text:
|Publication number
|Publication type
|Application number
|Publication date
|Mar 8, 2005
|Filing date
|Oct 9, 1998
|Priority date
|May 7, 1997
|Fee status
|Also published as
|Publication number
|Claudio I. Zanelli, Michael J. Rosinko, Gregg W. Stone, Steven A. Daniel
|Original Assignee
|Eclipse Surgical Technologies, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (32), Non-Patent Citations (9), Referenced by (20), Classifications (15), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation-in-part of U.S. patent application Ser. No. 08/852,977 filed May 7, 1997, entitled "ULTRASOUND DEVICE FOR AXIAL RANGING", incorporated herein by reference
1. Field of the Invention
The present invention relates generally to catheters, MIS or other surgical apparatus for therapeutic applications. More particularly, the invention relates to a catheter, MIS or other elongated body including an ultrasound transducer that makes it particularly suited for determining the depth of dynamic tissue in beating heart laser-assisted transmyocardial revascularization (TMR), but not limited to such application. As the ultrasound transducer is fired, an acoustic wave is generated and a signal is reflected back to the transducer from anatomical structures, thus providing information on the position of the catheter, MIS or other surgical apparatus in relation to the anatomical structure.
2. Description of Related Art
Transmyocardial Revascularization
In the treatment of heart disease, one method of improving myocardial blood supply is called transmyocardial revascularization (TMR), the creation of channels in the myocardium of the heart. The procedure using needles in a form of surgical Amyocardial acupuncture has been used clinically since the 1960s. Deckelbaum. L. I., Cardiovascular Applications of Laser Technology, Lasers in Surgery and Medicine 15:315-341 (1994). The technique relieves ischemia by allowing blood to pass from the ventricle through the channels either directly into other vessels communicating with the channels or into myocardial sinusoids which connect to the myocardial microcirculation.
Numerous surgical TMR studies have been performed, including early studies using needles to perform myocardial acupuncture, or boring, to mechanically displace and/or remove tissue. Such studies have involved surgically exposing the heart and sequentially inserting needles to form a number of channels through the epicardium, myocardium, and endocardium to allow blood from the ventricle to perfuse the channels. The early studies using needles showed that the newly created channels were subject to acute thrombosis followed by organization and fibrosis of clots resulting in channel closure. Interest in TMR using needles waned with the knowledge that such channels did not remain open. However, interest in TMR procedures recurred with the advent of medical lasers used to create TMR channels. Histological evidence of patent, endothelium-lined tracts within laser-created channels shows that the lumen of laser channels can become hemocompatible and resists occlusion. Additionally, recent histological evidence shows probable new vessel formation adjacent collagen occluded transmyocardial channels, thereby suggesting benefits from TMR with or without the formation of channels which remain patent.
Surgical TMR procedures using laser energy have been described in the prior art. U.S. Pat. No. 4,658,817 issued Apr. 21, 1987 to Hardy teaches a method and apparatus for surgical TMR using a CO2 laser connected to an articulated arm having a handpiece attached thereto. The handpiece emits laser energy from a single aperture and is moved around the epicardial surface of the heart to create the desired number of channels. U.S. Pat. No. 5,380,316 issued Jan. 10, 1995 to Aita et al. purports to teach the use of a flexible lasing apparatus which is inserted into the open chest cavity in a surgical procedure. A lens at the distal end of the flexible apparatus is used to focus laser energy, and the apparatus is moved about the epicardial surface of the heart to create the desired number of channels.
Since TMR involves creating channels through the endocardium into the lower left chamber of the heart, it is also desirable to create TMR channels percutaneously, i.e., by extending a catheter through the vasculature into the ventricle and creating the channels through endocardial surfaces and into myocardium. Performing such percutaneous TMR is desirable for a number of reasons. Percutaneous catheter procedures are typically less traumatic to the patient compared to surgical procedures. Adhesions between the pericardial sac and epicardium are eliminated. Percutaneous TMR with a catheter also offers an alternative solution to persons who are not candidates for surgical procedures.
TMR procedures generally involve creating a plurality of channels within the myocardium. In performing the procedure, particularly percutaneously, it is desirable to have information relating to the depth of channels created, placement of the channels relative to the heart walls and wall thickness of the beating heart. None of the TMR or atherosclerosis devices described above or elsewhere provide such information.
Ultrasound systems are widely used in medical applications. Sound waves above the frequency normally detectable by the human ear, that is, about 16 to 20 kHz are referred to as ultrasonic waves.
U.S. Pat. No. 4,576,177 issued Mar. 18, 1986 to Webster, Jr. teaches a catheter for removing arteriosclerotic plaque. The apparatus comprises a catheter having an optical fiber for transmitting laser energy and an ultrasound transducer. One embodiment of the device is operated in two different modes—a pulse-echo mode and a pulsed-Doppler mode. In the pulse-echo mode an electrical impulse delivered to the transducer transmits an ultrasound pulse, returning echoes thereof causing electrical signature signals. In the pulsed-Doppler mode, ultrasonic echoes from tone bursts generated in response to electrical bursts transmitted to the ultrasound transducer are used to determine the blood flow velocity at two selected distances from the catheter tip. The tissue signature and the change in blood flow velocity are used to determine the presence of occlusions in blood vessels.
U.S. Pat. No. 4,658,827 issued Apr. 21, 1987 to He et al teaches an ultrasound scanner for tissue characterization. A method and system are disclosed for simultaneously obtaining accurate estimates of the attenuation coefficient of the tissue and an index describing the heterogeneity of the scatterers within the tissue. According to the invention, there is provided a method and apparatus for tissue characterization by transmitting ultrasound energy into the sample tissue, and receiving and processing return echo signals.
U.S. Pat. No. 4,672,963 issued Jun. 16, 1987 to Barken teaches an apparatus and method for computer controlled laser surgery using an ultrasound imaging system.. The position of the laser energy delivery device is monitored with an ultrasound probe. The probe, in conjunction with a computer system, provides a multiplicity of cross-section images of the portion of body tissue within the range of emitted destructive radiation.
U.S. Pat. No. 5,109,859 issued May 5, 1992 to Jenkins teaches an ultrasound guided laser angioplasty system. This system is also directed to the removal of atherosclerotic plaque from coronary arteries of patients with heart disease. A probe with a phased-array ultrasound transducer will produce images of vascular tissue acquired in a plane that is 30� forward of the tip of the catheter to prevent vascular perforation. As above, the catheter provides primarily lateral imaging.
U.S. Pat. No. 5,158,085 issued Oct. 27, 1992 to Belikan et al. teaches a lithotripsy ultrasound locating device using both a locating and a therapy transducer in a fixed relationship. One or more locating ultrasound transducers, each axially rotatable and extendable, generate a signal representing the distance between the locating transducer and the focus of the second transducer, used to transmit therapeutic amounts of ultrasound for fragmentation of a concretion. The locating transducers can have two or more crystal rings, thus having two or more focal ranges, and operate according to annular phased-array principles.
U.S. Pat. No. 5,313,950 issued May 24, 1994 to Ishikawa et al. teaches another ultrasound probe. A rotor moves and/or rotates a transducer and/or a reflector and is driven by a stator outside the object under examination. Both forward as well as lateral firing of ultrasound is taught for obtaining sectional views. However, such rotating mirror technology is distinctly different from the ranging application disclosed herein.
U.S. Pat. No. 5,350,377 issued Sep. 27, 1994 to Winston et al. teaches a medical catheter using optical fibers that transmit both laser energy and ultrasound imaging signals. An external transducer couples to the optical fibers and pulse echoes are received and transmitted back to the transducer along the same optical fibers. Visualization is limited to images as to the configuration, location and character of the tissue in the area of atherosclerotic plaques.
As is evident by a review of the ultrasound imaging prior art, including the foregoing, catheters and other tools for TMR having axial ranging capability, in the sense of determination of the distance from the tip of the firing laser delivery means at a first wall of the heart to a second wall of the heart are virtually unknown. Determination of tissue depth viewed in a forward direction, such as in myocardial tissue for forming TMR channels, would be highly advantageous so as to prevent unwanted perforation of a heart wall and/or to form channels of selected depths.
Thus, it is an advantage of the present invention to provide a catheter, MIS or other surgical apparatus and method of use for percutaneous and other surgical procedures, including percutaneous, MIS and surgical TMR, stimulation procedures, agent delivery procedures including but not limited to the delivery of an angiiogensis stimulation agent, which overcome the limitations of the prior art.
It is another advantage of the present invention to provide such an apparatus with an ultrasound guidance system to provide visualization, depth determination, in particular apparatus tip-to-tissue wall distance in tissue for controlled treatment as desired, in particular to prevent perforation of epicardial tissue in percutaneous TMR.
An additional advantage of the present invention allows determination/visualization of the spatial dynamics of the tissue of a beating heart, i.e., one in which the wall depth is constantly changing.
It is a further advantage of the present invention to provide such an apparatus wherein the ultrasound guidance system is small, compact and durable, and either integral with the tip of an elongated body or modular to, interchangeable with and replaceable on an elongated body.
Yet a further advantage of the present invention is to provide such an apparatus for percutaneous, MIS or other surgical placement within a heart chamber, organ aperture or other body opening, the apparatus having at least one central lumen extending along at least part of the length of the tool for guiding a laser delivery means or other functional device to selected surfaces of the heart chamber, organ aperture or other body opening for laser or other treatment thereon, particularly adapted for laser-assisted transmyocardial revascularization (TMR).
One more advantage of the present invention is to provide such ultrasound apparatus with ranging visualization means enabling visualization of piercing of a heart wall, advancement of the piercing tip to a selected depth within myocardium, confirmation of such depth, and controlled, visualized withdrawal of the firing tip during laser activation for TMR.
Another advantage of the present invention is an ultrasound and ECG electrode apparatus that can be used to determine tissue thickness and and changes in thickness during the cardiac cycle. The thickness can be measured during a fixed period of the cardiac cycle or the ultrasound transmission can be synchronized with the ECG signal from the patient.
These and other advantages are obtained in an apparatus for treatment of body tissue that includes a treatment apparatus configured to be inserted into a patient. The treatment apparatus has a distal portion, a distal end, a proximal portion and an axis. An ultrasound transducer is positioned at the distal portion of the treatment apparatus. The ultrasound transducer includes a distal ultrasound energy delivery surface and a proximal ultrasound energy delivery surface. An ultrasound energy attenuator member is positioned adjacent to the proximal ultrasound energy delivery surface and is made of a material to at least partially attenuate ultrasound energy from the proximal ultrasound energy delivery surface.
In another embodiment of the invention, an apparatus for treatment of body tissue includes a treatment apparatus configured to be inserted into a patient. The treatment apparatus includes a distal portion, a distal end, a proximal portion and an axis. An ultrasound transducer is positioned at the distal portion of the treatment apparatus. The ultrasound transducer has a distal ultrasound energy delivery surface and a proximal ultrasound energy delivery surface. An electrode is positioned adjacent to the ultrasound transducer at the treatment apparatus distal end.
In yet another embodiment of the invention, an apparatus for treatment of body tissue includes a treatment apparatus configured to be inserted into a patient. The treatment apparatus has a distal portion, a distal end, a proximal portion, a treatment apparatus lumen and an axis. An ultrasound transducer is positioned at the distal portion of the treatment apparatus. The ultrasound transducer includes a distal ultrasound energy delivery surface and a proximal ultrasound energy delivery surface. A retractable energy delivery device is retractably positioned in the treatment apparatus lumen.
Numerous other advantages and features of the present invention will become readily apparent from the following detailed description of the invention and the embodiments thereof, from the claims and from the accompanying drawings.
FIG. 10(a) illustrates a determination of wall thickness when the distal tip is in contact with the wall.
FIG. 10(b) illustrates the determination of distance between the distal tip of the apparatus and a front boundary surface of a tissue site, as well as the return echo from the back boundary surface of the tissue site.
FIG. 10(c) illustrates the return echos from the front and back boundary surfaces of a tissue site are not detected when the angle between the longitudinal axis of the distal tip and the tissue surface falls outside of an acceptance range.
The distal tip 102 can be modular and independent of the elongated body 103 as illustrated in FIG. 2. The distal tip 102 includes a mounting face 130 with a mounting flange 132. The mounting face 130 and mounting flanges 132 provide a means for coupling the distal tip 102 to the distal end of an elongated body 103 such as conventional or other type catheter, MIS or other surgical apparatus. It will be understood by those known in the art that such coupling means includes, and is not limited to, bayonet and other quick connect mounts, screw on or snap on adhesive couplings, etc. Thus, in the preferred embodiment, the distal tip 102 is modular and can be coupled with an elongated body to provide an operable apparatus 100. A suitable elongated body includes, but is not limited to, a conventional catheter, a steerable catheter with a deflectable end portion, an MIS or other surgical apparatus, a modular fiber advance handpiece unit, and other functional devices including fiber advance depth control mechanism, visualization means, drug delivery apparatus, etc.
Although the distal tip 102 is illustrated as being modular in
The backing 114 can attenuate the ultrasound waves traveling in the direction indicated by the arrow 101. Attenuation can be achieved by constructing the backing from materials as described in U.S. Pat. No. 4,528,652, which is incorporated herein by reference in its entirety. In one embodiment, the backing 114 is made of a material that attenuates sound in the range of 10-30 dB/MHz-cm, preferably in the range of 15-25 dB/MHz-cm and more preferably about 20 dB/MHz-cm. The material for backing 114 is preferably made of at least two materials of different densities and can be a mixture of epoxies and microspheres in proportions selected to provide the ranges in the preceding sentence. A suitable epoxy mixture is EPO-TEK 301, commercially available from Epoxy Technology, Inc., Billerica, Mass. A commercially available microsphere is EXTENDOSPHERES, available from PQ Corporation of Valley Forge, Pa.
At electrically insulating housing 116 in the shape of a cup holds the backing 114. Suitable materials for the housing 116 are preferably soft materials including, but not limited to, polycarbonate, acrylic, PVDF, and the like. The housing 116 provides the backing 114 with a tapered geometric configuration. Tapering increases an echo return path of sound, extends the path of wave travel and provides greater attenuation. Backing 114 substantially eliminates echoes reflected off surface 146 that are larger than −40 dB relative to echoes from a target surface in front of the transducer 104. In one embodiment, the target surface is substantially flat. The backing preferably has a length of 3 mm or less and more preferably 2 mm or less.
The coaxial cable 112 extends through the housing 116. It will be understood that the coaxial cable 112 is but one possible signal interface which couples signals sent to and received from the ultrasound transducer 104 with a signal processing component. The coaxial cable can have a diameter of about 0.0075″ as currently available and manufactured by companies such as Temp-Flex Cable, Inc., located in Graften, Mass., to reduce the diameter of the apparatus 100. The coaxial cable 112 is comprised of, from the inside out, a conductor 160, an insulation layer 162, a shielding layer interface 164 and an outer jacket 166. The stripped central conductor 160 is inserted through housing 116 into the backing 114 which is preferably conductive. When the backing is not conductive, the central conductor 160 can extend through the backing and be coupled with the distal side of the transducer 104. The shielding layer interface 164 is coupled with the sputtered metal gold trace 106. The shielding layer interface 164 and the central conductor 160 can be electrically connected to the respective gold trace 106 and backing 114 using simple contact technology, conventional solder, silver or indium epoxy, etc. Applying a voltage across the transducer 104 can actuate the transducer 104 to create an acoustic wave traveling in the direction generally indicated by the arrow 115. Upon reflection of the wave off an anatomical surface, the acoustic echo will return to the transducer 104 and create a signal which can be detected and amplified. It will be understood that the transmitter and receiver combination may be a conventional design and/or may be a single, combined module.
The distal tip 102 can also include an electrode 121. Suitable materials for the electrode 121 include, but are not limited to, gold, platinum and titanium. The electrode 121 has sufficient size to detect electrical currents produced within the body of the patient. Suitable sizes for the electrode include, but are not limited to, 1.0-15 mm2, 1.5-12 mm2 and preferably 5-10 mm2. When it is desirable to increase the size of the electrode 121 to increase sensitivity, the electrode 121 can extend at least partially along the sides of the distal tip 102. The electrode 121 can be a unipolar electrode which would require an external ground reference or a bipolar electrode which could be separated into electrically isolated sections.
The electrode 121 is illustrated as being electrically coupled with the coaxial cable via the gold trace 106 but can be directly coupled with the coaxial cable 112. As a result, electrical signals from the body and from the ultrasound echo travel on the coaxial cable. The electronics for processing the signals from the coaxial cable 112 can include a high pass filter (fc=5 MHz) for the ultrasound echoes and a low pass filter (fc=100 Hz) for the signals from the body.
As will be discussed in more detail below, the electrode 121 can be electrically coupled with a processing unit with logic for processing signals from the electrode 121. These signals can be used at least in part to determine the ECG of the patient. The laser means can also be coupled with the processing unit. As a result, the delivery of laser energy from the laser means can be adjusted in response to the ECG.
An optional coating 123 can cover at least a portion of the distal tip 102. Suitable materials for the coating 123 include, but are not limited to, PARYLENE and TEFLON. The coating can be electrically conductive and cover the electrode or can be electrically insulative and be positioned between the electrode 121 and the matching layer 110 as illustrated in FIG. 3.
It will be understood that the material of construction as well as the shape of the ultrasound transducer 104 can be changed and that a generally flat annulus shaped transducer 104 is but one of many embodiments. Numerous types of ultrasound transducer materials are known, and the class of materials known as piezoelectrics are but one. Likewise, different shaped transducers are known and readily available, the different shapes having different signal propagating and receiving characteristics. As an example but not to limit the scope of the apparatus, the transducer 104 element may have a slightly parabolic shape. Furthermore, a single transducer 104 element can be divided into sections or replaced with a plurality of transducer 104 elements, optionally configured in an array such as a phased array or other. Such configurations comprising more than one transducer 104 element will have associated electrical couplings, drivers, etc.
Additionally, the ultrasound device may be used with curved or pre-bent catheters for delivery of a single optical fiber with or without a lens device for operatively, selectively and/or controllably directing laser energy.
As shown, such selected amplified signals 218 can be received by RFout buffer 220 for recording the information, etc. A video detector sufficient to cover the range of possible frequencies used in the ultrasound system, such as between about 5 and 20 MHz and more preferably about 15 MHz, provides a signal used to create an A-mode scan for viewing on display 260. It will be understood that the display means 260 can be an oscilloscope, computer monitor, or can be input to a computer and stored. It will further be understood that software processing of emitted pulse data and echo data to calculate signal delay, for determination of depth to a tissue boundary surface, or depth of myocardium from wall to wall, can be achieved using various or custom software.
One Embodiment of a Preferred Methodology
It is well understood that the time for return of an echo from a distance D is given by the following equation:
where Vs is the velocity of longitudinal sound waves, i.e., approximately 1540 m/s in myocardial tissue. Therefore, algorithms for generating the A trace consist of detecting the envelope of the received RF signal. These algorithms are known in the areas of echo ranging with ultrasound and radar. In a preferred embodiment, the analytic magnitude is used, which consists of computing the Fourier transform, taking the real part of the result, and computing the inverse Fourier transform. If the original echo signal is called f(t), then the amplitude A(t) is computed according to the following equation:
Referring again to
An additional peak 270 is observable at the right side of the display 260. As the ultrasound wave propagates through the tissue, an additional returning echo signal will indicate a structural interface or tissue boundary surface at the position which correlates with the distance to the back wall of the penetrated structure 271, i.e., in this case, the epicardial surface 271 of the heart. However, as is well known, the contractions of the beating heart will tend to cause the wall of the heart to contract in direction B, as well as expand in the opposite direction, thereby causing the wall peak 270 to move in direction C. Thus, an additional wall peak 270 a will be observable, which will be a transient peak moving between the positions indicated for peaks 270 and 270 a. Thus, as shown, the distance D can be correlated with the length of the channel 272 and the distance E can be correlated with the distance moved by the epicardial surface 271 during contraction of the heart muscle. Therefore, it will be understood that distance F will be correlated with the amount of remaining myocardial tissue between the end of the TMR channel 269 and the epicardial surface 271. This information regarding remaining depth of tissue is vital to the cardiologist in performing TMR. With regard to percutaneous TMR, by controllably forming TMR channels initiating at an endocardial surface, perforation of the epicardium can be avoided.
The apparatus 100 will be usefully operated at pulse repetition frequencies between about 100 Hz and about 10 KHz. Such frequencies will be fast enough to allow real-time display of the thickening and thinning of the heart wall brought about as a result of the changes between the systole and diastole components of the beating heart cycle.
As illustrated in
Referring now to FIG. 10(a), a portion of distal tip 102 is in physical contact with a heart tissue front boundary surface. The angle between the longitudinal axis of the distal tip 102 and the normal to the front boundary surface is 30� or less and a returning echo from the rear boundary surface is detected. In this manner, the depth of the tissue surface at the point of contact between distal tip 102 and the front boundary surface is determined. In FIG. 10(b), the distance between distal tip 102 and the front boundary surface is determined prior to contact. Finally in FIG. 10(c), the angle between the longitudinal axis of the distal tip 102 and the normal to the front boundary surface is too large and the return echo from the back boundary surface is not detected.
When the angle between the longitudinal axis of the device and a line perpendicular to the tissue surface is larger than the angle of acceptance of the apparatus 100, the reflected echo signal will not impinge on the transducer 104, resulting in no signal on the display. Since it is preferable to have contact occur within a limited angular range in order to ensure penetration, the lack of echo signal is interpreted as improper positioning, thus avoiding the firing of the laser means under unfavorable conditions.
Referring to
As another method for confirming and/or controlling specific heart function, pacing of the heart has been described. When pacing the heart with an external pulse generator during a TMR procedure, there is often no positive confirmation that the heart has beat properly or even at all. Therefore, this positive confirmation of heart function, albeit compromised or otherwise imperfect, may be accomplished by using the ultrasound ranging device and methods described herein to detect the heart beat. The ultrasound device could monitor the measured thickness of the myocardium and determine when a change has occurred. The change in thickness of myocardium can be correlated with contraction and/or expansion of the heart. This signal indicating that the heart has beat could be used such that the TMR laser would not fire unless and until this signal is received. Such pacing of the heart is more fully described in co-pending U.S. patent application Ser. No. 16684-702], filed Feb. 3, 1997 entitled "REVASCULARIZATION WITH HEART PACING", as well as U.S. patent application Ser. No. , filed concurrently therewith entitled "REVASCULARIZATION WITH LASER BURSTS AND REVASCULARIZATION WITH HEARTBEAT VERIFICATION", both of which are incorporated herein by reference.
Therefore, when a device or method in which a pre-set number of laser pulses, such as a burst of 5 pulses, is used to create the TMR channels, that pre-set number of pulses can be automatically reduced when heart wall thicknesses are reduced to below a predetermined threshold, such as 5 millimeters. This heart wall thickness measurement can be made in essentially real time by the axial ranging devices and methods of using the apparatus 100.
One embodiment of apparatus 100 shows real time indications of wall thickness as well as thickness changes during the cardiac cycle. Additionally, apparatus 100 can be used to measure the thickness during a fixed period of the cardiac cycle. Further, the ultrasound transmission of apparatus 100 can be synchronized to the ECG signal from the patient. Synchronization allows the signal to reduce the effect of the cardiac cycle on the readings from a ranging system.
Referring now to
In one specific embodiment of ranging display system 800, an HP3314 signal generator creates a pulse of RF energy. This pulse can be 12 MHz sine wave with an amplitude of 100 mV peak to peak. The duration of the pulse is 8 complete cycles or 667 nsec and the repetition frequency is 100 Hz. The computer controls the initialization and setup of parameters for the signal generator via a GPIB bus. The signal is amplified by an ENI RF amplifier which has a gain of 50 dB. This generates a signal with a peak power of 2.5 watts (with a transducer impedance of 50 ohms). The amplified signal is transmitted to the connector on the front of the unit to which the transducer 104 is connected.
Echos are returned from the transducer 104. These echos will correspond to either the epicardium or the distal tip of the fiber. The signals from the transducer 104 are amplified and filtered through an RF receiver and digitized. The gain is selected by the operator. The filter in the receiver has a 12 MHz center frequency and a 3 MHz 3 dB bandwidth. The amplified and filtered signals are digitized using an 8 bit, 40 MHz data acquisition system.
A computer controls the setup and initialization of the signal generator, and reads the signals from the data acquisition system. The processing of the digitized signals includes rectification, peak detection and temporal and spatial filtering. The processed signals are displayed on the LCD and comprise the A-trace data.
Several algorithms exist for generating an A-trace which is the envelope of the received RF signal (f(f)) from an ultrasound system. The sample algorithms include:
additionally, a conventional solution exists in which the A-trace is calculated as follows:
An alternative algorithm is as follows:
where ΔT is chosen to be an integer multiple of the sample period. This algorithm has the advantage of easy computation similar to equation (3) and better performance approaching equation (5). This method will give similar results to the optimal algorithm (5) in the case where the sample rate is equal to a multiple of 4� the fundamental frequency of the RF and the frequency of the envelope wave form is low in comparison to the fundamental frequency. In order to maintain the optimal performance a variable sample rate could be implemented to maintain the preferred relationship. In each case the parameter ΔT is chosen to as close to (1/4)(1/frf) where frf is the fundamental frequency. This gives a delay of 90� in phase between the two samples used in the equation.
Algorithm for Processing Ultrasound Echos #2
A computer simulation was performed in which a ramp function modulated by a fundamental frequency (frf) is sampled at a 40 MHz rate and processed by algorithm 6. The output of the algorithm is compared to the input wave form and a RMS error is computed. This simulation was repeated for frequencies from 9-16 MHz and ΔT=1, 1 and Z � the sample interval. For ΔT=O the algorithm is equivalent to equation (1) and is used as a baseline for comparison. The table below shows the results of the simulation.
RMS Error RMS Error RMS Error frf ΔT = 0 ΔT = 1Ts ΔT = 2Ts Error % 9 .223 .031 .199 13.9 10 .377 .015 .377 3.9 11 .223 .030 .199 13.4 12 .251 .056 .156 22.3 13 .220 .083 .110 37.7 14 .228 .107 .060 26.3 15 .266 .129 .026 9.77 16 .250 .153 .059 23.6
The table includes values of ΔT=0, 1Ts and 2Ts where Ts=the sampling interval. The % error is calculated by comparing to the baseline values (ΔT=0). For the higher fundamental frequencies (ff greater than 12 MHz), a delay of 2�Ts gives a lower error than 1�Ts and the algorithm could choose the desired delay as a function of frequency. This comparison shows that for the non-optimal fixed sample frequency case the RMS error is reduced to at worst 37% of algorithm (1). An advantage of the above formulae are the provision of a smooth signal with rapid processing which allows for an increased sampling rate and variable sampling.
Advantages of the preceding embodiment include improved accuracy, ease of use and implementation with a need for a less powerful computer, a higher process flow rate, an increase in sampling rate with a fixed amount of memory and higher resolution and ease in writing the code.
By the present disclosure, it will be apparent to those skilled in the art that audible or visual alarms may be incorporated into the apparatus 100. Audible or visual alarms will give the cardiologist advance notice of achievement of threshold TMR channel depth penetration. Such alarms can also be integrated with mechanical as well as electronic interlock systems for the laser, thereby enhancing efficacy and safety of the apparatus 100 and methods described herein. Thus, the apparatus 100 may also be configured to include means to automatically stop fiber advance based on the calculated or otherwise determined axial distance of the firing tip of the laser delivery means from the back wall. Such means includes, but is not limited to mechanically or electronically controlled interlock with feedback loop, electrophysiology signal, etc. The present invention will assist the cardiologist in visualizing the tip of the catheter or surgical apparatus and the distal tip of the laser delivery means, as well as the endocardial wall, in a percutaneous, intra-ventricle procedure, so that identification of contact between the distal tip of the apparatus 100 or laser delivery means and the heart surface can be made. Such contact identification will allow the operator or cardiologist to avoid applying excessive force upon the heart, and thus avoid excessive arrhythmagenic forces thereby.
The present invention is intended for use with any medical laser. In particular, the Holmium, YAG or excimer lasers are particularly suited to the present invention. However, any suitable laser source, pulsed or otherwise, could provide laser energy to the laser delivery means for performing the disclosed methods. Furthermore, other interventional systems, in addition to lasers, which are included within the scope of the apparatus 100, other radio frequencies thermal or mechanical intervention. Based on the disclosure herein, control of these types of interventional modalities will be known to those skilled in the art.
Likewise, the catheter and surgical equipment, including laser delivery means, referred to in the present document as well as that known and used in medicine and other disciplines today and in the future, will be included in the scope of this disclosure. Such laser delivery means include, but are not limited to, individual optical fibers, fibers or fiber bundles with lens tips as well as bundles of fibers with and without piercing tips and with or without firing tips, fiber ends having shaped or contoured end faces for selectively diverging the laser beam or other laser energy diverging means, rods, mirrors configurations and other laser delivery means with and without focusing lens and the like. It will also be understood that the apparatus and method as described herein including the novel combination or use with of any conventional mechanism or method which are known to those skilled in the art, are included within the scope of this invention. Furthermore, with regard to non-laser TMR, a cannula or trocar assembly may be extended into the tissue of the left ventricle, with or without use of a mechanical piercing tool.
It will further be understood that while the present invention has been described for performing TMR on endocardial surfaces in the left ventricle, the apparatus and methods described herein are equally intended for use in any suitable procedure, including but not limited to procedures where any device need be extended through a guide catheter to a given surface on a given structure and extended into the structure a selected and controlled distance, for any medical procedures including laser treatment, tissue or organ visualization, biopsy, etc. AStimulation@, for example, is performed by using laser energy to create zones or pockets, optionally interconnected at least initially by small channels ablated through the tissue, for the introduction of blood born growth and healing factors and stimulated capillary growth surrounding the lased zones or pockets to create an increased supply of oxygen to the tissue and thus a revitalization of the heart muscle. Methods and apparatus for causing stimulation are more fully described in co-pending U.S. patent application Ser. No. 08/664,956 filed Jun. 13, 1996.
The present invention is applicable to TMR, stimulating angiogenesis, introducing an angiogenesis stimulation agent, determining that location of a distal portion of a catheter or other surgical device relative to a surface of a targeted body structure, and the like.
Suitable angiogenesis stimulation agents include recombinant proteins, specific inhibitors of protein-protein interactions, tyrosine kinase inhibitors, gene transfer agents and the like. More specifically, the angiogenesis stimulation agent can be a basic fibroblast growth factor (bFGF) or a vascular endothelial growth factor (VEGF).
The FGF growth factor family includes eight structurally-related polypeptides: basic FGF, acidic FGF, int 2, hst 1/k-FGF, FGF-5, FGF-6, keratinocyte growth factor, AIGF (FGF-8) and a glia-activating factor, heparin-binding growth factor purified from the culture supernatant of a human glioma cell line, (Miyamoto, M. et al., Mol. and Cell. Biol., 13(7):4251-4259 (1993). The genes for each are cloned and sequenced. FGF-1 and FGF-2 are characterized as acidic and basic fibroblast growth factors.
Bacterial Expression and Purification of FGF-13 Protein
The DNA sequence encoding FGF-13 ATCC #97148, is initially amplified using PCR oligonucleotide primers corresponding to the 5′ sequences of the processed protein (minus the signal peptide sequence) and the vector sequences 3′ to the gene. Additional nucleotides corresponding to the gene are added to the 5′ and 3′ sequences respectively. The 5′ oligonucleotide primer 5′ GCCAGACCATGGAGAATCACCCGTCTCCTAAT 3′ (SEQ ID NO:3) contains a Nco restriction enzyme site. The 3′ sequence 5′ GATTTAAGATCTCGTGAGGGGCTGGGGCCG3′ (SEQ ID NO:4) contains complementary sequences to a BglII site and is followed by 18 nucleotides of FGF-13 coding sequence.
The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth, Calif.91311). pQE-60 encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-60 was then digested with NcoI and BglII. The amplified sequences are ligated into pQE-60 and are inserted in frame with the sequence encoding for the histidine tag and the ribosome binding site (RBS). The ligation mixture is then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory manual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (KaW). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA is isolated and confirmed by restriction analysis. Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml)and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O. D. sup 600) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalactopyranoside") is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression. Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chapotropic agent 6 Molar Guanidine HCl. After clarification, solubilized FGF-13 is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)). The proteins are eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mm olar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the proteins are dialyzed to 10 mmolar sodium phosphate.
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added. This is then incubated at 37 degree(s) C. for approximately one week. At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads. The cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively. The 5′ primer containing an EcoRI site and the 3′ primer having contains a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the EcoRI and Himd III fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is used to transform bacteria HB101, which are then plated onto agar-containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+am 12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells). Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.
The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.
To synthesize a protein having the mammalian bFGF amino acid residue sequence by recombinant DNA, a double-stranded DNA chain which encodes bFGFs synthetically constructed. In addition to the bFGF-encoding sequences, the DNA chain that is synthesized may contain additional sequences, depending upon vector construction considerations. Typically, the DNA chain is synthesized with linkers at its ends to facilitate insertion into restriction sites within a cloning vector. The DNA chain may be constructed so as to encode the bFGF amino acid sequences as a portion of a fusion polypeptide; and if so generally contains terminal sequences that encode amino acid residue sequences that serve as proteolytic processing sites, whereby the bFGF polypeptide may be proteolytically cleaved from the remainder of the fusion polypeptide. The terminal portions of the synthetic DNA chain may also contain appropriate start and stop signals.
To assemble a bFGF-encoding DNA chain, oligonucleotide are constructed by conventional methods, such as procedures described in T. Manatis et al., Cold Spring Harbor Laboratory Manual, Cold Spring Harbor, N.Y. (1982) (hereinafter, CSH). Sense and antisense oligonucleotide chains, up to about 70 nucleotide residues long, are synthesized, preferably on automated synthesizers, such as the Applied Biosystem Inc. model 380A DNA synthesizer. The oligonucleotide chains are constructed so that portions of the sense and antisense oligonucleotide overlap, associating with each other through hydrogen binding between complementary base pairs and thereby forming double stranded chains, in most cases with gaps in the strands. Subsequently, the gaps in the strands are filled in and oligonucleotides of each strand are joined end to end with nucleotide triphosphates in the presence of appropriate DNA polymerases and/or with ligases.
As an alternative to construction of a synthetic DNA chain through oligonucleotide synthesis, cDNA corresponding to bFGF may be prepared. AcDNA library or an expression library is produced in a conventional manner by reverse transcription from messenger RNA (mRNA) from a bFGF-producing mammalian cell line. To select clones containing bFGF sequences, hybridization probes (preferably mixed probes to accommodate the degeneracy of the genetic code) corresponding to portions of the bFGF protein are produced and used to identify clones containing such sequences. Screening of the expression library with bFGF antibodies may also be used, alone or in conjunction with hybridization probing, to identify or confirm the presence of bFGF-encoding DNA sequences in DNA library clones.
The double-stranded bFGF-encoding DNA chain is constructed or modified with insertion into a particular appropriate cloning vector in mind. The cloning vector that is to be recombined to incorporate the DNA chain is selected appropriate to its viability and expression in a host organism or cell line, and the manner of insertion of the DNA chain depends upon factors particular to the host. For example, if the DNA chain is to be inserted into a vector for insertion into a prokaryotic cell, such as E.coli, the DNA chain will be inserted 3′ of a promoter sequence, aShine-Delgarno sequence (or ribosome binding site) that is within a 5′ non-translated portion and an ATG start codon. The ATG start codon is appropriately spaced from the Shine-Delgarno sequence, and the coding sequence is placed in correct reading frame with the ATG start codon. The cloning vector also provides a 3′ non-translated region and a translation termination site. For insertion into a eukaryotic cell, such as a yeast cell or a cell line obtained from a higher animal, the bFGF-encoding oligonucleotide sequence is appropriately spaced from a capping site and incorrect reading frame with an ATG start signal. The cloning vector also provides a 3′ non-translated region and a translation termination site.
Prokaryotic transformation vectors, such as pBR322, pMB9, Col E1, pCR1, RP4 and lambda-phage, are available for inserting a DNA chain of the length which encodes bFGF with substantial assurance of at least some expression of the encoded polypeptide. Typically, such vectors are constructed or modified to have a unique restriction site(s) appropriately positioned relative to promoter, such as the lac promoter. The DNA chain may be inserted with appropriate linkers into such a restriction site, with substantial assurance of production of bFGF in a prokaryotic cell line transformed with the recombinant vector. To assure proper reading frame, linkers of various lengths may be provided at the ends of the bFGF-encoding sequences. Alternatively, cassettes, which include sequences, such as the 5′ region of the lac Z gene (including the operator, promoter, transcription start site, Shine Delgarno sequence and translation initiation signal), the regulatory region from the tryptophane gene (trp operator, promoter, ribosome binding site and translation initiator), and a fusion gene containing these two promoters called the trp-lac (Tac) promoter are available into which the synthetic DNA chain may be conveniently inserted and then the cassette inserted into a cloning vector of choice.
Similarly, eukaryotic transformation vectors, such as the cloned bovine papilloma virus genome, the cloned genomes of the murine retro viruses, and eukaryotic cassettes, such as the pSV-2 gpt system (described by Mulligan and Berg, Nature 277, 108-114, 1979) the Okayama-Berg cloning system (Mol.Cell Biol., 2: 161-170, 1982), the expression cloning vector described by Genetics Institute (Science 228: 810-815, 1985), are available which provide substantial assurance of at least some expression of bFGF in the transformed eukaryotic cell line. Production of bFGF or a polypeptide of a similar length can be achieved by producing the polypeptide initially as a segment of a gene-encoded fusion polypeptide. In such case, the DNA chain is constructed so that the expressed polypeptide has enzymatic processing sites flanking the bpFGF amino acid residue sequences. A bFGF-encoding DNA chain may be inserted, for example, into the beta-galactosidase gene for insertion into E. coli, in which case, the expressed fusion polypeptide is subsequently cleaved with proteolytic enzymes to release the bFGF from beta-galactosidase peptide sequences.
Frozen bovine pituitaries are homogenized with a Waring blender for 5 minutes in 0.15M ammonium sulfate (4 liter/kg tissue). The pH is adjusted to 4.5 with HCl and the homogenate stirred vigorously for 2 hours. After centrifugation (18,000�g, 30 minutes) the supernatant is retained. 230 g ammonium sulfate per liter of supernatant are added, the pH is adjusted to 6-6.5 with NaOH and the precipitation is allowed to proceed for 15 hours. After centrifugation of the reaction mixture (18,000�g, 30 min), the supernatant is retained. 300 g ammonium sulfate is added to each liter of the supernatant. The mixture is stirred for two hours. After centrifugation of the reaction mixture (18,000�g, 30 min) the pellet is retained, and the cumulative pellets from 3 kg starting tissue dissolved in 200 ml distilled water and dialyzed against 20 liters of distilled water overnight. The pH of the dialyzed retentate is then adjusted to 6, and the solution clarified by centrifugation (12,000�g, 30 min). The dialyzed retentate constitutes a dialyzed extract.
Basic FGF is subsequently isolated from the dialyzed, clarified extract using three successive protocols. Two of the protocols employ conventional ion-exchange and reverse phase HPLC purification steps. The third method uses heparin-Sepharose affinity chromatography in a key purification step as detailed as follows in the order in which they are performed.
(A) CM-Sephadex (C50) Ion-Exchange Chromatography
A 7�9 cm column of carboxymethyl Sephadex (C50) is washed with 1 liter of 50 mM sodium phosphate, 1.5M sodium chloride, pH 6.0 and then equilibrated with 0.1M sodium phosphate, pH 6 slashed zero. The dialyzed extract from 3 kg bovine pituitaries is loaded onto the column, and the column is washed sequentially with 0.1M sodium phosphate, pH 6.0 containing a) no NaCl, b) 0.2M NaCl and c) 0.65M NaCl, allowing the OD sub 280 to reach a minimum value before initiating each new wash. Fractions of 18 ml are collected at 3 ml/min at 4 degree(s) C. and subjected to radioimmunoassay.
(B) Heparin-Sepharose Chromatography
The 0.65M NaCl eluate from CM-Sephadex chromatography is loaded onto a 3�3 cm column of heparin-Sepharose (Pharmacia) previously equilibrated with 10 mM Tris-HCl, 0.6M NaCl, pH 7.0 at room temperature. The column is then washed sequentially with 10 mM Tris-HCl, pH 7.0 containing a) 0.6M NaCl and b) 1.1M NaCl, allowing the OD280 to reach a minimum value with each wash. The basic FGF is then eluted with a linear gradient in 10 mM Tris-HCl, pH 7.0 containing 100 ml 1.1M NaCl and 100 ml 2M NaCl. Fractions of 5 ml are collected at 0.8 ml/min and subjected to radioimmunoassay.
(C) Reverse Phase Liquid Chromatography
The basic FGF from heparin-Sepharose chromatography is pumped onto a Vydac C-4 (0.46�25 cm) reverse phase column using a 0.1% trifluoroacetic acid (TFA)/acetonitrile solvent system and eluted at 0.6 ml/min. with a 90 min. gradient of 23% to 35% acetonitrile. Fractions of 3 ml are collected at room temperature and subjected to radio-immunoassay.
In the above mentioned Radioimmunoassays (RIA) for basic FGF, antibodies are generated against a synthetic analog of the amino terminal sequence of basic FGF, [Tyr sup 10 ]bFGF(1-10) which is conjugated to bovine serum albumin, and subsequently used to develop the radioimmunoassay for basic FGF, as described in A. Baird et al. Regulatory Peptides 10, 309-317 (1985). Because it is not possible to quantitate unmodified cysteine by amino acid analysis, cysteine residues are modified either by reduction and alkylation with [sup 14 C] iodoacetamide (New England Nuclear) or oxidization with performic acid. The bFGF in 0.1% TFA/acetonitrile is dried in a 1.5 ml polypropylene microfuge tube in a Speed Vac vacuum centrifuge (Savant, Inc.) just prior to modification.
The reduction and alkylation of cysteine residues is performed in order to radioactively label cysteine residues, making it possible to determine which fragments of subsequent cleavage reactions contain cysteine residues. The dried bFGF is dissolved in 0.1 ml deoxygenated 0.5M Tris-HCl pH 7.7, 10 mM EDTA, 6M guanidine-HCl. Dithiothreitol is added to a final concentration of 5-10 mM, and the reduction is allowed to proceed at 37 degree(s) C. for 30 min. A 0.5-fold molar excess of [sup 14C]i odoacetamide (24 mCi/mmole) over total sulfhydryl groups is added, and the incubation continued at 37 degree(s) C. for 60 min. in the dark. The alkylation is terminated by addition of a large excess of dithiothreitolover iodoacetamide, and the alkylated bFGF purified by reverse phase-high performance liquid chromatography.
Performic acid oxidation of cysteine converts cysteine to cysteic acid, and the cysteic acid content of the protein is measurable by amino acid analysis. Performic acid is generated by incubating 9 ml distilled formic acid with 1 ml 30% H2O2 at room temperature in a tightly capped tube for 1 hour. 0.25 ml of this solution is employed to dissolve the dried bFGF (5-15 nmoles), and the oxidation permitted to continue at 0 degree(s) C. for 2.5 hours. Four lyophilizations from distilled water are employed to remove reaction by-products.
Basic FGFs (with cysteines modified by each method described above) are proteolytically and chemically digested to obtain fragments for further analysis, including sequence analysis. Prior to any digestion, the bFGF is dried in a polypropylene microfuge tube in a Speed Vac vacuum centrifuge from volatile RP-HPLC solvents.
In order to obtain multiple, overlapping bFGF fragments, three types of proteolytic digestions of bFGFs, with cysteines modified by each method described above, are performed as follows. The dried bFGF (1-5 nmoles) is dissolved in 0.01 ml 0.5M Tris-HCl pH 7.7, 10 mM EDTA, 6M guanidine-HCl and then diluted to 1 ml with 1% NH4HCO3. Submaxillaris protease or chymotrypsin is added in a 1/50 (w/w) ratio while digestions with Staphylococcus aureus V8 employed a 1:35 (mol:mol) ratio of enzyme to substrate. Submaxillaris protease cleaves at the C-terminus of arginine; Staphylococcus aureus V8 cleaves at the C-terminus of glutamic acid; and chymotrypsin cleaves at the C-terminus of several amino acid residues having bulky aromatic or hydrophobic groups. Incubations are allowed to proceed overnight at 37 degree(s) C. Digestion with cyanogen bromide, which cleaves proteins at the C-terminus of Met, are performed on bFGFs, with cysteines modified by each method described above, as follows. The dried, alkylated bFGF (5-6 nmoles) is dissolved with 0.05 ml 70% formic acid and reduced in a solution of 2.9MN-methylmercaptoacetamide in 7% formic acid (R. Heighten et al. Methods in Enzymol. (eds. Hirs., C. & Timasheff, S.) 91: Academic Press, N.Y., pp.549-559 (1983)) for 24 hours at 37 degree(s) C. The alkylated, reduced bFGF is purified by RP-HPLC, dried in a Speed Vac vacuum centrifuge and redissolved in 0.1 ml deoxygenated 70% formic acid. A 100-fold excess of cyanogen bromide is added and the incubation continued at room temperature in the dark overnight.
Reverse phase-high performance liquid chromatography purifications of modified bFGFs and their digestion fragments are accomplished using a Brownlee RP-300 reverse phase column (0.46�25 cm) and a 0.1%TFA/acetonitrile or a 0.1% heptafluorobutyric acid (HFBA)/acetonitrile solvent system. PhNCS-(sup 14 C)-carboxyamidomethylcysteine is identified during sequence analysis by liquid scintillation counting of the residues from the sequencer. The identification of cysteic acid in a given cycle is accomplished by comparison of the amino acid composition of the peptide and the remainder of its sequence as determined by Edman degradation. Carboxypeptidase Y is obtained from Pierce and utilized according to the manufacturer's recommendations. Carboxyl terminal analysis via tritium incorporation is accomplished as previously described (H. Matsuo et al. Protein Sequence Determination (ed., Needleman, S. B.) Springer-Verlag, N.Y., pp. 104-113 (1979)).
Synthesis of a bFGF-encoding DNA chain is accomplished by synthesizing oligonucleotides on an Applied B10 Systems automatic synthesizer with overlapping complementary sequences.
The overlapping oligonucleotides are fused to form a double-stranded DNA chain, gaps are filled in with DNA polymerase and with T4 ligase. Immediately 5′ of the FGF-encoding sequence in the sense strand is provided an ATG start signal, which results in an extraneous methionine being added to the N-terminus of the expressed polypeptide. Immediately 3′ of the bFGF-encoding sequence is a stop signal. At the 5′ end is a Eco RI overhang and at the 3′ end is a Sal I overhang, whereby the synthetic DNA strand is directly insertable in the Eco RI and Sal I site of the plasmid pUC8, described by Vieira er al. Gene 14: 259-268 (1982). The DNA strand is annealed into the pUC8 plasmid where it is under the control of the betagalactosidase promoter with the ATG start signal and the Shine Delgarno sequence retained in their natural orientation and association with the promoter.
The recombinant vector, designated bFGF, is transformed into the DH-1 strain of E. coli by the calcium chloride procedure, CSH, supra. The transformed E. coli is cultured in L broth, and ampicillan- resistant strains are selected. Because the DNA chain was inserted into the plasmid in an orientation which could be expected to lead to expression of protein product of the DNA chain, the ampicillan-resistant colonies are screened for reactivity with antiserum raised against bFGF extracted from the pituitary. These colonies are screened by the immunological method of Healfman et al., Proc. Natl. Acad. Sci. USA 80: 31-35 (1983), and colonies reacting positively with bFGF antibody are further characterized. The cells separated from their culture media are lysed, and their supernatant obtained. Supernatant from transformed cells is determined by RIA to be reactive with antibody raised against bFGF. 100 ml. of cell supernatant is obtained, and bFGF is purified therefrom using heparin-Sepharose as described above. Approximately 0.01 mg of bFGF, purified to upwards of 98% by weight of total protein, is produced. The biological activity of the synthetic bFGF, which contains the extraneous N-terminal methionine residue, is tested for biological activity by the ability of the synthetic bFGF to stimulate the proliferation of adult bovine aortic arch endothelial cells in culture, as described in J.Cell Biol. 97: 1677-1685 (1983). Briefly, cells (at passage 3-10) are seeded at a density of 2�10 sup 3 cells/dish on plastic tissue culture dishes and exposed to Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. Test samples, at a dilution ranging from 10 sup −1 to 10 sup −3, are added on day 0 and day 2 to the dishes. On day 4, triplicate dishes are trypsinized and counted in a Coulter counter. Background levels are ordinarily 10 sup 5 cells/dish, while those exposed to optimal concentrations of the growth factor can contain as much as 5 to 8�10 sup 5 cells. For a potency assay, a log response curve was established. For this purpose, 10 microliter-aliquots of a dilution (ranging from 10 sup −1 to sup −5) of the original solution made in 0.5%BSA/DMEM were added in triplicate.
The biological (mitogenic) activity of synthetic bFGF is substantially identical to natural, purified bFGF from bovine pituitary cells. The superfluous N-terminal residue is removable by partial chemical digestion with cyanogen bromide or phenyl isothiocyanate followed by treatment with a strong anhydrous acid, such as trifluoroacetic acid.
While the principles of the invention have been made clear in illustrative embodiments, there will be immediately obvious to those skilled in the art many modifications of structure, arrangement, proportions, the elements, materials, and components used in the practice of the invention, and otherwise, which are particularly adapted to specific environments and operative requirements without departing from those principles. The appended claims are intended to cover and embrace any and all such modifications, with the limits only of the true spirit and scope of the invention.
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|Dec 28, 1998
Owner name: ECLIPSE SURGICAL TECHNOLOGIES, INC., CALIFORNIA
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A novel by Darin Bradley
Unemployment has ravaged the U.S. economy. People struggle everywhere, exhausted by the collapse that destroyed their lives. Benjamin Cade is an expert in cognition, and before the flatlined economy caught up to him, he earned his living as a university instructor. Now, without income, he joins the millions defaulting on their loans - in his case, the money he borrowed to finance his degrees. But there are consequences. Using advances in cognitive science and chemical therapy, Ben's debtors can reclaim their property - his education. The government calls the process "Repossession Therapy". The data Ben's repossession will yield is invaluable to those improving the "indexing" technology - a remarkable medical advance that has enabled the effective cure of all mental disorders. By disassembling his mind, doctors will gain the expertise to assist untold millions. But Ben has no intention of losing his mind without a fight, so he begins teaching in the park, distributing his knowledge before it's gone in a race against ignorance. And somewhere in Ben's confusing takedown, Chimpanzee arrives. Its iconography appears spray-painted around town. Young people in rubber chimpanzee masks start massive protests. As Ben slowly loses himself, the Chimpanzee movement seems to grow. And all fingers point to Ben.
Similar Books by other authors...
Used availability for Darin Bradley's Chimpanzee
See all available used copies of this book at Abebooks UK or Abebooks US
Hardback Editions
August 2014 : USA Hardback
|Title: Chimpanzee: A Novel
Author(s): Darin Bradley
ISBN: 1-63023-000-6 / 978-1-63023-000-5 (USA edition)
Publisher: Underland Press
Availability: Amazon Amazon UK Amazon CA
Paperback Editions
August 2015 : USA Paperback
|Title: Chimpanzee: A Novel
Author(s): Darin Bradley
ISBN: 1-63023-014-6 / 978-1-63023-014-2 (USA edition)
Publisher: Underland Press
Availability: Amazon Amazon UK Amazon CA
Audio Editions
October 2014 : USA Audio edition
|Title: Chimpanzee
Author(s): Darin Bradley
Publisher: Resurrection House
Availability: Amazon
October 2014 : UK Audio edition
|Title: Chimpanzee
Author(s): Darin Bradley
Publisher: Resurrection House
Availability: Amazon UK
Kindle Editions
August 2014 : Canada, UK Kindle edition
|Title: Chimpanzee: A Novel
Author(s): Darin Bradley
Publisher: Underland Press
Availability: Amazon UK Amazon CA
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Please confirm that you want to add Adobe Acrobat XI to your Wishlist.
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Work files for this course can be downloaded from the first lecture.
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Heritage Bank to Aussie Women and their Superannuation Savings
Successful women usually find themselves juggling multiple responsibilities, both at home and in the workplace. With such a hectic life, it's often hard to find the time to think about the future.
But it's especially important for women to think about their superannuation and what it means for life after they finish work.
Women tend to live longer than men, are often paid less, and may have to take time out of the workforce to look after children in their early years. What that can mean is that women end up with a lower level of superannuation than men when they eventually retire.
Recent research by the Association of Superannuation Funds of Australia showed that the average superannuation account balance for women was around $40,000, while for men it was around $71,000. The average retirement benefit payout (at age 60-64) for women was $112,000 while for men it was $198,000.
There are a few simple things that successful women can do to close the gap and make sure they set themselves up for the future properly.
Keep an eye on your superannuation balance and look into steps you can take to maximise the benefit of superannuation savings. Super can offer you excellent tax advantages.
Of course, you also don't want to lock too much money away in super too early and not be able to use the money when you are looking to buy a house or start a family.
To find out more about superannuation rules, advantages and disadvantages, it's a good idea to consult a financial planner who can provide expert advice.
Heritage Bank has a range of financial planners across south east Queensland who can help you put in place the right financial plan for your circumstances. To set up an appointment, call the Heritage Contact Centre on 13 14 22, go to www.heritage.com.au or visit your nearest branch.
Contact Profile
Heritage Bank
Heritage Bank - customer-owned bank, offering Australians award winning home loans / mortgages, term deposits, credit cards, personal loans, banking accounts and much more.
W: www.heritage.com.au/


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Print, Web, & Mobile Publishing Photo & Video Workflows Adobe Software
Live, in-person, hands-on
choose from list at left or our calendar page
We teach Adobe Creative software, but also classes in photographic workflows, and several on "the basics" to get you started in web or desktop publishing. Our luxuriously small class sizes allow us adapt a course on the fly to accommodate that day's attendees. We teach people, not workstations.
We've all encountered experts who can't impart their knowledge. That isn't the case here.
New Classes:After Effects Essentials
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Achieve Motion Mastery!
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plan then publish Desktop Publishing Overview
A very good place to start Illustrator CC Level 2
stretch your vector skills
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We're honored & grateful to the many who have learned with us. Here are just a few:
We are now offering Adobe After Effects and Adobe Muse classes!
Steve presented to the Seattle Area Photoshop Users Group on April 18. In his talk, titled "Photoshop Adjustments: The State of the Art in 2017", Steve illuminated just how many ways there are to make Photoshop adjustments. View some outtakes here.
Weekend Classes! We've had some requests and are answering with a WordPress Level 1 offering Sunday August 13.
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Once again, we're thinking of offering evening classes. Please, let us know what you'd like to see.


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Long Shackle Torque Bolt; 41-63 Willys and Jeep Models
This long greasable replacement leaf spring pivot bolt and nut from Omix-ADA fits 41-63 Willys models. Front leaf spring to shackle.
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Bethel University's College of Arts & Sciences is a great place to finish your undergraduate education. Here, you'll receive a Christ-centered, liberal arts education with a tradition of excellence.
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Cindy Anschutz Barbieri
Based on a 2,000 calorie dietSee full nutrition
Ready In
This was very tasty. I haven't had truffles before so I am not quite sure what this oil should taste like... it added a slightly earthy musky taste, that I liked. I do have some black truffle s...


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Let us know when your receive great advice so we can thank our lawyers and point others with similar questions in the right direction.Got it!
They state that after 4 days you must fill out the paperwork for fmla and your employment status is changed to inactive effective from the first day you missed. It was my understanding that fmla was a benefit that I could ask to use?
I would contact the US Department of Labor's Wage and Hour Division. This agency enforces the law and would be able to assist you. I have inserted a link below for you.
This is meant as general legal information and is not to be construed as legal advice in an attorney client relationship.Ask a similar question
The employer may not force you to take such leave if your illness does not qualify under the FMLA. Thus, if it is not a serious health condition, the employer may not force you to take such leave. If it does, you may have a claim for unlawful interference under the act.
CAUTION: There is a divide among the circuits courts on this so the above statement may NOT apply in all states.....
Post a free question on our public forum.Ask a Question
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text:
Popular movies week 2 October 1932
Here's the list of the most popular movies in the week 02/October - 08/October - 1932. Do you remember Maedchen in Uniform by Leontine Sagan or Grand Hotel starring Greta Garbo, John Barrymore, Joan Crawford ?
What else happened in that week of 1932?
Discover what the world looked like on:


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Listen to free music played by lucaspinh
Blip.fm is internet radio made social. It's easy to search for, play, and discover free music recommended by real people. Join today to create your own free station and start listening to @lucaspinh.
Anjo de Resgate – Manda Teus Anjos| play
Jan 23, 2011 at 11:52 pm via YouTube | AMAZON | ITUNES
lucaspinh @Jungstir: "I know this may mess the flow.>:)stuff up gotta move somebody almost done with 2009 back later!" (reblip)
Third Eye Blind – Non-Dairy Creamer| play
Jan 8, 2011 at 2:50 pm via YouTube | AMAZON | ITUNES
TIRIRICA REMIX – DJ ALYSSON GUTEMBERG| play
Jan 8, 2011 at 2:44 pm via YouTube | AMAZON | ITUNES


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Yes, please contact the Potter Park Zoological Society at 342-2702 for details.
Bald Eagles can lift up to 4 pounds!
"My son would say that PPZ Rocks because he can see tigers there."
- Craig D, Lansing
Get events and information delivered to your inbox. No spam... promise.
Potter Park Zoo
1301 S. Pennsylvania Ave.
Lansing, MI 48912
Contact Us |


text:
THAI authorities have reportedly charged two men accused of killing an Australian travel agent Michelle Smith in Phuket in June.
Ms Smith, 60, of Perth, was fatally stabbed in Phuket on June 20 when two men on a motorbike tried to snatch her handbag.
The ABC says Surasak Suwannachote has confessed to the stabbing and Surin Tadthong has confessed to riding the motorbike during the crime.
They have been charged with cooperating to kill a person for personal benefit, taking property with a weapon, causing death and injury, and carrying a weapon in a public place.
They could face the death penalty, but the suspects' cooperation and admissions make a life sentence more likely if they are found guilty, the ABC says.
Originally published as Two men charged for travel agent death


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Will sharks keep Cape tourists at bay?
The great white sharks are back, and Cape business operators are split about whether they will draw tourists or drive them away.
"A lot of our customers are people who come for the weekends," said Jimmy Mitrokostas, the owner's son at Cooke's Seafood. "When they can't take advantage of the beaches, they don't come down."
But Ed Maas, owner of Orleans Waterfront Inn, said, "A lot of people like to come for the excitement."
|The article you requested has been archived
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- Cape Cod


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The background paper is from a Disney Finished in a Flash kit. The sticker in the corner is from Jolee's. The title is cut with the Mickey Font on the Cricut and then embossed.
The journaling is from the beginning of Wishes - it's the part the blue fairy says and the first part of the song.


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Slider Station's National Sliders
I passed by slider yesterday to try out the new Tahreer slider to find out they also offer Kuwaiti sliders. This one was named Kuwait Slider, it has been available for a few days now, comes with patty cheese, smoky aioli and coleslaw.
Soor Al Kuwait Slider also comes with patty cheese, smoky aioli and coleslaw but with ma'booch(معبوج) to add the Kuwaiti flavour. This has also been in slider for around two days now, they are both worth trying.
I really liked the Tahreer Slider, for those of you looking for a light slider or a small meal on the side, I recommend Tahreer. They gave it an Egyptian feel by adding flafel, cucumber and a tomato slice with taheena sauce on top, which is the traditional Egyptian sauce. I think all sliders are available until the end of the month, not sure though need to check with them.


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Learner's permit take three
I've taken the test twice and failed both times (and the reasons given contradict each other, but moving on). I had to get it renewed recently, because I couldn't pass the test in time before it expired.
Name: Chris
Want out of life: Fulfillment and self sufficiency.
Location: PA, USA


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Question: Define prior period errors How are they reported
Define prior- period errors. How are they reported?
View Solution:
View Solution:
Answer to relevant QuestionsDefine accumulated other comprehensive income (loss). Why is it a separate component of shareholders' equity? What are the two basic sources of shareholders' equity? Explain each. H& R Block is a well- known name, especially during income tax time each year. The company serves more than 25 million taxpayers in more than 10,000 offices in Canada, Australia, England, and the United States. The ...Average Corporation has the following shares outstanding at the end of 2013: Preferred shares, $ 3, no par value; 8,000 outstanding shares Common shares, no par value; 30,000 outstanding shares On October 1, 2014, the ...The annual report of Prodimax Ltd. for fiscal year 2014 included the financial statements for fiscal year 2013, which were restated to reflect the effects of a fraudulent action by a former employee. The restated financial ...
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Apple just doubled the basic storage on the iPhone SE, bumping it to 32GB while keeping the price the same, at $400. If you've wanted the phone's amazing battery life but balked at the 16GB basic storage option, this phone's for you.
Russia's antitrust investigators have ruled that Apple illegally engaged in price fixing and strong-armed its retail partners into going along with its plan. The company could face a fine of up to 15% of its sales in Russia.
It's been 10 years since Apple debuted the first iPhone. We review the devices' history, its achievements, and where Apple might go from here.
The FBI has acknowledged it can't crack all iPhone's — just the iPhone 5c and some earlier devices. While this is good news for Apple security, it's not the end of the issue.
Apple's new iPhone SE is a hand-sized device for anyone, with a design that echoes Apple's iPhone 5 family and the lowest price tag of any iDevice. Except for the storage capacity, it's a great-looking phone.
The first major update to iOS just landed — we take it for a test-spin on a just-upgraded device.
Apple's new iOS 9 is dropping on Wednesday — will you be grabbing it out of the gate, or playing it safe?
Apple has released a major point update for iOS with support for the Apple Watch, but the real question is whether it will also improve stability — a major issue as of late.
On September 17 — tomorrow — Apple will release iOS 8 for the iPhone 5S, 5C, and 4S, along with the iPad 2, 3, 4, iPad Air, and iPad Mini. If you want to install iOS 8 right now, before the official release, follow the instructions below. We will also include instructions how to officially update to iOS 8 when it is released tomorrow.
Apple is gearing up for the launch of the iPhone 6 in September, and has reportedly placed a massive order of between 70 and 80 million units — far more than any previous iPhone launch. As we've previously reported, the iPhone 6 will step up to a much larger 4.7-inch screen with a sapphire front panel — but curiously, there's now a very solid rumor that there will also be an iPhone 6 with a 5.5-inch screen. At first blush, a 5.5-inch iPhone 6 sounds utterly crazy, but it makes a little more sense following Apple's earnings call on Tuesday: Last quarter, Apple's tablet sales dropped a massive 19% sequentially and 9% year-over-year. Perhaps fading iPad sales are the reason Apple is finally looking at large-screen phones?
Late yesterday, Apple announced record earnings of $57.6 billion for the last quarter of 2013. The company sold a record number of iPhones (51 million), a record number of iPads (26 million), both of which contributed to a very healthy profit of $13.1 billion. By almost every measure of financial success, Apple is doing extremely well. Why, then, on the back of record earnings, did Apple's stock price tumble almost 10%? Why, after being one of the most successful companies in the world for the last 10 years, has its stock price been trending downwards for almost two years? For some reason, despite being one of the most popular, well-known, and desirable brands in the world, nothing Apple does ever seems to be good enough.
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text: Is Desmond one of the main characters in season 6 or not? Cuz ABC said that he's bumped down to recurring but he was listed as a main character in the opening credits and he was at the premiere in Hawaii so I'm confused. Is he or is he not a main character in season 6? Also on the ABC website he is listed as a main character with all the other main characters of season 6 and has a little bio.


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Saga Pearl II
0 out of 5 stars
Read the best port reviews and tips for this cruise itinerary.
|Day 1
|Southampton (London), England
|Day 2
|At Sea
|Day 3
|At Sea
|Day 4
|Day 5
|Stockholm, Sweden
|Day 6
|Helsinki, Finland
|Day 7
|St. Petersburg, Russian Federation
|Day 8
|St. Petersburg, Russian Federation
|Day 9
|Tallinn, Estonia
|Day 10
|At Sea
|Day 11
|At Sea
|Day 12
|Warnemunde (Berlin), Germany
|Day 13
|At Sea
|Day 14
|Ijmuiden (Amsterdam)
|Day 15
|Southampton (London), England
A roll call is a space for members who are signed up for the same cruise (same ship & departure date) to connect.
Make friends BEFORE you sail, exchange tips on what to do onboard and in the ports and maybe even organize a shore excursion with your ship mates.


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Vatican Peace Building conference spreads best practices in conflict resolution
In fact that's why these representatives gathered in Rome for a conference with the Catholic Peacebuilding Network. They were joined by Cardinal Peter Turkson of Ghana who listened to many of their stories.
Card. Peter Turkson
President, Pontifical Council for Justice and Peace
"In Congo we had conflict generated about mineral resources available and on a lot of renegade leaderships. Leaderships surrounded by armies who wanted to do their own thing.â?
The conference didn't focus only on violent conflict. They were also representatives speaking about disputes over religious freedom and how they could find a common ground with government.
Card. Peter Turkson
President, Pontifical Council for Justice and Peace
"If you start at United States, there is stuff going on there between the White House and the Catholic Church over the health care bill, that's something that calls for a certain amount of settlement.â?
During this two day seminar, many of these Catholic leaders shared their stories of success and failure in conflict resolution. Their hope is to learn from these different approaches and each take them back to their own country.


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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
I just reached 100 submissions. Will I need to upgrade?Asked by sketchy on April 01, 2015 at 12:56 AM
My free form just reached it's 100 limit, now my form is disabled. I only needed it until April 1st, and then not again until next year so I do not want tp upgrade if I do not need it again for 12 months. My question is will my account be restarted April 1st still for free or do I have to upgrade? Please reply because I am confused...sketchy
From my past experience with reaching the free form limit, the quota is usually reset on the 1st of every month, so in your case you should have a new quota for this month.
Please refer to this thread, the Jotform support team have provided a comprehensive explanation
Thank you for your explanation.
As mentioned by cyberhopper the account limits will be reset at the beginning of April, and you will have normal access to your account and forms. And your account will remain free if you do not wish to upgrade.
You can consult here the monthly limits of each plan.
Let us know if we can assist you further.


text: EnvironmentPakistan and UN ask for $350m in flood aid Prime minister says country needs help responding to disaster that has affected six million people. 18 Sep 2011 18:15 GMT | Environment, Humanitarian crises, Asia, Pakistan " /> Pakistan's prime minister says his country needs help responding to a flood disaster that has affected six million people. Yousaf Raza Gilani's government and the United Nations have asked for more than $350m dollars in foreign donations, as a UN team says nearly every district in the southern Sindh province has seen flooding. Al Jazeera's Imtiaz Tyab reports from Mirpur Khas in Lower Sindh.Source: Al Jazeera


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This desk and chair combo are ready to boldly go to work. Super-Fan Builds created a USS Enterprise desk from the saucer section with a matching chair that sits between the warp nacelles. Super-fan Jack Geiger is one lucky Trekkie.
See it come together in the video after the break.


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"Phoenix in Manchester"
About: Phoenix Futures / Trafford drug and alcohol services Phoenix Futures Trafford drug and alcohol services Manchester M16 0LN
Posted by gm (as ),
I was pointed in the wrong direction, heading downhill with alcohol coming to rule my life, and my relationships, as an omnipresent escape from every other difficulty I was facing.
Along with my GP, my psychiatrist, and wonderful family members, Phoenix have helped me turn things around. Being so friendly and welcoming, and providing a place I feel able to be open and honest about absolutely everything going on in my head, I feel much more confident that I'll be able to regain a grip on my life and start climbing back up that hill.
Response from Sara MacGregor, Locality Manager, Trafford, Phoenix Futures 6 years ago
Firstly my apologies for my late response I have had issues with logging in.
I am very pleased to read that your experience has been positive at Phoenix Futures in Trafford, also that your transition to the service was successful.
Many thanks for your positve feedback. I wish you every success in your Recovery.
Best wishes
- {{helpful}} of {{total()}} people think this response is helpful
- Was this response helpful? Yes | No
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Rain SummersSubscribe
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Rain Summers is a hot babe. Her bio will be updated as more information becomes available.
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