A transmitter (3) for generating a DQPSK-modulated optical signal, including: a splitter (7) for dividing an optical carrier signal into a first and second branch (8a, 8b), a first and second Mach-Zehnder interferometer (9, 10) in the first and second branch (8a, 8b), respectively, a phase shifter (11) in one of the branches (8b) generating a nominal phase shift of .pi./2, and a combiner (7′) for combining the optical output signals of the two branches (8a, 8b). The transmitter (3) has a feedback circuit (12) generating at least a first and second bias signal (15.1 to 15.3) for adjusting a bias of at least the first and second Mach-Zehnder interferometers (9, 10), the feedback circuit (12) includes a detector for generating at least a first and second feedback signal from a sample signal extracted from the optical signal after the combiner (7′), and for each bias signal: a local oscillator generating an auxiliary signal modulating the bias signal (15.1 to 15.3) at a pre-defined frequency (f1, f2, f3), a lock-in detector determining a phase difference between the feedback signal and the auxiliary signal, and a bias circuit for generating the bias signal (15.1 to 15.3) from an output signal of the lock-in detector, wherein the pre-defined frequencies of the auxiliary signals (f1, f2, f3) are different from one another (f1.noteq.f2.noteq.f3) and preferably no integer multiples of each other.

Differential Quaternary Phase Shift Keying (DQPSK) is an advanced new modulation format that has received great attention as a promising candidate for high spectral efficiency optical networks operable in high-speed transmissions (43 Gbit/s and beyond). However, the generation and reception of DQPSK signals is quite complex, as several feedback signals have to be generated to stabilize the transmitter and receiver, respectively.
In the following, a quick overview of the DQPSK generation and reception will be given for a better understanding of the invention, starting from an explanation of the PSK and QPSK modulation schemes.
Phase shift keying (PSK) is a digital modulation scheme for transmitting data by modulating the phase of a reference signal (carrier wave). Each of the phases is assigned a unique pattern of binary bits, in the following referred to as "symbol", represented by that particular phase. In QPSK, the symbols are arranged equally spaced around a circle in the complex plane, each symbol corresponding to a pattern of two bits ("dibits": "00", "10", "01" and "11") as shown in FIG. 2a. In Differential Phase Shift Keying (DPSK), information is not directly encoded in the phase of the symbols, but in their phase difference. Therefore, in the DQPSK modulation scheme the symbols of FIG. 2a are encoded in a pulse phase difference of π/4, 3/4 π, −3/4 π, −π/4, respectively.
Although QPSK (and consequently DQPSK) can be viewed as a quaternary modulation, it is easier to see it as two independently modulated quadrature carriers. The even (odd) bits are then used to modulate the in-phase component of the carrier, while the odd (even) bits are used to modulate the quadrature-phase component of the carrier. Therefore, for generating a DQPSK signal, a binary bit stream has to be divided into two sequences corresponding to the in-phase and quadrature-phase components, respectively, such that the generation of a DQPSK modulated signal is more difficult compared to classical modulation schemes as e.g. On-Off Keying (OOK).
A fiber optical transmission system 1 for DQPSK-modulated optical signals shown in FIG. 1 has a pre-coder 2 for pre-coding a binary bit stream by generating the two sequences described above and forming two corresponding electrical modulation signals. These are provided to a transmitter 3 for generation of a DQPSK-modulated optical signal therefrom. The optical signal is then transmitted via a fiber line 4 to a receiver 5 which demodulates the DQPSK-modulated optical signal by reconstructing the in-phase and quadrature-phase components.
The following description will be focused on the transmitter 3 and receiver 5, as the pre-coding performed in the pre-coder 2 is of minor relevance for the invention and its mode of functioning is well-known to the person skilled in the art.
The DQPSK transmitter 3 of FIG. 1 is shown in greater detail in FIG. 3a and comprises a laser source 6 (laser diode) for generating an optical carrier signal, an optical splitter 7 for splitting the optical carrier signal into a first and second branch 8a, 8b with equal intensities, a first Mach-Zehnder Modulator (MZM) 9 in the first branch 8a, a second Mach-Zehnder Modulator 10 and an optical π/2 phase shifter 11 in the second branch 8b, and a signal combiner 7′ to produce a single DQPSK output signal from the optical signals of the two branches 8a, 8b. The first and second MZMs 9, 10 are operated as phase modulators and are driven each with an electrical modulation signal uk, vk corresponding to the in-phase and quadrature-phase sequences as described above. The electrical signals are provided with a data rate of 20 Gbit/s each, leading to an overall data rate of the optical signal of 40 Gbit/s.
In the transmitter 3, two imperfections have been pointed out: bias deviations of the Mach-Zehnder Modulators 9, 10 used as phase modulators and phase shift errors of the π/2 phase shifter 11, both of which will be described in greater detail below.
For modulating the optical carrier signal with the electrical modulation signals uk, vk, the Mach-Zehnder Modulators 9, 10 being simple two-wave interferometers are driven with an electrical drive voltage leading to a sinusoidal transfer function as shown in FIG. 4. When working properly, a bias voltage of the MZMs 9, 10 is set at the minimum of transmission of the interferometer such that the electrical drive for +/−Vπ, respectively, correspond to two adjacent maximum transmission points, as is the case for the solid curve of FIG. 4. Then a high bit rate signal is applied which is centred around 0 V and has a 2 Vπ amplitude. However, if the bias voltage is not adjusted properly, a bias deviation ΔV may occur, leading to a shift of the transmission curve such that the maximum optical transmission is no longer attained for +/−VπV but for a higher voltage, as is the case for the dashed curve in FIG. 4.
The bias deviation ΔV, defined as a shift of the bias voltage from its proper value, leads to a modification of the symbol constellation by moving them from their ideal positions in the complex plane as shown by the arrows in FIG. 2b. A bias deviation in the MZM 9 of the first branch 8a shifts the symbols horizontally whereas a bias deviation in the MZM 10 of the second branch 8b containing the π/2 phase shifter 11 shifts symbols vertically (only the first case being shown in FIG. 2b). In both cases, symbols are closer to each other than in the idealistic case, such that back-to-back sensibility is lower.
The modification due to the bias deviation leads to an amplitude reduction in the DQPSK modulated signal as well as to phase errors, as modulated phases are not equal to 0 and π/2, see the time domain representation of FIG. 10a for an optical signal with a bias deviation equal to π/10, wherein the amplitude of the optical power and phase are represented by a solid and dashed line, respectively. The amplitude level of the modified signal is reduced compared to the ideal case, as the interferences of the signals from the two branches 8a, 8b in the combiner 7′ of FIG. 3a are not totally constructive. Furthermore, some artefacts (spikes etc.) appear in the phase and amplitude curves as the maximum transmission is not centred on voltages −Vπ and +Vπ, respectively.
The degradation of the performance of an optical transmission system caused by the bias deviation can be gathered from FIG. 11a, showing the Q-factor penalty (solid line) and output power deviation (dashed line) measured in dB in dependence of the bias deviation for an optical signal-to-noise ratio (OSNR) of 14.5 dB. It can be gathered from FIG. 11a that even though system performance is not strongly affected for low bias deviations, large penalties are caused for higher deviations, leading to a 1 dB penalty for a deviation of 0.6 rad. Therefore, it is mandatory to find a way of reducing the bias deviations in the MZMs 9, 10 for stabilizing the transmitter 3 of FIG. 3a.
Furthermore, the DQPSK transmitter 3 of FIG. 3a has to be stabilized with respect to impairments caused by the π/2 phase shifter 11. If the phase shift of the phase shifter 11 is not equal to π/2, interferences in the optical combiner 7′ or differential interferences at the demodulation part of a subsequent receiver are no more fully constructive or destructive. As can be gathered from FIG. 2c, representing the DQPSK symbols in the complex plane, a phase shift error leads to a dissymmetry in the DQPSK constellation resulting in different distances between the symbols, thus leading to different amplitude levels of the DQPSK optical signals generated in the transmitter 3, as can be gathered from FIG. 10b showing a time domain representation of an optical signal for a phase shift error of π/10. The main impairment of the π/2 phase shift error is the imperfection of interferences in the optical combiner 7′ at the output of the transmitter 3 of FIG. 3a. As a result, in addition to a first (normalized) power amplitude level 16, there is a second amplitude level 17 in the DQPSK optical signal (both marked by circles in FIG. 10b) such that detection at the receiver 5 of FIG. 1 is penalized. This is especially serious when using differential interferences for the demodulation, as the imperfect interferences between consecutive pulses affect system performances, as shown in FIG. 11b representing the Q-factor (solid line) which is penalized by a phase shift error at the transmitter, such that a 1 dB penalty is obtained for a 0.3 rad (corresponding to π/10) phase shift error.
In addition to the impairments of the transmission system 1 of FIG. 1 which are due to the transmitter 3 and have been described above, there are further impairments due to the receiver 5 shown in greater detail in FIG. 5a.
The receiver 5 comprises a splitter 30 for splitting a received DQPSK optical signal in two equal parts, each of which is introduced to one of two branches 31a, 31b. In the following, only the components in the first branch 31a will be described in detail, like components of the second branch 31b being assigned primed reference numerals. The first branch 31a comprises a differential interferometer 32 for making two consecutive pulses interfere together and is followed by a balanced receiver 33. The differential interferometer 32 splits the optical signal into a first and second branch 36a, 36b, the first of which comprises a time delay 34, the second of which comprises phase shifter 35 with a nominal phase shift of +π/4. Both branches are recombined at the output of the interferometer 32 and split again before being introduced to photodiodes in respective branches of the differential receiver 33, transforming the two optical input signals of the photodiodes into one electrical output signal.
The second branch 31b differs from the first branch 31a only in that the phase shifter 35′ generates a nominal phase shift of −π/4 instead of +π/4. The total phase difference between the two phase shifters 35, 35′ of π/2 is the reason why the balanced receiver 33 of the first branch 31a decodes the in-phase components Ik of the DQPSK optical signal, whereas the balanced receiver 33′ of the second branch 33′ decodes the quadrature-phase components Qk.
The main impairment in the DQPSK receiver 5 described above consists in a phase shift which is not equal to π/4, resulting in a multi-level eye-diagram and lower sensibility at detection. This phase shift error may be due to the following origins: laser detuning, imperfections from the π/4 phase shifter, or a time delay T not equal to one bit time T.
All there causes can be summed up in a phase term Δφ:Δφ=Δφπ/4shift+2πΔvlaserT+2πΔTv0,wherein:Δφπ/4 is the phase shifter mismatchingΔvlaser is the laser detuning from its central wavelength v0 ΔT is the bit time delay error
In the following, the term "phase shift error" designates the phase term Δφ which is due to all the imperfections described above.
As is the case with the phase shift error of the transmitter, phase shift mismatching in the receiver also leads to imperfect interferences and consequently to a 4-level eye-diagram, i.e. 4 different amplitude levels. The time domain representation of the normalized amplitude of the voltage of the electrical signal at the output of the balanced receiver 33 is shown in FIG. 10c for the ideal case (solid line) and a phase shift error equal to π/10 (dashed line). It is remarkable that variations from the ideal signal are about 50% of the normalized intensity value, such that four distinct levels appear in this case instead of two levels when the receiver is optimized. Consequently, a logical "1" which should be encoded with a nominal amplitude level of 1.0 is encoded in two different amplitude levels, the first one 18 equal to 0.7, the second one 19 equal to 1.2.
As expected, FIG. 11c shows that the tolerance to phase shift error measured as Q-factor penalty (in dB) at the receiver side is very low: 1 dB is obtained for ±π/40 (solid line). For comparison, a phase shift equal to π/40 corresponds to Δvlaser=500 MHz. Concerning a laser source, usual wavelength instability is around 0.01 nm, i.e. 1.2 GHz.
In summary, the transmitter as well as the receiver have to be stabilized in order to counteract the impairments illustrated above. Feedback signals have to be found for this purpose from which appropriate bias signals for the Mach-Zehnder modulators and phase shifters can be generated. The best prior art solution is to set modulator (transmitter) and demodulator (receiver) by using the Bit Error Rate (BER) as a feedback signal. The idea of this solution is to find optimal settings by minimizing the BER. However, the disadvantage of this solution for transmitter and receiver settings is the cost of BER monitoring, being a rather complex algorithm. Furthermore, the BER is monitored at the receiver side which may be located several hundreds of kilometers away from the transmitter.

Fiber optical transmission system, transmitter and receiver for DQPSK modulated signals and method for stabilizing the same


A fluid regulating device includes a regulator valve having an inlet, an outlet, and a valve port disposed between the inlet and the outlet. An actuator is coupled to the regulator valve and includes a valve disc that displaces between a closed position and an open position. The device also includes an overpressure protection device adapted to stop flow from the inlet to the outlet when pressure in a control cavity reaches a predetermined level. The overpressure protection device includes a sensing tube having a first end in fluid communication with the control cavity and a second end in fluid communication with the outlet. The sensing tube has a first portion extending parallel to the flow axis. One or more apertures are disposed in the sensing tube adjacent to the second end, and each of the apertures has a centerline that is perpendicular to the flow axis.

The pressure at which typical gas distribution systems supply gas may vary according to the demands placed on the system, the climate, the source of supply, and/or other factors. However, most end-user facilities equipped with gas appliances such as furnaces, ovens, etc., require the gas to be delivered in accordance with a predetermined pressure, and at or below a maximum capacity of a gas regulator. Therefore, gas regulators are implemented into these distribution systems to ensure that the delivered gas meets the requirements of the end-user facilities. Conventional gas regulators generally include a closed-loop control actuator for sensing and controlling the pressure of the delivered gas.
In addition to a closed loop control, some conventional gas regulators include a balanced trim to improve the reaction of the gas regulator to variations in the downstream pressure. The balanced trim is adapted to reduce the influence of the upstream pressure on the performance of the gas regulator. The upstream pressure is placed in fluid communication with a balancing diaphragm to apply a force to the control element of the gas regulator in the opposite direction as the force of the downstream pressure. Accordingly, as the upstream pressure varies, a corresponding force is applied to balance the force created by the upstream pressure as described further below so that the gas regulator acts in response to the downstream pressure only.
Some conventional gas regulators also include secondary monitoring devices (overpressure protection devices), such as overpressure monitoring devices, slam shut devices, token alarms and the like, that perform a responsive action if a sensed input pressure, such as a pressure downstream of the regulator, varies from a predetermined normal operating pressure range. An overpressure protection device controls the pressure downstream of the regulator in the event that the regulator fails, thereby allowing the downstream pressure to increase to undesired levels. In the event the regulator fails and the downstream pressure rises above a predetermined monitor setpoint pressure, the overpressure protection device operates to close the valve port of the regulator valve and cut off the flow of gas to the downstream components of the gas distribution system. As demand increases and/or the problem with the regulator is resolved and the downstream pressure drops, the overpressure protection device opens the valve port and thereby allows gas flow downstream.
Typically, an overpressure protection device, such as an overpressure monitoring device, responds in the event that the downstream pressure (i.e., pressure in the outlet) exceeds a cutoff pressure that is sensed by a diaphragm. Specifically, a bottom surface of the diaphragm of the overpressure monitor partially defines a control cavity such that pressure in the control cavity acts on the diaphragm. In one embodiment of an overpressure monitoring device, a control line extends from the control cavity to a portion of the outlet of the regulator valve, and the control line is disposed external to a both a housing of the overpressure monitoring device and portion of the regulator valve defining the valve outlet. So configured, downstream pressure is accurately communicated to the control cavity. However, such a configuration may be expensive to install and maintain.
Alternatively, overpressure protection devices may include an internal sense tube that extends from the control cavity to the outlet of the regulator valve and that is disposed through internal portions of the housing of the overpressure protection device and a portion of the body of the regulator valve defining the valve outlet. The internal sense tube may have a generally cylindrical shape having a constant cross-section. The internal sense tube may also have an open terminal end that extends into the regulator valve outlet, and the pressure of the fluid flowing within the valve at or adjacent to the outlet is sensed through the open terminal end of the internal sense tube. A portion of the internal sense tube that includes the terminal end may extend obliquely into the outlet or may extend parallel to the general direction of fluid flow through the outlet. However, as flow velocities and/or flow demand changes, the pressure communicated to the control cavity by the internal sense tube may correspondingly vary. Typically, a pressure lower than the actual control pressure is communicated to the control cavity via the open terminal end of the internal sense tube, thereby artificially driving the valve to induce boost in regulating pressures. More specifically, as flow passes over the end of the internal sense tube, a low pressure zone is created at the open terminal end which results in the registered pressure being lower than the actual pressure. As a result, the accuracy of the pressure measurement changes as the flow rate changes, thereby negatively impacting the ability of the overpressure protection device to protect the downstream system.

Dynamic pressure registration device for internally registered actuators and over pressure protection devices

EMERSON PROCESS MANAGEMENT REGULATOR TECHNOLOGIES, INC.


Genes-encoding a delta-9 CoA desaturase from Aspergillus nidulans have been isolated. The proteins encoded by genes, when expressed in a plant, can alter the saturate levels of the oil.

This invention relates to the preparation and use of nucleic acid fragments or genes which encode fungal palmitoyl-CoA xcex94-9 desaturase enzymes to create transgenic plants having altered oil profiles.
Plant-produced oils can be found in a wide variety of products including lubricants and foods. Interestingly, different plant species synthesize various oil types. For example, coconut and palm plants produce oils that are abundant in fatty acids having medium chain lengths (10-12 carbon atoms). These oils are used in manufacturing soaps, detergents and surfactants, and represent a U.S. market size greater than $350 million per year. Other plants, such as rape, produce oils abundant in long chain fatty acids (22 carbon atoms) and are used as lubricants and anti-slip agents. Additional applications of plant oils include their use in plasticizers, coatings, paints, varnishes and cosmetics (Volker et al., (1992) Science 257:72-74; Ohlrogge, (1994) Plant Physiol. 104:821-826). However, the predominant use of plant oils is in the production of food and food products.
Over the years, vegetable-derived oils have gradually replaced animal-derived oils and fats as the major source of dietary fat intake. However, saturated fat intake in most industrialized nations has remained at about 15% to 20% of total caloric consumption. In efforts to promote healthier lifestyles, the United States Department of Agriculture(USDA) has recently recommended that saturated fats make up less than 10% of daily caloric intake. To facilitate consumer awareness, current labeling guidelines issued by the USDA now require total saturated fatty acid levels be less than 1.0 g per 14 g serving to receive the xe2x80x9clow-satxe2x80x9d label and less than 0.5 g per 14 g serving to receive the xe2x80x9cno-satxe2x80x9d label. This means that the saturated fatty acid content of plant oils needs to be less than 7% and 1.75% to receive the xe2x80x9clow satxe2x80x9d and xe2x80x9cno satxe2x80x9d label, respectively. Since issuance of these guidelines, there has been a surge in consumer demand for xe2x80x9clow-satxe2x80x9d oils. To date, this has been met principally with canola oil, and to a much lesser degree with sunflower and safflower oils.
The characteristics of oils, whether of plant or animal origin, are determined predominately by the number of carbon and hydrogen atoms, as well as the number and position of double bonds comprising the fatty acid chain. Most oils derived from plants are composed of varying amounts of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) fatty acids. Conventionally, palmitic and stearic acids are designated as xe2x80x9csaturatedxe2x80x9d since the fatty acid chains have 16 and 18 carbon atoms, respectively, and no double bonds. They therefore contain the maximal number of hydrogen atoms possible. However, oleic, linoleic, and linolenic are 18-carbon fatty acid chains having one, two, and three double bonds, respectively, therein. Oleic acid is typically considered a mono-unsaturated fatty acid, whereas linoleic and linolenic are considered to be poly-unsaturated fatty acids.
Saturated fatty acids are linear molecules and tend to form self-stacked structures thereby resulting in high melting temperatures; a characteristic that is quite desirable when producing foods like chocolate. Animal fats, which are also solid at room temperature, are another readily available source of saturated fatty acids. However, use of said oil is often discouraged due to the high levels of cholesterol associated therewith. In comparison, unsaturated fatty acid chains are nonlinear due to bending induced by double bond insertion. The bending of the molecule impedes the ability of the fatty acid chains to stack thus causing them to remain fluid at lower temperatures. Vegetable oils, for example, are high in unsaturated fatty acids, and therefore are typically liquid at room temperature. Furthermore, saturated fatty acid can be modified to become unsaturated fatty acids by removal of hydrogen atoms and insertion of double bonds between two carbon atoms on the fatty acid chain. Desaturation can be achieved either enzymatically or chemically and decreases melting points due to the inability of the fatty acid molecules to self-stack.
The total saturated fatty acid level of corn oil, averaging about 13.9%, does not meet the current labeling guidelines discussed above. On average, corn oil is comprised of 11.5% palmitic acid, 2.2% stearic acid, 26.6% oleic acid, 58.7% linoleic acid, and 0.8% linolenic acid. Corn oil also contains 0.2% arachidic acid, a twenty-carbon saturated fatty acid (Dunlap et. al., (1995) J. Amer. Oil Chem. Soc. 72:981-987). The composition of corn oil instills it with properties that are most desirable in edible oils. These include properties such as heat stability, flavor, and long shelf life. However, consumer demand for xe2x80x9clow satxe2x80x9d oils has resulted in a significant decrease in corn oil utilization and thus decreased market share. Therefore, a corn oil with modified levels of saturated fatty acids is highly desirable and would have practical use in that it would meet the consumer demand for healthier oils while having most or all of the properties that made corn oil a popular and preferred oil in the past.
Corn is typically not considered to be an oil crop as compared to soybean, canola, sunflower and the like. In fact, the oil produced and extracted from corn is considered to be a byproduct of the wet milling process used in starch extraction. Because of this, there has been little interest in modifying the saturate levels of corn oil until those efforts disclosed herein.
As disclosed herein, saturate levels of fatty acids comprising plant oils can be altered by expressing a fungal palmitate-CoA xcex94-9 desaturase within a plant cell. These proteins most likely enzymatically desaturate palmitate-CoA molecules by removing two hydrogen atoms and adding a double bond between the 9th and 10th carbon atoms from the CoA portion of the molecule, thus producing palmitoleic-CoA (16:1xcex949) . The palmitoleic-CoA is ultimately incorporated into seed oil thus lowering the total saturate levels of said oil.
In the present invention, a gene encoding a fungal palmitate-CoA xcex94-9 desaturase has been isolated and cloned from Aspergillus nidulans. The saturate level of oils found in plant cells can be altered by expressing said palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans.
One aspect of the disclosed invention is a gene encoding said palmitate-CoA xcex94-9 desaturase, said gene being isolated and purified from Aspergillus nidulans.
An additional aspect of the present invention relates to producing a gene wherein the codon bias of a gene from a non-plant source has been modified to look similar to genes from a plant source.
Another aspect of the invention relates to altering oil saturate levels within a plant cell by expressing said genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans. Genes disclosed herein can be used to alter saturate levels by placing said genes in the sense orientation. Plants cells being transformed with genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans in the sense orientation results in the oils of said plants having increased 16:1 levels and decreased total saturate levels over non-transformed plants.
An additional aspect of the present invention is the production of chimeric genes using the genes disclosed herein encoding for palmitoyl CoA-xcex94-9 desaturase in combination with promoter regulatory elements and the use of said chimeric genes within a plant cell.
Yet an additional aspect of the present invention is the transformation of plant species disclosed herein with said chimeric genes.
Other aspects, embodiments, advantages, and features of the present invention will become apparent from the following specification.
The present invention relates to methods and compositions for obtaining transgenic plants wherein plant oils produced thereby have altered saturate levels. The following phrases and terms are defined below:
By xe2x80x9caltered saturate levelsxe2x80x9d is meant that the level of total saturated fatty acids of a plant oil produced by a modified plant is different from that of a normal or non-modified plant.
By xe2x80x9ccDNAxe2x80x9d is meant DNA that is complementary to and derived from a mRNA.
By xe2x80x9cchimeric DNA constructionxe2x80x9d is meant a recombinant DNA containing genes or portions thereof from one or more species.
By xe2x80x9ccomplementarityxe2x80x9d is meant a nucleic acid that can form hydrogen bond(s) with other nucleic acid sequences either through traditional Watson-Crick or other non-traditional types of base paired interactions.
By xe2x80x9cconstitutive promoterxe2x80x9d is meant promoter elements that direct continuous gene expression in all cell types and at all times (i.e., actin, ubiquitin, CaMV 35S, 35T, and the like).
By xe2x80x9cdevelopmental specificxe2x80x9d promoter is meant promoter elements responsible for gene expression at specific plant developmental stages, such as in early or late embryogenesis and the like.
By xe2x80x9cenhancerxe2x80x9d is meant nucleotide sequence elements which can stimulate promoter activity such as those from maize streak virus (MSV) protein leader sequence, alfalfa mosaic virus protein leader sequence, alcohol dehydrogenase intron 1, and the like.
By xe2x80x9cexpressionxe2x80x9d as used herein, is meant the transcription and stable accumulation of mRNA inside a plant cell. Expression of genes also involves transcription of the gene to create mRNA and translation of the mRNA into precursor or mature proteins.
By xe2x80x9cforeignxe2x80x9d or xe2x80x9cheterologous genexe2x80x9d is meant a gene encoding a-protein whose exact amino acid sequence is not normally found in the host cell, but is introduced by standard gene transfer techniques.
By xe2x80x9cgenexe2x80x9d is meant to include all genetic material involved in protein expression including chimeric DNA constructions, genes, plant genes and portions thereof, and the like.
By xe2x80x9cgenomexe2x80x9d is meant genetic material contained in each cell of an organism and/or virus and the like.
By xe2x80x9cinducible promoterxe2x80x9d is meant promoter elements which are responsible for expression of genes in response to a specific signal such as: physical stimuli (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites, chemicals, stress and the like.
By xe2x80x9cmodified plantxe2x80x9d is meant a plant wherein the gene, mRNA, or protein from Aspergillus nidulans palmitate-CoA xcex94-9 desaturase is present.
By xe2x80x9cplantxe2x80x9d is meant a photosynthetic organism including both eukaryotes and prokaryotes.
By xe2x80x9cpromoter regulatory elementxe2x80x9d is meant nucleotide sequence elements within a nucleic fragment or gene which controls the expression of that nucleic acid fragment or gene. Promoter sequences provide the recognition for RNA polymerase and other transcriptional factors required for efficient transcription. Promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express gene constructs. Promoter regulatory elements are also meant to include constitutive, tissue-specific, developmental-specific, inducible promoters and the like. Promoter regulatory elements may also include certain enhancer sequence elements and the like that improve transcriptional efficiency.
By xe2x80x9ctissue-specificxe2x80x9d promoter is meant promoter elements responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (i.e., zein, oleosin, napin, ACP, globulin and the like).
By xe2x80x9ctransgenic plantxe2x80x9d is meant a plant expressing a chimeric gene introduced through transformation efforts.
In plant cells, fatty acids are made as acyl-acyl carrier protein (acyl-ACP) substrates and are elongated by various enzymes through the addition of malonyl-ACP to make acyl-ACP molecules ranging in length from 2 to 18 carbon atoms. Afterwards, acyl-ACP thioesterases catalyze the hydrolytic cleavage of palmitic acid, stearic acid, and oleic acid from ACP, in a somewhat selective although not specific manner, thus producing a free fatty acids. The fatty acid molecules move out of the plastid into the cytoplasm where they are eventually modified into acyl-CoA molecules. Said molecules are then incorporated onto the triglyceride oil fraction. It has been discovered by applicants as disclosed herein that desaturation of an acyl-CoA molecule, wherein said molecule is preferably stearoyl-CoA and most preferably palmitate-CoA, can reduce saturate levels in the triglyceride oil fraction. Said desaturation most preferably results in the production and accumulation of palmitoleic acid (16:1xcex94-9). Said desaturation may also result in a decrease in palmitic and stearic acid in the triglyceride oil fraction.
In corn seed oil, the predominant fatty acids are linoleic acid (18:2 at about 59%), oleic acid (18:1 at about 26%) and palmitic (16:0 at about 11%), with stearic acid (18:0) generally comprising about 2.5% or less (Glover and Mertz, (1987) in: Nutritional Quality of Cereal Grains: genetic and agronomic improvement., p.183-336, (eds. Olson, R. A. and Frey, K. J.) Amer. Soc. Agronomy, Inc., Madison, Wis.; Fitch-Haumann, (1985) J. Am. Oil. Chem. Soc. 62:1524-1531). Biosynthesis of fatty acids in plant cells is initiated in the plastids where they are synthesized as acyl-ACP thioesters by a fatty acid synthase complex. More specifically, fatty acid production is accomplished by a series of condensation reactions involving addition of malonyl-ACP sequentially to a growing fatty acid-ACP chain by the enzyme xcex2-ketoacyl-ACP synthase I (KAS I). Most fatty acid-ACP units reach carbon chain lengths of 16 and are then elongated to 18 carbon units by KAS II. The vast majority of C18 fatty acids become desaturated by stearoyl-ACP xcex94-9 desaturase at the C9 position from the carboxyl end to produce oleyl-ACP.
Both saturated and unsaturated fatty acid-ACP units are hydrolyzed by acyl-ACP thioesterases to produce free fatty acids. These free fatty acids then cross the plastid membrane to the cytosol of the cell where they are modified by addition of a CoA moiety. Afterwards, said fatty acids are incorporated into plant oils (Somerville and Browse, (1991) Science 252:80-87; Browse and Sommerville (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:467-506; Harwood (1989) Critical Reviews in Plant Sci. 8:1-43; Chasan (1995) Plant Cell 7:235-237; Ohlrogge (1994) Plant Physiol. 104:821-826).
The palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans desaturates palmitic acid at the C9 position relative to the carboxyl end most likely after the point of modification with Co-A. In plant cells, this most likely occurs before being incorporated into the triglyceride fraction of the oil. Therefore, expressing palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans in plant-cells will cause a decrease in the saturate levels of the oil produced by said plant.
The palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans disclosed herein can be used to modify saturate levels in oil in both monocotyledonous and dicotyledonous plants. In dicotyledonous plants, expression of said desaturase preferably results in a decrease in 16:0 and 18:0 levels found in oil derived from said plants. More preferably, expression of said desaturase results in increased levels of 16:1 fatty acid in said oil. In monocotyledonous plants, expression of said desaturase preferably results in decreased levels of 18:0 and more preferably, increased levels of 16:1 found in the said oil. It is not applicants intention, however, to limit said gene expression exclusively to plants in that said desaturase and genes thereof can be expressed and used to modify lipid contents in both yeast and bacteria.
As further described herein, an Aspergillus palmitate-CoA xcex94-9 desaturase can be used to modify the saturate levels in oils produced by transgenic plants. Preferably, genes and nucleic fragments encoding the palmitate-CoA xcex94-9 desaturase are derived from Aspergillus nidulans. More preferably, genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans are those disclosed herein as SEQ ID NO:5 and SEQ ID NO:12, said genes encoding a protein having an amino acid sequence as disclosed herein as SEQ ID NO:6.
One method by which plant oils can be modified is by expressing the palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans in a dicotyledonous plant. This can be achieved by placing the genes or nucleic acid fragments encoding said proteins in the sense orientation 3xe2x80x2 to a promoter regulatory element of choice followed by a transcriptional terminator at the 3xe2x80x2 end of said gene thus producing a chimeric gene construct. These chimeric genes can then be transformed into plants, thereby producing plant oils having altered saturate levels relative to nontransformed controls. Expressing the palmitate-CoA xcex94-9 desaturase as disclosed herein from Aspergillus nidulans in dicotyledonous plants results in plant oils derived therefrom having 16:1 levels as a percentage of the total fatty acid from about 0.23 to about 4.65%; preferably from about 3.01 to about 4.65%; more preferably from about 4.07 to about 4.65%, with about 4.65% being most preferred. The total saturate levels range preferably from about 9.8 to about 12.5% with about 9.8% being most preferred.
Another method by which plant oils can be modified is by expressing the palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans in a monocotyledonous plant. As with dicotyledonous plants, this can be achieved by placing the genes or nucleic acid fragments encoding said proteins in the sense orientation 3xe2x80x2 to a promoter regulatory element of choice followed by a transcriptional terminator at the 3xe2x80x2end of said gene thus producing a chimeric gene construct. These chimeric genes can then be transformed into plants, thereby producing plant oils having altered saturate levels relative to nontransformed controls. Expressing the palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans in monocotyledonous plants results in plant oils derived therefrom to have 16:1 levels from about 0.4 to about 3.2%; preferably from about 1.2 to about 3.2%, with about 3.2% being most preferred.
As further disclosed herein, chimeric gene constructs encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans can be transformed in other oilseed crops to modify the saturate levels therein. Said oilseed crop plant species which may be modified include but are not limited to soybean, Brassicaceae sp., canola, rape, sunflower, flax, safflower, coconut, palm, olive, peanut, cotton, castor bean, coriander, Crambe sp., Cuphea sp., Euphorbia sp., Oenothera sp., jojoba, Lesquerella sp., marigold, Limnanthes sp., Vernonia sp., Sinapis alba, and cocoa, with maize being most preferred. Most if not all of these plant species have been previously transformed by those having ordinary skill in the art.
To obtain high expression of heterologous genes in plants it may be preferred to reengineer said genes so that they are more efficiently expressed in the cytoplasm of plant cells. Maize is one such plant where it may be preferred to reengineer the heterologous gene(s) prior to transformation to increase the expression level thereof in said plant. Therefore, an additional step in the design of genes encoding said palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans is the designed reengineering of a heterologous gene for optimal expression.
One reason for the reengineering the xcex94-9 Co-A desaturase gene from Aspergillus nidulans for expression in maize is due to the non-optimal G+C content of the native gene. For example, the very low G+C content of many native bacterial gene(s) (and consequent skewing towards high A+T content) results in the generation of sequences mimicking or duplicating plant gene control sequences that are known to be highly A+T rich. The presence of some A+T-rich sequences within the DNA of gene(s) introduced into plants (e.g., TATA box regions normally found in gene promoters) may result in aberrant transcription of the gene(s). On the other hand, the presence of other regulatory sequences residing in the transcribed mRNA (e.g., polyadenylation signal sequences (AAUAAA), or sequences complementary to small nuclear RNAs involved in pre-mRNA splicing) may lead to RNA instability. Therefore, one goal in the design of genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans for maize expression, more preferably referred to as plant optimized gene(s), is to generate a DNA sequence having a higher G+C content, and preferably one close to that of maize genes coding for metabolic enzymes. Another goal in the design of the plant optimized gene(s) encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans is to generate a DNA sequence in which the sequence modifications do not hinder translation.
The table below (Table 1) illustrates how high the G+C content is in maize. For the data in Table 1, coding regions of the genes were extracted from GenBank (Release 71) entries, and base compositions were calculated using the MacVector(trademark) program (IBI, New Haven, Conn.). Intron sequences were ignored in the calculations.
Due to the plasticity afforded by the redundancy of the genetic code (i.e., some amino acids are specified by more than one codon), evolution of the genomes in different organisms or classes of organisms has resulted in differential usage of redundant codons. This xe2x80x9ccodon biasxe2x80x9d is reflected in the mean base composition of protein coding regions. For example, organisms with relatively low G+C contents utilize codons having A or T in the third position of redundant codons, whereas those having higher G+C contents utilize codons having
G or C in the third position. It is thought that the presence of xe2x80x9cminorxe2x80x9d codons within a mRNA may reduce the absolute translation rate of that mRNA, especially when the relative abundance of the charged tRNA corresponding to the minor codon is low. An extension of this is that the diminution of translation rate by individual minor codons would be at least additive for multiple minor codons. Therefore, mRNAs having high relative contents of minor codons would have correspondingly low translation rates. This rate would be reflected by subsequent low levels of the encoded protein.
In reengineering genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans for maize expression, the codon bias of the plant has been determined. The codon bias for maize is the statistical codon distribution that the plant uses for coding its proteins and the preferred codon usage is shown in Table 2. After determining the bias, the percent frequency of the codons in the gene(s) of interest is determined. The primary codons preferred by the plant should be determined as well as the second and third choice of preferred codons. Afterwards, the amino acid sequence of palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans is reverse translated so that the resulting nucleic acid sequence codes for exactly the same protein as the native gene wanting to be heterologously expressed. The new DNA sequence is designed using codon bias information so that it corresponds to the most preferred codons of the desired plant. The new sequence is then analyzed for restriction enzyme sites that might have been created by the modification. The identified sites are further modified by replacing the codons with second or third choice with preferred codons. Other sites in the sequence which could is affect transcription or translation of the gene of interest are the exon:intron 5xe2x80x2 or 3xe2x80x2 junctions, poly A addition signals, or RNA polymerase termination signals. The sequence is further analyzed and modified to reduce the frequency of TA or GC doublets. In addition to the doublets, G or C sequence blocks that have more than about four residues that are the same can affect transcription of the sequence. Therefore, these blocks are also modified by replacing the codons of first or second choice, etc. with the next preferred codon of choice.
It is preferred that the plant optimized gene(s) encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans contain about 63% of first choice codons, between about 22% to about 37% second choice codons, and between about 15% to about 0% third choice codons, wherein the total percentage is 100%. Most preferred the plant optimized gene(s) contains about 63% of first choice codons, at least about 22% second choice codons, about 7.5% third choice codons, and about 7.5% fourth choice codons, wherein the total percentage is 100%. The preferred codon usage for engineering genes for maize expression are shown in Table 2. The method described above enables one skilled in the art to modify gene(s) that are foreign to a particular plant so that the genes are optimally expressed in plants. The method is further illustrated in pending PCT application WO 97/13402, which is incorporated herein by reference.
In order to design plant optimized genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans, the amino acid sequence of said protein is reverse translated into a DNA sequence utilizing a non-redundant genetic code established from a codon bias table compiled for the gene sequences for the particular plant, as shown in Table 2.
The resulting DNA sequence, which is completely homogeneous in codon usage, is further modified to establish a DNA sequence that, besides having a higher degree of codon diversity, also contains strategically placed restriction enzyme recognition sites, desirable base composition, and a lack of sequences that might interfere with transcription of the gene, or translation of the product mRNA. Said sequence produced using the methods described herein is disclosed as SEQ ID NO:12.
In another aspect of the invention, genes encoding the palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans are expressed from transcriptional units inserted into the plant genome. Preferably, said transcriptional units are recombinant vectors capable of stable integration into the plant genome and selection of transformed plant lines expressing mRNA encoding for said desaturase proteins are expressed either by constitutive or inducible promoters in the plant cell. Once expressed, the mRNA is translated into proteins, thereby incorporating amino acids of interest into protein. The genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans expressed in the plant cells can be under the control of a constitutive promoter, a tissue-specific promoter or an inducible promoter as described herein.
Several techniques exist for introducing foreign recombinant vectors into plant cells, and for obtaining plants that stably maintain and express the introduced gene. Such techniques include acceleration of genetic material coated onto microparticles directly into cells (U.S. Pat. No. 4,945,050 to Cornell and U.S. Pat. No. 5,141,131 to DowElanco, now Dow AgroSciences). In addition, plants may be transformed using Agrobacterium technology, see U.S. Pat. No. 5,177,010 to University of Toledo, U.S. Pat. No. 5,104,310 to Texas AandM, European Patent Application 0131624B1, European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications 116718, 290799, 320500 all to Max is Planck, European Patent Applications 604662,627752 and U.S. Pat. No. 5,591,616 to Japan Tobacco, European Patent Applications 0267159, and 0292435 and U.S. Pat. No. 5,231,019 all to Ciba Geigy, now Novartis, U.S. Pat. Nos. 5,463,174 and 4,762,785 both to Calgene, and U.S. Pat. Nos. 20 5,004,863 and 5,159,135 both to Agracetus. Other transformation technology includes whiskers technology, see U.S. Pat. Nos. 5,302,523 and 5,464,765 both to Zeneca. Electroporation technology has also been used to transform plants, see WO 87/06614 to Boyce Thompson Institute, U.S. Pat. Nos. 5,472,869 and 5,384,253 both to Dekalb, W09209696 and W09321335 both to Plant Genetic Systems. Furthermore, viral vectors can also be used in produce transgenic plants expressing the protein of interest. For example, monocotyledonous plant can be transformed with a viral vector using the methods described in U.S. Pat. No. 5,569,597 to Mycogen and Ciba-Giegy, now Novartis, as well as U.S. Pat. Nos. 5,589,367 and 5,316,931, both to Biosource. All of these transformation patents and publications are incorporated herein by reference.
As mentioned previously, the manner in which the DNA construct is introduced into the plant host is not critical to this invention. Any method which provides for efficient transformation may be employed. For example, various methods for plant cell transformation are described herein and include the use of Ti or Ri-plasmids and the like to perform Agrobacterium mediated transformation. In many instances, it will be desirable to have the construct used for transformation bordered on one or both sides by T-DNA borders, more specifically the right border. This is particularly useful when the construct uses Agrobacterium tumefaciens or Agrobacterium rhizogenes as a mode for transformation, although T-DNA borders may find use with other modes of transformation.
Where Agrobacterium is used for plant cell transformation, a vector may be used which may be introduced into the host for homologous recombination with T-DNA or the Ti or Ri plasmid present in the host. Introduction of the vector may be performed via electroporation, tri-parental mating and other techniques for transforming gram-negative bacteria which are known to those skilled in the art. The manner of vector transformation into the Agrobacterium host is not critical to this invention. The Ti or Ri plasmid containing the T-DNA for recombination may be capable or incapable of causing gall formation, and is not critical to said invention so long as the vir genes are present in said host.
In some cases where Agrobacterium is used for transformation, the expression construct being within the T-DNA borders will be inserted into the plasmid pDAB1542 as described herein or into a broad spectrum vector such as pRK2 or derivatives thereof as described in Ditta et al., (PNAS USA (1980) 77:7347-7351 and EPO 0 120 515, which are incorporated herein by reference. Included within the expression construct and the T-DNA will be one or more markers as described herein which allow for selection of transformed Agrobacterium and transformed plant cells. The particular marker employed is not essential to this invention, with the preferred marker depending on the host and construction used.
For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed Agrobacterium for sufficient time to allow transformation thereof. After transformation, the agrobacteria are killed by selection with the appropriate antibiotic and plant cells are cultured with the appropriate selective medium. Once calli are formed, shoot formation can be encourage by employing the appropriate plant hormones according to methods well known in the art of plant tissue culturing and plant regeneration. However, a callus intermediate stage is not always necessary. After shoot formation, said plant cells can be transferred to medium which encourages root formation thereby completing plant regeneration. The plants may then be grown to seed and said seed can be used to establish future generations as well as provide a source for oil isolation. Regardless of transformation technique, the gene encoding palmitoyl-CoA xcex94-9 desaturase from Aspergillus nidulans is preferably incorporated into a gene transfer vector adapted to express said gene in a plant cell by including in the vector a plant promoter regulatory element, as well as 3xe2x80x2 non-translated transcriptional termination regions such as Nos and the like.
In addition to numerous technologies for transforming plants, the type of tissue which is contacted with the foreign genes may vary as well. Such tissue would include but would not be limited to embryogenic tissue, callus tissue types I, II, and III, hypocotyl, meristem, and the like. Almost all plant tissues may be transformed during dedifferentiation using appropriate techniques described herein.
Another variable is the choice of a selectable marker. Preference for a particular marker is at the discretion of the artisan, but any of the following selectable markers may be used along with any other gene not listed herein which could function as a selectable marker. Such selectable markers include but are not limited to aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II) which encodes resistance to the antibiotics kanamycin, neomycin and G418, as well as those genes which encode for resistance or tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin (bialophos); imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such as chlorsulfuron; bromoxynil, dalapon and the like.
In addition to a selectable marker, it may be desirous to use a reporter gene. In some instances a reporter gene may be used with or without a selectable marker. Reporter genes are genes which are typically not present in the recipient organism or tissue and typically encode for proteins resulting in some phenotypic change or enzymatic property. Examples of such genes are provided in K. Weising et al. Ann. Rev. Genetics, 22, 421 (1988), which is incorporated herein by reference. Preferred reporter genes include the beta-glucuronidase (GUS) of the uida locus of E. coli, the chloramphenicol acetyl transferase gene from Tn9 of E. coli, the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria, and the luciferase genes from firefly Photinus pyralis. An assay for detecting reporter gene expression may then be performed at a suitable time after said gene has been introduced into recipient cells. A preferred such assay entails the use of the gene encoding beta-glucuronidase (GUS) of the uida locus of E. coli as described by Jefferson et al., (1987 Biochem. Soc. Trans. 15, 17-19) to identify transformed cells.
In addition to plant promoter regulatory elements, promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promoter regulatory elements of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S and 19S), 35T (which is a re-engineered 35S promoter, see PCT/US96/1682; WO 97/13402 published Apr. 17, 1997) and the like may be used. Plant promoter regulatory elements include but are not limited to ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter, heat-shock promoters and tissue specific promoters.
Other elements such as matrix attachment regions, scaffold attachment regions, introns, enhancers, polyadenylation sequences and the like may be present and thus may improve the transcription efficiency or DNA integration. Such elements may or may not be necessary for DNA function, although they can provide better expression or functioning of the DNA by affecting transcription, mRNA stability, and the like. Such elements may be included in the DNA as desired to obtain optimal performance of the transformed DNA in the plant. Typical elements include but are not limited to Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coat protein leader sequence, the maize streak virus coat protein leader sequence, as well as others available to a skilled artisan.
Constitutive promoter regulatory elements may also be used thereby directing continuous gene expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S, and the like). Tissue specific promoter regulatory elements are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin and the like) and these may also be used.
Promoter regulatory elements may also be active during a certain stage of the plants"" development as well as active in plant tissues and organs. Examples of such include but are not limited to pollen-specific, embryo specific, corn silk specific, cotton fiber specific, root specific, seed endosperm specific promoter regulatory elements and the like. Under certain circumstances it may be desirable to use an inducible promoter regulatory element, which is responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites; chemical; and stress. Other desirable transcription and translation elements that function in plants may be used. Numerous plant-specific gene transfer vectors are known in the art.
One of the issues regarding exploiting transgenic plants having altered saturate levels is the expression of multiple chimeric genes at once. European Patent Application 0400246A1 describes transformation of two Bt genes in a plant; however, these could be any two genes or fragments thereof in either the sense or antisense orientation. For example, commercially available hybrids have now been produced having stacked traits such as herbicide and insect resistance. The options could include but are not limited to genes and fragments encoding the palmitoyl-CoA xcex94-9 desaturase from Aspergillus nidulans with acyl-ACP thioesterase genes or genes encoding proteins such as stearoyl-ACP desaturase, xcex2-ketoacyl synthase II and the like, as well as genes to impart insect control or herbicide resistance. Another way to produce a transgenic plant having multiple traits is to produce two plants, with each plant containing the oil modifying gene of interest. These plants can then be back-crossed using traditional plant breeding techniques available and well-known to those skilled in the art to produce plants wherein phenotypic characteristics are related to the presence of more than one chimeric gene.
The particular embodiments of this invention are further exemplified in the Examples. However, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

Modification of fatty acid composition in plants by expression of a fungal acyl-CoA desaturase


In one embodiment, an enterprise network is provided that includes a mobility agent 150 associated with a switch/server 100 or 104. The mobility agent creates a visitor application record 172 associated with a second communication device 140b. The visitor application record is associated with a first subscriber. A second subscriber is normally associated with the second communication device, and the first subscriber with a first communication device 136a. In response to creating the visitor application record, the native communication device record is associated with a virtual communication device having the same public and/or private numbers as the second communication device 140b.

Enterprise user mobility, or the ability of subscribers to use stations located across the enterprise network while still being able to receive consistent phone service, is highly desirable in the global marketplace. Many large companies are commonly decentralized with large campuses and branch offices located around the world. Preferably, enterprise user mobility provides subscribers with the continued access to features and other functionality located on the subscriber's home, or assigned, station.
Enterprise user mobility is currently being offered in Voice over Internet Protocol or VoIP products of various vendors, such as Extension Mobility™ by Cisco. The Cisco product has an extensive Application Program Interface or API that allows access to third party applications to setup the home and visited, or remote, VoIP stations for remote access without the subscriber needing to touch either handset. To effect the reconfiguration of the remote station, the system administrator uses the API to enter a User Identifier or UI and Personal Identification Number or PIN. When the subscriber logs into the visited station and is successfully verified, the visited station automatically reconfigures with the subscriber's home station profile information. Where more than one switch is involved at the locations of the home and visited station, call processing is performed at the home switch and not the visited switch because the visited switch simply acts as a router.
These products, however, are limited to VoIP stations and are not adaptable to circuit-switched or Time Division Multiplexed or TDM telephones. IP networks generally provide an excellent infrastructure for geographically distributing components of a telecommunication system. The underlying IP network is optimal for transmission for control signaling, and, when bandwidth is available, can provide an acceptable Quality of Service (or QoS) or Grade of Service (or GOS) for voice communications. However when insufficient network resources are available for voice communications or one or more IP network components are down, voice communications can be adversely impacted. While great strides have been made to provide improved levels of voice quality in VoIP communications, rapid and unexpected deteriorations in the QoS or GOS still occur with some frequency and are unacceptable for many businesses.
For this reason, many businesses prefer wired circuit-switched or TDM communication devices. Although intra-switch wired mobility, such as hoteling or hot-desking, is known, inter-switch enterprise user wired mobility is not available for circuit-switched or TDM communication devices, particularly wired mobility in which the subscriber has close to the full functionality that the subscriber would have if he or she were at his or her home station. As will be appreciated, hoteling or hot-desking is a function that allows subscribers, without a native station, to be assigned anywhere in the local area network controlled by a common enterprise switch. Additionally, even with IP enabled mobility the native subscriber normally associated with the remote IP phone taken over by the traveling subscriber is no longer associated with that IP phone. This can create problems with the native subscriber having remote access to the features of the IP phone, such as abbreviated dial, bridged appearance, dial intercom, and the like.


A power transmission circuitry transmits power wirelessly to a plurality of power reception circuitry simultaneously. The power transmission circuitry includes, a communication circuit that wirelessly receives information on receiving power of the plurality of power reception circuitry, and a transmitting power control circuit that controls transmitting power based on the information on receiving power of the plurality of power reception circuitry received by the communication circuit.

In recent years, attention has been paid to a wireless power transmission (which may be also called a wireless power transfer, a contactless power transmission, or a wireless power feeding) technology for wirelessly transmitting power in order to supply power to a device or charge a device with power. For example, research and development (R&D) has been performed on wireless power transmission circuitry that wirelessly transmit power to power reception circuitry such as various electronic devices and home appliances including mobile terminals, notebook computers and personal computers, and power infrastructure devices such as automobiles.
In the related art, techniques using electromagnetic induction or radio waves have been applied for such wireless power transmission circuitry. In recent years, it has been expected to use magnetic field resonance capable of wirelessly transmitting power while keeping a distance between a power transmission circuitry (power transmitter) and a power reception circuitry (power receiver: an electronic device) to some extent.
Since a power transmission circuitry using the magnetic field resonance may transmit power of several W or more to a place distant from several centimeters to several tens of centimeters, the power transmission circuitry may be applied to, for example, electronic devices, home appliances, or power infrastructure devices. The application of this embodiment is not limited to the wireless power transmission using strongly-coupled resonance such as magnetic field resonance or electric field resonance. For example, electromagnetic induction may be used as well as long as the power can be wirelessly transmitted to plural power reception circuitry simultaneously.
In the related art, various proposals have been made to perform a wireless power transmission using the magnetic field resonance.
Related techniques are disclosed in, for example, Japanese Laid-Open Patent Publication Nos. 2013-034367, 2014-138507, and 2013-055871.

Power transmission circuitry, power reception circuitry, wireless power transmission system, and wireless power transmission method


A capacitive detection control interface device is provided, including at least one measurement electrode including an active surface, a guard made from an electrically conductive material placed adjacent to the measurement electrodes, the guard is excited up to an alternating electric potential substantially identical to that of the measurement electrodes, a first electronic component for exciting the electrodes and processing the measurement signals from the capacitive coupling of the electrodes with an object laced adjacent thereto, the electronic component is at least partly referenced to the electric potential of the guard, and a second electronic component for performing another functional display, placed adjacent to the active surface which second electronic component is at least partly referenced to the electric potential of the guard.

It is more and more attempted in virtual interfaces to have a proximity detection function, which enables new man-machine interaction modes to be created without any contact. Sensors must then be able to detect displacements or shapes within several centimetres, in a sufficiently accurate and determined manner to be able to transform them into controls.
More generally, so-called virtual, that is gestural and/or tactile and vocal man-machine interfaces, as opposed to interfaces where the user acts on mechanical sensors such as keyboard keys or switches, are most often 3D cameras in the field of gestural interface and active surfaces, often transparent and integrated on the display screens, in the field of tactile interface. These tactile interfaces are widespread under the name of "touchpad" for multiple industrial and consumer applications, such as for example smartphones, home automation or game interfaces.
The screen enables to display for example a keyboard or the software interface the image of which varies in response to actions from the user, which generally consist of a displacement, a tapping or even a prolonged contact of the finger or a stylus onto the screen surface in order to perform controls.
More recently, so-called gestural interfaces have been developed to meet the increasingly great complexity of digital products emanating from the convergence of communication and entertainment information technologies. These interfaces are most often based on a 3D camera and an image processing capable of interpreting at a long distance, up to 5 metres, the movements of the body or the hand to interact with the screen.
Among virtual controls, can also be mentioned the vocal control which enables the request to be performed from the voice.
Among existing tactile technologies, capacitive technologies are frequently used because:
they do not require exerting any mechanical action onto the screen surface unlike resistive techniques for example,
they are well-adapted to the meshing of the screen surface by a sensor network directly integrated to the surface thereof, which enables a much more compact and robust integration than with optical techniques for example which require a network of transmitters-receivers raised with respect to the detection surface.
It is more and more attempted in virtual interfaces to have a proximity detection function, which enables new man-machine interaction modes to be created without any contact. Sensors must then be able to detect displacements or shapes within several centimetres, in a sufficiently accurate and determined manner to be able to transform them into controls.
If optical technologies can not be dispensed with when accurately detecting movements at a very long distance (beyond 40 centimetres), capacitive technologies turn out to be very well adapted to interfaces without proximity contact, in particular because all sensors can be integrated to a non-planar active surface, for example a screen, without dead area or without requiring external devices (cameras).
Amongst capacitive tactile interfaces, the most used techniques are generally based on the charge transfer principle. They enable sensitivity in the order of one picofarad to be obtained but they are not adapted to make a gestural interface. Indeed, their sensitivity does not enable the approach of a finger to be detected because the capacity generated does not then exceed a few hundredths picofarads. Stray capacitances present in this type of sensor and in electronics prevent any sensitivity improvement.
Moreover, these techniques are very sensitive to electromagnetic disturbances, stray electrical charges and electric conduction of the dielectric covering the electrodes. When the air relative humidity is higher than 60%, most dielectrics become slightly electrically conducting and charges generated by the electronics are modified, which disturbs the capacitive measurement.
Among technical solutions employed to make non-tactile capacitive interfaces, some are known which implement a pseudoguard enabling the stray capacitances to be strongly reduced in the sensor and electronics. However, these techniques only enable at most one order of magnitude on sensitivity to be gained, which enables the presence of a finger to be detected at only a few mm of the sensitive surface of the sensor.
Regarding capacitive gestural interfaces, it is known from document U.S. Pat. No. 6,847,354 to Vranish, capacitive sensors implemented with an active guard. This active guard is created using a unity gain amplifier which generates in the guard a voltage with an amplitude identical to that of the measurement electrode.
The drawback of this method is that the electronics generates by a physical principle stray capacitances which correspond to the sum of the input capacitances of the amplifier to generate the active guard, also called pseudoguard, and of the circuit to excite the measurement electrode. These stray capacitances easily reach one picofarad and add up to the capacitance to be measured, which only represent one hundredth of this total value.
Moreover, the capacitance measurement is not direct because the Vranish electronics obtains the image of the capacitance to be measured by measuring its current via a reference resistor. This resistor generates a stray phase shift of the electrical current which strongly degrades the detection quality, or of demodulation of the signal representing the capacitance to be measured. Another drawback is the crosstalk level between different electrodes. Indeed, each electrode is excited by an operational amplifier the gain of which is approximately a unity one. Any gain deviation between different amplifiers causes a high further stray capacitance.
These drawbacks do not enable the position of an object such as a finger or a hand to be detected and located at several centimetres or even tens of centimetres with each sensor.
On the other hand, capacitive technologies implemented for making gestural interfaces have been developed most often in an attempt to be integrated to screens or at least substantially planar surfaces. The structures of sensors are arrays, as in U.S. Pat. No. 6,847,354, and interfaced with electrode structures disposed as a grid X-Y. These technologies are poorly adapted to the instrumentation of surfaces with more complex shapes.
In particular, they are hardly applicable to devices for virtual controls or gestural interfaces based not on an instrumented planar surface but on other types of geometries including cavities, reliefs, undulations simulating for example keys, buttons or wrapping the user wherein sensors can be disposed as various geometries, sometimes under dielectric materials with a high thickness, and must sometimes be able to be disposed and managed independently of one another.
More generally, the sensor devices for virtual interfaces are usually in the form of a detection array fastened to the front face on a viewing screen the display of which behaves as a function of actions from the user. The control electronics of the screen is placed at the rear of the sensor, and the measurement electrodes are protected from electric disturbances by the active guard.
The application field of these virtual interfaces is not restricted to screen instrumentation but can advantageously be extended to the equipment of keyboards, panels, etc. with various shapes which can be gathered as active surfaces. It is thus desirable to integrate further functions to these active surfaces, such as for example a lighting, tactile feedback devices, displays, to confirm a control command or accompany the user.
Integrating these electric functions at the capacitive electrodes, without degrading the capacitive measurement, is a major issue. In devices of prior art, these functions can only be performed by means of electrical and electronic circuits referenced to an electric ground identical to that of the target object. Consequently, all the circuits are seen by the capacitive electrodes also as target objects, which strongly disturb and degrade the sensitivity of the capacitive sensor.
Document FR2884349 to Roziere is known, which discloses a system based on capacitive sensors including a single measurement electrode. The device enables objects to be detected beyond 10 cm with each electrode, thanks to a floating bridge electronics. The measurements from the electrodes are sequentially read by means of a scrutinising system. However, this device, intended to equip walls of movable instruments, is specifically designed to cover anti-collision applications, and does not cover the gestural interface applications. For the purpose of restricting to the most the stray capacitances giving rise to disturbances, this electronics has the feature to include a floating part referenced to the alternating potential of the guard, which is identical to that of the electrode.
The purpose of the present invention is to provide a capacitive device for detecting a body or an object in a detection space, able to integrate complementary functions at the electrodes without the performances of the capacitive measurement being notably altered.

Capacitive control interface device having display integration


An example operation may include one or more of connecting, by a broker node, to a blockchain comprised of an arranger node and a plurality of client nodes, retrieving from the blockchain, by the broker node, a request for information sent by a client node of the plurality of the client nodes, decrypting, by the broker node, the request for the information with a private key of the broker node, extracting, by the broker node, a public key of a client associated with the client node from the decrypted request for the information, selecting, by the broker node, a set of client properties based on the public key of the client, generating, by the broker node, a modified request for information based on the request for the information and the set of the client properties, and sending, by the broker node, the modified request for the information to the arranger node.

A centralized database stores and maintains data in one single database (e.g., database server) at one location. This location is often a central computer, for example, a desktop central processing unit (CPU), a server CPU, or a mainframe computer. Information stored on a centralized database is typically accessible from multiple different points. Multiple users or client workstations can work simultaneously on the centralized database, for example, based on a client/server configuration. A centralized database is easy to manage, maintain, and control, especially for purposes of security because of its single location. Within a centralized database, data redundancy is minimized as a single storing place of all data also implies that a given set of data only has one primary record.
However, a centralized database suffers from significant drawbacks. For example, a centralized database has a single point of failure. In particular, if there are no fault-tolerance considerations and a hardware failure occurs (for example, a hardware, firmware, and/or a software failure), all data within the database is lost and work of all users is interrupted. In addition, centralized databases are highly dependent on network connectivity. As a result, the slower the connection, the amount of time needed for each database access is increased. Another drawback is the occurrence of bottlenecks when a centralized database experiences high traffic due to a single location. Furthermore, a centralized database provides limited access to data because only one copy of the data is maintained by the database. As a result, multiple devices cannot access the same piece of data at the same time without creating significant problems or risk overwriting stored data. Furthermore, because a database storage system has minimal to no data redundancy, data that is unexpectedly lost is very difficult to retrieve other than through manual operation from back-up storage.
Conventionally, a centralized database is limited by low search capability, lack of security and slow speed of transactions. As such, what is needed is a solution to overcome these significant drawbacks.
Computing nodes in a blockchain network may be used to request and receive information about products available from arrangers (i.e., sellers or providers) of such products. This information about certain structures of these products may be complex and difficult to evaluate. If such product information were more transparent, it would have been easier for the clients interested in these products to evaluate the products. However, the arrangers of the products, typically, do not to disclose such information without determining an identity of a client interested in the product. In other words, the client has to reveal his identity to the arranger, which may not be suitable to many of clients.
Accordingly, a method and system for efficient information transparency control that may balance the requirements of the clients, who need product information transparency, and arrangers, who do not wish to disclose product information for evaluating product risks, is desired.

International Business Machines Corporation


A method of manufacturing an embossed metal gasket includes the steps of supplying a metal gasket preform having opposed first and second planar surfaces and at least one sealing port defined therein, laminating the opposed planar surfaces with a chemical resist material, masking the chemical resist material on the first planar surface in such a manner so as to delineate a raised surface area of a bead circumscribing the sealing port, masking the chemical resist material on the second planar surface in such a manner so as to delineate a recessed surface area of the bead circumscribing the sealing port, curing the unmasked chemical resist material on the opposed planar surfaces of the preform, removing the uncured chemical resist material from the opposed planar surfaces, and exposing the gasket preform to a chemical etching agent to reduce the thickness of the gasket preform in areas which are absent cured chemical resist material.

1. Field of the Invention
The subject invention is related to automotive gasket technology, and more particularly, to a method of manufacturing an embossed metal engine gasket by chemical etching.
2. Background of the Related Art
Metal gaskets are traditionally used to form a seal between two mating components of a mechanical system or device such as an internal combustion engine, to prevent leakage of combustion gases, cooling water, lubricating oil, or the like. A common application involves placement of a gasket between the engine block and cylinder head. Cylinder head gaskets typically extend around the cylinder bores of an engine to seal high pressure combustion gases within the cylinder bores. Once installed, the gasket bears the load from a bolted connection of the engine components and relies upon this load to provide an adequate seal therebetween.
To function as a reliable seal, it is known to provide a raised bead in a metal gasket in the area which surrounds a cylinder bore. The bead elastically deforms by tightening a bolt connection, and the elastic restoring force of the bead provides the required sealing effect. Traditional manufacturing techniques consist of forming a beaded metal gasket by press-forming the bead contour using male and female die halves or molds. This technique requires expensive tooling and can become particularly costly during the design and prototype stages of gasket manufacture.
There is a need in the metal gasket manufacturing art to provide an efficient method of producing an embossed metal gasket which does not require the fabrication of expensive tools and dies.

Embossed metal gasket and method of manufacturing the same


This specification describes technologies relating to detecting anomalous user accounts. A computer implemented method is disclosed which evaluates an unknown status user account. The method described compares features associated with a plurality of known anomalous user accounts stored in a database to features present in the unknown account. A correlation value corresponding to the probability of a specific feature occurring in a particular anomalous user account is calculated and a dependence value corresponding to the degree of dependence between the given feature and at least one other feature is also calculated. A subset of features in the unknown account is generated comprising those features that possess a correlation value less than a threshold value and a dependence value below a maximum correlation value. A risk score for the unknown account is calculated by selecting those features from the subset that maximizes the correlation value. The unknown account is then reviewed by an account reviewer if the risk score exceeds a threshold value.

A modern feature of the Internet is that many Internet service providers, publishers, and social networks provide user accounts. These user accounts allow users to exchange communications inside the network or with other networks or services. This is a feature that is useful and important to the overall marketability and usability of the service. However, some users obtain accounts with the intent to engage in malicious or unwarranted activity. Unsolicited mail accounts, or spam accounts, are used to direct advertisements and online marketing promotions to users who have not expressed any interest in the product, nor have asked to be put on a general product mailing list. It is a difficult and time consuming task to distinguish these anomalous accounts from valid accounts with similar features.
As used herein, the term "Internet account" refers to, generally, user accounts provided by a service provider. Internet accounts may include, for example, e-mail accounts, web site access accounts, comment systems for articles, reviews for music, video and graphics, information feeds, or other material. Also as used herein the term "internet service provider" refers, generally, to a provider of Internet services over a communication network, such as the Internet. For example, an internet service provider may provide an account which includes access to content (e.g., articles, discussion threads, music, video, graphics, information feeds, web sites, etc.), and the ability to contact other members of the service or a different service. In another example, a publisher may provide a message sending service. In yet another example, a service provider grants access to a social network web site.
The term "anomalous account" refers, generally, to an account which is engaged in actions that constitute a violation of the terms and conditions of the Internet Service, or that is engaged in unwarranted, disruptive, abusive, or other actions or activities contrary to proposed usage guidelines, rules or laws. Non-limiting examples of anomalous accounts are spam email accounts, spam member accounts, fraudulent social network accounts.

Detection of anomalous instances through dynamic feature selection analysis


A method and system is provided to enable redundancy in the communication between a plurality of peripheral devices and redundant hosts through redundant switches. The peripheral devices and the host are connected through a Peripheral Component Interconnect Express (PCI-Express) architecture in a data processing system. In an embodiment of the invention, the system includes a switch, a redundant switch, and switch-level exchanging means. The switch-level exchanging means enables the exchange of data packets between the peripheral devices and the host, through an available switch. The available switch is either the switch or the redundant switch. In another embodiment of the invention, the system also includes a redundant host and host-level exchanging means. The host-level exchanging means enables the exchange of data packets between an available host and the available switch. The available host is either the host or the redundant host.

The invention relates to the field of redundancy in systems. More specifically, the invention relates to enabling redundancy in Peripheral Component Interconnect Express (PCI-Express) architecture in a data processing system.
PCI architecture typically enables communication between a plurality of peripheral devices and a host in a data processing system. The host detects and initializes the peripheral devices on the data processing system and enables communication between the peripheral devices.
In conventional data processing systems, the PCI architecture used is a shared bus topology, providing lanes for communication between the peripheral devices and the host. A PCI bus (PCI or PCI-X) is used to enable the shared bus topology. The PCI bus is a parallel multiplexed address/data/control bus, with either 32-bit or 64-bit data paths. However, in current data processing systems, the PCI-Express architecture uses a point-to-point topology, in which a switch replaces the bus as the single shared resource by means of which the peripheral devices communicate. Unlike a shared bus topology, where the peripheral devices collectively arbitrate among themselves for the use of the bus, each peripheral device in a point-to-point topology has direct and exclusive access to the switch. PCI-Express is therefore a serialized version of the PCI bus, with standard PCI-Express transactions being encoded into packets that are transmitted serially in a single-bit stream.
In embedded applications such as network appliances, routers or telecom equipment, the reliability of PCI-Express is a concern, since there is no provision for redundancy; for example, if the switch fails, the data processing system also fails.
Further, in current data processing systems, communication between the peripheral devices fails when no host is operational. Even communication between peripheral devices with processors fails, when no host is operational.
In light of the above discussion, there is a need for a system that provides a robust PCI-Express architecture. This system should provide redundancy when the host or the switch is not operational, to improve the reliability of the PCI-Express architecture. Further, the system should enable communication between—peripheral devices as well as external access to the system, when no host is operational.

System and method for enabling redundancy in PCI-Express architecture


The invention includes a method and apparatus for providing programs to a client device. The method includes providing a first video program stream over a first channel where the first video program stream conveys a first video program having a first quality level, and providing a plurality of second video program streams over a second channel where each of the second video program streams conveys a second video program having a second quality level below the first quality level. The first video program stream and second video program streams are adapted for use by the client device in locally switching from presenting the first video program at the first quality level to presenting a selected one of the second video programs at the second quality level.

In existing video program distribution networks, a channel change request (for switching between television channels) initiated by a user at a client device may be communicated from the client device to a video server that is the source of the television channels. In response to the channel change request, the video server identifies the requested television channel and begins streaming the video program shown on the requested television channel. Due to network delay associated with the propagation of the channel change request from the client device to the video server, as well as network delay associated with distribution of the video stream of the requested channel from the video server to the client device, the user experiences a delay (known as a "zapping" delay) before the program associated with the requested television channel is presented to the user. This delay is exacerbated when the user switches between television channels in rapid succession.
In an attempt to reduce the delay experienced by users during channel change operations, some video program providers cache video content within the video program distribution network. Disadvantageously, however, network caching solutions provide only slight improvements in the delay experienced by the user during a channel change operation. Furthermore, such network caching solutions are expensive in terms of the network storage required to cache numerous video programs, as well as in terms of network resources consumed in continuously updating the video content cached within the video program distribution network, and are not tailored to the preferences of the individual users. Moreover, other solutions that attempt to reduce the delay experienced by users during channel change operations also provide only slight improvements in the delay experienced by the user during a channel change operation.

Method and apparatus for reducing delays due to channel changes


The present invention relates to a snap ring pliers. As to the entire structure of the present invention, a lock member is simply turned to control gripping members to hold or release deformation of a snap ring. The lock member is turned for a stop of a limit member to hold against the upper surface of an elastic plate to block a hook portion from moving upward, so that the elastic plate can be made with a thinner plate. The hook portion is flexibly held by teeth of a ratchet rail. A lower protrusion of the elastic plate is held by an upper protrusion of the lock member with less resistance. This enhances the lock member to be turned in a labor-saving or flexible way.

1. Field of the Invention
The present invention relates to snap ring pliers, and more particularly to snap ring pliers provided with a lock member which is operated in a labor-saving flexible way.
2. Description of the Prior Art
A snap ring is shaped as C and used to retain elements (e.g., bearings or shafts) within a bore. The snap ring has certain elasticity itself to be expanded or contracted. In order to install the snap ring on a desired article, special pliers are required to contract or expand the snap ring so as to position the span ring on a workpiece. For conventional snap ring pliers, the user grips two handles of the pliers to operate the snap ring. The gripping members of the jaws of the pliers are opened or closed to expand or contract the snap ring. The operation is controlled by the user's feeling. That is to say, when the snap ring is contracted or expanded to a predetermined deformation, the user has to control the exerting force exactly. When the operation is inappropriate, the snap ring may fall before positioned. The stored elastic energy of the deformed snap ring may cause the snap ring to fly, hit and hurt the individual who holds the snap ring pliers.
The applicant invented snap ring pliers, disclosed in Taiwan Utility Model Reg. No. M409924 (U.S. Pat. No. 8,347,471), overcome the shortcomings of the conventional snap ring appliers. The snap ring pliers are operated conveniently and save for use. For the hook portion of the elastic plate to engage with the teeth stably and firm, a thick elastic plate is required.
However, the operation for the lock member to hold the elastic plate upward is laborious. If a thin elastic plate is used, the hook portion cannot be compressed or limited to cause the hook portion to bounce unexpectedly or disengagement. Thus, the need for improvement still exists.


Aspects of the present invention disclose a candleholder that may be manipulated by a user to advance a candle within a candleholder to facilitate optimal burning. Embodiments describe a candleholder having a platform to move a candle within a holder. Embodiments disclose a prepackaged semi-soft candle, which is readily movable against a candleholder's inner surface. Embodiments of the present invention disclose a handle permitting a user to manipulate platform height, allowing a candle to be raised or lowered within a candleholder. Embodiments disclose a disc, sized larger than the platform, preferably formed of a soft rubber sponge which stabilizes the candle and prevents candle wax from moving below the platform's surface. An alternative embodiment omits the disc, the platform being sealed by a seal around the platform's perimeter.

Candleholders have been known in the art for some time, and traditional wax candles utilize wax candles in a glass candle holder which are sold full. Over time the candle's wax and wick are consumed, resulting in the candle surface becoming progressively lower relative to the candleholder's surface opening. As a result, certain challenges are presented. Candles burning low in a candleholder are more difficult to light, may allow less air and oxygen to the flame. Further, as a candle burns, the wick may be frayed and discolored, and a user desires to trim the candle's wick. Where the wick position is low in a candleholder, this task is more difficult. Further, many candles are scented, and burning a scented candle releases scent into the surroundings. To optimize scent dissipation, it is useful to have a candle burn near a candleholder's surface—not deep within a candleholder. Further, wax candles tend to be relatively hard and immobile within a candleholder. Lastly, candles burning deep within candleholders typically leave unsightly black soot or carbon deposits on the candleholder's interior making the candleholder, and often candle, black, unsightly, and sooty appearing.

This patent is subject to a terminal disclaimer.


According to the present invention, there is provided a stirred tank for storing yeast slurry to be supplied to fermentation tanks where fermented foods such as beer are fermented, is characterized in that a stirring impeller is provided within the stirred tank and so constructed that a maximum diameter of a rotation body defined by the rotation of the stirring impeller is 60-90% of the inner diameter of the stirred tank, and the height of the rotation body is 70% or more of a standard depth of the yeast slurry normally stored in the stirred tank.

Commonly, in the process of manufacturing fermented products such as beer, yeast recovered from a fermentation tank is stored in the stirred tank, and then returned as seed yeast to the fermentation tank for reuse.
Yeast stored in the stirred tank settles down on a bottom of the stirred tank as the time lapses, resulting in non-uniform yeast concentration in the stirred tank or non-uniform temperature when cooled. Hence, there arises a necessity to stir yeast slurry for overcoming such non-uniformity.
However, likewise butter, soap, etc., the yeast slurry is a non-Newtonian fluid. It is known that in such non-Newtonian fluid, stirring effect in proportion to the increase of the stirring force cannot be necessarily produced even with enhanced force unlike Newton fluid, in which the stirring effect is enhanced in proportion to the stirring force.
On the other hand, there also exists a necessity to protect yeast from damages due to stirring, along with the necessity to perform stirring for uniform yeast concentration and uniform temperature of yeast slurry.
In such a conventional process of manufacturing beer, an inclined paddle impeller, propeller or the like has been mainly used as a stirring impeller provided within the stirred tank for storing yeast slurry to be supplied to the fermentation tank.
However, in case that yeast slurry which is a non-Newtonian fluid is stirred by using such a conventional stirring impeller, there arises a problem that uniformly mixing of the entire yeast slurry is unlikely to be achieved through low-speed stirring.
On the other hand, there arises another problem in high-speed and strong stirring which is performed for improved uniformity in the yeast concentration and temperature of the yeast slurry, that yeast may be damaged and destroyed, and hence its biological activity may be lowered.

Agitation tank for storing beer yeast slurry