|Publication number||US7469834 B2|
|Application number||US 11/333,644|
|Publication date||Dec 30, 2008|
|Filing date||Jan 17, 2006|
|Priority date||Jan 17, 2005|
|Also published as||CN1808059A, CN100385199C, DE102005002190A1, DE102005002190B4, US20060158662|
|Publication number||11333644, 333644, US 7469834 B2, US 7469834B2, US-B2-7469834, US7469834 B2, US7469834B2|
|Inventors||Uwe Schelinski, Michael Scholles, Alexander Wolter|
|Original Assignee||Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V.|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (33), Non-Patent Citations (11), Referenced by (3), Classifications (5), Legal Events (5)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This application claims priority from German Patent Application No. 10 2005 002 190.5, which was filed on Jan. 17, 2005, and is incorporated herein by reference in its entirety.
1. Field of the Invention
The present invention relates to the technical field of contactless detection or measurement, respectively, of three-dimensional objects. In particular, the present invention relates to the technical sub-field of scanners for detecting a surface relief by means of optical scanning.
2. Description of Prior Art
Known scanners for short object distances mainly use the method of triangulation, as it is illustrated in
Recently, with the so-called micro scanner mirrors new elastically suspended micro optical members electro-statically excited close to their natural resonance and the associated control electronics are available, as it is for example explained in more detail in the following documents:
A New Driving Principle for Micromechanical Torsional Actuators
H. Schenk, P. Dürr, D. Kunze, H. Kück; Micro-Electro-Mechanical System, MEMS-Vol. 1, Conf.: 1999 int. Mech. Eng. Congr. & Exh., 14-19 Nov. 1999, Nashville, p. 333-338, 1999
A Novel Electrostatically Driven Torsional Actuator
H. Schenk, P. Dürr, H. Kück Proc. 3rd Int. Conf. On Micro Opto Electro Mechanical Systems, Mainz, 30. Aug.-1. Sep. 1999, page 3-10, 1999
Micromirror Spatial Light Modulators
P. Dürr, A. Gehner, U. Dauderstädt, 3rd International Conference on Micro Opto Electro Mechanical Systems (Optical MEMS) Proc. MEMS 1999, Mainz, 1999, S. 60-65
A Resonantly Excited 2D-Micro-Scanning-Mirror with Large Deflection
H. Schenk, P. Dürr, D. Kunze, H. Lakner, H. Kück Sensors & Actuators, 2001 Sensors & Actuators, A 89 (2001), Nr. 1-2, ISSN 0924-4247, S. 104-111
Large Deflection Micromechanical Scanning Mirrors for Linear Scans and Pattern Generation
H. Schenk, P. Dürr, T. Haase, D. Kunze, U. Sobe, H. Lakner, H. Kück Journal of Selected Topics of Quantum Electronics 6, (2000), Nr. 5 ISSN 1077-260X, S. 715-722
An Electrostatically Excited 2D-Micro-Scanning-Mirror with an In-Plane Configuration of the Driving Electrodes
H. Schenk, P. Dürr, D. Kunze, H. Lakner, H. Kück Proc. MEMS 2000, 13th Int. Micro Electro Mechanical Systems Conf, Miyazaki, Japan, page 473-478, 2000
Mechanical and Electrical Failures and Reliability of Micro Scanning Mirrors
E. Gaumont, A. Wolter, H. Schenk, G. Georgelin, M. Schmoger 9th Int. Symposium on the physical and failure analysis of integrated circuits (IPFA 9), 8-12 Jul. 2002, raffles City Convention Centre, Singapore, Proc. New York, IEEE Press, 2002, ISBN 0-7803-7416-9, S. 212-217
Improved Layout for a Resonant 2D Micro Scanning Mirror with Low Operation Voltages
A. Wolter, H. Schenk, E. Gaumont, H. Lakner, SPIE Conference on MOEMS Display and Imaging Systems (mf07), 28-29 Jan. 2003, San Jose, Calif., USA, Proceedings, Bellingham, Wash.: SPIE, 2003 (SPIE Proceedings Series 4985) ISBN 0-8194-4785-4, S. 72-74
Mikromechanisches Bauelement (Micro-mechanical device)
WO002000025170A1, Mikromechanisches Bauelement Mit Schwingkorper (Micro-mechanical Device With Vibrating Body) EP000001123526B1, US000006595055B1
Mikromechanisches Bauelement Mit Einstellbarer Resonanzfrequenz (Micro-mechanical Device With Settable Resonance Frequency)
Driver ASIC for Synchronized Excitation of Resonant Micro-Mirror
K.-U. Roscher, U. Fakesch, H. Schenk, H. Lakner, D. Schlebusch, SPIE Confernece on MOEMS Display and Imaging Systems (mf07), 28-29 Jan. 2003, San Jose, Calif., USA, Proceedings, Bellingham, Wash.: SPIE, 2003 (SPIE Proceedings Series 4985) ISBN 0-8194-4785-4, S. 121-130
The class of MOEMS (MOEMS=Micro Opto Electromechanical Systems) allow to deflect light beams in an electronically controlled way one- or two-dimensional so that using points-shaped light sources or detector elements, respectively, an area or a solid angle may sequentially be scanned or sweeped (scanning).
For projection purposes the use of resonant micro-mirrors already represents a known solution, which may for example be seen from the following documents:
DE 000019615685 A1
Low Cost Projection Device with a 2-Dimensional Resonant Micro Scanning Mirror
K.-U. Roscher, H. Grätz, H. Schenk, A. Wolter, H. Lakner MEMS/MOEMS display and imaging systems II (2004), pp. 22-31
WO002003032046A1, Projektionsvorrichtung US020040218155A1,
Also the projection purposes mirrors are used in another way, for example moved in a rotating way according to DE 000010304187A1, DE000010304188A1 and WO002004068211A1 or be already mentioned DMD members according to EP 000000927334B1, US00000661134B1 or DE 000019810495A1 for generating light patterns.
One possibility for a one-dimensional detection of a position of a light beam was presented in “Torsional stress, fatigue and fracture strength in silicon hinges of a micro scanning mirror” of A. Wolter, H. Schenk, H. Korth and H. Lackner (SPIE Bellingham Wash. 2004; Proceedings of SPIE Vol. 5343). This one-dimensional detection of a position of a light beam only offers a coarse and delayed possibility for a determination of the position of the light beam, as the described method requires a complete pass of the light beam between two oscillation amplitude maxima of a travel path of the light beam.
Further, for the application area of the following invention, still the further documents are relevant:
EP000000999429A1 Messinstrument für 3D Form mit Laser Scanner und Digitalkamera (Measurement Instrument for 3D-form with laser scanner and digital camera
US020030202691A1 Calibration of multiple cameras for a turntable-based 3D scanner
US000006486963B1 Precision 3D scanner base and method for measuring manufactured parts
DE000019846145A1 Verfahren und Anordnung zur 3D-Aufnahme (Method and Arrangement for 3D-Recording)
DE000019613978A1 Verfahren zum Zusammenfügen der Messdaten unterschiedlicher Ansichten und Objektbereiche bei der optischen 3D-Koordinatenmeβtechnik mittels flächenhaft und auf der Basis von Musterprojekten arbeitenden Triangulationssensoren (Method for joining the measurement data of different views and object areas in the optical 3D-coordinate measurement technology by means of triangulation sensors operating in a laminar way and on the basis of sample projection)
DE000019536287A1 Verfahren zur geometrischen Kalibrierung von optischen 3D-Sensoren zur dreidimensionalen Vermessung von Objekten und Vorrichtung hierzu (Method for a geometric calibration of optical 3D sensors for a 3-dimensional measurement of objects and devices for the same)
DE000019536294A1 Verfahren zur geometrischen Navigation von optischen 3D-Sensoren zum dreidimensionalen Vermessen von Objekten (Method for a geomectrical navigation of optical 3D sensors for a 3-dimensional measurement of objects)
EP000001371969A1 Ausrichtungsverfahren zum Positionieren von Sensoren für 3D-Meβsysteme (Alignment method for positioning sensors for 3D measurement systems)
WO002000077471A1 Vorrichtung zur Berührungslosen Dreidimensionalen Vermessung von Körpern und Verfahren zur Bestimmung eines Koordinatensystems für Messpunktkoordinaten (Device for a contactless 3-dimensional measurement of bodies and methods for determining a coordinate system for measurement point coordinates)
EP000000916071B1 Triangulation-Based 3D Imaging And Processing Method And System
US000005546189A Triangulation-based 3D imaging and processing method and system
US000005654800A Triangulation-based 3D imaging and processing method and system
WO001998005923A1 Triangulation-Based 3D Imaging And Processing Method And System
CA000002365323A1 Method Of Measuring 3D Object And Rendering 3D Object Acquired By A Scanner
DE000019721903C1 Verfahren und Anlage zur meβtechnischen räumlichen 3D-Lageerfassung von Oberflächenpunkten (Method and apparatus for a measurement-technical spatial 3D position detection of surface points)
CA000002376103A1 Active Structural Scanner For Scanning In 3D Mode Data Of Unknown Structures
However, all these prior-art approaches are of disadvantage in that they require either an area camera or at least a line camera for detecting the image reflected by the object or may only detect a position of the image by means of a complicated control of micro mirrors having micromechanical electric motors in order to detect the three-dimensional surface relief therefrom. In addition, the solution of the complicated control of the micro mirrors has the additional disadvantage that this control is mechanically sensitive, area-consuming and, additionally, expensive. Also, using an area or line camera requires considerable space and is also expensive.
Thus, it is an object of the present invention to provide a possibility of detecting a three-dimensional surface relief, wherein this possibility should, compared to the prior art, have a better resolution and be cheaper, mechanically more robust and consume less space.
In accordance with a first aspect, the present invention provides a scanner for providing a possibility of detecting a surface relief of an object, having: a projector configured to guide a light beam in an illumination line over the surface relief to obtain an illuminated location on the surface relief, the projector also being configured to output a projection signal from which a position of the light beam in the illumination line is derivable; and a collector having a collector micro mirror stimulatable to oscillate in two dimensions and a point-shaped light detector, the collector micro mirror being oscillatingly arranged in a first direction of the illumination line and in a second direction differing from the first direction such that a reflection of the illuminated location within a scan area of the collector micro mirror is imagable by it onto the point-shaped light detector, and the collector being configured to output a detection signal from which a position of the illuminated location in the first and second directions is derivable.
In accordance with a second aspect, the present invention provides a method for operating a scanner for providing a possibility of detecting a surface relief of an object, the scanner having a projector configured to guide a light beam in an illumination line over the surface relief to obtain an illuminated location on the surface relief, the projector further being configured to output a projection signal from which a position of the light beam in the illumination line is derivable; and having a collector having a collector micro mirror stimulatable to oscillate in two dimensions and a point-shaped light detector, the collector micro mirror being oscillatingly arranged in a first direction of the illumination line and in a second direction differing from the first direction such that a reflection of the illuminated location within a scan area of the collector micro mirror is imagable by it onto the point-shaped light detector, and the collector being configured to output a detection signal from which a position of the illuminated location in the first and second directions is derivable, wherein the method has the following steps: providing and guiding, in the shape of lines, the light beam in an illumination line over the surface relief of the object to obtain an illuminated location on the surface relief, the step of guiding the light beam also including determining a position of the light beam in the illumination line and outputting a projection signal from which the position of the light beam in the illumination line is derivable; and detecting a position of the illuminated location using the collector micro mirror stimulated to oscillate in two dimensions and the point-shaped light detector, the step of detecting including imaging a reflection of the illuminated location onto the light detector by means of the collector micro mirror oscillating in the first and second directions, and the step of detecting also including outputting a detector signal from which the position of the illuminated location in the first and second directions is derivable.
In accordance with a third aspect, the present invention provides a computer program having a program code for performing the above-mentioned method when the computer program runs on a computer
The present invention is based on the idea of illuminating in the shape of lines, by the projector, the surface relief to be detected by a light beam in an illumination line such that a light point is guided over the surface relief in the shape of lines. It is now possible by means of the collector having the collector micro mirror stimulatable to oscillate in two dimensions and the point-shaped light detector to detect the position of the light point on the surface relief in a direction of the illumination line and also in a direction arranged perpendicularly thereto in a reference plane of the object, wherein, by means of the alterable height profile of the surface relief, the reflection of the light point on the surface relief cannot only be detected in the direction of the illumination line, but also in a direction perpendicular to the illumination line. Knowing the position of the light beam in the illumination line, the height profile of the surface relief may be calculated from the offset of the light point compared to an axis defined by the illumination line.
Advantageously, the surface relief of an object can be detected through knowing the position of the reflection of the light point on the surface relief and information on a position of the light beam in the illumination line, and at the same time this detection of the surface relief may be executed in an area-saving, cheap and mechanically robust way. The inventive approach may be realized in an area-saving way particularly because area cameras or line cameras need no longer be used, but a very compact setup of the collector is possible through the collector mirror stimulatable to oscillate in connection with the point-shaped light detector. Another advantage of the inventive approach is that a complicated and thus expensive control of the collector micro mirror, as would, for example, be possible by means of small electric motors, is no longer required due to the usage of a collector micro mirror stimulatable to oscillate. Furthermore, the inventive approach has the advantage that, by avoiding the area or line camera, a considerably cheaper detection unit, such as, for example, in the form of an individual photodiode, may be employed, which is how the manufacturing costs of a corresponding collector may be reduced. The fact that a complicated control by means of, for example, micromechanical electric motors, may be omitted, allows a considerably mechanically more robust construction.
In the following, some embodiments of the present invention are explained in more detail with reference to the accompanying drawings, in which:
In the Figures, same or similar elements are designated by same or similar reference numerals, wherein a repeated description for those elements is omitted.
The first embodiment of the inventive scanner is schematically illustrated in
According to the first embodiment of the present invention, micro mirrors may thus be used, when applying the known triangulation method, as a scanner for the 3D detection of objects. Micro scanner mirrors find application according to the first embodiment both for a projection of a structured illumination onto the object and within the light receiver (i.e. the collector 16) for the detection of the back-scattered light.
What is new, in particular, is that, for the image acquisition in the collector, a point-shaped light detector 18 (or also light sensor) is used in combination with a two-dimensional oscillating micro mirror which defines the respective “viewing direction” of the detector via its momentary displacement. Corresponding to
In order to detect the surface relief of the object to be scanned, there will further be used a procedure as explained in more detail in the following. When using micro mirrors, for example for the illumination of the object and for the detection of the backscattered light, what must basically be taken into consideration is that each of the oscillating mirrors is only directed to exactly one point (spot) of the object at any time. Therefore, it is preferred that both mirrors are controlled such that the detector may detect the spot generated by the projector on the surface relief of the object.
In order to detect this generated spot (i.e. the illuminated place marked with the reference numerals 22 or 22′, respectively, in
The principle of scanning with preferably two mirrors parallel to the triangulation plane may thus be represented as follows, wherein the term “triangulation plane” means the plane defined by the centers of the image field in the reference plane and both mirrors and/or by the triangulation angle:
At the collector, a framelike shutter provided with LEDs according to
A similar shutter is attached to the projector which, however, carries two opposing photodiodes instead of the LEDs, which, when illuminated by the spot, provide a signal serving to calculate amplitude and phase of the y oscillation of the projector micro mirror and, at the same time, limiting the displacement of the spot.
A second step 112 involves outputting a projection signal from which the position of the light beam in the illumination line may be derived.
Subsequently, a third step 114 involves detecting an illuminated place of the surface relief using a micro mirror in the collector stimulated to oscillations.
A fourth step 116 involves outputting a detection signal from which a position of the illuminated place on the surface relief may be derived. A final step 118 of the embodiment of the inventive method involves processing the projection signal and the detection signal to acquire the surface relief therefrom.
In summary, it may be said that a novel 3D scanner is disclosed herein provided with a projector (preferably with a point-shaped light source and a micro scanner mirror) and a collector, wherein the collector includes a micro scanner mirror and a point-shaped light detector on which a reflection of a light point from a surface relief of an object to be detected may be projected by the micro scanner mirror. Further, a corresponding electronic circuit with an interface to a host computer for controlling and further processing of the obtained data may be provided. Furthermore, a method for stimulating the two micro scanner mirrors of the 3D scanner is disclosed herein such that the spot generated by the projector micro scanner mirror may actually be found in the detection area via the collector micro scanner mirror and the light detector, by way of the projector mirror oscillating only in a direction perpendicular to the triangulation plane with a defined frequency and amplitude, the collector micro mirror being stimulated preferably synchronously and with the same amplitude preferably also perpendicular to the triangulation plane, and the collector mirror furthermore performing a second oscillation in the triangulation plane at the same time to detect the displacement of the spot caused by the measuring principle.
Furthermore, a device, for example for the 3D scanner, is disclosed herein, preferably allowing a synchronization of the oscillations of both mirrors in the direction perpendicular to the triangulation plane according to the stimulation method, consisting of a shutter with photodiodes in the optical train of the projector and/or a shutter designed analogously but provided with LEDs in the optical train of the collector by which signals on the momentary amplitudes and phases of one or both oscillations of the micro scanner mirrors may be obtained. Furthermore, an electronic circuit is disclosed which may influence the control of the mirrors in a regulating way, for example to influence control of the frequency, phase or amplitude of a stimulation of a micro mirror, for example to increase a line density of the Lissajous figure and thereby increase the probability of finding the reflection of the light point.
The invention described herein therefore has the advantage of being able to operate without area or line camera so that no area or line image sensors and no corresponding associated complex mapping optics are required. Furthermore, a micro scanner mirror is small, mechanically robust and may be manufactured at a low price, whereby the central advantages of the present invention present themselves in a reduction of the space requirements and the manufacturing costs as well as an increase in mechanical robustness. The described circuit for processing a faint light signal further allows an increase in resolving power. Thus, the described 3D scanner may continue to be constructed in a spatially very compact way. The required signal processing, for example the detection of the laser spot in the sensor data stream, may, at least in part, be realised in hardware and may, for example, be integrated in the scanner, whereby the corresponding computational effort for processing the obtained data to a controlling host computer may be significantly reduced and thus the detection of the surface relief of the corresponding object may be significantly accelerated and the complexity of corresponding algorithms to be performed in the host computer becomes possible. Thus, image processing operations for the extraction of areas of interest from a 2D image are not necessary.
Depending on the circumstances, the inventive method for operating a scanner may be implemented in either hardware or software. The implementation may be carried out on a digital storage medium, particularly a disk or CD with control signals that may be read out electronically, which may cooperate with a programmable computer system so that the corresponding method is performed. Generally, the invention thus also consists in a computer program product with a program code stored on a machine readable carrier for performing the inventive method, when the computer program product runs on a computer. In other words, the invention may thus be realised as a computer program with a program code for performing the method, when the computer program runs on a computer.
While this invention has been described in terms of several preferred embodiments, there are alterations, permutations, and equivalents which fall within the scope of this invention. It should also be noted that there are many alternative ways of implementing the methods and compositions of the present invention. It is therefore intended that the following appended claims be interpreted as including all such alterations, permutations, and equivalents as fall within the true spirit and scope of the present invention.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US5546189||May 19, 1994||Aug 13, 1996||View Engineering, Inc.||Triangulation-based 3D imaging and processing method and system|
|US5654800||Jul 29, 1996||Aug 5, 1997||General Scanning Inc,||Triangulation-based 3D imaging and processing method and system|
|US6215481 *||Sep 16, 1998||Apr 10, 2001||Minolta Co., Ltd.||Display apparatus|
|US6421132 *||Oct 15, 1999||Jul 16, 2002||Vladimir M. Brajovic||Method and apparatus for rapid range imaging|
|US6486963||Jun 20, 2000||Nov 26, 2002||Ppt Vision, Inc.||Precision 3D scanner base and method for measuring manufactured parts|
|US6501554||Jun 20, 2000||Dec 31, 2002||Ppt Vision, Inc.||3D scanner and method for measuring heights and angles of manufactured parts|
|US6595055||Dec 15, 1998||Jul 22, 2003||Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten||Micromechanical component comprising an oscillating body|
|US6611343||Aug 29, 1997||Aug 26, 2003||Gf Messtechnik Gmbh||Method and device for 3D measurement|
|US20030202691||Apr 24, 2002||Oct 30, 2003||Paul Beardsley||Calibration of multiple cameras for a turntable-based 3D scanner|
|US20040183149||Jul 26, 2001||Sep 23, 2004||Harald Schenk||Micromechanical device|
|US20040218155||Apr 5, 2004||Nov 4, 2004||Fraunhofer-Gesellschaft zur Forderungder Angewandten||Projection apparatus|
|CA2365323A1||Dec 14, 2001||Jun 14, 2003||Cedara Software Corp.||Method of measuring 3d object and rendering 3d object acquired by a scanner|
|CA2376103A1||Jul 20, 2000||Mar 1, 2001||Sonem Gmbh||Active structural scanner for scanning in 3d mode data of unknown structures|
|DE10149750A1||Oct 9, 2001||Sep 19, 2002||Tecmath Ag||Imaging, measuring at least part of surface of at least one three-dimensional object involves computing 3D information continuously using multiple acquisition units and self-calibration data|
|DE10304187A1||Jan 29, 2003||Aug 19, 2004||Iqsun Gmbh||3D-Scanner|
|DE10304188A1||Jan 29, 2003||Aug 19, 2004||Iqsun Gmbh||Three-dimensional scanner has rotor consisting at least partly of transparent material and multiple parts and inclined rotatable mirror in form of mirroring on surface of rotor part|
|DE19536294A1||Sep 29, 1995||Apr 3, 1997||Daimler Benz Ag||Geometric navigation of optical three=dimensional sensors for 3-D measurement of objects|
|DE19613978A1||Apr 9, 1996||Oct 16, 1997||Gom Ges Fuer Optische Mestechn||Joining together method of measurement data of different views and object ranges|
|DE19615685A1||Apr 19, 1996||Oct 23, 1997||Conrad Electronic Gmbh||Three-dimensional scanner with CCD cameras|
|DE19721688A1||May 23, 1997||Sep 10, 1998||Willytec Konstruktion Und Vert||Surface detection unit for obtaining 3D data using the triangulation method|
|DE19721903C1||May 26, 1997||Jul 2, 1998||Aicon Industriephotogrammetrie||Spatial three dimensional position detection method of surface points using scanner and electronic scanner|
|DE19810495A1||Mar 11, 1998||Sep 23, 1999||Bernd Schleifenbaum||Optical three-dimensional sensor with very high measurement speed and accuracy for three-dimensional measurement of work piece surfaces|
|DE19846145A1||Oct 1, 1998||Apr 20, 2000||Klaus Koerner||Three-dimensional imaging device for shape measurement has transmitter array whose elements move in straight, parallel lines|
|EP0999429A1||Nov 4, 1999||May 10, 2000||Masahiro Watanabe||Three-dimentional shape measuring instrument with laser scanner and digital camera|
|EP1371969A1||May 12, 2003||Dec 17, 2003||General Electric Company||Sensor alignment method for 3D measurement systems|
|WO1998005923A1||Aug 5, 1996||Feb 12, 1998||View Engineering, Inc.||Triangulation-based 3d imaging and processing method and system|
|WO1998012501A1||Aug 29, 1997||Mar 26, 1998||Gf Messtechnik Gmbh||Method and device for 3d measurement|
|WO2000025170A1||Dec 15, 1998||May 4, 2000||Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.||Micromechanical component comprising an oscillating body|
|WO2000077471A1||Jun 6, 2000||Dec 21, 2000||MPT Präzisionsteile GmbH Mittweida||Device for the contactless three-dimensional measurement of bodies and method for determining a co-ordinate system for measuring point co-ordinates|
|WO2003010545A1||Jul 26, 2001||Feb 6, 2003||Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.||Micromechanical component|
|WO2003032046A1||Oct 5, 2001||Apr 17, 2003||Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.||Projection device|
|WO2004068211A1||Jan 22, 2004||Aug 12, 2004||Faro Technologies Gmbh||3d scanner|
|WO2004092745A1||Apr 15, 2003||Oct 28, 2004||Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.||Micromechanical component having an adjustable resonance frequency|
|1||Alexander Wolter et al.: "Improved layout for a resonant 2D Micro Scanning Mirror with low operation voltages", MOEMS Display and Imaging Systems, Proceedings of SPIE, vol. 4985, SPIE 2003, pp. 72-82.|
|2||Alexander Wolter et al.: "Torsional stress, fatigue and fracture strength in silicon hinges of a micro scanning mirror", SPIE Symposium, Jan. 26-28, 2004, 10 pages.|
|3||Eric Gaumont et al.: "Mechanical and electrical failures and reliability of Micro Scanning Mirrors", 9th Int. Symposium on the physical and failure analysis of integrated circuits, Singapore, Jul. 8-12, 2002, IEEE Press, 2002, pp. 212-217.|
|4||Harald Schenck et al.: "A Novel Electrostatically Driven Torsional Actuator", Proc. 3rd Int. Conf. On Micro Opto Electro Mechanical Systems, Mainz, Aug. 30-Sep. 1, 1999, pp. 3-10.|
|5||Harald Schenk et al.: "A New Driving Principle For Micromechanical Torsional Actuators", Micro-Electro-Mechanical System , MEMS-vol. 1, Conf.: 1999 Int. Mech. Eng. Congr. & Exh., Nov. 14-19, Nashville (1999), pp. 333-338.|
|6||Harald Schenk et al.: "A Resonantly Excited 2D-Micro-Scanning-Mirror With Large Deflection", Sensors & Actuators, vol. A89, 2001, No. 1-2, pp. 104-111.|
|7||Harald Schenk et al.: "An Electrostatically Excited 2D-Micro-Scanning-Mirror With An In-Plane Configuration Of the Driving Electrodes", Proc. MEMS 2000, 13th Int. Micro Electro Mechanical Systems Conf., Miyazaki, Japan, 2000, pp. 473-478.|
|8||Harald Schenk et al.: "Large Deflection Micromechanical Scanning Mirrors for Linear Scans and Pattern Generation", Journal of Selected Topics of Quantum Electronics, vol. 6, 2000, No. 5, pp. 715-722.|
|9||Kai-Uwe Roscher et al.: "Driver ASIC for synchronized excitation of resonant Micro-Mirrors", MOEMS Display and Imaging Systems, Proceedings of SPIE, vol. 4985, SPIE 2003, pp. 121-130.|
|10||Kai-Uwe Roscher et al.: "Low cost projection device with a 2-dimensional resonant micro scanning mirror", MEMS/MOEMS display and imaging systems II, 2004, pp. 22-31.|
|11||Peter Dürr et al.: "Micromirror Spatial Light Modulators", 3rd Int. Conference on Micro Opto Electro Mechanical Systems (Optical MEMS), Proc. MEMS 1999, pp. 60-65.|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US9285210||Feb 4, 2011||Mar 15, 2016||Poly-Clip System Gmbh & Co. Kg||Method for measuring a storage frame|
|US9494418||Aug 3, 2009||Nov 15, 2016||SIRONA DENTAL SYSTEMS, GmbH||3D dental camera for recording surface structures of an object measured by means of triangulation|
|US20110196649 *||Feb 4, 2011||Aug 11, 2011||Poly-Clip System Gmbh & Co. Kg||Method for measuring a storage frame|
|U.S. Classification||235/462.35, 235/462.25|
|May 16, 2007||AS||Assignment|
Owner name: FRAUNHOFER-GESELLSCHAFT ZUR FOERDERUNG DER ANGEWAN
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SCHELINSKI, UWE;SCHOLLES, MICHAEL;WOLTER, ALEXANDER;REEL/FRAME:019301/0227
Effective date: 20060130
|Aug 13, 2012||REMI||Maintenance fee reminder mailed|
|Nov 15, 2012||FPAY||Fee payment|
Year of fee payment: 4
|Nov 15, 2012||SULP||Surcharge for late payment|
|Jun 28, 2016||FPAY||Fee payment|
Year of fee payment: 8


text: Riding on the success of its Phantom Series case designs, particularly of the ones that are white with a dash of black, NZXT unveiled the Switch 810 ATX full-tower chassis (model: CA-SW810-W1) at CES. The Switch 810 uses many of the "sharp angles" design elements of the Phantom series. It is characterized by a large acrylic window on its side panel, through which you can admire your creation. According to the placard, it measures 235 x 595 x 585 mm (W x H x D), its motherboard tray is capable of holding large XL-ATX and EATX motherboards. It has four 5.25" exposed drive bays, NZXT populated two of these in its demo rig with Sentry Mix fan-controllers (not included with the case). The bottom-most of these includes a 3.5"/2.5" docking bay to hold and hot-swap one drive. The dock is detachable to vacate the 5.25" bay. The front-panel cluster is located behind a door above the 5.25" bays. Internally, the case is said to have about six 3.5"/2.5" bays. Moving on to ventilation, the case has provision for two 140 mm intake fans in the front, one is included with the case; up to three 140 mm exhaust fans on the top, one included; one 140 mm rear exhaust (included); two internal circulatory fans at the HDD bays, one included; and provision for two fans on the bottom. As an XL-ATX capable case, the Switch 810 has 9 expansion slot bays. This case will be launched early next week.

domain: hobby


text:
Colombian Mines Corporation (TSXV:CMJ) has signed a subscription agreement with International Finance Corporation whereby IFC will make a non-brokered private placement in the Company for gross proceeds of $1 million dollars.
As quoted in the press release:
The placement will consist of 3,333,333 units (the “Units”) at a price of $0.30 per Unit for gross proceeds of $1 million dollars. Each Unit will consist of one common share of stock and one full share purchase warrant. Each full warrant will entitle the holder to purchase one additional share of the Company’s common stock for five years at an exercise price of $0.45 per share. The shares will be subject to a mandatory four month hold period.
|Otis Gold Corp. (TSXV:OOO) is a near-term producer with an NI 43-101 resource 520,000 ounces of drill-indicated gold at its Kilgore Project in Idaho. Work is scheduled to begin soon at the North Target area of the Kilgore Gold Project. Connect with Otis Gold Corp and never miss a catalyst.|

domain: news


text:
Chapter 7: The Media, Government and Politics
8.1|What is the Media?
- Describe the power problem
- What's the main problem within Congress?
- How was Congress designed to act?
- Because Congress was intended as the first branch of government by the Founders what were the two effects of that decision?
- How has the power of Congress evolved over time?
- What two responsibilities does modern Congress focus on?
- What are the reasons Congress lost power to the Executive branch?
- Describe the separation of powers as it relates to Congress, lawmaking and the other branches
- Powers that Senate has
- Enumerated v. implied powers
- Be familiar with the list of enumerated powers that are most relevant to modern America
- What article of the Constitution lists Congress' powers?
- Necessary and proper clause/elastic clause
- McCulloch v. Maryland

domain: law


The Ukrainians are a British band, which plays traditional Ukrainian music, heavily influenced by western post-punk.
The Ukrainians were formed in 1990 by Wedding Present guitarist Peter Solowka, with singer/violinist Len Liggins and mandolin player Roman Remeynes, after all three had played on the Wedding Present's Ukrainian John Peel Sessions recordings (Ukrayinski Vystupy v Johna Peela, released 1989). Following the success of that release, the trio began composing and recording in Ukrainian as a separate band.
In 1991, their first EP, Oi Divchino, was awarded Single of the Week by British music weekly, NME. Notably, the video for this release was filmed in pre-revolution Kiev making them the first western band to produce a video entirely in Eastern Europe.
In the same year, Solowka left the Wedding Present, later claiming that he had been kicked out, the success of the Ukrainian project making him the scapegoat for the band's lack of mainstream success.
Since 1991, the band have released five studio albums as well as various live albums and EPs, all without chart success in the UK. They continue to tour regularly, especially in Poland and England. They celebrated 20 years of the band in 2011 with a tour of British Ukrainian clubs.


text:
President Bush apologized to troops face to face on Friday for shoddy conditions they have endured at Walter Reed Army Medical Center. He shook the artificial hand of a lieutenant and cradled a newborn whose daddy is nursing his remaining, severely injured leg back to health....But Steve Robinson with Veterans for America tells a different story.
"I was at Walter Reed yesterday. Within 10 minutes I was encircled by about 15 soldiers having problems with their medical discharge, telling me they needed to get in touch with their congressman or their senator," Robinson said. "The system is broke," he said. "We need him (Bush) to be personally affected by it."


text:
STORY PLANET is a non-profit storymaking centre dedicated to helping young people discover and develop their voices. Their innovative programs ignite students’ imaginations, fuel their curiosity and equip them with the tools they need to launch their stories. Their retail store, with an outer space and extraterrestrial theme, helps to fund their creative writing and arts programs for neighbourhood kids.
They are currently revamping their retail space and seeking to work with handmade vendors who can supply items which work with their theme (like aliens, rockets, of space-themed wares), who might like to rent space in the store or sell on consignment with the fee going back into their kids writing and arts programming. They are especially keen on finding local vendors since they are all about helping the community. If you're interested, please contact Mahira Karim!
STORY PLANET is in the Bloor and Dufferin neighbourhood:
1165 Bloor Street West, Toronto Ontario, M6H 1M9


text:
Barely a day as Imo State Governor, Newly sworn in Imo State Governor, Emeka Ihedioha commenced the demolition of monuments built by the immediate past governor, Rochas Okorocha.
This is his first action as Governor of the state.
The Akachi tower which is one of the many tourist attractions built by Rochas Okorocha is being demolished as at the time this report is filed.
The tower was built on a reclaimed dumpsite that was abandoned by previous administrations.
The demolition was supervised by soldiers who stopped Journalists from getting close or covering the exercise.
The monument was one of the structures commissioned by Vice President, Yemi Osinbajo barely a week ago.
The monument, which is a massive tower with the symbol of a hand on a zenith, representing the hand of God on the state, sits on an expansive plot of land, reserved for religious purposes.
There are fears that the Governor will also demolish other monuments in Owerri, especially the famous Statues built by ex Governor Rochas Okorocha.
Pictures from the demolition exercise below


text:
This suggests that PDGF A and its receptor may be a paracrine ligand receptor pair involved in myofibroblast differentiation
Subsequently, sections had been This suggests that PDGF A and its receptor may be a paracrine ligand receptor pair involved in myofibroblast differentiation, This suggests that PDGF A and its receptor may be a paracrine ligand receptor pair involved in myofibroblast differentiation, This suggests that PDGF A and its receptor may be a paracrine ligand receptor pair involved in myofibroblast differentiation blocked in a 5% non fat dry milk for one h and then incubated with the rat anti CD68 antibody right away at four C. Since TLR4 mice are character ized by lowered expression of mucosal Cox 2 and PGE 2, we hypothesized that exogenous administration of PGE two would bypass the defense towards colitis related tumorigenesis in TLR4 mice. We tried two dif ferent doses of PGE 2 remedy for the duration of the recovery period of time. The doses of PGE two have been determined primarily based on our previ ous examine, which showed that 200 ug of PGE 2 was adequate to induce intestinal epithelial mobile proliferation in TLR4 deficient mice. 1st, we examined the incidence of dysplasia at working day fifty six. Substantial dose but not lower dose PGE 2 therapy resulted in an improve in dysplasia incidence in TLR4 mice. When compared to 28. six% of PBS treated TLR4 mice that build dysplasia, 75% of the large dose group and 33. three% of the lower dose group devel oped dysplastic lesions. In comparison, the incidence of dysplasia in WT mice was 92. 3%. When the amount of dysplastic lesions for each mouse was examined, a considerable boost of dysplastic lesions was noticed in the higher dose group.
Nevertheless, this increase in the amount of dysplastic lesions was not found in the minimal dose team. Up coming we examined regardless of whether PGE 2 remedy influenced the dimensions of the dysplastic lesion. PGE two remedy enhanced the dimensions of the dysplastic lesions. The common dimensions of the lesions in the high dose team was substantially higher than that in the PBS treated controls. These dysplastic lesions, how at any time, were nonetheless more compact than the lesions in WT mice. Each lesion in the minimal dose group was larger than any lesion located in PBS taken care of controls, but the big difference did not produce statistical importance. These benefits sug gest that TLR4 mediated up regulation of mucosal PGE 2 in the course of the recovery phases of colitis might be responsible for the improvement and progress of colitis linked neoplasia. PGE 2 supplementation during ongoing mucosal harm does not affect the improvement of dysplasia in TLR4 mice We have previously proven that PGE two supplementation restores the defective mucosal restore of TLR4 mice throughout acute DSS treatment. When we when compared mucosal PGE two generation amongst the acute phase and the continual period of colitic WT mice, the mice in the acute period of colitis had substantially larger generation of mucosal PGE two than the mice in the long-term inflammatory section.
Consequently, improved mucosal PGE 2 production may possibly have diverse roles throughout the acute and the long-term phases of colitis. We as a result examined whether exogenous administration of PGE 2 for the duration of DSS treatment also enhanced the incidence of colitis connected neopla sia. Twenty % of the mice handled with PGE two in the course of DSS treatment method produced dysplasia, even though 28. 6% of PBS dealt with management mice experienced dys plasia. The average number of dysplastic lesions for each mouse was . 6 1. 3 and was not statistically differ ent from the quantity in PBS dealt with mice. The size of the lesions was also equivalent in between PGE 2 dealt with mice and PBS dealt with controls.

domain: medical


text:
Delicious, delicate and beautiful. Zucchini Blossoms are subtly sweet and add a graceful addition to any plate. Get out of your comfort zone any try adding something extra special to your next dinner menu! There are a number of ways you can prepare these beauties, but here is a sublime stuffed version which perfecty showcases how magnificent these blossoms are. Using the vegetable oil gives the petals a delicious caramelized crunch which beautifully encases your savory filling! For best results; use the blossoms right away as they don’t have a long shelf life.
What you will need:
- 8-10 Zucchini blossoms; rinsed
- 2 strips of cooked bacon; roughly chopped
- 1 small cooking onion; finely chopped (approx ¼ cup)
- ⅓ cup feta cheese; crumbled
- 2 tbsp. vegetable oil
Prep Time: 30 minutes ◊ Cook Time: 15 minutes
- Begin by rinsing your blossoms gently, then set aside to dry thoroughly.
- In a medium sized skillet, cook the bacon until somewhat crispy. Set aside on paper towel.
- In the same pan, add chopped onions and sauté on medium heat until golden brown.
- In a small bowl, combine feta, onions and chopped bacon. Mix thoroughly.
- Now to stuff your blossoms. Because of the delicate nature of the blossoms, we found this an easier task to do with two people. Have one person gently hold open the blossoms, while the other uses a small spoon to fill the insides. Have the person holding the blossoms, carefully pack the filling mixture down inside.
- Once the blossom is filled, twist the tops of the blossom petals gently to seal.
- Add vegetable oil to a large skillet and heat to medium temperature. Once oil is heated fully, place blossoms into the hot oil and cook for approximately 8-9 minutes. Turn every 2 minutes until all sides are golden brown.
- Remove blossoms from pan and place on a paper towel briefly before serving.

domain: food


text:
Many harsh phrases are traded on Twitter every day. Dick Costolo, the previous CEO of the corporate, added a few of his personal tonight.
Costolo was responding to a debate that has consumed tech’s elite on Twitter over the previous few days, an argument about whether or not the business ought to actively promote social justice causes and encourage political conversations within the office. Costolo’s feedback had the impact of dumping a jerrycan’s price of gasoline on the hearth. “Me-first capitalists who suppose you’ll be able to separate society from enterprise are going to be the primary individuals lined up in opposition to the wall and shot within the revolution,” he tweeted. “I will fortunately present video commentary.”
Costolo’s extraordinary language—suggesting he’d prefer to see a few of his friends face a firing squad—was jarring and bolstered the profoundly uncommon and troubling circumstances which have enveloped company America this yr. Companies have confronted a lethal pandemic; a world recession; and the best reckoning over civil rights in a number of generations, sparked by George Floyd’s dying whereas in coverage custody final Could. By flip, firms and the CEOs working them have been left questioning what function—if any—they need to take within the motion, inaction on their half generally resulting in the unhappiness of their staff.
Whereas the fiery controversy had largely been diminished to embers over latest weeks, it was sparked anew on Twitter by Coinbase CEO Brian Armstrong’s sternly worded blog post from Sunday, a prolonged treatise that tried to attract a agency line within the sand: Coinbase would take no half in any social justice motion. Usually, Armstrong appeared to say, he was anticipating his staff to maintain their heads down and concentrate on their job of constructing out the cryptocurrency alternate. “We’ve seen what inner strife at corporations like Google
“We don’t interact right here when points are unrelated to our core mission,” Armstrong continued, “as a result of we consider impression solely comes with focus.” In ending, Armstrong made it clear that anybody who didn’t agree together with his profits-before-politics mentality ought to go away. On Wednesday, he reinforced that, encouraging any worker made sad by his coverage to make the most of a newly created seperation package deal, a buyout he promised was “beneficiant.”
Instantly, Armstrong’s feedback drew in supporters—and loads of aghast critics. Among the many most notable of those that rushed to Armstrong’s facet was the enterprise capitalist Paul Graham. “But once more, @brian_armstrong leads the way in which,” Graham tweeted. “I predict most profitable corporations will comply with Coinbase’s lead. If solely as a result of those that do not are much less more likely to succeed.”
However cryptocurrency by nature is antagonistic to the established order, a disruptive drive meant to overturn techniques of custom and entrenched considering. For these causes, Armstrong’s phrases urging his workers to disregard this mentality in terms of racial justice struck many as perplexing—or, worse nonetheless, as somebody bent on commercialism over ideas. Field founder Aaron Levie was one of the vital vocal critics. “When there is a vacuum of management from the federal government in coping with these subjects, companies typically want to guide greater than ever,” he tweeted Tuesday afternoon. The thought was nonetheless on his thoughts later that night time after watching the tumultuous presidential debate on Tuesday’s night. “So like do Coinbase staff simply faux that debate didn’t occur tomorrow?” Levie asked on Twitter.
Costolo finds himself squarely on the identical facet as Levi. He characterised Armstrong’s anti-politics dictum as “the abdication of management. It is the equal of telling your staff to ‘shut up and dribble.’ ” He additional advised that Coinbase would turn out to be “a financial institution with a mission no one actually believes.”
How he selected to ship this message—on Twitter—is each emblematic of immediately’s political period and the state of recent political discourse. Twitter has grown from “microblogging website,” the phrase typically used to explain the platform when he captained it from 2010 to 2015, to an all-important portal for info, the place, naturally, the place President Trump and Democratic nominee Joe Biden went after the controversy to additional press their messages to voters.
It’s the place Trump has made feedback that courted gun-point violence, selling Twitter to censor or append corrections to the president’s tweets a number of instances this previous summer time. And with the president of america normalizing such remarks, it was maybe inevitable that individuals on the other facet—Costolo on this case—would copy his tone.
Mike Cernovich, a proper wing media character with a 721,700-person following on Twitter, was fast to criticize Costolo. “This tweet is an precise violation of twitter TOS for glorifying violence,” he wrote on Twitter. “That apart, thanks for publicly endorsing the mass homicide of your poltical [sic] adversaries.” (Twitter didn’t instantly return a request to remark about whether or not Costolo’s tweet did in truth violate its phrases of servie—or whether or not it deliberate to take any motion.)
Geoff Lewis, a enterprise capitalist and former government at libertarian billionaire Peter Thiel’s Founders Fund, was left sputtering for words: “ ‘I will fortunately present video commentary’ ????? Wow.”


Winchester continues to supply principle features found successful in pre-1964 variants of this latest long-range rifle. A claw-type extractor delivers controlled-round feed functionality for enhanced performance through the entire loading and unloading process of the turn-bolt action. Feed lips within the receiver are included to improve function, too.
The rifle's receiver is made from steel, drilled-and-tapped and treated with a matte finish. The barrel, three sling studs, hinged floorplate and bolt handle are matte black as well. The bolt features jeweling, which is an attractive detail that further enhances functionality. Winchester supplies its well-regarded M.O.A. trigger system that works in conjunction with the bolt-mounted, three-position safety.
For the Model 70 Long Range MB, Winchester sourced a premium option from Bell & Carlson, which supplied a composite, crossover-style stock that sports a tan finish with black, spider web texturing. An aluminum bedding system works in concert with proprietary molding and use of composite, hand-laid materials to create a rigid gunstock. Four ventilation cuts found along the fore-end reduce the stock’s weight and promote barrel cooling. Additionally the fore-end features a wide, flat bottom for added stability, and two sling swivel stud are included for versatility.
Winchester is set to supply its Model 70 Long Range MB with 24” steel barrels that come fluted and capped with a muzzle brake, regardless of chambering. Eight chamberings are available for 2021 that include: .22-250 Rem., .243 Win., 6.5 Creedmoor, 6.5 PRC, 6.8 Western, .270 WSM, .300 WSM and .308 Win. Capacities vary from three to five rounds, depending upon chamber selection. Overall length of the rifle is 44” and rifle’s weight will deliver 7.5 lbs. in the hand.
To learn more about Winchester Model Long Range MB please visit the company website HERE.


Preparing to Ship Heavy Equipment in Pennsylvania
Transporting heavy equipment to or from Pennsylvania or the surrounding area? By using Uship, you can save time and money by being quickly connected to the best Pennsylvania heavy haul shipping companies. Uship uses a feedback system to deliver you top transport companies who compete for your business.
You get the best experience shipping your heavy equipment when you use uShip. Using uShip allows top-rated heavy equipment carriers in Pennsylvania to bid on your heavy equipment shipment instantly. When choosing a carrier for your heavy equipment transport, make sure to review the carrier's insurance, customer reviews, and authority.


Marie Jay's Metal Products Co., Ltd
Sumith Perera Apr 28, 2019 5Most valuable product Rolling Wire Rods Rebars I have ever puchased and I really suggest you guys to have it as well.
Pabitra Kumr Sarker Apr 14, 2019 5Marie Jay's Metal Products Co., Ltd is a very good company. Very professional.
Antonio Pascotto Apr 09, 2019 5Great, I'm very satisfied with the quality of this Rolling Wire Rods Rebars . My product has been in production for half a month. My products are also very popular with customers, I really appreciate Marie Jay's Metal Products Co., Ltd .
Funmilola Adedigba Apr 08, 2019 4I am really happy that I got this Rolling Wire Rods Rebars with a nice deal price as well.
Mostak Ahmed Mar 30, 2019 5A very good thing. high quality Rolling Wire Rods Rebars . thank you seller.
Vince Goss Mar 27, 2019 5The Rolling Wire Rods Rebars price is reasonable and help us a lot.Recommend !
Steve Mcgarry Mar 18, 2019 5thank you very much for fast and efficient delivery. A lot of sellers could learn from you. Highly recommend Marie Jay's Metal Products Co., Ltd company!
Sue Mar 12, 2019 5It’s worthy.
Yaketta Vaughn Feb 22, 2019 4Rolling Wire Rods Rebars match the description. Greatful.
Jessie Lam Feb 14, 2019 4Nice Rolling Wire Rods Rebars , appears to have good quality.
Zazreen Kirana Feb 12, 2019 5Had Rolling Wire Rods Rebars for about 2 weeks now and it comes with everything i need. Would come and buy again.
B.d.dube Feb 11, 2019 5The Rolling Wire Rods Rebars is good, pay a lot of money. after That EVERYTHING This ok.
YOU MAY LIKE
Good quality Wire shelf rack,rolling wire racks,wire racks on wheels
Trade Assurance Wire shelf rack wire rack shelf wire racks on wheels General Information 1 Name wire shelf for sale 2 Item No JS WS04 3 Material High quality raw steel material 4 Layers 1 6 Layers Accetable Layer hight can be adjusted freely 5 Wheels Avaliable 6 Loading Capacity 30 200 kgs per...
carbon steel wire rods
ms wire rod
sae 1008 wire rod
5.5mm sae 1008 wire rods
steel wire rods in coils
9.5mm steel wire rods
wire rod steel coil
nail steel wire rods
sae 1010 wire rod
hot rolled alloy steel wire rod
steel wire rod sae1008
EA1008B wire rods / sae steel wire rod
sae 1012b carbon wire rods
cold rolled steel wire rods
sae 1010 carbon steel wire rod
China supplier high tensile steel rods / wire rods with best price
China supplier 6.5mm 1008 wire rods with best price
mild steel wire rods
mild steel wire rods High Quality Stainless Steel Round Bar 1 HIgh Quality 2 Resonable price 3 Ontime delivery We strive to be the best in our industry We care about our customers our suppliers and partners We do our absolute best to honor our commitments We strive to always act with integrity and...
SEND US A MESSAGE
We didn't put all products on website. If you can't find the product you're looking for, please contact us for more information.
Copyright © 2019 Marie Jay's Metal Products Co., Ltd All rights reserved.


text:
Oncofetal genes are expressed in embryos or fetuses, are downregulated or undetectable in adult tissues, and then re-expressed in tumors. Known oncofetal genes, such as AFP, GCB, FGF18, IMP-1 and SOX1, often have important clinical applications or pivotal biological functions. To find new oncofetal-like genes, we used the public information of expressed sequence tags to systematically analyze gene expression patterns and identified a novel oncofetal-like gene, LRRC16B. It increased the proliferation, anchorage-independent growth and tumorigenesis of transformed cells in xenografts, possibly through its effects on cyclin B1 protein levels. These findings exemplify the feasibility of using bioinformatics to find new oncofetal-like genes and suggest that more genes with important functional roles will be uncovered in the candidate gene list.
Tumorigenesis is caused by various factors that upset the balance between cell proliferation and cell death, which results in uncontrolled cell division and tumor formation (Gilboa, 1999; Vogelstein and Kinzler, 2004). A tumor may be described as chaotic organ genesis, and tumor-related genes, like genes in the Wnt (Giles et al., 2003; Katoh, 2007) or Sonic hedgehog pathways (Ding and Schultz, 2004; Morton et al., 2007), may be important in human embryonic development.
Oncofetal genes are defined as genes expressed in the embryos or fetuses, which are turned off or suppressed in adult tissue, but re-expressed in tumor cells (Cleynen et al., 2007; Sarandakou et al., 2007). Most are clinically useful markers or functionally interesting genes. The prototype oncofetal gene/protein is α-fetoprotein (AFP), discovered in human fetal serum in 1960 (Bergstrand and Czar, 1956) and now widely used as a serum marker for hepatocellular carcinoma (HCC) and yolk sac tumor (Trojan et al., 1995). Other oncofetal genes/proteins include β-human chorionic gonadotropin (a placental glycoprotein) (Farghaly, 1992; McNamee, 1995), glypican 3 (Grozdanov et al., 2006; Hishinuma et al., 2006), H19 (Gabory et al., 2006; Matouk et al., 2007), the IMP family (IGF2 mRNA-binding protein) (Nielsen et al., 1999; Kato et al., 2007) and high-mobility group A 2 (Cleynen et al., 2007).
Most oncofetal genes were discovered as targets for some other research purposes (that is, embryogenesis or metabolism). There is only one previous report (Monk and Holding, 2001) of a specific search for oncofetal genes; it found the OCT4 gene. However, for a more comprehensive search, it is necessary to collect embryonic tissue at various developmental stages, adult tissue that represent most tissue types and tissue from many different kinds of human tumors. These tissue samples can then be subjected to high-throughput technologies for biomarker discovery (Aouacheria et al., 2006; Campagne and Skrabanek, 2006; Niemann et al., 2007, Xu et al., 2007). Fortunately, this gigantic research project has been largely accomplished through the expressed sequence tags (EST). So far, some 1.5 million human EST are stored in the National Center for Biotechnology Information (NCBI) dbEST database, which contains at least 7000 human EST cDNA libraries (Boguski and Schuler, 1995; Colantuoni et al., 2000).
In this study, to show that systemic analysis of the oncofetal patterns in the dbEST can lead to the discovery of genes with interesting functions and potential clinical use, we used the number of AFP, the prototype of oncofetal genes/proteins, as the threshold for discovering genes with potential oncofetal expression patterns.
Bioinformatic analysis of EST database for oncofetal genes and literature review of the resulting gene list
The bioinformatic analysis was carried out as described in the Materials and methods section and summarized in Figure 1. The final gene list included 29 genes with known functions and 44 genes with unknown functions. The frequencies of the 29 known genes in the mature, tumor and immature groups are listed in Supplementary Table 1. Three known oncofetal genes (AFP, GCB and IMP-1) were rediscovered. Two other genes (FGF18 and SOX1) had been shown (Alexander, 1970; Shimokawa et al., 2003) to have oncofetal expression patterns, but were not clearly designated as oncofetal genes/proteins in the literature. Three (DPYSL5, MDFI and TITF1) were tissue-specific genes or genes with limited tissue distribution (Chen et al., 1996; Fukada et al., 2000; Garcia-Barcelo et al., 2007). Twelve genes (AXIN2, ASCL1, BMP4, DKK2, EPHB3, GDF11, KISS1, MYB, MYCN, PGF, POLS and SOX2) were related to oncogenesis or embryogenesis (Boiani and Scholer, 2005; Esteve and Bovolenta, 2006; Dao et al., 2007; Katoh and Katoh, 2007; Sugimori et al., 2008). Only nine genes did not have interesting expression patterns or functions (Table 1).
Reverse transcription–polymerase chain reaction screening of the unknown genes for potential oncofetal expression patterns
As the known genes have a high percentage of interesting functions, we studied the unknown genes. Five unknown genes were evaluated for their expression patterns in various tumor cell lines, as well as in a panel of normal tissue cDNAs (BD Clontech, Mountain View, CA, USA) with three known oncofetal genes. The tumor cell lines were used as a more inclusive first-line screening. AFP was expressed only in Huh-7, HepG2 and SW480 cells. IMP1 (known control), FGF18 (known control), LOC129607 (unknown gene) and LRRC16B (unknown gene) were detected in the majority of tumor cell lines (Figure 2a). In normal tissue RNA, RT–PCR analysis showed that AFP was expressed only in fetal liver tissue. The unknown LOC129607 gene was ubiquitously expressed in normal tissue. The three known oncofetal genes, AFP, IMP-1 and FGF18 (Shimokawa et al., 2003; Kato et al., 2007), showed expected expression patterns, with higher expression in tumor cell lines and fetal tissue, and lower expression in normal tissue. The unknown LRRC16B gene showed an expression pattern very similar to that of IMP-1, with the exception in adult brain tissue (Figures 2b and c). Notably, both IMP-1 and LRRC16B were expressed in testis tissue. The remaining three unknown genes, FLJ10156, GPR153 and ZNF500, were neither oncofetal nor tissue specific (data not shown). FLJ10156, like LOC129607, was ubiquitously expressed, and GPR153 and ZNF500 were expressed in various normal tissue types.
Downregulation of LRRC16B in adult tissue
Real-time quantitative RT–PCR confirmed the expression pattern of LRRC16B. LRRC16B was highly expressed in fetal brain and lung tissue. It was also expressed in fetal spinal cord, kidney, stomach, liver and colon (4.4–0.2% of fetal brain expression) tissue. Skeletal muscle and heart tissue showed the lowest expression (0.0026–0.0014% of fetal brain expression) (Supplementary Table 2A). Most adult tissue samples also showed very low expression of LRRC16B (Supplementary Table 2B). Adult brain tissue had a LRRC16B/GAPDH ratio of 5.94 × 10−7, which was not much higher than that for fetal liver tissue (3.22 × 10−7).
Adult testis, liver, spinal cord, thyroid gland and thymus tissue had expression levels comparable to the lowest ones in fetal tissue. LRRC16B expression was downregulated 207-fold from fetal to adult brains. The lungs showed much more significant downregulation from fetus to adult (32 651-fold, −log 4.5), whereas the liver showed the least downregulation (21-folds, −log 1.3). Skeletal muscle, kidney, spinal cord, colon and heart tissue showed downregulation of 96 099- (−log 4.9), 7204- (−log 3.8), 2181- (−log 3.3), 507- (−log 2.7) and 266-fold (−log 2.4), respectively (Figure 2d).
RT–PCR using rat embryos and adult rats also showed downregulation of rat LRRC16B in the adult organs (Supplementary Figure 1). Similar to humans, the adult rat brain also expresses rat LRRC16B.
Expression of LRRC16B in ovarian, hepatic and colorectal cancers
To investigate LRRC16B expression in cancerous tissue, RT–PCR (upper) and real-time PCR (bottom) were used in tumor and non-tumor tissue pairs, respectively, from 10 ovarian, hepatic and colorectal cancers (Figure 3). The non-tumor tissue of the ovarian cancer came from the contralateral uninvolved ovary. Fifty percent of ovarian cancers (Figure 3a; cases 2, 4, 7, 8 and 9) and 60% of colorectal cancers (Figure 3b; cases 1 and 3–7) showed significant LRRC16B upregulation in cancerous tissue. The result of hepatoma was mixed, with both up- and downregulation observed in the tumors (Figure 3c). These results showed that LRRC16B was upregulated in certain cancerous tissue, such as ovarian and colorectal cancers.
Overall, LRRC16B was expressed in many fetal tissue types, downregulated in adults and upregulated in certain tumors, which conforms to the general definition of an oncofetal gene.
LRRC16B gene cloning, sequence analysis and in vitro expression
The LRRC16B gene was predicted to produce a 4.7-kb mRNA and encode a 1372-aa protein with a predicted molecular weight of 150 kDa (Figure 4a). Northern blotting on five different tumor cell lines using a 3′ probe showed a ∼4.7-kb transcript (Figure 4b). RT–PCR of fetal brain total RNA produced four overlapping fragments that pieced together into a 4770-bp cDNA, including the 4119-bp coding region, and 323-bp 5′- and 328-bp 3′-untranslated regions. The full-length LRRC16B was fused to enhanced green fluorescent protein (EGFP) and myc tag, cloned into pMSCVpuro vectors and expressed in human embryonic kidney 293T and baby hamster kidney (BHK) cells as stable pools.
Western blotting showed that LRRC16B-EGFP and LRRC16B-myc had molecular weights of ∼177 and ∼150 kDa, respectively (Figures 4c and 6a). Stable pools expressing LRRC16B-EGFP were created to determine the subcellular localization of LRRC16B protein in 293T cells. These showed that the majority of LRRC16B-EGFP was detected in the cytoplasm, whereas some were observed as small nucleus aggregates (Supplementary Figure 2).
LRRC16B promoted cell proliferation in 293T cells
XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) tests were carried out on 293T and BHK stable pools expressing LRRC16B-EGFP fusion proteins. Within 72 h, the growth curve had significantly increased by 90% in 293T stable pool cells compared with control cells expressing EGFP only (Figure 5a). The growth curve was slightly increased by 15% in BHK cells overexpressing LRRC16B-EGFP. The effect on growth regulation was further evaluated using bromodeoxyuridine (BrdU) incorporation. Both 293T and BHK cells overexpressing LRRC16B-EGFP showed significant increases of BrdU incorporation (Figure 5b).
Cell cycle distribution of LRRC16B-EGFP stable pools was analyzed using a flow cytometer. After 72 h of incubation, only 20% of 293T cells overexpressing LRRC16B-EGFP were retained in the G2/M phase, compared with 50% in control cells (Figure 5c). The BHK stable pools, however, did not show significant changes in cell cycle distribution (Figure 5c).
In terms of LRRC16B-EGFP effect, there were no significant changes in cyclin D1 or cyclin A levels in either 293T or BHK stable pools. Both stable pools showed elevated c-Myc protein levels. Only 293T cells overexpressing LRRC16B-EGFP showed a significant increase of cyclin B1 protein levels (Figure 5d). These results indicated that cyclin B1 protein levels might correlate with changes in cell cycle distribution.
The effect of LRRC16B-EGFP on 293T cells was reconfirmed using a different fusion tag. The 293T stable pool overexpressing LRRC16B-myc was created and subjected to XTT assays and western blotting. The molecular weight of the expressed fusion protein was as predicted, and the results were essentially the same as for LRRC16B-EGFP (Figures 6a and b), with one exception in western blot analysis. Only cyclin B1 increased; there was no significant change in the c-Myc protein level (Figure 6c).
Knocking down endogenous LRRC16B inhibited proliferation
To explore the role of LRRC16B in cancer cells, short hairpin RNAs (shRNAs) were used to knock down LRRC16B expression in BG1 and Huh-7 cells. Four LRRC16B shRNAs were designed, and two, LRRC16B-RNAi-1 and LRRC16B-RNAi-2, significantly reduced the amount of protein product of co-transfected LRRC16B-EGFP in 293T cells after 48 h (Supplementary Figure 3). BG1 and Huh-7 stable pools expressing LRRC16B-RNAi-1 and LRRC16B-RNAi-2 were established and subjected to XTT assays and western blotting. The proliferation rates of BG1-LRRC16B-RNAi-1 and BG1-LRRC16B-RNAi-2 stable pools were significantly reduced (Figure 7a left panel). There were no significant changes in Huh-7 stable pools (Figure 7b right panel). Western blotting showed that cyclin B1 was reduced in BG1, but not in Huh-7 stable pools (Figure 7b). On the other hand, there was no increased cleavage of caspase-3 or LC3-I in BG1 stable pools (Figure 7c), which indicated that LRRC16B knockdown did not affect apoptosis or autophagy while decreasing the proliferation of BG1 cells. The inhibition of endogenous LRRC16B against BG1 cell proliferation correlated with the decreased cyclin B1 protein level, which was consistent with the results of LRRC16B overexpression in 293T cells.
The transforming potential of LRRC16B in vitro and in vivo
The transforming potential of LRRC16B on 293T was gauged using a soft agar assay. The number of 293T cell colonies overexpressing LRRC16B-EGFP or LRRC16B-myc significantly increased compared with 293T cells overexpressing EGFP or treated with mock transduction or vector alone (Figure 8a). However, the numbers were much less than that produced by 7-4 cells, a ras-overexpressing 3T3 cell line that was a positive control for the assay. The sizes of the colonies increased with LRRC16B fusion gene expression (Figure 8b).
Using LRRC16B-RNAi-1 and -2 to inhibit endogenous LRRC16B in BG1 cells significantly decreased the number and sizes of the colonies in the soft agar assay. There were no changes in the number and size of the colonies for LRRC16B knockdown Huh-7 cells (Figures 8c and d).
To test whether LRRC16B promoted tumor growth in vivo, control and LRRC16B 293T stable pool cells were subcutaneously injected into NOD/LtSZ Prkdc mice. Mice given LRRC16B 293T stable pool cells grew local tumors significantly larger and heavier than mice given control cells during the 35-day observation. Routine histological staining showed no morphological changes in these mice (Figure 8e).
By systematically analyzing the EST database for oncofetal expression patterns, a previously unknown gene, LRRC16B, was shown to have transforming enhancing capabilities. This exemplifies the feasibility of a bioinformatics approach for finding functionally interesting genes. There may be more oncofetal genes, tissue-specific genes or genes related to oncogenesis, embryogenesis or both to be uncovered in the gene list, including 39 unknown genes that have not yet been examined.
The bioinformatics algorithm used in this study is uncomplicated and works well enough as a screening tool for further experimental confirmation. More sophisticated analysis may result in a more comprehensive gene list. For example, a combined profile of several known oncofetal genes, instead of AFP alone, may be used as the cutoff criterion. It is also possible to reiterate the cyclic process of bioinformatics analysis, experimental confirmation, discovering new oncofetal genes, using a new set of oncofetal genes to set a new cutoff, and then repeating the analysis until no new gene can be found.
Two of the five genes tested in this study were ubiquitously expressed, even though the bioinformatics analysis predicted them to be oncofetal. Most likely, they are under-represented in the selected mature group libraries.
The choice of cDNA libraries in this study was based on their size and the magnitude of the EST projects; we presumed that bigger EST projects have better annotation quality for their cDNA libraries. Unfortunately, this may not always be true. It will be helpful if someone with better knowledge of the annotation qualities of cDNA libraries chooses the cDNA libraries and has them classified. A more accurate classification of the cDNA libraries may result in a better gene list. Knowledge of the libraries will be particularly helpful in the mature group. In this study, libraries without any descriptions for disease status or age have been classified as mature (normal adult) because of the sheer lack of other helpful information.
Cell lines were used in the first step of experimental screening; however, a gene expressed in the cell lines may not be expressed in the tumor tissues. We used the cell lines as a more inclusive screening test. If a gene was not expressed in any of the cell lines tested, it would be less likely to be expressed in tumor tissues. By excluding such genes, we might have missed some highly tissue-specific oncofetal genes, but the genes we tested happened to be expressed in the majority of the cell lines; therefore, we believe that this did not occur.
An oncofetal gene is not strictly defined in the literature. It is generally defined as embryonic genes downregulated in adults and re-expressed in tumors. However, the magnitude of downregulation is not specified. LRRC16B is downregulated from 21- to 96 099-fold among various organs and, as such, it is proposed as an oncofetal-like gene. Even though it is expressed in the adult central nervous system, there is a 207-fold reduction of its expression from the fetal to the adult brain. LRRC16B is expressed in purified mouse neurons, but not in glia cells (unpublished data). In practice, it can be regarded as an oncofetal marker in organs or body fluids outside the central nervous system owing to the blood–brain barrier.
The expression of LRRC16B in the central nervous system is intriguing. Its promotion of cell proliferation and transforming activity should play no role in adult neurons. More studies are warranted to unravel its function in neurons.
It would be interesting to know which cell types express LRRC16B in which organs. Supplementary Figure 4 shows preliminary immunohistochemical results using a not-fully-characterized antibody obtained through the Human Protein Atlas portal (www.proteinatlas.org). The antibody highlighted the neurons, but not glial cells in the human fetal brain. There were signals in the epithelial cells of fetal colon, kidney and lung tissue. There were also immunohistochemical signals in the mesenchymal cells in the lamina propria of fetal colonic mucosa.
From a protein structural modeling analysis (MotifScan; http://myhits.isb-sib.ch/cgi-bin/motif_scan), the novel protein LRRC16B shows some sequences that belong to the leucine-rich repeats domain. Leucine-rich repeats domains are involved in protein–protein interaction, such as ribonuclease-inhibitor, GTPase-activating protein (Kobe and Kajava, 2001), SDS22-like subfamily and cysteine-containing subfamily (Kutay and Guttinger, 2005). They not only take part in many important bio-functions, such as signal transduction, cell metastasis, immune responses and tumorigenesis (Qiao et al., 2007; Barker et al., 2009), but are also involved in cell polarization (Imamura et al., 2006), apoptosis, cell cycle control (Adachi-Yamada et al., 2005; Park et al., 2009), cell migration, proliferation, development (Liu et al., 2005; Cowin et al., 2007; Hossain et al., 2008; Wang et al., 2009) and actin polymerization (Eisenmann et al., 2007; Kopecki and Cowin, 2008). The bio-function of the LRRC16B protein influences cell proliferation, cell cycle control and transformation.
Sequence analysis predicts that LRRC16B has actin-binding domains and nuclear localization signals. Confocal microscopy shows that some overexpressed LRRC16B-EGFP fusion proteins localize in the nucleus as small dots. Phalloidin staining indicates that LRRC16B-EGFP fusion proteins colocalize with actin (unpublished data). Whether LRRC16B binds directly with actin, and whether it interacts with importin, the key player for nuclear transportation, remains to be investigated.
In this study, 293T and BHK cells were used for soft agar assays. The more common cell lines for such an experiment are NIH3T3 and 293 cells. However, NIH3T3 and 293 cells expressing LRRC16B fusion genes quickly die (data not shown); therefore, stable pools were never established. LRRC16B appears to be more of promoter rather than gatekeepers in oncogenesis.
The transformation-enhancing capability of LRRC16B may be tumor-type specific and may be more important in ovarian cancer rather than in HCC. We found a trend of LRRC16B overexpression in ovarian cancer, but not in HCC. The knockdown experiment showed reduced proliferation and colony formation in BG1 cells, an ovarian cancer cell line, with a concomitant reduction of cyclin B1 protein levels. LRRC16B RNA interference (RNAi), however, had no effect on Huh-7, an HCC cell line. More clinical samples are needed to verify this initial observation.
In summary, we presented an effective method of finding interesting genes through a systematic bioinformatics analysis of the EST database for embryonic or fetal genes that are downregulated in adult tissue and re-expressed in tumors. LRRC16B is one such gene that has transformation-enhancing capability, possibly mediated by cyclin B1. Future investigations should focus on the other unexamined genes in the gene list and on LRRC16B functions.
Materials and methods
All EST were collected from the Cancer Genome Anatomy Project (http://cgap.nci.nih.gov/). To classify each library, a certified pathologist read each library's description, which contained its tissue origin, developmental stage (which included ‘fetus’, ‘infant’, ‘adult’ and ‘not specified’), and its histology or disease status (‘normal’, ‘tumor’, ‘other diseases’ and ‘not specified’). Excluding SAGE libraries, there were 7066 EST libraries in the file ‘library.report’ downloaded in 2005. From these, 6021 were initially chosen. To expand the coverage of fetal and normal mature groups, additional 162 normal mature and 63 fetal libraries were added; 128 of the 6246 libraries were later removed for various reasons. All subtraction libraries and all non-tumor libraries from tissue with inflammatory diseases were also excluded.
The 6118 EST libraries finally used were classified into three groups. The immature group had 77 libraries from fetal tissue, 10 libraries from infant tissue, 394 libraries from the placenta and two libraries from cord blood, for a total of 483 libraries. The tumor group contained 3911 libraries from benign and malignant tumors. The remaining 1724 libraries formed the mature group, including those without information about age or disease status.
Information for gene expression profiles was obtained from the file Hs_ExprData.dat. This contained three columns: UniGene cluster ID, library ID and frequency. There were data for the frequency of each unigene in a library. For each group (mature, immature and tumor), IDs were evaluated for their constituent libraries and unigenes. The frequency of the unigene expressed for a specific group of libraries was calculated.
The calculated frequencies of the AFP gene in each group of the libraries (immature group: 175; mature group: 10; and tumor group: 108) were used as references to set the thresholds of bioinformatics analysis. To exclude genes with higher expression than AFP in the adult tissues, the first step of the bioinformatics analysis was to eliminate any gene with a frequency >10 in the mature group (Figure 1). Subsequent steps were less stringent. Any gene with frequencies >5 in both of the immature and tumor groups was retained. For genes with frequencies ⩾2 (and ⩽10) in the mature group, the ratio between tumor and mature groups (tumor/mature) was then calculated, and only those with a ratio >5 were collected. The 73 genes collected at the end of analysis were subjected to a literature review.
The various human cancer cell lines included those for ovarian cancer (PA-1, NIH:OVCAR-3, SK-OV-3, BG1, TOV-21G and ES-2), renal carcinoma (786-O and ACHN), liver cancer (Huh-7 and HepG2), bladder cancer (T24, TSGH-8301), breast cancer (MCF-7), lung cancer (A549), colon cancer (SW480), clone 7-4 (NIH3T3 overexpressed in Ha-ras), 293T and BHK-21. These were maintained in various culture media supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Grand Island, NY, USA), under 5% CO2 at 37 °C.
Cancer samples were from the Department of Pathology, NCKU Hospital. Their use had been approved by the Institutional Review Board of NCKU Hospital with signed informed consent.
Normal and fetal cDNAs were screened for gene expression using panels of multiple normal total RNAs (Master Panel II; BD Clontech) and multiple fetal total RNAs (Human Fetal Normal Tissue Total RNA; BioChain, Hayward, CA, USA). Various tumor cell lines, embryos, P1 rat tissue and adult rat tissue were titrated using a homogenizer. Ten frozen 20-μm tissue slices were collected using TRIzol RNA isolation reagent (Invitrogen, Carlsbad, CA, USA). The quality of RNA was confirmed on agarose gel. The concentration was determined using a spectrophotometer.
Five micrograms of total RNA from each sample was used for reverse-transcription using oligo(dT)12−18 primer and Superscript II reverse transcriptase (Invitrogen), according to the manufacturer’s protocol. The total RT product was diluted 10 times in TE buffer and stored at −20 °C. Quality was checked using two housekeeping genes, β-actin and GAPDH, and PLA in the PCR system, with loading amounts adjusted according to different cDNA, with 5 μl of each cDNA per 25 μl of PCR mixture (BV buffer, 2.5 U of Platinum Taq, 0.4 mM dNTP and 1 μM of each primer). Conditions for touchdown PCR reactions involved the touchdown protocol (one cycle of initial denaturation at 95 °C for 2 min, three cycles of 94 °C for 30 s, 63 °C for 30 s, 70 °C for 30 s, three cycles of 94 °C for 30 s, 61 °C for 30 s, 70 °C for 30 s, three cycles of 94 °C for 30 s, 59 °C for 30 s, 70 °C for 30 s, and 25 cycles of 94 °C for 30 s, 58 °C for 30 s, 70 °C for 30 s and 70 °C for 10 min). Supplementary Table 3 shows primer sequences for the examined genes.
Quantitative RT–PCR was carried out using the Light-Cycler TaqMAn Master (Roche, Mannheim, Germany). The number of transcript copies per microgram of total RNA from each tissue was calculated from standard curves of amplified cDNA clone. The expression level of LRRC16B was normalized as a relative ratio of its signal to that of GAPDH or PLA.
Thirty micrograms of total RNA from each sample was electrophoretically separated on 1.2% formaldehyde gel, and then transferred to Hybond-N+ nylon membrane and fixed by baking it at 80 °C for 2 h. The membrane was pre-hybridized with hybridization buffer 1 h at 42 °C, and then hybridized with random-primed 32P-labeled cDNA probe (106 c.p.m./ml) at 42 °C for 16 h. After it had been hybridized, the membrane was washed at room temperature with 2 × standard saline citrate plus 0.1% sodium dodecyl sulfate for 20 min; 0.5 × SSC plus 0.1% sodium dodecyl sulfate for 20 min; and twice with 0.2 × SSC plus 0.1% sodium dodecyl sulfate at 42 °C for 10 min. The blots were auto-radiographed at −80 °C for one month.
Full-length and small interfering RNA plasmid construction and generation of stable clones
RT–PCR was used to clone full-length human LRRC16B from normal brain total RNA (BD Clontech). Their products were cloned into pGEM-T Easy vector (Promega, Madison, WI, USA), and the insert was confirmed using DNA sequencing. The pGEM-T-LRRC16B was subcloned, using restriction enzyme digestion, into pEGFP-C1, pEGFP-N1 and pMYC to generate the LRRC16B-EGFP and LRRC16B-myc fusion genes. The plasmid expressing LRRC16B RNAi was constructed by ligating an shRNA sequence containing both sense and antisense strands.
Retroviral plasmid: pMSCVpuro (BD Clontech), pMSCV-EGFP, pMSCV-LRRC16B-EGFP, pMSCV-LRRC16B-myc and pSUPERretro vectors were co-transfected into GP2-293T package cells with VSV-G plasmids using the calcium phosphate method for 48 h. The 293T, BHK, BG1 and Huh-7 were seeded in 1 × 105 cells per well in a 6-cm dish and incubated overnight in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin and 100 mg/ml of streptomycin under 5% CO2 at 37 °C. Retroviral supernatant was added with 5 μg/ml of polybrene (Sigma, St Louis, MO, USA), and then the cells were incubated overnight at 37 °C in 5% CO2 with 5 μg/ml of puromycin selection.
Western blotting and fluorescent microscopy
Lysis buffer (100 μl per well) (Complete Lysis M, EDTA free, Roche) was added to each stable pool of cells, incubated for 5 min at room temperature and centrifuged at 14 000 g for 15 min. Protein concentration was determined using Bradford reagent (BioRad, Hercules, CA, USA). Fifty micrograms of protein from each sample was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis with 10% polyacrylamide gels. Polyacrylamide gel electrophoresis-separated proteins were electroblotted onto polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and incubated with mouse monoclonal anti-GFP, anti-Myc (Millipore), anti-cyclin A and anti-cyclin B1 (Santa Cruz, Santa Cruz, CA, USA) rabbit polyclonal anti-cyclin D1 antibody (Millipore), anti-caspase-3 and anti-LC3 (Cell Signaling, Boston, MA, USA). Goat anti-mouse and anti-rabbit IgG-HRP antibodies (Calbiochem, Darmstadt, Germany) were the secondary antibodies.
Cells were seeded onto glass slides, grown for 2 days, fixed at 4 °C with paraformaldehyde, permeabilized, counterstained with 4′,6-diamidino-2-phenylindole and fluorescent images taken using a confocal microscope (Olympus FV1000, Tokyo, Japan).
In vitro and in vivo growth property analysis
XTT proliferation and BrdU incorporation assays
pMSCV-EGFP, pMSCV-LRRC16B-EGFP, pMSCV-LRRC16B-myc, BG1 RNAi stable pools and Huh-7 RNAi stable pools were seeded in 12-well plates for 96 h. Triplicate wells were plated for each time point and taken at 24-h intervals for 4 days. The number of viable cells for each time point was determined using the XTT reagent standard protocol (Roche).
Cell proliferation was measured using an ErdU incorporation assay (Invitrogen). Results are expressed as the percentage of BrdU-positive cells.
Fluorescence-activated cell sorting analysis
The 293T and BHK stable pool cells (106 per dish) were first seeded and trypsinized for flow cytometric analysis on the third day. The remaining cells were trypsinized and placed together with growth media and phosphate-buffered saline. Cells were pelleted, resuspended in 75% ethanol and then stored overnight at −20 °C. They were centrifuged, washed with phosphate-buffered saline, resuspended in phosphate-buffered saline containing 10 mg/ml of DNase-free RNase, and then incubated in 37 °C for 1 h. Finally, propidium iodide at a concentration of 0.05 mg/ml was added and the cells were incubated at room temperature for 20 min. Cell clumps were filtered and the DNA content was measured on FACscan flow cytometer (BD Clontech). The percentage of cells in each phase of the cell cycle was determined using WinMDI2.9.
Soft agar assay
The six-well plates (lower layer) were added 1 ml of 0.7% agar in culture medium and incubated for 30 min at 4 °C. Stable pool cells, 293T-pMSCV, 293T-EGFP, 293T-LRRC16B-EGFP, 293T-LRRC16B-myc, 293T, 7-4, BG1 RNAi stable pools and Huh-7 RNAi stable pools (1 × 104 cells per ml) were suspended in the upper layer of a six-well plate containing 0.35% agar in the medium. After 10 days (14 days for BG1 and Huh-7 stable pools), colony formation (>100 μm diameter) was microscopically examined after the samples had been stained with 0.005% crystal violet.
Induction of tumor xenografts in mice
For each experimental group, ten 5-week-old female NOD/LtSZ Prkdc mice were injected subcutaneously with 0.2 ml Dulbecco's modified Eagle's medium containing 1 × 106 293T clones. The appearance of tumors (tumor incidence) was monitored, and when they developed, their size was determined twice a week using the following formula: D × d2 × π/6, where D was the tumor diameter at its widest, and d at its smallest. The mice were treated in accordance with guidelines approved by the Institutional Animal Care and Use Committee of NCKU.
Statistical significance was evaluated using Student’s t-test for single comparisons and one-way analysis of variance for multiple comparisons. Significance was set at P<0.05.
Adachi-Yamada T, Harumoto T, Sakurai K, Ueda R, Saigo K, O'Connor MB et al. (2005). Wing-to-leg homeosis by spineless causes apoptosis regulated by Fish-lips, a novel leucine-rich repeat transmembrane protein. Mol Cell Biol 25: 3140–3150.
Alexander P . (1970). Mechanism of growth and dissemination of antigenic tumors in normal immunological carriers. Medicina (B Aires) 30: 176–183.
Aouacheria A, Navratil V, Barthelaix A, Mouchiroud D, Gautier C . (2006). Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues. BMC Genom 7: 94.
Barker N, Ridgway RA, van Es JH, van de Wetering M, Begthel H, van den Born M et al. (2009). Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457: 608–611.
Bergstrand CG, Czar B . (1956). Demonstration of a new protein fraction in serum from the human fetus. Scand J Clin Lab Invest 8: 174.
Boguski MS, Schuler GD . (1995). Establishing a human transcript map. Nat Genet 10: 369–371.
Boiani M, Scholer HR . (2005). Regulatory networks in embryo-derived pluripotent stem cells. Nat Rev Mol Cell Biol 6: 872–884.
Campagne F, Skrabanek L . (2006). Mining expressed sequence tags identifies cancer markers of clinical interest. BMC Bioinform 7: 481.
Chen CM, Kraut N, Groudine M, Weintraub H . (1996). I-mf, a novel myogenic repressor, interacts with members of the MyoD family. Cell 86: 731–741.
Cleynen I, Brants JR, Peeters K, Deckers R, Debiec-Rychter M, Sciot R et al. (2007). HMGA2 regulates transcription of the Imp2 gene via an intronic regulatory element in cooperation with nuclear factor-kappaB. Mol Cancer Res 5: 363–372.
Colantuoni C, Purcell AE, Bouton CM, Pevsner J . (2000). High throughput analysis of gene expression in the human brain. J Neurosci Res 59: 1–10.
Cowin AJ, Adams DH, Strudwick XL, Chan H, Hooper JA, Sander GR et al. (2007). Flightless I deficiency enhances wound repair by increasing cell migration and proliferation. J Pathol 211: 572–581.
Dao DY, Yang X, Chen D, Zuscik M, O'Keefe RJ . (2007). Axin1 and Axin2 are regulated by TGF- and mediate cross-talk between TGF- and Wnt signaling pathways. Ann N Y Acad Sci 1116: 82–99.
Ding S, Schultz PG . (2004). A role for chemistry in stem cell biology. Nat Biotechnol 22: 833–840.
Eisenmann KM, Harris ES, Kitchen SM, Holman HA, Higgs HN, Alberts AS . (2007). Dia-interacting protein modulates formin-mediated actin assembly at the cell cortex. Curr Biol 17: 579–591.
Esteve P, Bovolenta P . (2006). Secreted inducers in vertebrate eye development: more functions for old morphogens. Curr Opin Neurobiol 16: 13–19.
Farghaly SA . (1992). Tumor markers in gynecologic cancer. Gynecol Obstet Invest 34: 65–72.
Fukada M, Watakabe I, Yuasa-Kawada J, Kawachi H, Kuroiwa A, Matsuda Y et al. (2000). Molecular characterization of CRMP5, a novel member of the collapsin response mediator rotein family. J Biol Chem 275: 37957–37965.
Gabory A, Ripoche MA, Yoshimizu T, Dandolo L . (2006). The H19 gene: regulation and function of a non-coding RNA. Cytogenet Genome Res 113: 188–193.
Garcia-Barcelo MM, Lau DK, Ngan ES, Leon TY, Liu TT, So MT et al. (2007). Evaluation of the thyroid transcription factor-1 gene (TITF1) as a Hirschsprung's disease locus. Ann Hum Genet 71: 746–754.
Gilboa E . (1999). How tumors escape immune destruction and what we can do about it. Cancer Immunol Immunother 48: 382–385.
Giles RH, van Es JH, Clevers H . (2003). Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta 1653: 1–24.
Grozdanov PN, Yovchev MI, Dabeva MD . (2006). The oncofetal protein glypican-3 is a novel marker of hepatic progenitor/oval cells. Lab Invest 86: 1272–1284.
Hishinuma M, Ohashi KI, Yamauchi N, Kashima T, Uozaki H, Ota S et al. (2006). Hepatocellular oncofetal protein, glypican 3 is a sensitive marker for alpha-fetoprotein-producing gastric carcinoma. Histopathology 49: 479–486.
Hossain MS, Ozaki T, Wang H, Nakagawa A, Takenobu H, Ohira M et al. (2008). N-MYC promotes cell proliferation through a direct transactivation of neuronal leucine-rich repeat protein-1 (NLRR1) gene in neuroblastoma. Oncogene 27: 6075–6082.
Imamura F, Nagao H, Naritsuka H, Murata Y, Taniguchi H, Mori K . (2006). A leucine-rich repeat membrane protein, 5T4, is expressed by a subtype of granule cells with dendritic arbors in specific strata of the mouse olfactory bulb. J Comp Neurol 495: 754–768.
Kato T, Hayama S, Yamabuki T, Ishikawa N, Miyamoto M, Ito T et al. (2007). Increased expression of insulin-like growth factor-II messenger RNA-binding protein 1 is associated with tumor progression in patients with lung cancer. Clin Cancer Res 13: 434–442.
Katoh M . (2007). Networking of WNT, FGF, Notch, BMP, and Hedgehog signaling pathways during carcinogenesis. Stem Cell Rev 3: 30–38.
Katoh M, Katoh M . (2007). WNT antagonist, DKK2, is a Notch signaling target in intestinal stem cells: augmentation of a negative regulation system for canonical WNT signaling pathway by the Notch-DKK2 signaling loop in primates. Int J Mol Med 19: 197–201.
Kobe B, Kajava AV . (2001). The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 11: 725–732.
Kopecki Z, Cowin AJ . (2008). Flightless I: an actin-remodelling protein and an important negative regulator of wound repair. Int J Biochem Cell Biol 40: 1415–1419.
Kutay U, Guttinger S . (2005). Leucine-rich nuclear-export signals: born to be weak. Trends Cell Biol 15: 121–124.
Liu CI, Cheng TL, Chen SZ, Huang YC, Chang WT . (2005). LrrA, a novel leucine-rich repeat protein involved in cytoskeleton remodeling, is required for multicellular morphogenesis in Dictyostelium discoideum. Dev Biol 285: 238–251.
Matouk IJ, DeGroot N, Mezan S, Ayesh S, Abu-lail R, Hochberg A et al. (2007). The H19 non-coding RNA is essential for human tumor growth. PLoS ONE 2: e845.
McNamee D . (1995). Beta-hCG inhibits Kaposi's sarcoma. Lancet 345: 1169.
Monk M, Holding C . (2001). Human embryonic genes re-expressed in cancer cells. Oncogene 20: 8085–8091.
Morton JP, Mongeau ME, Klimstra DS, Morris JP, Lee YC, Kawaguchi Y et al. (2007). Sonic hedgehog acts at multiple stages during pancreatic tumorigenesis. Proc Natl Acad Sci USA 104: 5103–5108.
Nielsen J, Christiansen J, Lykke-Andersen J, Johnsen AH, Wewer UM, Nielsen FC . (1999). A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development. Mol Cell Biol 19: 1262–1270.
Niemann H, Carnwath JW, Kues W . (2007). Application of DNA array technology to mammalian embryos. Theriogenology 68 (Suppl 1): S165–S177.
Park TJ, Kim JY, Park SH, Kim HS, Lim IK . (2009). Skp2 enhances polyubiquitination and degradation of TIS21/BTG2/PC3, tumor suppressor protein, at the downstream of FoxM1. Exp Cell Res 315: 3152–3162.
Qiao M, Iglehart JD, Pardee AB . (2007). Metastatic potential of 21T human breast cancer cells depends on Akt/protein kinase B activation. Cancer Res 67: 5293–5299.
Sarandakou A, Protonotariou E, Rizos D . (2007). Tumor markers in biological fluids associated with pregnancy. Crit Rev Clin Lab Sci 44: 151–178.
Shimokawa T, Furukawa Y, Sakai M, Li M, Miwa N, Lin YM et al (2003). Involvement of the FGF18 gene in colorectal carcinogenesis, as a novel downstream target of the beta-catenin/T-cell factor complex. Cancer Res 63: 6116–6120.
Sugimori M, Nagao M, Parras CM, Nakatani H, Lebel M, Guillemot F et al. (2008). Ascl1 is required for oligodendrocyte development in the spinal cord. Development 135: 1271–1281.
Trojan J, Naval X, Johnson T, Lafarge-Frayssinet C, Hajeri-Germond M, Farges O et al. (1995). Expression of serum albumin and of alphafetoprotein in murine normal and neoplastic primitive embryonic structures. Mol Reprod Dev 42: 369–378.
Vogelstein B, Kinzler KW . (2004). Cancer genes and the pathways they control. Nat Med 10: 789–799.
Wang R, Kaul A, Sperry AO . (2009). TLRR (lrrc67) interacts with PP1 and is associated with a cytoskeletal complex in the testis. Biol Cell 102: 173–189.
Xu L, Geman D, Winslow RL . (2007). Large-scale integration of cancer microarray data identifies a robust common cancer signature. BMC Bioinform 8: 275.
This was supported by Grant NSC-95-2320-B-006-057-MY2 from the National Science Council, Taiwan, and Grant DOH99-TD-C-111-003 from the Department of Health, Taiwan. There is no competing financial interest in relation to this work.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
About this article
Cite this article
Hsu, C., Chiang, C., Cheng, H. et al. Identifying LRRC16B as an oncofetal gene with transforming enhancing capability using a combined bioinformatics and experimental approach. Oncogene 30, 654–667 (2011) doi:10.1038/onc.2010.451
- oncofetal genes
- expressed sequence tags
Journal of Assisted Reproduction and Genetics (2019)
Molecular and Cellular Biology (2017)
Molecular Biology of the Cell (2017)
Overexpression of the RNA-binding proteins Lin28B and IGF2BP3 (IMP3) is associated with chemoresistance and poor disease outcome in ovarian cancer
British Journal of Cancer (2015)

domain: biology


text:
Newer approaches may boost the number of conditions that gene therapies can treat, the Associated Press reports. It adds that there are currently about half a dozen approved gene therapies in the US and that there are some 400 gene therapy trials underway.
The main challenge facing gene therapy has been delivering the needed version of the gene to patients' cells, the AP adds, noting that therapies for blindness deliver the treatment right to the eye, while other therapies isolate patients' blood cells, alter them in the lab, and then return them to patients with a fixed gene.
Now, the AP says, other viral vectors, including HIV and AAV, are being explored as delivery vehicles. With this type of approach, it says that companies could largely just swap out the genes targeted to treat different diseases. "The only thing that's changing is the payload, the gene," Chris Mason, Avrobio's chief scientific officer, tells the AP.
The AP notes, though, that challenges remain. "Every time we think we have overcome the safety issues, we continue to butt up against them," Peter Marks from the US Food and Drug Administration tells it. "I have confidence that we will work through them ... but we can't get too sure of ourselves."


text:
BOSTON – The Federal Emergency Management Agency will be sending more than $13.2 million to the State of Vermont to reimburse it for the cost of testing both the public and public employees during the COVID-19 pandemic.
The $13,248,491 Public Assistance grant will go to the Vermont Agency of Human Services for the costs of administering 1,792 CUE LAMP and 269,629 PCR COVID-19 tests required for the safe opening and operations of state facilities, including employees and visitors at state buildings, between May 2021 and March 2022.
That also included inmates, staff, and visitors at correctional facilities; students, staff, and visitors at public and non-profit schools; residents, staff, and visitors at non-profit long-term care facilities; and staff, campers, and visitors at public and non-profit summer camps.
“FEMA is pleased to be able to assist the State of Vermont with these costs,” said FEMA Region 1 Regional Administrator Lori Ehrlich. “Providing resources for our partners on the front lines of the pandemic fight is critical to their success, and our success as a nation.”
FEMA’s Public Assistance program is an essential source of funding for states and communities recovering from a federally declared disaster or emergency. So far, FEMA has provided more than $413 million in grants to Vermont to reimburse the state for pandemic-related expenses.