivo (See Eberwine U.S. Pat. No. 5,514,545).
It is also advantageous to include quantitation controls within the sample to assure that amplification and labeling procedures do not change the true distribution of target polynucleotides in a sample. For this purpose, a sample is spiked with a known amount of a control target polynucleotide and the composition of polynucleotide probes includes reference polynucleotide probes which specifically hybridize with the control target polynucleotides. After hybridization and processing, the hybridization signals obtained should reflect accurately the amounts of control target polynucleotide added to the sample.
Prior to hybridization, it may be desirable to fragment the nucleic acid target polynucleotides. Fragmentation improves hybridization by minimizing secondary structure and cross-hybridization to other nucleic acid target polynucleotides in the sample or noncomplementary polynucleotide probes. Fragmentation can be performed by mechanical or chemical means.
The target polynucleotides may be labeled with one or more labeling moieties to allow for detection of hybridized probe/target polynucleotide complexes. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means. The labeling moieties include radioisotopes, such as 32P, 33P or 35S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like.
Exemplary dyes include quinoline dyes, triarylmethane dyes, phthaleins, azo dyes, cyanine dyes and the like. Preferably, fluorescent markers absorb light above about 300 nm, preferably above 400 nm, and usually emit light at wavelengths at least greater than 10 nm above the wavelength of the light absorbed. Specific preferred fluorescent markers include fluorescein, phycoerythrin, rhodamine, lissamine, and C3 and C5 available from Amersham.
Labeling can be carried out during an amplification reaction, such as polymerase chain and in vitro transcription reactions, or by nick translation or 5xe2x80x2 or 3xe2x80x2-end-labeling reactions. In one case, labeled nucleotides are used in an in vitro transcription reaction. When the label is incorporated after or without an amplification step, the label is incorporated by using terminal transferase or by kinasing the 5xe2x80x2 end of the target polynucleotide and then incubating overnight with a labeled oligonucleotide in the presence of T4 RNA ligase.
Alternatively, the labeling moiety can be incorporated after hybridization once a probe/target complex has formed. In one case, biotin is first incorporated during an amplification step as described above. After the hybridization reaction, unbound nucleic acids are rinsed away so that the only biotin remaining bound to the substrate is that attached to target polynucleotides that are hybridized to the polynucleotide probes. Then, an avidin-conjugated fluorophore, such as avidin-phycoerythrin, that binds with high affinity to biotin is added. In another case, the labeling moiety is incorporated by intercalation into preformed target/polynucleotide probe complexes. In this case, an intercalating dye such as a psoralen-linked dye can be employed.
Under some circumstances it may be advantageous to immobilize the target polynucleotides on a substrate and have the polynucleotide probes bind to the immobilized target polynucleotides. In such cases the target polynucleotides can be attached to a substrate as described above.
Hybridization and Detection
Hybridization causes a denatured polynucleotide probe and a denatured complementary target to form a stable duplex through base pairing. Hybridization methods are well known to those skilled in the art (See, for example, Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier Science, New York, N.Y. (1993)). Conditions can be selected for hybridization where exactly complementary target and polynucleotide probe can hybridize, i.e., each base pair must interact with its complementary base pair. Alternatively, conditions can be selected where target and polynucleotide probes have mismatches but are still able to hybridize. Suitable conditions can be selected, for example, by varying the concentrations of salt or formamide in the prehybridization, hybridization and wash solutions, or by varying the hybridization and wash temperatures.
Hybridization can be performed at low stringency with buffers, such as 6xc3x97SSPE with 0.005% Triton X-100 at 37xc2x0 C., which permits hybridization between target and polynucleotide probes that contain some mismatches to form target polynucleotide/probe complexes. Subsequent washes are performed at higher stringency with buffers, such as 0.5xc3x97SSPE with 0.005% Triton X-100 at 50xc2x0 C., to retain hybridization of only those target/probe complexes that contain exactly complementary sequences. Alternatively, hybridization can be performed with buffers, such as 5xc3x97SSC/0.2% SDS at 60xc2x0 C. and washes are performed in 2xc3x97SSC/0.2% SDS and then in 0.1xc3x97SSC. Stringency can also be increased by adding agents such as formamide. Background signals can be reduced by the use of detergent, such as sodium dodecyl sulfate, Sarcosyl or Triton X-100, or a blocking agent, such as sperm DNA.
Hybridization specificity can be evaluated by comparing the hybridization of specificity-control polynucleotide probes to specificity-control target polynucleotides that are added to a sample in a known amount. The specificity-control target polynucleotides may have one or more sequence mismatches compared with the corresponding polynucleotide probes. In this manner, whether only complementary target polynucleotides are hybridizing to the polynucleotide probes or whether mismatched hybrid duplexes are forming is determined.
Hybridization reactions can be performed in absolute or differential hybridization formats. In the absolute hybridization format, target polynucleotides from one sample are hybridized to the probes in a microarray format and signals detected after hybridization complex formation correlate to target polynucleotide levels in a sample. In the differential hybridization format, the differential expression of a set of genes in two biological samples is analyzed. For differential hybridization, target polynucleotides from both biological samples are prepared and labeled with different labeling moieties. A mixture of the two labeled target polynucleotides is added to a microarray. The microarray is then examined under conditions in which the emissions from the two different labels are individually detectable. Probes in the microarray that are hybridized to substantially equal numbers of target polynucleotides derived from both biological samples give a distinct combined fluorescence (Shalon et al. PCT publication WO95/35505). In a preferred embodiment, the labels are fluorescent labels with distinguishable emission spectra, such as a lissamine conjugated nucleotide analog and a fluorescein conjugated nucleotide analog. In another embodiment Cy3/Cy5 fluorophores (Amersham) Pharmacia Biotech, Piscataway, N.J. are employed.
After hybridization, the microarray is washed to remove nonhybridized nucleic acids and complex formation between the hybridizable array elements and the target polynucleotides is detected.
Methods for detecting complex formation are well known to those skilled in the art. In a preferred embodiment, the target polynucleotides are labeled with a fluorescent label and measurement of levels and patterns of fluorescence indicative of complex formation is accomplished by fluorescence microscopy, preferably confocal fluorescence microscopy. An argon ion laser excites the fluorescent label, emissions are directed to a photomultiplier and the amount of emitted light detected and quantitated. The detected signal should be proportional to the amount of probe/target polynucleotide complex at each position of the microarray. The fluorescence microscope can be associated with a computer-driven scanner device to generate a quantitative two-dimensional image of hybridization intensity. The scanned image is examined to determine the abundance/expression level of each hybridized target polynucleotide.
In a differential hybridization experiment, target polynucleotides from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengths. The relative abundances/expression levels of the target polynucleotides in two or more samples is obtained.
Typically, microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions. In a preferred embodiment, individual polynucleotide probe/target complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.
Expression Profiles
This section describes an expression profile using the composition of this invention. The expression profile can be used to detect changes in the expression of genes coding for SPPs. These genes include genes whose altered expression is correlated with cancer, immunopathology, neuropathology and the like.
The expression profile comprises the polynucleotide probes of the invention. The expression profile also includes a plurality of detectable complexes. Each complex is formed by hybridization of one or more polynucleotide probes to one or more target polynucleotides. At least one of the polynucleotide probes, preferably a plurality of polynucleotide probes, is hybridized to a target polynucleotide forming, at least one, preferably a plurality of complexes. A complex is detected by incorporating at least one labeling moiety in the complex. The labeling moiety has been described above.
The expression profiles provide xe2x80x9csnapshotsxe2x80x9d that can show unique expression patterns that are characteristic of a disease or condition.
Utility of the Invention
The composition comprising a plurality of polynucleotide probes can be used as hybridizable array elements in a microarray. Such a microarray can be employed in several applications including diagnostics and treatment regimens, drug discovery and development, toxicological and carcinogenicity studies, forensics, pharmacogenomics and the like.
In one situation, the microarray is used to monitor the progression of disease. Researchers can assess and catalog the differences in gene expression between healthy and diseased tissues or cells. By analyzing changes in patterns of gene expression, disease can be diagnosed at earlier stages before the patient is symptomatic.
Similarly, the invention can be used to monitor the progression of disease or the efficacy of treatment. For some treatments with known side effects, the microarray is employed to fine-tune the treatment regimen. A dosage will be established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or manifest symptoms, before altering the course of treatment.
Alternatively, animal models which mimic a disease rather than patients can be used to characterize expression profiles associated with a particular disease or condition. For example, a characteristic gene expression pattern for the graft versus host reaction can be generated using analogous reactions that occur when lymphocytes from one donor are mixed with lymphocytes from another donor. This gene expression data may be useful in diagnosing and monitoring the course of graft versus host reaction in a patient, in determining gene targets for intervention, and in testing novel immunosuppressants.
The microarray is particularly useful for diagnosing and monitoring the progression of diseases that may be associated with the altered expression of SPPs. The expression of SPPs is closely associated with cell proliferation. Thus, the microarray and expression profiles are particularly useful to diagnose a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma and teratocarcinoma. Such cancers include, but are not limited to, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid and uterus.
The expression of SPPs is also closely associated with an immune response. Therefore, the microarray can be used to diagnose immunopathologies including but not limited to AIDS, Addison""s disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitus, Crohn""s disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis, gout, Graves"" disease, hypereosinophilia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sjxc3x6gren""s syndrome, and autoimmune thyroiditis; viral, bacterial, fungal, parasitic, and protozoal infections and trauma.
Neuronal processes are also affected by the expression of SPPs. Thus, the microarray can be used to diagnose neuropathologies including but not limited to akathesia, Alzheimer""s disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia, cerebral neoplasms, dementia, depression, Down""s syndrome, tardive dyskinesia, dystonias, epilepsy, Huntington""s disease, multiple sclerosis, neurofibromatosis, Parkinson""s disease, paranoid psychoses, schizophrenia, and Tourette""s disorder.
The invention also allows researchers to develop sophisticated profiles of the effects of currently available therapeutic drugs. Tissues or cells treated with these drugs can be analyzed using the invention, and compared to untreated samples of the same tissues or cells. In this way, an expression profile of known therapeutic agents will be developed. Knowing the identity of sequences that are differentially regulated in the presence and absence of a drug will allow researchers to elucidate the molecular mechanisms of action of that drug.
Also, researchers can use the invention to rapidly screen large numbers of candidate drugs, looking for ones that have an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects. Thus, the invention provides the means to determine the molecular mode of action of a drug.
It is understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention.

Composition for the detection of signaling pathway gene expression